Biofilms - Saviours of the Intertidal Zone

Microbial biofilms found in intertidal zones are vital in conserving energy and materials, and maintaining this environment. Biofilms (composed of diatoms, protozoa, fungi, bacteria and archaea), are essential for supplying grazers and their predators with energy. They secrete extracellular polymeric substances (EPS), produced by photosynthesis and composed mainly of glucose. This is then passed on to the heterotrophic organisms, and acts as a stabiliser of the environment.

This regulation of energy transfer and recycling of material make biofilms extremely dynamic. Biofilms in the intertidal zone are also impacted by the stressors in this environment, such as light stress, desiccation, and extreme changes in temperature, salinity and pH. These factors have a major impact on the properties of the biofilms.

A review conducted by Van Colen et al. (2014) looks into the current knowledge on the ecological processes within biofilms, in particular looking at the production of EPS, photophysiological stress responses and the role of grazer interactions.

EPS is an integral part of the benthic microbial food web, and is a major carbon source. The carbon contained within this molecule can be traced into the phospholipid fatty acids and RNA of many bacterial groups within the biofilm. The activity of the bacteria using EPS may positively impact other microbial groups; however this is not fully understood. Past work has focused on EPS use in aerobic conditions. For more insight into microbial interactions, this paper stresses the necessity to understand the anaerobic pathways utilised by biofilms as well.

Light stress can affect biofilm primary productivity by increasing the amount of reactive oxygen species (ROS). This decreases primary productivity, and affects EPS production. Photoinhibition then occurs in two methods of photoprotection: an effective xanthophyll cycle and vertical migration. It is proposed that these methods allow the biofilms to successfully regulate their light exposure. While work has been completed using chlorophyll fluorescence imaging and inhibitors of the processes, the results have provided indirect evidence of the biogeochemical processes occurring within the biofilm. It is suggested that studies should focus on the specific photoprotection processes in order to understand how the biofilms avoid light stress, as well as further photoinhibition.

Recent studies have shown that the biofilms are able to stabilise their environment due to the production of EPS, which binds particles together. The biofilm biomass has been shown to have an effect on erosion thresholds, and so little/no biofilm equals increased erosion. The activities of grazers and predators can also disrupt biofilms, and so the environment is more susceptible to erosion. The macrobenthos population dynamics are very tightly linked to spatio-temporal dynamics of the biofilms, which means that biofilms are shown to play a massive role in understanding the bio-physical interactions that occur within the intertidal environment. This has revealed a much more complex and ever changing ecological system.

Recent findings will often make the marine microbial picture more complicated, and show how much more is left to determine. However, it is also important to note that new technologies allow for more microbial discoveries to be made than ever before. This paper is useful in reviewing our total biofilm knowledge, and shows how this field of microbial study can be moved forward during these environmentally uncertain times. What would be interesting to see in the future is how the ongoing anthropogenic activities impact biofilm processes, and if the environment is adapting to these introduced stressors.

Van Colen, C., Underwood, G., Serôdio, J., Paterson, D. M., (2014) 'Ecology Of Intertidal Microbial Biofilms: Mechanisms, Patterns And Future Research Needs'. Journal of Sea Research 92 : 2-5. Web.

http://www.sciencedirect.com/science/article/pii/S1385110114001166

Meet microbiologys newest genetic marker (RNRs and viral diversity)

The 16S rRNA gene is commonly used in marine microbiology as a genetic marker for a number of features. It’s highly conserved nature and presence across all taxa mean that it is key to the identification and classification of Bacteria and Archaea and provides vital information about their diversity. In spite of this, to date, no such phylogenetic marker has been associated with the marine virioplankton community. In fact, marker genes that provide insight into environmental viral diversity are thus far typically limited to specific viral taxa. However, a study by Sakowski et al may provide a gene analogous to 16S rRNA that reveals new insight into viral diversity.

The paper proposes the use of the genes for ribonucleotide reductases (RNRs) as a genetic marker for viral diversity due to the fact that it is widely distributed among diverse viral lineages (and therefore is evolutionarily ancient) and it is abundant within environmental viral assemblages. In addition to this the genes play an important role in viral biology, appear to have a single evolutionary origin and are phylogenetically informative. It should also be noted that they are well represented in reference databases. All of these criteria indicate that RNR gene products may be the ideal marker gene for marine viruses.

RNRs themselves are the only known enzymes capable of reducing ribonucleotides to deoxyribonucleotides which means they are an essential part of DNA synthesis. They are a key part of biosynthesis and have therefore been identified in most of the lytic marine phages which significantly influence nutrient cycles in the global ocean. RNRs are also biologically informative due to the fact that they form three classes according to reactivity with O₂ with each class being broadly indicative of certain viral characteristics. In fact the data from this study shows that RNR sequence diversity connects with phage morphological groups and can be predictive of ecological strategies used in the virioplankton.

The importance of this is that it provides insight into the ecological features of lytic phage populations. These viruses play a vital a vital role in marine ecosystems as top-down regulators of bacterial populations and agents of horizontal gene transfer. In addition to this they are key to marine nutrient cycling and yet little is understood about their diversity. Therefore, the thing to take away from this paper is really its methodology; by using RNR gene products as a proxy for phage population biology, it opens up a new way to investigate the marine viral community.

Sakowski, E. G, Munsell, E. V, Hyatt, M, Kress, W, Williamson, S. J, Nasko, D. J, Polson, S. W, and Wommack, K. E. (2014). Ribonucleotide reductases reveal novel viral diversity and predict biological and ecological features of unknown marine viruses. PNAS. 111, 15786-15791.

Are cyanobacteria pilfering credit for viral contributions?

High abundance (10⁹ – 10¹⁰ viruses L^-1) makes viruses huge contributors to marine microbial communities through infection of bacteria and phytoplankton. Infection of organisms may lead to cell death, but cell lysis also releases dissolved organic matter (DOM) creating a pathway for the regeneration of nutrients (the viral shunt). Synechococcus spp. are cyanobacteria which contribute up to 50% of total phososynthetic carbon fixation in the upper layer of oligotrophic waters.  In warm and summer weather Synechococcus spp. show distinct diel changes, with higher division rates and highest abundance at night. Grazing by pigmented nanoflaggelates appears to be the main regulating factor in Synechococcus spp. diel variations however, recent studies have investigated mortality resulting from viral lysis.

Tsai et al. (2014) investigated the significance of viral lysis in providing a source of recycled organic nutrients and the effect viral presence had on the daytime division frequency of Synechococcus spp. in a simple study using virus reduced and virus existing treatments. Serial filtering was used to provide a virus reduced (VD) treatment; and filtering through a 0.1 µm polycarbonate filter provided a grazer reduced virus existing (VE) treatment. In experiment 1: in-situ samples were taken every 3 hours for Aug 2012; experiment 2: incubations and in-situ samples were taken at 0, 20 and 44 hours for Sept 2012. Virus, bacteria and Synechococcus spp. were counted using epifluorescence. Virus were processed using SYBR Green I (molecular probes) while Bacteria were DAPI stained for identification.

Experiment 1 showed decreases in bacterial and Synechococcus spp. abundance during the night in VE experiments and greater increases in viral abundance in VE than VD treatments. Up to 30% of Synechococcus spp. were dividing during the day in the VE compared to approx. 5% in VD treatment. Experiment 2, was used to confirm experiment 1’s findings, together they indicate that viral presence significantly affects variations in Synechococcus spp. and bacterial abundance and the frequency of cell division in Synechococcus spp. with significantly more ammonium in VE cultures compared to VD cultures..

This study supports ideas that viral lysis leads to higher phytoplankton productivity due its important role in nutrient recycling. Moreover it is in favour of cell lysis stimulating the growth of the non-infected community. Studies in this area are vital in our understanding of the marine food web, without understanding of the norm we cannot investigate the effects of anthropogenic factors or longer term effects such as ocean acidification and warming. This study is important as it shows that although viral input causes mortality it can increase growth which could suggest nanoflagellate grazing has more of an input in phytoplankton abundance than viral activity. I think it would be interesting to complete this study during different seasons, do viruses increase in abundance seasonally reducing phytoplankton growth or does this effect still occur? 

Reference:

Tsai, A. Y., G. C. Gong, and Y. W. Huang, 2014: Importance of the viral shunt in nitrogen cycling in Synechococcus spp. growth in subtropical western Pacific coastal waters. Terr. Atmos. Ocean. Sci., 25, 839-846, doi: 10.3319/TAO.2014.06.11.01(Oc)

Macroalgal blooms lends a helping hand

Green tides, which are blooms of free-floating macroalgae, have been increasing in abundance over recent decades near coastal and estuarine areas due to increased nutrient loading. These macroalgal canopies release large amounts of dissolved organic carbon (DOC), fueling heterotrophic microbes, and further causing anoxic events to occur in our oceans waters. It is believed that the DOC released from the macroalgal canopies could be powering the energy-demanding process of fixing N₂ performed by diazotrophs and in return, the low-oxygen conditions could favour the diazotrophs during these blooms. 

This study by Zhang et al. (2015), looks at the contrasting community composition and abundance of diazotrophs in macroalgal canopy-covered and canopy-free waters in the Yellow Sea in China. This study involves measuring concentrations of macronutrients in blooms and using genetic techniques such as quantitative PCR to measure the copy numbers and phylogenetic affiliation of the nifH gene in a bloom. 

Overall, the results showed that the average nifH abundances were significantly higher in samples from canopy-covered areas (4.55 x 10⁶ copies 1^-1) compared to samples from the uncovered areas (2.49 x 10⁶ copies 1^-1) suggesting that the presence of macroalgal blooms was favouring the diazotrophic bacteria. Results showed that Gammaproteobacteria (in particular Vibrio-related) nifH operational taxonomic units (OTUs) dominated in both canopy-covered and uncovered samples. However, due to Fe-limited conditions, the DOC released by the macroalgae could be favouring the growth of the Gammoproteobacteria. Vibrios and members of Marinobacter can synthesize and secrete sidedophores called vibrioferrins, allowing a supply of iron to the macroaglae in exchange for DOM. High ratios of N:P indicated severe P-limitation for macroalgae and bacteria, however bacteria are effective competitors for orthophosphate where some can store inorganic polyphosphate in P-limiting situations. The dissolved organic phosphorus released by macroalgae, once regenerated into inorganic phosphorus under Fe-limiting factors, could be also taken up, allowing the bacteria to survive environmental stresses in low-phosphate and Fe-limited environments. 

I think this study is important in the fact it underlines how macroalgal blooms can control the community composition of N₂-fixing bacteria and how changes in this community were related to N:P ratio and bacteria-macroalgae interactions. However, the use of genetic techniques in this area of study should be further investigated as the presence of nifH genes doesn’t mean it is active and functioning, and therefore genetic expression and N₂-fixation rates should be further studied.

Reference: 

Zhang, X. et al., 2015. Macroalgal blooms favor heterotrophic diazotrophic bacteria in nitrogen-rich and phosphorus-limited coastal surface waters in the Yellow sea. Estuarine, Coastal and Shelf Science, Issue 163, pp. 75-81.

http://www.sciencedirect.com/science/article/pii/S0272771414003862

 

Prochlorococcus: Capable of more than we thought?

It is well known that Prochlorococcus Cyanobacteria are the most abundant marine photosynthetic microorganisms. Because of this they exist in a range of different light conditions and can be broadly classified into two ecotypes: low-light adapted and high-light adapted. Obviously these two ecotypes will face different limiting factors and therefore must have different adaptations to cope with external stressors. For instance, nitrogen availability often proves to be a limiting factor unless a microorganism is able to use inorganic sources of nitrogen. Currently though over one hundred single-cell-amplified partial genomes have been described for Prochlorococcus but not one was able to use nitrate as a source of nitrogen.

However, in a study by Astorga-Elo et al, it was found that certain uncultivated lineages of deep water Prochlorococcus populations showed nitrate assimilation rates. They were found to have the necessary genes for this process in both the global ocean sampling metagenomics database and in metagenomes of flow-cytometry-sorted populations. These uncultivated lineages thrive in Anoxic Marine Zones (AMZs) where oxygen concentration is below the threshold for detection by modern sensors and light is scarce but inorganic nutrients are abundant. It is therefore likely that the genomic potential for nitrate assimilation acts as an adaptation for survival in these relatively harsh conditions.

The paper reports the results of metagenomic analysis carried out on environmental sequences from samples collected within the AMZ of eastern tropical Pacific. The results showed that the microbial community was enriched in Prochlorococcus, the majority of which (~90%) were found to possess the genes for nitrogen assimilation. In addition to this, de novo assembled contigs found that there was a single contig that encodes the genes related to urea and nitrate uptake and assimilation. These genes were in synteny with those found in Synechococcus WH8102. In other words, the analysis suggests that the genetic potential for nitrate assimilation has not been recently obtained via horizontal gene transfer but is a characteristic retained from a common ancestor.

The results of this study are indicative of what is likely to be an adaptation of Prochlorococcus to the nutrient-rich environment of AMZs. I believe this study to be important as it provides new information on a major marine microorganism, helping us to further understand both its adaptive abilities and evolutionary history. And, by demonstrating that certain lineages of the cyanobacterium have retained this specific adaptive characteristic they show how environmental pressures can alter the process of genetic streamlining.

Astorga-Elo, M., Ramirez-Flandes, S., DeLong, E. F.. (2015). Genomic potential for nitrogen assimilation in uncultivated members of Prochlorococcus from an anoxic marine zone. The ISME Journal. 9, 1264-1267.

Oh the irony! EPS puts iron back on the microbial menu.

In ocean water, somewhere between particulate and dissolved organic matter, there exist gels of organic compounds, secreted or released by marine micro-organisms, that sink and conglomerate in the water column. These gels have a lot of important functions in the marine system but this blog will focus on how these gels influence the chemistry of dissolved iron.

Iron is a very important nutrient in the water column; required for processes like photosynthesis and nitrogen fixation. Despite this importance, iron is often a limiting factor for many phytoplankton. Organic ligands, such as porphyrins, humic acids and saccharides (exopolymeric substances) can be found in the aforementioned marine gels which bind to iron, making the nutrient more bioavailable. A study published this year by Hassler et al. investigates just how much EPS increases the bioavailablity of iron to phytoplankton. 

By adding two types EPS to populations of Chaetoceros simplex, the researchers found that the bioavailablity of iron was increased by 365% (+/-7) and 437% (+/-8) compared to dissolved iron not bound by organic ligands, showing that dissolved iron is more bioavailable to the model phytoplankton if it is bound in these organic gels. It should be noted that iron bound in bacterial EPS (made by Pseudoalteromonas sp.) was only 28% bioavailable compared to dissolved iron. The study also found that addition of EPS to natural populations stimulated the growth of both Prochlorococcus and Synechococcus, which have very important roles in cycling Carbon in ocean waters.

The study took EPS secreted by two phytoplankton: Phaeocystis antarctica and Emiliana huxleyi and a natural sample from  the Southern Ocean. The two EPSs were mixed with ⁵⁵Fe (a radioactive isotope).Liquid colonies of C.simplex were grown and then enriched with either the EPS or inorganic Carbon. Iron uptake was calculated relative to a form of iron that the scientists and previous studies had shown to 100% bioavailable (FeCl₃). The quench rate of the isotopic iron in the cells was also used to determine cellular concentration of iron.

To me, this is a very interesting study, it reminds me of the studies which seeded the oceans with iron, except this study suggests that the addition of iron bound to organic ligands might be more effective than just inorganic iron. Something that grabs my attention is that one of the types of iron they investigated; FeCl₃ which- to my knowledge- was not organic but was just as bioavailable as that bound in EPS. The iron bound in organic compounds was able to reach higher concentrations in seawater than inorganic iron (3.56nM vs. 2nM before precipitation), could there be a ferrous organic compound that can have an even higher concentration of iron? Furthermore, would it be even more accessible to phytoplankton? It might be worth researching more into finding more bioavailable forms of iron as opposed to increasing the concentration of iron in the seawater.

References:

This paper

Hassler, C.S. Norman,L. Nichols,C.A.M. Clementson,L.A. Robinson,C. Schoemann,V. Watson,R.J. Doblin,M.A. (2015) Iron associated with exopolymeric substances is highly bioavailable to oceanic phytoplankton. Marine Chemistry. Vol.173 pp.136-147.

Producer, Grazer and Predator - an Unlikely Alliance

Tritrophic mutualistic interactions are commonly seen in the plant-insect systems. A plant will secrete volatiles in response to a herbivore, which in turn attracts a predator and relieves the grazing. These interactions have not been widely studied within the marine environment; however the release of dimethylsulfide (DMS) has been shown to be an important infochemical within the marine systems for the grazers and predators, which could potentially mean that there may be a tritrophic link. For this to be mutualistic, the producer must also benefit.

A recent study conducted by Savoca & Nevitt (2014) suggests that within the Southern Ocean marine ecosystem, DMS-producing phytoplankton (eg. Phaeocystis antarctica) will be grazed upon by primary consumers (eg. Antarctic krill, Euphasia superba) and so will produce more DMS. This in turn will attract many carnivorous species, in particular the Procellariiform seabirds, which relieve the grazing.

However, the study also suggests that the marine ecosystem benefits due to iron being made available to the upper ocean surface in the form of the faecal matter from carnivores. In this way, the phytoplankton would benefit from the influx of vital iron, providing further evidence of tritrophic mutualism.

Over 3,000 individuals of 18 procellariiforms (with the phylogeny taken into account to control potential effects) had their stomachs analysed and it was found that DMS-tracking species foraged more on crustacea (as mentioned in the example above) than any other food group, whilst the non DMS-tracking species had an equal proportion of every food group.

The body mass, manoeuvrability and diet were taken into account, and the results suggested that “the DMS behavioural responsiveness is linked to the consumption of primary consumers, which themselves consume DMS-producing phytoplankton”. As the predators consume the grazers, it is implied that the phytoplankton benefit by no longer suffering grazing damage, thus supporting the idea that this interaction is an example of tritrophic mutualism.

However, the study also wanted to consider how iron from the carnivores’ faeces plays a role in supporting the growth of phytoplankton. As iron is vital for primary production in phytoplankton, the carnivores supplying a high proportion of iron (not usually abiotically introduced into the Southern Ocean) is necessary for the well being of the ecosystem.

This study used data from South Georgia to collate previous knowledge as well as their own, concluding that their work has supported the idea that seabird excrement has a beneficial effect on phytoplankton growth. The data can also provide further evidence for the tritrophic mutualistic interactions seen between primary producers and the top predators in the Southern Ocean.

I believe that this paper is introducing a new way of studying the interactions of the micro- and macro-scale interactions present in the oceans, as well as highlighting how important it is to look at the processes occurring at the microbial levels. I think that this study may be a useful starting point in understanding how microbial communities impact on the ecological hierarchy of the oceans, and how this could affect the marine environment in the future.

Savoca, Matthew S., and Gabrielle A. Nevitt. "Evidence that dimethyl sulfide facilitates a tritrophic mutualism between marine primary producers and top predators." Proceedings of the National Academy of Sciences 111.11 (2014): 4157-4161.

http://www.pnas.org/content/111/11/4157.full