Developing microbial biofilm as a robust biocatalyst and its challenges

Biocatalysts, such as bacteria, yeast, fungi and the enzymes they produce, have been used for many industrial applications since they function as effective and environmentally friendly tools. Whole cells have also been used in many sophisticated bioprocesses since a number of sequential reactions can be catalyzed within the cells. However, the use of whole cells in suspension in batch, fed-batch and continuous processes has some limitations. For instance, the cultures are non-reusable, they are sometimes sensitive to the toxicity of substrates or products, there can be issues with short-term stability, and each of these issues can impede biocatalyst regeneration, perturbing the downstream process and causing complexity in running large scale continuous culture. Recently, biofilms have emerged as a new generation of biocatalysts to solve these limitations in the production of many bio-based materials, including chemicals, antibiotics, enzymes, bioethanol, biohydrogen, and electricity production via microbial fuel cells. The establishment of industrial processes using biofilms has the potential for high benefit in terms of low-cost cell immobilization without the necessity of added polymers or chemicals. Many small-scale biofilm reactors have been developed for the production of value-added products, and it may be challenging to establish it on an industrial scale.     

Whole-cell biocatalysts for biodiesel fuel production

Biodiesel fuel (BDF), which refers to fatty acid alkyl esters, has attracted considerable attention as an environmentally friendly alternative fuel for diesel engines. Alkali catalysis is widely applied for the commercial production of BDF. However, enzymatic transesterification offers considerable advantages, including reducing process operations in biodiesel fuel production and an easy separation of the glycerol byproduct. The high cost of the lipase enzyme is the main obstacle for a commercially feasible enzymatic production of biodiesel fuels. To reduce enzyme associated process costs, the immobilization of fungal mycelium within biomass support particles (BSPs) as well as expression of the lipase enzyme on the surface of yeast cells has been developed to generate whole-cell biocatalysts for industrial applications.     

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.     

Use of ceramic-based cell immobilization to produce 1,3-propanediol from biodiesel-derived waste glycerol with Klebsiella pneumoniae

Aims: The feasibility of the continuous production of a valuable bioplastic raw material, namely 1,3‐propanediol (1,3‐PDO) from biodiesel by‐product glycerol, using immobilized cells was investigated. In addition, the effect of hydraulic retention time (HRT) was also analysed. Methods and Results: Ceramic balls and ceramic rings were used for the immobilization of a locally isolated strain; Klebsiella pneumoniae (GenBank no. 27F HM063413 ). HRT of 1 h is the best one in terms of volumetric production rate (g 1,3‐PDO l^?1 h^?1). The results indicated that ceramic‐based cell immobilization achieved a 2‐fold higher production rate (10 g 1,3‐PDO l^?1 h^?1) in comparison with suspended cell system (4·9 g 1,3‐PDO l^?1 h^?1). Conclusions: Continuous cultures with immobilized cells revealed that 1,3‐PDO production was more effective and more stable than suspended culture systems. Furthermore, cell immobilization had also obvious benefits especially for resistance of the production for extreme conditions (high organic loading rates, cell washouts). The results were important for understanding the significance of continuous immobilization process among other well‐known 1,3‐PDO fermentation processes. Significance and Impact of the Study: This work is a promising process for further studies, as the immobilized micro‐organism was able to reach high volumetric production rates at short HRT, it has an important role in tolerating and converting glycerol during fermentation. Therefore, HRT is a very significant operational parameter (P value <0·05) directly affecting the bioreactor performance and production rate.     

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.     

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.     

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.     

Progress in biocatalysis with immobilized viable whole cells: systems development,reaction engineering and applications

Viable microbial cells are important biocatalysts in the production of fine chemicals and biofuels, in environmental applications and also in emerging applications such as biosensors or medicine. Their increasing significance is driven mainly by the intensive development of high performance recombinant strains supplying multienzyme cascade reaction pathways, and by advances in preservation of the native state and stability of whole-cell biocatalysts throughout their application. In many cases, the stability and performance of whole-cell biocatalysts can be highly improved by controlled immobilization techniques. This review summarizes the current progress in the development of immobilized whole-cell biocatalysts, the immobilization methods as well as in the bioreaction engineering aspects and economical aspects of their biocatalytic applications.     

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2 h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.     

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.     

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.     

Biotechnological production of biodiesel fuel using biocatalysed transesterification: A review

Biotechnological production of biodiesel has attracted considerable attention during the past decade compared to chemical-catalysed production since biocatalysis-mediated transesterification has many advantages. Currently, there are extensive reports on enzyme-catalysed transesterification for biodiesel production; the related research can be classified into immobilised-extracellular and immobilised-intracellular biocatalysis and this review focusses on these forms of biocatalyst for biodiesel production. The optimisation of the most important operating conditions affecting lipase-catalysed transesterification and the yield of alkyl esters, such as the type and form of lipase, the type of alcohol, the presence of organic solvents, the content of water in the oil, temperature and the presence of glycerol, are discussed. However, there is still a need to optimise lipase-catalysed transesterification and reduce the cost of lipase production before it is applied commercially. Optimisation research of lipase-catalysed transesterification could include development of new reactor systems with immobilised biocatalysts, the use of lipases tolerant to organic solvents, intracellular lipases (whole microbial cells) and genetically modified microorganisms (intelligent yeasts). Biodiesel fuel is expensive in comparison with petroleum-based fuel and 60–70% of the cost is associated with feedstock oil and enzyme. Therefore ways of reducing the cost of biodiesel with respect to enzyme and substrate oils reported in literature are also presented.     

Cell immobilization for microbial production of 1,3-propanediol

Cell and enzyme immobilization are often used for industrial production of high-value products. In recent years, immobilization techniques have been applied to the production of value-added chemicals such as 1,3-Propanediol (1,3-PDO). Biotechnological fermentation is an attractive alternative to current 1,3-PDO production methods, which are primarily thermochemical processes, as it generates high volumetric yields of 1,3-PDO, is a much less energy intensive process, and generates lower amounts of environmental organic pollutants. Although several approaches including: batch, fed-batch, continuous-feed and two-step continuous-feed were tested in suspended systems, it has been well demonstrated that cell immobilization techniques can significantly enhance 1,3-PDO production and allow robust continuous production in smaller bioreactors. This review covers various immobilization methods and their application for 1,3-PDO production.     

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.     

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.     

Anaerobic fermentation of glycerol by Escherichia coli: a new platform for metabolic engineering

The worldwide surplus of glycerol generated as inevitable byproduct of biodiesel fuel and oleochemical production is resulting in the shutdown of traditional glycerol-producing/refining plants and new applications are needed for this now abundant carbon source. In this article we report our finding that Escherichia coli can ferment glycerol in a pH-dependent manner. We hypothesize that glycerol fermentation is linked to the availability of CO(2), which under acidic conditions is produced by the oxidation of formate by the enzyme formate hydrogen lyase (FHL). In agreement with this hypothesis, glycerol fermentation was severely impaired by blocking the activity of FHL. We demonstrated that, unlike CO(2), hydrogen (the other product of FHL-mediated formate oxidation) had a negative impact on cell growth and glycerol fermentation. In addition, supplementation of the medium with CO(2) partially restored the ability of an FHL-deficient strain to ferment glycerol. High pH resulted in low CO(2) generation (low activity of FHL) and availability (most CO(2) is converted to bicarbonate), and consequently very inefficient fermentation of glycerol. Most of the fermented glycerol was recovered in the reduced compounds ethanol and succinate (93% of the product mixture), which reflects the highly reduced state of glycerol and confirms the fermentative nature of this process. Since glycerol is a cheap, abundant, and highly reduced carbon source, our findings should enable the development of an E. coli-based platform for the anaerobic production of reduced chemicals from glycerol at yields higher than those obtained from common sugars, such as glucose.     

Application of a Burkholderia cepacia lipase-immobilized silica monolith to batch and continuous biodiesel production with a stoichiometric mixture of methanol and crude Jatropha oil

Background

The enzymatic production of biodiesel through alcoholysis of triglycerides has become more attractive because it shows potential in overcoming the drawbacks of chemical processes. In this study, we investigate the production of biodiesel from crude, non-edible Jatropha oil and methanol to characterize Burkholderia cepacia lipase immobilized in an n-butyl-substituted hydrophobic silica monolith. We also evaluate the performance of a lipase-immobilized silica monolith bioreactor in the continuous production of biodiesel.

Results

The Jatropha oil used contained 18% free fatty acids, which is problematic in a base-catalyzed process. In the lipase-catalyzed reaction, the presence of free fatty acids made the reaction mixture homogeneous and allowed bioconversion to proceed to 90% biodiesel yield after a 12 hour reaction time. The optimal molar ratio of methanol to oil was 3.3 to 3.5 parts methanol to one part oil, with water content of 0.6% (w/w). Further experiments revealed that B. cepacia lipase immobilized in hydrophobic silicates was sufficiently tolerant to methanol, and glycerol adsorbed on the support disturbed the reaction to some extent in the present reaction system. The continuous production of biodiesel was performed at steady state using a lipase-immobilized silica monolith bioreactor loaded with 1.67 g of lipase. The yield of 95% was reached at a flow rate of 0.6 mL/h, although the performance of the continuous bioreactor was somewhat below that predicted from the batch reactor. The bioreactor was operated successfully for almost 50 days with 80% retention of the initial yield.

Conclusions

The presence of free fatty acids originally contained in Jatropha oil improved the reaction efficiency of the biodiesel production. A combination of B. cepacia lipase and its immobilization support, n-butyl-substituted silica monolith, was effective in the production of biodiesel. This procedure is easily applicable to the design of a continuous flow-through bioreactor system.     

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.     

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.     

酶法合成生物柴油工业化研究进展

介绍了北京化工大学近年来酶法合成生物柴油工业化研究的结果。主要内容包括以下几个方面:高产脂肪酶菌株的选育、脂肪酶发酵工艺优化及放大、脂肪酶固定化方法、酶反应器放大、生物柴油分离精制及副产物甘油综合利用。该脂肪酶假丝酵母Candida sp.99-125在5 m3罐发酵活力不低于8 000 IU/mL,然后将该脂肪酶吸附固定在织物膜上并进行表面改性,用于搅拌罐式反应器生产每吨甲酯的需酶量仅为4.2 kg,产品经分离精制调质后,其各项指标完全符合德国生物柴油生产标准。副产物甘油可用于1,3-丙二醇发酵,30 L发酵罐中1,3-丙二醇的产量可达到76.1 g/L。