Sunday, 26 November 2017

too hot for my biofilm?

Microorganisms in marine environments inhabit surfaces and form biofilms. The formation of biofilms involves adsorption of organic and inorganic molecules to submerged surfaces. The role of first colonisers is important as they enable the biofilm to stay intact or detach. Biofilms can be altered by biotic and abiotic factors.

Global warming is becoming an increasing concern and an Increase in temperatures could influence the structure and functions of biofilms in coral reef systems. Increases in sea temperature can alter the distribution, abundance, function and community dynamics in marine microbial systems both positively and negatively by affecting the biofilm recruitment and inductive capabilities of invertebrate larvae. As a biofilm grows it develops a complex architecture that contains different micro habitats which facilitates the growth of diverse microbial communities and the settlement and growth of invertebrate larvae. Previous studies have shown that living in biofilms can be advantageous. Biofilms can provide nutrients and protection against external factors such as toxins, heavy metals, dehydration and UV-radiation. Gamma-proteobacteria have been found to produce chemical cues that facilitate the induction of coral larvae. Crustose algae harbour or produce biofilms that can produce morphogens and crustose coralline algae precipitate calcium carbonate which is Important in reef building.

The Arabian Gulf, one of the hottest bodies of water on the earth, is characterised by the highest variability in annual temperatures. Corals suffer thermal stress when water temperatures exceed 32 °C or drop below 18 °C but corals in the Gulf can survive extreme temperature fluctuations of 35 °C in summer and below 11 °C in winter. The coral spawning season in the Arabian Gulf occurs between may and august when temperatures are highest.  Because microbial biofilms provide cues for larvae to settle and metamorphose it is important to know how biofilms develop at these temperature extremes.

The experiment was conducted in the inshore reef system of Qit'at Benayah, north of the Arabian Gulf.  Glass slides were used as a substratum for biofilm growth. Replicates were collected every 2 weeks from both sides of the glass slides. The physical and chemical properties of the seawater were checked onsite and showed a continuous increase in water temperature during the sampling period.
Biofilms were removed from the glass slides and placed into petri dishes. Biofilms were divided into two and either used to measure the abundances of phototroughs and heterotroughs or used in molecular analysis.

The abundances of heterotroughs and phototroughs were investigated using total and viable count techniques using epifluorescence microscopy. Developed biofilms were plated onto marine agar to determine the culturable bacterial abundance and developed bacterial colonies were subcultured to purify them. PCR was then used with 16s r/RNA to identify the isolates.
Four Vibrio isolates, Vibrio harveyi, Vibrio owensii, Vibrio ponticus and Vibrio sinaloensis, were inoculated on a marine agar to assess the ability of vVbrio to precipitate calcium carbonate. Calcium carbonate crystals were removed and examined under an emission scanning microscope to assess size and number of crystals.

The results showed a linear increase in the number of heterotrophs on both sides of the glass slides as the age and temperature of the biofilm increased. Biofilms kept at 33 °C for longer times saw heterotroph numbers start to decrease. Phototroph numbers fluctuated overtime along with levels of phototroughs detected in seawater samples.
The study found that biofilms were similar in the diversity of microbes but were found grouped together by age. Biofilms of a younger age were seen to cluster together and remained separate from mature biofilms no matter which side of the substrata they were growing on suggesting the age groups had different bacterial communities.

Light microscopy was used to examine the diversity of phototrophs in the biofilms. Phototrophs which have the potential to produce substances involved in coral larvae settlement and metamorphosis was found colonizing the substrata. Bacillariophyceae (diatom) and Cyanobacteria dominated the biofilms. Bacillariophyceae dominated young biofilms but decreased in number with age whilst cyanobacterial abundance increased with age and dominated later.
For all biofilm age groups Gamma-Proteobacteria dominated with vibrio being the most dominant genus of that group. Invertebrate Larvae were found in 6-week-old biofilms suggesting that this may be the age the biofilm becomes mature. 
There were 20 Vibrio species isolated from the biofilm samples 4 of which could precipitate calcium. This calcification is important in providing stability for the biofilm, the Vibrio produced larger sized crystals at 30°C than at 23°C and there were more of them.

In conclusion, the study found that bio-films grown on glass substrata over 14 weeks contained bacterial and algal species capable of producing morphogens and stabilising biofilms through calcification. Natural elevations of prolonged temperature at 33°C did not affect structural diversity of the bacterial communities within the biofilm. The study found that the microbial community shifted as the bio-film aged with mature biofilms containing key species such as Vibrio important in calcification and the production of morphogens. During the study invertebrate larvae were found in 6-week-old bio-films but no coral larvae were found despite the experiment being carried out during coral spawning.

The paper stated that it may have no found coral larvae due to the method of removing the biofilms. They said they could have looked under a microscope to see if they were there but it would have meant they couldn’t have used other methods to assess the biofilm diversity. I feel that because this paper was based on corals they could have assigned extra slides just for looking at coral larvae settlement so they could see if they do settle or not and at what point which could have improved our knowledge on how these corals persist at higher temperatures.



Mahmoud (2015) ‘Variations in the abundance and structural diversity of microbes forming biofilms in a thermally stressed coral reef system’, Marine Pollution Bulletin 100, 710–718. doi.org/10.1016/j.marpolbul.2015.10.030

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