Date: 1 Dec 1997
From: Randy Reed <rreed@seafarm.com>
To: BRINE-L@UGA.CC.UGA.EDU
QUESTION 1:
I'm looking for information, references, or concerns about preserving
marine microalgae paste with glycerol. The preserved paste will later
be fed to bivalve shellfish, rotifers, shrimp, and larval finfish.
Any feedback would be very much appreciated.
Randy Reed
CEO, Reed Mariculture, Inc.
Santa Cruz, CA USA
e-mail rreed@seafarm.com
web http://www.seafarm.com
toll free 1-888-4-Seafarm (1-888-473-3276)
voice 1-408-768-9411
fax 408-287-2246
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Date: 2 Dec 1997
From: Graham Housefield <graham@bio.usyd.edu.au>
To: AQUA-L@killick.ifmt.nf.ca
QUESTION 2:
Can anyone please put us in touch with a supplier of paste/slurry or other feed supplement/emergency ration. We are trying to maintain mature oysters in a small recirculating research system.
We are culturing algae but would like access to feed back-up supplies.
Graham Housefield
Marine Ecology A11 2006
University of Sydney
Sydney, NSW, Australia
E-mail: graham@bio.usyd.edu.au
Tel 61 2 351 4854
Fax 61 2 351 6713
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COMMENTS 1:
Glycerol is one of the most used cryoprotective agents in microalgae
cryopreservation.
Glycerol is a good organic substrate for bacterial growth, for this reason you may want to avoid it in your paste.
I don't see any other point in using it. Are you thinking about freeze
drying or deep freezing your paste? If yes, there are a lot of other subjects you must concern about after using the glycerol. Nevertheless you may try a 10% v/v.
Joao Navalho <necton@mail.telepac.pt>
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COMMENTS 2:
Try Mike Heasman at Port Stephens. I am sure that he will be able to help you.
John Chapman <jchap@netadvantage.com.au>
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COMMENTS 3:
I'm not sure how long you want to preserve the paste for and have never tried glycerol. In our lab, we concentrated Nannochloropsis into a paste using and old centrifuge that we modified. The paste was stored in a fridge (4 C) for 2-3 weeks with no problem. Past that, and the paste started getting pretty nasty and the algae (when reconstituted) would settle quickly and generally smell real bad. We wouldn't use it. This didn't give us much long term storage, but did give us a little buffer when there were the inevitable algal production problems.
Randall Mager, <rcmager@ucdavis.edu>
Dept Animal Science
UC Davis
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COMMENTS 4:
I would recommend contacting Dr. Kelly Rusch at LSU. I do not have her direct contact information, though I am sure you can contact her via LSU's Department of Civil and Environmental Engineering at 1-504-388-8666.
Michael H. Schwarz
Aquaculture Specialist
Virginia Seafood Research and Extension Center
Virginia Tech
PO Box 369
Hampton, VA 23666, USA
757-727-4861 Fax: 757-727-4871
mschwarz@vt.edu
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COMMENTS 5:
Someone who might be able to help is cathy.johnston@marine.csiro.au - they have someone in the lab who is working on microalgal pastes. My microalgae keep well at very high densities at 4 degrees C for a week or so - I guess you've tried the fridge? Can you tell me/us how you concentrate your pastes and does the method kill or damage the cells?
This is an important subject. Do you have survival rates for your
concentrate? How do you measure that?
Joao Navalho
Necton - Companhia Portuguesa de Culturas Marinhas, SA
Belamandil
8700 Olhao
Portugal
voice/fax +351 89 703961
e-mail: necton@mail.telepac.pt
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COMMENTS 6:
In answer to your question about the survival of microalgal concentrates at 4C - I'm afraid I don't have any survival data - the method was recommended to me by a hatchery operator who used it to store concentrates. I tried it for concentrates of Chaetoceros gracilis and checked the cells after storage with a light microscope. The cells appeared fine - since I only used these stored cells as a back-up I didn't take the survival issue any further.
Dr. Paul Muir, <paul.muir@jcu.edu.au>
Department of Microbiology and Immunology
James Cook University
Queensland, Australia
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COMMENTS 7:
We keep our paste stored in a refrigerator (4 C). We have research to show that the nutritional quality remains for at least two months in a fridge, as long as it is kept air tight (with the air evacuated out). We store the paste in ziploc bags so that we can squeeze the air out. We have actually had a collaborator use three month old paste to start new cultures of Chaetoceros meulleri. This is with no preservatives. The problem with using preservatives is the effect on the organism that the paste is fed to.
As long as the paste is kept air-tight it should stay good for at least two months, maybe three to four. Sometimes a layer of fungus may form on the paste (if the container was not evacuated of air), but this can usually be scraped off and the paste underneath is still good.
Mike Christensen, <mchris@unix1.sncc.lsu.edu>
Louisiana State University, USA
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COMMENTS 8:
What speeds (rpm) are required for centrifuging algae? Is this a batch process or is there a way to set up centrifuge for continuous flow?
"Philip S. Kemp Jr." <skemp@coastalnet.com>
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COMMENTS 9:
It's common to add glycerol to bacterial cells to prevent cellular damage during cryopreservation (to keep the cells viable after thawing). More folks are using DMSO now for the same purpose because it's easier (to prevent contamination by other strains of bacteria, glycerol needed to be sterilized by autoclaving while DMSO can be used straight from the bottle).
When you say preserved paste, do you mean refrigerated or do you mean frozen? I can imagine glycerol helping if you were freezing the algae but don't see how glycerol by itself would help if the algae paste was simply refrigerated. Wouldn't frozen paste be better because of the longer shelf life?
Shiao Y. Wang
University of Southern Mississippi
sywang@whale.st.usm.edu
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COMMENTS 10:
Sterility isn't an issue if the preserved algae are being used as food,
right? If you want to store frozen stocks for future cultures, I can see
the need to autoclave glycerol (as I do when I freeze down bacteria in lab.)
Does anyone have any comments on the concentration range of glycerol that is most likely to be effective at preventing ice crystal damage to frozen algae? Any other comments about freezing technique? Is it necessary to freeze them down in liquid nitrogen and store in at -80 C, or do people just add enough glycerol to keep the cells from freezing at -20 C?
Craig Bingman <cbingman@netcom.com>
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COMMENTS 11:
You are right. I did not mean to suggest that the glycerol needed to be sterile for algae.
Shiao Y. Wang
University of Southern Mississippi
sywang@whale.st.usm.edu
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COMMENTS 12:
What was the recommended amount of glycerol to use? ppm? percent?
Do you remember Bob Engel's study of feeding cornmeal to oysters? He had problems with bacteria in the cornmeal slurry causing it to go bad before it could be fed. A salt brine solved the problem, but I don't
remember the salt concentration. Maybe your algae could be preserved in a salt solution as well. It would also lower the freezing point.
"Philip S. Kemp Jr." <skemp@coastalnet.com>
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COMMENTS 13:
I'd like to find out if anyone has concerns about using food grade
propylene glycol as a preservative in marine microalgae paste. The
paste will be fed to zooplankton (rotifers, brine shrimp, etc) which
will in turn be fed to aquacultured finfish and other animals meant
for human consumption. The paste will also be fed directly to
shellfish which are raised for human consumption. I'd really
appreciate feedback on any concerns or suggestion you might have.
Randy Reed
CEO, Reed Mariculture, Inc.
Santa Cruz, CA USA
e-mail: rreed@seafarm.com
web: http://www.seafarm.com
toll free: 1-888-4-Seafarm (888-473-2327)
voice: 1-408-768-9411
fax: 1-408-287-2246
***************
COMMENTS 14:
Microalgae can be cryopreserved in liquid nitrogen for many years
with protection from cryoprotectants such as glycerol, DMSO, ethylene glycol or propylene glycol etc. Cryoprotectants have toxic effect on shellfish embryos only if the concentration is higher than that embryos can tolerate. Embryos of some species of shellfish have also been successfully cryopreserved into liquid nitrogen using
similar cryoprotectants as preserving algae. Study on oyster
embryos showed that embryos of all developmental stages can survive up to 1M common used cryoprotectants including DMSO, ethylene glycol etc. I give you some reference paper found in my EndNote library, hope it can help.
Wang, <W.B.Wang@newcastle.ac.uk>
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