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Arthrospira platensis is a cyanobacterium known for its widespread use as nutraceutical and food additive. Besides a high protein content, this microorganism is also endowed with several bioactivities related to health benefits in humans that make it a candidate for functional foods. These properties are strain and culture condition dependent. We compared, in terms of biomass productivity and protein, C-phycocyanin, and polysaccharide content, two A. platensis strains, A. platensis F&M-C256 and A. platensis F&M-C260, characterized by morphological differences. The organisms were grown in annular photobioreactors with light-emitting diodes (LED) as light source in fed-batch and semi-continuous regimes. No significant differences in biomass productivity were found between the two strains. Both strains showed a protein content >55% in all culture conditions. C-phycocyanin content was higher in A. platensis F&M-C260 in semi-continuous regime. Cellular polysaccharide (PS) content, which included intracellular polysaccharide and those bound to the cell wall, was higher in A. platensis F&M-C256 during semi-continuous cultivation. In both strains, a higher release of polysaccharide was observed at the end of the fed-batch regime. A. platensis F&M-C256 showed the advantage to form clumps which facilitate harvesting, behavior not observed in A. platensis F&M-C260 and probably related to the different predominant monosaccharide found in the PS of the two strains (i.e., rhamnose in A. platensis F&M-C256 and glucose in A. platensis F&M-C260). The results show that the two strains are suitable for commercial production of high-value products, such as protein and C-phycocyanin, while for polysaccharide production, A. platensis F&M-C256 is preferable.

Microalgae are photosynthetic microorganisms that can reach higher areal productivity than terrestrial plants, do not require arable land or fresh water, and can use fertilizers with almost 100% efficiency. Microalgae-derived products are therefore considered a promising sustainable source of food and other commodities.Microalgae are commonly grown exploiting their photoautotrophic capacity (henceforth referred to as autotrophic), in which cells harvest light energy, use CO2 as a carbon source, and release O2 as a byproduct. CO2 and aeration are provided to prevent carbon limitation and oxygen accumulation. Some microalgae can also be grown heterotrophically, in which organic carbons, such as sugars and organic acids, are used as carbon sources in the absence of light. In this trophic mode, O2 is consumed and CO2 is released as a by-product. Autotrophic and heterotrophic cultivation of microalgae can be combined in mixotrophic cultivation in which light and reduced organic carbons are simultaneously exploited within a single microalgal monoculture.In this thesis we designed a novel mixotrophic process called “oxygen balanced mixotrophy” in which there is no net O2 production. This was possible by tuning the supply of organic carbon to the rate of photosynthesis. When illuminated, the culture did not required any aeration. Due to the internal gas recirculation, about the 90% of the organic carbon provided was converted into biomass making the process almost carbon neutral. The presence of two complementary growth modes within a microalgal monoculture led to doubled biomass productivity and concentration in comparison with an autotrophic reference.Oxygen balanced mixotrophy has been successfully applied to with two industrially relevant microalgal strains: Chlorella sorokiniana and Galdieria sulphuraria. G. sulphuraria had a protein content of over 60% w/w and compared favorably with the FAO dietary requirements for adults regarding amino acid composition. Moreover G. sulphuraria contains a high proportion of sulphurated amino acids compared with Chlorella, Spirulina and soybean protein. Due to its attractive amino acid profile and high protein content, G. sulphuraria proved to be a good candidate for food and feed applications to overcome sulphurated amino acid deficiencies.Mixotrophic cultivation of G. sulphuraria demonstrate to be also a good strategy to produce the blue pigment phycocyanin with superior acid- and thermal stability compared to phycocyanin extracted in Spirulina.Finally the insights of this thesis were combined in a techno-economic model. Projections were made on biomass production costs for a hypothetical 100-hectare facility located in southern Spain. Our projections indicated that mixotrophic cultivation of Chlorella sorokiniana would half microalgal production cost mainly due to the doubling of biomass productivity under mixotrophy. For Galdieria sulphuraria, because of the expected low efficiency of CO2 uptake and the doubling of biomass productivity, mixotrophic biomass production costs would be three times cheaper than autotrophic production. Microalgal protein yield per hectare is expected to be 30-60 times higher than for soy beans while it would require 25-50 times less water. If glucose is used as a substrate for mixotrophic cultivation, the land and water consumption of sugar production substantially increases the overall water and land usage. However, when we consider the land and the water needed for sugar beet production, mixotrophic cultivation still requires 4 times less land and 7 times less water than soy beans. Altogether, this thesis successfully designed and applied oxygen balanced mixotrophy with two industrially relevant microalgal strains proving its effectiveness in reducing microalgal production costs. We are on the right track to achieve an economically feasible protein production from microalgae for food and feed purposes.

G. sulphuraria is a polyextremophilic microalga that can tolerate low pH, high temperature and high osmotic pressure. We cultivated G. sulphuraria ACUF 064 in chemostat at a biomass concentration of 134 to 243 g⋅m-2 aiming for maximal pigment content without compromising biomass productivity. Autotrophy was compared to ‘oxygen balanced’ mixotrophy with intracellular recirculation of oxygen and carbon dioxide. No differences were found in C-phycocyanin (C-PC) and protein content between autotrophic and mixotrophic cultures. In mixotrophy the biomass productivity and concentration were doubled compared to the photoautotrophic counterpart. In mixotrophy aeration was not needed and 89% of the substrate carbon was converted into biomass. Mixotrophically grown biomass contained 10% w/w C-PC which, combined with its high areal biomass productivity (49 g⋅m-2⋅day-1), sums up as one of the highest C-PC areal productivities ever reported (5 g⋅m-2⋅day-1) under 24 h/24 h illumination. C-PC extracted from G. sulphuraria was more stable than the currently used C-PC extracts from Spirulina. No significant loss of color was observed down to a pH of 3 and up to a temperature of 55 ◦C. G. sulphuraria had a protein content of 62% w/w and compared favorably with FAO dietary recommendation of adults regarding amino acid composition. G. sulphuraria contains a high proportion of essential, sulfur amino acids compared to Chlorella, Spirulina and soybean protein. Due to its attractive amino acid profile and high protein content, G. sulphuraria is a good candidate for food and feed applications to overcome sulfur amino acid deficiencies. In addition, oxygen balanced mixotrophy allows for efficient and productive cultivation of G. sulphuraria biomass.

Microalgae productivity was doubled by designing an innovative mixotrophic cultivation strategy that does not require gas–liquid transfer of oxygen or carbon dioxide. Chlorella sorokiniana SAG 211/8K was cultivated under continuous operation in a 2 L stirred-tank photobioreactor redesigned so that respiratory oxygen consumption was controlled by tuning the acetic acid supply. In this mixotrophic setup, the reactor was first operated with aeration and no net oxygen production was measured at a fixed acetic acid supply rate. Then, the aeration was stopped and the acetic acid supply rate was automatically regulated to maintain a constant dissolved oxygen level using process control software. Respiratory oxygen consumption was balanced by phototrophic oxygen production, and the reactor was operated without any gas–liquid exchange. The carbon dioxide required for photosynthesis was completely provided by the aerobic conversion of acetic acid. Under this condition, the biomass/substrate yield was 0.94 C-molx·C-molS–1. Under chemostat conditions, both reactor productivity and algal biomass concentration were doubled in comparison to a photoautotrophic reference culture. Mixotrophic cultivation did not affect the photosystem II maximum quantum yield (Fv/Fm) and the average-dry-weight-specific optical cross section of the microalgal cells. Only light absorption by chlorophylls over carotenoids decreased by 9% in the mixotrophic culture in comparison to the photoautotrophic reference. Our results demonstrate that photoautotrophic and chemoorganotrophic metabolism operate concurrently and that the overall yield is the sum of the two metabolic modes. At the expense of supplying an organic carbon source, photobioreactor productivity can be doubled while avoiding energy intensive aeration. Paid Open Access

Different species of microalgae are highly efficient in removing nutrients from wastewater streams and are able to grow using flue gas as a CO2 source. These features indicate that application of microalgae has a promising outlook in wastewater treatment. However, practical aspects and process of integration of algae cultivation into an existing wastewater treatment line have not been investigated. The Climate-KIC co-funded Microalgae Biorefinery 2.0 project developed and demonstrated this integration process through a case study. The purpose of this paper is to introduce this process by phases and protocols, as well as report on the challenges and bottlenecks identified in the case study. These standardized technical protocols detailed in the paper help to assess different aspects of integration including biological aspects such as strain selection, as well as economic and environmental impacts. This process is necessary to guide wastewater treatment plants through the integration of algae cultivation, as unfavourable parameters of the different wastewater related feedstock streams need specific attention and management. In order to obtain compelling designs, more emphasis needs to be put on the engineering aspects of integration. Well-designed integration can lead to operational cost saving and proper feedstock treatment enabling algae growth.

The effect of light quality on cell size and cell cycle, growth rate, productivity, photosynthetic efficiency and biomass composition of the marine prasinophyte Tetraselmis suecica F&M-M33 grown in 2-L flat panel photobioreactors illuminated with light emitting diodes (LEDs) of different colors was investigated. Biomass productivity and photosynthetic efficiency were comparable between white and red light, while under blue and green light productivity decreased to less than half and photosynthetic efficiency to about one third. Differences in cell size and number correlated with the cell cycle phase. Under red light cells were smaller and more motile. Chlorophyll content was strongly reduced with red and enhanced with blue light, while carotenoids and gross biomass composition were not affected by light quality. The eicosapentaenoic acid content increased under red light. Red light can substitute white light without affecting productivity of T. suecica F&M-M33, leading to smaller and more motile cells and increased eicosapentaenoic acid content. Red LEDs can thus be profitably used for the production of this microalga for aquaculture.

The effect of light quality on cell size and cell cycle, growth rate, productivity, photosynthetic efficiency and biomass composition of the marine prasinophyte Tetraselmis suecica FM-M33 grown in 2-L flat panel photobioreactors illuminated with light emitting diodes (LEDs) of different colors was investigated. Biomass productivity and photosynthetic efficiency were comparable between white and red light, while under blue and green light productivity decreased to less than half and photosynthetic efficiency to about one third. Differences in cell size and number correlated with the cell cycle phase. Under red light cells were smaller and more motile. Chlorophyll content was strongly reduced with red and enhanced with blue light, while carotenoids and gross biomass composition were not affected by light quality. The eicosapentaenoic acid content increased under red light. Red light can substitute white light without affecting productivity of T. suecica FM-M33, leading to smaller and more motile cells and increased eicosapentaenoic acid content. Red LEDs can thus be profitably used for the production of this microalga for aquaculture.