By

Dr. Ahmed Z. Abdel Azeiz

College of Biotechnology
Misr University for Science and Technology
E-mail: alrahman3@hotmail.com
ahmed.abdelaziz@must.edu.eg
Tel:002- 01228188759
Contents:
1- Definitions.
2-Bioremediation related topics.
3- Stages of a biodegradation study.
4- Cells and enzymes immobilization.
5- Phytoremediation.
6- Bioremediation of metals contaminated environment.
7- Biosurfactants
8- Bioremediation techniques:
a) Soil bioremediation.
b) Water and gasses bioremediation.
 Bioremediation is any process that uses organisms
(microorganism, algae and plant) or their enzymes to return
the polluted environment to its original condition.

 Biodegradation is the use of these organisms in the

degradation of different pollutants.

 Xenobiotic compounds are chemical compounds found in an

organism but it are not normally produced or expected to be
present in it.

 Cometabolism: in this process the microorganism produces

an enzyme to utilizes its nutrients, but by chance this enzyme
can degrade a pollutant.
 Bioremediation is a triple-corners process:
Solid
Inorganic Organic Liquid
Pollutants
Gas

Environments Organisms

Soil Microorganisms
Water Plants
Air Enzymes
 Environments

Organisms Pollutants
Bioremediation of
Phytoremediation metals polluted
environment
Bioremediation
Enzymes
Cells immobilization
immobilization
Bioremediation
Biosurfactants techniques
Enzymes separation
and identification
1- Isolation of the microorganism

2- Purification of the obtained isolates

3- Identification of the microbial isolate

4- Optimization of the biodegradation
conditions
5- Determination of the biodegradation
efficiency
6- Identification of the biodegradation products.

7- Cell or enzyme immobilization.

8- Enzyme identification.
- Isolation of bacteria and fungi is performed on Mineral Salt
Medium (MSM) supplemented with the pollutant to be biodegraded
as a sole source of carbon (enriched technique).

- This medium can be used as liquid or solid.

Control
Soil pollutant Control
Isolation of petroleum biodegrading bacteria Isolation of pesticide
from soil and petroleum pollution. biodegrading bacteria from soil.
 A B

Fig. 2: Isolation of fungi (A) and bacteria (B) from the pollutant on solid MSM.

 The medium has a pH 7 is preferred for isolation of

bacteria, while pH 4.5– 5.5 for fungi.
 This is simply performed by streaking of a lope of the
MSM microbial culture or by pouring dishes
technique.
 The used medium is solid MSM supplemented with
the pollutant as a sole source of carbon.

Purification by pouring Purification by streaking

1- Spectrophotometer.
2- HPLC
3- GC/MS
4- Using the redox indicator 2,6-dichlorophenol
indophenol (DCPIP).
- The principle of this technique is that, during the
microbial oxidation of the carbon source,
electrons are transferred to electron acceptors
such as O2, nitrates and sulphate.
- DCPIP is an electron acceptor.
- The efficiency is determined by observing the
color change of DCPIP from blue (oxidized) to
colorless (reduced).
  Immobilized enzymes (or cells) is defined as the enzyme that
physically or chemically confined in defined materials with
retention of its catalytic activity.

The immobilization
methods

1- Carrier-binding 2- Cross-linking
3- Entrapping methods.
methods. methods.

1) Covalent binding methods 1- lattice method.

2- Ionic binding methods. 2- Microcapsule.

3- Physical adsorption. 3- Membrane.

4- Bio-specific binding methods. 4- Reversed micelle.

1- Polysaccharides: cellulose, dextran and agarose
derivatives.
2- Proteins: gelatin, albumin.
3- Synthetic polymers: Polystyrene derivatives, ion
exchange resins, polyurethane.
4- Inorganic materials: glass, sand, ceramic and
magnetite.
1) Covalent binding methods include:
a) Cyanogen Bromide method (CNBr).
b) Acid-azide derivative method
c) Condensing reagent methods
d) Diazo coupling methods
e) Alkylation methods.

Figure1: Steps of CNBr enzyme immobilization method

 By cross-linking of the enzyme molecules by
reacting with glutaraldehyde.
a) Lattice type:
 polyacrylamide,
 calcium algenate,
 polyvinylalcohol polymers.

b) Microcapsule type:
 Interfacial Polymerization Method.
 Liquid drying method.
  Phytoremediation is use of plants for accumulation,
removal or conversion of pollutants.

Phytoremediation

Phytostabilization Phytovolatilization Phytostimulation

1-.

Phytotransformation Phytoextraction
 Approximately 400 plant species have been classified as
hyperaccumulators of heavy metals, such as grasses,
sunflower, corn, hemp, flax, alfalfa, tobacco, willow, Indian
mustard, poplar, water hyacinth, etc.
 The root exudates of these plants play an important
role in phytoremediation as it activate the surrounded
microorganisms.

 Genetic engineering are used as in case of BT protein

or insect pheromones producing plants to reduce the
use of pesticides.
 Metals bioremediation
mechanisms

Metal Solubilization
immobilization (Bioleaching)

- Organic acids.
Complexation - Siderophores.
(Bioaccomulation) Precipitation - Root exudates.
(Biosorption)

- Exopolysaccharide. - H2S producing bacteria

- Lipoproteins. - Siderophores.
- Metal reduction.
 Chemical structure of some siderophores

pyridine-2,6-bis(thiocarboxylic acid)

SEM images of selenium-siderophore

complex formed in P. stutzeri culture
filtrate.
 Biosurfactants
 The biosurfactants are chemical compounds
characterized by hydrophobic and hydrophilic (non-
polar and polar) regions in one molecule
(amphipathic molecules).

 Biosurfactants from bacteria, cyanobacteria, fungi

and yeast are classified into:
1) Glycolipids.
2) Lipopeptides.
3) Phospholipids.
4) Glycoproteins.
5) Polymeric biosurfactants.
 Chemical structure of some biosurfactants

Glucolipid from Alcalivorax sp.

Trehalose tetra ester from Arthrobacter sp.

Glucolipid from Alcaligens sp.

1- Increase the availability of hydrophopic compounds
2- Nutrient storage molecules.
3- Save the microbial cells from toxic substances.
4- Efflux of harmful compounds.
5- Extracellular and intracellular interactions such as
quorum sensing and biofilm.
 The microbe may access a poorly water-soluble
substrate that has been “pseudosolubilized” by the
biosurfactant.
 Reduce the adsorption of the non-polar pollutants to
the surface of soil particles.
A) Soil bioremediation:
(1) In-situ (without excavation).
(2) Ex-situ (with excavation).
Only ex-situ processes allow an efficient optimization of
incubation parameters (biostimulation), including:
pH,
Aeration,
Agitation,
Moistening
nutrients,
solvents or surfactants.

In addition to addition of microorganisms (bioaugmentation).

 The ex-situ technique includes:
1- Bioslurry reactor.
2- Biopile.
3- landfarming

Biopile.

Bioslurry reactor.

Biopile.
1- High density poly ethylene (HDPE)
2- Sump pump to collect leachate
3- Layer of pea gravel
4- Layer of polluted soil to be treated
5- Chopped alfalfa hay to retain moisture
6- Wheels on sprinkler piping system
7- Piping frame, aluminum or PVC pipes with
frequent holes, sufficient to allow water,
nutrients and bacteria to treat the land farm
plot
8- Flexible leachate collection hose
9- Bypass valve that allows leachate to be
circulated directly to water distribution tank,
10- Recirculation hose
11- Alken-Murray Bioactivator 2000,
bioreactor unit
12- Fresh water supply hoses
13- Pumps for fresh water
14- Treated water hose
15- Water distribution tank
16- Pump for distribution tank
 Biofiltration is a process, in which, microorganisms
supported on inert materials are used to degrade
organic pollutants for air, gas and water
bioremediation.

 Types of biofilters:
 1- Bioscrubbers.
 2- Biotrickling filters.
 3- Slow sand or carbon filters.
Bioscrubber filters
Slow sand or carbon filters work through the formation
of a gelatinous layer (or biofilm layer) on the top few
millimetres of the fine sand or carbon layer.

This layer contains bacteria, fungi, protozoa, rotifera

and a range of aquatic insect larvae (i.e. rotifers).
 Severalresearch funding agency
fund this type of applied research.

 Now each of us can start to work

in a bioremediation research.