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The microbial biocenosis in highly saline fluids produced from the cold well of a deep geothermal heat store located in the North German Basin was characterized during regular plant operation and immediately after plant downtime phases. Genetic fingerprinting revealed the dominance of sulfate-reducing bacteria (SRB) and fermentative Halanaerobiaceae during regular plant operation, whereas after shutdown phases, sequences of sulfur-oxidizing bacteria (SOB) were also detected. The detection of SOB indicated oxygen ingress into the well during the downtime phase. High 16S ribosomal RNA (rRNA) and dsrA gene copy numbers at the beginning of the restart process showed an enrichment of bacteria, SRB, and SOB during stagnant conditions consistent with higher concentrations of dissolved organic carbon (DOC), sulfate, and hydrogen sulfide in the produced fluids. The interaction of SRB and SOB during plant downtimes might have enhanced the corrosion processes occurring in the well. It was shown that scale content of fluids was significantly increased after stagnant phases. Moreover, the sulfur isotopic signature of the mineral scales indicated microbial influence on scale formation.

Injection of up-flow packed-bed bioreactors with excess volatile fatty acids and limiting concentrations of nitrate and sulfate gave complete reduction of nitrate from 0 to 5.5 cm and complete or near-complete reduction of sulfate from 3.2 to 11.5 cm along the bioreactor flow path. Most of the biomass (85%) and most of the genes for nitrate reduction (narG, 96%; napA, 99%) and for sulfate reduction (dsrB, 91%) were present near the inlet (0-5.5 cm) of the 37-cm-long bioreactor. Microbial community analysis by a combination of denaturing gradient gel electrophoresis and pyrosequencing of 16S rRNA amplicons indicated that nitrate-reducing Arcobacter and Pseudomonas species were located from 0 to 3.2 cm and throughout, respectively. Desulfobulbus species were the main sulfate reducers present and acetotrophic methanogens of the genus Methanosaeta predominated at 20-37 cm. Overall, the results indicated a succession of microbial communities along the bioreactor flow path. In the absence of nitrate, the sulfate reduction zone moved nearer to the bioreactor inlet. The sulfide concentration in the bioreactor effluent was temporarily lowered after nitrate injection was re-started. Hence, the bioreactor sulfide output could be disrupted by pulsed, not by constant nitrate injection, as demonstrated also previously in a low-temperature oil field.

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol(-1), i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80-85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10-15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol(-1), i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40-45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.

Thermophilic sulfate-reducing bacteria (tSRB) can be major contributors to the production of H(2)S (souring) in oil reservoirs. Two tSRB enrichments from a North Sea oil field, NS-tSRB1 and NS-tSRB2, were obtained at 58 degrees C with acetate-propionate-butyrate and with lactate as the electron donor, respectively. Analysis by rDNA sequencing indicated the presence of Thermodesulforhabdus norvegicus in NS-tSRB1 and of Archaeoglobus fulgidus in NS-tSRB2. Nitrate (10 mM) had no effect on H(2)S production by mid-log phase cultures of NS-tSRB1 and NS-tSRB2, whereas nitrite (0.25 mM or higher) inhibited sulfate reduction. NS-tSRB1 did not recover from inhibition, whereas sulfate reduction activity of NS-tSRB2 recovered after 500 h. Nitrite was also effective in souring inhibition and H(2)S removal in upflow bioreactors, whereas nitrate was similarly ineffective. Hence, nitrite may be preferable for souring prevention in some high-temperature oil fields because it reacts directly with sulfide and provides long-lasting inhibition of sulfate reduction.

The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga- Flexibacter- Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H(2) as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2-4x10(9) cells cm(-3) and 5x10(7) filaments cm(-3), respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.

This study was conducted to characterize the phylogenetic diversity of a corrosive marine biofilm based on 16S rDNA. Results of phylogenetic analysis indicated that, out of the 112 clones developed, 52 clones (46.4%) were affiliated with two families of sulfate-reducing bacteria: Desulfovibrionaceae and Desulfobacteriaceae. Another 44 clones (39.3%) were affiliated with the Clostridiaceae family of low G+C, gram-positive bacteria. Three clones (2.7%) were closely related to Chlorobium vibrioforme, a green sulfur bacterium.