Effect of enzyme producing microorganisms on the biomass of epigeic earthworms (eisenia fetida) in vermicompost

We analyzed the bacterial community structure of the intestines of earthworms and determined the effect of enzyme producing microorganisms on the biomass of earthworms in vermicompost. Fifty-seven bacterial 16S rDNA clones were identified in the intestines of earthworms by using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis. Entomoplasma somnilux and Bacillus licheniformis were the dominant microorganisms; other strains included Aeromonas, Bacillus, Clostridium, Ferrimonas, and uncultured bacteria. Among these strains, Photobacterium ganghwense, Aeromonas hydrophila, and Paenibacillus motobuensis were enzyme-producing microorganisms. In the mixtures that were inoculated with pure cultures of A. hydrophila WA40 and P. motobuensis WN9, the highest survival rate was 100% and the average number of earthworms, young earthworms, and cocoons were 10, 4.00-4.33, and 3.00-3.33, respectively. In addition, P. motobuensis WN9 increased the growth of earthworms and production of casts in the vermicompost. These results show that earthworms and microorganisms have a symbiotic relationship.     

Distribution of bacteria and fungi in the earthworm Libyodrillus violaceous (Annelida: Oligochaeta), a native earthworm from Nigeria

Earthworms are soil invertebrates that play a key role in recycling organic matter in soils. In Nigeria, earthworms include Libyodrillus violaceous. Aerobic and anaerobic bacterial counts, as well as fungal counts of viable microorganisms in soils and gut sections, were made on twenty L. violaceous collected from different sites on the campus of the University of Agriculture, Abeokuta, Nigeria. The samples were collected between April and November, 2002. Numbers of microorganisms were higher in castings and gut sections than in un-ingested soil samples. The guts and their contents also had higher moisture and total nitrogen contents than the un-ingested soils. Bacteria and fungi isolated from the samples were identified by standard microbiological procedures on the bases of their morphological and biochemical characteristics. Isolated bacteria were identified as Staphylococcus, Bacillus spp., Pseudomonas aeruginosa, Streptococcus mutans, Clostridium, Spirocheata spp., Azotobacter spp., Micrococcus lylae, Acinetobacter spp., Halobacterium for bacteria. Yeast isolates were identified as Candida spp., Zygosaccharomyces spp., Pichia spp., and Saccharomyces spp while molds were identified as, Aspergillus spp., Pytium spp., Penicillium spp., Fusarium spp and Rhizopus spp. Of the five locations examined, the refuse dump area had the highest numbers of both aerobic and anaerobic organisms, followed by the arboretum while the cultivated land area recorded the lowest counts. The higher numbers of microorganisms observed in the gut sections and casts of the earthworms examined in this work reinforce the general concept that the gut and casts of earthworms show higher microbial diversity and activity than the surrounding soil.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Microflora of the honeybee gastrointestinal tract

Microorganisms in the midgut and rectum of the honeybee were enumerated and characterized. Counts of aerobic microorganisms were distinctly lower than counts of anaerobes (10(5)-10(6) viable cells per g of intestinal content vs. 10(8)-10(9) per g). Total numbers of anaerobic microorganisms were almost identical with the count of anaerobic Gram-positive acid resistant rods. A higher number of coliform bacteria and Bacillus spp. was detected in the rectum (10(5) per g). Anaerobic and aerobic microorganisms, coliforms, enterococci, Bacillus spp., Pseudomonas spp. and yeasts were found in all bees; lactobacilli, staphylococci and moulds were not found.     

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.     

Cecal and fecal bacterial flora of the Mongolian gerbil and the chinchilla.

The Mongolian gerbil is being increasingly used as a laboratory animal and as a pet. Both chinchillas and gerbils are used as animal models for otitis media and other otic research. Previously, only incomplete information was available regarding the indigenous bacterial flora of the lower intestinal tracts of these coprophagic animals. Using the strict anaerobic methodology of the Virginia Polytechnic Institute Anaerobe Laboratory, we studied the predominant bacterial flora of the cecum and fecal pellets of the gerbil and the chinchilla and the bacterial flora of digesta pellets in the proximal colon. We found species of the following anaerobic genera in high dilutions of gerbil fecal pellets: Bifidobacterium, Clostridium, Propionibacterium, Lactobacillus, and Bacteroides. Only lactobacilli were found in high dilutions of digesta from the upper colon, although the cecum yielded Peptostreptococcus, Bifidobacterium, Clostridium, Lactobacillus, Propionibacterium, and Bacteroides species from high dilutions of cecal contents. The facultatively anaerobic and aerobic flora isolated consisted of species of Bacillus, Streptococcus, Staphylococcus, Acinetobacter, Alcaligenes, Escherichia, Pasteurella, and Pseudomonas plus several unidentifiable organisms. Species of Bifidobacterium, Bacteroides, Eubacterium, and anaerobic Lactobacillus were isolated from chinchillas.     

Denaturing gradient gel electrophoresis for nonlethal detection of Aeromonas salmonicida in salmonid mucus and its potential for other bacterial fish pathogens

Denaturing gradient gel electrophoresis (DGGE) of 16S rDNA was used to nonlethally detect Aeromonas salmonicida and other bacteria in salmonid skin mucus. Mucus samples from wild spawning coho salmon (Oncorhynchus kisutch) with endemic A. salmonicida and from cultured lake trout (Salvelinus namaycush) were tested by PCR-DGGE and were compared with mucus culture on Coomassie brilliant blue agar and internal organ culture. PCR-DGGE gave a highly reproducible 4-band pattern for 9 strains of typical A. salmonicida, which was different from other Aeromonas spp. Aeromonas salmonicida presence in mucus was evident as a band that comigrated with the bottom band of the A. salmonicida 4-band pattern and was verified by sequencing. PCR-DGGE found 36 of 52 coho salmon positive for A. salmonicida, compared with 31 positive by mucus culture and 16 by organ culture. Numerous other bacteria were detected in salmonid mucus, including Pseudomonas spp., Shewanella putrefaciens, Aeromonas hydrophila and other aeromonads. However, Yersinia ruckeri was not detected in mucus from 27 lake trout, but 1 fish had a sorbitol-positive Y. ruckeri isolated from organ culture. Yersinia ruckeri seeded into a mucus sample suggested that PCR-DGGE detection of this bacterium from mucus was possible. PCR-DGGE allows nonlethal detection of A. salmonicida in mucus and differentiation of some Aeromonas spp. and has the potential to allow simultaneous detection of other pathogens present in fish mucus.     

Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

The effect of antimicrobial compounds on the gastrointestinal microflora of rainbow trout, Salmo gairdneri Richardson

Populations of aerobic and anaerobic heterotrophic bacteria occurring in the gastrointestinal tract of healthy rainbow trout were estimated using a dilution plate technique. Data revealed a progressive decline in numbers of aerobic bacteria along the digestive tract from oesophagus to lower intestine. However, the highest numbers were recovered from the intestinal contents and faeces. Anaerobes were generally restricted to the upper intestine and intestinal contents. The aerobic component of the bacterial microflora from the digestive tract was equated with Acinetobacter calcoaceticus, Aeromonas hydrophila, Bacillus circulans, Bac. megaterium , coryneforms, Grampositive irregularly shaped rods, Flavobacterium sp., Kurthia sp., Microhacterium sp., Providencia stuartii, Pseudomonas spp., Ps.fluorescens and Ps. pseudoalcaligenes . Evidence from scanning electron microscopy pointed to a general lack of colonization of the gut wall: instead, microorganisms were abundant in the intestinal contents. Antimicrobial compounds, i.e. oxolinic acid, oxytetracycline and sulphafurazole (which are commonly used to combat infections by Gramnegative bacterial fish pathogens), caused an increase in bacterial numbers throughout the digestive tract, with maximal numbers in the lower intestine. The bacteria, comprising an essentially different range of taxa, were generally resistant to the antibiotics in use. Conversely, erythromycin and penicillin G, which are used to treat some diseases caused by Gram-positive bacteria, caused a rapid reduction in bacterial numbers within the gastrointestinal tract.     

Microbial populations in wetwood of European white fir (Abies alba Mill.)

Abstract A method for extraction of microbial populations from wood samples was worked out which gave good recovery of both aerobic and anaerobic microorganisms in agar shake dilution and plating enumerations. This method was applied to the quantification of microbial populations in three European white firs ( Abies alba Mill.) which were afflicted with the European fir disease. Low numbers of aerobic microorganisms (10²−10⁴ colony- forming units (cfu) per g fresh tissue) were detected in sapwood irrespective of the degree of affliction. Anaerobic bacteria were usually 1–2 orders of magnitude less frequent. Wetwood of highly diseased firs contained significantly higher numbers of aerobic microorganisms (10⁵−10⁷), whereas the number of anaerobes was not enhanced significantly. Among the prevalent aerobic microorganisms in wetwood were Protaminobacter, Pseudomonas strains, and a yeast. In anaerobic counts from wetwood, Klebsiella and Vibrio strains predominated. The sapwood contained Bacillus, Beijerinckia, Staphylococcus , and Clostridium spp. High numbers of aerobic microorganisms were also detected in the roots and lower stem of a diseased vine plant ( Vitis vinifera L.). The importance of microbial populations in wetwood formation and disease expression is discussed.     

Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)

AIMS: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine. METHODS AND RESULTS: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4',6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were < or =2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were < or =2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium. CONCLUSIONS: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish. SIGNIFICANCE AND IMPACT OF THE STUDY: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.     

Bacteria isolated from the duodenum, ileum, and cecum of young chicks.

Facultatively anaerobic and strictly anaerobic bacteria colonizing the intestinal tracts of 14-day-old chicks fed a corn-based diet were enumerated, isolated, and identified. Colony counts from anaerobic roll tubes (rumen fluid medium) or aerobic plates (brain heart infusion agar) recovered from homogenates of the duodenum, upper and lower ileum, and cecum varied appreciably among samples from individual birds. Anaerobic and aerobic counts from the duodenum and ileum were similar. Anaerobic counts were highest from the cecum (0.7 X 10(11) to 1.6 X 10(11)/g of dry tissue) and exceeded aerobic plate counts by a factor of at least 10(2). Facultatively anaerobic groups (Streptococcus, Staphylococcus, Lactobacillus, and Escherichia coli) comprised the predominant flora of the duodenum and ileum, although large numbers of anaerobes (9 to 39% of the small intestine isolates), represented by species of Eubacterium, Propionibacterium, Clostridium, Gemmiger, and Fusobacterium, were also recovered. Strict anaerobes (anaerobic gram-positive cocci, Eubacterium, Clostridium Gemmiger, Fusobacterium, and Bacteriodes) made up nearly the entire microbial population of the cecum. Scanning electron microscopy of the intestinal epithelia of chicks revealed populations of microbes on the duodenal, ileal, and cecal mucosal surfaces.     

Diversity and dynamics of bacterial species in a biofilm at the end of the Seoul water distribution system

To investigate changes in the bacterial species and hygienic safety of the biofilm at the end of the drinking water distribution system in Seoul (Korea), denaturing gradient gel electrophoresis (DGGE) and DNA sequencing were used to analyse the bacterial population in the biofilm of a semi-pilot galvanized iron pipe model. The presence of sequences from aerobic Sphingomonas sp., anaerobic Rhodobacter sp., and unculturable bacteria indicated that these organisms coexisted after 1 day of model operation, demonstrating the ease of biofilm formation on galvanized iron pipes in the end region of the water distribution system studied. Sequences similar to those of unculturable bacteria, E. coli, and anaerobic bacteria were detected during the course of succession on the biofilm. More complicated band patterns were observed after 70 days of operation. PCR-DGGE illustrated changes in the biofilm during succession as well as the possibilities of anaerobic conditions and faecal contamination of the drinking water system. PCR-DGGE and culture-dependent fatty acid methyl ester (FAME) analysis showed different patterns for the same samples (Lee & Kim 2003); however, PCR-DGGE showed less diversity than did FAME analysis. This study compared the culture-dependent FAME and culture-independent PCR-DGGE methods directly, and their use in promoting the hygienic safety of drinking water.     

滴水湖沉积物中可培养优势微生物种群初探

于滴水湖湖心采集底泥样品,对底泥中可培养优势菌种进行分离、纯化,并利用Biolog微生物自动分析系统进行鉴定。结果显示,滴水湖沉积物中菌落总数为2.43×104CFU/g,分离纯化后的8株优势菌种中,革兰氏阴性菌占87.5%,其中7株为GN-NENT(革兰氏阴性非肠道菌)、1株为GP-ROD SB(革兰氏阳性芽孢杆菌)。鉴定结果显示,8株菌种分别为:鳗鱼气单孢菌(Aeromonas encheleia)、乙酸钙不动杆菌/基因型1(Acinetobacter calcoaceticus/genospecies1)、舒氏气单胞菌(Aeromonas schubertiiDNA group12)、腐败希瓦氏菌B(Shewanella putrefaciens B)、维罗纳/温和气单胞菌(Aeromonas veronii/sobria DNA group8)、坎氏弧菌(Vibrio campbelli)、蕈状芽孢杆菌(Bacillus mycoides)和梅氏弧菌(Vibrio metschnikovii)。     

Biosynthesis of thiamin. Different biosynthetic routes of the thiazole moiety of thiamin in aerobic organisms and anaerobic organisms

The nitrogen atom of glycine was incorporated into the thiazole moiety of thiamin in the aerobic microorganisms Bacillus subtilis, Pseudomonas putida, Saccharomyces cerevisiae, Mucor racemosus, Neurospora crassa, and Emericella nidulans. It was not incorporated in the case of the facultative anaerobic microorganisms Escherichia coli and Enterobacter aerogenes, which, however, did incorporate the nitrogen atom of tyrosine. These results show that aerobic microorganisms and facultative anaerobic microorganisms have different biosynthetic pathways for the thiazole moiety of thiamin.     

Epiphytic bacteria biodiversity in Brazilian Cerrado fruit and their cellulolytic activity potential

The Cerrado is the second largest Brazilian biome, yet little is known about its wild fauna, flora and microbiota. This work aimed to identify epiphytic bacteria present in fruits native to three different regions of the Cerrado and to select cellulase-producing bacteria. Culture-dependent and culture-independent (PCR-DGGE) methods were used to characterize the microbiota from 32 native Cerrado fruits, and the selection of cellulase-producing bacteria was performed by a semi-quantitative test on carboxymethylcellulose agar medium. Analysis of the 16S rRNA gene sequences of 69 profile representatives showed that the isolates belonged to 29 bacterial genera (Arthrobacter, Bacillus, Paenibacillus, Pseudomonas, Serratia, Staphylococcus, Streptomyces, Enterobacter, Microbacterium, Aerococcus, Bradyrhizobium, Methylobacterium, Erwinia, Pantoea, Acidithiobacillus, Ochrobactrum, Stenotrophomonas, Curtobacterium, Clostridium, Lactobacillus, Xanthomonas, Delftia, Klebsiella, Enterococcus, Burkholderia, Escherichia, Streptococcus, Citrobacter and Achromobacter). Species in the genera Methylobacterium, Stenotrophomonas, Clostridium, Pantoea and Enterobacter were detected by both culture-dependent and culture-independent methods. The species Lactobacillus fermentum, Acinetobacter sp. and Methylomonas methanica were detected only by PCR-DGGE. Additionally, 30 % (178 isolates) of the bacteria tested were able to produce cellulase. The best producers belonged to the genera Bacillus, Streptomyces, Paenibacillus, Enterobacter and Burkholderia, indicating that this ecosystem could be an attractive source for the study of novel enzymes.     

Diversity of culturable heterotrophic aerobic bacteria in pristine stream bed sediments

More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.     

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.     

鲤科（Cyprinidate）鱼肠道菌群分析

对江苏大纵湖区的草鱼、白鲢、团头鲂和鲤鱼等4种鱼肠道菌群中的好氧菌或兼性厌氧菌进行分离鉴定.对每种鱼的肠道中菌群数量和种类组成进行了分析,发现在上述鱼肠道中存在哈夫尼亚菌属、致病杆菌属、气单胞菌属、柠檬酸菌属、假单胞菌属、链球菌属、葡萄球菌属等.