Successional Development of Sulfate-Reducing Bacterial Populations and Their Activities in a Wastewater Biofilm Growing under Microaerophilic Conditions

A combination of fluorescence in situ hybridization, microprofiles, denaturing gradient gel electrophoresis of PCR-amplified 16S ribosomal DNA fragments, and 16S rRNA gene cloning analysis was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within a biofilm growing under microaerophilic conditions (dissolved oxygen concentration in the bulk liquid was in the range of 0 to 100 μM) and in the presence of nitrate. Microelectrode measurements showed that oxygen penetrated 200 μm from the surface during all stages of biofilm development. The first sulfide production of 0.32 μmol of H₂S m⁻² s⁻¹ was detected below ca. 500 μm in the 3rd week and then gradually increased to 0.70 μmol H₂S m⁻² s⁻¹ in the 8th week. The most active sulfide production zone moved upward to the oxic-anoxic interface and intensified with time. This result coincided with an increase in SRB populations in the surface layer of the biofilm. The numbers of the probe SRB385- and 660-hybridized SRB populations significantly increased to 7.9 × 10⁹ cells cm⁻³ and 3.6 × 10⁹ cells cm⁻³, respectively, in the surface 400 μm during an 8-week cultivation, while those populations were relatively unchanged in the deeper part of the biofilm, probably due to substrate transport limitation. Based on 16S rRNA gene cloning analysis data, clone sequences that related to Desulfomicrobium hypogeium (99% sequence similarity) and Desulfobulbus elongatus (95% sequence similarity) were most frequently found. Different molecular analyses confirmed that Desulfobulbus, Desulfovibrio, and Desulfomicrobium were found to be the numerically important members of SRB in this wastewater biofilm.     

Analyses of spatial distributions of sulfate-reducing bacteria and their activity in aerobic wastewater biofilms.

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 O(2), H(2)S, NO(2)(-), NO(3)(-), NH(4)(+), 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 10(9) to 10(10) cells per cm(3) 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 10(8) to 10(9) cells per cm(3)). The result of microelectrode measurements showed that a high sulfate-reducing activity was found in a narrow anaerobic zone located about 150 to 300 microm below the biofilm surface and above which an intensive sulfide oxidation zone was found. The biogeochemical measurements showed that elemental sulfur (S(0)) was an important intermediate of the sulfide reoxidation in such thin wastewater biofilms (approximately 1,500 microm), 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.     

Phylogenetic identification and substrate uptake patterns of sulfate-reducing bacteria inhabiting an oxic-anoxic sewer biofilm determined by combining microautoradiography and fluorescent in situ hybridization.

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 [(14)C]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 H(2)-utilizing and (14)CO(2)-fixing microbial populations in this sewer biofilm, accounting for roughly 42% of total H(2)-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 H(2) 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.     

Successional development of sulfate-reducing bacterial populations and their activities in an activated sludge immobilized agar gel film

A combination of fluorescence in situ hybridization (FISH), microprofiles, and denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S rDNA fragments followed by hybridization analysis with specific probes was applied to investigate successional development of sulfate-reducing bacteria (SRB) community structure and in situ sulfide production activity within an activated sludge immobilized agar gel film. In this model biofilm system, since biases arising from biofilm heterogeneity can be ignored, the population dynamics of SRB in the agar gel is directly related to physiological capability and in situ activity of SRB. Microelectrode measurements showed that an anoxic zone was already developed at the beginning (0 day), a first sulfide production of 0.054 mumol H2S m(-2) x s(-1) was detected during the first week, and the rate increased gradually to 0.221 mumol H2S m(-2) x s(-1) in the fifth week. The most active sulfide production zone moved upward to the chemocline and intensified with time to form a narrow zone with high volumetric sulfide production rates. This result coincided with the shift of the spatial distributions of SRB populations determined by FISH. In situ hybridization with probe SRB385 for mainly general SRB of the delta Proteobacteria plus some gram-positive bacteria and probe 660 for Desulfobulbus indicated that the most abundant populations of SRB were primarily restricted to near the oxic/anoxic interface (chemocline). A close observation of the development of the vertical distributions of SRB populations revealed that the cell numbers of Desulfobulbus tripled (from 0.5 x 10(8) to 1.5 x 10(8) cells cm(-3)) near the oxic/anoxic interface. Similar growth (from 1.0 x10(8) to 4.5 x 10(8) cells cm(-3)) of Desulfovibrio-like SRB that hybridized with probe SRB385 was observed. PCR-DGGE followed by hybridization analysis revealed that one Desulfobulbus strain was detected from the beginning, and another strain appeared after 1 week, coinciding with the first detected sulfide production. In addition, three strains hybridizing with probe 687 (possibly Desulfovibrio) were also dominant SRB in the agar gel.     

Phylogenetic Identification and Substrate Uptake Patterns of Sulfate-Reducing Bacteria Inhabiting an Oxic-Anoxic Sewer Biofilm Determined by Combining Microautoradiography and Fluorescent In Situ Hybridization

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.     

Linked redox precipitation of sulfur and selenium under anaerobic conditions by sulfate-reducing bacterial biofilms

A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixed biofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200 micro M selenite from solution during growth on lactate and sulfate. Elemental selenium and elemental sulfur were precipitated outside SRB cells. Precipitation occurred by an abiotic reaction with bacterially generated sulfide. This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can take place under low redox conditions and in the dark. The reaction represents a new means for the deposition of elemental sulfur by SRB under such conditions. A combination of transmission electron microscopy, environmental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used to reveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium in relation to biofilm elements. When pregrown SRB biofilms were exposed to a selenite-containing medium, nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix. Selenite was therefore shown to pass through the biofilm matrix before reacting with bacterially generated sulfide. This constitutes an efficient method for the removal of toxic concentrations of selenite from solution. Implications for environmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potential action of SRB in selenium transformations is presented.     

Community structure and activity dynamics of nitrifying bacteria in a phosphate-removing biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(-), and NO(3)(-) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 microm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 microm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems.     

Linked Redox Precipitation of Sulfur and Selenium under Anaerobic Conditions by Sulfate-Reducing Bacterial Biofilms

A biofilm-forming strain of sulfate-reducing bacteria (SRB), isolated from a naturally occurring mixed biofilm and identified by 16S rDNA analysis as a strain of Desulfomicrobium norvegicum, rapidly removed 200 μM selenite from solution during growth on lactate and sulfate. Elemental selenium and elemental sulfur were precipitated outside SRB cells. Precipitation occurred by an abiotic reaction with bacterially generated sulfide. This appears to be a generalized ability among SRB, arising from dissimilatory sulfide biogenesis, and can take place under low redox conditions and in the dark. The reaction represents a new means for the deposition of elemental sulfur by SRB under such conditions. A combination of transmission electron microscopy, environmental scanning electron microscopy, and cryostage field emission scanning electron microscopy were used to reveal the hydrated nature of SRB biofilms and to investigate the location of deposited sulfur-selenium in relation to biofilm elements. When pregrown SRB biofilms were exposed to a selenite-containing medium, nanometer-sized selenium-sulfur granules were precipitated within the biofilm matrix. Selenite was therefore shown to pass through the biofilm matrix before reacting with bacterially generated sulfide. This constitutes an efficient method for the removal of toxic concentrations of selenite from solution. Implications for environmental cycling and the fate of sulfur and selenium are discussed, and a general model for the potential action of SRB in selenium transformations is presented.     

Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine arctic sediments

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 to Desulfosarcina 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(-1) day(-1)), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

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

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 10¹⁰ cells 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.     

Stratified Microbial Structure and Activity in Sulfide- and Methane-Producing Anaerobic Sewer Biofilms

Simultaneous production of sulfide and methane by anaerobic sewer biofilms has recently been observed, suggesting that sulfate-reducing bacteria (SRB) and methanogenic archaea (MA), microorganisms known to compete for the same substrates, can coexist in this environment. This study investigated the community structures and activities of SRB and MA in anaerobic sewer biofilms (average thickness of 800 μm) using a combination of microelectrode measurements, molecular techniques, and mathematical modeling. It was seen that sulfide was mainly produced in the outer layer of the biofilm, between the depths of 0 and 300 μm, which is in good agreement with the distribution of SRB population as revealed by cryosection-fluorescence in situ hybridization (FISH). SRB had a higher relative abundance of 20% on the surface layer, which decreased gradually to below 3% at a depth of 400 μm. In contrast, MA mainly inhabited the inner layer of the biofilm. Their relative abundances increased from 10% to 75% at depths of 200 μm and 700 μm, respectively, from the biofilm surface layer. High-throughput pyrosequencing of 16S rRNA amplicons showed that SRB in the biofilm were mainly affiliated with five genera, Desulfobulbus, Desulfomicrobium, Desulfovibrio, Desulfatiferula, and Desulforegula, while about 90% of the MA population belonged to the genus Methanosaeta. The spatial organizations of SRB and MA revealed by pyrosequencing were consistent with the FISH results. A biofilm model was constructed to simulate the SRB and MA distributions in the anaerobic sewer biofilm. The good fit between model predictions and the experimental data indicate that the coexistence and spatial structure of SRB and MA in the biofilm resulted from the microbial types and their metabolic transformations and interactions with substrates.     

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

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 to Desulfosarcina 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⁻¹ day⁻¹), decreased by a factor of 3 within the first 2 cm, and were relatively constant in deeper layers.     

Succession of internal sulfur cycles and sulfur-oxidizing bacterial communities in microaerophilic wastewater biofilms

The succession of sulfur-oxidizing bacterial (SOB) community structure and the complex internal sulfur cycle occurring in wastewater biofilms growing under microaerophilic conditions was analyzed by using a polyphasic approach that employed 16S rRNA gene-cloning analysis combined with fluorescence in situ hybridization, microelectrode measurements, and standard batch and reactor experiments. A complete sulfur cycle was established via S(0) accumulation within 80 days in the biofilms in replicate. This development was generally split into two phases, (i) a sulfur-accumulating phase and (ii) a sulfate-producing phase. In the first phase (until about 40 days), since the sulfide production rate (sulfate-reducing activity) exceeded the maximum sulfide-oxidizing capacity of SOB in the biofilms, H(2)S was only partially oxidized to S(0) by mainly Thiomicrospira denitirificans with NO(3)(-) as an electron acceptor, leading to significant accumulation of S(0) in the biofilms. In the second phase, the SOB populations developed further and diversified with time. In particular, S(0) accumulation promoted the growth of a novel strain, strain SO07, which predominantly carried out the oxidation of S(0) to SO(4)(2-) under oxic conditions, and Thiothrix sp. strain CT3. In situ hybridization analysis revealed that the dense populations of Thiothrix (ca. 10(9) cells cm(-3)) and strain SO07 (ca. 10(8) cells cm(-3)) were found at the sulfur-rich surface (100 microm), while the population of Thiomicrospira denitirificans was distributed throughout the biofilms with a density of ca. 10(7) to 10(8) cells cm(-3). Microelectrode measurements revealed that active sulfide-oxidizing zones overlapped the spatial distributions of different phylogenetic SOB groups in the biofilms. As a consequence, the sulfide-oxidizing capacities of the biofilms became high enough to completely oxidize all H(2)S produced by SRB to SO(4)(2-) in the second phase, indicating establishment of the complete sulfur cycle in the biofilms.     

A highly selective direct method of detecting sulphate-reducing bacteria in crude oil

AIMS: The aim of this work was to develop a highly selective method of detecting sulphate-reducing bacteria (SRB) in crude oil. METHODS: A pair of PCR primers was designed based on an alignment of the nucleotide sequence of the 16S rRNA genes from the Desulfovibrionaceae family. DNA extraction from crude oil was performed by the method using zirconia beads and a stool kit. RESULTS: The PCR specifically detected Desulfovibrio and Desulfomicrobium in a sediment sample. When nucleic acids extracted directly from crude oil were used for the PCR, 16S rRNA genes of Desulfovibrio and Thermodesulforhabdus norvegicus were detected. IMPACT OF STUDY: A simple direct method for detection of the SRB in crude oil using PCR was established.     

Identification and Phylogenetic analysis of thermophilic sulfate-reducing bacteria in oil field samples by 16S rDNA gene cloning and sequencing

Thermophilic sulfate-reducing bacteria (SRB) have been recognized as an important source of hydrogen sulfide (H2S) in hydrocarbon reservoirs and in production systems. Four thermophilic SRB enrichment cultures from three different oil field samples (sandstone core, drilling mud, and production water) were investigated using 16S rDNA sequence comparative analysis. In total, 15 different clones were identified. We found spore-forming, low G+C content, thermophilic, sulfate-reducing Desulfotomaculum-related sequences present in all oil field samples, and additionally a clone originating from sandstone core which was assigned to the mesophilic Desulfomicrobium group. Furthermore, three clones related to Gram-positive, non-sulfate-reducing Thermoanaerobacter species and four clones close to Clostridium thermocopriae were found in enrichment cultures from sandstone core and from production water, respectively. In addition, the deeply rooted lineage of two of the clones suggested previously undescribed, Gram-positive, low G+C content, thermophilic, obligately anaerobic bacteria present in production water. Such thermophilic, non-sulfate-reducing microorganisms may play an important ecological role alongside SRB in oil field environments.     

Structural and Functional Dynamics of Sulfate-Reducing Populations in Bacterial Biofilms

We describe the combined application of microsensors and molecular techniques to investigate the development of sulfate reduction and of sulfate-reducing bacterial populations in an aerobic bacterial biofilm. Microsensor measurements for oxygen showed that anaerobic zones developed in the biofilm within 1 week and that oxygen was depleted in the top 200 to 400 μm during all stages of biofilm development. Sulfate reduction was first detected after 6 weeks of growth, although favorable conditions for growth of sulfate-reducing bacteria (SRB) were present from the first week. In situ hybridization with a 16S rRNA probe for SRB revealed that sulfate reducers were present in high numbers (approximately 10⁸ SRB/ml) in all stages of development, both in the oxic and anoxic zones of the biofilm. Denaturing gradient gel electrophoresis (DGGE) showed that the genetic diversity of the microbial community increased during the development of the biofilm. Hybridization analysis of the DGGE profiles with taxon-specific oligonucleotide probes showed that Desulfobulbus and Desulfovibrio were the main sulfate-reducing bacteria in all biofilm samples as well as in the bulk activated sludge. However, different Desulfobulbus and Desulfovibrio species were found in the 6th and 8th weeks of incubation, respectively, coinciding with the development of sulfate reduction. Our data indicate that not all SRB detected by molecular analysis were sulfidogenically active in the biofilm.     

Distribution of sulfate-reducing bacteria, O2, and H2S in photosynthetic biofilms determined by oligonucleotide probes and microelectrodes.

The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen penetration, we incubated two 4-mm-thick biofilm samples in darkness or exposed to light at natural intensity. Gradients of O2, H2S, and pH were examined with microelectrodes during incubation. The samples were subsequently frozen with liquid nitrogen and sliced on a cryomicrotome in 20-microns vertical slices. Fluorescent-dye-conjugated oligonucleotides were used as "phylogenetic" probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed in the biofilm, being present in all states from single scattered cells to dense clusters of several thousand cells. To quantify the vertical distribution of SRB, we counted cells along vertical transects through the biofilm. This was done in a blind experiment to ascertain the reliability of the staining. A negative correlation between the vertical distribution of positively stained SRB cells and the measured O2 profiles was found. The distribution differed in light- and dark-incubated samples presumably because of the different extensions of the oxic surface layer. In both cases the SRB were largely restricted to anoxic layers.     

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Factors that facilitate the onset of black band disease (BBD) of corals remain elusive, though anoxic conditions under the complex microbial mat and production of sulfide are implicated in necrosis of underlying coral tissues. This study investigated the diversity and quantitative shifts of sulfate-reducing bacterial (SRB) populations during the onset of BBD using real-time PCR (RT-PCR) and cloning approaches targeting the dissimilatory (bi)sulfite reductase (dsrA) gene. A quantitativePCR (qPCR) assay targeting the 16S rRNA gene also provided an estimate of total bacteria, and allowed the relative percentage of SRB within the lesions to be determined. Three Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CPs) (comprising microbial communities unlike those of BBD lesions), were tagged and followed through time as CP developed into BBD. The dsrA-targeted qPCR detected few copies of the gene in the CP samples (<65 per ng DNA), though copy numbers increased in BBD lesions (>2500 per ng DNA). SRB in CP samples were less than 1% of the bacterial population, though represented up to 7.5% of the BBD population. Clone libraries also demonstrated a shift in the dominant dsrA sequences as lesions shifted from CP into BBD. Results from this study confirm that SRB increase during the onset of BBD, likely increasing sulfide concentrations at the base of the microbial mat and facilitating the pathogenesis of BBD.     

Sulfate-reducing bacteria in tubes constructed by the marine infaunal polychaete Diopatra cuprea

Marine infaunal burrows and tubes greatly enhance solute transport between sediments and the overlying water column and are sites of elevated microbial activity. Biotic and abiotic controls of the compositions and activities of burrow and tube microbial communities are poorly understood. The microbial communities in tubes of the marine infaunal polychaete Diopatria cuprea collected from two different sediment habitats were examined. The bacterial communities in the tubes from a sandy sediment differed from those in the tubes from a muddy sediment. The difference in community structure also extended to the sulfate-reducing bacterial (SRB) assemblage, although it was not as pronounced for this functional group of species. PCR-amplified 16S rRNA gene sequences recovered from Diopatra tube SRB by clonal library construction and screening were all related to the family Desulfobacteriaceae. This finding was supported by phospholipid fatty acid analysis and by hybridization of 16S rRNA probes specific for members of the genera Desulfosarcina, Desulfobacter, Desulfobacterium, Desulfobotulus, Desulfococcus, and Desulfovibrio and some members of the genera Desulfomonas, Desulfuromonas, and Desulfomicrobium with 16S rRNA gene sequences resolved by denaturing gradient gel electrophoresis. Two of six SRB clones from the clone library were not detected in tubes from the sandy sediment. The habitat in which the D. cuprea tubes were constructed had a strong influence on the tube bacterial community as a whole, as well as on the SRB assemblage.     

Effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film as determined by use of microelectrodes

Microelectrode, fluorescence in situ hybridization (FISH), and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate the effect of nitrite and nitrate on in situ sulfide production in an activated sludge immobilized agar gel film. Microelectrode measurements of O(2), H(2)S, NO(3)(-), NO(2)(-), and pH revealed that the addition of NO(2)(-) and NO(3)(-) forced sulfate reduction zones deeper in the agar gel and significantly reduced the in situ sulfide production levels. The sulfate reduction zone was consequently separated from O(2) and NO(2)(-) or NO(3)(-) respiration zones with increasing the concentrations of NO(2)(-) and NO(3)(-). These NO(2)(-) and NO(3)(-) treatments had only a transient effect on sulfide production. The in situ sulfide production quickly recovered to the previous levels when NO(2)(-) and NO(3)(-) were removed. The PCR-DGGE and FISH analyses revealed that 2-day-continuous addition of 500 microM NO(3)(-) did not change the metabolically active sulfate-reducing bacterial (SRB) community. On the basis of these data, it could be concluded that the addition of NO(2)(-) and NO(3)(-) did not kill SRB, but induced the interspecies competition for common carbon source (i.e., acetate) between nitrate-reducing heterotrophic bacteria and SRB and enhanced the oxidation of the produced sulfide, which were main possible causes of the suppression of in situ sulfide production in the agar gel.