Production of Omega-3 Polyunsaturated Fatty Acids from Cull Potato

1. Principal Investigators: Dr. Shulin Chen (Dept. of Biological Systems Engineering) and Dr. Ron Kincaid (Dept. of Animal Science).

2. Project Objectives: To develop a cost-effective algal cultivation process converting cull potato starch to -3 polyunsaturated fatty acids (-3 PUFA), i.e. eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), that have beneficial effects in treating and preventing human heart and immune diseases. The -3 PUFA enriched algal biomass could be used as feed additives for dairy to enhance the nutritional value of the milk.

The project addresses the 4th IMPACT priority of “developing value-added food systems and alternative crops and new industrial uses of agricultural products for the international marketplace”. Specific objectives include:(1) optimizing the algal culture Thraustochytrium aureum for DHA production through direct use of cull potato starch; (2) optimizing the algal culture Nitzschia laevis for EPA production from potato starch through a simultaneous scarification and fermentation (SSF) process; (3) developing a high cell density for algal cultivation, (4) pilot study of the algae cultivation process, and (5) assessing the possibility of using algal biomass as additives in cattle feed.

3. Methodology/Procedures:

(1). Optimizing the algal culture for DHA production through direct use of potato starch

Thraustochytrium aureum (ATCC 34304) will be used for DHA production since the alga can directly utilize starch as a carbon source for DHA production (Bajpai et al., 1991), thereby removing the need for and cost of converting starch into glucose. A medium for T. aureum growth will be prepared by boiling fresh cull potatoes in water to obtain a liquefied starch that will later be mixed with other nutrient solutions including some mineral salts, yeast extract and traces of vitamin B12 and biotin. Usually, the carbon source, at 3~4% of the medium mass, is the major cost contributor as all of the other components are used in a very low level that is well less than 0.1% of the total. Thus, using under-valued cull potato to replace glucose as a carbon source can significantly reduced the medium cost and ultimately the price of the whole process. In addition, optimization might eliminate the need for some of the added nutrient components as potato might be determined to be a capable provider.

The cultural conditions for the algal culture will be optimized by first adding the cooked cull potato material to solution at four levels (10, 20, 30, 40 g/l), and measuring the resulting corresponding DHA yield. Once the optimal potato concentration is determined, a complete statistically-based experimental design will be used to optimize the other medium components as well as temperature and pH. The design includes three major components: (1) selection of significant factors influencing DHA production by a Plackett-Burman design; (2) optimization of the significant factors by a central composite design; and (3) verification of optimal conditions. All of the above experiments will be conducted by flask cultivation. The algae will be harvested for analyzing their DHA content. The algal biomass will be freeze-dried and its lipids/fatty acids will be extracted by standard AOCS methods (The American Oil Chemists’ Society). The fatty acids will then be analyzed by a gas-chromatography (GC) at the Biological Systems Engineering Department at WSU.

(2) Optimizing the algal culture for EPA production from potato starch by SSF process

Unfortunately, for EPA production, there is no report regarding an algal species that can directly utilize starch, thus, in this project, two enzymes will be employed, -amylase and glucoamylase, to hydrolyze potato starch into glucose, and then use the glucose to support algal growth. The algal species to be used is Nitzschia laevis (UTEX 2047) which has been proven to be an excellent EPA producer. Industrial grades of enzymes will be used to reduce the cost of the process. End-product (glucose) inhibition is a common problem for enzymatic hydrolysis processes so to avoid this end-product inhibition, a simultaneous saccharification and fermentation (SSF) process will be employed. The medium composition will be the same as in (1) except that -amylase and glucoamylase will be added. The medium (along with -amylase) will be autoclaved at 121oC for 15 min and then inoculated with algal seeds. The temperature will be maintained at 30~35oC and glucoamylase will be added when the alga cells are added.

Experiments will be conducted in shaker flasks. A 23 central composite design will be employed for optimizing the initial starch concentration and -amylase and glucoamylase loadings. The experimental data will be correlated by a second-order poly-nominal equation, which will be used to determine the optimal value of each factor. The software STATISTICA (StatSoft Inc., OK, 1995) will be used for data analysis. Once the optimal values are obtained from the equation, they will be verified experimentally. Yeast extract will be proportionally increased with that of the potato starch being used, in order to avoid nitrogen imitation.

(3) Developing a high cell density for high level DHA/EPA production from cull potato

A fed-batch culture process will be developed (in a 5-L fermentor at the Bioresource Utilization Lab) in order to achieve high DHA/EPA production levels. This culture method is easy to operate and the equipment is simple and dependable. Based on the results in (1) and (2), a batch cultivation process will be performed as a control. Cell growth, nutrient utilization, and DHA/EPA production will be dynamically determined. In fed-batch culture, firstly, a suitable feed strategy will be developed by investigating the limiting factors in the medium, with any limiting factor correspondingly being enriched in the feed medium. Secondly, a “temperature shift” strategy will be developed as the optimal temperatures for algal growth and EPA formation are commonly different, that is, algal cells require higher temperatures for their growth while DHA/EPA formation require lower temperatures. The temperature will be maintained at a higher temperature (~30oC) for cell growth during the earlier stage of cultivation, and then shifted down to a lower level (~20oC). The algal biomass will be harvested at the end of fed-batch culture and DHA/EPA will be analyzed as in (1).

(4) Pilot study of DHA/EPA production from cull potato

The purpose of pilot study is to investigate the potential for commercial DHA/EPA production from cull potato. A pilot study is necessary in order to scale-up the process, and will be performed in a 25 L fermentor. First, the study will be conducted in batch mode to provide basic information such as cell growth and DHA/EPA production. The fermentor operation conditions (medium composition, pH, and temperature) will be the same as those obtained in (1) and (2). Based on the data of batch fermentation, a fed-batch cultivation process will be conducted. The operation condition (feeding strategy) will be the same as (3).

(5). Assessing the possibility of using algal biomass as additives in dairy feed.

A feasibility additive study will be performed by feeding the DHA/EPA enriched algal biomass to dairy cows. The purpose is to reduce the high purification cost of DHA/EPA by using cows as “DHA/EPA extractors” to extract the fatty acids from the complex algal biomass to make DHA/EPA in the form of milk. The dairy feeding study will be conducted at the WSUDairyCenter. The algal biomass obtained in (4) will be fed to the cows. Daily dry matter intake will be recorded and fatty acids in diet, milk and plasma will be compared. This will be a second or third year task.

4. Expected form of results:

The main result of this project will be a technology that can be employed to produce DHA/EPA using under utilized potato starch and recommendations for enhancing -3 PUFA content in milk. The technology will be summarized in a patent application entitled “DHA/EPA production from cull potato”. In addition, 2~3 peer-reviewed journal articles are expected to be published, along with an annual report and a final project report. These project outcomes will allow the research team to attract other funding to commercialize the process. Another outcome of the project will be the graduation of a doctoral student as main funding is requested to provide a graduate research assistantship.

5. Practical implications:

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are two important -3 PUFA. DHA is the component of the photoreceptor cells of infant retinas and is also involved in the development of infant brain tissue. The inclusion of supplementary DHA in infant formulas is strongly recommended by the World Health Organization. EPA possesses therapeutic activities against cardiovascular diseases, cancers, schizophrenia, bipolar disorder, and most recently noted, alzheimers. Both of these two fatty acids unfortunately cannot be synthesized by the human body. Fish oil is currently the conventional source of DHA/ EPA. However, there are some limitations of fish oil as a source such as peculiar taste, odor, stability problems, and the difficult and expensive purification of these acids from low-grade fish oil. In addition, the fish stocks are subject to seasonal and climatic variations. It is reported 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 microalgae.

The proposed project offers an attractive alternative for PUFA production. This approach is unique in that (1) it uses a heterotrophic process to produce -3 PUFA containing algae so that the production is not limited by light, (2) the process utilizes cull potato as an inexpensive carbon source to reduce the production cost, and (3) the algae will be fed directly to cows to produce -3 PUFA-enriched cow milk.

In addition to the cost of substrate for algal culture, the high purification cost of DHA/EPA from algal biomass is another limiting factor in the commercialization of microalgal EPA and DHA production. The proposed feeding of algae to cows avoids this high purification cost. Dietary supplements containing DHA/EPA for dairy cow could help promote marketing of milk based upon its DHA/EPA content. Also, the cow may benefit from higher intakes of algal biomass because algae usually contain a high level of protein/amino acids, lipids and vitamins, which are crucial for a cow’s growth. Thus, the project can benefit consumers because the regular consumption of EPA/DHA-containing cultured milk can eliminate the additional intake of EPA/DHA-containing capsules as dietary supplements.

6. Value to Washington exports:

WashingtonState grows ~165,000 acres of potatoes with harvesting averages of 60,000 pounds/acre. A 10% rate of cull translates to an average of 3 ton/acre. Cull potato costs the growers about $65/ton to grow, while farmers generally receive less than $10/ton for those culls. Consequently, the growers must pay the cost of growing these culls out of income from the marketable grades. Clearly, the inclusion of value-added components will be necessary in order to attain profit margins for farmers.

DHA and EPA processed from the fermentation of cull potatoes are capable of providing this new level of profitability for the farmers. If one takes EPA as an example and uses the following assumptions: there are three general cost streams involving feedstock, purification, and fermentation; the current world wide EPA market is estimated to be 300 tonnes/year (Lebeau and Robert, 2003); the cost of feedstock for algae utilizing cull potato as a carbon source is a conservative 10% of biomass vs. potato starch yield; the algae biomass contains 1% EPA; the conversion from cull potato to EPA is 0.1%; and the sale price of cull potatoes is $10/ton, then the feedstock cost would be $0.01/gram, the purification cost would be $150/gram (Molina et al., 1996), and the fermentation cost, conservatively assumed to be the same as the purification costs, would be $150/gram. Thus, the total manufacturing cost for 1 gram EPA would be $300.01 with purified EPA presently being manufactured commercially from cod liver at a cost of $369/gram (Lebeau and Robert, 2003). This $69/gram difference could translate into a better sale price for cull potatoes and better profitability for WashingtonState farmers.

The idea proposed in this project is to feed the algae biomass into cow feed and convert algal EPA into milk. The cost of this process is estimated as follows. Usually, about 30~40% of algal--3 PUFA can be converted into cow milk (Franklin et al., 1999), this means to produce the same amount of EPA as mentioned before, 2.5 to 3.3 times more potatoes should be used, thus, the potatoes/feedstock cost to produce one gram of EPA would be $0.025 - $ 0.033. The second cost factor, purification cost could be totally avoided as cow will extract algal DHA/EPA into the cow milk. For the fermentation cost, we still use the conservative estimation of $150/gram. Thus, the total cost proposed in this project would be $150.03/gram EPA, creating an even better margin for farmers.

In terms of improving Washington State exports both domestically and internationally, it is worthwhile to note and emphasize that two area industries, potato and dairy, will be positively impacted in terms of sales and exports by not only producing a more marketable product and attaining a greater sale price for their undervalued culls, but by developing a production process for fine nutraceuticals; providing much needed jobs and economic sustainability to rural communities.

Reference:

Bajpai PK, Bajpai P, Ward OP (1991) Optimization of production of docosahexaenoic acid (DHA) by Thraustochytrium aureum ATCC 34304. J. Am. Oil Chem. Soc. 68 (7): 509-514.

Franklin ST, Martin KR, Baer RJ, Schingoethe DJ, Hippen AR (1999) Dietary marine algae (Schizochytrium sp.) increases concentrations of conjugated linoleic, docosahexaenoic and transvaccenic acids in milk of dairy cows. J. Nutri. 129: 2048-2052.

Lebeau T, Robert JM (2003) Diatom cultivation and biotechnology relevant products. Part II: current and putative products. Appl. Micro. Biotechnol. 60: 624-632.

Molina GE, Robles MA, Gimenez GA, Ibanez GMJ (1996). Gram scale purification of eicosapentaenoic acid (EPA 20:5 n-3) from wet Phaeodactylum tricornutum UTEX 640 biomass. J. Appl. Phycol. 8: 359-367.

OGRD Budget

PI's Name: / Agency: / Deadline:
Dr. Shulin Chen / Year 1 / Cumulative
Dr. Ron Kincaid
SALARIES - 00
PhD-Step 76 / Salary / 19,134 / 19,134
QTR / -
Health / 1,499 / 1,499
1.5% / Med / 287 / 287
WAGES - 01
Student - Part-time
Undergraduate / Wages / 2,800 / 2,800
9% / Benefits / 252 / 252
Total Salary - 00 / 19,134 / 19,134
Total Wages - 01 / 2,800 / 2,800
Total Personnel (Salary & Wages) / 21,934 / 21,934
BENEFITS - 07 / 2,038 / 2,038
Total Salaries/Wages/Benefits - Combined / 23,972 / 23,972
NON CAPITAL EQUIPMENT - 16 ($500 - $4,999)
Materials and Supplies / 5,028 / 5,028
Total Non-Capital Equipment - 16 / 5,028 / 5,028
TRAVEL - 04
Domestic / 1,000 / 1,000
Total Travel - 04 / 1,000 / 1,000
TOTAL DIRECT COSTS / 30,000 / 30,000
BASE / 30,000 / 30,000
TOTAL F&A - 13 / Rate / 0.00%
TOTAL PROJECTS COSTS / 30,000 / 30,000
Year 1 - Allocation
Category / Object / Amount / Amount
Salaries / 00 / 19,134
Wages / 01 / 2,800
Personal Services Contracts / 02 / -
Goods/Services / 03 / -
Travel / 04 / 1,000
Computer Services / 05 / -
Equipment / 06 / - / -
Benefits / 07 / 2,038 / -
Stipends/Subsides / 08 / -
Phone / 11 / -
Subcontracts/Restricted / 14 / - / -
Non-Capital Equipment / 16 / 5,028
Total Direct Costs / 30,000 / -
F&A / 13 / - / 30,000
Total Costs / 30,000 / 0.00%
-
Cumulative - Allocation
Category / Object / Amount / Amount
Salaries / 00 / 19,134
Wages / 01 / 2,800
Personal Services Contracts / 02 / -
Goods/Services / 03 / -
Travel / 04 / 1,000
Computer Services / 05 / -
Equipment / 06 / - / -
Benefits / 07 / 2,038 / -
Stipends/Subsides / 08 / -
Phone / 11 / -
Subcontracts/Restricted / 14 / - / -
Non-Capital Equipment / 16 / 5,028
Total Direct Costs / 30,000 / -
F&A / 13 / - / 30,000
Total Costs / 30,000 / 0.00%

8.1. Detailed Expenditure Data:

Item (Add additional rows to the table as needed) / Amount
00 / Salaries / 19,134
1. Graduate Student / 19,134
2.
01 / Wages / 2,800
1. Student Employee / 2,800
2.
07 / Fringe Benefits / 2,038
1. Graduate Student / 1,786
2. Student Employee / 252
03 / Goods/Services / 5,028
1. Materials and Supplies / 5,028
a. Chemicals / 2,000
b. Glassware and Analytical Supplies / 2,000
c. Fermentor Supplies / 1,028
2. Publication Costs
a.
b.
4. All other Direct Costs
a.
b.
04 / Travel (List destination and $ amount for each trip) / 1, 000
1. Domestic (including Canada) / 1,000
a. Symposium on Bioproducts XXVI @ Tennessee / 1,000
b.
2. Foreign
a.
b.
06 / Equipment -- large. (List items and $ amounts.)
a.
b.
16 / Equipment – small. (List items and $ amounts.)
a.
b.

9.0 CREES Budget

UNITED STATES DEPARTMENT OF AGRICULTURE
COOPERATIVE STATE RESEARCH, EDUCATION, AND EXTENSION SERVICE

BUDGET

/ OMB Approved 0524-0039
Expires 03/31/2004
ORGANIZATION AND ADDRESS
Washington State University, Pullman, WA / USDA AWARD NO.
DURATION
PROPOSED
MONTHS: __12___
Funds Requested by Proposer / DURATION
PROPOSED
MONTHS: _____
Funds Approved by CSREES
(If different) / Non-Federal Proposed Cost-Sharing/
Matching Funds
(If required) / Non-federal Cost-Sharing/Matching Funds Approved by CSREES
(If Different)
PROJECT DIRECTOR(S)
Shulin Chen
Ron Kincaid
A. Salaries and Wages...... / CSREES-FUNDED WORK MONTHS
1. No. Of Senior Personnel / Calendar / Academic / Summer
a. ____(Co)-PD(s)......
b. ____Senior Associates......
2. No. of Other Personnel (Non-Faculty)
a. _____Research Associates/Postdoctorates......
b. ____Other Professionals......
d. __1__Graduate Students...... / 19,134
e. __1__Prebaccalaureate Students...... / 2,800
Total Salaries and Wages......  / 21,934
B.Fringe Benefits (If charged as Direct Costs) / 2,038
C.Total Salaries, Wages, and Fringe Benefits (A plus B) / 23,972
D.Nonexpendable Equipment (Attach supporting data. List items and dollar amounts for each item.)
E.Materials and Supplies / 5,028
F.Travel / 1,000
H.Computer (ADPE) Costs
I.Student Assistance/Support (Scholarships/fellowships, stipends/tuition, cost of education, etc. Attach list of items and dollar amounts for each item.)
K.Total Direct Costs (C through J)......  / 30,000
L.F&A/Indirect Costs (If applicable, specify rate(s) and base(s) for on/off campus activity. Where both are involved, identify itemized costs included in on/off campus bases.)
M. Total Direct and F&A/Indirect Costs (K plus L)  / 30,000
N. Other...... 
O.Total Amount of This Request......  / 30,000
P.Carryover -- (If Applicable) Federal Funds: $ Non-Federal funds: $ Total $
Q. Cost-Sharing/Matching (Breakdown of total amounts shown on line O)
Cash (both Applicant and Third Party).
- Non Cash Contributions (both Applicant and Third Party)
AME AND TITLE (Type or print) / SIGNATURE (required for revised budget only) / DATE

Project Director Shulin Chen

Authorized Organizational Representative

1