Utilizing Local Soap-Derived Biosurfactant for ...

Annals of Science and Technology - B, Vol. 1(1):43-51, 2016 Copyright: An Official Journal of the Nigerian Young Academy

ARTICLE

Utilizing Local Soap-Derived Biosurfactant for Degradation of Petroleum Hydrocarbon Polluted Soils: Sustainable Remediation In Focus Geoffery O. Anoliefo, Beckley Ikhajiagbe*, Pascal C. Okoye, Osayi Omoregie Environmental Biotechnology and Sustainability Research Group, Department of Plant Biology and Biotechnology, University of Benin, Nigeria

Received 17th January 2016, Accepted 3rd May 2016 DOI: 10.22366/ast.2016.01.007 Corresponding author * B. Ikhajiagbe email: [email protected] [email protected] Tel: +234 8037094470

Abstract The study investigated the use of soap solutions as a source of biosurfactants in the remediation of a petroleum hydrocarbon-polluted soil. Ten (10) kg of top soil was obtained and contaminated with spent engine oil (SEO) on a weight basis (10% w/w, oil-in-soil). Having already determined the water-holding capacity of the soil to be 170 ml/kg, the oil-polluted soil (10 kg) was moistened with 1700 ml/kg of soap solutions made from a branded synthetic soap, (“Canoe® soap” brand or BSS), a local soap with additives, (brand name “Dudu Osun®”, or SAD), and a locally made unbranded “black soap”, or SAB. Soap solution was prepared by carefully dissolving 100 g of the different soaps in 1.7 litres of water manually until a solution was obtained. The soaps were initially prepared as 1% w/v soap-in-water. The entire set up was exposed in a screen house. Results showed that there was an increase in surface-active compounds including rhamnolipids, trehalolipids, sophorolipids, emulsan, liposan and surfactin at the third month after application of soap treatments, compared with original soil concentrations. There were similar increases in the microbial consortia of the contaminated soil, hence, a general reduction in the total petroleum hydrocarbon (TPH) contents of the soil. The initial 11948.82 mg/kg of TPH of contaminated soil before pollution was significantly reduced to 5562.82 mg/kg after 3 months of using treatment SAB. Phytoassessment with common bean (Phaseolus vulgaris) showed high tolerance of seedling development in SAB compared with BSS while SAD showed zero tolerance to seed germination. The locally made black soap contained biosurfactants such as rhamnolipids, sophorolipids, surfactin, and emulsan. Although the application of soap solution in soil significantly enhanced accumulation of biosurfactants in the oil-polluted soil as well as microbial composition, which are both sine-qua-non to successful bioremediation of petroleum hydrocarbons, the unamended soils showed comparatively better remediation efficiency. Keywords: Biosurfactant, black soap, cowpea, emulsifier, hydrocarbons

1.0 Introduction Among the natural resources such as cocoa, groundnut, aluminum and coal in oil-producing countries like Nigeria, petroleum is the greatest contributor to the country’s per capita income. However, crude oil (hydrocarbon) pollution generated from exploration and processing of petroleum is a widespread environmental problem in Nigeria as well as other oil producing countries. The massive This journal is © The Nigerian Young Academy 2016

oil spill in the Gulf of Mexico is still fresh in memory. Oil spill on land would usually damage soil properties and plant communities due to the associated changes in soil conditions (Anoliefo et al., 2008) and dehydration. The hydrocarbon molecules that make up petroleum products are usually highly toxic to many organisms when in high concentrations including humans (Alexander, 1994).

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Anoliefo et al., 2016

Crude oil pollution adversely affects the soil ecosystem through adsorption to soil particles, the release of excess carbon that might be unavailable for microbial use and induction of a limitation in soil nitrogen and phosphorus (Atlas, 1981 and Amanchukwu et al., 1989). Odjegba and Sadiq (2002) reported that pollution from spent engine oil (SEO) is another important environmental problem and is more widespread than crude oil pollution. The SEO as a petroleum product contains potentially hazardous chemicals, particularly the polycyclic aromatic hydrocarbons (PAHs), heavy metals and chemicals additives including amines, phenol, and benzenes (Meinz, 1999). Ikhajiagbe and Anoliefo (2011) reported that SEO pollution becomes widespread when it is carried by run-off during rainfall to nearby farms. The concentration of PAHs in lubricating oil increases with time of usage and those with two and three rings accumulate rapidly to very high levels (Vwioko and Fashemi, 2005). The development of different techniques adopted for the reclamation of contaminated soils has been an area of research interest for decades. Conventional methods including the use of dispersants as well as physical methods like burning, booming and skimming, are highly capital intensive and most importantly, they are unsustainable approaches to remediation, as they may expose the environment to greater risks than the pollutants. Studies on sustainability have shown that biological approach towards oil spill remediation is the most environmentally friendly and cost effective way of cleaning oil polluted soil (Isikhuemhen et al., 2003; Das and Mukhjee, 2007; Adenipekun and Lawani, 2012). This approach is generally referred to as bioremediation. . This biologically-mediated approach involves the application of organisms and nutrients to the contaminated soil to enhance biodegradation. Micro-organisms used in bioremediation must have been tested and proven to be successful in laboratory studies. Bioremediation may be employed to attack specific soil contaminants such as degradation of chlorinated hydrocarbons by bacteria. During bioremediation, microbes utilize chemical contaminants in the soils as an energy source and through oxidation-reduction reactions, metabolize the target contaminant into viable energy for microbes, releasing by-products that are typically in less toxic form than the parent contaminant (Onwurah, 2000; Nester et al., 2001). Ikhajiagbe and Anoliefo (2011) reported that bioremediation can occur by natural attenuation. One biological strategy that can enhance contact between bacteria and water-insoluble hydrocarbon is emulsification of the hydrocarbon (Rosenberg, 1993). Microorganisms produce a large variety of surface-active materials known as biosurfactants (Rosenberg, 1993; Mulligan and Gibbs, 2004). Microorganisms that grow in the hydrocarbons produce a structurally diverse group of surface active compounds which 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). All biosurfactants are amphiphiles. Due to their amphiphilic structure, biosurfactants increase the surface area of water-insoluble substances such as engine oil, increasing the water bioavailability of such substances. Surface activity makes surfactants excellent emulsifiers, foaming and dispersing agents (Desai and Banat, 1997).

Sustainable Remediation in Focus

Tadros (2009) reported that surfactants are crucial in creating and maintaining the emulsion. However, synthetic soap is not a bioemulsifier. Nevertheless, native soap, in this case, defined as that obtained from homestead or local technology, may be regarded as bio-emulsifier since the constituent raw materials are not chemically oriented. There is a dearth of information on the implementation of bio-emulsifers other than the microbial source in bioremediation of crude oil cum spent engine oil polluted soils. Synthetic soap is obtained when a fatty acid is saponified using caustic soda or potassium hydroxide (base). Production of nonsynthetic soap also involves saponification but unlike the synthetic counterpart, natural raw materials–ash from palm bunch (base) and palm oil (fatty acid) are used. Both synthetic and nonsynthetic soap are emulsifiers which are used as cleansing agents except that their chemical content differentiates them. Experiments have shown that ash (an important component of non-synthetic soap) has been used in ameliorating crude oil polluted soil. Vwioko and Omamogho (2012) reported that wood ash improved crude oil removal from contaminated soil. Onyelucheya et al.(2013) also reported that palm bunch ash enhanced the rate of bioremediation of crude oil polluted soil at low levels of contamination. Having known that soaps are commodities used by the inhabitants of oil polluted regions in Nigeria to ensure laundry and other domestic cleaning activities, they are readily available. Also, embracing the fact that the production of native soap requires natural raw materials which are readily available and does not require sophisticated skills to accomplish, it is thought that perhaps an alternative source of bio-emulsifier could be established other than depending on bacteria which culturing (as in the case of introduction of exogenous bacteria during bioaugumentation) requires professional skills. This study has become necessary to ascertain if emulsification action of native soap could be successfully used as one of the environmental friendly tool for cleanup of petroleum oil polluted soils and water bodies in Nigeria and beyond. The success of this application is compared with that of a synthetic soap, Canoe® soap, manufactured by PZ Cussons Plc, Lagos; and an industrially manufactured local, but branded soap, Dudu Osun®, manufactured by Tropical Naturals Ltd, Ogba Industrial Estate, Ikeja, Lagos. The aim of the study was to investigate changes in total petroleum hydrocarbon (TPH) and microbial content of oil impact soil as well as the presence of biosurfactants after emulsification with soap in solutions.

2.0 Materials and Methods 2.1 Collection and Pollution of Soil Top soil (0 – 10 cm) was gathered at random spots on a plot of land opposite the Botanic garden, Department of Plant Biology and Biotechnology, University of Benin, Benin City, Nigeria; and pooled together to obtain a composite sample. Ten (10) kg of the sun-dried soil was measured each into thirty-nine (39) separate buckets of 30 cm-diameter with 8 perforations made with 2 mm nails at the bottom of each bucket. Spent petrol engine oil (SEO) was obtained as pooled collections from an auto-mechanic workshop for use. The soils were equally contaminated with SEO by thoroughly mixing SEO with the soils to obtain a 10% concentration on a weight basis (i.e. 10% w/w). This concentration of oil-in-soil was adopted (Ikhajiagbe, 2010).

Soaps are among the most commonly used anionic surfactants which are particularly effective in oil cleaning and oil/clay suspension (Zhang et al., 2009). 44 |Annals of Science and Technology 2016 vol. (1)43–51

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2.2 Preparation of ash sample Palm bunches were obtained from a palm oil mill at Egor Local Government Area, Edo State, Nigeria. These were sun-dried for 3 weeks. The dried palm bunches were burnt to ashes on clean corrugated iron sheets. The incompletely burnt materials were separated, burnt again until thoroughly burnt to ashes. The ash sample was allowed to cool and then packed in a black polyethylene bag. 2.3 Preparation of Local Black Soap Five hundred milliliters (500 ml) of distilled warm water was added to 250 g of ash sample and stirred for 30 min. The mixture was filtered using a mesh screen to obtain the filtrate (alkali extract). The extract (200 ml) was gradually added to 250 ml beaker containing 40g of thoroughly heated palm oil. The mixture was then heated and stirred. On cooling, the mixture formed a black solid mass (native black soap). This procedure was repeated until several quantities of the native black soap were produced. It was subsequently stored for use in the present study. 2.4 Preparation of Materials Measured quantities of 3 selected soaps; the locally made “black soap”, a semi-synthetic soap, Dudu Osun® manufactured by Tropical Naturals Ltd, Ikeja Lagos, and a synthetic soap, Canoe® soap, manufactured by PZ Cussons Plc, Lagos, were cut into bits and place differently in a clean bowl. Prior to this, the water holding capacity (WHC) of the soil was pre-evaluated to be 170 ml/kg. A Hundred grammes (100 g) of the different soap were dissolved in 1.7 litres (WHC of soil) of water and each concentration was made in triplicate. These were allowed to dissolve for 24 h after which the dissolution was enhanced by stirring and breaking of undissolved particles manually until a foaming solution was obtained.


Hexachloride I.P., and supplied by Gary Pharmaceutical Pvt. Limited (India), were both filtered through a 0.45μm syringe filter. Thereafter 3 μl of standard and sample solutions were injected into injector and chromatogram was recorded. A Gas Chromatograph (GC) model GC-2010 (Shimadzu) that is equipped with a split/splitless injector together with a flame ionization detector (FID) from Agilent Technologies Inc. was used in the present study. The separation was carried out on an Agilent OV-17 Capillary Column (30 m × 0.25 mm i.d., 0.25μm film thickness). Nitrogen (ultrapure) was obtained from Pci Nitrogen Generator (Model NAG-02) and used as carrier gas at a constant flow rate of 9.0 ml min-1. For the determination of biosurfactant composition of soil samples, soil samples were preserved in the refrigerator (-4 oC) in the laboratory immediately they were received to avoid any degradation of the analytes during the transport to the laboratory. The samples were later dried in a heater at 35 oC. The dried samples were milled and strained through a sieve to a particle size of less than 2 mm, and then Soxhlet-extracted and analyzed. Soil samples were extracted using a slightly-modified procedure described by Lara-Martin et al. (2006). The Hewlett Packard HP 5890 series II Gas Chromagraph with mass selective detection (GC-MS) system was used for this determination (Lara-Martin et al., 2006).

The polluted soils were each moistened with the prepared solutions (1.7 litres) of the soaps. The control soil was moistened with water. The entire set up were kept in a well-ventilated screen house for 3 months. Soil samples were collected after three (3) months from each level of amendment and from control in aluminum foil wraps and taken for analyses in the laboratory.

Microbial composition of soil was determined by the spread plate method. One (1) ml of the fourth level serially diluted portion (i.e. 10-4) of each soil sample was inoculated onto nutrient agar plates for bacteria and Potato dextrose agar plates for fungal counts. The plates were inoculated at room temperature for 24 h and 72 h respectively, for bacteria and fungi growth. After incubation, colonies were then counted and the colony forming unit (CFU/g) of the soil samples determined. The culturable bacterial and fungal isolates were identified using conventional microbiological and biochemical tests, including the colonial characteristic and microscopic examination of hyphal morphology as well as by structure and nature of the fruiting body (Cheesebrough, 1998, 2001). For isolation of bacterial and fungal oil degraders, the Bushnell- Haas (BH) medium (MgSO4, 0.20 g/I; CaCl2, 0.02 g/l; K2H,P04, 1 g/l; NH4NO3, 1 g/l; FeCl3, 0.05 g/l; KH2PO4, 1 g/l; pH 7.0 was used as the enrichment medium with 8 % (v/v) filter sterilized oil as the sole carbon source.

2.6 Soil Analyses

2.7 Phytoassessment

The soil sample was assayed for total volatile aromatic hydrocarbon (TVAH) and total aliphatic hydrocarbons (TAH), which, in this study add up to give the total petroleum hydrocarbons (TPH). Determination of TAH and TVAH contents of soil were according to methods adapted from APHA (1985), and Dean and Xiong (2000). A measured quantity (100 mg) of soil sample was taken in a separating funnel and extracted with 2 × 25 ml of chloroform. The residue was dissolved in HPLC-grade acetone. The solution was thereafter filtered through Whatman Filter Paper No.1. Before injection, the sample solution as well as the standard sample containing 1% w/v of Gama benzene

After three (3) months of wetting, ten (10) seeds of Phaseolus vulgaris were planted in each of the buckets and watered twice a day, keeping in mind the soil’s water-holding capacity. The time taken for the first emergence was noted. Percentage emergence and the height of the seedling were calculated. Percentage (%) germination = No of seed that emerged × 100 No of seeds sown

2.5 Wetting of Polluted Soils

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(1) The seedling height was measured every 7 days and this was carried out using a flexible measuring tape. Annals of Science and Technology, 2016, vol. (1), 43–51 | 45



2.8 Statistical Analysis Results were presented as the mean of 3 replicates. Means were statistically separated at 95% confidence limit, using the least significant difference. Computations were done with the aid of the SPSS® 16 statistical software. Other factors calculated were remediation efficiency and tolerance index.

2.9 Bioremediation Efficiency Bioremediation efficiency is regarded as the proportion (%) of contaminant that was bioremediated compared with a measured concentration at the start point (Ikhajiagbe et al., 2013b). In this present study, the reference point was immediately after pollution. This was calculated as; Efficiency (%) = measured concentration at 3 MAP × 100 Conc. immediately after pollution (2) 2.10 Tolerance Index Tolerance index was determined after 5 weeks, where plant height was used as the determining parameter. Tolerance index was computed according to Iyagba and Offor (2013). Tolerance index = Parameter in contaminant x 100 Parameter in control

(3)

3.0 Results and Discussion The composition of surface-active compounds, or biosurfactants, of the soaps used in the experiment, was determined and recorded

in Table 1. The locally-made black soap showed the better composition of the biosurfactants, with > 150 mg/kg concentration of rhamnolipids, sophorolipids, and emulsan. The most abundant biosurfactant in the black soap was sophorolipids, with a 399 mg/kg. Table 2 shows the total petroleum hydrocarbon (TPH) contents of soil at 3 months after application of the various treatments. The concentration of nonane immediately after pollution was 1828.08 mg/kg compared with 81.44 mg/kg in tricosane which was the lowest. Total aliphatic hydrocarbon (TAH) after pollution was 11947.19 mg/kg. Three (3) months after the application of treatments, the concentration of nonane ranged from 388.64 mg/kg in the unamended oil-polluted soil (NSA) to 1681.83 mg/kg in the polluted soil that was amended with Dudu Osun (SAD). The lowest remediation of nonane was obtained in the soil amended with Dudu Osun, whereas the highest was obtained in the soil that was not amended though polluted with oil after three months. Similarly, the concentration of hexadecane in the oil-polluted soil at 3 months after pollution (MAP) was below 1 mg/kg for the untreated oil-polluted soil (NSA), soil amended with synthetic soap (BSS), and the soil treated with local black soap (SAB) respectively. However, there was a minimal increase in hexadecane content compared with its concentration immediately after the exogenous application of spent engine oil in the soil amended with Dudu Osun. It is suggested that this may be as a result of comparatively lower total biosurfactant composition as well as poor microbial soil quality of the SADsoil (see Tables 3 and 4). These are requisite for successful bioremediation of hydrocarbon-polluted soils. Remediation efficiency was lowest in SAD soil (7.96%), compared with 68.31% in NSA, 54.36% in BSS and 53.36% in SAB respectively.

Table 1: Biosurfactant composition of the soaps used in the experiment

Dudu Osun

Synthetic soap

Parameter (mg/kg)

Rhamnolipids Trehalolipids Sophorolipids Emulsan Liposan Surfactin Glucose Mannose Galactose Glucuronic acid

Black Soap 236.35

85.86

227.04

40.67

145.04

161.06

128.71

68.48

38.85

20.67

16.43

8.75

0.82

0.44