Presence of heavy metals

Thank you Navindu Gupta.

The problem may be in sampling, blank or control. Is your medium contain any oil as one of the ingredients? If so, is that emulsified?

For lipase production I assume you're using an oil phase as (part of) your feedstock. As soon as you have a dispersed phase, O.D. won't be reliable for cell concentration measurements. Also, the properties of your dispersion (e.g. droplet size) are likely to be affected by scale up (e.g. due to differences in power input).

For production of lipase in fermenter, the best way of measuring biomass is dry weight method.

I would like to add another option, collect the sample remove the oil phase by chloroform then take OD

Karthikumar Sankar-The media I use is YPD media to grow Candida antarctica. And I use Olive oil as an Inducer. The concentration being 0.01ml. Do you think that such a small concentration can effect the O.D as a whole?

Maria C. Cuellar- The media is not an oil phase Maria. Had it been an oil based feedstock your solution might have been apt. YPD media is homogenous and water soluble. The only oil based substrate is the Inducer.

Selvaraju Sivamani-Yes sir, indeed. But I need to use this method in order to validate a previous experiment.

Oils may reduce superficial tension and for this they are also used as anti-foams but decreasing kLa. However, the amount you use (0.01 mL/3L ?) is too low so the problem may be other. If the OD were decreasing with time the reason could be the increase of biomass thus increasing the demand for oxygen and this could be solved by increasing the impeller speed and/or air pressure. But as you say that OD is fluctuating I guess the problem is OD probe calibration or the OD is broken. Also, if the impeller speed is low there is not a well-mixed system and OD values would be fluctuating.

Marcel Gutiérrez-Correa- A very thought provoking Idea, Sir. I am sure that OD measurement cannot be wrong as we've just calibrated the Spectrophotometer and it is state-of-the-art. I will address the impeller speed and air pressure. Thank you, Sir. 

Kindly follow the dry biomass  weight method as said by selvaraju sivamani. check the DO of the broth during fermentation.

saravanan.c Kalki-Thank you, sir. Certainly.

Revathi, try doing CFU and even if that fails then it may be a real concern. Sometimes OD at 600 nm may give you erroneous or misleading results.

Deepti- Yes, I will have to do it if the problem persists. I am skeptical towards the fact that OD measurement might be wrong because this is the second time i am observing this decreasing-increasing-decreasing trend in the same culture.

Revathi, can you give more information about “decreasing-increasing-decreasing trend“ and period of measuring of OD? For instance time of period of the changeover, is it standard? What does mean increasing decreasing in % of average OD? Have you try to look in to microscope? Is the morphology of strain standard? Isn’t there any specific contamination? What about correlation with other parameters of cultivation, for instance fluctuation in air concentration? Alternation of aerobic and anaerobic phase would be explanation. In aerobic phase Candida growth to such concentration that oxygen starts limiting and culture is in anaerobic phase and Candida died (with cells lyse and OD is decreasing) until concentration of oxygen starts to growth and Candida also until….. Try to change aeration. Periodical growth is possible generally.

autolysis at the end of fermentation.

In fact there are several reasons for this decline in absorbance valuesirst of all the cells might be separating off from medium due to aggregation or spore formation. Secondly, there could be lysis of the cells during late stationary phase. There could be a decline in the opacity of the medium itself due to again, several reasons. 

The opinion of Dr. Erban is very reasonable and it may explain the OD fluctuation. As I told before you may control OD by increasing impeller speed and/or air presure. I don't know which type of bioreactor you have but in some models the sowtware can do this control automatically when OD goes down a set point the speed is increased and viceversa. If yours doesn't have you have to do it nanually.

It appears that you recently changed the fermenter. Are the monitor and control systems working well for pH, oxygen, nutrients? Look for any residual inhibitors from previous runs. Is it inconsistency of od among various runs or do you see drastic od declines within a run? Watch out for phage infection as well.

Vladimír Erban- Sir, the OD was take for every 2 hours. The OD increase or decrease hasn't shown a definite trend. So the relative increase or decrease w.r.t % average OD wasn't calculated. Do you suggest we do it in order to have further insight on the problem? We haven't done the microscopic examination of the cells either. Cross contamination may have occurred so we are in the process of procuring a new culture.of the same strain. I will also look into the aerobic-anerobic phase change in the fermentor, Sir. I'll also check for O2 levels intermittently.

Chuang Xue - Madam,. Please elaborate.

Jai Ghosh - Sir, even if there is cell lysis then how will it explain the decrease in OD? Stationary OD or increasing OD, I understand. But DECREASING OD?

Sarita Chauhan- Madam, The control systems have been tested and were found to be working optimally. The pH is maintained at 9.0 and there is no drastic pH change observed during the fermentation though. I am skeptical about O2 levels as the DO electrode was damaged during the last fermentation and we were not able to monitor the DO levels.

May be cell lysis

Revathi, you needn’t exactly calculate OD. It suffices roughly initial OD, around maximal OD and around minimal OD. It helps to decide among possibilities of explanation of problem. When OD falls near the initial OD it may mean that it is question of selection of resistant fraction after phage infection as suggest Sarita Chauhan (microscopic picture can testify it) or spore formation as suggest Jai Ghosh when difference between initial a minimal value OD will not be very big (microscopic picture can confirm it). Next greater difference may be in relation cell lyses due to oxygen concentration as wrote Marcel Gutiérrez-Correa. Oxygen concentration or pH change or glucose concentration or CFU will help you confirm it. Small difference between OD max and OD min will speak for aggregation formation on opinion of Jai Ghosh. Different shapes curves of oxygen concentration or pH change or glucose concentration or CFU will be found.

Microscopic picture, oxygen concentration, concentration of nutrients, pH, CFU and so on helps you in deciding among possibilities. Individual parameters you can compare to individual colours of the rainbow. As more parameters you have, better rainbow (your understanding) you will have met.

Can somebody explain me how cell lysis can contribute to the decrease of OD?

It's easy: Bible OLD TESTAMENT Genesis 3:19 You will eat bread by the sweat of your brow until you return to the ground, since you were taken from it. For you are dust, and you will return to dust." Candida makes the same. OD of dust is negligible.

Is possible that candida increased the viscosity of fermentation medium due to production or release of biopolymers.

Vladimír Erban- Thank you so much Sir. Your explanations taught me great deal! I will check out the possible causes using the OD values I've obtained so far. 

César Augusto Piedrahíta Aguirre- We haven't seen any biopolymer production in the broth so far.

It would be worthy to control the dissolved oxygen concentration at the (aerobic) fermenter, what can be done having air flow and/or agitation speed as manipulated variable(s). The classic PID control algorithm can be used, but improved performance can be achieved trough adaptive control. I have investigated this approach, which compared favourably to PID, by applying the recursive least squares algorithm with forgetting factor to estimate (on-line) the parameters from the usual KLa correlation; Cf: C.M.G.A. Queirós, "Controlo do Oxigénio Dissolvido em Fermentadores para Minimização de Energia Consumida", MSc Thesis, Instituto Superior Técnico, Universidade Técnica de Lisboa, 1997 (in Portuguese):

Thesis Controlo do Oxigénio Dissolvido em Fermentadores para Minimi...

Carlos Araújo Queiroz- Yes, indeed. Thank you for sharing your work, Sir.

Revathi, I also carried out some research establishing proper growh conditions for the yeast genus Candida. I can sahre with you my thesis if you wish, though it is written in polish. As my tests showed pH value may be of some importance when growing Candida cells. Although I'm not sure, whether the same thing happens for all the species.As for the answer to your question about the cause of lower OD value - I completely go along with Vladimir Erban, dead cells won't contribute much to the OD value. 

Szymon Strnad- Thank you for offering to share your thesis. Unfortunately I don't know polish. I will be performing the same experiment soon keeping the above mentioned points in mind. Hoping for a smooth run.