UV radiation as a new tool to control microalgal bio-product yield and quality : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Industrial Biotechnology at Massey University, Palmerston North, New Zealand

Abstract

While ultraviolet (UV) radiation is most commonly known as an abiotic stress, various studies have shown targeted UV exposure increases bioproduct and biomass yields in microalgae. Microalgal cultivation processes face significant limitations in achievable bioproduct and biomass yields and thus improvements offered by targeted UV treatments during large-scale microalgae cultivation provide an opportunity for development of a novel UV treatment tool. Growing demand in microalgae (bio)products indicate there may be a substantial market for such UV treatment tools. No initiatives that explore the development of targeted UV treatments during large-scale microalgae cultivation have been found in the literature or in the industry. In collaboration with industrial partner BioLumic, a company specializing in applying targeted UV treatments in plants as a tool in agriculture, this PhD research examined if specific treatments of UV radiation (i.e. specific in UV waveband, irradiance and exposure duration) can reliably increase carotenoid accumulation in the microalga Dunaliella salina and if this new understanding can be feasibly used to develop an industrial system for UV treatment of microalgae. The PhD research was conducted utilizing D.salina after evaluation in four commercially relevant microalgae species: Arthrospira platensis, Chlorella vulgaris, Haematococcus pluvialis and D.salina. A UV-A induced carotenoid accumulated response was identified in D.salina (strain UTEX 1644). Targeted UV-A treatments reliably induced carotenoid accumulation in this species, and the magnitude of the response depended on the UV-A wavelength, UV irradiance, UV exposure duration, and UV dose. The UV-A carotenoid accumulation response was induced within 6 hours and was largely complete in 96 hours (24 h·d⁻¹ UV exposure). The highest UV-A dose tested induced the highest carotenoid accumulation rates and the highest total carotenoid concentrations after continuous UV exposure (24 h·d⁻¹) at the highest UV-A irradiance tested (30 W·m⁻²). Total carotenoid concentration increases of up to 162% were thus observed after 72 hr of UV-A exposure. UV-A exposure was associated with slowed or stopped cell proliferation as well as increased D.salina cell size (up to 15%) and altered intracellular structural organization. Carotenoid accumulation ceased and cell proliferation increased when UV-A exposure was stopped, leading to a subsequent resumption of cell proliferation. UV-A induced carotenoid accumulation was improved 51% during UV-A exposure concomitant with non-UV carotegenic stimuli (high PAR intensity and salinity) compared to UV-A exposure alone. The observations from experiments carried out in the thesis served as inputs in a techno-economic analysis (TEA) model developed to assess feasibility of large-scale UV treatment. The TEA model was developed to allow assessment of the most critical areas for improving profitability of large-scale UV treatment technology, rather than provide absolute economical outputs for revenue and profit. The TEA was based on two reference cultivation systems currently used for commercial D.salina cultivation. The TEA analysis considered four locations for the UV treatment system applied along the cultivation process: pre-cultivation stage (i.e. inoculum), main cultivation stage, post-cultivation stage (i.e. immediately prior to harvest) and during fluid transfer between stages. A dedicated post-cultivation UV treatment stage was shown to have a number of advantages over other treatment options. A model cultivation system for the case-study of D.salina was developed assuming an annual β-carotene production of 1,000 kg. The developed TEA model cultivation system and TEA UV treatment system were able to identify a potential increase in profitability generated from the application targeted UV treatment during large-scale D.salina cultivation. The maximum increase in profitability was achieved using a broad wavelength UV treatment system (irradiance = 30 W·m⁻², exposure duration = 24 h·d⁻¹, surface area coverage = 100%) applied during an intensive cultivation post-cultivation system. A relatively small contribution of the UV treatment system to CAPEX and OPEX to overall β-carotene production cost (i.e. < 10%) combined with the large increase in β-carotene production (711 kg·y⁻¹ and 895 kg·y⁻¹ for fluorescent UV tube and UV LED systems, respectively) leads to potentially large increases in profitability. The TEA analysis identified the magnitude of the UV-A induced carotenoid accumulation response to be the most important factor to influence the potential profitability. Moreover, the TEA indicated the increases in profitability are strongly influenced by optical efficiency, electrical efficiency and maximum optical power. The profitability estimates from the current TEA indicate that UV treatment during commercial microalgae cultivation has potential and justifies further research. To our knowledge the exploration of the fundamental UV photobiology in microalgae required to develop UV treatment regimes from discrete UV wavebands, complemented with a commercial microalgal-engineering insight, to produce UV treatment regimes and UV treatment technology for application during large-scale microalgae cultivation, has never been attempted. The multidisciplinary approach employed during this PhD research explored for the first time the development of a UV treatment system from laboratory observations to commercial cultivation. The current research described for the first time the UV exposure behaviour of D.salina (strain UTEX 1644) to varying UV waveband, UV irradiance, UV exposure durations as well as UV response interaction with PAR and salinity. The case-study of UV treatment during large-scale D.salina cultivation in this PhD research allowed recommendations to be made to the industrial partner BioLumic on potential areas of focus for continued research and development.