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.

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.
<ol>
	<li>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.</li>
	<li>Filter the sample in a batch of 100 mL through 0.22 &#181;m-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.</li>
	<li>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.</li>
	<li>Serially dilute the unfiltered water samples (106 dilution) in sterile double distilled water by adding 100 &#181;L of the collected water sample in 900 &#181;L of sterile water. This results in a 1:10 dilution. From this sample, take 100 &#181;L and add in 900 &#181;L 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.</li>
	<li>Spread 100 &#181;L of the diluted water sample individually on all growth media plates mentioned in step 3 in triplicates.</li>
	<li>Incubate the plates at 30 &#176;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 &#176;C. If isolating the samples from an environment with extreme temperatures, incubate the plates at the same temperature as that of the collection site.</li>
	<li>Next, pick the colonies using a sterile toothpick or pipette tip and perform quadrant streaking to get isolated colonies.</li>
	<li>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.</li>
	<li>Perform gram-staining of each pure culture23 and proceed with glycerol stock preparation.</li>
	<li>To prepare the glycerol stocks, inoculate a single colony in 3 mL of appropriate growth media and incubate at 30 &#176;C. From the overnight culture, take 700 &#181;L and add 300 &#181;L of 100% glycerol (sterilized by autoclaving) in cryovials24. Freeze the vials at -80 &#176;C for long-term storage.</li>
</ol>
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.
<ol>
	<li>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 &#176;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.</li>
	<li>The next day, pellet the cells at 2862 x g for 5 min at 4 &#176;C and discard the supernatant.</li>
	<li>Wash the pellet twice with 2 mL of autoclaved saline (0.9% NaCl) and spin at 2862 x g for 5 min at 4 &#176;C. 
	NOTE: Saline is isotonic and, thus, it maintains the osmotic pressure inside bacterial cells.</li>
	<li>Resuspend the pellet in 2 mL of liquid carbon-free basal medium (LCFBM). Measure the absorbance (OD600).</li>
	<li>Take two sterile Erlenmeyer flasks with 150 mL capacity for control and the experimental group. Label them as A and B.</li>
	<li>In the uninoculated /control group, (flask A), add 40 mL of LCFBM and styrene (5 mM).</li>
	<li>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 &#8776; 0.1 and make up the remaining volume with LCFBM up to 40 mL. Incubate the flasks at 30 &#176;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.</li>
	<li>Repeat the above for each additional strain that must be evaluated for hydrocarbon degradation.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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 &#176;C and 200 rpm. Harvest the culture at 4 &#176;C when OD600 reaches between 1.4-1.6 (i.e., in late exponential phase) by spinning at 4500 x g for 20 min.</li>
	<li>Wash the cell pellet with phosphate buffer (0.5 M, pH 7.5).</li>
	<li>Resuspend the cells in the above-mentioned phosphate buffer and adjust the final OD600&#160;&#8776; 1.0.</li>
	<li>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.</li>
	<li>Remove the cell debris and unbroken cells by centrifugation at 9,000 x g for 30 min, maintaining the cold temperature (4 &#176;C).</li>
	<li>Carefully pipette the clear supernatant. This fraction has the crude extract for enzyme assay.</li>
	<li>Determine the protein concentration of crude extract by either Bradford or Lowry&#39;s method29,30.</li>
	<li>To determine the activity of catechol 2,3-dioxygenase, measure the formation of the reaction end product (2-hydroxymuconic semialdehyde) by a spectrophotometer.</li>
	<li>Prepare the reaction mixture by adding 20 &#181;L of catechol (50 mM), 960 &#181;L of phosphate buffer (50 mM, pH 7.5), and 20 &#181;L of the crude extract.</li>
	<li>For the negative control, replace the crude extract with phosphate buffer and adjust the final volume to 1 mL.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
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.
<ol>
	<li>Isolation of genomic DNA from Gram-negative bacteria31.
	<ol>
		<li>Pick a single colony and inoculate in a fresh growth medium in sterile test tubes.</li>
		<li>Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at 30 &#176;C.</li>
		<li>The next day, pellet 1.5 mL of overnight grown culture at 12,400 x g for 3 min.</li>
		<li>Remove the supernatant and resuspend the pellet in 200 &#181;L of lysis buffer (40 mM Tris-acetate, pH 7.8, 20 mM sodium acetate, 1 mM EDTA, 1% SDS).</li>
		<li>Add 66 &#181;L of NaCl solution (5 M) and mix well.</li>
		<li>Pellet the resulting mixture at 12,400 x g for 10 min (4 &#176;C).</li>
		<li>Pipette the clear supernatant in a fresh microcentrifuge tube and add an equal volume of chloroform.</li>
		<li>Invert mix the solution multiple times until a milky solution is observed.</li>
		<li>Spin at 12,400 x g for 3 min and transfer the supernatant to a clean vial.</li>
		<li>Add 1 mL of ice-cold 100% ethanol; mix by inversion till white strands of DNA precipitate out.</li>
		<li>Centrifuge the precipitated DNA at 2,200 x g for 10 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the DNA pellet with 1 mL of 70% ethanol and allow the DNA pellet to dry for 5 min at room temperature.</li>
		<li>Once dried, resuspend the pellet in 100 &#181;L of 1x Tris-EDTA(TE) buffer, and store the DNA at -20 &#176;C.</li>
		<li>Measure the concentration (A260/280) using spectrophotometer and run the DNA on agarose gel (1%) to assess the quality of DNA24.</li>
	</ol>
	</li>
	<li>Isolation of genomic DNA from gram-positive strain32
	<ol>
		<li>Pick a single colony and inoculate in&#160;fresh growth medium in sterile test tubes.</li>
		<li>Place the tubes in an incubator shaker at 200 rpm and allow the bacteria to grow overnight at a suitable growth temperature.</li>
		<li>Next day, take 1.5 mL of the grown culture and centrifuge at 8,600 x g for 5 min.</li>
		<li>Remove the supernatant and resuspend the cells in TE buffer.</li>
		<li>Adjust the OD600 = 1.0 with TE buffer and transfer 740 &#181;L of the cell suspension to a clean microfuge tube.</li>
		<li>Add 20 &#181;L of lysozyme (100 mg/mL stock) and mix well by pipetting. Incubate at 37 &#176;C for 30 min (in a dry bath).</li>
		<li>Add 40 &#181;L of 10% SDS and mix well.</li>
		<li>Add 8 &#181;L of Proteinase K (10 mg/mL). Mix well and incubate at 56 &#176;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.</li>
		<li>Preheat CTAB/NaCl mixture at 65 &#176;C (in a dry bath) and add 100 &#181;L of this mixture to the cell suspension. Mix well.</li>
		<li>Incubate at 65 &#176;C for 10 min (in a dry bath).</li>
		<li>Add 500 &#181;L of chloroform:isoamyl alcohol (24:1) and mix well. Spin at 16,900 x g for 10 min at 25 &#176;C.</li>
		<li>Transfer the aqueous phase to a fresh microcentrifuge tube avoiding the organic phase (viscous phase at the bottom).</li>
		<li>Carefully add 500 &#181;L of phenol:chloroform:isoamyl alcohol (25:24:1) and mix well. Spin at 16,900 x g for 10 min at 25 &#176;C.</li>
		<li>Take the aqueous phase in a fresh microcentrifuge tube. Add 500 &#181;L of chloroform:isoamyl alcohol (24:1) and mix well.</li>
		<li>Transfer the aqueous phase and add 0.6 volume isopropanol (prechilled at -20 &#176;C).</li>
		<li>The precipitated DNA strands must be visible in the threadlike form. Incubate at -20 &#176;C for 2 h to overnight.</li>
		<li>Centrifuge at 16,900 x g for 15 min at 4 &#176;C to pellet the DNA.</li>
		<li>Decant the isopropanol carefully and wash the pellet with 1 mL of cold 70% ethanol (prechilled at -20 &#176;C) to remove any impurities.</li>
		<li>Centrifuge at 16,900 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
		<li>Allow the pellet to dry at room temperature for 20 min or keep the tube at 37 &#176;C. Make sure the pellet is not over-dried.</li>
		<li>Resuspend in 100 &#181;L of 1x TE buffer and store the DNA at -20 &#176;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 &#176;C for 15-20 min and resuspend again by pipetting.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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&#39;-AGAGTTTGATCMTGGCTCAG-3&#39;) and 1492R (5&#39;- TACGGYTACCTTGTTACGACTT-3&#39;)33.</li>
	<li>Prepare the PCR mix (25 &#181;L reactions) on ice with 18 &#181;L of autoclaved/nuclease-free water, 2.5 &#181;L of 10x buffer, 0.5 &#181;L of both forward and reverse primers (100 &#181;M stock), 2 &#181;L of the dNTPs mix (100 &#181;M stock), 1 &#181;L of DNA template (2-15 ng/&#181;L) and 1 U of Taq Polymerase.</li>
	<li>Use the following cycling conditions for 16S rRNA gene amplification: Initial denaturation at 94 &#176;C for 10 min, (final denaturation at 94 &#176;C for 40 s, primer annealing at 56 &#176;C for 1 min, extension at 74 &#176;C for 2 min) x 30 cycles, final extension at 74 &#176;C for 10 min.</li>
	<li>After the cycle ends, mix 5 &#181;L of sample and 1 &#181;L of 5x DNA loading dye. Run on 1% agarose gel to verify the amplification. Store the PCR products at 4 &#176;C for the short term or freeze them at -20&#176;C until further use.</li>
	<li>For 16S rRNA gene sequencing, set up the same reaction as mentioned above for higher volume (100 &#181;L).</li>
	<li>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.</li>
	<li>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.</li>
</ol>

The authors declare no conflicts of interest.

It is well established that only approximately 1% of bacteria on Earth can be readily cultivated in the&#160;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...

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.&#160;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&#160;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.

<table><tbody><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A4718</td><td>Gel electrophoresis</td></tr><tr><td>Ammonium chloride (NH&lt;sub&gt;4&lt;/sub&gt;Cl)</td><td>Sigma-Aldrich</td><td>A9434</td><td>Growth medium component</td></tr><tr><td>Ammonium sulphate</td><td>Sigma-Aldrich</td><td>A4418</td><td>Growth medium component</td></tr><tr><td>Bacto-Agar</td><td>Millipore</td><td>1016141000</td><td>Solid media preparation</td></tr><tr><td>Calcium chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>MERCK</td><td>C4901-500G</td><td>Growth medium component</td></tr><tr><td>Catechol</td><td>Sigma-Aldrich</td><td>135011</td><td>Hydrocarbon degradation assay</td></tr><tr><td>Cetyltrimethylammonium bromide, CTAB</td><td>Sigma-Aldrich</td><td>H6269</td><td>Genomic DNA Isolation</td></tr><tr><td>Chloroform</td><td>HIMEDIA</td><td>MB109</td><td>Genomic DNA isolation</td></tr><tr><td>Disodium phosphate (Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5136</td><td>Growth medium component</td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>E9884</td><td>gDNA buffer component</td></tr><tr><td>Ferrous sulphate, heptahydrate (FeSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;0)</td><td>Sigma-Aldrich</td><td>215422</td><td>Growth medium component</td></tr><tr><td>Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td>Growth medium component</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td>Growth medium component; Glycerol stocks</td></tr><tr><td>Isopropanol</td><td>HIMEDIA</td><td>MB063</td><td>Genomic DNA isolation</td></tr><tr><td>LB Agar</td><td>Difco</td><td>244520</td><td>Growth medium</td></tr><tr><td>Luria-Bertani (LB)</td><td>Difco</td><td>244620</td><td>Growth medium</td></tr><tr><td>Magnesium sulphate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>MERCK</td><td>M2643</td><td>Growth medium component</td></tr><tr><td>Manganese (II) sulfate monohydrate (MnSO&lt;sub&gt;4&lt;/sub&gt;.H&lt;sub&gt;2&lt;/sub&gt;0)</td><td>Sigma-Aldrich</td><td>221287</td><td>Growth medium component</td></tr><tr><td>Nutrient Broth (NB)</td><td>Merck (Millipore)</td><td>03856-500G</td><td>Growth medium</td></tr><tr><td>Peptone</td><td>Merck</td><td>91249-500G</td><td>Growth medium component</td></tr><tr><td>Phenol</td><td>Sigma-Aldrich</td><td>P1037</td><td>Genomic DNA isolation</td></tr><tr><td>Potassium phosphate, dibasic (K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4)&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3786</td><td>Growth medium component</td></tr><tr><td>Potassium phosphate, monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>P9791</td><td>Growth medium component</td></tr><tr><td>Proteinase K</td><td>ThermoFisher Scientific</td><td>AM2546</td><td>Genomic DNA isolation</td></tr><tr><td>QIAquick Gel Extraction kit</td><td>QIAGEN</td><td>160016235</td><td>DNA purification</td></tr><tr><td>QIAquick PCR Purification kit</td><td>QIAGEN</td><td>163038783</td><td>DNA purification</td></tr><tr><td>R2A Agar</td><td>Millipore</td><td>1004160500</td><td>Growth medium</td></tr><tr><td>SmartSpec Plus Spectrophotometer</td><td>BIO-RAD</td><td>4006221</td><td>Absorbance measurement</td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td>Genomic DNA isolation</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich</td><td>S9888</td><td>Growth medium component</td></tr><tr><td>Sodium dodecyl sulphate (SDS)</td><td>Sigma-Aldrich</td><td>L3771</td><td>Genomic DNA isolation</td></tr><tr><td>Styrene</td><td>Sigma-Aldrich</td><td>S4972</td><td>Styrene biodegradation</td></tr><tr><td>Taq DNA Polymerase</td><td>NEB</td><td>M0273X</td><td>16s rRNA PCR</td></tr><tr><td>Tris-EDTA (TE)</td><td>Sigma-Aldrich</td><td>93283</td><td>Resuspension of genomic DNA</td></tr><tr><td>Tryptic Soy Broth (TSB)</td><td>Merck</td><td>22092-500G</td><td>Growth medium</td></tr><tr><td>Yeast extract</td><td>Sigma-Aldrich</td><td>Y1625-1KG</td><td>Growth medium component</td></tr><tr><td>Zinc sulfate heptahydrate (ZnSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;0)</td><td>Sigma-Aldrich</td><td>221376</td><td>Growth medium component</td></tr></tbody></table>

isolation, propagation, and identification of bacterial species with hydrocarbon metabolizing properties from aquatic habitats

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.

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. Aft...

Watch this Scientific Journal Video about Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats at JoVE.com

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

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.

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

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4.2K Views.  Shiv Nadar University. Dit protocol schetst de isolatie, teelt en identificatie van koolwaterstofafbrekende micro-organismen uit ecologische habitats. Deze methode vereist geen geavanceerde instrumenten en kan gemakkelijk worden uitgevoerd in een standaard laboratoriumopstelling. Het kan ook gemakkelijk worden aangepast om andere substraten te bestuderen.Deze techniek kan eenvoudig worden vertaald naar verschillende ecologische niches en de afbraak van verschillende substraten kan worden beoordeeld. De proce...

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

Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to seasonality variation in pH and temperature, fluctuation in element composition is periodically observed within these extreme environments, influencing the environmental microbial communities. Extremophilic microorganisms that thrive in volcanic thermal vents have developed resistance mechanisms to handle several metal ions present in the environment, thus taking part to complex metal biogeochemical cycles. Moreover, extremophiles and their products have found an extensive foothold in the market, and this holds true especially for their enzymes. In this context, their characterization is functional to the development of biosystems and bioprocesses for environmental monitoring and bioremediation. To date, the isolation and cultivation under laboratory conditions of extremophilic microorganisms still represent a bottleneck for fully exploiting their biotechnological potential. This work describes a streamlined protocol for the isolation of thermophilic microorganisms from hot springs as well as their genotypical and phenotypical identification through the following steps: (1) Sampling of microorganisms from geothermal sites ("Pisciarelli", a volcanic area of Campi Flegrei in Naples, Italy); (2) Isolation of heavy metal resistant microorganisms; (3) Identification of microbial isolates; (4) Phenotypical characterization of the isolates. The methodologies described in this work might be generally applied also for the isolation of microorganisms from other extreme environments.

The isolation of heavy metal-resistant microbes from geothermal springs is a hot topic for the development of bioremediation and environmental monitoring biosystems. This study provides a methodological approach for isolating and identifying heavy metal tolerant bacteria from hot springs.

Geothermal springs are rich in various metal ions due to the interaction between rock and water that takes place in the deep aquifer. Moreover, due to ...

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Bromodeoxyuridine (BrdU) Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification of bacterial taxa that transform specific pools of DOC in nature poses a technical challenge. 
Here we describe an approach that couples bromodeoxyuridine (BrdU) incorporation, fluorescence activated cell sorting (FACS), and 16S rRNA gene-based molecular analysis that allows culture-independent identification of bacterioplankton capable of degrading a specific DOC compound in aquatic environments. Triplicate bacterioplankton microcosms are set up to receive both BrdU and a model DOC compound (DOC amendments), or only BrdU (no-addition control). BrdU substitutes the positions of thymidine in newly synthesized bacterial DNA and BrdU-labeled DNA can be readily immunodetected 1,2. Through a 24-hr incubation, bacterioplankton that are able to use the added DOC compound are expected to be selectively activated, and therefore have higher levels of BrdU incorporation (HI cells) than non-responsive cells in the DOC amendments and cells in no-addition controls (low BrdU incorporation cells, LI cells). After fluorescence immunodetection, HI cells are distinguished and physically separated from the LI cells by fluorescence activated cell sorting (FACS) 3. Sorted DOC-responsive cells (HI cells) are extracted for DNA and taxonomically identified through subsequent 16S rRNA gene-based analyses including PCR, clone library construction and sequencing.

Bromodeoxyuridine BrdU Labeling and Subsequent Fluorescence Activated Cell Sorting for Culture-independent Identification of Dissolved Organic Carbon-degrading Bacterioplankton

Environmental bacterioplankton are incubated with a model dissolved organic carbon (DOC) compound and a DNA labeling reagent, bromodeoxyuridine (BrdU). Afterward, DOC-degrading cells are separated from the bulk community based on their elevated BrdU incorporation using fluorescence activated cell sorting (FACS). These cells are then identified by subsequent molecular analyses.

Microbes are major agents mediating the degradation of numerous dissolved organic carbon (DOC) substrates in aquatic environments. However, identification ...

Biology

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.

Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. ...

Bioengineering

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Comprehensive Approach for Microbial Isolation from Hidradenitis Suppurativa Tunnels

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's dermal layers, causing significant discomfort, foul-smelling discharge, disfigurement, contractures, and scarring, which severely diminish the quality of life. HS is associated with alterations in the skin microbiome, impacting immune regulation and the skin's defense against harmful bacteria. Despite its prevalence, the contribution of the HS microbiome to disease pathology and the limited response to treatment remains largely unknown. To date, multiple 16S rRNA sequencing studies on HS tissue have only achieved genus-level granularity, identifying an increase in Gram-negative anaerobes and a decrease in skin commensals. A deeper understanding of microbial dysbiosis in individuals with HS is essential for optimizing treatment strategies. This requires a two-pronged approach to assessing the HS microbiome, including the isolation of bacterial species, which are often underutilized in translational studies focused on skin disorders. Isolating individual microorganisms from HS tissue is crucial for elucidating the role of bacteria in HS pathogenesis. Here, we highlight reproducible methods to successfully isolate anaerobic pathogens from HS tunnel tissue, providing the initial and most critical step in understanding bacterial role in HS. This method paves the way for targeted research into microbial contributions to HS and for developing more effective, personalized treatment strategies that address the complex microbial burden of this chronic condition.

We showcase a method to successfully isolate fastidious and anaerobic organisms from cutaneous sinus tracts (tunnels) in tissues excised from patients with Hidradenitis Suppurativa.

Hidradenitis Suppurativa (HS) is a debilitating condition marked by painful nodules and abscesses, progressing to sinus tracts (tunnels) within the skin's ...

Immunology and Infection

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an ...

Measuring the Migration and Biofilm Formation of Various Bacteria

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923&#160;and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Here, we present a practical method for the isolation and identification of microorganisms within the host. In this way, the physicochemical properties of microorganisms and possible ways of living in the host are clearly described.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their ...

Developing Synergistic Multispecies Biofilms from Rhizosphere Soil

An Approach to Constructing Multispecies Biofilm Communities from Rhizosphere Soil

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current understanding of it is limited. Here, we provide an approach to rapidly establish synergistic multispecies biofilm communities. The first step is to extract cells from rhizosphere soil using the differential centrifugation method. Afterward, these soil cells are inoculated into the culture medium to form pellicle biofilm. After 36 h of incubation, the bacterial composition of the biofilm and the solution underneath are determined using the 16S rRNA gene amplicon sequencing method. Meanwhile, high-throughput bacterial isolation from pellicle biofilm is conducted using the limiting dilution method. Then, the top 5 bacterial taxa are selected with the highest abundance in the 16S rRNA gene amplicon sequencing data (pellicle biofilm samples) for further use in constructing multispecies biofilm communities. All combinations of the 5 bacterial taxa were quickly established using a 24-well plate, selected for the strongest biofilm formation ability by the crystal violet staining assay, and quantified by qPCR. Finally, the most robust synthetic bacterial multispecies biofilm communities were obtained through the methods above. This methodology provides informative guidance for conducting research on rhizosphere multispecies biofilm and identifying representative communities for studying the principles governing interactions among these species.

Author Spotlight: Developing Synthetic Microbial Communities for Generating Second-Generation Biofertilizers

A rapid and standardized procedure for establishing synergistic multispecies biofilm communities from various rhizosphere soils is presented here. It is a unique protocol designed to probe and simulate the complex rhizosphere soil microbiota.

The multispecies biofilm is a naturally occurring and dominant lifestyle of bacteria in nature, including in rhizosphere soil, although the current ...

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

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