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Journal of Applied Geology, vol. 2(2), 2017, pp.

109–114
DOI: http://dx.doi.org/10.22146/jag.40862

Preliminary Study of Liquid Hydrocarbon Biodegradation By Indigineous


Bacteria Isolated from Wonocolo Village, Bojonegoro District, East Java
Province

Bramono Dwi Kusumo1 , Wahyu Wilopo∗1 , and Endah Retnaningrum2


1 Department of Geological Engineering, Faculty of Engineering, Universitas Gadjah Mada, Jl. Grafika 2 Yogyakarta
55281
2 Faculty of Biology, Universitas Gadjah Mada, JL. Teknika Selatan Sekip Utara, Yogyakarta, Indonesia, 55281

A BSTRACT. Aquatic environmental pollution due to petroleum waste can cause disruption
to the environment and damaging of flora and fauna. It has been reported that petroleum
contaminatin occurs in the Bengawan Solo river, East Java Province. Liquid hydrocarbon
waste pollution can be remediate through various processes, one of them is biodegrada-
tion. Biodegradation a part of bioremediation, is the process by which organic substances
are decomposed by microorganisms into simpler substances such as carbon dioxide, wa-
ter and ammonia. Bioremediation has minor side effects compare to other methods be-
cause it’s more effective, efficient, economical and eco-friendly through biological process.
This study aims to identify bacteria for liquid hydrocarbon degradation from the rivers
in Wonocolo Village, Bojonegoro District and to determine maximum percentage of in-
oculum to produce the highest efficiency of liquid hydrocarbons degradation. Based on
phenotypic characters, the selected bacteria was identified as a genus of Moraxella. Its bac-
terium with a concentration of 2 % can reduce hydrocarbons to a maximum of 0.67 % per
hour at the exponential phase growth.
Keywords: Water pollutan · Biodegradation · Moraxella · Wonocolo · East Java.

1 I NTRODUCTION flows to Bengawan Solo Riverin Batokan Vil-


Pollution of the aquatic environment due to hy- lage, Kasiman Sub-district, Bojonegoro District.
drocarbon waste discharges may cause distur- PT. Pertamina Aset IV as supervisors does not
bance to the environment and is a major dan- have any solution to mitigate of water pollu-
ger to the flora and fauna present in water. tion by liquid hydrocarbonin Wonocolo Village,
This is because petroleum contains 50 to 95% Kedewan Sub-district. The pollution from liq-
of toxic hydrocarbon compounds and in some uid hydrocarbon waste will have a negative im-
cases carcinogenic to plants, animals and hu- pact to environment such as damage the ecosys-
mans (Varjani et al., 2017). Pollution of liquid tem and pollute the ground water. Most of
hydrocarbon waste has been observed in Ben- people in this area use groundwater to sup-
gawan Solo River. Local people said that the port their daily life. According to the Head
crude oil waste come from traditional oil min- of the Bojonegoro Environmental Service, the
ing in some villages in Kedewan Sub-district, pollution will be widen further as the num-
such as Wonocolo, Beji and Hargomulyo vil- ber of illegal oil wells increase from 200 wells
lages. In this area there are around 200 tradi- to 722 wells. It is necessary to prevent the
tional oil wells and liquid hydrocarbon waste pollution by improving of liquid hydrocarbon
waste management in the upstream area where
∗ Corresponding author: W. W ILOPO , Department
the wells are located. Bioremediation is one of
of Geological Engineering, Universitas Gadjah Mada. methods to remediate contaminated river wa-
Jl. Grafika 2 Yogyakarta 55281. E-mail: wilopo_-
w@ugm.ac.id ter by liquid hydrocarbon waste. Bioremedi-

2502-2822/
c 2017 Journal of Applied Geology
K USUMO et al.

ation is a method of controlling pollution by stage is carried out directly 3 times using two
biological activity of microbes (Leisinger, 1981; 250 mL Erlenmeyer containing liquid BHMS
Wilopo et al., 2008). The contamination preven- and added by Tween 80 as much as 1 % and liq-
tion through a biodegradation process that is uid hydrocarbons as much as 0.5 % and added
part of bioremediation and has minor side ef- 10 % starter samples of microorganisms in liq-
fects because it’s more effective, efficient, eco- uid form (mL) put into 250 mL Erlenmeyer. To-
nomical and eco-friendly than other methods tal culture is carried out for 3 weeks with each
(Azubuike et al., 2016). Enzymes from these mi- stage of subculture is one week. After 3 weeks,
crobes are used to clean and neutralize chemi- inoculation was transferred into the agar media
cal compounds and waste safely (Jednak et al., with 2 repetitions for each subculture in two Er-
2017). lenmeyers. One mL of pure culture obtained
The bacteria used in the study are bacteria from the 3rd stage subculture was transferred to
found in rivers or from the aquatic environment solid media in a petri dish in the form of BHMS
and classified as hydrocarbonoclastic bacteria added by Tween 80 as much as 1 % and liquid
(Marzan et al., 2017). Based on this background, hydrocarbons as much as 0.5 % and added to
in this research will be examined about biore- 20 % agar through pour method plate. Before
mediation by bacteria which is expected to be being transferred, dilution was carried out on a
efficient in degrading liquid hydrocarbons. test tube containing 10−2 aquadest until it was
not cloudy. After dilution the subculture which
2 M ETHODS has been diluted as much as 10−2 is transferred
Bacterial isolate was used in this experiment to a petri dish containing agar medium (BHMS
come from the river in Wonocolo Village, Kede- agar) and waited for a week. If the colony is at-
wan Sub-district, Bojonegoro District, East Java tached, then use streak method to identify the
Province. River water sample as bacteria source various microorganism. This method is done
was collected by selecting liquid hydrocarbons by heating the ose to bunsen lamp and attach-
accumulated in calm water with content a ing to the colony, then transferred to the new
biofilm. The collection was carried out at 2 BHMS agar by streaking. The results of the
adjacent sample points and given the codes streak method will grow after 1 week following
as WCL1 and WCl2. River water samples the streak from the ose. The next step is take
as starter for WCL1 and WCL2 in glass bot- measurements of colonies using the TPC (Total
tles were inoculated 10 % each in the 250 mL Plate Count) method before inserting the isolate
Erlenmeyer. This Erlenmeyer glass already on the tilt agar.
contained sterile liquid media in the form of The results of pure culture or isolates that
225 mL of liquid Bushnell Haas Mineral Salt have been grown on BHMS agar were used for
(BHMS). This liquid medium was prepared by biodegradation analysis. Before the biodegra-
in each 1 L containing 0.2 g of MgSO4 ·7H2 O; dation test, the optimal isolate was selected us-
0.02 g CaCl2 is; 1 g KH2 PO4 ; 1 g K2 HPO4 ; 1 g ing the TPC (Total Plate Count) method for 84
NH4 NO3 ; 0.05 g FeCl3 ; aquadest as much as 1 L hours with 2 repetitions in the petri dish. The
and 0.5 % of liquid hydrocarbons (estimated results of pure isolates using ose from 3rd sub-
concentration in the polluted water) and 1 % culture has been inoculated to the petri dish via
Tween 80. Tween 80 is a polyoxyethylene fatty the pour plate method and refined to the petri
acid ester surfactant and useful as an enhancer dish containing agar with the streak method.
of oil solubility in water. This solution helps The microbes grown on the streak method were
bacteria to degrade hydrocarbons as a source taken using ose and put into liquid BHMS on
of metabolism of these bacteria (Madigan et al., 250 mL Erlenmeyer for each isolate to be com-
2012). pared to growth, after being added to the liq-
Isolation of bacteria using culture of bacte- uid BHMS medium, the medium containing
ria or microorganisms was carried out up to 3 the microbial isolates were homogenized, after
subculture stages at room temperature, it was the medium containing the isolate was homog-
intended to obtain a pure culture to be used enized and inoculated into BHMS agar in the
as isolates (Hadioetomo, 1985). Each culture petri dish using the pour plate method with

110 Journal of Applied Geology


P RELIMINARY S TUDY OF L IQUID H YDROCARBON B IODEGRADATION B Y I NDIGINEOUS B ACTERIA

10−4 , 10−5 , 10−6 dilution for each isolate with the result shown that DW2 is the optimal isolate
2 repetitions and counted as t-0. The method for biodegradation of liquid hydrocarbons.
was repeated with a interval of 12 hours. The
bacteria will grow for 2 days and the results of Table 1: Comparison of specific growth rates be-
the pour plate are calculated per colony for each tween strain of DW1 and DW2.
t (time) which will be displayed in the form of a
growth curve. DW1 DW2 Unit
The results of isolates or pure culture in the N0 390 × 104 2700 × 104 CFU/mL
isolation stage will be tested through biodegra- N 1570 × 104 1385 × 104 CFU/mL
dation test to find an efficiency of microorgan- K 0.055817 0.196591
isms in the degradation of liquid hydrocarbons. µ 0.038681 0.136237
The test was carried out for 84 hours based on N0 The bacteria number at t0 incubation
the results of the selected isolate growth curve. N The bacteria number at tn incubation
The experiment using 4 scenarios with different K The growth rate of bacteria
inoculums (2 %, 5 %, 10 % inoculum) and 0 % µ The specific growth rate of bacteria
inoculum as a control. Observation parameters
consist of TPH (Total Petroleum Hydrocarbon) Bacterial identification was carried out by
test using GC (Gas Chromatography), growth phenotypic identification. Testing of colonies
of bacteria test using TPC (Total Plate Count) morphology of DW2 was observed by a trinoc-
method, volume of liquid hydrocarbon, pH test ular microscope with a lens magnification of
using litmus paper and temperature. 100/1 as shown in Figure 2. The bacterial genus
was identified as Moraxella according to sev-
3 R ESULTS AND D ISCUSSION eral phenotypic parameters based on Breed et
After 3 weeks of incubation through 3 times al. (2012) in Table 2.
subculture, the bacterial starter that become Table 2: Phenotypic characters of strain DW1.
be blackened are indicates there are growth
of bacteria in the liquid medium and ready No. Characters Results
to be transferred to agar medium. After 1
week incubation on agar media, WCL1 showed 1. Morphology
Colony Convex elevation,
growth but was contaminated by fungi and
smooth margin, all round
WCL2 showed more various colonies. There- with white colour
fore, only pure isolates taken from WCL2 to use Cell cocobacil, gram (-)
for biodegradation test. 2. Biochemistry
Optimal selection of isolates is needed to ob- TSIA test (+)
tain optimal bacterial isolates to degrade liquid Catalase test (+)
hydrocarbons. The selection uses the TPC (To- Oxidase test (+)
tal Plate Count) method for 144 hours with an Indol test (-)
observation interval every 12 hours. TPC (To- Gelatin test (+)
Simmon (+)
tal Plate Count) is done by counting the num-
Citrate test
ber of available colonies for each isolate from
Identification result Moraxella
WCL2 (Isolate 1 with the name DW1 and isolate
2 with the name DW2). These growth observa-
tions can be seen in Figure 1. It can be seen that Biodegradation test results includes TPH (To-
DW2 is faster growth than DW1 following ex- tal Petroleum Hydrocarbon), hydocarbon vol-
ponential graph. The lifetime of the bacteria in ume, pH and temperature were summarized
DW2 is also longer than DW1. DW2 shows af- in Table 3. All samples with contains inocu-
ter 12 hours have equilibrium grouth, however lum have capability to degrade liquid hydrocar-
for DW 1 the growth still continue. Constant bons. However, 2 % inoculum is the best result
growth rate (m) of DW2 is greater than DW1 for degradation process. Figure 3 shows a de-
and reach 0.136, as shown in Table 1. Based on crease in the TPH value of in the 2 % inoculum
occurs during the exponial phase where the
bacteria growth at t-36 to t-48. Moraxella able to

Journal of Applied Geology 111


K USUMO et al.

1800

1600
Growth of Bacteria (CFU/mL)

1400

1200

1000

800

600

400

200

0
0 12 24 36 48 60 72 84 96
Time (hour)
Control DW 1 DW 2

Figure 1: The growth of strain DW1 and DW2 in the BHMS medium added 0.5% liquid medium at
84 hours incubation.

by bacteria. pH of liqud almost did not change


during experiment around 6 to 6.5. This pH
support for bacteria grow because close to ne-
tral conditions. In addition, the temperatur also
did not have a big variation during the experi-
ment.

4 C ONCLUSIONS
Based on phenotypic characters, bacteria iso-
Figure 2: Phenotypic identification: (a) Colony lated from oil polluted rivers in Wonocolo Vil-
morphology; (b) Cell morphology using a lage was identified as Moraxella. After 4 days in-
Binocular Microscope with lens magnification cubation, its Moraxella at the 2 % number could
of 100/1.25. reduce the amount of TPH in liquid hydrocar-
bons at values of 0.5–0.17 %. Its bacteria could
also reduce the volume of liquid hydrocarbons
degrade hydrocarbon with the rate is 0.67 % per of 32 %.
hour in the exponential phase shown in Figure
3. The volume of liquid hydrocarbon also de- A CKNOWLEDGEMENTS
creases during experiment, especially in the 2 % The authors thank to all members of Microbiol-
inoculum that reach the highest value is 32 % ogy Laboratories in Universitas Gadjah Mada,
from initial volume (Figure 4). Most of liquid LPPT Universitas Gadjah Mada for supporting
hydrocarbon will be degraded by Moraxella bac- this researach. The authors alaso thanks to De-
teria become water and carbon dioxide (Leahy partement of Geological Engineering, Universi-
and Colwell, 1990). During experiment showed tas Gadjah Mada for giving a funding to this re-
Eh value of anaerobic conditions decrease until search.
negative value which means reductive condi-
tion due to degradation of liquid hydrocarbon R EFERENCES

112 Journal of Applied Geology


P RELIMINARY S TUDY OF L IQUID H YDROCARBON B IODEGRADATION B Y I NDIGINEOUS B ACTERIA

Table 3: Biodegradation test results from DW2.

Liquid HC
t (hour) TPH (%) Volume pH Eh (mV) T (◦ C)
(mL/100mL)
0 0.5 0.5 6.5 87 28.0
0% 48 0.5 0.5 6.5 85 28.0
84 0.5 0.5 6.4 86 28.0
0 0.17 0.43 6.5 66 28.0
2% 48 0.13 0.35 6.0 56 29.0
84 0.17 0.34 6.0 32 28.9
0 0.15 0.41 6.5 38 28.0
5% 48 0.13 0.39 6.0 -32 28.9
84 0.19 0.395 6.0 -85 29.0
0 0.14 0.40 6.5 28 28.0
10% 48 0.14 0.40 6.0 -85 28.8
84 0.18 0.35 6.0 -125 29.0

% TPH Decrease (Total Petroleum Hydrocarbon)


1600 0.6
Growth of Bacteria (CFU/mL)

1400
0.5
1200
0.4
1000

800 0.3

600
0.2
400
0.1
200

0 0
0 12 24 36 48 60 72 84 96

Time (hour)
Growth of Moraxella Control TPH 2% Inoculum
TPH 5% Inoculum TPH 10% Inoculum

Figure 3: The correlation between bacterial growth and total petroleum hydrocarbon values in a
medium at 96 hours incubation.

Journal of Applied Geology 113


K USUMO et al.

Hydrocarbon Volume after Degradation (ml/100ml)


1600 0.6

1400
0.5
Growth of Bacteria (CFU/mL)

1200
0.4
1000

800 0.3

600
0.2
400
0.1
200

0 0
0 12 24 36 48 60 72 84 96
Time (hour)
Growth of Moraxella Control
HC Volume 2% Inoculum HC Volume 5% Inoculum
HC Volume 10% Inoculum

Figure 4: The correlation between bacterial growth and volume of liquid hydrocarbon in a medium
at 96 hours incubation.

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114 Journal of Applied Geology

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