Progress 08/15/19 to 04/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems: While the technical work and reporting were completed on schedule, the draw down of funds and the uploading of progress and financial reports to REEport were delayed due to changes in staff related to the COVID-19 pandemic. This included our comptroller and this resulted in missed deadlines and delays. 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?
Nothing Reported
Impacts
What was accomplished under these goals?
Impact Statement: This application describes research to further advance an innovative algae-based production system for the manufacture of a suite of novel biobased ingredients for sustainable and environmentally advantaged skin care products. Specifically, we propose the mass cultivation of genetically enhanced photosynthetic algae (cyanobacteria) in specialized photobioreactors (PBRs) to produce mycosporine-like amino acids (MAAs), which provide several benefits when included in skin care products, including boosting UV protection, antioxidant, and anti-inflammation properties. This is a significant opportunity because many of the chemically synthesized UV filters in current skin care products (e.g., sunscreens) have been determined to have adverse impacts on health and the environment (DiNardo and Downs, 2018; Hanigan et al., 2018; Scheider and Lim, 2018; Kim and Choi, 2014). It has been reported that the algae-produced natural compounds that can boost UV protection are more environmentally friendly and sustainable than the current petroleum-based UV filters (Derikvand et al., 2017). In addition to providing superior products, this approach also offers an opportunity for farming of algae in various regions, including dry, hot environments with limited or declining fresh water availability. To address Technical Question 1 we performed two lab-scale experiments; Task 1a and Task 1b. Task 1A Executive Summary: This study evaluated, in lab scale photobioreactors, the impact of cultivation medium and induction strategy on the culture growth and productivity. Three different zinc induction strategies in both a defined salt matrix and natural saline water sourced from a well on Algenol's site (NWW) were tested. This natural well water is readily available and economical; however, it contains high levels of calcium, magnesium and sulfate that can cause issues in the downstream MAA purification process. The defined salt matrix was a modified artificial seawater (mASW) with a five-fold reduction in the divalent ions. For MAA gene induction with ZnSO4, this experiment assessed a bolus induction at 7 and 5 OD750, respectively, and a split dose strategy at 3.5 and 7.0 OD750. The NWW reactors had the highest densities across induction treatments; and, the mASW reactors had a slightly reduced growth in the final 10-days of cultivation. There was a significant difference in MAA production by induction treatment, but not between NWW and mASW with the same induction strategy. In this experiment, providing a split dose of ZnSO4 resulted in a 28-34% increase in total MAA production versus the NWW control. The split dose mASW and NWW reactors produced the most MAA at 528 and 505 mg L-1 shinorine, respectively, versus 369 and 395 mg L-1 shinorine in the control dose mASW and NWW reactors. Overall, induction of the MAA pathway earlier in the cultivation produced more MAAs, and a split induction resulted in a prolonged higher productivity rate. While the mASW reactors did exhibit a turn-over in growth, the benefit of comparable MAA production and reduced divalent ion concentrations supports the use of this defined salt matrix for future cultivations. Task 1B Executive Summary: This evaluation focused onsemi-continuous operation to further increase productivity. The overall objective of this study was to test the shinorine-producing strain in an indoor semi-continuous cultivation. Reactors from an initial batch cultivation were either harvested to 1⁄2 volume or harvested to 1⁄4 volume followed by a dilution with fresh media. Both of these harvest strategies were studied in combination with two different induction strategies: immediate induction or no induction. It was determined that the cultures which were harvested to 1⁄4 volume with an immediate induction by a split dose, resulted in the best MAA production rate per day. However, in this semi-continuous evaluation, the MAA productivity was lower than the results of the initial batch cultivation of Strain A, both in terms of total MAA concentration or MAA rate per day. It was determined that semi-continuous operation strategies and associated induction parameters would have to be further evaluated and optimized in the future but are not currently worth pursuing. To address Technical Question 2 we performed an outdoor validation experiment; Task 2. Task 2 Executive Summary: MAA producing Strain A was cultivated outdoors and three induction strategies were examined. mASW has a defined salt composition with lower magnesium, calcium, and sulfate than found in seawater, and with half the salinity. The cultivation proceeded for 33 days post inoculation of all reactors. Total MAA produced reached over 500 mg L-1 by the end of the experiment for both split induction treatments, with the bolus treatment having a concentration just under of 500 mg L-1. Overall, using the split induction method resulted in higher MAA production. Further experimentation is needed to dial in the nutrients required for growth in order to optimize MAA production under outdoor conditions. To address Technical Question 3 we performed a downstream process development experiment, Task 3, using the MAA harvested from Task 2. Task 3 Executive Summary: The primary objective of this process development was to produce a concentrated MAA solution through the extraction and concentration of shinorine/porphyra-334 produced by Algenol's modified cyanobacteria. This evaluation utilized culture prepared in the SBIR Task 2 cultivation as the feed material for downstream extraction and concentration of shinorine. This culture was the first time an outdoor cultivation, grown in 50% mASW medium, was used for the production of MAAs. Thus, this was a major change for both cultivation and the downstream process development for MAA production, which was intended to help improve the process yields and reduce conductivity in the final product. The harvest from Task 2 consisted of 390 L of culture at 18.3 sOD750 and 490 mg L-1 shinorine, which was one of the highest outdoor culture yields to date. The nanofiltration process yielded 9.4 kg of a 1.32% MAA solution with 14.7 mS conductivity, which was the highest volume and MAA concentration obtained to date. The final nanofiltration yielded a 91% step yield from the drained retentate alone and provided a 78% overall MAA yield. Previous experience with MAA nanofiltration process, using a natural seawater medium, had resulted in membrane fouling due to the higher initial conductivity. Thus, use of the modified artificial seawater in cultivation helps to greatly improve the downstream process. Overall, the MAA downstream process changes implemented for this evaluation have shown significant process improvements at each stage. The modified salt medium used in cultivation helped to improve overall product conductivity, while maintaining MAA productivity. This defined salt medium likely improved the nanofiltration process, which helped deliver the 78% overall yield. To address Technical Question 4 we conducted a technoeconomic analysis (TEA), Task 4, using inputs from Tasks 2 and 3. Task 4 Executive Summary: As part of SBIR grant, Algenol attempted a techno-economic analysis (TEA) of Algenol's MAA production technology. Based on Algenol's proprietary cultivation systems, a TEA model was generated to analyze impacts of key performance indicators on the baseline economics of MAA production at a facility of varying scales. A conservative analysis indicated annual productivity of 1,500 to 3,000 kg for a 5,000 to 10,000 L capacity production facility. Work performed in this Phase 1 SBIR grant was insufficient to parameterize the capital and operational expenses (CapEx and OpEx). Additional experience in operating the downstream product recovery process is required to determine equipment size and cost as well as to estimate the labor required to operate a production facility.
Publications
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Progress 08/15/19 to 04/14/20
Outputs
Target Audience:
Nothing Reported
Changes/Problems:While the technical work and reporting were completed on schedule, the draw down of funds and the uploading of progress and financial reports to REEport were delayed due to changes in staff related to the COVID-19 pandemic. This included our comptroller and this resulted in missed deadlines and delays. 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?The activiities are complete.
Impacts
What was accomplished under these goals?
Impact Statement: This application describes research to further advance an innovative algae-based production system for the manufacture of a suite of novel biobased ingredients for sustainable and environmentally advantaged skin care products. Specifically, we propose the mass cultivation of genetically enhanced photosynthetic algae (cyanobacteria) in specialized photobioreactors (PBRs) to produce mycosporine-like amino acids (MAAs), which provide several benefits when included in skin care products, including boosting UV protection, antioxidant, and anti-inflammation properties. This is a significant opportunity because many of the chemically synthesized UV filters in current skin care products (e.g., sunscreens) have been determined to have adverse impacts on health and the environment (DiNardo and Downs, 2018; Hanigan et al., 2018; Scheider and Lim, 2018; Kim and Choi, 2014). It has been reported that the algae-produced natural compounds that can boost UV protection are more environmentally friendly and sustainable than the current petroleum-based UV filters (Derikvand et al., 2017). In addition to providing superior products, this approach also offers an opportunity for farming of algae in various regions, including dry, hot environments with limited or declining fresh water availability. To address Technical Question 1 we performed two lab-scale experiments; Task 1a and Task 1b. Task 1A Executive Summary: This study evaluated, in lab scale photobioreactors, the impact of cultivation medium and induction strategy on the culture growth and productivity. Three different zinc induction strategies in both a defined salt matrix and natural saline water sourced from a well on Algenol's site (NWW) were tested. This natural well water is readily available and economical; however, it contains high levels of calcium, magnesium and sulfate that can cause issues in the downstream MAA purification process. The defined salt matrix was a modified artificial seawater (mASW) with a five-fold reduction in the divalent ions. For MAA gene induction with ZnSO4, this experiment assessed a bolus induction at 7 and 5 OD750, respectively, and a split dose strategy at 3.5 and 7.0 OD750. The NWW reactors had the highest densities across induction treatments; and, the mASW reactors had a slightly reduced growth in the final 10-days of cultivation. There was a significant difference in MAA production by induction treatment, but not between NWW and mASW with the same induction strategy. In this experiment, providing a split dose of ZnSO4 resulted in a 28-34% increase in total MAA production versus the NWW control. The split dose mASW and NWW reactors produced the most MAA at 528 and 505 mg L-1 shinorine, respectively, versus 369 and 395 mg L-1 shinorine in the control dose mASW and NWW reactors. Overall, induction of the MAA pathway earlier in the cultivation produced more MAAs, and a split induction resulted in a prolonged higher productivity rate. While the mASW reactors did exhibit a turn-over in growth, the benefit of comparable MAA production and reduced divalent ion concentrations supports the use of this defined salt matrix for future cultivations. Task 1B Executive Summary: This evaluation focused onsemi-continuous operation to further increase productivity. The overall objective of this study was to test the shinorine-producing strain in an indoor semi-continuous cultivation. Reactors from an initial batch cultivation were either harvested to ½ volume or harvested to ¼ volume followed by a dilution with fresh media. Both of these harvest strategies were studied in combination with two different induction strategies: immediate induction or no induction. It was determined that the cultures which were harvested to ¼ volume with an immediate induction by a split dose, resulted in the best MAA production rate per day. However, in this semi-continuous evaluation, the MAA productivity was lower than the results of the initial batch cultivation of Strain A, both in terms of total MAA concentration or MAA rate per day. It was determined that semi-continuous operation strategies and associated induction parameters would have to be further evaluated and optimized in the future but are not currently worth pursuing. To address Technical Question 2 we performed an outdoor validation experiment; Task 2. Task 2 Executive Summary: MAA producing Strain A was cultivated outdoors and three induction strategies were examined. mASW has a defined salt composition with lower magnesium, calcium, and sulfate than found in seawater, and with half the salinity. The cultivation proceeded for 33 days post inoculation of all reactors. Total MAA produced reached over 500 mg L-1 by the end of the experiment for both split induction treatments, with the bolus treatment having a concentration just under of 500 mg L-1. Overall, using the split induction method resulted in higher MAA production. Further experimentation is needed to dial in the nutrients required for growth in order to optimize MAA production under outdoor conditions. To address Technical Question 3 we performed a downstream process development experiment, Task 3, using the MAA harvested from Task 2. Task 3 Executive Summary: The primary objective of this process development was to produce a concentrated MAA solution through the extraction and concentration of shinorine/porphyra-334 produced by Algenol's modified cyanobacteria. This evaluation utilized culture prepared in the SBIR Task 2 cultivation as the feed material for downstream extraction and concentration of shinorine. This culture was the first time an outdoor cultivation, grown in 50% mASW medium, was used for the production of MAAs. Thus, this was a major change for both cultivation and the downstream process development for MAA production, which was intended to help improve the process yields and reduce conductivity in the final product. The harvest from Task 2 consisted of 390 L of culture at 18.3 sOD750 and 490 mg L-1 shinorine, which was one of the highest outdoor culture yields to date. The nanofiltration process yielded 9.4 kg of a 1.32% MAA solution with 14.7 mS conductivity, which was the highest volume and MAA concentration obtained to date. The final nanofiltration yielded a 91% step yield from the drained retentate alone and provided a 78% overall MAA yield. Previous experience with MAA nanofiltration process, using a natural seawater medium, had resulted in membrane fouling due to the higher initial conductivity. Thus, use of the modified artificial seawater in cultivation helps to greatly improve the downstream process. Overall, the MAA downstream process changes implemented for this evaluation have shown significant process improvements at each stage. The modified salt medium used in cultivation helped to improve overall product conductivity, while maintaining MAA productivity. This defined salt medium likely improved the nanofiltration process, which helped deliver the 78% overall yield. To address Technical Question 4 we conducted a technoeconomic analysis (TEA), Task 4, using inputs from Tasks 2 and 3. Task 4 Executive Summary: As part of SBIR grant, Algenol attempted a techno-economic analysis (TEA) of Algenol's MAA production technology. Based on Algenol's proprietary cultivation systems, a TEA model was generated to analyze impacts of key performance indicators on the baseline economics of MAA production at a facility of varying scales. A conservative analysis indicated annual productivity of 1,500 to 3,000 kg for a 5,000 to 10,000 L capacity production facility. Work performed in this Phase 1 SBIR grant was insufficient to parameterize the capital and operational expenses (CapEx and OpEx). Additional experience in operating the downstream product recovery process is required to determine equipment size and cost as well as to estimate the labor required to operate a production facility.
Publications
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