Continued Bioremediation of Deepwater Horizon Oil Pollution Almost a Decade On

Oil spills can be catastrophic for affected marine ecosystems. Amongst the most devastating of these events was the Deepwater Horizon disaster which began on April 20^th, 2010. Damage to the Macondo Well led to the discharge of an estimated 779 million litres of oil and gas from the sea floor into the Gulf of Mexico. Much of the oil initially deposited onto the nearby sandy coastline was manually cleaned up soon, however, oiled-sand patties continue to be washed ashore to this day. Almost a decade on from the spill, these sand patties provide the most accessible samples of residual Macondo Well oil.   

 There is a natural attenuation of oil in the environment over time, largely through microbial degradation. Researchers found that the Macondo Well oil within deposited sand patties became heavily degraded only two years after the spill, with many residues dominated by oxygenated hydrocarbon degradation products. These oxygenated hydrocarbon products have previously been hypothesised as resistant to decomposition. If so, remaining Macondo Well oil pollution could persist for a long time to come. Consequently, one recent study attempted to identify whether or not microbes were continuing to degrade the remaining, highly weathered Macondo Well oil deposits on affected beaches.

 Bostic et al. collected both oiled and non-oiled sand patties for analysis from intertidal and supratidal zones of three previously studied beaches on the coasts of Mississippi, Alabama and Florida. Initially, samples were solvent extracted and tests were carried out to ensure that acquired oil residues had originated from the Macondo Well. Moreover, the samples were characterised by gas chromatography coupled with a mass spectrometer and a flame ionisation detector. Results confirmed that samples were both Macondo Well-derived and heavily weathered in concordance with previous studies. Furthermore, analysis via thin layer chromatography coupled with a flame ionisation detector revealed that most of the solvent extractable material within the oiled-sand patties comprised oxygenated hydrocarbons (53-69%).     

 Subsequently, microbial phospholipid fatty acids (PLFA) were extracted from both oiled and non-oiled sand patties for analysis of natural abundance radiocarbon (¹⁴C) content. PLFA degrade rapidly following cell death so offer valuable insight into the viable microbial community and their C sources at the time of sampling. By measuring ¹⁴C content within the PLFA, it is possible to identify microbial C sources as either petroleum or as modern, photosynthesis-derived organic matter; petroleum contains significantly depleted ¹⁴C compared with modern organic matter.

 ¹⁴C content measurements revealed that microbial PLFA extracted from oiled-sand patties primarily contained carbon which was petroleum-derived whereas microbial PLFA extracted from non-oiled-sand patties contained carbon consistent with modern organic matter. Accordingly, it is clear that microbial communities within the oiled-patties are utilising different carbon sources to the communities residing within the non-oiled-patties. Additionally, there was no significant difference in ¹⁴C measurements between intertidal and supratidal oiled-patties, suggesting that proximity to fresh carbon sources and moisture had no influence on the ¹⁴C content of microbial PLFA. Therefore, Bostic et al. were able to conclude that petroleum carbon is the primary source of carbon for the microbes inhabiting all studied oiled-sand patties.  

 The results of this study are promising, highlighting the potential for further bioremediation of Deepwater Horizon residual oil pollution, contrary to previous suggestions that such heavily weathered oil could be resistant to degradation. Nevertheless, this study delivers only a snapshot of weathered oil degradation, providing no insight into the degradation rates of the remaining oil. As such, further research should utilise incubation techniques in an attempt to quantify these degradation rates, permitting improved estimates to be made of when affected ecosystems will return to their pre-Deepwater Horizon states. Additionally, employing metagenomics could identify key OTUs in the degradation process of weathered oil, highlighting shifts in oil degrading bacterial communities as oil becomes more heavily degraded over time.

Reviewed Paper:

Bostic, J. T., Aeppli, C., Swarthout, R. F., Reddy, C. M. & Ziolkowski, L. A. (2018). Ongoing biodegradation of Deepwater Horizon oil in beach sands: Insights from tracing petroleum carbon into microbial biomass. Marine Pollution Bulletin, 126: 130-136

Interactions between probiotic, pathogen and protein sources

Probiotics are an integral part of aquaculture practices to increase yields of fish by limiting loss through disease. Modulation of the immune system is a commonly reported benefit, with high potency to stimulate immunity seen under in vitro and in vivo conditions. There are an assortment of common bacteria used including Carnobacterium. The probiotic is naturally occuring in the gut microbiota of salmonids and has displayed antimicrobial abilities, but to be a favourable probiotic it must be able to adhere and grow in the mucus or on the enterocyte surface of the digestive tract.

Hartviksen et al 2015 studied the adherence of a species of this genus, Carnobacterium divergens, and whether it could exclude or displace the common fish pathogen Aeromonas salmonicida using ex vivo methods. This has been utilised for it combines the best of both in vitro and in vivo methods. The use of the intestine environment means results are more applicable to reality, and the removal of it from the organism allowing for flushing etc. enables greater control over variables. There are limitations, for the tissue viability will decrease over time, leading to the use of only one hour incubation as any longer may result in its degradation. Intestines were exposed to either saline (the control) the probiotic, the pathogen, first the probiotic then pathogen or the reverse. The effects of diet, which the fish were given for nine weeks, were also investigated the four variants being: fishmeal (FM), pea protein concentrate (PPC), extracted sunflower (ESF) and feather meal (FeM). Diet was considered as it is known to influence intestinal microbiota and structural integrity.

General exposure to C. divergens showed improvement of the intestinal structure with lower frequencies of intraepithelial leukocytes and debris in the lumen, as well as higher frequency of healthy mitochondria. Intestines exposed to A. salmonicida had greater debris in the lumen with damaged microvilli and enterocytes across all feed types, with FeM having the greatest changes observed up to 7 micrographs per individual showing damage. Having the probiotic before the pathogen showed similar intestinal structuring to the control but the reverse showed in all dietary groups an increase in tissue oedema. Fish fed FeM had an apparent decrease in the prevalence of healthy mitochondria. In the control, diet seemed to have very little effect on the levels of both probiotic and pathogen.

Concerning adherence to the intestine in all treatments where C. divergens was present it increased compared to the saline exposed control; diet had no influence nor was there an interaction between diet and treatment. Levels of the probiotic would lower on exposure to the pathogen in comparison to when solitary, illustrating that A. salmonicida is an opportunistic bacterium that can displace the endogenous probiotic. However pre and post exposures of C. divergens would cause lower levels of the pathogen compared to its singular treatment, so the probiotic had some desired effect.

Alternative protein sources in the form of the varying diets can be seen to not significantly affect the uptake and adherence of both bacteria species. Nor was there any dramatic alterations to intestinal structure following the control or probiotic exposure. However the combination of feed especially FeM with A. salmonicida enhanced structural changes and damage. The mechanism is unknown however as with the control FeM caused no alterations to structure. It is therefore important to consider feed type not only in its appropriateness to the animal but also on whether it increases susceptibility to disease. However the use of C. divergens as a probiotic does seem promising, especially in that the protein source had no effect on its ability to establish so could possibly be supplemented alongside any feed type.

Paper reviewed:

Jose L Gonzalez Vecino, M. (2015). Probiotic and Pathogen Ex-vivo Exposure of Atlantic Salmon (Salmo Salar L.) Intestine from Fish Fed Four Different Protein Sources. Journal of Aquaculture Research & Development, 06(05).

DNA containing extracellular vesicles leading to confusion

The possibilities of cells to communicate and interact with each other and their surrounding are vast. Biofilms are a great example – communication appears on different levels. Cells interchange biomolecules via direct physical contact, release chemical molecules or regulate the expression of genes as a response to the surrounding cell density, known as quorum sensing (Miller and Bassler, 2001). Another way to communicate for microorganisms is the excretion of extracellular vesicles (EVs). Cells of all domains of life produce EVs. These tiny structures, defined by a lipid-membrane can contain lipids, proteins, nucleic acids such as DNA fragments or other biomolecules. The significance of these small “bubbles” regarding biological processes is impressive. Apart from enabling intercellular communication, EVs play a role in pathogenesis, the acquisition of nutrients, in biofilms or can even be part in the cellular defence mechanisms.

Due to the appearance and other characteristics EVs are hard to distinguish from viruses. When those biological particles were first discovered concerns arose in the world of viral research regarding a widely used method for the measurement of viral abundance in seawater. The basis of this standard approach is a fluorescent DNA-binding dye, often SYBR dye. DNA-containing EVs could have been detected via this method unknowingly leading to overestimated numbers of viral abundance.

In the presented study, the main aim was to find out more about DNA containing extracellular vesicles by measuring the size and frequency of fragments and to evaluate the standard method of DNA-binding epifluorescence for the assessment of viral abundance.

The organisms of interest were four different gram-negative heterotrophic bacteria species (Prochloroccocus, Salinicola, Alteromonas and Thalassospira), cultivated as axenic cultures.

Interestingly the DNA content varied significantly among the four marine bacteria. Each of the released DNA fragments possessed unique and species-specific size distribution, between 35bp and 10kb. With the use of SYBR-staining the researches observed a heterogeneous DNA distribution, meaning the fragments were not uniformly scattered among individual vesicles. Therefore only <0.1 vesicles were identified with that method. Exclusively vesicles with large DNA-fragments could be visualized.

The investigated differences in size and amount of DNA fragments on a species-level indicate that vesicles may have a high potential to act as agent of horizontal gene transfer. The researchers underline the importance to look deeper into this subject, because DNA-containing EVs may have a significant effect in the marine environment by acting as defence agents or “vehicles” of transport between targeted host cells of viruses leading to a change of sensitivity to those pathogens.

To assess the questioned method for viral counting and the applicability in the field, seawater samples were collected. Half of the samples were treated with chloroform, which disrupted the lipid-membrane structure of EVs, to enable a first separation of vesicles and viruses. The other half was kept as a control. Eventually the chloroform did not have a significant impact on counts of particles. In sum, the epifluorescence method only led to a fraction of visualized vesicles in the laboratory. Combined with the field-based work where a relatively small decrease in SYBR-bound particles appeared upon chloroform treatment, EVs do not impact the estimates of viral abundance in marine waters notably.

Conclusively, this study is a great example demonstrating that a high quality data collection and analysis needs suitable methods. With new findings previous methods should be questioned, examined, evaluated for they furthermore applicability. Regarding EVs more research is needed in order to understand the exact mechanisms of fragment and vesicle production and to work out the role of EVs on an ecological level to a greater extend.

Article reviewed:

Biller, S. J., McDaniel, L. D., Breitbart, M., Rogers, E., Paul, J. H., & Chisholm, S. W. (2017). Membrane vesicles in sea water: heterogeneous DNA content and implications for viral abundance estimates. The ISME journal, 11(2), 394.

References:
 

Miller, M. B., & Bassler, B. L. (2001). Quorum sensing in bacteria. Annual Reviews in Microbiology, 55(1), 165-199.

Schatz, D., & Vardi, A. (2018). Extracellular vesicles—new players in cell–cell communication in aquatic environments. Current opinion in microbiology, 43, 148-154.

Microbes munching on microplastics in mangrove mud

Microplastic pollution in marine ecosystems is a contemporary issue that has been at the forefront of environmental research and the media in recent years. Roughly 4.8 million tonnes of plastic enter the marine environment every year with microplastics constituting 92.4% of this waste. Microplastics are often made deliberately for use in cosmetics that then pass through wastewater treatment or by the weathering and breakdown of large plastic debris.

Microplastics have a global ocean distribution and we are still only just beginning to discover the ill effects that they cause to marine animals and environments. Microplastics are often found within filter feeding invertebrates, marine mammals and seabirds causing pathological stress, stunted growth, and false satiation and even facilitate the accumulation of heavy metals in marine sediments. Even with ever-increasing evidence of the damage to marine life microplastics cause, less attention is given to strategies for environmental remediation. Mangrove forests support a high diversity of microbes and are often subjected to high plastic pollution, could members of these microbial communities be a solution to our growing plastic problem?

The aims of the reviewed study were to provide a remediation solution to microplastic polluted environments, using bacterial isolates, and to evaluate the potential of marine bacteria to degrade microplastics.

Microplastic polyethylene, Polypropylene (PP), Polystyrene and Polyethylene terephthalate were collected and treated with UV-radiation. Sediment samples were taken from the top 4 cm from six mangrove forests around the Peninsular Malaysia from which bacteria were isolated, plated onto nutrient agar (NA) and incubated. Pure cultures were obtained after incubation for species identification and plating onto a mineral salt medium (MSM) with the UV-treated microplastics as the only carbon source. Species that could grow on the MSM were inoculated into an MSM broth containing one of the four microplastics and incubated for 40 days. Cell growth was monitored every 10 days and after 40 days microplastic weight loss, the rate of degradation and half-life was calculated. Changes in the plastic structure were assessed by Fourier transform infrared and scanning electron microscopy (SEM).

Only two isolates obtained from incubation could grow in the presence of plastics, these were Bacillus cereus and Bacillus gottheilii. Both species showed similar growth patterns over the 40-day incubations with exponential growth between day 0 and 20 followed by a die off. However, B. gottheilii could grow in the presence of PP where B. cereus could not. B. gottheilii shows a wider capacity to degrade microplastics than B. cereus while also degrading plastics at a higher rate. Microplastics were also shown to be oxidised when inoculated, these bacterial species are able to alter the chemical structure of plastic polymers to make them easier to adhere to and degrade. SEM observations also revealed that plastics inoculated with B. cereus and B. gottheilii had much rougher surfaces and bore crevices and holes.

This study has revealed two bacterial species with a capacity to degrade plastic and could serve as a solution to boost remediation of polluted mangrove sediments. Adding bacteria to sediments may be an “environmentally safe” method of plastic clean-up, but future work should focus on what effect if any, adding these microbes has on the community dynamics of the sediment. Also, subsequent work should try to establish the molecular and genetic pathways these microbes possess that are involved in microplastic degradation.

Paper reviewed:

Auta, H.S., Emenike, C.U., & Fauziah, S.H. (2017). Screening of Bacillus strains isolated from mangrove ecosystems in Peninsular Malaysia for microplastic degradation. Environmental pollution, 231, 1552-1559.

Reef Fish Farming Behaviour May Be Promoting Coral Disease

 Anthropogenic driven environmental change has led to the severe degradation of coral reefs worldwide. The occurrence of coral disease has increased significantly in recent years, resulting in mass coral mortality and subsequent habitat loss for countless species. Whilst the exact causes of coral disease continue to elude researchers, well studied coral diseases such as black band disease (BBD) are associated with a consortium of pathogenic microbes infecting the host. As such, gaining a more comprehensive understanding of the processes influencing microbial community structure and disease ecology within coral reef ecosystems is paramount to the efficacy of future conservation efforts.

 Previous research has emphasised the significance of coral-algae interactions in configuring reef microbial communities, yet few studies have addressed the roles that fish may play in mediating these interactions. Consequently, one recent study endeavoured to examine how the grazing activities of territorial damselfish may indirectly alter benthic microbial populations, potentially influencing the prevalence of coral disease.  

 In order to cultivate patches of palatable filamentous algae, damselfish farm the reef benthos, weeding out unfavourable algal species. Accordingly, damselfish engineer reef ecosystems, promoting a large biomass of low-diversity turf algae. However, previous work has illustrated turf algae to be detrimental to coral health, accommodating potentially coral-pathogenic bacteria as well as releasing harmful dissolved compounds. Therefore, it is possible that damselfish farming behaviour could prove damaging to reef health.     

 Casey et al. investigated the benthic microbial communities of shallow reefs surrounding Lizard Island, situated in the northern Great Barrier Reef. Sampling was carried out within the territories of two damselfish species, Stegastes nigricans and Stegastes apicalis, as well as within control plots devoid of territorial grazers. Initially, algal compositions were characterised inside the territories of both species, revealing assemblages dominated by rhodophytes (over 50% coverage from Polysiphonia sp.) with almost all macroalgae eliminated.

 Benthic microbial communities were subsequently characterised by analysing samples of epilithic algal matrix (EAM). EAM is the overriding component of benthos within damselfish territories and is made up largely of turf algae, detritus, and an array of associated microbes. Bacterial assemblages within EAM samples were characterised by 16S rDNA sequencing, revealing that damselfish territories have microbial communities distinct from control plots. EAM microbial communities within S. nigricans and S. apicalis territories, whilst distinct from one another displayed some similarities, probably due to the dominance of Polysiphonia sp. cultivated by both species. Similarly, overlaps were found between EAM microbial communities in S. apicalis territories and control samples. S. apicalis tend to select plots on more flattened areas of benthos, comparable to control plots, likely accounting for these overlaps. In contrast, S. nigricans cultivates algae on the branches of acroporid coral.  

 Analysis of bacterial phylotypes within samples revealed that damselfish territories contained two to three times more potential coral pathogens than control samples. These potential pathogens are cyanobacteria belonging to generas Leptolyngbya and Oscillatoria and have been previously connected to the pathogenicity of BBD. Furthermore, coral disease surveys found that staghorn coral, Acropora muricata, was significantly more likely to be afflicted with BBD within territories of S. nigricans than within control plots. Together, these findings stress the link between damselfish grazing activities altering reef benthos and increased prevalence of microbes associated with coral disease.     

 As damselfish abundance appears to be increasing as an indirect consequence of overfishing, we may expect to see a proliferation in benthos sculpted by their farming behaviours. This study effectively demonstrates the potential adverse consequences for coral reef health associated with such ecosystem alteration, making a valuable contribution to the current understanding of coral reef disease ecology. Nevertheless, research is required to gain a clearer understanding of the mechanisms underpinning such shifts in benthic microbial communities, perhaps also factoring in abiotic variables such as water temperature, known to strongly influence coral disease occurrence.  

Reviewed Paper:

Casey, J. M., Ainsworth, T. D., Choat, J. M. & Connolly, S. R. (2014). Farming behavior of reef fishes increases the prevalence of coral disease associated microbes and black band disease. Proceedings of the Royal Society, 281: 20141032.