Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers

ABSTRACT The response of natural microbial communities to environmental change can be assessed by determining DNA- or RNA-targeted changes in relative abundance of 16S rRNA gene sequences by using fingerprinting techniques such as denaturing gradient gel electrophoresis (DNA-DGGE and RNA-DGGE, respectively) or by stable isotope probing (SIP) of 16S rRNA genes following incubation with a 13C-labeled substrate (DNA-SIP-DGGE). The sensitivities of these three approaches were compared during batch growth of communities containing two or three Nitrosospira pure or enriched cultures with different tolerances to a high ammonia concentration. Cultures were supplied with low, intermediate, or high initial ammonia concentrations and with 13C-labeled carbon dioxide. DNA-SIP-DGGE provided the most direct evidence for growth and was the most sensitive, with changes in DGGE profiles evident before changes in DNA- and RNA-DGGE profiles and before detectable increases in nitrite and nitrate production. RNA-DGGE provided intermediate sensitivity. In addition, the three molecular methods were used to follow growth of individual strains within communities. In general, changes in relative activities of individual strains within communities could be predicted from monoculture growth characteristics. Ammonia-tolerant Nitrosospira cluster 3b strains dominated mixed communities at all ammonia concentrations, and ammonia-sensitive strains were outcompeted at an intermediate ammonia concentration. However, coexistence of ammonia-tolerant and ammonia-sensitive strains occurred at the lowest ammonia concentration, and, under some conditions, strains inhibited at high ammonia in monoculture were active at high ammonia in mixed cultures, where they coexisted with ammonia-tolerant strains. The results therefore demonstrate the sensitivity of SIP for detection of activity of organisms with relatively low yield and low activity and its ability to follow changes in the structure of interacting microbial communities.

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Stable-Isotope Probing Identifies Uncultured Planctomycetes as Primary Degraders of a Complex Heteropolysaccharide in Soil

Applied and Environmental Microbiology ◽

10.1128/aem.00055-15 ◽

2015 ◽

Vol 81 (14) ◽

pp. 4607-4615 ◽

Cited By ~ 49

Author(s):

Xiaoqing Wang ◽

Christine E. Sharp ◽

Gareth M. Jones ◽

Stephen E. Grasby ◽

Allyson L. Brady ◽

...

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

Soil Bacteria ◽

Stable Isotope Probing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Content Type ◽

Degrading Bacteria ◽

Aerobic Soil

ABSTRACTThe exopolysaccharides (EPSs) produced by some bacteria are potential growth substrates for other bacteria in soil. We used stable-isotope probing (SIP) to identify aerobic soil bacteria that assimilated the cellulose produced byGluconacetobacter xylinusor the EPS produced byBeijerinckia indica. The latter is a heteropolysaccharide comprised primarily ofl-guluronic acid,d-glucose, andd-glycero-d-mannoheptose.13C-labeled EPS and13C-labeled cellulose were purified from bacterial cultures grown on [13C]glucose. Two soils were incubated with these substrates, and bacteria actively assimilating them were identified via pyrosequencing of 16S rRNA genes recovered from13C-labeled DNA. Cellulose C was assimilated primarily by soil bacteria closely related (93 to 100% 16S rRNA gene sequence identities) to known cellulose-degrading bacteria. However,B. indicaEPS was assimilated primarily by bacteria with low identities (80 to 95%) to known species, particularly by different members of the phylumPlanctomycetes. In one incubation, members of thePlanctomycetesmade up >60% of all reads in the labeled DNA and were only distantly related (<85% identity) to any described species. Although it is impossible with SIP to completely distinguish primary polysaccharide hydrolyzers from bacteria growing on produced oligo- or monosaccharides, the predominance ofPlanctomycetessuggested that they were primary degraders of EPS. Other bacteria assimilatingB. indicaEPS included members of theVerrucomicrobia, candidate division OD1, and theArmatimonadetes. The results indicate that some uncultured bacteria in soils may be adapted to using complex heteropolysaccharides for growth and suggest that the use of these substrates may provide a means for culturing new species.

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Identification of a Stable Hydrogen-Driven Microbiome in a Highly Radioactive Storage Facility on the Sellafield Site

Frontiers in Microbiology ◽

10.3389/fmicb.2020.587556 ◽

2020 ◽

Vol 11 ◽

Author(s):

Sharon Ruiz-Lopez ◽

Lynn Foster ◽

Chris Boothman ◽

Nick Cole ◽

Katherine Morris ◽

...

Keyword(s):

16S Rrna ◽

Microbial Communities ◽

Nuclear Fuel ◽

Spent Nuclear Fuel ◽

Quantitative Polymerase Chain Reaction ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Bacterial Cells ◽

Spent Fuel

The use of nuclear power has been a significant part of the United Kingdom’s energy portfolio with the Sellafield site being used for power production and more recently reprocessing and decommissioning of spent nuclear fuel activities. Before being reprocessed, spent nuclear fuel is stored in water ponds with significant levels of background radioactivity and in high alkalinity (to minimize fuel corrosion). Despite these challenging conditions, the presence of microbial communities has been detected. To gain further insight into the microbial communities present in extreme environments, an indoor, hyper-alkaline, oligotrophic, and radioactive spent fuel storage pond (INP) located on the Sellafield site was analyzed. Water samples were collected from sample points within the INP complex, and also the purge water feeding tank (FT) that supplies water to the pond, and were screened for the presence of the 16S and 18S rRNA genes to inform sequencing requirements over a period of 30 months. Only 16S rRNA genes were successfully amplified for sequencing, suggesting that the microbial communities in the INP were dominated by prokaryotes. Quantitative Polymerase Chain Reaction (qPCR) analysis targeting 16S rRNA genes suggested that bacterial cells in the order of 104–106 mL–1 were present in the samples, with loadings rising with time. Next generation Illumina MiSeq sequencing was performed to identify the dominant microorganisms at eight sampling times. The 16S rRNA gene sequence analysis suggested that 70% and 91% from of the OTUs samples, from the FT and INP respectively, belonged to the phylum Proteobacteria, mainly from the alpha and beta subclasses. The remaining OTUs were assigned primarily to the phyla Acidobacteria, Bacteroidetes, and, Cyanobacteria. Overall the most abundant genera identified were Hydrogenophaga, Curvibacter, Porphyrobacter, Rhodoferax, Polaromonas, Sediminibacterium, Roseococcus, and Sphingomonas. The presence of organisms most closely related to Hydrogenophaga species in the INP areas, suggests the metabolism of hydrogen as an energy source, most likely linked to hydrolysis of water caused by the stored fuel. Isolation of axenic cultures using a range of minimal and rich media was also attempted, but only relatively minor components (from the phylum Bacteroidetes) of the pond water communities were obtained, emphasizing the importance of DNA-based, not culture-dependent techniques, for assessing the microbiome of nuclear facilities.

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Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.904-911.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 904-911 ◽

Cited By ~ 57

Author(s):

Hiroyuki Futamata ◽

Yayoi Nagano ◽

Kazuya Watanabe ◽

Akira Hiraishi

Keyword(s):

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

Competitive Inhibition ◽

16S Rrna Genes ◽

Rrna Genes ◽

Kinetic Properties ◽

Rrna Gene ◽

High Affinity ◽

Degrading Bacteria ◽

Tce Degradation

ABSTRACT A chemostat enrichment of soil bacteria growing on phenol as the sole carbon source has been shown to exhibit quite high trichloroethylene (TCE)-degrading activities (H. Futamata, S. Harayama, and K. Watanabe, Appl. Environ. Microbiol. 67:4671-4677, 2001). To identify the bacterial populations responsible for the high TCE-degrading activity, a multidisciplinary survey of the chemostat enrichment was conducted by employing molecular-ecological and culture-dependent approaches. Three chemostat enrichment cultures were newly developed under different phenol-loading conditions (0.25, 0.75, and 1.25 g liter−1 day−1) in this study, and the TCE-degrading activities of the enrichments were measured. Among them, the enrichment at 0.75 g liter−1 day−1 (enrichment 0.75) expressed the highest activity. Denaturing gradient gel electrophoresis of PCR-amplified 16S rRNA gene fragments detected a Variovorax ribotype as the strongest band in enrichment 0.75; however, it was not a major ribotype in the other samples. Bacteria were isolated from enrichment 0.75 by direct plating, and their 16S rRNA genes and genes encoding the largest subunit of phenol hydroxylase (LmPHs) were analyzed. Among the bacteria isolated, several strains were affiliated with the genus Variovorax and were shown to have high-affinity-type LmPHs. The LmPH of the Variovorax strains was also detected as the major genotype in enrichment 0.75. Kinetic analyses of phenol and TCE degradation revealed, however, that these strains exhibited quite low affinity for phenol compared to other phenol-degrading bacteria, while they showed quite high specific TCE-degrading activities and relatively high affinity for TCE. Owing to these unique kinetic traits, the Variovorax strains can obviate competitive inhibition of TCE degradation by the primary substrate of the catabolic enzyme (i.e., phenol), contributing to the high TCE-degrading activity of the chemostat enrichments. On the basis of physiological information, mechanisms accounting for the way the Variovorax population overgrew the chemostat enrichment are discussed.

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High Diversity in Iron Cycling Microbial Communities in Acidic, Iron-Rich Water of the Pyhäsalmi Mine, Finland

Geofluids ◽

10.1155/2019/7401304 ◽

2019 ◽

Vol 2019 ◽

pp. 1-17 ◽

Cited By ~ 10

Author(s):

Malin Bomberg ◽

Jarno Mäkinen ◽

Marja Salo ◽

Päivi Kinnunen

Keyword(s):

16S Rrna ◽

Microbial Diversity ◽

Microbial Communities ◽

Mine Water ◽

Ferrous Iron ◽

Great Part ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Iron Oxidizers

Microbial communities of iron-rich water in the Pyhäsalmi mine, Finland, were investigated with high-throughput amplicon sequencing and qPCR targeting bacteria, archaea, and fungi. In addition, the abundance ofLeptospirillumandAcidithiobacilluswas assessed with genus-specific qPCR assays, and enrichment cultures targeting aerobic ferrous iron oxidizers and ferric iron reducers were established. The acidic (pH 1.4–2.3) mine water collected from 240 m, 500 m, and 600 m depth from within the mine had a high microbial diversity consisting of 63-114 bacterial, 10-13 archaeal, and 104-117 fungal genera. The most abundant microorganisms in the mine water were typical acid mine drainage (AMD) taxa, such as acidophilic, iron-oxidizingLeptospirillum,Acidiphilum,Acidithiobacillus,Ferrovum, andThermoplasma. The fungi belonged mostly to the phylum Ascomycetes, although a great part of the fungal sequences remained unclassified. The number of archaeal 16S rRNA genes in the mine water was between 0.3 and 1.2 × 107copies mL−1in the samples from 500 m and 600 m, but only 3.9 × 103at 240 m and archaea were in general not enriched in cultures. The number of fungal 5.8S rRNA genes was high only in the mine water from 500 m and 600 m, where 0.2–3.4 × 104spore equivalents mL−1were detected. A high number ofLeptospirillum16S rRNA genes, 0.6–1.6 × 1010copies mL−1, were detected at 500 m and 600 m depth and in cultures containing ferrous iron, showing the importance of iron oxidizers in this environment. The abundance of bacteria in general was between 103and 10616S rRNA gene copies mL−1. Our results showed a high microbial diversity in the acid- and iron-impacted waters of the Pyhäsalmi mine, whereLeptospirillumbacteria were especially prominent. These iron oxidizers are also the main nitrogen-fixing microorganisms in this ecosystem.

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Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

Applied and Environmental Microbiology ◽

10.1128/aem.00455-08 ◽

2008 ◽

Vol 74 (15) ◽

pp. 4877-4888 ◽

Cited By ~ 57

Author(s):

Pedro A. Dimitriu ◽

Holly C. Pinkart ◽

Brent M. Peyton ◽

Melanie R. Mormile

Keyword(s):

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Community Composition ◽

Soda Lake ◽

Community Diversity ◽

16S Rrna Genes ◽

Rrna Genes ◽

Most Probable Number ◽

Rrna Gene ◽

Probable Number

ABSTRACT The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.

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Quantifying Microbial Diversity: Morphotypes, 16S rRNA Genes, and Carotenoids of Oxygenic Phototrophs in Microbial Mats

Applied and Environmental Microbiology ◽

10.1128/aem.65.2.422-430.1999 ◽

1999 ◽

Vol 65 (2) ◽

pp. 422-430 ◽

Cited By ~ 170

Author(s):

Ulrich Nübel ◽

Ferran Garcia-Pichel ◽

Michael Kühl ◽

Gerard Muyzer

Keyword(s):

16S Rrna ◽

Microbial Mats ◽

Denaturing Gradient Gel Electrophoresis ◽

Morphological Diversity ◽

Numerical Approach ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Specific Pcr ◽

Phototrophic Microorganisms

ABSTRACT We quantified the diversity of oxygenic phototrophic microorganisms present in eight hypersaline microbial mats on the basis of three cultivation-independent approaches. Morphological diversity was studied by microscopy. The diversity of carotenoids was examined by extraction from mat samples and high-pressure liquid chromatography analysis. The diversity of 16S rRNA genes from oxygenic phototrophic microorganisms was investigated by extraction of total DNA from mat samples, amplification of 16S rRNA gene segments from cyanobacteria and plastids of eukaryotic algae by phylum-specific PCR, and sequence-dependent separation of amplification products by denaturing-gradient gel electrophoresis. A numerical approach was introduced to correct for crowding the results of chromatographic and electrophoretic analyses. Diversity estimates typically varied up to twofold among mats. The congruence of richness estimates and Shannon-Weaver indices based on numbers and proportional abundances of unique morphotypes, 16S rRNA genes, and carotenoids unveiled the underlying diversity of oxygenic phototrophic microorganisms in the eight mat communities studied.

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Application of the stochastic labeling methods with random-sequence barcodes for simultaneous quantification and sequencing of environmental 16S rRNA genes

10.1101/072298 ◽

2016 ◽

Cited By ~ 1

Author(s):

Tatsuhiko Hoshino ◽

Fumio Inagaki

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Microbial Communities ◽

Copy Number ◽

Random Sequence ◽

Digital Pcr ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

The 16S Rrna Gene

AbstractNext-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5’ end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 × 103to 5.0 × 104copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

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Evaluating Bias of Illumina-Based Bacterial 16S rRNA Gene Profiles

Applied and Environmental Microbiology ◽

10.1128/aem.01451-14 ◽

2014 ◽

Vol 80 (18) ◽

pp. 5717-5722 ◽

Cited By ~ 136

Author(s):

Katherine Kennedy ◽

Michael W. Hall ◽

Michael D. J. Lynch ◽

Gabriel Moreno-Hagelsieb ◽

Josh D. Neufeld

Keyword(s):

16S Rrna ◽

Sample Preparation ◽

16S Rrna Gene ◽

Microbial Communities ◽

Massively Parallel Sequencing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Sample Sequence ◽

Multiple Pcr

ABSTRACTMassively parallel sequencing of 16S rRNA genes enables the comparison of terrestrial, aquatic, and host-associated microbial communities with sufficient sequencing depth for robust assessments of both alpha and beta diversity. Establishing standardized protocols for the analysis of microbial communities is dependent on increasing the reproducibility of PCR-based molecular surveys by minimizing sources of methodological bias. In this study, we tested the effects of template concentration, pooling of PCR amplicons, and sample preparation/interlane sequencing on the reproducibility associated with paired-end Illumina sequencing of bacterial 16S rRNA genes. Using DNA extracts from soil and fecal samples as templates, we sequenced pooled amplicons and individual reactions for both high (5- to 10-ng) and low (0.1-ng) template concentrations. In addition, all experimental manipulations were repeated on two separate days and sequenced on two different Illumina MiSeq lanes. Although within-sample sequence profiles were highly consistent, template concentration had a significant impact on sample profile variability for most samples. Pooling of multiple PCR amplicons, sample preparation, and interlane variability did not influence sample sequence data significantly. This systematic analysis underlines the importance of optimizing template concentration in order to minimize variability in microbial-community surveys and indicates that the practice of pooling multiple PCR amplicons prior to sequencing contributes proportionally less to reducing bias in 16S rRNA gene surveys with next-generation sequencing.

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RNA-Based Stable Isotope Probing and Isolation of Anaerobic Benzene-Degrading Bacteria from Gasoline-Contaminated Groundwater

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3586-3592.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3586-3592 ◽

Cited By ~ 135

Author(s):

Yuki Kasai ◽

Yoh Takahata ◽

Mike Manefield ◽

Kazuya Watanabe

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

Denaturing Gradient Gel Electrophoresis ◽

Stable Isotope Probing ◽

Contaminated Groundwater ◽

Rrna Gene ◽

Major Band ◽

Benzene Degradation ◽

Dgge Analysis ◽

Degrading Bacteria

ABSTRACT Stable isotope probing (SIP) of benzene-degrading bacteria in gasoline-contaminated groundwater was coupled to denaturing gradient gel electrophoresis (DGGE) of DNA fragments amplified by reverse transcription-PCR from community 16S rRNA molecules. Supplementation of the groundwater with [13C6]benzene together with an electron acceptor (nitrate, sulfate, or oxygen) showed that a phylotype affiliated with the genus Azoarcus specifically appeared in the 13C-RNA fraction only when nitrate was supplemented. This phylotype was also observed as the major band in DGGE analysis of bacterial 16S rRNA gene fragments amplified by PCR from the gasoline-contaminated groundwater. In order to isolate the Azoarcus strains, the groundwater sample was streaked on agar plates containing nonselective diluted CGY medium, and the DGGE analysis was used to screen colonies formed on the plates. This procedure identified five bacterial isolates (from 60 colonies) that corresponded to the SIP-identified Azoarcus phylotype, among which two strains (designated DN11 and AN9) degraded benzene under denitrifying conditions. Incubation of these strains with [14C]benzene showed that the labeled carbon was mostly incorporated into 14CO2 within 14 days. These results indicate that the Azoarcus population was involved in benzene degradation in the gasoline-contaminated groundwater under denitrifying conditions. We suggest that RNA-based SIP identification coupled to phylogenetic screening of nonselective isolates facilitates the isolation of enrichment/isolation-resistant microorganisms with a specific function.

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Development and Application of a Selective PCR-Denaturing Gradient Gel Electrophoresis Approach To Detect a Recently Cultivated Bacillus Group Predominant in Soil

Applied and Environmental Microbiology ◽

10.1128/aem.70.10.5801-5809.2004 ◽

2004 ◽

Vol 70 (10) ◽

pp. 5801-5809 ◽

Cited By ~ 8

Author(s):

Vesela A. Tzeneva ◽

Youguo Li ◽

Andreas D. M. Felske ◽

Willem M. de Vos ◽

Antoon D. L. Akkermans ◽

...

Keyword(s):

16S Rrna ◽

The Netherlands ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Physiological State ◽

Soil Samples ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

Gradient Gel Electrophoresis

ABSTRACT The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world.

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