Diagrama de estados

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6.1 Discussion and future perspective

Movile Cave is an isolated ecosystem that harbours a thriving community of

organisms that are not supported by photosynthetically fixed carbon. Instead, primary producers including chemolithoautotrophic and methanotrophic bacteria are believed to provide the organic carbon supporting the cave inhabitants. The aim of this project was to determine the presence of methane oxidising bacteria in Movile Cave, to determine which, if any, actively oxidise methane in the cave, to identify if methanotrophs provide a carbon source for other organisms in the Movile Cave environment and to isolate and characterise any methane oxidising bacteria. The study here has been able to fulfil all aims stated above through the use of cultivation and cultivation independent methods.

A metagenomic analysis was carried out on DNA extracted from microbial floating mat and water obtained from air bell 2 in Movile Cave. The sample was frozen only a few hours after been taken in order to maintain the original community of organisms present at the time of sampling. DNA extraction took place when the sample was first thawed. The metagenome was screened for several functional genes associated with methane oxidising bacteria. Methane monooxygenase genes required for the

production of both soluble and particulate methane monooxygenase were identified. Almost all of the methane monooxygenase sequences identified were genes encoding soluble methane monooxygenase components. By far the most abundant methane monooxygenase sequences identified from a single organism related very closely to the species Methylococcus capsulatus Bath. It is likely that the Methylococcus capsulatus Bath like organism is the most prolific methane oxidising bacterium in

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Movile Cave. Methane monooxygenase gene sequences closely relate to the organism Methylosinus trichosporium OB3B were also rather abundant among the MMO sequences suggesting that it too may be one of the more prolific

methanotrophs in Movile Cave. Other functional genes screened for including

methanol dehydrogenase, formate dehydrogenase and hexulose-6-phosphate synthase indicated Methylococcus capsulatus species gene sequences to be among the most highly represented. Interestingly, there were no gene sequences involved in the oxidation of methane related to Methylomonas species despite this being the only methanotroph to be isolated from the cave. More work could be done with the comparison of this metagenome with other metagenomes, more specifically metagenomes from other cave environments to find trends in community structure linked with this type of environment. As the methane monooxygenase sequences were heavily biased towards soluble methane monooxygenase it indicated that coverage of the community was not sufficient as there should have been more particulate methane monooxygenase sequences. This would likely warrant the re- sequencing of the Movile Cave metagenome on a larger scale with the aim of increased coverage.

The pmoA microarray gave a good indication of the in situ diversity among methanotrophs. The tool is limited as it only targets the particulate methane monooxygenase which means it would miss organisms like Methylocella and

Methyloferulla (Theisen et al., 2005; Vorobev et al., 2011). Contrary to the metagenome data set, the pmoA microarray identified that metanotrophs with

particulate methane monooxygenase were present in Movile Cave and that there was diversity among them. It also suggested that it might be Methylocystis species that are

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the most abundant methanotroph (owing to the semi-quantitative nature of the microarray), however, Methylococcus species were also highly represented.

Methylomonas was highlighted on the pmoA microarray although quite low in abundance compared with the other organisms. Having conducted both the

metagenome sequencing and the pmoA microarray, and both giving different insights into the diversity of methane oxidising bacteria in Movile Cave, it highlights the importance of having multiple sources of evidence as drawing conclusions from either of the techniques alone would have resulted in inaccurate conclusions being drawn. Ideally, these studies should be conducted as replicates to add robustness to the diversity of methanotrophs observed. It would also be advisable in future to conduct a longitudinal study to determine if the methanotroph community is generally stable or perhaps more dynamic.

Methylomonas LWB was the only methane oxidising bacterium to be isolated from Movile Cave. This organism showed no apparent tolerance to tetrathionate, a possible toxic compound found in Movile Cave due to the high amount of sulfurous

compounds present. The genome of Methylomonas LWB was sequenced to gain a more in depth understanding of the potential metabolisms that the organism employs to survive in the Movile Cave environment. Methylomonas LWB displayed

characteristic metabolic pathways for a Type I methane oxidising bacterium including the genes required for formaldehyde assimilation by the ribulose monophosphate pathway. The Methylomonas LWB genome contained genes for expression of both the soluble and particulate form of methane monooxygenase. More interestingly, the Methylomonas LWB genome contained genes required for the putative particulate methane monooxygenase enzyme encoded by the pXM genes

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pxmABC. The orientation of the genes in the pXM operon differs to pMMO as the pMMO operon genes are found in the order pmoCAB. The pxmA gene branches separately from the pmoA genes on a phylogenetic tree, grouping with the few pxmA

genes that have recently been identified. The function of pXM has not been

determined categorically but transcripts of the pxmA gene have been found during the growth of Methylomonas LW13 growing on methane as a sole carbon and energy source (Tavormina et. al., 2011). The Methylomonas LWB isolate may well be a new species but further characterisation experiments are required to determine this. In order to fully understand the potential metabolic processes of Methylomonas LWB, the genome will need to be completed.

DNA-Stable Isotope Probing was carried out using a sample of floating microbial mat and water from airbell 2. A microcosm was set up with the Movile Cave sample containing 13CH4 in order to label the DNA of any active methane oxidising bacteria.

Analysis of 16S rRNA gene sequences from 13C-labelled heavy DNA did not indicate any methane oxidising bacteria having incorporated the 13C-label. Most of the

bacteria identified from the 16S rRNA genes from the heavy DNA were

heterotrophic Proteobacteria. This suggested that the 13C-label had been incorporated by methane oxidisers and cross-fed into these other organisms, thus highlighting methanotrophs as primary producers supporting the growth of other organisms in Movile Cave. Functional gene analysis by way of a clone library targeting the pmoA

gene from the heavy 13C-labelled DNA indicated that Methylomonas, Methylocystis

and Methylococcus along with a bacterium closely related to Methylobacter were actively oxidising methane in the DNA-SIP experiment. There were a number of different clones identified for Methylomonas, Methylocystis and Methylobacter like

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sequences suggesting multiple species of each genus are present and active in Movile Cave. The clone library carried out here was too small to determine the true diversity of methane oxidising bacteria from the heavy DNA. If the experiment was to be repeated either a larger clone library would be constructed or high throughput sequencing would be used to analyse the pmoA PCR products. The mmoX analysis would need to be repeated to ensure that they were definitely not present in the heavy DNA. Knowing that the Methylomonas LWB isolate contains the putative particulate methane monooxygenase pxm genes, the heavy DNA should be analysed for the

pxmA functional gene as proxy for a potential third methane monooxygenase. Initially, in this study it was planned to carry out a time course experiment with the DNA-SIP but complications meant that this never happened, one of the factors being a lack of labelling in the first time point. This could be overcome by carrying out RNA-SIP as RNA molecules are labelled much faster than DNA as cell replication is not required as prerequisite for labelling. The multiple time point SIP, if realised, would potentially give higher resolution of the incorporation of the 13C- label into the methanotrophs and the cross-feeding into the other organisms in the environment.

Continuation of the project

Should the project be continued over the next few years an effort should be put into isolating more methanotrophs from Movile Cave. Methylomonas species were shown to be active methane oxidisers but were one of the lesser abundant methanotrophs present from the in situ community studies. Ideally, isolation and characterisation of

Methylococcus and Methylocystis species should be a priority. Characterisation of the molecular biology of the pXM protein from Methylomonas LWB needs further