Synergistic improvement in spring maize yield and quality with micro/nanobubbles water oxygation

Soil oxygen shortages in root areas have negative effects on crop growth and decrease crops yield and quality, and soil hypoxia conditions will be aggravated by application of subsurface drip irrigation (SDI). A two-year field experiment was conducted to evaluate the response of maize to micro/nanobubbles oxygation (MNBO) at three dissolved oxygen (DO) concentrations (10, 20 and 30 mg/L) and seven MNBO periods (vegetative stage, reproductive stage, filling and ripening stage, combination of two stages and the whole growth stage) in addition to a control treatment (CK, no oxygation during the growth period). Our results revealed that the MNBO treatments increased maize root dry weight, root length density and root surface area in 0–20 cm soil. The highest yield was obtained in O20A (MNBO at 20 mg/L DO during the growth period), with an increase of 11.66% relative to CK. Crude ash, starch and vitamin C were improved by application of MNBO at 20 mg/L DO. However, excessive oxygen adversely affected maize growth, decreasing the maize yield in 2013 relative to CK. The results suggest that application of MNBO at 20 mg/L DO during the growth period of spring maize was appropriate.

emitters is asymmetrical in all directions. The air most likely diffuses into the atmosphere through several preferred channels; this is called the "chimney affect" 23 , and results in a low gas utilization rate and a short contact time with the crop roots.
The size of micro/nanobubbles (MNBs) is between that of microbubbles and nanobubbles [24][25][26] . MNBs can be generated by ultrasonic cavitation, chemical reactions, electrolysis and other methods [27][28][29] . MNBs have some unique properties, including stability, persistence, large specific surface area, slow rise in water, and good mass transfer coefficient. Broken MNBs can generate shock waves and local high temperatures [30][31][32] , and thus have important applications in the metallurgical and environmental fields. Therefore, the combination of MNBs and oxygation is expected to solve the aforementioned problems. It was hypothesized that MNB oxygation (MNBO) through an SDI system could promote crop growth and root development and, improve the yield and nutritional quality of spring maize. The objectives of this paper were to: 1) investigate the effects of different MNBO periods and DO concentration on the growth, yield, IWUE and nutritional quality of spring maize; 2) reveal how MNBO regulates maize yield and quality; and 3) determine the optimal application pattern of MNBO for spring maize.

Results
Maize height and stem diameter. Measurements of maize height and stem diameter were collected every 15 days from the beginning of the experiment until harvest in 2013 ( Table 1). Analysis of variance (ANOVA) results indicated that MNBO stage had no significant effect on maize height except for at 30 days and 105 days. The maize stem diameter significantly differed between different MNBO treatments from 30 days to 105 days (p < 0.05), increasing by 7.01-24.77%, 6.82-26.89%, 8.41-27.83% and 8. .55% compared to CK for 30, 45, 60 and 75 days, respectively. ANOVA results indicated that maize height and stem diameter significantly increased at 60 days by 5.86-7.04% and 12.99-18.83%, respectively. There were no significant differences in maize height and stem diameter after 60 days in the DO treatments.
Root dry weight (RDW), root length density (RLD) and root surface area (RsA). Most of the maize root system is concentrated in the top 0.4 m soil layer 6,33 . The vertical distribution of RDW ( Fig. 1) in the different treatments was similar, mostly concentrated in the 0-10 cm soil layer (Fig. 1a), which accounted for 58.14-80.14% of the total RDW. The RDW in the soil below 10 cm decreased with increasing soil depth ( Fig. 1b-d), and below 40 cm, there was little difference between treatments. MNBO significantly improved maize RDW (p < 0.05) in the 0-40 cm soil layer. The effect of MNBO on RDW was most pronounced in the 10-20 cm soil layer (Fig. 1b), in which RDW increased by 0.55-2.36 times compared with CK. The vertical distribution of maize RDW in the 0-10 cm soil layer showed an initial increase followed by a decrease with increasing DO concentration. RDW O20A , was the highest, at 26.55 g, and increased by 68.04% and 8.59% relative to RDW O10A and RDW O30A , respectively. Moreover, the vertical distribution of maize RDW in the 0-10 cm soil layer revealed that the longer the duration of MNBO was, the more pronounced the promotion of RDW. www.nature.com/scientificreports www.nature.com/scientificreports/ The effects of MNBO period on RLD (Fig. 2) distribution indicated that the longer the MNBO duration was, the more concentrated the roots in the shallow soil layer. Compared to CK, MNBO treatments increased maize RLD by 2.14-47.64%. It was inferred that MNBO period had a significant effect on RLD in the 0-40 cm soil layer (p < 0.05) and DO concentration affected RLD in the 0-30 cm soil layer. The highest RLD in the soil layer of 0-40 cm was observed in O 20 A and accounted for 93.34% of the total RLD.
The larger the total RSA (Fig. 3) of the maize roots was, the larger the area from which roots could absorb nutrients and moisture from the soil, and the better growth ability of maize. MNBO remarkably impacted maize RSA in the 0-20 cm soil layer ( Fig. 3a,b), but below 20 cm (Fig. 3c,d), the differences between treatments were not obvious. The vertical distribution of maize RSA at different DO concentrations in the 0-20 cm soil layer revealed that RSA O20A was the largest in the 0-10 cm and 10-20 cm soil layers, followed by RSA O10A and RSA O30A , with respective declines of 21.70% and, 31.34% in the 0-10 cm soil layer and 100.00% and, 129.70% in the 10-20 cm soil layer compared with O 20 A.   30 A, which decreased the maize yield. The IWUE, RUE and WUE were determined by maize yield, irrigation quota, rainfall per hectare, and total water consumption. The trends of IWUE, RUE and WUE were consistent with maize yield due to the irrigation quota, rainfall conditions and total water consumption being the same.

Yield, irrigation water use efficiency (IWUE), rainfall use efficiency (RUE) and water use efficiency (WUE
Nutritional quality. The maize nutritional quality data are summarized in Fig. 4. According to the ANOVA of two years of data, the content of maize crude ash (Fig. 4a), fat (Fig. 4c), vitamin C (Fig. 4d), crude protein (Fig. 4e) and crude fiber (Fig. 4f) were significantly affected by MNBO stage (p < 0.05), and there was no obvious difference in starch (Fig. 4b). The contents of crude ash, vitamin C and crude fiber in the O 20 A treatment increased www.nature.com/scientificreports www.nature.com/scientificreports/ The effects of DO concentration on crude ash, fat and vitamin C of spring maize were significant (p < 0.05), but the effect on the starch content was not significant (p > 0.05). No significant difference in vitamin C content was observed among the O 10 A, O 30 A and CK treatments, but an obvious elevation of 28.11% was observed in O 20 A compared to CK (p < 0.05). The vitamin C content of maize in O 20 A was the highest and was significantly different from those in O 10 A and O 30 A, with increases of 15.97% and 28.08%. MNBO application decreased the crude protein content of the maize kernels. The fat content in maize grain decreased with increasing oxygen concentration, and LSD variance analysis showed no significant difference between O 10 A and CK. However, comparing to CK, the fat content in the maize grain decreased by 5.14% and 24.95% in O 20 A and O 30 A. In O 10 A and O 20 A, the formation of crude ash in maize grain increased by 8.39% and 18.46%.

Discussion
In this study, field experiment was conducted in two successive years to investigate the effects of the MNBO stage application and DO concentration on maize growth, yield and nutritional quality. The results showed that MNBO under an appropriate DO concentration promoted the growth of maize roots and increased the yield, IWUE and nutritional quality. Similar results have been reported when oxygation was applied to other crops 7,15,34 . Soil moisture and air play contradictory roles when providing water to crops under traditional irrigation methods. Soil moisture removes air from soil pores and reduces the oxygen content of the soil 35 . Moreover, forced ventilation into the soil when the soil moisture is low will exacerbate the decline in soil moisture 20 . Oxygation, which transports the water and air that crops need to the root area, is an expansion of traditional irrigation methods 36 . The soil permeability rate is improved while ensuring soil moisture and improving the oxygen environment in the root zone 37 . The increased soil gas permeability promotes root growth and lateral root formation 38,39 , enhances leaf photosynthesis and root respiration 40 , and relieves the adverse effects of environmental factor stress on crops 41 . Overall, MNBO application improved crop growth, leading to increased yield, WUE and quality 15,34,37,42 .
The results indicated that maize roots, yield, WUE, and partial nutrition indicators (crude ash, vitamin C, and crude fiber) under MNBO were superior to those under CK. O 20 A produced more maize grain than did MNBO at each pair of growth stages (O 20 VR, O 20 VF and O 20 RF) and MNBO application at single growth stage (O 20 V, O 20 F, O 20 R). Maize is considered an "intertilled crop", which means that maize demands substantial amounts of oxygen during the growth period. MNBO applied at the vegetative stages guaranteed the germination of maize seeds after sowing, affecting roots, leaves, and stem node differentiation. MNBO applied at the reproductive stages ensured a vigorous rhizome leaf growth and anthesis-silking interval differentiation and development, which www.nature.com/scientificreports www.nature.com/scientificreports/ could maintain the kernel emergence and rapid enrichment. It was indicated that the oxygen demand of maize would be better satisfied with MNBO application during the growth period rather than at other stages. Figure 5 shows the morphology of the maize roots on day 100 after sowing. The figure indicates that the longer the MNBO duration was, the greater the growth and development of spring maize roots. Comparison of the O 20 V, O 20 R, O 20 F and CK treatments indicated that the maize root system in O 20 VR, O 20 VF, O 20 RF and O 20 A exhibited characteristics of well-developed surface roots and prosperous lateral roots, exhibiting a multidirectional root system. In addition, oxygation increased the soil microbial abundance and soil enzyme activity 16,43,44 . The decomposition of organic matter in the soil was accelerated and the effectiveness of soil fertility was improved 45 . MNBO applied at different growth stages of maize ensured sufficient nutrient contents throughout the growth period, which promoted the transformation and absorption of nutrients and significantly enhanced the yield and quality of maize.
The effects of DO concentration in irrigation water on maize root growth, yield and nutritional quality were investigated considering three DO concentrations during the growth period. The O 10 A and O 20 A treatments developed shallow roots and exuberant lateral roots compared with those in O 30 A, which grew single roots with fewer lateral roots. The RDW of maize increased as DO concentration increased. However, the maize yield, crude ash, starch and vitamin C trended to increase initially and then decreased with increasing DO concentration. Under 10 mg/L DO, the optimal oxygen content required for maize could not be met, and 30 mg/L DO provided excess oxygen to the roots, causing physiological damage to plant and inhibiting leaf area expansion, relative leaf growth rate and crop growth rate 21,46 . These changes caused a spindling phenomenon in maize roots. Although a higher RDW was obtained, the root tissues were damaged, which affected the absorption of water and nutrients, resulting in crop failure 21 . Because of trial limits, the experiment studied only MNBO at different crop growth stages in the same DO concentration. Further exploration of whether the same DO concentration should be used at different growth stages of maize is necessary. The results demonstrated that MNBO under a high DO concentration adversely affected crop yield 47 . The specific threshold should be further refined according to crop variety and planting conditions.

Conclusions
The present study was conducted over a two-year period to evaluate the response of maize to MNBO considering MNBO stage and DO concentration. The results demonstrated that MNBO applied during the growth period under appropriate DO concentration significantly affected maize roots, yield, WUE and nutritional quality. The highest yield was obtained in O 20 A and was 11.66% greater than that in CK. Crude ash, starch and vitamin C were also improved. However, excess oxygen adversely affected maize growth that O 30 A treatment decreased the maize  (39°42′N,  116°42′E). The local climate is a continental warm temperate, semi-humid monsoon climate, affected by the winter and summer monsoons, exhibiting the characteristics of hot and rainy summers, cold and dry winters, and a short spring and autumn. The annual average temperature and rainfall are 11.3 °C and 620 mm, respectively. The maize plants were grown in clay loam soils. The experimental site is saline land in Beijing, China, with the characteristics of high soil bulk density and poor soil aeration. Soil characteristics for the 0-100 cm layers are provided in Table 3 Each replication occupied 126 m 2 (30 m × 4.2 m) consisting of six rows. The maize plants were cultivated in wide/narrow planting rows with a wide-row spacing of 80 cm and narrow-row spacing of 60 cm. The subsurface drip tape was cylindrical pipelines (16 mm in diameter) with a flow rate of 2.6 L/h, a wall thickness of 0.4 mm, and an emitter spacing of 30 cm. The drip tapes were parallel to the maize planting ridges and buried 10-15 cm beneath the surface. The initial flow rate of the drip tape was tested before the experiment, and no significant variation during initial flow was observed along the laterals.
The layout of micro/nanobubbles generator connection, SDI pipeline and buried drip laterals is shown in Fig. S1. The irrigation water pumped from groundwater aquifer was oxygenated by MNB generator with initial 4-5 mg/L DO. A fiber optic trace oxygen meter (Fibox 4 Trace, PreSens, Germany) was used to measure the DO during generator operation time. The device collected DO concentration data every five seconds and fed it back to the display screen. The irrigation water was continuously oxygenated until the setting DO concentration was detected by the device. The MNB water was transported to crop root zone through totally enclosed drip pipe network system and DO in the irrigation water based on in-situ measurement is shown in Fig. S2. The method used for generating MNBs of the generator was pressurized gas-liquid mixing. The mean size and numbers of bubbles in water were determined via Nano-Particle Tracking Analysis (NanoSight NS300, Marlern, UK). The bubbles suspension mean size was between 320.08 and 1215.55 nm. The bubble concentration was 3.27 × 10 8 particles/ml. The zeta potential of the MNB surface was −13.7 mV. www.nature.com/scientificreports www.nature.com/scientificreports/ Irrigation and fertilization regime. A meteorological station was set up at the experimental site to continuously observe the meteorological conditions during the study. During the experimental period, the precipitation during the maize growing was 203.7 mm and 348.8 mm in 2013 and 2014, respectively, which mainly occurred in July and August. The soil moisture content was monitored via TDR (time domain reflectometry) after sowing, which provided soil moisture (%) at soil depths of 10, 20, 40 and 60 cm. The soil moisture content was measured once per week and additionally after irrigation and rainfall. The irrigation quota in 2013 and 2014 were 286.5 mm and 270.9 mm. Figure S3 shows the daily precipitation and irrigation quotas over the two-year trial period. Each treatment was equipped with a water meter to accurately control irrigation quantity. The amount of fertilizer for the ten treatments was identical. All the agronomic cultivation management measures including the irrigation quota and fertilizer amount were the same throughout the experiments except for experimental factors.
Measurements. Fifteen typical maize plants were sampled at the beginning of experiment and tagged to measure the maize height and stem diameter every 15 days from the beginning of the experiment to the end. The maize height was measured from the plant base to the last opened leaf and the stem diameter was determined with a Vernier caliper 10 cm above the plant base. The roots were excavated with a 0.10 m diameter soil auger 100 days after sowing and each treatment was randomly sampled with five representative maize plants. Soil cores were extracted at six depths (0-10, 10-20, 20-30, 30-40, 40-50 and 50-60 cm) and mixed from three points (¼ row spacing, ½ row spacing and ½ line spacing). The samples were brought to the laboratory for analysis of RDW, RLD and RSA.
The maize yield was estimated by harvesting fifteen tagged maize plants at the end of the physiological maturity. The samples were dried in an open environment, and the yield was determined after threshing. Total grains from a single maize were weighted. The number of maize on the tagged plants was counted. IWUE, RUE and WUE were calculated using the following formulas: www.nature.com/scientificreports www.nature.com/scientificreports/ where Y is the maize yield (kg/ha), I is the irrigation quota (m 3 ), R is the rainfall per hectare (m 3 ), and W is the consumed water amounts per hectare (m 3 ). Five maize per treatment were sampled for the nutritional quality measurements. The threshed maize kernels were evenly mixed, and each nutritional quality test sample weighed 200 g. Crude fiber was measured via the intermediate filtration method. The fat content was determined via the Soxhlet extractor method. The starch, crude protein, and crude ash were determined via near-infrared absorption, the Kjeldahl method, and combustion at 550 °C, respectively. The vitamin C content was measured using the 2,6-dichloro-indophenol titration method. Data analysis. All data are presented as the mean ± standard deviation. ANOVA was performed using the SPSS 22.0 software package. The least significant difference (LSD) test at a p value of 0.05 was used to separate treatment means exhibiting significant differences.   Table 3. Selected physicochemical properties of the soil at depths 0-10, 10-20, 20-40, 40-60 and 60-80 cm prior to the start of the experiment in 2013 and 2014. Notes: Organic matter in g/kg. Total N and available N in %. Total P, total K, available P, available K in mg/kg.