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.     

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.     

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.     

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.     

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.     

Changes in Microbial Biofilm Communities during Colonization of Sewer Systems

The coexistence of sulfate-reducing bacteria (SRB) and methanogenic archaea (MA) in anaerobic biofilms developed in sewer inner pipe surfaces favors the accumulation of sulfide (H₂S) and methane (CH₄) as metabolic end products, causing severe impacts on sewerage systems. In this study, we investigated the time course of H₂S and CH₄ production and emission rates during different stages of biofilm development in relation to changes in the composition of microbial biofilm communities. The study was carried out in a laboratory sewer pilot plant that mimics a full-scale anaerobic rising sewer using a combination of process data and molecular techniques (e.g., quantitative PCR [qPCR], denaturing gradient gel electrophoresis [DGGE], and 16S rRNA gene pyrotag sequencing). After 2 weeks of biofilm growth, H₂S emission was notably high (290.7 ± 72.3 mg S-H₂S liter⁻¹ day⁻¹), whereas emissions of CH₄ remained low (17.9 ± 15.9 mg COD-CH₄ liter⁻¹ day⁻¹). This contrasting trend coincided with a stable SRB community and an archaeal community composed solely of methanogens derived from the human gut (i.e., Methanobrevibacter and Methanosphaera). In turn, CH₄ emissions increased after 1 year of biofilm growth (327.6 ± 16.6 mg COD-CH₄ liter⁻¹ day⁻¹), coinciding with the replacement of methanogenic colonizers by species more adapted to sewer conditions (i.e., Methanosaeta spp.). Our study provides data that confirm the capacity of our laboratory experimental system to mimic the functioning of full-scale sewers both microbiologically and operationally in terms of sulfide and methane production, gaining insight into the complex dynamics of key microbial groups during biofilm development.     

Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of Spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO₄²⁻ reduction rates and geochemical parameters. SO₄²⁻ reduction was rapid in marsh sediments with rates up to 3.5 μmol ml⁻¹ day⁻¹. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO₄²⁻ reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10⁷ ml⁻¹. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.     

Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions

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 H₂ 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. 2–4×10⁹ cells cm^–3 and 5×10⁷ 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.     

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

The cross-feeding of microbial products derived from ¹⁴C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [¹⁴C]bicarbonate, biofilm samples were incubated with and without NH₄⁺ as a sole energy source for 10 days. The transfer of ¹⁴C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the β-Proteobacteria showed significant uptake of ¹⁴C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH₄⁺ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized ¹⁴C-labeled products in the culture with NH₄⁺ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of ¹⁴C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.     

In Situ Activity and Spatial Organization of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria in Biofilms

We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH₄⁺ and NO₂⁻ consumption rates decreased from 0.68 and 0.64 μmol cm⁻² h⁻¹ at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm⁻² h⁻¹ at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH₄⁺ and NO₂⁻ and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O₂ or organic compounds, which consequently established suitable microenvironments for anammox bacteria.     

A Study of the Relative Dominance of Selected Anaerobic Sulfate-Reducing Bacteria in a Continuous Bioreactor by Fluorescence <Emphasis Type="Italic">in Situ</Emphasis> Hybridization

The diversity and the community structure of sulfate-reducing bacteria (SRB) in an anaerobic continuous bioreactor used for treatment of a sulfate-containing wastewater were investigated by fluorescence in situ hybridization. Hybridization to the 16S rRNA probe EUB338 for the domain Bacteria was performed, followed by a nonsense probe NON338 as a control for nonspecific staining. Sulfate-reducing consortia were identified by using five nominally genus-specific probes (SRB129 for Desulfobacter, SRB221 for Desulfobacterium, SRB228 for Desulfotomaculum, SRB660 for Desulfobulbus, and SRB657 for Desulfonema) and four group-specific probes (SRB385 as a general SRB probe, SRB687 for Desulfovibrioaceae, SRB814 for Desulfococcus group, and SRB804 for Desulfobacteriaceae). The total prokaryotic population was determined by 4′,6-diamidino-2-phenylindole staining. Hybridization analysis using these 16S rRNA-targeted oligonucleotide probes showed that, of those microbial groupings investigated, Desulfonema, Desulfobulbus, spp., and Desulfobacteriaceae group were the main sulfate-reducing bacteria in the bioreactor when operated at steady state at 35°C, pH 7.8, and a 2.5-day residence time with feed stream containing 2.5 kg m⁻³ sulfate as terminal electron acceptor and 2.3 kg m⁻³ acetate as carbon source and electron donor.     

Identification of a novel acetate-utilizing bacterium belonging to Synergistes group 4 in anaerobic digester sludge

Major acetate-utilizing bacterial and archaeal populations in methanogenic anaerobic digester sludge were identified and quantified by radioisotope- and stable-isotope-based functional analyses, microautoradiography-fluorescence in situ hybridization (MAR-FISH) and stable-isotope probing of 16S rRNA (RNA-SIP) that can directly link 16S rRNA phylogeny with in situ metabolic function. First, MAR-FISH with ¹⁴C-acetate indicated the significant utilization of acetate by only two major groups, unidentified bacterial cells and Methanosaeta-like filamentous archaeal cells, in the digester sludge. To identify the acetate-utilizing unidentified bacteria, RNA-SIP was conducted with ¹³C₆-glucose and ¹³C₃-propionate as sole carbon source, which were followed by phylogenetic analysis of 16S rRNA. We found that bacteria belonging to Synergistes group 4 were commonly detected in both 16S rRNA clone libraries derived from the sludge incubated with ¹³C-glucose and ¹³C-propionate. To confirm that this bacterial group can utilize acetate, specific FISH probe targeting for Synergistes group 4 was newly designed and applied to the sludge incubated with ¹⁴C-acetate for MAR-FISH. The MAR-FISH result showed that bacteria belonging to Synergistes group 4 significantly took up acetate and their active population size was comparable to that of Methanosaeta in this sludge. In addition, as bacteria belonging to Synergistes group 4 had high K_m for acetate and maximum utilization rate, they are more competitive for acetate over Methanosaeta at high acetate concentrations (2.5–10 m). To our knowledge, it is the first time to report the acetate-utilizing activity of uncultured bacteria belonging to Synergistes group 4 and its competitive significance to acetoclastic methanogen, Methanosaeta.     

Physiological Ecology of Clostridium glycolicum RD-1, an Aerotolerant Acetogen Isolated from Sea Grass Roots

An anaerobic, H₂-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii. Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments. Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H₂-CO₂, formate, lactate, or pyruvate. Growth on sugars or ethylene glycol yielded acetate and ethanol as end products. RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% [vol/vol]) of O₂ in the headspace of static, horizontally incubated culture tubes; the concentration of O₂ decreased during growth in such cultures. Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O₂. In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H₂ became significant end products when RD-1 was grown on glucose in the presence of O₂. Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H₂ were produced. When the concentration of O₂ in the headspace exceeded 1% (vol/vol), supplemental H₂ was not utilized. The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288^T, an organism characterized as a fermentative anaerobe. Comparative experiments with C. glycolicum DSM 1288^T demonstrated that it had negligible H₂- and formate-utilizing capacities. However, carbon monoxide dehydrogenase was detected in both RD-1 and C. glycolicum DSM 1288^T. A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C. glycolicum DSM 1288^T confirmed that RD-1 was a strain of C. glycolicum. These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O₂, (iii) oxic conditions favor the production of ethanol, lactate, and H₂ by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O₂ might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O₂.     

Successive Mineralization and Detoxification of Benzo[a]pyrene by the White Rot Fungus Bjerkandera sp. Strain BOS55 and Indigenous Microflora

White rot fungi can oxidize high-molecular-weight polycyclic aromatic hydrocarbons (PAH) rapidly to polar metabolites, but only limited mineralization takes place. The objectives of this study were to determine if the polar metabolites can be readily mineralized by indigenous microflora from several inoculum sources, such as activated sludge, forest soils, and PAH-adapted sediment sludge, and to determine if such metabolites have decreased mutagenicity compared to the mutagenicity of the parent PAH. ¹⁴C-radiolabeled benzo[a]pyrene was subjected to oxidation by the white rot fungus Bjerkandera sp. strain BOS55. After 15 days, up to 8.5% of the [¹⁴C]benzo[a]pyrene was recovered as ¹⁴CO₂ in fungal cultures, up to 73% was recovered as water-soluble metabolites, and only 4% remained soluble in dibutyl ether. Thin-layer chromatography analysis revealed that many polar fluorescent metabolites accumulated. Addition of indigenous microflora to fungal cultures with oxidized benzo[a]pyrene on day 15 resulted in an initially rapid increase in the level of ¹⁴CO₂ recovery to a maximal value of 34% by the end of the experiments (>150 days), and the level of water-soluble label decreased to 16% of the initial level. In fungal cultures not inoculated with microflora, the level of ¹⁴CO₂ recovery increased to 13.5%, while the level of recovery of water-soluble metabolites remained as high as 61%. No large differences in ¹⁴CO₂ production were observed with several inocula, showing that some polar metabolites of fungal benzo[a]pyrene oxidation were readily degraded by indigenous microorganisms, while other metabolites were not. Of the inocula tested, only PAH-adapted sediment sludge was capable of directly mineralizing intact benzo[a]pyrene, albeit at a lower rate and to a lesser extent than the mineralization observed after combined treatment with white rot fungi and indigenous microflora. Fungal oxidation of benzo[a]pyrene resulted in rapid and almost complete elimination of its high mutagenic potential, as observed in the Salmonella typhimurium revertant test performed with strains TA100 and TA98. Moreover, no direct mutagenic metabolite could be detected during fungal oxidation. The remaining weak mutagenic activity of fungal cultures containing benzo[a]pyrene metabolites towards strain TA98 was further decreased by subsequent incubations with indigenous microflora.     

Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes

Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined. Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge⁻¹ day⁻¹. After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K. Noto, T. Ogasawara, Y. Suwa, and T. Sumino, Water Res. 32:769–773, 1998), presumably mediated by different bacterial populations. Activity data showed that all NH₄-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments. In contrast, all available nitrite was oxidized to nitrate in compartment three. To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations. Compartment one showed dominant populations having 50 and 67% G+C contents. Compartment two was similar in structure to compartment one. The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level. The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea. However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different. Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system.     

Modeling Reduction of Uranium U(VI) under Variable Sulfate Concentrations by Sulfate-Reducing Bacteria

The kinetics for the reduction of sulfate alone and for concurrent uranium [U(VI)] and sulfate reduction, by mixed and pure cultures of sulfate-reducing bacteria (SRB) at 21 ± 3°C were studied. The mixed culture contained the SRB Desulfovibrio vulgaris along with a Clostridium sp. determined via 16S ribosomal DNA analysis. The pure culture was Desulfovibrio desulfuricans (ATCC 7757). A zero-order model best fit the data for the reduction of sulfate from 0.1 to 10 mM. A lag time occurred below cell concentrations of 0.1 mg (dry weight) of cells/ml. For the mixed culture, average values for the maximum specific reaction rate, V_max, ranged from 2.4 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹) at 0.25 mM sulfate to 5.0 ± 1.1 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 10 mM sulfate (average cell concentration, 0.52 mg [dry weight]/ml). For the pure culture, V_max was 1.6 ± 0.2 μmol of sulfate/mg (dry weight) of SRB · h⁻¹ at 1 mM sulfate (0.29 mg [dry weight] of cells/ml). When both electron acceptors were present, sulfate reduction remained zero order for both cultures, while uranium reduction was first order, with rate constants of 0.071 ± 0.003 mg (dry weight) of cells/ml · min⁻¹ for the mixed culture and 0.137 ± 0.016 mg (dry weight) of cells/ml · min⁻¹ (U₀ = 1 mM) for the D. desulfuricans culture. Both cultures exhibited a faster rate of uranium reduction in the presence of sulfate and no lag time until the onset of U reduction in contrast to U alone. This kinetics information can be used to design an SRB-dominated biotreatment scheme for the removal of U(VI) from an aqueous source.     

Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO₂⁻) (30%), nitrous oxide (N₂O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [¹⁵N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N₂O with two molecular mass ions: one at 44 Da, corresponding to ¹⁴N¹⁴NO, and the second at 45 Da, corresponding to ¹⁵N¹⁴NO. The nonlabeled N₂O could be formed only from -NO₂, whereas the ¹⁵N-labeled one was presumed to originate from a nitramine group (¹⁵N-¹⁴NO₂) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C₂H₅N₃O₃. When the experiment was repeated with ring-labeled [¹⁵N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-¹⁴C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in ¹⁴CO₂ (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO₂ and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Structural basis of polyamine-DNA recognition: spermidine and spermine interactions with genomic B-DNAs of different GC content probed by Raman spectroscopy

Four genomic DNAs of differing GC content (Micrococcus luteus, 72% GC; Escherichia coli, 50% GC; calf thymus, 42% GC; Clostridium perfringens, 27% GC) have been employed as targets of interaction by the cationic polyamines spermidine {[H₃N(CH₂)₃NH₂(CH₂)₄NH₃]³⁺} and spermine {[(CH₂)₄(NH₂(CH₂)₃NH₃)₂]⁴⁺}. In solutions containing 60 mM DNA phosphate (~20 mg DNA/ml) and either 1, 5 or 60 mM polyamine, only Raman bands associated with the phosphates exhibit large spectral changes, demonstrating that B-DNA phosphates are the primary targets of interaction. Phosphate perturbations, which are independent of base composition, are consistent with a model of non-specific cation binding in which delocalized polyamines diffuse along DNA while confined by the strong electrostatic potential gradient perpendicular to the helix axis. This finding provides experimental support for models in which polyamine-induced DNA condensation is driven by non-specific electrostatic binding. The Raman spectra also demonstrate that major groove sites (guanine N7 and thymine C5H₃) are less affected than phosphates by polyamine–DNA interactions. Modest dependence of polyamine binding on genome base composition suggests that sequence context plays only a secondary role in recognition. Importantly, the results demonstrate that polyamine binding has a negligible effect on the native B-form secondary structure. The capability of spermidine or spermine to bind and condense genomic B-DNA without disrupting the native structure must be taken into account when considering DNA organization within bacterial nucleoids or cell nuclei.     

Stable-Isotope-Based Labeling of Styrene-Degrading Microorganisms in Biofilters

Deuterated styrene ([²H₈]styrene) was used as a tracer in combination with phospholipid fatty acid (PLFA) analysis for characterization of styrene-degrading microbial populations of biofilters used for treatment of waste gases. Deuterated fatty acids were detected and quantified by gas chromatography-mass spectrometry. The method was evaluated with pure cultures of styrene-degrading bacteria and defined mixed cultures of styrene degraders and non-styrene-degrading organisms. Incubation of styrene degraders for 3 days with [²H₈]styrene led to fatty acids consisting of up to 90% deuterated molecules. Mixed-culture experiments showed that specific labeling of styrene-degrading strains and only weak labeling of fatty acids of non-styrene-degrading organisms occurred after incubation with [²H₈]styrene for up to 7 days. Analysis of actively degrading filter material from an experimental biofilter and a full-scale biofilter by this method showed that there were differences in the patterns of labeled fatty acids. For the experimental biofilter the fatty acids with largest amounts of labeled molecules were palmitic acid (16:0), 9,10-methylenehexadecanoic acid (17:0 cyclo9-10), and vaccenic acid (18:1 cis11). These lipid markers indicated that styrene was degraded by organisms with a Pseudomonas-like fatty acid profile. In contrast, the most intensively labeled fatty acids of the full-scale biofilter sample were palmitic acid and cis-11-hexadecenoic acid (16:1 cis11), indicating that an unknown styrene-degrading taxon was present. Iso-, anteiso-, and 10-methyl-branched fatty acids showed no or weak labeling. Therefore, we found no indication that styrene was degraded by organisms with methyl-branched fatty fatty acids, such as Xanthomonas, Bacillus, Streptomyces, or Gordonia spp.     

Application of Denaturing High-Performance Liquid Chromatography for Monitoring Sulfate-Reducing Bacteria in Oil Fields

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H₂S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10¹ to 6 × 10⁵ dsrB gene copies ml⁻¹. DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.