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Micro-algen kunnen functioneren als grondstof voor voedsel, energie enchemicaliën. Ze groeien sneller en produceren meer per oppervlakte-eenheiddan gewassen. Bovendien concurreren ze niet met landbouwgrond en nemenalle nutriënten uit het water op.

Photogranules are spherical aggregates formed of complex phototrophic ecosystems with potential for “aeration-free” wastewater treatment. Photogranules from a sequencing batch reactor were investigated by fluorescence microscopy, 16S/18S rRNA gene amplicon sequencing, microsensors, and stable- and radioisotope incubations to determine the granules’ composition, nutrient distribution, and light, carbon, and nitrogen budgets. The photogranules were biologically and chemically stratified, with filamentous cyanobacteria arranged in discrete layers and forming a scaffold to which other organisms were attached. Oxygen, nitrate, and light gradients were also detectable. Photosynthetic activity and nitrification were both predominantly restricted to the outer 500 µm, but while photosynthesis was relatively insensitive to the oxygen and nutrient (ammonium, phosphate, acetate) concentrations tested, nitrification was highly sensitive. Oxygen was cycled internally, with oxygen produced through photosynthesis rapidly consumed by aerobic respiration and nitrification. Oxygen production and consumption were well balanced. Similarly, nitrogen was cycled through paired nitrification and denitrification, and carbon was exchanged through photosynthesis and respiration. Our findings highlight that photogranules are complete, complex ecosystems with multiple linked nutrient cycles and will aid engineering decisions in photogranular wastewater treatment.

Wastewater characteristics can vary significantly, and in some municipal wastewaters the N:P ratio is as low as 5 resulting in nitrogen limiting conditions. In this study, the microbial community, function, and morphology of photogranules under nitrogen replete (N+) and limiting (N-) conditions was assessed in sequencing batch reactors. Photogranules under N- condition were nitrogen deprived 2/3 of a batch cycle duration. Surprisingly, this nitrogen limitation had no adverse effect on biomass productivity. Moreover, phosphorus and COD removal were similar to their removal under N+ conditions. Although performance was similar, the difference in granule morphology was obvious. While N+ photogranules were dense and structurally confined, N- photogranules showed loose structures with occasional voids. Microbial community analysis revealed high abundance of cyanobacteria capable of N2-fixation. These were higher at N- (38%) than N+ (29%) treatments, showing that photogranules could adjust and maintain treatment performance and high biomass productivity by means of N2-fixation.

Photogranules are a novel wastewater treatment technology that can utilize the sun's energy to treat (waste)water and at the same time generate valuable biomass. The microbial community consists of phototrophs (e.g.., microalgae, cyanobacteria) and non-phototrophic microorganisms (e.g., nitrifiers, denitrifiers, PAOs) that form dark-green spheroid agglomerates. Photogranules have excellent settling properties and are easy to harvest. Introducing photosynthesis to the treatment process has the advantage of in-situ oxygenation. Oxygen produced by phototrophs is directly available for heterotrophs (and nitrifiers). In turn, carbon dioxide produced by respiration is sequestered via photosynthesis. This reduces (or eliminates) the need for energy intensive external aeration and simultaneously minimizes greenhouse gas emissions. Additionally, photogranules can use both light and organic carbon for metabolic energy and consequently can have higher biomass productivity compared to conventional treatment systems. This makes them especially suitable for nutrient recovery application.The aim or this thesis was to advance the field of phototrophic wastewater treatment by systematically exploring the microbial community assembly and functions of photogranules. To achieve this, we operated bioreactors that allowed photogranulation and subsequently explored the functional potential of photogranules for wastewater treatment by changing operation conditions. We integrated various approaches and techniques to investigate the microbial community composition and function, physical structure, and microscale functionality of photogranules. Ultimately, we provided a holistic overview on the microbial ecology and process engineering of photogranular technology to pave the way for more sustainable wastewater treatment.In chapter 2 we generated photogranules under hydrodynamic conditions in lab-scale bioreactors operated in sequencing batch mode. We used a diverse and species-rich inoculum f

Microalgae is one of the most promising bio-based sources. It does not compete for arable land; can be cultivated in marine and fresh water and contains high amount of valuable biomolecules. However, microalgae production at a commercial scale is hindered by the high costs of cultivation and biorefinery. Currently, microalgae biorefineries focus on one-single product and specific markets (e.g. high value pigments and specialty chemicals), wasting and devaluating the remaining of the biomass. Therefore, novel technologies able to simplify the extraction process of multiple products and integrate different unit operations in an efficient and clean manner are needed. In this PhD thesis a liquid-liquid extraction technology named aqueous two phase systems (using ionic liquids as green solvents) was studied in its ability to extract and fractionate proteins, carbohydrates, pigments and lipids. The potential of this technology was demonstrated by its application in multiproduct and integrated biorefineries

Dit rapport kwantificeert mogelijkheden om de afhankelijkheid van soja-producten import voor veevoer (momenteel ongeveer 1800 kton/j sojaproducten per jaar, met daarin ongeveer 720kton eiwit) te verlagen, op basis van binnenlandse productie, met behoud van het huidige volume voedseleiwit productie. Veel ‘nieuwe bronnen’ worden als veelbelovend beschouwd voor verbetering van eiwitproductie. Met ‘verbeteren’ worden echter verschillende vormen bedoeld, zoals verhoging van voedingswaarde voor mens of dier, verbeteren van inzetbaarheid voor voeding of veevoer, ontwikkeling van een aantrekkelijk ‘niche’ product of verhoging van het bulk productiepotentieel. Voor de meeste opties is niet evident hoe groot de daadwerkelijke volume-potentie is, op welke termijn de ontwikkelingen opgeschaald kunnen zijn en welke technische ontwikkelstap en investeringen nog nodig zijn. Dit rapport geeft inzicht in die kwesties waardoor een realistisch perspectief op de mogelijkheden voor bulk productie ontstaat. In deze studie is naast de effecten van inzet van ‘nieuwe bronnen’ voor veevoer ook gekeken naar effecten op eiwitbehoefte als de (voornamelijk plantaardige) nieuwe eiwitbronnen direct worden ingezet voor voeding. Daarvoor hebben we gebruik gemaakt voor een omrekening van voedereiwitten naar voedseleiwitten: we schatten dat 1kg plantaardig voedseleiwit 5 kg veevoeder eiwit verdringt. De resultaten beschrijven primair de volume-potentie om afhankelijkheid van eiwitimport te beperken met instandhouding van totale productie voedingseiwit in Nederland. Hier en daar worden ook duurzaamheidseffecten genoemd en mogelijkheden en belemmeringen van 'nieuwe' eiwitbronnen en circulaire benutting van reststromen, maar die opsomming is niet compleet. De auteurs zijn zich ook bewust dat verschillende opties ook maatschappelijke aanpassingen vragen, maar ook die kwesties vallen buiten de scope van deze kwantitatieve studie. De potenties zijn kwantitatief ingeschat op basis van typische rendement-scha

Background: Adaptive laboratory evolution (ALE) is a powerful method for strain optimization towards abiotic stress factors and for identifying adaptation mechanisms. In this study, the green microalga Picochlorum sp. BPE23 was cultured under supra-optimal temperature to force genetic adaptation. The robustness and adaptive capacity of Picochlorum strains turned them into an emerging model for evolutionary studies on abiotic stressors such as temperature, salinity, and light.Results: Mutant strains showed an expanded maximal growth temperature of 44.6 °C, whereas the maximal growth temperature of the wild-type strain was 42 °C. Moreover, at the optimal growth temperature of 38 °C, the biomass yield on light was 22.3% higher, and the maximal growth rate was 70.5% higher than the wild type. Genome sequencing and transcriptome analysis were performed to elucidate the mechanisms behind the improved phenotype. A de novo assembled phased reference genome allowed the identification of 21 genic mutations involved in various processes. Moreover, approximately half of the genome contigs were found to be duplicated or even triplicated in all mutants, suggesting a causal role in adaptation.Conclusions: The developed tools and mutant strains provide a strong framework from whereupon Picochlorum sp. BPE23 can be further developed. Moreover, the extensive strain characterization provides evidence of how microalgae evolve to supra-optimal temperature and to photobioreactor growth conditions. With this study, microalgal evolutionary mechanisms were identified by combining ALE with genome sequencing.

This is the genome assembly of the wildtype Picochlorum sp. BPE23 that was isolated at Bonaire. For this microalga, optimal and maximum growth temperature, growth rate and yield on light were characterised. Also, this strain was used for an adaptive laboratory experiment in which the microalga was grown under supra-optimal conditions., This is the genome assembly of the wildtype Picochlorum sp. BPE23 that was isolated at Bonaire. For this microalga, optimal and maximum growth temperature, growth rate and yield on light were characterised. Also, this strain was used for an adaptive laboratory experiment in which the microalga was grown under supra-optimal conditions.

Background Microalgae are considered a promising platform for sustainable lipid production. Despite this, productivities need to be improved to facilitate an economically viable production process. This can be achieved through strain improvement, for instance by genetic engineering. Strain improvement strategies often deploy high-throughput screening platforms i.a. involving single-cell methodologies such as fluorescence-activated cell sorting (FACS) for the identification and isolation of better-performing strains. The heterokont microalga Nannochloropsis is a prospective candidate for the industrial production of lipids. Previous studies have reported the isolation of high lipid-producing Nannochloropsis strains by combining qualitative staining of lipid bodies using the fluorophoric dye BODIPY with FACS methodology. However, it has never been investigated how cellular physiology and different staining conditions hamper the reproducibility of this method as a quantitative screening procedure. Results Here we report the development of an optimized single cell lipid screening procedure for Nannochloropsis oceanica. Systematic assessment of different staining conditions revealed that treatment with 6% DMSO and 1.2 μg ml−1 BODIPY for 15 min is ideal for staining neutral lipids in an exponentially growing culture of N. oceanica. Cultures that are overproducing lipids, for example after exposure to external stimuli such as nutrient deprivation stress, require treatment with 10% DMSO and 1.2–1.6 μg ml−1 BODIPY for 36 min to facilitate complete staining of lipid bodies. We verify that DMSO is required to permeabilize the particularly tough cell barrier of Nannochloropsis and we show that exposure to 10% DMSO does not affect cell viability. Increasing concentrations of BODIPY, however, correlated with a decrease in viability when screening stressed cultures. Using the optimized protocol, reproductive viabilities can be expected to be ~91% and 83–82% for no

The common lipid extraction from microalgae involves sequential of energy-intensive processes and the use of harmful organic solvents. Eutectic solvents (ES), a novel class of designer solvents, hold a great potential as alternative solvents. Not only they are easy to prepare and have tailorable properties, but they are also able to permeabilize the cell wall of microalgae. However, the lack of vapor pressure makes the solvent regeneration difficult. Therefore, in this thesis, a semi-hydrophobic eutectic solvent was developed to extract lipids from microalgae, with a viable solvent regeneration step. Moreover, a preliminary biorefinery process was also explored.In Chapter 2, we screened several semi-hydrophobic ES. The combination of polar imidazole and nonpolar hexanoic acid showed tuneable hydrophobicity depending on the composition. At low imidazole presence, the mixture dissolved lipids and the lipid solubility decreased at higher imidazole content. This principle was then applied to separate the ES from the dissolved lipids. With this approach, about 75% of lipids could be recovered with a high purity (> 85%).Since retrieving imidazole from the altered ES was difficult, we explored other auxiliaries which can also shift the ES hydrophobicity (Chapter 3). Polar antisolvents, such as water, methanol, and ethanol, were observed to reduce the lipid solubility in the ES. The reduction of lipid solubility increases with the antisolvent polarity and amount. Furthermore, since the antisolvents were (moderately) volatile, a large amount of antisolvents could be loaded to obtain higher purity (up to 100%) and recovery (> 90%) when compared to the previous approach. With this approach, more than 90% of ES could be regenerated through evaporating the antisolvents. Methanol was selected as the best antisolvent as it offered the ease of regeneration without sacrificing the lipid recovery.The novel ES was then applied to intact Nannochloropsis oceanica for lipid extraction (C

The Arabian Peninsula, due to its climate, availability of non-arable land, seawater, and carbon dioxide, is one of the best global locations for commercial cultivation of algae and cyanobacteria. This work focused on the screening of multiple locally isolated strains for their capability to thrive under industrially relevant conditions of elevated temperatures (up to 40 ˚C) and carbon dioxide levels (up to 30%). Two strains, Leptolyngbya sp. and Picochlorum sp., grew well at temperatures of up to 40 ˚C, and also showed a tolerance towards elevated CO2 concentrations. Both microalgae isolated, T. subcordiformis and Picochlorum sp., presented significant amounts of lipids, including high-value omega-3 fatty acids EPA and DHA. On the other hand, both cyanobacteria, Leptolyngbya sp. and Chroococcidiopsis sp., presented levels of phycobiliproteins. The isolates, all very diverse in response and products, showed promising characteristics, making them valuable strains for further investigation towards commercial applications and CO2 capture.One of the identified cyanobacteria was further investigated for its potential to produce phycocyanin, a nutraceutical with high commercial value, under desert climate conditions. Under elevated temperatures and light intensities, of up to 40 ˚C and 1800 µmol photons·m-2·s-1, Leptolyngbya sp. biomass productivity was up to 45% higher as compared A. platensis, the commercially most commonly produced strain for phycocyanin. High temperatures were found to improve both the biomass productivity and phycocyanin content, with maxima of 1.09±0.03 gX·L-1·d-1 and 72.12±3.52 mgPC·gX-1, respectively. Furthermore, various cell disruption methods and buffers were tested for the efficient extraction of high-purity phycocyanin. The best results were found through bead-beating in phosphate buffer, which showed the highest combined phycocyanin yield (169.9±3.6 mgPC·gX) and purity (7.37±0.16). The extract purities obta

The work concerns large scale production of microalgae in photobioreactors and involves both technical-economic studies, productivity modeling and validation in industrial scale.Chapter 2 is an economical comparison of the 3 major microalgal production technologies: open raceway ponds, tubular photobioreactors and flat panel photobioreactors. The assessment of the operational performance of the three technologies was based on published productivity results from experimental photobioreactor and pilot plant studies. Effects of scale, site and various optimization options were included in a sensitivity analysis.Reducing auxiliar energy requirements for mixing with the two photobioreactor technologies was identified as a major challenge in the development of photobioreactor production of microalgal biomass, particularly for biofuel application. A standard hydraulic examination of auxiliary energy use with tubular- and flat panel photobioreactors was carried out in Chapter 3, resulting in general design recommendations. In chapter 4, a model of microalgal growth as a function of irradiance and temperature was developed in the laboratory and extended with an irradiation model for a specific flat panel reactor. The model was used to optimize the design of the flat panel photobioreactor and was validated in a study of a full-scale installation of the photobioreactor. Chapter 5 and 6 describes the design and development of operating techniques for a tubular photobioreactor for production of microalgal oil, based on recommendations in chapters 2 and 3. A laboratory protocol to compare the suitability for oil production in microalgal strains was developed and a laboratory method to determine the minimum biomass concentration in an outdoor tubular photobioreactor. On the basis of that, two strains, Neochloris oleoabundans and Chlorococcum littorale were selected for further work. A method, the inverse nitrogen quota, was developed to predict and steer microalgal oil production

Sponges are recognized as a treasure trove of potential new drugs, as they produce many biologically active compounds to interact with their surroundings and protect themselves. Despite all efforts and some promising results, attempts to develop sponge cell lines to produce compounds in a controlled environment were ultimately unsuccessful.In Chapter 2 we reported a long-awaited breakthrough: cells of 9 sponge species divided rapidly in amino acid-optimized nutrient medium M1, based on mammalian cell culture medium 199 (M199). Members of the genus Geodia, G. neptuni, Geodia sp., and G. barretti, showed the least individual variation. The Geodia spp. were subcultured 3 - 5 times over a period of 21 - 35 days and reached an average total of 6 population doublings. These finite cell lines represented the first real leads to develop continuous marine sponge cell lines.In Chapter 3 we established that G. barretti cells proliferated even more rapidly and to a higher density when cultured in OpM1 medium, the successor of M1 with added growth factors, lipids, vitamins, and other nutrients. G. barretti cells of 3 individuals could double nearly 100 times in OpM1, compared to 5 doublings cells of the same individuals reached in M1. These results brought us one step closer to sponge cells producing biopharmaceuticals at industrial scale.In Chapter 4 we compared genes expressed by G. barretti cells in fragments of an intact sponge, after being dissociated and cryopreserved, and when cultured in OpM1 medium. Telomerase reverse transcriptase, the gene that determines the number of times the genome can be replicated, was expressed in all samples, providing further evidence that sponge cells may be naturally immortal. Cultured cells reorganized their cytoskeleton, down-regulated genes needed to adhere to the extracellular matrix, synthesized more proteins and lipids, phagocytosed less and broke down fewer lipids and glycogen reserves for energy, likely favoring soluble energy sourc

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. Moreo

The global demand for food, feed and fossil-based chemicals is increasing due to the continuously growing world population. Excessive and unsustainable consumption is causing anthropogenic effects resulting in high carbon emission, pollution and depletion of natural resources. Currently, the main renewable feedstocks used as alternative to petrochemical processes are derived from agricultural crops. Higher plants are a great source of oils which can be used in food, feed, paint, lighting and fuel industries. However, cultivation of crops requires vast areas of arable land, fresh water and minerals. Additionally, depletion of petrochemical resources increases the demand for plant-based fuels, creating a direct competition with food and feed production. Therefore, novel renewable and sustainable alternatives are necessary to reduce the current environmental impact of traditional industrial processes.Microalgae are considered as a promising alternative for the production of commodities. They are capable of converting sunlight, water, CO2 and nutrients into biomass and products which attract interest from pharmaceutical, cosmetic, biofuel, food and feed industries. Furthermore, microalgae can reach significantly higher biomass concentrations and product yields per hectare compared to higher plants, they do not require arable lands and some species can be grown on wastewater or seawater.Several microalgal species can accumulate large amounts of fatty acids when cultivated under stress conditions. At a cellular level, lipids such as triacylglycerols (TAGs) and polyunsaturated fatty acids (PUFAs) can serve either as structural components or storage compounds. Therefore, microalgae are a source of valuable oils that can be used directly as nutritional products or as raw material for biofuel production.Although microalgae have the potential to become a feedstock for lipid production some limitations and challenges remain. In order to achieve an economically feasible process

Microalgae hold promise for sustainable production of commodities including food, feed, and green chemicals. Nannochloropsis oceanica is a particularly interesting candidate for industrial purposes and an emerging model organism for oleaginous. Although production of bulk commodities from N. oceanica is not yet economically feasible, strain improvement strategies could transform this microalga into a cell factory for cost-effective production of designer food, feed and bioproducts. This will require a comprehensive framework for metabolic engineering and synthetic biology as cornerstones to maximization of yields and productivities. These practices rely on a proper genome annotation, on an in-depth understanding of metabolic fluxes, and on a highly developed genetic manipulation toolset. Therefore, advancing our understanding of the metabolic network and expanding the toolbox for genetic engineering are of the utmost importance to establish N. oceanica as a commodity production platform.To improve the available tools for conducting forward genetic screens with N. oceanica, we developed a high-throughput screening for cellular neutral lipid content in chapter 2. Fluorescence activated cell sorting (FACS) was employed as a powerful single cell methodology. A robust and efficient neutral lipid staining procedure for N. oceanica was devised, utilizing the fluorophore BODIPY505/515. It was found that 6% DMSO, 1.2 µg ml-1 and 15 min staining duration are ideal for staining neutral lipids in exponentially growing N. oceanica cultures. For cultures that were stressed by nutrient deprivation, 10% DMSO, 1.2-1.6 µg ml-1 and 36 min duration were required. The combination of this staining method with FACS allowed quantitative prediction of cellular neutral lipid contents.Chapters 3-4 focused on developing novel tools for genetic manipulation of N. oceanica using CRISPR-Cas technology. Cas12a was employed as a signi

In order to reduce CO2 emissions and the use of fossil resources, microbial production platforms offer a vast range of possibilities. Many different compounds and/or precursors can be produced by micro-organisms from renewable resources and their production can be enhanced by means of metabolic engineering.Ethyl acetate is a popular solvent used in various applications but production mainly relies on petrochemical resources. Whereas yeast can naturally produce ethyl acetate, metabolism does not allow for a complete anaerobic process. Especially the production of inexpensive bulk chemicals, like ethyl acetate, need to proceed at high yields and rates to be economically viable. Therefore, anaerobic processes are desired as more carbons and electrons are directed to formation instead of biomass. With the identification of the bulk ethyl acetate producing enzyme ethanol acetyltransferase (Eat1) the path was finally cleared for targeted engineering approaches and the use of alternative production hosts for the bio-based production of ethyl acetate.This research explored different metabolic engineering approaches in Escherichia coli to improve the key parameters titer, rate, and yield (TRY). Three anaerobic scenarios were followed up on that differed in their use of electrons and carbons for the desired product. An integrated strategy on in situ product removal via gas stripping was included to benefit downstream processing and to prevent process limitations by product toxicity. The resulting bioprocess with optimizations on expression, protein and process level increased product yields to above 70 % of the pathway yield, and productivities by a factor 1.4. The thesis reflects on the results and discusses the next steps towards efficient bio-based ethyl acetate production

Several technologies have aimed to recover nitrogen directly from urine. Nitrogen recovery in these technologies was limited by the mismatch of the nitrogen-phosphorus molar ratio (N:P) of urine, being 30–46:1, and that of the final product, e.g., 1:1 in struvite and 16–22:1 in microalgae biomass. Additionally, the high nitrogen concentrations found in urine can be inhibitive for growth of microorganisms. Cyanobacteria were expected to overcome phosphorus (P) limitation in urine given their ability to store an N-rich polymer called cyanophycin. In this study, it was found that the model cyanobacterium Synechocystis sp. PCC6803 did not experience significant growth inhibition when cultivated in synthetic medium with concentrations of 0.5 g ammonium-N L−1. In the case of urea, no inhibition was observed when having it as sole nitrogen source, but it resulted in chlorosis of the cultures when the process reached stationary phase. Synechocystis was successfully cultivated in a medium with 0.5 g ammonium-N L−1 and a N:P ratio of 276:1, showing the N:P flexibility of this biomass, reaching biomass N:P ratios up to 92:1. Phosphorus starvation resulted in cyanophycin accumulation up to 4%. Dilution of the culture in fresh medium with the addition of 118 mg N L−1 and 1.5 mg P L−1 (N:P of 174:1) resulted in a rapid and transient cyanophycin accumulation up to 11%, after which cyanophycin levels rapidly decreased to 3%.

The effect of light on biomass and fucoxanthin (Fx) productivities was studied in two microalgae, Tisochrysis lutea and Phaeodactylum tricornutum. High and low biomass concentrations (1.1 and 0.4 g L−1) were tested in outdoor pilot‐scale flat‐panel photobioreactors at semi‐continuous cultivation mode. Fluorescence spectroscopy coupled with chemometric modeling was used to develop prediction models for Fx content and for biomass concentration to be applied for both microalgae species. Prediction models showed high R2 for cell concentration (.93) and Fx content (.77). Biomass productivity was lower for high biomass concentration than low biomass concentration, for both microalgae (1.1 g L−1: 75.66 and 98.14 mg L−1 d−1, for T. lutea and P. tricornutum, respectively; 0.4 g L−1: 129.9 and 158.47 mg L−1 d−1, T. lutea and P. tricornutum). The same trend was observed in Fx productivity (1.1 g L−1: 1.14 and 1.41 mg L−1 d−1, T. lutea and P. tricornutum; 0.4 g L−1: 2.09 and 1.73 mg L−1 d−1, T. lutea and P. tricornutum). These results show that biomass and Fx productivities can be set by controlling biomass concentration under outdoor conditions and can be predicted using fluorescence spectroscopy. This monitoring tool opens new possibilities for online process control and optimization.