Pectinase production by solid fermentation from Aspergillus niger by a new prescription experiment

The Ordos Plateau in China is covered with up to 300,000 ha of peashrub (Caragana) which is the dominant natural vegetation and ideal for fodder production. To exploit peashrub fodder, it is crucially important to optimize the culture conditions, especially culture substrate to produce pectinase complex. In this study, a new prescription process was developed. The process, based on a uniform experimental design, first optimizes the solid substrate and second, after incubation, applies two different temperature treatments (30 degrees C for the first 30 h and 23 degrees C for the second 42 h) in the fermentation process. A multivariate regression analysis is applied to a number of independent variables (water, wheat bran, rice dextrose, ammonium sulfate, and Tween 80) to develop a predictive model of pectinase activity. A second-degree polynomial model is developed which accounts for an excellent proportion of the explained variation (R-2 = 97.7%). Using unconstrained mathematical programming, an optimized substrate prescription for pectinase production is subsequently developed. The mathematical analysis revealed that the optimal formula for pectinase production from Aspergillus niger by solid fermentation under the conditions of natural aeration, natural substrate pH (about 6.5), and environmental humidity of 60% is rice dextrose 8%, wheat bran 24%, ammonium sulfate ((NH4)(2)SO4) 6%, and water 61%. Tween 80 was found to have a negative effect on the production of pectinase in solid substrate. With this substrate prescription, pectinase produced by solid fermentation of A. niger reached 36.3IU/(gDM). Goats fed on the pectinase complex obtain an incremental increase of 0.47 kg day(-1) during the initial 25 days of feeding, which is a very promising new feeding prospect for the local peashrub. It is concluded that the new formula may be very useful for the sustainable development of and and semiarid pastures such as those of the Ordos Plateau. (c) 2005 Elsevier Inc. All rights reserved.

Production of polysaccharidases in different carbon sources by Leucoagaricus gongylophorus Moller (Singer), the symbiotic fungus of the leaf-cutting ant Atta sexdens linnaeus

Leucoagaricus gongylophorus, the fungus cultured by the leaf-cutting ant Atta sexdens, produces polysaccharidases that degrade leaf components by generating nutrients believed to be essential for ant nutrition. We evaluated pectinase, amylase, xylanase, and cellulase production by L. gongylophorus in laboratory cultures and found that polysaccharidases are produced during fungal growth on pectin, starch, cellulose, xylan, or glucose but not cellulase, whose production is inhibited during fungal growth on xylan. Pectin was the carbon source that best stimulated the production of enzymes, which showed that pectinase had the highest production activity of all of the carbon sources tested, indicating that the presence of pectin and the production of pectinase are key features for symbiotic nutrition on plant material. During growth on starch and cellulose, polysaccharidase production level was intermediate, although during growth on xylan and glucose, enzyme production was very low. We propose a possible profile of polysaccharide degradation inside the nest, where the fungus is cultured on the foliar substrate.

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.

Diversity of endophytic enterobacteria associated with different host plants

Fifty-three endophytic enterobacteria isolates from citrus, cocoa, eucalyptus, soybean, and sugar cane were evaluated for susceptibility to the antibiotics ampicillin and kanamycin, and cellulase production. Susceptibility was found on both tested antibiotics. However, in the case of ampicillin susceptibility changed according to the host plant, while all isolates were susceptible to kanamycin. Cellulase production also changed according to host plants. The diversity of these. isolates was estimated by employing BOX-PCR genomic fingerprints and 16S rDNA sequencing. In total, twenty-three distinct operational taxonomic units (OTUs) were identified by employing a criterion of 60% fingerprint similarity as a surrogate for an OTU. The 23 OTUs belong to the Pantoea and Enterobacter genera, while their high diversity could be an indication of paraphyletic classification. Isolates representing nine different OTUs belong to Pantoea agglomerans, P. ananatis, P, stewartii, Enterobacter sp., and E. homaechei. The results of this study suggest that plant species may select endophytic bacterial genotypes. It has also become apparent that a review of the Pantoea/Enterobacter genera may be necessary.

Biopulping of Paddy Straw by Pleurotus eous

Biopulping being less energy intensive, inexpensive and causing lesser pollution, can be a viable alternative to chemical and mechanical pulping in paper and pulp industry. In view of shrinking forest reserves, agricultural residues are considered as an alternative raw material for making paper and board. By suitable treatment agriwaste can be converted into substrate for mushroom cultivation. Mushrooms of Pleurotus sp. can preferentially remove lignin from agriwaste with limited degradation to cellulose. The present study examines utilization of Pleurotus eous for biopulping of paddy straw by solid substrate fermentation. SMS, the mushroom growing medium that results from cultivation process, is a good source of fibre and can be pulped easily. Ligninases present in SMS were able to reduce lignin content to nearly half the initial amount by 21st day of cultivation. Highest cellulose content (% dry weight) was observed on 21st day, while cellulase production commenced from 28th day of cultivation. SEM images revealed that SMS fibres are still associated with non-cellulosic materials when compared to chemically (20% w/v NaOH) extracted fibres.

Estudo da influência de pré-tratamentos de dois resíduos lignocelulósicos (bagaço do pedúnculo de caju e casca de coco) utilizados como substrato na indução à síntese de enzimas celulolíticas

Nowadays generation ethanol second, that t is obtained from fermentation of sugars of hydrolyses of cellulose, is gaining attention worldwide as a viable alternative to petroleum mainly for being a renewable resource. The increase of first generation ethanol production i.e. that obtained from sugar-cane molasses could lead to a reduction of lands sustainable for crops and food production. However, second generation ethanol needs technologic pathway for reduce the bottlenecks as production of enzymes to hydrolysis the cellulose to glucose i.e. the cellulases as well as the development of efficient biomass pretreatment and of low-cost. In this work Trichoderma reesei ATCC 2768 was cultivated under submerged fermentation to produce cellulases using as substrates waste of lignocellulosic material such as cashew apple bagasse as well as coconut bagasse with and without pretreatment. For pretreatment the bagasses were treated with 1 M NaOH and by explosion at high pressure. Enzyme production was carried out in shaker (temperature of 27ºC, 150 rpm and initial medium pH of 4.8). Results showed that T.reesei ATCC 2768 showed the higher cellulase production when the cashew apple bagasse was treated with 1M NaOH (2.160 UI/mL of CMCase and 0.215 UI/mL of FPase), in which the conversion of cellulose, in terms of total reducing sugars, was of 98.38%, when compared to pretreatment by explosion at high pressure (0.853 UI/mL of CMCase and 0.172 UI/mL of Fpase) showing a conversion of 47.39% of total reducing sugars. Cellulase production is lower for the medium containing coconut bagasse treated with 1M NaOH (0.480 UI/mL of CMcase and 0.073 UI/mL of FPase), giving a conversion of 49.5% in terms of total reducing sugars. Cashew apple bagasse without pretreatment showed cellulase activities lower (0.535 UI/mL of CMCase and 0,152 UI/mL of FPase) then pretreated bagasse while the coconut bagasse without pretreatment did not show any enzymatic activity. Maximum cell concentration was obtained using cashew nut bagasse as well as coconut shell bagasse treated with 1M NaOH, with 2.92 g/L and 1.97 g/L, respectively. These were higher than for the experiments in which the substrates were treated by explosion at high pressure, 1.93 g/L and 1.17 g/L. Cashew apple is a potential inducer for cellulolytic enzymes synthysis showing better results than coconut bagasse. Pretreatment improves the process for the cellulolytic enzyme production

Palma forrageira (Opuntia ficus indica e Nopalea cochenillifera) como matéria-prima para produção de etanol celulósico e enzimas celulolíticas

The need for new sources of energy and the concern about the environment have pushed the search for renewable energy sources such as ethanol. The use of lignocellulosic biomass as substrate appears as an important alternative because of the abundance of this raw material and for it does not compete with food production. However, the process still meets difficulties of implementation, including the cost for production of enzymes that degrade cellulose to fermentable sugars. The aim of this study was to evaluate the behavior of the species of cactus pear Opuntia ficus indica and Nopalea cochenillifera, commonly found in northeastern Brazil, as raw materials for the production of: 1) cellulosic ethanol by simultaneous saccharification and fermentation (SSF) process, using two different strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11), and 2) cellulolytic enzymes by semi-solid state fermentation (SSSF) using the filamentous fungus Penicillium chrysogenum. Before alcoholic fermentation process, the material was conditioned and pretreated by three different strategies: alkaline hydrogen peroxide, alkaline using NaOH and acid using H2SO4 followed by alkaline delignification with NaOH. Analysis of composition, crystallinity and enzymatic digestibility were carried out with the material before and after pretreatment. In addition, scanning electron microscopy images were used to compare qualitatively the material and observe the effects of pretreatments. An experimental design 2² with triplicate at the central point was used to evaluate the influence of temperature (30, 40 and 45 °C) and the initial charge of substrate (3, 4 and 5% cellulose) in the SSF process using the material obtained through the best condition and testing both strains of S. cerevisiae, one of them flocculent (LNF CA-11). For cellulase production, the filamentous fungus P. chrysogenum was tested with N. cochenillifera in the raw condition (without pretreatment) and pretrated hydrothermically, varying the pH of the fermentative medium (3, 5 and 7). The characterization of cactus pear resulted in 31.55% cellulose, 17.12% hemicellulose and 10.25% lignin for N. cochenillifera and 34.86% cellulose, 19.97% hemicellulose and 15.72% lignin for O. ficus indica. It has also been determined, to N. cochenillifera and O. ficus indica, the content of pectin (5.44% and 5.55% of calcium pectate, respectively), extractives (26.90% and 9.69%, respectively) and ashes (5.40% and 5.95%). Pretreatment using alkaline hydrogen peroxide resulted in the best cellulose recovery results (86.16% for N. cochenillifera and 93.59% for O. ficus indica) and delignification (48.79% and 23.84% for N. cochenillifera and O. ficus indica, respectively). This pretreatment was also the only one which did not increase the crystallinity index of the samples, in the case of O. ficus indica. However, when analyzing the enzymatic digestibility of cellulose, alkali pretreatment was the one which showed the best yields and therefore it was chosen for the tests in SSF. The experiments showed higher yield of conversion of cellulose to ethanol by PE-2 strain using the pretreated N. cochenillifera (93.81%) at 40 °C using 4% initial charge of cellulose. N. cochenillifera gave better yields than O. ficus indica and PE-2 strain showed better performance than CA-11. N. cochenillifera proved to be a substrate that can be used in the SSSF for enzymes production, reaching values of 1.00 U/g of CMCase and 0.85 FPU/g. The pretreatment was not effective to increase the enzymatic activity values

Cellulolytic activity of wild type and mutant Trichoderma pseudokoningii

Strains of Trichoderma pseudokoningii are promising objects for genetic studies and cellulase production. Auxotrophic mutants with deficiencies in the biosynthesis of aminoacids, nucleotides and vitamins (up to five markers) in addition to morphological aspects like conidial colour were obtained from two strains of double auxotrophic mutants using UV radiation. In order to compare the cellulolytic capabilities of the T. pseudokoningii (wild type strain), some of its mutants and T. reesei QM9414 we performed semiquantitative cellulase assays and quantitative determination of the enzymes exoglucanase and endoglucanase. The semiquantitative test showed that the strains with minimal mycelial growth rate were better producers. Both tests revealed that two of the studied mutants, TG3 and TG4 presented a yield higher than the wild type, reaching 30% more exoglucanase and 70% more endoglucanase. These results indicate that the wild type was improved for cellulase production. Highly significant values of correlation were found for exoglucanase and endoglucanase activities, suggesting that these enzymes may be co-regulated in T. pseudokoningii.

Produção e avaliação de enzimas fibrolíticas exógenas na ensilagem de milho

Pós-graduação em Zootecnia - FCAV

Sugarcane Growth Promotion by the Endophytic Bacterium Pantoea agglomerans 33.1

The promotion of sugarcane growth by the endophytic Pantoea agglomerans strain 33.1 was studied under gnotobiotic and greenhouse conditions. The green fluorescent protein (GFP)-tagged strain P. agglomerans 33.1: pNKGFP was monitored in vitro in sugarcane plants by microscopy, reisolation, and quantitative PCR (qPCR). Using qPCR and reisolation 4 and 15 days after inoculation, we observed that GFP-tagged strains reached similar density levels both in the rhizosphere and inside the roots and aerial plant tissues. Microscopic analysis was performed at 5, 10, and 18 days after inoculation. Under greenhouse conditions, P. agglomerans 33.1-inoculated sugarcane plants presented more dry mass 30 days after inoculation. Cross-colonization was confirmed by reisolation of the GFP-tagged strain. These data demonstrate that 33.1:pNKGFP is a superior colonizer of sugarcane due to its ability to colonize a number of different plant parts. The growth promotion observed in colonized plants may be related to the ability of P. agglomerans 33.1 to synthesize indoleacetic acid and solubilize phosphate. Additionally, this strain may trigger chitinase and cellulase production by plant roots, suggesting the induction of a plant defense system. However, levels of indigenous bacterial colonization did not vary between inoculated and noninoculated sugarcane plants under greenhouse conditions, suggesting that the presence of P. agglomerans 33.1 has no effect on these communities. In this study, different techniques were used to monitor 33.1:pNKGFP during sugarcane cross-colonization, and our results suggested that this plant growth promoter could be used with other crops. The interaction between sugarcane and P. agglomerans 33.1 has important benefits that promote the plant's growth and fitness.

Proceedings of the Seventh Annual Biochemical Engineering Symposium

This is the seventh in a series of symposia devoted to talks by students on their biochemical engineering research. The first four meetings were held alternately at Kansas State University and the University of Nebraska–Lincoln, with participants from those two schools. The next two took place at Kansas State and then in conjunction with the 8lst American Institute of Chemical Engineers National Meeting in Kansas City, with attendees from Kansas State and Iowa State Universities. This meeting, at Iowa State, was the first to include participation from the University of Missouri–Columbia. Contents"Properties of Soluble and In:anoblized Dextransucrase," Hossein Kaboli and Yah Eric Chen, Iowa State University "Growth of Lipid-Producing Organisms on Formic and Acetic Acid-Containing Waste Waters," Lin-Chang Chiang, University of Missouri–Columbia "Design of an Automated Alkaline Copper Reducing Sugar Assay," Alfred R. Fratzke and James R. Frederick, Iowa State University "Determination of Oxygen Transfer Coefficients in Hydrocarbon Fermentations Using a Material Balance Method," Sarafin N. Sanchez and J. R. Gutierrez, Kansas State University "Oxygen Transfer Characteristics in One Stage and Two Stage Air-Lift Towers," Mark E. Orazem, Kansas State University "A Comparison of Biological Digestibility Tests for Cellulose," Dou-Houng Hwang, University of Missouri–Columbia "Mechanism of Enzymatic Hydrolysis of Cellulose," L. T. Fan, Yong-Hyun Lee, and Liang-Shih Fan, Kansas State University "Purification of Xylan-Hydrolyzing Enzymes," James R. Frederick, Alfred R. Fratzke, and Mary M. Frederick, Iowa State University "Cellulase Production from Bagasse and Pith," A. Ferrer, Y. Alroy, and I. Brito, Kansas State University

Production and characterization of cellulase-free xylanase from Trichoderma inhamatum

The production of extracellular cellulase-free xylanase from Trichoderma inhamatum was evaluated in liquid Vogel medium with different carbon sources as natural substrates and agricultural or agro-industrial wastes. Optimal production of 244.02 U/mL was obtained with xylan as carbon source, pH 6.0 at 25 degrees C, 120 rpm, and 60-h time culture. Optimal conditions for enzyme activity were 50 degrees C and pH 5.5. Thermal stability of T. inhamatum xylanolytic complex expressed as T(1/2) was 2.2 h at 40 degrees C and 2 min at 50 degrees C. The pH stability was high from 4.0 to 11.0.These results indicate possible employment of such enzymatic complex in some industrial processes which require activity in acid pH, wide-ranging pH stability, and cellulase activity absence.

PRODUCTION OF MICROBIAL ALKALINE CELLULASE AND STUDIES OF THEIR CHARACTERISTICS

In order to obtain cellulases that improve the detergency of laundry detergent products, two alkalophilic microorganims, Bacillus sp B38-2 and Streptomyces sp S36-2, were isolated from soil and compost by incubating samples in enrichment culture medium containing CMC and Na2CO3 at pH9.6. It was found that they secrete a constitutive extracellular alkaline carboxymethyl cellulase (CMCase) in high quantity. The maximum enzyme activity was observed between 48hr to 72 hr at 30-degrees-C for the Streptomyces and between 72hr to 96hr at 35-degrees-C for the Bacillus. The optimum pH and temperature of the crude enzyme activities ranged from 6.0 to 7.0 at 55-degrees-C for the Streptomyces and 7.0 to 8.0 at 60-degrees-C for the Bacillus. Two crude CMCases activities were termostable at 45-degrees-C for 1hr and the both crude enzyme activities of the Bacillus as of the Streptomyces were stable at pH 5.0 to 9.0 after pH treatments in various buffer solutions at 30-degrees-C for 24hr.

Production of a xylose-stimulated beta-glucosidase and a cellulase-free thermostable xylanase by the thermophilic fungus Humicola brevis var. thermoidea under solid state fermentation

Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of beta-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 +/- A 411.2 U g(-1), while beta-glucosidase production was increased about 2.6-fold, reaching 20.7 +/- A 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis beta-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for beta-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The beta-glucosidase maintained about 95 % of its activity after 26 h in water at 55 A degrees C, with half-lives of 15.7 h at 60 A degrees C and 5.1 h at 65 A degrees C. The presence of xylose during heat treatment at 65 A degrees C protected beta-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 A degrees C. Xylose stimulated beta-glucosidase activity up to 1.7-fold, at 200 mmol L-1. The notable features of both xylanase and beta-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.