Cellulolytic Microbes in the Yanbaru,a Subtropical Rainforest with an Endemic Biota on Okinawa Island,Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

Cellulolytic microbes in the Yanbaru, a subtropical rainforest with an endemic biota on Okinawa Island, Japan

Cellulolytic microbes in the soil of the Yanbaru, a subtropical forest with an endemic biota, on Okinawa Island, were isolated and characterized in a search for novel microbial strains with biotechnological potential. Soil samples of the Yanbaru were suspended in sterilized water, inoculated on mineral salt agar overlaid with a filter paper as carbon source, and cultivated aerobically at 30 °C. After 2 weeks of cultivation, emerging colonies were isolated and subjected to phylogenetic and enzyme analyses. The phylogenetic analyses revealed bacterial and fungal isolates belonging to nine and three genera respectively. All isolates possessed cellulase activity, and several strains showed strong activity comparable to Trichoderma cellulase. Many isolates also exhibited xylanase activity.     

Resuscitation of viable but non‐culturable bacteria to enhance the cellulose‐degrading capability of bacterial community in composting

Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable (VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.     

Disclosing a crypt: Microbial diversity and degradation activity of the microflora isolated from funeral clothes of Cardinal Peter Pázmány

A crypt can be considered as a particular environment where different microbial communities contribute to decomposition of organic materials present inside during a long interval of time. The textile remains of the funeral clothes (biretta and tunic) of Cardinal Pázmány, an important historic figure dead in Bratislava the 19th March 1637, conserved in this kind of environment were subjected to microbial investigation. The sampling comprised three different approaches and the use of various kinds of cultivation media. Two different PCR-based clustering methods, f-ITS and f-CBH, were employed in order to select the bacterial and fungal microfloras which were identified in a second step by the 16S rRNA and ITS sequencing respectively. The isolated microflora was tested for its proteolytic, keratinolytic and cellulolytic activities and for its ability to grow on Fibroin agar medium. The combination of cultural, molecular and biodegradative assays was able to isolate and characterize a bacterial community composed mainly by members of the phyla Firmicutes and Actinobacteria. The fungal community appeared more diversified, together with several Penicillium and Aspergillus strains, members belonging to the species Beauveria bassiana, Eurotium cristatum, Xenochalara juniperi, Phialosimplex caninus and Myriodontium keratinophilum were isolated. Bacteria, especially the Bacillus members, showed their strong ability to degrade keratin and gelatin and a large portion of them was able to growth on Fibroin agar. The fungal isolates displayed a widespread cellulolytic activity and fibroin utilization, although they possessed a weaker and slower proteolytic and keratinolytic properties respect to bacterial counterpart. The present study can be considered perhaps as the first or among the few microbial investigations which treated the textile biodegradation from such unusual environment.     

Cellulose- and xylan-degrading thermophilic anaerobic bacteria from biocompost

Nine thermophilic cellulolytic clostridial isolates and four other noncellulolytic bacterial isolates were isolated from self-heated biocompost via preliminary enrichment culture on microcrystalline cellulose. All cellulolytic isolates grew vigorously on cellulose, with the formation of either ethanol and acetate or acetate and formate as principal fermentation products as well as lactate and glycerol as minor products. In addition, two out of nine cellulolytic strains were able to utilize xylan and pretreated wood with roughly the same efficiency as for cellulose. The major products of xylan fermentation were acetate and formate, with minor contributions of lactate and ethanol. Phylogenetic analyses of 16S rRNA and glycosyl hydrolase family 48 (GH48) gene sequences revealed that two xylan-utilizing isolates were related to a Clostridium clariflavum strain and represent a distinct novel branch within the GH48 family. Both isolates possessed high cellulase and xylanase activity induced independently by either cellulose or xylan. Enzymatic activity decayed after growth cessation, with more-rapid disappearance of cellulase activity than of xylanase activity. A mixture of xylan and cellulose was utilized simultaneously, with a significant synergistic effect observed as a reduction of lag phase in cellulose degradation.     

Cellulose degradation by a mixed bacterial culture

Summary An integrated mixed bacterial culture consisting of four strains has been isolated by a batch enrichment technique. The cellulolytic member (strain D) is aCellulomonas sp. and the others are non-cellulolytic. The interaction between strains D and C is pronounced and appears to involve an exchange of reducing sugars and growth factors. The symbiotic relationship of this naturally occurring mixed culture is therefore one of mutualism. The filter paper cellulase and carboxymethyl cellulase activities in extracellular fluid are high, while -glucosidase activity is low. The mixed culture digests a variety of lignocellulosics efficiently and is of fundamental interest in the study of microbial interrelationships.     

Isolation and characterization of cellulolytic bacteria from the Stain house Lake,Antarctica

The main aim was to evaluate the occurrence of cellulolytic bacteria from the Stain house Lake, located at Admiralty Bay, Antarctica. Thick cotton string served as a cellulose bait for the isolation of bacteria. A total of 52 bacterial isolates were recovered and tested for their cellulase activity, and two of them, isolates CMAA 1184 and CMAA 1185, showed significant cellulolytic activity on carboxymethylcellulose agar plates. Phylogenetic analysis placed the isolates into the Bacillus 16S ribosomal RNA gene subclade. Both isolates produced a cold-active cellulase which may play a crucial role in this extreme environment.     

Isolation of fungal cellobiohydrolase I genes from sporocarps and forest soils by PCR

Cellulose is the major component of plant biomass, and microbial cellulose utilization is a key step in the decomposition of plant detritus. Despite this, little is known about the diversity of cellulolytic microbial communities in soil. Fungi are well known for their cellulolytic activity and mediate key functions during the decomposition of plant detritus in terrestrial ecosystems. We developed new oligonucleotide primers for fungal exocellulase genes (cellobiohydrolase, cbhI) and used these to isolate distinct cbhI homologues from four species of litter-decomposing basidiomycete fungi (Clitocybe nuda, Clitocybe gibba, Clitopilus prunulus, and Chlorophyllum molybdites) and two species of ascomycete fungi (Xylaria polymorpha and Sarcoscypha occidentalis). Evidence for cbhI gene families was found in three of the four basidiomycete species. Additionally, we isolated and cloned cbhI genes from the forest floor and mineral soil of two upland forests in northern lower Michigan, one dominated by oak (Quercus velutina, Q. alba) and the other dominated by sugar maple (Acer saccharum) and American basswood (Tilia americana). Phylogenetic analysis demonstrated that cellobiohydrolase genes recovered from the floor of both forests tended to cluster with Xylaria or in one of two unidentified groups, whereas cellobiohydrolase genes recovered from soil tended to cluster with Trichoderma, Alternaria, Eurotiales, and basidiomycete sequences. The ability to amplify a key fungal gene involved in plant litter decomposition has the potential to unlock the identity and dynamics of the cellulolytic fungal community in situ.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Preparation of mutants of Trichoderma reesei with enhanced cellulase production.

The development of an agar plate screening technique has allowed the isolation of a range of mutants of Trichoderma reesei capable of synthesizing cellulase under conditions of high catabolite repression. The properties of one of these mutants (NG-14) is described to illustrate the use of this technique. NG-14 produced five times the filter paper-degrading activity per ml of culture medium and twice the specific activity per mg of excreted protein in submerged culture when compared with the best existing mutant, QM9414. NG-14 also showed enhanced endo-beta-glucanase and beta-glucosidase production. Although these mutants were isolated as cellulase producers in the presence of 5% glycerol on agar plates, in similar liquid medium, NG-14 exhibits only partial derepression of the cellulase complex. Since the proportions of filter paper activity, endo-beta-glucanase, and cellobiase were not the same in mutants NG-14 and QM9414, and the yields of each enzyme under conditions repressive for cellulase synthesis were different, differential control of each enzyme of the cellulase complex is implied. These initial results suggest that the selective technique for isolating hyper-cellulase-producing mutants of Trichoderma will be of considerable use in the development of commercially useful cellulolytic strains.     

Response surface optimization of cellulase production from Aneurinibacillus aneurinilyticus BKT-9: An isolate of urban Himalayan freshwater

Due to their vast industrial potential, cellulases have been regarded as the potential biocatalysts by both the academicians and the industrial research groups. In the present study, culturable bacterial strains of Himalayan Urban freshwater lake were investigated for cellulose degrading activities. Initially, a total of 140 bacterial strains were isolated and only 45 isolates were found to possess cellulose degrading property. On the basis of preliminary screening involving cellulase activity assay on CMC agar (with clear zone of hydrolysis) and biosafety assessment testing, only single isolate named as BKT-9 was selected for the cellulase production studies. Strain BKT-9 was characterized at the molecular level using rRNA gene sequencing and its sequence homology analysis revealed its identity as Aneurinibacillus aneurinilyticus. Further, various physico-chemical parameters and culture conditions were optimized using one factor approach to enhance cellulase production levels in the strain BKT-9. Subsequently, RSM based statistical optimization led to formulation of cellulase production medium, wherein the bacterial strain exhibited ~60 folds increase in enzyme activity as compared to un-optimized culture medium. Further studies are being suggested to scale up cellulase production in A. aneurinilyticus strain BKT-9 so that it can be utilized for biomass saccharification at an industrial level.     

Antimicrobial and antifungal activity of soil actinomycetes isolated from coal mine sites

In the current study, twenty-eight soil samples were collected from coalmine sites of Telangana, India. The isolates were purified and identified based on their culture characterization on oatmeal agar, glycerol asparagine agar, yeast extract-malt extract agar, inorganic salt starch agar, and starch casein agar medium. Further, the supernatant of all the isolates were tested for antimicrobial and antifungal activities. The biochemical and microscopic studies of isolated strains results indicates the potential isolate strains belongs to Streptomyces genus. Among all the strains the biological activity of BHPL-KSKU5 showed higher anti-bacterial and anti-funagal activity. The molecular characterization of BHPL-KSKU5 16s rDNA gene sequence and phylogenetic tree showed that is mostly related to the Streptomysis felleus (S. felleus) strain. This isolate was submitted to gene bank NCBI with accession number MH553077. In addition, physiological studies such as utilization of carbon, nitrogen, amino acid sources of potential isolated were studied. Further, optimization, purification and characterization of the novel compound producing strain may be helpful for discovering the new therapeutic microbial agent.     

Novel and rapid agar plate methods for in vitro assessment of bacterial biocontrol isolates’ antagonism against multiple fungal phytopathogens

Biological control of plant diseases with antagonistic bacteria is a promising alternative to conventional chemical control strategies. In vitro screening for inhibition of mycelial growth of phytopathogenic fungi by bacterial isolates is the first step in selecting putative bacterial biocontrol agents. Dual culture plate assay is the most common method involved in this first-line selection process. However, it needs independent agar plates to test antagonism by a specific bacterial isolate against each of the fungal phytopathogen. Two modified in vitro antagonism tests are proposed here. Antagonistic activity of a putative biocontrol bacterial strain against four different fungal phytopathogens could be assessed in a single agar plate simultaneously. A comparison of the new methods with conventional dual culture plate assay was also done. The proposed methods are easy to perform and results of antagonism are obtained rapidly. Results of fungal inhibition were qualitatively comparable with that generated through dual culture plate assay. Quantity of resources such as agar medium and plates required for the modified antagonistic assays is several folds less than that required for dual culture plate assay.     

Extracellular hydrolase enzyme production by soil fungi from King George Island, Antarctica

Various microbial groups are well known to produce a range of extracellular enzymes and other secondary metabolites. However, the occurrence and importance of investment in such activities have received relatively limited attention in studies of Antarctic soil microbiota. In order to examine extracellular enzyme production in this chronically low-temperature environment, fungi were isolated from ornithogenic, pristine and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica during the austral summer in February 2007. Twenty-eight isolates of psychrophilic and psychrotolerant fungi were obtained and screened at a culture temperature of 4°C for activity of extracellular hydrolase enzymes (amylase, cellulase, protease), using R2A agar plates supplemented with (a) starch for amylase activity, (b) carboxymethyl cellulose and trypan blue for cellulase activity or (c) skim milk for protease activity. Sixteen isolates showed activity for amylase, 23 for cellulase and 21 for protease. One isolate showed significant activity across all three enzyme types, and a further 10 isolates showed significant activity for at least two of the enzymes. No clear associations were apparent between the fungal taxa isolated and the type of source soil, or in the balance of production of different extracellular enzymes between the different soil habitats sampled. Investment in extracellular enzyme production is clearly an important element of the survival strategy of these fungi in maritime Antarctic soils.     

Production of extracellular hydrolase enzymes by fungi from King George Island

Fungi are known to produce a range of extracellular enzymes and other secondary metabolites. Investment in extracellular enzyme production may be an important element of the survival strategy of these fungi in maritime Antarctic soils. This study focuses on fungi that were isolated from ornithogenic, undisturbed and human-impacted soils collected from the Fildes Peninsula, King George Island, Antarctica, during the austral summer in February 2007. We (1) describe fungal diversity based on molecular approaches, (2) describe the thermal characteristics of the fungal isolates, and (3) screen extracellular hydrolase enzyme production (amylase and cellulase) by the isolates. Soil samples were cultured using the Warcup soil plating technique and incubated at 4 and 25 °C to allow basic thermal classification. In total, 101 isolates were obtained. All the isolates were screened at culture temperatures of 4 and 25 °C in order to detect activity of extracellular hydrolase enzymes. At 25 °C, ornithogenic penguin rookery soils recorded the lowest diversity of fungi, with little difference in diversity apparent between the other soils examined. At 4 °C, an undisturbed site recorded the lowest and a human-impacted site the highest diversity of fungi. The majority of the fungi identified in this study were in the mesophilic thermal class. Six strains possessed significant activity for amylase and 13 for cellulase at 25 °C. At 4 °C, four strains showed significant amylase and 22 significant cellulase activity. The data presented increase our understanding of microbial responses to environmental temperature.     

Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.     

Strategies for the isolation of cellulolytic fungi for composting of wheat straw

It was shown that inoculation of straw with cellulolytic fungi offers potential for manipulating and improving the composting of cellulose waste, where the C:N ratio is not optimal for composting. In this paper we report on a screening strategy used to isolate novel cellulolytic fungi from field samples. The screen comprised of two phases. In phase I, 300 cellulolytic fungi were isolated to pure culture from field samples collected from Hawaii, China and the UK. Isolates were selected on the basis of high cellulolytic activities and growth rates on cellulose agar. A total of 137 lead isolates progressed to an unreplicated phase II screen to rapidly identify strains that improved quality of the resulting compost over and above that of the uninoculated control. Compost quality was assessed by measuring C:N ratio, water holding capacity, water content and potential and polysaccharide content of the resulting compost. Effect on the aggregate stability of soil and the growth of wheat seedlings was assessed when compost was added to a sandy loam soil. Performance of each isolate was quantified by allocating a utility score for each compost analysed. Utility scores were based on the sum of the logged ranked score in each assay. The 10 highest scoring isolates were subsequently processed through a replicated phase II screen and the best performing isolates identified by calculating utility scores as before. Significantly lower C:N ratios, higher water-holding capacities and improved aggregate stabilities were obtained with some inoculated treatments compared to the uninoculated control, whilst the results obtained for polysaccharide content and plant growth showed no significant differences. Isolate 304, isolated from decomposing vegetation obtained from Egham, Surrey, UK, and identifed as a Trichurus sp., appeared the most effective inoculant, significantly decreasing the C:N ratio by 36% and increasing the aggregate stability of soil by 54% compared to the uninoculated control. As a result of adopting this screening strategy, it has been possible to identify cellulolytic fungi that can, under non-sterile (laboratory) conditions, significantly improve the quality of compost. This screening approach therefore offers real possibilities for selecting microbial inoculants in low-tech agricultural practices.     

棉秸秆降解高温菌株的筛选及产酶分析

从新疆地区分离具有降解棉秸秆纤维素功能的菌株,得到4株耐高温真菌(50°C)。纤维素酶学性质分析表明,该4株菌的纤维素酶具有良好的耐酸性(最适pH为4.5)和耐高温性(最高达60°C)。以羧甲基纤维素钠(CMC-Na)、微结晶纤维素、棉花、滤纸、淀粉、果胶为底物测定酶活力,滤纸酶活力(FPA)最高达2.63 U/mL、淀粉酶活力最高达6.17 U/mL、果胶酶活力最高达5.86 U/mL。4株真菌酶学特性分析表明,该系列菌株在秸秆生物质利用方面有很大的应用潜力。     

Isolation and characterization of acid-tolerant, thermophilic bacteria for effective fermentation of biomass-derived sugars to lactic acid

Biomass-derived sugars, such as glucose, xylose, and other minor sugars, can be readily fermented to fuel ethanol and commodity chemicals by the appropriate microbes. Due to the differences in the optimum conditions for the activity of the fungal cellulases that are required for depolymerization of cellulose to fermentable sugars and the growth and fermentation characteristics of the current industrial microbes, simultaneous saccharification and fermentation (SSF) of cellulose is envisioned at conditions that are not optimal for the fungal cellulase activity, leading to a higher-than-required cost of cellulase in SSF. We have isolated bacterial strains that grew and fermented both glucose and xylose, major components of cellulose and hemicellulose, respectively, to l(+)-lactic acid at 50 degrees C and pH 5.0, conditions that are also optimal for fungal cellulase activity. Xylose was metabolized by these new isolates through the pentose-phosphate pathway. As expected for the metabolism of xylose by the pentose-phosphate pathway, [(13)C]lactate accounted for more than 90% of the total (13)C-labeled products from [(13)C]xylose. Based on fatty acid profile and 16S rRNA sequence, these isolates cluster with Bacillus coagulans, although the B. coagulans type strain, ATCC 7050, failed to utilize xylose as a carbon source. These new B. coagulans isolates have the potential to reduce the cost of SSF by minimizing the amount of fungal cellulases, a significant cost component in the use of biomass as a renewable resource, for the production of fuels and chemicals.     

Screening of some wild fungal isolates for cellulolytic activities

Six wild fungal strains, Trichoderma viride, T. harzianum, Gliocladium virens, Aspergillus terreus, A. niger and Tiarosporella phaseolina , isolated from decomposed jute stacks and diseased jute stem, were tested for their cellulolytic and hemicellulolytic activities and compared with T. reesei MCG 77. Filter paper cellulase production by all these wild strains were lower than those produced by T. reesei while some strains ( T. viride, T. harzianum and G. virens ) possessed carboxymethyl cellulase, β-glucosidase, xylanase and β-xylosidase activities comparable to T. reesei. A. terreus and A. niger produced 3·2 and 1·2 times respectively, greater β-glucosidase activity compared to T. reesei when grown on microcrystalline cellulose.