Correlations between maximum reductive dechlorination rates and specific biomass parameters in Dehalococcoides mccartyi consortia enriched on chloroethenes PCE, TCE, and cis-1,2-DCE

2021 ◽  
Author(s):  
B Matturro ◽  
M Majone ◽  
F Aulenta ◽  
S Rossetti
Keyword(s):  
16S Rrna ◽  
Reductive Dechlorination ◽  
Correlation Factor ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Activity Levels ◽  
Dehalococcoides Mccartyi ◽  
Gene Copies ◽  
Specific Biomass

Abstract One of the challenges to implementing the modeling of the biological reductive dechlorination (RD) process is the evaluation of biological parameters that represent the abundance/activity levels of the microorganisms involved in the biodegradation of chloroethenes. Here we report a combined analysis of kinetic and specific biomass parameters conducted on three dechlorinating consortia enriched on PCE, TCE, and cis-1,2-DCE. In these consortia, Dehalococcoides mccartyi (Dhc) represented ≥ 70% of the bacterial population identified via 16S rRNA gene amplicon sequencing. Quantitative biomolecular methods were used to generate specific biomass parameters targeting either the Dhc population (16S rRNA genes or cells) or specific genes encoding RD process-involved reductive dehalogenases. The correlation factor between the abundance of active Dhc cells or tceA gene copies and maximum RD rates allowed to predict an increment of 7E+09 of active Dhc cells or 5E+09 tceA gene copies L−1 under controlled conditions. Diversely, the utilization of gene transcripts as biomass parameters for RD modeling did not provide reliable correlations with kinetic performances. This study provides valuable insights for further modeling of the RD process through the utilization of specific biomass parameters.

Heterogeneity between 16S ribosomal RNA gene copies borne by one Desulfitobacterium strain is caused by different 100-200 bp insertions in the 5´ region

2007 ◽  
Vol 53 (1) ◽  
pp. 116-128 ◽  
Author(s):  
Richard Villemur ◽  
Philippe Constant ◽  
Annie Gauthier ◽  
Martine Shareck ◽  
Réjean Beaudet
Keyword(s):  
In Situ Hybridization ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Ribosomal Rna ◽  
16S Ribosomal Rna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Gene Copies

Strains of Desulfitobacterium hafniense, such as strains PCP-1, DP7, TCE1, and TCP-A, have unusual long 16S ribosomal RNA (rRNA) genes due to an insertion of approximately 100 bp in the 5' region. In this report, we analyzed the 16S rRNA genes of different Desulfitobacterium strains to determine if such an insertion is a common feature of desulfitobacteria. We amplified this region by polymerase chain reaction (PCR) from eight Desulfitobacterium strains (D. hafniense strains PCP-1, DP7, TCP-A, TCE1, and DCB-2; D. dehalogenans; D. chlororespirans; and Desulfitobacterium sp. PCE1) and resolved each PCR product by denaturing gradient gel electrophoresis (DGGE). All strains had from two to seven DGGE- migrating bands, suggesting heterogeneity in their 16S rRNA gene copies. For each strain, the 5' region of the 16S rRNA genes was amplified and a clone library was derived. Clones corresponding to most PCR–DGGE migration bands were isolated. Sequencing of representative clones revealed that the heterogeneity was generated by insertions of 100–200 bp. An insertion was found in at least one copy of the 16S rRNA gene in all examined strains. In total, we found eight different types of insertions (INS1–INS8) that varied from 123 to 193 nt in length. Two-dimensional structural analyses of transcribed sequences predicted that all insertions would form an energetically stable loop. Reverse transcriptase – PCR experiments revealed that most of the observed insertions in the Desulfitobacterium strains were excised from the mature 16S rRNA transcripts. Insertions were not commonly found in bacterial 16S rRNA genes, and having a different insertion in several 16S rRNA gene copies borne by a single bacterial species was rarely observed. The function of these insertions is not known, but their occurrence can have an important impact in deriving 16S rRNA oligonucleotidic fluorescence in situ hybridization probes, as these insertions can be excised from 16S rRNA transcripts.Key words: Desulfitobacterium, 16S ribosomal RNA genes, heterogeneity, gene insertions, fluorescence in situ hybridization.

scholarly journals Vertical variation in Vibrio community composition in Sansha Yongle Blue Hole and its ability to degrade macromolecules

2019 ◽  
Vol 2 (1) ◽  
pp. 60-72 ◽  
Author(s):  
Bei Li ◽  
Jiwen Liu ◽  
Shun Zhou ◽  
Liang Fu ◽  
Peng Yao ◽  
...  
Keyword(s):  
Organic Carbon ◽  
16S Rrna ◽  
Community Composition ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Tween 20 ◽  
Rrna Gene ◽  
Free Living ◽  
Blue Hole ◽  
Gene Copies

Abstract With the advantages of wide distribution, fast growth, and broad metabolic spectrum to organic carbon compounds, Vibrio may play an important role in organic carbon cycling. However, the ecological roles of Vibrio in many marine environments have not been explored. Here, the world’s deepest ‘blue hole’, the Sansha Yongle Blue Hole (SYBH) in the South China Sea, which is a geographically semi-enclosed environment featuring unique chemical characters, was investigated. The abundance, diversity and carbon source utilization capability of Vibrio were studied by quantification and high-throughput sequencing of Vibrio specific 16S rRNA genes and cultivation methods. The abundance of Vibrio in water column of the SYBH ranged from 3.78 × 104 to 7.35 × 106 16S rRNA gene copies L−1. Free-living Vibrio was more abundant than particle-associated Vibrio (~ 1.20 × 106 versus~ 2.68 × 105 gene copies L−1), indicating that Vibrio prefers a free-living life style. The Vibrio assemblages showed clear vertical stratification and could be divided into three groups: aerobic-transition, middle anaerobic and bottom anaerobic zones. Dissolved oxygen (DO), temperature, pH and salinity were the main environmental factors affecting the abundance and community composition. Cultivated Vibrio demonstrated a degrading capability to various macromolecular substrates, including starch, Tween 20/40/80, DNA, gelatin, alginate, casein, chitin, lecithin, κ-carrageenan, mannan, xylan and hyaluronic acid. This suggests that Vibrio could produce a variety of highly active extracellular enzymes. Our study provides new insights into the distribution pattern and possible role in carbon cycle of Vibrio in the unique environment of a ‘blue hole’.

Determination of Bifidobacterium and Lactobacillus in breast milk of healthy women by digital PCR

2016 ◽  
Vol 7 (4) ◽  
pp. 559-569 ◽  
Author(s):  
L. Qian ◽  
H. Song ◽  
W. Cai
Keyword(s):  
Breast Milk ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
Digital Pcr ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Gene Copy ◽  
Rrna Gene ◽  
Qrt Pcr ◽  
Gene Copies

Breast milk is one of the most important sources of postnatal microbes. Quantitative real-time polymerase chain reaction (qRT-PCR) is currently used for the quantitative analysis of bacterial 16S rRNA genes in breast milk. However, this method relies on the use of standard curves and is imprecise when quantitating target DNA of low abundance. In contrast, droplet digital PCR (DD-PCR) provides an absolute quantitation without the need for calibration curves. A comparison between DD-PCR and qRT-PCR was conducted for the quantitation of Bifidobacterium and Lactobacillus 16S RNA genes in human breast milk, and the impacts of selected maternal factors were studied on the composition of these two bacteria in breast milk. From this study, DD-PCR reported between 0-34,460 16S rRNA gene copies of Bifidobacterium genera and between 1,108-634,000 16S rRNA gene copies of Lactobacillus genera in 1 ml breast milk. The 16S rRNA gene copy number of Lactobacillus genera was much greater than that of Bifidobacterium genera in breast milk. DD-PCR showed a 10-fold lower limit of quantitation as compared to qRT-PCR. A higher correlation and agreement was observed between qRT-PCR and DD-PCR in Lactobacillus quantitation as compared to Bifidobacterium quantitation. Based on our DD-PCR quantitation, a low abundance of Bifidobacterium bacteria in breast milk was correlated to higher pre-pregnancy body mass index (BMI). However, no significant difference was observed for these two bacteria in breast milk between mothers who had vaginal deliveries and caesarean deliveries. This study suggests that DD-PCR is a better tool to quantitate the bacterial load of breast milk compared to the conventional qRT-PCR method. The number of breast milk Bifidobacterium bacteria is influenced by maternal pre-pregnancy BMI.

scholarly journals Phylogenetic Characterization of a Polychlorinated-Dioxin- Dechlorinating Microbial Community by Use of Microcosm Studies

2005 ◽  
Vol 71 (8) ◽  
pp. 4325-4334 ◽  
Author(s):  
Naoko Yoshida ◽  
Nobutaka Takahashi ◽  
Akira Hiraishi
Keyword(s):  
16S Rrna ◽  
Oxidative Degradation ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Aerobic Bacteria ◽  
Rrna Gene ◽  
Phylogenetic Characterization ◽  
Gene Copies ◽  
Primer Sets ◽  
Microcosm Studies

ABSTRACT Microcosms capable of reductive dechlorination of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) were constructed in glass bottles by seeding them with a polluted river sediment and incubating them anaerobically with an organic medium. All of the PCDD/F congeners detected were equally reduced without the accumulation of significant amounts of less-chlorinated congeners as the intermediate or end products. Alternatively, large amounts of catechol and salicylic acid were produced in the upper aqueous phase. Thus, the dechlorination of PCDD/Fs and the oxidative degradation of the dechlorinated products seemed to take place simultaneously in the microcosm. Denaturing gel gradient electrophoresis and clone library analyses of PCR-amplified 16S rRNA genes from the microcosm showed that members of the phyla Firmicutes, Proteobacteria, and Bacteroidetes predominated. A significant number of Chloroflexi clones were also detected. Quantitative real-time PCR with specific primer sets showed that the 16S rRNA genes of a putative dechlorinator, “Dehalococcoides,” and its relatives accounted for 0.1% of the total rRNA gene copies of the microcosm. Most of the clones thus obtained formed a cluster distinct from the typical “Dehalococcoides” group. Quinone profiling indicated that ubiquinones accounted for 18 to 25% of the total quinone content, suggesting the coexistence and activity of ubiquinone-containing aerobic bacteria. These results suggest that the apparent complete dechlorination of PCDD/Fs found in the microcosm was due to a combination of the dechlorinating activity of the “Dehalococcoides”-like organisms and the oxidative degradation of the dechlorinated products by aerobic bacteria with aromatic hydrocarbon dioxygenases.

scholarly journals Growth of Dehalobacter and Dehalococcoides spp. during Degradation of Chlorinated Ethanes

2006 ◽  
Vol 72 (1) ◽  
pp. 428-436 ◽  
Author(s):  
Ariel Grostern ◽  
Elizabeth A. Edwards
Keyword(s):  
16S Rrna ◽  
Reductive Dechlorination ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Anaerobic Bacteria ◽  
Organic Substrate ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Real Time Quantitative Pcr ◽  
Gradient Gel Electrophoresis

ABSTRACT Mixed anaerobic microbial subcultures enriched from a multilayered aquifer at a former chlorinated solvent disposal facility in West Louisiana were examined to determine the organism(s) involved in the dechlorination of the toxic compounds 1,2-dichloroethane (1,2-DCA) and 1,1,2-trichloroethane (1,1,2-TCA) to ethene. Sequences phylogenetically related to Dehalobacter and Dehalococcoides, two genera of anaerobic bacteria that are known to respire with chlorinated ethenes, were detected through cloning of bacterial 16S rRNA genes. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene fragments after starvation and subsequent reamendment of culture with 1,2-DCA showed that the Dehalobacter sp. grew during the dichloroelimination of 1,2-DCA to ethene, implicating this organism in degradation of 1,2-DCA in these cultures. Species-specific real-time quantitative PCR was further used to monitor proliferation of Dehalobacter and Dehalococcoides during the degradation of chlorinated ethanes and showed that in fact both microorganisms grew simultaneously during the degradation of 1,2-DCA. Conversely, Dehalobacter grew during the dichloroelimination of 1,1,2-TCA to vinyl chloride (VC) but not during the subsequent reductive dechlorination of VC to ethene, whereas Dehalococcoides grew only during the reductive dechlorination of VC but not during the dichloroelimination of 1,1,2-TCA. This demonstrated that in mixed cultures containing multiple dechlorinating microorganisms, these organisms can have either competitive or complementary dechlorination activities, depending on the chloro-organic substrate.

Chlorpyrifos degradation under the influence of climate factors and fertilizer regimes in a tropical vertisol

2020 ◽  
Vol 158 (1-2) ◽  
pp. 15-24
Author(s):  
Bharati Kollah ◽  
Usha Ahirwar ◽  
Neera Singh ◽  
Garima Dubey ◽  
Ashok Patra ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Degradation Rate ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Climate Factors ◽  
Available N ◽  
Organic C ◽  
Gene Copies

AbstractBiodegradation of chlorpyrifos under the influence of fertilizer application and climate factors such as elevated CO2, temperature and moisture was studied. Soybean was grown in control, inorganic, organic and integrated (both inorganic and organic) fertilized fields. Rhizospheric soils collected during the vegetative growth phase were amended with chlorpyrifos (10 μg/g soil) and incubated under different climate factors. The climate factors were CO2 concentration (400, 800 ppm), temperature (25, 45°C) and moisture-holding capacity (60, 100%). Chlorpyrifos degradation rate varied from 0.28 to 0.65 μg/g soil/d. The abundance of 16S rRNA gene copies of eubacteria varied from 13 × 106 to 7 × 105/g soil. Actinomycetes-specific 16S rRNA gene copies were in the range of 62.5 × 105 to 18.5 × 103/g soil. Microbial abundance was high in organic amended soil and low in control soil irrespective of climate factors. Elevated CO2 and high temperature inhibited (P < 0.05) chlorpyrifos degradation rate and the abundance of 16S rRNA genes of eubacteria and actinomycetes. Chlorpyrifos degradation followed as: organic > integrated > inorganic > control. The degradation rate was positively correlated (P < 0.01) with the soil organic C, available N, water-stable aggregates and mean weight diameter of the soil aggregates of soil. Principal component analysis denoted temperature and fertilizer as the major components of variation. The study highlights that elevated CO2 and temperature affect chlorpyrifos biodegradation; however, the effect can be alleviated by the amendment of organic fertilizer.

scholarly journals Sequential Reductive Dechlorination of meta-Chlorinated Polychlorinated Biphenyl Congeners in Sediment Microcosms by Two Different Chloroflexi Phylotypes

2005 ◽  
Vol 71 (12) ◽  
pp. 8085-8090 ◽  
Author(s):  
Sonja K. Fagervold ◽  
Joy E. M. Watts ◽  
Harold D. May ◽  
Kevin R. Sowers
Keyword(s):  
16S Rrna ◽  
Reductive Dechlorination ◽  
Polychlorinated Biphenyl ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Most Probable Number ◽  
Individual Species ◽  
Rrna Gene ◽  
Probable Number

ABSTRACT Three species within a deeply branching cluster of the Chloroflexi are the only microorganisms currently known to anaerobically transform polychlorinated biphenyls (PCBs) by the mechanism of reductive dechlorination. A selective PCR primer set was designed that amplifies the 16S rRNA genes of a monophyletic group within the Chloroflexi including Dehalococcoides spp. and the o-17/DF-1 group. Assays for both qualitative and quantitative analyses by denaturing gradient gel electrophoresis and most probable number-PCR, respectively, were developed to assess sediment microcosm enrichments that reductively dechlorinated PCBs 101 (2,2′,4,5,5′-CB) and 132 (2,2′,3,3′,4,6′-CB). PCB 101 was reductively dechlorinated at the para-flanked meta position to PCB 49 (2,2′,4,5′-CB) by phylotype DEH10, which belongs to the Dehalococcoides group. This same species reductively dechlorinated the para- and ortho-flanked meta-chlorine of PCB 132 to PCB 91 (2,2′,3′,4,6′-CB). However, another phylotype designated SF1, which is more closely related to the o-17/DF-1 group, was responsible for the subsequent dechlorination of PCB 91 to PCB 51 (2,2′,4,6′-CB). Using the selective primer set, an increase in 16S rRNA gene copies was observed only with actively dechlorinating cultures, indicating that PCB-dechlorinating activities by both phylotype DEH10 and SF1 were linked to growth. The results suggest that individual species within the Chloroflexi exhibit a limited range of congener specificities and that a relatively diverse community of species within a deeply branching group of Chloroflexi with complementary congener specificities is likely required for the reductive dechlorination of different PCBs congeners in the environment.

Differentiation Of Clarias Batrachus, C. Gariepinus And Heteropneustes Fossilis By Pcr-Sequencing Of Mitochondrial 16s Rrna Gene

2015 ◽  
Vol 41 (1) ◽  
pp. 51-58
Author(s):  
Mohammad Shamimul Alam ◽  
Hawa Jahan ◽  
Rowshan Ara Begum ◽  
Reza M Shahjahan
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Fish Larva ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Valuable Insight ◽  
Multiple Sequence ◽  
Pcr Rflp ◽  
Alternative Means

Heteropneustesfossilis, Clariasbatrachus and C. gariepinus are three major catfishes ofecological and economic importance. Identification of these fish species becomes aproblem when the usual external morphological features of the fish are lost or removed,such as in canned fish. Also, newly hatched fish larva is often difficult to identify. PCRsequencingprovides accurate alternative means of identification of individuals at specieslevel. So, 16S rRNA genes of three locally collected catfishes were sequenced after PCRamplification and compared with the same gene sequences available from othergeographical regions. Multiple sequence alignment of the 16S rRNA gene fragments ofthe catfish species has revealed polymorphic sites which can be used to differentiate thesethree species from one another and will provide valuable insight in choosing appropriaterestriction enzymes for PCR-RFLP based identification in future. Asiat. Soc. Bangladesh, Sci. 41(1): 51-58, June 2015

scholarly journals Microdiversity and phylogeographic diversification of bacterioplankton in pelagic freshwater systems revealed through long-read amplicon sequencing

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Yusuke Okazaki ◽  
Shohei Fujinaga ◽  
Michaela M. Salcher ◽  
Cristiana Callieri ◽  
Atsushi Tanaka ◽  
...  
Keyword(s):  
16S Rrna ◽  
Regional Scale ◽  
Scale Up ◽  
Amplicon Sequencing ◽  
Freshwater Ecosystems ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Long Read

Abstract Background Freshwater ecosystems are inhabited by members of cosmopolitan bacterioplankton lineages despite the disconnected nature of these habitats. The lineages are delineated based on > 97% 16S rRNA gene sequence similarity, but their intra-lineage microdiversity and phylogeography, which are key to understanding the eco-evolutional processes behind their ubiquity, remain unresolved. Here, we applied long-read amplicon sequencing targeting nearly full-length 16S rRNA genes and the adjacent ribosomal internal transcribed spacer sequences to reveal the intra-lineage diversities of pelagic bacterioplankton assemblages in 11 deep freshwater lakes in Japan and Europe. Results Our single nucleotide-resolved analysis, which was validated using shotgun metagenomic sequencing, uncovered 7–101 amplicon sequence variants for each of the 11 predominant bacterial lineages and demonstrated sympatric, allopatric, and temporal microdiversities that could not be resolved through conventional approaches. Clusters of samples with similar intra-lineage population compositions were identified, which consistently supported genetic isolation between Japan and Europe. At a regional scale (up to hundreds of kilometers), dispersal between lakes was unlikely to be a limiting factor, and environmental factors or genetic drift were potential determinants of population composition. The extent of microdiversification varied among lineages, suggesting that highly diversified lineages (e.g., Iluma-A2 and acI-A1) achieve their ubiquity by containing a consortium of genotypes specific to each habitat, while less diversified lineages (e.g., CL500-11) may be ubiquitous due to a small number of widespread genotypes. The lowest extent of intra-lineage diversification was observed among the dominant hypolimnion-specific lineage (CL500-11), suggesting that their dispersal among lakes is not limited despite the hypolimnion being a more isolated habitat than the epilimnion. Conclusions Our novel approach complemented the limited resolution of short-read amplicon sequencing and limited sensitivity of the metagenome assembly-based approach, and highlighted the complex ecological processes underlying the ubiquity of freshwater bacterioplankton lineages. To fully exploit the performance of the method, its relatively low read throughput is the major bottleneck to be overcome in the future.

scholarly journals Microbiological and Geochemical Heterogeneity in an In Situ Uranium Bioremediation Field Site

2005 ◽  
Vol 71 (10) ◽  
pp. 6308-6318 ◽  
Author(s):  
Helen A. Vrionis ◽  
Robert T. Anderson ◽  
Irene Ortiz-Bernad ◽  
Kathleen R. O'Neill ◽  
Charles T. Resch ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Volatile Sulfide ◽  
Rrna Gene ◽  
Gene Sequences ◽  
16S Rrna Gene Sequences ◽  
Geochemical Heterogeneity

ABSTRACT The geochemistry and microbiology of a uranium-contaminated subsurface environment that had undergone two seasons of acetate addition to stimulate microbial U(VI) reduction was examined. There were distinct horizontal and vertical geochemical gradients that could be attributed in large part to the manner in which acetate was distributed in the aquifer, with more reduction of Fe(III) and sulfate occurring at greater depths and closer to the point of acetate injection. Clone libraries of 16S rRNA genes derived from sediments and groundwater indicated an enrichment of sulfate-reducing bacteria in the order Desulfobacterales in sediment and groundwater samples. These samples were collected nearest the injection gallery where microbially reducible Fe(III) oxides were highly depleted, groundwater sulfate concentrations were low, and increases in acid volatile sulfide were observed in the sediment. Further down-gradient, metal-reducing conditions were present as indicated by intermediate Fe(II)/Fe(total) ratios, lower acid volatile sulfide values, and increased abundance of 16S rRNA gene sequences belonging to the dissimilatory Fe(III)- and U(VI)-reducing family Geobacteraceae. Maximal Fe(III) and U(VI) reduction correlated with maximal recovery of Geobacteraceae 16S rRNA gene sequences in both groundwater and sediment; however, the sites at which these maxima occurred were spatially separated within the aquifer. The substantial microbial and geochemical heterogeneity at this site demonstrates that attempts should be made to deliver acetate in a more uniform manner and that closely spaced sampling intervals, horizontally and vertically, in both sediment and groundwater are necessary in order to obtain a more in-depth understanding of microbial processes and the relative contribution of attached and planktonic populations to in situ uranium bioremediation.

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