Biohydrogen production from pretreated lignocellulose by <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

In consolidated bioprocessing (CBP), the difference in optimum temperature between saccharification and fermentation poses a significant technical challenge to producing bioenergy efficiently with lignocellulose. The thermophilic anaerobic strain of Clostridium thermocellum has the potential to overcome this challenge if hydrolysis and fermentation is performed at an elevated temperature. However, this strain is sensitive to structure and components of lignocellulosic materials. To understand biohydrogen production from lignocellulosic materials, C. thermocellum was examined for biohydrogen production as well as bioconversion from different cellulosic materials (Avicel, filter paper and sugarcane bagasse (SCB)). We investigated hydrolysis-inhibitory effects of the cellulosic material types on the substrate degradation and biohydrogen production of C. thermocellum 27405. Within 168 h, the substrate degradation ratios of Avicel, filter paper, and SCB were 83.01, 51.78, and 42.19%, respectively. The substrate utilization and biohydrogen production of SCB reached 81 and 89.77% those of filter paper, respectively, indicating that SCB is a feasible substrate for biohydrogen production. Additionally, optimizing fermentation conditions can improve biohydrogen production, with the optimal conditions being an inoculum size of 7%, substrate concentration of 2%, particle size of 0.074 mm, and yeast extract concentration of 1%. This research provides important clues in relation to the low-cost conversion of renewable biomass to biohydrogen.     

Nanoengineered cellulosic biohydrogen production via dark fermentation: A novel approach

The insights of nanotechnology for cellulosic biohydrogen production through dark fermentation are reviewed. Lignocellulosic biomass to sugar generation is a complex process and covers the most expensive part of cellulose to sugar production technology. In this context, the impacts of nanomaterial on lignocellulosic biomass to biohydrogen production process have been reviewed. In addition, the feasibility of nanomaterials for implementation in each step of the cellulosic biohydrogen production is discussed for economic viability of the process. Numerous aspects such as possible replacement of chemical pretreatment method using nanostructured materials, use of immobilized enzyme for a fast rate of reaction and its reusability along with long viability of microbial cells and hydrogenase enzyme for improving the productivity are the highlights of this review. It is found that various types of nanostructured materials e.g. metallic nanoparticles (Fe°, Ni, Cu, Au, Pd, Au), metal oxide nanoparticles (Fe₂O₃, F₃O₄, NiCo₂O₄, CuO, NiO, CoO, ZnO), nanocomposites (Si@CoFe₂O₄, Fe₃O₄/alginate) and graphene-based nanomaterials can influence different parameters of the process and therefore may perhaps be utilized for cellulosic biohydrogen production_. The emphasis has been given on the cost issue and synthesis sustainability of nanomaterials for making the biohydrogen technology cost effective. Finally, recent advancements and feasibility of nanomaterials as the potential solution for improved cellulose conversion to the biohydrogen production process have been discussed, and this is likely to assist in developing an efficient, economical and sustainable biohydrogen production technology.     

Comparative evaluations of cellulosic raw materials for second generation bioethanol production

Aims: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. Methods and Results: Dilute acid pretreatment (2% H₂SO₄) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60–75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment. Conclusions: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. Significance and Impact of the Study: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food‐based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.     

Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Biohydrogen production from lignocellulosic feedstock

Due to the recent energy crisis and rising concern over climate change, the development of clean alternative energy sources is of significant interest. Biohydrogen produced from cellulosic feedstock, such as second generation feedstock (lignocellulosic biomass) and third generation feedstock (carbohydrate-rich microalgae), is a promising candidate as a clean, CO₂-neutral, non-polluting and high efficiency energy carrier to meet the future needs. This article reviews state-of-the-art technology on lignocellulosic biohydrogen production in terms of feedstock pretreatment, saccharification strategy, and fermentation technology. Future developments of integrated biohydrogen processes leading to efficient waste reduction, low CO₂ emission and high overall hydrogen yield is discussed.     

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.     

<Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> as a potent biocatalyst for the bioconversion of pentose sugars to value-added products

Corynebacterium glutamicum, the industrial microbe traditionally used for the production of amino acids, proved its value for the fermentative production of diverse products through genetic/metabolic engineering. A successful demonstration of the heterologous expression of arabinose and xylose utilization genes made them interesting biocatalysts for pentose fermentation, which are the main components in lignocellulosic hydrolysates. Its ability to withstand substantial amount of general growth inhibitors like furfurals, hydroxyl methyl furfurals and organic acids generated from the acid/alkali hydrolysis of lignocellulosics in growth arrested conditions and its ability to produce amino acids like glutamate and lysine in acid hydrolysates of rice straw and wheat bran, indicate the future prospective of this bacterium as a potent biocatalyst in fermentation biotechnology. However, the efforts so far on these lines have not yet been reviewed, and hence an attempt is made to look into the efficacy and prospects of C. glutamicum to utilize the normally non-fermentable pentose sugars from lignocellulosic biomass for the production of commodity chemicals.     

Biohydrogen production from palm oil mill effluent using immobilized Clostridium butyricum EB6 in polyethylene glycol

A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium butyricum EB6 to improve biohydrogen (bio-H₂) production from palm oil mill effluent (POME). POME is used as a substrate that can act as a carbon source. The resulting PEG-immobilized cells were found to yield 5.35 LH₂/L-POME, and the maximum H₂ production rate was 510 mL H₂/L-POME h (22.7 mmol/L h). The Monod-type kinetic model was used to describe the effect of substrate (POME) concentration on the H₂ production rate. The acclimation of immobilized cells greatly improved H₂ production. Batch experiments demonstrated that particle size of PEG-immobilized cells for efficient H₂ production 3 mm. It is significant that this is the first report on whole-cell immobilization in PEG for H₂ production from POME.     

1-Ethyl-3-methylimidazolium tolerance and intracellular lipid accumulation of 38 oleaginous yeast species

Pretreatment with ionic liquids (IL) such as 1-ethyl-3-methylimidazolium chloride or acetate is an effective method for aiding deconstruction of lignocellulosic biomass; however, the residual IL remaining in hydrolysates can be inhibitory to growth of ethanologenic or oleaginous yeasts that have been examined in the literature. The aim of this study was to identify oleaginous yeasts that are tolerant of the IL [C₂C₁Im][OAc] and [C₂C₁Im]Cl using 45 strains belonging to 38 taxonomically diverse species within phyla Ascomycota and Basidiomycota. Yeasts were cultivated in laboratory medium supplemented with 0, 2, or 4% IL in 96-well plates. The eight most tolerant strains were then cultivated in 10-mL media with no IL, 242mM [C₂C₁Im][OAc], or 242mM [C₂C₁Im]Cl. The effects of [C₂C₁Im]⁺ exposure on cell mass production and lipid accumulation varied at the species and strain level. The acetate salt decreased cell biomass and lipid production more severely than did the chloride ion for six strains. Lipid output was not markedly different (2.1 vs. 2.3 g/L) in Yarrowia lipolytica UCDFST 51-30, but decreased from 5 to 65% in other yeasts. An equimolar concentration of the chloride salt resulted in much milder effects, from 25% decrease to 66% increase in lipid output. The highest lipid outputs in this media were 8.3 and 7.9 g/L produced by Vanrija humicola UCDFST 10-1004 and UCDFST 12-717, respectively. These results demonstrated substantial lipid production in the presence of [C₂C₁Im]Cl at concentrations found in lignocellulosic hydrolysates, and thus, these two strains are ideal candidates for further investigation.

     

Ethanol production from xylose in engineered Saccharomyces cerevisiae strains: current state and perspectives

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.     

Bioethanol,biohydrogen and biogas production from wheat straw in a biorefinery concept

The production of bioethanol, biohydrogen and biogas from wheat straw was investigated within a biorefinery framework. Initially, wheat straw was hydrothermally liberated to a cellulose rich fiber fraction and a hemicellulose rich liquid fraction (hydrolysate). Enzymatic hydrolysis and subsequent fermentation of cellulose yielded 0.41 g-ethanol/g-glucose, while dark fermentation of hydrolysate produced 178.0 ml-H₂/g-sugars. The effluents from both bioethanol and biohydrogen processes were further used to produce methane with the yields of 0.324 and 0.381 m³/kg volatile solids (VS)_added, respectively. Additionally, evaluation of six different wheat straw-to-biofuel production scenaria showed that either use of wheat straw for biogas production or multi-fuel production were the energetically most efficient processes compared to production of mono-fuel such as bioethanol when fermenting C6 sugars alone. Thus, multiple biofuels production from wheat straw can increase the efficiency for material and energy and can presumably be more economical process for biomass utilization.     

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.

     

Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain

The production of fuel ethanol from low‐cost lignocellulosic biomass currently suffers from several limitations. One of them is the presence of inhibitors in lignocellulosic hydrolysates that are released during pre‐treatment. These compounds inhibit growth and hamper the production of ethanol, thereby affecting process economics. To delineate the effects of such complex mixtures, we conducted a chemical analysis of four different real‐world lignocellulosic hydrolysates and determined their toxicological effect on yeast. By correlating the potential inhibitor abundance to the growth‐inhibiting properties of the corresponding hydrolysates, we identified furfural as an important contributor to hydrolysate toxicity for yeast. Subsequently, we conducted a targeted evolution experiment to improve growth behaviour of the half industrial Saccharomyces cerevisiae strain TMB3400 in the hydrolysates. After about 300 generations, representative clones from these evolved populations exhibited significantly reduced lag phases in medium containing the single inhibitor furfural, but also in hydrolysate‐supplemented medium. Furthermore, these strains were able to grow at concentrations of hydrolysates that effectively killed the parental strain and exhibited significantly improved bioconversion characteristics under industrially relevant conditions. The improved resistance of our evolved strains was based on their capacity to remain viable in a toxic environment during the prolonged, furfural induced lag phase.     

Consolidated bioprocessing performance of <Emphasis Type="Italic">Thermoanaerobacterium thermosaccharolyticum</Emphasis> M18 on fungal pretreated cornstalk for enhanced hydrogen production

Background

Biological hydrogen production from lignocellulosic biomass shows great potential as a promising alternative to conventional hydrogen production methods, such as electrolysis of water and coal gasification. Currently, most researches on biohydrogen production from lignocellulose concentrate on consolidated bioprocessing, which has the advantages of simpler operation and lower cost over processes featuring dedicated cellulase production. However, the recalcitrance of the lignin structure induces a low cellulase activity, making the carbohydrates in the hetero-matrix more unapproachable. Pretreatment of lignocellulosic biomass is consequently an extremely important step in the commercialization of biohydrogen, and for massive realization of lignocellulosic biomass as alternative fuel feedstock. Thus, development of a pretreatment method which is cost efficient, environmentally benign, and highly efficient for enhanced consolidated bioprocessing of lignocellulosic biomass to hydrogen is essential.

Results

In this research, fungal pretreatment was adopted for enhanced hydrogen production by consolidated bioprocessing performance. To confirm the fungal pretreatment efficiency, two typical thermochemical pretreatments were also compared side by side. Results showed that the fungal pretreatment was superior to the other pretreatments in terms of high lignin reduction of up to 35.3% with least holocellulose loss (the value was only 9.5%). Microscopic structure observation combined with Fourier transform infrared spectroscopy (FTIR) analysis further demonstrated that the lignin and crystallinity of lignocellulose were decreased with better holocellulose reservation. Upon fungal pretreatment, the hydrogen yield and hydrogen production rate were 6.8 mmol H₂ g^-1 pretreated substrate and 0.89 mmol L^-1 h^-1, respectively, which were 2.9 and 4 times higher than the values obtained for the untreated sample.

Conclusions

Results revealed that although all pretreatments could contribute to the enhancement of hydrogen production from cornstalk, fungal pretreatment proved to be the optimal method. It is apparent that besides high hydrogen production efficiency, fungal pretreatment also offered several advantages over other pretreatments such as being environmentally benign and energy efficient. This pretreatment method thus has great potential for application in consolidated bioprocessing performance of hydrogen production.

     

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.     

An effective method for bioconversion of delignified waste-cellulose fibers from the paper industry with a cellulase complex

An effective method for production of glucose was developed using enzymatic hydrolysis of waste-cellulose fibers by the cellulase complex from Trichoderma reesei. However, these cellulosic materials are strongly resistant to direct enzymatic hydrolysis (the degree of degradation after 48 h of enzyme treatment did not exceed 14%) apparently due to the presence of various chemical substances used in the process of paper production. This adhesive “envelope” around the cellulose fibers was effectively pretreated with 0.25% H₃PO₄ and after that the free cellulose mass was extensively hydrolyzed by the cellulase complex (degree of degradation more than 80%). The HPLC analyses of the enzyme hydrolysates revealed glucose as the main component as well as some cellobiose and xylose.     

Integrated system approach to dark fermentative biohydrogen production for enhanced yield,energy efficiency and substrate recovery

The challenges of climate change, dwindling fossil reserves, and environmental pollution have fuelled the need to search for clean and sustainable energy resources. The process of biohydrogen has been highlighted as a propitious alternative energy of the future because it has many socio-economic benefits such as non-polluting features, the ability to use diverse feedstocks including waste materials, the process uses various microorganisms, and it is the simplest method of producing hydrogen. However, the establishment of a biohydrogen driven economy has been hindered by low process yields due to the accumulation of inhibitory products. Over the past few years, various optimization methods have been used in literature. Among these, integration of bioprocesses is gaining increasing prominence as an effective approach that could be used to achieve a theoretical yield of 4 mol H₂ mol^?1 glucose. In batch integrated systems, dark fermentation is used as a primary process for conversion of substrates into biohydrogen, carbon dioxide, and volatile fatty acids. This is followed by a secondary anaerobic process for further biohydrogen conversion efficiency. This review discusses the current challenges facing scale-up studies in dark fermentation process. It elucidates the potential of batch integrated systems in biohydrogen process development. Furthermore, it explores the various integrated fermentation techniques that are employed in biohydrogen process development. Finally, the review concludes with recommendations on improvement of these integrated processes for enhanced biohydrogen yields which could pave a way for the establishment of a large-scale biohydrogen production process.     

Detoxification of model phenolic compounds in lignocellulosic hydrolysates with peroxidase for butanol production from <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In the present study, we investigated the peroxidase-catalyzed detoxification of model phenolic compounds and evaluated the inhibitory effects of the detoxified solution on butanol production by Clostridium beijerinckii National Collection of Industrial and Marine Bacteria Ltd. 8052. The six phenolic compounds, p-coumaric acid, ferulic acid, 4-hydroxybenzoic acid, vanillic acid, syringaldehyde, and vanillin, were selected as model fermentation inhibitors generated during pretreatment and hydrolysis of lignocellulose. The enzyme reaction was optimized as a function of the reaction conditions of pH, peroxidase concentration, and hydrogen peroxide to substrate ratio. Most of the tested phenolics have a broad optimum pH range of 6.0 to 9. Removal efficiency increased with the molar ratio of H₂O₂ to each compound up to 0.5–1.25. In the case of p-coumaric acid, ferulic acid, vanillic acid, and vanillin, the removal efficiency was almost 100% with only 0.01 μM of enzyme. The tested phenolic compounds (1 g/L) inhibited cell growth by 64–74%, while completely inhibiting the production of butanol. Although syringaldehyde and vanillin were less toxic on cell growth, the level of inhibition on the butanol production was quite different. The detoxified solution remarkably improved cell growth and surprisingly increased butanol production to the level of the control. Hence, our present study, using peroxidase for the removal of model phenolic compounds, could be applied towards the detoxification of lignocellulosic hydrolysates for butanol fermentation.     

Production of lactic acid and ethanol by Rhizopus oryzae integrated with cassava pulp hydrolysis

Cassava pulp was hydrolyzed with acids or enzymes. A high glucose concentration (>100 g/L) was obtained from the hydrolysis with 1 N HCl at 121 °C, 15 min or with cellulase and amylases. While a high glucose yield (>0.85 g/g dry pulp) was obtained from the hydrolysis with HCl, enzymatic hydrolysis yielded only 0.4 g glucose/g dry pulp. These hydrolysates were used as the carbon source in fermentation by Rhizopus oryzae NRRL395. R. oryzae could not grow in media containing the hydrolysates treated with 1.5 N H₂SO₄ or 2 N H₃PO₄, but no significant growth inhibition was found with the hydrolysates from HCl (1 N) and enzyme treatments. Higher ethanol yield and productivity were observed from fermentation with the hydrolysates when compared with those from fermentation with glucose in which lactic acid was the main product. This was because the extra organic nitrogen in the hydrolysates promoted cell growth and ethanol production.     

Metabolic pathway engineering based on metabolomics confers acetic and formic acid tolerance to a recombinant xylose-fermenting strain of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance.