Filter Paper Degradation Research Articles

Characterization of probiotic strains of Bacillus sp. from fermented palm wine (Nypa fructicans sp.) and exploration of cellulolytic potential for use as an addition in animal feed.

The main objective of this study was to assess cellulolytic probiotic strains from traditional fermented beverages such as palm wine in order to supplement the animal feed and strengthen the gut health of the animal for better digestibility and absorption. In the present study, different types of microbes were isolated from traditionally prepared palm wine and analyzed for their probiotic nature. For any microbe to be probiotic in nature, it has to sustain the harsh conditions of the human gastrointestinal tract such as acid tolerance, bile tolerance at the lower range of pH, and other properties like auto aggregation test, cell surface hydrophobicity test with non-polar hydrocarbons for evaluating its capabilities to adhere to the intestinal cells and antimicrobial nature against pathogens. Bacillus mycoides strain PR04 and Bacillus subtilis strain PR21 were found to be resistant to acid and bile in simulated artificial gastrointestinal tract model, found to be than 55% hydrophobic with xylene and n-hexadecane and also showed antimicrobial activity greater towards pathogenic strains like Pseudomonas aeruginosa, Staphylococcus aureus, Candida albicans, and Salmonella typhimurium respectively. The cellulolytic activity of the isolates PR04 and PR21 was evaluated in (0.2-2) % CMC (carboxymethyl cellulose) plate. Bacillus mycoides PR04 and Bacillus subtilis PR21 could degrade carboxymethyl cellulose, filter paper, and sugarcane bagasse. The degradation of sugarcane bagasse was confirmed by Scanning electron microscopy and filter paper degradation after 4days of incubation at 37°C. Cellulase gene of the identified Bacillus sp. strains was amplified by primers CF5'-ACAGGATCCGATGAAAACGGTCAATTTCTATTTT-3' and CR5'-ACTCTCGAGATTGGGTTCTGTTCCCAAT-3'. This study proposes potential probiotic Bacillus mycoides PR04 (Accession no. OR625070) and Bacillus subtilis PR21 (Accession no. OR625072) in the application as an animal feed additive to assist in its digestibility and encourage the gut health.

Efficient Degradation of Untreated Complex Cellulosic Substrates by Newly Isolated Aerobic Paenibacillus Species

A stable aerobic consortium was enriched to degrade crystalline cellulose (Whatman filter paper 1). The degradation efficiency of the consortium after 7 days of incubation was 91% compared to the control. One bacterial isolate, C7, capable of degrading various cellulosic substrates, was obtained from the consortium under aerobic conditions. The sequencing of 16s rDNA revealed that it was related to Paenibacillus sp. It degraded 83% of cotton after 3 days of incubation. The degradation efficiency of Paenibacillus sp. C7 for filter paper, cotton, and avicel was 90%, 90%, and 92% after 5 days of incubation compared to the control. It also degraded non-pretreated agricultural residues efficiently by 70% for rice straw and 46% for wheat bran in 10 days. Scanning electron micrographs (SEMs) of degraded filter paper after 2 days of incubation indicated smoother and thinner fabrics in its structure. It is a potential cheaper candidate for the degradation of lignocellulosic biomass without any pretreatment.

Degradation Potential of Biodegradable Plastics under Laboratory Conditions

Conventional plastics are one of the greatest environmental issues due to their excessive usage and slow degradation. Biodegradable plastics have been introduced as a promising solution to the issue. However, the degradation potential and the best exposure conditions for biodegradable plastics are not well understood in the local context. Therefore, the present study was conducted to evaluate the degradation potential of some commercially available single-use plastics labeled as ―biodegradable‖. Two brands of lunch sheets (LS1 and LS2) made from Polylactic acid (PLA), Polybutylene adipate terephthalate (PBAT), and corn starch, and one waste bag made from Low- density polyethylene (LDPE) and Linear low-density polyethylene (LLDPE) were tested against cellulose filter paper (FP) as a positive control. Degradation was tested using mass loss analysis, biogas production, and FTIR spectrum analysis together with visual analysis. The test materials were subject to different exposures, i.e., placed under seawater, beach sand, freshwater, sand with overlying freshwater, and under direct sunlight for 210 days, and anaerobic exposure for 60 days. LS1 had the highest mass loss of 73.3% in freshwater and the lowest of 9.5% in sunlight exposure. LS2 recorded the highest mass loss of 59.3% in compost exposure and the lowest of 2.9% in sunlight exposure. Waste bags recorded 1.5% as the highest mass loss in the sand with overlying freshwater and the lowest 0.3% under direct sunlight which is the lowest among all four samples. FP has shown the highest and lowest mass loss in composting and direct sunlight exposures at 79.7% and 6.2%. The degradation of FP indicates that the conditions required for biodegradation were present in the exposure environments. Direct sunlight exposure does not show a significant impact on the degradation of any of the test materials. The waste bag does not show considerable degradation under any exposure. The carbon dioxide gas production was 8.76×10-4 mol, 18.49×10-4 mol, and 2.88×10-4 mol in LS1, LS2, and waste bag, respectively. LS1 and LS2 showed visual damages and also changes in the FTIR spectrum. However, waste bags did not show any changes visually or in FTIR spectra. Therefore, it can be concluded that LDPE and LLDPE products did not undergo a detectable degradation though PLA, PBAT, and corn starch products show clear signs of degradation. The study needs to be repeated in situ to better understand the degradation behavior in actual exposure situations. 
 Keywords: Biodegradable plastics, Degradation, Plastic pollution

Isolasi dan Aktivitas Bakteri Selulolitik pada Limbah Diapers

This study aims to isolate cellulolytic bacteria that have the potential to degrade cellulose by taking home-scale diaper waste samples. This research was also conducted to isolate and determine the ability of bacteria to degrade cellulose based on the Congo red test and filter paper degradation test. This research method is descriptive qualitative. Isolation of cellulolytic bacteria was carried out using selective Carboxymethyl Cellulose (CMC) media. The characterization was carried out by growing selected pure isolates on CMC media and then dropping congored drops to test their cellulolytic potential (cellulolytic potential is indicated by the appearance of a clear zone around the colony). The results showed that the results of isolating bacteria obtained 12 isolates that were able to grow and utilize cellulose as a carbon source. In the Congo red test isolate DS07 cellulolytic bacteria of the Hafnia genus had the highest cellulolytic index value of 2.5 mm. Isolate DS03 with the genus Bacillus had the highest ability to degrade cellulose on the Whatman No. filter paper test. 1 of 16.66%.
 Keywords: Bacteria, Diapers, Isolation, Cellulolytic

Pretreatment of lignocellulosic biogas substrates by filamentous fungi

Decomposition of lignocellulosic plant biomass by four filamentous fungi was carried out to facilitate subsequent anaerobic degradation and biogas formation. Agricultural side products, wheat straw and corn stover and forestry energy plant willow chips were selected as plant biomass sources. The substrates were confronted by pure cultures of Penicillium aurantiogriseum (new isolate from rumen), Trichoderma reesei (DSM768), Gilbertella persicaria (SZMC11086) and Rhizomucor miehei (SZMC11005). In addition to total cellulolytic filter paper degradation activity, the production of endoglucanase, cellobiohydrolase, β-glucosidase enzymes were followed during the pretreatment period, which lasted for 10 days at 37 °C. The products of pretreatments were subsequently tested for mesophilic biogas production in batch reactors. All 4 strains effectively pretreated the lignocellulosic substrates albeit in varying degrees, which was related to the level of the tested hydrolytic enzyme activities. Penicillium aurantiogriseum showed outstanding hydrolytic enzyme production and highest biogas yield from the partially degraded substrates. Corn stover was the best substrate for biomass decomposition and biogas production. Scanning electron microscopy confirmed the deep penetration of fungal hyphae into the lignocellulosic substrate in all cases.

Design and application of an efficient cellulose-degrading microbial consortium and carboxymethyl cellulase production optimization

Microbial consortia with high cellulase activities can speed up the composting of agricultural wastes with high cellulose contents and promote the beneficial utilization of agricultural wastes. In this paper, rabbit feces and sesame oil cake were used as feedstocks for compost production. Cellulose-degrading microbial strains were isolated from compost samples taken at the different composting stages and screened via Congo red staining and filter paper degradation test. Seven strains, Trichoderma reesei, Escherichia fergusonii, Proteus vulgaris, Aspergillus glaucus, Bacillus mycoides, Corynebacterium glutamicum, and Serratia marcescens, with high activities of carboxymethyl cellulase (CMCase), filter paper cellulase (FPase), and β-glucosidase (β-Gase) were identified and selected for consortium design. Six microbial consortia were designed with these strains. Compared with the other five consortia, consortium VI composed of all seven strains displayed the highest cellulase activities, 141.89, 104.56, and 131.18 U/ml of CMCase, FPase, and β-Gase, respectively. The single factor approach and response surface method were employed to optimize CMCase production of consortium VI. The optimized conditions were: culture time 4.25 days, culture temperature 35.5°C, pH 6.6, and inoculum volume 5% (v/v). Under these optimized conditions, the CMCase activity of consortium VI was up to 170.83 U/ml. Fermentation experiment of rabbit feces was carried out by using the consortium VI cultured under the optimal conditions. It was found that the application effect was better than other treatments, and the fermentation efficiency and nutrient content of the pile were significantly improved. This study provides a basis for the design of microbial consortia for the composting of agricultural wastes with high cellulose contents and provides a support for beneficial utilization of agricultural wastes.

Isolation and Comparative Analysis of Culturable Cellulose degrading Gut Bacteria from a Coleopteran and Lepidopteran Insects

Insect gut microbiota has been reported to influence multiple biological functions of the host such as nutrition, digestion and immunity. In this study, nine and eight culturable bacteria were isolated from the gut of eri silkworm, Samia ricini and root grub, Anamola dimidiata. The gut bacteria were assayed for their ability to degrade filter paper.. The results indicated that filter paper digestion was faster in A. dimidiata compared to S. ricini reaching 50% within five incubation days. Cellulolytic activity indicated significant endoglucanase, exoglucanase and β-glucosidase activity in A. dimidiata compared to S. ricini, an indication that A. dimidiata harbours more robust cellulose degrading bacteria than S. ricini. The culturable gut bacterial isolates with cellulolytic activity may be good sources for preparing probiotic formulations to boost eri-nutrition as well as profiling cellulolytic enzymes or genes for biotechnological application..

Comparative analysis of paddy straw-degrading consortia in China using high-throughput sequencing

Nine paddy straw-degrading microbial consortia were isolated from 253 paddy straw samples from northern China and the Yangtze River Valley, which are two major rice production regions in China. The microbial consortia completely degraded filter paper within 15 d at 25 °C. The V4 region of the microbial consortia 16S rRNA gene was amplified, and the amplicons were sequencing using the Illumina Miseq high-throughput platform. Compilobacterota and Bacteroidota were the predominant phyla in the samples. At the genus level, the relative abundance of Macellibacteroides was the highest in Y2, and Arcobacter was predominant in the other samples. Analysis of the microbial community structure revealed that the diversity and richness in samples from the Yangtze River Valley were significantly higher compared with those of the samples from northern China, and the community structure of the four microbial consortia from the northern region had higher similarity than the five consortia from the Yangtze River Valley.

Comparative metagenomic discovery of the dynamic cellulose-degrading process from a synergistic cellulolytic microbiota

To reveal the dynamic process of cellulose biodegradation and explore more potential cellulases, a microbiota (FPDM) with cellulose-degrading ability was cultivated, and different stages of filter paper degradation were compared. Ion chromatography and comparative metagenomic sequencing revealed that the diversity of FPDM enhanced as the hydrolysate length diversity increased. Sporocytophaga and Cohnella dynamically dominated the synergistic degradation of cellulose in early-intermediate and intermediate-final periods, respectively. Moreover, 363 declining shifting and 231 progressive shifting unannotated genes were speculated to participate in the catabolism of cellulose to cellodextrin/cello-oligosaccharide and to cellobiose, respectively. Based on the dynamic changes in hydrolysates, community structure and gene abundance, a dynamic cellulose-degrading pathway of FPDM was predicted. Our work should provide a new perspective for subsequent identification of key cellulolytic strains and enzymes and clarification of the mechanism of cellulose biodegradation.

Efficacy and Metagenomic Analysis of the Stabilized Anaerobic Lignocellulolytic Microbial Consortium from Bubalus bubalis Rumen with Rice Straw Enrichment for Methane Production

Five different inocula from cow manure, goat manure, pig manure, and rumen fluid (RU) and microbial sludge from anaerobic digestion were screened to select anaerobic lignocellulolytic microbial consortium (ALMC) of high efficacy in degradation of filter paper (FP), microcrystalline cellulose (MCC), and rice straw (RS) to produce methane. RU from Bubalus bubalis demonstrated the highest growth rate RU inoculum (10.45 mgVS/day); fastest degradation rate of FP, MCC, and RS (125.00, 50.07, and 25.43 mgVS/day); and highest methane production rate (6.23 ml/day). To obtain the enriched and stabilized ALMC from RU, consecutive batch subculturing was used and reached at 25 passages, namely, E25. The efficacies of E25 in RS removal and methane production were 78%VS and 293 ml/gVSadded compared with initial inoculum (E0) at 46% VS and 138 ml/gVSadded, respectively, within 14 days of incubation time. The efficacies of E25 in RS removal and methane production were 1.7- and 2.12-folds higher than of E0, respectively. E25 showed high ability to utilize various carbon sources (68%) from Biolog EcoPlate assay. It was confirmed that E25 could degrade derivatives of cellulose, hemicellulose, and lignin. Metagenomic analysis by 16 s and shotgun sequencing of E25 found dominant species of hydrolytic/fermentative bacteria (Proteiniphilum acetatigenes, Pyramidobacter piscolens, Syntrophus aciditrophicus, Mesotoga prima, and Candidatus Cloacimonas acidaminovorans) and methanogens (Methanoculleus bourgensis, Methanoculleus marisnigri, Methanofollis liminatans, Methanosarcina mazei, and Methanosaeta harundinacea). This stabilized E25 with enrichment of lignocellulolytic and fermentative bacteria as well as hydrogenotrophic and acetoclastic methanogens shows its efficacy in accelerating RS degradation and methane production.

Analysis of cellulolytic bacterial flora in the rumen of inner Mongolian sheep

The cellulolytic bacterial flora present in the rumen of Inner Mongolian sheep are thought to have a high degree of cellulose-degrading activity because of their foraging feeding regimen. However, there are no report on the genetic and species composition of the cellulolytic bacterial flora. In this study, cellulolytic bacteria were isolated from the rumen of Inner Mongolian sheep using a combined method of transparent zone and filter paper degradation. Twenty-two strains were identified via morphological, physiological, and biochemical tests. Ten strains were further identified via DNA (G + C) mol%, together with 16S rDNA gene sequencing analysis. Four types of extracellular and total cellulase activities of representative strains were determined. The results demonstrated that the isolates included Butyrivibrio fibrisolvens, Rumincoccus albus, R. flavefaciens, Fibrobacter succinogenes, and Clostridium polysaccharolyticum. A big proportion of cellulolytic Butyrivibrio fibrisolvens was found in the rumen of Inner Mongolian sheep. This was the first study to analyze the cellulolytic bacterial flora in the rumen of foraging Inner Mongolian sheep. These results indicated that the rumen of Inner Mongolian sheep represents an attractive source for cellulolytic microorganisms and enzymes, and the research results have a certain guiding importance for the efficient degradation of cellulosic materials.

ISOLASI BAKTERI SELULOLITIK PENDEGRADASI SELULOSA DARI KOMPOS

This study aimed to isolate cellulolytic bacteria that have the potential to degrade cellulose taken compost samples from Temesi and Bondowoso, this study was also conducted to determine the ability of bacteria to degrade cellulose based on Congo red test and filter paper degradation test. All isolates were tested for cellulolytic activity using soluble Carboxymethyl Cellulose (CMC). Characterization was carried out by growing selected pure isolates on CMC media and then dripping 0.1% congo red to test the cellulolytic potential (cellulolytic potential was characterized by the emergence of clear zones around the colony). The results of isolation of bacteria obtained 38 isolates, namely 26 Bondowoso sample isolates and 12 Temesi isolates that were able to grow and utilize cellulose as a carbon source. But only fourteen isolates produced clear zones in the Congo red test with diameters ranging from 1.66 cm to 6.76 cm. Six isolates that have the largest diameters clear zone, were tested for degradation of filter paper (Whatman no. 1). Isolates bacteria of B2S8 obtained from Bondowoso compost samples has the highest ability to degrade cellulose on Whatman paper no. 1 as much as 51.30%. 
 Keywords: Compost, Isolation, Screening, Cellulolytic Bacteria.

Cloning, expression, and characterization of novel GH5 endoglucanases from Thermobifida alba AHK119

Thermobifida alba AHK119 exhibits sufficient filter paper-degradation activity in its culture supernatant. AHK119-bMs (1365bp) and AHK119-E5 (1425bp), which encode novel GH5 family endoglucanases, were cloned from the genomic DNA of T.alba AHK119. AHK119-bMs and AHK119-E5 consisted of 454 and 474 amino acid residues, respectively, in which the catalytic domain (CD) and carbohydrate-binding module (CBM) were connected by an accessary module (linker region). The amino acid sequences of CD and CBM of AHK119-bMs were most identical to those of endo-β-mannanases (Man5As) from Thermobifidafusca TM51, T. halotolerans YIM90462, and T.cellulosilytica TB100. In contrast, the amino acid sequences of CD and CBM of AHK119-E5 were most identical to those of endo-1,4-β-glucanases (cellulases; Cel5As) from T.fusca and T.halotolerans YIM90462. However, the linker region of both the genes shared low identities with those of Man5As and Cel5As. AHK119-bMs showed broader specificities toward cellulosic substrates than Man5As, whereas AHK119-E5 showed higher activity toward insoluble cellulosic substrates than toward soluble ones, which was conflicting when compared with other Cel5As. In addition, AHK119-bMs and AHK119-E5 showed different requirements for metal ions from those of Man5As and Cel5As, respectively. Therefore, both the enzymes were identified as novel GH5 endoglucanases, and the accessary modules seemed to play important roles in their enzymatic properties.

Processivity and enzymatic mechanism of a multifunctional family 5 endoglucanase from Bacillus subtilis BS-5 with potential applications in the saccharification of cellulosic substrates

BackgroundPresently, enzymes still constitute a major part of the cost of biofuel production from lignocellulosic biomass. Processive endoglucanases, which possess both endoglucanase and exoglucanase activity, have the potential to reduce the costs of biomass saccharification when used together with commercial cellulases. Therefore, the exploration of new processive endoglucanases has attracted much attention with a view to accelerating the industrialization of biofuels and biochemicals.ResultsThe endoglucanase EG5C and its truncated form EG5C-1 from Bacillus subtilis BS-5 were expressed and characterized. EG5C was a typical endoglucanase, comprised of a family 5 catalytic domain and family 3 carbohydrate-binding domain, and which had high activity toward soluble cellulosic substrates, but low activity toward insoluble cellulosic substrates. Importantly, the truncated form EG5C-1 was a processive endoglucanase that hydrolyzed not only carboxymethyl cellulose (CMC), but also insoluble cellulosic substrates. The hydrolytic activities of EG5C-1 towards CMC, phosphoric acid-swollen cellulose (PASC), p-nitrophenyl-β-d-cellobioside, filter paper and Avicel are 4170, 700, 2550, 405 and 320 U/μmol, respectively. These data demonstrated that EG5C-1 had higher activity ratio of exoglucanase to endoglucanase than other known processive endoglucanases. When PASC was degraded by EG5C-1, the ratio of soluble to insoluble reducing sugars was about 3.7 after 3 h of incubation with cellobiose and cellotriose as the main products. Importantly, EG5C-1 alone was able to hydrolyze filter paper and PASC. At 5% substrate concentration and 10 FPU/g PASC enzyme loading, the saccharification yield was 76.5% after 60 h of incubation. Replacement of a phenylalanine residue (F238) by an alanine at the entrance/exit of the substrate binding cleft significantly reduces the ability of EG5C-1 to degrade filter paper and Avicel, but this mutation has little impact on CMCase activity. The processivity of this mutant was also greatly reduced while its cellulose binding ability was markedly enhanced.ConclusionsThe processive endoglucanase EG5C-1 from B. subtilis BS-5 exhibits excellent properties that render it a suitable candidate for use in biofuel and biochemical production from lignocellulosic biomass. In addition, our studies also provide useful information for research on enzyme processivity at the molecular level.

Transcriptome and Zymogram Analyses Reveal a Cellobiose-Dose Related Reciprocal Regulatory Effect on Cellulase Synthesis in Cellulosilyticum ruminicola H1.

The rumen bacterium Cellulosilyticum ruminicola H1 efficiently hydrolyzes cellulose. To gain insights into the regulatory mechanisms of cellulase synthesis, comparative transcriptome analysis was conducted for cultures grown on 2% filter paper, 0.5 and 0.05% cellobiose, and 0.5% birchwood xylan. It was found that cellulose induced a majority of (hemi)cellulases, including 33 cellulases and a cellulosomal scaffoldin (1.3- to 22.7-fold); seven endoxylanases, two mannanases, and two pectatelyases (2- to 16-fold); and pyruvate formate-lyase (PFL, 1.5- to 7-fold). Noticeably, 3- and 2.5-fold increased transcription of a cellobiohydrolase and the cellulosomal scaffoldin precursor were detected in 0.05% than in 0.5% cellobiose. Consistently, 9- and 4-fold higher specific cellobiohydrolase activities were detected in the filter paper and 0.05% cellobiose culture. SDS- and native-PAGE zymograms of cellulose-enriched proteins from the filter paper culture displayed cellulase activities, and cellulolytic “complexes” were enriched from the filter paper- and 0.05% cellobiose-cultures, but not from the 0.5% cellobiose culture. LC-MS/MS identified the cellulosomal scaffoldin precursor in the “complexes” in addition to cellulase, hemicellulase, and PFL proteins. The addition of 0.5% cellobiose, but not 0.05% cellobiose remarkably inhibited strain H1 to degrade filter paper. Therefore, this work reveals a cellobiose-dose related regulatory mechanism of cellulase synthesis by lower for induction and higher for repression, which has extended our understanding of the regulation of microbial cellulase synthesis.

Characterization of a Cellulomonas fimi exoglucanase/xylanase-endoglucanase gene fusion which improves microbial degradation of cellulosic biomass.

Effective degradation of cellulose requires multiple classes of enzyme working together. However, naturally occurring cellulases with multiple catalytic domains seem to be rather rare in known cellulose-degrading organisms. A fusion protein made from Cellulomonas fimi exo- and endo- glucanases, Cex and CenA which improves breakdown of cellulose is described. A homologous carbohydrate binding module (CBM-2) present in both glucanases was fused to give a fusion protein CxnA. CxnA or unfused constructs (Cex+CenA, Cex, or CenA) were expressed in Escherichia coli and Citrobacter freundii. The latter recombinant strains were cultured at the expense of cellulose filter paper. The expressed CxnA had both exo- and endo- glucanase activities. It was also exported to the supernatant as were the non-fused proteins. In addition, the hybrid CBM from the fusion could bind to microcrystalline cellulose. Growth of C. freundii expressing CxnA was superior to that of cells expressing the unfused proteins. Physical degradation of filter paper was also faster with the cells expressing fusion protein than the other constructs. Our results show that fusion proteins with multiple catalytic domains can improve the efficiency of cellulose degradation. Such fusion proteins could potentially substitute cloning of multiple enzymes as well as improving product yields.

Heterologous expression of the multi-functional cellulase gene (mfc) from the mollusc Ampullaria crossean, in Volvariella volvacea

ABSTRACTVolvariella volvacea, or straw mushroom, is a widely cultivated and important edible fungus. In this paper, we used straw mushroom as a host to express an exogenous mfc gene encoding a multi-functional cellulase (MFC) cloned from the mollusc Ampullaria crossean. The purpose was to increase expression of MFC in the new host, as well as to improve cellulose degradation and increase future yields of V. volvacea. MFC is an enzyme with exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-xylanase activities. The A. crossean mfc gene was expressed in V. volvacea under the control of an endogenous promoter (gpd-Vvs) using the expression vector pgVvs-mfc, constructed by ligating the gpd-Vvs promoter with the mfc gene. The expression plasmid, pgVvs-afp, containing an afp gene encoding an anti-freeze protein (AFP) cloned from spruce budworm as the selective marker gene, was co-transformed into protoplasts of V. volvacea along with the vector pgVvs-mfc using polyethylene glycol (PEG)-mediated transformation. Putative transformants of V. volvacea were obtained by exposing the mycelia to regeneration medium (200 g l−1 potato extract, 20 g l−1 dextrose, 20 g l−1 agar, and 0.8 M D-mannitol) at 0°C. PCR and Southern blotting were used to identify positive transformants. The results indicated that the mfc gene had been integrated into the genome of V. volvacea. The total cellulose activity, based on filter paper degradation, and carboxymethyl cellulase and xylanase activities in the transformants were 3.92, 4.75, and 58.99 Units ml−1, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed an approx. 46 kDa band, which was equivalent to the expected size of the MFC protein of A. crossean. Thus, the mfc gene appeared to have been expressed heterologously in V. volvacea.

Cellulolytic potential of probiotic Bacillus Subtilis AMS6 isolated from traditional fermented soybean (Churpi): An in-vitro study with regards to application as an animal feed additive

The aim of the present study is to evaluate the probiotic attributes of Bacillus subtilis AMS6 isolated from fermented soybean (Churpi). This isolate exhibited tolerance to low pH (pH 2.0) and bile salt (0.3%), capability to autoaggregate and coaggregate. AMS6 also showed highest antibacterial activity against the pathogenic indicator strain Salmonella enterica typhimurium (MTCC 1252) and susceptibility towards different antibiotics tested. The isolate was effective in inhibiting the adherence of food borne pathogens to Caco-2 epithelial cell lines, and was also found to be non-hemolytic which further strengthen the candidature of the isolate as a potential probiotic. Further studies revealed B. subtilis AMS6 showed cellulolytic activity (0.54±0.05 filter paper units mL−1) at 37°C. The isolate was found to hydrolyze carboxymethyl cellulose, filter paper and maize (Zea mays) straw. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96h of incubation. A full length cellulase gene of AMS6 was amplified using degenerate primers consisting of 1499 nucleotides. The ORF encoded for a protein of 499 amino acids residues with a predicted molecular mass of 55.04kDa. The amino acids sequence consisted of a glycosyl hydrolase family 5 domain at N-terminal; Glycosyl hydrolase catalytic core and a CBM-3 cellulose binding domain at its C terminal. The study suggests potential probiotic B. subtilis AMS6 as a promising candidate envisaging its application as an animal feed additive for enhanced fiber digestion and gut health of animal.

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C.

Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925. Glycoside hydrolase family 7 reducing end acting cellobiohydrolase.

In vitro evaluation of celluloytic Bacillus amyloliquefaciens AMS1 isolated from traditional fermented soybean (Churpi) as an animal probiotic

A microorganism showing probiotic attributes and hydrolyzing carboxymethylcellulose was isolated from traditional fermented soybean (Churpi) and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens AMS1. The potentiality of this isolate as probiotic was investigated in vitro and it showed gastrointestinal transit tolerance, cell surface hydrophobicity, cell aggregation and antimicrobial activity. The isolate was found to be non-hemolytic which further strengthens its candidature as a potential probiotic. The maize straw digestion was confirmed by scanning electron microscopy studies. The isolate was able to degrade filter paper within 96 hours of incubation. This study explores the possibility of combining the cellulase degrading ability of a microbe with its probiotic attributes to enhance gut health of animal and digestibility of the feed.