Isolation of Cellulolytic Actinomycetes from Marine Sediments

The cellulolytic activity of 36 actinomycetes strains isolated from marine sediments was investigated by the cellulose-azure method. Approximately 50% of the isolates exhibited various degrees of cellulolytic activity.     

Hydrolytic enzyme activity in landfilled refuse

Extracellular hydrolytic enzyme activity was assayed in 28 refuse samples excavated from 14 bore holes in Fresh Kills Landfill, Staten Island, N. Y. Esterases, proteases and amylases were present in all of the samples. Enzyme screening assays utilizing the API-ZYM test system showed the incidence of enzymes in the order: specific phosphatases > esterases > glycosyl hydrolases. Measurement of cellulase by the cellulose-azure test detected activity in two out of 28 samples. Analysis for cellulase activity using the cellulose-azure test on refuse samples from landfills in Naples, Florida, and Tucson, Arizona, also showed a limited distribution of cellulases. Mineralization of [¹⁴C]cellulose, an independent measure of cellulase activity, ranged from < 5 to 23% in a 4-week incubation, which supports a highly variable cellulolytic activity in landfilled refuse. Correspondence to: A. C. Palmisano     

Improvement in Isolation of Gliding Bacteria by the Elimination of Soluble Sugars

The selectivity and efficiency for the isolation of cellulolytic gliding bacteria were improved by eliminating soluble sugars from the usual cellulose agar medium in which the cellulolytic bacteria were often overgrown by fungi. The new selective medium did not support the growth of non-cellulolytic fungi.     

Cellulolytic activity in leachate during leach-bed anaerobic digestion of municipal solid waste

The degradation of municipal solid waste (MSW) under mesophilic conditions can be enhanced by exchanging leachate between fresh waste and stabilised waste. The optimum point in time when leachate from an anaerobically digesting waste bed can be used to initiate degradation of another waste bed might occur when the leachate of the digesting waste bed is highly active with cellulolytic and methanogenic bacteria. In this study, the cellulolytic activity of the leachate was measured using the cellulose-azure assay. As products of hydrolysis are soluble compounds, the rate of generation of these compounds was estimated based on a soluble chemical oxygen demand (SCOD) balance around the fresh waste bed. It was found that once the readily soluble material present in MSW was washed out there was very little generation of SCOD without the production of methane, indicating that flushing leachate from a stabilised waste bed resulted in a balanced inoculation of the fresh waste bed. With the onset of sustained methanogenesis, the rate of SCOD generation equalled the SCOD released from the digester as methane. The experimental findings also showed that cellulolytic activities of the leachate samples closely followed the trend of SCOD generation.     

Einflußder Aufbewahrungsmethode auf die Stabilität der biotechnologischen Eigenschaften von Zellulose utilisierenden Pilzen

The influence of long-term storage on the stability of the cellulolytic enzyme and protein biosynthesis ability, was determined. Microorganisms belonging to genera Aspergillus, Fusarium, Penicillium, Sporotrichum, Allescheria and Mucor were used. The strains were stored on wort agar slants, on SAUNDER'S medium and on a medium according to MANDELS -WEBER , under paraffin oil or without it. After 3 years storage no influence of the storage method on the biosynthesis of cellulolytic enzymes (strain A. niger 33/2 and 33/20) was found. A systematic increase in the celluloslytic activity was exhibited by strains A.n. 33/2 and A.n. 33/20 on wort agar (40 and 98%, respectively) and on SAUNDER'S medium (79 and 66%, respectively), as well as by the new strain CEL 2 on Saunder's medium without paraffin oil.     

Rapid tube test for detecting fungal cellulase production.

A simple procedure is described for the detection of cellulolytic activity in fungi, utilizing dyed cellulose powder as substrate. The powder, incorporated into a basal medium containing agar, mineral salts, and growth factors, is layered onto basal medium minus cellulose. Cellulase production results in release of the dye which diffuses into the lower layer.     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Influence of dietary fiber on xylanolytic and cellulolytic bacteria of adult pigs.

Xylanolytic and cellulolytic bacteria were enumerated over an 86-day period from fecal samples of 10 8-month-old gilts that were fed either a control or a 40% alfalfa meal (high-fiber) diet. Fecal samples were collected from all pigs on days 0, 3, 5, 12, 25, 37, 58, and 86. Overall, the numbers of xylanolytic bacteria producing greater than 5-mm-diameter zones of clearing on 0.24% xylan roll tube medium after 24 to 36 h of incubation were 1.6 X 10(8) and 4.2 X 10(8)/g (dry weight) of feces for the control pigs and those fed the high-fiber diet, respectively. After 1 week of incubation, a large number of smaller zones of clearing (1 to 2 mm) appeared. Besides Bacteroides succinogenes and Ruminococcus flavefaciens, which produced faint zones of clearing in xylan roll tubes, three strains which closely resembled B. ruminicola hydrolyzed and used xylan for growth. The overall numbers of cellulolytic bacteria producing zones of clearing in 0.5% agar roll tube medium were 0.36 X 10(8) and 4.1 X 10(8)/g for the control pigs and those fed the high-fiber diet, respectively. B. succinogenes was the predominant cellulolytic isolate from both groups of pigs, and R. flavefaciens was found in a ratio of approximately 1 to 15 with B. succinogenes. Degradation of xylan and cellulose, measured by in vitro dry matter disappearance after inoculation with fecal samples, was significantly greater for pigs fed the high-fiber diet than that for the controls. These data suggest that the number of fibrolytic microorganisms and their activity in the large intestine of the adult pig can be increased by feeding pigs high-alfalfa-fiber diets and that these organisms are similar to those found in the rumen.     

Simple cultural test for relative cellulolytic activity of fungi

A simple method is described for determining the relative cellulolytic activity of fungi. Opaque columns of an agar medium containing a partially crystalline cellulose preparation were inoculated with the fungi. Depth of the clear zone that developed beneath the growing cultures provided a visual measure of cellulolytic activity on a continuous, cumulative basis. Depth of clearing (DC) was determined for 25 species of fungi differing widely in cellulolytic activity, and compared by correlation analysis with results of three other methods for measuring cellulolytic activity. Relatively high coefficients of correlation (greater than 0.6) were obtained between DC and weight loss of cotton sliver, loss in tensile strength of cotton duck, and carboxymethyl cellulase activity in culture filtrates. In comparison with conventional assay procedures, the clearing method offered several advantages: (i) results were at least as well correlated with the capacity to utilize native cellulose as a substrate; (ii) the method measured activity of growing cultures rather than culture filtrates, thus involving less risk of losses due to product inhibition, binding, or denaturation of enzymes; (iii) repeated measurements were made on the same experimental set up, so that errors due to arbitrarily selected times of harvest were avoided conveniently; and (iv) the method required less working time and very simple equipment, making it convenient for large-scale screening tests.     

Development of a simple cultivation method for isolating hitherto-uncultured cellulase-producing microbes

Although enrichment culture is typically employed to isolate cellulolytic microbes, this approach tends to favor fast-growing species and discriminates against all others. Therefore, efforts to prevent the overgrowth of fast-growing species are necessary to isolate novel cellulase-producing strains. In this study, we developed a simple culture method for isolating hitherto-uncultured microbes that possess cellulase activity, particularly exocellulase. In this method, the microbial source (a forest soil) was suspended in sterilized water and inoculated onto a mineral salts agar medium, which was then overlaid with filter paper to sandwich the microbial suspension between the agar surface and paper. The filter paper fibers served to immobilize the microbial cells and were the dominant carbon source. Following cultivation at 30°C for 2 weeks, emerging colonies were isolated based on their morphology and were then subjected to phylogenetic and enzyme analyses. Using this method, 2,150 CFUs/g dry soil were obtained, and the ratio of fungal to bacterial isolates was approximately 4:1. Phylogenetic analyses revealed that most fungal and bacterial isolates belong to ten and two genera, respectively. Notably, all isolates possessed exocellulase activity, and several strains showed strong activity that was comparable to Trichoderma cellulase. Many isolates also exhibited cellulase and xylanase activity, and several strains possessed laccase activity. It is expected that the culture method described here will be useful for the isolation of hitherto-uncultured cellulolytic microbes and the identification of novel cellulases.     

桑粒肩天牛幼虫肠道菌群的研究

Intestinal flora of 47 Apriona germari(Hope) larvae,collected from fields,had been isolated and identified.The results showed that the predominant bacteria were Staphylococcus.Its viable count was 7.63±0.21,and the detection rate was 100%.Meanwhile,a strain of cellulose-utilizing bacterium was isolated from the fore-midgut fluid of A.germari larvae with the cellulose-congo red agar medium.The bacterium was tentatively identified as Cellulomonas.The detection rate of the cellulolytic bacterium was 23.40%,and…     

Detection of cellulolytic activity of bacteria and fungi growing on agar surfaces

Summary Cellulolytic activity of four fungal species growing on solid medium containing acid-swollen cellulose could be detected much more easily if fungal growth was partly inhibited by the detergent Triton X-100. The dye, aniline blue-black, did not affect growth but increased the sensitivity of detection of cellulolytic activity of both fungi and bacteria. Separating fungi from cellulose fibres by a layer of agar or by filters showed that cell-fibre contact is not necessary for cellulose degradation. Such degradation is clearer when contact is prevented.     

Qualitative and quantitative agar invasion test based on bacterial colony/biofilm

Invasion of the culture medium is a feature frequently studied in yeasts, in which it has been related to a greater virulence, but it is practically unknown in bacteria. Recently, it has been demonstrated that several clinically relevant bacterial species were also able of invading agar media, so it was necessary to design a microbiological assay to study the expression of this character in bacteria. Accordingly, a bacterial agar invasion test based on colony/biofilm development was designed, which allows qualitative and quantitative characterization of bacterial growth into the agar culture medium. Once the culture conditions were optimized, the test was applied to 90 strains from nine bacterial species, validating its usefulness for differentiating invasive strains (positive) from those non invasive (negative). The test also allows sorting invasive strains according to agar invasion intensity (low, moderate, high) and topographic invasion pattern (peripheral, homogeneous, mixed). Moreover, an image analysis routine to quantify the invasion was developed. Implemented method enables direct measuring of two invasion parameters (invasion area and number of invasion dots), automated calculation of three relative variables (invasion relative area, invasion dots relative density, and invasion dot average area), and the establishment of strain specific frequency histograms.     

Biomethanation of a cellulose-based substrate in the presence and absence of a cellulolytic bacterium

The effect of co-culturing a methanogen isolated from a paper mill waste (PMW) with cellulolytic bacteria isolated from the intestinal fluids of the silver cricket (Lepisma saccharina) on the biomethanation of filter paper strips was examined. The autoclaved filter paper strips were subjected to biomethanation in AC 21 medium inoculated with methanogen PMW in the presence and in absence of a co-culture of cellulolytic bacteria. In spite of poor initial response, methane production in the presence of the cellulolytic co-culture were found to increase gradually upto 25 days, after which a reduction in methane production was observed. Analysis of the results in terms of increased cellulose degradation in the presence of cellulolytic bacteria has been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus)

Bats (Order Chiroptera) are a widely distributed group of mammals. Pteropus giganteus belongs to the Suborder Megachiroptera. This bat consumes fruits and leaves as their major food. Cellulose and xylan are the major composition of leaves. As they consume leaves in their diet, their digestive tract must contain cellulolytic and xylanolytic bacteria which help in the digestion of cellulose and xylan. The cellulolytic and xylanolytic bacteria were isolated and screened on Berg's agar containing cellulose and xylan. The bacteria isolated were characterized biochemically and found to be Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Serratia liquefaciens and Klebsiella oxytoca. These bacteria help in digestion of cellulose and xylan in the diet of the bat, P. giganteus. Here we show that leaves are also used as a carbohydrate source by these bats. An insectivorous bat, Hipposideros fulvus, was used as a control and does not possess cellulolytic and xylanolytic bacteria.     

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.     

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.     

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.