The Diversity of Methanotrophs

Aerobic methane oxidation has been documented in a variety of pelagic environments. Although it is known that there is significant spatial variation in the distribution of the process, little is known about the organisms which carry it out and together, these factors indicate potential differences in the community composition of methanotrophs - both spatially and temporally.

Three clades (OPU1, OPU3 and Group X) are proposed to contribute to oceanic methane oxidation and are distinct from the phylotypes described for methanotrophs found in marine sediments, suggesting that they are adapted to a planktonic lifestyle. This study aims to elucidate the distribution of aerobic methanotrophs and the abundances of the different lineages in relation to location.

Water column samples were taken from various depths at 3 distinct marine environments: an active methane seep, a near-shore submarine canyon and within an oligotrophic gyre. Benthic water samples were also taken from along the California continental margin, above a methane seeping canyon and at sites adjacent to a white smoker hydrothermal vent. A recent PCR-based method, monooxygenase intergenic spacer analysis (MISA) was carried out, using newly developed primers, to determine the differences in the composition of monooxygenase genes (used to identify methanotrophs) spatially and temporally. This can be used to infer the distribution of planktonic aerobic methanotrophs. Clone library and q-PCR analysis were also used to assess the abundance and distribution of the methanotroph lineages, OPU1, OPU3 and Group X.

Of the active seep sites, the methane seeping canyon was the most interesting. All three expected clades were present according to the MISA analysis, and two other clades were also found. One corresponded to the clone library as a divergent monooxygenase termed ‘Group W’, but the other was not discernable. This shows that with new primers more groups can be discovered and therefore further our knowledge of these important microbes. The abundance of each clade varied horizontally and vertically as the most numerous and diverse assemblages were found close to the seep, with less activity and abundance being found further away and towards the epipelagic.

The communities at the non-seep sites were more closely tied to the local methane maximum (200-300m) and they varied according to depth. The surveys of the bottom waters revealed the presence of all clades, albeit in varying abundances; and this, with the presence of OPU’s 1 and 3 along the California continental margin, suggests that they comprise a broadly distributed methane sink in the deep ocean. The oligotrophic gyre showed no detectable abundance of any of the clades, although a single peak represented a member of a super family termed ‘Group O’ whose role in methane oxidation remains to be discovered. However, this suggests that perhaps the monooxygenase community in oligotrophic open ocean systems is distinct from those found in deep waters and continental margins. Again, further work into producing new primers may reveal novel OTU’s which could play a vital role in the oxidation process.

Further evidence for community differences according to environment comes from the findings from the hydrothermal vent. Here, only a member of OPU3 was found suggesting that there may be site-specific OTU’s; resulting in habitat-related variation in the distribution of what is usually a broadly distributed clade.

The findings of this study imply a broad spectrum of monooxygenase diversity throughout different marine systems, including novel groups. As groups are still being found and characterised, it would be prudent to continue developing primers and techniques to further investigate the methane oxidation process as the recent MISA assay, described in this study, goes to show that a lot more can be found. Further understanding of the way methane is cycled may become incredibly useful in the future in the fight to reduce global warming and climate change, not to mention the fact that it is vitally important for life in the oceans.

A review of: Patricia L Tavormina, William Ussler III, Samantha B Joye, Benjamin K Harrison and Victoria J Orphan (2010) Distributions of putative aerobic methanotrophs in diverse pelagic marine environments. The ISME Journal 4, 700–710.

How important is nitrogen?

A review of: Capone, D. G. (2008) The marine nitrogen cycle. Microbe 3: 186-192.


This paper is a neat and informative review of the marine nitrogen cycle and its potential impact on biogeochemical cycling. It gives a helpful description of the history and development of this research area spanning from the initial inception of the idea of nitrogen fixation in the early 1800s, through to the current discoveries of the present day. Alfred Redfield is highlighted for his shrewd observation which identified the role of marine plankton in maintaining the ratio of phosphorus and nitrogen in the deep ocean – the Redfield ratio. He also outlined the debate over which nutrient is the primary limiting factor in our oceans, which is still a highly contested area of discussion today.

Various studies have implicated Trichodesmium as a key global contributor of oceanic nitrogen input. However, parallel geochemical investigations were advocating a larger nitrogen input than could be accounted for by Trichodesmium on its own. This implies that either alternative agents weren’t noticed, the input of Trichodesmium was underestimated, or both. Nevertheless, Trichodesmium is undoubtedly an example of the major role that diazotrophs hold in nitrogen input in the sea. Researchers are aware of many diazotrophs in the sea but how much nitrogen input they have is unknown. Recent discoveries of diazotrophs less than 10µm in size have been observed among plankton of tropical and sub-tropical waters. The assessment of abundance of other diazotrophs, like heterosystous cyanobacteria of the genus Richelia, is being investigated. It is believed that, even with low numbers, microbes of this size division with nitrogenase activity could produce a significant quantity of nitrogen across large volumes of the ocean. Unfortunately, methodological problems of culturing oceanic bacteria are making it difficult to determine these affects.

The nutrients that limit the productivity of nitrogen fixers seem to be subject to the different ocean regions and determined by the relative availability of phosphorous against iron. Experimental evidence of this can be observed in the Atlantic Ocean. Diazotrophs dwelling in the surface waters in the northern expanses of the Atlantic Ocean show an increased prevalence of phosphorus stress than iron stress. This is due to the input of iron in to the sea via atmospheric dust transported from the desert regions of North Africa. This raises concentrations of dissolved iron to high nanomolar levels, which subsequently drives the drawdown of phosphorous until it becomes limiting. Recent investigations have also advocated that dissolved concentrations of CO2 affect oceanic nitrogen fixation rates. By doubling the partial pressure of CO2 in culture to emulate predicted oceanic conditions that could occur over the next 100 years, it was reported that an increase in nitrogenase activity and growth rate in Trichodesmium can be observed.

Feedback processes are advocated to preserve levels of global fixed nitrogen within reasonably stringent boundaries. This can be concluded from geochemical models, field investigations and paleoecological evidence. It is suggested that global processes that remove biological nitrogen are being overestimated or inputs of the same element are being underestimated. The future of this research area lies in the application of new sampling and measurement technologies as well as the refinement of current modelling methods. I think that climate change scientists should incorporate marine nitrogen fixation in to their work, in order to combat anthropogenic pressures such as anticipated increases in oceanic temperature, CO2 concentration and the run off nitrogen based fertilisers produced to support our growing population.

The Multiple Hosts of Candidatus Endoriftia persephone

Riftia pachyptila and Tevnia jerichonana are both species of tubeworms found in the unique environment surrounding deep sea thermal vents. As discussed in some resent blogs, R. pachyptila relies on a endosymbiotic chemoautotrophic bacterium for its carbon fixation and energy production and is found in a specialised organ called the trophosome. Following this discovery many studies have examined this remarkable relationship and in 2008 the symbiont was named Candidatus Endoriftia persephone (Robidart et al 2008). However, T. Jerichonana has received very little attention and very little is known about its symbiont.

This study had the purpose of comparing the endosymbionts and the geochemical environment of both R. pachyptila and T. Jerichonana to discover how their symbionts vary physiologically. T. jerichonana are found at sites of newly erupted thermal vents and they seem to favour the low oxygen levels and relatively high concentrations of sulphide. Sulphide being the electron donor compound needed for both species of tubeworms and their symbionts. As the thermal vent ages and becomes less active the surrounding environment shows increased levels of oxygen but sulphide levels decrease. This change in chemistry seems to be more favourable for R. pachyptila as they replace any previous populations of T. jerichonana found at the same site. The presumption of this would be that the bacterial symbionts of these two species would also differ to reflect the different chemistry of their environment.

Both tubeworm specimens were removed from thermal vents on the East Pacific Rise and their trophosome tissues was removed. The bacteria were separated from the trophosome cells by density gradient centrifugation which allowed comparisons to be made of the genomes and major proteins of the isolates. Further measurements of the environment where the tubeworms were collected were also taken to compare the chemistry of the two sites.

Habitat chemistry showed what had been previously described with higher levels of sulphide (~2mM) being present at the site of T. jerichonana sampling compared to a much lower sulphide concentration (0.01mM) at the site where R. pachyptila were removed. Temperature and pH also varied greatly between the two sites. Metagenomics of the two isolates showed that 16s rRNA subunits of the two were identical and key metabolic genes were 99.9% homologous. Protein analysis also showed almost identical abundance of metabolic enzymes. These results strongly suggest that the two species of tubeworm share the same species of symbiont despite the very different chemical environments they inhabit. The fact that the bacteria genes involved with sulphide oxidation differ by only 0.1% also suggests that the two species of tubeworm are able to create an identical trophosome micro-environment that suit the needs of the bacterium for sulphide metabolism. If that is the case then it may be possible that Candidatus Endoriftia persephone is a symbiont in many other invertebrate species.

In my opinion this shows that Candidatus Endoriftia persephone is highly important to the thermal vents ecosystem. The fact that it forms a symbiosis with two species that range the life span of the vents themselves shows a remarkable adaptation for life in these small environments. I think this is also highlighted by a previous blog which describes how this bacterium can also live freely in the water column before ‘infecting’ tubeworm larvae. It would be very interesting if T. jerichonana symbionts were somehow released into the water column and went on to infect a R. pachyptila larva. I imagine this would be next to impossible to study though.


A Review of:
Gardebrecht, A. Markert, S. Sievert, S. Felbeck, H. Thurmer, A. Albrecht, D. Wollherr, A. Kabisch, J. Le Bris, N. Lehmann, R. Daniel, R. Liesegang, H. Hecker, M and Schweder, T (2011) Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics. The ISME Journal, 1-11.

Additional Reference:
Robidart, J. Bench, S. Feldman, R. Novoradovsky, A. Podell, S. Gaasterland, T et al (2008) Metabolic versatility of the Riftia pachyptila endosymbiont revealed through metagenomics . Environmental Microbiology, 3, 121-126.

Metabolic demands of chemoautotrophic metabolism

Up until the late 1970’s people were unaware of a radically different ecosystem that existed deep in the ocean at the site of hydrothermal vents. The depth, pressure, lack of light and oxygen associated with these areas was an indication that the species that lived there must have a way of surviving which was not dependant on plants or algae for primary production. Other than living conditions the high biomasses and sheer size of the dominant species present, such as tube worms, molluscs, indicated something was different about the metabolism of these organisms. In the early 1980’s the secret of these organisms success was discovered; an endosymbiotic relationship with chemoautotrophic microorganisms. The primary production of this metabolism was fueled by the oxidation of hydrogen sulphide. Another type of symbiosis has also been uncovered, methanotrophic symbiosis; however there is a very little literature on this type of metabolism as much is still unknown.

The authors compare photoautotrophic, chemoautrophic and methanotrophic metabolism throughout the paper and although there are various similarities such as the placement of symbionts in the host, near the surface, there are a massive number of differences. The uptake of inorganic carbon, the use of nitrites and the elimination of proton equivalents are just a few examples along with the toxicity problems caused by sulphide; which must be oxidised to thiosulphate in order to reduce toxicity. For chemoautrophy one of the most important functions is the host’s ability to support high oxygen demands, as it is critical for the oxidation of sulphide and carbon fixation.

After years of studying the physiological functioning of vent species studies showed that haemoglobins play a key role in binding sulphide and oxygen and allowing their transport to symbionts. The presence of these high affinity, tissue or free flowing haemoglobins seems a necessary and efficient way to support the high oxygen demand required to support symbiont metabolism (thiotrophic endosymbiosis), and is a crucial adaptation for chemoautotropic species. The characteristics required metabolically, clearly restrict the ability of certain taxa to evolve this kind of endosymbiosis, and is a proposed theory as to why the most diverse phylum for phototrophic endosymbiosis, Cnidarian, has been unable to develop this type of relationship.

A review of: Childress. J, Girguis. P (2011) The metabolic demands of endosymbiotic chemoautotrophic metabolism on host physiological capacities. The Journal of Experimental Biology. Vol. 214(2) 312-325

“Organic” cure for harmful algal blooms?

Slightly different subject to current ones but perhaps you might find it interesting…

Over the last several decades cultural eutrophication has led to change in structure and diversity of marine benthic communities, coastal regions throughout the world have experienced an escalation in the frequency of algal blooms that are toxic or harmful. Many attempts have been made in order to tackle the problem, however high costs and in particular the use of chemical agents such as copper sulphate does not appear to be the right move as their rather extensive use resulted in an even higher contamination risk to aquatic environments and toxicity towards non-target species. Biological agents such bacteria or viruses are now considered to have potentially direct or indirect supressing effect on algae, however the relationship and mode of action of bacteria on algae has not yet been fully understood. Previously studies have already isolated certain strains of bacteria that possess algicidal properties (i.e. can regulate the growth or toxin production) and these include large groups of Cytophaga/Flavobacterium/Bacteroidetes (CFB) or to the ɣ-proteobacteria group, and to the genera Cytophaga, Saprospira, Alteromonas and Pseudoalteromonas. Similarly Su and his colleagues investigated the relationship of Alexandrium tamarense spp, a bloom forming algae that are mainly associated with paralytic shellfish poisoning and the bacteria isolated from water samples where Alexandrium sp blooms where observed.

The simplest way of explaining the methods involved was isolating and purification of A. tamerense and bacteria present in the water and inoculating it with either bacterial cells, filtrates or cell suspension of bacterial isolates which genera were confirmed through amplification of 16S rRNA genes. They investigated the proposed anti-algal properties by monitoring the growth progression of Alexandrium spp. under the presence of those bacterial isolates.

They have managed to isolate over 100 types of bacteria which 9 of them showed some algicidal properties through inhibiting the growth of algae. Bacteria mostly belonging to the ɣ-proteobacteria group have showed to have indirect effect since algicidal activity was only detected in cell free supernatants but not the bacterial cells. Therefore bacteria have probably most likely to possess some sort of chemical compound which acted indirectly on the algae; however that was just an assumption.

The initial aim of the study was to improve the understanding and explain the mode of action of bacteria on algal communities however authors apart from finding new strains of bacteria didn’t really conclude nor explained the exact interaction between those species and most of summary was based on assumptions.

To quote “our results showed high diversity of algicidal bacteria against A. tamarense” (which being fair they have done) and “providing possible choices for controlling HABs by microbial strategies” which in turn is a quite a “big” statement because authors didn’t actually find out how those bacteria attack the algae, it’s all based on more assumptions and pretty much everything is possible. Another problem of my own with this study is that it is a laboratory based study therefore everything has been done under a close control so realistically; in real conditions other strains of bacteria might also be present and perhaps protect algae against bacteria that have negative effect on them. Also environmental conditions do change and mode of action of certain bacteria can also change, especially in the time of apparent climate change.

To summarise, the paper was fairly clearly written and quite interesting however I was expecting a little bit more from their findings, few of their statements has been a little bit miss-selling but that is just my personal opinion, perhaps other readers would not agree with me.

Ref: Su J., Yang X., Zhou Y., Zheng T., (2011), Marine bacteria antagonistic to the harmful algal bloom species
Alexandrium tamarense (Dinophyceae), Biological Control, 56 (2011), 132–138

Oceans of Archaea

This year-long study in the Pacific ocean, through monthly sampling of the water column (surface to 4,750 m), shows that the ocean's interior, earth's largest biome, is mostly an archaea dominated ecosystem.

Using single-cell, fluorescent in situ hybridization (FISH) with specific rRNA-targeted oligonucleotide probes for either pelagic archaea (crenarchaeota and euryarchaeota) or bacteria, the authors calculated that world’s ocean contains approximately 1.3x10²⁸ archaeal cells and 3.1x10²⁸ bacterial cells and that both archaeal groups as well as bacteria collectively, lack a clear seasonal trends in cellular abundance.

Bacteria generally tend to dominate the microbial population in the upper 150m of the water column, representing up to 90% of all picoplanktonic cells. Then they clearly decrease in relative abundance with increasing depth, and below 1,000m they represent only 35±40% of total cells.

Pelagic archaea instead, show different patterns of abundance in the open sea. Pelagic euryarchaeota occasionally appear with low numbers in the near surface layer, but they generally remains at a few per cent of the total count over the entire water column. By comparison, a large fraction of total marine picoplankton appear to be represented by one specific clade, pelagic crenarchaeota. These archaea, are a consistent and significant component of deep sea microbiota and they represent one of the ocean's single most abundant cell types. In surface layers, pelagic crenarchaeota are only present sporadically, and never abundant numerically. The major relative increase in their abundace occurs below the euphotic zone (>150m). Then, below 1,000m and throughout the entire mesopelagic and bathypelagic zones, the fraction of crenarchaeota constantly increases with depth, even to the extent that it can equal bacterial population in cell numbers.

On the other hand, the authors found that the total number of microbial cells, sharply decreases with depth (an order of magnitude between 150 and 1,000 m), so they came to the conclusion that with increasing depth, there may be a population shift to microbial groups better adapted to deep sea conditions, such as pelagic crenarchaeota. As a matter of fact, bacteria and crenarchaeota standing stocks are inversely proportional and crenarchaeota can often rival bacterial abundances in the meso and bathypelagic zones. Moreover, most of pelagic deep-sea microbes (archaea and bacteria) were found to contain significant amounts of rRNA, this means that they are metabolically active contributors to the ecosystem. Thus, archaea are an important component of world's deep oceans and their large distribution throughout the entire oceanic water column suggest that they are really far from being merely confined just to extreme habitats as we thought in the past.

Reference:

Karner, M.B., delong, E.F. and Karl, D.M. (2001) Archaeal dominance in the mesopelagic zone of the Pacific Ocean. Nature 409: 507–510.

Coral bleaching: bacteria are against zooxanthellae,not corals!

Coral bleaching is the loss of the pigmented zooxanthellae causing the coral turn white.

Vibrio shiloi has been proved to be the causative agent of bleaching the coral Ocrulina patagonica, which exibited massive bleaching in the eastern Mediterranean during recent summers, when seawater temperatures reached 30° C. The infectious process requires 5 sequential temperature dependent steps :

1. adhesion of V.shiloi to coral surface
2. penetration into coral epidermal layer
3. differentiation into viable but not culturable state
4. intracellular multiplication
5. production of extracellular toxins that inhibit photosynthesis,bleach and lyse zooxanthellae

The adhesion is the critical step (non adhering mutants are not virulent) and is mediated by a beta-D-galactopyranoside-containing receptor on the coral surface.

Authors investigated V. shiloi infectious process in 3 forms of O. patagonica: white cave colonies,bleached and normal healthy colonies. The results indicated that healthy corals produced large amount of mucus compared to cave and bleached colonies and V. shiloi adhered just to normal healthy coral, thus zooxanthellae are required for this process.

In adhesion experiments mucus was removed from the healthy pigmented coral and fixed. Overnight cultures of V. shiloi pregrown at different temperature were added to the mucus,and the chemotaxis to O. patagonica was detected through a tube capillary assay. Bacteria pregrown at 25°C showed a great higher chemotaxis compared to bacteria pregrown at 16 °C, and both of them were higher than the control. The same data were obtained with mucus removed from corals mantaining at 16° and 25°C, indicating the temperature dependance of the infectious process.

Methyl-beta-D-galactopyranoside blocked the adhesion,indicating that the beta-D-galactoside-containing receptor is found in the coral mucus.

In order to investigate the role of zooxanthellae in adhesion process, several pieces of coral were treated to remove mucus and incubated with DCMU to inhibit photosynthesis of zooxanthellae during adhesion. Results showed that mucus-depleted corals treated with photosynthesis inhibitor did not produce fresh mucus and no adhesion occurred, thus zooxanthellae must be present and photosynthetically active for synthesis or secretion of the receptor. These results were confirmed by other adhesion experiments using 5 different coral species showing that V. shiloi was found only in the three coral species possessing zooxanthellae.

These results are consistent with other data indicating that bacteria are against algae, in fact toxins produced does not damage coral tissue: coral bleaching is due to the loss of zooxanthellae,not directly to V. shiloi infection, and it occurs only during summer;when seawater temperature drops in the winter,V.shiloi is no longer present in the coral,and the symbiosis with zooxanthellae is restablished.

The presence of arabinose (a sugar not commonly found in animals) in the mucus suggestes that the zooxanthellae may contribute carbohydrates directly to the mucus.

Although this paper is relatively recent,I find it very interesting and useful because it contibuted to our knowledge of coral bleaching process focusing on the key interaction between bacteria and zooxanthellae.

A review of: Banin, E., Israely, T., Fine, M., Loya, Y. and Rosenberg, E. (2001), Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloi to its host. FEMS Microbiology Letters, 199: 33–37. doi: 10.1111/j.1574-6968.2001.tb10647.x