Rice straw hydrolysis using in-situ produced enzymes: Feedstock influences fungal enzyme composition and hydrolytic efficiency.

Trichoderma reesei RUT-C30 was cultivated on differentially pretreated rice straw and pure cellulose as a carbon source/inducer for cellulase production, and the enzymes were evaluated for hydrolysis of sequential acid and alkali pretreated rice straw. Growth on pretreated rice straw enhanced protein secretion and cellulase activities compared to pure cellulose as a carbon source. The yield of cellulolytic enzymes was higher for alkali pretreated rice straw (ALP-RS), while H2O2-treated (HP-RS) could not induce cellulases to a larger level compared to pure cellulose. Protein concentration was 3.5-fold higher on ALP-RS as compared to pure cellulose, with a maximum filter-paper cellulase (FPase) activity of 1.76 IU/ml and carboxy-methyl cellulase (CMCase) activity of 40.16 IU/ml (2.18 fold higher). Beta-glucosidase (BGL) activity was more or less the same with the different substrates and supplementation of heterologous BGL could result in a quantum jump in hydrolytic efficiencies, which in the case of ALP-RS induced enzymes was 34% (increased from 69.26% to 92.51%). The use of lignocellulosic biomass (LCB) itself as a substrate for the production of cellulase is advantageous not only in terms of raw material costs but also for obtaining a more suitable enzyme profile for biomass hydrolysis.

Potential of camel rumen derived Bacillus subtilis and Bacillus velezensis strains for application in plant biomass hydrolysis.

Rumen inhabiting Bacillus species possesses a high genetic potential for plant biomass hydrolysis and conversion to value-added products. In view of the same, five camel rumen-derived Bacillus strains, namely B. subtilis CRN 1, B. velezensis CRN 2, B. subtilis CRN 7, B. subtilis CRN 11, and B. velezensis CRN 23 were initially assayed for diverse hydrolytic activities, followed by genome mining to unravel the potential applications. CRN 1 and CRN 7 showed the highest endoglucanase activity with 0.4 U/ml, while CRN 23 showed high ß-xylosidase activity of 0.36 U/ml. The comprehensive genomic insights of strains resolve taxonomic identity, clusters of an orthologous gene, pan-genome dynamics, and metabolic features. Annotation of Carbohydrate active enzymes (CAZymes) reveals the presence of diverse glycoside hydrolases (GH) GH1, GH5, GH43, and GH30, which are solely responsible for the effective breakdown of complex bonds in plant polysaccharides. Further, protein modeling and ligand docking of annotated endoglucanases showed an affinity for cellotrioside, cellobioside, and ß-glucoside. The finding indicates the flexibility of Bacillus-derived endoglucanase activity on diverse cellulosic substrates. The presence of the butyrate synthesis gene in the CRN 1 strain depicts its key role in the production of important short-chain fatty acids essential for healthy rumen development. Similarly, antimicrobial peptides such as bacilysin and non-ribosomal peptides (NRPS) synthesized by the Bacillus strains were also annotated in the genome. The findings clearly define the role of Bacillus sp. inside the camel rumen and its potential application in various plant biomass utilizing industry and animal health research sectors.

A semi-continuous algal-bacterial wastewater treatment process coupled with bioethanol production.

Harnessing the biomass energy potential through biofuel production offers new outlets for a circular economy. In this study an integrated system which combine brewery wastewater treatment using algal-bacterial aggregates instead of activated sludge was developed. The use of algal-bacterial aggregates can eliminate the aeration requirements and significantly reduce the high biomass harvesting costs associated with algal monocultures. A sequencing batch reactor (SBR) setup operating with and without biomass recirculation was used to investigate pollutant removal rates, aggregation capacity and microbial community characteristics under a range of hydraulic retention times (HRTs) and solid retention times (SRTs). It was observed that biomass recirculation strategy significantly enhanced aggregation and pollutant removal (i.e., 78.7%, 94.2% and 75.2% for d-COD, TKN, and PO43--P, respectively). The microbial community established was highly diverse consisting of 161 Bacterial Operational Taxonomic Units (B-OTUs) and 16 unicellular Eukaryotic OTUs (E-OTUs). Escalation the optimal conditions (i.e., HRT = 4 d, SRT = 10 d) at pilot-scale resulted in nutrient starvation leading to 38-44% w/w carbohydrate accumulation. The harvested biomass was converted to bioethanol after acid hydrolysis followed by fermentation with Saccharomyces cerevisiae achieving a bioethanol production yield of 0.076 g bioethanol/g biomass. These data suggest that bioethanol production coupled with high-performance wastewater treatment using algal-bacterial aggregates is feasible, albeit less productive concerning bioethanol yields than systems exclusively designed for third and fourth-generation biofuel production.

Anaeropeptidivorans aminofermentans gen. nov., sp. nov., a mesophilic proteolytic salt-tolerant bacterium isolated from a laboratory-scale biogas fermenter, and emended description of Clostridium colinum.

An anaerobic bacterial strain, designated strain M3/9T, was isolated from a laboratory-scale biogas fermenter fed with maize silage supplemented with 5 % wheat straw. Cells were straight, non-motile rods, which stained Gram-negative. Optimal growth occurred between 30 and 40°C, at pH 7.5-8.5, and up to 3.9 % (w/v) NaCl was tolerated. When grown on peptone from casein and soymeal, strain M3/9T produced mainly acetic acid, ethanol, and isobutyric acid. The major cellular fatty acids of the novel strain were C16 : 0 and C16 : 0 DMA. The genome of strain M3/9T is 3757  330 bp in size with a G+C content of 38.45 mol%. Phylogenetic analysis allocated strain M3/9T within the family Lachnospiraceae with Clostridium colinum DSM 6011T and Anaerotignum lactatifermentans DSM 14214T being the most closely related species sharing 57.86 and 56.99% average amino acid identity and 16S rRNA gene sequence similarities of 91.58 and 91.26 %, respectively. Based on physiological, chemotaxonomic and genetic data, we propose the description of a novel species and genus Anaeropeptidivorans aminofermentans gen. nov., sp. nov., represented by the type strain M3/9T (=DSM 100058T=LMG 29527T). In addition, an emended description of Clostridium colinum is provided.

Tubular electrosynthesis of silica-coated magnetite and evaluation of magnetic nanobiocatalyst efficacy during biomass hydrolysis.

Magnetic nanobiocatalysts (MNBCs) are a promising immobilization approach to ease enzyme recovery during bioprocessing. However, industrial adoption of MNBCs is unfeasible because MNBC-synthesis involves complex and potentially expensive processing steps including synthesis of silica-coated superparamagnetic iron oxide nanoparticles (Si-SPIONs). We developed a single-step process for Si-SPION synthesis using a tubular electrochemical system (TES) and investigated the effect of concentration of the Na2SiO3 coating agent on Si-SPION properties. The Si-SPIONs were used as a support for attachment of polymer-cellulase conjugate to make MNBCs. The spherical Si-SPIONs were 8-12 nm in diameter including a 2-nm silica coating. Na2SiO3 concentration in the reactor did not affect Si-SPION morphology, but increasing Na2SiO3 concentration reduced SPION productivity in the reactor. Protective properties of the SPION silica coatings were demonstrated by showing that they prevented dissolution of SPIONs in an acid solution for 48 h. Enzyme attachment was quantified as protein adsorption on Si-SPIONs which reached 55 µg/mg Si-SPION. The MNBCs were recovered and reused four times. The use of TES for Si-SPION synthesis is promising to reduce MNBC production complexity.

Co-cultivation of T. asperellum GDFS1009 and B. amyloliquefaciens 1841: Strategy to regulate the production of ligno-cellulolytic enzymes for the lignocellulose biomass degradation.

The influence of fossil fuels on the environment focused on the development of new technology on biofuels. In this situation, lignocellulolytic hydrolysis enzymes such as Cellobiohydrolase, ß-Glucosidase, Endoglucanase, cellulase and xylanase have broad applications in the biofuel production. The Trichoderma have used for the production of cellulase and xylanase to hydrolyze the lignocellulose. Hence, in the present study, co-culture has been employed to induce the production of polysaccharide hydrolyzing enzymes under both induction and repression conditions. The enzyme activity and its gene expression were induced by the co-culture of T. asperellum and B. amyloliquefaciens compared to the monoculture. Further, the co-culture upregulated the transcription regulatory genes and downregulated the repressor genes under both repressor and inducer conditions, respectively. The crude enzyme produced by the co-culture and monocultures using the optimized medium containing molasses, cornmeal and rice bran were further used to hydrolyze the pretreated corn Stover, rice straw, and wheat straw. These results indicate that the co-culture of T. asperellum and B. amyloliquefaciens is a promising and inexpensive method to advance the innovation on the continuous production of cellulase and xylanase under different circumstances for the bioconversion of lignocellulosic biomass into glucose for the bio-fuels.

Variimorphobacter saccharofermentans gen. nov., sp. nov., a new member of the family Lachnospiraceae, isolated from a maize-fed biogas fermenter.

Strain MD1T is an anaerobic, Gram-stain-negative bacterium isolated from a lab-scale biogas fermenter fed with maize silage. It has a rod-shaped morphology with peritrichously arranged appendages and forms long chains of cells and coccoid structures. The colonies of MD1T were white, circular, slightly convex and had a smooth rim. The isolate is mesophilic, displaying growth between 25 and 45 °C with an optimum at 40 °C. It grew at pH values of pH 6.7-8.2 (optimum, pH 7.1) and tolerated the addition of up to 1.5% (w/v) NaCl to the medium. The main cellular fatty acids of MD1T are C14:0 DMA and C16:0. Strain MD1T fermented xylose, arabinose, glucose, galactose, cellobiose, maltose, maltodextrin10, lactose starch, and xylan, producing mainly 2-propanol and acetic acid. The genome of the organism has a total length of 4163427 bp with a G+C content of 38.5 mol%. The two closest relatives to MD1T are Mobilitalea sibirica P3M-3T and Anaerotaenia torta FH052T with 96.44 or 95.8 % 16S rRNA gene sequence similarity and POCP values of 46.58 and 50.58%, respectively. As MD1T showed saccharolytic and xylanolytic properties, it may play an important role in the biogas fermentation process. Closely related variants of MD1T were also abundant in microbial communities involved in methanogenic fermentation. Based on morphological, phylogenetic and genomic data, the isolated strain can be considered as representing a novel genus in the family Lachnospiraceae, for which the name Variimorphobacter saccharofermentans gen. nov., sp. nov. (type strain MD1T=DSM 110715T=JCM 39125T) is proposed.

Non-productive binding of cellobiohydrolase i investigated by surface plasmon resonance spectroscopy.

Future biorefineries are facing the challenge to separate and depolymerize biopolymers into their building blocks for the production of biofuels and basic molecules as chemical stock. Fungi have evolved lignocellulolytic enzymes to perform this task specifically and efficiently, but a detailed understanding of their heterogeneous reactions is a prerequisite for the optimization of large-scale enzymatic biomass degradation. Here, we investigate the binding of cellulolytic enzymes onto biopolymers by surface plasmon resonance (SPR) spectroscopy for the fast and precise characterization of enzyme adsorption processes. Using different sensor architectures, SPR probes modified with regenerated cellulose as well as with lignin films were prepared by spin-coating techniques. The modified SPR probes were analyzed by atomic force microscopy and static contact angle measurements to determine physical and surface molecular properties. SPR spectroscopy was used to study the activity and affinity of Trichoderma reesei cellobiohydrolase I (CBHI) glycoforms on the modified SPR probes. N-glycan removal led to no significant change in activity or cellulose binding, while a slightly higher tendency for non-productive binding to SPR probes modified with different lignin fractions was observed. The results suggest that the main role of the N-glycosylation in CBHI is not to prevent non-productive binding to lignin, but probably to increase its stability against proteolytic degradation. The work also demonstrates the suitability of SPR-based techniques for the characterization of the binding of lignocellulolytic enzymes to biomass-derived polymers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10570-021-04002-6.

Understanding the alteration in rumen microbiome and CAZymes profile with diet and host through comparative metagenomic approach.

Rumen microbial community harbors a distinct genetic reservoir of potent carbohydrate-active enzymes (CAZyme) that functions efficiently for the deconstruction of plant biomass. Based on this premise, metagenomics approach was applied to characterize the rumen microbial community and identify carbohydrate-active genes of Bos taurus (cow) and Bubalus bubalis (buffalo) fed on green or dry roughage. Metadata was generated from the samples: green roughage-fed cow (NDC_GR), buffalo (NDB_GR) and dry roughage-fed cow (NDC_DR), buffalo (NDB_DR). Phylogenetic analysis revealed the dominance of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and Fibrobacter in all the four samples, covering 90-96% of the total bacterial population. On finer resolution, higher abundance of bacterial genera Fibrobacter, Bacteroides, Clostridium, Prevotella and Ruminococcus involved in plant biomass hydrolysis was observed in NDB_DR. Functional annotation using dbCAN annotation algorithm identified 28.13%, 8.08% 10.93% and 12.53% of the total contigs as putatively carbohydrate-active against NDC_GR, NDB_GR, NDC_DR and NDB_DR, respectively. Additional profiling of CAZymes revealed an over representation and diversity of putative glycoside hydrolases (GHs) in the animals fed on dry roughage with substantial enrichments of genes encoding GHs from families GH2, GH3, GH13 and GH43. GHs of families GH45, GH12, GH113, GH128, GH54 and GH27 were observed exclusively in NDB_DR metagenome. A higher abundance of cellulases, hemicellulases, debranching and oligosaccharide hydrolyzing enzymes was revealed in NDB_DR metagenome. Accordingly, it can be concluded that buffalo rumen microbiome are more efficient in plant biomass hydrolysis. The present study provides a deep understanding of the shifts in microbial community and plant polysaccharide deconstructing capabilities of rumen microbiome in response to changes in the feed type and host animal. Activity-specific microbial consortia procured from these animals can be used further for efficient plant biomass hydrolysis. The study also establishes the utility of rumen microbiome as a unique resource for mining diverse lignocellulolytic enzymes.

Ruminiclostridium herbifermentans sp. nov., a mesophilic and moderately thermophilic cellulolytic and xylanolytic bacterium isolated from a lab-scale biogas fermenter fed with maize silage.

An anaerobic bacterial strain, designated MA18T, was isolated from a laboratory-scale biogas fermenter fed with maize silage. Cells stained Gram-negative and performed Gram-negative in the KOH test. The peptidoglycan type was found to be A1y-meso-Dpm direct. The major cellular fatty acids were C14 : 0 iso, C15 : 0 iso, anteiso and iso DMA as well as a C16 unidentified fatty acid. Oxidase and catalase activities were absent. Cells were slightly curved rods, motile, formed spores and measured approximately 0.35 µm in diameter and 3.0-5.0 µm in length. When cultivated on GS2 agar with cellobiose, round, arched, shiny and slightly yellow-pigmented colonies were formed. The isolate was mesophilic to moderately thermophilic with a growth optimum between 40 and 48 °C. Furthermore, neutral pH values were preferred and up to 1.2 % (w/v) NaCl supplemented to the GS2 medium was tolerated. Producing mainly acetate and ethanol, MA18T fermented arabinose, cellobiose, crystalline and amorphous cellulose, ribose, and xylan. The genome of MA18T consists of 4 817 678 bp with a G+C content of 33.16 mol%. In the annotated protein sequences, cellulosomal components were detected. Phylogenetically, MA18T is most closely related to Ruminiclostridium sufflavum DSM 19573T (76.88 % average nucleotide identity of the whole genome sequence; 97.23 % 16S rRNA gene sequence similarity) and can be clustered into one clade with other species of the genus Ruminiclostridium, family Oscillospiraceae, class Clostridia. Based on morphological, physiological and genetic characteristics, this strain represents a novel species in the genus Ruminiclostridium. Therefore, the name Ruminiclostridium herbifermentans sp. nov. is proposed. The type strain is MA18T (=DSM 109966T=JCM 39124T).

Characterization of a glucose tolerant ß-glucosidase from Aspergillus unguis with high potential as a blend-in for biomass hydrolyzing enzyme cocktails.

OBJECTIVES: Characterization of glucose tolerant beta glucosidase (GT-BGL) secreted by Aspergillus unguis NII 08123, determination of the gene and protein sequences of the enzyme and establishing its performance in blends for lignocellulose hydrolysis. RESULTS: Supplementation of A. unguis beta glucosidase (BGL) to cellulase released 1.6 times more sugar within 12 h during the hydrolysis of lignocellulosic biomass. The enzyme was determined to be similar to BGL-F from Emericella nidulans by MALDI-TOF analysis, and was found to be a GH3 family protein. Molecular Docking simulation studies showed that the enzyme has lesser affinity for glucose (- 5.7 kcal/mol) compared to its substrate cellobiose (- 7.5 kcal/mol). The residues present in the N-terminal domain are mostly involved in bond formation with both the substrate and the product, while the C-terminal domain contains the catalytic region. In-silico studies showed that its predicted structure is unlike that of previously reported BGLs, which might provide a clue to its exceptional catalytic activity. CONCLUSION: The GT-BGL from A. unguis NII 08123 was proven effective as a blend in for biomass hydrolyzing enzyme cocktails and the possible reasons for its glucose tolerance was determined through studies on its modeled structure.

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis.

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-ß-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters Km and Vmax were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0-8.0) and was highly thermostable at 55 °C for 72 h. Low Cu2+ concentrations (0.1-1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse "in natura", sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications.

Metabolic characterization of anaerobic fungi provides a path forward for bioprocessing of crude lignocellulose.

The conversion of lignocellulose-rich biomass to bio-based chemicals and higher order fuels remains a grand challenge, as single-microbe approaches often cannot drive both deconstruction and chemical production steps. In contrast, consortia based bioprocessing leverages the strengths of different microbes to distribute metabolic loads and achieve process synergy, product diversity, and bolster yields. Here, we describe a biphasic fermentation scheme that combines the lignocellulolytic action of anaerobic fungi isolated from large herbivores with domesticated microbes for bioproduction. When grown in batch culture, anaerobic fungi release excess sugars from both cellulose and crude biomass due to a wealth of highly expressed carbohydrate active enzymes (CAZymes), converting as much as 49% of cellulose to free glucose. This sugar-rich hydrolysate readily supports growth of Saccharomyces cerevisiae, which can be engineered to produce a range of value-added chemicals. Further, construction of metabolic pathways from transcriptomic data reveals that anaerobic fungi do not catabolize all sugars that their enzymes hydrolyze from biomass, leaving other carbohydrates such as galactose, arabinose, and mannose available as nutritional links to other microbes in their consortium. Although basal expression of CAZymes in anaerobic fungi is high, it is drastically amplified by cellobiose breakout products encountered during biomass hydrolysis. Overall, these results suggest that anaerobic fungi provide a nutritional benefit to the rumen microbiome, which can be harnessed to design synthetic microbial communities that compartmentalize biomass degradation and bioproduct formation.

Characterization of cellulolytic enzyme system of Schizophyllum commune mutant and evaluation of its efficiency on biomass hydrolysis.

Schizophyllum commune is a basidiomycete equipped with an efficient cellulolytic enzyme system capable of growth on decaying woods. In this study, production of lignocellulose-degrading enzymes from S. commune mutant G-135 (SC-Cel) on various cellulosic substrates was examined. The highest cellulase activities including CMCase, FPase, and ß-glucosidase were obtained on Avicel-PH101 while a wider range of enzymes attacking non-cellulosic polysaccharides and lignin were found when grown on alkaline-pretreated biomass. Proteomic analysis of SC-Cel also revealed a complex enzyme system comprising seven glycosyl hydrolase families with an accessory carbohydrate esterase, polysaccharide lyase, and auxiliary redox enzymes. SC-Cel obtained on Avicel-PH101 effectively hydrolyzed all agricultural residues with the maximum glucan conversion of 98.0% using corn cobs with an enzyme dosage of 5 FPU/g-biomass. The work showed potential of SC-Cel on hydrolysis of various herbaceous biomass with enhanced efficiency by addition external ß-xylosidase.

Dark-Fermentative Biological Hydrogen Production from Mixed Biowastes Using Defined Mixed Cultures.

Biological hydrogen (H2) production from the biowastes is widely recognized as a suitable alternative approach to utilize low cost feed instead of costly individual sugars. In the present investigation, pure and mixed biowastes were fermented by defined sets of mixed cultures for hydrolysis and H2 production. Under batch conditions, up to 65, 67 and 70 L H2/kg total solids (2%, TS) were evolved from apple pomace (AP), onion peels (OP) and potato peels (PP) using a combination of hydrolytic mixed culture (MHC5) and mixed microbial cultures (MMC4 or MMC6), respectively. Among the different combinations of mixed biowastes including AP, OP, PP and pea-shells, the combination of OP and PP exhibited maximum H2 production of 73 and 84 L/kg TS with MMC4 and MMC6, respectively. This study suggested that H2 production can be effectively regulated by using defined sets of mixed cultures for hydrolysis and H2 production from pure and mixed biowastes as feed even under unsterile conditions.

Integrative Approach for Producing Hydrogen and Polyhydroxyalkanoate from Mixed Wastes of Biological Origin.

In this study, an integrative approach to produce biohydrogen (H2) and polyhydroxyalkanoates (PHA) from the wastes of biological origin was investigated. A defined set of mixed cultures was used for hydrolysis and the hydrolysates were used to produce H2. The effluent from H2 production stage was used for PHA production. Under batch culture, a maximum of 62 l H2/kg of pure potato peels (Total solid, TS 2 %, w/v) and 54 l H2/kg of mixed biowastes (MBW1) was recorded. Using effluent from the H2 production stage of biowaste mixture (MBW1), Bacillus cereus EGU43 could produce 195 mg PHA/l and 15.6 % (w/w). Further, supplementation of GM-2 medium (0.1×) and glucose (0.5 %) in H2 production stage effluents, resulted in significant improvements of up to 11 and 41.7 % of PHA contents, respectively. An improvement of 3.9- and 17-fold in PHA yields as compared to with and without integrative H2 production from the MBW1 has been recorded. This integrative approach seems to be a suitable process to improve the yields of H2 and PHA by mixing biowastes.

Biodelignification of lignocellulose substrates: An intrinsic and sustainable pretreatment strategy for clean energy production.

Lignocellulosic biomass (LB) is a promising sugar feedstock for biofuels and other high-value chemical commodities. The recalcitrance of LB, however, impedes carbohydrate accessibility and its conversion into commercially significant products. Two important factors for the overall economization of biofuel production is LB pretreatment to liberate fermentable sugars followed by conversion into ethanol. Sustainable biofuel production must overcome issues such as minimizing water and energy usage, reducing chemical usage and process intensification. Amongst available pretreatment methods, microorganism-mediated pretreatments are the safest, green, and sustainable. Native biodelignifying agents such as Phanerochaete chrysosporium, Pycnoporous cinnabarinus, Ceriporiopsis subvermispora and Cyathus stercoreus can remove lignin, making the remaining substrates amenable for saccharification. The development of a robust, integrated bioprocessing (IBP) approach for economic ethanol production would incorporate all essential steps including pretreatment, cellulase production, enzyme hydrolysis and fermentation of the released sugars into ethanol. IBP represents an inexpensive, environmentally friendly, low energy and low capital approach for second-generation ethanol production. This paper reviews the advancements in microbial-assisted pretreatment for the delignification of lignocellulosic substrates, system metabolic engineering for biorefineries and highlights the possibilities of process integration for sustainable and economic ethanol production.

Improved ethanol production from biomass by a rumen metagenomic DNA fragment expressed in Escherichia coli MS04 during fermentation.

With the aim of improving current ethanologenic Escherichia coli strains, we screened a metagenomic library from bovine ruminal fluid for cellulolytic enzymes. We isolated one fosmid, termed Csd4, which was able to confer to E. coli the ability to grow on complex cellulosic material as the sole carbon source such as avicel, carboxymethyl cellulose, filter paper, pretreated sugarcane bagasse, and xylan. Glucanolytic activity obtained from E. coli transformed with Csd4 was maximal at 24 h of incubation and was inhibited when glucose or xylose were present in the media. The 34,406-bp DNA fragment of Csd4 was completely sequenced, and a putative endoglucanase, a xylosidase/arabinosidase, and a laccase gene were identified. Comparison analysis revealed that Csd4 derived from an organism closely related to Prevotella ruminicola, but no homologies were found with any of the genomes already sequenced. Csd4 was introduced into the ethanologenic E. coli MS04 strain and ethanol production from CMC, avicel, sugarcane bagasse, or filter paper was observed. Exogenously expressed ß-glucosidase had a positie effect on cell growth in agreement with the fact that no putative ß-glucosidase was found in Csd4. Ethanol production from sugarcane bagasse was improved threefold by Csd4 after saccharification by commercial Trichoderma reesei cellulases underlining the ability of Csd4 to act as a saccharification enhancer to reduce the enzymatic load and time required for cellulose deconstruction.

Efficient dark-fermentation biohydrogen production by Shigella flexneri SPD1 from biowaste-derived sugars through green solvents approach.

This study evaluated the dark-fermentative hydrogen (H2) production potential of isolated and identified Shigella flexneri SPD1 from various pure (glucose, fructose, sucrose, lactose, and galactose) and biowastes (coconut coir, cotton fiber, groundnut shells, rice-, and wheat-straws)-derived sugars. Among sugars, S. flexneri SPD1 exhibited high H2 production of up to 3.20 mol/mole of hexose using glucose (5.0 g/L). The pre-treatment of various biowastes using green solvents (choline chloride and lactic acid mixture) and enzymatic hydrolysis resulted in the generation of up to 36.0 g/L of sugars. The maximum H2 production is achieved up to 2.92 mol/mol of hexose using cotton-hydrolysate. Further, the upscaling of bioprocess up to 5 L of capacity resulted in a maximum yield of up to 3.06 mol/mol of hexose. These findings suggested that S. flexneri SPD1, a novel H2-producer, can be employed to develop a circular economy-based approach to produce clean energy.

Application of Thermomyces lanuginosus polygalacturonase produced in Komagataella phaffii in biomass hydrolysis and textile bioscouring.

In this work, the polygalacturonase (TL-PG1) from the thermophilic fungus Thermomyces lanuginosus was heterologously produced for the first time in the yeast Komagataella phaffii. The TL-PG1 was successfully expressed under the control of the AOX1 promoter and sequentially purified by His-tag affinity. The purified recombinant pectinase exhibited an activity of 462.6 U/mL toward polygalacturonic acid under optimal conditions (pH 6 and 55 ˚C) with a 2.83 mg/mL and 0.063 µmol/minute for Km and Vmax, respectively. When used as supplementation for biomass hydrolysis, TL-PG1 demonstrated synergy with the enzymatic cocktail Ctec3 to depolymerize orange citrus pulp, releasing 1.43 mg/mL of reducing sugar. In addition, TL-PG1 exhibited efficiency in fabric bioscouring, showing potential usage in the textile industry. Applying a protein dosage of 7 mg/mL, the time for the fabric to absorb water was 19.77 seconds (ten times faster than the control). Adding the surfactant Triton to the treatment allowed the reduction of the enzyme dosage by 50% and the water absorption time to 6.38 seconds. Altogether, this work describes a new versatile polygalacturonase from T. lanuginosus with the potential to be employed in the hydrolysis of lignocellulosic biomass and bioscouring.