Measuring Bacterial

Growth
Bacterial Division
• Bacteria divide by binary fission
• Alternative means
• Budding
• Conidiospores (filamentous bacteria)
• Fragmentation
Fig. 7.13
Generation Time
• Time required for cell to divide/for population to double
• Average for bacteria is 1-3 hours
• E. coli generation time = 20 min
• 20 generations (7 hours), 1 cell becomes 1 million cells!
Standard Growth Curve
 Microbial growth kinetics
1. Microbial growth kinetics
Microbial growth kinetics is the
relationship between the specific growth
rate (μ) of a microbial population and
the substrate concentration (s)
Microbial growth kinetics
Growth kinetics is an autocatalytic
reactionRate of growth is directly
proportional to the concentration of cell
Measuring Growth
• Direct methods – count individual cells
• Indirect Methods – measure effects of bacterial growth
 Microbial growth kinetics
1. Growth is affected by physical,
chemical and nutritional conditions*
Cell concentration
2. Direct methods. Dry weight,
Turbidity (optical density) or Plate
counts
3. Indirect methods Concentration of
proteins, ATP or DNA content
Fig. 7.17
Turbidity
Metabolic Activity
Dry Weight
 Batch Culture

2. Growth curve of batch reactor

❖ A growth curve is a graphical representation of how
a particular quantity increases over time.
❖ Growth curves are used in statistics to determine the
type of growth pattern of the quantity—be it linear,
exponential, or cubic
❖ Cell growth implies increase in its mass and physical
size controlled by physical, biological and chemical
environments.
Phases of growth: Lag phase
Phases of growth: Log phase
Phases of growth: Log phase
Phases of growth: Stationary phase
Phases of growth: Death phase
 Microbial growth
• Microbial growth is quantified by
increase in the macromolecular and
chemical constituents of the cell and
growth pattern of each microbe is
unique.
• During the lag phase dX/dt and dS/dt are
essentially zero.
• However as exponential growth phase
begins it is possible to measure dX/dt
and dS/dt values which are very useful
for defining important microbial kinetic
parameters.
Kinetics of products formation
• Classified based on the relationship between product synthesis and
energy generation in the cell:
• Growth associated
• Non-growth associated
• Mixed-growth associated
Growth associated product
• 2.1 Growth associated
• Growth linked products are formed
by growing cells and hence primary
metabolites. Figure 1 clearly shows
that product is formed
simultaneously with growth of
cells. That is product concentration
increases with cell concentration.
The formation of growth
associated product may be
described by Eq. (1);
• where P = concentration of
product, qp = specific rate of
product formation, X = biomass Figure 1 clearly shows that product is formed simultaneously
concentration. with growth of cells.
Growth associated product
• 2.2 Non-growth associated
• They are formed by cells which
are not metabolically active and
hence are called secondary
metabolitesThe formation of
Non-growth associated product
may be described by Eq. (2);
• Figure clearly shows that
product formation is unrelated
to growth rate but is a function Figure 2. Non-growth associated.
of cell concentration.
Growth associated product
• 2.3 Mixed-growth associated
• The product formation from the
microorganism depends on both
growth and Non-growth
associated. It takes place during
growth and stationary phases.
The formation of Mixed-growth
associated product may be
described by Eq. (3);
•
• In Figure 3, product formation is
a combination of growth rate
and cell concentration. Figure 3. Mixed growth associated.
Process development
process development
The objective of process development is the timely production of sufficient product to meet market demand
using a cost-effective and reliable process, which also meets safety and quality requirements.
The broad stages of process development are
1. Product Identification: Product identification is a set of activities to identify the potential product
starting from its metabolic pathway involved and its manipulation, physicochemical properties of product
relevant for recovery and its pharmacokinetic properties.
2. Process Identification: Process identification is a set of activities aiming to systematically define the
set of processes of a fermentation and establish clear criteria for prioritizing them
3. Product Validation: Process to demonstrate that the product is safe and efficacious for
biotherapeutic and diagnostic purposes
4. Scale-Up : Turning a laboratory process into a robust practical commercial process
5. Process Validation: Establishing documented evidence which provides high degree of assurance
that a specific process will consistently produce a product meeting its predetermined Quality specifications
and Quality characteristics.
Process Optimization
Process Optimization
The economic objective of process optimization is to produce the greatest quantity of saleable
products at the least cost to maximize profit.
❖ To affect this optimization requires a process model spanning upstream and downstream
processes, which can be used to define strategies for optimizing sub- processes.
❖ However, process optimization is applied to the major sub-processes which have the greatest
impact on the competing priorities of quality, productivity and cost (i.e. efficiency). Optimizing
yield is a preferred strategy to reducing operating costs.
❖
❖ Optimization strategy at design stage is best option for process development.
❖ The potential benefits are the economic benefits and a more robust and more reliable
process.
❖ Process development and process optimization are thus all part of the same process
improvement continuum.
❖Optimized processes require fewer resources (Fig 1).
Process Optimization
PD (Process development) and PO(Process optimisation)_ Strategies should
incorporate: Isolation of microorganisms of potential industrial interest
• Classical methods of isolation and selection of microbial strains are
expensive, time consuming and without any clear objectives.
• Cost can be reduced considerably by isolation of microorganisms from
variety of sources, with desired characteristics, which gives a selective edge over
existing strain.
• Important characteristics, such as
c. Growth on simple growth medium,
d. Growth at ambient temperature
e. Better resistant to contaminants,
f. Which will be of economic significance -may be screened and selected.
Optimization Techniques
• Optimization Techniques
• Response surface methodology (RSM): RSM, which uses factorial
designs to optimize the production processes of the desired metabolites.
• Artificial neural network: An artificial neural network (ANN) is a
mathematical or computational model that is influenced by the structural
and/or functional aspects of the biological neural networks.
• Genetic algorithm (GA): A trained mathematical model serves as a
fitness function in the determination of optimum concentration of the
medium components using GA.
• Nelder Mead (NM) simplex method is another statistical technique,
which has been found to be helpful in reducing the expenses of classical
optimizations and gives satisfactory results.
• Nelder Mead (NM) simplex
Response Surface Methodology (RSM)
• RSM, uses factorial designs to optimize the production processes of the
desired metabolites.
• RSM is a sturdy, robust and efficient mathematical approach which includes
statistical experimental designs and multiple regression analysis, for
seeking the best formulation under a set of constrained equations.
• RSM has often been applied to optimize the formulation variables and
optimization of fermentation process.
• RSM employs several phases of optimization and it can be performed in
three basic steps, i.e.,
• experiments designed for the screening of the factors
• path of steepest ascent/descent
• quadratic regression model is fitted and optimized using canonical
regression analysis method.
• One of the important inputs of RSM is representation of the yield, as
a surface plot.
• It can provide multiple responses at the same time by considering the
interactions between the variables, which is utmost necessary for
designing and process optimization.
• Since, the theoretical relationships between the independent and
dependent variables are not clear, multiple regression analysis can be
applied to predict
the dependent
variables.
Advantages:
• RSM model is simple, efficient, less time consuming
• RSM is used to determine the factor levels
• With the help of RSM we can predict the product properties
throughout the region, even at factor combinations not actually run
and to find conditions for the process stability.
Limitations:
• the metabolic complexity of the microorganisms.
• When a large number of variables are involved, the development of
rigorous models
• If large variations in the factors
• it is complicated to study the interactions of more than five variables
• the non-linear nature of the biochemical network interactions.
ANN
• An artificial neural network (ANN) is a mathematical or computational
model that is influenced by the structural and/or functional aspects of
the biological neural networks.
• ANN mimics the learning ability of the brain and consists of input (like
synapse), which are multiplied by weights (strength of respective
signals) and then computed by a mathematical function which
determines the activation of neuron.
• They are “trained” using a data set and then applied to predict new
data points.
• Prior knowledge or equations is not essential for this training as the
network and system remains as a black box to the user.
• Significant characteristics of ANNs:
• they can work smoothly with large
amounts of data,
• excel at complex pattern
recognition
• require no mechanistic description
of the system
• The architecture of the ANN
consists of three layers of
information known as neurons:
• a layer of “input” units is connected
to a layer of “hidden” units, which is
further connected to a layer of
“output” units
• ANNs have been widely applied with great success for bioprocess
system designing, modeling, optimization and control.
• due to its capacity to learn filter noisy signals and generalize
information through a systematic training procedure.
• Widely used in the fermentation industry.
• Ex: ANN technique used to optimize nutrient mist reactor for hairy
root growth and developed an efficient model for optimizing the
culture conditions and also predicted the biomass productivity
effectively under different culture conditions.
GA

• GA mimics the process of mutation and is based upon the principle

“survival of the fittest”.
• The GA repeatedly modifies a population of individual solutions.
• At each step, the GA selects some of the individual solutions at
random from the current population as parents.
• It then uses the selected ones to produce the off springs for the next
generation, thus over a successive generations, the population
“evolves” toward the most favorable solution.
• The GA follows mainly three types of rules at each step to create the
next generation from the current population:
• Selection rule selects the individuals, known as parents that
contribute to the population of the next generation. ’
• Crossover rule combines two parents to form children for the next
generation.
• Mutation rule applies random changes to individual parents to form
children.
• GA was successfully used to optimize medium composition for
rifamycin B production using mutant strain of Amycolatopsis
mediterranei at shake flask level
• GA can handle a large amounts of data.
• Advantages:
• Is faster and more efficient
• Provides a list of “good” solutions and not just a single solution.
• Always gets an answer to the problem, which gets better over the
time.
• More efficient for large data analysis.
• Disadvantages:
• GAs are not suited for all problems, especially problems which are
simple
• Being stochastic, there are no guarantees on the optimality or the
quality of the solution.
Nelder-Mead Simplex Method
• A popular direct search method for multidimensional unconstrained
optimization.
• Does not require derivative information, making it suitable for non-
smooth functions.
• Iteratively improves a simplex (geometric figure) in the search space
to find the minimum or maximum of a function.
Advantages
❖Simplicity: Easy to understand and implement.
❖Efficiency: Can be efficient for low-dimensional problems.
❖Robustness: Can handle noisy and discontinuous functions.
❖Versatility: Applicable to a wide range of optimization problems in
biotechnology.
Disadvantages
❖Slow Convergence: Can be slow to converge, especially for high-
dimensional problems.
❖Prone to Local Minima: May get stuck in local minima, depending on the
initial simplex and function landscape.
❖Sensitivity to Initial Conditions: The choice of the initial simplex can
significantly affect the performance.
❖Difficulty in Handling Constraints: Not well-suited for problems with
constraints.
Applications in Biotechnology
❖Parameter Estimation: Estimating kinetic parameters in biochemical models.
❖Experimental Design: Optimizing experimental conditions (e.g., temperature, pH,
substrate concentration).
❖Protein Structure Prediction: Finding the optimal conformation of a protein.
❖Drug Design: Optimizing drug molecules for desired properties.
❖Metabolic Engineering: Optimizing metabolic pathways for increased production
of target compounds.
 Optimize the strategy to optimize primary and secondary metabolite
production

• Applications of microbes for industrial production of primary and

secondary metabolites - Industrial Microbiology.
• Metabolism in microorganisms involves two pathways: Primary metabolic
pathways (PMPs, produced during the growth phase of the organism)
produce too few end products , while secondary metabolic pathways
(SMPs, produced during the stationary phase) produce a variety of
products.
• There are some similarities between the pathways that produce primary
and secondary metabolites:
❖The product of one reaction is the substrate for the next and
❖The first reaction in each case is the rate-limiting step.
❖Also the regulation of secondary metabolic pathways is interrelated in
complex ways to primary metabolic regulation.
Industrial fermentation based on the end-product application, can be categorized
into four types:

1. Biomass:The end-product is viable cellular material eg, single cell protein, baker’s
yeast, probiotic cultures.
2. Extracellular metabolites: Chemical compound intermediates of microbial
biochemical pathways are produced and can be divided two groups:
• Primary metabolites (produced during the growth phase of the organism, eg,
ethanol, citric acid, glutamic acid, lysine, vitamins and polysaccharides)
• Secondary metabolites (produced during the stationary phase, eg, penicillin,
cyclosporin A, gibberellin, and lovastatin).
• 3. Enzymes and other proteins (intracellular components): A key component
of this process is lysis of cells at the end of fermentation. Proteins are typical end
products and need to be purified and crystallized.
• 4. Substrate transformations: Raw material is biologically transformed into a
finished product. Generally used for steroid transformations, food fermentations
and sewage treatment.
Primary metabolites
• Primary metabolites
• Involved in growth, development and reproduction. Hence, essential for
survival and existence of the organism and reproduction.
• Formed at the same time as new cells. Production curve follows the growth
curve.
• Formed in trophophase during exponential growth as normal end products
of primary metabolism.
• Also called central metabolites as these maintain normal physiological
processes. Cells maintain optimum concentration of all macromolecules
(proteins, DNA, RNA etc.).
• Produced in adequate amount to sustain cell growth for example vitamins,
amino acids, nucleosides etc.
Primary metabolites
• Overproduction can be genetically manipulated. Auxotrophic (auxo,
“increase,” and trophos, ‘‘food’’) mutants having a block in steps of a
biosynthetic pathway for the formation of primary metabolite .
Growth rate slows down due to limited supply of any other nutrient.
• Metabolism does not stop but product formation stops. Industrially
important for example ethanol, acetone, lactic acid, CO2.
• Common food supplements, L-glutamate and L-lysine, are produced
and purified via the mass production Corynebacterium glutamicum
(Gram-positive) .
• Citric acid, commonly used in pharmaceutical and cosmetic industries
is produced by Aspergillus niger.
Secondary metabolites
❖Secondary metabolites are not produced until the microbe has
largely completed its logarithmic growth phase and entered the
stationary phase of the growth cycle. Period of production is called
idiophase and metabolites as idiolites.
❖In the idiophase, cells do not divide but are metabolically active.
❖Idiolites are organic compounds produced only after considerable
number of cells and a primary metabolite have accumulated (end or
near the stationary phase of growth). Rather it can be said that these
are produced under sub-optimal concentrations of O2 , deviations of
pH or when primary nutrient source is depleted.
Secondary metabolites
❖Though idiolites are a characteristic feature of fungal, yeast, actinomycetes
and bacterial growth but are not produced by a few strains of E. coli.
❖In some strains secondary metabolite are produced by further conversion
of a primary metabolite..
❖Not necessary for growth, development, and reproduction like primary
metabolites. Their production is influenced by environmental factors.
❖Secondary metabolites are synthesized for a finite period by cells that are
no longer undergoing balanced growth.
❖A single microbial type can produce very different metabolites.