Sequencing of multiple clostridial genomes related to biomass conversion and biofuel production

Modern methods to develop microbe-based biomass conversion processes require a system-level understanding of the microbes involved. Clostridium species have long been recognized as ideal candidates for processes involving biomass conversion and production of various biofuels and other industrial products. To expand the knowledge base for clostridial species relevant to current biofuel production efforts, we have sequenced the genomes of 20 species spanning multiple genera. The majority of species sequenced fall within the class III cellulosome-encoding Clostridium and the class V saccharolytic Thermoanaerobacteraceae. Species were chosen based on representation in the experimental literature as model organisms, ability to degrade cellulosic biomass either by free enzymes or by cellulosomes, ability to rapidly ferment hexose and pentose sugars to ethanol, and ability to ferment synthesis gas to ethanol. The sequenced strains significantly increase the number of noncommensal/nonpathogenic clostridial species and provide a key foundation for future studies of biomass conversion, cellulosome composition, and clostridial systems biology.     

Modeling the self-assembly of the cellulosome enzyme complex

Most bacteria use free enzymes to degrade plant cell walls in nature. However, some bacteria have adopted a different strategy wherein enzymes can either be free or tethered on a protein scaffold forming a complex called a cellulosome. The study of the structure and mechanism of these large macromolecular complexes is an active and ongoing research topic, with the goal of finding ways to improve biomass conversion using cellulosomes. Several mechanisms involved in cellulosome formation remain unknown, including how cellulosomal enzymes assemble on the scaffoldin and what governs the population of cellulosomes created during self-assembly. Here, we present a coarse-grained model to study the self-assembly of cellulosomes. The model captures most of the physical characteristics of three cellulosomal enzymes (Cel5B, CelS, and CbhA) and the scaffoldin (CipA) from Clostridium thermocellum. The protein structures are represented by beads connected by restraints to mimic the flexibility and shapes of these proteins. From a large simulation set, the assembly of cellulosomal enzyme complexes is shown to be dominated by their shape and modularity. The multimodular enzyme, CbhA, binds statistically more frequently to the scaffoldin than CelS or Cel5B. The enhanced binding is attributed to the flexible nature and multimodularity of this enzyme, providing a longer residence time around the scaffoldin. The characterization of the factors influencing the cellulosome assembly process may enable new strategies to create designers cellulosomes.     

Cellulase, clostridia, and ethanol.

Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.     

Cellulase, Clostridia, and Ethanol

Biomass conversion to ethanol as a liquid fuel by the thermophilic and anaerobic clostridia offers a potential partial solution to the problem of the world's dependence on petroleum for energy. Coculture of a cellulolytic strain and a saccharolytic strain of Clostridium on agricultural resources, as well as on urban and industrial cellulosic wastes, is a promising approach to an alternate energy source from an economic viewpoint. This review discusses the need for such a process, the cellulases of clostridia, their presence in extracellular complexes or organelles (the cellulosomes), the binding of the cellulosomes to cellulose and to the cell surface, cellulase genetics, regulation of their synthesis, cocultures, ethanol tolerance, and metabolic pathway engineering for maximizing ethanol yield.     

Bioconversion to single cell protein: A potential resource recovery from paper mill solid waste

Disposal of paper mill waste by bioconversion is a novel approach which allows a direct conversion of cellulosic solid waste into fungal biomass. A cellulolytic microorganism Myrothecium verrucaria has been used for protein production under batch conditions in flasks. Biomass yield of 0.375 g per g of substrate consumed with 50–60% solid waste usage was observed. The nutritional quality of the biomass in terms of fat and protein contents shows promise for feed supplementation of monogastric animals.     

Lignin Structural Alterations in Thermochemical Pretreatments with Limited Delignification

Lignocellulosic biomass has a complex and rigid cell wall structure that makes biomass recalcitrant to biological and chemical degradation. Among the three major structural biopolymers (i.e., cellulose, hemicellulose, and lignin) in plant cell walls, lignin is considered the most recalcitrant component and generally plays a negative role in the biochemical conversion of biomass to biofuels. The conversion of biomass to biofuels through a biochemical platform usually requires a pretreatment stage to reduce the recalcitrance. Pretreatment renders compositional and structural changes of biomass with these changes ultimately governing the efficiency of the subsequent enzymatic hydrolysis. Dilute acid, hot water, steam explosion, and ammonia fiber expansion pretreatments are among the leading thermochemical pretreatments with a limited delignification that can reduce biomass recalcitrance. Practical applications of these pretreatment are rapidly developing as illustrated by recent commercial scale cellulosic ethanol plants. While these thermochemical pretreatments generally lead to only a limited delignification and no significant change of lignin content in the pretreated biomass, the lignin transformations that occur during these pretreatments and the roles they play in recalcitrance reduction are important research aspects. This review highlights recent advances in our understanding of lignin alterations during these limited delignification thermochemical pretreatments, with emphasis on lignin chemical structures, molecular weights, and redistributions in the pretreated biomass.     

The biofuel potential of municipal solid waste

The world in the 21st century is facing a dual crisis of increasing waste and global climate change. Substituting fossil fuels with waste biomass‐derived cellulosic ethanol is a promising strategy to simultaneously meet part of our energy needs, mitigate greenhouse gas (GHG) emissions, and manage municipal solid waste (MSW). However, the global potential of MSW as an energy source is as yet unquantified. Here, we report increasing trends of MSW generation, and waste biomass‐derived cellulosic ethanol potentials in relation to socio‐economic development across 173 countries, and show that globally, up to 82.9 billion litres of waste paper‐derived cellulosic ethanol can be produced worldwide, replacing 5.36% of gasoline consumption, with accompanying GHG emissions savings of between 29.2% and 86.1%.     

Bioconversion of lignocellulosic biomass: biochemical and molecular perspectives

In view of rising prices of crude oil due to increasing fuel demands, the need for alternative sources of bioenergy is expected to increase sharply in the coming years. Among potential alternative bioenergy resources, lignocellulosics have been identified as the prime source of biofuels and other value-added products. Lignocelluloses as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world. To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary. A variety of microorganisms including bacteria and fungi may have the ability to degrade the cellulosic biomass to glucose monomers. Bacterial cellulases exist as discrete multi-enzyme complexes, called cellulosomes that consist of multiple subunits. Cellulolytic enzyme systems from the filamentous fungi, especially Trichoderma reesei, contain two exoglucanases or cellobiohydrolases (CBH1 and CBH2), at least four endoglucanases (EG1, EG2, EG3, EG5), and one β-glucosidase. These enzymes act synergistically to catalyse the hydrolysis of cellulose. Different physical parameters such as pH, temperature, adsorption, chemical factors like nitrogen, phosphorus, presence of phenolic compounds and other inhibitors can critically influence the bioconversion of lignocellulose. The production of cellulases by microbial cells is governed by genetic and biochemical controls including induction, catabolite repression, or end product inhibition. Several efforts have been made to increase the production of cellulases through strain improvement by mutagenesis. Various physical and chemical methods have been used to develop bacterial and fungal strains producing higher amounts of cellulase, all with limited success. Cellulosic bioconversion is a complex process and requires the synergistic action of the three enzymatic components consisting of endoglucanases, exoglucanases and β-glucosidases. The co-cultivation of microbes in fermentation can increase the quantity of the desirable components of the cellulase complex. An understanding of the molecular mechanism leading to biodegradation of lignocelluloses and the development of the bioprocessing potential of cellulolytic microorganisms might effectively be accomplished with recombinant DNA technology. For instance, cloning and sequencing of the various cellulolytic genes could economize the cellulase production process. Apart from that, metabolic engineering and genomics approaches have great potential for enhancing our understanding of the molecular mechanism of bioconversion of lignocelluloses to value added economically significant products in the future. JIMB 2008: BioEnergy - Special issue.     

利用废弃物原料生产燃料乙醇的研究进展

乙醇是一种十分重要的工业用途的化工原料。目前国内外学者纷纷采用不同的方法和手段对乙醇发酵进行研究,目前,利用废弃物为原料生产乙醇是热点。本文阐述了利用各种废弃原料生产乙醇的必要性,并分别论述了利用纤维质废弃物、淀粉质废弃物、糖质废弃物等生产乙醇的研究进展,着重论述了利用纤维质废弃物的生产情况,提出了需进一步研究和解决的问题。     

米根霉利用木糖与葡萄糖的代谢差异

[目的]木质纤维素是世界上储量最丰富、最廉价的可再生生物质资源,以米根霉为研究对象,探讨对木质纤维素中主要单糖成分--木糖和葡萄糖的代谢差异,为木质纤维素的高效利用提供科学依据.[方法]分别以木糖和葡萄糖为碳源,考察米根霉的生物量、细胞大分子组分、胞内还原力(NADH/NAD+)、ATP含量以及有机酸积累的差异.[结果...     

Inhibition of enzymatic cellulolysis by phenolic compounds

Phenolics derived from lignin and other plant components can pose significant inhibition on enzymatic conversion of cellulosic biomass materials to useful chemicals. Understanding the mechanism of such inhibition is of importance for the development of viable biomass conversion technologies. In native plant cell wall, most of the phenolics and derivatives are found in polymeric lignin. When biomass feedstocks are pretreated (prior to enzymatic hydrolysis), simple or oligomeric phenolics and derivatives are often generated from lignin modification/degradation, which can inhibit biomass-converting enzymes. To further understand how such phenolic substances may affect cellulase reaction, we carried out a comparative study on a series of simple and oligomeric phenolics representing or mimicking the composition of lignin or its degradation products. Consistent to previous studies, we observed that oligomeric phenolics could exert more inhibition on enzymatic cellulolysis than simple phenolics. Oligomeric phenolics could inactivate cellulases by reversibly complexing them. Simple and oligomeric phenolics could also inhibit enzymatic cellulolysis by adsorbing onto cellulose. Individual cellulases showed different susceptibility toward these inhibitions. Polyethylene glycol and tannase could respectively bind and degrade the studied oligomeric phenolics, and by doing so mitigate the oligomeric phenolic's inhibition on cellulolysis.     

Reactor scale up for biological conversion of cellulosic biomass to ethanol

The absence of a systematic scale-up approach for biological conversion of cellulosic biomass to commodity products is a significant bottleneck to realizing the potential benefits offered by such conversion. Motivated by this, we undertook to develop a scale-up approach for conversion of waste paper sludge to ethanol. Physical properties of the system were measured and correlations were developed for their dependence upon cellulose conversion. Just-suspension of solid particles was identified as the scale up criterion based on experiments at lab scale. The impeller speed for just solids suspension at large scale was predicted using computational fluid dynamics simulations. The scale-up strategy was validated by analyzing mixing requirements such as solid–liquid mass transfer under the predicted level of agitation at large scale. The scale-up approach enhances the prediction of reactor performance and helps provide guidelines for the analysis and design of large scale bioreactors based on bench scale experimentation.     

Effect of Maize Biomass Composition on the Optimization of Dilute-Acid Pretreatments and Enzymatic Saccharification

At the core of cellulosic ethanol research are innovations leading to reductions in the chemical and energetic stringency of thermochemical pretreatments and enzymatic saccharification. In this study, key compositional features of maize cell walls influencing the enzymatic conversion of biomass into fermentable sugars were identified. Stem samples from eight contrasting genotypes were subjected to a series of thermal dilute-acid pretreatments of increasing severity and evaluated for glucose release after enzymatic saccharification. The biochemically diverse set of genotypes displayed significant differences in glucose yields at all processing conditions evaluated. The results revealed that mechanisms controlling biomass conversion efficiency vary in relation to pretreatment severity. At highly severe pretreatments, cellulose conversion efficiency was primarily influenced by the inherent efficacy of the thermochemical process, and maximum glucose yields were obtained from cellulosic feedstocks harboring the highest cellulose contents per dry gram of biomass. When mild dilute-acid pretreatments were applied, however, maximum bioconversion efficiency and glucose yields were observed for genotypes combining high stem cellulose contents, reduced cell wall lignin and highly substituted hemicelluloses. For the best-performing genotype, glucose yields under sub-optimal processing regimes were only 10 % lower than the genotype-set mean at the most stringent processing conditions evaluated, while furfural production was reduced by approximately 95 %. Our results ultimately established that cellulosic feedstocks with tailored cell wall compositions can help reduce the chemical and energetic intensity of pretreatments used in the industry and improve the commercial and environmental performance of biomass-to-ethanol conversion technologies.     

Genetic improvement of C4 grasses as cellulosic biofuel feedstocks

C4 grasses are among the most productive plants and most promising cellulosic biofuel feedstocks. Successful implementation of cellulosic biofuel feedstocks will depend on the improvement of critical crop characteristics and subsequent conversion technologies. The content and composition of lignin, cellulose, and hemicellulose, their biomass yields, and their biotic and abiotic stress tolerances are critical factors which can be enhanced by molecular breeding methods, including marker-assisted selection and transgenic approaches. To maximize biomass yield, no flowering or late flowering and no grain set would be ideal for cellulosic biofuel crops. Reducing fecundity also reduces the risk of undesired gene transfer and invasiveness, thus accelerating deregulation processes and permitting faster implementation of highly improved genotypes in cellulosic feedstock production.     

Genetic modification of lignin biosynthesis for improved biofuel production

The energy in cellulosic biomass largely resides in plant cell walls. Cellulosic biomass is more difficult than starch to break down into sugars because of the presence of lignin and the complex structure of cell walls. Transgenic down-regulation of major lignin genes led to reduced lignin content, increased dry matter degradability, and improved accessibility of cellulases for cellulose degradation. This review provides background information on lignin biosynthesis and focuses on genetic manipulation of lignin genes in important monocot species as well as the dicot potential biofuel crop alfalfa. Reduction of lignin in biofuel crops by genetic engineering is likely one of the most effective ways of reducing costs associated with pretreatment and hydrolysis of cellulosic feedstocks, although some potential fitness issues should also be addressed.     

The cellulose paradox: pollutant par excellence and/or a reclaimable natural resource?

The various aspects of cellulose as a pollutant are considered in view of its lack of toxicity on the one hand and its recalcitrant durable nature on the other. The microbial degradation of cellulosics is discussed, and the contrast between its success in handling natural cellulosic wastes versus its failure to cope with man-made refuse is described. Research carried out in the past decade has demonstrated that cellulolytic organisms are provided with cell surface multifunctional multienzyme conglomerates, called cellulosomes, which are capable of solubilizing solid cellulosic substrates. The intriguing properties of such complexes include their cohesive nature, their many enzymatic components, and a characteristic glycosylated cellulose-binding, scaffolding component. The latter appears to serve as a substrate-targeting carrier, which delivers the other (hydrolytic) components to the cellulose. Progress in establishing efficient model systems for in vitro solubilization of purified cellulose or natural cellulosic substrates has been achieved using purified cellulosome preparations, fortified with -glucosidase and pectinase. The latter enzymes were required in order to alleviate the phenomenon of product inhibition which reduces the efficiency of the free cellulosome. Such combined enzyme systems are proposed as examples of future tailor-made cellulolytic systems for the degradation of natural cellulosics.     

Rapid adaptation for fibre degradation by changes in plasmid stoichiometry within Lactobacillus plantarum at the synthetic community level

The multi-enzyme cellulosome complex can mediate the valorization of lignocellulosic biomass into soluble sugars that can serve in the production of biofuels and valuable products. A potent bacterial chassis for the production of active cellulosomes displayed on the cell surface is the bacterium Lactobacillus plantarum, a lactic acid bacterium used in many applications. Here, we developed a methodological pipeline to produce improved designer cellulosomes, using a cell-consortium approach, whereby the different components self-assemble on the surface of L. plantarum. The pipeline served as a vehicle to select and optimize the secretion efficiency of potent designer cellulosome enzyme components, to screen for the most efficient enzymatic combinations and to assess attempts to grow the engineered bacterial cells on wheat straw as a sole carbon source. Using this strategy, we were able to improve the secretion efficiency of the selected enzymes and to secrete a fully functional high-molecular-weight scaffoldin component. The adaptive laboratory process served to increase significantly the enzymatic activity of the most efficient cell consortium. Internal plasmid re-arrangement towards a higher enzymatic performance attested for the suitability of the approach, which suggests that this strategy represents an efficient way for microbes to adapt to changing conditions.     

Consolidated Bioprocessing of Lignocellulosic Feedstocks for Ethanol Fuel Production

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.     

Engineering cellulolytic ability into bioprocessing organisms

Lignocellulosic biomass is an abundant renewable feedstock for sustainable production of commodities such as biofuels. The main technological barrier that prevents widespread utilization of this resource for production of commodity products is the lack of low-cost technologies to overcome the recalcitrance of lignocellulose. Organisms that hydrolyse the cellulose and hemicelluloses in biomass and produce a valuable product such as ethanol at a high rate and titre would significantly reduce the costs of current biomass conversion technologies. This would allow steps that are currently accomplished in different reactors, often by different organisms, to be combined in a consolidated bioprocess (CBP). The development of such organisms has focused on engineering naturally cellulolytic microorganisms to improve product-related properties or engineering non-cellulolytic organisms with high product yields to become cellulolytic. The latter is the focus of this review. While there is still no ideal organism to use in one-step biomass conversion, several candidates have been identified. These candidates are in various stages of development for establishment of a cellulolytic system or improvement of product-forming attributes. This review assesses the current state of the art for enabling non-cellulolytic organisms to grow on cellulosic substrates.