Bioremediation

Resna N K
Assistant professor
GEMS COLLEGE
BIOREMEDIATION
 Bioremediation refers to the process of using
microorganisms to remove the environmental
pollutants or prevent pollution.
 The removal of organic wastes by microbes for

environmental clean-up is the essence of

bioremediation.
 The other names used for bioremediation are

biotreatment, bioreclamation and biorestoration.

 Xenobiotics broadly refer to the unnatural,

foreign and synthetic chemicals such as

pesticide, herbicide & other organic compounds.
 "Remediate" means to solve a problem, and "bioremediate“
means to use biological organisms to solve an environmental
problem such as contaminated soil or groundwater.

 Bioremediation means to use a biological remedy to abate or

clean up contamination.
 Bioremediation is a waste management technique that

involves the use of organisms to remove or neutralize

pollutants from a contaminated site.

 According to the EPA, bioremediation is a “treatment that

uses naturally occurring organisms to break down hazardous
substances into less toxic or non toxic substances”.
 Bioremediation is the a biological degrading processes for the
treatment of contaminated soils, groundwater and/or sediments,
relying on microorganisms including bacteria and/or fungi to use
the contaminant(s) as a food source with resulting degradation of
the contaminant.

 Microorganisms used to perform the function of bioremediation are

known as bioremediators.

 Bioremediation is one of the most economic remedial techniques

presently available for treating most organic fuel based
contaminants such as coal tars and liquors, petroleum and other
carcinogenic hydrocarbons such as benzene and naphthalene, and
some inorganics
How Does It Work?

 Waste material is examined & certain bacteria are isolated based on their

efficacy at digesting and converting the waste.

 Indigenous or local bacteria is to be used!
 The bacteria then go through several steps of cultures and process for

performance testing.
 The suitable bacteria are placed back in the waste environment.
 They grow & thrive & in the process digest & convert the waste into

Carbon dioxide & water.

 The right temperature, nutrients, and food also must be present.
 Conditions may be improved by adding “amendments.”
Mechanism of Bioremediation
 microorganisms use the organic contaminants (nitrogen,
phosphorus, and minor nutrients such as sulfur and trace
elements) for their growths
 metabolism modes are broadly classified as aerobic and

anaerobic.
 Aerobic transformations occur in the presence of molecular

oxygen, with molecular oxygen serving as the electron acceptor.

This form of metabolism is known as aerobic respiration.
 Anaerobic reactions occur only in the absence of molecular

oxygen and the reactions are

 subdivided into------anaerobic respiration

 ------fermentation, and
 -------methane fermentation.
 different modes of Microbial transformations
of organiccompounds

◦ Degradation -----initial substrate no longer exists

◦ Mineralization ------- complete conversion of the
organic structure to inorganic forms such as CO2,
H2O, and Cl–.
◦ Detoxification ------transformation of the
compound to some intermediate form that is
nontoxic or less toxic.
◦ activation ----The process of forming toxic end
products or intermediate products
Microorganisms are capable of catalyzing a
variety of
reaction
 Hydrolysis— frequently conducted outside the microbial cell by exoenzymes.
Hydrolysis is simply a cleavage of an organic molecule with the addition of water.

 Cleavage— cleaving of a carbon–carbon bond ------- An organic compound is

split or a terminal carbon is cleaved off an organic chain.

 Oxidation— breakdown of organic compounds using an electrophilic form of

oxygen.
 Reduction— breakdown of organic compounds by a nucleophilic form of hydrogen
or by direct electron delivery.

 Dechlorination— the chlorinated compound becomes an electron acceptor; in this

process, a chlorine atom is removed and is replaced with a hydrogen atom.

 Dehydrogenation— an oxidation–reduction reaction that results in the loss of two

electrons and two protons, resulting in the loss of two hydrogen atoms.
 Dehydrohalogenation— results in the loss of a hydrogen and chlorine atom from
the organiccompound.
 Substitution— these reactions involve replacing one atom with another.
Factors affecting bioremediation
 Moisture :
 influences the rate of contaminant metabolism because it influences the kind and
amount of soluble materials that are available as well as the osmotic pressure and pH of
terrestrial and aquatic systems. A range of 50-80% is optimal for biodegradation.
 Nutrients :
 nitrogen, phosphorous, potassium, sulfur, copper, and trace element etc are required
 If nutrients are not available in sufficient amounts, microbial activity will become limited.
 Nitrogen and phosphorous are deficient in the contaminated environment. These are
usually added to the bioremediation system in a useable form (e.g., as ammonium for
nitrogen and as phosphate for phosphorous).
 Oxygen level ;
 increasing the concentration of electron acceptors and nutrients in ground water and
surface water.
 Oxygen is the main electron acceptor for aerobic bioremediation.
 pH :
 Soil pH may affect the availability of nutrients.
 pH of 6.5 to 8.5 is generally optimal for biodegradation in most aquatic and terrestrial
systems and values ranging
 Temperature
 Temperature directly influences the rate of biodegradation
by controlling the rates of enzyme catalysed reactions.
 Temperature of 15-650C is optimal for biodegradation.

 Chemical nature of pollutant

 In general, aliphatic compound are more easily degraded
than aromatic ones.
 Presence of cyclic ring structure and length chains or
branches decrease the efficiency of biodegradation.
 Water soluble compounds are more easily degraded.
 The presence of halogen inhibit biodegradation.
USE OF BACTERIA IN
BIOREMEDIATION
 Greatly affected by unstable climatic an environmental
factors from moisture to temperature.
 For examples, pH in soil is slightly acidic; petroleum

hydrocarbon degrading bacteria do not work well <

10º C.
 These microbes are usually thermophilic anaerobic.
 Fertilizers are needed. Seeding or bioaugmentation

could be useful too.

 They contain monooxygenases an dehydrogenases to

break down organic matters including most toxic

substances.
 PSEUDOMONAS
 Genetically engineered bacteri ( Pseudomonas)

with plasmid producing enzymes to degrade

octane and many different organic compounds
from crude oil.
 A selected list of genetically engineered

microorganisms
 Pseudomonas putida -Mono-and dichloro

aromatic compounds
 P.diminuta –Parathion

 P.oleovorans –Alkane

 P.cepacia -2,4,5-Trichlorophenol

 Acinetobacter species -4-Chlorobenzene

 Alcaligenes species -2,4-Dichlorophenoxy acetic

acid
USE OF FUNGI IN BIOREMEDIATION
 Candida can degrade formaldehyde.
 Gibeberella can degrade cyanide.

(Slurry-phase bioremediation is useful too but only for small

amounts of contaminated soil.Composting can be used to degrade
household wastes.)

White rot fungi can degrade organic pollutants in soil and

effluent and decolorize kraft black liquor, e.g
Phanerochaete

 chrysosporium can produce aromatic mixtures with its lignolytic

system.
 Pentachlorophenol, dichlorodiphenyltrichloroethane (e.g. DDT),

even TNT (trinitrotoluene) can be degraded by white rot fungi.

 Methods of bioremediation
 On the basis of removal and transportation of

the wastes for the treatment, basically there

are two methods:
  in-situ bioremediation
  ex-situ bioremediation.
In situ bioremediation
 In situ bioremediation involves a direct approach for the microbial
degradation of pollution (soil, ground water).

 Biostimulation------Addition of adequate quantities of

nutrients at the site promote microbial growth

 When microorganisms are imported to a contaminated site to

enhance degradation, the process is called as “Bio-augmentation

 applied for clean-up of oil spillages, beaches etc.

 There are two types of in situ bioremediation –

◦ # intrinsic
◦ # engineered.
Intrinsic bioremediation
 Conversion of environmental pollutants into the harmless forms through
the innate capabilities of naturally occurring microbial population

 The intrinsic that is inherent capacity of microorganisms to metabolize the

contaminants should be tested at the laboratory and field levels before use
for intrinsic bioremediation. Through site monitoring programmes progress
of intrinsic bioremediation should be recorded time to time.

 The conditions of site that favour intrinsic bioremediation 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.

 Bioremediation of waste mixture containing metals such as Hg, Pb, and

cyanide at toxic concentration can create problem

 The environmental factors such as pH, concentration, temperature and

nutrient availability determine whether or not biotransformation takes
place.
Engineered in situ bioremediation
 Intrinsic bioremediation is satisfactory at some places, but it is slow process
due to the poorly adapted microorganisms, limited ability of electron
acceptor and nutrients, cold temperature and high concentration of
contaminants.

 When site conditions are not suitable, bioremediation requires construction

of engineered system 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 NO3 -,
SO4 2-) and nutrients (e. g nitrogen and phosphorous) promote microbial
growth in surface.

 When contamination is deeper, amended water is injected through wells.

 But in systems both extraction and injection wells are used in combination to
control the flow of contaminated ground water combined with above ground
bioreactor treatment and subsequent reinjection of nutrients spiked effluent
are done.
 In situ bioremediation Techniques

  Bioventing
  Biosparging
  Bioslurping
  Phytoremediation
Bioventing
 It is a promising technology that stimulates the natural in situ biodegradation of an
aerobically degradable compounds in soil by providing oxygen to existing soil
microorganisms.
 It typically uses low air flow rates to provide only enough oxygen to sustain microbial
activity
 Oxygen is most commonly supplied through direct air injection into residual
contamination in soil.
 In addition to degradation of adsorbed fuel residuals, volatile compounds ar
biodegraded as vapors move slowly through biologically active soil.
 Bioventing techniques have been successfully used to remediate soils contaminated by
petroleum hydrocarbons, no chlorinated solvents, some pesticides, wood
preservatives, and other organic chemicals.
 However, several factors may limit the applicability and effectiveness of the process for
example highly saturated soils, extremely low moisture content or low permeability
soils negatively affect the bioventing performance.
Biosparging
 It involves the injection of a gas (usually air or oxygen) and
occasionally gas-phase nutrients, under pressure, into the saturated
zone to promote aerobic biodegradation.

 In air sparging, volatile contaminants also can be removed from the

saturated zone by desorption and volatilization into the air stream.
 Typically, biosparging is

achieved by injecting air

into a contaminated
Subsurface through
a speciall designed series of
injection wells.
 The air creates an inverted

cone of partially aerated soils

surrounding the injection point
 The air displaces pore water, volatilizes contaminants, and exits the
saturated zone into the unsaturated zone.

 While in contact with ground water, oxygen dissolution from the air
into the ground water is facilitated and supports aerobic
biodegradation.

 A number of contaminants have been successfully addressed with

biosparging technology, including gasoline components such as
benzene, toluene, ethyl benzene, and xylenes .

 Biosparging is most often recommended at sites impacted with mid-

weight petroleum hydrocarbon contaminants, such as diesel and jet
fuels. Lighter contaminants, such as gasoline, tend to be easily
mobilized into the unsaturated zone and physically removed.

 Heavier contaminants, such as oils, require longer remedial intervals

because of reduced microbial bioavailability with increasing carbon
chain length.
Bioslurping
 also known as multi-phase extraction is effective in removing
free product that is floating on the water table.

 Bioslurping combines the two remedial approaches of

bioventing and vacuumenhanced free-product recovery.

 Bioventing stimulates aerobic bioremediation of contaminated

soils in in situ, while vacuum-enhanced free-product recovery
extracts --light, nonaqueousphase liquids (LNAPLs) from
the capillary fringe and the water table

 Bioslurping is limited to 25 feet below ground surface as

contaminants cannot be lifted more than 25 feet by this
method.
 A bioslurping tube with adjustable height is lowered into a ground
water well and installed within a screened portion at the water table.

A vacuum is applied to the bioslurping tube and free product is

slurped” up the tube into a trap or oil water separator for further
treatment.

 Removal of the LNAPL results in a decline in the LNAPL elevation,

which in turn promotes LNAPL flow from outlying areas toward the
bioslurping well.

 As the fluid level in the bioslurping well declines in response to

vacuum extraction of LNAPL, the bioslurping tube also begins to
extract vapours from the unsaturated zone.

 This vapour extraction promotes soil gas movement, which in turn

increases aeration and enhances aerobic biodegradation.
Phytoremediation
 Phytoremediation is an in situ technique that uses plants to
remediate contaminated soils.
 Phytoremediation is most suited for sites where other remediation

options are not costs effective, low-level contaminated sites, or in

conjunction with other remediation techniques.

 Deep rooted trees, grasses, legumes, and aquatic plants all have
application in the phytoremediation field.
 Phytoremediation has been used to remove PAH, 2,4,6-

trinitrotoluene (TNT), hexahyro-1,3,5- trinitro-1,3,5 triazine etc.

 Plants are able to remove pollutants from the groundwater and store,

metabolize, or volatilize them.

 Also, roots also help support a wide variety of microorganisms in the

subsurface. These microorganisms can then degrade the contaminants.
 The roots also provide organic carbon sources to promote

cometabolism in the rizosphere.

 Advantages of in situ bioremediation

  Cost effective, with minimal exposure to

public or site personnel
  Sites of bioremediation remain minimally

disrupted
 Disadvantages of in situ bioremediation

  Very time consuming process

  Sites are directly exposed to environmental

factors such as temperature, oxygen supply

etc.
  Microbial degrading ability varies

seasonally
Ex situ bioremediation
 Ex situ bioremediation involves removal of
waste materials and their collection at the
place to facilitate microbial degradation.
 On the basis of phases of contaminate

materials ex situ bioremediation is classified

in to two

 # Solid phase system

 # Slurry phase system
Solid phase system
 Solid waste system includes organic wastes
(e. g leaves, animal manure and agriculture
wastes) and problematic wastes ( e. g.
domestic and industrial wastes, sewage
sludge and municipal solid wastes

 bioremediation techniques

  Composting
  Land farming
Composting
 Composting is aerobic, thermophilic treatment process in which
contaminated material is mixed with bioremediation microorganisms.

 This is a controlled biological process by which organic contaminants

(e.g., PAHs) are converted by microorganisms to safe, stabilized by
products.
 Typically, thermophilic conditions -----54 to 65°C

 pH ------6-9.

 In composting, soils are excavated and mixed with bulking agents

and organic amendments, such as wood chips and vegetative wastes,
to enhance the porosity of the mixture to be decomposed.
 Degradation of the bulking agent heats up the compost, creating

thermophilic conditions.
 Oxygen content usually is maintained by frequent mixing, such as

daily or weekly turning off windrows.

 Surface irrigation often is used to maintain moisture content.

 Temperatures are controlled, to a degree, by mixing, irrigation, and

air flow, but are also dependent on the degradability of the bulk
material and ambient conditions.
 Organisms involved in composting:
◦ actinomycetes (a filamentous type of bacteria)
◦ Fungi ( molds, yeasts) and
◦ protozoa,
◦ earth worms ,insects, mites and ants.
 Mechanism of composting
  The bacteria bring out the decomposition of macromolecules namely
proteins and lipids, besides generating energy. Fungi and actinomycetes
degrade cellulose and other complex organic compounds.
 Composting may be divided into 3 stages with refernce to changes in
temperature

 i. Mesophilic stage: The fungi(Aspergillus, Mucor. Penicillium) and acid

producing bacteria (Pseudomonas, Bacillus) are active in this stage, and the
temperature increases to 400C.
 ii.Thermophilic stage: As the composting proceeds, the temperature
rises from 40 0C to70 0C.
 Thermophilic bacteria(Thermus, Bacillus) ,Thermophilic fungi (Absidia)
and actinomycetes (Sterpyomyces,Micropolyspora)are active at this stage.
Thermophilic stage is associated with high rate and maximum degradation of
organic materials.
 iii.Cooling stage: The microbial degradative activity slows down and
the thermophilic organisms are replaced by mesophilic bacteria and fungi.
Cooling stage is associated with formation of water,pH stabilization and
completion of humeic acid formation
 Designs commonly applied for composting:

 i. Aerated static piles— Compost is formed into piles and

aerated with blowers or vacuum pumps.

 ii. Mechanically agitated in-vessel composting—

Compost is placed in a reactor vessel, in which it is mixed
and aerated.

 iii. Windrow composting— Compost is placed in long,

low, narrow piles (i.e., windrows) and periodically mixed
with mobile equipment.
 Composting has been successfully applied to soils and
biosolids contaminated with petroleum hydrocarbons (e.g.,
fuels, oil, grease), solvents, chlorophenols, pesticides,
herbicides, PAHs, and nitro-aromatic explosives.

 For TNT, complete mineralization has been difficult to

demonstrate via composting. TNT may bind to soil,
resulting in low microbial bioavailability and apparent
disappearance .

 Composting is not likely to be successful for highly

chlorinated substances, such as PCBs, or for substances
that are difficult to degrade biologically.
 Land farming

 also called Land treatment

 useful in treating aerobically degradable contaminants.

 This process is suitable for non-volatile contaminants at sites where

large areas for treatment cells are available.

 Land treatment of site-contaminated soil usually entails the tilling of

an 8-12 inch layer of the soil to promote aerobic biodegradation

of organic contaminants.
 The soils are periodically tilled to aerate the soil, and moisture is

added when needed.

 In some cases, amendments may be added to improve the tilt of the

soil, supply
nutrients, moderate pH,
or facilitate bioremediation.
Slurry phase treatment
 The contaminated solid materials ( soil, degraded sediments etc.),
microorganisms and water formulated into slurry are brought within a
bioreactor . a triphasic system involving three major components:
water, suspended particulate matter and air.

 water serves as suspending medium where nutrients, trace

elemnts,pH adjustment chemicals are dissolved.

 Suspended particulate matter includes a biologically inert substratum

consisting of contaminants (soil particles) and biomass attached to
soil matrix or free in suspended medium.

 Air provides oxygen for bacterial growth.

 Biologically thre are two types of slurry phase reactors:

 Aerated lagoons
 Low shear air lift reactors.
 Aerated lagoons

 commonly used for treatment of municipal waste

water.
 Nutrients and aeration are pumped 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 used

as single stage or
multistage operation.
 If the waste contains

volatiles ,this reactor

is not appropriate.
 Low shear air lift reactors:
 useful when the waste contains volatile components:
 These 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 arms are connected with blades which is used for resuspension of
coarse materials and 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 behaviour of

slurry-phase bioreactors.
 Pre treatment process includes size fractionation

of solids , soil washing,milling to reduce particle

size slurry preparation.
 Certain surfactants such as anthracene, pyrene etc

are added to enhance the rate of biodegradation.

These act as cosubstrate and utilize as carbon source.
Co substrates also induce the production of beneficial
enzymes.
 Advantages of ex situ bioremediation

◦ Better controlled and more efficient process.

◦ Process can be improved by enrichment with desired
microorganisms.
◦ Time required in short.

 Disadvantages of ex situ bioremediation

◦ Very coastly process.

◦ Sites of pollution are highly disturbed.
◦ There may be disposal problem after the process is complete
ADVANTAGES OF BIOREMEDIATION
 Bioremediation is a natural process and is therefore perceived
by the public.
 Bioremediation is useful for the complete destruction of a

wide variety of contaminants.

 Instead of transferring contaminants from one environmental

medium to another, for example, from land to water or air,
the complete destruction of target pollutants is possible.

 Bioremediation can often be carried out on site, often without

causing a major disruption of normal activities.
 Bioremediation can prove less expensive than other

technologies that are used for cleanup of hazardous waste.

DISADVANTAGES OF
BIOREMEDIATION
 Bioremediation is limited to those compounds that are
biodegradable. Not all compounds are susceptible to rapid and
complete degradation.

 There are some concerns that the products of biodegradation may be

more persistent or toxic than the parent compound.

 Biological processes are often highly specific. microbial populations,

suitable environmental growth conditions, and appropriate levels of
nutrients and contaminants
 .
 It is difficult to extrapolate (deduce) from bench and pilot-scale
studies to fullscale field operations.

 Bioremediation often takes longer than other treatment options.