Impact of 50 Hz Electromagnetic Field on the Growth of Chlorella vulgaris

Abstract

The paper presents the experimental study of the influence of a 50 Hz extremely-low-frequency (ELF) electromagnetic field (EMF) on the growth of microalgae Chlorella vulgaris in a BG11 culture medium. Comparative experimental determinations carried out under reference conditions (microalgae growth without exposure to EMF) and with exposure to a homogeneous 50 Hz EMF of various intensities highlighted the fact that EMF has a major impact on both the growth speed and the nitrogen and phosphorus content of the obtained algal mass. Through spectrophotometry and gravimetric determinations, it was found that the lag time was reduced from approximately 8 h (reference) to approximately 6 h for EMF of 2 V/m, 4.5 h for EMF of 5 V/m, 3.2 h for EMF of 10 V/m, and 2.5 h for EMF of 15 V/m. In the stimulation with 15 V/m EMF, the maximum biomass growth rate was 2.75 times higher than the reference, leading to a 2-fold increase in the rate of exhaustion of nutrients, especially phosphorus, in the culture medium. The specific chemical analyses for N-NO₃, total nitrogen TN_b, and total phosphorus Pt highlighted that the N-NO₃ content of the culture medium decreased by 58 mg/L/day at 15 V/m EMF compared to 43 mg/L for the reference. The Pt content decreased to 90% depletion after approximately 80 h for the reference culture medium, versus only 48 h of growth with exposure to 15 V/m ELF. The TN_b content of the algal suspension in BG11 under the influence of 15 V/m EMF for 96 h of growth increased 14 times compared to the reference. This shows that nitrogen metabolization in the dispersed air was significantly stimulated. It was also found that the 50 Hz EMF also influences the nitrogen and phosphorus content of the increased algal mass. The results show the potential of EMF stimulation of Chlorella vulgaris growth, leading to an increased efficiency of algae growth reactors.

1. Introduction

Algae are phototrophic microorganisms that, through various carbon fixation pathways, have the ability to convert solar energy into chemical energy through photosynthesis, and to synthesize organic material from CO₂ and water through enzymatically catalyzed photobiochemical processes. They are widespread in nature and make a major contribution to the retention of carbon from atmospheric CO₂ and the release of oxygen in the biosphere [1,2,3].

Due to their high protein, lipid, and carbohydrate contents, microalgae—especially Chlorella, Spirulina, and Dunaliella—are used as a food source. Microalgae are natural raw materials in the pharmaceutical industry [4] where they are widely used to obtain food supplements with beneficial effects on digestion (Chlorella stimulates the growth of intestinal Lactobacillus). Spirulina sp. and Dunaliella sp., due to their high content in carotenoids, have demonstrated anticancer effects [5,6,7]. Different studies have explored the proper conditions for efficient algal biomass growth [8,9,10], with wastewater proving to be a suitable environment. On the other hand, through their oil content, oil-rich algae represent a renewable energy source. Thus, the third-generation biofuels are based on algal biomass production [11]. In this context, the demand for algal biomass—a product with increased added value—on the world market shows an upward trend [7,11].

The photobiochemical processes are relatively slow, which makes the productivity of bioreactors, including microalgae growth photobioreactors, relatively low, which has a substantial influence on production costs [2,12]. In this context, it is considered that research aimed at stimulating the processes of metabolism and growth of microalgae represents a way to reduce production costs, thus presenting economic importance.

Numerous studies have highlighted the fact that electromagnetic fields (EMFs) can influence the mechanism and kinetics of cellular biochemical processes of microorganisms [13,14,15,16,17,18]. The exhaustive investigations of Ryan W. Hunt et al. [19] and, more recently, Beretta, G et al. [20] show that by exposing biomass to an electromagnetic field, under certain specific conditions (waveform, frequency, applied field intensity), certain biochemical processes can be significantly modified. The excellently documented study of Ruslan M. Sarimov et al. [21] regarding the influences of magnetic fields of natural origin (magnetic storms) and/or anthropogenic causes (electromagnetic pollution of the environment [22,23,24] with an extremely-low-frequency EMF in the 0–1 kHz range) demonstrated complex biological effects.

The changes produced by ELF-EMF exposure in the mechanism and kinetics of biochemical processes are correlated with dielectric relaxation in biological systems. Thus, they are selectively produced at a proper frequency given by the relaxation frequency of certain chemical species (polarized molecules—with dipole moment) present in the system [21]. Dielectric spectroscopy is a useful tool for studying the behavior of biological systems in a sinusoidal electric field [24,25,26,27]. The rapid method of determining the proper frequency at which changes occur in the behavior of biochemical systems is based on the technique of dielectric spectroscopy [28]. Thus, by studying the dielectric behavior of the active sludge suspension from wastewater treatment plants, proper frequencies of 49.9, 99.9, 130.8, and 150.4 Hz were found [17,27]. Through specific chemical determinations, it was found that the rate of metabolism of wastewater pollutants increases significantly (doubles) following the exposure of the active sludge suspension to a 5 V/m electric field of 50 Hz [17]. Based on these results, a pilot installation was developed for the stimulation by exposure to ELF-EMF of microbiological activity in the biological treatment of wastewater. The comparative experimentation of the pilot plant highlighted the fact that, by stimulating the microbial activity with ELF-EMF, the specific electricity consumption of the wastewater biological treatment stage is reduced by approximately 50%; this represents an electricity saving of approximately 33% from the wastewater treatment plant’s total energy consumption [29]. Similar results of ELF-EMF stimulation of microbial cultures have been reported with applications to hydrocarbon pollution remediation [30], as well as in various biotechnologies for the production of biofuel and bioenergy [19].

It was experimentally found that the exposure to 100 Hz EMF of up to 250 V/m stimulates the ATP synthesis in Escherichia coli [31]. On the other hand, excessive EMF of 3000 V/m inhibits lactose uptake in the case of Kluyveromyces marxianus [32]. Regarding microalgae, it was shown that under the influence of a periodic direct current, the algal biomass growth of Scenedesmus obliquus and its lipid yield are significantly improved [33].

Given these considerations, a fully automated device for increasing phototrophic microalgae production by exposing the culture medium to ELF-EMF was recently designed and made [34]. In this context, the aim of the work is to study the behavior of the microalgae Chlorella vulgaris in the presence of ELF-EMF of 50 Hz. The selection of the microalgae strain for the experimental study was based on several advantageous characteristics, such as metabolic robustness, simple life cycle, rapid reproductive capacity, and industrial relevance within its genus [35,36].

2. Materials and Methods

In order to evaluate the influence of 50 Hz EMF on the growth of Chlorella vulgaris, comparative determinations were made on samples collected from two identical photobioreactors operating with identical parameters (illumination level, temperature, flow rate of air/CO₂ mixture). A reference photobioreactor without exposing the culture medium to EMF was compared to another photobioreactor provided with polarizing electrodes for exposing the culture medium to 50 Hz EMF at various intensities. The experimental setup illustrated in Figure 1 was described in detail in [34]. The equipment diagram is shown in Figure 2.

The culture medium used was BG11 with the following composition: 1.5 g/L NaNO₃; 0.08 g/L K₂HPO₄; 0.075 g/L MgSO₄ × 7H₂O; 0.036 g/L CaCl₂ × 2H₂O; 0.006 g/L citric acid; 0.006 g/L Ferric ammonium citrate green 15% Fe; 0.001 g/L Na₂EDTA; 0.002 g/L Na₂CO₃; and compounds for providing microelements: B from 0.00072 g/L H₃BO₃, Mn from 0.00045 g/L MnCl₂ × 4H₂O, Zn from 0.00045 g/lLZnSO₄ × 7H₂O, Mo from 0.000098 g/L Na₂MoO₄ × 2 H₂O, Cu from 0.0002 g/L CuSO₄ × 5H₂O, and Co from 0.000013 g/L Co(NO₃)₂ × 6H₂O).

The inoculum used to inoculate the culture medium from the two photobioreactors in Figure 1 was Chlorella vulgaris Bienjerinck CCALA 269 obtained from the Culture Collection of Autotrophic Microorganisms, Třeboň, Czech Republic. The working temperature was kept to 25 ± 0.5 °C and was monitored by the temperature sensor incorporated in the type S423/C/OPT oxygen transducer. The intensity of the luminous flux was measured with a portable luxmeter HD2102.1 (equipped with a PHOTON FLOW, PAR LP471 PAR probe, measurement range 0.01–10,000 μmol/m²/s—from Delta Ohm, Padova, Italy) and adjusted using an LED driver (Figure 2) at the level of 310 ± 10 µmol/m²/s, light 400 to 700 nm.

The bubbles were generated by injecting air mixed with 5% (by volume) CO₂ through a porous hose at 2 L/min, thus resulting in a two-phase flow inside the reactors. The water represents the continuous phase, while the air/CO₂ mixture represents the dispersed phase. The gaseous mixture flow rate was controlled by flow transducers and electrovalves.

The intensity and frequency of the EMF applied to the culture medium was controlled by measuring/monitoring the output voltage of the sin-way ELF generator (which was designed and made by the authors) connected to the plane-parallel mounted graphite polarizing electrodes (Figure 2).

The experiments were simultaneously performed in the reference reactor and in the experimental reactor, where different electromagnetic field intensities were applied in order to study their influence on the algae growth. Each experiment was carried out on fresh (new) culture medium, both in the reference and in the experimental reactor, and lasted for 96 h. For each of the 4 EMF intensities, 3 determinations were made in the two reactors simultaneously. Thus, under reference conditions, 12 determinations were made.

During the growth of Chlorella vulgaris, the following parameters were monitored:

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Evolution over time of pH was monitored by a pH sensor S401 DIG/N type, produced by Chemitec.

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Evolution over time of the culture medium turbidity was achieved through the measurement of light absorption in suspension (optical density DO at a wavelength of 750 nm [37,38]) with a portable spectrophotometer (HACH, Berlin, Germany, DR3900).

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Evolution over time of algal mass concentration by filtering 100 mL of algal suspension on pre-weighted glass fiber filter paper (ROTILABO^®, CR261, Ø 47 mm, pore Ø 1.2 μm) washed with double distilled water, dried at 100 °C for 4 h, and cooled in a desiccator. The weight difference (measured with a Precisa balance, XR 125SM) after drying and cooling represented the dry weight (dw) of the microalgae.

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The time evolution of dissolved oxygen in the culture medium by measurements performed with a type S423/C/OPT oxygen transducer manufactured by Chemitec, Florence, Italy.

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The consumption of nutrients from the culture medium through analyses of samples collected before inoculation and after 96 h (4 days) of growth for the following: a. nitrogen concentrations in nitrate (N-NO₃—Cuvette test LCK 340, measurement range 5–35 mg/L N-NO₃, HACH, Berlin, Germany); b. total nitrogen (TN_b—Cuvette test LCK 238—measurement range 5–40 mg/L TN_b, HACH, Berlin, Germany); c. total phosphorus (P-PO4—Cuvette test LCK 350—range 2–20 mg/L P-PO₄ and LCK 348—range 0.5–5 mg/L P-PO₄, HACH, Berlin, Germany).

In order to quantify the effect of the EMF influence on Chlorella vulgaris, a growth stimulation coefficient k_s was calculated as (1):

k_s = 100 · (c − c_ref)/c_ref

(1)

3. Results and Discussions

The results of the respective spectrophotometric determinations, and the comparative absorbance evolution of the algal mass suspension in the liquid culture medium taken from the reference photobioreactor and from that exposed to growth stimulation by EMF of 2, 5, and 10, at 15 V/m, in the first 96 h after inoculation, are synthetically presented in Figure 3. Its analysis shows that at the beginning, in the first hours after inoculation, the absorbance did not change significantly during the lag period [39] (in the reference photobioreactor it lasted approximately 8 h) and was reduced as the 50 Hz EMF intensity increased, to approximately 6 h for EMF 2 V/m, 4.5 h for EMF 5 V/m, 3.2 h for EMF 10 V/m, and 2.5 h for EMF 15 V/m.

A similar observation of lag time shortening under the influence of EMF 50 Hz was also reported for the particularly diverse flora of microorganisms from active sludge from wastewater treatment plants [17]. After the lag period, following the multiplication and growth of microalgae, the absorbance of the medium increases accordingly, with the most pronounced increases being recorded in the case of the stimulation with 15 V/m EMF. It was also found that at 40–80 h after inoculation (depending on the intensity of the EMF 50 Hz applied) a limiting trend appears; this can be explained by the decrease in the nutrients’ concentration in the culture medium (approximately proportional to the biomass formed).

The absorbance is approximately proportional to the concentration of biomass in the suspension. By calculating the variation rate of the absorbance V_AU750 = Δ AU750/Δt, information is obtained regarding the growth rate evolution of the algal mass during the 96 h of cultivation. Based on the data presented in Figure 3, the evolution of the absorbance variation rate during the first 96 h of the algal mass growth was obtained and is shown in Figure 4.

Figure 4 shows that at the end of the lag period, the algal mass growth rate increases up to a maximum plateau, after which—as the concentration of nutrients in the culture medium decreases—it gradually decreases. The algal mass growth rate is minimal in the case of the reference bioreactor (V_AU750 = 0.41), where growth limitation begins after 80 h from inoculation. By applying ELF-EMF of 50 Hz, the maximum recorded values increase, the algal mass formed increases, and, as a consequence, the nutrient consumption from the culture medium is higher. In these conditions, the decreases in the algal mass growth rates are established faster—after 72 h for 2 V/m EMF, after 40 h for 5 V/m EMF, after 32 h for 10 V/m EMF, and after 24 h for 15 V/m EMF. By comparing the maximum growth rate, it is found that at 15 V/m the EMF is 2.75 times higher than the reference. These findings are important for the optimization of the operating parameters of the experimental setup for continuous flow production of algal mass. The results show that, for maximum productivity, it is suggested to harvest the algal mass daily in the case of growth stimulation with 15 V/m EMF. At each harvest/sample, it is suggested that the exhausted culture medium be compositionally corrected (by nutrient dosing and pH correction) and revalued by re-introduction into the photobioreactor. The results of the gravimetric determinations, regarding the weighting of the algal mass obtained by filtering 100 mL of suspension and drying the filter, are presented comparatively in

Table 1. The evolution of the biomass concentration (% dried mass) as a function of time at various intensities of stimulating fields.

Growth Time [hours]	Biomass Concentration c [gdw/L] and Stimulation Coefficient ks [%]
	Reference	EMF-2 V/m		EMF-5 V/m		EMF-10 V/m		EMF-15 V/m
	cref *	c	ks	c	ks	c	ks	c	ks
48	0.76 ± 0.037	0.78 ± 0.014	2.6 ± 0.14	0.82 ± 0.07	7.9 ± 0.78	0.96 ± 0.093	26.3 ± 2.85	1.02 ± 0.03	34.2 ± 1.95
72	1.29 ± 0.027	1.36 ± 0.015	5.4 ± 0.13	1.52 ± 0.02	17.8 ± 0.44	1.66 ± 0.017	28.8 ± 0.67	1.75 ± 0.08	35.7 ± 1.79
96	1.77 ± 0.11	1.88 ± 0.046	6.2 ± 0.42	2.12 ± 0.023	19.8 ± 1.25	2.29 ± 0.011	29.4 ± 1.83	2.41 ± 0.06	36.2 ± 2.42

* c_ref: [gdw/L] concentration of reference; mean ± SD (N = 12). Mean ± SD (N = 3).

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By analyzing the data in Table 1, it is found that after 96 h of culture, by stimulation with 15 V/m EMF, the algal mass concentration in the culture medium is 36.2% higher than that of the reference. Based on the data in Table 1, the average growth rates during the 96 h (4 days) of cultivation can be calculated as 0.47 gdw/L/day for EMF 2 V/m, 0.53 gdw/L/day for EMF 5 V/m, 0.57 gdw/L/day for EMF 10 V/m, and 0.60 gdw/L/day for EMF 15 V/m, compared to only 0.44 gdw/L/day obtained under reference conditions (without exposure of the culture medium to 50 Hz ELF-EMF).

The phototropic growth of the algal mass involves the metabolization of CO₂ available in the culture medium—an endothermic process that takes place through enzymatic photocatalysis (hυ) and through which the conversion of CO₂ into organic (algal) mass takes place with the release of O₂ (2):

2x · CO₂ + 2y · H₂O + hυ → 2C_xH_yO_z + (2x + y − z) · O₂↑

(2)

The oxygen formed by the process described by Equation (2) is dissolved in the culture medium—the total dissolved oxygen OD (limited by the maximum solubility at the given temperature of 25 ± 0.5 °C) being given by the oxygen released by (2) and part of the oxygen from the air/CO₂ bubbles. Under these conditions, the OD from the algal suspension in liquid culture medium is a parameter that characterizes the speed of the process (2). The results of the recordings regarding the time evolution of dissolved oxygen OD in the culture medium under experimental conditions are presented in Figure 5.

By analyzing Figure 5, it can be noticed that before the inoculation of the liquid culture medium, OD (given by the oxygen supply from the air/CO₂ bubbles) is approximately 7.64 mg/L. After the inoculation of the culture medium, due to the increase in the algal mass, following the development of the processes described by Equation (2), the OD increase is more pronounced in the first approximately 30 h; as it approaches the maximum solubility of oxygen under the given conditions, it shows a limiting trend. This limitation of OD is more pronounced in the case of algal mass suspension exposed to 15 V/m EMF (appears after approx. 24 h limitation to approx. 8.13 mg/L), explained by the maximum growth rate of algal mass in these conditions, as is also the case of the results from Figure 4. The bioreactor is open at the top. Thus, under the current experiment conditions, by injecting bubbles of an air/CO₂ mixture, a turbulent flow is generated throughout the reactor and at its surface, intensifying the oxygen transfer from the atmosphere to the culture environment. Thus, it is difficult to assume that the equilibrium is reached. The value of 8.13 mg O₂/L is actually the result of the two opposing processes occurring in the reactor: algal photosynthesis that generates O₂ and the bubbling system that injects a mixture of air and 5% CO₂.

In order to evaluate the consumption of nutrients from the culture medium according to the growth of the algal mass through specific chemical analyses, the initial concentrations c_i (before inoculation) and the final concentrations c_f after 96 h of growth of the algal mass for inorganic nitrogen (N-NO₃) were determined, with total nitrogen (TN_b) and total phosphorus (P_t). The difference between total nitrogen TN_b and inorganic nitrogen N-NO₃ represents the amount of nitrogen retained by the algal mass (organic nitrogen). The results of the comparative chemical analysis performed on the culture medium before inoculation c_i and after inoculation and 96 h of culture c_f (25 ± 0.5 °C, continuous lighting with 300 μmol/m²/s) are presented comparatively in

Table 2. Chemical analysis results *.

Parameter	Concentrations [mg/L]
	ci *	cf–reference * (0 V/m)	cf—2 V/m	cf—5 V/m	cf—10 V/m	cf—15 V/m
N-NO3	265 ± 1.1	125 ± 0.71	123 ± 2.83	119 ± 0.4	114 ± 2.83	90 ± 1.4
TNb (total nitrogen)	267 ± 1.2	275 ± 7.78	288 ± 2.12	324 ± 0.53	363 ± 5.7	382 ± 2.83
Organic nitrogen (Nalga)	2 ± 1.6	150 ± 7.82	165 ± 3.5	205 ± 0.66	249 ± 6.3	292 ± 3.2
Pt (total phosphorus)	14.5 ± 0.1	0.91 ± 0.003	0.87 ± 0.1	0.7 ± 0.001	0.68 ± 0.008	0.65 ± 0.003

* Average values for 3 determinations each; Mean ± SD (N = 3), c_f–reference *—final reference concentration (average value for 12 determinations); Mean ± SD (N = 12).

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By analyzing the values in Table 2, it can be noticed that during the growth of Chlorella vulgaris Bienjerinck CCALA 269, a significant part of the nitrogen (N-NO₃) and phosphorus content of the culture medium is retained/incorporated into the formed algal mass. Unlike the prokaryotes [40], green microalgae are unable to utilize atmospheric N₂ because it would require the usage of a consortium of nitrogen-fixing bacteria and microalgae to access such nitrogen species. As it is known, nitrogen serves as a crucial macronutrient that governs the metabolism, and consequently, the growth and biochemical composition of microalgae. As microalgae proliferate, they uptake nitrogen present in the growth medium to facilitate the biosynthesis of essential biological macromolecules like proteins, chlorophylls, and DNA [41]. The reproductive mechanism of the microalga C. vulgaris involves the production of asexual autospores through the division of the mother cell, resulting in 2–32 autospores, a process influenced by a range of environmental factors [36,42]. The objective of the study was to investigate the impact of 50 Hz sinusoidal alternating electromagnetic field intensity on the growth of C. vulgaris under non-limiting growth conditions, including temperature, nutrients, and light. The noticed enhancement in total nitrogen (TN_b) levels in the culture subjected to varying field intensities could potentially be attributed to the stimulated influence of the electromagnetic field on the metabolic processes of C. vulgaris (Table 2). It is interesting to note that TN_b increases as algal mass increases. Thus, in the 96 h of growth in the case of the reference, an increase in TN_b of only 8 mg/L was recorded, and in the case of growth stimulation by exposure to 50 Hz EMF, the nitrogen surplus increased significantly (21 mg/L at 2 V/m EMF, 57 mg/L at 5 V/m EMF, 96 mg/L at 10 V/m EMF, and 115 mg/L at 15 V/m EMF). These data indicate that, by exposing the culture medium mixed with air/CO₂ dispersed gas to 50 Hz EMF, the ability of Chlorella vulgaris to metabolize nitrogen from culture media increases significantly (more than 14 times at 15 V/m EMF).

In order to evaluate the depletion rate of P_t nutrients and inorganic nitrogen N-NO₃ during the four days of growth, periodic comparative determinations were made (from 24 to 24 h). The recorded results are presented in Figure 6 (for nitrogen) and Figure 7 (for phosphorus).

From the analysis of Figure 6, it is observed that after a first slow decrease during the lag period, the N-NO₃ concentration decreases linearly as the algal mass increases. The decrease is more accelerated (58 mg/L/day compared to 43 mg/L for the reference) under the influence of 15 V/m EMF when the algae growth rate is higher than the reference (Table 1).

From the analysis of Figure 7, it is observed that after a first slow decrease during the LAG period, the P_t concentration drops sharply as the algal mass increases, and the decrease is more pronounced under the influence of 15 V/m EMF when the algae growth rate is higher than the reference (Table 1). It is also found that after approx. 48 h of growth by stimulation with 15 V/m ELF, 90% of the P_t reserve of the culture medium is depleted (compared to the reference, when 90% depletion is reached after approx. 80 h of growth). Comparing Figure 6 and Figure 7 with Figure 4 leads to the conclusion that the limitations of the biomass growth rate in Figure 1 are due to the depletion in N-NO₃ and especially in P_t of the culture medium. It is noted that the presence of the phosphorus resource in the culture medium is determined in the biosynthesis of phospholipids that enter the structure of the cell membrane, the native rotary enzyme [18], and ATP [43], including chloroplast ATP [44], which together with the light harvesting complex have a determined role in the photosynthesis process (2) [45,46,47,48]. Under these conditions, the rapid decrease in the phosphorus content of the culture medium for growth stimulated in 15 V/m EMF (Figure 7) can be explained by the increase in the growth rate of the algal mass following the stimulation of ATP synthesis. This is in good agreement with the results reported in [31] for Escherichia coli.

By comparing the values from Table 1 (the concentration of biomass formed) with the values from Table 2, the nitrogen and phosphorus contents of the algal mass formed are calculated as:

C_Nitrogen = 100 · c_{algal mass}/N_algae [%]

(3)

C_fPhosphorus = 100 · c_{algal mass}/(c_iPt − c_{f Pt}) [%]

(4)

The main source of nitrogen in the culture medium is NaNO₃ and that for phosphorus is K₂ HPO₄. Through the metabolism of nitrogen and phosphorus from these nutrients, the culture medium becomes alkaline. The comparative evolution of pH during the 96 h of growth at various intensities of the applied electric field is shown in Figure 8.

The pH measurement during the 96 h of cultivation allowed the observation of the effect of 50 Hz EMF on the growth of C. vulgaris based on N-NO₃ and CO₂ consumption. The guidelines for preparing the BG11 medium included adjusting the pH to 7.1. Based on the data collected by the pH sensor, it was observed that following the commencement of aeration using a mixture consisting of 95% air and 5% CO₂ by volume, the pH values in each comparative experiment decreased from the initial range of 7.1–7.2 and stabilized within the range of 5.82–5.91. During the cultivation of C. vulgaris, the process of photosynthesis resulted in a gradual increase in the alkalinity of the culture medium and subsequent elevation of the pH. This phenomenon was consistently noted irrespective of the presence or absence of the electromagnetic field. However, under the influence of the 50 Hz EMF of 10 and 15 V/m, the pH evolution exhibited increased values in comparison to the reference.

Figure 9 shows the nitrogen and phosphorus contents of the algal mass grown under reference conditions (EMF intensity = 0) and at various applied 50 Hz EMF intensities, calculated according to (3) and (4), respectively.

By analyzing Figure 9, it can be noticed that by stimulating the algal mass growth with a 50 Hz ELF in the investigated culture medium, the nitrogen content obtained after 96 h of culture increases linearly depending on the applied EMF intensity from 8.47% (reference—without electric field (ELF)) to 12.12% (ELF stimulation 50 Hz—at 15 V/m). According to the data in Table 2, this increase of about 3% can be explained by the fact that the exposure to 15 V/m EMF of 50 Hz determines an increase in the ability of Chlorella vulgaris to metabolize nitrogen from air. Also, Figure 9 shows that by stimulating the algal mass growth with 50 Hz in the investigated culture medium, the phosphorus content obtained after 96 h of culture decreases depending on the applied EMF intensity from 0.77% (reference—without electric field) to 0.57% (ELF stimulation 50 Hz—at 15 V/m). This finding, correlated with data from Figure 7, suggests that the decrease in phosphorus content of the algal mass grown in 15 V/m EMF 50 Hz is due to the rapid depletion of the phosphorus resource in the culture medium.

4. Conclusions

Through experimental determinations, the influence of ELF-EMF of 50 Hz on the microalgae Chlorella vulgaris growth in BG11 culture medium was studied. The experimental determinations were made simultaneously in two photobioreactors with identical geometry at identical operating parameters (25 ± 0.5 °C temperature, 310 ± 10 µmol/m²/s—light 400 to 700 nm, 2 L/minute dispersed mixture of air with 5% CO₂ by volume), one reference and one equipped with graphite electrodes placed in parallel planes connected to a sin-way ELF generator with adjustable output voltage to create a controlled and homogeneous EMF on the culture medium.

The results of spectrophotometric and gravimetric determinations showed the following:

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The reference lag period lasts approximately 8 h and is reduced as the 50 Hz EMF intensity increases to approx. 6 h for EMF 2 V/m, 4.5 h for EMF 5 V/m, 3.2 h for EMF 10 V/m, and 2.5 h for EMF 15 V/m;

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The average growth rates in the 96 h of cultivation were 0.47 gdw/L/day for EMF 2 V/m, 0.53 gdw/L/day for EMF 5 V/m, 0.57 gdw/L/day for EMF 10 V/m, and 0.60 gdw/L/day for EMF 15 V/m, compared to only 0.44 gdw/L/day obtained under reference conditions;

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After the lag period, the growth rates of the algal mass increase to a maximum at 80 h from inoculation to the reference and faster at 72 h for 2 V/m EMF, 40 h for 5 V/m EMF, 32 h for 10 V/m, and 24 h for EMF 15 V/cm;

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The maximum growth rate of the algal mass at 15 V/m is 2.75 times higher than that recorded in the reference.

The registered values of dissolved oxygen concentration (OD) in the culture medium showed that after LAG time, OD has a period of growth in which the growth rates are consistent with the growth speed of the algal mass, after which it shows a trend of limitation to the maximum solubility of oxygen.

The results of the chemical analysis for the concentrations of N-NO₃, TN_b (total nitrogen), and Pt (total phosphorus) showed the following:

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Following the increase in algal mass, the N-NO₃ content of the BG11 culture medium decreases by 58 mg/L/day at 15 V/m EMF compared to 43 mg/L in the reference;

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Following the increase in algal mass, the P_t content of BG11 decreases to 90% depletion after approx. 80 h at baseline versus only 48 h of growth by exposure to 15 V/m ELF;

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Following the increase in algal mass, the increase in TN_b in 96 h was 8 mg/L and 115 mg/L at 15 V/m EMF (14 times higher), showing that the influence of the EMF increases with its intensity;

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The nitrogen content of the algal mass obtained after 96 h of culture increases linearly depending on the applied EMF intensity from 8.47% (reference) to 12.12% for 15 V/m EMF;

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The phosphorus content of the algal mass obtained after 96 h of culture decreases from 0.77% (reference) to 0.57% for 15 V/m EMF.

These results lead to the conclusion that the EMF of 50 Hz stimulates the growth of Chlorella vulgaris (maximum growth rate 2.75 times higher than the reference) in a BG11 culture medium, whose P_t reserves are depleted approx. 2 times faster than in reference conditions. The depletion of 90% of the P_t resource in the culture medium results in the cessation of the proliferation of algal biomass. The 15 V/m EMF stimulates the ability to accelerate the nitrate assimilation, the increase in TN_b being 14 times higher than in the reference. Exposure to EMF 50 Hz leads to changes in the phosphorus and nitrogen content (concentration) of the algal mass. Considering these conclusions, in order to establish the technological parameters of an industrial installation for the intensive production of algal mass, it is considered appropriate to continue the research to establish the consumption of microelements and nutrients from the culture medium, to perform elemental analyses on algal mass, and to establish the EMF intensity of stimulation for a given composition of the culture medium under continuous dosing of nutrients and microelements.

5. Patents

The work reported in this manuscript led to the elaboration of two Romanian patent applications: A/00600/2023, Growth stimulation process and photobioreactor for the phototropic growth of microalgae and A/00292/2024, Method and installation for reducing CO₂ consumption in the phototropic growth of microalgae.