Advances in Environmental Biology Pseudomonas aeruginosa

Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756
EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Fed-batch Production of Valuable Biosurfactant, Rhamnolipid, from Waste
Cooking Oil by Indigenously Isolate Pseudomonas aeruginosa USM-AR2
1,2Zainatul
1Ahmad
1
2
`Asyiqin Samsu, 1Siti Zulaikha Mohd Yusof, 1Mohd Syafiq Awang, 1Nur Asshifa Md Noh,
Ramli Mohd Yahya
School of Biological Sciences, University Science of Malaysia, 11800 Minden, Penang, Malaysia
Section of Bioengineering, University Kuala Lumpur MICET, 78000 Alor Gajah, Melaka, Malaysia
ARTICLE INFO
Article history:
Received 25 June 2014
Received in revised form
8 July 2014
Accepted 14 September 2014
Available online 27 September 2014
Keywords:
Fed-batch,
Rhamnolipid,
Waste
cooking oil, Pseudomonas aeruginosa
ABSTRACT
Background: Biosurfactant especially rhamnolipid had received attention due to their
potential application in various industry. It could be produce from cheaper substrate
such as waste cooking oil for lower production cost. Objective: Main objective is to
improve rhamnolipid production via fed-batch culture from waste cooking oil using
modified formulation. Results:Highest rhamnolipid production 14.39 g/L, with 0.120
g/Lh productivity and Yp/x of 3.05 g/g was obtained from fed-batch: SUR-based
feeding, an improvement of 5 fold in all aspects (rhamnolipid concentration,
productivity and yield) from batch culture. Conclusion: Rhamnolipid could be obtained
at higher yield and productivity when suitable feeding strategy of fed batch-culture is
being implemented.
© 2014 AENSI Publisher All rights reserved.
ToCite ThisArticle: Zainatul `Asyiqin Samsu, Siti Zulaikha Mohd Yusof, Mohd Syafiq Awang, Nurasshifa Md Noh, Ahmad Ramli Mohd
Yahya., Fed-batch Production of Valuable Biosurfactant, Rhamnolipid, from Waste Cooking Oil by Indigenously Isolate Pseudomonas
aeruginosa USM-AR2. Adv. Environ. Biol., 8(14), 33-38, 2014
INTRODUCTION
Biosurfactants are amphiphilic compounds produce by various type of microorganisms. The demand for
industrial production of biosurfactants are very high due to their environmental compatibility compare to
chemically synthesized surfactants. The demand for biosurfactant is expected to be around 500 thousand tons by
2018 globally and 21% of total consumption is from developing region such as Asia, Africa and Latin America
[1]. Though, their large scale production are hindered by high production cost and low biosurfactants yield.
Rhamnolipid, a glycolipid type of biosurfactants receive much attention for its' potential application in various
industry, namely cosmetics, foods, oil industry, pharmaceuticals and environment. It industrial production is
currently dominated by two USA companies, Jeneil Biotech and AGAE Technologies with production capacity
around 10 to 20 g/L only [2]. While the price for rhamnolipid is around 150 USD/g [3] compare to only 1 to 3
USD/Kg for alkylpolyglycosides [4].
Raw materials contribute about 50% of total production cost. The use of cheap substrate might contribute
for lowering the production cost [5]. About 15 million tonnes per year of waste cooking oil (WCO) were
generated around the world [6] and with 0.5 million tonnes per year comes from Malaysia itself. WCO might
introduce severe environmental pollution especially land and water pollution due to improper waste
management. Moreover, WCO could be hazardous to human health if it contain polar compounds more than
25% due to high temperature frying and repeatedly use[7]. Nevertheless, WCO also proven as a potential source
for production of biosurfactant particularly rhamnolipid [8,9]. So, WCO is used in this study as a low cost
substrate for rhamnolipid production whilst contribute in minimizing environmental pollution and risk on
human wellbeing.
Rhamnolipid yield could be enhanced through proper medium formulation. Formulation which include
sodium nitrate as nitrogen source and vegetable oil as carbon source was reported to produced highest
concentration of rhamnolipid in three years back [10]. Different cultivation strategies have been implemented
for rhamnolipids production. Batch culture was usually implemented in biosurfactant production due to its
simplicity compared to fed-batch and continuous culture. However for a higher rhamnolipid production, fedbatch was proven to increase rhamnolipid production[11,12,10].Therefore, in this study, medium formulation
Corresponding Author: Ahmad Ramli Mohd Yahya, School of Biological Sciences, University of Science Malaysia,
11800 Minden, Penang, Malaysia.
Tel: 604-653388; E-mail: [email protected]
34
Zainatul `Asyiqin Samsu et al, 2014
Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
from literature is selected to evaluate the capacity of the formulation to support high rhamnolipid production.
Batch and fed batch culture in 3.6 L bioreactor are then studied and their performance in terms of rhamnolipid
productivity and yield are compared.
MATERIALS AND METHODS
Inoculum preparation:
A glycerol stock was cultured in 250 ml shake flasks containing 50 ml nutrient broth at 25oC with 200 rpm
agitation on a shaker (Certomat  R, B. Braun). 2% v/v of inoculum at OD540 = 2.0 was used as seed culture for
production medium.
Medium Preparation:
In this study, medium formulation was adapted from Müller [13], Zhu [10] and Nur Asshifa [11] and
denoted as medium A, B and C. Waste cooking oil from local cafe was used a carbon source in all formulation.
Medium compositions for each medium were as follows:
Medium A: 15.0 g/L NaNO3, 0.5 g/L MgSO47H2O, 1.0 g/L KCl, 0.3 g/L K2HPO4, 1 mL trace elements and
250 g/L waste cooking oil. Trace elements composed of: 2 g/L sodium citrate2H2O, 0.28 g/L FeCl36H2O, 1.4
g/L ZnSO47H2O, 1.2 g/L CoCl26H2O, 1.2 g/L CuSO45H2O, 0.8 g/L MnSO4 x H2O.
Medium B: 4.0 g/L KH2PO4, 6.0 g/L K2HPO4, 3.0 g/L NaNO3, 1.1 g/L NaCl, 1.1 g/L KCl, 0.2 g/L
MgSO47H2O, 0.2 g/L anhydrous CaCl2, 5 mL trace elements and 80 g/L waste cooking oil. The compositions of
trace element were: 12 g/L FeSO47H2O, 3.0 g/L ZnSO47H2O, 1.0 g/L CoSO47H2O and 3.0 g/L MnSO42H2O.
Medium C: 6 g/L yeast extract, 0.5 g/L MgSO47H2O, 0.5 mL Tween 80 and 1.2 g/L waste cooking oil.
Shake flasks study:
Different experiments had been conducted in this study i.e evaluation of different formulation for
rhamnolipid production at original formulation and effect of different carbon to nitrogen ratio (C/N) at constant
nitrogen source.For C/N study, nitrogen source was maintain at 5.5 g/L, while carbon source was at 9.2g/L,
18.4 g/L and 27.6 g/L to give carbon to nitrogen ratio of 9, 18 and 27. Experiments were done in 500 ml shake
flasks with 100 ml working volume. The flasks was incubated at 25 oC with 200 rpm agitation (Certomat  R,
B. Braun) for five days.
Rhamnolipid production in 3.6 L bioreactor:
Rhamnolipid production in 3.6 L bioreactor (Labfors 4, Infors HT, Switzerland) was conducted in batch and
fed-batch culture. Modified medium from shake flask experiment with C/N = 18 was utilized in bioreactor study
except that 9.2 g/L of waste cooking oil was used for starting up of fed-batch culture. Two different feeding
strategies, pulse and substrate uptake rate-based feeding were employed for fed-batch culture. The conditions of
bioreactor for all experiment were maintained at 400 rpm agitation, temperature at 28°C and 0.3 vvm aeration
without pH control. Dissolved oxygen was monitored using polarographic sensor (InPro6820, Mettler Toledo)
and data was captured by IRIS software, Version 5.2.
Biomass determination:
Cell biomass was measured using turbidity method with spectrophotometer (Genesys 20, Model 4001-04,
USA) at 540 nm. Broth medium was centrifuged at 10,000 × rpm for 5 min, the cell pellet was washed twice
with acetone and distilled water. Cell concentration was calculated from cell dry weight standard curve.
Quantification of rhamnolipid via orcinol method:
Rhamnolipid production is quantified based on rhamnose (Acros) concentration using orcinol-sulphuric
method[14]. First, 0.19% (w/v) of orcinol (Acros) was dissolved in 53% (v/v) of sulphuric acid (QReC). Then,
0.3 ml cell-free broth was mixed with 2.7 ml of orcinol-sulphuric acid solution and heated at 80°C for 30
minutes. The absorbance of the mixture was determined at 421 nm using a UV-Vis spectrophotometer (UV1800 UV/VIS Spectrophotometer, Shimadzu) after the solution was allowed to cool at room temperature.
Rhamnolipid concentration referring as rhamnose equivalent was determine from standard curve.
Oil leftover measurement:
Oil leftover measurement was determined gravimetrically and adapted from Müller [13]. Cell free broth
was mixed with hexane at a ratio of (1:1, v/v). The mixture was then centrifuged at 7000 rpm (Universal 320R,
Hettich Zentrifugen, Germany) for 10 min. The upper phase was transferred in a preweighted centrifuge tube
and hexane was left to evaporate in fumehood. The tube with left oil was weighted again and the weight of oil in
g/L was calculated as:
35
Zainatul `Asyiqin Samsu et al, 2014
Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
Weight of tube with sample- weight of empty tube
×1000
Volume of cell free broth (ml)
RESULTS AND DISCUSSION
Rhamnolipid (g/L)
Shake flasks study:
Medium formulation evaluation:
In this study, three different formulations from literature were evaluated for rhamnolipid production by
indigenous isolate Pseudomonas aeruginosa USM-AR2 using waste cooking oil as carbon source. In first
experiment, the original formulation was employed and medium A was shown to support high production of
rhamnollipid (4.5 g/L) compare to 1.1 g/L rhamnolipid from medium B and 1.3 g/L rhamnolipid of medium C
(Figure 1a). For medium A, rhamnolipid was increased rapidly starting from 32 hour onwards. However, for
medium B and C, a sharp increased was observed from 48 to 56 hour, and afterwards the production was
constant until the end of incubation time. The productivity of medium A was 0.037 g/L/h, 3.1% and 2.7% higher
than productivity from medium B (0.009 g/L/h) and C (0.010 g/L/h). Nevertheless, for all medium, more
substrate was converted to cell biomass instead of product as shown by higher yield of biomass over substrate,
Yx/s, than yield of product over substrate, Yp/s(Table 1). A different trend was observed for biomass growth as
shown in Figure1b.
5
Medium A
Medium B
Medium C
4
3
2
1
(a)
0
0
Cell Dry Weight (g/L)
12
10
8
20
40
60
80
Time (h)
100
120
Medium A
Medium B
Medium C
6
4
2
(b)
0
-2 0
20
40
60
80
100
120
Time (h)
Fig. 1: (a) Rhamnolipid and (b) biomass production from three different formulation. Data obtained from
triplicate experiment.
The highest biomass was produced from medium C, with the maximum biomass value 9.4 g/L obtain at 48
hour of incubation, followed by medium A, 7.6 g/L, and the lowest biomass was from medium B, 6.0 g/L.
Although biomass was higher in medium C than medium A, but it did not support high rhamnolipid production
from this medium. For 1 g production of rhamnolipid, require 1.6 g/L of biomass from medium A, compared to
6.6 g/L biomass for medium C and 5.3 g/L biomass from medium B. Low consumption of oil was observed for
the three medium used where only 24%, 29% and 31% of oil was utilized from medium C, B and A. Since
different concentration of oil was added in each medium, which mean 77.9 g/L, 23.6 g/L and 0.3 g/L of oil was
utilized from medium A, B and C respectively. This might contribute to high rhamnolipid production from
medium A compared to the other two medium.
Effect of C/N ratio on rhamnolipid production:
In this study, three different ratios, 9, 18 and 27 mol C/mol N was tested with medium A only. Highest
rhamnolipid produced was 4.7 g/L with C/N = 18. This was four times higher than rhamnolipid produced at C/N
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Zainatul `Asyiqin Samsu et al, 2014
Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
Concentration (g/L)
= 9 (1.1 g/L) and rhamnolipid was slightly lower at C/N = 27 (4.0 g/L). Meanwhile, the biomass production was
not significantly different among different C/N ratio.
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
C/N=9
C/N=18
C/N=27
Rhamnolipid Cell Dry weight
Fig. 2: Effect of carbon to nitrogen ratio on rhamnolipid and biomass production. Data shown from triplicates of
experiment.
At constant N, productivity was increased sharply from 0.009 g/L/h at C/N = 9 to 0.039 g/L/h at C/N = 18
and slightly lower at C/N = 27 (0.033 g/L/h). Substrate was converted more to product compared to biomass at
C/N = 18 as revealed by higher Yp/s than Yx/s (Table 1). While at C/N = 9 and 27, shows on the reverse.
Table 1: Comparison of productivity and yield from different shake flask experiment.
Experiment 1
A
B
C
Productivity (g/L/h)
0.037
0.009
0.010
Yp/s
0.058
0.048
4.281
(g product/g substrate)
Yx/s
0.094
0.253
28.37
(g biomass/g substrate)
Yp/x
0.611
0.189
0.151
(g product/ g biomass)
C/N=9
0.009
0.127
Experiment 2
C/N=18
0.039
0.253
C/N=27
0.033
0.145
0.467
0.243
0.164
0.272
1.040
0.885
Comparison of rhamnolipid production in batch and fed batch culture:
In bioreactor study, the investigation on rhamnolipid production was conducted under batch and fed-batch
mode with different feeding strategy. Similar medium formulation that had been established in previous shake
flasks experiment was utilized in this study. The highest concentration of cell biomass and rhamnolipid
produced was 5.49 g/L at the 84 hour and 2.51 g/L at the 108 hour for batch culture. The overall productivity
was 0.023 g/L/h and yield of product over biomass was (Yp/x) 0.57 g/g.
In fed-batch study two types of feeding strategy were tested, pulse and substrate uptake rate (SUR)
based feeding. Fed-batch was started when dissolve oxygen profile showed a sharp increase indicating that
WCO had reach a maximum concentration that could be consumed (Figure 3). Immiscible carbon source such as
WCO will not completely utilized as evidence by leftover oil measured (data not shown). For pulse feeding
strategy, WCO was added in bulk according to total volume estimated based on measuring the volume of each
feeding and the time taken for the dissolved oxygen to rise. First feeding was estimated from volume of oil
added during batch mode divided by the time when DO shows sharp increase. During each feeding, the culture
was fed with an hour worth of waste cooking oil and given 3 to 6 hours interval before the next feeding. The
intervals vary since the feeding was done manually.
Fig. 3: Dissolved oxygen (DO) profile for pulse feeding strategy, excerpt from IRIS software showing decrease
in profile when WCO was added. Arrow indicate first time WCO was added.
In this strategy, maximum rhamnolipid and biomass obtained were 8.41 g/L and 5.02 g/L, both at 120 hour
of incubation, implying that rhamnolipid was three time higher than in batch culture (Figure 4). The overall
productivity, 0.070 g/L/h was three fold higher than productivity of batch culture and Yp/x was 1.68 g/g, three
time increase than batch culture.
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Zainatul `Asyiqin Samsu et al, 2014
Rhamnolipid (g/L)
Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
20
Batch
Fed-batch:pulse feeding
Fed-batch:SUR-based feeding
15
10
5
0
Cell dry weight (g/)
0
20
7
6
5
4
3
2
1
0
-1 0
40
60
80
Time (h)
100
120
Batch
Fed-batch:Pulse feeding
20
40
60
80
Time (h)
100
120
Fig. 4: Figure 4 Comparison of (a) rhamnolipid and (b) biomass production from batch and fed-batch culture.
Substrate uptake rate
(ml/h)
For SUR-based feeding, the culture was fed continuously according to the rate (ml/h) calculated from
previous pulse feeding strategy by setting up feed pump to certain value which correspond to the predetermined
rate. An average of the calculated rate was used to set the feed pump instead of the actual calculated rate as
shown in Figure (5). Similarly, SUR-based feeding was started when a sharp increased on dissolved oxygen
profile was observed (Figure 6). In this strategy, highest rhamnolipid was produced with maximum
concentration at the end of incubation time was 14.39 g/L. This is almost two and six time higher than pulse
feeding strategy and batch culture respectively. This could be due to more WCO was added (729.9 g/L) and
consumed (593.4 g/L) in the later strategy. While, only 268.6 g/L and 260.8 g/L was added an consumed in
pulse feeding strategy. Biomass concentration, 6.00 g/L was slightly higher in SUR-based feeding compared to
5.02 g/L in pulse feeding strategy. Whereas, overall productivity and Yp/x was almost twofold and fivefold
higher than pulse feeding and batch culture.
25
Calculated uptake rate
Average uptake rate
20
15
10
5
0
0
20
40
60
80 100 120
Time (h)
Fig. 5: Profile of substrate uptake rate (ml/h) calculated base on pulse feeding strategy. An average was taken to
set up the feed pump for SUR-based feeding strategy. SUR calculation = Total WCO add (ml)/(time of
DO start to rise – time of DO start to decrease, h).
Time (Date/hour)
Fig. 6: Dissolved oxygen (DO) profile for SUR-based feeding strategy, excerpt from IRIS software. Arrow
indicate first time WCO was added.
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Zainatul `Asyiqin Samsu et al, 2014
Advances in Environmental Biology, 8(14) Special 2014, Pages: 33-38
Highest rhamnolipid production reported in 5 years back was 70 g/L by Zhu[10] using soybean oil with fedbatch pH-stat strategy. High rhamnolipid obtained was supported by high biomass produced which was about 30
g/L. However, using soybean oil could impose higher production cost. Although there are studies base on WCO
had been conducted, not many reported on fed-batch production. Recently, study by Luo [9], shows maximum
rhamnolipid of 8.5 g/L could be produced when two times feeding of WCO at 72 hour interval was implemented
with an optimized medium for NO3- and Mg2+ content. Therefore, finding in this investigation and by other
researchers suggest that, rhamnolipid production is viable using low cost substrate such as WCO. Fed-batch
mode of production should be implemented and feeding strategy should be improved to ensure maximum
utilization of carbon source due to its immiscible properties.
Conclusion:
Medium modified from Müeller [13] was shown to support higher rhamnolipid production compared to the
other medium investigated with C/N ratio at 18. Highest rhamnolipid recorded was 4.7 g/L. The medium was
then utilized in bioreactor study with batch and fed-batch culture. Highest rhamnolipid obtained when SURbased feeding was implemented with 14.39 g/L concentration, 0.120 g/L/h productivity and 3.05 g/g of Yp/x.
While rhamnolipid production from pulse feeding fed-batch was 8.41 g/L concentration with 0.070 g/L/h of
productivity and 1.68 g/g Yp/x. Only 2.51 g/L of rhamnolipid with 0.023 g/L/h productivity and 0.57 g/g Yp/x
could be produced from batch culture. Thus, results of this study have shown that rhamnolipid could be
promisingly produced at higher yield and productivity from water immiscible substrate, WCO, when suitable
fed-batch feeding strategy being used.
ACKNOWLEDGEMENT
This work was supported by Postgraduate Research Grant Scheme (1001/PBIOLOGI/846010) from Universiti
Sains Malaysia.
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