Ubiquity and Diversity of Cold Adapted Denitrifying Bacteria Isolated From Diverse Antarctic Ecosystems.

Nitrogen cycle has been poorly investigated in Antarctic ecosystems. In particular, how extreme conditions of low temperature, dryness, and high radiation select the microorganisms involved in the cycle is not yet understood. Denitrification is an important step in the nitrogen cycle in which nitrate is reduced stepwise to the gases NO, N2O, and N2. Denitrification is carried out by a wide group of microorganisms spread in the phylogenetic tree. The aim of this work was to isolate and characterize denitrifying bacteria present in different cold environments from Antarctica. Bacterial isolates were obtained from lake, meltwater, sea, glacier ice, ornithogenic soil, and penguin feces samples from King George Island, Fildes peninsula in the Antarctic. Samples were taken during the deicing season in five sampling campaigns. From all the samples we were able to isolate denitrifying strains. A total of 199 bacterial isolates with the capacity to grow in anaerobic mineral media reducing nitrate at 4°C were obtained. The characterization of the isolates by 16S rRNA gene sequence analysis showed a high predominance of the genus Pseudomonas, followed by Janthinobacterium, Flavobacterium, Psychrobacter, and Yersinia. Other minor genera detected were Cryobacterium, Iodobacter, Kaistella, and Carnobacterium. The capacity to denitrify was not previously described for most of the bacteria related to our isolates and in many of them denitrifying genes were not present suggesting the presence of new genes in this extreme environment. Our work demonstrates the ubiquity of denitrification in the Maritime Antarctica and gives important information linking denitrification at cold temperature with taxa in an unequivocal way.

Identification of active denitrifiers by DNA-stable isotope probing and amplicon sequencing reveals Betaproteobacteria as responsible for attenuation of nitrate contamination in a low impacted aquifer.

Groundwater reservoirs constitute important freshwater resources. However, these ecosystems are highly vulnerable to contamination and have to rely on the resident microbiota to attenuate the impact of this contamination. Nitrate is one of the main contaminants found in groundwater, and denitrification is the main process that removes the compound. In this study, the response to nutrient load on indigenous microbial communities in groundwater from a low impacted aquifer in Uruguay was evaluated. Denitrification rates were measured in groundwater samples from three different sites with nitrate, acetate and pyrite amendments. Results showed that denitrification is feasible under in situ nitrate and electron donor concentrations, although the lack of readily available organic energy source would limit the attenuation of higher nitrate concentrations. DNA-stable isotope probing, combined with amplicon sequencing of 16S rRNA, nirS and nirK genes, was used to identify the active denitrifiers. Members of the phylum Betaproteobacteria were the dominant denitrifiers in two of three sites, with different families being observed; members of the genus Vogesella (Neisseriaceae) were key denitrifiers at one site, while the genera Dechloromonas (Rhodocyclaceae) and Comamonas (Comamonadaceae) were the main denitrifiers detected at the other sites.

Isolation and functional analysis of denitrifiers in an aquifer with high potential for denitrification.

Aquifers are among the main freshwater sources. The Raigón aquifer is susceptible to contamination, mainly by nitrate and pesticides, such as atrazine, due to increasing agricultural activities in the area. The capacity of indigenous bacteria to attenuate nitrate contamination in different wells of this aquifer was assessed by measuring denitrification rates with either acetate plus succinate or nitrate amendments. Denitrification activity in nitrate-amended assays was significantly higher than in unamended assays, particularly in groundwater from wells where nitrate concentration was 33.5 mg L(-1) or lower. Furthermore, groundwater denitrifiers capable of using acetate or succinate as electron donors were isolated, identified by 16S rRNA gene sequencing and evaluated for functional denitrification genes (nirS, nirK and nosZ). Phylogenetic affiliation of 54 isolates showed that all members belonged to nine different genera within the Proteobacteria (Bosea, Ochrobactrum, Azospira, Zoogloea, Acidovorax, Achromobacter, Vogesella, Stenotrophomonas and Pseudomonas). In addition, isolate AR28 that clustered separately from validly described species could potentially belong to a new genus. The majority of the isolates were related to species belonging to previously reported denitrifying genera. However, the phylogeny of the nirS and nosZ genes revealed new sequences of these functional genes. To our knowledge, this is the first isolation and sequencing of the nirS gene from the genus Vogesella, as well as the nosZ gene from the genera Acidovorax and Zoogloea. The results indicated that indigenous bacteria in the Raigón aquifer had the capacity to overcome high nitrate contamination and exhibited functional gene diversity.

Methylogaea oryzae gen. nov., sp. nov., a mesophilic methanotroph isolated from a rice paddy field.

A novel methanotroph, designated strain E10(T), was isolated from a rice paddy field in Uruguay. Strain E10(T) grew on methane and methanol as sole carbon and energy sources. Cells were Gram-negative, non-motile, non-pigmented, slightly curved rods showing type I intracytoplasmic membranes arranged in stacks. The strain was neutrophilic and mesophilic; optimum growth occurred at 30-35 °C with no growth above 37 °C. The strain possessed only a particulate methane monooxygenase (pmoA). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain was most closely related to the moderately thermophilic strains Methylocaldum szegediense OR2(T) (91.6 % sequence similarity) and Methylococcus capsulatus Bath (91.5 %). Comparative sequence analysis of pmoA genes also confirmed that strain E10(T) formed a new lineage among the genera Methylocaldum and Methylococcus with 89 and 84 % derived amino acid sequence identity to Methylococcus capsulatus Bath and Methylocaldum gracile VKM-14L(T), respectively. The DNA G+C content was 63.1 mol% and the major cellular fatty acid was C(16 :0) (62.05 %). Thus, strain E10(T) (=JCM 16910(T) = DSM 23452(T)) represents the type strain of a novel species within a new genus, for which the name Methylogaea oryzae gen. nov., sp. nov. is proposed.

Bacterial community analysis of the water surface layer from a rice-planted and an unplanted flooded field

The bacterial communities in floodwater, from a rice-planted and an unplanted field were characterized at the beginning (flooding stage) and at the end (harvest stage) of the rice cropping cycle. Most probable number estimations and plate counts of aerobic and anaerobic heterotrophic bacteria and of several metabolic bacterial groups (methanogens, sulfate-reducers, anaerobic sulfur and nonsulfur phototrophs, denitrifiers and ammonifiers) were similar in rice and unplanted floodwater at both sampling times. The analysis of denitrifiers and methanogens by fluorescent in situ hybridization revealed a shift in the phylogenetic affiliation only of the former group in the rice-planted floodwater. Terminal restriction fragment length polymorphism of 16S rRNA gene amplicons indicated that the bacterial communities of the rice-planted and unplanted soils were consistently diverse and strongly influenced by the season.

Bacterial community analysis of the water surface layer from a rice-planted and an unplanted flooded field.

The bacterial communities in floodwater, from a rice-planted and an unplanted field were characterized at the beginning (flooding stage) and at the end (harvest stage) of the rice cropping cycle. Most probable number estimations and plate counts of aerobic and anaerobic heterotrophic bacteria and of several metabolic bacterial groups (methanogens, sulfate-reducers, anaerobic sulfur and nonsulfur phototrophs, denitrifiers and ammonifiers) were similar in rice and unplanted floodwater at both sampling times. The analysis of denitrifiers and methanogens by fluorescent in situ hybridization revealed a shift in the phylogenetic affiliation only of the former group in the rice-planted floodwater. Terminal restriction fragment length polymorphism of 16S rRNA gene amplicons indicated that the bacterial communities of the rice-planted and unplanted soils were consistently diverse and strongly influenced by the season.

Microbial community succession and bacterial diversity in soils during 77,000 years of ecosystem development.

The origins of the biological complexity and the factors that regulate the development of community composition, diversity and richness in soil remain largely unknown. To gain a better understanding of how bacterial communities change during soil ecosystem development, their composition and diversity in soils that developed over c. 77 000 years of intermittent aeolian deposition were studied. 16S rRNA gene clone libraries and fatty acid methyl ester (FAME) analyses were used to assess the diversity and composition of the communities. The bacterial community composition changed with soil age, and the overall diversity, richness and evenness of the communities increased as the soil habitat matured. When analysed using a multivariate Bray-Curtis ordination technique, the distribution of ribotypes showed an orderly pattern of bacterial community development that was clearly associated with soil and ecosystem development. Similarly, changes in the composition of the FAMEs across the chronosequence were associated with biomarkers for fungi, actinomycetes and Gram-positive bacteria. The development of the soil ecosystem promoted the development of distinctive microbial communities that were reminiscent of successional processes often evoked to describe change during the development of plant communities in terrestrial ecosystems.

Sterolibacterium denitrificans gen. nov., sp. nov., a novel cholesterol-oxidizing, denitrifying member of the beta-Proteobacteria.

A bacterial strain (Chol-1S(T)) that is able to oxidize cholesterol to CO2 and reduce nitrate to dinitrogen was enriched and isolated from an upflow sludge bed (USB) anoxic reactor that treats sanitary landfill leachate from the city of Montevideo, Uruguay. Cells of strain Chol-1S(T) were gram-negative, rod-shaped to slightly curved, measured 0.5-0.6 x 1.0-1.3 microm and were motile by a single polar flagellum. Strain Chol-1S(T) grew optimally at 30-32 degrees C and pH 7.0, with a doubling time of 44-46 h when cholesterol was used as the sole carbon and energy source. The metabolism of strain Chol-1S(T) was strictly respiratory, with oxygen or nitrate as the terminal electron acceptor. The presence of ubiquinone Q-8 as the sole respiratory lipoquinone indicated that strain Chol-1S(T) belonged to the beta-subclass of the Proteobacteria. Phosphatidylethanolamine was the predominant polar lipid and the G + C content of the DNA was 65.3 mol%. The fatty acid profile of strain Chol-1S(T), cultivated under denitrifying conditions by using a defined mineral medium supplemented with cholesterol, was characterized by the following major components: summed feature 4 (C16:1 omega7c and/or iso C15:0 2-OH), C16:0, C18:1 omega7c and hydroxy acid C10:0 3-OH. Minor components included C10:0, C11:0, C12:0, C14:0, C15:0, C19:0, C19:0 10-methyl and hydroxylated acids C8:0 3-OH and C16:0 3-OH. Analysis of the 16S rDNA sequence showed that strain Chol-1S(T) represents a separate lineage within the Thauera, Azoarcus, Zoogloea and Rhodocyclus assemblage of the beta-Proteobacteria. Strain Chol-1S(T) had highest sequence similarity (96.5%) with strain 72Chol, a denitrifying beta-Proteobacterium. On the basis of polyphasic evidence, strain Chol-1S(T) (=DSM 13999T=ATCC BAA-354T) is proposed as the type strain of Sterolibacterium denitrificans gen. nov., sp. nov.