[show abstract][hide abstract] ABSTRACT: Benthic invertebrates affect microbial processes and communities in freshwater sediment by enhancing sediment-water solute fluxes and by grazing on bacteria. Using microcosms, the effects of larvae of the widespread midge Chironomus plumosus on the efflux of denitrification products (N2O and N2+N2O) and the diversity and abundance of nitrate- and nitrous-oxide-reducing bacteria were investigated. Additionally, the diversity of actively nitrate- and nitrous-oxide-reducing bacteria was analyzed in the larval gut. The presence of larvae increased the total effluxes of N2O and N2+N2O up to 8.6- and 4.2-fold, respectively, which was mostly due to stimulation of sedimentary denitrification; incomplete denitrification in the guts accounted for up to 20% of the N2O efflux. Phylotype richness of the nitrate reductase gene narG was significantly higher in sediment with than without larvae. In the gut, 47 narG phylotypes were found expressed, which may contribute to higher phylotype richness in colonized sediment. In contrast, phylotype richness of the nitrous oxide reductase gene nosZ was unaffected by the presence of larvae and very few nosZ phylotypes were expressed in the gut. Gene abundance of neither narG, nor nosZ was different in sediments with and without larvae. Hence, C. plumosus increases activity and diversity, but not overall abundance of nitrate-reducing bacteria, probably by providing additional ecological niches in its burrow and gut.
Systematic and Applied Microbiology 09/2013; · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacteria of the class Dehalococcoidia (DEH), phylum Chloroflexi, are widely distributed in the marine subsurface, yet metabolic properties of the many uncultivated lineages are completely unknown. This study therefore analysed genomic content from a single DEH cell designated 'DEH-J10' obtained from the sediments of Aarhus Bay, Denmark. Real-time PCR showed the DEH-J10 phylotype was abundant in upper sediments but was absent below 160 cm below sea floor. A 1.44 Mbp assembly was obtained and was estimated to represent up to 60.8% of the full genome. The predicted genome is much larger than genomes of cultivated DEH and appears to confer metabolic versatility. Numerous genes encoding enzymes of core and auxiliary beta-oxidation pathways were identified, suggesting that this organism is capable of oxidising various fatty acids and/or structurally related substrates. Additional substrate versatility was indicated by genes, which may enable the bacterium to oxidise aromatic compounds. Genes encoding enzymes of the reductive acetyl-CoA pathway were identified, which may also enable the fixation of CO2 or oxidation of organics completely to CO2. Genes encoding a putative dimethylsulphoxide reductase were the only evidence for a respiratory terminal reductase. No evidence for reductive dehalogenase genes was found. Genetic evidence also suggests that the organism could synthesise ATP by converting acetyl-CoA to acetate by substrate-level phosphorylation. Other encoded enzymes putatively conferring marine adaptations such as salt tolerance and organo-sulphate sulfohydrolysis were identified. Together, these analyses provide the first insights into the potential metabolic traits that may enable members of the DEH to occupy an ecological niche in marine sediments.The ISME Journal advance online publication, 22 August 2013; doi:10.1038/ismej.2013.143.
[show abstract][hide abstract] ABSTRACT: Emission of the greenhouse gas nitrous oxide (N(2) O) from freshwater and terrestrial invertebrates has exclusively been ascribed to N(2) O production by ingested denitrifying bacteria in the anoxic gut of the animals. Our study of marine molluscs now shows that also microbial biofilms on shell surfaces are important sites of N(2) O production. The shell biofilms of Mytilus edulis, Littorina littorea and Hinia reticulata contributed 18-94% to the total animal-associated N(2) O emission. Nitrification and denitrification were equally important sources of N(2) O in shell biofilms as revealed by (15) N-stable isotope experiments with dissected shells. Microsensor measurements confirmed that both nitrification and denitrification can occur in shell biofilms due to a heterogeneous oxygen distribution. Accordingly, ammonium, nitrite and nitrate were important drivers of N(2) O production in the shell biofilm of the three mollusc species. Ammonium excretion by the animals was found to be sufficient to sustain N(2) O production in the shell biofilm. Apparently, the animals provide a nutrient-enriched microenvironment that stimulates growth and N(2) O production of the shell biofilm. This animal-induced stimulation was demonstrated in a long-term microcosm experiment with the snail H. reticulata, where shell biofilms exhibited the highest N(2) O emission rates when the animal was still living inside the shell.
[show abstract][hide abstract] ABSTRACT: The importance of extracellular DNA (eDNA) in biofilm formation has become increasingly clear from research on clinically relevant bacteria. This study aimed to determine if the quantity of eDNA produced can be linked to the ability to form biofilm. We systematically quantified eDNA over time during planktonic growth and biofilm formation in Reinheimera sp. F8 and three other environmental isolates belonging to the genera Pseudomonas, Microbacterium, and Serratia. eDNA in biofilms was visualised by fluorescence microscopy and quantified by PicoGreen(®) labeling without further sample preparation, whereas eDNA in planktonic cultures was precipitated before labeling and quantification. The effect of eDNA removal was investigated by DNase treatment. eDNA appeared in the early exponential growth phase of planktonic batch cultures and the concentration peaked in the stationary phase. The concentration in biofilms differed substantially between strains and over time during biofilm development. eDNA was important for the initial attachment in all strains, and DNase treatment reduced biofilm formation in 3 of 4 strains. The extent to which eDNA accumulated in planktonic cultures or biofilms did not reflect its significance to biofilm formation, and even very low concentrations of eDNA affected biofilm formation strongly. The significance of eDNA for biofilm formation in nature may thus be more widespread than previously anticipated. This article is protected by copyright. All rights reserved.
[show abstract][hide abstract] ABSTRACT: Half of the microbial cells in the Earth's oceans are found in sediments. Many of these cells are members of the Archaea, single-celled prokaryotes in a domain of life separate from Bacteria and Eukaryota. However, most of these archaea lack cultured representatives, leaving their physiologies and placement on the tree of life uncertain. Here we show that the uncultured miscellaneous crenarchaeotal group (MCG) and marine benthic group-D (MBG-D) are among the most numerous archaea in the marine sub-sea floor. Single-cell genomic sequencing of one cell of MCG and three cells of MBG-D indicated that they form new branches basal to the archaeal phyla Thaumarchaeota and Aigarchaeota, for MCG, and the order Thermoplasmatales, for MBG-D. All four cells encoded extracellular protein-degrading enzymes such as gingipain and clostripain that are known to be effective in environments chemically similar to marine sediments. Furthermore, we found these two types of peptidase to be abundant and active in marine sediments, indicating that uncultured archaea may have a previously undiscovered role in protein remineralization in anoxic marine sediments.
[show abstract][hide abstract] ABSTRACT: Rumen methanogens are major sources of anthropogenic methane emissions, and these archaea are targets in strategies aimed at reducing methane emissions. Here we show that the poorly characterised Thermoplasmata archaea in bovine rumen are methylotrophic methanogens and that they are reduced upon dietary supplementation with rapeseed oil in lactating cows. In a metatranscriptomic survey, Thermoplasmata 16S rRNA and methyl-coenzyme M reductase (mcr) transcripts decreased concomitantly with mRNAs of enzymes involved in methanogenesis from methylamines that were among the most abundant archaeal transcripts, indicating that these Thermoplasmata degrade methylamines. Their methylotrophic methanogenic lifestyle was corroborated by in vitro incubations, showing enhanced growth of these organisms upon methylamine supplementation paralleled by elevated methane production. The Thermoplasmata have a high potential as target in future strategies to mitigate methane emissions from ruminant livestock. Our findings and the findings of others also indicate a wider distribution of methanogens than previously anticipated.
[show abstract][hide abstract] ABSTRACT: Organic crusts on liquid manure storage tanks harbor ammonia- and nitrite-resistant methane oxidizers and may significantly reduce methane emissions. Methane oxidation potential (0.6 mol CH(4) m(-2) d(-1)) peaked during fall and winter, after 4 months of crust development. Consequences for methane mitigation potential of crusts are discussed.
Applied and environmental microbiology 10/2012; · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oxygen consumption in marine sediments is often coupled to the oxidation of sulphide generated by degradation of organic matter in deeper, oxygen-free layers. Geochemical observations have shown that this coupling can be mediated by electric currents carried by unidentified electron transporters across centimetre-wide zones. Here we present evidence that the native conductors are long, filamentous bacteria. They abounded in sediment zones with electric currents and along their length they contained strings with distinct properties in accordance with a function as electron transporters. Living, electrical cables add a new dimension to the understanding of interactions in nature and may find use in technology development.
[show abstract][hide abstract] ABSTRACT: Freshwater macrophytes stimulate rhizosphere-associated coupled nitrification-denitrification and are therefore likely to influence the community composition and abundance of rhizosphere-associated denitrifiers and nitrate reducers. Using the narG gene, which encodes the catalytic subunit of the membrane-bound nitrate reductase, as a molecular marker, the community composition and relative abundance of nitrate-reducing bacteria were compared in the rhizosphere of the freshwater macrophyte species Littorella uniflora and Myriophyllum alterniflorum to nitrate-reducing communities in unvegetated sediment. Microsensor analysis indicated a higher availability of oxygen in the rhizosphere compared to unvegetated sediment, with a stronger release of oxygen from the roots of L. uniflora compared to M. alterniflorum. Comparison of narG clone libraries between samples revealed a higher diversity of narG phylotypes in association with the macrophyte rhizospheres compared to unvegetated sediment. Quantitative PCR targeting narG- and 16S rRNA-encoding genes pointed to a selective enrichment of narG gene copies in the rhizosphere. The results suggested that the microenvironment of macrophyte rhizospheres, characterized by the release of oxygen and labile organic carbon from the root system, had a stimulating effect on the diversity and relative abundance of rhizosphere-associated nitrate reducers.
Systematic and Applied Microbiology 10/2012; · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitrification in shell biofilms and denitrification in the gut of the animal accounted for N(2)O emission by Dreissena polymorpha (Bivalvia), as shown by gas chromatography and gene expression analysis. The mussel's ammonium excretion was sufficient to sustain N(2)O production and thus potentially uncouples invertebrate N(2)O production from environmental N concentrations.
Applied and environmental microbiology 04/2012; 78(12):4505-9. · 3.69 Impact Factor
[show abstract][hide abstract] ABSTRACT: Clone library-based studies have shown that almost all lumbricid earthworm species harbour host-specific symbiotic bacteria belonging to the novel genus Verminephrobacter in their nephridia (excretory organs). To date the only described representative from this genus is Verminephrobacter eiseniae, the specific symbiont of the earthworm Eisenia fetida. In this study two novel rod-shaped, non-endosporeforming, betaproteobacterial symbionts were isolated from the nephridia of two closely related earthworm species. Both isolates were affiliated with the genus Verminephrobacter by 16S rRNA gene sequence analysis. Similarly to V. eiseniae, the two isolates grew aerobically with a preference for low oxygen concentrations on a range of sugars, fatty acids and amino acids and fermentatively on glucose and pyruvate. These phenotypes match well with the conditions reported or inferred for the nephridial environment. Based on 16S rRNA gene similarity, DNA-DNA hybridization value and phenotypic characteristics the two isolates are clearly distinct from V. eiseniae. Phenotypic characteristics could not clearly differentiate the two strains as separate species but a low DNA-DNA hybridization value of 57.3%, their earthworm host specificity, differing temperature ranges and pH optima suggest that they represent two subspecies of a novel species of Verminephrobacter. For this species, the name V. aporrectodeae sp. nov. is proposed, with the two subspecies V. aporrectodeae subsp. tuberculatae (type strain, At4(T) = DSM 21361(T) = LMG 25313(T)) and V. aporrectodeae subsp. caliginosae (type strain, Ac9(T) = DSM 21895(T) = LMG 25312(T)) isolated from the nephridia of the earthworms Aporrectodea tuberculata and A. caliginosa, respectively.
Antonie van Leeuwenhoek 03/2012; 101(3):507-14. · 2.07 Impact Factor
[show abstract][hide abstract] ABSTRACT: Members of Epsilonproteobacteria and Deferribacteres have been implied in nitrate-induced souring control in high-temperature oil production facilities. Here we report on their diversity and abundance in the injection and production part of a nitrate-treated, off-shore oil facility (Halfdan, Denmark) and aimed to assess their potential in souring control. Nitrate addition to deoxygenated seawater shifted the low-biomass seawater community dominated by Gammaproteobacteria closely affiliated with the genus Colwellia to a high-biomass community with significantly higher species richness. Epsilonproteobacteria accounted for less than 1% of the total bacterial community in the nitrate-amended injection water and were most likely outcompeted by putative nitrate-reducing, methylotrophic Gammaproteobacteria of the genus Methylophaga. Reservoir passage and recovery of the oil resulted in a significant change in the bacterial community. Members of the thermophilic Deferribacteres were the second major fraction of the bacterial community in the production water (~30% of the total bacterial community). They were not found in the injection water and were therefore assumed to be indigenous to the reservoir. Additional diversity analysis and targeted quantification of periplasmic nitrate reductase (napA) genes indicated that most resident Deferribacteres possessed the functional potential to contribute to nitrate reduction in the system. In sum, the dominance of nitrate-reducing Deferribacteres and the low relative abundance of Epsilonproteobacteria throughout the production facility suggested that the Deferribacteres play a major role in nitrate-induced souring control at high temperatures.
Systematic and Applied Microbiology 02/2012; 35(3):165-74. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: While genomic erosion is common among intracellular symbionts, patterns of genome evolution in heritable extracellular endosymbionts remain elusive. We study vertically transmitted extracellular endosymbionts (Verminephrobacter, Betaproteobacteria) that form a beneficial, species-specific, and evolutionarily old (60-130 Myr) association with earthworms. We assembled a draft genome of Verminephrobacter aporrectodeae and compared it with the genomes of Verminephrobacter eiseniae and two nonsymbiotic close relatives (Acidovorax). Similar to V. eiseniae, the V. aporrectodeae genome was not markedly reduced in size and showed no A-T bias. We characterized the strength of purifying selection (ω = dN/dS) and codon usage bias in 876 orthologous genes. Symbiont genomes exhibited strong purifying selection (ω = 0.09 ± 0.07), although transition to symbiosis entailed relaxation of purifying selection as evidenced by 50% higher ω values and less codon usage bias in symbiont compared with reference genomes. Relaxation was not evenly distributed among functional gene categories but was overrepresented in genes involved in signal transduction and cell envelope biogenesis. The same gene categories also harbored instances of positive selection in the Verminephrobacter clade. In total, positive selection was detected in 89 genes, including also genes involved in DNA metabolism, tRNA modification, and TonB-dependent iron uptake, potentially highlighting functions important in symbiosis. Our results suggest that the transition to symbiosis was accompanied by molecular adaptation, while purifying selection was only moderately relaxed, despite the evolutionary age and stability of the host association. We hypothesize that biparental transmission of symbionts and rare genetic mixing during transmission can prevent genome erosion in heritable symbionts.
Genome Biology and Evolution 02/2012; 4(3):307-15. · 4.76 Impact Factor
[show abstract][hide abstract] ABSTRACT: Microsensor measurements of oxygen were combined with mRNA-targeted fluorescence in situ hybridization (FISH) to relate the expression of nitrite reductase (nirS) to oxygen concentrations in artificial biofilms of the denitrifier Pseudomonas stutzeri. A distinct zone of nirS transcript-containing cells was detected at the oxic-anoxic transition zone, below an oxygen threshold concentration of 0.7-2.5μM, depending on incubation conditions. Although not a routine technique yet, the possibility of coupling microsensor and mRNA-targeted FISH analyses described here opens for studies addressing microenvironment, identity, and actual activity of microbes in stratified environments at single cell resolution.
Systematic and Applied Microbiology 01/2012; · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Three Gram-negative bacterial strains were isolated from the biofilter of a recirculating marine aquaculture. They were non-pigmented rods, mesophiles, moderately halophilic, and showed chemo-organoheterotrophic growth on various sugars, fatty acids, and amino acids, with oxygen as electron acceptor; strains D9-3(T) and D11-58 were in addition able to denitrify. Phototrophic or fermentative growth could not be demonstrated. Phylogenetic analysis of the 16S rRNA gene sequences placed D9-3(T) and D11-58, and D1-19(T) on two distinct branches within the alpha-3 proteobacterial Rhodobacteraceae, affiliated with, but clearly separate from, the genera Rhodobacter, Rhodovulum, and Rhodobaca. Based on morphological, physiological, and 16S rRNA-based phylogenetic characteristics, the isolated strains are proposed as new species of two novel genera, Defluviimonas denitrificans gen. nov., sp. nov. (type strain D9-3(T)=DSM 18921(T)=ATCC BAA-1447(T); additional strain D11-58=DSM19039=ATCC BAA-1448) and Pararhodobacter aggregans gen. nov., sp. nov (type strain D1-19(T)=DSM 18938(T)=ATCC BAA-1446(T)).
Systematic and Applied Microbiology 09/2011; 34(7):498-502. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: This study investigated the abundance and distribution of key functional microbial populations and their activities in a full-scale integrated fixed film activated sludge-enhanced biological phosphorus removal (IFAS-EBPR) process. Polyphosphate accumulating organisms (PAOs) including Accumulibacter and EBPR activities were predominately associated with the mixed liquor (>90%) whereas nitrifying populations and nitrification activity resided mostly (>70%) on the carrier media. Ammonia oxidizer bacteria (AOB) were members of the Nitrosomonas europaea/eutropha/halophila and the Nitrosomonas oligotropha lineages, while nitrite oxidizer bacteria (NOB) belonged to the Nitrospira genus. Addition of the carrier media in the hybrid activated sludge system increased the nitrification capacity and stability; this effect was much greater in the first IFAS stage than in the second one where the residual ammonia concentration becomes limiting. Our results show that IFAS-EBPR systems enable decoupling of solid residence time (SRT) control for nitrifiers and PAOs that require or prefer conflicting SRT values (e.g. >15 days required for nitrifiers and <5 days preferred for PAOs). Allowing the slow-growing nitrifiers to attach to the carrier media and the faster-growing phosphorus (P)-removing organisms (and other heterotrophs, e.g. denitrifiers) to be in the suspended mixed liquor (ML), the EBPR-IFAS system facilitates separate SRT controls and overall optimization for both N and P removal processes.
Water Research 07/2011; 45(13):3845-54. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biological air filters represent a promising tool for treating emissions of ammonia and odor from pig facilities. Quantitative fluorescence in situ hybridization (FISH) and 16S rRNA gene sequencing were used to investigate the bacterial community structure and diversity in a full-scale biofilter consisting of two consecutive compartments (front and back filter). The analysis revealed a highly specialized bacterial community of limited diversity, dominated by a few groups of Betaproteobacteria (especially Comamonas) and diverse Bacteroidetes. Actinobacteria, Gammaproteobacteria, and betaproteobacterial ammonia oxidizers (Nitrosomonas eutropha/Nitrosococcus mobilis-lineage) were also quantitatively important. Only a few quantitative differences existed between the two filter compartments at the group level, with a lower relative abundance of Actinobacteria and a higher relative abundance of the Cytophaga-Flavobacteria group in the back filter compared to the front filter. These results confirmed the N. eutropha/Nc. mobilis-lineage as the main ammonia oxidizers in pig house air filters and allowed first hypotheses for the key organisms involved in odor removal.
Systematic and Applied Microbiology 07/2011; 34(5):344-52. · 3.29 Impact Factor
[show abstract][hide abstract] ABSTRACT: Nitrate is one of the chemicals often added to wastewater to control hydrogen sulfide production by sulfate-reducing bacteria (SRB). While the effect of nitrate in various SRB pure cultures is well documented, the effect observed in mixed microbial communities is not consistent. This study investigates the response of mixed SRB communities to nitrate, by examining the changes in activity and community composition of sulfidogenic wastewater biofilm over a 10-day period with 10 mmol L(-1) nitrate exposure. Biofilms were enriched in SRB belonging to the Desulfobacter, Desulfobulbus, Desulfomicrobium, and Desulfovibrio genera. Nitrate exposure decreased dsrB transcription within 4 h, and sulfate consumption within 10 days, but it did not fully eliminate sulfide production in the biofilms. The effect of nitrate on SRB was genus specific; Desulfobacter and Desulfobulbus disappeared while Desulfovibrio and Desulfomicrobium persisted in the biofilms. Nitrate exposure also led to the rapid proliferation of nitrate-reducing bacteria within the biofilms, and increased the biofilm thickness. Nitrate consumption began within 2 h of nitrate exposure and gradually increased in rate over time. Transcription of the nitrate reductase napA, and the diversity of nitrate reductase genes narG and napA also increased concurrently. Our results demonstrate that some SRB, presumably those able to tolerate or detoxify nitrite, will persist in sulfidogenic wastewater biofilms despite continuous exposure to high levels of nitrate. Nitrate is therefore unlikely to provide lasting hydrogen sulfide suppression in wastewater biofilms harboring Desulfovibrio or Desulfomicrobium populations.
Applied Microbiology and Biotechnology 05/2011; 91(6):1647-57. · 3.69 Impact Factor