BATANGAS STATE UNIVERSITY

COLLEGE OF ENGINEERING, ARCHITECTURE, and FINE ARTS

Mechanical and Petroleum Engineering Department

BIOTECHNOLOGY: SULFOLOBUS ACIDOCALDARIUS AS AN EFFECTVE

DESULFURING MICROBE AND BIOCATALYST FOR HEAVY OIL
CONTAINING DRILLING WELLS

A Research Project
Presented to Dr.
Sicily Tiu

In Partial Fulfillment of the Requirement

for Research Project in Modern Biology

BAGUI, CLARK JESTER

ILAGAN, MAUI JULLIAN
LLANOS, GIOVANNY EARL
VARGAS, MHYKEL BALDWIN

March 2018
 CHAPTER 1

INTRODUCTION

The use of oil and gas has become a widely need in human’s life. From simple

household items, medical use, and for transportation needs, oil and gas has been

well applied and became a necessity in human’s existence. According to the

Wintershall, Germany’s largest internationally active producer of crude oil and

natural gas, oil, gas and gas condensate are among the most important energy

sources in the world. One thing people know for sure is that oil is a necessity -

currently around 90 million barrels of crude oil a day. But, gathering these sources

are not as basic as how people use it. In the Bureau of Labor and Statistics, they

defined Petroleum Engineers as the one who design and develop methods for

extracting oil and gas from deposits below the Earth’s surface. Petroleum engineers

also find new ways to extract oil and gas from older wells. In line with this, they

must insure the safety of the operation and the environmental protection must

always be the forefront of the drilling design. Various techniques and different

technology is being applied in drilling oil and gas to recover a huge percentage of its

portion in wells.

Through the improvement of technology, many paths were opened for the

betterment of oil recovery. To have sustainable source is the foremost goal of oil and

gas industries to maintain the stability of oil supply. Thus, transformation of heavy

oils into light oils helped the industry to turn produce higher quantity, usable oil.

But, processing heavy oil into lighter ones needs to go through several processes
and stages to turn it to more productive oil. Other than that, some oil contains

different materials which are excluded in the process. In this case, removal of these

materials is needed.

Sulfur is the most abundant element in petroleum after carbon and hydrogen. Crude

oils with higher viscosities and higher densities usually contain higher amounts of

more complex sulfur compounds. In a statistical analysis stated in the “23 rd edition

of Marsh’s The 100 Largest Losses,” from 1999-2013, about US130$ million loss was

recorded due to explosions from petrochemicals, refineries and blow outs from oil

rigs. These resulted from corroded equipments such as pipe lines and various

mishandled oil containing tanks which caused leakages. This certifies the refining of

heavy oil to final products requires desulfurization of oil. Many petrochemicals

products are likewise produced to be almost sulfur free. Desulfurization in oil is

probably one of the central conversion requirements in most refineries and the

price of the crude oil is influenced by its sulfur content. Several processes have been

accompanied in removing sulfur in oil. Desulfurization is usually discussed in terms

of sulfur removal from lighter refinery cuts, such as naphtha, distillate, and light

vacuum gas oil fractions. Hydro desulfurization (HDS) accounts as one of the most

commonly used process in desulfuring oil. It is a technology capable of desulfuring

oil but their carbon footprints are substantial. The said technology involves high

temperature processing. In the sense of refining oil, the cost in these technology

increases as heavier and more sulfur-rich crude oils are being processed. Another

technique of desulfurization is through the means of adsorption. The method

includes sorbent materials such as activated carbon, zeolites,, amorphous silica-

aluminas, and metallic organic framework (MOF) were evaluated in desulfurization

of model oils but in spite of the probability to desulfurize oil under mild conditions,

the performance of even the most efficient of the adsorbents is still insufficient for

industrial applications. Thus, this leaded the researchers to step forward into a

more efficient way of desulfuring oil through the application of microorganisms by

the means of biotechnology.

Biotechnology branches out into different application through the means and use

of microorganisms. In the oil industry, various microorganisms were used as an

enzyme to act as a biocatalyst in oil refining. Biocatalysts increase the rate of

chemical reaction in a compound and have a possibility to remove sulfur from

petroleum. According to the St. Louis. Energy BioSystems Corp. (EBC) in Houston,

Texas, certain enzymes could remove the sulfur from petroleum as it is the first

plant to use such system and termed it as Biocatalytic Desulfurization. Moreover,

certain microorganisms can act as a biocatalyst to decrease the viscosity of oil to

increase the flow rate and rate of oil recovery in heavy oil containing wells.

As such, the researchers come up with the research topic “c,” that sought to

identify the effectiveness of Sulfolobus acidocaldarius, a thermoacidophilic

crenarchaeon that grows optimally at 75-80 °C. as a desulfuring microbe and an

effective Biocatalyst. The researchers hypothesize the effectiveness of the bacteria

among other commonly used thermophilic microbes such as Pseudomonas and

Achromobacter. This would be of big help among oil rich countries and industries
that conducts oil refining as projected action plans will be procured or implemented

as a part of the research process by the future researchers.

STATEMENT OF THE PROBLEM

In the published “23rd edition of Marsh’s The 100 Largest Losses,” with the use of

Nelson-Farrar Petroleum Plant Cost Index, which allows for an easy comparison of

property damage on a constant basis across the period analyzed, it was shown that a

total of accumulated value of the 100 largest losses is over US$34 billion dating

1974-2013. The two highest contributors is the Upstream which covered the 34% of

loss due to explosions from corrosions of pipelines, other equipments, and human

error and 29% comes from the refining industries with the same cause from the

latter part. This does not yet include the loss of oil companies from addressing

techniques to recover oil and costly desulfurization processes.

The Statement of the Problem suggests the possible questions that could be a

guide in answering the conducted study about the “Biotechnology: Sulfolobus

acidocaldarius an an Effective Desulfuring Microbe and Biocatalyst in Heavy Oil

Containing Wells”.

Specifically, the researchers sought to answer the following questions:

1. What type of microbiological screening will be used to test the property of

Sulfolobus acidocaldarius

2. What is the rate of effectiveness of Sulfolobus acidocaldarius as a desulfuring

microbe?
3. What is the rate of effectiveness of Sulfolobus acidocaldarius as a biocatalyst

Null Hypothesis

Sulfolobus acidocaldarius is not an effective desulfuring microbe and biocatalyst

for heavy oils as compare to the usual desulfuring microbes used (Tested at 0.05

Level of Significance)

Significance of the Study

The study focused mainly on the determination of effectiveness of Sulfolobus

acidocaldarius an an Effective Desulfuring Microbe and Biocatalyst in Heavy Oil

Containing Wells. Collection and preparation of materials is done for preliminary

tests of Sulfolobus acidocaldarius.

The Oil Industries By studying the “Biotechnology: Sulfolobus

acidocaldarius as an Effective Desulfuring Microbe and Biocatalyst in Heavy Oil

Containing Wells,” the researchers could propose specific action plans to lessen loss

of oil refineries from using various techniques to desulfurize heavy oil that would be

executed by the future researchers.

The Community By identifying the most effective microbe, the

researchers could lessen the loss of earnings from different sectors from different

oil companies. The researchers assume that the oil price would decrease as the

process of recovery and refining of oil becomes efficient. Thus, it would positively

affect the consumers by which oil products would be not costly.

The Researchers In application to Petroleum Engineering, the field of

researchers, the accomplishment of the research might help the proponents to solve

the existing problems within desulfuring heavy oils in oil refineries. Such research

would help the proponents to have more idea and learning about the processes and

might as well serve as an initial step to future opportunities waiting for the said

proponents.

Future Researchers This research could serve as a resource of

information for further studies conducted by other researchers. The

recommendations include the application of the conducted research to the target

proponents that would be executed by the future researchers.

SCOPE AND LIMITATION

This study is conducted to identify the “Biotechnology: Sulfolobus acidocaldarius

an Effective Desulfuring Microbe and Biocatalyst in Heavy Oil Containing Wells.” The

limitation of the study would be to the certain strain of microbe which Sulfolobus

acidocaldarius, extremophile bacteria that is to be tested in the University of the

Philippines Los Banos. The bacteria will be tested under controlled supervision and

conditions. Any information gathered will be reliable in significance to the research.

Conceptual Framework
Independent Variable Dependent Variable

Sulfolobus
acidocaldarius as a
desulfuring microbe
Heavy Oils as an
effectiveness indicator of
Sulfolobus acidocaldarius as a
desulfuring microbe and
biocatalyst

Sulfolobus
acidocaldarius as a
biocatalyst

Figure 1. Research Paradigm of the Study

The research paradigm shows the independent and dependent variables being

used. S. acidocaldarius will be the independent variable and the dependent variable

will be the heavy oil because it is the changing variable or indicator of the

effectiveness of the independent variable.

DEFINITION OF TERMS
Biocatalysts –Microbes or enzymes that speed up the chemical reaction of a certain

compound

Biotechnology- is an application of living organisms into the technology. It is now

considered as an effective and efficient way of solving solution without totally

harming the environment

Desulfurization- is the removal of sulfur to certain compounds. It is used in oil

refineries and petrochemical industries to ensure the quality and usability of the io

products that would be produced. With this process, corrosion, less oil yield, and etc.

would be mitigated.

Hydro desulfurization- is the usual used process in desulfuring heavy oils. It

requires the use of technologies that is certainly effective method but has a huge

impact in the environment because of its emission of too much carbon in the

atmosphere.

CHAPTER 2
 REVIEW OF RELATED LITERATURE AND STUDIES

This section will provide the related literatures and related studies that are used

in proving significant data for the “Biotechnology: Sulfolobus acidocaldarius an

Effective Desulfuring Microbe and Biocatalyst in Heavy Oil Containing Wells.”

Related Literature

Oil refining comes with the process of desulfurization or the removal of sulfur in

heavy oils to produce more usable oil. Strategies for heavy oil desulfurization is

critically observed for the better recovery and for maximum produce of various oil

products. Desulfurization methods for heavy oil include hydrodesulfurization,

extractive desulfurization, oxidative desulfurization, biodesulfurization and

desulfurization through alkylation, chlorinosis, and by using superficial water

(Javadli, 2012). Some of these processes stated above produce a viable or efficient

outcome but literally costly that pushed oil industries to discover other methods for

desulfurization.

Hydrodesulfurization (HDS)

Hydrodesulfurization (HDS) method is the commonly used way in desulfuring oil.

HDS is an efficient but costly process in terms of return of investments and

environmental impacts. With higher carbon footprint, a lot of environmental

challenges might be faced in the process. Such knowledge is of extreme importance

in considering the so called green house effect those results from the burning of
fossil fuels and the subsequent collection of the carbon dioxide in the atmosphere

(Speight, 2017).

Many countries set sulfur content limits such as in Europe with 10ppm and 15ppm

in US with a goal of <1ppm in the near future (Martinez et. al, 2015). The removal of

sulfur contributes a huge effect in the quality of oil products. In oil refineries,

exposure of equipments in sulfur results from corrosion which in the latter part

would cause great danger in the vicinity.

Biotechnology -Biocatalysis in Oil Refining

The application of biotechnology is now of a trend in the petroleum industry.

Many oil industries use microorganisms as enzymes for oil transformation to a

usable one. Biocatalysts are capable of selective sulfur removals without significant

conversions of the components in the fuel are desirable. Moreover, Biocatalytic

processes generate novel useful materials in the petrochemical area (Corredore &

Borote, 2007). With the help of enzymes or microbes, transformation of oil into

usable product would increase its rate. For biocatalysts involves in biological

removal of sulfur (Corredore & Borote). It is seemingly of note that microbial

desulfurization is becoming a recognized technology for desulfurization (Speight,

2017).

Sulfolobus acidocaldarius
Sulfolobus acidocaldarius or S. acidocaldarius is a thermophilic and acid based

bacteria that grows from 70 – 80 degree Celsius and a pH of 2-4 from various hot

springs and mud spots. With regards to oil refining, operation of desulfurization

process at high temperature and low ph values reduce the chance of contamination

and the reaction medium (Speight, 2017).

In the Philippines, challenges are forwarded in oil refineries when it comes to

desulfuring heavy oils. With higher sulfur content from heavy oils, companies must

address the techniques to desulfurize oil in an inexpensive manner. Philippine oil

refineries use HDS which has a lot of disadvantages. From being costly and does not

work well on refractory organosulfur, oil price hikes. Following the trend,

Biodesulfurization method (BDS) has come on its way in the Philippines (Mohebali,

2008). With BDS, it operates at ambient temperature and pressure with high

selectivity, resulting in decreased energy cost, low emission, and no generation of

undesirable side products As of now, Philippines is still attempting to isolate

bacteria capable of efficient desulfurization of oil fractions(Mohebali, 2018).

Related Studies

In a recent study conducted in Biodesulfurization of Petroleum by free cell

microorganisms, certain bacteria such as M. goodi X7B and R. erythropolis FSD-2

showed a corresponding 86% reduction in desulfuring diesel oil. After the

biotreatment, 84% of the sulfur content of distillation diesel oil, from 666 to 105

ppm, and 94% of that HDS- treated diesel oil, from 198 to 11 ppm, was removed in

two consecutive reactions. These results expressed a positive output with a

desulfurization rate increased by 35% when 0.5% Tween 80 was added. These

results were more noteworthy because the diesel oil had been treated by HDS so

that only organosulfur compounds that were relatively recalcitrant to HDS

remained. Furthermore, the methods for the preparation of a biodesulfurization

biocatalyst were also investigated to reduce the biodesulfurization cost.

Recent Biotechnology trend

In the “Petroleum biotechnology: Technology trends for the future” review, it has

been stated that up to 2030, an expected of 107 million barrels of oil will be

produced however, there is a decrease of it for the following year because of the

increase in heavy oil. Thus, biotechnology is pathway to mitigate these future

effects. With biotechnology, there is an estimated of 30% of oil should be

desulfurized. Biodesulfurization serves as an alternative for HDS that is expected to

play an important role in bio refining of oil products wth the presence of microbes

and enzymes.

Preliminary results from Oak Ridge national laboratory showed that a certain

thermophilic bacteria, Pseudomonas aeruginosa EGSOX desulphurized 95% of a

solution containing 6.4 ppm sulphur in 24 hr.

Furthermore, a comparative study involving BDS and HDS,, developed by Enchira

Biotechnology Corp. (EBC, formerly Energy BioSystems), demonstrated the minor

energy consumption and CO2 generation by the bioprocess to produce a 50 ppm

sulphur in diesel from two diesel pools containing 2000 and 50ppm sulphur.
Sulfolobus acidocaldarius

In a research conducted by the BioSystems Corp. , S. acidocaldarius was used to

desulfurize coal. The desulfurization process presented a 90% reduction of pyrilic

sulfur.

SYNTHESIS

Various processes where stated above to address the problem in resolving

desulfurization using an efficient and inexpensive method. It is proven that HDS

produce an efficient outcome when it comes to desulfuring heavy oil with a resulting

of 94% in sulfur reduction but is costly. Similarly, it is stated in the studies that

organosulfur compounds are irremovable in the process of HDS. The literature and

studies showed a similar difference which is BDS is more effective than HDS. With

the use of certain microorganisms, decrease in sulfur in diesel oil was proven

effective. With the literature and studies gathered, the researchers hypothesized

that the S. acidocaldarius used to desulfurize coal, is also an effective biocatalyst and

desulfuring microbe in heavy oil compared to other common microbes used (i.e.

Pseudomonas)
 CHAPTER 3

METHODOLOGY AND PROCEDURES

This section dealt with the research methodology employed in the study. In this

chapter, main sections contribute to the description of the research methodology.

RESEARCH DESIGN

Methods of Research Used

The proponents have used the Experimental Research Method wherein the study is

focused on examining the effect of independent variable, where the independent

variable is manipulated through treatment or intervention(s), and the effect of those

interventions is observed on the dependant variable. Both variable used in the

method will be under controlled conditions. The method is effective in determining

the effectiveness of Sulfolobus acidocaldarius as a desulfuring microbe and

biocatalysts for heavy oil.

RESEARCH LOCALE

The study will be conducted using a strain of microbe collected from hot spots

located near Mt. Makiling in Makiling, Laguna. The certain strain will be gathered in

the said vicinity because it is located near the University of the Philippines Los

Banos where the strain would be tested.

DATA GATHERING AND PROCEDURES

To identify the characterization of thermophilic bacteria, the microbe will

undergo DNA isolation and characterization. The method used in identifying the

bacteria is Polyemerase Chain Reaction (PCR) (method will be discussed in results

and discussion). Once the bacteria is identified as S. acidocaldarius, it will undergo

certain methods to identify its effectiveness as a desulfuring microbe and biocatalyst

for heavy oils.

The research is aimed at isolating and applying the bacteria for the removal of

H2S. The bacteria with the ability to oxidize thiosulfate will be isolated from heavy

oil and contaminated soil with sulfur compounds and be identified by physiological,

biochemical, morphological, and genetic techniques. The removal of thiosulfate and

production of sulfate will be measured by chromatography, a sensitive method, with

conductivity detection.

STATISTICAL TREATMENT AND ANALYSIS

Simple sample T-test will be used in order to identify the effectiveness of S.

acidocaldarius as a desulfuring microbe and biocatalyst. The results gathered from

the experiment/laboratory results will be analyzed and compared to a fixed value

from different commonly used bacteria in desulfuring heavy oils.

 BIBLIOGRAPHY

Adams, C. D., Scanlan, P. A., and Secrist, N. D. (1994). Oxidation and biodegradability
enhancement of 1,4-dioxane using hydrogen peroxide and ozone. Environ.
Sci. Technol. 28, 1812–1818. doi: 10.1021/es00060a010

Adegboye, M., and Babalola, O. (2013). “Actinomycetes: a yet inexhaustive source of

bioactive secondary metabolites,” in Microbial Pathogens and Strategies for
Combating Them: Science, Technology and Education, Microbiology Book
Series, ed. A. Mendez-Vilas (Badajoz: Formatex),786–795

Anteneh Y. S., and Franco C. M. (2019) “Whole Cell Actinobacteria as Biocatalysts”

Retrieved from: https://doi.org/10.3389/fmicb.2019.00077

Babu, V., Shilpi, and Choudhury, B. (2010). Nitrile-metabolizing potential

of Amycolatopsis sp. IITR215. Process Biochem. 45, 866–873. doi:
10.1016/j.procbio.2010.02.008

Beam H. W., and Perry J. J. (1999) “Microbial degradation of cycloparaffinic

hydrocarbons via cometabolism and commensalism”. Journal of
General Microbiology

Casati D. F., Franjevic D., Bobek J. (2019) “Actinobacteria, a Source of Biocatalytic

Tools” Retrieved from: https://www.spotlight.frontiersin.org

Izumi, Y., and Ohshiro, T. (2001). Purification and characterization of enzymes

involved in desulfurization of dibezothiophene in fossil fuels. J. Mol. Catal.
B Enzym. 11, 1061– 1064. doi: 10.1016/S1381-1177(00)00063-1

Van der Heul R. M., (2009) “Environmental degradation of petroleum

hydrocarbons”. Utrecht university.
(http://studenttheses.library.uu.nl/search.php?language=en&qry =Envir on
mental+Degradation+of+petroleum+hydrocarbons)

Van Beilen J. B. and Funhoff E. G., (2005) “Expanding the alkane oxygenase toolbox:
new enzymes and applications”. Current Opinion in Biotechnology).

Weng Lou (2019) “Advances in Enzyme technology” Retrieved from:

https://pdfs.semanticscholar.org/5515/3033100ef5dc816f6d8251ba83e44
330be9 b.pdf