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Int. J. Environ. Res. Public Health 2021, 18, 2226. https://doi.org/10.3390/ijerph18052226 www.mdpi.com/journal/ijerph
Review
Bioremediation of Total Petroleum Hydrocarbons (TPH) by
Bioaugmentation and Biostimulation in Water with Floating
Oil Spill Containment Booms as Bioreactor Basin
Khalid Sayed
1,
*, Lavania Baloo
1
and Naresh Kumar Sharma
2
1
Civil and Environmental Engineering Department, Universiti Teknologi Petronas, Seri Iskandar,
Perak 32610, Malaysia; khalid_19000239@utp.edu.my; lavania.baloo@utp.edu.my
2
Kalasalingam Academy of Research and Education, Krishnankoil, Srivilliputhur, Tamil Nadu 626128, India;
naresh@klu.ac.in
*
Correspondence:
khalid_19000239@utp.edu.my; Tel.: +60-0102547454
Abstract: A crude oil spill is a common issue during offshore oil drilling, transport and transfer to
onshore. Second, the production of petroleum refinery effluent is known to cause pollution due to
its toxic effluent discharge. Sea habitats and onshore soil biota are affected by total petroleum hy-
drocarbons (TPH) as a pollutant in their natural environment. Crude oil pollution in seawater, es-
tuaries and beaches requires an efficient process of cleaning. To remove crude oil pollutants from
seawater, various physicochemical and biological treatment methods have been applied worldwide.
A biological treatment method using bacteria, fungi and algae has recently gained a lot of attention
due to its efficiency and lower cost. This review introduces various studies related to the bioreme-
diation of crude oil, TPH and related petroleum products by bioaugmentation and biostimulation
or both together. Bioremediation studies mentioned in this paper can be used for treatment such as
emulsified residual spilled oil in seawater with floating oil spill containment booms as an enclosed
basin such as a bioreactor, for petroleum hydrocarbons as a pollutant that will help environmental
researchers solve these problems and completely clean-up oil spills in seawater.
Keywords: oil spill clean-up; oil spill treatment; crude oil; petroleum products; bacteria; fungi;
algae; agro-industrial wastes
1. Introduction
The world is dominated by five massive oceans and the three main seas, which to-
gether account for 71% of the Earth [1]. For thousands of years, the ocean has attracted
human attention. It is also the food chain's principal source and popular for its diverse
aquatic species [2–4]. Several researchers have warned about the dangers to oceans and
acknowledged the threat to human survival by bioaccumulation and biomagnifications
of toxic substances in petroleum hydrocarbons [4,5]. There are many forms of life in these
oceans, and for this reason specific laws and regulations are continually framed to take
care of this insubstantial marine environment. New approaches must, therefore, be devel-
oped for managing existing marine ecosystem resources in order to preserve human
safety from toxic petroleum hydrocarbons through bioaccumulation and biomagnifica-
tions in the food chain [4,6].
The largest group of environmental pollutants worldwide is produced from crude
oil-based hydrocarbons [7]. Processing activities in the hydrocarbon oil industry releases
hazardous aromatic organic compounds such as polyaromatic hydrocarbons (PAHs),
phenolic substances that are barely degradable by nature, chlorophenols and cresols tox-
ins from hydrocarbons into the environment [8–10]. On the other hand, crude oil spills
have intensified oil pollution problems during transportation and storage operations.
Citation: Sayed, K..; Baloo, L.;
Sharma N.K., Bioremediation of To-
tal Petroleum Hydrocarbons (TPH)
by Bioaugmentation and Biostimula-
tion in Water with Floating Oil Spill
Containment Booms as Bioreactor
Basin. Int. J. Environ. Res. Public
Health 2021, 18, 2226. https://doi.org/
10.3390/ijerph18052226
Academic Editors: Yu-Pin Lin and
Marta Otero
Received: 6 February 2021
Accepted: 17 February 2021
Published: 24 February 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional
claims in published maps and insti-
tutional affiliations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(http://creativecommons.org/licenses
/by/4.0/).
Int. J. Environ. Res. Public Health 2021, 18, 2226 2 of 27
Crude oil spill in seawater requires an effective clean-up treatment process. Various phys-
icochemical and biological treatment processes have been applied worldwide to eliminate
crude oil spill pollution from the seawater. A biological treatment process using bacteria,
fungi and algae for biodegradation of crude oil recently received much attention because
of its efficiency and lower cost. Researchers have used bioaugmentation i.e., single strain
and consortium micro-organisms to degrade the maximum part of the spilled crude oil as
a part of the treatment.
Presently, there is a trend towards enhancing and putting back micro-organisms with
high potential agro-industrial waste. A variety of low-cost substrates exist such as soybean
waste oil, paneer whey, solid-waste-date, corn-steep-liquor, molasses, etc. All these agro-
industrial wastes not only serve as nutrients for the growth of organisms, but also act as
the main source for potential micro-organisms generating biosurfactants [11–17]. Some
researchers have used other methods by applying N:P:K nutrient ratios [18,19], food
wastes [20]. Some researchers have applied it in the field [12,15,18,19,21–24]. A compre-
hensive and practical collection of guidelines for the application of this technology to sea-
water oil spill responders is urgently required to address questions such as when to use
bioremediation, what bioremediation agents should be used, how to apply them, and how
to track and evaluate the outcomes [25]. The application of micro-organisms for the bio-
remediation of petroleum hydrocarbon pollutants in this day and age is a priority in the
effort to establish green technology [22–26]. The lack of guidelines as to how and when to
use this technology is now one of the biggest obstacles for the implementation of oil bio-
remediation in marine water. One good and beneficial factor is the possibility of using
bioremediation methods where water movement is less in an encircled area. This can be
done with floating oil containment booms, usually used to contain oil spills in flowing
water and restrict moving water movement with oil spills that create an enclosed area,
known as the booming technique. Potential studies have been reviewed in this review
paper to completely clean-up crude oil spills, TPH and related petroleum products using
bioremediation as polishing treatment in combination with floating oil containment
booms like a bioreactor basin. The novelty here is that no researcher has used bioremedi-
ation in combination with a booming technique that can be used as a bioreactor-like float-
ing oil container basin where micro-organisms can break waste into stable products (car-
bon dioxide, water etc.).
1.1. Petroleum Hydrocarbons
TPH is a term used to represent petroleum (crude oil) that consists of a blend of thou-
sands of compounds. TPH is a chemical combination in this context. They are referred to
as hydrocarbons because almost all consist of hydrogen and carbon. Petroleum hydrocar-
bons account for 50–98% of crude oil and are considered an important component de-
pending on the source of petroleum [27]. The main composition of crude oil is illustrated
in Table 1.
Table 1. Elemental composition of crude oil.
Sr.No.
Elements
Percentage (%)
1.
Carbon
85–90
2. Hydrogen 10–14
3. Sulfur 0.2–3
4. Nitrogen <0.1–2
5.
Oxygen
1–1.5
6. Metals * <1
*
Hg, Au, Cu, Al, Ca, Co, K, Mg, Si, Sr, Mo, Ti, Mn, Li, Se, Rb, Ag, Ba, Pb, As, Cd, Cr, Fe, Ni, V, Zn.
Int. J. Environ. Res. Public Health 2021, 18, 2226 3 of 27
Crude oil is extracted from offshore oil rigs in seawater and transported to the shore.
Crude oil recovered from the sub surface is of no use directly, for this reason it must un-
dergo refining for a variety of applications. In a petroleum oil refinery crude oil undergoes
processes. The oil refinery methods and processes refine products like petrol, gasoline,
diesel, jet fuel, asphalt, wax, lubricating oil, tar, kerosene, and liquefied petroleum gas
(LPG), etc. The petroleum industry supplies a substantial quantity of world's energy de-
mands in addition to popular petro-chemical intermediates required for production of
extensive range of goods viz. solvents, dye stuffs, pharmaceuticals, polymers, and new
chemicals etc. All these goods generate environmental pollution when discharged in the
environment [9,28]. Figure 1 show the different products obtained from the petroleum
hydrocarbon refinery with their molecular carbon ranges. Carbon ranges illustrated may
differ from state to state. These ranges mentioned are the most common.
Figure 1. Petroleum hydrocarbon products and fractions by carbon ranges.
The stability and behavior of petroleum in seawater depends on its relative density
(the relationship between the density of petroleum and pure water) and the distillation
characteristics (definition of volatility, large quantities of resin, asphalt, wax, etc. reduces
the volatility of oil), viscosity (flow resistance that varies with temperature) and point of
pour [29]. Due to the formation of water or gas, or liquids and chemicals extracted during
operations, inorganic salts like sodium chloride, magnesium chloride and other inorganic
salts often follow crude oil from wells. Heavy crude oils produce large quantities of diffi-
cult to process complex hydrocarbons, such as polynuclear aromatic compounds (PNA),
PAHs, alkyl aromatic compounds, heteroatoms, and metal materials. Sulphur, oxygen,
nitrogen, and metal atoms are typical heteroatoms in hydrocarbons [21].
Int. J. Environ. Res. Public Health 2021, 18, 2226 4 of 27
1.2. Sources of Petroleum Hydrocarbons Intrusion
TPH is released to the environment through oil spill incidents, industrial releases or
by-products of private or commercial uses [26]. Crude oil spill in coastal waters is mainly
the result of ship operations, tanker accidents, oil exploration and production. The main
causes of all the spills are illustrated in Figure 2. In the previous half century, the statistics
on the incidence of oil spills have shown a marked downward trend, but still the volume
of oil spills is of concern for the environment. More than 7 million tons of hydrocarbon oil
from over 140 major spills have been released into the environment [30]. The estimated
amount of petroleum hydrocarbon oil lost from tanker discharges alone in 2020 was
around 1000 tons [31]. That is the same amount as in the year 2012 and 2019. The list of
global oil spills and current spills reveals more than 200 of these incidences in last 50 years
on both the offshore and inland waters [32]. In addition to the occurrence of anthropogenic
oil spills, millions of tons of petroleum enter the marine environment every year from
natural seepages [33].
Figure 2. Major causes of all crude oil spills
[34]
.
1.3. Properties of Crude Oil
Crude oil is primarily a natural, sticky and flammable liquid. The crude oils vary
greatly in chemical composition. It is usually dark brown or black (though it may be yel-
low or green in color). From an engineering point of view, crude oils are usually classified
according to their sources, gravity of the American Petroleum Institute (API) and amount
of Sulphur (S). Crude oil is considered “light” when its density is low and “heavy” when
it is dense. Crude oils with relatively low sulphur content are called “mild” crudes, while
those containing significant amounts of sulphur are called “acid” crudes. Crude oil is a
blend of various organic substances, mostly hydrocarbons, organic compound [35]. Petro-
leum components are divided into four main groups according to their different solubil-
ity’s in organic solvents [36,37]. The chemical composition of the crude oil contains the
following four main compounds saturated, aromatics, resins and asphaltenes [38]. This is
also named SARA [39,40]. Saturated hydrocarbons are regular and branched alkanes with
C
n
H
2n+2
(aliphatic) structure. It contains cyclic alkanes (chain lengths of 1 to 40 or more
carbons). Saturated hydrocarbons are the most prevalent constituents of crude oil. Aro-
matic hydrocarbons are aromatic monocyclic compounds (benzene, toluene, xylene, etc.)
and PAHs (naphthalene, anthracene, phenanthrene, etc.). Resins include nitrogen, sul-
phur, and oxygen-containing polar compounds (for example, pyridine and thiophene).
Finally, asphaltenes are poorly polymerized compounds and of high molecular weight.
32%
28%
14%
7%
6%
6%
5% 2%
MAJOR CAUSES OF ALL CRUDE OIL SPILLS
Loading/discharging
Spills
Routine operations
Hull failures
Groundings
Bunkering
Collision
Fire and explosion
Int. J. Environ. Res. Public Health 2021, 18, 2226 5 of 27
Asphaltenes are poorly characterized hydrocarbons, metals such as nickel, vanadium, and
iron also relate to asphalt.
1.4. Toxicity of Petroleum Hydrocarbons
Many factors affect the health effects of exposure to TPH. This involve the form of
organic compounds in the TPH, the duration of exposure and the number of chemical
substances in contact. Figure 3 illustrate few impacted areas due to petroleum oil spills in
the marine environment.
Figure 3. Few impacts of oil spills in the marine environment.
1.4.1. Effects on Marine Organisms
The petroleum hydrocarbons oil spill disaster has an impact on the marine environ-
ment and ecosystem [41,42]. As TPH is discharged directly into water bodies by oil spills,
petroleum hydrocarbons float on the surface of water and establish thin oily layer. Figure
4 illustrates a few ways a petroleum oil spill can affect organisms in the marine environ-
ment.
Figure 4. Oil spill in marine environment and ways to affect organisms.
In situations where the exposure fills the organism’s body with petroleum hydrocar-
bon oil, direct toxicity is attained and death by smothering takes place [43]. The shallow
coral reefs are significant habitat that has been affected by petroleum hydrocarbon oil
spills. Coral damage and death following petroleum hydrocarbon oil exposure have been
seen extensively [9]. The species have decreased resistance to other environmental
stresses, such as variations in temperature, infectious diseases and other pollutants be-
cause of petroleum oil that covers the mammals and birds [43].
Seabirds are particularly vulnerable because oil contact inhibits the ability to fly. The
resulting intake of infected food, inhalation, and repeated encounters with the interface of
the oil water result in severe personal poisoning with high mortality rates [9]. Ingested or
dissolved oil in the body via membranes, e.g., gill surfaces cause direct lethal toxicity,
sublethal effects and marine organisms reproductive failure [43]. Turtles trapped in oil
Int. J. Environ. Res. Public Health 2021, 18, 2226 6 of 27
spills are exposed to prolonged physical contact with both floating oils, largely petroleum-
saturated respiration air, and the ingestion of food polluted by oil or tar balls. Old and
young tortoises were found to be starving to death, as petroleum hydrocarbons blocked
their esophagus [9].
The loss of economic capital due to direct mortality, loss of habitat, and restrictions
on harvesting and fisheries closures affects the commercial and aquaculture industries [9].
There are negative effects on marketing of commercially valuable species in the aquacul-
ture industry. Similarly, oil taint makes products not suitable for market. Another prob-
lem is high concentrations of petroleum oil chemicals of concern for human health in
products make then unacceptable for the market [43].
1.4.2. Impacts on Humans
First and foremost, in any accident involving petroleum oil spills in the aquatic envi-
ronment, it is imperative to prevent, if necessary, and reduce the loss of human life and
the detrimental effects on human health of the response and clean-up staff and any nearby
people and human communities [9,43]. The TPH released on the soil flows into the
groundwater through the surface. Some of these chemicals are volatile and evaporate in
the air. A few dissolve into the groundwater and move away from the spill area. Most
substances bind with soil particles and remain in the soil for a long time, while microbes
that are present in the soil break down some hydrocarbons. Secondly, contact may occur
via dermal constant contact, inhalation, and ingestion, depending on the properties of the
chemical or media (i.e., air, water, soil, food) in which the chemical affects human activity
in and around that material [9]. Figure 5 show the population affected by an oil spill acci-
dent.
Figure 5. Population of people affected due to oil spill.
The damage caused by contact to petroleum hydrocarbons can be cancerous, or tem-
porary, or permanently non-cancerous [44]. The numerous chemicals used in dispersants
and crude oils poses some documented and alleged health risks [9,45]. Compounds of
various fractions of TPH influence the body in different ways. TPH compounds, especially
smaller compounds such as benzene, toluene and xylene (which are present in gasoline),
can affect the human central nervous system [9,15,46]. Death can occur if exposures are
high enough. Breathing toluene at concentrations greater than 100 parts per million (100
ppm) for more than a few hours may induce fatigue, headache, nausea and drowsiness
[46]. When the exposure stops, the symptoms will go away. However, if anyone is exposed
for a long time, irreversible damage to the central nervous system can result. One TPH
compound (n-hexane) can have a distinct effect on the central nervous system, inducing a
nervous disease termed “peripheral neuropathy” marked by numbness of the feet and
Int. J. Environ. Res. Public Health 2021, 18, 2226 7 of 27
legs and, in extreme cases, paralysis [46]. Swallowing certain petroleum products such as
diesel and kerosene causes inflammation of the mouth and stomach, weakness of the cen-
tral nervous system, trouble coughing, and pneumonia from breathing the fumes of the
liquid into the lungs [46]. Compounds in certain TPH fractions may also affect the blood,
immune system, liver, spleen, kidneys, developing foetuses, and lungs [46]. Many TPH
compounds can be harmful to the skin and eyes. TPH products such as certain mineral
oils are not very harmful and are used in food [9,46].
Researchers analyzed the effect of crude oil, dispersants on epithelial cells of human
airways and identified similar pathological modes of action for the development of vari-
ous lung diseases. Their research indicates synergistic effects of crude oil and dispersants
important for understanding physical health outcomes and the importance of respiratory
safety for particular clean-up crews operating immediately after a spill [45,47]. Other re-
searchers studied the influence of Deep water Horizon (DWH) oil, dispersed mixtures on
rodent health in a laboratory setting, with results showing increased influence of the mix-
ture on modifying white blood cells and platelet counts, and affecting liver and kidney
function [45,48]. Researchers have reported the acute human health effects among the first
responders to the 2007 Hebei Spirit oil spill off the Yellow Sea Coast of South Korea,
dumping 12,547 kiloliters of crude oil polluted with 167 km of shoreline and 13,978 hec-
tares of fishery and aquaculture infrastructure, and involving 563,761 clean-up duties [49].
Another study shows that about 442 of the most impacted first responders to the original
exposure symptoms were analyzed 1 year later to determine the durability of the toxic
effects. Decreased periods of symptoms recorded were eye symptoms (average 9.7
months), headaches (average 8.4 months), skin symptoms (average 8.3 months), neuro-
vestibular structures (average 6.9 months), respiratory symptoms (average 2.1 months)
and back pain (average 1.8 months) [9,50]. They further reported that it is important to
remember that the statistics are merely observational, and some of those who come into
contact with volatile compounds during the cleaning operation appear to suffer from
these supposed effects after 12 months, with headaches, eye symptoms, neuro-vestibular
symptoms, respiratory symptoms, skin symptoms, and back pain in that order.
2. Petroleum Hydrocarbon Treatments
Clean-up techniques of hazardous materials are highly influenced by a number of
factors such as oil content, oil spill site characteristics and even political considerations
[25]. A variety of methods to control oil spills in marine shorelines and freshwater ecosys-
tems have been established but still the problem exists. These methods were closely re-
searched and outlined in several technical documents [25,30,33,43,51–54]. Floating booms
and barriers, oil collection materials, oil collection vessels, absorbing materials, chemical
dispersants, surfactants, physical degradation, biodegradation and on-site oil combustion
are the most common methods and techniques for oil containment and removal at sea
[30]. Clean-up oil is mechanically extracted in significant time using physical techniques.
The in situ burning method will contribute to air pollution and, when used with the com-
bustion system, worsen the ambient air quality. Secondly, shoreline vegetation deterio-
rates as many people manually collect oil and no more than 10–15% of oil recovery take
place after a major spill [25,55,56]. The chemical methods of oil removal are faster than
physical ones and include toxic chemicals in most situations. Oil spill treatment additives
like chemical surfactants are most often harmful rather than oil itself [56–58]. Oil spill re-
sponse workers (OSRWs) are exposed to those operating in the post-emergency process
onshore for the purpose of cleaning of oil. OSRWs may be highly exposed to oil spill
chemicals by dermal routes and inhalation unless protected and procedures are not fol-
lowed [59]. Most of the techniques for the recovery or removal of the spilled oil in the
water are physical and chemical methods. Oil spill cleaning techniques such as mechani-
cal skimming, sorbents, dispersants, controlled combustion, high-pressure hosing, etc. are
quite effective in cleaning up the maximum amount of oil spilled in seawater, but these
techniques are not capable of removing emulsified oil left over after physicochemical
Int. J. Environ. Res. Public Health 2021, 18, 2226 8 of 27
techniques have been applied. Finally, the complete removal of oil by physical and chem-
ical methods is not achievable and there is remaining residual oil that can be treated with
bioremediation. Recent oil spill clean-up methods advantages, limitations and efficiencies
are discussed in Table 2.
Table 2. Comparison of oil spill clean-up methods.
Physical
Clean-up
methods Advantages Disadvantages
Maximum
Clean-up
Efficiency
Application References
Sorbents
Recovery of oil which
prevents wastage and
more pollution
After the oil absorption, it is diffi-
cult to retrieve sorbent materials;
Become heavier and sink, diffi-
cult to retrieve and sink to cover
benthic organisms
90%
Most effective in small oil
spills or leftover traces of a
larger spill
[60–64]
Washing
Remove the trapped
and weathered oil
from machinery-inac-
cessible areas.
Organisms that fall into the direct
spray zone are likely to be
harmed by hot water (170
0
C).
-
Mechanical removal methods
such as booms and skimmers
are inaccessible or unavaila-
ble for oil clean-up.
[25,30,54,6
0]
In-situ burning
Where it is difficult
to
deploy other methods
Burning sites pollute the air and
can impact ecosystem both on-
shore and offshore;
Residue from in situ burning
reaches coastlines or in worse
condition, sink to cover benthic
organisms;
Fire-
resistant booms are high in
cost, difficult in towing due to
size and heavy weight.
98%
Arctic or sub-Arctic environ-
ments (remoteness and sea ice
formations);
the oil slick thickness was
also adequate for the combus-
tion to continue;
Seawater was calm and oil
slick was not located in vul-
nerable areas, equipment or
facilities
[25,30,60,6
5,66]
Skimming
Recover oil without
changing its
physical
or chemical properties
by suction and adhe-
sion
Surface conditions: wind and
waves disperse oil in the water
(rough seas can stop skimmers
from effective functioning);
To get the equipment operating
and to the site on time (as the
spilled oil will quickly spread
over quite a few km
2
)
95% Less movement of water
[25,30,60,6
4–66]
Booming
Light weight, limited
storage space, non-cor-
rosive and fast pro-
cessing, highly effi-
cient where water
movement is lower
Low stability in strong winds and
currents (current velocity more
than 0.4 m/s, wind velocity
greater than 5.5 m/s or height of
waves more than 1m)
-
Oil is at one spot;
s
pillage is reachable within a
few hours, or the spill area
becomes too vast to handle.
[60,67–69]
Manual re-
moval (Wiping)
Economically viable
(unskilled personnel
can be employed with
minimum training)
Labour-intensive and time-con-
suming 15% Shorelines oil slick clean-up
[60,67,68]
Chemical
Dispersants
Break up oil slicks to
avoid the coastlines
and vulnerable habi-
tats covering vast vol-
umes of oil;
Not much manpower
required (cheaper than
physical methods)
Poisoning fish, corals, and other
marine species 90%
If the spilled oil cannot be
stopped by booms
and spread
over large areas;
May be used in rough seas,
slowing emulsion formation
from oil water, speeding up
natural biodegradation
[60,67–71]
Int. J. Environ. Res. Public Health 2021, 18, 2226 9 of 27
Solidifiers
Convert oil spill into
solid or semi-solid ma-
terials;
Not much manpower
required (cheaper
than
physical methods)
Oil recovery not possible (oil re-
covery with high viscosity not ef-
fective)
- May be used in rough seas
[63,68,69,7
2]
Demulsifiers
Impede the spread and
pollution of oil in
nearby areas;
Not much manpower
required (cheaper than
physical methods)
The gelatine used may pose a risk
of entangling or suffocating the
aquatic animals
- May be used in rough seas
[41,60,68,6
9]
Bioremediation
Natural attenu-
ation
Most cost-effective
and sustainable meth-
ods;
Not much manpower
required
Quite time-consuming and unre-
liable Yet to be
evaluated Areas close to the shoreline
[41,43,73–
80]
Bioaugmenta-
tion Quite time consuming
Biostimulation
Quite time consuming
The treatment steps are discussed in later sections. Figure 6 show the proposed pro-
tocol to treat or clean oil spills. Figure 7 show the recent methods used to treat or clean oil
spills.
Figure 6. Proposed steps for complete oil spill treatment/clean-up in seawater.
Figure 7. Total petroleum hydrocarbons (TPH) oil spill treatment/clean-up methods.
At present, one of the greatest challenges to the application of oil bioremediation in
marine water is the lack of guidance about when and how to use this technology [25].A
positive and beneficial aspect is that bioremediation methods may be used in situations
where there is less movement of water in the enclosed environment. This form of condi-
tion can be created by placing oil containment booms known as booming (Figure 7) on the
Oil Spill Booming/Barriers Physico-chemical
clean-up Bioremediation
Int. J. Environ. Res. Public Health 2021, 18, 2226 10 of 27
surface of the water, which are typically used to contain oil spills in moving water and
limit movements of moving water with oil spills resulting in an enclosed environment.
Floating booms and barriers as the best form of containment for oil spills, followed by oil
collection of materials and vessels, have been tested in most cases [30]. The use of oil spill
booms as floating barriers should comply with environmental, mechanical and opera-
tional constraints. Numerical boom behavior modelling methods may be used to prepare
or verify booming strategies that meet these limitations [81]. The residual oil (pollutant)
concentration after physicochemical treatment in seawater can be determined by onsite
TPH analyzers [82]. Researchers can select an appropriate study from this review article,
considering local conditions such as availability of culture of micro-organisms, biostimu-
lants (agro-industrial waste, surfactants etc.), type of TPH pollutants and time to complete
bioremediation work.
In several of the studies mentioned in this review, micro-organisms are either iso-
lated from seawater or enhanced in seawater so that they can be used effectively in their
natural environment. Researchers have reported several laboratory scale studies using bi-
oaugmentation (BA), biostimulation (BS) or both methods combined (BA-BS) in aqueous
media studies that can be applied on site even after considering the problem due to poor
bioavailability of pollutants, protozoan predation or competition from native microbiota,
etc. Bioremediation is commonly used as a polishing stage following the application of
traditional mechanical clean-up options and is often started from weeks to months fol-
lowing the oil spill [25]. In bioremediation, there is minimal physical damage and short-
lived detrimental effects, helping to eliminate certain hazardous elements, a simpler and
more rigorous approach, a lower labor intensity and a lower cost [56,75]. Some of the ben-
efits of using bioremediation techniques like BA, BS or both methods combined (BA-BS)
are that harmful petroleum hydrocarbons mixtures or combinations are eliminated in-
stead of merely transferred to another nearby environment. Complex processes not appli-
cable in all pollution situations cannot produce substantial short-term outcome and
should not be adapted individually to each polluted site as a protective first measures if
high concentrations of oil is present [56]. When correctly used in certain oil-contaminated
environments, bioremediation has proved to be a cost-effective treatment technique
[25,28]. After its successful application in the Exxon Valdez 1989 oil spill, bioremediation
has been among the most promising secondary treatment options for oil removal [25,28].
The decision to bioremediate a site depends on the objectives and on all factors, which are
present that influence its performance, including clean-up, rejuvenation and habitat
preservation.
3. Bioremediation
Bioremediation is a process using naturally occurring species to break down hazard-
ous substances into less harmful or non-toxic substances [83]. All substances in nature end
up breaking down or decay or transforms into less toxic compounds. In order to obtain
energy for their growth, micro-organisms break down many organic compounds in the
environment. Bioremediation is also used to reduce pollutant impacts using micro-organ-
isms in the polluted environment. The main reason for clean-up of oil spills is that the
toxic and/or hazardous components are reduced or eliminated, allowing flora and fauna
to occupy the food chain including single-cell organisms. Since its successful application
following the 1989 Exxon Valdez spill, bioremediation has become one of the most prom-
ising secondary oil removal treatment solutions [25,84]. While today’s popular chemical
dispersants eliminate other harmful aspects of the substance, the toxicity of the spill re-
mains a concern in the area and is sometimes aggravated through adding as dispersants
chemicals. The purpose of bioremediation is transform toxic substances to non-toxic sub-
stances, such as carbon dioxide
,water and fatty acids thereby completely removing petro-
leum hydrocarbons from the affected environment and returning the affected oil spill
zone to its original conditions [25]. The advantage of bioremediation is that the end prod-
uct is carbon dioxide, water and fatty acids breakdown of hydrocarbons [22,83]. The
Int. J. Environ. Res. Public Health 2021, 18, 2226 11 of 27
biological process is an alternate method to eliminate toxins, since this procedure does not
cause adverse environmental effects.
Petroleum hydrocarbons may be used by bacteria [10,33,52], yeasts [11,85,86], fungi
[33,87] and algae [78,88]. The regulation of the bioremediation cycle is a difficult process
with multiple optimization variables. The key aspect is the energy required for cell growth
depending on the metabolic rate of the micro-organism [89]. Cell growth depends on the
type of substrates available and consumed by micro-organism. There are basically three
types of substrate: primary organic, in which contaminant is considered the main sub-
strate and from this micro-organism consumes energy for further replication. If the pollu-
tant is used as the main substrate and this energy is used to multiply into more cells, it is
known as the primary substrate. The second type is secondary organic, where contami-
nant is known as the secondary substrate, is metabolized by enzymes and helps cells to
draw energy. The microbes working in bioremediation with the presence of carbon pro-
duces enzymes [8,22]. These enzymes facilitate to break the bonds of hydrocarbons. Vari-
ous enzymes are used to make this process possible because the metabolism pathways for
hydrocarbon reductions are different [22]. It is very important to correctly choose micro-
organism based on the enzyme it creates, since this helps to break the hydrocarbons bond.
There are different rates of biodegradation of various petroleum hydrocarbon products.
It depends, however, on the amount of time required for microbial activity breaking down
the hydrocarbons. Therefore, as enzymes help to metabolize [8] and extract energy from
the pollutant, the pollutant is known as a secondary substrate. The third type is co-metab-
olism, while cell energy is obtained from other transformable compounds that are oxi-
dized to sustain microbial growth. In co-metabolism other compounds are oxidized to
support microbial growth and energy from other transformable compounds is consumed.
Co-metabolism tends to occur when the enzyme formed by the organism can catalyze the
degradation of its growth-substrate to generate energy and the carbon from it is also ca-
pable of degrading additional compounds [22]. The benefit of co-metabolic bioremedia-
tion is also that pollutants can be degraded to trace concentrations, since the microbes in
this technique are not reliant on carbon or energy pollutants [90].
Micro-organisms need nutrients (for example nitrogen, phosphate and other trace
elements), carbon and energy to survive, as with all living organisms. The rate of biodeg-
radation action depends on the growth conditions of microbes such as nutrient and sub-
stratum bioavailability, oxygen availability, electron acceptors, temperature, pH, salinity
and pressure [35]. Microorganisms may lack enough nutrients (such as nitrogen, phos-
phorous, potassium, sulfur, or trace elements) to use the chemical as a source of food.
When we compare the elemental composition of petroleum hydrocarbons and micro-or-
ganisms, we find that petrochemical residues are not “balance nutritional” for micro-or-
ganisms (Table 3) [83]. Biostimulants help to provide the deficit nutrients. Table 3 illus-
trate the necessary macro-nutrients and Table 4 show micronutrients for a cell microbial
metabolism. The effectiveness of bioremediation has been affected by many factors, the
most significant being the site’s type of bacteria, the oil and its environment’s physical
and chemical conditions. This involves effective bioremediation:
(a) The oiled material is still in contact with nutrients; and
(b) The nutrient concentrations are adequate to help during the cleaning process the op-
timal growth rate of the oil degrading bacteria [65,74].
Int. J. Environ. Res. Public Health 2021, 18, 2226 12 of 27
Table 3. Comparison for elemental composition of a microbial cell with petroleum crude oil
[91]
.
Elements Microbial Cell Composition (%) Crude Oil Composition (%)
Carbon 50 85–90
Nitrogen 14 < 0.1–2
Oxygen 20 1–1.5
Hydrogen 8 10–14
Phosphorous
3
-
Sulphur 1 0.2–3
Potassium 1 -
Sodium 1 -
Calcium
0.5
-
Magnesium 0.5 -
Chloride 0.5 -
Iron 0.2 -
All others
0.3
< 1
Table 4. Micro-nutrients for cell growth and their cellular functions.
Micro Nutrients
Cellular Functions
Cobalt VitaminB12; transcarboxylase (propionic acid bacteria)
Copper
Respiration (cytochrome c oxidase); Photosynthesis (plastocyanin, some
superoxide dismutases)
Manganese
Acts as activator of various enzymes; occurs in some superoxide dis-
mutases and in the photolytic (water-splitting) enzyme in oxygenic
phototrophs(photosystem-II)
Molybdenum
Present in some flavin containing enzymes, nitrogenase, nitrate reduc-
tase, sulphide oxidase, some formate dehydrogenases.
Nickel Present in most hydrogenase enzymes; coenzyme of methanogenes;
carbon monoxide dehydrogenase; urease
Selenium
Occurs in formate dehydrogenase; certain hydrogenases: amino acid se-
lenocysteine
Tungsten In some formate dehydrogenases; oxotransferases of hyperthermo-
philes
Vanadium
Vanadium nitrogenase; bromoperoxidase.
Zinc In carbonic anhydrase; alcohol dehydrogenase; RNA and DNA poly-
merase; many DNA-binding proteins.
4. Bioremediation Methods
Biodegradation is an especially important process for the removal from the atmos-
phere of non-volatile oil components. Potential bacteria, fungi and algae present in the
water steadily break down certain TPH fractions through natural attenuation. That may
take months or years to degrade a large proportion of oil that is deposited in the sediments
in marine and/or freshwater environments. This is a relatively slow process. Hence, other
techniques are used to enhance the bioremediation process. The bioremediation process
is enhanced by methods such as bioaugmentation and biostimulation. Bioaugmentation
(BA) adds to the indigenous microbial population known oil-degrading microbes and bi-
ostimulation (BS) stimulates the growth of indigenous microbes by adding nutrients, elec-
tron donors, electron acceptors and other growth enhancing co-substrates and/or environ-
mental changes in conditions (for example, chemical surfactants, biosurfactants etc.) [25].
Natural attenuation (NA) or natural recovery is essentially an option without intervention
that allows the removal and natural deterioration of petroleum hydrocarbon oil. In the
early stages of oil spills, evaporation of volatile compounds is the most critical method for
Int. J. Environ. Res. Public Health 2021, 18, 2226 13 of 27
natural cleaning and the removal of lighter weight components in petroleum hydrocarbon
oil. Up to 50% of the more toxic, lighter oil weight components can evaporate within the
first 12 h after the oil spill, depending on the composition of the oil spill [25]. Sunlight
reacts with oil components by photo-oxidation [8,9,30,43]. Photo-oxidation allows more
complicated compounds to degrade into simpler compounds that are typically lighter and
more water soluble, so that they can be further extracted by other methods. Various kinds
of micro-organism are widely distributed in nature that can oxidize petroleum hydrocar-
bons [25,33]. For instance, Actinobacteria have recently been known viable for hydrocarbon
biodegradation analyses due to their high metabolic capabilities. Two properties in par-
ticular are of interest in this case; the first is number and variety of degradative pathways
for hydrocarbons, and second the development of secondary metabolites such as biosur-
factants and siderophores. These properties enable actinobacteria to function under a
wide range of environmental conditions and, by secreting metabolites, modify or even
alter local conditions [92]. Figure 8 illustrate the types of bioremediation.
Figure 8. Different types of bioremediation techniques.
The product schedule of the National Oil and Hazardous Substance Pollution Con-
tingency Plan (NCP), USA, lists dispersants, biological remediators, surface washing
agents and various oil spill control agents [93]. All of these are divided into three catego-
ries and are illustrated in Figure 9. The first category BA is a method of bioremediation
using non-native bacteria. The primary concern with these kinds of products is that intro-
ducing foreign species into a given ecosystem unpredictable and future problems may be
caused that may be noticeable for some time, although it is useful in controlled/contained
environments. The second type of BS consists of some agents that still supply nutrient
substrates in the spill area to sustain indigenous microorganisms. BA and BS types are
considered to be unsuitable for use in open-water environments [25]. This limitation is
due to the inability to hold inoculated micro-organisms culture and nutrients with hydro-
carbon pollutants that can be overcome by implementing the proposed method of floating
oil containment booms/barriers as proposed in this study. The third type, enzyme addi-
tives (EA), is a first reaction system of soil, water and closed environment rejuvenation for
open water, intertidal zones, sensitive estuary habitants. Bioremediation experience EA
type on the ground has developed in recent years as the technology protocols have dra-
matically progressed. It provides broad application for oil spillage responses under tem-
perature conditions as low as 28 °F in natural, brackish or marine environments [74]. In
addition, bioremediation may be used in some oil-contaminated areas as a proven alter-
native treatment method. Normally, after conventional mechanical clean-ups it is used as
a polishing method. It takes weeks to months to undertake the clean-up. Bioremediation
can be very cost-efficient if done correctly, although a detailed economic analysis has not
been carried out to date [65]. Bioremediation of polluted hydrocarbon sites can be carried
out using BA, BS or both together as BA-BS.
BIOREMEDIATION
NATURAL ATTENUATION
•EVAPORATION
•PHOTO-OXIDATION
•BIODEGRADATION
BIOAUGMENTATION
•CELL INOCULATION
BIOSTIMULATION
•NUTRIENTS (ELECTRON DONOR/ACCEPTOR)
•SURFACTANTS
•MEATBOLITES
•ENZYMES
Int. J. Environ. Res. Public Health 2021, 18, 2226 14 of 27
Figure 9. Bioremediation agents under National Oil and Hazardous Substance Pollution Contingency Plan (NCP), USA.
4.1. Bioaugmentation
The process of bioaugmentation is “oil-degrading bacteria are added to supplement
the existing microbial population” [65,74]. Bioremediation activities aim to increase the
degradation rates that are naturally present by adding exogenous micro-organisms (BA).
Bioaugmentation is known as a ‘polishing-up’ or ‘finishing’ process because the impact of
fresh oil spill is too slow to turn to less harmful components because the concentration of
fresh spilled oil is initially very high. When non-native micro-organisms are exposed to
hazardous oil spills, in order to avoid adverse effects to the toxicity of the spill, they seek
to release an appropriate amount of biosurfactant and separate from the spill. Petroleum
hydrocarbons degrading bacteria (both indigenous and non-indigenous) use intracellular
enzymes that allow the bacteria to transform the petroleum hydrocarbons into yet another
food source. Oil-degrading microbes produced on a petroleum hydrocarbon-containing
culture medium are concentrated microbial agents. The micro-organisms can in some
cases be colonized at the site of a spill in bioreactors. Such form of agent is intended to
supply the affected region with a substantial oil degrading microbial inoculum, thereby
increasing the population that degrades oil down to a point that the spilled oil is used as
the main energy source. Case studies included in this review show a good percentage of
hydrocarbon degradation by BA, BS or BA-BS in the aqueous medium. The experiments
mentioned below in this review were carried out under certain conditions of pH, salinity,
temperature, selected micro-organisms as a consortium and oxygen intake. Bioaugmen-
tation techniques are applied for the bioremediation of crude oil, TPH and associated pe-
troleum products in polluted water. Table 5 illustrates a few selected studies for petro-
leum hydrocarbon degradation using only bioaugmentation.
BIOREMEDIATION
AGENT
BIOAUGMENTATION
(BA)
BIOSTIMULATION
(BS)
ENZYME ADDITIVES
(EA)
Int. J. Environ. Res. Public Health 2021, 18, 2226 15 of 27
Table 5. List of selected studies for degradation of petroleum hydrocarbons using bioaugmentation (BA).
References
Pollutant Micro-Organisms Degraded Efficiencies Time
[94]
0.5% (v/v) petroleum oil
Pseudomonas, Rhodococcus and
Acinetobacter. 66% 15 days
[95] 1% (v/v) crude oil
Bacillus sp.,
Corynebacterium sp.,
Pseudomonas sp.,
Pseudomonas sp.
77% 25 days
[96]
1% (v/v) crude oil
Betaproteobacteria,
Gammaproteobacteria,
Bacillus subtilis
85.01% 7 days
[97] 1% (v/v)
crude oil
Acinetobacter,
Pseudomonas,
Gordonia,
Rhodococcus,
Cobetia,
Halomonas,
Alcanivorax,
Marinobacter,
Microbacterium
82% 7 days
[98] 2% (v/v) Cargo fuel
Alcanivoraxborkumensis,
Alcanivoraxdieselolei, Marinobac-
terhydrocarbonoclasticus,
Cycloclasticus sp.,
Thalassolituusoleivorans
79 ± 3.2% 14 days
[99] 2% (v/v) diesel
Pseudomonas aeruginosa,
Bacillus subtilis 87% 20 days
[100] 5% (v/v) kerosene
Citrobactersedlakii,
Entrobacterhormeachei,
Entrobacter cloacae
69% 7 days
[101] 1% (v/v) crude oil
Bacillus algicola (003-Phe1),
Rhodococcus soli (102-Na5),
Isoptericolachiayiensis (103-
Na4),
Pseudoalteromonas agar-
ivorans (SDRB-Py1)
>85% 14 days
[102] 1% (v/v) crude oil
Paraburkholderia sp.,
Alloprevotellatannerae,
Paraburkholderiatropica,
Ralstonia sp.,
Paraburkholderiafungorum,
Rhodococcus sp.,
Brevundimonas_diminuta,
Lactobacillus sp.,
Acidocella sp.,
Fungus Scedosporiumboydii
81.45% 7 days
[103]
20 (g/L) crude oil/water
Chlorella vulgaris
94% 14 days
[104] 10 mg/L crude oil polluted sea-
water Alcanivoraxborkumensis SK2 95% 20 days
Int. J. Environ. Res. Public Health 2021, 18, 2226 16 of 27
From the above Table 5 we can see that researchers have used single strain and con-
sortium micro-organisms to degrade petroleum hydrocarbons using the bioaugmentation
method. Most of the studies are performed using a consortium micro-organism. In the
above studies discussed in Table 5, micro-organisms have been isolated from the polluted
site, such as seawater, soil, etc. Pseudomonas aeruginosa and Bacillus subtilis genera are usu-
ally used for bioaugmentation by researchers. Researchers took different concentrations
of petroleum hydrocarbons in the biodegradability assay. Petroleum hydrocarbons in the
studies were crude oil, diesel, kerosene, gasoline, petroleum, lubricating oil, etc. The range
of different concentrations of petroleum hydrocarbons in the biodegradability assay
ranged from 0.5% to 5% in all the above studies mentioned in Table 5. The above studies
were conducted either in culture medium or seawater. Bioaugmentation-based micro-or-
ganisms have been successful in completely degrading petroleum hydrocarbons in some
studies and degraded some of the selected components in a few studies. From the above
listed studies in the Table 5, maximum degradation efficiency up to 5% (v/v) concentration
of petroleum hydrocarbons in aqueous medium is observed.
It took the consortium micro-organisms 7 days to degrade 85% of crude oil at a con-
centration of 1% v/v and the consortium used in this study consisted of Betaproteobacteria
(47.4%), Gammaproteobacteria (51.1%), Bacillus subtilis (51.1%) [96]. In a similar study, a con-
sortium of Bacillus algicol (003-Phe1), Rhodococcus soli (102-Na5), Isoptericolachiayiensis (103-
Na4), and Pseudoalteromonas agar-Ivorans (SDRB-Py1) degraded more than 85% of crude
oil with a concentration of 1% v/v [101]. In another study, a consortium consisting of Aci-
netobacter, Pseudomonas, Gordonia, Rhodococcus, Cobetia, Halomonas, Alcanivorax, Marinobac-
ter and Microbacterium took 7 days to degrade 82% of crude oil at a concentration of 1%
v/v [97]. Researchers observed 81.45% degradation for 1% v/v crude oil with the consor-
tium consisting of Paraburkholderia sp., Alloprevotella tannerae, Paraburkholderiatropica, Ral-
stonia sp., Paraburkholderiafungorum, Rhodococcus sp., Brevundimonas_diminuta, Lactobacillus
sp., Acidocella sp. and the fungus of Scedosporiumboydii [102]. The similar crude oil degra-
dation study was successful with 95% degradation in 20 days using single strain Al-
canivoraxborkumensis SK2 [104].With respect to diesel, 87% of diesel at a concentration of
2% v/v was degraded in 20 days by Pseudomonas aeruginosa and Bacillus subtilis [99]. The
micro-algae Scenedesmus obliquus GH2 can be used to create an artificial bacteria–microal-
gae consortium to degrade crude oil [78,105]. Regarding microalgae, Chlorella vulgaris de-
graded 94% of crude oil having 20g/l concentration in water [103]. A similar study of bio-
degradation of crude oil was examined by [106], using algae Chlorella vulgaris and
Scenedesmus obliquus. These authors found that both algae are cultured heterotrophically
by crude oil as the sole source of carbon and can effectively degrade crude oil when incu-
bated with low crude oil concentrations.
The enhanced bacteria need time to adapt to the fresh available petroleum hydrocar-
bon oil, environmental temperature, pH and nutrients, but other environmental factors
may cause adverse conditions that prevent the disintegration of the oil [22]. These factors
along with the unpredictable timescales of their phase of acclimation are partly responsi-
ble for the uncertainty associated with the first response clean-up procedure of the form
bioremediation BA. The movement of water leads to a totally inefficient dilution of the
water, which does not generate adequate biosurfactants, metabolites and enzymes for the
destruction of the hydrocarbon molecular structure. A positive and beneficial aspect is
that this BA form can be used where very minimal movement of water occurs in the en-
closed environment as proposed in this review with floating booms/barriers as an oil con-
tainment bioreactor basin [74].
Int. J. Environ. Res. Public Health 2021, 18, 2226 17 of 27
4.2. Biostimulation
In many situations, certain environmental conditions can be modified to enhance the
process of biodegradation [83]. The process of biostimulation “in which nutrients, or other
growth-enhancing, substances, are added to stimulate the growth of indigenous oil de-
graders” [65]. Bioremediation activities aim to increase the degradation rates by stimulat-
ing native micro-organisms (biostimulation (BS)) with nutrients, electron acceptors, elec-
tron donors, biosurfactants, metabolites, enzymes etc. Besides the risk of the spill and the
perceived ability to compete with already acclimated native bacteria, indigenous bacteria
are also more competitive [74]. Therefore, biostimulation has more benefit than bioaug-
mentation. In certain cases, nutrients are essential components of the effective biodegra-
dation of contaminants, including nitrogen, iron and phosphorus. Some of those nutrients
may become an inhibiting factor affecting the biodegradation process. Researchers have
mostly used fertilizers as biostimulants. This is because it has N, P, and K. Carbon comes
from organic sources (petroleum hydrocarbons), water supplies with hydrogen and oxy-
gen. In marine and freshwater environments, crude oil spills and the effluent from petro-
leum refineries cause dramatic increases in carbon levels and decreases in nitrogen and
phosphorus levels that may affect the process of biodegradation [38,65]. Nitrogen and
phosphorus are low in aquatic ecosystems and wetlands cannot provide nutrients due to
the high demands on plant nutrients. The introduction of nutrients is, therefore, necessary
to facilitate the biodegradation of pollutants. Similarly, nitrogen sources should be con-
sidered [13]. For certain situations, nitrogen, phosphorus and iron are important nutrients
for a successful process of biodegradation. The most popular additives that promote bac-
terial growth in the bacterial population are phosphate and nitrate salts. Higher tempera-
tures, (NH
4
)
2
SO
4
and K
2
HPO
4
also improve the growth of micro-organisms [19,107]. Ac-
cording to some research into biostimulation of existing oil degraders, there were no last-
ing gain effects with the introduction of petroleum hydrocarbon oil degrading bacteria
[74]. On the other hand, researchers have studied the same problem at lab scale and pub-
lished promising results, which can be used as a base study for on-site applications to
clean-up petroleum hydrocarbon oil spills.
Biostimulation alone is mostly practiced in soil remediation [108–111]. Indigenous
micro-organisms remain deprived of nutrients in this natural environment. The supply of
nutrients to these micro-organisms allows them to degrade the pollutants by carrying out
anabolism and catabolism. In a spill area containing toxic oil, nutrients or fertilizers can
be difficult to use to promote the development of a crude oil-eating microbial population.
The toxicity of the oil initially weakens and/or kills several species native to the spill area.
Due to the oil's toxicity, nutrients are usually prevented from stimulating the remaining
indigenous microbes. Where there is no tidal flush and the spilled oil area has reduced
toxicity to the degree that indigenous bacteria can be retained (floating booms/barriers as
oil containment bioreactor basin), the bioremediation category BS can be used effectively
[74].
4.3. Bioaugmentation-Biostimulation
Researchers have combined biostimulation and bioaugmentation to predict out-
comes when both methods are used together. Such studies have been performed either in
seawater or culture medium. Table 6 illustrates a few selected BA-BS studies for degrada-
tion of petroleum hydrocarbons.
Int. J. Environ. Res. Public Health 2021, 18, 2226 18 of 27
Table 6. List of selected studies for degradation of petroleum hydrocarbons using bioaugmentation–biostimulation (BA-
BS).
References Pollutant Micro-Organisms Degraded
(%) Time Stimulator
[112]
0.5% (v/v)
Crude oil
polluted sea-
water
Rhodococcuscorynebacterioides 60% 15 days Chitin and Chitosan flakes
(shrimp wastes)
[113]
0.1% (v/v)
weathered
crude oil in
seawater
Thalassolituus,
Alcanivorax,
Cycloclasticus
85% 30 days Nutrients (20 mg/L NH
4
NO
3
and 10 mg/L KH
2
PO
4
)
[114]
nC15–nC35
(TPH = 10
g/L)
Pseudomonas aeruginosa Asph2 80% 30 days Corn-steep-liquor
[18]
10% (v/v)
Crude oil
Aspergillus niger,
Pseudomonas aeruginosa 94.4% 8 week NPK 15:15:15
[115]
1000 ppm
polluted sea-
water
Alcanivoraxborkumensis SK2 95% 20 days KH
2
PO
4
0.077 g/L, NH
4
Cl 0.2 g/L
and NaNO
3
0.1 g/L
[13] 0.5% (w/v)
crude oil Pseudomonas 97% 28 days Solid-waste-dates
[13]
0.5% (w/v)
crude oil Pseudomonas 91% 28 days Corn-steep-liquor
[98]
2% (v/v)
Cargo fuel
oily seawater
Alcanivoraxborkumensis, Alcani-
voraxdieselolei, Marinobacterhy-
drocarbonoclasticus,
Cycloclasticus sp. 78-ME,
Thalassolituusoleivorans
73 ± 2.4% 14 days KH
2
PO
4
0.077 g/L, NH
4
Cl 0.2 g/L
and NaNO
3
0.1g/L
[101]
1% (v/v)
Crude oil
Bacillus algicola (003-Phe1), Rho-
dococcus soli (102-Na5), Isopter-
icola chiayiensis (103-Na4),
Pseudoalteromonas agar-
ivorans (SDRB-Py1)
>85% 14 days Biosurfactant assisted
[7,116] 1% (v/v)
Diesel oil Proteobacteria 20–99% 7 days Surfactant (Tween-80), biosurfac-
tant (rhamnolipids)
After looking at the effects of bioaugmentation and biostimulation separately, re-
searchers combined bioaugmentation and biostimulation and obtained better results in a
few experiments. From Table 6 it can be concluded that researchers used single strains,
and mainly consortia, in studies involving BA-BS. Second, the researchers used stimulants
containing predominantly N and P. Third, BA-BS together have demonstrated greater ef-
ficiency in degrading petroleum hydrocarbons. Table 6 show that researchers have stud-
ied many different combinations of single or consortium micro-organisms with biostimu-
lators like fertilizers, mineral nutrients, chitin and chitosan flakes produced from shrimp
waste, corn-steep-liquor, solid-waste-dates, and other materials containing N, K and P.
Good results are achieved with corn-steep-liquor, solid-waste-dates, corn-steep-liquor
and other materials containing N, K and P. Researchers have achieved 97% degradation
efficiency for 0.5% w/v crude oil in 28 days by using single strain bacteria Pseudomonas
and solid-waste-dates as biostimulants [13]. Another related work obtained the 91% deg-
radation by simply changing biostimulant to corn-steep-liquor [13]. The degradation effi-
ciency depends upon the type of TPH pollutant to be degraded.
Int. J. Environ. Res. Public Health 2021, 18, 2226 19 of 27
Light crude oil degrades more easily and faster than heavy crude oil [22]. Arabian
light crude oil (1000 ppm) polluted seawater was degraded by single strain Alcanivorax-
borkumensis SK2 assisted with KH
2
PO
4
0.077 g/ L, NH
4
Cl 0.2 g/L and NaNO
3
0.1 g/L in 20
days. Similarly, 10% v/v crude oil (Escravos light) was degraded 94.4% by Aspergillus niger
and Pseudomonas aeruginosa assisted with (NPK 15:15:15) in 98 weeks (56 days).
Regarding
diesel, almost complete degradation was archived within 7 days using Proteobacteria
as-
sisted with surfactant and biosurfactant [7,116].
4.4. Natural Attenuation versus Bioaugmentation versus Biostimulation versus
Bioaugmentation-Biostimulation
Natural attenuation refers to processes that naturally transform pollutants to less
harmful forms or immobilize pollutants so that they are less of a threat to the environment.
Bioaugmentation and biostimulation will not be undertaken in natural attenuation. Pol-
lution and natural attenuation of petroleum hydrocarbons needs strategies for remedia-
tion of polluted areas. Simultaneous experiments of NA, BA, BS, and BA-BS have been
carried out by researchers to compare the methods for the same petroleum hydrocarbon.
Table 7 show a few selected bioremediation outcome studies compared with NA, BA, BS,
and BA-BS.
Table 7. Comparison of different bioremediation outcomes on petroleum hydrocarbons.
Medium
Natural Attenuation %
BA %
BS %
BA-BS %
Time
References
Polluted water
50.7
-
94.4
-
8 weeks
[18]
BSM
#
38 66 - 91 28 days [13]
BSM
#
38 66 - 97 28 days [13]
Seawater 32 ± 3.2 - 73 ± 2.4
79 ± 3.2 14 days [98]
Seawater
-
95
80
-
20 days
[104]
MSM * - 81.45 - - 7 days [102]
#
Basal Salt Medium; * Mineral Salt Medium.
From Table 7, it can be concluded that BA, BS and BA-BS provide more degradation
efficiency. BA, BS and BA-BS experiments have shown positive results in comparison to
natural attenuation. Degradation efficiency of some studies using BA-BS is more than
twice the percentage of natural attenuation [13]. This pattern is the same for all research
in BSM, MSM, and seawater. It indicates that degradation performance increases with the
modification of conditions such as BA, BS and BA-BS. If optimal conditions prevail, this
efficiency may increase and take even less time than previous studies. The degradation
time and efficiencies in the above Table 7 varies with the type (light or heavy crude oil),
concentration of pollutant, and micro-organisms inoculated assisted with stimulators.
Researchers used BA and BS to treat crude oil polluted water using mixed microbial
cultures Aspergillus niger and Pseudomonas aeruginosa. Four samples of oil hydrocarbon-
polluted water were monitored for eight weeks using the following bioremediation tech-
niques: control (nutrient-free), A (nutrient NPK 15:15:15), B (nutrient-plus aeration), and
C (nutrient-free, aeration, and agitation). For the A, B and C samples respectively, reduc-
tions of TPH were 92.3%, 93.6% and 94.4%. The pH was within the range of 6–9 for all
samples [18]. Similar studies have been performed in the Gulf of Taranto (Italy) for the
actual oil spill sample. In April 2012, more than 20 metric tons of cargo fuel oil was dis-
charged by an unknown source, covering an area of about 800 m
2
. Approximately, 250 L
of oil-polluted seawater was collected and transported to a laboratory immediately after
24 h of the spillage. The research was conducted in a tank of size 62 cm × 40 cm × 30 cm
each and for 14 days. In order to compare NA, BS and BA-BS methods, 200 L of oily sea-
water was distributed in separate microcosms: (1) NA; (2) BS (nutrients: KH
2
PO
4
0.077
g/L, NH
4
Cl 0.2 g/L and NaNO
3
0.1 g/L); (3) BA-BS (consortium: Alcanivorax borkumensis,
Alcanivorax dieselolei, Marinobacter hydrocarbonoclasticus, Cycloclasticus sp. 78-ME and
Int. J. Environ. Res. Public Health 2021, 18, 2226 20 of 27
Thalassolituus oleivorans) and nutrients as in the BS treatment; (4) washing agent with oily-
seawater and nutrients as in the BS treatment. The degradation efficiencies for NA, BS and
BA-BS was 32 ± 3.2%, 73 ± 2.4%, and 79 ± 3.2% respectively [98]. Another study in seawater
was performed using tank experiments. In this study, seawater was lifted by direct pipe-
line from the Messina Strait. During the entire experimental phase, the seawater was aer-
ated and continuously stirred. The seawater was held at 18 ± 2 °C. The experiments were
performed in an 11,250 L (5000 cm × 150 cm × 150 cm) rectangular tank filled with 10,000
L of seawater. The experiments were performed in three separate tanks. BS (crude oil and
inorganic nutrients); BA1 (A. borkumensis SK2T); BA2 (A. borkuminsis SK2T + T. oleivorans
MIL-1B). In all experiments, sterile Arabic light crude oil (10 mg/L) and inorganic nutri-
ents were supplemented with seawater. The inorganic nutrients (sterile) were (final con-
centrations: KH
2
PO
4
0.077 g/L, NH
4
Cl 0.2 g/L and NaNO
3
0.1 g/L). The biodegradation
study found that the degradation of BA1 was the highest (95%) compared to BS (80%) and
BA2 (70%) [104]. These studies are yet to be evaluated under real on-site conditions as
indicated and proposed in this review by floating oil containment booms as a bioreactor
basin.
5. Conclusions
Physical and chemical oil spill clean-up methods are ineffective at completely clean-
ing up the petroleum hydrocarbons of oil spilled in seawater and are not capable of re-
moving emulsified oil left over after physico-chemical techniques have been applied.
The
complete removal of petroleum hydrocarbons oil by physical and chemical methods is not
achievable and there is remaining residual oil that can be treated with bioremediation.
The lack of guidance on the use of this technology is now one of the greatest challenges
for petroleum hydrocarbons oil bioremediation in marine waters. The possibility of bio-
remediation methods is a good and beneficial factor, where there is less water movement
in the area surrounded by water. It can be achieved by floating oil containment booms,
which are generally used to cover flowing water oil spills and to limit water movement
through the oil spills that generate a confined area. Bioremediation can be used in some
petroleum hydrocarbon polluted areas as a proven alternative clean-up/treatment method
in combination with floating oil containment booms to enclose the petroleum hydrocar-
bon polluted areas and act like a bioreactor basin. Several of the studies mentioned in the
article are laboratory-based studies that have the potential to be applied in the field (on-
site) and are still to be evaluated. This is an untapped area and has scope in the future. In
many of the studies mentioned in this article, micro-organisms are either isolated from
seawater or enhanced in seawater so that they can be used effectively in their native envi-
ronment (on-site). The biostimulants mentioned as low-cost substrates have a large po-
tential and have been proven in laboratory-based studies that can be used in petroleum
hydrocarbon remediation. BS and BA-BS techniques would lead to the use of agro-indus-
trial waste and to sustainable treatment. At the same time, two problems are resolved: the
pollution problem of oil spills treatment and the utilization of agro-industrial waste. The
disadvantages and difficulties that may be encountered in the use of these studies are
outlined in the future scope section of the article. It is difficult to mention all data from a
study in a table format. The outcomes of the studies are, therefore, shown for the primary
reference for bioremediation using BA,BS and BA-BS. Researchers may refer to the re-
quirements of the particular study referred to in this review paper based on their suitabil-
ity and use either BA, BS and BA-BS as a viable bioremediation technique in combination
with a booming technique to enclose the oil spill as in the bioreactor basin. Case studies
reviewed in this paper may help environmental researchers adopt an appropriate method
for the bioremediation of a petroleum hydrocarbon pollutant in seawater, estuaries, and
beaches for the cleaning of emulsified oil left over by using BA, BS and BA-BS methods.
Int. J. Environ. Res. Public Health 2021, 18, 2226 21 of 27
6. Future Scope
Due to the conditions discussed in this review paper, bioremediation (BA and BS
type) of open flowing water is not deemed appropriate. There is scope here to identify the
method or technology to be used (BA and BS type) for flowing water sources such as sea-
water and rivers. There are a few drawbacks of BA, BS and BA-BS as applied to moving
water bodies such as seawater and rivers. Some of these drawbacks can be overcome by
booms/barriers method as discussed in this review. The drawbacks are listed below:
Nutrients are instantly diluted in nearly background quantities which do not bind in
fresh or weathered hydrocarbons/oil, if nutrients are added to flowing water. It is
often difficult to collect or add nutrient substrates to oil spills, in windy and other-
wise adverse weather conditions, which cause waves.
In an oil spill pollution environment containing toxic oil, it is difficult to use addi-
tional nutrients for micro-organisms which eat hydrocarbons. From the beginning,
the toxicity of oil damages and/or kills several species native to the spill area. The
nutrients are typically prohibited from improving the other indigenous microbes be-
cause of the toxicity of oil.
However, it is a major problem to supply adequate amounts of deficit nutrients i.e.,
nitrogen and phosphorous, in an effort to increase the population of petroleum hy-
drocarbons degrading bacteria without raising the concentrations of nitrogen and
phosphorous to the amount that it is harmful to marine water life. The method of
improving indigenous organisms using nutrients and fertilizers is uncertain and
sometimes takes a long time, with the hope that there will be sufficient secretion of
biosurfactants, metabolites and enzymes to catalyze the bioremediation process. The
greatest challenge to the respondent is to create the right conditions for optimal bio-
degradation, i.e., to keep sufficient nitrogen and phosphorus concentrations in sea-
water always.
Normally, after conventional mechanical clean-ups, bioremediation is used as a pol-
ishing method. It takes weeks to complete the clean-up, which is quite slow. This can
be very cost-efficient if done correctly, although a detailed economic analysis has not
been carried out to date.
Author Contributions: Conceptualization, K. Sayed.; resources, K. Sayed.; writing—original draft
preparation, K. Sayed.; writing—review and editing, K. Sayed.; supervision, L, Baloo and N.K
Sharma. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Yayasan (YUTP), grant number 015LC0-152 and The APC
was funded by YUTP cost center: 015LC0-152.
Institutional Review Board Statement: Not applicable
Informed Consent Statement: Not applicable
Data Availability Statement: Not applicable
Acknowledgments: This research was supported and funded by Yayasan, Universiti Teknologi
Petronas, Malaysia (YUTP cost center: 015LC0-152). We are also grateful to the Institute for Self-
Sustainable Building, Civil and Environmental Engineering Department, Universiti Teknologi
Petronas, Malaysia to provide facilities required for performing literature review reported in this
paper.
Conflicts of Interest: The authors declare no conflict of interest.
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