Microbial Community Succession is Associated With Corn Straw Degradation in Microbial Consortium M44 During Subculture

To systematically analyze the succession of functional microbiota that plays an important role during culture of microbial consortia M44 and its relationship with straw degradation characteristics, we determined the straw degradation ratio and activities of cellulose, hemicellulose, lignin enzyme, and VFA content of M44 in different culture periods. We also used 16S rRNA gene sequencing to analyze the change in microbial community structure in M44 and explore the differences in microbial composition in the original sample. The results showed that at 15 ℃ for 21 days, the straw degradation rate, endoglucanase activity, and filter paper enzyme activity of M44 generally decreased with increasing culture age, reaching their highest values at F1. The activities of xylanase, laccase, and lignin peroxidase, as well as VFA content, were the highest at F5, showing a single-peak curve change with first an increase and then decrease. At the phylum level, Proteobacteria, Bacteroidetes, and Firmicutes were dominant in the original samples and in different culture stages. At the genus level, Devosia and Bacillus were dominant in the original sample. During subculture, the dominant bacteria in the first generation (F1) were Pseudomonas, Flavobacterium, Brevundimonas, Achromobacter, Chryseobacterium, and Devosia. The dominant genera in the last generation (F11) were Trichococcus, Acinetobacter, Dyssgonomonas, and Rhizobium. In conclusion, we identified changes in microbial community structure occurring in M44 during subculture, as well as similarities and differences in microbial communities from the original sample.

Evaluation of Cellulolytic Endo-1,4-β-D-Glucanase Activity in the Digestive Fluid of Adult Phytophagous Beetle Hoplasoma unicolor

Insects of the taxonomic order Coleoptera are recognised for considerable cellulolytic activity in their digestive fluid. The cellulolytic activity of the gut fluid in Hoplasoma unicolor, a member of Coleoptera, however, remains unexplored. In this study, we, for the first time, report the qualitative and quantitative analysis of cellulolytic activity in the digestive fluid of this insect. The cellulolytic endo-1,4-β-D-glucanase activity was confirmed in the supernatant of the insect’s digestive fluid by agar plate assay using carboxymethyl cellulose as the substrate. To determine the optimum pH, enzyme activity was further assessed in an acidic pH range of 5 to 6, and the highest activity was observed at pH 5.3. For quantitative analysis, endoglucanase activity was measured using 3,5-dinitrosalicylic acid method which revealed that the specific activity of the gut sample was 0.69 (±0.01) units per mg of protein. For further characterisation of the cellulases in the sample, SDS-PAGE and zymogram analysis were carried out. Two active cellulolytic bands were detected on the zymogram suggesting the presence of two distinct endoglucanases which completely disappeared upon heating the sample at 55°C. Our study, therefore, highlights prospect of the gut fluid of H. unicolor as an important source of cellulase enzymes that merits further investigations into their extensive characterisation for potential industrial applications.

Brewer’s spent grain as substrate for enzyme and reducing sugar production using Penicillium sp. HC1

Brewer’s spent grain (BSG) is the main solid waste from the brewing process. It is recognized as a valuable resource for biobased industries because of its composition, high availability, and low cost. The objective of this study was to employ BSG as a substrate to produce the enzymes endoglucanase, cellobiohydrolase, β-glucosidase, and xylanase, as well as reducing sugars using Penicillium sp. HC1. For enzyme production, we evaluated BSG submerged fermentation at different concentrations (1%, 3%, and 5%, w/v) and two sources of nitrogen (yeast extract and ammonium sulfate) on different days (6, 10, and 12) in a 100 mL Erlenmeyer flask. The highest enzyme activity was obtained after 10 days. The enzyme extract obtained using 3% BSG (w/v) and 5 g L-1 of ammonium sulfate showed the highest xylanase activity (25013 ± 1075 U L-1). Using BSG 5% (w/v) without nitrogen supplementation, the endoglucanase activity was 909.7±14.2 U L-1 while underthe same conditions but using BSG 3% (w/v), the β-glucosidase and cellobiohydrolase activity was 3268.6 ±229.9 U L-1 and 103.15±8.1 U L-1, respectively. Maximum reducing sugar concentrations using an enzyme dosage of 1000 U g-1 of xylanase were: 2.7 g L-1 xylose, 1.7 g L-1 arabinose, and 3.3 g L-1 glucose after 6 h of hydrolysis. Result s demonstrated it is possible to produce enzymes and reducing sugars using Penicillium sp. HC1 and BSG as substrate and BSG grinding only as pretreatment.

Endoglucanase Activity of Novel Aspergillus sydowii Isolate WIU-01 and Application for Hydrophyte Degradation

<p>Many eutrophic lakes contain rapidly growing hydrophytes. Overgrown biomass is usually me-chanically harvested and thrown away, leading to resource waste and secondary environmental pollution. Microbial degradation is an economically and environmentally friendly approach for managing hydrophytic waste, fuelling the search for efficient biomass degraders. Here, we present isolation and characterization of Aspergillus sydowii WIU-01, a novel cellulolytic fungus. Strain WIU-01 was isolated from air. The degradation rate (29.75 vs. 21.95%) and endoglucanase activi-ty (0.31 vs. 0.16 U mL–1) of the fungus were higher in Canna indica (emergent plant) medium than in Hydrilla verticillata (submerged plant) medium, accordingly. Further, fungal endoglucanase ac-tivity was significantly positively correlated with the degradation rate, neutral detergent fiber con-tent, and acid detergent fiber content of hydrophyte powder. Fungal biomass was significantly negatively correlated with reducing sugar and cellulose content of hydrophyte medium, but was significantly positively correlated with hemicellulose, acid detergent lignin, and ash content of the medium. Collectively, these observations indicate that A. sydowii decomposes emergent and sub-merged plant mass without acid–base sample pretreatment, albeit its endoglucanase activity is rela-tively low. This highlights the role of cellulolytic microorganisms in the natural environment and the notion that the environment can be a source of cellulolytic microorganisms for potential envi-ronmentally friendly applications.</p>

Isolation and Identification of Cellulolytic Bacteria Symbiont from Various Termites on Different Nest Type in Bukit Baka Bukit Raya National Park, West Kalimantan, Indonesia

The microbial symbiotic community in the digestive tract of termites is reportedly influenced by the taxonomy and feeding habit of the host. Both factors are strongly correlated with the nest type. This study aimed to isolate the cellulolytic bacteria from termite’s digestive tract on different nest types and characterize and identify the potential isolates. The research methods included termite sampling conducted in Bukit Baka Bukit Raya National Park (BBBRNP), Melawi, West Kalimantan, isolation of cellulolytic bacteria from termites’ gut, endoglucanase activity test, biochemical characterization, and DNA analysis based on the amplification of 16S rRNA gene. Thirty isolates from 6 different species of termites on three different nest types were successfully isolated. Sixteen potential endoglucanase bacterial isolates were tested in terms of their endoglucanase activity. The cellulolytic index measured from those isolates ranged from 1.162 - 4.894. Three isolates (MRH.13.S, MRH.13.AF, and MRH.13.O2) with the highest cellulolytic index on each nest type were identified. The analysis of 16S rRNA gene using BLAST (Basic Local Alignment Search Tool for Nucleotides) revealed that isolate MRH.13.S had the closest relationship with Bacillus tequilensis (99 % homology). Based on biochemical characterization, MRH.13.AF and MRH.13.O2 isolates were related to Bacillus spp. HIGHLIGHTS Potential cellulolytic bacteria from termite intestinal tract from different nests (i.e., soil, wood, and arboreal) were isolated and compared Termites were obtained from a lowland dipterocarp primary forest ecosystem in Bukit Baka Bukit Raya National Park, West Kalimantan Province, Indonesia Termite species collected were Termes comis, Dicuspiditermes garthwaitei, Synhamitermes quadriceps, Havilanditermes proatripennis, Bulbitermes borneensis, and Bulbitermes parapusillus Potential cellulolytic bacteria acquired were closely related with Bacillus tequilensis and Bacillus spp GRAPHICAL ABSTRACT

Production of Gloeophyllum trabeum Endoglucanase Cel12A in Nicotiana benthamiana for Cellulose Degradation

Lignocellulosic biomass from plants has been used as a biofuel source and the potent acidic endoglucanase GtCel12A has been isolated from Gloeophyllum trabeum, a filamentous fungus. In this study, we established a plant-based platform for the production of active GtCel12A fused to family 3 cellulose-binding module (CBM3). We used the signal sequence of binding immunoglobulin protein (BiP) and the endoplasmic reticulum (ER) retention signal for the accumulation of the produced GtCel12A in the ER. To achieve enhanced enzyme expression, we incorporated the M-domain of the human receptor-type tyrosine-protein phosphatase C into the construct. In addition, to enable the removal of N-terminal domains that are not necessary after protein expression, we further incorporated the cleavage site of Brachypodium distachyon small ubiquitin-like modifier. The GtCel12A-CBM3 fusion protein produced in the leaves of Nicotiana benthamiana exhibited not only high solubility but also efficient endoglucanase activity on the carboxymethyl cellulose substrate as determined by 3,5-dinitrosalicylic acid assay. The endoglucanase activity of GtCel12A-CBM3 was maintained even when immobilized on microcrystalline cellulose beads. Taken together, these results indicate that GtCel12A endoglucanase produced in plants might be used to provide monomeric sugars from lignocellulosic biomass for bioethanol production.

Biorefining: the role of endoglucanases in refining of cellulose fibers

With an annual production of more than 400 million tons, paper is the main product of the largest biorefinery process industrially implemented. Enzymes have been used for pulp refining to dramatically reduce energy consumption. However, exact mechanisms related to the individual enzymes are hardly understood. Yet, this knowledge would be important to predict enzyme performance in industrial processes. Three commercial refining enzyme formulations showed different endoglucanase (1.25 nkat mg−1–13.7 nkat mg−1), β-glucosidase (0.57 nkat mg−1–1.34 nkat mg−1) and xylanase activities (1.78 nkat ml−1–62.1 nkat mg−1) on model substrates. Additionally, distinct amounts of reducing sugars from hardwood sulfate pulp were released. Endoglucases were purified from each formulation by using hydrophobic interaction and anion exchange chromatography and showed molecular weights from 20 to 55 kDa and specific activities ranging between 3.11 and 26.3 nkat mg−1 according to endoglucanase specific derivatized cellopentaose (CellG5). Refining trials of hardwood sulfate pulp were conducted using a PFI laboratory mill and fiber properties such as degree of refining or fiber length and properties of formed sheets like tensile index were monitored. Thereby, enzymes were dosed based on identical endoglucanase activity on CellG5. Enzyme formulations and purified endoglucanases led to an increase of the degree of refining of up to 47.9 [°SR] at 6000 PFI revolutions while the tensile index was improved by up to 76.0 Nm g−1. In summary, refining effects can be primarily attributed to endoglucanases indicating activity on CellG5 being a suitable parameter for enzyme dosing.

Study of the influence of external factors on the inheritance of forms of dissociants Pseudomonas mandelii

Differences in proteolytic, antioxidant, and endoglucanase activity were detected. One of the factors for switching dissociants of the P. mandelii strain IB-Ki14 is the pH of the medium; switching and inheritance schemes are drawn up.

Change of Endoglucanase Activity and Rumen Microbial Community During Biodegradation of Cellulose Using Rumen Microbiota

Treatment with rumen microorganisms improves the methane fermentation of undegradable lignocellulosic biomass; however, the role of endoglucanase in lignocellulose digestion remains unclear. This study was conducted to investigate endoglucanases contributing to cellulose degradation during treatment with rumen microorganisms, using carboxymethyl cellulose (CMC) as a substrate. The rate of CMC degradation increased for the first 24 h of treatment. Zymogram analysis revealed that endoglucanases of 52 and 53 kDa exhibited high enzyme activity for the first 12 h, whereas endoglucanases of 42, 50, and 101 kDa exhibited high enzyme activities from 12 to 24 h. This indicates that the activities of these five endoglucanases shifted and contributed to efficient CMC degradation. Metagenomic analysis revealed that the relative abundances of Selenomonas, Eudiplodinium, and Metadinium decreased after 12 h, which was positively correlated with the 52- and 53-kDa endoglucanases. Additionally, the relative abundances of Porphyromonas, Didinium, unclassified Bacteroidetes, Clostridiales family XI, Lachnospiraceae and Sphingobacteriaceae increased for the first 24 h, which was positively correlated with endoglucanases of 42, 50, and 101 kDa. This study suggests that uncharacterized and non-dominant microorganisms produce and/or contribute to activity of 40, 50, 52, 53, and 101 kDa endoglucanases, enhancing CMC degradation during treatment with rumen microorganisms.