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

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 regul....

Protocol

1. Sample collection, processing, and analysis

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

1. Collect 500 mL of water sample in five sterile glass bottles from different sites of the water body. Measure the pH and temperature of each sample using a pH meter and thermometer, respectively.
   NOTE: The protocol is not site-specific and can be easily adapted to isolate organisms from hydrocarbon-contaminated water bodies too.
2. Filter the sample in a batch of 100....

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. Afte.......

Discussion

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

Materials

List of materials used in this article

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