Seeding & Attachment

Sporulation, Seeding, and Attachment

Sporulation

Macroalgae sporulation is the final stage of the spore production process before spores are attached to a substrate or buoy prior to deployment.

Running Tide needed to effectively and consistently increase sporulation yield as precursor to seeding and growing macroalgae at scale. Over the course of 2023 and into 2024 the team refined Ulva sporulation protocols. We achieved our most impressive results in the first half of 2024, with the best and most efficient sporulation yielding around 400 billion Ulva spores in a total volume of 500L. Throughout our induced sporulation efforts, we observed that the sporulation yield was highly variable. We suspected that yields were affected by both cultivation-based factors, such as light intensity, nutrient availability, temperature, and pH, and environmental factors, such as the moon phase.

In 2024, we were working on a model to describe the effects of different environmental factors on sporulation yield. For full-scale deployments, we would rely on the continuous production of Ulva spores. Thus, determining the effect of environmental variables on sporulation yield through modeling was crucial to identify factors which could drive down variability and ensure consistent, high-quality production. By predicting the effects of these factors with models, we could optimize cultivation conditions to maximize sporulation yield. This approach would not only enhance productivity but also ensure the reliability and scalability of spore production.

Our model was a penalized linear regression model that could describe the sporulation yield with high accuracy (Figure above). The most important contributor to the variability observed in the sporulation yield was found to be the moon cycle (Figure below).

Seeding

In addition to the sporulation yield, we needed to consider the spore viability and attachment of spores to substrates. Without viability or adequate attachment, macroalgae would not grow on the substrate. This step in the process is crucial.

Furthermore, we needed to investigate seeding methods, i.e. how the substrates were coated with the algae spores for optimal coverage, scalability and algae growth. Therefore, we conducted a series of seeding experiments where we investigated the effects of seeding methods (submerged vs. spray-on), seeding densities (e.g., 5.000, 10.000, 50.000 spores/mL), settlement time duration, settlement light duration, drying-out period length, and waves.

One of the key findings of these experiments was that Ulva spores can survive harsh conditions on land-seeded substrates for up to 48 hours, suggesting that land-based seeding prior to ocean deployment is a viable option.

The type of seeding method used has a significant impact on the engineering of the seeding process at scale. The use of submersion seeding required the substrate to be submerged in seawater containing a high number of spores for up to 24 hours prior to deployment, while spray-on seeding uses an adhesive algae binder that requires curing/drying for the algae spores to become attached to the substrate. Each form of spore attachment has its pros and cons, but through 2023 the macroalgae and engineering teams agreed that spray-on attachment would better suit the overall process vision with regards to scalability.

Attachment

The figure below shows how algae binder helps with attaching Ulva spores to substrate and how the algae attachment can be increased by increasing the spore concentration in the spore solution the substrate is seeded with. In the figures below the substrate is cotton, which was our “control” substrate to which other experimental substrates were compared.

Extensive work was conducted by the Running Tide macroalgae teams in Alda and Portland on testing different types of substrate for spore attachment. The substrates that received most attention were wooden and cementitious substrates and a combination of those two.

In the figures below Ulva fenestrata can be observed growing on an engineered substrate that was made in collaboration with BM Vallá in Iceland. This substrate showed often great spore attachment followed by impressive Ulva blade growth. This substrate contained a mixture of scalable carbon buoy ingredients such as wood, cement and other widely available materials. Although the material showed promise for macroalgae attachment and tunable float time and buoyancy, we never further developed the substrate to meet float time requirements.

An obvious choice of material for the manufacturing of carbon buoys is wood. Wood has adequate buoyancy, high carbon content and low cost. It took the macroalgae team longer than expected to figure out how attaching spores to wood could be done effectively. Many of the components of wood have inhibitory effects on organisms wanting to attach, and when submerged into water the wood leaches acidic compounds that can inhibit organisms. These issues were however mostly solved through 2023 and Q1 2024, though results for macroalgae attachment and growth could be inconsistent. In the figure and video below Ulva attachment and growth is shown on Paulownia, a fast-growing and highly buoyant wood species.

In the video above wooden substrate with Ulva attached is shown submerged in one of Running Tide's “wave tanks” at the company's Portland location. The open ocean is a high energy environment and conditions can vary greatly compared to that of normal indoor algae cultivation in controlled conditions, yet we attemted to approximate agitation to explore the effects of waves on buoy prototypes.