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

Nitric oxide (NO) is a non-traditional regulator for the growth of plant and phytoplankton. This study monitored the growth of ve 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 inuence parameters of the carbonate system in the culture media. NO participates in the regulation of marine microalgae photosynthesis, which may inuence the parameters of carbonate system.

). Sakihama et al. (2002) reported that NR is involved in the NO 2 − -dependent NO production in C. reinhardtii. Tischner et al. (2004) concluded that mitochondria can produce NO under anoxia and su cient NO 2 − 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. 2005Zhang et al. , 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(Zhang et al. , 2006b(Zhang et al. , 2006c

Materials And Methods
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 ltrated through a cellulose acetate lter (0.45 µm). All the bottles were treated by the following procedure: rst soaked in hydrochloric acid for 24 hours, then washed with ultra-pure water, and nally 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 ask containing 300 mL f/2 medium (Guillard, 1975 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 ve 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 −6 mol 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

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% ).

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 ful ll constraints of normal distribution and homogeneity of variance for t-test.

Results
The

Effects of SNP solutions on the parameters of the carbonate chemistry
The variations of the CO 2 parameters in the culture media of S. costatum on Day 8 are shown in Table 1.
The values of HCO 3 − , CO 3 2− , pCO 2 , CO 2 , 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.  Disscussion 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 nal 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 rst changed into NO 2 and then into NO 3 − or NO 2 − after it was added into the cultivation medium .
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 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 −1 caused by the NO solutions had little promotion effect on the growth of the ve 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 −9 mol 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 −7 and 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 in uencing 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 ). 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 Lamattina, 1999a, 1999b). A relatively lower concentration of NO enhances the photochemical e ciency, 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 modi cations 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 The decomposition of SNP yields NO and FeC 5 N 6 , 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 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 CO 2 assimilation and electron ow in the mitochondrial inner membrane of photosynthesizing organisms. Our examination of the parameters of the carbonate chemistry demonstrated that CO 2 assimilation was affected by different concentrations of NO, high concentrations of NO (100 μmol L −1 of SNP) restrained CO 2 assimilation, whereas low concentrations of NO (0.01-1.0 μmol L −1 of SNP) promoted CO 2 assimilation, which was consistent with the observations in plants.

Conclusions
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 ve 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-speci c characteristics of the microalgae. Con icts of Interest There are no con icts 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 rst 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 nal manuscript.