ABSTRACT
Biosurfactants are amphipathic compounds produced extracellularly by microorganisms on cell surfaces, or excreted extracellularly. They contain hydrophilic and hydrophobic moieties that reduce surface and interfacial tension between molecules at the surface and interface respectively. The present study was focused on development of economical methods for biosurfactant production by the use of unconventional substrates. The research investigated the potential of utilizing agroindustrial (red cashew pomace) wastes to replace synthetic media for cultivation of Pseudomonas aeruginosa and biosurfactant production. The organism was able to grow and produce surfactant. The pseudomonas strains were screened for biosurfactant activity using haemolysis and oil spreading test. The surfactant was able to form emulsions with various vegetable oils and hydrocarbons being more effective against palm oil (70.3 ±0.57), olive oil (65.3 ±0.57) and kerosene (60.0 ±0.57). The surface-active compound retained its properties during exposure to elevated temperatures (up to 100°C), relatively high salinity (8% NaCl) and a wide range of pH values (2-12). The biosurfactant was extracted after 10 days using chlorofoam: methanol and the dry weight was calculated as 1.0g/L. Preliminary characterization by the use of basic biochemical tests revealed that the compound is a glycolipid. The biosurfactant produced was used in this study to explore the possible potential for cleaning up pesticides (chlorpyrifos) residue in tomatoes. Different concentration of biosurfactant solution (5ppm, 10ppm, 20ppm and 40ppm) were able to reduce 100ppm pesticide (chlorpyrifos) contaminated tomatoes to below maximum residue limit of 0.5ppm. The results of this study suggest the possible use of red cashew pomace in biosurfactant production and its useful properties in environmental and industrial application.
CHAPTER ONE
INTRODUCTION
AND LITERATURE REVIEW
Biosurfactants are naturally surface-active compounds derived from microorganisms (Anandaraj and Thivakaran, 2010). They are amphiphilic compounds produced mostly on microbial cell surfaces or excreted extracellularly and contain hydrophobic and hydrophilic moieties that reduce surface and interfacial tensions between two immiscible fluids like oil and water (Anyanwu et al., 2011; Govindammal, 2013). Biosurfactants are classified based on their chemical structure, molecular weight, physico-chemical properties and mode of action and microbial origin (Calvo et al., 2009). Their chemical composition is very unique in that they contain a hydrophilic moiety, comprising an acid, peptide cations or anions, mono-, di- or polysaccharides and they also contain a hydrophobic moiety comprising of unsaturated or saturated hydrocarbon chains or fatty acids (Saharan et al., 2011). The upsurge on replacement of synthetic surfactant with their biological counterparts (Biosurfactants) is due to the latter’s better characteristics such as low toxicity, higher biodegradability and mild process conditions, higher foaming capacity, temperature, pH and salinity stability and synthesis under user-friendly conditions (Parveen et al., 2011; Chandran and Das, 2010). On the other hand, different microorganisms are known to synthesize different types of biosurfactants when grown on several carbon sources, therefore the type, quality and quantity of biosurfactant produced are also influenced by the nature of the carbon substrate and the culture conditions such as pH, temperature, agitation and dilution rate in continuous culture (Lakshmipathy et al., 2010). Considerable attention has been given in the past to the production of surface-active molecules of biological origin because of their potential utilization in food processing, pharmacology, cosmetic, biomedical and petroleum industries (Emine and Aysun, 2009).
In spite of their numerous advantages over synthetic surfactants, biosurfactant has not yet been employed in industries due to their relatively high production and recovering cost involved (Makkar et al., 2011), hence the need for inexpensive and renewable carbon sources and highly efficient microorganisms for biosurfactants production (Plaza et al., 2011). Certain substances are used as sources of energy for microbial fermentation with the aim of producing biosurfactants. In the bid to diversify these substances recent advances have focused on the use of agricultural products, byproducts and wastes. Red cashew (Anacardium occidentale) fruits are widely distributed and are rich in carbohydrate, vitamins, proteins and mineral salts (Akinhanmi and Atasie, 2008) which make them an interesting and inexpensive renewable carbon source for
microbial fermentation. A large percentage of the red cashew (Anacardium occidentale) fruits are wasted in Nigeria annually as people are only interested in the nuts, hence the need to harness these raw materials for biosurfactant production. Pseudomonas aeruginosa is one of the widely studied microorganisms used in the production of biosurfactants. It is a bacterium that is able to thrive in various environments and conditions. It can also use a wide range of organic materials as source of energy and carbon. Pseudomonas species has been identified to degrade hydrocarbons and produce biosurfactants predominately glycolipids (Beal and Betts, 2000). In the current study, biosurfactants produced by Pseudomonas aeruginosa in submerged fermentation system using red cashew fruit pomace as substrates will be characterized and applied in cleaning of insecticide residue in vegetables.
LITERATURE REVIEW
1.1 Biosurfactant and Classification
Biosurfactants are suface active compounds produced on microbial cell surfaces or excreted extracellularly by a wide variety of microorganisms (Priya and Usharani, 2009; Jamal et al., 2012). The classification of biosurfactants is dependent on their chemical structure and molecular weight. Based on their chemical structure, biosurfactants are determined by the different molecules forming the hydrophobic and hydrophilic moieties. The hydrophobic moieties may contain saturated or unsaturated fatty acids while the hydrophilic moieties may contain peptide anions or cations, mono-, di-, or polysaccharides, or amino acids (Makkar and Cameotra, 2002). Based on molecular weight, they are divided into low-molecular-mass biosurfactants which include glycolipids, phospholipids and lipopeptides and into high- molecular-mass biosurfactants containing amphipathic polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers. Low-molecular- mass biosurfactants are efficient in lowering surface and interfacial tensions, whereas high- molecular-mass biosurfactants are more effective at stabilizing oil-in-water emulsions (Calvo et al., 2009).
1.1.1 Glycolipids
Glycolipids are the most common types of biosurfactants. They consist of carbohydrates in combination with long chain aliphatic and hydroxyaliphatic acids and are further divided into rhamnolipids, trehalose-lipids and sophorolipids, of which rhamnolipids are of utmost importance. Rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa and some
other Pseudomonas strains. Rhamnolipids have rhamnose sugars as hydrophilic moiety and fatty acids as hydrophobic moiety. New technologies have been used to discover up to 28 homologues of rhamnolipids (Benincasa et al., 2004) with four of these being more important than others. These four homologues are usually designated as R1, R2, R3 and R4 (where R represents rhamnolipids) (see Fig. 1). These four rhamnolipids are distinct from each other by the amount of rhamnose sugar and fatty acid chain each one of them contains. They usually contain two or more important rhamnose and fatty acid chain (Lang and Wullbrandt, 1999). Rhamnolipids are said to enhance the degradation and dispersion of different classes of hydrocarbons by lowering surface tension. They emulsify hydrocarbons and vegetable oils and induce the growth of Pseudomonas on n-hexadecane (Whang et al., 2008). Trehalose lipids are produced from different species of Mycobacterium tuberculosis, Arthrobacter and Nocardia. They enhance the bioavailability of hydrocarbons (Franzetti et al., 2010). Sophorolipids are produced by different strains of the yeast, Torulopsis. The sugar unit is the disaccharide sophorose which consists of two β-1, 2-linked glucose units(Perfumo et al., 2010).
FIG 1.1: The Four Major Rhamnolipids. Source: Lang and Wullbrandt, (1999)
1.1.2 Phospholipids, Lipopeptides and Polymeric Biosurfactants
Phospholipids are major components of microbial membranes. They contain a phosphate group and fatty acid chain and are further divided into corynomycolic acid, spiculisporic acid and phosphotidylethanolamine. The level of phospholipids increases greatly (40-80% w/w) when some micro-organisms like bacteria, yeast, Acinetobacter species, Arthrobacter species, Aspergillus species are grown in hydrophobic substrates (Pooja and Cameotra 2004). Phospholipids promote the enhancement of bitumen recovery, removal of metal ions from aqueous solution and dispersion of hydrophilic pigments. They are utilized in the preparation of new emulsion-type organogels, super fine microcapsules (liposomes or vesicles) and heavy sequestrants. Phospholipids increase the tolerance of bacteria to heavy metals (Ishigami et al., 2000).
Lipopeptides are biosurfactants which are produced by organisms like Pseudomonas, Bacillus and Streptomyces species. They are comprised of fatty acids attached to an amino acid chain (Kiran et al., 2010). They are classified into surfactin and lichenysin. Lipopeptides enhance oil recovery, biodegradation of hydrocarbons and chlorinated pesticides, removal of heavy metals from a contaminated soil, sediment and water; thus, increasing the effectiveness of phytoextraction (Chakraborty et al., 2011).
Polymeric biosurfactants are very complex molecules which usually contain a backbone of three to four repeating sugars having fatty acid chains attached to them. They consist of lipopolysaccharides, lipoproteins, proteins and polysaccharides. Polymeric biosurfactants are classified into emulsan, alasan, biodispersan, liposan and mannoprotein. Polymeric biosurfactants are implicated with functions like stabilization of the hydrocarbon-in-water emulsions and dispersion of limestone in water (Toren et al., 2001).
1.2 Biosurfactant Producing Microorganisms
Biosurfactants produced by a variety of microorganisms mainly bacteria, fungi and yeasts are diverse in chemical composition and their nature and the amount depend on the type of microorganism producing a particular biosurfactant. Many microorganisms for industrial utilization for waste products have been isolated from contaminated soils, effluents and waste water sources. Thus, these have the ability to grow on substrates considered potentially noxious for other non-producing microorganisms (Saharan et al., 2011).
Table 1.1: List of biosurfactants producing organisms.
| S.NO | Biosurfactant | Microorganism(s) | Current economic importance | ||
| 1 | Cellobiose lipids | Ustilago maydis | Antifungal Compounds | ||
| 2 | Rhamnolipids | Pseudomonas aeruginosa, Pseudomonas chlororaphis, Serratia rubidea. | Bioremediation, Antimicrobial and biocontrol properties | ||
| 3 | Trehalose lipids | Rhodococcus erythropolis, Arthrobacter sp., Nocardia erythropolis, Corynebacterium sp., Mycobacterium sp | Dissolution of hydrocarbons | ||
| 4 | Sophorolipids | Candida bombicola, C. antartica, Torulopsis petrophilum C. botistae, C. apicola, C. riodocensis, C. stellata, C. bogoriensis | Antimicrobial, Antiviral, Spermicidal | ||
| 5 | Phospholipids | AAcicnienteotboabcatcetrer sp. | Bioremediation | Bioremediation | |
| 6 | Emulsan | A. calcoaceticus | Microbially enhanced oil recovery (MEOR ) | ||
| 7 | Alasan | A.Ar.ardaidoiroerseistisetnesns | Biodegradation of | Biodegradation of | |
| polyaromatic | polyaromatic | ||||
| compounds | compounds | ||||
| 8 | Peptide lipids | B. licheniformis | Antimicrobial properties | ||
| 9 | Carbohydrate lipids | P.fluorescens, Debaryomyces polmorphus | Bio-emulsifiers | ||
| 10 | Fatty acids /neutral lipids | Clavibacter | Bio-emulsifiers | ||
Source: Saharan et al., (2011)
1.3 Properties of Biosurfactants
Biosurfactants are of increasing interest for commercial use because of the continually increasing spectrum of available substances. There are various advantages of biosurfactants compared to their chemically produced counterpart. The major distinctive features of biosurfactants and a brief description of each property are given below:
1.3.1 Surface and Interface Activity
Biosurfactants are substances with very strong surface active characteristics which accumulate at the interface between two immiscible fluids or between a fluid and a solid. They have the ability to lower surface and interfacial tension in water, gases, liquids and solids. Biosurfactant activities depend on the concentration of the surface-active compounds until the critical micelle concentration (CMC) is obtained. The concentration at which the rate of surface tension reduction results in the formation of micelles and vesicles is known as the critical micelle concentration (CMC). This concentration determines the efficiency in the rate of surface tension reduction ability of biosurfactants. Biosurfactants have CMC values ranging from 1 to
200mg/L (Puntus et al., 2004) and are said to have 10-40 fold lower CMC value than synthetic surfactants, which means that less biosurfactant is required to decrease the surface tension. The most active biosurfactants can lower the surface tension of water from 72 to 30 mN·m−1 and the
interfacial tension between water and n-hexadecane from 40 to 1 mN·m−1 (Signh et al., 2006).
At concentrations above the CMC, biosurfactant molecules associate to form micelles, bilayers and vesicles. Micelle formation enables biosurfactants to reduce the surface and interfacial tension and increase the solubility and bioavailability of hydrophobic organic compounds (Whang et al., 2008). Micelle formation has a significant role in microemulsion formation (Nguyen et al.,2008). Microemulsions are clear and stable liquid mixtures of water and oil domains separated by monolayer or aggregates of biosurfactants. Microemulsions are formed when one liquid phase is dispersed as droplets in another liquid phase, for example oil dispersed in water (direct microemulsion) or water dispersed in oil (reversed microemulsion).
Biosurfactants are also identified as biologically active substances, having biocidal activity against some microbes like yeast, bacteria, viruses and fungi. This is expressed in the zone of inhibition or minimal inhibitory concentration (MIC) (Muthusamy et al., 2008). Biosurfactants achieve this effect by influencing the bacterial cell surface hydrophobicity (CSH). This ability has been reported by Al-Tahhan et al. (2000), who studied chemical and structural modifications in the cell surface hydrophobicity (CSH) of Pseudomonas aeruginosa by a rhamnolipid in the presence of hexadecane. Results of their study demonstrated that
rhamnolipid, at very low concentration, caused release of lipopolysaccharide (LPS) from the outer membrane resulting in an increase of cell surface hydrophobicity. In contrast, Sotirova et al. (2009) reported that rhamnolipid at the concentrations below CMC did not affect the LPS component of the bacterial outer membrane but instead changed the composition of outer membrane proteins (OMP). However, all of the changes in the structure of the bacterial cell surface cause increase of accessibility of hydrocarbons to microbial cells.
1.3.2 Temperature, pH and Ionic Strength Tolerance
Many biosurfactants and their surface activities are not affected by environmental conditions such as temperature and pH. McInerney et al., (1990) suggested that lichenysin produced by B. licheniformis was not affected by temperature (up to 50°C), pH (4.5–9.0) and by NaCl and Ca concentrations up to 50 and 25 g/l respectively. A lipopeptide produced by B. subtilis was stable after autoclaving (121°C/20 min) and after 6 months at –18°C; the surface activity did not change from pH 5 to 11 and NaCl concentrations up to 20% (Charkraborty et.al.,
2011).
1.3.3 Biodegradability
Unlike synthetic surfactants, microbial-produced compounds surfactants are easily degraded (Mohan et al., 2006) and chiefly suited for the environmental applications such as bioremediation (Mulligan, 2005) and dispersion of oil spills.
1.3.4 Low Toxicity
Very little data are available in the literature regarding the toxicity of biosurfactants. They are in general considered as low or non-toxic products and therefore are appropriate for pharmaceutical, food and cosmetic uses. A biosurfactant from P. aeruginosa was compared to a synthetic surfactant that is widely used in the industry, regarding toxicity and mutagenic properties. Both assays indicated a higher level of toxicity and mutagenic effect of the chemically derived surfactant, whereas the biosurfactant was considered to be slightly non-toxic and non mutagenic (Cooper and Cavalero, 2003). Experiment conducted by Anyanwu et.al.,
2011, lipopeptide biosurfactant was non-toxic to mice at the 5.0g/kg body weight dose tested, which was the highest dose recommended by the Food and Agricultural Organization/World Health Organization for food additives. This is indicative of its non-toxic nature even when used as food additive or accidentally consumed. The low toxicity of biosurfactants has been recommended as a veritable advantage over synthetic surfactants.
1.4 Factors Affecting Biosurfactant Production
Biosurfactants are produced by a number of microorganisms, predominantly during their growth on water-immiscible substrates. However, some yeast may produce biosurfactants in the presence of different types of substrates, such as carbohydrates. The use of different carbon sources alters the structure of the biosurfactant produced and its properties and can be exploited to get products with desired properties for particular applications. There are a number of studies in biosurfactant production involving the optimization of their physicochemical properties (Sarubbo et al., 2006). The composition and characteristics of biosurfactants are influenced by the nature of the nitrogen source as well as the presence of iron, magnesium, manganese, phosphorus and sulphur in the media (Sarubbo et al., 2001). Environmental factors are also extremely important in the yield and characteristics of the biosurfactant produced. In order to obtain large quantities of biosurfactant it is necessary to optimize the process conditions because the production of a biosurfactant is affected by variables such as pH, temperature, aeration and agitation speed.
1.4.1 Nature of Carbon Source
Till date, biosurfactants are unable to compete inexpensively with chemically synthesized compounds due to their high production costs and recovery system. These costs may be significantly reduced by the use of alternative sources of nutrients. Zinjarde and Pant (2002) demonstrated the biosynthesis of surfactant by Y. lipolytica NCIM 3589 using soluble carbon source such as glucose, glycerol and sodium acetate. Sarubbo et al. (2001) identified for the first time a biosurfactant produced by Y. lipolytica IA 1055 using glucose as carbon source and concluded that the induction of biosurfactant production is not dependent on the presence of hydrocarbons. Biosurfactant production by B. subtilis MTCC 2423 was monitored by measuring the reduction in surface tension of the cell-free broth. Surface tension reduction was better when glucose, sucrose, tri sodium citrate, sodium pyruvate, yeast extract, and beef extract were used as carbon sources. The maximum bioemulsifiers production was observed when the strain C. glabrata isolated from mangrove sediments was cultivated on cotton seed oil (7.5%) and
glucose (5.0%), reaching values of 10 g L-1 after 144 hr. The soy molasses, a byproduct from the
production of soybean oil, plus oleic acid were tested as carbon sources for the production of sophorolipids by the yeast C. bombicola (Solaiman et al., 2004). The purified SLs were obtained at 21 g l−1 and were 97% in lactone form. The surface properties of the SLs obtained from the
soy molasses/oleic acid fermentation had minimum surface-tension values of 37 mN m−1 (pH 6)
and 38 mN m−1 (pH 9), and critical micelle concentration values of 6 mg l−1 (pH 6) and 13 mg
l−1 (pH 9). The carbon sources such as glucose, glycerol, acetates and other organic acids, as well as pure n-alkanes are quite expensive and cannot reduce the cost of biosurfactant production. An approach to lessen the cost is partial or complete replacement of pure reagents with industrial/agricultural mixtures. The substrate does merely determine the amount of biosurfactants produced but also determines the kind of biosurfactant produced.
1.4.2 Nitrogen Source
Nitrogen is important in the biosurfactant production medium because it is an essential component of the proteins that are essential for the growth of microbes and for production of enzymes for the fermentation process. Several sources of nitrogen have been used for the production of biosurfactants, such as urea, peptone, ammonium sulphate, ammonium nitrate, sodium nitrate, meat extract and malt extract (Mata-Sandoval et al., 2001). Yeast extract is the most widely used nitrogen source for biosurfactant production, but its required concentration depends on the nature of microorganism and the culture medium to be used. The production of biosurfactants often occurs when the nitrogen source is depleted in the culture medium, during the stationary phase of cell growth (Thanomsub et al, 2004).
1.4.3 Effect of pH
Production of biosurfactants occurs best at a pH of 8.0, which is the natural pH of sea water. The reported pH for rhamnolipid production by Pseudomonas aeruginosa was all in the neutral range. Lower production with lower cell growth rates could occur as a result of the pH being lower than 6.5 or higher than 7.5. It is important to have a proper control of the pH throughout the production process to avoid retardation in the process (Chen et al. 2007).
1.4.4 Effect of Temperature
Most of the biosurfactant productions reported so far have been performed in a temperature range of 25 to 30˚C. Casas and Garcia-Ocho (1999) reported that the amount of sophorolipids obtained in the culture medium of C. bombicola at temperature of 25˚C or 30˚C is similar. Nevertheless, the fermentation at 25˚C presents a lower biomass growth and a higher glucose consumption rate in comparison to the fermentation at 30˚C. In the culture of C. antarctica, temperature causes variations in the biosurfactant production. The highest mannosylerythritol lipid production was observed at 25˚C for the production with both growing and resting cells (Kitamoto et al., 2001).
1.4.5 Effect of Agitation and Aeration on the Production of Biosurfactants
Aeration and agitation are important factors that influence the production of biosurfactants as both facilitate the oxygen transfer from the gas phase to the aqueous phase. It may also be linked to the physiological function of microbial emulsifier, it has been suggested that the production of bioemulsifiers can enhance the solubilization of water insoluble substrates and consequently facilitate nutrient transport to microorganisms. In Agitation rates between
50rpm and 250rpm, it was observed that the best production was achieved at 250rpm (Wei et al.,
2007).
1.4.6 Metal Ion Concentration
Metal ion concentrations play a very important role in the production of some biosurfactants as they form important cofactors of many enzymes. The overproduction of surfactin biosurfactant occurs in the presence of Fe2+ in mineral salt medium. The properties of surfactin are modified in the presence of inorganic cations such as overproduction (Wei et al., 2007).
1.5 Applications of Biosurfactants
Biosurfactants are implicated in a wide range of applications. Most biosurfactants produced by micro organisms are utilized in the remediation of crude oil and pesticide-contaminated soils, hydrocarbons and heavy metals, oil recovery and as emulsifiers in food industries and in skin conditioning (Suwansukho, 2008). They are also utilized in medicine, agriculture and petroleum industries.
1.5.1 Bioremediation Applications
In recent times, biosurfactants have been utilized in bioremediation. Bioremediation is the use of micro organisms’ metabolism to remove pollutants. This process is achieved due to certain properties which the biosurfactants possess. Such properties may include their low toxicity, ability to disperse a wide range of hydrophobic pollutants like crude oil, pesticides and other chemicals and biocompatibility (Makkar et al., 2011).
1.5.1.1 Application in Biodegradation Process
A promising method that can improve bioremediation effectiveness of hydrocarbon contaminated environments is the use of biosurfactants. They can enhance hydrocarbon bioremediation by two mechanisms. The first includes the increase of substrate bioavailability for microorganisms, while the other involves interaction with the cell surface which increases the hydrophobicity of the surface allowing hydrophobic substrates to associate more easily with
bacterial cells (Mulligan and Gibbs, 2004). By reducing surface and interfacial tensions, biosurfactants increase the surface areas of insoluble compounds leading to increased mobility and bioavailability of hydrocarbons. In consequence, biosurfactants enhance biodegradation and removal of hydrocarbons. Addition of biosurfactants can be expected to enhance hydrocarbon biodegradation by mobilization, solubilization or emulsification (see Fig. 2) (Nievas et al.,
2008). The mobilization mechanism occurs at concentrations below the biosurfactant CMC. At such concentrations, biosurfactants reduce the surface and interfacial tension between air/water and soil/water systems. Due to the reduction of the interfacial force, contact of biosurfactants with soil/oil system increases the contact angle and reduces the capillary force holding oil and soil together. In turn, above the biosurfactant CMC the solubilization process takes place. At these concentrations biosurfactant molecules associate to form micelles, which dramatically increase the solubility of oil. The hydrophobic ends of biosurfactant molecules connect together inside the micelle while the hydrophilic ends are exposed to the aqueous phase on the exterior. Consequently, the interior of a micelle creates an environment compatible for hydrophobic organic molecules. The process of incorporation of these molecules into a micelle is known as solubilization (Urum and Pekdemir, 2004).
FIG 1.2: Mechanisms of hydrocarbon removal by biosurfactants
Source: Urum and Pekdemir (2004).
Emulsification is a process that forms a liquid, known as an emulsion, containing very small droplets of fat or oil suspended in a fluid, usually water. The high molecular weight biosurfactants are efficient emulsifying agents. They are often applied as an additive to stimulate bioremediation and removal of oil substances from environments (Urum and Pekdemir, 2004).
1.5.1.2 Application in Microbial Enhanced Oil Recovery
Biosurfactants can be utilized in oil recovery in a process called Microbial Enhanced Oil Recovery (MEOR). Here, the microorganisms in the reservoir are stimulated thereby causing them to yield biosurfactants and polymers which lower interfacial tension at the oil-rock interface and thus, increasing the production of oil from subtly-producing reservoirs. The mechanism responsible for the release of oil is the acidification of the solid phase. Micro organisms like Pseudomonas aeruginosa, Bacillus subtilis and Torulopsisbombicola utilize crude oil and hydrocarbons as carbon sources and can be utilized in cleaning oil spillages while micro organisms produced in situ are provided with low-cost substrates like molasses and inorganic nutrients in order to improve their growth and biosurfactant production (Das and Mukherjee, 2007).
FIG 1.3: Mechanism of oil recovery by biosurfactants. Source: Das and Mukherjee (2007)
1.5.1.3 Application in Agriculture
Biosurfactants when applied as mobilizing agents increases the apparent solubility of hydrophobic organic contaminants (HOC) in the soil by enhancing solubility of lethal chemical compounds like polycyclic aromatic hydrocarbons (PAH). Biosurfactants also aid in adsorbing microorganisms to soil particles occupied by pollutants and thereby reducing the diffusion path length between the site of biouptake and the site of absorption by the microbes (Makkar and Rockne, 2003).
Surfactants are utilized for hydrophilization of heavy soils to obtain good wet ability and
to achieve even distribution of fertilizer in the soil. They also prevent the caking of certain fertilizer during storage and promote spreading and penetration of the toxicants in pesticides (Makkar and Rockne, 2003).The rhamnolipid biosurfactant, mostly produced by the genus Pseudomonas is known to possess potent antimicrobial activity. Further, no adverse effects on
humans or the environments are anticipated from aggregate exposure to rhamnolipid biosurfactants. Biosurfactants can also be applied as cleaning agent for pesticide residue in vegetables. Churdchai and Nguyen, 2010, explore the possible potential of biosurfactant for cleaning up cypermethrin residue in lettuce.
1.5.2 Therapeutic and Biomedical Applications
Biosurfactants present good opportunity to be developed as new antibiotics, although the first biosurfactants to be produced are now being produced as commercial antibiotics. Their antimicrobial activity has been reported against bacteria, fungi, algae and viruses. Biosurfactants have other applications as anti-cancer and anti-adhesive agents, agents for stimulating stem fibroblast metabolism, gene delivery and immunomodulatory action agents, immunological adjuvant (Gomaa, 2012).
1.5.3 Miscellaneous Applications Of Biosurfactants
Biosurfactants are also known to be applied in other areas other than bioremediation and biomedicine. They are equally implicated in having roles as anti-foaming, foaming, wetting, emulsifying, dispersing and cleaning agents in many products and applications such as cosmetics (toothpastes, hair shampoo and conditioner), biopesticides, quantum dot coatings, paints, detergents, emulsions, adhesives, laxatives, fabric softeners, inks, agro chemical formulations (some herbicides and insecticides), anti-fogs, leak detectors in pipelines, ferrofluids, ski and snowboard waxes etc. They are also utilized in pipelines as liquid drag reducing agent, in mobilizing oil in oil wells and in firefighting.
1.5.3.1 Application in Cosmetics Industries
Due to the emulsifying character of biosurfactants such as foaming, water binding capacity, spreading and wetting properties effect on viscosity and on product consistency, biosurfactant have been proposed to replace chemically synthesized surfactants in cosmetics industries. These surfactants are used as emulsifiers, foaming agents, solubilizers, wetting agents, cleansers, antimicrobial agents, mediators of enzyme action, in insect repellants, antacids, bath products, acne pads, anti dandruff products, contact lens solutions, baby products, mascara, lipsticks, toothpaste, dentine cleansers (Gharaei-Fathabad, 2011).
1.5.3.2 Application in Food Processing Industries
Biosurfactants have been used for various food processing applications but they usually play a role as food formulation ingredient and anti-adhesive agents, as food formulation
ingredients they promote the formation and stabilization of emulsion due to their ability to decrease the surface and interfacial tension. They are also used to control the agglomeration of fat globules, stabilize aerated systems, improve texture and shelf -life of starch-containing products, modify rheological properties of wheat dough and improve consistency and texture of fat-based products (Krishnaswamy et al., 2008).
1.5.3.3 Application in Commercial Laundry Detergent
Almost all surfactants, an important component used in modern day commercial laundry detergents, are chemically synthesized and exert toxicity to fresh water living organisms. Growing public awareness about the environmental hazards and risks associated with chemical surfactants has stimulated the search for ecofriendly, natural substitutes of chemical surfactants in laundry detergents. Biosurfactants such as Cyclic Lipopeptide (CLP) are stable over a wide PH range (7.0-12.0) and heating them at high temperature does not result in any loss of their surface-active property. They showed good emulsion formation capability with vegetable oils and demonstrated excellent compatibility and stability with commercial laundry detergents favouring their inclusion in laundry detergents formulation (Das and Mukherjee, 2007).
1.5.3.4 Application as Biopesticides
Conventional arthropod control strategy involves applications of broad-spectrum chemicals and pesticides, which often produce undesirable effects. Further, emergence of pesticide resistant insect populations as well as rising prices of new chemical pesticides have stimulated the search for new eco-friendly vector control tools. Lipopeptide biosurfactants produced by several bacteria exhibit insecticidal activity against fruit fly Drosophila melanogaster and hence are promising to be used as biopesticide (Mulligan, 2005).
1.6 Economic Factors of Biosurfactant Production
To overcome the expensive cost constraints associated with biosurfactant production, two basic strategies are generally adopted worldwide to make it cost-effective: (i) the use of inexpensive and waste substrates for the formulation of fermentation media which lower the initial raw material costs involved in the process; (ii) development of efficient and successfully optimized bioprocesses, including optimization of the culture conditions and cost-effective recovery processes for maximum biosurfactant production and recovery. As millions of tons of hazardous and non-hazardous wastes are generated each year throughout the world, a great need exists for their proper management and utilization. The residues from tropical agronomic crops
such as cassava (peels), soybean (hull), sugar beet (Onbasli, 2009), sweet potato (peel and stalks), potato (peel and stalks), sweet sorghum, rice and wheat (Krieger et al, 2010); hull soy, corn and rice; bagasse of sugarcane and cassava; residues from the coffee processing industry such as coffee pulp, coffee husks, spent coffee grounds; residues of the fruit processing industries such as pomace and grape, waste from pineapple and carrot processing, banana waste; waste from oil processing mills such as coconut cake, soybean cake, peanut cake, canola meal and palm oil mill waste; saw dust, corn cobs, carob pods, tea waste, chicory roots etc. have been reported as substrates for biosurfactant production. Additional substrates used for biosurfactant production include water-miscible wastes, molasses, whey milk or distillery wastes. The various substrates previously reported for biosurfactants production are listed (Table 2) with their advantages.
Table 1.2: Substrate for microbial surface active agents and their end products
| Source | Substrate part | End product(s) |
| Cassava | Flour | Biosurfactant |
| Soybean | Seed | Rhamnolipids |
| Sugar beet | Peels | Biosurfactant |
| Cashew apple juice | Pomace | Biosurfactant |
| Diary whey | Whey | Bioemulsifier |
| Sweet potato | Peels | Biosurfactant |
| Sugar bagasse | Stem husk | Biosurfactant |
Despite possessing many industrially attractive properties and advantages compared with synthetic ones, the production of biosurfactants on industrial scale has not been undertaken due to high investment costs. This necessitates their profitable production and recovery on a large
scale. Various aspects of biosurfactants, such as their biomedical and therapeutic properties (Cameotra and Makkar, 2004) their natural roles, their production on inexpensive alternative substrates and their industrial potential, have been reviewed. However their cost of production continues to remain very high. Using low-cost raw materials is a possible solution for this obstacle. Another approach is to use renewable low cost starting materials from various sources including industrial wastes from frying oils, oil refinery wastes, molasses, starch rich wastes, cassava waste water and disti
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PRODUCTION AND CHARACTERIZATION OF BIOSURFACTANT BY PSEUDOMONAS AERUGINOSA USING RED CASHEW POMACE AS SUBSTRATE
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