Production of sophorolipids by the yeast Candida bombicola using simple and low cost fermentative media

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Abstract

In the present study, low cost media based on sugarcane molasses and three different oils, viz. soybean oil, sunflower oil or olive oil, were evaluated for the production of sophorolipids (SLs) from the yeast Candida bombicola in batch shake flasks. At the end of 5 days fermentation period, medium containing only molasses and soybean oil gave a maximum SLs yield of 23.25 ± 1.07 g/l compared to other media containing sugarcane molasses and either sunflower oil or olive oil. Moreover, the obtained yield was comparable to that obtained using a conventional synthetic medium having yeast extract, urea, soybean oil and glucose. Emulsification activity and stability of the produced SLs using different oils, and the effect of pH and temperature on the same were also studied. And these properties of the biosurfactant indicated good potential of the cheaply produced SLs in the pretreatment of high fats and oils containing food industry wastewaters.

Introduction

India has approximately 90 different vegetable oil refineries located in different states of the country. The sources of vegetable oil manufacture are soybean, sunflower, olive, groundnut, rapeseed, safflower, cotton, sesame, coconut, palm, mustard, rice bran, watermelon, etc. (Pandey, Sanyal, Chattopadhyay, & Kaul, 2003). Besides the vegetable oil refinery, dairy industry is another major food industry in India, and India ranks first among the maximum milk producing nations (Tripathi & Upadhyay, 2003). The wastewaters generated from these vegetable oil refineries and dairy industries contain large amount of fats/grease and oils that makes such wastewaters not easily biodegradable (Cammarota and Freire, 2006, Willey, 2001). Further, high levels of fats and oils in these wastewaters cause gross pollution of land and water due to their high biochemical oxygen demand (BOD) and chemical oxygen demand (COD) (Cammarota & Freire, 2006). Also, the high content of fats and oils often interfere with normal wastewater treatment procedure resulting in significant increase in the process cost and time. Hence, removal or pretreatment of these fats and oils is necessary before subjecting the wastewater to biological treatment operations. A large number of pretreatment methods are employed to remove fats and oils from these wastewaters prior to biological treatment and these methods include grease-trap, tilted plate separators, dissolved air flotation systems and other physico-chemical treatment methods. However, the cost of these methods is considered high and removal efficiency of fat and oil is low (Tano-Debrah et al., 1999, Willey, 2001, Cammarota and Freire, 2006). There are few reports available for degradation of fats and oils by alkaline/acid/enzymatic hydrolysis (Masse et al., 2001, Sivalingam et al., 2003), but these methods are still not very cost effective. Alternative methods to deal with high fats and oils in wastewaters such as thermophilic processes and use of surfactants facilitate the biodegradation of fats and oils primarily by increasing its solubility. In fact, the use of surfactants to aid in the emulsification and removal of oils from wastewaters has been explored and used successfully for the treatment of wool-scouring wastewater and high strength pet food wastewater (Cail et al., 1986, Wipa et al., 1996, Nakhla et al., 2003). But compared to chemical surfactants biosurfactants can be considered environmentally safer, and besides this, they have several advantages over chemical or synthetic surfactants, such as high ionic strength tolerance, high temperature tolerance, higher biodegradability and lower toxicity, lower critical micelle concentration and higher surface activity (Bognolo, 1999). However, the major concern regarding the use of biosurfactants, in place of chemical surfactants, is their production costs, which can, however, be kept low by using cheap raw materials in the fermentation medium. And, another essential condition for using such biosurfactants is that they should be produced by non-pathogenic microorganisms. Sophorolipids (SLs), a group of extracellular biosurfactant produced by several non-pathogenic species of yeast Candida (Van Bogaert et al., 2007). Being a surfactant molecule it can interact with the phase boundary in heterogeneous systems and can solubilize lipophilic compounds. In comparison to other biosurfactants, e.g. rhamnolipids that are produced by pathogenic microorganisms, SLs can be produced by non-pathogenic Candida species and that too in much larger quantities (Van Bogaert et al., 2007). Also, they can be produced by utilizing low cost fermentative feedstocks such as animal fat (Deshpande & Daniels, 1995), deproteinized cheese whey (Daniel, Otto, Reuss, & Syldatk, 1998), soy molasses (Solaiman et al., 2004, Solaiman et al., 2007), restaurant waste oil, (Shah, Jurjevic, & Badia, 2007) etc. by the yeast in order to reduce its production cost. SLs and their derivatives have also shown promise as surfactants, emulsifiers, antimicrobials, and a source of specialty chemicals such as sophorose and hydroxylated fatty acids (Rau et al., 2001, Solaiman et al., 2007).

In the present study, production of SLs from the yeast Candida bombicola (Starmerella bombicola) was carried out by using low cost fermentative substrates (sugarcane molasses and either soybean oil, sunflower oil or olive oil) and properties of the produced SLs based on its emulsification activity and its stability with different oils and effect of environmental factors on the emulsification activity and stability are reported.

Section snippets

Microbial culture and its maintenance

The yeast used in this study S. bombicola NRRL Y-17069 (an equivalent strain of C. bombicola ATCC 22214) was procured from Agricultural Research Service (ARS-Culture collection), USDA, Peoria, USA. The strain was grown, according to the supplier’s instructions, for 48 h at 30 °C incubation on agar slants containing (g/l): glucose, 10; yeast extract, 3; peptone, 5; and agar, 20 (GYP-agar). The microorganism was sub-cultured in every 4 weeks and maintained at 4 °C in a refrigerator.

Chemicals and reagents

All chemicals and

SLs production using different media types

The time course of SLs production during the yeast fermentation using different media types investigated in this study is presented in Fig. 1; it can be observed that SLs production decreased when conventional synthetic medium containing costly glucose (media type I) was replaced with the low cost medium containing sugarcane molasses (either media type II or III). More precisely, the yeast was found to produce 29.37 ± 1.11 g/l of SLs when grown on synthetic medium (medium type I) compared to 12.67 ±

Conclusions

In the present study, low cost fermentative media based on sugarcane molasses and soybean oil was successfully evaluated for SLs production by the yeast C. bombicola. Costly glucose and nitrogen source (yeast extract and urea) were replaced by sugarcane molasses to reduce the cost of the production medium, which also resulted in high SLs production by the yeast. The stability and activity of the emulsion formed towards various oils were tested and a trend was observed. While neutral pH was

Acknowledgement

The authors gratefully acknowledge the funding received from the Department of Science and Technology (DST), India to execute this work.

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