Comparison of plant biostimulating properties of Chlorella sorokiniana biomass produced in batch and semi-continuous systems supplemented with pig manure or acetate.

Microalgae-derived biostimulants provide an eco-friendly biotechnology for improving crop productivity. The strategy of circular economy includes reducing biomass production costs of new and robust microalgae strains grown in nutrient-rich wastewater and mixotrophic culture where media is enriched with organic carbon. In this study, Chlorella sorokiniana was grown in 100 l bioreactors under sub-optimal conditions in a greenhouse. A combination of batch and semi-continuous cultivation was used to investigate the growth, plant hormone and biostimulating effect of biomass grown in diluted pig manure and in nutrient medium supplemented with Na-acetate. C. sorokiniana tolerated the low light (sum of PAR 0.99 ± 0.18 mol/photons/(m2/day)) and temperature (3.7-23.7° C) conditions to maintain a positive growth rate and daily biomass productivity (up to 149 mg/l/day and 69 mg/l/day dry matter production in pig manure and Na-acetate supplemented cultures respectively). The protein and lipid content was significantly higher in the biomass generated in batch culture and dilute pig manure (1.4x higher protein and 2x higher lipid) compared to the Na-acetate enriched culture. Auxins indole-3-acetic acid (IAA) and 2-oxindole-3-acetic acid (oxIAA) and salicylic acid (SA) were present in the biomass with significantly higher auxin content in the biomass generated using pig manure (> 350 pmol/g DW IAA and > 84 pmol/g DW oxIAA) compared to cultures enriched with Na-acetate and batch cultures (< 200 pmol/g DW IAA and < 27 pmol/g DW oxIAA). No abscisic acid and jasmonates were detected. All samples had plant biostimulating activity measured in the mungbean rooting bioassay with the Na-acetate supplemented biomass eliciting higher rooting activity (equivalent to 1-2 mg/l IBA) compared to the pig manure (equivalent to 0.5-1 mg/l IBA) and batch culture (equivalent to water control) generated biomass. Thus C. sorokiniana MACC-728 is a robust new strain for biotechnology, tolerating low light and temperature conditions. The strain can adapt to alternative nutrient (pig manure) and carbon (acetate) sources with the generated biomass having a high auxin concentration and plant biostimulating activity detected with the mungbean rooting bioassay.

Photosynthesis Monitoring in Microalgae Cultures Grown on Municipal Wastewater as a Nutrient Source in Large-Scale Outdoor Bioreactors.

Microalgae cultures were used for a WW treatment to remediate nutrients while producing biomass and recycling water. In these trials, raceway ponds (RWPs; 1 and 0.5 ha) were located next to a municipal (WW) treatment plant in Mérida, Spain. The ponds were used for continuous, all-year-round microalgae production using WW as a source of nutrients. Neither CO2 nor air was supplied to cultures. The objective was to validate photosynthesis monitoring techniques in large-scale bioreactors. Various in-situ/ex-situ methods based on chlorophyll fluorescence and oxygen evolution measurements were used to follow culture performance. Photosynthesis variables gathered with these techniques were compared to the physiological behavior and growth of cultures. Good photosynthetic activity was indicated by the build-up of dissolved oxygen concentration up to 380% saturation, high photochemical yield (Fv/Fm = 0.62-0.71), and relative electron transport rate rETR between 200 and 450 µmol e- m-2 s-1 at midday, which resulted in biomass productivity of about 15-25 g DW m-2 day-1. The variables represent reliable markers reflecting the physiological status of microalgae cultures. Using waste nutrients, the biomass production cost can be significantly decreased for abundant biomass production in large-scale bioreactors, which can be exploited for agricultural purposes.

Biologia Futura: potential of different forms of microalgae for soil improvement.

Products derived from microalgae have great potential in diverse field. As a part of the enhancing agriculture application, various forms of microalgae applications have been developed so far. They are known to influence soil properties. The various forms of application may enhance soil in more or less similar manner. They can help improve soil health, nitrogen, and phosphorus content, and even carbon sequestration. Thus, overall, it can enhance fertility of the soil.

Effect of storage on plant biostimulant and bioactive properties of freeze-dried Chlorella vulgaris biomass.

Microalgae are potential plant biostimulants and biocontrol agents. A major hurdle towards their commercialization is the production of large volumes of biomass at the correct time of year. Secondary metabolites are unstable and the "shelf-life" of bioactive microalgal biomass needs to be investigated. The aim of the study was to investigate the effects of storage conditions on freeze-dried microalgae to determine how long the biomass retained its growth promoting and bioactive properties under various temperature and light conditions. Chlorella vulgaris biomass was stored in the dark at - 70 °C, 10 °C, and 25 °C and in the light at 25 °C. Samples were tested every 3-4 months for 15 months. Storage time significantly influenced the rate of change in the bioactivity in the C. vulgaris biomass with storage temperature also having some effect. Rooting activity decreased in the mungbean rooting assay over time up to 12 months and then increased slightly. Antimicrobial activity increased against Staphylococcus aureus and Escherichia coli for up to 12 months and then declined. Antioxidant activity measured in the DPPH assay remained relatively stable for up to 12 months and then significantly decreased with longer storage. The change in bioactivity over time was attributed to the gradual breakdown of the rigid cell wall of C. vulgaris, thereby improving extraction efficiency but exposing the secondary metabolites to oxygen, thus quickening their degradation. Biomass produced for commercial purposes requires preliminary validation as the results of the present study showed that bioactive compounds are susceptible to degradation over time. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10811-021-02596-9.

Strain-Specific Biostimulant Effects of Chlorella and Chlamydomonas Green Microalgae on Medicago truncatula.

Microalgae have been identified to produce a plethora of bioactive compounds exerting growth stimulating effects on plants. The objective of this study was to investigate the plant-growth-promoting effects of three selected strains of eukaryotic green microalgae. The biostimulatory effects of two Chlorella species (MACC-360 and MACC-38) and a Chlamydomonas reinhardtii strain (cc124) were investigated in a Medicago truncatula model plant grown under controlled greenhouse conditions. The physiological responses of the M. truncatula A17 ecotype to algal biomass addition were characterized thoroughly. The plants were cultivated in pots containing a mixture of vermiculite and soil (1:3) layered with clay at the bottom. The application of live algae cells using the soil drench method significantly increased the plants' shoot length, leaf size, fresh weight, number of flowers and pigment content. For most of the parameters analyzed, the effects of treatment proved to be specific for the applied algae strains. Overall, Chlorella application led to more robust plants with increased fresh biomass, bigger leaves and more flowers/pods compared to the control and Chlamydomonas-treated samples receiving identical total nutrients.

Effect of cell disruption methods on the extraction of bioactive metabolites from microalgal biomass.

Microalgae synthesize a variety of potentially high-value compounds. Due to their robust cell wall, cell disruption is necessary to improve extraction of these compounds. While cell disruption methods have been optimized for lipid and protein extraction, there are limited studies for other bioactive compounds. The present study investigated the effect of freeze-drying combined with sonication or ball-milling on the extraction of antioxidant and plant biostimulating compounds from Chlorella sp., Chlorella vulgaris and Scenedesmus acutus. Both cell disruption methods resulted in higher extract yields from the biomass compared to freeze-dried biomass using 50% methanol as a solvent. Antioxidant activity of Chlorella extracts was generally higher than freeze-dried extracts based on the diphenylpicrylhydrazyl (DPPH) and ß-carotene linoleic acid assays. However, the effectiveness of each treatment varied between microalgae strains. Sonication resulted in the highest antioxidant activity in Chlorella sp. extracts. Ball-milling gave the best results for C. vulgaris extracts in the DPPH assay. Both cell disruption methods decreased antioxidant activity in S. acutus extracts. Plant biostimulating activity was tested using the mung bean rooting assay. Damaging the membrane by freeze-drying was sufficient to release the active compounds using water extracts. In contrast, both cell disruption methods negatively affected the biological activity of the extracts. These results indicate that bioactive compounds in microalgae are sensitive to post-harvest processes and their biological activity can be negatively affected by cell disruption methods. Care must be taken to not only optimize yield but to also preserve the biological activity of the target compounds.

Manipulation of nitrogen levels and mode of cultivation are viable methods to improve the lipid, fatty acids, phytochemical content, and bioactivities in Chlorella minutissima.

The effect of nitrogen (N: low = 2% N or moderate = 10% N) levels and cultivation (photoautotrophic or mixotrophic) modes on the biochemicals in Chlorella minutissima was evaluated using a mass culture system. Moderate N and mixotrophic cultures had higher biomass, protein, carbohydrate content and photosynthetic pigments than the low N and photoautotrophic treatments. In contrast, lipid and fatty acid content of the low N and photoautotrophic treatments were higher than in the moderate N and mixotrophic cultures. More phytochemicals were accumulated in moderate N and mixotrophic cultures which corresponded to better antioxidant capacity in the extracts. The most potent (0.7 mg · mL(-1) ) acetylcholinesterase inhibitory activity was displayed by moderate N and mixotrophic treatment. Approximately 60% of the extracts exhibited a noteworthy antimicrobial activity regardless of the N levels and cultivation modes. Thus, moderate N level enhanced the phytochemicals and biological activities of C. minutissima cultured under a mixotrophic system.

Auxin and cytokinin relationships in 24 microalgal strains(1).

Endogenous auxins and cytokinins were quantitated in 24 axenic microalgal strains from the Chlorophyceae, Trebouxiophyceae, Ulvophyceae, and Charophyceae. These strains were in an exponential growth phase, being harvested on day 4. Acutodesmus acuminatus Mosonmagyaróvár Algal Culture Collection-41 (MACC) produced the highest biomass and Chlorococcum ellipsoideum MACC-712 the lowest biomass. The auxins, indole-3-acetic acid (IAA) and indole-3-acetamide (IAM) were present in all microalgal strains. No other auxin conjugates were detected. IAA and IAM concentrations varied greatly, ranging from 0.50 to 71.49 nmol IAA · g(-1) DW and 0.18 to 99.83 nmol IAM · g(-1) DW, respectively. In 19 strains, IAA occurred in higher concentrations than IAM. Nineteen cytokinins were identified in the microalgal strains. Total cytokinin concentrations varied, ranging from 0.29 nmol · g(-1) DW in Klebsormidium flaccidum MACC-692 to 21.40 nmol · g(-1) DW in Stigeoclonium nanum MACC-790. The general trend was that cis-zeatin types were the predominant cytokinins; isopentenyladenine-type cytokinins were present in moderate concentrations, while low levels of trans-zeatin-type and very low levels of dihydrozeatin-type cytokinins were detected. Ribotides were generally the main cytokinin conjugate forms present with the cytokinin free bases and ribosides present in similar but moderate levels. The levels of O-glucosides were low. Only one N-glucoside was detected, being present in nine strains in very low concentrations. In 15 strains, the auxin content was 2- to 4-fold higher than the cytokinin content.

CHANGES IN ENDOGENOUS CYTOKININ CONCENTRATIONS IN CHLORELLA (CHLOROPHYCEAE) IN RELATION TO LIGHT AND THE CELL CYCLE(1).

Endogenous cytokinins were quantified in synchronized Chlorella minutissima Fott et Novákova (MACC 361) and Chlorella sp. (MACC 458) grown in a 14:10 light:dark (L:D) photoperiod. In 24 h experiments, cell division occurred during the dark period, and cells increased in size during the light period. Cytokinin profiles were similar in both strains, consisting of five cis-zeatin (cZ) and three N(6) -(2-isopentenyl)adenine (iP) derivatives. Cytokinin concentrations were low during the dark period and increased during the light period. In 48 h experiments using synchronized C. minutissima (MACC 361), half the cultures were maintained in continuous dark conditions for the second photoperiod. Cell division occurred during both dark periods, and cells increased in size during the light periods. Cultures kept in continuous dark did not increase in size following cell division. DNA analysis confirmed these results, with cultures grown in light having increased DNA concentrations prior to cell division, while cultures maintained in continuous dark had less DNA. Cytokinins (cZ and iP derivatives) were detected in all samples with concentrations increasing over the first 24 h. This increase was followed by a large increase, especially during the second light period where cytokinin concentrations increased 4-fold. Cytokinin concentrations did not increase in cultures maintained in continuous dark conditions. In vivo deuterium-labeling technology was used to measure cytokinin biosynthetic rates during the dark and light periods in C. minutissima with highest biosynthetic rates measured during the light period. These results show that there is a relationship between light, cell division, and cytokinins.