Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Aquaculture, aquatic and animal feed industries, papaya industry, producers of value-added agricultural by-products. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral student and 5 undergraduate students were trained in this project. The postdoc participated in all three objectives, while the undergraduate students mainly participated in activities under objective two. How have the results been disseminated to communities of interest?A peer-reviewed publication has been published to report the findings from our study of microbial conversion of plant oil to value-added specialty chemicals. Our progress in the single cell protein production for aquaculture feed has been disseminated via Center for Tropical & Subtropical Aquaculture (CTSA) Regional e-Notes. What do you plan to do during the next reporting period to accomplish the goals?For objective 2, we will conduct engineering characterization of the new rocking-tray fermenter system in terms of power consumption, oxygen transfer and mixing, and further evaluate its utility in cultivation of additional microbial species for expanded applications. We also plan to conduct a basic techno-economic analysis (TEA) of the microbial bioprocess that converts culled papaya into single cell proteins for fish feeds. For objective 3, we will further the genetic modification and molecular analysis to advance understanding of lipid metabolism in Y. lipolytica using lipid as the sole carbon source.
Impacts
What was accomplished under these goals?
For objective 1, we described in our last report a process to achieve efficient fractionation of whole papaya culls into juice pulp, peel, and undamaged seeds, by repurposing simple, off-the-shelf, fruit processing equipment traditionally used in wine/cider making, along with a sieving system. During this reporting period, we have focused mainly on objective 2, i.e., to develop new products and applications from processed/fractionated agri-wastes. In the previous report, we described cultivation of the yeast Yarrowia lipolytica in puree of culled papaya fruits in stationary tray fermenters. While we were able to achieve yeast growth in this fashion, the growth was slow, due mainly to poor oxygen transfer. We investigated enzyme-catalyzed decomposition of 3% aqueous hydrogen peroxide, as a novel method to supply oxygen to the stationary culture. In this case, we used dry baker's yeast or papaya puree as an inexpensive source of catalase to lower the cost of the operation. A continuous-flow enzymatic bioreactor with immobilized baker yeast cells fed with a dilute aqueous hydrogen peroxide solution was successfully developed for continuous production of oxygen to aerate the stationary culture. Without any culture mixing though, even with oxygen supplementation, yeast growth was still slow. We subsequently developed an improved tray fermenter system. In this system, multiple commercial food pans are suspended using a unique suspension/shaking mechanism to enable efficient mixing of the culture placed inside the food pans with a very low energy input. Cultivation of Y. lipolytica in a medium formulated using juice derived from culled papaya fruits, under batch and fed-batch modes, were successfully achieved in the multi-tier rocking-tray bioreactor system, with growth performance on a par with that achieved in shake flasks under comparable shaking speeds. Using this new fermenter design, we produced over 1 kg of dry yeast cell mass from waste papaya juice, with an apparent biomass yield of approximately 50%, which will be tested as a feed protein source to replace fishmeal in a fish feeding trial. An important advantage of the multi-tier rocking-tray bioreactor system over standard stirred fermenters stems from its simplicity, while still scalable, and hence it is amenable to operation even by low-skilled workers, and requires very low cost to set up and operate. We also trained three undergraduate students in developing a passive aeration system for solid state fermentation, in their senior capstone design, and two other students in undergraduate research of developing the enzyme-mediated oxygenation system. For objective 3, i.e., to engineer microbes and bioprocesses to expand the repertoire of value-added products from processed agri-wastes, our work has focused on developing Y. lipolytica strains that are capable of converting plant oils as a carbon source into high-value specialty oleochemical astaxanthin, and to fill key knowledge gaps in lipid metabolism of this important oleaginous yeast. Efficient cell growth and astaxanthin production by an engineered Y. lipolytica strain using plant oils, including waste papaya seed oil, as the sole carbon source was validated, and a considerable portion of astaxanthin was found excreted into the spent oil.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Li, N., Han, Z., ODonnell, T.J., Kurasaki, R., Kajihara, L., Williams, P.G., Tang, Y.J., and Su, W.W. 2020. Production and excretion of astaxanthin by engineered Yarrowia lipolytica using plant oil as both the carbon source and the biocompatible extractant. Applied Microbiology & Biotechnology, 104, 69776989.
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Aquaculture, aquatic and animal feed industries, papaya industry, producers of value-added agricultural by-products Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One postdoctoral student and 3 undergraduate students were trained in this project. The postdoc participated in all three objectives, while the undergraduate students mainly participated in activities under objective two. How have the results been disseminated to communities of interest?No publications to report in this report period. However, currently three manuscripts resulted from this study are either in review or in preparation. In addition, we plan to disseminate our research findings from objectives 1 and 2 via Center for Tropical & Subtropical Aquaculture (CTSA) workshops and CTSA Regional e-Notes. What do you plan to do during the next reporting period to accomplish the goals?We plan to focus our study on objectives 2 and 3. Specifically, for objective 2, we will continue to investigate fermentation of papaya culls for efficient production of Yarrowia yeast biomass as a new single-cell-protein ingredient for aquaculture feeds to replace fishmeals. For objective 3, we will use our newly developed engineered Yarrowia strains and genetic tools to probe Yarrowia lipid-utilization metabolism and to elucidate potential metabolic bottlenecks in astaxanthin production by Yarrowia utilizing lipid-based carbon sources.
Impacts
What was accomplished under these goals?
For objective 1, i.e., to innovate agro-waste processing to allow simple adoption by small producers, our work has focused on processing papaya culls (unmarketable papaya fruits) into nutrient substrates suited for fermentation by the yeast, Yarrowia lipolytica, to produce single-cell-proteins for aquculture feeds, using simple affordable mechanized processing that is amenable to common commercial operations, and adoptable by local growers and producers. We established and evaluated a process by repurposing simple, off-the-shelf, fruit processing equipment traditionally used in wine/cider making, along with a drying oven and a sieving system to achieve efficient fractionation of whole papaya culls into juice pulp, peel, and undamaged seeds. Due to the large gaps between the crushing blades in the fruit crusher, seeds are not damaged (and hence no release of the antimicrobial substance into the juice) while also allowing the whole fruit to be efficiently processed into chunky pulps. By eliminating the need for peeling from initial processing, our fractionation process does not require costly and specialized fruit processing machines such as the energy-intensive rotating-drum peeling machine and the fruit pulper/finisher. For objective 2, i.e., to develop new products and applications from processed/fractionated agro-wastes, we have developed a fermentation process to produce protein-rich Yarrowia lipolytica yeast biomass using waste papaya fruit pulp and seed oil as carbon sources. A major technical barrier encountered in prior efforts to enrich papaya fruit waste with microbial proteins for aquatic animal feed resulted from the use of submerged fermentation (SmF) that involves large quantities of water. The large volumes of water present in the submerged culture has created major problems associated with a high cost of liquid mixing and oxygen gas transfer during fermentation, requirements for large fermenters to accommodate the large volume of water, as well as costly and extremely time consuming dewatering after fermentation to harvest the yeast biomass. Aiming at overcoming such technical barriers so that papaya fruit waste can be utilized efficiently and cost effectively to produce microbial single cell proteins, cultivation of Y. lipolytica yeast in semi-solid state fermentation (semi-SSF) is investigated. This type of fermentation involves minimum free water in the culture (though with higher water content than the traditional solid state fermentations). The yeast cells are grown on a stationary bed of papaya puree in a growth chamber with temperature and humidity control. An important advantage of semi-SSF over SmF in culturing Yarrowia yeast on papaya puree is that a much denser yeast mass can be obtained and hence avoid costly dewatering required in SmF. We have confirmed the dense cells of Y. lipolytica could be developed on a bed of papaya puree loaded in large aluminum trays after 3-4 days of semi-SSF cultivation. No energy intensive mixing is needed during the fermentation. Culture scale-up can be achieved by using multiple trays to grow the yeast on papaya puree in a tray bioreactor with humidity and temperature control. Commercially available and economical heater/proofer commonly used in restaurants and bakeries can be adapted as a bioreactor for this type of cultivation. To further accelerate and enhance yeast growth in semi-SSF, we developed a novel oxygenation method to improve oxygen delivery to the yeast during semi-SSF. Aqueous hydrogen peroxide decomposes to pure oxygen and water. This reaction is catalyzed by the enzyme catalase. Many types of low-cost biomass can serve as sources of catalase. Our new oxygenation method exploits this interesting property, in combination with inexpensive hydrogen peroxide, to generate O2 in situ. We have examined a series of designs in incorporating this system in semi-SSF. One challenge is that hydrogen peroxide at high concentrations (>300 mM) could be toxic to the Yarrowia yeast, and hence it is necessary to segregate hydrogen peroxide from the Yarrowia yeast. To this end, we placed a 3% H2O2 solution and a small amount of raw catalase in LDPE (low density polyethylene) bags. Oxygen generated from H2O2 decomposition can effectively permeate through the LDPE and be released, while residual H2O2 is confined within the bag. The oxygen release rate can be adjusted by the amount of H2O2 and catalase loaded and the LDPE film thickness. We attached the oxygen generating bag system to the inner face of an aluminum lid used to cover a disposable aluminum foil pan in which a liter of papaya puree was applied evenly and inoculated with the Yarrowia yeast. The lid prevented excess water evaporation from the puree during fermentation, and the oxygen-generation system effectively enriched the atmosphere above the puree/culture with oxygen to improve culture aeration. Using this system, we currently can obtain about 20 g yeast dry weight per liter of puree in 3 days without stirring the culture. By the end of 3-day fermentation, over 90% of the initial reducing sugars were found to be consumed. After fermentation, the yeast/puree mixture was efficiently dried in a food dehydrator to remove over 90% of the remaining moisture before storage at 4°C. We will continue to optimize aeration and carbon and nitrogen source concentrations to further increase the biomass yield. We are targeting about 1 kg of yeast biomass to be produced for the tilapia feeding trial to be conducted during the following report period. For objective 3, i.e., to engineer microbes and bioprocesses to expand the repertoire of value-added products from processed agro-wastes, our work has focused on developing Yarrowia lipolytica strains that are capable of converting plant oils as a carbon source into high-value specialty oleochemical astaxanthin. We have tested several available genetic tools, and developed new ones, for metabolic engineering of Y. lipolytica and protein engineering of astaxanthin biosynthetic enzymes. The astaxanthin-producing ST7403 strain we used in our study is a leucine auxotroph and is sensitive to nourseothricin. We developed a series of episomal and integrative expression vectors based on Leu and/or nourseothricin selection. In one series of integrative vectors, the Leu2 marker is flanked by loxP and it can be looped out by expressing CreA recombinase on an episomal vector carrying the nourseothricin resistance marker nourseothricin N-acetyl transferase (NAT). This allows multiple rounds of gene integration into several different genome integration sites reported in the literature. The ST7403 strain has its mus51 gene deleted and hence is capable of efficient homologous recombination to enhance gene integration. We also developed several knockout mutant strains derived from ST7403, using CRISPR-Cas9-based techniques to test two strategies aimed at increasing the cytosolic acetyl-CoA pool which feeds the MVA pathway and subsequent carotenoid synthesis, by reducing the drain from acetyl-CoA. Knockouts were made by introducing a frameshift to the native sequence along with an XhoI cut site replacing the PAM site for the screening of the transformants. The vector pCRISPRyl was used for expressing sgRNA and a codon optimized Cas9 from Streptococcus pyogenes. The following knockout strains have been created and characterized: Dga1- and Dga2- (diacylglycerol acyltransferase, knockout individually or in combination), and Mls1- (malate synthase knockout). The Dga1-/Dga2- mutant contains only very small lipid bodies (LBs), while Mls1- mutant has more abundant carotenoid-filled LBs compared with the parental ST7403 strain. An improved astaxanthin titer in the Mls1- mutant compared with the parent ST7403 strain was noted in YnB minimal medium supplemented with ammonium sulfate, casamino acids and 6% safflower oil. The opposite was noted with the Dga1-/Dga2- mutant, though cell growth was similar.
Publications
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