Proposal Screening of Hydrocarbon-Utilizing Bacteria With Mercury-Resistance Properties

8/8/2019 Proposal Screening of Hydrocarbon-Utilizing Bacteria With Mercury-Resistance Properties

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1.0 Title

Screening of Hydrocarbon-Utilizing Bacteria with Mercury-Resistance Properties

2.0 Problem Statement

There are limitations in oil biodegradation. One of them is the presence of heavy metals, especially

mercury. Elemental mercury and mercury compounds occur naturally in geologic hydrocarbons

including coal, natural gas, gas condensates and crude oil. And these traces of mercury will leak together

to the environment with the oil through any sort of oil leakage or oil waste in any level (EPA, 2001).

Mercury can bind and inactivate essential thiols that are part of enzymes and proteins (Bruins et. al.,

2000), thus posing a problem in biodegradation of oil. There is a suppressed degradation of oil and an

enhanced toxicity because of the partitioning of the mercury in the oil phase (Walker and Colwell, 1976). According to Sorkhoh (2004), the oil attenuation efficiency decreases relative to higher concentration of

mercury chloride. Therefore there is a need to identify strains of bacteria that are able to degrade oil

while able to withstand the toxicity of mercury.

3.0 Objectives

The main objective of this study is to screen and identify hydrocarbon-utilizing bacteria that are

resistant towards mercury. Other objectives of this study are as follows:

1. To study the oil attenuation potential of the bacteria isolated

2. To study the level of mercury resistance of the bacteria isolated.

3. To study the correlation between the hydrocarbon-utilizing and mercury-resistance capabilities

of the isolated bacteria.



4.0 Introduction

Oil released into the environment is a well-known problem today. Oil spills affect many living

creatures in the environment, including humans. The search for effective and efficient methods of

oil removal from contaminated sites has intensified in recent years. One promising method that has

been researched is the biological degradation of oil by bacteria. While human takes on rice and

wheat as our carbon source, some bacteria have the ability to metabolize the carbon-rich oil in

much the same way humans convert food into energy.

Nevertheless, there are confines in oil biodegradation. One of them is the presence of heavy

metals, especially mercury. Elemental mercury and mercury compounds occur naturally in geologic

hydrocarbons including coal, natural gas, gas condensates and crude oil. 65% of mercury release in

the environment is from stationary combustion, of which coal-fired power plants are the largest

aggregate source. This includes power plants fueled with gas where the mercury has not been

removed. Leakage of oil, especially crude oil, would mean leakage of mercury as well.

There were studies had been done and published on oil-degrading and mercury-resistant

bacteria all around the world. There are places such as Minamata Bay in Japan, Chesapeake Bay and

Onodaga Lake in United States, and the desert in Kuwait which are rich in bacteria with such

properties since they are highly contaminated with mercury and oil. However, in Malaysia there

were very few studies on mercury, especially on the relation between mercury and oil

biodegradation. Studies were typically done by oil and gas companies which mostly only included

technical and engineering approach in removing mercury from crude oil since mercury in crude oil

above certain limits can be problematic to refining operations. Nevertheless, in environmental

management point of view, environmental impacts are of utmost importance because running

mercury-laden crudes can produce wastewater and solid waste streams having mercury

concentrations that exceed regulatory limits. And this also contributes to the high mercury content

in oil-contaminated sites which would inhibit microbial biodegradation of oil.

Minuscule amount of mercury may not have effect on microbial metabolism, but higher

concentration of this element would definitely be an inhibitor to most microorganisms. Therefore

this study is proposed to screen for bacteria that are resistant towards mercury, and at the same

time have the ability to effectively remediate the oil.



5.0 Importance of Study

The findings in this study may take oil biodegradation to another level where the effectiveness of

the degradation may be enhanced greatly. This will also facilitate the in situ bioremediation process

such as bioaugmentation. Strains of bacteria that are resistance towards heavy metals such as

mercury would be highly useful for many purposes. Gene recombinant technology makes it possible

to isolate and reinsert this mercury-resistance gene into existing strains of bacteria that lack such

property. Most importantly, combined potential of hydrocarbon utilization and mercury resistance

in bacteria is highly valuable for bioremediation process of such polluted environments. It is

important in oil and gas industry today which emphasizes on environmental sustainability.

6.0 Literature Review

Accidental leakages during hydrocarbon fuels transportation and other activities are inevitable,

making these hydrocarbons the most common global environmental pollutants (Ganesh and Lin,

2009). The removal of oil is connected with microbial degradation (Groudeva et. al., 2000).

However, the fact that hydrocarbons persist for months and even years following major oil spills

indicates that hydrocarbon biodegradation is slow in most natural environments (Rosenberg, 2006;

Bento et.al., 2005), therefore there is a need to level up the effectiveness of oil biodegradation.

Remediation of aafected areas with the use of microorganisms can offer a cost effective solution for

restoring the ecosystem and can ensure clean groundwater supplies (Mukherji et.al., 2004; Bento

et.al., 2003). Oil-degrading bacteria are generally present in most soils. However, the best place to

collect soil is from an area already contaminated with oil (Mukherji et al., 2004).

Most metals are nonessential and are potentially toxic to microorganisms. At high level, both

essential and nonessential metals can damage cell membranes, alter enzyme specificity, disrupt

cellular functions, and damage the structure of DNA (Bruins et. al., 1999). According to him as well,

mercury is especially toxic because it binds to and inactivates essential thiols that are part of

enzymes and proteins. Studies by Walker and Cowell (1974) and Sorkhoh et.al. (2009) also showed

that the presence of mercury somehow greatly reduced the oil degradation effectiveness of

bacteria.



Mercury-resistant bacteria comprise many genera well known for their hydrocarbon utilization

potential (Misra, 1992). Some bacteria contain a set of a Hg(II) (mer ) resistance operon (Caslake

et.al., 2005). This operon not only detoxifies Hg(II) but also transports and self-regulates resistance

(Misra, 1992). According to Bruins (2000), this same set of genes also encodes the production of a

periplasmic binding protein and membrane associated transport proteins. The periplasmic binding

protein collects Hg (II) in the surrounding environment and transport proteins, and take it to the

cytoplasm for detoxification. A study by Caslake et.al. (2005) also suggested that mercury resistance

bacteria may potentially ameliorate the negative effects that mercury elicits in macrophytes.

7.0 Scope of Research

This study will only utilize local sources of bacteria for screening. There was no known mercury

contaminated site ever reported in Malaysia. Therefore collection of samples so far will only be

focused on high oil-contaminated sites, be it water, soil, or oil itself. Then their resistance towards

mercury will be tested. Identification will be done through both biochemical and molecular

approach. The correlation between oil-utilization by bacteria and concentrations of mercury will be

studied later whereby quantification of oil and mercury will be carried out using gas-liquid

chromatography and cold vapor atomic absorption spectrometry respectively.

8.0 Methodology

8.1 Sample Collection

Water, soil and oil samples will be collected from high oil-contaminated sites which will be

determined later.

8.2 Isolation of Bacteria

The first stage of screening will be on Bushnell-Haas media (Bushnell and Haas, 1940) with

1% v/v oil (diesel) as its sole carbon source.

The second stage of screening will be on the same media, added with 5ppm of mercury

chloride (HgCl2) or mercury nitrate (Hg(NO3)2).



8.3 Bacterial Identification through Biochemical Approach

Gram staining will be done to further assist in confirmation of preliminary grouping of

bacteria by previous morphological observation. Then major biochemical approach in

bacterial identification will be done using Biolog Microlog MicroStation Semi-Automated

Microbial (ML3) Identification System (Biolog Inc., Hayward, USA). Discrete test reactions

involving patented redox chemistry will be done within a 96 well microplate. This chemistry,

based on reduction of tetrazolium, responds to the process of metabolism and will produce

a characteristic pattern or metabolic fingerprint of the bacteria. The database provided will

identify bacteria based on the patterns.

8.4 Bacterial Identification through Molecular Approach

8.4.1 DNA Extraction

The extraction procedure and the buffer used will be adopted from Kuske et al.

(1998) and Zhou et al. (1995). The pure cultures that grew on each plates will be

scooped out using inoculation loop and mixed with 200 L sterile dH2O in a 1.5 mL

microcentrifuge tube. Then 500 L of extraction buffer (2X CTAB) and 40 L of 10mg/mL

Proteinase K will be added into the microcentrifuge tube and vortex. Then the mixture

will be incubated at 37°C for 30 minutes. 30 L of RNase will be added later on and

incubated again at 37°C for 15 minutes. Next, 200 L of phenol/chloroform/isoamyl

alcohol (25:24:1) solution will be added into the mixture, followed by shaking and

vortexing for 15 minutes. Then the microcentrifuge tube will be centrifuged at 13,000

rpm. for 1 minute. The supernatant will be transferred into a new microcentrifuge tube

and equal volume of isopropanol will be added and placed at -20°C for 30 minutes.

Microcentrifuge tube containing supernatant and isopropanol will be centrifuged at

13,000 rpm. for 10 minutes. All the supernatant will be discarded and the tube will be

let to air-dry for approximately 2 hours before the DNA to be eluted with 50 L of TE

buffer for the next step.



8.4.2 DNA Amplification

DNA amplification will be done through polymerase chain reaction (PCR). PCR technique

involves several phases, which includes denaturation, primer annealing and also primer

extension. Those phases have different temperature profile and are combined to form a

cycle. The cycle is repeated 25 to 35 times which results to doubling of the number of

the copies of the target DNA fragment after each cycle.

Primers that will be used in this study are PA (forward primer) and PH (reverse primer).

These primers would specifically amplify the 16S rRNA sequences of the DNA templates.

DNA amplification will then be performed in the Mastercycler (Eppendorf), which at first

had been programmed according to the desired temperature cycling profile. The PCR

cycle is repeated for 30 cycles. Optimized annealing temperature used for the primers

will be 55C.

8.4.3 DNA sequencing and similarity searching

Purified PCR products will be run for DNA sequencing. Similarity searching for the DNA

sequence obtained will be done by using the Basic Local Alignment Search Tool (BLAST)

which is available at NCBI sequence database (www.ncbi.nlm.nih.gov). The sequences of

the samples were compared to the existing sequences in the database.

8.5 Study on Hydrocarbon-utilizing and Mercury-resistance Capabilities

8.5.1 Hydrocarbon Utilization Potential

Gas-Liquid chromatography (GLC) will be used to quantify the oil (Sorkhoh, 2004). Liquid

mineral medium aliquots of 200ml will be dispensed in 500-ml conical flasks, and

provided with oil. Flasks will be inoculated with 0.2ml aliquots of a common inoculum (1

loopful of 2 days culture in 5ml sterile water) and sealed. It will be incubated at 250C for

3 weeks before being recovered with 30ml pentane. 1µl of the solution will be analyzed

using GLC.



8.5.2 Mercury Resistance Potential

Bacteria will be cultured in media added with different concentrations of mercury.

Growth will be observed. Flameless atomic absorption spectrometry (AAS) will also be

used to quantify mercury (EPA, 1994) to see if mercury is actually utilized by bacteria.

`

8.5.3 Strains will then be tested with series of concentrations of HgCl2 with different

concentrations of oil to study the correlation between those two parameters.

GLC and AAS will be used to quantify oil and mercury respectively.

9.0 Ref erences

Bento, F. M., Camargo, F. A. O., Okeke, B. C. and Frankenberger, W. T. (2003). Bioremediation of Soil

Contaminated by Diesel Oil. Brazilli an Jour nal of Micr obiology 34, 65-68

Bento, F. M., Camargo, F. A. O., Okeke, B. C. and Frankenberger, W. T. (2005). Comparative

Bioremediation of Soils Contaminated with Diesel Oil by Natural Attenuation, Biostimulation and

Bioaugmentation. Bioresour ce t echnology 96, 1049-1055

Bruins, M. R., Kapil, S. and Oehme, F. W. (2000). Review: Microbial Resistance to Metals in the

Environment. E cotoxicology and E nvi r onment al Sa f ety 45, 198-207

Bushnell, L. D. and Haas, H. F. (1941) The Utilization of Certain Hydrocarbons by Microorganism. J.

Bact er iol. 41, 653-673

Caslake, L. F., Harris, S. S., Williams, C. and Waters, N. M. (2005) Mercury-Resistant Bacteria Associated

wth Macrophytes from a Polluted Lake. W at er, Ai r and Soil Pollution 174, 93-105

Ganesh, A. and Lin, J. (2009) Diesel Degradation and Biosurfactant Production by Gram-positive Isolates.

Af r ican Jour nal of Biot echnology 8(21), 5847-5854

Groudeva, V. I., Groudev, S. N. and Doycheva, A. S. (2000) Bioremediation of Waters Contaminated with

Crude Oil and Toxic Heavy Metals. Int. J. of Mineral Pr ocessing 62, 293-299

Kuske, C. R., Banton, K. L., Adorada, D. L., Stark, P. C., Hill, K. K. and Jackson, P. J. (1998). Small-Scale DNA

Sample Preparation Method for Field PCR Detection of Microbial Cells and Spores in Soil. Appl E nvi r on

Micr obiol. 64, 2463-2472

Misra, T. K. (1992). Bacterial Resistance to Inorganic Mercury Salts and Organomercurials. Pl asmid 27, 4-

16.



Mukherji, S., jagadevan, S., Mohapatra, G. and Vijay, A. (2004). Biodegradation of Diesel Oil by an

Arabian Sea Sediment Culture Isolated from the Vicinity of an Oil Field. Bioresour ce T echnology 95, 281-

286.

Rosenberg, E., Dworkin, M., Falkow, S., Schleifer, K. and Stackebrandt, E. (2006). The Prokaryotes

Volume 2: Ecophysiology and Biochemistry. Springer, 3rd edition.

Sorkhoh, N. A., Ali, N., Dashti, N., Al-Mailem, D. M., Al-Awadhi, H., Eliyas, M. and Radwan, S. S. (2010).

Soil Bacteria with the Combined Potential for Oil Utilization, Nitrogen Fixation, and Mercury Resistance.

Int er national Biod et er ioration and Biod egrad ation doi:10.1016/j.ibiod.2009.10.011

U.S. EPA (1994) Determination of Mercury in Water by Cold Vapor Atomic Absorption Spectrometry.

Environmental Monitoring Systems Laboratory, Cincinnati, Ohio.

U.S. EPA (2001)EPA/600/R-01/066 Mercury in Petroleum and Natural Gas: Estimation of Emissions from

Production, Processing, and Combustion. National Risk Management Research Laboratory, Research

Triangle Park, NC.

Walker, J. D. and Colwell, R. R. (1974). Mercury-Resistant Bacteria and Petroleum Degradation. Appli ed

Micr obiology 27, 285-287

Zhou, J., Bruins, M.A., Tiedje, J.M., 1995. DNA recovery from soils of diverse composition. Appl. E nvi r on.

Micr obiol. 62, 316322.



10.0 Proposed Project Schedule

Research Activities2010 2011

J J A S O N D J F M A M J J A S O N D

Literature Review X X X X X X X X X X X X X X X X X

Pilot study X X X

Sampling X X X

Screening of bacteria X X X

Morphological observation X X

Bacterial Identification:Biochemical approach

Gram Staining X X

Biolog Microbial IdentificationSystem

X X

Bacterial Identification:Molecular approach

DNA Extraction X X X

Polymerase Chain Reaction (PCR) X X X

DNA Sequencing X X X

Sequence Similarity Searching(BLAST)

X X

Studies of hydrocarbon utilizationand mercury resistance of bacterialstrains.

X X X X X

Writing Thesis X X X X X X X

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Proposal Screening of Hydrocarbon-Utilizing Bacteria With Mercury-Resistance Properties