Bio-hydrogen production by biodiesel-derived crude glycerol bioconversion: a techno-economic evaluation

Global biodiesel production is continuously increasing and it is proportionally accompanied by a huge amount of crude glycerol (CG) as by-product. Due to its crude nature, CG has very less commercial interest; although its pure counterpart has different industrial applications. Alternatively, CG is a very good carbon source and can be used as a feedstock for fermentative hydrogen production. Further, a move of this kind has dual benefits, namely it offers a sustainable method for disposal of biodiesel manufacturing waste as well as produces biofuels and contributes in greenhouse gas (GHG) reduction. Two-stage fermentation, comprising dark and photo-fermentation is one of the most promising options available for bio-hydrogen production. In the present study, techno-economic feasibility of such a two-stage process has been evaluated. The analysis has been made based on the recent advances in fermentative hydrogen production using CG as a feedstock. The study has been carried out with special reference to North American biodiesel market; and more specifically, data available for Canadian province, Québec City have been used. Based on our techno-economic analysis, higher production cost was found to be the major bottleneck in commercial production of fermentative hydrogen. However, certain achievable alternative options for reduction of process cost have been identified. Further, the process was found to be capable in reducing GHG emissions. Bioconversion of 1 kg of crude glycerol (70 % w/v) was found to reduce 7.66 kg CO₂ eq (equivalent) GHG emission, and the process also offers additional environmental benefits.     

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.     

The bioconversion of wood hydrolyzates to butanol and butanediol

Steam-exploded aspenwood chips were acid hydrolysed to their component sugars. Near theoretical solvent yields were achieved in both the acetone-butanol-ethanol (ABE) fermentation and 2,3-butanediol fermentation of these liberated sugars. When Clostridium acetobutylicum was grown on wood hydrolysates, final butanol yields of 9.0 g/L (0.26 g of butanol per g of sugar consumed) were obtained. When Klebsiella pneumoniae was grown on the wood hydrolysates, final butanediol concentrations exceeded 20 g/L, resulting in a bioconversion efficiency approaching 0.5 g of butanediol per g of sugar utilised.     

纤维燃料丁醇研究进展

随着能源危机与粮食安全问题的日趋加重,以纤维质为原料生产石油替代燃料已成为生物质能研究的重点。分析了丁醇作为燃料的优点,归纳了丁醇发酵微生物的种类与研究现状,综述了纤维原料生产燃料丁醇的研究进展,最后对我国纤维燃料丁醇的产业化优势和前景进行了分析与展望。     

Prospective and development of butanol as an advanced biofuel

Butanol has been acknowledged as an advanced biofuel, but its production through acetone–butanol–ethanol (ABE) fermentation by clostridia is still not economically competitive, due to low butanol yield and titer. In this article, update progress in butanol production is reviewed. Low price and sustainable feedstocks such as lignocellulosic residues and dedicated energy crops are needed for butanol production at large scale to save feedstock cost, but processes are more complicated, compared to those established for ABE fermentation from sugar- and starch-based feedstocks. While rational designs targeting individual genes, enzymes or pathways are effective for improving butanol yield, global and systems strategies are more reasonable for engineering strains with stress tolerance controlled by multigenes. Compared to solvent-producing clostridia, engineering heterologous species such as Escherichia coli and Saccharomyces cerevisiae with butanol pathway might be a solution for eliminating the formation of major byproducts acetone and ethanol so that butanol yield can be improved significantly. Although batch fermentation has been practiced for butanol production in industry, continuous operation is more productive for large scale production of butanol as a biofuel, but a single chemostat bioreactor cannot achieve this goal for the biphasic ABE fermentation, and tanks-in-series systems should be optimized for alternative feedstocks and new strains. Moreover, energy saving is limited for the distillation system, even total solvents in the fermentation broth are increased significantly, since solvents are distilled to ~ 40% by the beer stripper, and more than 95% water is removed with the stillage without phase change, even with conventional distillation systems, needless to say that advanced chemical engineering technologies can distil solvents up to ~ 90% with the beer stripper, and the multistage pressure columns can well balance energy consumption for solvent fraction. Indeed, an increase in butanol titer with ABE fermentation can significantly save energy consumption for medium sterilization and stillage treatment, since concentrated medium can be used, and consequently total mass flow with production systems can be reduced. As for various in situ butanol removal technologies, their energy efficiency, capital investment and contamination risk to the fermentation process need to be evaluated carefully.     

Acetylation of glycerol to biofuel additives over sulfated activated carbon catalyst

Oxygenated fuel additives can be produced by acetylation of glycerol. A 91% glycerol conversion with a selectivity of 38%, 28% and 34% for mono-, di- and triacetyl glyceride, respectively, was achieved at 120 °C and 3 h of reaction time in the presence of a catalyst derived from activated carbon (AC) treated with sulfuric acid at 85 °C for 4h to introduce acidic functionalities to its surface. The unique catalytic activity of the catalyst, AC-SA5, was attributed to the presence of sulfur containing functional groups on the AC surface, which enhanced the surface interaction between the glycerol molecule and acyl group of the acetic acid. The catalyst was reused in up to four consecutive batch runs and no significant decline of its initial activity was observed. The conversion and selectivity variation during the acetylation is attributed to the reaction time, reaction temperature, catalyst loading and glycerol to acetic acid molar ratio.     

Thermogravimetric kinetics of crude glycerol

The pyrolysis of the crude glycerol from a biodiesel production plant was investigated by thermogravimetry coupled with Fourier transform infrared spectroscopy. The main gaseous products are discussed, and the thermogravimetric kinetics derived. There were four distinct phases in the pyrolysis process of the crude glycerol. The presence of water and methanol in the crude glycerol and responsible for the first decomposition phase, were shown to catalyse glycerol decomposition (second phase). Unlike the pure compound, crude glycerol decomposition below 500 K leaves behind a large mass fraction of pyrolysis residues (ca. 15%), which eventually partially eliminate in two phases upon reaching significantly higher temperatures (700 and 970 K, respectively). An improved iterative Coats–Redfern method was used to evaluate non-isothermal kinetic parameters in each phase. The latter were then utilised to model the decomposition behaviour in non-isothermal conditions. The power law model (first order) predicted accurately the main (second) and third phases in the pyrolysis of the crude glycerol. Differences of 10–30 kJ/mol in activation energies between crude and pure glycerol in their main decomposition phase corroborated the catalytic effect of water and methanol in the crude pyrolysis. The 3-D diffusion model more accurately reproduced the fourth (last) phase, whereas the short initial decomposition phase was poorly simulated despite correlation coefficients ca. 0.95–0.96. The kinetics of the 3rd and 4th decomposition phases, attributed to fatty acid methyl esters cracking and pyrolysis tarry residues, were sensitive to the heating rate.     

Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway

Solventogenic clostridia are well-known since almost a century due to their unique capability to biosynthesize the solvents acetone and butanol. Based on recently developed genetic engineering tools, a targeted 3-hydroxybutyryl-CoA dehydrogenase (Hbd)-negative mutant of Clostridium acetobutylicum was generated. Interestingly, the entire butyrate/butanol (C₄) metabolic pathway of C. acetobutylicum could be inactivated without a severe growth limitation and indicated the general feasibility to manipulate the central fermentative metabolism for product pattern alteration. Cell extracts of the mutant C. acetobutylicum hbd::int(69) revealed clearly reduced thiolase, Hbd and crotonase but increased NADH-dependent alcohol dehydrogenase enzyme activities as compared to the wildtype strain. Neither butyrate nor butanol were detected in cultures of C. acetobutylicum hbd::int(69), and the formation of molecular hydrogen was significantly reduced. Instead up to 16 and 20 g/l ethanol were produced in glucose and xylose batch cultures, respectively. Further sugar addition in glucose fed-batch fermentations increased the ethanol production to a final titer of 33 g/l, resulting in an ethanol to glucose yield of 0.38 g/g.     

Bioconversion of crude glycerol to glycolipids in Ustilago maydis

Ustilago maydis is known to produce glycolipid-type biosurfactants. Here, we show that U. maydis is able to efficiently convert biodiesel-derived crude glycerol to glycolipids. We have optimized the medium composition and environmental factors for bioconversion of crude glycerol to glycolipids. The synthetic medium (MinCG) contains 50 g L⁻¹ crude glycerol and 20.3 mg L⁻¹ ammonium citrate as the carbon and nitrogen sources, respectively. The supplementation of trace amount of amino acids, Group-B vitamins and precursors of glycolipids, mannose and erythritol, also improved the final yield. At pH 4.0 and 30 °C, 32.1 g L⁻¹ total glycolipids was produced in a 8.2-day fed-batch bioprocess. Methanol at 2% or above severely inhibited cell growth and production of glycolipids. Our results suggest that U. maydis is an excellent host for the bioconversion of crude glycerol to value-added products.     

Effect of glucose on glycerol bioconversion by Lactobacillus reuteri

The impact of glucose on glycerol metabolism, especially on 3-hydroxypropionaldehyde (3-HPA) accumulation by resting cells of Lactobacillus reuteri has been investigated. Two systems were used in the study: MRS(-) (modified MRS - omitting glucose, acetate and Tween 80) and distilled water (H(2)O). In MRS(-), addition of glucose enhanced glycerol metabolism in resting cells of L. reuteri, consequently increasing the accumulation of 3-HPA by regulating the NAD/NADH ratio. Enhanced glycerol metabolism correlated positively with the concentration of glucose. NADH produced during glucose metabolism was preferentially reoxidized to NAD by the reduction of 3-HPA to 1,3-propanediol; an adequate supply of glycerol therefore outweighed the repression of glucose on the accumulation of 3-HPA. At a molar ratio of glucose to glycerol no greater than 0.33, accumulation of 3-HPA was favored. In non-growing medium (H(2)O), addition of glucose seemed to be counter-productive with respect to 3-HPA accumulation. Lactate had a positive impact on glycerol metabolism, presumably by altering the redox flux, resulting in enhanced 3-HPA accumulation in both MRS(-) and H(2)O systems.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

Bioconversion of crude glycerol by fungi

The production of synthetic glycerol from petrochemical feedstocks has been decreasing in recent years. This is largely due to increasing supplies of crude glycerol derived as a co-product from the oleochemical industry, especially biodiesel production. The price of glycerol is at historic lows, and the supply of crude glycerol is projected to grow faster than its industrial uses. This oversupply is driving the transition from glycerol as a product to glycerol as a precursor for new industrial applications, including its use as a substrate for bioconversion. This article reviews the use of fungi for the bioconversion of crude glycerol to the value-added products 1,2-propanediol, ethanol, single cell oil, specialty polyunsaturated fatty acids, biosurfactants, and organic acids. Information on the impurities of crude glycerol from different industrial processes is also included.     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

Fermentation of glycerol and production of valuable chemical and biofuel molecules

Glycerol has attracted the attention of scientific and industrial communities due to its generation in bulk quantities as a byproduct of biofuel industries. With the rapid growth of these industries in recent years, glycerol is frequently treated as a very low-value byproduct or even a waste product with a disposal cost associated to it. Glycerol is not only abundant and inexpensive but also can generate more reducing equivalents than glucose or xylose. This unique characteristic of glycerol offers a tremendous opportunity for its biological conversion to valuable products at higher yield. This review focuses on research efforts to utilize glycerol as a carbon source for the production of a variety of fuels and chemicals by both native and metabolically engineered microorganisms.     

From crude glycerol to carotenoids by using a Rhodotorula glutinis mutant

In this work eighteen red yeasts were screened for carotenoids production on glycerol containing medium. Strain C2.5t1 of Rhodotorula glutinis, that showed the highest productivity, was UV mutagenized. Mutant 400A15, that exhibited a 280 % increase in β–carotene production in respect to the parental strain, was selected. A central composite design was applied to 400A15 to optimize carotenoids and biomass productions. Regression analyses of the quadratic polynomial equations obtained (R² = 0.87 and 0.94, for carotenoids and biomass, respectively) suggest that the models are reliable and significant (P < 0.0001) in the prediction of carotenoids and biomass productions on the basis of the concentrations of crude glycerol, yeast extract and peptone. Accordingly, total carotenoids production achieved (14.07 ± 1.45 mg l^?1) under optimized growth conditions was not statistically different from the maximal predicted (14.64 ± 1.57 mg l^?1) (P < 0.05), and it was about 100 % higher than that obtained under un-optimized conditions. Therefore mutant 400A15 may represent a biocatalyst of choice for the bioconversion of crude glycerol into value-added metabolites, and a tool for the valorization of this by-product of the biodiesel industry.     

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.     

玉米黄浆水在丙酮-丁醇厌氧梭菌发酵中的应用

为降低丙酮-丁醇厌氧梭菌发酵生产丁醇的成本,研究了不同添加量玉米黄浆水对发酵的影响。与葡萄糖培养基相比,在发酵培养基中添加少量玉米黄浆水对发酵产量无显著影响。当添加体积分数为25％的玉米黄浆水时,丙酮、丁醇和乙醇的最终质量浓度分别是0．31、2．70和8．00g/L,总溶剂量为11．01g／L。通过成本核算,每生产1kg溶剂,添加体积分数25％的玉米黄浆水可比葡萄糖培养基节约成本2．11元。     

Fermentation of glycerol by Anaerobium acetethylicum and its potential use in biofuel production

Growth of biodiesel industries resulted in increased coproduction of crude glycerol which is therefore becoming a waste product instead of a valuable ‘coproduct’. Glycerol can be used for the production of valuable chemicals, e.g. biofuels, to reduce glycerol waste disposal. In this study, a novel bacterial strain is described which converts glycerol mainly to ethanol and hydrogen with very little amounts of acetate, formate and 1,2‐propanediol as coproducts. The bacterium offers certain advantages over previously studied glycerol‐fermenting microorganisms. Anaerobium acetethylicum during growth with glycerol produces very little side products and grows in the presence of maximum glycerol concentrations up to 1500 mM and in the complete absence of complex organic supplements such as yeast extract or tryptone. The highest observed growth rate of 0.116 h^?1 is similar to that of other glycerol degraders, and the maximum concentration of ethanol that can be tolerated was found to be about 60 mM (2.8 g l^?1) and further growth was likely inhibited due to ethanol toxicity. Proteome analysis as well as enzyme assays performed in cell‐free extracts demonstrated that glycerol is degraded via glyceraldehyde‐3‐phosphate, which is further metabolized through the lower part of glycolysis leading to formation of mainly ethanol and hydrogen. In conclusion, fermentation of glycerol to ethanol and hydrogen by this bacterium represents a remarkable option to add value to the biodiesel industries by utilization of surplus glycerol.     

Optimization of metabolic pathways for bioconversion of lignocellulose to ethanol through genetic engineering

The optimization of metabolic pathways is of fundamental importance for strategies aimed at improving the economics and yield of the lignocellulose-to-ethanol processes. Although Escherichia coli is capable of metabolizing a wide variety of substrates including hexoses and pentoses, its hexose metabolism is inferior to that of Zymomonas mobilis, an obligate, ethanologenic bacterium. We therefore inserted and expressed Z. mobilis genes encoding essential enzymes involved in the fermentation pathway, alcohol dehydrogenase II (adh II) and pyruvate decarboxylase (pdc), into E. coli, resulting in increased cell growth and ethanol production. Ethanol concentrations of > 30 g/L were obtained on 10% glucose. Additionally, since pyruvate is mainly assimilated through pyruvate formate lyase (pfl) and forms formic acid and acetyl coenzyme A, metabolic redirection was attempted through gene knockout by Red-mediated recombination to decrease the byproducts of pyruvate metabolism. Under microaerobic conditions, pflA- and pflB-mutants produced more ethanol (163% and 207%, respectively) relative to the parent strain, using glucose as a carbon source.