Dear Reeta,

I do not know your temperature of fermentation but to increase oxygen transfer in the medium you can play on temperature, perhaps reduce temperature won't affect so much your productivity. Then, be carefull with your salinity in the medium, if you can play on it you can increase your oxygen solubility. These points can be replicated at the industrial level. To finish just for laugh, an other "idea" is to put microalgae in co-culture with your filamentous fungi :)

Dear Reeta, 

I have no experience whith that, sorry. But this is curious and I will follow the discussion.

You already tried, as I understand, to add pure oxygen to enrich the air, and cannot use a higher pressure... that should work. 

What do you mean by oxygen vector? And what is your carbon source, could you use a more oxidized carbon source? Is it liquid or solid fermentation?

Regards, 

Júlio

Dear Prof Singhania.........Have you tried addition of auxiliary liquids such as n-dodecane in the broth?

Dear Reeta

Filamentous fungi are known for their  higher viscosity creating problems for mass transfer. When the rate of growth is very fast it is further difficult to cope up the oxygen supply. You can try:

 1. Pellet type growth of fungi which will reduce the problems as the viscosity will be low further it may help in the down stream processing also .

2. Control growth by optimizing the media . The control on growth may help in viscosity of the broth & supply of oxygen.

Again it is difficult to answer your question. Only thing I can say is that oxygen vectors will not work either, May be:

1. you need to reduce the biomass of the fungi significantly  OR

2. you need to look at the viscosity of the medium

Carvalho, its liquid fermentation, carbon source is usually avicel (cellulose) and wheat bran. By oxygen vector I mean some solvent or component having capacity to bind oxygen with it while remaining in solution. So if add that in my medium probably it might help in increasing or maintaining DO but do not know about its effect on fermentation.

Thank you Jeremy for your great Idea...it will be fun for fungi and algae together! give and take relationship!

Thank you Mr Tandon, I have got pelleted growth this time and is working well till now but still DO is not maintained.

Dear Reeta Rani,

I have no experience with that either. But this is curious for me, too.

I am sure you did it but I have to ask anyway. Are you sure your oxygen sensor works well? Is it full with ionization liquid? Did you perform calibration and polarization of the O2 probe? Because it is interesting that even O2-enriched-gas-feeding did not work. It just occured to me that there must be something wrong with the measuring system.

Dear Deniz,

Yes I check the probe always for ionization liquid before setting the fermenter for autoclave and calibrate it also. In the beginning it shows 100% DO value but as I said later it drops as soon as the the fungi grows as it is very aerobic.

Dear Reeta,

I have experience working in 2-phase fermentation systems using bacteria, where the organic phase act as oxygen vector, although we don't use it for that purpose. These points could be of help:

- Always calibrate dissolve oxygen probes in the presence of the organic solvent.

- Check for the chemical compatibility between the probe membrane and the solvent (the same goes for O-rings if you want to prevent pressure leakages or risk of contamination).

- Measure kLa to determine the effect of: different concentration of the solvent, different viscosity of the fermentation broth, normal parameters as stirring rate, airflow, volume.

- Consider that if you don't stir your fermentor, you may have not enough mixing, which could lead to phase separation, diminishing the effect of the oxygen vector in kLa.

If you have filamentous fungi in your fermentation, in addition to the listed controlling factors you can use feeding hydrogen peroxide solution with a small feeding rate. We used such a  method in penicillin fermentation.                                                                        By the way, in filamentous fermentation the dissolved oxygen is not a parameter, which  can guarantee the provision of microorganisms by oxygen. Even 100% saturation may be not enough.  We use "the method of maximal respiration rate" for the control of process.

Dear Reeta,

as fungi fermentations become more viscous during cultivation, can you ensure that do not have concentration gradients between near impeller zone and electrode?

Thank you Biryukov for your important answer! I hope this will be of help as I am going to try this next. 

Bruns, we cannot control viscosity near impeller and probe but I am sure this not a problem of electrode sensitivity.

DO can go low i.e. greater 20% saturation is normally acceptable.

The problem can be approached from two angles.

On the supply side where increased mass transfer can be improved by

1.increases pressure (i.e depth) in the reactor, raising partial pressure of oxygen

2. changing aeration scheme where airlift reactors may be a better approach to agitate non-newtonian fluids like fungal biomass

3. changing impeller arrangement

On the demand side

1. reduce metabolic activity, (lower temp, restricted feed of nutrient, i.e. fed batch approach)

regards

bob

Thank you Lovitt! for your useful reply. We have changed rushton impeller to marine impeller and it works better but the problem is not solved completely. I cant change to airlift reactors but shall definitely work on reducing metabolic activity.

Hi Reeta !

I know two oxygen "vectors" used in industrial scale:

1) oxygen itself to a filamentous fingi fermentation suppleid as liquid O2 and then evaporated to gas

2) hydrogen peroxide i a yeast fermentation.

The latter may not be "universal" solution.

The exact numbers I dont have for either.

Kindly, Ilkka

Dear Reeta,

Your problem could arise from:

(1) an issue with the electrode

(2) inefficient oxygen transfer to the cells

The proper maintenance of the DO electrode is essential. Try cleaning the electrode as described in the user's manual. If you have fermentors, which are capable of in-situ sterilization, you can test the calibration of the electrodes by checking, if the pO2 value is at 0% when the sterilization cycle is running (there is no dissolved O2 present at 121°C). Alternatively you can submerge the electrode to a flask with a narrow opening and flush it with nitrogen. Please also check the raw signal, if you are using e.g. a Mettler Toledo InPro 6800 amperometric sensor, the signal should be between 50-110 nA. This values varies for different models, check the manual. You can also consider using a more advanced optometric sensor.

If the sensor is okay, then you need to increase the oxygen transfer in your fermentor. I would not try to slow the metabolism of the fungi, as I believe you are trying to produce something with it. Slower metabolism would mean less or no product. Increasing the overpressure of your fermentor to 0.5 or 1 bar would increase the partial pressure of oxygen and help with the O2 transfer. However not all fermentors have digital pressure controllers, to achieve this.

Many fungi strains are resistant for the sheer force of stirring, it is possible that the increased stirring wouldn't effect the growth and production at all. Try starting with a low 200 rpm (for a bench scale fermentor) and use the DO controller to maintain a 20% DO level, by gradually increasing the RPM. Set a maximum RPM to 800 (or about 2/3 of the theoretical maximum). Check the specifications of the electromotor for exact numbers.

I hope this helps!

Cheers,

Andras

Thank you Ekka your suggestions are useful!

Hi Reeta

I have used n-dodecane and silicone oil as oxygen-vectors in cultures of filamentous fungi. The preliminary results, not yet published, indicate an increase in the values of kLa and I was able to control the dissolved oxygen  above the critical value, when combined with other strategies, as mentioned in answers above (impeller arrangement, growth limiting conditions, etc.).

Kindly

Lenara

Immiscible liquids are often added to enhance the oxygen solubility in fermentation broth. Normally hydrocarbons and other liquids where solubility is oxygen is high is being used for the purpose. Please ensure toxicity of those oxygen vector on your microbe plus the problem you are going to face during downstream processing prior to use those materials. You can read following articles:

1. Biotechnol Adv. 1989;7(1):1-14.

Enhancement of oxygen transfer rates in fermentation using oxygen-vectors.

2. Biotechnol Bioeng. 1990 Feb 20;35(4):427-35.

Mechanism of enhanced oxygen transfer in fermentation using emulsified oxygen-vectors.

Recently researchers are also trying to genetically modified strains for solving these type of issues, which they are naming as heme responsive operon. The following article may work as initiator for your thought process:

1.Appl Microbiol Biotechnol. 2013 Dec;97(23):10211-21. doi: 10.1007/s00253-013-4755-7

2.  J. Bacteriol. July 2008 vol. 190 no. 14,  4903-4911 doi: 10.1128/JB.00447-08

Please note that air lift fermenter is not going to work for viscous fungal fermentation. One has to use CSTR even considering the risk generated due to high shear. Pelleted cultures may be another option. From engineering perspectives, changing impeller type may also work.

Considering all known approaches and its limitations on fungal cultures ( viscosity), I would like to look at using Hydrogen peroxide in combination with purging of air under pressure. in case the culture cannot produce peroxidase, one has to feed peroxidase also. in principle this works fine, which can be cheaper also. in addition, if the idea is to use hydrocarbons as carbon source in the fungal fermentations,  the oxygen demand would be quite high and hence supply of H2O2 in doses might be a good idea.  other ideas like oxygen purging, low temperature ( if viscosity is not too much), would help typically.

Thanks to all for their valuable answers. Hope I would be able to solve this problem of mine and I may get back to you all for further querries. Thanks again for contributing to this question.

Reeta Rani Singhania.

I do not know all of your search. In my research on xanthan gum production had no problems with oxygenation. However the viscosity of the polyelectrolyte and its amount complicate the results.

Have you thought about testing (at the current condition of the research problem) sufficiently adequate amounts of H2O2 in the fermentation medium? I remember that there were references on this procedure. But I have not needed to use it.

I am not sure, but have you tried to adjust your PID values for your cascade control?

Adjusting the PID will make your control system behave better!

Maintain the DO level 30%. 

I've got increased production by adding hydrogen peroxide in the fermentation medium but am using only 10mM/L at different time intervals. I am wondering what are the possibilities by which hydrogen peroxide can have enhancing effect on protein production or its release?

Dear Reeta! 

I think it will be better to feed the hydrogen dioxide in fermentor continuously in order to maintain the necessary dissolved oxygen level.

It is important, that your culture produce some peroxidase activity. If not, you can add it into cultural liquid separately. 

The other question is that oxigen concentration itself is not proper parameter for control, if you have a viscous filamentous broth. In my opinion, the better parameter is respiration rate, which could be measured by means of gas analyzers of oxygen or carbon dioxide. It is necessary to maintain this parameter on  maximum level during the whole fermentation cycle. The testing experiments are necessary in this case - from time to time.

Best regards!

Thank you Biryukov! Your suggestions are useful and am sure I will be benefited by this. I am grateful to you!

1- completely agree with Robert W Lovvit, during scaling up the fermenter mas transfer has limitation and the only way is to decrease consumption of oxygen by microorganisms in order to keep DO>20%, and to decrease it is to decrease cell concentration together with the nutrients concentration or supply if it is fed-continues run, from this point of view any scale down experiments or developments should be done at Kla=300 around because no sense to develop the commercial process in the lab scale at Kla>300 (Rpm>300-400) because it cannot be done in the commercial scale, the same reason for usage of pure oxygen in lab scale - no sense because it will be never used in the commercial scale

2 - Could anyone give me a contact email of my first and best supervisor, V.V.Biryukov, who has put the answer on this question in this blog?

V. V. Biryukov · 16.70 · 11.81 · Moscow State University of Mechanical Engineering 

Dear Anatoly! I have recieved your letter and answered in Russian. My E-mail is ([email protected]). See you later!

Dear Zhivotchenko,

Yes I understand your point and as our protein is growth associated I was thinking how to get the maximum product which is the main aim of this study. I very well understand the constrains of commercialization of a process and you have raised very valid points. We constrict our studies to 200 rpm max. without oxygen but have been benefited by hydrogen peroxide addition.

I am sure for 100% that nobody will use H2O2 in commercial scale therefore no sense to use it, I would recommend you to get detail knowledge about DO-stat technique that will allow to increase cell concentration by controlling the nutrient feeding, for example, it is possible and I did it myself to get cell concentration of E.coli up to 120 units of OD-600 at Kla=300 while typical maximum cell concentration at this level of mass transfer of oxygen is 40-50 units of OD-600.

you have fungi fermentation that requires much less transfer of oxygen and you can get 30-40 g/L of dry weight easy if you use this DO-stat technique at KLa=300, I have got it in commercial fermenter of 15 000 L volume, the main idea of this technique is that you feed your nutrients based on DO=20%, and of cause if you overfed your nutrients then cell growth becomes too high and DO goes down to zero, it is not easy to understand therefore only your trials can give you more experience to use this technique

Thank you Zhivotchenko!

Your points are very convincing and will try to do it and get back to you as I know  will need your help in future too.

Dear Reeta!   I'm glad to meet my friend Zhivotchenko on your site concerning your interesting question and discussion about it. I have lost any touch with him over many years.

I do not think that Anatoly is absolutely right. For bacterial or yeast fermentation it is possible to  obtain a high concentration of biomass using conventional technique of oxygen supply. But in your question you listed all conventional  methods you tried.  Of course, if you decrease the substrate concentration, the oxygen level will increase.  But your aim is not DO , but the final product concentration. That is why my main opinion is that DO-stat technique is not the best method for filamentous fermentation. I can describe my method in more detail, if you send me your E-mail. My address is [email protected].

Dear Biryukov, I am so much thankful to you for your extended help.

My email id is [email protected]