Bioconversion Of Crude Glycerol Research Articles

A non-aseptic bioprocess for production and recovery of 2,3-butanediol via conversion of crude glycerol and corn steep liquor at pilot-scale

The production and recovery of 2,3-butanediol (BDO) through biodiesel derived glycerol valorization by Klebsiella oxytoca ACA-DC 1581 was holistically optimized with regard to the efficiency and cost of the bioprocess. The absence of thermal treatment of the substrate had no negative effect upon the growth of microorganism and the bioconversion of crude glycerol into BDO, enabling the development of a non-aseptic and lower-cost bioprocess. Both digestate and corn steep liquor (CSL), the main by-products of the biogas and corn industries respectively, successfully served as the sole source of nitrogen, contributing to the complete replacement of more expensive sources (e.g., yeast extract). The biochemical pathway of glycerol catabolism was examined under varying concentrations of dissolved oxygen and BDO production was optimized in a fully aerobic environment (volumetric mass transfer coefficient; kLa = 70.5 1/h.) The glycerol consumption rate was 2.80 g/L/h, the BDO productivity reached 1.12 g/L/h and the yield of BDO produced per unit of glycerol consumed was 0.46 g/g, with these values being among the highest ones reported in the literature for wild-type strains cultivated on crude glycerol. In all fed-batch fermentations, final BDO and acetoin concentration reached ∼80 g/L, while a plateau was observed at ∼68 g/L of BDO. Finally, the culture was carried out efficiently in the pilot-scale reactor (250 L). The salting-out extraction (SOE), consisting of ethanol (24 %) and K2HPO4 (25 %), recovered 91.7 % of BDO from the fermentation medium and was studied for the first time in a glycerol-based medium. The study suggests the potential industrialization of the bioprocess through sustainable, pilot-scale and low-cost bioconversion of biodiesel-derived crude glycerol and CSL or digestate into BDO.

Biochemical and molecular characterization of a novel glycerol dehydratase from Klebsiella pneumoniae 2e with high tolerance against crude glycerol impurities

BackgroundThe direct bioconversion of crude glycerol, a byproduct of biodiesel production, into 1,3-propanediol by microbial fermentation constitutes a remarkably promising value-added applications. However, the low activity of glycerol dehydratase, which is the key and rate-limiting enzyme in the 1,3-propanediol synthetic pathway, caused by crude glycerol impurities is one of the main factors affecting the 1,3-propanediol yield. Hence, the exploration of glycerol dehydratase resources suitable for crude glycerol bioconversion is required for the development of 1,3-propanediol-producing engineered strains.ResultsIn this study, the novel glycerol dehydratase 2eGDHt, which has a tolerance against crude glycerol impurities from Klebsiella pneumoniae 2e, was characterized. The 2eGDHt exhibited the highest activity toward glycerol, with Km and Vm values of 3.42 mM and 58.15 nkat mg−1, respectively. The optimum pH and temperature for 2eGDHt were 7.0 and 37 °C, respectively. 2eGDHt displayed broader pH stability than other reported glycerol dehydratases. Its enzymatic activity was increased by Fe2+ and Tween-20, with 294% and 290% relative activities, respectively. The presence of various concentrations of the crude glycerol impurities, including NaCl, methanol, oleic acid, and linoleic acid, showed limited impact on the 2eGDHt activity. In addition, the enzyme activity was almost unaffected by the presence of an impurity mixture that mimicked the crude glycerol environment. Structural analyses revealed that 2eGDHt possesses more coil structures than reported glycerol dehydratases. Moreover, molecular dynamics simulations and site-directed mutagenesis analyses implied that the existence of unique Val744 from one of the increased coil regions played a key role in the tolerance characteristic by increasing the protein flexibility.ConclusionsThis study provides insight into the mechanism for enzymatic action and the tolerance against crude glycerol impurities, of a novel glycerol dehydratase 2eGDHt, which is a promising glycerol dehydratase candidate for biotechnological conversion of crude glycerol into 1,3-PDO.

Economic and environmental evaluation for a closed loop of crude glycerol bioconversion to biodiesel

Crude glycerol, a byproduct of biodiesel production, was utilized as a carbon source to produce microbial lipids by the oleaginous yeast Rhodotorula toruloides in this study. The maximum lipid production and lipid content were 10.56 g/L and 49.52%, respectively, by optimizing fermentation conditions. The obtained biodiesel met the standards of China, the United States, and the European Union. The economic value of biodiesel produced from crude glycerol increased by 48% compared with the sale of crude glycerol. In addition, biodiesel production from crude glycerol could reduce 11,928 tons of carbon dioxide emissions and 55 tons of sulfur dioxide emissions. This study provides a strategy for a closed loop of crude glycerol to biofuel and ensures sustainable and stable development of the biodiesel industries.

EFFECTS OF SALTS IN BIODIESEL DERIVED CRUDE GLYCEROL ON VANCOMYCIN PRODUCTION FROM AMYCOLATOPSIS ORIENTALIS ATCC® 19795™

Bioconversion of crude glycerol (CG), the primary byproduct in biodiesel generation, is currently of great interest due to its promising potential in producing chemicals of high commercial interest. The efficient use of CG is still limited primarily due to the presence of impurities that may vary in composition based on the parent feedstock, biodiesel generation process, and any recovery/ purification conducted. In this study, laboratory scale fermentation of vancomycin using Amycolatopsis orientalis ATCC® 19795™ was analyzed based on the presence of salts as an impurity in biodiesel derived CG. Results showed that CG as the sole carbon source inhibited the cell growth, but the vancomycin production was approximately the same (158±2 mg L-1) as that of refined glycerol (RG)-based media at varying initial glycerol concentrations studied (5 g L-1 to 40 g L-1). NaCl as an impurity in biodiesel derived CG reduced both the growth yield coefficient (YX/S) and vancomycin formation at high concentrations. Among other salts tested are NaNO3 and KCl. NaNO3 (0.8 g L-1) supplemented media generated a higher YX/S (1.1 g biomass (g glycerol consumed)-1) and a similar vancomycin concentration (193 mg L-1) compared to the media with added NaCl (YX/S of 0.4 g biomass (g glycerol consumed)-1 and a vancomycin concentration of (190 mg L-1)). Relatively, both the YX/S (0.55 g biomass (g glycerol consumed)-1) and vancomycin concentration (135 mg L-1) was reduced in KCl (0.7 g L-1) supplemented media. The results suggest the potential of using CG (with salts i.e., NaCl as impurities) as an excellent low-cost alternative for RG in fermentation of vancomycin using Amycolatopsis orientalis ATCC® 19795™.

Dihydroxyacetone production via heterogeneous biotransformations of crude glycerol

In this work, several immobilization strategies for Gluconobacter oxydans NBRC 14819 (Gox) were tested in the bioconversion of crude glycerol to dihydroxyacetone (DHA). Agar, agarose and polyacrylamide were evaluated as immobilization matrixes. Glutaraldehyde crosslinked versions of the agar and agarose preparations were also tested. Agar immobilized Gox proved to be the best heterogeneous biocatalyst in the bioconversion of crude glycerol reaching a quantitative production of 50 g/L glycerol into DHA solely in water. Immobilization allowed reutilization for at least eight cycles, reaching four times more DHA than the amount obtained by a single batch of free cells which cannot be reutilized. An increase in scale of 34 times had no impact on DHA productivity. The results obtained herein constitute a contribution to the microbiological production of DHA as they not only attain unprecedented productivities for the reaction with immobilized biocatalysts but also proved that it is feasible to do it in a clean background of solely water that alleviates the cost of downstream processing.

Sustainable vancomycin production from industrial residues and the effects of inoculum control to ensure reproducibility

The conversion of biodiesel-derived crude glycerol into value-added products provides a sustainable solution for the biodiesel industry. This study proposed the bioconversion of crude glycerol and soy-based products into vancomycin by Amycolatopsis orientalis (ATCC 19795). The vancomycin yield with crude glycerol as a carbon source was not different from the yield with refined glycerol. The productivity of 32.10 g/L of defatted soy flour and 11.76 g/L of crude glycerol was 621.69 μg/L/h, which was comparable to that of 25 g/L of the yeast extract (679.25 μg/L/h). The medium prepared from crude glycerol and defatted soy flour was cost-effective (US$ 0.48/g vancomycin), and estimated to be 12.34% of the cost from refined glycerol and yeast extract (US$ 3.89/g vancomycin). The inoculum volume of 5% at 30 h allowed a higher yield and minimum error. Biodiesel-derived crude glycerol with defatted soy flour offers a potential feedstock for sustainable vancomycin production.

Adaptability of Klebsiella pneumoniae 2e, a Newly Isolated 1,3-Propanediol-Producing Strain, to Crude Glycerol as Revealed by Genomic Profiling.

Crude glycerol is largely generated as the main by-product of the biodiesel industry and is unprofitable for industrial application without costly purification. The direct bioconversion of crude glycerol into 1,3-propanediol (1,3-PDO) by microorganisms is a promising alternative for effective and economic utilization. In this study, Klebsiella pneumoniae 2e was newly isolated for the conversion of crude glycerol into 1,3-PDO. Batch fermentation analysis confirmed that crude glycerol and its main impurities had slight impacts on the growth, key enzyme activity, and 1,3-PDO production of K. pneumoniae 2e. The 1,3-PDO yield from crude glycerol by K. pneumoniae 2e reached 0.64 mol 1,3-PDO/mol glycerol, which was higher than that by most reported 1,3-PDO-producing Klebsiella strains. Genomic profiling revealed that K. pneumoniae 2e possesses 30 genes involved in glycerol anaerobic metabolism and 1,3-PDO biosynthesis. Quantitative real-time PCR analysis of these genes showed that the majority of the genes encoding the key enzymes for glycerol metabolism and 1,3-PDO biosynthesis were significantly upregulated during culture in crude glycerol relative to that in pure glycerol. Further comparative genomic analysis revealed a novel glycerol uptake facilitator protein in K. pneumoniae 2e and a higher number of stress response proteins than in other Klebsiella strains. This work confirms the adaptability of a newly isolated 1,3-PDO-producing strain, K. pneumoniae 2e, to crude glycerol and provides insights into the molecular mechanisms involved in its crude glycerol tolerance, which is valuable for industrial 1,3-PDO production from crude glycerol.IMPORTANCE The rapid development of the biodiesel industry has led to tremendous crude glycerol generation. Due to the presence of complex impurities, crude glycerol has low value for industry without costly purification. Obtaining novel microorganisms capable of direct and efficient bioconversion of crude glycerol to value-added products has great economic potential for industrial application. In this work, we characterized a newly isolated strain, Klebsiella pneumoniae 2e, with the capacity to efficiently produce 1,3-propanediol (1,3-PDO) from crude glycerol and demonstrated its adaptation to crude glycerol. Our work provides insights into the molecular mechanisms of K. pneumoniae 2e adaptation to crude glycerol and the expression patterns of its genes involved in 1,3-PDO biosynthesis, which will contribute to the development of industrial 1,3-PDO production from crude glycerol.

Process optimization and kinetic analysis of malic acid production from crude glycerol using Aspergillus niger

In the present work, optimization of crude glycerol fermentation to produce malic acid by using Aspergillus niger was investigated using response surface methodology and artificial neural network. Kinetic investigation of bioconversion of crude glycerol into malic acid using Aspergillus niger was studied using Monod, Mosser, and Haldane-Andrew models. Crude glycerol concentration, initial pH and yeast extract concentration were found to be significant compounds affecting malic acid production by Aspergillus niger. Both dry cell weight and malic acid titre were found decreased with increase in crude glycerol concentration. Haldane-Andrew model gave the best fit for the production of malic acid from crude glycerol with µmax of 0.1542 h−1. The maximum malic acid production obtained under optimum conditions was 92.64 + 1.54 g/L after 192 h from crude glycerol using Aspergillus niger.

Stability and oscillatory behavior of microbial consortium in continuous conversion of crude glycerol to 1,3-propanediol.

Microbial consortium is an alternative for bioconversion of crude glycerol to value-added products whereas concerns about the process stability in long-term operation existed. The aim of this study is to evaluate the feasibility of using an anaerobic microbial consortium as inoculum for continuous conversion of crude glycerol to 1,3-propanediol (1,3-PDO). Performances of continuous fermentations with the consortium inoculum were evaluated under different dilution rates and glycerol feed concentrations. The highest 1,3-PDO production of 57.86g/L was achieved with a productivity of 5.55g/(L·h). Analyses of kinetic data showed that the consortium maintained a consistent pattern for 1,3-PDO production under different operating conditions despite changes in community composition. The continuous fermentation by the consortium was able to operate for a longer period of time (31 volume changes) than that using pure culture (24 volume changes) with the average 1,3-PDO concentration of 53.52g/L and productivity of 6.69g/(L·h) under glycerol-excess condition, which could be contributed to the intraspecies diversity among Clostridium butyricum in the consortium. Under glycerol-limited conditions, however, a spontaneous oscillation of the consortium was observed after continuous operation for about 120h, along with severe fluctuations of the microbial community. The oscillatory behavior could be reduced by increasing the dilution rates and was likely the metabolic feature of C. butyricum.

Bioconversion of crude glycerol from waste cooking oils into hydrogen by sub-tropical mixed and pure cultures

Abstract This study compared the biohydrogen generation by sub-tropical mixed and pure cultures from the crude glycerol from the biodiesel production using waste cooking oils (WCO). The crude glycerol was pretreated by pH adjustment. The mixed culture was obtained from a subtropical granular sludge of the UASB (Upflow Anaerobic Sludge Blanket) reactor used in the treatment of vinasse from sugarcane of ethanol and sugar industry. It was heat treated in order to inactivate hydrogen-consuming bacteria, which was identified by Illumina MiSeq Sequencing with a relative abundance of 97.96% Firmicutes Philum, 91.81% Clostridia Class and 91.81% Clostridiales Order. The pure culture was isolated from a sub-tropical granular sludge from UASB reactor of treating brewery wastewater and identified as Enterobacter sp. (KP893397). Two assays were carried in anaerobic batch reactors in order to verify the hydrogen production from crude glycerol bioconversion with: (I) mixed culture and (II) pure culture. The experiments were conducted at 37 °C, initial pH of 5.5 for assay I and 7.0 for assay II, with 20 g COD L−1 of crude glycerol. The crude glycerol consumption was 56.2% and 88.0% for the assay I and II, respectively. The hydrogen yields were 0.80 moL H2 mol−1 glycerol for the assay I and 0.13 moL H2 mol−1 glycerol for the assay II. Enterobacter sp. preferred the reductive metabolic route, generating 1460.0 mg L−1 of 1,3-propanediol, and it showed to be more sensitive in the presence of methanol from crude glycerol than mixed culture that preferred the oxidative metabolic route with biohydrogen generation. The mixed culture was more able to generate H2 than pure culture from the crude glycerol coming from the biodiesel production using WCO.

Bioconversion of biodiesel-derived crude glycerol into lipids and carotenoids by an oleaginous red yeast Sporidiobolus pararoseus KM281507 in an airlift bioreactor

Here we tested the bioconversion of biodiesel-derived crude glycerol by the oleaginous red yeast Sporidiobolus pararoseus KM281507 in two bioreactors types (stirred-tank and airlift). High production yields (biomass, 10.62±0.21g/L; lipids, 3.26±0.13g/L; β-carotene, 30.64±0.05mg/L; total carotenoids, 46.59±0.07mg/L) were achieved in a 3.0L airlift bioreactor under uncontrolled pH regimes (initial pH 5.63). Under optimized conditions (6.0 vvm aeration rate; 60±5% constant dissolved oxygen [DO] maintained by flushing pure oxygen [O2] into the vessel; 10,000 Lux light irradiation) volumetric production in the airlift bioreactor was further increased (biomass, 19.30±1.07g/L; lipids, 6.61±0.04g/L, β-carotene, 109.75±0.21mg/L; total carotenoids 151.00±2.71mg/L). Production was also recorded at a S.pararoseus KM281507 growth rate of 0.16±0.00h-1 (lipids, 0.94±0.04g/L/d; β-carotene, 15.68±0.40mg/L/d; total carotenoids, 21.56±0.20mg/L/d). Lipids from S.pararoseus KM281507 had a high unsaturated fatty acid content, with oleic acid (C18:1) accounting for 80% of all fatty acids. This high oleic acid content makes S.pararoseus KM281507 well-suited as a third generation biodiesel feedstock. Our findings show that airlift bioreactors are suitable for bioconversion of crude glycerol into lipids and carotenoids using S.pararoseus KM281507. This approach is advantageous because of its ease of operation, cost efficiency, and low energy consumption.

Utilization of by-product glycerol from bio-diesel plants as feedstock for 2,3-butanediol accumulation and biosynthesis genes response of Klebsiella variicola SW3

The booming of biodiesel industry all over the world has led to generate a large amount (10% v/v) of crude glycerol, created an oversupply problem. Herein, we compared the product concentrations of major metabolic products attained from pure and crude glycerol biotransformation process using an adapted mutant strain Klebsiella variicola SW3. Real-time qPCR and glycerol dehydrogenase (GDH) enzyme activity assay revealed that the overexpression of GDH gene resulted in an increased GDH enzyme activity, led to a markedly boosted 2,3-butanediol (2,3-BD) production. Based on these results, the SW3 strain obtained from wild type strain Klebsiella variicola SRP3 displayed a 1.39-fold increased 2,3-BD production of 82.5 g/L from 59.3 g/L, yielding 0.62 g/g using pure glycerol. However, in a batch culture, a final 33.5 g/L of 2,3-BD was accumulated within 96 h from 50 g/L glycerol. Moreover, the strain SW3 withstanding high concentration (200 g/L) of crude glycerol displayed 64.9 and 29.25 g/L 2,3-BD in fed-batch and batch cultures respectively. Therefore, this bioconversion of crude glycerol to 2,3-BD –a value-added green product with potential industrial applications as a liquid fuel or fuel additive would represent a remarkable alternative to add value to the biodiesel production helping biodiesel industries development.

Bioconversion of crude glycerol to microbial lipid using a robust oleaginous yeast Rhodosporidium toruloides ATCC 10788 capable of growing in the presence of impurities

In this work, crude glycerol, a by-product from a biodiesel industry was characterized and converted to microbial lipid. The sample of crude glycerol was found to contain 44.56wt.% glycerol, 13.86wt.% methanol, 10.74wt.% of ash and 32.97wt.% of soap. Rhodosporidium toruloides ATCC 10788 was studied for the first time to determine its ability to grow on crude glycerol as a carbon source. Methanol concentration as low as 0.5% (w/v) in the medium containing pure glycerol inhibited biomass and lipid production by this strain. However, higher concentration of methanol (i.e. 1.5% w/v) in crude glycerol containing media did not have the same effect on the organism, with double the biomass (21.16g/L), triple the lipid concentration (11.27g/L) and high lipid content (53.28wt.%) produced at the end of 168h. Unlike many other oleaginous microorganisms, the ability of this yeast to consume other impurities present in crude glycerol enhanced the growth and lipid production in such a way that the effect of methanol was masked or became insignificant. The lipid obtained contained 47.16% of oleic acid (a monounsaturated fatty acid), making it a very good feedstock for some oleochemical industries and the production of biodiesel. This bioconversion process which involves the production of lipid from crude glycerol waste stream will be of great interest to the biodiesel industry as it can be easily integrated into the existing biodiesel production plant. The use of such robust strains to produce other value added products can also be explored.

Crude glycerol by transesterification process from used cooking oils: Characterization and potentialities on hydrogen bioproduction

This study evaluated the potential bioconversion of crude glycerol from biodiesel production, applying used cooking oil for biohydrogen production by fermentative bacteria consortia. The pretreatment of crude glycerol was made by pH adjustment. Heat treatment of the inocula and initial pH 5.5 were applied to select hydrogen-producing bacteria and inactivate hydrogen-consumers microorganisms. The inocula tested were: (I) granular sludge from the thermophilic UASB reactor used on the treatment of vinasse and (II) granular sludge from the UASB reactor used on the treatment of sanitary sludge for the assays (1) and (2), respectively. The characterization of crude glycerol presented high levels of alkalinity, methanol and soap that may be inhibitory to biologic processes of H2 production. The assays were carried in anaerobic batch reactors in order to verify the efficiencies of crude glycerol to H2 generations by the microbial consortia (20%) at 37 °C, initial pH 5.5, with 20.0 g COD L−1 glycerol. The cumulative production of hydrogen for the assays (1) and (2) were, respectively, (mmol H2 L−1) 28.49 ± 1.55 and 19.14 ± 1.67. The subsequent yields were obtained as follows: 2.2 moL H2 mol−1 glycerol and 1.1 moL H2 mol−1 glycerol, respectively. The used cooking oil was an efficient waste for bioconversion of crude glycerol to H2 production.

Surfactant mediated enhanced glycerol uptake and hydrogen production from biodiesel waste using co-culture of Enterobacter aerogenes and Clostridium butyricum

In the present study, Tween 80, a non-ionic surfactant, has been used for enhanced hydrogen production by crude glycerol bioconversion using co-culture of Enterobacter aerogenes and Clostridium butyricum. The purpose of introducing the surfactant was to decrease the crude glycerol viscosity, so that apparent solubility and bioavailability of glycerol could be improved at the expenses of pretreatment steps. Experiments were planned using central composite design (CCD); crude glycerol and Tween 80 concentrations were optimized whereas, hydrogen production, glycerol utilization and viscosity of the media were considered as responses. The response surface for quadratic model showed, Tween 80 concentration had significant effect (p < 0.05) on all the three responses. Using the optimized conditions at 17.5 g/L crude glycerol and 15 mg/L Tween 80, hydrogen production reached a maximum of 32.1 ± 0.03 mmol/L of medium. The increase in hydrogen production was around 1.25-fold in presence of Tween 80 in comparison to its absence with 25.56 ± 0.91 mmol/L production. Selected optimum conditions were also validated against absence of crude glycerol (4.69 ± 0.76), with pretreated crude glycerol (20.06 ± 0.51) and across mono-culture system (15.43 ± 0.79 to 22.14 ± 0.94). Introduction of Tween 80 to the fermentation medium improved the glycerol utilization rate, resulting in increased hydrogen production and eliminated pretreatment steps.

Consolidating biofuel platforms through the fermentative bioconversion of crude glycerol to butanol.

Economic realities for the rising industrial biofuel production have changed substantially during the low oil price period starting in the mid 2010's. Increased competition requires the sector to increase productivity through the reduction of low-value by-products and full utilization of all value and energy stored in their respective feedstock. Biodiesel is produced commercially from substrates such as animal fat and vegetable oil, generating approximately 10wt% crude glycerol as its main, currently underutilized, by-product. This crude glycerol is contaminated with catalyst, soap, free fatty acids, glycerides and methyl esters; hence only a small fraction enters the existing glycerol markets, while the purification costs for the majority of crude glycerol are simply too high. However, this presents a unique opportunity to generate additional value. One technical possibility is to use crude glycerol as a carbon source for butanol production, a compound of higher value and energy, a potential additive for gasoline and diesel fuels and bulk chemical commodity. Conversion facilities could be co-located with biodiesel plants, utilizing established infrastructure and adding significant value and productivity to the existing biodiesel industry. This review focuses on the current activities geared towards the bioconversion of crude glycerol to butanol.

Hydrogen production from biodiesel industry waste by using a co-culture of Enterobacter aerogenes and Clostridium butyricum

ABSTRACTValorization of crude glycerol (CG), a waste of biodiesel production process, was investigated for increased hydrogen production by co-culture system using Enterobacter aerogenes NRRL B-407 and Clostridium butyricum NRRL B-41122. The ability of E. aerogenes was evaluated as a replacement of expensive reducing agent to maintain anaerobic conditions for the growth of C. butyricum. The co-culture resulted in increased hydrogen production, reaching a maximum of 19.46 ± 0.95 mmol-H2/L-medium in comparison to monoculture of E. aerogenes (15.64 ± 0.47) and C. butyricum (17.44 ± 0.38) in the presence of reducing agent. The effect of inoculum ratio was investigated and hydrogen yield was 0.95 mmol-H2/mol-glycerol at 1:11 inoculum ratio with more than 85% of substrate utilization was comparable to other mixed and co-culture studies. In addition to hydrogen, value added by-products such as 1,3-propandiol, acetic acid, butyric acid and ethanol have also been produced. Co-culture indicated the possibility of valorization of CG as a stable functional consortium with higher conversion efficiency for increased hydrogen along with high value by-product production. Additionally, the study provided the evidence of medium composition controlled metabolic pathway shift during CG bioconversion. Valorization of low cost CG with efficient co-culture of hydrogen production can aid biodiesel in-house energy source.

Bioconversion of crude glycerol to polyhydroxyalkanoate by Bacillus thuringiensis under non-limiting nitrogen conditions

Glycerol has emerged as a cheap waste material due to blooming biodiesel manufacturing units worldwide. The need is to exploit the crude glycerol (CG) to produce useful products such as polyhydroxyalkanoate (PHA). Bacillus thuringiensis EGU45 was found to produce 1.5–3.5gPHAL−1 from feed containing 1–10% CG (vv−1) and nutrient broth (NB, 125mL) without any acclimatization. B. thuringiensis EGU45 could produce PHA at the rate of 1.54–1.83gL−1, from 1% CG (vv−1) on media having high nitrogen contents: (i) NB, (ii) NB+0.5% NH4Cl (wv−1), and (iii) peptone+yeast extract+0.5% NH4Cl (wv−1). B. thuringiensis EGU45 was able to produce co-polymer of P(3HB-co-3HV) with 13.4% 3HV content on high N containing feed supplemented with propionic acid. This is the first report demonstrating the abilities of B. thuringiensis to convert CG into PHA co-polymer under non-limiting N conditions.

Improved bioconversion of crude glycerol to hydrogen by statistical optimization of media components

Bioconversion of crude glycerol to hydrogen has gained importance as it addresses both sustainable energy production and waste disposal issues. Until recently, statistical optimizations of crude glycerol bioconversion to hydrogen have been greatly focused on pure strains. In this study, biohydrogen production from crude glycerol by an enriched microbial culture (predominated with Clostridium species) was improved by statistical optimization of media components. Plackett–Burman design identified MgCl2.6H2O and KCl with negative effect on hydrogen production and selected NH4Cl, K2HPO4 and KH2PO4 as significant variables. Box–Behnken design indicated the optimal region beyond design area and studies were continued by ridge analysis. Central composite face centered design envisaged a maximal hydrogen yield of 1.41 mol-H2/mol-glycerolconsumed at concentrations 4.40 g/L and 2.27 g/L for NH4Cl and KH2PO4 respectively. Confirmation experiment with the optimized media (NH4Cl, 4.40 g/L; K2HPO4, 1.6 g/L; KH2PO4, 2.27 g/L; MgCl2.6H2O, 1.0 g/L; KCl, 1.0 g/L; Na-acetate.3H2O, 1.0 g/L and tryptone, 2.0 g/L) revealed an excellent correlation between predicted and experimental hydrogen yield. Optimization of media components by design of experiments enhanced hydrogen yield by 29%.

Efficient bioconversion of crude glycerol from biodiesel to optically pured-lactate by metabolically engineered Escherichia coli

Biodiesel has attracted considerable attention as one of the best choices among alternative and renewable fuels. Large quantities of crude glycerol are produced as a main co-product with increasing biodiesel production. Currently, the problem of waste glycerol utilization needs to be crucially addressed, not only for environmental protection but also for the economy of the biodiesel industry. In this paper, the use of crude glycerol for the production of D-lactate by engineered Escherichia coli was investigated. Engineered E. coli B0013-070 with a homolactic pathway for D-lactate synthesis by elimination of byproduct pathways (ethanol, succinate, formate and acetate) could convert 20 g L−1 of crude glycerol to 13.6 g L−1 of D-lactate with a yield of 0.67 g g−1 glycerol. Overexpression of D-lactate dehydrogenase by a low-copy vector in E. coli B0013-070 resulted in the increased production and yield of D-lactate, in which 14.5 g L−1 of D-lactate was produced with a yield of 0.72 g g−1 glycerol from crude glycerol. The effect of temperature on D-lactate fermentation by the engineered strain E. coli B0013-070-pTHldhA was also investigated, and 34 °C and 40 °C were found to be the optimal temperatures for cell growth and lactate production, respectively. The engineered strain B0013-070-pTHldhA produced 100.3 g L−1 of D-lactate with 99.97% optical purity from 531.5 g of crude glycerol with an overall productivity of 2.78 g L−1 h−1 and a yield of 75.4 g per 100 g glycerol (0.77 mol mol−1) using two phase fermentation combined with a temperature shifting strategy in a 7 L bioreactor. In summary, this paper shows that crude glycerol could be directly converted to D-lactate without any prior purification.