Bioprocess engg. & technology

Experiment 4

Principle

Practical 4 (a)

Fermentation process features cell cultivation in a suitable medium and ideal environmental conditions under aseptic conditions to facilitate higher growth which may lead to increased turnover of substrate to produce important metabolites. It can be classified as anaerobic cultivation of cells in which cells grow strictly in the absence of air particularly using metabolic intermediates as their final electron acceptors in the culture metabolism. Aerobic cultivation of cells, however, features vigorous growth of microorganisms in the presence of oxygen which acts as final electron acceptor. Higher growth of cells is achieved in aerobic cultivation than in anaerobic cultivation. In some cultivation the product of interest is accumulated with-in the cell (intracellular) and eventually the cell wall is raptured to isolate and purify the product. Another variety of cultivation features extracellular product formation in which substrate diffuses in to the cell, reacts with different enzymes to produce metabolites which is then excreted out of the cell in the fermentation broth. In this case cells are removed from the fermentation broth after the cultivation process and thereafter product isolation protocols e.g., extraction, distillation, precipitation etc are employed for harvesting the product from the fermentation broth.

Batch Microbial Cultivation

Batch cultivations are generally employed to grow cells for desired metabolite production as it is simple and involves least requirement of labor and equipments as opposed to other modes of cultivation (Fed-batch/ Continuous cultivations). However these cultivations work as closed system and thereby feature highly dynamic growth conditions and have less yield and productivity of desired product. This is particularly because of high non production time (Cleaning/Sterilization/Cooling etc) & due to significantly long Lag/Stationery phases in this mode of cultivation. Generally the higher activity featuring exponential growth (Balanced growth wherein all components of the cell grow by same proportion) of the culture is observed for only 25-40 % of total cultivation time of batch cultivation wherein the culture produces the metabolites of interest in growth associated fermentation processes. It is, therefore, desirable to study the culture growth and product formation characteristics (Kinetics) in detail and establish whether it is substrate or product inhibited system. If the kinetics turns out to be substrate inhibited (wherein, by increasing the initial substrate concentration in the bioreactor leads to significant decrease in growth) then it is desirable to design a fed-batch cultivation system such that higher initial substrate concentration in the bioreactor is replaced by gradual slow feeding of substrate such that at no point of time it reaches the inhibitory level. This will feature non limiting and non inhibitory nutrient availability cultivation conditions and result in increased product formation. If cultivation of growth in the presence of different product concentrations demonstrates slower growth at higher concentrations (product inhibition) then batch cultivation will not lead to higher rates of product formation as accumulated product will stay in the reactor and inhibit the growth. At significant higher product concentrations it may even stop the growth (and eventually product formation may cease) even under the conditions of high substrate (and associated nutrient) availability situations in the fermentation broth. To eliminate product inhibition growth conditions it is desirable to design either a plug flow or continuous reactor cultivations which features continuous feeding of nutrients and simultaneous withdrawal of fermentation broth along with the inhibitory product. Models are highly instrumental to design suitable reactors and their operation strategies to optimize any fermentation process.

Practical 4 (b)

Fed-batch cultivation

The most simple category of fermentation is batch cultivation where in the substrate is taken in the beginning of cultivation and nothing is added or withdrawn during the fermentation.

However the yield and productivity is lower in these cultivations mainly because, either the substrate &/or product inhibition occurs and the product accumulation in never optimal. Fed-batch cultivation can provide the solution to substrate inhibition problem by slow feeding of nutrients to the bioreactor; however it can still not address the severe inhibition problem due to accumulating high product concentrations. The optimal design of fed-batch cultivation has to take in to account several factors in to consideration for example time to start the fresh nutrient feed (in the end or when the culture is exponentially growing) what should be the substrate concentration in the feed and its rate of addition and when to finish the nutrient feeding so that the highest concentration of product is produced and no unconverted substrate when the reactor is full. It is rather impossible to do trial and error experimentation with so many “open ended” variables (as described above) which may play key role in the overall performance of the fed-batch cultivation.

Different types of Fed-batch cultivation

Following scenario of nutrient feeding can contribute in the elimination of substrate inhibition to yield high productivity of the product.

Add substrate when low

This is the simplest type of fed-batch cultivation where in the fresh feeding of the nutrient is done when substrate has become limiting towards the end of the batch cultivation. At this point of time if no feeding of fresh nutrient is done for some time, the culture dies out. A step input of substrate (predesigned concentration and its rate) is identified by the mathematical model and is administered to the dying culture in the bioreactor which instantaneously raises the concentration of substrate and thereby gives an “installment” of life to the starving culture for few hours which results in product formation also for some more time than the batch cultivation. This cycle can be repeated number of times till the reactor is full of medium. In fact different combinations of substrate concentration, its rate and time of feeding can be chosen and used in the mathematical model to arrive at the best possible cultivation protocol for highly productive fermentation. The best offline simulated protocol can then be taken in to the lab and implemented to optimize the production.

Constant feeding of substrate

Significant Improvement in product concentration and improvement in yield /productivity is possible if the nutrient feeding is done during the exponential phase of the cell growth when the maximum cell population is young and growing. This may be suitably selected by the study of batch kinetics. The mathematical model can then be used to simulate number of possibilities of start /stop time of nutrient feed, substrate concentration, its rate and so on. The simplest feeding profile could be constant feeding of suitably selected nutrient concentration and its pre identified rate such that it does not yield increased concentration of substrate than the initial substrate concentration at any time during the feeding in the bioreactor. Model can very easily facilitate the identification of above specific feeding strategy. The advantage of above strategy is that it is very simple it does not required computer coupled peristaltic pump to implement the feeding strategy. But the disadvantage is that it leads to build up of substrate which need to fermented by yet another batch cultivation so that in the end no unconverted substrate is left in the bioreactor.

Linearly or exponentially increasing nutrient feeding strategy

In this fed-batch cultivation the nutrient feed is linearly or exponentially increased at predetermined time of cultivation. If the feeding rate coincides with the growth rate in the bioreactor by model simulations it may be possible to achieve non limiting non inhibitory concentration of substrate during the feeding period. However after the termination of feeding it may be necessary to do a secondary batch cultivation to consume the residual substrate in the bioreactor.

Decreasing rate nutrient feeding strategy

The disadvantage of above feeding strategy can be overcoming by suitable selection of nutrient feeding strategy where in the feeding starts in exponential phase of fermentation and nutrient feeding rate is so designed that feeding is high when the culture is young and there after the rate is gradually decreased as the culture gets older & is diluted by incoming feed nutrients. It is possible to arrive at a simulation where the feeding rate of nutrient gradually decreases & stops when the reactor is full. In this there will not be any requirement of secondary batch fermentation.

Pseudo steady state of substrate or biomass

It is possible to design the nutrient feeding at a particular substrate concentration so that its concentration is neither high enough to inhibit the fermentation nor it is too low to limit the growth in the bioreactor. To arrive at the feeding profile, which might result the constant substrate concentration in the broth, the first derivative (say ds/dt) is made zero and the model equations (because for S to be constant its first derivative has to be made zero) are rearranged to calculate the corresponding feeding rate of the substrate The feeding of the nutrient thus calculated can then be implemented to achieve pseudo steady state of any variable e.g., S, X etc.

Practical 4 (c)

Basic concepts and need of continuous cultivation

Continuous Cultivation of microorganism

Continuous cultivation of microorganism are open systems which features addition of nutrients at a constant rate and simultaneous with drawl at the same rate. This mode of cultivation is particularly useful as it results in significant improvement in productivity of fermentation. Also it is rather easy to implement process control for these systems. However some disadvantages of this cultivation e.g, development of mutants and contamination free cultivation for longer time limits its common usage. However it is a best tool to study the physiology of cultivation as there is a perfect steady state cultivation condition at a particular dilution rate (= sp. growth rate) in the bioreactor.

Transients in Chemostats

The overall response of any continuous cultivation can be simulated by the mathematical model however it is rather interesting to see the culture behavior in transients in cultivation (Shift up / Shift down in dilution rates) It has been observed that Monod model is unable to perfectly simulate the transients in Continuous cultivations because the model assumes dependence of growth on the instantaneous value of substrate concentration. In shift up of dilution rates (i.e., increasing the feeding rate of nutrient) the metabolism switches from “famine” condition to “feast” condition, meaning suddenly the substrate concentration see a significant change against the nutrient limiting / dying culture and there is no proportionate increase in the cell growth as proposed by Monod’s model. It is therefore necessary to incorporate the “physiological state marker” in the model which can not only quantitatively describe the metabolic reactions of the cells but adequately graduate changes in nutrient limiting and nutrient rich situations in transient conditions of continuous cultivations. The transients are particularly important in microbial cultivations as it may lead to significant increase in the product concentrations which are not available elsewhere. This is particularly important because the culture experiences a major shift in nutrient availability and thereby it leads to quick change over of the culture metabolism.

Wash out condition

In any cultivation it is always necessary to devise strategies which might result in high productivity. Productivity in continuous cultivation is dependent on not only the concentration of biomass /product but also on its dilution rate (Productivity = DP or DX). It is therefore necessary to increase both D and X to increase the productivity of fermentation. However in actual practice, it is observed that at lower dilution rate, unconverted substrate is low & biomass concentration is high (Productivity = High Biomass x Low D) and on the contrary at high dilution rates, unconverted substrate is high & biomass concentration is low. (Productivity = Low Biomass x High D). Therefore in either of the two cases the multiplication of DX results in lower productivity. Also if dilution rate of the bioreactor is further enhanced then it may lead to “wash out” of the biomass from the reactor. The substrate concentration in the reactor will then be equal to the inlet concentration of feed substrate. For optimization of the productivity, suitable Dilution rate (Dmax out put) is identified which when used during cultivation conditions gives best value of steady state biomass accumulation in the bio reactor there by increasing the productivity. It is also possible to improve the process productivity by using cell retention / recycle system which allow build up of biomass in the bioreactor and it becomes possible to operate the reactor at higher dilution rates (D=Feed rate/Volume) with-out wash out.

Materials required

• Sorbitol - commercial grade

• NRRL B-72 strain of Acetobacter suboxydans

• sorbitol

• yeast extract powder

• ammonium dihydrogen phosphate,

• magnesium sulphate,

• agar

• rotary shaker (Adolf Kuhner, Switzerland)

Procedure

Practical 4 (a)

Bioconversion of sorbitol to sorbose –

Bioconversion of sorbitol to sorbose is an intermediate step in the commercial production of L-ascorbic acid (Vitamin-C) (Kulhanek, 1970, Bourdant, 1990). It involves the microbial oxidation of D-sorbitol to L-sorbose byAcetobacter suboxydans. Chemical oxidation of D-sorbitol leads to formation of both the enantiomers of sorbose whereas microbial oxidation produces only L-sorbose and therefore it is necessary to focus on optimization of fermentation for economic production of sorbose.

Microorganism and Maintenance

Sorbitol - commercial grade (70% w/w) is to be used. NRRL B-72 strain of Acetobacter suboxydans facilitates bioconversion of sorbitol to sorbose Cultures are to be maintained on agar slants having the composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; agar 20. The initial pH has to be kept as 6.0. Take samples after 48 h growth at 30º C and preserve the cultures at 4ºC.

Inoculum Development

A loop full of microorganism from the slant has to be transferred into 10 mL medium in test tubes having the composition (g/L): Sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH 6.0. The test tubes are to be incubated at 30º C for 72 h. The bacterial growth is characterized by the appearance of a thick pellicle on the surface of the medium and uniform turbidity. It is then ready for transfer to next stage. The growing inoculums from the test tube is then transferred into 1.0 liter capacity flasks containing 100 mL medium of composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH, 6.0. The flasks are then incubated in a rotary shaker (Adolf Kuhner, Switzerland) at 30º C and 250 rpm. Subsequent transfer of inoculums from the shake flask to the fermenter has to be done when the biomass concentration in shake flask is about 2.8 to 3.0 g/L.

Batch Fermentation

The microbial batch cultivation can be carried out in a 7.0 litre capacity fermenter (Bioengineering A.G., Switzerland) / or equivalent equipped with two sets of flat blade turbine impellers. The working volume can be kept as 4.5 litres. The medium is prepared and filled in the bioreactor. The reactor is autoclaved in-situ and the medium is allowed to cool down to room temperature. The actively growing inoculum is transferred from shake flask to the bioreactor The aeration rate and agitation speed have to be kept at 2.2 vvm and 700 rpm respectively. The temperature is to be maintained at 30º C and the pH at 6.0 by the automatic addition of 3 N NaOH and 3 N HCl. Intermittant samples are taken to assess the biomass concentration, unconverted substrate (sorbitol) and product (sorbose).

Video on microbial cultivation for batch fermentation: http://209.211.220.205/model/bmc/animation.html.

Link for stimulation: http://209.211.220.205/model/bmc/simulator.html

practical 4 (b).

Fed-batch Cultivation

A number of fed-batch cultivation can be designed using the mathematical model. As has been indicated, fed-batch cultivation features availability of disappearing nutrients particularly during exponentially growing phase of the culture. The batch cultivation is allowed to proceed till it reaches the exponential growth phase and then a number of feeding strategies (constant feed, linearly increasing / decreasing feed rate, exponential feed rates) can be simulated off line and selected few (which give high product concentration and productivity) can be shortlisted for experimental validation. The advantages/disadvantages of adoption of one nutrient feeding strategy over other has been described in detail in the theory section of the experiment.

To execute a fed-batch cultivation, start the cultivation as batch with an initial sorbitol concentration of 100 g/L. The concentration (g/L) of other nutrients have to be kept as; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0 and magnesium sulphate, 1.0. Sorbitol concentration in the feed bottle has to be kept as high as possible so that it gives minimum dilution to the fermentation broth in the bioreactor and the contents of the reactor are not excessively diluted by the incoming feed. (Usually 600 g/L sorbitol can be used in the feed bottle). The rest of the medium components have to be increased in same proportion so that they are not limiting during the cultivation. The fed-batch cultivation has to be stopped when all the sorbitol in the bioreactor is totally consumed as unconverted substrate (sorbitol) is a loss to the company and also it give rise to problems in recovery of product (sorbose). The completion of sorbitol (end of the fermentation) is reflected by increased DO signal in the bioreactor.

Video on microbial cultivation for fed-batch fermentation: http://209.211.220.205/model/fbmc/animation.html

Link for stimulation of fed-batch fermentation: http://209.211.220.205/model/fbmc/simulator.html

practical 4 (c)

Microorganism and Maintenance

Sorbitol - commercial grade (70% w/w) is to be used. NRRL B-72 strain of Acetobacter suboxydans facilitates bioconversion of sorbitol to sorbose Cultures are to be maintained on agar slants having the composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; agar 20. The initial pH has to be kept as 6.0. Take samples after 48 h growth at 30º C and preserve the cultures at 40ºC.

Inoculum Development

A loop full of microorganism from the slant has to be transferred into 10 mL medium in test tubes having the composition (g/L): Sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH 6.0. The test tubes are to be incubated at 30º C for 72 h. The bacterial growth is characterized by the appearance of a thick pellicle on the surface of the medium and uniform turbidity. It is then ready for transfer to next stage. The growing inoculums from the test tube is then transferred into 1.0 liter capacity flasks containing 100 mL medium of composition (g/L): sorbitol, 5.0; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0; magnesium sulphate, 1.0; pH, 6.0. The flasks are then incubated in a rotary shaker (Adolf Kuhner, Switzerland) at 30º C and 250 rpm. Subsequent transfer of inoculums from the shake flask to the fermenter has to be done when the biomass concentration in shake flask is about 2.8 to 3.0 g/L.

Batch Fermentation

The microbial batch cultivation can be carried out in a 7.0 litre capacity fermenter (Bioengineering A.G., Switzerland) / or equivalent equipped with two sets of flat blade turbine impellers. The working volume can be kept as 4.5 litres. The medium is prepared and filled in the bioreactor. The reactor is autoclaved in-situ and the medium is allowed to cool down to room temperature. The actively growing inoculum is transferred from shake flask to the bioreactor. The aeration rate and agitation speed have to be kept at 2.2 vvm and 700 rpm respectively. The temperature is to be maintained at 30º C and the pH at 6.0 by the automatic addition of 3 N NaOH and 3 N HCl. Intermittant samples are taken to assess the biomass, sorbitol and sorbose concentrations.

Continuous Cultivation

The continuous cultivation is initiated as a batch with an initial sorbitol concentration of 100 g/L. The concentration (g/L) of other nutrients have to be kept as; yeast extract powder, 5.0; ammonium dihydrogen phosphate, 3.0 and magnesium sulphate, 1.0.. After reaching the exponential growth phase of the culture feeding of fresh of substrate and nutrient is initiated. The selection of particular dilution rate of continuous cultivation is highly dependent on what is the objective of cultivation. For example if low biomass concentration and high growth rates are required under continuous cultivation condition then it would be desirable to operate the reactor at D= F/V= µ (where µ is the specific growth rate during the exponential growth phase). On the contrary if high biomass concentration and low growth rates are required under continuous cultivation conditions, then it would be advisable to operate the reactor at D= F/V =µ (where µ is the specific growth rate during the stationery phase of the cultivation). After selection of a particular dilution rate the feed rate is calculated as D = Feed rate / Volume of the reactor. The feeding of fresh nutrient is initiated at the desired feed rate in the reactor & at the same time the fermentation broth is withdrawn at a rate equal to fresh feed rate. The feeding is continued in the reactor till steady state (i.e., passage of at least three reactor volumes is completed) with respect to different process variables (Biomass, Sorbitol and sorbose) is achieved in the bioreactor.

Continuous fermentations are highly labor intensive and therefore mathematical model simulation can be highly useful to guide “the” specific cultivation which will feature high product concentration and productivity. In fact number of simulations can be done to figure out the maximum possible productivity of biomass &/or product in the bioreactor. The selected few simulations can then be implemented in the bioreactor.

Video on microbial cultivation for continuous fermentation: http://209.211.220.205/model/cmc/animation.html

Link for stimulation of continuous fermentation process: http://209.211.220.205/model/cmc/simulator.html

Result

Results showed that different factors affects production of secondary metabolite during large scale fermentation.

Conclusion

By performing practical 4, we can optimize best fermentation to be used for microbial cultivation and for bulk production of biotechnological products.

Reference Material

Video on microbial cultivation for batch fermentation: http://209.211.220.205/model/bmc/animation.html

Link for stimulation of batch fermentation: http://209.211.220.205/model/bmc/simulator.html

Video on microbial cultivation for fed-batch fermentation: Vhttp://209.211.220.205/model/fbmc/animation.html

Link for stimulation of fed-batch fermentation: http://209.211.220.205/model/fbmc/simulator.html

Video on microbial cultivation for continuous fermentation: http://209.211.220.205/model/cmc/animation.html

Link for stimulation of continuous fermentation process: http://209.211.220.205/model/cmc/simulator.html

Questions

1. What is batch fermentation?

2. what is the difference between fed batch and batch fermentation?

3. which method is best among batch, fed batch and continuous fermentation?