Annual Progress Report s1

ANNUAL PROGRESS REPORT

TITLE: Production of Omega-3 Polyunsaturated Fatty Acids from Cull Potato

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]

REPORTING PERIOD: 1/1/2006-12/31/2006

ACCOMPALISHMENTS

Four objectives were proposed for this project, including: (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. The research focus of this reporting period (second project year) was Objective 2 although some work on Objective 3 was also initiated. In the study for developing high cell density culture process, it was found that the growth of the DHA producing algae strain, Schizochytrium limacnum SR21, can be divided into two stages: (1) cell number increasing stage: cell reproduction and rapid cell number increasing with little increase in the size and weight of each cell and (2) cell size increasing stage: cells stopped reproduction but cell size enlarged due to lipids accumulation. Notably, the optimal culture conditions for the two stages were different. Based on this discovery, separate optimizing culture conditions of the two stages were developed, and by using a “shifting” strategy between these phases, significant improvement of algae biomass production was made. More than 100 g/L algae dry biomass was obtained in the lab scale experiment. This process was developed with the advances in the following aspects:

Oxygen supply protocol optimization The oxygen uptake rate of Schizochytrium limacnum SR21 in the different growth stage 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 concentration was required in the cell reproduction stage, while low oxygen concentration was required in the fatty acid accumulating stage. An optimized oxygen protocol was determined to produce more cells with dissolved oxygen controlled at above 50% level of saturation, and then providing a low dissolved oxygen concentration of no more than 5% saturation for fatty acid accumulation. With this protocol, 37.9 g/L dry algae biomass was produced.

Fed-batch culture protocol development A fed-batch culture protocol was developed, since the increased cell number need more nutrients to accumulate lipids to produce more biomass. In the fed batch culture, 25% of the initial nutrients were supplemented to the culture medium 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 102g dry algae biomass per liter of culture broth.

1 High cell density culture is obligate for a production process to be cost effective. Before this, there were only two reports on the algae cultured in a cell density of more than 100 g/L. This is a very important breakthrough in our research, and this showed that it is promising for this process to be industrialized.

Besides the impacts to the industry, this discovery of two-stage growth of Schizochytrium could improve the general understanding of the relationship between biomass production and lipid accumulation in lipid-producing microorganisms, especially microalgae. Previously, it was considered that lipid accumulation in the cell required the exhaustion of some essential nutrients in the culture medium, and limiting them is an effective way for inducing lipid accumulation (Granger, et al., 1993). However, this procedure also resulted in decreases in total productivity due to the overall effects on growth by the nutrient limitation. With our discovery of the two- stage growth, however, cell proliferation and lipid accumulation can be optimized separately with a two-stage culture, rather than one stage in which lipid accumulation conflicts with cell proliferation. This two-stage culture process could be used not only in the omega-3 fatty acids production process, but probably more widely, in all single cell oil (SCO) producing processes, including biodiesel production from algae, which is the largest potential of SCO application.

RESULTS

1. Verification of two-stage growth of Schizochytrium limacnum SR21

1) Cell condensing experiment The cells were cultured with the condition optimized in the previous experiment. At the time points shown in the table I, cells in 2 or 3 flasks were centrifuged and combined into 1 flask, cultured with the original medium in only 1 flask of the original 2 or 3. The maximum production was 42.7 g/L, which was far more than the control, which is 29.8 g/L. This indicates the nutrients in the original culture were enough to support more biomass production, and the reason why biomass production was stopped was because not enough number of cells was produced at the original culture.

Table I. the dry cell weight of algae biomass in the fermentation broth

Combined Dry cell weight Dry cell weight Dry cell weight time after combining 5 days 10 days (g/L) (g/L) (g/L) control 5.6 14.5 29.8 2 in 1 40 hrs. 11.2 21.3 33.2 3 in 1 16.8 25.7 42.7 2 in 1 66 hrs. 16.4 23.0 38.7

2) Difference between increasing of cell number and dry cell weight The variation of cell number and dry cell weight during the culture process was investigated. It was interesting to observe that the cell number stopped increasing after 24 hours culture, but the dry cell weights (g/L) kept increasing (Figure 1). The only explanation of this phenomenon was that the “body weight” of each cell was increased. This was also proved with the enlarged cell size, which was observed under microscope. With the understanding of two-stage growth, thus, improving the biomass production could be done from two aspects: producing more cells in the first stage, and grow bigger cells in the second stage.

2 flask DCW 12 BR DCW 140 flask cell number 120 10 BR cell number ) L m / s )

100 l l L / 8 e g c

6 t ^ h 80 0 g i 1 (

r l e l b e 60 c

m y u r

40 l e C 2 20

0 0 0 20 24 28 33 44 time (hr)

Figure 1. variation of cell number and dry cell weight in the culture process

2. The oxygen’s effect in the Schizochytrium limacnum SR21 culture

1) Higher dissolved oxygen (DO) culture After inoculation, the algae cells reproduced rapidly and the cell number stopped increasing after 48 hr. To investigate the effect of oxygen to the cell reproducing, the culture in bioreactor (BR) and shake flask was compared. The DO in the bioreactor was controlled at 50% with a fixed aeration rate of 1 VVM and agitation speed between 300-500 rpm, which was cascaded to DO. The 250ml flask with 50 ml broth was stopped with a cotton stopper, and placed in a shaker with a 175 rpm rotation speed.

Table II. The culture results with different D.O. control at 48 hr. Dissolved oxygen Dry Cell Weight cell density cell body weight (g/L) (10 6 cells (mg/106cells) /mL) Bioreactor (50%) 19.2 146 0.13 Shake Flask (175 rpm) 7.4 52 0.14

With a higher DO, the culture in bioreactor produced 146× 106 million cells/ml, but the culture in shake flask produced only 52 (Table II). This indicated that the cell reproduction required higher DO.

2) The oxygen consumption in the culture The oxygen uptake rate (OUR) in the bioreactor culture was also investigated. The OUR reached its maximum at 16th hr. The specific oxygen uptake rate (SOUR) reached its maximum at 8th hr, and kept decreasing dramatically (Figure 2). This indicated that the large amount of oxygen was consumed in the cell reproduction stage. The OUR decreased to a very low level at 40th hour, while the SOUR in term of cell number decreased to a very low level at 24th hour. This indicated that when the cells stopped growth, there was only a little oxygen consumed.

3 OUR SOUR 0.90 0.09 0.80 0.08 0.70 0.07 n i 0.06 ) 0.60 m n / i s l m l

/ 0.05

0.50 e L / c

6 0.04 m 0.40 ^ ( 0

g 0.03 U 0.30 m O 0.20 0.02 0.10 0.01 0.00 0.00 0 20 40 60 0 20 40 60 time (hr) time (hr)

Figure 2. Oxygen uptake rate in the high DO culture

3) The culture in bioreactor with high D.O. during all the process

culture in bioreactor w ith 60% DO pH in the 60% DO culture

bioreactor 25 8 bioreactor flask 7 20 flask ) ) 6 L L / / g g ( (

5 t t 15 h h g g i i 4 w w

l l l l 10 e e 3 c c

y y r r 2 d d 5 1

0 0 0 50 100 150 200 0 90 114 144 tim e (hr) tim e (hr)

Figure 3. The culture with high DO

The DO in the bioreactor culture was controlled at 60% by altering the agitation speed. After 72 hours, 50 mL broth was taken out to the shake flask, and the culture was continued with a lower DO, while the DO in bioreactor was maintained at 60%. The cell density in bioreactor was 15.7 g/L at maximum, while the culture in flask was increased to 20.6 g/L. The pH in flask maintained at around 6.7, while the pH in bioreactor decreased dramatically at the later phase, indicating acid was produced (Figure 3). After reproduction, the algae cells start to accumulate lipids inside the cells. In this stage, too high oxygen concentration probably drove cells to metabolite strongly, but not to accumulate lipids to storage energy, as this culture did. So, lower DO may be better for the lipid accumulation stage.

4) Shift strategy study The cells were cultured in bioreactor with DO controlled at 50% or in the 250ml-shake flask for 2 days at 25 ºC. After that, supplemented with another 100 g/L carbon source and transferred all of them to shake flasks and continued the cultures at 20 ºC. After another 8

4 days culture, the dry cell weight from bioreactor culture had little increase, but that of flask culture increased from 7.4 g/L to 20.7 g/L, due to the increase of cells’ body weight, from 0.14 to 0.32 mg/106 cells (Table III). The cease in increasing of cells’ body weight from bioreactor culture could be due to either insufficient nutrients in the culture medium or inhibition.

Table III. The shift culture from bioreactor and shake flask Initial time Dry cell wight cell density Cell body weight culture (days) (g/L) (106cells /ml) (mg/106 cells) BR 2 19.2 146 0.13 10 20.9 163 0.13 flask 2 7.4 52 0.14 10 20.7 64 0.32

3. Fed batch culture 1) Effect of medium changing and shifting time To avoid nutrient depletion or metabolites inhibition, after the seed cells culture in bioreactor or flask for 48 hours, the cells were transferred to fresh medium and cultured another 8 days. The culture transferred to flask without medium changing as the control, with 100 g/L carbon source supplemented.

Table IV. Effect of medium changing in shifting cultures Culture Feeding protocol Dry cell cell density cell body weight vessel weight (g/L) (106 cells /ml) (mg/106 cells) carbon source 20.9 163 0.13 Bioreactor supplemented Medium changed 36.2 162 0.22

Medium changed 32.1 67 0.48 Flask carbon source 20.7 64 0.32 supplemented

The medium changing enhanced the biomass production greatly, from 20.9 g/L to 36.2 g/L, which due to only by cell body weight increasing, but not cell densities (Table IV). This showed that fresh medium provided a better culture condition than just adding 100g/L carbon source. Analysis of the broth from both of the cultures with ion chromatography (IC) didn’t reveal obvious organic acid (data not shown), which were usual inhibitosr in fermetation process, indicating that there might be no inhibition. If so, the better result from fresh medium changing culture should be due to the supplementation of nutrients, but not removing the inhibitor.

Compared to the cells from flask, the body weight of cells from bioreactor was far less than that from flask. This may be due to the average nutrients provided to each single cell from flask was higher than that of bioreactor, which was in a higher cell density. This also indicated that the biomass production with cells from bioreactor can be further enhanced if culture condition were further optimized.

The optimal shifting time was also investigated (Table V). It was found that 40~48 hr was the best time for the cells to be shifted to accumulate lipids, since cells number was still increasing before this time.

5 Table V. Effect of shifting time Shift time Final Dry cell weight cell density cell body weight (hr) (g/L) (106 cells /ml) (mg/106 cells) 18 24.6 106 0.23 24 25.3 118 0.21 30 29.3 124 0.24 40 37.9 140 0.27 48 36.2 162 0.22

2) Optimization of feeding protocol Since no inhibitor was determined in the IC analysis, the limited biomass production was probably not caused by inhibition, but by nutrient depletion. To verify this, two cultures without medium changing but with nutrient feeding were conducted. The control was fed with only carbon and nitrogen source, while the salts feeding culture also fed with mineral salts in the original medium, besides carbon and nitrogen source. All of their feeding rates were 25% of original amount daily.

50 ) L / g (

t h g i

e 40 w

l salts feeding 1 l e c

salts feeding 2 y r

D 30 control 1 control 2

20 4 6 8 10 12 14 Time (days)

Figure 4. The culture with nutrient feeding

The results showed that fed culture with only carbon and nitrogen source produced biomass of less than 40 g/L, at the same level as in the former experiments with medium change. While the cultures with also salts feeding produced 55 g/L biomass (Figure 4). This indicated that not only carbon and nitrogen sources, but also mineral salts are needed to be added to obtain higher biomass production.

3) Effect of oxygen in the fatty acid accumulation stage Although high biomass production was obtained in the culture fed with all nutrients in original medium, the time was too long (Figure 4). As a whole, the productivity was very low. It was assumed that oxygen might be the limiting factor in this culture, since although specific oxygen uptake rate (SOUR) was low in the lipid accumulation stage, the too high cell density in the broth would require a lot of oxygen supply as a whole. Adequate oxygen which probably was not able to be provided in the shake flask culture. To verify this, cultures in both shake flask and a bioreactor with DO controlled at 5% were conducted. The culture temperatures were 20 ºC. Cultures were fed with 25% original medium nutrients daily.

6 60 BR with 5% DO

50 flask

40 ) L / g ( 30 W C D 20

0 0 2 4 6 Time (day) Figure 5. Effect of oxygen in the lipid accumulation stage

The result showed that the biomass production in bioreactor increased rapidly. After transferring, it took only 6 days to reach the maximum 55 g/L (Figure 5), much faster than the previous experiment in flask, which took 12 to 14 days. This indicated that oxygen was the limiting factor in the flask culture. Although low oxygen concentration is required in the lipid accumulation, too low oxygen transfer rate could be a limiting factor in this high cell density culture.

4. High cell density culture

100 ) L / g

t h g i

e 60 w

l l e c 40 y r D 20

0 0 2 4 6 8 Time (day)

Figure 6. High cell density culture

55 g/L biomass was produced from 200× 106cells /ml before shifting and feeding, with the optimized feeding and oxygen control protocol. It was assumed that even more biomass could be produced with more seed cells. To verify this, a culture with higher cell density of 360× 106cells /ml at initial was conducted. The result showed that 102.4 g/L algae biomass was produced in 7 days after shifting and feeding (Figure 6). This high cell density culture

7 makes it possible for high efficiency production of DHA in algae biomass, which is very promising to be industrialized.

PUBLICATIONS Zhanyou Chi, Bo Hu, Yan Liu, Craig Frear, Zhiyou Wen, Shulin Chen, Production of Omega-3 Polyunsaturated Fatty Acids from Cull Potato Using an Algae Culture Process, Applied Biochemistry and Biotechnology (in press)

PRESENTATIONS & REPORTS Quarterly report to potato commission, 10/1/2006 Quarterly report to potato commission, 4/1/2006