TITLE: Production of Omega-3 Polyunsaturated Fatty Acids from Cull Potato
YEAR INITIATED: 2005-2006. CURRENT YEAR: 2007-2008
TERMINATING YEAR: 2008
PERSONNEL:
Principal Investigators:
Dr. Shulin Chen, Professor, Department of Biological Systems Engineering, Washington State University
99164-6120 Phone: (509)335-3743; Fax: (509) 335-2272 [email protected]
Co PI Ron Kincaid, Department of Animal Sciences, Washington State University, Pullman WA
99164-6351, Phone (509) 335 2457 Fax (509) 335-1082, [email protected]
Co PI Zhanyou Chi, Department of Biological Systems Engineering, Washington State University,
Pullman, WA 99164-6120 Phone: (509)335-6239 Fax: (509) 335-2272;[email protected]
JUSTIFICATION:
The purpose of this 3-year project is to develop a cost-effective algal cultivation process converting cull
potato to docosahexaenoic acid (DHA), one of important -3 polyunsaturated fatty acids (-3 PUFA),
which have beneficial effects in preventing and treating human heart and immune diseases. The DHA
enriched algal biomass will be used as feed additives for dairy to enhance the nutritional value of the
milk. Satisfactory progress was made in the first two years in developing a viable process, and the
proposed effort in the next year will produce the technical information for the commercialization of the
technology.
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 (WHO). Also, research continues to demonstrate the need for DHA
beyond infancy. Studies 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.
Table I. Potential DHA Market for Use as Dietary Supplement in U.S.
$ in millions
Infant Formula
200
Dairy drinks
820
Cheese
500
Beverage (ex dairy)
770
Snacks/candy/cookies/crackers
625
Bread
510
Cereal/Breakfast food
465
Yogurt
70
Other
1,500
Total
5,460
*International market opportunity estimated to be approx. 2x U.S. market opportunity
(UBS Global Life Sciences Conference, September 27, 2006)
Besides using as the feed additive to cattle, the produced omega-3 enriched algae biomass could provide
an inexpensive source of omega-3 that does not originate from fish meal. In fact, Omega-3, although
widely used in the nutraceutical, food, and pharmaceutical industries, owes its biggest market share to
aquaculture fish feed. At present, fish oil production amounts to about 1 million tons annually, of which
70~80 % is utilized for the production of fish feed for farmed fish (Guzman, 2006, Chemical Marketing
Reporter). Currently, farm-raised fish obtain omega-3 only from fish meal. As the aquaculture feed
demand increases and ocean fishery resources decline, using fish meal to support aquaculture growth
becomes non-sustainable. In addition, the development of an organic fish movement requires an omega-3
source that does not originated from fish meal. Therefore, feeding an organic diet supplemented with
enriched omega-3 algae becomes almost the only future option for the aquaculture industry.
The proposed project offers such an attractive alternative with the PUFA production being derived from
cull potatoes that are used as feedstock for microalgae growth. The proposed project is unique in that: (1)
it reduces the cost of feedstock for algal cultivation by using cull potatoes, (2) it offers a high value use of
a low value potato product (cull potato, or potato processing water), and (3) it avoids the cost of purifying
-3 PUFA from algal biomass by using cow as “extractor”.
OBJECTIVES:
The objectives of this project include: (1) optimizing the algal culture of Schizochytrium limacnum SR21
for DHA production through the use of cull potato hydrolyzed broth; (2) developing a high cell density
for algal cultivation, (3) pilot study of the algae cultivation process, and (4) assessing the possibility of
using the algal biomass as additives in cattle feed. Objectives (3) and (4) will be addressed during this
funding year (04/01/2007-03/31/2008), and the project will be completed before 03/31/2008.
PROGRESS SUMMARY:
In the past year’s work on objective (1) and (2), the culture condition of Schizochytrium limacinum SR 21
was optimized and a fed batch culture process was developed. The results of these experiments and the
major accomplishments are summarized below.
1. Culture condition optimization
Optimization of the culture of Schizochytrium limacinum SR21 at a flask scale showed that cull potato
can in fact be an effective carbon and nitrogen source for production of DHA. 21.7 g/L of algae was
produced which contains 5.35 g/L of DHA when using a media of 50% potato hydrolyzed broth
supplemented with 20g/L glucose and without any extra-added nitrogen source.
2. Oxygen supply protocol optimization
The oxygen uptake rate at the different growth stages of this alga was investigated with both continuous
culture and batch culture in a fermentor with dissolved oxygen (DO) control. It was found that high
oxygen consumption was required in the cell propagating stage, when cell number increased with little
increase cell size. Low oxygen uptake was observed at the second stage of cell growth when the fatty acid
accumulation occurred. An optimized oxygen protocol has been developed to produce more cells (control
at 50% DO) and then provide a best condition (<5% DO) for fatty acid accumulation. With this protocol,
37.9 g/L dry algae biomass was produced.
3. 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, 25% of the initial nutrients
were supplemented to the culture daily, and the final cell density was increased to 55.6 g/L. With more
seed cells as initial cells, a bioreactor culture with fed batch protocol eventually attained a cell density of
102 g dry algae biomass per liter of broth. High cell density culture is critical for a production process
to be cost effective. Previously, there were only two reports that indicated that the algae could be cultured
at a cell density more than 100 g/L. This is a very important breakthrough in our research, as it
demonstrated that it is promising for our process to be industrialized.
PROCEDURES FOR THE PROPOSED STUDY NEXT YEAR:
Pilot study of the algae cultivation process
The main research focus for the last project year is to scale up the process for performance evaluation.
The results from the scale-up studies will provide the design parameters for commercial applications.
Scale-up will be accomplished using 1 L, 25L, and 125L fermentors within the Biomass Processing and
Bioproduct Laboratory at WSU.
The dissolved oxygen (DO) concentration will become the limiting nutrient in this process, since the cell
density in the later stage of this algae culture process is more than 100 g/L and although less oxygen is
consumed for the fatty acid accumulating step, such high cell density, on the whole, will require a large
amount of oxygen. The dissolved oxygen (DO) in the broth is limited by both oxygen uptake rate (OUR)
on cells and oxygen transfer rate (OTR). The mass balance of oxygen can be described as:
dCo/dt = OTR - OUR = KLa ∙(C*-C) - (Qo2∙ Cx)
In a specific fermentor and medium, the OTR value mainly depends on the air flow rate and the agitation
speed. In the above equation, KLa represents the ability for oxygen transfer in a specific fermentation
system and often serves to compare the efficiency of bioreactors and mixing devices as well as being an
important scale-up factor. In fact, fixing KLa values is a commonly used criterion for scale-up of aerobic
fermentations.
For the scale up, the OUR, OTR, and KLa values in both the seed cell producing stage and fatty acid
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 find out 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.
The produced algae in the pilot study will be washed and dried by a spray dryer. Then, the components in
the algae dry biomass will be analyzed for protein, carbohydrates, fatty acid profile, vitamins, sterols, and
amino acid profile. The price of the produced algae biomass will be evaluated based on their quality with
comparison to present market prices.
The cost of raw materials, energy, labors, as well as the process productivities etc. in the pilot study will
be recorded and studied. A techno-economic assessment of the feasibility of DHA enriched algae biomass
production will be conducted using Matlab Simulink software using the laboratory and scale-up results
and standard chemical engineering plant design and costing concepts as summarized by Peters and
Timmerhaus (Peters, M.S. and K.D. Timmerhaus. 1991. Plant design and economics for chemical
engineers. New York: McGraw-Hill publishing company, Inc.).
Assessing the possibility of using the algal biomass as additives in cattle feed
A feasibility additive study will be performed by feeding the DHA enriched algal biomass to dairy cows.
The purpose is to reduce the high purification cost of DHA by using cows as “DHA extractors” to extract
the fatty acids from the complex algal biomass to make DHA in the form of milk. The dairy feeding study
will be conducted at the WSU Dairy Center.
Dairy cows in mid-lactation will be randomly assigned to the experimental groups corresponding to 3
different feeding. Each group of cows will be fed with 4 kg concentrate/cow/day (the control group),
concentrate mixed with 750 g algae dry biomass and concentrate mixed with 1500 g algae dry biomass.
Feed samples will be analyzed for dry material, crude protein, crude fat and ash according to AOAC and
neutral detergent fiber, acid detergent fiber and acid detergent lignin. Composite milk samples will be
collected from four consecutive milkings on the 2 last days of each period. Milk composition (fat, protein)
then will be determined and compared, in terms of both saturated and unsaturated fatty acids.
To evaluate the economical viability of this cattle feeding process, the value of feeding dry algal biomass
will be compared with the added value of milk enriched with DHA than the milk without DHA.
ANTICIPATED BENEFITS:
Cull potatoes are currently a negative-value product for the growers, costing the growers $70 to $120 per
ton to grow, and even if farmers receive as high as $60/ton for those culls, they still must pay the
remaining negative cost of growing these culls out of income from the marketable grades. Producing
omega-3 fatty acids from the cull potatoes by algal fermentation provides a new level of profitability for
the farmers.
It was reported that the production cost of algae dry biomass has been reduced to less than $5/kg, and
even as low as $2/kg (Gladue and Maxey, 1994, Journal of Applied Phycology, 131-141). At this low cost,
the raw material cost would account for higher ratio of the total production cost. Current commercialized
algae culture processes use glucose or corn syrup as the carbon sources, which is the biggest part of raw
material cost, and another major cost element is the nitrogen source. Cull potato can provide both of
carbon and nitrogen source, as well as some salts and trace elements. Thus, using cull potato could
significantly further decrease the production cost, and make it competitive.
Washington State grows ~156,000 acres of potatoes with harvesting averages of 60,000 pounds/acre. A
10% rate of cull translates to an average of 3 ton/acre. Thus, the total volume of cull potato is about
500,000 tons, which at most could produce 50,000 tons of algae biomass and 15,000 tons DHA. It was
estimated that 100,000 tons of DHA demand in the aquaculture industry, and the fish oil originally DHA
need to be substituted gradually (Sijtsma, L.,2004, Applied microbiology technology, 146-153). For the
infant formula market, 100~200 tons of DHA demanded for the European annually (Lewis, 1999, Marine
Biotechnology, 580-587), and the demand from US will no less than this number. Thus, there is more than
enough of a market to meet the capability of this algae-omega-3 production process.
INFORMATION TRANSFER:
During the whole project period, the WSPC and the state potato growers will be kept updated on the
results/progress of the project by quarterly/annual/final report. The PIs will also submit at least 2 growerdirect articles to the WSPC newsletter, Potato Progress. In addition, PI and Co-PI will attend WSPC
annual meeting to communicate with WSPC members and local potato growers.
PROJECT TIMELINE: Note: FY starts from April of each year.
FY 2005-2006
FY 2006-2007
FY 2007-2008
Quarter
Quarter
Quarter
Procedures
1
Pilot study
Cattle feeding
2
3
4
1
2
3
4
1
2
3
4
4
7
10
1
BUDGET:
FY 2005-2006
Salaries 1 Post Doctoral Research
Associate
Time-slip 1
FY 2006-2007
FY 2007-2008
$17,712
$17,712
$17,712
$800
$800
$4,400
$4,373
$4,373
$10,430
Goods & Services
Algal species
1,000
1,000 0
Chemicals (including cull potatoes,
industrial enzymes etc.)
Glassware & flasks
1,500
1,500 1,225
300
300 350
Fermenter supplies
-
-
2,100
Centrifuge supplies (for algal biomass
separation)
Other expense (materials shipping,
correspondence etc.)
Fatty acids standard
-
-
700
500
500 350
300
300
GC column
600
600 280
Other analytical supplies (solvent,
pure H2, N2 and He gas etc.)
173
173 245
Animal care
3,780
Diet analysis, blood sampling,
general supplies
1,400
Travel 2
$1,000
$1,000
$2,000
Employee Benefits
$6,115
$6,115
$6,475
Regular benefits
Benefit- time-slip employee(s)
(10%)
Total:
1
3
6,035
6,035
6,035
80
80
440
$30,000
$30,000
$41,017
Salaries: Post Doc Salaries (8 months in years 3).
Timeslip: Undergraduates to work for 80 hours at $10/hour for Year 1 and 2. In Year 3 the students will
work on sample analysis and cow feeding experiments.
2
Travel: FY 2005-2006: visit local potato growers, introduce the project to local farmers, collecting
culls.
FY 2006-2007; visit local potato farmers, attend the Washington State Potato Commission
annual meeting (the meeting will benefit the growers by updating the progress of the project).
FY 2007-2008; multiple field trips to dairy farm for algal-feeding experiments; attend the
Washington State Potato Commission annual meeting.
3
Year 2:
Year one figures are actual expenses where available plus anticipated figures for a total
allocation of $30,000.
Projected Expenditures (by quarter)
Time Period
WSPC Funds
Total Funds
Jan-Mar
2007
7,500
10,000
Apr-Jun
2007
10,254
12,822
July-Sept
2007
10,254
12,822
Oct-Dec
2007
10,254
12,822
Jan-Mar
2008
10,254
12,822
Apr-Jun
2008
0
0
Comprehensive current (FY 2007-2008) budget (required) a:
Other Support of Project
Expenditure
Salaries
Time-slip
Goods &
Services
Travel
Equipment
Employee
Benefits
Total
WSPC
Request
State/Federal Grant funds
base funds
Source:
WSU Center for
Sustaining Agriculture
& Natural Resources
In-Kind
Support
Source: b
Total
Cost
$17,712
$5,314
$ 5,946
$28,972
$4,400
$1,320
$5,720
$10,430
$3,129
$13,559
$2,000
$600
$2,600
-00-
0
$0
$6,475
$1,943
$2,022
$10,440
$41,017
$12,305
$7,968
$61,290
Note: a Budget data provided in 'Other Support of Project' is for informational purposes, only, for the
Potato Commission to understand the scope of the project. These estimated costs are not
presented as formal cost-sharing and therefore do not constitute a cost-share obligation on the
part of Washington State University. Moreover, there is no requirement for WSU to document
this Other Support of Project as part of any cost-share or matching obligation.
b
Dr. Shulin Chen, PI of the project, will allocate 5% of his 9 month appointment
to this project.