Ex situ Bioremediation

Ex situ bioremediation involves removal of waste materials and their collection at a place to facilitate microbial degradation. Ex situ bioremediation technology includes most of disadvantages and limitations. It also suffers from costs associated with solid handling process e.g. excavation, screening and fractionation, mixing, homogenizing and final disposal. On the basis of phases of contaminated materials under treatment ex situ bioremediation is classified into two : (i) solid-phase system (including land treatment and soil piles) i.e. composting, and (ii) slurry-phase systems (involving treatment of solid-liquid suspensions in bioreactors). 

Solid-phase Treatment
Solid-phase system includes organic wastes (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 informal processing of the organic materials and production of composts which may be used as soil amendment. 
(i) Composting. Composting is a self-heating, substrate-dense, managed microbial system, and one solid-phase biological treatment technology which is suitable to the treatment of large amount of contaminated solid materials. However, many hazardous compounds are resistant to microbial degradation due to complex chemical structure, toxicity and compound concentration that hardly support growth. Microbial growth is also affected by moisture, pH, inorganic nutrients and particle size. Because composting of hazardous wastes typically involves the bioremediation of contaminated substrate-sparse soils, support of microbial self-heating needs incorporation of proper amount of supplements. The hazardous compounds reported to disappear through composting includes aliphatic and aromatic hydrocarbons and certain halogenated compounds. The possible routes leading to disappearance of hazardous compounds include volatilization, assimilation, adsorption, polymerization and leaching (Hogan, 1998).

Composting can be done in open system i.e. land treatment, and in closed system. The open land system can be inexpensive treatment method, but the temperature fluctuates from summer to winter. Therefore, rate of biodegradation of waste materials declines. Secondly, land treatment system may become oxygen limited, depending on amount of substrate, depth of waste, application, etc. However, efficiency of open treatment system can be increased by passing air (Fig. 21.2). 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 volatilization 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 (Fig. 21.3). Ventilators supply oxygen and remove heat through evaporation of water.

(ii) Composting Process. As composting is a solid-phase biological treatment, target compounds must be either solid or a liquid associated with a solid matrix. The hazardous compounds should be biologically transformed. To achieve this goal, the waste material should be suitably prepared so that biological treatment potential should maximize. This is done by adjustment of several physical, chemical and biological factors (Fig. 21.4). The hazardous wastes must be well solubilized so that they may be bioavailable. The hazardous compounds and soil organic matters serve the source of carbon and energy for microorganisms. Microbial enzymes secreted during growth phase degrade toxic compounds. However, proper maintenance of water, O₂, inorganic nutrients and pH increase the rate of decomposition.
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. Organic amendment also stabilize microbial population in inhibitory environment. Secondly, presence of sufficient amount of water enhances microbial growth. Addition of inorganic nutrients influences microbial growth and rate of decomposition of hazardous wastes. Under nitrogen limiting conditions Phanerochaete chrysosporium produces extracellular lignin peroxidase that degrades benzopyrene and 2,4,6-trinitrotoluene.

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.

Gradual colonization of organic materials is done by indigenous microflora, but hazardous chemicals may inhibit microbial growth. Therefore, bioaugmentation (i.e. use of commercial or GEMs) of wastes is also recommended.

To provide experimental proof of biodegradation during composting, a common hazardous contaminant pesticide, ¹⁴C-labelled Carbaryl was added in sewage sludge-wood chip mixture at 1.3 - 2.2 ppm concentration. After 18-20 days in laboratory composting apparatus, 1.6 - 4.9 per cent of Carbaryl was recovered as ¹⁴CO₂ and remaining bound to soil organic matter.

Slurry-phase Treatment
The contaminated solid materials (soil, degraded sediments, etc), microorganisms and water formulated into slurry are brought within a bioreactor i.e. fermenter. Thus slurry-phase treatment is a triphasic system involving three major components: water, suspended particulate matter and air. Here water serves as suspending medium where nutrients, trace elements, pH adjustment chemicals and desorbed contaminants are dissolved. Suspended particulate matter includes a biologically inert substratum consisting of contaminants (soil particles) and biomass attached to soil matrix or free in suspending medium. Air provides oxygen for bacterial growth. Slurry-phase reactors are new design in bioremediation. The objectives of bioreactor designing are to (i) alleviate microbial growth limiting factors in soil environment such as substrate, nutrients and oxygen availability, (ii) promote suitable environmental conditions for bacterial growth such as moisture, pH, temperature, and (iii) minimize mass transfer limitations and facilitate desorption of organic material from the soil matrix (Christodoultos and Kontsospyros, 1998).

Biologically there are three types of slurry-phase bioreactors : aerated lagoons, low-shear airlift reactor, and fluidized-bed soil reactor. The first two types are in use of full scale bioremediation, while the third one is in developmental stage.

(i) Aerated Lagoons. Fig. 21.5 shows the slurry-phase lagoon system which is very similar to aerated lagoon used for treatment of small common municipal waste water. 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. If the waste contains volatiles, this reactor is not appropriate.

(ii) Low-shear Airlift Reactors (LSARs). The LSARs are useful when waste contains volatile components; tight process control and increased efficiency of bioreactors are required.

Fig. 21.6 shows a low-shear airlift slurry-phase bioreactor. LSARs are cylindrical tanks which is made up of stainless steel. In this bioreactor pH, temperature, nutrient addition, mixing and oxygen can be controlled as desired. Shaft is equipped with impellers. It is 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 placed radially along the rake arm. Airlift provides to bottom circulation of contents in reactor. Baffles make the hydrodynamic behavior of slurry-phase bioreactors. Pre-treatment process includes size fractionation of solids, soil washing, milling to reduce particle size and slurry preparation. Certain surfactants such as anthracene, pyrene, perylene, etc. are added to enhance the rate of biodegradation. These act as co-substrate and utilize as carbon source. Co-substrates also induce the production of beneficial enzymes (Christodoultos and Kontsospyros, 1998).

Factors Affecting Slurry-phase Biodegradation
Factors that affect slurry-phase biodegradation are

1. pH (optimum 5.5-8.5),
2. moisture content,
3. temperature (20-30°C), oxygen (aerobic metabolism preferred),
4. aging,
5. mixing (mechanical and air mixing),
6. nutrients (N, P and micronutrients),
7. microbial population (naturally occurring microorganisms are satisfactory, genetically engineered microorganisms for layer compound may be added), and
8. reactor operation (batch and continuous cultures).