02 Yudono.cdr


Vol.11, No.3, September 2017, p 81-88
DOI: 10.5454/mi.11.3.2

Oil Recovery Test using Bio Surfactant of Halo Tolerant Bacteria Brevundimonas 

diminuta and Bhurkholderia glumae at Variation of NaCl Salt Concentrations

1* 2 3
BAMBANG YUDONO , MUHAMMAD SAID , SRI PERTIWI ESTUNINGSIH , 

1
AND AULIA KARIMA

1 2 3
Department of Chemistry; Department of Chemical Engineering; Department of Biology, 

Universitas Sriwijaya, Jalan Raya Palembang-Prabumulih. Km 32 Inderalaya, South Sumatera 30662.

  Oil recovery test has been done by using crude biosurfactant from Brevundimonas diminuta and 
Bhurkholderia glumae -the indigenous halo tolerant bacteria- with the variation of NaCl salt concentration 0; 1.5; 
3; 4.5; 6; and 7.5%. Oil recovery test was obtained by determining % TPH (Total Petroleum Hydrocarbon). The 
sample concentration was 28.19% TPH. It was extracted by using biosurfactant of  B. diminuta and B. glumae 
bacteria, the optimal salinity conditions were at 3, 4.5% salt concentrations with the value oil recovery as much as 
50.41 and 69.97%, respectively. Oil components which extraction by biosurfactant were analyzed by using GC-
MS (Gas Chromatography-Mass Spectrophotometry). From the result of GC-MS analysis, it could be concluded 
that bacteria B. diminuta could dissolve hydrocarbon compounds short chain carbon atom at fraction <C –C  10 14
and long chain carbon atom at fraction >C , <C , C -C , and C -C ; whereas B. glumae could dissolve 22 10 11 14 15 17
hydrocarbon compounds short chain carbon atom at fraction <C –C  and long chain carbon atom at fraction >C  10 14 22
according to the retention time.

 
  Key words: Bhurkholderia glumae, Brevundimonas diminuta, crude biosurfactant, indigenous halo tolerant 

bacteria, oil recovery

  Uji coba rekoveri minyak telah dilakukan dengan menggunakan biosurfaktan ekstrak kasar dari 
Brevundimonas diminuta dan Bhurkholderia glumae yang merupakan bakteri  halo toleran asli Indonesia dengan 
konsentrasi garam NaCl 0; 1.5; 3; 4.5; 6; dan 7,5%. Uji perolehan minyak diperoleh dengan cara menentukan % 
TPH (Total Petroleum Hydrocarbon). Konsentrasi sampel adalah 28,19% TPH. Sampel diekstraksi dengan 
menggunakan biosurfaktan bakteri B. diminuta dan B. glumae, kondisi salinitas yang optimal berada pada 
konsentrasi garam 3 dan 4,5% dengan nilai perolehan minyak masing-masing sebanyak 50,41 dan 69,97%. 
Komponen minyak yang diekstraksi dengan biosurfaktan dianalisis dengan menggunakan GC-MS (Gas 
Chromatography-Mass Spectrophotometry). Hasil analisis GC-MS dapat disimpulkan bahwa bakteri B. 
diminuta dapat melarutkan senyawa hidrokarbon rantai pendek atom karbon pada fraksi <C –C  dan atom 10 14
karbon rantai panjang pada fraksi >C , <C , C -C , dan C -C ; sedangkan B. glumae bisa melarutkan senyawa 22 10 11 14 15 17 
hidrokarbon atom karbon rantai pendek pada fraksi <C –C  dan atom karbon rantai panjang pada fraksi >C  10 14 22
sesuai waktu retensi.

 
  Kata kunci: bakteri halo toleran asli Indonesia, Bhurkholderia glumae, biosurfaktan ekstrak kasar, 

Brevundimonas diminuta, perolehan minyak

MICROBIOLOGY
INDONESIA

Available online at
http://jurnal.permi.or.id/index.php/mionline

ISSN 1978-3477, eISSN 2087-8575

 *Corresponding author: Phone: +62-8153821994, Fax:+62-
711-580056; Email: yudonob@hotmail.com

provide the thrust to the oil to flow to the surface (Lazar 

et al. 2007; Shibulal et al. 2014).

 In order to increase cumulative oil production this 

acquisition should be increased as much as possible. 

The way is by applying the EOR (Enhanced Oil 

Recovery) method to the reservoir. The principle is to 

use external power to the reservoir that the power can 

help lifting the oil to flow to the surface. The EOR is 

chemical surfactant and one of the EOR techniques 

developed now is by utilizing microbes known as 

Microbial Enhanced Oil Recovery (MEOR). The 

MEOR method is more favorable than the EOR 

method because this method can extract petroleum 

more efficient and environmentally friendly. The 

 World's petroleum demand will increase from year 

to year along with the rapid development of global 

economy. Therefore the oil industry players must 

continue to work to meet these demands. But the 

problem is that oil production is limited by the value of 

recovery factor (RF) that is a ratio showing the amount 

of oil that can be produced on the surface. This value 

will limit the amount of oil that can be produced by the 

primary recovery mechanism. The oil production is 

very dependent on the characteristics of reservoir and 

fluid and the type of driving mechanism that helps 



potential of microorganisms to degrade heavy crude oil 

to reduce viscosity is considered highly effective in 

MEOR (Lazar et al. 2007 The basic ; Enas 2007). 

principle of MEOR is the utilization of microbial 

secondary metabolite products to help increase the 

remaining oil gain or that is still trapped in the 

reservoir.

 Bacterial decomposition by MEOR can include 

organic, inorganic acids, gases, water and bio 

surfactants. Bio surfactant is one of the products 

produced by microbes and plays an important role in oil 

recovery because it reduces surface tension between 

two-phase fluids, thereby increasing oil mobilization 

.(Sea and Dhail 2013; Chandankere et al. 2013)

 The ability of bacteria to produce bio surfactants is 

related to the ability of bacteria to use hydrocarbons as 

their substrate. Microorganisms with large bio 

surfactant production generally have a great ability 

also in decomposing hydrocarbons. Types of growth 

substrate, bacteria type, source of nutrition, and 

environment are the main factor of the researcher's 

attention in optimizing bio surfactant production. Salt 

is an important environmental factor for bacterial 

growth and development. If the salt content of the 

environment is not compatible with microbial enzyme 

activity, bacteria can not be metabolized properly so it 

does not grow optimally (Sarafin et al. 2014; Shin et al. 

2001; Garcia-Blanco et al. 2007).
 Bhurkholderia glumae and Brevundimonas 

diminuta are potential bacteria as biosurfactant and 

degradation producer (Yudono  2010; Yudono  et al. et al.

2011). The presence or absence of crude biosurfactants 

that have been produced from bacteria with a density of 
7 -1

± 10  cells mL  was tested using hemolysis tests (Dhail 

2012). Potential bacteria in producing biosurfactant, 

were chosen based on the size of the inhibit zone/clear 

zone formed of colony of bacteria on blood agar. Both 

bacteria is known to have the ability to produce a good 

biosurfactant because it has a clear zone that is 25.96 

mm. A clear zone formed over 20 mm indicates that the 

bacteria tested have good potential for producing 

biosurfactants (Yudono and Estuningsih 2013). The 

growth of both of these bacteria depends on the 

concentration of NaCl so it is potentially also possible 

in producing optimal bioproducts at certain salt levels 

(Shin  2001). Each bacterium has resistance to et al.

different salts while oil wells in Indonesia mostly have 

salinity characteristics (up to 40 000 ppm) (Almeida et 

al. et al. 2004; Hao  2008). For certain concentrations of 

NaCl also causes a decrease in water-surface tension by 

surfactants, this is because the chemical bonds that 

make up NaCl are ionic bonds capable of affecting 

hydrophobic and hydrophilic groups in lowering inter-

phase surface tension (Mnif and Ghribi 2015; Rufino et 

al. 2014). Both bacteria have good tolerance to salinity 

concentration up to 7.5%.

 This research will be conducted in variation of salt 

content of 0, 1.5, 3, 4.5, 6, and 7.5% in biosurfactant for 

recovery in petroleum. The resulting biosurfactant was 

tested with a sludge sample, then the oil recovery was 

calculated using TPH calculation (Total Petroleum 

Hydrocarbon). To see the components of the degraded 

hydrocarbon compounds, a GC (Gas Chromatography) 

analysis of sludge produced the best percentage 

recovery (at the best salt level) of each bacterium 

(Ibrahim  2013). et al.

MATERIALS AND METHODS

 Materials. The Brevundimonas diminuta and 

Bhurkholderia glumae bacteria were isolated from oil 

field Babat Toman Village, sludge oil (obtained from 

Babat Toman Village, Musi Banyuasin, South 

Sumatera). Molasses was taken from sugar waste of PT 

Cinta Manis, Tanjung Raja, Ogan Ilir, South Sumatra).

 Maintainance of Culture. A total of 1 ose of each 

bacterium B. diminuta and B. glumae were inoculated 

to solid medium NA with zig zag movement. The 

bacteria that have been inserted into the NA medium are 

incubated in the incubator for 24 h. After incubation 

bacteria are ready to use.

 Medium Preparation. Zobell media was prepared 

by dissolving 5 g of peptone, 1 g of yeast extract, 0.012 

g of K HPO , and 0.01 g of FeSO  in aquadest with a 2 4 4
volume of 1000 mL solution. The mixture is boiled over 

the hotplate and homogenized with a magnetic stirrer. 

After boiling the mixture was sterilized with autoclave 

at 121 °C for 15 min (Sunitha et al. 2007;  Behlugil 

2002).

 Bacterial Starter. B. diminuta and B. glumae 

culture cultures were taken 5 ose, then subcultured into 

each Erlenmeyer flask containing 100 mL of medium 

Zobell, then aerated for 24 h, and after that it stopped. 

One hundred mL of medium was added to the mixture 

until the total volume is 200 mL and the mixture is re-

aerated until the shortest generation time of B. diminuta 

and B. glumae for 12 h.

 Production of Crude Biosurfactants. The 

production process of crude biosurfactant is done by 

mixing bacterial starter, Medium Zobell, and 15% 

molasses concentration for B. diminuta bacteria and 

20% for B. glumae bacteria with a total volume 

82   YUDONO ET AL. Microbiol Indones



Volume 11, 2017 Microbiol Indones     83

composition of 200 mL. The mixture was incubated 

based on the shortest generation time of each bacteria ie 

12 h.

 Biosurfactant Preparation with Variation of Salt 

Level Concentrations. The process of making salt-

containing biosurfactant solution is done by mixing 

crude biosurfactant from B. diminuta bacteria and 

variation of NaCl 0, 1.5, 3, 4.5, 6, and 7.5%. The same 

treatment was performed using B. diminuta and B. 

glumae bacteria.

 Crude Biosurfactant Application on Sludge for 

Oil Recovery. A total of 25 g sludge (obtained from 

Babat Toman village, Musi Banyu Asin, South 

Sumatra) (b/v), were incorporated into each 

biosurfactant with variations of 0, 1.5, 3, 4.5, 6, and 

7.5% (w/v against biosurfactant) to a total volume of 

200 mL. The mixture is then aerated for 10 d. The 

mixture is then filtered with filter paper. The residue 

from the filtration result is then calculated by the final 

percentage of TPH.

 TPH Measurement. Two hundreds fifty mL 

boiling water with a socket-shake extract to be used was 

dried in the oven, then cooled in the desiccator. A total 

of 10 g sludge was inserted using a filter paper of the 

appropriate size. The filter paper containing the sludge 

sample was then inserted into the socket tube, then the 

top of the socket tube was connected to the condenser 

and the bottom was connected to a boiling flask 

containing n-hexane solvent with the volume wetting 

the entire filter paper. The sludge sample then was 

extracted until the solvent descends back into the 

boiling flask until it is clear. 

 GC Analysis. GC analysis was performed on 

sludge which yields the largest % oil recovery in each 

bacteria by analyzing GC sludge chromatogram before 

and after treatment at 0-7.5% salt. The type of column 

used is TG-5MS with a column length of 30 m and a 

diameter of 0.25 mm (Yudono 1994). The samples were 

taken and injected to a Thermo Scientific GC tool with a 

pre-program temperature of 40 °C maintained for 4 

min, the temperature was raised 5 °C every 1 min until 

the temperature reached 300 °C.

 Data Analysis. The data to be obtained from this 

research are; the initial sludge TPH measurement data 

to determine the percentage of hydrocarbon compounds 

present in sludge prior to treatment and final TPH data 

after the treatment of salinity variation and then 

compared with the initial TPH values t​​ o see the 

biosurfactant ability to dissolve the hydrocarbon 

compound on sludge. 

 The data obtained from the experiment was 

processed by using ANOVA (Analysis of Variance) 

analysis to test the difference of salt and oil recovery.

 RESULTS

 Total Petroleum Hydrocarbons (TPH). TPH 

were measured to determine the percentage of 

hydrocarbon compounds present in petroleum. 

Measurements of Total Petroleum Hydrocarbons 

(TPH) in oil-contaminated sludge were used as samples 

and parameters to determine the bio surfactant's ability 

to reduce further TPH values. Initial TPH 

measurements on sludge samples were performed by 

solvent extraction using n-hexane and using 

sochletation method. The initial TPH data generated 

from the measurement of 28.19%. The samples were 

treated with the biosurfactants of both bacteria that had 

varied NaCl salt by 0, 1.5, 3, 4.5, 6, and 7.5%. The 

results are characterized by reduced oil sludge levels 

after treatment. In this study, the biodegradation process 

is known from the calculation of heavy TPH oil sludge 

from residual weight (oil residual) sludge weight, then 

compared with the initial weight added by gravimetric 

method, the results were presented in Table 1.
 Based on Table 1, the final% TPH value after 

biosurfactant treatment with variation of salt 

concentration of NaCl has different TPH value, 

respectively. As result, the salt concentration can affect 

the work of biosurfakan in ddissolving sludge.
 Result of Analysis of Petroleum Hydrocarbon 

Compound on Sludge using GC-MS. The following 

Table 1 The average percentage of TPH extracted from 28.19% TPH Samples

%TPH(average)
Salinity NaCl (%) B. diminuta B. glumae

0
1.5
3

4.5
6

7.5

8.3283
10.8486
14.1992
7.7921
9.3863
8.3641

6.7539
9.8157
7.2273
19.7209
14.2114
7.7767



84   YUDONO ET AL. Microbiol Indones

chromatograms show the initial components of sludge 

(before the treatment of variation of NaCl salt content), 

sludge chromatogram with control, chromatogram 

sludge which having optimal NaCl content yielding the 

largest petroleum recovery, and a histogram of oil 

abundance in each bacterium. 
 From the data in Table 1 and described in Figure 2 

showed that the two bacteria produced different 

biosurfactant yields at each salt concentration, in which 

the B. glumae. bacteria produced a more optimal 

biosurfactant compared with B. diminuta bacteria.
 Data analysis were done by using data analysis 

with ANOVA method, it shows that F value count on B. 

diminuta and B.a glumae bacteria bigger than F critical 

value, where F arithmetic for B. diminuta bacteria has 

value of F count 59, 24098 while bacteria B. glumae 

has a value of F arithmetic 87, 5864. The value of Least 

Significant Different test value is higher for each 

bacteria, where it can be concluded that any difference 

in salt content of NaCl gives a very significant oil 

recovery.
 Result of Petroleum Recovery Test by Using 

Control. Petroleum Recovery Test using aquades is 

used as a control to be applied to a sludge sample that 

aims to determine the biosurfactant's ability to dissolve 

petroleum. The result of oil recovery at control that is 

equal to 4.7321%. The results of the recovery using the 

aquades will then be analyzed using GC to see 

components of the hydrocarbon compound after 

treatment, and used as control.
 After adding biosurfactant further decreases the 

peak area of ​​the chromatogram (Fig 1). The peak areas 

decrease in chromatogram are caused by the 

breakdown of hydrocarbons into simple compounds. 

This is largely determined by the type of bacteria, 

where each bacteria has different capabilities in 

degrading the petroleum sludge. From the GC-MS data 

obtained, soluble and insoluble hydrocarbon analyzes 

that are left on the residue are shown (Fig 3) and the 

histograms of percent abundance of each bacteria are 

presented (Fig 4).
 The percent difference of abundance by subtracting 

percent abundance after addition of crude biosurfactant 

(At) from B. diminuta bacteria minus percent of peak 

abundance before addition of crude biosurfactant (A ) 0
(Fig 3). A positive reduction results indicate that the 

hydrocarbon compound dissolves in the biosurfactant. 

An increased histogram shows that short-chain 

hydrocarbon compounds dissolve in biosurfactant, so 

that at the top of the chromatogram there is a missing 

carbon chain and breaks down into short chains. This 

histogram explains that the biosurfactant of the B. 

diminuta based on its retention time is capable of 

dissolving short C chain hydrocarbons, i.e., the <C -C  10 14
atomic chain at 40-140 °C and dissolving the long C 

chain hydrocarbons ie the atomic chain >C  at a 22
o

temperature of 265-290 C but the biosurfactants of the 

B. diminuta bacteria are not able to dissolve the long C 

chain hydrocarbons C -C  and C -C  at the 15 17 18 21
o

temperature of 140-265 C.
 This histogram shows that the treatment with 

biosurfactant from B. glumae bacteria based on 

retention time is not capable of dissolving long chain C 

hydrocarbon compounds ie C -C , C - C , and > C  at 15 17 18 21 22
 o

a temperature of 140-290 C so that the component 

remains as residue. Furthermore, biosurfactant can 

dissolve the hydrocarbon chain compound of short C 

atom, i.e., the atomic chain <C , C -C , and C -C  at 10 11 14 15 17
o

temperature 40-140 C (Fig 4).

DISCUSSION
 

 The optimal NaCl concentration was 3% for 

Brahmanimonas diminuta and 4.5% for Bhurkholderia 

glumae bacteria. The higher levels of NaCl salt that 

affect will result in different percentages of petroleum 

recovery, where salinity is one of the natural 

environmental factors present in the sludge sample 

area  (Shin et al. 2001; Garcia-Blanco et al. 2007).
 This indicates that B. glumae is an effective 

bacteria for oil recovery because the resulting 

biosurfactant can increase the solubility of 

hydrophobic compounds so as to increase and 

accelerate the rate of degradation by microbes. 

Appropriate salt content will lead to a faster 

biodegradation process from bacterial culture. 

Similarly, the type of bacteria itself will affect changes 

in salt levels obtained from bacterial metabolism 

activities. And vice versa if the level of salt is not 

suitable then the activity of bacteria will decrease that 

will affect the total of bacterial cells (Shin et al. 2001). 

It is also revealed by Charlena (2010) that salt 

conditions affect the growth of bacteria in degrading 

petroleum hydrocarbons. Optimum salt conditions will 

enhance the species' ability to degrade hydrocarbons.
 GC-MS analysis is only performed for the 

treatment of the largest petroleum recovery results 

using biosurfactant with optimal saline NaCl content 

obtained from each bacterium. In addition, GC analysis 

was conducted on sludge with treatment using aquades 

that served as a control to see the working ratio between 

biosurfactant and aquadest. Objective GC-MS analysis 



Volume 11, 2017 Microbiol Indones     85

Fig 1 Early TPH chromatogram prior to the treatment of crude biosurfactant (A), final TPH chromatogram after 
treatment using crude bosurfactant with variation of 3% salt on Brevundimonas diminuta bacteria (B), final 
TPH chromatogram after treatment using crude bosurfactant with variation in salt content 4.5% on the 
Bhurkholderia glumae bacteria (C).

(A)

(B)

(C)

can provide information on the amount of the 

constituent components of petroleum before extraction 

and after the extraction using crude biosurfactant with 

variation of salt content (Ibrahim et al. 2013; 

Jovančićević et al. 2004).
 Early TPH chromatogram prior to the treatment of 

crude biosurfactant in obtaining the petroleum 

constituent component before treatment showed the 

presence of 16 peaks of the petroleum compound based 

on a total retention time of 56 min (Fig 1A), whereas 

final TPH chromatogram after treatment using crude 

bosurfactant with a variation of 4.5% salt on B. 

diminuta bacteria showed the presence of 15 petroleum 

compound peaks based on a total retention time of 56 

min (Fig 1B). TPH chromatogram after treatment using 

crude bosurfactant with a 3% salt content variation in 



86   YUDONO ET AL. Microbiol Indones

Fig 2 Graph of petroleum recovery results after addition of salt content of NaCl in Brevundimonas diminuta and 
Bhurkholderia glumae bacteria.

Fig 3 Histogram changes of dissolved hydrocarbons before and after addition of crude biosurfactants from 
Brevundimonas diminuta bacteria.

Fig 4 Dissolved hydrocarbon histogram before and after addition of crude biosurfactant from Bhurkholderia 
glumae bacteria.

Table 2 Chain fraction C based on its temperature

The identified chain C

fraction

Temperature

Range

<100 C
100-150 C
150-200 C
200-250 C
250-300 C

(Yudono 2014)

<C10
C -C11 14
C -C15 17
C -C18 21
>C22



the B. glumae bacteria showed 21 peaks of the 

petroleum compound based on a total retention time of 

56 min (Fig 1C). It can be concluded if viewed from the 

peak amount after the treatment compared before the 

treatment of the addition of the peak amount that 

appears on the chromatogram. To see the degradability 

of hydrocarbon compounds can be seen from the 

change in the concentration of the initial hydrocarbon 

compound before treatment and the end after 

treatment. Concentration changes can be analyzed 

using gas chromatography in the form of peak areas. Of 

these differences may indicate a change in peak areas at 

baseline and after addition of biosurfactant (Penet et al. 

2006).
 If the chromatogram (A) is compared with (B) it 

can be seen that there is a decrease in ​​the peak area 

detected at retention time 25-50 min. Then at 5-25 min 

retention periods small peaks indicate the presence of 

newly undetectable compounds. The increase is 

considered to be the result of degradation of a high 

molecular weight compound which then dissolves into 

a low molecular weight so that it appears at an earlier 

mooring time, resulting in a decrease in viscosity. This 

decrease in viscosity causes oil mobility to increase (Li 

et al. 2002) (Hao et al. 2008). While in figure (B) is a 

chromatogram of petroleum component of B. diminuta 

bacteria with the best salt concentration of 4.5% NaCl 

compared with chromatogram of petroleum 

constituent components before treatment (A) there is a 

decrease of peak area areas at retention time 25-55 min, 

the decline in area is suspected because the bacteria can 

directly use and degrade the hydrocarbon compound 

into a simple compound. While at 5-25 min retention 

time new peaks appear degradation of C length to C 

short with the addition of wide peak area (Penet et al. 

2004).
 The soluble component in the biosurfactant and the 

residual component can be explained through the 

histogram of the abundance change. Based on the 

histogram, the x axis is the temperature calculated from 

the retention time and the y-axis is the change of 

abundance. The histogram was derived from obtaining 

initial chromatogram data of the compound and after 

treatment of variation of salt NaCl content in each 

bacteria, by comparing initial abundance before 

treatment and after variation of salt content of NaCl 

based on retention time. Changes in abundance are 

indicated by ΔA =% At-% Ao if the resulting data is a 

positive number so it indicates the hydrocarbon chain 

component dissolved in biosurfactant, and if it is a 

negative number it indicates that the hydrocarbon 

chain component becomes residue. The carbon chain 

fraction contained in the chromatogram can be 

identified based on the program temperature 

methodology (Yudono et al. 2010). The carbon chain 

fractions can be grouped according to the temperature 

range of the program as shown in Table 2.
 The chromatogram ratio of petroleum and recovery 

chromatograms with biosurfactants from each 

bacterium has a peak change. Changes in the peaks of 

chromatograms can occur due to the degredation 

process by bacteria. The degredation results may 

indicate a missing, emerging peak, larger peak height, 

or peak height reduction. Missing peaks show that the 

hydrocarbon chains demineralized into CO  dan H O 2 2
(Bharali et al. 2011).

ACKNOWLEDGMENT

 Thanks to Directorate General Research and 

Higher Education of Indonesia for supporting and 

funding this research, and University of Sriwijaya 

which had given the facilities to do this research.

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