Effects of Nitric Oxide On The Growth of The Marine Microalgae And The Parameters of Carbonate Chemistry

doi:10.21203/rs.3.rs-521371/v1

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Effects of Nitric Oxide On The Growth of The Marine Microalgae And The Parameters of Carbonate Chemistry

https://doi.org/10.21203/rs.3.rs-521371/v1

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Nitric oxide (NO) is a non-traditional regulator for the growth of plant and phytoplankton. This study monitored the growth of five marine phytoplankton species, namely, Platymonas helgolandica var. tsingtaoensis, Platymonas subcordiformis, Skeletonema costatum, Gymnodinium sp., and Prorocentrum donghaiense, and examined the parameters of the carbonate system in the culture media after adding different concentrations of NO and sodium nitroprusside (SNP, NO donor) solutions. The different concentrations of exogenous NO had various roles in the growth of these microalgae. The two food algae,namely, P. helgolandica var. tsingtaoensis, P. subcordiformis, and three red tide algae, namely S. costatum, Gymnodinium sp., and P. donghaiense showed different responses to the same NO concentration. The red tide algae were more sensitive to exogenous NO than the food algae. NO with the concentration of 1.4 × 10^− 6 mol L^− 1 had the optimum stimulatory effect on the growth of the microalgae, the cell density increased by 9.8 ~ 38.3%. SNP solution with the concentration of 100 µmol L^− 1 inhibited the growth of the two food algae, the cell density decreased by 38.8 ~ 84.3%. Meanwhile, 10 µmol L^− 1 SNP solution additions to the three red tide algae declined the cell density by 95.3 ~ 99.9%. Low concentrations of SNP (0.1 µmol L^− 1 for the two food algae and 0.01 µmol L^− 1 for the red tide algae) promoted the growth of microalgae slightly. Different concentrations of exogenous NO could also influence parameters of the carbonate system in the culture media. NO participates in the regulation of marine microalgae photosynthesis, which may influence the parameters of carbonate system.

Behavioral Ecology

Marine and Freshwater Biology

Marine microalgae

Nitric oxide (NO)

Growth

Carbonate system

Dual role

Photosynthesis

Nitric oxide (NO) is an important signaling molecule that regulates a wide range of physiological, cytological, and molecular processes in animals and plants (Wendehenne et al. 2001; Li and Moore, 2007; Lushchak 2011; Domingos et al. 2015). In comparison with animals and plants, the knowledge about NO in the marine systems is still limited. NO is an important intermediate in the marine nitrogen cycle. The sources of NO in seawater are mainly involved in photochemistry (Zafiriou and True 1979; Olasehinde et al. 2009) and biological activity (Lipton et al. 1993; Martens-Habbena et al. 2014). Sunlight photolyzes NO₂⁻ in surface seawater and generates a low concentration of NO. The concentration of NO in the surface seawater was about 10^− 12 to 10^− 10 mol L^− 1 (Zafiriou and McFarland 1981; Olasehinde et al. 2009; 2010; Anifowose and Sakugawa 2017; Tian et al. 2020), moreover, NO showed higher concentration levels in coastal waters than in open sea (Tian et al. 2020).

Previous research focused on the production of NO and its physiological functions of NO on marine phytoplankton. Several reports have suggested that algae and cyanobacteria could produce NO (Mallick et al. 1999, 2000; Sakihama et al. 2002; Tischner et al. 2004). We also detected NO concentrations in the culture media of several species of marine microalgae during their growth. NO with the concentrations of 10^− 9−10^− 8 mol L^− 1 was observed in the algal culture pond of Daya Bay (1–300 m³) before the algal density reached its maximum, revealing the possible relationship between the NO concentration and algal bloom (Zhang et al. 2006c). The NO₃⁻ reductase (NR) pathway and a NO synthesis enzyme might be responsible for the NO generation of algae (Kim et al. 2006, 2008; Tang et al. 2011). Sakihama et al. (2002) reported that NR is involved in the NO₂⁻-dependent NO production in C. reinhardtii. Tischner et al. (2004) concluded that mitochondria can produce NO under anoxia and sufficient NO₂⁻ supply in C. sorokiniana. However, the NO production mechanism in microalgae and cyanobacteria is still unclear.

Microalgae and cyanobacteria can not only generate endogenous NO, but also respond to exogenous NO. It has been documented that NO can control the growth of microalgae depending on the local concentration of NO (Leshem et al. 1998; Zhang et al. 2005, 2006b; Lehner et al. 2009; Misra et al. 2014). Tang et al. (2011) pointed out the promotion effect of NO on the growth of M. aerugrinosa and the correlation of NO with the outbreak of cyanobacterial bloom was revealed to some extent. NO is an indicator of microalga growth and a signal molecule of the stress response (Zhang et al. 2006a, 2006b, 2006c). Previous studies showed that NO was involved in the protective effects under abiotic and biotic toxicity stresses including copper ion, sodium selenium, lead nitrate, methomyl, and ultraviolet (UV) radiation stress in marine algae (Mackerness et al. 2001; Mittler et al. 2004; Chen et al. 2003; Singh et al. 2004; Liu et al. 2008; Feng et al. 2011; Xiao et al. 2012; Li et al. 2013; Misra et al. 2014). Moreover, the function and role of NO in marine ecosystem requires more research and exploration.

Direct addition of NO and NO donors were used to investigate the effects of NO on the microalgae. NO is a short life bioactive molecule with the half-life ranging from 5 to 13 s (Gupta et al. 2011), however, it is relatively stable at very low concentration in seawater and plants (Zafiriou and Mcfarland 1981; Planchet et al. 2005). NO is a signaling molecule with high diffusion rate and high reactivity. As a free radical molecule, NO diffuses readily through biological membranes and reacts with metal complexes, radicals, and biomolecules such as nucleic acids, proteins and lipids (Wink and Mitchell 1998). NO donors are compounds which can continuously release NO when applied to biological systems to mimic endogenous NO-related responses. Sodium nitroprusside (SNP) is the most commonly used NO donor with the half-life of 12 h (Floryszak-Wieczorek et al. 2006).

The oceanic carbonate system, closely related to the photosynthetic process of phytoplankton, is important in many branches of marine chemistry and biology (Wilhelm et al. 2006; Chi et al. 2011). Exploring the factors that influence marine phytoplankton photosynthesis is important to the study of the global carbon cycle. Hill and Bennett (1970) reported that carbon dioxide (CO₂) assimilation rates are inhibited by the increased NO gas for the fumigation of trees. Yamasaki (2000) demonstrated that excessive NO directly inhibits CO₂ assimilation and electron flow in the mitochondrial inner membrane of photosynthesizing organisms. Despite the recognized importance of the role of marine phytoplankton in the global carbon cycle, which yield approximately 50% of the total global primary productivity, few studies have investigated the influence of NO on the carbonate system in seawater.

This study investigated the modulating function of NO on the growth of five marine phytoplankton species, namely, Platymonas helgolandica var. tsingtaoensis, P. subcordiformis, S. costatum, Gymnodinium sp., and Prorocentrum donghaiense, and corresponding variations of parameters of CO₂ system in the S. costatum culture media after adding different concentrations of SNP. Marine green algae P. helgolandica var. tsingtaoensis and P. subcordiformis are widely used as high-quality plant baits for seafood larvae. The red tide species S. costatum, Gymnodinium sp., and P. donghaiense frequently cause blooms in the East China Sea in early summer. The present research sheds light on the effect of NO on the growth of the marine algae and the parameters of carbonate system.

Algal cultivation

The strains of P. helgolandica var.tsingtaoensis, P. subcordiformis, S. costatum, Gymnodinium sp., and P. donghaiense were obtained from the Marine Pollution Eco-Chemistry Laboratory, the Ocean University of China, Qingdao. The seawater used in the experiment was collected from the East China Sea, then sterilized in a LDZX-II Autoclaves Sterilizer (Shanghai Shenan Medical Instrument Factory, China) for 20 min after being filtrated through a cellulose acetate filter (0.45 µm). All the bottles were treated by the following procedure: first soaked in hydrochloric acid for 24 hours, then washed with ultra-pure water, and finally sterilized in a Autoclaves Sterilizer as seawater. All cultures were axenic and unialgal. The strain was inoculated by adding 100 mL of the stock culture media during the exponential growth phase to a 500 mL Erlenmeyer flask containing 300 mL f/2 medium (Guillard, 1975). No nutrients were added during the experiment. The media were initially adjusted to pH 8.100 ± 0.001 with HCl. Each group had three replicates. All cultures were kept at 25.0 ± 0.1 °C in an illumination incubator (GXZ-380B, Ningbo Jiangnan Instrument Factory, Ningbo, China). The light condition was set as 14 h light and 10 h dark with an average photon flux density of 134 µmol m⁻² s⁻¹ (Liu et al. 2014b). The flasks with sterilized filter paper were shaken twice a day and randomly replaced on the shelves to avoid minor differences in irradiance exposure.

Preparation of nitric oxide and sodium nitroprusside solutions

Two milliliters of Milli-Q water sealed in a 10 mL vial was bubbled with high purity nitrogen gas (99.999%, Heli Industry Gas Center, Qingdao, China) at 10 mL min⁻¹ (6 mm diameter tube) for at least 30 min to remove oxygen in the water. The deoxygenated water was then bubbled with high purity NO gas (99.9%, Date Gas Ltd, Dalian, China) for 30 min until reaching NO saturation with the concentration of about 1.4 × 10⁻³ mol L⁻¹ (Lantoine et al. 1995; Zhang et al. 2003). Standards were freshly made for each experiment, and different concentrations of NO solutions were obtained by diluting the saturated solution using Milli-Q water (18.2 MW cm, Millipore Company, USA).

A total of 1.4790 g of sodium nitroprusside (SNP) (Sigma-Aldrich company, USA) was weighed accurately and poured into 100 mL of Milli-Q water that was subjected to high-pressure sterilization. A SNP stock solution of 50 mmol L⁻¹ was obtained. Stock solutions were freshly made for each experiment.

Experimental design

The experimental cultures were exposed to different concentrations of NO or SNP solutions after inoculation. Some saturated or diluted NO solutions were added into the cultivation of five marine microalgae rapidly with a gas-tight syringe. The initial concentrations of NO in the culture media were 0 (the control), 1.4 × 10⁻⁹, 1.4 × 10⁻⁸, 1.4 × 10⁻⁷, and 1.4 × 10⁻⁶mol L⁻¹, respectively. Preliminary experiment results showed that the two food algae and the three red tide algae have different responses to the same concentration of SNP. Therefore, the initial concentrations of the SNP solutions in the cultures were set to 0, 0.1, 1.0, 10, and 100 μmol L⁻¹ for the two food algae and 0, 0.01, 0.1, 1.0, and 10 μmol L⁻¹ for the three red tide algae. Different volumes of the SNP stock solution were added immediately into the culture media.

Biological analysis

An Olympus CX-31 microscope (Olympus Corporation, Tokyo, Japan) was used for Algal cells counting. An aliquot of 5 mL culture media was sampled and then preserved in Lugol’s buffer solution. After that the cell were counted in triplicate by 1 mL Sedgwick-Rafter counting chambers (Liu et al. 2014a).

Determination of carbonate system parameters in the culture media

The pH value in the culture samples was analyzed using a Ross Orion combination electrode (Ross-8102) and a Fisher pH meter (AR15) on a NBS scale (precision: ± 0.005) under the cultivation temperature. The DIC was measured by a DIC analyzer (AS-C2, Apollo SciTech Inc., Georgia, USA) (Cai and Wang, 1998; Hu et al. 2015). An aliquot of a 0.5 mL sample was acidified by the addition of 10% H₃PO₄ solution (0.5 mL). The extracted CO₂ gas was then determined through a nondispersive infrared CO₂detector (LI-6262, Li-COR Inc., USA) with a precision of 0.1%–0.2%. Certified reference materials from the A.G. Dickson of the Scripps Institution of Oceanography were used for calibration. The CO2SYS software (Pierrot et al. 2006) was used for calculating the concentrations of bicarbonate (HCO₃⁻) and pCO₂ from the values of pH and DIC. The equilibrium constants for calculation was from Mehrbach et al. (1973) refit by Dickson and Millero (1987) with KHSO₄ from Dickson (1973) as described in the report by Hu et al. (2015). The precision of calculated pCO₂ was 2.5 μatm (Grasshoff et al. 1999).

Determination of NO concentration of the media

The concentration of NO in the culture media was determined by an ISO-NO Mark II NO meter (WPI Inc., Sarasota, USA) connected with an ISO-NOPMC microsensor. A DUO18 two-channel data-acquisition system (WPI Inc., Sarasota, USA) connected to a PC computer was used for the analog signal digitizing (Zhang et al. 2006c). The detection limit of the method was 4.2×10⁻¹⁰ molL⁻¹. The experiment of precision showed that the relative standard deviation was 6.3% (Xing et al. 2005).

Statistics

Each group had three replicates. All data were expressed as mean ± standard deviation. Statistical analysis was performed with origin 8.0 software (OriginLab Corp. Northampton, Massachusetts, USA). Comparison of two groups was performed via t-test. Mann Whitney Rank Sum test was used if datasets did not fulfill constraints of normal distribution and homogeneity of variance for t-test.

The variations of NO concentration in the f/2 medium and Gymnodinium sp. cultures after adding 1.4 × 10^− 6mol L^− 1of NO

In order to understand the variations of NO when added into the culture media, we monitored the concentrations of NO after 1.4 × 10^− 6 mol L^− 1 of NO was added into the f/2 medium and Gymnodinium sp. cultures (Fig. 1). Results showed that the concentration of NO decreased rapidly in both f/2 medium and Gymnodinium sp. culture medium. The concentrations of NO dropped to 10^− 8 mol L^− 1 in these two culture media after 2 hours, furthermore, it decreased more rapidly within 2 hours in the Gymnodinium sp. culture media, implying that microalgae might utilize exogenous NO. Then the concentrations of NO declined continually until it reaching 10^− 9 mol L^− 1 order of magnitude and were similar in both media. After 6 hours the concentrations of NO were higher in the Gymnodinium sp. culture media than those of f/2 media (t(3) = 3.31, p༜0.05), showing that Gymnodinium sp. could produce more NO endogenously with the concentration of about 10^− 9 mol L^− 1. Moreover, the concentrations of NO in the Gymnodinium sp. culture media and f/2 media remained in the order of magnitude of 10^− 9 mol L^− 1. The magnitude of NO concentration was consistent with that observed in the natural environment with the range from 10^− 9 to10^− 8 mol L^− 1 in the algal culture pond of Daya Bay (Zhang et al. 2006c).

Effects of NO solutions on the growth of the marine microalgae

Though the concentration of NO declined rapidly in the culture media, the effect of NO on the growth of microalgae was not only instantaneous. The addition of NO to the incubation culture only once even influenced the whole growth of the microalgae (Zhang et al. 2005). To avoid the instantaneous disturbances of NO, the exponential phase (Day 8) was chosen to investigate the effects of NO on the growth of microalgae. The cell densities of the five cultured microalgae on Day 8 were shown in Fig. 2. The algal growths in the case of P. helgolandica var. tsingtaoensis, P. subcordiformis, and P. donghaiense were promoted when different concentrations of NO were added during the cultivation. That is, 1.4 × 10^− 6 mol L^− 1 of NO had the optimum stimulatory effect on the growth of microalgae with an increase of 18.9%, 38.3%, and 23.4% in cell density for P. helgolandica var. tsingtaoensis, P. subcordiformis, and P. donghaiense, respectively. Moreover, 1.4 × 10^− 9–1.4 × 10^− 7 mol L^− 1 of NO solutions slightly promoted the growth of these three microalgae. The cell densities increased by 8.4–11.3%, 12.9–31.1%, and 7.5–15.0%, respectively. The promotion was obvious when the concentration of NO solution was high. In S. costatum and Gymnodinium sp. culture media, 1.4 × 10^− 6 mol L^− 1 of NO marked the increase in the growth of microalgae with cell density increases of 10.1% and 9.8%, respectively. The other concentrations of NO showed minimal effect on the growth of these microalgae, and no significant difference was observed.

Effects of SNP solutions on the growth of the marine microalgae

The cell densities of P. helgolandica var. tsingtaoensis, P. subcordiformis, S. costatum, Gymnodinium sp., and P. donghaiense on Day 8 are shown in Fig. 3. P. helgolandica var. tsingtaoensis and P. subcordiformis showed a significant decrease of 84.3% and 38.8% in cell density, respectively, after adding 100 µmol L^− 1 of SNP solution compared with those of the control groups. The SNP with 0.1 µmolL^− 1 concentration had little effect on the growth of P. helgolandica var. tsingtaoensis. However, 10.8% and 13.1% increases were observed when 1.0 and 10 µmol L^− 1 SNP solutions were added, respectively. When the SNP concentrations were 0.1, 1.0, and 10 µmol L^− 1, the growth of P. subcordiformis was promoted and the cell densities increased by 8.3%, 10.8%, and 14.3%, respectively.

For the three red tide algae, namely S. costatum, Gymnodinium sp., and P. donghaiense, SNP with the concentration of 10 µmol L^− 1 strongly inhibited the growth of microalgae. The decreases in cell densities were 95.3%, 99.9%, and 97.5%, respectively, which indicated that no exponential growth was observed during cultivation. A slight decrease in final cell density compared to the control was observed in the S. costatum culture at 1.0 µmol L^− 1 of SNP. However, a slight increase was observed when the culture media was supplemented with 0.1 µmol L^− 1 of SNP. The SNP concentration of 0.01 µmol L^− 1 presented little influence on the growth of S. costatum. A similar phenomenon was observed in the culture of P. donghaiense after adding different concentrations of SNP solutions. An obvious decline in the cell density of Gymnodinium sp. was observed, except when 0.01µmol L^− 1 of SNP was added. The decreases were 26.7% and 84.3% for 0.1 and 1 µmol L^− 1 of SNP, respectively.

Effects of SNP solutions on the parameters of the carbonate chemistry

The variations of the CO₂ parameters in the culture media of S. costatum on Day 8 are shown in Table 1. The values of HCO₃⁻, CO₃²⁻, pCO₂, CO₂, and TA were calculated from pH and DIC. The initial pH value was 8.195 ± 0.001 and the initial DIC value was 1969 ± 13 µmol kg^− 1. The pH value decreased to 8.079 ± 0.078 and DIC increased to 2000 ± 62 µmol kg^− 1 when 10 µmol L^− 1 of SNP was added to the culture media, which strongly inhibited the growth of microalgae. The variations of pH were less than 0.1 units after the supplement of 0.01, 0.1, and 1.0 µmol L^− 1 of SNP compared with the control. The DIC values are similar when exposed to 0.01, 0.1, and 1.0 µmol L^− 1 of SNP, respectively. The values of pCO₂ were less than 25 µatm in the algal culture media with the addition of 0.01, 0.1, and 1 µmol L^− 1 of SNP. However, pCO₂ increased to 533 ± 2 µatm when 10 µmol L^− 1 of SNP was added to the culture media. The initial percentage of HCO₃⁻, CO₃²⁻, and CO₂ concentration was 86.1%, 13.5% and 0.4% respectively. Meanwhile, the percentage of HCO₃⁻, CO₃²⁻, and CO₂ concentration was 91.4%, 7.8%, and 0.8% respectively when 10 µmol L^− 1 of SNP was added to the culture media. The variation characteristic of HCO₃⁻ was similar with that of the DIC considering that HCO₃⁻ is the main component of DIC in seawater. However, the percentage of HCO₃⁻ in the S. costatum culture media with the supplement of 0, 0.01, 0.1, and 1.0 µmol L^− 1 of SNP was 48.8%, 57.1%, 55.8%, and 54.4%, respectively. The corresponding percentage of CO₃²⁻ was 51.0%, 42.9%, 44.2%, and 45.6%, respectively. These observations suggest that the parameters of carbon system could be influenced by the addition of NO.

Table 1

Variations of the parameters of carbonate chemistry in the *S. costatum* culture media after adding different concentrations of SNP solutions, DIC means dissolved inorganic carbon, pCO₂ means partial pressure of CO₂, TA means total alkalinity.
SNP concentration (µmol L^− 1)	pH	DIC (µmol kg^− 1)	TA (µmol kg^− 1)
initial	8.195 ± 0.001	1969 ± 13	2339 ± 16
0	9.019 ± 0.071	1250 ± 88	2211 ± 60
0.01	9.023 ± 0.062	1232 ± 66	2040 ± 32
0.1	9.047 ± 0.088	1219 ± 58	2045 ± 28
1.0	9.072 ± 0.092	1214 ± 82	2062 ± 56
10.0	8.079 ± 0.078	2000 ± 62	2208 ± 49

Effects of NO on the growth of the marine microalgae

The present study employed NO and SNP solutions to investigate the effect of NO on the growth of the marine microalgae. Previous study suggested that the real NO concentrations released by 5, 10, and 100 μmol L⁻¹ of SNP are approximately 6 × 10⁻⁹, 9 × 10⁻⁹, and 2 × 10⁻⁷ mol L⁻¹ and with the release time of 4, 5.5 and 7.5 h, respectively (Liu et al. 2010). However, high reactivity of NO reduces its concentration rapidly in the culture media when direct NO solution was added to the media. Therefore, the addition of NO and SNP solutions represented the short- and long-term effects of NO on algae growth, respectively. The most fundamental reason is the effect of the final NO concentration on the growth of microalgae. It is noteworthy that the function of NO in the algae growth is not in such a way that NO first changed into NO₂ and then into NO₃⁻ or NO₂⁻ after it was added into the cultivation medium (Zhang et al. 2005).

The results showed that the growth of algae was promoted when 10⁻⁹–10⁻⁶ mol L⁻¹ of NO solutions were added to the algae culture media. That is, the addition of 10⁻⁶ mol L⁻¹ of NO solution exhibited the optimum stimulatory effect on all the five microalgae. The other concentrations of NO had less influence on the growth of algae. Moreover, different algae had different responses to the same concentration of NO. No significant difference on the effect of NO solution was identified between the food and red tide algae. The present study was in consist with previous study that different phytoplankton has different optimum NO concentration (Zhang et al. 2005). However, food algae and red tide algae showed different response to the same concentration of NO, which was not observed in the present study when different concentrations of NO solutions were added to the five microalgae cultures. Such difference might be caused by the different temperature, light irradiance, NO addition type and species of microalgae.

However, a distinct difference in the responses to the SNP solution was revealed between these two types of algae. SNP with the concentration of 100 μmol L^–1 strongly inhibited the growth of the two food algae. 10 μmol L^–1 of SNP had a similar effect on the growth of the red tide algae, however, a slight promotion effect on the two food algae was observed with the same concentration of SNP. Similar results were found in the experiment with 1 μmol L^–1 of SNP addition, which promoted the growth of the food algae. Different results were found for the three red tide algae, indicating that the red tide algae were more sensitive to exogenous NO than food algae.

The NO concentration released by 10 μmol L⁻¹ of SNP was equivalent to 9 × 10⁻⁹ mol L⁻¹ of NO for about 5.5 hours (Liu et al. 2010). The short stimulation of 10⁻⁹ mol L⁻¹caused by the NO solutions had little promotion effect on the growth of the five examined microalgae (Fig. 2). The long stimulation of 10⁻⁹ mol L⁻¹ caused by the 10 μmol L⁻¹ of SNP solution promoted the growth of the food algae. However, it greatly inhibited the growth of the red tide algae. Meanwhile, similar results were found when the microalgae were exposed to 1.0 μmol L⁻¹ of SNP. Thus, SNP with the concentration of 1.0 and 10 μmol L⁻¹ stimulated the growth of food algae while inhibited the growth of red tide algae. In the present study, the real NO concentration, which promoted the growth of red tide algae, was less than 10⁻⁹mol L⁻¹ given the NO release rules of the SNP and was much lower than the concentration of the direct NO addition. This difference may be related to the duration function of the NO released by SNP in the culture media. SNP with the concentration of 100 and 10 μmol L⁻¹ can produce 2 × 10⁻⁷and 9 × 10⁻⁹ mol L⁻¹ NO and maintain 7.5 and 5.5 hours, respectively (Liu et al. 2010). Such concentration of NO produced by SNP can roughly maintain a stable concentration within a certain period, however, the direct addition of NO can rapidly disappear because of its instability. These results indicated that the function of NO on microalgae was closely related to the stimulation time and the concentration range. Moreover, the effect of SNP on the food algae was different from that on the red tide algae.

Previous studies suggested that NO could modulate the cell growth of microalgae by influencing the activity of the enzymes (Lehner et al. 2009). Exogenous NO increased photosynthesis rate of P. tricornutum, especially under high light environment, which could be explained that NO protected cell structure from high light damage (Wang et al. 2013). Moreover, Nagase et al. (2001) found that little NO was oxidized in the medium before its uptake by algal cells and NO mostly permeated directly into the cells by diffusion. Therefore, NO can be considered as a signaling molecule in marine microalgae as found in plant.

Physiological functions of NO

NO was once regarded as a poisonous air pollutant, nowadays it is mostly considered as a signaling hormone in many physiological processes in animals and plants. It is a key signaling molecule that controls plant growth and development, however, when concentrations of NO are too high, it is toxic to cells (Beligni and Lamattina, 1999a, 1999b). A relatively lower concentration of NO enhances the photochemical efficiency, increases the net photosynthesis and ameliorates the stress effects on chloroplasts. Misra et al. (2004) proved that NO could act as a regulator of photosynthetic electron transport and regulate the activity of the photosynthetic electron transport either by modifications of the oxygen evolving complex or by activation of the cyclic electron transport around PSII.

The protective mechanism of NO to counteract the abiotic and biotic stress including heavy metal, salinity, UV, herbicide might be associated with the ability of scavenging reactive oxygen species (ROS). The role of NO was achieved by up-regulating the activity of antioxidant enzymes such as Superoxide Dismutase (SOD), guaiacol peroxidase, and glutathione reductase (Mackerness et al. 2001; Uhida et al. 2002; Kopyra and Gwóźdź 2003; Shi et al. 2005; Qian et al. 2009).

The decomposition of SNP yields NO and FeC₅N₆, the latter of which decomposes to cyanide (CN) and Fe. Lehner et al. (2009) clearly showed that the changes induced by SNP can be ascribed to NO action and not to a release of CN. NO could inhibit the activities of enzymes involved in the secretory pathway, such as Glyceraldehyde-3-phosphate dehydrogenase via S-nitrosylation of the cysteine residue and, consequently, modulates cell growth of green alga M. denticulata (Lehner et al. 2009). Until now, the effect of NO on the marine phytoplankton is still unclear. However, more information revealed that NO acted as molecular messenger as observed in plants (Kumar et al. 2015).

Inorganic carbon parameters in marine microalgae culture media

Biological photosynthesis can absorb CO₂ in seawater, thereby resulting in increased seawater pH and decreased DIC and pCO₂. Note that the absorption of CO₂ of the marine microalgae was underestimated because of the low pCO₂ during the incubation period. The concentrations of HCO₃⁻ decreased significantly in the cultures of S. costatum when supplemented with 0.01–1.0 μmol L⁻¹ of SNP. A slight increase of DIC value was observed in the culture of 10 μmol L⁻¹ of SNP, which was consistent with the growth of S. costatum. These results suggested that no carbon was consumed, in contrast, CO₂ may be absorbed by the culture media or S. costatum released certain concentration of inorganic carbon to the culture media leading to a rise of DIC. Despite of slight increase in the cell density in the S. costatum culture after the addition of 0.1 μmol L⁻¹ of SNP, carbon parameters showed no significant difference in comparison with the control group, suggesting there may exist a time lag for the carbon parameter variations.

Some researchers have suggested that NO is associated with the photosynthesis process. The fumigation of NO immediately reduces the rate of photosynthesis in plants (Hill and Bennett 1970; Caporn 1989). Yamasaki (2000) found that excessive NO directly inhibits CO₂ assimilation and electron flow in the mitochondrial inner membrane of photosynthesizing organisms. Our examination of the parameters of the carbonate chemistry demonstrated that CO₂ assimilation was affected by different concentrations of NO, high concentrations of NO (100 μmol L⁻¹ of SNP) restrained CO₂ assimilation, whereas low concentrations of NO (0.01–1.0 μmol L⁻¹ of SNP) promoted CO₂ assimilation, which was consistent with the observations in plants.

The experimental results of adding NO and SNP solutions into the marine microalgae culture media showed that exogenous NO had different effects on the growth of five marine microalgae. The direct NO and SNP additions showed different patterns of growth for the three red tide algae, which can be explained as that SNP results in a long NO action on algae and exhibits different behaviors. The two food algae and three red tide algae showed different responses to the same concentration of NO. Moreover, the red tide algae were more sensitive to SNP additions than the food algae. These results might be caused by the species-specific characteristics of the microalgae.

NO with the concentration of 1.4 × 10^− 6 mol L^− 1 had the optimum stimulatory effect on the growth of microalgae. While the other concentrations of NO showed little effect. SNP solution with the concentration of 100 µmol L^− 1 inhibited the growth of the two food algae, however, 10 µmol L^− 1 SNP solution additions to the three red tide algae inhibited the growth greatly. Low concentrations of SNP (0.1 µmol L^− 1 for the two food algae and 0.01 µmol L^− 1 for the red tide algae) promoted the growth of microalgae. The parameters of carbon system could be influenced by NO addition, suggesting that NO could regulate CO₂ assimilation of algae. However, the regulation function of NO on the photosynthesis of marine microalgae and the function mechanism need further study.

Funding This research was supported by the National Natural Science Foundation of China (grant nos. 41676065 and 40706040) and the National Key Research and Development Program of China (grant no. 2016YFA0601301).

Conflicts of Interest There are no conflicts of interest for this submission.

Ethics approval The authors conform that the work described was original research that has not been published previously, and not under consideration for publication elsewhere. Citations to appropriate and relevant literature are used throughout.

Data availability statement The datasets of the current study are available from the corresponding author on reasonable request.

Author contribution All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Zhao Min. The first draft of the manuscript was written by Li Peifeng and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Effects of Nitric Oxide On The Growth of The Marine Microalgae And The Parameters of Carbonate Chemistry

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