Low-dose copper and blue light increases the yield of value-added biomolecules in Kirchneriella contorta (Chlorophyceae)

Microalgae are a natural source of valuable compounds with a wide range of applications. Given their physiological plasticity, strategies aiming at increasing the content of biomolecules have been proposed. In this study, low-dose copper and blue/red lights were combined and used as a manipulative strategy to induce biomolecule accumulation in Kirchneriella contorta. Cultures were exposed to a 1.6 × 10–9 to 1.7 × 10–8 mol L−1 free copper (Cu2+) concentration range under white light for 48 h. Afterwards, the white light was replaced with blue or red lights for up to 96 h. We evaluated population growth through growth rates, photosynthesis through pulse amplitude modulated fluorescence (PAM) and biomolecules by quantifying chlorophyll a, carotenoids, carbohydrates and proteins. The results showed that at 3.8 × 10–9 mol L−1 Cu2+ and blue light, 2 × more carotenoids, 4 × more carbohydrates and 3 × more proteins were accumulated in the cells compared to the control. Among the light colors, the blue light most significantly interfered with the algal metabolism compared to the other colors. Although growth rates and maximum and operational quantum yields were not affected, non-photochemical quenching (NPQ) increased under blue light, showing that its higher energy compared to red and white lights may have impacted the photosynthetic apparatus. This study contributes to the biotechnology of microalgae by proposing a manipulative strategy that triggered the build-up of biomolecules in K. contorta.


Introduction
Microalgae are primary producers in aquatic environments and changes in their biochemical composition or population numbers can impact higher trophic levels.In addition, microalgae produce a wide variety of compounds such as proteins, antioxidants, anti-inflammatories, carbohydrates, omega-3 fatty acids, and others that can be used in the biotechnology industry (Borowitzka et al. 2016).
Industrial production relies on high biomass and a favorable cost/benefit ratio, but so far this has been achieved for just a few species considering the diversity of microalgae.Examples of commercial strains include Dunaliella salina, Chlorella spp., Haematococcus pluvialis and Arthrospira (Spirulina) platensis.Strategies to increase the biomass yield of microalgae and/or biomolecules per unit of biomass can be a way to increase the number of species that can be cultivated for commercial purposes.To this end, the biochemical manipulation of algal cultures has been the subject of extensive studies.Light quality (Abiusi et al. 2014;da Fontoura Prates et al. 2020), major nutrients such as nitrogen and phosphorus, trace metals such as cadmium and copper (Chia et al. 2013(Chia et al. , 2015(Chia et al. , 2017)), and different temperatures (Gacheva and Gigova 2014;Menegol et al. 2017) have all been used to stress algal cells and increase Th content of certain biomolecules.Chia et al. (2013) reported that nitrogen depletion in combination with cadmium magnified biomolecule accumulation in Chlorella vulgaris.They showed that exposing the cells to 2.9 × 10 -6 mol L −1 N and 10 −7 mol L −1 Cd, 4 × more total proteins, 2.5 × more carbohydrates and 2 × more lipids compared to the control were obtained.Similarly, low phosphorus in combination with cadmium was also shown to potentialize biomolecules content in C. vulgaris (Chia et al. 2017).However, Lombardi and Wangersky (1991) showed that phosphorus deprivation can cause cell membrane fragility because it affects phospholipids, a membrane lipid class, which can become a problem.This is usually overlooked but can lead to the loss of the compounds of interest during cell harvesting due to membrane disruption.
As a micronutrient, copper plays an important role in microalgal physiology, affecting biological functions through its depletion or excess (Lombardi and Maldonado 2011).It is not uncommon to have copper as a stimulating factor for biomolecule accumulation in microalgae; it depends on the exposure concentration of the metal.Investigating the effects of copper (4.7 × 10 -7 to 4.0 × 10 -6 mol L −1 ) on the physiology of Chlorolobium braunii, Baracho et al. (2019) showed increased proteins, carbohydrates, and lipids, and Silva et al. (2018) showed higher carbohydrates and proteins in Scenedesmus quadricauda.Therefore, as a micronutrient, copper can be proposed as a manipulating agent.
Light quality is another manipulating agent that has been widely investigated (Müller et al. 2001;Keeling 2013).Light is characterized by its emission wavelength (or its color) and has been shown to affect the production of biomolecules (Yuan et al. 2020) and growth rates in microalgae (Teo et al. 2014b), thus stimulating specific metabolic routes.Changes in the composition of microalgae photoreceptors (Müller et al. 2001), smaller cells and higher growth rates have been shown to occur in cells exposed to red light (Keeling 2013).Under blue light, effects in gene expression have been reported (Ruyters 1984).McGee et al. (2020) reported up to 9 × increase in lipids synthesis under green light, twice as many carbohydrates under blue light, and 1.6 × increase in proteins under white light.Yuan et al. (2020) showed that the red light led to a greater accumulation of carbohydrates while the blue light of lipids, in addition to both favoring growth rates.
The use of different light colors and metals to stimulate the synthesis of biomolecules can affect the photosynthetic machinery of the microalgae.Lombardi and Maldonado (2011) showed that in semi-continuous cultures even environmentally significant copper concentrations (10 -11 to 10 -10 mol L −1 ) decreased maximum photosynthetic quantum yield in Phaeocystis cordata, but not the operational yield.Similarly, Dauda and Lombardi (2023) showed that low copper levels (10 -9 moL −1 ) stimulated photochemical quenching (qP), and that NPQ gradually increased with increasing copper in Monoraphidium sp.Pereira and Otero (2019) showed that the qP of Dunaliella salina, and the qP and NPQ of Dunaliella tertiolecta varied according to the quality of light provided (blue or red).Additionally, a decrease in the operational quantum yield was reported for both microalgae when cultivated with red light.
The aim of achieving an increase in biomolecules in microalgae by mild stress caused by exposure to a low dose of toxicant is attractive because this can be achieved by preserving or slightly altering the population growth rates (Agathokleous and Guo 2022).However, such a stimulus is less intense regarding the accumulation of biomolecules (Silva et al. 2018) than stress induced by, for example, the depletion of major nutrients (Chia et al. 2013).This means that in the first situation, a combination of manipulating agents may be needed to magnify the accumulation of biomolecules.Here, we combined the effects of copper at a low dose with a red or blue light aiming at an amplification of biomolecules in a freshwater Chlorophyceae.We showed that the combination of low-dose copper and blue light resulted in higher yields of biomolecules in Kirchneriella contorta compared to the red light and the controls.

Cultures, growth rates and photosynthesis
Kirchneriella contorta (Schmidle) Bohlin 1 (SisGen AD7D518) was obtained from the Freshwater Microalgae Culture Collection of the Botany Department, Federal University of São Carlos (São Paulo, Brazil), where it is kept in WC culture medium (Guillard and Lorenzen 1972).The Collection is registered in the World Data Center for Microorganisms under number 835.
The microalga was grown in laboratory-controlled conditions.Sterile WC culture medium with initial pH 7.0, at 25 ± 1 °C and 12:12 h light:dark cycle were used.In order to obtain the I k (saturating irradiance for the algal strain), we performed rapid light curves (RLC) using the Phyto PAM (Walz, Germany) settings.Twenty pulses of photosynthetically active radiation (PAR) of increasing intensity were applied onto the samples at 20 s intervals and the operational quantum yield was recorded.Multiplying PAR pulses by their respective operational quantum yield, the relative electron transport rate (rETR) was obtained and plotted as function of PAR.The data were mathematically fitted according to Platt et al. (1980) and the saturation irradiance was calculated as maximum rETR/α (α corresponds to the initial slope of the curve obtained after fitting the data).The I k obtained was 200 μmol photons m −2 s −1 and this light intensity was used for all cultures, independent of the light colors.Cultures were monitored daily for population density (cells mL −1 ) and maximum photosynthetic quantum yield (Φ M ).Cell counts were done on a MUSE flow cytometer (Millipore, USA) and Φ M using a PHYTO-PAM (Walz, Germany).Growth rates (day −1 ) were determined by means of linear regression plotting the natural logarithm of cell mL −1 vs experimental time (days) from 0 -48 h; the line slope represents the specific growth rate for exponentially growing cells.Samples for total dry weight (mg L −1 ) were filtered through previously dried (48 h, 40 °C) and pre-weighed 0.45 μm acetate filters (Sartorius Stedim Biotech; Germany).After filtering, the algal biomass was dried under the same conditions and transferred to a desiccator to cool before determining the dry weight mass.For this, an analytical scale balance with 1 μg precision and 1.5 μg repeatability (Mettler Toledo AG, Switzerland) was used.Cell biovolume (µm 3 ) was determined in 30 cells following Hillebrand et al. (1999) at 48 h and 96 h using an optical microscope (Nikon Eclipse, Japan) connected to a computer.
Photosynthesis related determinations were done using pulse amplitude modulated fluorescence following the methodology described in Lombardi and Maldonado (2011).For the maximum quantum yield, cells were dark acclimated for 20 min, after which the sample was excited by a continuous background light (2 µmol photons m −2 s −1 and frequency 25 Hz) to detect the minimal fluorescence (F O ).The maximum fluorescence (F M ) was obtained after applying a saturating light flash of 2000 µmol photons m −2 s −1 intensity and 700 ms duration.The maximum quantum yield (Φ M ) was calculated as described in Eq. 1.For measurement of steady state fluorescence (F S ) and maximum fluorescence at light adapted state (F M ' ), the samples were exposed to sequential pulses of actinic light of 200 µmol photons m −2 s −1 applied every 20 s for 10 min.Thus, it is possible to correlate the activation of the xanthophyll cycle and the increase in NPQ with the maintenance of qP.The effective quantum yield of PSII (Φ M ' ) was determined following Eq.2, the photochemical (qP) as in Eq. 3, and non-photochemical (NPQ) quenching as in Eq. 4. The equations used for calculations of the parameters are presented below.

Manipulation strategy
The manipulation strategy we used entails two steps.First, the cells are exposed to low dose copper and then to We began by defining the range of copper concentrations that would not influence growth rate or that would do it minimally.Thus, in three experimental replicates and using 96-well microplates following the protocol described in Stablein et al. (2021), we performed a copper screening test whereby the cells were exposed to 3 × 10 -8 -3 × 10 -5 mol L −1 nominal copper (copper added to the cultures) concentration range for 120 h.The microplates were covered with Parafilm, incubated under white light from below at 12:12 h light:dark cycle and population growth was monitored by measuring absorbance at 684 nm (Epoch Microplate Spectrophotometer, U.S.A.).The choice of absorbance of 684 nm as OD instead of 750 nm for growth evaluation is due to absorption by chlorophyll.
The 750 nm would be absorbed by other particulate material as well, in addition to the fact that the interference that may occur at 684 nm would be mainly due to the age of the culture, cell morphology and cell biochemical composition (Hofstraat et al. 1992;Griffiths et al. 2011), but microplate cultures were short-term and healthy.A commercial copper standard solution (AAS/ICP, 1000 mg L −1 , 38,996 Sigma-Aldrich, USA) was used for copper addition into culture medium 24 h before microalgae inoculation.
The initial cell density in cultures were 10 -4 cells mL −1 to reduce problems in batch cultures such as self-shading that can start 24 to 30 h after inoculation and thus interfere with sampling for biomolecules (Lombardi and Maldonado 2011).Controls contained the usual nominal copper concentration of WC culture medium (3 × 10 -8 mol L −1 ).From this copper screening test, the nominal copper concentration range that would not negatively affect growth rates was defined as 3 × 10 -8 to 3 × 10 -6 mol L −1 .This copper range was used for the manipulation experiment and the free copper ions to which the cells were exposed was determined before cells inoculation.The equivalent range of free copper ions was 1.6 × 10 -9 to 1.7 × 10 -8 mol L −1 free copper ions (Cu 2+ ).The manipulation experiment began with cells exposed to 1.6 × 10 -9 to 1.7 × 10 -8 mol L −1 copper range (Table 1) and white LED for 48 h, after which they were exposed to red and blue lights for another 48 h.Thus, the total manipulation 2.2 × 10 -9 1 × 10 -7 3.8 × 10 -9 1 × 10 -6 1.4 × 10 -8 3 × 10 -6 1.7 × 10 -8 process lasted 96 h.The emission spectra of the lights were recorded using an Ocean Optics UBS 2000 model spectroradiometer equipped with a BIF-600-UV-VIS optical fiber and OIBase32 collection software (USA).The spectra obtained confirm the different wavelengths with peaks at white 455/566 nm, blue 445 nm and red 652 nm, as shown in Fig. 1S (supplemental material).The commercial information of the LEDs used are 5630 IP20, 14.4 W m −1 , 15,000 lm.Cultures were performed in 250 mL polystyrene tissue culture flasks kept vertically, thus with 3 cm optical path, illuminated by the side and internal light intensity was kept ~ 200 µmol photons m −2 s −1 .The light:dark cycle was 12:12 h and cultures were bubbled with filtered air (0.22 µm; Chromafil Xtra PUDF 20/25, Germany).They were inoculated with 5 × 10 4 cells mL −1 from exponentially growing cells whose physiological condition was confirmed by maximum quantum yield (Φ M ) ~ 0.70 (Lombardi and Maldonado 2011).
Biomolecules, e.g., total carbohydrates and proteins, chlorophylls a, and total carotenoids were determined in 96 h cultures.Total carbohydrates were determined following Albalasmeh et al. (2013) and total proteins according to Slocombe et al. (2013).For these analyses, culture samples were centrifuged in a refrigerated centrifuge for 20 min at 3920 ×g.The supernatant was discarded and the pellet stored at -22 °C until analysis, which occurred within 15 days of sampling.The concentrations of chlorophyll a and total carotenoids were determined according to Wellburn (1994) in culture samples (3 mL) filtered through 0.45 μm acetate membranes (Sartorius Stedim Biotech; Germany) and dissolved in dimethylsulfoxide (DMSO).Absorbance (Abs) at 665 nm and 649 nm was measured to determine chlorophyll a and b, necessary for total carotenoids calculations in addition to measurement at 480 nm.The equations used for these calculations are shown below.

Determination of free copper ions (Cu 2+ )
Free copper ions were determined according to Lombardi et al. (2007) in the manipulation cultures only, and before cells inoculation.Since no Cu determination was performed in the screening test, we refer to it as nominal copper, e.g., copper added to the culture medium.At least 7 days would be needed to quantify free Cu 2+ in all the screening samples and this, per se, would affect Cu speciation, introducing to errors.Additionally, by knowing the nominal copper, it was possible to nearly reproduce the desired concentration in the manipulation experiment.Nevertheless, the importance of determining free Cu 2+ ions in the manipulation experiment relies on knowing the bioavailable fraction.This is important because of presence of EDTA in WC culture medium that chelates trace metals.A copper ion selective electrode (ISE-Cu, Thermo Scientific Orion 9429BN) was used as working electrode and a Thermo Scientific Orion 900,200 double junction electrode (D/J safe flow) as the reference electrode.For the calibration of the ISE system, copper buffers were used to extend the detection limit down to 10 -10 mol L −1 .The metallic buffer comprised sodium borate (Sigma-Aldrich, Germany), sodium nitrate (Sigma-Aldrich, Germany), copper standard (38,996 Sigma-Aldrich, USA) and nitrilotriacetic acid (Sigma-Aldrich, Germany).All measurements were performed in a filtered air cabinet and under controlled temperature (24 ± 1 °C), and a calibration curve for ISE-Cu system was performed at pH 5. It was made from serial dilution of a commercial monoelementary copper standard AAS/ICP (1000 mg L −1 , 38,996 Sigma-Aldrich, USA).Table 1 shows nominal copper and free copper ions concentrations (mol L −1 ) used for the manipulation experiment.

Data analysis
Statistical analyses of algal growth rates obtained for the copper screening test and maximum growth rates under the white light (0 -48 h) were done using one-way ANOVA (Minitab, version 17 for Windows), while twoway ANOVA and Tukey's Test were used to compare data obtained at 96 h.Normality and homoscedasticity assumptions were verified before analysis using the Shapiro-Wilk test and Levene test.

Results
The higher end of copper concentration in the screening test resulted in negative effect on growth curves (Fig. 2S).Thus, we defined the cell-supportable copper concentration range for this species to be within 1.6 × 10 -9 to 1.7 × 10 -8 mol L −1 and it was used for the biochemical manipulation experiment.
Figure 1 shows the growth curves for the biochemical manipulation experiment.Growth rates are shown in Table 2.
Under white light, growth rate was not significantly different among copper treatments (~ 0.96 day −1 ), except for 2.2 × 10 -9 mol L −1 Cu 2+ , which was lower (0.86 day −1 ).Cell viability provided by the Muse Cell Analyzer was similar among treatments, remaining above 95%, regardless of light color or Cu concentration (data not shown).Different from literature results (Teo et al. 2014a, b), we showed that, in general, blue or red lights did not affect the growth rates of K. contorta.
Figure 2a shows that cultures exposed to 3.8 × 10 -9 mol L −1 under red or blue lights had 43% higher biomass (ANOVA, p < 0.05) than white light under the same free copper concentration or the respective light color control.In relation to cell biovolume, K. contorta cells in cultures exposed to white and red lights had similar biovolume that were generally higher than cells under blue light.Considering photochemical quenching (Fig. 2c), it was similar among treatments (0.75-0.8; p > 0.05), except for treatment with free copper dose of 2.2 × 10 -9 mol L −1 under white light, where it was 0.73 (p < 0.05).In relation to NPQ, it was systematically higher under the blue light (ANOVA p < 0.05).No statistical difference and no trend that could be related to either copper or light colors were obtained for maximum (0.72 -0.74) and operational quantum yields (0.52 -0.56) (Fig. 3S -supplemental material).
In general, higher amounts of biomolecules were obtained in cells exposed to the combination of blue light and 3.8 × 10 -9 mol L −1 Cu 2+ .Compared to the control (1.6 × 10 -9 mol L −1 Cu 2+ white light), the treatment that combined 3.8 × 10 -9 mol L −1 Cu 2+ and blue light, accumulated 4 × more carbohydrates.The effect of light colors in carbohydrates accumulation is further corroborated by the ~ 3 × higher carbohydrates in cells exposed to either blue or red lights without extra copper.Similarly, the productivity of proteins (Fig. 3b) in K. contorta was affected by copper and light colors.Although red and blue lights resulted in ~ 1.7 × higher proteins in treatments without extra copper, it was the combination of blue light and 3.8 × 10 -9 mol L −1 Cu 2+ that increased proteins the most (3 × higher than the control -1.6 × 10 -9 mol L −1 Cu 2+ white light).Considering the pigments, the chlorophyll a data (Fig. 3c) followed a similar behavior to proteins, with the highest value (~ 1.5 × higher) in 3.8 × 10 -9 mol L −1 free copper and the blue light.The total carotenoids (Fig. 3d) were highest under the blue light and 3.8 × 10 -9 mol L −1 Cu 2+ treatment, a situation where it doubled in contrast to the white light control (1.6 × 10 -9 mol L −1 Cu 2+ ).

Discussion
The similarity in growth rates for the copper and white light treatments was expected as a carefully selected copper concentration range based on the screening test was used.According to Davies and Bennett (1985) the copper concentrations we used are considered environmentally significant, thus commonly found in natural aquatic environments and the cause of a lack of influence on K. contorta growth.Different from our results, others have shown that the blue light can lead to increased growth rates in microalgae.Teo et al. (2014a, b) showed higher growth rates for Nannochloropsis sp. and Tetraselmis sp.exposed to blue light compared to white light controls.According to the authors, the blue light has greater penetration in the  culture when compared to the white light, thus improving cell division and microalgae growth rates.In the present study, it is possible that the short optical path together with low density cultures that we used may have made the blue light effect unnoticeable, as both the white and red lights have penetrated thoroughly into the culture flasks.Furthermore, the light intensity inside the cultures was the same regardless of its color.The usually higher biomass in cultures exposed to 3.8 × 10 -9 mol L −1 and red/blue light means that in this research, K. contorta was stimulated by the combined effect.Literature data show that light color can affect microalgae biomass yield, but this did not happen in the present study.Abiusi et al. (2014) showed a 2.5-fold increase in Tetraselmis suecica biomass under red compared to the white light.They proposed that the red light is better absorbed and used by the photosynthetic pigment, chlorophyll a, than the white light.Baba et al. (2012) showed a higher growth rate (and biomass) in Botryococcus braunii cultures under a red light compared to white and blue lights and attributed it to higher chlorophyll a content under the red light.On the other hand, Asuthkar et al. (2016) showed that a blue light promoted greater accumulation of dry biomass in cultures of Chlorella pyrenoidosa compared to red, green and white lights.According to Baer et al. (2016), the effect of different light qualities on microalgae varies with the species, highlighting the importance of investigating different strains and the desired biomolecules.
Cell biovolume and morphology are other characteristics that can be affected by light quality or its spectrum.Schulze et al. (2016) observed that Nannochloropsis oculata cells in treatments with white light had the larger cell surface area, followed by red and blue lights, while for Tetraselmis chuii the descending order of cell surface area was blue, red and white lights.Similar to our results, Aidar et al. (1994) obtained variation on cell biovolume between species exposed to different light colors.They showed greater cell volumes for Tetraselmis gracilis in exposure to red light compared to blue light and the opposite for Cyclotella caspia.Nwoba et al. (2021) showed that the biovolume of the microalgae Dunaliella salina under blue light was ~ 57% larger compared to white light.Based on the work by Oldenhof et al. (2004), Nwoba et al. (2021) proposed that blue light can slow down DNA replication and cell division by affecting cyclin-dependent kinases, while the opposite occurs for red light.However, much uncertainty still exists about this subject in the literature, with different results depending on the strain studied (Kim et al. 2014).
Related to photosynthesis, our results agree with Zhong et al. (2018).They showed no variation in maximum quantum yield, but higher NPQ under the blue light for C. vulgaris and C. pyrenoidosa.Additionally, under low copper we obtained a gradual NPQ increase for red (twofold) and blue lights (2.4-fold), but constant and high NPQ at the highest copper concentration, suggesting that the light reactions of photosynthesis were affected at the highest copper concentration.It is known that NPQ, a heat related dissipation process, is a means of protecting photosynthetic microalgae against photodamage by high light (Baer et al. 2016).Therefore, its increase indicates that photoprotection mechanisms were triggered under both blue and red light.This means that cells in 1.7 × 10 -8 mol L −1 free copper could have been facing oxidative stress.
To understand the effects of light colors in microalgae cultures, several aspects should be considered.First, cell density and optical path of the culture flask can have a significant effect as the wavelengths of red and blue lights and the energy they carry per photon are significantly different.Red light (~ 650 nm) is less energetic than the blue light (~ 440 nm), affecting the extent to which it penetrates in the cultures.The energy of blue light is calculated to be 1.44 × higher per photon than the red light, thus able to induce higher NPQ (Fu et al. 2013).Higher penetration of blue light and its higher energy per photon can lead to higher NPQ and carotenoids as a protection mechanism (Katsuda et al. 2004;Costa et al. 2013).In this research, because we used cultures with low density and flasks with short optical path, the effects of different colors of light on the NPQ may have been highlighted, as seen in Fig. 2c.Considering that we assured the same light intensity regardless of its color inside the culture flasks, the higher NPQ under the blue light can be related to the higher energy carried by their photons than the white and red lights.
Considering biomolecules, literature results show a great variation on the effects of light colors on carbohydrates in microalgae.Our results are in line with those of Schulze et al. (2016), who showed that Nannochloropsis oculata produced more carbohydrates under red and blue lights, while Tetraselmis chuii under a red light.However, Abiusi et al. (2014) observed no effect of red/ blue lights on carbohydrate content in Nannochloropsis sp, but higher levels were detected in Tetraselmis suecica under white/ blue lights.Corroborating our results, Lv et al. (2022) and Markou (2014)  They propose that protein biosynthesis may play a role in copper exposure, protecting the cells against toxicity.Studying Chlorella ellipsoidea exposed to white, green, red and blue lights, Baidya et al. (2021) showed that it was the blue light that resulted in the highest protein increase and concluded that this light color was the best for the production of nutritionally rich biomass.Marchetti et al. (2013) reported greater protein synthesis for the microalgae Isochrysis sp.under blue light and proposed that this color allows greater incorporation of photosynthetic carbon in the synthesized proteins, in addition to be related to the increase in the light collection system, the structural protein of the PSII (Rivkin 1989;Zhou et al. 2009).According to Payer et al. (1969), this may be related to an increase in the efficiency of CO 2 fixation and its incorporation into proteins under blue light.We propose that for K. contorta in the present research, at very low copper (3.8 × 10 -9 mol L −1 ), the cells were subject to a physiological event by which they were brought to a subtle oxidative condition that permitted them to still photosynthesize and divide.Under this condition, cells can be more sensitive to light and further exposure to the blue light whose photons carry high energy, has triggered the synthesis of biomolecules as proteins, carbohydrates and carotenoids.
The results we obtained for chlorophyll a showed that copper was more important in determining their concentration than light color.Similarly, Baracho et al. (2019) and Silva et al. (2018) also showed higher levels of chlorophyll a in green microalgae exposed to copper.These authors attributed the results to a protection mechanism against copper toxicity.Hamed et al. ( 2017) reported a decrease of chlorophyll a at a copper concentration of 2.5 × 10 -5 mol L −1 for Chlorella sorokiniana.They hypothesized that the chloroplasts were affected by the metal and that the related stress caused inhibition of the electron transfer, thus reducing the formation of photosynthetic pigments (Wong et al. 1994;Yan and Pan 2002;Charles et al. 2006).Considering light colors without extra copper, different from our results, the literature shows that exposure of microalgae to blue light can increase chlorophyll a. Vadiveloo et al. (2015) showed this in Nannochloropsis sp., Rebolledo-Oyarce et al. (2019) in D. tertiolecta (14% higher Chl a) and Baidya et al. (2021) in Chlorella ellipsoidea.Thus, intracellular effects, which may include modulation of photosynthesis and cell division processes that led to higher biomolecules and biomass, supports the combined effect of blue light and 3.8 × 10 -9 mol L −1 copper in K. contorta.
Considering carotenoid accumulation in microalgae, our results agree with the literature, which shows that specific light colors can increase carotenoid production in microalgae, but this can be species-specific.Suyono et al. (2015) obtained 8 × higher carotenoids in Haematococcus pluvialis upon exposure to blue light.Different from the present results, Han et al. (2019) investigated D. salina, and showed that the red light increased carotenoid production by 13%.Fu et al. (2013) combined red and blue lights and reported 2.3 × higher carotenoid biosynthesis in D. salina.
According to Chory (2010), the variation in the composition of photoreceptors is crucial for microalgae adaptation to the natural variation of light in the environment and can be expected to occur under different light colors.Therefore, the mechanism of pigment production in green microalgae can be favored using different colors of light and, as we demonstrated, the potential of increasing photosynthetic pigments such as carotenoids can be magnified by combining the blue light and low dose copper exposure.
Finally, our suggested explanation for the combined effect of the blue light and low copper (3.8 × 10 -9 mol L −1 ), which increased carbohydrates, proteins, and carotenoids in K. contorta, is that at low doses the metal caused minor oxidative stress in the cells, making them more sensitive to the blue light.As a response, the cells accumulated biomolecules.

Conclusions
This study showed that exposure of K. contorta to copper at low doses under the white light was enough to induce effects in the cells, but blue/red lights without extra copper were not as effective.The combination of blue light and 3.8 × 10 -9 mol L −1 free copper magnified biomolecules in the cells.Approximately 4 × higher carbohydrates, 3 × higher proteins and 2 × higher carotenoids were detected.We showed that the photosynthetic apparatus was more sensitive to changes in light color than to increasing copper at sublethal levels.This research demonstrated the viability of a manipulation strategy by combining the copper and blue light to magnify biomolecules in green microalgae.

Fig. 1
Fig. 1 Growth curves of Kirchneriella contorta exposed to selected free copper concentrations and different light colors during the manipulation process.Error bars represent the standard deviation from the mean (n = 3)

Fig. 2
Fig. 2 Variables obtained in the manipulation experiment of K. contorta at 96 h.(a) dry biomass (mg L −1 ), (b) cell biovolume (µm 3 ) and (c) fluorescence quenchings, where qP is represented by the dashed line and NPQ by the bars.White bars refer to control, grey to red light and hatched to blue light.Error bars represent the standard deviation from the mean (n = 3).Letters above bars represent statistical evaluation (same letter indicates no significant difference, ANOVA p > 0.05) obtained higher carbohydrates in microalgae exposed to blue light in comparison to white light.Lv et al. (2022) observed 3 × more carbohydrates for Isochrysis zhanjiangensis and Markou (2014) for Arthrospira platensis.A possible reason for such differences might be the adaptations and evolutionary histories of each species, in addition to culture densities associated to its optical path.McGee et al. (2020) evaluated Rhodella sp.and Phaeothamnion sp.under green, blue, red and white lights, and observed that blue light led to a twice as high carbohydrate content.According to the authors, the shorter wavelength of blue light provides greater energy for biomass and biomolecule production.The results we obtained for proteins agree with the literature, where increased proteins in other Chlorophyceae exposed to copper ions are shown.Silva et al. (2018) exposed Scenedesmus quadricauda to copper and showed protein increase.Shakya et al. (2022) investigated sub-lethal copper concentrations in Chlorella sp. and showed modulation of proteins involved in energy production pathways, such as photosynthesis, carbon fixation, glycolysis, and oxidative phosphorylation.

Fig. 3
Fig. 3 Biomolecule productivity (µg mL −1 day −1 ) obtained in the manipulation experiment of K. contorta at 96 h.Total proteins (a), carbohydrates (b), chlorophyll a (c), and carotenoids (d).White bars refer to control, grey to red light and hatched to blue light.Error bars

Table 2
Growth rates (day −1 ) at 48 and 96 h in different concentrations of free copper (mol L −1 ) and light colors.The numbers in parentheses represent the standard deviation from the mean (n = 3).