I am using microscopic algae, and I've found the method for extracting the oil from the algae (AOAC 996.06).  However, I'm not sure how to get the algae ready for GC.  Will I be filtering it off and freezing it? Is GCMS the best method? or is gravimetrical a better way to go?

 E,

I think you are asking how to harvest the oil from the culture.  Various ideas have been suggested.  For a test process, a centrifuge is the logical choice if you have access to one. 

Hello Ebarkel,  it sounds like you are trying to extract the oil for analysis; am I right?  AOAC 996.06 would work for you to extract the fatty acids.  I'm not sure if AOAC 996.06 only refers to petroleum ether but hexane is pretty much the same thing and would work as well.  Although these solvents work well to get most of the lipids, if you are trying to perform a complete analysis, there are polar lipids that won't be extracted unless you add a more polar solvent to the pet ether or hexane.  A mixture of hexane and alcohol such as propanol has been done in the past (NREL ASP annual report 1989).

One other thing about extracting fatty acids from algae is you need a good method of lysing the cells prior to extraction.  The petroleum ether and heat will not be enough to break the cells sufficiently so without proper lysis the majority of fatty acids will remain in the cells after extraction.  If you have a good homogenizer such as a french pressure cell or ultrasonicator I would use that.  Get the algae as concentrated as possible in liquid medium and then homogenize with the method you are able to use.  If you don't have access to a homogenizer, one way that works is to place the dried algae into a mortar and pestle, add some dry ice, and grind away.  It takes a bit of grinding but you will get the majority of your algae cells broken up for full extraction of lipids.   

gcms should work well as long as you have access to and are experienced with the equipment.  

For a quicker and simpler method you can use TLC to analyze the lipids.  I have an article on the complete separation and analysis of lipid classes but need to look through my files to find the citation.  If you are interested let me know and I'll try to get you the article or at least the citation.

I'm not familiar with gravimetrical analysis so I can't offer any help on that. 

hello, i am new to this site but very interested in learning to analyze the lipid content of local algae. i would appreciate any hints on how to do this or laboratories i can talk to that do this. i have access to alot of florida algae(it grows everywhere here) and need to select a high lipid locally growing strain. thank you

can you also explain to me what (AOAC 996.06).  is?

jack

do you have access to a fluorometer or a fluorescence microscope?

you can stain the algae with red nile dye and visually analyze the lipid content.  You can measure the area of fluorescence compared to the area of the cells and determine the relative percentage.  It is not a perfect method of quantification but you can at least see which strains have more lipids than others to get an idea of which species produce more oil. 

if you can only measure fluorescence using a fluorometer, you will need to have separate cultures of different species to distinguish their lipid content.  you will need to put an exact amount of algae in solution in a cuvette, stain the lipids and measure the fluorescence.  This will allow you to compare relative amounts of fluorescence depending on the lipid content.  Again, not a perfect method but can help you to distinguish relative lipid content between species

one of the more simple methods is to extract the lipids using a solvent and then compare the mass of the extract to the mass of dried algae.  Since extraction efficiency isn't always 100% this method will not be perfect but again, you can determine lipid content and confidently compare species.

there are other methods but these are the three I am most familiar with and have done myself.  If you want to see some literature or more information let me know

jackflorida:

can you also explain to me what (AOAC 996.06).  is?

I normally don't refer to procedures by numbers and codes like that so I am not completely sure.  I believe it is a procedure using pertroleum ether as the solvent in a soxhlet apparatus.  A soxhlet is an extraction apparatus that percolates solvent through the algae several times in an hour or two.

This is what my Neochloris culture looks like when dyed and fluoresced.

 by using the imaging software of the microscope I was working on, I calculated the area of fluorescent regions and then also calculated the area when normal light was applied to the same slide.  Comparing these two calculations resulted in a lipid percentage of 17%.  

 If you want more details, just ask.  I'll try to do what I can to help

 I have more red nile images and I have a few different species if you are interested to see.  I just have to figure out how to convert TIFF images to jpeg so I can upload them.  The scenedesmus images look like the surface of the burning sun they fluoresce so much.

Jack,

You will have to kiss a lot of frogs before you find a prince.

I suggest you start with a known species from a culture collection or from Marc. 

liberty1:

Jack,

You will have to kiss a lot of frogs before you find a prince.

I suggest you start with a known species from a culture collection or from Marc. 

 

I wouldn't rule out wild algae too quickly...  I thought the same thing initially but for an experiment took algae samples from a local river downstream from a wastewater treatment plant. (Chicago River).  I actually found that more than one of the species I collected had a high lipid content.  One of the diatoms had over 50% lipid.  I'd say it is worth a look to gather algae that is found in local waterways.  What if you found that the dominant species in an algal bloom could produce a significant amount of oil? 

 Grtful,

Patrick also found a candidate diatom in Richmond. 

Of the 100,000 known species only about 300 are high oil, so both you and Patrick were very lucky.  (Patrick followed upstream from some foam, so he had a clue something was there.)

 I agree with you that we should not give up on local species, but, for someone just getting started, they are more likely to find success with known species.

 Liberty, you definately have a valid point.  While I did find some high oil-yielding species, I did not isolate and try to grow those; I grew cultures that I bought from utex for an inexpensive price since I am at a university.  I guess there is just something inside of me that says I should have isolated the strains I found in the river and cultured those but oh well.  Either way, people are going to come up with species that can produce the oils we are looking for.  I am getting optimisic lately considering the attention algae has gotten in the past few months.  I don't know if it's just the popular buzz of the moment, but I have a feeling some research dollars are going to flow in the right direction.       

I have read that the most effective method for breaking the cell walls and releasing the lipid is by "glass bead beating."  I believe this is a method where glass beads made for the purpose are placed in a test tube with the suspension (liquid form) to be mascerated.  The tube is agitated by placing the base in the rubber cup of high speed circular motion test tube agitator, also made for this purpose.  I have used this method to mascerate biological indicators (bacterial spores innoculated on thick filter paper).  It is very effective for grinding tough paper.  According to the article it was found to be the most effective physical method for mascerating individual algal cells as well, though the abstract did not say what other methods were tested or how long the tube must be agitated.  Following masceration the oil was separated using chemical methods though centrifugal separation as suggested above is a simple physical method.

Any suggestions out there for larger volumes out there? 

 I have also heard that technique is a good one.  I wanted to try it but I couldn't find the glass beads in a small enough size.  I think that you need beads that are .5mm in diameter; did the article say what size beads to use?  For scaling it up, I have thought about using a bead mill.  I also work with ceramic and for processing some clays and glazes we use a bead mill.  Basically it is just a big version of a rock tumbler.  I could imagine that you could use a large tumbling drum with the glass beads to process large amounts of algae.  I think we should try this method out and see how successful it is.

I suppose it would take more time to just roll a drum now that I think about it. You would have to leave it tumbling for at least a day I'm sure.  One thing that might work well is one of those paint mixers like they have at hardware stores.  In a metal 5 gallon drum you could put the glass beads and algae and then agitate the mixture with that machine.  It would be like a big version of homogenizing using a test tube and a vortex agitator.   

Can a Juiceman do the job in small scale? I had one apart many years ago and Im thinking it was some type of centrifuse correct me if Im wrong.

Guess the Juiceman idea was laughable by the huge amounts of responses

A friend from Morton WA, Bud Pitts (username Buuuuud ), has plans for a homemade centrifuse on the biodiesel.infopop SVO forum. Im now wondering how many G's are required to rupture the algae cells, a question for Froggy since he his so darn good with math.

I have seen this CF in action so I know it works for separating trash from the oil. Bud has made posts suggesting it can also dewater WVO. Could it be possible to rupture the cells and dewater at the same time?

My thoughts would be to trickle feed this CF based on algae production/growth in an attempt to create a continuos fed system.

Any thoughts?

Grtfulbuster:

 I have also heard that technique is a good one.  I wanted to try it but I couldn't find the glass beads in a small enough size.  I think that you need beads that are .5mm in diameter; did the article say what size beads to use?  For scaling it up, I have thought about using a bead mill.  I also work with ceramic and for processing some clays and glazes we use a bead mill.  Basically it is just a big version of a rock tumbler.  I could imagine that you could use a large tumbling drum with the glass beads to process large amounts of algae.  I think we should try this method out and see how successful it is.

I suppose it would take more time to just roll a drum now that I think about it. You would have to leave it tumbling for at least a day I'm sure.  One thing that might work well is one of those paint mixers like they have at hardware stores.  In a metal 5 gallon drum you could put the glass beads and algae and then agitate the mixture with that machine.  It would be like a big version of homogenizing using a test tube and a vortex agitator.   

 1/4" to 3" cast acrylic spheres from Tap Plastics

http://www.tapplastics.com/shop/product.php?pid=135&PHPSESSID=200806161712471457607806

Use a cement mixer from a local hardware for a cheap tumbler, a plastic one should work very well.

AlgaeKing 

Whoops...sorry for beating a dead horse in my last post.

I searched the forum for centrifuse and was surprised to find nothing then I realized my spelling.

Clint, those who have the answer for you know what you meant.     You're contributing to an informative thread.  It's not the form, it's the function.  :-) 

Gary

Gary,

Not sure if I was clear in my apology. Meaning...I searched the archives with the wrong spelling which returned no results so I posted. Some members of this forum find it annoying to keep repeating the same ol stuff since thats what the archives are for. At first I didnt see why anyone would give a hoot and simply not reply but I presume some have set their options to notify by email only to be bombarded with repeats in the archives. Buy the way, is anyone else having probs with the "notify be email" feature? I cant get it to work.

On a different note, Im a machinist by trade and concidering building a centrifuge after reading about its many uses since they are easily constructed. Im still interested in knowing how many G's it would take to rupture the cells (if at all possible) so I would know what diameter/rpm to build and run it at.

Any info appreciated and thanks for reading

 I have been so busy that I haven't had time to post lately.  Sorry about that.  I think the juicer idea isn't a bad one.  My roommate just got an omega juicer and have definately thought about trying it out.  I haven't been able to decide whether to use the household vegetable juicer with my algae though...Sounds like my roommates might not like that idea so much.

The only problem may be the small size of the algae.  The types of algae I have grown do not lend themselves to squeezing the oil out.  I think the type of press that seems to be best at this point involves shear forces caused by abrupt changes in pressure. I originally made many assumptions about getting oil out of algae because it sounds by the way everyone talks about algae that they are like pudgy pillows of oil that can be squeezed to get the oil out.  It seems that each algae cell, though it may have a large percentage of oil, Is mostly cellular material.  It takes a lot of algae to get a little bit of oil and it is not easy to separate that oil from the algae "dust" that goes along with it.

I cannot remember the exact numbers for the amount of force required to break algal cells but it is several thousand pounds of pressure.  When researching the use of presses for algae, I think I found that I would need a 20ton press to squeeze the cells enough to break.  I wouldn't bet on that number by any means though...I would feel more comfortable if I could pull up the article I read to back it up. 

 For coccal algae and/or coccal cyanobacteria - 18000 Psi in French-pressure cell is sufficient. With cell morphology it differ a lot filamentous, branching types...

 Drying of algae and usage of mortar and pestle it's also a good way - quite boring but working fine.

To get all lipid classes use Folch method or Blight-Dyer (found on www.cyberlipid.org).

Gravimetric analysis won't tell you much about constituents of your sample.

D.