32 Bioremediation of Organic Contaminants

Dr Abhijeet Joshi

1.Introduction

Organic chemical compounds like volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) have shown increasing concerns due to their pollution in industrial gasses and effluents. The release of chemicals in environment has caused several diseases like cancer, allergy, asthma, respiratory problems etc. Petroleum products contain various kinds of volatile organic compounds (VOC’s). VOC’s are chemical compounds having low boiling points and can get mixed in the atmospheres contaminating it. VOC’s are derived from fuels combustion and solvent usage in gasoline combustion, aircraft fuel and chemical synthetic reactions employing solvents (1). VOCs are also known as key precursor for ozone formation. Depending on the type and number of industries the emission of VOCs is variable. Alkanes, cycloalkanes, halogenated compounds, cyclic aromatic structures like benzene, xylene, toluene etc. are the most common VOCs. European Environment Agency classifies these as priority pollutants. VOC emissions globally are reported to be in the range of 1012 kg/y. Polycyclic aromatic hydrocarbons (PAH) are commonly occurring pollutant which contaminates the environment. They are formed from incomplete combustion of organic compounds either naturally or due to human activities. Industrially obtained waste products act as the major sources of PAH pollution. PAH possess toxic properties, cancer causing properties mutation inducing properties. They can also act as stimulators of allergic reactions on the skin. Since they contain several cyclic rings are difficult to degrade biologically and hence accumulate in soil, fishes and other marine organisms. Owing to this they can easily enter the food chain and can be consumed by humans making them prone to diseases. PAHs have also been reported to cause toxicity to immunity, genetic mutations, and reproductive problems apart from the carcinogenic potential. Oil industry is the most significantly contributing economy of all the countries. Production, refining, transportation and consumption of petroleum products are increasing day by day. Contamination of hydrocarbon products in soil and water -bodies is the result of accidents or poor management practices during production, processing and transportation in the oil industry. Huge amounts of petroleum products are liberated into environment during combat wars, explosions during boring and from leaky tankers. Millions of gallons of oil are spilled annually in seas (2). The number of medium to large volume oil tanker accidents are increasing day by day due to an increase in marine modes of transportation. It is highly critical to address oil pollution in water as it has potential to harm fishery industry, which ultimately affects the wellbeing of organisms that use these contaminated fishes as their dietary source. Apart from the transportation of oils on seas, several billion tons of crude oil is transported on roads. Soil contaminated with petroleum has severe health dangers to plants, animals and human beings (3).

Figure 1: Oil contamination in soil and marine environments, Oil Contamination Found in the Largest National Reserve in Peru January 27, 2014 (4)

Petroleum products in soil contaminate ground water supplies limiting supply of consumable water, economic loss and decrease in agricultural productivity of soil. Health risk can arise from direct exposure, exposure to volatile fumes, and contamination of ground water (5).

2.Organic Contaminants

 2.1. VOCs and PAHs

VOCs are chemical compounds having a low boiling point in the range of (50-100 °C) with respect to the vapor pressure more than 102 kPa at 25 °C. Several VOCs are toxic in nature and can be fatal to human population. Majority of VOCs are developed from petrochemical sources and emission from vehicles. Almost all VOCs are sensitive and form ozone when they are exposed to oxides of nitrogen.

NOx + VOC + Sunlight  —–>  O3 + NOx + other products

Ozone formation is thus dependent of VOCs and Nitrogen oxides resulting in ground level ozone formation and smog. VOCs also occur from solvents used in paints, photocopying machines, carpets, plastics etc. Formaldehyde is another VOC which is found in furnishing of wood and fiber boards.

Polycyclic aromatic hydrocarbons contain carbon, hydrogen in the form of fused cyclic structures. The cyclic ring structures can be have several arrangements like linear, cluster or angular. PAH are also characterized by bay and K-regions which form epoxides when metabolized. Epoxide entities are highly reactive species and can cause severe toxicity. PAH’s having lower molecular weights are volatile in nature and can be degraded comparatively easily (6). PAH’s having high molecular weight with many rings are more stable against degradation by microbes. Due to their hydrophobic nature the Log P values lie in the range of 3-5 enough to cause toxicity to cells.

2.2. Oil Contaminants

Petroleum hydrocarbons are known to be toxic to all microorganisms, plants, animals and humans. They have been used as weed killers owing to their oil dissolving property in lipidic cytoplasmic membrane which results in leakage of cell contents and ultimately cell death. Crude oil contains complex mixture of different chemical compounds and possesses unique composition (5). Petroleum hydrocarbons can be divided into four comprehensive groups namely saturated oils, aromatics compunds, asphaltenes and resins (2).

3.Strategies to Counter Contamination by Organic Contamination 3.1. Chemical Processes

Majority of metabolic degradation pathways in plants and animals are already known to mankind and can be used as tools for bioremediation. The process of metabolism of hydrocarbons by bacteria and fungi utilize an oxidation step by set of enzymes known as oxygenases. This step requires molecular oxygen in the vicinity of the enzymic reaction catalyzing the degradation. Carboxylic acids are formed when alkanes are metabolized by Alkanes by entering into β oxidation cycle (Beta-oxidation step leads to usage of fats and lipids forming acetate which can further be metabolized by tricarboxylic acid cycle). Compounds with aromatic rings are converted to diols using a hydroxylation reaction forming catechols. Catechols can then be broken down to form intermediates readily taken up in TCA cycle. Fungi and bacteria are known to form the intermediates with different stereochemistries and with different optical properties. Fungi are known to form trans-diols and bacteria are known to form cis-diols. Trans-diols are reported to be carcinogenic whereas cis-diols are biologically inactive. Thus bacterial degradation of hydrocarbon products leads to biologically inactive products, complete neutralization and without producing any carcinogenic products. Complete degradation of hydrocarbons produce CO2, water and cell biomass (Proteins) all of which can be safely utilized in food chain.

3.2. Bioremediation of Organic Contaminants

3.2.1. Oil Contamination

Bioremediation can be explained as a procedure by which organic compounds are metabolized by bacteria, fungi, or any other species of microorganisms (7). This strategy has successfully been used for treatment of soil, sea-water, fresh water, terrestrial areas, and any waste water in a controlled manner (8). Oil polluted water can be addressed using oil degrading bacteria or fungi to prevent any potential damage to fishery business and it consequent hazardous effects on human or animal population. Several methods like environmental control of nutrients and addition of other ingredients for promoting the growth of indigenous population of bacterial can be stimulated. The process of introduction of additional exogenous bacteria is called as bio-augmentation. Genetically modified bacterial tuned to degrade hydrocarbons can also be used for improving the scale and rate of bioremediation. Nutritional needs of the chosen bacteria must be carefully understood and met by providing correct mix of fertilizer, aeration, temperature etc.

3.2.2.   Bioremediation of PAHs

Bioremediation of PAH pollution in marine sources is somewhat common, however the pollution in land/ soil is not addressed to that extent. PAH’s being hydrophobic in nature bind with soil which makes them unavailable for degradation by microbes. Many bacterial strains have been discovered which can degrade PAH’s. These microbes use PAH as a source of carbon and energy. The degradation mechanisms for PAH by microbes can be either aerobic or anaerobic. Aerobic degradation is the primary mechanism of degradation of PAH by microbes which involve oxidation of cyclic rings by enzyme called as oxygenases forming intermediates which can undergo ring breakage and further useful for metabolism. Anaerobic biodegradation can occur slowly in comparison to the aerobic degradation. Microorganisms of Pseudomonas and Mycobacterium have been known to degrade PAH’s. Microorganisms from Geobacter, Achromobacter, Bacillus, Arthrobacter, and Phanerocheate genus have also been reported to degrade petroleum products. Fungi from Penicillum, Aspergillus, and Fusarium have also been described with PAH degrading properties (9). The competition from indigenous bacteria also limits the growth of inoculated microbes. It is believed that introduction of multiple species of bacteria with different degradation pathways for pollutant can serve in a better manner than introducing a single species. Bioremediation of PAHs is determined by several factors like microorganism strain used, chemical structures and concentrations in question, type of surface acting agent and its concentration etc (9). Some environmental factors also affect the biological degradation which includes pH, temperature, salinity, oxygen levels, sediment type, light intensity, season, etc. Microorganisms require electron acceptors for metabolism which take part in the reduction reaction during metabolism of PAHs. PAHs undergo metabolism using different mechanisms like bacterial degradation, lignolytic fungal degradation and non lignolytic bacterial degradation. All these mechanisms involve oxidation of the ring structure of PAHs.

3.2.3.   Bioremediation of VOCs

Bioremediation technique uses microbiological activity to metabolize and nullify the toxic effects of chemical substances. The process is relatively inexpensive and has a high community acceptance. Biological treatment of VOCs is less commonly used, but can offer multitude of advantages over conventional processes. These processes require a lower energy as they employ microorganisms to degrade the VOCs. Microorganisms operate at ambient temperature and pressure conditions bringing down the energy requirements. Biological method of removal of VOCs includes use of biofilters, bioscrubbers, bioreactors of membrane and suspended type. Major important conditions which need to be controlled include temperature, pH, nutrients and moisture content (1). Easily biodegradable VOCs include esters, phenols, and benzene. Lower VOC concentrations can be very effectively treated using biological methods of treatment. Bio-filtration is based on the principle that the VOCs are transferred from air medium to a stationary medium where it is metabolized to form carbon dioxide, biomass and water.

3.3. Strategies for Improving Bioremediation

a) Seeding with Cultures of Microbes

An important approach considered during bioremediation of oil pollutants after an oil leakage is the seeding or inoculation of microbes that have the capability to degrade

hydrocarbons present in the oils. The seeded inoculum can be a combination of non-indigenous microorganisms from contaminated environments, especially nominated and developed for their oil-degrading properties. It can also be a blend of oil-degrading microorganisms selected from different sites which need remediation and then cultured in in bioreactors/ labs. Seed cultures are also supplemented with necessary nutrients. The necessities for seeding are challenging than nutrient supplementation. Innoculation of microbes should have the capability to destroy petroleum hydrocarbons in a manner which is superior to indegenous microbes and also counter the defence mechanism of indigenous microbes to compete for growth in a mixed population of organisms. Coping with physical environmental parameters (like temperature, salinity, and chemistry) and attack by other species present in the vicinity also comes into picture in case of seeding of microbes (1). The time frame for metabolizing hydrocarbons also comes into picture because they need to rapidly degrade the hydrocarbons in comparison to indigenous microbes. Very few claims prove this benefit of seeding cultures over indigenous microbes. Seed cultures are invariably lyophilized forms which act as dormant forms before they can be activated to become dynamic. Seeded cultures are also needed to be genetically consistent, non-pathogenic and should not produce any toxic chemicals. Seed cultures also must be genetically constant, must not be pathogenic, and must not produce toxic metabolic products. However, since hydrocarbon-metabolizing microbes are extensively dispersed in varied environments like marine water, soil habitats, fresh water streams and introducing seed cultures has proved to be less effective in comparison to added fertilizers and ensuring adequate aeration. Some institutions claim that seeding of microbes with blends of bacterial populations, which are custom made for different types of oils with individualized nutritional needs can be developed (10). However, most examinations have showed that seed cultures are of little benefit over the naturally occurring microorganisms at a dirty site for the biodegradation. Seed innoculums will be more suitable in conditions were native microbes are not able to metabolized the hydrocarbons or they are present in the latent forms. This can occur in case of hydrocarbon spills of difficult to degrade nature like aromatic compounds with multiple rings.

b)Seeding with Genetically Engineered Microorganisms: Seeding with Genetically Engineered Microorganisms is another alternative of seeding microbes in contaminated fields (11). It has not been demonstrated to be superior in the field but the first organism to be patented was an oil degrading bacteria. The preliminary results of genetically engineered microorganism was first realized when it was designed by University of Tennessee in collaboration with Oak Ridge National Laboratory. The Pseudomonas fluorescens strain HK44GEM was designed from a original Pseudomonas fluorescens strain. The designed strain was contained in a soil environment. This genetically engneered strain was isolated from a gas plant hugely polluted sites with multiple aromatic rings containing compounds. Such genetically engineered microbes can be beneficial for faster degradation of petroleum products that are not degradable by other microbial population or are adaptable in the contaminated environment to survive better than the indigenous or seeded population of microbes (12). In order to be effective, these species need to supersede all the hurdles of seeding using non indigenous bacteria. The development and use of genetically modified microbes for environmental cleaning up is still limited owing to obstacles related to scientific, regulatory, economic, and perception of utility regarding bioengineered micro-organisms. Lack of sophisticated infrastructures and the limited companies undertaking these activities, existence of only small companies and red tape-ism are hurdles to be solved to obtain profitable genetically designed organisms

c) Environmental Modification

Degradation of oils in marine surroundings is inadequate due to nutritional requirements of bacterial population like abiotic features like nitrogen, oxygen and phosphate concentrations. Petroleum biodegradation kinetics are found to be negligible for cases of oxygen-less sediments as oxygen requirement for initial step of oxygenation is critical in hydrocarbon metabolism. Aerated marine environment show a higher rate of degradation of petroleum products. Marine surroundings have minimal concentrations of essential nutrients which are required for cell mass growth. Among the factors limiting the speed of petroleum degradation lack of supply of nutrients is most critical. This parameter can be easily addressed by nutrient enrichment. Scientists believe nutrient enrichment as a great strategy of bioremediation. This strategy uses the incorporation of those essential nutrients, which reduces bioremediation kinetics, however no additional micro-organisms are added in the contaminated sites (13). The rationale in this method is that hydrocabon metabolizing mirobes are sufficiently large in concentrations in oceanic environments. These colonies have already adaptation to environmental stresses at that site. Leakage of large quantities of oils limits the availability of nutrients to the microorganisms. The addition of nutrients like phosphorus and nitrogen are done to decrease the perils and allow for petroleum metabolism with sufficiently fast rate (14). This approach has been utilized since 1973 successfully. Use of fertilizer has been found to improve the biodegradation rates by naturally occurring microorganisms. An innovative aspect of this application has been investigated wherein two parallel trenches to the beach are constructed which can be used to distribute fertilizers. In such a structure nutrients will dissolve with the approaching waves and will get pulled to the sea, enabling a fine distribution of nutrients in comparison to a single point supply of fertilizers. Although nutrient enrichment and environmental modification is the commonest technique of bioremediation it is very difficult to conclude that providing nutrients will be operative under all environments and circumstances or it can be more beneficial tool than other oil contamination solutions or natural processes.

d) Anaerobic Degradation

In some sites, oxygen levels are very low where permeation of oxygen is not enough to replace the consumed O2 by micro-organisms. Deep oceans having sediments or and soil sites may be subject to anaerobic degradation of hydrocarbons. Land farming is one of the methods in which spoiled soil is distributed to form a bed along with fertilizers which are mixed and re-distributed again. Composting is another process where in contaminated soil is piled containing organic manure or agricultural waste products (14, 15). The organic substances support the microbial growth due to presence of necessary nutrients for effective biodegradation.

e) Phyto-remediation

Phytoremediation method uses plants and their microbes for removing the pollutants present in the soil. This method is an ecologically attractive and environmentally friendly technique of removing organic contamination from soil. Removal of high concentrations of pollutants is more challenging using phytoremediation as the pollutant themselves inhibit the growth of the plants. Slower growth of plants causes the rate of removal of organic pollutants to be very slow and is affected by weather and soil properties. VOCs are also removed by plants through stomata and cuticles (16). Formaldehyde, methyl isobutyl ketone benzene and toluene were found to be removed by stomata in light enviroment. Different plant species which have been used for removal of VOCs include Nerium indicum through stomata. Cuticular removal of VOCs like benzene, toluene, ethyl benzene and xylene is observed in plants cuticle by Vitis vinifera, Malus domestica, Z. zamiifolia, and Acer campestre. Apart from these wax mediate uptake of VOCs has been found to occur in Dracaena sanderiana. Stomatal, cuticular or wax based uptake may be variable based on VOCs in question. After uptake in the plants the compound may be stored, metabolized or eliminated. Cleaning indoor air can be achieved using plant based uptake of VOCs by planting these species inside buildings (16, 17).

f) Rhizo-remediation

Rhizoremediation is another technique used for biodegradation of PAHs where in rhizospheric and endophytic bacteria can be used as an inoculation system for soils. Rhizosphere technology is often combined with agronomic techniques so that both processes from plants and rhizo-bacteria can be synergistically utilized for PAH degradation. Rhizo-remediation is also advantageous due to a high surface area obtained by attachment of roots and the treatment occurs deep within the soil as deep as the roots penetrate. Essential nutrients are provided by roots and only enzyme activating co-factors may be needed to be added for an efficient degradation. Combination of plant microbe pairs needs to be properly identified for the success of rhizo-remediation. Bioaugmentation using mirobes although attractive is limited as high bacterial number needs to be maintained at the contaminated site but the nutrient to support such a large number of microorganisms are scarce. The inoculated microorganisms also do not penetrate deeper in to the soil limiting the bioremediation potential of inoculated bacteria. Plasmids coding for pollutant degradation can also get damaged in the soil. These facts make rhizo-remediation popular for soil bioremediation in comparison to bio-augmentation (18).

3.4 Factors affecting Biodegradation

Bacteria and fungi are commonly employed for VOC degradation, among them fungi have the potential for operation at extreme conditions of pH and nutrients. VOCs can be utilized by microbes for synthesis of energy or synthesis of anabolic compounds. Pseudomonas, Candida, Mycobacterium, Alcaligenes, Exophiala, Acetinobacter, Fusarium, Cladosporium, Rhodococus, Aspergillus and mucor are some commonly used VOCs degrading microorganisms.

Attachment, metabolism and detachment of microorganisms in the biofiltration systems produce slimy organic structures called biofilms. A gradient of biomass is formed in the systems highest at the inlet and reducing towards the outlet of gas. Biofilm thickness controls the rate of degradation of VOCs. Environmental variables greatly affects the rate and extent of bioremediation. Most of the environmental variables can be altered at the organic contamination sites to increase the natural process of biodegradation. Salinity is generally not controllable and depends on the site of the oil spills. These variables and lack of knowledge about their effects are the major reasons of successful or un-successful biodegradation process hampering any kind of predictability of the process (11).

a) Oxygen

Oxygen is an important requirement for microbial degradation. Its availability is critical in determining the rate of degradation. Many microbes employ oxygen-fixing enzymes to propogate reaction with hydrocarbons. Anaerobic metabolism of some hydrocarbons occur at minimal speeds. Microorganisms employ anaerobic degradation where oxygen content is low and may take alternative chemical paths. Oxygen is required for the preliminary initiation of hydrocarbons and consequent reactions. Consequent reaction also require direct inclusion of oxygen. Conversion of hydrocarbons may require a huge demand of oxygen which may be about 4 parts of oxgyen for degradation of 1 molecule of hydrocarbon to convert to carbon dioxide and water. As at the surface of oceans sufficeint oxygen in present, presence of oxygen does not occur as a rate limiting step. Due to presence of waves and tides the oxygen concentrations keep on getting replenished. Reduction of oxygen levels decrease the biodegradation rates, therefore sunken oil concentrations will take lot of time to degrade (11). Oxygen concentrations in deep sediments are determined by water column and turbulence and presence of organisms dwelling at the sea bottoms. Sediments and beaches having low tidal energy have depleted oxygen levels indicating reduced biodegradation rates in these areas. Similarly, excessive oil makes the oxygen permeation to hinder in the deeper levels.

b) Nutrients

Nutrients like nitrogen, phosphorus, and iron were found to have an important role than the oxygen in controlling the bioremediation in marine conditions. Insufficient supply of vital nutrients results in a reduced degradation kinetics (19). Petroleum is a rich source of carbon for growth of microorganisms but minerals are needed to support the growth of bacteria. Such nutrients are scarce in marine environments because they get consumed by non-oil degrading microorganisms (e.g. phytoplankton). Phosphates get sedimented after precipitation as calcium phosphate at sea water pH. Iron is present to a greater extent in coastal water rich in sediment when compared to offshore water. Thus presence of nitrogen, phosphorous and iron is required for bacterial growth and need to be provided externally as nutrients. Adjusting nutrient levels by addition of fertilizers (nitrogen and phosphorous rich) can stimulate the rate of degradation. Proper methods of application of nutrients also influences the result of extent of bioremediation.

c) Temperature

The water present in the seas over the world occurs in the temperature range of -2 °C to 35°C. Degradation of oils has been observed in all the above temperature conditions. As the temperature increases the biodegradation rates become faster, and when the temperature drops the degradation rates usually decrease. In an experiment it is indicated that reduction in temperature to an extent of 25 °C can reduce the rates to an extent of ten times. Reduction in temperatures causes a drop in the rate of biodegradation due to lesser possibility of volatilization of light oils causing a subsequent toxicity to the oil degrading microbes. The viscosity of the heavy oils also increases at low temperatures causing lesser degradation rates. Increasing viscosity also provides less surface area for colonization of bacteria. The changing climates across the year also produce significant changes in biodegradation (7).

d) Bio-surfactants

Bio-surfactants are chemical structures produced by hydrocarbon degrading bacteria which are chemically polysaccharides, fats, lipids, peptides and proteins. These molecules reduce the surface tension between the molecules making the emulsification of oils an easy process. Most oil degrading strains are known to produce biosurfactants in both water and soil (19). An advantage of presence of biosurfactants is that they are derived from the bacteria and thus they are non-toxic and biodegradable and do not cause a contamination by themselves in comparison to externally added chemical agents. This makes the bioremediation process cost effective.

e) pH and Salinity

In marine sources of hydrocarbons pH does not fluctuate to a great extent and stays between 7.6-8.1. This does not produce any significant effect on biodegradation rates in marine conditions. However extremes of pH affect micro-organisms ability to degrade hydrocarbons. In marshy lands with high salt content may have a pH in the range of 5-7, which may cause a slowing of biodegradation rates (8). Microbes are typically adapted to different conditions salinity across seas present in the world. Scientists have provided very little evidence that alinity can affect biodegradation rates. In case of Estuaries salinity values, oxygen and nutrients available may be different in comparison to adjacent water bodies.

f) Moisture Content

Water is an essential component for microbial growth and enzymatic or biochemical activities. It is important for uptake of elements by microbes using absorption and transportation processes across cell membrane. Amount of water present determines the availability of nutrients to the microbes. About 50-80 % of moisture content is optimal for aerobic bioremediation (8). Low moisture content below 10 % hinders the growth of microbes due to lack of nutrient supply and very high moisture content causes water clogging significantly reduces bioremediation rates.