The Improvement of Spinach Growth by Nano-anatase TiO₂ Treatment Is Related to Nitrogen Photoreduction

Abstract

The improvement of spinach growth is proved to relate to N₂ fixation by nano-anatase TiO₂ in this study. The results show that all spinach leaves kept green by nano-anatase TiO₂ treatment and all old leaves of control turned yellow white under culture with N-deficient solution. And the fresh weight, dry weight, and contents of total nitrogen, ${\text{NH}}^{ + }_{4}$, chlorophyll, and protein of spinach by nano-anatase TiO₂ treatment presented obvious enhancement compared with control. Whereas the improvements of yield of spinach were not as good as nano-anatase TiO₂ treatment under N-deficient condition, confirming that nano-anatase TiO₂ on exposure to sunlight could chemisorb N₂ directly or reduce N₂ to NH₃ in the spinach leaves, transforming into organic nitrogen and improving the growth of spinach. Bulk TiO₂ effect, however, was not as significant as nano-anatase TiO₂. A possible metabolism of the function of nano-anatase TiO₂ reducing N₂ to NH₃ was discussed.

Introduction

As a photocatalyst, nano-TiO₂ under light could cause an oxidation-reduction reaction [1]. Therefore, we have firstly studied the relation between nano-TiO₂ and photosynthesis. Our previous results showed that nano-TiO₂ treatment could markedly promote aged seeds’ vigor and chlorophyll biosynthesis of spinach, particularly, the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) activity and the photosynthesis efficiency. The nano-TiO₂ treatment also has obvious effects on the improvement of growth and development in spinach. However, bulk TiO₂ treatment shows little effect [2]. The researches on improving photosynthesis of spinach suggested that nano-TiO₂ could increase light absorbance, accelerate transport and transformation of light energy, protect chloroplasts from aging, and prolong photosynthetic time of chloroplasts [3–5]. Our recent study proved that the complex of Rubsico and Rubisco activase could be induced in nano-TiO₂-treated spinach, which promoted Rubsico carboxylation and increased the rate of photosynthetic carbon reaction [6].

Nitrogen is an important composition of chlorophyll, amino acid, protein, etc. The synthesis of those will be inhibited under nitrogen-deficient in vivo. Having an improvement of spinach growth and obvious increase of the chlorophyll and protein contents by nano-TiO₂ treatment [2–5], the effects of nano-TiO₂ on nitrogen metabolism of spinach were studied in our previous work [7]. It demonstrated that the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic-pyruvic transaminase were obviously increased by nano-TiO₂ treatment. And nano-TiO₂ treatment could promote spinach to absorb nitrate, accelerate inorganic nitrogen (such as ${\text{NO}}^{ - }_{3} - {\text{N}}$ and ${\text{NH}}^{ + }_{4} - {\text{N}}$) to be transformed into organic nitrogen (such as protein and chlorophyll), and enhance spinach yield [7].

We hypothesize that the improvement of spinach growth and the increase of N-compound contents might be related to the generation of N₂ fixation by nano-TiO₂. As known to all, biological N₂ fixation deoxidizes N₂ to NH₃ by nitrogenase [8–11]. Schrauzer et al. [12] demonstrated that bulk TiO₂ (rutile) could reduce N₂ to NH₃ or N₂H₄ and C₂H₂ to C₂H₄ under ultraviolet radiation in the 1970s. Then they found that rutile in desert surface could reduce N₂ from the air to NH₃ and traces of N₂H₄ on exposure to sunlight [13]. This finding had an important theory and ecology significance. The total amount of N₂ fixed by rutile was previously estimated to be 10 × 10⁶ tons per year, corresponding to about a third of the N₂ that is normally oxidized by lightning discharges and to about 10% of N₂ reduced biologically. However, they did not investigate N₂ fixation of rutile on plants under sunlight. According to their results, we hypothesize that nano-TiO₂ in spinach leaves might reduce N₂ to NH₃ on exposure to sunlight, and accelerate it to be translated into organic nitrogen, thus promote spinach growth. But all of these still need to be proved by experiments. The reaction equations of rutile catalyzing N₂ fixation advanced by Schrauzer are defined in Eqs. a, b and c [12, 13].

It is well-known that the quantum effect and the photocatalysis activity of nano-TiO₂ are greater than bulk TiO₂, i.e., nano-TiO₂ could induce an oxidation-reduction reaction and release an energetic electron under light, which might reduce N₂ to NH₃ directly. Therefore, we believe that the effect of catalyzing N₂ fixation of nano-TiO₂ might be more significant than that of bulk TiO₂. The N₂ fixation of nano-TiO₂ in spinach leaves was studied in the paper. The results showed that all leaves of nano-TiO₂ treatment kept green, one part of the old leaf surface turned green, and the other was yellowish white by bulk TiO₂ treatment, whereas all old leaf surface of control displayed yellowish white under nitrogen-deficient condition, demonstrating that nano-TiO₂ had N₂-fixation effect in spinach leaves.

Materials and Methods

Material Treatment and Culture

Experimental material was Spinacia oleracea. The seeds were purchased from a local seed company. Nano-anatase TiO₂ was prepared via controlled hydrolysis of titanium tetrabutoxide. The details of the synthesis are as follow [14]: colloidal titanium dioxide was prepared via controlled hydrolysis of titanium tetrabutoxide. In a typical experiment, 1 ml of Ti (OC₄H₉)₄ dissolved in 20 ml of anhydrous isopropanol was added dropwise to 50 ml of double-distilled water adjusted to pH 1.5 with nitric acid under vigorous stirring at room temperature. Then, the temperature was raised to 60°C and kept 6 h for better crystallization of nano-TiO₂ particles. The resulting translucent colloidal suspension was evaporated using a rotary evaporator yielding a nanocrystalline powder. The obtained powder was washed three times with isopropanol and dried at 50°C until complete evaporation of the solvent. The average grain size calculated from broadening of the (101) XRD peak of anatase (Fig. 1) using Scherrer’s equation was ca 5 nm. Spinach seeds were soaked with 0.25% nano-anatase TiO₂ and bulk TiO₂ suspension for 48 h at 10°C under light (1,200 μmol m^-2 s^-1 of light intensity), respectively, and with deionized water for control. And the seeds were whole surrounded with the suspension. Bulk TiO₂ (rutile) was purchased from Shanghai Chem. Co.; the average grain size was 10–15 μm. Then, the seeds were carefully selected and planted in a perlite-containing pot and placed in porcelain dishes, which were respectively added with 500 ml of the following culture solutions: 1) Hoagland nutrient solution; 2) nitrogen-deficient Hoagland nutrient solution. Hoagland nutrient solution and nitrogen-deficient Hoagland nutrient solution were prepared as described in [9]. These were placed in a glasshouse under sunlight in spring (1,200 μmol m^-2 s^-1 of light intensity) for 35 days. Spinach seedlings in the stage of two leaves and three leaves were sprayed with 0.25% nano-anatase TiO₂ and bulk TiO₂ suspension, respectively, and deionized water for control.

Fig. 1

XRD pattern of nano-anatase TiO₂

Assay of Physiological and Biochemical Indexes

The fresh weight and dry weight of spinach were weighted at the 35th day. The contents of total nitrogen were measured by Kjeldahl’s method [15]. The chlorophyll contents were determined by Arnon’s method [16]. The protein contents were set out using the Lowry method [17]. The contents of ${\text{NH}}^{ + }_{4} - {\text{N}}$ were measured as in earlier reports [18, 19]. The oxygen evolution of spinach leaves was measured with an Oxygraph oxygen electrode (Hansatech instruments, UK). The assay medium contained 0.5 M sorbitol, 10 mM KCl, 0.5 mM MgCl₂, 0.05% (w/v) bovine serum albumin (BSA), 10 mM NaHCO₃, and HEPES-KOH (pH 7.6).

Results and Discussion

Growth of Spinach Under N-Deficient Condition

As shown in Fig. 2a, nano-anatase TiO₂ could greatly improve spinach growth. The leaf area of spinach treated by nano-anatase TiO₂ was larger than those of bulk TiO₂-treated groups and the control under culture with Hoagland solution, respectively. The single fresh and dry weights of the nano-anatase TiO₂-treated-groups were enhanced by 86.14% and 88.32% as compared with the bulk TiO₂-treated groups, and by 91% and 99% as compared with control, respectively (Figs. 3 and 4).

Fig. 2

Efect of nano-anatase TiO₂ on growth of spinach. a Cultured by Hoagland solution. b Cultured by N-deficient Hoagland solution

Fig. 3

Effect of nano-anatase TiO₂ on the fresh weight of spinach

Fig. 4

Effect of nano-anatase TiO₂ on the dry weight of spinach

From Fig. 2b, all leaves of nano-anatase TiO₂ treatment kept green. After bulk TiO₂ treatment, one part of the old leaf surface turned green, and the other was yellowish white. Whereas all old leaf surface of control displayed yellow white under culture with N-deficient Hoagland solution. We can see from Fig. 2b that the growth of spinach was inhibited under N-deficient condition, e.g., the single fresh weight and dry weight of control, bulk TiO₂, and nano-anatase TiO₂ treatment in N-deficient Hoagland solution were much lower than those of control in Hoagland solution (Figs. 3 and 4). Whereas the single fresh weight and dry weight of nano-anatase TiO₂-treated spinach were as 3.7 and 2.0, 4.1 and 2.2 times those of control and bulk TiO₂ treatment under N-deficient condition, respectively (Figs. 3 and 4), indicating that nano-anatase TiO₂ treatment could decrease the inhibition of N-deficient on spinach growth.

As known to all, nitrogen is the main composition of chlorophyll, which cannot form without it. And it is known as chlorosis that the leaves of plants turn yellowish white. Nitrogen element in the old leaves can be transported to the young leaves under N-deficient condition. Therefore, it is the cardinal symptom of plants under N-deficient condition that the blade area is small, the old leaves turn yellowish white, the young leaves are green, and the plant is short [9]. All leaves were still green and obvious improvement of spinach growth by nano-anatase TiO₂ treatment in N-deficient Hoagland solution, implying that nano-anatase TiO₂ had a N₂ fixation effect in spinach leaves. But bulk TiO₂ effect was not as significant as nano-anatase TiO₂. We believe that spinach yield fell greatly and the old leaves displayed chlorosis under N-deficient condition that is related to the inhibition of synthesis of organic nitrogen compounds (such as chlorophyll, protein) and the damage of photosynthesis closely.

The Contents of Total Nitrogen of Spinach Under N-Deficient Condition

To demonstrate N₂ fixation of nano-anatase TiO₂ in spinach leaves under sunlight, the experiments assayed the contents of total nitrogen of spinach. The results, in Fig. 5, showed that the contents of total nitrogen of spinach with nano-anatase TiO₂ and bulk TiO₂ treatment were 23.35% and 10.38% higher than that of control, separately, under culture with Hoagland solution. On the other hand, the contents of total nitrogen of spinach with nano-anatase TiO₂ and bulk TiO₂ treatment were 2.03 and 1.33 times that of control, respectively, under culture with N-deficient Hoagland solution. In Hoagland solution, the enhancement of total nitrogen contents of spinach with nano-anatase TiO₂ and bulk TiO₂ treatment was caused by the increase of absorption of inorganic nitrogen (which was proved by our previous studies). And it was also related to the generation of N₂ fixation. However, the significant enhancement of total nitrogen contents of spinach with nano-anatase TiO₂ and bulk TiO₂ treatment were caused by N₂ fixation under N-deficient condition, e.g., there was an obvious N₂ fixation in spinach treated by nano-anatase TiO₂. Although the treatments of spinach were different, the contents of total nitrogen of seeds were same.

Fig. 5

Effect of nano-anatase TiO₂ on contents of total nitrogen of spinach

The Contents of ${\text{NH}}^{ + }_{4} - {\text{N}}$ and O₂-Evolving Rate of Spinach Under N-Deficient Condition

${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ of most plants was reduced from ${\text{NO}}^{{\text{ - }}}_{{\text{3}}} - {\text{N}}$. Leguminous plants could form ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ by N₂ fixation. It is shown in Fig. 6 that ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ contents of nano-anatase TiO₂-treated spinach were decreased by 9.93% and 2.39% compared with those of bulk TiO₂ and control in Hoagland solution. However, ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ contents of nano-anatase TiO₂-treated spinach were increased by 9.48% and 17.51% compared with those of bulk TiO₂ and control under culture with N-deficient Hoagland solution, respectively.

Fig. 6

Effect of nano-anatase TiO₂ on the contents of ${\text{NH}}^{ + }_{4} - {\text{N}}$ of spinach

${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ contents of nano-anatase TiO₂-treated spinach were lower than those of the control under Hoagland solution condition, which is because it was related to the enhancement of activity of key enzymes in ammonia assimilation and make ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ transform to organic nitrogen (such as proteins and chlorophyll) quickly, which was proved by our previous work [7].

There was no nitrogen nutrition (such as ${\text{NO}}^{{\text{ - }}}_{{\text{3}}} - {\text{N}}$) in N-deficient Hoagland solution. Thus, on the one hand, ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ in spinach came from ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ accumulation and the reduction of ${\text{NO}}^{{\text{ - }}}_{{\text{3}}} - {\text{N}}$ in seeds. On the other hand, it also might come from N₂ fixed in spinach leaves by nano-anatase TiO₂ and bulk TiO₂.

It is well-known that photosynthesis is composed of photocatalyzed chemical reaction and enzyme-catalyzed chemical reaction. Oxygen evolution is an important part of photosynthesis, and the photosynthesis is inhibited under N-deficient environment. Figure 7 displayed that nano-anatase TiO₂ and bulk TiO₂ treatment could significantly increase the oxygen-evolving rate of spinach chloroplast in Hoagland solution, which was enhanced by 43.41%, 16.95% compared with control. However, the oxygen-evolving of spinach was seriously inhibited under culture with N-deficient Hoagland solution, e.g., the oxygen-evolving rates of control, bulk TiO₂ treatment and nano-anatase TiO₂ treatment were 54%, 65.14%, and 85.61% of that of control under culture with Hoagland solution. But it can be seen that the oxygen-evolving rate of nano-anatase TiO₂-treated spinach was higher than that of control and bulk TiO₂ treatment under N-deficient condition, demonstrating that nano-anatase TiO₂ treatment could significantly decrease the inhibition of N-deficient on the oxygen evolution of spinach.

Fig. 7

Effect of nano-anatase TiO₂ on O₂-evolving rate of spinach

Our previous work confirmed that the enhancement of oxygen evolution rate from nano-anatase TiO₂-treated spinach was closely related to the enhancement of light absorption and photochemical reaction activity, which was consistent with improvement of chlorophyll synthesis and sensitized nano-anatase TiO₂ by chlorophyll in chloroplast [2–6, 20]. Nano-anatase TiO₂ and bulk TiO₂ can promote the pigments of chloroplast absorbing and transferring light energy, and excite energy-enriched electrons, which cause water photolysis [3–5].

Considering ${\text{NH}}^{{\text{ + }}}_{{\text{4}}} - {\text{N}}$ formation and the enhancement of oxygen-evolving rate of spinach by nano-anatase TiO₂ and bulk TiO₂ treatment, it can be explained by the reaction equation according to Schrauzer’s researches defined as in Eq. d [12, 13].

The reaction equation is linked to the ability of nano-anatase TiO₂ and bulk TiO₂ to chemisorb both H₂O and N₂ and closely related to the photolysis of H₂O in spinach leaves. The photolysis of H₂O on nano-TiO₂ and bulk TiO₂ has been described in line with current concepts of the electronic of semiconducting solid [1, 21, 22]. Upon illumination with near-UV light electrons from the valence band are excited into the lowest conduction band; the band gap of rutile is in the order of 2.9–3.2 eV (390–420 nm) [23], corresponding to between 70 and 80 kcal, thus sufficient for reaction to occur as defined in Eq. e [12, 13].

The positive holes generated in the valence band provide the sites for oxygen production or other oxidation reactions in spinach chloroplast. The electrons of nano-anatase TiO₂ and bulk TiO₂ in the conduction band can be utilized for the reduction of substrates such as H⁺ and N₂ in spinach chloroplast. It is also possible, however, to describe the electrons in the conduction band as excited titanium atoms in lower oxidation states at which the reduction of the substrates occurs. N₂ inhibits the H₂ evolution and thus may be assumed to be reduced at the same site or sites in spinach chloroplast. As H₂ is formed in reactions involving the transfer of two electrons and the production of C₂H₄ from C₂H₂ is inhibited by N₂, Schrauzer assumed that chemisorbed N₂ is reduced, at least in part, via chemisorbed diimide [12, 13].

As mentioned above, the oxygen-evolving rate of spinach with nano-anatase TiO₂ and bulk TiO₂ treatment was higher than that of control either in Hoagland solution or in N-deficient Hoagland solution. And the NH₃ formation and the oxygen evolution of nano-anatase TiO₂-treated spinach were more significant than that of bulk TiO₂-treated spinach, because the average grain size of nano-anatase TiO₂ (5 nm) is much smaller than that of bulk TiO₂, which entered spinach cell more easily.

Considering the improvement of spinach growth and oxygen evolution by nano-anatase TiO₂ treatment, we believed that nano-anatase TiO₂ entered spinach cell and bound to thylakoid membrane of chloroplast, especially photosystem II (PSII) or chlorophyll, and sensitized nano-anatase TiO₂ by chlorophyll in chloroplast. Li et al. showed that the sensitized nano-TiO₂ (rutile or anatase) by chlorophyll in vitro could strongly absorb light not only in ultraviolet region, but also in visible region from 400 to 800 nm [20]. PSII is capable of water splitting and oxygen evolution, which is a key site of electron transport. Therefore, N₂ fixation and oxygen evolution of nano-anatase TiO₂ in spinach chloroplast are schemed as follows (Scheme 1): two membrane complexes—PSII and Cytb6f—are shown. The transfer of electrons from nano-TiO₂ by Nano-TiO₂ entered spinach cell and bound to photosystem II (PSII) or chlorophyll directly, and sensitized nano-anatase TiO₂ by chlorophyll in chloroplast. Nano-TiO₂ chemisorbed N₂ and H₂O, N₂ is reduced to NH₃ and H₂O is photolyzed to O₂ upon exposure to sunlight. Parts of electrons from nano-TiO₂ by oxidation-reduction reaction are transferred to PSII reaction center and then to other electron carriers, such as PQ (plastoquinone), Cytb₆f (cytochrome b₆f complex), and PC (plastocyanin). The reaction center chlorophyll of PSII, P680, undergoes light-induced oxidation to produce the strong oxidant, P680+, which can oxidize water, releasing O₂. The electrons from P680 are transferred to PQ, Cytb₆f, PC, etc. Accompanying these electron transfers, a proton gradient is established across the membrane. This electrochemical gradient is ultimately utilized for the synthesis of ATP by the ATP synthase. Oxidation-reduction reaction is illustrated with yellow lines; the transfer of electrons produced from water is illustrated with red lines; the translocation of proton is illustrated with blue lines.

Scheme 1

Nano-TiO₂ on N₂ fixation and oxygen evolution in spinach chloroplast

The Protein and Chlorophyll Contents of Spinach Under N-Deficient Condition

To further prove inorganic nitrogen transformation, the protein and chlorophyll contents of spinach were assayed. The results in Fig. 8 indicated that the chlorophyll contents of nano-anatase TiO₂-treated spinach and bulk TiO₂-treated spinach were 37.48% and 13.34% higher than those of control in Hoagland solution. However, the chlorophyll contents of spinach were inhibited seriously under N-deficient condition, e.g., the chlorophyll contents of control; bulk TiO₂- and nano-anatase TiO₂-treated spinach were as much as 94.51%, 64.01%, and 31.81% less than those of the control under Hoagland solution, respectively. On the other hand, the chlorophyll contents of nano-anatase TiO₂-treated spinach under N-deficient condition were higher than those of control and bulk TiO₂-treated spinach, which lead to keeping green of all the leaves of nano-anatase TiO₂-treated spinach. It was obvious that the chlorophyll formation of spinach by nano-anatase TiO₂ treatment under N-deficient condition was caused by the N₂ fixation that occurred in spinach. There was little chlorophyll in control leaves under N-deficient condition, which was formed by parts of nitrogen in seeds, and led to chlorosis of spinach leaves. The increase of chlorophyll contents in spinach leaves with bulk TiO₂ treatment also came from N₂ fixation under culture with N-deficient Hoagland solution.

Fig. 8

Effect of nano-anatase TiO₂ on the chlorophyll contents of spinach

The increase of the protein contents of nano-anatase TiO₂- and bulk TiO₂-treated spinach under Hoagland solution is observed in Fig. 9, which enhanced by 17.55% and 6.33%, respectively, suggesting that nano-anatase TiO₂- and bulk TiO₂-treatment could promote the synthesis of protein. It can also be seen from Fig. 9, the synthesis of protein in spinach was also inhibited seriously under N-deficient Hoagland solution, whereas the reduction of the protein contents of nano-anatase TiO₂-treated spinach was lower than that of control and bulk TiO₂-treated spinach, i.e., the protein contents of control, bulk TiO₂- and nano-anatase TiO₂-treated spinach under N-deficient condition had a reduction of 77.03%, 61.61%, and 31.77% compared with those of control under Hoagland solution, respectively.

Fig. 9

Effect of nano-anatase TiO₂ on the protein contents of spinach

The results mentioned above obviously suggested that nano-anatase TiO₂ treatment supplied more nitrogen to the synthesis of chlorophyll and protein of spinach than control and bulk TiO₂ treatment, which led to notable promotion of spinach growth.

Conclusions

The study of the experiments showed that all spinach leaves kept green by nano-anatase TiO₂ treatment and all old leaves of spinach of control turned yellowish white under N-deficient condition. Nano-anatase TiO₂ treatment resulted in obvious increase in the fresh weight, dry weight, and contents of total nitrogen, ${\text{NH}}^{ + }_{4}$, oxygen, chlorophyll, and protein of spinach. After bulk TiO₂ treatment, one part of the old leaf surface of spinach turned green, and the other was yellowish white. The improvements of yield and N compounds, oxygen contents of spinach were not as good as nano-anatase TiO₂ treatment under N-deficient condition. It proved that nano-anatase TiO₂ on exposure to sunlight could chemisorb N₂ directly or reduce N₂ to NH₃ in spinach, which was transformed into organic nitrogen (such as chlorophyll, protein) and improve the growth of spinach. However, bulk TiO₂ effect was not as significant as nano-anatase TiO₂. According to Schrauzer’s reaction equation, a possible metabolism of the function of nano-anatase TiO₂ catalyzing N₂ to NH₃ was advanced in Eq. f.

This is a pathbreaking study on the N₂ fixation by nano-anatase TiO₂ in spinach, which is an important theoretical foundation and technical approach for the future of agricultural applications of nano-TiO₂.