scholarly journals Nutrient Amendments in Soil DNA Stable Isotope Probing Experiments Reduce the Observed Methanotroph Diversity

2006 ◽  
Vol 73 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Aur�lie C�bron ◽  
Levente Bodrossy ◽  
Nancy Stralis-Pavese ◽  
Andrew C. Singer ◽  
Ian P. Thompson ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Stable Isotope Probing ◽  
Population Diversity ◽  
Type I ◽  
Mineral Salts ◽  
Bacterial Populations ◽  
Soil Dna ◽  
Gradient Gel Electrophoresis ◽  
Soil Water Conditions

ABSTRACT Stable isotope probing (SIP) can be used to analyze the active bacterial populations involved in a process by incorporating 13C-labeled substrate into cellular components such as DNA. Relatively long incubation times are often used with laboratory microcosms in order to incorporate sufficient 13C into the DNA of the target organisms. Addition of nutrients can be used to accelerate the processes. However, unnatural concentrations of nutrients may artificially change bacterial diversity and activity. In this study, methanotroph activity and diversity in soil was examined during the consumption of 13CH4 with three DNA-SIP experiments, using microcosms with natural field soil water conditions, the addition of water, and the addition of mineral salts solution. Methanotroph population diversity was studied by targeting 16S rRNA and pmoA genes. Clone library analyses, denaturing gradient gel electrophoresis fingerprinting, and pmoA microarray hybridization analyses were carried out. Most methanotroph diversity (type I and type II methanotrophs) was observed in nonamended SIP microcosms. Although this treatment probably best reflected the in situ environmental conditions, one major disadvantage of this incubation was that the incorporation of 13CH4 was slow and some cross-feeding of 13C occurred, thereby leading to labeling of nonmethanotroph microorganisms. Conversely, microcosms supplemented with mineral salts medium exhibited rapid consumption of 13CH4, resulting in the labeling of a less diverse population of only type I methanotrophs. DNA-SIP incubations using water-amended microcosms yielded faster incorporation of 13C into active methanotrophs while avoiding the cross-feeding of 13C.

scholarly journals Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil

2005 ◽  
Vol 71 (3) ◽  
pp. 1202-1209 ◽  
Author(s):  
David R. Singleton ◽  
Sabrina N. Powell ◽  
Ramiah Sangaiah ◽  
Avram Gold ◽  
Louise M. Ball ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Separation Efficiency ◽  
Stable Isotope Probing ◽  
Rrna Gene ◽  
Clone Libraries ◽  
Bacterial Populations ◽  
E Coli ◽  
Gradient Gel Electrophoresis ◽  
Heavy Fractions ◽  
K 12

ABSTRACT [13C6]salicylate, [U-13C]naphthalene, and [U-13C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from Escherichia coli K-12 was added to each sample as an internal indicator of separation efficiency. While E. coli was not detected in most analyses of heavy DNA, a low number of E. coli sequences was recovered in the clone libraries associated with the heavy DNA fraction of [13C]phenanthrene incubations. The number of E. coli clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the Pseudomonas and Ralstonia genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the Acidovorax genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the 13C during the incubation did not appear to be an issue in this experiment.

scholarly journals RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny

2002 ◽  
Vol 68 (11) ◽  
pp. 5367-5373 ◽  
Author(s):  
Mike Manefield ◽  
Andrew S. Whiteley ◽  
Robert I. Griffiths ◽  
Mark J. Bailey
Keyword(s):  
Microbial Community ◽  
Stable Isotope ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Phenol Degradation ◽  
Stable Isotope Probing ◽  
Equilibrium Density ◽  
Community Function ◽  
Reverse Transcription Pcr ◽  
Gradient Gel Electrophoresis ◽  
Labeled Rna

ABSTRACT Identifying microorganisms responsible for recognized environmental processes remains a great challenge in contemporary microbial ecology. Only in the last few years have methodological innovations provided access to the relationship between the function of a microbial community and the phylogeny of the organisms accountable for it. In this study stable-isotope-labeled [13C]phenol was fed into a phenol-degrading community from an aerobic industrial bioreactor, and the 13C-labeled RNA produced was used to identify the bacteria responsible for the process. Stable-isotope-labeled RNA was analyzed by equilibrium density centrifugation in concert with reverse transcription-PCR and denaturing gradient gel electrophoresis. In contradiction with findings from conventional methodologies, this unique approach revealed that phenol degradation in the microbial community under investigation is dominated by a member of the Thauera genus. Our results suggest that this organism is important for the function of this bioreactor.

The comparison of the diversity of activated sludge plants

1998 ◽  
Vol 37 (4-5) ◽  
pp. 71-78 ◽  
Author(s):  
Thomas P. Curtis ◽  
Noel G. Craine
Keyword(s):  
Cluster Analysis ◽  
Activated Sludge ◽  
Treatment Process ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Optimal Sampling ◽  
Bacterial Populations ◽  
Large Numbers ◽  
Probable Presence ◽  
Gradient Gel Electrophoresis ◽  
Sampling Regime

The explicit engineering of bacterial populations requires that we know which organisms perform which tasks. The comparison of the bacterial diversity of activated sludge plants may give important information about the functions of different bacteria. This difficult task may be made easier by the use of technologies based on 16S rRNA based techniques. In this study we have used denaturing gradient gel electrophoresis (DGGE) to determine the optimal sampling regime for comparative studies and used cluster analysis to show how plants may be quantitatively compared. We sought evidence of spatial, diurnal and intrasample variation in a number of sites. No evidence for variation was found in the plants studied and we concluded that a single sample of an activated sludge plant was sufficient for a plant to plant comparison. The cluster analysis was able to distinguish between plants, though further work is required to find the most appropriate basis for such comparisons. We found organisms from raw sewage in the mixed liquor samples, these organisms may have no functional significance in the treatment process and thus complicate plant to plant comparisons as will the probable presence of heteroduplex rDNA products. Nevertheless we believe that these drawbacks do not outweigh the advantages of being able to take and compare relatively large numbers of samples.

PCR-DGGE analysis of denitrifying bacteria in a metallurgic wastewater treatment process

2002 ◽  
Vol 46 (1-2) ◽  
pp. 333-336 ◽  
Author(s):  
N. Noda ◽  
S. Yoshie ◽  
T. Miyano ◽  
S. Tsuneda ◽  
A. Hirata ◽  
...  
Keyword(s):  
Fluidized Bed ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Precious Metals ◽  
Packed Bed ◽  
Population Diversity ◽  
Operating Conditions ◽  
Industrial Wastes ◽  
Wastewater Treatment Process ◽  
Dgge Analysis ◽  
Gradient Gel Electrophoresis

The wastewater generated from the processes of recovering precious metals from industrial wastes contains high concentrations of acids such as nitric acid and of salts. Biological nitrogen removal from this wastewater was attempted by using a circulating bioreactor system equipped with an anoxic packed bed or an anoxic fluidized bed and an aerobic three-phase fluidized bed. The system was found to effectively remove nitrogen from the diluted wastewater (T-N; 1,000–4,000 mg litre−1). The microbial population structure of activated sludge in an anoxic reactor was analyzed by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. DGGE analysis under different operating conditions demonstrated the presence of some distinguishable bands in the separation pattern, which were most likely derived from many different species constituting the microbial communities. Furthermore, the population diversity varied in accordance with the nitrate-loading rate, water temperature and reactor condition. Some major DGGE bands were excised, reamplified and directly sequenced. It was revealed that the dominant population in the anoxic reactor were affiliated with the β subclass of the class Proteobacteria.

scholarly journals Molecular methods for exploring the intestinal ecosystem

2002 ◽  
Vol 87 (S2) ◽  
pp. S199-S201 ◽  
Author(s):  
G. W. Tannock
Keyword(s):  
Intestinal Microflora ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Molecular Methods ◽  
Bacterial Populations ◽  
Disease States ◽  
Large Numbers ◽  
Gradient Gel Electrophoresis ◽  
Health And Disease ◽  
Polymerase Chain

Molecular methods have provided renewed impetus for the analysis of the composition of the intestinal microflora in health and disease. The polymerase chain reaction coupled with denaturing gradient gel electrophoresis provides a method whereby the bacterial communities in large numbers of samples can be compared efficiently and effectively. Altered bacterial populations associated with disease states can then be targeted for further investigation. In the long-term, an ‘abnormal microflora’ might be rectified by the use of probiotics or prebiotics.

Methanotrophic diversity in high arctic wetlands on the islands of Svalbard (Norway) - denaturing gradient gel electrophoresis analysis of soil DNA and enrichment cultures

2003 ◽  
Vol 49 (10) ◽  
pp. 602-612 ◽  
Author(s):  
Ingvild Wartiainen ◽  
Anne Grethe Hestnes ◽  
Mette M Svenning
Keyword(s):  
Methane Emission ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
The Arctic ◽  
Rrna Gene ◽  
Climatic Warming ◽  
Soil Dna ◽  
Enrichment Cultures ◽  
Arctic Soil ◽  
Gradient Gel Electrophoresis

The methanotrophic community in arctic soil from the islands of Svalbard, Norway (78°N) was analysed by combining group-specific PCR with PCR of the highly variable V3 region of the 16S rRNA gene and then by denaturing gradient gel electrophoresis (DGGE). Selected bands were sequenced for identification. The analyses were performed with DNA extracted directly from soil and from enrichment cultures at 10 and 20 °C. The two genera Methylobacter and Methylosinus were found in all localities studied. The DGGE band patterns were simple, and DNA fragments with single base differences were separated. The arctic tundra is a potential source of extensive methane emission due to climatic warming because of its large reservoirs of stored organic carbon. Higher temperatures due to climatic warming can cause increased methane production, and the abundance and activity of methane-oxidizing bacteria in the arctic soil may be important regulators for methane emission to the atmosphere.Key words: methanotrophic diversity, Svalbard, arctic wetland, denaturing gradient gel electrophoresis.

scholarly journals Effect of DNA Extraction Method on the Apparent Microbial Diversity of Soil

2010 ◽  
Vol 76 (10) ◽  
pp. 3378-3382 ◽  
Author(s):  
Özgül İnceoǧlu ◽  
Eelco F. Hoogwout ◽  
Patrick Hill ◽  
Jan Dirk van Elsas
Keyword(s):  
Extraction Method ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Agricultural Soils ◽  
Extraction Methods ◽  
The Novel ◽  
Soil Dna ◽  
Dna Extraction Method ◽  
Gradient Gel Electrophoresis ◽  
Novel Method ◽  
Microbial Groups

ABSTRACT Four extraction methods, including a novel one, were compared for their efficiencies in producing DNA from three contrasting agricultural soils. Molecular analyses (PCR-denaturing gradient gel electrophoresis [DGGE] and clone libraries) focusing on different microbial groups were used as assessment criteria. Per soil, the DNA yields differed between extraction methods. Clear effects of method on apparent richness and community structure were found. Actinobacterial diversity based on soil DNA produced by two divergent methods revealed that a hitherto-undescribed group was obtained by the novel method.

Are some putative glycogen accumulating organisms (GAO) in anaerobic : aerobic activated sludge systems members of the α-Proteobacteria?

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2267-2275 ◽  
Author(s):  
Michael Beer ◽  
Yun H. Kong ◽  
Robert J. Seviour
Keyword(s):  
Activated Sludge ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Batch Reactors ◽  
Sequencing Batch Reactors ◽  
Bacterial Populations ◽  
Removal Capacity ◽  
Large Numbers ◽  
Gradient Gel Electrophoresis ◽  
Polyphosphate Accumulating Organisms

Activated sludge plants designed to remove phosphorus microbiologically often perform unreliably. One suggestion is that the polyphosphate-accumulating organisms (PAO) are out-competed for substrates by another group of bacteria, the glycogen-accumulating organisms (GAO) in the anaerobic zones of these processes. This study used fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) to analyse the communities from laboratory-scale anaerobic : aerobic sequencing batch reactors. Members of the genus Sphingomonas in the α-Proteobacteria were present in large numbers in communities with poor phosphorus removal capacity where the biomass had a high glycogen content. Their ability to store poly-β-hydroxyalkanoates anaerobically, but not aerobically, and not accumulate polyphosphate aerobically is consistent with these organisms behaving as GAO there. No evidence was found to support an important role for the γ-Proteobacteria as possible GAO in these communities, although these bacterial populations have been considered in other studies to act as possible competitors for the PAO.

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