Concentrated biogas slurry enhanced soil fertility and tomato quality

Abstract

Biogas slurry is a cheap source of plant nutrients and can offer extra benefits to soil fertility and fruit quality. However, its current utilization mode and low content of active ingredients limit its further development. In this paper, a one-growing-season field study was conducted to assess the effects of concentrated biogas slurry on soil property, tomato fruit quality, and composition of microflora in both nonrhizosphere and rhizosphere soils. The results showed that application of concentrated slurry could bring significant changes to tomato cultivation, including increases in organic matter, available N, P, and K, total N and P, electrical conductivity, and fruit contents of amino acids, protein, soluble sugar, β-carotene, tannins, and vitamin C, together with the R/S ratios and the culturable counts of bacteria, actinomycetes, and fungi in soils. It was concluded that the application is a practicable means in tomato production and will better service the-is area of sustainable agriculture.

Keywords:

• Biogas fermentation residue
• fruit nutrition
• microflora
• organic manure
• soil property
• sustainable agriculture

Introduction

Biogas dregs and slurry are by-products of biogas production generated from cattle dung. These residues, especially biogas slurry, are a good source of plant nutrients and can improve soil properties (Smith & Elliot, 1990; Prasad & Power, 1991; Pathak et al., 1992; Garg et al., 2005). Owing to the propagation of household-scale anaerobic digester and biogas plants in many Asian countries, including China, the amount of biogas slurry has drastically increased (Angelidaki & Ellegaard, 2003; Abraham et al., 2007). Nowadays, there are approximately 16 million households using anaerobic digesters in China, and the output of slurry was more than 450 million tons in 2008. What's more, China's 2003–2010 National Rural Biogas Construction Plan was announced in 2003. The proposal was to increase biogas use to a total of 20 million households by 2010. At that time, the projected amount of slurry produced will soar to about 600 million tons.

Digested slurry contains organic nitrogen (mainly amino acids), abundant mineral elements, and low-molecular-mass bioactive substances (e.g., hormones, humic acids, vitamins, etc.) (Liu et al., 2008), and could be used as organic manure in the sowing season and as a source of water in other seasons. Seeds submerged in slurry germinate better and the seedlings grow stronger than those not subjected to this treatment (Pathak et al., 1992). Used as a spray for plants, the slurry inhibits disease and boosts yields (Liu et al., 2007b; Zhao et al., 2007). Its use as soil amendment offers a promising win-win opportunity and, at the same time, prevents adverse environmental impacts of waste disposal (Garg et al., 2005). Besides, application of biogas slurry or organic nutrient solutions formulated from it could indeed improve crop and fruit qualities (Yu et al., 2006; Liu et al., 2007a; Liu et al., 2008).

However, at present there are two major problems in the utilization of biogas slurry. First, too simple and large application mode. Traditionally, farmers just directly sprayed it as organic manure or submerged seeds in it to stimulate their germination and growth, etc. Although there are commercially available concentrated biogas slurry-based products made by conventional evaporation technology, such as Zhaobao produced by Powered Technology Limited Liability Company (Wuhan, Hubei Province, China), the huge energy consumption, ease of inactivation of active ingredients, and relatively higher cost seriously restrict their development. Secondly, the low content of active ingredient. Owing to the majority of slurry being water (>99%, w/v), its efficacy is rather weak and short-lived as compared with chemical counterparts. The effective compensation of soil fertility and control of crop pests and disease could not be fulfilled by use of slurry only. What's more, although there are a few publications, such as Liu's (Liu et al., 2008), that reported that application of organic nutrient solutions formulated from biogas slurry dilution and other ingredients (e.g., amino acids, chemical pesticides, Chinese herbal medicines, etc.) is an effective means for enhancing pesticidal efficacy and soil fertility, and reducing nitrate concentrations in leafy vegetables, etc., the establishment of a novel, low-cost concentration technology still seems to be a key and should be appreciated.

The objectives of this research are to (1) investigate the feasibility of concentrated biogas slurry as a nutrient source to cultivate tomato (Lycopersicum esculentum Mill.), and (2) examine its possible effects on soil property, tomato fruit quality, and culturable microflora in rhizosphere and nonrhizosphere soils, etc. The results obtained from this study will provide information for better utilization of biogas slurry, and contribute to the sustainable development of agriculture.

Materials and methods

Site and season

Studies were carried out at a research farm (Pingshan Farm) of the Institute of Environmental Technology, Zhejiang Forestry University, near the city of Linan (30.25°N latitude, 119.44°E longitude), located in north-west Zhejiang Province. Linan has a subtropical humid monsoon climate with an annual precipitation of 1408 mm in 2003 and 1449 mm in 2007, and a mean annual air temperature of 16.6 °C with 206 frost-free days. The experimental site was level. Cumulative precipitation and mean temperature (meteorological data from Linan Atmospheric Administration, Hangzhou) from April to August were 840 mm and 24.2 °C, respectively, in 2008. The soil of the experimental sites was a sandy loam (sand 46%, silt 35%, clay 19%) with organic matter 4.64%, total N 0.83 g kg⁻¹, total P 0.29 g kg⁻¹ (P₂O₅), total K 0.22 g kg⁻¹ (K₂O), available K 64.32 mg kg⁻¹, available N 73.97 mg kg⁻¹, available P 24.31 mg kg⁻¹, and pH 5.43 in the top 0.10–0.20-m soil layer. Previously, the field had a one-year cropping history of tomato production, with a typical double-cropping system.

Concentrated biogas slurry

Biogas slurry was sampled from Banqiao livestock farm, Linan. Nowadays, there are about 80 cattle and 12 pigs on site. With a novel biogas slurry-concentration technique, about 10-or-more-times concentration effect of the major nutrient parameters, such as total N, P, and K, , organic matter, and 10 kinds of metal ions, could be achieved. Briefly, the biogas slurry was first passed through a 20-mm stainless steel sieve, and settled in an 8-m³ plastic tank; and then the effluent was sequentially passed through a sand filter and a membrane filter equipped with membranes which could retain 400–100000 Da molecular-weight substances. The relevant documents and materials have been submitted to China's State Intellectual Property Office for an invention patent, the current status of which is under review with application no. 2008103053062. The concentration effect is shown in Table I. The samples were analysed in the National Analytical Centre, Guangzhou, China, according to the standard methods of the American Public Health Association (American Public Health Association, 1995).

Table I. The concentration effect of a novel process analysed in the National Analytical Centre, Guangzhou, China. The data were reported as means (n=3).

Item	Biogas slurry	Concentrated biogas slurry
Total N (g L^−1)	0.069	0.71
Total P (g L^−1)	0.048	15.5
Total K (g L^−1)	0.34	2.1
(mg L^−1)	159	1250
EC^a(25 °C) (dS m^−1)	6.51	30.3
CODCr (mg L^−1)	491	25 400
Na (mg kg^−1)	110	1074
Mg (mg kg^−1)	36	434
Al (mg kg^−1)	<1^b	2
Ca (mg kg^−1)	25	526
Ti (mg kg^−1)	<1	4.6
Mn (mg kg^−1)	<1	10
Fe (mg kg^−1)	<1	23
Zn (mg kg^−1)	<1	8.5
Rb (mg kg^−1)	<1	4.8
Sr (mg kg^−1)	<1	7.3
Ba (mg kg^−1)	<1	4.3
^aEC: electrical conductivity. ^bThe result was lower than the limit of detection of the inductively coupled plasma mass spectrometry (ICP-MS).

Experimental design

A field experiment was conducted at 25±5 °C under natural illumination during March and August of 2008. Tunnels were covered with transparent polyethylene (PE) (0.18 mm thickness) film and kept open on both sides. Tomatoes (Lycopersicum esculentum Mill., var. Hezuo 903) were first planted on seedbeds covered with black PE mulch on March 15, and uniformly irrigated with furrow irrigation. 4-Week-old tomato seedlings (April 16), uniform in size, were transplanted by hand into each plot on double rows, 1 m apart between double rows with 0.5 m between plants, and with 2.2 m between seedbed centres. All tomato seedlings were irrigated after transplanting, and again at second reviving stage, then the differential treatments began. The experiment consisted of four treatments: (1) blank control (CT); (2) biogas slurry (BS); (3) concentrated biogas slurry (CBS); and (4) conventional management (CM). In CT, plants were watered with tap water only (equivalent conductivity EC = 0.5 dS m⁻¹) until harvest. During the whole growing season, the tomato plants were irrigated several times depending on weather condition. For BS and CBS, only irrigation water and slurry were applied. The application amounts of BS were the same as with CBS. The 200–800 mL slurry per plant was applied two or three days after each irrigation according to the plant's growth status. To ensure the total input amounts of N, P, and K of CM were the same as for CBS, chemical fertilizers, either three-nutrient compound fertilizer (N-P-K, 15-15-15) or pilled urea (46%), were applied following the surveyed results. In total, 3.6 L of concentrated biogas slurry per plant was applied (8 times). The first application date was 28th April, 2008. The total input amounts of total N, P, and K per plant were about 2.56, 55.8, and 7.56 g according to the data listed in Table I. The main growing point will be picked at the 6th fruit cluster set. Weeds and pests were controlled as required. In treatment CM, chlorothalonil, 9 g m⁻² on June 12, and Score®, 0.03% on June 29, were applied to control tomato leaf mildew disease. All treatments were in a randomized complete-block design with split-plot arrangements with three replications. The plot size was 12 square metres (6×2 m).

Soil and tomato fruit analysis

Composite soil samples were collected from the 10–20-cm soil layer either before tomato transplantation in April 2008 or at the first harvest. The first harvest was on July 16 and the last was on August 28. Soil samples were air-dried, ground, and passed through a 2-mm sieve and used for analysis. Soils sampled were analysed for organic matter content (Ryan et al., 2001), pH (McLean, 1982), and electrical conductivity of the saturation paste (Page et al., 1982). For determination of N, P, and K from soil samples, recommended methods (Ryan et al., 2001) have been followed. Total P and K were determined using the vanadomolybdophosphoric acid yellow colour method and flame photometry (Yoshida et al., 1976), respectively, and total N was determined by the Tecator (1981) method.

After ripening, tomato fruits with the same position were picked by hand from an area 4×2 m in each plot and weighed at every harvest event. Sub-samples of fruit were sent to the Research Institute of Subtropical Forestry (Fuyang, Zhejiang Province) within 6 h for quality analysis. The measured items of fruit quality were as follows: protein, vitamin C, soluble sugar, fat, amino acids, nitrate, beta-carotene, and tannins, analysed according to the previously used standard methods (Yu et al., 2006). What's more, the tomato leaf mildew disease was rated according to the following scale: 1-light leaf brown, 2-moderate plant stunting and leaf curling, and 3-severe plant stunting and leaf curling. The disease index (DI) was calculated from Equation (1) where n _i is the number of diseased plants in each grade, and N the total number of plants. Disease was rated at three stages, that is: vegetative growth, 50% flowering, and fruit maturation.

The data collected were analysed statistically following methods described by Steel & Torrie (1980).

Estimation of microflora in soil and rhizosphere zone of tomato plants

To study the microflora in the rhizosphere zone, the plants were uprooted with great care to obtain the intact root system. Rhizosphere soils were sampled and serial-diluted according to the known methods (Johnston & Booth, 1993). For counting the number of fungi, actinomycetes, and bacteria, Martin's, Gause's No. 1, and Nutrient agar media (Martin, 1950; Zhou et al., 2006; Liu et al., 2007b) were used, respectively. The microorganisms were identified on the basis of their colonial and morphological features, i.e., size, elevation, and colour. The average number of colonies was multiplied by the dilution factor to obtain the number of colonies per gram dry weight in the original sample. The rhizosphere soil was dried at 105 °C for 24 h to estimate its dry weight.

To study the soil microflora apart from the effects of roots, 10 g of soil in the root-free area of each treatment were taken and transferred to an Erlenmeyer flask (250 mL) containing 24 mL of phosphate buffer (0.01 M, pH 7.4). The microflora were counted and calculated as mentioned above.

Statistical analysis

The software of Data Processing System 2000 (Tang & Feng, 1997) was used for all statistical analyses. Mean values were calculated for each of the measurements, and analysis of variance (ANOVA) was used to assess the effects on the measured variables, followed by mean comparison at a probability ≤5% of Duncan's Multiple Range Test (DMRT).

Results

Biogas slurry in treatments BS and CBS significantly improved contents of soil-available N, P, and K, and ECs as compared with treatments CT and CM, while treatment CBS also significantly improved contents of total N and P (Table II). Biogas slurry and chemical fertilizer applications (treatments BS, CBS, and CM) clearly increased electrical conductivity compared with treatment CT. Available N and P concentrations of treatments CT and CM are statistically defferent at P < 0.05. Compared with the initial soil sample, sampled on April 12, the organic matter contents were improved wby different degrees, and the pH-values of treatments all declined from 5.43 to 5.22 or so. Besides, the total N and P concentrations of treatment BS were respectively reduced by 4.82 and 3.45%, demonstrating that biogas slurry solution could not fully compensate the change in soil fertility by itself.

Table II. Effects of additions of different biogas slurries and chemical fertilizers on soil properties at 10–20-cm depth in the field experiment carried out at the Pingshan Farm, Linan.

Treatment	Available N (mg kg^−1)	Available P (mg kg^−1)	Available K (mg kg^−1)	Total N (g kg^−1)	Total P (g kg^−1)	Organic matter (%)	pH	EC (s cm^−1)
CT	54.8d	12.9c	41.4c	0.56c	0.25c	4.72c	5.21b	89.4c
BS	83.2b	27.4a	66.0b	0.79b	0.28b	5.01b	5.22b	118b
CBS	88.4a	28.1a	69.2a	0.87a	0.30a	5.24a	5.24a	157a
CM	57.8c	14.3b	43.9c	0.60c	0.27b	4.80c	5.22b	113b
The data were reported as means (n=5). Means sharing the same letters in columns are statistically nonsignificant at P = 0.05 by DMRT. CT, applied with tap water only; BS, only irrigation water and biogas slurry were applied; CBS, only irrigation water and concentrated biogas slurry were applied; CM, only irrigation water and three-nutrient compound fertilizer (N-P-K, 15-15-15) or pilled urea (46%) were applied.

Compared with other treatments, application of concentrated biogas slurry (CBS) significantly increased contents of 16 kinds of amino acids, protein, β-carotene, soluble sugar, vitamin C, and tannins in tomato fruit (Table III), while the concentration of nitrate was also significantly improved up to 25.59 µg mg⁻¹. The examined results of nitrite concentration were lower than the limit of detection, and no significant difference in fat content was found among treatments. The mean fruit weights of treatments CT, BS, and CBS were lower than that of CM, while its protein, soluble sugar, vitamin C, and β-carotene contents were lowest, demonstrating that chemial fertilizer application could effectively improve the tomato production but not the quality.

Table III. Effects of additions of different biogas slurries and chemical fertilizers on fruit quality.

Item	CT	BS	CBS	CM
Aspartic acid (mg g^−1)	15.8b	16.0b	17.1a	14.9c
Serine (mg g^−1)	2.93d	3.09c	3.52a	3.20b
Glutamic acid (mg g^−1)	34.0c	33.9c	38.2a	35.7b
Glycine (mg g^−1)	3.62b	3.55b	3.96a	3.91a
Histidine (mg g^−1)	2.94c	3.12b	3.29a	3.02c
Arginine (mg g^−1)	5.55b	5.46c	5.89a	5.50b
Threonine (mg g^−1)	2.75c	2.91b	3.17a	2.90b
Alanine (mg g^−1)	3.83c	3.94b	4.17a	3.69d
Proline (mg g^−1)	3.03b	2.95b	3.17a	2.98b
Cystine (mg g^−1)	2.57b	2.61b	2.96a	2.18c
Tyrosine (mg g^−1)	2.95b	2.80d	3.03a	3.05a
Valine (mg g^−1)	3.71c	4.10b	4.41a	4.14b
Methionine (mg g^−1)	0.93a	0.88b	0.88b	0.87b
Lysine (mg g^−1)	3.65c	3.95b	4.17a	3.21d
Isoleucine (mg g^−1)	3.77d	4.20b	4.47a	4.12c
Leucine (mg g^−1)	4.90d	5.15c	5.53a	5.29b
Phenylalanine (mg g^−1)	3.94c	4.27b	4.43a	4.49a
Total amino acids (mg g^−1)	101c	103b	112a	103b
Protein (g kg^−1)	134c	140b	147a	133c
Soluble sugar (%)	3.08c	3.40b	3.74a	2.97c
Fat (%)	0.12a	0.11a	0.12a	0.12a
Vitamin C (mg g^−1)	20.2c	21.0b	22.9a	18.5d
Nitrate (g g^−1)	21.3c	20.2d	25.6a	23.6b
Nitrite (g g^−1)	ND	ND	ND	ND^1
Tannins (g g^−1)	69.0c	76.2b	97.4a	78.5b
-Carotene (g g^−1)	52.4c	54.7b	67.9a	51.1c
Fruit weight (g)	114c	123b	121b	132a
The data were reported as means (n = 10). Data within a row followed by different small letters mean a significant difference at P = 0.05 by DMRT. CT, applied with tap water only; BS, only irrigation water and biogas slurry were applied; CBS, only irrigation water and concentrated biogas slurry were applied; CM, only irrigation water and three-nutrient compound fertilizer (N-P-K, 15-15-15) or pilled urea (46%) were applied. ^1ND: not determined. The results were lower than the limit of detection.

In the vegetative growth and 50% flowering stages, tomato plants did not show symptoms, except for treatment CT at 50% flowering (data not shown). At the third stage (fruit maturation), tomato plants started to develop the disease, and the values of DI ranged from 1.11 to 11.33. However, no significant difference was observed in the treatments BS, CBS, and CM. The rhizosphere microbial counts (R) of all treatments were clearly higher than their respective counts (S) in the nonrhizosphere soil (Figure 1). In CBS, the total counts of three kinds of culturable microorganisms either in rhizosphere or nonrhizosphere soils were at maximum values at the first harvest. In addition, the ratios of R and S were calculated (data not shown). The R/S ratios of biogas slurry application treatments (BS and CBS) are obviously higher than those of treatments CT and CM.

Figure 1.  Effects of additions of different biogas slurries and chemical fertilizers on average total counts of culturable microorganisms per gram dry soil in rhizosphere and nonrhizosphere soils. The data were reported as means. CFU, colony-forming unit. Data within a cluster marked with different small letters mean a significant difference at P = 0.05 based on Duncan's Multiple Range Test. CT, applied with tap water only; BS, only irrigation water and biogas slurry were applied; CBS, only irrigation water and concentrated biogas slurry were applied; and CM, only irrigation water and three-nutrient compound fertilizer (N-P-K, 15-15-15) or pilled urea (46%) were applied.

Discussion

It is well known that biogas slurry is a good source of plant nutrients and can improve crop yield and soil properties. However, to our knowledge, this is the first study on the effects of concentrated biogas slurry application on tomato cultivation. As shown in Table I, about a 10-or-more-times concentration effect of the major nutrients was obtained. Among these, the most enriched item is total P, about 320-fold. Experimental data showed that concentrated biogas slurry could significantly increase the soil fertility as compared with other treatments (Table II), and there is a consistent positive correlation between soil electrical conducitivity (EC) and soil fertility, but not yield (data now shown). Traditionally, the agricultural application of soil electrical conductivity was as a means of measuring soil salinity (Corwin & Lesch, 2005). Owing to it being a quick, reliable, and easy-to-take measurement, it is among the most frequently used tools in agricultural research. Our result presented here is in accord with the previously reported one (Corwin et al., 2003). Just as Corwin et al. (2003) had concluded that because of the influence of soil properties (e.g., salinity, water content, texture, etc.) and the poorly captured temporal component of yield variability by only one state variable such as soil EC, the yield wihin a particular field may or may not be influenced.

However, more attention should also be paid to other analysed items, such as Na, Mg, Ca, and Zn (Table I), due to the potential risk for secondary soil salinization by long-term successive application (Yao et al., 2007). Traditionally, in practice, the major interest in slurry application to soils is its nutritional value, N in particular, to crops, and its merits in improving soil physical properties, while its salt content that could be harmful to crop growth and soil quality is generally ignored. What's more, the environmental behaviour of trace metals (copper, zinc, arsenic, etc) should also be taken into consideration (L'Herroux et al., 1997; Nicholson et al., 2003). These metals are essential nutrients and are required in very small amounts by plants, but their accumulation is toxic to plants and microorganisms (Bolan et al., 2003; Bernhard et al., 2005). Therefore, a rational crop-rotation system and application together with inorganic fertilizers are still recommended (Yao et al., 2007), and different application modes (spraying and root irrigation) should be further evaluated for better utilization.

There have been concerns over the presence of nitrates and nitrites in food, especially in vegetables, as they could be metabolized to potentially carcinogenic N-nitroso compounds (Penttilä et al., 1990; Wang & Li, 2003). For this reason, the tomato-fruit contents of nitrate and nitrite were examined. As shown in Table III, the nitrate concentrations of treatments are in the range of 20.2–25.59 µg g⁻¹, which were quite below the limit (432 µg g⁻¹) proposed by FAO/WHO (1998) and safe for human consumption. Yu et al. (2005) suggested that application of biogas slurry could reduce nitrate accumulation in vegetables, and they gave their explanation as follows: 1) compared with chemical fertilizer, the biodegradation of organic matter in slurry is a slow process which is better for nutrient assimilation by the plant; and 2) this organic matter could accelerate the soil nitrification process which will lessen the nitrate accumulation in soil and further decrease uptake. However, several authors, such as Li et al. (2007), also reported that biogas slurry application would stimulate both nitrate and salt accumulation in soil. Our result is not in accord with the previous two. The nitrate concentrations of CBS and BS are 25.59 µg g⁻¹ and 20.2 µg g⁻¹, which are, respectively, significantly higher and lower than the others. We suggest that there is an optimal level of slurry application for low nitrate accumulation in terms of cost efficiency, which is a topic that needs further investigation.

Tomato (Lycopersicum esculentum Mill.), which was domesticated in ancient Peru, has become the most popular and widely consumed vegetable in the world today, due to its flavour, nutritional value (high in vitamins C and A), short growth cycle, and relatively high yield (Maršić et al., 2005; Ortiz et al., 2007). However, it also contains a number of antinutritional factors. Different studies led to the conclusion that these antinutritional factors (including tannins) may bind proteins and some essential dietary minerals and make them unavailable or only partially available for absorption (Maga, 1982; Welch, 1999). At the same time, tannins have protective effects against cancer and cardiovascular diseases (Santos-Buelga & Scalbert, 2000). For these reasons, the fruit contents of vitamin C and tannins were examined. The data indicated that CBS could significantly improve the concentrations (Table III). Owing to the uncertainty of the comprehensive effect of tannins on human health, the relevant conclusion could not be easily drawn at present.

Several authors reported that biogas slurry has different inhibitory effects on crop pests and pathogens (Yu et al., 2006; Zhao et al., 2007). However, owing to the above-mentioned reasons, they also suggested that chemical pesticide and fungicide are still indispensable. In our field trial, no significant difference was found in the control effects of tomato leaf mildew disease among treatments CBS, BS, and CM (data not shown). But, owing to the fact that it is just the second year for tomato production and the duration is only one growing season, more field research should be carried out. What's more, the application of slurry greatly influences the microflora in both nonrhizosphere and rhizosphere soils (Figure 1). The total culturable microbial counts and the R/S ratios of CBS and BS are obviously higher, demonstrating that interactions between plant and soil microorganisms were activated (Rovira, 1965).

Acknowledgements

We acknowledge with thanks financial support from the National “863” High Technology Research (2006 AA062344).

Additional information

Notes on contributors

Xi-Ping Luo

Fang-Bo Yu and Xi-Ping Luo contributed equally to this work