Dekkera/Brettanomyces yeasts for ethanol production from renewable sources under oxygen-limited and low-pH conditions

Industrial fermentation of lignocellulosic hydrolysates to ethanol requires microorganisms able to utilise a broad range of carbon sources and generate ethanol at high yield and productivity. D. bruxellensis has recently been reported to contaminate commercial ethanol processes, where it competes with Saccharomyces cerevisiae [4, 26]. In this work Brettanomyces/Dekkera yeasts were studied to explore their potential to produce ethanol from renewable sources under conditions suitable for industrial processes, such as oxygen-limited and low-pH conditions. Over 50 strains were analysed for their ability to utilise a variety of carbon sources, and some strains grew on cellobiose and pentoses. Two strains of D. bruxellensis were able to produce ethanol at high yield (0.44 g g⁻¹ glucose), comparable to those reported for S. cerevisiae. B. naardenensis was shown to be able to produce ethanol from xylose. To obtain ethanol from synthetic lignocellulosic hydrolysates we developed a two-step fermentation strategy: the first step under aerobic conditions for fast production of biomass from mixtures of hexoses and pentoses, followed by a second step under oxygen limitation to promote ethanol production. Under these conditions we obtained biomass and ethanol production on synthetic lignocellulosic hydrolysates, with ethanol yields ranging from 0.2 to 0.3 g g⁻¹ sugar. Hexoses, xylose and arabinose were consumed at the end of the process, resulting in 13 g l⁻¹ of ethanol, even in the presence of furfural. Our studies showed that Brettanomyces/Dekkera yeasts have clear potential for further development for industrial processes aimed at production of ethanol from renewable sources.     

广谱碳源产油酵母菌的筛选

对10株酵母菌利用不同单糖为碳源条件下菌体内积累油脂的能力进行了初步考察,并对菌油进行了分离和脂肪酸组成分析。实验发现,以葡萄糖为唯一碳源时有9株菌油脂含量超过自身细胞干重的20%,可以界定为产油微生物。其中6#菌(T.cutaneumAS2.571)利用葡萄糖发酵菌体油脂含量达到65%(W/W)。所有实验菌株都能同化多种单糖,其中1#菌(L.starkeyiAS2.1390)、4#菌(R.toruloidesAS2.1389)和11#菌(L.starkeyiAS2.1608)表现出对碳源利用的广谱性,能转化五碳糖木糖和阿拉伯糖并在菌体内积累油脂,油脂含量最高达到26%。脂肪酸组成分析结果表明,菌油富含饱和及低度不饱和长链脂肪酸,其中棕榈酸、油酸和亚油酸三者之和占总脂肪酸组成的90%以上,脂肪酸组成分布类似于常见的植物油。这些结果对利用产油微生物转化木质纤维素水解混合糖获取油脂资源的研究具有重要意义。     

Regulation of D-xylose utilization by hexoses in pentose-fermenting yeasts

The aldopentose D-xylose is one of the most abundant sugars in plant biomass and its efficient microbial utilization is of fundamental importance in the overall bioconversion of lignocellulosic materials into liquid fuels and chemicals. The discovery of pentose-fermenting yeasts in the early 1980's led to world wide interest because of the perceived potential for improved D-xylose fermentation to enhance the prospect of biomass conversions. However, the utilization of D-xylose by pentose-fermenting yeasts can be adversely affected by the hexoses, mainly D-glucose and D-mannose, which are usually present in high amounts in lignocellulosic hydrolysates. Research in the past several years has uncovered some of the regulatory effects of D-glucose on D-xylose utilization. However, much remains unknown about the mechanisms responsible for these effects. This review summarizes the current state of knowledge on the induction, repression and inactivation of D-xylose utilization in pentose-fermenting yeasts.     

Ethanol productions from D-xylose and cellobiose by Kluyveromyces cellobiovorus

Yeasts capable of fermenting both D-xylose and cellobiose to ethanol were screened. Of 213 species of yeasts surveyed, Kluyveromyces cellobiovorus sp. nov., a new species belonging to genus of Kluyveromyces, was selected as the sole strain. This strain accumulated 32, 22, and 19 g/L of ethanol from 8% glucose, D-xylose, and cellobiose, respectively. It was also shown that this strain produced ethanol from the enzymatic bagasse hydrolysate containing hexoses and pentoses more efficiently than Saccharomyces cerevisiae.     

A modified Saccharomyces cerevisiae strain that consumes L-Arabinose and produces ethanol

Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase. Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose. With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight). h(-1) under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.     

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.     

Fermentation to ethanol of pentose-containing spent sulphite liquor

Ethanolic fermentation of spent sulphite liquor with ordinary bakers' yeast is incomplete because this yeast cannot ferment the pentose sugars in the liquor. This results in poor alcohol yields, and a residual effluent problem By using the yeast Candida shehatae (R) for fermentation of the spent sulphite liquor from a large Canadian alcohol-producing sulphite pulp and paper mill, pentoses as well as hexoses were fermented nearly completely, alcohol yields were raised by 33%, and sugar removal increased by 46%. Inhibitors were removed prior to fermentation by steam stripping. Major benefits were obtained by careful recycling of this yeast, which was shown to be tolerant both of high sugar concentrations and high alcohol concentrations. When sugar concentrations over 250 g/L (glucose: xylose 70:30) were fermented, ethanol became an inhibitor when its concentration reached 90 g/L. However, when the ethanol was removed by low-temperature vacuum distillation, fermentation continued and resulted in a yield of 0.50 g ethanol/g sugar consumed. Further improvement was achieved by combining enzyme saccharification of sugar oligomers with fermentation. This yeast is able to ferment both hexoses and pentoses simultaneously, efficiently, and rapidly. Present indications are that it is well suited to industrial operations wherever hexoses and pentoses are both to be fermented to ethanol, for example, in wood hydrolysates.     

Fermentation of D-xylose by free and immobilized Saccharomyces cerevisiae

With D-xylose (50 g l ) as sole carbon substrate, aerobic cultures of S. cerevisiae consumed significant amounts of sugar (26.4 g l ), producing 4.0 g xylitol l but no ethanol. In the presence of a mixture of glucose (35 g l ) and xylose (15 g l ), yeasts consumed 1.6 g xylose l that was converted nearly stoichiometrically to xylitol. Anaerobic conditions lessened xylose consumption and its conversion into xylitol. Traces of ethanol (0.4 g l ) were produced when xylose was the only carbon source, however. Agar-entrapped yeasts behaved as anaerobically-grown cultures but with higher specific rates of xylose consumption and xylitol production.     

Mucor indicus: Biology and industrial application perspectives: A review

Mucor indicus, one of the most important strains of zygomycetes fungi, has been the subject of several studies since a couple of hundred years ago. This fungus, regarded as a non-pathogenic dimorphic microorganism, is used for production of several beers and foods. Morphology of the fungus can be manipulated and well controlled by changing a number of parameters. Furthermore, M. indicus can grow on a variety of substrates including lignocellulosic hydrolysates which are mixtures of hexoses, pentoses, and different severe fermentation inhibitors. Indeed, high yield ethanol production is among the most important features of this strain. Presence of considerable amounts of chitosan in the cell wall is another important aspect of the fungus. Besides production of ethanol and chitosan, the biomass of this fungus has shown a great potential to be used as a rich nutritional source, e.g. fish feed. The fungus is also among the oleaginous fungi and produces high amounts of polyunsaturated fatty acids particularly γ-linolenic acid. Furthermore, the biomass autolysate has a high potential for yeast extract replacement in fermentation by the fungus. Additionally, the strain has shown promising results in heavy metal removal from wastewaters. This review discusses different aspects of biology and industrial application perspectives of M. indicus. Furthermore, open areas for the future basic and applied levels of research are also presented.     

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.     

Isolation and characterization of Cupriavidus basilensis HMF14 for biological removal of inhibitors from lignocellulosic hydrolysate

The formation of toxic fermentation inhibitors such as furfural and 5-hydroxy-2-methylfurfural (HMF) during acid (pre-)treatment of lignocellulose, calls for the efficient removal of these compounds. Lignocellulosic hydrolysates can be efficiently detoxified biologically with microorganisms that specifically metabolize the fermentation inhibitors while preserving the sugars for subsequent use by the fermentation host. The bacterium Cupriavidus basilensis HMF14 was isolated from enrichment cultures with HMF as the sole carbon source and was found to metabolize many of the toxic constituents of lignocellulosic hydrolysate including furfural, HMF, acetate, formate and a host of aromatic compounds. Remarkably, this microorganism does not grow on the most abundant sugars in lignocellulosic hydrolysates: glucose, xylose and arabinose. In addition, C. basilensis HMF14 can produce polyhydroxyalkanoates. Cultivation of C. basilensis HMF14 on wheat straw hydrolysate resulted in the complete removal of furfural, HMF, acetate and formate, leaving the sugar fraction intact. This unique substrate profile makes C. basilensis HMF14 extremely well suited for biological removal of inhibitors from lignocellulosic hydrolysates prior to their use as fermentation feedstock.     

Biotechnological strategies to overcome inhibitors in lignocellulose hydrolysates for ethanol production: review

One of the major challenges faced in commercial production of lignocellulosic bioethanol is the inhibitory compounds generated during the thermo-chemical pre-treatment step of biomass. These inhibitory compounds are toxic to fermenting micro-organisms. The ethanol yield and productivity obtained during fermentation of lignocellulosic hydrolysates is decreased due to the presence of inhibiting compounds, such as weak acids, furans and phenolic compounds formed or released during thermo-chemical pre-treatment step such as acid and steam explosion. This review describes the application and/or effect of biological detoxification (removal of inhibitors before fermentation) or use of bioreduction capability of fermenting yeasts on the fermentability of the hydrolysates. Inhibition of yeast fermentation by the inhibitor compounds in the lignocellulosic hydrolysates can be reduced by treatment with enzymes such as the lignolytic enzymes, for example, laccase and micro-organisms such as Trichoderma reesei, Coniochaeta ligniaria NRRL30616, Trametes versicolor, Pseudomonas putida Fu1, Candida guilliermondii, and Ureibacillus thermosphaericus. Microbial and enzymatic detoxifications of lignocellulosic hydrolysate are mild and more specific in their action. The efficiency of enzymatic process is quite comparable to other physical and chemical methods. Adaptation of the fermentation yeasts to the lignocellulosic hydrolysate prior to fermentation is suggested as an alternative approach to detoxification. Increases in fermentation rate and ethanol yield by adapted micro-organisms to acid pre-treated lignocellulosic hydrolysates have been reported in some studies. Another approach to alleviate the inhibition problem is to use genetic engineering to introduce increased tolerance by Saccharomyces cerevisiae, for example, by overexpressing genes encoding enzymes for resistance against specific inhibitors and altering co-factor balance. Cloning of the laccase gene followed by heterologous expression in yeasts was shown to provide higher enzyme yields and permit production of laccases with desired properties for detoxification of lignocellulose hydrolysates. A combination of more inhibitor-tolerant yeast strains with efficient feed strategies such as fed-batch will likely improve lignocellulose-to-ethanol process robustness.     

Prefermentation improves xylose utilization in simultaneous saccharification and co-fermentation of pretreated spruce

Background  

Simultaneous saccharification and fermentation (SSF) is a promising process option for ethanol production from lignocellulosic materials. However, both the overall ethanol yield and the final ethanol concentration in the fermentation broth must be high. Hence, almost complete conversion of both hexoses and pentoses must be achieved in SSF at a high solid content. A principal difficulty is to obtain an efficient pentose uptake in the presence of high glucose and inhibitor concentrations. Initial glucose present in pretreated spruce decreases the xylose utilization by yeast, due to competitive inhibition of sugar transport. In the current work, prefermentation was studied as a possible means to overcome the problem of competitive inhibition. The free hexoses, initially present in the slurry, were in these experiments fermented before adding the enzymes, thereby lowering the glucose concentration.     

Biotechnological production of ethanol: Biochemistry,processes and technologies

The majority of environmental problems arise from the use of conventional energy sources. The liability of such problems along with the reduction of fossil energy resources has led to the global need for alternative renewable energy sources. Using renewable biofuels as energy sources is of remarkable and continuously growing importance. Producing bioethanol through conversion of waste and residual biomass can be a viable and important perspective. In the first part of this review, general concepts, approaches and considerations concerning the utilization of the most important liquid biofuels, namely biodiesel and bioethanol, are presented. Unlike biodiesel (specifically first generation biodiesel), the production of bioethanol is exclusively based on the utilization of microbial technology and fermentation engineering. In the second part of this review, the biochemistry of ethanol production, with regards to the use of hexoses, pentoses or glycerol as carbon sources, is presented and critically discussed. Differences in the glycolytic pathways between the major ethanol‐producing strains (Saccharomyces cerevisiae and Zymomonas mobilis) are presented. Regulation between respiration and fermentation in ethanol‐producing yeasts, viz. effects “Pasteur”, “Crabtree”, “Kluyver” and “Custers”, is discussed. Xylose and glycerol catabolism related with bioethanol production is also depicted and commented. The technology of the fermentation is presented along with a detailed illustration of the substrates used in the process and in pretreatment of lignocellulosic biomass, and the various fermentation configurations employed (separate hydrolysis and fermentation, simultaneous saccharification and fermentation, simultaneous saccharification and co‐fermentation and consolidated bioprocessing). Finally, the production of bioethanol under non‐aseptic conditions is presented and discussed.     

Production of 2,3-butanediol from D-xylose by Klebsiella oxytoca ATCC 8724

It is known that 2,3-butanediol is a potentially valuable chemical feedstock that can be produced from the sugars present in hemicellulose and celluose hydrolysates. Klebsiella oxytoca is able to ferment most pentoses, hexoses, and disaccharides. Butanediol appears to be a primary metabolite, excreted as a product of energy methabolism. The theoretical maximum yield of butanediol from monosaccharides is 0.50 g/g. This article describes the effects of pH, xylose concentration, and the oxygen transfer rate on the bioconversion of D-xylose to 2,3-butanediol. Product inhibition by butanediol is also examined. The most important variable affecting the kinetics of this system appears to be the oxygen transfer rate. A higher oxygen supply favors the formation of cell mass at the expense of butanediol. Decreasing the oxygen supply rate increases the butanediol yield, but decreases the overall conversion rate due to a lower cell concentration.     

APJ1 and GRE3 homologs work in concert to allow growth in xylose in a natural Saccharomyces sensu stricto hybrid yeast

Creating Saccharomyces yeasts capable of efficient fermentation of pentoses such as xylose remains a key challenge in the production of ethanol from lignocellulosic biomass. Metabolic engineering of industrial Saccharomyces cerevisiae strains has yielded xylose-fermenting strains, but these strains have not yet achieved industrial viability due largely to xylose fermentation being prohibitively slower than that of glucose. Recently, it has been shown that naturally occurring xylose-utilizing Saccharomyces species exist. Uncovering the genetic architecture of such strains will shed further light on xylose metabolism, suggesting additional engineering approaches or possibly even enabling the development of xylose-fermenting yeasts that are not genetically modified. We previously identified a hybrid yeast strain, the genome of which is largely Saccharomyces uvarum, which has the ability to grow on xylose as the sole carbon source. To circumvent the sterility of this hybrid strain, we developed a novel method to genetically characterize its xylose-utilization phenotype, using a tetraploid intermediate, followed by bulk segregant analysis in conjunction with high-throughput sequencing. We found that this strain's growth in xylose is governed by at least two genetic loci, within which we identified the responsible genes: one locus contains a known xylose-pathway gene, a novel homolog of the aldo-keto reductase gene GRE3, while a second locus contains a homolog of APJ1, which encodes a putative chaperone not previously connected to xylose metabolism. Our work demonstrates that the power of sequencing combined with bulk segregant analysis can also be applied to a nongenetically tractable hybrid strain that contains a complex, polygenic trait, and identifies new avenues for metabolic engineering as well as for construction of nongenetically modified xylose-fermenting strains.     

Repeated-batch fermentations of xylose and glucose-xylose mixtures using a respiration-deficient Saccharomyces cerevisiae engineered for xylose metabolism

Xylose-fermenting Saccharomyces strains are needed for commercialization of ethanol production from lignocellulosic biomass. Engineered Saccharomyces cerevisiae strains expressing XYL1, XYL2 and XYL3 from Pichia stipitis, however, utilize xylose in an oxidative manner, which results in significantly lower ethanol yields from xylose as compared to glucose. As such, we hypothesized that reconfiguration of xylose metabolism from oxidative into fermentative manner might lead to efficient ethanol production from xylose. To this end, we generated a respiration-deficient (RD) mutant in order to enforce engineered S. cerevisiae to utilize xylose only through fermentative metabolic routes. Three different repeated-batch fermentations were performed to characterize characteristics of the respiration-deficient mutant. When fermenting glucose as a sole carbon source, the RD mutant exhibited near theoretical ethanol yields (0.46 g g(-1)) during repeated-batch fermentations by recycling the cells. As the repeated-batch fermentation progressed, the volumetric ethanol productivity increased (from 7.5 to 8.3 g L(-1)h(-1)) because of the increased biomass from previous cultures. On the contrary, the mutant showed decreasing volumetric ethanol productivities during the repeated-batch fermentations using xylose as sole carbon source (from 0.4 to 0.3 g L(-1)h(-1)). The mutant did not grow on xylose and lost fermenting ability gradually, indicating that the RD mutant cannot maintain a good fermenting ability on xylose as a sole carbon source. However, the RD mutant was capable of fermenting a mixture of glucose and xylose with stable yields (0.35 g g(-1)) and productivities (0.52 g L(-1)h(-1)) during the repeated-batch fermentation. In addition, ethanol yields from xylose during the mixed sugar fermentation (0.30 g g(-1)) were higher than ethanol yields from xylose as a sole carbon source (0.21 g g(-1)). These results suggest that a strategy for increasing ethanol yield through respiration-deficiency can be applied for the fermentation of lignocellulosic hydrolyzates containing glucose and xylose.     

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

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 degrees 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%.     

Fermentation of D-xylose, xylitol, and D-xylulose by yeasts

Fifteen yeasts which can assimilate D-xylose were examined for the ability to convert this pentose to ethanol. In six of the seven genera investigated the conversion was enhanced when air had access to the medium. Therefore, the ability to convert D-xylose to ethanol under these conditions is probably common among yeasts. Growth under the same conditions on xylitol, a putative catabolite of D-xylose, led to only traces of ethanol. The effects of growth on another putative catabolite, D-xylose, were complex, but some of the strains which were among the better producers of ethanol from D-xylose produced less from D-xylulose.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.