Small Cells, Big Trouble for Toxic Mercury Bioaccumulation

Many trace metals are utilised by microbes in metabolic reactions. Mercury is one of these elements. Mercury is used in demethylation reactions of substrates and results in the formation of methylmercury for which there are two forms, mono-methylmercury and di-methylmercury, both are toxic to humans. Methylmercury concentrations have been steadily increasing in coastal waters as increasing amounts of mercury are deposited into the sea either by polluted rivers or contamination from coal-fired power stations. This in turn leads to increased methylmercury formation by marine bacteria.

Higher marine methylmercury levels are a major concern because it biomagnifies in the marine food webs. Organisms consume microbes contaminated with methylmercury, which is easily absorbed but very difficult to excrete due to it being more soluble in lipids than water. The result is an increasing build up of methylmercury as it moves through the food web, from bacteria to piscivorous fish. If contaminated fish (tuna for example) are consumed by humans it can have a number of detrimental effects to health. The developing nervous systems of unborn babies are at particular risk.

Methylmercury levels in coastal regions have been well reported. However, a number of studies have concentrated on methylmercury concentration in the open oceans. Even though such areas are far away from potential mercury contamination sources, oceanic levels are also increasing. Heimburger et al (2010) conducted a 20 month study in the North-western Mediterranean, at a site called DYFAMED to investigate why this could be happening. This site was chosen because it is separated from coastal waters by the Ligurian current while being relatively close to the shore (~ 50 km). The hypothesis of this study was that methylmercury production in such an environment is seasonally dependent on the type of primary production. This was examined by measuring the methylmercury concentrations and the distribution of phytoplankton throughout the water column, reaching down to the sea floor (2350m).

Methylmercury concentrations showed a double peak of 0.22 pM and 0.82 pM at a depth of approximately 50m (euphotic zone) and 400m (aphotic zone) respectively. The upper euphotic zone (0-10m) showed the lowest concentrations (< 0.10 pM). The levels seasonally changed with the highest peaks in the autumn months. This correlated with blooms of nano- and picophytoplankton in the euphotic zone during oligotrophic conditions.

The authors concluded that the high methylmercury concentrations at 50m in the autumn months were caused by the larger surface to volume ratio of the nano- and picophytoplankton compared to larger photosynthetic microbes. This enables a higher rate of mercury intake and mercury methylation. The even higher concentrations in the aphotic zone was attributed to the sinking of POM from the nano- and picophytoplankton reaching the deeper water were hetrotrophic microbes feed upon them, starting the methylmercury accumulation. The small size of the nano- and picophytoplankton POM aggregates also sink more slowly and are more easily fed from, increasing activity in the microbial loop and further increasing biomagnification. Despite having less input of mercury (mainly atmospheric) than coastal areas, the oligotrophic conditions of the open ocean seem to favour mercury methylation as nano- and picophytoplankton are more common. Although it must be said that some of this is still just speculation.

I reviewed this article because it possibly has a link with my first blog from last year which talks about a shift in marine phytoplankton to smaller sized species as a result of global warming. This coupled with high amounts of atmospheric release of mercury from power stations, could increase the amount of methylmercury biomagnification in marine organisms caught for human consumption.

A Review of:
Heimburger L, Cossa D, Marty J, Migon C, Averty B, Dufour A and Ras J (2010) Methyl mercury distribution in relation to the presence of nano- and picophytoplankton in an oceanic water column (Ligurian Sea, North-western Mediterranean). Geochimica et Cosmochimica Acta. 74: 5549-5559.

Degrading crude oil in the marine environment

Oil spills are an increasing worry for marine ecosystems, especially after the recent BP spill in the Gulf of Mexico. It is known that many marine bacteria, such as Alcanivorax borkumensis, are able to biodegrade oil and other hydrocarbons and these bacteria are often used in clean-up missions. Although there are marine microorganisms that are capable of breaking down the hydrocarbons in crude oil some of the better known microbes used in bioremediation are terrestrial species and most studies have found them to degrade slowly in saline conditions, it is therefore important to investigate whether a micro-organism can still function adequately in high saline conditions before they are used in a marine environment. These organisms degrade more slowly in saline conditions for a number of reasons including: disruption to the cell membrane, the denaturing of proteins and a change in osmotic pressure.

                In this study the ability of Fusarium sp. F092 to degrade three types of crude oil, of two different concentrations, under saline conditions was tested. Agar plugs containing the fungus were added to the different treatments and the degradation of crude oil was determined at 15, 30 and 60 days. There was also a control in which no fungus was added to the treatment.

The compounds which make up crude oil are generally grouped into four fractions depending on their solubility (aliphatics, aromatics, asphaltenes and resins). As the aliphatic fraction is the largest component of fossil fuels the percentage of the crude oil which was composed of aliphatic hydrocarbons was also tested. The fractions of the crude oil were separated using n-hexane and then further separated using chromatography and gas chromatography using helium as the carrier gas.

                The aliphatic fraction of oil type-3 was found to be 75%, which was much higher than types-1 and 2 which was less than 31%. At the lower concentrations of crude oil type-3 was most efficiently degraded (98% of aliphatic fraction) and type-1 was least efficiently degraded (49%). Oil type-2 was about half way between the two (72%). At the higher concentration of crude oil the fungi degraded less than 40% of the aliphatic fraction. It is thought that this decrease in degradation may be due to an increase in toxicity or due to a nutrient or oxygen limitation. It was also found that Fusarium sp. F092 degraded short and long carbon compounds more efficiently than it did intermediate carbon compounds.

Reference: Hidayat, A., & Tachibana, S. (2012). Biodegradation of aliphatic hydrocarbon in three types of crude oil by Fusarium sp. F092 under stress with artificial sea water. Journal of environmental science and technology, 5(1), 64-73.

My old Nan...

 Sialic acids are a family of structurally distinct nine carbon amino sugars which are present in mucous regions of the human body. Previous studies have shown that many pathogenic bacteria can utilize these sialic acids in a number of ways. However there has been very little research on pathogens utilizing them as a carbon source. This study examines three genes needed for the catabolism of sialic acids (nanA, nanE and nanK) known as the Nan cluster. It examines the distribution and evolution of the Nan cluster among bacteria and determines whether bacteria containing the Nan cluster can utilize sialic acids as a sole carbon source. Although they looked at many bacteria I will mainly concentrate on the vibrios.

 To examine the distribution of the Nan cluster among bacteria DNA sequences were obtained from GenBank. Phylogenetic trees of NanA, NanE and NanK were constructed to examine its evolutionary history. They also examined the growth of four Vibrio spp. on minimal media supplemented with sialic acid.

 The Nan cluster was present in 46 of the approx. 1900 bacterial genomes examined. Only three Vibrio spp. contained the cluster (Vibrio cholerae, Vibrio vulnificus and Vibrio fisheri). The cluster was found in all tested strains of V.vulnificus and V.fisheri however was only found in pathogenic strains of V.cholerae. Phylogenetic analysis of nanA suggests that V.cholerae and V.vulnificus obtained the gene from commensal gut bacteria possibly through horizontal gene transfer and was very closely related to eukaryotes. Phylogenetic analysis of nanE and nanK suggests that V.cholerae and V.vulnificus evolved the genes separately from V.fisheri. In the growth experiment only and all vibrios containing the Nan cluster were able to grow on the sialic acid media.  

 The results strongly suggest that pathogenic vibrios have the ability to catabolise sialic acid and use it as a sole carbon source. It also gives tremendous insight into how marine bacteria have evolved to compete in the hostile human gut. I found it interesting that Vibrio parahaemolyticus cannot catabolise sialic acid yet V.fischeri can. It is however a bit annoying that the paper doesn’t mention all the vibrios tested, only the Nan positive species. The high correlation between the Nan cluster and pathogens suggests that genes needed for survival in the human body could be potential genetic marker for virulence.

Almagro-Moreno, S. and Boyd, E. (2009) Insights into the evolution of sialic acid catabolism among bacteria. BMC Evolutionary Biology. 9, 118.

Friendly, non - polar, biofilm inhibitors, derived form the symbionts of seagrasses.

Bintang Marhaeni, Ocky Karna Radjasa3, Miftahuddin Majid Khoeri, Agus Sabdono, Dietriech G. Bengen, Herawati Sudoyo (2011) Antifouling Activity of Bacterial Symbionts of Seagrasses against Marine Biofilm-Forming Bacteria Journal of Environmental Protection, 2, 1245-1249

Biofouling, as mentioned in many of the reviews is a major problem in the marine environment and is costly to marine industries. The formation of biofilms on the bottom of boats and other machinery has led to the production of chemicals such as TBT to help remove such fouling but these can create a more dangerous environmental problem. This review focusses on a journal that investigates a potential environmentally friendly solution using bacterial symbionts of seagrasses as antifoul. The report here focusses on seagrasses Thalassia hemprichii and En- halus acoroides after reports on the potential of other seagrasses to remove biofilm. Research into the bacteria associated with the seagrasses could, therefore, offer an alternative commercial anti – foulant.

Both the bacteria associated with the seagrass and the biofilm – forming bacteria were isolated, purified and identified in the lab using morphological features. Antifouling activity of the bacterial symbionts was determined using an overlay method. Each biofilm bacteria was mixed with soft agar medium which was then poured onto agar surfaces previously inoculated with bacterial symbionts. Also twenty microliters of each extract onto paper discs that had previously been placed on agar surfaces contained the biofilm – forming bacterium. Any antibacterial activity was described by inhibition around the bacterial colonies.

Biofilm – forming bacteria are a primary target for anti - fouling products as they provide important targets or cues for larval settlement and therefore the development of the large biofilms unwanted in the marine industry. Four bacterial symbionts (3 from E. acoroides and 1 Thalassia hemprichii) were able to inhibit the growth of the biofilm bacteria however crude extracts from said bacteria, here, were found to be much better at inhibiting the growth of biofilm - forming bacteria. Finding an environmentally friendly biofilm inhibitor is a large very popular area in marine microbiology at the moment, but there are some benefits to the endophtye and epiphytes of the seagrasses investigated in this paper. Firstly, as extracts were shown to inhibit growth more effectively it suggests that there is a diverse number of chemicals produced by the secondary metabolites of the seagrass’ symbionts. This could increase the viability of these symbionts as future anti - fouling products. Secondly, many of these extracts were found to be non – polar, therefore it is less likely they will be washed away with ease in a marine environment, another attractive feature. Furthermore, sustainable seagrass ecosystems provide the perfect habitat for growing such symbiont making the bacteria available for large scale commercial use.

The secondary metabolites mentioned have not yet been characterised or purified however this would be an obvious next step in the research here, enabling, possibly the production of a successful environmentally friendly biofilm inhibitor.

A Potential Super Star

Pollution is one of the most significant factors reducing biodiversity in marine ecosystems. Microbial bioremediation is a natural process which can be utilised to reduce pollution levels. The Persian Gulf sits on top of the largest oil reserve in the world it is being heavily exploited which having a detrimental effect to the local marine life. Water evaporation, drilling and oil extraction are all rapidly increasing pollution in this marine ecosystem. Heavy metals and poly aromatic hydrocarbons (PAHs) are the most important pollutants in marine environments because of their persistence they can remain in marine species tissues for long periods. This study is aims were to identify a species of bacteria that has the ability to absorb copper and degrade phenanthrene.

Samples were taken from contaminated sediment in the Persian Gulf. They isolated bacteria using an enrichment method and isolated 10 marine bacteria from these sediment samples. They determined their tolerance to harmful compounds by inoculating the growth medium with phenanthrene and copper and monitoring survival success. They found one of these bacteria could tolerate high concentrations of both these compounds. It was then identified morphologically using gram staining and through a range of biochemical tests such as oxidase-catalase test and showed that the bacterium was gram negative and belongs to Pseudomonas sp. Further studies were then conducted to assess the bacterium’s suitability as a tool for bioremediation.

To monitor the biodegradation capability of the strain 500µl of cell suspension was inoculated with 100ml of phenanthrene and the decreasing concentration of phenanthrene was detected by monitoring UV absorbance at 24hr intervals over 6 days. The bacterium showed no lag phase and immediately degraded the compound by utilising it as a carbon energy source. The strain reduced phenanthrene concentration by 96.52% in 120 hours. The specialised enzyme TpbA is suggested to be responsible for this rapid biodegradation.

To monitor the biosorption capability of the strain 1ml of cell suspension was inoculated with 100ml of copper solution and the absorption of copper was monitored by measuring the remaining copper concentrations after inoculation. The copper concentration was measured at 30 min intervals by the atomic absorption spectroscopy. The bacterium was capable of reducing the copper concentration by 70.3% in just 30 minutes. The negatively charged cell wall surface and the numerous absorption sites on the bacteria are the cause of the efficient absorption.

This strain of bacteria was shown to be a very promising tool which could be effectively used in water treatment. It is clear that this strain is highly efficient at degrading phenanthrene and absorbing copper by the rapid reduction of both compounds immediately after inoculation. The rapid growth rate and natural ability of the Pseudomonas species to adapt to environmental change will also contribute to this efficacy of the strain. Problems associated with the introduction of foreign bacteria into ecosystems could potentially be less problematic as the strain originates the region that it is being used to treat; The Persian Gulf. However there is still the potential for this bacterium to be introduced to foreign contaminated marine systems if it is intelligently applied.
A review of: Safahieh, A., Abyar, H., Roostan, Z., Zolgharnein, H., Mojoodi, F. (2012). Isolation and characterization of Pseudomonas resistant to heavy metals and poly aromatics hydrocarbons (PAHs) from Persian Gulf sediments. African Journal of Biotechnology: 11(19) 4418-4423

Bacterial symbionts of sea grasses: potential environmentally friendly anti-fouling agents

A review of: Marhaeni B, Radjasa OK, Khoeri MM, Sabdono A, Bengen DG, Sudoyo H (2011) Antifouling activity of bacterial symbionts of sea grasses against marine biofilm-forming bacteria. Journal of Environmental Protection, 2: 1245-1249.

Biofouling is a natural process that arises as a result of organism growth on water submerged surfaces, which can result in economic losses to marine industries. Bacteria are considered the primary and dominant colonizers, facilitating the attachment and development of fouling communities. The use of anti-fouling paints like tributyl-tin can disrupt the growth of biofilms, but long term use is a serious environmental concern to marine ecosystems. The European Union, International Marine Organization and the Maritime Environment Protection Committee have restricted the use of hazardous anti-fouling agents, thus the search and development of alternative and environmentally friendly anti-foulants has gained momentum in recent years.

Sea grasses are productive coastal ecosystems and are a rich source of secondary metabolites with possible ecologically important roles in preventing surface fouling. It has been reported previously that sea grasses Cymodocea serrulata and Syringodium isoetifolium are able to inhibit the growth of marine biofilm-forming bacteria, most likely as a consequence of the association between specific marine bacteria and sea grasses, which could provide an alternative to commercial metal-based antifouling coatings. Using antibacterial assays, the study conducted by Marhaeni et al (2011) aimed to investigate the potential of marine bacteria associated with sea grasses Thalassia hemprichii and Enhalus acoroides for controlling the growth of marine biofilm-forming bacteria.

Four bacterial symbionts of the two sea grasses were found to inhibit biofilm-forming bacteria ranging from one to six different species, with three isolates from E. acoroides and one isolate from T. hemprichii. The active bacterial isolates were closely related to the members of the genus Bacillus and Virgibacillus, with 98-99% gene homology. Several studies have also revealed broad spectrum inhibition of growth by Bacillus and Virgibacillus symbionts isolated from sea sponge Pseudoceratina purpurea and soft coral Sinularia sp. Moreover, the Virgibacillus isolate from Sinularia sp. was found to be effective at inhibiting the growth of multi-drug resistant strains of Staphylococcus aureus.

The study elucidates the potential of bacterial symbionts from sea grasses E. acoroides and T. hemprichii as an alternative source of environmentally friendly marine antifouling agents. The secondary metabolite(s) responsible for the desired biological activity remain uncharacterised and the progression from the described investigation would be to isolate and purify the active antifouling compounds and to test them on a wide range of biofilm producing marine organisms to determine specificity.

An eco-friendly way to prevent biofouling

Guezennec, J., Herry, J.M., Kouzayha, A., Bachere, E., Mittelman, M.W., Noelle, M., Fontaine, B., (2012), Exopolysaccharides from unusual marine environments inhibit early stages of biofouling, International Biodeterioration & Biodegradation, 66(1):1-7

Marine biofouling causes numerous problems for water-contacting structures. These detrimental effects are due to microbial biofilm formation and successional colonisation of macroorganisms. Prevention techniques rely on antimicrobial agents, which despite being successful, also show toxicity to non-target organisms and other non-toxic surface modification techniques have shown limited efficiency with some causing partial detachment of the biofilm, leading to a high risk of microbial-induced corrosion. It is therefore necessary to develop an effective and environmentally compatible technology to prevent such problems.

One approach is through the coating of surfaces with specific exopolysaccharides (polymers secreted by microorganisms), who’s physical and chemical nature could be important in the prevention of the attachment process, therefore inhibiting biofilm formation and thus biofouling. This study evaluates the antifouling potential of several exopolysaccharides (EPS) produced under laboratory conditions by several bacteria. They were tested for their ability to form a stable film in order to prevent biofilm formation on a glass plate which was immersed in natural flowing seawater containing samples of EPS.

The 5 EPS producers used were; Alteromonas, Pseudoalteromonas, Vibrio and Pyrococcus sp. Once the EPS samples were recovered through centrifugation and ultrafiltration, its composition, including sugars, uronic acids and non-carbohydrate substituents were determined using colometric methods and HPLC analysis. Antimicrobial activity against several bacteria and cellular toxicity was also analysed.

The samples contained uronic acids, sugars including mannose and glucose, along with acetate, lactate and pyruvate, which are of high relevance to the structure-function relationships. Despite several studies suggesting that EPS have antimicrobial effects against various bacteria, this study found no evidence of any antimicrobial activity or cytotoxicity,. This could be due to the different methodologies used, including culture conditions or the use of purified EPS rather than crude exopolymeric substances used in previous studies.

In the absence of any treatments, after 5 days of immersion, the glass surfaces were covered around 70% by a biomass mainly comprised of bacteria and few diatoms. However, after pre-conditioning the glass with the different EPS, bacterial colonisation did not exceed 20%, with less than 11% of the surface colonised with most of the samples. SEM showed that biopolymers remained on the surface after 72hours, suggesting the presence of a homogenous film.

The stability of the film is thought to be due to the chemical composition of EPS, with the possible high uronic acid content encouraging formation of the film on the glass. Similarly, sugars, charged polysaccharides and proteins are known to mediate mechanical stability of biofilms (Flemming and Wingender 2010). The author’s note that based on their preliminary data, it is difficult to correlate a particular EPS composition with the inhibition of bacterial attachment and biofilm formation, therefore further work should be done to deduce the key components, including molecular weight and spatial orientation leading to possible steric hindrance. Additionally, the presence of a polymeric film on the surface can induce changes in the hydrophobic/hydrophilic balance and interactions between bacterial cells and surfaces, suggesting microbial adhesion strongly depends of the hydrophobic–hydrophilic structure of interacting surfaces and should be further analysed.

Overall, there are clear advantages to using natural polymers, especially bacterial EPS for antifouling purposes through permanent coating. They prevent bacterial adhesion and subsequent biofilm formation and moreover, do not contain any toxic molecules which could affect the local ecosystem and are easily produced with simple cultivation. The specific mechanisms and compositions should be further explored.

Additional reference: Flemming, H.C., Wingender, J., (2010), The biofilm matrix, Nature Reviews Microbiology, 8:623-633