Progress 07/01/21 to 04/04/22

Outputs
Target Audience:As an SBIR grant, the primary target audience impacted by these efforts is the grantee, KnipBio Inc. Partners in this work also directly benefited. Finally, the US aquaculture sector will also benefit as KnipBio makes progress to bring new solutions to some of the industry's most pressing issues. The efforts undertaken in this work were based on in vivo animal testing, both in research settings as well as in a commercial farm. Changes/Problems:Freshwater Institute Several attempts were made to raise the off-flavor concentration in the culture systems. Unfortunately, despite the best effort from the FI staff, these remained unsuccessful, and the trial was transferred to a commercial farm. Commercial farm Unfortunately, there has been a number of mistakes at the farm that impacted our work: Feeding KBM feed to a group of 3 tanks instead of a single tank for 30 days. Once realized, 1 tank of these tanks was reverted back to the regular commercial feed while the third tank was taken out of the trial. Fortunately, there was a separate tank with fish of a similar size, that had never been exposed to KBM, and for which samples had been taken at the same time the initial samples were taken in the other tanks. A portion of these samples were made available for geosmin analysis, and this tank was used as the new control tank for the entire trial. Still, the new control tank was on a different water treatment loop (different RAS), so redistribution of tanks from the original design introduced a confounding factor in the data which cannot be controlled statistically. Water samples were not taken/correctly processed, and only 3 samples were taken at the day-30 mark. Since this coincides with the reassignment of tanks, the absence of water data makes any meaningful comparison in geosmin and 2-MIB tissue contents impossible. Consequently, the following description of the results will be mostly based on the comparison of the KBM and the "reverted KBM" treatments. Though not a mistake per se, fish from the control tank were harvested just before the end of the trial. Additionally, new fish were moved into a neighboring tank to the control tank. This introduces a significant bias for the trial at the last time point, which therefore does not include the control group. Unaccomplished goal Sensory panel - A significant overage in costs was incurred during the attemps at raising off-flavor in culture systems at FRI and from moving the trial to its new location. After completion and considering the issues detailed above, we realized that the sensory panel would not have yielded meaningful data, and that the fillet geosmin levels would provide the necessary information. As such, the sensory panels were not conducted. What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?As this is proprietary, confidential research, the results of this work have not been shared outside our direct partners, the Freshwater Institute and the commercial farm. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Accomplished goals 1- Feed production: the KBM was successfully produced and used to manufacture the feeds necessary to conduct the trial. There has been no issue with either productions, and the resulting feeds were of high quality. 2- Feeding trial: As documented in the annual report, we were unsuccessful in raising the water geosmin levels in the culture systems at the Freshwater Research Institute (FRI). In spite of many steps taken, it was finally decided to stop the activities at FRI and move the project to a commercial salmon producer where off-flavor issues are routinely observed. There, the trial was successfully conducted. The fish were fed the diets, though a mistake from the farm staff forced us to adapt the experimental protocol. The trial continued nonetheless, and samples were collected. 3- Sample analysis: All samples were sent for analysis as planned, and the results were reported back to us. 4- Data analysis and reporting: All data were analyzed statistically using a combination of analyses of variance and regression analyses. Reports were generated and shared internally between KnipBio and project collaborators. Key outcome Generally, the observations were positive, confirming the potential of the product in reducing the bioaccumulation of geosmin in the fillet of RAS-raised Atlantic salmon. Unfortunately, variability in the off-flavor measurements and the mistakes that occurred during the trial prevented us from reaching more firm conclusions. Nevertheless, it has become clear that the time during which the fish receive the KBM-containing feed is a critical factor to the reduction of off-flavor in the fillet. Impact statment Despite the difficulties experienced at the FRI in the initial phase of the project, we have gathered valuable information that will help us move forward as a business. First, with FRI we have gained knowledge in the dynamics and factors affected the rise (or not) of off-flavor compounds in a culture system. This knowledge is currently being incorporated into methods for future work. Second, with the commercial farm we demonstrated the potential of the product, though it is equally clear that more work is necessary before it can be brought to market. We have strengthened an existing relationship: they support our work and expressed interest in using feeds containing our product in the near future. Therefore, we have seen changes both in knowledge and in action as a result of this phase I project. As a result, we will submit our phase II application to continue this work.

Publications

Progress 07/01/21 to 02/28/22

Outputs
Target Audience: Nothing Reported Changes/Problems:First, weekly brushing and cleaning protocols, which are generally applied to manage biofilm growth on submerged RAS surfaces, were abruptly discontinued on June 7. It is important to note that discontinuation of standard cleaning protocols effectively supported rapid geosmin accumulation up to > 14 ng/L during a previous onsite study; albeit, in different fish production systems, i.e., partial reuse systems that exchanged large volumes of water and that lacked a biofilter. Conversely, after one and a half months without cleaning the six replicated RAS intended for use in the SBIR trial, geosmin and MIB levels averaged < 2 ng/L in RAS water, i.e., levels that were low and insufficient for the project. Other adjustments were then attempted including addition of biofilter sand from another onsite RAS that demonstrated higher off-flavor levels in the fish culture water (July 19) where introduction of off-flavor-producing bacteria that resided in the microbial biofilms surrounding sand particles was expected. Prior to adding sand to each of the six RAS biofilters, pumps were turned on and off several times to loosen and disrupt existing microbial biofilms. These procedures appeared to lead to positive trends for off-flavor accumulation; however, geosmin and MIB levels increased very slowly and not to levels that were supportive of the project. As such, a final attempt was made to further disrupt the seemingly robust microbial stability of the replicated RAS. On August 11 water flushing was increased nearly ten-fold and an ultraviolet light that treats the incoming spring water to these RAS was turned off. Periodic disruption of biofilm within the fluidized sand biofilter was also attempted via more frequent on/off cycling of the water recycle pumps. Trends for increasing off-flavor levels continued over the next two weeks and relatively high levels of MIB were achieved in some RAS on 9/7/21. However, analysis of water samples collected on 9/20/21 showed a significant drop in average geosmin and MIB concentrations to approximately 2-3 ng/L suggesting that base microbial populations had acclimated to the changes. Freshwater Institute scientists now hypothesize that microbially mature RAS that include fluidized sand biofilters that have been operated without shutdown for long periods of time (> 1 year) may develop a robust microbial consortium where opportunistic bacteria such as off-flavor-producers are crowded out or excluded. Anecdotally, these trends provide interesting and useful information for the RAS sector at large; however, the general lack of off-flavor production was not supportive of a study designed to evaluate the dietary effect of a novel single cell protein on off-flavor levels in Atlantic salmon flesh. It was therefore decided to terminate the efforts at the Freshwater Institute and transfer the project to a different facility where off-flavors were known to occur routinely. What opportunities for training and professional development has the project provided?Although the results described above precluded us from completing the trial as initally intended, the effort has provided valuable information regarding the onset of geosmin production in the system. 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?The Freshwater Institute has been replaced by AquaBounty as the partner for this project, and where the fish will be fed the experimental diets. AquaBounty is a commercial salmon farm where off-flavor are a common issue. We will also request a no-cost extension in order to allow enough time to complete the project.

Impacts
What was accomplished under these goals? In support of research described in the USDA-SBIR funded project, "Off-flavor Mitigation in Atlantic Salmon Grown in RAS via the Use of a Novel Single Cell Protein Based Feed" (Award # 2021-33530-3453), the Freshwater Institute attempted to create elevated levels of naturally produced off-flavor in six replicated RAS via strategic adjustments to system operation. Unfortunately, geosmin and 2-methylisooborneol levels did not increase as expected, and sufficient concentrations were not created for purposes of the trial.

Publications