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The genus Aloe (family: Xanthorrhoeaceae) encompasses 490 shrubby-succulent-perennial species which are native to Africa. Among these, Aloe vera has grabbed the limelight in the arena of herbal medicine due to the presence of unique phytochemicals such as Aloin A and B, Homonataloin, Aloe emodin, acemannan etc. in its latex. Moreover, its leaf extract contains 99% water, and 75 active compounds including vitamins (A, B1, B2, B3, B6, B9, B12, C, and E), minerals (Zn, Se, Na, Mn, Mg, K, Cu, Cr, Ca), amino acids, and enzymes (peroxidase, lipase, cellulase, catalase, carboxypeptidase, bradykinase, amylase, alkaline phosphatase). Thus, Aloe vera is revered as a ‘wonder plant’ as it possesses multiple pharmaceutical properties such as anti-oxidant, anti-inflammatory, anti-tumor, anti-fungal, anti-diabetic, anti-ulcer, anti-hypertensive and its juice helps to cure gastrointestinal disorders, and dermal diseases, it also strengthens the immune system. Being a xerophyte, Aloe vera is commercially cultivated in many countries which include South Africa, America, Australia, India etc. The Aloe gel, is meticulously extracted, processed and stabilized before using it in preparation of various health care products. This review focuses on the explicit information on origin, botanical description, phytochemistry, nutritional and medicinal benefits, with special emphasis on value-added products of Aloe vera. The review revealed 46 value-added Aloe-products that are being manufactured by different companies worldwide. Thus, sustainable Aloe-cultivation must be ensured to meet the rising global demands for Aloe vera gel and allied products.

Resistance against antibiotics mainly due to their misuse and overuse is an emerging health issue, worldwide. Antibiotics release active antibiotic residues in the environment during their production. Bacteria encounter these active antibiotic residues and the genes present in them; resultantly, they acquire resistance against antibiotics. The current study was conducted to determine the prevalence of multidrug-resistant (MDR) bacterial strains, isolated from hospital wastewater. Using standard procedures, bacterial resistance patterns against different classes of antibiotics were analysed and their species level identification was made. The disc diffusion method was used to determine the bacterial activity against antimicrobial agents. Clear zones were measured separately in millimeters around each disc. Five wastewater samples were collected from different drainage regions of hospitals situated in Multan. A total of 45 bacterial strains were isolated. Out of these 45 bacterial strains, 13 (29%) were found resistant against two or more than two classes of antibiotics. All the bacterial strains (100%) isolated from samples 2 and 3 were MDR. Twenty-five bacterial strains (55.5%) belonged to the Bacillus species and others belonged to Enterococcus species, Micrococcus species, Staphylococcus species, and Streptococcus species, respectively. The presence of resistant bacterial strains in hospital waste demands the availability of effectual treatment plants to treat the waste before it is disposed of into hospital waste lines.

Anthracnose disease caused by Colletotrichum lindemuthianum (Sacc. and Magnus) Lams- -Scrib is one of the most devastating seed-borne diseases of common bean (Phaseolus vulgaris L.). In the present study, we evaluated the antifungal activity of Bunium persicum essential oil (EO) and its main constituents on mycelial growth, sporulation and spore germination inhibition of C. lindemuthianum. The main objective of this study was to investigate the effect of EO and its main constituents on decreasing the activity of cell wall degrading enzymes (CWDEs) produced by C. lindemuthianum, which are associated with disease progress. Also, the effects of seed treatment and foliar application of EO and its main constituent, cuminaldehyde, on anthracnose disease severity was investigated. The essential oil of B. persicum, was obtained by using a clevenger apparatus and its major constituents were identified by gas chromatography-mass spectrometry (GC-MS). The EO was characterized by the presence of major compounds such as cuminaldehyde (37.7%), γ-terpinene (17.1%) and β-pinene (15.4%), which indicated antifungal effects against C. lindemuthianum. This pathogen did not grow in the presence of EO, cuminaldehyde and γ-terpinene, β-pinene at 1,500; 1,010 and 1,835 ppm concentrations, respectively. Also, sporulation and spore germination of C. lindemuthianum was completely inhibited by EO and cuminaldehyde. Synergistic effects of the main constituents showed that combing γ-terpinene with cuminaldehyde induced a synergistic activity against C. lindemuthianum and in combination with β-pinene caused an additive effect. Activities of pectinase, cellulase and xylanase, as main CWDEs, were decreased by EO and its main constituents at low concentration without affecting mycelial growth. Seed treatment and foliar application of peppermint EO and/or cuminaldehyde significantly reduced the development of bean anthracnose. We introduced B. persicum EO and constituents, cuminaldehyde and γ-terpinene, as possible control agents for bean anthracnose.

The quest for novel broad spectrum bioactive compounds is needed continuously because of the rapid advent of pathogenic multi drug resistant organisms. Actinomycetes, isolated from unexplored habitats can be a solution of this problem. The motive of this research work was isolation of actinomycetes having potential antimicrobial activities from unexplored regions of Devbag and Tilmati beach. The isolated actinomycetes were screened against pathogenic microbes for antimicrobial activities through cross streak method. Enzyme production activity was checked for these actinomycetes for amylase, protease, cellulase and lipase enzymes. Further antimicrobial activity of ethyl acetate extract of the potent strain KS46 was performed. The strain KS46 was identified with 16S rRNA gene sequencing and secondary structure was analysed. Gas chromatography-Mass spectrometry (GC-MS) profiling was conducted to ascertain the presence of bioactive metabolites in the ethyl acetate extract. The collected samples were pre-treated and 70 actinomycetes were isolated. The Streptomyces sp. strain KS46 showed the best antimicrobial activity in primary screening. Ethyl acetate extract of the strain KS46 revealed antimicrobial activity against S. aureus, B. subtilis, B. cereus, E. faecalis, K. pneumoniae, E. coli, S. flexneri, C. albicans and C. glabrata. The 16S rRNA gene sequencing identified the strain KS46 as Streptomyces levis strain KS46. The GC-MS metabolite profiling of the ethyl acetate extract revealed the availability of 42 compounds including fatty acid esters, fatty acid anhydrides, alkanes, steroids, esters, alcohols, carboxylic ester, etc. having antibacterial, antifungal, antiproliferative, antioxidant activities. This study indicated that Devbag and Tilmati beaches being untapped habitats have enormous diversity of promising antimicrobial metabolite producing actinomycetes. Therefore, further exploration should be carried out to characterize the potential actinomycetes, which can be optimistic candidates for generation of novel antimicrobial drugs.

This study provides a detailed analysis of the lignocellulolytic activity of a new isolate Stereum gausapatum F28, a Serbian autochthonous fungi, on beechwood sawdust supplemented with cheap waste, sugar beet molasses still age. Advanced multiple response optimization techniques were applied to improve ligninolytic and reduce hydrolytic activity as a requirement for potential biorefinery use. The applied techniques were supposed to select cultivation conditions that would give manganese peroxidase and laccase activities above 0.84 and 0.12 U g(-1) substrate, respectively, and cellulase and xylanase activities below 1.12 and 1.4 U g(-1) substrate. The optimal cultivation conditions that met the set requirements included molasses stillage concentration of 10 %, substrate moisture content of 53 %, incubation temperature of 23.5 degrees C, and pH 5.2. The research showed that the addition of molasses stillage had a positive effect on enzyme production and that the optimal stillage concentration differed depending on the enzyme type (for laccase it was lt 5 %, manganese peroxidase approximate to 12 %, cellulase approximate to 21 % and xylanase approximate to 16 %), which should be taken into consideration when optimizing the desired process..

To achieve an economically viable substitutive strategy for lignocellulosic residues use, cellulose degradation by efficient Trichoderma strains become a key alternative. This study aimed to explore the effects of various cultural conditions on the degradation of different lignocellulosic substrates (corn straw and licorice residues) by Trichoderma longibrachiatum and Trichoderma afroharzianum in solid state fermentation. The strains were isolated from the rhizospheric soils of medicinal plants. In addition, the best fermentation combination was selected to evaluate the degradation results. This study confirmed that fermentation conditions and different components of corn straw and licorice residues were both crucial factors affecting the cellulase activity and spore production of two Trichoderma strains. A combination of T. afroharzianum degrading corn straw was selected for Response Surface Methodology optimization. The best fermentation conditions were fermentation time of 5.72 days, initial material liquid ratio of 1:4.58, initial pH of 6.32 and fermentation temperature of 28.8 ℃, with a filter paper cellulase activity of 0.91 U/mL. After continuous fermentation, the spore production reached 6.18 × 109 spores/g, the crude fiber degradation rate reached 23.5% with the reducing sugar content maintained at 11 mg/g. These results highlight the great potential of T. afroharzianum degrading corn straw for organic amendment.

Polyphenols display potential functional activities especially for rosmarinic acid (RosA) and caffeic acid (CafA). In this study, two different methods including ultrasonic-assisted ethanol extraction (60%) and ultrasound-assisted cellulase (≥ 15000 U/g, 2%) hydrolysis were applied for the extraction of total phenolics from 44 species of Perilla frutescens. Folin-Ciocalteu method detection showed that the content of total phenolics extracted by cellulase hydrolysis was the highest and attained to 28.00 mgGAE/gextracts for ZB1. Continuously, the extracts were purified by XDA-8 macroporous resin and medium-pressure liquid chromatography (MPLC), and the content of total phenolics improved to 66.62 mgGAE/gextract. High-performance liquid chromatography (HPLC) assay showed that the total polyphenols were mainly composed of gallic acid, caffeic acid, rosmarinic acid, luteolin and apigenin. Besides, a sequential XDA-8 macroporous resin combined with high-speed counter-current chromatography (HSCCC)/MPLC system was established for simultaneously isolation and preparation of RosA (purity 98.29%) and CafA (purity 97.01%) from the extracts. Further, antibacterial activities of the total polyphenols were evaluated by disc diffusion method and scanning electron microscope (SEM) observation. The results verified that the total polyphenols had effective antibacterial capacity on three kinds of bacteria including E. coli, S. aureus, and B. subtilis in a concentration-dependent manner. All these results demonstrated that the ultrasound-assisted cellulase hydrolysis extraction of total polyphenols and proposed three-step separation of RosA and CafA had a high yield and good purity, and they exhibited an effective antibacterial ability.

Enhanced hydrogen evolution was pursued in this work. Rhodobacter sp. (Rb) and Rhodopseudomonas palustris (Rp), single or mixed were used to extract hydrogen molecules from Chlorella fusca biomass. To elevate their fermentable contents, Chlorella was grown at nitrogen and/or phosphorus deprivation. Besides, cellulase and/or macerozyme, Triton X100 or sonicated yeast were applied for further biohydrogen fermentation. Utilizing hydrolysates of mineral deprived Chlorella cultures, Rb exhibited relatively higher cumulative hydrogen (4200 ml L−1) than Rp (2500 ml L−1) while mixed cultures attained significantly higher levels (4700 ml L−1). Triton or enzymes significantly enhanced hydrogen evolution, with more effectiveness of macerozyme than cellulase. A novel use of sonicated yeast, as enzymes pool, induced the highest significant collective H2 (up to 47 times that of microalgal supernatant). Sonicated yeast induced a remarkable hydrolysis of algae, as inferred from increased reducing sugars. However, hydrogen evolution efficiency exhibited poor proportionality with reducing sugars, indicating fermentation of other metabolites.

Endoglucanases are carbohydrate-degrading enzymes widely used for bioethanol production as part of the enzymatic cocktail. However, family 5 subfamily 5 (GH5_5) endoglucanases are still poorly explored in depth. The Trichoderma reesei representative is the most studied enzyme, presenting catalytic activity in acidic media and mild temperature conditions. Though biochemically similar, its modular structure and synergy with other components vary greatly compared to other GH5_5 members and there is still a lack of specific studies regarding their interaction with other cellulases and application on novel and better mixtures. In this regard, the threedimensional structure elucidation is a highly valuable tool to both uncover basic catalytic mechanisms and implement engineering techniques, proved by the high success rate GH5_5 endoglucanases show. GH5_5 enzymes must be carefully evaluated to fully uncover their potential in biomass-degrading cocktails: the optimal industrial conditions, synergy with other cellulases, structural studies, and enzyme engineering approaches. We aimed to provide the current understanding of these main topics, collecting all available information about characterized GH5_5 endoglucanases function, structure, and bench experiments, in order to suggest future directions to a better application of these enzymes in the industry.

Saccharification of lignocellulose is prerequisite for the biorefinery. Clostridium thermocellum is a promising candidate for the hydrolysis of lignocellulose. A study on the saccharification by Clostridium thermocellum combined with surfactant and cellulase addition was investigated. The reducing sugars content increased with increasing substrate loading in the fermentation of C. thermocllum with isooctyl alcohol polyoxyethylene ether (IAPE) addition. Based on IAPE addition, the glucose content of two-step saccharification was higher than that of the batch saccharification. Besides, the addition of IAPE and Triton X-100 had the same effect on saccharification. The saccharification performance was related to the substrate, especially the lignin content. For the fed-batch hydrolysis of 20 % (w/v) rice straw pretreated by hydrogen peroxide-acetic acid (HPAC), 66.9 g/L glucose and 19.5 g/L xylose were obtained by using C. thermocellum DSM 1313 and 1 FPU/g substrate cellulase with 0.1 % (w/v) IAPE addition, and the glucose and xylose yield reached 63.2 % and 37.4 % respectively.

Numerous transcription factors (TFs) in ascomycete fungi play crucial roles in cellular processes; however, how most of them function is poorly understood. Here, we identified and characterized a novel TF, CxrC (POX01387), acting downstream of the key TF CxrA, which is essential for plant-biomass-degrading-enzyme (PBDE) production in Penicillium oxalicum. Deletion of cxrC in P. oxalicum significantly affected the production of PBDEs, as well as mycelial growth and conidiospore production. CxrA directly repressed the expression of cxrC after about 12 hr following switch to Avicel culture. CxrC bound the promoters of major PBDE genes and genes involved in conidiospore development. CxrC was found to bind the TSSGTYR core sequence (S: C and G; Y: T and C; R: G and A) of the important cellulase genes cbh1 and eg1. Both N- and C-terminal peptides of CxrC and the CxrC phosphorylation were found to mediate its homodimerization. The conserved motif LPSVRSLLTP (65-74) in CxrC was found to be required for regulating cellulase production. This study reveals novel mechanisms of TF-mediated regulation of the expression of PBDE genes and genes involved in cellular processes in an ascomycete fungus.

Invasive plants offer an interesting and unconventional source of protein and the considerable investment made towards their eradication can potentially be salvaged through their revalorisation. To identify viable sources, effective and high-throughput screening methods are required, as well as efficient procedures to isolate these components. Rigorous assessment of low-cost, high-throughput screening assays for total sugar, phenolics and protein was performed, and ninhydrin, Lever and Fast Blue assays were found to be most suitable owing to high reliability scores and false positive errors less than 1%. These assays were used to characterise invasive Scottish plants such as Gorse (Ulex europeans), Broom (Cystisus scoparius) and Fireweed (Chamaenerion angustifolium). Protein extraction (alkali-, heat- and enzyme assisted) were tested on these plants, and further purification (acid and ethanol precipitation, as well as ultrafiltration) procedures were tested on Gorse, based on protein recovery values. Cellulase treatment and ethanol precipitation gave the highest protein recovery (64.0 ± 0.5%) and purity (96.8 ± 0.1%) with Gorse. The amino acid profile of the purified protein revealed high levels of essential amino acids (34.8 ± 0.0%). Comparison of results with preceding literature revealed a strong association between amino acid profiles and overall protein recovery with the extraction method employed. The final purity of the protein concentrates was closely associated to the protein content of the initial plant mass. Leaf protein extraction technology can effectively raise crop harvest indices, revalorise underutilised plants and waste streams.

The present study reports the characterization of an extracellular polymer produced by a strain of Lactobacillus helveticus in potato waste medium and the subsequent application of the biopolymer for protecting farm-bruised pomegranates against fungal infection following harvest. Chemical, thermogravimetric analysis, FTIR and scanning electron micrographs of the purified biopolymer revealed it to be a polysaccharide with good thermal stability and a compact structure. Purified biopolymer exhibited strong antifungal properties against the fungal pathogen Penicillium implicatum. In tissues of pomegranates, subjected to fungal challenge, pectinase, cellulase and xylanase activity indicative of infectivity was not observed upon storage for 14 days at 28 °C. Besides visual discoloration of pomegranates, characteristic of soft rot and fungal growth of tissue extracts could not be detected on PDA plates. Postharvest parameters, physiological loss of weight, TA, TSS, TSS/TA ratio, ascorbic acids, total sugars and sensory attributes in the biopolymer dipped bruised pomegranates challenged with P. implicatum, showed no significant (p > 0.05) change following storage. P. implicatum challenged bruised fruits which lacked dipping treatment was spoiled by 48 h. Farm-bruised pomegranates dipped with biopolymer offered complete protection of the bruised pomegranates upon storage for 14 days at ambient temperature (28 °C). Overall, the results of this study suggest a sustainable use of potato processing wastes for the reutilization of 5–16% of damaged pomegranates, economically beneficial to farmers. Besides, the process will have strong importance in reducing and recycling potato industry wastes for innovative postharvest applications for other horticultural produces.

PurposeThe purpose of this research is to produce “cempedak” juice using enzyme aided-liquefaction by examining the effects of enzyme types (Pectinex® Ultra SP-L, Celluclast® 1.5 L and Fungamyl® 800 L), enzyme concentrations (0–1.5% v/w), incubation time (0–2.5 h) and incubation temperature (35–60 °C) on juice yield and viscosity, total soluble solids and color of fruit puree.Design/methodology/approachRipe “cempedak” pulp from CH28 fruit was first pureed in a blender and then homogenized with water at 1:2 ratio. The diluted puree was then liquefied with the enzymes separately to reduce its viscosity. Analyses such as juice yield, viscosity, total soluble solids and color of the liquefied “cempedak” puree were then carried out.FindingsResults indicated that the optimized use of 1.2% (v/w) Celluclast® 1.5 L (Novozymes, Denmark), a cellulase preparation, at 45 °C and 1 h produced juice with the lowest viscosity (349.4 cP) and the highest juice yield (82.3% v/w). Liquefied “cempedak” juice was darker (with L* value of 51.17) and more yellowish (b* value of 38.88) compared to “cempedak” juice without liquefaction (control). When compared to untreated “cempedak” juice, the droplet size of “cempedak” juice obtained after liquefaction under optimized conditions was found to be lower, regardless of whether the juice was filtered (with a total reduction of 23% of droplet size) or not filtered (with a total reduction of 16% of droplet size). The results indicate the possibility of employing Celluclast® 1.5 L to produce “cempedak” juice that can be further processed such as for the production of “cempedak” fruit powder.Originality/valueThis paper provides information on the enzyme concentration, incubation time and temperature for liquefying “cempedak” pulp such that the liquefied material produced can be used as a base feed for spray-drying to produce “cempedak” fruit powder.

Effective pretreatment is crucial for cellulase production from sugarcane bagasse. Pretreatment with tamarind extract could reduce the hazardous effect associated with chemical pretreatment. The present work investigated tamarind (Tamarindus indica) extract in combination with H2SO4 and thermal pretreatment of sugarcane bagasse for cellulase production by Aspergillus flavus. The sugarcane bagasse was pretreated with tamarind extract pH 2 and pH 4, followed by 1% H2SO4 and thermal treatment at 121°C for 15 min. The pretreatment slurry was analysed for reducing sugar while solid bagasse was analysed for weight loss. Aspergillus flavus grew on sugarcane bagasse under solid state fermentation and the Carboxy Methyl Cellulase (CMCase) and Filter Paper Assay (FPA) activities were compared on the various pretreatments. The pretreatments changed the visible morphology of the sugarcane bagasse observed by the swelling, fibrous appearance and colour change. Pretreatment slurry yielded highest soluble reducing sugar at 60.01 mg/ml in tamarind extract (pH 4/1% H2SO4 ) and highest weight loss of solids at 73.70% in tamarind extract (pH 2/1% H2SO4 /thermal 121°C). Aspergillus flavus performed better on tamarind extract (pH 2/1% H2SO4 ) by producing optimal CMCase and FPA activities at 0.100 U/ml and 0.409 U/ml respectively after 3 days of fermentation. Cellulase was maximally active at temperature of 50 °C. The tamarind extract pretreatment successfully proved to be an alternative organo-chemical pretreatment of sugarcane bagasse as evidenced by the physical properties, soluble reducing sugars and cellulase activities. Keywords: Aspergillus flavus, Cellulase, Pretreatment, Sugarcane bagasse, Tamarind extract

Management of lignocellulosic wastes in and around the municipality area requires special consideration. Continuous deposition of these wastes to the nearby areas led to gradual deterioration of the environment. The objective of this study was to produce cellulase from the bacteria isolated from the unexplored rainforest of NE-India for lignocellulosic waste hydrolysis. Based on carboxymethyl cellulose utilization and the congo red test, Novosphingobium sp. Cm1 was found to be the most promising strain out of 114 bacterial isolates and the strain was selected for further study. The optimization of the fermentative conditions for maximum enzyme activity was carried out using one factor-at-a-time strategy and the optimum pH, temperature and incubation time was recorded as pH 5, 37 °C and 96 h respectively. The maximum β-1,4-endoglucanase activity was observed with 1.5% CMC (5.1 ± 0.05 U/mL) and 0.25% yeast extract (7.6 ± 0.72 U/mL). The bacterial waste hydrolysis ability was investigated using three wastes where vegetable waste showed maximum activity of 3.4 ± 0.48 U/mL. Bacterial interaction and waste utilization were verified using Scanning Electron Microscope and Fourier-Transform infrared spectroscopy analysis. The present study confirmed the promising ability of Novosphingobium sp. to waste hydrolysis. Further investigations may lead to new possibilities for low-cost enzyme production that will help to meet the rising cellulase demand.

A rhizospheric bacterium belonging to the genus Pseudomonas isolated from the rhizosphere of maize was evaluated for the plant growth promoting and biocontrol traits. The strain was subjected to the different PGP and biocontrol traits such as ammonia, IAA, siderophores, phosphate solubilisation, ACC deaminase, HCN production, cellulase, pectinase and chitinase activity. 16S rRNA sequencing showed maximum homology with the strain Pseudomonas indoloxydans IPL-1(NR115922). In vivo plant growth promotion assay with the strains on maize seedlings showed a significant increase in the biometric parameters with respect to the number of shoots (32%) and roots (27%) and its fresh weight (20%) and dry weight (54%). Different formulations of the strain tested in fields revealed that the maize plants treated with the talc and lignite formulations showed a considerable increase in the growth of maize when compared to the plants without any treatment. The study concluded that the rhizosphere strains Pseudomonas indoloxydans (F3- 47) could be a potential plant growth promoter for future exploration in agriculture.

Our previous study on the binding activity between Cel5H and clay minerals showed highest binding efficiency among other cellulase enzymes cloned. Here, based on previous studies, we hypothesized that the positive amino acids on the surface of Cel5H protein may play an important role in binding to clay surfaces. To examine this, protein sequences of Bacillus licheniformis Cel5H (BlCel5H) and Paenibacillus polymyxa Cel5A (PpCel5A) were analyzed and then selected amino acids were mutated. These mutated proteins were investigated for binding activity and force measurement via atomic force microscopy (AFM). A total of seven amino acids which are only present in BlCel5H but not in PpCel5A were selected for mutational studies and the positive residues which are present in both were omitted. Of the seven selected surface lysine residues, only three mutants K196A(M2), K54A(M3) and K157T(M4) showed 12%, 7% and 8% less clay mineral binding ability, respectively compared with wild-type. The probable reason why other mutants did not show altered binding efficiency might be due to relative location of amino acids on the protein surface. Meanwhile, measurement of adhesion forces on mica sheets showed a well-defined maximum at 69 ± 19 pN for wild-type, 58 ± 19 pN for M2, 53 ± 19 pN for M3, and 49 ± 19 pN for M4 proteins. Hence, our results demonstrated that relative location of surface amino acids of Cel5H protein especially positive charged amino acids are important in the process of clay mineral-protein binding interaction through electrostatic exchange of charges.

This study investigated the effects of applying cellulase and starch on the fermentation characteristics and microbial communities of Napier grass silage after ensiling for 30 d. Three groups were studied: No additives (control); added cellulase (Group 1); and added cellulase and starch (Group 2). The results showed that the addition of cellulase and starch decreased the crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF) and pH significantly (p < 0.05) and increased water-soluble carbohydrate (WSC) content (p < 0.05). The addition of additives in two treated groups exerted a positive effect on the lactic acid (LA) content, lactic acid bacteria (LAB) population, and lactic acid / acetic acid (LA/AA) ratio, even the changes were not significant (p > 0.05). Calculation of Flieg’s scores indicated that cellulase application increased silage quality to some extent, while the application of cellulase and starch together significantly improved fermentation (p < 0.05). Compared with the control, both additive groups showed increased microbial diversity after ensiling with an abundance of favorable bacteria including Firmicutes and Weissella, and the bacteria including Proteobacteria, Bacteroidetes, Acinetobacter increased as well. For alpha diversity analysis, the combined application of cellulase and starch in Group 2 gave significant increases in all indices (p < 0.05). The study demonstrated that the application of cellulase and starch can increase the quality of Napier grass preserved as silage.

Recent investigations verified CaO2 accelerated hydrolysis of lignocellulose, but its residue caused a harmful oxidizing environment. This study therefore proposed the combined CaO2 pretreatment and zero-valent iron (ZVI) addition to enhance high-solid anaerobic digestion of wheat straw, trying to eliminate negative effect of residual oxides. Effects of CaO2 pretreatment without/with ZVI addition on digestion performance were investigated. Results showed that in non-ZVI added digesters, as CaO2 dosage increased, total production of SCOD and VFAs increased linearly, while activities of coenzyme F420 and electron transport system were negatively correlated. Pretreatment using appropriate CaO2 dosage indirectly increased biogas production by promoting hydrolysis and acidification. Comparably, initial ORP in ZVI-added digesters sharply declined below −350 mV, which was suitable for methanogen growth. ZVI significantly improved activities of cellulase, hemicellulase, acetic kinase and coenzyme F420, with the highest improvement percentages of 138%, 48%, 35% and 58%, respectively. Since ZVI could provide electrons for conversion of CO2 to CH4, compared with non-ZVI added digesters (R0-R4), methane content in ZVI-added digesters (RZ0-RZ4) increased by 8.1%, 9.0%, 11.2%, 17.0% and 18.0% successively. Consequently, 0.6 g/g VS CaO2 pretreatment combined with 0.4 g/g VS ZVI addition achieved the maximum methane production, increasing by 35.6% compared to control digester.

Fungal spoilage and mycotoxin contamination are two of the greatest hazards of silage. The present work was carried out to evaluate the impact of Lactobacillus bulgaricus and cellulolytic enzymes on the maize silage (MS) quality. Fungal analysis of different MS samples showed different mycotoxigenic fungi. The highest frequency (62.8%) was associated with Fusarium spp. Four species with different relative densities were found: F. graminearum (71.1%), F. culmorum (15.2%), F. proliferatum (11.2%), and F. oxysporum (2.50 %). High-performance liquid chromatography analysis showed the presence of trichothecene, nivalenol, zearalenone, and fumonisins mycotoxins in MS inoculated by F. graminearum. The inhibition % of trichothecene, nivalenol, and zearalenone synthesis was 50.2%, 47.5%, and 23.5%, respectively, in MS inoculated by Lactobacillus bulgaricus after a 30 d incubation period. Trichoderma harzianum succeeded in producing cellulolytic enzymes, i.e., carboxymethyl cellulase, manganase peroxidase, and laccase, with a maximum production of 350 µg/mL, 5.47 µg/mL, and 16.0 µg/mL, respectively, after 21 d using MS as the substrate. Treatment by the extracted cellulolytic enzyme with L. bulgaricus enhanced unfavorable conditions for MS fungal contamination, i.e., the production of lactic acid, a lowered pH, and increased L. bulgaricus colony-forming units, compared to the addition of enzyme extract or L. bulgaricus alone.

Two genes encoding lytic polysaccharide monooxygenases (LPMOs) CsLPMO9A and CsLPMO9B were cloned from the white-rot fungus Ceriporiopsis subvermispora and successfully expressed in Komagataella phaffii. The oxidative degradation of phosphoric acid swollen cellulose by the LPMOs was conducted using ascorbic acid as an electron donor. Analysis with high performance anion-exchange chromatography and matrix assisted laser desorption/ionization-time-of-flight mass spectrometry indicated that the two enzymes cleaved the polysaccharides by oxidization of the C1 carbon atom of the glucose unit. Synergistic effects between CsLPMO9A or CsLPMO9B and a commercial cellulase cocktail from Trichoderma reesei were observed in the saccharification of delignified wheat straw. The amounts of glucose, cellobiose and xylose released from the substrate by the combined enzymes were increased by 83 %, 16 % and 47 % (for CsLPMO9A), or 79 %, 52 % and 81 % (for CsLPMO9B), compared to the cellulase alone, respectively. The two new enzymes displayed promising potential in industrial bioconversion of lignocellulosic biomass.

Fungal pre-treatment and fermentation could improve nutritional value of wet corn distillers’ grains and solubles (WDGS), soybean hull (SH), and their blend in swine and poultry diets, which can potentially increase revenues for corn-ethanol and soybean processing industries while reducing feed cost in swine and poultry production systems. This study evaluated the effectiveness of pre-treatment of SH, WDGS, and their mixture by Trichoderma reesei and fermentation by Aspergillus oryzae to improve the nutritional profile and digestibility of these ingredients. T. reesei produced cellulase and xylanase, resulting in structural carbohydrates reduction by 69.2% while concentrating amino acids from 6.8% to 11% in SH. Fermentation with A. oryzae degraded phytate by over 50% and improved in vitro digestibility of amino acids in WDGS and its mixture with SH. Fermenting T. reesei pre-treated SH by A. oryzae showed higher in vitro dry matter digestibility than non-fermented substrate. Moreover, the proportion of key amino acids (arginine, threonine, methionine, and lysine) in both T. reesei and A. oryzae fermented substrates were significantly improved. The results demonstrated the feasibility of combining T. reesei and A. oryzae in improving feeding value of WDGS and SH for potential use as feed ingredients for monogastric animals.

A codon optimized cellobiohydrolase (CBH) encoding synthetic gene of 1188 bp from a thermophilic mold Myceliophthora thermophila (MtCel6A) was cloned and heterologously expressed in Escherichia coli for the first time. In silico analysis suggested that MtCel6A is a GH6 CBH and belongs to CBHII family, which is structurally similar to Cel6A of Humicola insolens. The recombinant MtCel6A is expressed as active inclusion bodies, and the molecular mass of the purified enzyme is ~ 45 kDa. The rMtCel6A is active in a wide range of pH (4–12) and temperatures (40–100 °C) with optima at pH 10.0 and 60 °C. It exhibits T1/2 of 6.0 and 1.0 h at 60 and 90 °C, respectively. The rMtCel6A is an extremozyme with organic solvent, salt and alkali tolerance. The Km, Vmax, kcat and kcat/Km values of the enzyme are 3.2 mg mL−1, 222.2 μmol mg−1 min−1, 2492 s−1 and 778.7 s−1 mg−1 mL−1, respectively. The product analysis of rMtCel6A confirmed that it is an exoenzyme that acts from the non-reducing end of cellulose. The addition of rMtCel6A to the commercial cellulase mix (Cellic CTec2) led to 1.9-fold increase in saccharification of the pre-treated sugarcane bagasse. The rMtCel6A is a potential CBH that finds utility in industrial processes such as in bioethanol, paper pulp and textile industries. Graphical Abstract

Herein, we cloned and expressed an endo-β-1,4-glucanase gene (celA1805) from Bacillus subtilis B111 in Escherichia coli. The recombinant celA1805 contains a glycosyl hydrolase (GH) family 8 domain and shared 76.8% identity with endo-1,4-β-glucanase from Bacillus sp. KSM-330. Results showed that the optimal pH and temperature of celA1805 were 6.0 and 50°C, respectively, and it was stable at pH 3-9 and temperature ≤50°C. Metal ions slightly affected enzyme activity, but chemical agents generally inhibited enzyme activity. Moreover, celA1805 showed a wide substrate specificity to CMC, barley β-glucan, lichenin, chitosan, PASC and avicel. The Km and Vmax values of celA1805 were 1.78 mg/ml and 50.09 μmol/min/mg. When incubated with cellooligosaccharides ranging from cellotriose to cellopentose, celA1805 mainly hydrolyzed cellotetrose (G4) and cellopentose (G5) to cellose (G2) and cellotriose (G3), but hardly hydrolyzed cellotriose. The concentrations of reducing sugars saccharified by celA1805 from wheat straw, rape straw, rice straw, peanut straw, and corn straw were increased by 0.21, 0.51, 0.26, 0.36, and 0.66 mg/ml, respectively. The results obtained in this study suggest potential applications of celA1805 in biomass saccharification.

The filamentous fungi Rhizopus oryzae are well known for their ability to hydrolyze plant polysaccharides. Representatives of this species are widely used for the production of various products, such as organic acids (lactic acid, fumaric acid), ethanol, and hydrolytic enzymes (glucoamylases, polygalacturonases). Various carbon sources such as pentose and hexose sugars are used for the synthesis of R. oryzae exoproducts. Expensive plant sugarcontaining raw materials are used in the lactic acid production by the microbiological method, which significantly increases the final cost of the product. The article demonstrates the possibility of using cheaper by-products of pulp and paper production such as sulphite liquors as an alternative source of carbon for microbiological synthesis of lactic acid. Sulphite liquors contain a large amount of carbohydrates – products of incomplete hydrolysis of cellulose such as hemicelluloses, mono- and oligomeric sugars. The composition of sulfite liquors allows us to consider them as a potential substrate for the synthesis of lactic acid by using different microorganisms. The paper considers the dependence of the lactic acid yield on the composition of the nutrient medium based on sulfite liquor and the cultivation method of the fungus R. oryzae F-1030. As additional sources of nitrogen and phosphorus, (NH4)2SO4 and KH2PO4 were introduced to the nutrient medium. The fungus R. oryzae F-1030 was cultivated by two methods: the semicontinuous culture method, when the nutrient medium was completely replaced after depletion of the reducing sugars in it; the batch culture method, when the lack of reducing sugars in the medium was compensated by the addition of concentrated sulfite liquor. Since the nutrient medium based on sulfite liquor contains the products of incomplete cellulose hydrolysis and xylans, the xylanase and cellulase activities of the fungus R. oryzae F-1030 were measured in order to determine its absorption degree of carbohydrates, contained in the nutrient medium. It was found that it is more expedient to use the semicontinuous method for the fungus R. oryzae F-1030 cultivation on a nutrient medium based on sulfite liquor in order to obtain more synthesized lactic acid. The addition of mineral sources of nitrogen and phosphorus has practically no effect on the final yield of lactic acid.For citation: Mingazova L.A., Kryakunova E.V., Kanarskaya Z.A., Kanarsky A.V. Applying Sulfite Liquors as a Nutrient Medium for Cultivation of Lactic Acid Producer Rhizopus oryzae F-1030. Lesnoy Zhurnal [Russian Forestry Journal], 2021, no. 5, pp. 163–173. DOI: 10.37482/0536-1036-2021-5-163-173Acknowledgements: The work was carried out using the equipment of the Center for Collective Use “Ecology, Biotechnology and Processes for Producing Environmentally Friendly Energy Carriers” of the Volga State University of Technology.

Rice straw is an important low-cost feedstock for bio-based economy. This report presents a study in which rice straw was used both as a source for isolation of bacteria producing the biodegradable polyester polyhydroxyalkanoate (PHA), as well as the carbon source for the production of the polymer by the isolated bacteria. Of the 100 bacterial isolates, seven were found to be positive for PHA production by Nile blue staining and were identified as Bacillus species by 16S rRNA gene sequence analysis. Three isolates showed 100% sequence identity to B. cereus, one to B. paranthracis, two with 99 and 100% identity to B. anthracis, while one was closely similar to B. thuringiensis. For use in PHA production, rice straw was subjected to mild alkaline pretreatment followed by enzymatic hydrolysis. Comparison of pretreatment by 2% sodium hydroxide, 2% calcium hydroxide and 20% aqueous ammonia, respectively, at different temperatures showed maximum weight loss with NaOH at 80 °C for 5 h, but ammonia for 15 h at 80 °C led to highest lignin removal of 63%. The ammonia-pretreated rice straw also led to highest release of total reducing sugar up to 92% on hydrolysis by a cocktail of cellulases and hemicellulases at 50 °C. Cultivation of the Bacillus isolates on the pretreated rice straw revealed highest PHA content of 59.3 and 46.4%, and PHA concentration of 2.96 and 2.51 g/L by Bacillus cereus VK92 and VK98, respectively.

Microbial enzymes are crucial for material biotransformation during the composting process. In this study, we investigated the effects of adding bamboo charcoal (BC) (i.e., at 5%, 10%, and 20% corresponding to BC5, BC10, and BC20, respectively) on the enzyme activity levels during chicken manure composting. The results showed that BC10 could increase the cellulose and urease activities by 56% and 96%, respectively. The bacterial community structure in BC10 differed from those in the other treatments, and Luteivirga, Lactobacillus, Paenalcaligenes, Ulvibacter, Bacillus, Facklamia, Pelagibacterium, Sporosarcina, Cellvibrio, and Corynebacterium had the most important roles in composting. Compared with other treatments, BC10 significantly enhanced the average rates of degradation of carbohydrates (D-xylose (40%) and α-D-lactose (44%)) and amino acids (L-arginine (16%), L-asparagine (14%), and L-threonine (52%)). We also explored the associations among the bacterial community and their metabolic functions with the changes in the activities of enzymes. Network analysis demonstrated that BC10 altered the co-occurrence patterns of the bacterial communities, where Ulvibacter and class Bacilli were the keystone bacterial taxa with high capacities for degrading carbon source, and they were related to increases in the activities of cellulase and urease, respectively. The results obtained in this study may help to further enhance the efficiency of composting.

Aims Investigate the impact of highly adapted bacterial strains and their ability in waste degradation under a wide range of temperatures. Methods and results Bacteria isolated from soil and food waste were grown in various media under fluctuated temperatures. After screening for organic compound degradation, the seven strongest bacterial strains have been selected for further experiments. Their enzyme activities were expressed in terms of the size of the hydrolysis zone in a wide temperature range of 2.5-70 oC. The enzyme production assay was carried out for each protease, cellulase, and amylase. The waste degradation was determined with a maximum 80% decrease in the volume of food waste in 21 days compared to the control in lab-scale with enriched bacterial cultures and soil bacteria as additives at room temperature around 18 oC-20 oC. Conclusion These seven bacteria are promising candidates for food waste biodegradation in composting especially in the winter without heating expense for maintaining ambient temperature. Significance and impact of the study It is necessary to coax the uncultured bacteria from the various environments into the laboratory for investigating their valuable functions. Herein, using enrichment culture of consortium and additive of soil have illustrated the significant mean in food waste degradation.

Biocontrol of plant pathogens is considered an environmentally friendly strategy and it is preferred over the use of chemicals which cause environmental pollution. Lysobacter enzymogenes is a bacterium that has been identified as an agriculturally important biocontrol agent. L. enzymogenes possess antagonistic activity against numerous phytopathogens such as fungi, oomycetes, bacteria and nematodes. Its antagonistic activity is conferred by its ability to produce bioactive secondary metabolites such as the Heat-Stable Anti-Fungal Factor (HSAF), Heat-Stable Degrading Metabolite (HSDM) and WAP-8294A2. It can also produce abundant lytic enzymes such as; chitinases, proteases, glucanases and cellulases that can degrade fungal cell walls and therefore inhibit their growth. To design effective biocontrol strategies employing L. enzymogenes, it is important to understand its antagonistic mechanisms. This review crystalizes information on the biosynthesis mechanisms and biocontrol mechanisms of various antimicrobial compounds produced by L. enzymogenes, this information is essential in designing biocontrol strategies against phytopathogens. Further, this review highlights the uncharacterized HSDM and proposes the need for its future characterization, determination of its biosynthetic gene cluster and characterization of its antagonistic mechanisms against various phytopathogens. Also, the mechanism of clp regulation of lytic enzymes biosynthesis needs to be further studied.

This study was aimed to evaluate the utilization of hydrolyzed Indigofera zolingeriana by celullase enzyme as the feed ingredient of gourami fish. This study used a completely randomized design which contained three steps, whereas each step contained four treatments and four replications. The first step performed by evaluating the Indigofera leaf meal (ILM) added with cellulase enzyme of 0 g/kg (control), 0.4 g/kg, 0.8 g/kg, and 1.2 g/kg. The second step was the digestibility test of ILM on gourami seeds. The third step was feed evaluation added with ILM as much as 0% (control), 15%, 30%, and 45% against the growth performance on gourami seeds. The gourami seeds used in the second and third steps with a weight of 13.65 ± 0.39 g/seed and 5.95 ± 0.15 g/seed, respectively. The addition of 0.8 g/kg and 1.2 g/kg cellulase enzyme could significantly decrease the crude fiber of ILM with 43.33%, besides having the best value of total, ingredient, protein, lipid, and energy digestibility. The growth performance of gourami seeds given 15% ILM added feed had the best value and insignificantly different from the control feed without ILM addition based on the specific growth rate (SGR), protein retention (PR), and feed efficiency (FE). Keywords: Cellulase, feed, hydrolyze, Indigofera zolingeriana, Osphronemus gouramy. ABSTRAK Penelitian ini dilakukan untuk mengevaluasi pemanfaatan daun Indigofera zolingeriana yang dihidrolisis enzim selulase sebagai bahan baku pada pakan benih ikan gurami. Penelitian ini menggunakan rancangan acak lengkap yang terdiri atas tiga tahap, dimana masing-masing tahap terdiri dari empat perlakuan dan empat ulangan. Pada tahap pertama dilakukan evaluasi tepung daun Indigofera (TDI) yang ditambahkan enzim selulase sebesar 0 g/kg (kontrol), 0.4 g/kg, 0.8 g/kg, dan 1.2 g/kg. Pada tahap kedua dilakukan uji kecernaan bahan TDI pada benih ikan gurami. Pada tahap ketiga dilakukan evaluasi pakan yang ditambahkan TDI sebesar 0% (kontrol), 15%, 30% dan 45% terhadap kinerja pertumbuhan pada benih ikan gurami. Benih ikan gurami yang digunakan pada penilitian tahap kedua dengan bobot 13.65 ± 0.39 g/ekor dan 5.95 ± 0.15 g/ekor. Penambahan enzim selulase pada dosis 0.8 g/kg dan 1.2 g/kg secara signifikan mampu menurunkan serat kasar TDI sebesar 43.33 % dan memberikan nilai terbaik terhadap nilai kecernaan total, kecernaan bahan, kecernaan protein, kecernaan lemak dan kecernaan energi. Kinerja pertumbuhan benih ikan gurami yang diberikan pakan yang ditambahkan TDI sebesar 15% memilki nilai terbaik dan tidak berbeda nyata terhadap pakan kontrol tanpa TDI dari aspek laju pertumbuhan spesifik (LPS), retensi protein (RP) dan efisiensi pakan (EP). Kata kunci: Hidrolisis, Indigofera zolingeriana, Osphronemus gouramy, pakan, selulase.

Background Cellulolytic microorganisms are considered a key player in the degradation of plant biomass in various environments. These microorganisms can be isolated from various environments, such as soils, the insect gut, the mammalian rumen and oceans. The Red Sea exhibits a unique environment in terms of presenting a high seawater temperature, high salinity, low nutrient levels and high biodiversity. However, there is little information regarding cellulase genes in the Red Sea environment. This study aimed to examine whether the Red Sea can be a resource for the bioprospecting of microbial cellulases by isolating cellulase-producing microorganisms from the Red Sea environment and characterizing cellulase genes. Results Three bacterial strains were successfully isolated from the plankton fraction and the surface of seagrass. The isolated strains were identified as Bacillus paralicheniformis and showed strong cellulase activity. These results suggested that these three isolates secreted active cellulases. By whole genome sequencing, we found 10 cellulase genes from the three isolates. We compared the expression of these cellulase genes under cellulase-inducing and non-inducing conditions and found that most of the cellulase genes were generally upregulated during cellulolysis in the isolates. Our operon structure analysis also showed that cellulase genes form operons with genes involved in various kinds of cellular reactions, such as protein metabolism, which suggests the existence of crosstalk between cellulolysis and other metabolic pathways in the bacterial isolates. These results suggest that multiple cellulases are playing important roles in cellulolysis. Conclusions Our study reports the isolation and characterization of cellulase-producing bacteria from the Red Sea. Our whole-genome sequencing classified our three isolates as Bacillus paralicheniformis, and we revealed the presence of ten cellulase orthologues in each of three isolates’ genomes. Our comparative expression analysis also identified that most of the cellulase genes were upregulated under the inducing conditions in general. Although cellulases have been roughly classified into three enzyme groups of beta-glucosidase, endo-β-1,4-glucanase and exoglucanase, these findings suggest the importance to consider microbial cellulolysis as a more complex reaction with various kinds of cellulase enzymes.

Rhizoctonia solani is the causal agent of rice sheath blight, which is one of the most serious diseases of rice worldwide. In this study, seventeen Egyptian isolates of sheath blight pathogen, R. solani AG1 IA, were identified using morphological and molecular characterization by the internal transcribed spacer (ITS) sequences. Extracellular enzymes were assessed in in vitro and in infected rice samples. Also, pathogenicity tests for all isolates were done on two rice cultivars under greenhouse conditions. Growth behavior by RS13 isolate was studied using two rice cultivar Sakha 101 (Japonica type) and Egyptian Yasmine (Indica type). The obtained results exhibited a wide range of variability in the morphological and virulence characteristics of isolates. Two isolates RS10 and RS13 (AB5) were sequenced and found aligned with Egyptian and Chinese isolates. RS13 and RS10 isolates exhibit the highest production of cellulase, pectinase and amylase enzymes in plate culture (in vitro). While, on the plant sample tissue cellulase and pectin methylesterase (PME) activity enzymes are relating to development of rice sheath blight disease. Under the artificial condition, SK 101 rice proved the most susceptible to infection with sheath blight pathogen compare with E. Yasmine. The hyphae growth on SK101 cultivar was more than in the E. Yasmine cultivar. Furthermore, we examined the morphological developments of hyphae and sclerotial on both cultivars using light and scanning electron microscopy (SEM). It was observed that the fungal pathogen could intercept host surface structures for improving cell penetration and anchorage.

This paper studies the characteristics of the kinetic interaction of lytic polysaccharide monooxygenases (LPMOs) from Thielavia terrestris (TtLPMO), Penicillium verruculosum (PvLPMO), and Trichoderma reesei (TrLPMO) with purified cellobiohydrolases (CBH I and CBH II) and endoglucanase II (EG II) of P. verruculosum during the enzymatic destruction of microcrystalline (Avicel) and amorphous cellulose. TtLPMO belongs to the C1-type of LPMOs, while PvLPMO and TrLPMO are of the mixed C1/C4-type, according to the generally accepted classification of this class of oxidases. Under the action of any of the three LPMO together with CBH II or EG II on Avicel or amorphous cellulose, respectively, the enzymes displayed synergism, which was manifested in an increase in the yield of reducing sugars (RS). The synergism was expressed to the greatest extent in the initial reaction period. The mixtures of PvLPMO and TrLPMO with CBH I also demonstrated synergism on Avicel as a substrate, while TtLPMO, on the contrary, had an inhibitory effect on RS formation from cellulose with CBH I. The observed synergism and antagonism between LPMOs and CBH II/CBH I can be explained in terms of a simple kinetic scheme, in which the first enzyme forms additional ends of polysaccharide molecules that serve as a substrate or inhibitor for the second enzyme.

A novel expansin-like protein (CxEXL22) has been identified and characterized from newly isolated Arthrobotrys sp. CX1 that can cause cellulose decrystallization. Unlike previously reported expansin-like proteins from microbes, CxEXL22 has a parallel β-sheet domain at the N terminal, containing many hydrophobic residues to form the hydrophobic surface as part of the groove. The direct phylogenetic relationship implied the genetic transfers occurred from nematode to nematicidal fungal Arthrobotrys sp. CX1. CxEXL22 showed strong activity for the hydrolysis of hydrogen bonds between cellulose molecules, especially when highly crystalline cellulose was used as substrate. The hydrolysis efficiency of Avicel was increased 7.9-fold after pretreating with CxEXL22. The rupture characterization of crystalline region indicated that CxEXL22 strongly binds cellulose and breaks up hydrogen bonds in the crystalline regions of cellulose to split cellulose chains, causing significant depolymerization to expose much more microfibrils and enhances cellulose accessibility.

Plant endophytic microorganisms, which live symbiotically within their plant host, are a novel resource of active secondary metabolites. The current study was carried out to isolate fungal endophytes from 11 mangrove plants, to investigate their antifungal activity against phytopathogenic fungi and to assess their enzyme production. The ethyl acetate crude extracts of selected endophytic fungi were determined for their antioxidant and anti-mutagenic activities and the active compounds present in the extracts were identified. Of the 37 isolates of endophytic fungi, 30 were isolated from leaves and 7 from stems. For anti-pathogenic activity, all endophytes were tested against 5 plant pathogens. The results revealed that AmL-02 and CtL-06 had the highest growth inhibition of Curvularia sp. and Fusarium sp. respectively and they were chosen for phytochemical analysis and to explore further their anti-mutagenic activities. A preliminary qualitative phytochemical analysis of the fungal crude extracts revealed the presence of constituents such as alkaloids, tannins and coumarins. Gas chromatography-mass spectrometry analysis of the ethyl acetate extract of AmL-02 revealed the presence of various chemical compounds with the highest amount present being 1,3-butanediol (11.37%), while the highest amount of compound present in CtL-06 was kojic acid (95.6%). The antimutagenic activities were determined of AmL-02 and CtL-06 against the mutagenic substances 3-amino-1-methyl-gamma-carboline (Trp-P-2) and 7,12-dimethylbenzo[a]anthracene (DMBA) based on an Ames test using the Salmonella Typhimurium strains TA98 and TA100 which revealed that the extracts of AmL-02 and CtL-06 had strong antimutagenic activity against both mutagens. All the endophytic isolates were assessed for their potential role in producing extracellular enzymes on solid media. The results showed that the endophytic isolates had the capability of producing lipase (78%) cellulase (30%) protease (22%) and pectinase (19%), but none could produce amylase. One of the endophytic isolates (XmL-02) isolated from leaves had significant maximum lipase enzyme activity based on extracellular enzyme production ratio values (3.56 mm). Isolate XmL-02 had the highest lipase activity (82.22 U/ml) in the culture media for 72 hours at 30°C and pH 3. Hence, the three isolates, AmL-02, CtL-06 and XmL-02, were identified based on a combination of their morphological characteristics and internal transcribed spacer (ITS) sequences. The results revealed that CtL-06, AmL-02 and XmL-02 were as Aspergillus luchuensis, Xylaria feejeensis and Colletotrichum gloeosporioides, respectively. These results indicated that the mangrove plants at this location harbored rich sources of fungal endophytes with different types of bioactivity.

This study aims to produce a significant quantity of fermentable sugars from tubers sourced from northern Thailand to boost bioethanol production. Bioethanol production from Amorphophallus spp. tubers was compared using biological and chemical approaches. The tubers were sliced into small pieces (1–2 cm), dried in a solar oven, and powdered before hydrolyzing with cellulase enzymes. The results revealed the fermentable sugar content of Amorphophallus spp. tuber increased from 2.6 g/L–19.01 g/L after enzymatic hydrolysis. Furthermore, under the ideal condition, the total sugar concentration was 33.22 g/L. Enzymes help to speed up the hydrolysis process, and biological methods are also less expensive and more ecologically friendly than chemical equivalents. After the alcoholic fermentation, the highest ethanol content was obtained, 8.68 ± 0.91 g/L, using S. cerevisiae for 48 h. Konjac receives little attention in the biofuel industry due to its irritating nature to the mouth and throat when swallowed, implying that competition between the fuel and food sectors is lower than for other feedstocks such as cassava and corn. Improved fermentable sugars may be used in the near future for bioethanol production to address the worldwide issue of declining fossil fuel consumption.

Cellulases are a group of hydrolytic enzymes capable of hydrolyzing the most abundant organic polymer that means cellulose to smaller sugar components including glucose subunits. The aim of this study was to screen cellulase producing oyster mushroom collected from Eucalyptus tree bark to evaluate the in vitro production of cellulase by Pleurotus ostreatus using different lignocellulosic substrates, and to characterize the cellulase produced with respect to changes in pH, temperature, and concentration of substrates. A total of ten mushroom specimens were randomly collected from Eucalyptus tree bark in the premise of Holetta Agricultural Research Center campus. All of the collected mushroom specimens were identified morphologically and biochemically as Pleurotus ostreatus and also screened for their ability to produce cellulase by detecting and measuring zone of hydrolysis on commercial media containing Carbxymethyl Cellulose (CMC) as the sole carbon source. These mushroom specimens were cultivated using both solid state fermentation and submerged fermentation systems supplemented with different lignocellulosic substrates (wheat straw, teff straw, bean straw, wood fiber and Eucalyptus tree bark) to identify the most suitable medium for the production of cellulase. The highest enzyme production was obtained on bean straw and wheat straw which resulted in 0.191 U/ml, 0.868 U/ml and 0.389 U/ml; and 0.216 U/ml, 0.444 U/ml, and 0.245 U/ml of FPase, CMCase, and β-glucosidase in solid state fermentation. The lowest values were, however, obtained in media containing wood fiber in both solid state fermentation and submerged fermentation. Comparison of the lignocellulosic substrates revealed that wheat straw was selected for further growth parameter optimization. The production of cellulase was higher at the 5th day of incubation period, and the optimum pH and incubation temperature required for maximum cellulase production were 4 and 30°C, respectively. Sucrose and Yeast extract at 1% concentration were found to be the most preferred carbon and nitrogen sources for cellulase production by Pleurotus ostreatus. The optimum pH and temperature for cell_free cellulase activity on were found to be 4 and 50°C, respectively. Generally the cellulases produced by Pleurotus ostreatus were stable and active at temperatures ranging from 20-50°C. These characteristics hopefully would make this enzyme potentially attractive in a variety of industrial applications including animal feed treatments. There was a linear relationship between cellulase and its substrate concentration for there was an increase in activity with increase in substrate concentration. The relationship between rate of reaction and substrate concentration depended on the affinity of the enzyme for its substrate. Finally the cellulase was tested for its ability to saccharify agricultural wastes and the results showed the highest release of sugars from wheat straw.

Removal of xylan in plant biomass is believed to increase cellulose hydrolysis by uncovering cellulose surfaces for cellulase adsorption and, in turn, catalysis reaction. Herein, we describe an eco-friendly method by culturing a xylanolytic Bacillus firmus K-1 on rice straw to remove xylan. The bacterium was grown on 2.5% (w/v) rice straw with different biomass particle sizes for two days at 37 °C. We found that the particle sizes ranged from

Single cultures and co-cultures of Bacillus licheniformis and Bacillus paralicheniformis isolated from compost were evaluated for their carboxymethyl cellulase (CMCase) and filter paperase (FPase) production potential. Using a medium supplemented with microcrystalline cellulose (MCC), in the co-culture, CMCase and FPase activities increased 8.87- and 2.28-fold and 10.15- and 3.20-fold over B. licheniformis and B. paralicheniformis monocultures, respectively. The synergistic behavior of the two isolates might be due to the consumption of hydrolysis product (glucose, cellobiose) by one or both of the isolates, which improved their metabolic performance for cellulase secretion. Optimal conditions for cellulase production by this co-culture were a temperature of 45 °C, and pH 7 at 180 rpm in a medium containing rice bran at 1% (w/v) and chicken manure as nitrogen supplement at 2% (w/v). The maximum CMCase and FPase produced under the above conditions were 79.8 U/mL and 12.5 U/mL, respectively. This corresponds to 257.4- and 59.5-fold enhancement in CMCase and FPase activity, respectively, over B. licheniformis monoculture, and 306.9- and 83.3-fold increase with respect to the B. paralicheniformis monoculture. These results indicate that improved cellulase production can be achieved through co-culture and chicken manure nitrogen-supplement.

Some anaerobic bacteria, particularly Clostridium species, produce extracellular cellulolytic and xylanolytic enzymes as multienzyme complexes (MECs). However, an amylolytic/xylanolytic/cellulolytic multienzyme complex (AXC-MEC) from anaerobic bacteria is rarely found. In this work, the glycoprotein AXC-MEC, composed of subunits of amylolytic, xylanolytic, and cellulolytic enzymes, was isolated from crude extracellular enzyme of the mesophilic anaerobic bacterium Clostridium manihotivorum CT4, grown on cassava pulp, using a milled cassava pulp column and Sephacryl S-500 gel filtration chromatography. The isolated AXC-MEC showed a single band upon native-polyacrylamide gel electrophoresis (native-PAGE). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed at least eight protein bands of the multienzyme complex which predominantly exhibited amylolytic enzyme activity, followed by xylanolytic and cellulolytic enzyme activities. The AXC-MEC is highly capable of degrading starch and non-starch polysaccharides present in cassava pulp into glucose and oligosaccharides, without conventional pretreatment. Base on the genomic analysis of C. manihotivorum CT4, we found no evidence of the known structural components of the well-known multienzyme complexes from Clostridium species, cellulosomes such as scaffoldin, cohesin, and dockerin, indicating that AXC-MEC from strain CT4 exhibit a different manner of assembly from the cellulosomes. These results suggest that AXC-MEC from C. manihotivorum CT4 is a new MEC capable of hydrolyzing cassava pulp into value-added products, which will benefit the starch industry. KEY POINTS: • Glycoprotein AXC-MEC was first reported in Clostridium manihotivorum. • Unlike cellulosomes, AXC-MEC consists of amylase, xylanase, and cellulase. • Glucose and oligosaccharides were hydrolysis products from cassava pulp by AXC-MEC.

The rise in world industrialization and the cost of importing enzyme by local industries have led to arise in the search for novel and native enzyme producing microorganisms. Cellulase is an enzyme that catalyzes the breaking down of carbon chains in cellulose and hemicellulose, this research therefore aimed at studying fungal cellulase produced by Penicillium expansum grown on malus domestica (apple fruits). Fresh apple fruit was allowed to deteriorate under laboratory condition until there was visible mould growth. The mould with desired features of the organism of interest was subcultured by direct plating on PDA plates to which 10 % streptomycin has been added to prevent bacterial contaminants. The plates were incubated at 28±2 0C for 7 days until a visible mass of blue mycelia appear. The isolate was further subcultured onto freshly prepared media until pure culture was obtained. Characterization and identification of isolate were done using macroscopy and microscopy techniques. The isolate was re-inoculated into healthy apple fruits and the fruits were incubated at temperature of 28±2 oC for 8 days. Cellulolytic activity was examined every day throughout the incubation period. Crude enzyme was extracted each day using standard methods. Carboxyl methyl cellulose was used as standard for the crude cellulase activity assay after extraction from the infected apple fruits using Dinitrosalicylic acid (DNSA). Culture parameters like pH and temperature were also optimized to determine their effect on cellulolytic activity of the fungus. Cellulase activity was defined as the amount of glucose produced in μmol/mg/min under the assay condition. The highest cellulase activity of 86.84±0.52 μmol/mg/min was observed on day 6 of incubation at 28±2 oC and at pH 7. In conclusion, it is evident from this research that P. expansum isolated could be used as potential novel organism for industrial production of cellulase under optimized fermentation conditions.

Large amount of agro wastes is produced in Rwanda each year. The global annual potential bioethanol production from the major vegetables wastes such as carrot peel, onion peel, potato peel and sugar beet peel were estimated. Those wastes processing was successfully used as raw materials for the production of bioethanol, employing by cellulase produced from various filamentous fungi including Cladosporium cladosporioides was used for hydrolysis and the fermentation of the hydrolyzed samples was done using Saccharomyces cerevisiae. The fermented product was purified by primary distillation process at 79°C and the fraction was collected. The ethanol is then determined by specific dichromate method and Gas Chromatography. Instantaneous saccharification and fermentation process yielded maximum ethanol in the substrate of carrot peel was 16.9% at 21st day and further confirmed by Gas chromatography and the yield of ethanol obtained was 15.8%.

More than 70% of heat resistance enzymes, such as lipase, cutinase, cellulase, Alpha amylase, L-aspartas, are being used in industries which are producing by thermophillic microorganisms. The purpo...

In this study, citrus pulp residue were treated to improve their nutritional compositions through solid-state fermentation using Candida utilis and Bacillus subtilis. The crude and soluble protein contents increased by 54.83 % and 866.50 %, reaching 242.50 ± 6.50 mg/g and 39.53 ± 1.77 mg/g after fermentation, respectively. The contents of essential amino acids and nonessential amino acids significantly increased by 230 % and 402 %, especially leucine and phenylalanine (8.3 % and 5.1 %, based on total protein). Moreover, antinutritional factors, such as pectin and crude fiber, were reduced by 43.10 % and 20.21 %, respectively. Correlation analysis showed that the activities of fiveenzymes, including acid protease, neutral protease, pectinase,xylanase and cellulase, were significantly correlated with the variation in the contents of soluble protein, total sugars and reducing sugars (p

Recently, a hyphae-dispersed type of filamentous fungus Aspergillus oryzae was constructed via genetic engineering, and industrial applications are expected due to the ease of handling and to the level of protein production properties. In this study, we constructed cellulase-expressing strains using wild-type and hyphae-dispersed strains to investigate the correlation between protein productivity and metabolism. Compared with the original strain, the hyphae-dispersed cellulase-expressing strain showed elevated cellulase activity, rapid glucose consumption, increased mycelial dry weight, an increased expression of cellulase genes, and activated respiration activity. Comparative metabolomic analysis showed fewer metabolites in the glycolysis and TCA cycles in the dispersed strains than in the original strains. These results indicate that the flux of carbohydrate metabolism in the hyphae-dispersed strains is smoother than that in the original strains. Such efficient metabolic flux would contribute to efficient energy conversion and to sufficient energy supply to anabolisms, such as mycelial growth and protein production. Our findings suggest that the hyphae-dispersed strains could be a useful host not only for protein production but also for the biological production of various chemicals such as organic acids.

Microbial communities involved in hydrogen (H2) production from enzymatic hydrolysates of agave bagasse were analyzed through 16S rRNA sequencing. Two types of reactor configurations and four different enzymatic hydrolysates were evaluated. Trickling bed reactors led to highly-diverse microbial communities, but low volumetric H2 production rates (VHPR, maximum: 5.8 L H2/L-d). On the contrary, well-controlled environments of continuous stirred-tank reactors favored the establishment of low diverse microbial communities composed by Clostridium-Sporolactobacillus leading to high-performance H2-production (VHPR maximum: 13 L H2/L-d). Cellulase-Viscozyme and Celluclast-Viscozyme hydrolysates led to the co-dominance of Clostridium and Sporolactobacillus, possibly due to the presence of xylose and hemicellulose-derived carbohydrates. Cellulase hydrolysates were linked to communities dominated by Clostridium, while maintaining low abundance of Sporolactobacillus. Stonezyme hydrolysates favored microbial communities co-dominated by Clostridium-Lachnoclostridium-Leuconostoc. Moreover, contrary to the prevailing theory, it was demonstrated that H2 production performance was inversely related to microbial diversity.