Progress 06/01/14 to 01/31/16
Outputs
Target Audience:Prairie Aquatech's target audience is aquaculture feed manufacturers. Ultimately these feed diets are provided to a producer, but the manufacturer and formulator must create a proper diet; which often encompasses omega oils. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has allowed for engagement with feed manufactuers and fish nutritonist to further understand the markets and needs in the industry. The opportunities have lead to a commericial understanding and gaps that need to be filled in the market place. The focus of the project has allowed for our preliminary understanding to become a more thorough and validated understanding. How have the results been disseminated to communities of interest?The results and developments have been shared with University students. Prairie Aquatech has had interns and students participate in laboratory observations to further enahnce their interests in science and technology. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Original work plan: The first generation Omega-3 DDGS (distillers' dried grains with solubles) was anticipated to have >1.5% of both DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) and >45% protein. Part 1: Producing DHA in DDGS The algae Schizochytrium limacinum SR21 (ATCC MYA-1381) was used in the studies. The cells were maintained in 3.6% sea salt medium containing 5 g/l glucose, 1.0 g/l yeast extract and 1.0 g/l peptone. The pH of the sea salt medium is ~7.5. The medium was autoclaved at 121oC for 15 min before use. The inoculum size was 10% of the total liquid volume in each flask. The cells were grown in 250-ml Erlenmeyer flasks each containing 100 ml of medium and incubated at 25oC in an orbital shaker set to 200 rpm. Subcultured cells were used as inoculum for future studies. For each experimental condition, two replicates were used, and the standard deviation was calculated. Optimize the solid loading rate To get the highest volume yield of DHA, different solid loading rates (SLR) were conducted. The 5%, 10%, 15%, 20% of DDGS were mixed with distilled water. The pH of the medium was adjusted to pH 7 with 10 M NaOH. The medium was autoclaved before incubation. The inoculum size was 10% of the total liquid volume. The flasks were fermented at 25oC incubator with 200 rpm shaking speed for 5 days. All the following experiments will follow the same incubation conditions. Table 1 Sample DHA content and DHA yield with different solid loading rate SLR% DHA in DDGS % STDERROR DHA conc in liquid (mg/l) STDERROR 5 0.25 0.01 1.225 0.025 10 0.40 0.02 3.95 0.15 15 0.42 0.04 6.225 0.525 20 0.31 0.07 6.1 1.3 Table 1 shows that the DHA content in Omega-3 DDGS and DHA concentration in liquid vary when solid loading rate is different. At 10% and 15% SLR, DHA content of the final product are not significant different, but both of them are much higher than that at 5% and 20% SLR. As to the DHA concentration in liquid, 15% and 20% SLR are not significant different, but much higher than 5% and 10% SLR. Considering the two factors, 15% SLR is preferred for the future studies. Schizochytrium growth needs enough nutrients. The low SLR could not provide as much nutrients as the high SLR. Especially when the oxygen supply is not the influencing factor for Schizochytrium growth in this experiment condition (Chi et al., 2007), comparing higher SLR could be the better choice in this research. But too high SLR have the dilution effect on the final DHA content, which would bring down the product quality. Plackett-Burman design for different minerals effect A Palckett-Burman design was used to screen the minerals and pH having significant effects on the DHA yield on DDGS. The variables evaluated are listed in Table 2 (not pictured). The choices of the variables were based on the artificial sea water composition suggested by Chi (2007) with modifications. Each independent variable was investigated at a high (+) and a low (-) level. The low levels (-) of medium components were taken as 0. In summary, there were 20-runs of experiment, with 13 variables and 6 dummy variables (D1-D6) (Table 3). In this work, the software Design-Expert 8.0.6 (Stat-Ease Inc.) was used for analyzing the significant (P) level through F-test. The Plackett Burman design was used to screen the medium composition of DDGS. Schizochytrium was grown in 20 different conditions, with DHA content in the final content as the response factor (Table 3, not pictured). The effects of the variables on the responses, and their significant level (P) were calculated and shown in table 4. Based on the effects of dummy variables, the standard error of the Plackett Burman design was 0.09%. The effects with P<0.5 were accepted as significant. Thus NaCl and pH will be the significant influencing factors. Figure 1 confirmed the significant positive effect of the variables were NaCl and pH. Summary of Part 1 The highest DHA content we can obtain is around 0.83% which is from the Plackett Burman design run 1, with the fermentation condition as following: NaCl 2g/l; MgSO4.7H2O 2.44 g/l; KCl 0.6 g/l; CaCl2.2H2O 0.3 g/l; Tris base 1 g/l; sodium acetate 0.6 g/l; pH 8; solid loading rate 5%. From the Plackett Burman design, the NaCl concentration and pH are the two significant parameters to influence DHA content in the S. limacinum fermentation process with DHA as the substrate. Also, based on the experiment results about solid loading rate, the carbohydrate and nitrogen sources in DDGS limit S. limacinum growth. Saccharification is recommended to be used to release more nutrients from DDGS before fermentation. And aeration is recommended to use while using the enzyme saccharification. Part 2: EPA production in thin stillage Pythium growth in thin stillage solution The fungus Pythium irregular (ATCC 10951) was used. The fungus was grown on PDA plate at 25oC for 5 days. The PDA plates were then washed with distilled water and mycelium was dislodged with inoculating loop. The mycelium solution was used as the inoculum. Fifty milliliter thin stillage solution was put in 250 ml flasks. The concentration of thin stillage was set at 20, 50, 70 and 100%. The composition is listed in table 5. The thin stillage solution was adjusted to pH 7 with 10 M NaOH. The solution was autoclaved at 121oC for 20 min. After cooling down, 10% inoculum was inoculated. The flaks were incubated at room temperature in an orbital shaker set at 200 rpm. Two control samples were also set up: Pythium growth in 30 g/l glucose + 10 g/l yeast extract and in 30 g/l glycerol + 10 g/l yeast extract. Figure 2 (not pictured) shows the Pythium growth in different concentration of thin stillage and also the comparison of its growth in thin stillage to glucose and glycerol. Pythium could grow in 100% thin stillage. The higher the thin stillage concentration, the longer it required to grow. But for the higher concentration, the whole biomass was larger. Pythium could grow in 30 g/l glycerol, plus yeast extract, but its growth was not so good as in glucose and thin stillage. For clearance of 50 ml solution, 20% TS needs 2d, 50% 3d, 70% 5d, 100% 6d. Fed-batch fermentation of Pythium in thin stillage in flasks The reason to do the fed batch is that the inoculum size is very important for Pythium growth. It doesn't grow very well in large volume. The idea for fed-batch is that continually using the growth as the inoculum for the next step fermentation. Fifty milliliter total volume was started for the fed batch flask. The concentration of thin stillage at the starting point was 20%. The inoculum size was 10% (v/v). After the solution was clear, 12.5 ml sterilized thin stillage (pH 7) was added to the flask. After the 2nd round solution was clear, continually add 15.625 ml, 19.53 ml, 24.41 ml thin stillage. The total thin stillage percentage is 69.57%. Also the 63.5% thin stillage, with pH7, 10% inoculum was set up at the same time as the control experiments. The sequence of adding thin stillage was listed in table 6. The experiment was recorded in table 7 and the comparison between the fed batch flask and the control flask was shown in figure 3. Reference Chi Z., Pyle D., Wen Z., Frear C., Chen S. 2007. A laboratory study of producing docosahexaenoic acid from biodiesel-waste glycerol by microalgal fermentation. Process Biochemistry. 42: 1537-1545.
Publications
- Type:
Other
Status:
Published
Year Published:
2014
Citation:
14//506,385
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Progress 06/01/14 to 05/31/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems: To scale up the P. irregulare growth in thin stillage in the 5 L fermenters with fed-batch, we have met the contamination problems. We will try to use the aeration fermenters, with the batch mode fermentation to avoid the contamination from adding thin stillage. The other problem is the protein content of P. irregulare (30%), which is much lower than the protein we expected (45%). We will try different other cultures and grow them in DDGS or soy meal. What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals? To achieve our goal of 1.5% DHA and EPA we will evaluate additional variables during the no-cost extension. For DHA, our results indicate that carbon, nitrogen, sodium chloride, and pH are the most significant factors regulating growth S. limacinum and production of DHA. Thus, we will apply pretreatments and enzymatic saccharification to DDGS prior to incubation to release more sugars for microalgae growth. We expect that jointly optimizing these factors will result in a DHA content higher than 1.5% in the final Omega-3 DDGS. S. limacinum will also be acclimated to a chlorine dioxide based antimicrobial to reduce contamination issues that could affect subsequent scale up. For EPA, P. irregulare biomass production will be optimized by further evaluating fed batch production with thin stillage, while optimizing aeration and agitation. We may also explore addition of supplemental inexpensive sources of carbon. Here we also expect to achieve EPA levels that will exceed 1.5% in the final Omega-3 DDGS. The final Omega-3 DDGS will then be evaluated in fish feeding trials, with the goal of replacing at least 30% of fish meal and fish oil.
Impacts
What was accomplished under these goals?
The microalgae Schizochytrium limacinum and the fungus Pythium irregular are being used to convert nutrients in DDGS and thin stillage into DHA and EPA, respectively. Our preliminary trials achieved 0.24% DHA in the Omega-3 DDGS. At present we are working to optimize the composition of the production medium to enhance DHA content. Different solid loading rates (SLR) were tested (5, 10, 15, 20%) and the 15% SLR gave us the highest DHA content and the highest productivity. The addition of mineral was evaluated with Plackett Burman design, and we were able to boost DHA content in the Omega-3 DDGS from 0.24% to 0.83%. Initial trials with P. irregulare resulted in 0.91% EPA. To further enhance EPA production we are currently assessing fed-batch fermentation, which has worked well at the flask level.
Publications
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