Use of Fungi in the Bioremediation of Crude Oil in Wake of the Deepwater Horizon Oil Spill

Since the 1970’s the amount of worldwide oil spills has decreased from around 24 per year to around 10 per year in 2010. The use of microbes in the bioremediation of crude oil is an attractive alternative to chemical dispersants or physical burning because of the negative impacts on ecosystem function these more conventional methods have. Crude oil consists of paraffinic, cyclo-paraffinic and aromatic hydrocarbons, heavy metal compounds and nitrogen, oxygen and sulphur containing compounds. This complexity in composition gives crude oil a huge range of physiochemical properties that make it extremely difficult to break down. The degradation of oil by fungi is well established, but, with the estimates of fungal diversity estimated at between 1.5 million to 3.5-5.1 million species studies exploring the use of them in oil degradation is merely scratching the surface.

This study by Simister et al (2015) set out to examine fungi isolated from oil soaked sand patties from beaches in the Gulf of Mexico that were affected by the 2010 deepwater horizon spill in order to better understand taxonomic and functional diversity.

 Patties were collected from Gulf shores in 2012 and fungi aseptically transferred to agar plates. For fungal characterisation, DNA was isolated and BLAST searches carried out. Fungi were assessed to determine whether they could utilize crude oil as a sole carbon source and then degradation experiments in triplicate for each species were carried out at 20, 30 or 40˚C. Degradation of individual components of the oil was determined and the results statistically analysed.

Isolates degraded variable quantities of oil (32-65%) with the preferential degradation of short chain n-alkanes (90-99%) and long chain n-alkane degradation even more variable (7-87%). They found a preference for degradation of low molecular weight polycyclic aromatic hydrocarbons (PAH) over high molecular weight PAHs. They found no significant or consistent trends with species at certain temperatures.

This study produces some expected and some possibly new results. The authors further hypothesise that factors such as nutritional status may have a more important impact on the ability of fungi to degrade oil than temperature. They identified 3 species of Ascomycota fungi; Fusarium, Scopulariopsis and Aspergillus all of which are commonly known to degrade petroleum hydrocarbons. This study provides further evidence for the successful use of fungi in the bioremediation of crude oil and provides the basis for further study in the area. It also raises the question of nutritional importance on the impact of crude oil degrading fungi, an interesting area and potential for future study.

Reviewed Paper:

Simister, R.L., Poutasse, C.M., Thurston, A.M., Reeve, J.L., Baker, M.C. and White, H.K., 2015. Degradation of oil by fungi isolated from Gulf of Mexico beaches. Marine pollution bulletin, 100(1), pp.327-333.

Antarctic algal-fungal associations

Antarctic lakes, soils and ice have shown the complex communities associated with plants, however there has not been much research on the algal-fungal association in the Antarctic marine environments. Godinho et al, 2013 aim to assess the diversity and distribution of fungal communities and explore their association with cold adapted macroalgae across the Antarctic Penninsula.

Thalli of eight macroalgal species were collected during December 2010 and January 2011 at intertidal transects along a rocky coastline along the Antarctic Peninsula. Macroalgae were identified and fungal specimens were isolated and incubated for 60 days before being purified. Fungi were identified to species using amplification of the ITS region and b-tubulin sequences. Diversity, richness and dominance of the fungal taxa were determined and assays for antimicrobial activity were carried out.

DNA sequences of the ITS region and b-tubulin gene from 148 fungal isolates, recovered from 391 tissue fragments from the eight macroalgal species identified they were from 21 different genera within the phyla Ascomycota, Basidiomycota and subphylum Mortierellomycotina The number of fungal taxa and diversity differed among macroalgae however the most represented order in majority of communities was Eurotiales and Penicillium species was the most frequent fungal taxa identified with 35.8% of samples. Overall there were high levels of fungal diversity associated with the Antarctic macroalgae however the values of diversity differed among the macroalgae species. Penicillium species were able to produce bioactive extracts, which may enable them to survive the extreme environment of the Antarctic. Both species of Penicillium showed antifungal activity against different species of fungi, which may have lead to the dominance of this fungus.

This paper has provided supporting evidence that the Antarctic fungi diversity is much higher than previously anticipated. This paper also highlights the ecological importance of macroalgae and associated fungi and possible future research based on this paper may also provide insight into the benefits of these algal-fungal interactions, including the potential into new insights into the biological mechanisms resulting in the tolerance of the macroalgae to the extreme marine polar regions.

Godinho, V., Furbino, L., Santiago, I., Pellizzari, F., Yokoya, N., Pupo, D., Alves, T., S Junior, P., Romanha, A., Zani, C., Cantrell, C., Rosa, C. and Rosa, L. (2013). Diversity and bioprospecting of fungal communities associated with endemic and cold-adapted macroalgae in Antarctica. The ISME Journal, 7(7), pp.1434-1451.

OMG, Vibrios produce OMVs

The production and release of outer membrane vesicles or OMVs is an important trait which all domains of life have conserved. OMVs are used mainly in transporting cell signalling compounds, DNA/RNA and toxins.  However, they have been shown to contain the content of the cytoplasm. OMVs allow the biochemical compound to be delivered to the target location at high concentrations with relatively little chemical from a safe distance. This allows bacteria to initiate disease processes in their targets without coming into contact with potentially fatal antimicrobial compounds. Despite this their ecological importance has just begun to be studied. Vibrio shilonii is an important marine pathogen and a well studied gram negative bacteria. It is most well studied in is ability to induce coral bleaching in Oculina patagonica by damaging the zooxanthellae symbionts. This study tests whether Vibrio shilonii AK1 are able to produce and release OMVs and if so address the fitness advantage for V.shilonii and their influence on the coral holobiont.  Examining the effect of temperature on OMV release in V.shilonii at 20 and 30 degrees centigrade.

The confirmation of OMVs was done using a transmission electron microscope. The OMVs were then isolated and purified. The majority of OMVs had a single membrane but occasionally vesicles with two membranes were observed. There was no significant difference observed between the amount of OMVs at the two different temperatures. Using MaxQuant analysis the authors discovered 1,405 different proteins present in the OMVs. Proteins which were identified in multiple replicates were then selected for further analysis. Intensity based absolute protein quantification or iBAQ was used to quantify the amount of each protein present. The results ranged from 10^6 to 10^9. Of the 50 most abundant proteins to be identified, these were predicted to be membrane proteins. 598 common proteins were collected at the two temperature ranges and 91 showed significant difference. 81 of which were more abundant at the lower temperature and were predicted as cytoplasmic membrane proteins. 334 were exclusive to the lower temperature whilst only 27 were exclusive to the higher temperature. Most of the proteins exclusive to the lower temperature also showed the lowest abundance in the iBAQ.  Gene ontogeny analysis showed that the majority of the proteins found at both temperatures were related to catalystic, substrate binding and transporter activity.

Screening using the agar diffusion method revealed significant quorum sensing activity in the supernatant both with and without the OMVs at both temperatures. Of all the enzymes measured, V.shilonii AK1 showed high activity. There was an increase of 2-4 fold in cultures grown at the higher temperature. What is very interesting is lipase activity increased 100 fold at the higher temperature but the lipase activity of the OMVs was similar at the two temperatures.

This study is conclusive evidence that AK1 produce OMVs but unlike similar studies there was not a significant increase in OMVs at an elevated temperature. This is interesting as an increase in sea temperature may not necessarily mean that AK1 are able to cause more disease to coral. One way in which temperature stressed bacteria rid themselves of misfolded proteins is through OMV release. So an increase in temperature could lead to more stressed pathogens which are unable to rid themselves of waste. This could potentially positively effect disease of corals. I would be interested to see how this relates in further studies.

Li, J., Azam, F. and Zhang, S. (2016). Outer membrane vesicles containing signalling molecules and active hydrolytic enzymes released by a coral pathogen Vibrio shilonii AK1. Environmental Microbiology, 18(11), pp.3850-3866.

Phycosphere fad

The phycosphere is the region around the outside of cells which is rich in organic matter and surrounds an algal cell. This area is a small niche in which bacterial communities have been shown to inhabit, utilise the extracellular products and show chemotaxis towards. Some bacteria have been shown to cause lysis or inhibit growth of harmful algal bloom (HAB) species. This is a dynamic interaction affected by different environments. Yang et al (2013) looked at the effect of algicidal bacteria on two species of red tide causing algae, Skeletonema costatum and Scrippsiella trochoidea. They looked to build upon research by Zhang et al (2010) and aimed to visualise algal morphology under SEM as well as characterising the use of different carbon forms by the communities.

When a medium that favoured bacterial growth was added the algae lysed within 72 hours, demonstrating a finite balance between the algae and its phycosphere occupiers. The morphology of the two species was similar after 48 hours, with the cells being severely deformed. S. costatum differed in bacteria being attached to the algal surface, unlike S. trochoidea. The bacterial community of S. trochoidea utilised were more diverse than S. costatum, reflecting the dynamic change in the community. The phycosphere microbial diversity indices were significantly lower than those of natural environments, suggesting a potentially more simple community structure within the phycosphere. During the lysis period, the density of bacterial cells dramatically increased, demonstrating that a large number of cells is require to induce lysis.

Biolog ECO microplates were used to assess the community level physiology of the two red tide causing species. S. trochoidea bacterial community also differed by utilising carbon more quickly than S. costatum. Both species’ sources of carbon varied significantly during the lysis of the algae and the main sources (polymers, carboxylic acids, amino acids and carbohydrates) were similar, but Skeletonema costatum carbon source use is more complex. There was an increase in the use of carboxylic acids and amino acids which may indicate the production of extracellular enzymes. These may be used to take up nutrients and causing lysis.  

Overall, I like this paper, I think it is an exciting area of research and one I had not previously considered prior to reading about in a review by Worden et al (2015). Although I feel their discussion leaves a bit to be desired and is a little messy, by not following the same clear structure given throughout the rest of the paper.  I do find myself wondering what the algicide bacteria is, therefore I think it would be interesting to use 16s rRNA sequencing to identify what the dominant species is during the lysis phase. It would also be useful for authors to further study the phycosphere in relation to biogeochemical cycles, such as carbon.

Reviewed paper:

Yang, Y., Hu, X., Zhang, J., & Gong, Y. (2013). Community level physiological study of algicidal bacteria in the phycospheres of Skeletonema costatum and Scrippsiella trochoidea. Harmful algae, 28, 88-96.

Worden, A. Z., Follows, M. J., Giovannoni, S. J., Wilken, S., Zimmerman, A. E., & Keeling, P. J. (2015). Rethinking the marine carbon cycle: Factoring in the multifarious lifestyles of microbes. Science, 347(6223), 1257594.

Zhang, J., Yang, Y. F., Gong, Y. X., Zhang, J. Y., & Jiang, J. L. (2010). The lytic effect of bacteria in the phycosphere of Skeletonema costatum and Scrippsiella trochoidea. Acta Sci Circumst, 30, 1271-1279.

Diazotroph diversity in the Arctic Ocean

Anthropogenic causes of climate change are contributing to enhanced greenhouse gases. As a result of climate change Artic sea ice is melting at alarming rates and this decline in sea ice cover has enhanced phytoplankton primary production due to the water being exposed to higher light intensities.  Nitrogen availability is usually a limiting factor of primary production in these areas, however recent understanding indicate there is an imbalance between the supply, demand and export of nitrogen in the Arctic Ocean, suggesting nitrogen fixation may be involved however little is known about the presence and role of diazotrophs in these regions.

Fernández-Méndez et al. aimed to assess the significance of nitrogen fixing organisms and their contribution to the nitrogen budget in the Arctic Ocean. They used PCR to carry out targeted analysis of nifH gene, which is responsible for the coding of the nitrogenase enzyme involved in nitrogen fixation, in 26 samples of melt ponds, sea ice and surface waters of the Central Arctic Ocean in summer 2012. Bacteria community was then identified through the analysis of the 16s rRNA region.. The effect of temperature and nutrient availability on the role and occurrence of diazotrophs was also investigated.

Results showed that of the total of 529 sequences retrieved the sequences were clustered into 43 clusters at 92% amino acid similarity. Oragnisms detected and identified within samples included both cyanobacterial and non-cyanobacterial phylotypes.  Sequences identified were associated with Proteobacteria, Firmicutes, Cyanobacteria, members of the Archaea, putative anaerobes including sulfate reducing genera of the Deltaproteobacteria, genera such as Clostridium and several uncultivated microorganisms. nifH paralogs that are thought to function in metabolic processes other than nitrogen fixation were also identified from sea ice and melt pond samples. Vast majority of sequences retrieved across the different environments of the Central Arctic Ocean belonged to non-cyanobacterial diazotrophs. Temperature gradients between sample sites did seem to have an effect on the diazotroph communities due to the optimum temperatures of different organisms. Nutrient availability also influenced the assemblage and occurrence of diazotrophs as cyanobacterial organisms were associated with high phosphate environments, therefore affecting their abundance and distribution.

Overall I believe this to be an important paper because it highlights the potential for nitrogen fixation in Arctic environments where they have not been previously detected. Understanding these diazotrophs role and distribution in the environment is vital to dictate the implications and relevance to climate change and the influence it has on the Arctic’s primary production. I believe this paper to be a key stone paper for future research of nitrogen fixers in polar environments and to assess their importance and contribution to the ecosystem.

Fernández-Méndez, M., Turk-Kubo, K., Buttigieg, P., Rapp, J., Krumpen, T., Zehr, J. and Boetius, A. (2016). Diazotroph Diversity in the Sea Ice, Melt Ponds, and Surface Waters of the Eurasian Basin of the Central Arctic Ocean. Frontiers in Microbiology, 7.

From parasites to saprotrophs the important role of Chytrids and other fungi members on the Arctic ecosystem

Climate change has been shown to be reducing sea ice coverage in the Arctic this reduction of ice coverage is increasing light penetration into the Arctic Ocean and is predicted to increase primary production of phytoplankton, however this increase in light can also lead to photoinhibition and cause stress to phytoplankton, this increase stress in organism can increase the susceptibility to disease. In addition to this changes in sea ice cover allowing higher light penetration resulting in changes to environmental conditions this combined with availability of nutrients affects the seasonal composition of marine fungi.  A study by Hassett and Gradinger (2016) wanted to look at the role parasitic fungi belonging to chytridiomycota may have on phytoplankton as a response to this changing light penetration and how the community structure may shift with season and the impacts this may have to the food web.

Method

•   In order to access the impact of reduce snow coverage five areas each 5m²  were cleared of snow.
•     Samples form the experimental sites and controlled sites were collected every other day using ice cores and the photosynthetic yield was accessed.
•  To assess the abundance and diversity of the fungi community cell counts and DNA sampling were also performed.

Findings

Based on a 3 year study the authors found that the life history and abundance of Arctic marine fungi are closely link to seasonal difference in light penetration, which may trigger phytoplankton blooms but may also lead to stress in the phytoplankton. The study was the first to detail the important functional role fungi play in the Arctic marine environment and showed how the fungi community structure changed with season. The study found seasonality and host specificity of the parasitic chytirds on diatoms with parasitism occurring in April near the height of the algae bloom, Along with cell counts the authors also sequenced DNA barcodes to assess the seasonal diversity and abundance of fungi, The results showed that members of the Chytridiomycoata seasonally dominated sea ice and sediment fungal communities however distinct communities were present with different seasons.

The fungal community structure was driven by availability of nutrition and environmental conditions which resulted in major community shifts. During January and June the sea ice community was dominated by members of the group Dikarya. Dikaryotic fungi belonging to Aureobasidium and Cladosporium are halotolerant which explains how they tolerate the hyersaline condition during the period when diatom host concentrations were low. Another group included the saprotrophic Zygomycota it appears they are especially important in June when they are high in abundance allowing them to redirect carbon away from higher tropic levels and into the microbial loop highlighting the importance if fungi in marine nutrient cycling.

I think the findings of this paper are important in showing us the role parasitic fungi and other fungi groups play in the Arctic environment and their impact on the food web. The paper demonstrates for the first time the seasonality and functionality of parasitic chytrid fungi, and is very relevant in helping us understand the impact that reduce ice coverage may have in the Arctic ecosystem and how it might lead to a restructuring of the food web. I think it provides a good case for the impacts of parasitism to be incorporated into models of the marine environment which may be especially important on local scales such as the Arctic Ocean.

Hassett, B. and Gradinger, R. (2016). Chytrids dominate arctic marine fungal communities. Environmental Microbiology, 18(6), pp.2001-2009.

The Deep Water Horizon Spill and its Microbial Companions.

Hydrocarbons enter the marine environment through both natural and anthropogenic pathways, however the local hydrocarbon input of natural sources pales in comparison to the amount discharged during an event such as the Deepwater Horizon Accident (DWH), which released at least 4 million barrels of oil into the environment. The DWH spill resulted in a unique microbial response due to the release of both oil and natural gas at such significant depths, these conditions drastically altered the diversity and distribution of hydrocarbon degrading bacteria.

It is well documented that microbes aid in the degradation and remediation of hydrocarbons, however due to the rare occurrence of such a massive influx of oil, studies of bacteria communities’ in-situ are rare. Most published studies are carried out in mesocosms which focus on sediment, beach sand, or surface water, while the pelagic microbial response is often overlooked.

In the study Redmond and Valentine, carried out environmental 16s rRNA sequencing as well as stable isotope probing (SIP), allowing them to survey the diversity of organisms present and map the links between bacterial taxa and the respective hydrocarbon they degrade.

Deep sea samples were collected on three cruises between May to Sepetember 2010, sampling locations between 2 – 385km away from the wellhead. After the initial accident in April, the first deep sea samples were collected in May. These revealed that Oceanospirillales were dominant with 16 other groups of Gammaproteobacteria enriched in the samples. This diversity changed in June, with Oceanospirillales being replace with Gammaproteobacteria, Colwellia and Cycloclasticus as the dominant taxa. The well was capped in mid-July and samples collected two months after, had methylotrophs taking over while both Colwellia and Cycloclasticus were less abundant. Aswell as the deep sea samples, surface slicks were also analysised revealing that none of the dominant deep sea bacteria were present, indicating a possible link to temperature as both Oceanospirillales and Colwellia are psychrophiles, organisms that growth and reproduction in cold temperatures (−20 °C to +10 °C). This was followed up in lab tests which showed Colwellia was far more abundant in samples incubated at 4co, rather than room temperature, reinforcing the idea of temperature controlled proliferation.

Stable Isotope Probing was conducted which identified Colwellia as a key oxidiser of ethane, propane, and benzene while methylotrophs prefer methane. This indicates that Colwellia was a key component in the breakdown of ethane and propane, both key components of natural gas, however there is some indication of the possible degradation of higher molecular weight hydrocarbons. DWH was characterized by the release of large quantities of natural gas which likely helped the proliferation of Colwellia and Oceanospirillales compared to normal oil spills with less natural gas.

The conducted study was a success, revealing the direct links between oil-degrading microbes  and their associated hydrocarbons, these initial steps into mapping bacteria - hydrocarbon interactions could be vitally important in future bio-remediation techniques. The study of microbial responses to mass influxes of hydrocarbons is challenging due to the rarity and the unpredictable nature of such events, making preliminary work difficult, meaning measurements before an event are often missing leading teams to work without an accurate control.

In regards to further research , the mapping of microbe and hydrocarbon interactions should be heavily perused as well as further study analysing oil in the pelagic environment. This is more relevant than ever due to the practice of pumping dispersant into the broken well-head, roughly 5,000 feet below the surface. Resulting in the oil becoming suspended rather than floating. Suspended oil will have completely different microbial interactions that are critically under explored.

Redmond, M. and Valentine, D. (2011). Natural gas and temperature structured a microbial community response to the Deepwater Horizon oil spill. Proceedings of the National Academy of Sciences, 109(50), pp.20292-20297.

Drunk on algal toxins

Harmful algal blooms (HABs) pose a significant threat to coastal economies and human health. Moreover, evidence suggest that HABs are increasing in intensity and frequency. Copepods have a key role in the transfer of toxins in food webs, as they generally are the first consumers of the algae and act as vectors for the toxins. To date, research on copepod responses to HABs has mostly been limited to studying algae rejection, algae avoidance and incapacitation of the copepod. In their paper, Lasley-Rasher et al. (2016) examined the effect of HABs on the swimming behaviour of copepods and if the changes affected encounter rates with predators. 

Specimens of the neurotoxic alga Alexandrium fundyense and the copepod Temora longicornis were collected from the Damariscotta River estuary in the Gulf of Maine. The dinoflagellate produces toxins (e.g. saxitoxins) that cause paralytic shellfish poisoning and red tides. HPL-chromatography showed the total amount of saxitoxins in the A. fundyense culture to be 2.9 pg/cell. Copepods were placed in a tank with filtered seawater and fed with either i) A. fundyense, ii) a 50/50 mixture containing A. fundyense and Rhodomonas lens (a non-toxic control alga) or iii) R. lens. After a 2h exposure period the copepods were transferred to other tanks. Incubation lasted for another 15h during which the copepods were fed with Tetraselmis sp. (another non-toxic alga) to minimize variation due to hunger levels. Subsequently the swimming behaviour of T. longicornis was tracked using the LABTRACK software to generate 3D tracks, of which only the first 10s of movement were analysed. Survivorship, ingestion and egg production were tested separately in beakers.

After being exposed to A. fundyense, the copepods swam faster (25%) and displayed straighter swimming paths than those conspecifics fed only on the control alga. Survival was not affected by exposure the harmful alga, nor were there significant differences in the amount of food ingested or in the egg production. Moreover, the copepods did not seem to avoid A. fundyense or become incapacitated.

Unexpectedly, exposure to A. fundyense had no significant effects on either survival, ingestion or egg production and even stimulated swimming behaviour. The authors theorize that this T. longicornis strain may be resistant to saxitoxins, as the characteristic blocking of sodium channels was not observed in this study. However, a growing number of evidence suggests that saxitoxins can sometimes stimulate grazers. Along with path straightness, faster swimming increases the probability of dispersal and of an encounter with a predator. Analysis using a model predicted the 25% increase in swimming speed to theoretically lead to an 56% increase in encounter rates with predators. This has considerable implications for HABs as it could potentially decrease grazing pressure while simultaneously increasing toxin uptake by copepod predators. So far, the bioaccumulation of saxitoxins in T. longicornis has not been studied and they are generally thought to be less likely to bioaccumulate than shellfish

.

In conclusion, the paper demonstrates how HABs can alter the behaviour of their grazers. In addition to more direct harmful impacts, the effects on grazer behaviour also have to be addressed when examining HABs. While the paper has not been cited yet, it has garnered quite a bit of attention online. On the whole, it was an interesting paper that had a good structure, was easy to follow and didn’t try to do too much. 

Reviewed Paper:

Lasley-Rasher, R. S., Nagel, K., Angra, A., & Yen, J. (2016, April). Intoxicated copepods: ingesting toxic phytoplankton leads to risky behaviour. In Proc. R. Soc. B (Vol. 283, No. 1829, p. 20160176). The Royal Society. http://rspb.royalsocietypublishing.org/content/283/1829/20160176