Fermentation design & types

Fermentation technology

1. Fermentation - Design & Types Mr. Dilip O. Morani. Asst. Prof., Shri D. D. Vispute College of Pharmacy and Research Center, Panvel .

2. Ferment a tion Fermentation is the process of growing microorganisms in a nutrient media by maintaining physico- chemical conditions and thereby converting feed into a desired end product Fermentation technology is the use of org a nis m s to pharmaceuticals produ c e and food, al c oh o lic beve r a g es on a large s cale in d ust r ial basis.

3. Major fermentation products Group Product Organism Industrial ch e m i c a l s Ethanol Lactic acid Saccharomyces cerevisiae Lactobacillus bulgaricus Enzymes  -amylase Proteases Lipases Bacillus subtilis Bacillus species Saccharomyces lipolytica Antibiotics Penicillin Streptomycin Chlorampenicol Penicillium chrysogenum Streptomyces griseus Streptomyces venezuelae Vitamins Riboflavin Vitamin B12 Ashbya gossypi Pseudomonas dentrificians

4. DESIGN OF FERMENTER A fermentation process requires a fermenter for successful production . Fermentor is the large vessel containing considerable quantities of nutrient media by maintaining favourable conditions. The design and nature of the fermentor varies depending upon the type of fermentation carried out. Invariably all the fermentors provide the following facilities for the process such as contamination free environment, specific temperature maintenance, maintenance of agitation and aeration, pH control, monitoring Dissolved Oxygen (DO), ports for nutrient and reagent feeding (antifoam agents, alkali or acid), ports for inoculation and sampling, provide all aseptic conditions at the time of sample withdrawal and addition of innoculum complete removal of broth from the tank and should be easy to clean It should be designed in such away that it consumes less power, have less evaporation, can be used for long periods of operation

5. FigI. An Ideal fermenter DESIGN OF FERMENTER

6. Types of fermenter Available in various sizes According to the sizes classified as Small lab and research fermenter :1-50L Pilot plant fermenter: 50-1000 L Large size industrial production scale fermenter: more than 1000 L Broadly fermentes are also claified as surface fermenters Tray fermenter Packed bed column fermenter Submerged fermenters Simple fermenters (batch and continuous) Fed batch fermenter Air-lift Bubble fermenter Cyclone column fermenter Tower fermenter Other more advanced systems, etc

7. Types of fermenter Surface fermenters Microbial cells cultured on surface layer of the nutrient medium (solid/liquid) held in dish or tray Aspergillus niger and nicotinic Used for produc t ion of ci t ric acid acid from f r om Aspergillus terrus Microbial films can be developed on the surfaces of suitable packing medium, may be in the form of fixed bed, stones or plastic sheets

8. Tray fermenter TRAY FERMENTER one of the simplest and widely used fermenters. Its basic part is a wooden, metal, or plastic tray, often with a perforated or wire mesh bottom to improve air circulation. A sha l l ow la y er of less t h a n . 15 m dee p, p re t r e a t e d substrate is placed on the tray for fermentation. T e m pe r at ur e an d hu m idit y - c o nt r ol le d c ha m be rs a re u se d for keeping the individual trays or stacks. A spaci n g of at l east o ne t r a y h ei g ht is u su ally all owe d between stacked trays. Ch e e s eclo t h m ay b e u s ed t o c o v er t h e t r a ys t o r edu ce contamination. Ino c ula tio n a nd o c c a si on al mi x in g a r e d on e man u a ll y , often by hand.

9. Tray fermenter Solid as well as liquid medium are used If liquid medium, cells are allowed to float easily and to make a process continuous If solid medium is used the mic ro -o r g an i sms are allowed grow on moist solid materials, process is called Solid State Fermentation Method (SSF).

10. Solid State Fermentation (SSF) Solid State Fermentation Method (SSF) SSF defined as the growth of the micro-organisms on (moist) solid material in the absence or near-absence of free water Used for production of antibiotics, enzymes, alkaloids, organic acids bio-pharmaceutical products y i elds th a n su b mer g ed liqu i d Advantages : Pro d uce higher fermentation Possibilities of contamination by bacteria and yeast is very less All natural habitats of fungi are easily maintained in SSF culture media very simple , provides all nutrients for growth of micro-organisms Simple design of fermenter and low energy

11. SSF Disadvantages: Causes problems in monitoring of the process parameters such as pH, moisture content, and oxygen concentration Despite some automation, tray fermenters are labor intensive Di f ficulties w i th pr o cessing hu n dr e ds o f t r ays lim it their scalability Aeration may be difficult due to high level of solid content S u bstrates requ i r e pre t r eat m ent such a s size reductio n, chemical or enzymatic hydrolyses

12. Submerged fermenters The microorganisms are dispersed in liquid nutrient medium at maintained environmental conditions. on the mechanism of agitation Submerged fermenters grouped as follows: Mechanically stirred fermenter batch operate fermenter continuous stirred tank fermenter Forced convection fermenters Air –lift fermenter Bubble column Sparged tank fermenter Pneumatic fermenter Fluidized bed reactor

13. These are equipped with a mechanical agitator so as to maintain homogencity and rapid dispersion and mixing of materials stirred tank Examples includes fermenter (bat c h or continuous operated) , multistage fermenter, paddle wheel reactor, and stirred loop reactor Mechanically stirred fermenter

14. Stirred tank fermenter (STF) stirred tank fermenter b a tch o p erated fermenter agitators consists of one or more impellers mounted on the shaft It is rotates with the help of electric motor of this flexibility in Ad v antage fermenter design Used i n t he ran g e o f 1- 100 ton capacity sizes Stirred tank fermenter

15. A continuous stirred tank fermenter consists of a cylindrical vessel with motor driven central shaft that supports one or more agitators (impellers). The shaft is fitted at the top of the bioreactor (ref. fig.). The number of impellers is variable and depends on the si z e of the f erm e nter Continuous stirred tank fermenter (CSTF) Continuous stirred tank fermenter

16. Continuous stirred tank fermenter I n this fresh medium is added continuously in the fermenter vessel On the other end the medium is withdrawn for the recovery of fermentation products As it is a continuous fermenter the Steady state conditions can be achieved by either Chemostatic or Turbidostatic principles.

17. Continuous stirred tank fermenter(CSTF ) Different types of continuous fermenter are Single stage: single fermenter is inoculated and kept in continuous operation by balancing the input and output culture media Recycle continuous fermentation: a re s idual unus e d substrate plus portion o f the withd r awn culture or the withdrawn culture is recycled

18. CSTF c. Multistage c o nt i n u o u s operation: involves two or more stages with the fermenter being operated in sequence multistage

19. STF Advantages of batch operated Less risk of contamination because of short growth period Process is more economical and simple Raw material conversion level is high Disadvantages: Low productivity due to time required fro the sterilizing, filling, cooling, emptying and cleaning s u b c u l tu r es for i n o c u l at i o n, l a b o r Mo r e expense s ar e r e q u ir e d for and process control

20. STF Advantages of continuous operated Less labor expenses due to automation of fermentation process Less toxicity risk to operator by toxins producing microorganisms High yield and good quality product due invariable operating parameters and automation of the process Less stress on the fermenter as sterilization is not frequent Disadvantages: Higher investment costs in control and automation equipment More risk of contamination and cell mutation

21. Air lift fermenter Airlift fermenter (ALF) is generally classified as forced convection fermenters without any mechanical stirring arrangements for mixing. The turbulence caused by the fluid (air/gas) flow ensures adequate mixing of the liquid. The baffle or draft tube is provided in the reactor. A baffle or draft tube divides the fluid volume of the vessel into 2 inter-connected zones. Only one of the 2 zones is sparged with air or other gas. The sparged zone is known as " riser", the zone that receives no gas is "downcomer“. Air lift fermenter

22. Air lift fermenter Mainly 2 types Internal-loop airlift bioreactor (ref Fig) has a single container with a central draft tube that creates interior liquid circulation channels. These bioreactors are simple in design, with volume and circulation at a fixed rate for fermentation. External loop airlift bioreactor (ref fig) possesses an external loop so that the liquid circulates through in d e p e n d e nt sepa r ate cha n n els. Th e se r e act o r s can be suitably modified to suit the requirements of different fermentations. Internal loop External loop

23. Air lift fermenter Advantages The airlift bioreactors are more efficient than bubble columns, particularly for more denser suspensions of microorganisms as the mixing of the contents is better compared to bubble columns. Commonly employed for aerobic bioprocessing technology. They ensure a controlled liquid flow in a recycle system by pumping. Due to high efficiency, airlift bioreactors are sometimes preferred e.g., methanol production, waste water treatment, single-cell protein production

24. There are two different process of fermentation viz.: (1) Batch fermentation (2) Continuous culture. Batch fermentation: Nutrients are added in the fermentation for the single time only and growth continues until the particular nutrients are exhausted

25. In the batch process when the microorganism is added into a medium which supports its growth, the culture passes through number of stages known as ‘growth curve’ A typical growth curve consists of following stages Lag phase Acceleration phase Log or exponential phase Deceleration phase Stationary phase Death phase

26. (a) Lag phase: Immediately after inoculation, there is no increase in the numbers of the microbial cells for some time and this period is called lag phase. In this is phase the organisms adjust to the new environment in which it is inoculated into. (b) Acceleration phase: The period when the cells just start increasing in numbers is known as acceleration phase. (c) Log phase: This is the time period when the cell numbers steadily increase. (d) Deceleration phase: The duration when the steady growth declines.

27. (e) Stationary phase: The period where there is no change in the microbial cell number is the stationary phase. This phase is attained due to depletion of carbon source or accumulation of the end products. (f) Death phase: The period in which the cell numbers decrease steadily is the death phase. This is due to death of the cells because of cessation of metabolic activity and depletion of energy resources. Depending upon the product required the different phases of the cell growth are maintained. For microbial mass the log phase is preferred. For production of secondary metabolites i.e. antibiotics, the stationary phase is preferred.

28. Growth kinetics of batch culture The number of living cells (population of growth rate dN/dt)varies with time in a batch system as shown below:

29. w h ere; LAG Phase: Number of bacteria does not change with time in lag phase. LOG Phase: Number of bacteria increases exponentially in log phase.

30. During log phase the number of organisms in the reactor at any time t can be calculated, by using rate equation shown bel ow:

31. According to last equation, number of bacteria in the reactor at any time t during log phase can be calculated, as it is seen in the graph. This rate equation can be integrated:

32. STATIONARY Phase: There is no net change in number of bacteria with time in stationary phase. Bacteria divide but also die at equal rate. Most of the important biological products (especially secondary metabolites like antibiotics) or biomass are produced during this phase. The biomass concentration at stationary phase is determined by following equation X = Y. S R X=cell concentration Y= yield factor for limiting nutrient S R = original nutrient concentration in the medium

33. Y’ measures the efficiency of a cell in converting nutrients into biomass So the biomass at a particular time in the during the fermentation is given by the following equation. X = Y (S R - s) S= nutrient concentration at particular time thus ‘Y’ is represented by the following equation Y = X/ (S R - s)

34. Continuous operations Continuous fermentation: The growth rate and physiological conditions of microorganisms can be maintained by using a process of continuous culture (chemostat ) In this the products are removed continuously along with the cells and the same is replenished with the cell girth and addition of fresh culture media. This results in a steady or constant volume of the contents of the fermenter. This type of fermentation is used for the production of single cell protein (S.S.P), antibiotics and organic solvents.

35. CONTINUOUS fermentation process The dilution rate is the ratio of inflowing amount of medium to the volume of the culture. Thus D = F / V D= dilution rate F= Flow rate V=Volume

36. The change in cell concentration of cells at perticular time period is expressed by the following equation dx/dt= growth rate – output Or dx/dt = μ x - Dx In the process of continuous culture technique the output is balanced by growth hence, μ x = Dx μ – D Dx / dt= D

37. The biomass concentration in the chemostat is determined by the following equation X = Y(S R - s) X= steady state concentration S= steady state residual concentration in the medium

38. Advantages and disadvantages of batch and continuous operations BATCH SYSTEMS easy to operate and control genetic stability of organism could be controlled if it is genetically engineered biocatalyst. lower contamination risk non-productive down time is a disadvantage batch to batch variability is problem accumulation of inhibitory products is problem CONTINUOUS SYSTEMS degeneration of biocatalyst higher contamination risk is a disadvantage efficient, higher productivity product is obtained with uniform characteristics; quality of the product is almost same from time to time no accumulation of inhibitory products.