Abatement of polyaromatic hydrocarbons and other xenotoxic waste compounds that are generated during the refinement and recovery of petroleum and its byproducts.

2. Petroleum hydrocarbons
 Petroleum Hydrocarbons (PHCs) is the
name given to a very broad range of
chemicals that comprise oil and products
refined from oil, such as gasoline and
diesel.
 Petroleum hydrocarbons are complex
substances formed from hydrogen and
carbon molecules and sometimes contains
other impurities such as oxygen, sulfur,
nitrogen and trace amounts of metals such
as iron, nickel, copper and vanadium.
 Petroleum hydrocarbons can be divided
into four classes: the saturates, the
aromatics, the asphaltenes (phenols, fatty
acids, ketones, esters, and porphyrins),
and the resins (pyridines, quinolines,
carbazoles, sulfoxides, and amides).
https://www.sciencedirect.com/topics/earth-and-planetary-sciences/petroleum-hydrocarbon 2

3.  Petroleum has a wide range of applications
 In vehicle fuel
 As heating source for homes and industries
 for electricity generation
 In basic industrial operations
 Used as raw material for many chemical products, including
pharmaceuticals, fertilizers, pesticides, and plastics There is,
therefore, an enormous demand for the production and
movement of oil. Due to its high-energy density, easy
transportability, and relative abundance, crude petroleum
has become the world’s most important source of energy
since the mid-1950s.
 Leaks and accidental spills occur regularly during the
exploration, production, refining, transport, and storage of
petroleum and petroleum products.
Okafor, N. (2014). Environmental microbiology of aquatic and waste systems. Springer. 3

4. Hazardous effects of petroleum
hydrocarbons on environment
Parameters Hazardous effects
Agriculture Reduction of soil fertility, affects physiological properties
of soil, adverse effect
on seed germination.
Aquatic Life Death of natural flora and fauna (oil spill cause anaerobic
condition),
aquatic birds suffer from hypothermia, drowning, loss
in flight, poisoning,
reproductive impairment in fish.
Human Severe diseases (skin erythema (reddening), skin
cancer, sinonasal cancer,
gastrointestinal cancer and bladder cancer, effect on
CNS, depression,
irregular heartbeats.
Ecosystem Imbalance in marine ecosystem, physical and chemical
alteration of
natural habitats, imbalance in food chain.
Plants Plants covered by oil are unable to photosynthesize.
Animals Crude oil exposure may cause damage to lungs, liver,
kidneys, intestines
and other internal organs.

5.  When petroleum products are burned as fuel, they give off
carbon dioxide (CO2), a greenhouse gas that is linked with
global warming. The use of petroleum products also gives
off pollutants-Carbon monoxide (CO), nitrogen oxides,
particulate matter and unburned hydrocarbons-that pollute
the air. Sulfur oxides released into the atmosphere during
combustion of oil are a major pollutant.
 Release of hydrocarbons into the environment whether
accidentally or due to human activities is a main cause of
water and soil pollution .
 Soil contamination with hydrocarbons causes extensive
damage of local system since accumulation of pollutants in
animals and plant tissue may cause death or mutations.
5

6. Removal of petroleum hydrocarbons
 Remedial methods for the treatment of petroleum
contaminated environment include various physiochemical
and biological methods. Due to the negative consequences
caused by the physiochemical methods, the bioremediation
is widely adapted and considered as one of the best process
for the treatment of petroleum contaminated environment.
 The process of bioremediation, defined as the use of
microorganisms to detoxify or remove pollutants owing to
their diverse metabolic capabilities.
 It is an evolving method for the removal and degradation of
many environmental pollutants including the products of
petroleum industry .
6

7.  Hydrocarbons differ in their susceptibility to microbial
attack. The susceptibility of hydrocarbons to microbial
degradation can be generally ranked as follows: linear
alkanes > branched alkanes > small aromatics > cyclic
alkanes.
 Aliphatic hydrocarbons are easily degraded
by microorganisms, but large branched aliphatic chains are
not easily degraded; therefore, they persist in the
environment. Likewise, aromatic hydrocarbons are difficult
to degrade because of their complex structures.
 Some compounds, such as the high molecular weight
polycyclic aromatic hydrocarbons (PAHs), may not be
degraded at all.
Colwell RR, Walker JD, Cooney JJ. Ecological aspects of microbial degradation of petroleum in the
marine environment. Critical Reviews in Microbiology. 1977;5(4):423–445.
7

8. Mechanism of Petroleum Hydrocarbon
Degradation
W. Fritsche and M. Hofrichter, “Aerobic degradation by microorganisms,” in
Environmental Processes- Soil Decontamination, J. Klein, Ed., pp. 146–155,
Wiley-VCH, Weinheim, Germany, 2000.
8
 The most rapid and
complete degradation of
the majority of organic
pollutants is brought about
under aerobic conditions.
 Figure shows the main
principle of aerobic
degradation of
hydrocarbons . The initial
intracellular attack of
organic pollutants is an
oxidative process and the
activation as well as
incorporation of oxygen is
the enzymatic key reaction
catalyzed by oxygenases
and peroxidases.

9. R. K. Hommel, “Formation and phylogenetic role of biosurfactants,”Journal of Applied Microbiology, vol. 89,
no. 1,
pp. 158–119, 1990.
 Peripheral degradation pathways convert organic pollutants
step by step into intermediates of the central intermediary
metabolism, for example, the tricarboxylic acid cycle.
Biosynthesis of cell biomass occurs from the central
precursor metabolites, for example, acetyl-CoA, succinate,
pyruvate. Sugars required for various biosyntheses and
growth are synthesized by gluconeogenesis.
 The degradation of petroleum hydrocarbons can be
mediated by specific enzyme system. Figure shows the
initial attack on xenobiotics by oxygenases . Other
mechanisms involved are
(1) attachment of microbial cells to the substrates
(2) production of biosurfactants
9

10. Factors Influencing Petroleum Hydrocarbon
Degradation
 Biodegradation of petroleum hydrocarbons is a complex
process that depends on the nature and on the amount of the
hydrocarbons present.
1. Temperature plays an important role in biodegradation of
hydrocarbons by directly affecting the chemistry of the
pollutants as well as affecting the physiology and diversity of
the microbial flora.
 Temperature also affects the solubility of hydrocarbons
 Although hydrocarbon biodegradation can occur over a wide
range of temperatures, the rate of biodegradation generally
decreases with the decreasing temperature.
 The highest degradation rates that generally occur in the range
30–40◦C in soil environment, 20–30◦C in some freshwater
environment and 15–20◦C in marine environment.
A. D. Venosa and X. Zhu, “Biodegradation of crude oil contaminating marine shorelines and freshwater wetlands,” Spill Science and
Technology Bulletin, vol. 8, no. 2, pp. 163– 178, 2003.
10

11. 2. Nutrients are very important ingredients for successful
biodegradation of hydrocarbon pollutants especially nitrogen,
phosphorus, and in some cases iron.
 When a major oil spill occurred in marine and freshwater
environment, the supply of carbon was significantly
increased and the availability of nitrogen and phosphorus
generally became the limiting factor for oil degradation.
 Freshwater wetlands are typically considered to be nutrient
deficient due to heavy demands of nutrients by the plants.
Therefore, additions of nutrients were necessary to enhance
the biodegradation of oil pollutant.
 Therefore, additions of nutrients were necessary to enhance
the
biodegradation of oil pollutant . On the other hand,
excessive nutrient
concentrations can also inhibit the biodegradation activity.
C. H. Chaˆıneau, G. Rougeux, C. Y´epr´emian, and J. Oudot, “Effects of nutrient concentration on the
biodegradation of crude oil and associated microbial populations in the soil,” Soil Biology and Biochemistry,
vol. 37, no. 8, pp. 1490–1497, 2005. 11

12. 3. Oxygen availability in the soil depends on microbial
oxygen consumption rates and soil type, whether soil is
water-logged, and the useable substrates presence which
can drive to oxygen depletion.
 The aerobic biodegradation of PHCs occurs at faster rate
than the anaerobic biodegradation.
 Substrate oxidation by oxygenases in the catabolism of all
aliphatic, cyclic and aromatic compounds by microbes is
considered a key step in the biodegradation process .
12

13. Enzymes Participating in Degradation of
Hydrocarbons
 Cytochrome P450 alkane
hydroxylases constitute a
super family of Heme-
thiolate Monooxygenases
which play an important
role in the microbial
degradation of oil,
chlorinated hydrocarbons,
fuel additives, and many
other compounds .
 Depending on the chain
length, enzyme systems
are required to introduce
oxygen in the substrate
to initiate biodegradation.
J. B. Van Beilen and E. G. Funhoff, “Alkane hydroxylasesinvolved in microbial alkane
degradation,” Applied Microbiology and Biotechnology, vol. 74, no. 1, pp. 13–21, 2007. 13
Enzymatic reactions involved in the processes of hydrocarbons
degradation.

14. 14
Enzymes involved in biodegradation of
petroleum hydrocarbons

15. Uptake of Hydrocarbons by microorganisms
producing Biosurfactants
 Biosurfactants are heterogeneous group of surface active
chemical compounds produced by a wide variety of
microorganisms. Surfactants enhance solubilization and
removal of contaminants.
 Biosurfactants increase the oil surface area and that amount of
oil is actually available for bacteria to utilize it.
 Biosurfactants can act as emulsifying agents by decreasing the
surface tension and forming micelles. The micro droplets
encapsulated in the hydrophobic microbial cell surface are
taken inside and degraded.
 Pseudomonads are the best known bacteria capable of utilizing
hydrocarbons as carbon and energy sources producing
biosurfactants. Among Pseudomonads, P. aeruginosa is widely
studied for the production of glycolipid type biosurfactants.
However, glycolipid type biosurfactants are also reported from
some other species like P. putida and P. Chlororaphis.
A. Daverey and K. Pakshirajan, “Production of sophorolipids by the yeast Candida bombicola using simple and
low cost fermentative media,” Food Research International, vol. 42, no. 4, pp. 499–504, 2009. 15

16.  Pseudomonas aeruginosa, has shown the ability to break
hexadecane, octadecane, heptadecane as well as nonadecane
after 28 days of incubation. The degradation ability of this
bacterium is due to the production of a biosurfactant.
 Pseudomonas aeruginosa can effectively degrade a range of
hydrocarbons like 2-methylnaphthalene, tetradecane and
pristine. The rhamnolipid content of Pseudomonas
aeruginosa is extensively characterized for its hydrocarbon
degradation ability.
 In one of the experiment, the hydrocarbon contaminated soil
was inoculated with Acinetobacter
haemolyticus and Pseudomonas ML2 (biosurfactant producing
strains) and the degradation of hydrocarbons were studied,
after the completion of the 2 months period of incubation, a
tremendous reduction of hydrocarbons (39–71%) and (11–
71%), was achieved by Acinetobacter
haemolyticus and Pseudomonas ML2 respectively. These results
suggested that cell free biosurfactant produced by bacteria had
the remarkable hydrocarbon degradation ability.
16

17. K. Muthusamy, S. Gopalakrishnan, T. K. Ravi, and P. Sivachidambaram, “Biosurfactants: properties, commercial
production and application,” Current Science, vol. 94, no. 6, pp. 736–747, 2008. 17
Biosurfactants Microorganisms
Sophorolipids Candida bombicola
Rhamnolipids Pseudomonas
aeruginosa
Lipomannan Candida tropicalis
Surfactin Bacillus subtilis
Glycolipid Aeromonas sp.
Biosurfactants produced by microorganisms

18. Commercially Available Bioremediation Agents
 Microbiological cultures, enzyme additives, or nutrient additives
that significantly increase the rate of biodegradation to mitigate
the effects of the discharge were defined as bioremediation
agents.
 Inipol EAP22 is listed on the NCP (National Oil and Hazardous
Substances Pollution Contingency Plan) Product Schedule as a
nutrient additive and probably the most well-known
bioremediation agent for oil spill cleanup. This nutrient product
is a microemulsion-containing urea as a nitrogen source,
sodium laureth phosphate as a phosphorus source, 2-butoxy-1-
ethanol as a surfactant, and oleic acid to give the material its
hydrophobicity. The claimed advantages of Inipol EAP22 include
(1) preventing the formation of water-in-oil emulsions by
reducing the oil viscosity and interfacial tension
(2) providing controlled release of nitrogen and phosphorus
for oil biodegradation
(3) exhibiting no toxicity to flora and fauna and good
biodegradability
W. J. Nichols, “The U.S. Environmental Protect Agency: National Oil and Hazardous Substances Pollution
Contingency Plan, Subpart J Product Schedule (40 CFR 300.900),” in Proceedings of the International Oil
SpillConference, pp. 1479–1483, American Petroleum Institute,Washington, DC, USA, 2001. 18

19. 19
Abbreviations of product type:
MC: Microbial Culture EA: Enzyme Additive NA: Nutrient Additive.
Bioremediation agents in NCP product schedule (Adapted from USEPA, 2002).
Oil Spill Eater II is another
nutrient product listed on
the NCP Schedule. This
product is listed as a
nutrient/enzyme additive
and consists of “nitrogen,
phosphorus, readily
available carbon, and
vitamins for quick
colonization of naturally
occurring bacteria.

20. Phytoremediation
 Phytoremediation is an
emerging technology that uses
plants to manage a wide
variety of environmental
pollution problems, including
the cleanup of soils and
groundwater contaminated
with hydrocarbons and other
hazardous substances. The
different mechanisms, namely,
hydraulic control,
phytovolatilization,
rhizoremediation, and
phytotransformation could be
utilized for the remediation of
a wide variety of
contaminants.
20
Advantages and disadvantages of phytoremediation over
traditional technologies.

21.  Grasses were often planted with trees at sites with organic
contaminants as the primary remediation method. Tremendous
amount of fine roots in the surface soil was found to be
effective at binding and transforming hydrophobic
contaminants such as TPH, BTEX, and PAHs.
 Grasses were often planted between rows of trees to provide
soil stabilization and protection against wind-blown dust that
could move contaminants offsite. Legumes such as alfalfa
(Medicago sativa), alsike clover (Trifolium hybridum), and peas
(Pisum sp.) could be used to restore nitrogen to poor soils.
Fescue (Vulpia myuros), rye (Elymus sp.), clover (Trifolium
sp.), and reed canary grass (Phalaris arundinacea) were used
successfully at several sites, especially contaminated with
petrochemical wastes. Once harvested, the grasses could be
disposed off as compost.
 Microbial degradation in the rhizosphere might be the most
significant mechanism for removal of diesel range organics in
vegetated contaminated soils.
R. K. Miya and M. K. Firestone, “Enhanced phenanthrene biodegradation in soil by slender oat root exudates and root
debris,” Journal of Environmental Quality, vol. 30, no. 6, pp. 1911–1918, 2001. 21

22. Genetically Modified Bacteria
J. L. Ramos, A. Wasserfallen, K. Rose, and K. N. Timmis, “Redesigning metabolic routes: manipulation of TOL
plasmid pathway for catabolism of alkylbenzoates,” Science, vol. 235, no. 4788, pp. 593–596, 1987. 22
Genetic engineering for biodegradation of contaminants.
Applications for genetically engineered microorganisms
(GEMs) in bioremediation have received a great deal of
attention to improve the degradation of hazardous wastes
under laboratory conditions. The use of genetically
engineered bacteria was applied to bioremediation process
monitoring, strain monitoring, stress response, end-point
analysis, and toxicity assessment.

23. Advantages of bioremediation
 It is an environment friendly approach and is therefore
accepted by the public as a remedy in the treatment of
contaminants
 It is believed to be noninvasive and relatively cost-effective
 It can be done on site and site disruption is minimal
 It eliminates waste and also eliminates the chance of future
liability associated with treatment and disposal of
contaminated material
 The microorganisms involved in the degradation of
contaminant increases in their number till the contaminant
is present. After the degradation of contaminant the
microbial population itself decreases naturally
 It transforms the toxic substances to harmless products
such as CO2 (utilized by plants in photosynthesis), H2O
and fatty acids
23

24. SYMBOL OF TRUST