Sunday 15 April 2012

Monitoring sewage pollution with sea fans

A review of: Baker, D. M., Jorda’n-Dahlgren, E., Maldonado, M. A. and Harvell, C. D. 2010. Sea fan corals provide a stable isotope baseline for assessing sewage pollution in the Mexican Caribbean. Limnology and oceanography, 55(5), 2139-2149.

As populations continue to rise, especially on the coastlines, sewage pollution is becoming a greater threat to coastal marine systems. The input of contaminants, especially nutrients such as nitrogen and phosphate can greatly disturb the environment. Increases in primary productivity can smother critical species such as sea grasses and reef building corals, pathogens can cause disease especially in corals and there is a generally common result of lowered biodiversity. While monitoring of this kind of activity is important everywhere, it is especially important that it is studied and controlled in developing regions which are often dependent on their ecosystem’s health and wellbeing.

This study hypothesised that sewage-derived nitrogen inputs are detectable and more severe in developed areas along the Mesoamerican barrier reef of Mexico. To test their hypothesis in this area they compared the stable nitrogen isotope (δ15N) values from the common Caribbean sea fan, Gorgonia ventalina, collected from a developed and undeveloped are of the coastline. Akumal coast was selected as the developed site as there are a great number of residents and a huge influx of tourists. The shoreline of Mahahual was selected to be the undeveloped site since there were few residents and tourists and a sewage treatment infrastructure.

The isotopic ratio of 15N:14N is regarded as an effective and direct indicator of human nitrogen pollution. Enriched isotope values arise from the accumulation and degradation of human and animal wastes and are easily distinguishable from other sources. Perpendicular to the shore samples of sea fans were taken 1km from the shore. 2cm-squared fragments were cut that are
likely to represent the previous year of growth. Stable isotope analysis was performed on the samples. The prevalence of Enterococcus was sampled at sites adjacent to where the sea fans were sampled. Enterococcus assays were used to determine if the nitrogen isotope analysis values were correlated with sewage pollution. Positive results for this test would rule out the possibility of enrichment due to denitrification. Statistical analysis was then performed.

Results found that samples from the developed site sea fans were enriched in δ15N (as high as 7.7‰ near shore) and were ≈3.5‰ greater than sea fan samples from the undeveloped site. The δ15N values were also positively correlated with faecal Enterococcus counts from the seawater. This confirms that the enrichments are associated with sewage and not denitrification. This study suggests that data collected from the undeveloped site which is relatively pristine could now be used as an isotopic baseline for monitoring the Mesoamerican barrier reef at sites where increased development is planned or underway. Another interesting find of this study was that the highest Enterococci counts were found to be from a lagoon popular with bathing tourists. The counts were approximately 59CFU per 100mL. The presence of faecal Enterococci is well above US Environmental Protection Agency standards for recreational waters which is 35CPU per 100mL.

This method of stable isotope analysis in sea fans could be a promising tool for monitoring changes in the contribution of human nitrogen sources to nearby ecosystems, especially in developing regions where water quality monitoring programs are not established.

Not just for the Bathroom...


Marine sponges are well known for harbouring a wide variety of microbes which have been found to have a mixture of roles, including protection, pathogens and competitors.  Sponges are also the most prolific marine producers of novel compounds, many of which are of pharmaceutical and biotechnological importance. The structural similarity between the compounds found in sponges and compounds found in sponge microbiota suggest that they could in fact be of microbial origin. Sponge-associated microorganisms have recently received renewed attention, with much research focusing on the production of bioactive compounds. This research has found that bacteria belonging to the actinobacteria genera are the largest producers of these secondary metabolites.

The aim of this investigation is to analyse the compounds produced by bacteria from two species of sponge (Suberites carnosus – non-calcarious, and Leucosolenia sp. - calcarious) in an effort to produce novel antibacterials to combat drug resistant pathogens, which are becoming a big problem in the medical industry.

Samples of the sponge species were collected at a 15m depth from Lough Hayne, Co. Cork in November 2008. Bacteria from these samples were cultivated on agar over a period of two months, during which time they were incubated at 18oC and checked regularly for distinguishable colonies.  After the incubation period, the cultivated bacteria underwent deferred antagonism and well diffusion assays, PCR and phylogenetic analysis of the 16S rRNA gene.

For both sponge species, analysis of 16S rRNA sequences revealed that the dominant phylum of bacteria found was Protebacteria, and the most abundant in both cases was γ – Proteobacteria. The genetic identity of over 98% of the isolates found in both sponge species are already known to science. However, some isolates, especially from Leucosolenia sp. are not. A result like this is not unexpected, as calcarious sponges have been subject to little research before now. A wide range of the bacteria found in both sponge species displayed antimicrobial activity during analysis, though more of the isolates from the sponge species S. carnosus showed this than those from Leucosolenia sp. This can be explained by different genera and species isolated in both, as the dominant genera of bacteria found was different in each species of sponge (Pseudoalteromonas and Vibrio in Leucosolenia sp., and Pseudovibrio and Spongiobacter in S. carnosus).

The authors summarised that in both sponge species, a high level of anti-microbial activity was found. Leucosolenia sp was found to produce more effective antifungal compounds, whereas S. carnosus appeared to contain more microbes with antibacterial properties. The researchers did struggle to isolate the compounds responsible for this activity in many cases however, meaning that the paper lends itself easily to further research. Overall, the findings from this paper may prove to be useful in future, particularly to the medical industry.

A review of Flemer B., Kenedy J., Margassery L.M., Morrissey J.P., O’Gara F. and Dobson A.D.W. (2011) Diversity and Antimicrobial Activities of Microbes from Two Irish Marine Sponges, Suberites carnosus and Leucosolenia sp., Journal of Applied Microbiology, 112, 289-301

Nitrification in the OMZ of the Arabian Sea (OMG!)


A review of: Newell SE, Babbin AR, Jayakumar A, Ward BB (2011) Ammonia oxidation rates and nitrification in the Arabian Sea. Global Biogeochemical Cycles, 25: 1-10.

A major rate limiting nutrient in the marine environment is nitrogen and oxidation of ammonia derived from organic matter is postulated to support 12-32% of primary production globally. The involvement of ammonia oxidising archaea (AOA) from the phylum Crenarcheota in nitrogen recyling was a relatively recent discovery. It was previously thought that ammonia oxidation was restricted to groups of β and γ proteobacteria (AOB), however more recent studies reveal AOA numbers that are considerably higher than AOB. The rates of archaea and bacteria ammonia oxidation in the oceans have not been quantified separately and archaea metabolism in the mesopelagic zone is yet to be fully elucidated. The Arabian Sea oxygen minimum zone (OMZ) is the largest in the world and the nitrogen cycle within this body of water has been the focus of many recent studies. The aim of the investigation by Newell et al (2011) was to explore the factors that influence the rates of ammonia oxidation and the abundance of AOA and AOB on the surface of the Arabian Sea, in low oxygen regions and in the mesopelagic zone below the OMZ.

Specific primers to identify the gene that encodes the ammonia monooxygenase enzyme (amoA) were used to estimate abundance of AOA and AOB using qPCR. Bacteria and archaea amoA genes were detected at every sampling depth in three locations, revealing AOA to be 35 to 216 times more abundant than AOB. Ammonia oxidation rates ranged from undetectable to 21.1 ± 0.1 nmol l-1 d-1; with the highest measurement near the surface layer (40 to 80 m in depth). The rates of ammonia oxidation within the oxycline (95-136 m in depth) above the OMZ were between 1.5 ± 0.2 and 4.6 ± 0.4 nmol l-1 d-1, while a low average of 0.07 ± 0.05 nmol l-1 d-1 was detected  in the mesopelagic zone (900 to 1000 m in depth)directly below the OMZ.

AOA appear to be the dominant ammonia oxidisers in the Arabian Sea, significantly contributing to nitrite production within the primary nitrite maximum (PNM) zone above 100 m and may be responsible for 10-100% of nitrite supply required to maintain the PNM. The role of ammonia oxidising Crenarchaeota in the deep ocean remains unclear however amoA gene abundance of 105 copies ml-1 at around 1000 m indicates a large population that substantially impacts nitrogen and carbon cycling. AOA nitrification in the photic zone provides recycled nitrate for primary production and may be a significant source of nitrous oxide (a greenhouse gas) which warrants further investigation. The role of AOA in OMZs and the metabolic pathways that sustain growth in hypoxic and anoxic conditions also requires elucidation, along with microbial diversity (via 16S gene analysis) and community dynamics. Furthermore, the abundance of amoA genes in the mesopelagic zone suggests approximately 50% of microbial biomass could be autotrophic and nitrification may be the main process that utilises most of the ammonium produced from organic carbon fluxes.         

Bioactive potential of Seagrass bacteria against human bacterial pathogens

The discovery of drugs from natural sources is an increasing trend. Researchers are searching for more novel chemical entities, in the hope they will provide new leads for disease treatment. The marine environment is particularly interesting for researchers because of the harsh physical and chemical conditions experienced by many organisms; yet despite this and the biodiversity of the oceans far outweighing terrestrial biodiversity research in this area remains in its infancy.
In particular, marine plants are known to produce a large number of structurally diverse secondary metabolites. Seagrasses are the only angiosperms to successfully grow in sub-tidal and tidal conditions. Several species of Seagrass have obligate microbial populations within their roots, leaves and rhizomes. Some medicines and chemicals are already prepared from Seagrasses and their associates.
An even greater concern than bacteria which are resistant to a single antibiotic is bacteria which are resistant to multiple antibiotics. As antibiotic resistance has developed, researchers have developed alternative antibiotics and combination therapies. However, the constant overuse of antibiotics in humans and their livestock has led to many bacteria being resistant to many antibiotics. The problem of resistance demands renewed efforts to seek antibacterial agents effective against pathogenic bacteria.
Two species of Seagrass from the SE coast of India were sampled and returned to the lab under sterile conditions, for the isolation of epiphytic and endophytic heterotrophic bacteria. 32 strains of endo/epiphytic bacteria were tested for their antagonistic activities against 5 antibiotic resistant human pathogens, of these, 10 were found to be antagonistic against one or more human pathogens. The authors go on to discuss the minimum inhibitory concentrations (MIC) as well as minimum bacterial concentrations (MBC) for the 32 tested strains.
The outcome of this research is that the endophytic bacteria isolated from the Seagrasses showed maximum sensitivity against several of the human pathogens compared with the epiphytic bacteria. And also that the bioactive compounds from the endophytic bacteria show maximum sensitivity with MIC than the bioactive compounds from the epiphytic bacteria.
From the outcomes of this research steps have been taken to find out the reason for the maxium activity of endophytic bacteria from Seagrasses.

There were two main reasons I choose to review this paper 1) The use of natural sources in the fight against humans pathogens is something that I find interesting and 2) In another module I have studies Seagrasses and like to see an over-lap/relation between modules. That being said, I found this paper very hard to read. It appears to me that the authors first language is defiantly not English, the paper did not read smoothly and the sentences seemed very disjointed, this I feel should have been addressed further. There were also seemingly simple mistakes made, for example species names were not italicised – I think these should have been picked up on. Simple changes would have made for a better read.
Despite this, the results of the paper are encouraging and the above mentioned points shouldn’t distract from the outcome. I hope that more antibiotics which can be used against both human and animal pathogens are found, as the problem of antibiotic resistance does appear to be rising.

A review of:
Ravikumar, S., Thajuddin, N., Suganthi, P., Inbaneson, S. J. and Vinodkumar, T. (2010) Bioactive potential of seagraa bacteria against human bacterial pathogens. Journal of Environmental Biology. 31 387-389.

Biofilms lead the way

Biofilms are thought to stimulate the attachment of invertebrates and algae to marine surfaces. This is known as biofouling, which refers to the accumulation of organisms and biogenic structures on marine surfaces. In particular sessile organisms compose the fouling assemblage. Many organisms such as barnacles and tubeworms produce shells and other firm structures during growth, which allow for the attachment of other organisms. This results in a multilayered fouling community. A central issue to the build up of biofouling on ships is an increases fractional drag, biofouling covers oceanographic equipment, coats floating structures and promotes structural deterioration. Biofouling costs industry billions of dollars per year due to prevention costs, maintenance and additional fuel consumption. Biofouling begins with the formation of biofilms followed by the aggregation of other diatoms and other micro-organisms bound together by extracellular polymeric substances. Biofilms can form within hours of immersion and rapidly increase in density and structural complexity. Furthering this, algae and invertebrates and aggregate. Biofouling is thought to be facilitated by the detection of appropriate sub strata and adhesion of larvae. It is thought that chemical cues play a pivotal role in invertebrate settlement. Microbial biofilms produce chemical signals that attract settlement. Antifouling research has provided information about the structure and function of biofilms, in particular with reference to understanding the adhesion and settling of invertebrate influence by microbial films. Zardus et al.'s research investigates the influence of microbial films on the adhesion of newly settled invertebrate larvae, they compared the removal rate of settlers from glass surfaces with and without natural biofilm coatings after exposure to controlled forces of shear. Their research was carried out on four marine fouling organisms: polychaete worm Hydroides elegans, barnacle Balanus Amphitrite, bryozoan Bugula neritina and a tunicate Phallusia nigra.

Larvae of the four invertebrate species were obtained from field collected adults and cultures were maintained in the laboratory following standard protocols. Trials were carried out in a turbulent channel flow apparatus and replicates of treatments were tested on the different invertebrates.

Larvae settlement was much greater on biofilm glass than on clear glass surfaces for Hydroides elegans, same was the case for Balanus amphitrite. Biofilms also had a positive effect on the adhesion strength for some of the settlement stages. Therefore bioflims increase invertebrates shear strength.

Adhesion of larvae is facilitated by microbial biofilms providing a connection between biofouling and biofilms. The mechanisms that cause this relationship require further research. Invertebrates use viscoelastic gels that have an adhesive nature in the presence of biofilms and form complex interactions, the adhesive strength is dependent on the taxa involved. It is suggested that biofilms may also stimulate increases and decreases in the adhesive produced by invertebrates, which has consequences for its structural strength. The trend observed was that with age invertebrates showed stronger adhesive forces. This discovery that biofilms facilitate invertebrate adhesion requires further research. Investigations are called for into the physical mechanisms of the biofilm invertebrate connection, paying particular attention on how these adhesives are modulated.

It is understandable that biofouling is undesirable for ships, however if structures such as marine renewable energy devices attract biofouling, this may have trophic food chain benefits supporting local biodiversity. This may be a desirable outcome provided biofilms don't cause any structural corrosion.

References:

Zardus, J. D., Nedved, B. T., Huang, Y., Tran, C. & Hadfield, M. G. (2008). "Microbial biofilms facilitate adhesion in biofouling invertebrates." The Biological bulletin 214: 91-98

Sorry Gastroenteritis. Theres just no relationship between us. From Entrococci

The purpose of this study was to evaluate the risk of exposing bathers to human pathogens in sub tropical recreational marine waters with non-point source of sewage and other pollutants. The authors also examined possible relationships between microbial densities and random symptoms in human subjects by questioning bathers at random and following up with microbial monitoring.

A group of regular adult bathers were recruited and divided into two separate groups : bathers and non bathers. Bathers were required to spend 15 minutes in knee deep water across the beach and submerge their heads every 5 minutes. Water samples were also collected by the bathers which were assayed for enterococci by membrane filtration. The non bathers group were not allowed to enter the water and were restricted to sitting on a plastic seat in a covered roped off area distant from water and sand exposure for 15 minutes at a time. This was followed up 7 days later with a phone questionnaire to determine if any of the bathers and non bathers had any illnesses.

From the water sampling it was determined that all bathers were exposed to an average of 71 enterococci/100ml of water however there was a very large range between samples. Symptoms reported after a week included gastrointestinal, skin, eye and ear infections which would normally be associated with exposure to contaminated bathing waters. Bathers reported more gastrointestinal, respiratory and skin infections compared to the non-bathers group and also reported a significantly shorter onset time of illness.

This paper confirms that people bathing are more likely to acquire infections from subtropical recreational marine waters compared to those that do not bathe even when there is no source point.

It was also found that the skin illnesses reported were dose dependant, as expected, however there was no relationship between the degree of exposure to enterococci and severity of the respiratory and gastrointestinal infections with the causative agents unknown. This could have further implications as, currently, many U.S. authorities monitor water systems using gastrointestinal illnesses as an indicator to water quality. Therefore, if there is no direct correlation between dose and outcome of gastrointestinal illness, the advice given by these authorities may not be completely accurate.

Although this was the first paper of its kind, all the participants were previously healthy adults and does not represent children or anyone with a compromised immune system.

A review of: Fleisher, J.M., et.al.; 2010; The BEACHES Study: health effects and exposures from non-point source microbial contaminants in subtropical recreastional marine waters; Int. J. Epidemiol.; 39(5): pp1291-98.

Rhamnolipid, another application

I have spoken about the uses of rhamnolipids, a biosurfactant, before and this paper goes on to talk about their potential to disrupt biofilms from surfaces exposed to the marine environment, a simple but generally unexplored area. Specifically this paper focuses on the biofilm crated by Bacillus pumilus and the effect of rhamnolipid on its ability to adhere to steel. Many Bacillus species are resistant to harsh environmental conditions such as low nutrient availability and UV radiation and this hardiness also means they are able to resist anti- fouling biocides. Adhesion to steel and the production of sticky exopolysaccharides and organic acids by Bacillus biofilm leads to increased corrosion and thousands of dollars’ worth of damage. Primary colonizing bacteria are the first to adhere to such surfaces and so the killing and ideally the removal of such is an important problem in need of a solution, biosurfactants could act as that solution.

B. pumilus were allowed to adhere for 4 hours at 30°C. Different concentrations (0.05 – 100mM) of rhamnolipid were then added to the polystyrene microtitre plates and left for a further one hour. After staining the results were taken as a percentage of cell adhesion compared to control plates which were not treated with rhamnolipid.

Exopolymeric substances (EPS), which have been investigated in other blogs, are the basis for adhesion of cellular substances to surfaces underwater. They allow for the attachment of other species leading to a community of microbes ultimately creating a damaging biofilm. Rhamnolipids have been shown to have antimicrobial as well as surfactant properties against other bacteria such as B. subtilis and Staphylococcus epidermis at low minimum inhibitory concentrations (<1.6 mM MIC) however the growth of B. pumilis in this case was not inhibited until >1.6mM and therefore higher concentrations of rhamnolipid are needed compared to other Gram negative bacteria. In contrast to this, at low concentrations, rhamnolipid significantly inhibited the adhesion of 46 – 99% of B. pumilus, after one hour there was at least 80% inhibition of adhesion to the polystyrene surfaces which may be enough to prevent the formation of biofilm. Other tests with different bacteria also show the effectiveness of rhamnolipid as an anti – adhesive when it comes to biofilm.

EPS is thought to neutralize antimicrobial agents and therefore assist the microbial community form dangerous biofilm. Therefore the disruption of such EPS could stop such formation. At higher concentrations than the MIC value there was a significant dose – dependent increase in biofilm disruption after treatment with rhamnolipid. After one hour there was significant EPS disruption and 24hours treatment led to destruction of microcolonies. One problem with this report is the suggestion that rhamnolipid usage would be more successful with the help of biocides. In the marine environment the use of biocides in unattractive due to other environmental hazards this brings about. However, the treatment of rhamnolipids does have potential and should be investigated further.

Review of Dusane et al (2010) Rhamnolipid mediated disruption of marine Bacillus pumilus biofilms

Fugu no longer toxic!

A review of: Noguchi, T., Arakawa, O. and Takatani, T. 2006. Toxicity of pufferfish Takifugu rubripes cultured in netcages at sea or aquaria on land. Comparative Biochemistry and Physiology Part D: Genomics and Proteomics, 1(1), 153-157.

Marine pufferfish of the family Tetraodontidae can be known to possess a neurotoxin (TTX/Tetrodotoxin). This toxin is poisonous to humans, causing paralysis and even death. In Japan ‘Fugu’ is a popular dish which is made from these pufferfish. Many believe the liver to be the tastiest part, however this is also the most toxic. Safe preparation of this dish requires a great amount of training and the serving within restaurants is strictly controlled. The serving of the liver has even been banned due to a frequent occurrence of food poisonings. No detailed studies have been made on the mechanisms of uptake, accumulation, metabolism and excretion of TTX in pufferfish. While they do have a TTX-secreting gland or cells within their skin which is thought to be a defence mechanism, only a small amount if any is ever produced and this does not intoxicate the liver. Previous studies have suggested that the pufferfish do not synthesise the TTX by themselves. Instead they are thought to accumulate it through the food chain. So this study hypothesised that non-toxic pufferfish should be able to be produced if cultured with a TTX-free diet.

Over 5000 species of the pufferfish, Takifugu rubripes were cultured by netcages at sea, and aquaria on land for up to three years. As they are known to accumulate the toxin mainly in the liver and ovary following ingestion, the liver was focussed on for testing, but other parts were also used. Toxicity was assessed using the Japanese official mouse assay method for TTX and also LC/MS analysis. Each tissue was extracted with 0.1% acetic acid and then examined for toxicity through intraperitoneal injection into male mice.

The results indicated that all parts of all pufferfish tested were considered to be ‘non-toxic’ in both the mouse assay where levels were less than 2MU/g and the LC/MS analysis where levels were less than 0.1MU/g. The expression MU is used where 1 MU is defined as the amount of toxin required to kill a mouse in 30 minutes after injection. Anything less than 10MU/g is considered to be non-toxic in food hygiene. This provides solid evidence that the pufferfish become intoxicated through the food chain and non-toxic fish can be successfully produced by netcage or land culture irrespective of culture area and season. If cultured in this way, the fish and in particular the liver can be considered safe to consume.

Lava Eaters

The aim of this study was to assess the abundance, species richness and phylogenetic diversity of endolithic and epilithic microbial communities inhabiting young, unsedimented ocean crust at the sea floor. Hence, basaltic lavas of various ages and alteration states were sampled by the authors from the East Pacific Rise (EPR) and around Hawaii and then analyzed using quantitative PCR, FISH and microscopy. The PCR measurements of the glassy rinds of lava flows showed that the total bacterial and archaeal cell densities were ranging from 3x106 to 1x109 cells/g and that bacteria were dominating (88–96%) all the rock samples examined. This results were then confirmed by the FISH analysis, which revealed dense populations of Bacteria (significantly more abundant than Archaea) exhibiting cell abundances of 3-4 orders of magnitude greater than in the overlying deep sea waters (8x103 - 9x104 cells/ml) where half of the cells were instead, Archaea.

Subsequently, in order to evaluate in more detail the community composition, the authors used fulllength 16S ribosomal RNA gene clone libraries constructed from basaltic lavas and surrounding sea water samples. These phylogenetic analyses revealed that both the basalt-hosted biospheres (EPR and Hawaii), were harbouring high-richness bacterial communities and that community membership was shared between these sites. A statistical approach was then used to evaluate the species richness (number of operational taxonomic units) as compared to other oceanic environments analyzed in other studies (e.g. an hydrothermal white smoker, the upper water column of the Sargasso Sea, hydrothermal fluids from the Mid-Atlantic Ridge and deep-subsurface sediments from the Nankai Trough). These comparative analysis, revealed that abundance, phylogenetic diversity and richness of Bacteria in these other deep-sea environments were clearly lower and much different than EPR and Hawaii deep-sea basalts. The 21 taxonomic groups recovered from basalt were dominated by Proteobacteria (68% and 66% of all sequences in EPR and Hawaii respectively), while non-Proteobacteria groups included Plantomycetes (8%/5%), Actinobacteria (7%/8%), Bacteroidetes (4%/1%), Acidobacteria (3%/4%) and Verrucomicrobia (2%/2%). Interestingly, the OTU richness for the two geographically separated basalt communities (EPR and Hawaii) showed considerable overlap in community membership, suggesting that oceanic basalt microbes are widely distributed among this biotope.

These differences in phylogenetic diversity, species richness, and total biomass between the basaltic lavas and overlying sea water raised questions about what energy source fuel this biosphere. Potential energy sources capable of sustaining microbial life in ocean crust include hydrothermal input of manganese and iron (chemolithoautotrophic growth) and dissolved organic carbon in sea water or hydrothermal fluids (heterotrophic growth). However, according to the authors, the most plausible explanation is that oceanic lithosphere exposed at the sea floor undergoes seawater-rock alteration reactions and these reactions are capable of supplying sufficient energy for chemolithoautotrophic microbial growth. Lava surfaces in fact, are composed predominantly of volcanic glass, a highly reactive rock component that contains reduced elemental species such as iron, sulphur and manganese. Oxygen and nitrate in deep sea water oxidize these  constituents and chemolithoautotrophic microorganisms can potentially exploit the free energy changes associated with these redox reactions for their metabolic requirements. Laboratory studies have already demonstrated that iron-oxidizing bacteria isolated from the sea floor are able to use rock and minerals, including glassy basalt, for metabolism and growth. The authors estimated that about 6x107-6x109 cells per g basalt may be supported through these reactions and actually, cell densities in EPR basalts were falling exactly within this range. So in conclusion, alteration reactions in the upper ocean crust may fuel microbial ecosystems at the sea floor, which constitute a trophic base of the basalt biotope, with important implications for deep-sea carbon cycling and chemical exchange between basalt and sea water. This hypothesis supports the understanding of the phylogenetically rich and distinct nature of the basalt biotope. The enrichment of taxa from diverse metabolic groups may result from the establishment of chemical microenvironments within or on rock cavities and surfaces during alteration, mineral precipitation and biofilm formation. This niche creation would allow for a greater variety of redox reactions and metabolic pathways (e.g. heterotrophic, anaerobic, or reductive) including those supporting complex organotrophic and mixotrophic communities. 

Reference:
Santelli, C.M., B.N. Orcutt, E. Banning, W. Bach, C.L. Moyer, M.L. Sogin, H. Staudigel, and K.J. Edwards. (2008). Abundance and diversity of microbial life in ocean crust. Nature 453:653-656.  

New Species of Bacteria Isolated from the RMS Titanic

A review of: Sánchez-Porro, C., Kaur, B., Mann, H., and Ventosa, A. (2010) Halomonas titanicae sp. nov., a halophilic bacterium isolated from the RMS Titanic. International Journal of Systematic and Evolutionary Microbiology 60:2768-2774

A new species of bacterium isolated from the RMS titanic is described in this paper. The bacterium was isolated from the rusticles on the ship, bioconcretious structures that look like icicles but are formed from oxidised iron. Various microorganisms live within these structures, which are the by-product of the microorganisms effectively feeding off the metal of the ship.

In this study, the authors used phylogenetic analysis alongside other methods including obtaining 16s rRNA though PCR to perform a BLAST search in order to try and identify the strain. The results of these methods showed that the strain isolated was most closely related to the genus Halomonas. Its most closely related species included Halomonas neptunia (98.6% sequence similarity), Halomonas variabilis (98.4%), Halomonas boliviensis (98.3%) and Halomonas sulfidaeris (97.5%), amongst others confirming it belonged in the Halomonas genus. However, several important differences, including phenotypic and chemotaxonomic differences features that confirmed the strain was a separate and distinct species not previously identified. The paper goes on to describe the new bacterium in detail.

The genus halomonas is heterogeneous, containing more than 60 species to date and are a member of the salt-loving Halomonadaceae family which are able to grow at salinities of 5 - 10% and who are considered generally non-pathogenic aerobes. The new bacterium is also gram negative, flagellated and motile.The discovery of this new bacterium is of particular interest as it may contribute more to the understanding of the mechanisms of rusticles, how they form etc. It also can have wider implications in understanding how to protect other submerged metal structures such as oil pipeline, oil rigs and the disposal of ships at sea for example.

Additional reference: BBC. (06/12/2010). New species of bacteria found in Titanic 'rusticles'.Available: http://www.bbc.co.uk/news/science-environment-11926932.

Prebiotics help for soybean meal bases, do they work?

Prebiotics like probiotics are used in aquaculture to maintain the health of a species, however prebiotics unlike probiotics are a non-digestible food ingredient which can benefit the host by stimulating the growth and activity of bacteria residing in the colon, promoting a healthier host organism. This study investigates the effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by the Red Drum Sciaenops ocellatus (Linnaeus).

The authors used a recirculation system for the 35 sub adult Red Drum which were fed a control diet containing 40% crude protein, exclusively from menhaden fish meal, 0% lipid, and an estimated available energyof14.6 kJ g-1. Five experimental diets were to be similar to the control diet, but with approximately 50% of the protein supplied by menhaden fish meal and 50% provided by soybean meal. To four of the experimental diets, prebiotics were singularly added at 1% of dry weight in place of cellulose while the basal diet had no prebiotic supplementation. The prebiotics in use for the trial (a mix or individually) were mannanoligosaccharide (MOS), galactooligosaccharide (GOS) and inulin. The faecal matter was used for nutrient analysis.

The results showed that the basal diet mixed with MOS and GOS significantly increased protein (82% for the three tanks) and organic matter (69, 64 & 66% for the three tanks) in comparison to the control diet which showed 69% for protein and 49% for organic matter. However the lipid values were significantly decreased for fish fed with MOS, GOS and inulin (63, 61 & 61%) compared to the control of 77%. Energy values were the same for the fish fed with inulin as the control diet of 54%.

This is the first study to demonstrate that nutrient and energy digestibility of soybean-meal-based diets can be enhanced by prebiotics. The wider significance of this investigation is paramount to replacing a higher percentage of fishmeal in fish diets with soybean and or other protein replacements. The side effects such as gastroenteritis can be treated with probiotics and the protein enhancement can be treated with prebiotics, thus a mixture of both in diets would be the next step in further research.

A review of: Burr, G., Hume, M., Neill, W. H., & Gatlin III, D. M. (2008). Effects of prebiotics on nutrient digestibility of a soybean-meal-based diet by red drum Sciaenops ocellatus (Linnaeus). Aquaculture Research, 39, 1680-1686.

The occurrence of enteric viruses in shellfish

A review of: Suffredini, E., Corrain, C., Arcangeli, G., Fasolato, L., Manfrin, A., Rossetti, E., Biazzi, E., (2008), Occurrence of enteric viruses in shellfish and relation to climatic-environmental factors, Applied Microbiology, 47(5):467-474

Shellfish are considered to be potential vectors of foodborne diseases, due to their accumulation of pathogenic microorganisms through filter-feeding. Norovirus (NoV) is an enteric virus that has been linked to shellfish-associated disease outbreaks and is responsible for 60-80% of human gastroenteritis outbreaks. Similarly, shellfish are linked to the transmission of hepatitis A virus (HAV), and its consumption has been reported in 69% of HAV infected patients. In Italy, there have been numerous cases of these diseases related to seafood consumption registered in several cities.

It has been established that climate has direct and indirect effects on the occurrence of enteric viruses. For example, high precipitation can cause flooding and sewage runoff which are key factors in contamination of coastal water and shellfish harvesting areas.

The aim of this study was to assess the prevalence of HAV and NoV in shellfish harvested in a specific national production area, the deltic area of the river Po (North Italy). They also looked at the effect that environmental factors have on viral contamination in the production area.

A survey was carried out for 1 year on samples of shellfish (mussels and clams), taken every 15days from two areas, A (sea area) and B (lagoon area). Environmental parameters such as temperature, pH and salinity were noted as well as bacteriological analysis (for E.coli and Salmonella) and virological analysis (for NoV and HAV), using various PCR methods.

The results showed no significant differences in environmental paramaters in the two areas, with some predictable fluctuations due to seasonal variations. No Salmonella was detected and E.coli numbers were always below legislation limits. There was also no HAV found in both areas but NoV was detected in 10 of the 120 samples, all from area B. NoV was also present in samples ranging throughout the sampling period, with the majority of positive samples in spring and summer. This led the researchers to conclude that a definite association between NoV clinical cases arising during the winter period and shellfish cannot be established.

They also noted that the increase in viral contamination could be in relation to the flow of the tributary river, which could transport further viruses into the harvesting areas and moreover mixes and lifts the sediment at the bottom of the lagoon where viruses can deposit and survive for long periods.

The study highlights the importance of accurate classification of harvesting areas in assuring the safety of shellfish for direct consumption and is useful in helping to establish suitable prevention techniques, especially after meteorological events.

Novel adenovirus isolated from sea lions

Viruses of the family Adenoviridae have genomes consisting of double stranded DNA. They infect various species of vertebrates, including humans. Adenoviruses were first isolated in 1953 from human adenoids. Two types of canine adenoviruses are well known, type 1 and 2. Type 1 causes infectious canine hepatitis, a potentially serious disease involving vasculitis and hepatitis. Type 1 infection can also cause respiratory and eye infections. Canine adenovirus 2 (CAdV-2) is one of the potential causes of kennel cough. Viral hepatitis associated with adenoviral infection has been previously seen in free-ranging California sea lions Zalophus californianus. However previous isolation of viruses were unsuccessful and identification of this virus was ceased and no specific virus was documented. However because the morphological features seen under the microscope were quite similar to that of canine infectious hepatitis and since the virus has a wide host range, it was thought that perhaps the virus responsible for disease was CAdV-1.

The presence of adenoviral DNA was examined in tissue samples from 2 live stranded California sea lions that were admitted to the rehabilitation facility at Californian Marine Mammal Centre. The two rescued animals died with serious symptoms , first was diagnosed with arteritis , pneumonia and pulmonary haemorrhage. Also eosinophilic intranuclear inclusion bodies (sign of adenoviral infection) were discovered in few organs including lymph nodule and lungs. The other case died from severe viral hepatitis with intranuclear eosinophilic inclusions found within hepatocytes.

Tissues from both animals were first examined under electron microscope and then analysed using PCR with the use of specific primers (sense: 5’-GCG CAC TTA CTC ATC CAT TTC C-3’, antisense: 5’-GCT ATT TCT CCA CGC AGC GG-3’). The virus was isololated and compared with known adenoviruses.

The examination of lymph node from first animal and liver of the second one revealed the presence of adenoviral-like particles (70 to90 nm icosahedral) , within the nucleus of affected endothelial cells .The sera from both animals were negative for antibodies against both CAdV-1 and 2. The PCR to detect CAdV-1 and 2 was also negative. Sequencing confirmed the presence of a fragment of the DNA polymerase gene of a novel adenovirus and comparison of the sequence to known adenoviruses in GenBank showed that this was a novel virus from the Mastadenovirus genus. This virus was similar in 77% to tree shrew adenovirus 1 (TSAdV-1) CAdV-1 (72%) and 2 (74%) however in overall is was treated as independent lineage and species.

This was very interesting study and quite exciting that they managed to find new virus , although the more analysis is needed in order to establish if this virus is a primary pathogen causing death. It is quite surprising that although the symptoms of this disease were quite similar to those reported in many other cases , nobody actually managed to identify this virus before.

A review of Goldstein et al.2011 . Isolation of a novel adenovirus from California sea lions Zalophus californianus. Dis Aquat Org. 94: 243–248.

Phytoplankton Numbers Plummet due to Global Warming

A review of: Boyce, D. G., Lewis, M. R., and Worm, B. (2010) Global phytoplankton decline over the past century. Nature 466: 591-596

Recent research has suggested that phytoplankton numbers are plummeting. Marine phytoplankton are important marine organisms, strongly influential in major climatic process and biogeochemical cycles as well as being responsible for over half of global primary production. A decline in phytoplankton numbers does not just threaten ocean processes and diversity. Three billion people depend on seafood as part of their diet and nearly one tenth of the world’s population depend on the fisheries industry.

Perhaps the most obvious suggestion for this decline can be found in global warming. More than 90% of the heat retained by the Earth due to greenhouse gases end up in the sea. This causes surface waters to warm and as they do so, become less dense, preventing colder nutrient-filled waters to rise to the surface. This, in turn prevents the waters mixing and so, the phytoplankton in the warmer waters run out of nutrients required for growth. In this article, the authors compile and analyse in-situ chlorophyll (Chl) and ocean transparency measurements collected over the last century, to investigate the changes in phytoplankton biomass and to see whether this is in fact happening.

The authors found that in 59% of the data used, there was a decline in phytoplankton numbers. It was also observed that Chl declined more rapidly, the further the distance from land. They suggest this may be due to an increasing intensity of vertical stratification and ocean warming. The global oceans were then split into ten regions in which to evaluate regional trends and found that there was phytoplankton decline in 8 of the 10 regions. The largest declines were found in the South and equatorial Atlantic regions.

All in all, the analysis suggested that global Chl concentration and therefore phytoplankton biomass had declined over the last century, and has declined by almost 40% since the 1940s. Evidence from the analysis related primarily to climate variation, particularly to rising sea surface temperatures. These results are therefore consistent with the hypothesis that increasing ocean warming is changing the marine ecosystem with implications for both biogeochemical cycling and population numbers. The authors hope that this study provides an incentive for greater observation and remedial action.

Additional reference: Holmes, B. (2012) Too-blue oceans: The invisible famine. New Scientist 2859

Plenty of extracellular DNA makes your community stronger

Biofilms are formed by aggregates of bacteria and are held together by extracellular polymeric substances (which consists of a mixture of substrates of bacterially produced polymers), one of these constituents is extracellular DNA (eDNA), it's role is not clearly understood. The advantages of biofilms are thought to be: protection against desiccation, mechanical sheer strength and chemical toxins. The composition of EPS is varied and is dependent on the species present in the biofilm and the growth conditions. Generally speaking it mainly consists of three compounds: polysaccharides, proteins and extracellular DNA. Recent research indicated eDNA is frequently a component of many biofilms. Documented species that produce eDNA are: Pseudomonas aeruginosa, Neisseria gonorrhoeae, Staphyloccocus epidermidis, Shewanella sp., Acinetobacter calcoaceticus and Bacillus subtilus. eDNA is present in both natural and synthetic environments and comprises of up to 70% of total DNA pool in a biofilm and typically ranges from 2 mg g-1 to 300 mg g-1. It is thought to originate from either cell lysis or be secreted from live cells. The production of eDNA in P.aeruginosa have been linked to quorum sensing. Research indicates eDNA has several roles: a nutrient source during low nutrient conditions, phosphorus cycling in sea sediments, horizontal gene transfer, and structural strength. The abundance and importance of eDNA in bacterial complexes is not well documented, the authors attribute this to a lack of stable methods for quantitative in situ studies. Methods that are used are qPCR, spectrophotometric detection, DAPI, SYTO, DDAO and PI stains. The aim of Dominiek et al.'s research was to further develop in situ techniques for quantitative analysis of eDNA in mixed biofilms and activated sludge flocks. Their technique was simplistic and combined refining staining methods with in situ hybridisation (fish) for the identification of microbes.

Their staining results indicate that most of the eDNA was found around living cells, more specifically there was increased abundance around microcolonies of certain species of bacteria, which suggests there is variation in the production of eDNA among species. This supports the hypothesis that eDNA originates from live cells, however, it is also suggested that it may provide evidence that sub microcolonies are releasing eDNA during lysis. Confocal FISH images of extracellular floc before and after a 60 minute DNase 1 incubation, showed there was a loss in eDNA as a result of its degradation. Their quantitative results indicate eDNA ranges from 50 to 300 mg g-1. Fish results reveal that some species in particular produce high levels of floc eDNA: Curvibacter, Thauera, Nitrospira, Accamulibacter and Campetibacter. These were found in three Danish waste water treatments. Results also demonstrated that eDNA provided structural integrity for the biofilms.

The main discussion points are: 1. eDNA is an important component of ESP. 2. The different contents of eDNA in activated sludge in different tested treatment plants is most likely due to different microbial populations. 3. Most eDNA was found around living cells, suggesting active production of this polymer. 4. DNase 1 had substantial defloculating effects on the entire biofilm floc and also indicated low levels of eDNA impacted the EPS matrix and had consequences to floc strength. 5. Structural biofilm strength resulted in better performance in pressure dewatering conditions and gravity drainage, finally 6. The species composition influences the properties of the EPS and thus its function.

The methods they developed to quantify the abundance of eDNA proved effective. New evidence was gathered about particular bacteria that are strong floc producers. This research presents further detailed insight into the structure of constituent parts of biofilms. What I found surprising is that DNA holds a key structural role, I would have thought that cheaper to produce macromolecules would have been used. This leads me to postulate that eDNA plays other roles within biofilms e.g. transformation and nutrient cycling and cellular communication.

Dominiak, D. M., Nielsen, J. L., Nielsen, Per H., (2011) Extracellular DNA is abundant and important for microcolony strength in mixed microbial biofilms. Environmental Microbiology 13(3), 710-721

Oil Bioremediation

A review of: Lü, J. C., Li, Z. T., Hussain, K., and Yang, G. K. (2011) Bioremediation: The New Directions of Oil Spill Cleanup. Middle-East Journal of Scientific Research 7(5): 738-740

Oil pollution is an increasing concern, particularly with the ever increasing demand. With this increasing level of pollution, an effective solution for the environment is required; bioremediation is one such effective method. This paper looks at current knowledge in bioremediation including its techniques, advantages and disadvantages, particularly as a pollution control mechanism.

Bioremediation is the breakdown of dangerous or hazardous chemical and contaminants using microorganisms to break down and detoxify by transforming them into less harmful substances. This can take advantage of a naturally occurring metabolic pathway or through the genetic modification of the microorganism. Bioremediation can be used both in marine and terrestrial pollution and can take place in-situ (treated at the site of contamination) or ex-situ (the contaminated material is removed for treatment elsewhere). For marine environments, in-situ treatment is the only really viable option in most circumstances. Bioremediation can also be done both aerobically (through the addition of oxygen either by injection or the addition of nutrients) and anaerobically. Techniques outlined in this paper include; bioangmentation (involves the addition of microorganisms that can degrade a particular contaminant, biostimulation (the addition of nutrients to increase the level of activity) and bioventing (the addition of oxygen).

The advantages of bioremediation generally outweigh the disadvantages. The authors state that bioremediation is generally more economical than using other methods of treatment since it can be performed in situ and, in marine environments, there are plenty of microorganisms present for bioremediation to occur. It also has the advantage of being a natural process and therefore is expected to have minimal environmental impact. However, bioremediation may not be an instantaneous process, mostly it isn’t. It can take weeks or months depending on the amount of pollution and additional nutrients and/or microorganisms may be required for this method to be effective. As well as this, some contaminants may not be biodegradable, limiting the practical uses of this method.

Bioremediation is therefore an effective, low cost, naturally occurring method that can be of particular use in certain circumstances of pollution, such as in the marine environment. However, its scope of use is limited and so can only really be used for certain types of contaminants, such as oil pollution. The authors mention the use of microorganisms to degrade metals and research on this would certainly be valuable to increase the usefulness of this method and to help introduce methods of pollution control that are ‘environmentally friendly’.

Saturday 14 April 2012

Marine isoprene-degrading bacteria

A review of: Alvarez, L.A., Exton, D.A., Timmis, K.N., Suggett, D.J. & McGenity, T.J., (2009), Characterisation of marine isoprene-degrading communities, Environmental Biology, 11(12): 3280-3291

Isoprene is the second most abundant natural hydrocarbon in the atmosphere. It is volatile and can result in the formation of tropospheric ozone, the third most powerful greenhouse gas. Marine algae are known as a significant source of isoprene, whilst some freshwater bacteria are able to degrade the compound. Despite its high energy content, only hints of isoprene degradation in marine and coastal environments have been observed. Significant proportions of gases like methane are oxidised before they can enter the atmosphere and models of marine isoprene flux assume a bacterial sink, however clear evidence is still lacking. This study aims to determine the extent of isoprene degradation in marine environments, whilst identifying the organisms responsible and their relationship with the microalgae.

All samples were taken from various points in an estuary. Isoprene-degrading bacteria obtained from the samples were cultured on agar in isoprene-saturated atmosphere and analysed by DGGE and pyrosequencing of the 16s rRNA genes. Some were also incubated with phytoplankton species to analyse their relationship.

They found that Isoprene concentrations decreased from the head of the estuary to the mouth. They noted that the higher nutrient levels at the head of the estuary elevated microalgal production in the water column which may have led to the higher levels. Isoprene added to the samples of the marine and estuarine water and sediment slurry was significantly degraded, with degradation at least an order of magnitude faster in sediments then water samples and the onset and rate of biodegradation being more rapid at the head of the estuary then at the mouth. Also interestingly, biodegradation was more than 10-fold faster in all samples when isoprene was added at 0.001% than when added at 0.1% v/v, suggesting that degradation is favoured at lower concentrations. It could also be that the high concentrations may have killed the degraders, as isoprene and its degradation products can be toxic.

The degrading bacteria were mainly Actinobacteria, such as Rhodococcus, which were found in all samples and seem to be the main organism responsible for isoprene degradation in coastal samples. Bacteroidetes and Psuedomonas were also present, which were not previously known as isoprene-degraders. Studies generally suggest that Bacteriodetes benefit from the extracellular polymeric substances produced by algae, however this study explains that isoprene is also an important algal product that supports bacterial growth.

They also found that isolated isoprene-degrading bacteria are nutritionally versatile and most degrade n-alkanes for carbon and energy. They explain that isoprene-degrading capacity is widespread in diverse phyla, questioning whether such microbes are specialist isoprene degraders or generalists. In contrast to specialist methane-oxidizing microbes and many coastal microorganisms, these degraders opportunistically utilize a wide range of compounds from the dissolved organic carbon pool. Suggesting that, in the absence of spilled petroleum hydrocarbons, algal production of isoprene could maintain viable populations of hydrocarbon-degrading microbes.

This study confirms that Isoprene-producing algae support the growth of a mixture of isolates which utilise the carbon source and act as marine isoprene-sinks, overall providing a clearer picture of the relationship between consuming bacteria and producing algae and how this affects the isoprene flux and the proportion emitted into the atmosphere.

Silver nanoparticles as a cause of oxidative stress in microalgae


            Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials in products across industries. They are often used for their antimicrobial activity in medicine and are also often found in detergents. Unfortunately these nanoparticles are highly mobile and easily transported into aquatic systems and although AgNPs are beneficial to us, in the products previously discussed, they have been shown to have a negative impact on marine ecosystems, the extent of which is not yet known. As the nanoparticles have such a large surface area to volume ratio it is thought that AgNP react strongly with compartments within and outside of the cell which may cause problems such as an increase in free radical production, causing oxidative stress, which may fatally damage the cells

            This paper investigates the detrimental effects of AgNPs on two species of microalgae; Chlorella vulgaris, a freshwater species, and Dunaliella tertiolecta, a marine species. The two species of algae were exposed to varying concentrations of AgNPs (0 mg/l, 0.01 mg/l, 0.1 mg/l, 1 mg/l and 10 mg/l) for 24 hours. They investigated the damage done to the different algae and different exposures using a number of methods. Any morphological changes between the different exposures were recorded. As well as this the total chlorophyll, amount of viable cells, amount of reactive oxygen species (ROS) formation and amount of lipid peroxidation were measured.

            It was found that both algal species showed cell aggregate formation (at 0.1 mg/l AgNPs) compared to the control (o mg/l) and it was also found that at 10 mg/l AgNPs C. vulgaris formed large aggregates. It was also found that the total chlorophyll count decreased with increasing concentrations of AgNPs in both species of algae. The total chlorophyll in D. tertiolecta was 75% (of the total chlorophyll in the control) at 10 mg/l in comparison to 50% in C. vulgaris. The number of viable cells also decreased with increasing AgNP concentration and there was a drastic decrease in viable cells at 10 mg/l with 4% (of the viable cells in the control) in D. tertiolecta and a 12% in C. vulgaris. The amount of lipid peroxidation and ROS levels (both per viable cells) increased with increasing concentration. It was also found that the amount of lipid peroxidation and ROS levels in D. tertiolecta were much higher than those in C. vulgaris.

            The results of this paper clearly indicate that both of the species of algae are badly affected by the presence of AgNPs at concentrations as low as 0.01 mg/l and that at 10 mg/l the production of ROS increases dramatically.  D. tertiolecta, the marine species, is more severely affected by AgNPs, It is however thought that this may be because more chloride is present in the seawater growth medium, in comparison to the freshwater growth medium, and that this chloride augments the toxic effects of silver. As algae play a vital role in oxygen production and are involved in the food chain the presence of AgNPs may have a huge effect on the wider aquatic community and it is therefore clear that something needs to be done to reduce the amount of AgNPs being leaked into the environment.


Reference: Oukarroum, A., Bras, S., Perreault, F., & Popovic, R. (2012). Inhibitory effects of silver nanoparticles in two green algae, Chlorella vulgaris and Dunaliella tertiolecta. Ecotoxicology and Environmental Safety, 78, 80-85.