3 Phages Walk Into a Vibrio

Apostichopus japonicas, commonly known as the Japanese sea cucumber is a temperate shallow water species found along the coasts of south east Asia. Its common use is as an edible consumable and as such is considerably over fished. Sea cucumbers aquaculture is a rapidly growing, extremely profitable industry. The industry has the largest economic output for any single marine species in the whole of China. As with any aquaculture, disease has a serious negative effect on both yield and profit. Skin ulceration syndrome is one of the more serious diseases that affects cucumbers and is commonly caused by Vibrio species, in particular Vibrio splendidus.

At present antibiotics are used to control bacterial diseases, however, phage therapy is an alternative method which does not result in environmental pollution or antibiotic resistance. Li et al., sought to isolate and test the efficiency of phages that protect cucumbers against V.splendidus infection. Being the first study to assess the potential of a phage cocktail lytic to V.splendidus. 3 phages were isolated from raw sewage obtained from cucumber hatcheries. The sewage samples were introduced to pellets of V.splendidus which were then filtered through a 0.22 μm filter after a 24 hour incubation period. The presence of phages was then confirmed using the Double-Layered Agar Method. The phages were inspected using a Transmission Electron Microscope and their morphological features including head, tail and tail fibers were recorded. Cultures of V.splendidus were incubated in a 96 well plate for 6 hours with the turbidity of plates being visually examined to calculate the lytic capability of the 3 phages. The inhibition of bacterial growth was then tested using either a cocktail of all 3 phages in a ratio of 1:1:1 or each phage separately. A 96 well plate was inoculated and each phage dilution was added with a control for plate sterility, phage suspension sterility and bacterial culture included. All plates were incubated for 16 hours before the optical density being measured at 600 nm.

360 cucumbers were divided into 6 groups. Each group was divided into 3 tanks each housing 20 cucumbers. The cucumbers were fed for 60 days with each treatment being fed a separate diet of either an ‘unsupplemented’ control, antibiotic, all 3 phages or each phage separately. At days 20, 40 and 60 the levels of acid phosphatase and nitric oxide synthase were measured. 90 fresh cucumbers from the same source were selected and separated into 3 treatments; a blank control, an untreated group and a treated group. The treatment was phage injection by coelomic injection and was done to test the survival rate and enzyme activity in the coelomic fluid.

The electron microscopy of the phages revealed that all 3 belong to the order Caudovirales, with two belonging to the family Myoviridae and one belonging to the family Siphoviridae. The effects of each of the 3 phages on V.splendidus showed a significant decrease in the optical density at 600 nm compared to the control culture. For the ‘unsupplemented’ diet the survival rate was 18% whereas both antibiotic and phage cocktail treatments had 82% survival rates. The 3 phages individually showed far higher survival rates than the ‘unsupplemented’ treatment but not as high as the 3 combined. There was a significant difference detected between phage PVS-1 and PVS-3 but not between PVS-2 and the other phages. After the 60 days, there was no significant difference in weight gain or ingestion rate across any of the treatments. The levels of enzymes did significantly increase in cucumbers fed phages over the ‘unsupplemented’ treatment. There was however, no significant difference between antibiotic and ‘unsupplemented’ cucumber enzyme levels.

This study is very interesting and very useful for combatting current global problems. It follows on from a lot of previous studies and provides further evidence to support the move away from antibiotic use. Whilst showing that phage therapy is just as effective as antibiotics the authors also discovered 3 new bacteriophages lytic to V.splendidus and imaged them. The study uses proven microbiological techniques which are very robust and provide good, usable results in my opinion. My main qualm with this study is the size of it. I feel whilst done very well the authors attempt to do too much in one study. If each experiment was done separately or less was done, then more time and resources could have potentially been spent on individual elements. Whilst this study serves its purpose well, allowing for the potential disease reduction of aquaculture species and the reduction of antibiotics in the environment, the potential for broader future study is low. 

Li, Z., Li, X., Zhang, J., Wang, X., Wang, L., Cao, Z. and Xu, Y. (2016). Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus. Fish & Shellfish Immunology, 54, pp.302-311.

http://www.sciencedirect.com/science/article/pii/S1050464816301772

Hurricane Irene wipes her salty shoes on the Ocean's welcome mat

It has only been in the last 20 years that the idea of microbes forming colonies on surfaces in shallow, intertidal and deeper waters, has come to light. But now we are faced with exciting research showing the complex nature of microbial mats and how their diversity and interactions with the microhabitat around them are formed. These delicate systems are placed under many pressures everyday such as light, nutrient and oxygen availability. Preisner et al looked into microbial mats and how taxa within these diverse systems can provide stable biological reservoirs that are influenced by environmental stressors such as salinity brought on by natural disasters, in this instance, Hurrican Irene which hit the coast at the end of August 2011. Microbial mats can arguably have some of the highest diversity which, these in turn, influences foundations of ecological processes so any additional stressors should be recognised and taken into account when understanding these delicate microbial ecological interactions. Much of the greatest diversity is within low abundance taxa, which this study takes into account. While much of the biomass of microbial mats is common within a few dominant taxa, both the dominant and ‘rare’ taxas show different activity levels within environmental pressures.

The paper studied a microbial mat on San Salvador Island in the Bahamas on August 1^st-2^nd in 2011 (Pre Hurricane Irene) and again on August 1^st – 2^nd in 2012 (Post Hurricane Irene). This microbial mat consisted of well-defined layers, which as we understand from general knowledge of microbial mats, can have steep gradients of oxygen, light and sulfide availability. These gradients were taken into account to an extent in this paper, however it is not the primary focus.

Post disturbance the reduction in salinity showed that general community composition was not affected significantly diurnally however general archaea decreased from 57% to 33% and Bacteria increased from 43% to 67%. Most taxa showed low activity across conditions but activity levels in general did increase after disturbance. This highlights how these communities are sensitive to environmental change. Pre-disturbance showed to favour Halobacteria as they are shown to thrive in lower salinities, Halobacteria held 48% of the community biomass predisturnbance however these results were favouring towards one OTU halobacteriaceae. Halobacteriaceae are defined by their halophilic nature and are abundant in hypersaline conditions. Post disturbance showed abundance and activity decrease in Halobacteria from 48% to 31%, however abundance and activity increased in Proteobacteria, Cyanobacteria, Thermoplasmata, Thaumarchaeota and Crenarchaeota.

Cyanobacteria are known to be tolerant of a range of salinities however results did show a decrease in its nitrogen fixating abilities. Cyanobacteria are important for the formation of microbial mats, filamentous Cyanobacteria such as Phormidium are dominant members in the mats and although not mentioned in this paper, it is important to remember the interaction of this microorganism with microbial mats so when disturbance occurs we can understand any impacts it will have on healthy mats.

The sample site chosen is often disturbed by natural disasters like hurricanes, so is therefore used to environmental pressures such as wide ranges of salinity, high irradiance and high temperatures. This study site does seem ideal for studying larger communities of microbial diversity in relation to salinity, however it does suggest that these communities are already well adapted to cope with pressures, therefore it would be interesting to see the diversity and activity rates in communities in other, less pressurised areas.

Paper reviewed: Preisner, E. C., Fichot, E. B., & Norman, R. S. (2016). Microbial Mat Functional and Compositional Sensitivity to Environmental Disturbance. bioRxiv, 063370. http://www.biorxiv.org/content/biorxiv/early/2016/07/12/063370.full.pdf

Fuel for Bacteria

During the last two decades, alternative hydrocarbon fuels have gained popularity. These alternative fuels are produced by biological processes such as agriculture and anaerobic digestion. Not to be confused with biodiesel and ethanol biofuels, these alternative fuels are similar to conventional hydrocarbon fuels and can be used in regular vehicles. Despite being more environmentally friendly in production, alternative hydrocarbon fuels can also have significant impacts on the environment. In their paper, Ruiz et al. (2015) investigated how differences in hydrocarbon composition affected bacterial communities. 

Coastal seawater containing sediments was collected from Florida. Samples were set up in tanks and monitored over 90 days. Petroleum types were chosen based on their prevalence in civilian and military transport. The impacts of conventional fuels, F-76 (military petroleum marine diesel) and JP-5 (petroleum jet propellant) as well as camelina-derived renewable jet propellant (HRJ-5) and a 50/50 mixture of JP-5 and HRJ-5 (Blend) were studied. Bacterial isolates were cultured and bacterial growth was monitored using quantitative Real-Time PCR. Individual fuel degradation profiles were analysed by gas chromatography-mass spectrometry (GC-MS). 

Analysis of the unexposed seawater samples showed a diverse and mainly uncharacterized bacterial community. The diversity was higher in the sediment fractions, and sequencing coverage was not sufficient to detect all bacteria present. Proteobacteria were the most abundant group in both liquid and sediment, with Bacteroidetes and an unclassified group coming in second and third. Firmicutes and Lentisphaerae were also detected. Within each group, most of the bacteria were unclassified at the genus level.

Exposure to fuels decreased the biodiversity, however, the impact of the diesel F-76 was less severe than that of the jet fuels. Proteobacteria abundance was enhanced by all fuels, while Bacteroidetes decreased with fuel presence. Exposure to JP-5 and Blend promoted Firmicutes and Lentisphaerae, respectively. In Proteobacteria, JP-5 increased the abundance of the Marinobacter and Dusulfovibrio genera. HRJ enhanced Hyphomonas. F-76 promoted the growth of Rhodovulum and unclassified genera, while the Blend mainly promoted unclassified groups. The differences in communities were presumably connected to compositional differences in the fuels. 

Subsequently, the hydrocarbon-degradation profiles of two bacterial strains were examined. GC-MS analysis showed Marinobacter hydrocarbonoclasticus N19 mainly degraded short chained n-alkanes and specific aromatics. In contrast, Rhodovulum sp. NI22 primarily degraded naphthalene, light branched and n-alkanes as well as aromatics. These differences were attributed to different degradation enzymes such as alkane monooxygenase and naphthalene dioxygenase in M. hydrocarbonoclasticus and Rhodovulum sp., respectively. 

Metagenomic analysis showed that different fuels increased the growth and abundance of specific bacterial groups. In a separate experiment, M. hydrocarbonoclasticus and Rhodovulum sp. were cultured together with Halobacillus sp. and exposed to different fuels. After ten days, Marinobacter dominated all samples. HRJ supported the highest total abundance of bacteria and enhanced Marinobacter as well as Rhodovulum. Presumably, the abundance of light alkanes in HRJ promoted the growth of these bacteria. JP-5 seemed to impact all three groups negatively. Along with metabolic flexibility, competition between hydrocarbon degraders may influence bioremediation. Once the dominant bacteria have degraded their compounds, out-competed bacteria might grow back by consuming different hydrocarbon compounds. The authors suggest that bioremediation might also benefit from the systemic addition of specific bacteria once some compounds have been degraded.

In conclusion, this paper provides useful information for the development of new fuels and to maximise bioremediation. The main finding that different fuels enhance growth and abundance of specific bacteria is perhaps not very surprising. However, the experimental design could only show culturable bacteria of which most were unclassified. Confusingly, different fuels were used in different experiments without giving any justification. In my review, I consciously chose to refer only to the four fuels used in the first experiment.

Reference:

Ruiz, O. N., Brown, L. M., Striebich, R. C., Smart, C. E., Bowen, L. L., Lee, J. S., ... & Gunasekera, T. S. (2015). Effect of Conventional and Alternative Fuels on a Marine Bacterial Community and the Significance to Bioremediation. Energy & Fuels, 30(1), 434-444. http://pubs.acs.org/doi/abs/10.1021/acs.energyfuels.5b02439

When the usual suspect is not the suspect…?

Crassostrea gigas (pacific oyster) has been characterised by a number of successive outbreaks, resulting in dramatic losses of production in France, which has consequently lead to subsequent decline within the industry. The herpes virus (OsHV-1μvar) has often been associated with the deaths of juvenile oysters, along with members of the Vibrionales. The pathogenicity of OsHV-1μvar, as well as several Vibrio, has been demonstrated by experimental infections; however, our understanding of the complexity of these infections is still limited. This study uses specific-pathogen-free (SPF) oysters, infected in estuarine environments, to study the diversity and dynamics of cultured microbial populations during disease expression.

Wild oyster spat was collected and moved to grow in areas within Northern Brittany between 2012 and 2014. Oysters were exposed to disease during spring 2012, and suffered >50% mortality. In 2014, 60 individuals were transferred to experimental facilities, and treated with an antibiotic (chloramphenicol) for 5 days prior to maturation; then reared in controlled conditions until reaching a mean individual wet mass between 0.5 and 3g. qPCR was used to quantify the herpes virus at different developmental stages.

SPF oysters were maintained in another region, during a disease outbreak. Total mortalities resulting in the field were recorded. For cohabitation experiments, field-exposed oysters (“donors”) were brought back to the lab and cohabited in a tank with SPF oysters (“recipient”) under controlled experimental conditions. Chloramphenicol was added to tanks every 2 days to remove any cultivable microbiota, with the number of dead or alive donors being daily counted.

After being killed, oysters had their digestive glands removed and were ground. The total cultivable bacteria and Vibrio microbiota were quantified (cfu/mg of tissue) using serial dilutions on marine agar and TCBS (a type of agar culture plate used in microbiology laboratories to isolate Vibrio sp) selective media. Additionally, the haemolymph from randomly selected colonies surviving either longer than 9 days or less than 24hr was non-destructively acquired and amplified with PCR using the partial hsp60 gene.

OsVH-1 DNA was quantified from ground tissue using PCR. Bacteria were grown under constant agitation at a set temperature for 24h. Intramuscular injections of (10⁷ cfu) were made into the oysters, and bacterial concentration confirmed by conventional dilution plating on agar.

Results

Both adults and juveniles act as reservoirs of pathogens which are able to induce mortalities of young. Only juveniles were shown to transmit the herpes virus, suggesting that the transmitted diseases between the two are distinct and that virus infection is not a prerequisite for juvenile mortality outbreaks. A not-so-surprising discovery was that farmed oysters seem to present higher transmission risks than wild oysters. Additionally, geographic origin of oysters also influences the consortium of pathogens.

Rapid Vibrio colonisation followed by viral replication occurs before oyster death, and no correlation was determined between Vibrio concentration and viral load in co-infected oysters. Instead, the quantity of viral DNA can be used as a predictor of mortality, with OsHV-1 DNA detected in the haemolymph of oysters which died within the first day found to be significantly higher ( up to 4 log units) than those of oysters which lived up to 9 days. Interestingly, the absence of bacteria meant that even a high load of herpes virus is insufficient to induce full disease expression. Juvenile mortalities can occur in the absence of herpes virus, strongly indicating that the herpes virus is neither essential nor sufficient to induce juvenile death, whilst bacteria are essential for disease. Lastly, this paper highlights that the geographic origin, cultivation method and age are all influential factors in disease expression.

I feel that this paper is an interesting read, as it opens up an almost-settled debate on the origin of pathogenicity in Oysters. Herpes virus’ in many papers has been proven as the etiological agent; however, this paper challenges this and shows that it is, in fact, the presence of bacteria, not the herpes virus, which is most important.

Reviewed paper

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4491618/

                                                                                                                                           

Petton, B., Bruto, M., James, A., Labreuche, Y., Alunno-Bruscia, M. and Le Roux, F., 2015. Crassostrea gigas mortality in France: the usual suspect, a herpes virus, may not be the killer in this polymicrobial opportunistic disease. Frontiers in microbiology, 6.

Checking out the Molecular Barcodes of Loricate Choanoflagellates

In recent years, a sharp increase in the capacity of high throughput DNA sequencing (HTS) has been met with a concomitant decrease in its cost (Caporaso et al, 2012). As a result, vast databases of marine microbial metagenomes and -barcodes have been gathered from research cruises across the global oceans. One such cruise was the Tara Oceans expedition (2009-2013), which sampled 210 oceanic stations. Like other HTS studies, Tara Oceans revealed swathes of OTU molecular barcodes unidentified at high taxonomic resolutions. This problem highlights a key issue in the HTS age of marine microbiology: the paucity of curated sequences coupled to morphological and taxonomic descriptions. Nitsche and colleagues (2016) set out to remedy this problem, by creating a database of curated rDNA barcodes from acanthoecid (loricate) choanoflagellates. Unlike their naked cousins, acanthoecid choanoflagellates are strictly marine and synthesise a complex, silica lorica with species-specific morphology. As abundant bacterivorous nanoflagellates, choanoflagellates can too have a considerably effect on marine carbon flow. Therefore, the ecological importance and well-defined morphology of this group make the acanthoecid choanoflagellates good candidates as a proof of concept to resolve the identity of unknown HTS barcodes.

Although 115 species of this group have been described, <10% of them have been sequenced. Therefore, the authors collected choanoflagellates from Danish coastal waters and manipulated mixed cultures at the single cell level, to characterise both lorica morphology and ribosomal DNA sequence. Their curated findings supplemented and re-described previous species characterisations, which covered 45 morphospecies. For the first time, 9 species and 5 genera were characterised at the barcode level. The authors used their sequences to mine the Tara Ocean database for acanthoecid V9 barcodes and make a startling discovery – that one of their characterised species (Calliacantha natans) represented the second most abundant choanoflagellate in the global oceans, which was previously unidentified. This study, with relatively little effort, doubled the curated sequence database of this potentially biogeochemically important clade and showed that locally collected choanoflagellates from Danish waters have a global distribution and abundance.

On the surface, this study is taxonomically dense and offers little to those not passionately interested in details of acanthoecid classification. However, this study acts as a proof of concept to stress the importance of curating large HTS databases. The identification of the second most globally abundant choanoflagellate from the Tara Oceans is a big achievement and, while monocultures were not achieved by this study, I believe a fascinating next step would be to isolate C. natans and explore its cell biology and trophodynamics in the lab. This could yield real insights into global marine carbon flow. As well as this, I think a great candidate for future work of this type would be the tintinnid ciliates, who share with the acnathoecids a distinct loricate morphology and global distribution (Dolan et al, 2012).

Overall this study is a small, but impressive, step forward into coupling morphological and HTS barcode data and offers a simple technique to answer some of the larger questions surrounding unidentified barcoded taxa.

Reviewed Paper: Nitsche, F., Thomsen, H. A., & Richter, D. J. (2016). Bridging the gap between morphological species and molecular barcodes− exemplified by loricate choanoflagellates. European Journal of Protistology. http://www.sciencedirect.com/science/article/pii/S0932473916301080

HTS: Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., ... & Gormley, N. (2012). Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME journal, 6(8), 1621-1624. http://www.nature.com/ismej/journal/v6/n8/abs/ismej20128a.html

Tintinnids: Dolan, J. R., Montagnes, D. J., Agatha, S., Coats, D. W., & Stoecker, D. K. (Eds.). (2012). The biology and ecology of tintinnid ciliates: Models for marine plankton. John Wiley & Sons. 

The role coral reefs may play in the sea surface microlayer

The sea surface microlayer (SML) is important in the exchange of gases between the atmosphere and the oceans, the enrichment of organic compounds in the SML has been shown to suppress gas exchange. The SML is home to a distinct microbial community most studies looking into microorganisms in the SML have been focused in temperate regions with much less conducted in tropical regions before the study by Nakajima et al (2013), there were no studies conducted focusing on the SML in coral reef areas.

In coral reef ecosystems scleractinian corals release a large amount of organic matter which is known as coral mucus. Corals have been found to release up to half of the organic carbon provided by zooxanthellae into the surrounding seawater. The gel like mucus contains gas bubbles which result in positive buoyancy as the mucus travels up the water column it traps various organic particles which allows it to become enriched in microorganisms. The authors looked at the difference in enrichment of microorganisms in the SML compared to SSW over coral reefs, if this microbial abundance and production varied with coral cover and how microbial biomass in the SML of coral reefs compared with other marine ecosystems.

The study investigated a fringing coral reef at Bidong Island, Malaysia samples were collected over the course of 3 days using a metal mesh sampler which collected the upper 250 μm of the water layer. The sites were categorised into higher live coral coverage (HCC) and lower coral coverage (LCC) samples were collected from both the SML (surface microlayer) and SSW (subsurface water).

Findings

With the exception of bacterial production and growth rate enrichment in the SML was significant compared to the SSW for every biological parameter. With increasing coral coverage enrichment factor (EFs) also increased. This higher bacterial abundance in the SML could be due to production of bacteria in the SML or supply of bacteria from the SSW through adhesion to rising bubbles. The result of the paper suggest that it is likely the latter which explains this trend, although bacteria production was slightly higher in the SML the enrichment factor was lower than those for bacterial abundance suggests the SSW is likely the major source of bacteria. A reason why the bacterial growth rate may have been lower in the SML is that bacterial growth may be inhibited in the SML due to higher exposure to UV radiation this would be especially important in the lower latitudes where this coral reef was found. In the HCC sites due to higher coral coverage resulting in a higher release of coral mucus this explains why there is a higher enrichment factor for the HCC sites however the study did not directly measure particulate organic matter which would of allowed finding to be more quantitative.

Heterotrophic nanoflagellates (HNF) were the most enriched microorganisms in the SML showing a significantly higher abundance in the SML. HNF abundances were found to increase exponentially as numbers of bacteria and cyanobacteria increased which suggest that HNF grazed these organisms and therefore increased. The ratio of bacteria/HNF was found to be lower in the SML compared to the SSW indicating that grazing rates are higher in the SML HNF/bacteria and HNF/cyanobacteria rates were also lower at the HCC site suggesting that grazing of flagellates on bacteria and cyanobacteria is enhanced in the SML with high coral coverage.

The microbial composition of the SML is important because higher microbial heterotrophic metabolism in the SML is known to cause a  CO2 saturation which drives CO2 emission at the SML this is important to the findings of this study because the higher microbial abundance in the SML of coral reefs may result in a higher metabolic activity which may potential influence gas exchange between the ocean and the atmosphere furthermore higher coral coverage which results in a higher abundance of microbes may further increase this process. Due to differences in methods used it was hard for the authors to compare the EFs from their study to others one big method difference was that most recent studies use flow cytometry in contrast to the use of microscopy for cell counting in older studies. However comparing results showed that the EFs for the abundance of bacteria cyanobacteria and HNF were considerably higher in the coral reef sites compared to other sea environments.

I found this paper interesting as it looked at the impact coral mucus may have on the MSL and its part in effecting the gas exchange between the ocean and atmosphere, It showed that coral mucus does appear to play a role in influencing the community in the SML through it buoyant behaviour and it source of microbes to the SML. I think further study is needed as this paper only looked at 2 coral sites and an offshore coral site. A disadvantage I found with this paper was I do not think it’s very well structured I found that it tended to repeat itself a number of times and was not very concise.  

Nakajima, R., Tsuchiya, K., Nakatomi, N., Yoshida, T., Tada, Y., Konno, F., Toda, T., Kuwahara, V., Hamasaki, K., Othman, B., Segaran, T. and Effendy, A. (2013). Enrichment of microbial abundance in the sea-surface microlayer over a coral reef: implications for biogeochemical cycles in reef ecosystems. Marine Ecology Progress Series, 490, pp.11-22.

Mediterranean Oil Spill Mycobiota

Although occurring less, oil spills have huge environmental impacts. Several different techniques have been used to removal oil from marine ecosystems, one with potential is bioremediation. Previous work in this areas has focused largely on Bacteria and those who have looked into the use of fungi have not, as yet, demonstrated if fungi isolated from contaminated sites, can use crude oil as their sole carbon source. Bovio et al (2017) made use of a spill off the coast of Sicily, in 2013 to help provide some insight into this question as well as investigating the mycobiota post disaster.

Three days after the spill, seawater and sediment was collected and marine fungi were cultured. The numbers of Colony Forming Units were estimated and fungi were identified. A fluorochrome was used to evaluate if fungi were actively growing. Actively growing colonies were isolated and exposed to crude oil within the medium and the percentage of stimulation was calculated. Four strains that grew the best with crude oil were assessed for the ability to use oil as their sole carbon source.

Nearly all of the taxa identified in the water column and sediment were Ascomycota (94%), this was in accordance with previous research. But, a lower biodiversity was observed within the sediment. Several unrecorded species were present, 12 species in the water column and 14 in the sediment. All 142 fungi isolated could grow on crude oil but differed in efficiencies; not all were positively affected, 27% (water column) and 33% (sediment) were inhibited. For the four strains that responded positively to crude oil (Aspergillus terreus MUT 271; Lulworthiales sp. MUT 263; Penicillium citreonigrum MUT 267 75; Trichoderma harzianum MUT 290), biomass was higher in the presence of crude oil. These strains also showed moderate ability to remove petroleum hydrocarbons, greater than n-C30. Other species within these genera have been attributed to oil degradation previously.

I think this is a good paper, helping to add to the promising field of bioremediation, with possible oil degrading strains of fungi for future research. One element of this paper which is of concern to me is how quickly the sample was taken after the oil spill. Although focusing on another domain of life, Kimes et al (2014) found that Obligate hydrocarbonoclastic bacteria took a little while to increase in abundance. Although increases occurred shortly after, this did not represent a long term oil affected community. Despite this, I think it is also important to know how the fungal community is affected short term too. Therefore I think it would be interesting to resample this previously unstudied system to gain a better understanding of how this dynamic system changes with a major damaging environmental change, such as an oil spill.

Bovio, E., Gnavi, G., Prigione, V., Spina, F., Denaro, R., Yakimov, M., ... & Varese, G. C. (2017). The culturable mycobiota of a Mediterranean marine site after an oil spill: Isolation, identification and potential application in bioremediation. Science of The Total Environment, 576, 310-318.

Kimes, N. E., Callaghan, A. V., Suflita, J. M., & Morris, P. J. (2014). Microbial transformation of the Deepwater Horizon oil spill—past, present, and future perspectives. Frontiers in microbiology, 5.