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The effect of salinity on the activity of nitrifying bacteria, floc characteristics, and microbial community structure accessed by fluorescent in situ hybridization and polymerase chain reaction–denaturing gradient gel electrophoresis techniques was investigated. Two sequencing batch reactors (SRB1 and SBR2) treating synthetic wastewater were subjected to increasing salt concentrations. In SBR1, four salt concentrations (5, 10, 15, and 20 g NaCl/L) were tested, while in SBR2, only two salt concentrations (10 and 20 g NaCl/L) were applied in a more shock-wise manner. The two different salt adaptation strategies caused different changes in microbial community structure, but did not change the nitrification performance, suggesting that regardless of the different nitrifying bacterial community present in the reactor, the nitrification process can be maintained stable within the salt range tested. Specific ammonium oxidation rates were more affected when salt increase was performed more rapidly and dropped 50% and 60% at 20 g NaCl/L for SBR1 and SBR2, respectively. A gradual increase in NaCl concentration had a positive effect on the settling properties (i.e., reduction of sludge volume index), although it caused a higher amount of suspended solids in the effluent. Higher organisms (e.g., protozoa, nematodes, and rotifers) as well as filamentous bacteria could not withstand the high salt concentrations., Biotechnology, Applied Sciences

The ammonia-oxidizing bacterial community (AOB) was investigated in two types of laboratory-scale bioreactors performing partial oxidation of ammonia to nitrite or nitrate at high (80 mM) to extremely high (428 mM) concentrations of ammonium bicarbonate. At all conditions, the dominant AOB was affiliated to the Nitrosomonas europaea lineage as was determined by fluorescence in situ hybridization and polymerase chain reaction in combination with denaturing gradient gel electrophoresis. Molecular analysis of the mixed populations, based on the 16S rRNA and cbbL genes, demonstrated the presence of two different phylotypes of Nitrosomonas, while microbiological analysis produced a single phylotype, represented by three different morphotypes. One of the most striking features of the AOB populations encountered in the bioreactors was the domination of highly aggregated obligate microaerophilic Nitrosomonas, with unusual cellular and colony morphology, commonly observed in nitrifying bioreactors but rarely investigated by cultural methods. The latter is probably not an adaptation to stressful conditions created by high ammonia or nitrite concentrations, but oxygen seems to be a stressful factor in these bioreactors., Biotechnology, Applied Sciences

The long- and short-term effects of salt on biological nitrogen and phosphorus removal processes were studied in an aerobic granular sludge reactor. The microbial community structure was investigated by PCR-denaturing gradient gel electrophoresis (DGGE) on 16S rRNA and amoA genes. PCR products obtained from genomic DNA and from rRNA after reverse transcription were compared to determine the presence of bacteria as well as the metabolically active fraction of bacteria. Fluorescence in situ hybridization (FISH) was used to validate the PCR-based results and to quantify the dominant bacterial populations. The results demonstrated that ammonium removal efficiency was not affected by salt concentrations up to 33 g/liter NaCl. Conversely, a high accumulation of nitrite was observed above 22 g/liter NaCl, which coincided with the disappearance of Nitrospira sp. Phosphorus removal was severely affected by gradual salt increase. No P release or uptake was observed at steady-state operation at 33 g/liter NaCl, exactly when the polyphosphate-accumulating organisms (PAOs), “Candidatus Accumulibacter phosphatis” bacteria, were no longer detected by PCR-DGGE or FISH. Batch experiments confirmed that P removal still could occur at 30 g/liter NaCl, but the long exposure of the biomass to this salinity level was detrimental for PAOs, which were outcompeted by glycogen-accumulating organisms (GAOs) in the bioreactor. GAOs became the dominant microorganisms at increasing salt concentrations, especially at 33 g/liter NaCl. In the comparative analysis of the diversity (DNA-derived pattern) and the activity (cDNA-derived pattern) of the microbial population, the highly metabolically active microorganisms were observed to be those related to ammonia (Nitrosomonas sp.) and phosphate removal (“Candidatus Accumulibacter”)., Biotechnology, Applied Sciences

Anaerobic enrichment cultures with elemental sulfur as electron acceptor and either acetate or propionate as electron donor and carbon source at pH 10 and moderate salinity inoculated with sediments from soda lakes in Kulunda Steppe (Altai, Russia) resulted in the isolation of two novel members of the bacterial phylum Chrysiogenetes. The isolates, AHT11 and AHT19, represent the first specialized obligate anaerobic dissimilatory sulfur respirers from soda lakes. They use either elemental sulfur/polysulfide or arsenate as electron acceptor and a few simple organic compounds as electron donor and carbon source. Elemental sulfur is reduced to sulfide through intermediate polysulfide, while arsenate is reduced to arsenite. The bacteria belong to the obligate haloalkaliphiles, with a pH growth optimum from 10 to 10.2 and a salt range from 0.2 to 3.0 M Na? (optimum 0.4–0.6 M). According to the phylogenetic analysis, the two strains were close to each other, but distinct from the nearest relative, the haloalkaliphilic sulfur-reducing bacterium Desulfurispirillumalkaliphilum, which was isolated from a bioreactor. Onthe basis of distinct phenotype and phylogeny, the soda lake isolates are proposed as a new genus and species, Desulfurispira natronophila (type strain AHT11T = DSM22071T = UNIQEM U758T)., Biotechnology, Applied Sciences

An anaerobic enrichment with pyruvate as electron donor and thiosulfate at pH 10 and 0.6 M Na? inoculated with pasteurized soda lake sediments resulted in a sulfidogenic coculture of two morphotypes of obligately anaerobic haloalkaliphilic endospore-forming clostridia, which were further isolated in pure culture. Strain AHT16 was a thin long rod able to ferment sugars and pyruvate and to respire H2, formate and pyruvate using thiosulfate and fumarate as electron acceptors and growing optimally at pH 9.5. Thiosulfate was reduced incompletely to sulfide and sulfite. The strain was closely related (99% sequence similarity) to a peptolytic alkaliphilic clostridium Natronincola peptidovorans. Strain AHT17 was a short rod with a restricted respiratory metabolism, growing with pyruvate and lactate as electron donor and sulfite, thiosulfate and elemental sulfur as electron acceptors with a pH optimum 9.5. Thiosulfate was reduced completely via sulfite to sulfide. The ability of AHT17 to use sulfite explained the stability of the original coculture of the two clostridia—one member forming sulfite from thiosulfate and another consuming it. Strain AHT17 formed an independent deep phylogenetic lineage within the Clostridiales and is proposed as a new genus and species Desulfitisporum alkaliphilum gen. nov., sp. nov. (=DSM 22410T = UNIQEM U794T)., Biotechnology, Applied Sciences

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range., Biotechnology, Applied Sciences

Evidence on the utilization of simple fatty acids by sulfate-reducing bacteria (SRB) at extremely haloalkaline conditions are practically absent, except for a single case of syntrophy by Desulfonatronum on acetate. Our experiments with sediments from soda lakes of Kulunda Steppe (Altai, Russia) showed sulfide production with sulfate as electron acceptor and propionate and butyrate (but not acetate) as an electron donor at a pH 10–10.5 and a salinity 70–180 g l?1. With propionate as substrate, a highly enriched sulfidogenic culture was obtained in which the main component was identified as a novel representative of the family Syntrophobacteraceae. With butyrate as substrate, a pure SRB culture was isolated which oxidized butyrate and some higher fatty acids incompletely to acetate. The strain represents the first haloalkaliphilic representative of the family Desulfobacteraceae and is described as Desulfobotulus alkaliphilus sp. nov., Biotechnology, Applied Sciences

A possibility of dissimilatory MnO2 reduction at extremely high salt and pH was studied in sediments from hypersaline alkaline lakes in Kulunda Steppe (Altai, Russia). Experiments with anaerobic sediment slurries demonstrated a relatively rapid reduction of colloidal MnO2 in the presence of acetate and formate as electron donor at in situ conditions (i.e., pH 10 and a salt content from 0.6 to 4 M total Na+). All reduced Mn at these conditions remained in the solid phase. A single, stable enrichment culture was obtained from the slurries consistently reducing MnO2 at pH 10 and 0.6 M total Na+ with formate. A pure culture of a haloalkaliphilic Mn-reducing bacterium obtained from the positive enrichment was phylogenetically closely related to the anaerobic haloalkaliphilic Bacillus arseniciselenatis isolated from Mono Lake (CA, USA). Bacillus sp. strain AMnr1 was obligately anaerobic, able to grow either by glucose fermentation, or respiring few nonfermentable substrates by using MnO2 as the electron acceptor. Optimal growth by dissimilatory MnO2 reduction was achieved with glycerol as electron donor at pH 9.5–10 and salt content between 0.4 and 0.8 M total Na+., Biotechnology, Applied Sciences

Neutrophilic iron-oxidizing bacteria (FeOB) are important catalysts of iron cycling in wetland environments. However, little is known about their diversity and distribution in various environments. The aim of this study was to develop a PCR-DGGE assay enabling the detection of neutrophilic iron oxidizers in wetland habitats. Gradient tubes were used to enrich FeOB. From these enrichments, a clone library was established on the basis of the almost complete 16S rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly - and -Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, followed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella sp. On the basis of their nucleotide sequences, four groups could be identified that were comparable to the DGGE banding pattern obtained before with the same PCR products as used for the second clone library. Using these Gallionella-specific 16S rRNA gene-based primers, in combination with DGGE, first insights into the diversity and distribution of these bacteria in wetland soils were obtained., Neutrophilic iron-oxidizing bacteria (FeOB) are important catalysts of iron cycling in wetland environments. However, little is known about their diversity and distribution in various environments. The aim of this study was to develop a PCR-DGGE assay enabling the detection of neutrophilic iron oxidizers in wetland habitats. Gradient tubes were used to enrich FeOB. From these enrichments, a clone library was established on the basis of the almost complete 16S rRNA gene using the universal bacterial primers 27f and 1492r. This clone library consisted of mainly - and -Proteobacteria, among which two major clusters were closely related to Gallionella spp. Specific probes and primers were developed on the basis of this 16S rRNA gene clone library. The newly designed Gallionella-specific 16S rRNA gene primer set 122f/998r was applied to community DNA obtained from three contrasting wetland environments, followed by Denaturing Gradient Gel Electrophoresis (DGGE) analysis. A second 16S rRNA gene clone library was constructed using the PCR products from one of our sampling sites amplified with the newly developed primer set 122f/998r. The cloned 16S rRNA gene sequences all represented novel culturable iron oxidizers most closely related to Gallionella sp. On the basis of their nucleotide sequences, four groups could be identified that were comparable to the DGGE banding pattern obtained before with the same PCR products as used for the second clone library. Using these Gallionella-specific 16S rRNA gene-based primers, in combination with DGGE, first insights into the diversity and distribution of these bacteria in wetland soils were obtained.

In a survey on the presence of archaea in a number of European lakes, it was found that known archaeal primer sets for PCR were not suited for use in freshwater environment, as some lack selectivity, while others were too selective. A nested PCR was developed for denaturing gradient gel electrophoresis (DGGE) with primer sets 21F–958R and Parch519f–Arch915r, respectively. After sequencing of the DGGE bands obtained by this nested method, 93% of the sequences were of archaeal origin. More diverse archaeal DGGE patterns were found as compared with other PCR methods. The nested PCR-DGGE method presented here is therefore a reliable tool to analyze the archaeal diversity in freshwater habitats, revealing even more widespread diversity of the archaea., In a survey on the presence of archaea in a number of European lakes, it was found that known archaeal primer sets for PCR were not suited for use in freshwater environment, as some lack selectivity, while others were too selective. A nested PCR was developed for denaturing gradient gel electrophoresis (DGGE) with primer sets 21F–958R and Parch519f–Arch915r, respectively. After sequencing of the DGGE bands obtained by this nested method, 93% of the sequences were of archaeal origin. More diverse archaeal DGGE patterns were found as compared with other PCR methods. The nested PCR-DGGE method presented here is therefore a reliable tool to analyze the archaeal diversity in freshwater habitats, revealing even more widespread diversity of the archaea.

The occurrence of cryptic and pseudocryptic species, often living in sympatry, is widespread among microalgae. This phenomenon raises important questions about niche partitioning between these closely related species. To date, however, few studies have addressed the ecological mechanisms underlying sympatry in cryptic and pseudocryptic species. As a result, we have only a limited understanding of the factors that govern their distribution along environmental gradients. Here, we used the ribosomal internal transcribed spacer (ITS), 18S rRNA gene, and the RUBISCO LSU (rbcL) chloroplast gene sequence data together with cell wall morphology to show that estuarine populations of the widespread and common benthic diatom Navicula phyllepta Kutz. consist of pseudocryptic species. Growth rate measurements in function of salinity showed that N. phyllepta strains assigned to the different species differed in their tolerance to low salinities (<5 practical salinity units, psu), whic! h was reflected by their different (but widely overlapping) distribution in the Westerschelde estuary (the Netherlands). Multiple regression analyses of the factors determining the abundance of the different species in field samples revealed that, in addition to salinity, sediment type and ammonium concentrations were probably equally important. Our results show that N. phyllepta sensu lato comprises different species with specialized ecophysiological characteristics rather than generalists with a broad adaptability to different environmental conditions., The occurrence of cryptic and pseudocryptic species, often living in sympatry, is widespread among microalgae. This phenomenon raises important questions about niche partitioning between these closely related species. To date, however, few studies have addressed the ecological mechanisms underlying sympatry in cryptic and pseudocryptic species. As a result, we have only a limited understanding of the factors that govern their distribution along environmental gradients. Here, we used the ribosomal internal transcribed spacer (ITS), 18S rRNA gene, and the RUBISCO LSU (rbcL) chloroplast gene sequence data together with cell wall morphology to show that estuarine populations of the widespread and common benthic diatom Navicula phyllepta Kutz. consist of pseudocryptic species. Growth rate measurements in function of salinity showed that N. phyllepta strains assigned to the different species differed in their tolerance to low salinities (<5 practical salinity units, psu), whic! h was reflected by their different (but widely overlapping) distribution in the Westerschelde estuary (the Netherlands). Multiple regression analyses of the factors determining the abundance of the different species in field samples revealed that, in addition to salinity, sediment type and ammonium concentrations were probably equally important. Our results show that N. phyllepta sensu lato comprises different species with specialized ecophysiological characteristics rather than generalists with a broad adaptability to different environmental conditions.

A hydrogenotrophic, sulfate-reducing bacterium, designated strain SB1(T), was isolated from sulfidogenic sludge of a full-scale synthesis-gas-fed bioreactor used to remediate wastewater from a zinc smelter. Strain SB1(T) was found to be an abundant micro-organism in the sludge at the time of isolation. Hydrogen, formate, pyruvate, lactate, malate, fumarate, succinate, ethanol and glycerol served as electron donors for sulfate reduction. Organic substrates were incompletely oxidized to acetate. 16S rRNA gene sequence analysis showed that the closest recognized relative to strain SB1(T) was Desulfovibrio gigas DSM 1382(T) (97.5 % similarity). The G+C content of the genomic DNA of strain SB1(T) was 62.2 mol%, comparable with that of Desulfovibrio gigas DSM 1382(T) (60.2 mol%). However, the level of DNA-DNA relatedness between strain SB1(T) and Desulfovibrio gigas DSM 1382(T) was only 56.0 %, indicating that the two strains are not related at the species level. Strain SB1(T) could also be differentiated from Desulfovibrio gigas based on phenotypic characteristics, such as major cellular fatty acid composition (anteiso-C(15 : 0), iso-C(14 : 0) and C(18 : 1) cis 9) and substrate utilization. Strain SB1(T) is therefore considered to represent a novel species of the genus Desulfovibrio, for which the name Desulfovibrio paquesii sp. nov. is proposed. The type strain is SB1(T) (=DSM 16681(T)=JCM 14635(T))

Citrate-containing wastewater is used as electron donor for sulfate reduction in a biological treatment plant for the removal of sulfate. The pathway of citrate conversion coupled to sulfate reduction and the microorganisms involved were investigated. Citrate was not a direct electron donor for the sulfate-reducing bacteria. Instead, citrate was fermented to mainly acetate and formate. These fermentation products served as electron donors for the sulfate-reducing bacteria. Sulfate reduction activities of the reactor biomass with acetate and formate were sufficiently high to explain the sulfate reduction rates that are required for the process. Two citrate-fermenting bacteria were isolated. Strain R210 was closest related to Trichococcus pasteurii (99.5% ribosomal RNA (rRNA) gene sequence similarity). The closest relative of strain S101 was Veillonella montepellierensis with an rRNA gene sequence similarity of 96.7%. Both strains had a complementary substrate range

Anaerobic fermentation by an open mixed culture was investigated at different pH values (4–8.5) and with three substrates (glucose, glycerol and xylose). The populations established in each condition were assessed by denaturing gradient gel electrophoresis analysis of the 16S ribosomal RNA gene fragments. The fermentation pattern and the composition of the microbial population were also evaluated when operational variations were imposed (increase of substrate concentration or introduction of a second substrate). The experimental results demonstrated that at low and high pH values, a clearly different fermentation pattern was associated with the dominance of a specialised group of clostridiae. At intermediate pH values, the product spectrum was rather variable and seemed to be sensitive to variations in the microbial community. Different substrates resulted in the establishment of different microbial communities. When fed with a mixture of two substrates, mixotrophic microorganisms (capable of degrading both substrates) were found to overgrow the originally dominant specialists. Overall, the experiments have shown that some operational variables have a clear impact on the fermentation pattern and on the population established. However, a uniform relationship between the process characteristics (associated to a metabolic response) and the microbial population present is not always possible., Biotechnology, Applied Sciences

Anaerobic enrichments with H2 as electron donor and thiosulfate/polysulfide as electron acceptor at pH 10 and 0.6 M total Na+ yielded two non sulfate-reducing representatives of reductive sulfur cycle from soda lake sediments. Strain AHT 1 was isolated with thiosulfate as the electron acceptor from north–eastern Mongolian soda lakes and strain AHT 2—with polysulfide as the electron acceptor from Wadi al Natrun lakes in Egypt. Both isolates represented new phylogenetic lineages: AHT 1—within Clostridiales and AHT 2—within the Deltaproteobacteria. Both bacteria are obligate anaerobes with respiratory metabolism. Both grew chemolithoautotrophically with H2 as the electron donor and can use thiosulfate, elemental sulfur and polysulfide as the electron acceptors. AHT 2 also used nitrate as acceptor, reducing it to ammonia. During thiosulfate reduction, AHT 1 excreted sulfite. dsrAB gene was not found in either strain. Both strains were moderate salt-tolerant (grow up to 2 M total Na+) true alkaliphiles (grow between pH 8.5 and 10.3). On the basis of the phenotypic and phylogenetic data, strains AHT 1 and AHT 2 are proposed as new genera and species Dethiobacter alkaliphilus and Desulfurivibrio alkaliphilus, respectively., Department of Biotechnology, Applied Sciences

An enrichment culture from saline soda soils, using acetate as carbon and energy source and 2- phenylpropionitrile as nitrogen source (PPN) at pH 10, resulted in the isolation of strain ANL-?CH3. The strain was identified as a representative of the genus Halomonas in the Gammaproteobacteria. The bacterium was capable of PPN utilization as a nitrogen source only, while phenylacetonitrile (PAN) served both as carbon, energy and nitrogen source. This capacity was not described previously for any other haloalkaliphilic bacteria. Apart from the nitriles mentioned above, resting cells of ANL- ?CH3 also hydrolyzed mandelonitrile, benzonitrile, acrylonitrile, and phenylglycinonitrile, presumably using nitrilase pathway. Neither nitrile hydratase nor amidase activity was detected. The isolate showed a capacity to grow with benzoate and salicylate as carbon and energy source and demonstrated the ability to completely mineralize PAN. These clearly indicated a potential to catabolize aromatic compounds. On the basis of unique phenotype and distinct phylogeny, strain ANL-?CH3 is proposed as a novel species of the genus Halomonas—Halomonas nitrilicus sp. nov., Biotechnology, Applied Sciences

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 Desulfovibriolike 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 accounte, Biotechnology, Applied Sciences

A chemolithoautotrophic sulfur-oxidizing bacterium (SOB) strain ALCO 1 capable of growing at both near-neutral and extremely alkaline pH was isolated from hypersaline soda lakes in S-W Siberia (Altai, Russia). Strain ALCO 1 represents a novel separate branch within the halothiobacilli in the Gammaproteobacteria, which, so far, contained only neutro-halophilic SOB. On the basis of its unique phenotypic properties and distant phylogeny, strain ALCO 1 is proposed as a new genus and species Thioalkalibacter halophilus gen. nov. sp. nov. ALCO 1 was able to grow within a broad range of salinity (0.5–3.5 M of total sodium) with an optimum at around 1 M Na+, and pH (7.2–10.2, pHopt at around 8.5). Na+ was required for sulfur-dependent respiration in ALCO 1. The neutral (NaCl)-grown chemostat culture had a much lower maximum growth rate (?max), respiratory activity and total cytochrome c content than its alkaline-grown counterpart. The specific concentration of osmolytes (ectoine and glycine-betaine) produced at neutral pH and 3 M NaCl was roughly two times higher than at pH 10 in soda. Altogether, strain ALCO 1 represents an interesting chemolithoautotrophic model organism for comparative investigations of bacterial adaptations to high salinity and pH., Department of Biotechnology, Applied Sciences

Deposition of dredged harbor sediments in relatively undisturbed ecosystems is often considered a viable option for confinement of pollutants and possible natural attenuation. This study investigated the effects of deposition of heavy-metal-polluted sludge on the microbial diversity of sandy sediments during 12 months of mesocosm incubation. Geochemical analyses showed an initial increase in pore-water metal concentrations, which subsided after 3 months of incubation. No influence of the deposited sediment was observed in denaturing gradient gel electrophoresis (DGGE) profiles of bacterial 16S rRNA genes, whereas a minor, transient impact on the archaeal community was revealed. Phylogenetic analyses of bacterial 16S rRNA clone libraries showed an abundance of members of the Flavobacteriaceae, the ?- and ?-Proteobacteria, in both the muddy and the sandy sediments. Despite the finding that some groups of clones were shared between the metal-impacted sandy sediment and the harbor control, comparative analyses showed that the two sediments were significantly different in community composition. Consequences of redeposition of metal-polluted sediment were primarily underlined with cultivation-dependent techniques. Toxicity tests showed that the percentage of Cd- and Cu-tolerant aerobic heterotrophs was highest among isolates from the sandy sediment with metal-polluted mud on top., Biotechnology, Applied Sciences

In this paper we describe denitrification at extremely high salt and pH in sediments from hypersaline alkaline soda lakes and soda soils. Experiments with sediment slurries demonstrated the presence of acetate-utilizing denitrifying populations active at in situ conditions. Anaerobic enrichment cultures at pH 10 and 4 M total Na+ with acetate as electron donor and nitrate, nitrite and N2O as electron acceptors resulted in the dominance of Gammaproteobacteria belonging to the genus Halomonas. Both mixed and pure culture studies identified nitrite and N2O reduction as rate-limiting steps in the denitrification process at extremely haloalkaline conditions., Biotechnology, Applied Sciences

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na(+) and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H(2) and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5-10, and grew in soda brines containing 1-4 M total Na(+) (optimum at 1.0-2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1(T) and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6(T) from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov

Thiopaq biotechnology for partial sulfide oxidation to elemental sulfur is an efficient way to remove H2S from biogases. However, its application for high-pressure natural gas desulfurization needs upgrading. Particularly, an increase in alkalinity of the scrubbing liquid is required. Therefore, the feasibility of sulfide oxidation into elemental sulfur under oxygen limitation was tested at extremely haloalkaline conditions in lab-scale bioreactors using mix sediments from hypersaline soda lakes as inoculum. The microbiological analysis, both culture dependent and independent, of the successfully operating bioreactors revealed a domination of obligately chemolithoautotrophic and extremely haloalkaliphilic sulfur-oxidizing bacteria belonging to the genus Thioalkalivibrio. Two subgroups were recognized among the isolates. The subgroup enriched from the reactors operating at pH 10 clustered with Thioalkalivibrio jannaschii¿Thioalkalivibrio versutus core group of the genus Thioalkalivibrio. Another subgroup, obtained mostly with sulfide as substrate and at lower pH, belonged to the cluster of facultatively alkaliphilic Thioalkalivibrio halophilus. Overall, the results clearly indicate a large potential of the genus Thiolalkalivibrio to efficiently oxidize sulfide at extremely haloalkaline conditions, which makes it suitable for application in the natural gas desulfurization.

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 ac

Thiopaq biotechnology for partial sulfide oxidation to elemental sulfur is an efficient way to remove H2S from biogases. However, its application for high-pressure natural gas desulfurization needs upgrading. Particularly, an increase in alkalinity of the scrubbing liquid is required. Therefore, the feasibility of sulfide oxidation into elemental sulfur under oxygen limitation was tested at extremely haloalkaline conditions in lab-scale bioreactors using mix sediments from hypersaline soda lakes as inoculum. The microbiological analysis, both culture dependent and independent, of the successfully operating bioreactors revealed a domination of obligately chemolithoautotrophic and extremely haloalkaliphilic sulfur-oxidizing bacteria belonging to the genus Thioalkalivibrio. Two subgroups were recognized among the isolates. The subgroup enriched from the reactors operating at pH 10 clustered with Thioalkalivibrio jannaschii–Thioalkalivibrio versutus core group of the genus Thioalkalivibrio. Another subgroup, obtained mostly with sulfide as substrate and at lower pH, belonged to the cluster of facultatively alkaliphilic Thioalkalivibrio halophilus. Overall, the results clearly indicate a large potential of the genus Thiolalkalivibrio to efficiently oxidize sulfide at extremely haloalkaline conditions, which makes it suitable for application in the natural gas desulfurization, Biotechnology, Applied Sciences

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The 18S ribosomal DNA (rDNA) isolated from sulfidic Holocene sediments and particulate organic matter in the water column of the stratified Small Meromictic Basin (SMB) in Ellis Fjord (eastern Antarctica) was analyzed to identify possible biological sources of organic matter. Previous work had shown that the sediments contained numerous diatom frustules and high contents of a highly branched isoprenoid (HBI) C25:2 alkene (which is a specific biomarker of certain species of the diatom genera Navicula, Haslea, Pleurosigma, or Rhizosolenia), so we focused our search on preserved fossil 18S rDNA of diatoms using sensitive polymerase chain reaction (PCR) approaches. We did not find diatom-derived fossil 18S rDNA using general eukaryotic primers, and even when we used primers selective for diatom 18S rDNA, we only identified a Chaetoceros phylotype, which is known to form cysts in the SMB but is not a likely source of the C25:2 HBI. When we used PCR/denaturing gradient gel electrophoresis methods specific to phylotypes within the HBI-biosynthesizing genera, we were able to identify three phylotypes in the sediments related to HBI-producing strains of the genera Haslea and Navicula. The ancient DNA data thus provided a limited, but valuable, view of the diversity of late Holocene primary producers with a particular bias to specific components of the biota that were better preserved such as the Chaetoceros cysts. This use of paleogenetics also revealed unexpected possible sources of organic matter such as novel stramenopiles for which no specific lipid biomarkers are known and thus would not have been identified based on traditional lipid stratigraphy alone., Applied Sciences

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Applied Sciences

Applied Sciences

Phototrophic biofilms occur on surfaces exposed to light in a range of terrestrial and aquatic environments. Oxygenic phototrophs like diatoms, green algae, and cyanobacteria are the major primary producers that generate energy and reduce carbon dioxide, providing the system with organic substrates and oxygen. Photosynthesis fuels processes and conversions in the total biofilm community, including the metabolism of heterotrophic organisms. A matrix of polymeric substances secreted by phototrophs and heterotrophs enhances the attachment of the biofilm community. This review discusses the actual and potential applications of phototrophic biofilms in wastewater treatment, bioremediation, fish-feed production, biohydrogen production, and soil improvement., Biotechnology, Applied Sciences

The composition and seasonal dynamics of biofilm-associated eukaryotic communities were analysed at the metre and kilometre scale along a salinity gradient in the Westerschelde estuary (The Netherlands), using microscopy and a genetic fingerprinting technique (PCR-DGGE). Microphytobenthic biomass, measured as chlorophyll a (chl a), varied seasonally over 2 orders of magnitude, being highest in spring. Communities were dominated by epipelic diatoms, in particular by members of the genus Navicula. In spring, a few smaller epipelic diatom species dominated during biomass peaks, while during the rest of the year, communities were more diverse and were characterised by larger species. The microphytobenthic community collapsed when grazers appeared, which happened concomitantly with a rise in temperature. Spring biomass development was associated with marked changes in porewater nutrient concentrations, especially towards the estuary mouth. In the DGGE data, diatoms, ciliates, amoebae, copepods, nematodes, annelids and platyhelminthes were detected. Ordination analysis of the species counts and DGGE data were largely congruent and indicated that on the scale of the whole estuary (i.e. km scale), taxonomic turnover in microphytobenthos composition was mainly associated with the salinity gradient. At smaller spatial scales, the position of sampling localities along the tidal exposure gradient appeared to be the main determinant of species turnover, in particular in the brackish reaches of the estuary., BT/Biotechnology, Applied Sciences

Here we describe the diversity and activity of sulfate reducing bacteria along a salinity gradient in four different soda lakes from the Kulunda Steppe (South East Siberia, Russia). For this purpose, a combination of culture-dependent and independent techniques was applied. The general bacterial and SRB diversity were analyzed by denaturing gradient gel electrophoresis (DGGE) targeting the 16S rDNA gene. DNA was used to detect the microbial populations that were present in the soda lake sediments, whereas ribosomal RNA was used as a template to obtain information on those that were active. Individual DGGE bands were sequenced and a phylogenetic analysis was performed. In addition, the overall activity of SRB was obtained by measuring the sulfate reduction rates (SRR) and their abundance was estimated by serial dilution. Our results showed the presence of minor, but highly active microbial populations, mostly represented by members of the Proteobacteria. Remarkably high SRR were measured at hypersaline conditions (200 g L-1). A relatively high viable count indicated that sulfate reducing bacteria could be highly active in hypersaline soda lakes. Furthermore, the increase of sodium carbonate/bicarbonate seemed to affect the composition of the microbial community in soda lakes, but not the rate of sulfate reduction., Biotechnology, Applied Sciences

Applied Sciences

The composition and seasonal dynamics of biofilm-associated eukaryotic communities were analysed at the metre and kilometre scale along a salinity gradient in the Westerschelde estuary (The Netherlands), using microscopy and a genetic fingerprinting technique (PCR-DGGE). Microphytobenthic biomass, measured as chlorophyll a (chl a), varied seasonally over 2 orders of magnitude, being highest in spring. Communities were dominated by epipelic diatoms, in particular by members of the genus Navicula. In spring, a few smaller epipelic diatom species dominated during biomass peaks, while during the rest of the year, communities were more diverse and were characterised by larger species. The microphytobenthic community collapsed when grazers appeared, which happened concomitantly with a rise in temperature. Spring biomass development was associated with marked changes in porewater nutrient concentrations, especially towards the estuary mouth. In the DGGE data, diatoms, ciliates, amoebae, copepods, nematodes, annelids and platyhelminthes were detected. Ordination analysis of the species counts and DGGE data were largely congruent and indicated that on the scale of the whole estuary (i.e. km scale), taxonomic turnover in microphytobenthos composition was mainly associated with the salinity gradient. At smaller spatial scales, the position of sampling localities along the tidal exposure gradient appeared to be the main determinant of species turnover, in particular in the brackish reaches of the estuary., The composition and seasonal dynamics of biofilm-associated eukaryotic communities were analysed at the metre and kilometre scale along a salinity gradient in the Westerschelde estuary (The Netherlands), using microscopy and a genetic fingerprinting technique (PCR-DGGE). Microphytobenthic biomass, measured as chlorophyll a (chl a), varied seasonally over 2 orders of magnitude, being highest in spring. Communities were dominated by epipelic diatoms, in particular by members of the genus Navicula. In spring, a few smaller epipelic diatom species dominated during biomass peaks, while during the rest of the year, communities were more diverse and were characterised by larger species. The microphytobenthic community collapsed when grazers appeared, which happened concomitantly with a rise in temperature. Spring biomass development was associated with marked changes in porewater nutrient concentrations, especially towards the estuary mouth. In the DGGE data, diatoms, ciliates, amoebae, copepods, nematodes, annelids and platyhelminthes were detected. Ordination analysis of the species counts and DGGE data were largely congruent and indicated that on the scale of the whole estuary (i.e. km scale), taxonomic turnover in microphytobenthos composition was mainly associated with the salinity gradient. At smaller spatial scales, the position of sampling localities along the tidal exposure gradient appeared to be the main determinant of species turnover, in particular in the brackish reaches of the estuary.

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime. © 2006 The Authors.

A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 108). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30¿35% and 25¿35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20¿25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15¿20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor.

Soda lakes are naturally occurring highly alkaline and saline environments. Although the sulfur cycle is one of the most active element cycles in these lakes, little is known about the sulfate-reducing bacteria (SRB). In this study we investigated the diversity, activity, and abundance of SRB in sediment samples and enrichment cultures from a range of (hyper)saline soda lakes of the Kulunda Steppe in southeastern Siberia in Russia. For this purpose, a polyphasic approach was used, including denaturing gradient gel electrophoresis of dsr gene fragments, sulfate reduction rate measurements, serial dilutions, and quantitative real-time PCR (qPCR). Comparative sequence analysis revealed the presence of several novel clusters of SRB, mostly affiliated with members of the order Desulfovibrionales and family Desulfobacteraceae. We detected sulfate reducers and observed substantial sulfate reducing rates (between 12 and 423 µmol/dm3 day¿1) for most lakes, even at a salinity of 475 g/liter. Enrichments were obtained at salt saturating conditions (4 M Na+), using H2 or volatile fatty acids as electron donors, and an extremely halophilic SRB, strain ASO3-1, was isolated. Furthermore, a high dsr gene copy number of 108 cells per ml was detected in a hypersaline lake by qPCR. Our results indicate the presence of diverse and active SRB communities in these extreme ecosystems.

Applied Sciences

Applied Sciences