Accumulation of recombinant cellobiohydrolase and endoglucanase in the leaves of mature transgenic sugar cane

A major strategic goal in making ethanol from lignocellulosic biomass a cost-competitive liquid transport fuel is to reduce the cost of production of cellulolytic enzymes that hydrolyse lignocellulosic substrates to fermentable sugars. Current production systems for these enzymes, namely microbes, are not economic. One way to substantially reduce production costs is to express cellulolytic enzymes in plants at levels that are high enough to hydrolyse lignocellulosic biomass. Sugar cane fibre (bagasse) is the most promising lignocellulosic feedstock for conversion to ethanol in the tropics and subtropics. Cellulolytic enzyme production in sugar cane will have a substantial impact on the economics of lignocellulosic ethanol production from bagasse. We therefore generated transgenic sugar cane accumulating three cellulolytic enzymes, fungal cellobiohydrolase I (CBH I), CBH II and bacterial endoglucanase (EG), in leaves using the maize PepC promoter as an alternative to maize Ubi1 for controlling transgene expression. Different subcellular targeting signals were shown to have a substantial impact on the accumulation of these enzymes; the CBHs and EG accumulated to higher levels when fused to a vacuolar-sorting determinant than to an endoplasmic reticulum-retention signal, while EG was produced in the largest amounts when fused to a chloroplast-targeting signal. These results are the first demonstration of the expression and accumulation of recombinant CBH I, CBH II and EG in sugar cane and represent a significant first step towards the optimization of cellulolytic enzyme expression in sugar cane for the economic production of lignocellulosic ethanol.     

Novel enzymes for the degradation of cellulose

ABSTRACT: The bulk terrestrial biomass resource in a future bio-economy will be lignocellulosic biomass, which is recalcitrant and challenging to process. Enzymatic conversion of polysaccharides in the lignocellulosic biomass will be a key technology in future biorefineries and this technology is currently the subject of intensive research. We describe recent developments in enzyme technology for conversion of cellulose, the most abundant, homogeneous and recalcitrant polysaccharide in lignocellulosic biomass. In particular, we focus on a recently discovered new type of enzymes currently classified as CBM33 and GH61 that catalyze oxidative cleavage of polysaccharides. These enzymes promote the efficiency of classical hydrolytic enzymes (cellulases) by acting on the surfaces of the insoluble substrate, where they introduce chain breaks in the polysaccharide chains, without the need of first "extracting" these chains from their crystalline matrix.     

Metagenomic analysis reveals a dynamic microbiome with diversified adaptive functions to utilize high lignocellulosic forages in the cattle rumen

Rumen microbiota play a key role in the digestion and utilization of plant materials by the ruminant species, which have important implications for greenhouse gas emission. Yet, little is known about the key taxa and potential gene functions involved in the digestion process. Here, we performed a genome-centric analysis of rumen microbiota attached to six different lignocellulosic biomasses in rumen-fistulated cattle. Our metagenome sequencing provided novel genomic insights into functional potential of 523 uncultured bacteria and 15 mostly uncultured archaea in the rumen. The assembled genomes belonged mainly to Bacteroidota, Firmicutes, Verrucomicrobiota, and Fibrobacterota and were enriched for genes related to the degradation of lignocellulosic polymers and the fermentation of degraded products into short chain volatile fatty acids. We also found a shift from copiotrophic to oligotrophic taxa during the course of rumen fermentation, potentially important for the digestion of recalcitrant lignocellulosic substrates in the physiochemically complex and varying environment of the rumen. Differential colonization of forages (the incubated lignocellulosic materials) by rumen microbiota suggests that taxonomic and metabolic diversification is an evolutionary adaptation to diverse lignocellulosic substrates constituting a major component of the cattle’s diet. Our data also provide novel insights into the key role of unique microbial diversity and associated gene functions in the degradation of recalcitrant lignocellulosic materials in the rumen.Subject terms: Bacterial genetics, Metagenomics     

Pretreatments to enhance the digestibility of lignocellulosic biomass

Lignocellulosic biomass represents a rather unused source for biogas and ethanol production. Many factors, like lignin content, crystallinity of cellulose, and particle size, limit the digestibility of the hemicellulose and cellulose present in the lignocellulosic biomass. Pretreatments have as a goal to improve the digestibility of the lignocellulosic biomass. Each pretreatment has its own effect(s) on the cellulose, hemicellulose and lignin; the three main components of lignocellulosic biomass. This paper reviews the different effect(s) of several pretreatments on the three main parts of the lignocellulosic biomass to improve its digestibility. Steam pretreatment, lime pretreatment, liquid hot water pretreatments and ammonia based pretreatments are concluded to be pretreatments with high potentials. The main effects are dissolving hemicellulose and alteration of lignin structure, providing an improved accessibility of the cellulose for hydrolytic enzymes.     

Application of next-generation sequencing methods for microbial monitoring of anaerobic digestion of lignocellulosic biomass

The anaerobic digestion of lignocellulosic wastes is considered an efficient method for managing the world’s energy shortages and resolving contemporary environmental problems. However, the recalcitrance of lignocellulosic biomass represents a barrier to maximizing biogas production. The purpose of this review is to examine the extent to which sequencing methods can be employed to monitor such biofuel conversion processes. From a microbial perspective, we present a detailed insight into anaerobic digesters that utilize lignocellulosic biomass and discuss some benefits and disadvantages associated with the microbial sequencing techniques that are typically applied. We further evaluate the extent to which a hybrid approach incorporating a variation of existing methods can be utilized to develop a more in-depth understanding of microbial communities. It is hoped that this deeper knowledge will enhance the reliability and extent of research findings with the end objective of improving the stability of anaerobic digesters that manage lignocellulosic biomass.     

木质纤维素预处理技术研究进展

详细评述了木质纤维素的预处理工艺研究进展,特别是浓酸低温水解-酸回收工艺、稀酸二阶段水解工艺、金属离子在稀酸水解过程中的助催化作用以及水蒸汽爆裂、氨纤维爆裂、CO2爆裂、酶催化水解等方法的研究进展情况。木质纤维素原料预处理技术发展为发酵生产乙醇技术的研究开发奠定了坚实基础。     

Evaluation of industrial Saccharomyces cerevisiae strains as the chassis cell for second-generation bioethanol production

To develop a suitable Saccharomyces cerevisiae industrial strain as a chassis cell for ethanol production using lignocellulosic materials, 32 wild-type strains were evaluated for their glucose fermenting ability, their tolerance to the stresses they might encounter in lignocellulosic hydrolysate fermentation and their genetic background for pentose metabolism. The strain BSIF, isolated from tropical fruit in Thailand, was selected out of the distinctly different strains studied for its promising characteristics. The maximal specific growth rate of BSIF was as high as 0.65 h⁻¹ in yeast extract peptone dextrose medium, and the ethanol yield was 0.45 g g⁻¹ consumed glucose. Furthermore, compared with other strains, this strain exhibited superior tolerance to high temperature, hyperosmotic stress and oxidative stress; better growth performance in lignocellulosic hydrolysate; and better xylose utilization capacity when an initial xylose metabolic pathway was introduced. All of these results indicate that this strain is an excellent chassis strain for lignocellulosic ethanol production.     

Hydrolysis of lignocellulosic materials for ethanol production: a review

Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus increasing the yield and rate of cellulose hydrolysis.     

Label free quantitative proteomic analysis of secretome by Thermobifida fusca on different lignocellulosic biomass

Solid state fermentation of lignocellulosic biomass by filamentous microorganisms to induced enzyme production has been recognized as an attractive and cost effective technology. The secretion profile of lignocellulolytic enzymes by thermostable filamentous Thermobifida fusca (T. fusca) in solid state fermentation of different lignocellulosic biomasses, such as corn stover, hay; saw dust; sugarcane bagasse; wood chips; and un-dried green plant were explored using label-free exponentially modified protein abundance index (emPAI) based quantitative proteomics. Comparative analyses of T. fusca secretion profiles between cellulose and the various lignocellulosic biomasses showed induced expression of cellulolytic proteins by cellulose, and expression of hemicellulose, pectin and lignin degrading enzymes were induced by lignocellulosic biomasses. The solid state fermentation by T. fusca on lignocellulosic biomasses also revealed increased expressions of various transport proteins and hypothetical proteins. The Bray-Curtis similarity indices, clustering, and multidimensional scaling plot explicated differential protein expressions by T. fusca on different lignocellulosic biomasses, indicating that protein secretion by T. fusca is reliant on substrate complexity.     

Bioconversion of lignocellulosic biomass to hydrogen: Potential and challenges

No comprehensive review on the bioconversion of lignocellulosic biomass to hydrogen is presented. This paper provides an up-to-date review on recent research development in biotechnology-based lignocellulosic biomass-to-H₂ conversion. Bioconversion of lignocellulosic prehydrolysate, hydrolysate or cellulose to hydrogen was discussed in terms of the involved microorganisms and the bioaugmentation tactics. To achieve fully the utilization of biomass, the integrated approaches composed of coupled dark–photo fermentation and the dark fermentation and bioelectrohydrogenesis were sketched. Additionally, this review sheds light on the perspectives on the lignocellulosic biomass conversion to hydrogen, and on the scientific and technical challenges faced for the lignocelluloses bioconversion.     

木质纤维素资源生物精炼技术研究进展

开发利用可再生性的木质纤维素资源来生产液体燃料和大宗化学品,对于解决人类发展面临的资源与环境危机具有重要的意义。然而,作为其代表性工艺的纤维素乙醇生产却因为经济上无法过关而迟迟不能真正实现产业化。采用生物精炼技术,充分利用木质纤维素材料中各种组分,生产包括部分高值产品的多种产品,是克服其转化技术产业化经济可行性问题的有效措施。综述了木质纤维素原料生物精炼技术的研究发展现状,着重阐述了玉米芯的生物精炼技术产业化进展,并对木质纤维素的生物精炼前景进行了展望。     

Quantitative secretomic analysis of Trichoderma reesei strains reveals enzymatic composition for lignocellulosic biomass degradation

Trichoderma reesei is a mesophilic, filamentous fungus, and it is a major industrial source of cellulases, but its lignocellulolytic protein expressions on lignocellulosic biomass are poorly explored at present. The extracellular proteins secreted by T. reesei QM6a wild-type and hypercellulolytic mutant Rut C30 grown on natural lignocellulosic biomasses were explored using a quantitative proteomic approach with 8-plex high throughput isobaric tags for relative and absolute quantification (iTRAQ) and analyzed by liquid chromatography tandem mass spectrometry. We quantified 230 extracellular proteins, including cellulases, hemicellulases, lignin-degrading enzymes, proteases, protein-translocating transporter, and hypothetical proteins. Quantitative iTRAQ results suggested that the expressions and regulations of these lignocellulolytic proteins in the secretome of T. reesei wild-type and mutant Rut C30 were dependent on both nature and complexity of different lignocellulosic carbon sources. Therefore, we discuss here the essential lignocellulolytic proteins for designing an enzyme mixture for optimal lignocellulosic biomass hydrolysis.     

On the Determination of Water Content in Biomass Processing

Processing of lignocellulosic materials to fuels such as methane and bioethanol may involve several processing steps including pretreatment, saccharification, fermentation, and anaerobic digestion. The amounts of substrate used in these processes are usually based on dry matter content, and the processes themselves typically lead to a change in dry matter content. Thus, it is of great importance to be able to measure dry matter accurately. Dry matter content is commonly determined by measuring loss of water during oven drying. We have used Karl Fischer (KF) titration to measure the water content in a wide range of biomass fractions and have compared these data to results obtained by oven drying. This revealed considerable differences for all tested materials. For lignocellulosic materials, oven drying tends to overestimate dry matter content for untreated material. On the other hand, oven drying generally underestimates dry matter content in pretreated materials due to loss of organic volatiles. These differences have major consequences for the calculation of mass balances and yields in bioprocessing. The KF method gives more accurate water determination than oven drying due to the unique selectivity of the analysis. The method is suitable for the analysis of lignocellulosic biomasses and is particularly useful for determination of water content in pretreated materials, where oven drying usually underestimates the dry matter content due to loss of volatiles.     

Designed for deconstruction--poplar trees altered in cell wall lignification improve the efficacy of bioethanol production

? There is a pressing global need to reduce the increasing societal reliance on petroleum and to develop a bio-based economy. At the forefront is the need to establish a sustainable, renewable, alternative energy sector. This includes liquid transportation fuel derived from lignocellulosic plant materials. However, one of the current limiting factors restricting the effective and efficient conversion of lignocellulosic residues is the recalcitrance of the substrate to enzymatic conversion. ? In an attempt to assess the impact of cell wall lignin on recalcitrance, we subjected poplar trees engineered with altered lignin content and composition to two potential industrial pretreatment regimes, and evaluated the overall efficacy of the bioconversion to ethanol process. ? It was apparent that total lignin content has a greater impact than monomer ratio (syringyl : guaiacyl) on both pretreatments. More importantly, low lignin plants showed as much as a 15% improvement in the efficiency of conversion, with near complete hydrolysis of the cellulosic polymer. ? Using genomic tools to breed or select for modifications in key cell wall chemical and/or ultrastructural traits can have a profound effect on bioenergy processing. These techniques may therefore offer means to overcome the current obstacles that underpin the recalcitrance of lignocellulosic substrates to bioconversion.     

Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass

Room temperature ionic liquids (RTILs) are emerging as attractive and green solvents for lignocellulosic biomass pretreatment. The unique solvating properties of RTILs foster the disruption of the 3D network structure of lignin, cellulose, and hemicellulose, which allows high yields of fermentable sugars to be produced in subsequent enzymatic hydrolysis. In the current review, we summarize the physicochemical properties of RTILs that make them effective solvents for lignocellulose pretreatment including mechanisms of interaction between lignocellulosic biomass subcomponents and RTILs. We also highlight several recent strategies that exploit RTILs and generate high yields of fermentable sugars suitable for downstream biofuel production, and address new opportunities for use of lignocellulosic components, including lignin. Finally, we address some of the challenges that remain before large-scale use of RTILs may be achieved.     

Compositional analysis of lignocellulosic materials: evaluation of methods used for sugar analysis of waste paper and straw

To determine the overall efficiency of processes designed to convert lignocellulosic polysaccharides to ethanol, it is first necessary to determine the composition of the lignocellulosic substrates. Three standard methods routinely referenced in the literature for this purpose are monoethanolamine, trifluoroacetic acid and concentrated sulphuric acid-based methods. However, in the course of our studies, the suitability of these standard methods for analysis of wastepaper and wheat straw came into question. This paper details our investigations in this area, together with recommendations for appropriate modifications to one of the standard methods for reproducible and representative lignocellulosic compositional analysis of waste paper and cereal straw.     

High‐yield lipid production from lignocellulosic biomass using engineered xylose‐utilizing Yarrowia lipolytica

Lignocellulosic biomass shows high potential as a renewable feedstock for use in biodiesel production via microbial fermentation. Yarrowia lipolytica, an emerging oleaginous yeast, has been engineered to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into lipids for lignocellulosic biodiesel production. Yet, the lipid yield from xylose or lignocellulosic biomass remains far lower than that from glucose. Here we developed an efficient xylose‐utilizing Y. lipolytica strain, expressing an isomerase‐based pathway, to achieve high‐yield lipid production from lignocellulosic biomass. The newly developed xylose‐utilizing Y. lipolytica, YSXID, produced 12.01 g/L lipids with a maximum yield of 0.16 g/g, the highest ever reported, from lignocellulosic hydrolysates. Consequently, this study shows the potential of isomerase‐based xylose‐utilizing Y. lipolytica for economical and sustainable production of biodiesel and oleochemicals from lignocellulosic biomass.     

Metabolic engineering for the utilization of carbohydrate portions of lignocellulosic biomass

The petrochemical industry has grown to meet the need for massive production of energy and commodities along with an explosive population growth; however, serious side effects such as greenhouse gas emissions and global warming have negatively impacted the environment. Lignocellulosic biomass with myriad quantities on Earth is an attractive resource for the production of carbon-neutral fuels and chemicals through environmentally friendly processes of microbial fermentation. This review discusses metabolic engineering efforts to achieve economically feasible industrial production of fuels and chemicals from microbial cell factories using the carbohydrate portion of lignocellulosic biomass as substrates. The combined knowledge of systems biology and metabolic engineering has been applied to construct robust platform microorganisms with maximum conversion of monomeric sugars, such as glucose and xylose, derived from lignocellulosic biomass. By comprehensively revisiting carbon conversion pathways, we provide a rationale for engineering strategies, as well as their features, feasibility, and recent representative studies. In addition, we briefly discuss how tools in systems biology can be applied in the field of metabolic engineering to accelerate the development of microbial cell factories that convert lignocellulosic biomass into carbon-neutral fuels and chemicals with economic feasibility.     

Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae

Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources toward engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, recent advances in metabolic engineering of yeast is summarized to address bottlenecks on xylose assimilation and to enable simultaneous co-utilization of xylose and other substrates in lignocellulosic hydrolysates. Distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals are also highlighted. Although many challenges remain, recent research investments have facilitated the efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding xylose-induced metabolic rewiring in engineered yeast has encouraged the use of xylose as a carbon source for producing various non-ethanol bioproducts. To boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.     

Pretreatment and Lignocellulosic Chemistry

Lignocellulosic materials such as wood, grass, and agricultural and forest residues are promising alternative energy resources that can be utilized to produce ethanol. The yield of ethanol production from native lignocellulosic material is relatively low due to its native recalcitrance, which is attributed to, in part, lignin content/structure, hemicelluloses, cellulose crystallinity, and other factors. Pretreatment of lignocellulosic materials is required to overcome this recalcitrance. The goal of pretreatment is to alter the physical features and chemical composition/structure of lignocellulosic materials, thus making cellulose more accessible to enzymatic hydrolysis for sugar conversion. Various pretreatment technologies to reduce recalcitrance and to increase sugar yield have been developed during the past two decades. This review examines the changes in lignocellulosic structure primarily in cellulose and hemicellulose during the most commonly applied pretreatment technologies including dilute acid pretreatment, hydrothermal pretreatment, and alkaline pretreatment.