Methanotrophic microbiomes from North Sea sediment

Methane is the most abundant organic greenhouse gas in our atmosphere, and has a strong infrared absorbance, being 25 to 30 times more effective than carbon dioxide on a 100 years scale. Methane therefore plays an important role in the climate warming regulation. Methanotrophs are microorganisms that can consume methane and utilize it as their sole source of carbon and energy. These organisms are the most important biological sink of methane. Their importance is especially demonstrated in marine ecosystems. While the oceans have the potential to produce enormous quantities of methane, a series of very effective microbiological oxidation processes results in the ocean being one of the smallest net global methane sources. Hence, it is of vital importance to understand who is there and what factors may positively or negatively impact the methane-oxidizing activity of these organisms. Despite the importance of the aerobic methane oxidizing bacteria (MOB) in the marine ecosystems currently only a limited amount of ex situ cultures is available. Nevertheless, they are indispensable to link physiology to genomic features and expand our knowledge about the specific habitat preferences of marine MOB. This study focused on aerobic marine MOBs and aimed at designing a large-scale enrichment and isolation strategy to retrieve a maximal MOB diversity from marine sediments. First, the effect of adhesion material and headspace composition on the methane oxidation activity in marine sediment enrichments were investigated. The addition of sterilized natural sediment as well as acid-washed silicium dioxide significantly positively influenced methane oxidation. The exact mechanism of this positive effect needs further investigation but might be the facilitation of methane, carbon dioxide and oxygen gradients in addition to adhesion. Use of adhesion material might thus facilitate the cultivation and subsequent enrichment of members of this functional guild. Next, using these insights, a large scale isolation strategy was performed from sediment collected at six different stations in the North Sea along a transect from estuary to open sea. An initial enrichment step with serial subcultivations was followed by miniaturized extinction culturing mimicking a range of nitrogen and oxygen microniches. A clear decreasing trend of cultivability and detectability was observed along the investigated transect. Furthermore widely applied pmoA primers failed to amplify biomarkers in a large number of active methanotrophic cultures, suggesting enormous underestimation of methanotrophs in situ in PCR-based molecular surveys. Unfortunately, despite the numerous attempts we were not able to obtain axenic methanotrophic cultures, most likely due to tight mutualistic interactions with heterotrophic bacteria.Shot gun sequencing of four methane-oxidizing enrichment cultures revealed the presence of a novel gammaproteobacterial MOB belonging to the deep-sea cluster 2 in two cultures and a novel alphaproteobacterial MOB belonging to the recently described methylotrophic genus Methyloceanibacter in the other two cultures. Methyloceanibacter methanicus represented the first MOB found in an exclusively methylotrophic genus, the first marine type II MOB and only the third taxon in which solely sMMO was resoponsible for methane oxidation. A targeted isolation using methanol as carbon source led to a axenic culture of the MOB, in addition to three closely related novel strict methylotrophic species, M. superfactum, M. stevinii, M. marginalis. Together with the previously described M. caenitepidi, these species exemplify an extreme niche differentiation, with a wide ecotypic variation related to growth kinetics on methanol, and preferences for nitrogen, pH, temperature and salt. Furthermore, the most striking difference of the deep-sea cluster 2 representatives demonstrated a striking difference with other gammaproteobacterial MOB in its lack of a calcium dependent methanol dehydrogenase encoded in the genome. The genome solely contained the genes for xoxF5 for the lanthanide-containing methanol dehydrogenase.Lastly, a preservation protocol was optimized for the long term storage of marine bacteria in order to successfully store the enrichments and axenic cultures obtained throughout this dissertation. Fastidious nitrite-oxidizing bacteria were used as model organisms. They demonstrated that optimal preservation conditions were strain-dependent whereby marine strains, appeared to be more sensitive to freezing than non-marine strains. Nevertheless, a general cryopreservation protocol using 10% dimethyl sulfoxide as cryoprotective agent with or without ten-fold diluted trypticase soy broth and trehalose as a preservation medium allowed successful preservation of all tested strains. Applying the same protocol on whole marine sediment samples allowed successful storage of different key players in the carbon and nitrogen cycle.In conclusion marine MOBs are notoriously difficult to cultivate and isolate. Despite numerous attempts, I was only able obtain one axenic culture, in addition to over 200 enrichment cultures, from the oxygenated zones of North Sea sediments. The availability of a successful preservation protocol allows the storage of this axenic culture, ensuring that novel diversity does not get lost, but also further guaranties archiving of the obtained enrichment cultures and environmental samples. As such these are available for future isolation when novel insights in marine MOB and isolation strategies/techniques become available

Draft genome sequences of eight obligate methane oxidizers occupying distinct niches based on their nitrogen metabolism

The genome sequences of Methylomonas methanica (NCIMB 11130T, R-45363, and R-45371), Methylomonas koyamae (R-45378, R-45383, and R-49807), Methylomonas lenta (R-45370), and Methylosinus sp. (R-45379) were obtained. These aerobic methanotrophs were isolated from terrestrial ecosystems, and their distinct phenotypes related to nitrogen assimilation and dissimilation were previously reported

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps

Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites

Exploring rhizobia in wild native legumes in Belgium

Rhizobia are symbiotic bacteria capable of establishing root or stem nodules on various leguminous plants. An increasing number of nodule-forming bacteria have been reported, including bacteria from the alpha-, beta- and gammaproteobacteria (Chen et al., 2001; Chen et al., 2003; de Lajudie et al., 1994; de Lajudie et al., 1998; Jarvis et al., 1997; Jordan, 1982; Moulin et al., 2001; Rivas et al., 2002; Zakhia & de Lajudie, 2001). In Western Europe, relatively few native legume species have been studied in detail and therefore potentially a large part of the legume symbiont diversity remains to be discovered. In Belgium, legumes belong to 30 genera and comprise 113 plant species. They vary from annual and perennial herbs to shrubs and trees. In the present work, the diversity of bacteria isolated from root nodules from legumes in the area around Brakel (Belgium) was investigated. The sampling area comprised different habitat types, from nature reserves to industrial areas. Human activities are responsible for pollution and have a great influence on the plants. For example, in the more industrial habitats several times a year the area is sprayed with herbicides, such as Round up and Biofix. The aim was to get a first insight in the diversity present in these wild Belgian leguminous plants and to compare the diversity in the different habitats and plants. A total of 439 samples were taken covering 28 different plant species. We used the rep-PCR technique as a first screening to reduce the number of isolates, and 16S rDNA partial gene sequencing as an initial identification tool. To gain insights in the nodulation, fixation capacity and host range of the strains, NodC and NifH were sequenced. Our study revealed that two possible new species were found. Isolate B462 was identified as Bradyrhizobium sp. (97.3%) according to partial 16S rDNA sequencing and as Bradyrhizobium cancariensis (87.3%) according to recA sequencing. The strains B1209, B1215T11 and B1215T22 were identified as Sinorhizobium morelense (98.7%) according to partial 16S rDNA and as Rhizobium gallicum (88.8%) according to recA. Further investigation is necessary to reveal the true identity of the strains. Remarkable is the discovery of new associations between rhizobia and legumes, namely the presence of Mesorhizobium sp. in Medicago lupulina, the presence of Rhizobium indigoferae in Trifolium campestre, the presence of M. caraganae in Securigera varia and Bradyrhizobium canariense in Robinia pseudoacacia. Also noticeable is the rather small diversity in symbiotic genes present in the strains. This is most apparent when comparing the recA phylogeny and the nodC/nifH phylogeny. Our result did not reveal significant differences in nodule symbionts for the same plant species in different habitat types. Future research will focus on broadening the sampling areas, hopefully revealing much more diversity in the nodule symbionts and interesting features of the association with native legumes

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

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

Arcobacter peruensis sp. nov., a Chemolithoheterotroph Isolated from Sulfide- and Organic-Rich Coastal Waters off Peru

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