Progress 09/01/19 to 04/30/20
Outputs
Target Audience:The AGU Ocean Sciences Meeting 2020 was attended by Maddelyn Harden who presented the poster, described below, on the joint work between researchers at USC and Carlsbad Aquafarm. Poster Presentation: Establishing Aquaculture Frameworks for the Methane-Mitigating Rhodophyta, Asparagopsis taxiformis: Abstract:The ruminant livestock industry currently accounts for nearly a quarter of all global methane emissions, pointing to the urgent need to identify methods for methane mitigation in the farming of ruminants. One potential avenue for such mitigation efforts can be found through the use of the Rhodophyta genusAsparagopsis, which has been observed to reduce ruminant methane production by up to 98%1 when used as a cattle feed supplement. A series of in vitro assays have revealed that the California naturalized speciesAsparagopsis taxiformis (At)can reduce simulated enteric methane emissions by up to 50% when used as a 1% by weight feed supplement2. Through a NIFA SBIR grant with Carlsbad Aquafarm, we have established research developing methods for vegetatively propagating the tetrasporophyte stage ofAt, while simultaneously exploring approaches to open ocean cultivation and genetic identification of traits and population structures. The biomass produced is intended to be used in subsequent in vivo research trials and will continue establishing baseline research into efficient downstream applications for the commercial cultivation ofAt. Changes/Problems:The biggest challenge our team faced during Phase I growth trials was the limited growing season of Asparagopsis taxiformis. Researchers were only able, due to permitting, to collect along coastal California where only one site has been identified to collect Asparagopsis taxiformis, tetrasporophytes. Moving forward, researchers will ideally need the summer months when the tetrasporophyte life-cycle stage is abundant to collect and maintain growth trails. What opportunities for training and professional development has the project provided?Both Andrew Change, Carlsbad Aquafarm and Maddelyn Harden, University of Southern California are considered young career professionals who garnered a wealth of training opportunities throught the Phase I stage of this report. Andrew became better-versed in the realm of terrestrial aquaponics and flow-through systems though his training with Production Manager, Matthew Steinke; Maddelyn Harden was able to develop her molecular bench work skills through the DNA isolation and identification of different Rhodophyta strains which culminated in a poster presentation at the AGU Ocean Sciences Meeting in 2020. 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?
BIOMASS MEASUREMENTS: We first took weights on 10/29/19. Biomass was removed from each carboy system individually with a dip net and placed into a cleaned graduated container. Once removed, the biomass is gently drained of excess water.The biomass is then weighed in the Carlsbad Aquafarm lab. After weights were taken, the raceway system was restocked with 45-55g in each of the five carboys. We then had a remaining mass of 263g. The bioreactors in the green house began to look like a viable option to maintain higher temperatures during the day and throughout the night. We filled tube #3 with 163g and 150L of UV 5 micron filtered seawater, dosed with 80 mL of f/2, placed an airline and stone inside, positioned 4 T-8 full spectrum fluorescent lights, 3 heat lamps, and frost cloth along the outside bottom of the green house to enhance the system. The lights would be set to turn on at 1700 and turn off at 0700. The remaining 163g was placed in an opaque 5 gallon bucket with an airline and air stone in the Carlsbad Aquafarm Algae room. The algae room was the best temperature controlled room on the farm and has 24 hours of fluorescent lighting, thus thought to be a good choice for continuing this cultures growth. It was established that we would maintain a nitrate level between 30 and 40 ppm. Weights were then taken again 11/3, and over the course of 4 days, there was an increase of 2-4 grams in the carboy system. Now that we can quantify growth, through increases of weight, we can correlate it with our nutrient inputs (nitrate, phosphate, photoperiod, etc.) to determine optimal growth conditions. For an undetermined reason the system in the algae room had lost 50% of its mass, we continued to monitor this system. The bioreactor system had increased in biomass, 80% of the biomass in the bioreactor weight was 505g; this was likely due to a dose of f/2 that put nitrate levels at 80 ppm. Though the bioreactors proved to be a good grow out system, it should be noted that there is a dead space with little to no flow at the bottom where the system's drainage is. Biomass would accumulate and degenerate in this area causing a loss in biomass. The A.t. would accumulate on the air line at the surface of the water, a potential fix for both of these issues would be to fit the air into the drainage valve at the bottom of the system. With this increased growth in the bioreactor, we split the biomass into 2 more bioreactors with 163g to try and replicate the results. Weights were taken for the bioreactors that had just been stocked and they showed a similar increase. We were able to stock four more bioreactors with 163g each. 2 of these bioreactors were filled with 200L UV and 5 micron filtered seawater, and 2 with 100L with the double dose of f/2, 100 mL and 50 mL respectively. It was decided that the carboy system would be maintained as stock cultures. The carboys would now be on a water change routine of every 3 weeks with a 10 mL dose of f/2 once a week. The bioreactors were also switched over to a schedule of a water change every 3 weeks with culture media doses once a week. During the next 3 months, November through January, temperatures declined and we experienced a number of low pressure systems with rain, with a steady drop in temperature towards the end of October through November. There was a decrease in growth during this period of time. While performing water changes using the UV, 5 micron filtered water we saw an increase in fouling, in the form of Ulva lactuca. It was noted that during this time span was the time which U. lactuca releases its spores, which survive the UV treatment and pass through the 5 micron bag filtration. During water changes the U. lactuca was removed by hand as best as possible before restocking the bioreactors. After switching to the 3 week water change schedule it was seen that in the original bioreactors allowed for the At to settle on the rough surface on the interior. The newly stocked bioreactors have a smooth interior and provided less surface area for the At to settle onto. One of the new bioreactors crashed, on 1/12/20, due to some type of algal contaminant that turned the entire biomass a deep green color, we lost an approximate mass of 789g. GROWTH PARAMETERS: We are currently running the 5 carboys as our stock cultures, 6 bioreactors with approximately 3,600g of A.t. in total. Moving forward temperature is still the most fluctuating variable. An option is to reduce the volume of each sample container, while increasing the number of samples, and performing the study in a temperature controlled facility with a water table to reduce fluctuations. Future plans are to work on methods for sporulation so that we can make headway in upscaling production. Our ultimate goal is to seed grow out lines to be planted in our lagoon for mass production. MOLECULAR RESULTS: A new protocol was designed and streamlined for Rhodophyta DNA isolation. (See below). Samples of At were frozen and shipped from Hawaii where USC researchers conducted a DNA Isolation protocol on samples from Carlsbad and on the HI strains.DNA from each sample was then sequenced by Laragen Inc. Sequenced regions were compared to Gene Bank Accessions of the Cox 2-3 intergenic spacer region sequences. The HI strains, were an 85% accurate match for this known region of the At genome, whereas Carlsbad strains were not a match. Asparagopsis taxiformis DNA isolation protocol & PCR Amplification of DNA marker regions: modified from Andreakis et al. 2004 & Zuccarello et al. 1999 by Maddelyn Harden, 2020 DNA Extraction and Purification: Prep: I.Turn water bath on to 65 C II.Put isopropanol on ice III.Pre-cool centrifuge IV.Mix extraction buffer (below) Extraction Buffer (CTAB) modification: Making 2mL CTAB (for 2, 200mg samples) 1%=1g/100mL 1% w/v 10%=10g in 100mL 100mg x 2= 200mg of tissue (made a 10% stock of SDS & PVP (?) by putting 0.01g in 1mL) Step 1.) 100mg frozen tissue/700uL extraction buffer mix Mechanically Lyse using a tissue lyser. Add 700 uL of extraction buffer (below) to your mixture Incubate (water bath) your mixture at 65 C for 45 minutes vortexing every 5 minutes Extraction Buffer Mix Volume 2% w/v CTAB 0.04g 1.4 M NaCl (start 2M) 1.4 mL NaCl 20mM EDTA 80uL 100mM Tris-HCL, pH 8.0 30uL 0.2% w/v PVP (10% stock solution) 40uL 0.01% w/v SDS (10% stock solution) 2uL B-mercaptoethonal 4uL Step 2.) Extract DNA with an equal volume of chloroform/isoamyl alcohol (CIA: 24:1: v/v/) Centrifuge in a table-top microfuge at max speed for 10 min Collect aqueous phase, re-extract with CIA and centrifuge as above Step 3.) Mix aqueous phase with NaCl to 1.66M (You will need to calculate this concentration based on the new volume of your sample) Mix solution with an equal volume of ICE COLD 100% isopropanol Let sit on ice for 5 minutes Centrifuge in 4 C centrifuge at max speed for 15 min Step 4.) Wash DNA pellets in 300uL 70% v/v ethanol Centrifuge at max speed for 10 minutes Decant ethanol and allow to air dry Step 5.) Allow DNA pellets to dissolve overnight in 50uL of sterile water PCR amplification and sequencing of DNA marker regions forward primer (cox2-for) 5'-GTACCWTCTTTDRGRRKDAAATGTGATGC-3' reverse primer (cox3-rev) 5'-GGATCTACWAGATGRAAWGGATGTC-3'? PCR Mix Volume Template DNA See above note Water Fill to 50uL 5x HF Buffer 10 10uM dNTP 1 PI Primer (100uM starting) 0.5 P2 Primer (100uM starting) 0.5 MgCl2 (50uM) 2.5 Phusion HF Taq 0.5 PCR Amplification: The mitochondrial cox2 - 3 spacer was PCR amplified in 50 ml PCR reaction medium containing template DNA and 1mM of forward and reverse primers each, described in Zuccarello et al. (1999). 94.8 C for 4 min followed by 35 cycles of 93.8 C for 1 minute, 48.8 C for 1 min, and 72.8 C for 1.5 minutes followed by an extension cycle at 728 C for 5 minutes. Quantity and length of PCR products were examined by 1% gel electrophoresis.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Harden, M. (2020). Establishing Aquaculture Frameworks for the Methane Mitigating
Rhodophyta, Asparagopsis taxiformis. AGU Ocean Sciences Meeting February, 2020.
|
Progress 09/01/19 to 04/17/20
Outputs
Target Audience:The AGU Ocean Sciences Meeting 2020 was attended by Maddelyn Harden who presented the poster, described below, on the joint work between researchers at USC and Carlsbad Aquafarm. Poster Presentation: Establishing Aquaculture Frameworks for the Methane-Mitigating Rhodophyta, Asparagopsis taxiformis: Abstract:The ruminant livestock industry currently accounts for nearly a quarter of all global methane emissions, pointing to the urgent need to identify methods for methane mitigation in the farming of ruminants. One potential avenue for such mitigation efforts can be found through the use of the Rhodophyta genusAsparagopsis, which has been observed to reduce ruminant methane production by up to 98%1 when used as a cattle feed supplement. A series of in vitro assays have revealed that the California naturalized speciesAsparagopsis taxiformis (At)can reduce simulated enteric methane emissions by up to 50% when used as a 1% by weight feed supplement2. Through a NIFA SBIR grant with Carlsbad Aquafarm, we have established research developing methods for vegetatively propagating the tetrasporophyte stage ofAt, while simultaneously exploring approaches to open ocean cultivation and genetic identification of traits and population structures. The biomass produced is intended to be used in subsequent in vivo research trials and will continue establishing baseline research into efficient downstream applications for the commercial cultivation ofAt. Changes/Problems:The biggest challenge our team faced during Phase I growth trials was the limited growing season of Asparagopsis taxiformis. Researchers were only able, due to permitting, to collect along coastal California where only one site has been identified to collect Asparagopsis taxiformis, tetrasporophytes. Moving forward, researchers will ideally need the summer months when the tetrasporophyte life-cycle stage is abundant to collect and maintain growth trails. What opportunities for training and professional development has the project provided?Both Andrew Change, Carlsbad Aquafarm and Maddelyn Harden, University of Southern California are considered young career professionals who garnered a wealth of training opportunities throught the Phase I stage of this report. Andrew became better-versed in the realm of terrestrial aquaponics and flow-through systems though his training with Production Manager, Matthew Steinke; Maddelyn Harden was able to develop her molecular bench work skills through the DNA isolation and identification of different Rhodophyta strains which culminated in a poster presentation at the AGU Ocean Sciences Meeting in 2020. 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?
BIOMASS MEASUREMENTS: We first took weights on 10/29/19. Biomass was removed from each carboy system individually with a dip net and placed into a cleaned graduated container. Once removed, the biomass is gently drained of excess water.The biomass is then weighed in the Carlsbad Aquafarm lab. After weights were taken, the raceway system was restocked with 45-55g in each of the five carboys. We then had a remaining mass of 263g. The bioreactors in the green house began to look like a viable option to maintain higher temperatures during the day and throughout the night. We filled tube #3 with 163g and 150L of UV 5 micron filtered seawater, dosed with 80 mL of f/2, placed an airline and stone inside, positioned 4 T-8 full spectrum fluorescent lights, 3 heat lamps, and frost cloth along the outside bottom of the green house to enhance the system. The lights would be set to turn on at 1700 and turn off at 0700. The remaining 163g was placed in an opaque 5 gallon bucket with an airline and air stone in the Carlsbad Aquafarm Algae room. The algae room was the best temperature controlled room on the farm and has 24 hours of fluorescent lighting, thus thought to be a good choice for continuing this cultures growth. It was established that we would maintain a nitrate level between 30 and 40 ppm. Weights were then taken again 11/3, and over the course of 4 days, there was an increase of 2-4 grams in the carboy system. Now that we can quantify growth, through increases of weight, we can correlate it with our nutrient inputs (nitrate, phosphate, photoperiod, etc.) to determine optimal growth conditions. For an undetermined reason the system in the algae room had lost 50% of its mass, we continued to monitor this system. The bioreactor system had increased in biomass, 80% of the biomass in the bioreactor weight was 505g; this was likely due to a dose of f/2 that put nitrate levels at 80 ppm. Though the bioreactors proved to be a good grow out system, it should be noted that there is a dead space with little to no flow at the bottom where the system's drainage is. Biomass would accumulate and degenerate in this area causing a loss in biomass. The A.t. would accumulate on the air line at the surface of the water, a potential fix for both of these issues would be to fit the air into the drainage valve at the bottom of the system. With this increased growth in the bioreactor, we split the biomass into 2 more bioreactors with 163g to try and replicate the results. Weights were taken for the bioreactors that had just been stocked and they showed a similar increase. We were able to stock four more bioreactors with 163g each. 2 of these bioreactors were filled with 200L UV and 5 micron filtered seawater, and 2 with 100L with the double dose of f/2, 100 mL and 50 mL respectively. It was decided that the carboy system would be maintained as stock cultures. The carboys would now be on a water change routine of every 3 weeks with a 10 mL dose of f/2 once a week. The bioreactors were also switched over to a schedule of a water change every 3 weeks with culture media doses once a week. During the next 3 months, November through January, temperatures declined and we experienced a number of low pressure systems with rain, with a steady drop in temperature towards the end of October through November. There was a decrease in growth during this period of time. While performing water changes using the UV, 5 micron filtered water we saw an increase in fouling, in the form of Ulva lactuca. It was noted that during this time span was the time which U. lactuca releases its spores, which survive the UV treatment and pass through the 5 micron bag filtration. During water changes the U. lactuca was removed by hand as best as possible before restocking the bioreactors. After switching to the 3 week water change schedule it was seen that in the original bioreactors allowed for the At to settle on the rough surface on the interior. The newly stocked bioreactors have a smooth interior and provided less surface area for the At to settle onto. One of the new bioreactors crashed, on 1/12/20, due to some type of algal contaminant that turned the entire biomass a deep green color, we lost an approximate mass of 789g. GROWTH PARAMETERS: We are currently running the 5 carboys as our stock cultures, 6 bioreactors with approximately 3,600g of A.t. in total. Moving forward temperature is still the most fluctuating variable. An option is to reduce the volume of each sample container, while increasing the number of samples, and performing the study in a temperature controlled facility with a water table to reduce fluctuations. Future plans are to work on methods for sporulation so that we can make headway in upscaling production. Our ultimate goal is to seed grow out lines to be planted in our lagoon for mass production. MOLECULAR RESULTS: A new protocol was designed and streamlined for Rhodophyta DNA isolation. (See below). Samples of At were frozen and shipped from Hawaii where USC researchers conducted a DNA Isolation protocol on samples from Carlsbad and on the HI strains.DNA from each sample was then sequenced by Laragen Inc. Sequenced regions were compared to Gene Bank Accessions of the Cox 2-3 intergenic spacer region sequences. The HI strains, were an 85% accurate match for this known region of the At genome, whereas Carlsbad strains were not a match. Asparagopsis taxiformis DNA isolation protocol & PCR Amplification of DNA marker regions: modified from Andreakis et al. 2004 & Zuccarello et al. 1999 by Maddelyn Harden, 2020 DNA Extraction and Purification: Prep: I.Turn water bath on to 65 C II.Put isopropanol on ice III.Pre-cool centrifuge IV.Mix extraction buffer (below) Extraction Buffer (CTAB) modification: Making 2mL CTAB (for 2, 200mg samples) 1%=1g/100mL 1% w/v 10%=10g in 100mL 100mg x 2= 200mg of tissue (made a 10% stock of SDS & PVP (?) by putting 0.01g in 1mL) Step 1.) 100mg frozen tissue/700uL extraction buffer mix Mechanically Lyse using a tissue lyser. Add 700 uL of extraction buffer (below) to your mixture Incubate (water bath) your mixture at 65 C for 45 minutes vortexing every 5 minutes Extraction Buffer Mix Volume 2% w/v CTAB 0.04g 1.4 M NaCl (start 2M) 1.4 mL NaCl 20mM EDTA 80uL 100mM Tris-HCL, pH 8.0 30uL 0.2% w/v PVP (10% stock solution) 40uL 0.01% w/v SDS (10% stock solution) 2uL B-mercaptoethonal 4uL Step 2.) Extract DNA with an equal volume of chloroform/isoamyl alcohol (CIA: 24:1: v/v/) Centrifuge in a table-top microfuge at max speed for 10 min Collect aqueous phase, re-extract with CIA and centrifuge as above Step 3.) Mix aqueous phase with NaCl to 1.66M (You will need to calculate this concentration based on the new volume of your sample) Mix solution with an equal volume of ICE COLD 100% isopropanol Let sit on ice for 5 minutes Centrifuge in 4 C centrifuge at max speed for 15 min Step 4.) Wash DNA pellets in 300uL 70% v/v ethanol Centrifuge at max speed for 10 minutes Decant ethanol and allow to air dry Step 5.) Allow DNA pellets to dissolve overnight in 50uL of sterile water PCR amplification and sequencing of DNA marker regions forward primer (cox2-for) 5'-GTACCWTCTTTDRGRRKDAAATGTGATGC-3' reverse primer (cox3-rev) 5'-GGATCTACWAGATGRAAWGGATGTC-3'? PCR Mix Volume Template DNA See above note Water Fill to 50uL 5x HF Buffer 10 10uM dNTP 1 PI Primer (100uM starting) 0.5 P2 Primer (100uM starting) 0.5 MgCl2 (50uM) 2.5 Phusion HF Taq 0.5 PCR Amplification: The mitochondrial cox2 - 3 spacer was PCR amplified in 50 ml PCR reaction medium containing template DNA and 1mM of forward and reverse primers each, described in Zuccarello et al. (1999). 94.8 C for 4 min followed by 35 cycles of 93.8 C for 1 minute, 48.8 C for 1 min, and 72.8 C for 1.5 minutes followed by an extension cycle at 728 C for 5 minutes. Quantity and length of PCR products were examined by 1% gel electrophoresis.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Harden, M. (2020). Establishing Aquaculture Frameworks for the Methane Mitigating
Rhodophyta, Asparagopsis taxiformis. AGU Ocean Sciences Meeting February, 2020.
|