ORIGINAL ARTICLE

Enhanced rhamnolipid production by Pseudomonasaeruginosa USM-AR2 via fed-batch cultivation based onmaximum substrate uptake rateN.A. Md Noh, S. Mohd Salleh and A.R.M. Yahya

School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia

Significance and Impact of the Study: This study highlights the significance of an effective fed-batchstrategy for rhamnolipid production in a submerged fermentation using a water-immiscible substrate,based on maximum substrate uptake rate. The impact of this strategy ensured that the substrate was sup-plied at the rate matching the maximum substrate utilization by the cells without excess feeding, leadingto increased rhamnolipid production, yield and productivity.

Keywords

diesel, fed-batch, maximum substrate uptake

rate, Pseudomonas aeruginosa, rhamnolipid.

Correspondence

Ahmad Ramli Mohd Yahya, School of Biologi-

cal Sciences, Universiti Sains Malaysia,

11800 Penang, Malaysia.

E-mail: armyahya@usm.my

2013/1901: received 18 September 2013,

revised 23 January 2014 and accepted 10

February 2014

doi:10.1111/lam.12236

Abstract

A fed-batch strategy was established based on the maximum substrate uptake

rate (MSUR) of Pseudomonas aeruginosa USM-AR2 grown in diesel to produce

rhamnolipid. This strategy matches the substrate feed rates with the substrate

demand based on the real-time measurements of dissolved oxygen (DO). The

MSUR was estimated by determining the time required for consumption of a

known amount of diesel. The MSUR trend paralleled the biomass profile of

Ps. aeruginosa USM-AR2, where the MSUR increased throughout the

exponential phase indicating active substrate utilization and then decreased

when cells entered stationary phase. Rhamnolipid yield on diesel was

enhanced from 0�047 (g/g) in batch to 0�110 (g/g) in pulse-pause fed-batch

and 0�123 (g/g) in MSUR fed-batch. Rhamnolipid yield on biomass was also

improved from 0�421 (g/g) in batch, 3�098 (g/g) in pulse-pause fed-batch to

3�471 (g/g) using MSUR-based strategy. Volumetric productivity increased

from 0�029 g l�1 h�1 in batch, 0�054 g l�1 h�1 in pulse-pause fed-batch to

0�076 g l�1 h�1 in MSUR fed-batch.

Introduction

Biosurfactants are produced by bacteria, yeasts and fungi

growing on different carbon substrates, especially in

hydrocarbon-rich medium (Ron and Rosenberg 2001;

Singh et al. 2006). They concentrate at the interface

between two immiscible phases, reducing the surface and

interfacial tensions which consequently increase the sur-

face areas of insoluble compounds. This leads to increased

solubility, bioavailability and subsequent biodegradation

(Muthusamy et al. 2008; Chrzanowski et al. 2012).

The most widely studied biosurfactants are rhamnoli-

pid produced by Pseudomonas aeruginosa strains (Singh

et al. 2006; Medina-Moreno et al. 2011). It was first

described in 1949 by Jarvis and Johnson, who showed

that rhamnolipid was secreted into the medium during

the stationary phase of growth upon nitrogen exhaustion

(Guerra-Santos et al. 1984; Abdel-Mawgoud et al. 2011).

Rhamnolipid offers special advantages compared with

other biosurfactants because of its potent emulsifying

activity, high surface activities and low critical micelle

concentration (Maier and Soberon-Cha’vez 2000; Raza

et al. 2007).

Economic obstacles for rhamnolipid production, such

as low productivity, expensive raw material and prohibi-

tively high costs for downstream processing, prevented

them from being applied in bulk applications. It is there-

fore prudent to increase the productivity. Fed-batch

cultivation is one of the most effective strategies for

achieving high productivity, achieved by maintaining low

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology 1

Letters in Applied Microbiology ISSN 0266-8254

concentrations of substrate and growth rates by control-

ling feeding rates (Abdel-Mawgoud et al. 2011).

Traditionally, hydrocarbons have been the substrate of

choice to produce biosurfactants, as they are produced to

aid in oil metabolism. Although water-soluble carbon

sources, such as glycerol, glucose and molasses, can be

used to produce rhamnolipids, production is generally

much lower (Patel and Desai 1997; Maier and Soberon-

Cha’vez 2000; Muthusamy et al. 2008). In this work,

diesel was used as the main feed as it was proven to be

effective in rhamnolipid production by Ps. aeruginosa

USM-AR2 compared with glucose, glycerol, kerosene,

olive and corn oil. The production of rhamnolipid using

olive oil and corn oil was comparable. However, their

higher cost makes them less economically attractive, as

shown in Table 1 (Nurul Akma 2007; Nur Asshifa 2009).

Although diesel is not a renewable raw material and

does not assure sustainability, the production of rhamn-

olipid by Ps. aeruginosa USM-AR2 favours diesel com-

pared with other carbon sources tested. Consequently, it

was chosen as a model insoluble substrate in fed-batch

cultivation. Parallel work using waste cooking oil is cur-

rently carried out using the strategy established with

diesel. It is anticipated that the fed-batch protocol using

diesel can be a reference to other fed-batch works using

cheaper water-immiscible substrate.

It is thus necessary to develop an effective fed-batch

strategy, particularly due to the very low solubility of die-

sel. The proper development of a suitable feeding strategy

can lead to significant cost savings in terms of wasted

substrates and improved process kinetics. This work was

carried out to establish an effective feeding strategy based

on the maximum substrate uptake rate (MSUR) of

Ps. aeruginosa USM-AR2 on diesel to enhance rhamnoli-

pid production. The MSUR is a measurement of the max-

imum potential substrate-consuming ability of the cells,

expressed as a rate (Oh et al. 1998). The strategy depends

on the real-time measurements of substrate consumption

rate, estimated from the dissolved oxygen (DO) measure-

ments. The amount of time required for substrate deple-

tion, indicated by a marked rise in DO, is used to

estimate the MSUR of a microbial culture.

Fed-batch fermentations of other Ps. aeruginosa strains

producing rhamnolipid have been reported elsewhere.

Pseudomonas aeruginosa S2 cultivations recorded a

repeated pH-stat and constant feeding strategy using glu-

cose, which resulted in 6�06 and 3�04 g l�1 of rhamnoli-

pid, respectively (Chen et al. 2007). The resting cells of

Pseudomonas sp. DSM 2874 under nitrogen-limiting con-

ditions resulted in a rhamnolipid concentration of up to

45 g l�1 in a fed-batch process with rapeseed oil as the

carbon source (Trummler et al. 2003). Intermittent feed-

ing strategy applied by Lee et al. (2004) on Ps. aeruginosa

BYK-2 KCTC 18012P cultivation using fish oil resulted in

22�7 g l�1 of rhamnolipid. Although these cultivations

resulted in significant rhamnolipid production, the sub-

strates used are very expensive.

Nonetheless, the application of MSUR strategy in

rhamnolipid production using water-immiscible substrate

has been never reported, which highlights the novelty of

this work. A similar approach has been reported by Henes

and Sonnleitner (2007), albeit using soluble substrate

(glucose) and different organisms (E. coli, Saccharomyces

cerevisiae). It is important to note that the feedback

response time of DO towards soluble (e.g. glucose) and

insoluble (e.g. oil) substrates is different. The response

time of a soluble substrate is immediate compared with

an insoluble substrate, as bacterial cells need to emulsify

or break down the hydrocarbon chain before it can be

utilized. The rheology of the culture broth in this oil–water system is also different due to emulsification of oil

at different biosurfactant concentrations throughout the

fermentation. In addition, the oil dispersion and hence

homogeneity in the liquid medium varies with agitation.

The underlying principle of this strategy is to avoid

starvation and overfeeding by supplying the feed at the

maximum substrate utilization rates. It ensures a low sub-

strate concentration throughout the cultivation with a

sustained supply for microbial growth and rhamnolipid

production. Maintaining a low concentration is important

to prevent phase separation as diesel is water insoluble.

The use of rhamnolipid has been granted a patent for

facilitating crude oil distillation process (MY-147418-A)

(Nur Asshifa et al. 2012). Therefore, any bioprocessing

improvement in rhamnolipid production may assist the

process more effectively.

Results and discussion

During batch fermentation, rigorous growth took place in

4–24 h with little, if any, rhamnolipid production as

shown in Fig. 1. The highest growth was recorded at

9�5 g l�1 with the highest rhamnolipid concentration at

2�61 g l�1. The rate of rhamnolipid production was gen-

erally constant during the exponential (0–c. 30 h) and

Table 1 Cost of rhamnolipid production on different carbon sub-

strates in shake flask cultures

Carbon substrate Yield p/s (g/g) Cost impact ($)

Glucose 0�020 6�76Glycerol 0�045 2�62Olive oil 0�058 0�93Corn oil 0�048 0�75Diesel 0�054 0�16Kerosene 0�025 0�48

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology2

Development of fed-batch strategy N.A. Md Noh et al.

stationary (c. 30–50 h) growth phases. The production of

rhamnolipid stabilizes during the decline of growth.

Fed-batch is an option to increase productivity as the

high proportion of down time in batch operation reduces

the overall process productivity. Inavailability of nutrients

other than carbon is known to promote the production

of rhamnolipid (Guerra-Santos et al. 1984; Abdel-Maw-

goud et al. 2011). Thus, the cultivation strategy is aimed

at inducing rhamnolipid biosynthesis by feeding only

carbon source while limiting nitrogen.

Fed-batch cultivation was initiated by conducting a

pulse-pause feeding technique, using DO as an indicator

for substrate exhaustion, as oxygen was needed by the

cells to metabolize carbon source (Giridhar and Srivastava

2000). This step is crucial to establish a proper fed-batch

strategy based on MSUR. The aim was to feed the

growth-limiting substrate (diesel) at almost the same rate

as substrate utilization by the cells. Diesel was fed during

cultivation as prompted by any DO rise. Shortly after

feeding, DO would decrease as oxygen was consumed in

aerobic carbon metabolism.

During the test phase, agitation was reduced from 500

to 400 rpm to increase DO sensitivity to oxygen uptake

by the culture. At 500 rpm cultivation, DO values were

typically high (80–85% saturation). The agitation was

changed back to 500 rpm after the test phase. The feed

rate was then set to the latest calculated MSUR value. A

new feeding rate was used after each test phase to meet

the change in substrate requirement at different phases of

growth, which typically reflected the change in cell popu-

lation.

The DO profile of pulse-pause fed-batch for MSUR

determination is shown in Fig. 2. From 24 h onwards, DO

values fluctuated within the range of 70–98% throughout

the cultivation. The oxygen uptake rate (OUR) varied dur-

ing fermentation, with the highest OUR occurring during

the exponential phase at 3�53 mmol l�1 h�1. The highest

specific OUR (qO2) was 0�59 mmol g�1 h�1 (data not

shown). This value is low compared with the typical respi-

ration rates in other bacterial cultures where the qO2 values

range from 2 to 12 mmol g�1 h�1 (Shuler and Kargi

2002). Other strains of Ps. aeruginosa were also reported to

prefer microaerobic condition for growth and metabolite

formation, especially when grown in hydrocarbons (Chay-

abutra and Ju 2000; Sabra et al. 2002).

As depicted in Fig. 3, most of the rhamnolipid was

produced after growth had ceased, and increased through-

out the stationary phase, reaching the maximum produc-

tion of 18�9 g l�1. A subsequent fed-batch fermentation

was conducted using the estimated MSUR values from

the previous pulse-pause run. Agitation was fixed at

500 rpm throughout the cultivation without performing

test phases. Given the finite minimum flow rate, it was

not possible to feed diesel continuously without causing

excess feeding. Unlike soluble substrates, excess immisci-

ble substrate will be floating on the culture broth if fed

faster than its consumption rate, which can interrupt

mass transfer. Instead, diesel was fed intermittently.

Substrate feed rates was plotted with growth and

rhamnolipid production in Fig. 4. Diesel uptake rate

increased as the cells entered exponential phase. The rate

decreased as the cells entered stationary phase. The high-

est uptake rate was 5�6 ml h�1 at 54 h during the expo-

nential phase. Throughout the stationary phase, which

started at 72 h, the substrate uptake rate decreased to 3�6,1�8 ml h�1 and finally reached a plateau at 0�4 ml h�1.

The highest growth at 15�9 g l�1 was achieved after 60 h

of cultivation. This is quicker compared with previous

runs, assumed to be due to the higher oxygen transfer at

0·0

1·0

2·0

3·0

4·0

5·0

6·0

7·0

8·0

9·0

10·0

0 12 24 36 48 60 72 84 96Cel

l dry

wei

ght,

Rha

mno

lipid

con

c. (

g l–

1 )

Time (h)

Figure 1 Growth ( ) and rhamnolipid ( ) production in a batch cul-

ture of Pseudomonas aeruginosa USM-AR2.

40

50

60

70

80

90

100

110

0 24 48 72 96 120

144

168

192

216

240

264

288

312

336

360

Dis

solv

ed o

xyge

n sa

tura

tion

(%)

Time (h)

Figure 2 Dissolved oxygen and feeding profile in Pseudomonas aeru-

ginosa USM-AR2 pulse-pause fed-batch cultivation. *Arrows indicate

feeding at test phases.

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology 3

N.A. Md Noh et al. Development of fed-batch strategy

higher agitation without interruption of the test phases.

Rhamnolipid concentration also reached the highest at

23�6 g l�1 in a shorter time than the previous run

(Fig. 4).

The trend in the MSUR paralleled the growth profile of

Ps. aeruginosa USM-AR2, where active growth coincided

with high substrate utilization during the early cultiva-

tion. Similarly, MSUR declined as growth entered station-

ary phase indicating other substrate limitation, believed to

be nitrogen. This has been verified based on runs using a

mixed substrate feeding with diesel and yeast extract. In

this case, growth was enhanced further, reaching 28 g l�1.

However, rhamnolipid production did not improve

significantly (data not shown).

In the fed-batch rhamnolipid profiles, a sharp increase

in production was observed when growth entered the

stationary phase. Many postulated this was due to nitro-

gen limitation (Maier and Soberon-Cha’vez 2000; Raza

et al. 2007). Therefore, Kjeldahl analysis was performed

to detect the nitrogen content (from yeast extract) in the

culture broth. Figures 3 and 4 show that nitrogen was

consumed rapidly within 48 h and remained minimal

throughout the cultivation. Contrary to popular postu-

lates, rhamnolipid production did not necessarily start

upon nitrogen exhaustion. Although nitrogen limitation

may contribute to rhamnolipid production, it cannot

explain the sudden sharp increase in rhamnolipid. Conse-

quently, further experiments were conducted to investi-

gate this observation.

Rhamnolipid emulsifies hydrocarbon and stabilizes oil-

in-water emulsions (Chrzanowski et al. 2012). The inter-

facial activity of rhamnolipid was assessed by determining

0

0·1

0·2

0·3

0·4

0·5

0·6

0·7

0·8

0·9

0

2

4

6

8

10

12

14

16

18

20

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384

Nitr

ogen

con

tent

(g

l–1 )

CD

W, R

ham

nolip

id c

onc.

(g

l–1 )

Time (h)

Figure 3 Growth ( ), rhamnolipid ( )

production and leftover nitrogen ( ) in pulse-

pause fed-batch culture.

0

0·2

0·4

0·6

0·8

1

1·2

0

2

4

6

8

10

12

14

16

18

20

22

24

26

Nitr

ogen

con

tent

(g

l–1 )

CD

W, R

ham

nolip

id c

onc.

(g

l–1 )

Die

sel f

eed

rate

s (m

l h–1

)

Time (h)

0 24 48 72 96 120 144 168 192 216 240 264 288 312

Figure 4 Growth ( ), rhamnolipid ( )

production, leftover nitrogen ( ) and diesel

feed rates ( ) in maximum substrate uptake

rate (MSUR)-based fed-batch culture. A few

points were added to nitrogen content data

in Figs 3 and 4, and the unit was changed

from % to g l�1 as suggested by reviewer.

The trend of the graph was the same as

before. The chart type for diesel feed rate in

Fig. 4 was changed for more accurate

feeding profile.

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology4

Development of fed-batch strategy N.A. Md Noh et al.

its emulsification index on diesel, the limiting substrate in

this fermentation system. Diesel emulsification at different

rhamnolipid concentrations is shown in Table 2. It is

obvious that the uptake of diesel is correlated with its

emulsification by rhamnolipid. At concentrations lower

than 2�5 g l�1, no activity was observed. However,

beyond this, emulsifying activity increased with increasing

rhamnolipid concentration.

The emulsification result reflected the rhamnolipid pro-

duction profile in Figs 3 and 4, where a sharp increase in

rhamnolipid occurred when rhamnolipid concentration

reached c. 4–5 g l�1. Similarly, a significant increase in

emulsification index was also observed when rhamnolipid

concentration reached 5�0 g l�1. This suggests that emul-

sification has to occur before diesel becomes available to

the cells, indicated by the sudden increase in rhamnolipid

production. Better emulsification facilitates diesel uptake

by the cells, thereby resulting in more rhamnolipid secre-

tion. Diesel uptake during early cultivation was much

slower as rhamnolipid was insufficient for adequate diesel

emulsification.

Pertinent values of Ps. aeruginosa USM-AR2 fermenta-

tion runs are summarized in Table 3. The product yield

on biomass was calculated as the ratio of rhamnolipid

concentration to the corresponding biomass, not by the

highest biomass obtained. The table shows that the volu-

metric and specific productivity were enhanced from

batch to fed-batch cultivation using MSUR-based strategy.

The higher productivity in MSUR compared with the

batch culture was expected and in agreement with the

fed-batch principle. Moreover, rhamnolipid yield on die-

sel was enhanced from 0�047 (g/g) in batch to 0�110 (g/g)

in pulse-pause fed-batch and 0�123 (g/g) in MSUR fed-

batch. Rhamnolipid yield on biomass was also improved

from 0�421 (g/g) in batch to 3�098 (g/g) in pulse-pause

fed-batch and finally 3�471 (g/g) via feeding at the MSUR.

This depicted higher substrate utilization rates by the cells

and an effective strategy in improving rhamnolipid

vproduction by Ps. aeruginosa USM-AR2.

Overall, growth, rhamnolipid production, yield and

productivity have improved from batch to fed-batch culti-

vation based on the MSUR of Ps. aeruginosa USM-AR2

on diesel. However, conducting a pulse-pause feeding

strategy was a crucial step before establishing a proper

MSUR-based fed-batch.

Materials and methods

A wild-type strain, Ps. aeruginosa USM-AR2, was isolated

and identified as a hydrocarbon-utilizing and rhamnoli-

pid-producing bacterium (Nur Asshifa 2009; Nur Asshifa

et al. 2012). A stirred-tank bioreactor was retrofitted with

a foam collector on its gas outlet. Foam, containing bio-

surfactants, was allowed to flow out of the bioreactor

from the air outlet and left to collapse into liquid in a

separate vessel, and pumped back into the fermentation

vessel (Salwa et al. 2010). The modified bioreactor was

able to address the foaming problem without the addition

of any antifoam agent. Typically, foam production in

Ps. aeruginosa USM-AR2 culture is extremely quick and

severe, making antifoam addition ineffective (Nur Asshifa

2009; Salwa et al. 2010).

Production of biomass and rhamnolipid in bioreactor

Experiments were performed in a 3�0-l bioreactor (BioFlo115, New Brunswick, Hamburg, Germany) with 1�5 l

working volume at 27°C, 500 rpm agitation, aerated at an

air flow rate of 0�5 vvm and a medium initial pH of 5.

The pH was left uncontrolled. A production medium was

used to cultivate Ps. aeruginosa USM-AR2 containing the

ingredients as follows per litre (Nur Asshifa 2009): 0�6%

Table 2 Emulsification index of diesel at different rhamnolipid

concentrations

Rhamnolipid conc. (g l�1) E24 (%)

Control 0

0�1 0

0�2 0

0�5 0

2�5 0

5�0 57�57�5 62�5

10�0 72�514�0 97�5

Table 3 Summary of biomass and rhamnolipid production by Pseudomonas aeruginosa USM-AR2 fermentation at different cultivations

Cultivation

Maximum

biomass (g l�1)

Biomass at

highest

RL (g l�1)

Highest

RL (g l�1)

Volumetric

productivity

(g l�1 h�1)

Specific

productivity (h�1)

Yield (p/s)

(g/g)

Max. yield (p/x)

(g/g)

Batch 9�5 6�2 2�61 0�029 0�0047 0�047 0�421Pulse-pause fed-batch

(to determine MSUR)

9�5 6�1 18�9 0�054 0�0089 0�110 3�098

MSUR-based fed-batch 15�9 6�8 23�6 0�076 0�0116 0�123 3�471

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology 5

N.A. Md Noh et al. Development of fed-batch strategy

(w/v) yeast extract, 0�05% (w/v) MgSO4, 0�05% (v/v)

Tween� 80, 20 ml initial diesel. Diesel served as the sole

carbon source and the feed substrate, while Tween� 80

was added to increase diesel solubility in water and conse-

quently assist in the diesel uptake by Ps. aeruginosa USM-

AR2. Seed culture was prepared in nutrient broth before

inoculation into the production medium. A 2% (v/v) cell

suspension of a 24-h culture (OD540 = 2) was used as the

inoculum.

Different cultivations were performed, and results were

compared between batch, pulse-pause fed-batch for

MSUR determination and MSUR-based feeding strategy.

During the course of the fermentation, samples of the

culture broth were periodically collected and analysed for

biomass growth and rhamnolipid production.

Biomass quantification

Cells were harvested by centrifugation at 10 000 g for

5 min (Eppendorf Centrifuge 5424; Eppendorf AG, Ham-

burg, Germany), treated with acetone to remove the

adhering hydrocarbon (diesel) and washed twice with dis-

tilled water to remove traces of nutrients. The cell pellet

was suspended in 3 ml distilled water, and the biomass,

expressed in dry weight (g l�1), was obtained by compari-

son with a calibration curve. Bacterial growth was moni-

tored by OD540 with a spectrophotometer (Genesys 20,

Model 4001-04; Thermo Scientific, Waltham, MA, USA).

Rhamnolipid quantification: orcinol assay

Rhamnolipid was quantified indirectly, using rhamnose as

a reference (Jeong et al. 2004). To 0�3 ml of each sample,

2�7 ml of a solution containing 0�19% orcinol (in 53%

H2SO4) was added. After heating for 45 min at 70°C, themixtures were cooled at room temperature and the OD421

was measured using a spectrophotometer. The rhamnoli-

pid concentrations were calculated from a standard curve

prepared with L-rhamnose and expressed as rhamnolipid

equivalent (RE).

Nitrogen determination by Kjeldahl analysis

Nitrogen content in the broth was determined by the

Kjeldahl method and estimated by the equation below:

%N ¼ ðVstd acid � VblankÞ � Nacid � 1�4007Msample

N = normality.

Vblank = volume in millilitres of standard acid needed

to titrate a reagent blank.

The minimum detection limit is 0�01 g l�1.

Maximum substrate uptake rate determination

The principle of MSUR is that the oxygen uptake by

micro-organism is dependent on the conversion of car-

bon. The MSUR determination was initiated at the

instance of complete diesel depletion detected by a rapid

increase in DO concentration. At that instant, a known

amount of diesel was fed into the culture. This caused

DO to decrease and remained at a low value for a certain

period as carbon metabolism ensued. DO eventually rise

again upon complete depletion of diesel. This whole step

is called the ‘test phase’. The MSUR was calculated using

the equation below (Oh et al. 1998):

MSURml

h

� �¼ S

t2 � t1

where

t1 = time at which a known amount of carbon sub-

strate is fed, following the first rise in DO (h).

t2 = time at which the second rise in DO occurs (h).

S = amount of substrate fed (diesel) (ml).

The test phase was performed whenever deemed neces-

sary during the course of the fermentation to give an

accurate assessment of MSUR at different stages of the

cultivation. During the test phase, DO observation and

feeding were done manually and the data were recorded

and compared. The smallest tubing gave a minimum flow

rate of 0�067 ml s�1.

Acknowledgements

The authors are grateful to Universiti Sains Malaysia

(USM) for supporting this research through USM APEX

Research Grant (1002/PBIOLOGI/910308) and USM

Incentive Grant (1001/PBIOLOGI/821090).

Conflict of interest

No conflict of interest declared.

References

Abdel-Mawgoud, A.H., Hausmann, R., L�epine, F., Muller,

M.M. and D�eziel, E. (2011) Rhamnolipids: detection,

analysis, biosynthesis, genetic regulation, and

bioengineering of production. In Biosurfactants,

Microbiology Monographs 20 ed. Soberon-Cha’vez, G. pp.

13–55. Springer-Verlag: Berlin Heidelberg.

Chayabutra, C. and Ju, L.K. (2000) Degradation of

n-hexadecane and its metabolites by Pseudomonas

aeruginosa under microaerobic and anaerobic

denitrifying conditions. Appl Environ Microbiol 66,

493–498.

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology6

Development of fed-batch strategy N.A. Md Noh et al.

Chen, S.Y., Wei, Y.H. and Chang, J.S. (2007) Repeated pH-stat

fed-batch fermentation for rhamnolipid production with

indigenous Pseudomonas aeruginosa S2. Appl Microbiol

Biotechnol 76, 67–74.

Chrzanowski, L., Lawniczak, L. and Czaczyk, K. (2012) Why

do microorganisms produce rhamnolipids? World J

Microbiol Biotechnol 28, 401–419.

Giridhar, R. and Srivastava, A.K. (2000) Fed-batch sorbose

fermentation using pulse and multiple feeding strategies

for productivity improvement. Biotechnol Bioprocess Eng 5,

340–344.

Guerra-Santos, L., Kappeli, O. and Fiechter, A. (1984)

Pseudomonas aeruginosa biosurfactant production in

continuous culture with glucose as carbon source. Appl

Environ Microbiol 48, 301–305.

Henes, B. and Sonnleitner, B. (2007) Controlled fed-batch by

tracking the maximal culture capacity. J Biotechnol 132,

118–126.

Jeong, H.S., Dong-Jung, L., Sun-Hee, H., Soon-Duck, H. and

Jai-Yul, K. (2004) Rhamnolipid production by

Pseudomonas aeruginosa immobilized in polyvinyl alcohol

beads. Biotechnol Lett 26, 35–39.

Lee, K.M., Hwang, S.H., Ha, S.D., Jang, J.H., Lim, D.J. and

Kong, J.Y. (2004) Rhamnolipid production in batch and

fed-batch fermentation using Pseudomonas aeruginosa

BYK-2 KCTC 18012P. Biotechnol Bioprocess Eng 9,

267–273.

Maier, R.M. and Soberon-Cha’vez, G. (2000) Pseudomonas

aeruginosa rhamnolipids: biosynthesis and potential

applications. Appl Microbiol Biotechnol 54, 625–633.

Medina-Moreno, S.A., Jimenez-Islas, D., Gracida-Rodriguez,

J.N., Gutierrez-Rojas, M. and Diaz-Ramirez, I.J. (2011)

Modeling rhamnolipids production by Pseudomonas

aeruginosa from immiscible carbon source in a batch

system. Int J Environ Sci Technol 8, 471–482.

Muthusamy, K., Gopalakrishnan, S., Ravi, T.K. and

Sivachidambaram, P. (2008) Biosurfactants: properties,

commercial production and application. Curr Sci 94,

736–747.

Nur Asshifa, M.N. (2009) The effects of Pseudomonas

aeruginosa USM-AR2 culture on crude oil biomodification

and its potential in facilitating distillation. M.Sc. Thesis

(Biotechnology). Malaysia: Universiti Sains Malaysia.

Nur Asshifa, M.N., Amirul, A.A., Mohamad Nasir, M.I. and

Ahmad Ramli, M.Y. (2012) Rhamnolipid produced by

Pseudomonas aeruginosa USM-AR2 facilitates crude oil

distillation. J Gen Appl Microbiol 58, 153–161.

Nurul Akma, M.R. (2007) Production of biosurfactant in

Pseudomonas aeruginosa USM-AR2 culture. B.Sc. Thesis

(Biotechnology). Malaysia: Universiti Sains Malaysia.

Oh, G., Moo-Young, M. and Chisti, Y. (1998) Automated fed-

batch culture of recombinant Saccharomyces cerevisiae

based on on-line monitored maximum substrate uptake

rate. Biochem Eng J 1, 211–217.

Patel, R.M. and Desai, A.J. (1997) Biosurfactant production by

Pseudomonas aeruginosa GS3 from molasses. Lett Appl

Microbiol 25, 91–94.

Raza, Z.A., Rehman, A., Khan, M.S. and Khalid, Z.M. (2007)

Improved production of biosurfactant by a Pseudomonas

aeruginosa mutant using vegetable oil refinery wastes.

Biodegradation 18, 115–121.

Ron, E.Z. and Rosenberg, E. (2001) Natural roles of

biosurfactants. Environ Microbiol 3, 229–236.

Sabra, W., Kim, E.J. and Zeng, A.P. (2002) Physiological

responses of Pseudomonas aeruginosa PAO1 to oxidative

stress in controlled microaerobic and aerobic cultures.

Microbiology 148, 3195–3202.

Salwa, M.S., Nur Asshifa, M.N., Amirul, A.A. and Ahmad

Ramli, M.Y. (2010) Different feeding strategy for the

production of biosurfactant from Pseudomonas aeruginosa

USM-AR2 in modified bioreactor. Biotechnol Bioprocess

Eng 14, 763–768.

Shuler, M.L. and Kargi, F. (2002) Bioprocess Engineering: Basic

Concepts. New Jersey: Prentice Hall.

Singh, A., Van Hamme, J.D. and Ward, O.P. (2006)

Surfactants in microbiology and biotechnology: Part 2.

Application aspects. Biotechnol Adv 25, 99–121.

Trummler, K., Effenberger, F. and Syldatk, C. (2003) An

integrated microbial/enzymatic process for production of

rhamnolipids and L-(+)-rhamnose from rapeseed oil with

Pseudomonas sp. DSM 2874. Eur J Lipid Sci Technol 105,

563–571.

Letters in Applied Microbiology © 2014 The Society for Applied Microbiology 7

N.A. Md Noh et al. Development of fed-batch strategy