PRODUCTION AND CHARACTERIZATION OF BIOSURFACTANT BY PSEUDOMONAS AERUGINOSA USING RED CASHEW POMACE AS SUBSTRATE

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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