are doing heterotrophic culture? try to add 1 drop of 1% FeCl3.... or maybe the algae need time for the adaptation process...

How does your pH change over the time period?

if you question contamination perhaps take a swab and culture in a plate culture? Check under microscope should clear up if contamination is an issue

Dear Hossein,

In order to diagnose this properly, some more details are necessary: there are different versions of 'proteose medium' each with different composition, are you culturing in light or not, what light conditions, type of culture vessel, do you add CO2? 

Algae will not become white due to presence of bacteria but due to lack of something, some bacteria can even be growth-promoting for Chlorella. Therefore I suspect that some important macro or micro nutrient (carbon, iron?) has become limiting. 

If presence of bacteria is not wanted (i.e. in the case of heterotrophic culture) you should indeed check your media and check whether you are working with an axenic strain.

Good luck,

Anthony

High temperature and excess light can also cause "bleaching" of the cells. As mentioned in earlier posts - more information is required.

Turning colour from green to yellow in leaf of plants is commonly attributed by nutrient deficiency  since the plants is unable to synthesize chlorophyll.  However, biosynthesis in plants is really "complicated multi-steps mechanism"  which involving various factors.  In my view, among other factors, light intensity above that required could possibly be a factor that inhibit biosynthesis. Suggestion, vary light intensity and observe color changes 

آقای امینی عزیز سلام وقت به خیر

من نیز بر روی کلرلا تحقیق می کنم ... زرد شدن تدریجی جلبک کلرلا ولگاریس می تواند به دلیل تمام شدن مواد غذایی درمحیط کشت یا عدم دسترسی جلبک به منبع کربن ، تغییر PH ، شدت نور بالا ویا تاریکی ممتد ودرجه حرارت بالای محیط صورت بگیرد ...

مناسبترین دما  23-25 درجه سانتیگراد برای Chlorella vulgaris  است .

-PH=7- مناسبترین دوره نوری 16-8 .... h16 روشنایی و8 ساعت تاریکی  با 3500 lux

 برای تامین Co2 ,O2 از پمپ هوا (پمپ آکواریوم) استفاده کنید حتمآ از فیلترسر سرنگی 0.22  در ورودی شلنگها برای جلوگیری از ورود باکتریها وآلودگیهای قارچی استفاده کنید.

چنانچه هوادهی نمی کنید منبع کربن را به صورت شیمیایی در محیط کشت فراهم کنید مثل بی کرنات سدیم و...( کلرلا میکسوتروف) است . اگر ازمحیط کشت  مورد نظر جواب نمی گیرید پیشنهاد میکنم از محیط کشت Bg11 ,MOH202 استفاده کنید ( ما اینجا جواب گرفتیم)

حتما عناصر غذایی ماکرو ومیکروالمانها(Hutner Trace elemant) را رعایت کنید..

با آرزوی موفقیت برای شما ....

Hi,

I agree with the above comments, it looks like the algae had not enough nutrients. Baybe CO2 is limiting factor? It is important how do you culture your algae: in what kind of vessels? are they aerated? If not  - do you supplement cultures with bicarbonate? How is the air/media ratio in the vessel? By the way, did you try to change the media? It could help to distinguish between nutrient/CO2 limitation.

Anna

Thank you all for your responses.

I am culturing them in some shake flasks with 70-100 umole/m2 s of light intensity and the temperature ranges in room temperature. The proteose medium is based on what UTEX suggests since the chlorella is bought from them. I inject the air through needles into the medium and the thing is that because I desire to use this medium in wastewater treatment in future works, I don't care they get contaminated. However I autoclave the medium before using it. One of the problems may be that the CO2 is not provided in excess and I have to deal with that since I think other factors seem fine. because some times I see the pH goes up to 8.5. Although I may inject some CO2 in, the pH will decrease for a short time and will go up again during day time. I will give it a shot to see what happens. The question about the nutrients is that, what other nutrients should be monitored aside form Phosphorus and Nitrate? because when I check them, they are in enough amount.

Hi,

that is strange. If N and P are in excess, air-bubbling should be enough to promote Chlorella growth at room temp. provided that cultures are not too dense. The light and pH also seem OK (8.5 is not so bad). By the way: why do you use proteose medium instead of pure bristol's medium? Are you going to culture Chlorella mixotrophically? As Anthony proeviously suggested, with proteose your cultures may be heavily contaminated.

Anna

Dear Hossein,

Proteose medium means that it is not a selective medium for Chlorella. It could be told that the situation is nearly opposite: ~ all aerobic bacteria will love this mixture. Chlorella does not have (I am not sure) proteolytic activity, so at least peptides are not acceptable. 

This situation is not simple at all. Longterm cultivation w/o sanitization and re-inoculation will select enemies from the environment (Paramecium with Ch PB-1 virus, Bdellovibrio - or Vampirovibrio chlorellavorus etc.). So I guess better to make inoculum in mineral medium, and then put Chlorella to the waste water model media.

Istvan

Istvan:

Then if you say Proteose is not good, what is best fresh water medium for this strain. On the other hand, I have selected this medium because UTEX has suggested that

Hi,

I used pure Bold Basal Medium (bristol's medium) for Chlorella + air-bubbling and I had no problems with  cultures.

Anna

Anna:

Thanks a lot. I will try it to see what I get. I think it will take me about 10 days to get some new results. I will let you know...

Anna:

Can I ask about the initial cell density you use? (per ml). I was wondering if this also could happen because of low cell density as the pre-culture

Dear Hossein,

The Bold Basal medium what Anna suggested should be good. I used something similar (Tamiya medium, https://www.jstage.jst.go.jp/article/shokueishi1960/10/2/10_2_95/_pdf)

These are selective autotroph media. But you should consider further on the presence of other bugs, just one article:  http://www.sciencedirect.com/science/article/pii/S0273117705003595

Hello every one;

As Istvan and Anna suggested, I started using Bold medium. It's been a week and I am gonna wait about 10-14 days more to see what happens. Until now, every thing seems better than proteose medium...

Hello Hossein,

I am sorry, I missed your question about cells density. I keep the algae on the solid agar slants and then I transfer them to the liquid media so the pre-culture is actually not dense (about 100 000 cell/ml, sometimes less) and the light density is about 80-100 umol photons/m2 *s. You said that your algae grown very fast during first days of experiment - I think that if you had too low density of pre-culture, algae would die at the beginning.

Good luck,

Anna

Hello every one:

I used Bold medium instead of Proteose medium and it worked really great! Thanks to Anna and Istvan for your really good recommends. As far as I experienced, I think proteose medium is really bad for growing Chlorella vulgaris (or even other strains) because it gets contaminated by bacteria very easily...