Tuesday, 29 October 2013

Could microbes be the answer to our plastics problem?

During the last 60 years plastics have become one of the major sources of pollution on the planet and with annual production of plastic globally being approximately 245 metric tonnes per year this problem is likely to continue.  It is hardly surprising then that some of this discarded plastic finds its way into our oceans, accumulating on our beaches and also in the centre of ocean gyres where it is held by currents.  This paper looks at the effect that these accumulations of plastic have on microbial communities, in particular they are concerned about the longevity of plastic within the oceans when compared to more natural substances such as wood. 
Zetter and his team were particularly interested in the plastic accumulation in the North Atlantic Subtropical Gyre as it does not appear to have increased in size since 1980 despite the rate at which plastic is being disposed of.  Given that the hydrophobic surface of the plastic promotes microbial colonisation and biofilm formation this study wanted to show that the microbial communities found on the plastic would be distinct from those found in the surrounding waters.

Sampling and Visual Identification.
Three types of sample were taken: a sample of seawater surrounding the plastic debris, pieces of polyethylene (PE) and pieces of polypropylene (PP).  The plastic chosen for the sample are two of the most common used in commercial packaging.  After fixing the plastic samples were inspected using Scanning Electron Microscopy (SEM) which revealed 50 different morphotypes of  eukaryotic and bacterial cells living on both types of  plastic.  The two most common organisms were diatoms and filaments however the third most common which is as yet unidentified was found embedded in pits on the surface of the plastic. 

Molecular Analysis
Through molecular analysis it was shown that there were distinct communities associated with each type of plastic and the seawater, (Fig 1).  For the communities within the plastic the data showed the presence of phototrophes such as the cyanobacteria Phormidium and Rivularia, biofilm forming species including Navicula and NitzschiaVibrio species were also detected within the community but on the rRNA sequences used it was not possible to fully identify which species were present giving rise to concerns that the Vibrio strains could be harmful to humans or animals, this is of greater concern given the length of time that plastic can remain in the ocean and the distances it may cover.

Figure 1.  Venn diagram showing bacterial OTU overlap for pooled PP, PE, and seawater samples; n = number of sequenced reads per group. Numbers inside the circles represent the number of shared of unique OTU’s for a gives substrate.

Of particular interest were the bacteria found only on the plastics that were known to be capable of degrading hydrocarbons.  These were shown to be part of a widespread network within the microbial community linking to other microbes that have previously been found in areas of hydrocarbon contamination.  Together with the visual examination of the plastic which showed individual cells embedded in pits within the plastic, this suggests that they may be working as a whole to possibly providing a sink for the recalcitrant carbon which is held on these plastic islands.

It will be interesting to follow the journey that this research team takes as it tries to establish if the microbial communities found within the plastic debris could be the solution to the problem.

Zettler, E. R., Mincer, T. J., & Amaral-Zettler, L. A. (2013). Life in the ‘Plastisphere’: Microbial communities on plastic marine debris. Environmental science & technology.

The Microbial Community In The Palm Of Your Hand!

Fierer et al used the bacterial diversity on 51 volunteers’ palms to discover the variability between the hands of an individual and the variability between individuals. There is a huge array of bacteria found upon the surface of the human body, with each area of the body having it’s own unique community of microbes.  The presence of a community is dictated by frequency of skin shedding, UV exposure, host anti-microbial defence, moisture availability and exposure to detergents and soaps. This study was conducted upon the dominant and non-dominant hands of each volunteer (handedness).

The 16S rNA genes were amplified using PCR techniques, before being analysed using a pyro-sequencing run; this was conducted for each palm of each volunteer.  This technique, coupled with various bar-coding techniques, provided the most comprehensive dataset of a human skin bacteria diversity to date. 
The study found that the average human palm contains >150 distinct species-level bacteria.  This is a radical difference to previous studies which are noted by the author to have been largely under-estimated. The bacterial richness found in the swabs from a palm were >3 times higher than previous swabs conducted on elbow and forearm skin.  It was hypothesized that the increased bacterial diversity and richness may be due to inoculation due to contact with the surrounding environment though it was suggested that the depth of the survey, which produced rarer bacteria often un-documented, was the root-cause of the heightened richness and diversity.  The results were compared with a previous study that documented the diversity of the throat, stomach and fecal bacteria, collected using similar pyro-techniques; the palms were found to be higher.
               The most abundant genera were found to be Proprionbacterium, Streptococcus, Staphylococcus, Corynebacterium and Lactobacillus which accounted for 94% of the total population.  These are believed to be the most common skin residents.  The other genera were deemed to be either transient, short-term residents or permanent residents that were simply present in low quantities or were only present due to specific host factors.
               Despite similarities, across the 102 palms swabbed, 4,742 distinct bacterial phylotypes were documented; only 5 of which were shared across all hands, showing a huge range of inter-variability.  There was a significant difference in the communities found on the dominant and non-dominant hands, with the dominant hand having a very different community to the non-dominant hand.
               The sex of the volunteer was also found to significantly affect the diversity and abundance of bacteria, with women harbouring 1573% more abundance of several species.  Women were also found to have a higher diversity of bacteria than men.  There were several hypotheses for why this may be; reduced pH in men, increased skin thickness in men, different hormone secretion and frequency of cosmetics applied. It was also reported that women wash their hands more often than men.

               This study explores the techniques involved with microbial identification whilst helping to express the particularity and definition of identification of the skin’s bacterial community.  The technique detailed in this study could be applied to deeper meta-genomic studies.  This study also could help with understanding the behaviours associated with increased hygiene and health. 

Fierer et al (2008) The influence of sex, handedness and washing the diversity of hand surface bacteria. PNAS. 105:46 pp.17994-17999

Science gets weird: Microbial Bebop, creating music from complex dynamics in microbial ecology

'Here, we combine music and biology to generate musical compositions from microbial ecology data.'

A bizarre one, this. A team of American scientists has decided to tackle the issue of ineffective communication of scientific ideas to the general public by taking the only logical course of action, the conversion of complex biological data sets into jazz music. 
Microbial bebop makes use of the patterns seen in microbial ecological data sets and transforms them into patterns of notes and chords to be used in jazz bebop improvisation. This study generated four compositions from data collected by a marine monitoring system in the Western English Channel, each composition (apparently) represents the relationships between microbial community structure and a range of environmental parameters.

Inspired by the lack of scientific awareness displayed by the American public in recent surveys, the authors explain their obligation to explore more effective ways to convey ecological science.  Proposed as an alternative to the graph-and-chart style of data communication, their use of jazz music capitalises on repeated patterns and the complex interactions seen between microbial populations and their environment. It is not the first time music has been used as a possible way to communicate data, amino acid and genomic sequences have both been converted into musical scores, though the linearity of the data makes for less aesthetic compositions (honestly, I’m not making this up). One proposed advantage of musical compositions is the shear amount of biological data they can convey relative to a graph. A picture may tell a thousand words but the authors have calculated there are 6.88×10109 unique combinations possible using standard compositions, offering almost unlimited interpretations of the same data set using different environmental parameters.

For those of you more versed in music theory than I am, the complete methodology for transforming data to music is in the paper. In layman’s terms, a melody represents one element from the biological dataset, for example nitrate or chlorophyll A concentration, these are ‘plotted’ against the chords, which represent another column of data, such as monthly measures of temperature or salinity. In one of their compositions ‘Fifty Degrees North, Four Degrees West’, a chord progression of 12 chords used in the chorus represent the changes in salinity and temperature data over the 12 months of the year.

Figure 1: The general approach to Microbial music is summarised using a hypothetical data set.

Though this may all sound very farfetched, the authors genuinely propose Microbial Bebop as one approach to engage the non-scientific community in ecological science. Perhaps one serious point they make in their conclusion is that the use of human intuitive understanding to solve problems that computers find it hard to deal with has precedents, and that the innate human ability to detect patterns and subtle changes in music may make the handling and interpretation of complex biological data sets easier with this technology. 

They claim in their final note that the ‘possible permutations of data transformed into music are nearly infinite’. I personally do not see it becoming part of the mainstream in scientific literature, ‘Fig 1: an MP3 of our dataset’ is not likely to be the future of ecological research. However they do raise the point that more effective methods of engaging the public are needed. Just perhaps not in the form of bebop jazz improvisation.

All in all a surreal and yet interesting area of research to look in to, and if nothing else, it proves that you can receive funding to do just about anything (so long as the words ‘climate change’ are in there somewhere).

Larsen, P., & Gilbert, J. (2013). Microbial Bebop: Creating Music from Complex Dynamics in Microbial Ecology. PloS one8(3), e58119.

Sunday, 27 October 2013

Could gut microbes determine how long their hosts live?

   Whilst the life extending effects of calorie restriction (CR) are known in many mammalian groups (including humans), the mechanisms of this phenomenon are not. Changes in urinary bacterial metabolites have been associated with CR in monkeys and dogs, flagging gut microbiome changes as a potential suspect and inspiring this study.

   The trial fed mice either low-fat diet (LFD) or a high-fat diet (HFD) and sub-grouped into calorie restricted (CR) with/without exercise and calorie unrestricted (CU) with/without exercise. Faecal and serum sampling tested for changes in endotoxin load from the gut microbiota and for associations of improved lifespan with certain microbial community compositions. All CR mice were significantly longer lived and healthier than controls, most dramatically in LFD mice; exercise had no significant effect on longevity.

   Of 34 distinct microbial phylotypes, 16 increased and 18 decreased in abundance between LFD and LFD + CR mice; Lactococcus phylotype abundances were lower in CR mice, whereas Lactobacillus dominated CR communities, but was almost absent in non-CR mice. As the mice aged, their gut communities developed distinctly, with the familiar probiotic Bifidobacterium thriving in CR mice and the obesity/inflammation-associated Desulfovibrionaceae being more populous in non-CR mice.

   HFD and LFD microbial shifts were different; CR was associated with fewer phylotype differences in HFD than in LFD mice. Most differences were unique to each diet; only 3 phylotypes showed the same response to CR in HFD and LFD mice.
In LFD mice Lactobacillus members showed the strongest correlation with increased longevity and the 30 phylotypes associated with lower lifespan were from the Phyla of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria and TM7. In HFD mice, more similar numbers of phylotypes were correlated either positively or negatively with lifespan and most were from Bacteroidetes or Firmicutes.

   Additionally, lipopolysaccharide-binding proteins (LBP) were lower in CR mice, indicating a lower antigen load from the gut microbiome, suggesting that the benefits of calorie restriction may be related to altered interactions between the host's immune system and gut microbiota, for example, CR gut communities may be better at excluding opportunistic pathogens, thereby reducing antigen load.
Gender is known to affect lifespan and there are known gender associated gut microbiota in mammals, which is why this study used only male mice; a replica of this study using female mice could provide another insight into the relationship between gut microbes and longevity.
The study proposes that calorie restriction allows the host to extract more fat and protein from food, leaving proportionately more indigestible polysaccharides (dietary fibre) for gut microbes to digest, favouring the growth of beneficial phylotypes; perhaps you are what your microbes eat.

   Clearly mammals, and probably all animals, have a dynamic, complex and obligatory relationship with their gut microbiota, one which we should increase our understanding of, as it will likely provide powerful new tools for improving human and animal health. The implications of this study extend to marine mammals and probably all marine animals; it also could impact aquaculture probiotic techniques and algae cultures, since edible algae contain diverse lipopolysaccharides, which could likely be beneficial for the microbe communities of humans and grazers.

Zhang, C., Li, S., Yang, L., Huang, P., Li, W., Wang, S., ... & Zhao, L. (2013). Structural modulation of gut microbiota in life-long calorie-restricted mice. Nature communications, 4.

Viruses as masters of the phytoplankton?

Virus-driven nitrogen cycling enhances phytoplankton growth

Last lecture Colin highlighted the importance of the viral shunt in the nutrient cycling and I chose this paper to go into more detail with this.

As already mentioned in several blog entries before, viruses have a great impact on the ecology of aquatic ecosystems and they are far more abundant than any other biological “beings” in the ocean. They account for roughly 20 – 40% of bacterial death through cell lysis every day and thereby make nutrients available to the environment, which is referred to as the “viral shunt”. The free organic carbon can then be used by other bacteria, which means that viruses have a major role in the recycling of carbon in the microbial community. Furthermore, the lysis of cells releases amino acids into the water column as a source of organic Nitrogen and contributes to the Nitrogen cycling.

Evidence for this has been found in the Gulf of Mexico and the Mediterranean where growth rates and division of cyanobacterial cells from Synechococcus were found as highest when viruses were present and lowest when viruses were removed or reduced. Additionally, it was discovered that the ammonium (NH4+) concentration of a water column decreased with the removal of viruses which subsequently led to a decrease in phytoplankton growth.

For assessing the production of NH4+ and phytoplankton growth, water samples were taken from two completely different environments; (1) Vancouver, Canada, with a salinity of ~ 25-26 ‰, low chlorophyll α, eutrophic conditions, (2) Indian Ocean, with salinity of ~ 35 ‰, low chlorophyll α, oligotrophic conditions.
The two water samples were subject to two conditions of viral concentration (+V = presence, -V = absence) and both set-ups were kept at in situ concentrations of bacterial cells as well as environmental conditions. Bacterial cells and viruses were stained with SYBR Green and counted using flow cytometry, phytoplankton cells were counted unstained. The chlorophyll α and NH4+ content of the samples were established using fluorometry and results showed a much higher NH4+ concentration from samples with viruses compared to the ones where the viruses had been removed. In addition to this, the +V treated samples had a much higher chlorophyll content than –V treated ones (see Figure 1).


Fig. 1: Difference in chlorophyll α concentration and picoalgal abundance from both water samples (IO = Indian Ocean; FC = Vancouver, Canada) when viruses present (+V) and viruses absent (-V). 

From these results, assumptions of viral-induced NH4+ production resulting in higher phytoplankton growth rate (= increased chlorophyll α content) could be confirmed. This study strongly underlines the importance of viruses in the aquatic ecosystem as they greatly influence the nutrient cycling. The authors examined two very different ecosystems (Canada and Indian Ocean), however, I suggest that future studies also need to look at the most productive regions such as the Antarctic waters where phytoplankton growth during summer months is very high. In my first blog post (“Virus genes in the Arctic marine bacteria identified by metagenomic anaylsis”) I highlighted that viral mortality of bacteria in Antarctic waters is the highest of any marine environment and it would be interesting to see how reliant the phytoplankton production would be upon the presence of lysing viruses.

Shelfold, E.J., Middelboe, M., Møller, E.F., Suttle, C.A., 2012. Virus-driven nitrogen cycling enhances phytoplankton growth. Aquatic Microbial Ecology, 66, pp.41-46.

Friday, 25 October 2013



Viruses are the main killers of bacteria. However, bacteria increasingly get resistant to multiple strains of viruses. Thus, there is a trade-off between viral-induced bacterial mortality and bacterial resistance against viruses. Apart from mortality, viruses also induce evolution and diversification of bacteria via multiple processes. Both the bacteria and their viruses influence evolution of each other. In other words, they co-evolve continuously where the host bacteria evolve resistance against viruses and viruses evolve in order to conquer host’s resistance. Thus, it is a microbial arms race or an antagonistic co-evolution.  
This study investigated co-evolution between Synechococcus (key primary produces of the marine environment) and cyanophages (myoviruses). Significant co-evolutionary cycles between Synechococcus and cyanophages, were observed by the authors. This includes Synechococcus evolving resistance against the cyanophage which subsequently conquer the resistance of evolved Synechococcus and so on..., in such a way persistence of an antagonistic co-evolutionary cycle.
Authors noted that although, genotypes of both the hosts and its viruses were altering together (co-evolving), the phenotypic diversity of the host Synechococcus was greater than the viruses. Authors also found that co-occurring host phenotypes had different viral-resistance capabilities. They linked this to the fitness cost of viral resistance, previously reported in both the   Synechococcus and Prochlorococcus.
Surprisingly, genome sequencing of the evolving viruses could not find mutations in the tail fiber genes, implicated in determining the host range. Rather, half of the mutations occurred in another gene of unknown functions which could be involved in the host-viral interactions. As significant mutations were detected in that gene, in numerous tested viruses; authors suggested that it could be under strong positive selection pressure. It might be involved in determining the viral host range. Nevertheless, they argued that multiple genes in addition to this one might be involved in the host range determination.   
Adaptive mutations were observed in the Synechococcus genome. Viral resistance is an intricate trait, involving multiple loci of the Synechococcus. Remarkably, they evolved resistance to some viruses in multiple ways. In other words, mutations in totally different genes provided resistance to the same virus. This finding which was not observed in viruses, support the hypothesis that parallel evolution within a gene arises more frequently in bacteriophages, compared to their hosts.
To test the relevance of this experimental study to the viral-host co-evolutionary patterns occurring in nature, authors challenged the evolved population of Synechococcus with the genetically distinct myoviruses isolated from Rhode Island waters and found Synechococcus cells getting resistant to them, over a period of time. Pleiotrophy was observed in the Synechococcus population.  Resistance to one virus also provided resistance to other viruses whereas some of them showed opposite trend where getting resistant to one virus concurrently made them susceptible to other viruses.
Overall, this study explores antagonistic co-evolution between bacteria (Synechococcus) and their bacteriophages, highlighting its complexity. Particularly, it underlines how bacteria and their viruses change continuously in order to conquer each other and in doing so generate and sustain diversity of these key primary producers and also their viruses. Additionally, this co-evolution has potential implications in understanding viral activity in the oceans’ nutrient cycling.  

Marston M.F., Pierciey F.J., Shepard A., Gearin G., Qi J., Yandava C., Schuster S.C., Henn M.R. & Martiny J.B.H. (2012) Rapid diversification of coevolving marine Synechococcus and a virus; Proceedings of the National Academy of Sciences, 109(12):4544-4549.

Thursday, 24 October 2013

Harbouring Oil-Degrading Bacteria is a Potential Mechanism of Adaptation and Survival in Corals

Coral reef systems are very diverse and complex habitats, harbouring a huge diversity of microbes in their tissues, mucus and skeleton; known as a holobiont system. Corals are currently under threat from anthropogenic stressors such as crude oil pollution released from ships, oil tankers and damaged pipelines. Occasionally, oil can also be released naturally through cracks in the seabed. When exposed to oil pollution, the coral secretes excess of mucus to try and slough off the oil, in turn leaving their tissues bare thus more prone to degradation. Abnormalities in reproduction have also been recorded, as well as an increase in mucus cell size which causes them to rupture; resulting in a lack of their most important defence mechanism.

Despite these negative effects of oil on corals, a phenomenon has been found north of the Arabian Gulf surrounding Quaro Island. Corals in this area are said to be some of the healthiest and most developed; yet are subjected to continuous natural oil seepage. This study shows how harbouring hydrocarbon degrading bacteria helps these corals to survive.

Samples were collected from corals near two islands, Quaro Island and Umm Al-Maradim Island. Tissue and mucus samples were taken from Porites compressa and Acropora clathrata from both sites. The tissue and mucus samples were put in microcosms with 2 different quantities of crude oil and incubated. At stages of 1, 4, 8, 12 and 16 weeks, samples were removed for denaturing gradient gel electrophoresis (DGGE). Between and within site comparisons were completed with a non-parametric Mann-Whitney U test and Kruskal Wallis test.

Results showed that the two coral species from both sites had an equivalent number of oil-degrading bacteria in their tissues; but a significantly higher number in their mucus. In Qaro, the higher polluted area, corals produced a greater quantity of mucus in response compared to Umm Al-Maradim, the less polluted area. Coral mucus in Qaro also contained a higher number oil-degraders. Corals inhabiting Umm Al-maradim depend mainly on bacteria in their tissues as mucus production has a very high energy cost, therefore less efficient in the low polluted area. Comparisons within sites found that P. compressa uses sloughing off and oil-degrading bacteria in their tissues whereas A. clathrata depends on only the latter.

During incubation in microcosms it was expected that the number of oil-degraders would decrease over time, however the opposite was observed. Even though oil has toxic effects on bacteria it seemed to stimulate metabolic activities resulting in an increase in tolerant species. Effects were more pronounced when the higher concentration of oil was present. The DGGE results showed that the microbial population shifted to the most effective and tolerant groups; suggesting that corals can adapt to their surrounding environment by selecting the most beneficial bacteria.

A higher diversity of oil-degraders was found in the Qaro corals. It has been suggested that this is due to oil components being degraded by tolerant bacteria into more toxic compounds thus stimulating the growth of bacteria that can utilize them. Ummm Al-Maradim corals receive a lower concentration of oil and therefore not as toxic, so this process doesn’t occur.

In conclusion, this study shows that oil-degrading bacteria inhabit corals where there is a continuous release of crude oil and give them an advantage to survive and adapt. The benefits of hydrocarbon metabolism genes are currently being studied, including the role of viruses transferring the DNA between bacteria.
Al-Dahash, L.M. and Mahmoud, H.M. (2013) Harboring oil-degrading bacteria: A potential mechanism of adaptation and survival in corals inhabiting oil-contaminated reefs. Marine Pollution Bulletin 72: 364-374

Tuesday, 22 October 2013

Investigating the Photoprotective Capacities of Bacteria collected from Deep Sea Hydrothermal Vents in the Mid-Atlantic Ridge

Overexposure to Ultraviolet (UV) radiation is harmful to organisms and can result in skin cancer. Therefore some form of photoprotection is needed to block this damage, so some natural compounds in organisms are known to act as a natural sunscreen, such as melanin, and there has been high commercial interest from several industries in the bioactive compounds present in marine microorganisms. The extreme conditions found in the marine environment has led to the development of a wider range of compounds in organisms  and there is high demand to exploit this, as there aren’t yet any available natural anti-UV sunscreens on the market.

To obtain the highest diversity of these compounds, scientists are looking for marine microbial resources from the most extreme sources, such as hydrothermal vents. Marine prokaryotes were collected at four vents in the Mid-Atlantic Ridge (MAR) to discover more about the diversity of functional taxonomic groups of free-living microorganisms, and their biotechnological potential.

New Marine Prokaryotes Isolates from MAR Vents:
289 marine prokaryotes were successfully isolated from the sites and grown in standardised culturing conditions, even though it was previously thought that complex culturing methods were needed for microorganisms, particularly in the deep sea (Alain & Querellou, 2009). These prokaryotes were categorised into three phenotypic operational groups (Groups I, II and III) depending on oxygen demand and temperature for optimum growth.

MAR Vents Prokaryotic Biodiversity
A polyphasic characterisation approach was applied in conjunction with a strain clustering strategy for the 246 marine prokaryotes isolated to investigate biodiversity, and used methods such as PCR fingerprinting, which involved whole-cell protein profiling. Shannon (J’) and Simpson (D’) biodiversity indices were also obtained for each of the phenotypic groups. The authors used two molecular techniques to obtain a more robust dataset, which led to 23 clusters of phonetically similar isolates being identified, and the observation that phenetic diversity decreases from groups I to III. A subset of isolates from the clusters were also identified by 16S rRNA gene sequencing, and the results was suggested that almost half of the collected samples were composed of new species.

MAR Vents Bacteria Extract Possess Photoprotective Capacity
A yeast-based assay was exposed to either UV-A or UV-C lethal radiation doses and  it was found that the yeast cells injected with extracts with a photoprotective capacity were able to proliferate and form colonies, but those without didn’t grow. Two extracts, MGMS241O2 and RBRS241O2 isolates, were also found to possess protective capacity under UV-B as well. Further gene sequencing identified MGMS241O2 as a new strain belonging to the species Shewanella algae, which is known to produce melanin for multiple roles, potentially for photoprotection. RBRS251O2 was also found to be a new strain that belonged to Vibrio fluvialis, although this work was the first to identify photoprotection in this species.

The attraction to the study of bioactive compounds in marine microorganisms is due to their already established photoprotective ability, and many industries are keen to exploit this trait and develop anti-UV natural sunscreen products. Considering the high commercial value of these microorganisms, the authors have managed to portray the importance of biological products in marine prokaryotes, which allow them to tolerate stressful conditions in extreme marine environments, by developing a novel and industrial-suited marine bacteria collection, as well as managing to grow these microorganisms under standard cultivation methods, which is controversial to previous work.

Martins, A., Tenreiro, T., Andrade, G., Gadanho, M., Chaves, S., Abrantes, M., Calado, P., Tenreiro, R., & Vieira, H. (2013) Photoprotective Bioactivity Present in a Unique Marine Bacteria Collection from Portuguese Deep Sea Hydrothermal Vents. Marine Drugs, 11, 1506-1523

Monday, 21 October 2013

Estimating atmospheric nitrogen fixed by cyanobacteria at the ecosystem scale.

It has long been known that diazotrophic cyanobacteria are capable of fixing atmospheric nitrogen into biomass for their own nutrition, conferring the advantage that they are free from dependence on dissolved organic/inorganic nitrogen (DON and DIN) supplies. It is also known that this process may contribute significantly to the amount of nitrogen available in an environment. This study aimed to quantify the transfer of diazotrophic nitrogen into an estuarine ecosystem during a summer bloom of Nodularia spumigena.

The relative contribution of diazotrophs to total nitrogen input has already been shown to vary greatly between aquatic systems, dependent on the trophic status of the ecosystem, as well as the seasonality, frequency and size of algal blooms. Studies in lake systems have produced estimates ranging from 6-82% of annual nitrogen input, whereas estuaries have been the focus of fewer studies, with estimates range from 3-39% of annual nitrogen input being fixed by diazotrophs. The conventional view is that diazotrophic cyanobacteria are less successful in estuarine systems due to increased grazing pressure from zooplankton and environmental stress.
The experimental method used in this study is promoted as a more cost-effective way of estimating the diazotrophic nitrogen contribution to an environment. The authors explain the expenses and limitations of the mesocosm-scale incubation assays and the 15N labelled N2 tracers used in past studies, and endorse their own in-situ techniques as an economically viable alternative. A comparison of stable nitrogen isotopes 15N and 14N in pre- and post-bloom samples, together with measurements of plankton abundance using chlorophyll fluorescence, among other techniques, were used to ultimately determine diazotroph contribution.
These techniques generated a conservative estimate of diazotrophic nitrogen flux of 146 tons, accounting for 177% of the total summer nitrogen-load. The authors acknowledge the difficulties of placing this result in the context of total annual nitrogen flux, yet by using data from a study the previous year, they estimated a total annual nitrogen yield of 695 tons, taking into account fluvial, atmospheric and diazotrophic sources. They therefore state that this ‘back of the envelope calculation’ suggests diazotrophic cyanobacteria are responsible for 22% of the annual total nitrogen load.
However, it had been shown in a previous study that N. spumigena are un-grazed in the estuary studied, so the results may offer limited inferences for other estuarine systems where cyanobacteria are more heavily grazed, as well as estuaries that experience less severe algal blooms. These limitations, along with any sources of potential methodological error are acknowledged by the authors.<>

Despite the differences between estuarine systems, the result of 22% contribution to total nitrogen load by diazotrophs sits directly central in the range of previous estimates. This study thus does not challenge the conventional view in its field, its contribution is rather in its suggestion that the experimental method used is the most cost effective technique for further research in the field.
Woodland, R. J., & Cook, P. L. (2013). Using stable isotope ratios to estimate atmospheric nitrogen fixed by cyanobacteria at the ecosystem-scale.Ecological Applications.