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: james.winterburn@manchester.ac.uk (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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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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576

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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