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This work aimed to introduce a new technique for improving sustainable biodiesel from Microchloropsis salina by examining the impact of various nitrogen sources, salt stress, and carbon enrichment, individually or in combination, to determine the best treatment conditions for biomass and lipid productivity as well as biodiesel quality. M. salina effectively utilizes nitrate (NaNO3 and KNO3) and organic nitrogen (especially yeast extract and glycine) but not ammonium for growth. The highest biomass and lipid productivity were observed on yeast extract (0.11 g L−1 day−1 and 52.11 μgmL−1 day−1, respectively). Lipid production enhanced by 80% under 300 mM of NaCl, while growth increased only by 30%. Acetate could not be used to cultivate the alga mixotrophically; only glucose and glycerol were effective. The ideal organic carbon supply values for glucose and glycerol were 3 g L−1 and 2 g L−1, respectively. Nutrition by 2 g L−1 glycerol promoted biomass and lipid productivities by 62% and 95%, respectively, while biomass productivity was reduced by 16% under combined and lipid productivity increased by 80%. All treatments significantly improved the biomass and biodiesel quality-related fatty acid profile. [ABSTRACT FROM AUTHOR]

Dissolved exometabolites mediate algal interactions in aquatic ecosystems, but microalgal exometabolomes remain understudied. We conducted an untargeted metabolomic analysis of nonpolar exometabolites exuded from four phylogenetically and ecologically diverse eukaryotic microalgal strains grown in the laboratory, freshwater Chlamydomonas reinhardtii, brackish Desmodesmus sp., marine Phaeodactylum tricornutum, and marine Microchloropsis salina, to identify released metabolites based on relative enrichment in the exometabolomes compared to cell pellet metabolomes. Exudates from the different taxa were distinct, but we did not observe clear phylogenetic patterns. We used feature-based molecular networking to explore the identities of these metabolites, revealing several distinct di- and tripeptides secreted by each of the algae, lumichrome, a compound that is known to be involved in plant growth and bacterial quorum sensing, and novel prostaglandin-like compounds. We further investigated the impacts of exogenous additions of eight compounds selected based on exometabolome enrichment on algal growth. Of these compounds, five (lumichrome, 5'-S-methyl-5'-thioadenosine, 17-phenyl trinor prostaglandin A2, dodecanedioic acid, and aleuritic acid) impacted growth in at least one of the algal cultures. Two of these compounds (dodecanedioic acid and aleuritic acid) produced contrasting results, increasing growth in some algae and decreasing growth in others. Together, our results reveal new groups of microalgal exometabolites, some of which could alter algal growth when provided exogenously, suggesting potential roles in allelopathy and algal interactions. IMPORTANCE Microalgae are responsible for nearly half of primary production on earth and play an important role in global biogeochemical cycling as well as in a range of industrial applications. Algal exometabolites are important mediators of algal-algal and algal-bacterial interactions that ultimately affect algal growth and physiology. In this study, we characterize exometabolomes across marine and freshwater algae to gain insights into the diverse metabolites they release into their environments ("exudates"). We observe that while phylogeny can play a role in exometabolome content, environmental conditions or habitat origin (freshwater versus marine) are also important. We also find that several of these compounds can influence algal growth (as measured by chlorophyll production) when provided exogenously, highlighting the importance of characterization of these novel compounds and their role in microalgal ecophysiology.

Microalgae produce specific chemicals indicative of stress and/or death. The aim of this study was to perform non-destructive monitoring of algal culture systems, in the presence and absence of grazers, to identify potential biomarkers of incipient pond crashes. Here, we report ten volatile organic compounds (VOCs) that are robustly generated by the marine alga, Microchloropsis salina, in the presence and/or absence of the marine grazer, Brachionus plicatilis. We cultured M. salina with and without B. plicatilis and collected in situ volatile headspace samples using thermal desorption tubes over the course of several days. Data from four experiments were aggregated, deconvoluted, and chromatographically aligned to determine VOCs with tentative identifications made via mass spectral library matching. VOCs generated by algae in the presence of actively grazing rotifers were confirmed via pure analytical standards to be pentane, 3-pentanone, 3-methylhexane, and 2-methylfuran. Six other VOCs were less specifically associated with grazing but were still commonly observed between the four replicate experiments. Through this work, we identified four biomarkers of rotifer grazing that indicate algal stress/death. This will aid machine learning algorithms to chemically define and diagnose algal mass production cultures and save algae cultures from imminent crash to make biofuel an alternative energy possibility.

Abstract Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day–night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mgDHA gCDW −1, and 23 mgEPA gCDW −1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures. Graphical Abstract

Featured Application: Automated and scalable high-throughput development of photoautotrophic bioprocesses with microalgae. Scalable lab-scale photobioreactors are needed for the exploration of new and improved photoautotrophic bioprocesses. Microbioreactor systems in which parallel bioreactors operate automatically are frequently employed to increase the speed of strain selection as well as the bioprocess-based exploration of heterotrophic fermentation processes. To enable the photoautotrophic operation of a commercially available parallel microbioreactor system with 48 stirred-tank bioreactors, LED illumination modules were designed to allow for individual light supply (400–700 nm) for each of the parallel bioreactors automated by a liquid handling station that performs both individual pH control and OD750 detection. The illumination modules enable dynamic variation of the incident light intensities of up to 1800 µmol m−2 s−1. Automated liquid level detection and volume control of each individual mL-scale gassed photobioreactor has to be established to compensate for evaporation because of the long process times of several days up to weeks. Photoautotrophic batch processes with Microchloropsis salina that employ either varying constant incident light intensities or day and night dynamics resulted in a standard deviation of OD750 of up to a maximum of 10%, with the exception of high-photoinhibiting incident light intensities. The established photoautotrophic microbioreactor system enables the automated investigation of microalgae processes in up to 48 parallel stirred photobioreactors and is thus a new tool that enables efficient characterization and development of photoautotrophic processes with microalgae. [ABSTRACT FROM AUTHOR]

Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection. [ABSTRACT FROM AUTHOR]

Marine microalgae have received much attention as a sustainable source of the two health beneficial omega-3-fatty acids docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5). However, photoautotrophic monocultures of microalgae can only produce either DHA or EPA enriched biomass. An alternative may be the photoautotrophic co-cultivation of Tisochrysis lutea as DHA-producer with Microchloropsis salina for simultaneous EPA production to obtain EPA- and DHA-rich microalgae biomass in a nutritionally balanced ratio. Photoautotrophic co-cultivation processes of T. lutea and M. salina were studied, applying scalable and fully controlled lab-scale gas-lift flat-plate photobioreactors with LED illumination for dynamic climate simulation of a repeated sunny summer day in Australia [day–night cycles of incident light (PAR) and temperature]. Monocultures of both marine microalgae were used as reference batch processes. Differences in the autofluorescence of both microalgae enabled the individual measurement, of cell distributions in co-culture, by flow cytometry. The co-cultivation of T. lutea and M. salina in artificial sea water with an inoculation ratio of 1:3 resulted in a balanced biomass production of both microalgae simultaneously with a DHA:EPA ratio of almost 1:1 (26 mgDHA gCDW−1, and 23 mgEPA gCDW−1, respectively) at harvest after depletion of the initially added fertilizer. Surprisingly, more microalgae biomass was produced within 8 days in co-cultivation with an increase in the cell dry weight (CDW) concentration by 31%, compared to the monocultures with the same amount of light and fertilizer. What is more, DHA-content of the microalgae biomass was enhanced by 33% in the co-culture, whereas EPA-content remained unchanged compared to the monocultures. [ABSTRACT FROM AUTHOR]

Algae and microalgae are used as a source of different biomolecules, such as lipids and carbohydrates. Among carbohydrates, polysaccharides, such as β-glucans, are important for their application as antioxidants, antisepsis, and immunomodulators. In the present work, the β-glucans production potential of Microchloropsis salina was assessed using two different culture conditions: a high-density batch and a modeled high-density fed-batch. From the biochemical parameters determined from these two cultures conditions, it was possible to establish that the modeled high-density fed-batch culture improves the biomass growth. It was possible to obtain a biomass productivity equal to 8.00 × 10−2 ± 2.00 × 10−3 g/(L × day), while the batch condition reached 5.13 × 10−2 ± 4.00 × 10−4 g/(L × day). The same phenomenon was observed when analyzing the β-glucans accumulation, reaching volumetric productivity equal to 5.96 × 10−3 ± 2.00 × 10−4 g of product/(L × day) against the 4.10 × 10−3 ± 2.00 × 10−4 g of product/(L × day) obtained in batch conditions. These data establish a baseline condition to optimize and significantly increase β-glucan productivity, as well as biomass, adding a new and productive source of this polymer, and integrating its use in potential applications in the human and animal nutraceutical industry. [ABSTRACT FROM AUTHOR]

Scalable lab-scale photobioreactors are needed for the exploration of new and improved photoautotrophic bioprocesses. Microbioreactor systems in which parallel bioreactors operate automatically are frequently employed to increase the speed of strain selection as well as the bioprocess-based exploration of heterotrophic fermentation processes. To enable the photoautotrophic operation of a commercially available parallel microbioreactor system with 48 stirred-tank bioreactors, LED illumination modules were designed to allow for individual light supply (400–700 nm) for each of the parallel bioreactors automated by a liquid handling station that performs both individual pH control and OD750 detection. The illumination modules enable dynamic variation of the incident light intensities of up to 1800 µmol m−2 s−1. Automated liquid level detection and volume control of each individual mL-scale gassed photobioreactor has to be established to compensate for evaporation because of the long process times of several days up to weeks. Photoautotrophic batch processes with Microchloropsis salina that employ either varying constant incident light intensities or day and night dynamics resulted in a standard deviation of OD750 of up to a maximum of 10%, with the exception of high-photoinhibiting incident light intensities. The established photoautotrophic microbioreactor system enables the automated investigation of microalgae processes in up to 48 parallel stirred photobioreactors and is thus a new tool that enables efficient characterization and development of photoautotrophic processes with microalgae.

Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection.

Algae and microalgae are used as a source of different biomolecules, such as lipids and carbohydrates. Among carbohydrates, polysaccharides, such as β-glucans, are important for their application as antioxidants, antisepsis, and immunomodulators. In the present work, the β-glucans production potential of Microchloropsis salina was assessed using two different culture conditions: a high-density batch and a modeled high-density fed-batch. From the biochemical parameters determined from these two cultures conditions, it was possible to establish that the modeled high-density fed-batch culture improves the biomass growth. It was possible to obtain a biomass productivity equal to 8.00 × 10−2 ± 2.00 × 10−3 g/(L × day), while the batch condition reached 5.13 × 10−2 ± 4.00 × 10−4 g/(L × day). The same phenomenon was observed when analyzing the β-glucans accumulation, reaching volumetric productivity equal to 5.96 × 10−3 ± 2.00 × 10−4 g of product/(L × day) against the 4.10 × 10−3 ± 2.00 × 10−4 g of product/(L × day) obtained in batch conditions. These data establish a baseline condition to optimize and significantly increase β-glucan productivity, as well as biomass, adding a new and productive source of this polymer, and integrating its use in potential applications in the human and animal nutraceutical industry.

Featured Application: Open microalgae mass culture with water recycling as a sustainable feedstock for feed, fuels or chemicals. (1) Background: Recycling of water and non-converted nutrients is considered to be a necessity for an economically viable production of microalgal biomass as a renewable feedstock. However, medium recycling might also have a negative impact on algal growth and productivity due to the accumulation of growth-inhibiting substances. (2) Methods: Consecutive batch processes with repeated water recycling after harvesting of algal biomass were performed with the saline microalga Microchloropsis salina in open thin-layer cascade photobioreactors operated at a physically simulated Mediterranean summer climate. The impact of water recycling on culture performance was studied and the composition of the recycled water was analyzed. (3) Results: Water recycling had no adverse effect on microalgal growth and biomass productivity (14.9−21.3 g m−2 d−1) if all necessary nutrients were regularly replenished and KNO3 was replaced by urea as the nitrogen source to prevent the accumulation of K+ ions. Dissolved organic carbon accumulated in recycled water, probably promoting mixotrophic growth. (4) Conclusion: This study shows that repeated recycling of water is feasible even in high-density cultivation processes with M. salina of more than 30 g L−1 cell dry weight, increasing culture performance while reducing nutrient consumption and circumventing wastewater production. [ABSTRACT FROM AUTHOR]

Studies on microalgal lipid production as a sustainable feedstock for biofuels and chemicals are scarce, particularly those on applying open thin-layer cascade (TLC) photobioreactors under dynamic diurnal conditions. Continuous lipid production with Microchloropsis salina was studied in scalable TLC photobioreactors at 50 m2 pilot scale, applying a physically simulated Mediterranean summer climate. A cascade of two serially connected TLC reactors was applied, promoting biomass growth under nutrient-replete conditions in the first reactor, while inducing the accumulation of lipids via nitrogen limitation in the second reactor. Up to 4.1 g L−1 of lipids were continuously produced at productivities of up to 0.27 g L−1 d−1 (1.8 g m2 d−1) at a mean hydraulic residence time of 2.5 d in the first reactor and 20 d in the second reactor. Coupling mass balances with the kinetics of microalgal growth and lipid formation enabled the simulation of phototrophic process performances of M. salina in TLC reactors in batch and continuous operation at the climate conditions studied. This study demonstrates the scalability of continuous microalgal lipid production in TLC reactors with M. salina and provides a TLC reactor model for the realistic simulation of microalgae lipid production processes after re-identification of the model parameters if other microalgae and/or varying climate conditions are applied.

Microalgae produce specific chemicals indicative of stress and/or death. The aim of this study was to perform non-destructive monitoring of algal culture systems, in the presence and absence of grazers, to identify potential biomarkers of incipient pond crashes. Here, we report ten volatile organic compounds (VOCs) that are robustly generated by the marine alga, Microchloropsis salina, in the presence and/or absence of the marine grazer, Brachionus plicatilis. We cultured M. salina with and without B. plicatilis and collected in situ volatile headspace samples using thermal desorption tubes over the course of several days. Data from four experiments were aggregated, deconvoluted, and chromatographically aligned to determine VOCs with tentative identifications made via mass spectral library matching. VOCs generated by algae in the presence of actively grazing rotifers were confirmed via pure analytical standards to be pentane, 3-pentanone, 3-methylhexane, and 2-methylfuran. Six other VOCs were less specifically associated with grazing but were still commonly observed between the four replicate experiments. Through this work, we identified four biomarkers of rotifer grazing that indicate algal stress/death. This will aid machine learning algorithms to chemically define and diagnose algal mass production cultures and save algae cultures from imminent crash to make biofuel an alternative energy possibility.

(1) Background: Recycling of water and non-converted nutrients is considered to be a necessity for an economically viable production of microalgal biomass as a renewable feedstock. However, medium recycling might also have a negative impact on algal growth and productivity due to the accumulation of growth-inhibiting substances. (2) Methods: Consecutive batch processes with repeated water recycling after harvesting of algal biomass were performed with the saline microalga Microchloropsis salina in open thin-layer cascade photobioreactors operated at a physically simulated Mediterranean summer climate. The impact of water recycling on culture performance was studied and the composition of the recycled water was analyzed. (3) Results: Water recycling had no adverse effect on microalgal growth and biomass productivity (14.9−21.3 g m−2 d−1) if all necessary nutrients were regularly replenished and KNO3 was replaced by urea as the nitrogen source to prevent the accumulation of K+ ions. Dissolved organic carbon accumulated in recycled water, probably promoting mixotrophic growth. (4) Conclusion: This study shows that repeated recycling of water is feasible even in high-density cultivation processes with M. salina of more than 30 g L−1 cell dry weight, increasing culture performance while reducing nutrient consumption and circumventing wastewater production.

Studies on microalgal lipid production as a sustainable feedstock for biofuels and chemicals are scarce, particularly those on applying open thin-layer cascade (TLC) photobioreactors under dynamic diurnal conditions. Continuous lipid production with Microchloropsis salina was studied in scalable TLC photobioreactors at 50 m2 pilot scale, applying a physically simulated Mediterranean summer climate. A cascade of two serially connected TLC reactors was applied, promoting biomass growth under nutrient-replete conditions in the first reactor, while inducing the accumulation of lipids via nitrogen limitation in the second reactor. Up to 4.1 g L−1 of lipids were continuously produced at productivities of up to 0.27 g L−1 d−1 (1.8 g m2 d−1) at a mean hydraulic residence time of 2.5 d in the first reactor and 20 d in the second reactor. Coupling mass balances with the kinetics of microalgal growth and lipid formation enabled the simulation of phototrophic process performances of M. salina in TLC reactors in batch and continuous operation at the climate conditions studied. This study demonstrates the scalability of continuous microalgal lipid production in TLC reactors with M. salina and provides a TLC reactor model for the realistic simulation of microalgae lipid production processes after re-identification of the model parameters if other microalgae and/or varying climate conditions are applied. [ABSTRACT FROM AUTHOR]

• Batch production of 6.6 g L−1 lipids in open thin-layer cascade photobioreactors. • Accumulation of up to 46% (w/w) lipids in Microchloropsis salina. • Continuous production of 3.0 g L−1 lipids in a cascade of two open photobioreactors. • Stable lipid space-time-yield of 0.2 g L−1 d−1. • Control of alkalinity to 10 mM resulted in high CO 2 conversion efficiency of 84%. Microalgal biomass is considered as the most promising feedstock for sustainable production of liquid fuels. Lipid production with Microchloropsis salina was studied in open thin-layer cascade (TLC) photobioreactors with a surface area of 8 m2 applying a physically simulated Mediterranean summer climate. High lipid concentrations of up to 6.6 g L−1 with 46% (w w−1) total lipids in dry cell mass were achieved in two-phase batch processes applying a nitrogen limitation. The two-phase batch process was transferred into a continuously operated reactor cascade of two TLC photobioreactors. Microchloropsis salina cells were produced continuously in the first photobioreactor, whereas continuous lipid production was enabled in the second, nitrogen-limited TLC photobioreactor resulting in continuous production of 3.0 g L−1 lipids with a high overall lipid space-time-yield of 0.2 g L−1 d−1. The control of alkalinity to about 10 mM resulted in high CO 2 conversion efficiencies of 84–87%. [ABSTRACT FROM AUTHOR]