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THE EFFECTS OF LIMITED AERATION
ON EXPANDED BED
BIOLOGICAL WASTEWATER TREATMENT
by
Joshua D. Shrout
A Thesis submitted to the
Faculty of the Graduate School,
Marquette University,
in Partial Fulfillment of
the Requirements for
the Degree of
Master of Science
in Civil and Environmental Engineering
Milwaukee, Wisconsin
May, Nineteen Hundred and Ninety-Eight
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PREFACE:
Many industrial wastewaters contain a high concentration of organics (chemical
oxygen demand or COD) and sulfate concentrations which are too high to be treated by
conventional biological anaerobic treatment methods. Additionally, anaerobic biological
processes may be subject to inhibition under some conditions by constituents or products
of a high-sulfate waste stream. There is a need for biological wastewater treatment
processes which can treat high COD and high sulfate levels and achieve effluent
discharge requirements.
Recent research has examined expanded bed biological treatment of industrial
wastewaters. Expanded bed treatment has been shown as a highly effective anaerobic
treatment technology allowing for removal of high COD levels in a small reactor volume.
Other research has shown the ability of traditionally anaerobic (devoid of oxygen)
biological cultures to exist and perform in the presence of low amounts of oxygen
(microaerobic). Lastly, methanogenic treatment of high sulfate wastewaters has been
shown to be successful in systems which utilize some form of aeration.
The research performed involved a study of expanded bed reactors. Laboratory
techniques and analytical skills were utilized to perform a mass balance of COD and
sulfur constituents in EBR biological treatment systems. The collected data were
analyzed and organized to determine the significance of findings.
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ACKNOWLEDGEMENT:
I would like to thank the Graduate School and Water Quality Center of Marquette
University for funding this research and Dr. Daniel Zitomer for his direction to perform
and complete this work. My sincerest gratitude is also extended to Mr. Don Gamble for
his time and expertise utilized to address the myriad of tasks required to complete this
research. Lastly, I would like to thank my friends and family for their love and support
of my efforts throughout the execution of this project.
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TABLE OF CONTENTS
CHAPTER 1: CRITICAL REVIEW OF EXPANDED BED BIOLOGICAL
WASTEWATER TREATMENT .....................................................................................1
INTRODUCTION ...............................................................................................................1OVERVIEW OF TREATMENT USES ..............................................................................1REACTOR DESIGN ...........................................................................................................8BIOMASS..........................................................................................................................11TRENDS AND FUTURE RESEARCH............................................................................17
CHAPTER 2: STUDY OF MIXING, AERATION, AND ALKALINITY
REQUIREMENTS...........................................................................................................18
INTRODUCTION .............................................................................................................18MATERIALS AND METHODS.......................................................................................19
RESULTS AND DISCUSSION........................................................................................23CONCLUSIONS................................................................................................................30
CHAPTER 3: TROPHIC STUDY OF METHANOGENIC OXYGEN-LIMITED
TREATMENT..................................................................................................................32
INTRODUCTION .............................................................................................................32MATERIALS AND METHODS.......................................................................................36RESULTS AND DISCUSSION........................................................................................40CONCLUSIONS................................................................................................................46
CHAPTER 4: AERATED METHANOGENIC EXPANDED BED BIOLOGICALTREATMENT OF HIGH-COD HIGH-SULFATE WASTEWATER.......................48
INTRODUCTION .............................................................................................................48MATERIALS AND METHODS.......................................................................................50RESULTS AND DISCUSSION........................................................................................51CONCLUSIONS................................................................................................................58
REFERENCES.................................................................................................................60
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LIST OF FIGURES
2-1 EBR Configuration ..............................................................................................19
2-2 Modified EBR Configuration .............................................................................22
2-3 Alkalinity Added over time to maintain pH~7 at OLR=28g/LAd .................252-4 Fluorescein Tracer Dye Concentration over 3 HRTs.......................................272-5 KLa(33C)of Initial EBR Configuration ...............................................................282-6 KLa(33C)of Modified EBR Configuration ..........................................................29
3-1 Anaerobic-Aerobic Culture Methanogenic Activity.........................................41
3-2 Aerobic-Anaerobic Culture Methanogenic Activity.........................................42
4-1 COD Removal by Anaerobic Control EBR.......................................................53
4-2 COD Removal by Anaerobic Control EBR.......................................................53
4-3 COD Removal by Anaerobic Control EBR.......................................................54
4-4 COD Removal by Anaerobic Control EBR.......................................................54
4-5 Sulfur Speciation Balance ...................................................................................56
LIST OF TABLES
1-1 COD Removal of Various EBRs ...........................................................................3
1-2 Denitrification by Various EBRs..........................................................................5
1-3 Chloro- and Other Phenolic Removal by Various EBRs ...................................7
1-4 EBR Physical Effects on Biofilm ........................................................................14
2-1 Macro and Micro-nutrients in Influent Feed ....................................................20
2-2 Reactor Characteristics for Loading of 8g COD/LA
d ....................................242-3 Electron Equivalent Balance During OLR of 8g COD/LAd ..........................242-4 Reactor Characteristics for Loading of 28g COD/LAd ..................................262-5 Electron Equivalent Balance During OLR of 28g COD/LAd ........................262-6 Estimated Cost of Alkalinity versus Aeration ...................................................30
3-1 Oxygen-Limited, Methanogenic Biotransformation of Sucrose......................43
3-2 Methanogenic Activity.........................................................................................45
4-1 Reactor Oxygenation Data ..................................................................................52
4-2 Reactor Characteristics .......................................................................................55
4-3 Electron Equivalent Balance...............................................................................58
LIST OF EQUATIONS
4-1 Sulfide Production Stoichiometry ......................................................................57
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CHAPTER 1: CRITICAL REVIEW OF EXPANDED BED BIOLOGICAL
WASTEWATER TREATMENT
INTRODUCTION
Expanded bed reactors (EBRs) are currently utilized to achieve several biological
wastewater treatment goals. Also called fluidized bed reactors, EBRs were originally a
chemical engineering tool used to perform phase transformations, reactions, and
diffusions of various chemicals existing in solid, liquid, and vapor phases. With the
concept of maximum diffusion and maximum chemical reaction within a minimum
volume in mind, EBRs have been adapted to perform biological wastewater treatment
and are utilized in several process configurations. Examples of sole-standing EBRs,
EBRs in series, or EBRs as one part of a series of treatment steps may be found in the
literature. Most of these configurations may further be found to be operating
anaerobically, aerobically, or in some combination.
OVERVIEW OF TREATMENT USES
In many of the wastewaters amenable to biological treatment, the primary concern
is removal of chemical oxygen demand (COD) from the wastewater. COD sources in
wastewater can act as both a carbon source and electron donor to a microbial consortium
in many EBR biological treatment processes. In EBR treatment, the presence of high
biomass concentrations upon a carrier material allows for faster utilization of COD per
unit volume than many other types of biological treatment (Heijnen et al., 1989).
Numerous industrial and municipal wastewaters are suitable for EBR treatment,
including waste streams from brewery, dairy, ice-cream manufacturers, pharmaceutical
manufacturers, and paper mills. Usage of EBR treatment may also be found for process
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waters. For example, a closed-circuit recirculating papermill processing water was
treated by an anaerobic EBR with a hydraulic retention time of 12 hours to achieve up to
75% removal of COD which would otherwise promote biological growth in the system
fouling process systems and deteriorating product quality (Barascud et al., 1992).
Examples of wastewaters treated by anaerobic and aerobic EBRs are included in
Table 1-1.
Nitrogen Waste Constituents
EBRs may also effectively remove some non-COD constituents from waste
streams. An aerated biological EBR achieved 90% oxidation of ferrous iron in high-acid
mine drainage (Omura et al., 1991). EBRs have also been operated for the removal of
phosphorous from domestic wastewater (e.g. Piekema and Gaastra, 1993; Rensink et al.,
1991). Numerous examples also exist in the literature for denitrification. EBRs used for
denitrification are primarily run under anoxic conditions; however, several unique low-
aeration configurations have been utilized. Fdz-Polanco et al. (1994) utilized a two-stage
EBR with an aerobic zone at the top of the reactor. The anoxic zone present at the
bottom portion of the reactor achieved denitrification while the top portion oxidized
reduced Kjeldahl nitrogen (TKN) forms to nitrate (Fdz-Polanco et al., 1994). The
successful overall denitrification of TKN is achievable in one reactor because of the high
recycle ratio (23:1 of the influent) utilized to suspend the carrier medium--wastewater is
exposed to each of the two zones 23 times before discharge from the reactor. Therefore
concurrent nitrification and denitrification occurs, but at different locations within the
system.
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ReactorConfiguration
LoadingRate(g/L-d)
InfluentCODs(mg/L)
CODRemoval(%)
Wastewater HRT(hr)
pH Temp
(C)Reference
AnaerobicEBR
70 5,000 80 winedistillery
ng ~7 35 Holst et al.,1997
Anaerobic/Aerobic EBR
30 3,600 65 industrial 1.4 7 36 Heijnen etal. 1991
AnaerobicEBR
16 5,200 94 ice-cream 8 7.1 35 Borja andBanks,1995
AnaerobicEBR
14 47,000* 95 wort-brewery
24 7 53 Kida et al.,1991
AnaerobicEBR
10 6,000 41 biologicalsludge
2 7 35 Poggi-Varaldo etal.,1986
AnaerobicEBR
10** 390 100 municipal 1.5 7.4 10 Sanz andFdz-Polanco,1990
Aerobic EBR 8 188 82 synthetic 0.5 7 29 Tavares etal., 1995
EBR woxygenatorcolumn
3 830 80 simulateddairy
24 ng ng Forster etal., 1986
Anaerobiczone/Aerobiczone EBR
0.5 175 80 municipal 24 7 ng Fdez-Polanco, etal, 1994
Table 1-1: COD Removal of Various EBRsCODs- Soluble COD, i.e. filtered*-TOC(mg/L)**Total COD, i.e. unfilteredng-not given
Werner and Kayser (1990) utilized a unique three phase configuration for
denitrification. Biogas containing 60% methane from a landfill and a high nitrate
leachate wastewater were supplied to the EBR. The biogas methane (and possibly other
VOC constituents) was utilized as a carbon-source and electron donor. While the exact
pathway among assumed methanotrophic, methylotrophic, denitrifying, and other
organisms was not determined, denitrification of the wastestream was achieved, and
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methane was consumed (Werner and Kayser, 1990). An additional unique denitrification
design was utilized in a pilot study performed in Dresden, Germany to address high
nitrate concentrations in groundwater. Floatable carrier material was fluidized by a
downward flow in an anoxic EBR. Carrier material which escaped the reactor was
separated from the effluent (by floatation), and was returned to the EBR after a shearing
removal of biomass (Boehler et al., 1994). The cleaning of particles on a continuous
basis allowed for a pseudo-steady state condition where a less-thick more-active biomass
was selected (an important operational issue to be addressed in following sections).
Examples of denitrification EBRs are included in Table 1-2.
Sulfate/Sulfide Waste Constituents
Another potential waste stream to be treated by EBRs are high sulfate/sulfide
waste streams. Janssen et al. (1997) oxidized sulfide to sulfate and elemental sulfur in an
EBR supplied with dissolved oxygen in a recycle line aeration tank. The maximum
sulfide load reached was 14g HS-/Ld (Janssen et al., 1997). Other pertinent recent
research addressing sulfate/sulfide waste streams has been reported, however EBRs have
not been utilized. Anaerobic filter reactors have achieved removal of sulfate and COD
for influent COD loadings of 5 g/Ld at a COD to sulfur ratio of 8:1 (Parkin et al., 1991).
Modifications to biological filters have been made to aerate recycle flows, air strip
hydrogen sulfide, and achieve speciation to elemental sulfur from hydrogen sulfide laden
reactor contents (Guiot et al., 1997). Examples of successful treatment of high-sulfate,
high-COD wastewaters which were diluted prior to treatment (e.g. Fox and
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Venkatasubbiah, 1996) suggest that a modified EBR capable of handling higher COD
and sulfate loadings should be investigated.
Nitrogen Loading
(g NO3-N/Ld)
COD:N
(mass basis)
COD
Removal(%)
pH of
Reactor
Effluent
Products
Reference
70 1.7:1 ng ng 98% N2 Green et al., 1994
30 3.8:1 86 ng 10% N2,1% NO2,89% N2
Chen, S.D., et al.,1996
18.7 4.9:1 ng 7.0** 100% N2 Hirata and Meuta,1996
9.7 1.2:1 95 ng 98%N2,2% NOx
Germopre et al., 1992
8.3 0* ng 8.0 97% N2 Lazarova et al., 1994
7.8 0.68:1 76 6.3 79% N2,21% NO3
Boehler et al., 1994
0.55 0.82:1 ng ng 100% N2 Werner and Kayser,1990
Table 1-2: Denitrification by Various EBRs*NaHCO3utilized as carbon source**pH of influentng-not given
Specific Organic Constituents
EBR reactors have also been successful in treating waste streams containing
potentially toxic specific organics. Papermill wastewaters which potentially contain
chlorinated phenolics (e.g. Hakulinen and Salkinoja-Salonen, 1982; Puhakka et al., 1994)
and sulphite chlorine pulp bleaching agents (e.g. Fahmy et al., 1994a) have been
converted to mineralized products by EBRs. An aerobic reactor maintained at 3mg/L
oxygen was used to dechlorinate 2,4,6-trichlorophenol synchronously with nitrification
of ammonia present in bleached kraft pump mill effluents (Nevalainen et al., 1993).
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Further investigations of chlorophenol wastewaters indicated the availability of 4-
chlorophenol as a carbon source for denitrification, however, removals of both 4-
chlorophenol and nitrate were incomplete, achieving a maximum of 82% and 60%,
respectively (Melin et al., 1993).
To address the limitations of strictly anaerobic treatment, such as the formation of
rate-limiting tri-chlorophenolic intermediates (e.g. Tseng and Lin, 1994), anaerobic-
aerobic conditions have been studied. The phasing of an anaerobic EBR followed by an
aerobic EBR achieved biodegradation of 2,4,6-trichlorophenol, 2,4-dichlorophenol and 4-
chlorophenol under the highest loadings compared to anaerobic and aerobic control
EBRs which did not achieve biodegradation at high loadings (Fahmy et al., 1994b). The
use of an anaerobic EBR utilizing granular activated carbon (GAC) as the carrier medium
successfully converted greater than 99% pentachlorophenol to mono-chlorophenolic
compounds (Wilson et al., 1994). Degradation of phenols has also been achieved by
three-phase aerated EBRs which receive oxygen (e.g. Shishido et al., 1995; Wu and
Wisecarver, 1990). The successful partial conversion of chlrorinated-phenolic and other
phenolic compounds during anaerobic and aerobic steps would suggest that research of
anaerobic and aerobic phasing, or limited-aeration steps should be further investigated.
For while the use of GAC as an interactive carrier medium proved successful, the partial-
biodegradation partial-chemical exchange process generates GAC material which must
be regenerated or disposed rather than generation of all mineral products by complete
biodegradation. Systems that do not require carbon regeneration would be advantageous.
Table 1-3 includes examples of chlorophenolic and phenolic EBR wastewater treatment.
EBR Configuration Influent Effluent Species Reference
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anaerobic EBR/aerobic EBR
2,4,6-trichlorophenol,2,4-dichlorophenol,4-chlorophenol
no chlorophenols orphenols
Fahmy et al., 1994a,b
limited-aerobic EBR 2,4,6-trichlorophenol mineralized products Nevalainen et al.,1993
anaerobic EBR pentachlorophenol 2,3,6-trichlorophenol,2,4,6-trichlorophenol,mineralized products
Hakulinen andSalkinoja-Salonen,1982
anoxic EBR 4-chlrophenol 4-chlorophenol andmineralized products
Melin, et.al., 1993
anaerobic EBR 2,4,5-trichlorophenol 3,4-dichlorophenol +other chlorophenolsand mineralizedproducts
Tseng and Lin, 1994
aerobic EBR phenol mineralized products Wu and Wisecarver,1990
draft tube EBRwith aeration
phenol mineralized products Shishido et al., 1995
anaerobic EBRwith GAC
pentachlorophenol monochlorophenols Wilson et al.,1994
Table 1-3: Chloro- and Other Phenolic Removal by Various EBRs
Biodegradation of chlorinated ethenes is also of great interest. An anaerobic EBR
process showed biodegradation of tetrachloroethene (PCE) to vinyl chloride and ethene
when hydrogen partial pressure conditions were raised such that dechlorinating
microorganisms were able to out-compete methanogens for electron donors (Ballapragda
et al., 1997). An EBR supplied with oxygenated influent achieved biodegradation of 1,2-
dichloroethane and dichloromethane further indicating the possible benefits of anaerobic-
aerobic staging or phasing within EBR biological treatment systems to achieve complete
biodegradation of partially chlorinated or perchlorinated organics (Herbst and Wiesmann,
1996). As dehalogenation pathways, mechanisms, and kinetics continue to be more
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greatly understood, EBRs will continue to be an efficient treatment option to address high
concentration wastewaters.
REACTOR DESIGN
The capacity of a biological treatment system is often measured by using organic
loading rate (OLR). The OLR, expressed in units of COD mass per volume per time
(gCOD/Ld), allows comparison beyond influent COD concentration to help determine
the reactor volume required to achieve a required effluent concentration. In this manner,
EBRs are distinguished from many other biological treatment methods as sustaining
relatively high loading rates. For example, anaerobic filter or contact process systems
can typically operate at OLRs between 1-5 gCOD/Ld and aerated activated sludge
systems often operate at OLRs less than 1 gCOD/Ld (Speece, 1996). Conversely,
aerobic EBRs can treat between 1-5g COD/Ld (e.g. Forster et al., 1986) and anaerobic
EBRs can typically perform effective treatment at loadings of 40g COD/Ld or greater
(Totzke, 1997). EBRs generally exhibit a lower HRT, improved COD removal
efficiency, and lower sludge production than comparable treatment options (Tavares et
al., 1995). It should be noted that the OLR of biological systems is expressed in terms of
active reactor volume--a distinction not necessary for suspended growth systems.
However, for biofilm systems such as the EBR, the active volume constitutes that volume
within the whole reactor which contains carrier material sustaining microbial adhesion.
The ability of an EBR to effectively operate under high-OLR conditions is
influenced by the composition of the wastewater. Non-soluble suspended solid COD
forms are more difficult to treat using high load EBR processes. Suspended solids should
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generally be limited to less than 10% of the influent COD (Totzke, 1997) and also be
below 500 mg/L of the influent COD (Holst et al., 1997). However, high influent solids
concentrations comprising approximately 50% of influent COD have been found to be
successfully treated by EBR reactors (Sanz and Fdz-Polanco, 1990; Fdz-Polanco et al.,
1994).
The form of soluble COD is also very important to EBR performance. Typically
microbiological consortia present in EBRs are able to convert COD to methane when
influent COD is in readily degradable forms such as alcohols and volatile fatty acids
(Speece, 1996). More complex COD-form conversion to methane often requires raising
the HRT to allow time for the rate-limiting hydrolysis or acidogenesis reactions to occur.
Polysaccharide and simple sugars are typically best treated by inclusion of acidification
tanks as a first step in EBR systems or by the phasing of two EBR reactors in series
(Speece et al., 1997, Heijnen et al., 1989). Phasing refers to the development of unique
biomass in each reactorwhich promotes a two step conversion of COD to methane--
hydrolysis and acidogenesis in the first phase followed by methanogenesis in the second
phase (Speece et al., 1997). Additional benefits of phasing include a potentially greater
stability of pH, biofilm thickness, and sludge stability (Heijnen et al., 1989). While
phasing does require an additional tank or EBR, the total volume required to achieve
treatment is typically the same as a single-phase less-stable EBR (Heijnen et al., 1989).
The pH of the reactor is an important operational issue. A relatively high
concentration of un-buffered acids in the EBR can lead to biological inhibition (Speece,
1996), biomass detachment, and an overall decrease in COD removal efficiency (Meraz,
1997). Addition of alkalinity is typically required to maintain a buffered system at an
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uninhibitory pH. The operating pH of the reactor is typically based upon additional
removal efficiency achieved by raising the pH versus alkalinity addition costs. If
removal of sulfur and nitrogen compounds are a treatment goal, then the pH of the
reactor may frequently be operated at a pH other than 7 to achieve optimum treatment of
non-COD constituents (e.g. Boehler et al., 1994; Zuo Jiane et al., 1997). In an extreme
case, Omura and Umita et al. (1991) determined that a pH of 2.0 or less was required to
prevent reactor clogging by ferric hydroxide precipitate from oxidation of ferrous iron in
mine drainage.
Anaerobic EBRs containing predominantly mesophilic microorganisms are
typically operated at 35C. Significant OLR reductions must often be made for anaerobic
systems treating wastewaters at lower temperatures. Alternately, the EBR can be heated
to maintain high COD removal rates. Anaerobic EBR temperature problems are also due
to the greater temperature sensitivity of methanogens in comparison to acetogens. This
may lead to volatile fatty acids build-up in lower temperature reactors resulting in
inhibition of methanogenesis (Speece, 1996). Anaerobic EBRs can recover from
temperature shocks (e.g. Borja and Banks, 1995) which is an advantage over biological
treatment systems which have less reserve biomass and may be more greatly affected by
drastic swings in temperature. Aerobic processes are typically less sensitive to
temperature decreases, and aerobic EBRs have been shown to operate at temperatures of
10C and lower (Sanz and Fdz-Polanco, 1990). In instances of high temperature
wastewaters, thermophilic anaerobic EBR systems can also operate quite efficiently at
55to 65C for treatment of high temperature wastewaters (e.g. Kida et al., 1991).
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Shape of the EBR is a design consideration. Operation of a tall small-diameter
EBR containing the same liquid volume will have a greater upflow velocity. High
upflow velocity increases expansion of carrier material and shear stress at biomass/ liquid
interfaces. EBR diameter also effects the bubble size of gases which rise through the
system. A greater diameter EBR will lead to smaller bubble diameter and lower bubble
gas flow at a given height of the EBR (Shiau and Lin, 1991). Tapering of the EBR shape,
deviating to a cone from a perfect cylinder, affects mixing and bed height. Furthermore,
Peclet numbers (a measure of reactor mixing addressed in Chapter 2) decrease with an
increase in taper angle and fluidization becomes more violent (Webster and Perona,
1990). Shape, liquid flow, and gas flow are understood to inderdependently determine
the expansion and solid, liquid, and gas volume fractions within the EBR and may be
predicted from modeled relationships (Yu and Rittmann, 1997).
BIOMASS
An advantage of EBRs over other biological treatment methods is the high
loading capacity made possible by the adhesion and growth of microbiological organisms
upon the suspended medium. Organisms which are utilized for biological treatment are
usually a mixture of bacteria and archaea microorganisms. Mixed cultures provide the
advantages of COD transformation from polysaccharides to sugars to acids and hydrogen
with a final transformation to methane and carbon dioxide. Each of these transformation
steps is achieved by different organisms (Speece, 1996). Under anaerobic conditions,
methanogens must compete with other organisms in the mixed culture for carbon sources
which may be utilizing an alternative electron donor such as sulfate (Matsui et al., 1993).
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Characteristics of the wastewater, EBR design, and EBR operation are all factors in the
COD removal and product formation of EBR biological treatment. Overall success of the
EBR is determined by the development of an active biofilm on EBR carrier media which
transforms the desired wastestream constituents without producing undesired products.
For this reason, consideration must be given to the previously discussed physical
characteristics of the EBR. Most important, however, is the consideration of known
behavior of the biofilms to be developed, and the tendencies which may be exploited or
avoided.
Organisms present in a mixed culture utilized in biological suspended growth
systems (e.g. anaerobic digester, activated sludge, anaerobic contact process, and
sequencing batch reactor) do not behave in the same manner when the same culture is
introduced into an EBR system. EBR systems rely upon biomass adhesion to a carrier
material. Under some conditions in anaerobic EBRs, methanogens will adhere to carrier
material while acidifiers tend to remain suspended in the liquid (Heijnen et al., 1989).
In previously studied EBR systems, well before a steady state with respect to
solids retention time (SRT) was reached, specific activity of adhered biomass declined
(Imai et al., 1994). The specific activity of biomass in an EBR system is defined as the
COD removal from the wastestream which occurs per unit mass of biomass, typically
measured by volatile solids. Several mathematical models have been developed or
modified to predict the substrate utilization kinetics of EBR biofilms (e.g. Schwarz et al.,
1996; Uluatam, 1994).
Biofilm density is a function of upflow velocity and decreases with increasing
upflow velocity. Biofilm thickness, however, is independent of upflow velocity
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achieving a steady state thickness which is unaffected by upflow velocity (Araki and
Harada, 1994). Biofilm composition was shown to remain independent in anaerobic
EBR treatment of a sucrose and skim milk wastewater where acidifiers and acetoclastic
methanogens always predominated in contrast to other known methanogenic
microbiological pathway organisisms at various upflow velocities (Araki and Harada,
1994).
Biofilms are affected by substrate flux in EBRs. Rittmann et al. (1992) found that
when flux was large, biofilms became deep and biomass at the attachment surface
approaches zero activity. When flux was small, the biofilm on carrier material was thin
and approached complete substrate penetration. Biofilm accumulation is both a function
of substrate flux and specific detachment of biomass off the carrier material which may
be modified by changing particle shear. Active biofilm portions, however, are
determined solely by substrate flux, remaining independent of specific detachment rates
(Rittmann et al., 1992). An increase in substrate flux increases biofilm activity as well as
observed growth yield and oxygen uptake in aerobic EBRs. At low substrate
concentrations, a low bed expansion with a high upflow velocity is preferred to
encourage a maximum liquid-solid mass transfer, while at high substrate concentrations,
a higher expansion may be preferred to prevent diffusion limited conditions due to
increased biofilm (Ruggeri et al., 1994). Modifications to bed expansion without
changing upflow velocity are made by changing the density of carrier material. Biomass
growth upon carrier media typically causes the overall particle to become less dense as
well as causing an increase in Peclet number (indicating more plug-flow conditions) and
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a decrease in HRT as the available liquid volume in the reactor is decreased (Turan and
Ozturk, 1996). Table 4 includes effects of EBR conditions on biofilms.
Control Parameter Biofilm Thickness Biofilm Density Biofilm Activity Reference
increase detachmentrate
decreases increases unchanged Rittmann et al.,1992
increase substrateflux
increases increases increases Rittmann et al.,1992
increase liquidupflow velocity
unchanged decreases unchanged Araki andHarada, 1994
increase gas upflow
velocity
decreases not examined increases Tavares et al.,
1995Table 1-4: EBR Physical Effects on Biofilm
Biofilm effectiveness is typically measured by activity. If the biomass continues
to transform more COD or other constituent, then increases in biofilm thickness or
effluent suspended solids concentration may be acceptable. However, activity, thickness,
density, and detachment are interdependent. It is possible for an EBR operating with
high biomass and a low activity to produce a greater suspended solids effluent
concentration than a higher-activity low-thickness biofilm operating at the same SRT
achieving the same removal (Rittmann et al., 1992). If an EBR system is not initially
operated at ideal substrate flux and upflow velocity to achieve a higher activity biofilm,
the EBR may be modified to achieve the biofilm desired by physical or mechanical
alteration of the EBR system. Investigations into carrier media cleaning to limit biofilm
thickness have proven successful. The EBR design is modified to promote rise and
escape of fluidized carrier media for capture. The carrier is agitated to remove biofilm
and then replaced in the EBR preventing excessively thick biofilms from accumulating
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(e.g. Safferman and Bishop, 1996). Methods of increasing shear to decrease biofilm
thickness include increasing gas upflow velocity by adding more gas. Studies with
aerobic EBRs show that more active biofilms are achievable by utilizing aeration to shear
biofilms and maximize biofilm activity (Rittmann et al., 1992). For while high SRTs are
inherent to EBR systems, too high a SRT promotes accumulation of excessive thick
biofilm, decreasing substrate removal (Bousfield and Hermanowicz, 1984).
Choosing the EBR carrier material is also an important consideration. Sand and
glass beads are often utilized due to their simplicity in structure, availability, and cost.
However, carrier material specifically designed for use in EBRs appears to hold distinct
advantages. Biomass has been shown to adhere better to more porous carriers with more
crevices and coarse surface area making the biofilm less susceptible to washout (Chang
and Rittmann, 1989). Also, most engineered carrier materials are less dense than sand or
glass which allows for greater fluidization with less upflow velocity (Prakash and
Kennedy 1996). Plastic carrier materials utilize a greater surface area compared to the
same mass of a sand or glass material due to their low density and promote fast
accumulation of biomass to decrease start-up time for the EBR (Tavares et al., 1995).
Some plastic carrier materials in use are less dense than water and are used in downflow
EBRs as particles become fluidized as media is forced down into the reactor from a
floating position (e.g. Meraz et al., 1997; Boehler et al., 1994). Particles which are too
small tend to be problematic in EBRs. Diatomaceous earth, for example, indicated good
biomass adhesion properties but proved extremely susceptible to entrapment in recycle
lines causing fouling and clogging (Converti et al., 1993). Particle size also influences
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the consistency of expanded particles. Larger glass beads of 0.044 cm and 0.121 cm
diameter were found to promote disk-shaped voids which traveled upward through the
EBR throughout operation, whereas, smaller 0.0114 cm diameter glass beads remained
expanded in a homogeneous manner (Webster and Perona, 1990). Numerous EBR
designs have utilized and recommended the use of granular activated carbon (GAC) as a
carrier material. The GAC provides a suitable site for biological growth while
synchronously acting chemically as an exchange site to remove chlorinated and other
specific organics from many industrial waste streams (e.g. Safferman and Bishop, 1996;
Tseng and Lin, 1994). It should also be noted, however, that chemical exchange
properties will likely change with time, indicating the need for regeneration or
replacement to avoid a decrease in removal efficiency.
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TRENDS AND FUTURE RESEARCH
The ability of EBR reactors to remove COD constituents in high concentration
from wastewaters is well documented. However, further research into temperature and
pH dependence, as well as investigation into modifications which broaden the use of
EBR biological treatment would have significant value. Numerous high-COD
wastewaters, other than those examples currently found in the literature, are likely
candidates for EBR treatment.
One prevalent research area to be addressed is combined anaerobic/aerobic
systems. Combined nitrification denitrification, and systems which reduce sulfate to
sulfur or oxidize sulfide to sulfur would address high-sulfate wastewaters. Similarly,
chlorinated organics which have been shown to degrade under sequential anaerobic-
aerobic steps (e.g. Zitomer and Speece, 1993; Gerritse et al., 1995) should be
investigated for amenability to EBR treatment. Dual-phase and dual-stage anaerobic-
aerobic EBR treatment should be further investigated to address these issues (Speece, et
al, 1997).
Research in biofilm optimization with respect to activity has suggested that
pseudo-steady state conditions created by medium-cleaning or modifying process
parameters will achieve a more active biofilm. These principles should be specifically
applied to strictly anaerobic treatment to address the benefits of increased gas upflow
velocity by use of inert gases. Inert gas addition to the EBR as a method of maximizing
biofilm activity, on both a continuous or periodic basis should be investigated to assess
possible cost savings and benefits in suspended solids effluent quality.
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CHAPTER 2: STUDY OF LIMITED-AERATION, ALKALINITY, AND
EXPANDED BED REACTOR PERFORMANCE
INTRODUCTION
The use of methanogenic expanded bed reactors (EBRs) reactors for wastewater
treatment has been increasing due to the relatively high loading rates possible (e.g. >40 g
COD/m3-day) and the associated small reactor size required (Totzke, 1997; Heijnen et
al., 1989). For this reason, it is interesting to consider process modifications that may
extend the applicability of methanogenic EBR technology, such as limited air addition.
When the oxygen transfer rate is relatively low, concurrent methane production and
oxygen utilization is easily attainable (Zitomer, 1998). In addition, COD removal rates in
suspended growth systems are not adversely affected by limited aeration under some
conditions, and may even increase (Zitomer and Shrout, 1997). Some aerated
methanogenic cultures have been shown to achieve lower effluent COD concentrations
and more rapid pH recovery after carbohydrate shock-loading as compared to strictly
anaerobic cultures (Zitomer and Shrout, 1997). Biofilms developed in EBR reactors with
high gas flows are typically dense and thin (Trinet et al., 1991). The turbulence imparted
by high gas flow shears less dense microbial mass, resulting in selection of a dense
culture. The dense biofilm generally resists washout and exhibits a higher specific
substrate removal rate as compared to a less dense film (Rittmann et al., 1992).
Other benefits of partial aeration of methanogenic cultures include a reduction in
alkalinity supplementation. The cost of alkalinity addition to nutralize carbonic acid and
transient increases in volatile fatty acid intermediates in methanogenic processes
typically ranges from $68 to $670 per ton of alkalinity chemical added (Speece, 1996).
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Air stripping of CO2from a reactor may be an economical approach under some
conditions.
The objectives of this research were to (1) determine the feasibility of low-
aeration methanogenic EBR treatment, (2) determine the reduced alkalinity requirements
due to CO2stripping in aerated EBR reactors under different aeration rates, and (3)
determine the mixing characteristics and oxygen transfer of a bench scale EBR system.
MATERIALS AND METHODS
Four expanded bed reactors (EBRs) were operated at 35C (2C) for over 170
days. EBR configuration is detailed in Figure 2-1. Seed organisms were obtained from
the Brookfield, Wisconsin anaerobic digesters and activated sludge mixed liquor.
Methanol (Fisher Chemicals, Fair Lawn, NJ) was employed as a primary substrate during
Figure 2-1: EBR Configuration
Effluent
SamplePort
Air In
Activevolume
Gas
Glass beads InfluentPum
RecyclePum
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startup to encourage rapid growth of methanogens. After 15 days, an ethanol/propionate
mixture (1:1 as COD) was fed (Fisher Chemicals, Fair Lawn, NJ and Aldrich Chemical,
Milwaukee, WI), and methanol addition was discontinued. The feed medium also
contained nitrogen, phosphorus, iron, and other trace nutrients as suggested for
methanogenic cultures as outlined in Table 2-1 (Speece, 1996).
Constituent Concentration in Feed (mg/L)
NH4ClKCl
MgSO47H2OCaCl22H2O(NH4)2HPO4FeCl24H2OSodium CitrateCoCl26H2OKIMnCl24H2ONH4VO3CuCl22H2O
Zn(C2H3O2)2AlCl36H2ONaMoO42H2OH3BO3NiCl22H2ONa2SeO3Cysteine
400400
400508042610100.50.50.5
0.50.50.50.50.50.510
Table 2-1:Macro and Micro-nutrients in Influent Feed (Adapted from Speece 1996)
The influent was stored at 4C to and was pumped at 3 mL/min to all EBRs. The
recycle ratio was 300:1. All systems were operated at organic loading rates from 8 to 50
grams COD per liter of active volume per day (g COD/LAd). Each reactor contained
275 g of a solid medium (Celite Bio-Catalyst Carrier R-632) to support biofilm growth;
this comprised an active volume of 0.75L when expanded. Effluent gas was measured by
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a wet-tip gas meter (Rebel Instrument Company, Nashville, TN). One reactor was
operated as a non-aerated anaerobic control, whereas the remaining three reactors
received 60 milliliters of air per minute (mL air/min) 120 mL air/min, and 225 mL
air/min respectively, regulated by rotometers (Gilmont Instruments, Barrington, IL).
After 130 days, alkalinity in each EBR was added independently to assess the
requirements of each reactor to maintain a pH near 7.
The following parameters were measured: influent and effluent COD; effluent
volatile fatty acids, bicarbonate alkalinity, pH, total and volatile suspended solids; and
total gas volume and methane content.
One additional EBR as previously described was operated without
microbiological innoculum to investigate mixing charactersitics and oxygen transfer. An
influent flow of Milwaukee tap water was maintained at 3 mL/min and the recycle ratio
was 300:1. The reactor contained 275 g of a solid medium (Celite Bio-Catalyst Carrier
R-632-Lompoc, California) which comprised an active volume of 0.75 L when expanded.
The reactor was operated in a temperature controlled room maintained at 35C (2C).
A tracer dye study was performed on an EBR receiving no air by injecting 7 milliliters of
75 mg/L (525 g) of flourescein. Effluent samples were collected over time and
measured for luminescence. By measuring the recovery of a tracer dye over time, the
mixing characteristics of the reactor were described with relation to ideal plug-flow and
completely-mixed flow models. The Peclet number (ranging from 0 [completely-mixed]
to [ideal plug-flow]) is an indication of actual mixing conditions for the reactor. The
Peclet number was determined by utilizing the difference of first and second moments of
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tracer dye mass recovered with respect to the hydraulic residence time of the reactor
(Levenspiel, 1972).
The transfer of oxygen at several air flow rates to Milwaukee tap water was
measured over time using a portable dissolved-oxygen probe and meter (YSI, Inc.-
Yellow Springs, Ohio). Effluent gas was measured by a wet-tip gas meter (Rebel
Instruments-Nashville, Tennessee). The EBR was modified to examine differences in
aeration due to poor transfer efficiency and high turbulence created by the original EBR
configuration (Figure 2-1). EBR aeration was modified by suspending a diffuser above
the active volume of the reactor. This EBR modification is detailed in Figure 2-2. The
transfer of oxygen at several air flow rates was measured for the modified EBR.
Figure 2-2: Modified EBR Configuration
RecyclePump
InfluentPump
Glass beads
Gas
Activevolume
Air In
SamplePort
Effluent
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Analyses. Measurements of pH were performed using a combination probe and
pH meter. Volatile suspended solids, chemical oxygen demand (COD), volatile fatty
acids, and bicarbonate alkalinity were determined according to standard methods
(APHA-AWWA, 1995).Gas samples for methane content analysis were collected using a
microsyringe (Hamilton Instruments, Reno, NV) and injected into a gas chromatograph
(Autosystem, Perkin-Elmer Corp., Norwalk, CT) with a flame ionization detector. An 8-
ft. by 0.125-in. o.d. stainless steel column packed with 1% SP-1000 on 60/80 Carbopack
B (Supelco, Inc., Bellefonte, PA) accomplished separation.
Luminescence of EBR effluent in the abiotic reactor for the tracer dye study was
measured by a flourimeter (Turner Designs, Sunnyvale, CA) calibrated to a known
concentration of flourescein.
RESULTS AND DISCUSSION
Table 2-2 presents influent COD, methane production, and and volatile fatty acid
effluent results for the four reactors during the initial days of operation when ethanol and
propionate served as primary substrates at an OLR of 8 g/LA-d utilizing aeration
delivered from the bottom of the EBR (Figure 2-1). All reactors produced methane and
aqueous
soluble COD removal averaged 71% for these reactors. Under the conditions studied,
limited aeration did not have an adverse effect upon COD removal as effluent COD
concentrations and effluent VFA concentrations were not statistically differentiable using
the t-test for a 90% confidence interval. The COD electron equivalent mass balance for
EBRs operated under the loading rate of 8 g/LA-d is included as Table 2-3. All oxygen
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CODremoved(%)
Methanegenerated(L/d)
effluentVFA(mg COD/L)
pH of reactor
Reactor 1(anaerobic)
72 (31) 1.3 (50) 276 (49) 6.8
Reactor 2(60mL air/L-min)
74 (23) 0.3 (114) 289 (48) 7.0
Reactor 3(120mL air/L-min)
69 (22) 0.6 (102) 300 (49) 7.1
Reactor 4(225mL air/L-min)
69 (25) 0.4 (127) 322 (51) 7.1
Table 2-2: Reactor Characteristics for Loading of 8g COD/LAd(Coefficient of Variation)=Standard Deviation/mean100
transferred to the EBRs was assumed to be utilized as the resazurin indicator was clear
indicating no dissolved oxygen in these systems. Synthesis estimates were calculated
from observed effluent volatile suspended solids (VSS) concentrations assuming all
effluent VSS was due to growth.
Decimal % COD Donor Used
e-AcceptorReactor 1(anaerobic)
Reactor 2(60mL air/L-min)
Reactor 2(120mL air/L-min)
Reactor 3(225mL air/L-min)
Unused(Effluent)
0.28 0.26 0.31 0.31
CO2 0.44 0.11 0.20 0.14
O2 -- 0.11 0.18 0.24
Synthesis 0.01 0.02 0.02 0.01
Balance 0.73 0.50 0.71 0.70
Table 2-3: Electron Equivalent Balance During OLR of 8 g COD/LA-d
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During periods of higher loading, reactors receiving air required significantly less
alkalinity addition to maintain a reactor pH near 7 (Figure 2-3). Alkalinity requirements
of each EBR decreased with time, however the EBR receiving the most air (225 mL
air/LR-min) continously required the lowest alkalinity addition to maintain a desired pH
level. This is presumably due to the increased stripping of CO2as a result of the higher
air flows. In additon, aerobic oxidation of volatile fatty acids may have been occuring.
0
1000
2000
3000
4000
5000
6000
120 130 140 150 160 170 180 190 200 210 220
Time (days)
AlkalinityAd
ded(mg/LNaHCO3)
Anaerobic Reactor
60mL/min Reactor
120mL/min Reactor
225mL/min Reactor
Figure 2-3: Alkalinity Added over time to maintain pH~7 at OLR=28g/LAd
Reactors did not achieve high COD removal during the high load period studied
(Table 2-4). However, significant methane production was observed. It is possible that
acetogens show a greater adaptability to oxygen than methanogens which becomes
pronounced under high load conditions and was not directly observed under the start-up
conditions when COD loading was 8 g/LAd. This may explain the high concentration of
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VFAs in aerated reactor effluents. An electron equivalent mass balance of COD during
the 28 g/LA-d is included as Table 2-5. All oxygen transferred to the EBRs was assumed
to be utilized as the resazurin indicator was clear indicating no dissolved oxygen in these
systems. Synthesis estimates were calculated from observed effluent volatile suspended
solids (VSS) concentrations assuming all effluent VSS was due to growth.
CODremoved(%)
Methanegenerated(L/d)
effluentVFA(mg COD/L)
pH of reactor
Reactor 1(anaerobic)
64 (36) 5.6 (19) 1163 (24) 7.1
Reactor 2(60mL air/L-min)
40 (43) 4.1 (10) 1355 (41) 7.3
Reactor 3(120mL air/L-min)
32 (52) 3.7 (16) 2201 (8) 6.6
Reactor 4(225mL air/L-min)
30 (58) 1.7 (33) 1639 (41) 7.1
Table 2-4: Reactor Characteristics for Loading of 28g COD/LAd(Coefficient of Variation)=Standard Deviation/mean100
Decimal % COD Donor Used
e-Acceptor Reactor 1
(anaerobic)Reactor 2(60mL air/L-min)
Reactor 2(120mL air/L-min)
Reactor 3(225mL air/L-min)
Unused(Effluent)
0.26 0.60 0.68 0.70
CO2 0.52 0.41 0.32 0.15
O2 -- 0.03 0.05 0.07
Synthesis 0.02 0.01 0.03 0.02
Balance 0.80 1.05 1.08 0.94
Table 2-5: Electron Equivalent Balance During OLR of 28 g COD/LA-d
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Results of the tracer dye study performed using the abiotic reactor indicated
largely-dispersed plug-flow (Levenspiel, 1972). The Peclet number is estimated to be
9.05 (unitless) for the 1.75 L EBR reactor having a hydraulic retention time of 9.7 hours.
Recovery of flourescein tracer dye was 43%. This poor recovery of tracer dye is
attributed to the porous nature of the carrier media in the reactor which likely adsorbed
the balance of flourescein. Figure 2-4 displays the tracer dye recovery data over a period
of three hydraulic retention times.
Figure 2-4: Fluorescein Tracer Dye Concentration over 3 HRTs
The oxygen transfer efficiency of the aerated abiotic EBR utilizing bubbled air
delivered at the base of the EBR ranged from 1.2% to 2.1% at 33C for the ranges tested.
The KLa(33)for aeration rates of 25 mL air/LR-min, 100 mL air/LR-min, and 200 mL
air/LR-min are 0.7 hr
-1
, 4.3 hr
-1
, and 6.0 hr
-1
, respectively. Figure 2-5 shows theKLa(33)
versus air delivery rate for the EBR configuration detailed in Figure 2-1. Oxygen
transfer efficiency of the aerated EBRs utilizing the modified aeration design to deliver
air above the active volume indicated decreasing transfer efficiency with increasing
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delivery. Efficiencies ranged from 4.3% to 1% for the air deliveries examined. The
KLa(33) foror
aeration rates of 50 mL air/LR-min, 250 mL air/LR-min, and 500 mL air/LR-min are 2.4
hr-1, 5.5 hr-1, and 6.8 hr-1, respectively. Perceived disadvantages in transfer efficiency
from this modified design however were compensated by the increased mass of oxygen
transferred which could reasonably be achieved by the EBR modification detailed in
Figure 2-2. Due to high turbulence created by large bubbles created in the EBR utilizing
aeration at the base of the reactor, fluidization of particles was turbulent and non-
homogeneous. Aeration was limited to prevent the carrier material from escaping the
reactor and entering the recycle line causing clogging. Fluidization during operation of
the modified EBR utilizing aeration above the active volume of the reactor promoted a
much less turbulent fluidization of particles at any aeration rate. The use of higher
aeration rates (even at a lower transfer efficiency) can achieve an overall higher oxygen
aeration rates of 50 mL air/LR-min, 250 mL air/LR-min, and 500 mL air/LR-min are 2.4
hr-1, 5.5 hr-1, and 6.8 hr-1, respectively. Perceived disadvantages in transfer efficiency
from this modified design however were compensated by the increased mass of oxygen
transferred which could reasonably be achieved by the EBR modification detailed in
Figure 2-2. Due to high turbulence created by large bubbles created in the EBR utilizing
aeration at the base of the reactor, fluidization of particles was turbulent and non-
homogeneous. Aeration was limited to prevent the carrier material from escaping the
reactor and entering the recycle line causing clogging. Fluidization during operation of
the modified EBR utilizing aeration above the active volume of the reactor promoted a
much less turbulent fluidization of particles at any aeration rate. The use of higher
aeration rates (even at a lower transfer efficiency) can achieve an overall higher oxygen
Figure 2-5: KLa(33C)of Initial EBR Configuration
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transfer to the EBR system. Figure 2-6 shows the KLa(33)curve calculated from the
oxygen transfer analysis for the EBR configuration detailed in Figure 2-2.
R2= 0.782
0
1
2
3
4
5
6
7
8
0 100 200 300 400 500 600 700
Air De liver y (m L/m in)
K
a(1/hr)
Figure 2-6 KLa(33C)of Modified EBR Configuration
During periods of higher loading, reactors receiving air required significantly less
alkalinity addition to maintain a reactor pH of nearly 7. Interestingly, aerated EBRs
requiring lower alkalinity addition exhibited higher effluent VFA concentration. Further
research is required to investigate the relationship of methanogens, acetogens, and
alkalinity in limited-aeration EBR systems. Air stripping of CO2from a methanogenic
EBR reactor may be an economical approach to reduce alkalinity additon costs under
some conditions. However, cost estimates of alkalinity addition versus aeration indicated
that aeration was more costly for the system studied for this research. Table 2-6 outlines
these cost estimates.
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EBR Alkalinity cost($/yr)*
Aeration Cost($/yr)**
Total Cost($/yr)
Reactor 1(anaerobic)
2.92
0
2.92
Reactor 2(60mL air/L-min)
1.83
8.76
10.59
Reactor 3(120mL air/L-min)
1.83
14.24
16.07
Reactor 4(225mL air/L-min)
1.01
18.98
19.99
Table 2-6 Estimated Cost of Alkalinity versus Aeration*$400/ton HCO3(Speece, 1996)**1.8kg O2 transferred/kWh (Metcalf and Eddy, 1991), $0.05/kWh
CONCLUSIONS
EBRs maintained under low aeration rates produced significant ammounts of
methane and achieved COD removals comparable to those of an anaerobic control under
moderate load conditions (8 g COD/LAd).
The EBR system has a low oxygen transfer efficiency. Analysis of two aeration
configurations for EBRs indicated transfer efficiencies ranging from 1% - 4.3% of
oxygen delivered at 33C. Both aeration at the bottom of the EBR and above the active
volume of the EBR have physical limitations. Carrier media propagate larger bubble
sizes decreasing transfer efficiency when oxygen is supplied at the bottom of the EBR;
howevever, oxygen delivery provided from aeration above the active volume becomes
limited by the number of bubbles which pass through the recycle. To achieve a greater
transfer efficiency, it is speculated that a lighter carrier material should be used to
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promote greater expansion (without raising upflow velocity) and allow bubbles formed at
the bottom of the active volume to remain small, enhancing transfer efficiency.
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CHAPTER 3: TROPHIC STUDY OF METHANOGENIC OXYGEN-LIMITED
TREATMENT
INTRODUCTION
Oxygen, which is classically considered to be the nemesis of methanogenic
processes, may potentially enhance methanogenesis under some conditions. Although
even low levels of dissolved oxygen are considered to be extremely toxic to
methanogens, they have been found to survive short periods in the presence of dissolved
oxygen and to coexist with aerobic or microaerophilic organisms. For example, methane
production by the methanogenMethanothrix soehngeniidid not decline after oxygen was
administered for 48 hours and then removed (Huser et al., 1982). Other methanogens,
includingMethanosarcina barkeri(e.g. Kiener and Leisinger 1983),Methanobacterium
bryantii(e.g. Kirby et al., 1981),Methanothrix soehngenii(e.g. Huser et al., 1982),
Methanobacterium thermoautotrophicum, andMethanobrevibacter arboriphilus(e.g.
Field et al., 1995) all exhibit limited tolerance to low oxygen levels.
Mixed cultures of methanogenic, acetogenic, and obligate aerobic
microorganisms have been described, and methane production has been sustained over a
wide range of oxygen supply rates (Gerritse et al., 1990). Obligate aerobes in these
systems have been found to oxidize acetate and propionate produced by acetogenic
anaerobes during lactose fermentation (Gerritse et al., 1992), and to transform acetamide
in synergistic association with methanogens (Guyot et al., 1995). Stoichiometric
equations for propionate- and ethanol-fed methanogenic, oxygen-limited batch cultures
have been developed using electron equivalent balances (Zitomer, 1998). The oxygen
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utilized by various cultures ranged from 10 to 30% of the added COD, whereas the
remainder of removed COD was converted to methane.
Survival of methanogens in oxygen-limited mixed cultures is often attributed to
formation of reduced microniches in otherwise oxic environments (Field et al., 1995). For
instance, facultative microorganisms on the surface of granular sludge particles from an
upflow anaerobic sludge blanket reactor consumed oxygen before it diffused into the
inner-particle region (Kato et al., 1993). Similarly, porous support media such as calcium
alginate beads have been employed to co-culture strict aerobes (outer-bead region) and
anaerobes (inner-bead region) (Kurosawa and Tanaka, 1990). This oxygen shielding
effect may explain the viability of a full-scale methanogenic bioreactor (Hormel Packing
Plant, Austin, MN) that has been operating since 1960 which achieves effluent BOD
concentrations consistently below 20 mg/l even though biomass is aerated prior to
thickening and recycle (Speece, 1996). Shielding may also explain the presence of
methanogens in aerobic activated sludge (Lens et al., 1995) and the cultivation of
anaerobic granular sludge in a reactor seeded with aerobic activated sludge (Wu et al.,
1987). Interestingly, others have found that the addition of 4 mg O2/LR-day to essentially
anaerobic cultures doubled methane production when algae was the primary substrate
(Pirt and Lee, 1983).
It is significant that both reductive and oxidative biotransformations may occur
concomitantly under oxygen-limited conditions. The ability of anaerobic systems to
reduce highly chlorinated compounds that are relatively recalcitrant to aerobic
biotransformation is documented (Zitomer and Speece, 1993). On the other hand, the
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ability of aerobic processes to oxidize aromatic organics resistant to anaerobic
transformation is also apparent. At the same time, aerobic cometabolic oxidation of
chlorinated organics may occur in the presence of methane and some other primary
substrates (Oldenhuis et al., 1989).
Similarly, the mineralization of DDT, polycyclic aromatic hydrocarbons (Field et
al., 1995), highly chlorinated solvents, and other organics often proceeds through a
sequence of anaerobic, aerobic, or anoxic biotransformations (Zitomer and Speece,
1993). For example, the mineralization rate of a chlorinated insecticide, Methoxychlor,
was significantly increased when a single mixed culture was incubated under sequential
anaerobic/aerobic conditions (Fogel et al., 1982). Mineralization was not apparent under
strictly anaerobic nor aerobic conditions alone. When considering in-situ technologies for
soil/groundwater remediation, it may be infeasible to divide the system into two locales
containing different cultures. Consider that PCB congeners (Aroclor 1242) in sediment
are biotransformed when biologically active soil samples are incubated under anaerobic
conditions, and then under aerobic conditions (Anid and Vogel, 1992). The same initial
culture performs both reductive and oxidative steps. It has also been reported that
transition from anaerobic to aerobic conditions is required for particular cultures to
reductively dechlorinate tetrachloroethylene and trichloroethylene (Kastner, 1991).
Other possible benefits of methanogenic, oxygen-limited systems relate to gross
pollutant removal in wastewater treatment. Combined systems are potentially more
energy-efficient than conventional aerobic systems, requiring less energy for blower
operation and producing significantly less biosolids to be handled, transported, and
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biological reactions (Zitomer and Speece, 1993), and degradation of specific organics
through cometabolic, methanotrophic reactions (Gerritse et al. 1995).
MATERIALS AND METHODS:
Serum bottle batch reactor studies. Seed organisms for the mixed cultures were
obtained from anaerobic digesters of the South Shore Wastewater Treatment Plant,
Milwaukee, Wisconsin. Fifty millileters of anaerobic sludge was added to 160-mL serum
bottles. The bottles were then flushed with methane gas and sealed with rubber septa.
All cultures contained 50 mL of active volume, and were maintained in a batch
mode having a two-day wasting/feeding cycle. A solids retention time (SRT) of 10 days
was maintained by wasting 10 mL of culture every two days. Subsequently, 10 mL of
medium containing approximately 50 mg of COD in the form of sucrose, ammonia-
nitrogen, phosphorus, and other trace nutrients was added. An oxidation-reduction
indicator dye, resazurin, was also added. Nitrate-nitrogen, nitrite-nitrogen, and sulfate
concentrations were very low such that COD oxidation associated with reduction of these
electron acceptors was negligable.
A factorial approach was used in which two oxygenation conditions and three
oxygen doses were maintained. Cultures maintained under the first oxygenation
condition received oxygen at the time of medium addition (aerobic/anaerobic cultures),
whereas cultures maintained under the second condition received oxygen 1 day into the
2-day feeding cycle (anaerobic/aerobic cultures). Pure oxygen was injected into serum
bottles using syringes fitted with needles. Oxygen volumes injected to various cultures
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included 4, 12, and 48 mL every two days. These addition rates correspond to 10, 30, and
125% of the added COD respectively. A strictly anaerobic control was also maintained.
Excess headspace gas was wasted daily using a 50-mL glass syringe with a wetted barrel.
Microorganisms were acclimated for at least three SRTs (30 days) before data were
collected. All culture conditions were maintained at 35C on a shaker table in the dark.
Methanogenic activity assays were performed by withholding oxygen the day of
the assay (to maintain anaerobic conditions) and adding 400 mg/L of acetate (as calcium
acetate) to each mixed culture. Culture wasting and medium addition was not performed
for 7 days, and gas production was measured daily using a 50-mL glass syringe with a
wetted barrel. Under the studied conditions, the concentration of extraneous electron
acceptors, such as nitrate and sulfate, were negligible. Therefore, under anaerobic
conditions biogas was generated theoretically containing 30% carbon dioxide and 70%
methane.
Specific methanogenic activity ( mL biogas per g-VSS per day) was estimated as
the maximum rate of gas production determined from the initial slope of a plot of
cumulative gas production versus time, and is similar to the approach employed by other
researchers (Araki and Harada, 1994). The unitless relative activity (R) of a culture was
calculated as the estimated initial pseudo zero-order rate of gas produced by the culture
of interest divided by the initial rate of gas production exhibited by the strictly anaerobic
culture. The value of R indicates the acetoclastic methanogen activity in a given culture
relative to the strictly anaerobic control. Aerated cultures demonstrating R values less
than one have a lower acetoclastic methanogen activity, whereas R values greater than
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one indicate a higher acetoclastic methanogen activity, relative to a strictly anaerobic
culture.
Soluble chemical oxygen demand (COD) was measured by first filtering the
sample through a 45 m glass fiber filter, and then measuring COD according to standard
methods (APHA-AWWA, 1995). Mixed liquor volatile suspended solids (MLVSS) were
determined according to standard methods (APHA-AWWA, 1995). The pH of reactor
content aliquots was measured using a probe and pH meter.
Expanded Bed Reactor studies. Four expanded bed reactors (EBRs) were operated
at 35C for 120 days. Seed organisms were obtained from the Brookfield, Wisconsin
anaerobic digesters. Methanol was employed as a primary substrate during startup to
encourage rapid growth of methanogens. After 15 days, an ethanol/propionate mixture
(1:1 as COD) was fed, and methanol addition was discontinued. The feed medium also
contained nitrogen, phosphorus, iron, and other trace nutrients as suggested for
methanogenic cultures (Speece, 1996).
The influent flow rate to all FBRs was 3 mL/min and the recycle ratio was 300:1.
Each reactor contained 275 g of a solid medium (Celite Bio-Catalyst Carrier R-632) to
support biofilm growth; this comprised an active volume of 0.75L when expanded.
Effluent gas was measured by a wet-tip gas meter. One reactor was operated as a non-
aerated anaerobic control, whereas the remaining two reactors received 60, 120, and 225
milliliters of air per liter EBR per minute (mL air/LR-min), respectively. All systems were
operated at an organic loading rates of 8 grams COD per liter active volume per day (g
COD/LAday).
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Aliquots containing fifty milliliters including biofilm coated carrier material from
each EBR were placed in 160-mL serum bottles for trophic assays. Bottles were flushed
with 30% CO2and 70% N2gas, sealed with rubber septa, then fed either acetate or
hydrogen under anaerobic conditions. An additional 50 mL of N2gas (at STP conditions)
was added to serum bottles receiving hydrogen to ensure a measurable excess gas volume
throughout the experiment. Gas production or utilization was subsequently measured
using a 100-mL wetted barrel glass syringe, and biogas methane content was determined
by gas chromatography. The acetate dose (5,000 mg/L as acetate in the form of sodium
acetate) and hydrogen dose (4.38 mmol/L as hydrogen gas in headspace) employed are at
least 20 times the reported half-saturation constants for methanogens (Yamaguchi, 1997).
Therefore, methanogenic activity was not substrate-limited.
Specific activity values (SA) of aceticlastic and hydrogenotrophic methanogens
from each MFB reactor were caluculated as the initial rate of methane production per
gram of VSS. Calculated values were compared to reported specific activity of pure
cultures (SAP) (Yamaguchi, 1997; Araki and Harada, 1994). Relative activity indices
(RAIs) were calculated as the quotent of measured SA and reported pure-culture specific
activity (RAI = SA/SAP) as described elsewhere (Araki and Harada, 1994). Serum
bottles were maintained at 35C on a shaker table in the dark. All tests were run in
triplicate or greater.
Analyses Gas samples for methane content analysis were collected using a
microsyringe (Hamilton Instruments, Reno, NV) and injected into a gas chromatograph
(Autosystem, Perkin-Elmer Corp., Norwalk, CT) with a flame ionization detector. An 8-
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ft. by 0.125-in. o.d. stainless steel column packed with 1% SP-1000 on 60/80 Carbopack
B (Supelco, Inc., Bellefonte, PA) accomplished separation. A gas chromatograph (GC)
with a thermal conductivity detector (Series 150, GOW-MAC Instrument Company,
Lehigh Valley, PA) was used to quantify oxygen in batch reactor headspace gas. A 6-foot
concentric stainless steel column having a 0.125-in. o.d. inner column packed with a
porous polymer mixture, and a 0.125-in. o.d. outer column packed with activated
molecular sieve ( CRT I column, Alltech Assoc., Inc., Deerfield, Ill.) accomplished
separation. The helium carrier gas flow rate was 40 mL/min, and the oven temperature
was 20C.
Measurements of pH were performed using a combination probe and pH meter.
Chemical oxygen demand (COD) and total and volatile suspended solids were
determined according to standard methods (APHA-AWWA, 1995).
RESULTS AND DISCUSSION
Serum bottle studies.
Oxygen-limited and anaerobic serum bottle cultures utilizing sucrose substrate
were maintained for over 5 months under sustainable conditions. The dissolved oxygen
concentration in systems receiving oxygen doses of 0, 10, and 30% of the added COD
was typically zero as indicated by a lack of pink color exhibited by the resazurin dye in
the basal medium. During brief periods (approximately 30 minutes) immediately after
oxygen addition, however, the probable presence of dissolved oxygen was indicated by
the pink color of resazurin. In addition, the systems receiving an oxygen dose of 125% of
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the COD were typically pink at all times, indicating more oxidized conditions, yet
approximately 0.1% methane was detected in the headspace of these systems. More
importantly, the results of methanogenic assay tests indicate that a significant population
of acetotrophic methanogens was present in all cultures (Figures 3-1 and 3-2), and that
the specific methanogenic activity (R) determined as described in the Methods section,
Figure 3-1: Anaerobic-Aerobic Culture Methanogenic Activity
was highest in systems which received 125% oxygen (Table 3-1). Therefore, oxygen
addition did not prevent the growth of methanogens, but increased their initial activity
under the specific conditions studied. The relatively low percent of methane in the
headspace in comparison to the relatively high methanogenic activity may be attributable
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to aerobic oxidation of methane (methanotrophic reactions). However, more work is
required to confirm this.
Figure 3-2 Aerobic-Anaerobic Culture Methanogenic Activity
Maximum biomass yields of oxygen-limited cultures ranged from 0.13 to 0.07 g
VSS/g sucrose as COD (Table 1), and are more typical of strictly methanogenic, rather
than aerobic processes. Lower-than-expected yields have also been calculated for
propionate- and ethanol-fed methanogenic cultures under oxygen-limited conditions
(Zitomer, 1998).
Higher COD removal efficiencies and lower residual COD concentrations were
exhibited by batch systems under oxygen-limited conditions (Table 3-1). Sucrose may
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have been oxidized directly by aerobic or microaerophilic processes. It is also possible
that aerobic oxidation of intermediates such as propionic acid, acetic acid, and hydrogen
occurred. The possibility of lower concentrations of these intermediates in the more oxic
environments may also explain the relatively high activity of methanogens in cultures
that received the most oxygen. Elevated concentrations of these intermediates may
Culture Condition OxygenAdded(%COD)
pH MLVSS
(mg/L)
EffluentCOD(mg/L)
CODRemoval(%)
Yobs
)CODg
VSSg(
R*
1 anaerobic - 6.8 330 1970 61 0.11 1.0
2 aerobic/anaerobic
10 6.7 310 1300 74 0.08 0.80
3 aerobic/anaerobic
30 6.9 270 1290 74 0.07 0.80
4 aerobic/anaerobic
125 7.1 520 540 89 0.12 1.2
5 anaerobic/aerobic
10 6.8 290 1330 74 0.08 1.0
6 anaerobic/
aerobic
30 6.8 430 1300 74 0.12 0.70
7 anaerobic/aerobic
125 7.1 535 590 90 0.13 1.2
Table 3-1: Oxygen-Limited, Methanogenic Biotransformation of Sucrose*R= Relative Rate of Methanogenic Activity
potentially inhibit methane production (Speece, 1996). Other reactor configurations,
such as UASB and separate, staged acetogenic and methanogenic processes may be much
more efficient. Methanogenesis with limited oxygen under these conditions should be
investigated.
Although survival of methanogens under low-aeration conditions is usually
attributed to formation of anaerobic microniches, the cultures described herein existed as
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dispersed, suspended growth. Large flocs or pellets were not apparent, and a
predominance of free-swimming bacteria was observed microscopically. Therefore,
exceptional spatial variation in redox conditions is unlikely. Others have reported
ephemeral methane production by pure cultures of methanogens in the presence of
dissolved oxygen (Roberton and Wolfe, 1970). Although a decline in production was
evident, it was a "relatively slow process" under some conditions. It has been reported
that some methanogens exhibit an oxygen-consuming activity, and, therefore are
potentially more resistant to low oxygen levels (Leadbetter and Breznak, 1996).
Similarly, some researchers have considered methanogenesis under aerobic (or
microaerophilic) conditions when investigating the aerobic marine environment, whereas
more traditional concepts, such as the presence of anaerobic microenvironments, were
described as unlikely based upon conservative estimates (Rudd and Taylor, 1980). The
possibility of sustained methanogenesis under very low dissolved oxygen conditions is
even more intriguing when considering that superoxide dismutase (an oxygen
"detoxifying" enzyme) has been detected in methanogens (Kirby et al., 1981), and other
oxygen-adapting phenomenon have been reported for methanogens (Zinder, 1993).
EBR Studies
The anaerobic and three aerated EBRs previously fed ethanol and propionate
contained a significant population of hydrogenotrophic methanogens (Table 3-2). Mean
hydrogenotrophic methanogen specific activity (SAH) was highest for biomass from
Reactor 4 which was exposed to the highest aeration rate, and the mean SAHvalues for
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Reactor 4 and Reactor 1 (anaerobic control) biomass were significantly different when
measured by the t-test (n = 7, P = 0.90). However, the mean SAHvalues for Reactor 2
(lowest aeration) and Reactor 1 (anaerobic control) biomass, and Reactor 3 (intermediate
aeration) and Reactor 1 (anaerobic control) were not significantly different.
Approximately 0.04 to 0.14% of the VSS from all reactors was composed of
hydrogenotrophs as indicated by the RAIHvalues (Table 3-2).
Hydrogenotrophic MethanogenicActivity
Acetoclastic MethanogenicActivity
Specific Activity
)biomassxhg
producedMethanemmole(
Relative
ActivityIndex*(%)
Specific Activity
)biomassxhg
producedMethanemmole(
Relative
ActivityIndex**(%)
Reactor 1
(anaerobic)0.058 (68) 0.09 2.44 (10) 41
Reactor 2
(60mL air/L-min)0.026 (237) 0.04 1.72 (36) 28
Reactor 3
(120mL air/L-min)0.072 (25) 0.11 0.13 (10) 2.1
Reactor 4
(225mL air/L-min)0.088 (30) 0.14 1.09 (23) 18
Table 3-2: Methanogenic Activity*RA (Hydrogenotrophic Methanogens) = 62.5 mmole CH4/g biomassh (Yamaguchi et al., 1997)
**RA (Acetoclastic Methanogens) = 5.95 mmole CH4/g biomassh (Araki and Harada, 1994)
(Coefficient of Variation)=Standard Deviation/mean100
The specific activity of aceticlastic methanogens (SAAc) was highest in cultures
from the anaerobic control (Reactor 1) (Table 3-2); however, all aerated reactor cultures
also exhibited significant aceticlastic methanogen activity. The SAAcof Reactor 3
(intermediate aeration) showed the lowest aceticlastic methanogen activity. However,
this value is due to the inability to adequately buffer the pH near 7. The mean SAAc
values for aerated cultures were significantly different from the anaerobic control
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(Reactor 1) when measured by the t-test (n = 7, P = 0.90), and the two aerated cultures
demonstrated decreasing SAAcvalues with increasing aeration rates.
CONCLUSIONS
Biological systems that employ more than one electron acceptor have been used
for many years in full-scale applications. For example, combined nitrification (oxygen as
electron acceptor) and denitrification (nitrate as electron acceptor) are used to remove
nitrogen from wastewater. Similarly, sequential anaerobic (methanogenic) and aerobic
processes have been used to treat industrial wastewaters with high BOD concentrations.
However, in these systems the biological reactions are spacially separated, or temporarily
separated in phases in sequencing batch reactors.
In contrast, a single biological culture can synchronously employ oxygen as an
electron acceptor and produce methane. Under oxygen-limited conditions,
methanogenesis and oxygen reduction with sucrose as the primary substrate was
achieved concurrently in a single mixed culture maintained without pure culture
precaution using digester and activated sludge as innoculum. Methanogenesis is
sustainable in oxygen-limited cultures. Even though dissolved oxygen is present in the
bulk liquid at all times, methane may still be produced and methanogenic activity can be
significant. Under the studied conditions, the relative methanogenic activity was higher
in some cultures that received oxygen than it was in parallel, strictly anaerobic cultures.
Low aeration rates do not necessarily cause a decrease in COD removal in
essentially anaerobic processes. Overall COD removal efficiencies for oxygen-limited,
strictly anaerobic, and strictly aerobic cultures were comparable under the complete-mix,
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suspended growth conditions investigated. In one case, effluent COD values were lower
for a methanogenic system under low aeration as compared to a strictly anaerobic system.
It may be possible to achieve the low COD values associated with aerobic biological
processes while producing the minimal waste biomass associated with anaerobic systems.
Other reactor configurations, such as UASBR and separate, staged acetogenic and
methanogenic processes would likely be more efficient. Methanogenesis with limited
oxygen under these conditions should be investigated.
In the future, methanogenesis under limited-aeration may be employed as an
energy efficient treatment option to achieve low final COD concentrations, minimal
biosolids generation, and mineralization of a broad range of organics. It is probable that
the optimum dissolved oxygen concentration in these systems is always a non-detectable
concentration close to zero and dissolved oxygen measurement is of little or no value.
For this reason, oxidation-reduction potential (Eh) measurement appears most promising
for monitoring and control. Continued research is required to determine optimum ORP
and to address technology application and process control issues.
Biofilms developed in EBRs under low-aeration conditions exhibited
hydrogenotrophic methanogen specific activities that were the same or greater than the
activity exhibited by a non-aerated control. In contrast, aceticlastic methanogen activity
decreased with increasing aeration rate, but remained significant in all reactors.
Additional interest lies in identifying methanotrophic and aerobic organisms which
influence the end COD balance of the EBR systems.
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CHAPTER 4: AERATED METHANO