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Production of poly(3-hydroxybutyrate) from a complete feedstock derived from

biodiesel by-products (crude glycerol and rapeseed meal)

Apilak Salakkam1,2* and Colin Webb1

1 Satake Centre for Grain Process Engineering, School of Chemical Engineering and

Analytical Science, University of Manchester, Oxford Road, M13 9PL, United Kingdom

2 Present address: Department of Biotechnology, Khon Kaen University, Khon Kaen, 40002,

Thailand

* Corresponding author

Contact details:

Apilak Salakkam: apilsa@kku.ac.th, Tel: +66 43363121

Colin Webb: colin.webb@manchester.ac.uk, Tel: +44 1613064379

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Highlights

A complete microbial feedstock has been produced entirely from biodiesel by-products.

Rapeseed meal proved to be a feasible source of nitrogen for PHB production.

Impurities in crude glycerol did not affect PHB synthesis in Cupriavidus necator.

C. necator produced 86% (w/w) PHB by dry weight from the produced feedstock.

A conversion process for biodiesel by-products to PHB, with mass balance, is proposed.

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Abstract

A combination of crude glycerol and rapeseed meal from biodiesel production can be utilised

to produce a complete microbial feedstock for value-added chemicals production. In this

study, rapeseed meal was transformed into a solution rich in free amino nitrogen and was used

as a nitrogen source, in combination with crude glycerol as carbon source, to produce poly(3-

hydroxybutyrate) (PHB). Using Cupriavidus necator in fed-batch fermentation, PHB

concentration at 24.75 g/L with a productivity of 0.21 g·L−1·h−1, and a yield of 0.32 g-PHB/g-

glycerol were obtained. Based on these results, a process for bioconversion of biodiesel by-

products to PHB is proposed. In this process, 3.41 L of crude glycerol and 0.72 kg of rapeseed

meal are all that is required for the production of 1 kg of PHB. The study demonstrates clearly

that a complete microbial feedstock with no requirement for further nutrient supplements can

be derived directly from the principal by-products of a conventional biodiesel process.

Keywords: Crude glycerol; Rapeseed meal; Biodiesel by-products; Microbial transformation;

Cupriavidus necator; Poly(3-hydroxybutyrate) (PHB)

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1. Introduction

The persistence of plastics in the environment causes environmental problems all over the

world. Accumulations of plastic wastes are found in oceans, landfills, and other terrestrial

environments, affecting not only wildlife but also human health [1]. Biodegradable plastics

provide a partial solution to this problem. Poly(3-hydroxybutyrate) or PHB is biodegradable

and can be synthesised by a number of microorganisms e.g. Cupriavidus necator [2]. It is a

natural polyester that has similar thermoplastic properties to polyethylene (PE) and

polypropylene (PP) but can be fully degraded in the environment [3]. Despite this advantage,

commercial use of PHB is limited due to its high production cost. Recently, the price of PHB

was reported to be around 3.5 USD/kg [4]. Although this is a dramatic decrease from the 11-

13 USD/kg price in 2006, it is still higher than the current price for PE and PP, which is

around 1.2-1.3 USD/kg [5]. To popularise the use of PHB, it will therefore be necessary to

continue to lower its price. Since raw materials account for 35% of the total PHB production

cost [6], using cheaper raw materials would lead to lower PHB sales price. Several low-cost

waste streams have been assessed for their usability for PHB production. It has been shown

through techno-economic analysis that the PHB production cost can be reduced to 1.6-1.9

USD/kg when low-quality biodiesel (saturated fatty acid esters) and wastes from animal

processing industry, e.g. offal materials, and meat and bone meal, are used [7]. Alternatively,

crude glycerol (CG) obtained from biodiesel production process can be used to reduce the

PHB production cost by 70% [8], compared to glucose, to 2.4-2.6 USD/kg [8, 9], which is

more competitive than the current price.

CG is a by-product of the transesterification of lipids to produce fatty acid alkyl ester (bio-

diesel). Theoretically, around 10 kg of CG is obtained for every 100 kg of biodiesel [10]. The

Organisation for Economic Co-operation and Development (OECD) and the Food and

Agriculture Organization of the United Nations (FAO) project that the world production of

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biodiesel will increase rapidly from 36 billion litres in 2016 to 40 billion litres in 2020 [11].

This will inevitably generate massive amounts of CG, leading to a glut in the market.

Industries, like cosmetics, pharmaceuticals, food and drinks, cannot use CG directly [12]

because it contains impurities such as methanol (0.5-28%), soap (9-19%), and salts (5-7%)

[13]. And since purification of CG is costly and difficult, the use of CG in its original

condition is more attractive [10, 14]. Owing to its high glycerol content (up to 90%) [13], CG

can be used as carbon source in fermentation processes to produce various products, for

example 1,3-propanediol, hydrogen, docosahexaenoic acid, lipids, and PHB [14]. The

production of PHB from glycerol is reported to yield 0.2-0.36 g-PHB/g-glycerol [15, 16] with

PHB content up to 70% of microbial dry weight [17].

Rapeseed meal is another attractive biodiesel by-product. It is a low-cost residue left after the

extraction of rapeseed oil. In the European Union, rapeseed oil is the main raw material for

biodiesel production. It is projected that the production of rapeseed oil for biodiesel will reach

over 9 million metric tons by 2020 [18]. The use of rapeseed meal for human consumption is

extremely limited due to the presence of antinutritional components such as glucosinolates

and phytic acid [19]. So, due to its high protein content, it is typically used as a low-cost

organic fertiliser and animal feed supplement [20]. The value of rapeseed meal can be

enhanced by converting it to products of industrial value. A sequential process consisting of

solid-state fermentation (SSF) and hydrolysis of the fermented solids has been demonstrated

to be effective in transforming various solid substrates to hydrolysates containing suitable

nutrients for microbial growth. Fermentations of such hydrolysates yield, for example,

succinic acid, lipids, ethanol, and hydrogen, depending on the solid substrate entering the

process and microorganism used [19, 21-24]. Rapeseed meal has also been reported being

used in this process for microbial oil [25] and poly(3-hydroxybutyrate-co-3-hydroxyvalerate,

P(3HB-co-3HV)) [2] production. With the carbon-rich nature of CG and nitrogen-rich nature

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of rapeseed meal, valorisation of both into PHB would improve not only the price

competitiveness of PHB to conventional plastics but also the economics of the biodiesel

industry.

The objective of the present study was to investigate the use of CG and rapeseed meal for

PHB production. Rapeseed meal was used as the sole substrate for the production of nutrient-

rich hydrolysate through SSF followed by hydrolysis of the fermented meal. The rapeseed

meal hydrolysate was then supplemented with CG to produce microbial feedstock for PHB

production. Pure glycerol (PG) was also used in this study to investigate the effect of

impurities present in CG on PHB production. A process for the bioconversion of CG and

rapeseed meal to PHB, along with an indicative with mass balance, is subsequently proposed.

2. Materials and methods

2.1 Microorganisms and media

Aspergillus oryzae was used in SSF of rapeseed meal. This fungal strain was isolated at the

University of Manchester from a soy sauce starter supplied by Amoy Food Ltd., Hong Kong.

Spore suspensions were prepared by growing the fungus at 35°C for 3-4 days on sporulation

medium consisting of 30-50 g/L rapeseed meal and 2.5% (w/v) agar. The spores were scraped

and suspended in sterile distilled water before being stored at −80°C until use. For SSF,

inoculum size was controlled at about 1×106 spores/g-meal on a dry basis (db).

Cupriavidus necator DSM4058 was used for PHB production. It was previously adapted to

grow at 50 g/L glycerol [26] and stored at −80°C. Prior to conducting experiments, the stock

culture was thawed and cultivated at 30°C in mineral medium consisting of (per L) 1 g

(NH4)2SO4, 1.5 g KH2PO4, 4.5 g Na2HPO4.2H2O, 0.2 g MgSO4.7H2O and 1 mL trace element

solution [27] with 50 g/L of pure glycerol (>99%, Sigma) as a carbon source. The bacterium

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was sub-cultured once after 48 h in the mineral medium and incubated at 30°C for 18 h prior

to inoculation.

2.2 Crude glycerol and rapeseed meal

Crude glycerol was obtained from Double green Ltd., Hull, UK. It was stored in sealable glass

bottles at room temperature. Preliminary determination of its composition revealed that it

contained 916 g/L glycerol (72.7 % v/v) and 98 g/L methanol (12.4% v/v). The acid value of

crude glycerol, as an indicator of free fatty acid, was 7.8 mg-KOH/g-crude-glycerol.

Rapeseed meal was obtained from Brocklebank Oilseed Processing Division of Cargill PLC,

Liverpool, UK. The meal was kept in an air-tight plastic container and stored at room

temperature until use. It contained 0.37 mg-glucose/g, 0.65 mg-free-amino-nitrogen (FAN)/g,

and 38.87% (w/w) protein (total nitrogen × 6.25). Other compositions of the meal were

reported in a previous publication [19].

2.3 Solid-state fermentation and further hydrolysis of the fermented biomass for rapeseed

meal hydrolysate production

In this study, rapeseed meal was used as the sole substrate in SSF following the method of

Wang et al. [19]. The meal was moistened with tap water to 65% (w/w) moisture content then

sterilised at 121°C for 45 min. After being left to cool to room temperature, the meal was

inoculated with approximately 1×106 spores of A. oryzae/g-meal (db). The content was mixed

thoroughly and transferred into 9-cm Petri dishes before being incubated at 35°C for 3 days.

After that, the fermented biomass was further hydrolysed by suspending it in distilled water at

10-15 g/L (db). The suspension was blended using a kitchen blender then transferred into

screw-capped bottles and incubated in a water bath at 55°C for 3 days. No air supply was

provided to the mixture in order to create oxygen-limited conditions. After the hydrolysis, the

hydrolysate (HL) was filtered through 0.45 µm membranes (Millipore, Durapore PVDF) for

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use in the PHB production experiment. Samples were taken to follow fungal growth, the

production of protease and FAN. Sampling procedures were described previously by

Salakkam et al. [21].

2.4 Fed-batch fermentation for PHB production

In this study, PG and CG were used as carbon sources with the HL as the nitrogen source, to

examine the effect of impurities in the CG on the production of PHB. The fermentation was

carried out in a 1.5-L bioreactor (Electrolab, model 351 equipped with 300 stirrer control)

with a working volume of 1 L. Dissolved oxygen (DO) (Oxyprobe D140, Broadley James

Corporation, USA) and pH probe (Sterprobes, Sentek, UK) were connected to the bioreactor

and calibrated prior to sterilisation at 121°C, 15 min. After being cooled to 30°C, the sterile

medium was aseptically inoculated with 100 mL of 18-h inoculum. Aeration rate was set at

1.5 vvm. DO set point was 25% while agitation speed (300-1,200 rpm) was controlled by the

DO level. The temperature and pH were controlled at 30°C and 6.8 respectively. Inlet and

outlet airstreams were filter sterilised using 0.2 µm membranes (Midisart 2000, Sartorius,

Germany). Fed-batch fermentation was conducted by feeding a required amount of stock

carbon source (PG or CG) to the bioreactor every 24 h to achieve a concentration of around

50 g/L. No nitrogen source was supplied in order to create nitrogen-limited conditions.

Samples (10 mL) were taken at time intervals until the fermentation was terminated at 120 h.

These were used for cell dry mass (CDM) measurement as well as determining FAN, glycerol

and PHB concentrations.

2.5 Analytical methods

Growth of A. oryzae was monitored in terms of weight reduction ratio (WRR) as described

previously [19]. Protease activity was assayed using casein as the substrate following the

method of Kiran et al. [25]. One unit of protease activity was defined as the enzyme required

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for releasing 1 μg FAN per min. FAN was used in this study as an indicator for the production

and utilisation of nitrogen substrate. It was determined using the ninhydrin colorimetric

method as described by Abernathy et al. [28]. Modelling of A. oryzae growth, protease and

FAN productions during SSF were by the modified Gompertz model:

(1)

where Y is WRR (%) or protease activity (U/g) or FAN (mg/g), Ym is maximum WRR or

protease activity or FAN, Rm is maximum rate of WRR or protease activity or FAN

increment, e is Euler’s number (2.7183), λ is lag time, and t is fermentation time (h).

Glycerol and methanol concentrations were determined using a GL6 analyser and reagent kits,

GMRD-185 and GMRD-125 respectively (Analox instruments Ltd., UK). Acid value of crude

glycerol was determined by titrating the sample with 1 M KOH using phenolphthalein (1%

(w/v) in a 50% ethanol solution) as an indicator. It is reported in a unit of mg-KOH per gram

of crude glycerol. Total CDM measurement was performed using a gravimetric method. Five

mL of culture broth was centrifuged at 10,000 rpm for 10 min then the pellet was washed

twice with distilled water. It was then suspended in 5-10 mL of acetone and transferred to a

pre-dried and pre-weighed universal bottle. The suspension was dried at 55°C overnight then

moved to a desiccator to cool to room temperature and weighed. Residual biomass (non-PHB

biomass) concentration was determined as the difference between CDM and PHB

concentration.

Determination of PHB concentration was conducted using dry cells obtained from CDM

measurement using a method reported by Riis and Mai [29]. Gas chromatography (model CP-

3800, Varian, Inc., USA) assembled with an autosampler (Combi/Pal, USA) was used.

Software used was Varian Star Workstation version 6.20. Column, detector and carrier gas

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were Poraplot Q-HT 10×32 mm column, flame ionisation detector and helium respectively.

Injection volume was 1 µL. The injection, detection and initial temperatures were 230°C,

200°C and 120°C respectively. The standard PHB used for calibration curve generation was

produced in-house and extracted using a method described by Hahn et al. [30].

3. Results and discussion

3.1 Solid-state fermentation and further hydrolysis of the fermented biomass

Despite only small amounts of reducing sugar and FAN being present in the meal, the growth

of A. oryzae was initiated successfully. Fig. 1 shows the growth of the fungus on the rapeseed

meal during 72 h of fermentation. During the first 6 h, the fungus was in the lag phase, which

is a period of moisture uptake for spore swelling and germination [19]. Also, during this

period, the production and secretion of growth-associated extracellular hydrolytic enzymes

take place [31]. A rapid increase in WRR, from 0.16% to 2.4% between 6 and 30 h, indicated

that the fungus was in the exponential growth phase. The modified Gompertz model fitted the

data very well. The lag time and maximum specific growth rate, determined by the model,

were 6.2 h and 0.14 1/h, respectively. However, the model only fitted the graph satisfactorily

for the first 48 h of fermentation since it does not have a term for the declining phase. The

fungus entered stationary phase after 30 h and stayed in this stage for 18 h, after which the

WRR decreased, eventually reaching 1.72%.

Fig. 1

Enzyme production in A. oryzae is associated with growth [19]. In order to take up

surrounding nutrients, the fungus secretes extracellular enzymes such as amylases and

proteases to hydrolyse large nutrient molecules (e.g. starch and protein) into smaller units

[32]. Protease activity was determined over the course of the SSF. It was found that the

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profile of protease activity was very similar to that of fungal growth. It has been reported that

the ability of A. oryzae to produce extracellular protease is inducible [33, 34] and it was

confirmed for this strain by Wang et al. [35]. From Fig. 1, it is clearly seen that protease

activity increased sharply during the exponential growth phase (between 6 and 30 h) to

148±17 U/g. The lag time and maximum protease production rate were 8.5 h and 10.6

U·g−1·h−1 respectively. After the fungus entered the stationary phase, no more protease activity

was detected and it declined to 118±5 U/g by the end of experiment. This was possibly due to

the deterioration of the enzyme.

In this study, FAN was used as an indicator to follow the production of assimilable

nitrogenous substances. It was found that as soon as protease activity appeared to increase,

FAN content rose, along with the enzyme activity (Fig. 1). The FAN concentration at the end

of the fermentation was 15.3±0.6 mg/g. Although the fungus utilised the FAN as its nitrogen

source, high levels of enzyme activity sustained the production of FAN over the period of the

study [19]. The rate of FAN production decreased at around 48 h due to the reduction in

protease activity. However, as seen in the figure, FAN concentration continued to increase

until the end of the experiment. This suggests that most of the protease released earlier was

still active and continued to digest the protein in the meal. Kinetic parameters estimated by

the modified Gompertz model (Eq. (1)) for WRR, protease production, and FAN production

are summarised in Table 1.

Table 1

Hydrolysis of the fermented solids resulted in degradation, releasing FAN into the

surrounding liquid. During incubation under oxygen-limited conditions, not only did

hydrolysis take place, but also the autolysis of the fungal cells occurred. During autolysis,

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many enzymes such as protease [21, 36], phosphatase, nuclease [37], glucanase and chitinase

[38] work synergistically to digest building blocks in fungal cells and cell walls. This results

in release of intracellular nutrients such as amino acids, peptides, nucleotides, phosphorus,

vitamins and some trace elements, into the hydrolysate [22]. As a result of combined

hydrolysis and fungal autolysis, solids concentration decreased exponentially from 11.7 to

8.15 g/L (db) within 72 h (Fig. 2). The slow rate of hydrolysis observed towards the end of

the experiment might be because of product inhibition caused by very high FAN

concentration as suggested by Wang et al. [19]. The hydrolysis rate of the fermented biomass

was estimated to be 0.045 1/h using an equation proposed by Moresi et al. [39]:

(2)

where W is the dry solids concentration (g/L), W0 is the initial dry solids concentration (g/L),

D is the difference between initial and final dry solids concentration, k is a first-order reaction

constant (1/h), and t is reaction time (h).

Fig. 2

The production of FAN was considered to be linked to the activity of both extracellular

protease released during the SSF and intracellular protease from autolysis. At the beginning of

the reaction, the mixture contained 56.4±1.2 mg-FAN/g (represented as zero in Fig. 2). At the

end of the experiment, FAN concentration was 84.5±7.8 mg/g (28.1 mg-FAN/g production),

having risen sharply during the first 24 h and then reaching a plateau with not much more

being produced (Fig. 2). This is probably due to the inhibition of enzyme by product

inhibition as described earlier. The first-order reaction constant for FAN production during

the hydrolysis stage was 0.071 1/h. This was estimated using an equation proposed by

Koutinas et al. [40]:

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(3)

where X is the concentration of FAN (mg/g), Xf is the final concentration of FAN (mg/g), k is

the first-order reaction constant (1/h), and t is the reaction time (h).

3.2 Fed-batch fermentation for PHB production

Fig. 3 shows the performance of C. necator in the hydrolysate with pure glycerol (HL+PG)

(Fig. 3(A)) and with crude glycerol (HL+CG) (Fig. 3(B)) as carbon source. The biomass

production in the two fermentations was similar although there were surges of glycerol

concentration during the processes. This implies that the bacterium can tolerate high glycerol

concentration up to around 70 g/L. Log-linear plots of CDM against fermentation time (data

not shown) revealed that the cells entered both exponential growth phase and stationary phase

at nearly the same time. From the results, it can be seen that the total biomass increased

steadily for about 45 h (between 3 h and 48 h). Biomass productivities during this period in all

fermentations were very close (0.45 g·L−1·h−1 for HL+PG and 0.40 g·L−1·h−1 for HL+CG).

This indicates that the impurities that were present in the media had only subtle effect on

biomass production. Even methanol that was found to be as high as 5 g/L in the HL+CG

experiment did not have a large effect on the growth. These results agree well with our

previous report where a significant effect was observed only beyond 10 g/L methanol [26].

From the figure, it can be seen that PHB accumulation increased rapidly after the depletion of

FAN while non-PHB biomass tended to decrease. For this reason, the increase in CDM was

considered due to PHB accumulation. Final biomass concentrations measured at 120 h for

HL+PG and HL+CG were similar at 28.9±0.7 and 28.9±0.6 g/L, respectively.

FAN utilisation in all fermentations was also very similar. Total FAN utilisation of 91.6% and

93.6% were observed in HL+PG and HL+CG respectively. These resulted in residual FAN

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concentrations of 50 and 38.4 mg/L. From the result, it was found that C. necator

accumulated PHB at 87.4% and 82.3% of its dry weight by the end of the experiment in

HL+PG and HL+CG, respectively. Since the bacterial elemental formula (without PHB) is

C4H8O2N and the monomer of PHB is C4H6O2 [41], the formula for cells containing 87.4%

and 82.3% PHB can be calculated as C1.3H2 O0.7N0.04 and C1.3H2.1O0.7N0.05, respectively. On this

basis, the cellular nitrogen will account for 1.8% and 2.4%. In order to achieve 28.9 g/L

biomass (for both fermentations), 0.51 and 0.71 g nitrogen would therefore be required,

respectively. Surprisingly, the calculated figure for HL+CG appeared to be higher than the

total FAN available (ca. 0.6 g/L). However, since the HL also contains larger nitrogenous

molecules such as di-, oligo- or polypeptides, as reported by Kiran et al. [25], it is likely that

the bacterium utilised these. C. necator was grown successfully on soy cake, which is rich in

protein [42], under SSF to produce PHB [43]. It has been reported to be able to synthesize

ATP-dependent serine protease that can degrade polypeptides to yield small peptide

fragments (http://www.uniprot.org/uniprot/Q0KBK2). These help confirm that this bacterium

is able to produce the extracellular proteolytic enzymes necessary to hydrolyse protein

substrates for its growth.

Fig. 3

The key results for PHB production from this study along with those from various other

publications are shown in Table 2. The PHB productivity and yield of PHB on substrate can

be as high as 2.42 g-PHB·L−1·h−1 and 0.43 g-PHB/g-substrate respectively. However, while

the productivity varies depending on fermentation conditions, the yield, in most systems, is

found to fluctuate over only a narrow range (0.3-0.36 g/g). This agrees well with the results

shown in Fig. 3 that the media composition did not have significant effect on PHB synthesis

in C. necator. From the table, it can be seen that the system used in this study (rapeseed

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hydrolysate supplemented with glycerol of different quality) gave relatively low biomass

concentration and therefore PHB productivity. This was possibly because the biomass

production before the commencement of PHB accumulation was low and the fermentation

times were long. Mozumder et al. [44] showed that PHB production could be increased by

delaying the shift to the nitrogen-limited phase of the fermentation. However, this resulted in

decreased PHB content.

Table 2

3.3 Proposed process for PHB production from rapeseed biodiesel by-products

Based on the results obtained from the HL+CG experiment, a process for using CG and

rapeseed meal to produce PHB is proposed (Fig. 4). With yields of PHB on glycerol and FAN

of 0.32 g/g and 43.64 g/g respectively, a total of 0.72 kg rapeseed meal and 3.41 L CG are

required to produce 1 kg of PHB. In this process, rapeseed meal is fermented by A. oryzae

under SSF to produce protease. After that, the fermented biomass is hydrolysed to produce a

hydrolysate containing 0.023 kg FAN (the conversion is 8.4%). The hydrolysis time of 24 h is

used in order to reduce the production cost. The hydrolysate is then mixed with CG (3.13 kg

of glycerol) and then fermented by C. necator to produce PHB.

Fig. 4

4. Conclusions

In this study, rapeseed meal was transformed into a hydrolysate and used, with a

supplementation of crude glycerol, to produce poly(3-hydroxybutyrate) (PHB). The results

showed that solid-state fermentation followed by hydrolysis of the fermented solids was

effective in transforming rapeseed meal to hydrolysate containing a high concentration of free

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amino nitrogen. Through the use of C. necator, impurities present in crude glycerol did not

have observable effects on PHB yield, and a yield of 0.32 g-PHB/g-glycerol was obtained.

This leads to an outlook that PHB can be produced from low-cost biodiesel by-products

without the need for additional nutrients.

5. Acknowledgement

A. Salakkam would like to acknowledge the Royal Thai Government, Thailand, for financial

support. The authors would also like to dedicate this article to the memory of Dr. RuoHang

Wang who passed away in July 2010.

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Table 1 Kinetic parameters for WRR, protease production, and FAN production during

A. oryzae fermentation of rapeseed meal.

Variable Ym Rm λ R2

WRR 2.5% 0.14 h−1 6.2 h 0.9075

Protease activity 148.5 U/g 10.6 U·g−1·h−1 8.5 h 0.9628

FAN 16.0 mg/g 0.41 mg·g−1·h−1 13.2 h 0.9993Ym is maximum WRR or protease activity or FAN, Rm is maximum rate of WRR or protease activity or FAN increment, λ is lag time

Table 2 Comparison of key results in the production of PHB by C. necator in systems

containing sugar and glycerol as carbon source.

Carbon source CDM (g/L)

PHB (g/L)

Productivity(gPHB·L−1·h−1)

Yield (gPHB/gsubstrate)

Fermentation mode Reference

Pure glycerol 28.86 25.22 0.21 0.29 Fed-batch This work

Crude glycerol 28.86 24.75 0.21 0.32 Fed-batch This work

Pure glycerol 7.7 4.8 0.11 0.3 Batch [8]

Crude glycerol 7.85 5.26 0.15 0.34 Batch [45]

Pure glycerol 82.5 51.2 0.6-1.5 0.36 Fed-batch [15]

Crude glycerol 68.8 26.1 0.84 0.34 Fed-batch [15]

Pure glycerol 75 53 0.92 0.2 Fed-batch [16]Pure glycerol and glucose 68.56 44.25 0.76 0.34 Fed-batch [46]

Glucose 7.1 4.2 0.1 0.4 Batch [8]

Glucose 164 121 2.42 0.3 Fed-batch [27]

Glucose 75.4 45.2 0.29 0.43 Fed-batch [47]

Glucose 63.8 27.3 0.35 - Fed-batch [44]

Glucose 42.4 30.5 1.23 0.36 Continuous [48]

Fructose 19.7 10.9 0.18 - Batch [49]

Fructose 36.2 16.8 0.48 - Fed-batch [49]

Fructose 27.7 5.5 0.55 - Continuous [50]

23

Fig. 1 Changes in weight reduction ratio (WRR), protease activity, and free amino nitrogen

(FAN) during A. oryzae fermentation of rapeseed meal. Solid lines represent the values

estimated by the modified Gompertz model (Eq. (1)).

24

Fig. 2 Reduction in solid concentration and production of free amino nitrogen during the

hydrolysis of fermented biomass. The solid and broken lines represent the values estimated by

Eq. (2) and (3), respectively.

25

Fig. 3 Growth and PHB production of C. necator in rapeseed hydrolysate supplemented with

pure glycerol (A) and crude glycerol (B) in fed-batch fermentation. RB is residual biomass.

Green arrows indicate re-feeds.

26

Fig. 4 Proposed bioconversion process of crude glycerol and rapeseed meal to PHB, based on

the results of the present study.