In order to avoid pigments' interference, try using a wavelenght of 750 nm.

I recommend this article for more details: https://www.sciencedirect.com/science/article/pii/S016770121100056X

Thank you Mara for your advise. My trials with Dunaliella and Asteromonas using 680 nm gave satisfactory fittness. Searching for the best I'll repeat it with 750 nm.

It is better to check wavelength between 400-800 nm to find the maximum absorbance for your species. than, you can use this maximum absorbance for calculating of biomass.

750nm is the wavelength that has less interference with pigment concentration. as microalgae tend to regulate its pigment during its growth by environmental condition. To me dry wight is the best method.

My experience in this regard is mainly with green microalgae, but I believe that the what I describe here holds true to other algal groups.

Chlorophyll, both as extracted chlorohyll and indirectly as absorbance measurements, is a good proxy for cell counts and biomass, even considering natural variations in chlorophyll across growth phases, except during extended stationary phase (more on that below). (I'm sorry for the lack of references, I'm in a bit of a hurry now).

Here is what we do in our lab for unialgal suspensions of cells, with good fit for the absorbance x cell counts curve (usually R2 > 0.9):

- First, check absorbance maxima wavelength, as suggested by Amirreza Zarekarizi, and record the measured absorbance value at this wavelength. This maxima should correspond to the absorbance of the predominant pigment. For green microalgae the maxima (chlorophyll-a) is around 678 - 684 nm.

- Record the absorbance at 750 nm. This corresponds to 'particles' in suspension, which not necessarily means cells. However, some researchers use only this value as a proxy for cell counts with good results.

- Subtract the absorbance at 750 from the absorbance at the wavelength you determined as the maxima.

The obtained value should have a good correlation to the number of cells across the growth curve, as it is mostly related to the chlorophyll content.

Something to keep in mind is that the fit of absorbance vs. cell counts and especially biomass will substantially decrease when your culture reaches stationary phase. Following the onset of nutrients limitation, microalgal cells tend to accumulate storage compounds (carbohydrates and lipids), cease cell division, and the production of chlorophyll become limited, all of which will cause deviation in this relationship.

Excellent analysis dear Guilherme, clear and concise. Thank you very much.