Microbial conversion of glycerol: present status and future prospects

Biodiesel has emerged as a potential alternate renewable liquid fuel in the past two decades. Total annual production of biodiesel stands at 6.96 million tons and 11.2 million tons in USA and Europe, respectively. In other countries, Asia and Latin America, biodiesel production has increased at unprecedented rate. Despite this, the economy of biodiesel is not attractive. An obvious solution for boosting the economy of the biodiesel industry is to look for markets for side products of the transesterification process of biodiesel synthesis. The main by-product is glycerol. However, this glycerol is contaminated with alkali/acid catalyst and alcohol, and thus, is not useful for conventional applications such as in toothpaste, drugs, paints and cosmetics. Conversion of this glycerol to value-added product is a viable solution for effective and economic utilization, which would also generate additional revenue for the biodiesel industry. Intensive research has taken place in area of conversion of glycerol to numerous products. The conventional catalytic route of glycerol transformation employs prohibitively harsh conditions of temperature and pressure, and thus, has slim potential for large-scale implementation. In addition, the selectivity of the process is rather small with formation of many undesired side products. The bioconversion processes, on the other hand, are highly selective although with slower kinetics. In this review, we have given an assessment and overview of the literature on bioconversion of glycerol. We have assessed as many as 23 products from glycerol bioconversion, and have reviewed the literature in terms of microorganism used, mode of fermentation, type of fermentor, yield and productivity of the process and recovery/purification of the products. The metabolic pathway of conversion of glycerol to various products has been discussed. We have also pondered over economic and engineering issues of large-scale implementation of process and have outlined the constraints and limitations of the process. We hope that this review will be a useful source of information for biochemists, biotechnologists, microbiologists and chemical engineers working in the area of glycerol bioconversion.     

Anaerobic fermentation of glycerol: a path to economic viability for the biofuels industry

Although biofuels such as biodiesel and bioethanol represent a secure, renewable and environmentally safe alternative to fossil fuels, their economic viability is a major concern. The implementation of biorefineries that co-produce higher value products along with biofuels has been proposed as a solution to this problem. The biorefinery model would be especially advantageous if the conversion of byproducts or waste streams generated during biofuel production were considered. Glycerol-rich streams generated in large amounts by the biofuels industry, especially during the production of biodiesel, present an excellent opportunity to establish biorefineries. Once considered a valuable 'co-product', crude glycerol is rapidly becoming a 'waste product' with a disposal cost attributed to it. Given the highly reduced nature of carbon in glycerol and the cost advantage of anaerobic processes, fermentative metabolism of glycerol is of special interest. This review covers the anaerobic fermentation of glycerol in microbes and the harnessing of this metabolic process to convert abundant and low-priced glycerol streams into higher value products, thus creating a path to viability for the biofuels industry. Special attention is given to products whose synthesis from glycerol would be advantageous when compared with their production from common sugars.     

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.     

Current Prospects on Production of Microbial Lipid and Other Value-Added Products Using Crude Glycerol Obtained from Biodiesel Industries

Production of microbial lipids using crude glycerol from the biodiesel industry is reviewed in this paper. Approximately 10 wt.% of crude glycerol is obtained for every batch of biodiesel. The crude glycerol accumulated contains various impurities and hence cannot be used for any commercial applications without further purification. Its conversion via biological and chemical routes into valuable products has been studied by different researchers. Varieties of fungal, yeasts, and algal species have been used to produce microbial lipids from crude glycerol. However, research focus on screening a robust industrial oleaginous strain capable of doing this is still on-going. Due to its chemical similarity to vegetable oils, microbial lipids are considered a potential renewable feedstock for biodiesel production and for applications in food and pharmaceutical industries. Its conversion to polyols and subsequently to biobased polymers is also being explored. The rising price of vegetable oils, increasing energy demands, growing environmental concerns, and availability of crude glycerol as a cheap carbon substrate result in considerable potential for the application of these processes in the future.     

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.     

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.     

Pyrolysis of glycerol for the production of hydrogen or syn gas

Biodiesel has high potential as alternative liquid transportation fuel because it is renewable and CO(2) neutral, and has similar properties as diesel fuel. Glycerol is a by-product obtained during the production of biodiesel. Canadian government has planned to produce 500 million litres of biodiesel by 2010. An increase in biodiesel production would decrease the market price of glycerol. The objective of this study is to pyrolyse glycerol for the production of clean fuels such as H(2) or a feedstock such as syn gas for additional transportation fuel via Fisher-Tropsch synthesis. The pyrolysis of glycerol was carried out at various flow rates of N(2) (30-70 mL/min), temperatures (650-800 degrees C) and types and sizes of packing material in a tubular reactor at atmospheric pressure. The products were mostly gas, essentially consisting of CO, H(2), CO(2), CH(4) and C(2)H(4). It was observed that temperature, carrier flow rates and particle diameter of packing material had profound effects on the conversion of glycerol as well as product distribution. Composition of product gas ranged between syn gas 70-93 mol%, CH(4) 3-15 mol% and C(2)H(4) 2-12 mol% and heating value ranged from 13 to 22 MJ/m(3). This study indicates that the bio-glycerol has potential in making syn gas and medium heating value gases.     

An overview of enzymatic production of biodiesel

Biodiesel production has received considerable attention in the recent past as a biodegradable and nonpolluting fuel. The production of biodiesel by transesterification process employing alkali catalyst has been industrially accepted for its high conversion and reaction rates. Recently, enzymatic transesterification has attracted much attention for biodiesel production as it produces high purity product and enables easy separation from the byproduct, glycerol. But the cost of enzyme remains a barrier for its industrial implementation. In order to increase the cost effectiveness of the process, the enzyme (both intracellular and extracellular) is reused by immobilizing in a suitable biomass support particle and that has resulted in considerable increase in efficiency. But the activity of immobilized enzyme is inhibited by methanol and glycerol which are present in the reacting mixture. The use of tert-butanol as solvent, continuous removal of glycerol, stepwise addition of methanol are found to reduce the inhibitory effects thereby increasing the cost effectiveness of the process. The present review analyzes these methods reported in literature and also suggests a suitable method for commercialization of the enzymatic process.     

Biofuels in China: opportunities and challenges

With rapid economic development, energy consumption in China has tripled in the past 20 yr, exceeding 2.4 billion tons of standard coal in 2006. The search for new green energy as substitutes for the nonrenewable energy resources has become an urgent task. China has a variety of climates and is rich in potential biofuel plant species. Corn and cassava are used as the main raw materials for bioethanol production in China. At the end of 2005, bioethanol productivity had increased to 1.02 million tons produced by four companies, and bioethanol-blended petrol accounted for 20% of the total petrol consumption in China. According to the Mid- and Long-term Development Plan for Renewable Energy, the consumption of biodiesel in China will reach 0.2 million tons in 2010 and 2.0 million tons in 2020. This review is intended to provide an introduction to the distribution and development of biofuel crops and biofuel industry in China.     

Integrated production for biodiesel and 1,3-propanediol with lipase-catalyzed transesterification and fermentation

Biodiesel, a renewable alternative to fossil energy, has shown great prospects for global proliferation in the past decade. Lipase catalyzed transesterification for biodiesel production, as a biological process with many advantages has drawn increasing attention. As a by-product, glycerol accounts for about 10% w/w of biodiesel during the process of biodiesel production. As a result, the conversion of glycerol has become a common problem which has to be resolved if considering large amount of biodiesel production. Glycerol can be fermented into 1,3-propanediol, a high value added chemical with a promising future in the polymers, for example, polytrimethylene terephthalate, and also fermentation approaches for 1,3-propanediol production which have drawn more and more attention due to advantages such as relatively low investment, mild reaction conditions and using renewable sources as the starting materials. Based on the latest technology advancements in lipase-mediated transformation for biodiesel production, the aerobic fermentation technology and genetic engineering for 1,3-propanediol production, and the integrated production of 1,3-propanediol from crude glycerol could be a promising way to improve the profit of the whole process during biodiesel production.     

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.     

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).     

Energy recovery from lipid extracted,transesterified and glycerol codigested microalgae biomass

The production of methane (CH₄) via the anaerobic digestion of microalgae biomass residues from the biodiesel production process has the potential to meet some of the energy requirements of the primary biomass to fuel conversion process. This paper investigates the practical CH₄ yields achievable from the anaerobic conversion of the microalgae residues (as well as codigestion with glycerol) after biodiesel production using both the conventional and in situ transesterification methods. Results demonstrate that the type of lipid extraction solvent utilized in the conventional transesterification process could inhibit subsequent CH₄ production. On the basis of actual CH₄ production, a recoverable energy of 8.7–10.5 MJ kg^?1 of dry microalgae biomass residue was obtained using the lipid extracted and transesterified microalgae samples. On codigesting the microalgae residues with glycerol, a 4–7% increase in CH₄ production was observed.     

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.     

Impurities of crude glycerol and their effect on metabolite production

Glycerol is a valuable raw material for the production of industrially useful metabolites. Among many promising applications for the use of glycerol is its bioconversion to high value-added compounds, such as 1,3-propanediol (1,3-PD), succinate, ethanol, propionate, and hydrogen, through microbial fermentation. Another method of waste material utilization is the application of crude glycerol in blends with other wastes (e.g., tomato waste hydrolysate). However, crude glycerol, a by-product of biodiesel production, has many impurities which can limit the yield of metabolites. In this mini-review we summarize the effects of crude glycerol impurities on various microbial fermentations and give an overview of the metabolites that can be synthesized by a number of prokaryotic and eukaryotic microorganisms when cultivated on glycerol.     

Enhancing biodiesel production by immobilized whole cells by optimizing reaction conditions and adding glycerol and water

In this study, several methods were devised and evaluated to enhance biodiesel production by whole cells immobilized onto the polyurethane foam coated with activated carbon. Biodiesel conversion was increased to 76.4% with the increase in the number of polyurethane foam until it occupied 18.0 or 2.4% of reaction mixture based on apparent or actual volume of supports, respectively. Stepwise methanol addition to prevent methanol inhibition on the immobilized whole cells was optimized in terms of number of aliquot and feeding interval. When 4.5 molar ratio of methanol to soybean oil was divided into 4 equal aliquots and each aliquot was fed to the reaction mixture every 24 h, the highest final biodiesel conversion of 82.4% was achieved. Chemical treatment of the immobilized cells with 0.1% of chloroform for 2 h enhanced biodiesel conversion to 90.5%. The initial addition of 5% glycerol in the fresh reaction mixture increased biodiesel conversion to 90.3% while the removal of glycerol during biodiesel production barely increased biodiesel conversion. The biodiesel conversion was increased with the increase of initial water content in the fresh reaction mixture and the highest value was 92.7% at 3.0% of water content, but decreased thereafter. The effects of co-addition of glycerol and water on biodiesel production were also investigated, and the co-addition of 3.125% of glycerol and 1.875% of water relative to soybean oil substantially increased biodiesel conversion to 95.0%. By these optimization of reaction conditions and co-adding glycerol and water, initial biodiesel production rate and final biodiesel conversion were remarkably enhanced by 26.8 and 24.1%, respectively.     

Process design and evaluation of value-added chemicals production from biomass

Three different biodiesel production processes were simulated using the SuperPro Designer program. The process for producing biodiesel from soybean oil and methanol was designed using commercial chemical catalysts. This chemical process was compared with the biological process catalyzed by immobilized enzymes. In addition, a hybrid process consisting of catalytic biodiesel production and enzymatic glycerol carbonate production was designed and simulated for the conversion of waste glycerol to value-added chemical. Finally, the economics and productivity of these processes were evaluated to determine economic feasibility.     

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.     

Bioconversion of lignocellulose in solid substrate fermentation

In this review the state of the art of lignocellulose bioconversion by solid substrate fermentation (SSF) is presented. The most important lignocellulolytic fungi and their properties are described, and their application in novel solid state bioreactors with on-line process control is discussed. The most important bioconversion products, biofuels, enzymes, animal feeds, biofertilizers, biopesticides, biopromoters, secondary metabolites, and the economy of their production by SSF is discussed. The use of SSF in the pulp and paper industry and in integrated crop management is illustrated.     

Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae

Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process.