Progress report for LNE21-431R
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 ~6 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Instead, US farmers rely upon provision of tenuous wild-sourced seed to cultivate juvenile sporophytes for product, an outdated approach subject to natural vagaries in environmental conditions and stochastic changes in reproductive timing, fecundity, and seed quality.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.
The proposed study seeks to develop long-term maintenance technologies for current (Sugar kelp - Saccharina latissima) and emerging seaweeds (Winged kelp – Alaria esculenta, Irish moss – Chondrus crispus, and Dulse – Palmaria palmata) in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Laboratory experiments will be used to develop cryopreservation approaches and will be initiated by harvesting wild broodstock (spores) of commercially emergent seaweeds 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. Using live-cultured spores, the traditional culturing technique will serve as a control and will be compared to revival culturing using preserved/thawed seed stocks. Accurate indication of viability is critical to developing quality control measures for nurseries seeking to offer cryopreservation services. We plan to engage both a hatchery/nursery (Atlantic Sea Farms) and a seaweed farm (Maine Sea Farms) to test the feasibility and ease of implementation of cryogenic protocols as well as provide seeded lines for grow-out tests of both brown and red algae for on-farm trials.
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.
Research goal: To develop long-term maintenance technologies for current (sugar kelp - Saccharina latissima) and emerging seaweeds in the Northeast by testing the efficacy of various sterile cryopreservation techniques for storage of vegetative cultures and spores.
Although funding was received for this new NE SARE’s Research for Novel Approaches project in March 2021 – our research formally began in the Fall of 2021. To begin this project we established a cryogenic center with an auto-fill liquid nitrogen dewar for the storage of cryogenically preserved seaweed seed/spores.
During the fall of 2021 we targeted two phenotypes of sugar kelp (Saccharina latissima), which included skinny and wide blade phenotypes. Sporulation and cryogenic freezing activities were conducted in October and November 2021 for each phenotype, respectively. For each sporulation of seed the following methodology was applied: Natural sources of sugar kelp (Saccharina latissima) reproductive tissue were obtained by the co-PIs from of mature sporophytes collected from two locations along the midcoast, Maine and key morphological features were recorded (stipe length, blade length and width). For each phenotype 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.
For the “skinny kelp” phenotype two cryoprotectants mixed 1:1 in 2 mL cryovials were used and 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 vapor. For the “wide blade” phenotype three cryoprotectants were used and mixed 1:1 in 2 mL cryovials and frozen via CRF. For each phenotype the following number of vials were collected and stored in Liquid Nitrogen:
Skinny blade Kelp: cryoprotectant 1 = 97 vials, cryoprotectant 2 = 96 vials
Wide blade Kelp: cryoprotectant 1 = 75, cryopretectant 2 = 76, & cryoprotectant 3 = 55.
The number of vials cryogenically frozen depended on the meisopore density during each sporulation event. Also in order to minimize the genetic variation between different kelp blades, the meiospores from several fronds were combined for each sporulation event and downstream cryogenic freezing.
In February 2022 we will test the vitality of these cryoprotectants used and we plan to revive the sugar kelp meiospores using 3 thawing techniques. The survival and vitality will be estimated by enumerating the fraction of meiospores that are swimming via microscopy (for kelp species) and vital staining using flow cytometry.
No formal results at this time.
No formal conclusions at this time.
Education & Outreach Activities and Participation Summary
Over the course of the past year, tours at Bigelow Laboratory have taken place for both the general public and for educational purposes (high school students and science educators) - during these tours the aspects of this seaweed project are highlighted and demonstrations of kelp reproduction have been presented.