Proofing Mycelium-based Buoys in Aquaculture Applications

Treatments

The primary factor in the success of the myco buoys is how long they are able to last in a dynamic ocean environment. The treatments will test one waterproof 100% bio-based coating against an uncoated control and a commercial marine paint. Previous field trials have indicated potential seasonal longevity for uncoated buoys if they were allowed to grow longer than one week enabling the strain of fungus used to further develop its own hydrophobic outer layers. Our hope is that this may satisfy the five-six month season used to float oysters near the ocean surface. If sealants are required to guarantee longevity of mycobuoys for oysters, we strive for 100% biocompatibility in the coatings such that any future composting or degradation of the buoys will not harm marine ecosystems. In fact, our goal is to compost the buoys as added nutrients on land at the end of their useful marine life. A third treatment using a proven lobster buoy paint will serve as a control to see if the mycelium-based biocomposite can replace carcinogenic polystyrene and polypropylene. Biofouling for each treatment will be recorded to ascertain the antifouling properties of the coatings used. Based on prior testing, we have determined that the following treatments are most likely to yield comparative data that will inform future iterations.

1. Uncoated - grown for 2 weeks before drying (Control) These were grown between 9 and 12 days before drying with the "flip" day occurring on days 5 or 6.

2. Two coats soy paint - two coats are necessary to maintain surface integrity

3. One coat commercial sealant - current practice for lobster buoys. Two coats were needed to completely seal irregular edges of all three buoy designs.

Methods

From prior research, testing, and farmer input, we identified three unique buoy designs that serve the needs of farmers in aquaculture. A second orange-pigmented fungal strain comprised a fourth subset of 13 mooring design buoys to affirm Van Hook’s earlier successful research with this species. In the first phase of this project, we focused on buoys for oyster cultivation applications. In year one, 244 buoys were grown and deployed in practical application by 8 participating farmers:

• 12 8" diameter cylinder by 30" height, 24 with each treatment
• 75 24" x 16"x 3" rectangular flats, 24 with each treatment
• 144 32" x 4" x 3" half-domes, 25 with each treatment
• 13 mooring buoys of three shapes and sizes

Buoys were monitored by Key Individuals on a bi-weekly basis. This interval is congruent with the regular operations of oyster farmers, who flip and check their cages every two weeks.

Based on results in year one, we repeated the four buoy designs from the first year using a different substrate supplier, namely Lambert Spawn Co. Their substrate was identical to the substrate used in Van Hook’s original research between 2011 and 2016 that performed well for 4-6 months in the ocean. We grew 180 flat buoys, 240 half-dome buoys, 33 hollow cylinder buoys and 16 mooring ball buoys and 135 bullet buoys and distributed them among 10 oyster farmers. However, new thermoformed tooling was used to make the cylinders, half-domes and flats the second year.

Buoys were again monitored by Key Individuals on a bi-weekly basis.

Data Collection

Data collected during monitoring included:

1. a) Scoring of surface degradation of mycelium buoys
2. b) Presence or absence of biofouling on immersed buoy surfaces
3. c) photo documentation
4. d) end of field trial success/failure score

Revised Methods Year One

Tooling for all three designs presented a set of challenges. The 50 plastic tubs with lids that were ordered for making the rectangular flat buoys arrived but did not come close to meeting the measurements reported on the label. We ordered 400 feet of 2 x 4 lumber and assembled 36 wooden frames with 24 x 16" inner dimensions. A consequence of this change was that the outer dimensions increased to 24 x 18" so our grow racks could fit only two buoys per shelf rather than three. Our growing capacity was therefore brought down from 50 to 36 buoys. The frames were primed with white enamel paint so that they could be washed between uses (Figure 9). We fashioned bottoms by taping mylar plastic between uses, and lids by taping a 3 ml plastic film taut across the top of the frames. Two 1.5-inch wide hemp fabric straps, to serve as attachments to cedar crate oyster cages, were evenly spaced lengthwise across the bottom and short ends of the frame before filling them (Figure 10). We poked needle-sized holes at 3-inch intervals across the upper film to allow for air exchange (Figure 10). All components were washed and sterilized in a 10% bleach bath between uses. This greatly increased the time spent to make each buoy from the anticipated 10 minutes to 45 minutes. We made a total of 80 rectangular flat buoys distributed between two oyster farmers using the cedar wooden crates design, Smithereen Farm received 15 and North Haven Oysters received 60. A third of these were left uncoated after drying as a control, a third were painted with two coats of red soy paint, and a third were painted with two coats of yellow or orange lobster buoy paint (Figure 11).

The forms for the large hollow cylinder buoys were constructed using aluminum sheet metal taped together along the seam for the outside and mylar cylinders internally supported by two adjustable metal rings. The cavity between the two cylinders, measuring 8″ at the outer diameter and 31″ in height (Figures 12,13), was filled with 4 ⅓ bags of inoculated substrate atop a perforated metal tray. The trays were placed upright in a baker’s rack beneath a plastic humidity tent and were allowed to grow for 6 days. On the sixth day the inner mylar cylinder was gently removed to allow for complete colonization inside the cylinder. The cylinders were removed from the baker’s racks on the 13^th the remaining contents as it could not be evenly distributed into the buoy mixture. An average of 5 bags were discarded per day due to poor colonization or evidence of bacterial contamination (Figure 14).

For the flat rectangular buoys, the assembly and disassembly and cleaning of these forms added many hours to production. We spent an average of 45 minutes per day to begin air-drying.

The half-dome buoys were all grown at the North Spore facility between April 25^th and May 18^th. A supply chain delay for commercial metal rain gutters forced us to fabricate alternative forms from aluminum sheeting held with two galvanized steel end caps (Figure 15). Buoys were removed from their forms at irregular intervals as a result of staffing availability between 5 and 8 days and placed flat side down on racks within a humidity tent to allow for full colonization on all surfaces (Figure 16). After 12-14 days of total colonization, the buoys were placed on racks and rolled to the boiler room for drying where the average temperature was 110°F.

Mooring buoys were grown in the Myco Lab in Pembroke using stainless steel restaurant bowls in three different sizes. Half-inch holes were drilled in the center of the bottom of the bowls to accommodate placement of manila rope. Two of the first three buoys were grown using 4 bags of substrate of the GL strain in 13 quart bowls and the third in the 8 quart bowl using 2.33 bags of material (Figure 17). Buoys 4-6 were grown in the same bowls using the same quantities of the TC orange-pigmented strain. We placed 16 oz Eco Choice compostable paper soup cups inside the bowls atop the substrate and even with the upper surface with a short dowel rod inserted to maintain a central opening for the manila rope to pass through after colonization was complete for the second round of making mooring buoys (Figure 18). The buoy halves were grown for 6 days before being mated and returned to the humidity tent to grow together along with a length of manila rope passed through from one end to the other (Figure 19). After an additional 7 days, the buoys were air-dried.

Air-drying the buoys was a prolonged process due to inadequate facilities and wet spring weather conditions. Air-drying took place in three locations according to availability on an active farm in the spring season. In all three locations, heaters, fans and dehumidifiers were run to hasten the drying process that averaged three weeks (Figures 20-22). Buoys made in Pembroke were determined fully desiccated when the mass did not change over 3 successive days as measured on a Globe GS30 30 lb capacity scale.

Flat rectangular, large hollow cylindrical, and mooring buoys were first lightly sanded on all exterior surfaces and with attention to rounding the edges. They were then coated with two coats of either the red soy paint or yellow or orange lobster buoy paint in the greenhouse in Pembroke (Figure 23). The paint was allowed to dry fully for 24 hours before the second coat was applied. The half-dome buoys were sanded and painted at North Spore’s facility in Westbrook (Figure 24). Prior to painting, large fungal primordia were cut off the half-dome flat surfaces.

All buoys were assigned numbers using a random selection and a random group generator program. Buoys were tagged with their corresponding strain and number codes before distribution to the 8 oyster farmers on June 19^th and 20^th.

Farmers reported results using the data collection sheet provided to record and photodocument the damage and biofouling every two weeks.

Methods Year Two

We were excited for the second year of the project knowing that Ecovative’s mycocomposite substrate would prolong the life of the MycoBuoys compared to our results the first year using North Spore’s substituted material. For example, one large 65L kelp buoy made prior to the start of the grant using Ecovative’s mycocomposite was deployed on September 22, 2023 (Figure 44), and is seen here after floating for 3 months with two coats of the soy paint at the Downeast Institute facility in Beals, Maine (Figure 45). Additionally, four cylindrical uncoated buoys made at the Smithereen Farm workshop in April, 2022 using Ecovative’s material successfully floated a plastic-coated wire oyster farm for 5 months (Figure 8).

A new facility was rented for the second year that has temperature controlled cold storage to house 6600 lbs of Ecovative’s mycocomposite material that was received on December 1, 2023 (Figure 46). An 8x10x8 foot Flow Clean Room was purchased and erected to create a HEPA-filtered space for mixing and filling the three shapes of buoy forms. Van Hook ordered new tooling for the three designs (flats, half-domes and hollow cylinders) from the Construction and Engineering Research Lab (CERL) at the University of Maine, Orono (Figure 47). To remedy the prolonged air drying time, MycoBuoys purchased a Hogentogler Convection Drying Oven that allowed us to control temperature and humidity cycles to fully dry the material within 2-3 days instead of 2-3 weeks.

One critique MycoBuoys received after presenting at Ocean Exchange Neptune Finalists business competition in Fort Lauderdale, Florida on October 23rd (Figure 48) was that the business was too focused on Maine aquaculture. Van Hook enlisted other oyster growers in Provincetown MA., the Billion Oyster Project in New York harbor, and the Brant Marine Hatchery in Nantucket, MA. to add to six of the previous year’s farmers who field tested buoys during the 2024 season.

In 2024, we used the rectangular flats (Figure 49) and half-domes (Figure 50) thermoformed tooling from the Construction and Engineering Research Lab (CERL) at the University of Maine, Orono. 

In between each use, we washed all tooling and tools in a mild solution using Thieves essential oils and air dried them. We entered the cleanroom, we wiped down all surfaces, tools, and bags of substrate with 99% ethanol.We emptied 2 bags of substrate into the cement mixer and added 1 cup of white wheat flour per batch ( ½ cup per 10 lb bag).

We tumbled for 30 seconds per batch, and then dispensed into a 20 quart tupperware tub with a sanitized mylar sheet held to direct outflow. We ran the mixer for 2 rotations to dispense the mixture. We took moisture measurements from 1 to 3 batches per day using a Torbal ATS120 balance scale precision moisture meter. The moisture content of the bags ranged from 63% to 87.8% and averaged 68%.

The flat rectangular molds measured 17.25” x 24” and 3.5” deep, and had a 2.25” rim. They weighed between 2.94 and 3.2 pounds, with an average of 3.032 lbs. We started by filling the flats with 8.08 lbs, but increased to 9 lbs starting with the 3rd batch to increase the thickness of the buoy. The halves molds measured 33” x 6.25” and 5” deep, and had a 2.25” rim. They weighed between 1.84 and 1.94 pounds, with an average of 1.902 lbs. Then we transferred the mold to the work table and distributed the substrate evenly, breaking apart clumps to ensure even growth. We tamped and beveled the edges of the substrate, then placed the sanitized lid on the flats in a convex position and clipped the edges using binder clips.  For the halves, we placed the lids in a concave position and used a combination of clips and painter’s tape to seal the edges.

After putting all the molds on the shelves, we labeled each buoy with the batch number and buoy number with blue masking tape (e.g., a flat buoy made in batch 2 that was the 23rd flat made would be F2-23). We grew the buoys on stainless steel racks. The flats grew 2 per shelf, and the halves grew 4 per shelf (Figures 49,50). The buoys grew for 7 days in the molds.

On the 7th day, we flipped them out of the molds (Figure 51).  We placed 3 buoys per shelf, running our fingers in between each to make sure they did not touch and grow together. The long half buoys were malleable, so we had to adjust them to make sure they were straight. We transferred all of the buoys' labels from the molds onto their racks. We covered the racks with plastic grow tents and added a stainless steel tray to the bottom rack filled with 3 liters of tap water and grew them for 7 more days (Figure 52).

On the 14th day we took the tents off of the racks. We dried the buoys in the Hogentogler Convection Drying Oven (Figure 53) with a ramp from 90°F to 180°F until the moisture as read by a Klein Tools digital moisture meter was between 0% and 2.5% on both ends and in the middle. The halves were placed with the flat side down and the flats with the wider side down to allow moisture to escape.

During the grant period, MycoBuoys was awarded a subcontract from Deer Isle Oyster Company to make hollow cylinder and mooring ball buoys under a Sustainable Oyster Aquaculture Research (SOAR) grant from The Nature Conservancy and Pew Charitable Trust. We grew the cylinders in new custom tooling from CERL using a tubular sheet metal insert to create the void space for each half of the cylinder. After 7 days in tool, the inserts were removed and the halves were placed in the convex position on racks in a humidity tent to further mycelial colonization of the substrate (Figures 54-56). On day 10, two halves were mated under compression using three 2 inch mylar strips wrapped around the two halves and secured with blue painter’s tape (Figure 57). Between days 16-20 the buoys were removed from the humidity tent and allowed to air dry in front of a sunny window until the moisture content was reduced to 1.5-3.5%. Final processing, after coating all cylinders with 2 layers of soypaint, 1 layer of sprinkled ground oyster shells, and a third layer of soypaint, included caulking the seam with General Electric 100% waterproof supreme silicone.

Hollow Cylinders

Mooring Balls

Mooring ball buoys were grown in stainless steel bowls that had a central hole cut into the middle of the bowl bottom through which we could feed a manila rope. To decrease the density and increase the buoyancy, we placed cardboard soup cups in the bowls to create a cavity (Figure 58). After 7 days growing inside a plastic trash bag to which air was added, the two halves were mated after removing the soup cups and threading the rope grown into one half through the opening left by a dowel rod place holder in the second half. A circular 8 pound weight was added atop the mated buoy to compress the growth of the two halves during the second phase in the humidity tent (Figure 59). The buoys were air dried in a sunny window for 2-3 weeks and oven dried for 3 days after which they were painted and sealed as above (Figure 60). The manila ropes were soaked in 200F 50:50 mixture of pine tar and linseed oil for 3 minutes after buoys were painted.

Bullet Buoys

We had a special request from the Brant Marine Hatchery in Nantucket, MA. to grow bullet shaped buoys to support their spat collection lines. This project provided another opportunity to grow the buoys in recycled 3L soda bottles with Environmental Science students at Cambridge Central School (CCS). Students made 30 of the 135 bullet buoys delivered to the Hatchery. A 75 m length of manila rope was threaded through the spout of the bottles that had their bottoms cut out. The bottles were filled to within 1 cm of the opening and were sealed shut by inserting a plastic quart yogurt container lid upside down (Figure 61). Bottles were tied to rods to allow colonization to occur for 4 days. On day 4 the lids were removed and the buoys slid out. They were tied to the rod and securely covered 5-7 on one rod with plastic sheeting to create the humidity tents for the skinning over phase of growth. On the 7th day, the sheeting was removed and the buoys were allowed to air dry. Half these buoys were coated as above (Figure 62). The ropes were treated in a 200F mixture of 50:50 pine tar to linseed oil for 3 minutes to preserve their life in the sea.

All buoys grown in year two were coated with 2 layers of soypaint, 1 layer of sprinkled ground oyster shells, and a third layer of soypaint and were branded “MycoBuoys.com” using a stencil and black soyink.

MycoRafts

Without the coated buoys lasting an entire oyster season, we sought to find a more appropriate application for the 5 month duration properties of the material. Van Hook and team grew rafts from Ecovative’s material in April, 2024 and in late January, 2025, the team grew 7 four foot diameter hexagonal rafts that measured 4.5 inches thick using a removable frame designed and fabricated for us. Into the first two rafts, we grew 6 five inch segments of a 2” diameter hollow bamboo pole, one placed 3 inches inside each of the 6 corners of the hexagon (Figure 63).

These were delivered to Dr. Alyssa Novak, marine ecologist, at Boston University. She deployed one of the rafts from October through November, 2024 in an estuary in Essex, MA. To the raft she stapled rhizomes of seedling eelgrasses (Figure 64).  

Two additional rafts had only 1 central bamboo insert (Figure 65), a fifth raft was grown to a frame of 1 x 6 inch rough sawn pine (Figure 66).  A sixth raft had 12 bamboo inserts places evenly such that it could be cut into 12 smaller hexagons and tested laterally for the mechanical force necessary to pull the bamboo out of the raft (something that happened to the first raft in one corner that was secured too tightly and was not allowed to float up and down with the tides) (Figure 67).

Material Testing

The mycocomposite buoy material was tested for nitrate and phosphorus leaching by high school environmental studies students at Cambridge Central School in 2023 and by Maxwell Bleyle at the University of New England in 2024 under the direction of Dr. Carrie Byron in the School of Marine and Environmental Programs. Hach test kits were used to test water samples over a 2 week period by the high school students and over 6.5 weeks at UNE.

Dr. Ron Bucinelli, Principal Engineer at Innotech International, LLC at Union College designed and performed strength testing on the bamboo inserts in 12 hexagonal cutouts from one MycoRaft (Figures 68-70).