DEVELOPMENT OF BIOSURFACTANTS-IRON OXIDE NANOPARTICLES- BIOCHAR FORMULATION FOR REMEDIATION OF CRUDE OIL CONTAMINATED SOIL

ABSTRACT

Biosurfactant-mediated degradation of contaminants is practical and safe environmental remediation agent. In addition, iron oxide nanoparticles and biochar are bioremediation agents with great potentials due to their strong adsorption capacity, microbial growth enhancement and chemical inertness. Therefore, a combination of biosufactant, iron oxide nanoparticles and biochar could produce a very desirable and efficient alternative to conventional environmental treatment of contaminants. This study focused on the development of biosurfactants-ironoxide nanoparticles-biochar (BS/NP/BC) formulation for clean-up of crude oil polluted soil. A potential biosurfactant producing bacterium, previously isolated from the soil  was obtained from the Microbiology Department, Federal Univeristy of Technology, Minna and confirmed as Alcaligenes faecalis strain ADY25. The isolate was screened to confirm its biosurfactant producing ability and used to produce biosurfactantat with a yield of 4.5 g/L. Iron oxide nanoparticles and biochar were synthesized using corn silk extract and plantain trunk respectively. Biosurfactant was produced using Alcaligenes faecalis strain ADY25 and the functional groups were determined using Raman spectroscopy, which confirmed the produced biosurfactant as Lipoprotein. UV spectroscopy of the synthesized nanoparticles showed peak at a range of 262-269 nm, which is a characteristic wavelength for iron oxide nanoparticles. Brunauer-Emmett-Teller (BET) analysis revealed that the produced biochar has an average surface area of 209.106 m2/g, micropore volume of  0.074 cc/g and an average pore width of 6-522 nm at anadsorption energy of 3.987 kJ/mol. The synthesized biosurfactant/ironoxide/biochar nano-composites was utilized to bioremediate soil contaminated with crude oil (10 %w/w of soil) for a period of 35 days and total microbial count was determined at seven days intervals. Statistical analysis for total bacterial growth revealed that there is no significant difference among the treatments for week 0 and 1 and a significant difference was observed from week 3 to 5 while fungal growth had significant difference at all weeks. The highest growth was observed with treatment BS/NP/BC (1:1:1a) at week 3 for both bacteria and fungi. The rate of biodegradation was determined at the end of the treatment period and treatment BS/NP/BC (1:1:1a) gave the highest degradation rate of 75 %. This study revealed that biosurfactants-ironoxide-biochar nano-composites can be used to bioremediate crude oil polluted soil and at 1:1:1 formulation ratio of 100mg each for best result.

CHAPTER ONE

1.0       INTRODUCTION

1.1       Background to the Study

Crude oil is a fossil fuel, which contains different hydrocarbon mixture obtained from the remains of plants and animals that existed for several millions of years ago. This fuel is liquid in nature found in underground reservoirs, in spaces inside sedimentary rocks and close to the surface of tar sands (Bennet, 2016). Petroleum oil is the main energy source used by most industries in the world. The main energy source and feedstock in the chemical industries is the petroleum hydrocarbon, which is known to be extensively used by these industries and have led to an increased attention on soil pollution as well as it’s environmentally harmfully effects in the world. Various activities involved in oil exploration, sabotage, transportation and accidental oil seepage or leakage involved in  several  oil  recovery  processes  have  led  to  the  discharge  of  extremely large  quantity of hydrocarbon pollutants in the environment that result to serious pollution (Peng et al., 2008; Almeida et al., 2016; Patowary et al., 2018).

Hydrocarbon pollutants are recalcitrants, extremely hydrophobic and persist in the environment as they are naturally very difficult for environmental degradation. Plants that grow in hydrocarbon polluted sites often take up these pollutants and are therefore transferred to animals and humans when they feed on them (Alagic et al., 2015; Patowary et al., 2018). Crude oil contains polyaromatic hydrocarbons (PAHs), which is one of the toxic substances with their possible mutagenic and carcinogenic properties. This substance is placed in position 9 of Agency for Toxic Substances and Disease Registry list (Yoon et al., 2007). Thus, it is highly important that strategies for their level reduction are ensured in the environment. Environmental consequences have resulted to researchers continuous research on sustainable approach that involve the use of biodegradable  substances  acquired  from  living  organisms  (known  as  biosurfactants)  for the cleanup of sites contaminated with hydrocarbon (Chaprão et al., 2018).

Biosurfactants are biomolecules synthesized by microorganisms, consisting of both hydrophobic and hydrophilic moieties, whose action is between two liquids that are different in polarities (for example, water   and oil), penetrating hydrophobic substances by increasing the contact area between two immiscible compounds as well as enhancing their mobility and bioavailability, resulting in the degradation of the substrate (Chaprão et al., 2018). These characteristics make biosurfactants to be able to decrease surface and interfacial tension and also form microemulsions, which enable hydrocarbons to be soluble in water. Properties such as emulsification, foaming capacity, lubrication and phase dispersion make biosurfactants to be applicable in industries. In comparison to the synthetic biosurfactants, biosurfactants are stable over wide conditions of the environment, safe and biodegradable.

Biosurfactant producing microorganisms can be found in different environments. Several strains of microorganisms such as Serratia mercencis, Pseudomonas sp., Bacillus subtilis, Mycobacterium sp., Candida sp., Rhodococcus sp., Arthrobacter sp. are known with the potential of producing biosurfactant either naturally or as a result of response to stress (Patowary et al., 2018). The large production of biosurfactants is the commercial success of microbial surfactants and it is presently hindered by high production cost. Optimization of the conditions of growth by the use of cheap, sustainable substrates coupled with novel and effective downstream processing approaches  could result in the production of  cheap and economically feasible biosurfactants, resulting in success of large scale production (Almeida et al., 2016; Patil and Pratap, 2018; Patowary et al., 2018).

Surfactin biosurfactants are the most broadly studied surfactants produced by Bacillus subtilis, sorpholipids by Candida antartica (Kitamoto et al., 1993) and rhamnolipid by Pseudomonas aeruginosa (Maier and Soberón-Chávez, 2000). Different researches are of the opinion that biosurfactants are necessary for microorganisms to grow and survive in a wide range of environments. Alcaligenes faecalis strains have been reported to produce biosurfactants but their biosurfactants are yet to be studied extensively and characterized (Pleaza et al., 2005; Chikere et al., 2009; Bharali et al., 2011).

In comparison to the conventional physical and chemical approaches in remediating hydrocarbon polluted environment, bioremediation has attracted an increased attention because of its ecologically friendly and economical characteristics. Among the numerous methods (incineration, land filling, and chemical treatments) used for remediating oil polluted environment, bioremediation using biosurfactants is a promising approach as it is environmentally compatible, less toxic, cheap and biodegradable as compared to the physical and chemical based approaches, which are costly, toxic and non-biodegradable (Guntupalli et al., 2016; Patowary et al., 2018). Several researches have reported that microorganisms from oil polluted sites are able to clean up oil polluted environment but with low degradability due to their recalcitrant nature and low availability of such organic compounds to microbes.  Therefore, biosurfactant helps to speed up the biodegradation process (Bharali et al., 2011; Zhang et al., 2014; Patowary et al., 2018). One of the limitations of using biosurfactants is their low penetration in soil interface. The search for more efficient  approach  for the remediation of polluted environment  has led to the use of nanoremediation.

Nanotechnology has aroused a significant interest because of its perceived impact in areas of catalysis and its extensive use to improve several reactions as reductants and/or catalysts in chemistry as a result of their large surface areas and properties. These nanomaterials have the capacity to act as carrier of larger molecules and seep through tiny pores in the soil subsurface, reaching locations/areas farther than where larger particles such as biosurfactants can reach. Nanotechnology is an emerging field for the production of nanoscale substances whose reactivity is more effective with larger surface area than its bulk phase. These special properties of nanoparticles provide huge potential for them to be applied in the cleanup of organic compounds, pesticides and metals contaminants. Nanoparticles (NPs) are used directly for removing organic pollutants by adsorbtion or chemical modification (Kumari and Singh, 2016) or to facilitate microbial degradation of pollutants either by stimulating the growth of microorganisms by the immobilization of the agents of remediation or by inducing the production of microbial enzymes used in remediation.

Use of nanoparticles to enhance biosurfactants production in microbes also improve the solubility of the hydrophobic substrates, thereby, creating a conducive conditions for the degradation of the substance by microorganisms in the environment (Kumari and Singh, 2016). Nanoparticles are able to penetrate through tiny pores in soil or they can remain suspended in groundwater. However, due to agglomeration and adsorption processes, the nanomaterials have been reported to have a limited radius of influence. Notwithstanding, these nanoparticles have a huge potential in the remediation of the environment. From the most commonly reported nanoparticles, iron nanoparticles and iron oxide nanoparticles are the commonly used ones and they have demonstrated to be very effective and efficient for removing wide varieties of pollutants like pharmaceutical products, chlorinated solvents and metals (Crane and Scott, 2012; Machado et al., 2015).

Although iron nanoparticles possess high reactivity in the soil but this alone is not enough for successful and effective field application. Control of particles aggregation, movement in permeable environments, chemical reaction and long lasting effect in the subsurface environment are important dominant factors for an efficient and effective clean up of polluted environments (Cecchin et al., 2016).

As early as 90s, iron has been explored and used as a reducing agent for contaminants and the main focus has been on treating water polluted with persistent chlorinated hydrocarbons (Cecchin et al., 2016). The first field trials of zerovalent ironnanoparticles (nZVI) was its use as a filling material in permeable reactive barriers. Several researchers’ have reported the use of zerovalent iron as a reducing agent to clean up environment polluted with aromatic polycyclic hydrocarbons, polychlorinated biphenyls, herbicides, pesticides and metals. It has been established that the use of zerovalent iron in its nano-size can significantly increase the surface area and reactivity (Sakulchaicharoen et al., 2010; Cecchin et al., 2016).

Several modifications of surface have been assessed by scientists in the search for preventing particles agglomeration as well as efficient delivery into the soil. Coating of particles using polymeric substances to act as a barrier in preventing particle agglomeration have been reported. Stabilized particles when compared to non stabilized (agglomerated) particles, offer several important advantages. Non stabilized nanoparticles aggregate quickly while stabilized nanoparticles is left in the nanoscale and are therefore, delivered into the soil sediment. Stabilized nanoparticles present significant reactivity than non-stabilized equivalents, which lead to more efficient and total dechlorination. The use of stabilizers of different physico-chemical properties (e.g. functionality, molecular weight, degree of substitution, matrix type and viscosity) enhance physical dispersibility, chemical reactivity and longevity of stabilized nanoparticles (Zhao et al., 2016).

1.2       Statement of Research Problem

According to Nwilo and Badejo (2006), about 1,820,410.5 barrels equivalent to (77 %) of oil spilled between 1976 and 1996 were not recovered and are therefore, left in the environment while about 549,060 barrels equivalent to 23.17 % of the total oil spilt were recovered. This has led to loss of soil fertility resulting from soil microbes’ destruction, groundwater pollution, alteration of geotechnical properties of the contaminated soil, poisoning of sea foods and other aquacultures, (Ahmadu, 2013; Ejiba et al., 2016). Contamination of the soils with harmful and persistent compounds constitutes lot of hazards to human and the environment (Cocârtă et al., 2017). Apart from the environmental impacts posed by oil spill, loss of oil, cost of cleanup and compensation, damage to agricultural lands, fishery and wildlife are some economic challenges caused by this spill as well as social impact, which includes conflicts, violence and frustration between communities, leading to tourism reduction and militancy (Baghebo et al., 2012).

Crude oil contains polyaromatic hydrocarbons (PAHs) with possible mutagenic and carcinogenic property. Physical and chemical methods for the removal and cleaning up of PAHs are expensive, rarely successful and generate toxic byproducts. Synthetic surfactants used in petroleum industries for oil cleanup are toxic, unsafe with low degradability, necessitating the need for an alternative method (Guntupalli et al., 2016). The use of single technology to remediate persistent pollutants is costly and inefficient necessitating the need to search for and make use of several technologies such as bioremediation and nanoremediation. Thus, this research is to develop technologies to surmount the differences in each of the technologies and make their processes cost effective.

Physicochemical techniques have been used in synthesis of iron nanoparticles of particular sizes and configurations. These techniques utilise lethal chemicals such as stabilizing agents, non- biodegradable reducing agents or organic solvents which are hazardous to the earth and organic systems and are costly and time consuming (Wang et al., 2014). Therefore, it is necessary to search for an alternative and efficient approach to overcome the drawback, resulting in the use of green approach (Campos et al., 2015). Pollution of land, water and air resulting from dumping and burning of agro wastes have been a major environmental problem (Nagendran, 2011). When corn is harvested and consumed, the remains such as the husk, silk and cob are discarded mostly thrown on the street or dump sites, which cause environmental pollution. When matured plantain plants are harvested, the trunks and other parts are left to decompose or dry up and later burnt resulting in air pollution.

1.3       Justification for the Study

The methods currently in use for the cleanup of oil polluted soils are classified into physical, chemical and biological processes. However, physical and chemical methods are not very effective as biological methods for the treatment of hazardous organic compounds (Patowary et al., 2018). Bioremediation has been known to be the most promising approach because of its low production cost, safety, pollutants bioavailability and biodegradability. Bioremediation of the soil refers to a process of degrading organic pollutants by soil microorganisms and the conversion of these pollutants into harmless and non toxic products like methane, water and carbon (iv)oxide (Erdogan and Karaca, 2011).

Biosurfactants are known to decrease both surface tension and interfacial tension and form microemulsions, which solubilise hydrocarbons in water (Patowary et al., 2018). Biosurfactant is non  toxic  and  biodegrable  unlike  the  chemical  surfactants  and  with  the  high  demand  for biosurfactant in petroleum industry, there is need to explore microorganisms for biosurfactant production. Several researches have reported microorganisms from oil polluted sites are able to clean up oil polluted environment utilizing the oil as sole carbon and energy source but with low degradability due to the high recalcitrant and low availability of such organic compounds to microorganisms. However, biosurfactant helps to speed up the biodegradation process by breaking the oil into microemulsions and increasing the oil bioavailability and biodegradability to microorganisms (Pei et al., 2010; Patowary et al., 2018).

The main limitation of using biosurfactants is their low penetration in soil interface. The use of biosurfactants  together with iron nanoparticles is a more efficient approach since nanoparticles have the capacity to act as carrier of larger molecules and penetrate tiny pores in the soil subsurface and have been reported to remove hydrocarbon pollutants through adsorption, chemical modification and facilitating microbial cleanup of pollutants either by promoting   microbial growth,  immobilizing the agent of remediation  or by enhancing microbes to produce enzymes involved in bioremediation (Kumari and Singh, 2016). Among other nanoparticles, iron oxide nanopaticles have been reported to be very effective in removing of a wide variety of pollutants and safe (Machado et al., 2015; Cecchin et al., 2017).

Biological method is regarded as an effective green approach for nanoparticles production because it is simple, cheap, eco-friendly, requires short reaction time and result in more stable nanomaterials compare to physico-chemical approaches. Plants materials used for reduction, capping and stabilization of nanoparticles are in abundance (Machado et al., 2015). Therefore, corn silk extract could be a good biological agent for iron oxide nanoparticles production, since it contains polyphenolic compounds and acts as antioxidant.

Several reports have shown that the use of biochar is very useful in improving soil organic carbon, water holding capacity, increasing microbial population size and diversity, decreasing nutrient to the  groundwater,  availability  and  retention  of  nutrients,  increased  in  soil  aeration  and pH.   Therefore, use of biosurfactant, iron oxide nanoparticles and biochar are far more effective bioremediation strategy of petroleum polluted soil.  Corn silk and plantain trunk are regarded as agricultural waste and use of these wastes in the synthesis of nanoparticles and biochar could be considered as waste management process; a beneficial way of managing agro wastes in the environment.

1.4       Aim and Objectives of the Study

1.4.1    Aim: The aim of this study was to develop biosurfactants-ironoxide nanoparticles-biochar formulation for remediation of crude oil contaminated soil.

1.4.2    Objectives: The objectives of this study were to

I. confirm the identity of a potential biosurfactant producing bacterium

II. confirm the biosurfactant production potential of the bacterium

III. produce and characterize biosurfactant from the bacterium isolate

IV. produce and characterize iron oxide nanoparticles using corn silk extract

V. produce and characterize biochar from plantain trunk

VI. remediate crude oil polluted soil using the produced biosurfactant, biochar and iron oxide nanoparticles formulation VII. determine the rate of biodegradation of crude oil

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