Selective Enrichment and Isolation of Oil-Degrading Marine Bacteria from Seawater Samples

Abstract

Source: Villela, H. D. M., et al. Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation. J. Vis. Exp. (2019)

This video demonstrates the selective isolation of oil-degrading marine bacteria by enriching seawater samples on oil-based media, enabling the recovery of pure strains with potential applications in crude oil bioremediation and environmental cleanup.

Protocol

1. Water and coral collection and storage for microbial isolation

NOTE: It is essential to take the coordinates and temperature of the sampling sites. If possible, metadata such as salinity, pH, depth, and light intensity can also help in finding fine-tuned cultivation approaches and future interpretation of data. For reliable results, keep the samples stored for the minimum length of time possible. The water/coral microbiomes may change considerably if the samples are not kept at the right temperature and/or are stored for long periods. If the isolation step is not performed instantly after collection, it is crucial to maintain samples at 4 °C until processing. The longer the samples are stored, even at 4 °C, the more the microbial community will change.

1. Sample and store seawater.
   1. Collect 500 mL of water samples in at least triplicate from each targeted sampling site. Preferably, use sterile bottles with screw caps.
   2. If processing the water instantly after collection, keep the bottles at RT (room temperature) for a short interval. If sample processing is happening later, keep the bottles at 4 °C.
2. Sample and store the coral.
   1. Use a sterile pair of pliers to cut coral fragments from the same sampling site as the water samples. To avoid contamination, touch corals only with sterile gloves.
   2. Rinse the sampled coral fragment using 20 mL sterile saline solution (3% NaCl (sodium chloride) in distilled water) or artificial seawater to get rid of the loosely attached free-living bacteria of the seawater.
   3. Using forceps, place each coral fragment into a sterile 250−500 mL container with a screw cap containing sterile saline solution.
   4. In the laboratory, using sterile forceps and pliers, weigh 5 g of coral fragments using sterile 100 mm x 20 mm Petri dishes on a weighing scale.
   5. Transfer the 5 g of coral sample to a sterile mortar and macerate it using a sterile pestle.
   6. Using a sterile spatula, transfer the macerated sample to a sterile culture flask containing 45 mL of 3% NaCl sterile solution and 10−15 glass beads of 5 mm. Use some of the 45 mL sterile saline solution to wash the mortar and recover the maximum amount of the macerate.
   7. Keep the flasks under constant agitation (150 x g) for 16 h at the water temperature of the sampling site.
      NOTE: For shallow-water corals the optimum temperature will range from 24−28 °C. This step will detach microorganisms from different coral compartments, such as those attached to the host cells, or the ones living inside the tissue and the skeleton. After this step, the coral macerates should not be stored, and the isolation step must be instantly performed.

2. Isolation of oil-degrading bacteria from seawater and/or corals

1. Prepare a minimum medium containing an oil water-soluble fraction (oWSF) and oil water-insoluble fraction (oWIF) as the only carbon source.
   1. Add 1−2% crude oil to 500 mL of sterile distilled water. Use a filter flask opened on the bottom to take the soluble fraction out without disturbing the upper layer of the insoluble fraction.
   2. Keep the mixture under constant agitation at 24−28 °C at 150 x g for 48 h.
   3. Place the filter flask containing the crude oil fractions on a stable surface and wait 10−20 min to allow soluble and insoluble fraction separation.
   4. Transfer the oWSF to a new sterile flask, saving the oWIF by opening the bottom filter flask and taking out the soluble fraction.
   5. Using all the oWSF recovered in the previous step (~400 mL), prepare 1 L of BH (Bushnell Haas) agar minimum medium containing oWSF as the only carbon source.
   6. Using the oWIF remaining in the flask from step 2.1.4, prepare 1 L of BH agar minimum medium containing oWIF as the only carbon source.
2. Isolate oWSF- and oWIF-degrading bacteria.
   1. Using water and coral macerate from steps 1.1.2 and 1.2.7, respectively, dilute the samples up to 10^-6 in sterile saline solution as below.
3. Perform serial dilutions up to 10^-9 in sterile saline solution for coral samples and up to 10^-6 for water samples. Pipette dilutions up and down 5x before discarding the tip. Vortex samples for 5 s every time before performing the next serial dilution.
4. Pipette 100 µL of each dilution on Petri dishes containing BH-oWSF and BH-oWIF agar media and plate them.
5. Incubate the plates for 1−3 days at the target temperature (e.g., 26 °C). Check the plates once a day.
6. 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
500 mL PYREX Media Storage Bottle	thomas scientific/Corning	1743E20/1395-500	Used to sample water.
500 mL Aspirator Bottles	thomas scientific/Corning	1234B28/1220-2X	Used to separate the oil fractions.
6-inch wire cutter plier	thomas scientific/Restek	1173Y64/23033	Used to cut coral fragments.
Agar	Himedia	PCT0901-1KG	Used to make solid media.
Bushnell Haas Broth	Himedia	M350-500G	Used as minimum media to be supplemented with carbon sources.
Erlenmeyer Flask	thomas scientific/DWK Life Sciences (Kimble)	4882H35/26500-125	Used to incubate coral macerate with glass beads.
Laminar Flow Hood			Needed to work at sterile conditions.
Luria Bertani Broth, Miller (Miller Luria Bertani Broth)	Himedia	M1245-1KG	Used as rich media to grow bacteria.
Marine Agar 2216 (Zobell Marine Agar)	Himedia	M384-500G	Used as rich media to grow bacteria.
Orbital-Shaker Incubator			Used to incubate liquid media and oil.
Plates Incubator			Used to incubate plates.
Porcelain Mortar and Pestle	Thomas scientific/United Scientific Supplies	1201U69/JMD150	Used to macerate coral fragments.
Refrigerated Centrifuge			Used to centrifuge bacterial cultures.
Spectrophotometer			Used to measure optical density of bacterial cultures.
Wizard Genomic DNA Purification kit	Promega	A1120	Used for microbial strains DNA extraction.