scholarly journals Searching for Mesophilic Thermotogales Bacteria: “Mesotogas” in the Wild

2010 ◽  
Vol 76 (14) ◽  
pp. 4896-4900 ◽  
Author(s):  
Camilla L. Nesbø ◽  
Rajkumari Kumaraswamy ◽  
Marlena Dlutek ◽  
W. Ford Doolittle ◽  
Julia Foght
Keyword(s):  
Low Temperature ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Oil Reservoirs ◽  
Rrna Gene ◽  
Enrichment Cultures ◽  
In The Wild ◽  
16S Rrna Gene Primer

ABSTRACT All cultivated Thermotogales are thermophiles or hyperthermophiles. However, optimized 16S rRNA primers successfully amplified Thermotogales sequences from temperate hydrocarbon-impacted sites, mesothermic oil reservoirs, and enrichment cultures incubated at <46°C. We conclude that distinct Thermotogales lineages commonly inhabit low-temperature environments but may be underreported, likely due to “universal” 16S rRNA gene primer bias.

scholarly journals Anaerobic microbial LCFA degradation in bioreactors

2008 ◽  
Vol 57 (3) ◽  
pp. 439-444 ◽  
Author(s):  
D. Z. Sousa ◽  
M. A. Pereira ◽  
J. I. Alves ◽  
H. Smidt ◽  
A. J. M Stams ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Cloning And Sequencing ◽  
16S Rrna Gene Analysis ◽  
Sulfate Reducing ◽  
Bacterial Consortia ◽  
Enrichment Cultures ◽  
The Family ◽  
Insight Into

This paper reviews recent results obtained on long-chain fatty acids (LCFA) anaerobic degradation. Two LCFA were used as model substrates: oleate, a mono-unsaturated LCFA, and palmitate, a saturated LCFA, both abundant in LCFA-rich wastewaters. 16S rRNA gene analysis of sludge samples submitted to continuous oleate- and palmitate-feeding followed by batch degradation of the accumulated LCFA demonstrated that bacterial communities were dominated by members of the Clostridiaceae and Syntrophomonadaceae families. Archaeal populations were mainly comprised of hydrogen-consuming microorganisms belonging to the genus Methanobacterium, and acetate-utilizers from the genera Methanosaeta and Methanosarcina. Enrichment cultures growing on oleate and palmitate, in the absence or presence of sulfate, gave more insight into the major players involved in the degradation of unsaturated and saturated LCFA. Syntrophomonas-related species were identified as predominant microorganisms in all the enrichment cultures. Microorganisms clustering within the family Syntrophobacteraceae were identified in the methanogenic and sulfate-reducing enrichments growing on palmitate. Distinct bacterial consortia were developed in oleate and palmitate enrichments, and observed differences might be related to the different degrees of saturation of these two LCFA. A new obligately syntrophic bacterium, Syntrophomonas zehnderi, was isolated from an oleate-degrading culture and its presence in oleate-degrading sludges detected by 16S rRNA gene cloning and sequencing.

scholarly journals Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

2010 ◽  
Vol 76 (11) ◽  
pp. 3545-3553 ◽  
Author(s):  
Javier A. Izquierdo ◽  
Maria V. Sizova ◽  
Lee R. Lynd
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Cellulose Degradation ◽  
Glycosyl Hydrolase ◽  
Rrna Gene ◽  
Cellulolytic Activity ◽  
The Novel ◽  
Glycosyl Hydrolase Family ◽  
Enrichment Cultures ◽  
Hydrolase Family

ABSTRACT The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.

scholarly journals Microbial Communities Involved in Anaerobic Degradation of Unsaturated or Saturated Long-Chain Fatty Acids

2006 ◽  
Vol 73 (4) ◽  
pp. 1054-1064 ◽  
Author(s):  
Diana Z. Sousa ◽  
M. Alcina Pereira ◽  
Alfons J. M. Stams ◽  
M. Madalena Alves ◽  
Hauke Smidt
Keyword(s):  
Fatty Acid ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Enrichment Culture ◽  
Methane Yield ◽  
Rrna Gene ◽  
Microbial Composition ◽  
Dgge Profile ◽  
Enrichment Cultures ◽  
Long Chain

ABSTRACTAnaerobic long-chain fatty acid (LCFA)-degrading bacteria were identified by combining selective enrichment studies with molecular approaches. Two distinct enrichment cultures growing on unsaturated and saturated LCFAs were obtained by successive transfers in medium containing oleate and palmitate, respectively, as the sole carbon and energy sources. Changes in the microbial composition during enrichment were analyzed by denaturing gradient gel electrophoresis (DGGE) profiling of PCR-amplified 16S rRNA gene fragments. Prominent DGGE bands of the enrichment cultures were identified by 16S rRNA gene sequencing. A significant part of the retrieved 16S rRNA gene sequences was most similar to those of uncultured bacteria. Bacteria corresponding to predominant DGGE bands in oleate and palmitate enrichment cultures clustered with fatty acid-oxidizing bacteria withinSyntrophomonadaceaeandSyntrophobacteraceaefamilies. A low methane yield, corresponding to 9 to 18% of the theoretical value, was observed in the oleate enrichment, and acetate, produced according to the expected stoichiometry, was not further converted to methane. In the palmitate enrichment culture, the acetate produced was completely mineralized and a methane yield of 48 to 70% was achieved from palmitate degradation. Furthermore, the oleate enrichment culture was able to use palmitate without detectable changes in the DGGE profile. However, the palmitate-specialized consortia degraded oleate only after a lag phase of 3 months, after which the DGGE profile had changed. Two predominant bands appeared, and sequence analysis showed affiliation with theSyntrophomonasgenus. These bands were also present in the oleate enrichment culture, suggesting that these bacteria are directly involved in oleate degradation, emphasizing possible differences between the degradation of unsaturated and saturated LCFAs.

scholarly journals Populations Implicated in Anaerobic Reductive Dechlorination of 1,2-Dichloropropane in Highly Enriched Bacterial Communities

2004 ◽  
Vol 70 (7) ◽  
pp. 4088-4095 ◽  
Author(s):  
Kirsti M. Ritalahti ◽  
Frank E. Löffler
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Anaerobic Bacteria ◽  
Community Analysis ◽  
Pcr Primers ◽  
Rrna Gene ◽  
Enrichment Cultures ◽  
Specific Pcr ◽  
Shed Light ◽  
Detoxification Process

ABSTRACT 1,2-Dichloropropane (1,2-D), a widespread groundwater contaminant, can be reductively dechlorinated to propene by anaerobic bacteria. To shed light on the populations involved in the detoxification process, a comprehensive 16S rRNA gene-based bacterial community analysis of two enrichment cultures derived from geographically distinct locations was performed. Analysis of terminal restriction fragments, amplicons obtained with dechlorinator-specific PCR primers, and enumeration with quantitative real-time PCR as well as screening clone libraries all implied that Dehalococcoides populations were involved in 1,2-D dechlorination in both enrichment cultures. Physiological traits (e.g., dechlorination in the presence of ampicillin and a requirement for hydrogen as the electron donor) supported the involvement of Dehalococcoides populations in the dechlorination process. These findings expand the spectrum of chloroorganic compounds used by Dehalococcoides species as growth-supporting electron acceptors. The combined molecular approach allowed a comparison between different 16S rRNA gene-based approaches for the detection of Dehalococcoides populations.

scholarly journals Enumeration and Characterization of Iron(III)-Reducing Microbial Communities from Acidic Subsurface Sediments Contaminated with Uranium(VI)

2003 ◽  
Vol 69 (12) ◽  
pp. 7467-7479 ◽  
Author(s):  
Lainie Petrie ◽  
Nadia N. North ◽  
Sherry L. Dollhopf ◽  
David L. Balkwill ◽  
Joel E. Kostka
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Contaminated Sediments ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Enrichment Cultures ◽  
Cell Counts ◽  
Subsurface Sediments ◽  
Reducing Bacteria

ABSTRACT Iron(III)-reducing bacteria have been demonstrated to rapidly catalyze the reduction and immobilization of uranium(VI) from contaminated subsurface sediments. Thus, these organisms may aid in the development of bioremediation strategies for uranium contamination, which is prevalent in acidic subsurface sediments at U.S. government facilities. Iron(III)-reducing enrichment cultures were initiated from pristine and contaminated (high in uranium, nitrate; low pH) subsurface sediments at pH 7 and pH 4 to 5. Enumeration of Fe(III)-reducing bacteria yielded cell counts of up to 240 cells ml−1 for the contaminated and background sediments at both pHs with a range of different carbon sources (glycerol, acetate, lactate, and glucose). In enrichments where nitrate contamination was removed from the sediment by washing, MPN counts of Fe(III)-reducing bacteria increased substantially. Sediments of lower pH typically yielded lower counts of Fe(III)-reducing bacteria in lactate- and acetate-amended enrichments, but higher counts were observed when glucose was used as an electron donor in acidic enrichments. Phylogenetic analysis of 16S rRNA gene sequences extracted from the highest positive MPN dilutions revealed that the predominant members of Fe(III)-reducing consortia from background sediments were closely related to members of the Geobacteraceae family, whereas a recently characterized Fe(III) reducer (Anaeromyxobacter sp.) and organisms not previously shown to reduce Fe(III) (Paenibacillus and Brevibacillus spp.) predominated in the Fe(III)-reducing consortia of contaminated sediments. Analysis of enrichment cultures by terminal restriction fragment length polymorphism (T-RFLP) strongly supported the cloning and sequencing results. Dominant members of the Fe(III)-reducing consortia were observed to be stable over several enrichment culture transfers by T-RFLP in conjunction with measurements of Fe(III) reduction activity and carbon substrate utilization. Enrichment cultures from contaminated sites were also shown to rapidly reduce millimolar amounts of U(VI) in comparison to killed controls. With DNA extracted directly from subsurface sediments, quantitative analysis of 16S rRNA gene sequences with MPN-PCR indicated that Geobacteraceae sequences were more abundant in pristine compared to contaminated environments,whereas Anaeromyxobacter sequences were more abundant in contaminated sediments. Thus, results from a combination of cultivation-based and cultivation-independent approaches indicate that the abundance/community composition of Fe(III)-reducing consortia in subsurface sediments is dependent upon geochemical parameters (pH, nitrate concentration) and that microorganisms capable of producing spores (gram positive) or spore-like bodies (Anaeromyxobacter) were representative of acidic subsurface environments.

scholarly journals Identification and Isolation of Anaerobic, Syntrophic Phthalate Isomer-Degrading Microbes from Methanogenic Sludges Treating Wastewater from Terephthalate Manufacturing

2004 ◽  
Vol 70 (3) ◽  
pp. 1617-1626 ◽  
Author(s):  
Yan-Ling Qiu ◽  
Yuji Sekiguchi ◽  
Hiroyuki Imachi ◽  
Yoichi Kamagata ◽  
I-Cheng Tseng ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Pure Culture ◽  
Initial Step ◽  
Anaerobic Sludge ◽  
Rrna Gene ◽  
Enrichment Cultures ◽  
Cluster Group ◽  
Degrading Bacterium ◽  
High Concentrations

ABSTRACT The microbial populations responsible for anaerobic degradation of phthalate isomers were investigated by enrichment and isolation of those microbes from anaerobic sludge treating wastewater from the manufacturing of terephthalic acid. Primary enrichments were made with each of three phthalate isomers (ortho-, iso-, and terephthalate) as the sole energy source at 37�C with two sources of anaerobic sludge (both had been used to treat wastewater containing high concentrations of phthalate isomers) as the inoculum. Six methanogenic enrichment cultures were obtained which not only degraded the isomer used for the enrichment but also had the potential to degrade part of other phthalate isomers as well as benzoate with concomitant production of methane, presumably involving strictly syntrophic substrate degradation. Our 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization revealed that the predominant bacteria in the enrichment cultures were affiliated with a recently recognized non-sulfate-reducing subcluster (subcluster Ih) in the group ‘Desulfotomaculum lineage I' or a clone cluster (group TA) in the class delta-Proteobacteria. Several attempts were made to isolate these microbes, resulting in the isolation of a terephthalate-degrading bacterium, designated strain JT, in pure culture. A coculture of the strain with the hydrogenotrophic methanogen Methanospirillum hungatei converted terephthalate to acetate and methane within 3 months of incubation, whereas strain JT could not degrade terephthalate in pure culture. During the degradation of terephthalate, a small amount of benzoate was transiently accumulated as an intermediate, indicative of decarboxylation of terephthalate to benzoate as the initial step of the degradation. 16S rRNA gene sequence analysis revealed that the strain was a member of subcluster Ih of the group ‘Desulfotomaculum lineage I', but it was only distantly related to other known species.

scholarly journals Prokaryotic Community Structure and Sulfate Reducer Activity in Water from High-Temperature Oil Reservoirs with and without Nitrate Treatment

2009 ◽  
Vol 75 (22) ◽  
pp. 7086-7096 ◽  
Author(s):  
Antje Gittel ◽  
Ketil Bernt S�rensen ◽  
Torben Lund Skovhus ◽  
Kjeld Ingvorsen ◽  
Andreas Schramm
Keyword(s):  
Community Structure ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Oil Reservoirs ◽  
Rrna Gene ◽  
Prokaryotic Community ◽  
Nitrate Treatment ◽  
Nitrate Reducers ◽  
Prokaryotic Abundance ◽  
Major Player

ABSTRACT Sulfate-reducing prokaryotes (SRP) cause severe problems like microbial corrosion and reservoir souring in seawater-injected oil production systems. One strategy to control SRP activity is the addition of nitrate to the injection water. Production waters from two adjacent, hot (80�C) oil reservoirs, one with and one without nitrate treatment, were compared for prokaryotic community structure and activity of SRP. Bacterial and archaeal 16S rRNA gene analyses revealed higher prokaryotic abundance but lower diversity for the nitrate-treated field. The 16S rRNA gene clone libraries from both fields were dominated by sequences affiliated with Firmicutes (Bacteria) and Thermococcales (Archaea). Potential heterotrophic nitrate reducers (Deferribacterales) were exclusively found at the nitrate-treated field, possibly stimulated by nitrate addition. Quantitative PCR of dsrAB genes revealed that archaeal SRP (Archaeoglobus) dominated the SRP communities, but with lower relative abundance at the nitrate-treated site. Bacterial SRP were found in only low abundance at both sites and were nearly exclusively affiliated with thermophilic genera (Desulfacinum and Desulfotomaculum). Despite the high abundance of archaeal SRP, no archaeal SRP activity was detected in [35S]sulfate incubations at 80�C. Sulfate reduction was found at 60�C in samples from the untreated field and accompanied by the growth of thermophilic bacterial SRP in batch cultures. Samples from the nitrate-treated field generally lacked SRP activity. These results indicate that (i) Archaeoglobus can be a major player in hot oil reservoirs, and (ii) nitrate may act in souring control—not only by inhibiting SRP, but also by changing the overall community structure, including the stimulation of competitive nitrate reducers.

scholarly journals Impact of Preservation Method and 16S rRNA Hypervariable Region on Gut Microbiota Profiling

mSystems ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Zigui Chen ◽  
Pak Chun Hui ◽  
Mamie Hui ◽  
Yun Kit Yeoh ◽  
Po Yee Wong ◽  
...  
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Gut Microbiota ◽  
16S Rrna Gene ◽  
Rrna Gene ◽  
Preservation Solution ◽  
Hypervariable Regions ◽  
Preservation Solutions ◽  
Stool Samples ◽  
16S Rrna Gene Primer

ABSTRACT Proper preservation of stool samples to minimize microbial community shifts and inactivate infectious agents is important for self-collected specimens requiring shipment to laboratories when cold chain transport is not feasible. In this study, we evaluated the performance of six preservation solutions (Norgen, OMNI, RNAlater, CURNA, HEMA, and Shield) for these aspects. Following storage of human stool samples with these preservatives at room temperature for 7 days, three hypervariable regions of the bacterial 16S rRNA gene (V1-V2, V3-V4, and V4) were amplicon sequenced. We found that samples collected in two preservatives, Norgen and OMNI, showed the least shift in community composition relative to −80°C standards compared with other storage conditions, and both efficiently inhibited the growth of aerobic and anaerobic bacteria. RNAlater did not prevent bacterial activity and exhibited relatively larger community shift. Although the effect of preservation solution was small compared to intersubject variation, notable changes in microbiota composition were observed, which could create biases in downstream data analysis. When community profiles inferred from different 16S rRNA gene hypervariable regions were compared, we found differential sensitivity of primer sets in identifying overall microbial community and certain bacterial taxa. For example, reads generated by the V4 primer pair showed a higher alpha diversity of the gut microbial community. The degenerate 27f-YM primer failed to detect the majority of Bifidobacteriales. Our data indicate that choice of preservation solution and 16S rRNA gene primer pair are critical determinants affecting gut microbiota profiling. IMPORTANCE Large-scale human microbiota studies require specimens collected from multiple sites and/or time points to maximize detection of the small effects in microbe-host interactions. However, batch biases caused by experimental protocols, such as sample collection, massively parallel sequencing, and bioinformatics analyses, remain critical and should be minimized. This work evaluated the effects of preservation solutions and bacterial 16S rRNA gene primer pairs in revealing human gut microbiota composition. Since notable changes in detecting bacterial composition and abundance were observed among choice of preservatives and primer pairs, a consistent methodology is essential in minimizing their effects to facilitate comparisons between data sets.

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