This is an interesting question. What are the other parameters for your growth? i.e. are you growing in flasks or reactors, are you using a light/dark cycle, do you shut the CO2 off at all, how bright are the lights, how often are you measuring pH, what is the concentration of tris and ammonium in the system? If the pH is around 6.5 it would most likely be an issue with the pH being on the lower limit of what the organism likes. Ammonium has a toxicity concern at higher pH ranges when the concentration of free ammonia increases.

Thank you for your answer Everett!

I tried to grow the algae in photobioreactors and in flasks. I tried different light intensities from 50µE to 450µE. Algae are illuminated 24/24. Tris concentration is 20mM (same concentration as in the usually used TAP medium). I don't turn off CO2 supplementation. Ammonium concentration is 9mM (same concentration as NO3 in bold 3N medium).

About the pH issue : The fact is that the cells grow very very well until they reach a plateau and die. If the pH was a problem, they wouldn't grow well at the beginning... What do you think?

Do you track the pH throughout the experiment? I think tris buffer has a pKa close to 8, which would indicate that a pH of 6.5 is below the buffering range of tris. The problem with growth on ammonium, is that during ammonium uptake the cell translocates a proton into the medium to maintain cell neutrality, effectively acidifying the medium. I would suggest changing the buffer you are using to Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES), which has a pka of 6.8, or a similar buffer with a lower pka. You can look at the article "Growth, nitrogen utilization and biodiesel potential for two chlorophytes grown on ammonium, nitrate or urea" at http://link.springer.com/article/10.1007/s10811-013-0008-5 or https://www.researchgate.net/publication/237198009_Growth_nitrogen_utilization_and_biodiesel_potential_for_two_chlorophytes_grown_on_ammonium_nitrate_or_urea?ev=prf_pub , which has looked at growth on ammonium in Scenedesmus.

Data Growth, nitrogen utilization and biodiesel potential for two...

Now that´s an interesting problem. Even with pH alteration (and you should indeed track the pH throughout the experiment, as Everett suggests), a sudden bleaching should not occur. Have you analyzed your samples for bacterial contamination? Most microalgal cultures are not axenic, and perhaps after this plateau you have some sort of apoptotic effect or the development of a competing population of bacteria. The latter should be easy to verify.

I have tracked the pH throughout the experiment and it remains stable until the cells death. It is difficult to determine if the pH fall causes cells death or if the pH fall is caused by cells death as it happens at the same time...

But i think that you're right Everett : At the beginning, Tris can compensate for the low acidification caused by NH4+ uptake. Due to the exponential growth, the acidification process is also exponential and when culture reach a high cell concentration, Tris cannot compensate the high acidification induced by high NH4+ uptake. And at this point, pH falls to 3...

I will try PIPES (have you tried MES?)!

Concerning bacterial contamination, it is not the case as my cultures are axenic and i observed this phenomenon with different strains on a long time scale so i don't think this could explain the bleaching...

Thank you both for your answers and by the way, nice paper Everett!

Hi Thomas,

Everett is correct, ammonia uptake is going to acidify the media, and the CO2 is going to acidify it also. 3% CO2 is much higher than is needed for a gentle boost of growth (1000 fold higher that atmospheric levels!). you could get by with a lower 0.5% and it should be just fine.

one way to get around this a little bit is to try using nitrate instead of ammonia. Nitrate will increase the pH during uptake. a problem with this and Chlamydomonas is that many of the standard lab strains cannot utilize nitrate due to a defect in the nit-1 gene.

Consult the Chlamydomonas center for strain information.

Another way around this is to use urea (which does not donate either H+ or OH-) during uptake. However, you will not get the nice buffering from the nitrate.

Another option is to pulse the CO2 into the cultures. This can be done using a small solenoid valve on a timer. I would suggest using a little 6v valve from clippard (www.clippard.com/‎) and turning it on every half hour for a minute or two. This way you can bubble with air all of the time and occasionally give the cells a boost of CO2. you should be able to control the valve using an arduino microcontroller and a small 5v power supply.

Good luck!

Oh ya. for Chlamy and CO2, try HS minimal media the excess phosphates have way more buffering capacity than TAP!!!!!

I agree with Julio and Everett on the buffer issue. After how many generations does the culture crash? Another thought I had was to adjust your photoperiod to a period closer to natural (possibly 16:8) and turn off CO2 during the dark phase. With the low buffering capacity as mentioned above your 24h dark phase may just be enough trigger cell death due due to acidification as carbon uptake is limited.

I tested MES, PIPES and MOPS with Monoraphidium braunii in the nineties. Both PIPES and MOPS worked right. MES buffer has a slightly lower pKa and cells grow slowly, but they still do. Nevertheless, I never used so high NH4 concentrations (9 mM could be rather toxic even at neutral pHs).

pH drops because there is a huge extrusion of H+. With addition of high levels of CO2 NH4 converts to NH3 and the cells are not able to control this entrance since it is a gas. This leads to an internal unbalance, a more medium acidification, more NH3 present which triggers the degradation of chlorophyll and the bleaching you see

Dear Thomas,

Add NaHCO3 with an equivalent quantity to NH4NO3. Green algae like NH4+ more than NO3-. They grow well in the beginning, but not to the end. At pH < 7 some of NO3- are in form of HNO3. You have to neutralize it with equivalent amount of NaHCO3. Green algae do not need other buffers.

Georgi

Hi Thomas,

An interesting observation and although I have only worked with saltwater media we also encounter these sorts of problems. If your cultures reach stationary phase then clearly something is limiting growth. It could be either a limiting factor (nutrient, light) or a toxic factor building up. For example in nitrate grown cultures I have seen nitrite get very high and it is much more toxic. Some twenty five years ago when preparing artificial media I was using such high purity ingredients that we generated Se deficiency in batch cultures. It took months to figure out what the problem was because the cells morphologically looked like they were being exposed to high copper concentrations. If you can reproduce the phenomenon then you can add a wide range of other essential nutrients as the cells approach stationary phase and look to see which one might be limiting. I agree with your other respondents that the combination of high biomass, high NH4 and high CO2 could easily lead to complications in media chemistry that might be revealed if you analyze for more components of the acid base system. You have a lot of factors contributing to increasing acidity. Finally in any culture system with an exponentially growing biomass the addition of a constant rate of carbon will eventually be insufficient. Without seeing the data and crunching the numbers it is hard to know when that will happen for your system, although given the high CO2 concentration the rate of flow seems more likely to be the (possible) limitation.

This is a typical problem with using NH3. When the algae grows very fast, the media pH will very reduce very fast. You lost your culture most likely due to the medium acidification. This is worst under warmer conditions.

Algae have the habit of growing well and then rolling over and dying without warning. You get used to it but biotechnology types never seem to realise that it is the nature of the beasts.

The 9 mM ammonia level is unnecessarily high.

The sudden bleaching indicates to me an intracellular pH problem. Think of the plasmalemma membrane potential and the consequences of the Nernst Equation and the nasty habit of NH4+ acting as a K+ analogue. Under your conditions the cells are struggling to keep NH4+ out of the cells and maintain intracellular pH at about 7.3. They are not actively taking up NH4+. When the cells are overwhelmed by the influx of NH4+ their pHi regulation mechanisms fails, the chloroplasts bleach because of the acid intracellular and your cells die.

Learn something about membrane potentials, bioenergetics and the Nernst equation.

Though I am not an algologist ,being a microbiologist : can you try to pulse feed NH+ ions?

I fully agree with Raymond. Well said.

I think the best way to supply ammonia would be through the air supply. Put a few ml of NH4OH in a gas passing jar in the airline. Algae can usually pull NH4+ down to immeasureably low concentrations (nM) using active transport mechanisms and so I think supplying them with NH3 in the airline would work very well with little danger of poisoning them. If you pump NH3 into even slightly acid media it will immediately form NH4+ and stay there. You would be able to work out exactly how much NH3 they need for continuous growth as a bonus and adjust the NH3 supply accordingly.

I have done this with solid ammonium bicarbonate in a gas passing jar. That worked fine. When it evaporates you put in some more.

Feeding nutrients as gas rather than a drip feed is a lot easier to keep clean.

Its probably nitrite accumulation: there are cheap test strips available for nitrite detection.

I think this is most likely to be caused by low internal or external pH. The high CO2 treatment will tend to depress external pH and ammonium assimilation leads to the generation of protons that will decrease pH further. You could check this by monitoring pH and bringing it back to, say pH 7- 7.5, during the growth of the culture. If this works a stronger buffer may be the solution.

What are you able to measure? There is quite a bit of guessing in the answers posted here--many of the suggestions could be right or completely irrelevant. Do you have data for nitrite or DIC? How low does the pH go down to at night? With constant 3% CO2 injection I assume you are not continuously monitoring pH, (could be too much at first and not enough later). Do you know what PON the culture is at when it fails? How much NH4 and phosphate is left int the media at this point? pH of 6.5 is pretty low. OD is a fairly useless measurement. Without data, we are all just guessing.

I think pH of 6.5 is low. Maybe higher pH would make the algae grow longer? Nitrite may accumulate to toxic levels. It is fairly easy to measure using a standard colorimetric technique og maybe a commercial aquarium test kit.

Wow, i am surprised by the numbers of responses! Thank you!

Unfortunately, i don't have the time to test all your tricks...

As i told you, i already did a lot of experiments with a wild-type strain on Bold 3N (NO3). I have to use NH4 media because i need to study mutant strains (PGRL1, Ptox,...) which are nit- (and sometimes cell wall less, which is even worse with the stress caused by bubbling in photobioreactor...). So, i cannot change everything right now or i will have to repeat everything... No please! :-)

Concerning nutrients deprivation mentioned by Peter Thompson, i already tried to add each elements separately to see if something was missing but it is not the case (cultures crashes at the same OD).

I have just added PIPES (pka=6,76) to my medium. I will start cultures this afternoon and compare growth on TRIS-NH4 medium and PIPES-TRIS-NH4 medium. If they grow well with PIPES, the problem is solved! So let's try it and we will see if other analysis are needed afterwards!

High ammonium concenrarion inside the cell will turn into NH3 (ammonia) that is a respiration toxin. The pH rise and Mg is leaving the chlorophile molecule/

Thank you for this suggestion! However, this should not be the case here because at the beginning, algae grow very well! And there is more ammonium in the culture at the beginning than after...

Well, yesterday, i started cultures in 2 media with a CO2 supplementation : TMP-NH4 (TRIS buffered) and TMP-NH4-PIPES (TRIS and PIPES buffered). Both media were at pH 7,2 before inoculation with algae.

Both cultures are green and grow at the same speed. However, the pH in the TRIS-PIPES buffered medium is at 7 while it is already at 4 in the TRIS buffered medium. I expect the latter culture to bleach in the next hours... So i think that the problem is solved (i will tell you tomorrow)!

I have 2 questions :

1) what concentration of buffer do you use? I added 20 mM PIPES because i usually use 20mM TRIS. However, i can see a precipitate (and PIPES is expensive). Do you think 10mM would be enough?

2) do you think that TRIS in still useful in combination with PIPES? I don't think so.

For whatever it is worth: I have grown C. reinhardtii sucessfully on NH4Cl as nitrogen source for extended periods of time in turbidostat systems as well as in batch cultures. The buffer was a simple phosphate buffer at pH 6.75-6.8. Most of the time I was supplying CO2 by ordinary air bubbling but at occasions I went to 5% CO2 without any problems. If you want to try, the reference for the complete media is in Falk et al. (1990) Temperature-dependent photoinhibition and recovery of photosynthesis in the green alga Chlamydomonas reinhardtii acclimated to 12 and 27 C. Physiologia Plantarum 78: 173-180.

Good luck

Stefan

Yes thank you for your suggestion Stefan. However, as i told in the discussion, i already done many experiments on Bold (for 1 project) and TMP (for another project). I don't want to change all the medium composition right now or i will have to repeat everything with a new medium...

Concerning the cultures i launched two days ago, you can see that the 2 cultures without PIPES bleached (on the right) and that the 2 cultures with PIPES (on the left) are growing well :-)

Problem is solved! Thank everybody!

This may be due to production of autotoxoins which many of the Volovocalean algae are known to excrete at the end of log phase.

Sorry for spelling errors:

This may be due to production of autotoxins which many of the Volvocalean algae are known to excrete at the end of log phase.

Yes, the pH low may be reason.or else at decline phase you would have tranfer the parent culture to dilution at low pH level.

Thomas - I am looking to supplement AF6 media with ammonium to raise a Chlamydomonad. Can you give me a sense of the ranges used? Are you just adding NH4+ directly? Thanks

Hello Ryan. I add NH4Cl to my medium. The final concentration is 9mM. Have a nice day!

Hi Thomas,

It is most likely pH effects. Once the buffering capacity is reached, the pH will drop quickly.

Try less CO2, 3% is ~100 fold higher than air. you could get away with using far less.

you can sparge with the CO2 intermittently, use this valve and control it with an arduino.

http://www.clippard.com/part/EC-2-6-H

let me know if you need a hand but it is fairly straight forward

You can also use HS media, it has a large buffering capacity.

You can also use urea as an N source. It won't lower the pH.

Of course using Nit+ lines are the way to go!

Cheers

Ben

I now about this and I have never really understood why.  I have always thought it was some sort of redox or pH effect.  One thing I have noticed is that if you add a very small amount of nitrate (say a few micromolar NO3) it does not seem to happen.

Any similar experiences? 

i don't know about this problem, but i grow chlamydomnas on bold with urea and doing good job is it right?

It may be due to autoinhibiting substances produced by algae

DId you measure the concentration of NH4-N? High concentrtaion inhibits algae growth.

In entirely agree with Ben. For me it is a pH matter although you didn't notice it. When I mean pH is the one very difficult to detect, around the algae cells.

Check your media pH. You will notice that you will have a fast drop in pH in a fast growing microalgae culture when grown in high NH3.

I still say it is because of autoinhibitory substances produced which results is sudden bleaching and death of cells.

Dear Thomas, 

If it is not pH effect, then it might be the ammonium toxicity. To some plants which are not adapted to ammonium as the sole N source, ammonium may become toxic, particularly at high concentration. The ammonium concentration used (9 mM) in your culture appears to be high. Just try with lower ammonium concentration, may be 1 or 2 mM or alternatively use a mixed N source (ammonium + nitrate).  

A part of the nitrogen is removed by insufflation, so the crops start a nitrogen starvation phase before than occurs with nitrate. You should check the ammonia concentration in the medium.