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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

Abstract

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

Introduction

Hydrocarbons (HC) are extensively used both as fuels and in chemical applications. Aromatic hydrocarbons such as benzene, toluene, and xylene are used widely as solvents1. Alkenes such as ethylene and propylene serve as precursors in the synthesis of polyethylene and polypropylene polymers, respectively. Polymerization of another hydrocarbon, styrene forms polystyrene. Anthropogenic activities introduce hydrocarbons into the environment during their production and transport. Hydrocarbon contamination of soil and water has serious concerns for the environment and human health. Microbes play a major role in maintaining the ecosystem by regulating the biogeochemical cycles and utilizing a wide range of substrates, which include pollutants and xenobiotics as well, converting them into carbon and energy source. This process of detoxification of environmental contaminants by microorganisms is known as bioremediation3,4,5,6,7.

Microorganisms with the capability to degrade hydrocarbons are found in aquatic and soil habitats8,9,10. Many bacteria with the potential to degrade alkanes and aromatic HCs have been identified, such as Pseudomonas, Acinetobacter, Rhodococcus, Marinobacter, and Oleibacter11. The development of technologically advanced culture-independent approaches has helped discover novel HC-degrading microbial communities12. Genomic material directly isolated from source samples is amplified and sequenced by high throughput methods such as Next Generation Sequencing (NGS) followed by analysis eliminating the need to cultivate microorganisms. NGS methods, such as metagenome analysis, are expensive and suffer from drawbacks related to the amplification process13. Cultivation techniques such as selective enrichment culture14 that target isolation of hydrocarbon-degrading microbes are still useful as they allow researchers to probe and manipulate metabolic pathways in bacterial isolates.

Genomic DNA isolation and subsequent sequencing of the genomic material reveals valuable information about any organism. Whole-genome sequencing helps in the identification of genes that code for antibiotic resistance, potential drug targets, virulence factors, transporters, xenobiotic-metabolizing enzymes, etc15,16,17. Sequencing of 16SrRNA encoding gene has been proven to be a robust technique to identify bacterial phylogeny. Conservation of the gene sequence and function over the years makes it a reliable tool for identifying unknown bacteria and comparing an isolate with the closest species. In addition, the length of this gene is optimum for bioinformatics analysis18. All these features along with the ease of gene amplification using universal primers and improvement in gene sequencing technology make it a gold standard for the identification of microbes.

Here, we describe a procedure to recover cultivable microorganisms with HC-degrading potential from environmental samples. The method described below outlines the collection and identification of HC-degrading bacteria and is divided into five sections: (1) collection of bacteria from water samples, (2) isolation of pure cultures, (3) exploring HC-degrading capability of bacterial isolates (4) genomic DNA isolation, and (5) identification based on 16S rRNA gene sequencing and BLAST analysis. This procedure can be adapted to isolate bacteria for many different biotechnological applications.

Protocol

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 method29,30.
  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 (A260/280) 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 (A260/280) 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 sequencing24,34 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.

Results

The schematic outlining the entire procedure for isolation and screening of bacteria from aquatic habitats and their subsequent identification by 16S rRNA analysis is represented in Figure 1. Water samples from a wetland in Dadri, India were collected in sterile glass bottles and immediately taken to the laboratory for processing. The samples were passed through filter sheets with 0.22 µm pore size, and the filter papers were kept in contact with different media plates....

Discussion

It is well established that only approximately 1% of bacteria on Earth can be readily cultivated in the laboratory6. Even among the cultivable bacteria, many remain uncharacterized. Improvements in molecular methods have given a new dimension to the analysis and evaluation of bacterial communities. However, such techniques do have limitations, but they do not make the culture analyses redundant. Pure culture techniques to isolate individual bacterial species remain the primary mechanism for t...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

We thank Dr. Karthik Krishnan and members of the RP lab for their helpful comments and suggestions. DS is supported by SNU-Doctoral fellowship and Earthwatch Institute India Fellowship. RP lab is supported by a CSIR-EMR grant and start-up funds from Shiv Nadar University.

Materials

NameCompanyCatalog NumberComments
AgaroseSigma-AldrichA4718Gel electrophoresis
Ammonium chloride (NH4Cl)Sigma-AldrichA9434Growth medium component
Ammonium sulphateSigma-AldrichA4418Growth medium component
Bacto-AgarMillipore1016141000Solid media preparation
Calcium chloride (CaCl2)MERCKC4901-500GGrowth medium component
CatecholSigma-Aldrich135011Hydrocarbon degradation assay
Cetyltrimethylammonium bromide, CTABSigma-AldrichH6269Genomic DNA Isolation
ChloroformHIMEDIAMB109Genomic DNA isolation
Disodium phosphate (Na2HPO4)Sigma-AldrichS5136Growth medium component
EDTASigma-AldrichE9884gDNA buffer component
Ferrous sulphate, heptahydrate (FeSO4.7H20)Sigma-Aldrich215422Growth medium component
GlucoseSigma-AldrichG7021Growth medium component
GlycerolSigma-AldrichG5516Growth medium component; Glycerol stocks
IsopropanolHIMEDIAMB063Genomic DNA isolation
LB AgarDifco244520Growth medium
Luria-Bertani (LB)Difco244620Growth medium
Magnesium sulphate (MgSO4)MERCKM2643Growth medium component
Manganese (II) sulfate monohydrate (MnSO4.H20)Sigma-Aldrich221287Growth medium component
Nutrient Broth (NB)Merck (Millipore)03856-500GGrowth medium
PeptoneMerck91249-500GGrowth medium component
PhenolSigma-AldrichP1037Genomic DNA isolation
Potassium phosphate, dibasic (K2HPO4)Sigma-AldrichP3786Growth medium component
Potassium phosphate, monobasic (KH2PO4)Sigma-AldrichP9791Growth medium component
Proteinase KThermoFisher ScientificAM2546Genomic DNA isolation
QIAquick Gel Extraction kitQIAGEN160016235DNA purification
QIAquick PCR Purification kitQIAGEN163038783DNA purification
R2A AgarMillipore1004160500Growth medium
SmartSpec Plus SpectrophotometerBIO-RAD4006221Absorbance measurement
Sodium acetateSigma-AldrichS2889Genomic DNA isolation
Sodium chloride (NaCl)Sigma-AldrichS9888Growth medium component
Sodium dodecyl sulphate (SDS)Sigma-AldrichL3771Genomic DNA isolation
StyreneSigma-AldrichS4972Styrene biodegradation
Taq DNA PolymeraseNEBM0273X16s rRNA PCR
Tris-EDTA (TE)Sigma-Aldrich93283Resuspension of genomic DNA
Tryptic Soy Broth (TSB)Merck22092-500GGrowth medium
Yeast extractSigma-AldrichY1625-1KGGrowth medium component
Zinc sulfate heptahydrate (ZnSO4.7H20)Sigma-Aldrich221376Growth medium component

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