Evolution of microbial community diversity and enzymatic activity during composting

The composting of organic material is dependent on microbial activity. However, the dynamics of the microbial community during the composting process remain obscure. Here, denaturing gradient gel electrophoresis of 16S rDNA amplicons in a chicken manure-based compost was applied to characterize the components of the microbial community during the composting process. In addition, the activity of key microbial enzymes was monitored. Arcobacter spp. and Marinospirillum spp. were the dominant species prior to composting, whereas Thermotogae spp. became more strongly represented as the composting process proceeded. Bacillus and Cohnella spp. were featured at various phases. Correlation analysis showed that the diversity of the microbial community was positively correlated with the compost pH, its total nitrogen level, its carbon-to-nitrogen ratio and the activity of protease, and negatively correlated with its organic carbon content and seed germination indices.     

Microbial diversity during Rotary Drum and Windrow Pile composting

This study investigates the prevailing microbial communities during the composting of vegetable waste, cattle manure and saw dust, in a household (250 l) batch scale Rotary Drum composter and Windrow Pile. Physico-chemical parameters were analyzed to study the organic matter transformations. Total organic matter reduced from 63.8% to 36.2% in rotary drum and 39.6% in windrow pile composting. The C/N ratio decreased from 26.52 to 8.89 and 14.33 in rotary drum and windrow pile composting. The indigenous population of total heterotrophic bacteria decreased in rotary drum and windrow pile composting after 20 days. However, total fungal load initially increased within initial 4 days, then subsequently reduced in final composts. The average number of fecal coliforms and fecal Streptococci showed decrement with time, in both composting systems. Escherichia coli and Salmonella species number deduced during the study. Composting cycle started with Gram positive rods but ended up with the dominance of Gram negative bacilli shaped bacteria. Transformation of organic compounds during the biodegradation of organic waste, difference in the utilization of nutrients (organic matter) by the different group of microbes and high temperature could be cited as a possible reason of the above changes. Scanning electron microscopy has been used to obtain the surface structures of the cultured mycoflora. Results of the study revealed that higher diversity of microbes prevailed in rotary drum as compared to windrow pile, yielding more stable and pathogenic free compost in lesser period of composting.     

Application of thermotolerant microorganisms for biofertilizer preparation.

BACKGROUND AND PURPOSE: Intensive agriculture is practised in Taiwan, and compost application is very popular as a means of improving the soil physical properties and supplying plant nutrition. We tested the potential of inoculation with thermotolerant microorganisms to shorten the maturity and improve the quality of biofertilizer prepared by composting. METHODS: Thermotolerant microorganisms were isolated from compost and reinoculated for the preparation of biofertilizer. The physical, chemical and biological properties of the biofertilizer were determined during composting. The effects of biofertilizer application on the growth and yield of rape were also studied. RESULTS: Among 3823 colonies of thermotolerant microorganisms, Streptomyces thermonitrificans NTU-88, Streptococcus sp. NTU-130 and Aspergillus fumigatus NTU-132 exhibited high growth rates and cellulolytic and proteolytic activities. When a mixture of rice straw and swine manure were inoculated with these isolates and composted for 61 days, substrate temperature increased initially and then decreased gradually during composting. Substrate pH increased from 7.3 to 8.5. Microbial inoculation enhanced the rate of maturity, and increased the content of ash and total and immobilized nitrogen, improved the germination rate of alfalfa seed, and decreased the content of total organic carbon and the carbon/nitrogen ratio. Biofertilizer application increased the growth and yield of rape. CONCLUSIONS: Inoculation of thermotolerant and thermophilic microorganisms to agricultural waste for biofertilizer preparation enhances the rate of maturity and improves the quality of the resulting biofertilizer. Inoculation of appropriate microorganisms in biofertilizer preparation might be usefully applied to agricultural situations.     

Production of lignocellulolytic enzymes by Trametes gallica and detection of polysaccharide hydrolase and laccase activities in polyacrylamide gels

White rot fungus Trametes gallica was studied for the production of lignocellulolytic enzymes: cellulase, xylanase, laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). The results demonstrated that low-nitrogen (2.2 mM N) and surface stationary cultivation favored production of extracellular MnP. MnP activity reached 118.1 UL(-1) while T. gallica was grown in a low-nitrogen culture containing phenylalanine. However, laccase levels observed in high-nitrogen (22 mM N) agitated cultures were much greater than those seen in low-nitrogen. The N source experiments seemed to reveal that NH4+ plays an important role in inducing MnP and laccase of the fungus. Results showed that T. gallica produces a series of the lignocellulolytic enzymes, and needs high N to produce all the enzymes during solid-state fermentation of wheat straw. This paper also presents a modified zymogram procedure to detect xylanase and laccase of T. gallica in polyacrylamide gel. Xylanase in crude enzyme of T. gallica was displayed by contacting protein gel strips with xylan substrate gels and by staining with iodine. By immersing the protein gel strips in o-tolidine solution, the blue-green zones representing laccase activity were visualized against a colorless background.     

Effects of differing temperature management on development of Actinobacteria populations during composting

Actinobacteria are believed to play a major role in organic matter degradation and humification processes in composts. In this study, the effects of different temperature regimes on the succession of Actinobacteria populations during composting were investigated in a laboratory reactor. Phospholipid fatty acid (PLFA) was used to investigate quantitative changes in the overall microbial biomass and community structure, and in the size of Actinobacteria populations. Qualitative changes were determined using PCR-DGGE (denaturing gradient gel electrophoresis) and sequencing of 16S rRNA genes with Actinobacteria-specific primers. The peak in total microbial biomass was roughly twice as high and delayed in trials where the maximum temperature was 40 degrees C, compared to those where it was 55 or 67 degrees C. There was a shift from members of Corynebacterium, Rhodococcus and Streptomyces at the onset to species of thermotolerant Actinobacteria in the cooling phase, e.g. Saccharomonospora viridis, Thermobifida fusca and Thermobispora bispora. In conclusion, temperature was an important selective factor for the development of Actinobacteria populations in composts, and they constituted a substantial part of the community in the later compost stages.     

Baker's asthma due to xylanase and cellulase without sensitization to alpha-amylase and only weak sensitization to flour

BACKGROUND: The baking additives xylanase and cellulase were described as baking additives causing baker's asthma. It is not known whether monosensitization to these enzymes may occur. METHODS: We present a case report of a baker with work-related asthma evaluated by skin prick test (SPT), enzyme-linked immunosorbent assay (EAST), immunoblot, EAST and immunoblot inhibition, and specific bronchial challenge. Fungal xylanase and alpha-amylase were measured by two-site enzyme immunoassays in products used by the patient at work. RESULTS: Allergy to xylanase and cellulase was demonstrated by SPT, EAST, immunoblot and specific bronchial challenge (for xylanase only). No sensitization to alpha-amylase could be demonstrated, but there was a weak flour allergy as documented by EAST and immunoblot and a positive occupational-type challenge with high concentrations of rye flour. Four baking additives contained measurable amounts of fungal alpha-amylase and xylanase, without a correlation between these enzymes. CONCLUSIONS: We conclude that occupational asthma due to the baking additives xylanase and cellulase may occur without concomitant sensitization to alpha-amylase and only weak sensitization to flour.     

Cloning of two cellulase genes from endophytic Paenibacillus polymyxa GS01 and comparison with cel 44C-man 26A

Endophytic bacteria are acknowledged as a new source of genes, proteins and other biochemical compounds, which are often used in biochemical processes. In this study, Paenibacillus polymyxa GS01 was isolated from the interior of the roots of Korean cultivars of ginseng (Panax ginseng C. A. Meyer). Two cellulase genes, cel 5A and cel 5B, were cloned from GS01, and encode 334 aa and 573 aa proteins, respectively. Cel5A and Cel5B each contain a glycosyl hydrolase family 5 (GH5) catalytic domain. The molecular mass of Cel5A and Cel5B were estimated to be 33 kDa and 61 kDa, respectively, by CMC-SDS-PAGE. When purified from Escherichia coli Cel5A and Cel5B both displayed cellulase activity with pH optima of 7.0 and 6.0, respectively and shared a temperature optimum of 50 degrees C. Neither enzyme had detectable xylanase, lichenase, or mannase activity, in contrast to the multifunctional Cel44C-Man26A enzyme of P. polymyxa which displays cellulase, xylanase, lichenase and mannanase activities. However, Cel5A and Cel5B exhibited higher specific cellulase activity than Cel44C-Man26A (120% and 140%, respectively). Cel5A and Cel5B mutants with alanine substitutions at a conserved glutamic acid in the GH5 domain (Glu 179 of Cel5A and Glu184 of Cel5B) lacked cellulase activity, suggesting that this residue is important for GH5 domain function.     

Allergy from cellulase and xylanase enzymes

Modern biotechnical methods have enabled production of many new types of potentially allergenic proteins. Enzymes have long been known to be respiratory allergens, but relatively few cases of skin allergy have been reported. Here we describe four patients who developed occupational allergic respiratory symptoms, three with bronchial asthma and one with allergic rhinitis, caused by cellulase and/or xylanase enzymes. Each patient also had urticarial symptoms after skin contact with these enzymes. In addition, one of the patients had allergic contact dermatitis from cellulase, and one from xylanase. Allergic contact dermatitis was verified by positive patch tests with the enzymes, and the immediate allergy was revealed by skin prick tests, specific IgE determinations (RAST) and RAST-inhibition tests. All patients had positive RASTs to both cellulase and xylanase. In the RAST inhibition test 20 microliters of cellulase brought about a 94% inhibition, indicating the specificity of the RAST. Xylanase (20 microliters, 5% w/v) gave an 92% inhibition of cellulase RAST, indicating cross-reactivity between cellulase and xylanase. Three patients have been able to continue at their previous places of work, but at different worksites. One of the patients requires continuous medication for asthma and had to change her job because of persistent symptoms.     

Produktion von Xylanase durch Bacillus subtilis

Bacillus subtilis forms a xylanase complex which does not depend on induction by xylane or xylose in the medium. The enzyme production is influenced by the kind of the carbon source, probably due to catabolic repression. The regulation of enzyme formation is complex in nature, because the C/N/P-Relation in the medium, cell mass, and growth phase influence the amount of enzyme in the culture fluid. Maximal enzyme production was achieved at high but balanced amounts of C/N/P, although growth was strongly reprimed under these conditions. A formation of cellulase, aryl-β-xylosidase and aryl-β-glucosidase could not be observed.     

Production of laccase as the sole phenoloxidase by a Brazilian strain of Pleurotus pulmonarius in solid state fermentation

The production of laccase by a Brazilian strain of Pleurotus pulmonarius was studied in solid state fermentation using wheat bran as substrate. Among oxidative and hydrolytic enzymes tested (laccase, aryl alcohol oxidase, lignin peroxidase, Mn peroxidase, xylanase and cellulase), laccase was the main enzyme produced by P. pulmonarius. The most suitable condition for maximum production of laccase (8,600 U/g substrate) was initial moisture content of 75% and 5 days of cultivation at 30 degrees C. The optimum pH and temperature for laccase activity were found to be 6.5 and 50 degrees C, respectively. P. pulmonarius laccase was stable at 50 degrees C for more than 6 hours, and it retained about 73% and 18% of its activity when heated for 1 h at 55 and 60 degrees C, respectively. The enzyme was greatly stable at alkaline pH, but not at acidic pH. The laccase activity appear to be correlated with the ability of crude extract to decolourize several industrial dyes.     

Solid state bioconversion of oil palm biomass for ligninase enzyme production

A laboratory-scale study of bioconversion of local lignocellulosic material, oil palm biomass (OPB) was conducted by evaluating the enzyme production through microbial treatment in solid state bioconversion (SSB). OPB in the form of empty fruit bunches (EFB) was used as a solid substrate and treated with the white-rot fungus, Phanerochaete chrysosporium, to produce ligninase. The results showed that the highest ligninase activity of 400.27 U/liter was obtained at day 12 of fermentation. While the optimum study indicated the enzyme production of 1472.8 U/liter with moisture content of 50%, 578.7 U/liter with 10% v/w of inoculum size, and 721.8 U/liter with co-substrate concentration of 1% (w/w) at days 9, 9 and 12 of fungal treatment, respectively. The parameters glucosamine and reducing sugar were observed to evaluate the growth and substrate utilization in the experiment.     

Haloferax sulfurifontis GUMFAZ2 producing xylanase‐free cellulase retrieved from Haliclona sp. inhabiting rocky shore of Anjuna,Goa‐India

Salt stable cellulases are implicated in detritic food webs of marine invertebrates for their role in the degradation of cellulosic material. A haloarchaeon, Haloferax sulfurifontis GUMFAZ2 producing cellulase was successfully isolated from marine Haliclona sp., a sponge inhabiting the rocky intertidal region of Anjuna, Goa. The culture produced extracellular xylanase‐free cellulase with a maximum activity of 11.7 U/ml, using carboxymethylcellulose‐Na (CMC‐Na), as a sole source of carbon in 3.5 M NaCl containing medium, pH 7 at 40°C and produced cellobiose and glucose, detectable by thin‐layer chromatography. Nondenaturing polyacrylamide gel electrophoresis of the crude enzyme, revealed a single protein band of 19.6 kDa which on zymographic analysis exhibited cellulase activity while corresponding sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a molecular weight of 46 kDa. Unlike conventional cellulases, this enzyme is active in presence of 5 M NaCl and does not have accompanying xylanase activity, hence can be considered as xylanase‐free cellulase. Such enzymes from haloarchaea offer great potential for biotechnological application because of their stability at high salinity and is therefore worth pursuing.     

Xylanolytic complex from Aspergillus giganteus: production and characterization

An Aspergillus giganteus strain was isolated as an excellent producer of xylanase associated with low levels of cellulase. Optimal xylanase production was obtained in liquid Vogel medium containing xylan as carbon source, pH 6.5 to 7.0, at 25 degrees C and under shaking at 120 rpm during 84 h. Among the several carbon sources tested, higher xylanase production was verified in xylan, xylose, sugar-cane bagasse, wheat bran and corn cob cultures, respectively. Optimal conditions for activity determination were 50 degrees C and pH 6.0. The xylanolytic complex of A. giganteus showed low thermal stability with T(50) of 2 h, 13 min and 1 min when it was incubated at 40, 50 and 60 degrees C, respectively, and high stability from pH 4.5 to 10.5, with the best interval between 7.0 to 7.5. This broad range of stability in alkali pH indicates a potential applicability in some industrial processes, which require such condition. Xylanolytic activity of A. giganteus was totally inhibited by Hg(+2), Cu(+2) and SDS at 10 mM. The analysis of the products from the oat spelts xylan hydrolysis through thin-layer chromatography indicated endoxylanase activity, lack of debranching enzymes and beta-xylosidase activity in assay conditions.     

Microbial biodegradation of cellophane

The microbial biodegradation of cellophane (U.C.B. - Division Sidac) was studied. Preliminary experiments with pure cultures of seven cellulolytic microorganisms (Aspergillus sp., Penicillium sp., Chaetomium crispatum, Ch. globosum, Sclerotium rolfsii and two actinomycetes) revealed that the substrate as such was very recalcitrant, probably due to the occurrence of insoluble coating agents. Therefore, mixed cultures of the above mentioned cellulolytic microorganisms were used as inoculum. The cellophane showed a slow microbial degradation which starts only after 37 days of incubation. This long lag-phase is due to the unaltered presence of the coating agents. However, when the coating agents are extracted with tetrahydrofuran, the biodegradation starts after 10 days, resulting in a biodegradation rate of 85% after 52 days of incubation and a protein content of 30%. The endproduct (30% protein, 60% soluble sugars, 10% residual substrate) will probably be useful as compost.     

Influence of inoculum type,inorganic salt and nitrogen to carbon ratio on sclerotium formation and carotenoid production in surface culture of Penicillium sp. PT95

This study examined the respective effect of inoculum type, inorganic salt and nitrogen to carbon ratio on sclerotium formation and carotenoid production in surface culture of Penicillium sp. PT95. Neither the spore inoculum nor the mycelial pellet inoculum could result in the formation of sclerotium on a modified Czapek agar medium after incubation of 28 days, whereas the inoculum in the form of sclerotium caused the formation of numerous orange, sand-shaped sclerotia after incubation of 14 days. Among four inorganic salts tested, K(2)HPO(4) was more essential to the sclerotium formation and carotenoid production of strain PT95 as compared to KCl, MgSO(4) or FeSO(4). It was also shown that the combination of K(2)HPO(4), KCl and MgSO(4) could produce the best positive cooperation and give the highest sclerotia biomass (782 mg/plate) and carotenoid content in sclerotium (420 microg/g of dry sclerotia) as well as pigment yield (328 microg/plate). The medium containing 0.24 approximately 0.48 g/l sodium nitrate-nitrogen was effective to both the sclerotium formation and carotenoid production of strain PT95 when available maltose-carbon concentrations were at 5.26 approximately 21.05 g/l. The optimal N:C ratio was found to be 1:25.     

Utilization of carbon sources in the rice rhizosphere and nonrhizosphere soils with different long‐term fertilization management

Carbon (C) plays an important role in the interaction between plant and rhizosphere microbial communities, but there is still limited information about how C source utilization soil microbial structure responds to soil fertility changes under the double‐cropping rice (Oryza sativa L.) system in Southern China paddy fields. Therefore, the effects of long‐term (33 years) fertilizer regimes on the characteristics of C utilization in both rhizosphere and nonrhizosphere soils under double‐cropping rice fields in Southern China were investigated by using the metagenome sequencing technology. The experiment began in 1986, and included five fertilizer treatments: without fertilizer input (CK), chemical fertilizer alone (MF), rice straw residue and chemical fertilizer (RF), 30% organic matter, and 70% chemical fertilizer (LOM), and 60% organic matter and 40% chemical fertilizer (HOM). The results showed that the relative abundance of Gemmatimonadetes and Planctomycetia in both the rhizosphere and nonrhizosphere soils was increased by application of rice straw residue and organic manure, whereas the relative abundance of Gammaproteobacteria and Nitrospira was promoted by application of inorganic fertilizers. The largest group of clusters of orthologous groups of proteins categories was “amino acid transport and metabolism” with 16.46% unigenes, followed by “general function prediction only” (12.23%). Regarding the gene ontology categories, biological process were the largest category (174 949, 46.40%), followed by cellular component (126 766, 33.62%), and molecular function (110 353, 29.26%). The principal coordinate analysis indicated that different parts of the root zone were the most important factors affecting the variation of C source utilization bacteria community, and the different fertilizer treatments were the second important factor affecting the variation of C source utilization bacteria community. As a result, the application of fertilization practices had significant effects on the abundance and community composition of C source utilization microbes in paddy soils. The results showed that the combined application of rice straw residue or organic manure with chemical fertilizer practices significantly increases the C source utilization of soil microorganisms in double‐cropping rice fields.     

Zerlegung von hemicellulosereichem biologischem material mit wasser in einem strömungsreaktor

Biological materials, rich in hemicelluloses (birch and pine wood, wheat straw and rice hulls) were disintegrated batchwise in a flow reactor with pure water at elevated temperatures and pressures. The resulting extractives and residual fibre compounds were characterized. Because of rapid removal of carbohydrate based extractives from the hot reaction zone, the degradation of pentosans and hexosans to furfural and hydroxymethylfurfural could be limited to only a small amount. Contrary to normal prehydrolysis conditions employing a sealed batch reactor, temperatures of 190 to 210°C and residence times of goods of 60 min are the optimum. Thus, hemicelluloses are dissolved and isolated with up to more than 90% yield. At temperatures above 225°C severe destruction of hemicelluloses and solvolysis of cellulose starts. Depending on disintegration temperatures, pentosan average molecular weights (M?_w) vary in case of birch wood between 3800 at 150°C and 900 at 225°C. The content of monomers rises with temperature and reaches a maximum of 45% at 225°C. DP _w of residual cellulose is also temperature dependent and varies in case of birch wood and wheat straw between 1500 and 450. Lignin, which is mostly not dissolved, can be removed almost quantitatively by further treatment of the residue with ethanol/water at 250°C, thus allowing the isolation of relatively pure cellulose.     

Bacillus subtilis α-amylase,lichenase, β-mannanase and xylanase: Screening for their induction by agro-industrial byproducts and precipitation by organic solvents

Current research focuses on the utilization of low value agro-industrial byproducts for targeting B. subtilis towards different exoenyzeme lines. This includes α-amylase, β-mannanase, xylanase, and lichenase. As an orientation step, growth and α-amylase activity were monitored in three different nutritional media. A medium which showed low levels of catabolite repression and spore development was selected as a basal fermentation medium. Different patterns of exoenzyme induction were obtained when beet pulp, corn cob, rice husk, wheat bran, and wheat straw were separately used to partially replace the nutrient contents of the selected medium. α-Amylase and lichenase were maximally expressed in the presence of corn cob or beet pulp. On the other hand, considerable levels of the four polysaccharide hydrolyzing enzymes were induced by wheat bran. β-mannanase and xylanase expression responded more significantly towards inducers than α-amylase and lichenase. The effects of five different organic solvents as precipitative agents on these exoenzymes were also studied.     

α-L-rhamnosidase and β-D-glucosidase activities in fungal strains isolated from alkaline soils and their potential in naringin hydrolysis

α-L-Rhamnosidases (EC 3.2.1.40) and β-D-glucosidases (EC 3.2.1.21) obtained from several microbial sources are potential catalysts in food, beverage, and pharmaceutical industries. However, the enzyme preparations currently used have limitations related to the stability and activity of the enzyme as well to their reuse. A microtiter screening was carried out in 55 fungal strains isolated from alkaline soils, to obtain active α-L-rhamnosidases and β-D-glucosidases at pH 9.0. While α-L-rhamnosidase activity was detected in 45% of the strains tested, β-D-glucosidase activity was found only in 27%. Based on the fungal ability to produce α -L-rhamnosidase activity, cultures were supplemented with naringin to study the activities of the enzymes and the potential of the fungal strains on naringin hydrolysis. About 70% of the fungal strains tested increased the activities of both enzymes in the naringin-supplemented cultures as compared to non-supplemented ones. This effect was higher in Acrostalagmus luteo-albus LPSC 427 (15.3 fold) for α-L-rhamnosidase activity and Metarrhizium anisopliae LPSC 996 (51.1 fold) for β-D-glucosidase activity. All the enzyme preparations tested hydrolyzed naringin at pH 9.0, being that obtained from Acremonium murorun LPSC 927 cultures the one which showed highest hydrolysis. Here, different fungal species are reported for the first time for their ability to produce α-L-rhamnosidase and β-D-glucosidase activity at alkaline pH.     

Indirect effects of soluble nitrogen on growth of Ochlerotatus triseriatus larvae in container habitats

We conducted experiments in laboratory microcosms to simulate input of stemflow nutrients and flushing of metabolites in the tree hole habitats of larval Ochlerotatus triseriatus (Say). In the first experiment, we simultaneously examined the effects of nutrient additions (nitrogen, phosphorus, glucose, or combination) and flushing (removal of one-half of water volume replaced by deionized water) on mosquito production. The combination of nutrients had the greatest positive effects on mosquito production, with nitrogen (N) likely accounting for most of the increase in adult emergence and adult mass. Dilution of the nutrient pool via simulated flushing reduced mosquito growth, suggesting that the primary effect of stemflow input was nutrient addition as opposed to dilution of any latent toxic metabolites. In a second experiment, N additions were crossed with larval presence or absence to examine effects on key microbial processes. N increased leaf decay rates, soluble carbohydrate concentrations, fungal biomass and leaf-associated carbohydrase activity, but it did not stimulate bacterial productivity. Leaf decay was enhanced and bacterial production on leaves and in the water column was depressed in the presence of larvae. We conclude that the inputs of soluble N stimulated fungal growth, which made more fungal biomass available because of both its absolute increase and via the softening of the leaf particulate matter that could allow direct ingestion by larvae.