CHAPTER II

REVIEW OF RELATED LITERATURE AND STUDIES

In this chapter of the study the researchers will discuss the related

literature. It is composed of facts and information from the other sites and

references which is relevant to the present study. This chapter will also

provide information related from a variety of studies that provide

explanation to further understand the study and investigatory project.

Oil spills have the potential to cause major environmental and

economic effects. The health of the marine organisms and humans on site

are at risk if an oil spill happens and it is not reacted upon quickly. Although

there are multiple methods on how to clean oil spills, most are either difficult

to do or not as effective as other methods.

Because of that, the researchers came up with an effective way to

improve biostimulation, a method to render bioremediation which is to clean

up oil spills called bioremediation, to be more effective and relevant.

 Related Literature

Bioremediation

Bioremediation is a process that uses mainly microorganisms or plant

enzymes. It is a process that includes biodegration, which is a partial or total

transformation or detoxification of contaminants by microorganisms and

plants. Bioremediation enhances the rate of the natural microbial

degradation of contaminants supplementing the on-site bacteria with

nutrients, carbon sources or electron donor by adding an enriched culture of

microorganisms that have specific characteristics that allow the on-site

bacteria capable of bioremediation to degrade the contaminant at a quicker

rate. (Surajit, 2014)

Biostimulation

Biostimulation involves the modification of the environment to

stimulate the bacteria capable of bioremediation. This can be done by adding

nutrients and electron acceptors such as phosphorus, nitrogen, oxygen or

carbon directly or contained in a medium. (Kanissery & Sims, 2011)

Bioaugmentation

Bioaugmentation is the process of introducing organisms to enhance

the existing population of bacteria present in the site to help reduce any

contaminats within the site. . (Artiola, et. al., 2004)

Pros and Cons of Bioremdiation

Bioremediation like unlike other methods for detoxification of a site, it

is not labor intensive or expensive, it can be used on-site, it can break down

the harmful components of petroleum, it is more environmentally-friendly

and it does not disrupt the site or the economy within the site. Although it

has a lot of pros, it also has some cons, one is the inability to produce a fast

response, therefore it cannot be used as a primary response, knowledge

about bioremediation is also needed to conduct any actions using

bioremediation, and each formula has to be specifically tailored for each site

since not all bacteria can be introduced to the polluted site. (Kiat, et. al.,

2013)

Oil

Oil is a liquid that is formed deep underground from the decomposition

of organisms over millions of years under heat and pressure. It is the world’s

most important fuel and underpins our high standard of living. It provides

convenience and freedom of movement as it is crucial to public and private

transport systems. Since the 1850s, oil has been produced commercially and

refined.

Oil Spills

Oil spills happen when oil floats on water and spreads out rapidly

across the surface, forming a thin layer called an oil slick, As the oil
continues spreading, the layer becomes thinner and thinner until it becomes

a very thin layer called a sheen. When an oil spill happens, the animals and

the actual body of water are harmed. Seabirds are also a victim, if oil gets

on a bird’s feather, they get heavier and if the bird tries to clean the oil out,

the bird may ingest it and get poisoned. Once oil has spilled, the federal

government and volunteer organizations respond to the incident, depending

on the situation, one out of a variety of methods is used manage the

incident.

Methods of cleaning the oil

Booms, which are floating barriers, may be placed around the oil to surround

and isolate it to a smaller space.

Skimmers, which are boats that skim or scoop the spilled oil from the water

can be used.

Sorbents are big sponges used to absorb oil.

Chemical dispersants and biological agents break down the oil into smaller

and smaller droplets for easier natural cleanup by the bacteria on that can

biodegrade the oil.

In situ burning is a method where freshly spilled oil is burned.

Vacuum trucks can vacuum spilled oil off of beaches or the water near the

surfaces of the shore

Shovels and road equipment can be used to separate the oil contaminated

sand from the clean sand and then the oil contaminated sand is cleaned

using a series of hoses and tumbling.

The method used is dependent on the event, the weather, the type and

amount of oil spilled, the distance from the shore, whether or not the area is

inhabited are factors used to determine what method is best for the incident.

Bacteria

Bacteria are single celled microbes. The cell structure has no nucleus

or membrane bound organelles. Instead their control center containing the

genetic information is contained in a single loop of DNA. Some bacteria have

plasmid, which is an extra circle of genetic material. The plasmid often

contains genes that give the bacterium an advantage over other bacteria.

For example, a plasmid may contain a gene that makes the bacterium

resistant to a certain antibiotic.

Types and Structure of Bacteria

Bacteria are microscopic organisms that have single cell. They can live

in different media. They can survive in an acidic environment, saline

environment etc. Depending on their types.

The structure of bacteria is very simple compared to eukaryotic cells.

It consist of cell wall that are made of peptidoglycan. Capsules are mainly
present in pathogenic bacteria, protecting the bacterium from the immune

system of the host. Flagella are responsible for the motility of the bacteria. A

bacterium can have one flagellum or a cluster of flagella.

Based on different survival conditions, bacteria are classified into two

different types. Eubacteria thrive in normal conditions. Archaebacteria thrive

in extreme conditions such as very high or low temperature. Bacteria can

also be classified based on Gram Staining. Gram-positive bacteria give a

positive result to the gram staining test indicating the presence of a thick

cell wall. Gram negative bacteria are the opposite, they do not give a result.

It can also be based on structure. Cocci are spherical in shape. Bacilli have

cylindrical or rod shaped structures. Spirilla are spiral shaped bacteria. Vibro

have the shape of a comma.

Nutritional Requirements of Cells

At the basic level, the nutritional requirements of a bacterium are

revealed by the cell’s elemental composition, which consists of Carbon,

Hydrogen, Oxygen, Nitrogen, Sulfur, Phosphorus, Potassium, Magnesium,

Calcium, Manganese and traces Zinc, Cobalt, Copper and Molybdenum.

These elements are found in the form of water, inorganic ions, small

molecules, and macromolecules which serve either a structural or functional

role in the cells.

 The major elements, their sources and functions in bacterial cells are

as goes:

Carbon. 50% of dry weight, comes from organic compounds or CO2, and

serves as the main component for the cellular material.

Oxygen. 20% of dry weight, comes from water, organic compounds, CO2

and O2, it also serves as a component for cell material, as well as cell water

and is an electron acceptor in aerobic respiration

Nitrogen. 14% of dry weight, comes from NH3, NO3, organic compounds

and N2, serves as component of amino acids, nucleic acids, nucleotides and

coenzymes.

Hydrogen. 8% of dry weight, comes from water, organic compounds and H2,

functions as the main component of organic compounds and cell water.

Phosphorus. 3% of dry weight, comes from inorganic phosphates, serves as

a component of nucleic acid, nucleotides, phospholipids, LPS and teichoic

acids.

Sulfur. 1% of dry weight, comes from So4, H2S, S0 and organic sulfur

compounds, serves as a component of cysteine, methionine, glutathione and

several coenzymes.

Potassium. 1% of dry weight, comes from Potassium salts, serves as the

main cellular inorganic cation and cofactor for certain enzymes.

Magnesium. 0.5% of dry weight, comes from Magnesium salts, also serves

as an inorganic cellular cation and a cofactor for certain enzymatic reactions.

Calcium. 0.5% of dry weight, comes from Calcium salts, also serves as an

inorganic cellular cation, a cofactor for certain enzymes and a component of

endospores.

Iron. 0.2%, comes from Iron salts, serves as component of cytochromes and

certain nonheme iron-proteins and a cofactor for some enzymatic reactions.

(Todar, 2008)

Related Studies

Extraction and Characterization of Oil Degrading Bacteria

The present study was conducted to determine the isolation,

identification and characterization of efficient oil degrading bacterial strains

and to study the effect of different concentration of crude oil on the growth

of bacterial strain. Two samples soil and water were collected from the crude

oil contaminated areas for the isolation and characterization of efficient

crude oil degrading strains. Sub culturing technique was employed to isolate

43 numerically dominant bacteria that had the ability to grow on 1.0% crude

oil on nutrient agar plates. The isolates were then subjected to different

concentrations of crude oil on nutrient agar, mineral salt agar media

containing Phosphorous, Nitrogen and trace elements with glucose (PNTG)

and without glucose (PER). These isolates showed rich growth on nutrient
agar media along with crude oil. Out of 43 isolates 7 were able to grow up to

2.0% crude oil and were named as AA-1 to AA-7. These strains were also

able to grow on mineral salt agar media with and without glucose but with

different susceptibility to different concentrations of crude oil. Finally 3

prospective strains AA-1, AA-2 and AA-3 were selected for further studies.

These strains exhibited good growth in PNTG containing 1.0% crude oil as

evident by increase in Optical Density (OD) after every 24 h for five days.

These isolated strains were identified by morphological and biochemical tests

and were found to belong to genus Bacillus. These strains were subjected to

shake flask transformation of crude oil in mineral salt media (PNTG) with

glucose for 15 days. Marked change in crude oil color was observed for these

isolates, indicating their biodegradative ability. These isolated strains were

able to use crude oil as the sole source of carbon and energy even under

stressed environmental conditions. Thus these strains have bright potential

for biodegradation of crude oil resulting in cleanup of oil spills. (Asif, et. al.,

2006)

Hydrocarbon‐degrading bacteria: the oil‐spill clean‐up crew

Around three million tons of oil enters the sea each year, of which

about 20% originates from oil‐pumping operations, transport and refining

activities and 25% from non‐tanker shipping and natural seepages. More

than half originates from illegal activities that include the dumping of ballast

water and oil residues as well as accidents . Hydrocarbons are also produced
continuously by living cells as natural oils and fats . The observation that the

oceans are not covered with an oily layer is a testimony to the activity of the

hydrocarbon‐degrading microorganisms .

Several bacteria are even known to feed exclusively on hydrocarbons .

For these hydrocarbon degraders the occasional supertanker oil spill forms

an occasional carbon banquet. They play an important role in the clean‐up

after an oil spill and form the biological basis for the natural oil‐degrading

capacity of the ecosystem. Studies have focused on identifying and

characterizing these oil‐eating microbes, as well as how they cope with the

oil/water interface, and how to improve this capacity.

Degradation of hydrocarbons

Specifically in marine environments, low phosphorous and nitrogen

levels may limit growth of oil‐degrading microorganisms and thus rapid oil

consumption. In open‐sea environments, dilution of soluble nutrients quickly

occurs, and the administration of insoluble or hydrophobic fertilizers is

thought to enhance biostimulation effectiveness. Biosurfactants seem

another promising form of biostimulation. Biosurfactants increase the oil‐

surface area and with that the amount of oil actually available for attack by

bacteria.

Depending on the particular local circumstances, tilling of beaches may

be applied to expose the sequestered oil. However, tilling itself is disruptive

for many coastal plants and animals and, as the oil was beyond the reach of

the biota in the first place, may do more harm than good. Local conditions,

in general, have a big effect on the efficiency of oil breakdown, such as

temperature, waves, availability of oxygen, and of course the composition of

the spilled oil. These should be taken into account when devising an

intervention strategy.

Diversity and metabolism of oil‐degrading bacteria

A great number of bacteria have been identified that help clean up the

hydrocarbon compounds in the aftermath of oil spills. Evolution has created

some bacteria that dine exclusively on hydrocarbons, including obligate

hydrocarbon degraders of the genera Oleispira, Oleiphilus, Thalassolituus,

Alcanivorax and Cycloclasticus. (Brooijimans, et. al., 2009)

Hydrocarbon-Degrading Bacteria and the Bacterial Community

Response in Gulf of Mexico Beach Sands Impacted by the Deepwater

Horizon Oil Spill

The objectives of this study were to identify and characterize

predominant oil-degrading taxa that may be used as model hydrocarbon

degraders or as microbial indicators of contamination and to characterize the

in situ response of indigenous bacterial communities to oil contamination in

beach ecosystems. This study was conducted at municipal Pensacola Beach,

FL, where chemical analysis revealed weathered oil petroleum hydrocarbon

(C8 to C40) concentrations ranging from 3.1 to 4,500 mg kg−1 in beach

sands. A total of 24 bacterial strains from 14 genera were isolated from oiled

beach sands and confirmed as oil-degrading microorganisms. Isolated

bacterial strains were primarily Gammaproteobacteria, including

representatives of genera with known oil degraders (Alcanivorax,

Marinobacter, Pseudomonas, and Acinetobacter). The abundance of bacterial

SSU rRNA gene sequences was ∼10-fold higher in oiled (0.44 × 107 to 10.2

× 107 copies g−1) versus clean (0.024 × 107 to 1.4 × 107 copies g−1)

sand. Community analysis revealed a distinct response to oil contamination,

and SSU rRNA gene abundance derived from the genus Alcanivorax showed

the largest increase in relative abundance in contaminated samples. The

researchers conclude that oil contamination from the DH spill had a profound

impact on the abundance and community composition of indigenous bacteria

in Gulf beach sands, and our evidence points to members of the

Gammaproteobacteria (Alcanivorax, Marinobacter) and Alphaproteobacteria

(Rhodobacteraceae) as key players in oil degradation there. (Kostka, et. al.,

2011)

Oil Degrading Microorganisms

These microorganisms metabolize oil and it's product as a source of

carbon and energy. The microorganisms were selected from soils where oil

had been spilled and from contaminated fuels. The change in composition in
the hydrocarbon feed due to the microbial activity was analyzed by gas

chromatography. (Rodrigues, et. al., 1996)

Bioaugmentation for Enhancing Biological Wastewater Treatment

In laboratory investigation, bioaugmentation usually failed, whereas at

full scale it was often successful, probably due to the imposition of steady

state at laboratory scale. (Stephenson & Stephenson, 1992)

Comparative Studies of Biostimulation and Phytoremediation in the

Mitigation of Crude Oil Toxicity in Tropical Soil

Relative investigations of biostimulation utilizing nearby materials and

phytoremediation in the moderation of unrefined poisonous quality on

tropical soil of the Niger-Delta of Nigeria were done. 5% contamination

dimension of unrefined petroleum were done nearby the control.

Remediation medicines were done following multi week of contamination

utilizing the accompanying materials: Batch A : 20g of NPK 15:15:15; bunch

B: 50g of poultry manure; clump C: 5 seeds of Vigna unguiculata developed

on the contaminated soil (phytoremediation); group D: control

(contamination however no remediation); and bunch E: twofold control (no

contamination and no remediation treatment). Soil examinations result

demonstrated that the diverse remediation medications have possibilities to

improve unrefined petroleum poisonous quality at various degrees on

account of their capacity to build the supplement substance and reduction

the aggregate hydrocarbon substance of the dirt. There was no factual

contrast in the dirt pH, Zinc and Copper substance between and inside

medicines all through the examination time frame. Most astounding raw

petroleum biodegradation and enhancement in supplement substance of the

dirt were seen in NPK 15:15:15 and phytoremediation medicines

demonstrating that these are great therapeutic treatment alternatives in the

alleviation of raw petroleum poisonous quality. (Tanee, Kinako, 2010)

Bibliography

Das, Surajit, ed. MICROBIAL BIODEGRADATION AND BIOREMEDIATION.

S.l.: ELSEVIER, 2017. 2014. Accessed November 15, 2018.

Kanissery, Ramdas Gopinath, and Gerald K. Sims. "Biostimulation for the

Enhanced Degradation of Herbicides in Soil." Applied and Environmental Soil

Science 2011 (2011): 1-10. Accessed November 15, 2018.

doi:10.1155/2011/843450.

Artiola, Janick F., Ian L. Pepper, and Mark L. Brusseau. Environmental

Monitoring and Characterization. Amsterdam: Elsevier. Academic Press,

2018.

Kiat, Chun, Samuel Nicholas, Han Jian, and Jenny. "Types of Bioremediation;

Pros & Cons of Bioremediation." Bioremediation of Oil Spills. August 01,

2013. Accessed November 15, 2018. http://embt-assignment-

blog.tumblr.com/post/57083798567/types-of-bioremediation-pros-cons-of.
"What Is Oil?" APPEA | Oil and Gas in Everyday Life. Accessed November 15,

2018. https://www.appea.com.au/oil-gas-explained/oil-and-gas/what-is-oil/.

"How Do Spills Happen?" Exxon Valdez Oil Spill. Accessed November 15,

2018. https://response.restoration.noaa.gov/training-and-

education/education-students-and-teachers/how-do-spills-happen.html.

"About Microbiology – Bacteria." Microbiology Online. Accessed November

17, 2018. https://microbiologyonline.org/about-microbiology/introducing-

microbes/bacteria.

Todar, Kenneth. Impact of Microbes on the Environment. 2008. Accessed

November 17, 2018. http://textbookofbacteriology.net/nutgro.html.

"Types and Structure of Bacteria." ScienceAid. Accessed November 18,

2018. https://scienceaid.net/Bacteria.

Khalida Khan, M. Naeem, M. Javed Arshed, and M. Asif. "Extraction and

Characterization of Oil Degrading Bacteria." Journal of Applied Sciences 6,

no. 10 (2006): 2302-306. Accessed November 18, 2018.

doi:10.3923/jas.2006.2302.2306.

Brooijimans, Rob J. W., Margreet I. Pastink, and Roland J. Siezen.

"Hydrocarbon‐degrading Bacteria: The Oil‐spill Clean‐up Crew." Microbial

Biotechnology 2, no. 6 (November 2009): 587-94. Accessed November 18,

2018. doi:10.1111/j.1751-7915.2009.00151.x.
Kostka, Joel E., Om Prakash, Will A. Overholt, Stefan J. Green, Gina Freyer,

Andy Canion, Jonathan Delgardio, Nikita Norton, Terry C. Hazen, and Markus

Huettel. "Hydrocarbon-Degrading Bacteria and the Bacterial Community

Response in Gulf of Mexico Beach Sands Impacted by the Deepwater Horizon

Oil Spill." Applied and Environmental Microbiology 77, no. 22 (2011): 7962-

974. Accessed November 18, 2018. doi:10.1128/aem.05402-11.

Rodrigues, E.g.c., E.p. Menezes, L.m.m. Santa Anna, R.l. Vital, and G.v.

Sebestian. "Oil Degrading Microorganisms." International Biodeterioration &

Biodegradation 37, no. 1-2 (1996): 119. Accessed November 18, 2018.

doi:10.1016/0964-8305(96)84329-0.

Stephenson, Dianne, and Tom Stephenson. "Bioaugmentation for Enhancing

Biological Wastewater Treatment." Biotechnology Advances 10, no. 4

(1992): 549-59. Accessed November 18, 2018. doi:10.1016/0734-

9750(92)91452-k.

Tanee, Fbg, and Pds Kinako. "Comparative Studies of Biostimulation and

Phytoremediation in the Mitigation of Crude Oil Toxicity in Tropical Soil."

Journal of Applied Sciences and Environmental Management 12, no. 2

(2010). Accessed November 18, 2018. doi:10.4314/jasem.v12i2.55552.