Effectiveness of Biochar in the Short-Term Abatement of GHGs and NH₃ Emissions from Digestate and Slurry

Management of livestock wastes significantly contribute to gaseous losses in the form of N₂O, CH₄ and NH₃, causing threats to soil health and climate. Several strategies reducing the environmental impact of wastes storage are available today. Among them, we tested the effectiveness of adding biochar alone and in combination with bio acidification on gaseous emissions from livestock slurry and digestate in a short-term laboratory simulation. We simultaneously analyzed CO₂, CH₄, N₂O and NH₃ emissions and the main microbial groups involved in their production, and the impact of different storage treatments. Digestate showed twice NH₃ emissions and half CH₄ emissions than slurry, according to the NH₄⁺ and C availability of the substrates and abundance of microbial communities. Biochar favoured oxidative conditions that reduced CH₄ emissions slightly from slurry and largely from digestate, but increased CO₂ losses from slurry. The combination of biochar with lactic acid was effective in reducing NH₃ emissions from both slurry and digestate. However, it triggered C losses (CO₂ and CH₄ emissions) due to a fast microbial response to increased labile C availability.

Manure management is responsible for 5.7 % and 4.3 % of global livestock methane (CH₄) and nitrous oxide (N₂O) emissions, respectively [1]. Additional losses take place in the form of ammonia (NH₃) emissions that have a severe impact on utrophication and acidification processes, and in particulate matter formation [2]. On the other side, livestock wastes are valuable sources of nutrients and organic matter that maintain soil fertility and crop production [3]. The potential use of such wastes as alternatives to mineral fertilizers open new opportunities in a circular economy perspective [4]. Several techniques that prevent emissions and preserve N in livestock wastes are today available, as reviewed by Ambrose et al. [5] for CH₄ and by Kupper et al. [6] for NH₃. Among them, anaerobic digestion is designed to optimize conversion of available carbon (C) into biogas and obtain an ammonium (NH₄⁺) enriched product, producing low N₂O [7], but high NH₃ emissions during storage [6,8,9]. Gaseous emissions are the result of microbial processes directly involved in CH₄, N₂O and NH₃ [10] production and consumption. Few research focused comprehensively on environmental feedback of livestock wastes [11] and a better understanding of biological processes and microbial functioning is fundamental to better setup ad hoc solutions

Biochar was successfully used to reduce CH₄, N₂O and NH₃ emissions from manure [12]. However, clear evidence on the net balance of gaseous emissions are still scarce, and contrasting effects on each gas were reported [11]. Schmidt [13] suggested the combined use of biochar and lactic acid bacteria to treat liquid slurry to obtain several environmental benefits. Laboratory- scale testing was successful to compare different treatments in short-term simulated storage [11,14-16]. This work evaluated at laboratory-scale the effectiveness of adding biochar alone and in combination with bio-acidification on gaseous exchanges from livestock slurry and digestate. Despite the short-term duration of the experiment, the focus was directed to comprehensively analyze gaseous losses and microbial communities and processes which are at the base of gas production.

Lab-scale set up

The experiment was conducted in a controlled environment (20^°C Temperature) at Fondazione Minoprio (Italy) in February - March 2022. Eighteen tanks of 20 L capacity and hermetical closure were prepared and positioned within the greenhouse in a randomized design. Six combination treatments in tri-replicates were set up: slurry, digestate, slurry + biochar (SluB), digestate + biochar (DigB), slurry + biochar + lactic acid (SluLacB) and digestate + biochar + lactic acid (DigLacB). 10 L of digestate or slurry were used in each tank, 500 g of biochar (DigB and SluB) and a mix with 0.03 g Lactobacillus plantarum 14D/CSL (Lactosil 3.0, CLS srl), lactic acid (20 mL), 25 g glucose and 25 g saccarose (DigLacB and SluLacB). Characteristics of digestate, slurry and biochar are reported in Lagomarsino et al. [17]. Production of CO₂, CH₄, N₂O and NH₃ was monitored for two weeks, at 0, 0.8, 1, 2, 3, 4, 7, 9, 11 and 14 days from the substrate’s addition. Temperature within the tanks was measured at every single measurement time with portable instruments (Hanna thermometer, Part Code HI7661).

GHGs emissions measurements

Gas measurements were conducted with a DX4040 FTIR Gas Analyzer (Gasmet Technologies Oyd), detecting gaseous compounds by absorbance of infrared radiation at 10 s intervals (Powell and Vadas, 2016). The tanks were left open between each measurement to avoid saturation and were closed during measurements by a lid equipped with two valves for instrument connection via two Teflon tubes. The tank headspace air was pulled into the FTIR and then returned back into the tank through the outlet with an on-board sample pump (maximum pressure is 1.0 bar and maximum flow is 2.0 l min-1), creating a dynamic chamber with a closed loop air circulation required for measuring cumulative gas concentrations [15], which allowed for reliable NH₃ measurements [9]. Consecutive measurements of the individual gas concentrations over time were performed until NH₃ saturation in the chamber’s headspace was observed, at least for 10 minutes, reading gas concentrations every minute. Gas fluxes were calculated from linear increase of gas concentration (R² > 0.75) versus time plot, headspace volume, and emitting volume. Significant differences in GHGs productions were assessed by analysis of variance (ANOVA) followed by Fisher LSD post-hoc test (p<0.05) using Statistica 7 (StatSoft).

GMicrobial analyses

Total DNA was extracted from 0.5 mg of slurry or digestate samples collected at the end of experimentation using the Fast DNA Spin Kit for soil (Biomedicals). The microbial communities were analyzed by denaturing gradient gel electrophoresis (DGGE) using primers for the bacterial and methanogenic archaeal 16S rRNA gene [18,19]. DGGEs were carried out and analyzed as previously described by Pastorelli et al. [20]. Real-time PCR was used for microbial groups quantification [21]. Bacteria and methanogenic archaea were quantified using primers for 16S rRNA gene [22,23]. Denitrifiers were quantified using primers for nirK and nosZ marker genes [24]. Nitrifiers were quantified using primer for amoA marker gene, distinguishing archaea and bacteria [25]. Significant differences were assessed by ANOVA.

Gas production

CO₂ emissions increased sharply in the first 24 hours of incubation (Figure 1, top). The highest peaks were observed from SluLacB and DigLacB. The initial peaks declined rapidly in the first 24 hours. The effects of treatments remained consistent along measurements, with the maximum emissions from DigLacB and SluLacB > SluB > Slurry > DigB and Digestate (Table 1). CH₄emissions raised after the third day in all treatments, except for DigB that peaked at the beginning but then remained very low throughout the storage period, with a reducing effect of biochar (Figure 1, middle). The maximum rates were observed from SluLacB (from 24 hours onwards) and DigLacB (from 72 hours onwards). CH₄ emissions from slurry and digestate were reduced by sole biochar addition but strongly increased with the combination of biochar and lactic acid (Table 1). NH₃ emission rates were on average higher in the first 24 hours of storage, with a progressive reduction after the fourth day of storage (Figure 1, bottom). Independent of treatments, emissions from digestate were about double those from slurry throughout the monitoring period. The effect of treatments (Table 1) was clear from the beginning with a constant reduction of emissions from digestate and slurry with biochar plus lactic acid combination. N₂O emissions remained very low, around 0 values throughout the 2 weeks of monitoring, without significant effects of treatments and were not reported.

Table 1: Percentage effects of biochar and biochar + lactic acid treatments on untreated slurry or digestate on of CO₂, CH₄ and NH₃ fluxes at each measurement time. *p<0.05, **p<0.01, ***p<0.001.

Microbial community

The analysis of similarity (ANOSIM) conducted on DGGE profiles showed that the type of substrate (slurry or digestate) has a significant role in shaping the bacterial and methanogenic archaeal communities (R=1, p<0.001 and R=0.9, p<0.001, respectively) regardless the addition of biochar or lactic acid (R=0.13, p>0.05 and R=-0.04, p<0.001, respectively). The canonical correspondence analysis (CCA) showed the microbial communities of slurry or digestate, clearly and significantly separated with respect to axis 1. The microbial communities of digestate were mainly correlated to NH₃ emissions, while CH₄ emissions were the least relevant variable in separating the bacterial and archaeal communities (Figure 2). Untreated slurry showed the highest bacterial and methanogenic archaeal 16S rRNA gene copy values while the lowest values were recorded in SluLacB. Treated and untreated digestate samples showed similar 16S rRNA gene copy values for both bacteria and methanogenic archaea (Table 2). The nirK and nosZ genes showed a similar trend with the highest values found in the slurry and the lowest in the digestate (Table 2). However, in no case significant differences were recorded.

The amoA gene showed values below the detection threshold.

Table 2: Real time PCR results (gene copy gr^-1). Values are means with standard error in parenthesis.

Even if anaerobic digestion is considered one promising technique to reduce NH₃ emissions from untreated slurry [26], increasing rates of NH₃ emission from digestate than slurry were detected, driven by the higher NH₄⁺ content, as observed by Zilio et al. [27] and reviewed by Kupper et al. [6]. Conversely, digestion of livestock wastes was an effective strategy to reduce CH₄ emissions [5], as confirmed by a contextual lower abundance of methanogenic communities in digestate than in slurry. Similar results were obtained by Aguirre-Villegas et al. [7] that observed higher NH₃ and lower CH₄ emissions from digestate than untreated manure. N₂O fluxes were negligible during all storage period, independent of treatments and waste type, demonstrating a minor contribution of this gas during storage (lower than 3%), which was however prevalent after application to soil [17]. Accordingly, the amoA gene at the end of the incubation period was below the detection limit for both nitrifying bacteria and archaea, which may limit the generation of N₂O [28], while contributing to NH₃ emission [29]. Several studies report negligible values of N₂O in the absence of a dry encrusted surface [30-32], and Park et al. [33] suggested to ignore N₂O emissions from non-aerated manure storage in GHG inventories.

The use of biochar during the storage of livestock wastes to reduce their environmental impact is rapidly increasing, thanks to the biochar capacity to absorb gases and liquids [12,13]. The effect of biochar varied depending on
a. The type of livestock waste (slurry or digestate)
b. The type of gas and
c. The storage time, highlighting the complexity of a system where feedbacks are interacting each other.

Overall, an increase of CO₂ emissions from slurry and a decrease of CH₄ emissions from either slurry or digestate was evident. Biochar is reported to improve oxidative conditions [34] and reduced methanogenic archaea (Table 2), which drove the CH₄ reduction. Ammonia emissions were little affected by biochar, unless an increasing trend from digestate after the first week, probably supported by the release of NH₄⁺ initially sorbed [35]. Indeed, biochar was found to reduce NH3 emissions from manure, but this effect was mostly related to composting [12] or surface application [11]. The manipulation of the balance between NH3 and NH₄⁺ by adding acids and lowering the pH value of the manure successfully reduced NH₃ emissions [32,36] and CH₄ [37]. Moreover, promising results on NH₃ emissions reduction have been recently obtained through biochar activation with additives such as acids or oxidants that can improve sorption capacities [38-40]. The decrease of NH₃ observed in our simulation supported those findings, confirming the combination of biochar and lactic acid as a promising strategy for both slurry and digestate. Conversely, lactic acid increased both CO₂ and CH₄ emissions, this last with huge peaks, suggesting the occurrence of a fast microbial response to availability of labile C substrates added, which triggered microbial activity and C losses. In fact, this increase was not related to a larger bacteria or methanogenic communities, which remained stable among treatments.

This research activity has been funded by Regione Lombardia with PSR 2014–2020, Operazione 1.2.01 of FEASR (Fondo europeo per l’agricoltura e lo sviluppo rurale), Projects N-CONTROL (2020– 2022) and AMMOCHAR (2022-2024).

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