Improved enrichment culture technique for methane-oxidizing bacteria from marine ecosystems: the effect of adhesion material and gas composition.

Cultivation of microbial representatives of specific functional guilds from environmental samples depends largely on the suitability of the applied growth conditions. Especially the cultivation of marine methanotrophs has received little attention, resulting in only a limited number of ex situ cultures available. In this study we investigated the effect of adhesion material and headspace composition on the methane oxidation activity in methanotrophic enrichments obtained from marine sediment. Addition of sterilized natural sediment or alternatively the addition of acid-washed silicon dioxide significantly increased methane oxidation. This positive effect was attributed to bacterial adhesion on the particles via extracellular compounds, with a minimum amount of particles required for effect. As a result, the particles were immobilized, thus creating a stratified environment in which a limited diffusive gas gradients could build up and various microniches were formed. Such diffusive gas gradient might necessitate high headspace concentrations of CH4 and CO2 for sufficient concentrations to reach the methane-oxidizing bacteria in the enrichment culture technique. Therefore, high concentrations of methane and carbon dioxide, in addition to the addition of adhesion material, were tested and indeed further stimulated methane oxidation. Use of adhesion material in combination with high concentrations of methane and carbon dioxide might thus facilitate the cultivation and subsequent enrichment of environmentally important members of this functional guild. The exact mechanism of the observed positive effects on methane oxidation and the differential effect on methanotrophic diversity still needs to be explored.

Nitrous oxide emission by the non-denitrifying, nitrate ammonifier Bacillus licheniformis.

BACKGROUND: Firmicutes have the capacity to remove excess nitrate from the environment via either denitrification, dissimilatory nitrate reduction to ammonium or both. The recent renewed interest in their nitrogen metabolism has revealed many interesting features, the most striking being their wide variety of dissimilatory nitrate reduction pathways. In the present study, nitrous oxide production from Bacillus licheniformis, a ubiquitous Gram-positive, spore-forming species with many industrial applications, is investigated. RESULTS: B. licheniformis has long been considered a denitrifier but physiological experiments on three different strains demonstrated that nitrous oxide is not produced from nitrate in stoichiometric amounts, rather ammonium is the most important end-product, produced during fermentation. Significant strain dependency in end-product ratios, attributed to nitrite and ammonium, and medium dependency in nitrous oxide production were also observed. Genome analyses confirmed the lack of a nitrite reductase to nitric oxide, the key enzyme of denitrification. Based on the gene inventory and building on knowledge from other non-denitrifying nitrous oxide emitters, hypothetical pathways for nitrous oxide production, involving NarG, NirB, qNor and Hmp, are proposed. In addition, all publically available genomes of B. licheniformis demonstrated similar gene inventories, with specific duplications of the nar operon, narK and hmp genes as well as NarG phylogeny supporting the evolutionary separation of previously described distinct BALI1 and BALI2 lineages. CONCLUSIONS: Using physiological and genomic data we have demonstrated that the common soil bacterium B. licheniformis does not denitrify but is capable of fermentative dissimilatory nitrate/nitrite reduction to ammonium (DNRA) with concomitant production of N2O. Considering its ubiquitous nature and non-fastidious growth in the lab, B. licheniformis is a suitable candidate for further exploration of the actual mechanism of N2O production in DNRA bacteria and its relevance in situ.

Regulation of nitrogen metabolism in the nitrate-ammonifying soil bacterium Bacillus vireti and evidence for its ability to grow using N2 O as electron acceptor.

Bacillus vireti is a nitrate-ammonifying bacterium and a partial denitrifier, reducing NO3 (-) , NO2 (-) , NO and N2 O with NarG, NrfA, CbaA and NosZ respectively. Growth is optimized through successive use of the electron acceptors O2 and NO3 (-) , followed by NO2 (-) , NO and N2 O. Fermentation takes place simultaneously with anaerobic respiration. When grown in batch culture with 5 mM initial NO3 (-) , transcription of nrfA was high and most NO3 (-) was reduced to NH4 (+) . With 20 mM initial NO3 (-) , nrfA transcription was lower and more than 50% of the nitrate was recovered as NO, N2 O and N2 . Analysis of gene transcription patterns and corresponding gas kinetics indicated that O2 and NO2 (-) or NO are main controllers of nrfA, nirB, cbaA and nosZ transcription. This was corroborated by analyses of putative binding regions for specific transcriptional regulators. Furthermore, we demonstrate that N2 O reduction in B. vireti supports growth. The high nosZ transcription but low N2 O production seen at 5 mM NO3 (-) implies that this organism can use N2 O reductase to scavenge N2 O from other organisms in the soil, thus possibly acting as a net sink for N2 O.

New Methyloceanibacter diversity from North Sea sediments includes methanotroph containing solely the soluble methane monooxygenase.

Marine methylotrophs play a key role in the global carbon cycle by metabolizing reduced one-carbon compounds that are found in high concentrations in marine environments. Genome, physiology and diversity studies have been greatly facilitated by the numerous model organisms brought into culture. However, the availability of marine representatives remains poor. Here, we report the isolation of four novel species from North Sea sediment enrichments closely related to the Alphaproteobacterium Methyloceanibacter caenitepidi. Each of the newly isolated Methyloceanibacter species exhibited a clear genome sequence divergence which was reflected in physiological differences. Notably one strain R-67174 was capable of oxidizing methane as sole source of carbon and energy using solely a soluble methane monooxygenase and represents the first marine Alphaproteobacterial methanotroph brought into culture. Differences in maximum cell density of >1.5 orders of magnitude were observed. Furthermore, three strains were capable of producing nitrous oxide from nitrate. Together, these findings highlight the metabolic and physiologic variability within closely related Methyloceanibacter species and provide a new understanding of the physiological basis of marine methylotrophy.

Genome Characteristics of Two Novel Type I Methanotrophs Enriched from North Sea Sediments Containing Exclusively a Lanthanide-Dependent XoxF5-Type Methanol Dehydrogenase.

Microbial methane oxidizers play a crucial role in the oxidation of methane in marine ecosystems, as such preventing the escape of excessive methane to the atmosphere. Despite the important role of methanotrophs in marine ecosystems, only a limited number of isolates are described, with only four genomes available. Here, we report on two genomes of gammaproteobacterial methanotroph cultures, affiliated with the deep-sea cluster 2, obtained from North Sea sediment. Initial enrichments using methane as sole source of carbon and energy and mimicking the in situ conditions followed by serial subcultivations and multiple extinction culturing events over a period of 3 years resulted in a highly enriched culture. The draft genomes of the methane oxidizer in both cultures showed the presence of genes typically found in type I methanotrophs, including genes encoding particulate methane monooxygenase (pmoCAB), genes for tetrahydromethanopterin (H4MPT)- and tetrahydrofolate (H4F)-dependent C1-transfer pathways, and genes of the ribulose monophosphate (RuMP) pathway. The most distinctive feature, when compared to other available gammaproteobacterial genomes, is the absence of a calcium-dependent methanol dehydrogenase. Both genomes reported here only have a xoxF gene encoding a lanthanide-dependent XoxF5-type methanol dehydrogenase. Thus, these genomes offer novel insight in the genomic landscape of uncultured diversity of marine methanotrophs.

Initial community evenness favours functionality under selective stress.

Owing to the present global biodiversity crisis, the biodiversity-stability relationship and the effect of biodiversity on ecosystem functioning have become major topics in ecology. Biodiversity is a complex term that includes taxonomic, functional, spatial and temporal aspects of organismic diversity, with species richness (the number of species) and evenness (the relative abundance of species) considered among the most important measures. With few exceptions (see, for example, ref. 6), the majority of studies of biodiversity-functioning and biodiversity-stability theory have predominantly examined richness. Here we show, using microbial microcosms, that initial community evenness is a key factor in preserving the functional stability of an ecosystem. Using experimental manipulations of both richness and initial evenness in microcosms with denitrifying bacterial communities, we found that the stability of the net ecosystem denitrification in the face of salinity stress was strongly influenced by the initial evenness of the community. Therefore, when communities are highly uneven, or there is extreme dominance by one or a few species, their functioning is less resistant to environmental stress. Further unravelling how evenness influences ecosystem processes in natural and humanized environments constitutes a major future conceptual challenge.

Exploring methane-oxidizing communities for the co-metabolic degradation of organic micropollutants.

Methane-oxidizing cultures from five different inocula were enriched to be used for co-metabolic degradation of micropollutants. In a first screening, 18 different compounds were tested for degradation with the cultures as well as with four pure methane-oxidizing bacterial (MOB) strains. The tested compounds included pharmaceuticals, chemical additives, pesticides, and their degradation products. All enriched cultures were successful in the degradation of at least four different pollutants, but the compounds degraded most often were sulfamethoxazole (SMX) and benzotriazole (BTZ). Addition of acetylene, a specific methane monooxygenase (MMO) inhibitor, revealed that SMX and BTZ were mainly degraded co-metabolically by the present MOB. The pure MOB cultures exhibited less degradation potential, while SMX and BTZ were also degraded by three of the four tested pure strains. For MOB, copper (Cu(2+)) concentration is often an important factor, as several species have the ability to express a soluble MMO (sMMO) if the Cu(2+) concentration is low. In literature, this enzyme is often described to have a broader compound range for co-metabolic degradation of pollutants, in particular when it comes to aromatic structures. However, this study indicated that co-metabolic degradation of the aromatic compounds SMX and BTZ was possible at high Cu(2+) concentration, most probably catalyzed by pMMO.

The nitrate-ammonifying and nosZ-carrying bacterium Bacillus vireti is a potent source and sink for nitric and nitrous oxide under high nitrate conditions.

Several Gram-positive bacteria carry genes for anaerobic reduction of NO3(-) via NO2(-) to NH4(+) or gaseous nitrogen compounds, but the processes are understudied for these organisms. Here, we present results from a whole-genome analysis of the soil bacterium Bacillus vireti and a phenotypic characterization of intermediate and end-products, formed under anoxic conditions in the presence of NO3(-). Bacillus vireti has a versatile metabolism. It produces acetate, formate, succinate and lactate from fermentation and performs dissimilatory nitrate reduction via NO2(-) to ammonium (DNRA) using NrfA, while NirB may detoxify NO2(-) in the cytoplasm. Moreover, it produces NO from an unknown source and reduces it via N2O to N2 using two enzymes connected to denitrification: an unusual NO reductase, qCuA Nor encoded by cbaA, and a z-type N2O reductase, encoded by nosZ. In batch cultures, B. vireti reduced all NO3(-) to NO2(-) before the NO2(-) was reduced further. The quantities of all products varied with the initial NO3(-) concentration. With 5 mM NO3(-) , 90% was reduced to NH4 (+) while with ≥ 20 mM NO3(-), 50% was reduced to NO, N2O and N2. This organism is thus an aggressive NO2(-) accumulator and may act as a net source and sink of NO and N2O.

Pathway of nitrous oxide consumption in isolated Pseudomonas stutzeri strains under anoxic and oxic conditions.

The microbial consumption of nitrous oxide (N2O) has gained great interest since it was revealed that this process could mitigate the greenhouse effect of N2O. The consumption of N2O results from its reduction to dinitrogen gas (N2) as part of the denitrification process. However, there is ongoing debate regarding an alternative pathway, namely reduction of N2O to NH4(+), or assimilatory N2O consumption. To date, this pathway is poorly investigated and lacks unambiguous evidence. Enrichment of denitrifying activated sludge using a mineral nitrogen-free medium rendered a mixed culture capable of anoxic and oxic N2O consumption. Dilution plating, isolation and deoxyribonucleic acid fingerprinting identified a collection of Pseudomonas stutzeri strains as dominant N2O consumers in both anaerobic and aerobic enrichments. A detailed isotope tracing experiment with a Pseudomonas stutzeri isolate showed that consumption of N2O via assimilatory reduction to NH4(+) was absent. Conversely, respiratory N2O reduction was directly coupled to N2 fixation.

Niche differentiation in nitrogen metabolism among methanotrophs within an operational taxonomic unit.

BACKGROUND: The currently accepted thesis on nitrogenous fertilizer additions on methane oxidation activity assumes niche partitioning among methanotrophic species, with activity responses to changes in nitrogen content being dependent on the in situ methanotrophic community structure Unfortunately, widely applied tools for microbial community assessment only have a limited phylogenetic resolution mostly restricted to genus level diversity, and not to species level as often mistakenly assumed. As a consequence, intragenus or intraspecies metabolic versatility in nitrogen metabolism was never evaluated nor considered among methanotrophic bacteria as a source of differential responses of methane oxidation to nitrogen amendments. RESULTS: We demonstrated that fourteen genotypically different Methylomonas strains, thus distinct below the level at which most techniques assign operational taxonomic units (OTU), show a versatile physiology in their nitrogen metabolism. Differential responses, even among strains with identical 16S rRNA or pmoA gene sequences, were observed for production of nitrite and nitrous oxide from nitrate or ammonium, nitrogen fixation and tolerance to high levels of ammonium, nitrate, and hydroxylamine. Overall, reduction of nitrate to nitrite, nitrogen fixation, higher tolerance to ammonium than nitrate and tolerance and assimilation of nitrite were general features. CONCLUSIONS: Differential responses among closely related methanotrophic strains to overcome inhibition and toxicity from high nitrogen loads and assimilation of various nitrogen sources yield competitive fitness advantages to individual methane-oxidizing bacteria. Our observations proved that community structure at the deepest phylogenetic resolution potentially influences in situ functioning.

Methylomonas lenta sp. nov., a methanotroph isolated from manure and a denitrification tank.

Two methanotrophic bacteria, strains R-45377(T) and R-45370, were respectively isolated from a slurry pit of a cow stable and from a denitrification tank of a wastewater treatment plant in Belgium. The strains showed 99.9 % 16S rRNA gene sequence similarity. Cells were Gram-negative, motile rods containing type I methanotroph intracytoplasmic membranes. Colonies and liquid cultures appeared white to pale pink. The pmoA gene encoding particulate methane monooxygenase (pMMO) and the nifH gene encoding nitrogenase were present. Soluble methane monooxygenase (sMMO) activity, the presence of the mmoX gene encoding sMMO and the presence of the pxmA gene encoding a sequence-divergent pMMO were not detected. Methane and methanol were utilized as sole carbon sources. The strains grew optimally at 20 °C (range 15-28 °C) and at pH 6.8-7.3 (range pH 6.3-7.8). The strains grew in media supplemented with up to 1.2 % NaCl. The major cellular fatty acids were C16 : 1ω8c, C16 : 1ω5c, C16 : 1ω7c, C14 : 0, C15 : 0 and C16 : 0 and the DNA G+C content was 47 mol%. 16S rRNA gene- and pmoA-based phylogenetic analyses showed that the isolates cluster among members of the genus Methylomonas within the class Gammaproteobacteria, with pairwise 16S rRNA gene sequence similarities of 97.5 and 97.2 % between R-45377(T) and the closest related type strains, Methylomonas scandinavica SR5(T) and Methylomonas paludis MG30(T), respectively. Based on phenotypic characterization of strains R-45377(T) and R-45370, their low 16S rRNA gene sequence similarities and the formation of a separate phylogenetic lineage compared with existing species of the genus Methylomonas, we propose to classify these strains in a novel species, Methylomonas lenta sp. nov., with R-45377(T) ( = LMG 26260(T) = JCM 19378(T)) as the type strain.

Methyloparacoccus murrellii gen. nov., sp. nov., a methanotroph isolated from pond water.

Two novel methanotrophic strains, R-49797(T) and OS501, were isolated from pond water in South Africa and Japan, respectively. Strains R-49797(T) and OS501 shared 99.7% 16S rRNA gene sequence similarity. Cells were Gram-stain-negative, non-motile cocci with a diplococcoid tendency and contained type I methanotroph intracytoplasmic membranes. The pmoA gene encoding particulate methane monooxygenase was present. Soluble methane monoooxygenase (sMMO) activity, the mmoX gene encoding sMMO and the nifH gene encoding nitrogenase were not detected. Methane and methanol were utilized as sole carbon source. The strains grew optimally at 25-33 °C (range 20-37 °C) and at pH 6.3-6.8 (range 5.8-9.0). The strains did not support growth in media supplemented with 1% (w/v) NaCl. For both strains, the two major fatty acids were C(16 : 1)ω7c and C(16 : 0) and the DNA G+C content was 65.6 mol%. The isolates belong to the family Methylococcaceae of the class Gammaproteobacteria and cluster most closely among the genera Methylocaldum, Methylococcus and Methylogaea, with a 16S rRNA gene sequence similarity of 94.2% between strain R-49797(T) and its closest related type strain (Methylocaldum gracile VKM 14L(T)). Based on the low 16S rRNA gene sequence similarities with its nearest phylogenetic neighbouring genera, the formation of a separate lineage based on 16S rRNA and pmoA gene phylogenetic analysis, and the unique combination of phenotypic characteristics of the two isolated strains compared with the genera Methylocaldum, Methylococcus and Methylogaea, we propose to classify these strains as representing a novel species of a new genus, Methyloparacoccus murrellii gen. nov., sp. nov., within the family Methylococcaceae. The type strain of Methyloparacoccus murrellii is R-49797(T) ( = LMG 27482(T) = JCM 19379(T)).

Customized media based on miniaturized screening improve growth rate and cell yield of methane-oxidizing bacteria of the genus Methylomonas.

The growth of twelve methanotrophic strains within the genus Methylomonas, including the type strains of Methylomonas methanica and Methylomonas koyamae, was evaluated with 40 different variations of standard diluted nitrate mineral salts medium in 96-well microtiter plates. Unique profiles of growth preference were observed for each strain, showing a strong strain dependency for optimal growth conditions, especially with regards to the preferred concentration and nature of the nitrogen source. Based on the miniaturized screening results, a customized medium was designed for each strain, allowing the improvement of the growth of several strains in a batch setup, either by a reduction of the lag phase or by faster biomass accumulation. As such, the maintenance of fastidious strains could be facilitated while the growth of fast-growing Methylomonas strains could be further improved. Methylomonas sp. R-45378 displayed a 50 % increase in cell dry weight when grown in its customized medium and showed the lowest observed nitrogen and oxygen requirement of all tested strains. We demonstrate that the presented miniaturized approach for medium optimization is a simple tool allowing the quick generation of strain-specific growth preference data that can be applied downstream of an isolation campaign. This approach can also be applied as a first step in the search for strains with biotechnological potential, to facilitate cultivation of fastidious strains or to steer future isolation campaigns.

An rpoD gene sequence based evaluation of cultured Pseudomonas diversity on different growth media.

The last decade has shown an increased interest in the utilization of bacteria for applications ranging from bioremediation to wastewater purification and promotion of plant growth. In order to extend the current number of micro-organism mediated applications, a continued quest for new agents is required. This study focused on the genus Pseudomonas, which is known to harbour strains with a very diverse set of interesting properties. The aim was to identify growth media that allow retrieval of a high Pseudomonas diversity, as such increasing the chance of isolating isolates with beneficial properties. Three cultivation media: trypticase soy agar (TSA), potato dextrose agar (PDA) and Pseudomonas isolation agar (PIA) were evaluated for their abilities to grow Pseudomonas strains. TSA and PDA were found to generate the largest Pseudomonas diversity. However, communities obtained with both media overlapped. Communities obtained with PIA, on the other hand, were unique. This indicated that the largest diversity is obtained by sampling from either PDA or TSA and from PIA in parallel. To evaluate biodiversity of the isolated Pseudomonas members on the media, an appropriate biomarker had to be identified. Hence, an introductory investigation of the taxonomic resolution of the 16S rRNA, rpoD, gyrB and rpoB genes was performed. The rpoD gene sequences not only had a high phylogenetic content and the highest taxonomic resolution amongst the genes investigated, it also had a gene phylogeny that related well with that of the 16S rRNA gene.

Rapid and simple cryopreservation of anaerobic ammonium-oxidizing bacteria.

A quick and simple protocol for long-term cryopreservation of anaerobic ammonium-oxidizing bacteria (anammox bacteria) was developed. After 29 weeks of preservation at -80°C, activity recovery for all tested cultures under at least one of the applied sets of preservation conditions was observed. Growth recovery was also demonstrated for a single-cell culture of "Candidatus Kuenenia stuttgartiensis."

Safeguarding bacterial resources promotes biotechnological innovation.

Environmental research delivers valuable bacterial resources for biotechnology. We believe that systematic long-term preservation of bacteria will promote future biotechnological innovations, by safeguarding the accessibility of bacteria already recognized to have interesting features and providing a "pool" of bacterial resources for novel applied research. To this end, we want to advocate the incorporation of preservation tests in environmental or applied microbiological research. This paper introduces non-specialists to different preservation methods for bacteria. Several parameters that influence long-term storage of bacterial resources are explained and practical tips and guidelines are formulated. Also, the vital role of public culture collections is highlighted and the state-of-the-art of preservation of non-pure cultures is described.

Trend analysis of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli isolated from Belgian pork and poultry meat products using surveillance data of 2004-2009.

The purpose of this study was to analyze and compare antimicrobial resistance in Campylobacter spp. isolated from pork and poultry carcasses, and pork and poultry meat (at slaughterhouse level, during meat cutting, and at retail) in Belgium, using available surveillance data over the period 2004-2009. The susceptibilities of 1724 Campylobacter isolates for ampicillin, ciprofloxacin, nalidixic acid, tetracycline, erythromycin, and gentamicin were tested by E-test. Gentamicin resistance was low (near 0%) until 2007, with an increase to over 20% by 2009 for all species-matrix combinations. Resistance to tetracycline fluctuated around the same level during the entire study period and was significantly higher (p-value of <0.05) in C. coli than in C. jejuni. Erythromycin resistance was low and showed a slight decrease between 2004 and 2007, but increased from 2007 until 2009. Fluoroquinolone and ampicillin resistance was significantly higher in isolates derived from poultry, compared to pork-related isolates. This correlates with the higher use of these antimicrobials in poultry husbandry. A total of 25% of C. coli isolates from poultry showed the most apparent multiresistance (resistance to four or more antimicrobials). Approximately 1% of the poultry-derived isolates (both C. coli and C. jejuni) showed resistance to all tested antimicrobials, while none was found in pork products.

Denitrification in Gram-positive bacteria: an underexplored trait.

Denitrifying organisms are essential in removing fixed nitrogen pollutants from ecosystems (e.g. sewage sludge). They can be detrimental (e.g. for agricultural soil) and can also produce the greenhouse gas N2O (nitrous oxide). Therefore a more comprehensive understanding of this process has become increasingly important regarding its global environmental impact. Even though bacterial genome sequencing projects may reveal new data, to date the denitrification abilities and features in Gram-positive bacteria are still poorly studied and understood. The present review evaluates current knowledge on the denitrification trait in Gram-positive bacteria and addresses the likely existence of unknown denitrification genes. In addition, current molecular tools to study denitrification gene diversity in pure cultures and environmental samples seem to be highly biased, and additional novel approaches for the detection of denitrifying (Gram-positive) bacteria appear to be crucial in re-assessing the real diversity of denitrifiers.

Lessons learned from the management of a national outbreak of Salmonella ohio linked to pork meat processing and distribution.

During the summer of 2005, an increase in reports of human cases of Salmonella enterica serovar Ohio infection was observed in Belgium. During 11 weeks, between 1 July and 13 September, 60 cases of laboratory-confirmed Salmonella Ohio infection were reported to the National Reference Centre for Salmonella, with a peak onset of symptoms in the third week of July. All clinical isolates caused self-limiting gastroenteritis; both genders (32 males and 28 females) and all age groups (three children <5 years of age, three children 5 to 14 years of age, 32 adults 15 to 64 years of age, and 22 adults >65 years of age) were affected. The isolates were distributed throughout Belgium but a cluster of several cases was observed around Brussels. At the same time, an increase in the incidence of this serovar was observed in the Salmonella isolates originating from the official surveillance campaign conducted by the Federal Agency for the Safety of the Food Chain, which identified pork as a likely source of the outbreak strain. Pulsed-field gel electrophoresis typing confirmed the clonal relationship between the human isolates, the isolates from samples collected in the cutting plants, and the isolates from pork meat in distribution. Further epidemiological investigations indicated that one particular slaughterhouse was involved. In that slaughterhouse, the carcasses were contaminated during the evisceration process because of contaminated equipment and uncontrolled environmental conditions. This study highlights the importance of a centralized surveillance laboratory in the management of outbreaks and the need of strict implementation of hygienic rules to avoid this type of outbreak.