Eating Oil… Not as Crude as You’d Think!

Due to sea water being poor in biogenic mineral nutrients, carbon and other sources of energy, marine microbial communities have adapted to survive in such oligotrophic conditions. However, when there is a large influx of nutrients, for example in the case of an oil spill, some microbes find it very stressful and difficult to cope with. On the contrary, other microbes can rapidly adapt to these situations and thrive, thus out-competing their competitors. An example group of organisms which are able to do this are the Obligate Hydrocarbonoclastic Bacteria (OHCB), containing organisms from the genera Cycloclasticus, Thalassolituus, Oleiphilus, amongst others. OHCB are able to grow in these short period, high nutrient conditions, and rapidly deplete them. Once depleted, the OHCB become inactive, making them potential organisms used in bioremediation techniques.

Having observed promising results of bioremediation in similar studies, the authors of this paper decided to look at the changes in marine flagellate and ciliate communities in a controlled mesocosm experiment. They did this by filling 2 500l mesocosms with seawater, followed by the addition of 2.5l Bunker C heavy fuel oil. Samples were taken from the mesocosms throughout the duration of the experiment, and the microbial communities within these were identified.

The results of this experiment revealed that there was an increase in the numbers of marine protozoa. This protozoan growth was continually observed throughout the experiment in both mesocosms. There were two initial blooms, where protozoan numbers fluctuated between 300 and 3000 cells per millilitre. Protozoa development paralleled the depleting levels of oil, oil emulsion and biofilm aggregates observed in the mesocosms. The majority of protozoa which were observed in the mesocosms were in the Ciliatia. Cilliates are commonly found in experiments involving oil degradation as they are well adapted to graze on oil-degrading communities. The ciliates in this study were predominantly Scuticocilitia,

Finally, in the second part of this experiment, an abundance of marine fungi  and yeasts were observed. This was reported as the ability for Candida yeasts to grow on gas oil has been acknowledged for several years, and a high number of these and related species are efficient degraders of fatty acids, polyaromatic hydrocarbons and oil. Marine fungi and yeasts have been used on a large scale for single cell protein production from oil paraffins.

I believe that this paper gives a good insight into marine microbiological degradation of oil spillages. I think, however that the difference between a controlled mesocosm experiment and a spill in the ocean could show large differences in results. I understand that studies have been carried out in controlled, closed-water environments, however this could still show different results than if the same were to be tested at sea.

Gertler, C., Näther, D. J., Gerdts, G., Malpass, M. C. & Golyshin, P. N. (2010) A Mesocosm Study of the Changes in Marine Flagellate and Cilliate Communities in a Crude Oil Bioremediation Trial. Environmental Microbiology. 60: 180-191.

It's snot impossible to break down biofilms

Chronic Rhinosinusitis is one of the most common respiratory tract disorders in Europe, affecting 10% of the population. This disease is characterised by inflammation of the paranasal sinuses and nasal airway and is classified as chronic if it lasts for 12 consecutive weeks or more. Current therapy involves topical steroids and nasal douching for chronic disorders and antibiotics for acute, however these methods have been criticised as ineffective and in some exacerbated cases surgery may be warranted to remove mucin build up and nasal blockage. This recalcitrance to medical therapy is due to the biofilm forming capabilities of the bacteria responsible (mostly Staphylococci spp.) which enables a 1000fold increase in resistance to antibiotics. Several novel methods have been proposed for treating biofilms, most predominant of which involves degrading the extracellular DNA (eDNA) which provides stabilisation of the biofilm structure, enhanced adhesion capabilities and enhanced exchange of genetic material. Extracellular deoxyribonucleases (eDNAses) exist which catabolise the eDNA, thus disrupting the existing biofilm.

The authors of this study collected mucin from patients suffering from Chronic Rhinosinusitis and applied the afore mentioned eDNAse, called NucB, isolated from a marine strain of the gram positive bacterium Bacillus licheniformis. Confirmation of biofilm formation and structure was obtained using transmission electron microscopy. The authors applied the NucB to the infected mucus and observed the subsequent degradation of the biofilm. The results of this study show no extracellular biofilm observed in the treated samples.

The authors conclude that this test shows a novel therapeutic methodology which shows promising prospects for the future and whilst I agree with this, I can’t help but point out that this study is only in vitro and clinical trials are a very lengthy and very costly process, often ending prematurely and in disappointment. Nevertheless, ultimately all medical therapies must start somewhere and this study shows that NucB undoubtedly has potential for the control of biofilms. It will be interesting to see the results of the in vivo study on the same enzyme, which is currently being undertaken by the same research group in Newcastle (this is stated in the paper and as far as I know is still underway). 

REF: Shields, R.C., Mokhtar, N., Ford, M. et al (2013) Efficacy of a Marine Bacterial Nuclease against Biofilm Forming Microorganisms Isolated from Chronic Rhinosinusitis. PLoS One, 8 (2), e55339. (Published online)

Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575374/

Quorum sensing in Acinetobacter sp.

Detection of Quorum Sensing Signal Molecules and Identification of an Autoinducer Synthase Gene among Biofilm Forming Clinical Isolates of Acinetobacter spp.

Quorum sensing allows bacteria to communicate and monitor their own population density, having this ability enables them to monitor and decide on the correct size of biofilm to produce depending on the environment and the general function the biofilm will undertake. It has been seen that numerous gram-negative bacteria use N-acyl homoserine lactones as sensing molecules. This study looked at a particular gram negative aerobic coccobacilli, Acinetobacter to see whether it too used quorum sensing molecules.

This type of bacteria is under investigation here as they are commonly observed in the hospital environment and are known to cause numerous nosocomial infections. Some common infections they have been isolated from and seen to cause are, septicemia, pneumonia and urinary and wound infections.

The method undertaken here was that fifty isolates of the Acinetobacter spp. were isolated and were monitored using a Chromobacterium violaceum CV026 biosensor monitor system. Mass spectrometry was also used to observe whether AHL’s were produced by any of these isolates. They also incubated some of the isolates to see if this inhibited or increased biofilm formation.

The results saw that 60% of the isolates formed significant biofilms after a prolonged period of incubation and that time incubated appears to have a significant effect as those incubated for 48 hours had much larger biofilms compared to those at 24 hour periods. The incubation test, also known as the microtiter plate method was repeated three times and so the results were validated and replicated thus increasing the strength of the conclusions made. Regarding the AHL production, this was not as significant, all of the 50 isolates produced colourless colonies in the CV026 induction test and this means that none of the isolates here produce short chain AHL’s however seven of the isolates did produce long chain AHL’s. Mass spectrometric analysis revealed that five of these isolates produced N-decanoyl homoserine lactone and two isolates produced acyl-homoserine lactone with a chain length equal to C₁₂.

Due to the small number of isolates producing AHL’s the authors then decided to look and try and identify whether a specific gene was present in the seven isolates to determine the producer of AHLs within. The abaΙ gene was identified and a tetracycline mutant of the abaΙ gene was created and the inhibition in biofilm formation in the mutant was shown, so it appears that this gene causes the communication to occur and without it the biofilm formation is very much hindered, if not completely inhibited.

The conclusions made from this study therefore state that these signal molecules are of great significance in biofilm formation and therefore show possible ways to alter these isolates so as to not use this gene therefore reducing biofilm formation and aiding in pathogenic resistance, thus increasing the health and such of hospitals and medical facilities, anywhere that has been affected by these isolates.

Therefore this is extremely useful in today’s life and will provide immeasurable steps forward in increasing the cleanliness of many areas, and hopefully reducing disease and infections because of this species.

 

The paper is available at:

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0036696

 

Reference:

Anbazhagan D, Mansor M, Yan GOS, Md Yusof MY, Hassan H, et al. (2012) Detection of Quorum Sensing Signal Molecules and Identification of an Autoinducer Synthase Gene among Biofilm Forming Clinical Isolates of Acinetobacter spp. PLoS ONE 7(7): e36696. doi:10.1371/journal.pone.0036696

Recruiting: Bacterial Genes Needed!

Recruiting: Bacterial Genes Needed!

Most benthic marine organisms have a planktonic larval phase in their life-cycle and, as a consequence, larval settlement is an important factor determining distribution patterns of benthic marine organisms.  Biofilms (defined as a community of organisms on the surface of an inanimate object or living organism) provide chemical cues for settling larvae and are therefore markedly important in the marine environment. Biofilms are also particularly relevant in the commercial sector where bio“fouling” can be economically expensive. Given the financial incentive from the commercial sector; research to increase the understanding of biofilms has exploded, with over 1000 published papers on the topic to date. Despite much speculation, the role of bacterial cues in biofilms and recruitment is unknown. Furthermore the role of molecular inducers in general is poorly understood. Huang et al. (2012) aimed to fill the described knowledge gap by investigating the molecular cues and mechanisms in which an important marine tube worm larvae (Hydroides elegans) is induced to settle on bacterial biofilms of the abundant Pseudoaltermonas luteoviolacea.

Wilde-type P.luteoviolacea is known to induce the settlement of H.elegans, however the mechanisms of induction are unstudied. Huang et al. created mutants of P.luteoviolacea via random transposon insertion and screened for non-inducing strains. Two strains which did not induce the settlement of H.elegans were isolated for further investigation (Plm9 and Plm45). Analysis of the isolated mutant’s Open Reading Frames (ORFs) revealed possible genes important for settlement induction. Using the isolated mutants, deletion mutants were created (strain with deletion of the genes in which the transposon was inserted), allowing the confirmation that the genes disrupted by transposon insertion were responsible for the lack of induction.  The growth rate of mutants was compared to that of wild-type cells to confirm lack of induction was not simply down to reduced mutant growth rate. In addition, phenotypic parameters such as biofilm cell density, thickness and exopolymeric substance biomass were not different in mutant and wild-types biofilms. The lack of induction by deletion mutants suggests that the deleted gene products are important and in some way provide cues for settlement. BLAST analysis of the deleted ORFs revealed some putative information about the potential function of protein products, of particular interest was a gene coding for Type VI secretion systems (secretion in bacteria means the transport or translocation of effector molecules).

The reviewed study represents the first evidence that bacterial genes are important in the settlement of macrofaunal marine organisms. Whilst the paper is in no way conclusive, it does provide a solid preliminary understanding into the possible role of molecular cues in settlement. Increasing our understanding of bacterial biofilm induction cues is not only important in the broad areas of benthic ecology and micro-macro interactions, but is also potentially important for the development of solutions to biofouling.

Huang, Y., Callahan, S., & Hadfield, M. G. (2012). Recruitment in the sea: bacterial genes required for inducing larval settlement in a polychaete worm. Scientific reports, 2, 228.

http://www.nature.com/srep/2012/120118/srep00228/full/srep00228.html

 

Early microbial biofilm formation on marine plastic debris

Plastic debris in marine environments can cause multiple problems for the marine ecosystems. The problems include; ingestion by animals, transporting bound organic pollutants and animal entanglement. Pollution risks from plastics are determined by behaviours of plastic debris, such as buoyancy. Most plastics are positively buoyant and are dispersed by wind and ocean currents, however some become neutrally buoyant and sink below the surface. It has been found that biofueling can play a significant role in controlling plastic debris buoyancy.

Marine biofilms, which can lead to biofueling, can occur on any surface in the marine environment. The degree of biofueling can be increased via the attachment of specific invertebrates and algae. The objective of this study was to characterise early biofilm formation on plastic debris with the aim of contributing to a systemic understanding that maybe useful for biofueling.

Polyethylene plastic food bags were secured to weighted Perspex boards and suspended 2 m below the water’s surface. Samples were taken weekly for 3 weeks. The plastic samples were stained, washed and dried and then the optical density was measured. The buoyancy of the plastic pieces was determined. Attached biofilms were dislodged, diluted and plate on Marine Agar 2216 and polyethylene marine agar plates. The hydrophobicity of the plastic’s surface was measured.

Biofilm formation was visibly apparent on submerged plastic after 1 week and continued to increase throughout the experiment. Along with biofilm formation, the surface of the plastic became less hydrophobic during the experiment. Surface hydrophilicity significantly increased after 2 weeks. The buoyancy assessment showed that after the plastic was initially very hydrophobic and remained at the seawater-air interface. By the end of the experiment, the plastic started to sink below the surface and showed signs of neutral buoyancy.

The number of culturable heterotrophic bacteria on the submerged plastic increased from 1.4 x 10⁴ cells cm^-2 after the first week to 1.2 x 10⁵ cells cm^-2 by the end of the experiment. No polyethylene-degrading organisms had grown under the incubation conditions provided. Previously it has been shown, two strains of the common soil bacteria genera, Pseudomonas and Arthrobacter, have been able to degrade plastic. These were obtained from enrichments that had been maintained for 12 weeks, which was longer than the time that plastic was submerged for in this study (3 weeks).

Plastic debris in marine environments is now an international problem, the factors that influence the behaviour of the plastic debris in the environment need to be identified. It has been shown that microbial biofilms develop rapidly on submerged, concordant with significant changes in the physicochemical properties of plastic. There is still no evidence of potential plastic-degraders during early attachment. I think more studies should concentrate on this subject as it is such a huge problem. Maybe one such study could be one similar to this but over a longer time frame to see if the previously mentioned strains of plastic degrading bacteria become present.

Lobelle D. and Cunliffe M. 2011. ‘Early microbial biofilm formation on marine plastic debris’. Marine Pollution Bulletin. 61 (1). 197-200.

http://www.sciencedirect.com/science/article/pii/S0025326X1000473X

Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spill

The Deepwater Horizon (DH) oil spill in 2010 resulted in huge amounts of oil and gas being spilled into the Gulf of Mexico, a large quantity of which reached the surface water and was subsequently transported to the shoreline. Once oil reaches the shoreline it is likely to have negative effects on the economy and the environment.

Sands in the gulf are covered in a diverse biofilm of bacteria and the number of bacteria in the sand is much higher than in the sea water itself.  The sand is also highly permeable and very important to nutrient cycling in shallow waters. The majority of oil hydrocarbons that enter the environment will be broken down by indigenous bacteria. There is little information available on the specific bacteria that catalyze the breakdown of oil in the environment at a spill site and the environmental parameters that control the activity of these bacteria therefore it is difficult to predict the activity of oil degrading bacteria in situ. Kostka et al (2011) had two objectives for this study; firstly to characterize the main oil-degrading taxa that can be used as model hydrocarbon degraders or microbial indicators of contamination and secondly to the environmental response of indigenous bacteria to contamination of the ecosystem.

The study was conducted on samples collected from Pensacola Beach, Florida which was exposed to heavy oil pollution after the Horizon oil spill. Samples were collected approximately 18 weeks after the start of the spill.  Visual observations of oil contamination levels were carried out and hydrocarbon levels verified using gas chromatography –mass spectrometry.  Results were compared to source oil from DH wellhead to determine depletion levels. These samples were also used to carry out most probable number counts (MPN) of cultivatable hydrocarbon-degrading bacteria. Bacteria capable of using oil as the only source of carbon were isolated under aerobic and anaerobic conditions and genes sequencing and phenotypic characterization were carried out to identify the bacterial strains.  

Hydrocarbon concentrations were found to range from 3.1 to 4,500 mg kg^-1 in beach sands and comparison to carbon concentrations found at the wellhead showed substantial degradation had occurred; those remaining hydrocarbon compounds were found to mainly be large aliphatic and aromatic compounds which have previously been shown to degrade more slowly. Smaller hydrocarbons had been reduced to near the detection limit.  MNP counts showed samples taken from oil contaminated sands were 3-4 orders of magnitude higher than those taken from clean sands and rRNA analysis backed up MNP counts showing bacterial rRNA gene abundance was 10 times higher in oiled than non-oiled sands. 24 bacterial strains from 14 genera were isolated and confirmed as oil-degrading microorganisms. Strains were mainly found to be Gammaproteobacteria, including bacteria from genera Alcanivorax, Marinobacter, Pseudomonas, and Acinetobacter which are known oil degraders therefore a clear response was seen to oil contamination in the form of a shift in bacterial community structure.

The authors concluded that oil contamination from the DH spill had a marked impact on the abundance and community composition of indigenous bacteria in beach sands. Results from this study indicate that Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria (Rhodobacteraceae) are some of the most important bacteria to the degradation of oil in this region.

This study is important as few previous studies have used quantitative molecular techniques in situ to assess numbers of hydrocarbon degrading bacteria. The authors also propose Alcanivorax spp. of the Gammaproteobacteria as indicators of the early stages of hydrocarbon degradation and members of the Alphaproteobacteria as indicators of later stages.  Future studies are needed to fully identify the specific role of each.  This study also produced some pure cultures of these strains to be used as model organisms for further work.

Kostka, J., Prakash, O., Overholt, A., Green,W., Freyer, G. Canion, A., Delgardio,J., Norton, N., Hazen, T., Huttel,M.  2011.  Hydrocarbon-Degrading Bacteria and the Bacterial Community Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon Oil Spil. Journal of Applied Microbiology.  77; 7962-7974.

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills

Boronated tartrolon antibiotic produced by symbiotic cellulose-degrading bacteria in shipworm gills

J. Hinchcliffe

Shipworms rely on gill symbionts to survive in their habitat, they are well known for the ability to burrow into wood. As in other animals that consume wood, it is thought that the shipworm’s microbial symbionts facilitate the degradation of cellulose, which without the presence of microbial symbionts the animal is unable to digest.  Teredinibacter turnerae, a symbiont species, has been isolated from different shipworm hosts collected around the world; it secretes wood degrading enzymes thought to assist the host indigestion. An interesting point is that the symbiotic bacteria live in the gill but cellulose is degraded in the digestive tract in the cecum. Although the gut is an excellent habitat for microbes in organisms, the cecum in shipworms contains very few bacteria. This absence is striking, because cellulose digestion increases the availability of glucose, which is an excellent nutrient source for microbes. The genome of one strain, T. turnerae T7901, was sequenced and revealed, in addition to genes that encode enzymes specific for lignocellulose degradation and nitrogen fixation, at least nine regions encode enzymes for the biosynthesis of polyketides and nonribosomal peptides.

Elshahawi et al (2013) therefore hypothesized that some of the secondary metabolites produced by T. turnerae might contribute to reducing the bacterial population in the cecum to prevent glucose scavenging and that secondary metabolites might play a significant role in microbial competition among symbionts in the gill. Elshahawi and co described the polyketide tartrolons, antibiotics that are produced by T. turnerae that were detected in shipworms. These and other antibiotics from shipworm symbionts may help structure the symbiont community, possibly even enabling the unique lignocellulose digestion strategy found in shipworms. This study reports the secondary metabolites identified from T. turnerae and their bioactivities, describes the biosynthetic gene cluster linked to them, and presents evidence that these metabolites are produced in the symbiotic state.

In summary, Elshahawi et al identified two molecules called macro diolides from the marine shipworm symbiont T. turnerae T7901, that had anti-bacterial activity. They also identified a biosynthetic gene cluster that will shed more light on the biosynthesis of other active natural products in this class. Moreover, tartrolons were detected in the shipworm host and in other T. turnerae strains, which suggests that it plays a role in the bioactive metabolite symbiosis of the shipworm.

So in my opinion….. T. turnerae plays a major role in the shipworm symbiosis. Its genome contains information enabling this bacterium to have a facultative endosymbiotic or even a free-living lifestyle, yet T. turnerae has only been found in intracellular symbiotic association with the molluscs. The trt gene cluster that was found in this investigation has a potential role in the shipworm–microbial symbiosis. The antibacterial macrodiolides produced by the shipworm symbionts in the gills might contribute to bacterial suppression in the cecum, this suppression could allow the host to maximize efficient uptake of the glucose liberated by the breakdown of lignocellulose. But the mechanism by which products of the symbionts in the gill could be translocated to the cecum is unknown. How do the symbionts get in the microbes in the first place? Much remains unknown but I feel this study contributes significantly to the field.

http://www.pnas.org/content/110/4/E295.abstract