Production and characterization of poly‐3‐hydroxybutyrate from biodiesel‐glycerol by Burkholderia cepacia ATCC 17759

Glycerol, a byproduct of the biodiesel industry, can be used by bacteria as an inexpensive carbon source for the production of value‐added biodegradable polyhydroxyalkanoates (PHAs). Burkholderia cepacia ATCC 17759 synthesized poly‐3‐hydroxybutyrate (PHB) from glycerol concentrations ranging from 3% to 9% (v/v). Increasing the glycerol concentration results in a gradual reduction of biomass, PHA yield, and molecular mass (M_n and M_w) of PHB. The molecular mass of PHB produced utilizing xylose as a carbon source is also decreased by the addition of glycerol as a secondary carbon source dependent on the time and concentration of the addition. ¹H‐NMR revealed that molecular masses decreased due to the esterification of glycerol with PHB resulting in chain termination (end‐capping). However, melting temperature and glass transition temperature of the end‐capped polymers showed no significant difference when compared to the xylose‐based PHB. The fermentation was successfully scaled up to 200 L for PHB production and the yield of dry biomass and PHB were 23.6 g/L and 7.4 g/L, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Optimization of the Process for the Production of L(+)‐Lactic Acid from Cull Potato by Rhizopus oryzae

Cull potato is currently an under‐utilized biomass in the potato processing states of the USA. L(+)‐Lactic acid production by three Rhizopus strains and one homofermentative, facultative anaerobic Lactobacillus amylophilus strain was investigated using potatoes as the sole nutrient supply in the culture medium. Rhizopus oryzae NRRL 395 was chosen as the strain for further studies because it showed the highest lactate yield. The fermentation conditions for seed cultures were studied for three treatment structures using a completely randomized design. Optimum conditions for the seed culture were determined to be 2 % potato medium, 10⁴ spores/mL concentration, and 24 h of fermentation. Plackett‐Burman and central composite designs were used to screen and optimize the factors for lactic acid production. Substrate (potato) concentration, fermentation temperature, and shaking speed were found to be the most significant factors affecting both the yield and concentration of lactate. Optimum values for substrate concentration, fermentation temperature, and shaking speed were 10 %, 27 °C, and 170 rpm, respectively. Under these optimum conditions, the lactate concentration was predicted by the model to be 35.5 g/L, which was verified by the experimental data (33.3 g/L). The results indicate that cull potato can be an effective feedstock for R. ryzae NRRL 395 in the production of lactic acid.     

Screening of bacterial strains capable of converting biodiesel‐derived raw glycerol into 1,3‐propanediol, 2,3‐butanediol and ethanol

The ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PD_max concentration of ～32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ～22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ～0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.     

Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains

In the present report, crude glycerol, waste discharged from bio‐diesel production, was used as carbon substrate for three natural Yarrowia lipolytica strains (LFMB 19, LFMB 20 and ACA‐YC 5033) during growth in nitrogen‐limited submerged shake‐flask experiments. In media with initial glycerol concentration of 30 g/L, all strains presented satisfactory microbial growth and complete glycerol uptake. Although culture conditions favored the secretion of citric acid (and potentially the accumulation of storage lipid), for the strains LFMB 19 and LFMB 20, polyol mannitol was the principal metabolic product synthesized (maximum quantity 6.0 g/L, yield 0.20–0.26 g per g of glycerol consumed). The above strains produced small quantities of lipids and citric acid. In contrast, Y. lipolytica ACA‐YC 5033 produced simultaneously higher quantities of lipid and citric acid and was further grown on crude glycerol in nitrogen‐limited experiments, with constant nitrogen and increasing glycerol concentrations (70–120 g/L). Citric acid and lipid concentrations increased with increment of glycerol; maximum total citric acid 50.1 g/L was produced (yield 0.44 g per g of glycerol) while simultaneously 2.0 g/L of fat were accumulated inside the cells (0.31 g of lipid per g of dry weight). Cellular lipids were mainly composed of neutral fraction, the concentration of which substantially increased with time. Moreover, in any case, the phospholipid fraction was more unsaturated compared with total and neutral lipids, while at the early growth step, microbial lipid was more rich in saturated fatty acids (e.g. C16:0 and C18:0) compared with the stationary phase.     

γ-decalactone production by Yarrowia lipolytica and Lindnera saturnus in crude glycerol

Flavor compounds are commonly obtained from chemical synthesis or extracted from plants. These sources have disadvantages, such as racemic mixture generation, more steps to yield the final product, low yield, and high cost, making the microbial fermentation an alternative and potential way to obtain flavor compounds. The most important lactone for flavor application is γ-decalactone, which has an aroma of peach and can be obtained by ricinoleic acid biotransformation through yeast peroxisomal β-oxidation. The aim of this work was to use crude glycerol, a residual biodiesel industry, for the production of bioaroma from two different yeasts. Yarrowia lipolytica CCMA 0357 and Lindnera saturnus CCMA 0243 were grown at different concentrations (10, 20, and 30% w/v) of substrates (castor oil and crude glycerol) for γ-decalactone production. L. saturnus CCMA 0243 produced higher concentration of y-decalactone (5.8?g/L) in crude glycerol, whereas Y. lipolytica CCMA 0357 showed a maximum production in castor oil (3.5?g/L). Crude glycerol showed better results for γ-decalactone production when compared to castor oil. L. saturnus CCMA 0243 has been shown to have a high potential for γ-decalactone production from crude glycerol.     

Purified crude glycerol by acid treatment allows to improve lipid productivity by Yarrowia lipolytica SKY7

In this study, crude glycerol with high potassium concentration was purified using acid treatment and used as carbon source for lipid production using Yarrowia lipolytica SKY7. The crude glycerol was purified using phosphoric acid (pH 2) followed by centrifugation. When purified glycerol was used as carbon source for fermentation, higher biomass productivity (0.54 g/L/h) and lipid productivity (0.2 g/L/h) was observed at 96 h compared to crude glycerol. Results indicated that 6.32 g/L potassium in crude glycerol medium was inhibitory for cell growth and lipid production by Y. lipolytica. Yield coefficients, productivities and specific growth rates were calculated for each glycerol medium. The process performance with purified glycerol medium was comparable to that of pure glycerol medium. A higher lipid yield was obtained in purified glycerol medium (0.21 g/g glycerol) than crude glycerol medium (0.124 g/g glycerol). During purification of crude glycerol, KH₂PO₄ was also produced as by-product. This study provides a way for valorization of crude glycerol with high potassium concentration for microbial lipid production.     

Optimization of biosurfactant production using waste from biodiesel industry in a new membrane assisted bioreactor

The main byproduct of biodiesel production is glycerol. Here, crude glycerol – byproduct of biodiesel industry – was evaluated as sole carbon source in rhamnolipids production by Pseudomonas aeruginosa. The optimal concentration of crude glycerol and sodium nitrate was assessed using response surface methodology, resulting in about 40–50 mg/L.h of rhamnolipids, which was about four times higher than previously reported in the literature. Fermentation parameters were similar to those observed with commercial glycerol as sole carbon source. The optimized medium was suitable for production using simple (22.9 mg/L.h) and fed-batch (32.4 mg/L.h) fermentation in oxygen-controlled bioreactor without foaming formation. Composition and relative abundance of rhamnolipid congeners showed that crude glycerol had little effect on metabolic pathways involved in their production. CMC values were approximately 130 mg/L and 230–260 mg/L for rhamnolipids from crude and commercial glycerol fermentation, respectively, which were about 2–6 times lower than CMC values of synthetic surfactants.     

Production of 3‐Fucosyllactose in Engineered Escherichia coli with α‐1,3‐Fucosyltransferase from Helicobacter pylori

3‐Fucosyllactose (3‐FL), one of the major oligosaccharides in human breast milk, is produced in engineered Escherichia coli. In order to search for a good α‐1,3‐fucosyltransferase, three bacterial α‐1,3‐fucosyltransferases are expressed in engineered E. coli deficient in β‐galactosidase activity and expressing the essential enzymes for the production of guanosine 5′‐diphosphate‐l ‐fucose, the donor of fucose for 3‐FL biosynthesis. Among the three enzymes tested, the fucT gene from Helicobacter pylori National Collection of Type Cultures 11637 gives the best 3‐FL production in a simple batch fermentation process using glycerol as a carbon source and lactose as an acceptor. In order to use glucose as a carbon source, the chromosomal ptsG gene, considered the main regulator of the glucose repression mechanism, is disrupted. The resulting E. coli strain of ?LP‐YA+FT shows a much lower performance of 3‐FL production (4.50 g L^?1) than the ?L‐YA+FT strain grown in a glycerol medium (10.7 g L^?1), suggesting that glycerol is a better carbon source than glucose. Finally, the engineered E. coli ?LW‐YA+FT expressing the essential genes for 3‐FL production and blocking the colanic acid biosynthetic pathway (?wcaJ) exhibits the highest concentration (11.5 g L^?1), yield (0.39 mol mol^?1), and productivity (0.22 g L^?1 h) of 3‐FL in glycerol‐limited fed‐batch fermentation.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process

The aim of the present study was to investigate the production of 1,3-propanediol (PDO) under non-sterile fermentation conditions by employing the strain Clostridium butyricum VPI 1718. A series of batch cultures were performed by utilizing biodiesel-derived crude glycerol feedstocks of different origins as the sole carbon source, in various initial concentrations. The strain presented similarities in terms of PDO production when cultivated on crude glycerol of various origins, with final concentrations ranging between 11.1 and 11.5 g/L. Moreover, PDO fermentation was successfully concluded regardless of the initial crude glycerol concentration imposed (from 20 to 80 g/L), accompanied by sufficient PDO production yields (0.52–0.55 g per gram of glycerol consumed). During fed-batch operation under non-sterile culture conditions, 67.9 g/L of PDO were finally produced, with a yield of 0.55 g/g. Additionally, the sustainability of the bioprocess during a continuous operation was tested; indeed, the system was able to run at steady state for 16 days, during which PDO effluent level was 13.9 g/L. Furthermore, possible existence of a microbial community inside the chemostat was evaluated by operating a polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis, and DGGE results revealed the presence of only one band corresponding to that of C. butyricum VPI 1718. Finally, non-sterile continuous cultures were carried out at different dilution rates (D), with inlet glycerol concentration at 80 g/L. Maximum PDO production was achieved at low D values (0.02 h⁻¹) corresponding to 30.1 g/L, while the elaboration of kinetic data from continuous cultures revealed the stability of the bioprocess proposed, with global PDO production yield corresponding to 0.52 g/g.     

The correlation between glycogen–glycolysis metabolite pyruvate and vancomycin antibiotic productions of Amycolatopsis orientalis grown in glucose medium

The effect of glucose concentration in the growth medium on the relationship between glycolysis, glycogen accumulation and vancomycin production of Amycolatopsis orientalis was investigated depending on the incubation time. After a lag phase, bacterial growth of A. orientalis began and biomass concentration increased continuously up to 36th or 48th hours while glucose concentration in the culture medium was consumed rapidly in the same time of incubation. In addition, increase in glucose concentrations of the growth medium lead to increase intracellular glucose as well as glycerol levels. Intracellular pyruvate levels increased significantly up to 15 g/L while extracellular pyruvate levels with respect to increases in glucose concentration. A positive correlation between glucose kinase activities and glucose concentration was determined during the incubation period. Pyruvate kinase activity increased up to 15 g/L glucose and 48th hour of incubation. As a glycopeptide antibiotic, vancomycin production increased with the increases in glucose concentrations up to 15 g/L. These results indicated that glycogen accumulation with respect to glucose concentration of the growth medium was concomitant with the sporulation of A. orientalis. When the initial glucose concentration exceeded 15 g/L, pyruvate excretions as well as intracellular glycogen and glycerol productions were supported in spite of repression in vancomycin production of A. orientalis.     

High tolerance to glycerol and high production of 1,3‐propanediol in batch fermentations by microbial consortium from marine sludge

1,3‐Propanediol (1,3‐PD) is a versatile bulk chemical and widely used as a monomer to synthesis polymers, such as polyesters, polyethers and polyurethanes. 1,3‐PD can be produced by microbial fermentation with the advantages of the environmental protection and sustainable development. Low substrate tolerance and wide by‐product profile limit microbial production of 1,3‐PD by Klebsiella pneumonia on industrial scale. In this study, microbial consortia were investigated to overcome some disadvantages of pure fermentation by single strain. Microbial consortium named DL38 from marine sludge gave the best performance. Its bacterial community composition was analyzed by 16S rRNA gene amplicon high‐throughput sequencing and showed that Enterobacteriaceae was the most abundant family. Compared with three K. pneumonia strains isolated from DL38, the microbial consortium could grow well at an initial glycerol concentration of 200 g/L to produce 81.40 g/L of 1,3‐PD with a yield of 0.63 mol/mol. This initial glycerol concentration is twice the highest concentration by single isolated strain and more than the critical value (188 g/L) extrapolated from the fermentation kinetics for K. pneumonia. On the other hand, a small amount of by‐products were produced in batch fermentation of microbial consortium DL38,  especially no 2,3‐butanediol detected. The mixed culture of strain W3, Y5 and Y1 improved the tolerance to glycerol and changed the metabolite profile of single strain W3. The batch fermentation with the natural proportion (W3: Y5: Y1 = 208: 82: 17) was superior to that with other proportions and single strain. This study showed that microbial consortium DL38 possessed excellent substrate tolerance, narrow by‐product profile and attractive potential for industrial production of 1,3‐PD.     

Biodiesel-derived crude glycerol bioconversion to animal feed: a sustainable option for a biodiesel refinery

This study examined the potential of producing an edible fungus, Rhizopus microsporus var. oligosporus, on biodiesel-derived crude glycerol. Prolific fungal growth was observed with a fungal biomass yield of 0.83 ± 0.02 (g biomass increase/g initial biomass) under optimal cultivation conditions (e.g. nonsterile crude glycerol at a concentration of 75% (w/v) with nutrient supplementation and without pH control). The potential of utilizing front-end processed banagrass (Pennisetum purpureum) juice as a source of nutrients for crude glycerol fermentation was evaluated with a 2.3-fold improvement in the fungal biomass yield. The glycerol-derived fungal biomass showed high amounts of threonine, one of the main limiting amino acids in non-ruminant feeds. An inexpensive fungal protein has the potential to reduce meat product prices by lowering the production costs of animal feeds. The application of fungal technology thus provides a unique sustainable option for biodiesel refineries by providing an additional source of revenue from fungal products.     

Monacolin K production by citrinin‐free Monascus pilosus MS‐1 and fermentation process monitoring

Monacolin K (MK) is a naturally occurring hypocholesterolemic agent that specifically inhibits HMG‐CoA reductase. As a natural source of MK, Monascus‐fermented products are of special interest; however, some Monascus strains could produce citrinin, which is a nephrotoxin, as a contaminant in Monascus‐derived products. A Monascus pilosus strain (MS‐1) that produces high amounts of MK, but no citrinin, was screened in previous investigations. Herein, liquid‐state fermentation parameters of the MS‐1 strain were optimized using statistical methods to maximize the MK yield with potato juice as a basic medium. The maximum MK yield (326.74 μg/mL) was predicted with 50 mL of medium in a 250‐mL conical flask containing 30 g/L sucrose, 38.75 g/L soybean flour, 0.00105 mol/L Mg²⁺ at pH 5.48, and 8% v/v seed inoculum precultured for 42 h at 30°C, incubated at 30°C for 3 days, followed by further incubation for 11 days at 24.7°C. The verified MK yield was 390.68 μg/mL and the MK yield increased to 565.64 μg/mL after 21 days of fermentation. No citrinin was detected in MS‐1‐fermented products. The results suggest that citrinin‐free MK can be obtained from natural medium through liquid‐state fermentation in an economical way. This method will be of practical value to the industrial production of MK.     

Fermentation of crude glycerol from biodiesel production by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis>

Clostridium pasteurianum can utilize glycerol as the sole carbon source for the production of butanol and 1,3-propanediol. Crude glycerol derived from biodiesel production has been shown to be toxic to the organism even in low concentrations. By examination of different pretreatments we found that storage combined with activated stone carbon addition facilitated the utilization of crude glycerol. A pH-controlled reactor with in situ removal of butanol by gas stripping was used to evaluate the performance. The fermentation pattern on pretreated crude glycerol was quite similar to that on technical grade glycerol. C. pasteurianum was able to utilize 111 g/l crude glycerol. The average consumption rate was 2.49 g/l/h and maximum consumption rate was 4.08 g/l/h. At the maximal glycerol consumption rate butanol was produced at 1.3 g/l/h. These rates are higher than those previously reported for fermentations on technical grade glycerol by the same strain. A process including pretreatment and subsequent fermentation of the crude glycerol could be usable for industrial production of butanol by C. pasteurianum.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation

Crude glycerol is the primary by-product in the biodiesel industry, which is too costly to be purified into to higher quality products used in the health and cosmetics industries. This work investigated the potential of using the crude glycerol to produce docosahexaenoic acid (DHA, 22:6 n-3) through fermentation of the microalga Schizochytrium limacinum. The results showed that crude glycerol supported alga growth and DHA production, with 75–100 g/L concentration being the optimal range. Among other medium and environmental factors influencing DHA production, temperature, trace metal (PI) solution concentration, ammonium acetate, and NH₄Cl had significant effects (P < 0.1). Their optimal values were determined 30 mL/L of PI, 0.04 g/L of NH₄Cl, 1.0 g/L of ammonium acetate, and 19.2 °C. A highest DHA yield of 4.91 g/L with 22.1 g/L cell dry weight was obtained. The results suggested that biodiesel-derived crude glycerol is a promising feedstock for production of DHA from heterotrophic algal culture.     

Intra- and extra-cellular flux distributions of TCA and glyoxalate cycle and vancomycin production of <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> grown in different glycerol concentration

The relationship between tricarboxylic acid (TCA) and glyoxalate cycle and the effect of their metabolites levels on the vancomycin production of Amycolatopsis orientalis were investigated in different concentration of glycerol (2.5–20 g/l). Intracellular glycerol levels increased with respect to increases in glycerol concentrations of the growth medium. Extracellular glycerol levels decreased slowly up to 24 h while uptake rates were increased during 36–48^th h for 10 and 15 g/l and during 36–60^th h at 20 g/l of glycerol. Intracellular citrate, α-ketoglutarate, fumarate levels increased up to 10 g/l glycerol concentration. However, intracellular succinate and malate levels were increased up to 15 g/l glycerol. Extracellular citrate, α-ketoglutarate, succinate and malate levels increased with respect to increases in glycerol concentration. The highest α-ketoglutarate dehydrogenase activity was determined at 15 g/l glycerol. Isocitrate lyase activity showed a positive correlation with the increases in glycerol concentration of the growth medium. Vancomycin production increased with the increases in glycerol concentration from 5 to 10 g/l. These results showed that A. orientalis grown in glycerol containing medium used glyoxalate shunt actively instead of TCA cycle which supports precursors of many amino acid which are effective on the antibiotic production.     

Enhanced hydrogen and 1,3‐propanediol production from glycerol by fermentation using mixed cultures

The conversion of glycerol into high value products, such as hydrogen gas and 1,3‐propanediol (PD), was examined using anaerobic fermentation with heat‐treated mixed cultures. Glycerol fermentation produced 0.28 mol‐H₂/mol‐glycerol (72 mL‐H₂/g‐COD) and 0.69 mol‐PD/mol‐glycerol. Glucose fermentation using the same mixed cultures produced more hydrogen gas (1.06 mol‐H₂/mol‐glucose) but no PD. Changing the source of inoculum affected gas production likely due to prior acclimation of bacteria to this type of substrate. Fermentation of the glycerol produced from biodiesel fuel production (70% glycerol content) produced 0.31 mol‐H₂/mol‐glycerol (43 mL H₂/g‐COD) and 0.59 mol‐PD/mol‐glycerol. These are the highest yields yet reported for both hydrogen and 1,3‐propanediol production from pure glycerol and the glycerol byproduct from biodiesel fuel production by fermentation using mixed cultures. These results demonstrate that production of biodiesel can be combined with production of hydrogen and 1,3‐propanediol for maximum utilization of resources and minimization of waste. Biotechnol. Bioeng. 2009; 104: 1098–1106. © 2009 Wiley Periodicals, Inc.     

Metabolic engineering of Escherichia coli for efficient free fatty acid production from glycerol

Crude glycerol, generated as waste by-product in biodiesel production process, has been considered as an important carbon source for converting to value-added bioproducts recently. Free fatty acids (FFAs) can be used as precursors for the production of biofuels or biochemicals. Microbial biosynthesis of FFAs can be achieved by introducing an acyl–acyl carrier protein thioesterase into Escherichia coli. In this study, the effect of metabolic manipulation of FFAs synthesis cycle, host genetic background and cofactor engineering on FFAs production using glycerol as feed stocks was investigated. The highest concentration of FFAs produced by the engineered stain reached 4.82 g/L with the yield of 29.55% (g FFAs/g glycerol), about 83% of the maximum theoretical pathway value by the type II fatty acid synthesis pathway. In addition, crude glycerol from biodiesel plant was also used as feedstock in this study. The FFA production was 3.53 g/L with a yield of 24.13%. The yield dropped slightly when crude glycerol was used as a carbon source instead of pure glycerol, while it still can reach about 68% of the maximum theoretical pathway yield.