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PubMed1998

A Comparison of the Use of In Vitro-Transcribed and Native rRNA for the Quantification of Microorganisms in the Environment.

Abstract Nearly full-length, small subunit (SSU) rRNA was transcribed in vitro from clones of SSU rDNA genes. Comparing the use of in vitro-transcribed and native rRNA indicated that, when in vitro-transcribed rRNA was used as a standard for quantitative hybridizations with oligonucleotide probes, the population was consistently underestimated. The population abundance was expressed as a percentage of specific target SSU rRNA (determined with a specific oligonucleotide probe), relative to the total SSU rRNA (measured with a universal probe). Differences in hybridization signals could be related to specific probe target locations and rRNA denaturation conditions, suggesting that higher order structure is important in quantitative membrane hybridizations. Therefore, in vitro-transcribed rRNA cannot always be used for the absolute quantification of microbial populations, but can be employed as a standard to quantify shifts in population abundance over time, and to compare community structure in various environments.

Journal Article↗

PubMed1997

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

Bacteria, Anaerobic↗

PubMed2004

Stable isotope probing of rRNA and DNA reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice field soil.

Stable isotope probing (SIP) is a novel technique to characterize structure and in situ function of active microbial populations, which is based on the incorporation of 13C-labelled substrates into nucleic acids. Here, we have traced methylotrophic members of a rice field soil microbial community, which became active upon continuous addition of 13C-methanol (< 22 mM) as studied in microcosms. By combining rRNA- and DNA-based SIP, as well as domain-specific real-time PCR detection of templates in fractions of centrifugation gradients, we were able to detect 13C-labelled bacterial rRNA after 6 days of incubation. Fingerprinting and comparative sequence analysis of 'heavy' bacterial rRNA showed that mostly members of the Methylobacteriaceae and a novel clade within the Methylophilaceae formed part of the indigenous methylotrophic community. Over time, however, the Methylophilaceae were enriched. Unexpectedly, nucleic acids of eukaryotic origin were detected, mostly in intermediately 13C-labelled gradient fractions. These eukaryotes were identified as fungi mostly related to Fusarium and Aspergillus spp., and also Cercozoa, known as predatory soil flagellates. The detection of fungi and protozoa in 13C-enriched nucleic acid fractions suggests a possible involvement in either direct assimilation of label by the fungi, or a food web, i.e. that primary 13C-methanol consuming methylotrophs were decomposed by fungi and grazed by protozoa.

Animals↗

PubMed2003

Substrata effects on bacterial biofilm development in a subsurface flow dairy waste treatment wetland.

Biofilm development on two distinct rock substrata was investigated both in vitro and in a subsurface flow wastewater treatment wetland in order to determine the effect of hydrophobicity on initial bacterial adsorption, tertiary biofilm development and microbial population structure. Two commonly used wetland rock types, slag (a hydrophobic by-product of the steel smelter industry) and greywacke (a more hydrophilic sedimentary rock) were evaluated. In vitro investigations of initial microbial adsorption trends showed that the more hydrophobic slag displayed rapid bacterial adsorption rates compared to greywacke. Mean microbial adsorption rates of a mixed wetland bacterial population over 5 hours, described using a first order kinetics model, were 1.3 x 10(-12) m/sec for slag and consistently lower at 8.7 x 10(-13) m/sec for greywacke. Pristine rock studs of the two substrata were also exposed to wetland microbial communities during a six week field trial using confocal scanning laser microscopy to determine tertiary biofilm structure and fluorescent in situ hybridisation to investigate bacterial populations. During the first five weeks of growth CSLM analysis revealed that 75% of biofilms on slag were thicker and had greater coverage compared with those grown on greywacke. After six weeks of growth over 50% of the tertiary biofilms were structurally very similar on both rock types and only 25% of those grown on slag were larger than those on greywacke. In situ hybridisation analysis of bacterial populations revealed very little difference in population structure between biofilms grown on slag and those grown on greywacke. Eubacteria were present as a very high proportion of total bacteria throughout biofilm development (74.3%). The beta subgroup was the most populous of the Proteobacteria (31.4%) followed by the gamma subgroup (13.4%) and the alpha subgroup (1.3%). The results of this study suggest that slag, as a more hydrophobic substratum, promotes the initial adsorption of bacteria during early biofilm growth and better supports mature biofilm structures when used in wetlands. This study has implications for the design and construction of wastewater treatment wetlands.

Adsorption↗

PubMed2001

Comparative effects of a genetically engineered insect virus and a growth-regulating insecticide on microbial communities in aquatic microcosms.

The effects of a genetically engineered insect baculovirus on indigenous aquatic microbial communities were determined in closed, recirculating aquatic microcosms, and compared with the effects of a natural strain of the virus and of a growth-regulating insecticide, Dimilin. The recombinant virus was a nuclear polyhedrosis virus (NPV) of the spruce budworm (Choristoneura fumiferana (Cf)) with a lacZ marker gene inserted into the egt region of the CfNPV. The natural virus was Ireland strain CfNPV. Microbial measurement endpoints included decomposition activity (mass loss of organic material), respiration on two different substrates (O2 consumption), heterotrophic bacterial abundance (plate counts), and microbial community metabolic profiles (carbon source utilization patterns in Biolog GN microplates). Viral DNA of both the natural strain and the recombinant viruses, detected by polymerase chain reaction techniques, settled out of the microcosm water and accumulated on bottom substrates within 3 days of the microcosm inoculations. The viral DNA persisted in bottom substrates for the duration of the 21-day experimental period, although there was some evidence that the recombinant virus was less stable than the natural strain in particulate organic matter. No significant changes in microbial decomposition or respiration activity, bacterial abundance, or average metabolic responses were detected by a time trend analysis in microcosms inoculated with either the lacZ recombinant virus or the natural Ireland strain CfNPV. Significant effects on microbial decomposition and respiration activity were detected in microcosms treated with the growth-regulating insecticide at, and above, the expected environmental concentrations. Despite significant effects on microbial community functional attributes in Dimilin-treated microcosms, there were no detectable changes in community structure in terms of metabolic profiles or bacterial abundance.

DNA, Viral↗

PubMed2001

Biology of extremophilic and extremotolerant methanotrophs.

This review summarizes recent findings on the biology of obligate methanotrophic bacteria living in various extreme environments. By using molecular ecology techniques, it has become clear that obligate methanotrophs are ubiquitous in nature and well adapted to high or low temperature, pH and salinity. The isolation and characterization of pure cultures has led to the discovery of several new genera and species of extremophilic/tolerant methanotrophs. Their major physiological role is participation in the methane cycle and supplying C(1) intermediates and various metabolites to other members of microbial communities in extreme ecosystems. To survive under extreme conditions, methanotrophs have developed diverse structure-function adaptive mechanisms including cell-surface layer formation, changes in cellular phospholipid composition and de novo synthesis of organic osmolytes such as ectoine, 5-oxoproline and sucrose. However, despite the above advances, basic knowledge of other stress protectants, as well as bioenergetic and genetic aspects of methanotroph adaptation, is still lacking. This information is necessary for better understanding the molecular mechanisms underlying the versatility of methanotrophs and for the development of novel biotechnological processes.

Cold Temperature↗

PubMed2003

Scanning transmission X-ray, laser scanning, and transmission electron microscopy mapping of the exopolymeric matrix of microbial biofilms.

Confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and soft X-ray scanning transmission X-ray microscopy (STXM) were used to map the distribution of macromolecular subcomponents (e.g., polysaccharides, proteins, lipids, and nucleic acids) of biofilm cells and matrix. The biofilms were developed from river water supplemented with methanol, and although they comprised a complex microbial community, the biofilms were dominated by heterotrophic bacteria. TEM provided the highest-resolution structural imaging, CLSM provided detailed compositional information when used in conjunction with molecular probes, and STXM provided compositional mapping of macromolecule distributions without the addition of probes. By examining exactly the same region of a sample with combinations of these techniques (STXM with CLSM and STXM with TEM), we demonstrate that this combination of multimicroscopy analysis can be used to create a detailed correlative map of biofilm structure and composition. We are using these correlative techniques to improve our understanding of the biochemical basis for biofilm organization and to assist studies intended to investigate and optimize biofilms for environmental remediation applications.

Bacteria↗

PubMed2003

Linking floc structure and settling properties to activated sludge population dynamics in an SBR.

Over a period of 227 days properties of activated sludge grown in an sequencing batch reactor (SBR) operated under stable conditions were analyzed. Settling properties (sludge volume index (SVI)) of the activated sludge were compared with on-line measurements of floc size and size distribution obtained by using a laser light scattering technique (Malvern Mastersizer/S, Malvern, UK), and with measurements of microbial community dynamics analyzed by denaturing gradient gel electrophoresis (DGGE) patterns of 16S rRNA genes. In addition, microscopical observations were used to confirm the results. Three distinct stages in the SBR evolution were observed. In the first stage the structural floc properties showed predominant presence of floc-forming bacteria in the activated sludge. A good correlation between floc size, properties and microbial community evolution was observed. The second stage showed a good balance between floc-forming and filamentous bacteria, with good settling properties and a highly dynamic community in the SBR. In the third stage, an increase in the filamentous bacteria, which became predominant in the system was observed. Again, a good correlation between settling properties and floc size distribution was obtained and a new dominant species was observed in the DGGE patterns, which can be assumed to be a filamentous organism.

Bioreactors↗

PubMed1998

Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes.

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge-1 day-1. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769-773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH4-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea. However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.

Journal Article↗

PubMed2005

Analysis of community structures in anaerobic processes using a quantitative real-time PCR method.

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.

Acetates↗

PubMed2004

Genomic and mechanistic insights into the biodegradation of organic pollutants.

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.

Bacteria↗

PubMed2001

Bacterial inoculum enhances keratin degradation and biofilm formation in poultry compost.

Native microbial populations can degrade poultry waste, but the process can be hastened by using feather-degrading bacteria. Strains of Bacillus licheniformis and a Streptomyces sp. isolated from the plumage of wild birds were grown in a liquid basal medium and used to inoculate feathers in compost bioreaction vessels. Control vessels had only basal medium added to the feathers, litter and straw. Temperature, ammonia, carbon and nitrogen were monitored for 4 weeks. Scanning electron microscopy of the feather samples showed more complete keratin-degradation, more structural damage, and earlier microbial biofilm formation on inoculated feathers than on uninoculated feathers. A diverse community of aerobic bacteria and fungi were cultured early, but declined rapidly. Thermophilic B. licheniformis and Streptomyces spp. were abundant throughout. Enteric gram-negative bacteria, (e.g., Salmonella, E. coli) originally found on waste feathers were not recovered after day 4. Vessel temperatures reached 64-71 degrees C within 36 h and stabilized at 50 degrees C. When tumble-mixed at day 14, renewed activity peaked at 59 degrees C and quickly dropped as available carbon was used. Feathers soaked in an inoculum of B. licheniformis and Streptomyces degraded more quickly and more completely than feathers that were not presoaked. Inoculation of feather waste could improve composting of the large volume of feather waste generated every year by poultry farms and processing plants.

Animals↗

PubMed1999

Structure and function of the methanogenic archaeal community in stable cellulose-degrading enrichment cultures at two different temperatures (15 and 30 degrees C).

Methanogenic cultures were enriched from an air-dried rice field soil and incubated under anaerobic conditions at 30 degrees C with cellulose as substrate (ET1). The culture was then transferred and further incubated at either 15 degrees C (E15) or 30 degrees C (E30), to establish stable cultures that methanogenically degrade cellulose. After five transfers, the rates of CH(4) production became reproducible. At 30 degrees C, CH(4) production rates were (mean+/-S.D.) 15.2+/-0.7 nmol h(-1) ml(-1) culture for the next 16 transfers and at 15 degrees C, they were 0.38+/-0.07 nmol h(-1) ml(-1) for the next six transfers. When E30 was assayed at temperatures between 5-50 degrees C, CH(4) production rates increased with the temperature, reached a maximum at 40 degrees C and then decreased. The same temperature optimum was observed in E15, but with a lower maximum CH(4) production rate. The apparent activation energies of CH(4) production were similar (about 120 kJ mol(-1)4 mM at the beginning of the assay. The structure of the archaeal community was analyzed by molecular techniques. Total DNA was extracted from the microbial cultures before the transfer to different temperatures (ET1) and afterwards (E15, E30). The archaeal small subunit (SSU) ribosomal RNA-encoding genes (rDNA) of these DNA samples were amplified by PCR with archaeal-specific primers and characterized by terminal restriction fragment length polymorphism (T-RFLP). After obtaining a constant T-RFLP pattern in the cultural transfers at 15 and 30 degrees C, the PCR amplicons were used for the generation of clone libraries. Representative rDNA clones (n=10 for each type of culture) were characterized by T-RFLP and sequence analysis. In the primary culture (ET1), the archaeal community was dominated by clones representing 'rice cluster I', a novel lineage of methanogenic Euryarchaeota. However, further transfers resulted in the dominance of Methanosarcinaceae and Methanosaetaceae at 30 and 15 degrees C, respectively. This dominance was confirmed by fluorescence in situ hybridization (FISH) of archaeal cells. Obviously, different archaeal communities were established at the two different temperatures, but their activities nevertheless exhibited similar temperature optima.

Journal Article↗

PubMed2006

Spatial scaling of microbial biodiversity.

A central goal in ecology is to understand the spatial scaling of biodiversity. Patterns in the spatial distribution of organisms provide important clues about the underlying mechanisms that structure ecological communities and are central to setting conservation priorities. Although microorganisms comprise much of Earth's biodiversity, little is known about their biodiversity scaling relationships relative to that for plants and animals. Here, we discuss current knowledge of microbial diversity at local and global scales. We focus on three spatial patterns: the distance-decay relationship (how community composition changes with geographic distance), the taxa-area relationship, and the local:global taxa richness ratio. Recent empirical analyses of these patterns for microorganisms suggest that there are biodiversity scaling rules common to all forms of life.

Animals↗

PubMed1990

Genetic diversity in Sargasso Sea bacterioplankton.

Bacterioplankton are recognized as important agents of biogeochemical change in marine ecosystems, yet relatively little is known about the species that make up these communities. Uncertainties about the genetic structure and diversity of natural bacterioplankton populations stem from the traditional difficulties associated with microbial cultivation techniques. Discrepancies between direct counts and plate counts are typically several orders of magnitude, raising doubts as to whether cultivated marine bacteria are actually representative of dominant planktonic species. We have phylogenetically analysed clone libraries of eubacterial 16S ribosomal RNA genes amplified from natural populations of Sargasso Sea picoplankton by the polymerase chain reaction. The analysis indicates the presence of a novel microbial group, the SAR11 cluster, which appears to be a significant component of this oligotrophic bacterioplankton community. A second cluster of lineages related to the oxygenic phototrophs--cyanobacteria, prochlorophytes and chloroplasts--was also observed. However, none of the genes matched the small subunit rRNA sequences of cultivated marine cyanobacteria from similar habitats. The diversity of 16S rRNA genes observed within the clusters suggests that these bacterioplankton may be consortia of independent lineages sharing surprisingly distant common ancestors.

Animals↗

PubMed2006

Phylogenetic and functional biomakers as indicators of bacterial community responses to mixed-waste contamination.

Few studies have demonstrated changes in community structure along a contaminant plume in terms of phylogenetic, functional, and geochemical changes, and such studies are essential to understand how a microbial ecosystem responds to perturbations. Clonal libraries of multiple genes (SSU rDNA, nirK, nirS, amoA, pmoA, and dsrAB) were analyzed from groundwater samples (n = 6) that varied in contaminant levels, and 107 geochemical parameters were measured. Principal components analyses (PCA) were used to compare the relationships among the sites with respect to the biomarker (n = 785 for all sequences) distributions and the geochemical variables. A major portion of the geochemical variance measured among the samples could be accounted for by tetrachloroethene, 99Tc, No3, SO4, Al, and Th. The PCA based on the distribution of unique biomarkers resulted in different groupings compared to the geochemical analysis, but when the SSU rRNA gene libraries were directly compared (deltaC(xy) values) the sites were clustered in a similar fashion compared to geochemical measures. The PCA based upon functional gene distributions each predicted different relationships among the sites, and comparisons of Euclidean distances based upon diversity indices for all functional genes (n = 432) grouped the sites by extreme or intermediate contaminant levels. The data suggested that the sites with low and high perturbations were functionally more similar than sites with intermediate conditions, and perhaps captured the overall community structure better than a single phylogenetic biomarker. Moreover, even though the background site was phylogenetically and geochemically distinct from the acidic sites, the extreme conditions of the acidic samples might be more analogous to the limiting nutrient conditions of the background site. An understanding of microbial community-level responses within an ecological framework would provide better insight for restoration strategies at contaminated field sites.

Bacteria↗

PubMed2002

Metabolic shifts in hypersaline microbial mats upon addition of organic substrates.

The responses of hypersaline microbial mats to the addition of acetate, glycolate or glucose were investigated using oxygen, pH and sulphide microsensors. Changes in community structure were investigated with molecular techniques. Acetate addition inhibited respiration in the photic zone, stimulated respiration in the aphotic zone and had no effect on gross photosynthesis. Glycolate addition strongly increased both respiration and gross photosynthesis in the photic zone. Thus, glycolate and acetate were probably consumed in those regions of the mat where these substrates are usually formed. Moreover, photosynthesis was only stimulated by increased respiration and concomitant CO2 production in the photic zone which indicates that the photosynthetic and respiratory populations must be present in close proximity to each other. Glucose addition had an unexpected negative effect on the microbial population, strongly inhibiting both respiration and gross photosynthesis within hours. After four days, oxygen profiles in the light were equal to those measured in the dark. After replacing the water phase with unamended water, photosynthesis and respiration recovered within a week. None of the physiological changes were accompanied by detectable shifts in the cyanobacterial or the overall microbial community. The mechanism of inhibition of photosynthesis by glucose requires further investigation.

Acetates↗

PubMed2006

The microbial degradation of azimsulfuron and its effect on the soil bacterial community.

AIMS: Azimsulfuron is a recently introduced sulfonylurea herbicide useful in controlling weeds in paddy fields. To date very little information is available on the biodegradation of this pesticide and on its effect on the soil microbial community. The aim of this work was to study its biodegradation both in slurry soil microcosms and in batch tests with mixed and pure cultures. METHODS AND RESULTS: Azimsulfuron was applied to forest bulk soil in order to study its effect on the structure of the bacterial soil community, as detectable by denaturant gradient gel electrophoresis (DGGE) analyses. Biodegradation and abiotic processes were investigated by HPLC analyses. In addition, a microbial consortium was selected, that was able to use azimsulfuron as the sole energy and carbon source. One of the metabolites produced by the consortium was isolated and identified through LC-MS analyses. Cultivable bacteria of the consortium were isolated and identified by 16S rDNA sequencing (1400 bp). CONCLUSIONS: Azimsulfuron treatment seems to have the ability to cause changes in the bacterial community structure that are detectable by DGGE analyses. It is easily biodegraded both in microcosms and in batch tests, with the formation of an intermediate that was identified as 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on the biodegradation of azimsulfuron and its effects on the soil microbiota.

Bacteria↗