Enhanced production of cellulases byPenicillium citrinum in solid state fermentation of cellulosic residue

Penicillium citrinum, using rice husks in a solid state fermentation, produced maximum cellulase yields (37 Units/g) after 12 days with a cellulose utilization of more than 70%. Enzyme yields were three times higher than in shake-flask cultures.     

Cellulase and protein production by Chaetomium cellulolyticum strains grown on cellulosic substrates

Summary Fermentation with Chaetomium cellulolyticum was carried out on media containing either Avicel cellulose or newspaper. Production of free cellulolytic enzymes, cellulose degradation and the formation of cell protein were studied with the original strain and a mutant strain.     

典型产纤维小体梭菌的遗传改造及其在纤维素乙醇中的应用研究进展简

以解纤维梭菌（ Clostridium cellulolyticum）和热纤梭菌（ Clostridium thermocellum）为代表的产纤维小体梭菌可以直接完成从木质纤维素原料到乙醇的生物转化,是用于通过整合生物加工技术生产纤维素乙醇的优良候选菌株。然而,这些产纤维小体梭菌的纤维素降解效率及乙醇产量尚不能满足工业化生产的要求,其遗传改造技术的不成熟严重制约了通过定向代谢工程改造提高生产性能的进程。针对这些典型的产纤维小体菌株,各国科学家近年来在基于二类内含子的嗜中温及嗜高温遗传改造平台建立方面取得了较大突破,并通过靶向代谢工程改造,显著提高纤维素乙醇的产量。笔者对这些前期研究工作以及国内外相关研究成果进行系统的总结,并对构建的遗传改造工具的应用前景进行展望。     

Formation of cellulases by co-culturing ofTrichoderma reesei andAspergillus niger on cellulosic waste

A cellulase system possessing high hydrolytic and -glucosidase activity was obtained by co-culturingTrichoderma reesei andAspergillus niger by a new approach using semi-solid fermentation of lignocellulosic materials. Various types of pretreatments were used for making the cellulose easily accessible to enzymatic attack. The optimal water content for maximum activity of the mixed fermentation was investigated. A more concentrated enzyme preparation could be obtained by semi-solid state fermentation than by conventional submerged fermentation.     

Evaluation of holocellulase production by plant-degrading fungi grown on agro-industrial residues

Agaricus brasiliensis CS1, Pleurotus ostreatus H1 and Aspergillus flavus produced holocellulases when grown in solid and submerged liquid cultures containing agro-industrial residues, including sugar cane bagasse and dirty cotton residue, as substrates. These isolates proved to be efficient producers of holocellulases under the conditions used in this screening. Bromatological analysis of agro-industrial residues showed differences in protein, fiber, hemicellulose, cellulose and lignin content. Maximal holocellulase activity (hemicellulase, cellulase and pectinase) was obtained using solid-state cultivation with 10% substrate concentration. In this case, remarkably high levels of xylanase and polygalacturonase activity (4,008 and 4,548 IU/l, respectively) were produced by A. flavus when grown in media containing corn residue, followed by P. ostreatus H1 with IU/l values of 1,900 and 3,965 when cultivated on 5% and 10% sugar cane bagasse, respectively. A. brasiliensis CS1 showed the highest reducing sugar yield (11.640 mg/ml) when grown on medium containing sugar cane bagasse. A. brasiliensis was also the most efficient producer of protein, except when cultivated on dirty cotton residue, which induced maximal production in A. flavus. Comparison of enzymatic hydrolysis of sugar cane bagasse and dirty cotton residue by crude extracts of A. brasiliensis CS1, P. ostreatus H1 and A. flavus showed that the best reducing sugar yield was achieved using sugar cane bagasse as a substrate.     

The potential of cellulases and cellulosomes for cellulosic waste management

Lignocellulose is the most abundant plant cell wall component of the biosphere and the most voluminous waste produced by our society. Fortunately, it is not toxic or directly harmful, but our major waste disposal facilities--the landfills--are rapidly filling up with few realistic alternatives. Because cellulose is pure glucose, its conversion to fine products or fuels has remained a romantic and popular notion; however, the heterogeneous and recalcitrant nature of cellulosic waste presents a major obstacle for conventional conversion processes. One paradigm for the conversion of biomass to products in nature relies on a multienzyme complex, the cellulosome. Microbes that produce cellulosomes convert lignocelluose to microbial cell mass and products (e.g. ethanol) simultaneously. The combination of designer cellulosomes with novel production concepts could in the future provide the breakthroughs necessary for economical conversion of cellulosic biomass to biofuels.     

Some comparisons of cellulases from two different strains of aspergillus fumigatus

Michaelis-Menten kinetics for exoglucanase, endoglucanase and β-glucosidase activities from two different strains of Aspergillus fumigatus were compared in the absence and the presence of ammonium ions. Inhibitory effects, evident in only one strain, were quantified, suggesting non-competitive inhibition for endoglucanase and β-glucosidase, but competitive inhibition of exocellulase. Possible reasons are discussed.     

Enhanced root production in Haplopappus gracilis grown under spaceflight conditions

The production and growth of roots in two aseptically maintained clonal populations of Haplopappus gracilis (family Compositae), each with a distinctive pattern of root production, were studied after they had been exposed to space for 5 days aboard a NASA Space Shuttle. Total root production of both populations was 67-95% greater when compared with their Earth-grown controls. Roots were generated: (1) laterally from pre-formed roots, the tips of which had been severed at the time of plantlet insertion into a "horticultural foam" substrate supplied with a nutrient solution; (2) adventitiously from the basal or cut-end portion of shoots; (3) de novo, i.e. from primordial which were non-existent at the outset of the experiment. Roots grew in all directions in space but were uniformly positively gravitropic in ground controls. In space and on Earth, both clonal populations maintained their clone-specific root formation and growth characteristics and produced an equivalent amount of tissue when compared to each other. As on Earth, and as expected, there were fewer and shorter roots on plantlets that formed floral buds. The significance of altered moisture distribution in the "horticultural foam" substrate in space for root growth and the significance of our findings for growing plants in altered gravity environments are discussed.     

Production of cellulases by thermophilic fungi grown on Leptochloa fusca straw

Seven indigenous thermophilic fungi were screened for cellulase and xylanase production when grown on Leptochloa fusca (kallar grass) straw. Aspergillus fumigatus produced the highest activities of 0.4, 2.5, 3.5 and 0.14 U/ml of filter paper cellulase, CM-cellulase, xylanase and -xylosidase, respectively. Sporotrichum thermophile produced 0.47 -glucosidase/ml. Chaetomium thermophile, Humicola grisea and Torula thermophila had lower activities than the other thermophilic fungi.The authors are with the National Institute for Biotechnology & Genetic Engineering, P.O. Box 577, Faisalabad, Pakistan.     

Joint effect of nitrogen and phosphorus concentrations on citric acid production by different strains of Aspergillus niger grown on an effluent

Summary The joint effects of the initial concentration of N and P on the production of citric acid by 11 strains of Aspergillus niger on an effluent are studied by means of orthogonal full-factorial 2² designs. The results allow the definition of a series of empirical equations describing well differentiated types of behaviour. This demonstrates the usefulness of the procedure followed in order to establish selection criteria for the more suitable strains, to determine the tendencies that permit approaching optimum production and evaluate the viability of different culture options characterized by discontinuous feeding of one or another nutrient.     

Lignin peroxidase production by strains of Phanerochaete chrysosporium grown on glycerol

Summary Lignin peroxidase production by several strains of Phanerochaete chrysosporium was determined during growth on glycerol under conditions of nitrogen sufficiency. Fungal strains which grew poorest on glycerol produced the highest titres of lignin peroxidase whereas enzyme levels were much lower when marginally greater biomass values were recorded. In the case of P. chrysosporium strain INA-12, the nature of the nitrogen source had a pronounced effect on both growth and enzyme production. Highest biomass values were obtained when l-glutamate or l-glutamine served as the major nitrogen source but enzyme synthesis was normally repressed completely. Lignin peroxidase activity in this strain was maximal when the initial pH of the culture medium was adjusted to pH 5.0.     

Enhanced polygalacturonase production by Aspergillus niger NRRL-364 grown on supplemented citrus pectin

Enhanced levels of extracellular polygalacturonase activity were obtained when Aspergillus niger NRRL-364 was grown on pectic substances as sole carbon sources in a submerged culture. Among the factors affecting enzyme production those of carbon source concentration, nitrogen source, initial pH and time of cultivation were found to be the most important ones. Under optimum growth and activity conditions yields as high as 14.5 U (measured as reducing groups) ml^-1 of growth medium were obtained, comparing favourably with those reported for fungi grown under similar conditions and used in food processes.     

Hydrolytic enzyme(s) production in Micrococcus roseus growing on different cellulosic substrates

Micrococcus roseus (G12) isolated from higher termite Odontotermes obesus gut exhibited cellulose digesting properties. A lignocellulosic substrate, rice husk induced endoglucanase, β-glucosidase, β-xylanase and β-xylosidase production. Besides rice husk, CMC also induced endoglucanase production. β-Glucosidase activity was quite pronounced when rice husk was supplemented with CMC or cellobiose. Both β-xylanase and β-xylosidase activities could be induced by xylan as well as xylobiose, whereas CMC induced partial activity. Endoglucanase and β-xylanase enzymes were secreted into the culture medium, whereas β-glucosidase and β-xylosidase activities were intracellular in nature. Enzyme production was subject to end product inhibition. The extracellular enzyme(s) possessed the potential to saccharify rice husk, xylan and CMC to reducing sugars.     

Enhanced production of plasmid DNA by engineered Escherichia coli strains

Escherichia coli strains VH33 (PTS? GalP? strain displaying a strongly reduced overflow metabolism) and VH34 (additionally lacking the pyruvate kinase A) were evaluated for the production of a plasmid DNA (pDNA) vaccine. The parent (W3110) and mutant strains were cultured using 10 g of glucose/L. While the specific growth rates of the three strains were similar, they presented differences in the accumulation of acetate. W3110 accumulated up to 4 g/L of acetate, VH33 produced 1.4 g/L, and VH34 only 0.78 g/L. VH33 and VH34 produced 76% and 300% more pDNA than W3110. Moreover, VH34 demanded 33% less oxygen than VH33 and W3110, which can be advantageous for large-scale applications.     

True cellulase production by Clostridium thermocellum grown on different carbon sources

Summary Clostridium thermocellum produced different levels of true cellulase (Avicelase) depending on the carbon source used for growth. In defined medium with fructose, the cellulase titer was seven times higher than with cells growing on cellobiose and four times higher than cells growing with glucose. During the lag phase on fructose, the differences were even more dramatic, i.e. 60 times higher than in cells growing on cellobiose and 40 times that of cells lagging or growing in glucose. In an attempt to detect factors that might contribute to these differences, we considered intracellular ATP, chemical potential (pH), electrical potential (Y), proton motive force (p), growth rate, and rates of uptake of inorganic phosphate and sugars. We noted a direct correlation between cellulase production and intracellular ATP levels and an inverse relationship of cellulase production with Y and p values. It thus appears that cellulase is best produced by cells high in ATP and low in Dp and its electrical component DY. There was no obvious relationship between the cellulase titer and the other parameters. Although the physiological significance of such correlations is unknown, the data suggest that further investigation is warranted.     

Glycosidases of the rumen anaerobic fungus Neocallimastix frontalis grown on cellulosic substrates

The rumen anaerobic fungus Neocallimastix frontalis was grown on cellulosic substrates, and the cellular distribution and types of glycosidases produced by the organism were studied. Fungal cultures were fractionated into extracellular, insoluble (membrane), and intracellular fractions and assayed for glycosidase activity by using Avicel, carboxymethylcellulose, xylan, starch, polygalacturonic acid, and the p-nitrophenyl derivatives of galactose, glucose, and xylose as substrates. Enzymic activity was highest in the extracellular fraction; however, the membrane fraction also displayed appreciable activity. The intracellular fraction was inactive towards all substrates. Polygalacturonic acid was the only substrate not hydrolyzed by the active fractions, indicating that pectinase was absent. The results show that N. frontalis, a common rumen anaerobic fungus, produces enzymes for degrading cellulose and hemicellulose, key components of plant fiber.