WTC Final Report
Title: Development of Omega 3 Rich Algae from Biodiesel Waste for Use as an Animal Feed supplement (Phase I)
Project manager:
Dr. Shulin Chen, Professor, Department of Biological Systems Engineering, WashingtonState University 99164-6120 Phone: (509)335-3743; Fax: (509)
Progress Summary:
1. Simple pretreatment of crude glycerol
A general procedure has been developed to pre-treat the crude glycerin to obtain a working 100 g/L concentration. This pretreatment involves dilution with water to approximate 20% glycerin content, followed by acid addition to neutralize the pH and convert contained soap into fatty acids which can then be separated out as they reside in a top-level organic layer. The resulting solution has the needed salts, nitrogen and water added to make a 10% solution of glycerin. The 10% solution has been targeted because of laboratory results which showed that this concentration was optimal for fermentation on the utilized algal species.
2. Culture condition optimization
Optimization of the process at a flask scale showed that crude glycerin can in fact be an effective carbon source for production of DHA enriched algae of the strain Schizochytrium limacinum SR21. Optimization results show that 23.84 g/L of algae can be produced which contains 5.27 g/L of DHA when using a media of 100 g/L pre-treated crude glycerin at 18.96C, and containing appropriate levels of artificial seawater and nitrogen source. These results show a slightly reduced algae growth concentration but a considerably higher DHA production rate than was theoretically predicted prior to experimentation.Later optimization and improvements as discussed below have lead to much higher production rates and yields.
3. Oxygen supply protocol optimization
The oxygen uptake rate in the different growth stage of this alga was investigated with both continuous culture and batch culture in a fermentor with a dissolved oxygen (D.O.) control. It was found that high oxygen consumption is required in the cell propagating stage, when cell number increases but little increase occurs in the size of the cell. A second stage of cell growth occurs and it is at this stage with low oxygen uptake that the fatty acid accumulates. An optimized oxygen protocol has been determined to produce more cells (control at 50% D.O.) and then provide a best condition (<5% D.O.) for fatty acid accumulation. With this protocol, 37.9 g/L dry algae biomass was produced.
4. Fed-batch culture protocol development
A fed-batch culture protocol was developed, since the increased cell number needs more nutrients to accumulate fatty acids and reach higher cell density. In this fed batch culture, 25g/L glycerol and 25% of the initial nitrogen and salts were supplemented to the culture daily, and the final cell density was increased to 55.6 g/L. Another protocol, repeat batch culture, in which all the spent medium is replaced by fresh medium at the end of culture, also caused an increase, with the final cell density becoming56.1 g/L. With more seed cells as initial cells, a bioreactor culture with fed batch protocol eventually attained acell density of 102gdry algae biomass per liter broth. High cell density culture is obligate for a production process to be cost effective. Before this, there were only two reports on the algae can be cultured in a cell density more than 100 g/L. this is a very important breakthrough in our research, and this showed the promising of this process to be industrialized.
There were four tasks in the original proposal: optimizing culture conditions, fed batch culture development, pilot study and fish feeding experimentation. In this phase I work, we optimized the culture conditions, and developed a fed batch protocol, with which a high cell density culture was realized in the lab-scale fermentor. The pilot study and fish feeding experiment was not carried out. The fish feeding experiment was not completed because not enough algal biomass was available due to the lack of time/funding to complete a pilot-scale fermentor and the fact that there is considerable research already in print showing that cultured Schizochytrium algae biomass has nutritional value for farmed fish and cattle with an ability for the omega-3 fatty acids to accumulate within the meat and/or milk. Also, spray-dried Schyzochitrium has been commercialized and is presently being marketed as a live algae replacement and DHA supplement to aquaculture diets (
The participation of company partner
American Premix Technologies Inc.(APT) was the company partner as the original proposal; however APT’s involvement in regard to the original vision of the project was curtailed because of their necessary concerns in regard to their business start-up and the fact that the fish study for which they were a large player was not accomplished. Additionally, all research, including the work done during the WSU requested extension period, was done without APT cash as the required funding from the commercial partner was never received. Despite the reduced interaction between WSU and APT because of the aforementioned reasons, two important meetings were accomplished to discuss project results/concerns and possible future commercialization realities/concerns. WSU, though, is actively considering and pursuing new potential collaborators on future phase work because of APT’s inability to meet their financial duty.
Publication and invention disclosures
A paper entitled “Production of Omega-3 Fatty Acids from Algae Fermentation with Crude Glycerol from Biodiesel Industry” has been developed and is the process of submission. Additionally, an invention disclosure entitled“co-production of carotenoids and DHA with culture of Schizochytrium with the crude glycerol from biodiesel industry” has been filed with the WSU Office of Intellectual Property Administration, but patenting has not been completed due to on-going investigation into existing patent rights, concerns about patent costs and concerns about needing more extensive research and breakthroughs in production. Please note that all disclosures were based upon original WSU research ideas formulated prior to the study as well as refinements learned during the research with all refinements accomplished by WSU.
Potential commercial and economic viability of the concept as well as potential intellectual property protection and licensing
The algae will be grown in bioreactor tanks not ponds. For the industrial production, 50 or 100 ton (m3) fermentors are preferred. From our present data, 1 ton crude glycerol contains about 700 kg pure glycerol, with the rest of the mass beingimpurities. At the current production rate (note that we are confident that we can continue to improve the yield and productivity) this 700 kg pure glycerol can produce 250 kg (550 lbs) of dry algal biomass (the conversion rate is 36%) that contains 50 kg (110 lbs) of DHA.
It is envisioned that the process will be done in fed batch mode with each batch conservatively lasting 7 days (this time probably will be shortened to 4 days). For each batch, 40 tons of crude glycerol will be fed into a 100-ton fermentor, and will produce 10 tons of dry algae biomass. The crude glycerol has a density of approximately 1.1 kg/L. So this amount of crude glycerol would take up a volume of about 36 m3(9,500 gallons). Assuming that 40 weeks in one year (52 weeks) can be used for production, there would be 40 batches per year. Then, this production system could process 1,440 m3 (380,000 gallons) crude glycerol per year.
An invention disclosure on DHA production process with crude glycerol from biodiesel industry has been made by WSU. It is anticipated that a more detailed patent will be filed after the accomplishment of the research on the shift-strategy and fed-batch and additional proof that the work being done is significantly different than previous work accomplished or other patents filed by other inventors.
Potential phase II objectives
It is has been proven in the phase I work that it is feasible to feed the crude glycerol to the algae as the carbon source to produce DHA and a high cell density culture (more than 100 g/L) has been realized at the lab-scale. These results show promise for additional workon the project; however, to industrialize this process, there are still some problems to be solved. Thus, the objectives for phase II will be: (1) refining the medium components; (2) scaling-up the laboratory scale DHA production process to pilot scale; and (3) assessing the safety of using the algae as a feed additive for fish.