This protocol outlines the isolation, cultivation, and identification of hydrocarbon-degrading microorganisms from ecological habitats. This method does not require sophisticated instruments, and can be easily performed in a standard laboratory setup. It can also be easily adapted to study other substrates.
This technique can easily be translated to various ecological niches, and the degradation of different substrates can be assessed. Demonstrating the procedure will be Deepa Sethi, a doctoral student from Dr.Richa Priyadarshini's laboratory. To begin, collect 500 milliliters of water sample in five sterile glass bottles from different water body sites, and measure each sample's pH and temperature.
Then filter the sample under aseptic conditions in batches of 100 milliliters through 0.22 micron pore size filter sheets. And place the sheets over different nutrient media plates, placing one paper per plate. After two hours, peel the paper off using sterile forceps.
Next, dilute the unfiltered water samples by adding 100 microliters of the collected water sample to 900 microliters of sterile double distilled water. Continue the tenfold dilutions until a dilution ratio of one to 1, 000, 000 is reached. Mix by pipetting, and ensure that the final volume of each dilution is one milliliter.
Spread 100 microliters of the diluted water samples individually on all growth media plates, in triplicates. Incubate the plates at 30 degrees Celsius for 24 to 48 hours, depending on the growth of colonies. To get isolated colonies, pick a colony using a sterile toothpick or pipette tip.
Perform quadrant streaking, and incubate the plate overnight. The next day, screen the colonies based on their morphological features, such as color, texture, shape, size, margin, and elevation. Re-streak the colonies to obtain pure cultures.
After performing gram staining of each pure culture, prepare glycerol stocks by inoculating a single colony in three milliliters of appropriate growth media, and incubating at 30 degrees Celsius. The following day, add 700 microliters of the overnight culture, and 300 microliters of 100%glycerol in cryo vials. Freeze the vials at minus 80 degrees Celsius for long-term storage.
From a freshly-streaked plate, pick a colony and inoculated it in five milliliters of triptyc soy broth or nutrient broth. Grow the culture overnight at 30 degrees Celsius, with shaking at 200 RPM, til the absorbance reaches approximately two. The next day, pellet the cells.
Discard the supernatant, and wash the pellet twice with two milliliters of autoclaved saline. Spin the cells again, then re-suspend the pellet in two milliliters of liquid carbon-free basal medium, or LCFBM, and measure the absorbance. Next, label two sterile 150 milliliter Erlenmeyer flasks as A and B, for the control and experimental group respectively.
In flask A, add 40 milliliters of LCFBM, followed by a styrene at a five millimolar final concentration. In flask B, add 35 milliliters of LCFBM and styrene. Then add the cell suspension with a final absorbance of approximately 0.1.
After bringing up the volume in flask B to 40 milliliters with LCFBM, incubate both flasks at 30 degrees Celsius with shaking at 200 RPM for 30 days. Measure the absorbance of each flask every five days, and plot a growth curve. Increase the incubation by up to 45 days if the bacteria can utilize styrene.
An increase in absorbance indicates that the bacterium can metabolize styrene. To isolate genomic DNA from gram-negative bacteria, pick a single colony and inoculate it in a fresh growth medium in a sterile test tube. After overnight incubation in a shaking incubator, pellet 1.5 milliliters of the grown culture.
Remove the supernatant and resuspend the pellet in 200 microliters of lysis buffer. Then add 66 microliters of five-molar sodium chloride solution, and mix well. After centrifuging the resulting mixture, pipette the clear supinate in a fresh micro centrifuge tube, and add an equal volume of chloroform.
Mix the solution by inverting it multiple times, until a milky solution is observed. Spin the tube again, and transfer the supernatant to a clean vial. Next, add one milliliter of ice cold 100%ethanol, and mix by inversion, til white strands of DNA precipitate out.
Centrifuge the precipitated DNA. Discard the supernatant. And wash the pellet with one milliliter of 70%ethanol.
After discarding the wash, allow the DNA pellet to dry for five minutes at room temperature. Once dried, re-suspend the pellet in 100 microliters of Tris-EDTA buffer. Measure the DNA concentration using a spectrophotometer, and run the DNA on a 1%agarose gel to assess its quality.
To identify the strains, amplify the DNA isolated from the pure bacterial cultures by PCR, with universal primers targeting the 16S ribosomal RNA sequence for bacteria. Prepare 25 microliters of the PCR mix on ice, add one microliter of the DNA template, and set the cycling conditions for gene amplification. After the PCR, mix five microliters of the sample with one microliter of five times DNA loading dye.
And run it on a 1%agarose gel to verify the amplification. For 16S ribosomal, rNA gene sequencing, set up the same reaction as described earlier, but in a higher volume. Then, to purify the amplicons for Sanger sequencing, mix the entire sample with DNA loading dye, and load it on an agarose gel to perform the gel extraction method.
Once the sequencing is completed, convert the results file into faster format, and check the sequence similarity using the BLAST tool on NCBI. Bacterial colonies isolated from water samples from a wetland in Dadri, India are shown here. The isolated bacteria were individually grown in respective media, with liquid styrene as a sole source of carbon.
The hydrocarbon-degrading potential of the bacterial isolates was assessed using a catechol degradation assay. A representative assay for one of the isolates is shown here. The integrity of the genomic DNA was confirmed by visualizing a small DNA sample on an agarose gel.
And the 16S ribosomal rNA gene was amplified using universal primers. A phylogenetic tree was constructed to depict the relatedness among exiguobacterium strains isolated from a wetland, with the known exiguobacterium species. A critical step of the protocol is to check the utilization of the substrate being tested.
Depending on growth characteristics, the microbe may not immediately adapt to utilizing the substrate being tested, and may require an enrichment process. This method focuses on the cultivable bacterial population in environmental samples. Researchers can further perform experiments, such as bacterial whole-genome sequencing, and metabolic profiling, that can provide valuable insights into genes and pathways involved in hydrocarbon degradation.
