Substrates for Biosurfactants Production

Conventional Substrates for Microbial Surfactants Production

The carbon source used in growth medium primarily affects the quantity and quality of biosurfactants produced by microorganisms (Sen 1997; Abouseoud et al. 2008). Different microbes have been reported to use wide range of carbon sources, such as alkanes, pyruvate, fructose, succinate, citrate, glycerol, mannitol, lactose, n-paraffin, hexadecane, and glucose for production of biosurfactants (Robert et al. 1989; AbuRuwaida et al. 1991; Pruthi and Cameotra 2003). The commonly used organic and inorganic nitrogen supplements for biosurfactants production includes urea, beef extract, yeast extract, casein hydrolysate, ammonium nitrate, potassium nitrate, sodium nitrate, and ammonium chloride (Abouseoud et al. 2008; Aparna et al. 2012). Although, use of these conventional carbon and nitrogen sources is suitable for lab scale studies but is not economically viable to support production at commercial scale. Thus, there is a need to explore possibilities of using various low-cost agro-industrial waste or by-products as nutrient supplements in fermentation media to lower the cost of biosurfactant production.

Low-Cost Substrates: Cheap and Value-Added Alternatives

Farming and agricultural production worldwide support basic food requirements of mankind, and processing of agricultural raw materials in industries produces a considerable amount of solid waste and by-products. The processing of wheat, rice, corn, sorghum, and barley grains in food industries releases large amounts of wastewater rich in carbohydrates in the environment. Thus, studies have been carried out to explore possibilities of using low-cost and renewable agro-industrial wastes including distillery wastes, plant oils, oil wastes, starchy substances, and whey as substrates for cost effective production of biosurfactant (Deleu and Paquot 2004; Ferreira 2008; Montoneri et al. 2009). The agricultural residues of various crops and industrial wastes reported as substrates for biosurfactant production are presented in Table 18.3.

18.4.2.1 Starchy Substrates

The carbohydrate-enriched crops, such as corn, wheat, rice, cassava, and potatoes, are being grown worldwide. Starch obtained from these crops is a major industrial

Table 18.3 Agro-industrial wastes utilized for biosurfactant production

product used as a thickener in food industries for making puddings, custards, soups, noodles, pastas, etc. and as a nonfood product in paper and clothing industries. Potato is a rich source of starch (9.1–22.6 g/100 g), protein (0.8–4.2 g/100 g), micronutrients (iron, potassium, magnesium, phosphorus, and calcium), and vitamin C (Lutaladio and Castaldi 2009). The potato peel containing about 40 % dietary fibers and 28–51 % starch depending upon the peeling process is usually discarded as waste (Camire et al. 1997). Moreover, it is estimated that 41 % of the potato crop cannot be utilized for making consumable products due to its overproduction/spoilage and thus used in animal feed and alcohol production (Natu et al. 1991; Fox and Bala 2000).

Fox and Bala (2000) utilized waste from potato processing industry to produce biosurfactant by B. subtilis ATCC 21332 in shake flask studies. The production of biosurfactant reduced the surface tension of growth medium to 28.3 mN/m from

71.3 mN/m with critical micellar concentration of 0.10 g/l. Similarly, Das and Mukherjee (2007) reported biosurfactant (lipopeptides) production by two Bacillus subtilis strains DM-03 and DM-04 using powder of potato peels as substrate in solid state fermentation (SSF) and submerged fermentation (SmF). Bacillus subtilis DM-03 supported 67.0 mg/gram dry substrate (mg/gds) to 80 mg/gds in SSF and SmF systems, respectively. Zhu et al. (2012) reported production of 50.01 mg/gds lipopeptides by Bacillus amyloliquefaciens XZ-173 by utilizing starch-rich rice straw (3.67 g) and soybean flour (5.58 g) as substrates in SSF after 2 days of incubation.

Cassava is cultivated as an annual crop in tropical and subtropical regions for its edible starch-rich tuberous roots. The cassava wastewater is a rich source of protein, fructose, glucose, maltose, nitrate, phosphorus, and minerals (Nitschke and Pastore 2006). Nitschke and Pastore (2003, 2004, 2006) used cassava wastewater for surfactin production by Bacillus subtilis ATCC 21332 and B. subtilis LB5a. The yield of surfactin was 2.2 g/l and 3.0 g/l respectively during shake flask studies in the fermentation medium. While, Barros et al. (2008) reported production of a biosurfactant by Bacillus subtilis LB5a in a 40 l pilot scale batch bioreactor by using cassava wastewater. The foam was collected simultaneously by fractionation which was precipitated to yield 2.4 g/l of biosurfactant during the fermentative process. The microbial surfactant supported good surface activity with reduction in surface tension of growth medium to 27 mN/m and the CMC of 11 mg/l. Further, Costa et al. (2009) reported use of cassava wastewater as a substrate for the production of rhamnolipids by P. aeruginosa in lab scale shake flask studies. The rhamnolipid production was 660 mg/l with reduction in surface tension of growth medium up to 30 mN/m and CMC of 26.5 mg/l.

Corn steep liquor, a by-product obtained after separation of starch by wet milling of corns, is generally used as a nutritive source in animal feed as it contains protein (47 %), lactic acid (26 %), dextrose (2.5 %), nitrogen (7.5 %), and fat (0.4 %). It can be potentially used as a low-cost nutrient for microbial growth (White and Johnson 2003). Sobrinho et al. (2008) reported a low-cost medium containing 5.0 % (w/v) groundnut oil refinery residue and 2.5 % (w/v) corn steep liquor as substrates for biosurfactant (4.5 g/l) production by yeast Candida sphaerica (UCP 0995). Recently, Luna et al. (2013) reported 9 g/l biosurfactant production by using same

Table 18.4 Fatty acid composition of different vegetable oils

strain of C. sphaerica (UCP 0995) in medium supplemented with 9.0 % corn steep liquor and 9.0 % groundnut oil refinery residue. The biosurfactant with CMC of

0.25 mg/ml lowers the surface tension of water from 70 to 25 mN/m. Thus, these studies indicated the potential of wastes/by-products containing starch/others micronutrients as low-cost substrates for economical production of biosurfactants.

18.4.2.2 Oils and Oil Wastes as Substrates

Vegetable Oils

The production of oil and fats around the world from animal and plant sources was more than three million tons, and out of that 75 % of the oil is obtained from plants (Haba et al. 2000). According to a recent report, the total worldwide production of vegetable oils in 2008–2009 was around 133 million tons (USDA 2009). The vegetable oils are comprised of varied ratios of different fatty acids such as palmitic acid (C16:0), linoleic acid (C18:2), oleic acid (C18:1), lauric acids (C12:0), stearic acids (C18:0), and myristic acids (C14:0) as described in Table 18.4.

Mercade et al. (1988) reported production of 7,10 dihydroxy-8(E)-octadecanoic acid by Pseudomonas strain 42A2 by utilizing by-product of vegetable oils mixtures containing 98 % (w/w) of oleic acid. Kitamoto et al. (1993) reported use of soybean oil as substrate for Candida antarctica T-34 which supported production of mannosylerythritol lipids (MEL-A and B) having antimicrobial properties against gram-positive bacteria. Vollbrecht et al. (1999) used oleic acid-rich oils, rapeseed oil, and sunflower oil individually as substrates for Tsukamurella sp. DSM 44370 and obtained better growth and glycolipid production than that obtained in synthetic media. The sunflower oil supported overall yield of 5 g/l of glycolipids GL1, GL2, and GL3 with reduction in surface tension of water from 72 mN/m to 35 mN/m, 23 mN/m and 24 mN/m respectively. CMC value of glycolipids was 10 mg/l. Similarly, Casas and Garcia-Ochoa (1999) used sunflower oil as a substrate for Candida bombicola and reported 120 g/l production of sophorolipid after 8 days of incubation. Mata-Sandoval et al. (1999) reported improved production of rhamnolipid (100–165 mg/g substrate) by Pseudomonas aeruginosa UG2 using corn oil as a sole carbon source in the medium instead of succinic acid and glucose as these supported only 12–36 mg rhamnolipid per gram substrate. Rau et al. (2001) reported economical production of sophorolipid with overall yield of 57 g/l/day in feed-batch and 76 g/l/day in continuous mode using rapeseed oil along with glucose as carbon source. Trummler et al. (2003) obtained overall yield of 45 g/l biosurfactant mixtures of monoand dirhamnolipids by using rapeseed oil as substrate for Pseudomonas sp. DSM 2874. Camargo-de-Morais et al. (2003) used 1 % (v/v) olive oil as carbon source for Penicillium citrinum which supported production of a glycolipid. Pekin and Vardar-Sukan (2005) reported capability of Candida bombicola ATCC 22214 to produce 400 g/l of sophorolipids in a 3 l bioreactor using Turkish corn oil, glucose, and cheap market honey as carbon sources. Thaniyavarn et al. (2006) studied the production of biosurfactant by using 2 % (v/v) of different vegetable oils as a carbon sources in the growth medium for Pseudomonas aeruginosa A41. The maximum biosurfactant yield of 6.58 g/l was obtained with olive oil followed by 2.91 g/l with palm oil and 2.93 g/l with coconut oil. Thaniyavarn et al. (2008) further reported use of 4 % (v/v) soybean oil as carbon source for production of sophorolipid by a thermotolerant strain Pichia anomala PY1. The biosurfactant lowered the surface tension of growth medium to 28 mN/m and possesses crude oil displacement property of 69.43 cm2 for a potential application in extraction of crude oil from oil wells. Coimbra et al. (2009) reported production of biosurfactant by six Candida strains utilizing mixture of oil-rich substrates including groundnut, oil refinery residue, and soybean oil with corn steep liquor and glucose. The biosurfactant produced was able to remove 90 % of the hydrophobic contaminants from sand. Daverey and Pakshirajan (2009) used soybean, sunflower, and olive oil in combination with sugarcane molasses as a low-cost medium and reported production of 24 g/l sophorolipids from the yeast Candida bombicola. In subsequent fermentor (3 l) studies using 50 g/l of sugarcane molasses with 50 g/l of soybean oil as the substrates under optimal conditions the authors achieved 60 g/l of sophorolipids production (Daverey and Pakshirajan 2010).

Waste Vegetable Oil

Vegetable oil wastes generated from food industries, oil refineries, soap industries, and kitchen are generally disposed in sewers or drains which causes pollution and blockages in waterways. Thus, oil waste residues, such as olive oil mill effluents (OOME), soybean oil refinery wastes, and fried oils, are being exploited as lowcost substrates for the production of rhamnolipid, sophorolipid, mannosylerythritol, and lipopeptides (Trummler et al. 2003; Rahman et al. 2002; Pekin and Vardar-Sukan 2005).

Olive oil mill effluents, a water-soluble fraction released after extraction of oil, contain toxic polyphenols and are not suitable for human consumption but can be used as a nutrient-rich substrate for microbes to produce biochemicals of commercial importance. OOME contains sugars (20–80 g/l), nitrogen (12–24 g/l), organic acids (5–15 g/l), and residual oil (0.3–5 g/l) and was reported for first time in 1993 as a substrate for production of rhamnolipids (0.058 g/g OOME) by Pseudomonas sp. JAMM (Mercade et al. 1993). The biosurfactant produced was able to lower the surface tension of growth medium from 40 mN/m to about 30 mN/m.

The used frying oils of sunflower, canola, soybean, olive, rice bran, corns, etc. are being released at the large scale in the environment. The restaurants in the United States are producing waste frying oil at an average rate of 100 billion liters per week (Shah et al. 2007). The nutritional value of frying oils varies depending upon food products fried and number of times it has been reused. The used frying oils have 30 % higher polar hydrocarbons (triacylglycerol oligomers, triacylglycerol dimmers, oxidized triacylglycerol monomers, and diacylglycerols) than the fresh oil and can be exploited as a low-cost substrate for production of microbial products (Marmesat et al. 2007). There is a considerable difference in the composition and chain lengths of fatty acids in fresh and fried oils. Haba et al. (2000) reported that used olive and sunflower oils have 22.52 % more myristic acid and lauric acid as compared to the standard unused oils. They reported production of biosurfactants by Pseudomonas spp., Bacillus spp., Rhodococcus sp., Acinetobacter calcoaceticus, and Candida spp. in medium supplemented with 2 % (v/v) used olive oil and sunflower oil as carbon source. All the cultures lowered the surface tension of growth medium to 40 mN/m from 62 mN/m. Pseudomonas aeruginosa strain 47T2 produced 2.7 g/l of rhamnolipid using waste frying sunflower and olive oil as substrates (Haba et al. 2000). Abalos et al. (2001) reported production of new rhamnolipids (9.5 g/l) by Pseudomonas aeruginosa AT110 using soybean oil refinery wastes, and the CMC of biosurfactant was 122 mg/l. Shah et al. (2007) studied biosurfactant production by C. bombicola in fed-batch fermentations using restaurant oil waste and reported 34 g/l of sophorolipids production. Vedaraman and Venkatesh (2011) reported production of surfactin, a cyclic lipopeptides (CLP) by Bacillus subtilis MTCC 2423 in submerged batch cultivation using waste frying sunflower oil and rice bran oil. The overall yield of biosurfactant was 14.9 g/kg and 11.0 g/kg of frying sunflower oil and rice bran oil respectively. Wadekar et al. (2012) reported production of rhamnolipid mainly dirhamnolipids by Pseudomonas aeruginosa (ATCC 10145) using waste frying oil. The yield of biosurfactant was 2.8 g/l and 7.5 g/l respectively before and after activated earth treatment of waste frying oil. The acid activated earth upon mixing with 50 g of heated (90 °C) waste frying oil adsorbed the peroxide, hydroperoxides, and low-molecular-weight aldehydes and ketones from the oil. The treatment increased the consumption of linoleic acid from 58 to 75 % by P. aeruginosa which indicated that presence of peroxides in frying oil might have restricted the consumption of linoleic acid and decreased the yield of rhamnolipids.

Spent Lubricating Oils

The worldwide estimated use of lubricating oils, such as hydraulic oil, motor oil, transmission oil, brake fluids, crankcase oil, and gear box oil generated from different industries, was 42 million tons in 2010 (IETC 2012). The lube oil is composed of synthetic base oils (90 %), such as polyalphaolefin (PAO), polyalkylene glycols, phosphate esters, silicate esters, and synthetic esters, and variety of oil additives (10 %), such as emulsifiers/demulsifiers, friction modifiers, and corrosion inhibitors (Bartels 2005). The difference between fresh and used lube oil is that polyaromatic hydrocarbons (PAHs) are generally undetectable in unused oil while these are abundant in all used lubricating oils. Moreover, concentrations of alkanes are higher in used oils by 2–3 folds than fresh lube oils. The used lube oil is either disposed of by burning on-site in permitted hazardous waste incinerators or used as fuel in industrial furnaces and boilers. However, only 38 % is recycled or properly managed and remaining is a potential source of pollution in the environment. Mercade et al. (1996) reported the screening and isolation of microorganisms utilizing waste lubricating oil as sole carbon source from hydrocarbon-contaminated soil for production of biosurfactants. The biosurfactant produced by Rhodococcus sp. and Bacillus sp. was characterized as trehalose, glycolipids, and lipopeptide. Thavasi et al. (2008) used a mixture of waste motor lubricant oil and low-costing peanut oil cake a by-product obtained from peanut oil manufacturing as substrates for glycolipid biosynthesis by Bacillus megaterium, Azotobacter chroococcum, and Corynebacterium kutscheri.

18.4.2.3 Waste of Dairy Industries

The wastes from animal origin, such as whey from dairy industries, animal fat, and tallow from meat processing industries, are generally being discarded through the effluent treatment systems. These wastes being rich in proteins, amino acids, lipids, vitamins, and minerals can be utilized as medium supplements to support microbial growth and biosurfactant production (Deshpande and Daniels 1995; Dubey and Juwarkar 2001).

Daniel et al. (1998) reported two-stage fed-batch (3 l) cultivation process using deproteinized whey and rapeseed oil as substrates for production of sophorolipids by Candida bombicola ATCC 22214 and Cryptococcus curvatus ATCC 20509. The first stage involved consumption of lactic acid in deproteinized whey (1.5 l) by Candida bombicola ATCC 22214 in stirred fed-batch. Thereafter cells of Candida bombicola in the medium were homogenized at high pressure to release crude cell debris and single cell-oil in the medium. This medium was then supplemented with rapeseed oil (100 g/l) and inoculated with Cryptococcus curvatus ATCC 20509 for production of sophorolipids. This two-stage fed-batch system yielded 422 g/l sophorolipids. Dubey and Juwarkar (2001) used mixture of distillery and whey wastes as substrate for production of biosurfactant by Pseudomonas aeruginosa strain BS2 which supported overall yield of 0.92 g/l during the stationary growth phase. The biosurfactant with CMC of 0.028 mg/ml reduced the surface tension of water from 72 to 27 mN/m. Further, a decrease in chemical oxygen demand (COD) by 81.0 % and 87.0 % of distillery and whey wastes respectively was observed indicating ability of cells to assimilate these waste as carbon and nitrogen sources. Daverey and Pakshirajan (2010) used deproteinized whey and glucose as substrate for sophorolipids production by Candida bombicola. The supplements supported up to 33.3 g/l of biosurfactant production by yeast in bioreactor. The CMC of biosurfactant was 27.17 mg/l and able to reduce surface tension of water down to 34.18 mN/m. The biosurfactant was able to solubilize nonaqueous phase liquids (n-hexane, sunflower oil, and olive oil) indicating its potential application in bioremediation of hydrocarbon polluted sites. Jahanshah et al. (2013) used whey to enrich microbial diversity present in municipal waste compost and isolated two biosurfactant producers viz. Bacillus sp. and Streptomyces sp. The biosurfactant produced by these isolates resulted in >50 % removal of heavy metals (lead, nickel, chromium, and cadmium) during bioremediation. The anionic biosurfactants form a complex with positively charged metal ions to remove them from the negatively charged humus and organic matters of compost matrix. Thus, removal of heavy metals using crude biosurfactant decreased the bio-toxicity of polluted site and enhanced the decomposition rate of organic matter by 42.3 %.

18.4.2.4 Waste of Sugar Industries

The sugar industries release molasses as a by-product after crystallization of the sugar from liquid extracts of sugarcane or sugar beet as further extraction of sugar from molasses is not economical (Maneerat 2005). Sugarcane molasses is a complex mixture of 48–56 % total sugar (mainly sucrose), 9–12 % nonsugar organic matter, 2–4 % protein, 1.5–5 % potassium, 0.4–0.8 % calcium, 0.06 % magnesium, 0.6–2.0 % phosphorus, 1.0–3.0 mg/kg biotin, 15–55 mg/kg pantothenic acid, and 2,500– 6,000 mg/kg inositol, and 1.8 mg/kg can be an effective growth medium supplement to support microbial growth (Makkar and Cameotra 1997). Soy molasses, a by-product of soybean oil processing, is rich in fermentable carbohydrate (30 % w/v) mainly glucose, arabinose, sucrose, raffinose, and stachyose. Due to increase in demand of soy protein-based foods and drinks, soy molasses is available as by-product and can be used as a medium supplement for supporting microbial growth (Deak and Johnson 2006). The cost of soy molasses is approximately 20 % lower than glucose, thus reducing the overall cost of biosurfactant production (Lynd et al. 1999).

Solaiman et al. (2004) used 333 g/l soy molasses equivalent to 100 g/l of carbohydrates and 90 ml/l oleic acid as substrates to produce 21 g/l of pure sophorolipids by Candida bombicola. The yield of sophorolipids increased to 55 g/l in 12 l benchtop fermentor with 4 l of working volume using same fermentative medium (Solaiman et al. 2007). Rashedi et al. (2005) used sugar beet molasses as carbon source to support rhamnolipid production by Pseudomonas aeruginosa. The yield of rhamnolipid per gram biomass was 0.003 g, 0.009 g, 0.053 g, 0.041 g, and 0.213 g in medium supplemented with 2 %, 4 %, 6 %, 8 %, and 10 % (v/v) molasses, respectively. The rhamnolipid was able to form stable emulsions with aromatics, n-alkanes, olive oil, and crude oil. Rodrigues et al. (2006) used sugarcane molasses and cheese whey as alternative substrates to synthetic media for Lactococcus lactis 53 and Streptococcus thermophilus. The authors observed 1.2–1.5 time increase in biosurfactant production per gram cell dry weight and 75 % reduction in overall cost of production process at lab scale. Raza et al. (2007) reported gamma ray-induced Pseudomonas aeruginosa EBN-8 mutant utilizing clarified blackstrap molasses as a sole carbon and energy source for production of rhamnolipid-based surfactant with a yield of 1.45 g/l after 96 h of incubation. Moldes et al. (2007) reported ability of Lactobacillus pentosus to produce 0.71 g of biosurfactant per gram of biomass while growing in medium supplemented with hemicellulosic sugar hydrolyzates derived from trimming vine shoots as carbon source. Moldes et al. (2011) used biosurfactant for bioremediation of soil contaminated with octane by mobilizing the target contaminant from the soil surface to make it bioavailable for the microbial population. They reported 62.5 % degradation of octane by Lactobacillus pentosus in presence of biosurfactant as compared to 24 % achieved in absence of biosurfactant after 15 days of treatment. Abdel-Mawgoud et al. (2008) reported 1.12 g/l of surfactin production by Bacillus subtilis BS5 in medium having of 16 % (w/v) molasses and 5 g/l NaNO3. Joshi et al. (2008) reported lipopeptide biosurfactant production based upon decrease surface tension (29 mN/m) of growth medium by Bacillus licheniformis K51, B. subtilis 20B, B. subtilis R1, and Bacillus strain HS3 using 5.0–7.0 % (w/v) of molasses as a sole source of nutrition at 45 °C. Onbasli and Aslim (2009) reported rhamnolipid biosurfactant production by Pseudomonas luteola B17 and Pseudomonas putida B12n using 1–5 % (w/v) sugar beet molasses supplements to growth medium.

Glycerol, a trihydroxy alcohol, syrup is abundant in nature and can be utilized by many microorganism as a carbon source (Syldatk et al. 1985). The glycerol is obtained economically from renewable resources by hydrolysis of animal fat and vegetable oil and also as a coproduct of biodiesel production as every 100 kg of biodiesel production generates 10 kg of glycerol. The depleting natural fuel resources, and increasing price and demand of fuel have increased production of biodiesel with average annual growth rate of 38 % during 2005–2010 (REN21 2011). There are reports regarding use of glycerol obtained from bio-refineries without pretreatment or purification to support enhanced production of biosurfactants. Ashby et al. (2005) used biodiesel coproduct stream (BCS) containing crude glycerol, free fatty acids (FAA), and fatty acyl methyl esters (FAME) as a feedstock for synthesis of sophorolipids (SL) by Candida bombicola with an overall yield of 60 g/l which was significantly higher than 9 g/l obtained using pure glycerol. This may be due to easy availability of FAA and FAME and other micronutrients in BCS required for SL synthesis. Samadi et al. 2007 isolated Brevibacterium sp. strain S-34 capable of utilizing glycerol as sole carbon source and supported yield of 2.4 g/l glycolipid after 72 h of incubation. Dos Santos et al. (2010) reported two strains of Pseudomonas spp. capable of using vegetable oils and glycerol as alternative lowcost carbon sources supporting production of biosurfactant as evident by decrease in surface tension and mineral oil emulsification efficiency of cell-free culture broths ranging from 50 to 59 %. Sousa et al. (2011) used coproduct of biodiesel production as carbon and energy source for Pseudomonas aeruginosa MSIC02 and reported the production of rhamnolipids with overall yield of 1.2 g/l. The biosurfactant obtained was reported to possess emulsifying properties with emulsification index (IE24) = 65 % and form stable emulsions with mineral and vegetable oils.

 
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