Industrial Microbiology INDM Lecture 10 24/02/04

Transcription

1 Industrial Microbiology INDM 4005 Lecture 10 24/02/04

2 4. INFLUENCE OF PROCESS VARIABLES Overview Nutrient Limitation Cell Immobilisation

3 Overview 4. Influence of process variables 4.1. Kinetics and technology of nutrient limitation Types of continuous culture; Kinetics of continuous culture; Typical pattern of biomass and substrate levels in continuous culture fermenter Influence of growth constants on biomass behaviour in continuous culture Application of continuous culture; Advantages / disadvantages of continuous culture Modifications of basic chemostat; 4.2. Nutrient limitation also applied in fed-batch Fed-batch Industrial application of fed-batch 4.3. Nutrient limitation and cell composition 4.4. Use of continuous culture for calculation of growth kinetics

4 Batch Cultures Closed systems microorganisms undergo a predictable pattern of growth characterised by 4 phases Describe the 4 phases of growth and the factors influencing them Understand the mathematics of exponential growth Define and apply growth parameters (t d, m, m max, k, Y s ) Describe the Monod relationship and the meaning of k s

5 4.1. KINETICS AND TECHNOLOGY OF NUTRIENT LIMITATION Type of culture; Batch; m varies during culture cycle Fed-batch; m is controlled or regulated after a certain time Continuous; m is controlled m reflects the physiological state or intracellular environment control m control intracellular environment

6 Growth in Continuous Culture Scientists are trained to conduct experiments in which only one variable is changed at any one time Continuous culture methods enable constant cell numbers to be maintained in a constant chemical environment at specified growth rates for prolonged periods of time In this lecture we will focus on the theoretical and practical aspects of growth in flow-through systems

7 Fresh medium from reservoir Set up for Continuous culture Sterile air Flow-rate regulator Stirrer Culture Overflow Effluent

8 TYPES OF CONTINUOUS CULTURE Method of control; Chemostat - regulated by control of concentration of limiting nutrient Turbidostat - regulated by biomass using optical density (photoelectric cell) Biostat - regulated by systems monitoring biomass other than optical density (e.g CO 2 production)

9 How can the population density and growth rate be controlled? To regulate the growth rate and density it is necessary to control the influx of nutrients per unit time A distinctive feature of a chemostat is that one nutrient (C, N, P, energy source, growth factor) is at a low conc By selecting the concentration of substrate we can predetermine a certain microbial growth rate After a period of adjustment a steady-state equilibrium is achieved Changing the initial substrate concentrations alters the population density but growth rate remains unaltered at the new steady-state

10 Fermenter configuration STR Up-flow Plugflow Single-stage Multi-stage Cell recycle Draw diagrams and make notes on the above

11 CASE STUDY Re Continuous Culture draw a diagram of a typical pilot/ laboratory system and an industrial system

12 The development of growth in a chemostat Steady State Growth rate equals loss of cell biomass Cell Number Nutrient limitation causes decrease in m Population density increases Inoculation m max Time in Hours

13 Mathematical relationships of growth in chemostats Relationship between growth rate or specific growth rate and medium flow can be described mathematically The medium flow through the system is represented by the term dilution rate D = F V D = dilution rate (h -1 ) V = culture volume (l) F = flow rate (l h -1 )

14 KINETICS OF CONTINUOUS CULTURE Thus: Mass balance or the rate of change of cells in reactor = RATE of ACCUMULATION minus RATE of LOSS dx /dt = m.x - D.x Mass balance of the substrate = INPUT minus LOSS DUE TO OUTFLOW minus SUBSTRATE USED BY BIOMASS ds / dt = D. Sr - D. S - m. X / Y X = Total biomass D = Dilution rate x = Biomass concentration m = Specific growth rate Y = Yield S = Substrate conc in fermenter S r = Substrate conc in reservoir

15 INCORPORATE MONOD MODEL The empirically derived equation for the relationship between specific growth rate and [S] is Monod equation D = m max. S / (Ks + S) This is the most basic model for continuous culture NOTE; When dx / dt = 0 (at steady state) then D = m This equation demonstrates how the steady state substrate concentration in the chemostat is determined by the dilution rate

16 Batch versus Chemostat Exponential phase Chemostat of batch culture operating in steady-state Growth rate of culture Specific growth rate of culture Biomass Available nutrients Culture volume Toxic metabolites Increasing Constant Increasing Decreasing Constant Increasing Constant Constant Constant Constant Constant Constant Constant, Variable, Increasing, Decreasing

17 CASE STUDY A chemostat operating in steady-state at a dilution rate of 0.25 h -1 sets a limiting nutrient concentration of 0.6 micromoles l -1. Determine the Monod constant in suitable units if m max for the organism is 0.25 h -1

18 D = m max. S / (Ks + S) Rearrange the equation K s = s m max - D D K s = 0.6 ( ) 0.25 K s = 0.6 x 1.4 K s = 0.84 micromoles l -1

19 THE PERFECT MODEL WOULD REQUIRE AN UNREALISTIC AMOUNT OF INFORMATION Simplifying assumptions are made, for example, Assume that population density has no effect If D = 0 then batch culture - but no lag period predicted Transient conditions predicts either stable condition or wash-out Assumes all substrate goes to biomass (maintenance!) No allowance for substrate or product inhibition In more advanced models these areas must be considered

20 TYPICAL PATTERN OF BIOMASS AND SUBSTRATE LEVELS IN CONTINUOUS CULTURE FERMENTER CASE STUDY Plot; steady state substrate concentration steady state biomass concentration steady state product concentration against dilution rate (m) Page 15 Stanbury & Whitaker

21 INFLUENCE OF GROWTH CONSTANTS ON BIOMASS BEHAVIOUR IN CONTINUOUS CULTURE Influence of low vs high Ks or m max on biomass or substrate level Influence of low vs high Ks or m max of different populations on competition DEVIATIONS FROM IDEAL BEHAVIOUR may be due to Maintenance energy Synthesis of reserve polymers Switch to less efficient pathways Imperfect mixing Substrate toxicity Second substrate becomes limiting

22 APPLICATION OF CONTINUOUS CULTURE INDUSTRY; Waste-treatment Single-cell protein Continuous beer production Continuous amino acids, organic acids production Continuous ethanol Continuous bakers yeast

23 RESEARCH - more important Physiology and biochemistry growth rate control Influence of environmental / process factors on growth and product formation. Induction, repression, growth rate, influence of temperature, ph etc. Microbial ecology Selection of slow growing populations Prey-predator interactions Competition (e.g plasmid +/-) Kinetics Calculation of growth constants, fermentation data

24 CASE STUDY From the literature record some applications of continuous culture to studies in microbial physiology and ecology

25 4.1.6 ADVANTAGES / DISADVANTAGES OF CC Advantages Uniformity of operation Process demands are constant i.e. continuous cycle of sterilisation, fermentation, harvesting, extraction Once in steady-state demands re process control are constant i.e. oxygen demand Disadvantages Susceptibility to contamination Duration of run is longer increased chance of contamination Strain degeneration arising from large number of generations Contamination with "fitter" competitor e.g. lower Ks

26 OBJECTIVES IN INDUSTRIAL APPLICATION? CONTINUOUS PROCESSING Advantage? example beer Residence time of "pint" in brewery same. example waste-treatment definite advantage. EXERT PHYSIOLOGICAL CONTROL Can use fed-batch which is less demanding

27 MODIFICATIONS OF BASIC MULTI-STAGE CHEMOSTAT Different environments or growth rates in the various reactors (e.g. 1st biomass, 2nd product) SINGLE STAGE WITH CELL RECYCLE Application in activated sludge waste-treatment Relationship between D and m different when recycle used. EFFECT OF FEEDBACK; 1. Increase biomass conc. in fermenter - lower in effluent 2. Decrease residual substrate 3. Maximise rate of product formation 4. Dcrit is increased - useful when substrate is dilute

28 Chemostats in series F 1 S R X 1 S 1 V 1 F O2 S R2 X 2 S 2 V 2 F 2

29 CONTINUOUS CULTURE PRINCIPLES Also applied in; UP-FLOW REACTORS (often with immobilised cells) PLUG-FLOW SYSTEMS

30 4.2. NUTRIENT LIMITATION ALSO APPLIED IN FED-BATCH Fed-Batch Takes advantage of fact that residual substrate concentration may be maintained at very low levels Type of continuous culture but volume is not constant. Quasi-steady state achieved.

31 CLASSIFICATION OF FED-BATCH OPERATION Without feed-back control - programmed feed-rate 1. Intermittent addition 2. Constant rate 3. Exponentially increased rate With feed-back control 1. Indirect feed-back e.g. respiration rate, dissolved oxygen, ph 2. Direct feed-back concentration of substrate in culture exerts control

32 4.2.2 INDUSTRIAL APPLICATION OF FED-BATCH Penicillin Glucose, phenyl acetic acid, ammonia source Cephalosporin Glucose, methionine Streptomycin Glucose, ammonia source Glutamic acid Urea, ethanol, (acetic acid) Amylase Carbon source Bakers Yeast Glucose Citric acid Glucose, ammonia

33 4.3 NUTRIENT LIMITATION and CELL COMPOSITION Media can be designed to allow limitation on any essential nutrient NUTRIENT LIMITATION EFFECT CARBON energy supply NITROGEN or SULPHUR protein synthesis PHOSPHORUS Nucleic acid synthesis MAGNESIUM or POTASSIUM Nucleic acid and or membrane synthesis

34 4.3 NUTRIENT LIMITATION and CELL COMPOSITION THE DEGREE OF LIMITATION INFLUENCES THE CELL COMPOSITION, for example CELL SIZE NUCLEIC ACIDS CONSEQUENTLY CELLS BEHAVE DIFFERENTLY UNDER DIFFERENT LIMITATION CONDITIONS; Repression mechanisms may be removed, for example, antibiotic production or pigment production under phosphate limitation

35 4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF GROWTH KINETICS (1) Calculation of Ks and m max (2) Determine variation in yield with growth rate (3) Calculation of Yg and m, endogenous respiration (4) m /m max to compare growth under different conditions NOTE; growth rate becomes an independent variable in continuous culture

36 4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF GROWTH KINETICS Use of higher dilution rates can lead to higher biomass productivity But result in higher substrate concentrations in the effluent and lower biomass concentrations in the reactor due to wash-out when the dilution rate exceeds the critical dilution rate then washout occurs

37 4.4. USE OF CONTINUOUS CULTURE FOR CALCULATION OF GROWTH KINETICS These factors have a number of consequences e.g in continuous wastewater treatment processes The minimum reactor volume is set by the critical dilution rate High dilution rates will lead to an effluent containing high concentration of substrate and the effluent will therefore contain substrates/wastes and not have been treated properly Low cell concentrations at high dilution rates will also make the reactor sensitive to inhibitors in the feed. Inhibitors would cause the specific growth rate of the cells to drop and cause the cells to washout. The lower the conc of cells, then the faster the cells will washout

38 Conclusions In this lecture we have seen that a chemostat is a means of providing nutrient limitation an important process variable Mathematical relationships can be used to predict growth and determine growth parameters such as m max, K s, Y List the differences between growth in batch and in continuous culture Understand the terms steady-state, dilution rate, growth limiting substrate, Monod constant, Describe the principles of fed-batch, biomass feedback, and multi-stage cultivation Give applications for continuous cultivation techniques Describe the main practical problems encountered in chemostat operation