Progress 01/01/99 to 12/31/03
Outputs
Results from a study to evaluate the feasibility of utilizing rice bran as solo substrate for Pythium irregulare submerged culture to produce important polyunsaturated fatty acids indicated that the fungus grew well in rice bran media. The results showed rice bran served well as solo substrate for P. irregulare submerged culture to produce eicosapentaenoic acid and arachidonic acid, with the bioconversion rates of 0.42 percent and 0.16 percent, respectively after appropriate length of culture. Meanwhile, the majority of rice bran components had been utilized or removed by P. irregulare at the end. Results of a study to examine the effects of media composition, composed of 1-4 percent glucose and 0.25-1 percent yeast extract serving as carbon and nitrogen sources, and the corresponding carbon/nitrogen (C/N) ratio, indicated that the interaction of carbon and nitrogen sources played a significant role in biomass synthesis. The optimal C/N for biomass synthesis was 12/24
and the substrate utilization rates were higher if the glucose level was no higher than 2 percent. Nitrogen limitation occurred if the C/N reached 32, which turned to trigger the oil accumulation. Media composition and C/N didn't change the main fatty acid type but did the amount in the oil. Individual fatty acids yield to biomass had positive relationship with the oil content. The optimal media composition for EPA and AA production was 2 percent glucose and 0.25 percent yeast extract, of which the C/N was 32. The EPA and AA yields to biomass were reached 20 mg/g and 14.7 mg/g under that condition, respectively. The growth of Pythium irregulare and fatty acid synthesis were temperature dependent. Optimal temperature for growth was 25 degress Celisus and the biomass peak appeared at the 4th day, while those of the other two temperatures (13 degrees Celisus and 9 degrees Celisus) were the 6th and 8th day, respectively. At all three temperatures, the growth fit the Pirt Model fairly
well. Lipid was the mixer of the primary and secondary metabolites of the microorganism. The lipid peak appeared at or slightly later than the biomass peak. For the target polyunsaturated fatty acids, EPA and AA synthesis, 25 degrees Celisus also was the optimal temperature, at which the EPA (eicosapentaenoic acid) and AA (arachidonic acid) yields were 124 mg/L and 75 mg/L, respectively. Lower temperature didn't help to increasing the target fatty acids yields. Suitable temperature shifting benefited the lipid and fatty acids synthesis only if temperature shifting was taken when the nitrogen limitation occurred. In our research, the suitable temperature shifting strategy was incubating the culture at 25 degrees Celisus for 5 days and dropped the temperature to 13 degrees Celisus for 2 days.
Impacts
Value-added uses for byproducts from sources such as rice, sweet potato and sugarcane have great economic potential in contrast to the high waste treatment costs incurred from disposal of these materials.
Publications
- Zhu, H. 2002. Utilization of Rice Bran by Pythium irregulare for Lipid Production. M.S. Thesis, Louisiana State University.
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Progress 01/01/02 to 12/31/02
Outputs
The byproducts from rice have demonstrated excellent potential as a substrate for the filamentous fungi, Pythium irregulare capable of producing omega-3 enriched oils when grown at optimal conditions. The oils are easily extracted with supercritical CO2 and are essentially odor-free compared to fish oils containing similar fatty acids (Walker et al., 1999). Several graduate thesis projects were completed in 2002. The initial phase of the supercritical fluid extraction study on rice bran oil and fungal oil was completed. Results showed that particle size of both rice bran and fungal biomass were important for extraction kinetics. The kinetic study indicated that oil extracted more slowly from the fungal biomass than from the rice bran control samples. A second masters thesis was completed for determining optimal growth conditions of Pythium irregulare on rice bran including temperature, time of batch culture, and C/N ratio. Room temperature growth yielded the highest
growth rate and oil production. Significant loss of omega-3 fatty acids was evident after 7, 9 and 11 days for growth at 25, 19 and 14 degrees Celisus, respectively. The conversion products are still unknown. One Ph.D. project is in progress to examine potential use of rice ash as an absorbent compound for primary purification and fractionation of rice bran oil subjected to supercritical carbon dioxide.
Impacts
Potential market value could exceed the nutraceutical value of rival oils such as fish, flax (linseed) and borage oils currently sold at approximately $7 per 100g (100 tablets). Value-added uses for the byproducts from sources such as rice, sweet potato and sugarcane have great economic potential in contrast to the high waste treatment costs incurred from disposal of these materials.
Publications
- Walker, T.H. 2002. Bioprocessing technologies for production of Nutraceuticals from food and agricultural byproducts. Proceedings of the AIT International Conference on Innovations in Food Processing Technology and Engineering. Bangkok, TH.
- Patel, P. and Walker, T.H. 2002. Rice hull ash: potential use as an adsorbent. Volume 3. IBE proceedings, Baton Rouge, LA.
- Badal, R. and Walker, T.H. 2002. Supercritial fluid extraction of bioconverted rice bran oil. Volume 3. IBE Proceedings.
- Kim, C. S., Walker, T. H. and Drapcho, C. M. 2002. Introduction of finite element methods (FEM)to biological engineering core courses. Volume 3. IBE proceedings, Baton Rouge, LA.
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Progress 01/01/01 to 12/31/01
Outputs
The proposed experiments from funding year 2000 gave preliminary results to assess the extractability of rice bran oil and the feasibility of bioconversion of rice byproducts to omega-3 polyunsaturated fatty acid (omega-3 PUFA) enriched oil followed by extraction. Rice bran (from Cyprus var.) milled with a pilot-scale Satake rice mill in the Biological and Agr. Engr. Dept. initially containing 50 g/L COD decreased to less than 6 g/L after 6 days of bioconversion using Pythium irregulare, which indicated significant ability for the fungi to remove waste while increasing the total oil content from 18 to 30 wt %. Further studies in 2001 investigated the effect of carbon to nitrogen ratio (C/N) on fungal growth and PUFA production and supercritical fluid extraction (SFE) of rice bran and/or bioconverted oil using carbon dioxide. The optimum C/N ratio was about 32, which is close to that of natural rice bran media. This indicated that the nitrogen amount was adequate for
omega-3-PUFA production, and no additives were needed. The rates of extraction from 0.1 mm particles resulted in 50% total fungal oil extracted compared to 70% total rice bran oil extracted by SFE after 2 hours at 200 mL/min flow, 4000 psi, 40 degrees Celsius, which suggested more resistance to oil extraction in the fungal mycelia cell walls. Diffusion coefficients are currently being evaluated. Sweet potato waste may serve as another excellent source of carbon, nitrogen, and antioxidants that may be utilized to high-value end products before being considered a waste material. Enzymatic conversion of polysaccharide material to simple sugars was investigated for potential production of fuel-grade ethanol considered to replace environmentally hazardous MTBE in gasoline. Bioprocessing pilot-scale equipment including an 80L bioreactor, 2L SFE unit and 2L high-pressure stirred reactor were recently obtained from a Board of Regents enhancement grant. Plans for 2002 include modeling the
kinetics of fungal growth, oil production and SFE oil recovery and conducting pilot-scale fermentation and SFE studies. Utilization of rice hull ash as a possible absorbent material in SFE and fractionation processes will also be investigated.
Impacts
The byproducts from rice have demonstrated excellent potential as a substrate for the filamentous fungi, Pythium irregulare capable of producing these omega-3 enriched oils when grown at optimal conditions. The oils are easily extracted with supercritical carbon dioxide and are essentially odor-free compared to fish oils containing similar fatty acids. Other components including antioxidants in the oil fractions may add significant value while preserving the oil from oxidation reactions. Currently, fish oils containing omega-3 polyunsaturated fatty acids that are similar to the fungal oils are sold in tablet form at a price of $500/lb of oil. Value-added uses for the byproducts from sources such as rice, sweet potato and sugarcane have great economic potential in contrast to the high waste treatment costs incurred from disposal of these materials. For instance, value-added technology in processing, marketing and transportation increased rice revenues in the state of
Louisiana by $60 million in 1995, adding 23% to the total amount of revenue ($260 million) generated by the rice industry.
Publications
- Lima, M., Drapcho, C., Walker, T., Bengtson, R. and Verma, L. 2001. A model for integrating communication skills across the biological engineering curriculum. Int. J. Eng. Ed. 17(1):67-74.
- Walker, T. H. 2001. ASEE BAED News. "USDA Challenge Grant News" Spring Issue http://www.bae.lsu.edu/newsletter/newsletter.htm
- Kim, C.S., Walker, T.H. and Drapcho, C.M. 2001. Supercritical fluid extraction of polyunsaturated fatty acids (PUFA): Modeling and Simulation. Proceedings ASAE 01-7103. Sacramento, CA. ASAE.
- Zhu, Hui, Drapcho, C.M. and Walker, T.H. 2001. Bioconversion of rice bran to omega-3 fatty acids of Pythium irregulare submerged culture. ASAE Paper 01-7020. Sacramento, CA: ASAE.
- Zhu, Hui, Walker, T.H. and Drapcho, C.M. 2001. Kinetic modeling and media optimization for fungus Pythium irregulare submerged culture. ASAE Paper 01-7044. Sacramento, CA: ASAE.
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Progress 01/01/00 to 12/31/00
Outputs
The proposed experiments from funding year 2000 gave preliminary results to assess the extractability of rice bran oil and the feasibility of bioconversion of rice byproducts to omega-3 enriched oil followed by extraction. The results from this experiment compared oil extraction by conventional hexane and supercritical carbon dioxide methods at the laboratory scale. Recovery of oils from rice bran and the fungal biomass resulted in high yields (30 wt%). Initial rice bran from long grain (Cypress var.) rice milled using the Satake Rice Mill located in the Department of Biological and Agricultural Engineering contained approximately 18 wt% oil. After 5 days of bioconversion using Pythium irregulare inoculum the oil wt% increased to 24 wt% and after 6 days reached 30 wt% and leveled off. The results were very encouraging in that the bran media cleared (from a suspended cloudy state) after 5 days showing that this microorganism contains enough of the necessary enzymes for
breaking down complex carbohydrates including amylose, cellulose and hemicellose components. The waste initially containing 50 g/L substrate showed that COD decreased to less than 6 g/L, which indicated significant ability to remove waste while increasing the total oil content from 18 to 30 wt%. The composition of omega-3 fatty acid increased over the 9-day fermentation period. Oil has been successfully extracted from similar substrates using supercritical carbon dioxide with recoveries up to 99% (Walker et al. 1999; Kuk and Dowd, 1998; Garcia et al., 1996). Implementation of this technology could be crucial to the economic viability of rice bran oil and bioconverted products, which has yet to fulfill its full potential in this regard. Supercritical carbon dioxide solvent proves to extract greater than 90% oil at 100 ml/min in less than one hour showing great potential compared to 4 hours needed with conventional hexane extraction. Therefore, further investigation on improvements in
operating conditions are merited to reduce cost and examine the alternative bioconversion process for added value, particularly at the pilot-scale level. The Biological and Agricultural Engineering Department at LSU has recently been granted significant funding to purchase bioprocessing pilot-scale equipment (bioreactors, SFE unit, etc.) to continue this study at the pilot scale.
Impacts
A potential use for rice byproducts is bioconversion of the substrate material to higher-value oils containing omega-3 polyunsaturated fatty acids (PUFA) used in functional foods or nutriceuticals. The byproducts serve as an excellent source of substrate for fermentation using filamentous fungi capable of producing these oils when grown at optimal conditions. The oils are easily extracted with supercritical carbon dioxide and are essentially odor-free compared to fish oils containing similar fatty acids (Walker et al., 1999). Currently, fish oils containing omega-3 polyunsaturated fatty acids that are similar to the fungal oils are sold in tablet form at a price of $500/lb of oil.
Publications
- King, J.M. and Walker, T.H. 2000. Ozone: New weapon for fighting food hazards. Louisiana Agriculture 43(2):12-13.
- King, J.M., Walker, T.H., Njapau, H., Park, D.L. and Damann, Jr., K.K. 2000. Managing aflatoxin contamination in corn. Louisiana Agriculture 43(2):20-21.
- Walker, T.H., Cochran, H.D. and Hulbert, G.J. 1999. Supercritical carbon dioxide extraction of lipids from Pythium irregulare. JAOCS. 76(5):595-602.
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Progress 01/01/99 to 12/31/99
Outputs
Objectives of this project include the development of bioprocessing techniques for the recovery and utilization of byproducts important to the state of Louisiana. Four areas of progress were initiated during the first year of this project: (1) A supercritical extraction (SFE) system capable of obtaining accurate solubility data for determination of extractability of important (high-value) components with the use of green solvents such as carbon dioxide has been developed. The SFE unit has been tested with the extraction of rice bran oil that is presently being analyzed by GC techniques. (2) An ozonation system capable of producing high concentrations of ozone, which is also known in the food industry as a friendly oxidizer, is under construction and nearly complete. Several projects have been initiated in collaboration with the Food Science department and other investigators in the Muscle Foods Research group, LSU Agricultural Center. Efficacy techniques have been
developed for determination of ozone destruction of off-flavor components in catfish fillets and aflatoxin degradation in corn. Safety tests using Ames assay have been developed for the aflatoxin study. (3) A bioreactor system for the bioconversion of byproducts to high-value components that are concentrated using supercritical extraction techniques is presently under construction. Experimentation with the current system will begin summer 2000, with rice byproducts. (4) Development of a biodegradable packaging film from rice byproduct components and chitosan is underway. Preliminary tests indicate that 20% chitosan yields the best film quality indicated by tensile strength tests using an Instron testing machine. Detailed analysis of gas permeability using Fick's relationships is presently underway with newly installed Mocon package testing units.
Impacts
Ozonation of aflatoxin-contaminated corn and off-flavor catfish could potentially limit losses. Safety analysis for oxidation byproducts is an important issue. Development of clean, safe, and economical extraction systems for concentration of nutriceuticals from bioconverted rice, sweet potato, and shellfish byproducts would impact these industries by creating value-added products much needed as commodity prices continue to drop with higher yields.
Publications
- Walker, T.H., Chaar, J.M. and Guarana, C.M. 1999. Brazil's super fruit for the caffeinated beverage industry. Ed. T.H. Parliament, C.T. Ho and P. Schieberle. Chapter 31: The Chemistry of Guarana. Symposium Series No. 754. Caffeinated Beverages: Health Benefits, Physiological Effects, and Chemistry. ISBN: 0-8412-3654-2.
- Walker, T.H., Chambers, Z., Taylor, M. and Baker, A.J. 1999. Finite element analysis for biological engineering: a web-based distant education course. 1999 ASEE Proceedings. In Print.
- Walker, T.H., Cochran, H.D. and Hulbert, G.J. 1999. Supercritical carbon dioxide extraction of lipids from Pythium irregulare. JAOCS. 76(5): 595-602.
- Bal, M. and Walker, T.H. 1999. Cellulose-based composite (CBC) edible films from rice by-products for modified atmosphere packaging materials. Paper No. 99-6143. Toronto, Ontario: ASAE/CSAE-SCGR Annual International Meeting.
- Laio, W.C., Hulbert, G.J. and Walker, T.H. 1998. Two-phase cross-flow microfiltration model development and application to fungal biomass solutions. Proceedings Inst. Bio. Eng. 1: A1-A15.
- Cheng, M.H., Walker, T.H. and Hulbert, G.J. 1998. Fungal production of eicosapentaenoic acid from soybean oil in an external-loop airlift bioreactor. Bioresource Technol. 67:101-110.
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