Effects of ozone-treated domestic sludge on hydroponic lettuce growth and nutrition

YANG Peng, GUO Yan-zhi, QIU Ling

1 College of Agronomy, Northwest A&F University, Yangling 712100, P.R.China

2 Agro-environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, P.R.China

3 Institute of Food and Nutrition Development, Ministry of Agriculture, Beijing 100081, P.R.China

1.Introduction

Sludge produced during the municipal wastewater treatment process contains numerous nutrients such as nitrogen,phosphorus and small amounts of potassium (Keskin et al.2010), organic matter (Bitton 2005) and other trace elements.Therefore, this sludge could be used as a nutritional amendment to improve soil physical characteristics, fertility,microbial diversity and enzyme activity.It has been shown that the agricultural utilization of sludge is one of the most economical and effective options for sludge treatment (Oliver et al.2005).However, sludge is also rich in organic matter,pathogenic bacteria, parasitic eggs, heavy metals and other toxic and harmful substances that was difficult to degrade biologically (Appels et al.2010; Bratina et al.2016).Such sludge could not be used in raw or dried form for agricultural application and composting, leading to the secondary environmental pollution caused by improper disposal of sludge (Bratina et al.2016).Ozone is a functional disinfectant, oxidizer, and reducer when used to treat sludge (Tian et al.2015; Zhang et al.2016).Furthermore,the sludge mineralization process during ozone treatments could decompose organic matter into inorganic materials(Murat et al.2010).Significant research efforts have been devoted to the application of ozone in sludge treatment (Chu et al.2009; Zhang et al.2016; Chacana et al.2017), which promote cell cracking and reduce the number of excess sludge, thus reducing the waste disposal of activated sludge(even to zero) and decreasing the chemical oxygen demand(COD), chromaticity, and turbidity in conventional activated sludge process with ozone treating partial backflow sludge.Ozone has also been shown to be a superior disinfectant compared to conventional disinfectants such as chlorine and its derivatives (Chong et al.2011; Mecha et al.2017).When microorganisms were exposed to ozone, their cell walls were destroyed and internal organic matter was oxidized.The ozone could directly disrupt viral and bacterial metabolism and reproduction by breaking down macromolecule polymers.Most studies have focused on ozone treatments on sewage sludge derived from the municipal wastewater treatment plants where wastewater was collected from industrial and domestic sources (Chu et al.2009; Zhang et al.2016; Chacana et al.2017; Chekli et al.2017).Very little research has been focused on the agricultural utilization of solely domestic sludge, which avoids the environmental pollution introduced by industrial sludge.

2.Materials and methods

2.1.Raw materials

The sludge was collected from the digester of the Jizhuangzi Wastewater Treatment Plant in Tianjin, China.The water content of the sludge was 99.5%.The sewage treatment plant only deals with domestic sewage, and the sludge is not doped with industrial sources.After testing, the selected experimental sludge met the specific requirements of the control standard of pollutants in agricultural utilization of sludge(GB4284-84 1984).The sludge contains numerous nutrients,meeting the plant growth to a certain extent.Through ozone treatment, the sludge organic pollutants and micro pollutants which were detrimental to both plants and human health were degraded (Bougrier et al.2006; Neumann et al.2016).The sludge represented a viable alternative nutrient and water source for plants.Therefore, hydroponics was used in this experiment, which was a widely used technique for growing plants without soil, and could eliminate all the problems associated with soil culture.There were many hydroponic lettuce (Lactuca sativa) researches reported (Park et al.2016;Awad et al.2017; Sylwester et al.2017), especially, in terms of ecotoxicological testing, lettuce has been recommended by the Environmental Protection Agency of USEPA (1982) and the Food and Drug Administration, USFDA (1987).Modified Hoagland nutrition and 2-fold ozone-treated sludge dilution solutions were used in hydroponic lettuce here, which was simple, rapid and economic.

The domestic sludge was treated continuously by ozone until the concentration of fecal Escherichia coli was less than 1 000 colony forming units 100 mL-1, that is the water quality criterion in GB 5084 (2005).The dilution ratio of the ozonetreated domestic sludge in the irrigation was optimized for lettuce growth.This study works on the scientific exploration of vegetable planting fertilized by the low-cost domestic sludge.

This experiment was carried out from November 1, 2015 to January 9, 2016 in the greenhouse (39°40´N, 117°06´E)of Agro-environmental Protection Institute, Ministry of Agriculture, China over a period of 70 d.Lettuce seeds of the cultivar Dasusheng were used in all experiments.The lettuce seeds were grown in the artificial substrate and irrigated with water.Three weeks later, when the seedlings grew to four or five true leaves, they were transplanted(Hawrylak et al.2013).Plant roots were washed with distilled water before planted.The average temperature is 15.6°C in the greenhouse, and the air relative humidity is 41.3%.And the characteristics of ozone-treated domestic sludge on the lettuce growth improvement in the water cultivation process were investigated.

2.2.Ozone treatment device

The core of the ozone-treated device is consist of an Oxygen Generator (ZY-A30, BAIDUO, China), a High-Pressure Ozone Generator (ZY-A30, BAIDUO, China) and an Ozone Gas-Liquid Mixing Reactor which was made of a stainless steel container with a volume of 120 L,30-cm diameter and 170-cm in height.During the ozone treatments, the gas flown from the bottom of the titanium microporous aerator to the ozone oxidation reactor at a flow rate of 25 L min-1and an O3content of 65-85 mg L-1.A boric acid solution of 20 g L-1was used as the absorption liquid.After the ozone generator was turned on, the timer was activated after the starch potassium iodide reagent turned to blue.The ozone exhausts are absorbed into boric acid absorption liquid and KI absorption liquid, and the ozone in the exhaust gas is completely removed by ozone destructor (Fig.1-A and B).Considering the effects of equipment energy consumption and the conversion of organic matter, and combining with the ozone flux level of ozone equipment selected by the experiment, 120 min was choosen as the ideal aeration time (Yang et al.2015).

2.3.Hydroponic device

The cultivation bed was fabricated from a U-shape PVC tube with an 8-cm diameter and 123.5-cm length.The inlet endof the U-shape tube was connected to an elbow-shaped upward funnel for liquid feeding from the inlet bucket.The outlet end was connected to another elbow-shaped tube facing downward to discharge liquid into the outlet bucket.Fourteen holes with 5-cm diameter were made at 8.5-m interval along the U-shape tube surface to place the seedling.Every four U-shape tubes were placed horizontally on a 1.5 m×1.1 m×0.94 m cuboid bracket (Fig.2).The water was recycled from an outlet bucket to the inlet bucket through a 1.5-cm diameter plastic tube by a water pump,controlled by a timer (Yang et al.2015).

2.4.Experimental design

The hydroponic lettuce under control treatment (CK)was cultivated with modified Hoagland nutrition solution(Hoagland and Arnon 1950).While, other hydroponic lettuces under T1, T2, T3, and T4 treatments were cultivated using 1-, 2-, 3-, and 4-fold ozone-treated sludge dilution, respectively.And the nutrition contents in modified Hoagland and sludge solutions were shown in Table 1.Each treatment was repeated three times, and 40 plants were planted on each hydroponic shelf.The water loss caused by evaporation was supplemented by distilled water every 2 d.In order to ensure the nutrient solution was evenly dispersed in the cultivation bed, the hydroponic device was recirculated 12 times per day, and 10 min per time.

2.5.Analysis and methods

The representative five plants were removed from each hydroponics at 15 d intervals, and the leaf number, plant height, root length, root weight, fresh weight and dry weight were determined, respectively.The plant roots were cut off and weighted in a Thousandth Balance (METTLER TOLEDO, China) after the surface water was absorbed by filter paper.The fresh weight was the weight of the whole plant and measured with Thousandth Balance (METTLER TOLEDO) too.

The chlorophyll content was determined directly using the SPAD-502 Plus Portable Chlorophyll Meter (Konica Minolta, China).

The soluble sugar was determined by the anthrone colorimetric method, and glucose is used to make the standard curve (Buysse et al.1993).One gram of plant samples was ground into homogenate, transferred to 20-mL scale test tube, kept in boiling water for 10 min.After the homogenate was cool, it was filtrated into volumetric flask.Then, 1-mL filtrate was put into 20-mL tube with stopper and mixed with 1 mL water, 0.5 mL anthrone reagent (Kernel,Canada) and 5 mL concentrated sulfuric acid (H2SO4),then kept in a boiling water bath for 10 min.At last, optical density value was measured using Spectrophotometer(Macy Instruments Inc., China) under the wavelength of 620 nm (APHA et al.1998).

The content of ascorbic acid (Vc) was determined by 2,6-dichlorophenol titration.A total of 200 g lettuce leaves were crushed and added to the same amount of 2% oxalic acid, then shaked for 5 min.And the solution was fixed to 100 mL with 1% oxalate.Then 10 mL of the above solution was taken and mixed with 10 mL 1% oxalic acid and 1 mL chloroform, then titrated to the end with 2,6-dichlorophenol indophenol dye (APHA et al.1998).

Fig.2 Seedlings and hydroponics equipment.A, float tray.B, mobile hydroponic shelf.

Table 1 The physical-chemical properties comparison of pretreatment sludge and modified Hoagland nutrition solution

The nitrates were determined by Ultraviolet (UV)Spectrophotometry (UV-2802, UNICO, Japan).A total of 0.51 g samples of lettuce leaves were ground into homogenate with some distilled water, then the homogenate was washed into 100-mL volumetric flask with 30 mL distilled water.Another volumetric flask with 30 mL distilled water only was used as control.Then, 5 mL ammonia buffer (pH=9.6), 30 mL distilled water, and 0.5 g powdered activated carbon were added into both flasks and mixed well.Subsequently, protein precipitating agent was added, mixed, and filtered after the sample and control flasks were kept still for 20 min.The absorbance was determined by the quartz color dish in the UV Spectrophotometer (219 nm) (Armstrong 1963; Cawse 1967).

The number of fecal E.coli was measured using enzyme substrate method (Edberg et al.2000).The sample was incubated at 44.5°C for 24 h, then the number of E.coli was got by UV irradiation.And the Colilert Reagent was provided by IDEXX, UK.

2.6.Data processing

The differences of the experimental data were determined based on the Tukey test at 0.05 level using the DPS 16.5 Statistical Software (Ruifeng, China) (Tang and Zhang 2013).

3.Results

3.1.The effect of the ozone-treated sludge dilution on the agronomic traits of lettuce

Table 2 showed the lettuce leaf number, plant height, root length, fresh weight and dry weight of the hydroponic lettuce.Using biomass as a performance determinant, the lettuce under CK treatment showed the best performance,followed by the lettuces of T2, T1, T3 and T4 treatments.This trend was observed for all of the agronomic traits determined with the exception of root weight.Root weight was followed the order of CK＞T2＞T3＞T1＞T4.The lettuce under CK had obvious advantages in biomass growth,however, its stalk was spindling, which might be caused by excessive nitrogen supply, small temperature differences and lower phosphorusus supply.The common nitrogen and phosphorus contents were 49-210 and 15-198 mg L-1, respectively (Jones 2016; Award et al.2017).In this study, the nitrogen content in modified Hoagland nutrient solution was excessive in comparison with the phosphorus content.Using an analysis of variance, the lettuce cultured by modified Hoagland nutrition solution showed a significant advantage over the others cultured by ozone-treated sludge dilution treatments at all dilutions in the leaf number, plant height, fresh weight, and dry weight (P＜0.05).Among all the ozone-treated sludge dilution treatments, the T2 was significantly better than the other three treatments (P＜0.05)in the leaf number, plant height, root length, fresh weight,and dry weight.Therefore, from the point of view of lettuce agronomic traits, T2 was the most suitable treatment for lettuce growth and development among all ozone-treated sludge dilution solutions.First, the nitrogen and phosphoruscontents in T2 dilution were within suitable range.Second,there were some copper (Cu＜100 μg L-1) and zinc (Zn＜200 μg L-1) in the hydroponics solution.The results also indicated that T1 had excessive Cu and Zn that limited the lettuce growth.It was also reported by other researches(Lamb et al.2010; Park et al.2016).

Table 2 Agronomic traits of lettuce in the experimental treatments1)

The lettuce biomass results implied that the nutrients in the ozone-treated sludge dilution solutions could improve lettuce growth at low dilutions and inhibit lettuce growth at high concentration applications.This result might be attributed to the favorable buffering of the nutrient solution around plant roots in the low dilutions, which was similar to that in modified Hoagland nutrition solution.On one hand,it was deduced that the release of organic matter during sludge degradation caused a lack of oxygen to the root tissue.On the other hand, the heavy metal contents in T1 solution were higher than those in CK.In other words, Cu and Zn contents in the nutrition solution of CK were lower than 100 and 200 μg L-1, respectively.The root elongation bioassay was one of the most straightforward test methods used for environmental monitoring in terms of simplicity,rapidity and economy (Park et al.2016).Nitrogen and phosphorus contents in T1 nutrition solution was higher than those in CK, but the root length of lettuce in T1 was lower than that in CK.The result was caused by the higher heavy metal contents in T1 solution.There was no significant difference between CK and T2 on the root length,however, the dry biomass of CK was higher than that of T2.It was as a result of lower nitrogen content in T2 than in CK.Additionally, high heavy metal concentrations in the sludge were also harmful, resulting in the decrease of root enzyme activity and the inhibition of root growth.

3.2.Effect of the ozone-treated sludge dilution solutions on the lettuce chlorophyll content

As shown in Fig.3-A, the effect of the ozone-treated sludge dilution solutions on the lettuce chlorophyll content was the greatest in T2, followed by T1, T3, CK and T4.There was a significantly higher chlorophyll content in T2 treatment than that in CK and T4 treatments (P＜0.05).Similarly, among the ozone-treated sludge dilution treatments, T2 treatment was significantly higher than T4 treatment (P＜0.05).No significant difference was found among the T1, T2, and T3 treatments.This observation indicated that the appropriate ozone-treated sludge dilution ratio (e.g., T2) could greatly improve the chlorophyll content of lettuce.

The increased chlorophyll content in lettuce could be attributed to many factors.One factor could be that a large amount of nutrients sewage sludge could provide beneficial trace elements required for chlorophyll synthesis in plants,such as calcium (Ca), magnesium (Mg) and Zn (Chekli et al.2017).Another factor was that the trace elements accelerated the transportation each other by promoting plant growth and metabolism.These were the advantage for the use of sludge hydroponic solution, therefore sludge solution could be used directly after diluted without adding any elements.The third explanation could be that when the lettuce was subjected to the high concentrations of the ozone-treated sludge, the chlorophyll content in plant increased through screening ions and resisting the external osmotic stress.

3.3.The effect of the ozone-treated sludge dilution sulutions on lettuce soluble sugar content

The soluble sugar contents in the lettuce cultured with the ozone-treated sludge dilution solutions and the CK nutrition solution was determined (Fig.3-B).The soluble sugar contents of lettuce leaves in T2 treatment were the greatest, then followed by T3, T4, T1 and CK.According to the ANOVA analysis, the soluble sugar content of lettuce in T2 treatment showed significantly higher than that of other treatments (P＜0.05); while the soluble sugar contents in the T3 treatment showed significantly higher than that of CK,T1 and T4.There was no significant difference for CK, T1 and T4 treatments.

Fig.3 Chlorophyll (A), soluble sugar (B), ascorbic acid (Vc, C) and nitrate (D) contents in lettuce leaves.CK treatment was cultured with modified Hoagland nutrition solution.T1, T2, T3, and T4 were treated with 1-, 2-, 3- and 4-fold ozone-treated sludge dilution solutions, respectively.Bars mean SD.The different letters mean significant differences at P=0.05.

The trend of soluble sugar contents was similar to the trend of chlorophyll contents for all treatments.As was known to all, chlorophyll absorbed energy from the light, and then used the energy to turn carbon dioxide and water into carbohydrates.The high soluble sugar content of lettuce in T2 may be the result of an optimal sludge concentration causing overall improvements in plant metabolic activity,transport of trace elements and enzyme activity.These improvements could accelerate adenosine tri-phosphate(ATP) synthesis and other enzymatic reactions, leading to increased photosynthesis where soluble sugar was the primary by product from this process.Cu and Zn were the most likely toxicant elements in lettuce when exposed in the sludge dilution solution (Park et al.2016).The biological toxicity of ozone-treated sludge dilution solution in T1 was presented, which was caused from the higher toxic compounds (Cu and Zn) (Park et al.2012).The Cu and Zn contents were over the range of control standards,which resulted in the decrease of soluble sugar contents in T1 when compared with that in T2.

3.4.The effect of the ozone-treated sludge dilution solutions on lettuce Vc content

The Vc content in lettuce cultured by various ozone-treated sludge dilution solutions and the CK nutrition solution were calculated by ascorbic acid measurements (Fig.3-C).The Vc content of lettuce in T2 was the highest among all the treatments, then followed by T4, T1, T3 and CK.And the Vc content of lettcue in T2 was significantly higher (P＜0.05)than that of other treatments.No significant difference was observed among the T1, T3, and T4 ozone-treated sludge dilution treatments.The trend of Vc was similar to the trend of chlorophyll and soluble sugar contents for all treatments.

Vc was especially found in vegetables and fruits and an essential nutritional element for human and cannot be synthesized within the human body.This vitamin must be obtained from external sources such as food.If the Vc storage content in human body was less than 300 mg,some symptoms would appear, such as increased capillary fragility and subcutaneous haemorrhaging to some degree(Yue 2008).Therefore, Vc was an important index for lettuce quality evaluation.The Vc content in the lettuce from the T2 treatment was significantly higher than that of other treatments, demonstrating the advantages of the nutritional value in ozone-treated sludge applications when compared to the modified Hoagland nutrient solution.

3.5.The effect of the ozone-treated sludge dilution solutions on nitrate content in the lettuce leaves

The nitrate contents in the lettuce leaves cultured by the ozone-treated sludge dilution and the CK nutrition solutions were determined.The results demonstrated that the nitrate content in the lettuce leaves under CK was the highest among all the treatments, followed by T1, T2, T3, and T4 treatments (Fig.3-D).Based on the ANOVA results, there were significant differences (P＜0.05) between CK and the ozone-treated sludge dilution treatments on the nitrate content in lettuce.Among the ozone-treated sludge dilution treatments, nitrate content in T1 showed significantly higher than that in the T2, T3, and T4 treatments (P＜0.05).The nitrate contents of the cultured lettuce in the T1, T2, T3 and T4 were 34.3, 53.93, 55.34, and 68.60% lower than that in CK treatment, respectively.

Nitrate content is one of the most important indices in vegetable safety evaluation.Normally, a certain amount of nitrate in vegetables is harmless to human health.However,nitrate can be converted into nitrite by plants, fungi and human gut bacteria, resulting in the formation of compound nitrosamine that is a strong carcinogen (Bedale et al.2016;Lu et al.2016).In addition, excessive absorption of nitrate can lead to mental and developmental retardation, low levels of methemoglobin, or even death (Du 2011).According to the requirements of criterion in GB18406.1 (2001), the nitrate content must be below 600 mg kg-1in fruits and vegetables, 1 200 mg kg-1in root vegetables, and 3 000 mg kg-1in leafy vegetables.Because 80% of the nitrogen in modified Hoagland nutrition solution is nitrate, the cultivated plant will absorb and accumulate excessive nitrate after the ammonium nitrogen is exhausted (Rosales et al.2010).While, the plant can utilize ammonium nitrogen directly when cultivated by the ozone-treated sludge dilution solution due to the main nitrogen source of this solution (Kramer 1983).Thus, the nitrate contents of lettuce treated by the ozonetreated sludge dilution solution satisfied the nitrate limit(3 000 mg kg-1) in leafy vegetables set by the GB18406.1-2001 (2001).As shown in Fig.3-D, nitrate contents in all the treatments of this study were within security scope, and the nitrate contents of lettuce in T1-T4 were significantly lower than that in CK, which indicated that the lettuces cultivated with demestic sludge were security when evaluated with nitrate index only.The result was consisted with the nitrate contents in the nutrition solution, and the nitrate in CK was the highest.However, the leaves of lettuce in T4 are pale green or yellow, which is typical of nitrogen deficiency.Therefore, T2 and T3 were suitable solutions in terms of nitrogen requirements and security of plant.

4.Discussion

In this hydroponic experiment, we found that 2-fold ozonetreated sludge dilution solution could improve the quality of hydroponic lettuce more compared to the modified Hoagland nutrient solution.The lettuce cultured using the 2-fold ozonetreated sludge dilution solution showed significantly higher contents of chlorophyll, soluble sugar and Vc.The 2-fold ozone-treated sludge dilution solution improved the quality of hydroponic lettuce compared to the modified Hoagland nutrient solution used traditionally.It was also observed that unsuitable concentrations of ozone-treated domestic sludge dilution solution could inhibit lettuce growth.High sludge concentrations inhibited lettuce growth and at low sludge concentrations the nutrition provided was insufficient to satisfy the lettuce growth demand (Chekli et al.2017).

The leaf number, plant height, fresh weight and dry weight in the lettuce cultured by the modified Hoagland nutrient solution were significantly higher than those cultured by any of the ozone-treated sludge dilution solutions.While the nitrate content in the lettuces cultured using the 1-, 2-, 3-,and 4-fold ozone-treated sludge dilution solutions were 34.3,53.93, 55.34, and 68.60% lower than that in the modified Hoagland nutrient solution treatment, respectively, although the chlorophyll, soluble sugar and Vc contents increased.

The various physiological indexes of lettuce were not independent and there was a certain relationship between them (Table 3).As shown in Table 3, nitrate content had a significant or extremely significant correlation with leaf number, plant height and dry weight, and the correlation coefficient were 0.99, 0.86, 0.87, respectively.The result indicated that the nitrate content would increase with the leaf number, plant height and dry weight increasing.Thus, the leaf number, plant height and dry weight could be appraisal indexes of nitrate content.So, the nitrate content could be controlled within the scope of the security by the leaf number,plant height and dry weight.And dry weight had a significant or extremely significant correlation with leaf number, plant height and root length, with the correlation coefficient of 0.89,1.00, and 0.90, respectively.This was consistent with others reports (Blasco 2010; Lucini 2015).But the correlation for chlorophyll or soluble sugar content with other physiological indexes was not significant.Additinally, Vc had a significant correlation with leaf number.

Some researches showed that the significant correlation among physiological indexes and quantitative trait loci(QTLs) of corresponding physiological indexes may exist chain effect (Zhu et al.2004).Therefore, the correlation among physiological indexes could be understood further by the correlation analysis among physiological indexes.The results could provide basic theory for genetic analysis of lettuce stress resistance in the future.

The ozone-treated domestic sludge dilution solutions investigated in this study were thought to be an efficient liquid fertilizer substitute.Here, improvements in terms of physiological, biochemical and nutritional characteristics and safety in the lettuce growth process were observed.Before the sludge was treated by ozone, the number of fecal E.coli bacteria was measured.The results showed that the number of the feces in the sludge before ozone treating ismore than 1×105L-1, which was measured with the most probable number (MPN) method, while the number of the feces in the modified Hoagland nutrient solution is zero.And the fecal E.coli was not detected in the 1-, 2-, 3-, and 4-fold ozone-treated sludge dilution solutions after ozone treated for 120 min, so ozone treatment showed strong bactericidal ability, which was beneficial to the effective control of pathogenic microorganisms.But heavy metal distribution in the sludge solution and lettuce cultivated with sludge must be studied in subsequent research.And further studies will also be performed on food safety.Using ozonetreated sludge dilution solution as fertilizer in hydroponic systems has the potential to outperform traditional fertilizers and to cut costs for growers, however, further reserarch is necessary.

Table 3 Correlation analysis on physiological indexes of lettuce for all treatments

5.Conclusion

In this study, hydroponic lettuce with demestic sludge without industrial pollution domestic sludge was carried out, and correlation analysis among the lettuce physiological and biochemical indexes was done, which including leaf number,plant height, fresh weight, dry weight, root length, root weight,chlorophyll, soluble sugar, Vc and nitrate contents.The results showed that the modified Hoagland nutrient solution could provide the plant abundant nitrogen source, and the plant cultivated with that were more vigorous, the biomass was larger, and the physiological indexes were better than that cultivated wth sludge.But from the perspective of the nutrition of the food, the chlorophyll, soluble sugar and Vc contents of the lettuce cultivated with suitable ozone-treated sludge dilution solution were better than that cultivated with the modified Hoagland nutrient solution.Thus, the suitable ozone-treated sludge dilution solution can be as a hydroponic nutrition solution.Particularly, the nitrite content in lettuce plants cultivated with ozone-treated sludge dilution solution were significantly lower than that cultivated with the modified Hoagland nutrient solution nitrate level and is far lower than the national standard requirements, so the ozone-treated sludge dilution solution the is more safe and reliable.Moreover, the pathogenic microorganisms in the sludge solution can be killed by ozone, so the security is protected.At the same time, the suitable ozone-treated sludge dilution solution was found in the study, which will provide a useful reference for the application of sludge as fertilizer to the production of lettuce.As to the risk of heavy metals in sludge, we will carry out further studies.

Acknowledgements

The work was financially supported by the Special Fund for Agro-scientific Research in the Public Interest, China(20130310104), and the Key Technology R&D Program of Tianjin, China (12ZCZDNC09600).

APHA (American Public Health Association), AWWA (American Water Works Association), WPCF (Water Pollution Control Federation), WEF (Water Environment Federation).1915.Standard Methods for the Examination of Water and Wastewater (vol.2).American Public Health Association,USA.

Appels L, Degrève J, van der Bruggen B, Van Impe J, Dewil R.2010.Influence of low temperature thermal pre-treatment on sludge solubilisation, heavy metal release and anaerobic digestion.Bioresource Technology, 101, 5743-5748.

Armstrong F A J.1963.Determination of nitrate in water ultraviolet spectrophotometry.Analytical Chemistry, 35,1292-1294.

Awad Y M, Lee S E, Ahmed M B M, Vu N T, Farooq M, Kim I S, Kim H S, Vithanage M, Usman A R A, Al-Wabel M,Meers E, Kwon E E, Ok Y S.2017.Biochar, a potential hydroponic growth substrate, enhances the nutritional status and growth of leafy vegetables.Journal of Cleaner Production, 156, 581-588.

Edberg S C L, Rice E W, Karlin R J, Allen M J.2000.Escherichia coli: The best biological drinking water indicator for public health protection.Journal of Applied Microbiology, 88,106-108.

Bedale W, Sindelar J J, Milkowski A L.2016.Dietary nitrate and nitrite: Benefits, risks, and evolving perceptions.Meat Science, 120, 85-92.

Bitton G.2005.Wastewater Microbiology.John Wiley and Sons,Hoboken, New Jersey.

Blasco B, Rios J J, Cervilla L M, Sánchez-Rodríguez E, Rubio-Wilhelmi M M, Rosales M A, Ruiz J M, Romero L.2010.Photorespiration process and nitrogen metabolism in lettuce plants (Lactuca sativa L.): Induced changes in response to iodine biofortification.Journal of Plant Growth Regulation,29, 477-486.

Bougrier C, Albasi C, Delgenès J P, Carrère H.2006.Effect of ultrasonic, thermal and ozone pre-treatments on waste activated sludge solubilisation and anaerobic biodegradability.Chemical Engineering and Processing:Process Intensification, 45, 711-718.

Bratina B, Šorgo A, Kramberger J, Ajdnik U, Zemljič L F, Ekart J, Šafarič R.2016.From municipal/industrial wastewater sludge and FOG to fertilizer: A proposal for economic sustainable sludge management.Journal of Environmental Management, 183, 1009-1025.

Buysse J A N, Merckx R.1993.An improved colorimetric method to quantify sugar content of plant tissue.Journal of Experimental Botany, 44, 1627-1629.

Chacana J, Alizadeh S, Labelle M A, Laporte A, Hawari J, Barbeau B, Comeau Y.2017.Effect of ozonation on anaerobic digestion sludge activity and viability.Chemosphere, 176, 405-411.

Cawse P.1967.The determination of nitrate in soil solutions by ultraviolet spectrophotometry.Analyst, 1094, 311-315.

Chekli L, Kim J E, El Saliby I, Kim Y, Phuntsho S, Li S, Ghaffour N, Leiknes T, Shon H K.2017.Fertilizer drawn forward osmosis process for sustainable water reuse to grow hydroponic lettuce using commercial nutrient solution.Separation and Purification Technology, 181, 18-28.

Chong M N, Jin B, Saint C P.2011.Bacterial inactivation kinetics of a photo-disinfection system using novel titaniaimpregnated kaolinite photocatalyst.Chemical Engineering Journal, 171, 16-23.

Chu L, Yan S, Xing X H, Sun X, Jurcik B.2009.Progress and perspectives of sludge ozonation as a powerful pretreatment method for minimization of excess sludge production.Water Research, 43, 1811-1822.

Du L F, Zhao T K, Zhang C J, An Z Z, Wu Q, Liu B C, Li P,Ma M T.2011.Investigations on nitrate pollution of soil,groundwater and vegetable from three typical farmlands in Beijing Region, China.Agricultural Sciences in China,10, 423-430.

GB 18406.1-2001.2001.Safety Qualification for Agricultural Product-Safety Requirements for Non-Environmental Pollution Vegetable.General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China.(in Chinese)

GB 4284-1984.1984.Control Standard of Pollutants in Agricultural Utilization of Sludge.General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China.(in Chinese)

GB 5084-2005.2005.Standards for Irrigation Water Quality.Agro-Environmental Protection Institute, Ministry of Agriculture of China.(in Chinese)

Hawrylak-Nowak B.2013.Comparative effects of selenite and selenate on growth and selenium accumulation in lettuce plants under hydroponic conditions.Plant Growth Regulation, 70, 149-157.

Hoagland D R, Arnon D I.1950.The water-culture method for growing plants without soil.California Agricultural Experiment Station, 347, 357-359.

Jones J J B.2016.Hydroponics: A Practical Guide for the Soilless Grower.CRC Press, USA.

Keskin B, Bozkurt M A, Akdeniz H.2010.The effects of sewage sludge and nitrogen fertilizer application on nutrient and(Bromus inermis Leyss.).Journal of Animal and Veterinary Advances, 9, 896-902.

Kramer P J.1983.Water Deficit and Plant Growth.Academic Press, New York.p.342.

Lamb D T, Ming H, Megharaj M, Naidu R.2010.Relative tolerance of a range of Australian native plant species and lettuce to copper, zinc, cadmium, and lead.Archives of Environmental Contamination and Toxicology, 59, 424-432.

Lu J, Zhang L, Lewis R S, Bovet L, Goepfert S, Jack A M, Dewey R E.2016.Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco-specific nitrosamine accumulation in cured leaves and cigarette smoke.Plant Biotechnology Journal, 14, 1500-1510.

Lucini L, Rouphael Y, Cardarelli M, Canaguier R, Kumar P,Colla G.2015.The effect of a plant-derived biostimulant on metabolic profiling and crop performance of lettuce grown under saline conditions.Scientia Horticulturae, 182,124-133.(in Chinese)

Mecha A C, Onyango M S, Ochieng A, Momba M N.2017.Evaluation of synergy and bacterial regrowth in photocatalytic ozonation disinfection of municipal wastewater.Science of the Total Environment, 601, 626-635.

Murat C, Merih O U, Isil B.2010.Treatment of E.coli HB101 and the tetM gene by Fenton’s reagent and ozone in cow manure.Journal of Environmental Management, 91, 1-4.

Neumann P, Pesante S, Venegas M, Vidal G.2016.Developments in pre-treatment methods to improve anaerobic digestion of sewage sludge.Reviews in Environmental Science and Bio-Technology, 15, 173-211.

Oliver I W, Mclaughlin M J, Merrington G.2005.Temporal trends of total and potentially available element concentrations in sewage biosolids: A comparison of biosolid surveys conducted 18 years apart.Science of the Total Environment,337, 139-145.

Park J, Yoon J H, Depuydt S, Oh J W, Jo Y M, Kim K, Brown M T, Han T.2016.The sensitivity of an hydroponic lettuce root elongation bioassay to metals, phenol and wastewaters.Ecotoxicology and Environmental Safety, 126, 147-153.

Park J S, Brown M T, Han T.2012.Phenol toxicity to the aquatic macrophyte Lemna paucicostata.Aquatic Toxicology, 106,182-188.

Tang Q Y, Zhang C X.2013.Dat a processing system (DPS)software with experimental design, statistical analysis and data mining developed for use in entomological research.Insect Science, 20, 254-260.(in Chinese)

Tian X, Wang C, Trzcinski A P, Lin L, Ng W J.2015.Interpreting the synergistic effect in combined ultrasonication-ozonation sewage sludge pre-treatment.Chemosphere, 140, 63-71.

USEPA (U.S.Environmental Protection Agency).1982.Seed Germination/Root Elongation.Toxicity Tests.EG-12, Office of Toxic Substances, Washington, D.C.

USFDA (U.S.Food and Drug Administration).1987.Environmental Assessment Technical Handbook.Center for Food Safety and Applied Nutrition and Center for Veterinary Medicine, Washington, D.C.

Yang P, Wang D Q, Qiu L, Zhang K Q.2015.The effect of digested sludge pretreated with UV-O3on hydroponic Gynura cusimbua yield and quality.Journal of Argo-Environmental Science, 9, 1653-1658.(in Chinese)

Yue S B.2008.Study on hydroponics with biogas slurry as nutrient solution.Ph D thesis, Huazhong Agricultural University, Wuhan.p.26.(in Chinese)

Zhang J, Zhang J, Tian Y, Li N, Kong L, Sun L, Yu M, Zuo W.2016.Changes of physicochemical properties of sewage sludge during ozonation treatment: Correlation to sludge dewaterability.Chemical Engineering Journal,301, 238-248.

Zhu S, Rossnagel B G, Kaeppler H F.2004.Genetic analysis of quantitative trait loci for groat protein and oil content in oat.Crop Science, 44, 254-260.