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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  â€“  to  â€“  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   Î²-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  â€“  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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