i
RECOVERY OF SURFACE ACTIVE MATERIAL
FROM MUNICIPAL WASTEWATER ACTIVATED
SLUDGE
by
Flor Yunuén García Becerra
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Department of Chemical Engineering and Applied Chemistry
University of Toronto
© Copyright by Flor Yunuén García Becerra 2010
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Recovery of surface active material from municipal Wastewater Activated Sludge
Doctor of Philosophy, 2010
Flor Yunuén García Becerra
Department of Chemical Engineering and Applied Chemistry
University of Toronto
Abstract
Wastewater activated sludge is produced during the biological treatment of wastewater.
After treating the sewage, the sludge is allowed to settle. Part of the settled material is
returned to the treatment process as return activated sludge (RAS) and the excess is removed
as waste activated sludge (WAS). The handling and disposal of the sludge are energy and
capital-intensive treatments, with a significant environmental impact. This work studies the
possibility to utilize RAS (an example of wastewater sludge) as a source of surface active
agents. The results indicate that higly surface active materials can be extracted from RAS,
and that the RAS extract has potential applications as a detergent and wood adhesive. The
results also suggest that recovering a suite of products from RAS, a biological heterogenous
source, can be technically feasible.
An effective alkaline treatment was developed (at pH>12) that can extract up to 75% of
the sludge’s organic matter, a yield higher than previously reported. Increasing the
extraction pH increased the extract surface activity, which is linked to increasing the amount
of higher molecular weight molecules and the presence of phospholipids. Increasing the
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extraction pH beyond 11 was also related to extensive cell lysis, increasing significantly the
amount of recovered material and the surface activity of the extract.
The alkaline extract has properties comparable to commercial detergents. Without further
purification, the extract has a low surface tension (37 mN/m on average) and performs
similarly to synthetic detergents. Further assessment of the RAS extract (insensitivity to pH,
surface tension, interfacial tension) suggests that it may be suitable for commercial
applications.
The RAS extract can also be formulated into wood adhesives using glutaraldehyde as a
crosslinker. The extract fraction with 10-50 kDa constituents at pH 9 achieves high adhesive
shear strengths (4.5 MPa on average, at 30% relative humidity and 25°C) with 40% of wood
failure. The adhesive strength of RAS-based adhesives is strongly correlated to its protein
content.
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Acknowledgments
I would like to express my sincere gratitude and appreciation to the following people and organizations:
• I am especially grateful to Professor D. Grant Allen and Professor Edgar J. Acosta for their supervision, encouragement, support, and patience. In their own way, both have inspired me with their admirable qualities as individuals and scientist. I feel fortunate to have had such talented mentorship as a graduate student.
• Professors Levente Diosady and Steven Liss for their thoughtful guidance and
dedication as members of my reading committee. • Professors Mohini Sain and Amarjeet S. Bassi, for their interest in my research and
valuable feedback. • CONACyT (Mexican Advisory Board of Science and Technology) and the
Environmental Consortium Members of the Pulp and Paper Centre at University of Toronto for their financial support.
• The administrative and technical staff in the Department of Chemical Engineering
and Applied Chemistry for their kind assistance through out my stay in this department.
• The summer students and 4th year thesis students that helped me through out my
experiments, many thanks for all your hard work. • My colleagues in Professor Acosta’s lab: Carol, Jackie, Jessica, Sumit, Suniya; and
Professor Allen’s lab: Alex, Chris, Candida, Elena, Ivy, Nalina, Tyler, and Yaldah. Thank you for your support, feedback and friendship.
• Elena, Fleur, Maida, Megan and Sabina for their invaluable experience as graduate
students, brilliant scientists, and lovely friendship.
• All those friends who made my life richer and made me stronger as an individual. • Dr. Levine for providing me the tools to move forward and mi Angelito to help me
appreciate the life in me. • Jesus and Palas Atenea for our childhood friendship and dreams of becoming
someone great.
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• My mother, Silvia Aurora Becerra Langarica and my father, Martin Garcia Hernandez for their love, support, inspiration, the family they gave me and for providing me with the best gift in my life: my sister Silvia Patricia Garcia Becerra.
• To the beautiful Patricia of my heart, my sister of blood and soul.
• Finalmente, le doy gracias a Dios por sus bendiciones, por las oportunidades de
crecimiento, sonrisas brindadas. Finally, I would like to thank God for the blessings, growth opportunities, and the smiles that have been given to me.
No te salves Don’t save yourself (by Mario Benedetti)
No te quedes inmóvil al borde del camino no congeles el júbilo
no quieras con desgana no te salves ahora
ni nunca
No te salves no te llenes de calma
no reserves del mundo sólo un rincón tranquilo
no dejes caer los párpados pesados como juicios
no te quedes sin labios no te duermas sin sueño no te pienses sin sangre no te juzgues sin tiempo
Pero si
pese a todo no puedes evitarlo y congelas el jubilo
y quieres con desgana y te salvas ahora
y te llenas de calma y reservas del mundo
sólo un rincón tranquilo y dejas caer los párpados
pesados como juicios y te secas sin labios
y te duermes sin sueño y te piensas sin sangre y te juzgas sin tiempo y te quedas inmóvil al borde del camino
y te salvas entonces
no te quedes conmigo
Don't stay motionless by the roadside don't freeze joy
or love halfheartedly don't save yourself now
or ever
Don't save yourself don't become serene
don’t make of the world only a safe place
don't let your eyelids close heavy as judgements
don't stay without lips don't sleep without dreams, imagine youself bloodless or judge yourself in haste
But if
in spite of everything you can't help it
and you freeze joy and you love halfheartedly
and you save yourself, become serene,
and you make of the world only a safe place,
and let your eyelids drop heavy as judgements and stay without lips
and sleep without dreams, imagine yourself bloodless,
judge yourself in haste and stay motionless
by the side of the road and you save yourself
then don't stay with me.
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Table of contents
Table of contents..................................................................................................................................................vi
List of tables .........................................................................................................................................................ix
List of figures ........................................................................................................................................................x
CHAPTER 1 INTRODUCTION ................................................................................. 1
1.1 Statement of research ............................................................................................................................1
1.2 Industrial significance of the resulting products .................................................................................2
1.3 Hypotheses and objectives .....................................................................................................................4 1.3.1 Hypotheses ..........................................................................................................................................4 1.3.2 Research objectives .............................................................................................................................4
1.4 Thesis outline ..........................................................................................................................................5
1.5 Publications and conference participations derived from this thesis.................................................8
CHAPTER 2 LITERATURE REVIEW..................................................................... 10
2.1 Potential of wastewater activated sludge as a raw material of value added products....................10
2.2 Wastewater activated sludge constituents..........................................................................................11
2.3 Microbial exopolymeric polysaccharides and proteins .....................................................................14 2.3.1 Microbial exopolysaccharides ...........................................................................................................14 2.3.2 Microbial exoproteins........................................................................................................................15
2.4 Biologically based surface active agents.............................................................................................17 2.4.1 Biological emulsifiers........................................................................................................................17 2.4.2 Biological adhesives..........................................................................................................................18
2.5 Analytical extraction of EPS from wastewater sludge ......................................................................19
2.6 Extraction of value added products from wastewater sludge ..........................................................22
2.7 Product recovery ..................................................................................................................................23 2.7.1 Biopolymer fractionation using ultrafiltration...................................................................................26
2.8 Significance of this research ................................................................................................................28
CHAPTER 3 ALKALINE EXTRACTION OF WASTEWATER ACTIVATED SLUDGE BIOSOLIDS ............................................................................................. 30
3.1 Abstract.................................................................................................................................................30
vii
3.2 Introduction..........................................................................................................................................30
3.3 Materials and Methods ........................................................................................................................34 3.3.1 Materials............................................................................................................................................34 3.3.2 Methods.............................................................................................................................................35 Determination of extraction pH range. ............................................................................................................35 Determination of cell lysis during alkaline extraction. ....................................................................................35 Effect of extraction pH on extract yield and composition ...............................................................................36
3.4 Results and Discussion .........................................................................................................................39 3.4.1 Long-term extraction studies. ............................................................................................................39 3.4.2 Short-term extraction studies (48 h). .................................................................................................42 3.4.3 Effect of extraction pH on extract yield and composition .................................................................44 3.4.4 Extraction kinetics and yield .............................................................................................................45 3.4.5 Chemical composition (protein, polysaccharide, and lipid content)..................................................48 3.4.6 Physical properties of the extracts .....................................................................................................53 3.4.7 Fractionation and NMR characterization of the extract.....................................................................56
3.5 Conclusions ...........................................................................................................................................59
3.6 Acknowledgments ................................................................................................................................60
CHAPTER 4 SURFACTANT-LIKE PROPERTIES OF ALKALINE EXTRACTS FROM WASTEWATER BIOSOLIDS....................................................................... 61
4.1 Abstract.................................................................................................................................................61
4.2 Introduction..........................................................................................................................................62
4.3 Materials and Methods ........................................................................................................................66 4.3.1 Materials............................................................................................................................................66 4.3.2 Methods.............................................................................................................................................66 Effect of extraction pH on surface activity ......................................................................................................66 Interfacial activity and detergency performance of the pH 12.6 extract. .........................................................68
4.4 Results and Discussion .........................................................................................................................70 4.4.1 Effect of extraction pH on surface activity........................................................................................70 4.4.2 Surface activity, interfacial activity and detergency performance of pH 12.6 extract.......................77 4.4.3 Potential applications and outlook.....................................................................................................83
4.5 Acknowledgments ................................................................................................................................84
CHAPTER 5 WOOD ADHESIVES BASED ON ALKALINE EXTRACTS FROM WASTEWATER BIOSOLIDS .................................................................................. 85
5.1 Abstract.................................................................................................................................................85
5.2 Introduction..........................................................................................................................................85
5.3 Materials and Methods ........................................................................................................................88 5.3.1 Materials............................................................................................................................................88 5.3.2 Methods.............................................................................................................................................89 Production of RAS extract...............................................................................................................................89
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Preparation of alkali-modified MSPI...............................................................................................................91 Characterization of the RAS extract, RAS fractions, and modified MSPI ......................................................92 Formulation of adhesives.................................................................................................................................93
5.4 Results and Discussion .........................................................................................................................94 5.4.1 Chemical and physical characterization of RAS extract, extract fractions, and modified MSPI.......94 5.4.2 Rheological properties of formulated adhesives................................................................................98 5.4.3 Analysis of adhesive strength on wood ...........................................................................................101
CHAPTER 6 PRELIMINARY FEASIBILITY ASSESSMENT OF THE PRODUCTION OF DETERGENTS AND ADHESIVES FROM MUNICIPAL RETURN ACTIVATED SLUDGE (RAS)................................................................ 106
6.1 Process description.............................................................................................................................106 6.1.1 Recovery of detergents from RAS...................................................................................................106 6.1.2 Recovery of Adhesives from RAS ..................................................................................................109
6.2 Preliminary analysis for the production of value-added surface active agents from RAS ..........109
6.3 Recommendations for future work...................................................................................................111 6.3.1 Extraction ........................................................................................................................................112 6.3.2 Recovery..........................................................................................................................................112 6.3.3 Formulation .....................................................................................................................................113 6.3.4 Additional issues .............................................................................................................................114
6.4 The importance of utilizing RAS as a resource ...............................................................................114
CHAPTER 7 OVERALL DISCUSSION AND SIGNIFICANCE OF RESEARCH FINDINGS.............................................................................................................. 116
7.1 Effective extraction of biopolymers from a heterogeneous culture ...............................................117
7.2 Potential of RAS to produce surface active agents..........................................................................118 7.2.1 Detergents........................................................................................................................................120 7.2.2 Adhesives ........................................................................................................................................121
7.3 Additional Comments ........................................................................................................................124
CHAPTER 8 CONCLUSIONS AND RECOMMENDATIONS ............................... 125
8.1 Conclusions .........................................................................................................................................125
8.2 Recommendations ..............................................................................................................................127
REFERENCES ...................................................................................................... 129
APPENDICES........................................................................................................ 139
ix
List of tables
Table 2-1 Characterization of EPS from municipal activated sludges with respect to protein and polysaccharides
....................................................................................................................................................................12
Table 2-2 Effect of polysaccharide composition on physical properties (Sutherland, 2001) ...............................14
Table 2-3 Effect of molecular properties of proteins on physical properties (Magdassi, 1996) ...........................16
Table 2-4 Surface tension, γ, of commercial hydrocolloids and proteins (Bhattacharjee, 1994; Garti & Leser,
2001). ..........................................................................................................................................................18
Table 2-5 Shear strengths of various biopolymers as adhesives (Haag et al., 2006; Haag et al., 2004)...............19
Table 2-6 Comparison of selected EPS extraction methods.................................................................................20
Table 2-7 Separation techniques for biotechnological products commonly used for industrial applications
(Ghosh, 2003; Henry & Yonker, 2006). .....................................................................................................25
Table 4-1 Extraction parameters for material recovered at pH 12.0, 12.6, and 12.9 from municipal aerobic return
activated sludge. RAS Sample collected in 2007. The concentration of the extracts are given in grams of
total organic carbon (TOC) per L. Yield is defined on the basis of dry mass total organic carbon (TOC) of
the concentrated RAS sample. Errors indicate the 95% confidence intervals...........................................70
Table 4-2 Elemental analysis (metallic content) of the 2008 extract. All values in mg/L. The Na concentration
in the blank (pH 12.6) is 2267 mg/L. Other metals that were assayed are not reported as they were all
below the detection limit.............................................................................................................................71
Table 5-1 Mass balance on Total Organic Carbon (TOC) bases for the downstream process suggested in
Chapter 4. The yield is calculated with respect to the RAS extract. ..........................................................91
Table 5-2 Rheological parameters of RAS extract and fractions..........................................................................99
Table 6-1 Historical average values (TOC basis) of the concentrated RAS and RAS extract (2005-2009).......107
Table 6-2 Calculations for the production of 1 kg of detergent or adhesives from RAS. The prices of NaOH is
of January 2010 at $0.17/kg (Anonymous, 2010a) and glutaraldehyde at $310/kg (Duvic, 2010)...........110
x
List of figures
Figure 1-1 Simplified diagram of the Activated Sludge Treatment........................................................................1
Figure 1-2 Experimental approach for this thesis ..................................................................................................5
Figure 1-3 Distribution of the experimental phases in the thesis............................................................................7
Figure 3-1 Influence of extraction pH on extraction yield (a) and surface tension of the extract (b). The extract
yield is presented as grams of Total Organic Carbon (TOC) content in the supernatant (extract) obtaining
from treating the equivalent of 100 grams of TOC in the return activated sludge (RAS). Extraction time:
5 weeks. Controls in surface tension measurements: Distilled Water, 70.5 mN/m; NaOH at pH 12.6, 65
mN/m. RAS sample collected in January 2006, Initial TOC = 3500 mg/L ................................................40
Figure 3-2 Effect of extraction time and extraction pH on TOC extraction yield (a) and DNA release from the
extracts (b). The extract yield is presented as grams of TOC content in the supernatant (extract) obtaining
from treating the equivalent of 100 grams of TOC in RAS. Extraction conditions: pH range from 9 to 13;
extraction time up to 48h; room temperature. Total DNA extracted from the concentrated RAS was
measured to be 45 mg/L. RAS sample collected in March 2009, Initial TOC = 6500 mg/L. .....................43
Figure 3-3 Sodium hydroxide (NaOH) required to increase the pH of the concentrated RAS sample. TOC in
concentrated RAS sample: 5.8±0.7 g/L, samples obtained in June-July of 2007. Error bars indicate the
95% confidence intervals. ...........................................................................................................................44
Figure 3-4 Extraction kinetics of alkaline and CER extractions. TOC content in the extracts (supernatants) as a
function of extraction time. Extraction conditions for alkaline extraction: pH range from 12.0 to 12.9;
extraction time up to 24 h; room temperature. TOC in concentrated RAS sample: 5.8±0.7 g/L, samples
obtained in June-July of 2007. Error bars indicate the 95% confidence intervals ......................................45
Figure 3-5 Extraction yield in log10-log10 plot. Influence of extraction time on the extraction yield. .................47
Figure 3-6 Extraction yield towards protein (a), polysaccharides (b), and lipids (c) as a function of extraction
time. These yields were calculated as grams of TOC of the particular fraction measured obtained from the
equivalent of 100 grams of TOC in the concentrated RAS. For proteins the surrogate compound used in
the calculations was BSA and for carbohydrates, D-glucose. TOC in concentrated RAS sample: 5.8±0.7
xi
g/L, samples obtained in June-July of 2007. The TOC of the extract corresponding to each extraction
condition are presented in Figure 3-4. Error bars indicate the 95% confidence intervals ..........................49
Figure 3-7 FAME composition profile at different extraction conditions. The C# ranges presented at the right
side represent the number of carbons in the fatty acid methyl esters (FAME) observed in the
chromatographs. Error bars indicate the 95% confidence intervals ............................................................52
Figure 3-8 Size exclusion chromatograms of alkaline and CER extracts collected after 1 min (dashed line), 4 h
(gray solid line) and 24 h (black solid line) of treatment. (a) pH 12.0; (b) pH 12.3; (c) pH 12.6; (d) pH
12.9; (e) CER. The retention times from BSA (66.43 kDa) and D-glucose (MW 180 Da) are 6.5 min and
13 min, respectively ....................................................................................................................................54
Figure 3-9 31P NMR spectrum of P containing constituents from pH 12.6 extract; a = diester P, b=diester and
teichoic P, c=monoester and inorganic P. ...................................................................................................57
Figure 4-1 Surface tension – concentration (expressed in mg of total organic carbon, TOC, of the extract per
liter of solution) curves for extracts recovered at pH 12.0, 12.6, and 12.9 from return activated sludge
(RAS) samples collected in May of 2007. Error bars indicate the 95% confidence intervals.....................72
Figure 4-2 Surface tension of extracts recovered at pH 12.0, pH 12.6, and pH 12.9 from May 2007 RAS
samples and neutralized with HCl to pHs 11, 9, 7, 4 and 2. Error bars indicate the 95% confidence
intervals.......................................................................................................................................................73
Figure 4-3 Surface tension – concentration curves for extracts recovered at pH 12.0, 12.6, and 12.9 from RAS
samples collected in May of 2007, and neutralized to pH 7. The surfactant solutions, including sodium
dodecyl benzene sulfonate (SDBS), are diluted in 1% NaCl solution. Error bars indicate the 95%
confidence intervals. ...................................................................................................................................74
Figure 4-4 Chromatograms (retention time vs. mV signal) of fatty acid methyl esters (FAMEs) derived from the
extracts obtained at pH 12.0, 12.6, and 12.9 from RAS samples collected in May of 2007. The number of
carbons in the fatty acid chains of the FAMEs of characteristic peaks are annotated in the Figure. ..........76
Figure 4-5 Surface tension – concentration curves for extracts recovered at pH 12.6 from RAS samples
collected in May of 2007 and June of 2008, and neutralized to pH 7. The surfactant solutions are diluted
in 1% NaCl solution. The solid lines are guides for the eye to illustrate the location of CMC. The gray
region represents the range of concentrations were one could define the CMC of these mixtures. ............77
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Figure 4-6 Surface tension – concentration curves for the extract recovered at pH 12.6 from RAS samples
collected in June of 2008, and neutralized to pHs 11, 4, and 7. The concentrated extract was diluted with
1% NaCl solutions to evaluate the effect of neutralization pH on the surface activity of the samples. Error
bars indicate the 95% confidence intervals. ................................................................................................79
Figure 4-7 Interfacial tension of 3.4 gTOC/L solutions of the pH 12.6 extract (neutralized to various pHs)
against heptanes, hexadecane, and toluene. The electrolyte concentration in all the samples was adjusted
to 1% NaCl. As a reference, the interfacial of SDBS against these oils, and at pH 7, was 9.9 mN/m for
heptane, 8.9 mN/m for hexadecane, and 7.2mN/m for toluene...................................................................80
Figure 4-8 Increment (∆) in % of hexadecane removal from cotton swatches using the surfactant formulation
over water, as a function of surfactant concentration expressed in terms of CMC. For the detergency tests
using the 2007 extracts (using aged stains) 19% of hexadecane was removed using water-only wash. For
the detergency tests using the 2008 extracts (using freshly stained swatches) 44% of hexadecane was
removed using water-only wash. Washing solutions (extract and SDBS solutions) were at neutral pH,
and contained 1% NaCl. .............................................................................................................................81
Figure 4-9 Swatches before (soiled) and after the wash cycle using different washing solutions: distilled water,
1 CMC extract (recovered at pH 12.6) and 1 CMC SDBS. Washing solutions (extract and SDBS
solutions) were at neutral pH and 1% NaCl during the wash step. .............................................................82
Figure 4-10 Unsoiled swatches washed with 1 CMC of extract recovered at pH 12.6 (left) and with deionized
water (right). The swatch washed with the extract shows some sign of “yellowing”. ................................83
Figure 5-1 Fractionation Scheme. The RAS extract was fractionated through 4 stages, two ultrafiltration stages
(UF1 and UF2) and two diafiltration (washing) stages. The RAS extract, Permeate 1, Retentate 2, and
Retentate 4 were used for the formulation of wood adhesives....................................................................91
Figure 5-2 Total Organic Carbon and Total Nitrogen content with respect to the analytes’ total solids. Error
bars indicate the 95% confidence intervals. ................................................................................................95
Figure 5-3 Protein and Polysaccharide content with respect to the analytes’ total solids. Error bars indicate the
95% confidence intervals. ...........................................................................................................................96
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Figure 5-4 Size exclusion chromatograms of RAS extract, extract fractions, and modified MSPI. (a) RAS
extract; (b) Permeate 1; (c) Retentate 2; (d) Retentate 4; (e) modified MSPI. The retention times from
BSA (66.43 kDa) and D-glucose (MW 180 Da) are 8.14 min and 19.27 min, respectively. ......................97
Figure 5-5 Surface tension of solutions at 4g/L. Error bars indicate the 95% confidence intervals. The surface
tension of deionized water (3 µS/cm) measured under the same conditions was 70.9±0.2 mN/m. ............98
Figure 5-6 Effect of addition of glutaraldehyde on viscosity. Viscosity vs. shear rate (semi-log10 scale) of 15%
w/w modified MSPI solutions: a) with 0.5%w/w glutaraldehyde, and b) without glutaraldehyde. ............99
Figure 5-7 Viscosity vs. shear rate (semi-log10 scale) of adhesive formulations with glutaraldehyde: (a)
Retentate 2 (b) RAS extract at 30%, (c) Permeate 1 at 15%, and RAS at 15%. .......................................100
Figure 5-8 Effect of curing conditions on the formulations’ adhesive strength. Permeate 1 formulations only
exhibited adhesiveness under hot press conditions. RAS extract formulations did not present adhesiveness
at the cold dead weight curing conditions. The MSPI formulations were not tested under the cold press
conditions. The average adhesive strength for TitebondTM is 5.5 ±0.8 MPa. The Error bars indicate the
95% confidence intervals of 8 replicates. .................................................................................................102
Figure 5-9 Correlation between the protein and polysaccharide content in the formulations and the adhesive
strength at hot press curing conditions (all adhesive formulations are at 15% w/w of RAS extract/fraction
or MSPI). In the adhesive strength vs. protein/polysaccharide % plot, the linear R-squared values for
protein and polysaccharide contents are 0.96, and 0.12, respectively.......................................................103
Figure 6-1 Block flow diagram of the alkaline treatment to extract highly surface active material from return
activated sludge.........................................................................................................................................107
Figure 6-2 Titration curves of return sludge (100mL) with 0.1M NaOH conducted at room temperature, 20-
25°C (2 replicates), and in icebath, 0°C. The blank is the titration of distilled H2O (100mL) at 20-22°C.
..................................................................................................................................................................108
Figure 6-3 Block flow diagram of the alkaline treatment to produce detergents from RAS, including mass
balance calculations. The density of the concentrated RAS and RAS is similar to that of water (1 kg/L).
..................................................................................................................................................................108
Figure 6-4 Block flow diagram of the alkaline treatment and recovery of wood adhesive raw material from
RAS, including mass balance calculations................................................................................................109
1
1 Chapter 1 Introduction
1.1 Statement of research
Activated sludge is produced during of the biological activated sludge treatment of
wastewater (Kroiss, 2004; Ødegaard et al., 2002)(Figure 1-1). After treating the sewage, the
sludge is allowed to settle. Part of the settled material is returned to the treatment process as
return activated sludge (RAS) and the excess is removed as waste activated sludge. Due to
its composition, RAS has the potential to be harvested for industrial applications. It is made
up predominantly of water and organic matter, mainly microorganisms and the biopolymers
they produce during flocculation and consumption of the organic contaminants in
wastewater. The principal constituents of RAS solids include cells, proteins,
polysaccharides, humic substances, and lipids (Frølund et al., 1996). Consequently,
wastewater sludge could have the potential to be utilized as a source of biologically derived
compounds.
Disposal,
~50% of total operation costs
Aeration
Tank
Waste Activated
SludgeReturned Activated Sludge
(RAS)
WastewaterSettling
Tank
Treated Wastewater
Disposal,
~50% of total operation costs
Aeration
Tank
Waste Activated
SludgeReturned Activated Sludge
(RAS)
WastewaterSettling
Tank
Treated Wastewater
Figure 1-1 Simplified diagram of the Activated Sludge Treatment.
2
The biological products being sought after in this work are surface active agents,
specifically emulsifiers and adhesives. Materials from wastewater sludge flocs exhibit
important surface active properties as they enable intra and extracellular processes to take
place, which are mostly interfacial phenomena. They play an essential role in microbial
physiological behavior, such as cellular motility, cell-cell interactions (aggregates and
biofilm formation, quorum sensing), cellular differentiation and maturation, and substrate
accession (Van Hamme et al., 2006). Further, it has been suggested that microorganisms
from wastewater treatment plants may have evolved for producing biosurfactants capable of
degrading complex substrates found in wastewater (Mercade & Manresa, 1994).
1.2 Industrial significance of the resulting products
The biological production of surface active agents can be done through fermentation or
using a feedstock derived from biological sources and chemically modifying such source to a
more conventional surfactant. Biologically derived surfactants, or bio-based surfactants,
have been studied for a range of industrial applications (Schramm et al., 2003; Singh et al.,
2007; Van Hamme et al., 2006) and have shown to offer important advantages over chemical
surfactants, including higher biodegradability, lower toxicity, higher foaming and high
surface activity at extreme temperatures, pH, and salinity (Desai & Banat, 1997). Most
importantly, they can be synthesized from renewable feedstocks, including waste biomass
(Desai & Banat, 1997; Mercade & Manresa, 1994), which addresses the growing need for
natural, bio-based products in the surfactant and adhesives industries (McCoy, 2008).
In addition, utilizing wastewater sludge to produce bio-based surface active agents has
the potential of reducing the net cost and environmental impacts of its disposal (Ødegaard et
al., 2002). Handling and disposal of activated sludge represents 50% of the operation costs
3
in wastewater treatment plants. The environmental impact of its disposal (landfill,
incineration, etc.) is also considerable (Kroiss, 2004). This is only expected to worsen in
places like Toronto, Canada, where its production is projected to increase by 80% over the
next 30 years (Schramm et al., 2003; Vyhnak, 2008). Further, extracting these chemicals
from wastewater sludge enhances its dewatering and could help in the recovery of the treated
water (Y. Liu, 2003).
The overall objective of this PhD thesis is to recover a range of value-added surface
active materials from municipal activated sludge. It is based on the notion of producing a
few basic chemicals from a heterogeneous raw material, a parallel concept to the production
scheme in the petrochemical industry. In this case, a biological raw material, RAS, is to be
converted to bulk chemical products and finally to fine chemicals, a concept sometimes
referred to as biorefinery (Kamm et al., 2006). This is in contrast with current biological
production processes that focus on the recovery of a particular product. RAS is used as
equivalent to WAS, which is a waste by product of the activated sludge treatment of
wastewater.
It is expected that this research will make three main contributions. First, it will
develop an effective procedure for extracting biopolymers from a heterogeneous culture.
Second, it will explore the potential of RAS to produce bio-based surface active agents,
specifically emulsifiers and adhesives. Thirdly, it will determine the physicochemical
properties of RAS constituents in the context of industrially relevant surface active agents
(detergents and adhesives).
4
1.3 Hypotheses and objectives
1.3.1 Hypotheses
The hypotheses tested in this work are:
• Constituents from RAS have properties of surface active agents that can be exploited
as surface active material, specifically emulsifiers and adhesives.
• Due to the physicochemical properties of surface active agents found in RAS, they
can be recovered using a combination of charge and molecular weight based
fractionation methods.
1.3.2 Research objectives
The objectives are:
1. Design an extraction process to recover a range of compounds from RAS according to
their charge and molecular weight.
2. Determine the chemical characteristics of the extracts and relate them to their
performance as detergents and/or adhesives.
3. Determine the physical properties of the extracts as emulsifying/detergency agents.
4. Determine the physical properties of the extracts as adhesive agents.
This project is divided into 4 experimental phases to achieve the mentioned objectives
and test the hypotheses. Figure 1-2 illustrates this approach and the experimental overview
in each phase.
Phase I. Extraction of surface active agents from wastewater sludge: develop an effective
extraction technique based on molecular charge and weight fractionation of wastewater
constituents, depending on the final application of the extracts.
Phase II. Extract characterisation: analyze the chemical composition of the extract and relate
it to physical properties of detergents and/or adhesives.
5
Phase III. Assess extract detergency performance: determine the extract’s surface
tension, interfacial tension, and detergency performance at various conditions (pH,
salinity, and concentration).
Phase IV. Formulate extract into wood adhesives: fractionate the extract constituents
recovering the most hydrophobic and with highest molecular weight; and evaluate the
adhesive strength of the hydrophobic fraction in different wood adhesive formulations.
Phase IV
AdhesiveFormulation
Phase IBiopolymer Extraction
Phase II
Extract Characterization
Phase III
Detergent Characterization
Measure•Surface Tension•Interfacial Tension •Detergency Performance
Benchmark: sodium dodecylbenzene-sulfonate(SDBS)
Measure•Total Organic Carbon (TOC)•Total Nitrogen (TN)•Proteins•Carbohydrates•Lipids•Molecular weight range•Cell lysis (as DNA)
•Design purification process•Formulate wood adhesive•Measure adhesive strength
Benchmark: commercial soybean-based adhesive
Phase IV
AdhesiveFormulation
Phase IBiopolymer Extraction
Phase II
Extract Characterization
Phase III
Detergent Characterization
Phase IV
AdhesiveFormulation
Phase IBiopolymer Extraction
Phase II
Extract Characterization
Phase III
Detergent Characterization
Measure•Surface Tension•Interfacial Tension •Detergency Performance
Benchmark: sodium dodecylbenzene-sulfonate(SDBS)
Measure•Total Organic Carbon (TOC)•Total Nitrogen (TN)•Proteins•Carbohydrates•Lipids•Molecular weight range•Cell lysis (as DNA)
•Design purification process•Formulate wood adhesive•Measure adhesive strength
Benchmark: commercial soybean-based adhesive
Figure 1-2 Experimental approach for this thesis
1.4 Thesis outline
The thesis is reported as a collection of manuscripts. The document is divided into
eight chapters: a general introduction; a general literature review; three manuscripts that
include the main findings of this research; a preliminary feasibility analysis of the proposed
extraction/recovery scheme and recommendations for future work to enhance the scheme’s
productivity; an overall discussion of the findings and their significance; and the main
conclusions of the findings and recommendations.
6
Chapter One contains the thesis overview and the document outline. Chapter Two
provides a general literature survey for this research. It reviews the constituents commonly
present in municipal wastewater sludge in order to understand the type of carbon compounds
available for exploitation. The chemical and physical nature of the principal RAS
constituents (exopolysaccharides and proteins) are reviewed to learn about the molecular
elements that make them surface active. Also, to understand the mechanism and expected
qualities of the extracted products from RAS, literature on commercial biological emulsifiers
and adhesives is reviewed. Current RAS extraction methodologies are identified and
compared to provide the basis to develop an extraction technique able to recover surface
active material from RAS. Further, since isolation and purification of biological products are
technical and economical roadblocks in bioprocessing, bioseparation techniques are
discussed focusing on ultrafiltration as the selected biotechnological unit operations for this
project. The review in Chapter 2 is aimed at understanding the general theoretical basis for
this work and show the potential of wastewater activated sludge as a raw material of value
added products. Chapter 2 also mentions the projected significance of the research findings.
More in-depth literature reviews are given in Chapters Three, Four and Five, which showcase
this research’s main findings in manuscript format.
Figure 1-2 depicts how the results of the experimental phases of this research are
reported in Chapters Three through Five. Chapter 3 describes the alkaline extraction
technique that was developed to recover surface active material from RAS and analyses its
extraction kinetics and the physical and chemical characterization of the resulting extracts.
Chapter 3 is based on a manuscript that has been accepted for publication in the Bioresource
Technology Journal. Chapter Four describes the surface and interfacial activity of the RAS
7
extract and assesses its detergency. This work has been accepted for publication in the
Journal of Surfactants and Detergents. The RAS extract performance as a wood adhesive is
reported in Chapter Five. The research conducted in Chapter 5 includes developing an
ultrafiltration fractionation scheme to recover high-molecular-weight and hydrophobic
constituents from the RAS extract. These fractions are then formulated into glutaraldehyde-
crosslinked adhesives and their adhesive strengths assessed. The work in Chapter Five will
be submitted for review to the Journal of the American Oil Chemists’ Society.
AdhesiveFormulation
Biopolymer Extraction
Extract Characterization
Detergent Characterization
Chapter 3
Chapter 5Chapter 4
Figure 1-3 Distribution of the experimental phases in the thesis
Chapter Six presents a preliminary assessment of the feasibility of implementing the
developed extraction/recovery scheme at an industrial scale and suggests future work that
could improve the productivity of such scheme. Chapter Seven includes an overall
discussion of the results from Chapters 3 through 5, and highlights their significance.
Finally, Chapter Eight lists the main conclusions of the results from this thesis, and contains
recommendations for future research topics to further understand the physicochemistry and
surface activity of RAS.
8
1.5 Publications and conference participations derived from this thesis
Publications
Garcia-Becerra, F. Y., Acosta, E. J., & Allen, D. G. (2009). Surfactant-like properties of
alkaline extracts from wastewater biosolids. Journal of Surfactants and Detergents,
13(3), 261-271.
Garcia-Becerra, F. Y., Acosta, E. J., & Allen, D. G. (2010). Alkaline extraction of
wastewater activated sludge biosolids. Bioresource Technology, 101(18), 6983-6991.
Garcia Becerra F.Y., Allen D.G., Acosta E.J. (2010). Chapter 9: Surfactants from Waste
Biomass, in Surfactants from Renewable Resources. Edited by M. Kjellin and I.
Johansson, John Wiley & Sons, Ltd., Wiltshire, Great Britain, 167-185.
Conference oral presentations
Garcia Becerra F.Y., Acosta E.J, Allen D.G. Production of Detergents from Wastewater
Sludge. Presented at the 8th World Conference of Chemical Engineering, Montreal,
Quebec. August 24, 2009
Garcia Becerra F.Y., Acosta E.J., Allen D.G. Extraction of Adhesives and Emulsifiers from
Wastewater Biosolids. Presented at the 58th Canadian Chemical Engineering
Conference. October 22, 2008.
Garcia Becerra F.Y., Acosta E.J, Allen D.G. Production of Biosurfactants from Wastewater
Sludge. Presented at the 82nd Colloid and Surface Science Symposium from the
American Chemical Society Division of Colloid and Surface Science. June 16, 2008.
Garcia Becerra F.Y., Xuan X.Y., Maniyali Y., Acosta E.J, Allen D.G. Extraction and Uses
of Biopolymers from Wastewater Sludge. Presented at the 57th Canadian Chemical
Engineering Conference. October 31, 2007.
9
Conference poster presentations
Garcia Becerra F.Y., Acosta E.J, Allen D.G. Extraction of Value Added Products from Waste
Activated Sludge. Poster session presented at the 82nd Annual Water Environment
Federation Technical Exhibition Conference, Orlando, Florida. October 12, 2009.
10
2 Chapter 2
Literature review
2.1 Potential of wastewater activated sludge as a raw material of value added products
Wastewater sludge can be a reliable source of carbon compounds, and producing
marketable materials from it could add value to this waste stream. Municipal RAS
production ranges from 20 to 40 kg dry matter per population equivalent per year1 (Kroiss,
2004). However, most of the sludge generated is currently directed to incineration,
landfilling or disposed in the sea (Hospido et al., 2005). In turn, the operating costs for
sludge handling and disposal are about 40% of the total wastewater treatment operating costs,
followed by the expense of sludge stabilisation representing 8 to 10% of total operating costs
(Kroiss, 2004). By utilising wastewater sludge there is the potential to reduce the economic
and environmental impact of RAS disposal. In addition, biopolymers obtained from this
source offer important advantages, including biodegradability and reduced reliance on
petroleum (McWilliams, 1991).
With respect to the available market for the resulting products, the emulsifiers and
adhesives industries are one of the fastest growing markets in the chemical field. The global
demand for surfactants was estimated to be $20.8 billion in 2009 while the adhesives and
sealants global market was valued at $61 billion USD in 2009 with a growth rate of about
3%/year in volume (Coons, 2009). The primary reasons for these high rates are the health
and environmental hazards of current formulations, such as solvent-based ones (Anonymous,
1 Population equivalent (in wastewater treatment) refers to the amount of oxygen-demanding substances whose O2 consumption during biodegradation equals the average oxygen demand of the wastewater produced by one person. For practical calculations, it is assumed that one unit equals 54 grams of BOD per 24 hours (United Nations, 2001).
11
2000; Coons, 2009). The resulting products from this research target the mentioned driving
causes since they will be produced from a renewable source (i.e., active sludge) and will be
water-based formulations (i.e., biopolymers).
2.2 Wastewater activated sludge constituents
Activated sludge extracellular polymeric substances (EPS) have been reported as major
RAS components. EPS are the building blocks of activated sludge flocs and biofilms and
represent the main constituents of the organic fraction for these microbial aggregates
(Sutherland, 2001). They have two different origins, from metabolism/lysis of
microorganisms and the wastewater itself. The compounds found in the EPS matrix include
proteins, polysaccharides, humic substances, DNA, lipids, and uronic acids (Garnier et al.,
2005). Other important floccomponents are microorganisms and up to 98% weight water
(Keiding et al., 2001). The roles of EPS include protecting microorganisms from a hostile
environment, supporting cells with nutrients, and allow communication between cells. The
gel-like EPS network also permits cells to adhere to diverse surfaces in natural and
engineered systems, such as biological wastewater treatment processes (Sutherland, 2001).
The most important constituents in flocs/biofilms are polysaccharides and proteins,
either alone or in associations with other compounds, such as glycoproteins, rhamnolipids,
lipoproteins, etc. These two types of biopolymers play important roles in determining the
physical properties and structures of the microbial agglomeration. Exopolysaccharides are
believed to mainly have the role of hydrocolloids and proteins are believed to be responsible
of hydrophobic and covalent interactions in the formation and adhesiveness of flocs and
biofilms (Sutherland, 2001). Further, associations of proteins and polysaccharides, such as
glycoproteins, are believed to be capable of stabilising oil-in-water emulsions due to their
12
amphiphaticity (Garti & Leser, 2001). The molecular masses of EPS polysaccharides and
proteins range from a few thousands to several million Daltons and their components contain
a large number of negatively charged functional groups including carboxyl, amino, sulphate
and phosphate (Garnier et al., 2005). Consequently, exopolysaccharides and proteins have a
significant role in adhesion phenomena, and in the formation of expolymeric networks as the
supporting matrix of microbial aggregates (Görner et al., 2003; Wilen et al., 2003).
Table 2-1 lists the amounts and the main types of exopolymers present in municipal
wastewater sludge. Total solids (TS) and volatile solids (VS) are a common measure of the
mass extractable from wastewater sludge. VS is taken as a measure of the organic content,
and thus the amount of EPS/product that could be extracted from wastewater sludge. A more
complete analysis of the studies presented in the following table is in the Appendix.
Table 2-1 Characterization of EPS from municipal activated sludges with respect to protein and polysaccharides
Activated Sludge
TotalSolids g/L
Volatile Solids g/L
VS/TS EPS Extractedtotal / Method
Carbohydrates Proteins Reference
16 activated sludges
1.89 1.56 0.83 14%TS 17%VS CER
22.63 mg/g VS average
135.98 mg/g VS average
(Urbain et al., 1993)
1 municipal activated sludge
1.6 1.1 0.69 CER 55 mg/g VS average
236 mg/gVS average
(Görner et al., 2003)
5 municipal activated sludges, 2 industrial activated sludges
NR NR NR 10 to 30%VS CER
50-120 mg/g VS 270-500 mg/g VS
(Wilen et al., 2003)
1 municipal activated sludge
NR NR 0.88 6% TS 7% VS Sonication
159.1 mg/g VS 395.45 mg/g VS
(Guibaud et al., 2005)
2 municipal activated sludges
NR NR 0.59 to 0.63
20-25%TS 33-42%VS CER
179-181 mg/g VS
462-523 mg/g VS
(Frølund et al., 1996)
NR: Not reported
13
In general, the following trends were observed:
1. In municipal wastewater sludges organic content (VS) ranges from 59% to 88% of
the total solids (dry weight) as indicated in the VS/TS column in Table 2-1, with an
average of 70% TS.
2. The average amount of extracted EPS is 20% of the total organic content (with a wide
range of 10 to 42%) as indicated in the fifth column of the table above.
Carbohydrates represent 11% and proteins 36% of this extracted fraction, as shown in
the sixth and seventh column (table above).
From the second point above, there is still on average 80% of organic content that is not
recovered in current analytical extraction procedures. Assuming an average value of biosolid
availability of 30 kg of dry matter (TS) per population equivalent per year, 21 kg of organic
material per population equivalent per year could be harvested. Since our technique will be
developed for production purposes it will aim at recovering significantly higher yields than
current analytical methods.
Considering the types and amount of exopolymers found in EPS, RAS has the potential
to be a source of biopolymers such as polysaccharides and proteins. Further, studies by
Garnier et al., 2005 and Görner et al., 2003 observed that wastewater treatment plants have a
chromatographic fingerprint, i.e. a consistent profile of chromatographic peaks, at stable
operating conditions. This might suggest that once an extraction procedure is implemented,
the quality of exopolymeric output is likely to be uniform throughout time from wastewater
treatment plants operated at a steady operation mode. Such feature is important for the
industrial production of chemicals and reinforces the potential of wastewater sludge as a
possible feedstock for the production of value added biopolymers.
14
2.3 Microbial exopolymeric polysaccharides and proteins
Identifying the chemical and physical characteristics of microbial EPS is important
since our proposed extraction method is intended to separate RAS constituents according to
their charge and molecular weight.
2.3.1 Microbial exopolysaccharides
Chemical Properties. Most microbial polysaccharides are either homopolysaccharides
composed of a single sugar unit, or heteropolysaccharides where regular repeat units are
formed from 2 to 8 possible monosaccharides. Acyl groups or inorganic substituents such as
phosphate or sulphate may also be present. Bacterial polysaccharides contain mainly
hexoses or methylpentoses commonly found together with uronic acids. Due to the uronic
acids and inorganic subtituents, polysaccharides may be either neutral or polyanionic in
charge. Exopolysaccharides can display a broad range of linkage types and acylation
patterns as indicated in Table 2-2. Consequently, considerable differences in physical
properties are observed.
Table 2-2 Effect of polysaccharide composition on physical properties (Sutherland, 2001)
Component Effect Properties affected Example
Neutral sugars Uncharged polymer Solubility Cellulose, floc and biofilm EPS
Uronic acids Polyanionic Solubility and ion binding Xanthan gum, alginates Pyruvate Polyanionic Ion binding and transition Xanthan gum,
galactoglucans Methylpentoses Lipophilicity Solubility Floc and biofilm EPS Acetylation Solubility Gelation, reduced ion binding Alginates, gellan Side chains Various Solubility Scleroglucan, xanthan gum 1,3 or 1,4 linkages Rigidity Solubility Curdian, cellulose 1,2 linkages Flexibility Solubility, stability Dextrans
Physical Properties. Due to their macromolecular nature, microbial polysaccharides
can form gels or hydrocolloids on their own, in the presence of multivalent cations, or when
mixed with other polysaccharides. According to their functional groups some of these
15
polysaccharides are effective lipopolysaccharide-like, amphipathic molecules capable of
stabilizing oil-in-water emulsions (Sutherland, 2001).
In an indirect manner, the adhesiveness of exopolysaccharides has been analyzed
(Tsuneda et al., 2003). Cell adhesion onto a glass bead surface was correlated with the
amount of EPS components and cell surface characteristics such as zeta potential and
hydrophobicity using 27 heterotrophic bacterial strains isolated from a wastewater treatment
reactor. It was observed that amounts of hexose and pentose exhibited good correlations
with cell adhesiveness for exopolymer-rich strains, indicating that hexose and pentose,
facilitate cell adhesion onto glass beads. Also, there was no correlation between protein
content with adhesiveness, and between protein content and hydrophobicity. Thus,
exopolysaccharides can mediate both cohesion and adhesion of phenomena, and play a
crucial role in maintaining structural integrity in flocs and biofilms.
2.3.2 Microbial exoproteins
Chemical Properties. Contrary to the case of exopolysaccharides, the role and
characteristics of microbial exopolymeric proteins in RAS has not been extensively studied.
This is mostly because prior to the year 2000, proteins were not considered the dominant
components in EPS wastewater flocs and biofilms (Neyens et al., 2004). In general, proteins
are typically amphiphilic polymeric substances made of up to 20 possible amino acid
residues, combined in definite sequences by peptide bonds (primary structure). In many
cases polypeptide chains are present in helical or βsheet configuration (secondary structure),
which are stabilized by intramolecular bonding, such as sulphide or hydrogen bridging. The
tertiary structure is determined by the folding of the polypeptide chains to more or less
compact globules (subunits), maintained by hydrogen bonding, van der Waals forces,
16
disulfide bonds, etc. Further, the subunits can associate into small clusters, which is known
as the quaternary structure.
Physical properties. The main molecular properties of proteins responsible for their
physical properties are size, charge, and features of structure and stability. Table 2-3
illustrates the effect that different chemical properties have on the physical properties of
proteins. Matrix characteristics can also greatly influence protein physicochemical
properties; they include pH, ionic strength, temperature, redox state, and presence of
interfaces (Magdassi, 1996).
Table 2-3 Effect of molecular properties of proteins on physical properties (Magdassi, 1996)
Property Effect Main properties affected* Example
Chemical composition (primary structure)
Balance of polar, non polar, charged and neutral amino acid side chains
Solubility and amphipacity Variety in composition allows proteins to bind with surfaces of different chemical nature at different conditions.
Molecular size Multipoint binding Adsoption Considerable difference between activation energies for the adsorption and desorption processes
Charge (density and distribution)
Net charge and nonuniform distribution of ionic patches
Surface activity Greater surface activity near the isoelectric point, due to minimizationof electrostatic interactions between molecules.
Protein structure (tertiary and quaternary structure)
Conformational stability
Rearrangement at interface (molecule rigidity)
“Soft” proteins can undergo structural rearrangement and interact at a greater degree than “rigid” proteins.
* Other functional properties include water adsorption and binding, rheology modification, emulsifying activity, emulsion stabilization, gel formation, foam formation, and stabilization, and fat adsorption In the case of microbial flocs and biofilms, a high content of negatively charged amino
acids has been found in exopolymeric proteins. It could then be suggested that proteins may
be more involved than sugars in electrostatic bonds with multivalent cations, underlining
their key role in floc and biofilm structure and adhesion (Frølund et al., 1996; Sutherland,
2001).
17
2.4 Biologically based surface active agents
Biological emulsifiers and adhesives were reviewed in order to understand the modes
of action of the possible products from wastewater sludge. In addition, the physical
properties of commercially available bio-based products were researched to have an idea of
the expected qualities of the extracted products from wastewater sludge.
2.4.1 Biological emulsifiers
Many industrial products (e.g. food, pharmaceuticals, agro-chemical, and cosmetics)
are categorized as oil-in-water emulsions, which consist of small lipid droplets dispersed in
an aqueous medium. However, emulsions are thermodynamically unstable systems prone to
destabilisation. Emulsifiers are surface-active ingredients that are widely used to improve the
stability of oil-in-water emulsions. Emulsifiers can readily adsorb at water and oil interfaces,
lowering the interfacial tension and facilitating emulsion formation (McClements, 2004).
There are differences in the mode of action between polysaccharide and protein
emulsifiers. In the case of polysaccharides, those that are low-molecular-weight, water-
soluble, and can adsorb onto molecules and reduce surface and interfacial tensions are termed
hydrocolloid-emulsifiers. As opposed to proteins, hydrocolloids do not posses hydrophobic,
flexible moieties. Thus, their adsorption onto solid or liquid interfaces is rather weak.
Nevertheless, stabilisation seems to be steric due to weak adsorption of the hydrocolloid onto
the oil droplets. The adsorption can form a thick gel-like semi-organized layer, which
exhibits strong birefringency (Garti & Leser, 2001).
Proteins, on the other hand, are considered high-molecular-weight surfactants. Proteins
adsorb to the surfaces of freshly formed oil droplets created by homogenisation of oil-water-
protein mixtures, where they facilitate further droplet disruption by lowering the interfacial
tension and retard droplet coalescence by forming protective membranes around the droplets
18
(McClements, 2004). Proteins can then stabilise droplets against flocculation and coalesce
during long-term storage through their abilities of generating repulsive interactions (steric
and electrostatic) between oil droplets and forming an interfacial membrane resistant to
rupture. It is important to note that hydrocolloids in conjunction with proteins
(polysaccharide-protein mixtures) are also known to be efficient emulsifiers.
Table 2-4 shows the surface tension of various commercial exopolymeric surface active
compounds. As reference, commercial petroleum counterparts at 1% w/w are able to achieve
surface tensions >40 mN/m (Cooper et al., 1981) which implies an improved performance by
biopolymers.
Table 2-4 Surface tension, γγγγ, of commercial hydrocolloids and proteins (Bhattacharjee, 1994; Garti & Leser, 2001).
Surfactant Surfactant weight% γγγγ (nN/m)
Tragacanth 0.6 42 Xanthan 0.6 43 Guar 0.7 55 LBG (locus bean gum) 0.7 50 Fenugreek 0.7 48 Casein 0.06 51 Bovine serum albumin 0.04 53 Rhamnolipid (glycolipid from P. aeruginosa)* 0.77 31 Surfactin (lipopeptide from B. subtilis) * 0.0013 27 * These products are in the development stage for their commercialization (Sen & Swaminathan, 2005; Wei et al., 2005).
2.4.2 Biological adhesives
The chemistry of industrial adhesive bonding is generally of two types: high energetic
(covalent or chelate) or a collection of weaker, non-covalent interactions. In biological
systems, it has been suggested that adhesiveness is dependent on weaker noncovalent
interactions across the interface. These interactions include charge-charge, hydrogen bonds,
dipole-dipole, induced dipole-dipole, and nonpolar coupling, among others. The mentioned
interactions are short ranged and good adsorption is required for a strong adhesive joint.
19
Several mechanisms and theories of bioadhesion exist. These include the electronic theory
(cross-linking), adsorption theory (non-covalent interactions), wetting theory, and diffusion
theory (entanglement) (Berglin et al., 2005; Geraghty et al., 1997).
Table 2-5 summarizes the adhesive strength (shear strength) of commercially available
bacterially derived adhesives. These products have a carbohydrate content of 95% dry
weight. As reference, commercial petroleum-based wood adhesives used at room
temperature range from 1 to 50 MPa, depending on the substrate, joint design, and rate of
loading (Haag et al., 2006; Haag et al., 2004).
Table 2-5 Shear strengths of various biopolymers as adhesives (Haag et al., 2006; Haag et al., 2004).
Product Source Use Shear strength
MB Adhesive, 30%w/w in water
Bacterial fermentation Wood bonding applications
<20 MPa (53%RH, 22°C)
SB Adhesive, 36%w/w in 44% ethanol (aq)
Bacterial fermentation Wood bonding applications
30 MPa (53%RH, 22°C)
RH: Relative Humidity
2.5 Analytical extraction of EPS from wastewater sludge
This project aims to extract surface active compounds from RAS. Thus, EPS extraction
techniques were reviewed to determine the extraction conditions to be used in this project.
Four comparative studies of the principal extraction methods currently performed for
wastewater sludge EPS were reviewed. All comparative studies used municipal wastewater
sludge as the EPS source and performed the same quantitative analyses, the Anthrone method
for carbohydrates and the Lowry method for proteins. A protein extraction performance
value, PEP, was assigned within the context of each study. An assigned value of 1 implied
that the greatest amount of EPS was extracted using the given methodology in a presented
study. In order to analyse the selectivity of each technique towards carbohydrates
20
(polysaccharides) or proteins, a ratio was calculated between the extracted values of
carbohydrates and proteins, the C/P value.
The detailed examination of the mentioned comparative studies can be found in the
Appendix in Table A-2.1-1. A summary of the findings is in Table 2-6 (below). Other
popular methods were not analyzed such as ultrasonication. These techniques were not
included since their yields tend to be lower than 20% VS and cannot be compared in a
straightforward manner (not reported in TS/VS basis, protein and polysaccharides
determined with different techniques, etc) (Guibaud et al., 2005; Matias et al., 2003).
Table 2-6 Comparison of selected EPS extraction methods
Method PT* Treatment time Recovered protein PEP C/P Reference
CER (cation exchange resin)
Yes 3 h 68±4 mg/g TS 1.0 0.16 (Wuertz et al., 2001)
CER (cation exchange resin)
Yes 17 h 243±7 mg/g VS 1.0 0.20 (Frølund et al., 1996)
CER (cation exchange resin)
No 1 h 17.6±0.9 mg/g VS 0.32 0.72 (Liu & Fang, 2002)
NaOH Yes 3 h 126.6 mg/g dry cells 1.0 0.06 (Sheng, et al., 2005)
NaOH Yes 1 h 96±4 mg/g VS 0.39 0.23 (Frølund et al., 1996)
Formaldehyde +NaOH
No 3 h 54.6±2.0 mg/g VS 1.0 0.74 (Liu & Fang, 2002)
Heating (80°C) Yes 1 h 121±3 mg/g VS 0.49 0.07 (Frølund et al., 1996)
Heating (70°C) Yes 1 h 37.7 mg/g VS 0.30 0.27 (Sheng et al., 2005) EDTA Yes 3 h 58.4 mg/g dry cells 0.46 0.11 (Frølund et al.,
1996) EDTA No 3 h 22.9±0.5 mg/g VS 0.41 0.54 (Liu & Fang, 2002) EDTA Yes 3 h 21±1 mg/g TS 0.31 0.14 (Wuertz et al.,
2001) Centrifugation, 15000g
Yes 15 min 6.2 mg/g dry cells 0.05 0.66 (Sheng et al., 2005)
Centrifugation, 20000g
No 20 min 7.9 mg/g VS 0.14 0.97 (Liu & Fang, 2002)
*PT: Pretreatment
21
In general, the following conclusions can be made:
1. The average operating condition for sludge pretreatment (before extraction) is washing
and concentration. The sludge is pelleted, washed with distilled or ultrapurified water
and pelleted again. Both centrifugations are carried out at 2000g for 15 min on average.
2. The average operating condition for EPS recovery after the extraction treatment is
centrifugation at 16000g for 16 min.
3. The length of the treatment has a positive correlation with the amount of exopolymers
extracted. As illustrated in Table 2-6 (fourth and sixth columns), the longer the treatment
time, the more protein is recovered.
4. The extraction yield of sludge exopolymers in quantity and quality is dependent of the
extraction procedure. This conclusion that has also been consistently stated throughout
the literature.
5. Carbohydrate recovery, indicated in the Table 2-6 as the C/P ratio, is greater when the
sludge is not pretreated (i.e., washed with water). This could be due to carbohydrates’
greater hydrophilicity with respect to proteins. The importance of the washing step has
also been indicated previously (Esparza-Soto & Westerhoff, 2001; Jahn & Nielsen, 1998;
Zhang et al., 1999).
6. On average, the cation exchange resin (CER) and NaOH techniques have the greatest
EPS extraction capability. As observed in Table 2-6, these techniques have a PEP of 1.0
in most of the studies they appear in.
The effectiveness of the cation exchange resin and NaOH extraction techniques has
been confirmed in other works (Jorand et al., 1998; McSwain et al., 2005). In CER
extraction, the resin exchanges its monovalent cations, Na+, with divalent cations, mainly
22
Ca2+ and Mg 2+, which are believed to be responsible for the cross-linking of charged
compounds in the EPS matrix. The repulsion of EPS components is then increased along
with their water solubility. In the case of the alkaline treatment, NaOH causes charged
groups, such as carboxylic groups in proteins and polysaccharides, to be ionized since their
isoelectric points are typically below pH 6. Moreover, as in the previous case, this causes
repulsion among EPS components and increases their water solubility. Despite the improved
extraction performance with NaOH, previous studies have reported that boiling and addition
of NaOH causes severe cell lysis. The best results with alkaline extraction have been
achieved with the conditions presented in Sheng et al. (2005) and Liu & Fang (2002) where
the temperature is kept at 4°C.
The mass balance of polysaccharides in cell biomass and total sludge/biofilm, indicated
that 40-90% of polysaccharides are extracellular (Jahn & Nielsen, 1998). However, from a
C/P of 0.6-0.25 in the biofilm only a C/P of 0.06 to 0.21 was detected in the extracted
material. In addition, in this same study where sludge is pretreated –washed- the measured
amount of polysaccharides from EPS is only 21% of the total polysaccharides found in its
corresponding activated sludge (Jahn & Nielsen, 1998). Since we would like to extract as
much organic material as possible, the wash step will not be carried out.
2.6 Extraction of value added products from wastewater sludge
Currently, wastewater activated sludge is utilized in the production of fertilizers and
biofuels. Wastewater biosolids can be used as fertilizers, but the use of these biosolids is
limited because of their heavy metal content (Dewil et al., 2006). In the case of biofuels,
wastewater treatment facilities have included cogeneration systems in their operations, where
wastewater activated sludge is further processed in anaerobic digesters to produce methane
23
and hydrogen (Ghosh et al., 1975). Another biofuel related application has been the use of
pyrolysis to transform this waste biomass into liquid fuels (Kim & Parker, 2008; Konar et al.,
1994). More recently there have been some efforts in using various organic and supercritical
fluid extraction techniques to recover the lipid fraction and turn them into biofuels (Dufreche
et al., 2007). Unfortunately, with the exception of biogas production, the effort into the
conversion of wastewater biofuels into liquid biofuels is not economical yet because the lipid
fraction typically represents approximately 6% of the dry biomass obtained from municipal
wastewater biosolids (Dufreche et al., 2007).
Another active area of research is the production of poly hydroxyl alkanoates (PHAs)
from wastewater sludge as an alternative to propylene in the manufacture of plastics.
However, the economics of the production of PHAs is an issue as it includes costly processes
such as cell culture isolation and complex product recovery (Wallen & Rohwedder, 1974;
Yan et al., 2008).
Considering that there are multiple constituents in RAS, it is worthwhile to consider
multiple value-added products (i.e. a biorefinery approach) to extract from wastewater
biosolids (Ødegaard et al., 2002). We are proposing that these biosolids can be a source of
carbon based compounds, mainly microbial biopolymers (Garnier et al., 2005; Jahn and
Nielsen, 1998). In this research we focus on the application as surface active agents for these
biopolymers, mainly as emulsifiers and adhesives.
2.7 Product recovery
Most biological products need to be purified before they can be used, and in the case of
biorefining, purification and fractionation of different products from a given raw material is
critical (García-Ochoa et al., 2000). Since this project aims to recover valuable products
24
from a waste source, the production scheme of EPS should be competitive with respect to the
disposal cost of wastewater sludge and should avoid generating more complex waste streams.
Precipitation appears to be the preferred technique for recovery of microbial adhesives
and emulsifiers (García-Ochoa et al., 2000). However, this technique is not recommended
for the recovery of products from RAS. The cost of precipitating solvents like alcohols and
the inevitable production of more complex waste streams can reduce the benefits and cost-
effectiveness of producing biopolymers from waste biosolids. A viable alternative appears to
be ultrafiltration, even though not enough research has been conducted for proper application
of this technique for microbial surfactants and adhesives (Ghosh, 2003; Magdassi, 1996).
Ultrafiltration is discussed further in the following section.
Isolation and purification of biotechnological products from the product streams of
bioreactors and other biological feed streams is widely recognised to be technically and
economically challenging. The main reasons for these challenges are the properties that are
common in many biological products (García-Ochoa et al., 2000; Ghosh, 2003):
• Present at very low concentrations in biological feed streams
• Present in the product stream along with large numbers of impurities, some of which are
only slightly different from the products themselves.
• Most are thermolabile.
• Sensitive to harsh operating conditions (e.g. shear stress, pH and salt concentration).
• Sensitive to chemicals, such as surfactants and solvents.
• Product quality requirements are frequently demanding.
An ideal bioseparation technique must combine high productivity along with high
selectivity of separation and must be carried out at mild operating conditions. According to
25
the degree of purification achieved the techniques can be classified in to high productivity-
low resolution, low productivity-high resolution, and high productivity-high resolution.
Table 2-7 lists the most commonly used bioseparation techniques in industry.
Table 2-7 Separation techniques for biotechnological products commonly used for industrial applications (Ghosh, 2003; Henry & Yonker, 2006).
Type Technique Uses Methods
Cell disruption Recover intracellular products Physical methods (colloid mill, French press, ultrasonication) Chemical methods (detergents, enzymes, solvents)
Precipitation Partial purification from the bulk liquid (usually followed by centrifugation or filtration)
Use of salts (ammonium sulphate, sodium chloride), organic solvents, and concentrated acids or alkali.
Centrifugation Separate precipitates Spinning at a range of 1000 to 10000 revolutions per minute
Liquid-liquid extraction
Extract products from liquid phase
Solvent extraction
Microfiltration Separate micron-sized particles from fluids
Membrane based separation through microporous membranes.
Ultrafiltration Separate macromolecules from fluids
Membrane based separation through pore size ranging from 10-8 to 10-6 m.
High productivity-low resolution
Supercritical fluid extraction
Extract products sensitive to chemical and thermal degradation
Supercritical fluids can have solvating powers similar to organic solvents, with higher diffusivities, lower viscosity, and lower surface tension
Ultracentrifugation Separate macromolecules in solution
Spinning samples at >30000 revolutions per minute
Column Chromatography
Fractionate macromolecules based on the affinity between stationary and mobile phase.
Columns: packed beds, packed capillary, open tubular, monolith. Separation chemistries: ion exchange, reverse phase, hydrophobic interaction, size exclusion.
Low productivity-high resolution
Electrophoresis Fractionate macromolecules based on eletrophoretic mobility
Gel or liquid phase electrophoresis.
Fluidised bed chromatography Monolith column chormatography
Fractionate macromolecules based on the affinity between stationary and mobile phases.
Special cases of column chromatography
Membrane chromatography
Fractionate macromolecules based on convection separation
Use of synthetic microporous membranes
High productivity-high resolution
Ultrafiltration Fractionate macromolecules based on membrane separation (pressure differences, molecular size and charge)
Ultrafiltration with optimised operating parameters (pH, salt concentration, permeate flux, system hydrodynamics)
26
High productivity-low resolution techniques tend to be used for the downstream
separation of biotechnological products from the product streams coming straight from
bioreactors. High productivity-high resolution techniques tend to be used for the purification
and polishing of products that require elevated purity compliance standards such as those
from the biopharmaceutical industry. Lastly, low productivity-high resolution techniques
tend to be used for analytical purposes, such as quality control sampling (Ghosh, 2003).
Ultrafiltration has the advantage that it can be applied in all levels of productivity and
resolution.
2.7.1 Biopolymer fractionation using ultrafiltration
Fractionation using ultrafiltration is achievable, although it is significantly more
challenging and more of a recent development (Ghosh, 2003). Since this work aims to
extract multiple value-added products (emulsifiers and adhesives), ultrafiltration can be a
promising downstream purification process in order to recover a range of products from RAS
(biorefinery approach).
Ultrafiltration is a pressure-driven separation process in which membrane of different
materials and with pore sizes ranging from 10 to 1000 Å are used for the filtration of
macromolecules, smaller molecules and even particulate matter. It has been widely used for
concentration, desalting, and clarification of biological product solutions.
Selectivity of macromolecule fractionation can be enhanced in the following ways on the
technical side (Ghosh, 2003):
• Proper membrane selection and membrane surface modification
• pH and salt concentration optimisation
• Concentration/polarisation control
27
• Optimisation of permeate flux and mass transfer coefficient and consequently system
hydrodynamics
On the side of the macromolecules, the following generalised behaviours have been observed
during the fractionation via ultrafiltration (Ghosh, 2003):
• Transmission of macromolecules, especially proteins, is highest at their isoelectric point
• Effect of pH on the transmission of macromolecules is negligible at high salt
concentrations
• Oppositely charged macromolecules interact to form complexes, which result in lower
transmission of both
• The transmission behaviour of a macromolecule is altered by the presence of other
macromolecules in solution
• Intermolecular interactions can be minimised using high salt concentration
• Similar charge on macromolecule and membrane results in electrostatic repulsion and
thus lower transmission
• Adsorption of proteins on the membrane surface and within the pores can dramatically
alter the flux and transmission properties of membranes.
The major advantages of ultrafiltration over other fractionation techniques include high
throughput of product, relative ease of scale-up (in both the technical and economic aspects),
and ease of equipment cleaning and sanitisation. Another important advantage is that
membrane separation (micro and ultrafiltration) has been used at wastewater treatment
facilities since the 1990’s as membrane bioreactor technology (Visvanathan et al., 2000).
This may make the industrial-scale application of the designed process in wastewater
treatment facilities more feasible from both the economical and technological perspective.
28
2.8 Significance of this research
Currently, extraction of RAS constituents is performed for analytical purposes. This
study is the first one to extract surface active material from RAS for production purposes.
This work will aim at recovering a greater amount of the RAS organic content than current
analytical techniques, without significantly compromising the constituent’s ability to be used
as commercial surface active agents.
The literature review indicated that RAS floc constituents, mainly protein and
polysaccharides, play a crucial role in maintaining the floc structural integrity and have
important physicochemical properties. However, their actual roles and surface active
properties remain largely unknown. In this study these two issues are further explored in
order to establish the potential of RAS as a source of bio-based emulsifiers and adhesives.
For this, the extracted constituents will be chemically characterized, correlated with their
physicochemical properties (surface tension, interfacial tension, and detergency/adhesiveness
capabilities) and their performance as detergents and adhesives enhanced if required.
While there are significant market and environmental needs for biotechnological
(microbial) products, their production tends to be expensive. They are usually produced by
batch-wise fermentation in stirred tanks under sterile conditions, using isolated cultures.
Further, glucose and sucrose are commonly used as the carbon and energy sources (Haag,
2006). Thus, cheaper microbial biopolymers, such as fermentation waste or by-products
would be advantageous and have been explored with various degrees of success (Weimer, et
al., 2003; Weimer et al., 2005). This work attempts to recover microbial products from a
waste and highly heterogeneous source. Additionally, the recovery of biotechnological
products requires extensive downstream purification processing which increases costs and
29
reduces yield. To address this issue, we are proposing to recover a range of products based
on their physical performance as detergents or adhesives, not a specific target compound.
30
3 Chapter 3
Alkaline extraction of wastewater activated sludge
biosolids∗∗∗∗
3.1 Abstract
Activated sludge produced by wastewater treatment facilities is a sub-utilized by-product,
with the sludge handling and disposal representing significant costs to these facilities. In this
work, we introduced a simple and effective alkaline extraction technique that extracts up to
75% of the sludge's organic matter into a liquor containing potentially useful organic material
(proteins, carbohydrates, etc.). The results suggest that at pH 11 and above, cell lysis occurs,
liberating substantial quantities of organic material into the alkaline solution. When
compared to a cation exchange resin (CER) extraction developed for analytical purposes, the
alkaline extraction recovered 3x more organic material. The alkaline extract was highly
surface active, despite containing a relatively small fraction of lipids. At pH 12 and above the
lipid fraction was enriched with C15-C16 fatty acid residues, likely associated with cell
membrane phospholipids as suggested by nuclear magnetic resonance spectroscopy (31P
NMR). Size exclusion chromatography studies show that the extract is enriched with
biopolymers or assemblies of molecular weights in the order of tens of thousands of Daltons.
Potential uses for the extract are discussed.
3.2 Introduction
Wastewater biosolids from activated sludge treatment is a waste by-product from
biological wastewater treatment processes. Its handling/disposal represents approximately
50% of total wastewater treatment operating costs (Kroiss, 2004). Contrary to common
∗ This chapter is based upon the manuscript titled “Alkaline extraction of wastewater activated sludge biosolids”, which has been accepted for publication in the journal Bioresource Technology (March 2010).
31
belief, only a fraction of wastewater biosolids can be used as fertilizers, partly due to
environmental regulations that limit the use of these biosolids because of their heavy metal
content (Dewil et al., 2006). Its environmental impact is considerable as the generated sludge
is directed to landfills, incineration or, to a lesser degree, disposed in the sea (Hospido et al.,
2005; Sponza, 2004). However, these biosolids can be a source of carbon based compounds,
mainly microbial biopolymers (Garnier et al., 2005; Jahn & Nielsen, 1998).
The principal components in wastewater sludge flocs are polysaccharides and
proteins, in pure form or in association with other compounds, including glycoproteins and
lipopolysaccharides, etc. Both play major roles in the physical properties and structure of the
microbial agglomeration, including the adhesion phenomena and formation of biopolymeric
networks (Görner et al., 2003; Wilen et al., 2003). Other floc constituents include lipids,
humic substances, DNA and uronic acids (Garnier et al, 2005).
The physicochemical properties of polysaccharides and proteins make them
potentially suitable as surface active agents, such as emulsifiers and adhesives. Naturally
occurring polymers have important advantages like maintaining their effectiveness under
critical conditions (extreme humidity, salinity/pH), lower toxicity and higher
biodegradability than their petrochemical counterparts, as well as reduced reliance on
petroleum (Banat et al., 2000; Ginsburg & Prasso, 2001). Commercial production of
microbial emulsifiers and adhesives has been studied (Haag et al., 2006; Sutherland, 2001).
Their industrial applications include oil recovery/drilling, lubricants, and bioremediation of
water-insoluble pollutants (Banat et al., 2000). Additional applications being studied include
drug delivery systems, cosmetic and detergent formulations, paints, wood adhesives, etc
(Banat et al., 2000; Haag, 2006; Sandford et al., 1984).
32
Lipids and humic substances are also part of the surface active material present in
wastewater sludge. In the case of lipids, low molecular weight phospholipids are highly
surface active and have shown potential as commercial surfactants with surface tensions
ranging from 34 to 21 mN/m (at critical micelle concentration, neutral pH) (Tausk et al.,
1974; Weschayanwiwat et al., 2005). Humic substances have also shown to have surface
tensions as low as 44.2 mN/m (at 2% w/v, pH 12.7) (Chen & Schnitzer, 1978).
Wastewater activated sludge has been studied for the production of biofuels
(Dufreche et al., 2007; Ghosh et al., 1975; Kim & Parker, 2008; Konar et al., 1994) and
bioplastics (Wallen & Rohwedder, 1974; Yan et al., 2008). Unfortunately, with the exception
of biogas, transforming wastewater into biofuels or bioplastics is not economical yet. Thus, it
is worthwhile to consider extracting multiple value-added products such as surface active
agents (i.e. a biorefinery approach) from wastewater biosolids.
Extraction of wastewater biosolids, in particular extracellular polymeric substances, is
routinely performed for analytical purposes using a variety of techniques. The most common
technique is the cation exchange resin (CER) extraction (Frølund et al., 1996) where a
cationic resin exchanges its monovalent cations, Na+, with divalent cations present in
wastewater sludge flocs (mostly Ca2+ and Mg2+) believed to be responsible for cross-linking
charged compounds in the flocs, thus increasing their aqueous solubility (Frølund et al.,
1996). Previous studies ( Frølund et al., 1996; Görner et al., 2003; Urbain et al., 1993; Wilen
et al., 2003) where municipal activated sludges were treated under similar conditions using
the technique by Frølund et al. (1996), have observed that the average extraction yield is
close to 20% (based on volatile solids, VS) and the average extracts’ protein to carbohydrate
ratio (P:C) is approximately 6. Alkaline treatment (mainly using NaOH) is another effective
33
extraction technique. NaOH ionizes charged groups in proteins and polysaccharides,
increases repulsion among polymeric matrix components, and increases their water
solubility. Despite the alkaline extraction’s effectiveness, it has been avoided as it disrupts
the analyzed biopolymers and induces extensive cell lysis (Brown & Lester, 1980; McSwain
et al., 2005). Previous studies (Brown & Lester, 1980; Frølund et al., 1996; H. Liu & Fang,
2002; McSwain et al., 2005; Sheng et al., 2005) have treated municipal wastewater sludge
with alkaline solutions, and obtained the P:C values of 1.4 to 16.7.
In various fields, alkaline extractions have shown to recover high yields of surface
active extracts. Proteins have been historically extracted with strong alkali from waste
livestock bone and hide, and most recently from waste beef bone rendering and tallow by-
products, and fishing and legume processing, to be used as adhesives and binders (Arnesen &
Gildberg, 2006; Arnesen & Gildberg, 2002; Boles et al., 2000; Haag et al., 2006).
Polysaccharides have also been recovered from agricultural waste by-products and used as
emulsion/foam stabilizers and gelling agents (Daiuto et al., 2005; Hromádková, et al., 1999;
Madaeni et al., 2007; Somboonpanyakul et al., 2006). Most importantly, for these
applications, there is no reduction on the physical performance of the recovered biopolymers,
suggesting that the degradation by strong alkali may be neglected (Boles et al., 2000;
Mwasaru et al., 1999; Sun et al., 1998).
The purpose of this study was to develop a scalable (i.e. non-analytical) alkaline
extraction technique to recover surface active extracts from wastewater biosolids. In this
work, return activated sludge (RAS) is used as equivalent to waste activated sludge, which is
the by-product of the activated sludge treatment of wastewater. The reason for not using
waste wacitvated sludge directly is that a number of additives are used to stabilize the sludge
34
before final disposal, additives that may mask the real potential of the sludge extract. To this
end, the effect of extraction pH and extraction time on extraction yield and extract
composition and properties (particularly surface tension) were evaluated. As a comparison,
a common analytical extraction technique, CER was also considered.
3.3 Materials and Methods
3.3.1 Materials
All reagents used in this experiment were reagent or analytical grade and purchased
from Sigma Aldrich (ON, Canada), Thermo Fisher Scientific Inc. (IL, USA), and EM
Science (NJ, USA). Alkaline extractions used NaOH 50% Solution in H2O (Sigma Aldrich);
the cation exchange resin was Sodium Form DOWEX Marathon® Cation Exchange Resin
(Sigma Aldrich). The buffer used for the resin was Dubecco’s Phosphate Buffered Saline
(PBS) without CaCl2 and MgCl2 (Sigma Aldrich). Protein content was determined using the
Pierce BCATM Protein Assay kit (Thermo Fisher Scientific Inc.) which included the reference
protein Bovine Serum Albumin (BSA). For polysaccharide analysis, Phenol, 99% (Sigma
Aldrich) and Sulfuric acid, 95%-98% (EM Science) were used with D-glucose, 99.5%
(Sigma Aldrich) as the reference sugar. Determination of lipids (derivatized into fatty acid
methy esters, FAMEs) used NaOH pellets, HCl 35-37%, hexane, methyl tert-butyl ether, and
methanol (all from Sigma Aldrich). All solvents were HPLC grade. GLC-90 fatty acid
methyl ester (FAME) Supelco®standard mix (Sigma Aldrich) was used as the reference
FAMEs. Phosphorous containing constituents (phospholipids, humic substances) were
analyzed with nuclear magnetic resonance spectroscopy (31P NMR) using D2O and NaOD
(Sigma Aldrich).
35
3.3.2 Methods
Determination of extraction pH range.
To determine the optimal extraction pH for wastewater sludge, various samples were
incubated at different pHs. Municipal wastewater aerobic Return Activated Sludge (Sludge
Retention Time: 2.5 days; Aeration Time: 6-8 h) from the North Toronto Wastewater
Treatment Plant, City of Toronto (average capacity of 34,000 m3/day) was incubated for 5
weeks at pHs 7, 9, 10, 11, 12.3, and 12.6, by adding 50% concentrated NaOH, and kept under
continuous agitation (500 rpm) the first week. Incubations were carried out in closed
Nalgene bottles at room temperature and 4°C. Incubated samples were then centrifuged
(Beckman Coulter Centrifuge) at 3000 RPM (559 g) for 13 min at 4°C, and their
supernatants collected. The supernatants are considered to be the extracts as it is assumed
they contain the solubilized organic compounds from wastewater sludge flocs. Total Organic
Carbon (TOC) content was measured in the alkalinized sludges and their extracts using the
Shimadzu TOC analyzer (VCPH/CPN standard model). The extracts’ surface tension was
determined using the KSV/Instruments tensiometer (Sigma 700 model) with 24X50 mm
glass plates.
Determination of cell lysis during alkaline extraction.
A shorter term extraction experiment (48 h) was conducted to evaluate the extraction
yield, cell lysis and endogenous degradation (loss) of the extracted material as a function of
extraction pH. Aerobic RAS samples were collected from the Ashbridges Bay Wastewater
Treatment Plant, City of Toronto (1400 Population Equivalent; Average Capacity: 725,000
m3/day; Sludge Retention Time: 2.5 days; Aeration Time: 6-8 h). Samples were kept in ice
bath and transported to the laboratory within 1 h. After 1.5 h since its collection, the water
overhead of the settled activated sludge (decant) was discarded.
36
The concentrated RAS was incubated for 48 h at pH 9, 10, 11, 12 and 13 by adding
50 % wt NaOH and maintaining continuous agitation (500 rpm). Incubations were carried
out in closed Nalgene bottles at room temperature. To determine cell lysis, deoxyribonucleic
acid (DNA) content in the extracts was determined using the Flourescent DNA Quantitation
Kit from Bio-Rad, which included calf thymus DNA as the reference standard. Fluorescent
spectrophotometric readings were measured with the multiwell-plate reader Infinite M200
from TEC (excitation: 355 nm; emission: 460 nm; flashes: 25; gain: 90; integration time: 25
µs) using i-Control 1.4 as interface. DNA in the original concentrated RAS was extracted
using the UltraCleanTM Soil DNA Isolation Kit and measured using the NanoDrop®
Spectrophotometer ND-1000. The extraction kinetics were followed measuring the TOC and
DNA in the extracts as mentioned above at 5 min, 0.5 h, 2.0 h, 4.0 h, and 48.0 h.
Effect of extraction pH on extract yield and composition
Sampling and sample preparation. Aerobic RAS samples were collected from the
Ashbridges Bay Wastewater Treatment Plant, transported, and concentrated as described
above.
Alkaline extraction. Half of the concentrated RAS was used for alkaline treatment. It
was divided into 4 aliquots of equal volume and 50% NaOH was added under constant
stirring to each aliquot to reach a specific pH: 12.0, 12.3, 12.6, and 12.9. The aliquots were
covered with GLAD Press'n Seal® wrap to avoid contamination from outside sources, and
incubated at room temperature under constant stirring (500 RPM). The alkalinized samples
were centrifuged at 3000 RPM (559g) for 13 min at 4°C, and the supernatant collected.
Cation exchange resin (CER) extraction. The other half of the concentrated RAS was
treated according to the CER procedure (Frølund et al., 1996). This extraction was carried
37
out in triplicates. Corning Ltd. mixers used consisted of 3 blades (diameter: ¾ in) and were
calibrated to 900 RPM using a light sensitive tachometer (Extech Instruments Model # L
381874).
Both alkaline and CER extractions were performed simultaneously and conducted for
24 h. Extracts (supernatants) of each extraction were taken throughout the 24h-treatment to
study the extraction kinetics: 1min, 1h, 2h, 3h, 4h, and the 24h. Sampling times were
determined based on previous work (Frølund et al., 1996). All collected supernatants were
stored at -20°C.
Mass characterization of RAS and extracts. TOC was determined as mentioned
above.
Chemical characterization of extracts. Carbohydrates were measured using the
phenol-sulfuric acid method with D-glucose as standard (Masuko et al., 2005). Proteins were
measured using both BCATM Protein Assay kit and a modified Lowry method (Fryer et al.,
1986; Haff, 1978), using BSA as standard. Spectrophotometric readings were measured with
the multiwell-plate reader ThermoU Spectra III A-5082 from SLT-Labinstruments. Both
techniques gave similar results, but the BCA method achieved a lower error, therefore those
are the results presented here. The lipid (FAME) composition was analyzed with the MIDI
method (Microbial Identification System, Microbial ID Inc.) (Smid & Salfinger, 1994) using
gas chromatography (GC). A Perkin Elmer GC (Auto System XL) apparatus, equipped with
a Z5(poly (5% phenyl/ 95% dimethylpolysiloxane) capillary column (0.25 mm, 30 m, 0.25
µm), and flame ionization detector was used. The carrier gas was hydrogen. Fatty acids
were identified according to their retention time using a standard mix of reference FAMEs
(C13, C15, C17, C19, and C21).
38
Physical characterization of extracts. The molecular size distribution of the extract
was determined through size exclusion chromatography with the Dionex ICS-3000 apparatus
equipped with the 300 X 7.7 mm Nucleogel® GFC 300-8 column by Macherey-Nagel
GmbH & Co. Mobile phase (flowrate 0.5mL/min) was double distilled de-ionized water.
The eluent was analyzed with ultraviolet-visible (230nm) and conductivity detectors
(conductivity chromatograms not shown here). D-glucose (180 Da) and BSA (66.430 kDa)
were used as standards. The viscosity was measured with Gilmont® Instruments viscometer
Model GV-2200 of the falling ball type (glass ball size #3, GF-1332 P) (data not presented).
The surface tension of the extracts was determined using the KSV/Instruments Sigma 700
tensiometer with a platinum 10X19.62 mm Wilhelmy plate.
Characterization of extract with 31P NMR. To understand the effect of different
constituents on the extracts’ surface activity, the presence of phospholipids and humic
substances (phosphorous containing compounds) was analyzed in the extract of lowest
surface tension and NaOH content. The extract was fractionated and diafiltered until pH 9.0
with distilled water using ultrafiltration hydrophilic polysulfone membranes (A screen, Mini
Biomax membrane, Millipore) of 10 kDa nominal molecular-weight limit. The limit was
selected to separate humic substances (usually lower than 10 kDa) (Perminova et al., 2003)
from macromolecules like phospholipids (above 106 kDa). Retentate and permeate were
lyophilized and grounded to powder. The surface tensions were determined as stated above
for both retentate and permeate in solution at 4g/L, beyond their critical micelle
concentration (Garcia-Becerra et al., 2009).
The presence of phosphor-containing compounds in the retentate was evaluated using
31P NMR. The spectrum was obtained after dissolving 200 mg of retentate powder in 6 mL
39
of D2O and the pH adjusted to 12.6 using aliquots of NaOD. Residual insoluble material was
removed by centrifuging the sample (22°C, 30 min). Sample was prepared one hour prior to
acquiring data and placed in a 10 mm NMR tube (Norel) .The spectrum was run on a Bruker
Avance III spectrometer operating at 162.00 MHz for 31P, equipped with a 5 mm variable
temperature PFG BBO probe. The spectrum was acquired at 25°C over a 104166 Hz spectral
window with 262144 points and 32768 transients. A 0.1s delay time was implemented and 4
dummy scans were acquired prior to data acquisition. The data was processed and 50 Hz line
broadening applied with MestreNova 6.0.3 software.
Statistical analyses. The long and short-term extraction studies and the 31P NMR
measurement were not replicated. The experiments for the development of the extraction
technique (effect of pH on extraction yield and composition) were replicated four times. All
physical and chemical analyses in each experiment were performed in triplicate. With the
exception of the FAME GC profiles, the statistical analyses were carried out with Microsoft
Excel software. This work reports the average values along with the 95% confidence
interval. FAME measurements are in the form of GC chromatograms, not in terms of a
single value. Thus, they were subjected to analysis of variance (3-way ANOVA: replicate,
extraction time, and FAME category according to molecular weight) with the Bonferroni
approach, significance level of 0.05, using SPS software.
3.4 Results and Discussion
3.4.1 Long-term extraction studies.
In order to determine the optimal extraction pH, samples of RAS were incubated at
different pHs at room temperature and 4°C for a period of 5 weeks to approach equilibrium
(saturation) conditions. Figure 3-1 shows the extraction yield (Figure 3-1a) and extract
40
surface tension (Figure 3-1b) as a function of extraction pH. As shown in Figure 3-1a, the
extraction yield increases substantially beyond pH 11. These findings are consistent with
recent technologies developed to reduce wastewater biosolids from industrial wastewater
treatments (Mizoguchi et al., 2008). In that extraction technology, pH values of 11 -12 were
used to extract a maximum of 30% of the biosolids. In their case, the solubilized biosolids
were neutralized and fed back into the wastewater treatment system.
With respect to the fraction of TOC extracted at pH values lower than pH 12, it is
important to keep in mind that some of the reduction in TOC after 5 weeks could be due to
further degradation (mineralization) of organic matter. In fact, a foul smell was detected in all
incubations at pHs<12, which supports the idea of endogenous degradation for those
systems.
0
20
40
60
80
100
6 7 8 9 10 11 12 13
TO
C E
xtra
ctio
n Y
ield
(g
/10
0g
)
.
4°C Room Temperature
30
40
50
60
70
80
6 7 8 9 10 11 12 13
Su
rfa
ce
Te
nsi
on
(m
N/m
)
Extraction pH
4°C Room Temperature
(a)
(b)
Figure 3-1 Influence of extraction pH on extraction yield (a) and surface tension of the extract (b). The extract yield is presented as grams of Total Organic Carbon (TOC) content in the supernatant (extract) obtaining from treating the equivalent of 100 grams of TOC in the return activated sludge (RAS). Extraction time: 5 weeks. Controls in surface tension measurements: Distilled Water, 70.5 mN/m; NaOH at pH 12.6, 65 mN/m. RAS sample collected in January 2006, Initial TOC = 3500 mg/L
41
There are several physicochemical processes that may explain the increase in
extraction yield with increasing pH. First, in alkaline media organic compounds tend to
become ionized (negatively charged). This excess negative charge inside the floc weakens
the internal binding, promoting its dissociation. Another process that might be relevant is the
hydrolysis of organic compounds, especially polysaccharides and proteins. Also, the
ionization of the lipids that form cell membranes, such as phospholipids, glycolipids, and
steroids, may also occur at high pHs. Finally, another aspect that is necessary to consider is
that the solubility of positive ions holding together the floc, such as calcium, decreases at
high pHs as they tend to form neutral species (Al-Anezi & Hilal, 2007). In the case of
calcium hydroxide, Ksp = 8.0 M, at pH 11 the solubility is 8.0X10-2 M, but at pH 13 the
solubility reduces to 8.0X10-4 M. Other metal ions such as Fe2+ and Fe3+ also have a
significant reduction in their solubilities at high pHs (Waite, 2002). That is, at extraction
pH≥12, the biopolymer extraction may be enhanced by the removal of divalent cations. The
results also show that at pH≥11, recovered biopolymers from RAS are physically functional
(exhibit low surface tensions).
The surface tension values presented in Figure 3-1b reflect the changes in extraction
yield observed in Figure 3-1a. At pH values of 11 or higher, the significant decrease in
surface tension may be due to the neutralization of fatty acids to form soaps and potential
ionization of phospholipids that are also highly surface active.
These initial long-term studies were suitable to understand the range of pHs that
produce substantial biomass solubilization into the extraction media. However, five weeks of
extraction time is not practical for full scale processes. Furthermore, it is important to reduce
potential endogenous degradation observed in systems with pH<12. Therefore, the next sets
42
of studies consider the pH of extraction as well as the extraction time, and evaluate the
potential effect of pH on cell membrane disruption.
3.4.2 Short-term extraction studies (48 h).
Figure 3-2 presents the effect of extraction time and extraction pH on the extraction
yield expressed in terms of TOC (Figure 3-2a), and the DNA content in the extracts as a
function of time (Figure 3-2b). Despite the fact that the samples of Figure 3-2 were collected
at a different time than the sample employed in Figure 3-1, it is remarkable to note that the
TOC extraction yields obtained for the various extraction pHs at 48 h of treatment (Figure
3-2) are similar to those at 5 weeks (Figure 3-1). In addition to the significant advantage that
this represent in terms of producing an scalable process (smaller residence time, smaller
reactors), this also suggests that the amount of TOC lost to endogenous decay in Figure 3-1 is
likely to be small. In addition, at extraction pH levels 12, up to 50% of TOC may be
recovered in the supernatant after 4 h of treatment.
According to the DNA recovered from the supernatant (Figure 3-2b), we obtain a
high and nearly constant concentration of DNA in the supernatant at pH 11 or higher. This
suggests that at pH 11, the cell membrane breaks, releasing the content of the cell to the
aqueous solution. According to Figure 3-2b, this cells lysis seems to take place fully within
the 4 h of extraction. This implies that using alkaline extractions with pH 11 or higher, may
make it possible to recover cell membrane material, as well as intracellular constituents,
which could explain the ability of these extracts to reach low surface tensions.
43
0
20
40
60
80
100
0 10 20 30 40 50
TO
C E
xtra
ctio
n Y
ield
(g
/10
0g
)
Extraction Time (h)
0
15
30
45
60
0 10 20 30 40 50
DN
A in
Ext
ract
(m
g/L
)
Extraction Time (h)
pH 9 pH 10 pH 11 pH 12 pH 13
(a)
(b)
Figure 3-2 Effect of extraction time and extraction pH on TOC extraction yield (a) and DNA release from the extracts (b). The extract yield is presented as grams of TOC content in the supernatant (extract) obtaining from treating the equivalent of 100 grams of TOC in RAS. Extraction conditions: pH range from 9 to 13; extraction time up to 48h; room temperature. Total DNA extracted from the concentrated RAS was measured to be 45 mg/L. RAS sample collected in March 2009, Initial TOC = 6500 mg/L.
To interpret the effect of extraction pH it is helpful to consider the example of
lecithin, a phospholipid, which has two pKa values, 3 and 7. In between these pH values, the
lecithin is neutrally charged and above that range the molecule become increasingly
negatively charged (Price & Lewis, 1933). However, the same authors indicate that these
pKa values are similar to those of phosphoric acid, but that ortho-phosphoric acid has one
more, pKa3 ~ 12. This suggests that the transitions observed at pH 11-12 may be linked to a
decomposition of the phospholipids and full ionization of the phosphor–containing groups in
wastewater sludge.
44
3.4.3 Effect of extraction pH on extract yield and composition
The RAS samples considered in this part of the study were collected during the
summer (June-July) of 2007. Because the extraction studies discussed above show that
indeed, the best extraction yield and extracts with the lowest surface tensions are obtained at
pH 12 or higher, this part of the study concentrated on that pH range. Figure 3-3 presents the
ratio of mass of sodium hydroxide per mass of carbon (TOC) in the extracted (concentrated)
RAS sample. As it can be expected, the higher the extraction pH, the more sodium
hydroxide required. At higher pH values, our instrumental error was greater and therefore
larger error bars are associated with the extraction at pH 12.9. Even when those error bars are
considered, the amount of sodium hydroxide required to reach pH values near 12.9 are
significantly higher than the amount of sodium hydroxide required to extract the biomass at
pH 12.3 or 12.6.
0
0.5
1
1.5
2
2.5
11.8 12 12.2 12.4 12.6 12.8 13
g N
aOH
/g T
OC
in C
on
cen
trat
ed
RA
S
Extraction pH
Figure 3-3 Sodium hydroxide (NaOH) required to increase the pH of the concentrated RAS sample. TOC in concentrated RAS sample: 5.8±0.7 g/L, samples obtained in June-July of 2007. Error bars indicate the 95% confidence intervals.
45
3.4.4 Extraction kinetics and yield
To follow the mass balance we have measured the TOC content of the extracts and
RAS at different stages during the extraction processes. TOC measurements (before and
after the extractions) are then used to determine the kinetics and yield for both alkaline and
CER techniques. Figure 3-4 shows the extraction, in g/L of TOC of extracted biomass for
NaOH and CER extractions. The graph includes the average values of the extractions
performed in quadruplicate.
0
1
2
3
4
5
0 4 8 12 16 20 24
TO
C o
f E
xtra
ct (
g/L
)
Extraction Time (h)
pH 12.0 pH 12.3 pH 12.6 pH 12.9 CER
Figure 3-4 Extraction kinetics of alkaline and CER extractions. TOC content in the extracts (supernatants) as a function of extraction time. Extraction conditions for alkaline extraction: pH range from 12.0 to 12.9; extraction time up to 24 h; room temperature. TOC in concentrated RAS sample: 5.8±0.7 g/L, samples obtained in June-July of 2007. Error bars indicate the 95% confidence intervals
Considering that the average TOC content in the concentrated RAS is 5.8 g/L, the
extraction curves indicate that as early as the first minute of alkaline extraction a significant
amount of biomass is extracted. Within this first minute, an average of 75% of the amount of
extractable biomass at 24 h is dissolved in the alkaline solution. The rapid effect of NaOH
has been previously observed (Hromádková et al., 1999) where up to 20-37% of biomass (on
dry weight basis) was recovered during the first 10 min of treatment. Figure 3-4 also
46
confirms the short term extraction studies (Figure 3-2) where there is a positive correlation
between extraction rate and extraction pH. The average extraction yields at 24h in the
alkaline extractions are: pH 12.0: 58%, pH 12.3, 69%, pH 12.6, 74%; and pH 12.9, 75%.
These results are in good agreement with the short-term studies conducted using RAS
samples collected for Figure 3-2 (March 2009). In contrast with the alkaline extraction, the
CER extraction only reaches a maximum average extraction yield of 25%, which agrees with
the previous literature (Frølund et al., 1996; H. Liu & Fang, 2002; McSwain et al., 2005;
Sheng et al., 2005). In interpreting the CER data is necessary to consider that the CER
method was specifically designed to extract extracellular biomass exclusively at neutral pH,
and that it is only at pH values higher than 11, as shown by the DNA data of Figure 3-2, that
the cell membranes are disrupted and the contents of the cells are likely to be liberated into
the alkaline extraction solution.
The extraction kinetics can also give us an idea of the type of polymeric network
RAS flocs and the effect the extraction process may have on it. The results from Figure 3-4
are expressed in terms of yield versus extraction time and plotted in log10-log10 curves in
Figure 3-5. These curves are analyzed by fitting them to Equation 2 (below) which is
derived from Equation 1. Equation 1 has been proposed by (Ritger & Peppas, 1987) as a
semi-empirical equation based on Fickian diffusion to describe the general release behavior
of constituents within a polymer matrix.
nt ktYieldExtraction ==
∞µ
µ Equation 1
tnkYieldExtraction t10101010 loglogloglog +=
=
∞µ
µ Equation 2
47
Where µ t, is defined as the mass of constituents released at time t; µ∞ is the mass of
constituents released as time approaches infinity (for this work, µ∞ is at time 24 h); k is a
constant incorporating characteristics of the macromolecular network system and the
constituents; and n is indicative of the transport mechanism. According to Ritger et al.
(1987), Fickian diffusion is defined by n ≤ 0.50 and non-Fickian by n > 0.50. Equation 2 is
of the linear type y=a+bx, where log10k indicates the y-intercept of the curves and n the slope
of the curve.
-0.6
-0.4
-0.2
0
-2 -1 0 1 2
log
10
µt/
µ2
4h
log10 Extraction Time
pH 12.0 pH 12.3 pH 12.6 pH 12.93 CER
Figure 3-5 Extraction yield in log10-log10 plot. Influence of extraction time on the extraction yield.
After the linear regression of the curves in Figure 3-5, the n values for the alkaline
extractions are 0.06 to 0.05 and for the CER extraction n is 0.17. Since all calculated values
for n are smaller than 0.5, it can be assumed that in both types of extraction, the biopolymeric
network is porous enough to allow the release of constituents to follow Fickian diffusion. It
can be observed in Figure 3-5 that all the alkaline extractions fit closely to the proposed
linear behavior. However, the CER extraction exhibits 2 types of linear sections during the
48
extraction, before and after the first hour of treatment. This might suggest that different
processes take place at different times throughout this extraction. After the first hour, the n
value increases to 0.3. This could suggest that the biopolymer matrix begins to have an effect
on the release mechanisms, which might indicate that at this point, constituents embedded
into the matrix begin to be extracted. The idea that the floc has various layers has been
proposed before (Liao et al., 2002). However, after 24h the CER extraction has only
recovered 25% of the organic material in RAS, suggesting that despite the change in the n
value, it may not be effective in disrupting the core of the flocs and recovering the
constituents deeply inbedded in the polymeric network. Since the alkaline extractions
present a similar release behavior across time, it could imply that high pH values are able to
disrupt the floc polymeric network more effectively, which could explain why more product
is extracted overall.
3.4.5 Chemical composition (protein, polysaccharide, and lipid content)
After the extracts were collected the extract protein, polysaccharide and lipid content
was determined and reported on TOC basis. For proteins, the BCA™ assay report values of
proteins in equivalent mg/L of BSA. For carbohydrates the results are expressed in terms of
equivalent mg/L of D-glucose. For lipids, the analysis produces the equivalent fatty acid
methyl ester composition. For each of these compounds, the equivalent carbon (TOC)
concentration in the sample was calculated based on their molecular formula. The yields
reported in Figure 3-6 were calculated as the grams of equivalent TOC of each species
(protein, carbohydrate or lipid) in 100 grams of TOC of concentrated RAS extracted.
49
0
10
20
30
0
6
12
18
0
0.8
1.6
2.4
0 4 8 12 16 20 24
Extraction Time (h)
pH12
pH12.3
pH12.6
pH12.9
CER
Lip
id T
OC
yie
ld (
g/1
00g)
Carb
ohyd
rate
TO
C
yie
ld (
g/1
00
g)
Pro
tein
TO
C y
ield
(g/1
00
g)
(c)
(b)
(a)
0
10
20
30
0
6
12
18
0
0.8
1.6
2.4
0 4 8 12 16 20 24
Extraction Time (h)
pH12
pH12.3
pH12.6
pH12.9
CER
Lip
id T
OC
yie
ld (
g/1
00g)
Carb
ohyd
rate
TO
C
yie
ld (
g/1
00
g)
Pro
tein
TO
C y
ield
(g/1
00
g)
(c)
(b)
(a)
Figure 3-6 Extraction yield towards protein (a), polysaccharides (b), and lipids (c) as a function of extraction time. These yields were calculated as grams of TOC of the particular fraction measured obtained from the equivalent of 100 grams of TOC in the concentrated RAS. For proteins the surrogate compound used in the calculations was BSA and for carbohydrates, D-glucose. TOC in concentrated RAS sample: 5.8±0.7 g/L, samples obtained in June-July of 2007. The TOC of the extract corresponding to each extraction condition are presented in Figure 3-4. Error bars indicate the 95% confidence intervals
50
Between 13 to 23% of the TOC content in the initial concentrated RAS can be
recovered as proteins with alkaline treatments after 24 h, and up to 6% with CER extractions
(Figure 3-6a). For polysaccharides, an extraction yield of 6 to 12% was obtained after 24 h,
while CER extracts achieve a maximum of 3% yield (Figure 3-6). The average protein:
carbohydrate ratios (P:C) recovered are: 2.2± 0.2 at pH 12, 2.3 ±0.4 at pH 12.3:, 2.4±0.5 at
pH 12.6, 2.8±0.3 at pH 12.9, and 2.4±0.3 with CER.. The P:C values of both alkaline and
CER extracts are similar, which suggests a similar ability to extract proteins and
polysaccharides among NaOH and CER treatments. However, the P:C values are lower than
the average P:C in previous studies (Frølund et al., 1996; Görner et al., 2003; Liu & Fang,
2002; Sheng et al., 2005; Wilen et al., 2003). The average protein extract content in the
alkaline extraction at high pH is almost twice the average protein content in previous works,
while the polysaccharide extract content is almost 5 times higher. The fact that the content of
protein and polysaccharides is similar in both alkaline and CER suggests that the low P:C
ratio is characteristic of the Ashbridges Bay sludge. It may also be because these works
wash the RAS pellet before the extraction treatment, eliminating hydrophilic constituents like
low molecular weight polysaccharides, resulting in higher P:C values than ours.
The lipid (FAME) results indicate significant differences between alkaline and CER
extractions. Average alkaline lipid yields are at least ten times more than the CER lipid yield
(Figure 3-6c). This difference in lipid content is likely due to the contribution of the cell
membranes lipids released into the alkaline extraction solutions at high pH values. However,
it is necessary to consider that the lipid (FAME) fraction reported in Figure 3-6c is lower
than the lipid content of 2% to 11% reported in other studies (Conrad et al., 2003; Réveillé et
al., 2003). The reasons for the lower compositions may be related to the fact that
51
approximately 31% of the total fats in municipal wastewater sludge are unsaponifiable
(Vriens et al., 1989) and cannot be detected with the MIDI technique. In addition, the
previous studies (Conrad et al., 2003; Réveillé et al., 2003) conducted extensive lipid
extractions using organic solvents. Furthermore, the lipid content in this Asbridges Bay
sludge can be characteristic of the operational conditions of that facility. However, despite
the low fatty acid content, the extract is highly surface active, as shown in Figure 3-1b. This
suggests that there could be other surface active species in the extract that are not directly
related to the lipids detected by the FAME test.
Besides the lipid yield, another point of interest is the composition of those lipids.
Figure 3-7 shows the relative composition of the lipid fraction obtained using different
extraction conditions and after 24hrs of extraction. According to Figure 3-7, higher
molecular-weight lipids (most likely from intracellular or cell membrane sources) are present
in the alkaline extracts, but those fractions are not found in CER extracts. Also, higher
extraction pHs lead to higher molecular-weight lipids. However, since cells make up
approximately 10-15% of activated sludge flocs (Frølund et al., 1996), the total amount of
lipids extracted at different pHs remains relatively the same (Figure 3-6c). Another
important observation from Figure 3-7 is that the lipids obtained with alkaline extractions are
enriched with C15-C17 fatty acid fractions, which is consistent with the fact that microbial
cell membranes are enriched with palmitic (C16) fatty acid (Zelles, 1999).
52
0
25
50
75
100
CER pH 12.0 pH 12.3 pH 12.6 pH 12.9
Extraction Conditions
Lip
id C
on
ten
t in
Ex
trac
ts (
%)
C9 -C13
C13 -C15
C15 -C17
C17 –C19
C19 –C21
C21+
0
25
50
75
100
CER pH 12.0 pH 12.3 pH 12.6 pH 12.9
Extraction Conditions
Lip
id C
on
ten
t in
Ex
trac
ts (
%)
C9 -C13
C13 -C15
C15 -C17
C17 –C19
C19 –C21
C21+
Figure 3-7 FAME composition profile at different extraction conditions. The C# ranges presented at the right side represent the number of carbons in the fatty acid methyl esters (FAME) observed in the chromatographs. Error bars indicate the 95% confidence intervals
In alkaline extractions, the statistical analysis of the protein, polysaccharide and lipid
assays suggests that the type of chemicals recovered are mostly dependent on the extraction
pH. Furthermore, the ANOVA analysis of FAME GC profiles indicate that there is no
significant difference among FAME profiles across time at a given extraction condition.
That is, despite the length of the treatment, the produced extracts at a given pH do not vary
significantly in chemical composition, only in the amount of lipid recovered. This is
consistent with the earlier findings from Figure 3-5, a given pH is able to distabilize the RAS
floc to a certain degree and recover a certain type of constituents which are realeased into the
extract. A higher pH would disrupt the polymeric network further and constituents that are
burried deeper into the matrix, such as high molecular weigh lipids, are released. The
ANOVA analysis also indicates that there is no significant difference among the four
53
experimental trials (quadruplicates), which might suggest that the alkaline extraction
technique is robust.
3.4.6 Physical properties of the extracts
Size exclusion chromatography (SEC) was used to assess the molecular weight distribution
of the extracted compounds in the alkaline extraction. Figure 3-8 depicts the chromatograms
obtained from alkaline and CER extractions after 1 min, 4 h and 24 h of extraction. To
interpret these chromatograms it is important to consider that larger molecules may not be
trapped in the reticular structure of the column, elute earlier than lower molecular species and
may not be detected. In addition, the resolution of this elution is poor at extremely low or
high molecular weight. The column used to produce the chromatograms of Figure 3-8 is
capable of separating the range of molecular weights between 100Da and 100kDa.
Overall, as the extraction pH increase, the area of a peak observed at 7-8 min of
retention time in Figure 3-8a-d also increases. There are no evident differences in the
features of the chromatogram for pH 12.3 and 12.6, but the chromatogram at pH 12 show
various peaks at higher residence times, suggesting the presence of lower molecular weight
species. For the case of CER, there is a peak at smaller molecular weights (~ 11 min
retention time) that does not appear in the alkaline extractions. On the other hand, the
extraction pH of 12.9 produces an increase in peaks of higher molecular weight with
retention times between 6-7 min. This observation is consistent with SEC studies of alkaline
humic extracts where increasing the extraction pH produce assembled structures of large
molecular weight (Piccolo, 2002).
54
(a) (b) (c)
(d) (e)
Figure 3-8 Size exclusion chromatograms of alkaline and CER extracts collected after 1 min (dashed line), 4 h (gray solid line) and 24 h (black solid line) of treatment. (a) pH 12.0; (b) pH 12.3; (c) pH 12.6; (d) pH 12.9; (e) CER. The retention times from BSA (66.43 kDa) and D-glucose (MW 180 Da) are 6.5 min and 13 min, respectively
In this article we refrain from specifying a range of molecular weights of the extract
because translating residence times into actual molecular weights requires knowing what
type of molecule one is exploring since the hydrodynamic radius (which controls the
retention time in SEC) is a function of the internal structure or folding of the molecules.
However, as a reference it is relevant to mention that a protein like BSA has a retention time
of 6.5 min and that D-glucose (180 Da) has a retention time of 13 min. It is also important to
consider than in SEC some molecules can be associated in clusters (e.g. micelles) which
produce higher apparent molecular weight distributions. The data, however, shows that
increasing the concentration of the alkali extracts higher molecular weight species. The
ability of high alkaline extractions to recover high molecular weight species has been
previously observed (Somboonpanyakul et al., 2006; Sun et al., 1998).
55
Despite the fact that a significant portion of the species had an intermediate molecular
weight (in the order of tens of thousands of Daltons), the viscosity of all the extracts within
the range of pH 12 – 12.9, at extraction times larger than 1 min ranges from 1.5 to 2 cP.
Perhaps this relatively low viscosity is to be expected given the relatively low concentration
of the extract (less than 4 g/L, see Figure 3-4). When compared to 50 kDa pullulan solutions
at a concentration of about 4 g/L, the viscosity of those solutions is approximately 1.1 -1.2 cP
(Nishinari et al., 1991). While this is consistent with the approximate molecular weight of
some of the extract, it also suggests that the extract is not a highly effective viscosity
modifier. It may be possible that the extraction protocol is not suitable to extract high
molecular weight species and that the substrate itself does not have large molecular size
carbohydrates that impart viscosity modifying properties to other biomass extracts (e.g.
Xantham gum).
The potential use of the extracts as surface active agents was evaluated through the
effect of extraction conditions on the extracts’ surface tension. The average surface tension
of the extracts after 24 h of treatment are: 44.5 mN/m at pH 12.0; 41.1 mN/m at pH 12.3;
37.8 mN/m at pH 12.6; 34.4 mN/m at pH 12.9; and 45 mN/m with CER. Overall, the
alkaline extracts have a lower surface tension than CER extracts. Further, the higher the
extraction pH, the lower the extract surface tension. Figure 3-7 indicates that higher
molecular weight (more hydrophobic) lipids are present at higher extraction pHs, which
could explain the lower surface tensions obtained in those extracts. CER and pH 12 extracts
are enriched with low molecular weight lipids, and therefore are not expected to be as surface
active. The extract TOC content (Figure 3-4) may also explain the low surface tensions
found in the alkaline extracts. The alkaline extracts are able to achieve a surface tension
56
range of 44 to 34 mN/m with a TOC content range of 3.2 to 4.1 g TOC/L, which is
comparable to commercial products that reach 35mN/m at ~3 g/L (Bernhard et al., 2000).
3.4.7 Fractionation and NMR characterization of the extract
The fractionation of the pH 12.6 (4hr) extract with a 50 kDa polysulfonate membrane
was introduced to separate salts and low molecular weight materials. The surface tensions of
the reconstituted retentate (at 4 g/L, pH 9) was 43.2mN/m and for the reconstituted permeate
(at 4 g/L, pH 12.6) was 52.7 mN/m. These results suggest that the more surface active
compounds remain associated with the larger molecular weight compounds. This observation
is consistent with the current view that in humic extracts, and in protein extracts, surface
active lipids associate with the hydrophobic groups in the extracted material to form
assemblies that resemble the structure of giant micelles (Le Maire, et al., 2000; Piccolo,
2002).
Considering that lower surface tensions in the extract coincide with membrane
disruption (DNA release at pH>11), one could suggest that phospholipids from the cell
membrane could reassemble in the extracts to produce large surface active assemblies.31P
NMR spectrum (Figure 3-9) was used to assess the presence of phosphorus compounds (that
could include phospholipids) in the retentate. The assignment of peaks in the 31P NMR
spectrum was made on the basis of previous reports (Bartoszek et al., 2008; Cade-Menun,
2005; Hinedi et al., 1989). The sample pH during NMR analysis is close enough to the
reference pH that the shift number is not affected significantly and the comparison is
appropriate (Crouse et al., 2000). The retentate 31P NMR spectrum is characterized by high
organic P content in the form of P-diesters, P-monoesters, being P-diester the main peak, and
lower inorganic P content. The spectrum showed a broad resonance in the P-diester region
57
(-1.5 to -5 ppm). The P-diester peak in this study can be related to the presence of
phospholipids, as the DNA P diesters are found in the chemical shift at 0 and -.37 nm
(Makarov et al., 2002) . A broad P-diester peak has also been observed in aerobic municipal
sewage sludges (Hinedi et al., 1989). The low peak resolution in P-diester area may be due
to presence of proteins in the extract and their interaction with phospholipids as well as the
mixture of phospholipids found in the sample (Yeagle et al., 1977). Additional evidence that
we are collecting phospholipids and other cell membrane constituents in the extract is the
peak from 1.3 to 0.5 ppm as the coumpounds assigned to this peak are phosphatidyl choline,
(0.78 ppm), and teichoic acids (2.5 to 1.2 ppm). A smaller and broader peak is detected from
3.5 to 5.5 ppm. This range of shifts is attributed to orthophosphates (5.7-6.1 ppm) and
orthophosphate monoesters (3 to 6 ppm). These type of P compounds have been found in
humic substances from sewage sludge (Bartoszek et al., 2008).
-10-505101520
Chemical Shift (ppm)
a
b
c
Figure 3-9 31P NMR spectrum of P containing constituents from pH 12.6 extract; a = diester P, b=diester and teichoic P, c=monoester and inorganic P.
58
The presence of phospholipids in the extract can partially explain the surface activity
of the extract. Phospholipids are capable of reducing the surface tension of solutions to
levels ranging from 25mN/m (for phospholipids) to 45 mN/m (for lysophospholipids) at the
CMC. The CMC of di-C16-phospholipids is in the range of 10-10 M (Tanford, 1978) and in
the range of 10-5 M for mono-C16 phospholipids (lysophospholipids) (Stafford et al., 1989).
Considering the typical molecular weight of these phospholipids, their CMC is within 10-7 to
10-2 g/L, a range of concentration that is lower than the concentration of lipids at the CMC of
the extract (~ 1g/L see Garcia-Becerra et al., 2009).
The 13C NMR and 1H NMR spectra for the pH 12.6 (4hr) extract were also obtained
(data not shown) but the heterogeneity of organic compounds in the extract produced broad
peak distribution that prevented us from identifying chemical structures for the compounds in
the extract. However, these distributions were consistent with the work of Bartoszek et al.
(2008) for treated municipal sludge samples. In the 13C spectrum there are two main regions,
one assigned to aliphatic carbons (50-110 ppm) and one assigned to carbons associated with
oxygen and nitrogen (160-200 ppm). These regions are consistent with the presence of
proteins, polysaccharides and lipids in the extract. The 1H NMR spectrum presents three
characteristic areas, one associated with hydrogen associated with alkanes (0-3 ppm), one
with hydrogen associated to carbons that are also associated with oxygen and nitrogen (3-5.5
ppm), and a small proportion of hydrogen associated to aromatic carbons (6-8.5 ppm)
(Bartoszek et al., 2008).
These fractionation and chemical structure studies further support the idea that the
extracted components associate in species that resemble the structure of giant micelles,
possibly with surface active phospholipids or other lipids adsorbed on the surface of these
59
assemblies (Le Maire et al., 2000; Piccolo, 2002). Unfortunately, this association prevents us
from assigning the surface activity of the extract to a specific set of compounds. Regardless
of the composition-property relationship, the surface activity of these extracts have been
explored in more detail (Garcia-Becerra et al., 2009). The prospect of using the extract in
some suitable form of cleaning formulation is an attractive proposition from the point of view
of producing “green” cleaning products. The rich chemistry of the extracts opens the doors
to recover a range of valuable products from this waste material. Issues such as heavy metals
content in the extract should be further studied. With regards to heavy metals, it is relevant to
highlight the findings of (Stendahl & Jäfverström, 2004) who observed that in activated
sludge residues, heavy metals precipitate at high (alkaline) pHs. The same authors propose
the use of lime in the process to recover the sodium hydroxide used in the extraction (a
similar process is used in Kraft recovery cycles).
3.5 Conclusions
The alkaline extraction for wastewater sludge biosolids is a feasible and productive
extraction method to recover surface active compounds. As a reference, this alkaline
extraction achieves approximately 2 to 3 times higher yields than the CER technique.
However, it is important to remember that CER is not meant to be used as a separation
technique for production purposes, but rather as an analytical extraction method for
extracellular polymeric substances. Both techniques have similar extraction selectivity for
proteins and polysaccharides but the alkaline method recovers approximately 10 times more
lipids, most likely as a result of lysing cell membranes. The analysis of the extraction kinetics
agrees with this, since it could be suggested that the alkaline extraction is able to disrupt RAS
flocs more effectively than the CER technique. As a result, deeply imbedded constituents
60
like intracellular material can be released into the extract. The presence of phospholipids and
teichoic acids, which are found in microbial cell walls, was detected in the 31P NMR of the
extract recovered at pH 12.6 and the size-exclusion chromatograms indicate that the alkaline
method recovers compounds of molecular weights in the order of ten of thousands of
Daltons. Further, the alkaline extracts achieves an average surface tension of 37 mN/m with
an average TOC content of 3.2 g/L, comparable to commercial products (35mN/m at ~3 g/L).
Further work is being conducted to determine the most suitable usage of the recovered
materials.
3.6 Acknowledgments
This work was supported by CONACyT (Mexican advisory board of science and
technology), the Environmental Consortium of the Pulp and Paper Centre (University of
Toronto), and by NSERC (Natural Sciences and Engineering Research Centre, Government
of Canada).
61
4 Chapter 4
Surfactant-like properties of alkaline extracts from
wastewater biosolids♣♣♣♣
4.1 Abstract
In order to assess the potential for utilizing wastewater biosolids as a source of useful
substances, the surface activity of materials extracted from wastewater biosolids (activated
sludge) by simple incubation with sodium hydroxide solutions at room temperature was
assessed. The surface activity, measured by surface and interfacial tension methods, of the
extracts was shown to be dependent on the extraction pH and the concentration of the organic
matter solubilized in the alkaline solution. Increasing the extraction pH increased the surface
activity of the extract (lower surface tensions), which is linked to the presence of more
hydrophobic species in the extract. After adjusting the pH of the extract to more acidic
values (e.g. pH=4), the extract retained their surface activity. The apparent CMC of pH 12.6
extracts was approximately 1000 mg/L (based on total organic carbon or TOC), and the
surface tension after CMC approximately 35 mN/m. While the CMC of the extract is
significantly high when compared to a conventional surfactant, sodium dodecyl benzene
sulfonate (SDBS, CMC ~ 25 mg/L), its surface tension after CMC was comparable with the
surface tension of SDBS. Above its CMC, the pH 12.6 extract had similar interfacial
tensions than SDBS against toluene, heptane and hexadecane. Furthermore, the extract and
SDBS had similar detergency performance for the removal of hexadecane from cotton. The
potential use of these extracts in commercial products is discussed.
♣ This chapter is based upon the manuscript titled “Surfactant-like properties of alkaline extracts from wastewater biosolids” which was accepted for publications in the Journal of Surfactants and Detergents (November 2009).
62
4.2 Introduction
Surfactants derived from biological sources, including waste biomass, using
biological means (i.e. fermentation) are conventionally known as biosurfactants (Alvarez et
al., 2002). These surfactants have been studied for a range of industrial applications for a
number of years, due to their high surface activity (low CMC, low surface tensions after
CMC), their biocompability, and the fact that they are derived from renewable resources
(Desai & Banat, 1997; Hayes, 2009; Mercade & Manresa, 1994; Singh et al., 2007; Van
Hamme et al., 2006). Despite their multiple advantages and desirable properties, there are
limitations to their industrial production, including the relatively slow fermentation, low
concentration in the fermentation broth (thus the need for separation), and the costs of
substrate and nutrients. Another approach to overcome the increasing need for surfactants
based on renewable resources is the use of a feedstock derived from a biological source
(biomass, e.g. grains) and chemically modifying such source to prepare more conventional
surfactants. The latter alternative can be classified as bio-based surfactants. Currently these
bio-based materials have found ample use in various surfactants and detergent products
(Hayes, 2009).
Despite the renewable nature of the feedstock used in these bio-based surfactants,
their sustainability is another aspect that needs to be considered. Various groups have argued
that the deforestation and ecosystem changes due to the high intensity cultivation of
oleaginous crops (that produce the vegetable oil feedstock for surfactant production) make
such biological feedstocks unsustainable (Brown & Jacobson, 2009). In this work we
63
explore the use of an arguably more sustainable feedstock, waste activated sludge, a waste
biomass, for the production of surfactant-like material. Work on waste biomass feedstock for
surfactant-like material production is relatively recent.
The work of Montoneri and collaborators at the University of Torino deserves special
mention (Montoneri et al., 2008; Montoneri et al., 2009; Quagliotto et al., 2006; Savarino et
al., 2007). Montoneri and collaborators started their work based on the earlier work that
reported that humic material extracted from decayed organic matter was capable of reducing
the surface tension, forming micelles, and increasing the solubility of organic material in
water (Guetzloff & Rice, 1994). Montoneri and collaborators extracted municipal solid
waste compost using an alkaline extraction method carried out in a nitrogen-rich atmosphere
at 65°C for approximately 24 hours, at a pH above 10 (Montoneri et al., 2008). The
surfactant –like material is recovered from the alkaline supernatant by precipitating the
extracted material at pH 2. The yields from that process are close to 12% of the dry biomass.
The CMC of the extracted surfactant-like material have been reported to range between 400
mg/L and 1000 mg/L and the surface tension after CMC has been reported to be about 36
mN/m (Montoneri et al., 2008; Quagliotto et al., 2006). These values suggest that these
alkaline extracts from municipal solid waste are more surface active than more conventional
humic material, which has CMC values close to 8 g/L and surface tensions after the CMC of
nearly 48 mN/m (Guetzloff & Rice, 1994).
In this article, a different waste biomass feedstock is extracted using a simplified
alkaline extraction method. Here, wastewater biosolids (activated sludge), is the substrate of
interest. Wastewater activated sludge is a by-product from the biological treatment of
wastewater that has the potential to be utilized as a source of biomass feedstock. It is
64
composed predominantly of water and organic matter, mainly microbial cells and the
biopolymers they produce during flocculation and consumption of the organic contaminants
in wastewater. The principal constituents of wastewater sludge solids include cells, proteins,
polysaccharides, humic substances, and lipids (Frølund et al., 1996) that have the potential to
be harvested for industrial applications (Kroiss, 2004). These wastewater sludge constituents
exhibit important surface active properties that enable intra and extracellular processes, such
as cellular motility, cell-cell aggregation, biofilm formation, cellular differentiation and
maturation, and substrate accession (Van Hamme et al., 2006). It has been suggested that
microorganisms from wastewater treatment plants may have evolved to produce
biosurfactants capable of degrading complex oily substrates (Mercade & Manresa, 1994).
Utilizing wastewater sludge to produce surface active agents has the potential of
reducing the net cost and environmental impacts of its disposal. Handling and disposal of
activated sludge represents approximately 50% of the operation costs in wastewater
treatment plants, and the environmental impact of its disposal (landfill, incineration, etc.) is
also considerable (Kroiss, 2004). It is estimated that in populations served by wastewater
treatment plants that produce activate sludge residues, nearly 30 grams of dry sludge are
produced per day per person (Von Sperling, 2007). This suggests that an average city of 3 to
4 million inhabitants could produce 100 tons of dry sludge every day. This is a significant
amount of biomass that could be utilized to produce a variety of products, including
surfactant-like material, which could improve the overall life cycle of surfactant-based
products. Activated sludge is currently utilized as feedstock for the production of biogass,
and has been explored as the source of lipids (between 2-30% of the dry biomass) for liquid
fuels (Boocock et al., 1992).
65
A number of facilities already use alkaline solutions to treat wastewater sludge to
stabilize the sludge (inactivate pathogens), to dissolve a fraction of the organic matter to
facilitate anaerobic digestion, or to reduce the heavy metal content in the dry sludge (Dewil
et al., 2006; Lin et al., 1997; Mizoguchi et al., 2008). In order to solubilize wastewater sludge
produced after treating crude oil process water, alkaline solutions with pH 11-12 have been
used to dissolve between 30-40% of the organic matter in the sludge (Mizoguchi et al.,
2008). The dissolved organic matter was recycled into the treatment facility to improve the
overall organic matter removal and reduce the solid waste.
We have evaluated the use of a simple alkaline extraction method carried out at room
temperature and in containers exposed to air to solubilize the organic matter from municipal
activated sludge (Garcia-Becerra et al., 2010). That method produced yields of solubilized
material of up to 60% within one hour of extraction at pH≥12. At that high extraction pH the
cell walls are disrupted, liberating most of the organic content into the aqueous solution. The
extract produced at those high pHs have a relatively low surface tension (~ 36 mN/m) and a
concentration (based on total organic carbon) ranging between 3 to 4 g/L. The relatively low
surface tension of these alkaline extracts suggests that they may have desirable surface active
properties. The purpose of this work is to evaluate the surface activity of the alkaline extracts
recovered at different pHs by determining their surface and interfacial tension, using
appropriate dilutions to determine their CMCs. To investigate their surface activity further,
their potential use as detergents is evaluated by determining the fraction of oil (hexadecane)
removed from oil-stained cotton swatches. The results from these studies are discussed in
light of the potential application of the extracts.
66
4.3 Materials and Methods
4.3.1 Materials
The following reagents were purchased from Sigma-Aldrich (ON, Canada) and used without
further purification: aqueous solution of 50% wt NaOH, reagent grade, used in the extraction
protocol), NaOH pellets (reagent grate, used in the MIDI lipid protocol), HCl (35-37% wt.,
reagent grade), hexane (HPLC grade), hexadecane (99%+), toluene (99%+), methyl tert-
butyl ether (HPLC grade), methanol (HPLC grade), GLC-90 fatty acid methyl ester (FAME)
Supelco® standard mix, NaCl (99+%), acetone (reagent grade), Sudan red III, sodium
dodecylbenzene-sulfonate (SDBS, 80% wt.). Heptanes (99+%) were purchased from
Caledon Laboratory Chemicals (ON, Canada). An ICP QC standard 4 solution (multi-
element, 5% HNO3) was purchased from Plasma Cal (QC, Canada).
4.3.2 Methods
Effect of extraction pH on surface activity
Alkaline extraction. Surface active material was recovered from aerobic return
activated sludge (RAS) collected from the metropolitan Ashbridges Bay Wastewater
Treatment Plant (1400 Population Equivalent; Average Capacity: 725,000 m3/day; Sludge
Retention Time: 2.5 days; Aeration Time: 6-8 h). There are two RAS sampling events
included in this study, the first one was taken in May of 2007, and the second one in June of
2008. After each collection, the RAS samples were kept in ice bath and transported to the
laboratory. After 1.5 h of settling, the supernatant (clear) water was decanted. The leftover
sludge (concentrated RAS) was later extracted. 50% NaOH solution was added to the
concentrated RAS to raise its pHs to 12.0, 12.6, and 12.9. These mixtures were incubated for
four hours under continuous agitation (500 rpm) at room temperature. The incubated
67
samples were then centrifuged (Beckman Coulter Centrifuge) at 3000 RPM (559Gs) for 13
minutes at 4°C, and the supernatants (i.e. the extracts) were collected. The extracts were
stored at 4˚C. To determine the amount of material extracted, the Total Organic Carbon
(TOC) content was measured in the concentrated RAS, as well as in the extracts using a
Shimadzu TOC-VCHS analyzer. In addition, the 2008 extract’s trace metals content were
assayed directly by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP AES)
using the Perkin Elmer Model Optima 3000DV ICP AEOS apparatus; the Total Nitrogen and
Total Carbon contents were determined using the Shimadzu TOC-VCHS analyzer; and the
total solids concentration was determined by drying the extract to constant weight at 50°C for
4 h.
Surface tension measurements. The surface tension of the extracts and SDBS
solutions was measured with a Sigma 700 tensiometer (KSV Instruments, Helsinki, Finland)
using the Wilhelmy (platinum) plate method. The measurements were carried out at room
temperature, using a stabilization time of 10 min. The surface tension of deionized water (3
µS/cm) measured under the same conditions was 71.5±0.5 mN/m. The surface tensions of the
extracts were analyzed as a function of extract concentration (diluting the original extract
with deionized water). In another set of experiments, the pH of the extracts was reduced by
addition of HCl, and their surface tension was measured as a function of pH. In order to
determine the CMC of the extracts at constant electrolyte concentration, the surface tension
of the extracts diluted in a 1% wt. NaCl solution was measured as a function of the total
organic carbon (TOC, mg/L) in the solution. The critical micelle concentration (CMC) was
determined graphically from a semi log plot of surface tension vs. concentration , identifying
68
the point of transition to constant surface tensions (after CMC) . The same studies to
determine CMC and surface tension after CMC were conducted using SDBS as a benchmark.
Lipid composition of the extracts. The lipid (FAME) composition was analyzed using
the MIDI method (Microbial Identification System, Microbial ID Inc.) (Smid & Salfinger,
1994). Briefly, the extract’s lipids were saponified by heat and the addition of a strong base.
Once the fatty acids were cleaved from these lipids, they were derivatized into their
corresponding FAME, extracted in an organic solvent and analyzed by gas chromatography.
A Perkin Elmer GC (Auto System XL) gas chromatograph, equipped with a Z5(poly (5%
phenyl/ 95% dimethylpolysiloxane) capillary column (0.25 mm, 30 m, 0.25 µm), and flame
ionization detector was used. Hydrogen was used as carrier gas. Fatty acids were identified
according to their retention time using a standard mix of reference FAMEs. All samples
were spiked with 3 ppm hexadecane as internal standard.
Interfacial activity and detergency performance of the pH 12.6 extract.
As it will be shown later, the extract at pH 12.6 has similar activity to the extract of
pH 12.9 but requires substantially less sodium hydroxide. Thus, the interfacial activity of
these extract as well as their performance as detergents was evaluated and compared to a
more conventional surfactant, sodium dodecyl benzene sulfonate (SDBS).
Interfacial tension. The spinning drop interfacial tensiometer (Temco Inc., Model
500) was used to measure the interfacial tension between the pH 12.6 extract neutralized to
various pHs (pH 4, 7, 11, and 12.6) in 1% NaCl, at a concentration equivalent to ~ 3 times
the CMC. Anhydrous heptane, toluene, and hexadecane were used as the oil phases,
representing a wide range of hydrophobicity. As a reference, the interfacial tension of SDBS
(above its CMC) was also measured using similar conditions but at pH 7 only.
69
Detergency performance. The soiling and detergency procedures of the fabric were
adapted from the literature (Acosta et al., 2003; Tongcumpou et al., 2003). Briefly, the fabric
used was 100% cotton prewashed to remove possible contaminants, dried under a fume hood
overnight, and cut into 12 cm by 8 cm swatches. The soiling was performed by immersing
the swatches for 1 h in a 20% v/v hexadecane in acetone solution with 500 ppm of red Sudan
III and dried overnight in a fume hood at room temperature. The detergency tests followed
the ASTM standard D3050-07 (ASTM, 2007). A Terg-O-Meter (Mechanical Components
Corp., Model 7243ES) was used for the studies. One liter of extract solution containing 1%
NaCl at neutral pH was put into each of the stainless steel containers of the Terg-O-Meter
along with four soiled swatches. The washing cycle consisted of 20 min wash, 3 min first
rinse with deionized water and 2 min second rinse with deionized water as well. The wash
and rinse cycles were conducted at 25°C and 110 rpm. The washed swatches were then dried
in a fume hood overnight.
To evaluate the percentage of oil removed, the specular reflectance of soiled and
washed swatches (sA), the soiled unwashed swatches (sB), and unsoiled swatches (sC) was
measured with an Ocean Optics (HR2000 model) spectrophotometer equipped with a fiber
optic reflectance probe. The reflectance probe was illuminated by a Tungsten halogen light
source (360-2000 nm). The probe was set at a 90° angle from the surface. The reflection
spectrum was captured and analyzed using the OOI Base 32TM software. The removal of
sudan red III-hexadecane was evaluated using the ratio of reflectances {(sA-sB)/(sC-
sB)}*100% (ASTM, 2000; Tongcumpou et al., 2003).
70
Statistical Analysis. All physical analyses were conducted in quadruplicates. This
work reports the average values along with the 95% confidence interval using the Microsoft
Excel software.
4.4 Results and Discussion
4.4.1 Effect of extraction pH on surface activity
The extraction parameters for activated sludge collected in 2007 and incubated for
four hours in alkaline solutions (pH 12.0, 12.6, and 12.9) are presented in Table 4-1 (similar
values were obtained for the 2008 samples). The extraction yields indicate that more of the
organic material is solubilized in the alkaline solution at higher extraction pH. These values
of yield are substantially higher than the 30% -40% solubilization reported for industrial
activated sludges treated with pH 11-12 alkaline solutions (Mizoguchi et al., 2008).
Table 4-1 Extraction parameters for material recovered at pH 12.0, 12.6, and 12.9 from municipal aerobic return activated sludge. RAS Sample collected in 2007. The concentration of the extracts are given in grams of total organic carbon (TOC) per L. Yield is defined on the basis of dry mass total organic carbon (TOC) of the concentrated RAS sample. Errors indicate the 95% confidence intervals
Extraction pH Yield (%) Concentration (gTOC/L) Surface Tension (mN/m)
12.0 55 ± 4 3.0 ± 0.3 41.6 ± 1.9 12.6 61 ± 3 3.4 ± 0.3 37.2 ± 0.7 12.9 64 ± 1 3.6 ± 0.4 34.5 ± 0.6
The reasons for the high extraction yields obtained at pH values higher than 12 likely
involve the disruption of cell membranes and the ionization of the constituents of the floc
(Garcia-Becerra et al., 2010). In order to interpret the values of extraction yield presented in
Table 4-1 it is necessary to keep in mind that not all the components extracted in the alkaline
solution are necessarily surface active. It has been found that those extracts are enriched in
proteins (~30% of the TOC), carbohydrates (~ 10% of the TOC) and approximately 3% of
C8-C20 fatty acids recovered as methyl esters (Garcia-Becerra et al., 2010). However, in this
work, no further separation or purification is considered since these extracts have surface
71
tensions that range between 34 to 37 mN/m, values that are comparable to the surface tension
after the CMC of conventional surfactants (Rosen, 2004). Further characterization of the
2008 extract metal content is found in Table 4-2. The principal metal is Na, which can be
attributed to the NaOH added during the extraction process. The rest of the predominant
metals in the extract are: K, S, Ca, Al, Fe, Si, Mg, and Cu. The presence of the divalent
metals is expected as they could have been extracted along with the extracellular polymeric
substances in the wastewater flocs (Alvarez et al., 2002). The presence of Si is also expected
due to the clays found in the open air secondary treatment in Ashbridges’s Bay’s facilities.
Overall the values of the metals are below the concentrations found in the wastewater
sludges, which may suggest that it is not hazardous with respect to its heavy metal content.
Table 4-2 Elemental analysis (metallic content) of the 2008 extract. All values in mg/L. The Na concentration in the blank (pH 12.6) is 2267 mg/L. Other metals that were assayed are not reported as they were all below the detection limit.
Constituent mg/L
Na* 2201.64 K 58.56 S 45.17 Ca 12.26 Al 10.74 Fe 6.29 Si 5.48 Mg 2.81 Cu 1.49 Zn 0.53 B 0.18 Ba 0.06 Mn 0.05 Co 0.02 Total Nitrogen 657.32 Total Carbon 3188.64 Total Organic Carbon 2932.16 Total Solids 8318.0
All surface, interfacial, and CMC values are reported in mgTOC/L. This allows the
results from the 2007 and 2008 extracts to be compared more accurately as in each extraction
different amounts of inorganic constituents are found, as well as different amounts of NaOH
72
were added to reach the target pH values. The surface tensions of the 2007 extracts obtained
at different extraction pHs is presented in Figure 4-1 as a function of the concentration of the
extract. To construct Figure 4-1, the extracts were diluted in deionized water. These surface
tension values are approximately constant at extracts concentrations above 1000 mgTOC/L,
but the surface tension increases as the extract concentration decreases below 1000 mg/L.
This suggests that under these conditions the CMC of the extracts is close to that
concentration. It is also important to recognize that the surface tension after this apparent
CMC is lower for the extracts obtained at pH 12.6 and 12.9.
30
35
40
45
50
55
100 1000 10000
Su
rfa
ce T
en
sio
n (
mN
/m)
Concentration (mgTOC/L)
pH 12.0 pH 12.6 pH 12.9
Su
rfa
ceTe
nsi
on
(m
N/m
)
Figure 4-1 Surface tension – concentration (expressed in mg of total organic carbon, TOC, of the extract per liter of solution) curves for extracts recovered at pH 12.0, 12.6, and 12.9 from return activated sludge (RAS) samples collected in May of 2007. Error bars indicate the 95% confidence intervals.
One point of concern is that these highly alkaline solutions are not compatible with
most cleaning applications. Instead, the surface activity should be evaluated at more neutral
pH. To this end, the extracts of Figure 4-1 were neutralized using HCl to pH 11, 9, 7, 4, and
2. The surface tensions of these neutralized extracts (all above their apparent CMC) are
73
presented in Figure 4-2. The surface tension at pH 11 and below were important because
during the development of the extraction procedure extracts with low surface tensions were
only recovered with pH values higher than 11 (Garcia-Becerra et al., 2010). As Figure 4-2
shows, reversing the pH to low values does not affect the surface tension of the extract. This
suggest that the extraction process is irreversible and that the extracts are stable (in terms of
surface tension) at a wide range of pHs.
30
35
40
45
50
1 3 5 7 9 11 13
Su
rfa
ce T
en
sio
n (
mN
/m)
pH
pH 12.0 pH 12.6 pH 12.9
Su
rfa
ceTe
nsi
on
(m
N/m
)
Figure 4-2 Surface tension of extracts recovered at pH 12.0, pH 12.6, and pH 12.9 from May 2007 RAS samples and neutralized with HCl to pHs 11, 9, 7, 4 and 2. Error bars indicate the 95% confidence intervals.
The CMC of the extracts obtained at different pHs was reassessed by neutralizing the
extracts with HCl (to pH 7±0.05). In the process of neutralizing the extracts, a substantial
amount of dissolved salts, mainly NaCl, are produced. Since the CMC of surfactants is
dependent of the electrolyte concentration, it is important that in these cases when the
concentrated surfactant has a significant amount of salt, the electrolyte concentration is kept
constant (Rosen, 2004). In Figure 4-3, the surface tension of the 2007 extracts obtained at
74
different pH values (but adjusted to neutral pH) was evaluated as a function of the surfactant
concentration in 1% NaCl solutions. The trends in Figure 4-3 are close to that in Figure 4-1,
mainly that the CMCs even in these conditions of different pH and electrolyte are close to
1000 mgTOC/L, and that the surface tension of the pH 12 extract is significantly higher than
the surface tensions obtained with pH 12.6 and 12.9. In practical terms, the data of Figure 4-3
indicate that the surface activity of the pH 12.9 extract is not superior to that of the pH 12.6
extract. This is important because the amount of sodium hydroxide required to achieve pH
12.9 is almost twice the required to achieve pH 12.6 (Garcia-Becerra et al., 2010).
25
40
55
70
1 10 100 1000 10000
Su
rfa
ce T
en
sio
n (
mN
/m)
Concentration (mgTOC/L)
pH 12.0
pH 12.6
pH 12.9
SDBS
Figure 4-3 Surface tension – concentration curves for extracts recovered at pH 12.0, 12.6, and 12.9 from RAS samples collected in May of 2007, and neutralized to pH 7. The surfactant solutions, including sodium dodecyl benzene sulfonate (SDBS), are diluted in 1% NaCl solution. Error bars indicate the 95% confidence intervals.
Figure 4-3 also incorporates the surface tension – concentration curves for SDBS,
used as benchmark in this study. The CMC of SDBS in the 1% NaCl solution (~ 0.17 M
NaCl) is approximately 25 mgTOC/L , a value that compares well with CMC values for SDBS
in saline solutions (Rosen, 2004). The surface tension after CMC for this SDBS solution is
75
slightly lower than the common value of 30-35 mN/m measured for similar solutions,
probably due to the presence of impurities in the technical grade SDBS used in this study.
Repeated measurements of the surface tension of deionized water (71-72 mN/m) were used
to validate the instrument. Certainly, the CMC of SDBS is substantially lower than that of
the extract. Since detergency, solubilization and other properties of surfactant formulations
are a function of the CMC, this suggests that higher concentrations of the extract are
necessary to obtain similar performance to that of a conventional surfactant like SDBS.
Once that concentration is achieved, the properties of the extract (for example, surface
tension in Figure 4-3) and the properties of the conventional surfactant seem to be
comparable.
In order to understand the effect of extraction pH on the surface active properties of
the surfactant, the FAME composition has been thoroughly analyzed (Garcia-Becerra et al.,
2010). Figure 4-4 only presents 3 FAME chromatograms to illustrate the changes in lipid
composition induced by the alkaline extraction. Each chromatogram was obtained from
2007 extracts at pH 12, 12.6 and 12.9. It is important to keep in mind that the actual height
of each FAME peak is not important since different total lipid concentrations were injected
each time. Instead, one should concentrate on comparing the relative heights of the peaks.
When comparing pH 12.6 with the pH 12 chromatogram, one notices that the fraction of C8-
C10 FAMEs reduces in comparison to the fraction of C16+ fractions. Furthermore, at pH
12.9 there are fractions of C20+ that begin to appear in the chromatogram. Overall,
increasing the extraction pH allows the extraction of increasingly more hydrophobic fractions
from the sludge. The fact that the extracts are enriched with C16 fatty acids is typical for
microbial cultures found in wastewater sludge (Conrad et al., 2003; Réveillé et al., 2003).
76
The data in Figure 4-4 has been presented to illustrate the changes induced with increasing
extraction pH, but it does not mean that the fatty acids in the extracted material are the only
ones responsible for the surfactant-like properties of the extract. For example, the group of
Torino has identified a complex humic-like material structure from NMR studies of their
extracts (Montoneri et al., 2008; Quagliotto et al., 2006).
pH 12.9
pH 12.6
pH 12.0
Internal standard
C18 fatty acid esters
C16 fatty acid esters
C20 fatty acid esters
C14
C12
C10
C8
Figure 4-4 Chromatograms (retention time vs. mV signal) of fatty acid methyl esters (FAMEs) derived from the extracts obtained at pH 12.0, 12.6, and 12.9 from RAS samples collected in May of 2007. The number of carbons in the fatty acid chains of the FAMEs of characteristic peaks are annotated in the Figure.
77
4.4.2 Surface activity, interfacial activity and detergency performance of pH 12.6 extract
Surface activity. Given the relatively high surface activity of the extract at pH 12.6
with relatively low sodium hydroxide (~0.45 g NaOH/g TOC in the concentrated RAS)
consumption when compared to pH 12.9 extract, the surface activity of this extract was
further explored. Figure 4-5 presents the surface tension versus surfactant concentration close
to the CMC of the pH 12.6 extracts obtained in 2007 and 2008, neutralized to pH 7, and in
the presence of 1% NaCl. It is worth noting that the surface activity of the extracts can vary
according to the origin or collection time of the RAS sample. Considering the variability in
the composition of the sludge it is, however, noteworthy the fact that even after one year the
CMC of the extract is relatively close. The surface tension after the CMC was, however,
slightly higher in 2008. For some samples collected during the winter of 2007, the CMC and
the surface tension after CMC were higher than the values obtained for the samples collected
in June of 2008.
30
35
40
45
50
100 1000 10000
Su
rfa
ce
te
ns
ion
(m
N/m
)
Concentration (mg TOC/L)
pH 12.6 - 2008
pH 12.6 - 2007
Figure 4-5 Surface tension – concentration curves for extracts recovered at pH 12.6 from RAS samples collected in May of 2007 and June of 2008, and neutralized to pH 7. The surfactant solutions are diluted in 1% NaCl solution. The solid lines are guides for the eye to illustrate the location of CMC. The gray region represents the range of concentrations were one could define the CMC of these mixtures.
78
The values of the CMC for the samples collected in 2007 and 2008, and presented in
Figure 4-5, cannot be determined precisely using the graphical method. The surface tension
curves have a number of kinks that are typically observed in mixtures of surfactants and
polyelectrolytes, and may reflect the interaction between low molecular weight surfactant-
like materials and proteins and polysaccharides (Goddard & Hannan, 1976). The values of
CMC, according to Figure 4-5, may be best reported as an interval between 800 to 1400
mg/L. For simplicity, 1000 mg/L (or 1g/L) was used as the CMC of the extract for the
purposes of evaluating the interfacial activity and detergency performance of the formulation
and comparing to the performance of SDBS. The range of CMC values, and surface tension
after CMC reported in Figure 4-5 are lower than those for sodium lignosulfonates. These
lignosulfonates have CMCs in the order of 5-10 g/L, and surface tensions after CMC in the
order of 42-45 mN/m (Askvik et al., 1999). While the extract seems to be more surface
active than lignosulfonates, is still less surface active than most biosurfactants (Desai &
Banat, 1997; Makkar & Cameotra, 2002).
In order to confirm the early findings that neutralizing the extract at different pH does
not affect the surface activity, the surface tension versus extract concentration data is plotted
in Figure 4-6 for the pH 12.6 extracts obtained in 2008 and neutralized to pH 11, 7, and 4.
The data in Figure 4-6 show that the surface tension – concentration data for the four pH
values considered coincides almost in the same curve. This observation is consistent with the
data presented in Figure 4-2 for the 2007 extracts, where it was concluded that changing pH
after extraction has a minimal influence in the surface activity of the extract.
79
25
40
55
70
1 10 100 1000 10000
Su
rfa
ce te
ns
ion
(m
N/m
)
Concentration (mg TOC/L)
pH 12.6 Extract @ pH 4
pH 12.6 Extract @ pH 7
pH 12.6 Extract @ pH 12.6
pH 12.6 Extract @ pH 11
Figure 4-6 Surface tension – concentration curves for the extract recovered at pH 12.6 from RAS samples collected in June of 2008, and neutralized to pHs 11, 4, and 7. The concentrated extract was diluted with 1% NaCl solutions to evaluate the effect of neutralization pH on the surface activity of the samples. Error bars indicate the 95% confidence intervals.
Interfacial activity of the extract In order to evaluate the interfacial activity of the
extract at oil/water interfaces, the interfacial tension of the extract above its CMC was
measured against toluene, heptane, and hexadecane. These are oils that represent a wide
range of hydrophobicity (Acosta et al., 2003). The interfacial tensions were measured as a
function of the pH of the solution containing 1% NaCl, and are presented in Figure 4-7. In
general, lower values of interfacial tension mean a better match of the hydrophilic-lipophilic
nature of the oil and the surfactant, and better detergency performance can be achieved
(Tongcumpou et al., 2003). Contrary to the response of surface tension in Figure 4-2 and
Figure 4-6, the pH of the solution plays a significant role on the interfacial activity of the
extract. For heptane and hexadecane, as the pH of the solution reduces from 11 to 7 the
interfacial tension doubles, from 7 to 14 mN/m. This change can be interpreted based on the
fact that the alkaline extraction ionizes numerous species with a negative charge that makes
80
them water-soluble. It could be suggested that as the pH of the solution reduces, close or
below the pKa of the species, the charges are neutralized, and the hydrophobicity of the
extracted material (e.g. fatty acids) increases. These hydrophobic species partition into the oil
phase, and in the process they lose their interfacial activity, thus increasing the interfacial
tension. In the case of toluene, the same effects are also observed, but the only difference is
that increases from pH 12.6 to pH 7, which might be due to the fact that toluene is
compatible with polar oils, and therefore more amenable to dissolve the amphiphilic species
present in the extract. The interfacial tensions of the pH 12.6 extract neutralized to pH 7 are
comparable to that the interfacial tensions SDBS against these oils (Figure 4-7), confirming
that above the CMC the surface activity of the extract is comparable to that of the
conventional surfactant. Furthermore, the interfacial tension observed with the neutralized
extract is within the upper range of interfacial tension values obtained with biosurfactants
(Makkar & Cameotra, 2002).
5
7
9
11
13
15
3 5 7 9 11 13
Inte
rfa
cia
l Te
nsi
on
(m
N/m
)
pH
Hexadecane
Heptane
Toluene
Inte
rfa
cia
l Te
nsi
on
(m
N/m
)
Figure 4-7 Interfacial tension of 3.4 gTOC/L solutions of the pH 12.6 extract (neutralized to various pHs) against heptanes, hexadecane, and toluene. The electrolyte concentration in all the samples was adjusted to 1% NaCl. As a reference, the interfacial of SDBS against these oils, and at pH 7, was 9.9 mN/m for heptane, 8.9 mN/m for hexadecane, and 7.2mN/m for toluene.
81
Detergency performance of the extract. Figure 4-8 presents the detergency
performance of the pH 12.6 extract neutralized to pH 7 in the presence of 1% NaCl. The
detergency performance in Figure 4-8 is presented as the %detergency obtained with the
surfactant solution (SDBS or the extract) minus the % detergency obtained with water alone.
Plotting this difference allows the comparison of the detergency performance obtained with
the extract obtained in 2007 and the extract obtained in 2008. The two data sets also
employed different aging of the swatches before washing. In the set of data of the 2007
extract, the swatches were aged for one week before washing, but for the washing studies of
2008 the swatches were prepared fresh (less than 12 hours of aging) before washing. Water
alone removed 19% of the oil (hexadecane) from the 2007 samples. However, water alone
removed 44% of hexadecane from the 2008 samples.
0
10
20
30
40
50
60
0 0.5 1 1.5 2 2.5
% R
em
ov
al -
% R
em
ov
al-
wa
ter
Surfactant Concentration (CMC)
SDBS- 2008
pH 12.6 Extract - 2008
SDBS-2007
pH 12.6 Extract - 2007%R
em
ov
al-
%R
em
ov
al w
ate
r
Figure 4-8 Increment (∆) in % of hexadecane removal from cotton swatches using the surfactant formulation over water, as a function of surfactant concentration expressed in terms of CMC. For the detergency tests using the 2007 extracts (using aged stains) 19% of hexadecane was removed using water-only wash. For the detergency tests using the 2008 extracts (using freshly stained swatches) 44% of hexadecane was removed using water-only wash. Washing solutions (extract and SDBS solutions) were at neutral pH, and contained 1% NaCl.
82
Aged stains are more difficult to remove, which helps explain the wider gap between
the performance of the extract and the performance of SDBS in the 2007 samples (Chi &
Obendorf, 1998). In the 2008 samples that gap is small, and supports the early hypothesis of
the similarity in the detergency performance of the extract and the conventional surfactant
when compared on the basis of their CMC. Figure 4-9 presents visual evidence (picture of
swatches) of this detergency performance.
Another point of interest is that the extracts have a brownish-reddish color after
extraction. Even at pH 7 they still retain a brownish –yellowish appearance. One area of
concern is the potential yellowing of the substrate after being exposed to the solution. Figure
4-10 presents visual evidence of an unsoiled swatch washed with deionized water and an
unsoiled swatch washed with the pH 12.6 extract at 1 CMC. The red –green –blue levels
obtained using the histogram tool of Corel’s Paint Shop ™ Pro 9 were R/G/B = 211/207/207
(as a reference, white R/G/B = 255/255/255) for the extract-washed swatch and 200/208/227
for the water-washed swatch. This suggest that the extract-washed swatch turned lightly
reddish (increase in red level) and lost some of its brightness (decrease in blue levels).
Soiled
Swatch
Distilled
Water
1 cmc
Extract
1 cmc
SDBS
Figure 4-9 Swatches before (soiled) and after the wash cycle using different washing solutions: distilled water, 1 CMC extract (recovered at pH 12.6) and 1 CMC SDBS. Washing solutions (extract and SDBS solutions) were at neutral pH and 1% NaCl during the wash step.
83
Unsoiled, pH
12.6 extract
Unsoiled,
water
Figure 4-10 Unsoiled swatches washed with 1 CMC of extract recovered at pH 12.6 (left) and with deionized water (right). The swatch washed with the extract shows some sign of “yellowing”.
4.4.3 Potential applications and outlook
Extracts from wastewater sludge (or RAS) have shown potential as surface active
material. The alkaline extraction method can recover up to 64% of the organic material in
wastewater sludge after 4 h of treatment. The simplicity of this alkaline extraction should
yield high throughput and reasonable production costs. These “raw” extracts have CMCs
comparable to purified extracts from solid waste treatment, which are lower than the CMC of
lignosulfonates and humic extracts. From the production standpoint, the availability of the
feedstock is not an issue, and it is possible that other wastewater sludges (with higher lipid
content) may produce even more surface active material. The surface activity of the extract
approaches but does not completely match the performance of conventional surfactants used
in the detergent industry.
The alkaline extraction method has other benefits with respect to the quality of the
extract; it disrupts the cell walls of the microorganisms, possibly killing residual bacterium
and viruses in the extract, and it may contribute to precipitate heavy metals from the extract.
However, there might be some limitations to using sludge-derived surface active agents, such
as long-term toxicity and presence of residual heavy metals, aspects that should be further
explored. Also, the extract's low interfacial tension values may limit its use in oil recovery
84
processes. At this point the extract may be more suitable for applications with minimal
human contact such as car washing, washing of exterior windows, or in environmental
remediation. The alkaline extraction technique may also be relevant to industrial wastewater
sludge.
4.5 Acknowledgments
This work was supported by CONACyT (Mexican advisory board of science and
technology), the Environmental Consortium of the Pulp and Paper Centre (University of
Toronto), and by NSERC (Natural Sciences and Engineering Research Centre, Government
of Canada).
85
5 Chapter 5
Wood adhesives based on alkaline extracts from
wastewater biosolids♣♣♣♣
5.1 Abstract
Wood adhesive formulations based on wastewater activated sludge, a renewable resource,
have been explored. The activated sludge was treated using a simple alkaline extraction
method. The effect of molecular weight, composition and pH on the extract’s adhesive
strength was assessed, as well as the effect of crossliking using glutaraldehyde. Mustard seed
protein isolate was used as a benchmark for these studies. Shear strengths of up to 4.5 MPa
for bonding maple were obtained at 30% relative humidity and 25°C, achieving up to 40% of
wood failure. It was determined that the adhesive strength was strongly correlated to the
microbial protein content in the adhesive formulas.
Keywords: wood adhesives, novel adhesives, water based adhesives; microbial adhesives;
wastewater sludge usages, biorefinery; ultrafiltration fractionation
5.2 Introduction
Urea-formaldehyde and phenol-formaldehyde resins, derived from petroleum or natural
gas, dominate the wood adhesive industry because of performance advantages and lower
costs (Wang et al., 2008). However, due to the uncertainty on the continuous availability of
petrochemical products, environment issues and health concerns, biological based adhesives
from renewable feedstocks have become desirable alternatives for wood adhesives (Haag,
2006; Kalapathy et al., 1995; Park et al., 2000; Wang et al., 2007).
♣ This chapter is based upon the manuscript titled “Wood adhesives based on alkaline extracts from wastewater biosolids” to be submitted to the Journal of the American Oil Chemists' Society
86
Historically, adhesives from biological sources have been produced from animal (hide,
casein) and plant (soy) proteins, and plant polysaccharides (starch) (A. P. Haag et al., 2004).
Lately, agricultural wastes or by-products have been used (Odozi & Agiri, 1986). These
include: soy meal, chicken feather protein, flavonoid-glycosides from orange mesocarp, red
onion skin tannin, and meat and bone meal (Akaranta & Wankasi, 1998; Jiang et al., 2008;
Kalapathy et al., 1995; Park et al., 2000; Wang et al., 2008). More recently, microbial
biopolymers have also been considered (Combie et al., 2004; Haag et al., 2006; Haag et al.,
2004; Weimer et al., 2003; Weimer et al., 2005). Microorganisms, like bacteria and algae,
excrete polymeric substances that enable them to get anchored in place (Combie et al., 2004;
Haag et al., 2006). Their strong attachment to a variety of surfaces in aqueous environments
has led to exploration and development of bacterial exopolymers as commercial wood
adhesives (Combie et al., 2004; A. P. Haag, 2006). However, microbial products tend to be
expensive. They are usually produced by batch-wise fermentation in stirred tanks under
sterile conditions. Further, glucose and sucrose are commonly used as the carbon and energy
sources (A. P. Haag, 2006). Thus, cheaper microbial biopolymers, such as fermentation
waste or by-products would be advantageous and have been explored (Weimer et al., 2003;
Weimer et al., 2005).
In this article we use the byproduct from the biological treatment (fermentation) of
municipal wastewater activated sludge, for the production of wood adhesives. Specifically,
for this work we use return activated sludge (RAS) as equivalent to waste activated sludge
that requires disposal in activated sludge treatment facilities. The principal constituents of
wastewater sludge solids include cells, and proteins and polysaccharides, either in pure form
or in conjugation with other compounds, such as glycoproteins, rhamnolipids, lipoproteins,
87
etc. (Frølund et al., 1996) that have the potential to be harvested for industrial applications
(Kroiss, 2004). Utilizing RAS to produce adhesives has the potential of reducing the net cost
and environmental impacts of its disposal. Activated sludge handling and disposal represents
up to 50% of the operation costs in wastewater treatment plants, and the environmental
impact of its disposal (landfill, incineration, etc.) is also considerable (Kroiss, 2004).
In general, biopolymer based adhesives are useful but have limited applications due to
moisture sensitivity, thermal instability, or processing difficulties (Haag et al., 2006). In
order to enhance their performance, biopolymers are modified to increase their
hydrophobicity and molecular weight, change their conformation, and/or to expose or
derivatize their functional groups (Wang et al., 2007). Dispersion and unfolding of proteins
is enhanced using sodium hydroxide, urea, guanidine, and hydrochloric acid (Wang et al.,
2008; Wang et al., 2007). In the case of polysaccharides, they can be made more
hydrophobic by derivatizing their hydroxyl groups into esters or ethers (Haag et al., 2006).
Crosslinking also improves the mechanical properties and water stability of biopolymers
(Haag, 2006; Reddy et al., 2008; Wang et al., 2007). The most common cross linking agents
are aldehydes, carboxylic acids and enzymes (Haag, 2006).
In aldehydes, the method of action is believed to be the formation of crosslinkages both
within and between biopolymers that contain amino groups, such as proteins and some
polysaccharides (i.e. chitosan) (Haag, 2006). They form links between the soluble
(hydrophilic) biopolymers and the structural biopolymers to form a more hydrophobic and
heterogeneous network (Hopwood, 1969). Of the aldehydes, glutaraldehyde has been
extensively studied as a crosslinking agent due to its effectiveness with biopolymers (Haag,
2006; Hopwood, 1969), as it can also react with aromatic groups forming strong
88
intermolecular crosslinks (Hopwood, 1969). Glutaraldehyde can also react to crosslink the
hydroxyl groups of the cell wall polymers in wood. Wood modification with glutaraldehyde
has been shown to increase wood’s anti-swell efficiency, dimensional stability, and
resistance against fungi decay (Xiao et al., 2010).
For this work, RAS was treated using a simple alkaline extraction method that has been
developed at room temperature (Garcia-Becerra et al., 2010). At that high pH we are able to
solubilize both extracellular and intracellular constituents producing the RAS extract, a
heterogeneous alkaline aqueous mix of dispersed and denatured biopolymers and other
organic material. The aim of this paper is to evaluate the performance of the extract as a
wood adhesive. We investigate the effect of molecular weight and pH on the extract’s
adhesive strength, as well as the effect of crossliking using glutaraldehyde. Mustard seed
protein isolate (MSPI) produced according to (Marnoch & Diosady, 2006) was used as a
positive control for our studies; and TitebondTM Original Wood Glue was used as the
commercial benchmark.
5.3 Materials and Methods
5.3.1 Materials
NaOH solution (50% wt., reagent grade) used in the extraction protocol and
glutaraldehyde solution (50% w/w, reagent grade) were purchased from Sigma Aldrich (ON,
Canada) and used without further purification. Protein content was determined using the
Pierce BCATM Protein Assay kit from Thermo Fisher Scientific Inc. (IL, USA), which
included the reference protein Bovine Serum Albumin (BSA). For polysaccharide analysis,
Phenol, 99% (Sigma Aldrich) and Sulfuric acid, 95%-98% (EM Science, NJ, USA) were
used with D-glucose, 99.5% (Sigma Aldrich) as the reference sugar. Lyophilized mustard
89
seed soluble protein isolate (MSPI) was kindly donated by Professor Diosady, University of
Toronto.
5.3.2 Methods
Production of RAS extract
On June 2009, the RAS aerobic return activated sludge (RAS) sample was collected
from the metropolitan Ashbridges Bay Wastewater Treatment Plant (1400 Population
Equivalent; Average Capacity: 725,000 m3/day; Sludge Retention Time: 2.5 days; Aeration
Time: 6-8 h), Toronto, Canada, kept in ice bath and transported to the laboratory. After 1.5 h
of settling, the supernatant (clear) water was decanted. The leftover sludge (concentrated
RAS) was treated with 50% NaOH solution to reach a pH of 12.6 and incubated for four
hours under continuous agitation (500 rpm) at room temperature. The incubated sample was
then centrifuged (Beckman Coulter Centrifuge) at 6000 RPM (559 g) for 6 minutes at 4°C.
The supernatant (i.e. the extract) was collected and stored at 4˚C.
Fractionation of the RAS extract. Prior to membrane fractionation, the extract was
filtered through a Millistack+® Pod D0HC disposable filter to remove suspended
particulates. The extract was then fractionated using ultrafiltration (UF) hydrophilic
polysulfone membranes (A screen, Mini Biomax membrane, Millipore) of 50kDa and 10 kDa
nominal molecular-weight limits (NMWL). Hydrophylic membranes were chosen to reduce
membrane fouling and possibly reduce the transmission of more hydrophobic constituents
(Musale & Kulkarni, 1998; Musale & Kulkarni, 1997). Higher molecular weight and more
hydrophobic biopolymers have shown to have enhanced adhesive strength (Wang et al.,
2007). Further, the molecular weight limits were selected based on previous size exclusion
chromatography studies specifically for these extracts (Garcia-Becerra et al., 2010) and the
90
suggested molecular weight range of the adhesins found in RAS flocs (12 to 30 kDa) (Brei,et
al., 2009).
The extract was first concentrated to 30% of its initial volume at 50 kDa NMWL and
0.7 bar. The retentate was diafiltered at 10 kDa NMWL until pH 9.0 with 11 volumes of
distilled water. The permeate was further concentrated at 10 kDa NMWL and 0.6 bar to 30%
of its initial volume and diafiltered until pH 8.9 with 9 volumes of distilled water. Figure 5-1
shows the fractionation scheme of the extract.
The fractions’ pH was lowered from 12.6 to reduce their hazardousness and analyze
the effect of pH on their adhesive strength. They were taken to pH 8.9-9.0 because it has
been observed that alkali-modified soy protein can be brought down to 9 or 8 without
adversely affecting its adhesive strength or hydrophobic properties (Hettiarachchy et al.,
1995). The membrane operating conditions were based on work done for the ultrafiltration
method development. Reducing the extracts’ volume to less than 30% from its initial volume
reduced considerably the biopolymer yield (data not shown).
The RAS extract, Permeate 1, Retentate 2, and Retentate 4 were the chosen fractions
to formulate wood adhesives. These fractions were collected, frozen at -80oC, lyophilized at
0.12 mBar and -80oC in a Labconco FreeZone 2.5 Plus freeze dryer. Once dried, the analytes
were grounded in a mortar for uniformity, and stored at room temperature.
91
Retentate 4
pH 8.9
Permeate 1
pH 12.6
Retentate 2
pH 9.0
Stage 1 Concentration (UF1)50 kDa
Stage 2 Diafiltration(washing)10 kDa
Permeate 3
Permeate 2
Permeate 4
Filter
Stage3 Concentration (UF2)50 kDa
Stage 4 Diafiltration(washing)10 kDa
RAS Extract
pH 12.6
Water
Water
Retentate 3pH 12.6
Retentate 1pH 12.6
Retentate 4
pH 8.9
Permeate 1
pH 12.6
Retentate 2
pH 9.0
Stage 1 Concentration (UF1)50 kDa
Stage 2 Diafiltration(washing)10 kDa
Permeate 3
Permeate 2
Permeate 4
Filter
Stage3 Concentration (UF2)50 kDa
Stage 4 Diafiltration(washing)10 kDa
RAS Extract
pH 12.6
Water
Water
Retentate 3pH 12.6
Retentate 1pH 12.6
Figure 5-1 Fractionation Scheme. The RAS extract was fractionated through 4 stages, two ultrafiltration stages (UF1 and UF2) and two diafiltration (washing) stages. The RAS extract, Permeate 1, Retentate 2, and Retentate 4 were used for the formulation of wood adhesives.
The mass balance for the recovery scheme above was carried out for RAS extract,
Permeate 1, Retentate 2 and Retentate 4 on TOC bases in Table 5-1.
Table 5-1 Mass balance on Total Organic Carbon (TOC) bases for the downstream process suggested in Chapter 4. The yield is calculated with respect to the RAS extract.
Volume (L) Concentration (gTOC/L) Total mass (gTOC)
RAS Extract 22 4.5 99.4 Yield from RAS
Extract
Permeate 1 15.4 3.5 54.6 54.9% Retentate 2 6.6 6.8 44.8 45.1% Retentate 4 1 1.0 1.0 1.0%
Preparation of alkali-modified MSPI
The MSPI was produced from yellow mustard seed with the technique developed by
Manorch and Diosady (Marnoch & Diosady, 2006). Briefly, the protein was extracted from
defatted yellow mustard seed at pH 11, ultrafiltrated, diafiltrated, precipitated at pH 5, and
freeze-dried. The MSPI powder was then modified according to the alkaline treatment by
92
(Hettiarachchy et al., 1995). The pH of the suspended MSPI solution was adjusted to 12.0
with NaOH and incubated at 40oC for 1 h. The modified MSPI solution was frozen,
lyophilized, grounded, and stored at room temperature.
Characterization of the RAS extract, RAS fractions, and modified MSPI
The RAS extract, chosen fractions, and modified MSPI were analyzed for the following:
Chemical Characterization. The Total Organic Carbon (TOC) and Total Nitrogen
(TN) contents were measured using a Shimadzu TOC-TN VCHS analyzer. Carbohydrates
were measured using the phenol-sulfuric acid method according to (Masuko et al., 2005)
using D-glucose as standard. Proteins were measured using bicinchoninic acid with the
BCATM Protein Assay kit. The spectrophotometric readings were measured with the
multiwell-plate reader ThermoU Spectra III A-5082 from SLT-Labinstruments.
Physical Characterization. The molecular size was determined using size exclusion
chromatography with Dionex ICS-3000 apparatus equipped with the 300 X 7.7 mm
Nucleogel® GFC 300-8 column by Macherey-Nagel GmbH & Co, and Chromeleon
interface. Mobile phase (flowrate 0.3mL/min) was double distilled de-ionized water and the
detection was carried out at 230 nm. The conductivity was also measured (conductivity
chromatograms not shown). D-glucose (180 Da) and BSA (66.430 kDa) were used as
standards. Prior to injection, the RAS extract and Retentate 2 solutions were filtrated through
Whatman filter paper 1 to eliminate suspended particles to avoid plugging the column.
Surface tension was measured with a Sigma 700 tensiometer (KSV Instruments, Helsinki,
Finland) using the Wilhelmy (platinum) plate method. The measurements were carried at
room temperature with a stabilization time of 10 min. All the analyte solutions were at 4 g/L,
beyond the observed critical micelle concentration (Garcia-Becerra et al., 2009).
93
Formulation of adhesives
The grounded freeze-dried samples were dispersed in distilled water at 15% w/w and
stirred for 30 min. These solutions were divided into two aliquots. 50% Glutaraldehyde
solution was added to one aliquote to 0.5%w/w and stirred for 30 min. Due to the low TOC
content in the RAS extract and Permeate 1 (approximately half of other analytes) another set
of formulations were prepared at 30% w/w of analyte, with 0.5% and 1%w/w of
glutaraldehyde. That way, the formulations of the different fractions could be at similar TOC
concentration, since it has been observed that the variation of adhesive strength with protein
glue concentration is high (Kalapathy et al., 1995). The adhesive formulations were based on
the work by (Park et al., 2000) and optimized in preliminary studies for the RAS extract and
fractions to achieve high adhesive strength and low viscosity.
Rheological measurements of adhesive formulations. The rheological properties of
the formulated glues were measured using a TA Instruments Carri-Med Rheometer (Model
CSL2 500, TA Instruments Rheology Solutions Software Data V 1.2.2) with a 4 cm
diameter, 1'59˚ cone angle geometry with solvent trap (truncation: 59 µm; stress factor:
0.0597; rate factor: 28.7). Experiments were conducted at 20°C.
Adhesive strength on wood. The adhesives were analyzed for their bond strength
following the modified lap shear test method developed by (Kalapathy et al., 1995). Wood
pieces were made from maple wood, cut into 2.54X 10.16 X 0.32 cm pieces, their surface
sanded, and preconditioned for 5 days at 25˚C and 30% relative humidity (RH) in a
controlled temperature and RH chamber. 120 mg of adhesive was placed on each of the
extremes on one side of a wood piece and spread on the two marked areas (2.54X2.54 cm),
this amount is based on (Wang et al., 2007). Two additional wood pieces of similar size
were superimposed on the glued areas and allowed to set for 10 min. The glued pieces were
94
then subjected to three different curing conditions. Hot press: 0.26 MPa press at 150˚C for
10 min. Cold press: 0.26 MPa press at room temperature for 2 h. Cold dead weight: 0.04
MPa press at room temperature for 12 h. The hot and cold press conditions were based on
(Wang et al., 2007) and (Kalapathy et al., 1995), respectively. For the hot and cold press
treatments the Dieffenbacker North America Inc. Type 450 press was used along with the
Pressman Press Control System and the Pressman Logger V 0.78 software. In the case of the
modified MSPI adhesive formulations the cold press condition was not tested.
The cured glued pieces were then equilibrated at 25˚C and 30% RH for 7 days in a
temperature and temperature controlled chamber. The adhesive performance was determined
by measuring the force required to break glued joints using an Instron Universal Testing
Machine (Model 4500, with MTS Sintech Test WorksTM V 2.1 software) by pulling apart the
two edges at a loading rate of 1.27 mm/min. All the adhesive strength data reported are
means of 8 replicates.
Statistical Analysis. With the exception of the adhesive strength measurements on
wood substrates, all analyses were conducted in triplicates. This work reports the average
values along with 95% confidence intervals using the Microsoft Excel software.
5.4 Results and Discussion
5.4.1 Chemical and physical characterization of RAS extract, extract fractions, and modified MSPI
The TOC and TN contents of RAS extract, Permeate 1, Retentate 2, Retentate 4, and
modified MSPI were measured (Figure 5-2). The analytes at higher pH (RAS and Permeate
1) have approximately half the TOC and TN contents than the other samples. The TOC and
TN contents are lower most likely because they were not desalted and the total solids include
a significantly greater amount of NaOH.
95
0
0.1
0.2
0.3
0.4
RAS Extract Permeate 1 Retentate 2 Retentate 4 MSPI
mg T
OC
or
TN
/ m
g T
ota
l S
oli
ds
TOC
TN
Figure 5-2 Total Organic Carbon and Total Nitrogen content with respect to the analytes’ total solids. Error bars indicate the 95% confidence intervals.
The protein and polysaccharide composition of the analytes are shown in Figure 5-3.
Figure 5-3 also depicts the fractionation of the extract. The RAS extract composition is
consistent with previous observations (Garcia-Becerra et al., 2010). Permeate 1 has a similar
protein and polysaccharide profile to the RAS extract. During the first concentration (UF1,
Figure 5-1), it was observed that a substantial amount of material migrated to the permeate
implying that the majority of the molecules in the RAS extract are smaller than 50 kDa
NMWL, which was used for the initial UF. Retentate 2 shows a greater concentration of
both protein and polysaccharides the RAS extract, which can be related to the diafiltration it
was subjected to. During the washes with 10 kDa NMWL (Stage 2, Figure 5-1), smaller and
more hydrophilic molecules (such as humic substances) were eliminated and the protein and
polysaccharide contents thus increased. Retentate 4 is the fraction with the highest protein
content. This is as expected from the working NMWL range, which was selected in order to
recover the RAS adhesins and other higher molecular weight constituents. The Retentate 4
96
carbohydrate content is higher than Permeate 1, but lower than Retentate 2. This could be
due to the additional processing done to Retentate 4. Polysaccharides smaller than 10 kDa
would of had a longer time to permeate and leave Retentate 4, than in the case of Retentate 2.
The protein and carbohydrate content of the MSPI is as expected (Marnoch & Diosady,
2006). Further, TN values in Figure 5-2 are consistent with the protein values found in
Figure 5-3.
0
0.3
0.6
0.9
RAS Extract Permeate 1 Retentate 2 Retentate 4 MSPI
mg
Pro
tein
or
Po
lysa
cch
ari
de
/ m
g T
ota
l S
oli
ds Protein
Polysaccharide
Figure 5-3 Protein and Polysaccharide content with respect to the analytes’ total solids. Error bars indicate the 95% confidence intervals.
The molecular size distribution of the analytes is shown in Figure 5-4. The retention times of
the main peaks are 9.01 min for RAS extract; 9.15 min for Permeate 1; 8.46 min for
Retentate 2; and 9.02 min for Retentate 4. Retentate 2 has the peak with the highest
molecular weight, consistent with its recovery from the 50 kDa NMWL (UF1,Figure 5-1),
while Permeate 1 has the peak with the lowest molecular weight since it would include all
molecules lower than 50 kDa NMWL. Also, as the number of UF steps increase, the
97
broadness of the peaks decrease, with Retentate 4 having the narrowest peaks among the
RAS extract fractions. It is important to consider that both RAS extract and Retentate 2 were
filtered to remove suspended solids and those larger molecules were not accounted for in the
spectra in Figure 5-4 and it may be possible that their peaks could be much broader.
Although protein and polysaccharides are the measured constituents in this work, activated
sludge is a highly heterogenous mix of free and conjugated lipids, proteins and
polysaccharides, as well as humic substances. These molecules can be associated in clusters
(e.g. micelles) which produce higher apparent molecular size distributions, which may make
the extract’s fractionation with respect to molecular size and identification using SEC less
effective. According to the results, while the fractionation scheme did not modify
significantly the molecular size of the constituents in each fraction, it was able to modify the
protein and polysaccharide composition of the fractions significantly (Figure 5-3). The
modified MSPI chromatogram has two main peaks at 8.75 min and 9.43 min, which may be
related to the partial degradation MSPI could have experienced with the alkaline treatment.
0
0.1
0.2
0.3
0.4
0 5 10 15 20
Retention Time (min)
0
0.1
0.2
0.3
0.4
0 5 10 15 20
Retention Time (min)
0
0.1
0.2
0.3
0.4
0 5 10 15 20
Retention Time (min)
0
0.5
1
1.5
0 5 10 15 20
Retention Time (min)
0
0.02
0.04
0.06
0.08
0 5 10 15 20
Retention Time (min)
(a) (b) (c)
(d) (e)
Figure 5-4 Size exclusion chromatograms of RAS extract, extract fractions, and modified MSPI. (a) RAS extract; (b) Permeate 1; (c) Retentate 2; (d) Retentate 4; (e) modified MSPI. The retention times from BSA (66.43 kDa) and D-glucose (MW 180 Da) are 8.14 min and 19.27 min, respectively.
98
The surface tension of the analyte solutions at 4g/L are presented in Figure 5-5. The
surface tension of the RAS extract is similar to previous studies (Garcia-Becerra et al., 2009).
Permeate 1 surface tension is higher than the rest of the other extract fractions. The reason
could be that during UF1 the more hydrophobic elements (such as proteins and lipids)
migratedt to the retentate, resulting in a higher surface tension in Permeate 1 from the RAS
extract. An inverse relation between molecular size and surface tension can also be observed
in the fractions with higher molecular weight constituents (RAS Extract and Retentate 2),
which have the lower surface tensions.
0
10
20
30
40
50
60
RAS Extract Permeate 1 Retentate 2 Retentate 4 MSPI
Su
rfa
c T
ensi
on
(m
N/m
)
Figure 5-5 Surface tension of solutions at 4g/L. Error bars indicate the 95% confidence intervals. The surface tension of deionized water (3 µS/cm) measured under the same conditions was 70.9±0.2 mN/m.
5.4.2 Rheological properties of formulated adhesives
The viscosity of the analyte solutions increased when glutaraldehyde was added,
which has been observed in other works (Haag, 2006; Wang et al., 2007). Figure 5-6 shows
the increase in viscosity of the modified MSPI solution with the addition of glutaraldehyde.
99
0
0.05
0.1
0.15
0.2
0.25
1 10 100 1000 10000
Vis
cosi
ty (P
a*
s)
Shear Rate (1/s)
0
5
10
15
20
25
30
0.1 1 10 100 1000
Vis
cosi
ty (P
a*
s)
Shear Rate (1/s)
(a) (b)
Figure 5-6 Effect of addition of glutaraldehyde on viscosity. Viscosity vs. shear rate (semi-log10 scale) of 15% w/w modified MSPI solutions: a) with 0.5%w/w glutaraldehyde, and b) without glutaraldehyde.
The rheological parameters (yield stress, τ0; consistency index, K; flow behavior
index, n) of RAS extract/fractions adhesives were evaluated by the Herschel Bulkley model:
τ=τ0+Kγn, where τ is the shear stress (Pa) and γ is the shear rate (s-1). The method of least
squares was used to find the best fitting equation for the experimental data by using
Microsoft® Excel Solver analysis tool, Table 5-2 presents the calculated values. The MSPI
formulations along with all the measured RAS extract/fractions based adhesives presented a
shear thinning beviour, with n<1 (Table 5-2).
Table 5-2 Rheological parameters of RAS extract and fractions
Adhesive formulation RAS Extract, 30%
RAS Extract 15%
Permeate 1, 15%
Retentate 2, 15%
MSPI
Yield stress, τ0 (Pa) 5.5 0.8 1.0 20.4 0.8 Consistency index, k (Pasn) 0.2 0.02 0.01 50.00 0.03 Flow behavior index, n (no units) 0.8 0.9 0.9 0.4 0.9
The protein content did not seem to have a significant effect in increasing the viscosity;
MSPI with the highest protein content does not have the greatest “k” value. Among the RAS
extract/fractions formulas, what seems to have affected the viscosity was the molecular
weight (Retentate 2 with the highest molecular weight constituents > 50 kDa shows the
100
highest yield stress). The amount of analyte in the formulation also increased the viscosity
(RAS extract 30% vs. 15% k index values) but not as significantly as the molecular weight.
0
0.05
0.1
0.15
0.2
1 10 100 1000 10000
Vis
cosi
ty (P
a*
s)
Shear Rate (1/s)
Permeate 1, 15%
RAS Extract, 15%
0
500
1000
1500
2000
2500
3000
0.01 0.1 1
Vis
cosi
ty (P
a*
s)
Shear Rate (1/s)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 10 100 1000
Vis
cosi
ty (P
a*
s)
Shear Rate (1/s)
(b)(a)
(c)
Figure 5-7 Viscosity vs. shear rate (semi-log10 scale) of adhesive formulations with glutaraldehyde: (a) Retentate 2 (b) RAS extract at 30%, (c) Permeate 1 at 15%, and RAS at 15%.
As a result, the solutions of the analytes with higher molecular weight constituents
(RAS extract at 30% w/w, Retentate 2) thickened considerably with the addition of
glutaraldehyde (Figure 5-7 a). Also, as previously observed (Kalapathyet al., 1996; Wang et
al., 2008), the presence of NaOH reduces the viscosity of the formulations. The RAS extract
and Retentate 2 formulations have high molecular weight constituents, but with the high
NaOH content, the RAS extract has a much lower consistency index (Table 5-2). Retentate 4
and Permeate 1 at 30% adhesive formulations could not be measured due to lack of enough
samples. However, anecdotally it can be mentioned that Retentate 4 formulation had a
101
similar consistency to the RAS extact at 15%; while the formulation of Permeate 1 at 30%
has a similar consistency to the RAS extract formulation at 30% (Figure 5-7).
5.4.3 Analysis of adhesive strength on wood
Effect of glutaraldehyde on adhesive strength. For RAS extract and its fractions,
none of the formulations exhibited measurable adhesive strength without glutaraldehyde. It
could be that the strong basic conditions the biopolymers and organic molecules where
extracted at (pH 12.6, 4h) affected their adhesive ability, as suggested in (Hettiarachchy et
al., 1995; Wang et al., 2008). The positive effect of glutaraldehyde on the RAS-based wood
adhesive formulas is most likely related to its ability to crosslink the biopolymers in the RAS
extract/fractions, improving the formulas mechanical properties. Glutaraldehyde can also
crosslink the hydroxyl groups of the cell wall polymers in wood (Xiao et al., 2010), further
enhacing the adhesiveness of the formulas.
In the case of modified MSPI cured under the cold dead weight curing conditions, the
formulation without glutaraldehyde achieved a slightly higher average adhesive strength than
MSPI with glutaraldehyde (Figure 5-8). This could be due to a higher viscosity in the
formulation with glutaraldehyde (Figure 5-6), which could reduce adhesive strength
(Kalapathy et al., 1996).
Effect of curing conditions on adhesive strength. From Figure 5-8, it can be seen that
the all the formulations achieved their highest adhesive strengths under hot press curing
conditions. This is in agreement with the observations from (Wang et al., 2007), where the
physical attraction and chemical bonding took place during the thermal setting procedure
using glutaraldehyde as the crosslinking agent. The increase in adhesive strength may be
related to increasing the molecular weight of the extract biopolymers through glutraldehyde
102
crosslinking. The increase in adhesiveness may also be due to the modification of wood
polymers crosslinking with glutaraldehyde among themselves or with the extract
biopolymers. Further, the effect of exerted pressure (during the curing conditions) on the
adhesive strength can be seen in Retentate 2 and Retentate 4 (Figure 5-8). Despite applying
0.047 MPa of pressure during 12 h, the adhesive strength was higher with a higher curing
pressure of 0.26 MPa for 2 h.
0 2 4 6 8
MSPI, 15%
MSPI 15% + Glu
Retentate 4, 15%
Retentate 2, 15%
Permeate 1, 15%
Permeate 1, 30%
RAS Extract, 15%
RAS Extract, 30%
Adhesive Strength (MPa)
Cold Dead Weight
Cold Press
Hot Press
Figure 5-8 Effect of curing conditions on the formulations’ adhesive strength. Permeate 1 formulations only exhibited adhesiveness under hot press conditions. RAS extract formulations did not present adhesiveness at the cold dead weight curing conditions. The MSPI formulations were not tested under the cold press conditions. The average adhesive strength for TitebondTM is 5.5 ±0.8 MPa. The Error bars indicate the 95% confidence intervals of 8 replicates.
Effect of pH on adhesive strength. Formulations at higher pH (NaOH) conditions
(RAS extract and Permeate 1) were sensitive to moisture without the hot curing conditions.
After being treated with the different curing conditions their formulations seemed to have
glued the wood sample. However, after 7 days at 30%RH the glue joint was rehydrated and
103
presented extensive cohesive failure. The decrease in adhesive strength at higher ionic
concentrations has been seen before and has been suggested to be a result of weakening
interactions between the polar groups of the proteins and the polar groups in wood
(Kalapathy et al., 1996).
Effect of protein content on adhesive strength. Most of the microbial biopolymer
based adhesives in the literature have been composed primarily of polysaccharides (Combie
et al., 2004; Haag et al., 2006; Haag et al., 2004; Weimer et al., 2003; Weimer et al., 2005).
However, the RAS extract and fractions are heterogeneous mixtures of polymers (Garcia-
Becerra et al., 2010) and the formulations adhesives strength can be correlated to the protein
content (Figure 5-9), while the polysaccharide content vs. adhesive strength has a linear
correlation value of 0.12. The formulation of Retentate 4, which has the highest amount
protein among the RAS derived adhesives, achieved the highest adhesive strength with 40%
of wood failure during the adhesive strength testing.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 1 2 3 4 5 6
% P
rote
n o
r P
oly
sacc
ha
rid
e (i
n T
ota
l So
lid
s)
Adhesive Strength (MPa)
Protein
Polysaccharide
Figure 5-9 Correlation between the protein and polysaccharide content in the formulations and the adhesive strength at hot press curing conditions (all adhesive formulations are at 15% w/w of RAS
104
extract/fraction or MSPI). In the adhesive strength vs. protein/polysaccharide % plot, the linear R-squared values for protein and polysaccharide contents are 0.96, and 0.12, respectively.
It is important to note that the protein to adhesive strength correlation in the RAS
extract and Permeate 1 formulations seem to be negative (Figure 5-8). This could be
explained with the higher biopolymer concentration at the 30% formulations than the 15%
formulations. Doubling the biopolymers amount in the 30% w/w formulations increased the
viscosity when compared to the 15% w/w formulations. This may have created larger
structures most likely with a higher hydrophobicity. If these parameters are increased, they
could be detrimental to the adhesive strength (Wang et al., 2007). This observation was also
made in the MSPI adhesive formulations with and without glutaraldehyde. The MSPI
formula with glutaraldehyde has a significantly higher viscosity (Figure 5-6) and lower
adhesive strength (Figure 5-8) than the MSPI formula without glutaraldehyde. However, it is
important to note that rheological properties could not be correlated in a straight forward
manner with the adhesive strength across all glue formulas in this study.
Overall, the range of values obtained with the RAS extract and fractions is
comparable to adhesive strengths of other biologically based adhesives (Akaranta et al.,
1996; Akaranta & Wankasi, 1998; Combie et al., 2004; Haag, 2006; Jiang et al., 2008; Liu &
Li, 2002; Wang et al., 2008; Weimer et al., 2003; Zhang & Hua, 2007). Specifically, the
Retentate 4 adhesive formulation was strong enough to produce up to 40% of wood failure
during the lap shear strength test, while the formulation presented a low viscosity. Moreover,
the formulations in this work do not use as hazardous chemicals as formaldehyde or other
volatile organic compounds like phenol. The adhesive strengths achieved in this work also
suggest that a heterogeneous mix of biopolymers can produce useful wood adhesives at
105
mildly humid (30% RH), and that both RAS and MSPI have the potential to be a source of
wood adhesives mostly for indoor applications.
106
6 Chapter 6
Preliminary feasibility assessment of the production of detergents and adhesives from municipal return
activated sludge (RAS)
The alkaline extraction technique developed in this work is able to recover highly
surface active constituents from return activated sludge (RAS). The results indicate that the
alkaline RAS extract can be used in the production of detergents and wood adhesives. The
extract can perform similarly to commercial detergents without further purification, while
strong wood adhesives can be formulated with glutaraldehyde after fractionating (molecular
weight range 10-50 KDa) and diafiltrating the extract to pH 9.
Due to the encouraging results from this research, in this section we look at the
possibility of implementing at industrial scale the proposed extraction and recovery scheme.
Since the recovery scheme and the formulation of wood adhesives are still in the
development stage, the preliminary cost estimation only included the cost of raw materials
and the price of the products. All other costs, including capital, operational (other than raw
materials), and maintenance costs were not considered in this assessment. The
recommendations for future work are provided addressing the extraction, fractionation and
formulation stages separately, as well as technical issues that have not been considered
before. The details of the calculations of this chapter are found in the Appendix.
6.1 Process description
6.1.1 Recovery of detergents from RAS
The developed alkaline treatment is presented in Figure 6-1. The wastewater sludge
is dewatered to approximately 5-6 g/L (on Total Organic Carbon basis) prior to the alkaline
107
treatment. The concentrated RAS is then put into contact with concentrated sodium
hydroxide at pH 12.6 for 4 hours under constant agitation. This stage produces an alkaline
liquor (supernatant) with solubilized material from RAS. The alkalinized concentrated RAS
is centrifuged and the supernatant separated from the pellet. At this point the extract
(supernatant) can perform as a detergent (Chapter 4).
Extraction at pH 12.650% NaOH
Concentrated Sludge
Pellet
RAS ExtractExtraction at pH 12.650% NaOH
Concentrated Sludge
Pellet
RAS Extract
Figure 6-1 Block flow diagram of the alkaline treatment to extract highly surface active material from return activated sludge.
The mass balance for the alkaline treatment described above was carried out on Total
Organic Carbon (TOC) basis (Table 6-1). The historical average values over 4 year of
research of concentrated RAS, RAS extract, and yield were considered in these calculations.
Table 6-1 Historical average values (TOC basis) of the concentrated RAS and RAS extract (2005-2009)
Volume (L) TOC concentration (g/L) TOC Content (gTOC)
Concentrated RAS 16 5-6 80-96 Yield (TOC)
RAS extract 15 4-3 45-60 56-63%
To reach pH 12.6, the consumption of NaOH during the alkaline treatment was on
average 2.1g per g of TOC in concentrated RAS in the range from 5 to 6 gTOC/L. If the RAS
is less concentrated, then it needs more NaOH to recover the same amount of solids because
a larger volume has to be treated. This is because the NaOH added during the alkaline
extraction not only ionizes/solubilizes RAS constituents, but also increases the pH of the
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solution and ionizes the carbonates dissolved in the solution as suggested in Figure 6-2.
According to this Figure close to 1/3 of the mass of NaOH is used to increase the pH of
water to 11 (blank data). This fraction increases even further to pH values of 12 and larger.
6.00
7.00
8.00
9.00
10.00
11.00
0.00 5.00 10.00 15.00 20.00
0.1M NaOH (mL)
pH
Experiment 8Experiment 7Experiment 6Blank-Experiment 6
Ice bath
Room temperature
Room temperature (bis)
Blank
6.00
7.00
8.00
9.00
10.00
11.00
0.00 5.00 10.00 15.00 20.00
0.1M NaOH (mL)
pH
Experiment 8Experiment 7Experiment 6Blank-Experiment 6
Ice bath
Room temperature
Room temperature (bis)
Blank
Figure 6-2 Titration curves of return sludge (100mL) with 0.1M NaOH conducted at room temperature, 20-25°C (2 replicates), and in icebath, 0°C. The blank is the titration of distilled H2O (100mL) at 20-22°C.
Considering a RAS concentration of 5-6 gTOC/L, the mass balance for processing 1 kg of
concentrated RAS into the RAS extract (detergent) is presented in Figure 6-3.
Extraction at pH 12.650% NaOH20.8-24.96 g
Concentrated Sludge1 kg (at 5-6 gTOC/L)
Pellet0.06 kg
RAS Extract0.94 kg (at 3-4 gTOC/L)
Extraction at pH 12.650% NaOH20.8-24.96 g
Concentrated Sludge1 kg (at 5-6 gTOC/L)
Pellet0.06 kg
RAS Extract0.94 kg (at 3-4 gTOC/L)
Figure 6-3 Block flow diagram of the alkaline treatment to produce detergents from RAS, including mass balance calculations. The density of the concentrated RAS and RAS is similar to that of water (1 kg/L).
109
6.1.2 Recovery of Adhesives from RAS
The supernatant can also be used in the production of bio-based wood adhesives. The
membrane separation technique from Chapter 5 produces extract fractions that can be
formulated into wood adhesives of various strengths. These fractions are collected,
lyophilized and ground. The fraction powder can then be formulated as wood adhesive at
15% w/w and 0.25% w/w glutaraldehyde, with the rest made up of water. Based on the
yields of the membrane processing from Chapter 5, the mass balance for processing 1 kg of
concentrated RAS into Retentate 2 and Retentate 4 is presented in Figure 6-4. Permeate 1 is
not included since it did not show considerable adhesive strength.
Extraction at pH 12.650% NaOH20.8-24.96 g
Concentrated Sludge1 kg (at 5-6 gTOC/L)
Pellet0.06 kg
RAS Extract 0.94 kg
(at 3-4 gTOC/L)
FractionationDiafiltration
Freeze-drying
Retentate 40.03-0.04 gTOC
Retentate 2 1.27-1.69 gTOC
Water for diafiltration(10X wash volume)
Extraction at pH 12.650% NaOH20.8-24.96 g
Concentrated Sludge1 kg (at 5-6 gTOC/L)
Pellet0.06 kg
RAS Extract 0.94 kg
(at 3-4 gTOC/L)
FractionationDiafiltration
Freeze-drying
Retentate 40.03-0.04 gTOC
Retentate 2 1.27-1.69 gTOC
Water for diafiltration(10X wash volume)
Figure 6-4 Block flow diagram of the alkaline treatment and recovery of wood adhesive raw material from RAS, including mass balance calculations.
It is important to note that the process in Figure 6-4 is not suitable for production
purposes. It was developed for analytical purposes in order to select the fractions and pH
conditions that made strong adhesives from the RAS extract.
6.2 Preliminary analysis for the production of value-added surface active agents from RAS
The economics of producing detergents and adhesives from RAS was assessed by
comparing the cost of the raw materials (NaOH and glutaraldehyde) to produce 1 kg of
110
product (either detergents or adhesives) and the potential revenue of selling 1 kg of product
(Table 6-2). The costs associated with the actual production (capital cost, operation and
maintenance) were not estimated since the recovery scheme and the formulation of wood
adhesives are still in the development stage.
Table 6-2 considers the average ratios from Figure 6-3 and Figure 6-4, 11.4 g of NaOH
(22.9 g of 50% NaOH) to produce 940g of RAS extract (detergent solution) or 3.3 g of
detergent (dry), 1.5 g of Retentate 2, and 0.035 g of Retentate 4. The suggested prices at
which the products can be sold at were obtained through personal communication with Mr. J.
Jevric, from LV Lomas Ltd. (March 17, 2010).
Table 6-2 Calculations for the production of 1 kg of detergent or adhesives from RAS. The prices of NaOH is of January 2010 at $0.17/kg (Anonymous, 2010a) and glutaraldehyde at $310/kg (Duvic, 2010).
Product Amount of NaOH required
NaOH Cost ($0.2/kg)
Glutaraldehyde cost ($310/kg)
Product Price
Revenue minus Raw material cost
Detergent 3.48 kg/kg dry detergent $0.6 - $6.6/kg $6.04 Retentate 2 7.73 kg/kg of Retentate 2 $1.3 $5.7 $4.0/kg - $2.94 Retentate 4 326.9 kg/kg of Retentate 4 $53.9 $5.7 $11.0/kg - $42.92
The preliminary assessment in Table 6-2 indicates that with the schemes proposed in
Figure 6-3, producing detergents may have the potential to create revenue. However, the
recovered extract/detergent in Figure 6-3 would require further processing to increase its
concentration since liquid detergents cannot be sold at such low dilutions. In contrast, the
production of adhesives according to Figure 6-4 does not show potential. If the extract RAS
could be utilized as wood adhesive with minimal or without further downstream processing,
it may also generate revenue, although further work is required in the recovery and
formulation of adhesives from RAS.
The alkaline extract can be sold as a raw material for the production of commercial
detergents and adhesives, the alkaline treatment operating costs are not expected to increase
111
significantly the net operating costs within a wastewater treatment facility. This is because
wastewater sludge must undergo certain treatment steps before its disposal or further use, in
particular disinfection. Sludge treatment is considered as an integral part of treatment of
wastewater and most wastewater treatment facilities already have the capability of
treating/stabilizing sludge (Fytili & Zabaniotou, 2008).
A cost comparison study was carried out for different RAS treatment alternatives for
the Gardermoen Wastewater Treatment Plant (Sludge load: 3.73 tonnes of dry solids per
year) (Ødegaard et al., 2002). The alternatives included thermophilic aerobic digestion with
anaerobic digestion, pre-pasteurization with anaerobic digestion, anaerobic digestion and
thermal drying, reactor composting, as well as quick-lime treatment of dewatered sludge (2h
at pH≥12). The quick-lime technique can be considered somewhat equivalent to the alkaline
treatment proposed in this work, as they have similarities in their operations. The cost of the
alkaline treatment was found within the range of other treatments, and in fact is lower than
other commonly used techniques such as anaerobic digestion with thermal drying and reactor
composting. At the same time, it should be highlighted that the costs associated with
concentrating/drying the alkaline extract have not been assessed and these may increase the
net operating costs considerably.
6.3 Recommendations for future work
The recommendation for future work in this section are towards the improvement and
optimization of the production scheme with the intent to make the recovery of value-added
surface active agents from RAS more effective and/or competitive.
112
6.3.1 Extraction
Recycling NaOH after the alkaline treatment should be explored as it is represents the
principal expenditure in raw materials (Table 6-2). An economic viability study of large-
scale implementation of the alkaline extraction technique by (Sarmiento, 2010) concluded
that NaOH is the main cost of raw materials, in agreement with the simpler analysis from
Table 6-2. The variability in the cost of caustic soda is also a significant issue. In January
2009 it was selling at approximately $1000/tonne and by July its price was down to
$250/tonne, mostly due to the cyclical nature of the caustic soda industry (Anonymous,
2010b).
Using an alternative base to NaOH in the alkaline treatment would also be beneficial
since caustic soda tends to be expensive with unstable pricing. A possible alternative to
NaOH is soda ash at $20-$90/ton in 2009. However, soda ash only reaches pH 12 (at 30%
solution) (Anonymous, 1991). This means that the extraction technique would have to be
modified to a lower pH working value. According to the kinetic analysis, lower pH
extraction values require more time to recover products, so the new scheme should also
include recirculation to increase the contact time and achieve similar conversion rates.
Reducing the extraction pH can also be an option when using NaOH. Both options will have
to be further explored.
6.3.2 Recovery
The fractionation scheme needs to be modified to maximize the yield of the fractions
that produced the stronger adhesives. Results from Chapter 4 suggest that the fractions with
higher protein content and possibly lower pH values produce stronger adhesives. Therefore,
the membrane fractionation scheme could be optimized to only recover these fractions.
113
Another option is to further study the adhesive strength of the RAS extract without
fractionation. The fractionation could be eliminated as the adhesive strength of the RAS
extract is similar or even higher than the strength of other fractions when formulated into
wood adhesives (Chapter 5). The RAS extract could be concentrated and diafiltrated to pH 9
with membranes of NMWL lower than 10kDA or with dialysis membranes. The formulation
of the desalted RAS extract would most likely need to be modified as well.
The recovery could also include processing the pellet (Figure 6-1). Preliminary
analysis suggested that the pellet contains lipids, nitrogen containing and other organic
(TOC) compounds (data not shown). It is likely that these constituents are higher molecular
weight products with respect to those found in the RAS extract and possibly more
hydrophobic compounds as they do not remain in suspension after the alkaline treatment.
6.3.3 Formulation
The need to lyophilize the RAS extract and fractions prior to formulation could also
be reconsidered. Freeze-drying the fractions may be avoided since the formulations require
them to be at a concentration of 15% Total Solids (or 10% TOC), not to be completely dried.
Reducing the volume can be done by further concentrating the fractions through
ultrafiltration. It will have to be assessed how this affects the adhesive strength of the
formulations.
The formulations can also be further tailored for each fraction to enhance their
adhesiveness. For example, since Retentate 2 recovers almost half of the initial TOC in the
RAS extract it could be an important fraction to develop as a strong adhesive. Although its
adhesiveness was not the strongest, its consistency index was approximately 100 times
greater than other formulations, which has been shown to be detrimental to the performance
114
of adhesives. Therefore its formulation could be modified to reduce its viscosity, which in
turn may be able to improve its adhesiveness.
The curing conditions of the RAS based adhesives could be further optimized.
Currently the conditions that produced the strongest adhesives are 0.26 MPa at 150˚C for 10
min. These conditions, specially the high temperature, can limit the application of the
adhesive. The hot-press conditions for bio-based formulations have shown to have the
following ranges 0.5 to 6 MPa, 5 to 15 min and 100 to 180°C.
6.3.4 Additional issues
In order to obtain a more complete picture of the capital and operating costs of the
proposed alkaline extraction, additional processes need to be further developed. The process
of drying/concentrating the extract should be optimized. Also, the disposal and reuse of
waste streams or by-products should be studied.
The use of the RAS extract in other applications could be explored. Due to the
extract’s high surface activity, once the extract has been desalted, it could be used as a paper
binder or dewatering/coagulating agent. The use of the developed adhesives could also be
studied in paper and cardboard applications.
6.4 The importance of utilizing RAS as a resource
The potential to utilize RAS as a raw material of industrially relevant products (fuels,
fertilizer, chemicals) is enhanced by the fact that handling of sludge is one of the most
significant challenges in wastewater management. Currently there are great efforts towards
addressing this issue as multiple regulatory bodies around the globe are significantly limiting
wastewater sludge disposal. For example, the European Union’s target to reduce final
wastewater sludge disposal by 50% compared to 2000 by 2050 includes the strategy of waste
115
recovery through reuse, recycling and energy recovery. At the same time, the most common
management practices (land application and disposal to landfills) are increasingly regarded as
insecure and being phased out. Further, sludge production is expected to increase
significantly in the future due to propulation growth (Fytili & Zabaniotou, 2008). Even if
there is no monetary profit in the production of value-added materials from RAS, the need to
utilize wastewater sludge and reduce the volume to be disposed of is an important driver of
this project.
116
7 Chapter 7
Overall discussion and significance of research findings
The overall objective of this project was to recover a range of potentially useful
surface active materials from wastewater sludge (RAS). This suggested application for
wastewater sludge is novel as previous works have focused mainly on the recovery of
biofuels and fertilizers from wastewater sludge, and to a lesser extend on bioplastics.
Utilizing wastewater sludge to produce bio-based surfactants has the potential of reducing the
net cost and environmental impacts of its disposal. In addition, biologically derived
surfactants have shown to offer important advantages over petrochemical ones, including
higher biodegradability, lower toxicity, and high surface activity at extreme conditions
(temperature, pH, salinity).
This study expands the existing literature on characterization of wastewater
sludge/sludge flocs by documenting the interfacial/surface active properties and lipid content
of the extracted sludge constituents. Also, this study has shown the correlation between RAS
extract protein content and adhesive strength, a novel finding in the field of microbially-
derived adhesives, which so far has been focused primarily on polysaccharides. The three
main contributions of this work are: (a) the development of an alkaline extraction, which is
able to ionize/solubilize the RAS biomass leading to the disruption of cell membranes and a
high yield of a surface active extract under conditions (time, temperature, concentration) that
are potentially scalable in wastewater treatment systems; (b) determining that the alkaline
RAS extracts have surface active properties similar to conventional surfactants and
detergents (most likely due to the presence of ionized lipids in the extract), opening the
117
possibility for an alternative use of this waste biomass extract; and (c) determining that these
extracts can also be used as crosslinkable adhesives and that this property seems to be
dominated by the presence of proteinic material in the extract.
7.1 Effective extraction of biopolymers from a heterogeneous culture
In this research, a scalable and effective alkaline extraction technique was developed
to recover surface active material from RAS (Chapter 3). Previous studies have proposed
various techniques to extract biopolymers from RAS for analytical purposes but not with an
aim for production. Also, the method in this work explored the extraction of RAS
biopolymers at pH values higher than 9, which had not been considered for activated sludge
before. The results show that this technique can extract up to 75% of the sludge’s organic
matter into a liquor containing potentially useful constituents (proteins, carbohydrates, etc.).
The high extraction yield can be explained by the treatment’s high pH. It was
observed that increasing the extraction pH increases the extraction yield. The results indicate
that above pH 12 the constituents can be solubilised and extracted at yields higher than
previously reported. From the comparison between the alkaline and CER treatments, it could
be suggested that in extraction techniques at pH values lower than 12, RAS macromolecules
(proteins, polysaccharids, lipids) cannot be recovered in the aqueous phase as effectively.
Gas chromatography (GC) and size exclusion chromatography (SEC) data showed that
higher extraction pH values also recovered larger molecules. Increasing the pH most likely
increases the degree of ionization of RAS constituents that have ionizable functional groups
like carboxylic acid (-COOH) and phenolic groups (-OH). This in turn can make the
constituents more water soluble, and thus increases the recovery of these molecules.
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The alkaline extraction technique was able to disrupt RAS flocs extensively and
recover deeply imbedded constituents like intracellular material (phospholipids) that are
highly surface active. The results also indicate that above pH 12 the RAS constituents can be
solubilised and extracted at yields higher than previously reported.
The simplicity of the alkaline extraction method is an encouraging element towards
the development of industrial processes for the production of surface active agents from
RAS. The operations used to recover the RAS detergents and adhesives were selected
because they are already used in wastewater treatment facilities and their documented ease of
scale-up in other production schemes. The selected operations are mixing,
centrifuging/filtering, and ultrafiltration, all of which are conducted at ambient conditions
and do not include complex mass transfer procedures as in the case of liquid-liquid
extractions for example. The process has has been taken from laboratory scale (processing
100-200 mL) to semi-pilot scale (60-80 L) without difficulty in achieving the set operation
conditions, and replicating the yield and quality of the extracts. During the implementation
of the extraction technique at industrial scale, the main issue to address is mainly related to
the large volume of water that will require processing.
7.2 Potential of RAS to produce surface active agents
This study showed that RAS can be a source of surface active agents comparable to
commercial detergents and adhesives. Despite the variability in RAS and the RAS extract
compositions, the extract performance did not show significant variability throughout the
length of this work (4 years). The surface tension and surface tension profiles of the extracts
remained similar during the 4 years of conducting extractions (Figure A-1 in the
119
Appendices). These factors increase the potential of RAS to become a raw material of value-
added products.
The resulting alkaline extracts were highly surface active. The low surface tension
(44 to 34 mN/m) could be linked to the extract’s constituents and the ionization/hydrolysis of
the extracted (solubilised) material. The extract contains proteins, polysaccharides, and
lipids (most likely as free and conjugated molecules) with a range of molecular weights
between 100 Da to 100 kDa. These amphipathic biomolecules can be highly surface active
materials in solution, on their own or in mixtures. High molecular weight polysaccharides
and lipopolysaccharides have high density of polar functionalities which can interact with
surfaces and interfaces. Similarly, polar and apolar groups in fatty acids, phospholipids and
proteins, can make these macromolecules highly surface active. It has been previously
shown that the major types of microbial surfactants include cross linked fatty acids,
glycolipids, lipopolysaccharides, lipoproteins/lipopeptides, and phospholipids. In general,
their structure includes a hydrophilic moiety consisting of amino acids, peptides (anions or
cations) and mono- or polysaccharides, and a hydrophobic moiety consisting of unsaturated,
saturated, or fatty acids. In this work, it was observed that as the surface tension decreased
the content of high molecular weight lipids increased, as well as the protein to polysaccharide
ratio in the extracts.
The alkaline treatment could produce low surface tension extracts probably because it
can induce hydrolysis and loss of molecular structure (e.g. unfolding of proteins) of RAS
components. This relates to increasing the extracts surface activity by exposing the polar and
non polar groups of macromolecules or modifying their hydrophilicty. Moreover, the
presence of phospholipids and humic substance (known surfactants) was suggested by
120
nuclear magnetic resonance (NMR) spectroscopy and by the fact that beyond extraction pH
11 extensive cell lysis occurs. Low surface tension may also be due to the saponification of
lipids during the alkaline treatment.
Overall, in this work it was found that the presence of proteins and phospholipids in
the RAS extract was correlated with the extract’s high surface activity. It was observed that
increasing the phospholipids and protein content and molecular weight improved the surface
activity in the RAS extracts. This is relevant since the surface activity of RAS flocs have
been mainly attributed to polysaccharides, and industrial microbial surfactants are currently
produced mainly from microbial polysaccharides or rhamnolipids. The results from this
research also indicate that heterogeneous mixes of biopolymers from RAS can perform
comparably to synthetic detergents and adhesives. These findings suggest that the
purification of specific compounds may not be necessary if the physical performance is the
main characteristic of a given product is its surface activity rather than its composition.
7.2.1 Detergents
In Chapter 4, the extracts surface active behavior was studied in detail to understand
their mode of action and establish the optimal extraction conditions (pH 12.6 for 4h). The
recovered extract was characterized as a detergent and its performance measured. The high
extraction pH (>12.3) was selected since it produced extracts with the lowest surface tension.
The extraction time was selected at 4h, since any additional time would imply a high capital
cost due to increased volume (retention time) with little recovery gain. Further, it was
observed that without further purification, the extract performed similarly to synthetic
detergents. This reduces the need of further purification, which is widely recognized to be
technical and economical challenging for biotechnological products.
121
In Chapter 4, as in Chapter 3, the extracts surface activity (measured as surface and
interfacial tension) was shown to be dependent on the extraction pH. Increasing the
extraction pH increased the surface activity of the extracts, which is linked to the presence of
more components and higher molecular weight lipids in the extract. After adjusting the pH
of the extract to more acidic values, the extracts retained their surface activity, implying that
it can be used at a wide pH range (pH 4 to 12.6), which is convenient for their application as
commercial detergents.
To characterize the extract as a detergent its critical micelle concentration (CMC) and
detergency performance was evaluated. The apparent CMC of pH 12.6 extracts was
approximately 1000 mg/L (total organic carbon basis), and the surface tension after CMC
was approximately 37 mN/m. While the extract CMC is significantly higher than that of the
conventional surfactant, sodium dodecyl benzene sulfonate (SDBS, CMC ~ 25 mg/L), its
surface tension after CMC was similar to that of SDBS. Above its CMC, the pH 12.6 extract
had comparable but higher interfacial tensions to SDBS against toluene, heptane and
hexadecane, implying that the extract is more hydrophilic than SDBS. Furthermore, the
extract and SDBS had similar detergency performances for the removal of hexadecane from
cotton.
7.2.2 Adhesives
The results from Chapter 5 indicate that the alkaline extract could be used in the
production of wood adhesives. The range of values obtained with the RAS extract and its
fractions are comparable to adhesive strengths of other biological based adhesives. The
formulations in this work have the additional advantage that they do not use as hazardous
chemicals as formaldehyde or other volatile organic compounds like phenol.
122
The extract recovered at pH 12.6 showed that it can be formulated into strong wood
adhesives using glutaraldehyde as a crosslinker. The effect of composition, pH and
molecular weight (>50 kDa, and 10-50 kDa) was assessed and discussed in Chapter 5. It was
determined that the adhesive strength was strongly correlated to the microbial protein content
in the adhesive formulas. It was also shown that high pH (high ionic content) reduces the
extract adhesive strength. The extract fraction with 10-50 kDa constituents at pH 9 achieves
the highest adhesive shear strengths (4.5 MPa with maple wood at 30% relative humidity and
25°C) with 40% of wood failure.
The presence of denatured proteins from the alkaline treatment in the RAS extract
could be related to its adhesive strength. Denatured proteins are surface active, flexible, and
high molecular weight molecules that make them strong adhesives. Denatured proteins have
shown to have a more labile surface which might facilitate their ability to penetrate the
adhesive substrate more easily. Further, it has been observed that strong protein-based
adhesives consist of relatively large flexible and interworven polymer chains. Higher
molecular weight molecules usually have increased hydrophobicity and better water
resistance; therefore, they tend to have higher adhesive strength. During fractionation with
hydrophilic membranes, the retentate (RAS extract) became enriched with higher molecular
weight constituents, resulting in an increase in protein content and thus, adhesives strength of
their corresponding formulations.
Regarding the effect of pH on the formulations adhesive strength, it was observed that
those at high pH (12.6) were sensitive to moisture (glue joint was rehydrated after curing)
and presented extensive cohesive failure. It has been previously suggested that high ionic
concentrations result in weaker interactions between the polar groups of proteins and polar
123
groups in wood. The excess ions (high pH) could be related to increasing the formulation
hydrophilicity, which may reduce the hydrophobic interactions between the adhesive and
substrate, thus, reducing the adhesive strength.
The effect of molecular weight on adhesive strength is less clear. It was expected that
the formulations with the highest molecular weight would be the most hydrophobic and
would have a stronger adhesive strength. However, the fractions with higher molecular
weight constituents did not show the highest adhesive strength, most likely because they
produced the most viscous formulations. Viscosity is a property of the adhesive’s functional
behavior and physicochemical nature as diffusion through the substrates affects its adhesion
properties. Usually, higher viscosity is not desired because it reduces the degree of chemical
wetting of the wood surface, increasing the possibility of developing voids in the bond-line,
resulting in a reduction of adhesive strength. Moreover, the low molecular weight
constituents did not make stronger adhesives either, most likely because they exhibited low
protein content. Overall, it has been observed that the factors that influence adhesive
strength (hydrophobicity, pH, etc.) tend to reach an optimum after which the adhesiveness of
the biopolymers no longer improves or even decreases.
Our work also shows a strong correlation between the adhesive strength of the RAS
extract formulations and their protein content. This is important because most microbial-
biopolymer-based adhesives in the literature have been composed primarily of
polysaccharides. The alkaline technique can then be applied in other industrial wastewater
sludges with higher lipid or protein content to produce more surface active material and
better performing detergents and adhesives.
124
The work from Chapter 5 has shown that RAS can be fractionated by molecular
weight and protein content to recover useful products. This is an important finding as the
fractionation of complex heterogenous mixes of biopolymers is still a recent field of research
(Bhattacharjee, 1994). In addition, the work from this thesis indicates that biorefinery,
recovering a suite of products from a biological heterogenous source, is technically feasible
in the case of RAS with the extraction and recovery scheme proposed in this work.
7.3 Additional Comments
There are multiple advantages of utilizing wastewater sludge as a raw material of
industrially relevant products. Environmentally, a renewable feedstock (RAS) is being used
to produce commercially surface active agents. Production of value-added materials from
RAS might also result profitable, although the recovery scheme stills needs optimization.
However, even if there would be little or no monetary gain in the utilization of RAS, the need
to reduce the volume of RAS to be disposed of is an important driver of this project.
125
8 Chapter 8
Conclusions and recommendations
8.1 Conclusions
In this work the potential of municipal return activated sludge (RAS) as source of value
added surface active agents was explored. The principal conclusions from this research are
the following:
1. A readily scalable and effective alkaline extraction technique was developed to
recover surface active material from RAS.
The high pH (pH 12.6) treatment can extract up to 75% of the sludge’s organic matter, a
yield higher than previously reported since past studies had proposed analytical
extraction techniques not for production purposes. The alkaline extraction’s scalability
suggests that it has potential to produce commercially surface active agents from RAS.
2. Highly surface active agents can be recovered from RAS with the developed alkaline
extraction, and have properties comparable to commercial detergents.
Without further purification, the RAS extract has a low surface tension (37 mN/m on
average) and performs similarly to synthetic detergents. The assessment of the alkaline
RAS extract as a detergent (insensitivity to pH, surface tension, interfacial tension)
suggests that the extract may be suitable for commercial applications.
126
3. The surface activity of extracts obtained from alkaline extraction of RAS is
dependent on the extraction pH.
Increasing the extraction pH increased the surface activity of the extracts, which is linked
to increasing the amount of higher molecular weight lipids, and the presence of
phospholipids and humic substances. Increasing the extraction pH beyond 11was also
linked to extensive cell lysis, increasing significantly the amount of recovered material
and the surface activity of the extracts.
4. The RAS extract can be formulated into strong wood adhesives using
glutaraldehyde as a crosslinker.
The range of adhesive strengths obtained with the RAS extract and its fractions are
comparable to adhesive strengths of other biological based adhesives. Specifically, the
extract fraction with 10-50 kDa constituents at pH 9 achieves the highest adhesive shear
strengths (4.5 MPa with maple wood at 30% relative humidity and 25°C) with 40% of
wood failure.
5. The adhesive strength of RAS-based adhesive formulas is strongly correlated to the
formulas’ microbial protein content.
The adhesive strength versus protein content of the RAS-based adhesives has a linear
correlation (R-squared) of 0.96. This is contrary to previous works where most
microbial-biopolymer-based adhesives in the literature have been composed primarily of
polysaccharides.
127
6. Recovering a suite of products from RAS, a biological heterogenous source, can be
technically feasible with the extraction and recovery scheme proposed in this work.
8.2 Recommendations
The following future research topics are recommended to further understand the surface
activity of the RAS extract:
1. Study the structure of the micelle/molecular associations of the RAS extract. This can
give further information of the molecular interactions between RAS constituents, which
may be related to their interactions in wastewater flocs.
2. Refine the analysis of the extract’s composition. Identify complex molecules (humic
substances, glycoproteins, lipoproteins, etc) not just proteins, polysaccharides and fatty
acids.
3. Determine the hydrophobicity of the extract and its fractions. This may provide
additional information on the surface and interfacial interactions of the extract as a
detergent and adhesive.
4. Establish the extent of protein/molecular degradation with alkaline treatment. This may
indicate how these compounds are modified, and further explain their physicochemistry.
5. Determine the presence of polyhydroxyalkanoate in alkaline extract. This may indicate
the potential use of alkaline treatment and/or alkaline RAS extract for the production of
bioplastics.
6. Study the application of the alkaline extraction on industrial wastewaters, especially those
containing high levels of proteins and lipids, in order to recover high surface active
extracts from other waste streams.
128
Additional recommendations of applied research projects were discussed in Chapter 6,
Section 6.3 which. These recommendations focus on the optimization of the extraction,
recovery and formulation of value-added surface active agents from RAS. They are
summarized below:
• Extraction: explore the possibility of i) recycling NaOH after the alkaline treatment as it
represents the principal cost in raw materials; and ii) using an alternative base to NaOH
in the alkaline treatment.
• Recovery: optimize fractionation scheme to maximize the yield of fractions with higher
protein content and lower pH values; study the adhesiveness of the RAS extract without
fractionation (only concentratated and desalted); study the adhesiveness of the pellet from
the alkaline extraction.
• Formulation: optimize the formulation for each RAS extract fraction (reduce viscosity);
optimize curing conditions for each adhesive formula.
• Additional issues: optimize the drying and concentration processes; explore the potential
to reuse waste streams/by-products from the alkaline treatment and fractionation scheme.
• Additional applications: explore the potential to use the desalted RAS extract as a paper
binder, and paper and carboard adhesives.
129
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Appendices
140
Appendix for Chapter 2 Wastewater sludge EPS characterization Activated sludge extracellular polymeric substances have been reported as a major sludge floc component. EPS have two different origins, from metabolism or lysis of microorganisms and from the wastewater itself. The types of biopolymers found in the EPS matrix are: proteins, polysaccharides, associations of proteins and polysaccharides, DNA, humic substances, and lipids.
Table A-1 EPS characterization
Reference Analyte TS g/L
VS, g/L
VS/TS Extracted EPS, %VS / Method
Wash step
Carbohydrates Proteins DNA Other findings
(Urbain et al., 1993)
16 activated sludges
1.89 1.56
0.83 14%TS 17%VS CER
Yes 22.63 mg/gVS average
135.98 mg/gVS average
16.70 mg/L --
(Görner et al., 2003)
1 municipal activated sludge
1.6 1.1 0.69 CER Yes 55 mg/gVS average
236 mg/gVS average
NM Polysaccharide-protein associations were observed
(Wilen et al., 2003)
5 municipal activated sludges, 2 industrial activated sludges
NR NR NR 10 to 30%VS CER
Yes 50-120 mg/gVS 270-500 mg/gVS
1-19 mg/VS EPS are very hydrophilic and negatively charged.(0.9% Rel. Hydrophobicity, -0.80 surface charge meq/gMLSS)
(Guibaud et al., 2005)
1 municipal activated sludge
NR NR 0.88 6% TS 7% VS Sonication
No 159.1 mg/gVS 395.45 mg/g VS
52.27 Extracted EPS from sludge have a greater capacity for complexing metals than those from pure cell cultures
(Frølund et al., 1996)
2 municipal activated sludges
NR NR 0.59 to 0.63
20-25%TS 33-42%VS CER
Yes 179-181 mg/gVS 462-523 mg/g VS
NM 50% of total protein in sludge was extracted from EPS vs. 10% of total carbohydrates. Cell biomass accounted for 10% of the organic fraction in sludge*.
* This value has also been found in 2S, with cell biomass accounting for 15% of the organic matter in biofilms from municipal biofilms. NM: Not measured NR: Not reported
141
Appendix for Chapter 6 Calculations for Figure 6-3 Historical average of NaOH consumed during extraction 2.08 g NaOH/gTOC in concentrated RAS Historical average TOC in concentrated RAS 5 and 6 g/L Historical average of TOC in RAS extract 3 and 4 g/L Historical ratio of Concentrated RAS to RAS extact 16 L of Conc RAS to 15 L of RAS extract Mass Balance for 1 L of concetrated RAS (= 1kg of concentrated RAS)
Amount of NaOH consumed during the extraction
for 5g /L of TOC for 6g /L of TOC
10.4 12.48 average 11.44 Amount of 50% NaOH consumed during the extraction
for 5g /L of TOC for 6g /L of TOC
20.8 24.96 average 3.29 Amount of average of NaOH/ dry extract 3.48
142
Appendix for Chapter 7 Similarity between different extracts produced through 2006-2010
25
40
55
10 100 1000 10000 100000
TOC (mg/L)
Su
rfa
ce T
en
sio
n (
mN
/m)
pH12.6 Summer
pH 12.6 Winter
SDBS
25
40
55
10 100 1000 10000 100000
TOC (mg/L)
Su
rfa
ce T
en
sio
n (
mN
/m)
pH12.6 Summer
pH 12.6 Winter
SDBS
Figure A-1 Seasonal variability in the surface tension profiles of the alkaline RAS extracts throughout the 4 years of this study (2006 – 2010). The variability is found in actual CMC value (between summer and winter) but not on ultimate minimum surface tension achieved by the different extracts.
