Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization

ABSTRACT We simultaneously determined the phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria (SRB) inhabiting a sewer biofilm with oxygen, nitrate, or sulfate as an electron acceptor by combining microautoradiography and fluorescent in situ hybridization (MAR-FISH) with family- and genus-specific 16S rRNA probes. The MAR-FISH analysis revealed that Desulfobulbus hybridized with probe 660 was a dominant SRB subgroup in this sewer biofilm, accounting for 23% of the total SRB. Approximately 9 and 27% of Desulfobulbus cells detected with probe 660 could take up [14C]propionate with oxygen and nitrate, respectively, as an electron acceptor, which might explain the high abundance of this species in various oxic environments. Furthermore, more than 40% of Desulfobulbus cells incorporated acetate under anoxic conditions. SRB were also numerically important members of H2-utilizing and 14CO2-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H2-utilizing bacteria hybridized with probe EUB338. A comparative 16S ribosomal DNA analysis revealed that two SRB populations, related to the Desulfomicrobium hypogeium and the Desulfovibrio desulfuricans MB lineages, were found to be important H2 utilizers in this biofilm. The substrate uptake characteristics of different phylogenetic SRB subgroups were compared with the characteristics described to date. These results provide further insight into the correlation between the 16S rRNA phylogenetic diversity and the physiological diversity of SRB populations inhabiting sewer biofilms.

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Microbial ecology of sulfate-reducing bacteria in wastewater biofilms analyzed by microelectrodes and fish (fluorescent in situ hybridization) technique

Water Science & Technology ◽

10.2166/wst.1999.0323 ◽

1999 ◽

Vol 39 (7) ◽

pp. 41-47 ◽

Cited By ~ 3

Author(s):

Satoshi Okabe ◽

Hisashi Satoh ◽

Tsukasa Itoh ◽

Yoshimasa Watanabe

Keyword(s):

In Situ Hybridization ◽

Sulfate Reduction ◽

Sulfate Reducing Bacteria ◽

Hybridization Technique ◽

Concentration Profiles ◽

Reduction Zone ◽

Sulfate Reducing ◽

Reducing Bacteria ◽

Culture Dependent

The vertical distribution of sulfate-reducing bacteria (SRB) in microaerophilic wastewater biofilms grown on fully submerged rotating disk reactors (RDR) was determined by the conventional culture-dependent MPN method and in situ hybridization of fluorescently-labelled 16S rRNA-targeted oligonucleotide probes for SRB in parallel. Chemical concentration profiles within the biofilm were also measured using microelectrodes for O2, S2-, NO3- and pH. In situ hybridization revealed that the SRB probe-stained cells were distributed throughout the biofilm even in the oxic surface zone in all states from single scattered cells to clustered cells. The higher fluorescence intensity and abundance of SRB probe-stained cells were found in the middle part of the biofilm. This result corresponded well with O2 and H2S concentration profiles measured by microelectrodes, showing sulfate reduction was restricted to a narrow anaerobic zone located about 500 μm below the biofilm surface. Results of the MPN and potential sulfate reducing activity (culture-dependent approaches) indicated a similar distribution of cultivable SRB in the biofilm. The majority of the general SRB probe-stained cells were hybridized with SRB 660 probe, suggesting that one important member of the SRB in the wastewater biofilm could be the genus Desulfobulbus. An addition of nitrate forced the sulfate reduction zone deeper in the biofilm and reduced the specific sulfate reduction rate as well. The sulfate reduction zone was consequently separated from O2 and NO3- respiration zones. Anaerobic H2S oxidation with NO3- was also induced by addition of nitrate to the medium.

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Abundance and spatial organization of Gram-negative sulfate-reducing bacteria in activated sludge investigated by in situ probing with specific 16S rRNA targeted oligonucleotides

FEMS Microbiology Ecology ◽

10.1111/j.1574-6941.1998.tb00459.x ◽

1998 ◽

Vol 25 (1) ◽

pp. 43-61 ◽

Cited By ~ 194

Author(s):

Werner Manz ◽

Michael Eisenbrecher ◽

Thomas R. Neu ◽

Ulrich Szewzyk

Keyword(s):

16S Rrna ◽

Activated Sludge ◽

Spatial Organization ◽

Sulfate Reducing Bacteria ◽

Gram Negative ◽

Sulfate Reducing ◽

Reducing Bacteria

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Development of a group-specific 16S rRNA-targeted probe set for the identification of Marinobacter by fluorescence in situ hybridization

Deep Sea Research Part II Topical Studies in Oceanography ◽

10.1016/j.dsr2.2013.10.009 ◽

2016 ◽

Vol 129 ◽

pp. 360-367 ◽

Cited By ~ 9

Author(s):

Luke J. McKay ◽

Tony Gutierrez ◽

Andreas P. Teske

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Probe Set

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Development of a 16S rRNA-targeted probe set for Verrucomicrobia and its application for fluorescence in situ hybridization in a humic lake

Systematic and Applied Microbiology ◽

10.1016/j.syapm.2009.12.005 ◽

2010 ◽

Vol 33 (3) ◽

pp. 139-148 ◽

Cited By ~ 34

Author(s):

Julia Arnds ◽

Katrin Knittel ◽

Ulrike Buck ◽

Matthias Winkel ◽

Rudolf Amann

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Fluorescence In Situ Hybridization ◽

Humic Lake ◽

Probe Set

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Community Structure, Cellular rRNA Content, and Activity of Sulfate-Reducing Bacteria in Marine Arctic Sediments

Applied and Environmental Microbiology ◽

10.1128/aem.66.8.3592-3602.2000 ◽

2000 ◽

Vol 66 (8) ◽

pp. 3592-3602 ◽

Cited By ~ 198

Author(s):

Katrin Ravenschlag ◽

Kerstin Sahm ◽

Christian Knoblauch ◽

Bo B. Jørgensen ◽

Rudolf Amann

Keyword(s):

Community Structure ◽

16S Rrna ◽

Sequence Similarity ◽

Blot Hybridization ◽

Sulfate Reducing Bacteria ◽

Slot Blot ◽

Sulfate Reducing ◽

Slot Blot Hybridization ◽

Reducing Bacteria

ABSTRACT The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburgfjorden, Svalbard) was characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes. The SRB community was dominated by members of the Desulfosarcina-Desulfococcus group. This group accounted for up to 73% of the SRB detected and up to 70% of the SRB rRNA detected. The predominance was shown to be a common feature for different stations along the coast of Svalbard. In a top-to-bottom approach we aimed to further resolve the composition of this large group of SRB by using probes for cultivated genera. While this approach failed, directed cloning of probe-targeted genes encoding 16S rRNA was successful and resulted in sequences which were all affiliated with the Desulfosarcina-Desulfococcus group. A group of clone sequences (group SVAL1) most closely related toDesulfosarcina variabilis (91.2% sequence similarity) was dominant and was shown to be most abundant in situ, accounting for up to 54.8% of the total SRB detected. A comparison of the two methods used for quantification showed that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5 mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface. Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell−1day−1), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.

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Analyses of Spatial Distributions of Sulfate-Reducing Bacteria and Their Activity in Aerobic Wastewater Biofilms

Applied and Environmental Microbiology ◽

10.1128/aem.65.11.5107-5116.1999 ◽

1999 ◽

Vol 65 (11) ◽

pp. 5107-5116 ◽

Cited By ~ 123

Author(s):

Satoshi Okabe ◽

Tsukasa Itoh ◽

Hisashi Satoh ◽

Yoshimasa Watanabe

Keyword(s):

In Situ Hybridization ◽

Vertical Distribution ◽

Reduction Rate ◽

Sulfate Reducing Bacteria ◽

Sulfur Cycle ◽

Sulfate Reducing ◽

High Sulfate ◽

Reducing Bacteria ◽

The Vertical Distribution

ABSTRACT The vertical distribution of sulfate-reducing bacteria (SRB) in aerobic wastewater biofilms grown on rotating disk reactors was investigated by fluorescent in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. To correlate the vertical distribution of SRB populations with their activity, the microprofiles of O2, H2S, NO2 −, NO3 −, NH4 +, and pH were measured with microelectrodes. In addition, a cross-evaluation of the FISH and microelectrode analyses was performed by comparing them with culture-based approaches and biogeochemical measurements. In situ hybridization revealed that a relatively high abundance of the probe SRB385-stained cells (approximately 109 to 1010cells per cm3 of biofilm) were evenly distributed throughout the biofilm, even in the oxic surface. The probe SRB660-stained Desulfobulbus spp. were found to be numerically important members of SRB populations (approximately 108 to 109 cells per cm3). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 μm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S0) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 μm), which accounted for about 75% of the total S pool in the biofilm. The contribution of an internal Fe-sulfur cycle to the overall sulfur cycle in aerobic wastewater biofilms was insignificant (less than 1%) due to the relatively high sulfate reduction rate.

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