bioremediation

Bioremediation


Naturally occurring bioremediation and phytoremediation have been used for centuries. For example, desalination of agricultural land by phytoextraction has a long tradition.


CONTENTS:

• Invention
• Definition
• Types of bioremediation
o In situ
o Ex situ
• In situ
o Definition
o Advantages
o Disadvantages
o Types-
o Intrinsic
o Engineered

• Ex situ
o Definition
o Disadvantages
o Types
-Solid phase treatment
Composting
Composting process
-Slurry phase treatment
Aerated lagoons
Low shear airlift reactors
o Factors affecting
• Bioremediation of hydrocarbons
• Bioremediation of industrial waste
• Bioremediation of dyes
• Bioremediation of coal waste
• Bioremediation of heavy metals





Invention
Bioremediation technology using microorganisms was reportedly invented by George M. Robinson. He was the assistant county petroleum engineer for Santa Maria, California. During the 1960's, he spent his spare time experimenting with dirty jars and various mixes of microbes.

Definition
It is the use of living microorganisms to degrade environmental pollutants and to prevent pollution.
It is the technology to remove pollutants from environment restoring contaminated sites and preventing future pollution.

Types of Bioremediation-
Is the enormous natural capacity of microorganisms to organic compounds which could further be improved by genetic engineering.

The toxic waste material remain in vapour, liquid or solid phases, therefore, Bioremediation technology varies accordingly whether waste material involved is in its natural surroundings or is removed and transported into a fermenter. On the basis of removal and transportation of waste for treatment, basically there are are two methods-

• In situ bioremediation
• Ex situ. bioremediation

IN SITU BIOREMEDIATION-
In situ bioremediation involves treating the contaminated material at the site. It is the clean up approach which directly involves the contact between microorganisms and the dissolved and the sorbed contaminants for biotrasformation.

Advantages
• minimal site disruption
• simultaneous treatment of contaminated soil and ground water
• minimal exposure of public and site personnel
• low cost

Disadvantages
• time consuming
• seasonl variation of microbial activity resulting fron direct exposure to prevailing environmental factors and lack of control of these factors.
• Problematic applications if treatment additives.

The microorganisms work well only when waste material help them to generate energy and nutrients to build up more cells.

In situ bioremediation is of two types
• Intrinsic
• Engineered

Intrinsic bioremediation- Conversion of environmental pollutants into the harmless forms through the innate capabilities of naturally occurring microorganisms is called intrinsic bioremediation. The intrinsic i.e. inherent capacity of microbes to metabolize the contaminants should be tested at laboratory and field levels before use for intrinsic bioremediation. Through site monitoring programmes progress of intrinsic bioremediation should be recorded time to time.

Conditions of site that favour intrinsic bioremediation are-

• Ground water flow throughout year.
• Carbonate minerals to buffer acidity produced during biodegradation.
• Supply of electron acceptors and nutrients for microbial growth.
• Absence of toxic compounds.
• Other environmental factors such as pH, concentration, temperature and nutrient availability

Determine whether biotransformation takes place.

Bioremediation of waste mixtures containing metals such as Hg, Pb, As and cyanide at toxic concentrations can create problem.

The ability of surface bacteria to degrade a given mixture of pollutants in ground water is dependant on the type and concentration of compounds, electron acceptor and duration of bacteria exposed to contamination. Therefore, ability of indigenous bacteria degrading contaminants can be determined in laboratory by plate count and microcosm studies.

Limitations of intrinsic bioremediation-

• It is slow process due to poorly adapted microorganisms.
• Limited ability of electron acceptor and nutrients
• Cold temperature
• High concentration of contaminants.

Engineered in situ bioremediation- When site conditions are not suitable, bioremediation requires construction of engineered systems to supply materials that stimulate microorganisms. Engineered in situ bioremediation accelerates the desired biodegradation reactions by encouraging growth of more microorganisms via optimizing physico chemical conditions. Oxygen and electron acceptors (e.g. nitrate and sulphate ions) and nutrients (e.g. nitrogen and phosphorus) promote microbial growth in surface. When contamination is deeper, amended water is injected through wells. But in some in situ bioremediation systems both extraction and injection wells are used in combinations to control the flow of contaminated ground water combined with above ground bioreactor treatment and subsequent re-injection of nutrients spiked effluent are done.

EX SITU BIOREMEDIATION-
Ex situ bioremediation involves removal of waste materials and their collection at a place to facilitate microbial degradation.

Limitations- Suffers from the cost 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 types:

• Solid phase system (including land treatment and soil piles) i.e. composting
• Slurry phase treatment ( 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.

Composting- Composting is 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.

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. 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 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. Ventilators supply oxygen and remove heat through evaporation of water.

Composting process- As composting is a solid phase biological treatment, target compound 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. The hazardous waste must be solubilized so that they may be bioavailable. The hazardous waste 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, oxygen, inorganic nutrients and pH increase rate of decomposition.













































Outline of composting treatment sequence


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, the 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-trinitotoluene. It has also been noted that a pH range of 5.0 to 7.8 promoted the highest rates of biodegradation 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 GMMs) of wastes is also recommended.

To provide experimental proof of biodegradation during composting, a common hazardous contaminant pesticide, 14C labeled 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 % 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 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:

• Alleviate microbial growth limiting factors in soil environment such as substrate, nutrients and oxygen availability.
• Promote suitable environmental conditions for bacterial growth such as moisture, pH, temperature and
• Minimize mass transfer limitations and facilitate desorption of organic material from soil matrix.

Biologically there are three types of slurry phase bioreactors:

• Aerated lagoons
• Low shear airlift reactor
• Fluidized bed soil reactor

The first two types are in full use of full scale bioremediation while third is in developmental stage.

Aerated lagoons: the slurry phase lagoon system which is very similar to aerated lagoons used for the treatment of small common municipal waste water. Nutrients and aeration are supplied to 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 multi stage operation. If the wastes contains volatiles, the reactor is not appropriate.

Low shear airlift reactors (LSARs)- LSARs are useful when waste contains volatile components; tight process control and increased efficiency of bioreactors are required. LSARs are cylindrical tanks which is madeup of stainless steel. In this bioreactor, pH, nutrient addition, temperature mixing and oxygen can be controlled as desired. Shaft is equipped with impellers. It is driven by motor set up at top. The rake arms are connected with blades which is used for resuspension of coarse materials that tend to settle on bottom of bioreactor. Air diffusers are placed radially along rake arm. Airlft provides to bottom circulation of contents in bioreactor. Baffles make hydrodynamic behaviour of slurry phase bioreactors. Pretreatment 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 rate of biodegradation. These act as co-substrate and utilizes as carbon source. Co-substrates also induce production of beneficial enzymes.

Factors affecting slurry phase biodegradation- Factors that affect slurry phase biodegradation are-
• pH (optimum 5.5-8.5)
• moisture content
• temperature (20-30°C)
• oxygen (aerobic metabolism preferred)
• aging
• mixing ( mechanical and air mixing)
• nutrients ( N,P and micronutrients)
• microbial population (naturally occurring microorganisms are satisfactory, genetically engineered microorganisms for layer compound may be added)
• reactor operation (batch and continuous cultures)

BIOREMEDIATION OF HYDROCARBONS-

Petroleum and its products are hydrocarbons. These two have much economic importance. Oil constitutes a variety of hydrocarbons viz., xylanes, naphthalenes, octanes camphor etc. if present in the environment these cause pollution. For example, during cold war between iraq and America, millions of gallons of petroleum was leaked into sea which resulted in fish mortality. In addition, leakage of oil and petrol in marine is usual phenomenon.
In toxic environment microbes act only if the conditions e.g. temperature, pH and nutrients are adequate. Oil is insoluble in water and less dense. It floats on water surface and forms slicks. It should be noted that in bulk storage tank microbial growth is not possible provided air and water are supplied. The microbes which are capable of degrading petroleum includes pseudomonads, various corynebacteria, mycobacteria and some yeasts.

However, there are two methods for bioremediation of hydrocarbons/ oil spills,:

• By using mixture of bacteria
• By using genetically engineered microbial strains.

Use of mixture of bacteria- A large number of bacteria resides in interfaces of water and oil droplets. Each strains of bacteria consumes a very limited range of hydrocarbons, therefore, methods have been devised to introduce mixture of bacteria. Moreover, mixture of bacteria have been successfully been used to control oil pollution in water supplies or oil spills from ships. Bacteria living in interface degrade oil at a very slow rate. The rate of degradation could not be accelerated without human intervention. Artificially well characterized mixture of bacterial strains along with inorganic nutrients such as phosphorus and nitrogen are pumped into ground or applied to oil spill areas as required. This increases the rate of bioremediation significantly. For example, in the Exxon Valdez spill, accelerated bioremediation of oil washed upon beaches was noticed after spraying bacteria with admixture of inorganic nutrients.

Swaranjit singh and his group (IMTECH, Chandigarh) have isolated both bacterial and fungal cultures from the petroleum sludge. The fungal culture could degrade 0.4% sludge in 3 weeks. Degradation of petroleum sludge occurred within two weeks when the bacterial culture (Bacillus circulans CI) was used. Moreover, significant degradation of petroleum sludge was noticed in 10 days when the fungus + B. circulans and a prepared surfactant were exogenously added to petroleum sludge.

Use of genetically engineered bacterial strains- In 1979, for the first time Anand Mohan Chakrabarty, an Indian borne American scientist obtained a strain of Pseudomonas putida that contained the XYL and NAH plasmid as well as a hybrid plasmid derived by recombinant parts of CAM and OCT (these are incompatible plasmid and can not coexist as separate plasmid in same bacterium). This strain could grew rapidly on crude oil because it was capable of metabolizing hydrocarbons more efficiently than any other single plasmid. In 1990, the USA Government allowed him to use this Super bug for cleaning up of an oil spill in water of State of Texas. Superbug was produced on large scale in laboratory, mixed with straw and dried. The bacteria laden straw can be stored until required. When the straw was spread over oil slicks, the straw soaked up the oil and bacteria broke up the oil into non polluting and harmless products.

BIOREMEDIATION OF INDUSTRIAL WASTES-

A variety of pollutants are discharged in the environment from a large number of industries/mills. For example, textile industry alone contributes a significant amount of pollutants to water bodies such as enzymes, acids, alkali, alcohols, phenols, dyes, heavy metals, radionucliods etc. traces of zinc, cadmium, mercury, copper, chromium, lead are found in dyes.
It has been reported that actinomycetes show a higher capacity to metal ions as compared to fungi and bacteria. In addition, uptake mechanism of living and dead cells differ. Due to these differences they have potential application in industries. The living microbial cells accumulate intracellularly at a higher concentration, whereas dead cells precipitate metals in around cell walls by several metabolic processes. Dead biomass immobilized on polymeric membrane absorbs Uranium well, and immobilized Aspergillus oryzae cells on reticulated foam particles have been used for Cd removal. Aspergillus niger biomass contains upto 30 % of chitin and glucan. Chitin phosphate and chitosan phosphate of fungi absorb greater amount of U than Cu, Cd, Mn, Co, Mg and Ca.

BIOREMEDIATION OF DYES-

There is limited supply on microbial degradation of azo and reactive dyes. Maximum number of dyes undergo degradation through reduction.

• Kulla discussed azo-reductase of Pseudomonas strains in the chemostat culture. This enzyme catalyses azo linkage of dye. During degradation process of azo, NAD(P) acts as electron donor.

• Srivastva et.al. (1995) observed degradation of black liquor pulp mill effluents by the strains of Psuedomonas putida. Some anaerobic bacteria, Streptomyces and fungi e.g. Phaenerochaete chrysosporium have been characterized for decolouration of chromogenic dyes. The enzymes involved in dye degradation are lignases (lignin peroxidase), Mn (II) dependant peroxidase and glyoxal oxidase. These enzymes are well associated with lignin degrading system.

BIOREMEDIATION OF COAL WASTES THROUGH VAM FUNGI-

Bioremediation of coal waste land through VAM fungi is gaining importance in recent years. Selected fungi are introduced through plants in coal mine areas. Extensive infection of most plant species colonizing coal waste has been observed in India and other countries. It has been found that VAM fungi improved the growth and survival of desirable revegetation species. Increased growth of red maple, maize, alfalfa and several other plants inoculated with VAM fungi growing in coal mine soil has been recorded.

BIOREMEDIATION OF HEAVY METALS-

Bacteria, algae, fungi, actinomycetes and higher plants accumulate high amount of heavy metals in their cells.

Algae- The species of Chlorella, Anabaena inaequalis, Weatiellopsis prolifica, Stigeoclonium tenue, Synechococcus sp. tolerate heavy metals. However, several species of Chlorella, Anabaena, marine algae have been used for the removal of heavy metals. But the operational conditions limit the practical application of these organisms. Rai.et.al. studied biosorption i.e. both adsorption and absorption of Cd++ by a capsulated nuisance cyanobacterium, Microcystis both from field and laboratory. The naturally occurring cells showed higher efficiency for Cd++ and Ni++ as compared to laboratory cells.

Fungi- Fungi are also capable of accumulating heavy metals in their cells. However, several mechanisms operate in them for the removal of heavy metals from the solution; a few of these have been discussed below:

• Metabolism independent accumulation- The positively charged ions in the solution are attracted to negatively charged ligands in cell materials. Biosorption of metal ion occur on microbial cell surface. But composition of biomass and other factors affect biosorption. For example, in Rhizopus arrhizus depends on ionic radius of Li3+, Mn2+, Cu2+, Zn2+, Cd2+, Ba2+, Hg2+, Pb2+. However, binding of Hg2+, Ag2+, Cd2+, Al3+, Ni2+, Cu2+ and Pb2+ strongly depends on concentration of yeast cells.

• Metabolism dependent accumulation- In fungi and yeast, heavy metals ions are transported into the cells through cell membrane. However, because of metabolic processes ions are precipitated around the cells, and synthesized intracellularly as metal binding proteins. Energy dependent uptake of Cu2+, Zn2+,Cd2+ Ni2+ by fungi has been demonstrated. Moreover, intracellular uptake is influenced by certain external factors such as pH, cations, anions and organic materials, growth phase etc. metal uptake by growing batch culture was found maximum during lag phase and early log phase in Aspergillus niger, Penicillium spinulosum and Trichoderma viride.

• Extracellular precipitation and complexation- Fungi produce several extracellular products which can complex or precipitate heavy metals. For example, many fungi and yeast release high affinity Fe binding compounds that chelate iron. It is called siderophores. The Fe3+ chelates which are formed outside cell wall are taken up into the cell. In Saccharomyces cerevisiae removal of metals is done by their precipitation as sulphides e.g. Cu2+ is precipitated as CuS.