Isolation and identification of sulfate reducing bacteria (SRB) from the sediment pond

after a coal mine in Samarinda, East Kalimantan

Eko Kusumawati, Sudrajat, and Junita Susilaning Putri

Citation: 1813, 020006 (2017); doi: 10.1063/1.4975944

View online: http://dx.doi.org/10.1063/1.4975944
View Table of Contents: http://aip.scitation.org/toc/apc/1813/1
Published by the American Institute of Physics
 Isolation and Identification of Sulfate Reducing Bacteria
(SRB) From the Sediment Pond after a Coal Mine in
Samarinda, East Kalimantan
Eko Kusumawati1*, Sudrajat2, Junita Susilaning Putri3
1,2,3
Microbiology and Genetic Molecular Laboratory, Department of Biology
Faculty of Mathematics and Natural Sciences, Mulawarman University, Indonesia 75123
*)
Corresponding author: eko.kusumawati11@gmail.com

Abstract. Title isolation and identification of sulfate reducing bacteria (SRB) of sediment pond former coal mine in
Samarinda, East Kalimantan. Sulfate reducing bacteria (SRB) is a group of microbes that can be used to improve the
quality of sediment former coal mine. In the metabolic activities, the SRB can reduce sulfate to H2S which immediately
binds to metals that are widely available on mined lands and precipitated in the form of metal sulfides reductive. Isolation
and identification of sulfate reducing bacteria carried out in the Laboratory of Microbiology and Molecular Genetics,
Faculty of Mathematics and Natural Sciences, University of Mulawarman, Samarinda. Postgate B is a liquid medium
used for isolation through serial dilution. Physiological and biochemical characterization was done by Bergey's Manual
of Determinative Bacteriology. Six isolates of sulfate reducing bacteria were isolated from the sediment pond former coal
mine in Samarinda. Several groups of bacteria can grow at 14 days of incubation, however, another group of bacteria
which takes 21 days to grow. The identification results showed that two isolates belong to the genus Desulfotomaculum
sp., and each of the other isolates belong to the genus Desulfococcus sp., Desulfobacter sp., Desulfobulbus sp. and
Desulfobacterium sp.

INTRODUCTION
Coal is one of the natural resources that can be exploited through mining activities. Mining activities can provide
a series of interrelated effects, namely decreasing pH, nutrient availability and balance in the sediment is disturbed,
and the solubility of micronutrients which is generally a metal element increases. According to Widyawati [6]
showed that the content of sulfate in sediments former coal miner PT. Bukit Asam in South Sumatra was 60,000
ppm, pH 2.8 and metals content well above the threshold for clean water. Thus the quality of aquatic environments
can affect people's health and life.
One method that environmentally friendly is bioremediation, a process by using microorganisms, fungi, green
plants or enzymes to restore the environment by eliminating contaminants. Group of microbes that can be used to
improve the quality of sediment former coal mine is the sulfate reducing bacteria (SRB). In the metabolic activities
sulfate reducing bacteria (SRB) can reduce sulfate to H2S. This gas will immediately bind to the metals found in
many mined lands and precipitated in the form of metal sulfide reductive [2].
The purpose of this study was to determine the types of sulfate reducing bacteria obtained from sediment pond
former coal mine in Samarinda. The information obtained will be used for the development of bacteria as a potential
bioremediation agent that environmentally friendly.

RESEARCH METHODS
The study was conducted at Microbiology and Genetic Molecular Laboratory Department of Biology, Faculty of
Mathematics and Natural Sciences, Mulawarman University, Samarinda. The research was experimental research.

The 1st International Conference on Mathematics, Science, and Computer Science (ICMSC) 2016
AIP Conf. Proc. 1813, 020006-1020006-4; doi: 10.1063/1.4975944
Published by AIP Publishing. 978-0-7354-1481-5/$30.00

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 Isolation Sulfate Reducing Bacteria (SRB)
Sulfate reducing bacteria (SRB) were isolated from sediments former coal mine. Samples were taken from
sediments (bottom) pool. Samples was taken as many as 1 g sample is introduced into a test tube and added to saline
(0.85%) to 10 mL sterile, then homogenized with a vortex. Furthermore, the sample were diluted until g L-3, and
took 1 mL inserted into a test tube, added Postgate B sterile medium (g L-1) Na lactate (3.5), Mg.SO4 (2.0), NH4Cl
(0.2), KH2PO4 (0.5), FeSO4. 7H2O (0.5) and pH 4 slowly until full and tightly closed. Incubated at 35 C. Growing
BPS marked by the changing media into black (with the formation of sulfides) which shows activity BPS.
Observations were made when the color changes to black the entire media. Isolates were grown were scored density
black color.

Identification Sulphate Reducing Bacteria (SRB)

A total of 1 mL isolates was grown aseptically transferred by micropipette into the empty sterile petri dish and
then filled Postgate B for sterile media that has been given the additional antifungal ketoconazole to inhibit mold
growth on the media. Microbial suspension and the media so that the homogenized evenly. Cultures were incubated
at 35 C for more than 7 days [7]. Inoculated colonies grown on selective media slant for pure bacterial stock.
Further identification of the bacteria was done by physiological and biochemical tests:
1. Gram Stain
Bacteria of isolates tested was smeared on a glass slide, fixed spreads heat carefully, then stained with crystal
violet dye (Cat A) for one minute and rinsed with distilled water. Subsequent staining with iodine (Cat B) for two
minutes before rinsing with 95% ethanol (Cat C) for 30 seconds and was washed with distilled water. Furthermore
smear stained with safranin (Cat D) for 30 seconds, then rinsed of excess color before observed in the microscope.
Gram-positive will be blue or purple, while Gram-negative would be pink.
2. SIM (Sulfide Indol Motility) Test
One ose isolates were taken from stock and then inoculated on SIM medium upright. Furthermore, incubated was
performed at temperature of 37 C for 2 x 24 hours. A positive result (motile) if there is creeps around ose needle
marks that indicate the movement of the BPS.
3. TSIA (Triple Sugar Iron Agar) Test
Bacterial isolates 1 Ose then inoculated into medium TSIA by means inserted into the medium until it reaches
the upright (butt). Furthermore taken 1 ose culture and scratched on the surface of the media. Furthermore,
incubated was performed at temperature of 37 C for 2 x 24 hours. The color changes to yellow indicates acidic
medium, the red color signifies medium becomes alkaline, black color indicates the formation of H 2S and if medium
that lifted indicates such microbes capable of producing gas.
4. MR-VP (Methyl Red-Voges Posquer) Test
One ose isolates were taken from stock and then inoculated in a liquid medium MR-VP. Furthermore, incubated
was performed at temperature of 37 C for 5 x 24 hours. After incubation, Methyl-red was added about 5 drops of
the above preparations of bacterial isolates. A positive result, if the complex is formed of pink to red indicating that
the microbes that produce acid.
5. Peroxide Test
First 7 mL reagent H2O2 was inserted into a test tube, then 1 ose review bacterium was inserted into a test tube.
An observed change is a positive result if gas bubbles are formed.

Data Analysis
Data such as result of the isolation, characterization of morphology and types of sulfate reducing bacteria were
presented in tables and descriptive images.

RESULT AND DISCUSSION

The SRB was isolated from sediment pond after a coal mine in Samarinda. The isolation results showed that the
nine point after coal mining sediment pond at Jl. Pelita 7, District Speech, Samarinda, East Kalimantan obtained 6

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isolates different, namely P1T13 (Sp 1), P2T12 (Sp 2), P2T33 (Sp 3), P3T22 (Sp 4), P3T23 (Sp 5) and P3T31 (Sp 6)
(TABLE 1).

TABLE 1. Growth of sulfate reducing bacteria (SRB) isolated from sediment pond after coal mining in Samarinda in a liquid
medium Postgate B
No. Sample Code Time to Growth (Days) The Intensity Level
1 P1T13 14 +++
2 P2T12 21 +
3 P2T33 14 ++
4 P3T22 14 +++
5 P3T23 21 +++
6 P3T31 14 ++
Note : + = Black thin; ++ = Black was; +++ = Pitch black

A conditions pond which contains sulfate and low pH is a suitable habitat for the growth of the SRB. However
that group of bacteria has different character, some of bacteria can grow after 14 days incubation, but there are also
found a group of bacteria which takes 21 days to grow. The ability of bacteria to reduce sulfate groups is also
different. The ability to reduce the concentrations of sulfate indicated solution and black color on the test tubes. In
accordance with the reduction reaction of sulfate, SO 42- reduced by sulfate reducing bacteria into 2-, and reacts with
metal ions to form metal sulphides black and insoluble. Therefore, more and more metal sulfides are formed, the
solution will be more concentrated in the tube (FIGURE 1).
The diversity of the sulfate reducing bacteria is strongly influenced by environmental conditions, environmental
unfriendliness, the depth of the sediments, the availability of energy from organic materials, and the content of
sulfate [5]. This is consistent with other research that suggested that environmental factors affect the diversity of the
types and characteristics of sulfate reducing bacteria include acidity environment [1], the depth of sediment [3], the
availability of energy from organic materials [5], and the content of sulfate [4]. Icgen and Harrison [4] reported that
sulfate content will determine the dominant groups of sulfate reducing bacteria grow in an ecosystem.

FIGURE 1. Indication of sulfate reduction by sulfate reducing bacteria isolated from sediment pond after a coal mine in
Samarinda

Of the nine points after the pond sediment coal mining, which is used as a sample, obtained six sample indicated
the occurrence of sulfate reduction by sulfate reducing bacteria. Of the six samples were then planted and
manufacture of pure cultures of sulfate reducing bacteria were tested at a later stage.
Identification of the SRB was done by Bergey's Manual of Determinative Bacteriology, which was based on
several characteristics, among others forms colonies, forms the edge of colonies, contour, surface colony, colony
color, Gram stain, cell shape, motility, sporulation, and resources carbon. The results of the identification of sulfate

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reducing bacterial isolates that has been taken from nine points after coal mining sediment pond which has been
purified is presented in TABLE 2 as follows:

TABLE 2. Characteristics of the isolates sulfate reducing bacteria isolated from sediment pond after coal mining in Samarinda
Sulphate Reducing Bacteria Type
Character
Sp 1 Sp 2 Sp 3 Sp 4 Sp 5 Sp 6
Form Colonies Irregular Irregular Irregular Irregular Irregular Irregular
Margin Colonies Flat Flat Flat Flat Flat Flat
Elevation Flat signage Flat signage
Flat signage Flat signage Flat signage Flat signage
Colonies
Colour Colonies black white white-cream white-cream white-cream white
Gram Stain Negative Positive Negative Negative Negative Positive
Cell Shape coccus coccus basil basil coccus coccus
Motility + + - + + +
Producing
+ - - + + +
sulfide
Producing Gas + - - + + +
Indol test - - + - - -
MR test + - + - - +
VP test + - + - + +
Peroxide test + - + + - -
Spesies (sp) Desulfococcus Desulfotomaculum Desulfobacter Desulfobulbus Desulfobacterium Desulfotomaculum

CONCLUSIONS
Six isolates pure sulfate reducing bacteria were isolated from the sediment pond after a coal mine in Samarinda.
Several groups of bacteria were able to grow at 14 days of incubation. However, it was found a group of bacteria
which takes 21 days to grow. The identification results showed that two isolates belong to the genus
Desulfotomaculum sp., and each of the other isolates belong to the genus Desulfococcus sp., Desulfobacter sp.,
Desulfobulbus sp. and Desulfobacterium sp. were found.

ACKNOWLEDGMENTS
We acknowledged Mulawarman University, Indonesia (Grant: Dana BOPTN Faculty of Mathematics and
Natural Sciences, Mulawarman University Rectors Decision No. 397/SK/2016, 2016).

REFERENCES
1. Bractova S., Groudev S., Georgiev P. The effect of some essential environmental factors on microbial
dissimilatory sulphate reduction. Annual of the University of Mining and Geology St Ivan Ritski, Vol 44-45,
Part II, Mining and Mineral Processing. pp. 123-127 (2002).
2. Hards B.C., J.P. Higgins. Bioremediation of Acid Rock Drainage Using SRB. Jacques White Environment
Limited. Ontario (2004).
3. Hoehler T.M., Alperin M.J., Albert D.B., Martens, C.S. Apparent minimum free energy requirements for
methanogenic archaea and sulfate-reducing bacteria in an anoxic marine sediment. FEMS Microbiology and
Ecology, 38: 33-41 (2001).
4. Icgen B., Harrison, S. Identification of population dynamics in sulfate-reducing consortia on exposure to
sulfate. Res Microbiol, 157: 922-927 (2006).
5. Liamleam W., Annachhatre A.P. Electron donors for biological sulfate reduction.
Biotechnol Adv, 25: 452-463 (2007).
6. Widyati E. Bioremediasi Tanah Bekas tambang Batubara dengan Sludge Industri Kertas untuk Memacu
Revegetasi Lahan. Disertasi Doktor. Sekolah Pascasarjana IPB. Bogor (2006).
7. Yusron M., Lay B.W., Fauzi A.M., Santoso D.A. Isolasi dan Identifikasi Bakteri Pereduksi Sulfat pada Area
Pertambangan Batubara Muara Enim, Sumatera Selatan. Jurnal Matematika, Sains dan Teknologi 9(1): 26-35
(2009).

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