In this article we will discuss about:- 1. Introduction to Bioremediation 2. Principles of Bioremediation and Biodegradation 3. Concept and Requirement 4. Bioremediation of Hydrocarbons 5. Advantages and Limitations.
Contents:
- Introduction to Bioremediation
- Principles of Bioremediation and Biodegradation
- Concept and Requirement of Bioremediation
- Bioremediation of Hydrocarbons
- Advantages and Limitations of Bioremediation
1. Introduction
to Bioremediation:
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Bioremediation is the process of removal of complex material by degrading environmental pollutants using living micro-organisms. It is a method to remove out pollutants from the environment, restoring contaminated sites and preventing future pollution. Bioremediation activity depends on natural capacity of micro-organisms to degrade organic compounds.
This capacity could be improved by providing optimum growth conditions to micro-organism or by applying the genetically modified micro-organisms (GMMs). This technology has been used to eradicate environmentally hazardous chemicals and detoxify them into nontoxic forms.
Micro-organisms play important role in this toxic removal technology, several members of microbial group like algae, fungi and bacteria are known to solubilise, transport and deposit the metals, and detoxify dyes and complex chemicals. The toxic waste materials are present in vapour, liquid or solid phases; therefore, bioremediation technology varies depending upon nature of toxic material.
2. Principles of Bioremediation and Biodegradation
:
Biodegradation:
Biodegradation is the process in which micro-organisms reduce complex organic pollutants into smaller chemical compounds. Most of the biodegradable matters are usually organic, and are generally derivatives of plant and animal matter. The micro-organisms take this matter as food material and convert them to smaller compounds by enzymatic or metabolic processes.
Type of Biodegradation:
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Biomineralisation:
Biodegradation is basically categories in to two type. The first category is called as biomineralisation. Mineralisation is the process in which micro-organisms feed on organic compounds and by a chemical process, reduce them to inorganic material such as water carbon dioxide, and other such inorganic compounds. In mineralisation process total degradation of the organic matter occurs.
Biotransformation:
The second category of bioremediation is called biotransformation. Biotransformation essentially differs from mineralisation. In biotransformation process, organic matter is not degraded totally. Some part of it is degraded and another part is converted into other smaller chain organic compounds.
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The converted smaller chain organic compounds may be either toxic or non-toxic. In the case of the pesticide Dichloro Diphenyl Trichloroethane (DDT), the biotransformation yields more toxic compound. Another example of biotransformation is the fermentation process, in which sugar, a long chain organic compound, is transformed into ethanol.
Relative Biodegradability of Different Compounds:
Order of biodegradation is as follows from simple to complex compounds.
Simple hydrocarbons and petroleum fuels → Aeromatic hydrocarbon → Alcohol and esters → Nitrobenzenes → Clorinated hydrocarbons → Pesticides
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Bioremediation utilise abilities of micro-organisms for degradation of toxic pollutants. These include natural attenuation, bioaugmentation or biostimulation. Bioremediation may be enhanced by engineered techniques, either by addition of selected micro-organisms or by stimulation, where nutrients are added for this purpose.
Genetic engineering is also used to improve the biodegradation capabilities of micro-organisms. There are several factors affecting the efficiency of bioremediation process and risks associated to the use of genetically modified micro-organisms.
Role of Micro-Organisms in Biodegradation of Pollutants:
Biodegradation is the natural way of recycling wastes, or breaking down organic matter into simple nutrients that can be used and reused by other organisms. In the microbiological sense, ‘biodegradation’ means degradation of all organic materials by bacteria, yeast and fungi, and possibly other organisms.
Bioremediation and biotransformation methods are naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons, polychlorinated biphenyls, polyaromatic hydrocarbons, radionuclides and metals.
Some Biodegradable Pollutants:
Due to industrial development to produce a range of products, highly toxic organic compounds have been synthesised and released into the environment for direct or indirect application over a long period of time. These products are different fuels, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, pesticides and dyes.
There are some other synthetic chemicals like radionuclides and metals those are not biodegradable using native flora compared with the naturally occurring organic compounds that are easily degraded in the natural environment. Hydrocarbons, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pesticides, dyes, radionuclides, heavy metals are categorise as biodegradable pollutants.
3. Concept and Requirement of Bioremediation
:
Type of Bioremediation:
On the basis of removal and transportation of wastes for treatment, basically there are two methods of bioremediation:
1. In situ bioremediation.
2. Ex situ bioremediation.
1. In Situ Bioremediation:
In situ bioremediation is the cleaning up approach where micro-organisms feed on contaminants and dissolve contaminants for biotransformation. Biotransformation is a very complex process. Minimal site disruption, simultaneous treatment of contaminated soil and ground water, minimal exposure of public, site personnel, and low costs are potential advantages of in situ bioremediation method.
Limitations of in Situ Bioremediation:
Following are limitations of in situ bioremediation:
1. Time consuming method as compared to other remedial methods.
2. Seasonal variation of microbial activity resulting from direct exposure to prevailing environmental factors, and lack of control of these factors.
3. Problematic utilisation of treatment additives as nutrients, surfactants and oxygen. The micro-organisms act well only when the waste materials help them to generate more cells. When the native micro-organisms lack biodegradation capacity, genetically engineered micro-organisms may be added to the site during in situ bioremediation.
There are two types of in situ bioremediation, intrinsic and engineered in situ bioremediation.
1. Intrinsic in Situ Bioremediation:
Intrinsic bioremediation is conversion of environmental pollutants into the harmless forms through the natural capabilities of naturally occurring microbial population. There is increasing interest on intrinsic bioremediation for control of all or some of the contamination at waste sites.
The inherent capacity of micro-organisms to degrade the contaminants should be analysed and tested at laboratory and in field trails before use for intrinsic bioremediation. There are several conditions of site that favour intrinsic bioremediation.
These conditions are ground water flow throughout the year, carbonate minerals to buffer acidity produced during biodegradation, supply of electron acceptors and nutrients for microbial growth and absence of toxic compounds. The other environmental factors such as pH, concentration, temperature and nutrient availability determine whether or not biotransformation takes place.
Presence of metals such as Hg, Pb, As and cyanide at toxic concentration can create problem for microbial growth during bioremediation of that waste. Degradation of pollutants using bacteria in ground water is dependent on the type and concentration of compounds, electron acceptor and duration of bacteria exposed to contamination. Therefore, ability of bacteria used to degrade contaminants must be determined in laboratory by microbial studies before use.
2. Engineered/Accelerated in Situ Bioremediation:
Intrinsic bioremediation is giving good results at some places, but it is slow process due to poor availability and growth of micro-organisms, limited ability of electron acceptor and nutrients, cold temperature and high concentration of contaminants. When site conditions are not matching with microbial growth requirement, in this case bioremediation requires engineered systems to supply materials that stimulate micro-organisms growth.
Engineered in situ bioremediation accelerates the desired biodegradation reactions by encouraging growth of more micro-organisms under optimum physico-chemical growth conditions.
2. Ex Situ Bioremediation:
Ex situ bioremediation involves removal of waste materials and their collection from the contaminated site or place to facilitate microbial degradation. Ex situ bioremediation technology includes most of disadvantages and limitations as it is costly process due to costs associated with solid handling process, e.g., excavation, screening and fractionation, mixing, homogenising and final disposal. Contaminated material may be in liquid or solid form.
On the basis of phases of contaminated materials under treatment ex situ bioremediation is classified into two part as per following:
1. Solid-phase system (including land treatment and soil piles), i.e., composting.
2. Slurry-phase systems (involving treatment of solid-liquid suspensions in bioreactors).
Solid-Phase Treatment:
Solid-phase system includes organic wastes which is in solid form (e.g., leaves, animal manures and agricultural wastes), and problematic wastes (e.g., domestic and industrial wastes, sewage sludge and municipal solid wastes). The traditional clean-up practice involves the processing of the organic materials and production of composts which may be used as soil conditioning.
Composting:
Composting is a self-heating, substrate-dense, managed microbial degradation system used to digest organic matter. This is solid-phase biological treatment technology which is suitable to the treatment of large amount of contaminated solid materials.
It has been seen that many hazardous compounds are non-degradable using micro-organisms due to complex chemical structure, toxicity and compound concentration that do not support to microbial growth. Microbial growth is also affected by other factors such as moisture, pH, inorganic nutrients and particle size. During composting of hazardous wastes, micro-organisms requires proper amount of supplements for support of microbial self-heating.
The aliphatic, aromatic hydrocarbons and certain halogenated compounds considered as hazardous compounds disappear through composting process. Volatilisation, assimilation, adsorption, polymerisation and leaching are the possible steps leading to disappearance of hazardous compounds present as contaminants.
Composting can be done in open system, i.e., land treatment, and in closed system also. The open land system can be inexpensive treatment method, but limitation is the control of temperature fluctuation from summer to winter. Therefore, rate of biodegradation of waste materials decreases. Another limitation is land treatment system which becomes oxygen limited, depending on amount of substrate, depth of waste, application, etc.
The efficiency of open treatment system can be increased by passing air through blower. This approach is referred to as engineered soil piles and forced aeration treatment. The closed treatment system is preferred over the open land treatment system because controlled air is supplied to maintain the microbial activity.
As a result of microbial growth and volatilisation of hazardous compounds, internal temperature gradually rises. Therefore, use of blowers for air circulation and exhaust for removal of toxic volatiles are set up in closed treatment system. Ventilators supply oxygen and remove heat through evaporation of water.
Composting is a solid-phase biological treatment therefore target compounds must be either solid or a liquid associated with a solid matrix. The hazardous compounds should be biologically transformed. To do this, the waste material should be pretreated or prepared so that biological treatment potential should maximise. This is done by adjustment of several physical, chemical and biological factors.
The hazardous wastes must be solubilised so that they may be available to micro-organisms very easily. The hazardous compounds and soil organic matters act as source of carbon and energy for micro-organisms. Enzymes secreted by micro-organisms during growth phase are used to degrade toxic compounds. Availability of water, O2, inorganic nutrients and pH, increase the rate of decomposition of hazardous compounds.
If there is low substrate-density or site-specific conditions, analogue or non-analogue, non-hazardous carbon sources that can stimulate microbial growth and enzyme production can be added to compost. Presence of sufficient amount of water enhances microbial growth. Addition of inorganic nutrients influences microbial growth and rate of decomposition of hazardous wastes.
It has also been noted that a pH range of 5.0-7.8 promoted the highest rates of degradation of hazardous wastes. But lignin degradation has been found the most rapid at pH of 3.0-6.5. This shows that optimal pH levels can be species, site and waste specific.
Soil Treatment by in Situ Bioremediation:
Bioventing is a advance technology that stimulates the natural in situ biodegradation of any biodegradable compound in soil by supplying oxygen to existing soil micro-organisms. In comparison to soil vapour vacuum extraction, bioventing uses low air flow rates to provide only enough oxygen to accelerate microbial activity. Oxygen is supplied through direct air injection into contaminated soil site.
Degradation of adsorbed fuel residuals as well as volatile compounds are degraded as vapours and move slowly through biologically active soil. Bioventing technique has been successfully used to remediate soils contaminated by petroleum hydrocarbons, non-chlorinated solvents, some pesticides, wood preservatives, and other organic chemicals.
On other hand bioremediation cannot degrade inorganic contaminants, bioremediation can be used to change the valence state of inorganics and cause adsorption, uptake, accumulation, and concentration of inorganics in micro or macro-organisms.
Biodegradation of contaminants occurs in presence of micro-organisms naturally at the site of contamination. These naturally present micro-organisms on the contaminated site, called indigenous micro-organisms. In some cases, if indigenous microbial populations may not be capable of degrading the wide range of potential substrates present in complex mixtures such as petroleum or that they may be in a stressed state as a result of the recent exposure to the site, then bioaugmentation technique is used to solve this problem.
Bioaugmentation may also be used when the indigenous hydrocarbon- degrading population is low, the speed of degradation is the primary factor, and when seeding may reduce the lag period to start the bioremediation process.
For successful application of bioaugmentation, the seed micro-organisms must be able to degrade most contaminants, maintain genetic stability and viability during storage, survive in foreign and hostile environments. This method effectively compete with indigenous micro-organisms, and move through the pores of the sediment to the contaminants.
Different microbial species have different enzymatic abilities and preferences for the degradation of contaminants. Some micro-organisms degrade linear, branched, or cyclic alkanes. Others prefer mono or polynuclear aromatics, and others jointly degrade both alkanes and aromatics.
The study of microbes in bioremediation systems makes possible the selection of micro-organisms with potential for the degradation and production of compounds with biotechnological applications in the oil and petrochemical industry.
Bioaugmentation treatments depend on the use of inocula consisting of microbial strains or microbial consortia that have been well adapted to the site to be remediated. Foreign micro-organisms have been applied successfully but their efficiency depends on ability to compete with indigenous microorganisms, predators and various abiotic factors.
Factors affecting proliferation of micro-organisms used for bioaugmentation including the chemical structure and concentration of pollutants, the availability of the contaminant to the microorganisms, the size and nature of the microbial population and the physical environment should be taken into consideration when screening for microorganisms to be applied.
Bioaugmentation involves the introduction of micro-organisms isolated from the contaminated site or genetically modified to support the remediation of contaminated sites based on the confirmation that indigenous organisms within the contaminated site cannot biodegrade contaminants.
Biosparging is the method in which the atmospheric air is injected into the aquifer. Biosparging is used in both saturated and unsaturated soil zones. The technique was developed to reduce the consumption of energy. The injection of air into the aquifer results the formation of small channels for the air to move to the unsaturated soil zone.
In order to form the several branches in these channels to supply the air in to soil biosparging results the transportation of volatile contaminants to the unsaturated zone. Soil vapour extraction is used to extract the developed volatile vapours and then treat them at the surface.
For effective biosparging, the sparge points must be installed below the contamination zone because air always flows in upward direction. The upflow of air will form a cone. The degree of branching and the angle of the cone are determined by the amount of air pressure during the injection.
Monitoring wells are installed around the point and then the groundwater level and dissolved oxygen content are measured to determine the zone of influence for the sparge point. In order to effectively remove contaminants from the soil using biosparging, the soil should be relatively homogeneous throughout the contamination zone.
Reactors for Bioremediation:
Slurry-phase lagoon system which is very similar to aerated lagoon used for treatment of small common municipal wastewater. For maximising growth of micro-organisms, nutrients and aeration are supplied to the reactor. Mixers are fitted to mix different components and form slurry, whereas surface aerators provide air required for microbial growth.
The process may be used as single-stage or multistage operation depending upon requirement. This reactor is not appropriate for treatment of waste containing volatile materials or components. This is the limitation of slurry phase lagoon system.
Low-Shear Airlift Reactors (LSARs):
To solve the limitation of slurry phase lagoon in case of volatiles containing waste, low shear airlift reactor has been developed. The LSARs are useful when waste contains volatile components; tight process control and increased efficiency of bioreactors are required.
Figure 10.9 shows a low-shear airlift slurry-phase bioreactor. LSARs are like cylindrical tank which is made up of stainless steel. In this bioreactor pH, temperature, nutrient addition, mixing and oxygen can be controlled as per requirement. Impellers are mounted on shaft to fulfill the need and driven by motor set up at the top.
The rake arms are connected with blades which is used for resuspension of coarse materials that tend to settle on the bottom of the bioreactor. Air diffusers are also arranged along the rake arm. Airlift provides to bottom circulation of contents in reactor.
Baffles maintains the hydrodynamic behaviour of slurry-phase bioreactors. Contaminated material should be pre-treated using size fractionation of solids, soil washing, milling to reduce particle size and slurry preparation. To enhance the rate of biodegradation, some surfactants such as anthracene, pyrene, perylene, etc., are added to waste. These act as co-substrate and utilise as carbon and energy source. Co-substrates also induce the production of beneficial enzymes.
Factors Affecting Slurry-Phase Biodegradation:
Following factors play important role in slurry phase biodegradation:
1. pH (optimum 5.5-8.5)
2. Moisture content
3. Temperature (20-30°C)
4. Ageing
5. Mixing
6. Nutrients (N, P, micronutrients)
7. Microbial population (naturally occurring micro-organisms are satisfactory, genetically engineered micro-organisms for layer compound may be added).
8. Reactor operation (batch and continuous cultures).
4. Bioremediation of Hydrocarbons
:
Petroleum and its products are best examples of hydrocarbons and have much economic importance. Oil is made up of a variety of hydrocarbons, viz., xylanes, naphthalenes, octanes, camphor, etc. If these are present in more amount in the environment, these cause pollution.
In toxic environment micro-organisms perform, if the growth conditions, e.g., temperature, pH and inorganic nutrients are as per requirement. Oil is insoluble in water and is less dense. It floats on water surface and forms slicks. It has been seen that in storage tank microbial growth is not possible although water and air are supplied.
The micro-organisms which are capable of degrading petroleum include pseudomonas, various corynebacteria, mycobacteria and some yeasts. However, there are two methods for bioremediation of hydrocarbons/oil spills, by using mixture of bacteria, and using genetically engineered microbial stains.
Use of Mixture of Bacteria:
A large number of bacteria live in interfaces of water and oil droplets. Each strain of bacteria consumes a selective type of hydrocarbons, so, methods have been developed to introduce mixture of bacteria not a single strain. Mixture of bacteria have been used successfully to control oil pollution in water or oil spills from ships.
Bacteria living in interface degrade oil at a very slow rate. The rate of degradation could be accelerated with human efforts. Artificially well-developed mixture of bacterial strain along with inorganic nutrients such as phosphorus and nitrogen are pumped into the ground or applied to oil spill areas as required for treatment. This increases the rate of bioremediation at target site.
Use of Genetically Engineered Bacterial Strains:
Anand Mohan Chakrabarty, an India borne American scientist in 1979, obtained a strain of Pseudomonas putida that contained the XYL and NAH plasmid as well as a hybrid plasmid derived by recombining parts of CAM and OCT (these are incompatible and cannot co-exist as separate plasmids in the same bacterium). This strain could grow rapidly on crude oil because it had capability of metabolising hydrocarbons more efficiently than any other single plasmid.
5. Advantages and Limitations of Bioremediation:
Advantages of Bioremediation:
Bioremediation is a natural process and accepted by the public as waste treatment process for contaminated material such as soil. Microbes able to degrade the contaminant, increase in numbers and release harmless products. The residues for the treatment are usually harmless products such as carbon dioxide, water, and cell biomass.
Bioremediation is useful for the complete destruction of a wide variety of contaminants. Many hazardous compounds can be transformed to harmless products. This reduces the chance of future liability associated with treatment and disposal of contaminated material.
3. On Site Treatment:
Bioremediation can be carried out on site treatment, without causing a major disruption of normal activities. This removes the need to transport huge quantities of waste off site and thus reduce potential harm to human health and the environment that can arise during transportation.
4. Cost Effective Process:
Bioremediation is less expensive compare to other methods that are used for removal of hazardous waste.
Limitations of Bioremediation:
Limited up to biodegradable compounds-Bioremediation is limited to those compounds that are biodegradable. This method is susceptible to rapid and complete degradation. Products of biodegradation may be more persistent or toxic than the parent compound.
1. Specificity:
Biological processes are highly specific. Important site factors required for success include the presence of metabolically capable microbial populations, suitable environmental growth conditions, and appropriate levels of nutrients and contaminants.
2. Scale Up Limitation:
It is difficult to scale up from bench and pilot scale studies to full scale field operations.
3. Technological Advancement:
Research is needed to develop and engineer bioremediation technologies that are suitable for sites with complex mixtures of contaminants that are not evenly distributed in the environment. It may be present as solids, liquids, and gases.
4. Time Taking Process:
Bioremediation takes longer time compare to other treatment options, such as excavation and removal of soil from contaminated site.
5. Regulatory Uncertainty:
We are not certain to say that remediation is 100% completed, as there is no accepted definition of clean. Due to that performance evaluation of bioremediation is difficult, and there is no acceptable endpoint for bioremediation treatments.