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.     

Use of biodiesel‐derived crude glycerol for vancomycin production by Amycolatopsis orientalis XMU‐VS01

Crude glycerol is a primary by‐product in the biodiesel industry. Microbial fermentation on crude glycerol for producing value‐added products provides opportunities to utilize a large quantity of this by‐product. This study investigates the potential of using the crude glycerol to produce vancomycin (glycopeptide antibiotics) through fermentation of Amycolatopsis orientalis XMU‐VS01. The results show that crude glycerol was the most effective carbon source for mycelium growth and vancomycin production, with 40–60 g/L glycerol concentration as optimal range. Among other culture medium components, potato protein (nitrogen source) and the phosphate concentration had significant effects (p<0.05) for vancomycin production. A Box‐Behnken design and response surface methodology were employed to formulate the optimal medium. Their optimal values were determined as 52.73 g/L of glycerol, 17.36 g/L of potato protein, and 0.1 g/L of dipotassium phosphate. A highest vancomycin yield of 7.61 g/L with biomass concentration of 15.8 g/L was obtained after 120 h flask fermentation. The yield of vancomycin was 3.5 times higher than with basic medium. The results suggest that biodiesel‐derived crude glycerol is a promising feedstock for production of vancomycin from A. orientalis culture.     

Utilization of glycerol and crude glycerol for polysaccharide production by an endophytic fungus Chaetomium globosum CGMCC 6882

Abstract

Crude glycerol is becoming a financial and environmental liability due to its surplus production from biodiesel industry, and its utilization as a fermentation feedstock for value-added chemicals production has been widely studied. In present work, the capacity of an endophytic fungus, Chaetomium globosum CGMCC 6882, using glycerol and crude glycerol for polysaccharide production was investigated. Results showed that the polysaccharide titers from glucose and glycerol were 1.85 and 3.8?g/L, respectively. Moreover, spore morphology of C. globosum CGMCC 6882 was favorable for polysaccharide production. Meanwhile, impurities in crude glycerol have no effect on polysaccharide production by C. globosum CGMCC 6882. Finally, characteristic results of polysaccharides produced from glucose, glycerol, and crude glycerol have suggested that metabolic flux might be a determinant factor on polysaccharide structure. Taken together, this research provided an innovative approach of utilizing crude glycerol produced from the biodiesel production process.     

l-Lactate Production from Biodiesel-Derived Crude Glycerol by Metabolically Engineered Enterococcus faecalis: Cytotoxic Evaluation of Biodiesel Waste and Development of a Glycerol-Inducible Gene Expression System

Biodiesel waste is a by-product of the biodiesel production process that contains a large amount of crude glycerol. To reuse the crude glycerol, a novel bioconversion process using Enterococcus faecalis was developed through physiological studies. The E. faecalis strain W11 could use biodiesel waste as a carbon source, although cell growth was significantly inhibited by the oil component in the biodiesel waste, which decreased the cellular NADH/NAD⁺ ratio and then induced oxidative stress to cells. When W11 was cultured with glycerol, the maximum culture density (optical density at 600 nm [OD₆₀₀]) under anaerobic conditions was decreased 8-fold by the oil component compared with that under aerobic conditions. Furthermore, W11 cultured with dihydroxyacetone (DHA) could show slight or no growth in the presence of the oil component with or without oxygen. These results indicated that the DHA kinase reaction in the glycerol metabolic pathway was sensitive to the oil component as an oxidant. The lactate dehydrogenase (Ldh) activity of W11 during anaerobic glycerol metabolism was 4.1-fold lower than that during aerobic glycerol metabolism, which was one of the causes of low l-lactate productivity. The E. faecalis pflB gene disruptant (Δpfl mutant) expressing the ldhL1_LP gene produced 300 mM l-lactate from glycerol/crude glycerol with a yield of >99% within 48 h and reached a maximum productivity of 18 mM h⁻¹ (1.6 g liter⁻¹ h⁻¹). Thus, our study demonstrates that metabolically engineered E. faecalis can convert crude glycerol to l-lactate at high conversion efficiency and provides critical information on the recycling process for biodiesel waste.     

A comparative study of solvent-assisted pretreatment of biodiesel derived crude glycerol on growth and 1,3-propanediol production from Citrobacter freundii

The rapidly growing biodiesel industry has created a scenario, where it is both important and challenging to deal with the enormous amount of crude glycerol generated as an inherent by-product. With every 100 gallons of biodiesel produced, 5-10 gallons of the crude glycerol is left behind containing several impurities which makes its disposal difficult. The objective of the present investigation was to evaluate the impact of biodiesel-derived crude glycerol upon microbial growth and production of 1,3-propanediol by Citrobacter freundii. Five different grades of crude glycerol (obtained from biodiesel preparation using jatropha, soybean, sunflower, rice bran and linseed oils) were used. Crude glycerol caused significant inhibition of microbial growth and subsequently 1,3-propanediol production as compared to pure glycerol. Therefore, a process was developed for the treatment of crude glycerol using solvents before fermentation wherein four different non-polar solvents were examined yielding different grades of pretreated glycerol. Subsequently, the potential toxic effects of pretreated glycerol on the growth and 1,3-propanediol production by C. freundii was evaluated. In case of petroleum ether-treated crude glycerol obtained from jatropha & linseed and hexane-treated crude glycerol obtained from rice bran, the yields obtained were comparable to the pure glycerol. Similarly, soybean-derived glycerol gave comparable results after treatment with either hexane or petroleum ether.     

Effect of impurities in biodiesel-derived waste glycerol on the performance and feasibility of biotechnological processes

The rapid development of biodiesel production technology has led to the generation of tremendous quantities of glycerol wastes, as the main by-product of the process. Stoichiometrically, it has been calculated that for every 100 kg of biodiesel, 10 kg of glycerol are produced. Based on the technology imposed by various biodiesel plants, glycerol wastes may contain numerous kinds of impurities such as methanol, salts, soaps, heavy metals, and residual fatty acids. This fact often renders biodiesel-derived glycerol unprofitable for further purification. Therefore, the utilization of crude glycerol though biotechnological means represents a promising alternative for the effective management of this industrial waste. This review summarizes the effect of various impurities-contaminants that are found in biodiesel-derived crude glycerol upon its conversion by microbial strains in biotechnological processes. Insights are given concerning the technologies that are currently applied in biodiesel production, with emphasis to the impurities that are added in the composition of crude glycerol, through each step of the production process. Moreover, extensive discussion is made in relation with the impact of the nature of impurities upon the performances of prokaryotic and eukaryotic microorganisms, during crude glycerol bioconversions into a variety of high added-value metabolic products. Finally, aspects concerning ways of crude glycerol treatment for the removal of inhibitory contaminants as reported in the literature are given and comprehensively discussed.     

High yield conversion of a crude glycerol fraction from biodiesel production to hydrogen by photofermentation

Present biodiesel manufacturing processes inevitably produce a crude glycerol side fraction. Projected future volumes of biodiesel will generate enormous quantities of glycerol of a magnitude suggesting that conversion to a fuel is the only viable route. Here we have shown that the photosynthetic bacterium Rhodopseudomonas palustris is capable of the photofermentative conversion of glycerol, both pure and a crude glycerol fraction, to hydrogen, a proposed future fuel. Relatively high yields, up to 6 moles H₂/mole glycerol (75% of theoretical, 8 moles of H₂/mole glycerol) were obtained. Even the crude glycerol fraction, at the concentrations used here, was readily converted to hydrogen with no apparent evidence of inhibition or toxicity. We show that the concentration of added nitrogen can be used to modify both rates and yields of hydrogen production with an apparent trade-off between the two. Finally, some factors are identified that might be examined in future studies in attempts to increase rates and/or yields.     

Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens

The rapidly expanding market for biodiesel has increased the supply and reduced the cost of glycerol, making it an attractive sustainable feed stock for the fuel and chemical industry. Glycerol-based biorefinery is the microbial fermentation of crude glycerol to produce fuels and chemicals. A major challenge is to obtain microbes tolerant to inhibitors such as salts and organic solvents present in crude glycerol. Microbial screening was attempted to isolate novel strain capable of growing on crude glycerol as a sole carbon source. The newly isolated bacteria, identified as nonpathogenic Kluyvera cryocrescens S26 could convert biodiesel-derived crude glycerol to ethanol with high yield and productivity. The supplementation of nutrients such as yeast extract resulted in distinguished enhancement in cell growth as well as ethanol productivity under anaerobic condition. When glycerol fermentation is performed under microaerobic condition, there is also a remarkable improvement in cell growth, ethanol productivity and yield, compared with those under strict anaerobic condition. In batch fermentation under microaerobic condition, K. cryocrescens S26 produced 27 g/l of ethanol from crude glycerol with high molar yield of 80% and productivity of 0.61 g/l/h.     

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 10 wt% 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.     

High-level production of 1,3-propanediol from crude glycerol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> AKR102a

The aim of this study was to optimize a biotechnological process for the production of 1,3-propanediol (1,3-PD) based on low-quality crude glycerol derived from biodiesel production. Clostridium butyricum AKR102a was used in fed-batch fermentations in 1-L and 200-L scale. The newly discovered strain is characterized by rapid growth, high product tolerance, and the ability to use crude glycerol at the lowest purity directly gained from a biodiesel plant side stream. Using pure glycerol, the strain AKR102 reached 93.7 g/L 1,3-PD with an overall productivity of 3.3 g/(L*h). With crude glycerol under the same conditions, 76.2 g/L 1,3-PD was produced with a productivity of 2.3 g/(L*h). These are among the best results published so far for natural producers. The scale up to 200 L was possible. Due to the simpler process design, only 61.5 g/L 1,3-PD could be reached with a productivity of 2.1 g/(L*h).     

Transcriptome profiling of Zymomonas mobilis under ethanol stress

Biodiesel is a promising alternative, and renewable, fuel. As its production increases, so does production of the principle co-product, crude glycerol. The effective utilization of crude glycerol will contribute to the viability of biodiesel. In this review, composition and quality factors of crude glycerol are discussed. The value-added utilization opportunities of crude glycerol are reviewed. The majority of crude glycerol is used as feedstock for production of other value-added chemicals, followed by animal feeds.     

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.     

Biomass and lipid production of heterotrophic microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> by using biodiesel-derived crude glycerol

Microalgal lipids may be a more sustainable biodiesel feedstock than crop oils. We have investigated the potential for using the crude glycerol as a carbon substrate. In batch mode, the biomass and lipid concentration of Chlorella protothecoides cultivated in a crude glycerol medium were, respectively, 23.5 and 14.6 g/l in a 6-day cultivation. In the fed-batch mode, the biomass and lipid concentration improved to 45.2 and 24.6 g/l after 8.2 days of cultivation, respectively. The maximum lipid productivity of 3 g/l day in the fed-batch mode was higher than that produced by batch cultivation. This work demonstrates the feasibility of crude biodiesel glycerol as an alternative carbon substrate to glucose for microalgal cultivation and a cost reduction of carbon substrate feed in microalgal lipid production may be expected.     

Biodiesel biorefinery: opportunities and challenges for microbial production of fuels and chemicals from glycerol waste

ABSTRACT: The considerable increase in biodiesel production worldwide in the last 5 years resulted in astoichiometric increased coproduction of crude glycerol. As an excess of crude glycerol hasbeen produced, its value on market was reduced and it is becoming a "waste-stream" insteadof a valuable "coproduct". The development of biorefineries, i.e. production of chemicals andpower integrated with conversion processes of biomass into biofuels, has been singled out asa way to achieve economically viable production chains, valorize residues and coproducts,and reduce industrial waste disposal. In this sense, several alternatives aimed at the use ofcrude glycerol to produce fuels and chemicals by microbial fermentation have beenevaluated. This review summarizes different strategies employed to produce biofuels andchemicals (1,3-propanediol, 2,3-butanediol, ethanol, n-butanol, organic acids, polyols andothers) by microbial fermentation of glycerol. Initially, the industrial use of each chemical isbriefly presented; then we systematically summarize and discuss the different strategies toproduce each chemical, including selection and genetic engineering of producers, andoptimization of process conditions to improve yield and productivity. Finally, the impact ofthe developments obtained until now are placed in perspective and opportunities andchallenges for using crude glycerol to the development of biodiesel-based biorefineries areconsidered. In conclusion, the microbial fermentation of glycerol represents a remarkablealternative to add value to the biodiesel production chain helping the development ofbiorefineries, which will allow this biofuel to be more competitive.     

Microbial utilization of crude glycerol for the production of value-added products

Energy fuels for transportation and electricity generation are mainly derived from finite and declining reserves of fossil hydrocarbons. Fossil hydrocarbons are also used to produce a wide range of organic carbon-based chemical products. The current global dependency on fossil hydrocarbons will not be environmentally or economically sustainable in the long term. Given the future pessimistic prospects regarding the complete dependency on fossil fuels, political and economic incentives to develop carbon neutral and sustainable alternatives to fossil fuels have been increasing throughout the world. For example, interest in biodiesel has undergone a revival in recent times. However, the disposal of crude glycerol contaminated with methanol, salts, and free fatty acids as a by-product of biodiesel production presents an environmental and economic challenge. Although pure glycerol can be utilized in the cosmetics, tobacco, pharmaceutical, and food industries (among others), the industrial purification of crude glycerol is not economically viable. However, crude glycerol could be used as an organic carbon substrate for the production of high-value chemicals such as 1,3-propanediol, organic acids, or polyols. Microorganisms have been employed to produce such high-value chemicals and the objective of this article is to provide an overview of studies on the utilization of crude glycerol by microorganisms for the production of economically valuable products. Glycerol as a by-product of biodiesel production could be used a feedstock for the manufacture of many products that are currently produced by the petroleum-based chemical industry.     

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 a Novel Two-step Process Comprising Re-esterification and Transesterification in a Single Reactor for Biodiesel Production Using Waste Cooking Oil

Waste cooking oil (WCO) has attracted attention as a non-edible feedstock for biodiesel. Although an alkali catalyst has several advantages over an acid catalyst in biodiesel production, biodiesel conversion from WCO is only 5.2% when using an alkali catalyst (NaOH), owing to its high free fatty acid (FFA) content of 4.2%. In this study, a novel two-step process in a single reactor, comprised of re-esterification of the FFAs with crude glycerol, using a Tin (II) chloride (SnCl₂) catalyst, and subsequent transesterification with methanol, using an alkali catalyst, was adopted, and each step was optimized. This study revealed that the FFA content after re-esterification should be approximately 1.5%, not only to save glycerol and the catalyst involved in the re-esterification, but also to achieve high biodiesel conversion during the transesterification. An alkaline catalyst was successfully used to produce biodiesel in the second step, and a 92.8% conversion to biodiesel was achieved under the optimized conditions (0.6% catalyst relative to WCO, 0.2mL-methanol/WCO, 70ºC, 3 h). Overall, this novel two-step process achieved highly enhanced biodiesel conversion (4.0% to 92.8%) with significantly reduced reaction time (12 h to 4 h) and methanol requirements (15 mL/g-WCO to 0.2 mL/g-WCO).     

Impact of impurities in biodiesel-derived crude glycerol on the fermentation by <Emphasis Type="Italic">Clostridium pasteurianum</Emphasis> ATCC 6013

During the production of biodiesel, crude glycerol is produced as a byproduct at 10% (w/w). Clostridium pasteurianum has the inherent potential to grow on glycerol and produce 1,3-propanediol and butanol as the major products. Growth and product yields on crude glycerol were reported to be slower and lower, respectively, in comparison to the results obtained from pure glycerol. In this study, we analyzed the effect of each impurity present in the biodiesel-derived crude glycerol on the growth and metabolism of glycerol by C. pasteurianum. The crude glycerol contains methanol, salts (in the form of potassium chloride or sulfate), and fatty acids that were not transesterified. Salt and methanol were found to have no negative effects on the growth and metabolism of the bacteria on glycerol. The fatty acid with a higher degree of unsaturation, linoleic acid, was found to have strong inhibitory effect on the utilization of glycerol by the bacteria. The fatty acid with lower or no degrees of unsaturation such as stearic and oleic acid were found to be less detrimental to substrate utilization. The removal of fatty acids from crude glycerol by acid precipitation resulted in a fermentation behavior that is comparable to the one on pure glycerol. These results show that the fatty acids in the crude glycerol have a negative effect by directly affecting the utilization of glycerol as the carbon source, and hence their removal from crude glycerol is an essential step towards the utilization of crude glycerol.     

3-Hydroxypropionaldehyde production from crude glycerol by <Emphasis Type="Italic">Lactobacillus diolivorans</Emphasis> with enhanced glycerol uptake

Background

In their quest for sustainable development and effective management of greenhouse gas emissions, our societies pursue a shift away from fossil-based resources towards renewable resources. With 95% of our current transportation energy being petroleum based, the application of alternative, carbon-neutral products—among them biodiesel—is inevitable. In order to enhance the cost structure of biodiesel biorefineries, the valorization of the crude glycerol waste stream into high-value platform chemicals is of major importance.

Results

The purpose of this study is the production of 3-hydroxypropionaldehyde (3-HPA) from biodiesel-derived crude glycerol by Lactobacillus diolivorans. Particular focus is given on overcoming potential limitations of glycerol transport into the cell, in order to use the cells’ total glycerol dehydratase capability towards the formation of 3-HPA as the main product. Recombinant overexpression of the endogenous glycerol uptake facilitating protein PduF results in a significant increase of glycerol conversion by a factor of 1.3. Concomitantly, glycerol dehydratase activity increased from initially 1.70 ± 0.03 U/mg protein to 2.23 ± 0.11 U/mg protein. With this approach, an average productivity of 4.8 g_3-HPA/(g_CDM h) yielding up to 35.9 g/L 3-HPA and 0.91 mol_3-HPA/mol_Glycerol have been obtained.

Conclusion

Lactobacillus diolivorans proves to be a valuable cell factory for the utilization of crude glycerol delivering high-value C3 chemicals like 3-HPA, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP). Enhancing the glycerol influx into the cell by genetic engineering was successful paving the way towards the commercial production of 3-HPA.