BioDieselNow - Renewable biodiesel fuel

I would like to start a thread dedicated to growing botryococcus Braun ii. I recently received a second 15ml sample from UTEX collection (after killing a first sample with Miricle Gro) and I am soliciting the observations of other members who have had or are having experience growing this species. I had selected it because its lipids are very closely linked to crude oil and have, in the lab, been processed into gasoline.

The current sample is growing now about 5 days in my 'soil water' and is doing well. It looks like the cells have multiplied 100% in that time. The cells act strangely. Some times they lie on the bottom, sometimes they are free floating thruout the medium, and sometimes they all gather into a sphere. I have a co2 enriched atmosphere pumping into the flasks. The current produced doesn't explain this behavoir I see. Allways looking for any explanation for this.

The cells range from a bright green to a dull green. they tend to clump up, strings of cells attached to one another, and almost seem to propel themselves (not going along with current in flasks) at times.

I hope to hear from other so please post often with as much insight as you may want to share.

I measured the co2 in my incubating chamber, it's 2726 ppm vs a normal atmosphere of 350 to 450. Quite a difference, almost5.5x normal atmosphere. Co2 is produced bu baking soda and vinegar mix once a day, with stirring done twice a day. It is having a positive result. Chamber temp is 91 deg F, same as outside, illuminated by a 40 watt florescent light places aprox 4 in from incubator chamber (16 qt clear rubber maid container with lid), on 24 hr a day, surrounded by aluminum foil to reflect light into incubator. There seems to be much more haze in the flasks, like hundreds of little cells (?), smaller than the strings of Bb. I don't think this is contamination because the entire apparatus is enclosed. I hope these a re new cells forming up.

Sgt,

You need to be agitating the culture - that can be an outside motion or a bubbler. 

Yes, OK. I've had an aquarium pump set within the container to move the CO2 rich atmosphere within the containers. Today the algea is going olive brown. The medium is alternating between cloudy color and going clear. The cloudy color is white. There seems to be more growth, as small clumps of cells are visible in one of the flasks, much more than I originally put in. The color change bothers me. My first sample did a color change before dying. And the additional cells in the second flask confuses me. All I will do is continue to watch and see if either flask shuts down. The first sample cell groups fell apart to almost individual cells before settling to the bottom. These cells are not doing this. I'll keep my fingers crossed. If this cell group dies than I have a serious problem. I'll think about finding a local "pond skum" floating sample to work on until I can find out where I've gone wrong with these samples. These algae are touchey critters.

Hi Sgt,

 Don't give up brother and I hear and share in your frustration. These suckers are touchy little buggers.

 I am growing Dunaliella Salina in a saline solution and have had a few bottles crash on me. I have kept a 1 litre sample going since early February but as soon as I try and scale it up to 15 litre bottle it seems to change character.

In it's defence however, I have to say I am very much time poor and do not give the samples enough attention.

I have just set up a sugar water and yeast Co2 generator which I pipe to a box in which the aquarium pumps are set up so hopefully I am now getting a better, maybe not the best, volume of Co2. Air pumps run 24 hours to maintain circulation within the bottles.

After 2 days the two 15 litre bottles look greener so hold thumbs they stay improving.

sgt,

What is the volume of your algae cultures?  How long do you think the algae can grow in the flask before you need to feed them again?

Slippery,

Excellent design of a CO2 generator, I have been making beer for months now and it never occured to me use the CO2 produced - yeast are my new best friends!

AT

I don't know long before recharging the sample should be enough. I'll give it till next week to recharge with a shot of soil water. Things have stabalized. The sample vessels are still cloudy with new cell growth and the old clumps have green to them. It was scarry there for a few days.

I picked up some local algae to work with on the side. Ya gotta see this algae. There is a water leak from a city provided water line under a sun drenched street. It's a slow leak, maybe two gallons a minute. This wild algae grows in this cholrine city water, drenched by harsh sunlight all day, clings to concrete, and is an intense blue green. I have it in a sample flask with soil water and it looks like it is growing, although slowly. If it were possible to "cross pollinate" the Bb with this local variety, WOW!! That would be something. I don't think it is possible, but I can dream.

you need to be carefull when pumping co2 into a culture ...too much co2 will drop the ph suddenly and dramatically and kill the algae.. its better to use atmospheric co2 when starting  your cultures after all nature designed algae and its intent was to use atmospheric co2 not high concentrations particularly in small cultures yes co2 is a co2  bioremediator but under natural conditions ot in carefully monitored rates not in  high concentrations. that is probably why your algae is not doing well youve shocked it.. I bont mess with botyrococcus because the oils are complex chains that arent transesterifiable and require  treatment more akin to petroleum cracking.besides it very slow growing from what ive  heard.

any how watch out for too much co2 when working with small quantities of cultures.

Marc

 Marc,

BB does require a different process, but you can get gasoline, diesel, or jet fuel.  For a backyard grower, it would be more difficult, but for a large industrial process, it produces products that sell into several different markets.  It could be a money maker.

Samples are doing OK. It is amazing how the BB sample water is cloudy , like a white mist is hanging in the water, even though it is turbulent with air pumping in. Both BB samples have visible free floating cell groups along with original clumps. One of the samples has started to have cell clumps attach to the vessel wall. The mongrul (just the hardy local street wise algae) is very clear. The mongrul looks like it is growing with more independent cells floating around. The mass of original clumps together. No matter how many times I break it up with air pumped in, it seems to find its pieces and cling together, exclusive of the newer cells. The soil water stays crystal clear. Amazing. Sometimes I wish I would have discover algae 40 years ago.

sgtrock, do you have a microscope? They can be had fairly cheap, even with a camera. Working with algae, a microscope is a must have.

sgtrock101:

  intense blue green  

sgtrock, I think you should be very careful and cautious about playing with things you dont know about. Some blue-green algaes can be highly toxic.

sgtrock101:

   If it were possible to "cross pollinate" the Bb with this local variety, WOW!! That would be something. I don't think it is possible, but I can dream. 

I dream that the spicegirls would recruit me as their roadie sex slave. But ofc neither your nor mine is likely to ever happen. Infact... scientifically, my dream is statistically more probable to come true than yours. Why? Because by definition, a species is one that cannot be bred into. Thus, I can cling to my dream...

Froggy,

Thanks for your concern. However, it is self evident that I know little about algae. If I knew a lot, I would be at a university trying to persue my thoughts. As it is, I am retired, and I do NOT, rub algea on my skin, inject, injest, inhale or in any other way, fool with the algea save obtain samples and watch them grow-observe. The algea is in soil water mediumand will stay there.

 Both samples (Bb and mongrul)  are growing. My inspection this evening revealed the Bb samples are  clearing the medium (becomming less cloudy), the free cells banded into clumps and are deffinately multiplying. As I have read as much literature as is available to me on Bb, this activity has not been mentioned. I think it will takes several weeks of observation to confirm this is a normal activity. I am sure there will be more and more cell activity that is not noted in any scientific journal or research. I am a "layman wanna be" algea farmer. I am willing, able and doing what I may to study these creatures. Knowledge comes from many sources, including those outside the university/corperate settings. I am happy to add to that knowledge base, if I may, in a non competetive/combative environment.

As to the mongrul sample, it too, is multiplying. However, it does not cloud the soil medium, the free floating cells are very distinct, and are beginning to form new clumps. The mongrul's ability to survive in a harsh urban environment, chemical laden city water, and intense ultaviolet lighting from sunlight intrigues me. I know enough about genetics to conclude that algae do NOT pollinate and I have a rudimentary foundation in cell division. It was a oblique reference to genetic engineering. 

One of these days, soon as my tax rebate check comes, I will buy a better microscope that will allow me to more easily see the samples and photograph them. It's kind of exciting, thinking that there are so many different algea and that some of them may hold the key to our society's ability to survive and prosper, instead of self destructing. Thanks again and your posts are always welcome.

sgtrock101:

I know little about algae.  ...    Knowledge comes from many sources   ...   I am happy to add to that knowledge base  

If I could suggest an approach seldom used but seems obvious.

As my friend Mike Briggs rightly suggests, a limiting factor to an 'algae world' is the high cost of the infrastructure to grow algae. PBR's and huge elaborate high science is much too expensive to raise pond scum and make $. Very low tech veg pond with a slightly up higher tech breeding system seens like an excellent design. Huntley has such a design that you might be interested in.

The approach twist is to apply the local conditions to the existing designs. This means that one would build a benchtop model of the fullscale system and measure the local parameters within that system. These include seasonal, climate, cloud cover/various times of year, weed organisms, temps, temps in the system... all of the parameters that algae need. Once one has a full understanding of the operational parameters of the system, then one can start looking for algae to fit that set of parameters.

As you have proven to yourself, growing algae isnt a cake walk. Its not about the algae, its about the system. Design the system for your part of the world and apply algae to that system.

Ofc you should be growing algae in vessels just to get better acquainted with our little green friends. But the real 'boots on the ground' IMO is design of the system, not the algae that grows in them. If you build it, they will come.

 Froggy,

Is the darkfield feature worth the extra $140 or so?

(not a fair comparision since this one does not have the PC connection feature) 

http://cgi.ebay.com/PROFESSIONAL-BINOCULAR-COMPOUND-DARKFIELD-MICROSCOPE_W0QQitemZ200228767249QQihZ010QQcategoryZ48741QQcmdZViewItem

or:

http://tinyurl.com/4b76kk

 Sgt,

What you are working toward is exactly what we need - a simple set of instructions for growing algae that can be understood by new researchers, farmers, and other people without a degree in microbiology.

Keep up the good work in figuring out inexpensive ways to grow algae.

 Froggy,

Michael Borowitzka has already laid out how to build algae ponds - Chapter 14 - Culturing Microalgae in Outdoor Ponds - Pages 205 - 218 of Algal Culturing Techniques edited by Robert A. Andersen.

Perhaps it would be worthwhile for Sgt. to vaidate those instructions. 

liberty1:

  Is the darkfield feature worth the extra $140 or so? 

Depends on how serious you are. How much $ you got. What you need to get done. I would rather have a cam than dark field if I had to pick.

Dark field will offer you techniques that light wont but there are many ways to skin the cat of characterization. I would say it like this, 'in phycology, dark field is not a must have but a microscope is'.  

 Froggy,

Thanks for the judgement call - sounds like a good feature, but not a necessary one. 

Bb update. Both Bb samples are doing well, each is growing in spurts. The sample in the 150ml flask is doubled to about 4x the  sample size initially started with. The second sample, aprox 500ml in a plastic jar, has really done well. I estimate that it is the same size as the 150ml flask sample. It started out the same day as the flask with barely visible cells. It definately grew much faster than the 150ml sample. The only difference has been the size of the containers. The cells have turbulence, though much less because of the second samples jar sample, more so because of the 150ml flask size.  So I am concluding that turbulence is absolutely necessary but, the amount of turbulance is greatly influencing growth rate. I don't quite know where to go from there. Maybe bigger size jars with the same turbulence in each to see which size causes a deminishing return. Just a thought.

I am considering starting a second experiment. I want to use waste water from my canine septic system. I want to take a small sample from the 150 ml jar, split in two, and place it in the waste water, aprox 500ml size plastic jars. One will be filtered and boiled, the second just filtered. Before doing so, I'd like some comment as to how concentrated a sample of waste water. I will attempt to see if the boiled and unboiled water affects the sample growth as well as seeing if the waste water will increase the growth rate, now estimated as doubling every 8 or 9 days.

 The mongroul sample is doing well. It has almost doubled. It grows slower than Bb. The water stays clear, but when the cells devide they come out as a small cloud, free float, are very distict and visible, then join into a clump after one day, or so. Then things settle down for a couple of days, and the process starts over again. I was expecting that all the cells would devide closely timed. That is not how it happens. Only a small portion devide at any one time. The process is not continous, or at least it is not visible with the unaided eye. It is like the group of cells (colony) need a rest period befor some will devide. Fascinating to me. I suspect the Bb devides the same way, just not as visible and the cells (new) cloud the water with a white mist.

I look forward to comments, especially about the anticipated experiment with waste water.