Biofuels produced from various lignocellulosic materials, such as wood, agricultural, or forest residues, have the potential to be a valuable substitute for, or complement to, gasoline. Many physicochemical structural and compositional factors hinder the hydrolysis of cellulose present in biomass to sugars and other organic compounds that can later be converted to fuels. The goal of pretreatment is to make the cellulose accessible to hydrolysis for conversion to fuels. Various pretreatment techniques change the physical and chemical structure of the lignocellulosic biomass and improve hydrolysis rates. During the past few years a large number of pretreatment methods have been developed, including alkali treatment, ammonia explosion, and others. Many methods have been shown to result in high sugar yields, above 90% of the theoretical yield for lignocellulosic biomasses such as woods, grasses, corn, and so on. In this review, we discuss the various pretreatment process methods and the recent literature that...

http://ucce.ucdavis.edu/files/datastore/234-1388.pdf

Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(06)00254-X.pdf

Bio-ethanol--the fuel of tomorrow from the residues of today.

Hemicelluloses for fuel ethanol: A review.

Simultaneous saccharification and fermentation (SSF) is one process option for production of ethanol from lignocellulose. The principal benefits of performing the enzymatic hydrolysis together with the fermentation, instead of in a separate step after the hydrolysis, are the reduced end-product inhibition of the enzymatic hydrolysis, and the reduced investment costs. The principal drawbacks, on the other hand, are the need to find favorable conditions (e.g. temperature and pH) for both the enzymatic hydrolysis and the fermentation and the difficulty to recycle the fermenting organism and the enzymes. To satisfy the first requirement, the temperature is normally kept below 37°C, whereas the difficulty to recycle the yeast makes it beneficial to operate with a low yeast concentration and at a high solid loading. In this review, we make a brief overview of recent experimental work and development of SSF using lignocellulosic feedstocks. Significant progress has been made with respect to increasing the substrate loading, decreasing the yeast concentration and co-fermentation of both hexoses and pentoses during SSF. Presently, an SSF process for e.g. wheat straw hydrolyzate can be expected to give final ethanol concentrations close to 40 g L-1 with a yield based on total hexoses and pentoses higher than 70%.

/pdf/a-short-review-on-ssf-an-interesting-process-option-for-41evfqreo5.pdf

A short review on SSF - an interesting process option for ethanol production from lignocellulosic feedstocks.

Production of bioethanol from forest and agricultural products requires a fermenting organism that converts all types of sugars in the raw material to ethanol in high yield and with a high rate. This review summarizes recent research aiming at developing industrial strains of Saccharomyces cerevisiae with the ability to ferment all lignocellulose-derived sugars. The properties required from the industrial yeast strains are discussed in relation to four benchmarks: (1) process water economy, (2) inhibitor tolerance, (3) ethanol yield, and (4) specific ethanol productivity. Of particular importance is the tolerance of the fermenting organism to fermentation inhibitors formed during fractionation/pretreatment and hydrolysis of the raw material, which necessitates the use of robust industrial strain background. While numerous metabolic engineering strategies have been developed in laboratory yeast strains, only a few approaches have been realized in industrial strains. The fermentation performance of the existing industrial pentose-fermenting S. cerevisiae strains in lignocellulose hydrolysate is reviewed. Ethanol yields of more than 0.4 g ethanol/g sugar have been achieved with several xylose-fermenting industrial strains such as TMB 3400, TMB 3006, and 424A(LNF-ST), carrying the heterologous xylose utilization pathway consisting of xylose reductase and xylitol dehydrogenase, which demonstrates the potential of pentose fermentation in improving lignocellulosic ethanol production.

Towards industrial pentose-fermenting yeast strains

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

Background: Fermentation of lignocellulosic biomass is an attractive alternative for the production of bioethanol. Traditionally, the yeast Saccharomyces cerevisiae is used in industrial ethanol fermentations. However, S. cerevisiae is naturally not able to ferment the pentose sugars D-xylose and L-arabinose, which are present in high amounts in lignocellulosic raw materials. Results: We describe the engineering of laboratory and industrial S. cerevisiae strains to coferment the pentose sugars D-xylose and L-arabinose. Introduction of a fungal xylose and a bacterial arabinose pathway resulted in strains able to grow on both pentose sugars. Introduction of a xylose pathway into an arabinose-fermenting laboratory strain resulted in nearly complete conversion of arabinose into arabitol due to the L-arabinose reductase activity of the xylose reductase. The industrial strain displayed lower arabitol yield and increased ethanol yield from xylose and arabinose. Conclusion: Our work demonstrates simultaneous co-utilization of xylose and arabinose in recombinant strains of S. cerevisiae. In addition, the co-utilization of arabinose together with xylose significantly reduced formation of the by-product xylitol, which contributed to improved ethanol production.

/pdf/co-utilization-of-l-arabinose-and-d-xylose-by-laboratory-and-4y6nr08nfi.pdf

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Bioethanol produced by microbial fermentations of plant biomass hydrolysates consisting of hexose and pentose mixtures is an excellent alternative to fossil transportation fuels. However, the yeast Saccharomyces cerevisiae, commonly used in bioethanol production, can utilize pentose sugars like l-arabinose or d-xylose only after heterologous expression of corresponding metabolic pathways from other organisms. Here we report the improvement of a bacterial l-arabinose utilization pathway consisting of l-arabinose isomerase from Bacillus subtilis and l-ribulokinase and l-ribulose-5-P 4-epimerase from Escherichia coli after expression of the corresponding genes in S. cerevisiae. l-Arabinose isomerase from B. subtilis turned out to be the limiting step for growth on l-arabinose as the sole carbon source. The corresponding enzyme could be effectively replaced by the enzyme from Bacillus licheniformis, leading to a considerably decreased lag phase. Subsequently, the codon usage of all the genes involved in the l-arabinose pathway was adapted to that of the highly expressed genes encoding glycolytic enzymes in S. cerevisiae. Yeast transformants expressing the codon-optimized genes showed strongly improved l-arabinose conversion rates. With this rational approach, the ethanol production rate from l-arabinose could be increased more than 2.5-fold from 0.014 g ethanol h−1 (g dry weight)−1 to 0.036 g ethanol h−1 (g dry weight)−1 and the ethanol yield could be increased from 0.24 g ethanol (g consumed l-arabinose)−1 to 0.39 g ethanol (g consumed l-arabinose)−1. These improvements make up a new starting point for the construction of more-efficient industrial l-arabinose-fermenting yeast strains by evolutionary engineering.

Codon-optimized bacterial genes improve L-Arabinose fermentation in recombinant Saccharomyces cerevisiae.

The present invention relates to a S. cerevisiae strain expressing both arabinose and xylose utilization pathways, and in particular to a S. cerevisiae strain fermenting both arabinose and xylose to ethanol, and more particularly a S. cerevisiae strain with overexpression or upregulation of xylose- or aldose reductase (XR, AR) with xylitol dehydrogenase (XDH) together with overexpression or upregulation of genes forming an arabinose utilization pathway. The invention encompasses both laboratory and industrial strain having these properties.

Arabinose- and xylose-fermenting Saccharomyces cerevisiae strains

The present invention relates to the use of nucleic acid molecules coding for a bacterial xylose isomerase (XI), preferably coming from Clostridium phytofermentans, for reaction/metabolization, particularly fermentation, of recombinant microorganisms of biomaterial containing xylose, and particularly for the production of bioalcohols, particularly bioethanol, by means of xylose fermenting yeasts The present invention further relates to cells, particularly eukaryotic cells, which are transformed utilizing a nucleic acid expression construct which codes for a xylose isomerase, wherein the expression of the nucleic acid expression construct imparts to the cells the capability to directly isomerize xylose into xylulose Said cells are preferably utilized for reaction/metabolization, particularly fermentation, of biomaterial containing xylose, and particularly for the production of bioalcohols, particularly bioethanol The present invention also relates to methods for the production of bioethanol, and to methods for the production of further metabolization products, comprising the metabolization of media containing xylose

Beate Wiedemann

Papers

Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae.

Co-utilization of L-arabinose and D-xylose by laboratory and industrial Saccharomyces cerevisiae strains

Codon-optimized bacterial genes improve L-Arabinose fermentation in recombinant Saccharomyces cerevisiae.

Arabinose- and xylose-fermenting Saccharomyces cerevisiae strains

Prokaryotic Xylose Isomerase for the Construction of Xylose Fermenting Yeasts