Phylogeny of Acetate-Utilizing Microorganisms in Soils along a Nutrient Gradient in the Florida Everglades

ABSTRACT The consumption of acetate in soils taken from a nutrient gradient in the northern Florida Everglades was studied by stable isotope probing. Bacterial and archaeal 16S rRNA gene clone libraries from eutrophic and oligotrophic soil microcosms strongly suggest that a significant amount of acetate is consumed by syntrophic acetate oxidation in nutrient-enriched soil.

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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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Fatty Acid-Oxidizing Consortia along a Nutrient Gradient in the Florida Everglades

Applied and Environmental Microbiology ◽

10.1128/aem.72.4.2400-2406.2006 ◽

2006 ◽

Vol 72 (4) ◽

pp. 2400-2406 ◽

Cited By ~ 40

Author(s):

Ashvini Chauhan ◽

Andrew Ogram

Keyword(s):

Soil Microbial Communities ◽

Stable Isotope Probing ◽

Florida Everglades ◽

Rrna Gene ◽

Clone Libraries ◽

Fermentation Products ◽

Bacterial Phyla ◽

Soil Microbial ◽

Nutrient Gradient ◽

And Function

ABSTRACT The Florida Everglades is one of the largest freshwater marshes in North America and has been subject to eutrophication for decades. A gradient in P concentrations extends for several kilometers into the interior of the northern regions of the marsh, and the structure and function of soil microbial communities vary along the gradient. In this study, stable isotope probing was employed to investigate the fate of carbon from the fermentation products propionate and butyrate in soils from three sites along the nutrient gradient. For propionate microcosms, 16S rRNA gene clone libraries from eutrophic and transition sites were dominated by sequences related to previously described propionate oxidizers, such as Pelotomaculum spp. and Syntrophobacter spp. Significant representation was also observed for sequences related to Smithella propionica, which dismutates propionate to butyrate. Sequences of dominant phylotypes from oligotrophic samples did not cluster with known syntrophs but with sulfate-reducing prokaryotes (SRP) and Pelobacter spp. In butyrate microcosms, sequences clustering with Syntrophospora spp. and Syntrophomonas spp. dominated eutrophic microcosms, and sequences related to Pelospora dominated the transition microcosm. Sequences related to Pelospora spp. and SRP dominated clone libraries from oligotrophic microcosms. Sequences from diverse bacterial phyla and primary fermenters were also present in most libraries. Archaeal sequences from eutrophic microcosms included sequences characteristic of Methanomicrobiaceae, Methanospirillaceae, and Methanosaetaceae. Oligotrophic microcosms were dominated by acetotrophs, including sequences related to Methanosarcina, suggesting accumulation of acetate.

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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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Microbial Community Dynamics and Stability during an Ammonia-Induced Shift to Syntrophic Acetate Oxidation

Applied and Environmental Microbiology ◽

10.1128/aem.00166-14 ◽

2014 ◽

Vol 80 (11) ◽

pp. 3375-3383 ◽

Cited By ~ 83

Author(s):

Jeffrey J. Werner ◽

Marcelo L. Garcia ◽

Sarah D. Perkins ◽

Kevin E. Yarasheski ◽

Samuel R. Smith ◽

...

Keyword(s):

16S Rrna ◽

16S Rrna Gene ◽

Community Dynamics ◽

Ammonia Concentration ◽

Stable Operation ◽

Rrna Gene ◽

Acetate Oxidation ◽

16S Rrna Gene Analysis ◽

Phylogenetic Structure ◽

Syntrophic Acetate Oxidation

ABSTRACTAnaerobic digesters rely on the diversity and distribution of parallel metabolic pathways mediated by complex syntrophic microbial communities to maintain robust and optimal performance. Using mesophilic swine waste digesters, we experimented with increased ammonia loading to induce a shift from aceticlastic methanogenesis to an alternative acetate-consuming pathway of syntrophic acetate oxidation. In comparison with control digesters, we observed shifts in bacterial 16S rRNA gene content and in functional gene repertoires over the course of the digesters' 3-year operating period. During the first year, under identical startup conditions, all bioreactors mirrored each other closely in terms of bacterial phylotype content, phylogenetic structure, and evenness. When we perturbed the digesters by increasing the ammonia concentration or temperature, the distribution of bacterial phylotypes became more uneven, followed by a return to more even communities once syntrophic acetate oxidation had allowed the experimental bioreactors to regain stable operation. The emergence of syntrophic acetate oxidation coincided with a partial shift from aceticlastic to hydrogenotrophic methanogens. Our 16S rRNA gene analysis also revealed that acetate-fed enrichment experiments resulted in communities that did not represent the bioreactor community. Analysis of shotgun sequencing of community DNA suggests that syntrophic acetate oxidation was carried out by a heterogeneous community rather than by a specific keystone population with representatives of enriched cultures with this metabolic capacity.

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Syntrophic acetate oxidation in two-phase (acid–methane) anaerobic digesters

Water Science & Technology ◽

10.2166/wst.2011.748 ◽

2011 ◽

Vol 64 (9) ◽

pp. 1812-1820 ◽

Cited By ~ 36

Author(s):

T. Shimada ◽

E. Morgenroth ◽

M. Tandukar ◽

S. G. Pavlostathis ◽

A. Smith ◽

...

Keyword(s):

Phylogenetic Analysis ◽

Anaerobic Digestion ◽

Full Scale ◽

Laboratory Scale ◽

Gene Clone ◽

Rrna Gene ◽

Acetate Oxidation ◽

Two Phase ◽

Anaerobic Digesters ◽

Syntrophic Acetate Oxidation

The microbial processes involved in two-phase anaerobic digestion were investigated by operating a laboratory-scale acid-phase (AP) reactor and analyzing two full-scale, two-phase anaerobic digesters operated under mesophilic (35 °C) conditions. The digesters received a blend of primary sludge and waste activated sludge (WAS). Methane levels of 20% in the laboratory-scale reactor indicated the presence of methanogenic activity in the AP. A phylogenetic analysis of an archaeal 16S rRNA gene clone library of one of the full-scale AP digesters showed that 82% and 5% of the clones were affiliated with the orders Methanobacteriales and Methanosarcinales, respectively. These results indicate that substantial levels of aceticlastic methanogens (order Methanosarcinales) were not maintained at the low solids retention times and acidic conditions (pH 5.2–5.5) of the AP, and that methanogenesis was carried out by hydrogen-utilizing methanogens of the order Methanobacteriales. Approximately 43, 31, and 9% of the archaeal clones from the methanogenic phase (MP) digester were affiliated with the orders Methanosarcinales, Methanomicrobiales, and Methanobacteriales, respectively. A phylogenetic analysis of a bacterial 16S rRNA gene clone library suggested the presence of acetate-oxidizing bacteria (close relatives of Thermacetogenium phaeum, ‘Syntrophaceticus schinkii,’ and Clostridium ultunense). The high abundance of hydrogen consuming methanogens and the presence of known acetate-oxidizing bacteria suggest that acetate utilization by acetate oxidizing bacteria in syntrophic interaction with hydrogen-utilizing methanogens was an important pathway in the second-stage of the two-phase digestion, which was operated at high ammonium-N concentrations (1.0 and 1.4 g/L). A modified version of the IWA Anaerobic Digestion Model No. 1 (ADM1) with extensions for syntrophic acetate oxidation and weak-acid inhibition adequately described the dynamic profiles of volatile acid production/degradation and methane generation observed in the laboratory-scale AP reactor. The model was validated with historical data from the full-scale digesters.

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Using DNA-based stable isotope probing to reveal novel propionate- and acetate-oxidizing bacteria in propionate-fed mesophilic anaerobic chemostats

Scientific Reports ◽

10.1038/s41598-019-53849-0 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 4

Author(s):

Hui-Zhong Wang ◽

Xiao-Meng Lv ◽

Yue Yi ◽

Dan Zheng ◽

Min Gou ◽

...

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

16S Rrna Gene ◽

High Throughput Sequencing ◽

Anaerobic Fermentation ◽

Stable Isotope Probing ◽

Rrna Gene ◽

Metabolic Characteristics ◽

Rate Limiting Step ◽

Propionate Degradation

AbstractPropionate is one of the most important intermediates of anaerobic fermentation. Its oxidation performed by syntrophic propionate-oxidizing bacteria coupled with hydrogenotrophic methanogens is considered to be a rate-limiting step for methane production. However, the current understanding of SPOB is limited due to the difficulty of pure culture isolation. In the present study, two anaerobic chemostats fed with propionate as the sole carbon source were operated at different dilution rates (0.05 d−1 and 0.15 d−1). The propionate- and acetate-oxidizing bacteria in the two methanogenic chemostats were investigated combining DNA-stable isotope probing (DNA-SIP) and 16S rRNA gene high-throughput sequencing. The results of DNA-SIP with 13C-propionate/acetate suggested that, Smithella, Syntrophobacter, Cryptanaerobacter, and unclassified Rhodospirillaceae may be putative propionate-oxidizing bacteria; unclassified Spirochaetaceae, unclassified Synergistaceae, unclassified Elusimicrobia, Mesotoga, and Gracilibacter may contribute to acetate oxidation; unclassified Syntrophaceae and Syntrophomonas may be butyrate oxidizers. By DNA-SIP, unclassified OTUs with 16S rRNA gene abundance higher than 62% of total Bacteria in the PL chemostat and 38% in the PH chemostat were revealed to be related to the degradation of propionate. These results suggest that a variety of uncultured bacteria contribute to propionate degradation during anaerobic digestion. The functions and metabolic characteristics of these bacteria require further investigation.

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Identification of a Toluene-Degrading Bacterium from a Soil Sample through H218O DNA Stable Isotope Probing

Applied and Environmental Microbiology ◽

10.1128/aem.05689-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 5995-5999 ◽

Cited By ~ 13

Author(s):

Angela Woods ◽

Maribeth Watwood ◽

Egbert Schwartz

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

16S Rrna Gene ◽

Stable Isotope Probing ◽

Rrna Gene ◽

Soil Dna ◽

Content Type ◽

Degrading Bacterium ◽

Rhodococcus Jostii ◽

Oxygen Atoms

ABSTRACTDNA stable isotope probing (DNA-SIP) with H218O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H218O, H216O, H216O and 48 ppm toluene, or H218O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H216O. With extracts from soils to which only H218O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H218O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H218O into metabolic intermediates to form nucleic acidsde novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader,Rhodococcus jostiiRHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to beRhodococcus jostiiRHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene ofRhodococcus jostiiRHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H218O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.

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Stable Isotope Probing Analysis of Interactions between Ammonia Oxidizers

Applied and Environmental Microbiology ◽

10.1128/aem.01964-09 ◽

2010 ◽

Vol 76 (8) ◽

pp. 2468-2477 ◽

Cited By ~ 40

Author(s):

Maria Tourna ◽

Thomas E. Freitag ◽

James I. Prosser

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

Microbial Communities ◽

Denaturing Gradient Gel Electrophoresis ◽

Ammonia Concentration ◽

Stable Isotope Probing ◽

16S Rrna Genes ◽

Rrna Genes ◽

Rrna Gene ◽

High Ammonia

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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Putative glycogen-accumulating organisms belonging to the Alphaproteobacteria identified through rRNA-based stable isotope probing

Microbiology ◽

10.1099/mic.0.28445-0 ◽

2006 ◽

Vol 152 (2) ◽

pp. 419-429 ◽

Cited By ~ 121

Author(s):

Rikke Louise Meyer ◽

Aaron Marc Saunders ◽

Linda Louise Blackall

Keyword(s):

Stable Isotope ◽

16S Rrna ◽

16S Rrna Gene ◽

Phosphorus Removal ◽

Stable Isotope Probing ◽

Metabolic Phenotype ◽

Rrna Gene ◽

Biological Phosphorus Removal ◽

16S Rrna Gene Analysis

Deterioration of enhanced biological phosphorus removal (EBPR) has been linked to the proliferation of glycogen-accumulating organisms (GAOs), but few organisms possessing the GAO metabolic phenotype have been identified. An unidentified GAO was highly enriched in a laboratory-scale bioreactor and attempts to identify this organism using conventional 16S rRNA gene cloning had failed. Therefore, rRNA-based stable isotope probing followed by full-cycle rRNA analysis was used to specifically identify the putative GAOs based on their characteristic metabolic phenotype. The study obtained sequences from a group of Alphaproteobacteria not previously shown to possess the GAO phenotype, but 90 % identical by 16S rRNA gene analysis to a phylogenetic clade containing cloned sequences from putative GAOs and the isolate Defluvicoccus vanus. Fluorescence in situ hybridization (FISH) probes (DF988 and DF1020) were designed to target the new group and post-FISH chemical staining demonstrated anaerobic–aerobic cycling of polyhydroxyalkanoates, as per the GAO phenotype. The successful use of probes DF988 and DF1020 required the use of unlabelled helper probes which increased probe signal intensity up to 6·6-fold, thus highlighting the utility of helper probes in FISH. The new group constituted 33 % of all Bacteria in the lab-scale bioreactor from which they were identified and were also abundant (51 and 55 % of Bacteria) in two other similar bioreactors in which phosphorus removal had deteriorated. Unlike the previously identified Defluvicoccus-related organisms, the group identified in this study were also found in two full-scale treatment plants performing EBPR, suggesting that this group may be industrially relevant.

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