Unlocking the Potential of Green Gravel Production for Efficient Kelp Restoration: How Seeding Density Affects the Development of the Golden Kelp Laminaria ochroleuca

Abstract

Kelp forests are facing a global decline due to climate change and human-induced stressors, underlining the urgency for proactive interventions. Among the most used restoration methods, “green gravel” has emerged as a promising solution for the recovery of degraded kelp forests. While initial findings suggest its potential effectiveness, green gravel remains a novel approach that requires fine-tuned protocols and optimisation across all stages of the process. In this study, we assessed the performance of two different seeding densities for kelp growth on green gravel to optimise the use of seeding material. Our results show that, while the juveniles in the high-density treatment grew faster, they also had a higher mortality rate during green gravel production. This was a hypothesised outcome, as growing sporophytes reared under laboratory conditions allows for faster production of a large number of individuals, increasing competition for space, which may drive higher mortality rates. Comprehensive experimentation is essential to unlock the full potential of green gravel and ensure its efficiency in all process steps, to achieve successful kelp forest restoration. Well-defined and optimised protocols are indispensable for minimising production costs, simplifying logistics, and allowing future efforts to scale up.

1. Introduction

Kelp forests, mainly formed by species of large brown seaweeds in the orders Laminariales and Fucales, are complex natural structures that can create a wide variety of habitats and provide several ecosystem services [1,2]. Among these, some of the most relevant include providing shelter and food to a multitude of species, increasing associated biodiversity, drawing carbon from the atmosphere, and helping reduce marine nutrient pollution [1,3]. During the last decades, areas covered by kelp forests have been on the decline globally, with some regions experiencing near-total disappearances of these critical ecosystems [4,5]. The causes of these declines are multifaceted, ranging from local stressors, like pollution and the effect of herbivory, which can also be amplified by broader global impacts, particularly those associated with climate change [6,7]. Given their characteristic rapid growth rates, kelp forests possess great potential and relevance for successful restoration efforts [8]. With their ecological importance as ecosystem engineers, kelp reforestation initiatives are paramount for the marine habitat restoration goals established by the EU Biodiversity Strategy for 2030.

Kelp restoration methods can follow four main approaches: (i) herbivore control, either by introducing a natural predator for the grazer species or physically excluding them from the restoration area (e.g., controlled harvesting, introduction of natural predators, exclusion); (ii) seeding with microscopic stages; (iii) transplanting the sporophytes, either while juveniles or as adults; and (iv) addition of artificial reefs to provide adequate substrate or to increase its three-dimensionality [2].

Green gravel falls within the seeding strategies, and it is a novel approach recently developed to restore depleted kelp forests [9]. This technique has an ecological advantage as it does not demand the removal of individuals from the donor population, but only involves harvesting the reproductive material, lowering its impact. The green gravel protocol involves sowing kelp spores or gametophytes onto small stones in the laboratory, maintaining them under controlled conditions until recruits are visible to the naked eye, and then deploying them at the chosen reforestation location. Green gravel was proposed as an economic and sustainable alternative to current restoration efforts, which were limited by the need for high investment or by the difficulty in both investment and logistics for scaling them up [2,9]. The cost of this tool, in fact, is about 7 USD m⁻², which is significantly lower than other restoration techniques, including transplantation methods and recruitment enhancement [9].

The easiness of the deployment methods also has the potential to allow for treating large, degraded areas. The low impact on donor populations is also an important feature potentiating the technique’s scalability [9]. However, before this technique can be applied at an impactful scale, there is a strong need to gather and consolidate existing knowledge and establish optimised protocols for each step, from reducing the cultivation phase time and expenses to improving the logistics of deployment and monitoring and fine-tune the criteria for choosing restoration areas. Setting these guidelines and protocols is essential to increasing the success rate of restoration actions.

Our research is focused on the golden kelp Laminaria ochroleuca. This brown macroalga is a temperate-water Iberian species, which can be found from Morocco to the southern UK, inhabiting both intertidal and subtidal rocky habitats [10,11]. In recent decades, the effects of climate change (e.g., changes in the intensity and frequency of upwelling events and seawater warming) have caused alterations in kelp distribution and abundance [5,11,12]. In Portugal, several kelp species, including L. ochroleuca, have already experienced contractions of their distribution range and local disappearance, mainly due to climate change and other stressors, such as herbivory [13]. Considering the growing interest in active restoration, aiming to preserve and recover the depleted kelp forests, and the peculiar traits of the species (e.g., fast growth and quick recovery after perturbations), L. ochroleuca represents an ideal choice for reforestation efforts. In this study, we aimed to evaluate the effect of different seeding densities in green gravel production using the golden kelp L. ochroleuca. This is an important step in green gravel production optimisation as, although the impact on donor populations is lower than removing entire individuals, the constant collection of reproductive material has the associated risk of driving the spore density at the donor location below the threshold that would allow its maintenance. For these reasons, and with the possibility of scaling up in mind, the reproductive material needs to be used in the most efficient manner.

2. Materials and Methods

Reproductive tissue was collected from fertile Laminaria ochroleuca individuals (n = 15) in Viana do Castelo (Portugal, 41°41′46.77″ N, 8°51′1.79″ W) in April 2023, and spore release was carried out in the lab on the same day, as described in Pereira et al. (2011) [14]. The spore solution was maintained under white light (AquaBar T Series, 7000 K, white LEDs, 40 µmol m⁻² s⁻¹) to induce germination and allow for a quicker development into gametophytes. Spores were observed daily to assess development and after one week the stock culture was transferred to low red light (12 µmol m⁻² s⁻¹, 700 nm) to impair reproduction while maintaining vegetative growth. Since, with vegetative growth, gametophytes are able to produce a larger number of gametes, this is an important step in using reproductive material efficiently. After about 3 months growing vegetatively, the healthy gametophyte mass was separated into smaller fractions using a handheld electric blender. This fractioning can also trigger reproduction, speeding up the whole cultivation phase. As granite is one of the most common substrates in rocky shores, particularly in Portugal, this material was used as a substrate. The solution was thus sprayed onto small granite stones (3–5 cm) at two different seeding densities: high (120 gametophytes mL⁻¹) and low (90 gametophytes mL⁻¹), to assess the impact of density on recruitment mortality and growth.

Light was provided by LED lamps (AquaBar T Series, 7000 K, white LEDs, Fiji, UK), with a photoperiod of 12:12 h dark–light and a light intensity of 100 μmol m⁻² s⁻¹, to simulate the environmental conditions found in Northern Portugal in spring, matching L. ochroleuca recruiting season. Eight tanks (40 × 60 cm, 50 L) were used, with four replicates for each seeding density treatment. Small pumps, with a 950 L/h flow, (Sicce, Syncra Silent, Pozzoleone, Italy) were added to the tanks to provide appropriate water flow, making them more resistant and allowing a better adaptation to open sea conditions. The seawater temperature was maintained at 15 °C by a cooler (Aqua Medic Titan 150, Bissendorf, Germany), to ensure the optimum temperature for golden kelp growth. Provasoli’s Enriched Seawater (PES) was added weekly to ensure the supply of nutrients from then on. Germanium dioxide (GeO₂) was also added to the tanks to inhibit the growth of contaminating diatoms during the first two weeks [15]. Water in the whole system was also changed once a week, not only to rid seedlings of metabolites, but also to drive the accumulation of polysaccharides.

Two weeks after the seeding, when sporophytes were visible under the stereo microscope (Leica EZ4W, Wetzlar, Germany), their growth (i.e., the maximum length, measured from the base to the tip of the blade, of the recruits on gravel) was monitored weekly through photographic sampling (125 individuals for each tank per week). The density of the juveniles (i.e., number of individuals cm⁻²) was also assessed, considering 25 photographic replicas for each tank per week. The experience lasted 7 weeks, until the recruits reached a suitable size for deployment in the field (i.e., less than 1 cm). Allowing the recruits to grow over 1 cm in length, in fact, often results in more fragile stipes and a reduced ability to adhere to the natural substrate, especially in coastal areas with strong hydrodynamics, such as those along the Portuguese coastline.

Assumptions of data normality were assessed using the Shapiro–Wilks test and data were log-transformed when necessary [16]. To explore the effect of the different seeding densities on the green gravel over time, the two variables of sporophyte growth (i.e., length and density of the recruits) were analysed. A two-way ANOVA (analysis of variance), with seeding density (fixed, two levels: high and low) and time, in weeks (random, 6 levels), was performed.

All analyses were performed in the R statistical environment (R Core Team, 2020, version 4.3.2).

3. Results

The two-way ANOVA carried out on the data of juvenile lengths highlighted a significant difference (F₍₅₎ = 237.4, p < 0.001) in the interaction between the seeding density and time. In the first weeks, the length of the recruits was slightly higher in the low-density tanks (Week 3: 0.559 ± 0.019 mm) compared to those in the high-density ones (0.436 ± 0.013 mm) (Figure 1). This trend appeared to reverse from week 4 onward, with the individuals in the high-density treatment reaching the maximum length at the end of the experiment (Week 7: 3.419 ± 0.082 mm). This value was almost thrice the average length of low-density individuals in the same week (Week 7: 1.392 ± 0.033 mm).

The ANOVA carried out on recruit density showed significant differences in the interaction between the seeding density and week (F₍₅₎ = 4.906, p < 0.001). The density of the recruits was significantly higher in the first two weeks in the high-density individuals (Week 2: 97.100 ± 17.949 ind. cm⁻² vs. 185.024 ± 29.008 ind. cm⁻², Week 3: 128.808 ± 39.718 ind. cm⁻² vs. 259.179 ± 39.498 ind. cm⁻², low density vs. high density; Figure 2). After the first two weeks, the recruit density showed a decreasing trend throughout the experiment, reaching minimum values in the last week, with the low-density recruits showing values six times higher than the high-density recruits (Week 7: 74.140 ± 10.499 ind. cm⁻² vs. 12.096 ± 1.636 ind. cm⁻²; Figure 2).

4. Discussion

Cultivating gametophytes and microscopic sporophytes under controlled conditions usually allows fast production of a large number of individuals, but often with extremely high mortality rates (about 1 out of 100,000 embryonic sporophytes develop into a mature individual) [17]. Previous studies highlighted how, at high densities, gametophytes may compete with each other for limited resources, such as light and nutrients, which can hinder their growth and survival [18]. Competition for nutrients among gametophytes of the same age and species seems to be more important than competition for light, as shading effects are negligible under thin layers of germlings [19]. Our results highlighted a significant effect of the seeding density on the development and survival of the recruits, which resulted, after 7 weeks, in the length of the recruits in the high-density treatment on average being almost three times larger than those in the low-density treatment. Conversely, recruits in the high-density treatment suffered higher mortality, resulting in a recruit density, after 7 weeks, that was a sixth of those cultivated at low-density. Nevertheless, “self-thinning”, i.e., the reduced probability of survival due to intraspecific competition in dense macroalgae stands, is a phenomenon that has already been observed in brown macroalgae for both adults and germlings [20,21].

A previous experiment with green gravel highlighted how sporophyte density on green gravel changed over time from many microscopic sporophytes covering the gravel at the beginning of cultivation to only 1–4 juvenile sporophytes on each stone after 12 months [22]. Taking into account the high mortality of the individuals during the first months, we suggest that the best solution to ensure an adequate number of individuals for deployment in the field would be to reduce the intraspecific competition among the recruits during the cultivation phase, using a lower seeding density. This would also allow for reducing the costs associated with green gravel production and minimise the impact on donor populations, making the technique more sustainable.

Despite its logistical convenience, green gravel still presents some potential limits, such as the lack of adequate monitoring protocols for pre- and post-deployment phases. Environmental assessments to ensure the suitability of the chosen sites and regular monitoring of the success rates of green gravel projects are absolutely essential to identifying patterns and areas for the improvement of the technique.

Marine habitat restoration is a critical element of the EU Biodiversity Strategy for 2030, as well as other initiative such as the UN Decade of Restoration and the UN Decade of Ocean Science for Sustainable Development. By promoting sustainable practises, and supporting research and funding, these programmes aim to collectively manage and reverse the decline in ocean health, aiming to halt biodiversity loss and restore healthy, resilient marine ecosystems that can thrive in the face of environmental challenges. One of their most ambitious goals has been the scaling up of restoration techniques in order to address the alarming rate of marine ecosystem degradation.

Compared to other restoration methods, green gravel represents a promising tool for marine habitat restoration, offering several important advantages, including cost-effectiveness, ease of deployment, scalability, and reduced impact on donor populations [23]. The simplicity and affordability of green gravel make it an attractive option for large-scale restoration projects, allowing for broader application across various marine environments. Nevertheless, its novelty also means that further research is necessary to address its limitations and ensure its effective application in diverse marine environments and conditions. A proper use of green gravel requires optimisation of all phases and clear protocols. This includes selecting the best types of gravel and fine-tuning other cultivation variables, as well as identifying suitable deployment conditions [24]. Continued investigation into these factors is essential to maximise the success chances of green gravel and fully realise its potential as a viable marine restoration technique.

5. Conclusions

By evaluating various seeding densities, this study sought to develop effective protocols for the use of green gravel in large-scale marine restoration projects, ensuring efficient and sustainable reforestation of kelp habitats. Costs and usage of resources represent one of the major constraints in scaling up reforestation efforts, undermining the feasibility of actions on a large spatial and temporal scale.

In our experiment, the green gravel sown with a higher gametophyte density resulted in faster growth of the kelp sporophytes, but a lower recruit density after 7 weeks. This suggests that while a higher seeding density can accelerate early growth stages, it might not be sustainable in the long term due to limited space, light, and nutrients, which could inhibit the survival of young kelps. To ensure an adequate kelp abundance at the end of the cultivation phase and maximise the likelihood of successful deployments, the most recommended procedure would be to sow the green gravel with a low seeding density. This approach would not only ensure the availability of enough space and resources for each individual to grow, but would also lower production costs, thus helping scale-up future reforestation actions. Customising green gravel methods by adjusting the seeding density can help suit the technique to specific local environmental conditions based on local water currents, wave action, and grazing pressures. Additionally, integrating green gravel with other restoration techniques, such as artificial reefs and grazer management, could enhance the overall effectiveness of the restoration efforts. Deploying green gravel inside Marine Protected Areas (MPAs), where stressors like overfishing and pollution are reduced, can also provide a safer environment for the young kelp to grow and thrive. This comprehensive and multifaceted approach to marine restoration could significantly increase the resilience and success rates of reforestation projects. Furthermore, implementation of long-term studies and pilot projects is crucial to gather data on the effectiveness and ecological impact of green gravel in various environmental conditions. These studies will help build a robust track record, providing valuable insights into the method’s large-scale and long-term viability. Understanding how green gravel performs over extended periods and in different marine settings will be essential for refining the technique and ensuring it can be reliably used in future marine restoration initiatives.