Production of Omega-3 Polyunsaturated Fatty Acids
from Cull Potato and Biodiesel Waste Glycerol
Principal Investigator: Dr. Shulin Chen, Professor, Department of Biological Systems Engineering, Washington State University 99164-6120; Phone: (509)335-3743; Fax: (509) 335-2272
Co-PI Zhanyou Chi, Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164-6120 Phone: (509)335-3241; Fax: (509) 335-2272;
Requested duration: 2 years
Amount requested for the first year: $46,484 ($97,040 total for both years)
Emerging Issue(s) being addressed
The purpose of this project is to develop a two-stage culture process of heterotrophic microalgae to produce a nutraceutical (omega-3 fatty acids) with cull potato and crude glycerol as the raw materials. This project addresses the emerging issues of the RFP in the category of Value-added agriculture production systems. Specifically, this project first addresses Issue No. 8 (the impact of the developing biofuels economy on Washington’s agricultural economy) since crude glycerol, a waste stream from the biodiesel industry, will be used in this process as a raw material. Crude glycerol is a low value by-product from biodiesel industry which is too costly to be purified. Glycerol will become a liability to biodiesel producers if alternative uses are not developed. Converting this waste to high value omega-3 fatty acid will change this burden to a higher profit margin. Secondly, this project addresses Issue No. 10 (improving farm profitability through new uses of crops, livestock, and by-products) because cull potato is used in this process as another raw material. Development of new markets for the culls is essential to the economics bottom line of the potato producers. Thirdly, this project addresses Issue No. 9 (consumer preferences and acceptance of evolving biotechnologies and functional foods aimed at improving health and nutrition) since DHA enriched algae biomass will be produced with this process. DHA has significant beneficial effects in the treatment of neurological and cardiovascular diseases as well as in infant brain and ocular development. Inclusion of supplementary DHA in infant formulas is recommended by the World Health Organization, and DHA is becoming an adult dietary supplement in food and beverage to improve brain health.
Problem statement
The problem of cull potato There are 579,000 acres of potatoes grown in the Pacific Northwest with average yield of 60,000 pounds per acre. For every acre of potatoes harvested 10-15% of the crop is graded as culls. This includes undersized tubers, bruised, damaged and deformed tubers, and tubers unfit for market. In addition, if the tubers have low specific gravities/total solids, hollow heart, internal discoloration, disease, etc. the crop is considered unsuitable for processing. The potato growers generally receive less than $10 per ton for their culls. However, it costs the growers about $65/ton to grow their crop (Personal Communication WA Potato Commission). Consequently, the growers must pay the cost of growing these culls out of income from the marketable grades.
The problem of biodiesel waste glycerol During a typical biodiesel production process oils from oilseeds or fats from the oleo-industry are mixed with methyl alcohol and alkaline catalysts to produce biodiesel, with glycerol as a primary waste product. The mass conversion of this process is approximately described by 100 units oil/fat plus 10 units alcohol to produce 100 units biodiesel and 10 units of glycerol. The results are that for every 7.35 pounds of oil/fat converted, 1 gallon of biodiesel and 1 pound of crude glycerol (80-70% pure) are produced (Thompson and He, 2005). Clearly, the biodiesel industry will be in need of alternative uses for waste glycerol. For example, it was estimated that one billion pounds of biodiesel glycerol will be introduced into market by 2007, but only half of that has an existing market and that is for a refined glycerol, not the crude glycerol being produced here with numerous contained impurities. (USDOE, 2004).Consequently, a situation exists where there is not just a surplus of glycerol, but a surplus of crude glycerol that is expensive to be purified to higher quality. With the rapid development of biodiesel industry, glycerol price has dropped significantly. It is known from the biodiesel producers in Washington State that the price of crude glycerol at present is $0.05/lbs, and this price is decreasing, probably becoming negative (Personal Communication Seattle Biodiesel, 2007).
Omega-3 polyunsaturated fatty acids and DHA Omega 3 polyunsaturated fatty acids (w-3 PUFAs) are a group of fatty acids containing two or more double bonds, of which the last double bond is located at the third carbon atom from the methyl terminal. Docosahexaenoic acid (DHA, 22:6) is one of the more important w-3 PUFAs, with a 22 carbon chain and 6 double bonds and is known to have particular beneficial effects in fetal and infant brain and ocular development (Innis, 1994). The inclusion of supplementary DHA in infant formulas is strongly recommended by the World Health Organization (WHO). Also, research continues to demonstrate the need for DHA beyond infancy. Studies have suggested a positive correlation between DHA consumption and the reduced risk of age related neurological disorders, such as Alzheimer’s and dementia. As a result, DHA is not only used as additives in infant formulas, but also in adult dietary supplement in food and beverage. Example foods are cheeses, yogurts, spreads and dressings, and breakfast cereals. Other markets include foods for pregnant and nursing women and applications in cardiovascular health. These markets may have much greater growth potential than infant formulae (Ward, 2005).
DHA production with algae heterotrophic culture It is known that fish, like humans, are not capable of synthesizing PUFA de novo. Much of their PUFA is derived from the primary producer in the oceanic environment.The best microbial sources of DHA are from the genus Thraustochytrium and Schizochytrium. Schizochytrium species with higher growth rates have been isolated to grow to high biomass densities (200 g/L) in short fermentation cycles (90–100 h), accumulating 40–45 g/L DHA and hence DHA productivities of >10 g/(L day) (Bailey et al., 2003). However, although advances have been made and some commercial processes are now present within the market, fish oil and its subsequent purification of DHA still represents a substantial competitive threat to the algae fermentation process (Martek Annual Report, 2004). Thus, it is still an indispensable work to decrease the cost of heterotrophic algae production.
Progresses of this group on DHA production with algae culture The study of algal cultivation for converting cull potato to DHA has been conducted in the PI’s research group. Schizochytrium limacinum SR21 was selected as the better producing strain. Used as both a carbon and nitrogen source, an optimal ratio of hydrolyzed potato broth (HPB) in the culture medium was determined as 50%, with which the highest production of 21.7 g/L dry algae biomass and 5.35 g/L DHA was obtained, with extra glucose supplemented.
Studies with crude glycerol as carbon source showed that it supported alga growth and DHA production, with 75 to 100 g/L concentration being the optimal range. Temperature, trace metal (PI) solution concentration, ammonium acetate, and NH4Cl had significant effects to the DHA production process. A DHA yield of 4.91 g/L with 22.1 g/L cell dry weight was obtained with the optimized condition in this experiment. The results suggested that biodiesel-derived crude glycerol is a promising feedstock for production of DHA from heterotrophic algal culture.
The technical challenge and innovative solution of this process Although great progress has been made in the past studies, the cost of DHA from algae culture is still higher than that from fish oil. To compete with fish oil, even higher biomass concentration and DHA productivity is required. However, it was ignored in traditional algae culture processing that the growth of Schizochytrium can be divided to two stages: (1) cell number increasing stage (cell proliferation and rapid cell number increase with little increase in the size and weight of each cell) and (2) cell size increasing stage (cells stopped proliferation and enlarged due to fatty acids accumulation). With the recognition of the two-stage process, it became obvious that improving the biomass concentration must start from two aspects, increasing both cell density and cell size. Based on the knowledge that different optimal culture conditions are required in the two stages, a two-stage culture process which aims to provide optimal culture condition for the two stages separately was proposed by the PI’s research group. Our initial work significantly increased the cell culture density from 100 million cells/ml to 400 million cells/ml and growth of the culture increased to the point that a cell density of more than 100 g/L dry algae biomass was achieved within 60 hours.
It needs to be pointed out that this result was obtained without optimization of the culture conditions of these two stages. Optimizing the culture condition for the cell number increasing stage will result in even higher cell density; similarly optimizing the culture condition for the cell size increasing stage will shorten the culture period, i.e., leading to even higher productivity. The final “cell body weight” obtained from our current process is 0.25 mg/million cells. If the same cell body weight can be maintained, but while also increasing the cell density to 1,000 million cells/ml thorough culture condition/process optimization, 250 g/L of dry biomass and 5 g/L/hr of biomass productivity can be obtained. This productivity will be higher than any other existing processes. Our previous experience on the culture of this algal strain tells us that this cell density (1,000 million cells/ml) is only a conservative target. If we can also optimize the size increasing stage to shorten the time to reach high biomass concentration and to increase the DHA content in the biomass at the same time, the productivity will be further increased, making this process more competitive than other processes.
Objectives:
The goal of this project is to further improve algal productivity based on the above approach. The specific objectives of this project include: (1) optimizing the cell number increasing stage of Schizochytrium limacnum SR21 to obtain an ultra-high cell density, (2) optimizing the feeding strategy of the cell size increasing stage, and (3) conducting a pilot study of the algae cultivation process. Cull potato hydrolyzed broth will be used in the first stage to provide carbon source, nitrogen source and trace elementals; while crude glycerol will be used in the second stage as carbon source, with other nitrogen source added.
Research methodology and approach
Algae strain and culture medium
The algae strain Schizochytrium limacinum SR 21 (ATCC, MYA-1381) will be used in this study. Culture medium for this alga will be prepared by supplementing a certain ratio of hydrolyzed potato broth and other nutrients such as mineral salts and vitamins. To prepare the hydrolyzed potato broth, cull potato will be boiled and minced, mixed with certain volume of water and placed in a 5-L tank with agitation. Two enzymes, a-amylase and glucoamylase will be employed to hydrolyze the potato starch into glucose. The temperature used in the hydrolysis process will be 55 ºC. After 4 days, the hydrolyzed broth will be harvested and centrifuged to remove the solids. The glucose concentration in the liquid phase will be determined and then diluted to 100 g/L with water.
Task 1. Optimizing the cell number increasing stage (use potato hydrolyzed broth) of Schizochytrium limacnum SR21 to obtain an ultra-high cell density
Cull potato will be used in the first stage since it provides a better carbon source (glucose) than the crude glycerol, which contains less energy. Additionally, it provides nitrogen and other elements to the cell reproduction. A Plakett-Burman design will be conducted to investigate the effect of every element upon the performance of the culture in this stage. The details of the Plackett-Burman (PB) method are described by Chi et al (2008). In this experiment, the cultures with high and low level of each element will be compared, and statistical analysis will be made to determine whether each factor is significant. The algae cell density, described as cells/ml, will be the responses in the statistical analysis. The effect of each variable (Ei) on response will be determined by subtracting the average response of the low level from the high level. The significant factors will be determined as the confidence levels is above 90%, then, further studies will be conducted to optimize their concentration with a central composite design.
Task 2. Optimize feeding strategy of the cell size increasing stage with crude glycerol as carbon source
With the increased cell density, an optimal feeding strategy will be developed to reach the high biomass concentration, high DHA content and high DHA productivity. Temperature, dissolved oxygen, and nitrogen concentration are the most important factors to affect this process. To optimize these factors, crude glycerol will be used as the carbon source in the feeding stage, and adequate nitrogen concentration will be maintained by continuously adding nitrogen sources. Dry cell weight, nutrient utilization, and DHA production will be dynamically determined and used as the response. A “temperature shift” strategy will be developed as the optimal temperatures for the size increasing state and DHA formation stage are different. It has been demonstrated that algal cells require higher temperatures for their size growth while DHA formation requires lower temperatures. The temperature will be maintained at a higher level (~30oC) for cell growth during the earlier stage of cultivation, and then shifted down to a lower level (~20oC). The biomass will be harvested at the end of fed-batch culture and then the DHA content will be analyzed using a GC.
Task 3. Pilot study of the algae cultivation process and economic viability assessment
Scale-up the two-stage culture process with the optimal conditions determined from the above research tasks will be accomplished using 1L, 25L, and 125L fermentors within the Biomass Processing and Bioproduct Laboratory at WSU. To study the oxygen transfer, the OUR, OTR, and KLa values in both the seed cell producing stage and DHA accumulating stage will be investigated at various aeration and agitation rates in a 1 liter lab scale fermentor (0.1–1.0 vvm and 200–500 rpm, respectively) as well as in a 25 L fermentor (0.1 –1.0 vvm and 200–500 rpm, respectively). The KLa values of both scales will be compared to determine a control condition that gives the same KLa for scale-up study. The effect of KLa to the algae’s growth and fatty acid accumulation in the 1L, 25 L, and 125L fermentors will be recorded and compared. Problems such as oxygen concentration gradients in the fermentor in this scaling up process will be recorded and investigated if encountered.