I am culturing algae, utilising a batch regime (800 ml culture media) over five days. I am sampling N, P, COD, dry cell weight and cell counts. Thus is there a maximum Volume/Volume % sample volume that may be withdrawn over the five days without affecting my results?
Depending on the size and shape of the container, and the phase of growth at the time of sampling some things may change minorly. I know we harvest batch cultured algae in late exponential growth phases (up to 25% of an 18L batch culture) and the following day mean cell sizes are up to 20% larger, and doubling seems to occur much quicker than in earlier phases. Guessing this is due to increased S.A. for gas exchange... We do not observe such results when sampling ~2 ml per day ( over a week or two) of the same alga in 1L media in a fernbach flask to get growth rates. If your are using a container with parallel sides the s.a. and incident light:volume wouldn't change much, but in a more or less conical flask this might have some effect. just some thoughts....
Difficult to answer if I dont know what you want and where you want to go (experimental design)
As an artificial environment, the success in algal cultures depends on multiple factors. One of those is the critical watch on the appropriate sub-samples volumetric ratio to the culture's total volume. Then we can say that such and such V/V% is appropriate! But such an affirmation needs a good knowledge of the growth physiology of the studied species. Secondly to check the bottle effect (+ or -) on the growth of the algae. The bottom line is that there is a minimum of 3D pretest "algae - growth - bottle volume" mandatory for any culturing algae (provided all major media factors be kept stable).
Tim
Even removing 10% by volume over five days would reduce the overall volume by about half and change the culture conditions quite radically. Why don't you try using semi-continous cultures instead which are much more reproducible?
To calculate the volume you need to remove daily, measure the culture density (OD750) and use the following expression:
v = V - (V.ODt0 / ODt1)
For example, using your culture violume, if your initial density (ODt0) is 0.2 and the density after 24 hours (ODt1) is 0.4 then:
v = 800 - (800 x 0.2 / 0.4)
v = 800 - (160/0.4) = 400
So you can remove 400 ml for a culture dividing once per day and replace this with an equal volume of fresh medium to restore the original culture volume (i.e. 800 ml) and return the culture conditions to the starting set up. The standard OD you use is up to you and would depend on how much material you need for analysis.
Hope this is useful
Growing algal cultures in batch always requires the reduction of volume over time. If you are using a vertical tube reactor the affects of removing volume is fairly insignificant. However, if you are using flasks then there is more concern due to changes in multiple parameters. The important thing to remember is to remove only the amount of volume required for tests. N, P and cell counts only require 2 or 3 mL depending on equipment used for Nutrient analysis. I am not sure about volume required for COD. Dry cell weight will typically require 20 mL per filter for dilute samples. If it is a thick culture 5 or 10 mL is acceptable. This means that you will be removing roughly 45 mL of sample per day. This should have minimal effect on your results; however, without more detail on your experimental design and what the experimental parameters I cannot say this with absolute certainty.
I agree with Everett. the geometry of your culture system is key. The volume required for COD may be rather small, or fairly large, depending on the type of medium (at a guess, you are working with wastewater, but the level of previous treatment and the method you chose for determination determines the volume required for analysis). I would suggest, like Everett did, that if you use a cylindrical reactor or a flat panel (see Ryan's reply) you would be safe with 5 to 10 ml/day, and even larger (up to 20) might be feasible,with a well mixed culture
Some excellent answers here. For sterile experiments run in batch culture, I usually sample less than 2% of the total volume if a test runs 3-5 days, or have sufficient smaller volume replicates that I can sacrifice the whole sample at each sample interval (see Damian Carrieris response). This reduces the chance contamination (maybe not such an issue for N and P?) and preserves axenic conditions.
From 800 ml of culture volume, you are allowed to withdraw 2 ml intermittently for estimating N, P and other elements. Absolutely no problem, BUT please ensure that your CONTROL also grown in identical condition (100 %) as that of your samples. Then you are very well allowed to express and interpret your results with respect to control. Whatever growth/metabolic/micro-environmental changes happened to the samples the same changes might have occurred for the control as well.
All the best.. happy experimentation.
hi Tim. No sweat and I guess we enjoyed the sequence of the different answers to your query. The idea of a control culture mentioned by the colleague Arumugam stands. No treatment will survive the lack of a controlt, so make sure that your design has a control or better a series of control cultures.
best luck with your work
Hi Domenico, I enjoyed the answers too, its great to have this platform to tap into knowledge, I have been working in isolation really in sunny Birmingham. However I really like the interaction that Researchgate is giving me. Also it shows that my questions are relevant, which is always good for your confidence.
Thanks again for your help.
Tim
No problem Tim. I have more or less left the field of microalgae cultures and work mainly on metal contamination, but I have an accumulated experience of more than 30 years in your field. So, if you feel isolated, do not hesitate either to post more questions here or to write directly. [email protected] or [email protected].
cheers