Final report for LNE21-431R
Project Information
Seaweed farming is the second largest aquaculture industry worldwide, with global production used in cuisine, manufactured foods, fertilizer, and animal feed. Sea vegetables (the edible subset) are a $6 billion market worldwide and a growing industry, particularly in the Northeast US where commercial species are native. In the Northeast there are >200 established seaweed farms currently in operation as well as ~2 seaweed nurseries that supply these farms. Techniques for growing sea vegetables (both brown and red algae) are well established despite a complex two-part life cycle, but the US industry has been slow to adopt the more technically advanced germ line cultivation techniques used in Asia and Northern Europe. Instead, US farmers rely upon provision of tenuous wild-sourced seed to cultivate juvenile sporophytes for product, an outdated approach subject to natural vagaries in environmental conditions and stochastic changes in reproductive timing, fecundity, and seed quality. To increase production for novel emerging US seaweed markets, an investment into the technological methods for mechanization of seed stock development and preservation are needed. Annually, sea vegetable nurseries require access to sterilized seawater and sufficient lighting, space for care and maintenance of seed stocks, and expertise, making seedling cultivation a barrier to entry for most new aquaculturists. With the development of seed or broodstock cryopreservation, this provides ‘insurance’ for common and specialized strains, minimizing manual labor associated with long-term cultivation and shortened turnaround times for the production of healthy, viable, juvenile sporophytes on ‘seeded-lines’ that can be sold to farmers.
This study developed long-term maintenance technologies for Sugar Kelp - (Saccharina latissima) in the Northeast region by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Laboratory experiments examined cryopreservation approaches and were initiated by harvesting wild broodstock (spores) of commercially produced sugar kelp and subjecting these spores to different freezing and thawing rates and cryoprotectants. Initial results for sugar kelp (Saccharina latissima) spores indicate that controlled-rate-freezing (CRF) is more effective than flash freezing. Accurate indication of viability is critical to developing quality control measures for nurseries seeking to offer cryopreservation services. We engaged a hatchery/nursery (Atlantic Sea Farms) to test the feasibility and ease of implementation of cryogenic protocols. Atlantic Sea Farms also assisted in the development of seeded lines for grow-out tests that could be used by seaweed farmers in the future.
Edible seaweeds are a $6 billion USD market worldwide. Globally, the seaweed aquaculture sector is growing 7.7% annually, and a rate twice that in the Northeast US where many commercial species are native. To increase production for novel emerging US seaweed markets, an investment into the technological methods for mechanization of seed stock development and seed preservation are required. We propose to develop and optimize a well-established method for cryopreserving (freezing), thawing and growing juvenile seaweed vegetable species in biosecure nurseries. The method maintains frozen cultures of the microscopic life stage (spores) of current farmed seaweed vegetables and emerging species.
The cultivation of plants from seed for land-based agriculture systems was established thousands of years ago when man first domesticated plants. Since then Asian countries like China and Japan have been ‘farming’ seaweeds for hundreds of years, however, it wasn’t until the 1940s that the full life cycle of seaweeds (macroalgae) and alternation of generations between the visible edible sporophyte and the microscopic spores/gametophyte stages was elucidated. Over the past ~80 years, the number of total cultivated seaweeds has increased rapidly, yet globally we still have yet to develop a systematic approach to accumulating and storing seaweed ‘seed’.
In the Northeast US, >200 aquaculture leases are permitted to farm seaweed and 2 nurseries supply these farms. Techniques for long-line cultivation in nearshore environments are well established, but are not without biosecurity concerns. Seaweed diseases threaten farms in Europe and Asia, but have not yet impacted US operations; biofouling issues are much more common. Further, farmers must rely on tenuous wild-sourced spores from nearby natural populations to create seeded-line. This outdated approach is subject to vagaries in environmental conditions and stochastic changes in reproductive timing, fecundity, and seed quality. The probability of vertical transmission of bacterial or fungal disease from natal broodstock to spores is unknown, but contamination of nurseries by diatoms and ciliates carried over from the wild broodstock is common and disruptive to seed production. Creating on-demand cryopreserved seed from farmed broodstock reduces the probability of contamination that plagues long-term live culture and allows for maintenance of a selected proprietary strain within a farm.
Some cryopreservation protocols are available for kelp (brown macroalgae) species, but they vary widely in the media and cryoprotectant used, life stage frozen, freezing process, thawing process, and tend to be very species-specific; optimizing the protocol can lead to an improvement from <10% to >80% viability of the thawed stock and still needs to be explored in detail for any red macroalgae species. Cryopreservation minimizes manual labor associated with long-term cultivation and shortens turnaround times for the production of healthy, viable juvenile sporophytes on ‘seeded lines’ that can be made available directly to farmers. Currently, profitability for seaweed farmers is limited to a short harvest period using the wild-sourced seed approach. With the availability of a seed cryopreserved source throughout the year, farmers could sequentially seed to maximize production annually and extend their growing season to year-round.
In order to increase safe production of emerging seaweeds, an investment into the technological methods for mechanization, broodstock development, and preservation is required for year-round cultivation. Here we developed and optimized an established method for cultivating seaweed species. The method maintains frozen cultures of the microscopic life stage of sugar kelp (Saccharina latissima) to facilitate year-round production. During this process we were able to generate contaminant-free cryogenic meiospore seed of sugar kelp that is favorable for commercial markets.
Cooperators
- (Researcher)
Research
Research goal: To develop long-term maintenance technologies for current (sugar kelp - Saccharina latissima) and emerging seaweeds (Winged Kelp – Alaria esculenta) in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Development of cryogenic freezing, thawing and re-growth, protocol for Sugar Kelp meiospores
Our research formally began in the Fall of 2021, and as part of this project we established a cryogenic center at Bigelow Laboratory dedicated to this project, that contained an auto-fill liquid nitrogen dewar for the vapor storage of cryogenically preserved seaweed seed/meiospores.
During the fall of 2021 and 2023 we targeted two phenotypes of sugar kelp (Saccharina latissima), skinny and wide blade. Sporulation and cryogenic freezing activities were conducted in October and November of 2021 and 2023 for each phenotype. Natural sources of reproductive tissue from mature sporophytes were collected from different geographic locations along the mid-coast, Maine. These sporophytes were collected by the project team in 2021 via diving and by our nursery partner (Atlantic Sea Farms) in 2023. The key morphological features were recorded (stipe length, blade length and width) after collection.
Sporulation:
To induce sporulation of the reproductive sorous tissue, the surface of the reproductive tissue was soaked in sterile seawater for 5–10 min, cleaned with a damp cloth, scraped with a clean razor blade then stored on paper towels in the dark for 24 hours at 10°C. The following day the meiospores were harvested following the procedures of Flavin et al 2013 and Redmond et al 2014.
Cryogenic (and Non-Cryogenic) Meiospore Freezing:
In 2021 two different phenotypes (skinny and wide blade kelp) of S. latissima were tested using cryogenic freezing. For the skinny kelp two cryoprotectants were mixed 1:1 (meiospore solution to cryoprotectant) in 2 mL cryovials. The meiospores were frozen using a ThermoFisher CryoMed controlled rate freezer (CRF) at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen (LN) vapor using an auto-fill LN2 dewar. For the wide blade kelp three cryoprotectants were tested, and mixed 1:1 in 2 mL cryovials and frozen via CRF (see above). For each phenotype the following number of vials were collected and stored:
Skinny blade Kelp: Cryo-1 = 97 vials, Cryo-2 = 96 vials
Wide blade Kelp: Cryo-1 = 75, Cryo-2 = 76, & Cryo-3 = 55 vials
The number of vials frozen depended on the meiospore density (optimal > 1 million per mL) for each of the sporulation events. To minimize the effects of genetic variation between different kelp blades, the meiospores from several kelp fronds were combined during each sporulation event for cryogenic freezing. There are advantages and disadvantages to doing this, as each kelp frond has a different meiospore vitality (unpublished data), but in order to test different cryoprotectants we chose to combine meiospores from different blades (from the same geographic location) in order to generate many vials for downstream vitality testing (see protocol below), and differentiation into gametophytes and sporophytes.
In 2023 our team worked in collaboration with Matthew (Thew) Suckiewicz at Atlantic Sea Farms (ASF), a project cooperator/seaweed nursery, to obtain meiospores for cryogenic freezing of both phenotypes of sugar kelp (wide and skinny blade). Please note that other species of kelp (ie. Alaria) were not collected by ASF in 2023 so our primary focus was to optimize cryogenic freezing protocols for S. latissima. Over the course of the fall of 2023, three sporulation events were conducted at ASF. A controlled rate freezer (CRF) and liquid nitrogen tank was brought on site to ASF in Biddeford, ME so meiospores could be frozen quickly after sporulation. Similar to 2021 and to minimize genetic variability, multiple fronds from one geographic location along mid-coast Maine were combined to obtain a single mixture of meiospores for each sporulation and cryopreservation event. Based on our previous results for vitality (see vitality methods below) in 2021, two cryoprotectants, Cryo-1 and Cryo-3 were used for all three sporulation events at ASF. In order to optimize and increase the number of frozen meiospores the volume per cryovial was increased from 1 mL to 1.8 mL. For each sporulation event, 10 mL of live meiospores was transported back to Bigelow Laboratory for LIVE meiospore vitality testing. The remaining meiospore solution was cryopreserved via CRF. To verify that CRF was the optimal freezing method, a few vials of meiospores with each cryoprotectant were frozen at -20 C (using a standard freezer).
ASF Sporulation Event 1 - October 3, 2023, Sugar Kelp - wide blade, source – mid-coast Maine, Lot # 275A1-23. For this sporulation event, the initial spore density was ~ 1.6 million per mL, and 220 vials were frozen (110 per cryoprotectant) using CRF (at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen vapor). Afterwards, vials were thawed using a water bath (see method below) and we attempted to seed spooled lines (for eventual farm out-planting). Growth and differentiation was monitored by ASF of both parent and cryopreserved seeded spools.
ASF Sporulation Event 2 - October 18, 2023, Sugar Kelp - wide blade, source mid-coast Maine. Lot #291A1-23. For this sporulation event, the initial spore density was ~1.7 million per mL, 302 vials were frozen (151 per cryoprotectant) using CRF (at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen vapor). Afterwards, we attempted (again) to seed spooled lines post-cryogenic freezing. In this case, we incubated the spools in the meiospores seeding solution for various lengths of time, then the experimental spools were inspected and transferred to the recirculating tanks with the same parent stock. Growth and differentiation was monitored by ASF of both parent and cryopreserved seeded spools. In addition, from this seed lot, the remaining frozen vials (from both cryoprotectants) returned to Bigelow Laboratory for long-term vitality testing and meiospore differentiation.
ASF Sporulation Event 3 - October 29, 2023, Sugar Kelp - skinny blade, source mid-coast Maine. Lot #302B1-23. For this sporulation, the initial spore density was ~400,000 per mL, ~176 vials were frozen (88 per cryoprotectant) using CRF (at a rate of 1°C per min to a final temperature of -40°C followed by storage in liquid nitrogen vapor). In addition, all the remaining frozen vials (from both cryoprotectants) returned to Bigelow Laboratory for long-term vitality testing and meiospore differentiation (no cryogenic seed spool attempts were made on site at ASF).
Meiospore Thawing (post cryogenic freezing):
For the revival of meiospores (post-freezing), thawing methods are important aspects of meiospore survival and downstream differentiation. It is very important to remove or dilute the cryoprotectants soon after thawing as long-term exposure to the cryoprotectant will be detrimental to the spore vitality. In 2022, two thawing methods were examined for our two Saccharina latissima phenotypes (skinny and wide blade), 1) rapid thawing in a water bath and 2) CryoThaw: a simple centrifuge tube adapter to expedite and automate thawing.
For rapid thawing in a water bath, each frozen meiospore cryovial is held in a 20 C water bath until a pea sized pellet appears and then the vial contents is transferred into a 9 mL vial of chilled (4 C) Prov50/2 media and inverted 5 times then evaluated for vitality using Fluorescein Diacetate (FDA, see vitality testing below). Ideally a dilution of 1:10 or greater is recommended.
CryoThaw (Medax Intl. Inc.; Beddall et. al, 2016) uses a small tube adaptor for 15 mL centrifuge tubes that holds the frozen uncapped meiospore cryovial at the entrance of the centrifuge tube containing 9 mL Prov50/2 media (chilled to 4 C). The centrifuge is programmed to 22 C, 1500 RPM, for 5 min. Once the vial thaws the meiospore pellet enters the culture media within minutes and is diluted rapidly within a controlled environment. Afterwards, the 15 mL vial is removed and inverted 5 times prior to analysis for vitality. The goal of this method is to provide a controlled environment and standardized method for thawing and diluting meiospores.
In 2023 when working with ASF for attempted cryogenic seeding onto spools, only the rapid thaw water bath method was used, as obtaining a large centrifuge would be challenging for farmers and nurseries. Since all frozen meiospore vials generated at ASF contained 1.8 mL each frozen meiospore vials were placed in a 22 C water bath for 2.5 mins until a pea sized pellet appeared and then the vial contents was emptied into chilled seawater and diluted. The thawed meiospores were used to seed spooled lines in aquaria with seawater containing diluted cryogenically preserved meiospores.
Meiospore Vitality Testing
As a part of this research, flow cytometric vitality testing was used to rapidly assess meiospore vitality before and after freezing using Fluorescein Diacetate (FDA). To prepare for flow cytometric staining, 5 mg of FDA was diluted into 1 mL of acetone (primary stock). A working stock of FDA was prepared by adding 40 μl of primary FDA stock to 1 mL cold deionized water and kept on ice in the dark until use. To assess vitality during each experiment, vitality was assessed before and after freezing. For this assessment, 1 mL of live or thawed meiospores were stained with 10 μL of FDA working stock and incubated at 10 C for 20 mins prior to flow cytometric analysis on a BioRad ZE5 Cell Analyzer. All vital meiospores fluoresce green (525/30 BandPass) with blue laser excitation (488 nm) and are counted. The total number of meiospores within each vial is determined using red fluorescence (692/30 BandPass), as an indicator of chlorophyll in all meiospores. The percent of vital cells was determined and recorded for each vial and cryoprotectant after each cryogenic test and over time to assess vitality with prolonged storage in LN2 vapor. A negative control during each experiment, 1 mL of live or thawed meiospores is heat killed at 50 C for 30 min and stained using the FDA protocol above.
In 2023, we determined if vitality changed over time while being stored in LN2 vapor. For this testing two phenotypes of Sugar Kelp (wide and skinny blade) were examined using two cryoprotectants (Cryo-1 and Cryo-3) ~ every 15 days for up to ~ 60 days and then periodically for up to one year.
Motility Testing and Microscopic Examination
After thawing, each thawed solution is observed under the microscope using a hemocytometer to determine if any of the thawed cells are motile – an indication of vitality.
Meiospore Differentiation Post-Freezing
To assess if differentiation after thawing the diluted seed solution (1:10 dilution) was placed into a 6-well tissue culture plate and incubated on a 12/12 light/dark cycle and monitored for meiospore differentiation into gametophytes and eventual sporophyte formation over a period of 4-6 weeks. The 6 plates were monitored for differentiation approximately 1-2 times per week after thawing. This was done in triplicate using both cryoprotectants.
Development of Winged Kelp (Alaria esculenta) Sporulation for Future Cryopreservation
In fall of 2022 a protocol for releasing Winged Kelp (Alaria esculenta) spores from reproductive fronds was developed in order to begin testing spore cryopreservation techniques. The sporophylls were collected from natural sources (Pemaquid Point, ME), and returned to the lab on ice for processing. The sporophylls were removed from the fronds and scraped with a razor blade to remove mucus and contaminates from the surface. Afterwards the sporophylls were divided into two treatments to induce spore release. 1) Betadine dip: sporophylls were dipped in a Betadine solution (500 μL of Betadine to 500 mL of DIW) for 2 mins then removed into a tray of filtered seawater (FSW) and scraped with a razor blade again, sandwiched between paper towels, and incubated overnight in the dark at 12 C. 2) FSW treatment: sporophylls were dipped in FSW and scraped with a razor blade to remove mucus and surface contaminants, afterwards all scraped sporophylls were dipped in FSW for 30 seconds then removed and scraped again with a razor blade. All sporophylls were sandwiched between paper towels and incubated overnight at 12 C. Of the two treatments the one with the most success (largest spore release) was the Betadine dip. Unfortunately, all the reproductive tissue in the field had been harvested when we attempted a sporulation for cryopreservation. To date (both in 2022 and 2023) we were unable to obtain additional reproductive fronds of Winged Kelp and diverted our focus to the two phenotypes of Sugar Kelp.
References:
Beddall, M., Chattopadhyay, P. K., Kao, S. F., Foulds, K., & Roederer, M. 2016. A simple tube adapter to expedite and automate thawing of viably frozen cells. Journal of immunological methods, 439, 74–78.
Flavin, K., Flavin, N., Flahive, B. 2013. Kelp Farming Manual: A Guide to the Processes, Techniques, and Equipment for Farming Kelp in New England Waters. Ocean Approved, Portland, Maine.
Redmond, Green S. L., Yarish C., Kim J., and Neefus C. 2014. New England Seaweed Culture Handbook-Nursery Systems. Connecticut Sea Grant CTSG‐14‐01. 92 pp.
Cryoprotectant Optimization and Freezing methods for Sugar Kelp
Using vials preserved in 2021 using wide blade sugar kelp (Saccharina latissima) we examined vitality using three cryoprotectants and two thawing methods. Of the three cryoprotectants tested, Cryo-3 had the highest FDA vitality after thawing (Figure 1). When comparing both phenotypes of sugar kelp, Cryo 1 had slightly higher vitality compared to Cryo2. (Figure 1 & 2). Based on these initial experiments we decided to use two cryoprotectants, Cryo-1 and Cryo-3 for all future experimental work. For both cryoprotectants, freezing at -20 C using a standard laboratory freezer showed 0% vitality after thawing. Early results of the two thawing methods (Figure 1 & 2) were inconclusive and would require much more replication and experimentation for validation. Therefore, considering the ease of use (at a farm or nursery), the rapid thawing using a water bath procedure was prioritized and used for the remainder of all experiments. In nearly all cases, no meiospore motility was observed using microscopy after cryogenic freezing.
Longevity of Sugar Kelp Meiospore Vitality using different cryoprotectants
Between 2023 to 2024 the longevity of FDA vitality observed in cryogenically preserved meiospores was examined for the course of one year. Two ASF sporulation Lots, wide and skinny kelp phenotypes (Lots #291A1-23 & #302B1-23) respectively, were compared. High FDA vitality, ranging from 83-89 % vital, was observed for live meiospores in both meiospore Lots prior to cryopreservation (Figure 3. A & B). For each cryoprotectant the percent vitality stayed relatively constant over time. When comparing the two cryoprotectants to one another there was a significant difference in vitality. Overall, Cryo-3 had higher vitality ranging between 18 - 50% vital for both ASF Lots whereas Cryo-2 ranged from 3.5 - 8 % vital (Figure 3).
Meiospore Differentiation (post-thawing) and Seeding
For each thawing event between 2023-2024, the diluted sugar kelp spore material was plated and incubated as previously described, and gametophyte and sporophyte formation was assessed over a 4 to 6-week period. In all cases, gametophyte and sporophyte formation was observed after thawing for meiospores that were cryogenically preserved using CRF, even after a year in liquid nitrogen vapor storage (see Image 1).
In 2023, during our collaboration with ASF, we attempted to seed spools using meiospores both before and after cryopreservation. For the first and second sporulation events at ASF on Oct 3rd and Oct 18th, 2023, respectively, the parent (live) stock was seeded using the industry standard method and the thawed cryogenic meiospores stock were seeded using a proprietary method (iEdison invention disclosure 1796801-25-001). After 3-4 weeks of monitoring by ASF, normal sporophyte growth was observed on the parent stock spools that were seeded with live meiospores, however, no sporophyte growth was observed on the spools using cryogenically preserved meiospores. We decided for the final sporulation event in 2023 (Lot #302B1-23) and with frozen vials from the first and second spore Lots to focus on developing a new proprietary method for seeding after cryopreservation in the laboratory.
Since meiospore differentiation was previously observed (using plates) after cryopreservation (see Image 1), our team began to develop a method of setting cryogenically preserved meiospores onto line using a proprietary method. Part of this work was to determine if meiospore differentiation (gametophyte and sporophyte formation) onto line was possible after spore cryopreservation, the other aspect of this work was to test out-planting on a sea farm to ensure re-growth of mature sporophytes on seeded line.
In order to develop the new method we used cryopreserved meiospores from the ASF Sporulation Lots #291A1-23 & #302B1-23 (each representing the wide and skinny phenotypes, respectively). Preliminary results using this new seeding method showed limited differentiation to the gametophyte stage using spores sourced from wide blade kelp (Lot #291A1-23); however, analysis is still underway and will continue during the summer of 2025 as part of an undergraduate research project. Preliminary results obtained from the skinny kelp phenotype (Lot #303B1-23), showed high meiospore differentiation to both gametophytes and sporophytes (data not shown for proprietary reasons).
To assess sporophyte density on the seeded lines, all sporophytes were removed from the surface as well as all floating sporophytes and counted for each replicate treatment. Preliminary sporophyte results show a higher average sporophyte density for the Cryo-1 (43 sporophytes) compared to Cryo-3 (18 sporophytes). Further examination of kelp biomass (image analysis) is required to assess the effect of the cryoprotectants on meiospore differentiation and will be continued during the summer of 2025.
As a final note, the seeded line using our proprietary method was out-planted in East Boothbay, ME in early January 2025 using a Limited Purposed Aquaculture (LPA) License in collaboration with the Boothbay Sea and Science Center. The out-planting will assess if the new proprietary seeding method produces adult kelp fronds for aquaculture production. Final re-growth assessment will take place in the late spring of 2025.
In order to increase the long-term safe production of emerging seaweeds, an investment into the technological methods for mechanization, broodstock development, and preservation is required for year-round cultivation. Here, we developed and optimized an established method for cultivating seaweed species, specifically sugar kelp (Saccharina latissima). The method maintains frozen cultures of the microscopic life stage of sugar kelp to facilitate year-round production. During this process we were able to generate contaminant-free cryogenic meiospore seed of sugar kelp that is favorable for commercial markets using two different cryoprotectants.
The use of cryopreservation is essential for the future seaweed industry as sourcing wild-seed stocks will become challenging if not prohibitive in the future. With the cryogenic preservation of seed stocks, the establishment of an edible seaweed seed bank for the Northeast edible seaweed industry is possible. Seed banking and re-growth will be a key mechanism for establishing gametophyte cultures for use within the edible seaweed industry. Additionally, strains that are developed using animal husbandry methods or genetically modified for adaptations to environmental conditions (ie. temperature) could be maintained in near perpetuity for future use in a cryogenic state.
This work emphasizes the need for cryopreservation method standardization and continued optimization for edible seaweeds. Optimization is a continuous process, as there are many variables that affect the success of cryopreservation. However, during this work, we minimized certain variables (thawing methods) based on practicality and ease of use within the industry, but optimization could continue in areas, such as meiospore sporulation techniques and CRF timing.
A major outcome of this work was the development of a standard working protocol for effective cryopreservation of Saccharina latissima (sugar kelp) meiospores. This protocol could be used be used by industry (ie. seaweed nurseries) or by seed banks (ie. National Center for Marine Algae and Microbiota at Bigelow Laboratory) for the assimilation of seaweed spores into public or private collections. Currently, the cost of maintaining seaweed gametophyte cultures (on farms or in culture collections) is currently between 2 to 3 times higher than maintaining cryopreserved seed stocks within seed banks. As a long-term strategy for the seaweed industry, the use of meiospore cryopreservation will be key to making seaweed strains/phenotypes available to nurseries or farmers in perpetuity.
Education & Outreach Activities and Participation Summary
Educational activities:
Participation Summary:
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Presentations
MEDIA & FIELD TRIPS 2022
2023
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Learning Outcomes
Unfortunately, Bigelow Laboratory does not have an IRB committee, and there were no social scientists involved in the study, so surveys were not an appropriate tool in this research program.
Project Outcomes
At this time cooperators /nurseries that assisted and were a part of this research (providing reproductive tissue and grow out of seeded line) has been modified from our original proposal. A list of current cooperators are presented within this report. Unfortunately, one of our farmers at Maine Fresh Sea Farms passed away in 2022.