Microbial growth kinetics and fermentation

1 Microbial growth kinetics and fermentation Measurement of Microbial growth 3. Measurement of Microbial growth A. Measurement of cell numbers microscopic observation on specially etched slides (hemacytometers). cell counts involve plating diluted samples (using a pour plate or spread plate). B. Measurement of cell mass-may be used to approximate the number of microorganisms (Suitable parameters may be dry weight, light scattering in liquid solutions, or biochemical determinations of specific cellular constituents such as protein, DNA). If plated on a suitable medium, each viable unit grows and forms a colony. Each colony that can be counted is called a colony forming unit (cfu) and the number of cfu's is related to the viable number of bacteria in the sample 1 OD600 nm = 109 cells/ml (E. coli Bacterial colonies growing on a plate of nutrient agar.)

2 Saccharomyces cerevisiae 1 OD600 nm = 4-9 x 10^7 cells/ml DILUTION PLATING. Beer-Lambert Law Beers Law states that absorbance is proportional to concentration over a certain concentration range A = log(IO/I) = cl A = cl A = absorbance; = molar extinction coefficient (M-1 cm-1 or mol-1 L cm-1); c =. concentration (M or mol L-1); l = path length (cm) (width of cuvette). A = Absorbance (optical density). IO = Intensity of light on the sample cell I = Intensity of light leaving the sample cell c = molar concentration of solute l = length of sample cell (cm). = molar absorptivity (molar extinction coefficient). Intensity of spectral lines sample I I0 I. Transmittance T . I0. [J] Molar concentration Length Molar absorption coefficient (extinction coefficient) : L mol-1 cm-1. Beer-Lambert law I I0 10 [ J ].

3 I A J . Absorbance A A log 0. I. Note that ~ (cm2 mol-1) ~ cross-section for absorption Plating methods Measure number of viable cells Population size is expressed as colony forming units (CFU). Simple and sensitive Widely used for viable counts of microorganisms in food, water, and soil Inaccurate results obtained if cells clump together plate dilutions of population on suitable solid medium . count number of colonies . calculate number of cells in population Membrane filtration methods Especially useful for analyzing aquatic samples Measurement of Cell Mass Dry weight time consuming and not very sensitive Quantity of a particular cell constituent protein, DNA, ATP, or chlorophyll useful if amount of substance in each cell is constant Turbidometric measures (light scattering). quick, easy, and sensitive Effects of Physical and chemical parameters on cell Physiology Effect of pH.

4 Acidophiles growth optimum between pH 0 and pH Neutrophiles growth optimum between pH and pH 7. Alkalophiles growth optimum between pH and pH most acidophiles and alkalophiles maintain an internal pH near neutrality The plasma membrane is impermeable to protons some synthesize proteins that provide protection , acid-shock proteins many microorganisms change pH of their habitat by producing acidic or basic waste products most media contain buffers to prevent growth inhibition Temperature organisms exhibit distinct cardinal growth temperatures minimal maximal optimal -Dissolved oxygen (DO). Effect of factors: aerobic growth is more efficient. - aerobic fermentation requires oxygen - oxygen gas is sparingly soluble in water - specific growth rate may be limited by DO if DO is below a critical oxygen concentration.

5 growth rate becomes independent of DO concentration. bacteria and yeast: 5%-10% of the saturated DO. mold: 10%-50% of the saturated DO. The saturated DO in aqueous solution is 7 ppm at 25oC and 1. atm. Media Optimization: Diauxic growth The biphasic response of a culture of micro-organisms based on a phenotypic adaptation to the addition of a second substrate; characterized by a growth phase followed by a lag after which growth is resumed. growth of Microbial cells Introduction: Batch growth Characteristics growth Stages, Effects of Environmental Conditions, Product Formation, Mathematical Models Fed-batch: No substrate inhibition, desired growth rate, minimization of inhibitory product formation, High cell density Continuous growth Characteristics Dilution Rate, optimum Operation Microbial growth in Batch Region 1: Lag phase microbes are adjusting to the new substrate (food source).

6 Region 2. Exponential growth phase, log [X]. microbes have 1 2 3 4. acclimated to the conditions Region 3. Stationary phase, limiting substrate or electron acceptor limits the growth rate Time Region 4. Decay phase, substrate supply has been exhausted Lag Phase Cell growth is the primary response of viable cells to substrates and nutrients. Substrates/nutrients + cells products + more cells Product formation is a secondary response. Exponential Phase The second major phase of Microbial growth in a batch fermentation ferment process which is also known as the logarithmic growth phase. Cells have adjusted to their new environment The cells are dividing at a constant rate resulting in an exponential increase in the number of cells present. This is known as the specific growth rate and is represented mathematically by first order kinetics as the following: where X is the cell concentration, is the cell growth rate, and kd is the cell death rate.

7 The term can be referred to as net . kd ( net) The cell death rate is sometimes neglected if it is considerably low (smaller than the cell growth rate). fermentation fermentation is a biological reaction in which both electron- donor and electron-acceptor are organic molecules In industrial microbiology, fermentation refers to any large scale Microbial process ,whether or not, it is biochemically a fermentation fermentation processes Batch fermentation Continuous fermentation Fed-batch fermentation Batch fermentation Reasons for Batch Popularity Equations were for cell mass (or other growth -associated product). Many industrial applications are for non- growth associated products. Selective pressure of a chemostat is detrimental to engineered organisms Batch is more mechanically reliable Batch system is more more flexible Batch fermentation OPEN SYSTEM CLOSED SYSTEM.

8 All components that compose All components that the system may enter and leave compose the system may Eg. Continuous flow cultures not enter and leave. Biomass balances the output Batch culture which rate. consists of an essential Establishment of steady state limited amount of nutrient may occur medium. growth rate tends to zero System is always in transient state. growth kinetics of batch fermentation Introduction - Autocatalytic reaction: The rate of growth is directly related to cell concentration substrates + cells extracellular products + more cells S + X P + nX. S: substrate concentration (g/L); X: cell mass concentration (g/L);. P: product concentration (g/L); n: increased number of biomass. Net specific growth rate (1/time): 1 dX. net . X dt t: the time Batch growth kinetics Exponential growth phase net R m m is the maximum specific growth rate (1/time).

9 Doubling time of cell mass: the time required to double the Microbial mass: ln X / X 0 ln 2 d . net net net Fed-Batch fermentation Fed-Batch fermentation Initially starts in a batch mode and is then fed according to one of the following feed strategies: i) Medium used to establish the batch culture is added ii) Solution of limiting substrate of same concentration as in the initial medium is used iii) Concentrated solution of the limiting substrate is used Fed-Batch Nutrients are continuously or semi-continuously fed, while effluent is removed discontinuously. Overcome substrate inhibition or catabolite repression by intermittent feeding of substrate by maintaining low substrate concentration. Used for production of secondary metabolites antibiotics, lactic acid, E. Coli making proteins from recombinant DNA technology.

10 Preliminary knowledge required to implement fed-batch Before starting a fed-batch process, a batch fermentation should be implemented to "get to know" the fermentation of the microorganism. From a batch fermentation , the operator should have a knowledge of: Best abiotic conditions such as temperature, light, agitation, pH, growth medium, etc. Specific needs of precursors, inducers or other enrichment factors The different growth phases and the consumed (substrate) and produced components (product of interest and by-product). The relationship between the biomass and product formation ( growth or non- growth associated product) and the oxygen uptake rates Limiting substrate for growth and the relationship between the specific growth rate and the limiting substrate concentration Eventual inhibitions from the substrate and/or product Productivity of Fed-Batch fermentation Though batch fermentation is considered simple, fed-batch fermentation offers the convenience of better control over substrate concentration variations and differentiation of growth leading to improved overall productivity with essentially the same equipment used for batch fermentation .