Biosurfactants producing organisms: classification and application in environmental biotechnology.
Polycyclic aromatic hydrocarbons (PAHs) are the world’s largest class of carcinogens known to date, not only because of their ability to cause gene mutation and cancer, but due to their persistency in the environment. They are particularly recalcitrant due to their molecular weight, hydrophobic nature and thus, accumulate in various matrices in the environment.
PAHs, also known as polyarenes or polynuclear aromatic hydrocarbons, are formed and released into the environment through natural and anthropogenic sources. Natural sources include volcanoes and forest fires while anthropogenic sources include, majorly, incomplete combustion of fossil fuels, wood burning, municipal and industrial waste incineration. PAHs containing two or three fused benzene rings are classified as low molecular weight (LMW) PAHs and are more water soluble while those with four or more benzene rings are referred to as high molecular weight (HMW) PAHs. They tend to adsorb onto soil and sediment thus, making them recalcitrant in the environment. Sixteen of these organic compounds have been identified as priority pollutants due to their hazardous properties, with HMW PAHs being considered as potential human carcinogens, by the United State Environmental Protection Agency .
The cost of biodegradation technology and the low bioavailability including mass transfer limitations of PAHs, especially those with high molecular weight, from several matrices into the aqueous phase for effective enzyme-based microbial biodegradation still constitute major challenges. However, current research efforts have focused on the combined use of biosurfactants and enzymes produced from renewable resources such as agricultural by-products and/or agro-industrial waste, through assisted biostimulation and bioaugmentation, for biodegradation of PAHs. Such methods are relatively inexpensive and less invasive as compared to physico-chemical remediation processes [2-4]. The application of crude biosurfactants produced by
The synergistic effect of biostimulation combined with bioaugmentation using fungal strains of
2. Bioavailability of HMW PAHs
2.1. PAHs bioavailability — Definitions and intrinsic factors
Polycyclic aromatic hydrocarbon mobilization and biodegradation are contingent upon their bioavailability from various matrices. Given the legal and regulatory implications of the bioavailability concept as part of a risk assessment framework, the term must be clearly understood in order to establish the minimum level permissible for contaminants like PAHs in the environment. The understanding of bioavailability is also important to be able to assess and evaluate the overall success of PAHs biodegradation. Researchers, however, differ in their opinion as to what the exact definition of bioavailability should be .
The following definitions for bioavailability were compiled in Technical Reports published by the European Centre for Ecotoxicology and Toxicology of Chemicals  and the United State National Research Council : (i) “The ability of a substance to interact with the biosystem; (ii) “The fraction of the contaminants in the environment that is potentially available for biological action” ; (iii) “The amount/percentage of a compound that is actually taken up by an organism as the outcome of a dynamic equilibrium of organism-bound sorption processes, and soil particle-related exchange processes, all in relation to a dynamic set of environmental conditions” ; (iv) “The fraction of a chemical accessible to an organism for absorption, the rate at which a substance is absorbed into a living system, or a measure of the potential to cause a toxic effect”. In pharmacology and toxicology, the term relates to the systemic availability of a xenobiotic after intravenous or oral dosing . Although these concepts are useful, making direct parallels from the pharmacological usage to contaminants in soil and sediment or biota can be problematic. For example, microorganisms do not have a digestive tract, target organs, or a circulatory system . These concepts may also not be appropriate as contaminants in soil or aquatic environments, being controlled by, for example, the rate of desorption and mobilization from solid matrices, are often continuously supplied to organisms gradually rather than at an acute dose. Environmental scientists often consider bioavailability to represent the accessibility of a soil-bound chemical for assimilation and possible toxicity [18, 19] and thus, have tried to adapt the use of bioavailability concept when considering human exposure to soil-borne contaminants. For example, Ruby  including Kramer and Ryan  suggested that the bioavailable portion is the amount of compound that is removed from soil through desorption processes under physiological conditions. Another view of bioavailability is represented by the contaminant crossing a cell membrane, entering a cell, and becoming available at a site of biological activity. Others might think of bioavailability more specifically in terms of contaminant binding to or release from a solid phase.
Obviously, the different definitions of the term bioavailability by scientists in various disciplines are capable of causing semantic confusion and thus, garner more attention than proffering solution to its challenges. The authors, in this chapter, have compiled these opinions in order to present a simple and workable definition to the term bioavailability as it is important to estimateF; the extent of contaminants desorption from the sorbed phase, the non-aqueous phase residue as against the minimum level required in the environment for such contaminants, and thus assessing the overall success of biodegradation. Considering these opinions about bioavailability, two words are common to almost all, which are; uptake or absorbed and available. Based on this observation, bioavailability can be defined as the amount of available contaminants in the environment that can be absorbed by microorganisms and /or biological products. Other clauses such as the fraction of contaminants taken up or absorbed, the fraction of contaminant that is potentially available, the mobilization or transportation of contaminants from the sorbed phase, the amount desorbed from the soil matrices, etc., which are often included in the definition of bioavailability and thus causing confusion, are intrinsic factors or features of bioavailability. However, these intrinsic factors are influenced by the physico-chemical properties of the contaminants and those of the sorbent.
Hence, studies on bioavailability are very crucial in order to link the quantity of PAHs taken up by microorganism with the actual amounts that are available to cause adverse effects in the environment. Many factors have been known to affect PAH bioavailability, which are [12, 22, 23];
Physical and chemical properties of PAHs,
Soil properties (soil organic matter, dissolved organic matter, moisture content, etc.),
Aging of PAHs in soil and receptor microorganism.
2.2. Effects of physical and chemical properties of PAHs on bioavailability
Bioavailability is influenced by the molecular structure and size of PAHs. LMW PAHs are removed faster by physico-chemical and biological processes due to their higher solubility, volatility and the ability of many microorganisms to use them as sole carbon sources in comparison to the HMW PAHs . Bioavailability changes with time and weathering . Aging is a central part concerning availability and refers to the process of organic compounds in soil becoming less susceptible to degradation, extractability and other related processes in a time dependent manner [26, 18]. Aging increases sorption propensity of soil contaminants making them more recalcitrant to diffusion and mobility which consequently lead to low bioavailability. Both the physico-chemical properties of the contaminant and the soil characteristics influence aging, which may include several steps and processes such as oxidation thus incorporating the contaminant into natural organic matter [27, 28], slow diffusion into small pores and absorption into organic matter, or entrapment due to the formation of semi-rigid films around non-aqueous-phase liquids (NAPL) with a high resistance toward NAPL-water mass transfer .
2.3. Effects of soil or sediment and dredging properties on PAHs bioavailability
Soil properties such as organic matter content, soil texture, soil depth, particle size, pH, porosity, intrinsic permeability, liquid limit, and cation exchange capacity, influence PAH bioavailability. Soil properties can vary greatly from one region to another. They can even vary within the same region spatially and with depth . Microorganisms have different levels of tolerance to these factors, which affect their growth and other metabolic activities. Soil structure such as aggregation has been found to decrease PAH availability through physical sequestration of PAHs in the interior of aggregates . For instance, Nam
2.4. Effects of mass transfer on bioavailability of HMW PAHs
The overall PAHs biodegradation can be conceptually divided into the following steps: desorption to the eqeuous phase; mass transfer to biologically accessible regions; and biological uptake and transformation [35, 18], as shown in Figure 1. These steps occur sequencially, such that the overall biodegradation rate can be controlled by any of these steps. Given the sequential nature of the process, the impact of desorption rate on overall biodegradation is expected to be greatest in the case where biodegradation rates are higher relative to desorption rates. This occurs when the active microorganisms are capable of high biodegradation rates and either the media is porous thus having a high capacity for the solute, or media has large diffusion distances [36, 37]. Based on this, it is important to examine how the physical morphology of surface and subsurface soils can impact the biodegradation of sorbed organic chemicals. Naturally, soil contains porous particles of different sizes, many of which are smaller than the size of microorganisms. For example, analysis of one of the coarser sand sizes from the Borden aquifer in Ontario (Canada), indicated that, roughly 50% of the intraparticle pore volume resides in pores that are less than 0.1 µm in diameter .
Pores with diameter larger than 1 µm comprised about 12% of the total pore space, and only about 5% of the pore volume was attributed to pores larger than 2 µm. Considering that most indigenous bacteria found in soil are 0.5 to 1.0 µm in diameter , bacteria will be physically excluded from attaching to intraparticle pores of these grains. The mean diameter of intraparticle pores occupied by bacteria has been estimated to be typically larger than 2 µm , and this is likely to be larger than intraparticle porespaces of many natural sorbent solids. However, for those pores which are accessible to bacteria, slow mass transfer of contaminants from the pore interior as well as from the pore surface can limit the extent of microbial growth and consequently biodegradation.
3. Kinetic models and assessment of bioavailability of PAHs
3.1. First–order kinetics
Biodegradation rate of PAHs in the aqueous phase and the rate constant (
3.2. Michaelis-Menten kinetic model
The most commonly assumed relationship for the assessment of bioremediation is done using the biodegradation rate and the concentration
At low PAH concentrations, such as when the concentration of the contaminant is in the part per billion ranges usually encountered for underground water contaminations, insufficient energy and carbon source availability can become a limiting factor for microbial growth and maintenance. That is, a threshold may exist below which microbial biomass growth cannot be sustained. Such a minimum concentration for sustainable growth is defined as the concentration at which microbial growth is balanced by decay [36, 43]. Also at such a concentration, the biodegradation rate reverts back to that described by first-order kinetics. However, the application of suitable microorganisms and/or their products can be used to enhance the concentration of PAH in the aqueous phase through increasing the desorption rate and mobilization from the sorbed phase. At such an enhanced concentration, Michaelis–Menten rate kinetics becomes appreciably applicable and from its linearized plot (Figure 3), the maximum PAH degradation rate can easily be determined. The rate equations can also be simplified by making certain assumptions. Particularly, the steady-state approximation which assumes a negligible change in the concentration of the enzyme-substrate complex during the course of the reaction. Furthermore, the Michaelis–Menten reaction mechanism presupposes that catalysis is irreversible and that the enzyme is not subject to any product inhibition. This limits the suitability of using this model to predict biodegradation rates where chemical surfactants have been known to inhibit product formation .
3.3. Freundlich adsorption isotherm
The Freundlich isotherm is an adsorption isotherm which relates the concentration of a solute on the surface of an adsorbent to the concentration of the solute in the liquid with which it is in contact. The isotherm is mathematically represented as:
For which the linearized form is:
The isotherm can also be represented in terms of sorbent pore pressure
At high pressure, the extent of adsorption is independent of pressure (i.e. 1/n = 0), at low pressure, it is dependent on pressure.
In a recent study of biodegradation of phenanthrene and pyrene in a slurry soil system using
3.4. Enhanced bioavailability model
Using integration, the concentration of PAHs in the aqueous phase at any particular time during the biodegradation process can be determined as:
The bioavailability factor (
3.5. Effects of temperature on bioavailability
Studies have demonstrated that temperature optimization had a positive effect on biodegradation of hydrophobic organic compounds especially for PAHs in a soil historically contaminated with these hydrocarbons. These findings indicated that in-situ remediation processes are accelerated with elevated temperature in a range between mesophilic and thermophilic temperatures. However, at a high temperature, the activity of fungal and bacterial populations is reduced . Temperatures in the thermophilic range (50 to 60°C) are shown to greatly accelerate decomposition of organic matter, in general . Microbial utilization of hydrocarbons can occur at temperatures ranging from -2 to 70°C. Iqbal
Temperature elevation coupled with moisture optimization was also determined to enhance the bioremediation of such contaminated soil. Raising the temperature also decreases adsorption, which makes more organic material available for microorganisms to degrade. With the synergistic effects of elevated temperature and sufficient moisture content, White
Furthermore, temperature plays a role when nutrients are added for biodegradation. Previous studies indicate that at 10°C, biodegradation rates were not affected by the addition of phosphorus or nitrogen. However, at 20°C, biodegradation was increased by the addition of phosphorus . This suggests that temperature optimization needs to be combined with sufficient and suitable nutrient amendments. Sartoros
In a bioaugmentation system, where allochthonous or genetically engineered microorganisms are added into contaminated soil to aid the operation of indigenous microbes, microbial population can decline rapidly following their introduction into such natural soil, and growth of the engineered populations in microbiologically undisturbed soils is a rare phenomenon due to microbiostasis [56, 57]. Furthermore, temperature variability is one of the principal contributors leading to the decline of microorganisms introduced into a 'hostile' soil microenvironment. In high temperature treatments, the response and survival of the microorganisms will be compromised compared to ambient temperature treatments. The microbial concentration increases under ambient temperature thus significantly increasing remediation activity. Results suggest that the stimulated growth and activity of indigenous microbes with elevated temperature in non-inoculated treatment indicates a positive relationship between optimum temperatures and better biodegradation performance.
4. Enhancing bioavailability of HMW PAHs using biosurfactants from renewable resources
4.1. Effects of biosurfactants on sorbed PAHs
Biosurfactants are surface active agents produced by microorganisms. Table 1 shows some of these microorganisms and the different groups of biosurfactant they produce. All biosurfactants consist of two parts, a polar (hydrophilic) moiety and a non polar (hydrophobic) group. 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 . Due to their amphiphilic structure, biosurfactants show a wide range of properties, including the lowering of surface and interfacial tension of liquids, the ability to form micelles and micro-emulsions between two different phases, the ability to increase the surface area of hydrophobic water-insoluble substances, and thus increase the water bioavailability of such substances. In comparison to their chemically synthesized equivalents they have many advantages; they are environmentally friendly, biodegradable, less toxic and non-hazardous. They have better foaming properties and higher selectivity; they are active at extreme temperatures, pH and salinity, and can be produced from wastes and from various by-products. This feature makes the production of cheap biosurfactants possible and the concomitant effects of utilizing waste substrates and reducing their environmental pollution [59-63]. Biosurfactants increase the bioavailability of PAHs resulting in enhanced growth of the degrading microorganism and the biodegradation of the contaminants.
An important feature of the physico-chemical properties of surfactants is their hydrophilic-lipophilic balance (HLB) [64, 65]. The HLB value indicates whether a surfactant will produce a water-in-oil or oil-in-water emulsion. Emulsifiers with lower HLB values of 3 to 6 are lipophilic and promote water-in-oil emulsification, while emulsifiers with higher HLB values between 10 and 18 are more hydrophilic and promote oil-in-water emulsion formation . A classification based on HLB values has been used to evaluate the suitability of different surfactants for various applications. For example, it has been reported that successful surfactants are those with the ability to promote desorption of contaminants from contaminated soils and are normally those with HLB values above 10 .
|Glycolipids||Rhamnolipids||Enhancement of the degradation, dispersion, emulsification of different classes of hydrocarbons and vegetable oils; removal of metals from soil.||[69, 71]|
|Trehalolipids||Enhancement of the bioavailability of hydrocarbons.|||
|Sophorolipids||Recovery of hydrocarbons from dregs and muds; removal of heavy metals from sediments; enhancement of oil recovery.||[73, 74, 69]|
|Fatty acids, phospholipids and neutral lipids||Corynomycolic acid||Enhancement of bitumen recovery.|||
|Spiculisporic acid||Removal of metal ions from aqueous solution; dispersion action for hydrophilic pigments; preparation of emulsion-type organogels, microencapsulation|||
|Phosphati-dylethanolamine||Increasing the tolerance of bacteria to heavy metals.|||
|Lipopeptides||Surfactin||Enhancement of the biodegradation of hydrocarbons and chlorinated pesticides; removal of heavy metals from a contaminated soil, sediment and water; increasing the effectiveness of phytoextraction.|||
|Lichenysin||Enhancement of oil recovery|||
|Polymeric biosurfactants||Emulsan||Stabilization of the hydrocarbon-in-water emulsions.|||
|Biodispersan||Dispersion of limestone in water.|||
|Liposan||Stabilization of hydrocarbon-in-water emulsions.|||
Another feature is the Critical Micelle Concentration (CMC); which is the concentration above which the formation of micelles is thermodynamically favored . The mobilization mechanism occurs at concentrations below the biosurfactant CMC. At such concentrations, biosurfactants reduce the surface and interfacial tension between air-water and soil-water systems. Due to the reduction of the interfacial force, contact of biosurfactants with a soil-oil system increases the contact angle and reduces the capillary force holding the oil and soil together. Above the biosurfactant CMC, the solubilization process takes place. At these concentrations, biosurfactant molecules aggregate to form micelles, which dramatically increase the solubility of the oil. The hydrophobic part of the biosurfactant molecules interconnect inside the micelle while the hydrophilic ends are then exposed to the aqueous phase on the exterior. Consequently, the interior of a micelle creates an environment compatible for hydrophobic organic molecules. The process of incorporation of these molecules into a micelle is known as solubilization . The formation of micelles leads to a significant increase in the apparent solubility of hydrophobic organic compounds, even above their water solubility limit, as these compounds can partition into the central core of a micelle. The effects of such a process are the reduction of surface and interfacial tension, enhancement of mobilization and mass transfer of contaminants from soil particles into the aqueous phase, and consequently the bioavailability of the hydrophobic contaminants for microbial attack [69, 70].
4.2. Biosurfactants production using agricultural and industrial wastes
The bioconversion of waste materials is considered to be of importance for the development of sustainable biotechnology processes in the near future because of its favorable economics, low capital and energy cost, reduction in environmental pollution, and their relative ease of operation [86, 10, 87, 88]. Producing usable products from agricultural and industrial waste is therefore a feasible and favorable option [89, 90]. Modern society produces high quantity of waste materials through activities related to industries such as those in the forestry, agriculture and municipal sectors [86, 91].
The use of the alternative substrates such as agro-based industrial wastes is one of the attractive strategies for the economic production of biosurfactants to enhance biodegradation of environmental hydrophobic contaminants. It has been suggested that successful approaches to more economical production technologies of biosurfactants will be a collaborative approach involving process development and sustainable raw material supplies. According to Marchant and Banat , emphasis should be on the cost effective management of downstream processing. These inexpensive agro-industrial wastes substrates include olive oil mill effluent, plant oil extracts and waste, distillery and whey wastes, potato process effluent and cassava wastewater . These waste materials are some examples of food industry by-products or waste that can be used as feedstock for biosurfactants production. Similarly, vegetable oil wastes can be used for biosurfactant production as they are lipidic carbon sources and are mostly comprised of saturated or unsaturated fatty acids with 16 to18 carbon atoms chain.
Studies involving the application of a variety of vegetable oils for biosurfactants production from canola, corn, sunflower, safflower, olive, rapeseed, grape seed, palm, coconut, fish and soybean oil have been reported. The world production of oils and fats is about 2.5 to 3 million tons, 75% of which are derived from plants and oil seeds . The high content of fats, oils and other nutrients in these wastes make them invaluable cheap raw materials for industries involved in useful metabolite production. Furthermore, from an economical point of view, nutrient rich agricultural residues can be employed for producing useful biological products such as biosurfactants. These materials are among the most abundant organic carbon available on earth  and they are the major components of different waste streams from various industries.
In recent times, studies have been focused on the application of agro-industrial wastes or by-products for the production of biosurfactants which are used in crude form or the direct use of surfactants producing strains for biodegradation processes. This is due to the high cost of biosurfactant purification and the stability including sustainability provided by these biosurfactants producing strains in biodegradation processes. The production and properties of a biosurfactant, synthesized by
4.3. Application of biosurfactants for enhancing PAHs bioavailability
Only limited numbers of microorganisms are capable of degrading HMW PAHs. Hence their biodegradation is limited by their low bioavailability to the microorganisms, which is due to their hydrophobicity, low aqueous solubility and strong adsorptive capacity in soil [99, 100]. Berg
The efficiency of biosurfactants in the remediation of soil contaminated by phenanthrene and polychlorinated biphenyls (PCBs) was also reported . In an investigation of the capacity of PAH-utilizing bacteria to produce biosurfactants using naphthalene and phenanthrene, Deziel
Microbially produced biosurfactants were studied to enhance crude oil desorption and mobilization in model soil column systems . The results showed that the ability of biosurfactants from
The capability of biosurfactants and biosurfactant-producing microorganisms to enhance organic contaminants’ availability and biodegradation rates was reported by several authors [109, 110, 104]. Kang
Although environmental biotechnology is regarded as an eco-friendly technology for the clean-up of PAH-contaminated ecosystems, the successful application of this technology is still restricted by the enormous costs of its operation and the limited bioavailability of the contaminants to degradative micro-organisms, especially HMW PAHs, due to sorption and sequestration. Many bacteria, fungi and algae and their products have been applied to degrade a range of LMW PAHs, such as naphthalene, fluorene and phenanthrene; however, their activity towards HMW PAHs containing five or more fused benzene rings, such as Benzo(a)pyrene and Benzo(ghi)perylene, is limited. Application of biosurfactants produced using agricultural and industrial wastes may be promising for reducing the costs of biodegradation technology as well as enhancing bioavailability of HMW PAHs. However, there is a dearth of research on this subject. Bioavailability model could be a vital tool to estimate and assess the success of bioremediation; therefore, more research is needed in this area, considering variations in environmental conditions that could limit field simulation of laboratory results. The synergistic effects of increased temperature and moisture with biosurfactants application have been shown to enhance biodegradation efficiency; however, kinetic investigation is important under these conditions to provide an understanding of biodegradation rate in situation where these conditions cannot be controlled such as in a typical field operation.