Mitigating Nitrous Oxide Emissions from Tea Field Soil Using Bioaugmentation with a Trichoderma viride Biofertilizer

Land-use conversion from woodlands to tea fields in subtropical areas of central China leads to increased nitrous oxide (N2O) emissions, partly due to increased nitrogen fertilizer use. A field investigation of N2O using a static closed chamber-gas chromatography revealed that the average N2O fluxes in tea fields with 225 kg N ha−1 yr−1 fertilizer application were 9.4 ± 6.2 times higher than those of woodlands. Accordingly, it is urgent to develop practices for mitigating N2O emissions from tea fields. By liquid-state fermentation of sweet potato starch wastewater and solid-state fermentation of paddy straw with application of Trichoderma viride, we provided the tea plantation with biofertilizer containing 2.4 t C ha−1 and 58.7 kg N ha−1. Compared to use of synthetic N fertilizer, use of biofertilizer at 225 kg N ha−1 yr−1 significantly reduced N2O emissions by 33.3%–71.8% and increased the tea yield by 16.2%–62.2%. Therefore, the process of bioconversion/bioaugmentation tested in this study was found to be a cost-effective and feasible approach to reducing N2O emissions and can be considered the best management practice for tea fields.


Introduction
Three typical land-uses occur in most areas of southern subtropical China: woodlands in the mountains, uplands on slopes, and paddy fields in the lowlands [1]. In China, 1.3 M ha of tea plantations has been established during the last 50 yrs., due to rapid economic development [2]. Most of these recent plantations occupy large areas that were originally woodlands and are continuing to expand [3]. Landuse conversion is typically characterized by frequent tillage and use of large amounts of nitrogenous fertilizer, which contributes significantly to losses of N as nitrous oxide (N 2 O) emissions to the atmosphere [4]. N 2 O emissions are, on average, >10 times higher from tea fields than from forests. N 2 O has an ozone-depleting potential similar to that of hydrochlorofluorocarbons and a global warming potential 300 times that of CO 2 [3,5,6].
In China, annual production of rice straw was about 1.74 × 10 8 t in 2000, containing about 2.5 − 4.0 × 10 4 t N [7]. Due to lack of feasible conversion technologies, until the 2000s, on-site burning was traditionally used to dispose of crop residues, resulting in severe environmental pollution including greenhouse gases and nitrogen oxides [8]. Recently, straw return has become the preferred method of rice straw disposal because of its positive effects on crop yield and soil properties, such as increased soil organic carbon content. However, straw decomposition in soils takes time; over the short-term decomposition, straw incorporation can hamper crop root penetration, causing N deficiencies and contributing to disease and weed problems [9,10], directly limiting its application in agriculture. Therefore, it is important to develop a method for enhancing straw decomposition before or after incorporation.
Many studies have attempted to hasten straw decomposition via microbial processes. Trichoderma spp. is the bestknown cellulolytic fungi that can accelerate the process of rice straw decomposition and produce more soil-available nutrients such as labile organic matter and inorganic N/P/S [11][12][13]. However, a major challenge in implementing bioconversion of rice straw is identifying a suitable carrier substrate to 2 The Scientific World Journal amplify the target organism, promote survival of Trichoderma spp. in the rice straw, and enhance colonization in the soil near plant roots. Fortunately, sweet potato starch wastewater (SPSW) from local starch production plants could be used as an appropriate culture medium for microbial growth because of its high organic content. A few studies have been published on production of microbial biomass through starch industry wastewater with various microbes such as Trichoderma viride [14].
Compared to chemical fertilizers and protectants, some strains of Trichoderma spp. provide additional benefits in terms of plant growth and productivity, such as systemic resistance to disease and abiotic stresses such as water deficits and salt stress [15,16]. If Trichoderma spp. colonize the roots, they strongly affect plant physiology by changing plant gene expression, providing season-long benefits to the plants [17,18]. Another major benefit is induction of increased N use efficiency (NUE) in plants [19], potentially reducing the N fertilizer application rate by 30%-50% through bioconversion/bioaugmentation with no reduction in yield. Therefore, the lower N inputs to soil and higher NUE may reduce N 2 O emissions.
The objectives of this study were to (1) develop a costeffective technology for using SPSW and rice straw for large-scale production of Trichoderma viride biofertilizer, (2) evaluate the feasibility of biofortification of crops through application of T. viride N fertilizers to reduce N 2 O emissions, and (3) minimize synthetic N fertilizer input via recycling of N to achieve a sustainable agricultural system.

Methods and Materials
2.1. Site Description. The field experiment was carried out at the Changsha Research Station for Agricultural and Environmental Monitoring (CRSAEM), Changsha, Hunan, in subtropical central China (28 ∘ 32 50 N, 113 ∘ 10 58 E). The region has a subtropical monsoon climate with a mean annual air temperature of 17.5 ∘ C and a mean annual precipitation of 1330 mm . The red soils of this area are classified as ultisols (USDA soil taxonomy) [20]. The selected site is a typical hilly, agricultural catchment with pine forests (woodlands), paddy fields (lowland), and tea fields (upland) as the three primary land-use types, accounting for 65.5, 25.1, and 3.4% of the total catchment area (135 km 2 , Figure 1), respectively. The other small upland areas in the catchment are cropped sweet potato (<0.5%). The land-use distribution in 1990 shows substantial conversion from woodlands dominated by Masson pine and bamboo to large-scale tea plantations (Camellia sinensis L., Figure 1). Each land-use type is fertilized other than the woodlands. The average annual application rate of N fertilizers was 450, 300, and 250 kg N ha −1 yr −1 for tea fields, paddy fields, and sweet potato fields, respectively.

Raw Materials and Starter
Inoculum. SPSW, containing approximately 25 g L −1 COD and 1 g L −1 total N, was obtained from a local sweet potato starch production company (Xiangfeng Corp., Hunan, China) as a by-product of sweet potato starch processing. Rice straw (total organic carbon (TOC), 56.5%; total nitrogen (TN), 0.46%) was collected after harvest from local paddy fields and ground to 5-8 mm lengths for the solid fermentation culture of Trichoderma viride.
The strain T. viride EBL13 was isolated from the soil and was found to be active against phytopathogenic fungi in our laboratory [21]. The starter cultures were obtained by inoculating 500 L of a spore suspension at 10 8 spores mL −1 into 20 mL potato dextrose broth (PDB; Sigma-Aldrich, St. Louis, MO) and incubating at 25 ∘ C in orbital agitation at 150 rpm in the dark for 48 h.

Mass Production of T. viride Mycelium.
The starter cultures (2 mL) were transferred to a 500-mL Erlenmeyer flask containing 150 mL sterile sweet starch industry wastewater (121 ± 1 ∘ C for 15 min) and were incubated in a rotary shaker at 28 ± 1 ∘ C and 200 ± 5 rpm for 48 h. The mycelium was collected by centrifugation at 4000 g for 10 min, the biomass fresh weight was determined, and then the fungal biomass was prepared for solid-state fermentation (SSF) with rice straw.

Production of Biofertilizer by Solid-State Fermentation.
The homogenized mycelial mat (10 g dewatered mycelium with 10 mL fermentation supernatant) was transferred to a 500-mL Erlenmeyer flask containing 10 g sterile rice straw (121 ± 1 ∘ C for 15 min). Afterwards, the flasks were incubated at 28±1 ∘ C for 148 h until mass production of T. viride conidia occurred. The conidia concentration was measured as cfu g −1 air-dried cotton stalk substrate, using a modified conidia assessment method [22].

Biofertilizer Application to the Tea Plantation.
Biofertilizer was compared with chemical fertilizer (N) using a completely randomized design in an ongoing trial of about 3-year-old tea (Camellia sinensis cv.) from November 2010 to May 2012 at a tea plantation in Jinjing. There were four treatments in the field experiment: (1) unfertilized (CK0), (2) chemical fertilizer (CNH450, urea applied at 450 kg N ha −1 yr −1 ; CNL225, fertilizer applied at 225 kg N ha −1 yr −1 ), (3) biofertilizer (BFH225, biofertilizer applied at 50000 kg ha −1 yr −1 , with the total N adjusted to that of CNL225; BFL113, biofertilizer applied at 25000 kg ha −1 yr −1 , with the total N adjusted to half that of CNL225), and (4) raw materials (RMH225, sweet starch industry wastewater and rice straw at 50000 kg ha −1 yr −1 for fermentation of biofertilizer; RML113, sweet starch industry wastewater and rice straw at 25000 kg ha −1 yr −1 ). The fertilizer was applied in three stages each year: 20% of the total N on November 12, 60% on March 1, and 20% on October 1, tilled 10-15 cm under the soil surface. The experimental plot was 60 m 2 (10 × 6 m 2 ) for each fertilizer treatment.
The Scientific World Journal 3 2.4. Field Measurement of N 2 O Fluxes. Soil N 2 O fluxes were measured using a static closed chamber and gas chromatography (GC), as described by Li et al. [3] and Zheng et al. [23], from March 2011 to April 2012. The closed minichambers were constructed of polyvinylchloride, 0.15 m in diameter and 0.18 m in length, with sharpened ends and screw lids fitted with rubber septa for gas sampling. The chambers were gently inserted vertically into the soil to a depth of 0.05 m using the sharpened ends. Headspace gas samples were collected from 09:30 to 10:30 a.m. once a week during tea harvest seasons (spring tea: mid-March to mid-April; autumn tea: mid-September to mid-October). For each treatment, 3 replicate gas samples were collected from the headspace into preevacuated 12-mL vials (Exetainers, Labco, High Wycombe, UK) 0 and 30 min after the lid was closed. After gas sampling, the air temperature in each chamber was measured for subsequent correction of the flux calculation. The N 2 O concentrations in the gas samples were analyzed using a gas chromatograph (Agilent 7890A, Agilent, Santa Clara, CA) fitted with a 63 Ni-electron capture detector and an automatic sample injector system [3]. N 2 O fluxes (FLUX30, g N ha −1 d −1 ) were calculated using the following equation: where 30 − 0 is the difference in the N 2 O concentration in the headspace of the mini-chamber 0 and 30 min after the lid was closed (ppmv); N 2 O is the molecular weight of N 2 O (g mol −1 ); 0 is the molecular volume of N 2 O under standard conditions (temperature = 273 K and pressure = 1013 hPa), 22.4 × 10 −3 m 3 ; 0 = 273 K; N is the atomic weight of nitrogen (g mol −1 ); ℎ is the chamber height (m); Δ is the incubation period (0.5 h); air is the air temperature inside the mini-chamber ( ∘ C); and 10,000, 1000, and 24 are conversion factors for m 2 to ha, mg to g, and h to days, respectively.

Auxiliary Field Measurements.
In addition to measuring gas fluxes, ammonium-N and nitrate-N in the soil were measured along with tea plant growth. Four 0-20 cm soil samples were collected from random locations in each plot using a soil auger during each field measurement of N 2 O fluxes. Each fresh soil sample was manually homogenized and analyzed for soil ammonium-N (NH 4 + -N), nitrate-N (NO 3 − -N), and total N using an automated flow injection analyzer (FIAStar 5000, Foss Tecator, Hoganas, Sweden) as described by Liu et al. [24]. Tea plant growth was monitored by recording the weight of a fresh tea bush (bud with two leaves) during the tea harvest seasons (spring tea: mid-March to mid-April; autumn tea: mid-September to mid-October).

Statistical Analyses.
All statistical analyses were conducted using SPSS 12.0 (SPSS China, Beijing, China) and Origin 8.0 (Origin Lab Ltd., Guangzhou, China). The statistical significance of the results was determined using Duncan's multiple-range test ( < 0.05). Simple correlation coefficients between soil N 2 O flux and NH 4 + -N and NO 3 − -N contents were calculated using the same statistical package.

Effects of Land-Use Changes on N 2 O Emissions from Hilly
Areas of Subtropical Central China. Figure 1 depicts changes in land-use distribution patterns using a geographic information system since 1955. In the late 1950s, the land-use pattern was simple and mainly comprised of woodlands and paddy fields with 63.05 and 29.38% of the total land, respectively. With rapid economic development and population increases, rapid land-use and land-cover changes have taken place in these areas beginning in the 1990s; there has been a large decline in forest cover due to tea plantation expansion. Overall, 398 ha of woodland areas has been converted to tea crop cultivation, about 2.98% of the total area of the district. Woodland cover (60.3%) has decreased by 4. ) application were 17.7 ± 3.4 and 9.4 ± 6.2 times higher than those from woodlands, respectively. Many studies have reported that land-use changes can impact N 2 O emissions and that N 2 O emissions significantly increase when woodlands are converted into pastures [25], orchards [26], or cropland [27]. Merino et al. [28] measured N 2 O releases from cropland and a pasture (7.40 and 13.15 g N ha −1 d −1 ) and found that they were 3 and 6 times higher than those from a forest (2.19 g N ha −1 d −1 ), respectively. We have previously reported the high annual variability of N 2 O emissions as well as its spatial variability and distribution from a tea field in 2010 [3,29]. Many studies have shown that agricultural land has much higher N 2 O emissions than forests because of generally higher N in soils that may be further enhanced through intensification of the N cycle through application of synthetic N fertilizer [30,31].

Bioconversion of Sweet Potato Starch Wastewater and Rice Straw into Biofertilizer by Trichoderma viride.
In the studied catchment, paddy fields accounting for 25.1% of the total land area produced 3.02 × 10 4 t yr −1 straw containing 9.07 × 10 3 t C and 1.21 × 10 2 t N ( Table 1). Sweet potatoes were cultivated in the upland area, making up 0.25% of the total land area of the catchment. The sweet potatoes were processed to produce   [15,16,18,21]. The biological activity of Trichoderma spp. could replace some functions of synthetic nitrogen to minimize synthetic inputs of fertilizer in tea cultivation.  [26,[32][33][34]. In addition, the magnitude of the N 2 O emissions from our tea fields was much higher than previously reported values for paddy fields and uplands with N fertilization. Accordingly, it is urgent to establish technologies and management practices

Mitigation Options for Reducing N 2 O Emissions.
The Scientific World Journal 5 Note: in each row, means followed by the same letter are not significantly different at < 0.05.

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The Scientific World Journal for mitigating N 2 O emissions from tea fields while sustaining or increasing tea production. Improved management using biofertilizer instead of synthetic N fertilizer significantly decreased N 2 O emissions ( Figure 2). The BFH225 treatment reduced N 2 O emissions by 33.3%-71.8% and increased the tea yield by 16.2%-62.2% compared to the CNL225 treatment, depending on the season. The RMH225 treatment also reduced N 2 O emissions by 43.6%-80.0%; however, the yield of 2012 spring tea decreased and some tea plants died. There have been contradictory reports on the effects of organic waste materials on N 2 O emissions. Some studies have shown an inhibitory effect [35][36][37], while others have reported stimulation [38,39]. Based on our results, direct application of the organic waste materials (rice straw and starch industry wastewater) to the tea field carried significant risks to the tea plants. However, transferring these waste materials into biofertilizer by fermentation was a feasible approach for decreasing N 2 O fluxes from tea fields.
In addition, compared to the high application rate of chemical N fertilizer (CNL450), the BFH225 treatment that reduced the annual N fertilization rate by 50% did not affect the tea yield (Table 2) but significantly decreased average N 2 O emissions by 71.6% (Figure 2). This decrease may be ascribed to three possible mechanisms. First, because Trichoderma viride could promote plant growth, mineral nitrogen in the soil may be more quickly taken up by the vigorously growing tea plants in the presence of Trichoderma spp. [19], thereby reducing the N in the substrate available for N 2 O production. Second, application of biofertilizer with a high C/N ratio (>30) results in temporarily improving soil N immobilization and thus a low availability of soil N for N 2 O emission [40].
Finally, the buffering and loosening effect of the organic matter in the biofertilizer resulting in higher pH and oxygen in the soils may have favored N 2 as an end product rather than N 2 O in denitrification [41].  The Scientific World Journal = 0.72, and < 0.01, Figure 4(a)) and a similar effect was observed between N 2 O fluxes and soil NO 3 − -N contents (y = 1.24x + 0.96, 2 = 0.67, and < 0.01, Figure 4(b)).

Effects of Soil
In several previous studies, soil temperature, soil moisture, and NH 4 + -N or NO 3 − -N contents were identified as the main environmental drivers of N 2 O fluxes [26,34,42], affecting either nitrification or denitrification and thus N 2 O production. The temperature and humidity of the soil, which were significantly correlated with N 2 O fluxes, are controlled by natural factors such as seasons and precipitation. However, another key factor affecting N 2 O flux, the N content in the soil, can be controlled by changing the type of fertilizer applied, the fertilization rate, and the timing of application, providing a feasible method of controlling the N 2 O flux.

Conclusions
Land-use conversion from woodlands to tea fields in subtropical areas of central China leads to increased N 2 O emissions, partly due to increased N fertilizer use. By liquidstate fermentation of SPSW and solid-state fermentation of paddy straw by T. viride, tea plantations can be provided 8 The Scientific World Journal with biofertilizer that can replace some functions of synthetic N, minimizing synthetic inputs of fertilizer in tea cultivation. Improved management using biofertilizer rather than synthetic N fertilizer can significantly decrease N 2 O emissions while sustaining or increasing tea production. Thus, the process described here using SPSW and rice straw for mass-scale production of T. viride biofertilizer is a feasible and cost-effective approach for minimizing synthetic inputs of fertilizer, reducing cumulative N 2 O emissions and developing the best management practices for N in soils.