Abstract · Web viewPseudomonas aeruginosa PAO1 was supplied by the School of Biomedical Sciences,...
Transcript of Abstract · Web viewPseudomonas aeruginosa PAO1 was supplied by the School of Biomedical Sciences,...
Biorefining palm oil agricultural refinery waste for added
value rhamnolipid production via fermentation
Mohd Nazren Radzuana,c, Ibrahim M. Banatb and James Winterburna*
aSchool of Chemical Engineering and Analytical Science, The University of Manchester,
Manchester, United Kingdom
bSchool of Biomedical Sciences, Faculty of Life and Health Sciences, Ulster University,
Northern Ireland, United Kingdom
cDepartment of Biological and Agricultural Engineering, Faculty of Engineering, Universiti
Putra Malaysia, Malaysia.
*Corresponding author: [email protected] (Tel: 0161 306 4891)
Abstract
Rhamnolipids (RL) production by Pseudomonas aeruginosa PAO1 is a potentially attractive
route to add value to palm oil refinery agricultural by-products; palm fatty acid distillate
(PFAD) and fatty acid methyl ester (FAME). The results showed maximum RL concentration
of 3.4 and 2.5 g L−1 when using 10 g L−1 PFAD and FAME respectively; while using 20 g L−1
PFAD and FAME, the RL concentrations achieved were 3.2 and 3.1 g L−1, respectively. The
predominant congener produced was identified as dirhamnolipid, Rha-Rha-C10-C10. The RL
produced reduced surface tension to 29-32 mN m−1 with a CMC value of 19 mg L−1. A high
emulsion index with kerosene, 40 % and sunflower oil, 46 % were measured. This work
demonstrates the potential for the utilisation of palm oil refinery agricultural by-products,
PFAD and FAME, as low cost and renewable substrates for RL production in integrated palm
oil biorefinery systems.
1List of Abbreviations: Rhamnolipids (RL), Palm fatty acid distillate (PFAD), Fatty acid methyl ester (FAME), Critical micelle concentration (CMC), Emulsion index (EI), Crude palm oil (CPO), Protease Peptone Glucose Ammonium Salt Media (PPGas).
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Keywords
Pseudomonas aeruginosa PAO1, fermentation, rhamnolipid, palm fatty acid distillate
(PFAD), fatty acid methyl ester (FAME), biorefinery
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1. Introduction
Biosurfactants are extracellular microbial surfactants that are amphipathic and have
high emulsifying activity and surface activity (Ji et al., 2016). Biosurfactants have several
advantages compared to synthetic surfactants, such as biodegradability, low toxicity, stability
and effectiveness at extreme pH, salinity and temperature and their potential to be produced
from renewable substrates (George and Jayachandran, 2013). These features give
biosurfactants potential for use as green alternatives to chemical surfactants that can be
applied in environmental remediation, as antimicrobial agents, as emulsifiers and stabilisers
in the food industry, in enhanced oil recovery processes and medical applications (Elshikh et
al., 2016; Moya Ramírez et al., 2015).
Chemical structure plays a major role in biosurfactant classification into glycolipids,
lipopeptides, phospholipids, fatty acids and polymeric compounds (Lourith and
Kanlayavattanakul, 2009). Rhamnolipids are one of the low molecular mass glycolipid type
biosurfactants, being produced mainly from Pseudomonas aeruginosa strains that are being
intensively studied by researchers (Irorere et al., 2017). There are two main types of
rhamnolipids with one (monorhamnolipid) and two (dirhamnolipid) rhamnose sugar moieties,
attached to one or two β-hydroxy fatty acid chains (Gudiña et al., 2016). To date, several
companies have been producing rhamnolipids in relatively large quantities such as
Rhamnolipid Incorporated (http://rhamnolipid.com/), Rhamnolipid Holdings
(http://www.rhamnolipidholdings.com/) and AGAE Technologies
(http://www.agaetech.com/), thus showing rhamnolipids have enormous potential for
commercialisation into various products and application areas.
There are several factors that affect rhamnolipid production which include the carbon
source, nitrogen source and environmental factors. Pseudomonas aeruginosa is capable of
using water immiscible free fatty acid containing substrates such as sunflower oil, olive oil
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and soybean oil to produce rhamnolipid. Glucose, glycerol, ethanol and mannitol are
commonly used hydrophilic carbon sources, with results showing that hydrophobic substrates
give better rhamnolipid production compared to hydrophilic substrates (Nitschke et al.,
2011). Nitrogen sources also play an important role in rhamnolipid production. It has been
reported that when the nitrogen became limiting there was overproduction of rhamnolipid and
cell growth become stationary (Reis et al., 2011). Furthermore, environmental factors such as
pH, temperature, agitation speed, strain type and age, oxygen availability and culture media
affect rhamnolipid production and cell growth (Banat, 1997).
Despite intensive research efforts into the development of large scale biosurfactant
production processes, there still exists the main challenge of high production costs that limit
the wider use of biosurfactants (Dobler et al., 2016). The high cost of rhamnolipids
production mainly arises from the expensive substrate and downstream processes, making the
use of rhamnolipids costly, especially for low added value applications such as environmental
remediation (Banat et al., 2014). One of the ways to reduce the production cost is the
utilization of some industrial/agro-industrial waste or by-product which could provide a low
cost fermentation substrate and an opportunity to generate added value, as well as making use
of wastes that can potentially have a harmful impact on the environment (Moya Ramírez et
al., 2015).
The Malaysian palm oil industry is one of the most important contributors to the
Malaysian economy with substantial export earnings of Malaysian Ringgit (RM) 60 × 109 and
high employment rate. Globally, 34 % of palm oil production is obtained from Malaysia,
where in 2015 the planted area was 5.64 × 106 hectares (MPOB, 2016). Palm oil industry
made Malaysia the second largest producer in the world with crude palm oil (CPO)
production in 2016 of 17.3 × 106 tonnes (MPOB, 2017). The refining process of CPO
produces a by-product called palm fatty acid distillate (PFAD) that is low value, renewable
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and abundant waste substrate. It is estimated that 6.2 × 105 tonnes of PFAD are produced
annually, which accounts for 3.6 % of the total CPO processed. Recently, PFAD has been
used as a feedstock for the oleochemical industry, animal feed and, as a source of vitamin E
in the cosmetics industry and has also been extensively studied for the production of biodiesel
(Abdul Kapor et al., 2017; Hosseini et al., 2015).
PFAD is a by-product of palm oil refining, produced in agricultural palm oil refinery
mills. At room temperature, PFAD is a yellow solid, which turns to a dark brown liquid at
temperatures above the melting point of ≈40 °C and FAME (fatty acid methyl ester) is the
biodiesel that can be derived from PFAD via the esterification process (Chabukswar et al.,
2013). The esterification of PFAD to produce FAME, an existing valorisation route,
significantly affects its physical properties, changing from solid to liquid at room
temperature. PFAD and FAME are mainly composed of fatty acids such as oleic acid,
pentadecanoic acid, tridecylic acid, palmitic acid, stearic acid, palmitic acid and others with
carbon chain lengths from C11 to C18 (Nazren Radzuan et al., 2016). There is much interest in
biodiesel (FAME) produced from PFAD because of its comparable properties to the diesel
fuel produced from petroleum and the increasing demand for biofuels by both developing and
developed countries (Yadav et al., 2010). The high production cost of biodiesel, however, is a
big challenge that inhibits its industrial growth (Lokman et al., 2014).
There are many studies in which free fatty acids derived from waste or low-value by-
products, such as olive mill waste, waste frying oil, glycerol and soap stock, have been used
for rhamnolipid production (Lovaglio et al., 2015). De Faria et al., (2011) used raw glycerol
produced from biodiesel production as a sole carbon sources for rhamnolipid production
which yield 1.36 g L-1 meanwhile Moya Ramírez et al., (2015) used olive mill waste produce
much lower rhamnolipid production of 0.56 g L-1. George and Jayachandran, (2013) present a
study that produce 1.97 g L-1 of rhamnolipid from used coconut oil meanwhile soap stock
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from refining vegetable oil process was used as substrate for rhamnolipid production by
Benincasa et al., (2004) producing 15.8 g L-1. All the studies mentioned used different types
of P. aeruginosa strain thus it shows its ability to used fatty acid as main substrate for
rhamnolipid production, and the production from water-immiscible substrate such as fatty
acid has better production than water -soluble substrate like glucose and glycerol (Banat,
1997).
In this present research, the high fatty acid content of PFAD and FAME have been
investigated as sole carbon sources for rhamnolipid (RL) production using Pseudomonas
aeruginosa PA01 in a minimal culture medium. Cell growth, RL production, RL yields and
RL characteristics were determined and compared to other reports of value added
biosurfactant production routes using agricultural wastes. The novelty of this study is the
production of significantly increased amounts of RL when using minimal media, compared to
our previous study, using PFAD as the sole carbon source, demonstrating the technical
feasibility of producing meaningful amounts of RL from palm oil refinery mill by-product. In
a broader biorefining strategy, it is advantageous to have alternative valorisation routes for
PFAD and FAME which can be utilised based on the prevailing market conditions. One
option is to use the FAME as a substrate for RL production, thus providing an alternative
valorisation route that could be employed as part of a wider palm oil biorefinery industry.
This investigation expands our knowledge and understanding of the usage of palm oil
refinery agricultural waste for RL production and its potential to be transformed into a
valuable product that can be used in various applications.
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2. Materials and Methods
2.1 Esterification of PFAD
Palm fatty acid distillate (PFAD) was converted into fatty acid methyl ester (FAME)
by esterification. The FAME produced was subsequently used as a substrate to produce
rhamnolipids by fermentation. PFAD was obtained from Sime Darby-Jamolina and The
Cucurbit Company Sdn. Bhd., Malaysia and dried in a drying oven at 70°C for one day. The
esterification was then carried out using a 10:1 ratio of methanol to PFAD, with the addition
of sulfuric acid (2.5% weight of PFAD) as a catalyst. The reaction was then carried out at
100C for 1 hour, using a reflux condenser. The product was then cooled, transferred to a
separating funnel and washed with hot distilled water until the bottom water layer becomes
evident and the pH reached 7.
2.2 Microorganism
Pseudomonas aeruginosa PAO1 was supplied by the School of Biomedical Sciences,
Ulster University from their culture collection. The strain was stored at −80°C as master
stock and working culture was preserved at 4°C on nutrient agar plates.
2.3 Media and Culture Condition
To prepare cultures P. aeruginosa PAO1 was first spread onto a nutrient agar petri
dish and incubated at 37°C for 24 h. There were two types culture medium used in this
fermentation. The first culture medium used for seed culture was the Protease Peptone
Glucose Ammonium Salt (PPGas) medium which consisted of 0.5 g L−1 MgSO4.7H2O, 10 g
L−1 peptone, 19 g L−1 Tris-HCl, 1.5 g L−1 KCl, 1 g L−1 NH4Cl and 1% glucose. The second
culture medium used was minimal medium (MM) which consisted of 0.5 g L−1
MgSO4.7H2O, 1.0 g L−1 KCl, 0.3 g L−1 K2HPO4, 1.0 g L−1 NaNO3. Trace elements for MM
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were 2 g L−1 C6H5Na3O7·2H2O, 0.28 g L−1 FeCl3.6H2O, 1.4 g L−1 ZnSO4.7H2O, 1.2 g L−1
CoCl2.6H2O, 1.2 g L−1 CuSO4.5H2O and 0.8 g L−1 MnSO4.H2O.
Fermentation experiments were conducted in 5 L shake flasks in triplicate, using 1L
of MM supplemented with the desired amount of glucose, PFAD or FAME and were carried
out at 37C over three days. Inocula were prepared in two stages; in stage 1 50 mL of PPGas
medium containing 1% glucose in a 250 mL flask was inoculated with one loop of bacteria
into and grown at 37C for 24 h. In stage 2, 40 mL of stage 1 culture were transferred to 400
mL PPGas media with 1% of glucose in a 2 L flask and grown for 24 h. For the preparation
of the final inoculum, 100 mL of stage 2 culture was centrifuged for 10 min and the cell
pellet resuspended in 100 mL of sterile distilled water. This was then used to inoculate the 1
L MM in 5 L shake flask. The initial concentration of the fermentation was maintain at 0.2 g
L−1.
2.4 Growth Measurement
Cell growth was quantified by measuring optical density (OD) from which dry cell
weight (DCW) was calculated using a linear correlation DCW =0.4639(OD)+0.0276 with
R2=0.87 for glucose and DCW =0.5871(OD)+0.1014 with R2=0.92 for fatty acid substrate.
PFAD and FAME were removed from samples by adding 0.5 mL of n-hexane to 1 mL
fermentation broth and centrifuging at 13000 g using a Minispin Centrifuge (Eppendorf). Cell
biomass was re-suspended in 0.7% sodium chloride solution (physiological saline) and the
OD determined by measuring absorption at 600 nm using a UVmini-1240 Spectrophotometer
(Shimadzu, USA).
2.5 Rhamnolipid Extraction
Rhamnolipids were extracted from 10 mL samples of fermentation broth. Firstly
samples were centrifuged for 10 min and the supernatant taken and acidified with1 M
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hydrochloric acid to form a precipitate at pH 3. The acidified supernatant was then mixed
with an equal volume of ethyl acetate and shaken vigorously. The ethyl acetate washing
process was repeated three times, and lastly, traces of water present in the RL containing
ethyl acetate layer were removed by using 0.5 g of magnesium sulphate per 100 mL. Finally,
the samples were filtered, and the solvent evaporated using a rotary evaporator (Cole-Parmer
Ltd, model RE300) at 70 °C to give a crude rhamnolipid biosurfactant extract. The RL
concentration was then determined gravimetrically.
2.6 Biosurfactant Identification
Mass Spectrometry-Electrospray Ionization (MS-ESI) was used for biosurfactant
identification. An Agilent 6510 Q-TOF LC/MS equipped with Agilent 1200 Liquid
Chromatography (LC) was used with 5 µL of crude rhamnolipids extract, diluted in
methanol, injected using 50% ACN with 0.1% formic acid as an eluent with electrospray
(ESI) in negative mode (Smyth et al., 2016).
2.7 Biosurfactant Characterization
A Krüss K11 Tensiometer equipped with a De Nöuy ring was used to measure
equilibrium surface tension and determine the critical micelle concentration (Smyth et al.,
2016). The crude rhamnolipid extract was diluted with 0.1 M Tris-HCl pH 8.0 solution, from
an initial concentration of 1000 mg L−1, and the equilibrium surface tension determined. The
emulsion index was measured over 24 h and calculated as the percent of the height of the
emulsified layer relative to the total height of the liquid. A solution of 3 mL of dissolved
crude rhamnolipids, initial concentration 1000 mg L−1 in 0.1 M Tris-HCl pH 8.0, was mixed
with 3 mL of sunflower oil or kerosene and shaken vigorously for 1 min to obtain maximum
emulsification.
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2.8 Gas chromatography analysis
To characterise the PFAD and FAME, 0.2 g of the PFAD, FAME and emulsified
PFAD after fermentation respectively were vortex mixed with hexane and 0.1 mL methanolic
potassium hydroxide for 30 s and the mixture was centrifuged for 10 min. After centrifuging,
0.2 mL of the top layer was mixed with 2 mL of hexane and 0.1 µL was injected into GC-MS
for analysis. A BPX 70 capillary column (SGE, length: 60 cm, ID: 0.22 mm and film
thickness: 0.25 µm) was used for separation of FAME compounds. The GC injection port
was set at 155°C, and the detector temperature was 220°C. The GC oven was programmed
with a temperature ramp from 155°C to 180°C at 2°C/min and then from 180°C to 220°C at
4°C/min.
3. Results and Discussion
3.1 Esterification of PFAD
Biodiesel or fatty acid methyl ester (FAME) has attracted much research and
industrial interest due to depletion of petroleum reserves, an increasing awareness of
environmental issues and drove towards sustainability (Hosseini et al., 2015). FAME can be
produced from renewable resources that contain a high percentage of free fatty acids, such as
PFAD (Lokman et al., 2014). One of the methods to derive FAME from renewable resources
is esterification, using methanol in the presence of an acid as a catalyst (Yadav et al., 2010).
In this work, esterification using methanol and sulfuric acid was used to convert PFAD into
FAME. The esterification reaction significantly transforms the physical properties of PFAD
from solid to liquid FAME at room temperature. Due to this change, it was expected that the
availability of FAME in culture medium for fermentation process would be higher than that
of solid PFAD, thus promoting improved rhamnolipid production by P. aeruginosa PAO1.
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In order to assess the availability of the substrates to the microorganism, three
different substrate types; PFAD, FAME and emulsified PFAD after fermentation were
analysed using GC-MS to determine any significant differences in fatty acid composition
between the substrates. Table 1 showed the main fatty acid components of PFAD, FAME and
emulsified PFAD after fermentation. As expected, PFAD and FAME contain the same fatty
acid components, as the methyl esters are derived from the fatty acids present in the PFAD.
The main elements of PFAD/FAME are stearic acid 50.18%, pentadecanoic acid 16.98% and
palmitic acid 16.91%. Meanwhile, the emulsified fraction of PFAD after fermentation was
42.9% stearic acid and 32.4% palmitic acid, which is consistent with Gapor Md Top (2010).
This leads to the understanding that for PFAD substrate, primarily stearic palmitic acid was
available to P. aeruginosa PAO1 for growth and rhamnolipid production, with both fatty
acids emulsified in the culture medium. Other fatty acids might have formed sticky solids
clumps during the fermentation process, the formation of solid lumps which accumulated on
the surface of the culture media was observed during shake flask experiments. Meanwhile
for FAME, being liquid at room temperature may help to increase the availability of the free
fatty acid content to the microbial culture for growth and rhamnolipid production, mitigating
issues with poor emulsification of the PFAD substrate
PFAD and FAME have the potential to be utilised as renewable substrates for strain
growth and rhamnolipid production as they contain various types of free fatty acids at high
concentrations (Nazren Radzuan et al., 2016). In 2016, several researches reported the use of
fatty acid containing substrates for rhamnolipid production with P. aeruginosa strains. For
example Reddy et al. (2016) used mango kernel waste, Ji et al. (2016) used olive oil, Gudiña
et al. (2016) and Moya Ramírez et al. (2016) used olive oil mill wastewater and Lotfabad et
al. (2016) used soybean oil waste as the sole carbon sources for rhamnolipid production. This
high number of publications highlighted using fatty acid waste as substrates shows the
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potential in promoting rhamnolipid production, through utilising low-cost substrates and
transforming fatty acid rich wastes into added value products.
3.2 Effect of type and concentration of carbon source on rhamnolipid production
3.2.1 General observations
A series of 5 L shake flask fermentation experiments were carried out using glucose,
PFAD and FAME as the sole carbon sources, to determine and compare the rhamnolipid
production and fermentation kinetics. The fermentation broth showed a significant change in
colour from colourless to green in all experiments. Pyocyanin pigment has been reported to
be responsible for the green colour change in the culture medium, and this pigment also has a
positive correlation to the growth of this strain (El-Fouly et al., 2015). At the end of
fermentations, the foam was observed accumulating on the top of the culture medium which
is caused by the production of extracellular rhamnolipids, the surface-active target product
(Junker, 2007). The occurrence of foaming during rhamnolipid production has also been
observed by other researchers such as Funston et al. (2016), Díaz De Rienzo et al. (2016) and
Lotfabad et al. (2016).
3.2.2 Glucose as the carbon source
Rhamnolipid production using 10 g L−1 and 20 g L−1 of glucose as the sole carbon
source under batch fermentation conditions is presented in Error: Reference source not
found(a). This experiment acts as a baseline control to allow for the major rhamnolipid
congeners produced to be identified and to compare growth, production, and yields to those
obtained when using fatty acids from PFAD and FAME (Zhang et al., 2012). Steady growth
and rhamnolipid production were observed during the experiment to 72 h and 84 h at initial
glucose concentrations of 10 g L−1 and 20 g L−1 respectively, after which time the growth rate
decreased until the end of fermentation. In detail, using 20 g L−1 of glucose the final dry cell
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weight (DCW) was 1.2 g L−1, and the final rhamnolipid (RL) concentration was 2.0 g L−1.
While with 10 g L−1 of glucose, lower DCW and RL of 1.0 g L−1 and 1.34 g L−1 were reached
respectively. Tiso et al. (2016) and Varjani and Upasani (2016) reported that when using 10 g
L−1 of glucose as, P. putida KT2440 and P. aeruginosa NCIM 5514 were able to produce 3.0
g L−1 of RL while Moya Ramírez et al. (2016) showed that using P. aeruginosa PAO1, a
lower RL production of 0.045 g L−1 was achieved when using 2 g L−1 of glucose. This result
also suggests that by using P. aeruginosa PAO1 in batch fermentation, up to a point, the
higher the concentration of glucose the better the cell growth and RL production, which is
consistent with Clien et al. (2007). Clien et al. (2007) also reported that P. aeruginosa S2
could significantly increase its RL production from 0.5 g L−1 to 2.5 g L−1 when doubling the
initial glucose concentration from 20 g L−1 and 40 g L−1. However, Varjani and Upasani
(2016) observed the inverse pattern for RL production by P. aeruginosa NCIM 5514 which
decreased from 3.0 g L−1 to 1.0 g L−1 when increasing the initial glucose concentration from
10 g L−1 to 50 g L−1. The differences in RL production with varying initial glucose
concentrations may be caused by the varying characteristic of the different strains and the
influence of culture conditions, which potentially affect the growth kinetics and yields.
3.2.3 Palm fatty acid distillate (PFAD) and Fatty acid methyl ester (FAME) as sole carbon
sources
PFAD and FAME have similar free fatty acid content but have significant differences
regarding physical characteristics, as discussed in Section 3.1. PFAD is the raw, solid form of
the by-product from palm oil agricultural refinery mill. Meanwhile FAME is biodiesel
derived from PFAD via esterification process. Both substrates have been used as sole carbon
sources in this experiment to understand their suitability for use as substrates and the
determine differences in cell growth, RL production and characterisation. PFAD was also
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used as the sole carbon source for RL production in minimal media, leading to increased
production in comparison to earlier reports (Nazren Radzuan et al., 2016).
PFAD was used as the sole carbon source at initial concentrations of 10 g L−1 and 20 g
L−1. Error: Reference source not found (b) presents the fermentation kinetics observed, in
which better growth and RL production were observed at 10 g L−1 initial PFAD compared to
the case with 20 g L−1 PFAD. Growth and RL production increased to a maximum level at 72
and 84 h for 10 g L−1 and 20 g L−1 respectively. The maximum DCW and RL concentration
for 10 g L−1 of PFAD were 3.3 g L−1 and 3.4 g L−1 while at 20 g L−1 of PFAD 2.6 g L−1and 3.1
g L−1 of DCW and RL were produced respectively.
FAME produced from the esterification of PFAD was used as the sole carbon source
with initial concentrations of 10 and 20 g L−1. Error: Reference source not found (c) shows
growth and more RL production at higher FAME concentration. With 10 g L−1 of FAME, the
DCW and RL concentrations were 3.1 g L−1 and 2.5 g L−1, reaching the maximum level at 72
h, while with 20 g L−1 of FAME the DCW and RL concentration was 2.8 g L−1 and 3.0 g L−1 ,
respectively, which was the maximum level reached after 84 h.
P. aeruginosa PAO1 can grow and produce biosurfactant in minimal culture medium
using PFAD and FAME as sole carbon sources with a maximum crude RL concentration in
the range 3-3.5 g L−1, which is in the middle range of reported RL production based on Table
2. The different effects of varying substrate concentration on RL production were seen in
Error: Reference source not found, which were mainly due to the different physical
characteristics of PFAD, being solid at the fermentation temperatures compared with FAME
which was in liquid form. This plays an important role in the availability of the substrate to
the strain and also how the carbon sources are converted into RL.
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3.2.4 Comparison of Yields
Table 2 shows the comparison of results obtained from this study to those reported by
other researchers, in terms of maximum biomass produced (DCWmax), maximum rhamnolipid
produced (RLmax), biomass formed related to initial substrate (*YX/S, g g−1), product yield
related to initial substrate (*YP/S, g g −1) and volumetric productivity (PRL, g L−1 h−1) at
different concentrations of glucose, PFAD, FAME and other substrates used. In this study,
maximum RL production and the time taken to reach this point for each substrate
concentration were the criteria used to determine the *YX/S, *YP/S, YP/X and PRL. Generally, the
*YX/S,*YP/S, and PRL observed for glucose were significantly lower compared to PFAD and
FAME because using glucose shows lower growth and RL production throughout the
fermentation process. PFAD can be potentially used as low-cost renewable substrate for
rhamnolipid production using P. aeruginosa PAO1 as it shows better growth, substrate
conversion into the product and high productivity.
The most favourable free fatty acid substrate and concentration found in this study for
growth and substrate conversion to RL is 10 g L−1 of PFAD with the highest *YX/S of
0.337 g g−1 and *YP/S of 0.343 g g−1, compared to other renewable substrates such as mango
kernel oil, crude oil, olive mill waste, olive oil and soybean oil soap stock (Gudiña et al.,
2016; Ji et al., 2016; Lotfabad et al., 2016; Reddy et al., 2016; Varjani and Upasani, 2016).
However, the PRL for 10 g L−1 of PFAD is 0.048 g L−1 h−1 is the second highest value
compared the PRL from 40 g L−1 of olive oil which is 0.130 g L−1 h−1, mainly because of
higher RL production by P. aeruginosa M408 during the fermentation process (Ji et al.,
2016).
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3.3 Rhamnolipid Identification
The mass spectra (MS) of crude RL produced by P. aeruginosa PAO1 using glucose,
PFAD and FAME all showed similar peaks with various types of RL congeners being present
with molecular weight in the range 400 to 700 m/z and a high abundance of molecular ions at
475, 503, 529, 621, 649 and 647 m/z. As seen in Table 3, the m/z values are consistent with
the molecular structure of Rha-C8-C10, Rha-C10-C10, Rha-C12:1-C10, Rha-Rha-C8-C10, Rha-Rha-
C10-C10 and Rha-Rha-C8-C12 respectively. Specifically, the monorhamnolipid (Rha-C10-C10)
was present in a significantly lower abundance compared to dirhamnolipid (Rha-Rha-C10-
C10). This result is consistent with other researchers (George and Jayachandran, 2013; Nazren
Radzuan et al., 2016). Table 3 shows the ratio of mono- to di- rhamnolipids produced during
the fermentation experiments. Whilst dirhamnolipid were more abundant for all substrates
used the ratio of Rha-C10-C10 (mono) to Rha-Rha-C10-C10 (di) was greater in the case of
production from PFAD and FAME compared to glucose. For PFAD and FAME ratios of
approximately 1:2 and greater were observed, compared to a ratio of 1:1.3 for glucose. This
suggests that the substrate can be selected to favour the production of particular rhamnolipid
congeners. It was demonstrated that the strain could produce monorhamnolipid and
dirhamnolipid, with dirhamnolipid being the most abundant at the end of the fermentation
process. However, the type of congener and overall rhamnolipids mixture composition is
typically affected by many factors such as types of strain, type of carbon source, the age of
culture and culture conditions (Aparna et al., 2012).
3.4 Rhamnolipid characterization
The crude RL extracted from experiments with glucose, PFAD and FAME were
characterised by their capability to reduce the surface tension of a solution, the critical
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micelle concentration and the ability to emulsify kerosene and sunflower oil. Figure 2 shows
the ability of crude RL product by P. aeruginosa PAO1 in this study to reduce the surface
tension of Tris-HCl pH 8.0 buffer to values between 29-32 mN m−1. Figure 2 also shows that
the crude rhamnolipid extract gave the lowest CMC value of 8 mg L−1 when glucose was used
as a substrate. Meanwhile, PFAD and FAME gave the same CMC value of 19 mg L−1. The
comparison of surface tension and CMC values are shown in Table 4, which demonstrates
that despite the variability observed, the surface tension values reported in this study are
comparable with those of others (Aparna et al., 2012; Lan et al., 2015; Varjani and Upasani,
2016). The CMC value for rhamnolipids produced from PFAD and FAME were lowest when
compared to those produced on other substrates such as waste cooking oil, glycerol, molasses
and sodium citrate, as shown in Table 4.
The emulsion index (EI) of crude RL extracts at a concentration of 1 g L−1are in the
range of 40-50 % for an emulsion of Tris-HCl pH 8.0 solution with either kerosene or
sunflower oil. The percentage of emulsification decreased as the crude rhamnolipids
concentration decreased, as shown in Figure 3. When compared to other literature results,
Table 4, the EI for this study is lower when compared with rhamnolipids produced from other
substrates such as crude oil and waste cooking oil (Varjani and Upasani, 2016).
The results of this study vary when compared with other reported research listed in in
Table 4, suggesting that the crude rhamnolipid produced by different Pseudomonas strains
and substrates are different regarding component and concentration of each congener, and
degree of purity thus affecting the surface tension, CMC and emulsion index characteristics.
The concentration of the crude rhamnolipid has a significant effect on the functionality of the
biosurfactant as an emulsifier.
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3.5 Effect of culture media on growth and production
In this section, we discuss the effect using either PPGas or minimal media on P.
aeruginosa PAO1 growth, rhamnolipid production and characteristics, yields and the cost of
culture media in 5 L shake flask fermentation using 20 g L−1 of PFAD as the sole carbon
source. A comparison between this study and a previous study by Nazren Radzuan et al.,
2016 is noteworthy. Figure 4 (a) shows the dry cell weight for both media were almost the
same, 2.52 g L−1 using PPGas media and with minimal media, DCW 2.66 g L−1. Meanwhile,
significant differences were found in rhamnolipid production on minimal media with 3.19
g L−1 of rhamnolipids, 8.4 times higher than the rhamnolipid concentration attained using
PPGas media. This significant increase in RL production is suggested mainly because of
nitrogen limitation. PPGas media contains peptone, a source of amino acids, peptides and
protein, meaning the media contains a high amount of nitrogen in excess of the nitrogen
present in minimal medium, which contains 1 g L-1 of nitrogen from NaNO3. Nitrogen
limitation potentially stimulated over production of rhamnolipid and caused the cell growth to
enter stationary phase (Banat, 1997). In Figure 4 (b), the *YP/S and PRL from using minimal
media are 0.160 g g−1 and 0.038 g L−1 h−1, higher when compared to PPGas media, *YP/S 0.019
g g−1 and PRL 0.0014 g L−1 h−1. The comparison of rhamnolipid characteristics regarding
surface tension, CMC and EI is shown in Figure 4 (c).
The ability of the rhamnolipids produced from PPGas media and minimal media to
reduce surface tension were similar, 29.00 mN m−1 and 31.73 mN m−1.The EI with kerosene
and sunflower oil of rhamnolipid produced from minimal media also showed a higher result
of 40% and 45%, compared to rhamnolipids produced from PPGas with an EI of 25% and
30% for kerosene and sunflower oil respectively. Lastly, Figure 4 (d) shows the lower media
cost of rhamnolipid production using minimal media of £0.07 per litre, compared £2.40 per
litre when using PPGas media. This indicates that rhamnolipids produced from minimal
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media performed better than those from PPGas media regarding production, yields,
characteristic and cost. The minimal media is the best for scale up in the bioreactor and has
greater potential to be commercialised for rhamnolipid production by P. aeruginosa PAO1
using PFAD as the sole carbon source.
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4. Conclusion
The use PFAD and FAME as sole carbon sources for rhamnolipid production by P.
aeruginosa PAO1 using minimal media significantly improved rhamnolipid production from
1 g L-1 to 3 g L-1 with a *YX/S of 0.3 g g−1, a*YP/S of 0.3 g g−1and PRL of 0.4 g L−1 h−1.
Furthermore, both substrates PFAD and FAME have the potential to be used as renewable
substrates in larger scale RL production. Based on the results of this study, using minimal
media an estimated of 2 × 105 tonnes yr−1 of RL can be produced from the PFAD available
from palm oil refining, which at current RL prices can generate an estimated £200 million
gross income. Since PFAD is a low-cost by-product from palm oil refinery mills, this will
significantly decrease RL production cost. In addition, FAME can be further utilised by
providing a new value-added route for rhamnolipid production, rather than limiting use to
only the production of biofuels for transportation. This would be advantageous in a palm oil
biorefinery where the overall production strategy could be tailored to maximise added value,
with the flexibility to further convert FAME to biosurfactant in the face of market variation
for biodiesel. This flexibility is crucial for future palm oil biorefineries economic feasibility.
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Acknowledgements
This study is financially supported by Ministry of Education, Malaysia and Department of
Biological and Agricultural Engineering, Universiti Putra Malaysia under “Hadiah Skim
Latihan IPTA (SLAI)”. The authors would also like to thank Sime Darby Refinery-Jamolina
and The Cucurbit Company Sdn. Bhd. for providing the PFAD used in this research.
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Figure Captions
Error: Reference source not found Time course profiles of P. aeruginosa PAO1 cell
growth and rhamnolipid production at 200 rpm and 37 °C by using (a)
Glucose, (b) PFAD and (c) FAME as carbon sources. Solid line is 10 g L−1 and
dashed line is 20 g L−1 of substrate fed. (■) is dry cell weight and (●) is
rhamnolipid production.
Figure 2 Critical micelle concentration (CMC) of rhamnolipid from glucose, PFAD and
FAME. (■) Glucose, (●) PFAD and (▲) FAME.
Figure 3 Emulsion Index of rhamnolipids with (a) kerosene and (b) sunflower oil. (■)
Glucose, (●) PFAD and (▲) FAME.
Figure 4 Bar chart comparison of (a) Growth and Production, (b) Yields, (c)
Rhamnolipid Characteristics and (d) Cost for different culture medium in 5 L
shake flask fermentation using 20 g L−1 of PFAD as sole carbon source. ( )
denotes PPGas and ( ) Minimal Media
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Table 1 Fatty acid content of PFAD, FAME and emulsified PFAD fraction
Substrates ComponentChemical
formula
Percentage
(%)
PFAD and
FAME
Stearic acid C18H36O2 50.18
Pentadecanoic acid C15H30O2 16.98
Palmitic acid C16H32O2 16.91
Linoleic acid C20H35ClO2 5.59
Squalene C30H50 4.16
Margaric Acid C17H34O2 2.95
Tridecanoic acid C15H30O2 0.93
Myristic acid C15H30O2 0.84
2,6,10,14,18-Pentamethyl-2,6,10,14,18-
eicosapentaene C25H42 0.57
Arachidic acid C20H40O2 0.46
Oleic acid C18H34O2 0.28
2-Nonadecanone C19H38O 0.15
Emulsifie
d PFAD
Stearic acid C18H36O2 42.49
Palmitic acid C16H32O2 32.41
Squalene C30H50 11.99
2,6,10,14,18-Pentamethyl-2,6,10,14,18-
eicosapentaene C25H42 5.99
Pentadecanoic acid C15H30O2 3.56
Tridecanoic acid C15H30O2 3.56
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Table 2 Comparison of maximum biomass produced (DCWmax), maximum rhamnolipid produced (RLmax), biomass formed related to
initial substrate (*YX/S, g g−1), product yield related to initial substrate (*YP/S, g g −1), product yield related to biomass formed
(YP/X, g g−1) and volumetric productivity (PRL, g L−1 h−1) on different concentration of glucose, PFAD and FAME.
Types of
fermentationMicroorganism Substrate
Concentration
(g L−1)Timemax
(h)
DCWmax
(g L−1)
RLmax
(g L−1)
*YX/S (g
g −1)
*YP/S (g g
−1)
PRL
(g L−1 h−1)References
5 L flaskP. aeruginosa
PAO1
Glucose10 72 1.033 1.340 0.103 0.134 0.019
This study
20 84 1.244 2.040 0.062 0.102 0.024
PFAD10 72 3.371 3.433 0.337 0.343 0.048
20 84 2.665 3.190 0.133 0.160 0.038
FAME10 72 3.113 2.537 0.311 0.254 0.035
20 84 2.818 3.090 0.141 0.155 0.037
250 mL flaskP. aeruginosa
DR1
Glucose 10 72 1.430 1.050 0.143 0.105 0.015
(Reddy et al.,
2016)
Palmitic
Acid10 72 0.960 0.350 0.274 0.035 0.005
Stearic 10 72 1.610 0.800 0.161 0.080 0.011
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573
574
575
Acid
Oleic Acid 10 72 1.800 0.880 0.180 0.088 0.012
Mango
Kernel Oil10 72 1.560 1.810 0.156 0.181 0.025
250 mL flaskP. aeruginosa
NCIM 5514
Glucose 10 96 3.872 2.946 0.387 0.295 0.031 (Varjani and
Upasani,
2016)Crude Oil 10 96 2.482 1.917 0.248 0.192 0.020
250 mL flaskP. aeruginosa
#112
Olive Mill
Waste150 168 - 5.030 - - 0.030
(Gudiña et
al., 2016)
Shake flask
(100 mL
medium
P. aeruginosa
M408Olive oil 40 96 - 12.440 - - 0.130
(Ji et al.,
2016)
250 mL flaskP. aeruginosa
MR01
Soybean
Oil Soap80 240 3.000 18.000 0.0375 0.225 0.075
(Lotfabad et
al., 2016)
*YP/S and *YX/S are using initial substrate fed, calculated only for this study
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577
Table 3 Chemical composition and mono- to di-rhamnolipid ratio of rhamnolipid
mixture produced by P. aeruginosa PAO1 from mass spectrometry analysis.
Ratio of mono- to di- rhamnolipid (Rha-C10-
C10: Rha-Rha-C10-C10) Rhamnolipid congeners
(Pseudomolecular Ion, m/z)
Carbon source Ratio
Glucose 1:1.28 Rha-C8-C10 (475)
Rha-C10-C10 (503)
Rha-C12:1-C10 (529)
Rha-Rha-C8-C10 (621)
Rha-Rha-C10-C10 (649)
Rha-Rha-C10-C12 (677)
PFAD 1:2.52
FAME 1:2.38
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Table 4 Comparison of effect of different carbon sources and microorganism on surface
tension (mN m−1), critical micelle concentration (CMC, g L−1) and emulsion
index (E24, %) with previous research.
Microorganis
m
Substrat
e
Surface
tension
reductio
n (mN
m−1)
CM
C
(mg
L−1)
Emulsion Index (%)
Reference
sRhamnolipid
Concentratio
n (g L-1
Sunflowe
r Oil
Kerosen
e
P. aeruginosa
PAO1
Glucose 32.24 8
1.00
42.26 44.64
This
studyPFAD 31.73 19 40.47 46.43
FAME 29.60 19 39.28 46.43
P. aeruginosa
NCIM 5514
Glucose 29.42 -
-
- 82.34 (Varjani
and
Upasani,
2016)
Crude
Oil36.70 - - 53.60
Pseudomonas
SWP-4
Waste
Cooking
Oil
24.1 27 10.50 - 62.40(Lan et
al., 2015)
P. aeruginosa
DS10-129
Glycero
l27.90 22 - - 71.30
(Rahman
et al.,
2010)
Pseudomonas
sp. 2B
Molasse
s30.14 100 0.02 84.00 67.50
(Aparna
et al.,
2012)
32
580
581
582
P. aeruginosa
B-59188
Sodium
Citrate-
56.0
01 5.00 -
(Hošková
et al.,
2015)
33
Figure 1 Time course profiles of P. aeruginosa PAO1 cell growth and rhamnolipid
production at 200 rpm and 37 °C by using (a) Glucose, (b) PFAD and (c)
FAME as carbon sources. Solid line (—) is 10 g L-1 and dashed line (----) is
20 g L−1 of substrate fed. (■) is dry cell weight and (●) is rhamnolipid
production.
34
(a)
(b)
(c)
583
584
585
586
587
588
589
590
591
592
593
594
595
596
597
598
599
600
601
602
603
604
605
606
607
608
Figure 2 Critical micelle concentration (CMC) of rhamnolipid from glucose, PFAD and
FAME. (■) Glucose, (●) PFAD and (▲) FAME.
35
609
610
611
612
613
614
615
616
617
618
619
620
621
Figure 3 Emulsion Index of rhamnolipids with (a) kerosene and (b) sunflower oil. (■)
Glucose, (●) PFAD and (▲) FAME.
36
(b)
(a)
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
644
645
Figure 4 Bar chart comparison of (a) Growth and Production, (b) Yields, (c) Rhamnolipid Characteristics and (d) Cost for different
culture medium in 5 L shake flask fermentation using 20 g L−1 of PFAD as sole carbon source. ( ) denotes PPGas and ( )
Minimal Media
37(d)(c)
(b)(a)
646
647
648
38
649
651
652
653