Identification of Isolate PP Bacteria in Illegal Gold Mining

in Simpi, Belitang Hilir District, Sekadau, West

Kalimantan

Abdullaha*, Luqmanul Hakima, Edi fitriandia, dan Rahmawatia

a
Program Study of Biology, Faculty of Mathematic and Natural Science, Tanjungpura University, Indonesia
bStreet on Prof. Dr. H. Hadari Nawawi, Pontianak 78124, West Kalimantan, Indonesia
a
Email: Abdullah.dullah13@student.untan.ac.id

Abstract

Gold mining activities conducted in Simpi, Sekadau noteworthy because in the process still use mercury metal
as a purifying agent of gold. Mercury is one of the most dangerous heavy metals and is in the environment in
various forms of compounds. The use of mercury (Hg) as a separator between gold seeds and rocks (gold
purifier) can lead to environmental damage. The contamination of mercury heavy metal waste causes certain
types of microorganisms to evolve, thus having a high resistance mechanism to these metals such as bacteria.
Bacteria that have this ability can basically come from a different kind. The purpose of this study was to identify
the most potent mercury degrading bacteria in the Unlicensed Gold Mining area in Simpi Village, Belitang Hilir
sub-district, Sekadau District, West Kalimantan. The research methods included biochemical tests of PP isolates
and identification based on manual identification books. The results showed that PP isolates had the ability to
ferment glucose and produce a catalase enzyme. For motility and indool testing showed a negative reaction.
Isolate with PP code that shows the most resistant to the stress of mercury and allegedly have the ability to
degrade mercury metal. These bacteria belong to the genus of Trichococcus.

Keywords: Illegal Gold mining; Mercury; Isolate PP

------------------------------------------------------------------------
* Corresponding author.
E-mail address: Abdullah.dullah13@student.untan.ac.id

1. Introduction

Indonesia is one of the countries that fall into the category of the level of gold mining is quite large since 1990
until 2011. Some community groups are interested to participate in mining activities both legal corporations and
[1]
non-legal such as illegal gold mining activities . Increasing the amount of unlicensed gold mining occurred in
one of the areas in Sekadau District, West Kalimantan.

1
Sekadau is one of the regencies located in the province of West Kalimantan with an area of 544,430 Ha[2]. In
this area, there is an area of Simpi village, Belitang Hilir sub-district which is known to have potential gold
mining producer which should be taken into account its existence. Gold processing activities conducted in the
Village Simpi still done traditionally with the use of mercury (Hg) in the process of processing. The results
show that mercury contamination is bad for the environment and a threat to human health, especially the people
around the location.

The natural environment created by God Almighty has been equipped with a mechanism to balance life in it. the
environment has the ability to degrade pollutants into one of them through a biological process of
biodegradation. Mechanism of biodegradation by utilizing microorganism activity one of them is bacteria
through transformation process of structure of a compound so that there is change of integrity and toxicity of the
compound is reduced or become not toxic at all. Some bacterial spersis has the ability to decompose the metal
mercury naturally because it has a gene factor capable of decomposing the mercury compounds in nature into
simple particles. To address the problem, it is necessary to conduct research related microorganisms, especially
bacteria that have the ability to cope with contamination of heavy metal mercury the area.

2. Material and Method

2.1 Study Area

This study was conducted for 2 months from May to June 2018 which included data collection, identification,
and data analysis. Sampling was conducted in Unlicensed Gold Mining (PETI) area of Simpi Village, Belitang
Hilir Sub-district, Sekadau. Further research was conducted at Microbiology Laboratory, Biology Study
Program, Faculty of Mathematics and Natural Sciences, Tanjungpura University, Pontianak and West
Kalimantan.

Figure 1: Research Location

2
2.2 Tools and Materials

The tools used in this research is test-tube, tube threaded, needle ose, rack test tubes, petri dishes, clamp test
tubes, glass beaker, bunsen, plastic puppets, vortex,tweezers, skapel, scissors, Biology Safety Cabinet ( BSC),
incubator, colony counter, hot plate, magnetic stirrer,spray bottle, dropper drops, Erlenmeyer flask, analytical
balance, oven, autoclave, shaker, measuring cup, light microscope, Nutrient Broth (NA), alcohol 70%, 96%
alcohol, sodium chloride (NaCl), spirit, crystal violet, Lugol's solution (iodine), oil immersion (immersionoil),
Triple Sugar Iron Agar ( TSIA), Sulfide Indol Motility (SIM), Kovacs reagent, Methyl red (MR), hydrogen
peroxide (H2O2) and Simon Citrate Agar (SCA).

2.3 Research Procedures

2.3.1 Purification of Isolate

Isolate PP isolate has been purified again in order to obtain bacterial culture in optimum metabolic state prior to
testing. Purification was carried out by taking as much as 1 ose from a bacterial colony that has grown later in
inoculation into amedium Nutrient Agar (NA)that has been enriched with ahgclmixture2 1 mg / Lusing astreak
method. Then incubated in the incubator for 2x24 hours at 37ᵒC.

2.3.2 Identification

The Identification is done with two steps is Biochemistry test :

a. Citrate
Test The citrate test was performed by 1 ose pure bactericidisate inoculated onmedium Simmons citrate solidand
incubated for 2x 24 hours at 37ᵒC. Positive results are marked by the change of color on the media from green to
blue [3].

b. Motility and Indole Test

A total of 1 ose isolates from the culture stock were inoculated by stabbing on amedium sulfide indole motility
(SIM) upright, then incubated at 37ᵒC within 2x 24 hours. Positive results when there are rambatan around the
needle puncture on the medium and the negative results if there are no rambatan direk puncture ose needle
puncture on the medium. Into each isolate were added a few drops of kovacks compounds, to see the production
of indol compounds. Positive results marked gterbentuknya red rings and negative results if there is no change
in color [3].

c. TSIA

3
Test Htest was2Sperformed by isolate bacteria inoculated by stabbing on the butt and scratched on the slant on
Triple Sugar Iron Agar (TSIA) medium for 3x 24 hours at 37ºC. The change observed after incubation is the
color of the medium to yellow indicates acid, the red color signifies the medium being base, the color becomes
black signifies the formation of H2S and when the medium is raised indicates that the microbe is capable of
producing gas [3].

d.Test Methyl Red (MR)

A total of 1 ose (ose round) bacterial isolate was extracted from culture stock and inoculated by scratch
onmedium Christensens Urea tilting, then incubated at 37 ° C for 5x24 hours. Red color means a positive
reaction.

e. Catalase
Test The catalase test is carried out by isolating mercury-resistant bacteria smeared on a glass preparation using
ose. Isolate bacteria are dripped with hydrogen peroxide 3% by 1-2 drops. Positive catalase test is characterized
by the formation of air bubbles [3].

6. Oxygen Requirement Test A

total of 1-2 ose pure isolate baktyeri were inoculated inmedium Nutrient Broth (NB) then incubated for 1x 24
hours at 37ᵒC. After that it was observed that bacterial colonies growing on the surface of the medium meant the
bacteria were aerobic obligate, if bacterial colonies growing on the bottom of the tube meant anaerobic obligate
and if grown in the middle meant facultative anaerobic [3].

2.4 Data Analysis

The data were analyzed descriptively on the basis of identification using a morphology of cell physiology
comparison of Bergey's Manual of Determinative Bacteriology [4].

3. Result and Discussions

Based on the research be able to discuss as follows:
3.1 Biochemical Test

Characterization of bacteria based on biochemical test needs to be done considering the cell activity of each type
[5]
of bacteria would be different. It is also suggested by that each bacterium has different metabolic and
enzymatic activities. As for some biochemical tests performed such as citrate test, motility and indole test,
methyl red (MR) test, TSIA test, catalase test, urease test and oxygen demand test (Table 1).
Table 1: Characterization of Bacteria with Biochemical Test
Karakter Morfologi Sel
Isolate of Bacteria PP10-4
dan Biokimia
Morfologi Sel
Gram Positive (+) (Determinated)
Cell Formation Coccus, long chain
(Determinated)

4
 Biochemical Test
TSIA Test
- Glucosa +
- Sucrosa -
- Lactosa -
- H2 S -
Methyl Red -
Motility -
- Indole -
Catalase +
Citrat +
Oxygen Demand A/nF

TSIA test is one of the tests that aims to determine the ability of microbial isolates in the process of fermentation
of glucose, sucrose and lactose, red and ferrosulfate indicator and production of fermented and hydrogen
sulphide (Hgas2S)[6]. The positive test results fermented glucose marked by discolouration on theportion butt
TSIAmediumbeing yellow in thesection slant showing a negative reaction because it is still red which means the
isolates can not ferment sucrose and lactose. The TSIA medium is also not raised or broken which means it does
not produce gas and color on the base of the black colored medium which signifies not producing gass of H2S.

Methyl Red (MR) is one of the test aimed to determine whether there is a mixed acid product of glucose
fermentation makromer form of lactic acid, acetic acid, formic acid, succinic acid by bacteria. The results show
[7]
a negative reaction characterized by a media color that does not change to red. According that the
accumulation of acid compounds on the media resulting from bacterial metabolism activity is not formed, so the
pH of the medium remains stable and does not change color.

Motility test is one of the tests to determine the ability of bacteria in moving position because of the existence of
a stimulus that comes from the environment in the form of chemical substances so that the bacteria akab move
[8]
closer to the direction of stimulation .In addition, motility tests are also used to determine the presence or
absence of motion in bacteria. The results of the PP10isolate test-4 showed a negative reaction characterized by
the absence of propagation of the puncture on the medium. Indole test is a bacteria test in order to see the
bacteria in doing a revamp of the amino acid tryptophan[9]. The test results showed a negative reaction
characterized by the non-forming of a red colored ring on the medium after it was spilled withreagent Kovac's.
This suggests that bacteria do not have tryptophanase enzymes that are unable to produce indole compounds,
pyruvic acid and ammonia.

5
The catalase test is one of the tests that aims to determine the ability of bacteria capable of producing catalase
[10]
enzymes. According katalse is one of the enzymes used by microbes to break down the toxic compound is
hydrogen peroxide (H2O2)into water (H2O) and oxygen (O2).Hasi shows a positive reaction characterized by the
formation of gas bubbles after being metitized with a solution of hydrogen peroxide. This indicates that
microbes grown in aerobic environments are able to decompose certain toxic substances [11].

Citrate test is one of the tests to determine the ability of a surviving bacteria to survive in environmental
conditions that have a single carbon and nitrogen content[12]. The results of the PP isolate showed a positive
reaction that is marked by the change of medium with the initial medium color of the green being blue. This is
because at the krebs cycle stage, the citrate compound is converted to oxaloacetic acid and acetic acid with the
help of citrate enzyme then converted again to pyruvic acid and carbon dioxide which then reacts with water and
sodium on the medium of Simmons Citrate Agar to form sodium carbonate compound. Because of the existence
of this compound the medium can change the nature of a base and able to change the color to blue. In bacteria
[10]
there is a citrate enzyme enzyme that makes the reaction positive. According to that the use of citrate by
bacteria causes the acidic properties of the culture to disappear so that there is an increase in pH in culture which
causes the medium to turn purple.

Oxygen demand test is one of the tests conducted to determine the properties of microbial based on the need of
oxygen. The results showed that there was bacterial growth on the basis ofmedium, Nutrient Broth (NB) so it
was known that the bacteria had anaerobic facultative characteristics. Facultative anaerobes are conditions in
which microbes are able to grow with relatively fewer oxygen levels or even no oxygen at all.

3.2 Identification
Table 2: Identification of Bacteria based on Bergey's Manual of Determinative Bacteriology
Karakter Morfologi Sel Genus Trichoccus
Isolat Bakteri PP10-4
dan Biokimia (Holt et al., 1994)
Morfologi Sel
Gram Positive (+) Positive (+)
Cell formation Coccus, long chain Coccus, long chain
Biokimia
TSIA
Glucosa + +
Sucrosa - ND
Lactosa - ND
H2S - ND
Methyl Red - ND
Motility - -
Indole - -
Catalase + +
Citrate + ND
Oxygen Demand A/nF A/nF
Description: ND : Not Determinant

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Based on the results of adjustment to the physiological character of the biochemical test (Table 2), bacterial
isolates of PP belong to the genusgroup Trichococcus. The results showed that bacteria of the genus
Trichococcus included as bacteria that have the ability to degrade the metal mercury. This is in accordance with
previous studies by [13]that the bacteria of the genus Trichococcus became one of the dominant microorganisms
in the process of decomposition of Pb2+ and Hg2+ to form acetic acid, formate and lactate compounds to
facilitate the reduction of sulfate compounds and to withstand the environment with Pb2+ and Hg2+ in the longer
term compared to other bacterial groups.

3.5 Conclusion

Based on the results of the research that has been done, it has been obtained one pure isolate with PP code that
shows the most resistant to the stress of mercury and allegedly have the ability to degrade mercury metal. These
bacteria belong to the genus of Trichococcus.

References

[1]. Kementerian Perindustrian Republik Indonesia, ‘Peran ekspor kelompok industri penghasil emas, perak,
logam mulia, perhiasan dan lain-lain terhadap total ekspor hasil industry, Kementerian Perindustrian
Republik Indonesia’ http://www.kemenperin.go.id/statistik/peran_-kelompok.php?kel=6&ekspor=1. 18
November 2017 (17:30), 2012.

[2] Situs Resmi Pemerintah Kabupaten Sekadau, ‘Keadaan Geografis’,

sekadaukab.go.id/v1/profile/daerah/geografis.html. 28 November 2017 (14:00), 2012.

[3] Waluyo, ‘Metode Analisis Mikrobiologi. Universitas Muhammadiyah Malang Press. Malang’2008.

[4] J. G. Holt, NR. Krig. P. Sneath. J. Staley. dan S. Williams. ‘Bergey’s Manual Of Determinative
Bacteriology, 9th Edition.’, 1994.

[5] S. T. Cowan, k. J. Steel, G. I. Barrow, and Feltham, R,K,A, 1993, ‘Cowan and Steel’s Manual for The
Identification of Medical Bacteria 3rd Edition’, 1993.

[6] B. W. Lay, ‘Analisis Mikroba di Laboratorium’, 1994.

[7] R. S. Hadioetomo, ‘Mikrobiologi Dasar Dalam Praktek: Teknik dan Prosedur Dasar Laboratorium’, 1990.

[8] P. Singleton, ‘Introduktion to Bacteria’, 1992.

7
[9] J. G. Cappucino, and N. Sherman, N, ‘Microbiology: A Laboratory Manual’, 1983.

[10] A. Ulfa, E. Suarsini, dan M.H. I. Muhdhar, ‘Isolasi dan Uji Sensitivitas Merkuri pada Bakteri dari Limbah
Penambangan Emas di Sekotong B, arat Kabupaten Lombok Barat: Penelitian Pendahuluan’, Proceeding
Biology Education Conference, vol. 3, pp. 398-399, 2018.

[11] H. Prescott, ‘Laboratory Exercise in Microbiology’, 2002.

[12] G. W. Gokel, ‘Dean’s Handbook Of Organic Chemistry’, 2004.

[13] L. Zhang, X. Lin. J. Wang. F. Jiang. L. Wei. G. Chen. dan X. Hao, ‘Effects of Lead and Mercury on
Sulfate-Reducing Bacterial Activity in a Biological Process for Flue Gas Desulfurization Wastewater
Treatment’, Scientific Reports. Vol. 6, no. 3.2016.