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 10−9 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 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.