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Subject: Information on duration limited volume culture
From: Michael Agbeti <[log in to unmask]>
Reply-To:[log in to unmask]
Date:Sun, 30 Jan 2000 19:14:35 -0800

text/plain (190 lines)

Hi Colleagues:
Please following are the responses I had to my request on the above
subject.  A number of people have expressed interest; thus, I have decided
to post the results net-wide.
Thanks to all who have responded.


Dear Michael,  Last year we tried the same thing both in culture vessels in
the lab (2 litre or 15 litre) and in a lake.  The results were very
disappointing in that added P on its own was not sufficient to give us
results.  Also, do not use Enteromorpha as it sporulates and accurate
biomass measurements cannot be made.   Trying to retain Chaetomorpha in a
lake for a period of even a week is also difficult as it fragments.  Good
luck,  Adrienne Grant

Dear Michael,
        We found that P or N alone or even in combination, were
to increase growth rates compared to controls, over a period of 13 days in
laboratory culture.  We used levelsof P and N that were within the range
measured in water collected from  the lake environment from which the algae
were collected.
        We concluded that these nutrients are insufficient on their own to
account for the periodic algal blooms that are observed in natural
situations.  It is more likely to be a combination of several plants and
organisms growing together with the release of nutrients from sediment also
playing a part.   Adrienne Grant



Lehman and I published a paper in L&O in 1983 that is appropriate for you
to look at regarding uptake of P spikes in natural systems.  I would guess
that the spike of dissolved P you add will be gone (i.e.  unmeasureable
because it is already inside algae and bacteria) within just a few hours;
maybe even faster.  My opinion: if the added nutrient isn't gone this
quickly: 1) that nutrient was already present in excess (i.e. wasn't
growth-limiting), or 2) you have added way too much; the concentration is
unnaturally high and hence the experimental design if flawed.  From all
the nutrient bioassay experiments I have done on lake Michigan and small
lakes, I would expect you to get a metabolic response to the
growth-limiting nutrient (altered respiratory rates and primary production
rates) within 12-24 hours, and a usable biomass response (as chlorophyll
or carbon) after 3-5 days, depending on water temperature.  "Container
effects" (wall growth, gas limitation, clumping)  are real concerns in
these experiments, so use the shortest incubation time possible.

There is a considerable literature on these sorts of experiments.  Try
looking up papers by FP Healey and DRS Lean from the 1970's and early
1980's.  Many were published in the J. Phycology and Can J. Fish. Aq Sci.

Good luck, craig
Dr. Agbeti,
The duration of the experiment depends upon the type of response that you
are interested in seeing.  From what you have said, you are looking for
biomass and/or nutrient changes.  From experience, I can say you will
likely have problems with such small volumes and long incubation times.
The surface area/volume ratio will not be in favor of seeing plankton
changes but more likely development of a the benthic (periphytic)

If you are restricted to that volume (2 L), the shorter the incubation time
the better.  I would say a week, 10 days tops.

I can follow up if you have more specific questions.

Hudson DeYoe
Dear Michael,

Problems you may encounter with extended incubations include massive
mortality, changes in pH, nutrient (P) depletion below the concentration
threshold for thermodynamically possible uptake, and contamination.
Monitoring these may enable you to extend your "reliable" incubation


We have performed many experiments using batch cultures to investigate the
phytoplankton biomass response to different p levels. We usually looked at
one organism at a time though. for example a unialgal culture of a
particular cyanobacteria. In some cases organisms are able to store P and
you should really starve your organisms from P at first . It may be
difficult to evaluate results if you do not do this as how do you know
weather the increase in growth is due to the stored P or not. You dont know
how much the organism has stored. We also kept all other nutrients the same
so we were looking at P response only. We ran the cultures for 30 days so
they progressed from Lag, log and stationary phases. we could then look at
the different growth rares for each P concentration and see if different P
concentrations were limiting etc.

If you need any further information regarding this work please do not
hesitate to email and I will try and help if i can.

Dear Michael
        I have done literally thousands of bioassays in limited volume cultures.
Some of the results are reported in  "Thompson P. A. 1998. Spatial and
temporal patterns of factors limiting phytoplankton in a salt wedge
estuary, the Swan River, Western Australia. Estuaries. 21:801-817" but also
in marine waters.    There are a number of factors that you should

light;   you need some suitable irradiance, 50- 100 uEin for optimal growth
OR simulated in situ (if lower than 50 growth will be slow)

temperature:  you need a suitable temperature, perhaps 16-18 degrees
although you cannot shift to far from in situ, but if this is low then
growth will be slow

condition of the initial biomass:  if it is in physiologically weak
condition due to prolonged nutrient stress (summer) or temperature (winter)
then you will have to wait for a response, there seems to be an initial lag
in growth

amount of initial biomass:  very small inoculums (< 0.2 ug chla per liter
seem to take a long time to show a measurable response

species present:  some (particularly BG) have relatively slow growth rates
and may take more time to grow

Having said all that,  in almost all cases biomass had plateaued in 1 week.
 However in situations (rare in my studies) where N-fixing BG were present
and in the absence of N the cultures sometimes peaked during week 2.

Samples with high biomass were usually diluted prior to starting the

all the best

Hi Michael,
I would be concerned that you will have periphyton growing all over the
flask in a couple weeks and that 3 d would be more than enough to see which

compartment and how much will be transformed.  We see 30 ug/L SRP disappear

in 2 L recirculating chambers with small amounts of periphyton in about 1

I hope this helps.

Michael D. Agbeti, Ph.D., CLM
Bio-Limno Research & Consulting
8210-109 Street, PO Box 52197
Edmonton, Alberta
Telephone/Fax: (780) 439-1558
e-mail: [log in to unmask]

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