Effects of irrigation water and N fertilizer types on soil microbial biomass and enzymatic activities

Shang Fangze, Yang Peiling, Ren Shumei, Huang Yuefei, Sun Xiaoling, Xu Hongliang

Effects of irrigation water and N fertilizer types on soil microbial biomass and enzymatic activities

Shang Fangze1,2, Yang Peiling3, Ren Shumei3, Huang Yuefei2, Sun Xiaoling1, Xu Hongliang1

(1..,518102,; 2.,100084,; 3.,,100083,)

Effects of irrigation water and N fertilizer types on soil microbes and enzymes are poorly understood. This study aimed to evaluate the effect of different irrigation water and N fertilizer types on soil microbial biomass and enzymatic activities. A 2-year experiment (2015—2017) was conducted at the Tongzhou Experimental Station of Tongzhou District, Beijing, China(39°42′6.93″N, 116°41′2.31″E). The experiments utilized a factorial randomized complete block design with 8 treatments (2×4) in 3 replicates. The first factor had 2 irrigation water types (fresh water (FW) and reclaimed water (RW)), and the 2nd factor had 4 N fertilizer types (no nitrogen, urea, ammonium sulfate, and slow-release urea). Physicochemical, microbe and enzyme analyses from soil and water samples were determined. The results showed the contents of SOC, SON, DOC, DON, TN, NO3--N, and NH4+-N in different treatment soils were significantly different across crop seasons(<0.05). The average contents of DON in soils irrigated with RW are significantly higher than that in soils irrigated with FW (<0.05). Fertilization increased the SOC, SON, DON, TN, and NO3--N contents in the soils significantly (<0.05). No significant interaction between irrigation and fertilization on the soil SOC, SON, DOC, DON, TN, NO3--N, and NH4+-N contents. the DON content in the soils irrigated with RW was average 8.33% significantly higher than in the soils irrigated with FW (<0.05). The SOC, SON, DON, TN and NO3--N contents in the soils fertilized with inorganic N was on average 8.35%, 10.13%, 11.75%, 11.60% and 92.99% significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05). Soil bacterial biomass under different treatments ranged from 9.59 to 12.18 nmol/g. The soil actinobacterial biomass values under different treatments ranged from 1.01 to 1.24 nmol/g. The soil G+ bacterial biomass values under different treatments were 6.10- 6.73 nmol/g. Compared with FW and RW irrigation significantly increased the soil bacterial, actinobacterial, and gram-positive (G+) bacterial biomasses and total phospholipid fatty acids (PLFAs) by 7.60%, 10.48%, 4.97% and 4.88%, respectively. N fertilization significantly increased soil bacterial biomass by 13.42%-17.34% and increased the total PLFAs by 8.12%-11.19%. Compared with FW, RW irrigation did not significantly increase soil urease, catalase and invertase activities. N fertilization did not significantly increase soil urease, catalase and invertase activities. Soil microbes and enzymes were more active in the soils fertilized with slow-release urea. These results indicate that actinobacteria and G+ bacteria grew more quickly and could more efficiently utilize the DON introduced by the RW and that the soil bacteria could more efficiently utilize NO3--N introduced by the fertilizers than the other microbes. RW irrigation and slow-release urea fertilization were more effective to increase the soil microbes and enzymes. For better soil quality, higher yields and save water resources, drip irrigation with RW under slow-release urea fertilization was recommended for summer maize-winter wheat crop rotation.

fertilization; irrigation; soils ; microbial biomass; enzymatic activities; reclaimed water

0 Introduction

Irrigation and fertilization are two of the most important agricultural management practices that have impact on soil quality. Soil C and N contents are traditional parameters used to assess the impact of agricultural practices on soil quality[1]. In addition, biological characteristics such as soil microbes and enzymes are also sensitive to soil management practices. Soil microbial biomass is strongly dependent on nutritional and other chemical and physical conditions of soil[2].Soil microbes play a key role in organic matter decomposition, nutrient and energy cycling[3]. As biocatalysts, soil enzymes can promote various reactions and metabolic processes in biogeochemical cycles of nutrients, and maintain soil structure[4]. Soil microbes and enzymes can rapidly respond to soil management practices such as irrigation and fertilization[5-8], and are thus considered to be sensitive indicators of nutrient cycling processes and soil quality[9].

The scarcity of fresh water (FW) has become a worldwide problem, especially in arid and semiarid regions. In all the industries, agriculture consumes 90% of the total water usage[10]. Thus, the use of reclaimed water (RW) for irrigation has become a common practice to alleviate water shortages. Compared to FW, RW contains greater amounts of inorganic nutrients such as NO3--N, NH4+-N, total K, and total P; organic nutrients such as organic C and N, microbes, organic pollutants and heavy metals[11].Although irrigation with RW can reduce the use of FW[12-13], it also can increase the amounts of salts, heavy metals and xenobiotic organic compounds in the soil[14-16].

The effects of long-term irrigation with RW on soil quality are still controversial. Some studies have found that organic components in RW provide organic carbon that stimulates microbial growth, but short-term RW irrigation could not alter soil microbial biomass, and microbial biomass increases when RW is used for long time[17-21]. In contrast, some studies have confirmed that RW inhibits the growth of soil microbes due to the toxicity of heavy metals and organic molecules in RW[22]. In addition, studies have found that long-term irrigation of crops with RW can significantly increase the activity of dehydrogenase in soil[23-24]. With the improvement of water treatment technology, the content of salt, heavy metals and xenobiotics in RW has been reduced in recent years[25]. In addition, drip irrigation is one of the most effective ways to save water and provide nutrients for crops[26]. Therefore, the impact of RW irrigation using soil drip irrigation technology on soil quality in recent years needs to be re-evaluated.

The effects of fertilizer application on soil microbes and enzymes activities depend on the nature and quantity of the fertilizer, initial soil fertility status, and duration of fertilization[27]. Fertilizers have different effects on soil microbes in different area. Some researchers found that inorganic N fertilizer increases the amount of soil microbes when the soil C content reaches a certain amount[28-29]. Some studies have found that inorganic nitrogen fertilizers reduce soil microbial biomass[30-31]. At the same time, other studies found that fertilizer addition has no effect on the microbial index[32-33]. Soil enzymatic activities were correlated with the long-term total C inputs, and short-term inorganic N applications have limited effects on soil enzymatic activities[34]. Although some studies have analyzed the effects of N fertilizer application on soil microbes and enzymes, few studies have been able to quantify the impact of N fertilizer, RW and drip irrigation techniques on soil microbial biomass and enzymatic activities.

The North China Plain produces more than 76% and 29% of the nation’s wheat and maize. However, this region experiences severe water crisis, with approximately 70% of the irrigation water being groundwater[35]. The improvement of water treatment technology has greatly improved the water quality of RW. The use of RW for drip irrigation is the optimal choice to solve the water crisis in this region. However, few studies focus on the effects of RW drip irrigation on soil quality, especially soil microbial biomass and enzymatic activities. To promote the sustainable development of agriculture in the region, the research questions of this study were: 1) to determine the effect of RW drip irrigation on soil microbial biomass, enzyme activities and soil quality; 2) to analyze the effect of different types of nitrogen fertilizer on soil microbial biomass, enzyme activities, soil quality and crop yield; 3) to examine the interaction of irrigation water and N fertilizer on soil microbial biomass and enzyme activities.

1 Materials and methods

1.1 Site description

A 2-year experiment (2015—2017) was conducted at the Tongzhou Experimental Station of Tongzhou District, Beijing, China (39°42′6.93″N, 116°41′2.31″E). The annual mean precipitation is 620 mm. The annual mean temperature is 11.3 ℃ and annual mean sunshine is 2 460 h. The local climate is warm temperate continental semi-humid monsoon[36]. The physical and chemical properties of soil within the top 10 cm were as followed: sand 51.1%, silt 34.5%, clay 14.4%, bulk density 1.45 g/cm3, field water holding capacity 0.31 m3/m3, saturated water content 0.42 m3/m3, soil pH value 8.1, soil electrical conductivity (EC) 0.33 dS/m, soil organic matter 19.36 g/kg, soil NO3--N 44.88 mg/kg, soil NH4+-N 8.15 mg/kg, soil organic carbon (SOC) 11.22 g/kg, soil organic nitrogen (SON) 1.01 g/kg, soil total nitrogen (TN) 1.12 g/kg, soil total phosphorus (TP) 0.62 g/kg, and soil total potassium (TK) 1.05 g/kg.

1.2 Field experimental design

The experiment was carried out in a 240 m×240 m farmland with a total of 24 plots, and each plot was 10 m× 10 m. The experiments utilized a factorial randomized complete block design with 8 treatments (2×4) in 3 replicates.

The first factor had 2 irrigation water types (FW and RW), and the second factor had 4 N fertilizer types (no nitrogen, urea, ammonium sulfate, and slow-release urea). In this study, drip irrigation with FW and RW was used during the summer maize and winter wheat rotation. FN, FU, FA and FS represented treatments irrigated with FW and fertilized with no nitrogen, urea, ammonium sulfate, and slow-release urea fertilizer, respectively. RN, RU, RA and RS represented treatments irrigated with RW and fertilized with no nitrogen, urea, ammonium sulfate, and slow-release urea fertilizer, respectively. The detailed irrigation date and amount included: 1) irrigation 53.2 mm on July 3, 2015; 2) irrigation 46 mm on August 10, 2015; 3) irrigation 35.7 mm on September 26, 2015; 4) irrigation 44.6 mm on October 20, 2015; 5) irrigation 46.5 mm on April 6, 2016; 6) irrigation 33.4 mm on April 16, 2016; 7) irrigation 31.1 mm on April 28, 2016; 8) irrigation 45.9 mm on May 13, 2016; 9) irrigation 35.8 mm on May 20, 2016; 10) irrigation 34.2 mm on May 29, 2016; 11) irrigation 34.3 mm on June 30, 2016; 12) irrigation 26.5 mm on July 15, 2016; 13) irrigation 30.5 mm on August 6, 2016; 14) irrigation 33.1 mm on August 26, 2016; 15) irrigation 21 mm on September 18, 2016; 16) irrigation 17.6 mm on September 28, 2016; 17) irrigation 61.2 mm on November 28, 2016; 18) irrigation 46.8 mm on April 2, 2017; 19) irrigation 42 mm on May 9, 2017.

The sowing date of the summer maize in 2015 was June 26, and the harvest date was October 6. The sowing date of the summer maize in 2016 was June 25, and the harvest date was October 15. The summer maize in 2015 and 2016 was sown manually at the rate of 50 kg/hm2with 60 cm width per row. The application of both P and K fertilizer was at a rate of 105 kg/hm2before planting for all the treatments. For summer maize, the N fertilization were:1) for no nitrogen fertilizer treatments, no nitrogen fertilizer; 2) for the urea fertilizer treatments, urea was applied at 140 kg/hm2before planting, and on August 4 and 27, urea was applied with drip irrigation at 80 kg/hm2; 3) for the ammonium sulfate fertilizer treatments, ammonium sulfate was applied at the same N rate and the same date as urea; 4) for the slow-release urea fertilizer treatments, slow-release nitrogen was applied at 300 kg/hm2before planting. The sowing date of the winter wheat was October 8, 2015, and the harvest date was June 15, 2016. The sowing date of the winter wheat was October 18, 2016, and the harvest date was June 14, 2017. The winter wheat was sown manually at a rate of 160 kg/hm2with 15 cm row spacing. For winter wheat, the N fertilization were as follows: 1) for no nitrogen fertilizer treatments, no nitrogen fertilizer; 2) for the urea fertilizer treatments, urea was applied at 100 kg/hm2before planting, and on April 4 and May 11, 2014, urea was applied with drip irrigation at 70 kg/hm2; 3) for the ammonium sulfate fertilizer treatments, ammonium sulfate was applied at the same N rate and the same date as urea; 4) for the slow-release urea fertilizer treatments, slow-release nitrogen was applied at 240 kg/hm2before planting.

The FW was taken from a well at the experimental station. The RW was obtained by the method of deep cell wastewater reclamation and reuse from the Beijing Bishui Wastewater Treatment Plant. For the summer maize crop, the FW and RW irrigation systems consisted of 2 tanks (600 L) and 32 drip tubes (8 tubes in each fertilization treatment), respectively. The tank was on the ground and installed with a pump. The drip tubes with 0.2 m emitter intervals were placed at the center of each row. For the winter wheat crop, the FW and RW irrigation systems consisted of 2 tanks (600 L) and 64 drip tubes (16 tubes in each fertilization treatment), respectively. The drip tubes with 0.2 m emitter intervals were placed at the center of 2 rows. The quality of irrigation water is shown in Table 1. Rainfall, irrigation and nutrients input into soils by irrigation during the experiments are shown in Table 2.

Table 1 Chemical parameters of fresh water (FW) and reclaimed water (RW) used in this study

Note: TN, total nitrogen; TP, total phosphorus; TK, total potassium; EC, electrical conductivity; DOC and DON are dissolved organic C and N;

Table 2 Rainfall, irrigation and nutrients input into the soils by irrigation during experiments

1.3 Sampling

Soil samples were taken from the surface layer (0-10 cm) during the field experiment. The initial soil samples were collected on June 25, 2015. The 6 sampling dates were October 6, 2015 (summer maize harvest), April 2, 2016 (winter wheat jointing stage), June 15, 2016 (winter wheat harvest), August 24, 2016 (summer maize earring stage), October 15, 2016 (summer maize harvest), and June 14, 2017 (winter wheat harvest). All of the soil samples were taken from 5 positions using the “S” form sampling method and then mixed to form a composite soil for each plot. The soil samples were sieved through a 2 mm sieve, and one-third of them were dried for measuring soil physical index, one-third of them were preserved in a 4 ℃ refrigerator for measuring soil enzymes and chemical index, and the others were preserved in a −18 ℃freezer for measuring soil microbes.

1.4 Soil physicochemical, microbe and enzyme analyses

SOC, SON, DOC and DON are the 4 main parameters reflecting organic matter conditions in soil. The soil TN, NO3--N, and NH4+-N are the 3 primary parameters reflecting the nitrogen conditions in the soil.

The soil total phospholipid fatty acids (PLFAs) reflects the total amount of microbial biomass. The values of the soil total PLFAs were the sum of the values of soil bacterial, actinobacterial, and fungal biomasses, and the other microbial types. Analyzing the total PLFAs is helpful to understand the effects of the different treatments on the whole microbial biomass.

Urease regulates the rates of N mineralization. Catalase activity is responsible for the breakdown of hydrogen peroxide, yielding water and molecular oxygen. Invertase regulates the rates of C decomposition and mineralization.

In the soil, soil pH was measured by a pH meter using distilled water (2.5:1 of water-soil mass ratio). The SOC was analyzed using a VarioMax CN dry combustion Analyzer (Elementar GmbH, Germany)[37]. The soil TN was quantified by the micro-Kjeldahl technique with a Gerhardt Automatic N Analyzer (Model KB8S, Germany). The soil texture was analyzed using a Malvern laser particle size analyzer (Hydro2000Mu). The soil bulk density was measured using the conventional core method[38]. The soil EC was measured with a conductivity meter using distilled water (5:1 of water-soil ratio). The soil TP and TK were determined by digestion with a mixed acid containing HF, HNO3and HClO4[39]. The soil NO3--N and NH4+-N were determined using a continuous flow analyzer (TRACCS-2000) with a 1 M KCl extraction. The SON was calculated by the formula [SON]=[soil TN]-[soil NO3--N]-[soil NH4+-N]. The soil total dissolved nitrogen (TDN) and soil dissolved organic carbon (DOC) were measured by a TOC/TN Multi N/C 3000Analyzer (Analytik JenaAG, Germany) with a 1 M KCl extraction. The soil dissolved organic nitrogen (DON) was calculated by the formula [Soil DON]=[soil TDN]-[soil NO3--N]-[soil NH4+-N].

In the water, the concentrations of TN and DOC were measured by a TOC/TN Multi N/C 3000 Analyzer (Analytik JenaAG, Germany), and the NO3--N, NH4+-N concentrations were determined using a continuous flow analyzer (TRACCS-2000); DON was calculated by the formula [water DON]=[water TN]-[water NO3--N]-[water NH4+-N]. The water pH was measured by a pH meter. The water EC was measured by a conductivity meter. The water TK was measured by a flame photometric method[40]. The water TP was determined by a spectrophotometer method[41].

PLFAs were extracted from freeze-dried soil samples (0.75–1.5 g) using a modified Bligh-Dyer method[42]. The concentrations of individual PLFAs were calculated based on the 19:0 internal standard concentrations. The peaks were assigned using bacterial standards and identification software from the Microbial Identification System. The fatty acid nomenclature was used[43]. PLFAs indicative of 5 different soil microbial communities were quantified as follows[43-45]: bacteria, actinobacteria, gram-positive(G+) bacteria, gram-negative(G-) bacteria, and fungi. Thus, iso/anteiso methyl-branched fatty acids are considered as markers for G+ and cyclic forms and the monoenoic18:1ω7 fatty acid is a marker for G- bacteria, whereas 10-methyl-substituted fatty acids are characteristic of actinobacteria. The PLFA 18:2ω6 represents saprotrophic fungi[1]. The total biomass was estimated as the sum of all the extracted PLFAs. A detailed grouping of the bacteria biomarkers are as follow: i14:0, 15:0, i15:0, a15:0, i16:0, 16:1ω7, 17:0, i17:0, a17:0, 18:1ω7, cy17:0, cy19:0, 10Me16:0, 10Me17:0, 10Me18:0. The actinobacteria biomarkers are as follows: 10Me16:0, 10Me17:0, 10Me18:0. The G+ bacteria biomarkers are as follows: i14:0, i15:0, a15:0, i16:0, i17:0, a17:0. The G- bacteria biomarkers are as follows: 16:1ω7, 18:1ω7, cy17:0, cy19:0, while the fungi biomarkers include18:2ω6.

In this study, 3 soil enzymes involved in C cycling (invertase), N cycling (urease), and the most widely studied soil oxidoreductases (catalases) were monitored. Urease activity (EC 3.5.1.5) was assayed using a 10% urea solution as a substrate, and the sample (2.5 g of fresh soil) was incubated at 37 ℃ for 24 h. After the addition of a phenol Na hypochlorite solution, 0.5 mL of the extract solution was diluted to 10 mL, and the activity was determined as the NH4+released in the hydrolysis reaction[46]. Catalase activity (EC 1.11.1.6) was assayed using the titration method[47-48]. Five grams of fresh soil were placed at 0—4 ℃ for 30 min, and after adding 25 mL of 3% H2O2, the sample was placed at 0—4 ℃for 30 min again, before terminating the reaction using the addition of 25 mL of 1 M H2SO4. After filtration, 4 mL of 0.5 M H2SO4was added to 1 mL of filtrate, using 20 mM KMnO4to measure the O2absorbed. Invertase activity (EC 3.2.1.26) was assayed using a fresh 5 g of soil, and the soil was added to a sucrose solution, incubated for 24 h at 37 ℃, and its activity was mg glucose equivalents per g soil during 24 h[49].

1.5 Data analysis

The figures were drawn using Origin 8.0 (Origin Lab Corporation, USA). The tests for the significant differences were conducted using SPSS17.0 software (IBM Corporation, Armonk, NY, USA). Two-way fixed factor analysis of variance (ANOVA) and one-way ANOVA were used to test the effect of irrigation water types and N fertilizer types on soil nutrient contents, microbial biomass and enzymatic activities in the study soils. The multiple comparisons for the mean values were carried out using a Turkey procedure (<0.05).

2 Results and Analysis

2.1 Soil C and N contents

The variances of soil C and N contents over time under different treatments are shown in Table 3. One-way ANOVA showed that the contents of SOC, SON, DOC, DON, TN, NO3--N, and NH4+-N in different treatment soils were significantly different across crop seasons (7 sampling dates) (<0.05). Two-way ANOVA showed no significant interaction between irrigation and fertilization on the soil SOC, SON, DOC, DON, TN, NO3--N, and NH4+-N contents (Table 3).

The average contents of DON in soils irrigated with RW were significantly higher than that in soils irrigated with FW (<0.05). Compared to no fertilizer, fertilization increased the SOC, SON, DON, TN, and NO3--N contents in the soils significantly (<0.05) with the order of slow-release urea < ammonium sulfate < urea, but no significant difference was found among different fertilizer types.

Specifically, the DON content in the soils irrigated with RW was average 8.33% significantly higher than in the soils irrigated with FW (<0.05). The SOC, SON, DON, TN and NO3--N contents in the soils fertilized with inorganic N was on average 8.35%, 10.13%, 11.75%, 11.60% and 92.99% significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05).

2.2 Soil microbial biomass

2.2.1 Soil bacteria, actinobacteria and G+ bacteria

The values of soil bacterial biomass, actinobacterial biomass and G+ bacterial biomass are shown in Fig.1a, Fig.1b and Fig.1c, respectively. The initial values of soil bacterial, actinobacterial and G+ bacterial biomasses were small, and increased during the first rotation of the summer maize and winter wheat, and reached the maximum at the first harvest time of the winter wheat. After that, the values decreased during the second rotation of the summer maize, but increased during the second rotation of the winter wheat again. The changes in the soil bacteria, actinobacteria and G+ bacteria had a similar trend during the experiment.

Table 3 Soil SOC, SON, DOC, DON, TN, NO3--N and NH4+-N contents and statistical analysis

Note: Different lower case letters represent significant different between treatments (<0.05).

Fig.1 Change of soil microbial biomass during experiment

The soil bacterial biomass under different treatments ranged from 9.59 to 12.18 nmol/g (Table 4). The bacterial biomass in the soils irrigated with RW was on average 7.60% significantly higher than that in the soils irrigated with FW (<0.05). The bacterial biomass in the soils fertilized with urea, ammonium sulfate and slow-release urea was on average 13.42%, 16.10% and 17.34% significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05).

The soil bacterial biomass values under different treatments ranged from 9.59 to 12.18 nmol/g (Table 4). The actinobacterial biomass in the soils irrigated with RW was on average 10.48% significantly higher than that in the soils irrigated with FW (<0.05). It showed that RW irrigation improved the actinobacterial biomass in soils significantly (<0.05). No significant difference were found between fertilized and unfertilized soils.

The soil G+ bacterial biomass values under different treatments ranged from 6.10 to 6.73 nmol/g (Table 4). The G+ bacterial biomass in the soils irrigated with RW was on average 4.97% significantly higher than that in the soils irrigated with FW (<0.05). Fertilization had no significant influence on soil G+ bacterial biomass(>0.05), while RW irrigation increased it significantly (<0.05).

2.2.2 Soil G- bacteria and fungi

The measured values of soil G- bacterial biomass and soil fungal biomass are shown in Fig.1d and Fig.1e respectively. Statistical analysis of the soil G- bacterial biomass and fungal biomass is shown in Table 5. The soil G- bacterial biomass was relatively stable during the summer maize and winter wheat rotations, while the soil fungal biomass slightly increased during the experiment. Different from the soil bacteria and G+ bacteria, neither of the RW irrigation and fertilization had significant influence on the G- bacterial and fungal biomasses.

2.2.3 Soil total PLFAs

Variations of the soil total PLFAs over time under different treatments are shown in Fig.1f, and the measured values had the same trend as the variations of soil bacterial biomass, actinobacterial biomass and G+ bacterial biomass.

The total PLFAs values under different treatments ranged from 18.15 to 20.82 nmol/g (Table 4). The total PLFAs in the soils irrigated with RW was on average 4.88% significantly higher than that in the soils irrigated with FW (<0.05). The total PLFAs in the soils fertilized with urea, ammonium sulfate and slow-release urea was on average 8.12%, 8.69% and 11.19% significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05). Both of the RW irrigation and fertilization increased the soil total PLFAs significantly (<0.05).

Thus, the bacterial, actinobacterial, G+ bacterial biomass and total PLFAs in the soils irrigated with RW was on average 7.60%, 10.48%, 4.97% and 4.88% significantly higher, respectively, than that in the soils irrigated with FW (<0.05). The bacterial biomass and total PLFAs in the soils fertilized with inorganic N was on average 13.42%-17.34% and 8.12%-11.19% significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05).

One-way ANOVA showed that the soil bacterial, actinobacterial, G+ bacterial, G- bacterial, and fungal biomasses and the total PLFAs were significantly different between different crop seasons (<0.05). Two-way ANOVA showed no significant interaction between irrigation and fertilization on the soil bacterial, actinobacterial, G+ bacterial, G- bacterial, and fungal biomasses and the total PLFAs (Table 4 and Table 5).

Table 4 Statistical analysis of soil bacterial, actinobacterial, G+ bacterial biomass and Total PLFAs (nmol·g-1)

Note: Different lower case letters within a row represent significant difference between irrigation with fresh water and reclaimed water (<0.05). Different capital letters within a column represent significant difference among different fertilization treatments (<0.05).

Table 5 Statistical analysis on soil G- bacterial and fungal biomass

Note: d, degree of freedom;,value;, significant value.

Some studies have found that soil microbial biomass is increased when irrigating with RW over the short-term and long-term[18,20]. However, others found that RW irrigation has no influence or reduces soil microbes, because the soil microbes are susceptible to the toxic effects of some of the organic molecules in the RW[22,50]. In our 2-year experiment, the total amount of the DOC, DON and TN input into the soils under RW irrigation was 6.877, 3.056 and 18.956 g/m2, respectively, which was 5-, 10- and 14-fold higher than that under FW irrigation (Table 2). Thus, we found a clear causal relationship that, compared to FW irrigation, RW irrigation brought 10 times the amount of DON into the soils, which led to an increase in the soil DON of 8.33%. The increased DON was utilized by soil microbes and promoted their growth. Correspondingly, the soil bacterial, actinobacterial, and G+ bacterial biomasses increased by 7.60%, 10.48%, and 4.97%, respectively, compared to the FW irrigation (Fig.2). Many studies have emphasized the importance of C and N nutrients on soil microbes[19], and our study further pointed out that especially the DON plays an important role in the growth of soil microbes. It should be noted that the actinobacteria and G+ bacteria were faster growing and more competitive towards the available substrates (especially DON) brought by the RW than the G- bacteria and fungi.

N fertilization can supply inorganic N and change the ratio of C to N. Some studies have found that N fertilization increases the soil microbial biomass[29], while others found that inorganic N fertilization has no influence on the bacteria or reduced the soil microbial biomass[31-32]. This study found that N fertilization significantly increased the soil bacterial biomass and total PLFAs, and a similar result was found in a grassland[3].We also found a clear causal relationship that the N fertilization treatments brought 500 kg/hm2each year into the soils, which led to an increase in the soil SOC, SON, DON, and TN contents by 8.35%-11.75% and the NO3--N content by 92.99%, respectively. The increased C and N contents (especially NO3--N) were utilized by the soil bacteria, and correspondingly, the soil bacterial biomass increased by 15.62% compared to the no nitrogen treatment (Fig.2). Unlike the results where the N fertilizer increases the G+ bacteria and reduces the G- bacteria[51], our study found that the N fertilizer had no significant influence on the soil G+ bacteria, G- bacteria, actinobacteria and fungi. First, the effects of fertilizer management may depend on the nature and quantity of the fertilizer added, initial soil fertility status, and the duration of fertilization[27]. Second, short-term inorganic N applications had limited effects on the soil microbial biomass C. Although the N fertilizer increased the soil bacterial biomass and the total PLFAs, there was no significant difference between the urea, ammonium sulfate and slow-release urea treatments on the soil microbial biomass. Because the chemical formula of the urea and slow-release urea is CO(NH2)2, and the chemical formula of ammonium sulfate is (NH4)2SO4, these three N fertilizers all belong to ammonium type nitrogen fertilizers. However, the soil microbial biomass was higher in the soils fertilized with slow-release urea. The way that slow-release urea slowly releases inorganic N into soils resulted in higher amount of microbial biomass.

a. Path modeling of soil microbes under reclaimed water irrigation compared with fresh water irrigation

b. Path modeling of soil microbes under N fertilization compared with no N fertilization

Fig.2 Path modeling diagram of soil microbes response to reclaimed water irrigation and N fertilization

2.3 Soil enzymatic activities

Soil urease activity values are shown in Fig.3a. It slowly increased during the first rotation of summer maize and winter wheat, and dramatically decreased during the second rotation of summer maize, followed with an improvement at the end of winter wheat. The average values under different treatments ranged from 1.448 to 1.561 mg/(g·d). The measured values of soil catalase activity are shown in Fig.3b. Soil catalase activity fluctuated largely during the experiments. The average values under different treatments ranged from 10.224 to 11.400 mg/(g·d). Soil invertase activity values are shown in Fig.3c. They were obviously increased during the first rotation of summer maize and winter wheat, and reached a maximum at the first harvest time of winter wheat, then dramatically decreased during the second rotation of summer maize, and increased again at the end of winter wheat.The average values under different treatments ranged from 32.40 to 37.29 mg/(g·d). One-way ANOVA showed that the soil urease, catalase and invertase activities were all significantly different between different crop seasons (<0.05). Two-way ANOVA showed no significant interaction between irrigation and fertilization on the activities of soil urease, catalase and invertase (Table 6).

Fig.3 Changes in the soil enzymatic activities during experiment

Table 6 Statistical analysis of soil enzymatic activities

Some studies have found that soil enzymatic activities are higher in the soils irrigated with RW[23]. However, others have found that RW irrigation has no influence or reduced enzymatic activities[23,52]. One of the reasons is the presence of metals such as Cr and Co in RW that inhibit the enzymes[20]. In our study, the values of enzymatic activities were very close in the soils irrigated with RW and FW. Because the RW used in this experiment was secondary treated and contained almost no heavy metals, the RW could not inhibit the activities of the soil enzymes. Thus, the most likely reason that the RW did not increase the activities of the soil enzymes is that the duration of the irrigation is relatively short and the amount of nutrient input into the soils by the RW does not effectively stimulate soil enzymatic activities. The second most likely reason is that drip irrigation inputs nutrients and water at a lower level each time but more frequently. The characteristics of drip irrigation input in terms of nutrients and water cause the soil enzymes to be stable.

Some studies have found that long-term N fertilization increased soil enzymatic activities[53]. Others found that short-term N applications had limited soil enzymatic activities[34]. In this study, there was no significant difference among the 3 types of N fertilization on soil enzymatic activities, because from the aspect of the chemical formulas, these 3 N fertilizers are all ammonium type nitrogen fertilizers.

2.4 The relationship between soil C, N, microbes and enzymes

The correlations between 16 parameters including the soil C and N contents, soil microbial biomass and enzymatic activities were analyzed (Table 7). Our results showed that soil nutrients, especially nitrogenous ones, had a positive correlation with the soil bacteria, actinobacteria and fungi. However, the soil microbial biomass is inhibited by NH4+-N to some extent. Therefore, in order to boost microbial growth, measures that appropriately reduce the NH4+-N forms of N fertilizer and the addition of NO3--N forms of N fertilizer are necessary in farmland. Our results also indicated that the C and N nutrients had different influences on different enzymatic activities. The soil urease activity was significantly enhanced by the SON, DOC, TN and NO3--N, while the soil invertase activity was significantly enhanced by SON, DOC, DON, TN and NO3--N. The soil urease activity was inhibited by SOC and NH4+-N, but soil invertase activity was just inhibited by NH4+-N (Table 7).

The different microbes themselves had significant positive correlations, and that the different enzymes themselves also had significant positive correlations (Table 7). The positive correlation coefficients between the soil bacteria and urease, and invertase ranged from 0.374 to 0.566 (<0.01). The positive correlation coefficients among the soil actinobacteria, G+ bacteria, G- bacteria, fungi and enzymes parameters ranged from 0.276 to 0.777 (<0.01). The positive correlation coefficients between the total PLFAs and urease, and invertase indicators ranged from 0.333 to 0.494 (<0.01). Thus, except for the relationship between soil bacteria and catalase, and soil total PLFAs and catalase, other soil microbes and enzymes were significantly influenced by each other.

2.5 Crop yield

The crop yields of summer maize and winter wheat under different treatments are shown in Table 8. The crop yields of summer maize fertilized with urea, ammonium sulfate and slow-release urea were on average 23.96%, 20.81% and 17.62% (average, 20.80%) significantly higher, respectively, than that in the soils fertilized with no nitrogen (<0.05). The crop yields of the winter wheat fertilized with urea, ammonium sulfate and slow-release urea were on average 20.34%, 16.32% and 27.79% (average, 21.45%) significantly higher, respectively, than those in the soils fertilized with no nitrogen (<0.05). The crop yields of the summer maize in the soils irrigated with RW were not significantly different from those in the soils irrigated with FW under no N fertilization and N fertilization, respectively. These results indicated that N fertilization significantly increased crop yields, but RW irrigation did not greatly affect crop yields. Similar results of N fertilization on maize yield and RW irrigation on maize and wheat yields were found by other studies[54-55].

Table 7 Correlations among soil C, N, microbes and enzymes

Note: *<0.05, **<0.01

Table 8 Crop yields of the summer maize and winter wheat

Note: Different lower case letters within a column represent significant difference (<0.05).

Soil quality is defined as the ‘continued capacity of soil to function as a vital living system, within ecosystem and land use boundaries, sustain biological productivity, to promote the quality of air and water environments, and to maintain plant, animal and human health’[56]. The soil C, N, microbial and enzymes are some of the soil properties that are used to assess soil quality. The soil microbial biomass is a small but key component of the active soil C and N pool and serves as a source and sink of soil nutrients[57]. Soil enzymes act as a catalyst to sustain soil health and its fertility[58]. Thus, soil microbial biomass and enzymatic activities are particularly important to take into consideration when evaluating soil quality. Many studies showed that soil microbial and enzyme are the suitable parameters to assess agricultural soil quality[59-62]. The increase of soil microbial biomass and the enhanced enzyme activity improve the agricultural soil quality. At the same time, crop yield was positively correlated with soil nutrient levels, microbial biomass and enzyme activity[63].

RW irrigation significantly increased the soil DON. RW irrigation increased soil microbial biomass. In terms of environment, RW irrigation can save freshwater, reuse nutrients in wastewater, and avoid the over-exploitation of scarce groundwater resources.

N fertilization significantly increased the soil organic matter and inorganic nitrogen. N fertilization increased the soil microbial biomass and enzymatic activities to some extent. In addition, the soil microbial biomass is the highest under slow-release urea fertilization. N fertilization significantly increased the crop yields of the summer maize and winter wheat, and the crop yield of the winter wheat is the highest under slow-release urea fertilization.

Considering the soil C and N contents, soil microbial biomass and crop yields, drip irrigation with RW and fertilization with slow-release urea can achieve a better soil quality and higher yields, and more environmentally friendly.

3 Conclusions

The results showed that RW irrigation significantly increased the soil DON content by 8.33%, and that N fertilization significantly increased the soil SOC, SON, DON, TN contents by 8.35%-11.75% and the NO3--N content by 92.99%. The bacterial, actinobacterial, G+ bacterial biomasses and total PLFAs in the soils irrigated with RW were on average 7.60%, 10.48%, 4.97% and 4.88% significantly higher, respectively, than those irrigated with FW. Urease, catalase and invertase activities in the soils irrigated with RW were not significantly different from those irrigated with FW. The RW drip irrigation increased the microbial biomass, but had no influence on soil enzymes. The RW drip irrigation promoted the soil quality. The bacterial biomass and total PLFAs in the soils fertilized with inorganic N were on average 13.42%-17.34% and 8.12%-11.19% significantly higher, respectively, than those fertilized with no N fertilizer. The application of different types of nitrogen fertilizer showed a similar impact on soil microbes, but the values were higher in soils fertilized with slow-release urea. Thus slow-release urea was less harmful to the soil while ensuring crop yield. The irrigation water and N fertilizer types had no significant interaction impact on soil microbial biomass and enzyme activities. These results indicated that the actinobacteria and G+ bacteria were faster growing and more efficient at utilizing the DON introduced by the RW, and the soil bacteria were more efficient at utilizing the NO3--N introduced by the fertilizers than were the other microbes. DON plays an important role in the growth of soil microbes, and NO3--N is important for the growth of soil bacteria. RW irrigation and slow-release urea fertilization were more effective at increasing the microbial biomass and enzymatic activities under drip irrigation. For better soil quality, high yields and saving water resources, we recommended drip irrigation with RW and fertilization with slow-release urea for summer maize-winter wheat crop rotation.

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灌水和氮肥类型对土壤微生物量和酶活性的影响

商放泽1,2，杨培岭3，任树梅3，黄跃飞2，孙小玲1，徐宏亮1

（1. 中电建生态环境集团有限公司，深圳 518102；2. 清华大学土木水利学院，北京 100084；3. 中国农业大学水利与土木工程学院，北京 100083）

灌水和施氮肥类型对土壤微生物和酶的影响尚不清楚。该研究旨在评估不同灌水和氮肥类型对土壤微生物生物量和酶活性的影响。结果表明，与清水灌溉相比，再生水灌溉显著增加了土壤细菌、放线菌、革兰氏阳性菌的生物量以及总磷脂脂肪酸，增加幅度分别为7.60%、10.48%、4.97%和4.88%。施氮肥显著提高了土壤细菌生物量和总磷脂脂肪酸，增加幅度范围分别为13.42%～17.34%和8.12%～11.19%。与清水灌溉相比，再生水灌溉并没有显著增加土壤脲酶、过氧化氢酶和蔗糖酶活性。施氮肥也没有显著提高土壤脲酶、过氧化氢酶和蔗糖酶活性。土壤微生物和酶在施缓释尿素肥的土壤中更为活跃。研究结果表明，与其他土壤微生物相比，放线菌和革兰氏阳性菌生长得更快，能更有效地利用再生水灌溉带入的可溶性有机氮；而细菌能更有效地利用氮肥带入的硝态氮。再生水灌溉和施缓释尿素肥在增加土壤微生物生物量和酶活性方面更有效。为了获得更好的土壤质量，更高的作物产量和可持续性利用水资源，建议夏玉米-冬小麦轮作种植利用再生水滴灌并施用缓释尿素肥。

施肥；灌水；土壤；微生物量；酶活性；再生水

Shang Fangze, Yang Peiling, Ren Shumei, Huang Yuefei, Sun Xiaoling, Xu Hongliang. Effects of irrigation water and N fertilizer types on soil microbial biomass and enzymatic activities[J]. Transactions of the Chinese Society of Agricultural Engineering (Transactions of the CSAE), 2020, 36(3):107－118.doi：10.11975/j.issn.1002-6819.2020.03.014 http://www.tcsae.org

商放泽，杨培岭，任树梅，黄跃飞，孙小玲，徐宏亮. 灌水和氮肥类型对土壤微生物量和酶活性的影响[J]. 农业工程学报，2020，36(3)：107－118. (in English with Chinese abstract) doi：10.11975/j.issn.1002-6819.2020.03.014 http://www.tcsae.org

2019-06-27

2020-01-22

National Natural Science Foundation of China (51679239), The China Postdoctoral Science Foundation (2017M610906)

Shang Fangze, Senior engineer, Ph.D, mainly focus on water resources and water environment research. Email：shangfangze@126.com

10.11975/j.issn.1002-6819.2020.03.014

S154; S273

A

1002-6819(2020)-03-0107-12