ABSTRACT
During the bioprocess of the biosurfactant production (by the consortium), the volume of CO2  evolved was 28.23 ± 5.08 cm3, which is equivalent to 0.056 ± 0.01g CO2  per 100 ml of broth. In terms of gCO2/1000Kg biosurfactant, this gave a value of 4545 ± 817.93g  CO2  in  this  bioprocess.  Following  from  above,  the  life  cycle  impact assessment (LCIA) of biosurfactant production by this consortium, based  on global warming  potential  (GWP)  was  0.046  tonnes/1000Kg  biosurfactant.  Other  impact values calculated for acidification and eutrophication potentials were 0.008 tonnes / 1000 Kg and 0.0014 tonnes/1000Kg of biosurfactant. These values were considered insufficient  in terms  of  environmental  pollution  when  compared  with the regular methods of surfactants production. In this work also, the consortium of Pseudomonas sp/Azotobacter vinelandii produced 1.22 ± 0.04 mg biosurfactant per 100ml of cell – free broth.  However,  the individual  organisms  (Pseudomonas  sp. and Azotobacter vinelandii) produced 1.03 ± 0.02 and 0.08 ± 0.001 mg of biosurfactants per 100 ml cell – free broth respectively. The microbial growth kinetics during the production of the biosurfactant by the consortium gave a specific maximum growth (µmax) of 1.306 ± 0.201 hr-1), a saturation constant (Ks) of 0.017 ± 0.007 mg/l, an Inhibition constant (Ki) of 121.83 ± 21.18 mg/land a Death constant (Kd) of 0.017 ± 0.006 mg/l. These values  when  compared  with  those  of  individual  organisms  shows  that  using  a consortium for the bioprocess is more sustainable.
CHAPTER ONE
1.0 Introduction
Crude oil exploration and exploitation activities have resulted in inadvertent oil spill incidences. In Nigeria, increase in oil spill have been reported due to sabotages by the host communities in the Niger Delta region. Hence crude oil waste abound in the Niger Delta areas of Nigeria. Oil wastes generated in the oil industries can be channeled into various biotechnological tools. Environmental research is often technology dependent with new advances in thinking. Biotechnological conversion of the petroleum oily wastes into biosurfactant by a consortium of Azotobacter vinelandii and Pseudomonas sp is a welcome idea. In this work, we evaluated the environmental impact of biosurfactant production from oily waste by this consortium. There are on-going biotechnological research activities for clean – up and utilization of these crude oil contaminated environment. Onwurah and Nwuke (2004) reported the production of surface active agents by this consortium during enhanced bioremediation of crude oil.
The use of biosurfactants has gained ground due to the following advantages it possesses over the chemical surfactant. These include its biodegradability (El- Sheshtawy and Doheim, 2014), low toxicity and availability of raw materials (Waghmode et al., 2014). Biosurfactants also have some wide range of application in food and cosmetics industries and enhanced oil recovery, and in bioremediation of polluted ecosystem (Marchant and Banat, 2012). Due to these applications and demand, there is an increase in the use of microorganism in the production of biosurfactants. Different organisms produces different types of surfactants.In this study, the environmental impact of producing biosurfactant from the consortium of Azotobacter vinelandii andPseudomonas sp.was evaluated using Life Cycle Assessment (LCA) tools. In this assessment, a Gate – to – Gate Life Cycle Assessment was considered.
1.1 Biosurfactants.
Surfactants are amphiphillic molecules that accumulate at interfaces, decrease interfacial tensions and form aggregate structures such as micelles (Van – Hamme et
al., 2006). They normally possess both hydrophilic and hydrophobic moieties which confers the ability to accumulate at the oil and water interface. Biosurfactants on the other hand are a structurally diverse group of surface-active substances produced by microorganisms. All biosurfactants are amphiphiles.They consist of two parts—a polar (hydrophilic) moiety and non-polar (hydrophobic) group that enables them to interact with the hydrophobic phase as well as the hydrophilic phase (Matvyeyeva et al., 2014). A hydrophilic group consists of mono-, oligo- or polysaccharides, peptides or proteins and a hydrophobic moiety usually contains saturated, unsaturated and hydroxylated fatty acids or fatty alcohols (Lang, 2002). They are known to have surfactant activities which make them an interesting group of materials for application in many areas such as agriculture, pharmaceutical, cosmetic industries, waste utilization, and environmental pollution control such as in degradation of hydrocarbons present in soil.
1.2 Biosurfactant classification
Biosurfactant are classified based on the nature of their polar groups.They are categorized mainly by their chemical structure and their microbial origin. In general, their structure includes a hydrophilic moiety consisting of amino acids or peptides, anions or cations; mono-, di-, or polysaccharides; and a hydrophobic moiety consisting of unsaturated, saturated, or hydroxylated fatty acids. The major classes of biosurfactants include glycolipids, lipopeptides and lipoproteins, phospholipids and fatty acids, polymeric surfactants, and particulate surfactants (Desai and Banat, 1997).
1.2.1 Glycolipids
Most known biosurfactants are glycolipids. Just as the name implies, glycolipids contains carbohydrates moiety in combination with long-chain aliphatic acids or hydroxyaliphatic acids. The most common glycolipids are rhamnolipids, trehalolipids, and sophorolipids.
1.2.1.1 Rhamnolipids:
Rhamnolipids consist of one or two molecules of rhamnose linked to one or two molecules of β-hydroxydecanoic acid. They are normally produced from Pseudomonas aeruginosa (Jarvis and Johnson, 1949; Henkel et al., 2014). L- Rhamnosyl- L rhamnosyl – β – hydroxydecanoyl – β – hydroxydecanoate and L –
rhamnosyl – β- hydroxydecanoyl – β – hydroxydecanoateare the principal glycolipids produced by P. aeruginosa. Four of these are most predominant which are usually referred to as R1, R2, R3 and R4 (Rhamnolipids). These forms differ in the amount of rhamnose sugar and fatty acid chain present, with one or two rhamnose and fatty acid chains (C8-C12) being predominant (Benincasa et. al., 2004; Peter and Singh, 2014).
Figure 1. Structures of rhamnolipids (monorhamnolipid and dirhamnolipid)
1.2.1.2 Trehalolipids:
The trehalolipid biosurfactants consist of trehalose (a disaccharide of two glucose molecules) connected to long chain fatty acids. Disaccharide trehalose linked at C6 and C9 to mycolic acids is associated with most of the species. Trehalolipids produced from different organisms might differ in the size and structure of mycolic acid, the number of carbon atoms, and the degree of unsaturation (Asselineau and Asselineau, 1978). Trehalolipids have been isolated from Mycobacterium tuberculosis, Rhodococcus erythropolis, Arthrobacter sp., Nocardia sp., and Corynebacterium sp. (Franzetti et al., 2010).
Figure 2. The structure of Trehalolipid
1.2.1.3 Sophorolipids:
Sophorolipids is made up of a dimeric carbohydrate sophorose connected to a glycosidic bond through a hydroxyl group at the penultimate position of an 18-carbon fatty acid. This type of biosurfactant occurs as a mixture of macrolactone and open- chain (free acid) forms and may be acetylated at the primary hydroxyl positions of the sophorose (Daverey and Pakshirajan, 2009). They have been isolated from microorganisms such as Torulopsis bombicola, Torulopsis petrophilum, Torulopsis apicola (Baviere et al., 1994; Pesce, 2002 and Whang et al., 2008).
Figure 3. Structure ofsophorolipids and derivatives (Fu et al,, 2008).
1.2.1.4 Mannosylerythritol-lipids:
Mannosylerythriol – lipid are normally produced from a fungi (yeasts), which according to Kitamoto et al., (1993) are receiving much attention due to their biomedical applications (Kitamato et. al., 2002). Mannosylerythritol lipid (MEL), 2,3- di-O-alka(e) noyl-β-D- mannopyranosyl-(1→4)-Omeso- erythritol partially acetylated at C4 and/or C6, is a glycolipid that contains mannose and the sugar alcohol erythritol as hydrophilic moiety and acetyl groups as well as fatty acids as the hydrophobic moiety
Figure 4. Structure of mannosylerythritol lipids produced by Candida antarctica. (Im et al., 2001),
Other glycolipids are cellobiolipids and oligosaccharide lipids (Deasi and Banat,
1997), glucose lipid (Wattanaphon et al., 2008) sugar-based bioemulsifiers (Kim et al., 2000) hexose lipids (Golyshin et. al., 1999).
1.2.2 Lipopeptides and lipoproteins
A large number of cyclic lipo-peptides, including decapeptide antibiotics (gramicidins) and lipo-peptide antibiotics (polymyxins) are produced. These consist of a lipid attached to a polypeptide chain (Rosenberg and Ron, 1999).
1.2.2.1 Surfactin
Surfactin is composed of a seven amino-acid ring structure coupled to a fatty-acid chain via lactone linkage. It is one of the most powerful biosurfactants. It was found out by Arima et al., (1968) that it lowers the surface tension from 72 to 27.9mNm at concentrations as low as 0.005%. Furthermore, it possesses anti-bacterial, antiviral, anti-fungal, antimycoplasma and hemolytic activities (Singh et al., 2014). This lipopeptide consists of a hexapeptide lactonised to a hydroxyl fatty acid (Shoeb et al., 2013)
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LIFE CYCLE ASSESSMENT OF THE ENVIRONMENTAL IMPACT OF BIOSURFACTANT PRODUCTION FROM OIL WASTE BY A DICULTURE OF AZOTOBACTER VINELANDII AND PSEUDOMONAS SP
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