Bioprocessing

Elmer Ametefe (PhD)

eametefe@ug.edu.gh
Outline

• Introduction to Bioprocessing:
• Evolution of food processing
• impact and integration of Biotechnology in Processing
• Food and Beverages processing
• Fermentation
• Enzyme applications
• Value addition
• public perceptions
Evolution of Food /Bioprocessing
• Food chain origins
• Production agriculture
• Consumer utilization
• Food biotechnology integrates
• Biological knowledge
• Current bioengineering principles
• Techniques in processing and preservation
• Impact of biotechnology
• Agronomic
• Non – Agronomic
Introduction to Bioprocessing
Food and Beverage processing
• one of the most rapidly growing industries
• Food bioprocessing generally include
• Fermentations
• Enzyme application
Stages in Bioprocessing
• Upstream Process: defined as the entire process from
early cell isolation and cultivation, to cell banking and
culture expansion of the cells until final harvest
• Inoculum Devt, Culture preparation
• Sources of microorganisms
• Isolation of new microoganisms
• Media enrichment
• Substrate
• Toxic analogues
• Bioactive activity tests
• Strain improvement
• Naturally occurring variants
• Genome manipulation
 Fermentation and Downstream process
• Operations in industrial fermentation
• Fermentor design and operation
• Types of fermenters
• Fermentor operation
• Types of cultivation processes
• Product extraction
• Extraction of
• Solids, primary products
• Purification
• Preservation
• Perimeter design operation
• Facility selection and construction
• Equipment
• Sanitation
Bioreactor/Fermentor design
• Definition:
• A fermentor is a vessel for the growth of microorganisms which, while not permitting contamination,
provides conditions necessary for optimum production of the desired product
• Suitable containment system for each biotech process varies and is necessary to give the
correct environment for optimisation of organism growth and metabolic activity
• There is no universal fermentor
• Non-aseptic systems – for brewing, effluent disposal (traditional systems)
• Aseptic system – operate with pure cultures eg antibiotics, vitamins, polysaccharides. (More modern
systems, challenging)
• The design and model of a fermentor mainly depends on: the production organism,
the optimal operating condition required for the target product formation, product
value and scale of production.
• The design also takes into consideration capital investment and running costs
• Performance of any fermenter depends on key factors:
• Agitation rate
• Oxygen transfer
• pH
• Temperature
• Foam production
 General requirements
• Sterility from start to finish
• Optimal mixing with low uniform shear
• Adequate mass transfer
• Clearly defined flow conditions
• Feeding substrate with prevention of under or over dosing
• Suspension of solids
• Gentle heat transfer
• Compliance with design regulations such as: sterilization ability,
simple constuction, simple measuring, control regulating
techniques, scale up, flexibility, long term stability, compatibility
with up/downstream process and antifoam measures.
 Types of fermentors
• Liquid (submerged) fermentors
• Solid state (surface) fermentors
• They vary in size and complexity
from a 10 ml volume in a test
tube to computer controlled
fermenters with liquid volumes
greater than 100 m3.
Main fermentor types

• Batch fermentor
• Continuous stirred tank fermentor
• Airlift / loop (recycle) fermentor
• Tubular or Tower fermentor
• Fluidized bed fermentor
• Solid state fermentor
Aerated batch fermentor
• Upright closed cylindrical
tank
• Fitted with 4 or more
baffles
• Waterjacket or coil for
heating / cooling
• Aeration device
• Agitator
• Inlets for microbes and
nutrients
• Outlets for samples, gas
exhaust
• Monitors.
• Cleaning / sterilization
system
Continuous stirred tank bioreactor
• Advantages
• Continuous operation
• Good temperature control
• Simplicity of construction
• Low operating cost
• Easy to clean
• Disadvantages
• Need for shaft seals and bearings
• Size limitations
Airlift / loop bioreactor
• Advantages
• Simple design
• Easier sterilization
• Low energy requirement
• Greater heat removal
• Low cost.
• Disadvantages
• Higher pressures needed
• No bubble breaker
• Inefficient foam break
Fluidized bed fermentor
• Advantages
• Uniform particle mixing
• Uniform temperature
gradient
• Ability to operate in
continuous state
• Disadvantages
• Increased reactor vessel size
• Pumping requirements,
• Particle entrainment
• Erosion of internal
components
• Pressure loss /drop
 .
• Substrate/slurry
stream enters one end
and leaves thru the
other.
• Advantages
• Can run for long
periods
• Heat transfer can be
optimised
• Disadvantages
• Hard to control
temperature
• Expensive to maintain

Assignment 1: operating principles of the above mentioned bioreactors

 Microbial growth and bioprocessing
• Quantity of biomass t0 = 1.5h:
• Gravimetrically (dry weight, wet weight,) N = 8.4x101,
log10 N = 1.92
• Numerically (Doubling time /generation time) t = 8.5h:
N = 3.39x108,
• growth rate constant µ : log10 N = 8.53

Rate of increase of cells = µ x number of cells

ln Nt - ln N0 = µ(t - t0)
log10 N - log10 N0 = (µ/2.303) (t - t0)
µ = ( (log10 N - log10 N0) 2.303) / (t - t0) Therefore in this case:
• mean generation time or "doubling time" (g) µ = ( (log10 N - log10 N0) 2.303) / (t - t0)
log10 Nt = log10 N0 + g log102 = ( (8.53 - 1.92) 2.303) / (8.5 - 1.5)
Nt = N0 x2 g
g = (log10 Nt - log10 N0) / log102 = (6.61 x 2.303) / 7
= 15.22 / 7
= 2.18 hour-1
g = (log10 Nt - log10 N0) / log102
Note that µ and g are related to each other: µ = ln2/g = 0.693/g
= (8.53 - 1.92) / 0.301
= 21.96 generations in 7 hours
= 7 / 21.96 = 0.32 hours (x 60 = 19.2 minutes)
Note that µ and g are related to each other: µ = ln2/g = 0.693/g
 Growing biomass in the bioreactor

• Batch culture :
• fixed volume of medium innoculated with microbes, nutrients consumed: end product formation.
• Optimise organism or biomass production, specific biochemical transformations (amino acids, enzymes)
(sewage treatment, bioremediation)
• Fed batch (intermitent addition of conc nutrient), developed out of yeast cultivation on malt. Now used
in production of antibiotics, amino acids, vitamins, glycerol, acetone, lactic acid.
• Modifications include: diffusion capsule, continuous addition of single or multiple media,
withdrawal of broth from vessel followed by immediate dilution with fresh medium.

• Perfusion (addition of medium – removal of used cell free medium) ( animal cell cultivation)
• Semicontinuous :simultaneous nutrient addition and outflow withdrawal intermittently.
• Near balanced growth, however lower productivity.
• Cyclic continuous (simultaneous removal of used and addition of fresh medium)
• Cell reuse (reinoculation with used cell)
• Continuous : simultaneous nutrient addition and corresponding withdrawal of medium + cells
continously.
• Little fluctuation in nutrients, metabolites, cell numbers and biomass.
• Steady environment, easily automated but expensive.
• Easily contaminated.
• Brewing, food and feed yeast production, vinegar
Scale up and process control
• Bioprocessing (Fermentation processes) normally develop in
three stages. Moving from one stage to the next is known as
scale up
• The initial stage involves basic screening procedures
• Simple microbiological processes
• Followed by a pilot plant investigation
• Optimal operation conditions
• Volume capacity etc
• Final stage involves transfer of the study to plant or
production scale and final economic realisation.

• Throughout these stages optimal environmental conditions

are maintained at all level of processing
• Chemical (substrate conc etc)
• Physical (mixing ability, mass transfer, power dissipation etc)
 Process control in a fermentor
• Aeration and agitation: supply of O 2 is critical for aerobic processes. For
maximum absorption O2 must be dissolved in aqueous solution. May be
sterile or non sterile air. Air is forced under pressure into the bottom of
the fermenter.
– Agitators help distribute incoming air as fine bubbles, mix organisms uniformly,
create local turbulence and ensure uniform temperature.
– Agitation can increase expenditure due to foam formation. (use of baffles)
• pH measurement and control
– Good yield depends on accurate control of pH of fermentation broth.
• Use of natural buffers in medium.
• Phosphates and CaCO3.
• Continuous sampling, testing and adjusting necessary.
• Sterilizable pH probes
• Automatic induction of alkali or acid into medium
• CO2 and O2 determination and control
• As an endproduct CO2 measurement helps determine the
course of the fermentation as well as carbon balance
– Infrared sensor
– Dil NaOH with phenyl red (Photocell)
– Thermal conductivity
• O2 conc in fermenting broth
– Back titration (winkler method)(cumbersome)
– Modern oxygen probes (DO)
• Polarographic method
• Galvanic method.
• Pressure is measured with a manometer.
• Computer control – reduces labour, human error, makes
tasks easier.
 Foam control
• Foams are dispersions of gas in liquid.
• Occur in bioprocessing as a result of agitation and
aeration
• Advantageous in beer industry, foam latex etc.
• Undesirable in most industrial processes
– Removal of cells from culture
– Physical changes
– Reduction in working volume
– Lower mass & heat ratios
– Invalid process data
– Decrease in sterility
3 ways to solve:
• Defined media – media with inorganic compounds do
not foam, protein media produce most stable foam,
larger broth volumes
• antifoams or defoamers – chemicals controlling
foaming
• mechanical foam breakers – physically disperse foams

Ideal antifoam
• Should disperse easily and have fast action on foam
• Should be active at low concentrations
• Should be long acting in preventing new foam
formation
• Should not be metabolized by microbe
• Should be nontoxic to microbe
• Should not cause any problem in the extraction step
• Should be cheap
• Should be sterilazable

Examples: Silicones, sulphonates, esters, fatty acids,

alcohols
 Fermentations
• Alcoholic Beverages
• Beers, Wines and Spirits
• Food and Ingredients
• Dairy
• Vegetables
• Cereal
• Meats
• Additives
• Traditional fermentations
• Starter culture Use/ Genetically modified organism.
In the Biochemical sense…..

• The term fermentation refers to the metabolic process in which

organic compounds (particularly carbohydrates) are broken down to
release energy without the involvement of terminal electron acceptor
such as oxygen. Partial oxidation of the substrate occurs so that only
relatively small amount of ATP energy is released compared with the
energy generated if a terminal electron acceptor is involved.
• Partial oxidation of a carbohydrate can give rise to a variety of organic
compounds. The compounds produced by microorganisms vary from
organism to organism and are produced via different metabolic
pathways.
• The term fermentation can also be applied to any industrial process
that depends on the activity of one or more microorganisms
Introduction of Fermented foods

• Fermented foods are an extremely important part of human diet and

worldwide may contribute to as much as one third of human diet.
• The scope of food fermentation ranged from producing alcoholic
beverages, fermented milk and vegetable products to genetically
engineered super bugs to carry out efficient fermentation to
treatment and utilization of waste and overall producing nutritious
and safe products with appealing qualities.
• produced by modification of
raw material of either animal
or vegetable origin by the
activities of microorganisms.
• Bacteria , yeast and molds can
be used to produce a diverse
range of products that differ
in flavor, texture and stability
from the original raw
material.
• Enzymes are also used to get
desirable biochemical
changes and cause significant
modification to food.
Benefits of fermented foods
• Increase in variety
• Use as ingredients
• Improvement in nutritional quality
• increases the level of essential amino acids.
• Increase the vitamin B content.
• Removal of anti nutritional factors such as phytase, glucosinolates and lectins
• increase in the availability of minerals.
• Preservation
• Improve digestibility
• Detoxification of raw materials:
• Health benefits ???
Microflora in fermented foods
• By tradition, lactic acid bacteria are the commonly used
microorganism for preservation of foods.
• Safe metabolic activity while growing in food utilizing available sugar for the
production of organic acids and other metabolites.
• Their common occurrence in the foods and feeds coupled with their long
lived use contributes to their natural acceptance as GRAS (Generally regarded
as safe) for human consumption.
• There are many kinds of fermented foods in which the dominating
processes and end products are contributed by a mixture of
endogenous enzymes and other microorganisms like yeasts and
molds.
• Very often, a mixed culture originating from the native micro flora of
the raw materials is in action in most of the food fermentation
processed
On an industrial scale……

• Defined starter culture is preferred so that the qualities of finished

product could be consistently maintained day after day.
• natural micro flora is either
• inefficient, uncontrollable, and unpredictable.
• destroyed during preparation of the sample prior to fermentation (e.g.
pasteurization).
• A starter culture can provide particular characteristics in a more controlled
and predictable fermentation.
• design and development of starter cultures using gene technology
• identification of organisms isolated from various fermented foods.
• Culture dependent methods
• Culture independent methods (molecular methods)
For example Lactic acid bacteria
• Major group of Fermentative organisms comprised of 11 genera of
gram-positive bacteria:
• Carnobacterium, Oenococcus, Enterococcus, Pediococcus,
Lactococcus, Streptococcus, Lactobacillus, Vagococcus, Lactosphaera,
Weissella and Leuconostoc
• Related to this group are genera such as Aerococcus, Microbacterium, and
Propionibacterium
• A loosely defined group
• Produce lactic acid from hexoses.
• they lack functional heme-linked electron transport systems or cytochromes
• they do not have a functional Krebs cycle.
• Energy is obtained by substrate-level phosphorylation while oxidising
carbohydrates
• Two groups based on the end products of glucose metabolism.
• Homofermentative (homolactics): produce lactic acid as the major or sole
endproduct
• Heterofermentative (heterolactics): produce equal amounts of lactic acid,
ethanol and CO2

• The homolactics are able to extract about twice as much energy from
a given quantity of glucose as the heterolactics
• The heterolactics are more important in producing flavour and aroma
components such as acetylaldehyde and diacetyl.
Applications of Enzymes in Food Industry
• Enzyme technology is concerned with the application of enzymes as
tools of industry
• biological catalysts
• bind specific substrates at their active sites.
• value of using enzymes over inorganic catalysts
• efficiency, selectivity and specificity
• Ability to operate at room temperature, atmospheric pressure and within
normal pH ranges
• biodegradable and no unwanted by-products.
• Source of enzymes
• Living things basically
• Fruits, animals, yeasts, bacteria etc.
• Enzymes break down specific components within fruit & vegetables
such as pectin, starch, proteins and cellulose which results in
increased yields, shortening of processing time and improving sensory
characteristics.
• Some examples: Pectinases and Cellulases are used to break down cell walls
in fruit and vegetables, resulting in improved extraction and increase in yield.
• They can also be used to decrease the viscosity of purees or nectars, and to
provide ‘cloud stability’ and texture in juices.

• In food processing, enzymes could be:

• Endogenous enzymes
• Exogenous enzymes
• Endogenous enzymes
• naturally exist in food and may affect the quality of the foods (either
positively or negatively) by their actions.
• pectin methyl esterase (PME), which is found in fruits and vegetables. It affects the
quality of the final products (such as fruit juices) made from these fruits and vegetables.
• rapidly hydrolyses the pectin esters to form methanol, which lowers the pH of the product.
• applied in texture modification of the thermo-processed fruits and vegetables.
• Eg in cold break tomato juice processing,
• involves heating the tomato to 60°C to activate the PME. The activated enzyme
depolymerizes pectin leading to loss of viscosity and cloudiness in the product. This
facilitates separation of solid from liquid components of the juice making the process
suitable for manufacturing juices such as tomato juice.
• Hot break processing on the other hand
• involves heat inactivation of the PME by heating the juice to 90°C. This results in
preservation of the pectin, which promotes and stabilizes cloud and viscosity of the
product. Hot break processing is utilized in manufacturing of pastes and gels, e.g. tomato
paste.
• Exogenous enzymes (exo-enzymes) are those enzymes that you add
into the food to promote a desirable effect.
• An example of the exogenous enzyme is pectin esterase (bacteria, yeasts)
• Breaks specific bonds in molecules of pectin.
• used in thinning of pastes/juice concentration and to clarify wines
 Diagnostics and public acceptance
• Rapid diagnostics for microbial contaminations and toxins
• Bacterial endotoxins and fungal mycotoxins
• Traditional procedures
• Modern methods (ELISA, DNA/RNA probe technology,)

• Public perceptions of Bioprocessed foods

• GRAS
• GMO
 Class work /Presentation
• Describe the current traditional processing of the following in Ghana,
using either microbial fermentation process or enzyme application.
Describe any optimization processes that would make the product
more economically viable and appealing to the consumer
• Beverage fermentations
• Beer
• Wine.
• Food fermentations
• Dairy
• Cereal
• Vegetable
Nutraceuticals
Nutraceuticals

• coined in 1989 by Stephen De Felice

• Pharmaceutical-grade and standardized nutrient
• FDA regulated (jurisdiction dependent)
• Classifications
• Dietary supplements (food derived nutrients concentrated as liquid or
capsules)
• Functional foods (enriched foods in natural state)
• Foods or medicines
• Time and cost for drug approval
• Patents for nutraceuticals
• Unlicensed herbal remedies
What are dietary supplements?

• Products intended to supplement the diet when taken by mouth as a pill, capsule,
tablet, or liquid
• Natural sources
• Synthetic
• "The Dietary Supplement Health and Education Act of 1994 (DSHEA) defines the term
“dietary supplement” to mean a product (other than tobacco) intended to supplement
the diet that bears or contains one or more of the following dietary ingredients: a
vitamin, a mineral, an herb or other botanical, an amino acid, a dietary substance for
use by man to supplement the diet by increasing the total dietary intake, or a
concentrate, metabolite, constituent, extract, or combination of any of the
aforementioned ingredients.
• Furthermore, a dietary supplement must be labeled as a dietary supplement and be intended for
ingestion and must not be represented for use as conventional food or as a sole item of a meal or
of the diet.
• In addition, a dietary supplement cannot be approved or authorized for investigation as a new
drug, antibiotic, or biologic, unless it was marketed as a food or a dietary supplement before such
approval or authorization.
• Types of supplements
• Vitamins
• Dietary minerals
• Proteins and amino acids
• Essential fatty acids
• Natural products
• Probiotics
• Quality and safety
• GMP
• International Organization for Standardization’s Quality Management System ISO
• Safe Quality Food (SQF)Certification
• HACCP
• Clinical studies
• Research
• Claims, Controversies
• Public health concerns Vs profitability for industries
Functional foods – what are they?
• Food with an added function
• Health promoting, disease preventing
• Addition of new ingredients, already existing ingredient.
• Bred into existind plants/ animal source.
• Pysiologically active compounds
• safety
• Let food be thy medicine and medicine be thy food,” – Hippocrates 2,500
years ago.
• Fastest growing segment in food industry. (10% CAGR)
• Explosion in consumer interest due to health claims
• Characteristics
• present in their naturally occurring form(Not a capsule, tablet, or powder)
• consumption in the diet daily;
• regulation of a biological process (preventing or controlling disease)
Types:
• Inorganic mineral - Minerals supplements
• Probiotics - Helpful bacteria
• Prebiotics - Digestive enzymes
• Dietary fibres - Fibres
• Antioxidants - Natural antioxidants
• Phytochemicals
• Fatty acids - Omega 3 fatty acids
• Phenolics - Tea polyphenols
• Isoprenoids - carotenoids
• Lipids - Sphingolipids
• Proteins - soyaproteins
• Herbs as functional food -
Sources and claims
• Plant sources
• Oats
• reduction of LDL cholesterol
• Reducing risk of Coronary heart disease
• First food specific health claim awarded in 1997 (Quaker oats company)
• For health claim, the recommended effective level of consumption is
• a minimum of at least 0.75 g of the soluble fiber β-glucan per serving and a daily intake of at least 3 g (four servings).
• A diet low in saturated fat and cholesterol in conjunction with the soluble fiber intake was specified
• Citrus fruits
• Protective against variety of human cancers
• Presence of limonoids – anticancer activities 9effective in chemically induced tumor rodents)
• Non toxic to humans
• Limonene – clinical trials for malignancies
• Soy, Tomato, Garlic, Cranberry, Tea, Wine, Grapes, Brocolli etc.
• Fish
• Omega-3 fatty acids
• Lower triglycerides but not LDL
• One serving of fish per week sig. reduced total cardiovascular mortality
• Beef, Dairy products
What are Probiotics?

• Probiotics are defined as live

microbes which when administered
in adequate amounts confer a
beneficial health effect on the host---
WHO 2002
• Lactobacillus species Bifidobacterium
species L. acidophilus, B. bifidum, L.
casei (rhamnosus), B. longum, L.
reuteri, B. breve, L. bulgaricus, B.
infantis, L. plantarum, B. lactis, L.
johnsonii, B. adolescentis, L. lactis.
• Others Bacillus cereus, Bacillus subtilis,
Non pathogenic Escherichia coli,
Saccharomyces cerevisiae, Enterococcus
faecalis, Streptococcus thermophilus.
History
• Ellie Metchnikoff, the first scientist
who proposed the therapeutic use of
lactic acid bacteria.
• Lactic acid bacteria were first
discovered by Pasteur in 1857

• Their isolation from rancid milk was

reported in 1878 by Lister. In 1889
Tissier discovered Bifidobacterium
spp.

• The first stable cultures of

Lactobacillus casei strain Shirota were
made in 1930 by Dr. Minoru Shirota
Features of Probiotics

• Non-pathogenic
• Nontoxic
• Resistant to gastric acid
• Adhere to gut epithelial tissue
• Produce antibacterial substances
• It should persist for short periods in the gastro-intestinal tract
• Able to resist the antibacterial mechanisms that operate in the gut
• Need to avoid the effects of peristalsis, which tend to flush out
bacteria with food
• Resistant to the bile acid
• Role of Probiotics in Various
Diseases
• Antibiotic-associated
Diarrhea
• Probiotics and Clostridium
difficile Infections, Helicobac
ter pylori Infections
• Inflammatory Bowel
Diseases (IBD), Probiotics
and Irritable Bowel
Syndrome (IBS)
• Multi-Organ Dysfunction
Syndrome
• Allergy and Immune
Response
 Mechanism of action of Probiotics
• Production of low-molecular-weight antibacterial
substance that inhibits both gram-positive and gram-
negative enteric bacteria
• Use of enzymatic mechanisms to modify toxin
receptors and block toxin- mediated pathology
• Prevent colonization of pathogenic microorganisms by
competitive inhibition for microbial adhesion sites
• Immune modulation
Biotechnological applications in product development
• Key role from traditional focus on energy providing foods to
biologically active non-nutritive components.
• Major breakthroughs in aspects of genetic engineering and
biotechnology
• Advances in microbial genetics
• genome sequence information, high-throughput analysis of expressed products,
transcripts and proteins and the application of bioinformatics
• Detection of pathogens /Mycotoxins
• PCR-based techniques, ELISA, sophisticated media (chromogenic or fluorogenic media)
• Challenges
• Frankenstein foods? Consumer acceptance
• Traditional sequential methods for product development (laboratory scale, regulations,
scale up, consumer acceptance)
• Labels and Technological constraints
Class Exercise

• Select a named product with nutraceutial claims, outline the claims

and highlight the methods for detecting the active components in the
product.
• Outline the possible mechanisms of action of the active
components/compounds.
Bioremediation
Introduction
• Bioremediation is the use of living organisms, primarily
microorganisms, for the degradation of hazardous chemicals in soil
sediments, water, or other contaminated materials into less toxic forms

• Microorganisms metabolize the chemicals to produce carbon dioxide

or methane, water and biomass

• Enzymatically transformed to metabolites that are less toxic or

innocuous
• However, in some instances, the metabolites formed are more toxic than the
parent compound. For example, perchloroethylene and trichloroethylene may
degrade to vinyl chloride.
Bioremediation organisms

• The genus Pseudomonas are the most predominant microorganisms

that degrade xenobiotics

• Xenobiotics----hydrocarbons, phenols, organo phosphates, polycyclic

aromatics and naphthalein

• Other microorganisms are Mycobacterium, Mycococcus,

Nitrosomonas, Nocardia, Penicillium, Phanerochaete etc
Mechanism of Bioremediation
• Microorganisms use the organic contaminants (nitrogen, phosphorus,
and minor nutrients such as sulfur and trace elements) for their
growths

• Metabolically they are classified as:

• Aerobic – Oxygen serving as the electron acceptor
• Anaerobic - occur only in the absence of molecular oxygen and the reactions
are subdivided into
• anaerobic respiration, fermentation, methane fermentation
Microbial transformations of organic compounds

• 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 reactions
• 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
organic compound.
• Substitution—these reactions involve replacing one atom with another.
Factors affecting bioremediation
Methods of bioremediation

• On the basis of removal and transportation of the wastes for the treatment, basically there are
two methods:

• in-situ bioremediation and

• 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 is done.
• 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 in situ bioremediation
• the innate capabilities of naturally occurring microbial population
• should be tested at the laboratory and field levels before use for intrinsic
bioremediation.
• progress should be recorded time to time through site monitoring.
• The conditions 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
• absence of toxic compounds
• Short falls
• it is slow process due to the poorly adapted microorganisms, limited ability of
electron acceptor and nutrients, cold temperature and high concentration of
contaminants
Engineered in situ bioremediation

• When site conditions are not suitable,

• construction of an engineered system to supply materials that stimulate
microorganisms.
• Accelerate 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.
• However, 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
any 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 are
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.
• This technique shows considerable promise of stabilizing or removing inorganics
from soil as it can induce changes in the valence state of inorganics and cause
adsorption, uptake, accumulation, and concentration of inorganics in micro or
macro organisms.
• 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 formation through a specially
designed series of injection wells. Biosparging system

• 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,
• gasoline components such as benzene, toluene, ethyl benzene, and xylenes .
• 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
• It is effective in removing free product that is floating on
the water table.
• Bioslurping combines the two remedial approaches of
bioventing and vacuum- enhanced free-product recovery.
• Bioventing stimulates aerobic bioremediation of
contaminated soils in situ, while vacuum-enhanced free-
product recovery extracts --light, nonaqueous- phase
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 rhizosphere.
• Water hyacinth (Eichhornia crassipes) Duckweed (Lemna minor) Water
velvet (Azolla pinnata) Indian mustard (Brassica juncea) Sun flower
(Helianthus annus) Alyssum.
• 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

• Involves removal of waste materials and their collection at the place

to facilitate microbial degradation.
• Two types based on the phases of contaminated materials
• 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)
• Composting
• Land farming
Composting
• An aerobic, thermophilic treatment process in which contaminated material is mixed
with bioremediation microorganisms.
• 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 about 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 reference 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 40 oC.
II. Thermophilic stage: As the composting proceeds, the temperature rises from 40 oC to70oC.
Thermophilic bacteria( Thermus, Bacillus) ,Thermophilic fungi (Absidia) and Actinomycetes
(Streptomyces, 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.
• Windrow composting is the least expensive method, but has the potential to emit
larger quantities of VOCs .
• In-vessel composting is generally the most expensive type, but provides for the
best control of VOCs.
• Aerated static piles, especially when a vacuum is applied, offer some control of
VOCs and are typically in an intermediate cost range, but will require off gas
treatment .
• Berms may also be needed to control runoff during composting operations.
• Runoff may be managed by retention ponds, provision of a roof.
• 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 tilth of the
soil, supply nutrients, moderate pH, or facilitate bioremediation.
• Typically, full-scale land treatment would be
conducted in a prepared-bed land treatment
unit—an open, shallow reactor with an
impermeable lining on the bottom and sides to
contain leachate, control runoff, and minimize
erosion and with a leachate collection system
under the soil layer .
• In some cases, hazardous wastes (such as highly
contaminated soils) or process wastes (such as
distillate residues) may be treated in land
treatment units. In these cases, the waste may
be applied to a base soil layer.
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 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
suspended medium.
• Air provides oxygen for bacterial growth.
• Biologically there 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.
• pH, temperature, nutrient addition, mixing and oxygen can be controlled.
• 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 and tend to settle on the bottom
of the bioreactor.
• Air diffusers are placed radially along the rake arm.
• Airlift provides bottom circulation of contents in reactor.
• Baffles create a hydrodynamic nature for 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 cosubstrates and
utilize as carbon source.
• Co substrates also induce the production of beneficial enzymes.
• Factors affecting slurry phase biodegradation
• pH(optimum 5.5-8.5)
• moisture content
• temperature( 20-300C)
• Mixing
• Nutrients
• Microbial population(naturally occurring microorganisms are satisfactory,
genetically engineered microorganisms for layer compound may be added)
• Reactor operation (batch and continuous)
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 costly process.
• Sites of pollution are highly disturbed.
• There may be disposal problem after the process is complete.
ASSIGNMENT
• Present a paper on the bioremediation of any Two of
the following in soil or water. ( Cite references)

• Crude oil and petroleum refined products, solvents,

detergents, pesticides, plastics, organometallic compounds

• Sources (examples, how are they formed, where are they found)
• Uses (in nature, in industry)
• Effect of exposure ( humans, environment)
• Degradation (microorganisms involved, method of degradation.)