2LTO laboratory, South China Sea Institute of Oceanography, Chinese Academy of Sciences, Guangzhou, 510301, China
3Laboratory of Atmospheric Environment, East China University of Technology, Nanchang, 330013, China
Keywords: coupling effect; Phaeodactylum tricornutum; Thalassiosira weissflogii; silicon utilization;
Iron, although in the crust the fourth element of abundance, has very low concentration in seawater due to its extremely solubility and easily being absorbed on the particulate matter surface in ocean. It plays an important role in phytoplankton’s physiological activities, such as electron transportation, oxygen metabolism, nitrogen fixation, photosynthesis and respiration [25,19]. Iron receives more attention than other trace elements because 40% of the world ocean has been thought to be Fe limited . Some iron can reach surface seawater with upwelling  and provide trace metal nutrition for coastal phytoplankton, while only dissolved iron is biologically effective [28,3]. Iron demand by marine phytoplankton is very high compared to its low content, indicating that iron may be one of limiting factors in ocean [20,5].
Iron deficiency has been considered to be the main reason for low biomass of phytoplankton in the high-nitrogen-lowchlorophyll (HNLC) area [17,2]. The limiting effect of iron on marine primary production has been testified by some field experiments [4,34]. And some laboratory culture experiments also seem to confirm the results of the iron fertilizing experiment in the wild . For instance, growth rate of Thalassiosira weissflogii and Prorocentrum minimum was significantly limited when iron concentration is less than 100 pM in culture experiments .
The researchers found some very interesting phenomena concerning iron restriction on marine phytoplankton. For example, under iron limiting conditions, phytoplankton consumption of silicon is much higher than nitrogen, resulting in a common restriction of iron and silicon [26,13,17]. Iron and light had co-limiting effect on the growth of phytoplankton in areas with low iron inputs such as HNLC area . However, due to the difficulty in identifying these factors, the phenomenon of the joint limitation of iron, silicon and other factors has not attracted enough attention as they deserve [49,50,24]. In the present work, we try to focus on the coupling effect of silicate and iron on the growth of Phaeodactylum tricornutum and Thalassiosira weissflogii through single and full factor experiments respectively, and to further analyze and identify the effects of different Abiotic factors, including silicate, iron, temperature, light intensity, salinity. In single factor experiment, there is only one factor is changed and the rest of the test factors remain unchanged, the specific role and influence of this factor is determined by observing the change of experimental system caused by changing this factor. The main purpose of single-factor experiment is to prepare the orthogonal test and provide a reasonable range of data for orthogonal experiment. Full factorial experiment design is involved in all levels of the experimental factors which are comprehensively combined to form different experimental conditions. Two or more independent experiments are repeated under these conditions. The greatest advantage of design is that a large amount of information can be obtained to accurately assess the size of the main effects of each factor and the magnitude of interactions between factors. Its biggest drawback is that it needs the most experiments and consumes more manpower, material resources and time.
The diatoms were inoculated in 500 mL polycarbonate conical flask with 300 mL f/2 medium and cultivated in light incubator under following conditions: temperature 21 ± 0.5oC, light intensity 81.25 μmol photons•m-2•s-1, light : dark cycle 12h:12h, and salinity 30‰. In the process of culture, algae cells growth was observed daily, and subculture was carried out regularly to check whether the cells are stained or contaminated with harmful organisms under microscopic.
Algae cells were harvested in the exponential period, centrifuged under 3000 rpm for 10 minutes and cleansed by 0.4 mol•L-1 NaCl solution three times to reduce the effect of residual nutrients on the next culture medium. Diatom cells was acclimated in iron-starvation status for 2 weeks before used in iron specific experiments to eliminate the influence of iron adsorbed by diatom cells.
In full factorial experiment, initial levels of silicate, iron, temperature, light intensity and salinity were setup using Minitab for statistical analysis (P < 0.05) as shown in table 1. The low level and the high level of each factor presented as “L” and “H”, respectively. Each match of two factors could be performed under four kinds of matches (“HH”, “HL”, “LH”, “LL”) to investigate their interaction effects comprehensively.
Bio-silicon was analyzed at the end of cultivation in order to investigate the utilization efficiency of diatom cells on silicate. Subsample of 30 mL algal culture medium was filtered through 0.2 μm polycarbonate membrane (Millipore, USA) at the end of cultivation. The membranes were folded and put into 50 mL plastic centrifuge tube, extracted for 10 minutes in a 95oC water bath after adding 10 mL of 0.2 mol.L-1 NaOH solution. The extract is cooled to room temperature, HCl neutralized and centrifuged, and the content of silicates was determined then.
Both diatom algae’s seemed had same optimum light intensity 231.25 μmol photons•m-2•s-1 for growth as our single factorial experiments revealed. In this light intensity, the cell density of Phaeodactylum tricornutum and Thalassiosira weissflogii at exponential stage amounted to 4.21×106 cells•cm-3 and 5.37×105 cells•cm-3 respectively. Either insufficient or excessive irradiance inhibited the growth of diatom cells, the slowest growth of two diatoms appeared under light intensity 81.25 μmol photons•m- 2•s-1.
It was revealed from figure1 that Phaeodactylum tricornutum was seemly less sensitive to salinity than Thalassiosira weissflogii. There was no obvious difference of growth rate and cell density of Phaeodactylum tricornutum under various salinity conditions in the early days of experiments. The effect of salinity on the growth of algae cells did not begin until 13th day, when the algae cells density in the medium with salinity of 35‰ was obviously higher than in culture solutions with other salinities. The cell density maximized 3.87×106 cells•mL-1 under salinity 35‰ and minimum cell density was 2.36×106 cells•mL-1 under salinity 15‰. For diatom Thalassiosira weissflogii, the effect of salinity on cells growth was another. The cells had a relatively different growth rate under different salinity conditions, and the cell density maximized 3.85×105 cells•mL-1 on 11th day under salinity 30‰, significantly higher than other four groups.
The inhibitory effect of low concentration of iron on these two diatoms growth was also evident. In iron concentration 5 nmol•L-1, 10 nmol•L-1 and control group, the growth of algal cells was almost stagnant, while the increase of iron concentration significantly accelerated the growth of algal cells. Algal cell density of Phaeodactylum tricornutum and Thalassiosira weissflogii reached maximum under the same initial iron concentration 10 μmol•L-1, amount to 5.07×106 cells•mL-1 and 7.96×105 cells•mL-1 respectively.
Figure (3a) revealed the interaction effects of temperature, light intensity, salinity and iron contents on cell density of Phaeodactylum tricornutum, and no interaction effect of silicate and iron was found. The specific performances of these interactive effects were shown in figure (3c) in detail. High temperature inhibited the growth of Phaeodactylum tricornutum in all interaction designs, while high iron concentrations significantly boosted the growth of diatom cells in all interaction designs. The coupling effects of low temperature and high iron concentration was relatively obvious. While the coupling effect of temperature and light intensity was not significant, and high light intensity inhibited the diatom cells growth slightly at high temperature indeed. As to biogenic silica contents diatom Phaeodactylum tricornutum cells, the interaction of temperature and silicate, temperature and iron had significant affects figure (3b). The interactive effect of temperature and contents of silicate and iron was that when temperature remained constant, the increase of iron concentration obviously promoted the biological uptake of silicate by algal cells, and this coupling effect was even more obvious in low temperature environments. Although the increase of silicate contents obviously promoted the synthesis of bio-silicon, the effect of the stimulant was more significant in relative high temperature. As shown in figure (3d), environmental temperature, iron concentration, and the coupling effect of temperature and light, temperature and iron contents had significant influence on the growth of algal cells. The decrease of temperature and the increase of iron concentration were beneficial to the growth of algal cells. The effect of light on algal cell concentration was not obvious at low temperature, but when the temperature rose, the increase of light intense significantly inhibited the growth of algae cells.
Initial factorial levels
231.25 μmol photons·m-2·s-1
81.25 μmol photons·m-2·s-1
Since light serves as the energy source for photosynthesis, light limitation is regarded as one of major reasons for low phytoplankton biomass, and light intensity and duration are also considered to be determining factors in the degree of photosynthetic activity . In addition, light intensity is considered to regulate the uptake and utilization of nutrients by phytoplankton . Experiments have shown that light intensity can be a major factor in controlling diatoms growth in good mixing conditions and in nutrient-rich waters . As with temperature, the growth of these two diatoms had slightly different manifestations of light intensity response. Our experiment found that the final algal cell density of both diatoms increased with the enhancement of illumination except for the group with the strongest light conditions (250 μmol photons•m-2•s-1). In other words, in a certain range, as the light intensity increase, the photosynthetic rate of algal cells increase, till at the compensation point when the rate of photosynthesis and respiratory is equivalent. As light intensity continues to strengthen, the rate of photosynthesis will gradually decrease, and the phytoplankton will exhibit photo inhibition effect after the light saturation (Chai, 2009).
The two species of diatom algae in our experiment showed strong salinity adaptability, even if their growth rate had different response to the change of salinity condition. Cell density of Thalassiosira weissflogii was found to reach its maximum between salinity 25-35‰ , which is very similar to our experimental results. The effect of salinity on algae growth is mainly that salinity can affect the osmotic pressure of algal cells, uptake of nutrients and suspension of algal cells themselves . Because both of these two diatoms have considerable salinity adaptability, they have a wide distribution from offshore to open sea.
Diatoms need silicate as substrate to meet their growth requirements, especially to produce frustules. The silicon demand of different species of diatom mainly depends on their differences in physiology . Therefore it is reasonable that high cell density can be found in high silicate cultures as our experiments. Iron is another limiting factor for marine diatoms [20,5]. Iron is essential for phytoplankton, involved in cellular processes such as photosynthesis . The importance of iron in photosynthesis stems from its high concentrations in the photo system I and II and the cytochrome b6f complex . Iron deficiency strongly influence electron transport kinetics, reduce the efficiency in electron transfer and partial inhibit the photo system . As shown in our experiments, final cell density of both Phaeodactylum tricornutum and Thalassiosira weissflogii increased with the amount of iron added to the medium, and there was a critical concentration of iron that allowed the algae to grow into the exponential phase. It was also found that different species of algae cells had different iron demands and different tolerance for iron deficiency. Unlike Phaeodactylum tricornutum, Thalassiosira weissflogii cells almost ceased to grow at low iron concentrations, including 10, 5 nmol•L-1, and control group, which indicated that Thalassiosira weissflogii demand more iron than Phaeodactylum tricornutum to sustain cell division.
When studying effects of iron on the growth of diatoms, the researchers often made such a hypothesis that diatom growth rate was limited by iron, and biogenic silica production rates and cellular silicon content was controlled by a combined influence of both iron and silicate . Our experiments further found the coupling effect of iron and silicate was closely related to the actual combination of temperature, salinity and illumination in the growing environments, and the combined effect of various conditional factors could be different due to different algae species and other environmental conditions. For instance, iron concentration, temperature, coupling interaction between temperature and iron concentration, coupling interaction temperature and light intensity were the major factors affecting final cell density of Phaeodactylum tricornutum, but no obvious interactions among salinity, light intensity, and silicate concentration were found in our experiments. Temperature and light intensity had an inhibitory coupling effect on the growth of Phaeodactylum tricornutum mainly because the dark respiration of algae was greatly enhanced under the combination of high temperature and strong illumination [52,33]. Our experiments also showed that the coupling of iron and silicate had an important influence on the growth of these two diatoms, even iron concentration itself could be acted as a separate influencing factor to control the final cell density of Phaeodactylum tricornutum. This promotive coupling effect of iron and silicate concentration on diatom Phaeodactylum tricornutum growth was even more pronounced at low temperatures.
Environmental conditions and their coupling effects also affected the efficiency of silicate utilization by diatom cells. However, the environmental factors and their coupling interaction that affecting silicate utilization and those affecting the growth of algae cells were different in some degree. Our experiments showed that temperature, concentration of iron, and concentration silicate were the main control factors affecting the algal silicate utilization, and for different algae, these three factors had different order of influence, and they have different forms of coupling. This indicated that in addition to the environmental temperature and the content of silicate itself, the content of iron in environment played an important role in controlling the silicate utilization by diatom cells in some degree. Temperature is undoubtedly considered to be the main factor affecting algae growth . For diatom, temperature not only affects the growth and photosynthesis of algae cells, but also affects the synthesis and accumulation of chemical constituents in cells . In addition, temperature has effects on the activity of enzyme in diatom cells, the uptake efficiency of nutrients and the cycle of cell division . Diatom plays an important role in silicon recycle. Diatom uptake silicate mostly during the period of sub cells from new shell, which indicates that accumulation and storage of bio-silicon is not in the life cycle but in the process of cell wall formation . Diatoms can accumulate large amounts of silicon, which can be between 30 and 350 times the silicate concentration of the medium therefore . Coupling interaction of iron and silicate was testified to be an important controlling factor in bio-silicon synthesis by both Phaeodactylum tricornutum and Thalassiosira weissflogii in our experiments. Even for algae Thalassiosira weissflogii, in addition to being coupled with the silicate concentration, iron concentration itself also affected silicates utilization by algae cells. It has been testified that iron deficiency has an important effect on the growth and photosynthesis of phytoplankton by many laboratory experiments and the rich iron experiment in high-nutrient-low-chlorophyll (HNLC) oceans . Iron deficiency will change the proportion of macronutrient uptake by phytoplankton, reducing the uptake of nitrogen and silicate uptake increased. At the same time, under the condition of iron deficiency, silicon will be utilized by the algae at a faster rate than nitrogen, eventually lead to the coupling effect of iron and silicate, resulting iron and iron co-limiting [26,17]. `Diatoms will adapt to iron limiting condition by reducing the cell volume, the increase of diatom cell volume and thicken of diffusion boundary will change the nutrients uptake rate. Compared to carbon and nitrogen contents, silicon contents in diatom cells under iron deficiency condition will increase accordingly .
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