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.
Subscription Required. Please recommend JoVE to your librarian.
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.
- 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. - 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. - 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.
- 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.
- Spread 100 µL of the diluted water sample individually on all growth media plates mentioned in step 3 in triplicates.
- 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. - Next, pick the colonies using a sterile toothpick or pipette tip and perform quadrant streaking to get isolated colonies.
- 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.
- Perform gram-staining of each pure culture23 and proceed with glycerol stock preparation.
- 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.
- 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. - The next day, pellet the cells at 2862 x g for 5 min at 4 °C and discard the supernatant.
- 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. - Resuspend the pellet in 2 mL of liquid carbon-free basal medium (LCFBM). Measure the absorbance (OD600).
- Take two sterile Erlenmeyer flasks with 150 mL capacity for control and the experimental group. Label them as A and B.
- In the uninoculated /control group, (flask A), add 40 mL of LCFBM and styrene (5 mM).
- 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. - Repeat the above for each additional strain that must be evaluated for hydrocarbon degradation.
- 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.
- 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.
- Wash the cell pellet with phosphate buffer (0.5 M, pH 7.5).
- Resuspend the cells in the above-mentioned phosphate buffer and adjust the final OD600 ≈ 1.0.
- 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.
- Remove the cell debris and unbroken cells by centrifugation at 9,000 x g for 30 min, maintaining the cold temperature (4 °C).
- Carefully pipette the clear supernatant. This fraction has the crude extract for enzyme assay.
- Determine the protein concentration of crude extract by either Bradford or Lowry's method29,30.
- To determine the activity of catechol 2,3-dioxygenase, measure the formation of the reaction end product (2-hydroxymuconic semialdehyde) by a spectrophotometer.
- 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.
- For the negative control, replace the crude extract with phosphate buffer and adjust the final volume to 1 mL.
- 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.
- Isolation of genomic DNA from Gram-negative bacteria31.
- Pick a single colony and inoculate in a fresh growth medium in sterile test tubes.
- Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at 30 °C.
- The next day, pellet 1.5 mL of overnight grown culture at 12,400 x g for 3 min.
- 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).
- Add 66 µL of NaCl solution (5 M) and mix well.
- Pellet the resulting mixture at 12,400 x g for 10 min (4 °C).
- Pipette the clear supernatant in a fresh microcentrifuge tube and add an equal volume of chloroform.
- Invert mix the solution multiple times until a milky solution is observed.
- Spin at 12,400 x g for 3 min and transfer the supernatant to a clean vial.
- Add 1 mL of ice-cold 100% ethanol; mix by inversion till white strands of DNA precipitate out.
- Centrifuge the precipitated DNA at 2,200 x g for 10 min at 4 °C and discard the supernatant.
- Wash the DNA pellet with 1 mL of 70% ethanol and allow the DNA pellet to dry for 5 min at room temperature.
- Once dried, resuspend the pellet in 100 µL of 1x Tris-EDTA(TE) buffer, and store the DNA at -20 °C.
- Measure the concentration (A260/280) using spectrophotometer and run the DNA on agarose gel (1%) to assess the quality of DNA24.
- Isolation of genomic DNA from gram-positive strain32
- Pick a single colony and inoculate in fresh growth medium in sterile test tubes.
- Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at a suitable growth temperature.
- Next day, take 1.5 mL of the grown culture and centrifuge at 8,600 x g for 5 min.
- Remove the supernatant and resuspend the cells in TE buffer.
- Adjust the OD600 = 1.0 with TE buffer and transfer 740 µL of the cell suspension to a clean microfuge tube.
- 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).
- Add 40 µL of 10% SDS and mix well.
- 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. - Preheat CTAB/NaCl mixture at 65 °C (in a dry bath) and add 100 µL of this mixture to the cell suspension. Mix well.
- Incubate at 65 °C for 10 min (in a dry bath).
- Add 500 µL of chloroform:isoamyl alcohol (24:1) and mix well. Spin at 16,900 x g for 10 min at 25 °C.
- Transfer the aqueous phase to a fresh microcentrifuge tube avoiding the organic phase (viscous phase at the bottom).
- 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.
- Take the aqueous phase in a fresh microcentrifuge tube. Add 500 µL of chloroform:isoamyl alcohol (24:1) and mix well.
- Transfer the aqueous phase and add 0.6 volume isopropanol (prechilled at -20 °C).
- The precipitated DNA strands must be visible in the threadlike form. Incubate at -20 °C for 2 h to overnight.
- Centrifuge at 16,900 x g for 15 min at 4 °C to pellet the DNA.
- Decant the isopropanol carefully and wash the pellet with 1 mL of cold 70% ethanol (prechilled at -20 °C) to remove any impurities.
- Centrifuge at 16,900 x g for 5 min at 4 °C. Discard the supernatant.
- 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.
- 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. - 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.
- 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.
- 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.
- 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.
- 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.
- For 16S rRNA gene sequencing, set up the same reaction as mentioned above for higher volume (100 µL).
- 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.
- 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.
Subscription Required. Please recommend JoVE to your librarian.
Representative 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. After 2 h, filter papers were removed, and the plates were incubated overnight at 30 °C for colonies formation (Figure 2). The next day, individual bacterial colonies were selected and streaked on fresh media plates (Figure 2). The pure culture generated was stored and subsequently used for further analysis. Using this method, we were able to create a library of more than 100 unique bacterial isolates. We aimed to identify bacterial isolates that can utilize hydrocarbons, especially styrene, which is the primary component of single-use plastic. The isolated bacteria were individually grown in respective media with the addition of liquid styrene as a sole source of carbon (Figure 3). We could identify four isolates that utilize styrene as a sole source of carbon. Two of the isolates were extensively characterized further for styrene degradation25.
The bacterial isolates were then tested for the presence of enzymatic pathways for the degradation of hydrocarbon metabolism. Hydrocarbon metabolism in some bacteria results in the production of catechols as intermediates, which are further degraded by ortho-cleavage and meta-cleavage pathways. Catechol 1,2- dioxygenase and catechol 2,3-dioxygenase enzymes are responsible for ring-cleavage reaction36. Environmental bacteria possessing these enzymes have been shown to metabolize several aromatic compounds. Thus, a catechol degradation assay was performed to assess the HC-degrading potential of bacterial isolates (Figure 4). A representative assay for one of the isolates is shown in Figure 4.
To identify the bacterial isolates, 16S rRNA sequencing was performed. A preliminary gram staining was performed to characterize the bacteria, which helps identify and troubleshoot subsequent steps. Gram-positive bacteria are usually recalcitrant to cell lysis buffers leading to low genomic DNA yield37. Thus, the results obtained from gram-staining38 before genomic DNA isolation would help in choosing the protocol for genomic DNA isolation. After DNA isolation, the integrity of genomic DNA was confirmed by visualizing a small sample of DNA on agarose gel (Figure 5A) and quantified by UV absorbance method using a spectrophotometer. 16S rRNA gene was amplified using universal primers sequence (Figure 5B). While 500 bp are essential for sequencing, ideal results are obtained with 1,300-1,500 bp39. To obtain the degree of relatedness among isolated strains, a phylogenetic tree was constructed using the phylogeny.fr software40 (Figure 6).
Figure 1: Schematic workflow of the study Please click here to view a larger version of this figure.
Figure 2: Images of bacterial colonies from water samples. The collected water sample was passed through 0.22 µm filter paper. The filter papers were kept over different media plates. The plates were incubated for 24-48 h until isolated colonies were observed. The single colonies were then streaked on fresh plates for pure culture isolation. Please click here to view a larger version of this figure.
Figure 3: Representative results of microbial degradation of styrene and screening hydrocarbon degradation potential of bacteria. The cells were grown in LCFBM supplemented with 5 mM styrene as a sole carbon source for 40 days at 30 °C and 200 rpm. OD600 was measured every 5 days. The control flask had only LCFBM. Please click here to view a larger version of this figure.
Figure 4: Representation of Catechol 2,3-dioxygenase enzyme assay to monitor the degradation of catechol. (A) The colorless substrate catechol is converted into a yellow-colored product by the action of catechol 2,3-dioxygenase. The reaction mixture contains catechol, phosphate buffer, and crude cell lysate. The formation of the product is detected by measuring absorbance at 375 nm. (B) Representative graph of catechol 2,3-dioxygenase enzyme assay with whole-cell lysate. The reaction mixture in negative control has buffer and catechol substrate without cell lysate. Absorbance was measured at 375 nm at an interval of 10 min. Please click here to view a larger version of this figure.
Figure 5: Genomic DNA isolation and 16S rRNA PCR. (A) Gel electrophoresis of isolated genomic DNA. Lane M: DNA size marker, Lane 1-2: Genomic DNA. (B) Verification of 16S rRNA gene amplification by 1% gel electrophoresis. Gels were visualized by staining with ethidium bromide; Lane M: DNA size marker (1 kb), Lane 1-4: Amplified PCR products from different strains. Please click here to view a larger version of this figure.
Figure 6: Analysis of the 16S rRNA gene sequencing results. Representative dendrogram construction using the phylogeny.fr program to depict the relatedness among Exiguobacterium strains isolated from a wetland (highlighted in red box) with the known Exiguobacterium sp. The 16S rRNA sequences of known Exiguobacterium sp. were obtained from NCBI. This figure has been taken from a previous paper (Chauhan et.al.) without any modification25. Please click here to view a larger version of this figure.
| Peptone Yeast Extract (PYE) | |
| Peptone | 2g |
| Yeast extract | 1g |
| 1M MgSO4 | 1 ml |
| 1M CaCl2 | 1 ml |
| Distilled water | Up to 1000 ml |
| Sterilize by autoclaving at 121°C and 15 PSI for 15 min. | |
| Reasoner’s 2A (R2A) | |
| Casein acid hydrolysate | 0.5 g |
| Yeast extract | 0.5 g |
| Protease peptone | 0.5 g |
| Dextrose | 0.5g |
| Starch, soluble | 0.5 g |
| K2HPO4 | 0.5 g |
| Distilled water | Up to 1000 ml |
| Sterilize by autoclaving at 121°C and 15 PSI for 15 min. | |
| M2G | |
| 10X M2 salts (1L) - | |
| Na2HPO4 | 17.4 g |
| KH2PO4 | 10.6 g |
| NH4Cl | 5.0 g |
| Autoclave 10X M2 salts at 121 °C and 15 PSI for 15 min. | |
| 10X M2 salts | 100 ml |
| 50mM MgCl2 | 10 ml |
| 30% glucose (w/v) | 10 ml |
| 1 mM FeSO4 in 0.8 mM EDTA, pH 6.8 | 10 ml |
| 50 mM CaCl2 | 10 ml |
| Distilled water | Up to 1000 ml |
| Filter sterilize. | |
| Lysogeny Broth (LB) | |
| Casein enzymic hydrolysate | 10 g |
| Yeast extract | 5 g |
| NaCl | 10 g |
| Distilled water | Up to 1000 ml |
| Sterilize by autoclaving at 121°C and 15 PSI for 15 min. | |
| Nutrient Broth (NB) | |
| Peptone | 15 g |
| Yeast extract | 3 g |
| NaCl | 6 g |
| Glucose | 1 g |
| Distilled water | Up to 1000 ml |
| Sterilize by autoclaving at 121°C and 15 PSI for 15 min. | |
| Tryptic Soy Broth (TSB) | |
| Pancreatic digest of casein | 17.0 g |
| Papaic digest of soyabean meal | 3 g |
| NaCl | 5 g |
| K2HPO4 | 2.5 g |
| Dextrose | 2.5 g |
| Distilled water | Up to 1000 ml |
| Sterilize by autoclaving at 121°C and 15 PSI for 15 min. | |
| M63 | |
| NH4Cl | 2g |
| KH2PO4 | 13.6 g |
| FeSO4.7H2O | 0.5 mg |
| 20% glycerol | 10 ml |
| 1M MgSO4 | 1 ml |
| Distilled water | Up to 1000 ml |
| M9 minimal media | |
| 5X M9 salts | |
| Na2HPO4.7H20 | 12.8 g |
| KH2PO4 | 3 g |
| NH4Cl | 1 g |
| NaCl | 0.5 g |
| Distilled water | Up to 200 ml |
| Autoclave 5X M9 salts at 121°C and 15 PSI for 15 min. | |
| 1X M9 media | |
| 5X M9 salts | 20 ml |
| 20% glucose | 2 ml |
| 1M MgSO4 | 200 µl |
| 1M CaCl2 | 10 µl |
| Autoclaved water | Up to 100 ml |
| NOTE – For solid media preparation, use 1.5% Bacto Agar (15 g/L) |
Table 1.
Subscription Required. Please recommend JoVE to your librarian.
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 the characterization of physiological properties. Soil and aquatic habitats harbor many bacteria with novel enzymes and pathways, which can be harnessed for biotechnological uses. This study describes a simple and inexpensive method for the isolation and characterization of bacteria from ecological samples.
Different bacteria have different nutritional requirements, hence various growth media were used to increase the probability of isolating diverse bacterial species. One major limitation of this method is that microbes with fastidious growth requirements may get excluded. Also, the main goal of this step is to maximize the number of bacterial species obtained from the sample. The number of bacterial species in the sample library would improve the chances of isolating microbes with bioremediation potential. Though we only varied growth media, varying growth temperature and oxygen concentration can also increase the chances of further expanding the sample library with unique species41,42.
A critical step of the protocol is to check the utilization of the substrate being tested (styrene in our case). It is important to design the experiment for such investigation with care to avoid false-negative results. Depending on growth characteristics, the microbe may not immediately adapt to utilizing the substrate being tested and may require an enrichment process. In our case, the bacterial growth is slow in the LCFBM medium used to test the utilization of hydrocarbon as the sole carbon source25. To circumvent this problem, initial cultures can be started by adding (1% v/v) TSB or NB to the LCFBM medium to support bacterial growth. Identification of the cultivated microbe is accomplished through 16S rRNA sequencing43. This method offers a robust and cost-effective method for microbial identification. However, 16S sequencing can only provide higher-level taxonomical identification. For specific species-level identification, other family-specific primers have to be used combined with various biochemical tests44,45.
Enzyme assay with whole-cell lysate requires using an efficient cell lysis method. Bacterial cell lysis is usually achieved by performing sonication. However, a freeze-thaw method is an alternative method for gentle cell lysis, which is believed to prevent denaturation of protein. The procedure consists of quickly freezing the cells at -80 °C and thawing at 4 °C in a sequential manner46. The addition of mild detergents such as NP-40 or Triton-X-100 also aids in cell lysis and does not denature the proteins, due to their non-ionic nature47. However, bacteria with thick cell walls such as cyanobacteria48 may not benefit from the gentle cell lysis method using detergents49 and thus, the lysis method for enzyme assay must be chosen accordingly.
By focusing on the cultivable bacterial population from environmental samples, researchers can quickly perform many different experiments. The methods described here do not require the use of very sophisticated instruments and can be easily performed in a standard laboratory setup. Since hydrocarbons and hazardous chemicals are used, the laboratory should be equipped with proper handling and disposal according to standard operating procedures. The approach described here can be easily adapted to study a variety of bacterial species for numerous biotechnological applications.
Subscription Required. Please recommend JoVE to your librarian.
Disclosures
The authors declare no conflicts of interest.
Acknowledgments
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
| Name | Company | Catalog Number | Comments |
| Agarose | Sigma-Aldrich | A4718 | Gel electrophoresis |
| Ammonium chloride (NH4Cl) | Sigma-Aldrich | A9434 | Growth medium component |
| Ammonium sulphate | Sigma-Aldrich | A4418 | Growth medium component |
| Bacto-Agar | Millipore | 1016141000 | Solid media preparation |
| Calcium chloride (CaCl2) | MERCK | C4901-500G | Growth medium component |
| Catechol | Sigma-Aldrich | 135011 | Hydrocarbon degradation assay |
| Cetyltrimethylammonium bromide, CTAB | Sigma-Aldrich | H6269 | Genomic DNA Isolation |
| Chloroform | HIMEDIA | MB109 | Genomic DNA isolation |
| Disodium phosphate (Na2HPO4) | Sigma-Aldrich | S5136 | Growth medium component |
| EDTA | Sigma-Aldrich | E9884 | gDNA buffer component |
| Ferrous sulphate, heptahydrate (FeSO4.7H20) | Sigma-Aldrich | 215422 | Growth medium component |
| Glucose | Sigma-Aldrich | G7021 | Growth medium component |
| Glycerol | Sigma-Aldrich | G5516 | Growth medium component; Glycerol stocks |
| Isopropanol | HIMEDIA | MB063 | Genomic DNA isolation |
| LB Agar | Difco | 244520 | Growth medium |
| Luria-Bertani (LB) | Difco | 244620 | Growth medium |
| Magnesium sulphate (MgSO4) | MERCK | M2643 | Growth medium component |
| Manganese (II) sulfate monohydrate (MnSO4.H20) | Sigma-Aldrich | 221287 | Growth medium component |
| Nutrient Broth (NB) | Merck (Millipore) | 03856-500G | Growth medium |
| Peptone | Merck | 91249-500G | Growth medium component |
| Phenol | Sigma-Aldrich | P1037 | Genomic DNA isolation |
| Potassium phosphate, dibasic (K2HPO4) | Sigma-Aldrich | P3786 | Growth medium component |
| Potassium phosphate, monobasic (KH2PO4) | Sigma-Aldrich | P9791 | Growth medium component |
| Proteinase K | ThermoFisher Scientific | AM2546 | Genomic DNA isolation |
| QIAquick Gel Extraction kit | QIAGEN | 160016235 | DNA purification |
| QIAquick PCR Purification kit | QIAGEN | 163038783 | DNA purification |
| R2A Agar | Millipore | 1004160500 | Growth medium |
| SmartSpec Plus Spectrophotometer | BIO-RAD | 4006221 | Absorbance measurement |
| Sodium acetate | Sigma-Aldrich | S2889 | Genomic DNA isolation |
| Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | Growth medium component |
| Sodium dodecyl sulphate (SDS) | Sigma-Aldrich | L3771 | Genomic DNA isolation |
| Styrene | Sigma-Aldrich | S4972 | Styrene biodegradation |
| Taq DNA Polymerase | NEB | M0273X | 16s rRNA PCR |
| Tris-EDTA (TE) | Sigma-Aldrich | 93283 | Resuspension of genomic DNA |
| Tryptic Soy Broth (TSB) | Merck | 22092-500G | Growth medium |
| Yeast extract | Sigma-Aldrich | Y1625-1KG | Growth medium component |
| Zinc sulfate heptahydrate (ZnSO4.7H20) | Sigma-Aldrich | 221376 | Growth medium component |
References
- Sirotkin, A. V. Reproductive effects of oil-related environmental pollutants. Encyclopedia of Environmental Health. , 493-498 (2019).
- Li, C., Busquets, R., Campos, L. C. Assessment of microplastics in freshwater systems: A review. Science of The Total Environment. 707, 135578 (2020).
- Siddiqa, A., Faisal, M. Microbial degradation of organic pollutants using indigenous bacterial strains. Handbook of Bioremediation. , 625-637 (2021).
- Hossain, F., et al. Bioremediation potential of hydrocarbon degrading bacteria: isolation, characterization, and assessment. Saudi Journal of Biological Sciences. , (2021).
- Wongbunmak, A., Khiawjan, S., Suphantharika, M., Pongtharangkul, T. BTEX biodegradation by Bacillus amyloliquefaciens subsp. plantarum W1 and its proposed BTEX biodegradation pathways. Scientific Reports. 10 (1), 17408 (2020).
- Bodor, A., et al. Challenges of unculturable bacteria: environmental perspectives. Reviews in Environmental Science and Bio/Technology. 19 (1), 1-22 (2020).
- Phale, P. S., Sharma, A., Gautam, K. Microbial degradation of xenobiotics like aromatic pollutants from the terrestrial environments. Pharmaceuticals and Personal Care Products: Waste Management and Treatment Technology. , 259-278 (2019).
- Brooijmans, R. J. W., Pastink, M. I., Siezen, R. J. Hydrocarbon-degrading bacteria: the oil-spill clean-up crew. Microbial Biotechnology. 2 (6), 587-594 (2009).
- Sorkhoh, N. A., Ghannoum, M. A., Ibrahim, A. S., Stretton, R. J., Radwan, S. S. Crude oil and hydrocarbon-degrading strains of Rhodococcus rhodochrous isolated from soil and marine environments in Kuwait. Environmental Pollution. 65 (1), 1-17 (1990).
- Chaillan, F., et al. Identification and biodegradation potential of tropical aerobic hydrocarbon-degrading microorganisms. Research in Microbiology. 155 (7), 587-595 (2004).
- Bôto, M. L., et al. Harnessing the potential of native microbial communities for bioremediation of oil spills in the Iberian Peninsula NW coast. Frontiers in Microbiology. 12, 879 (2021).
- Wang, Y., et al. A culture-independent approach to unravel uncultured bacteria and functional genes in a complex microbial community. PloS One. 7 (10), 47530 (2012).
- Jovel, J., et al. Characterization of the gut microbiome using 16S or shotgun metagenomics. Frontiers in Microbiology. 7, 459 (2016).
- Spini, G., et al. Molecular and microbiological insights on the enrichment procedures for the isolation of petroleum degrading bacteria and fungi. Frontiers in Microbiology. 0, 2543 (2018).
- Pightling, A. W., et al. Interpreting whole-genome sequence analyses of foodborne bacteria for regulatory applications and outbreak investigations. Frontiers in Microbiology. , 1482 (2018).
- Salipante, S. J., et al. Application of whole-genome sequencing for bacterial strain typing in molecular epidemiology. Journal of Clinical Microbiology. 53 (4), 1072-1079 (2015).
- Lovley, D. R. Cleaning up with genomics: applying molecular biology to bioremediation. Nature Reviews Microbiology. 1 (1), 35-44 (2003).
- Janda, J. M., Abbott, S. L. 16S rRNA gene sequencing for bacterial identification in the diagnostic laboratory: pluses, perils, and pitfalls. Journal of Clinical Microbiology. 45 (9), 2761-2764 (2007).
- Poindexter, J. S. Biological properties and classification of the Caulobacter group. Bacteriological Reviews. 28 (3), 231-295 (1964).
- Reasoner, D. J., Geldreich, E. A new medium for the enumeration and subculture of bacteria from potable water. Applied and Environmental Microbiology. 49 (1), 1-7 (1985).
- Pardee, A. B. The genetic control and cytoplasmic expression of “Inducibility” in the synthesis of β-galactosidase by E. coli. Journal of Molecular Biology. 1 (2), 165-178 (1959).
- Ely, B. Genetics of Caulobacter crescentus. Methods in Enzymology. 204, 372-384 (1991).
- R, C. Gram staining. Current Protocols in Microbiology. , Appendix 3 (2005).
- Green, M. R., Hughes, H., Sambrook, J., MacCallum, P. Molecular cloning: a laboratory manual. Molecular Cloning: A Laboratory Manual. , 1890 (2012).
- Chauhan, D., Agrawal, G., Deshmukh, S., Roy, S. S., Priyadarshini, R. Biofilm formation by Exiguobacterium sp. DR11 and DR14 alter polystyrene surface properties and initiate biodegradation. RSC Advances. 8 (66), 37590-37599 (2018).
- Takeo, M., Nishimura, M., Shirai, M., Takahashi, H., Negoro, S. Purification and characterization of catechol 2,3-Dioxygenase from the aniline degradation pathway of Acinetobacter sp. YAA and its mutant enzyme, which resists substrate inhibition. Bioscience, Biotechnology, Biochemistry. 71, 70079-70080 (2007).
- Nguyen, O. T., Ha, D. D. Degradation of chlorotoluenes and chlorobenzenes by the dual-species biofilm of Comamonas testosteroni strain KT5 and Bacillus subtilis strain DKT. Annals of Microbiology. 69 (3), 267-277 (2019).
- Hupert-Kocurek, K., Guzik, U., Wojcieszyńska, D. Characterization of catechol 2, 3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration. Acta Biochimica Polonica. 59 (3), (2012).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1-2), 248-254 (1976).
- Peterson, G. L., et al. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Analytical Biochemistry. 83 (2), 346-356 (1977).
- Chen, W. P., Kuo, T. T. A simple and rapid method for the preparation of gram-negative bacterial genomic DNA. Nucleic Acids Research. 21 (9), 2260 (1993).
- William, S., Feil, H., Copeland, A. Bacterial genomic DNA isolation using CTAB. Sigma. 50 (6876), (2012).
- Frank, J. A., et al. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Applied and Environmental Microbiology. 74 (8), 2461-2470 (2008).
- Sanger, F., Nicklen, S., Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences of the United States of America. 74 (12), 5463-5467 (1977).
- Johnson, M., et al. NCBI BLAST: a better web interface. Nucleic Acids Research. 36, suppl 2 5-9 (2008).
- Harayama, S., Rekik, M. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. Journal of Biological Chemistry. 264 (26), 15328-15333 (1989).
- Li, X., et al. Efficiency of chemical versus mechanical disruption methods of DNA extraction for the identification of oral Gram-positive and Gram-negative bacteria. The Journal of International Medical Research. 48 (5), 300060520925594 (2020).
- Coico, R. Gram staining. Current Protocols in Microbiology. , Appendix 3 (2005).
- Clarridge, J. E. III Impact of 16S rRNA gene sequence analysis for identification of bacteria on clinical microbiology and infectious diseases. Clinical Microbiology Reviews. 17 (4), 840 (2004).
- Dereeper, A., et al. Phylogeny. fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Research. 36, suppl 2 465-469 (2008).
- Wadowsky, R. M., Wolford, R., McNamara, A. M., Yee, R. B. Effect of temperature, pH, and oxygen level on the multiplication of naturally occurring Legionella pneumophila in potable water. Applied and Environmental Microbiology. 49 (5), 1197-1205 (1985).
- du Toit, W. J., Pretorius, I. S., Lonvaud-Funel, A. The effect of sulphur dioxide and oxygen on the viability and culturability of a strain of Acetobacter pasteurianus and a strain of Brettanomyces bruxellensis isolated from wine. Journal of Applied Microbiology. 98 (4), 862-871 (2005).
- Johnson, J. S., et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nature Communications. 10 (1), 1-11 (2019).
- Kim, E., et al. Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate. BMC Microbiology. 20 (1), 1-14 (2020).
- Poretsky, R., Rodriguez-R, L. M., Luo, C., Tsementzi, D., Konstantinidis, K. T. Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics. PLoS ONE. 9 (4), (2014).
- Johnson, B. H., Hecht, M. H. Recombinant proteins can be isolated from E. coli cells by repeated cycles of freezing and thawing. Bio/Technology. 12 (12), 1357-1360 (1994).
- Rodríguez-Carmona, E., Cano-Garrido, O., Seras-Franzoso, J., Villaverde, A., García-Fruitós, E. Isolation of cell-free bacterial inclusion bodies. Microbial Cell Factories. 9 (1), 71 (2010).
- Hoiczyk, E., Hansel, A. Cyanobacterial cell walls: News from an unusual prokaryotic envelope. Journal of Bacteriology. 182 (5), 1191 (2000).
- Erstad, S. M., Sakuragi, Y. Easy and efficient permeabilization of cyanobacteria for in vivo enzyme assays using B-PER. Bio-Protocol. 8 (1), (2018).
