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In Malaysia, the demand of water is expected to grow significantly by 2010 to approximately 17000Mld when the country's population reaches 30 million. Currently, surface water accounts for more than 98 percent of water consumed in Malaysia. This was a statement was taken from Sime Darby recent report towards supporting groundwater as a sustainable and reliable source of water. Fresh water being one for the basic necessities for the sustenance of life, many through the decades has striven to locate and develop it (Karanth 1987). However the level of dependence on surface water is unsustainable and needs urgently need to be addressed, especially when surface water is easily affected by extreme weather conditions. Groundwater, unlike surface water, is available in some quantity almost everywhere, is more dependable in periods of drought, and has many other advantages over surface water (Fetter 1999).
However, there are potential pollution problems that can affect the quality of groundwater such as migration of contaminants from the surface such as landfills, leakages from septic tank, chemical spills from industrial plant, fertilizer, hazardous waste sites and other chemical (Wycisk, Weiss et al. 2003). These contaminants includes hydrocarbons, biological parameters, organic chemicals, inorganic cations and anions and toxic metals (Iwamoto and Nasu 2001). Hence, once groundwater is contaminated it is an extremely costly operation to remove the contaminants.
Bioremediation, which involves the use of microbes to detoxify and degrade environmental contaminants whether in soil or groundwater, has received increasing attention as an effective biotechnological approach to clean up a polluted environment (Iwamoto and Nasu 2001). Bioremediation has been proved to be very effective particularly in dealing with NAPL contamination such as petroleum hydrocarbon (Balba, Al-Awadhi et al. 1998). Thus, study on the capability of bioremediation technology in treating NAPL and the potential of biosurfactants to enhance the removal of hydrophobic substances from homogeneous saturated porous media would contribute to significant understanding.
BACKGROUND TO RESEARCH
In NAPLs contaminated soil or groundwater, treating the hydrocarbon compounds are still evolving. A number of technologies are currently to treat porous media such as soil contaminated with NAPL constituents including excavation and containment, vapor extraction, stabilization and solidification, soil flushing, soil washing and incineration. Most of these technologies are either too expensive or do not result in the reduction of contaminant. Alternatively, bioremediation is an emerging technology for the treatment of soil and groundwater contamination dealing with wide range of NAPL constituents (Balba, Al-Awadhi et al. 1998). Bioremediation techniques used to remove NAPL compounds is typically less expensive that equivalent physical and chemical methods. It is also offer potential to remediated contaminated soil without the need for excavation. Hence, bioremediation can be implemented below existing buildings without the need to rebuild or reconstruct the existing infrastructure. However, the bioremediation is a slow process due to NAPLs found remain on water surface or adhered to soil particles due to their low solubility. In recent years, surfactant enhanced remediation techniques have become popular (McCray et al., 2001). Surfactants enhanced solubility of hydrophobic compounds by presenting a micelle with a hydrophobic core into which the organic compounds may partition (Zoller and Reznik, 2006). The surfactant enhanced aqueous phase, thus, has much higher affinity for the organic compounds (Pal et al., 2009). Surfactants increase the surface area of hydrophobic contaminants in soil or water and thus increase their aqueous solubility and consequently their bio-degradability.
The need of groundwater in Malaysia should be addressed as surface water is not dependable for sustainable water resources. The increasing numbers of contaminated land reported by the Department of Environment has increased rapidly throughout the years where neglected lands are becoming the centre of environmental pollution such as petrol stations, railways, fuel depots, vehicle workshops and industrial sites. Once these contaminants are intact with soil particles, especially NAPL contaminants, the degrading process would be complex. One of the main problems of remediating such site is that NAPLs are strongly adsorbed to solid particles and are trapped in porous media, thus difficult to flush out (Zoller and Reznik 2006). Thus, the ability of microbial communities to access and degrade NAPL compounds is highly dependable on the heterogeneity of the soil and morphology of the NAPL source (Cort, Davis et al. 2001). The penetration of NAPL into soil and groundwater and its migration of NAPLs into porous media such as soil occurs in various mechanism such as advection, dispersion (in aqueous medium within the porous soil matrix), and diffusion, resulting accumulation of entrapped NAPLs as residual in the saturated and unsaturated zones. These residual of hydrophobic fuel hydrocarbons entrapped in soil porous maxtrix would be biodegradable resistant. These conditions can be substantially affected the microbial growth and the biodegradation of NAPLs. Chemical surfactants have been used to some extent with some concerns. Apart from high cost associated with chemical surfactants, there are concerns on residue remaining on site after the remediation process Hence, investigation regarding the influence biodegradation during the transport in porous media (Brusseau, Hu et al. 1999), the need for the dissolution of the entrapped NAPL (Dickson and Thomson 2003), and its mobilization (Miller, Hill et al. 2000), bioavailability in soils (Ehlers and Luthy 2003) and the use of surfactant (Sabatini and Knox 1992) studied by many researchers can be use as the basis for further research and development of other biodegradable surfactants. . Biosurfactants have considerable potential for remediation application because biosurfactants are better biocompatibility to many contaminated sites. Challenges of entrapment of NAPLs in soil matrix should be addressed as problems would contribute to the efficiency of bioremediation.
The aims of research are as follows:-
To identify and develop potential biosurfactants from industrial and municipal sludges
To characterize the potential biosurfactant in degrading NAPLs contaminant
To establish degradation rate and the efficiency of biosurfactant
To create the optimization of condition for the degradation of NAPLs
To model the efficacy of biosurfactant and degradation rate behavior in degrading NAPLs contaminant
SIGNIFICANCE OF RESEARCH
The use of microbes to treat contaminated soil is rapidly evolving. Hence, the research is in line with the national biotechnology initiatives that encourages the exploration of new biodegradable surfactant. The intention of the research is treat NAPLs in soil using the bioremediation technology and suggest surfactant which can be degraded together with the NAPLs. Hence, the research will indirectly deals with tools for bioremediation to detect and enumerate target bacteria that are directly related to the degradation of contaminants and enable to monitor the changes in the bacterial community in detail. With the optimization of condition of biodegradation, such as added nutrients and biosurfactants, this should be able to speed up the rate of degradation. Previous study proved that biosurfactants can emulsify hydrocarbons, thus enhancing their water solubility and subsequently increase the displacement of hydrophilic substances from solid particles in saturated porous media. The increased in hydrophobic properties is attributed to decrease in surface tension. Biosurfactants have recently received much attention because their potential to become green technology alternative to conventional surfactants due to their biodegradability, low toxicity, renewable nature and functionality under extreme condition (Bodour, 2002; Makkar and Cameotra, 2002). The understanding the potential of biosurfactants would also contribute to the strategies in improving in situ bioremediation and reduce the existing environmental impact of bioremediation.
The bioremediation of soil contaminated with aromatic hydrocarbons and fossil fuels is limited by the poor availability of these hydrophobic contaminants to microorganisms (Rahman et al., 2002). Surfactants can help to release hydrocarbons sorbed to soil organic matter by solubilization or emulsification, and increase the aqueous concentrations of hydrophobic compounds, resulting in higher mass transfer rates. Recent studies indicate that biosurfactants can enhance hydrocarbon biodegradation by increasing microbial accessibility to insoluble substrates. Several researchers have investigated the addition of biosurfactants to enhance the biodegradation of hydrocarbons (Miller, 2000). Biosurfactants have been tested in environmental applications such as bioremediation and dispersion of oil spills, enhanced oil recovery and transfer of crude oil. Many of the biosurfactants known today have been investigated with a view toward possible technical applications
NON AQUEOUS PHASE LIQUIDS (NAPL)
Non Aqueous Phase Liquids can be divided into Dense Non Aqueous Phase Liquids (DNAPL) and Light Non Aqueous Phase Liquids (LNAPL). Dense non-aqueous phase liquidÂ orÂ DNAPLÂ is aÂ liquidÂ that isÂ denserÂ thanÂ waterÂ andÂ does not dissolve in water. ManyÂ chlorinatedÂ solvents (orÂ organochlorides), such as trichloroethylene, are DNAPLs. DNAPLs may contain otherÂ halogens, such asÂ bromine. DNAPLs can be broken down byÂ methanogens, but only inÂ anoxicÂ conditions. Â Light Non-Aqueous Phase LiquidÂ or LNAPL is groundwaterÂ contaminateÂ that is not soluble and has a lower density than water. Example of LNAPLs compounds are gasoline and other hydrocarbon (Miller, 2000).
Bioremediation Systems & Processes
Bioremediation technologies can be broadly defined as ex situ or in situ. Ex situ technologies are the treatments that remove contaminants at a separate facility. In situ bioremediation technologies involve the treatment of contaminants in the place itself. The in situ technologies offer several advantages over physical and chemical remediation. Microbes have an extensive capacity to degrade synthetic compounds; therefore, bioremediation can be applied to sites contaminated with variety of chemical pollutants (Iwamoto and Nasu 2001). Bioremediation process and system utilized are classified into the following category.
Bioventing is an application of soil vapor extraction by which air is introduced into contaminated zone to stimulate aerobic degradation. Bioventing can involve the pumping of vapor out of vadose zone, to induce a significant drawdown of the water table (Cort, Davis et al. 2001). Biosparging is the process injecting air into the saturated zone to deliver oxygen to the contaminated zone and enhance biodegradation of NAPLs. Depending on the soil type, direct injection of air can result in preferential pathways of sir flow. These preferential pathways will reduce oxygen delivery to the bulk of soil (Cort, Davis et al. 2001).
Bioattenuation method is the method of monitoring the natural process of degradation to ensure that contaminant concentration decreases with time at relevant sampling points. Bioattenuation is widely used as cleanup method for underground storage tank sites with petroleum contaminated soil and groundwater (Iwamoto and Nasu 2001).
Biostimulation is being applied if degradation does not occur or degradation is too slow. The environment has to be manipulated in such a way that biodegradation is stimulated and the reaction rates are increased. The measures to be taken including supplying environment with nutrients such as nitrogen and phosphorus, with electron acceptors (oxygen) with substrates such as methane, phenol and toluene. The chemical additives used as substrates, phenol and toluene, are well known toxic chemicals (Iwamoto and Nasu 2001). Thus the concentrations of these chemicals during biostimulation should be carefully monitored.
Bioaugmentation is the treatment which enhances the biodegradative capacities of contaminated site by inoculation of bacteria with desired catalytic capabilities. This is considered to be an effective approach in the case of very recalcitrant chemicals where bioattenuation or biostimulation does not work. However, the application of bioaugmentation needed to be pay much attention as large amounts of degradative bacteria added to contaminated sites, may effect human and environment (Gallego, Loredo et al. 2001).
Factors Influencing the Bioremediation of NAPLs
The biodegradation of NAPLs are influence by the heterogeneity of contaminants where contaminants can be found as liquid, solids, gases, free or tightly bound to the particulate matter. The amount of NAPLs concentration also affect the biodegradation of NAPLs for example, the presence of high concentration of NAPLs can be inhibitory or toxic to the microorganism while extremely low concentrations may not be adequate to support microbial activities. The variable site conditions such as soil depth, type and soil microorganism as well as physical condition such as pH, temperature, oxygen availability, redox potential, moisture content and substrate bioavailability should also be taken into consideration. These conditions can substantially affect the microbial growth and biodegradation or organic contaminant. Biodegrading is also a slow process and subject to constraints which influence its selection as the clean up technology, particularly with respect to the required cleanup standards and the pressure for immediate spill which do not allow enough time for process optimization (Balba, Al-Awadhi et al. 1998).
Biosurfactants are surface active compounds released by microorganism. Biosurfactants are amphiphilic compounds produced extracellularly or as part of the cell membrane by variety of yeast, bacteria and filamentous fungi (Chen et al., 2007; Mata-Sandoval et al., 2000) from various substances including sugars, oils and wastes. They are biodegradable non toxic and ecofriendly materials. The biosurfactant production depends on the fermentation conditions, environmental factors and nutrient availability. All surfactants have two ends namely, a hydrocarbon part which is less soluble in water (hydrophobic end). The hydrophobic part of the molecule is a long chain of fatty acids. The water soluble end (hydrophilic) can be a carbohydrate, amino acids, cyclic peptide, phosphate, carboxylic acid or alcohol. The unique properties of biosurfactants allow their use and possible replacement of chemically synthesized surfactants in a number of industrial operations (Kosaric, 1992) Biosurfactants reduce surface tension, critical micelle concentration (CMC) and interfacial tension in both aqueous solutions and hydrocarbon mixtures (Rahman et al., 2002).
types of biosurfactant
There are many types of biosurfactants each produced by specific microorganisms. Glycolipids are the most known biosurfactants. They consists of mon-, di-, tri- and tetrasaccharides which include glucose, mannose, galactose, glucuronic acid, rhamnose and galactose sulphate. The fatty acid component usually has a composition similar to that of phospholipids of the same microorganism (Veenanadig et al., 2000). Also they are made up of carbohydrates in combination with long chains aliphatic acids or hydroxyaliphatic acids. Among glycolipids, the best known are the rhamnolipids, trehalolipids and sophorolipids are dissacharides (Rosenberg and Ron, 1999).
Bacteria of the genus Pseudomonas are known to produce glycolipids surfactant containing rhamnose and 3-hydroxy fatty acids (Lang and Wullbrandt, 1999 ; Rahman et al., 2002).Rhamnolipids produced by Pseudomonas aeruginosa strains are among the most effective surfactants when applied for the removal of hydrophobic compounds from contaminated soil (Rahman et al., 2002).Sophorolipids are group of biosurfactant produced by Torulopsis sp. Sophorolipids (SLs) consist of a dimeric s ugar (sophorose) and hydroxyl fatty acid linked by a ï¢-glycosidic bond (Asmer et al., 1988). Trehalolipids are another groupd of glycolipids, the serpentine group seen in many members of the genus Mycobacterium is due to the presence of trehalose esters on the cell surface (Asselineau and Asselineau, 1978). Trehalolipids from different organism differ in the size and structure of mycolic acid, the number of carbon atoms and the degree of unsaturation (Desai and Banat, 1997).
Lipopeptides called surfactin are produced by Bacillus sp. containing seven amino acids bonded to a carboxyl and hydroxyl groups of a 14-carbon acid. Surfactin just as any other biosurfactant reduces surface tension making surfactin one of the most powerful biosurfactants. Fatty acids produced from alkanes as a result of microbial oxidations have been considered as surfactants (Rehn and Reiff, 1981). In addition to the straight chain acids, microorganisms produce complex fatty acids containing OH groups and alkyl branches. The hydrophilic or lipophilic balance of fatty acids is clearly related to the length of the hydrocarbon chain.
Phospholipids are known to form major components of microbial membranes. When certain hydrocarbon degrading bacteria or yeast are grown on alkane substrates, the level of phospholipids increases greatly. Phospholipids have been quantitatively from Thiobacillus thiooxidans that are responsible for wetting elemental sulphur necessary for growth (Desai and Banat, 1997). Emulsan, liposan, mannoprotein and polysaccharide protein complexes are known to be the best studied polymeric biosurfactants.
factors affecting biosurfactant production
Biosurfactant are usually produced extracellularly or as part of cell membrane by yeast, bacteria or filamentous fungi (Mata-Sandoval et al., 1999). Different kinds of bacteria have been employed by many researchers in producing biosurfactant using culture media. Most of such bacteria used are isolated from contaminated sites usually containing petroleum hydrocarbon by products and/or industrial waste (Rahman et al., 2006; Benincasa, 2007).
A number of factors affect the production of biosurfactants. These factors include environmental factors as well as source of carbon substrate among others. Biosurfactant production like any other chemical reaction is affected by a number of factors that either increase its productivity or inhibit it. Accordingly, environmental factors such as pH, salinity and temperature affect biosurfactant production (Rahman et al., 2002; Raza et al, 2007). During in situ application, bacteria for Microbially Enhanced Oil Recovery (MEOR) must be able to grow under extreme conditions encountered I oil reservoirs such as high temperature, pressure, salinity and low oxygen level. Desai and Banat (1997) also affirm the fact that environmental factors and growth conditions such as pH, temperature, agitation and oxygen availability also affect biosurfactant production through their effects on cellular growth or activity. Salt concentrations also affect biosurfactant production depending on its effect on cellular activity.
A number of carbon substrates have been used in many researches during biosurfactant production. Indeed the type, quality and quantity of biosurfactant production are affected and influenced by the nature of the carbon substrate (Raza et al., 2007). Diesel and crude oil were identified to be good sources of carbon for biosurfactant production by organisms (Ilori et al., 2005) Other water soluble compounds such as glucose, sucrose and glycerol have also been reported to be a source of carbon substrate for biosurfactant production (Desai and Banat, 1997; Rahman et al., 2002). It has become evident that the importance of carbon substrates does have a major role to play on the biosurfactant production. It was noted that carbon sources such as nutrient concentrations, pH and age of the culture affects the yield of biosurfactant.
BioSurfactants application on bioremediation
Surfactants enhance removal of NAPLs from porous media by two methods: solubilization and mobilization. Mobilization is caused by the reduction in NAPL water interfacial tension that occurs when surfactant molecules partition to NAPL surfaces. The reduction in interfacial tension decreases the capillary forces holding NAPL globules in place, thereby allowing mobilization of the globule. Mobilization may also occur at very low interfacial tensions. Mobilization is often considered inappropriate for remediation applications because hydraulic control of the mobilized NAPL can be difficult to maintain (Bai, Brusseau et al. 1997). For these reasons, it is typically recommended that surfactants enhanced remediation should promote solubilization rather than mobilization. This may be achieved by choosing specific surfactants that do not cause significant reductions in interfacial tension and by optimizing other system properties, such as ionic strength and hydrophilic lipophilic balance (Sabatini and Knox 1992).
Mobilization can be divided into displacement and dispersion. Displacement is the release of NAPL droplets from porous media owing to a reduction in interfacial tension
(Abdul and Gibson 1991). From a theoretical perspective, entrapped NAPL will undergo displacement if the interfacial tension between the aqueous and NAPL phase is reduced sufficiently to overcome the capillary forces that caused the formation of residual saturation (Bai, Brusseau et al. 1997).
Dispersion is the process in which the NAPL is dispersed into the aqueous phase as very small emulsions (Abdul and Gibson 1991). Emulsions are generally not thermodynamically stable. However, owing to kinetic constraints, they may remain stable for significant time periods. Dispersion is related to both the interfacial tension and the surfactant concentration, and is different from displacement in that the displacement process is only related to the interfacial tension between aqueous and NAPL phases and no emulsion forms (Bai, Brusseau et al. 1997)
Biosurfactants are surface active amphipathic molecules produced by a plethora of microorganisms. They have wide structural diversity, ranging from glycolipids, polymeric and particulate surfactants (Sen, Mukherjee et al. 2008). Biosurfactants can emulsify hydrocarbons, thus enhancing their water solubility, decreasing surface tension and increasing the displacement of oil substances from soil particles (Banat, Makkar et al. 2000). Biosurfactants have considerable potential for surfactant enhanced remediation application because biosurfactants are a naturally occurring, biodegradable product and, thus, may be acceptable for application at many waste sites. Furthermore, biosurfactants are generally nontoxic to microorganisms, especially hydrocarbon degrading microorganisms. Industrial production is likely to be cost effective relative to synthetic surfactants and it may be possible to induce in situ production of a biosurfactant at a hazardous waste site (Bai, Brusseau et al. 1997).
Although, bioremediation is a reliable approach to improve contaminated area, the limit understanding of the biological contribution to the effect of bioremediation and its impact on ecosystem, has been challenging to make the technology safer and reliable. The behavior of target bacteria related to the degradation of contaminants and the changes in the microbial communities during the process of bioremediation has been challenging since many environmental bacteria cannot be cultivated by conventional laboratory methods. Therefore the application of culture independent molecular biological techniques offers new opportunities to understand the dynamic of microbial communities. Biosurfactant application in enhancing bioremediation is still challenging. Since the composition of final products is affected by the nutrient and environmental factors, it is obvious to find the right surfactant for industrial scale up. The requirement of the purity of the biosurfactants depends on its application, for example the surfactants used for environmental remediation should be free from microbial loading, but the quality of the product could be compromised. Therefore, this research is intended in exploration of a novel, more active biosurfactant from microbes and finding alternatives substrates for growth derived from cheaper carbon sources such as agriculture waste.
Strains are isolated from industrial and municipal sludge and identified.
Characterize the potential biosurfactant. Strains with high potential in producing biosurfactant will be subjected to carbon source (substrate). Determine the optimum culture conditions, strain will be cultured at different temperature, substrate concentration and pH values.
Evaluate the surface active properties of biosurfactant where tests on emulsification and surface tensions will be conducted. Efficiency of biosurfactant in enhancing NAPLs will be determined using laboratory scale reactor with NAPLs contaminated saturated sand. Column test will be conducted to study the feasibility and effciency on removing hydrophobic compounds from soil matrix using biosurfactant.
Model the biosurfactant efficiency and degradation rate behaviour.
To identify and develop potential biosurfactant from industrial and municipal sludge.
To characterize the potential biosurfactant.
To establish degradation rate and efficiency of biosurfactant.
To create the optimization of condition for the degradation of NAPLs.
To model the efficacy of biosurfactant and degradation rate behaviour in degrading NAPLs containment.