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Screening genus Penicillium for producers of cellulolytic and xylanolytic enzymes.

For enzymatic hydrolysis of lignocellulosic material, cellulolytic enzymes from Trichoderma reesei are most commenly 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 filamentous 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.

Avena↗

Enzymatic saccharification and fermentation of xylose-optimized dilute acid-treated lignocellulosics.

The cellulose reactivity of two lignocellulosic feedstocks, switchgrass and poplar, was evaluated under straight saccharification (SS) and simultaneous saccharification and fermentation (SSF) conditions following dilute sulfuric acid pretreatments designed for optimum xylose yields. The optimum pretreatment conditions, within the constraints of the experimental system (Parr batch reactor), were 1.2% acid, 180 degrees C, and 0.5 min for switchgrass and 1% acid, 180 degrees C, and 0.56 min for poplar. The cellulase enzyme preparation was from Trichoderma reesei and fermentations were done with Saccharomyces cerevisiae. Time courses for SS were monitored as the sum of glucose and cellobiose; those for SSF as the sum of glucose, cellobiose, and ethanol. Percentage conversions under SS conditions were 79.1% and 91.4% for the pretreated poplar and switchgrass feedstocks, respectively. Analogous values under SSF conditions were 73.0% and 90.3% for pretreated poplar and switchgrass, respectively.

Cell Culture Techniques↗

Microbial synthesis of chitinase in solid cultures and its potential as a biocontrol agent against phytopathogenic fungus Colletotrichum gloeosporioides.

Antifungal activity of chitinase can be effectively utilized in biologic pest control strategies. Because solid-state cultivation has been termed a cost-effective means for fungal growth and metabolite production, chitinase production by Trichoderma harzianum was studied using wheat bran-based solid medium containing 1% colloidal chitin. Chitinase synthesis was found to be growth associated because maximum enzyme (5.4 U/g of dry substrate) and biomass production occurred at 72 h. Substrate moisture had a critical impact on chitinase production; five grams of medium having an initial moisture content of 68.4% when incubated for 72 h increased the enzyme yield to 9.3 U/g of dry substrate. Optimization of colloidal chitin concentration showed that improvements in chitinase yield and maximum activity were attained with a 2% (w/w) concentration. Supplementation of additional nitrogen sources also influenced enzyme production, and the best yield was obtained with yeast extract. The effect of crude chitinase on hyphal morphology of the phytopathogenic fungus Colletotrichum gloeosporioides was swelling as well as lysis of hyphal wall, depending on the age of the mycelium. Studies of pH and thermal stability showed that crude culture filtrate was active over pH 4.0-6.0 and retained about 48.2% activity after 40 min of incubation at 40 degrees C.

Biomass↗

Lactic acid production from cellulosic material by synergetic hydrolysis and fermentation.

The hydrolysis process on corncob residue was catalyzed synergetically by the cellulase from Trichoderma reesei and the immobilized cellobiase. The feedback inhibition to cellulase reaction caused by the accumulation of cellobiose was eliminated efficiently. The hydrolysis yield of corncob residue was 82.5%, and the percentage of glucose in the reducing sugar reached 88.2%. The glucose in the cellulosic hydrolysate could be converted into lactic acid effectively by the immobilized cells of Lactobacillus delbrueckii. When the enzymatic hydrolysis of cellulose and the fermentation of lactic acid were coupled together, no glucose was accumulated in the reaction system, and the feedback inhibition caused by glucose was also eliminated. Under the batch process of synergetic hydrolysis and lactic acid fermentation with 100 g/L of cellulosic substrate, the conversion efficiency of lactic acid from cellulose and the productivity of lactic acid reached 92.4% and 0.938 g/ (L.h), respectively. By using a fed-batch technique, the total concentration of cellulosic substrate and lactic acid in the synergetic process increased to 200 and 107.5 g/L, respectively, whereas the dosage of cellulase reduced from 20 to 15 IU/g of substrate in the batch process. The results of the bioconversion of renewable cellulosic resources were significant.

Bioreactors↗

Endocellulase and exocellulase activities of two Streptomyces strains isolated from a forest soil.

Two Streptomyces strains, M7a and M23, from a Brazilian forest soil were evaluated for the cellulase production of their supernatants after growth in a microcrystalline cellulose medium, using carboxymethylcellulose and filter paper as substrates at different temperatures and pH values. Endoglucanase and exoglucanase activities were compared to a commercial Trichoderma reesei cellulase using fluorogenic conjugated substrates. Similar specific activities were observed for the enzyme preparations of strain M23 and T. reesei. For M7a the activities were about seven times higher than those obtained for T. reesei. Extracellular or cell-associated cellobiase activities were not detected in both strains.

Brazil↗

Repeated solid-phase fermentation and extraction for enzyme production.

Solid-phase fermentation has been found to have a much higher productivity than the popular liquid submerged fermentation in producing cellulase enzymes. The highest reported productivity in the literature for cellulases by Trichoderma cultures in submerged fermentation is 158 filter paper units (FPU)/(h.L) of fermenting liquid. From preliminary experiments of solid-phase fermentation in 1000-mL flasks, a productivity of 234 FPU of cellulases/(h.L) of solid-bed volume was obtained. When two novel techniques--pressure pulsation and repeated extraction--were applied, a productivity of 806 FPU/(h.L) was achieved. The same techniques also greatly enhanced the productivity of other enzymes by fungal cultures in solid-phase fermentation.

Biotechnology↗

Improvements in titer, productivity, and yield using Solka-floc for cellulase production.

Researchers studying cellulase enzymes for the economical production of fuel ethanol envision cellulose as the carbon source. However, submerged Trichoderma reesei cultures grown on cellulose exhibit high run-to-run variability. Thus, an investigation of 30 batch cellulase production experiments was instrumental in determining fermentation conditions that improved enzyme titers, yields, and productivities. Eighteen of the 30 batch experiments experienced minimal process upsets and were classified into eight groups based on agitation rate, gas sparge rate, and the use of oxygen supplementation. Comparing corn steep liquor with yeast extract/peptone also tested the effect of different sources of nitrogen in the media. Average 7-d enzyme titers were doubled from 4 to 8 FPU/mL primarily by increasing aeration.

Biotechnology↗

Influence of operating conditions and vessel size on oxygen transfer during cellulase production.

The production of low-cost cellulase enzyme is a key step in the development of an enzymatic-based process for conversion of lignocellulosic biomass to ethanol. Although abundant information is available on cellulase production, little of this work has examined oxygen transfer. We investigated oxygen transfer during the growth of Trichoderma reesei, a cellulase-producing microorganism, on soluble and insoluble substrates in vessel sizes from 7 to 9000 L. Oxygen uptake rates and volumetric mass transfer coefficients (kLa) were determined using mass spectroscopy to measure off gas composition. Experimentally measured kLa values were found to compare favorably with a kLa correlation available in the literature for a non-Newtonian fermentation broth during the period of heavy cell growth.

Biomass↗

Production of recombinant bleaching enzymes from thermophilic microorganisms in fungal hosts.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host-vector expression system critical. We have developed two systems for the bulk production of bleaching enzymes from thermophiles. Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2mu-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Fusion and nonfusion vectors have been constructed using the strong cellobiohydrolase 1 (cbh1) promoter. The KEX2 protease cleavage site and a 6 x HIS-tag have been incorporated to facilitate both cleavage and purification of the mature foreign proteins.

Amino Acid Sequence↗

Cellulase production on bagasse pretreated with hot water.

Because pretreatment of biomass with hot water only in differential flow systems offers very digestible cellulose and potentially less inhibition by liquid hydrolysate, solids and liquid hydrolysate from bagasse pretreated with hot water were fed to a batch cellulase production system using the Rut C30 strain of Trichoderma reesei to determine the suitability of these substrates for cellulase production. The organism was found to be sensitive to inhibitors in the liquid hydrolysate but could be adapted to improve its tolerance. In addition, filtering of the material reduced inhibitory effects. The organism was also sensitive to some component in the solids, and they had to be washed heavily to achieve good growth and cellulase production rates. Even then, a lag was found before enzyme production would commence on pretreated solids whereas no such lag was experienced with Solka Floc. However, once enzyme production began, as high and even somewhat greater cellulase productivities were realized with washed pretreated solids. Adding lignin to Solka Floc delayed enzyme production, suggesting that lignin or other materials in the lignin solids could cause the lag observed for pretreated bagasse, but more studies are needed to resolve the actual reason for this delay.

Cellulase↗

Evaluation of filamentous fungi and inducers for the production of endo-polygalacturonase by solid state fermentation.

Aspergillus oryzae CCT 3940, Aspergillus awamori NRRL 3112 and a Trichoderma sp.) were compared for their capacity to produce endo-polygalacturonase (endo-PG) in solid state fermentation. Maximum pectinolytic activity was reached in 72 h of growth, the best two fungal strains being A. niger T0005007-2 and A. oryzae CCT 3940. Three types of commercial purified pectin and four of unprocessed pectin (tangerine, orange, Tahiti lime and sweet lime rind) were used to assess the effect of pectin on the production of endo-PG by A. niger T0005007-2. Maximum pectinolytic activity was achieved using 6 and 10% (w/w) of purified pectin as inducer. Depending on the origin of the commercial pectin used as inducer, maximum endo-PG levels varied from 223 to 876 units per gram of dry medium (one endo-PG unit (U) was defined as the quantity of enzyme which caused a reduction in viscosity of 50% in a 1% w/v solution of pectin in 30 min), indicating that care should be taken when choosing this component of the medium. When the crude pectins were used as inducers at the same concentration as purified pectin, maximum endo-PG activities were 250-300 units/g. However, by increasing the amount of Tahiti lime rind to 50% (w/w) maximum endo-PG was 919 U/g, thus opening up the possibility of a low cost medium for endo-PG production.

Aspergillus↗

Integrated application of some compatible biocontrol agents along with mustard oil seed cake and furadan on Meloidogyne incognita infecting tomato plants.

Experiments were carried out to study the effect of two fungal bioagents along with mustard oil cake and furadan against root knot nematode Meloidogyne incognita infecting tomato under greenhouse condition. Bioagents viz., Paecilomyces lilacinus and Trichoderma viride alone or in combination with mustard cake and furadan promoted plant growth, reduced number of galls/plant, egg masses/root system and eggs/egg mass. The fungal bioagents along with mustard cake and nematicide showed least nematodes reproduction factor as compared to untreated infested soil.

Animals↗

Structure of dried cellular alginate matrix containing fillers provides extra protection for microorganisms against UVC radiation.

Soil microorganisms in general and biocontrol agents in particular are very sensitive to UV light. The packaging of biocontrol microorganisms into cellular solids has been developed as a means of reducing loss caused by exposure to environmental UV radiation. The bacterial and fungal biocontrol agents Pantoea agglomerans and Trichoderma harzianum were immobilized in freeze-dried alginate beads containing fillers and subjected to 254 nm UV radiation (UVC). Immobilization of cells in freeze-dried alginate-glycerol beads resulted in greater survival after UV irradiation than for a free cell suspension. Adding chitin, bentonite or kaolin as fillers to the alginate-glycerol formulation significantly increased bacterial survival. Immobilization in alginate-glycerol-kaolin beads resulted in the highest levels of survival. The transmissive properties of the dried hydrocolloid cellular solid had a major influence on the amount of protection by the cell carrier. Dried alginate matrix (control) transmitted an average of 7.2% of the radiation. Filler incorporation into the matrix significantly reduced UV transmission: Alginate with kaolin, bentonite and chitin transmitted an average of 0.15, 0.38 and 3.4% of the radiation, respectively. In addition, the filler inclusion had a considerable effect on the bead's average wall thickness, resulting in a approximately 1.5- to threefold increase relative to beads based solely on alginate. These results suggest that the degree of protection of entrapped microorganisms against UVC radiation is determined by the UV-transmission properties of the dried matrix and the cellular solid's structure. It is concluded that for maximum protection against UV-radiation-induced cell loss, biocontrol microorganisms should be immobilized in alginate-glycerol beads containing kaolin.

Alginates↗

The site-directed mutagenesis of gastrodia anti-fungal protein mannose-binding sites and its expression in Escherichia coli.

Gastrodia anti-fungal protein (GAFP) displays strong inhibitory activity against certain fungal pathogens. Five GAFP analogues with different mutations at mannose-binding sites and the wild-type one were expressed and purified in Escherichia coli. The inhibitory analysis of the purified various GAFPs against the growth of Trichoderma viride indicates that single amino acid mutated-type GAFPs have inhibitory activity, but its activity is much less than the wild-type one. The double and triplicate amino acids mutated GAFPs have very low inhibitory activity. For the first time it was proved that GAFP mannose-binding sites play key role in anti-fungi process.

Antifungal Agents↗

Intake, digestibility, and composition of orchardgrass and alfalfa silages treated with cellulase, inoculant, and formic acid fed to lambs.

The objectives of this study were to determine the effect of a cellulase (from Trichoderma longibrachiatum) alone or combined with a bacterial inoculant (Lactobacillus plantarum and Pediococcus cerevisiae) or formic acid on composition, intake, and digestibility of orchardgrass (Dactylis glomerata L.) and alfalfa (Medicago sativa L.) silages. Orchardgrass and alfalfa were harvested at the early heading stage and at the early bloom stage of maturity and wilted to approximately 22 and 32% DM, respectively. Forages were then ensiled in 100-L sealed barrels for at least 60 d before they were fed to lambs. Silage treated with cellulase had lower (P < .001) pH and lower (P < .001) acetic acid and NH3 N concentrations than untreated silage of both plant species and a higher (P = .004) lactic acid concentration than the control treatment of alfalfa silage. Fermentation characteristics of cellulase-treated silages, especially of alfalfa, were further enhanced by use of inoculant. Formic acid addition increased (P < .001), reducing sugar concentration of cellulase-treated orchardgrass and alfalfa silage by 90 and 154%, respectively, and decreased (P < .001) NH3 N concentration of cellulase-treated alfalfa silage by 19%. Averaged across plant species, cellulase, combined with inoculant or formic acid, resulted in 8 and 13% greater (P = .03) DMI, respectively, than the control silage. Extensive enzymatic cell-wall degradation during ensiling decreased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 25% and decreased (P = .001) cellulose intake by 23%, when averaged across plant species. Addition of formic acid increased (P = .003) NDF intake of cellulase-treated orchardgrass silage by 19%. Averaged across species, cellulase application decreased (P < .05) silage NDF digestibility by 18%. Greater sugar and lower acetic acid, NH3 N, and NDF concentrations resulted in greater DMI of cellulase-treated silage than of control silage, when cellulase was combined with formic acid or inoculant.

Animal Feed↗

Resistance of feed enzymes to proteolytic inactivation by rumen microorganisms and gastrointestinal proteases.

Potential feed enzyme additives for ruminants were tested in vitro for their stability to ruminal microbial and gastrointestinal proteolysis. Four commercial preparations from Trichoderma longibrachiatum (A, B, C, and D) and one from an undisclosed source (E) were incubated up to 6 h with ruminal fluid taken from four lactating dairy cows before or 2 h after feeding. The stability of preparation B was also tested in the presence of pepsin at pH 3 and pancreatin at pH 7. Cellulase (EC 3.2.1.4), cellulose 1,4-beta-cellobiosidase (EC 3.2.1.91), beta-glucanase (EC 3.2.1.6), xylanase (EC 3.2.1.8), beta-glucosidase (EC 3.2.1.21), and beta-xylosidase (EC 3.2.1.37) activities were monitored throughout the incubations. Polysaccharidase activities of all enzyme preparations were remarkably stable in ruminal fluid taken after feeding. Ruminal fluid obtained before feeding inactivated the polysaccharidases in preparations B and D to a greater extent than ruminal fluid obtained after feeding. Cellulase and cellulose 1,4-beta-cellobiosidase activities were the least stable, declining (P < 0.05) by 35 and 60% for preparations B and D, respectively. Xylanase activity of preparation D decreased (P < 0.05) by up to 30% after 6 h of incubation, whereas beta-glucanase activity was not affected. The ability to degrade exogenous enzymes also differed among cows (P < 0.05). Pepsin and acid (pH 3.0) did not affect polysaccharidases in preparation B but decreased glycosidase activities by 10 to 15% (P < 0.05) after 1 h of incubation. Pancreatin, at the maximum concentration used, inactivated cellulase, cellulose 1,4-beta-cellobiosidase, and xylanase activities at a rate of 0.55, 1, and 0.45%/min, respectively. beta-Glucosidase and beta-xylosidase activities decreased by 1 and 0.75%/min, respectively. Partial proteolysis of cellulase, cellulose 1,4-beta-cellobiosidase, and xylanase by pancreatin produced a transient increase in activity. This twofold increase for cellulase and fourfold increase for cellulose 1,4-beta-cellobiosidase was directly proportional to pancreatin concentration. These results suggest that the enzyme feed additives tested were stable in the rumen of animals after feeding. Exogenous enzymes are likely to be more susceptible to the host gastrointestinal proteases in the abomasum and intestines than to ruminal proteases. However, exogenous polysaccharidases may survive for a considerable period of time in the small intestine and they probably maintain activity against target substrates in this environment.

Animal Feed↗

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro.

A series of in vitro studies was conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 microL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20 degrees C. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 microL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 microL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Animal Feed↗

Fungal colonisation in digital silicone rubber prostheses.

The fungal discolouration of silicone rubber prostheses is reported in four cases. In two of the cases, the discolouration was caused by the fungus Candida tropicalis. In the other two cases, two different fungal organisms, namely Trichoderma sp. and Scedosporium prolificans were incriminated. The non-porous silicone rubber layers create an enclosed environment in the suction cup of the prosthesis and preclude ventilation at the prosthesis-stump interface. The moisture as a result of sweat and body warmth in the stump assists fungal growth. Residual salts from the sweat, sebum from sebaceous glands and the residues from petroleum jelly (Vaseline) applied to facilitate donning, can adhere to the surfaces of the prosthesis and provide the nutrients for fungal growth. Prolonged continuous usages of the prosthesis, the presence of sweaty palms in the users, donning the prosthesis during manual physical activities which induce perspiration, washing of hands with the prosthesis on and warm humid climatic conditions have been identified as factors predisposing the prosthesis to fungal colonisation. The fungal growth caused a black discolouration and marred the aesthetic quality of the prostheses. As a preventative measure, daily immersion of the prostheses in denture cleaner such as benzalkonium chloride, or water at 60 degrees C for 15 minutes, or decontamination with 70% alcohol is recommended. Prior cleaning to remove organic matter before decontamination is emphasised.

Biocompatible Materials↗