Screening of potential antibiotic action of cellulolytic fungi

Twenty different strains of filamentous fungi were initially selected for evaluation of cellulolytic activity using a single test in a simple mineral salts culture medium with filter paper as the only carbon source. Those fungi strains that were capable of completely breaking the filter paper strip within 4–8 d were assayed also for antimicrobial action, using Staphyloccocus a ureus ATCC 6538P according to the so-called agar piece method. We screened three different strains with both capacities: the production of cellulolytic activity and antibiotic action. The experimental results suggest that the fungi Pinicillium sp. FOPCO1, Aspergillus sp. F0Q001, and Cephalosporium sp. F03800 have both capabilities because they grew rapidly on cellulose as the only carbon source and were able to produce an area of growth inhibition in S. aureus of approx 2.04, 1.57, and 2.39 cm, respectively, on agar plates using the agar piece method. Subsequently, the antibiotic production obtained with those cellulolytic strains was evaluated by submerged fermentation at the flask level, in a simple culture medium containing lactose without biosynthesis precursor, obtaining 3670, 2830, and 4060 antibiotic units/mL, referred to as penicillin G, whereas for cellulolytic activity, the results were 1.34, 1.81 and 0.57 FPU/mL, respectively.     

Isolation, Screening, and Optimization of the Fermentation Conditions of Highly Cellulolytic Bacteria from the Hindgut of Holotrichia parallela Larvae (Coleoptera: Scarabaeidae)

From the hindgut contents of Holotrichia parallela, 93 cellulolytic bacterial isolates were isolated after enrichment in carboxymethyl cellulose medium. Among these isolates, a novel bacterium, designated HP207, with the highest endoglucanase productivity was selected for further study. This bacterium was identified as Pseudomonas sp. based on the results of the 16S ribosomal DNA analysis, morphological characteristics, and biochemical properties. The production of the endoglucanase was optimized by varying various physical culture conditions using a submerged fermentation method. Under the optimized fermentation conditions, the maximum endoglucanase activity of 1.432?U?mL^?1 in bacterial cultures was obtained, higher than those of the most widely studied bacteria and fungi, which are the attractive candidates for the commercial producer of cellulase. And the crude endoglucanase enzyme was also highly thermostable; approximately 55?% of the original activity was maintained after pretreatment at 70?°C for 1?h. Thus, from the present study, the bacterium can be added up to the database of cellulolytic bacteria.     

Secretion of thermostable β-glucosidase by an intergeneric bacterial hybrid betweencellulomonas and bacillus subtilis

The intergeneric protoplast fusion hybrid (Bs/C 005) betweenCellulomonas sp. andBacillus subtilisproduced extracellular aryl β-glucosidase that is otherwise intracellular in parentalCellulomonassp. This extracellular aryl β-glucosidase was active at relatively higher temperature (60°C) and lower pH (pH 5.0) conditions than that ofCellulomonas enzyme. It also exhibited increased thermostability and stability over wide range of pH. Cellobiase activity, distinctly different from aryl β-glucosidase detected in bothCellulomonassp. Bs/C 005, was only intracellular. Cellobiase from Bs/C 005, however, was more thermostable than that ofCellulomonassp.     

Controlled composting of straw

Production of composts on the farm from surplus straw might provide a low-cost biotechnological approach for increasing the value of this lignocellulosic waste. Successful composting depends on the conversion of the polysaccharides (cellulose and hemicelluloses) of straw by inoculated microorganisms to products that can promote plant growth when applied to the land. None of the potentially useful products we have identified are produced by cellulolytic organisms. We have therefore studied mixed populations in which noncellulolytic bacteria depend for growth on the products of fungal cellulolysis. The nature and yield of bacterial products depends not only on conditions within the compost, but also on the microbial inoculants used. Under defined laboratory conditions, using pure cellulose, N₂ is fixed by the anaerobic bacteriumClostridium butyricum in association with a cellulolytic fungus such asTrichoderma sp. A similar association has been achieved on straw withPenicillium corylophilum as the cellulolytic fungus. Cellulolytic fungi can also provide available substrates for the production of bacterial polysaccharides that can improve the structure of unstable soils. The yield of polysaccharide and its efficacy in soil aggregate stabilization again varies with the inoculants used. Such composts can thus contribute to plant nutrition and to soil structure. The adoption ofTrichoderma spp. as the cellulolytic inoculants would further extend the potential value of the compost to include the biocontrol of plant pathogens.     

Enhancing the Cellulose-Degrading Activity of Cellulolytic Bacteria CTL-6 (Clostridium thermocellum) by Co-Culture with Non-cellulolytic Bacteria W2-10 (Geobacillus sp.)

The effect of a non-cellulolytic bacterium W2-10 (Geobacillus sp.) on the cellulose-degrading activity of a cellulolytic bacterium CTL-6 (Clostridium thermocellum) was determined using cellulose materials (paper and straw) in peptone cellulose solution (PCS) medium under aerobic conditions. The results indicated that in the co-culture, addition of W2-10 resulted in a balanced medium pH, and may provide the required anaerobic environment for CTL-6. Overall, addition of W2-10 was beneficial to CTL-6 growth in the adverse environment of the PCS medium. In co-culture with W2-10, the CTL-6 cellulose degradation efficiency of filter paper and alkaline-treated wheat straw significantly increased up to 72.45 and 37.79 %, respectively. The CMCase activity and biomass of CTL-6 also increased from 0.23 U ml^?1 and 45.1 μg ml^?1 (DNA content) up to 0.47 U ml^?1 and 112.2 μg ml^?1, respectively. In addition, co-culture resulted in accumulation of acetate and propionate up to 4.26 and 2.76 mg ml^?1. This was a respective increase of 2.58 and 4.45 times, in comparison to the monoculture with CTL-6.     

Cellulose production based on hemicellulose hydrolysate from steam-pretreated willow

The production cost of cellulolytic enzymes is a major contributor to the high cost of ethanol production from lignocellulosics using enzymatic hydrolysis. The aim of the present study was to investigate the cellulolytic enzyme production ofTrichoderma reesei Rut C 30, which is known as a good cellulase secreting micro-organism, using willow as the carbon source. The willow, which is a fast-growing energy crop in Sweden, was impregnated with 1–4% SO₂ and steam-pretreated for 5 min at 206°C. The pretreated willow was washed and the wash water, which contains several soluble sugars from the hemicellulose, was supplemented with fibrous pretreated willow and used for enzyme production. In addition to sugars, the liquid contains degradation products such as acetic acid, furfural, and 5-hydroxy-methylfurfural, which are inhibitory for microorganisms. The results showed that 50% of the cellulose can be replaced with sugars from the wash water. The highest enzyme activity, 1.79 FPU/mL and yield, 133 FPU/g carbohydrate, was obtained at pH 6.0 using 20 g/L carbon source concentration. At lower pHs, a total lack of growth and enzyme production was observed, which probably could be explained by furfural inhibition.     

Identification of Cellulase Gene from the Metagenome of Equus burchelli Fecal Samples and Functional Characterization of a Novel Bifunctional Cellulolytic Enzyme

The metagenomic approach has been used successfully to isolate novel biocatalyst gene from uncultured microorganisms. The gene encoding exo-1,4-??-glucanase avicelase was amplified from the metagenome of the Equus burchelli fecal sample and cloned. The gene was found to be of 1,007?bp of nucleotide which encodes a protein of 318 amino acids with a calculated MW of 36?kDa. The deduced amino acid sequence was homologous with cellulases belonging to the glycosyl hydrolases 6 superfamily. The expressed protein was active towards the substrates avicel and carboxymethyl cellulose, indicating that it has bifunctional cellulolytic enzyme activity. The recombinant protein showed an activity of 5.23?U with specific activity of 6.8?U?mg^?1 protein with the substrate avicel, while when CMC was used, an activity of 3.0?U with a specific activity of 4.2?U?mg^?1 protein was achieved. Its optimum pH was determined to be 7.0 and optimum temperature of 35°C.     

Production of Cellulolytic Enzymes by Fungi Acrophialophora nainiana and Ceratocystis paradoxa Using Different Carbon Sources

Although a number of filamentous fungi, such as Trichoderma and Aspergillus, are well known as producers of cellulases, xylanases, and accessory cellulolytic enzymes, the search for new strains and new enzymes has become a priority with the increase in diversity of biomass sources. Moreover, according to the type of pretreatment applied, biomass of the same type may require different enzyme blends to be efficiently hydrolyzed. This study evaluated cellulases, xylanases, and β-glucosidases produced by two fungi, the thermotolerant Acrophialophora nainiana and Ceratocystis paradoxa. Cells were grown in submerged culture on three carbon sources: lactose, wheat bran, or steam-pretreated sugarcane bagasse, a commonly used cattle feed in Brazil. Xylanase and endo-1-4-β-glucanase (CMCase) highest production were found in A. nainiana growing on lactose and reached levels of 2,200 and 2,016 IU/L, respectively. C. paradoxa showed highest activity for xylanase when grown on wheat bran and for β-glucosidase when grown on steam-treated bagasse, at levels of 12,728 and 1,068 IU/mL, respectively.     

Investigations on Hydrolytic Activities from Stachybotrys microspora and Their Use as an Alternative in Yeast DNA Extraction

Stachybotrys microspora is a filamentous fungus characterized by the secretion of multiple hydrolytic activities (cellulolytic and non-cellulolytic enzymes). The production of these biocatalysts was studied under submerged culture using glucose, cellulose, and wheat bran as carbon sources. Endoglucanases, pectinases, xylanases, β-glucanases, chitinases, and proteases were induced on cellulose-based medium and repressed on glucose in both strains with higher amounts produced by the mutant. β-glucosidases were roughly equally produced by both strains under glucose and cellulose conditions. The yield of chitinases, β-glucanases, and proteases produced by Stachybotrys strains was as much higher than the commercialized lysing enzyme called “zymolyase,” currently used in yeast DNA extraction. In this context, we showed that S. microspora hydrolases can be successfully applied in the extraction of yeast DNA.     

Potential of Mangrove-Associated Endophytic Fungi for Production of Carbohydrolases with High Saccharification Efficiency

The endophytic fungi represent a potential source of microorganisms for enzyme production. However, there have been only few studies exploiting their potential for the production of enzymes of industrial interest, such as the (hemi)cellulolytic enzymatic cocktail required in the hydrolysis of lignocellulosic biomass. Here, a collection of endophytic fungi isolated from mangrove tropical forests was evaluated for the production of carbohydrolases and performance on the hydrolysis of cellulose. For that, 41 endophytic strains were initially screened using a plate assay containing crystalline cellulose as the sole carbon source and the selected strains were cultivated under solid-state fermentation for endoglucanase, β-glucosidase, and xylanase enzyme quantification. The hydrolysis of a cellulosic material with the enzymes from endophytic strains of the Aspergillus genus resulted in glucose and conversion values more than twofold higher than the reference strains (Aspergillus niger F12 and Trichoderma reesei Rut-C30). Particularly, the enzymes from strains A. niger 56 (3) and A. awamori 82 (4) showed a distinguished saccharification performance, reaching cellulose conversion values of about 35% after 24 h. Linking hydrolysis performance to the screening steps played an important role towards finding potential fungal strains for producing enzymatic cocktails with high saccharification efficiency. These results indicate the potential of mangrove-associated endophytic fungi for production of carbohydrolases with efficient performance in the hydrolysis of biomass, thus contributing to the implementation of future biorefineries.     

Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes

For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commently used, but, there is a need for more efficient enzyme cocktails. In this study, the production of cellulolytic and xylanolytic enzymes was investigated in 12 filamento us fungi from genus Penicillium and compared with that of T. reesei. Either Solka-Floc cellulose or oat spelt xylan was used as carbon source in shake flask cultivations. All the fungi investigated showed coinduction of cellulolytic and xylanolytic enzymes during growth on cellulose as well as on xylan. The highest filter paper activity was measured after cultivation of Penicillium brasilianum IBT 20888 on cellulose.     

The Family 1 Glycoside Hydrolase from Clostridium cellulolyticum H10 is a Cellodextrin Glucohydrolase

The only family 1 glycoside hydrolase in Clostridium cellulolyticum H10 (CcGH1) is annotated as a beta-galactosidase but has high sequence homology with many beta-glucosidases. Given the possible importance of beta-glucosidase in cellulose utilization by C. cellulolyticum, the encoding open reading frame Ccel_0374 was cloned and expressed in E. coli as a soluble fusion protein with thioredoxin. After tag cleavage, the purified enzyme had a molecular mass of 52 kDa and was active in dimeric form on a broad range of substrates, including cellobiose, cellotriose, cellotetraose, p-nitrophenyl-beta-glucopyranoside, lactose, and o-nitrophenyl-beta-galactopyranoside. The enzyme showed lower K _m and higher catalytic efficiency (k _cat/K _m) on cellodextrins with degree of polymerization from 2 to 4 than on lactose, and the k _cat/K _m values on cellodextrins increased in the order of cellobiose < cellotriose < cellotetraose, suggesting that CcGH1 was a cellodextrin glucohydrolase (EC 3.2.1.74). The high K _m (69 mM) on cellobiose implies that CcGH1 likely has a minimal role in the intracellular hydrolysis of cellobiose in C. cellulolyticum. The three-dimensional structure model of CcGH1 generated by homology modeling showed a typical (α/β)₈ barrel topology characteristic of family 1 glycoside hydrolases.     

Production of cellulolytic enzymes by coculturing ofAspergillus ellipticus andAspergillus fumigatus grown on bagasse under solid state fermentation

Production of cellulolytic enzymes on bagasse under solid state fermentation by coculture ofAspergillus ellipticus andAspergillus fumigatus was studied. Cocultivation ofA. ellipticus andA. fumigatus showed improved hydrolytic and Β-glucosidase activities as compared to the occasions when they were used separately. Various pretreatment methods were used to make cellulose accessible to enzymatic attack. Best results were obtained through pretreatment with 2% (w/v) calcium hydroxide. Maximum enzyme production was obtained after 8 d of fermentation process.     

Influences of Media Compositions on Characteristics of Isolated Bacteria Exhibiting Lignocellulolytic Activities from Various Environmental Sites

Efficient isolation of lignocellulolytic bacteria is essential for the utilization of lignocellulosic biomass. In this study, bacteria with cellulolytic, xylanolytic, and lignolytic activities were isolated from environmental sites such as mountain, wetland, and mudflat using isolation media containing the combination of lignocellulose components (cellulose, xylan, and lignin). Eighty-nine isolates from the isolation media were characterized by analyzing taxonomic ranks and cellulolytic, xylanolytic, and lignolytic activities. Most of the cellulolytic bacteria showed multienzymatic activities including xylanolytic activity. The isolation media without lignin were efficient in isolating bacteria exhibiting multienzymatic activities even including lignolytic activity, whereas a lignin-containing medium was effective to isolate bacteria exhibiting lignolytic activity only. Multienzymatic activities were mainly observed in Bacillus and Streptomyces, while Burkholderia was the most abundant genus with lignolytic activity only. This study provides insight into isolation medium for efficient isolation of lignocellulose-degrading microorganisms.     

Bioconversion of cellulose and simultaneous production of thermoactive exo- and endoglucanases by <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

Saccharification of cellulose is a promising method for production of biofuels. However, low bioconversion efficiency of cellulose to soluble sugars is a major challenge. In this study, a cellulolytic strain of Fusarium oxysporum was cultivated on pure cellulosic substrates (avicel, α-cellulose, carboxymethylcellulose and methylcellulose) and conversion efficiency into glucose was investigated. Production of exo- and endoglucanases during the bioconversion process was evaluated. Influence of pH on saccharification of cellulose and enzyme production by F. oxysporum were determined. Highest yield of glucose (1.76 μmol/ml) was obtained from F. oxysporum on methyl cellulose at 192 h under basal conditions. Liberated glucose under optimized condition of pH 6.0 at 96 h of fermentation was 2.12 μmol/ml with maximum production of exo- and endoglucanases (23.70 and 34.72 U/mg protein, respectively). The crude exo- and endoglucanases had optimum activities at pH 8.0, 70 °C and pH 7.0, 50 °C, respectively. The enzymes were stable over pH of 4.0–7.0 with relative residual activity above 60% after 1 h incubation. Exoglucanase activity was enhanced by Ca²⁺ and Cu²⁺ at 5 mM and Mg²⁺ at 10 mM. Endoglucanase activity was greatly enhanced in the presence of Mn²⁺, Ca²⁺, Mg²⁺, Cu²⁺ and Fe³⁺ at 5 and 10 mM. Activities of both enzymes were inhibited in the presence of Hg²⁺ at 5 and 10 mM. Results show that F. oxysporum possessed good cellulolytic enzyme system for efficient conversion of cellulose. Exhibited thermotolerance of exoglucanase with the striking tolerance of endoglucanase to metal ions demonstrate potentials of enzymes for biofuel industry.     

Exploring Thermophilic Cellulolytic Enzyme Production Potential of Aspergillus fumigatus by the Solid-State Fermentation of Wheat Straw

Cellulases can be used for biofuel production to decrease the fuel crises in the world. Microorganisms cultured on lignocellulosic wastes can be used for the production of cellulolytic enzymes at large scale. In the current study, cellulolytic enzyme production potential of Aspergillus fumigatus was explored and optimized by employing various cultural and nutritional parameters. Maximum endoglucanase production was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. Addition of 0.3 % of fructose, peptone, and Tween-80 further enhanced the production of endoglucanase. Maximum purification was achieved with 40 % ammonium sulfate, and it was purified 2.63-fold by gel filtration chromatography. Endoglucanase has 55 °C optimum temperature, 4.8 optimum pH, 3.97 mM K _m, and 8.53 μM/mL/min V _max. Maximum exoglucanase production was observed at 55 °C after 72 h, at pH 5.5, and 70 % moisture level. Further addition of 0.3 % of each of fructose, peptone, and Tween-80 enhances the secretion of endoglucanase. It was purified 3.30-fold in the presence of 40 % ammonium sulfate followed by gel filtration chromatography. Its optimum temperature was 55 °C, optimum pH was 4.8, 4.34 mM K _m, and 7.29 μM/mL/min V _max. In the case of β-glucosidase, maximum activity was observed after 72 h at 55 °C, pH 5.5, and 70 % moisture level. The presence of 0.3 % of fructose, peptone, and Tween-80 in media has beneficial impact on β-glucosidase production. A 4.36-fold purification was achieved by 40 % ammonium sulfate precipitation and gel filtration chromatography. Optimum temperature of β-glucosidase was 55 °C, optimum pH was 4.8, K _m was 4.92 mM, and V _max 6.75 μM/mL/min. It was also observed that fructose is better than glucose, and peptone is better than urea for the growth of A. fumigatus. The K _m and V _max values indicated that endoglucanase, exoglucanase, and β-glucosidase have good affinity for their substrates.     

Influence of cultivation conditions on the production of cellulolytic enzymes withTrichoderma reesei Rutgers C30 in aqueous two-phase systems

Cellulolytic enzyme production in aqueous two-phase systems withTrichoderma reesei Rutgers C30 has been investigated. The influ ence of different phase systems, as well as addition of media compo nents and substrate on enzyme production have been studied. Extractive enzyme production in fed-batch cultivations was per formed in a phase system composed of PEG 8000 5%-Dextran T500 7% with 1% Solka-Floc BW 200 as substrate. The cellulolytic enzyme system was intermittently withdrawn with the top phase. Addition of media components every 24 h and cellulose every 72 h gave an aver age enzyme activity in the withdrawn top phase of 2.2 FPU/mL dur ing 170 h cultivation. The corresponding productivity was 18 FPU/lh. The productivity was increased to 24 FPU/l.h when media compo nents and cellulose were added every 72 h. The average enzyme con centration was then 1.6 FPU/mL. The results are discussed in relation to methods for cellulolytic enzyme production involving immobiliza tion and cell recycling.     

Screening,Growth Medium Optimisation and Heterotrophic Cultivation of Microalgae for Biodiesel Production

This article presents a study on screening of microalgal strains from the Peking University Algae Collection and heterotrophic cultivation for biodiesel production of a selected microalgal strain. Among 89 strains, only five were capable of growing under heterotrophic conditions in liquid cultures and Chlorella sp. PKUAC 102 was found the best for the production of heterotrophic algal biodiesel. Composition of the growth medium was optimised using response surface methodology and optimised growth conditions were successfully used for cultivation of the strain in a fermentor. Conversion of algal lipids to fatty acid methyl esters (FAMEs) showed that the lipid profile of the heterotrophically cultivated Chlorella sp. PKUAC 102 contains fatty acids suitable for biodiesel production.     

Characterization and Phylogenetic Analysis of Microbial Surface Active Compound-Producing Bacteria

Microbial surface active compounds (SACs) were isolated from various environmental sources in Thailand. Isolates were screened for SACs production in different carbon sources (crude glycerol, commercial sugar, decanter, glucose, molasses, used palm oil, and used lubricating oil) by using drop-collapsing test and emulsification activity. Molasses produced the highest number of positive results (23 of 25 isolates). Twenty-one isolate strains produced emulsions with xylene, and 15 exhibited high emulsion-stabilizing capacity, maintaining more than 50?% of the original emulsion volume for 24?h, and six isolate strains reduced the growth medium surface tension to 40?mN/m. The phylogenetic position of these 25 isolates was evaluated by 16S rRNA gene sequence analysis. The production of microbial SACs was determined for strains representative of 16 different bacterial genera, in which ten genera (Blastococcus, Erysipelothrix, Humicoccus, Methylophilus, Microlunatus, Nevskia, Pectinatus, Rubrimonas, Selenomonas, and Stenotrophomonas) were firstly reported as SAC-producing strains. Overall, the new SAC-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.