Influences of Air, Oxygen, Nitrogen, and Carbon dioxide Nanobubbles on Seeds Germination and Plants Growth -

Influences of Air, Oxygen, Nitrogen, and Carbon dioxide Nanobubbles on Seeds Germination and Plants Growth

Nanobubbles (NBs) hold promise in green and sustainable engineering applications in diverse fields (e.g., water/wastewater treatment, food processing, medical applications, and agriculture). This study investigated the effects of four types of NBs on seed germination and plant growth. Air, oxygen, nitrogen, and carbon dioxide NBs were generated and dispersed in tap water. Different plants including lettuce, carrot, fava bean, and tomato were used in germination and growth tests. The seeds in water containing NBs exhibited 6-25% higher germination rates. Especially, nitrogen NBs exhibited considerable effects in the seed germination, whereas air and carbon dioxide NBs did not significantly promote germination. The growth of stem length and diameter, leave numbers, and leave width were promoted by NBs (except air). Furthermore, the promotion effect was primarily ascribed to the generation of exogenous reactive oxygen species (ROS) by NBs and higher efficiency of nutrient fixation or utilization.

Methods and materials

Production and storage of NBs suspensions containing air, O₂, CO₂, and N₂

Different kinds of NBs including air nanobubbles (ANBs), oxygen nanobubbles (ONBs), carbon dioxide nanobubbles (CNBs), and nitrogen nanobubbles (NNBs) were generated by direct injection of compressed air (Ultra zero grade air, Airgas Inc.), oxygen (purity 99.999%, Airgas 105 Inc.), carbon dioxide (purity 99.99%, Airgas Inc.), and nitrogen (purity 99.999%, Airgas Inc.)  through a tubular ceramic membrane into tap water. The gases were injected continuously for 90 min under a pressure of 414 kPa and a flow of 0.45 L·m-1 to reach stable bubble size distribution and a saturation point. The hydrodynamic diameters of the produced NBs were measured by dynamic 110 light scattering (DLS) on a Zetasizer Nano ZS instrument (Malvern Instruments). The used tap water was firstly left at ambient temperature for 24 hours to allow the free residual chlorine to exit water. Fresh NBs suspension was used immediately for the plant growth tests. However, for germination tests, all types of NBs suspension were generated every three days, stored separately in closed 1-gallon water bottles and used daily. The pH for air nanobubbles (ANBs), oxygen nanobubbles (ONBs), carbon dioxide nanobubbles (CNBs), and nitrogen nanobubbles (NNBs) and tap water were between 6 and 7 and the pH for CNBs was around 4.5. A monitored the dissolved oxygen of the freshly and stored NBs suspension with Orion Star A329 118 Multi-Parameter Meters (Thermo-Fisher Scientific, USA).

Examination of ROS production in water suspension of NBs

 The generation of hydroxyl radicals (•OH) by NBs was detected by a photoluminescence (PL) technique with terephthalic acid as a probe molecule. Terephthalic acid readily reacts with •OH to produce highly fluorescent product, 2-hydroxyterephthalic acid. The intensity of the PL peak of 2-hydroxyterephtalic acid is in proportion to the amount of •OH radicals produced in water. This method relies on the PL signal at 425 nm of the hydroxylation of terephthalic acid with •OH generated by NBs. Compressed air, oxygen, nitrogen and carbon dioxide were separately purged into 300 mL of the 5×10^-4 M terephthalic acid solution with a concentration of 2×10^-3 127 M NaOH in a 1L beaker for 30 min at a constant temperature of 20 °C. To increase the collapse rate of NBs, an ultrasonic wave (100 W, 42 kHz±6%) was applied to the four paralleled samples of control and NB suspensions for different times (0 min, 0.5 min, 3 min and 6 min). After ultrasonication, the PL spectra of these liquid samples were measured on a Hitachi fluorescence spectrophotometer to determine the generated 2-hydroxyterephthalic acid.

Germination tests

Lettuce, carrot, and fava bean seeds were used in germination tests. For each seed type, five paralleled groups were prepared to investigate the effects of four different NBs on their germination rates, which were calculated daily by the percentage of germinating seed number to the total number on the Petri dish. Among the five groups, there was a control group using tap water without NBs. The other four groups were performed by watering the seeds with suspensions of different NBs including ANBs, ONBs, NNBs, and CNBs. Each group was composed of 25 seeds, and each 5 seeds were separately submerged in 10 mL of the tested NB suspension inside a non-sterilized petri dish. All petri dishes were kept at the same room temperature (~23 o 145 C) and natural light conditions. Germination tests for lettuce, carrot, and bean seeds lasted for about 6‒10 days. The seed’s sizes and weights were measured before and after the tests. The hypocotyl lengths were measured daily to compare hypocotyl elongation.

Plant growth tests

For growth study Fava bean (Vicia faba), carrot, and tomato san marzano (Solanum lycopersicum) were grown in the garden soil (Miracle-Gro soil). The height, width, and length of the planters were 16.75, 20.02, and 60.33 cm respectively. Five groups of each plant type were cultivated. All groups were subjected to irrigation every three days by saturated water suspension of ANBs, ONBs, NNBs, CNBs, and tap water only (control group). For each group, five seeds were inoculated in one planter with an apart distance of approximately 10 cm. For growth tests, the length or diameter (cm) of the leaves, stem, and root were measured depending on the growth rate with a Caliper.

Results

Effect of different types of NBs on the germination rates of vegetable seeds

After 6 days of submersion, the lettuce germination rate reached 100% with NNBs followed by CNBs, ONBs, ANBs, and tap water, which corresponded to the germination rates of 85%, 85%, 82%, and 80%, respectively. The same results were achieved on carrot and bean, for which the germination rates were highest under irrigation by NNB water. For carrot and bean, ANBs and CNBs did not significantly promote germination compared to tap water. Thus, NNBs had a considerable promotion effect on the germination rate, probably because of the effective delivery of nitrogen elements or other growth factors by NBs.

Effect of NB type on the hypocotyl length of vegetable seeds

ANBs appeared to slow down the growth rates of hypocotyls length compared to tap water, which is observed in last two days. Clearly, the promotion effects by NBs became evident on the 4^th and 6^th days of incubation. Seeds exposed to NBs had a higher germination rate and hypocotyl length than seeds treated with tap water.

Effect of NBs type on the vegetable growth

Compared to tap water, the numbers of leaves were increased with exposure to most of the tested NBs. NNBs led to significantly increase in tomato leaves number whereas ANBs did not increase but instead reduced the number of tomato leaves. Conversely, ANBs had a negative effect on tomato growth. NBs-treated beans grew faster with apparent leaves sprouting out of their buds, whereas the tap water-treated ones had no leaf sprout during the same initial growth period. Similar to the results of the leaf number, some of the results show that stem length and diameter were both increased by NBs. However, ANBs appeared to inhibit the growth of stem length for bean and tomato after 30 days compared to tap water. Stem length exhibited distinct time dependent growth. However, stem diameter did not change with exposure time and also the effect of NBs varied on three types of vegetables. For example, for bean’s stem diameter, NNBs promoted its growth and CNBs/ONBs’ effects were negligible, whereas ANBs had an inhibitory effect. For tomato, the stem diameter was considerably increased by ONBs, NNBs, and CNBs. For tomato, its leaf length and width were both enhanced by NNBs water, but inhibited by ANBs.

ROS generation by NBs and disolved oxygen measurement

The ultrasound could accelerate the aggregation process of NBs in an aqueous solution and promote collapse and ROS generation. Measured the •OH radicals in the water saturated with NBs under different sonication time. Without sonication, the PL intensity of different NB solutions is similar with the control group, indicating that terephthalic acid could self-decompose and there were no significant or detectable amounts of ROS in NB waters without sonication. •OH radicals were produced in the NBs water, and both ONBs and ANBs generated considerable amounts of •OH radicals. NNBs seemed to quench some radical formation resulted from the sonication, which produced some •OH radicals under sonication. CNBs did not produce considerable levels of •OH radicals in the solution under the sonication. ANBs water had almost the same DO concentration (8.7 mg·L^-1 on average) with tap water. NNBs and CNBs reduced dissolved oxygen levels below the level in tap water. The dissolved oxygen level in ONBs suspension progressively reduced from 41.8 to 15.0 mg·L^-1, whereas the level of dissolved oxygen increased in the water suspension of NNBs and CNBs, probably due to oxygen gas transfer from the ambient air. The water was re-spiked with NBs every three days, which replenished DO in ONBs suspension from 15.0 to 41.8 mg·L^-1.

Overall, NNBs showed a considerable promotion in both seeds germination and plant growth for all species similar with ONBs. Especially, NNB enhanced germination rate of lettuce seeds by 25% higher than tap water alone. ANBs and CNBs did not show an obvious enhancement on seeds germination, but CNBs did show a mild enhancement on plant growth, especially with bean and tomato. ONBs generated the highest concentration of ROS, followed by ANBs, CNBs and NNBs, which provide a partial explanation for the promotion on seeds germination. Additionally, NBs may more effectively deliver nutrients (e.g., nitrogen or oxygen) to plants or to nitrogen-fixer in roots environment due to the high surface area and mass diffusion rates. Also, it is interesting that ANBs have a lower promotion effect than both ONBs and NNBs. Clearly, speculate that nitrogen or oxygen exerts different mechanisms of plant growth enhancement. For instance, enhanced nitrogen delivery might be the governing factor for NNBs, while ONBs may play a different role such as enhancing the activity of aerobic root microorganisms and indirectly promote plant growth. By contrast, ANBs have substantially different properties from NNBs or ONBs, which could explain the resulted different phytoplankton effects. For example, ANBs has different ionization energy than NNBs, as well as different surface charges and zeta potentials in aqueous solutions. The effects of NBs vary slightly with the types of seeds or plants, which deserves future studies. Moreover, the potential applications of NBs may not be limited to plant growth promotion and agriculture, but also applicable to other chemical or industrial processes such as phytoremediation as an efficient, green and cost effective approach to boost up plant growth for pollution removal or remediation.

Reference:

Ahmed, A.K.A., Shi, X., Hua, L., Manzueta, L., Qing, W., Marhaba, T. and Zhang, W., 2018. Influences of air, oxygen, nitrogen, and carbon dioxide nanobubbles on seed germination and plant growth. Journal of Agricultural and Food Chemistry, 66(20): 5117-5124.