OR I G I N A L R E S E A R CH Microalgae from Nordic collections demonstrate biostimulant effect by enhancing plant growth and photosynthetic performance Erik Chovancˇek | Joa˜o Salazar | Sema Şirin | Yagut Allahverdiyeva Molecular Plant Biology, Department of Life Technologies, University of Turku, Turku, Finland Correspondence Yagut Allahverdiyeva, Molecular Plant Biology, University of Turku, Turku FI-20014, Finland. Email: allahve@utu.fi Funding information NordForsk Bioeconomy Program: NordAqua— Nordic Centre of Excellence consortium, Grant/Award Number: 82845 Edited by R.M. Rivero Abstract We investigated the biostimulant potential of six microalgal species from Nordic col- lections extracted with two different procedures: thermal hydrolysis with a weak solution of sulfuric acid accompanied by ultrasonication and bead-milling with aque- ous extraction followed by centrifugation. To this aim, we designed a phenotyping pipeline consisting of a root growth assay in the model plant Arabidopsis thaliana, complemented with greenhouse experiments to evaluate lettuce yield (Lactuca sativa L. cv. Finstar) and photosynthetic performance. The best-performing hydrolyzed extracts stimulated Arabidopsis root elongation by 8%–13% and lettuce yield by 12%–15%. The in situ measured photosynthetic performance of lettuce was upregu- lated in the efficient extracts: PSII quantum yield increased by 26%–34%, and thyla- koid proton flux increase was in the range of 34%–60%. In contrast, aqueous extracts acquired by bead-milling showed high dependence on biomass concentra- tion in the extract and an overall plant growth enhancement was not attained in any of the applied dosages. Our results indicate that hydrolysis of the biomass can be a decisive factor for rendering effective plant biostimulants from microalgae. 1 | INTRODUCTION The genetic diversity of microalgae is an unfathomed source of bio- active compounds for multivarious products, such as pharmaceuti- cals, nutraceuticals, biopesticides, and biostimulants (González-Pérez et al., 2021; Silva et al., 2022). Plant biostimulants facilitate nutrient uptake, improve crop performance and tolerance to abiotic stress, and therefore can have a crucial role in reducing dependency on synthetic fertilizers and plant protection products, and thus address agronomic sustainability challenges (Chiaiese et al., 2018). Though macroalgae have long been known for their beneficial influence on crop production, the interest in microalgae is on the rise due to recent technological development in microalgae cultivation, enabling nutrient-looped circular economies, and refineries rendering biomass with standard composition (Allahverdiyeva et al., 2021; Lacroux et al., 2023). Several microalgal and cyanobacterial species have been reported to stimulate germination, seedling growth, shoot, and root biomass of various crops (Bulgari et al., 2019; Gonçalves, 2021; Ronga et al., 2019). Microalgae with plant biostimulant effects have often been reported based on so called hormone-like effects that focus on hormonal evaluation (Garcia-Gonzalez & Sommerfeld, 2016; Navarro-Lopez et al., 2020). However, aside from hormones, many other components in microalgae with bioac- tive potential might remain unknown. Microalgal crude extracts (MCEs) can induce nutrient uptake and plant growth by triggering accumulation of metabolites in primary and secondary biosynthetic pathways, such as lipid and jasmonate synthesis (Mutale-Joan Received: 5 February 2023 Revised: 31 March 2023 Accepted: 6 April 2023 DOI: 10.1111/ppl.13911 Physiologia Plantarum This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2023 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society. Physiologia Plantarum. 2023;175:e13911. wileyonlinelibrary.com/journal/ppl 1 of 11 https://doi.org/10.1111/ppl.13911 et al., 2020). Microalgal polysaccharides have also improved the metabolite profile of azelaic acids, phytosterol, and other precursors in plant defense mechanisms (Rachidi et al., 2021). Although a wide range of bioactive compounds has been identi- fied in microalgae, including phytohormones, amino acids, poly- amines, glycosides, fatty acids, vitamins, terpenoids, and flavonoids (Mazepa et al., 2021; Mogor et al., 2018; Plaza et al., 2018; Puglisi et al., 2018; Refaay et al., 2021), the extract composition varies depending not only on microalgal strain, but also on cultivation method, harvesting time, and extraction technique (Aremu et al., 2015; Gitau et al., 2022; Mironiuk & Chojnacka, 2018). While extraction should be inexpensive and environmentally sustainable, bioactive compounds must be preserved (Kapoore et al., 2021). In this context, mechanical methods seem more favorable than chemi- cal or thermal methods. Among mechanical methods, bead milling is considered one of the most effective methods for cell disruption (Kapoore et al., 2018), and bead-milling has previously shown an effective increase in the antioxidant activity of Chlorella vulgaris and Scenedesmus acutus (Stirk et al., 2020). For the processing of macro- algal biomass, one of the most utilized extraction processes on the industrial level is chemical hydrolysis (El Boukhari et al., 2020). Microalgal extracts acquired by acid hydrolysis on a laboratory scale stimulated tomato shoot and root growth and increased nutrient efficiency uptake of the seedlings (Mutale-Joan et al., 2020). Though several studies have shown lettuce growth enhancement by microal- gal extracts (Kopta et al., 2018; Puglisi et al., 2020; Rouphael et al., 2017), the underlying physiological processes have not been studied thoroughly. In this study, we hypothesized that biomass of intensively grown microalgae could be utilized to produce efficient plant biostimulants on the condition that effective strains and extraction procedures are employed. We developed a cost-effective phenotyping pipeline, con- sisting of a rapid root growth assay and medium-term greenhouse experiments, that was used to compare two relatively inexpensive and scalable extraction methods—bead-milling and thermal hydrolysis. We also attempted to identify physiological processes responsible for observed effects by in situ evaluation of photosynthetic performance. Our research aims to further the understanding of microalgal plant growth-promoting activity and thus contribute to the development of effective plant biostimulants acquired from sustainably acquired microalgal biomass. 2 | MATERIALS AND METHODS 2.1 | Microalgae cultivation and harvesting Six microalgal species were acquired from the Norwegian Culture Col- lection of Algae (NORCCA) and the Microbial Domain Biological Resource Centre (HAMBI). The strains were used to inoculate 100 mL of BG11 medium (pH = 7.5) (Stanier et al., 1971), with or without the addition of seawater and vitamins if required by guidelines (see Table 1), and maintained at room temperature and continuous light (50 μmol m2 s1 photosynthetic photon flux density; PPFD). After 4 weeks, cultures were used for inoculation of glass cultivation bottles containing 4 L of BG11 (with or without the addition of seawater and vitamins) in duplicate batches. Each culture was cultivated in a growth chamber (Sanyo) with continuous light (130 μmol m2 s1 PPFD), at 25C and aerated with 1.5% CO2. Continuous mixing of CO2 with growth media was secured with magnetic stirrers. The culture growth was recorded spectrophotometrically at OD750 (Genesys 10S UV–vis, Thermo Scientific), each culture commencing with an OD750 = 0.1 ± 0.05. Cultures were harvested in the late exponential phase (Figure S1). Biomass was separated from the medium by centrifuga- tion (6000g, 15 min, 18C; Avanti JXN-26, Beckmann), freeze-dried overnight (Alpha 1-4LSC, Christ), and the dried biomass was stored at 20C. 2.2 | Preparation of microalgal crude extracts 2.2.1 | Aqueous crude extracts A sample of 67.5 mg of dried microalgal biomass was ground in a mor- tar and resuspended in 1.5 mL deionized water (Milli-Q; Merck). Cellu- lar disruption was performed at 4C with 0.5 mm ZrO beads filling the 2 mL Eppendorf tubes to one quarter (1 g) using a bead beater for 5 min at maximum speed (Bullet Blender Storm 24, Next Advance). Subsequently, the suspension was centrifuged (16,000 g, 5 min, 4C), and the supernatant was collected. The pellet was then again bead- milled with added MQ-water two more times to get 4.5 mL of con- centrated suspension (15 gDW L 1). The resulting aqueous crude extracts (ACEs) were stored at 20C and adjusted to the final con- centration with distilled water shortly before the experiments. TABLE 1 List of microalgae screened for plant-biostimulant activity. Microalga species Strain ID Growth medium Culture collection Chlorella sorokiniana NIVA-CHL176 BG11 NORCCA Coelastrum sp. K-0559 BG11 + vitaminsa NORCCA Porphyridum purpureum NIVA-1/92 50% BG11 in seawaterb NORCCA Scenedesmus sp. UHCC0027 BG11 HAMBI Tetradesmus obliquus NIVA-CHL107 BG11 NORCCA Tetraselmis subcordiformis NIVA-2/94 50% BG11 in seawaterb NORCCA ahttps://norcca.scrol.net/norcca-algal-culture-medium. bInstant Ocean, Aquarium Systems, France. 2 of 11 CHOVANCˇEK ET AL. Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 2.2.2 | Hydrolyzed crude extracts Thermal acid hydrolysis was performed according to Mutale-Joan et al. (2020) with modifications. In short, 0.75 g of freeze-dried microalgal biomass was ground in a mortar and hydrolyzed with 50 mL of 0.1 M H2SO4 (Scharlab, Spain) to have a biomass con- centration of 15 gDW L 1. The mixture was vortexed and heated for 2 h at 95C with constant stirring while interrupted every 30 min for 10 min bath sonication (Venus2; Polaris). After the hydrolysis, the suspension was cooled to room temperature and centrifuged (6000 g, 10 min, 4C). The supernatant was collected, and the pH was adjusted to 5.8 (KOH). The hydrolyzed crude extracts (HCEs) were stored at 20C and adjusted to experimen- tal concentration by adding distilled water. 2.3 | Arabidopsis rooting assay For the Arabidopsis rooting assay, we employed and modified the protocol described by Rayorath et al., 2008. Seeds of A. thaliana ecotype Columbia were sieved for size 280–300 μm to minimize heterogeneity, sterilized according to chlorine gas protocol (Lindsey et al., 2017), and transferred to square plates with auto- claved ½ MS media (Duchefa BV), 0.8% agar (Duchefa BV), 1% sucrose (Millipore) with pH adjusted to 5.8 (KOH). The seeds were placed in one line with ca., 3–4 mm apart and stratified in 4C dark for 48 h for similar germination timing. Then, plates were placed vertically in a growth chamber (Fitotron–Weiss) with ambient light (120 μmol m2 s1 PPFD, 16/8 h light–dark cycle) and constant temperature (22C). After 5 days, 15 seedlings with similar root lengths (17–20 mm) were carefully transferred in sterile conditions to another square plate with autoclaved ½ MS media, 5 mM MES (pH 5.8, Sigma-Aldrich), 0.8% agar. These plates were in the case of treatment groups supplemented with MCE in specific final doses (DW)—ACEs with 0.05, 0.1, 0.3, and 0.5 g L1, and HCEs with 0.1, 0.5, and 1 g L1. Seedlings were arranged in one line with 7 mm spaces, and the root tip position was marked with a fine tip permanent marker. Plates with the seedlings were placed vertically in the growth chamber with the previous settings. In one experimental batch, plates supplemen- ted with extracts in different concentrations were tested. Batches were repeated for each microalga and concentration to validate the reproducibility of results (n = 2  15). RGB images were taken on the 3rd, 5th, and 7th day after transfer with a Canon EOS 250 camera. Roots were analyzed with the ImageJ software (Schindelin et al., 2012). Primary root elongation was measured from the position on day 0, and lateral roots (length >2 mm) were counted on day 5 from the transfer. Statis- tical significance was evaluated within each day with the Kruskal–Wallis test (p < 0.05) followed by Dunn's multiple com- parison tests adjusted according to Benjamini et al. (2006) for false discovery rate (FDR) control. 2.4 | Greenhouse lettuce experiments To investigate the effects of MCE on yield and photosynthetic perfor- mance of a crop, we conducted experiments with lettuce in a semi- controlled glass greenhouse environment (Ruissalo Botanical Garden of the University of Turku, Finland) from May to October 2022. The environmental settings during the experiments were: 15–18C, natu- ral light supplemented with halogen light (300 μmol m2 s1 PPFD) in a 16/8 h diurnal cycle, 60% rel. humidity. Seeds of a crisp-type let- tuce variety, Lactuca sativa L. cv. Finstar (Helle Oy) were sown into 15-cell seedling trays with moist sphagnum peat substrate (VHM620 pH 6.0 R8060; Kekkilä). The presence of pests was prevented by applying the Steinernema sp. nematodes (Helle Oy) at the beginning of experiments. MCE-treated and control plants (untreated) were arranged in a completely randomized block design (n = 15) with at least one control plant in each block. MCEs were applied by soil drenching on the 14th, 21st, and 28th day after germination (DAG) in specific doses—ACE with 0.1, 0.3, 0.5, and 1 g L1, and HCE with 0.5 and 1 g L1. Trays were irrigated abundantly, and individual plants were supplemented with equal amounts of nutrient solution (Ferticare 7-9-32; Yara) on the 18th, 25th, and 32nd DAG. Plants were har- vested on the 35th DAG, and the fresh weight (FW) of the shoot parts was determined immediately. Statistically significant differences between the mean values were determined using one-way ANOVA fol- lowed by Fisher's least significant difference (LSD) post hoc test with a significance level of p < 0.05. 2.4.1 | Lettuce photosynthetic performance Spectroscopic measurements of photosynthetic parameters were per- formed with MultispeQ V2.0 (PhotosynQ LLC) using Photosynthesis RIDES 2.0 protocol based on pulse-amplitude modulation (PAM) fluo- rometry. The measurements were taken on the 32nd DAG with seven replicates per treatment. Notable parameters, that is, the relative chlo- rophyll content (SPAD), the quantum yield of PSII (ΦII), the thylakoid membrane proton conductivity (gH+), and proton flux (vH+), were determined under light acclimation (Kononchuk et al., 2022; Kuhlgert et al., 2016). Treatments were considered significantly different from control if they had a p < 0.05, based on one-way ANOVA followed by Fisher's LSD test, or for non-parametric testing—based on Kruskal– Wallis rank test followed by Dunn's comparison adjusted for FDR control according to Benjamini et al., 2006. Normality of residuals was tested using the Shapiro–Wilk test and the homogeneity of variances using the Brown–Forsythe test. 3 | RESULTS The workflow of the experiments is presented in Figure 1. Six microal- gal species acquired from NORCCA and HAMBI algal collections (Table 1) were cultivated in a laboratory setup until the late CHOVANCˇEK ET AL. 3 of 11 Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License exponential phase (Figure S1). The resulting biomass was freeze-dried and then either bead-milled with MQ-water or hydrolyzed with a weak solution of sulfuric acid to acquire crude extracts. Our experi- mental design of screening the microalgae for plant growth stimulat- ing effects consisted of two types of bioassays: a rapid rooting assay conducted on ½ MS agar media supplemented with MCE and green- house experiments for crop yield assessment. 3.1 | Hydrolyzed crude extracts from microalgae enhance Arabidopsis root growth A. thaliana is a small terrestrial plant with a short vegetative cycle and, therefore, suitable for rapid and large-scale screenings (Pavicic et al., 2017). Most pronounced stimulation effects on the Arabidopsis root elongation were observed on day 7 from transfer (mean ± SE and p-values for all treatment groups and each analyzed day are listed in Tables S2 and S3). Primary root growth was stimulated with several HCEs, especially at the 0.5 g L1 dose (Figure 2A). Scenedes- mus sp. HCE stimulated root elongation in all applied concentrations (0.5 g L1: p < 0.0001, 0.1 g L1: p = 4.9E  03, 1 g L1: p = 6.2E  03). A significant increase was also attained with a 0.5 g L1 dose HCE of P. purpureum and Coelastrum sp. (p < 0.0001). Interestingly, C. sorokiniana HCE increased primary root elongation at a lower dose (0.1 g L1; p = 2.8E  02) but showed slight inhibi- tion at 1 g L1 (p = 5.79E  02). T. subcordiformis HCE (0.5 g L1) enhanced root elongation on day 5 (p = 1.18E  02), but the stimu- lation effect faded on day 7 (p = 2.62E  01). The highest stimula- tion of the root elongation (13.35%) was observed with the P. purpureum HCE, 0.5 g L1. Effects of T. obliquus HCE were weak, and a 1 g L1 dose inhibited Arabidopsis root elongation (p = 1.33E  02). Though lateral root establishment is promoted by nutrient heterogeneity in the growth media, lateral roots are impor- tant for increasing the surface area of a healthy root system (Banda et al., 2019). Nevertheless, among hydrolyzed extracts, only C. sorokiniana (0.1 g L1) and Coelastrum sp. (0.5 g L1) demonstrated significant stimulation of lateral root establishment (Figure 2B; p = 1.19E  02 and p = 7.74E  03, respectively). Altogether, Coe- lastrum sp. HCE enhanced Arabidopsis root growth in horizontal and vertical directions. 3.2 | Effects of aqueous crude extracts are highly concentration-dependent ACEs of Chlorella sp. and Scenedesmus sp. microalga were previously reported for high antioxidant activity and enhancement of root forma- tion (Stirk et al., 2020). The rooting assay allowed us to determine F IGURE 1 The workflow of screening microalgae for plant biostimulant effects. (A) Six microalgae were cultivated in bottles with growth media, continuous light, and 1.5% CO2, harvested by centrifugation, and freeze-dried. (B) Microalgal crude extracts were acquired by either thermal acid hydrolysis with weak sulfuric acid (0.1 M) complemented with ultrasonication (hydrolyzed crude extracts) or by bead- milling followed by centrifugation (aqueous crude extracts). In both cases, the supernatant was collected and stored. (C) The phenotypic characterization for discerning growth-enhancing effects of the diluted microalgal crude extracts was performed via two plant bioassays: Arabidopsis rooting assay conducted on agar media and greenhouse experiments for evaluating relative chlorophyll content, photosynthetic performance, and crop yield of lettuce. The image was created with BioRender.com. 4 of 11 CHOVANCˇEK ET AL. Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License in vivo responses to similar extracts. A dose of 0.05 g L1 ACE of C. sorokiniana stimulated both primary root growth (p = 3.82E  02) and lateral root establishment (p = 5.2E  06; Figure 3A,B), while a higher concentration inhibited root elongation. Although similar trends emerged, none of the other extracts stimulated primary root formation. Lateral roots were further enhanced with a 0.05 g L1 and 0.1 g L1 dose of P. purpureum (p = 6.83E  05 and p = 3.01E  05, respectively) and a 0.05 g L1 dose of T. subcordiformis (p = 1.06E  04) ACE. In comparing the effects on the root phenotype of ACE (Figure 3C), C. sorokiniana, P. purpureum, and T. subcordiformis ACE at a dose of 0.05 g L1 promoted a healthy, branched root system. In contrast, 0.5 g L1 ACE of Coelastrum sp. and T. obliquus caused short- ening of the root complemented with distortion from normal growth similar to an overdose of ethylene or ABA (De Smet et al., 2003; Negi et al., 2008). 3.3 | Hydrolyzed microalgal crude extracts increase lettuce crop yield To assess the stimulating potential of the MCEs on crop yield and green biomass of plants, we applied the hydrolyzed and aqueous extracts to the soil with lettuce seedlings (L. sativa L. cv. Finstar) three times throughout a shortened vegetative cycle (35 days). The FWs were determined at harvest to assess crop yield perfor- mance. The results indicate that lettuces treated with ACE showed negligible differences from control plants or even growth inhibition (Figure 4A)—the ACE of Coelastrum sp., P. purpureum, and T. subcordiformis (all with 1 g L1 dose) displayed a decrease of green biomass FW (18.16%, 11.64%, and 13.20%, respec- tively). However, the yield significantly increased after applica- tion of the following HCE-dose (Figure 4B): T. obliquus: 1 g L1 (15.97%), Coelastrum: 0.5 and 1 g L1 (14.22% and 13.19%, F IGURE 2 Several hydrolyzed microalgal crude extracts significantly increase root elongation and lateral root establishment in Arabidopsis. Five-days old A. thaliana seedlings were transferred to ½ MS agar media (control) supplemented with crude extracts acquired from six screened microalgae in different concentrations (0.1 g L1, 0.5 g L1, and 1 g L1). (A) Primary root elongation analyzed by ImageJ on days 3, 5, and 7 after transfer. (B) Lateral roots counted on day 5 from transfer. Different letters above bars indicate significant differences within 1 day according to Dunn's test (FDR adjusted; p < 0.05). Bars represent mean ± SE (n = 28–30). CHOVANCˇEK ET AL. 5 of 11 Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License respectively), T. subcordiformis: 0.5 g L1 (13.34%), P. purpureum: 1 g L1 (12.78%), and Scenedesmus sp.: 0.5 g L1 was at the threshold of detectability (p = 0.05; 12.52%). Surprisingly, although a low dose of C. sorokiniana HCE stimulated Arabidopsis root growth, neither of the two applied doses enhanced lettuce yield significantly. F IGURE 3 Legend on next page. 6 of 11 CHOVANCˇEK ET AL. Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 3.4 | Effective microalgal crude extracts upregulated photosynthesis in lettuce Photosynthetic performance can delineate the general fitness, pro- ductivity, and the state of the underlying physiological processes (Kromdijk et al., 2016). On day 32 (DAG), we conducted spectropho- tometric measurements of photosynthetic parameters on lettuce leaves. The results of the photosynthetic measurements on lettuces treated with HCE indicate no significant variation in the relative chlo- rophyll content (Figure 5A). However, several parameters assessing the photosynthetic performance were increased after the HCE treat- ments. PSII quantum yield measured on light-acclimated leaves was significantly upregulated after six treatments with relative increases ranging from 25.96% with P. purpureum (0.5 g L1) to 33.90% with Scenedesmus sp. (1 g L1; Figure 5B). The thylakoid proton conductiv- ity in the lettuce leaves significantly increased after the HCE treat- ment of two microalga, C. sorokiniana (p = 3.34E  03; 1 g L1) and P. purpureum (p = 7.34E  03; 0.5 g L1; Figure 5C). Finally, the let- tuce thylakoid proton flux has increased after treatment with most of the hydrolyzed microalgal extracts (Figure 5D)—ranging from 33.99% with up to 59.87% (C. sorokiniana—0.5 g L1 and 1 g L1, respectively). 4 | DISCUSSION New effective plant biostimulants ought to be acquired from natural resources following a bottom-up approach where biological screen- ing represents the first step in the process (Povero, 2020). Despite abundant research on microalgal plant biostimulants, extensive screenings of the diverse microalga collections are largely under- scored. The main reason is probably a lack of cheap and relatively fast available methods. Therefore, many current screenings rely on assays for assessment of the hormonal activity of microalgal extracts (Masojídek et al., 2022; Rupawalla et al., 2022). However, this dimin- ishes the possibility of finding other bioactive compounds with plant-enhancing effects. In this work, we developed a rapid and affordable phenotyping pipeline for evaluating plant biostimulant effects of microalgae. Microalgae can be a feasible resource for pro- ducing high-value products if suitable strains, cultivation, and extrac- tion methods are utilized (Carnovale et al., 2021; Kapoore et al., 2021). For industrial potential, advantageous microalgae should meet the minimum requirement of scalability (Allahverdiyeva et al., 2021; Barone et al., 2019). Therefore, species with robust bio- mass growth and suitable for intensive cultivation were selected for our experiments. For screening purposes, we designed the cultiva- tion set-up to fit the laboratory scale and mimic intensive culturing. For further biomass processing, we followed a stepwise approach with freeze-drying in the first step and bead-milling or thermal acid hydrolysis in the second step. Bead-milling was followed by centrifu- gation, and hydrolysis was further accompanied by heating and ultra-sonication. Altogether, we tested 42 extracts from six microal- gal strains acquired with two extraction methods and applied in F IGURE 3 Aqueous microalgal crude extracts can inhibit Arabidopsis root growth. Five-days old A. thaliana seedlings were transferred to ½ MS agar media (control) supplemented with crude extracts acquired from six screened microalgae in four different concentrations (0.05 g L1, 0.1 g L1, 0.3 g L1, and 0.5 g L1). (A) Primary root elongation analyzed by ImageJ on days 3, 5, and 7 after transfer. (B) Lateral roots counted on day 5 from transfer. Different letters above bars indicate significant differences within 1 day according to Dunn's test (FDR adjusted; p < 0.05). Bars represent mean ± SE (n = 28–30). (C) Representative images of A. thaliana seedlings grown on agar media supplemented with aqueous crude extracts acquired from microalgae (gDW L 1). Scale bar = 20 mm. F IGURE 4 Best-performing microalgal crude extracts increase greenhouse-grown lettuce fresh weight. Lettuces treated with (A) aqueous crude extracts in 0.1 g L1, 0.3 g L1, 0.5 g L1, and 1 g L1 dose; (B) hydrolyzed crude extracts in 0.5 g L1 and 1 g L1 dose; control = untreated plants. Extracts acquired from six microalgal species were applied three times throughout the vegetative cycle by soil drenching in different concentrations. Green biomass was harvested and weighed 35 days after germination. Bars represent mean ± SE (n = 15) and asterisks indicate significant differences to the control plants as determined by protected Fisher's LSD test (ANOVA; p < 0.05). CHOVANCˇEK ET AL. 7 of 11 Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License different doses to identify optimal plant-enhancing effects by evalu- ating Arabidopsis rooting and lettuce crop performance. The processes responsible for root elongation in A. thaliana are viewed as a result of an intricate interplay of environmental conditions and plant innate regulation mechanisms (Band et al., 2012). Microalgae biomass contains significant amounts of plant hormones, such as auxins, cytokinins, gibberellins, abscisic acid (ABA), ethylene, and so on (Gonçalves, 2021; Kapoore et al., 2021). ACEs acquired by bead-milling have often displayed hormonal activity assuming stimula- tion of tissue-specific processes, such as germination, cotyledon expansion, and rooting of excised shoots (Ferreira et al., 2021; Navarro-Lopez et al., 2020; Stirk et al., 2020). Our results of the root- ing assays indicate that 7 days of intensive ACE exposure can inhibit root growth (Figure 3). Only C. sorokinana ACE in 0.05 g L1 dose sig- nificantly increased root elongation and lateral root development in Arabidopsis. High concentrations of ACE inhibited root growth and, in some cases, resulted in an altered root morphology (Coelastrum sp.; Figure 3C). The root development of Arabidopsis seedlings can be sus- ceptible to increased concentrations of plant hormones, especially ABA or ethylene (De Smet et al., 2003; Negi et al., 2008). Interest- ingly, decreasing the concentration of ACE to 0.05 g L1 did not have the expected root-stimulation outcome for most of the ACE. Also, application of ACEs did not increase the FW of lettuce in any of the applied concentrations (0.1–1 g L1; Figure 4A). Furthermore, treat- ment with Coelastrum sp., P. purpureum, and T. subcordiformis ACE resulted in a decrease in lettuce yield when applied at the highest con- centration. Some studies point out the importance of microalgal circa- dian rhythms and harvesting time in relation to balanced hormonal composition of the extracts (Stirk et al., 2011; Xu et al., 2016), which was not considered in this study due to the focus on intensive cultivation. Contrastingly, HCEs stimulated root elongation in four of the six HCE (Figure 2A). C. sorokiniana HCE enhanced both primary and lat- eral root development, while P. purpureum HCE rendered a distinctive root phenotype with increased primary root growth (Figure 2A) but slightly decreased lateral root development (Figure 2B). Coelastrum sp. and Scenedesmus sp. enhanced both observed aspects resulting in a healthy root system. The optimal concentration was 0.5 g L1 for Coelastrum sp., P. purpureum, Scenedesmus sp., and 0.1 g L1 for C. sorokiniana. HCE of C. sorokiniana displayed previously significant stimulation of tomato root and shoot growth, as well as metabolite stimulation (Mutale-Joan et al., 2020). Even though we observed a stimulation in lettuce photosynthetic performance, our analysis of green biomass did not show a significant increase with C. sorokiniana. However, all other HCE in this study improved lettuce crop yield by F IGURE 5 Hydrolyzed crude extracts acquired from six microalgae improved photosynthetic performance of lettuce leaves. Photosynthetic parameters were measured spectrophotometrically on the 32nd day after germination by MultispeQ device. (A) Relative chlorophyll content (SPAD value), (B) photosystem II quantum yield, (C) thylakoid proton conductivity, and (D) thylakoid proton flux. Control was acquired from untreated plants. Bars represent mean ± SE (n = 5–7) and asterisks indicate significant differences (p < 0.05) according to one-way ANOVA followed by Fisher's LSD test (B,D) or Kruskal–Wallis test followed by Dunn's multiple comparison test (FDR adjusted; A,C). 8 of 11 CHOVANCˇEK ET AL. Physiologia Plantarum 13993054, 2023, 2, D ow nloaded from https://onlinelibrary.w iley.com /doi/10.1111/ppl.13911 by U niversity of Turku, W iley O nline Library on [24/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on W iley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 12%–16% (Figure 4B). Interestingly, T. obliquus HCE stimulated let- tuce crop performance but failed to stimulate Arabidopsis root growth, indicating that extract effectivity can be plant- or tissue-specific. Regarding the photosynthetic performance of lettuce, a global enhancement was attained in the thylakoid proton flux parameter (Figure 5D), which can be attributed significantly to the ATP synthase activity. Congruently, in tomato plants treated with HCE, Mutale-Joan et al. (2020) observed an increase in pyridine-3-carboxamide, the pri- mary precursor of adenine dinucleotide (NAD+) and the substrate for ADP polymerization. Sulfuric acid, which was utilized in preparing our HCE, is in low concentration and is known to effectively solubilize and hydrolyze the microalgal biomass (Silva & Bertucco, 2017). The polysaccharide frac- tion of thermally hydrolyzed microalgal extracts has previously improved plant profiles of primary and secondary metabolites (Rachidi et al., 2020). Sugars and amino acids are common products of acid hydrolysis of organic biomass (Bin Hossain et al., 2015; Darragh & Moughan, 2005). Though plants are essentially primary producers, the uptake of amino acids and saccharides by roots is well-known in Ara- bidopsis (Tegeder & Rentsch, 2010; Yamada et al., 2011). Amino acids can be utilized in protein build-up or in regulatory pathways, while saccharides can contribute to metabolism bidirectionally. Still, regula- tory pathways, effective metabolites, and molecular mechanisms par- taking in the described effects of microalgal extracts are elusive. More fundamental knowledge about the mode of action of microalgal plant biostimulants is necessary to understand the underlying processes, synergies, and metabolic implications that can, in the end, help develop reliable formulations with described effects. 5 | CONCLUSIONS Here, we described a cost-effective phenotyping pipeline for evaluat- ing plant biostimulant potential of microalgal crude extracts that con- sists of Arabidopsis rooting assay and lettuce greenhouse experiments. Six screened microalgae showed potential to be utilized as future plant biostimulants on the condition of an effective biomass extrac- tion procedure. Hydrolyzed crude extracts enhanced Arabidopsis root development and demonstrated a superior potential for lettuce crop improvement with five of the six evaluated microalgae. Lettuce fresh weight increase was concomitant with increased photosynthetic per- formance. More fundamental research of microalgal plant biostimulant effects is essential to understand specific in planta modes of action and explain the synergistic effects of microalgal bioactive compounds. AUTHOR CONTRIBUTIONS Yagut Allahverdiyeva conceived the research and acquired funding. Erik Chovancˇek, Sema Şirin, Yagut Allahverdiyeva conceptualized and designed research. Erik Chovancˇek performed experiments, curated data, and drafted the manuscript. Joa˜o Salazar aided with experi- ments. All authors contributed to the discussion and revision of the manuscript draft. All authors read and approved of the published ver- sion of the manuscript. ACKNOWLEDGMENTS The authors would like to thank the Norwegian Culture Collection of Algae (NORCCA) and Microbial Domain Biological Resource Centre in Helsinki (HAMBI) for supplying microalgal specimens for the screen- ing. The authors would also like to thank the technical personnel of the Molecular Plant Biology Department and the Ruissalo Botanical Garden of the University of Turku. FUNDING INFORMATION NordForsk Bioeconomy Program: NordAqua—Nordic Centre of Excel- lence consortium, Grant/Award Number: 82845. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Erik Chovancˇek https://orcid.org/0000-0003-4575-8395 Joa˜o Salazar https://orcid.org/0000-0002-3666-8841 Sema Şirin https://orcid.org/0000-0001-8332-6695 Yagut Allahverdiyeva https://orcid.org/0000-0002-9262-1757 REFERENCES Allahverdiyeva, Y., Aro, E.M., van Bavel, B., Escudero, C., Funk, C., Heinonen, J. et al. (2021) NordAqua, a Nordic Center of Excellence to develop an algae-based photosynthetic production platform. Physiolo- gia Plantarum, 173(2), 507–513. 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