Elucidating the effect of CO2-nanobubbles on the growth and carbon uptake of the indigenous Hawaiian macroalgae, Ulva

Abstract

Macroalgae, also known as limu in Hawaiian, are ubiquitous and photosynthetic powerhouses with tremendous morphological and genetic diversity. Macroalgae grow quickly, have favorable characteristics for bioproducts (e.g. bioenergy, biomaterials), and can help drive a more sustainable blue economy. There are three main algal groups, the Chlorophyta (green algae), Rhodophyta (red algae), and Phaeophyta (brown algae), each of which are taxonomically classified based on their pigment composition, genetic lineage, and morphological features. One of the macroalgae in the Chlorophyta phylum, Ulva, is widespread globally and typically grows in shallow coastal waters attached to substrate. This genus exhibits extremely rapid growth rates with sufficient nutrients and light, causing green tides in stagnant waters. However, because of these very fast growth rates, Ulva has also been proposed as a promising macroalgal species to cultivate to capture carbon and create valuable bioproducts. Yet, without sufficient nutrients and carbon availability, they do not exhibit these high productivities. Nanobubbles (NB) are gas-filled bubbles within aqueous solutions, ranging from around 50 – 200 nm in diameter and exhibit unique physicochemical properties. Due to their miniscule size, they are able to stay within solutions for long periods of time, up to days to weeks. Moreover, they exhibit negative zeta potentials on the order of -20 – 30 mV, allowing for electrostatic repulsion effects and furthering their retention time in liquids. They can be generated with a multitude of methods, including pressure dissolution, electrolysis, membranes, among others, with varying gas species used depending on the application. It has been proposed previously that NB could be used for improving algal growth through the improved delivery and retention of CO2, thus boosting carbon availability within liquid mediums. However, the physicochemical properties (zeta potential, concentration, size, mass transfer, decay kinetics) and carbonate equilibria dynamics of CO2 delivered with NB technology have scarcely been characterized, as well as tested in whether carbon uptake is improved within algal cultivation. In addition, it is not known how CO2-NB affects the resulting macromolecular profile of Ulva, which could alter its resulting use cases. In this dissertation, we show that CO2 delivered with NB technology (NB) differ in physicochemical characteristics compared to CO2 delivered with a conventional diffuser (CON) based on the following results: 1) Gas-liquid mass transfer coefficients (kLa) with NB were improved by 4.87-fold and 2.58-fold in ultrapure water (UPW) and seawater (SW), respectively, while also extending dissolved CO2 retention times during the decay phase (0.10 h-1 in UPW and 0.14 h-1 in SW). Additionally, NB had nearly 2-fold higher bubble concentrations with less negative zeta potential than CON. 2) NB did not substantially modify carbonate equilibria compared to CON, with PHREEQC modeling showing overlapping carbonate speciation, mineral saturation trajectories, pH differences of <0.3 at peak CO2 input. This suggests that NB primarily improved CO2 gas-liquid mass transfer and CO2 retention times through physical mechanisms, rather than chemically driven processes. 3) When applied to algal cultivation, CO2 delivered via NB in mixed, shallow 145 L raceways supported reliable Ulva production, with fresh weight (FW) biomasses increasing from ~25 g per raceway on Day 1 to ~43 g FW by Day 10 (mean specific growth rates of 6 – 7% d-1). However, when compared to conventional CO2 bubbling at the same CO2 input rates, this biomass improvement was not statistically significant. Furthermore, in both the NB and CON CO2 supplied raceways, Ulva was able to rapidly assimilate daily nutrient additions, with dissolved inorganic nitrogen (DIN) and PO4 reaching concentrations of ≤ 0.05 mg N L-1 and ≤ 0.015 P L-1 (~98% and ~95% removal efficiencies for DIN and PO4, respectively). Dissolved inorganic and organic carbon tracking, coupled with the biomass measurements resulted in raceway scale carbon mass balances that showed ~15 – 17% of injected CO2 being retained overall, with 40 – 45% incorporated into Ulva biomass, 40 – 45% to dissolved organic carbon, and 12 – 14% to residual DIC. 4) Based on macromolecular analyses, NB did not substantially alter the composition of Ulva compared to CON, with around 39 – 47%, 16 – 17%, 4 – 7%, and 29 – 33% DW in carbohydrates, protein, lipids, and ash, respectively. We show this through conducting thorough mass transfer and decay analyses using a dissolved CO2 sensor, inorganic carbon measurements, nanoparticle tracking analysis, electrophoretic light scattering, and modeling in the USGS program PHREEQC. Furthermore, we also demonstrate a novel NB-integrated raceway that facilitates improved productivity of Ulva biomass while suggesting improvements to existing macromolecular protocols. To the extent of our knowledge, this is the first report that comprehensively examines the generation and decay of CO2 delivered with NB technology, benchmarked against a conventional diffuser in both ultrapure water and saltwater, while also utilizing it in production of Ulva biomass. Taken together, these studies provide valuable insights and knowledge into the fundamental physicochemical properties of CO2 delivered with NB technology and differentiates it from CO2 delivered through conventional diffusers, which will aid in many industrial sectors that require aqueous phase CO2, while showcasing a use-case proof-of-concept in the form of Ulva biomass production.