Progress 09/01/06 to 08/31/09
Outputs
OUTPUTS: The heterotrophic marine alga Crypthecodinium cohnii is capable of producing edible oil with a high fraction of DHA and less than 1% of the total lipid content from other long-chain PUFAs. Batch-production of DHA from C. cohnii has been conducted in bioreactors at a commercial level; however, the opportunity to improve upon a process that would yield a sustainable, vegetarian source of DHA exists. The objective of this study was to investigate growth parameters in bioreactor vessels that would cultivate the highest yields of C. cohnii with the greatest lipid fraction. High-density batch cultivation of C. cohnii in an environment-controlled bioreactor has been completed. The intent was to verify growth and lipid deposition under a controlled temperature and feeding strategy. Table 1 shows the accumulation of DHA by C. cohnii at 12 h intervals beginning at 24 h over a 137 h time frame. Table 1: Accumulation of DHA by C. cohnii in an environment-controlled bioreactor. High cell density was achieved by feeding a 25% glucose solution continuously at a rate of 0.78 ml/min. Temperature was maintained at ~27˚C for 24 h and then reduced to 15C until harvest (137 h). Hour 24 - 15.7% 41 - 24.98% 48 - 29.95% 65 - 28.32% 89 - 35.34% 113 - 35.27% 137 - 36.65% PARTICIPANTS: Deborah James: M.S. Graduate Student, Deniz Inan: M.S. Graduate Student, Christopher Moore: Undergraduate Lab Technician, Loren Bane: Undergraduate Lab Technician, Sarah Beamer: Research Assistant, Jacek Jaczynski: Associate Professor, Kristen Matak: Assistant Professor TARGET AUDIENCES: Efforts: Laboratory instruction. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The results of this trial and other trials whose results are not shown have been used to design a continuous growth, feeding and harvesting system. Many hours of labor have gone into retrofitting existing equipment, determining flow rates, strategies to maintain temperature, feeding and harvesting techniques, etc. The investigators are now confident that the processing strategies are capable of supporting continuous growth and harvest of C. cohnii. It is the objective of continuing research to ascertain processing strategies that would optimize C. cohnii biomass accumulation and DHA production. As a result of this research, two abstracts have been published and two posters have been presented at international conferences.
Publications
- Inan, D., Beamer, S., Jaczynski, J., Matak, K. 2009. Docosahexaenoic acid production by the marine alga Crypthecodinium cohnii in a continuous mode process. Abstract # 120-07. Institute of Food Technologists Annual Meeting, Anaheim, CA, June 2009.
|
Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: The purpose of this study was to investigate DHA production by C. cohnii using a novel, continuous-mode process. Cultivation of C. cohnii was conducted in two 15 L computer controlled bioreactor vessels. Temperature of both vessels was maintained at 27˚C during the growth mode of the study and standard media (25g/L glucose, 5.5g/L yeast, and 25 g/L salt) was administered to both bioreactors. After 40 h, the system was switched to "continuous" mode where one vessel was maintained as a growth vessel at 27˚C, and the other as a lipid accumulation vessel at 17˚C. In continuous mode, standard media was administered to the growth vessel and a 25% glucose solution was administered to the lipid accumulation vessel. Results showed that C. cohnii growth was maintained in continuous production. PARTICIPANTS: Deniz Inan, M.S. Graduate Student Sarah Beamer, Research Assistant Jacek Jaczynski, Co-P.I. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The results showed that C. cohnii growth could be maintained in continuous mode bioreactors with lipid and DHA productivity. More research is needed to optimize processing parameters in continuous mode so that biomass concentration and DHA production exceed reported values from batch mode processes. Future research will look at the effect of different temperatures and carbon sources on C. cohnii growth and DHA accumulation in a continuous mode process. Results of this study and its continuation will improve the continuous process to provide a sustainable and economical vegetarian source of DHA which could be incorporated in value-added food items.
Publications
- No publications reported this period
|
Progress 09/01/06 to 08/31/07
Outputs
The effect of different initial growth media on growth parameters of C. cohnii (ATCC 30772) were determined. C. cohnii was initially grown in complex ATCC media 460 and compared with initial growth in simple media (9g/L glucose, 2g/L yeast, and 27.8g/L salt). Cultures were then grown in standard growth media (25g/L glucose, 5.5g/L yeast, and 25g/L salt) for the duration of the study. Optical density (OD) was taken every hour for 60 hours. Conductivity, pH, and dissolved oxygen (DO2) tension were also measured. Results showed that C. cohnii grew equally in both media (P<0.05). There were no significant differences in pH, conductivity, or DO between the two media (P>0.05). The OD indicated a lag phase of growth for 13 h, followed by an exponential phase of growth until 48 hr. High-cell density cultivation of C. cohnii was performed in computer-controlled laboratory bioreactors (Biostat Twin Controller, Sartorius BBI Systems, Bethlehem, PA, USA). The initial medium
consisted of 5.5 g/L yeast extract, 25 g/L sea salt, 25 g/L glucose. Inoculum (4.6 % v/v) was transferred from flask into the above media and incubated at 30C for 24 hours. The computer program was set to maintain a dissolved oxygen (DO) concentration above 30% achieved by stirring (100-300 rpm) and aeration (1 L min-1) with filter-sterilized air. A commercial polysaccharide-hydrolyzing enzyme (Glucanex, Novo Nordisk, Neumatt, Switzerland) was added at the start of cultivation (0.5 g L-1) to reduce culture viscosity which will promote aeration. Foam production was suppressed with antifoam (Mallinckrodt Baker Inc., USA). The pH was maintained at 6.5+/-0.1 by automated addition of 1 N NaOH (BIO 1) and 10 N NaOH (BIO 2). Continuous cultivation mode began after 24 h. To stimulate DHA accumulation, the temperature of the second bioreactor (Bio2) was reduced to 15oC. Fresh media was pumped into the first bioreactor (Bio1) while alga-rich broth was pumped out of Bio1 and into Bio2 at a flow
rate that corresponds to a retention time of 48 h in Bio1 and 172 h in Bio2. This time has been shown to be adequate for maximum cell growth and lipid production. Samples were harvested and centrifuged every 24 h for 172 h. The results indicated that DHA production did not meet batch mode quantities; therefore, current studies will determine parameters that would increase DHA yields.
Impacts
Docosahexanoic acid is optimal in prenatal and neonatal stages of growth, particularly with the development of the nervous system and visual development, as well as reducing the risk of various diseases such as cardiovascular disease, inflammatory disorders, and thrombosis. C. cohnii is capable of producing edible oil with a high fraction of DHA. The current method of cultivating C. cohnii is limited to batch processing. A continuous method to produce DHA would increase DHA production and lower costs, allowing more companies to incorporate the DHA in commonly eaten foods allowing the general public to attain the favorable health benefits.
Publications
- James, D.L., Elizabeth R. Parsons, J. Jaczynski, and K.E. Matak. 2007. Effect of Different Initial Growth Media on Growth Parameters of Crypthecodinium cohnii ATCC 30772. Biotechnol Progress. In Review.
|
|