Anoxic Enrichment Research Articles

The Influence of O2 on the Enrichment and Activities of Acidophilic Fe(III) Reducing Bacteria

Microbiological Fe(III) reduction under oxic and acidic conditions in acid mine drainage (AMD)-impacted systems is surprising, given the greater energetic favorability of O2 reduction than Fe(III) reduction. A possible explanation for this observation is that the energetic favorability of Fe(III) reduction approaches that of O2 reduction at pH < ∼2. However, some microorganisms require O2 for optimal Fe(III) reduction, and the process has still been observed at pH ≥ 3.5 under oxic conditions. We set up acidophilic Fe(III) reducing enrichment cultures with and without O2. The time of initiation and extent of Fe(III) reduction in oxic and anoxic enrichments were identical, and Fe(III) reduction by the enrichment cultures occurred to similar extents with subsequent transfers of the cultures. Desulfosporosinus- and Clostridia-attributable phylotypes dominated the anoxic enrichment cultures and Alicyclobacillus- and Acidibacillus-attributable phylotypes dominated the oxic enrichment cultures. Our results indicate that while O2 does not impact Fe(III)-reducing activities, it does impact the types of organisms that can be enriched in its presence or absence. The ability of AMD-associated organisms to reduce Fe(III) under both oxic and anoxic conditions may allow them to adapt to fluctuating O2 levels that are encountered in AMD-impacted settings.

Anaerobic Degradation of Sulfated Polysaccharides by Two Novel Kiritimatiellales Strains Isolated From Black Sea Sediment.

The marine environment contains a large diversity of sulfated polysaccharides and other glycopolymers. Saccharolytic microorganisms degrade these compounds through hydrolysis, which includes the hydrolysis of sulfate groups from sugars by sulfatases. Various marine bacteria of the Planctomycetes-Verrucomicrobia-Chlamydia (PVC) superphylum have exceptionally high numbers of sulfatase genes associated with the degradation of sulfated polysaccharides. However, thus far no sulfatase-rich marine anaerobes are known. In this study, we aimed to isolate marine anaerobes using sulfated polysaccharides as substrate. Anoxic enrichment cultures were set up with a mineral brackish marine medium, inoculated with anoxic Black Sea sediment sampled at 2,100 m water depth water and incubated at 15°C (in situ T = 8°C) for several weeks. Community analysis by 16S rRNA gene amplicon sequencing revealed the enrichment of Kiritimatiellaeota clade R76-B128 bacteria in the enrichments with the sulfated polysaccharides fucoidan and iota-carrageenan as substrate. We isolated two strains, F1 and F21, which represent a novel family within the order of the Kiritimatiellales. They were capable of growth on various mono-, di-, and polysaccharides, including fucoidan. The desulfation of iota-carrageenan by strain F21 was confirmed quantitatively by an increase in free sulfate concentration. Strains F1 and F21 represent the first marine sulfatase-rich anaerobes, encoding more sulfatases (521 and 480, 8.0 and 8.4% of all coding sequences, respectively) than any other microorganism currently known. Specific encoded sulfatase subfamilies could be involved in desulfating fucoidan (S1_15, S1_17 and S1_25) and iota-carrageenan (S1_19). Strains F1 and F21 had a sulfatase gene classification profile more similar to aerobic than anaerobic sulfatase-rich PVC bacteria, including Kiritimatiella glycovorans, the only other cultured representative within the Kiritimatiellaeota. Both strains encoded a single anaerobic sulfatase-maturating enzyme which could be responsible for post-translational modification of formylglycine-dependent sulfatases. Strains F1 and F21 are potential anaerobic platforms for future studies on sulfatases and their maturation enzymes.

Enrichment of anaerobic heterotrophic thermophiles from four Azorean hot springs revealed different community composition and genera abundances using recalcitrant substrates.

DGGE analysis combined with a metagenomic approach was used to get insights into heterotrophic anoxic enrichment cultures of four hot springs of Vale das Furnas, Portugal, using the recalcitrant substrate spent coffee ground (SCG). Parallel enrichment cultures were performed using the major components of spent coffee ground, namely arabinogalactan, galactomannan, cellulose, and proteins. DGGE revealed that heterotrophic thermophilic bacteria are highly abundant in the hydrothermal springs and significant differences in community composition depending on the substrate were observed. DNA, isolated from enrichment cultures of different locations that were grown on the same substrate were pooled, and the respective metagenomes were analyzed. Results indicated that cultures grown on recalcitrant substrate SCG consists of a totally different thermophilic community, dominated by Dictyoglomus. Enrichments with galactomannan and arabinogalactan were dominated by Thermodesulfovibrio, while cultures with casein and cellulose were dominated by Thermus. This study indicates the high potential of thermophilic bacteria degrading recalcitrant substrate such as SCG and furthermore how the accessibility to complex polymers shapes the bacterial community.

Anaerobic Benzene Mineralization by Nitrate-Reducing and Sulfate-Reducing Microbial Consortia Enriched From the Same Site: Comparison of Community Composition and Degradation Characteristics.

Benzene mineralization under nitrate-reducing conditions was successfully established in an on-site reactor continuously fed with nitrate and sulfidic, benzene-containing groundwater extracted from a contaminated aquifer. Filling material from the reactor columns was used to set up anoxic enrichment cultures in mineral medium with benzene as electron donor and sole organic carbon source and nitrate as electron acceptor. Benzene degradation characteristics and community composition under nitrate-reducing conditions were monitored and compared to those of a well-investigated benzene-mineralizing consortium enriched from the same column system under sulfate-reducing conditions. The nitrate-reducing cultures degraded benzene at a rate of 10.1±1.7μMd-1, accompanied by simultaneous reduction of nitrate to nitrite. The previously studied sulfate-reducing culture degraded benzene at similar rates. Carbon and hydrogen stable isotope enrichment factors determined for nitrate-dependent benzene degradation differed significantly from those of the sulfate-reducing culture (ΛH/C nitrate=12±3 compared to ΛH/C sulfate=28±3), indicating different benzene activation mechanisms under the two conditions. The nitrate-reducing community was mainly composed of Betaproteobacteria, Ignavibacteria, and Anaerolineae. Azoarcus and a phylotype related to clone Dok59 (Rhodocyclaceae) were the dominant genera, indicating an involvement in nitrate-dependent benzene degradation. The primary benzene degrader of the sulfate-reducing consortium, a phylotype belonging to the Peptococcaceae, was absent in the nitrate-reducing consortium.

Deep Subsurface Life from North Pond: Enrichment, Isolation, Characterization and Genomes of Heterotrophic Bacteria.

Studies of subsurface microorganisms have yielded few environmentally relevant isolates for laboratory studies. In order to address this lack of cultivated microorganisms, we initiated several enrichments on sediment and underlying basalt samples from North Pond, a sediment basin ringed by basalt outcrops underlying an oligotrophic water-column west of the Mid-Atlantic Ridge at 22°N. In contrast to anoxic enrichments, growth was observed in aerobic, heterotrophic enrichments from sediment of IODP Hole U1382B at 4 and 68 m below seafloor (mbsf). These sediment depths, respectively, correspond to the fringes of oxygen penetration from overlying seawater in the top of the sediment column and upward migration of oxygen from oxic seawater from the basalt aquifer below the sediment. Here we report the enrichment, isolation, initial characterization and genomes of three isolated aerobic heterotrophs from North Pond sediments; an Arthrobacter species from 4 mbsf, and Paracoccus and Pseudomonas species from 68 mbsf. These cultivated bacteria are represented in the amplicon 16S rRNA gene libraries created from whole sediments, albeit at low (up to 2%) relative abundance. We provide genomic evidence from our isolates demonstrating that the Arthrobacter and Pseudomonas isolates have the potential to respire nitrate and oxygen, though dissimilatory nitrate reduction could not be confirmed in laboratory cultures. The cultures from this study represent members of abundant phyla, as determined by amplicon sequencing of environmental DNA extracts, and allow for further studies into geochemical factors impacting life in the deep subsurface.

Anaerobic utilization of toluene by marine alpha- and gammaproteobacteria reducing nitrate

Aromatic hydrocarbons are among the main constituents of crude oil and represent a major fraction of biogenic hydrocarbons. Anthropogenic influences as well as biological production lead to exposure and accumulation of these toxic chemicals in the water column and sediment of marine environments. The ability to degrade these compounds in situ has been demonstrated for oxygen- and sulphate-respiring marine micro-organisms. However, if and to what extent nitrate-reducing bacteria contribute to the degradation of hydrocarbons in the marine environment and if these organisms are similar to their well-studied freshwater counterparts has not been investigated thoroughly. Here we determine the potential of marine prokaryotes from different sediments of the Atlantic Ocean and Mediterranean Sea to couple nitrate reduction to the oxidation of aromatic hydrocarbons. Nitrate-dependent oxidation of toluene as an electron donor in anoxic enrichment cultures was elucidated by analyses of nitrate, nitrite and dinitrogen gas, accompanied by cell proliferation. The metabolically active members of the enriched communities were identified by RT-PCR of their 16S rRNA genes and subsequently quantified by fluorescence in situ hybridization. In all cases, toluene-grown communities were dominated by members of the Gammaproteobacteria, followed in some enrichments by metabolically active alphaproteobacteria as well as members of the Bacteroidetes. From these enrichments, two novel denitrifying toluene-degrading strains belonging to the Gammaproteobacteria were isolated. Two additional toluene-degrading denitrifying strains were isolated from sediments from the Black Sea and the North Sea. These isolates belonged to the Alphaproteobacteria and Gammaproteobacteria. Serial dilutions series with marine sediments indicated that up to 2.2×10(4) cells cm(-3) were able to degrade hydrocarbons with nitrate as the electron acceptor. These results demonstrated the hitherto unrecognized capacity of alpha- and gammaproteobacteria in marine sediments to oxidize toluene using nitrate.

Hitherto Unknown [Fe-Fe]-Hydrogenase Gene Diversity in Anaerobes and Anoxic Enrichments from a Moderately Acidic Fen

Newly designed primers for [Fe-Fe]-hydrogenases indicated that (i) fermenters, acetogens, and undefined species in a fen harbor hitherto unknown hydrogenases and (ii) Clostridium- and Thermosinus-related primary fermenters, as well as secondary fermenters related to sulfate or iron reducers might be responsible for hydrogen production in the fen. Comparative analysis of [Fe-Fe]-hydrogenase and 16S rRNA gene-based phylogenies indicated the presence of homologous multiple hydrogenases per organism and inconsistencies between 16S rRNA gene- and [Fe-Fe]-hydrogenase-based phylogenies, necessitating appropriate qualification of [Fe-Fe]-hydrogenase gene data for diversity analyses.

Benthic organic enrichment from suspended mussel ( Mytilus edulis) culture in Prince Edward Island, Canada

The effects of increased biodeposition within mussel ( Mytilus edulis) farms on the benthic environment were investigated using surface sediment samples collected from 11 coastal embayments in Prince Edward Island (PEI), Canada. The degree of organic enrichment, determined using geochemical indicators (total free sulfides (S), redox potentials (Eh NHE), water content (WC) and organic matter (OM)), was significantly greater in mussel leases compared to reference sites located both outside lease boundaries and in inlets not used to culture mussel (MANOVA, p < 0.001). Post-hoc tests showed that mean WC and OM, Eh NHE potentials and S concentration were all significantly different between lease and reference sites ( p ≤ 0.009). Temporal changes in organic enrichment conditions were detected by comparing our 2001 data to those previously reported from a 1997 survey of the same sampling sites (MANOVA, p < 0.001). This multivariate effect resulted primarily from a 39% increase in mean S concentration between 1997 and 2001 ( p = 0.029). Surface sediment variables at reference sites were similar between years. Benthic conditions were discriminated along an oxic–anoxic enrichment gradient using multidimensional scaling analysis. Mussel lease sites sampled in 1997 were clustered within predefined oxic sediment classifications along with the majority of reference sites, but were grouped farther along the enrichment gradient in 2001. The significant increase in hypoxic and sulfidic sediments within mussel farms between 1997 and 2001 is consistent with the 43% increase in PEI mussel production—a classic response to excessive organic biodeposition in shallow coastal inlets with relatively low dispersive capacity.

Natronobacillus azotifigens gen. nov., sp. nov., an anaerobic diazotrophic haloalkaliphile from soda-rich habitats

Gram-positive bacteria capable of nitrogen fixation were obtained in microoxic enrichments from soda soils in south-western Siberia, north-eastern Mongolia, and the Lybian desert (Egypt). The same organisms were obtained in anoxic enrichments with glucose from soda lake sediments in the Kulunda Steppe (Altai, Russia) using nitrogen-free alkaline medium of pH 10. The isolates were represented by thin motile rods forming terminal round endospores. They are strictly fermentative saccharolytic anaerobes but tolerate high oxygen concentrations, probably due to a high catalase activity. All of the strains are obligately alkaliphilic and highly salt-tolerant natronophiles (chloride-independent sodaphiles). Growth was possible within a pH range from 7.5 to 10.6, with an optimum at 9.5-10, and within a salt range from 0.2 to 4 M Na(+), with an optimum at 0.5-1.5 M for the different strains. The nitrogenase activity in the whole cells also had an alkaline pH optimum but was much more sensitive to high salt concentrations compared to the growing cells. The isolates formed a compact genetic group with a high level of DNA similarity. Phylogenetic analysis based on 16S-rRNA gene sequences placed the isolates into Bacillus rRNA group 1 as a separate lineage with Amphibacillus tropicus as the nearest relative. In all isolates the key functional nitrogenase gene nifH was detected. A new genus and species, Natronobacillus azotifigens gen. nov., sp. nov., is proposed to accommodate the novel diazotrophic haloalkaliphiles.

Physiological and phylogenetic characterization of a stable benzene-degrading, chlorate-reducing microbial community

A stable anoxic enrichment culture was obtained that degraded benzene with chlorate as an electron acceptor. The benzene degradation rate was 1.65 mM benzene per day, which is similar to reported aerobic benzene degradation rates but 20-1650 times higher than reported for anaerobic benzene degradation. Denaturing gradient gel electrophoresis of part of the 16S rRNA gene, cloning and sequencing showed that the culture had a stable composition after the seventh transfer. Five bacterial clones were further analyzed. Two clones corresponded to bacteria closely related to Alicycliphilus denitrificans K601. The three other clones corresponded to bacteria closely related to Zoogloea resiniphila PIV-3A2w, Mesorhizobium sp. WG and Stenotrophomonas acidaminiphila. DGGE analysis of cultures grown with different electron donors and acceptors indicated that the bacterium related to Alicycliphilus denitrificans K601 is able to degrade benzene coupled to chlorate reduction. The role of the other bacteria could not be conclusively determined. The bacterium related to Mesorhizobium sp. WG can be enriched with benzene and oxygen, but not with acetate and chlorate, while the bacterium related to Stenotrophomonas acidaminophila grows with acetate and chlorate, but not with benzene and oxygen. As oxygen is produced during chlorate reduction, an aerobic pathway of benzene degradation is most likely.

Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system

District heating systems (DHS) are extreme aqueous environments characterized by high temperatures, high pH (9.5-10.0), and low nutrient availability. Culture-independent and culture-dependent techniques showed that DHS may nevertheless harbour geno- and phenotypically diverse bacterial biofilm communities. Approximately 50% of the cells in biofilms growing on mild steel coupons in rotortorque reactors connected to the return line (40 degrees C) of a Danish DHS were detectable by FISH analysis and thus were probably metabolically active. A bacterial 16S rRNA gene clone library generated from the biofilms was dominated by proteobacterial phylotypes (closely related to known aerobic species) and by phylotypes affiliated to the anaerobic class Clostridia. Anoxic enrichment cultures derived from biofilms primarily contained 16S rRNA gene and dsrAB (encoding major subunits of dissimilatory sulfite reductase) phylotypes affiliated to the latter class. Alkalitolerant and neutrophilic anaerobic bacteria were isolated from the DHS, including novel Gram-positive and deltaproteobacterial sulfate-reducers and sulfite-reducers constituting novel Gram-positive lineages. In total, 39 distinct 16S rRNA gene phylotypes representing ten classes were identified. The detection of several alkalitolerant, sulfide-producing, and, thus, potentially biocorrosive species underlines the need to maintain a high water quality in the DHS in order to prevent the proliferation of these species.

Incorporation of Glucose under Anoxic Conditions by Bacterioplankton from Coastal North Sea Surface Waters

It has been hypothesized that the potential for anaerobic metabolism might be a common feature of bacteria in coastal marine waters (L. Riemann and F. Azam, Appl. Environ. Microbiol. 68: 5554-5562, 2002). Therefore, we investigated whether different phylogenetic groups of heterotrophic picoplankton from the coastal North Sea were able to take up a simple carbon source under anoxic conditions. Oxic and anoxic incubations (4 h) or enrichments (24 h) of seawater with radiolabeled glucose were performed in July and August 2003. Bacteria with incorporated substrate were identified by using a novel protocol in which we combined fluorescence in situ hybridization and microautoradiography of cells on membrane filters. Incorporation of glucose under oxic and anoxic conditions was found in alpha-Proteobacteria, gamma-Proteobacteria, and the Cytophaga-Flavobacterium cluster of the Bacteroidetes at both times, but not in marine Euryarchaeota. In July, the majority of cells belonging to the alpha-proteobacterial Roseobacter clade showed tracer incorporation both in oxic incubations and in oxic and anoxic enrichments. In August, only a minority of the Roseobacter cells, but most bacteria affiliated with Vibrio spp., were able to incorporate the tracer under either condition. A preference for glucose uptake under anoxic conditions was observed for bacteria related to Alteromonas and the Pseudoalteromonas-Colwellia group. These genera are commonly considered to be strictly aerobic, but facultatively fermentative strains have been described. Our findings suggest that the ability to incorporate substrates anaerobically is widespread in pelagic marine bacteria belonging to different phylogenetic groups. Such bacteria may be abundant in fully aerated coastal marine surface waters.

Manganese oxide reduction in laboratory microcosms

Manganese biogeochemistry holds special interest for the characterization of passive treatment systems designed to treat acidic mine waters while meeting enforceable effluent discharge limits set for manganese. In the present study, an initial anoxic enrichment culture was developed for use as an inoculum in experimental systems. Standard anoxic microcosms capable of reducing manganese from Mn4+ to Mn2+ were established from the initial enrichment and altered to study the effects of electron acceptor availability and inhibitors on manganese reduction. Manganese reduction was not significantly inhibited in aerobic and nitrate amended microcosms; however, systems amended with metabolic inhibitors (sodium azide or sodium molybdate) exhibited significant inhibition of manganese reduction relative to standard microcosms. The presence of iron was found to influence the partitioning of reduced manganese with adsorption becoming more important with increasing iron to manganese ratios.

Anaerobic degradation of cyclohexane-1,2-diol by a new Azoarcus species

A bacterium, strain 22Lin, was isolated on cyclohexane-1,2-diol as sole electron donor and carbon source and nitrate as electron acceptor. Cells are motile rods and are facultatively anaerobic. A phylogenetic comparison based on the total 16S rRNA gene sequence allowed the assignment of the isolate to the genus Azoarcus. Cyclohexanol, cyclohexanone, cyclohexane-1,3-diol, and cyclohexane-1,3-dione, which are oxidized by a different denitrifying strain, did not support denitrifying growth of isolate 22Lin. On the contrary, cyclohexanol (I50 = 37 μM) and cyclohexanone (I50 = 28 μM) inhibited growth on cyclohexane-1,2-diol, but not on acetate. NAD was reduced by crude extracts of strain 22Lin in the presence of cyclohexane-1,2-dione, but not in the presence of cyclohexanone or cyclohexane-1,3-dione. The formation of 6-oxohexanoate from cyclohexane-1,2-dione and of adipate during NAD reduction suggests that strain 22Lin possesses a carbon–carbon hydrolase that transforms cyclohexane-1,2-dione into 6-oxohexanoate. This pathway was once observed in an aerobic pseudomonad that was lost and could not be reisolated. Here, the application of strictly anoxic enrichment conditions enabled the reisolation of another strain (22Lin) that uses this pathway.

Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU.

Organosulfonates are important natural and man-made compounds, but until recently (T. J. Lie, T. Pitta, E. R. Leadbetter, W. Godchaux III, and J. R. Leadbetter. Arch. Microbiol. 166:204-210, 1996), they were not believed to be dissimilated under anoxic conditions. We also chose to test whether alkane- and arenesulfonates could serve as electron sinks in respiratory metabolism. We generated 60 anoxic enrichment cultures in mineral salts medium which included several potential electron donors and a single organic sulfonate as an electron sink, and we used material from anaerobic digestors in communal sewage works as inocula. None of the four aromatic sulfonates, the three unsubstituted alkanesulfonates, or the N-sulfonate tested gave positive enrichment cultures requiring both the electron donor and electron sink for growth. Nine cultures utilizing the natural products taurine, cysteate, or isethionate were considered positive for growth, and all formed sulfide. Two clearly different pure cultures were examined. Putative Desulfovibrio sp. strain RZACYSA, with lactate as the electron donor, utilized sulfate, aminomethanesulfonate, taurine, isethionate, and cysteate, converting the latter to ammonia, acetate, and sulfide. Strain RZATAU was identified by 16S rDNA analysis as Bilophila wadsworthia. In the presence of, e.g., formate as the electron donor, it utilized, e.g., cysteate and isethionate and converted taurine quantitatively to cell material and products identified as ammonia, acetate, and sulfide. Sulfite and thiosulfate, but not sulfate, were utilized as electron sinks, as was nitrate, when lactate was provided as the electron donor and carbon source. A growth requirement for 1,4-naphthoquinone indicates a menaquinone electron carrier, and the presence of cytochrome c supports the presence of an electron transport chain. Pyruvate-dependent disappearance of taurine from cell extracts, as well as formation of alanine and release of ammonia and acetate, was detected. We suspected that sulfite is an intermediate, and we detected desulfoviridin (sulfite reductase). We thus believe that sulfonate reduction is one aspect of a respiratory system transferring electrons from, e.g., formate to sulfite reductase via an electron transport system which presumably generates a proton gradient across the cell membrane.