Topic 1.Microbial growth curves and culture systems (2).pptx

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1. Microbial growth curves and culture systems Topic 1: Culture systems- Pure cultures, mixed cultures, batch cultures, continuous culture. Fermentation; Bioreactor and Bioprocess design- Outlines the basic structure, function and components of bioreactors. Different fermentation methods will be discussed giving their growth curves.

2. Introduction Industrial systems using microbes for fermentation processes utilise varying culturing systems. The systems aim for optimal production of the desired metabolic products from microbes. Hence varying culturing systems are used. These systems are scaled up for industrial production (high yields) by implementing the optimal conditons in which the microbe survives and thrives Equipment is also designed and automated to achive optimal yields. The culturing systems also allow for removal of inhibitory products and toxins.(down-stream processing)

3. Summary of sections covered 1. Definitions and basic concepts 2. Phases of growth curve 3.Pure culture system and batch culture systems 4. Fed-batch culture systems 5. Continuous culture system 6. Mixed culture system

4. Key words: Fermentation, culture systems, growth curves, batch culture, fed-batch culture, continuous fed-batch culture , mixed culture, bioreactors

5. Growth : Basic concepts

6. Definition: Microbial growth curve Bacterial growth is a complex process involving numerous 1. anabolic (synthesis of cell constituents and metabolites) and 2. catabolic (breakdown of cell constituents and metabolites) reactions. Ultimately, these biosynthetic reactions result in cell division as shown in slide 5. In a homogeneous rich culture medium, under ideal conditions, a cell can divide in as little as 10 minutes.

7. In coliform bacilli ( E. coli )and many other medically important bacteria, it takes about about 20 minutes, in tubercle bacilli ( Mycobacterium tuberculosis ) it is about 20 hours and in lepra bacilli ( Mycobacterium leprae )it is about 20 days. It is often difficult to grasp fully the scale of exponential microbial growth meaning it’s not universal.

8. If a suitable liquid medium is inoculated with bacterium and incubated, its growth follows a definitive course. To come with a growth curve, small samples are taken at regular intervals after inoculation and plotted in relation to time. A plotting of the data will yield a characteristic growth curve The changes of slope on such a graph indicate the transition from one phase of development to another.

9. Typical Growth curve phases 1. Lag 2. Log 3.Stationary 4. decline

10. Lag phase the growth rate is essentially zero When an inoculum is placed into fresh medium, growth begins after a period of time called the lag phase The lag phase is thought to be due to the physiological adaptation of the cell to the culture conditions May involve a time requirement for induction of specific messenger RNA (mRNA) and protein synthesis to meet new culture requirements lag phase may also be due to low initial densities of organisms that result in dilution of exo -enzymes (enzymes released from the cell) and of nutrients that leak from growing cells Normally, such materials are shared by cells in close proximity. But when cell density is low, these materials are diluted and not as easily taken up As a result, initiation of cell growth and division and the transition to exponential phase may be slowed.

11. 3. environmental factors such as temperature, pH,oxygen, water activity. Metabolic activities require optimal temperatures. 4. and nutrients present in the new medium. 5. The lag phase varies considerably in length with the species, 6. size of inoculum This initial period is the time required for adaptation to the new environment, during which the necessary enzymes and metabolic intermediates are built up in adequate quantities for multiplication to proceed. The lag phase is defined to transition to the exponential phase after the initial population has doubled

12. Exponential / log phase The cells start dividing and their numbers increase exponentially or by geometric progression with time The exponential phase is characterized by a period of the exponential growth—the most rapid growth possible under the conditions present in the batch system . During exponential phase 1. growth rate of the increasing of cells in the culture is proportional to the number of cells present at any particular time. (geometric progression of 2 n 2. The cells have well adapted to conditions in the vessel

13. . 3. Phase when cells are most active metabolically 4. and is preferred for industrial purposes e.g production of enzymes, fermentation products, antibiotics etc However, during their log phase of growth, microorganisms are particularly sensitive to adverse conditions. Exponential phase is of limited duration because of exhaustion of nutrients; accumulation of toxic metabolic end products; rise in cell density,

14. (iv) change in pH; and (v) decrease in oxygen tension (in case of aerobic organisms)

15. Stationery phase ending of the exponential phase occurs when any of the essential nutrients is depleted or toxic metabolite accumulated i n the system during this phase the growth rate is almost nil/insignificant(some split to decelerated phase ) iii) As the media start depleting, the concentration of metabolites increases the growth is inhibited. iv) The increase in cell mass ceases the cell enters stationary phase.

16. Decline phase/Death phase In the death phase where i ) the cells will start to lyses and ii) the cell density decreases (death phase is the period when the population decreases due to cell death) iii) Decline in the number of viable cells (Eventually the rate of death exceeds the rate of reproduction, and the number of viable cells declines.) iv) may also be caused by autolysis besides nutrient deprivation and buildup of toxic wastes

17. Sub-topic 2: Culture systems Several culturing systems are used in industry for desired products. The section will outline different systems used in culturing microbes including the types of bioreactors and the growth curves expected.

18. Microbial culture system A microbial culture system is a method of multiplying microbial organisms (increase in mass culture) by letting them reproduce in predetermined culture medium under controlled laboratory conditions. Measurable parameters of importance include: The substrates, The waste, the products,the biomass, aerobic/anaerobic conditions and time .

19. Culture systems Batch Culture Fed-batch Culture 3. Continuous Culture 4. Mixed culture

20. 1.Batch culture systems In a batch culture the growth of a single organism or a group of organisms, called a consortium, is evaluated using a defined medium to which a fixed amount of substrate (food) is added at the onset.

21. Batch culture systems-Bioreactor designs

22. Growth and product curves

23. Typically, to understand and define the growth of a particular microbial isolate, cells are placed in a liquid medium in which the nutrients and environmental conditions are controlled ( closed system ). If the medium supplies all nutrients required for growth and environmental parameters are optimal, the increase in numbers or bacterial mass can be measured as a function of time to obtain a growth curve

24. Discuss -Batch system advantages no need for close monitoring as the substrates and culture are added at once disadvantages no continuous addition of the substrate to obtain more yield as it is a closed system

25. APPLICATIONS It is beneficial for the construction of biomass (Baker’s yeasts) and primary metabolites (l actic acid, citric acid, acetic acid, or ethanol production). In food industries, organic acids are used as preservatives or acidifiers( lactic acids, citric acids, and acetic acids), alcoholic beverages (wine, beer, and distilled spirits i.e. brandy, whisky, and rum), and sweeteners (e.g., aspartate ) or amino acids used as flavoring agents (e.g., monosodium glutamate)are the various product manufactured by batch cultivation.

26. 2. Fed-batch culture systems

27. In fed-batch fermentation nutrients are added incrementally at various times, or regular interval of time no growth media and micro-organisms are removed until the end of process, this addition is prolonged to log phase and stationary phase, thereby increasing the biomass and the amount of metabolites.

28. Fed-batch culture system

29. Fed-batch advantages fill in disadvantages fill in

30. Comparison Question: What can you deduce from the graph when you compare and contrast the two systems?

31. APPLICATIONS Fed-batch culture is used to produce antibiotics such as penicillin , which requires controlled feeding of nutrients. Underfeeding or overfeeding of substrates may results in nutrient starvation and dilution of the product, respectively. Thus, the fed-batch provides a controlled flow of nutrients to achieve maximum production of desired products or to obtain high cell density. It is also used in the production of baker’s yeast in order to avoid the Crabtree effect (repress respiration by the fermentation pathway above the limit of substrate concentration).

32. 3. Continuous culture systems In continuous culture there is a steady influx of growth medium and substrate such that the amount of available substrate remains the same Growth under both batch and continuous culture conditions has been well characterized physiologically and also described mathematically . These products are often produced in large batches (up to 500,000 liters) also called large-scale fermentations.

33. To maintain cells in a state of continuous growth, i) nutrients must be continuously added ii) and waste products removed. This is called continuous culture or open system . Continuous culture uses a chemostat in which cells of a growing culture are continuously harvested and nutrients continuously replenished. The second type of continuous culture system, the turbidostat , has a photocell that measures the absorbance or turbidity of the culture in the growth vessel. The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity or cell density

34. a steady-state conditions of the total number of cells and the volume in the bioreactor is maintained Continuous culture can be operated over the long term because it is an open system with a continuous feed of influent solution that contains nutrients and substrate,

35. as well as a continuous drain of effluent solution that contains cells, metabolites, waste products , and any unused nutrients and substrate There is a control of the rate of growth, which can be used to optimize the production of specific microbial products.

36. Continuous culture growth curve

37. Advantages Continuous cultures are not used in small fermentations Continuously fermentation can be easily centrifuged/ seperated Avoids down time to batch time Physiological state of cell during continuous fermentation is more uniform thus production yield much consistence. Disadvantages Due to longer fermentation 500-600 h rs , some cells may lose viability Sterility maintenance is very difficult.

38. APPLICATIONS Continuous culture fermentation has been used for the production of single-cell protein, organic solvents, starter cultures, etc. It has been used in the production of beer, fodder yeast, vinegar, baker’s yeast, etc. In the industrial production of secondary metabolites (such as antibiotics from a Penicillium or a Streptomyces sp.) Continuous culture has been tested for L-lysine-producing C. glutamicum mutant B-6 It has been used in municipal waste treatment processes.

39. 4. Mixed culture Two or more organisms that can be cultured together in the same bioreactor and producing varying products without inhibition occuring The products can be seperated from each other the organisms may use the same carbon sources for different metabolic pathways some of the products may assist the symbiotic organism to grow and produce desired products faster

40. Mixed cultures Advantages Disadvantages

41. APPLICATIONS Product yield may be higher. Yogurt is made by the fermentation of milk with Streptococcus thermophilus and Lactobacillus bulgaricus . The number of S. thermophilus cells increased from 500 × 10 6 per milliliter to 880 × 10 6 per milliliter with L. bulgaricus The starter cultures used for most yoghurt production are a mixture of Streptococcus thermophilu s (ST) and Lactobacillus bulgaricus (LB ). Although they can grow independently, they are symbiotic and the growth rate and acid production is better if they are grown together. These bacteria are ultimately responsible for the flavour and aroma of the final product. To achieve this, they produce a number of products, such as: lactic acid, acetic acid, diacetyl, acetoin, and acetaldehyde

42. Mixed cultures are able to bring about multistep transformations that would be impossible for a single microorganism. Examples are the miso and shoyu fermentations in which Aspergillus oryzae strains are used to make koji. Koji produces amylases and proteases, which break down the starch in rice and proteins in soybeans. In the miso and shoyu fermentations, these compounds are then acted on by lactic acid bacteria and yeast to produce flavor compounds and alcohol.

43. Summary of microbial growth curves

44. Refr Suriender Kumar- 31pg Chapter 3- bacterial growth curves

45. Study questions 1. What is the importance of studying microbial growth curves? 2. Looking at a fed-batch culture ,continuous culture and batch culture systems i) draw all the components and discuss their functionality. 3 . Give the advantage/disadvantages of Batch cultures over continuous cultures? 4. How can sterility be maintained in all the reactor systems? 5. Draw a product and substrate curve for the above 4 systems.

46. Questions 1. Draw a table and give 3 advantages and 3 disadvantages of i. Batch culture ii. Fed-batch culture iii. Continuous culture iv. And mixed culture systems. (24) 2. Describe a closed system and an open system. (6) 3. In one graph ,draw a clearly labelled product curve, growth curve and substrate curve of a i. Fed-batch ii. Continuous culture iii.Batch culture systems.