Commercial Bioethanol Production Research Articles

Development of a Robust Saccharomyces cerevisiae Strain for Efficient Co-Fermentation of Mixed Sugars and Enhanced Inhibitor Tolerance through Protoplast Fusion.

The economical and efficient commercial production of second-generation bioethanol requires fermentation microorganisms capable of entirely and rapidly utilizing all sugars in lignocellulosic hydrolysates. In this study, we developed a recombinant Saccharomyces cerevisiae strain, BLH510, through protoplast fusion and metabolic engineering to enhance its ability to co-ferment glucose, xylose, cellobiose, and xylooligosaccharides while tolerating various inhibitors commonly found in lignocellulosic hydrolysates. The parental strains, LF1 and BLN26, were selected for their superior glucose/xylose co-fermentation capabilities and inhibitor tolerance, respectively. The fusion strain BLH510 demonstrated efficient utilization of mixed sugars and high ethanol yield under oxygen-limited conditions. Under low inoculum conditions, strain BLH510 could completely consume all four kinds of sugars in the medium within 84 h. The fermentation produced 33.96 g/L ethanol, achieving 84.3% of the theoretical ethanol yield. Despite the challenging presence of mixed inhibitors, BLH510 successfully metabolized all four sugars above after 120 h of fermentation, producing approximately 30 g/L ethanol and reaching 83% of the theoretical yield. Also, strain BLH510 exhibited increased intracellular trehalose content, particularly under conditions with mixed inhibitors, where the intracellular trehalose reached 239.3 mg/g yeast biomass. This elevated trehalose content contributes to the enhanced stress tolerance of BLH510. The study also optimized conditions for protoplast preparation and fusion, balancing high preparation efficiency and satisfactory regeneration efficiency. The results indicate that BLH510 is a promising candidate for industrial second-generation bioethanol production from lignocellulosic biomass, offering improved performance under challenging fermentation conditions. Our work demonstrates the potential of combining protoplast fusion and metabolic engineering to develop superior S. cerevisiae strains for lignocellulosic bioethanol production. This approach can also be extended to develop robust microbial platforms for producing a wide array of lignocellulosic biomass-based biochemicals.

Bioprospecting of Indigenous Microbial Species for Bioethanol Production from the Fruits of Some Selected Exotic Ornamental Plants

The study determined the ability of some selected indigenous microbial isolates, namely Bacillus subtilis, Zymomonas mobilis and Saccharomyces cerevisiae, in the production of bioethanol from the fruits of some exotic plants (i.e. Ixora coccinea, Duranta repens and Syzygium guineense). B. subtilis was isolated from soil, while Z. mobilis and S. cerevisiae were isolated from palm wine using cultural methods. The organisms were identified based on their colony characteristics and morphological features, which were observed by microscopic examination of the stained smears of the isolates. The identity of the organisms was confirmed through the molecular study of the 16S rRNA and 18S rRNA regions of the microbial genomes using Polymerase Chain Reaction – Denaturing Gradient Gel Electrophoresis (PCR - DGGE). The fruit samples were collected, washed, blended, filtered using a clean piece of cloth and pretreated using ultrasonication and autoclave. Concentrations of reducing sugars in the fruit filtrates were determined using DNSA-UV-visible spectrophotometry. Seven (7) conical flasks (500ml capacity) were arranged for each fruit sample in duplicate, and 250ml of the fruit filtrates were dispensed in the flasks accordingly. The fermenting organisms were inoculated in the flasks containing the fruit filtrates singly and in combination. Flasks were incubated in shaking incubator for seven days at 35oC. After incubation, the concentrations of bioethanol produced were determined using the UV-visible spectrophotometry. Highest reducing sugars concentrations of 43.7% and 43.6% were found in I. coccinea and S. guineense, respectively, while 36.4% was recorded for D. repens. Highest bioethanol yield of 27.5m/L was found in I. coccinea fermented by S. cerevisiae and Z. mobilis, while the lowest yield of 17.25m/L was found D. repens. The yield in bioethanol production was higher in samples containing a combination of fermenting organisms compared to ones with individual cells. It was recommended that the fruits of these exotic plants should be considered for commercial production of bioethanol.

Enhanced Bioethanol Production from Corn Stover Using Choline Chloride and Lactic Acid Pretreatment: A Gravimetric analysis Approach

The transition to renewable energy sources necessitates the development of efficient and sustainable bioethanol production methods. This study investigates the use of a deep eutectic solvent (DES) blend of choline chloride and lactic acid for the pretreatment of corn stover, a widely available lignocellulosic biomass, to enhance cellulose yield and bioethanol production. The gravimetric method was employed to quantify cellulose yield post-pretreatment, providing a direct assessment of pretreatment efficacy. The study further evaluated the subsequent steps of enzymatic hydrolysis, fermentation, and distillation to determine the overall bioethanol yield. Results demonstrated that the choline chloride and lactic acid blend effectively disrupted the lignocellulosic structure of corn stover, significantly increasing cellulose accessibility and fermentable sugar release. The optimized pretreatment process led to a notable improvement in bioethanol yield, highlighting the potential of this method for commercial bioethanol production. This research contributes to the advancement of biofuel technologies and supports the broader goal of achieving sustainable energy solutions by demonstrating a viable approach to enhancing the efficiency and sustainability of lignocellulosic bioethanol production.

Optimization of parameters for bio-ethanol production from sweet sorghum (Sorghum bicolor (L.) Moench) stalk juice and finger millet malt using Taguchi method

Bio-ethanol is a promising renewable energy but its production is expensive from high cost of feedstocks. In this study, sweet sorghum (Sorghum bicolor (L.) Moench) stalk juice was investigated for bio-ethanol production. Most reports on bio-ethanol productions use commercial Saccharomyces cerevisiae as yeast. However, this study used finger millet (Eleusine coracana) malt with already high adaptation to local conditions and high economic viability as it is being utilized by the indigenous communities. Five sweet sorghum varieties of IESV-92001-DL (V1), NTJ (V2), 15233-IESV (V3), 92008-DJ (V4) and IESV-92028-DL (V5) were planted at 0°3'45.4644" North, 34°17'16.1052" South, in Kenya. °Brix content of juice was determined at 11th to 16th weeks after sowing. Highest °Brix for all varieties were at 15th week where V1 was highest at 22.07. V1 was then harvested for the juice. Factors affecting fermentation; temperature, time, pH and yeast to substrate ratio were optimized using Taguchi method and were obtained as 30 ℃, 48 hours, pH 5 and 5 g/L, respectively. Kinetics parameters of Vmax and Km were 0.35 g/L/h and 12.56 g/L, respectively. The optimized and kinetic parameters were within literature values and therefore finger millet malt has a great potential, as a substitute yeast source, in commercial bio-ethanol production.
 KEY WORDS: Bio-ethanol, Sweet sorghum juice, Taguchi method
 Bull. Chem. Soc. Ethiop. 2024, 38(1), 55-67. DOI: https://dx.doi.org/10.4314/bcse.v38i1.5

The Production of Bioethanol from Lignocellulosic Biomass: Pretreatment Methods, Fermentation, and Downstream Processing

Bioethanol is the most widely used alternative transportation fuel to petrol. Bioethanol is considered a clean, renewable, and environmentally friendly fuel that can contribute to climate change mitigation, decreased environmental pollution, and enhanced energy security. Commercial bioethanol production is based on traditional agricultural crops such as corn, sugarcane, and sugarbeet, primarily used as food and feed. In order to meet the growing demand for this fuel and decrease competition in the food and biofuel sectors for the same feedstock, other raw materials and process technologies have been intensively studied. Lignocellulosic biomass is one of the most abundant renewable resources, with it being rich in compounds that could be processed into energy, transportation fuels, various chemical compounds, and diverse materials. Bioethanol production from lignocellulosic biomass has received substantial attention in recent decades. This review gives an overview of bioethanol production steps from lignocellulosic biomass and challenges in the production process. The following aspects of bioethanol production are covered here, including pretreatment methods, process strategies, strain development, ethanol isolation and purification, and technical hurdles.

Assessment of the TRX2p-yEGFP Biosensor to Monitor the Redox Response of an Industrial Xylose-Fermenting Saccharomyces cerevisiae Strain during Propagation and Fermentation.

The commercial production of bioethanol from lignocellulosic biomass such as wheat straw requires utilizing a microorganism that can withstand all the stressors encountered in the process while fermenting all the sugars in the biomass. Therefore, it is essential to develop tools for monitoring and controlling the cellular fitness during both cell propagation and sugar fermentation to ethanol. In the present study, on-line flow cytometry was adopted to assess the response of the biosensor TRX2p-yEGFP for redox imbalance in an industrial xylose-fermenting strain of Saccharomyces cerevisiae during cell propagation and the following fermentation of wheat-straw hydrolysate. Rapid and transient induction of the sensor was recorded upon exposure to furfural and wheat straw hydrolysate containing up to 3.8 g/L furfural. During the fermentation step, the induction rate of the sensor was also found to correlate to the initial ethanol production rate, highlighting the relevance of redox monitoring and the potential of the presented tool to assess the ethanol production rate in hydrolysates. Three different propagation strategies were also compared, and it was confirmed that pre-exposure to hydrolysate during propagation remains the most efficient method for high ethanol productivity in the following wheat-straw hydrolysate fermentations.

Bioprocessing of underutilized Artocarpus altilis fruit to bioethanol by Saccharomyces cerevisiae: A fermentation condition improvement study

Raw materials availability needed for commercial bioethanol production is one of the challenges against its global adoption. Identifying rich, cheap, underused, and readily available starch sources for bioethanol production could help address the problem. Thus, this current study investigated the bioconversion of underutilized Artocarpus altilis (breadfruit) starch to bioethanol using Saccharomyces cerevisiae. The effects of the essential fermentation conditions (fermentation time, breadfruit starch hydrolysate (BSH) concentration, pH, and inoculum size) and their interactions on bioethanol production were investigated. The central composite design was used to generate twenty-one experiments conducted under batch fermentation conditions in the laboratory. The breadfruit starch hydrolysis led to a BSH concentration of 108.9 g/L under a starch concentration of 122 g/L, microwave output of 720 W, and an incubation time of 6 min. For the fermentation of BSH, maximum bioethanol production of 4.99% (V) was reached under the cultivation conditions of BSH concentration of 80 g/L, medium pH of 4.7, inoculum size of 2% (V), and fermentation time of 20.41 h. Except for pH, the impact of each parameter on the bioethanol production was in this order: BSH concentration, inoculum size, and fermentation time. While for the interactions amongst the parameters, the impact is in this order: BSH concentration and inoculum size; BSH concentration and fermentation time; and fermentation time and inoculum size. The results of this study indicated breadfruit starch could be hydrolyzed using acid hydrolysis and microwave irradiation in a relatively short time. The BSH obtained could potentially add to other substrates for bioethanol production.

Progress and development of syngas fermentation processes toward commercial bioethanol production

AbstractSyngas is created through the thermochemical conversion of biomass using gasification or pyrolysis and from CO‐rich off‐gases obtained from industries such as steel mills. The Wood–Ljungdahl metabolic pathway, or one of its variations, is used by acetogenic bacteria to convert syngas components (CO, H2, and CO2) to alcohols and other compounds. Many factors affect how well syngas is fermented, including the bacteria species used, syngas composition, medium components, bioreactor type, operational parameters used and the gas–liquid mass transfer rate. These parameters impact carbon and electron flow in the bacteria, influencing the distribution, concentration and metabolic end‐product yield, which determines process feasibility. This article focuses on gas composition, microorganisms, gas–liquid mass transfer fermentation strategies, medium design and commercialization activities to develop the syngas fermentation processes. © 2023 Society of Industrial Chemistry and John Wiley & Sons Ltd.

Characterization and Composition Analysis of Municipal Solid Waste for Bioethanol Production

Every unwanted or non-useful solid substance generated in any human population is referred to as solid waste. Globally, the management of solid waste is an enormous challenge for municipalities. Previous studies have indicated that an increase in population with relating attendant urbanization dynamics and growth of continuous aggregation will further aggravate this challenge. This paper aims at determining the potentials of using municipal solid waste as a feedstock in the commercial production of Bioethanol. The research team which was made up of the coordinator, co-coordinators and research assistants carried out a preliminary survey which consist of mapping the study areas and obtaining some information through the observation of the various sites. The study area which was the residential areas of the University of Benin community were then mapped into Senior Staff Quarters (which comprised of Professorial quarters, senior staff quarters and Blocks of flats), junior staff quarters and Student Hall of Residence. A preliminary survey form was originated for this purpose. Printing of questionnaires, sample notes, pamphlets, stickers and tagging of bin baskets and wheelie bins were done. A three (3) Stage sorting and characterization of respondents’ waste were carried out. Composition of waste was determined after which the digital weighing balance was used to determine the mass of the waste which was recorded, before allowing proper disposal of the waste. Samples of sorted waste (organic) were collected into sample bottles and taken to the laboratory for microbial analysis. The solid waste generated is made of nine major components for which color coded bags was used to differentiate them (fruit, food, plastic, paper, glass, metals, combustibles, leaves, and ceramics). The study showed that 58.85% of the total solid waste generated in the residential areas of the University of Benin was made of biodegradable matter (food and fruit) which can be harnessed for the production of Bioethanol. The recyclables (Papers and Plastics) which are 36.34 % of the waste can also be recycled into other useful products. Efforts should be made by stakeholders to evolve sustainable waste management policies for disposal, waste reduction and recycling projects.

Environmental ramifications and economic viability of bioethanol production in Malaysia

This paper employs a partial equilibrium model to investigate the economic and environmental impact of commercial bioethanol production from biomass on the Malaysian agricultural markets. The results show that the palm oil mills can produce bioethanol commercially as the mills utilize the waste biomass created onsite and thus avoid the feedstock and hauling costs. Furthermore, by consuming biomass that recycles carbon dioxide from the atmosphere, a large bioethanol industry would help Malaysia reduce its GHG emissions and meet its GHG commitments in the Paris Agreement. With a potential to lead to greater forestation as landowners clear rainforests to enlarge oil palm plantations, such an industry could also be expected to expand the agricultural industry, reduce the agricultural prices, and raise agricultural employment.

Understanding Ethanol Tolerance Mechanism in Saccharomyces cerevisiae to Enhance the Bioethanol Production: Current and Future Prospects

The commercial production of bioethanol from lignocellulosic biomass is challenged by the repression of cell growth and compromised fermentation conditions. However, employing Saccharomyces cerevisiae is an economically feasible process resulting in the effective conversion of fermentable sugars to bioethanol. S. cerevisiae’s high productive feature is contributed by robust alcohol dehydrogenase and the ability to tolerate various stresses during the fermentation process. The present review employs various bioinformatics pipeline to assess the structural insights of ADH and interaction among the tolerance genes. Docking of S. cerevisiae’s ADH interaction shows the high binding affinity of − 2.81 Kcal/mol with acetaldehyde contributed by four zinc fingers. In inhibitor tolerance capacity of S. cerevisiae was explored. The STRING platform sheds light on the mechanism and interaction of ASR1 and other stress-responsive elements playing a pivotal role in ethanol tolerance. The stress-responsive genes such as HSPs and MSN2/4 provide balanced physiology under various stress conditions. The present review unravels the complex mechanism behind the inhibitor and ethanol tolerance, directing to several bottlenecks for the improvisation of S. cerevisiae’s performance.

Saccharomyces cerevisiae Bio-Ethanol Production as an Alternative Source of Sustainable Energy Ethanol Production using Saccharomyces cerevisiae

Current world energy demand is based on fossil fuels, which will vanish in coming decades. Renewable energy especially biofuels has attracted great interest as solutions to the current energy problem. Among available biofuel resources, bioethanol seems to be an efficient alternative thus, Saccharomyces cerevisiae a well-established organism for bioethanol production. However, during fermentation process, yeast cells experience various stress conditions and inhibitors hampering its efficacy for commercial bioethanol production. To overcome these yeast cells, adopt different signal transduction pathways. In this review, common and least explored carbon feedstock which can be readily converted into bioethanol are highlighted. The various protectants, genes, and pathways which can be tempered to engineer yeast strains are discussed. Thus, we have suggested strategies to utilize this lucrative alternative for sustainable bioethanol production.

Development and characterization of acidic-pH-tolerant mutants of Zymomonas mobilis through adaptation and next-generation sequencing-based genome resequencing and RNA-Seq

BackgroundAcid pretreatment is a common strategy used to break down the hemicellulose component of the lignocellulosic biomass to release pentoses, and a subsequent enzymatic hydrolysis step is usually applied to release hexoses from the cellulose. The hydrolysate after pretreatment and enzymatic hydrolysis containing both hexoses and pentoses can then be used as substrates for biochemical production. However, the acid-pretreated liquor can also be directly used as the substrate for microbial fermentation, which has an acidic pH and contains inhibitory compounds generated during pretreatment. Although the natural ethanologenic bacterium Zymomonas mobilis can grow in a broad range of pH 3.5 ~ 7.5, cell growth and ethanol fermentation are still affected under acidic-pH conditions below pH 4.0.ResultsIn this study, adaptive laboratory evolution (ALE) strategy was applied to adapt Z. mobilis under acidic-pH conditions. Two mutant strains named 3.6M and 3.5M with enhanced acidic pH tolerance were selected and confirmed, of which 3.5M grew better than ZM4 but worse than 3.6M in acidic-pH conditions that is served as a reference strain between 3.6M and ZM4 to help unravel the acidic-pH tolerance mechanism. Mutant strains 3.5M and 3.6M exhibited 50 ~ 130% enhancement on growth rate, 4 ~ 9 h reduction on fermentation time to consume glucose, and 20 ~ 63% improvement on ethanol productivity than wild-type ZM4 at pH 3.8. Next-generation sequencing (NGS)-based whole-genome resequencing (WGR) and RNA-Seq technologies were applied to unravel the acidic-pH tolerance mechanism of mutant strains. WGR result indicated that compared to wild-type ZM4, 3.5M and 3.6M have seven and five single nucleotide polymorphisms (SNPs), respectively, among which four are shared in common. Additionally, RNA-Seq result showed that the upregulation of genes involved in glycolysis and the downregulation of flagellar and mobility related genes would help generate and redistribute cellular energy to resist acidic pH while keeping normal biological processes in Z. mobilis. Moreover, genes involved in RND efflux pump, ATP-binding cassette (ABC) transporter, proton consumption, and alkaline metabolite production were significantly upregulated in mutants under the acidic-pH condition compared with ZM4, which could help maintain the pH homeostasis in mutant strains for acidic-pH resistance. Furthermore, our results demonstrated that in mutant 3.6M, genes encoding F1F0 ATPase to pump excess protons out of cells were upregulated under pH 3.8 compared to pH 6.2. This difference might help mutant 3.6M manage acidic conditions better than ZM4 and 3.5M. A few gene targets were then selected for genetics study to explore their role in acidic pH tolerance, and our results demonstrated that the expression of two operons in the shuttle plasmids, ZMO0956–ZMO0958 encoding cytochrome bc1 complex and ZMO1428–ZMO1432 encoding RND efflux pump, could help Z. mobilis tolerate acidic-pH conditions.ConclusionAn acidic-pH-tolerant mutant 3.6M obtained through this study can be used for commercial bioethanol production under acidic fermentation conditions. In addition, the molecular mechanism of acidic pH tolerance of Z. mobilis was further proposed, which can facilitate future research on rational design of synthetic microorganisms with enhanced tolerance against acidic-pH conditions. Moreover, the strategy developed in this study combining approaches of ALE, genome resequencing, RNA-Seq, and classical genetics study for mutant evolution and characterization can be applied in other industrial microorganisms.

Effects of acids pre-treatment on the microbial fermentation process for bioethanol production from microalgae

BackgroundMicroalgae are one of the promising feedstock that consists of high carbohydrate content which can be converted into bioethanol. Pre-treatment is one of the critical steps required to release fermentable sugars to be used in the microbial fermentation process. In this study, the reducing sugar concentration of Chlorella species was investigated by pre-treating the biomass with dilute sulfuric acid and acetic acid at different concentrations 1%, 3%, 5%, 7%, and 9% (v/v).Results3,5-Dinitrosalicylic acid (DNS) method, FTIR, and GC-FID were employed to evaluate the reducing sugar concentration, functional groups of alcohol bonds and concentration of bioethanol, respectively. The two-way ANOVA results (p < 0.05) indicated that there was a significant difference in the concentration and type of acids towards bioethanol production. The highest bioethanol yield obtained was 0.28 g ethanol/g microalgae which was found in microalgae sample pre-treated with 5% (v/v) sulfuric acid while 0.23 g ethanol/g microalgal biomass was presented in microalgae sample pre-treated with 5% (v/v) acetic acid.ConclusionThe application of acid pre-treatment on microalgae for bioethanol production will contribute to higher effectiveness and lower energy consumption compared to other pre-treatment methods. The findings from this study are essential for the commercial production of bioethanol from microalgae.

An Introduction to Bioethanol and Its Prospects in Bangladesh: A Review

This study aims to present a comprehensive review of bioethanol, its prospects, and also to estimate a potential source for commercial bioethanol production in Bangladesh. In Bangladesh, the primary source of fuel comes from natural gas and petroleum oil. But the total output of fossil fuel is incapable of meeting the annual fuel demand. And also, the natural reserves are insufficient to meet the energy demand for long-term economic development. To meet the demand every year Bangladesh needs to import a huge amount of petroleum oil from overseas markets. In this situation, to manage the future demand, production of renewable energy may be an alternative solution. Bioethanol production from potato and potato peel waste maybe a prospective source of renewable energy. It can use as an alternative clean-burning fuel by replacing with regular vehicle fuel. It can not only save huge foreign currency but also lessen greenhouse effect in the environment. In this scenario, there is a huge prospect to introduce bioethanol as an alternative fuel in Bangladesh.

Business models for commercial scale second-generation bioethanol production

Abstract Faced with the prospect of depletion of non-renewable sources of fossil fuels and concerns about the emission of substances that compromise the environment, biofuels, and especially bioethanol, have attracted the attention of researchers, companies and governments. Specifically, the second generation of bioethanol has critical stages in the production process which have been limiting for companies to achieve production on a commercial scale. Although research has sought to overcome such constraints by focusing on technological challenges, it has been recognized that the greater or lesser absorption capacity of technological innovations is related to the business model used. The purpose of this article was to analyze the business models used by Brazilian companies that have achieved commercial scale production of second generation bioethanol, studying their business models as commercial viability instruments of technologies, looking to subsidize companies of this segment in formulating their policies to reach commercial scale. The research was carried out through a methodology based on the content analysis technique, which provided the following categories that served as the basis for the analysis of the business models: company focus, raw material, costs, certification, technology and production. Among the six major second-generation commercial ethanol plants in the world - GranBio, Raizen, Poet-DSM, BetaRenewables, Abengoa (plant bought by Synata Bio) and DuPont - two are Brazilian: GranBio and Raizen, which mainly use sugar cane bagasse and straw as raw material. The study demonstrated that both companies have competitive advantages that are equivalent in most of the categories studied, albeit GranBio’s business model performs better in some other categories due to the strategic partnerships it has established with some of the main companies in the segment, thus, better exploring the benefits of the open innovation environment that characterizes the competitive arena.

Ethanol Production from Cassava Pulp by a Newly Isolated Thermotolerant &amp;lt;i&amp;gt;Pichia kudriavzevii&amp;lt;/i&amp;gt; LC375240

A total of 500 thermotolerant fermenting yeast isolates (100 from palm-wine and 400 from spoilt fruits) were screened for ethanol production at high temperatures. The best isolate that produced up to 4% ethanol from 10% glucose at 45°C was selected for further experiments. The optimum pH for ethanol production by the isolate was pH 6 at both 30°C and 42°C. The isolate was identified as Pichia kudriavzevii base on the 18s ribosomal DNA. Ethanol production from 200 g/L cassava pulp using Simultaneous Saccharification and Fermentation (SSF) method at 30°C and 42°C by the isolate was investigated. At 30°C, an ethanol concentration of 30 g/L was produced. This represents an ethanol yield of 0.15 g/g of cassava pulp and 58.8% of the theoretical yield. However at 42°C, the concentration of ethanol produced increased to 42 g/L representing an ethanol yield of 0.21 g/g of cassava pulp and 82.4% of the theoretical yield. The isolate produced slightly higher ethanol than the two typed strains NCYC 587 and NCYC 2791 at 42°C. This isolate has a good potential to be used for commercial bioethanol production since it can produce ethanol at 45°C without a significant drop in ethanol yield.

TALEN-mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield.

SummarySugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first‐ and second‐generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose and cell wall‐bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O‐methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass. Genome editing has been used in a number of crops for creation of loss of function phenotypes but is very challenging in sugarcane due to its highly polyploid genome. In this study, a conserved region of COMT was targeted with a single‐transcription activator‐like effector nuclease (TALEN) pair for multi‐allelic mutagenesis to modify lignin biosynthesis in sugarcane. Field‐grown TALEN‐mediated COMT mutants showed up to 19.7% lignin reduction and significantly decreased syringyl to guaiacyl (S/G) ratio resulting in an up to 43.8% improved saccharification efficiency. Biomass production of COMT mutant lines with superior saccharification efficiency did not differ significantly from the original cultivar under replicated field conditions. Sanger sequencing of cloned COMT amplicons (1351–1657 bp) revealed co‐editing of 107 of the 109 unique COMT copies/alleles in vegetative progeny of line CB6 using a single TALEN pair. Line CB6 combined altered cell wall composition and drastically improved saccharification efficiency with good agronomic performance. These findings confirm the feasibility of co‐mutagenesis of a very large number of target alleles/copies for improvement in crops with complex genomes.

Screening and evolution of a novel protist xylose isomerase from the termite Reticulitermes speratus for efficient xylose fermentation in Saccharomyces cerevisiae

BackgroundThe yeast Saccharomyces cerevisiae, a promising host for lignocellulosic bioethanol production, is unable to metabolize xylose. In attempts to confer xylose utilization ability in S. cerevisiae, a number of xylose isomerase (XI) genes have been expressed heterologously in this yeast. Although several of these XI encoding genes were functionally expressed in S. cerevisiae, the need still exists for a S. cerevisiae strain with improved xylose utilization ability for use in the commercial production of bioethanol. Although currently much effort has been devoted to achieve the objective, one of the solutions is to search for a new XI gene that would confer superior xylose utilization in S. cerevisiae. Here, we searched for novel XI genes from the protists residing in the hindgut of the termite Reticulitermes speratus.ResultsEight novel XI genes were obtained from a cDNA library, prepared from the protists of the R. speratus hindgut, by PCR amplification using degenerated primers based on highly conserved regions of amino acid sequences of different XIs. Phylogenetic analysis classified these cloned XIs into two groups, one showed relatively high similarities to Bacteroidetes and the other was comparatively similar to Firmicutes. The growth rate and the xylose consumption rate of the S. cerevisiae strain expressing the novel XI, which exhibited highest XI activity among the eight XIs, were superior to those exhibited by the strain expressing the XI gene from Piromyces sp. E2. Substitution of the asparagine residue at position 337 of the novel XI with a cysteine further improved the xylose utilization ability of the yeast strain. Interestingly, introducing point mutations in the corresponding asparagine residues in XIs originated from other organisms, such as Piromyces sp. E2 or Clostridium phytofermentans, similarly improved xylose utilization in S. cerevisiae.ConclusionsA novel XI gene conferring superior xylose utilization in S. cerevisiae was successfully isolated from the protists in the termite hindgut. Isolation of this XI gene and identification of the point mutation described in this study might contribute to improving the productivity of industrial bioethanol.

Isolation of Thermophilic Lignin Degrading Bacteria from Oil-Palm Empty Fruit Bunch (EFB) Compost

Empty Fruit Bunch (EFB) is a potential and sustainable feedstock for bioethanol production due to its high cellulosic content and availability in Malaysia. Due to high lignin content of EFB and the lack of effective delignification process, commercial bioethanol production from EFB is presently not viable. Enzymatic delignification has been identified as one of the key steps in utilising EFB as a feedstock for bioethanol conversion. To date, limited work has been reported on the isolation of lignin degrading bacteria. Hence, there is a growing interest to search for new lignin degrading bacteria with greater tolerance to temperature and high level of ligninolytic enzymes for more effective lignin degradation. This study aimed to isolate and screen thermophilic ligninolytic microorganisms from EFB compost. Ten isolates were successfully isolated from EFB compost. Although they are not capable of decolorizing Methylene Blue (MB) dye under agar plate assay method, they are able to utilize lignin mimicked compound – guaiacol as a sole carbon on the agar plate assay. This infers that there is no correlation of ligninolytic enzymes with dye decolourization for all the isolates that have been isolated. However, they are able to produce ligninolytic enzymes (Lignin peroxidase, Manganese peroxidase, Laccase) in Minimal Salt Medium with Kraft Lignin (MSM-KL) with Lignin Peroxidase (LiP) as the predominant enzyme followed by Manganese Peroxidase (MnP) and Laccase (Lac). Among all the tested isolates, CLMT 29 has the highest LiP production up to 8.7673 U/mL following 24 h of growth.