Lignocellulosic Biofuel Production Research Articles

Enhancing deep eutectic solvent systems for efficient fermentable sugar recovery from lignocellulosic fiber

Efficient conversion of sugars into fermentable sugars is a critical challenge in the cost-effective production of lignocellulosic biopolymers and biofuels. This study focuses on various sugar quantification techniques applied to Furcraea Foetida (Mauritius Hemp) samples, utilizing natural deep eutectic solvents (NADES) and deep eutectic solvents (DES) like urea, glycerol, citrates, pyrogallol (PY), and cetyltrimethylammonium bromide (CTAB). Employing a Taguchi-designed experiment, operational conditions were fine-tuned to evaluate the influence of time, concentration, and temperature on each deep eutectic solvent-based process. The emerging green solvent extraction approach demonstrated significant results, achieving notably high sugar yields compared to traditional techniques such as alkali, hot-water, and acid-mediated extraction. At a CTAB:PY molar ratio of 1:3, optimized for 60 min at 50 °C, the highest fermentable sugar (FS) yield of 0.6891 ± 0.0123 g FS/g LCB was attained—2 to 6 times higher than non-optimized values and 0.2 to 0.3 times higher than optimized traditional methods. In light of this, this research study emphasizes the pivotal significance of efficient sugar conversion through optimized deep eutectic solvent-based extraction methods, with a particular focus on Furcraea Foetida fibers, offering promising outcomes for the biofuel and biopolymer production industry.

Cold plasma pretreatment reinforces the lignocellulose-derived aldehyde inhibitors tolerance and bioethanol fermentability for Zymomonas mobilis

BackgroundLignocellulose-derived aldehyde inhibitors seriously blocked the biorefinery of biofuels and biochemicals. To date, the economic production of lignocellulose-based products heavily relied on high productivities of fermenting strains. However, it was expensive and time-consuming for the achievable rational modification to strengthen stress tolerance robustness of aldehyde inhibitors. Here, it aimed to improve aldehyde inhibitors tolerance and cellulosic bioethanol fermentability for the chassis Zymomonas mobilis ZM4 pretreated using energy-efficient and eco-friendly cold plasma.ResultsIt was found that bioethanol fermentability was weaker in CSH (corn stover hydrolysates) than that in synthetic medium for Z. mobilis, and thus was attributed to the inhibition of the lignocellulose-derived aldehyde inhibitors in CSH. Convincingly, it further confirmed that the mixed aldehydes severely decreased bioethanol accumulation through additional aldehydes supplementary assays in synthetic medium. After assayed under different processing time (10–30 s), discharge power (80–160 W), and working pressure (120–180 Pa) using cold atmosphere plasma (CAP), it achieved the increased bioethanol fermentability for Z. mobilis after pretreated at the optimized parameters (20 s, 140 W and 165 Pa). It showed that cold plasma brought about three mutation sites including ZMO0694 (E220V), ZMO0843 (L471L) and ZMO0843 (P505H) via Genome resequencing-based SNPs (single nucleotide polymorphisms). A serial of differentially expressed genes (DEGs) were further identified as the potential contributors for stress tolerance via RNA-Seq sequencing, including ZMO0253 and ZMO_RS09265 (type I secretion outer membrane protein), ZMO1941 (Type IV secretory pathway protease TraF-like protein), ZMOr003 and ZMOr006 (16S ribosomal RNA), ZMO0375 and ZMO0374 (levansucrase) and ZMO1705 (thioredoxins). It enriched cellular process, followed by metabolic process and single-organism process for biological process. For KEGG analysis, the mutant was also referred to starch and sucrose metabolism, galactose metabolism and two-component system. Finally, but interestingly, it simultaneously achieved the enhanced stress tolerance capacity of aldehyde inhibitors and bioethanol fermentability in CSH for the mutant Z. mobilis.ConclusionsOf several candidate genetic changes, the mutant Z. mobilis treated with cold plasma was conferred upon the facilitated aldehyde inhibitors tolerance and bioethanol production. This work would provide a strain biocatalyst for the efficient production of lignocellulosic biofuels and biochemicals.

One-pot synthesis of rod-like lignin@zeolitic imidazolate framework-8 with enhanced immobilization of β-glucosidase

Developing value-added lignin-based biomaterials for enzyme immobilization has drawn great attention. This study developed a cost-effective and eco-friendly one-pot strategy to synthesize the rod-like lignin@zeolitic imidazolate framework-8 (ZIF-8) hybrid nanomaterial by using lignin derived from lignocellulosic biofuel production. The incorporation of lignin into the ZIF-8 network not only altered the morphology, but also modified the surface properties of the material, making it an ideal support for enzyme immobilization. The synthesized nanoscale hybrid materials were used to immobilize beta-glucosidase (BG) with high immobilization capacity and about 92–166 mg/g of BG was immobilized through physical adsorption. The immobilized BG exhibited good stability, catalytic activity and recycling properties, and was reused under acidic conditions for more than 8 cycles with more than 60% activity kept. The as-prepared hybrid material can serve as a great carrier for immobilizing various biomolecules.

Genome-wide analysis of the Populus trichocarpa laccase gene family and functional identification of PtrLAC23.

Biofuel is a kind of sustainable, renewable and environment friendly energy. Lignocellulose from the stems of woody plants is the main raw material for "second generation biofuels". Lignin content limits fermentation yield and is therefore a major obstacle in biofuel production. Plant laccase plays an important role in the final step of lignin formation, which provides a new strategy for us to obtain ideal biofuels by regulating the expression of laccase genes to directly gain the desired lignin content or change the composition of lignin. Multiple sequence alignment and phylogenetic analysis were used to classify PtrLAC genes; sequence features of PtrLACs were revealed by gene structure and motif composition analysis; gene duplication, interspecific collinearity and Ka/Ks analysis were conducted to identify ancient PtrLACs; expression levels of PtrLAC genes were measured by RNA-Seq data and qRT-PCR; domain analysis combine with cis-acting elements prediction together showed the potential function of PtrLACs. Furthermore, Alphafold2 was used to simulate laccase 3D structures, proLAC23::LAC23-eGFP transgenic Populus stem transects were applied to fluorescence observation. A comprehensive analysis of the P. trichocarpa laccase gene (PtLAC) family was performed. Some ancient PtrLAC genes such as PtrLAC25, PtrLAC19 and PtrLAC41 were identified. Gene structure and distribution of conserved motifs clearly showed sequence characteristics of each PtrLAC. Combining published RNA-Seq data and qRT-PCR analysis, we revealed the expression pattern of PtrLAC gene family. Prediction results of cis-acting elements show that PtrLAC gene regulation was closely related to light. Through above analyses, we selected 5 laccases and used Alphafold2 to simulate protein 3D structures, results showed that PtrLAC23 may be closely related to the lignification. Fluorescence observation of proLAC23::LAC23-eGFP transgenic Populus stem transects and qRT-PCR results confirmed our hypothesis again. In this study, we fully analyzed the Populus trichocarpa laccase gene family and identified key laccase genes related to lignification. These findings not only provide new insights into the characteristics and functions of Populus laccase, but also give a new understanding of the broad prospects of plant laccase in lignocellulosic biofuel production.

CAZyme from gut microbiome for efficient lignocellulose degradation and biofuel production

Over-exploitation and energy security concerns of the diminishing fossil fuels is a challenge to the present global economy. Further, the negative impact of greenhouse gases released using conventional fuels has led to the need for searching for alternative biofuel sources with biomass in the form of lignocellulose coming up as among the potent candidates. The entrapped carbon source of the lignocellulose has multiple applications other than biofuel generation under the biorefinery approach. However, the major bottleneck in using lignocellulose for biofuel production is its recalcitrant nature. Carbohydrate Active Enzymes (CAZymes) are enzymes that are employed for the disintegration and consumption of lignocellulose biomass as the carbon source for the production of biofuels and bio-derivatives. However, the cost of enzyme production and their stability and catalytic efficiency under stressed conditions is a concern that hinders large-scale biofuel production and utilization. Search for novel CAZymes with superior activity and stability under industrial condition has become a major research focus in this area considering the fact that the most conventional CAZymes has low commercial viability. The gut of plant-eating herbivores and other organisms is a potential source of CAZyme with high efficiency. The review explores the potential of the gut microbiome of various organisms in the production of an efficient CAZyme system and the challenges in using the biofuels produced through this approach as an alternative to conventional biofuels.

Chemical-Anatomical Characterization of Stems of Asparagaceae Species with Potential Use for Lignocellulosic Fibers and Biofuels

During the last decades, the possibility of using species resistant to droughts and extreme temperatures has been analyzed for use in the production of lignocellulosic materials and biofuels. Succulent species are considered to identify their potential use; however, little is known about Asparagaceae species. Therefore, this work aimed to characterize chemically-anatomically the stems of Asparagaceae species. Stems of 10 representative species of Asparagaceae were collected, and samples were divided into two. One part was processed to analyze the chemical composition, and the second to perform anatomical observations. The percentage of extractives and lignocellulose were quantified, and crystalline cellulose and syringyl/guaiacyl lignin were quantified by Fourier transform infrared spectroscopy. Anatomy was observed with epifluorescence microscopy. The results show that there were significant differences between the various species (p < 0.05) in the percentages of extractives and lignocellulosic compounds. In addition, there were anatomical differences in fluorescence emission that correlated with the composition of the vascular tissue. Finally, through the characterization of cellulose fibers together with the proportion of syringyl and guaiacyl, it was obtained that various species of the Asparagaceae family have the potential for use in the production of lignocellulosic materials and the production of biofuels.

Biorefining of ethanol and methane from NaOH pretreated poplar residues: Mass balance and energy flow analyses

Poplar, a crucial player in lignocellulosic biofuel production worldwide, can be divided into five types. The feedstock variability would significantly impact the scale-up and commercialization of biofuel technologies from poplar. To date, few studies were found comparing ethanol and biogas production from different types of poplar. This study compared the conversion efficiency of NaOH pretreated five types of poplar residues to ethanol and biogas under three biological conversion processes (namely ethanol fermentation process, anaerobic digestion (AD) process, and cascading ethanol fermentation and AD process); the flow of mass, carbon, nitrogen and energy were also analyzed by material flow analysis. 48.4–60.1 % of theoretical ethanol yield, and the specific methane yield of 201 ± 11.0–270 ± 3.40 mL/g volatile solids were obtained under the co-production process, with the highest yield from Populus trichocarpa (N2). These results showed that the co-production process (cascading ethanol fermentation and AD process) of poplar N2 outperformed ethanol fermentation and AD in terms of energy conversion efficiency, which was 121 % and 42.9 % higher than the ethanol fermentation process and AD process, respectively. Mass and energy balance analysis showed that under the co-production process, 158 g ethanol and 103 g methane could be obtained from 1 kg pretreated poplar N2, corresponding to a total energy output of 9.5 MJ, with an energy conversion efficiency of 54.3 %. This study may provide new insights into the breeding of new poplar species for the current biorefinery process.

Multi-omic characterization of bifunctional peroxidase 4-coumarate 3-hydroxylase knockdown in Brachypodium distachyon provides insights into lignin modification-associated pleiotropic effects.

A bifunctional peroxidase enzyme, 4-coumarate 3-hydroxylase (C3H/APX), provides a parallel route to the shikimate shunt pathway for the conversion of 4-coumarate to caffeate in the early steps of lignin biosynthesis. Knockdown of C3H/APX (C3H/APX-KD) expression has been shown to reduce the lignin content in Brachypodium distachyon. However, like many other lignin-modified plants, C3H/APX-KDs show unpredictable pleiotropic phenotypes, including stunted growth, delayed senescence, and reduced seed yield. A system-wide level understanding of altered biological processes in lignin-modified plants can help pinpoint the lignin-modification associated growth defects to benefit future studies aiming to negate the yield penalty. Here, a multi-omic approach was used to characterize molecular changes resulting from C3H/APX-KD associated lignin modification and negative growth phenotype in Brachypodium distachyon. Our findings demonstrate that C3H/APX knockdown in Brachypodium stems substantially alters the abundance of enzymes implicated in the phenylpropanoid biosynthetic pathway and disrupt cellular redox homeostasis. Moreover, it elicits plant defense responses associated with intracellular kinases and phytohormone-based signaling to facilitate growth-defense trade-offs. A deeper understanding along with potential targets to mitigate the pleiotropic phenotypes identified in this study could aid to increase the economic feasibility of lignocellulosic biofuel production.

Development and characterization of efficient xylose utilization strains of Zymomonas mobilis

BackgroundEfficient use of glucose and xylose is a key for the economic production of lignocellulosic biofuels and biochemicals, and different recombinant strains have been constructed for xylose utilization including those using Zymomonas mobilis as the host. However, the xylose utilization efficiency still needs to be improved. In this work, the strategy of combining metabolic engineering and adaptive laboratory evolution (ALE) was employed to develop recombinant Z. mobilis strains that can utilize xylose efficiently at high concentrations, and NGS-based genome resequencing and RNA-Seq transcriptomics were performed for strains evolved after serial transfers in different media to understand the impact of xylose and differences among strains with different xylose-utilization capabilities at molecular level.ResultsHeterologous genes encoding xylose isomerase and xylulokinase were evaluated, which were then introduced into xylose-utilizing strain Z. mobilis 8b to enhance its capacity of xylose utilization. The results demonstrated that the effect of three xylose isomerases on xylose utilization was different, and the increase of copy number of xylose metabolism genes can improve xylose utilization. Among various recombinant strains constructed, the xylose utilization capacity of the recombinant strain 8b-RsXI-xylB was the best, which was further improved through continuous adaption with 38 transfers over 100 days in 50 g/L xylose media. The fermentation performances of the parental strain 8b, the evolved 8b-S38 strain with the best xylose utilization capability, and the intermediate strain 8b-S8 in different media were compared, and the results showed that only 8b-S38 could completely consume xylose at 50 g/L and 100 g/L concentrations. In addition, the xylose consumption rate of 8b-S38 was faster than that of 8b at different xylose concentrations from 50 to 150 g/L, and the ethanol yield increased by 16 ~ 40%, respectively. The results of the mixed-sugar fermentation also demonstrated that 8b-S38 had a higher xylose consumption rate than 8b, and its maximum ethanol productivity was 1.2 ~ 1.4 times higher than that of 8b and 8b-S8. Whole-genome resequencing identified three common genetic changes in 8b-S38 compared with 8b and 8b-S8. RNA-Seq study demonstrated that the expression levels of genes encoding chaperone proteins, ATP-dependent proteases, phage shock proteins, ribosomal proteins, flagellar operons, and transcriptional regulators were significantly increased in xylose media in 8b-S38. The up-regulated expression of these genes may therefore contribute to the efficient xylose utilization of 8b-S38 by maintaining the normal cell metabolism and growth, repairing cellular damages, and rebalancing cellular energy to help cells resist the stressful environment.ConclusionsThis study provides gene candidates to improve xylose utilization, and the result of expressing an extra copy of xylose isomerase and xylulokinase improved xylose utilization also provides a direction for efficient xylose-utilization strain development in other microorganisms. In addition, this study demonstrated the necessity to combine metabolic engineering and ALE for industrial strain development. The recombinant strain 8b-S38 can efficiently metabolize xylose for ethanol fermentation at high xylose concentrations as well as in mixed sugars of glucose and xylose, which could be further developed as the microbial biocatalyst for the production of lignocellulosic biofuels and biochemicals.

Revitalizing the ethanologenic bacterium Zymomonas mobilis for sugar reduction in high-sugar-content fruits and commercial products

The excessive consumption of sugars can cause health issues. Different strategies have been developed to reduce sugars in the diets. However, sugars in fruits and commercial products may be difficult to reduce, limiting their usage among certain populations of people. Zymomonas mobilis is a generally recognized as safe (GRAS) probiotic bacterium with the capability to produce levan-type prebiotics, and thrives in high-sugar environments with unique characteristics to be developed for lignocellulosic biofuel and biochemical production. In this study, the sugar reduction capabilities of Z. mobilis ZM4 were examined using two fruits of pear and persimmon and three high-sugar-content commercial products of two pear pastes (PPs) and one Chinese traditional wine (CTW). Our results demonstrated that Z. mobilis ZM4 can utilize sugars in fruits with about 20 g/L ethanol and less than 5 g/L sorbitol produced within 22 h using pears, and about 45 g/L ethanol and 30 g/L sorbitol produced within 34 h using persimmons. When PPs made from pears were used, Z. mobilis can utilize nearly all glucose (ca. 60 g/L) and most fructose (110 g/L) within 100 h with 40 ~ 60 g/L ethanol and more than 20 g/L sorbitol produced resulting in a final sorbitol concentration above 80 g/L. In the high-sugar-content alcoholic Chinese traditional wine, which contains mostly glucose and ethanol, Z. mobilis can reduce nearly all sugars with about 30 g/L ethanol produced, resulting in a final ethanol above 90 g/L. The ethanol yield and percentage yield of Z. mobilis in 50 ~ 60% CTW were 0.44 ~ 0.50 g/g and 86 ~ 97%, respectively, which are close to its theoretical yields—especially in 60% CTW. Although the ethanol yield and percentage yield in PPs were lower than those in CTW, they were similar to those in fruits of pears and persimmons with an ethanol yield around 0.30 ~ 0.37 g/g and ethanol percentage yield around 60 ~ 72%, which could be due to the formation of sorbitol and/or levan in the presence of both glucose and fructose. Our study also compared the fermentation performance of the classical ethanologenic yeast Saccharomyces cerevisiae BY4743 to Z. mobilis, with results suggesting that Z. mobilis ZM4 had better performance than that of yeast S. cerevisiae BY4743 given a higher sugar conversion rate and ethanol yield for sugar reduction. This work thus laid a foundation for utilizing the advantages of Z. mobilis in the food industry to reduce sugar concentrations or potentially produce alcoholic prebiotic beverages.Graphical

Development and characterization of a Napier grass (Cenchrus purpureus Schumach) mapping population for flowering‐time‐ and biomass‐related traits reveal individuals with exceptional potential and hybrid vigor

AbstractNapier grass is a tropical perennial C4 grass with superior biomass yield and quality. It is an important forage crop and a promising feedstock for lignocellulosic biofuel production. However, precise phenotyping and genotyping data to support the molecular breeding of Napier grass are scarce. A Napier grass F1 mapping population (segregating pseudo‐F2 population) was generated by hybridizing genetically distant parents (N190 × N122) with contrasting flowering‐time‐ and biomass‐related traits. True F1 hybrids were confirmed with a simple sequence repeat marker, vegetatively propagated, and phenotyped in replicate field plots for 2 years (2 harvests per year) in Citra, FL, USA (29.40N). Near‐normal distributions were observed for flowering date, plant height, number of tillers, stem diameter, and leaf width, confirming the quantitative nature of these traits. The annual dry biomass yield of F1 hybrids varied between 21.0 and 41.1 t ha−1. The highest‐yielding F1 hybrids showed remarkable hybrid vigor exceeding the annual biomass production of the highest‐yielding parental accession (26.9 t ha−1) by 52% and the biomass yield of the control “Merkeron” cultivar (29.2 t ha−1) by 41% over 2 years. F1 hybrids with delayed flowering time along with significantly increased biomass yield were also identified. Late‐flowering accessions allowed maximum biomass harvest before formation and dispersal of seeds, reducing invasion risk. The developed mapping population will be an excellent resource for identifying quantitative trait loci and candidate genes for flowering‐ and biomass‐related traits in Napier grass that will accelerate the improvement of this non‐domesticated bioenergy crop.

PdNF-YB21 positively regulated root lignin structure in poplar

Poplar is a key player in biomass resource and a dominant species for lignocellulosic biofuel production around the world. Today, the use of poplars is mainly concentrated in the aboveground parts. However, as an important part of trees, the poplar root system developed, roots account for about 20 % of the volume of the tree, and the root material volume is very considerable and its utilization potential is huge. The root lignin is central to biomass production in poplar. However, there are very few studies on root lignin structure. Recently, we found a root-specific transcription factor, NUCLEAR FACTOR Y subunit B21 (NF-YB21), is involved in regulating root growth and lignification. In the present study, physiology, transgenosis, transcriptomics, metabolome, Confocal Raman Microscopy (CRM) and Nuclear Magnetic Resonance (NMR) techniques were performed to further investigate the regulatory mechanisms of PdNF-YB21 on lignin structure in poplar roots. The results of transcriptome and metabolome showed that there were more genes and metabolites related to S-lignin synthesis in transgenic plants. Root firmness measurements, CRM and NMR results showed that overexpression of PdNF-YB21 dramatically improved root hardness, increased root lignin content and S/G ratio, and increased the concentrations of glucan, xylan and lignin in roots, which promoted root growth. In short, this present study not only illustrated the regulatory mechanisms of PdNF-YB21 on lignin structure in poplar roots, but also proposed a directive breeding of new poplar species for current biorefinery process.

NMR chemical shift assignments of a module of unknown function in the cellulosomal secondary scaffoldin ScaF from Clostridium thermocellum.

The cellulosome is a highly efficient cellulolytic complex containing cellulolytic enzymes and non-catalytic subunits, i.e. scaffoldins, which are assembled by the interactions between the dockerin modules of the enzymes and the cohesin modules of the primary scaffoldins. The cellulosome attaches to the cell surface via the S-layer homology (SLH) modules of the anchoring scaffoldins. Clostridium thermocellum DSM1313 is a thermophilic cellulosome-producing bacterium with great potential in lignocellulose bioconversion and biofuel production. The bacterium contains four anchoring scaffoldins ScaB, ScaC, ScaD and ScaF, among which ScaF is the only one that contains an additional module of unknown function (ScaF-X) between the cohesin and SLH modules. The gene of ScaF is located outside the scaffoldin gene cluster of scaA, scaB, scaC and scaD. Previous studies showed unique regulation properties and function of ScaF compared to other anchoring scaffoldins, which might be related to the additional ScaF-X module. Here we report the NMR chemical shift assignments of ScaF-X from C. thermocellum DSM1313. The well-dispersed NMR spectrum and the secondary structure prediction based on the chemical shifts of ScaF-X indicated that ScaF-X is a well-folded protein module. The chemical shift assignments provide the basis for future studies on the structure of this module and its function in cellulosomes.

Transcriptomic Changes Induced by Deletion of Transcriptional Regulator GCR2 on Pentose Sugar Metabolism in Saccharomyces cerevisiae.

Being a microbial host for lignocellulosic biofuel production, Saccharomyces cerevisiae needs to be engineered to express a heterologous xylose pathway; however, it has been challenging to optimize the engineered strain for efficient and rapid fermentation of xylose. Deletion of PHO13 (Δpho13) has been reported to be a crucial genetic perturbation in improving xylose fermentation. A confirmed mechanism of the Δpho13 effect on xylose fermentation is that the Δpho13 transcriptionally activates the genes in the non-oxidative pentose phosphate pathway (PPP). In the current study, we found a couple of engineered strains, of which phenotypes were not affected by Δpho13 (Δpho13-negative), among many others we examined. Genome resequencing of the Δpho13-negative strains revealed that a loss-of-function mutation in GCR2 was responsible for the phenotype. Gcr2 is a global transcriptional factor involved in glucose metabolism. The results of RNA-seq confirmed that the deletion of GCR2 (Δgcr2) led to the upregulation of PPP genes as well as downregulation of glycolytic genes, and changes were more significant under xylose conditions than those under glucose conditions. Although there was no synergistic effect between Δpho13 and Δgcr2 in improving xylose fermentation, these results suggested that GCR2 is a novel knockout target in improving lignocellulosic ethanol production.

Lignocellulosic acid hydrolysis inhibitor impact on 5-hydroxymethylfurfural biotransformation into 2, 5-furandicarboxylic acid using immobilised Burkholderia cells

Acid hydrolysis extensively applies as an economical pre-treatment to obtain monosaccharides from recalcitrant lignocellulosic residues. However, inhibitors are inevitably formed and reduce the downstream biofuel yield. Our bacterial isolate Burkholderia cepacia H-2 was capable of biotransforming 5-hydroxymethylfurfural (5-HMF), a common inhibitor, into valuable 2, 5-furan-dicarboxylic acid (FDCA). This isolate also has potential use for detoxifying other inhibitors derived from lignocellulosic acid hydrolysis. To integrate this strain into further lignocellulosic biofuel production processes, strain B. cepacia H-2 was immobilised to biotransform 5-HMF into FDCA and detoxify various inhibitors. Strain B. cepacia H-2 could biotransform 5-HMF into FDCA in the presence of inhibitors derived from lignocellulosic acid hydrolysis and detoxified formic acid, furfural and levulinic acid. The negative effect of approximately 500 mg L−1 inhibitor on 5-HMF biotransformation was as follows (from strongest to weakest): furfural > acetic acid > phenol > formic acid > levulinic acid.

Multi objective optimization of ethanol production based on regional resource availability

Production of lignocellulosic biofuels must respect the regional water availability constraints, while simultaneously balancing the social and economic objectives. This work has proposed a multi-objective optimization model to design regional biomass to energy system incorporating these factors. The goal is to design a bioenergy system that is constrained by district level land availability, regional water availability, and competing water demands. The allocation of agricultural land to selected crops is the key decision. Additionally, the number of biorefineries, their locations, and their feedstock procurement strategies are also optimised. Four different objectives, namely, minimization of ethanol cost, minimization of water footprint of ethanol, minimization of irrigation water, and maximization of farmers’ profit, are considered for meeting a specified monthly ethanol demand. The resulting multi-objective mixed-integer linear programming problem was applied to a case study of 33 districts in Maharashtra, India. The problem was solved using the ϵ-constraint method where all objectives, except one, were used as constraints. Results indicated a significant trade-off between ethanol cost and irrigation water requirement. Sorghum, wheat and cotton were the prominent crops recommended by the model at various regions in the Pareto front, while Jalna, Kolhapur and Pune were among the most frequently selected locations for setting up biorefineries. Ethanol cost varied from ₹47/l ($0.63/l) to ₹60/l ($0.80/l), while water footprint varied from 665 l/l to 301 l/l, irrigation water varied from 43m3/ha to 2768m3/ha, and farmers’ profit from ₹21059/ha ($281/ha) to ₹6825/ha ($91/ha).

Improving lignocellulosic biofuel production by CRISPR/Cas9‐mediated lignin modification in barley

AbstractBarley is a major cereal crop with a wide ecological range, and its lignocellulosic residues have the potential to be used as a feedstock for various purposes, including biofuel production. Lignocellulosic biomass is an abundant renewable source of carbon energy. However, its heterogeneous properties and intrinsic recalcitrance caused by cell wall lignification have lowered the biorefinery efficiency. The reduced lignin content and/or altered lignin structure have been desirable traits for lignocellulosic feedstock. We report the reduction of lignin content in barley by CRISPR/Cas9‐mediated mutagenesis of caffeic acid O‐methyltransferase 1 (HvCOMT1), the lignin biosynthetic gene responsible for lignin syringyl unit formation. The transgene‐free, homozygous HvCOMT1 mutant was generated and analyzed for its cell wall composition, saccharification efficiency, and bioethanol production performance. The mutant had 14% lower total lignin content and 34% higher fermentable glucose recovery rate, compared to the wild‐type (WT). The bioethanol concentration and yield from the hydrolysates of the mutant biomass were 14.3 g/L and 0.46 g/g total sugar, respectively. This result was 34% and 12% higher than those obtained from WT (10.7 g/L and 0.41 g/g total sugar). Under controlled environmental conditions, the overall growth performance of the HvCOMT1 mutant was similar to WT, with no distinct morphological variations between them. The HvCOMT1 mutant barley could offer improved quality lignocellulosic feedstock for efficient lignocellulosic biofuel production.

Assessment of Bacterial Community Structure in Saline Sediment Microcosms Exposed for the First Time to the Ionic Liquid 1-Ethyl-3-Methylimidazolium Chloride

Ionic liquids, such as 1-ethyl-3-methylimidazolium chloride ([Emim]Cl), have immense applicability as solvents in different industrial settings including lignocellulosic biofuel production. However, these solvents are toxic to most organisms. Moderate halophiles are an established microbial group that can tolerate [Emim]Cl by employing their general salt-tolerant mechanisms. Anticipating future exposure of [Emim]Cl to natural environments, the present study was designed to evaluate bacterial community structure in slightly saline (20 g L−1 or 2%) sediments of Sambhar Salt Lake, Rajasthan, India, after microcosm enrichment with [Emim]Cl. The sediment used does not have any native [Emim]Cl (100 mM)–tolerant bacteria. However, after a two-step enrichment of 25 days each with 0.5 M and 1.0 M [Emim]Cl, the tolerant colonies increased to 54.9% and 93.0%, respectively. But in similar NaCl enrichment, or control incubations, such colonies were absent. Bacterial 16S rRNA gene amplicon sequence-based alpha diversity in 50 days [Emim]Cl-enriched set was lower than the initial, control and NaCl-enriched sets (Shannon index of 6.71 vs. 8.0–8.26, respectively). Even its bacterial community structure was distinct from the remaining three sets, which clustered together. [Emim]Cl-enriched set has almost fourfold higher abundance of Proteobacteria (50.36%), with Gammaproteobacteria (32.25%) and particularly Enterobacteriaceae (24.19%) members being predominant with a predicted higher abundance of membrane transporters. Cyanobacteria, Actinobacteria and Bacteroidetes were most sensitive groups to [Emim]Cl enrichment. Although moderate halophiles can utilise their general NaCl tolerance mechanisms to resist toxicity of [Emim]Cl, abrupt exposure of the later disturbs microbial population structure and thus demands their meticulous use avoiding environmental contamination.

Metagenomics approach to identify lignocellulose-degrading enzymes in the gut microbiota of the Chinese bamboo rat cecum

Abstract Background Lignocellulose is considered a renewable organic material, but the industrial production of biofuel from lignocellulose is challenging because of the lack of highly active hydrolytic enzymes. The guts of herbivores contain many symbiotic microorganisms that have evolved to hydrolyze plant lignocellulose. Chinese bamboo rats mainly consume high-fiber foods, indicating that some members of the intestinal tract microbiota digest lignocellulose, providing these rats with the energy required for growth. Results Here, we used metagenomics to analyze the diversity and functions of the gut microbiota in Chinese bamboo rats. We identified abundant populations of lignocellulose-degrading bacteria, whose main functions involved carbohydrate, amino acid, and nucleic acid metabolism. We also found 587 carbohydrate-active enzyme genes belonging to different families, including 7 carbohydrate esterase families and 21 glycoside hydrolase families. The glycoside hydrolase 3, glycoside hydrolase 1, glycoside hydrolase 43, carbohydrate esterase 4, carbohydrate esterase 1, and carbohydrate esterase 3 families demonstrated outstanding performance. Conclusions The microbes and enzymes identified in our study expand the existing arsenal of proficient degraders and enzymes for lignocellulosic biofuel production. This study also describes a powerful approach for targeting gut microbes and enzymes in numerous industries. How to cite: Bai D, Lin X, Hu Y, et al. Metagenomics approach to identify lignocellulose-degrading enzymes in the gut microbiota of the Chinese bamboo rat cecum. Electron J Biotechnol 2021;50. https://doi.org/10.1016/j.ejbt.2020.12.001

Detailed kinetic mechanism of thermal decomposition of furyl radicals: Theoretical insights

This study presents the detailed kinetic mechanism of the thermal decomposition of furyl radicals, which play an essential role in the production of the second-generation lignocellulosic biofuels. In particular, the accurate composite method W1U was used to explore the main reaction pathways on which the temperature- and pressure-dependent kinetic behaviors were analyzed in the range of 500–2000 K and 1–76000 Torr using a complementary deterministic/stochastic Rice-Ramsperger-Kassel-Marcus based master equation rate model. Corrections of tunneling and hindered internal rotation treatments were included in the rate model. Detailed kinetic analyses, including time-resolved species profile and flux analysis, reveal that: (i) the decomposition pathways are more favorable than the (2-furyl ↔ 3-furyl) isomerization; and (ii) the well-skipping reactions, accounting for the formation of the products in a shorter time scale, are observed at high temperature and/or low pressure. The calculated thermodynamic and kinetic parameters are in good agreement with literature data; thus, the reported detailed kinetic mechanism can be confidently used to investigate furyl-related systems in different conditions. Also, among the selected composite methods, CBS-QB3 and CBS-APNO have shown to be cost-effective methods in replacing the higher level W1U for constructing the reaction mechanisms for larger similar systems.