Anaerobic Digestion and Storage Influence Availability of Plant

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Anaerobic digestion and storage influence availability of plant hormones in livestock slurry XIN LI, Jianbin Guo, Changle Pang, and Renjie Dong ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.5b00586 • Publication Date (Web): 13 Jan 2016 Downloaded from http://pubs.acs.org on January 14, 2016

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ACS Sustainable Chemistry & Engineering

Anaerobic digestion and storage influence availability of plant hormones in livestock slurry

Xin Li, Jianbin Guo∗, Changle Pang, Renjie Dong College of Engineering, China Agricultural University, Key laboratory for clean renewable energy utilization technology, Ministry of Agriculture, Beijing 100083, People’s Republic of China

Abstract Anaerobic digestion slurry (ADS) is typically applied as a liquid fertilizer and soil amendment because it contains high concentrations of plant nutrients. For many years, macronutrient such as nitrogen and phosphorus in ADS has been widely studied, whereas little attention has been paid to plant hormones. Lab-scale batch experiments were conducted to evaluate the effect of anaerobic digestion (AD) on plant hormones (gibberellic acid (GA3), indoleacetic acid (IAA), abscisic acid (ABA)) in three typical animal manures (chicken manure, dairy manure, and pig manure). Results from the lab-scale batch experiment revealed that the ADS contained higher concentrations of plant hormones than the original animal manures. Subsequently, quantitative analysis of samples from large-scale continuously fed biogas plants verified the high contents of plant hormones in ADS. However, the storage experiment demonstrated a ∗

Corresponding author. Tel.: +86-10-62737858; Fax: +86-10-62737885.

E-mail address: [email protected] 1 / 32

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significant loss of plant hormones in ADS prior to land application because of decomposition and transformation. Specifically, the results showed that the patterns of GA3, IAA, and ABA variations depended on the storage temperature, showing increased losses at increased storage temperatures. Consequently, it is important to assess plant hormones availability of ADS prior to indiscriminate application for efficient utilization.

Keywords: Anaerobic digestion slurry; storage; gibberellic acid; indoleacetic acid; abscisic acid

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Introduction With the rapid development of animal husbandry and biogas plants, numerous anaerobic digested slurry (ADS) have been produced, typically containing high contents of plant nutrients such as nitrogen, phosphate and potassium. 1 ADS is often applied as a liquid fertilizer to improve crop yield and quality by seed soaking, top dressing, foliar spraying and fertigation.2,3 Studies have shown that soaking of wheat seeds for 6-12 hours in ADS significantly increased germination percentage and root length of seedlings.4 Spraying ADS to wheat can not only increase the spikelet length, grain number, as well as thousand-grain weight and yield, but can also inhibit diseases and insects.5 Moreover, ADS can be directly used as a soil amendment. Many results showed that application of the ADS blending with inorganic fertilizer can increase contents of nitrogen, potassium, phosphorus and other nutrients in soil.6 Therefore, increasing the use of ADS is a promising way for consuming the increasing quantities of ADS and preventing adverse environmental impacts.

The existing literature commonly attributed the positive effect of ADS on plant growth to the high contents of macronutrients, such as nitrogen (0.03%~0.08%), phosphorus (0.02%~0.07%), and potassium (0.05%~1.40%).7,8 In addition to these macronutrients, some authors reviewed that ADS contained plant hormones, such as gibberellic acid (GA), indoleacetic acid (IAA), abscisic acid (ABA).9-11 Plant hormones positively influence many plant physiological processes, such as root system expansion, biomass accumulation, nutrient absorption and mobilization, 3 / 32



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tolerance to stress, as well as resistance to diseases and retardation of senescence.12,13 Previous studies have reported the presence of IAA at 23.5-33.0 nmol/g in ADS of instant coffee waste.14 Scaglia, et al. recently concluded that no GA-like effect was present in the ADS of pig manure, whereas hormone activity was due principally to the presence of IAA. 15 Apparently, the plant nutrients of ADS from the AD plant can vary widely, depending on various operating parameters (feedstock type and quality, organic loading rate, and hydraulic retention time).

In addition, previous studies showed that seed soaking of ADS at low concentrations (25%, 50% and 75%) showed optimal results for maximum germination and enhanced growth. By contrast, ADS exhibited a decreasing tendency in seed germination and seedling growth at high concentration (100%).16 Consistent with these findings, several studies showed that low concentration of IAA promoted, whereas high concentration of IAA suppressed the growth of root; in addition, a rise in IAA concentration led to the enhancement of suppression.17 A similar study has also been reported for ABA. Wang, et al. reported that a low concentration (2.5 mg/L) of ABA can alleviate Pb-induced toxicity in the leaves of macrocephala, whereas high concentrations (5 and 10 mg/L) of ABA improve Pb-induced toxicity.18 Meanwhile, response to exogenous plant hormones varies, depending on the mode of application (spraying or seed soaking),19 as well as other factors plant age and size, plant sensitivity, climate and type of soil.20 This finding emphasizes the importance of

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analyzing and assessing the plant hormone availability of ADS prior to its indiscriminate application as a fertilizer to agricultural land. In contrast to the well-studied nitrogen and phosphorus, information is limited regarding the quantitative analysis for plant hormones in the ADS of animal manures.

Furthermore, ADS is commonly collected from digesters and stored in uncovered storage tanks for 1-3 months in different seasons at various temperatures prior to field application. The reason is that ADS can only be applied to arable land during certain periods with the year in accordance with the growth rhythm of crops. In general, current studies have focused on examining the nutrient dynamics of ADS after application.21,22 However, knowledge concerning the fate of plant nutrients in ADS during storage before field application is limited. Affected by storage temperatures, the plant hormone composition could significantly change because of decomposition and transformation, which may affect availability to crops. Response to exogenous plant hormones varies depending on the mode of application (spraying or seed soaking), as well as other factors, including the plant age and size and plant sensitivity.19 Therefore, plant hormone availability in ADS should be evaluated in accordance with different storage season to synchronize the nutrient supply from ADS with crop requirements.

The objective of this study was to evaluate the availability of plant hormones in ADS during the anaerobic digestion and storage prior to field application. Batch 5 / 32



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experiments were conducted for synchronous analysis of dynamic changes in three plant hormones (GA3, IAA, and ABA) during AD of chicken, dairy and pig manures. Meanwhile, three types of ADS from continuously fed biogas plants were determined to verify the high contents of plant hormones in ADS. In addition, the effects of storage temperatures on the changes in plant hormones of ADS were evaluated. This type of research would be useful for the effective application of ADS.

Material and Method Batch experiment feedstock characteristics Dairy manure (DM) (Total solids (TS): 21.69% of fresh matter (FM); Volatile Solid (VS): 14.94% FM; pH: 8.68) and chicken manure (CM) (TS: 29.96% FM; VS: 20.89% FM; pH: 8.41) samples were collected from a livestock farm in China Agriculture University, Beijing. Pig manure (PM) (TS: 25.57% FM; VS: 18.23% FM; pH: 8.41) was obtained from a large-scale pig farm located in Shunyi District, Beijing. The inoculum (seed sludge) (TS: 4.73% FM; VS: 3.86% FM; pH: 7.36) was collected from another municipal wastewater treatment plant (Xiaohongmen WWTP, located in southern Beijing), where the sewage sludge was treated by AD. Prior to the experiment, the manure samples were frozen at -20 °C to prevent biological decomposition, and the frozen substrates were stored in a refrigerator at 4 °C for 1 day before use.

Batch digester start-up and experimental design The batch digestion experiments were performed in 3 L biogas reactors capped with 6 / 32


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natural rubber sleeve stoppers. The reactors have two ports on the top of the cap. One port was connected with the gas bag and the other port was connected with gas stopper. The working volume of the reactors was 2 L. Manure was filtered using a sieve having a mesh aperture of 0.9 mm to remove large particles prior to feeding into the reactor. The reactors were filled with 1.5 L inoculum and 500 g of CM, DM or PM samples. The deionized water was filled to the working volume. The headspace of the reactors was flushed with pure nitrogen for approximately 20 min to ensure anaerobic conditions. The anaerobic digester reactors were maintained at 37±1 °C in a temperature-controlled chamber. All batch experiments were performed in triplicate. The experiments lasted for 30 days. Samples were periodically removed using syringe for determination of

pH, volatile fatty acid (VFA), and plant hormones of ADS

every 2 day.

Biogas Plants sampling and analysis Three types of ADS were sampled from a large-scale pig farm biogas plant (located in Beilangzhong, Shuiyi district, Beijing), a large-scale dairy farm biogas plant (located in Donghuashan, Shunyi district, Beijing) and a large-scale chicken farm biogas plant (in Liuminying Daxing, Beijing), respectively. In order to obtain fresh and homogenized ADS, the ADS were collected directly from the liquid outlet of digester. The ADS in digester was discharged at a fixed time of the day. We collected 10 L of ADS after discharging for 1 minute. All of the biogas plants were operated semi-continuously for more than five years. The anaerobic digestion process

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parameters of the large-scale biogas plants were listed in Table 1. The three plant hormones (GA3, IAA and ABA) in these samples were measured.

Storage experimental design The ADS of pig manure originated from a biogas plant located in Shunyi district, Beijing, was chosen for storage experiment. The experiment was carried out at three different temperature (4 °C, 20 °C, and 37 °C) treatments for 88 days. The ADS was stored in 10 L containers which were maintained at 4±1 °C, 20±1 °C, and 37±1 °C in a temperature-controlled chamber. The ADS in the containers was stirred to produce a homogeneous mass before sampling. 20 mL ADS was periodically sampled from each container for determination of plant hormones every 3 days.

Analytical methods All standards used in the experiment were obtained from Sigma (St. Louis, MO, USA). Double distilled water was produced by Direct 8 Milli-Q deionization system and filtered through a 0.45 µm membrane filter (Montpellier, France).

The pH was measured using a digital pH meter (FE20, METTLER TOLEDO, Switzerland). The biogas volume was measured from gas bags using a calibrated wet-type gas flow meter (LML-1, China), and the CH4 content was determined using an EHEIM Visit 03 (Messtechnik Eheim, Germany) biogas analyzer every 1-3 days depending on the volume of the produced biogas enough for analyzing. The cumulative biogas production was calculated by sum up each daily biogas yield within 30 days. 8 / 32


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The pH of ADS was adjusted to 2.5 by adding formic acid to prevent the ionization of analytes. The samples were centrifuged at 8000 g for 20 min. The supernatant was percolated through a 0.45 µm nylon filter to remove suspended matters, and transferred into sample vials. The VFA concentrations were quantified using a gas chromatograph (Shimadzu, GC-2010 Plus, Kyoto, Japan) with a flame ionization detector and rtx-wax capillary column (30 m × 0.25 mm × 0.25 µm) with high purity nitrogen as the carrier gas at a flow rate of 40 mL/min and split ratio of 30. The column had an initial temperature of 60 °C (2 min holding time), which increased at 10 °C /min to 140 °C, and then further increased at 20 °C /min to 230 °C (5 min holding time). The temperatures of the injector and detector were 230 °C and 250 °C, respectively. Concentrations of the VFA were determined using a standard curve obtained by injecting standard solutions of acetic, propionic, isobutyric, n-butyric, isovaleric, nvaleric, and caproic acids.23

The plant hormones were analyzed using a Dionex Ultimate U3000 system (Dionex, Sunnyvale, USA), equipped with an on-line solid phase extraction (SPE) column and diode array detector. The column configuration consisted of an Acclaim® Polar Advantage II (PA II) C18 (150 mm × 4.6 mm, 5 µm), which was used as the analytical column for the clean-up of the sample and enrichment of the analytes, and PA II C18 guard column (10 mm × 4.6 mm, 5 µm), which was used as the on-line SPE column for sample trapping. System control, data acquisition, and data analysis 9 / 32



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were performed using the Chroméléon software (Dionex, Sunnyvale, USA). The mobile phase was a mixture of methanol and water, which was filtered in a millipore device through microfiber filters of medium pore diameter (4.5 µm; Phenomenon, Tianjin, China) before use. A gradient program was used after the ADS sample (20 µL) was injected into the SPE column. All physico-chemical analyses were carried out in triplicate and the mean value was calculated in each case. The results are expressed as mean ± standard deviation.

Statistical analyses and calculation Nutrient removal efficiencies were calculated using Eq. (1):

Ri = ( Si 0 − Sit ) / Si 0

(1)

Where: Ri is the removal efficiency of substrate i (GA3, ABA or IAA); Si0 is the initial concentration of i; and Sit is the final concentration of i after t days storage. Differences in results were evaluated by using single factor analysis of variances (ANOVA) in Excel software 2007.

Results and Discussion Batch reactor performance In this study, the pH, methane yield, and VFA concentration were used as performance indicators of the reactors.24

The batch reactors were operated for 30 days. As shown in Fig. 1, the pH of the CM reactor and the DM reactor remained relatively constant and slightly increased at the end of the experiment.

By contrast, the pH of the PM reactor decreased initially

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until a pH of 6.3 was obtained on day 12 (Fig. 1). Later, the pH of PM reactor also recovered on day 16 and was stable around 7.6 during the following days. In all reactors, the pH remained within 7.6-8.1 after 16 days, which was constant or slightly higher than the initial value, reflecting the accumulation of ammonia. All final pH values were considered healthy (above 7) and compatible with the normal growth of anaerobic microorganisms.25

As shown in Fig. 2, the methane production rate of the CM reactor and the DM reactor rapidly increased after 6 days, whereas the methane production rate of the PM reactor increased after 12 days (Fig. 2a, 2b, 2c). The results showed that the CM reactor and the DM reactor showed a significantly higher maximum methane production rate (0.028 L/g VSadded-d and 0.023 L/g VSadded-d, respectively) than the PM reactor (0.018 L/g VSadded-d). Furthermore, CM reactor gained the highest cumulative methane yield (0.18 L/g VSadded), follewed by the DM reactor (0.158 L/g VSadded), and the PM reactor (0.119 L/g VSadded). This is possibly because the pH in the CM reactor and the DM reactor exhibit greater stability compared to that in the PM reactor. The pH values below 7.0 is known to inhibit methanogenic bacteria,26 resulting in low gas production in the PM reactor.

The batch anaerobic digestion in this study can be roughly divided into an early acidogenic phase (0-8 day) and a later methanogenic phase (8-30 day). The acidogenic stage involves the hydrolysis and conversion of complex materials into simple ones (e.g. lactose into organic (volatile) acids, which is accompanied by an increase in VFA). In the methanogenic phase, the products of the acidogenic stage are 11 / 32



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finally converted to methane and CO2.27 Fig. 2 (d, e, and f) showed the changes in the concentrations of acetic acid and VFA during AD. Acetic acid was the predominant VFA in ADS. In the acidogenic phase, the acetic acid contents of the CM reactor, the DM reactor, and the PM reactor increased and peaked on day 6 (773.5 mg/L), day 8 (315.5 mg/L) and day 10 (407.0 mg/L), respectively. Simultaneously, the highest VFA concentration in the CM reactor (865.0 mg/L) correspond to the highest methane production rate (0.028 L/g VSadded-d) and the highest cumulative methane yield (0.18 L/g VSadded) in the CM reactor, indicating that the VFA produced by acetogenic bacteria in the CM reactor were timely converted to methane (Fig. 2a, 2d). In all reactors, as pH increased, the concentrations of acetic acid and VFA sharply decreased to approximately 100 mg/L in the methanogenic phase (Fig. 2d, 2e and 2f). These results suggested that the complex organic polymers had almost been broken down; meanwhile, VFA and acetic acid were timely converted to methane and other end-products. The reactors were considered operating normally.

Effect of AD on the plant hormones in ADS GA3 is essential for normal growth and affects a wide variety of plant developmental processes.28 Fig. 2g, 2h and 2i showed the dynamic changes in GA3 during AD of CM, DM, and PM. During AD, the concentration of GA3 significantly increased during the acidogenic phase, especially in the PM reactor (from 4.25 mg/L to 47.18 mg/L), indicating that GA3 was produced during AD. A rapid decline was subsequently obtained during the methanogenic phase. After AD, the final GA3 contents of the CM 12 / 32


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reactor (3.58 mg/L), DM reactor (5.15 mg/L) and PM reactor (5.21 mg/L) were similar to or slightly higher than the initial levels (Table 2). This is possibly because GA3 is typically unstable and can easily decompose in water.29

ABA controls plant development and growth, including embryo development, seed dormancy, inhibition of lateral root formation, transpiration, synthesis of proteins, and photosynthesis.30 As shown in Fig. 2g, 2h and 2i, the ABA content in the DM reactor and PM reactor significantly increased by 348.07% and 276.79% to 32.44 and 33.12 mg/L, respectively. These values are remarkably higher than the initial levels (7.24 mg/L and 8.79 mg/L for DM reactor and PM reactor, respectively) (Table 2). The final ABA content in the CM reactor was 24.55 mg/L after AD, which was much lower than those in the DM reactor and the PM reactor. However, no significant differences were indicated among their initial levels. The increasing rate of ABA content in the methanogenic phase after 16 days was significantly higher than that in the acidogenic phase (0-8 d), indicating that ABA was continuously produced during AD, especially in the methanogenic phase.

IAA is often used as a plant growth regulator associated with the promotion of growth, callus proliferation, and the induction of rooting.31 In the present study, IAA production started with a rapid increase in the first 8 days, and then followed by a relatively slow rate of increase until the end of the experiment (Fig. 2g, 2h and 2i). After AD, no significant differences in the final IAA concentration were observed between the DM reactor (23.18 mg/L) and the PM reactor (23.41 mg/L).The IAA 13 / 32



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concentration in the CM reactor was lowest at 13.30 mg/L (Table 2).

A number of studies indicated that IAA could be produced from tryptophan by the acetogen Clostridium.32 The rapid production of IAA in the acidogenic phase, which is in agreement with the decrease in pH and increase in acetic acid and VFA, confirmed that IAA can be produced as a consequence of the anaerobic bacteria such as Clostridium spp degradation of the tryptophan present in animal manures (Fig. 4b). The tryptophan cames from the polymeric breakdown of considerable amounts of proteins (12%-40%) in animal manure.33 These results are also consistent with those in the studies by Kostenberg, Marchaim, Watad and Epstein

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and Scaglia, Pognani

and Adani 15, who reported the presence of IAA in ADS. In terms of GA3 and ABA, the increased levels may be attributed to the bacteria producing and/or release due to degradation of manure constituents and changes in pH.

Contents of plant hormones in ADS from biogas plants ADS from three continuously fed biogas plants using CM, DM and PM, respectively, as feedstocks were sampled.

The concentration of GA3 in the ADS from biogas

plant (16.37-44.83 mg/L) was significantly higher than that in the ADS from lab-scale batch reactors (3.58-5.21 mg/L), indicating that GA3 was continuously produced when the anaerobic reactors were continuously fed (Table 2). Many studies demonstrated that the characteristics of plants, including total dry matter, net photosynthetic rate, relative growth rate, crude protein and the number of fruits per 14 / 32


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plant of the test plants were significantly enhanced by spraying or seed soaking with exogenous GA3 at 0.3-34.6 mg/L.13,34 GA3 application at 3.46 mg/L could mitigate the adverse effects of salinity stress on the overall performance and productivity of mustard.35 Thus, the ADS of animal manures contain effective GA3 concentrations of 16.37-44.83 mg/L in favor of promoting plant growth even though it was unstable.

In terms of IAA and ABA, no significant difference was observed between the ADS from biogas plants and lab-scale reactors. All types of ADS have higher contents of plant hormones than feedstocks, indicating that the plant hormones can be produced during AD. As shown in Table 2, the highest concentration of GA3 (44.83 mg/L) and IAA (36.84 mg/L) were found in the ADS of CM, whereas that of ABA (35.59 mg/L) were found in the ADS of PM. The significant differences in the plant hormones of ADS could be attributed to several factors, including physio-chemical and biological characteristics of feedstocks, and biogas plant operating parameters (feedstock type and quality, organic loading rate, hydraulic retention time, temperature). Previous studies have shown that ABA application at 10-26.4 mg/L can increase the antioxidant capacities and phenolic contents of fruits and vegetables,

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as well as

delay wilting and improve plants performance under drought stress.37,38 Foliar application of 15 ppm IAA to the sodium chloride stressed plants caused an alleviating effect on the salt stressed plants and increased crop yield.39 Another study showed that the root and shoot growth of metal-stressed plants were most effectively increased with 10-10 M IAA.40 This result suggests that after AD, all types of ADS 15 / 32



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from the CM reactor, DM reactor, and PM reactor contained sufficient IAA and ABA to regulate plant development.

Effect of storage temperature on plant hormone dynamics in ADS High level of GA3 (16.28-16.44 mg/L) was detected in the fresh ADS of PM from biogas plant (Table 3). It was noticed that GA3 presented significant variations among different storage temperatures (Fig. 3a). A significant degradation was observed for GA3 in ADS stored at 20 °C and 37 °C compared with that stored at 4 °C. In the first 20 days storage, the GA3 contents immediately decreased by 80.2% and 80.7%, respectively, for storage at 20 °C and at 37 °C (Table 3); GA3 declined more rapidly at 37 °C than at 20 °C. By contrast, only 10.8% of the losses in GA3 occurred in the ADS stored at 4 °C. The higher the temperature, the faster the decline in GA3. These results may be caused by the instability and degradability of GA3 in water.29 It is demonstrated that in aqueous solutions GA3 (I) forms the dicarboxylic gibberellenic acid (GEA) (II), which is also unstable and, on decomposing, provides a mixture of monocarboxylic aromatic acids consisting of allogibberic acid (AGA) (IV), 9-epiallogibberic acid (9-epi-AGA) (V), and dehydroallogibberic acid (∆9, 11-AGA) (VI) (Fig. 4a).41 Kuhr reported that in neutral and weakly alkaline media, its stability is considerably low and the decomposition of gibberellic acid in aqueous solutions is also markedly stimulated by higher temperatures.42 As shown in Fig. 1, the ADS is weak alkaline media with pH estimated at 7.6. Therefore, storage at high temperature for more than 20 days dramatically mitigated the GA3 availability in ADS for plant growth although at high level of GA3 is present in fresh ADS.

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Similarly, the concentration of IAA (21.17-22.02 mg/L) in ADS from biogas plant can sufficiently regulate the plant development.17 A significant variation in IAA in ADS at three storage temperatures was observed (Fig. 3c). After storage, the IAA concentration decreased by 26.2%, 48.1% and 70.5% at 4 °C, 20 °C and 37 °C, respectively (Table 3).

Studies have been conducted to illustrate that IAA can be converted to 3-methyl indole (skatole) by the action of Lactobacillus and Clostridium scatologenes.32,43 The skatole is a malodorous compound and can’t promote plant growth. Thus, in this study, the skatole was determined to assess the losses of IAA in ADS. As shown in Fig. 3d, a significant increase was observed in skatole concentration during the storage at storage temperatures. At the end of the storage period, the skatole significantly increased to 4.92 mg/L, 8.60 mg/L and 13.77 mg/L, compared with the initial values at 4 °C (0.68 mg/L), 20 °C (0.69 mg/L), and 37 °C (0.68 mg/L), respectively (Table 3). Specifically, the results showed that higher storage temperature exerted more significant effects on the degradation of IAA to skatole (Fig. 4b). Thus, the storage negatively affected the availability of IAA in ADS.

Changes in ABA followed a similar pattern to IAA (Fig. 3b). The ABA in ADS stored at 4 °C and 20 °C statistically remained unchanged along 90 days. Correspondingly, a slight decrease was observed at 37 °C (22.14%) (Table 3),, much lower compared to GA3 because of the stability of the ABA in an aqueous solution. Previous studies have shown that ABA application by foliar application at 10-25 mg/L can increase the antioxidant capacities and phenolic contents of fruits and vegetables, 17 / 32


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delay wilting and improve plant performance under drought stress.37,44 Thus, though there is a slight decrease in ABA content after 88 days storage, sufficient ABA to regulate plant development is present in the fresh ADS (35.50-35.68 mg/L) and ADS (27.78-35.41 mg/L) stored at different temperatures.

Assessment of the availability of plant hormones in ADS As to fertilizer properties, ADS contained higher contents of plant hormones with the potential to promote plant growth and to increase the tolerance to biotic and abiotic stress. From the results of this study, significant differences were observed in the plant hormones in ADS, depending on the origin of the organic waste used as feedstock, biogas plant operating parameters, and storage condition (time and temperature). Consequently, the optimal use of ADS is important for gaining appreciable fertilizing effect of ADS to minimize losses and increase efficiency.

Germination power and germination percentage of seeds as well as seed quality are increased by soaking seed with ADS.45 The most important plant hormones for seed germination are ABA and GA3, which exert inhibitory and stimulatory effects on seed germination, respectively. The GA3/ABA balance determines the seed ability to germinate.46 High GA3/ABA is favorable to seed germination. IAA can also affect seed germination by influencing the activity of enzymes resulting in increased rates of cell growth and development.47 However, other studies have shown that seed viability/germination are highly sensitive to anaerobic digestion and completely lost within a few days of incubation, depending on the plant species, temperature, and 18 / 32


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time.48 Therefore, fresh ADS with high concentration of GA3 is more efficient for soaking seed under appropriate soaking conditions.

The most common application method of ADS is as a foliar fertilizer. The crop with ADS spraying not only increases the spikelet length and grain number, the thousand-grain weight and yield; it can also inhibit diseases and insects.49 This effect may be attributed to the high concentration of GA3 and N in ADS, resulting in appreciable and significant additive increase in the shoot dry biomass of crops.50

Plants are frequently subjected to environmental stresses such as water deficit, freezing, heat, or salt stress. The ABA has been suggested to play a role in stress responses and/or adaptation.51 The results showed that more than 80% of GA3 in ADS was lost after storage at high temperatures (Table 3), whereas a low percentage of ABA was lost during storage. Thus, the ADS after storage is more appropriate for application in plants under abiotic stresses.

Based on the discussion, the ADS exhibits superior potential a replacement for inorganic fertilizers, not only contributing to nitrogen and phosphorus but plant hormones as well.

Conclusion The results obtained from the batch experiments indicated that the contents of plant 19 / 32



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hormones (GA3, IAA and ABA) in ADS increased 1-4 times after 30 days of fermentation. High contents of plant hormones in ADS from large-scale biogas plants were also observed, which were sufficient to improve plant growth. However, higher storage temperatures negatively affect the availability of plant hormones in ADS because of decomposition and transformation of plant hormones.

Acknowledgements This study was supported by the National Key Technology Research and Development Program of China during the 12th Five-Year Plan Period (Grant No. 2015BAD21B04), Beijing Science and Technology Program (Z151100001115010) and Ministry of Science and Technology of China within China-Italy cooperation on food waste energy utilization, (SQ2013ZOA000017). We likewise greatly appreciate the critical and constructive comments from the anonymous reviewers, which have helped improve this manuscript.

References

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figures and tables Table 1 The anaerobic digestion process parameters of the large-scale biogas plants Table 2 Plant hormones of raw manures and anaerobic digested slurry (ADS) from Lab-scale anaerobic digested reactor and from large-scale biogas plants using chicken manure (CM), dairy manure (DM), pig manure (PM) Table 3 The concentrations and total removal of plant hormones in anaerobic digested slurry (ADS) of pig manure after 88 days storage at different temperatures

Figure 1. The pH of chicken manure (CM) Reactor, dairy manure (DM) Reactor, pig manure (PM) Reactor during the anaerobic digestion process Figure 2. Changes in the methane production rate and methane cumulative yield (a, b, c), acetic acid and VFA (d, e, f), and plant hormones (g, h, i) during anaerobic digestion (AD) of chicken manure (CM), dairy manure (DM), and pig manure (PM) Figure 3. The changes in plant hormones of anaerobic digested slurry (ADS) during storage at different temperatures Figure 4. Degradation metabolism pathway of plant hormones in ADS

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Table 1 The anaerobic digestion process parameters of the large-scale biogas plants

Location

Beilangzhong

Donghuashan

Liuminying

Feedstock

Pig Manure

Dairy Manure

Chicken Manure

Processes

USR

CSTR

USR

Temperature of digester

26-35

32-35

30

hydraulic retention time (HRT) (d)

12

20

15

Organic Loading Rate (OLR) (kg/(m3·d))

4-5

4.0-4.7

3-4

USR: Upflow Solid Reactor, CSTR: Continuous Stirred Tank Reactor

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

1 2

Table 2 Plant hormones of raw manures and anaerobic digested slurry (ADS) from Lab-scale anaerobic digested reactor and from large-scale biogas plants using chicken manure (CM), dairy manure (DM), pig manure (PM) Plant

RCM

LADS-CM

PADS-CM

RDM

LADS-DM

hormones

(mg/L)

(mg/L)

GA3

1.45±0.65a

3.58±0.50b

IAA

4.44±0.03a

ABA

6.45±0.15a

PADS-DM

RPM

LADS-PM

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

(mg/L)

+146.90

44.83±1.68c

3.06±0.67b

5.15±2.21b

+68.30

38.53±1.40c

4.25±0.26b

5.21±3.69b

+22.59

16.37±2.16d

13.30±0.23c

+199.55

36.84±4.32f

7.05±0.92b

23.18±0.61d

+228.79

17.38±2.31e

4.37±0.02a

23.41±2.10d

+275.51

21.17±2.02d

24.55±9.15c

+280.62

13.23±2.82b

7.24±0.28a

32.44±1.29d

+348.07

23.53±2.27c

8.79±0.37a

33.12±3.74d

+276.79

35.59±3.42d

Variation(%)

3 4 5 6 7

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Variation(%)

PADS-PM Variation(%) (mg/L)

“-” means the total concentration decreased, and “+” means the total concentration increased Values are expressed as mean and figures in parentheses are standard deviations (n = 3); a,b Means different superscript letters differ (P < 0.05) in horizontal position RCM: raw chicken manure, LADS-CM: Lab-scale anaerobic digested slurry of chicken manure, PADS-CM: Plant-scale anaerobic digested slurry of chicken manure RDM: raw dairy manure, LADS-DM: Lab-scale anaerobic digested slurry of dairy manure, PADS-DM: Plant-scale anaerobic digested slurry of dairy manure RPM: raw pig manure, LADS-PM: Lab-scale anaerobic digested slurry of pig manure, PADS-PM: Plant-scale anaerobic digested slurry of pig manure

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

8 9


Table 3 The concentrations and total removal of plant hormones in anaerobic digested slurry (ADS) of pig manure after 88 days storage at different temperatures 4 ℃

10 11

20 ℃

37 ℃

initial Concentration

Final Concentration

Removal


Final Concentration

Removal


Final Concentration

Removal

(mg/L)

(mg/L)

(%)

(mg/L)

(mg/L)

(%)

(mg/L)

(mg/L)

(%)

GA3

16.37±0.16a

14.61±0.76a

10.75

16.44±0.23a

3.26±0.20b

80.17

16.28±0.18a

3.14±0.30b

80.71

IAA

21.17±0.02a

15.63±0.74b

26.17

21.49±0.58a

11.16±1.00c

48.07

22.02±0.24a

6.49±0.04d

70.53

ABA

35.59±0.02a

35.41±0.67a

0.50

35.50±0.35a

32.96±0.44b

7.15

35.68±0.17a

27.78±0.60c

22.14

skatole

0.68±0.03a

4.93±0.12b

-625

0.70±0.01a

8.60±0.17c

-1128.6

0.69±0.01a

13.78±1.43d

-1897.1

“-” means the total concentration increased. Values are expressd as mean±standard deviations (n=3); a,b Means different superscript letters differ (P < 0.05) in horizontal position.

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8.5

8.0

7.5

pH

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 32

7.0 CM reator DM reator PM reator

6.5

6.0 0

5

10

15

20

25

30

Time (d) Figure 1. The pH of chicken manure (CM) Reactor, dairy manure (DM) Reactor, pig manure (PM) Reactor during the anaerobic digestion process

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Methanogenic phase Acidogenic phase

PM Reactor

Methanogenic phase

Acidogenic phase

Methanogenic phase

.20

.030 .025

c

b

a

.15 .020 .10

.015 .010

.05 .005

Methane production rate Cumulative methane yield

0.00

0.000 1000 d

e

acetic acid VFA

800

Cumulative methane yield (L/g VSadded)

DM Reactor

CM Reactor Acidogenic phase

f

600 400 200 0 60

Concentration (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-1 -1 Concentration (mg/L) Methane production rate (Lg VSadded d )

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g

i

h

50 40

GA3 IAA ABA

30 20 10 0 0

5

10

15

20

25

30

Fementation Time (d)

350

5

10

15

20

25

30

35 0

5


10

15

20

25

30

35


Figure 2 Changes in the methane production rate and methane cumulative yield (a, b, c), acetic acid and VFA (d, e, f), and plant hormones (g, h, i) during anaerobic digestion (AD) of chicken manure (CM), dairy manure (DM), and pig manure (PM)

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25 GA 3 (a)

18

IAA Concentration (mg/L)

GA3 Concentration (mg/L)

20

16 14 12 10

4 20 37

8 6 4 2

IAA (c) 20

15

10 4 20 37

5

0

0 0

20

40

60

0

80

20

40

60

80

Strorage Time (d)

Storage Time (d) skatole Concentration (mg/L)

40

ABA Concentration (mg/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABA (b) 35

30

25 4 20 37

20

20 skatole (d) 15 4 20 37

10

5

0 0

20

40

60

Stroage Time (d)

80

0

20

40

60

80

Storage Time (d)

Figure 3. The changes in plant hormones of anaerobic digested slurry (ADS) during storage at different temperatures

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O H CO

9

OH

(a) OH COOH

CH3

CH

GA3 (I)

OH OH

COOHCOOH

CH

GEA (II)

CH3

COOH

N IAA

N

CH3

C C

(c)

OH

CH3 CH AGA (IV) 9-epi-AGA (V) 9, 11 -AGA (VI)

N Skatole

CH3

H H3C

COOH

CH3

CH2COOH

H Tryptophan

OH

CH

conjugated triene (III)

NH2 H2 C C COOH

(b)

OH

H

CH H C COOH

CH3

O ABA

Figure 4. Degradation metabolism pathway of plant hormones in ADS

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For Table of Contents Use Only Anaerobic digestion and storage influence availability of plant hormones in livestock slurry Xin Li, Jianbin Guo∗, Changle Pang, Renjie Dong Synopsis The plant hormones, as important growth regulators, were produced in the anaerobic process, and their changes during storage were discussed for effective application.

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Anaerobic Digestion and Storage Influence Availability of Plant

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