Experiment 3-5
Design a continuous bioreactor and the growth kinetic of Zymomonas mobilis in continuous culture.

Submitted to
Dr. Tatsaporn Todhanakasem
Faculty of Biotechnology
Assumption University

In partial fulfillment of the requirement of the course BT3014 Microbial Physiology

by
Syed Zia Nayeem 5045215

Date of experiment: 25 June, 2012

Introduction:
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.
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.
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 buildup 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.
In continuous fermentation, an open system is set up. Sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system. Two basic types of continuous fermentations can be distinguished:
Plug Flow Reactor - In this type of continuous fermentation, the culture solution flows through a tubular reactor without back mixing. The composition of the nutrient solution, the number of cells, mass transfer, and productivity vary at different locations within the system. At the entrance to the reactor, cells must be continuously added along with the nutrient solution.
In a continuous process under steady state conditions, cell loss as a result of outflow must be balanced by outgrowth of the organism.

Materials and methods: * 2 x 500ml Autoclaved Conical flasks * Sterile plastic tubing * Sterile Corks and Sartorius air pressure controller * 1 liter sterile bottle (Autoclaved) * Magnetic mixer and magnet bar * Micropipette and cuvette to collect sample for OD determination trials. * Paraffin film (for mouth sealings)
Procedure:
For Experiment 3: Design a continuous bioreactor i. Design my own continuous bioreactor by using available laboratory apparatuses. ii. Referring to the maximum specific growth rate of Z.mobilis in any media of the last experiment, calculate the optimum dilution rate for Z.mobilis continuous culture. iii. Assemble the continuous bioreactor for next part.
For Experiment 4 & 5: The growth kinetic of Zymomonas mobilis in continuous culture. (2 days experiment) i. Fill in the culture vessel and feed vessel with sterilized medium. (LB medium) ii. Inoculate 1% of Z.mobilis overnight culture into the culture vessel. iii. The culture will be grown in the batch culture mode until the OD reach the µm then switch to manipulate in continuous mode (D= µm). iv. The optical density (OD600) will be measured every 30 minutes in the beginning of the continuous culture mode and every 1 hour later on. v. Draw the graph of the cell density versus time (OD600 vs. time) vi. Discuss on the growth kinetic of the bacteria.

Results:

Flow rate of the gravity continuous bioreactor = 246 ml/hr or 2ml/min
Discussion:
After performing this experiment, the growth kinetic of Z.mobilis in continuous culture were analyzed by recording the optical density at 600nm at fixed time intervals of 30 minutes in the beginning and 1 hour later to inspect constant optical density to achieve an efficient continuous bioreactor system. It was observed from the results obtained it was determined that the continuous culture of Z.mobilis reached maximum lag phase or µm after 3 hours. The flow rate of fresh medium was calculated to be 2ml/min from the dilution rate formula (D=F/V) by taking µm=0.41 and volume of flask = 600m. After the OD reached its recorded peak of 0.711 at the 3rd hour of the experiment, the flow rate was regularly regulated manually by rotating knob on pipe (as we used gravity-based feeding system) to keep the cell activity in the continuous culture constant in terms of cell mass in flask also. This step is vital to run an efficient continuous bioreactor to achieve maximum yield of products. In case of this experiment the product from the fermentation process is ethanol.
Conclusion:
By doing this experiment, it can be concluded that a continuous bioreactor can be constructed using laboratory apparatuses maintaining strict aseptic techniques. The optical density can be recorded to regulate the constant conditions inside a continuous bioreactor. The feed in should be equal to the product formed to achieve maximum efficiency.
References:
http://www.ncbi.nlm.nih.gov/pubmed/18576479 http://www.gbanalysts.com/Reading%20Room/Situation%20Analysis/BiologicalTechs/fermentorsheteroflowbioreactors.html http://www.ncbi.nlm.nih.gov/pubmed/17596937

Appendix: Time(hr) | OD 600 nm ( Gravity continuous bioreactor) | 1 | 0.247 | 2 | 0.448 | 3 | 0.711 | 4 | 0.589 | 5 | 0.670 |

Ideal dilution rate: d = µ max= 0.41 Cell density from previous experiment V= 600 ml for the bioreactor flask D x V = 0.41 x 600 = 246 ml/ hr or 2ml/ min