Indonesian Journal of Chemical Research 

http://ojs3.unpatti.ac.id/index.php/ijcr Indo. J. Chem. Res., 10(1), 47-52, 2022 

 

DOI: 10.30598//ijcr.  47   
 

Biosurfactant Production from Pseudomonas aeruginosa ATCC27853 with Carbon Source from 

Crude Palm Oil for Oil Recovery 

 
Said Zul Amraini1*, Sri Rezeki Muria1, Bahruddin1, Irdoni HS1, Ulfa Dwi Artha1, Reno Susanto2  

1Chemical Engineering, Faculty of Engineering, University of Riau, Bina Widya Km 12,5, Pekanbaru, Indonesia  

2Chemical Engineering, Faculty of Industrial Engineering, Bandung Institute of Technology, Bandung, Indonesia 
*Corresponding Author: saidzulamraini@eng.unri.ac.id 

 
Received: November 2022 

Received in revised: December 2022 

Accepted: May 2022 

Available online: May 2022 

Abstract 

Biosurfactants are surfactants that are synthesized by microorganisms using organic 

materials and have biodegradable properties, making them environmentally friendly. 

One of the applications of biosurfactants in the recovery of petroleum. This study aims 

to determine the type of biosurfactant produced by Pseudomonas aeruginosa bacteria 

using crude palm oil as the main carbon source, to determine the effect of variations in 

pH and CPO concentration on surface tension reduction and emulsification, and 

compare the best biosurfactant with surfactant synthesis. The production of 

biosurfactants has 3 stages, namely the bacterial preparation, the biosurfactant 

production, and the analysis in the form of surface tension, emulsification, crude oil 

removal, and FTIR. The best biosurfactant was obtained at pH 7 and a carbon source 

concentration of 3% v/v with surface tension and emulsification values of 42.49 mN/m 

and 58%, respectively. The pH value and CPO concentration can affect the growth in 

the biosurfactant production process, thus also affecting the surface tension and 

emulsification values. The biosurfactants obtained were rhamnolipid biosurfactants. 

The biosurfactants produced in this study have lower crude oil recovery capabilities 

than synthetic surfactants with crude oil removal values of 57.78% and 79.34%, 

respectively. 

 

Keywords: Biosurfactant, CPO, pH, Pseudomonas aeruginosa, FTIR. 
 

 

INTRODUCTION 

The need for fuel is increasing as the population 

increases and technology develops  (Rezki et al., 

2017). In recent years, oil production in Indonesia has 

decreased (Sutapa et al., 2013; Bandjar et al., 2014). 

Oil production in Indonesia from 2016 to 2018 

decreased by 4%. In 2016, 2017, and 2018, oil 

production was 875 thousand, 837 thousand, and 808 

thousand barrels/year, respectively. One of the 

reasons is the condition of the oil field which is old. 

As much as 72% of crude oil production comes from 

old fields that have been producing for more than 30 

years, thus experiencing a decline in field production 

of 29% per year (Habibullah, 2018). 

 Enhanced oil recovery (EOR) technology is one 

of the technologies that can increase petroleum 

recovery by injecting a material into the reservoir 

(Gozan et al., 2014). One of the EOR techniques that 

can increase oil production by 30% from the reservoir 

that has been extracted from the oil is the surfactant 

injection technique (surfactant flooding) (Gudina et 

al., 2012). In general, the surfactants used in EOR 

technology are surfactants synthesized from 

petroleum such as petroleum sulfonate. However, the 

use of synthetic surfactants is not environmentally 

friendly because it contains petrochemicals (Ginting 

et al., 2017; Nugroho & Buchori, 2019). Currently, the 

development of EOR technology is being carried out 

towards more environmentally friendly biotechnology 

or known as microbial enhanced oil recovery (MEOR) 

technology. MEOR technology is a technology that is 

carried out by injecting a bioproduct in the form of a 

biosurfactant. The manufacture of biosurfactants 

focuses more on the use of raw materials and microbes 

to be used. The raw materials used are usually 

vegetable and animal oils which are cheaper and 

environmentally friendly (Kandasamy et al., 2019). 

 Several previous studies have been conducted on 

the Production of biosurfactants using Pseudomonas 

aeruginosa ATCC 9027 and varying temperatures of 

37 oC and 55 oC and pH 5, 6, 7, and 8. The optimum 

conditions for reducing surface tension and interfacial 

tension were at pH 8 and 37 oC with a surface tension 

value of 71 mN/ m to 34 mN/m and the interfacial 

tension from 41 mN/m to 8 mN/m (Kanna, 2017). The 

production of biosurfactants using the bacteria 



Said Zul Amraini et al. Indo. J. Chem. Res., 10(1), 47-52, 2022 

 

DOI: 10.30598//ijcr.  48   
 

Pseudomonas putida MTCC 2467 and crude palm oil 

as a carbon source obtained consists of two types of 

purity levels, namely low purity (raw) and high purity 

(pure). The efficiency of pure biosurfactant, crude 

biosurfactant, SDS, and Tween-80 at the same 

concentration was 79.40%; 46.84%; 64.10%; and 

36.91% (Kanna, 2018). 

 This study used the bacteria Pseudomonas 

aeruginosa and crude palm oil (CPO) or better known 

as crude palm oil as a carbon source by varying the 

concentration of the carbon source and the pH of the 

fermentation medium. The use of crude palm oil 

(CPO) as a carbon source is due to the production of 

CPO in Riau Province in 2006 as many as 7.43 million 

tons or about 23.58 percent of Indonesia's total 

production (Habibullah, 2018). Palm oil can be chosen 

as a raw material for biosurfactant production because 

of its fatty acid components, which make up 

triglycerides. Fatty acids with C16-C18 carbon chains 

can be applied because they have detergency 

properties and are able to play a good role in hard 

water, while fatty acids with C12-C14 carbon chains 

have a role in the foaming effect (Yuliasari et al, 

2014). The use of Pseudomonas aeruginosa bacteria 

are bacteria that have the ability to synthesize several 

compounds by biotransformation and degrade various 

types of hydrocarbons (Rengga et al., 2018). 

 This study aims to determine the type of 

biosurfactant produced by the bacterium 

Pseudomonas aeruginosa using crude palm oil as the 

main carbon source, to determine the effect of 

variations in pH and CPO concentration on the 

reduction of surface tension and emulsification, and to 

compare the best biosurfactant with synthetic 

surfactant  SDS in recovering petroleum. 

METHODOLOGY 

Materials and Instrumentals 

 The raw materials used in this study were 

Pseudomonas aeruginosa ATCC 27853 bacteria, 

Nutrient Agar, and Nutrient Broth from the Microbial 

Laboratory, University of North Sumatra, crude palm 

oil (SMART TBK), distilled water (Brataco). 

 

Methods 

Bacterial Preparation 

This bacterial preparation process consists of 

bacterial rejuvenation and bacterial cultivation. The 

bacterial rejuvenation process was carried out in a 

petri dish containing an agar plate. The agar plate 

media was made by dissolving 0.28 g of NA into 10 

mL of distilled water. The solution is to be stirred 

continuously while heated until it boils. Next, the agar 

solution was transferred into sterile Petri dishes and 

autoclaved at 121 °C for 15 minutes, then cooled to 

become gelatinous. A pure culture of P. aeruginosa 

ATCC 2785 was taken as much as 1 ose needle and 

inoculated on the agar plate aseptically, then 

incubated for 24 hours at 37 °C. 

 After completion of the bacterial rejuvenation 

process, the next process is the cultivation process 

which is carried out in a 250 mL Erlenmeyer 

containing liquid media. Liquid media was prepared 

by dissolving 1.3 gr of NB into 100 mL of distilled 

water. The solution was stirred until homogeneous 

and autoclaved at 121 °C for 15 minutes. Bacteria that 

have been rejuvenated are taken as many as 2 to 3 ose 

needles and inoculated on liquid media aseptically, 

then incubated in a shaker incubator at a speed of 120 

rpm, for 24 hours at 37 °C (Nurani & Marsudi, 2013; 

Gozan et al., 2014))  

Biosurfactant Production 

 The biosurfactant production process used a 

batch fermentation method in a fermenter with a 

capacity of 250 mL. The fermentation process was 

carried out by combining 100 mL of active bacterial 

culture and concentrations of crude palm oil (1, 3, and 

5% v/v). Fermentation was carried out for 72 hours (3 

days) and was shaken in an incubator shaker at 120 

rpm at 37 °C. For 72 hours, the pH of the growth 

medium was kept constant from 6, 7, and 8. After the 

fermentation was complete, the biomass was 

separated by centrifugation at 3300 rpm for 30 

minutes and filtered using filter paper. The solids that 

remain on the filter paper in the form of biomass are 

discarded, while the liquid that is filtered on the filter 

paper is a biosurfactant (Kanna, 2017; Gozan et al., 

2014). 

Analytical measurement 

Surface tension analysis is one of the test 

parameters for the ability of biosurfactants to reduce 

surface tension. This analysis was carried out using a 

tensiometer. 

Emulsification is one of the test parameters for 

the ability of biosurfactants to emulsify the oil. This 

analysis starts by mixing cooking oil, water, and 

biosurfactant with as much as 5 mL each in a test tube. 

The mixture was stirred with the highest speed vortex 

for 5 minutes. The mixture was allowed to stand for 

24 hours and measure the height of the emulsion layer 

and the total height of the solution. Calculation of % 

emulsification (IE24) can use the following          

Equation 1.  

 



Said Zul Amraini et al. Indo. J. Chem. Res., 10(1), 47-52, 2022 

 

DOI: 10.30598//ijcr.  49   
 

IE24 = 
High emulsion layer

Total height of solution
  × 100%                  (1) 

Crude oil removal analysis was carried out to see 

the efficiency of biosurfactants in EOR technology. 

This analysis starts with sand sieving with a sieve size 

of 50 mesh. After that, 40 grams of sand was put into 

a 250 mL Erlenmeyer and 10 mL of kerosene was 

added. The mixture of sand and kerosene is allowed to 

stand for ± 7 days. After 7 days, sand and kerosene 

were weighed as initial weight (Oi). Next, add 10 mL 

of biosurfactant into a 250 mL Erlenmeyer containing 

a mixture of sand and kerosene. The mixture of sand, 

kerosene, and biosurfactant was stirred with a rotary 

shaker with a rotation speed of 200 rpm for 20 minutes 

and allowed to stand for 24 hours. After settling, the 

mixture was washed with dichloromethane twice and 

heated at 50 oC, and weighed as the final weight (Or)   

(Costa et al.,2010). Calculation of % crude oil removal 

can use the following Equation 2. 

 

% Crude Oil Removal = 
𝑂𝑖−𝑂𝑟

𝑂𝑖
  × 100%                  (2) 

Where:  

Oi = weight of sand and kerosene (gr). 

Or = weight of sand after washing with 

dichloromethane (gr). 

FT-IR analysis was also carried out for 

biosurfactants which aim to determine the functional 

groups of the biosurfactants produced. 

RESULTS AND DISCUSSION 

Surface Tension and Emulsification 

The analysis of surface tension and 

emulsification aims to determine the best 

biosurfactant with the lowest surface tension value 

and the highest emulsification percentage value. The 

surface tension analysis used distilled water as a 

positive control and the emulsification analysis used a 

mixture of water and cooking oil. Figure 1 and Figure 

2 show the effect of increasing pH and concentration 

of carbon source on the surface tension and 

emulsification of a biosurfactant. The biosurfactant 

obtained at pH 7 and the carbon source concentration 

of 3% v/v had the lowest surface tension value of 

42.49 mN/m and the highest emulsification value of 

58%. The surface tension value of distilled water as  

positive control is 73.05 mN/m (Ikhwan, 2017) 

Based on the calculation, the decrease in surface 

tension of biosurfactants at pH 7 treatment and carbon 

source concentration of 3% v/v was 30.6 mN/m. pH 

and concentration of CPO can affect the growth of 

bacteria in the biosurfactant production process so it 

also affects the surface tension and emulsification. 

The low acidity (pH) of the growth media will result 

in bacteria not being able to efficiently produce 

biosurfactants and the pH of the media will always 

increase during the bacterial growth period (Saikia et 

al, 2012). In addition, the optimum pH for the growth 

of Pseudomonas aeruginosa bacteria is 6.6 to 7.0 

(Badal, 2018). The concentration of CPO is a nutrient 

source of bacterial carbon where excess nutrients can 

lead to the accumulation of toxic materials and the 

limited availability of dissolved oxygen can inhibit 

bacterial growth. However, a lack of nutrients can 

cause bacteria to compete for nutrients, resulting in 

inhibition of bacterial growth (Radzuan et al., 2016).  

Sari et al., (2018) have conducted research on the 

manufacture of biosurfactants using the bacterium 

Halomonas meridiana BK-AB4 with palm oil as a 
carbon source with a surface tension of 49.8 mN/m 

and emulsification of 75%. Meanwhile, the research 

conducted by  Ali et al., (2019) using Bacillus 

licheniformis ali5 bacteria with glucose and sucrose 

carbon sources obtained a surface tension of 26.21 

mN/m and emulsification of 66.4%. 

Figure 1. Effect of pH and Concentration Carbon 

Source Against (a) Surface Tension; (b) 

Emulsification 

 

 

 

a 

0%

10%

20%

30%

40%

50%

60%

70%

6 7 8

E
m

u
ls

if
ic

a
ti

o
n

(%
)

pH

Concentration 1% v/v Concentration 3% v/v Concentration 5% v/v

b 

0

10

20

30

40

50

60

6 7 8

S
u

rf
a
c
e
 T

e
n

si
o
n

 (
m

N
/m

)

pH

Concentration 1% v/v Concentration 3% v/v Concentration 5% v/v

a 



Said Zul Amraini et al. Indo. J. Chem. Res., 10(1), 47-52, 2022 

 

DOI: 10.30598//ijcr.  50   
 

Fourier Transform Infra-Red (FTIR)  

 In the Fourier transform infrared (FTIR) analysis, 

the sample used was the best biosurfactant obtained at 

pH 7 treatment with a carbon source concentration of 

3% v/v. Figure 2 shows the absorption band at a 

wavelength of 3347.60 cm-1 indicating the presence of 

O-H bonds in the rhamnopiranosil compound. The 

presence of rhamnopiranosil compounds in the 

resulting biosurfactant indicates that the biosurfactant 

is a rhamnolipid type  (Bordoloi & Kowar, 2009). In 

addition, in Figure 2, there is an absorption band at a 

wavelength of 1616.42 cm-1 which indicates the 

presence of -C=C bonds in carboxylate anion 

compounds (Saikia et al, 2012). In Figure 2, there is 

also an absorption band at a wavelength of 1634.74 

cm-1 which indicates the presence of C=O bonds in 

carbonyl compounds (Suryanti et al, 2014). 

 
Figure 2. FTIR Spectrum of Biosurfactants at pH 7 

and Carbon Source Concentration      

3% v/v 

 

Crude Oil Removal (COR) 
 In crude oil removal (COR) analysis, the 

biosurfactant used was the best biosurfactant obtained 

at pH 7 treatment with a carbon source concentration 

of 3% v/v. Figure 3 shows the comparison of crude oil 

removal values between biosurfactants and synthetic 

surfactants in the form of SDS (sodium dedocyl 

sulfate). The percentage value of crude oil removal on 

biosurfactants obtained from this study was 57.78%, 

while the percentage value of crude oil removal for 

synthetic surfactants in the form of SDS was 79.34%. 

The biosurfactant from this study had a lower %COR 

than the synthetic biosurfactant (SDS) because this 

biosurfactan is an impure product, while the synthetic 

surfactant in the form of SDS is a pure product 

(Ikhwan, 2017). Unpurified biosurfactant contains 

mostly water, dissolved biomass, and biosurfactants 

rhamnolipids (Rahayu, 2015). In addition, Kanna 

(2018) conducted research by producing 

biosurfactants from the bacterium Pseudomonas 

putida MTCC 2467 with crude palm oil as a carbon 

source. The percentage value of crude oil removal in 

crude biosurfactant is 46.84%, while the percentage 

value of crude oil removal in pure biosurfactants by 

79.40%. 

 
Figure 3. Crude Oil Value Comparison Removal  

Between Biosurfactants and Synthetic Surfactants 

CONCLUSION 

 The type of biosurfactant produced by 

Pseudomonas aeruginosa bacteria using crude palm 

oil as the main carbon source is rhamnolipid 

biosurfactant. The best results were obtained at pH 7 

with a carbon source concentration of 3% v/v, a 

surface tension value of 42.49 mN/m, and 

emulsification of 58%. The biosurfactant produced in 

this study had a lower crude oil recovery ability than 

the synthetic surfactant (sodium dodecyl sulfate/SDS) 

with crude oil removal values of 57.78% and 79.34%, 

respectively. 

 

ACKNOWLEDGMENT 

Thank you to LPPM Riau University who has 

funded this research with contract number 

696/UN.19.5.1.3/PT.01.03/2021 

 

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