Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

1. Sample collection, processing, and analysis

NOTE: Here, we present a protocol to isolate bacteria from aquatic habitats. Some of the isolates may be pathogenic, therefore, wear gloves and disinfect the work area before and after use.

1. Collect 500 mL of water sample in five sterile glass bottles from different sites of the water body. Measure the pH and temperature of each sample using a pH meter and thermometer, respectively.
   NOTE: The protocol is not site-specific and can be easily adapted to isolate organisms from hydrocarbon-contaminated water bodies too.
2. Filter the sample in a batch of 100 mL through 0.22 µm-pore size filter sheets, in aseptic conditions.
   NOTE: The diameter of the filter paper should not exceed the Petri dish diameter. For example, filter paper not exceeding 85 mm diameter is optimum for a 100-120 mm Petri dish.
3. Keep the filter papers over different nutrient media plates (PYE¹⁹, R2A²⁰, M9, LB, NB, TSB, M63²¹ and M2G²²). The different types of growth media allow the selection and enrichment of different microorganisms. Compositions of various growth media are listed in Table 1. Use one paper for each media plate and peel off after 2 h using sterile forceps.
4. Serially dilute the unfiltered water samples (10⁶ dilution) in sterile double distilled water by adding 100 µL of the collected water sample in 900 µL of sterile water. This results in a 1:10 dilution. From this sample, take 100 µL and add in 900 µL of sterile water to obtain 1:100 dilution. Repeat the dilution until the dilution fold is 1:1,000,000. Mix by pipetting. The final volume of each dilution will be 1 mL.
5. Spread 100 µL of the diluted water sample individually on all growth media plates mentioned in step 3 in triplicates.
6. Incubate the plates at 30 °C for 24 to 48 h depending on the growth of colonies.
   NOTE: Most of the environmental isolates grow at an optimum temperature of 30 °C. If isolating the samples from an environment with extreme temperatures, incubate the plates at the same temperature as that of the collection site.
7. Next, pick the colonies using a sterile toothpick or pipette tip and perform quadrant streaking to get isolated colonies.
8. Incubate the plates overnight. Next day, screen the colonies based on their morphological features such as color, texture, shape, size, margin, elevation, etc. Restreak the colonies to obtain pure cultures.
9. Perform gram-staining of each pure culture²³ and proceed with glycerol stock preparation.
10. To prepare the glycerol stocks, inoculate a single colony in 3 mL of appropriate growth media and incubate at 30 °C. From the overnight culture, take 700 µL and add 300 µL of 100% glycerol (sterilized by autoclaving) in cryovials²⁴. Freeze the vials at -80 °C for long-term storage.

2. Degradation of hydrocarbons

NOTE: The example below is to screen the isolates which can degrade styrene. It is a slight modification of the method adapted in a previous report²⁵. Follow the steps under aseptic conditions.

1. From a freshly streaked plate, pick a colony and inoculate in 5 mL of Tryptic soy broth (TSB)/Nutrient broth (NB). Grow the culture overnight at 30 °C with shaking at 200 rpm till the absorbance reaches ~2.
   NOTE: Other than TSB/NB, any growth medium can be chosen in which the bacteria reach high cell density.
2. The next day, pellet the cells at 2862 x g for 5 min at 4 °C and discard the supernatant.
3. Wash the pellet twice with 2 mL of autoclaved saline (0.9% NaCl) and spin at 2862 x g for 5 min at 4 °C.
   NOTE: Saline is isotonic and, thus, it maintains the osmotic pressure inside bacterial cells.
4. Resuspend the pellet in 2 mL of liquid carbon-free basal medium (LCFBM). Measure the absorbance (OD₆₀₀).
5. Take two sterile Erlenmeyer flasks with 150 mL capacity for control and the experimental group. Label them as A and B.
6. In the uninoculated /control group, (flask A), add 40 mL of LCFBM and styrene (5 mM).
7. In flask B, add 35 mL of LCFBM and styrene (adjust the final concentration of styrene to 5 mM). Add the cell suspension with a final OD₆₀₀ of cells ≈ 0.1 and make up the remaining volume with LCFBM up to 40 mL. Incubate the flasks at 30 °C with shaking at 200 rpm for 30 days.
   NOTE: Hydrocarbons in excess can be toxic for the microbes, therefore, start with low concentration and gradually increase it.
8. Repeat the above for each additional strain that must be evaluated for hydrocarbon degradation.
9. Measure the OD₆₀₀ of each flask every 5 days and plot a growth curve. Increase the incubation up to 45 days if the bacteria can utilize styrene. An increase in OD₆₀₀ indicates that the bacterium can metabolize styrene.

3. Screening of catechol degradation by bacterial isolates

NOTE: The degradation of aromatic hydrocarbons such as styrene, benzene, xylene, naphthalene, phenols, etc. produce catechols as reaction intermediates. The catechols are further metabolized by bacteria with the help of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase enzymes through the ortho- and meta-cleavage pathways, respectively²⁶. These enzymes are also involved in the degradation of other hydrocarbons such as chlorobenzene²⁷. The protocol mentioned below uses whole cell lysate for catechol 2, 3-dioxygenase enzyme assay²⁸. The same lysis method can be used to screen the activity of catechol 1, 2-dioxygenase. However, the composition of the reaction mixture will vary. Both the enzymes are inducible in nature and can be induced by the addition of phenol to the growth media.

1. With the help of a sterile loop, inoculate the bacterial colony from a freshly streaked plate into mineral salts medium (MSM) supplemented with 1-4 mM phenol. Incubate the culture at 30 °C and 200 rpm. Harvest the culture at 4 °C when OD₆₀₀ reaches between 1.4-1.6 (i.e., in late exponential phase) by spinning at 4500 x g for 20 min.
2. Wash the cell pellet with phosphate buffer (0.5 M, pH 7.5).
3. Resuspend the cells in the above-mentioned phosphate buffer and adjust the final OD₆₀₀ ≈ 1.0.
4. Lyse the cells by pulsed sonication for 1.5 min, the duration of each pulse being 15 s. After this step, the suspension must be clear or less turbid. If not, increase the number of pulses and check whether the suspension is clear. After each pulse, keep sample on ice to avoid protein degradation.
5. Remove the cell debris and unbroken cells by centrifugation at 9,000 x g for 30 min, maintaining the cold temperature (4 °C).
6. Carefully pipette the clear supernatant. This fraction has the crude extract for enzyme assay.
7. Determine the protein concentration of crude extract by either Bradford or Lowry's method^29,30.
8. To determine the activity of catechol 2,3-dioxygenase, measure the formation of the reaction end product (2-hydroxymuconic semialdehyde) by a spectrophotometer.
9. Prepare the reaction mixture by adding 20 µL of catechol (50 mM), 960 µL of phosphate buffer (50 mM, pH 7.5), and 20 µL of the crude extract.
10. For the negative control, replace the crude extract with phosphate buffer and adjust the final volume to 1 mL.
11. Incubate the reaction mixture for 30 min. At set time intervals, measure the absorbance at 375 nm. An increase in absorbance indicates the formation of the reaction end product, 2-hydroxymuconic acid semialdehyde (2-HMS). Perform the experiment in triplicates.
    ​NOTE: Catechol is light-sensitive and oxygen-sensitive. Store the reaction mixture in dark and close the tubes tightly to prevent the natural degradation of catechol.

4. Genomic DNA isolation of the pure culture

NOTE: This is the general protocol for the isolation of genomic DNA. Gram staining was performed during the sample collection, processing, and analysis step. Due to the variation in cell wall thickness of gram-positive and gram-negative bacteria, the cell lysis method is modified accordingly. Wear gloves while isolating and disinfect the workbench with 70% ethanol to avoid the nucleases from degrading DNA. Some of the chemicals mentioned below can cause severe burns on the skin and proper care must be taken while handling them.

1. Isolation of genomic DNA from Gram-negative bacteria³¹.
   1. Pick a single colony and inoculate in a fresh growth medium in sterile test tubes.
   2. Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at 30 °C.
   3. The next day, pellet 1.5 mL of overnight grown culture at 12,400 x g for 3 min.
   4. Remove the supernatant and resuspend the pellet in 200 µL of lysis buffer (40 mM Tris-acetate, pH 7.8, 20 mM sodium acetate, 1 mM EDTA, 1% SDS).
   5. Add 66 µL of NaCl solution (5 M) and mix well.
   6. Pellet the resulting mixture at 12,400 x g for 10 min (4 °C).
   7. Pipette the clear supernatant in a fresh microcentrifuge tube and add an equal volume of chloroform.
   8. Invert mix the solution multiple times until a milky solution is observed.
   9. Spin at 12,400 x g for 3 min and transfer the supernatant to a clean vial.
   10. Add 1 mL of ice-cold 100% ethanol; mix by inversion till white strands of DNA precipitate out.
   11. Centrifuge the precipitated DNA at 2,200 x g for 10 min at 4 °C and discard the supernatant.
   12. Wash the DNA pellet with 1 mL of 70% ethanol and allow the DNA pellet to dry for 5 min at room temperature.
   13. Once dried, resuspend the pellet in 100 µL of 1x Tris-EDTA(TE) buffer, and store the DNA at -20 °C.
   14. Measure the concentration (A_260/280) using spectrophotometer and run the DNA on agarose gel (1%) to assess the quality of DNA²⁴.
2. Isolation of genomic DNA from gram-positive strain³²
   1. Pick a single colony and inoculate in fresh growth medium in sterile test tubes.
   2. Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at a suitable growth temperature.
   3. Next day, take 1.5 mL of the grown culture and centrifuge at 8,600 x g for 5 min.
   4. Remove the supernatant and resuspend the cells in TE buffer.
   5. Adjust the OD₆₀₀ = 1.0 with TE buffer and transfer 740 µL of the cell suspension to a clean microfuge tube.
   6. Add 20 µL of lysozyme (100 mg/mL stock) and mix well by pipetting. Incubate at 37 °C for 30 min (in a dry bath).
   7. Add 40 µL of 10% SDS and mix well.
   8. Add 8 µL of Proteinase K (10 mg/mL). Mix well and incubate at 56 °C for 1-3 h (in a dry bath). The suspension should become clear now with increased viscosity, marking efficient cell lysis.
      NOTE: The suspension can be left overnight if the cells are not lysed properly.
   9. Preheat CTAB/NaCl mixture at 65 °C (in a dry bath) and add 100 µL of this mixture to the cell suspension. Mix well.
   10. Incubate at 65 °C for 10 min (in a dry bath).
   11. Add 500 µL of chloroform:isoamyl alcohol (24:1) and mix well. Spin at 16,900 x g for 10 min at 25 °C.
   12. Transfer the aqueous phase to a fresh microcentrifuge tube avoiding the organic phase (viscous phase at the bottom).
   13. Carefully add 500 µL of phenol:chloroform:isoamyl alcohol (25:24:1) and mix well. Spin at 16,900 x g for 10 min at 25 °C.
   14. Take the aqueous phase in a fresh microcentrifuge tube. Add 500 µL of chloroform:isoamyl alcohol (24:1) and mix well.
   15. Transfer the aqueous phase and add 0.6 volume isopropanol (prechilled at -20 °C).
   16. The precipitated DNA strands must be visible in the threadlike form. Incubate at -20 °C for 2 h to overnight.
   17. Centrifuge at 16,900 x g for 15 min at 4 °C to pellet the DNA.
   18. Decant the isopropanol carefully and wash the pellet with 1 mL of cold 70% ethanol (prechilled at -20 °C) to remove any impurities.
   19. Centrifuge at 16,900 x g for 5 min at 4 °C. Discard the supernatant.
   20. Allow the pellet to dry at room temperature for 20 min or keep the tube at 37 °C. Make sure the pellet is not over-dried.
   21. Resuspend in 100 µL of 1x TE buffer and store the DNA at -20 °C.
       NOTE: If the pellet becomes over-dried and is difficult to resuspend, incubate the microcentrifuge tube with DNA pellet and nuclease-free water at 37 °C for 15-20 min and resuspend again by pipetting.
   22. Measure the concentration (A_260/280) using a spectrophotometer after making 1:100 dilution in 1x TE buffer and run the DNA on agarose gel (1%) to assess the quality of DNA²⁴.

5. 16S rRNA sequencing

NOTE: The protocol outlined below is for amplification and sequencing of 16S rRNA for bacterial identification. Information derived from the 16S rRNA sequence is used for the identification of an unknown organism and to find the relatedness between different organisms.

1. To identify the strains, amplify the DNA isolated from the pure bacterial cultures by PCR with universal primers targeting 16S rRNA sequence for bacteria: 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'- TACGGYTACCTTGTTACGACTT-3')³³.
2. Prepare the PCR mix (25 µL reactions) on ice with 18 µL of autoclaved/nuclease-free water, 2.5 µL of 10x buffer, 0.5 µL of both forward and reverse primers (100 µM stock), 2 µL of the dNTPs mix (100 µM stock), 1 µL of DNA template (2-15 ng/µL) and 1 U of Taq Polymerase.
3. Use the following cycling conditions for 16S rRNA gene amplification: Initial denaturation at 94 °C for 10 min, (final denaturation at 94 °C for 40 s, primer annealing at 56 °C for 1 min, extension at 74 °C for 2 min) x 30 cycles, final extension at 74 °C for 10 min.
4. After the cycle ends, mix 5 µL of sample and 1 µL of 5x DNA loading dye. Run on 1% agarose gel to verify the amplification. Store the PCR products at 4 °C for the short term or freeze them at -20°C until further use.
5. For 16S rRNA gene sequencing, set up the same reaction as mentioned above for higher volume (100 µL).
6. Purify the amplicons for Sanger sequencing^24,34 using PCR product purification kit or mix the entire sample with DNA loading dye and load on an agarose gel to perform gel extraction method.
7. Once the sequencing is done, convert the results file in FASTA format and check the sequence similarity with the basic local alignment search tool (BLAST) on NCBI (http://www.ncbi.nlm.nih.gov/)³⁵.