Sampling Frequency Is a Key Factor to the Ecient Actuation of the Phytohormones in the Increase of Biomass and Macromolecules Production by Spirulina (Arthorspira)

Application of microalgae in industries is limited by their low biomass production and low macromolecule content. Phytohormones are being studied to solve these problems, however is necessary to dene conditions that enhance the results for this kind of cultivation strategy. The increasing of the contact time between substances and cells can be a factor of the improvement in the phytohormone uptake by microalgae. We hypothesize that by decreasing the sampling frequency for monitoring culture parameters, we would also be interfering less in the uptake of phytohormones by microalgae. Therefore, the present study aimed to investigate the inuence of sampling frequency on the production of biomass and biomolecules of Spirulina sp. LEB 18 in cultivations supplemented with trans-zeatin and indole-3-acetic acid (IAA). Treatments with lower sampling frequency and supplementation of 1 mg L − 1 IAA enhanced biomass accumulation by 75 %, carbohydrate content by 50 %, and protein content by 30 % as compared to control experiment daily sampling. In addition, sampling every 10 days with 0,01 mg L − 1 IAA supplementation increased the lipid content by 51 % as compared with the experiment without phytohormone and daily sample removal. Therefore, we developed a new strategy to supplement phytohormones and improve microalgal production. This investigation presented a cultivation system ecient, which can be easy to implement in industries because of no need to change the operational aspects of the cultivation. Furthermore, the strategy will can be very useful to enhance the production capacity of microalgae on a large-scale.


Introduction
Despite the potential applications of microalgae, their use in industry is limited owing to the low production capacity to their low biomass and low macromolecule content. As a result, microalgal biotechnology has focused on improving production systems in terms of e ciency, cost, and biomass composition [1]. The modi cation of biochemical factors, such as supplementation of phytohormones, is considered to be a promising strategy to assist in this goal [2].
Phytohormones are signaling molecules that regulate the growth and development of plants and algae [3], but also endogenous phytohormones are found in microalgae [4], performing a functional role in their physiology [5]. The principal growth phytohormones are indole-3-acetic acid (IAA), which is an auxin, and trans-zeatin (tZ), which is a cytokinin. They are natural phytohormones [6,7], and different proportions of these two phytohormones determine the kind of organ that will form during plant development [8].
Although phytohormone supplementation is considered a promising strategy for improving the e ciency of microalgae cultivation, it is necessary to optimize the concentration of these substances to each kind of microalgae because small variations in concentration can result in great changes to the cells [9,10]. In the literature, there are many relates to phytohormone supplementation in the cultivation of eukaryotic microalgae genres as Chlorella [11][12][13][14][15][16] and Scenedesmus [15,17,18]. Nevertheless, there are fewer studies about the use of this strategy to increase the cyanobacteria biomass [19][20][21], and it was found just one with Spirulina [22]. Spirulina is the most known cyanobacteria and one of the most-studied microalgae in the world [1]. Thus, it is essential to improve the e ciency of its cultivation system. In this way, to de ne the adequate concentration of phytohormones to cultivate the prokaryotic microalgae can be a tool very useful.
In addition, a question yet no study is the sampling frequency of the cultures supplemented with phytohormone. It decreases in the sampling frequency could be a factor that improves the phytohormone uptake mechanism by microalga because the contact time between the substances and the cells become higher. This relation is similar to the occurred at Dictyosphaerium pulchellum, Chlorella sp., Micractinium pusillum, Scenedesmus armatus, and Scenedesmus acutus cultivations, when the retention time was increased from daily to ten days to improve the nitrogen uptake mechanism. More contact time allowed that nitrogen concentration incorporated into algal cells was enhanced, as well as the biomass concentration [23]. Therefore, decreasing the sampling frequency of microalgae cultivation with phytohormones could allow more contact between cells and phytohormones. In the same way, could promote an increase in the concentration of microalgal biomass.
In this study, we investigated the effect of natural phytohormones supplementation on the growth of Spirulina. In addition, to determine if contact time is important to improve the phytohormone uptake by microalgae, we conducted experiments trying not to interfere in this process. Thus, we propose a decrease in sampling frequency to monitoring the parameters of the cultivations. In this way, the present study aimed to investigate the in uence of sampling frequency on the production of biomass and biomolecules of Spirulina sp. LEB 18 in cultivations supplemented with tZ and IAA.

Materials And Methods
Microalgae and cultivation conditions Spirulina sp. LEB 18 was obtained from the Culture Collection of the Laboratory of Biochemical Engineering (LEB) at the Federal University of Rio Grande (FURG). This strain was isolated from Mangueira Lagoon (latitude 33°31′ 08″ S and longitude 53°22′ 05″ W) [24] and cultivated in Zarrouk medium [25]. The commercial phytohormones tZ and IAA (98 %; Sigma-Aldrich, Brazil) were supplemented in the cultures on day of inoculation from stock solutions. These were prepared by dissolving the phytohormones in 0.1 % (v/v) NaOH to obtain a nal concentration of 0.1 mg mL −1 .
All experiments were performed in two replicates batch per treatment. The microalga was cultivated in 0.5 L Erlenmeyer photobioreactors (0.4 L useful volume), with an initial concentration of 0.2 g L −1 (dry weight, DW); sterile distilled water was added daily to replenish evaporation. The assays were carried out in a culture chamber at 30 °C, with a 12 h light/dark photoperiod [26], illumination of approximately 70 μmol photons m −2 s −1 (promoted by uorescent lamps), and agitation by air injection.
Primarily, to verify the effects of the supplementation in the kinetic parameters of the microalgae cultivation, an experiment was carried out for 15 days with supplementation of different concentrations of tZ and IAA (0.01, 0.1, 1, and 10 mg L −1 ). Sampling was performed daily (2 mL) to monitor biomass accumulation and assess kinetic parameters.
In the next set of experiments, the goal was to evaluate if decrease the sampling frequency is a successful strategy to improve the phytohormones uptake e ciency. The experiments were divided into two groups, one was 2 mL sampled daily (GD group), and the other was 5 mL sampled every ten days (G10 group). The GD group was supplemented with selected concentrations of tZ and IAA (Table 1), based on the results obtained from the previous experiments. The G10 group was supplemented with different concentrations of tZ and IAA (Table 1). In addition, these experiments also were investigated the effect of the combined supplementation between tZ and IAA in the cultivations. The proportions of tZ and IAA (Table 1) were determined from the results of biomass production and kinetic parameter analysis and were performed at the same G10 group conditions. Both groups had the cultivations were carried out for 30 days. Each group had a control assay (duplicate) without the addition of phytohormones, but which followed the sampling frequency conditions. At the end of the experiments, the biomass was recovered from the liquid medium by centrifugation (Hitachi, Himac CR-GIII, Japan) at 9690 g at 20 °C for 20 min. The biomass was then washed with distilled water to remove salts, frozen at −80 °C, lyophilized for 48 h, and stored at −20 °C until characterization.

Evaluation of growth responses
To determine the effect of phytohormones supplementation and sampling frequency in the development of the microalgae the growth responses were monitored daily for GD group and once in 10 days for G10 group. Cell morphology was examined using an electronic optical microscope (AxioCanERc 5s Microscope camera, Zeizz, Germany). Biomass concentration was determined by measuring the optical density (n=6) at a wavelength of 670 nm using a spectrophotometer (Shimadzu UV/VIS UVmin-1240 spectrophotometer, Japan). For this purpose, a calibration curve of optical density versus dry weight biomass was constructed (R² = 0,993) [27].
For all cultivations, the maximum biomass concentration (X max , g L −1 ) was determined by measuring the optical density of the cultures. Other parameters including maximum speci c growth rate (μ max , d −1 ), maximum biomass productivity (P max , mg L −1 d −1 ), and generation time (t g , d) were calculated for the 15day cultures. Speci c growth rate was calculated by linear regression of the logarithmic growth phase obtained from the graph of ln X as a function of time (d). Maximum biomass productivity and generation time was determined using the Equations 1 and 2, where X t is the biomass concentration (mg L −1 ) at time t (d), X 0 is the biomass concentration (mg L −1 ) at time t 0 (d), and μ max (d −1 ) is the speci c growth rate in the exponential growth phase.

Quanti cation of macromolecules
The macromolecules were quanti ed in all biomass generated from the experiments conducted. The analyses were performed with lyophilized biomass stored properly. To each treatment were made four extractions replicates (n=4) and twelve analyses replicates (n=12) to the characterization analyses. The nal composition was showed by mean percentage (%) of fraction macromolecule (g) in the nal microalgal biomass (g) calculated to fresh weight (FW).
Lipid analysis was performed for the lyophilized biomass using the method of Marsh and Weinstein [28].
Lipids were extracted using the organic solvents chloroform and methanol (chloroform-to-methanol ratio, 1:2), colorimetrically quanti ed, and compared with the standard curve of tripalmitin.
For carbohydrate and protein analyses, extracts made of lyophilized biomass (5 mg) dissolved in distilled water (10 mL) was sonicated for 10 cycles (59 s on/off) using an ultrasonic probe (Cole Parmer, CPX 130, EUA) to break the cell wall and release intracellular material. Total carbohydrates were quanti ed using the phenol-sulfuric acid method [29] and compared to the standard curve of glucose. Protein content was quanti ed using the colorimetric method described by Lowry et al. [30]. The biomass was subjected to thermal and alkaline pretreatment to solubilize the proteins; and bovine serum albumin was used as the standard.

Statistical analysis
The results were compared using analysis of variance (ANOVA) followed by Fisher's test at a 95 % (p ≤ 0.05) con dence level.

Biomass concentration and kinetic parameters of 15-day cultures
The rst set of analyses investigated the effect of phytohormones supplementation on the cultivations of Spirulina sp. LEB 18 for 15 days. Supplementation of 0.01 mg L −1 tZ showed signi cantly better kinetic parameters as compared to other concentrations of this phytohormone (Table 2). Therefore, this concentration was de ned as the most promising to supplement tZ in the next set of experiments. In the IAA concentration selection, the supplementation of 0.1 mg L −1 was the condition with more potential to continue the assays. This supplementation showed results signi cantly higher than the control experiment for all parameters analyzed ( Table 2).
The motivation to realized experiments with longer duration emerged through monitoring of the biomass concentration of Spirulina sp. LEB 18 with tZ and IAA supplementation obtained during cultured for 15 days (Fig. 1). Observing the results of this determination was possible to infer that the microalga was still growing during the last few days of cultivation because the biomass concentration was yet increasing.

Cell morphology
Phytohormones regulated the morphological development of Spirulina sp. LEB 18. Cultures supplemented with tZ (Fig. 2a) and IAA (Fig. 2b) was 5 mm in length, while normal trichomes of Spirulina exhibit a maximum length of 1 mm [31]. On the other hand, the cultivations supplemented with combinations of tZ and IAA no enhancement in microalga size (Fig. 2c), and its morphology was similar to the control experiment (Fig. 2d).

Macromolecular content
The composition of Spirulina biomass is an important factor governing its commercial use. Therefore, we quanti ed proteins, carbohydrates, and lipids present in the nal biomass of Spirulina sp. LEB 18 supplemented with different concentrations of tZ and IAA using spectrophotometry (Table 3).
In general, IAA supplementation was more adequate than tZ to increase the content of proteins, lipids, and carbohydrates in the biomass. In G10 groups supplemented with 1 mg L −1 and 10 mg L −1 IAA, the protein and carbohydrate concentrations were signi cantly increased (p ≤ 0.05), reaching approximately 70 % and 20 % of microalgal biomass, respectively. The lipid content in the biomass reached about 25 % in GD group supplemented with 0.1 mg L −1 IAA and in G10 group supplemented with 0.01 mg L −1 IAA ( Table 3). The values obtained represent an increase of 30.2 % for protein, 60.1 % for carbohydrates, and 55.7 % for lipids as compared to the GD control group. When the phytohormones were supplemented together, the macromolecule content was lower than or similar to that of control group (Table 3).

In uence of sampling frequency on the production of biomass and biomolecules
The combination of phytohormone supplementation and low sampling frequency was very e cient in increasing the nal biomass concentration of the cultivations. The maximum biomass concentration in G10 group supplemented with 1 mg L −1 IAA was 75 % higher as compared to control experiment of GD group, which simulated the usual way of microalgae cultivation, without phytohormone supplementation and with daily sampling (Fig. 3). The nal biomass concentration was 3.6 g L −1 in G10 group supplemented with 1 mg L −1 IAA, and only 2.1 g L −1 in GD control group.
The sampling frequency alone was also a signi cant factor in increasing biomass concentration independent of phytohormone supplementation. This can be proved by X max of the G10 control group (2.9 g L −1 ), which was 43.3 % higher than that of GD control group (2.1 g L −1 ) (Fig. 3). In addition, phytohormone supplementation alone could also positively in uence biomass concentration independent of sample removal frequency. Final biomass concentration of GD group supplemented with 0.01 mg L −1 tZ (2.6 g L −1 ), was 24.6 % higher than that of GD control group (2.1 g L −1 ) (Fig. 3). On the other hand, the combined supplementation between phytohormones was not successful. The supplementation of 0.1 mg L −1 tZ and 0.1 mg L −1 IAA resulted in a maximum biomass concentration equal to that in G10 control group. The combination of 0.01 mg L −1 tZ with 1 mg L −1 IAA and 1 mg L −1 tZ with 0.01 mg L −1 IAA resulted in lower biomass concentration as compared to the G10 control group.
In addition, in the results of macromolecules content there was a statistical difference (p ≤ 0.05) between GD and G10 groups, even at same IAA concentrations, indicating that the sampling frequency in uenced in the biomass composition. The supplementation of 0.1 mg L −1 IAA in the G10 group was 76.4 % and 108,9 % to proteins and carbohydrates, respectively, higher than to the same phytohormone concentration the GD group (Table 3). However, a contrasting result was obtained for lipid content. GD group supplemented with 0.1 mg L −1 IAA exhibited high lipid content (24.6 %), while G10 group supplemented with the same amount of IAA exhibited low lipid content (11.9 %). Meanwhile, the lipid content of G10 group supplemented with 0.01 mg L −1 IAA (23.9 %) was similar to that of GD group supplemented with 0.1 mg L −1 IAA.

Discussion
Our study was successful in proving that the sampling frequency is a key point to phytohormones e ciency, originating a new strategy to increase the biomass and macromolecule production of microalgae cultivation. The higher maximum biomass concentration of the G10 group compared to GD group results can be explained by the high sample removal frequency in the GD group. We hypothesize that during sampling, it is possible that a portion of the phytohormone as well as replicating cells could be withdrawn along with the sample, affecting phytohormone availability for microalgae growth.
For macromolecule content analyses there were observed different results between GD and G10 groups, even at same phytohormone concentrations, indicating that the sampling frequency in uenced the results too. In our results, we demonstrated that the lipid production promoted by IAA follows a restricted phytohormone concentration range that can be added in cultivation. Supplementation of phytohormones above this concentration decreases the lipid content and increases the content of other macromolecules. This fact was observed from our approach of sampling frequency since, from it, we can let the phytohormone more or less available for the microalga consumption. Therefore, the sampling frequency study may be useful for auxiliary to enhance the content of biomass and any macromolecule in the microalgal biomass.
In addition, the results of macromolecular quanti cation imply that supplementation of phytohormones can be a versatile strategy to enhance the content of any macromolecule in the microalgal biomass. By simply modifying the concentration of phytohormone supplemented in the cultivation, it is possible to direct the metabolic ux towards the synthesis of one particular macromolecule. This nding corroborates the results of Yu et al. [10], who reported that carbon transport is facilitated for synthesis of either lipids or carbohydrates depending on the concentration of phytohormone supplemented during cultivation. Nevertheless, the carbon partitioning mechanisms in microalgae are still not understood clearly.
When the phytohormones IAA and tZ were supplemented together, our results were unsatisfactory. The results of biomass concentration, macromolecular content, and the cell morphology were lower than or similar to that of the control experiment. From this, there is evidence to support the hypothesis that was no signi cant change in auxin-cytokinin balance, because according to Su et al. [8]is this factor that causes effects in the cells. Therefore, it is necessary to conduct new studies utilizing different proportions of tZ and IAA than that used in our study to a rm that there is no synergic action between the two phytohormones.
However, it was con rmed that the IAA and tZ supplementation in uenced the Spirulina nal biomass concentration in a dose dependent manner. Our results were showed that when the concentration of phytohormone increased, nal biomass concentration also increased. Nevertheless, it looks like this trend occurs until a limited supplementation and if higher concentrations of phytohormones are added there is a reverse effect, i.e., decreased the maximum biomass concentration. This nding also reported by Saygideger and Okkay [22], who tested several concentrations of the synthetic auxin 2,4dichlorophenoxyacetic acid (2,4-D) in Spirulina platensis cultivations. The researchers noted that higher concentrations of 2,4-D inhibited the growth of the microalga. The toxic effect of these substances manifests when they are used at higher concentrations than those tolerated by the organism to which they are added [32].
There evidence to suggest that one of the causes of the increase of microalga biomass production from tZ and IAA supplementation is given by the enlargement of trichomes. We observed this phenomenon by microscopic monitoring and believed that cells appear enlarged as they are stretching considerably for dividing. This result is in agreement with the work of Park et al. [2], who evaluated the effects of IAA, gibberellic acid, kinetin, 1-triacontanol, and abscisic acid at concentrations between 0.1 to 10 ppm in Chlamydomonas reinhardtii strain CC124 cultivations. The authors found that treatment with all phytohormones resulted in enlargement of the cell diameter from less than 10 μm to 20 µm, and also observed increase in the biomass concentration.

Conclusion
Phytohormone supplementation combined with low sampling frequency proved to be an e cient strategy for increasing the biomass and macromolecule content for cultivating Spirulina sp. LEB 18.
Supplementation with 1 mg L −1 IAA increased the biomass concentration by 75 %, protein content by 30 %, and carbohydrate content by 50 % as compared to the usual method of cultivation. In addition, by changing the concentration of phytohormones supplemented, it was possible to increase the production of any particular macromolecule, which demonstrates the versatility of the strategy. Our study provides an initial basis for improving the microalgal biomass production on a larger scale utilizing an e cient and easy to implement a strategy.

Declarations Funding
This study was nanced in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior -Brasil (CAPES) -Finance Code 001, and also had the support of MCTI (Ministry of Science, Technology, and Innovation). This research was developed within the scope of the Capes-PrInt Program (Process # 88887.310848/2018-00).

Con icts of interest/Competing interests
The authors con rm that there are no con icts of interest/Competing interests.

Availability of data and material
Not applicable.   Table 2 Maximum biomass concentration (X max ), maximum biomass productivity (P max ), maximum speci c growth rate (μ max ), and generation time (t g ) for 15-day Spirulina sp. LEB 18 cultures supplemented with different concentrations of trans-zeatin (tZ) and 3-indoleacetic acid (IAA)