Submitted in partial fulfilment of the requirement for the award of degree of
Masters of science Biotechnology (Hons.)
Submitted by:
Anisha kapoor11404349
Under the guidance of


Fig :-1 oil spill in soil
Hydrocarbons are one of the most used energy and primary energy resources in the world in terms of energy production. During routine operations of crude oil production, refining, distribution and as a result of accidental spills (1), which is one of the world’s problems in industrialized and developing countries and has generated a continuous research interest in this field. The most direct measure of the effectiveness of bioremediation is the monitoring of the disappearance rates of hydrocarbons (2), which are considered to be of biological origin because of their short and long chains which appear to be the exclusive origin of biological processes (3).

Pollution due to hydrocarbons is dangerous for the health of plants and are also carcinogenic, mutagenic and powerful immuno-toxic substances that pose a serious threat to human and animal health. (4)The biodegradation of oil and other hydrocarbons in the environment is a complex process, whose quantitative and qualitative transformations depend on the nature and quantity of the hydrocarbon present. Biodegradation rates generally increase as they increaseso that ecosystems exposed to extremely low temperatures degrade hydrocarbons very slowly. The microbial degradation of the oil in aquatic environments is mainly limited by nutrients such as nitrogen and phosphorus; salinity and pressure can be important in estuarine and deepwater regions respectively. Oxygen, nutrient concentrations,Humidity and pH are the predominant factors in determining soil biodegradation rates.

Petroleum is a complex mixture of different hydrocarbons that includes aliphatic (linear or branched), cycloalkanes, mono and polyaromatic, asphaltenes and resins and most of these compounds are stable, toxic and carcinogenic (5). Petroleum compounds such as alkanes, benzene, toluene, ethylbenzene and xylenes (BTEX) and some polycyclic aromatic hydrocarbons (PAHs) are biodegradable under appropriate environmental conditions (6). Non-biodegradable components can still represent a high risk in the surrounding environment. near the area where they remain. Therefore, petroleum hydrocarbons are important pollutants in the environment and cause damage to the surrounding ecosystems. (7)
Petroleum and coal contain a class of molecules known as hopanoids, which are commonly found in bacterial cell walls (8). These fuels at some point originated at least in part from

Fig :-2 Hydrocarbon biodegradation
microorganisms and that the biodegradation of these fuels has always occurred to a certain extent. The biodegradation of hydrocarbons from natural populations of microorganisms allows the conversion of hazardous substances in ways that are less toxic or non-toxic and is one of the main mechanisms by which oil and diesel products are economically eliminated from the environment (9). One of the most important characteristics of the hydrocarbon degrading bacteria is the ability to emulsify hydrocarbons in solution through the production of surfactants, such as biotensing agents (10) Biosurfactants are directly involved in the process of eliminating hydrocarbons from the environment through greater bioavailability and subsequent biodegradation of hydrocarbons by direct contact with cells (11). The addition of biosurfactants increases the availability of long chain hydrocarbons for microbes and makes them more accessible to the microbial enzyme system for their use (12). In some cases, when intrinsic bacteria fail to meet the required demand, specific degradants are used that have been selected to degrade various hydrocarbons. (13)
In many ecosystems there is already an adequate autochthonous microbial community, capable of a wide biodegradation of oil, on condition that the environmental conditions are favorable to the metabolic activity of the degradation of the oil. There are several advantages in relying on indigenous micro-organisms instead of adding micro-organisms to degrade hydrocarbons. First of all, natural populations must be developed over many years. These microorganisms are adapted for survival and proliferation in that environment.Secondly, the ability to use hydrocarbons is distributed among a diverse microbial population.There are two main approaches for bioremediation of oil spills: a) bioaugmentation, in which known degrading bacteria of the oil are added to supplement the existing microbial population e (b) biostimulation, in which the growth of indigenous oil degradants is stimulated by the addition of nutrients or other co-substrates that limit growth.Dangerous incineration or disposal methods of currently used burials can become prohibitive when the quantities of contaminants are high. The mechanical and chemical methods generally used to remove hydrocarbons from contaminated sites are of limited effectiveness and can be expensive. Bioremediation is the promising technology for the treatment of these contaminated sites, as it is profitable and will lead to complete mineralization
Review of literature
The biodegradation of petroleum hydrocarbons is a complete process that depends on the nature and quantity of the hydrocarbons present. Petroleum hydrocarbons can be divided into four classes: saturated, aromatic, asphaltenes (phenols, fatty acids, ketones, esters and porphyrins) and resins (pyridines, quinoline, carbazoles, sulfoxides and amides). Petroleum hydrocarbon compounds bind to soil components, and are difficult to remove or degrade 21. Hydrocarbons differ in their susceptibility to microbial attackand the susceptibility of hydrocarbons to microbial degradation can generally be classified as linear alkanes; small branched; aromatic alkanes; cyclic alkanes 6, 22.The hydrocarbons in the environment are mainly biodegraded by bacteria, yeasts and fungi. Many scientists have reported that mixed populations are required with great ability to degrade enzyme generally complex mixtures of hydrocarbons, such as crude oil in soil 33, fresh water 34 and 35 marine environments, 36. Bacteria are the most active agents in the degradation of oil and function as primary degradative oil poured into the environment 37, 38. It is known that many bacteria feed exclusively on hydrocarbons 39.

Paraffins to C10H22 CyclopentaneBenzene
Isobutene Cyclohexane Toluene
2-Methylbutane MethylcyclopentaneEthylbenzene2,3-dimethylbutane 1,1-dimethylcyclopentane Xylene
2-methylpentane Methylcyclohexane1,2,4-trimethylbenzene
3-methylpentane 1,3dimethylcyclohexane 2-methylhexane 1,2,4-trimethylcyclohexane 3-methylhexane 2,6-dimethylheptane 2-methyloctane 2-methylheptane Tab:- 1 example of crude oil composition
Das and Mukherjee 40 reported oil contaminated from the ground from northeastern India. Acinetobacter sp. It was discovered that it was able to use n-alkanes of the C10-C40 length chain as the sole carbon source 41. The bacterial genera, ie Gordonia, Brevibacterium, Aeromicrobium, Dietzia, Burkholderia and Mycobacterium isolated from oil-contaminated soils have proved to be potential organisms fordegradation of hydrocarbons 42.Crude oil is a mixture of hydrocarbons composed primarily of heteroatomic and non heteroatomic hydrocarbons 11. To date, many studies have reported the ability of microorganisms to use crude oil components as growth substrates. There are several microorganisms that have degradation potential for crude oil, including
Streptomyces albiaxialis , Marinobacter aquaeolei , Rhodococcus erythropolis , Dietzia maris ,Fusarium lateritium, Drechslera ,Cellulomonas , Bacillus , Dietzia , Halomonas , Rhodococcus , Gordonia , Dietzia , Pseudomonas, Actinopolyspora ,Bacillus Halomonas shengliensis , Halomonas , Pseudomonas , Haloferax , Halobacterium , Halococcus Amycolicicoccus subflavus , Marinobacter sedimentalis , Marinobacter falvimarisSeveral limiting factors have been recognized that influence the biodegradation of petroleum hydrocarbonsThe composition and the intrinsic biodegradability of petroleum hydrocarbon pollutants are the first and most important consideration in assessing the adequacy of a remediation approach. Among the physical factors, temperature plays an important role in the biodegradation of hydrocarbons, directly influencing the chemistry of contaminants and influencing the physiology and diversity of microbial flora. Atlas 54 found that at low temperatures, the viscosity of the oil increased, while the volatility of low molecular weight toxic hydrocarbons was reduced, delaying the onset of biodegradation.Nutrients are very important ingredients for the success of the biodegradation of hydrocarbon contaminants, in particular nitrogen, phosphorus and, in some cases, iron 34. Some of these nutrients could become limiting factors, influencing biodegradation processes in the event of significant spills in marine and freshwater environments, the carbon supply has increased significantly and the availability of nitrogen and phosphorus has generally become the factor limiting for oil degradation. be more pronounced due to the low levels of nitrogen and phosphorus. Therefore, nutrient additions were needed to improve the biodegradation of the oil contaminant 67, 68. On the other hand, excessive nutrient concentrations can also inhibit the activity of biodegradation

Fig :-3 Hydrocarbon degradation rates in soil, fresh water, and marine environments.

Temperature also affects the solubility of hydrocarbons 62. Although biodegradation of hydrocarbons can occur over a wide temperature range, the rate of biodegradation generally decreases with decreasing temperature. Figure 1 shows the highest degradation rates that generally occur in the 30-40 ° C range in soil environments, 20-30 ° C in some freshwater environments and 15-20 ° C in marine environments 33, 34. Venosa and Zhu 63 reported that the ambient temperature of the environment has influenced both the properties of the spilled oil and the activity of micro-organisms.

Fig :-4 Activity of bacterial consortium in degrading recalcitrant petroleum hydrocarbons and simultaneous production of biosurfactant.  ( HYPERLINK “https://www.frontiersin.org/articles/10.3389/fmicb.2016.01092/full” l “B11” Calvo et al., 2009)
Several microorganisms that degrade hydrocarbons in oil have been isolated from the soil and from marine sources, which are the two main environments affected by oil contamination from oil.43,44 Microorganisms are equipped with metabolic machinery to use petroleum products as a source of carbon and energy. The metabolic pathways used by heterotrophs that degrade hydrocarbons can be aerobic (that is, they use oxygen as the main electron acceptor) or anaerobic (that is, they use an alternative electron acceptor, such as nitrate or sulphate).Soil ecosystem: petroleum compounds bind to soil components and are difficult to remove or degrade 45. Contamination of the oil in the soil causes an imbalance in the carbon-nitrogen ratio at the spill site, since crude oil is essentially a mixture of carbon and hydrogen. This causes a lack of nitrogen in the soil soaked in oil, which delays the growth of bacteria and the use of carbon sources. Petroleum hydrocarbons in nature are degraded by various groups of microorganisms, which are able to use hydrocarbons as food 48. The degradation of complex mixtures of hydrocarbons such as oil and metals into the soil requires mixed populations with broad general enzymatic capabilities 43,49. Bacteria are the most active agents in oil degradation and function as primary degraders of oil poured into the environment 50,51. It is known that many bacteria feed exclusively on hydrocarbons 52.Aquatic ecosystem: in aquatic ecosystems, the dispersion and emulsification of oil in the spots appear to be the preconditions for rapid biodegradation. Large amounts of mousse, tar balls or high oil concentrations in quiescent environments tend to persist due to the limitedsurface areas available for microbial activity. When an oil spill occurs, a combination of recovery, disposal and containment of the oil is subsequently carried out. The conventionalmethods to remove oil from aquatic ecosystems include; mechanical cleaning, chemical cleaning and microbial degradation. Mechanical cleaning of spilled oil and dispersing is almost impossible in “protected” ecosystems. Microbial degradation is the main mechanism forelimination of hydrocarbons escaped and dispersed from the aquatic environment 64.The applications of genetically modified microorganisms (GEM) in bioremediation have received great attention to improve the degradation of hazardous waste under laboratory conditions. There are reports on
process monitoring, stress monitoring, stress response, endpoint analysis and toxicity assessment. The range of tested pollutants included chlorinated compounds, aromatic hydrocarbons and non-polar toxic substances. The combination of microbiological and ecological knowledge, biochemical mechanisms and engineering projects in the field are essential elements for the success of in situ bioremediation using genetically modified bacteria.

Microorganism Application Contaminants
A. eutrophus H850Lr process monitoring PCB
P. putida TVA8 process monitoring CE, BTEX
P. fluorescens HK44 process monitoring Naphthalene, anhraceneB. cepacia BRI6001L strain monitoring 2,4-D
P. fluorescens 10586s/pUCD607 stress response BTEX
A. eutrophus 2050 end point analysis Non polar narcotics

Table:- 2 Application of genetically modified bacteria for assessing the biodegradation process efficiency.

The mechanism of biodegradation has a high ecological importance that depends on the indigenous microorganisms to transform mineralizing organic pollutants. The microbial degradation process helps to eliminate oil leaking from the environment after the critical removal of large quantities of oil by various physical and chemical methods. This is possible because microorganisms have enzyme systems that degrade and use different hydrocarbons as a carbon and energy source.

In the present investigation, various soil samples from different oil contaminated soil areas were investigated for presence, isolation and identification of crude oil degrading bacteria
Main objectives were:
Collection of Soil Samples from different oil contaminated soil areas
Isolation of crude oil degrading bacteria from the soil samples.

Optimization of cultivation media, growth condition for the growth of isolated bacteria
Screening of the oil contaminated soil sample
Identification of the isolates.

This selection is meant for giving the details of all the methods and the specific tec. It is this systematic , theoretical analysis methods applied to field of study. Following are the methods that will be used during this study.

The soil sample was collected form oil contaminated soil from different areas such as
Oil spilled areas
Oil refineries
Mechanic workplace
Car repair shop
Medium used for Screening and isolation of crude oil degraders
The oil contaminated soil sample was use to isolate crude oil degrading microbes using the Bushnell Hass Mineral Salts (BHMS) medium comprising crude oil in various concentrations as sole carbon and energy source .
For the screening of oil contaminated soil samples , it will be suspended and agitated on vortex in sterile distilled water, this sample can be used as an inoculum for the isolation of oil degrading bacteria. The BHMS broth medium must be transferred to each flask and sterilized. The crude oil is used as the sole carbon source and incubated in an orbital agitator incubator at 370 ° C at 125 rpm for 5 days, 10 days and 15 days, respectively, for the detection of crude oil degradants. After the respective incubation days, the degradation of the crude can be studied by gravimetric assay.After the selection test, the mixed culture showing the greatest degradation of crude oil is taken for the isolation of powerful crude oil degradants for the isolation of individual colonies and incubated at 37 ° C for 72 hours and observed. The pure colonies are isolated based on the morphology of the colony.The BHMS broth can be added to each flask and sterilized. The crude oil was added to each flask and incubated for 5, 10, 15 and 20 days, respectively, in an orbital agitator incubator at 370 ° C at 125 rpm and degradation was investigated by gravimetric or gas chromatography.

Study of diesel degradation
Crude oil degradation can be studied by gravimetric analysis (Chang, 1998, Marquez-Rocha et al., 2001) or gas chromatography .
Gravimetric assay
After a time interval , the flasks is to betaken out and bacterial activities are stopped by adding 1% 1N-HCL. Crude oil extraction is done by , broth culture which is mixed with petroleum ether : acetone (1:1) in a separating funnel and is shaken vigorously to get a single emulsified layer. Acetone is to be added and shaken gently to break the emulsification, and will form three layers. Top layer is the mixture of petroleum ether, diesel oil and acetone; clumping cells make a middle layer and the bottom aqueous layer contains acetone, water and biosurfactant in soluble form. The lower two layers are spread out while top layer containing petroleum ether mixed with diesel oil and acetone is taken in a pre weighed clean beaker. The extracted oil is then passed through anhydrous sodium sulphate to remove moisture. The petroleum ether and acetone is evaporated on a water bath. The gravimetric estimation of residual oil left after biodegradation is made by weighing the quantity of oil in a tared beaker.

Gas chromatography
The oil cultures were incubated for 3 weeks at 37°C temperature with agitation of 130 rpm on a rotary shaker in an incubator. Growth can bemonitored by testing optical density (OD) at 620 nm after every 4th day. The
collected samples can be extracted and analyzed to check the total petroleum hydrocarbon degradation by gas chromatography.

Crude oil components are to be separated on a capillary column , nitrogen can be used as carrier gas at pressure of 2.0 kg/cm² measured at injector 20 ml par min with total flow of 33.6 ml per min. The hydrogen flow, 35 ml par min and air flow of 350 ml par min during running process. The initial column oven temperature was 35°C (for 1 to 5 min).The ramp 1 was 5 to 10°C par min to 300°C (for 20 min) and ramp 2 was 4 to
10°C par min to 280°C. This final temperature can be held for 20 min with maximum temperature exceeding 350°C. The total run time can be 74 min. Individual components, aliphatic and aromatic hydrocarbons, can be used as standards.
The concentrations of the petroleum components can be calculated from the peak area chromatograms then total aliphatic components and aromatic components is to be calculated to compare the percentage degradation at different time and intervals of the biodegradation experiment. The effects of different percentage on individual components is also analyzed in specific cases.
Identification of selected bacterial isolates
The identification of bacterial isolates with the ability to degrade crude oil can be performed on the basis of microscopic examination and biochemical tests (Holt, 1996).  The bacterial isolate can be identified based on morphology, surface, shape, size, margin and pigmentation on the nutrient agar. Microscopic examination included Gram staining, capsule staining and staining of the spores. Motility testing can also be performed.Biochemical tests including citrate utilization , oxidase production, starch hydrolysis, lipid hydrolysis, casein hydrolysis, methyl red, voges proskauer test (MR-VP), nitrate reduction test, gelatin liquefaction test, triple sugar test (TSI) for fermentation of lactose, dextrose, sucrose, glucose and mannitol, fermentation of carbohydrates, H2S production, indole production test and urease test according to standard procedures