Bacterial diversity and community structure in an aerated lagoon revealed by ribosomal intergenic spacer analyses and 16S ribosomal DNA sequencing

We investigated the bacterial community structure in an aerated plug-flow lagoon treating pulp and paper mill effluent. For this investigation, we developed a composite method based on analyses of PCR amplicons containing the ribosomal intergenic spacer (RIS) and its flanking partial 16S rRNA gene. Community percent similarity was determined on the basis of RIS length polymorphism. A community succession was evident in the lagoon, indicated by a progressive community transition through seven sample locations. The most abrupt changes in community structure were associated with a temperature change from 39 to 35 degrees C and with increases in dissolved oxygen. The temporal differences in community structure, based on summer and winter samplings, were greater than the spatial differences during either season. Clone libraries of rDNA-RIS amplicons were constructed from each of three summer samples. Among 90 clones analyzed (30 clones from each sample), 56 phylotypes were distinguished by restriction fragment length polymorphism. Indices of phylotype richness, evenness, and diversity all increased in clone libraries from the beginning to the end of the lagoon. A representative clone of each phylotype was phylogenetically analyzed on the basis of its partial 16S rRNA gene sequence (ca. 450 bp). Phylogenetic analysis confirmed the increase in diversity and further indicated increasing richness of bacterial divisions. Pioneers in the community spatial succession appeared to include thermotolerant, microaerophilic methanol-oxidizing bacteria related to the genus Methylobacillus, as well as thermotolerant, microaerophilic nitrogen-fixing bacteria related to the genus Azospirillum.     

Microbial diversity in ostrich ceca as revealed by 16S ribosomal RNA gene clone library and detection of novel Fibrobacter species

The ostrich (Struthio camelus) is a herbivorous bird and although the hindgut is known as the site for fiber digestion, little is known about the microbial diversity in the ostrich hindgut. Our aim was to analyze the microbial diversity in ostrich ceca using a 16S ribosomal RNA gene (rDNA) clone library approach. A total of 310 clones were sequenced and phylogenetically analyzed and were classified into 110 operational taxonomy units (OTUs) based on a 98% similarity criterion. The similarity of the sequences ranged from 86 to 99% and 95 OTUs had less than 98% similarity to the sequences in the public databases. Coverage and the Shannon–Wiener index (H′) of the library were 83.9% and 4.29, respectively. The sequences were assigned to the following 6 phyla: Firmicutes (50.9% of the total number of sequences), Bacteroidetes (39.4%), Fibrobacteres (6.5%), Euryarchaeota (1.9%), Spirochaetes (1.0%), and Verrucomicrobia (0.3%); approximately 90% of the sequences were affiliated with Firmicutes and Bacteroidetes. The only OTU of Fibrobacteres (OTU 107), had 93 and 90% similarity to Fibrobacter succinogenes and F. intestinalis, respectively, suggesting a new species of Fibrobacter in ostrich ceca. Clostridium coccoides and C. leptum formed major groups within the Firmicutes. There was no OTU with high similarity (≥98%) to the 16S rDNA of cultivated fibrolytic bacteria in our library. Although two OTUs were affiliated with Euryarchaeota, no sequence was affiliated with methanogenic Archaea. This study presents the very complex ostrich cecal microbial community, in which the majority of the bacterial species have not yet been cultivated.     

Archaeal diversity in tidal flat sediment as revealed by 16S rDNA analysis

During the past ten years, Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages. More recently, the presence of novel archaeal phylogenetic lineages has been discovered in coastal marine environments, freshwater lakes, polar seas, and deep-sea hydrothermal vents. Therefore, we conducted an investigation into the archaeal community existing in tidal flat sediment collected from Ganghwa Island, Korea. Phylogenetic analysis of archaeal 16S rDNA amplified directly from tidal flat sediment DNA revealed the presence of two major lineages, belonging to the Crenarchaeota (53.9%) and Euryarchaeota (46.1%) phyla. A total of 102 clones were then sequenced and analyzed by comprehensive phylogenetic analysis. The sequences determined in our samples were found to be closely related to the sequences of clones which had been previously obtained from a variety of marine environments. Archaeal clones exhibited higher similarities (83.25-100%) to sequences from other environments in the public database than did those (75.22-98.46%) of previously reported bacterial clones obtained from tidal flat sediment. The results of our study suggest that the archaeal community in tidal flat sediment is remarkably diverse.     

Microbial community of a volcanic mudspring in the Philippines as revealed by 16S rDNA sequence analysis and fluorescence in situ hybridization

Mt. Makiling Mudspring in Laguna, Philippines is a thermophilic, acidophilic environment that previously has been shown to harbor novel microorganisms. We assessed the microbial community that exists at this volcanic mudspring using 16S rRNA-based approaches. DNA was extracted from solfataric soils and sediments taken from Mudspring. The 16S rDNA was PCR amplified using universal (519F-1392R) and archaeal-specific (23FPL-1391R) primer pairs, cloned, and sequenced. Phylogenetic analysis of the cloned 16S rDNA showed that eleven clones clustered with, and therefore related to Sulfolobus tokodaii 7 and two clones clustered with S. solfataricu, S. shibatae and S. islandicus. Three clone sequences were related to those found in thermophilic chalcopyrite (CuFeS₂), a copper sulfuric ore from bioleaching reactors. One clone had low similarity (95% identity) with uncultured archaeon clone KOZ184. Fluorescence in situ hybridization (FISH) analysis revealed that about 71% of the microbial community present in the Mudspring belong to domain Archaea of which 63% were Crenarchaeota and 8% were Euryarchaeota. Seventeen percent (17%) of the population consisted of bacteria as indicated by the positive hybridization with the BACT338 probe, and the remaining 12% are unidentified. This study is the first attempt to use molecular techniques in any environment in the Philippines.     

Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.     

The phylogenetic diversity of thermophilic members of the genus Bacillus as revealed by 16S rDNA analysis

Abstract Sixteen thermophilic strains of the genus Bacillus , representing eight validly described and six invalidly described species, as well as one unassigned strain, were investigated by comparative 16S rDNA analyses and the sequences compared to the existing database for the genera Bacillus and Alicyclobacillus . The majority of strains were found to cluster in two groups represented by B. stearothermophilus and B. pallidus. Bacillus smithii, B. thermocloacae , and B. thermoruber are phylogenetically well separated and cluster within the radiation of mesophilic bacilli. The as yet undescribed taxon 'B. flavothermus' warrants species status. B. schlegelii and B. tusciae group peripherally with members of Alicyclobacillus and may be reclassified when more phenotypic data support their phylogenetic position.     

Picobenthic cyanobacterial populations revealed by 16S rRNA-targeted in situ hybridization

We report on the morphological identification of a population of benthic cyanobacteria from microbial mats, known previously only from molecular analyses of field samples, based on the retrieval of environmental 16S rRNA sequences. We used in situ hybridization with horseradish peroxidase-labelled oligonucleotide probes designed to target the 16S rRNA of our unidentified population. Two probes were designed and checked for target binding ability and specificity using membrane hybridization against electroblotted bands from a denaturant gradient gel electrophoresis (DGGE) fingerprint of 16S rDNA gene fragments from the original cyanobacterial community. Under in situ hybridization, these probes bound specifically to extremely small, unicellular, colony-forming cyanobacteria, 0.75-1 microm in diameter, which were embedded in abundant mucilaginous investments. We propose the term picobenthos, by analogy with picoplankton, to describe those unicellular benthic microbes around or less than 1 microm in diameter. Although picoplanktonic cyanobacteria are abundant in ocean and freshwaters, picobenthic (<1 microm) unicellular cyanobacteria are not typically recognized as a major component of microbial mats. The small size and low levels of photopigment autofluorescence from these cells probably rendered them cryptic or indistinguishable from heterotrophic bacteria in routine microscopic observations. It is not known how widespread picobenthic cyanobacteria may be in other environments.     

Effect of disodium fumarate on ruminal metabolism and rumen bacterial communities as revealed by denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA

The aim of the present study was to examine the effects of feeding diets with addition of disodium fumarate (DF) to goats on ruminal metabolism and changes of rumen bacterial communities. Four cannulated goats were used in a 4 × 4 Latin square design. The results showed that ruminal pH increased linearly (P<0.01)as the amount of DF added increased, while lactate production decreased linearly (P<0.01). DF addition did not affect the production of acetate, propionate, butyrate, TVFA and NH₃-N. The effect of DF on the changes in rumen bacterial-community structure of goats was analyzed using 16S rDNA-based approaches. Amplicons of the V6-V8 variable regions of bacterial 16S rDNA were analyzed by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing. Differences in rumen bacterial community structure were determined based on the Shannon index of diversity for pairwise comparison of the DGGE fingerprints and revealed significant changes in rumen microbiota after DF addition. As compared with those fed with the control diet, goats fed on the diets with DF addition showed a higher bacterial diversity. The sequences of seven amplicons in total 11 clones showed less than 97% similarity with those of previously identified or unidentified bacteria, suggesting that most bacteria in the gastrointestinal tract have not been cultured or identified. Amplicons related to Succinivibrio dextrinosolvens species were found in most DGGE fingerprints derived from goats on the diet containing DF, but not in goats on the control diet. These results demonstrated the ability of DF to improve the metabolism of rumen lactate fermentation and to influence the bacterial composition of the rumen in goats.     

A bioengineering system for in situ bioremediation of contaminated groundwater

Much of the past and current focus of bioremediation has been on laboratory studies of microbial processes. By necessity, early studies have ignored important field properties, parameters, and processes that control the ultimate success of in situ bioremediation of contaminated groundwater. This paper presents a bioengineering systems approach that examines the impact of some of these field variables on common bioremediation practices. Using simple systems, the niche of biostimulation is shown to be aquifers with high contaminant sorption. A novel gas-phase biostimulation filter and a novel resting-state bioaugmentation/biofilter approach which show promise for effective field implementation are discussed. Received 08 December 1995/ Accepted in revised form 30 July 1996     

Numerical dominance and phylotype diversity of marine Rhodobacter species during early colonization of submerged surfaces in coastal marine waters as determined by 16S ribosomal DNA sequence analysis and fluorescence in situ hybridization

Early stages of surface colonization in coastal marine waters appear to be dominated by the marine Rhodobacter group of the alpha subdivision of the division Proteobacteria (alpha-Proteobacteria). However, the quantitative contribution of this group to primary surface colonization has not been determined. In this study, glass microscope slides were incubated in a salt marsh tidal creek for 3 or 6 days. Colonizing bacteria on the slides were examined by fluorescence in situ hybridization by employing DNA probes targeting 16S or 23S rRNA to identify specific phylogenetic groups. Confocal laser scanning microscopy was then used to quantify and track the dynamics of bacterial primary colonists during the early stages of surface colonization and growth. More than 60% of the surface-colonizing bacteria detectable by fluorescence staining (Yo-Pro-1) could also be detected with the Bacteria domain probe EUB338. Archaea were not detected on the surfaces and did not appear to participate in surface colonization. Of the three subdivisions of the Proteobacteria examined, the alpha-Proteobacteria were the most abundant surface-colonizing organisms. More than 28% of the total bacterial cells and more than 40% of the cells detected by EUB338 on the surfaces were affiliated with the marine Rhodobacter group. Bacterial abundance increased significantly on the surfaces during short-term incubation, mainly due to the growth of the marine Rhodobacter group organisms. These results demonstrated the quantitative importance of the marine Rhodobacter group in colonization of surfaces in salt marsh waters and confirmed that at least during the early stages of colonization, this group dominated the surface-colonizing bacterial assemblage.     

Detection of Leptospiraceae by amplification of 16S ribosomal DNA

The polymerase chain reaction (PCR) was developed to detect Leptospiraceae. Primers were used to amplify 1 631 base-pair (bp) 5'-region of 16S rDNA. Representative strains from the species, Leptospira interrogans sensu stricto, L. borgpetersenii, L. noguchii, L. santarosai, L. weilii, L. inadai, L. meyeri and the single member strain of Leptonema were amplified. In contrast, strains representing the saprophytic species. L. biflexa, L. wolbachii and L. parva were not amplified. There was no PCR product from 23 phylogenetically unrelated species of bacteria. As little as 10-1 pg of purified DNA and as few as 10-1 leptospires could be detected using the PCR analysis. Isolates of leptospires from clinical sources gave a positive PCR band, but those from surface waters did not.     

Bacterial,archaeal and eukaryotic diversity in Arctic sediment as revealed by 16S rRNA and 18S rRNA gene clone libraries analysis

We studied the microbial diversity in the sediment from the Kongsfjorden, Svalbard, Arctic, in the summer of 2005 based on the analysis of 16S rRNA and 18S rRNA gene clone libraries. The sequences of the cloned 16S rRNA and 18S rRNA gene inserts were used to determine the species identity or closest relatives by comparison with sequences of known species. Compared to the other samples acquired in Arctic and Antarctic, which are different from that of ours, the microbial diversity in our sediment is much higher. The bacterial sequences were grouped into 11 major lineages of the domain Bacteria: Proteobacteria (include α-, β-, γ-, δ-, and ε-Proteobacteria); Bacteroidetes; Fusobacteria; Firmicutes; Chloroflexi; Chlamydiae; Acidobacteria; Actinobacteria; Planctomycetes; Verrucomicrobiae and Lentisphaerae. Crenarchaeota were dominant in the archaeal clones containing inserts. In addition, six groups from eukaryotes including Cercozoa, Fungi, Telonema, Stramenopiles, Alveolata, and Metazoa were identified. Remarkably, the novel group Lentisphaerae was reported in Arctic sediment at the first time. Our study suggested that Arctic sediment as a unique habitat may contain substantial microbial diversity and novel species will be discovered.     

Investigation of catechol 2,3-dioxygenase and 16S rRNA gene diversity in hypoxic, petroleum hydrocarbon contaminated groundwater

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of the contaminants under aerobic conditions. In this study, we investigated the diversity and distribution of Comamonadaceae family (Betaproteobacteria) related catechol 2,3-dioxygenase genes, which belong to the I.2.C subfamily of extradiol dioxygenase genes. These catabolic genes encode enzymes supposed to function under hypoxic conditions as well, and may play a notable role in BTEX degradation in oxygen limited environments. Therefore, their diversity was analyzed in oxygen limited, petroleum hydrocarbon contaminated groundwater by terminal restriction fragment length polymorphism and cloning. Subfamily I.2.C related catechol 2,3-dioxygenase genes were detected in every investigated groundwater sample and a dynamic change was observed in the case of the structure of C23O gene possessing bacterial communities. To link the metabolic capability to the microbial structure, 16S rRNA gene-based clone libraries were generated and it was concluded that Betaproteobacteria were abundant in the bacterial communities of the contaminated samples. These results support the opinion that Betaproteobacteria may play a significant role in BTEX degradation under hypoxic conditions.     

Genetic diversity of Colombian sylvatic Trypanosoma cruzi isolates revealed by the ribosomal DNA

American trypanosomiasis is a common zoonosis in Colombia and Trypanosoma cruzi presents a wide distribution throughout the country. Although some studies based on enzyme electrophoresis profiles have described the population structure of the parasite, very few molecular analyses of genotipic markers have been conducted using Colombian strains. In this study, we amplified the non-transcribed spacer of the mini-gene by PCR, typing the isolates as T. cruzi I, T. cruzi zymodeme 3 or T. rangeli. In addition, the internal transcribed spacers of the ribosomal gene concomitant with the 5.8S rDNA were amplified and submitted to restriction fragment polymorphism analysis. The profiles were analyzed by a numerical methodology generating a phenetic dendrogram that shows heterogeneity among the T. cruzi isolates. This finding suggests a relationship between the complexity of the sylvatic transmission cycle in Colombia and the diversity of the sylvan parasites.     

Relationships among the bdellovibrios revealed by partial sequences of 16S ribosomal RNA

Members of the genusBdellovibrio possess the unifying phenotypic trait of attacking and preying upon other Gram-negative bacteria. It has been suggested that this common lifestyle arose by convergent evolution. Physiological and G + C studies have led to the notion that bdellovibrios are a heterogeneous group of loosely related bacteria. We have inferred the phylogenetic relatedness of 12 strains ofBdellovibrio through the analysis of partial 16S ribosomal RNA sequences. Similarity and degree of homology were assessed, and a phylogenetic tree was constructed by the distance matrix method. One branch of the two-branched tree consisted ofB. bacteriovorus and related strains (W, 6-5-S, 109, 109D, 109J, 114, HI Ox9-2, and HI Ox9-3). The other branch was itself branched, withB. starrii, B. stolpii, and marine strain BM4 in separate sub-branches. AllBdellovibrio strains in turn clustered with representatives of the delta division of theProteobacteria. The results indicate that there are at least two subdivisions of the genusBdellovibrio and that present-day bdellovibrios arose from a common ancestor. The placement of the genusBdellovibrio within the delta division of theProteobacteria was confirmed.     

Uncultured bacterial diversity in a seawater recirculating aquaculture system revealed by 16S rRNA gene amplicon sequencing

Bacterial diversity in a seawater recirculating aquaculture system (RAS) was investigated using 16S rRNA amplicon sequencing to understand the roles of bacterial communities in the system. The RAS was operated at nine different combinations of temperature (15°C, 20°C, and 25°C) and salinity (20‰, 25‰, and 32.5‰). Samples were collected from five or six RAS tanks (biofilters) for each condition. Fifty samples were analyzed. Proteobacteria and Bacteroidetes were most common (sum of both phyla: 67.2% to 99.4%) and were inversely proportional to each other. Bacteria that were present at an average of ≥ 1% included Actinobacteria (2.9%) Planctomycetes (2.0%), Nitrospirae (1.5%), and Acidobacteria (1.0%); they were preferentially present in packed bed biofilters, mesh biofilters, and maturation biofilters. The three biofilters showed higher diversity than other RAS tanks (aerated biofilters, floating bed biofilters, and fish tanks) from phylum to operational taxonomic unit (OTU) level. Samples were clustered into several groups based on the bacterial communities. Major taxonomic groups related to family Rhodobacteraceae and Flavobacteriaceae were distributed widely in the samples. Several taxonomic groups like [Saprospiraceae], Cytophagaceae, Octadecabacter, and Marivita showed a cluster-oriented distribution. Phaeobacter and Sediminicola-related reads were detected frequently and abundantly at low temperature. Nitrifying bacteria were detected frequently and abundantly in the three biofilters. Phylogenetic analysis of the nitrifying bacteria showed several similar OTUs were observed widely through the biofilters. The diverse bacterial communities and the minor taxonomic groups, except for Proteobacteria and Bacteroidetes, seemed to play important roles and seemed necessary for nitrifying activity in the RAS, especially in packed bed biofilters, mesh biofilters, and maturation biofilters.