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Microbial degradation of petroleum

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Carlos Navarro Laguna

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 Bioresource Technology 223 (2017) 277–286

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Review

Microbial degradation of petroleum hydrocarbons

Sunita J. Varjani
School of Biological Sciences and Biotechnology, Indian Institute of Advanced Research, Gandhinagar 382007, Gujarat, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 Petroleum hydrocarbon pollutants

are classified as persistent organic
pollutants.
 These pollutants pose adverse effects
on human and environmental health.
 Combat environmental pollution due
to hydrocarbon pollutants is big issue
for scientists.
 Biodegradation is an eco-friendly and
economic method to control
hydrocarbon pollution.
 Oleophilic microorganisms can be
used for oil spill bioremediation.

a r t i c l e i n f o a b s t r a c t

Article history: Petroleum hydrocarbon pollutants are recalcitrant compounds and are classified as priority pollutants.
Received 18 September 2016 Cleaning up of these pollutants from environment is a real world problem. Bioremediation has become
Received in revised form 12 October 2016 a major method employed in restoration of petroleum hydrocarbon polluted environments that makes
Accepted 13 October 2016
use of natural microbial biodegradation activity. Petroleum hydrocarbons utilizing microorganisms are
Available online 15 October 2016
ubiquitously distributed in environment. They naturally biodegrade pollutants and thereby remove them
from the environment. Removal of petroleum hydrocarbon pollutants from environment by applying
Keywords:
oleophilic microorganisms (individual isolate/consortium of microorganisms) is ecofriendly and eco-
Biodegradation
Enzymes
nomic. Microbial biodegradation of petroleum hydrocarbon pollutants employs the enzyme catalytic
Crude oil activities of microorganisms to enhance the rate of pollutants degradation. This article provides an over-
Petroleum hydrocarbons view about bioremediation for petroleum hydrocarbon pollutants. It also includes explanation about
Oil spill hydrocarbon metabolism in microorganisms with a special focus on new insights obtained during past
Oleophilic couple of years.
Ó 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
2. Composition of crude petroleum oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 278
2.1. Aliphatics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.2. Aromatics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.3. Resins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
2.4. Asphaltenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
3. Petroleum hydrocarbons toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

E-mail address: drsvs18@gmail.com

http://dx.doi.org/10.1016/j.biortech.2016.10.037
0960-8524/Ó 2016 Elsevier Ltd. All rights reserved.
278 S.J. Varjani / Bioresource Technology 223 (2017) 277–286

4. Fate of petroleum hydrocarbons in environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279

5. Removal of petroleum hydrocarbon pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
5.1. Physico-chemical vs. biological methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
6. Biodegradation/bioremediation of petroleum hydrocarbons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
7. Petroleum hydrocarbons bioegradation: metabolic aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
7.1. Pathways for biodegradation of petroleum hydrocarbon pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
7.1.1. Aerobic biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 282
7.1.2. Anerobic biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
7.2. Genes involved in biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
8. Factors affecting biodegradation of petroleum hydrocarbon pollutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
9. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285

1. Introduction catabolic cooperation between different microbial groups during

Petroleum hydrocarbons are important energy resource and a
raw material for various industries. Increasing demand for petro-
leum products in day to day life may cause their scarcity and
increase their cost as suitable alternatives are still not found
(Varjani et al., 2015; Varjani and Upasani, 2016b). Petroleum
hydrocarbon pollutants are recalcitrant compounds and are classi-
fied as priority pollutants (ATSDR, 2011; Costa et al., 2012).
Anthropogenic activities such as industrial and municipal runoffs;
effluent release; offshore and onshore petroleum industry activi-
ties as well as accidental spills cause petroleum hydrocarbon pol-
lution. This pollution affects the environment and pose direct or
indirect health risk to all life forms on planet earth (Margesin
and Schinner, 2001; Deppe et al., 2005; Souza et al., 2014; Sajna
et al., 2015). Marine environment is considered as the ultimate
and largest sink for petroleum hydrocarbon pollutants, therefore
it is necessary to combat pollution problem (Ron and Rosenberg,
2014; Varjani and Srivastava, 2015). Remediation of hydrocarbon
pollutants and enhanced oil recovery are two main burning issues
of petroleum industry (Sajna et al., 2015; Varjani et al., 2015;
Varjani and Upasani, 2016a,c). To understand the scope and strate-
gies of pollutant bioremediation it is essential to first understand
properties of crude oil, environment of concern, fate of oil in that
environment, mechanisms of crude petroleum biodegradation
and factors that control its rate (Atlas, 1981; Boopathy, 2000;
Varjani, 2014).
Mainly petroleum is constituted by saturates/paraffins, aromat-
ics, resins and asphaltenes (Varjani, 2014). Crude oil is a mixture of
variety of simple and complex hydrocarbons which are degraded
by several indigenous microorganisms, each capable of breaking
down a specific group of molecules (Zanaroli et al., 2010).
Sugiura et al. (1997), and Ghazali et al. (2004), have reported that
same compound in different crude oil samples was degraded to dif-
ferent extents by same organisms/consortium, the reason could be
bioavailability of a particular compound in a crude oil sample and
not it’s chemical structure.
Bioremediation of crude oil polluted sites is often limited due to
poor biodiversity of indigenous microflora and/or scarcity of native
specialized microbes with complementary substrate specificity
required for degrading different hydrocarbons occurring at pol-
luted site (Ron and Rosenberg, 2014). There are various reports
available on metabolic versatility of mixed cultures that have
demonstrated superiority of mixed cultures to pure cultures to uti-
lize hydrocarbon pollutants in petroleum crude as sole carbon
source (Cerqueira et al., 2011; Das and Chandran, 2011; Varjani
et al., 2013). Bacterial and fungal co-culture(s) have shown
improved degradation rates of diesel oil and many polyaromatic
hydrocarbons (PAHs) when checked in laboratory conditions (Li
et al., 2008; Wang et al., 2012; Varjani and Upasani, 2013). Hence
biodegradation is very important (Atlas, 1981; Varjani et al., 2015).
The intent of present review is to expand bioremediation scope
of petroleum hydrocarbon pollutants. It includes consideration of
toxicity and fate of petroleum hydrocarbons in environment. It also
discusses factors affecting biodegradation rate, microbial metabo-
lism of petroleum hydrocarbon pollutants, pathways for hydrocar-
bon pollutants degradation and types of bioremediation
technologies as well as processes.
2. Composition of crude petroleum oil
Petroleum is produced by thermal decay of buried organic
material over millions of years. Crude oil (naturally occurring raw
oil) once extracted from subsurface is transported to refineries
where it undergoes distillation to produce various products
(Speight, 2007; Varjani, 2014). Petroleum, in Latin means ‘‘rock oil”,
which occurs as a dark, sticky, viscous liquid (Vieira et al., 2007).
Petroleum hydrocarbons mainly consists of varying proportions
carbon and hydrogen. However they also contain nitrogen, sulfur
and oxygen in some amount (Chandra et al., 2013; Varjani et al.,
2015). Crude oil can be classified as light, medium or heavy oil
based on relative proportions of heavy molecular weight con-
stituents present in it (Varjani, 2014). Crude oil composition may
vary with location and age of an oil field as well as depth of oil well.
About 85% components of all types of crude oil can be classified as
(a) asphalt base, (b) paraffin base and/or (c) mixed base (Atlas,
1981; Varjani, 2014).
Crude oil is categorized in four broad fractions (a) Saturates
(aliphatics), (b) Aromatics (ringed hydrocarbons), (c) Resins and
(d) Asphaltenes (Balba et al., 1998; Widdel and Rabus, 2001;
Speight, 2007; Chandra et al., 2013). Saturates are defined as
hydrocarbons without double bonds and represent the highest
percentage of crude oil constituents. They are categorized
according to their chemical structures into alkanes (paraffins)
and cycloalkanes (Abbasian et al., 2015). Aromatic hydrocarbons
have one or several aromatic rings usually substituted with dif-
ferent alkyl groups (Meckenstock et al., 2016). In comparison to
saturated and aromatic fractions, resin and asphaltenes contain
non-hydrocarbon polar compounds. Resins and asphaltenes have
very complex and mostly unknown carbon structures with addi-
tion of many nitrogen, sulfur and oxygen atoms (Harayama
et al., 2004; Chandra et al., 2013). Each component has a
unique chemical behavior that affects their biodegradability
(Costa et al., 2012). In structural arrangement of four main
hydrocarbon components of crude oil, saturates make up the
outermost layer, whereas asphalthenes being greater molar
mass component constitute the innermost portion of oil
(Speight, 2007; Varjani, 2014).
 S.J. Varjani / Bioresource Technology 223 (2017) 277–286
2.1. Aliphatics
Aliphatic hydrocarbons are saturated or unsaturated and linear
or branched open-chain structures such as n-alkanes, iso-alkanes,
cyclo-alkanes (naphthenes), terpenes and steranes (Rahman
et al., 2003). n-Alkanes are divided into four molecular weight
groups: (a) gaseous alkanes, (b) aliphatic hydrocarbons with lower
molecular weight (C8-C16), (c) aliphatic hydrocarbons with med-
ium molecular weight (C17–C28), and (d) aliphatic hydrocarbons
with high molecular weight (>C28) (Abbasian et al., 2015).
2.2. Aromatics
Aromatics are ringed hydrocarbon molecules. They are mainly
divided as (a) monocyclic aromatic hydrocarbons (MAHs) viz. BTEX
(benzene, toluene, ethylbenzene and xylenes) (Farhadian et al.,
2008; Costa et al., 2012), and (b) polycyclic aromatic hydrocarbons
(PAHs) (Chandra et al., 2013). PAHs contain more than one benzene
ring and those that are made up of two or three cyclic rings making
(several) hexagon chains with double bonds such as naphthalene
(two-ringed), phenanthrene and anthracene (three-ringed) are
referred to as low molecular weight or light PAHs (Wilkes et al.,
2016). PAHs made up of four rings and above such as pyrene and
chrysenes (four-ringed), fluoranthene and benzo[a]pyrene (five-
ringed) are referred to as high molecular weight or heavy PAHs
(Farhadian et al., 2008; Costa et al., 2012; Macaulay and Rees,
2014).
2.3. Resins
Resins contain numerous polar functional groups formed with
N, S, O and trace metals (Ni, V, Fe). Resins are amorphous solids
and truly dissolved in oil (Balba et al., 1998; Speight, 2007). Resins
contain aromatic compounds with long alkyl chain and are soluble
in n-heptane and n-pentane (Jada and Salou, 2002; Parra-Barraza
et al., 2003; Chandra et al., 2013). They are structurally similar to
surface-active molecules in crude oil and act as peptizing agents
(Jada and Salou, 2002; Chandra et al., 2013).
2.4. Asphaltenes
Asphaltenes like resins contain numerous polar functional
groups. They are dark brown, large and complex molecules that
are colloidally dispersed in saturates and aromatics (Balba et al.,
1998; Speight, 2007). They are soluble in light aromatic hydro-
carbons such as benzene and toluene (Parra-Barraza et al.,
2003). Asphaltenes are viscous and high molecular weight com-
pounds composed of polycyclic clusters, variably substituted
with alkyl groups, which contributes to their resistance to
biodegradation (Chandra et al., 2013). Peptizing agents i.e.
resins keep asphaltenes in suspension thereby promoting the
stability of crude oil (Parra-Barraza et al., 2003; Chandra
et al., 2013).
3. Petroleum hydrocarbons toxicity
Hydrocarbon pollutants are one of the persistent organic pollu-
tants. Due to their bio-magnification they cause extensive and/or
permanent damage to ecosystems (Chandra et al., 2013). Wide-
spread release of hydrocarbon pollutants through spillages and
leakage from underground tanks, steamers, unplugging of oil wells,
abandoned oil refinery sites cause contamination of surface soil,
groundwater and ocean (Saeki et al., 2009; Janbandhu and
Fulekar, 2011; Prince et al., 2013; Souza et al., 2014). Many con-
stituents of petroleum crude are recalcitrant and highly toxic due
279
to presence of hemotoxic, carcinogenic and teratogenic compo-
nents such as BTEX and PAHs (Zhang et al., 2011; Costa et al.,
2012; Chandra et al., 2013; Souza et al., 2014; Meckenstock
et al., 2016).
Acute or chronic as well as direct/indirect effects of petroleum
pollutants are reported earlier (Chandra et al., 2013;
Meckenstock et al., 2016). Suffocation, anoxia, stunted growth, dis-
turbances in metabolic reactions and hormone imbalance in life
forms is noted as direct/indirect effects (Walker, 2006; Souza
et al., 2014). Acute necrosis mortality, hypothermia, smothering,
drowning and ingestion of toxic compounds during preening are
some of the short-term impacts (Varjani, 2014; Desforges et al.,
2016). Long-term impacts include developmental abnormalities
of sea animals such as jaw reductions, lack of pigmentation and
unfused skulls (Van Meter et al., 2006; Alonso-Alvarez et al.,
2007; Varjani, 2014). These effects cause changes in a species pop-
ulation or community and thereby cause changes to an entire
ecosystem (Walker, 2006). Volatile organic compounds (VOCs) of
crude oil are extremely harmful to humans (Costa et al., 2012;
Souza et al., 2014). Crude oil component specifically PAHs have
potential to induce malignant tumors that primarily affect skin
and other epithelial tissue as they have a great affinity for nucle-
ophilic center of macromolecules like RNA, protein and DNA
(Perez-Cadahia et al., 2007; Costa et al., 2012; Desforges et al.,
2016).
Petroleum hydrocarbons are sub-divided into specified ranges
of equivalent carbon number or fraction (s) to assess human and
environmental health risk (CCME, 2008, 2010). These fractions
can be subdivided in sub-fractions and described according to their
physical, chemical, toxicological characteristics. Fraction 1 refers
range of equivalent carbon number from C6 to C10, representing
volatile fraction of most hydrocarbon mixtures. Fraction 2 can be
defined as range of equivalent carbon number from C > 10 to
C16, representing semi-volatile fraction. Fraction 3 encompasses
range of equivalent carbon number from C > 16 to C34, which is
considered as non-volatile fraction. Fraction 4 refers the com-
pounds with equivalent carbon number of C35+, they are consid-
ered as fraction with the lowest volatility and solubility (CCME,
2008).
4. Fate of petroleum hydrocarbons in environment
It is critical to gain knowledge about the fate of hydrocarbons
within environment in order to control and combat pollution
(Walker, 2006; CCME, 2010). Petroleum crude is subjected to sev-
eral weathering processes such as spreading, evaporation, disper-
sion, sinking, dissolution, emulsification, photo-oxidation,
resurfacing, tar ball formation and biodegradation, which naturally
degrades it’s hydrocarbon components (Al-Majed et al., 2012;
Souza et al., 2014). Effect of photo-oxidation is limited because it
takes place only in sunlight exposed oil (Widdel and Rabus,
2001). Weathering processes depends on environmental factors
such as temperature, ocean currents and weather conditions
(Atlas, 1981; Widdel and Rabus, 2001). Biodegradation of PAHs in
environment is generally the most important process which is
taken into consideration for intermediate to long-term changes
in substance levels over a period of time (CCME, 2010). Resistance
of hydrocarbon pollutants to microbial degradation in either soil or
water tends to increase with the type as well as molecular weight
and number of rings (incase of PAHs). Naphthalene is readily
biodegraded in most situations however PAHs with four, five, or
six rings tends to be degraded much more slowly. Generally aero-
bic biodegradation occurs much more rapidly than anaerobic
biodegradation (Widdel and Rabus, 2001; CCME, 2010; Abbasian
et al., 2015; Meckenstock et al., 2016).
280 S.J. Varjani / Bioresource Technology 223 (2017) 277–286
5. Removal of petroleum hydrocarbon pollutants
5.1. Physico-chemical vs. biological methods
Many conventional engineering based physico-chemical decon-
tamination methods are expensive due to the cost of excavation
and transportation of large quantities of contaminated materials
for ex-situ treatment viz. soil washing, chemical inactivation (use
of potassium permanganate and/or hydrogen peroxide as a chem-
ical oxidant to mineralize non-aqueous contaminants such as pet-
roleum) and incineration (Chaudhry et al., 2005; Farhadian et al.,
2008; Varjani and Srivastava, 2015). Other physico-chemical tech-
niques used for the same purpose are dispersion, dilution, sorption,
volatilization and abiotic transformations, etc (Varjani and
Upasani, 2012; Chandra et al., 2013). The increasing costs and lim-
ited efficiency of these traditional physico-chemical treatments
have spurred the development of alternative technologies for in-
situ applications, particularly based on biological remediation
capabilities of plants and microorganisms (Singh and Jain, 2003;
Farhadian et al., 2008). Green technologies for cleanup of pollu-
tants by biological means are used for bioremediation of petroleum
polluted site(s) (Rahman et al., 2003; Varjani et al., 2015). Bioreme-
diation can be defined as application of living organisms to
degrade/detoxify pollutants (Dua et al., 2002; Ron and Rosenberg,
2014; Varjani et al., 2014b; Sajna et al., 2015; Varjani and
Upasani, 2016c). This technology is an efficient, economic, versatile
and environmentally sound technique (Farhadian et al., 2008;
Chandra et al., 2013).
Bioremediation is an innovative technique, in which microor-
ganisms mitigate, degrade or reduce hazardous organic pollutants
to innocuous compounds such as CO2, CH4, H2O and biomass with-
out adversely affecting environment (Ron and Rosenberg, 2014).
Biodegradation is one of the primary mechanisms for bioremedia-
tion in which oleophilic microbes are used for elimination of
hydrocarbon pollutants from environment (Varjani et al., 2013;
Macaulay and Rees, 2014; Varjani and Upasani, 2016c). Hydrocar-
bons being natural energy-rich compounds, there are several
hydrocarbon degrading/utilizing organisms available in nature.
The use of individual indigenous microorganism(s)/consortium as
mitigatory tool employs the catalytic abilities of living organisms
to enhance the rate of pollutant degradation and can also be used
for Microbial Enhanced Oil Recovery (MEOR) (Varjani and Upasani,
2013, 2016a,c; Zhao et al., 2016). Partially purified rhamnolipid
produced by P. aeruginosa 5514 was successfully employed for
MEOR which showed 8.82% enhancement in oil recovery as% of
residual oil saturation (ROS) through ex-situ bioaugmentation in
laboratory simulation experiment (Varjani and Upasani, 2016a).
6. Biodegradation/bioremediation of petroleum hydrocarbons
Microbial bioremediation is widely used technique for treating
petroleum hydrocarbon pollution in both terrestrial and aquatic
ecosystems (Varjani and Upasani, 2012; Abbasian et al., 2015;
Varjani and Srivastava, 2015). Numerous research studies concern-
ing biodegradation of hydrocarbon pollutants has been done in last
decade (Farhadian et al., 2008; Sajna et al., 2015; Varjani et al.,
2015; Varjani and Upasani, 2016c). Many reviews have been pub-
lished outlining general mechanisms and pathways for hydrocar-
bon degradation (Watkinson and Morgan, 1990; Rojo, 2009;
Jaekel et al., 2013; Ron and Rosenberg, 2014; Abbasian et al.,
2015; Meckenstock et al., 2016; Wilkes et al., 2016).
Microorganisms play crucial role in maintaining ecosystem and
biosphere to develop sustainable environment (Widdel and Rabus,
2001; Varjani and Srivastava, 2015). They can be used in biomon-
itoring of ecosystem, bioremediation i.e. pollutant cleanup from
environment as well as mitigate adverse effects of pollutants
(Chandra et al., 2013; Varjani and Upasani, 2016c). Microrogan-
isms are widely distributed in water (fresh/marine), soil and air
(Varjani et al., 2013). It is not surprising that microorganisms are
isolated from hydrocarbon polluted soil, sediments or water and
have hydrocarbon utilizing/degrading ability as these compounds
are naturally occurring (Atlas, 1981; Batista et al., 2006; Ron and
Rosenberg, 2014; Varjani and Upasani, 2016b). In biodegradation
microorganisms gain energy during pollutant degradation that
can be utilized for metabolism also they obtain ‘‘Carbon” – an
essential component of all cellular constituents so it can be said
that biodegradation of petroleum hydrocarbons is energetically
favourable (Abbasian et al., 2015). Some microorganisms possess
ability to degrade alkanes and/or aromatics (Boonchan et al.,
2000; Rahman et al., 2003; Meckenstock et al., 2016; Varjani and
Upasani, 2016c). Microorganisms in polluted areas adapt according
to environment as a result genetic mutations are caused in subse-
quent generations preparing them to become hydrocarbon degra-
ders (Watkinson and Morgan, 1990; McDonald et al., 2006). Atlas
(1991), has reviewed that hydrocarbon degrading microorganisms
in unpolluted ecosystems consists of less than 0.1% of microbial
community, however this number may increase up to 1–10% of
total population in petroleum hydrocarbon polluted environment.
However it has been reported that in polluted environments over-
all microbial diversity declines (Atlas, 1981; Aislabie et al., 2006).
Indigenous hydrocarbon degrading microorganisms play a sig-
nificant role in bioremediation process (McDonald et al., 2006;
Varjani et al., 2015; Varjani and Upasani, 2016c). Microorganisms
such as bacteria, fungi, algae are reported for their ability to
degrade hydrocarbon pollutants (Boonchan et al., 2000;
Hendrickx et al., 2006; Varjani and Upasani, 2013; Wilkes et al.,
2016). Bacteria are reported as primary degraders and most active
agents in petroleum pollutant degradation (Atlas, 1981; Varjani
and Upasani, 2012; Abbasian et al., 2015; Meckenstock et al.,
2016).
Some microorganisms have ability to degrade aliphatics, some
can degrade monoaromatics or polyaromatics while others
degrade resins. Petroleum hydrocarbon pollutants degrading
microorganisms and the type of hydrocarbon degraded by them
is enlisted in Table 1. Bacterial sp. of genera Achromobacter, Acine-
tobacter, Arthrobacter, Azoarcus, Brevibacterium, Cellulomonas,
Corynebacterium, Flavobacterium, Marinobacter, Micrococcus, Nocar-
dia, Ochrobactrum, Pseudomonas, Stenotrophomaonas and Vibrio are
reported as hydrocarbon degraders (Widdel and Rabus, 2001; Roy
et al., 2002; Foght, 2008; Mittal and Singh, 2009; Chandra et al.,
2013; Varjani et al., 2015; Varjani and Upasani, 2016c). Fungi of
genera Aspergillus, Amorphoteca, Fusarium, Graphium, Neosartoria,
Paecilomyces, Penicillium, Sporobolomyces, Talaromyces and yeast
of genera Candida, Pichia, Pseudozyma Rhodotorula and Yarrowia
play vital role in petroleum hydrocarbon pollutant degradation
(Atlas, 1981; Leahy and Colwell, 1990; Boonchan et al., 2000;
Sajna et al., 2015; Wilkes et al., 2016). Yakimov et al. (2007), and
Brooijmans et al. (2009), have reported evolution of hydrocarbon-
oclastic bacterial genera Oleispira, Marinobacter, Thalassolituus,
Alcanivorax and Cycloclasticus from petroleum hydrocarbon pol-
luted sites. These indigenous bacteria were present at low or unde-
tectable levels before pollution but were found to dominate in oil
polluted sites. Alcanivorax strains grow on n-alkanes and branched
alkanes, while they cannot grow on any sugars or amino acids as
carbon sources. Cycloclasticus strains grow on aromatic hydrocar-
bons, naphthalene, phenanthrene and anthracene, whereas Oleis-
pira strains grow on aliphatic hydrocarbons, alkanoles and
alkanoates (Harayama et al., 2004). It is very crucial to assess
biodegradation in light of a multi-domain community in order to
understand complete metabolic potential of indigenous microbial
community (Widdel and Rabus, 2001).
 S.J. Varjani / Bioresource Technology 223 (2017) 277–286 281

Table 1 Varjani and Upasani, 2016b,c). Roy et al. (2002), have reported
Petroleum hydrocarbon pollutants degrading microorganisms. Pseudomonas, Mycobacterium, Micrococcus, Nocardia, Acinetobacter
Petroleum Name of microorganism References and Klebsiella as petroleum-degrading bacteria from survey study
hydrocarbon in surface water samples from five different locations in
compound Hooghly-Matla river mouth. Ghazali et al. (2004), have reported
Aliphatics Acinetobacter sp. Mittal and Singh (2009) and comparison of biodegradation by two consortia obtained from Cen-
Foght (2008) ter for Research in Enzymes and Microbiology (CREAM), Malaysia.
Alcanivorax sp. Harayama et al. (2004) and
Brooijmans et al. (2009)
They concluded that consortium 2 consisting of three Bacillus, two
Azoarcus sp. Widdel and Rabus (2001) P. aeruginosa and one Micrococcus strains, is more efficient for
Bacillus sp. Ghazali et al. (2004) and cleanup of medium- and long-chain alkanes in diesel-
Das and Mukherjee (2007) contaminated soil than consortium 1 consisting of one Bacillus
Brevibacterium Leahy and Colwell (1990)
and two Pseudomonas strains.
Desulfosarcina sp. Jaekel et al. (2013)
Desulfococcus sp. Jaekel et al. (2013)
Marinobacter sp. Yakimov et al. (2007)
Micrococcus sp. Roy et al. (2002) and
7. Petroleum hydrocarbons bioegradation: metabolic aspects
Ghazali et al. (2004)
Ochrobactrum sp. Varjani et al. (2015) Biodegradation of a pollutant involves series of steps using dif-
Oleispira sp. Harayama et al. (2004) and ferent enzymes (Abbasian et al., 2015). Hydrocarbons can selec-
Brooijmans et al. (2009)
tively be metabolized by individual strain of microorganism or
Pseudomonas sp. Mittal and Singh (2009),
Rocha et al. (2011), Sajna consortium of microbial strains belonging to either same or differ-
et al. (2015), Varjani et al. ent genera (Boopathy, 2000; Varjani et al., 2015; Varjani and
(2015) and Varjani and Upasani, 2016c). However, consortium has been proved to be more
Upasani (2016c) potential than individual cultures for metabolizing/degrading com-
Rhodococcus sp. Abbasian et al. (2015)
Stenotrophomonas sp. Varjani et al. (2015)
plete assortments of hydrocarbons (Deziel et al., 1996; Deppe et al.,
Thalassolituus sp. Brooijmans et al. (2009) 2005; Varjani et al., 2013, 2015). It has been reported that n-
Aspergillus sp. Wilkes et al. (2016) alkanes are preferred for biodegradation as compared to PAHs by
Candida sp. Leahy and Colwell (1990) microorganisms (Sugiura et al., 1997; Widdel and Rabus, 2001;
Penicillium sp. Atlas (1981)
Das and Mukherjee, 2007). This could be due to availability of pet-
Pseudozyma sp. Sajna et al. (2015)
roleum hydrocarbon (sole source of carbon and energy) com-
Monoaromatics Acinetobacter sp. Batista et al. (2006)
pounds subjected to bacterial decomposition, adaption of
Archaeoglobus fulgidus Wilkes et al. (2016)
Aromatoleum aromaticum Wilkes et al. (2016) hydrocarbon pollutant degraders to polluted environment and
Bacillus sp. Janbandhu and Fulekar presence of enzymes which contribute to various pathways for
(2011) biodegradation (Atlas, 1991; Boopathy, 2000; Thamer et al.,
Halomonas sp. Widdel and Rabus (2001) 2013; Meckenstock et al., 2016). In majority microorganisms
Pseudomonas sp. Mittal and Singh (2009) and
Meckenstock et al. (2016)
enzymes for biodegradation are encoded on plasmids. Acinetobac-
Rhodococcus sp. Leahy and Colwell (1990) ter sp. are exceptional with regards to presence of biodegradative
and Salleh et al. (2003) enzymes, in this sp. plasmids are located on chromosome (Atlas,
Sphingobacterium sp. Janbandhu and Fulekar 1995; Salleh et al., 2003). Whyte et al. (1998), suggested role of
(2011)
Q15 plasmid in hydrocarbon degradation. Various plasmids are
Polyaromatics Achromobacter insolitus Janbandhu and Fulekar involved in petroleum hydrocarbon pollutant degradation such as
(2011)
Q15, OCT, TOL, NAH7, pND140 and pND160 with presence of genes
Bacillus sp. Mittal and Singh (2009)
Cycloclasticus sp. Harayama et al. (2004) alkA, alkM, alkB, theA, LadA, assA1 and assA2 and nahA-M (Salleh
Phanaerochaete chrysporium Salleh et al. (2003) et al., 2003; Abbasian et al., 2015; Wilkes et al., 2016).
Pseudomonas sp. Widdel and Rabus (2001) The simpler compounds of crude oil can be degraded by a wide
and Meckenstock et al. variety of bacteria, but ability to degrade complex compounds
(2016)
Vibrio sp. Widdel and Rabus (2001)
(such as PAHs, resins and asphaltenes) is found in very few species.
Penicillium janthinellum Boonchan et al. (2000) A bacterial sp. specializes in utilization of few hydrocarbons as pre-
Resins Pseudomonas sp. Leahy and Colwell (1990)
ferred food source and in consortium they give synergistic effect
Member of family Chandra et al. (2013) (Sugiura et al., 1997; Peressutti et al., 2003; Varjani et al., 2013,
Vibrionaceae, Members of 2015). The degradation of petroleum hydrocarbons can be medi-
family Enterobacteriaceae, ated by specific enzyme system (Widdel and Rabus, 2001; Das
Moraxella sp.
and Chandran, 2011). Initial attack is generally achieved through
various mechanisms: (a) attachment of microbial cells to the sub-
strates and (b) production of biosurfactants / bioemulsifier,
biopolymers, solvents, gases and acids (Banat, 1995;
Mittal and Singh (2009), have reported eleven petroleum
Pornsunthorntawee et al., 2008; Saeki et al., 2009; Varjani and
hydrocarbon degrading bacterial isolates from oil production site
Upasani, 2016b). Crude oil is a mixture of simple and complex
of Lingala oil field, ONGC and nine isolates from oil contaminated
hydrocarbons which are degraded by several indigenous microor-
soil of local area at Haridwar. Bayoumi and Abul-Hamd (2010),
ganisms, each capable of breaking down a specific group of mole-
have reported isolation of one hundred and nine (109) pure bacte-
cules (Zanaroli et al., 2010; Varjani and Upasani, 2013; Varjani
rial cultures in mineral salt medium (MSM) supplemented with
et al., 2014b).
toluene and phenol as sole carbon and energy source. There are
several reports available on isolation and identification of P. aerug-
inosa, S. maltophilia, Rhodococcus sp., Bacillus sp., Acinetobacter sp., 7.1. Pathways for biodegradation of petroleum hydrocarbon pollutants
Ochrobactrum sp., and Exiguobacterium for hydrocarbon utiliza-
tion/degradation and biosurfactant production (Batista et al., Microorganisms either catabolize petroleum hydrocarbon pol-
2006; Pornsunthorntawee et al., 2008; Varjani et al., 2015; lutants to obtain energy or assimilate them into cell biomass
282 S.J. Varjani / Bioresource Technology 223 (2017) 277–286

(Leahy and Colwell, 1990). Fig. 1 represents schematic overview of
pathways for petroleum hydrocarbon utilization by hydrocarbon
degrading microorganisms. There are three possible ways for pet-
roleum hydrocarbon utilization: (a) Phototrophic, anoxygenic; (b)
Chemotrophic, aerobic; and (c) Chemotrophic, anaerobic (Fig. 1).
Petroleum hydrocarbon pollutants degradation by bacterial species
has been well-documented and metabolic pathways have been
elucidated (Leahy and Colwell, 1990; Hendrickx et al., 2006;
Abbasian et al., 2015; Meckenstock et al., 2016; Wilkes et al.,
2016). Hydrocarbon catabolism has long been considered as
strictly aerobic process; however certain microorganisms are
reported for anaerobic degradation of hydrocarbons (Widdel and
Rabus, 2001; Abbasian et al., 2015; Meckenstock et al., 2016). Var-
ious reactions viz. oxidation, reduction, hydroxylation and dehy-
drogenation are common for both aerobic and anaerobic
pathways of microbial degradation petroleum hydrocarbon pollu-
tants (Abbasian et al., 2015; Wilkes et al., 2016). Biodegradation
can be characterized as increase in relative abundance of polar
fractions as well as loss of saturated and aromatic hydrocarbons
(Mittal and Singh, 2009; Varjani et al., 2015; Varjani and
Upasani, 2016c).
7.1.1. Aerobic biodegradation
Petroleum hydrocarbon pollutants can be degraded by various
pathways such as terminal oxidation, sub-terminal oxidation, x-
oxidation and b-oxidation (Salleh et al., 2003; Abbasian et al.,
2015). Possible peripheral pathways for aerobic biodegradation of
n-alkane in microorganisms are shown in Fig. 2. Common pathway

Fig. 1. Schematic overview of potential pathways for petroleum hydrocarbons utilization by microorganisms.

Fig. 2. Three possible peripheral pathways for n-alkane degradation (aerobic) in microorganisms.
 S.J. Varjani / Bioresource Technology 223 (2017) 277–286
for hydrocarbon degradation include initial degradation of alkane
with oxidation of methyl group and results in formation of an alco-
hol, which is then dehydrogenated via aldehyde to corresponding
carboxylic acid, which can then be metabolized by b-oxidation
pathway of fatty acids (Das and Chandran, 2011; Abbasian et al.,
2015). Alkanes are commonly degraded by mono-terminal oxida-
tion (Salleh et al., 2003). Oxidation of terminal methyl to form pri-
mary alcohol by introduction of molecular oxygen into
hydrocarbon is the first step in this process (Li and Liu, 2002;
Abbasian et al., 2015). The primary alcohol formed is then oxidized
to aldehyde and fatty acid. This step is followed by b-oxidation,
which results in formation and removal of acetyl coenzyme-A by
which fatty acid is shortened to two-carbon atom compound.
Other oxidation pathways are di-terminal and sub-terminal oxida-
tion (Li and Liu, 2002; Das and Chandran, 2011). In di-terminal
pathway, oxidation of both ends of alkane molecule takes place
through x-hydroxylation (x position represents terminal methyl
group) of fatty acids, and then further converted into a di-
carboxylic acid & processed by b-oxidation (Abbasian et al.,
2015). However alkanes are oxidized to secondary alcohol and
then to corresponding ketone and ester in sub-terminal oxidation.
The ester is then hydrolyzed and generate an alcohol and a fatty
acid (Rojo, 2009). Zhang et al. (2011), investigated diesel, crude
oil, n-alkanes and PAHs degradation by P. aeruginosa DQ8 and
reported n-alkanes degradation via terminal oxidation pathway.
The aromatic hydrocarbons are less biodegradable than satu-
rated hydrocarbons They pose more deteriorating effects in envi-
ronment and life forms, they are the priority pollutants in
bioremediation processes (Salleh et al., 2003). Initial oxidative
attack followed by cleavage of benzene ring are the key steps in
degradation of aromatic hydrocarbon pollutants (Hendrickx et al.,
2006). The bacterial degradation of aromatics normally involves
formation of a diol, followed by ring cleavage and formation of
di-carboxylic acid. Fungi and other eukaryotes generally oxidize
aromatics using mono-oxygenases, forming a trans-diol (Zhang
et al., 2011). The most common way of initial oxidation is forma-
tion of cis-dihydrodiols by incorporation of both oxygen atoms of
an oxygen molecule and then to formation of catechols (Abbasian
et al., 2015). Benzene ring is cleaved by microorganisms in differ-
ent ways by appropriate enzymes (Li and Liu, 2002): ortho- or
meta-cleavage pathways leading to formation of central interme-
diates such as protocatechuates and catechols which are further
converted to tricarboxylic acid (TCA) cycle intermediates
(Abbasian et al., 2015). The ortho- and meta-cleavage pathways
differ in cleavage site. Ortho cleavage is catalyzed by intradiol
dioxygenases which are either homomultimers or composed of
two different subunits containing ferric iron. However meta cleav-
age is catalyzed by extradiol dioxygenases which are multimers of
a single subunit containing ferrous iron (Li and Liu, 2002). Genes
for meta-cleavage are located on plasmids. Ortho-cleavage genes
are located on chromosome whereas genetically modified
Table 2
Enzymes involved in microbial degradation of petroleum hydrocarbon pollutants.
Enzyme Petroleum hydrocarbon compound
Soluble/particulate C1-C8 alkanes, C1-C5 (halogenated)
Methane alkanes, Alkenes, Cycloalkanes
Monooxygenases
AlkB related Alkane C5-C16 alkanes, Alkyl benzenes,
Hydroxylases Cycloalkanes, Fatty acids
Eukaryotic P450 (CYP52) C10-C16 alkanes, Fatty acids
Bacterial P450 C5-C16 alkanes, Cycloalkanes
oxygenase system
(CY153)
Dioxygenases C10-C30 alkanes
283
ortho-genes are located on catabolic plasmids (Van der Meer
et al., 1992). Polyaromatic hydrocarbons are degraded one ring at
a time, by above described methods.
Cyclic alkanes are converted to cyclic alcohols and dehydro-
genated to ketones by an oxidase system. A mono-oxygenase sys-
tem followed by lactonates ring which is finally opened by a
lactone hydrolase (Abbasian et al., 2015). Two oxygenase systems
are almost never found in same bacterium, making it difficult to
isolate pure cultures able to degrade cycloalkanes (Bartha, 1986;
Abbasian et al., 2015). Alkenes may be attacked by (a) terminal
oxygenase, (b) sub-terminal oxygenase, (c) oxidation across double
bond to corresponding epoxide and (d) oxidation across double
bond to corresponding diol (Watkinson and Morgan, 1990).
7.1.2. Anerobic biodegradation
Understanding of anaerobic degradation is more recent as com-
pared to aerobic degradation (Jaekel et al., 2013; Meckenstock
et al., 2016). Hence less information is available about the genes
and enzymes involved in these pathways (Leahy and Colwell,
1990; Wilkes et al., 2016). The BTEX (Benzene, Toluene, Ethyl ben-
zene, Xylene) compounds are typically removed at about same rate
by both aerobic and anaerobic metabolism (Abbasian et al., 2015).
Under anaerobic metabolism these aromatic compounds are first
oxidized to phenols or organic acids, and then transformed to
long-chain volatile fatty acids, which are finally metabolized to
CH4 and CO2 (Heider et al., 1999; Abbasian et al., 2015; Wilkes
et al., 2016) .
Various electron acceptors viz. nitrate, ferrous iron, manganese
or sulfate ions are reported for anaerobic degradation of petroleum
hydrocarbons (Widdel and Rabus, 2001; Foght, 2008; Abbasian
et al., 2015). Widdel and Rabus (2001), as well as Jaekel et al.
(2013), reported anaerobic degradation of propane and butane by
sulfate-reducing bacteria enriched from marine hydrocarbon cold
seeps. They found that enriched cultures formed a distinct phylo-
genetic cluster affiliated with Desulfosarcina-Desulfococcus cluster
within d-Proteobacteria. Wilkes et al. (2016), proposed two bio-
chemical mechanisms i.e. addition of fumarate and carboxylation
as initial activation of alkanes. Heider et al. (1999), reported
Benzoyl-CoA as common intermediate in anaerobic catabolism of
many aromatic compounds. Biodegradation of non-substituted
aromatic hydrocarbons viz. benzene and some polyaromatic
hydrocarbons (naphthalene and phenanthrene) is not very well
understood (Widdel and Rabus, 2001; Meckenstock et al., 2016).
Toluene, alkyl benzene and ethylbenzene are oxidized to
benzoyl-CoA. Benzene and naphthalene are initially activated by
carboxylation but detailed mechanism remains unclear (Foght,
2008). Further degradation follows benzoyl-CoA or naphthalene-
CoA pathways (Foght, 2008; Meckenstock et al., 2016). Harwood
et al. (1998), and Wilkes et al. (2016), have reported that central
benzoyl-CoA pathways are different for many aspects in denitrify-
ing, phototrophic and fermenting bacteria.
Name of microorganism References
Methylocystis, Methylomonas, Methylocella, Methylobacter, McDonald et al. (2006) and
Methylococcus, Geobacillus hermodenitrificans, Methylomirabilis Abbasian et al. (2015)
oxyfera
Pseudomonas, Burkholderia, Rhodococcus, Mycobacterium Abbasian et al. (2015)
Candida maltose Salleh et al. (2003) and
Candida tropicalis Abbasian et al. (2015)
Yarrowia lipolytica
Acinetobacter, Caulobacter Watkinson and Morgan
Mycobacterium (1990) and Abbasian et al.
(2015)
Acinetobacter sp. Abbasian et al. (2015)
284 S.J. Varjani / Bioresource Technology 223 (2017) 277–286
7.2. Genes involved in biodegradation
The initial intracellular attack of organic pollutants is an oxida-
tive process. Oxygenases and peroxidases catalyse activation as
well as incorporation of oxygen. Peripheral degradation pathways
convert organic pollutants step by step into intermediates of cen-
tral intermediary metabolism i.e. tricarboxylic acid cycle
(Abbasian et al., 2015). Synthesis of cell biomass occurs from cen-
tral precursor metabolites such as succinate, acetyl-CoA and pyru-
vate (Fig. 1). Gluconeogenesis pathway give rise the synthesis of
sugars required for biosynthesis of molecules and growth (Das
and Chandran, 2011). Diverse degradation pathways and catabolic
genes for biodegradation of each hydrocarbon group are reported
earlier (Widdel and Rabus, 2001; Hendrickx et al., 2006; Foght,
2008; Meckenstock et al., 2016). Depending on the chain length
and type of petroleum hydrocarbon pollutants different enzymes
are required to introduce oxygen in substrate to start biodegrada-
tion. Details of enzymes involved in petroleum hydrocarbon pollu-
tant degradation along with hydrocarbon pollutant degraded and
microorganism for the same are depicted in Table 2. alkB gene
for alkane monooxygenase, xylE gene for catechol dioxygenase
and nahAc gene for naphthalene dioxygenase are major genes
involved in microbial degradation of hydrocarbon pollutants
(Whyte et al., 1998; Baldwin et al., 2003; Hendrickx et al., 2006;
Wilkes et al., 2016).
8. Factors affecting biodegradation of petroleum hydrocarbon
pollutants
Microorganisms are highly sensitive to growth environment
and respond to changes in their surrounding environment
(Boopathy, 2000). Biodegradation rates are influenced by many
factors viz. (a) pollutant characteristics viz. availability, type and
length of hydrocarbons, dispersion into aqueous phase and
volatilization (Chaudhry et al., 2005; Rojo, 2009; Beskoski et al.,
2011; Chandra et al., 2013), (b) microorganisms, cell metabolic
pathways and several structural changes from inclusions to com-
plex extracellular polymeric spheres (Baldwin et al., 2003; Rocha
et al., 2011; Meckenstock et al., 2016), (c) environmental condi-
tions, such as pH, temperature, water content, salinity; oxygen
availability and nutritional factors viz. carbon and nitrogen source
and other nutrients (Atlas, 1991; Boopathy, 2000; Aislabie et al.,
2006; Chandra et al., 2013; Varjani et al., 2015), and (d) physico-
chemical properties of soil such as density water holding capacity,
pH moisture and texture etc. (Leahy and Colwell, 1990; Sabate
et al., 2004; Beskoski et al., 2011). It is important to consider all
these factors before selecting any alternative for remediation pro-
cess (Atlas, 1981; Okoh, 2006). Relationship between soil condi-
tions and microbial activity have been reported earlier by Leahy
and Colwell (1990). The extent of hydrocarbon biodegradation in
contaminated soils critically depends on optimum environmental
conditions to stimulate biodegradative activity (Okoh, 2006;
Varjani et al., 2014b, 2015), predominant petroleum hydrocarbon
types in contaminated matrix (Ghazali et al., 2004) and bioavail-
ability of contaminants to microorganisms (Chaudhry et al.,
2005; Varjani and Upasani, 2016c).
Physico-chemical properties and bioavailability of hydrocar-
bon pollutants plays very important role for successful bioreme-
diation (Boopathy, 2000; Varjani et al., 2015; Varjani and
Upasani, 2016c). Bioavailability can be defined as amount of a
substance that is physico-chemically accessible to microorgan-
isms (Chaudhry et al., 2005; Varjani et al., 2014a, Varjani and
Upasani, 2016b). It has been reported that same compound in
different pollutants can be degraded to different extents by
same organisms/consortium, due to bioavailability of a particu-
lar compound and not it’s chemical structure (Sugiura et al.,
1997; Varjani et al., 2015). Different products viz. gases, biosur-
factants, biopolymers, solvents and acids are also produced by
hydrocarbon pollutant degrading microorganisms which
enhances bioremediation and oil recovery (Leahy and Colwell,
1990; Saeki et al., 2009; Varjani et al., 2014a; Varjani and
Upasani, 2016a,b). Among all such products biosurfactant is
very well studied as it plays critical role to enhance hydrocar-
bon pollutants bioavailability (Mulligan, 2005; Saeki et al.,
2009; Varjani and Upasani, 2016c). Atlas (1981), has reported
that hydrocarbon pollutant degradation depends on its composi-
tion. Biodegradability of hydrocarbons can be ranked as:
linear alkanes > branched alkanes > low-molecular-weight alkyl
aromatics > monoaromatics > cyclic alkanes > polyaromatics 
asphaltenes (Atlas, 1981; Leahy and Colwell, 1990). Biodegrada-
tion capacity of aliphatic and aromatic hydrocarbons of
petrochemical oily sludge in liquid medium by heterogeneous
bacterial consortium and five pure petroleum degrading bacte-
rial cultures was investigated and compared. Heterogeneous
bacterial consortium demonstrated best results with excellent
degradation capacity. Aliphatic and aromatic fractions were
reduced 90.7% and 51.8%, respectively by bacterial consortium
(Cerqueira et al., 2011).
Aislabie et al. (2006), and Okoh (2006), have reported that ele-
vated temperature increases solubility of hydrocarbon pollutants,
decrease viscosity and transfer long chain n-alkanes from solid
phase to water phase. Thamer et al. (2013), have reported that
salinity and high temperatures hamper microbial growth and their
products. Varjani et al. (2014b), have reported optimization studies
for hydrocarbon utilizing bacterial consortium (HUBC) and con-
cluded that 3%, v/v crude oil or 1%, w/v glucose, pH 7.2, incubation
at 37 °C at 180 rpm with 2%, v/v inoculum as optimum conditions
for its growth. Leahy and Colwell (1990), have reviewed that
although petroleum hydrocarbons are rich source of carbon and
energy they do not contain significant concentrations of other
nutrients (such as nitrogen and phosphorous) required for micro-
bial growth. Carbon/nitrogen/phosphorous/potassium (C-N-P-K)
ratios can be adjusted by addition of urea, phosphate, N-P-K fertil-
izers, ammonium and phosphate salts which accelerates biodegra-
dation of petroleum hydrocarbon pollutants (Boopathy, 2000; Ron
and Rosenberg, 2014). However, Varjani et al. (2014b), have
reported that nitrogen nutrition does not seem to play significant
role in biodegradation of petroleum crude components when it
was grown on 3%, v/v crude oil, which suggests that the nitrogen
present in Bushnell-Hass medium (used in their study) is sufficient
for microbial growth. They stated that their report is first where
3%, v/v crude oil being optimum for biodegradation by a consor-
tium. The consortium used by them was halo-tolerant growing
up to 5% w/v NaCl and they suggested its application in oil spill
bioremediation.
9. Conclusion
Petroleum hydrocarbon pollutants are priority pollutants as
they are resistant to degradation due to their low reactivity. These
persistent organic pollutants (POPs) pose serious threat to the
human and environmental health. Bioremediation is recognized
as an efficient, economic and versatile alternative to physico-
chemical treatment. Biodegradation of this pollutants can be per-
formed using oleophilic microorganisms either as individual
organism or consortium of microorganisms to control environmen-
tal pollution. Understanding factors affecting biodegradation is of
great research interest. Catabolic pathways involved in biodegra-
dation (aerobic/anaerobic) show the way to design efficient strate-
gies for bioremediation of petroleum hydrocarbon pollutants
impacted environments.
 S.J. Varjani / Bioresource Technology 223 (2017) 277–286 285

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