The diatom Phaeodactylum tricornutum as a sustainable microalgal cell factory: towards a biorefinery approach

Microalgae offer a means of fixing atmospheric CO2, thus providing realistic reductions in carbon emissions and producing valuable compounds and chemical precursors of high value. Phaeodactylum tricornutum is a well characterised model diatom and has been exploited commercially for single products; the nutraceuticals eicosapentaenoic acid (EPA) (omega-3 fatty acid for maintaining cardiovascular health), fucoxanthin (pigment for weight loss, antioxidant, and anticancer compound), and as a live feed additive for aquaculture. Natural pathways exist for the production of EPA and fucoxanthin in P. tricornutum but biomass and product yields are low.

The aim of this thesis was to utilise a biorefinery approach with the model diatom, P. tricornutum CCAP 1055/1, for the production of multiple products of interest; particularly fucoxanthin, EPA, and protein using non-GMO methods, to enable economic viability. In this thesis, the concept of P. tricornutum as a cell factory and biorefinery chassis along with the challenges to overcome have been discussed.

A cost-effective powdered media formulation (JWP) was revealed to involve low preparation time and outperformed conventional liquid f/2 medium. JWP resulted in a higher biomass productivity and fucoxanthin, EPA, and protein yield. JWP was subsequently used for cultivation of P. tricornutum indoors and outdoors (8-18°C) in a prototype airlift photobioreactor (PhycoLift) using a repeated fed-batch approach. Indoor cultivation resulted in a higher biomass and fucoxanthin yield (with a higher average protein, chlorophyll a, and fucoxanthin content). Outdoor cultivation resulted in a higher protein, carbohydrate, total fatty acid (TFA), and EPA productivity (with a higher TFA, EPA, and carbohydrate content). EPA content was found to be inversely correlated with temperature. Commensal bacteria (Marinobacter sp. and Halomonas sp.) were isolated from the cultures and identified. The total bacteria count showed a sinusoidal growth profile, and it was apparent that Halomonas predominated at low algae densities, but at higher algae densities there was a community shift to Marinobacter. EPA secretion into the extracellular matrix has been hypothesised as a bacterial control agent, but this was not observed in the present study.

A high throughput flask screening approach was used to optimise product yields, particularly fucoxanthin and EPA using f/2 medium as a baseline. A culture medium consisting of nitrate and phosphate at 4.41 mM and 0.18 mM (5-fold those employed in the traditional f/2 medium), salinity at 33 %, 2 % CO2 (v/v in air), and a transition to mixotrophy using glycerol (0.01 M) were found to result in increased biomass (0.20 g L-1 d- 1), fucoxanthin (1.78 mg L-1 d-1), EPA (7.54 mg L-1 d-1), and protein yields (15.65 mg L-1 d-1), up to ten-fold higher than the f/2 control medium.

Harvesting algae biomass from suspended cultures is a major bottleneck industrially and can account for up to 30 % of the production cost. A series of biobased flocculants were examined and a tannic acid-based derivative was found to flocculate the algal biomass effectively within 10 minutes, resulting in a high harvesting efficiency (>85 %), and a concentration factor ≥5.6, with promising economic potential for wider usage (cost savings of >US $61 ton-1 biomass compared with chemical and other biobased flocculants.
Finally, three different P. tricornutum strains; CCAP 1055/1, UTEX 646 and a commercial strain - N017 (Necton, Portugal) were screened to determine the influence of strain on product yields cultivated in brackish seawater. It was identified that P. tricornutum UTEX 646 resulted in the highest biomass productivity and highest EPA, TFA, protein, and carbohydrate yield. Whereas, the P. tricornutum N017 strain had the highest fucoxanthin productivity. Warm white light emitting diodes (LEDs) resulted in a higher biomass productivity, fucoxanthin, and carbohydrate yield, but a lower EPA, TFA, and protein yield compared with red: blue LEDs (2.25:1). The bacterial community composition was found to be similar under white and red: blue LEDs. Marinobacter sp. was present in all strains and dominated at most stages of cultivation indicating a commensal relationship. Halomonas sp. was only present in P. tricornutum CCAP 1055/1 and the P. tricornutum N017 strain. Algoriphagus sp. was only present in P. tricornutum CCAP 1055/1 and was low in number. Comparatively, Sphingorhabdus and Croceibacter spp. were present in the commercial P. tricornutum N017 strain. The total bacterial count was observed to be lower under red: blue LEDs for all three strains over the cultivation period, which may reveal potential to reduce bacterial loading.

In summary, UK outdoor cultivation under cold conditions utilising a biorefinery approach has been demonstrated to be feasible with an airlift PBR (Phycolift), but further improvements are needed for economic viability; a commercially viable and environmentally friendly solution to harvesting biomass using a biobased flocculant has been demonstrated; simple culture condition manipulation using a high-throughput standardised flask screening setup has been showcased to improve biomass, EPA, fucoxanthin, and protein yields; and commensal bacteria have been isolated and characterised. This thesis has real commercial application to rapidly screen treatments for elevating biomass and product yield, whilst decreasing the time taken for harvesting at reduced cost, for example, the production of fucoxanthin, and a depigmented by-product as an aquaculture additive.