substrate dependent production and isolation of an...

Philippine Journal of Science141 (1): 13-24, June 2012ISSN 0031 - 7683Date Received: 01 Apr 2011

Key Words: emulsion, emulsification activity, emulsification index, extracellular, substrate

*Corresponding author: [email protected]

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Virgie A. Alcantara1*, Irene G. Pajares1, Jessica F. Simbahan1,and Ma. Leah D. Rubio2

1Environmental and Industrial Biotechnology Program, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines, Los Baños

2Thesis Student, B.S. Biochemistry, University of the Philippines - Manila

Substrate Dependent Production and Isolationof an Extracellular Biosurfactant from

Saccharomyces cerevisiae 2031

Improvements of both biomass yield and emulsification activity are important criteria for profitable biosurfactant production. In this study, Saccharomyces cerevisiae 2031 gave the highest emulsification activity (E24 = 58%) by using Cooper and Paddock’s basal medium, containing glucose and waste cooking oil as carbon sources. Glucose and waste cooking oil were found to be essential for high biomass and emulsification activity. Emulsification activity of the biosurfactant increased to 76% after optimization of fermentation conditions. The optimum carbon source concentration for both glucose and waste cooking oil was 5%. Optimum pH for high biomass production was pH 5.0 – 8.0. Isolation of the biosurfactant by heat treatment of the S. cerevisiae 2031 cells effectively solubilised the extracellular biosurfactant.

INTRODUCTIONBiosurfactants or microbial surfactants are surface active biomolecules produced by microorganisms. These molecules are capable of reducing surface and interfacial tensions in both aqueous solutions and hydrocarbon mixtures (Ferraz et al. 2010). They include low-molecular-weight glycolipids, lipopeptides and high-molecular-weight polysaccharide-protein-fatty acid complexes (Ron and Rosenberg 2001). High molecular weight biosurfactants produce stable emulsions without lowering surface or interfacial tension and they are called bioemulsifiers (Bognolo 1999). The biosurfactant from S. cerevisiae 2031, possessed these aforementioned characteristics as shown by its high emulsification activity and stability (Edding 2009). Low molecular weight biosurfactants on the other hand, lower surface and interfacial tensions.

Emulsifiers and surfactants are used interchangeably (Sekhon et al. 2011, Navon-Venezia et al. 1995; Conlette 2011, Sarubbo 2006). Although nearly all emulsifiers are considered surfactants not all surfactants are emulsifier. Emulsifiers reduce interfacial tension between oil and water, minimizing surface energy through the formation of globules (Zajic and Seffens 1984).

Surfactants are widely used in pharmaceutical, cosmetic, petroleum and food industries. They are mostly petroleum-based and produced by chemical means. These compounds are toxic to the environment and their use may lead to environmental problems. Bio-accumulation, toxicity and biodegradability of surfactants are therefore issues of high concern. An alternative to chemical surfactants are biosurfactants/bioemulsifiers which possess good functional properties with low environmental impact. They can be synthesized from renewable feedstocks, typically sugars and vegetable oils,) and even have antimicrobial, antitumor and antiviral properties. They inhibit fibrin

Alcantara et al.: Substrate Dependent Production and Isolation of Extracellular Biosurfactant from S. cerevisiae 2031

Philippine Journal of ScienceVol. 141 No. 1, June 2012

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clot formation and they have ani-adhesive action against several pathogenic microorganisms (Cameotra and Makkar 2004; Singh and Cameotra 2004; Rodrigues et al. 2006). Biosurfactants and bioemulsifiers have higher biodegradability over chemical surfactants, high selectivity, higher foaming, lower toxicity, and stability at extreme temperatures, pH and salinity (Abouseoud et al. 2008; Desai and Banat 1997; Ilori and Amund 2001; Makkar and Cameotra 1999; Raza et al. 2007; Makkar and Cameotra 2002). They are useful surface active agents for emulsion polymerization, wetting, foaming, phase dispersion, emulsification and de-emulsification (Desai and Banat 1997).

Despite the numerous interesting properties of biosurfactants, high cost of production and low yields compared to commercially available surfactants, are major obstacles for their large scale application. Efforts are being directed towards reducing their production cost and increasing the yields by strain improvement, nutritional and environmental optimization or fermentor design, as well as using cheap and renewable substrates (Mulligan 2005).

Bioemulsifiers from yeasts are extracellular or bound to the cell wall (Cameron et al. 1988). Methods used to isolate/solubilize the bioemulsifier were heat treatment and Zymolyase digestion, SDS (sodiumdodecyl sulphate) or with reducing agents (Abramova et al. 2001; Moukadiri et al. 1997).

The carbon substrate is an important limiting factor in biosurfactant production (Sen 1997). The type of carbon substrate used for production influenced both the quality and quantity of biosurfactant (Robert et al. 1989; Panilaitis et al. 2007; Abouseoud et al. 2008; Das et al. 2009).

The potential commercial applications of bioemulsifiers/biosurfactants include bioremediation of oil-polluted soil and water, enhanced oil recovery, replacement of chlorinated solvents used in cleaning oil-contaminated pipes and formation of oil-in-water emulsions for the food and cosmetic industries.

The effects of carbon substrates (glucose and waste cooking oil) on biosurfactant production and emulsification activity and the most effective method of isolation of the extracellular biosurfactant were determined for S. cerevisiae 2031 in this study.

MATERIALS AND METHODSYeast Strains and Growth Conditions

Ten (10) Saccharomyces strains: 2003, 2008, 2010, 2012, 2013, 2014, 2015, 2020, 2023 and 2031 were obtained from the yeast collection of the Environmental

and Industrial Biotechnology Program, National Institute of Molecular Biology and Biotechnology (BIOTECH), University of the Philippines Los Baños (UPLB), Laguna. Y42 and Y49 were isolated from copra and water samples from an oil processing plant in Lucena, Quezon, Philippines. Yarrowia lipolytica, ATCC 8662, was used as reference strain. The strains were maintained in Yeast Extract-Malt Agar (YMAT) (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, Tween 80 10 mL, CaCl2 0.015%, Agar 1.5%) without glucose, supplemented with 1% (w/v) Tween 80 and 0.01% CaCl2 and stored at 4 ºC. Seed culture was prepared by transferring a loopful of cells grown for 24 hours in YMAT slants to 125 mL Erlenmeyer flasks containing 50 mL of Yeast Malt Extract Broth (YMB) 0.3% (w/v) malt extract, 0.5% (w/v) peptone and 1% glucose and incubating at ambient temperature (28-30 ºC) overnight.

Biosurfactant ProductionBiosurfactant production was carried out in 250 mL Erlenmeyer flasks containing 50 mL Cooper and Paddock’s medium (which contains 0.1% KH2PO4, 0.5% MgSO4·7H2O, 0.01% CaCl2, 0.01% NaCl, 0.5% yeast extract), 5% waste cooking oil and 8% technical grade glucose. The waste cooking oil was obtained from a fast food restaurant near UPLB. One mL of the seed culture was added and the flasks were incubated at ambient temperature (28-30 ºC) on a rotary shaker for 4 days. Cells were harvested by centrifugation at 3000 rpm for 10 minutes and boiled for 5 minutes.

The culture broth, supernatant, and cell suspension were tested for emulsification activity. The yeast strain with the highest emulsification activity was used to produce the biosurfactant for optimization of carbon source requirement and isolation.

Biomass concentrationBiomass concentration was determined by dry weight. Culture samples (1mL) were centrifuged at 3000 rpm for 10 min and the resulting cell pellets were washed with distilled water. Washed cells were then transferred to pre-weighed aluminum dishes and dried at 105 °C to constant weight.

Measurement of Emulsification activityEmulsification activity was measured using the method described by Cooper and Goldenberg (1986). Kerosene (6 mL) was added to 4 mL biosurfactant and vortexed at high speed for 2 mins. The emulsification index (E24) was obtained by dividing the height of emulsion layer by the total height, and multiplied by 100. Measurements were made using a vernier caliper after 24 hours.



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Isolation of BiosurfactantAfter 4 days of incubation, S. cerevisiae cells were harvested by centrifugation at 3000 rpm for 15 min, then suspended in 67 mM phosphate buffer, pH 7.0. The biosurfactant was isolated either by boiling the S. cerevisiae cells 100°C for 5 mins, autoclaving at 121°C for 5 mins, or incubating with Zymolyase 100T (0.021mg/mL) for 2 hours at 25 °C with gentle shaking. The cells were then centrifuged at 3000 rpm for 10 mins and the supernatants were collected and tested for emulsification activity.

One gram (1.0 g) of S. cerevisiae cells was used for all the treatments to isolate and extract the biosurfactant.

Optimization of Biosurfactant Production To determine the optimum conditions for biosurfactant production, S. cerevisiae 2031 was cultured using different concentrations of glucose and waste cooking oil ( 0 – 10% ) and pH values ( 1.0 – 12.0 ).

Biosurfactant production was carried out in 250 mL Erlenmeyer flasks containing 50 mL Cooper and Paddock’s basal medium amended with different concentrations of technical grade glucose and waste cooking oil. One mL of the isolate grown in YMB for 24 hours was used to inoculate the media and the flasks were incubated at ambient temperature with shaking for 4 days. Biomass and E24

were determined.

Residual Oil ConcentrationResidual oil concentration was estimated using n-hexane extraction method for oils and grease (US EPA, 1999). Culture broth was acidified with 1 mL 50% HCl (v/v) to break the emulsion and extracted with 5 mL n-hexane. Phase extraction was done using 250 mL separatory funnel. Extracts were dried with 0.1 g anhydrous Na2SO4 and filtered using Whatman No. 1 filter paper into pre-weighed beakers. Solvent was allowed to evaporate at room temperature in the fumehood and dried overnight in a vacuum oven at 70 °C. The extracts were then weighed.

Statistical AnalysisData were analyzed by one-way ANOVA to detect significance of treatment effect. Significance of treatment means were analyzed by Tukey’s test.

RESULTS AND DISCUSSION

Screening for Biosurfactant ProductionA total of 13 yeast strains were screened for biosurfactant production in Cooper and Paddock’s medium supplemented with 8% glucose and 5% waste cooking oil.

Only three isolates, S. coreanus 2023, S. cerevisiae 2031, and S. cerevisiae 2010, were found to produce biosurfactant with E24 of 53.25%, 62.44%, and 38.96%, respectively. During biosurfactant production, the flasks exhibited turbidity with visual disappearance of the oil layer and concomitant biomass and biosurfactant production. Biosurfactant from S. cerevisiae 2010 produced unstable emulsion unlike the stable emulsion produced by S. coreanus 2023 and S. cerevisiae 2031.

This ability of S. cerevisiae strains to produce biosurfactant indicates the potential of utilizing spent yeast, a by-product of fermentation industries, in the production of high value products like biosurfactants/bioemulsifiers (Cameron et al. 1988; Liu et al. 2008). Recovery of spent yeast would also reduce pollution from distilleries since most brewery liquid wastes end in wastewater disposal leading to contamination of natural water sources with organic material (Zechner-Krpan et al. 2010).

The standard organism used in this study was Yarrowia (Candida) lipolytica known to produce a polymeric biosurfactant , liposan (Zinjarde and Pant 2002). Liposan is an extracellular water soluble emulsifier synthesized by Candida lipolytica composed of 83% carbohydrate and 17% protein (Cirigliano and Carman 1984; Kappeli and Fiechter 1977). Among the 5 yeasts studied, Y42 produced the highest biomass (20.01 g/L) while S. coreanus 2023 produced the lowest biomass of 4.35 g/L (Figure 1). High biomass production was achieved with the addition of 8% glucose and 5% waste cooking oil to the medium. Furthermore, emulsification indices of the culture broths were very low or with no emulsification activity at all (0 - 3.79%) for all the yeast isolates. Boiling the S. cerevisiae cells for 5 minutes increased the E24 of the supernatants (19.59% - 56.88%) and cell suspensions (3.91% - 63%) for all the isolates (Figure 2). The resulting emulsions were thick, viscous and stable compared to the loose and unstable emulsions of the biosurfactant obtained without heating the cells. S. cerevisiae 2031 produced the highest E24 for both the supernatant and cell suspension among the strains tested. Therefore, this strain was used for further optimization studies.

The high emulsification activity of the cells after heat treatment proved that the biosurfactant is extracellular. The surface active compound was not released in the culture broth but remained associated in the cells and was extracted and solubilized only after heat treatment.

The biosurfactant produced by Saccharomyces cerevisiae is a polysaccharide-protein complex called mannoprotein. It has been shown to possess excellent emulsifier activity toward several oils, alkanes, and organic solvents (Cooper et al. 1988).



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Figure 1. Biomass of yeast isolates during biosurfactant production using Cooper and Paddock’s medium with 8% glucose and 5% waste cooking oil.

Saccharomyces coreanus 2023


Yarrowia lipolytica

Bio

mas

s (g/

L)

Y42 Y49

Figure 2. Emulsification index of broth, supernatant and cell suspension fractions of yeast isolates using Cooper and Paddock’s medium with 8% glucose and 5% waste cooking oil. Weight of cells = 1.0 g.


Yarrowia lipolytica

Emul

sific

atio

n In

dex

(E24

, %)

Y42 Y49

Saccharomyces coreanus 2023



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Several authors suggested that biosurfactants are produced as primary metabolite accompanying cellular biomass formation (Adebusoye et al. 2008; Abu-Ruwaida et al. 1991; Ilori et al. 2005). Parallel relationship between biomass production and emulsification activity was reported (Adebusoye et al. 2008) and also verified for S. cerevisiae 2031.

Optimization of Biosurfactant Production Concentration of glucose and waste cooking oil in Cooper and Paddock’s medium was optimized for growth of S. cerevisiae 2031. Biomass concentration increased linearly with increasing oil concentration but only up to 5% waste cooking oil, after which biomass production no longer increased (Figure 3a). Similar findings were made by

Yabes (2002) where biomass concentration of yeast strains increased linearly with increasing oil concentration but only up to 3% oil. Maximum biomass production of 9.62 – 10.51 g/L was obtained by using 5 - 10% waste cooking oil with glucose concentration kept constant at 8%. No significant difference in the biomass was seen at this range. On the other hand, biomass production was maximum, 8.72 – 10.57 g/L by using 5 – 9% glucose with waste cooking oil concentration kept constant at 5% (Figure 3b). Difference in the biomass was also not significant at this range.

Emulsification index (E24) was determined for varying combinations of cooking oil and glucose. When glucose was provided at 8% in combination with varying concentrations of cooking oil, no significant

Figure 3a and b. Biomass production of Saccharomyces cerevisiae 2031 during biosurfactant production using Cooper and Paddock’s medium with different amounts of ( •) waste cooking oil and (▲) glucose.

Biom

ass,

g/L

Biom

ass,

g/L

% Glucose

% waste cooking oil

12

10

8

6

4

2

0

12

10

8

6

4

2

00 2 4 6 8 10 12

0 2 4 6 8 10 12



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differences in E24 was observed (Figure 4). However when waste cooking oil at 5% was combined with different concentrations of glucose, E24 increased with increasing glucose concentration until 8%. Increasing the concentration of glucose above 8% with 5% waste cooking oil was not significant. The addition of glucose and waste cooking oil as carbon sources to Cooper and Paddock’s medium stimulated biomass production with a corresponding increase in emulsification activity. Based from these findings, 5% glucose and 5% waste cooking oil are optimum for high biomass production and emulsification activity.

Optimizing factors that affect growth of biosurfactant producing organisms with potential for commercial application is of paramount importance (Abouseoud et al. 2008).

Utilization of renewable and cheap substrates like waste cooking oil to produce a high value product will not only reduce waste generation and lower production cost (Dos Santos et al. 2010) but enable biosurfactants to be competitive with chemically synthesized surfactants.

Carbon sources from carbohydrates, hydrocarbons or vegetable oils, whether in combination or individually,

were found necessary to produce biosurfactants. Different microorganisms require different carbon sources for efficient biosurfactant production. Torulopsis bombicola ATCC 22214 utilized a carbohydrate in combination with vegetable oil (Cooper and Paddock 1984), Serratia marcescens favoured sunflower oil (Ferraz et al. 2002), Pseudomonas fluorescens preferred olive oil (Abouseoud et al. 2008), while Nocardia amarae gave high yields with corn oil or olive oil (Moussa et al. 2006). Candida bombicola and Pseudomonas aeruginosa utilized restaurant waste oil (Shah et al. 2008) and waste soybean oil (De Lima et al. 2008), respectively.

Biosurfactant produced by a marine bacterium was found dependent on the carbon substrates in terms of biomass yield and biomass production, surface tension reduction, emulsification index and antimicrobial action (Das et al. 2009).

The effect of varying the pH of the media to the biomass and emulsification activity of biosurfactant from S. cerevisiae 2031 was also evaluated. Maximum biomass production of 10.35 - 10.75 g/L was observed at pH 5 - 8. Biomass was lower at very acidic and highly alkaline pH's.

Figure 4. Emulsification index (E24) of biosurfactant from Saccharomyces cerevisiae 2031 using Cooper and Paddock’s medium with different amounts of glucose and waste cooking oil. Weight of cells = 0.2 g.



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The pH of the media played an important role in the production of the biosurfactants, sophorolipid from T. bombicola (Gobbert et al. 1984) and rhamnolipid from Pseudomonas sp. Rhamnolipid production was maximum at pH 6 - 6.5 and decreased sharply above pH 7.0 (Guerra-Santos et al. 1984). Optimum pH for biosurfactant production by Pseudomonas aeruginosa A41 using palm oil as carbon source was at pH 7 - 9 (Thaniyavarn et al. 2006).

Time-course study of biomass production of S. cerevisiae during biosurfactant production was monitored daily. Biomass was highest at day 6 (Figure 5). It was higher when the media was supplemented with glucose and waste cooking oil than glucose alone. Based from these results, waste cooking oil was utilized by S. cerevisiae 2031 as additional carbon source necessary for biomass production.

To determine the fate of waste cooking oil as carbon source during biosurfactant production, time course measurement of oil removal/utilization during biosurfactant production was done (Figure 6). Oil removal/utilization during biosurfactant production decreased with time with maximum oil utilization of 37.58% and 37.11% at day 6 and 7, respectively with no significant difference. Waste cooking oil utilization was confirmed by the decrease in the residual oil concentration per day (Figure 7). Maximum decrease in residual oil was also observed at day 6 when biomass production was high. This result

shows that the waste cooking oil was utilized by the microorganism for energy and biomass and product formation. Carbon and energy substrates are consumed not only for cellular growth but also for product formation (Gerhardt and Drew 1994). Biosurfactant production is triggered when the soluble carbon source had been consumed and the water –immiscible substrate is available (Banat 1995; Banat et al. 1991).

Isolation of Biosurfactant Methods for the isolation of biosurfactant depends on the nature of these compounds, whether it is water soluble or not, anionic or non-ionic, cell wall-bound or extracellular. A suitable method or a combination of methods for biosurfactant recovery for every new compound should be developed (Syldatk and Wagner 1987).

Emulsification activity of the biosurfactant isolated by heat treatments (boiling and autoclaving) and Zymolyase treatment was compared (Fig. 8). Boiling the S. cerevisiae cells gave the highest E24 of 79.44%. Autoclaving the cells at 121 ºC for 15 min resulted in a lower E24 of 73.49% which may have been caused by denaturation of some proteins resulting in lower emulsification activity. Zymolyase treatment gave the lowest E24 of 14.94%, possibly due to the incomplete digestion of cell wall glucans, releasing very little amount of mannoproteins.

Figure 5. Time-course of biomass production of Saccharomyces cerevisiae 2031.



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Days

0 2 4 6 8

% O

il R

emov

al

0

10

20

30

40

Figure 6. Oil removal/utilization during biosurfactant production by Saccharomyces cerevisiae 2031.

Days

0 1 2 3 4 5 6 7 8

Res

idua

l Oil

Con

cent

ratio

n (g

/L)

25

30

35

40

45

50

55

Figure 7. Residual oil concentration during biosurfactant production by Saccharomyces cerevisiae 2031.

Resid

ual O

il Co

ncen

tratio

n (g

/L)

Days

Days

% O

il Re

mov

al



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Boiling

Autoclaving

Zymolyase treatment

Em

ulsi

ficat

ion

Inde

x (%

)

0

20

40

60

80

100

Figure 8. Emulsification index (E24) of the biosurfactant using different extraction methods.

The protein component of mannoproteins played a significant role in emulsification activity (Sarubbo et al. 2006) and stability of emulsions (Iyer et al. 2006). The polysaccharide and protein components have no emulsification activity by themselves. However, the combination of polysaccharide and protein in mannoproteins led to efficient emulsification (Toren et al. 2001). The extracellular bioemulsifier from Acinetobacter calcoaceticus strain RAG-1 contains a non-covalently bound protein shown to significantly enhance the emulsifying activity of emulsan (Zosim et al. 1989). The active component of the biosurfactant/bioemulsifier of Acinetobacter radioresistens KA53 are the proteins (Toren et al. 2001).

The most common physical method of extracting biosurfactants from S. cerevisiae 2031 was heat-treatment at neutral pH (Peat et al. 1998) which solubilizes the structural mannoproteins in the cell wall. Mannoprotein was extracted in high yield from whole cells of baker’s yeast by autoclaving in neutral citrate buffer and by digestion with Zymolyase. (Cameron et al. 1988). Freimund et al. (2003), on the other hand, found that hot water extraction of yeast cell walls at 1250C was optimum.

CONCLUSIONThe carbon substrates, waste cooking oil and glucose, were found to affect biosurfactant production essential for high biomass yield and emulsification activity. These carbon substrates can be obtained from cheap alternatives. Waste cooking oil and glucose can be obtained from fast food restaurants and sugar processing industries respectively. Spent yeast from fermentation industries can also be utilized in the production of a high value product like biosurfactant. The use of inexpensive and renewable substrates make biosurfactant production economically viable. Considering the eco-friendly advantages and the various uses of biosurfactants, they can easily compete with chemical surfactants.

ACKNOWLEDGEMENTThis study is part of the BIOTECH-CORE project. The authors would like to thank Miss Buena R. Collado, Mrs. Teodora C. Ilagan, and Miss Victoria F. Gomez for the technical assistance.



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