Fusarium oxysporum: status in bioethanol production

Fermentation of lignocellulosic materials to ethanol and other solvents provides an alternative way of treating wastes and producing chemical feedstocks and fuel additives. Considerable efforts have been made in past 10 years to improve the process based on lignocellulosic biomass and hydrolysate that contains a complex mixture of sugars, decomposition products of sugars, and sometimes the inhibitory levels of soluble lignin. Despite the relative abundance of D-xylose in crop and forest residues it has not been found efficiently fermentable by most of the microorganisms. Recent research has revealed that D-xylose may be fermented to ethanol and organic acids. Recently, several strains of Fusarium oxysporum have been found to have potential for converting not only D-xylose, but also cellulose to ethanol in a one-step process. Distinguishing features of F. oxysporum for ethanol production in comparison to other organisms are identified. These include the advantage of in situ cellulase production and cellulose fermentation, pentose fermentation, and the tolerance of sugars and ethanol. The main disadvantage is the slow conversion rate when compared with yeast.     

Development of genetic tools for heterologous protein expression in a pentose-utilizing environmental isolate of Pseudomonas putida

Pseudomonas putida has emerged as a promising host for the conversion of biomass-derived sugars and aromatic intermediates into commercially relevant biofuels and bioproducts. Most of the strain development studies previously published have focused on P. putida KT2440, which has been engineered to produce a variety of non-native bioproducts. However, P. putida is not capable of metabolizing pentose sugars, which can constitute up to 25% of biomass hydrolysates. Related P. putida isolates that metabolize a larger fraction of biomass-derived carbon may be attractive as complementary hosts to P. putida KT2440. Here we describe genetic tool development for P. putida M2, a soil isolate that can metabolize pentose sugars. The functionality of five inducible promoter systems and 12 ribosome binding sites was assessed to regulate gene expression. The utility of these expression systems was confirmed by the production of indigoidine from C6 and C5 sugars. Chromosomal integration and expression of non-native genes was achieved by using chassis-independent recombinase-assisted genome engineering (CRAGE) for single-step gene integration of biosynthetic pathways directly into the genome of P. putida M2. These genetic tools provide a foundation to develop hosts complementary to P. putida KT2440 and expand the ability of this versatile microbial group to convert biomass to bioproducts.     

Conversion of pentoses to ethanol by yeasts and fungi

Fermentation of D-xylose is of interest in enhancing the yield of ethanol obtainable from lignocellulosic hydrolysates. Such hydrolysates can contain both pentoses and hexoses, and while technology to convert hexoses to ethanol is well established, the fermentation of pentoses had been problematical. To overcome the difficulty, yeasts and fungi have been sought and identified in recent years that can convert D-xylose into ethanol. However, operation of their cultures in the presence of the pentose to obtain rapid and efficient ethanol production is somewhat more complex than in the archetype alcoholic fermentation, Saccharomyces cerevisiae on D-glucose. The complexity stems, in part, from the association of ethanol accumulation in cultures where D-xylose is the sole carbon source with conditions that limit growth, by oxygen in particular, although limitation by other nutrients might also be implicated. Aspects of screening for appropriate organisms and of the parameters that play a role in determining culture variables, especially those associated with ethanol productivity, are reviewed. Performance with D-xylose as sole carbon source, in sugar mixtures, and in lignocellulosic hydrolysates is discussed. A model that involves biochemical considerations of D-xylose metabolism is presented that rationalizes the effects of oxygen on cultures where D-xylose is the sole carbon source, notably effects of the specific rate of oxygen use on the rate and extent of ethanol accumulation. Alternate methods to direct fermentation of D-xylose have been developed that depend on its prior isomerization to D-xylose, followed by fermentation of the pentulose by certain yeasts and fungi. Factors involved in the biochemistry, use, and performance of these methods, which with some organisms involves sensitivity to oxygen, are reviewed.     

Conversion of pentoses by yeasts

The utilization and conversion of D-xylose, D-xylulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: (1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. (2)The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol, D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. (3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. (4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. (5) Of the four substrates examined, D-xylulose was the perferred substrate, followed by D-xylose, L-arabinose, and xylitol. (6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Novel two‐stage fermentation process for bioethanol production using Saccharomyces pastorianus

Bioethanol produced from lignocellulosic materials has the potential to be economically feasible, if both glucose and xylose released from cellulose and hemicellulose can be efficiently converted to ethanol. Saccharomyces spp. can efficiently convert glucose to ethanol; however, xylose conversion to ethanol is a major hurdle due to lack of xylose‐metabolizing pathways. In this study, a novel two‐stage fermentation process was investigated to improve bioethanol productivity. In this process, xylose is converted into biomass via non‐Saccharomyces microorganism and coupled to a glucose‐utilizing Saccharomyces fermentation. Escherichia coli was determined to efficiently convert xylose to biomass, which was then killed to produce E. coli extract. Since earlier studies with Saccharomyces pastorianus demonstrated that xylose isomerase increased ethanol productivities on pure sugars, the addition of both E. coli extract and xylose isomerase to S. pastorianus fermentations on pure sugars and corn stover hydrolysates were investigated. It was determined that the xylose isomerase addition increased ethanol productivities on pure sugars but was not as effective alone on the corn stover hydrolysates. It was observed that the E. coli extract addition increased ethanol productivities on both corn stover hydrolysates and pure sugars. The ethanol productivities observed on the corn stover hydrolysates with the E. coli extract addition was the same as observed on pure sugars with both E. coli extract and xylose isomerase additions. These results indicate that the two‐stage fermentation process has the capability to be a competitive alternative to recombinant Saccharomyces cerevisiae‐based fermentations. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:300–310, 2014     

Induction of NADPH-linked D-xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D-xylose, L-arabinose, or D-galactose

Considerable interest in the D-xylose catabolic pathway of Pachysolen tannophilus has arisen from the discovery that this yeast is capable of fermenting D-xylose to ethanol. In this organism D-xylose appears to be catabolized through xylitol to D-xylulose. NADPH-linked D-xylose reductase is primarily responsible for the conversion of D-xylose to xylitol, while NAD-linked xylitol dehydrogenase is primarily responsible for the subsequent conversion of xylitol to D-xylulose. Both enzyme activities are readily detectable in cell-free extracts of P. tannophilus grown in medium containing D-xylose, L-arabinose, or D-galactose and appear to be inducible since extracts prepared from cells growth in media containing other carbon sources have only negligible activities, if any. Like D-xylose, L-arabinose and D-galactose were found to serve as substrates for NADPH-linked reactions in extracts of cells grown in medium containing D-xylose, L-arabinose, or D-galactose. These L-arabinose and D-galactose NADPH-linked activities also appear to be inducible, since only minor activity with L-arabinose and no activity with D-galactose is detected in extracts of cells grown in D-glucose medium. The NADPH-linked activities obtained with these three sugars may result from the actions of distinctly different enzymes or from a single aldose reductase acting on different substrates. High-performance liquid chromatography and gas-liquid chromatography of in vitro D-xylose, L-arabinose, and D-galactose NADPH-linked reactions confirmed xylitol, L-arabitol, and galactitol as the respective conversion products of these sugars. Unlike xylitol, however, neither L-arabitol nor galactitol would support comparable NAD-linked reaction(s) in cellfree extracts of induced P. tannophilus. Thus, the metabolic pathway of D-xylose diverges from those of L-arabinose or D-galactose following formation of the pentitol.     

Strain engineering of Saccharomyces cerevisiae for enhanced xylose metabolism

Efficient and rapid fermentation of all sugars present in cellulosic hydrolysates is essential for economic conversion of renewable biomass into fuels and chemicals. Xylose is one of the most abundant sugars in cellulosic biomass but it cannot be utilized by wild type Saccharomyces cerevisiae, which has been used for industrial ethanol production. Therefore, numerous technologies for strain development have been employed to engineer S. cerevisiae capable of fermenting xylose rapidly and efficiently. These include i) optimization of xylose-assimilating pathways, ii) perturbation of gene targets for reconfiguring yeast metabolism, and iii) simultaneous co-fermentation of xylose and cellobiose. In addition, the genetic and physiological background of host strains is an important determinant to construct efficient and rapid xylose-fermenting S. cerevisiae. Vibrant and persistent researches in this field for the last two decades not only led to the development of engineered S. cerevisiae strains ready for industrial fermentation of cellulosic hydrolysates, but also deepened our understanding of operational principles underlying yeast metabolism.     

Metabolic engineering of the yeast Hansenula polymorpha for the construction of efficient ethanol producers

Until recently, the methylotrophic yeast has not been considered as a potential producer of biofuels, particularly, ethanol from lignocellulosic hydrolysates. The first work published 10 years ago revealed the ability of the thermotolerant methylotrophic yeast Hansenula polymorpha to ferment xylose—one of the main sugars of lignocellulosic hydrolysates—which has made the yeast a promising organism for high-temperature alcoholic fermentation. Such a feature of H. polymorpha could be used in the implementation of a potentially effective process of simultaneous saccharification and fermentation (SSF) of raw materials. SSF makes it possible to combine enzymatic hydrolysis of raw materials with the conversion of the sugars produced into ethanol: enzymes hydrolyze polysaccharides to monomers, which are immediately consumed by microorganisms (producers of ethanol). However, the efficiency of alcoholic fermentation of major sugars produced via hydrolysis of lignocellulosic raw materials and, especially, xylose by wild strains of H. polymorpha requires significant improvements. In this review, the main results of metabolic engineering of H. polymorpha for the construction of improved producers of ethanol from xylose, starch, xylan, and glycerol, as well as that of strains with increased tolerance to high temperatures and ethanol, are represented.     

Direct fermentation of D-xylose to ethanol by a xylose-fermentating yeast mutant,Candida sp XF 217

Summary A mutant strain of Candida sp. XF 217, was found to produce ethanol from D-xylose aerobically as well as anaerobically. The rate of ethanol production under aerobic conditions was greater, indicating an oxygen requirement for the uptake of D-xylose in XF 217. Ethanol was also produced by XF 217 when D-glucose, D-fructose, sucrose or maltose were used as substrates. The D-xylose fermenting yeast strain is a potential organism to use for ethanol production from renewable biomass-derived hexoses and pentoses.     

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae

Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.     

Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

Ethanol production from olive cake biomass substrate

The inexpensive production of sugars from lignocellulose is an essential step for the use of biomass to produce fuel ethanol. Olive cake is an abundant by-product of the olive oil industry and represents a potentially significant lignocellulosic source for bioethanol production in the Mediterranean basin. Furthermore, converting olive cake to ethanol could add further value to olive production. In the present study, olive cake was evaluated as a feedstock for ethanol production. To this end, the lignocellulosic component of the olive cake was dilute-acid pretreated at a 13.5% olive-cake loading with 1.75% (w/v) sulfuric acid and heating at 160°C for 10 min. This was followed by chemical elimination of fermentation inhibitors. Soluble sugars resulting from the pretreatment process were fermented using E. coli FBR5, a strain engineered to selectively produce ethanol. 8.1 g of ethanol/L was obtained from hydrolysates containing 18.1 g of soluble sugars. Increasing the pretreatment temperature to 180°C resulted in failed fermentations, presumably due to inhibitory by-products released during pretreatment.     

Production of 2,3-butanediol by Klebsiella pneumoniae grown on acid hydrolyzed wood hemicellulose

Summary Previously steam explosion had been used to enhance the enzymatic hydrolysis of lignocellulosic substrates to glucose. The conditions for pretreating aspen wood chips were optimized so that highest amounts of undegraded hemicellulose could be obtained after washing the steam exploded chips. The hemicellulose rich water soluble fractions showing highest pentosan yields were then acid hydrolysed to their composite sugars. Approximately 65–75% of the total reducing sugars detected in the wood hydrolysates were in the form of monosaccharides with D-xylose being the major component. Klebsiella pneumoniae was grown in media containing these wood hydrolysates as the substrate and 2,3-butanediol yields of 0.4–0.5 g per g of monosaccharide utilised were obtained.     

Detoxification of Lignocellulose Hydrolysates: Biochemical and Metabolic Engineering Toward White Biotechnology

Chemical hydrolysis of lignocellulosic biomass (LB) produces a number of inhibitors in addition to sugars. These inhibitors include lignin-derived phenolics, carbohydrate-derived furans, and weak acids that have shown a marked effect on the productivities of various metabolites and the growth of biocatalysts in the fermentative reaction. In the past, a number of physicochemical and biological approaches have been proposed to overcome these fermentation inhibitors, including modified fermentative strategies. Additionally, the timely intervention of genetic engineering has provided an impetus to develop suitable technologies for the detoxification of lignocellulosics in biorefineries. However, the improvements in detoxification strategies for lignocellulose hydrolysates have resulted in significant loss of sugars after detoxification. Hydrolysis of myco-LB (LB after fungal pretreatment) has been recognized as a promising approach to avoid fermentation inhibitors and improve total sugar recovery. Biotechnological inventions have also made it possible to widen the range of suitable biocatalysts for biorefineries by microbial-routed induction of enzymatic expression for the elimination of inhibitors, eventually improving ethanol production from acid hydrolysates. This article aims to highlight the strategies that have been adopted to detoxify lignocellulosic hydrolysates and their effects on the chemical composition of the hydrolysates to improve the fermentability of lignocellulosics. In addition, genetic manipulation could widen the availability and variety of substrates and modify the metabolic routes to produce bioethanol or other value-added compounds in an efficient manner.     

Yeast strains for ethanol production from lignocellulosic hydrolysates during in situ detoxification

Yeast strains Y1, Y4 and Y7 demonstrated high conversion efficiencies for sugars and high abilities to tolerate or metabolize inhibitors in dilute-acid lignocellulosic hydrolysates. Strains Y1 and Y4 completely consumed the glucose within 24 h in dilute-acid lignocellulosic hydrolysate during in situ detoxification, and the maximum ethanol yields reached 0.49 g and 0.45 g ethanol/g glucose, equivalent to maximum theoretical values of 96% and 88.2%, respectively. Strain Y1 could metabolize xylose to xylitol with a yield of 0.64 g/g xylose, whereas Y4 was unable to utilize xylose as a substrate. Strain Y7 was able to consume sugars (glucose and xylose) within 72 h during hydrolysate in situ detoxification, producing a high ethanol yield (equivalent to 93.6% of the maximum theoretical value). Y1 and Y7 are the most efficient yeast strains yet reported for producing ethanol from non-detoxified dilute-acid lignocellulosic hydrolysates. These findings offer huge potential for improving the economics of bio-ethanol production from lignocellulosic hydrolysates.     

Use of catabolite repression mutants for fermentation of sugar mixtures to ethanol

Use of agricultural biomass, other than corn-starch, to produce fuel ethanol requires a microorganism that can ferment the mixture of sugars derived from hemicellulose. Escherichia coli metabolizes a wide range of substrates and has been engineered to produce ethanol in high yield from sugar mixtures. E. coli metabolizes glucose in preference to other sugars and, as a result, utilization of the pentoses in hemicellulose-derived sugar mixtures is delayed and may be incomplete. Residual sugar lowers the ethanol yield and is problematic for downstream processing of fermentation products. Therefore, a catabolite repression mutant that simultaneously utilizes glucose and pentoses would be useful for fermentation of complex substrate mixtures. We constructed ethanologenic E. coli strains with a glucose phosphotransferase (ptsG) mutation and used the mutants to ferment glucose, arabinose, and xylose, singly and in mixtures, to ethanol. Yields were 87-94% of theoretical for both the wild type and mutants, but the mutants had an altered pattern of mixed sugar utilization. Phosphotransferase mutants metabolized the pentoses simultaneously with glucose, rather than sequentially. Based upon fermentations of sugar mixtures, a catabolite-repression mutant of ethanologenic E. coli is expected to provide more efficient fermentation of hemicellulose hydrolysates by allowing direct utilization of pentoses.     

基因组改组及其在木质纤维素水解液乙醇发酵菌种选育中的应用展望

能高效代谢木质纤维素水解液中的可发酵糖、同时可耐受/分解发酵抑制剂的菌种, 是利用木质纤维素为原料生产燃料乙醇技术的关键。基因组改组技术是近些年发展起来的一项新型育种技术, 该技术已运用于食品和医药行业菌种的改良。本文综述了基因组改组技术的原理、方法、特点、及其运用, 并对其在木质纤维素水解液乙醇发酵菌种选育方面的应用进行了展望。     

Yeasts in sustainable bioethanol production: A review

Bioethanol has been identified as the mostly used biofuel worldwide since it significantly contributes to the reduction of crude oil consumption and environmental pollution. It can be produced from various types of feedstocks such as sucrose, starch, lignocellulosic and algal biomass through fermentation process by microorganisms. Compared to other types of microoganisms, yeasts especially Saccharomyces cerevisiae is the common microbes employed in ethanol production due to its high ethanol productivity, high ethanol tolerance and ability of fermenting wide range of sugars. However, there are some challenges in yeast fermentation which inhibit ethanol production such as high temperature, high ethanol concentration and the ability to ferment pentose sugars. Various types of yeast strains have been used in fermentation for ethanol production including hybrid, recombinant and wild-type yeasts. Yeasts can directly ferment simple sugars into ethanol while other type of feedstocks must be converted to fermentable sugars before it can be fermented to ethanol. The common processes involves in ethanol production are pretreatment, hydrolysis and fermentation. Production of bioethanol during fermentation depends on several factors such as temperature, sugar concentration, pH, fermentation time, agitation rate, and inoculum size. The efficiency and productivity of ethanol can be enhanced by immobilizing the yeast cells. This review highlights the different types of yeast strains, fermentation process, factors affecting bioethanol production and immobilization of yeasts for better bioethanol production.