Bacterially speaking: The application of quorum sensing inhibitors in marine biofouling

In the marine environment, all natural and artificial substrata are quickly colonised by micro- and macro- organisms, a process called biofouling. This can be particularly troublesome for ships, increasing drag, promoting metal corrosion and reducing transfer efficiency of heat exchangers. In 1987, mussel biofouling alone cost the US Navy more than 200 million dollars in the areas of hull scraping and excess fuel consumption from unnecessary drag on large vessels Morgan (1990). In the past, toxic coatings such as tributyltin (TBT) have been used to prevent biofouling, however, such coatings are toxic, causing significant harm to marine life. In light of this, much research has been put in to alternative, biologically friendly methods.

Dobretsov et al. (2011) tested 78 natural products for their ability to inhibit quorum sensing (QS), using a reporter, without causing toxicity. 24% of the chemicals tested inhibited QS without any measurable signs of toxicity, from these 24%, the most potent and abundant were further investigated using a luminescent LuxR-based reporter E. coli pSB1075. The cognate AHL was added in the presence of these chemicals and any reduction in luminescence was noted, results indicated that demoxy-encecalin, hymenialdisin at concentrations > 6.6 μM and 15 μM, respectively. Furthermore, luminescence was completely inhibited by Hymenialdisin and demoxy-encecalin, microlins A and B and kojic acid at concentrations of >0.2 μM, 2.2 μM, 1.5 μM, 15 μM and 36 μM respectively.

The ability to prevent microfouling by one compound, kojic acid at concentrations of 330 μM and 1 mM, was tested in controlled mesocosm experiments. Results showed that bacterial and diatom communities, on glass slides, where decreased in density when comparison with controls lacking kojic acid.

An earlier study by Dobretsov et al. (2007) also showed that using QS blockers can also control larval settlement indirectly by regulating the microbial community structure of biofilms and the density of bacteria, which in turn affects larval behaviour.

These two studies show that QS blockers have potential as biologically friendly anti-fouling compounds, through either the direct inhibition of QS, or indirectly, by altering laval settlement behaviour. I chose to review this paper because it recognises another application for QS inhibition, also known as quorum quenching (QQ). To me many scientists seem blinded by the possible application of QQ as a novel treatment for pathogenic bacteria, and forget that QS controls many different phenotypes, not just the presence of virulence factors.

Furthermore, if anyone else has found studies discussing alternative applications for QQ compounds, I would be very much interested.

Both studies can be found below:

1.           Dobretsov S, Dahms H-U, Yili H, Wahl M, Qian P-Y. 2007. The effect of quorum-sensing blockers on the formation of marine microbial communities and larval attachment. FEMS microbiology ecology 60:177–88.

2.           Dobretsov S, Teplitski M, Bayer M, Gunasekera S, Proksch P, Paul VJ. 2011. Inhibition of marine biofouling by bacterial quorum sensing inhibitors. Biofouling 27:893–905.

Morgan D. 1990. Two firms race to derive profits from mussels glue: despite gaps in their knowledge of how the mollusk produces the adhesive, scientists hope to recreate it. Scientist 4, 1

Mesocosm study of indigenous oil-degrading microbial consortia

The Deepwater Horizon oil spill in 2010 showed that there is still an urgent demand for the development and optimisation of offshore bioremediation techniques. The biggest challenge that scientists face is to develop a bioremediation technique with a universal applicability in different geographical locations. Gertler et al. (2012) aimed to determine the response of indigenous microbial consortia, from different geographical locations, to a simulated oil spill. They hypothesised that because of global exchanges of sea water and the relative stable properties of the seawater system, comparable oil-degrading microbial communities would be present at all locations despite distinct site characteristics. In fact, some species of obligate hydrocarbon-degrading bacteria (OHCB) have been linked to hydrocarbon producing microalgae, providing a natural niche for OHCB, which may explain the global distribution of these microorganisms. 

A standardised mesocosm experimental design was used with two different treatments, filtered and unfiltered sea water, in order to investigate the effect of reduced grazing. They used sea water samples from the Irish (Menai Bridge), North (Helgoland) ad Mediterranean (Messina) Seas, all differing in meteorological and hydrological parameters as well as in nutrient availability (N/P ratio). Fifteen millilitres of crude oil and some slow-release fertiliser were added to each system. Changes in microbial community composition were assessed by ARISA (Automated Ribosomal Intergenic Spacer Analysis) and DGGE fingerprinting and 16 S rRNA gene library analysis over a period of 50 days (30 for Helgoland mesocosms). 

Gertler et al. (2012) found that specific key stone species forming a central and integral part of the oil-degrading consortia were found at all sites and were related with Alcanivorax (=alcane eater) borkumensis. This finding is in accordance with previous studies that showed the overwhelming prevalence of microorganisms from the genus Alcanivorax in oil-degrading microbial consortia in temperate zones. The effective primary colonisation of oil/water interfaces by Alcanivorax in temperate zones is thought to be due to multiple alkane hydroxylases, strong mineral nutrient scavenging, biosurfactant production and biofilm formation capabilities. Apart from this common set of bacteria, 58 of 64 OTUs detected in the clone libraries were site specific, highlighting the complexity of microbial ecology in marine oil degradation. These site-specific microbial consortia were detected at early and late stages of the experiment, yet fingerprint patterns of all three locations showed intense convergence during oil degradation, consisting mainly of Alcanivorax. 

The authors highlight the complexity of indigenous marine oil-degrading microbial communities, questioning the necessity of bioaugmentation practises. Instead they propose that biostimulation of these readily available, locally adapted OHCB (by stimulating the growth of Alcanivorax without hindering the development of location-specific consortia) would be more beneficial compared with less cost-efficient bioaugmentation approaches. 

I think this study illustrates well how wide-spread OHCB are in the marine environment and that stimulating, rather than augmenting, these populations may help in the bioremediation of oil-polluted waters. Although similar studies have been carried out at each geographical location, lack of standardisation in methodology hindered direct comparison, Gertler et al. managed to provide a better insight in the community structures of different geographical locations, vital to develop universally applicable bioremediation techniques, designed to suit microbial consortia independently of both location and oil type. Unfortunately the filtration of sea water had no effect on the occurrence of marine protozoa (only larger protozoa were excluded), so future studies should modify their methodology to improve removal of grazers from the water samples. 

Gertler, C., Näther, D. J., Cappello, S., Gerdts, G., Quilliam, R. S., Yakimov, M. M. & Golyshin, P. N. 2012. Composition and dynamics of biostimulated indigenous oil-degrading microbial consortia from the Irish, North and Mediterranean Seas: a mesocosm study. FEMS microbiology ecology, 81, 520–36. Available at: http://www.ncbi.nlm.nih.gov/pubmed/22462472

Phenol toxicity in the tropics

Phenol, a compound that is toxic to a wide range of organisms including humans and microbes, has become a more widespread pollutant in Malaysia with the concentration of phenolic compounds exceeding the limit set down by The National Guidelines for Raw Drinking Water Quality (0.002mg L^-1). The authors of this paper have outlined the need to isolate microbes that can be used for degrading the phenol within the water for bioremediation purposes. This paper has been able to isolate a clade of phenol degrading microbes known as Rhodococcus sp. from soil samples that, when tested, registered growth of the microbe on phenol of concentrations up to 2000mg L^-1. It was found that the supernatant of the culture contained 2-hydroxymuconate semialdehyde which is the meta-degradation of phenol. This seems to indicate that the Rhodococcus both excretes this compound extracellularly and then absorbs the resulting carbon based compounds, or that it absorbs the phenol across the cell envelope and then deals with the toxin intracellularly. The authors have also reasoned that due to Rhodococcus’ ability to withstand starvation conditions Rhodococcus is very well adapted to be used as a bioremediator commercially.  

The strain isolated in this investigation was shown to exhibit a broad range of optimal temperatures for the growth on phenol, which can be a large advantage in commercial applications in many geographical regions. It is also stated within the paper that the presence of phenol and/or other toxic xenobiotics resulted in changes in the fluidity of the cell envelope of Rhodococcus which probably plays an important role in the resistance mechanism of Rhodococcus to the toxic effects of phenol. Specific growth rate was shown to be severely inhibited when this strain wasexposed to high phenol concentrations, despite this degradation was found to continue suggesting that the cellular processes were being directed more towards phenol degradation than growth.  There is the possibility that this could be used to grow Rhodococcus in lower phenol concentrations until the culture reaches a kind of critical mass which can then theoretically be used to more quickly degrade higher concentrations of phenol more efficiently. Overall this paper has shown that the Rhodococcus which was isolated has possible commercial applications for biodegradation of phenol concentrations within the ground water well of Malaysia, though this will need to be further researched before this can be implemented as a measure against phenol poisoning.

Arif NM., Ahmad SA., Syed MA., Shukor MY., 2013, Isolation and characterisation of a phenol-degrading Rhodococcus sp. strain AQ5NOL 2 KCTC 11961BP, Journal of Basic Microbiology, 53, 9-19

Temperature Related Mass Mortalities of Cyanobacteria-Harbouring Sponges: Could Symbiosis Breakdown be Responsible?

 

During recent decades, numerous mass mortality events involving sessile, epibenthic invertebrates of the temperate, Western Mediterranean Sea have been reported.  The geographical coverage and number of taxa involved in such events has varied, but a general, temperature related trend has been observed. Anomalously high temperatures have been reported as occurring simultaneously to (or on occasion previous to) mass mortality events.  Unfortunately, such observations have been difficult to examine thoroughly in the past, as mass mortality events are often difficult to quantify due to a lack of baseline, pre-event data.  However, proceeding extensive die-offs of shallow water sponge populations in two Marine Protected Areas (MPAs) of the Western Mediterranean Sea (Cabrera National Park (Cabrera NP) and Scandola Marine Reserve (Scandola MR)), quantitative assessments of sponge population status were already underway.  This circumstance provides the basis of an investigation reported by Cebrian et al. (2011).

Cebrian et al. (2011) conducted a comparative evaluation of the impact of two mortality events which occurred in consecutive summers (2008; 2009), focusing on two, phylogentically close species of keratose bacteriosponges, Ircinia fasciculata and Sarcotragus spinosulum.  I. fasciculata is known to harbour both heterotrophic bacteria and cyanobacteria, whereas S. spinosulum hosts the former, but not the latter.  The percentage of injured sponges (i.e. sponges displaying white pustules or areas of necrosis indicative to these mass mortality events), and sponge density, were recorded via randomised sampling conducted once at the beginning, and once at the end of summer (in July and October, respectively), from July 2007 to October 2010 in Cabrera NP, and from October 2008 to October 2010 in Scandola MR.  In situ Water-temperature was recorded hourly throughout these periods via autonomous sensors, facilitating calculation of the percentage of time sponges spent in temperatures above defined threshold values. 

In both MPAs, the percentage of injured I. fasciculata individuals was reported as having peaked in the Octobers of both 2008 and 2009, at 80-100% (of surveyed individuals).  Injured individuals did not exceed 30% during the other periods sampled.  This contrasted to the percentage of injured S. spinosulum individuals, which varied between 10-30% in both MPAs over the entire 2007-2010 period, with differences over time not being found as significant.  The authors also reported a dramatic decrease in I. fasciculata density in both MPAs, from a mean 7 (Cabrera NP) or 10 (Scandola RN) specimens per m² before the summer 2008 mortality event, to less than 1 specimen per m² at the end of autumn 2010.  Over the same time period, mean S. spinosulum densities in Cabrera NP decreased from 7 to 3 specimens per m², but density decreases in Scandola MR were not found to be significant.  Regression analyses between the percentages of time above maximum temperature thresholds (Cabrera NP: 26^oC; Scandola RN: 25^oC) during the summer period, and the percentage of injured I. fasciculata specimens, were positively correlated for each site (i.e. the greater percentage of summer time above a maximum threshold, the higher the impact on sponge populations).

The ultrastructure of healthy and injured tissue from a single I. fasciculata specimen was examined via Electron Transmission Microscopy.  Healthy tissue was observed as containing healthy cyanobacteria and heterotrophic ‘symbiotic’ bacteria.  In injured tissue, cyanobacteria were always degraded, and an unidentified, microorganism was frequently observed in cellular vacuoles of ‘disorganised’ choanocytes.  From these observations, the authors formed a working hypothesis: high temperatures may induce a breakdown of the cyanobacteria-sponge symbiosis present in I. fasciculata (potentially related to sponge mortality as cyanobacteria are thought to contribute to sponge carbon requirements).  The biological mechanism which might be involved in such a breakdown was then investigated via laboratory based experimentation.  The effect of temperature on the photosynthetic efficiency of Cyanobacteria harboured by I. fasciculate was estimated via measurement of chlorophyll florescence parameters (effective quantum yield and photosynthetic electron transfer).  I. fasciculata individuals were maintained in seawater at 16 ^oC (control), 23 ^oC (‘normal summer), and 26 ^oC (‘extreme summer’); Only I.  fasciculata individuals maintained at 26 ^oC showed significantly lower photosynthetic efficiencies (i.e. photosynthetic inhibition) than those in control conditions.

This paper particularly interested me due to both the subject matter (being previously unaware of a potential link between photosynthetic inhibition and symbiosis breakdown in sponges), and the logical progression of the investigation (from observation of correlated factors in the environment, through to experimental investigation of a potential mechanism for this correlation).  Cebrian et al. (2011) provide some convincing evidence for a relationship between the I. fasciculata mass mortalities observed in the summers of 2008 and 2009, and elevated temperatures.  However, much remains to be investigated regarding the mechanisms of such mass mortality events.  Although the authors provide some interesting ideas regarding a possible relationship between sponge mortality and the photoinhibition of symbiotic cyanobacteria, certain limitations regarding the experimental method indicate that further investigation into this theory is required. 

Unfortunately, the experiment undertaken appeared to suffer from pseudoreplicaiton (each temperature treatment was represented by a number of individuals in a single tank), and although Cebrian et al. (2011) discuss the probability of photoinhibition driven damage via the formation of reactive oxygen species (ROS), no investigations into such damage (e.g. antioxidant assays) were performed.  Similarly, although the TEM observations of heterotrophic bacteria particular to injured I. fasciculata tissues were attributed to an opportunistic spongin consumer, and the possibility of pathogen virulence/density increase with elevated temperatures speculated upon, no surveys of microbial diversity (e.g. DGGE analysis) were reported at any point.  Considering the importance of opportunistic pathogens and shifts in microbial populations in mass mortality events of other sessile invertebrates (e.g. corals; bivalves), this omission appears particularly problematic.  Nevertheless, Cebrian et al. (2011) have reported some interesting ideas on the potential role of temperature related I. fasciculata-Cyanobacteria symbiosis breakdown.  Further investigations including quantitative TEM studies of cyanobacteria density/condition in injured and recovered I. fasciculata tissues, antioxidant assays, and microbiota diversity surveys may assist the exploration of this theory.

 

Cebrian, E., Uriz, M. J., Garrabou, J. & Ballesteros, E. (2011).  Sponge mass mortalities in a warming Mediterranean Sea: are cyanobacteria-harboring species worse off? PloS one 6, e20211.

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3105983&tool=pmcentrez&rendertype=abstract

Symbiont shifts in deep-sea mussels

Bathymodiolus, a genus of deep-sea mussels found in hydrothermal vents and cold seeps worldwide, are colonised by chemosynthetic bacteria, obtained from the surrounding environment at an early life stage. As these symbionts oxidise sulphur and methane, it is thought they provide the mussels with additional nutrients. However the exact process through which colonisation occurs is unknown and was the aspect this study aimed to investigate.

Samples of B. azoricus and B. puteoserpentis (juveniles measuring between 4-21mm) were collected from two hydrothermal vents in the Mid-Atlantic Ridge located 3000km apart. Semi-thick sections of the whole juvenile were analysed using FISH to determine the distribution and specificity of symbionts colonising these two species of mussels. Three probes were used: a symbiont specific probe; a general eubacterial probe and a negative probe.

The symbiont specific probe revealed sulphur and methane oxidising symbionts in the gills of all samples. The smallest of the mussels also demonstrated these in the epithelial cells of the retractor muscle, mantle and foot. Overall, the gills showed the densest colonisation of symbionts. There was overlap between the symbiont specific probe and eubacterial probe in all samples, suggesting that these specific symbionts are the only colonising bacteria on the juvenile mussels. Furthermore, they found that the larger juveniles had symbionts only on their gills. To determine whether this was also true for adult Bathymodiolus they carried out further analysis on the mantle tissue attached to the gills of adult B. azoricus (measuring 55-100mm), and found symbionts only on the gills.

This was the first study that used FISH to prove that symbionts colonise a range of epithelial tissues of mussels at an early life stage, providing further evidence to support previous research that also stated this. They believe this to be an unusual occurrence since most other studies have demonstrated symbiosis to be limited to a specific tissue even at an early life stage. Since the gills develop after the foot and mantle, it is believed the reason for such widespread colonisation is due to the provision of additional nutrients for the mussel. Nonetheless, Bathymodiolus are filter feeders, so could use this alone to obtain a sufficient amount of nutrients, suggesting the symbionts colonising the foot and mantel may actually be irrelevant.  

They found the shift of the symbionts became restricted to the gill bacteriocytes when the mussels were at the developmental stage between 8.4-9mm. Since the foot and mantle epithelia are not directly next to the hemolymph lacuna, like the bacteriocytes of the gills, the symbionts are not able to supply a sufficient amount of nutrients in relation to the costs of maintaining them. This therefore provides a suitable explanation to the benefits of the symbionts being retained only in the gills. Furthermore, the large surface area and cilial ventilation of the gills means they can supply oxygen and reduced compounds required by the symbionts. Despite this, juveniles measuring less than 9mm have a thin layer of non-gill epithelia, which is sufficient in providing the requirements of the symbionts, and it was unclear of the difference that would make this insufficient in adult mussels. The paper then reveals that the early stage Bathymodiolus may simply be yet to have developed an immune system capable of preventing indiscriminate infection by the symbionts. Therefore, further study is required to understand the role of the immune system and the benefits the symbionts supply to the host in order to determine the exact process of colonisation and how this is maintained. 

http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej20135a.html

Shift from widespread symbiont infection of host tissues to specific colonisation of gills in juvenile deep-sea mussels 

Cecilila Wentrup, Annelie Wendeberg, Julie Y Huang, Christian Borowski and Nicole Dubilier

Oil Plume Fades As Microbes Degrade

The Deep-water Horizon oil spill is the second largest oil spill in the history of the petroleum industry. On 20^th April 2010, high pressure oil and gas caused the deep-water drilling rig in the Gulf of Mexico to explode, it is estimated that 4.1 million barrels of light crude oil and natural gas leaked into the Gulf. To tackle this pollution 1.8 million gallons of the chemical dispersant COREXIT was applied to the surface waters as well as directly into the wellhead (1500m below surface level). During the spill several research groups reported a plume of dispersed MC252 crude oil at 1100-1220mbsl (meters below surface level) at distances up to 35km from the wellhead. It is thought to have formed as a reaction to the application of COREXIT at the wellhead combined with the physical and chemical factors at 1100 mbsl depth.

The aim of this study was to investigate the succession of the microbial community and the formation of microbial flocs in deep sea water from the Gulf of Mexico. This aim was achieved by laboratory enrichments at cold temperatures (5˚C) with high concentrations of the MC252 oil and COREXIT. There were five enrichments tests using combinations of oil, COREXIT and FeCl₂ as well as three control enrichment tests, with additional to study floc formations. These investigations involved a source of uncontaminated water from the depth of the recorded plume, MC252 oil from the Discovery Enterprise drillship located directly above the wellhead and COREXIT 9500 dispersant which was provided. The authors also aimed to select and isolate specific members of the microbial community that are capable to degrade oil. The members were isolated and identified from 20 day incubated enrichment samples, using 16S rRNA-based identification and scanning electron microscopy. By monitoring the respiration and degradation of oil and COREXIT during incubations the authors were able to understand the pattern of succession and response of specific microbial populations when exposed to high concentrations of high hydrocarbons. This illustrates what happens in situations like the Deep-water Horizon oil spill or from oil seeps on the ocean floor.

The different hydrocarbon fractions of MC252 oil in the deep-water plume were previously found to be largely removed by a combination of dispersion and microbial degradation by indigenous microbes in the deep sea. The results showed that the bacterial communities adapted rapidly to the introduction of hydrocarbons with sequences representative of first Oleispira, then Colwellia becoming dominant. The findings suggest that Colwelliaceae and Oceanospirillales played a predominant role in hydrocarbons in deep sea; Colwellia could have a competitive advantage in the presence of high concentrations of oil and dispersant due to their ability to produce EPS and form flocs.

The deep-sea holds some oil degrading potential since there is natural oil seeps, so it is interesting to see how application of dispersants will affect this potential, especially in high concentrations. The fact that this paper identified a dominant crude oil degrader (Colwellia) means it could be utilised to perfect oil spill management. It would be interesting to compare this study with future studies in the Gulf of Mexico to track the succession of this species and if competition with Oleispira changes its dominance.

Baelum, J., Borglin, S., Chakraborty, R., Fortney, J.L., Lamendella, R., Mason, O.U., Auer, M., Zemla M., Bill, M., Conrad, M.E., Malfatti, S.A., Tringe S.G., Holman, H-Y., Hazen, T.C., Jansson, J.K., (2012), Deep-sea bacteria enriched by oil and dispersant from the Deepwater Horizon spill, Environmental Microbiology, Volume 14, Issue 9, September 2012, Pages 2405-2416

http://onlinelibrary.wiley.com/doi/10.1111/j.1462-2920.2012.02780.x/pdf

Streptomyces never tasted so good!

In recent years, fish meal as a component to animal feed in aquaculture has become more uncertain as it has become increasingly expensive. This prompted the authors of this paper to look for cheaper alternatives to fish meal. Prior to their study, there were some reports showing that some micro organisms incorporated into animal feed resulted in promising outcomes. For example, effective protection against white spot virus syndrome (WSSV) in Black Tiger Shrimp (Penaeus monodon), the inhibition of vibrio sp. biofilm formation and vibrio sp.-caused disease, and increased growth sizes in the juvenile prawn Macrobrachium idella. The micro organisms used in these examples were Streptomyces, a marine actinobacteria, which wasn’t given much attention in the poultry or aquaculture industries. The species chosen in this experiment to consume the altered animal feed were the ornamental fish species Red Swordtails (Xiphophorus helleri).The lack of attention in this micro organism combined with the fact that it has shown proof of effectiveness in other organisms is what lead the authors into conducting this study.

Four species of marine sponge (Callyspongia diffusa, Mycale mytilorum, Tedania anhelans and Dysidea fragilis) were collected by divers, and Streptomyces were extracted, isolated and characterized. They were characterized by means of nutritional, morphological, physiological and biochemical properties. Eight different feeds were produced; 7 probiotic feeds containing Streptomyces and the control feed. The control feed used ingredients of fish meal, rice bran, groundnut oil cake and chickpea flour, using a tapioca flour binder. These were ground up and mixed with water to form a dough which was steam cooked and allowed to cool. After cooling, the dough was put through a pelletiser, and the pellets were then dried. The probiotic feed was also a dough steam cooked and cooled, but with a known quantity of Streptomyces in it. Again the dough was put through a pelletiser, and the pellets were freeze dried. The Red Swordtails were divided into two tanks, with 20 individuals in each. One tank contained the probiotic feed, and the other contained the control feed. They were fed once a day for a duration of 50 days.

After 50 days the fish were measured, showing that all fish which consumed the probiotic feed containing Streptomyces had grown larger than those who fed on the control feed. This included significantly higher absolute growth rate, specific growth rate and relative growth rate. Arriving at this result, the authors state that the use of Streptomyces in probiotic feed has a significantly positive effect on the growth of the ornamental fish. They also state that Streptomyces in probiotics will be used, and be important in aquaculture nutrition.

This paper was a good read and it is interesting to see how different bacteria could bring a variety of advantages by being incorporated into animal feed. However, there were a few points and explanations that I think this paper missed out on. When suggesting the known examples of where micro organisms have had positive effects in animal feed, they did not specify which vibrio sp.-caused disease the bacteria stopped. The authors also failed to explain why fish meal in animal feed has become more ‘uncertain’, so I assumed that it was due to the rapid price increase that was also mentioned. Finally (and this might just be me being picky), I think their statement of ‘in the near future, applications of Streptomyces as probionts will play an important role in aquaculture nutrition’ is quite a bold statement. I know it is a great find and has had a good effect on some species, but it hasn’t been used on more commercially valuable species such as salmon, so to say it will be important is a bit bold in my opinion.

Dharmaraj, S. & Dhevendaran, K. (2010). Evaluation of Streptomyces as a Probiotic Feed for the Growth of Ornamental Fish Xiphophorus helleri. Food Technology and Biotechnology. 48 (4): 497-504.

Marine bacteria being utilised to aid in the medicinal field.

Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties.

 

Albeit this topic does not relate exactly to the topics we are studying currently, I thought it was a very interested topic. As the title suggests Sargassum sp. has already been studied regarding its anticoagulant and antioxidant properties, this study aims to see whether marine lactic acid bacteria (LAB) can be used as a starter culture to enhance these properties.

 

These bacteria also have a long history of being used to preserve types of foods. They not only do this just by adding taste and flavour but also by reducing the risk of pathogenic microbes by the internal production of lactic acid and other antimicrobial peptides. The Sargassum spp. are a group of brown alga and have been extensively exploited for their range of uses, which are not just limited to anti-coagulant and antioxidant properties.

 

Shobharani et al (2012) isolated this alga from the west coast of India and were then transported to the laboratory under iced conditions. The samples were then rinsed, dried and then mashed up and sieved to give a fine powder. Essentially the method used was that the fine powdered part of the seaweed was suspended in distilled water, and then either autoclaved to give the sample control or left alone to show the effect that having this active LAB would have on the properties presented. From each sample, an aliquot was drawn at a regular interval of 3 days and analysed for viable cell count, pH, total titratable acidity, total and reducing sugars.  For antioxidant activity they used alternate isolates and cultures to determine whether these had an effect as well.

 

The results revealed that a fermentation period of 12 days was effective with maximum culture viability and other desirable characteristics such as pH, total titratable acidity, total and reducing sugars. Under optimum fermentation period, the sample fermented with P1-2CB-w1 (Enterococcus faecium) exhibited maximum anticoagulation activity and antioxidant activity.  It was concluded quite clearly that this type of bacteria do in fact have a wider range of uses than just food preservation, and this can hopefully be utilised within the medicinal world sooner rather than later.

 

The results from this are once again contributing to an understudied area but the aims of the authors were to show and analyse the suitability of these bacteria for starter cultures, thus improving the final product and usefulness of this brown alga species, and they did this indeed demonstrating that under the right researched conditions this is indeed the case.

 

Reference:

 

Shobharani,P., Halami,P. and Sachindra,N. (2012). Potential of marine lactic acid bacteria to ferment Sargassum sp. for enhanced anticoagulant and antioxidant properties. Journal of Applied Microbiology, 1364-5072.

 

Availabe at:  http://onlinelibrary.wiley.com/doi/10.1111/jam.12023/pdf

DIY RNA

Firstly, I’d like to apologise as I know this is not strictly marine microbiology. But it is a keen interest of mine and it was a toss-up between yet another post on bio-augmentation of oil degraders and this… And this won!

The question of “Where did we come from?” is a prominent one throughout history. Many see the answer as the Darwinian definition; that we came from Prehistoric apes. Others may think that LUCA, the Last Common Universal Ancestor, is the answer. However the study of abiogenesis has led to many real answers about the true origin of life through the primordial soup system as evidenced by the key research of Miller & Urey (1952). This experiment showed that basic amino acids and certain proteins will self-assemble under the correct conditions and thus this theory has been generally accepted.

However Cafferty et al (2013) have recently shown that much larger, much more complex molecules can self-assemble in water, and are very similar to RNA (which many think was a precursor to DNA). These molecules consist of two weakly interacting low-molecular-weight monomers (cyanuric acid and a modified triaminopyrimidine), which form non-covalent supramolecule through spontaneous assembly, completely absent of intermediates. This occurs through several, negative gibbs energy reactions which are documented in the paper.

Previously, no other attempt at this kind of research has been able to successfully dimerise RNA in water, unaided, without a polymer backbone.

Although I do not fully understand the biochemistry, from what I do understand Cafferty et al (2013) have managed to create proposed “proto-RNA” bases which spontaneously assemble into gene length (up to 18000 molecules) linear stacks. These molecules were visualised using TEM and Atomic Force Microscopy and characterised using NMR spectroscopy. Using these methods, large (>1µm) linear and branched fibrilliar structures were observed, which the authors claim are “the longest supramolecular polymers generated to date by untethered, monocyclic monomers in waters”.

Many questions about the origin of life can be answered with the conclusions drawn from this study, and further research is essential to see if this methodology can be incorporated into other areas of research, such as nanotechnology and the formation of synthetic nanowires.

REF: Brian J. Cafferty, Isaac Gállego, Michael C. Chen, Katherine I. Farley, Ramon Eritja, Nicholas V. Hud. Efficient Self-Assembly in Water of Long Noncovalent Polymers by Nucleobase Analogues. Journal of the American Chemical Society, 2013; 135 (7) 2447

Accessed From: http://pubs.acs.org/doi/full/10.1021/ja312155v

‘Waste materials’: an economic and sustainable ‘fuel’ for hydrocarbon-loving marine microbes involved in oil remediation?

Contamination of oil pollutants in marine environments has long lasting, catastrophic effects. The current methods used to degrade the emollient spills are limited. Currently conventional treatments are: the application of blooms, skims, adsorbents and dispersants. However, these techniques have been found only recover approximately 10-15% of the oil impurity. Furthermore, research has suggested that using these dispersants; the particularly the surfactants involved, have an acute and chronic effect of the surrounding ecosystem (Singer et al. 2001), to add insult to injury; it’s expensive!

One alternative approach to ‘clean up’ these spills is the addition of biological systems. This process is called Bioremediation, it can be enhanced through bio-augmentation; addition of microbes and bio-stimulation: the supplementation of nutrients. For degradation to occur, there must be direct contact between microbes and the hydrocarbon substrate within the spill. This interaction enables marine microorganisms to metabolize the present oil compounds. Applied combinations of bio-augmentation and bio-stimulation have not been shown to be very effective at remediating oil spills; the mix can become diluted and washed away from tidal action. To over come this problem, the uses of ‘carriers’ as mediators in this mix have been considered. Carriers are materials that carry inoculants and nutrients to the oil spill. The use of carriers is thought to enhance the process of oil degradation by marine bacteria; it supplies more nutrients and counteracts the mixing of water, increasing the longevity of bioremediation. Characteristics of model carriers are: not soluble, highly stable, not readily degraded, natural and economical. Consequently, a natural waste product would be ideal.  

Simons et al. (2012) investigated the application of three different natural carrier materials: mussel shells, coir peat and a mussel shell and agar complex. This study wad conducted over a 30-day period. Six hydrocarbon-degrading microbes, previously isolated and characterised by Kadali et al. (2012) were used. The ID strains of the isolates were: Pseudomonas mendocina, Planomicrobuim alkanoclasticum, Bacillus sp, Bacillus sp, Arthrobacter pascens and Arthrobacter nitrogujacolicus. The samples were cultured until an OD₆₀₀ of 1.0 was obtained. For the carriers: waste mussel shells were acquired from Woolworths and ground, Coir peat bricks obtained from Bunnings and the mussel shell and agar complex was made by adding molten agar to ground mussel shells. Erlenmeyer flask biodegradation experiments were carried out. For each isolate and carrier material: the flasks contained mineral salts medium made in seawater containing weathered oil. To specify; the process by which an oil spill changes both physically and chemically is called oil weathering. Weathered oil was used in these experiments because the stage of which ‘weathered oil’ is degraded by marine microbes is responsible for eliminating last traces of an oil spill. At days: 0, 15 and 30 samples were collected to measure the Total Petroleum hydrocarbon (TPH). DGGE fingerprinting analysis and PCR amplification of 16s DNA was used to analyse the microbial community at those intervals.

Simons et al. (2012) considered whether the application of natural carriers to the process of bioremediation, would improve the efficiency of an important stage in the mechanism of oil remediation. The main findings in this study were that the flasks containing the carrier consisting of solely of mussel shells exhibited the greatest degree of oil degradation: 55% reduction of the weathered oil. The mussel and agar complex carrier closely followed this; with a reduction of 49%. Coir peat displayed a 36% decrease. The first two carriers were shown to be convincing, significantly different to the control flasks. Flasks in which just nutrients was added to the isolates was not shown to beneficial in improving the extent of degradation, it was only the combination of the carriers, nutrients and hydrocarbon-loving microbes that resulted in significant bioremediation.    

This particular paper was chosen because it advocates that inexpensive, accessible ‘waste’ materials could potentially be used as carriers for hydrocarbonoclastic bacteria to significantly degrade hydrocarbon contaminants in seawater oil spills. The paper also begins to investigate the community composition of these noteworthy microbes. This was a topic that was discussed in the previous post: “Obligate oil-degrading marine bacteria” Sophie suggested that more knowledge of population dynamics and ecophysiological functioning of marine oil-degrading communities are needed. Simons et al. (2012) have started to explore this. The DGGE analysis carried out during the experiments indicated that the there was an increase in community complexity over time. The microbial population was suggested to be adapting and growing in the presence of weathered oil. The presence of the carrier material was also shown to not to affect the community structure. Further studies could investigate what effects the previously discussed carriers could have on in situ spills. Other natural waste products could also be examined as potential carriers, which theoretically would make this method globally applicable.       

Simons, K. L., Ansar, A., Kadali, K. K., Bueti, A., Adetutu, E. M. and Bali, A. S. (2012)

“Investigating the effectiveness of economically sustainable carrier material complexes for marine oil remediation”

Bioresource Technology, 126: 202-207.

http://www.sciencedirect.com/science/article/pii/S1369527412000938

Kadali, K. K., Simons, K. L., Skuza, P. P., Moore, R. B. and Ball, A. S. (2012)

“A complementary approach to identify and assessing the remediation potential of hydrocarbonoclastic bacteria”

Microbiol. Methods, 88: 348-355.

Singer, M. M., Jacobson, S., Tjeerderm, R. S. and Sowby, M. (2001)

“Acute effects of fresh versus weathered oil to marine organisms: California findings”

Int. Oil Spill Conf, 2: 1263-1268.