We can isolate and assemble probiotics that can both promote coral health and at the same time mitigate impact from pollution and that can contribute to a reef's persistence in the changing world. The isolation and screening approach described can be used to select microorganisms presenting key metabolic activities such as the degradation of pollutants or other beneficial characteristics to marine organisms such as corals. This method can be applied to isolate microorganisms able to degrade any contaminants.
And additionally, other sources of material can be used in the other environmental samples such as plants, soil, water, anything. Study the literature and try to focus on strains that can be good candidates for your purpose. If possible, direct the isolation methods for those strains and avoid known pathogens.
And if you are aiming to use a consortium of bacteria, it is also very important to first investigate if the members of the consortium have antagonistic activity against each other. Because corals are under increasing risk of extinction, this methodology is starting to be started worldwide and there's a need to spread and standardize the method. As some coral researchers have no experience with conventional microbiology, that's why they have been frequently asking for advice on how to proceed with such approach.
To begin, from each targeted sampling site, use sterile bottles with screw caps to collect 500 milliliters of water in triplicate. If sample processing is happening later, keep the bottles at four degrees Celsius. Use a sterile pair of pliers and forceps to cut coral fragments from the same sampling site of the water samples.
Rinse the sample coral fragment using 20 milliliters of sterile 3%saline solution or artificial seawater to get rid of the loosely attached free-living bacteria of the seawater. Using forceps, place each coral fragment into a sterile container with a screw cap containing sterile saline solution. Using sterile forceps and pliers, weigh five grams of coral fragments in sterile Petri dishes on a weighing scale.
Transfer the five grams of coral sample to a sterile mortar and macerate it using a sterile pestle. Using a sterile spatula, transfer the macerated sample to a sterile culture flask containing 25 milliliters of 3%sodium chloride sterile solution and 10 to 15 glass beads of 2.5 millimeters. Use 20 milliliter sterile saline solution to wash the mortar into the tube to recover the maximum amount of the macerate.
To detach microorganisms from different coral compartments, keep the flasks under constant agitation at 150 times g for 16 hours at the water temperature of the sampling site. To select bacteria, first perform serial dilutions up to 10 to the negative nine in sterile saline solution for coral samples and up to 10 to the negative six for water samples. Pipette dilutions up and down five times before discarding the tip.
Then vortex each one. Vortex the samples for five seconds every time before performing the next serial dilution. Pipette 100 microliters of each dilution on Petri dishes containing 3%sodium chloride lysogeny broth agar medium and plate them.
Incubate the plates for one to three days at the target temperature, for example 26 degrees Celsius. Check the plates once a day. Select and isolate the colonies presenting distinct growth morphologies on new plates using the streak plate technique.
Repeat this step as many times as needed to have pure colonies growing on the plates. Store the isolates at four degrees Celsius or in glycerol as described in the manuscript. To perform EE2 degradation ability test, pick a single colony from the fresh plates and inoculate in two milliliters of sterile LB medium in a tube.
In case it is a glycerol stock, take 10 microliters of glycerol bacterial stock and streak onto LB agar plates. Incubate at 24 to 28 degrees Celsius overnight to have single colonies growing. Place the tube containing LB medium inclined under constant agitation at 150 times g at 24 to 28 degrees Celsius overnight.
After bacterial growth, pellet the cells by centrifuging them at 8, 000 times g for eight minutes at room temperature. Discard the supernatant. To wash the remaining LB broth, add two milliliters of saline water to resuspend the cells.
Repeat the washing twice to guarantee that there are no traces of carbon source. Inoculate the washed and resuspended cells in minimum Bushnell Haas culture medium containing EE2 as the only carbon source. Assess bacterial growth by optical density at 600 nanometers and our colony forming units on LB agar medium after 16 to 72 hours of incubation.
To prepare minimum media containing an oil-water soluble fraction and an oil-water insoluble fraction as the only carbon source, add 1-2%crude oil to 500 milliliters of sterile distilled water in a filter flask containing a valve on the bottom. Keep the flask under constant agitation at 24 to 28 degrees Celsius at 150 times g for 48 hours. After that, place the flask on a stable surface and wait 10 to 20 minutes to allow the soluble and insoluble fraction to separate.
Then open the bottom filter flask to transfer the oil-water soluble fraction to a new sterile flask saving the oil-water insoluble fraction. Then prepare two bottles of Bushnell Haas agar minimum medium. Autoclave it.
Combine with the oil-water soluble fraction as the only carbon source to make 500 milliliters of agar medium. Do the same with the oil-water insoluble fraction and transfer approximately 25 milliliters of each medium onto individual Petri dishes. To isolate oil-degrading bacteria, reuse previously diluted samples and pipette 100 microliters of each dilution onto the Petri dishes containing agar medium and plate them.
Remember to keep negative controls for your plates. Incubate the dishes for one to three days at the target temperature and repeat the isolation procedures as previously. In this protocol, microorganisms were isolated from different water sources and coral nubbins presenting putative BMC characteristics and capable to degrade different classes of contaminants.
Using water samples collected at a sewage treatment plant obtained from Experimental Center of Environmental Sanitation of the Federal University of Rio de Janeiro, 33 bacterial strains able to degrade EE2 were isolated. Additionally, using the technique for selecting oil-degrading bacteria, 20 strains able to degrade both oil-water soluble fraction and oil-water insoluble fraction were isolated. Putative BMC characteristics were screened in microorganisms, among them a strain presenting strong antagonistic activity against the coral pathogen Vibrio cholerae lyticus, strains able to degrade urea, a good catalase producer, and microorganisms presenting potentially beneficial genes were found.
Employing both bioremediation and BMC inoculation, it was possible to predict corals from oil exposure impacts. An oil bioremediator PBMC consortium isolated from the coral was inoculated on coral nubbins exposed to 1%oil. It showed a better preserved photochemical ability compared with the coral health with no inoculation.
Don't leave samples at room temperature for too long. Otherwise, the microbial community may change and disease-related microbes may overgrow others. Crude oil is toxic.
Please make sure you use appropriate personal protective equipment when working with this or any other contaminant you decide to use. The technique paved an innovative way in the field of coral restoration and ocean bioremediation can potentially be used to minimize economic, ecological and social impacts caused by global change.
