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 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.
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.
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.
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.
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.
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.
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.
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.
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.
The authors have nothing to disclose.
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.
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 |