Effects of Biochar and Clinoptilolite on Composition and Gaseous Emissions during the Storage of Separated Liquid Fraction of Pig Slurry

The storage of animal manure is a major source of gaseous emissions. The aim of this study was to evaluate the effects of biochar and clinoptilolite on the composition and gaseous emissions during the storage of separated liquid fraction of pig slurry. The experiment was carried out using containers with 6 L of pig slurry each. The additives biochar and clinoptilolite were added alone and mixed to the pig slurry at a rate of 2.5% each, in a total of four treatments with three replicates including the control. Gaseous emissions were monitored by a photoacoustic multigas monitor, and slurry samples were collected at 0 and 85 days and their composition assessed. Results showed that the addition of biochar could modify the physicochemical properties of the slurry. The addition of biochar did not reduce the E. coli during the experiment while clinoptilolite decreased its prevalence. The addition of biochar or clinoptilolite reduced significantly the NH3 emission during the storage of slurry, but no advantages were gained with their combination. The addition of biochar significantly reduced the CO2 and CH4 emissions relative to clinoptilolite, however N2O emissions and global warming potential did not differ among the additives. We conclude that the biochar and clinoptilolite are recommended as a mitigation measure to reduce gaseous emissions and preserve the fertiliser value at slurry storage.


Introduction
The strong increase in intensive livestock production has led to severe environmental problems due, essentially, to the management of the large amounts of slurry (liquid manure) resulting from this activity. The storage of animal manure is a major source of ammonia (NH 3 ), nitrous oxide (N 2 O), carbon dioxide (CO 2 ) and methane (CH 4 ) emissions, all with important impacts on climate change, acid rain and ozone formation in the troposphere [1][2][3]. Regarding the Directive 2016/2284 of the European Union, from 2030, the national NH 3 emission ceilings should be reduced at all stages of animal manure management, namely: 10% for feeding, 20% for housing, 40% for storage and 30% for soil application.
At storage stage, the NH 3 and CO 2 are originated by decomposition of urea present in slurry and the main characteristics that control NH 3 volatilisation are temperature, concentration of total ammoniacal N, pH and air velocity on slurry surface [4,5]. The majority of inorganic N present in slurry is in the form of ammonium (NH 4 + ). Nitrification can be performed by autotrophic and heterotrophic organisms under aerobic conditions whereas denitrification is the stepwise reduction of nitrate (NO 3 − ) to N 2 under anaerobic conditions [6]. Since the bulk of stored slurry is predominantly anaerobic with little chance for NH 4 + to be nitrified, N 2 O and N 2 through nitrification and denitrification are considered insignificant [3]. The availability of NH 4 + and NO 3 − are primary requirements for nitrification and denitrification processes, respectively but also the availability of easily degradable carbon influences these processes [6]. The anaerobic decomposition of the organic matter by methanogenic bacteria leads to CH 4 and CO 2 emissions. Organic matter is considered a major limiting factor for CH 4 production, once anaerobiosis is established, and CH 4 emissions are closely related to manure temperature [7]. Relative to stored slurry, management practices such as storage duration, agitation or mixing and emptying of storage tanks plays an important role in CH 4 emissions during storage [8].
Several practices aimed at reducing the environmental impact of slurry storage, and the techniques used, have been pointed out as an efficient solution to decrease gaseous emissions, namely: diet manipulation, design of the storage tank, fitting a covering material on the slurry store, application of additives to the slurries, for biodegradation of organic materials, lowering pH, bind/convert NH 4 + /NH 3 , down urea hydrolysis and inhibition of nitrification [3,[7][8][9][10][11]. Additionally, the solid-liquid separation of pig slurry has been reported as a good technique for manure management at farm scale, modifying the main characteristics of derived fractions, reducing the costs of storage and the environmental impacts [12][13][14][15]. The derived liquid fraction could be stored prior to soil application whereas the solid fraction could be composted and exported out of the farm [12]. Our hypothesis was that the application of adsorbing additives, like biochar and/or clinoptilolite, will modify initial slurry characteristics and consequently influence gaseous emission patterns during storage and after field application. Therefore, the treatment of slurry-derived liquid fraction either by biochar and/or clinoptilolite will minimise N (NH 3 + N 2 O) emissions, preserve fertiliser value and consequently increase the availability of N for crop utilisation after soil application. The aim of this study was to evaluate the effects of biochar and clinoptilolite on composition and emission of NH 3 , N 2 O, CO 2 and CH 4 during the storage of mechanical separated liquid fraction of pig slurry.

Experimental Design and Treatments
In order to follow the changes in slurry composition and gaseous emissions after application of additives at the storage of pig slurry, a laboratory experiment was conducted in which the slurry was amended with biochar and/or clinoptilolite. The following four treatments with three replications were considered: 1.
Slurry amended with biochar at a rate of 2.5% (w/w; 2.5 g of additive present in 100 g of slurry) (Biochar treatment); 3.
Slurry amended with biochar and clinoptilolite and each one at a rate of 2.5% (w/w) (Biochar + Clinoptilolite treatment).
Each container was closed at 0 days, leaving an open headspace (volume = 6 L) between the surface of the slurry and the container lid. One air inlet and one air outlet were positioned symmetrically in the container lid, being inserted a Teflon tube (3 mm internal diameter) that was fitted through one of the septa and its end kept 20 mm above the slurry surface. Airflow through the headspace of each container was achieved using one individual pump (Marina 100, Hagen, Leeds, UK), with a 2.5 L min −1 flowrate regulated by a needle valve coupled to a flow meter (AalborgTM FT10201SAVN, Aalborg, Denmark), located before each slurry container ( Figure 1). The outlet air from the 12 plastic containers was exhausted out of the climatic room by a fume hood.
Each container was closed at 0 days, leaving an open headspace (volume = 6 L) between the surface of the slurry and the container lid. One air inlet and one air outlet were positioned symmetrically in the container lid, being inserted a Teflon tube (3 mm internal diameter) that was fitted through one of the septa and its end kept 20 mm above the slurry surface. Airflow through the headspace of each container was achieved using one individual pump (Marina 100, Hagen, Leeds, UK), with a 2.5 L min −1 flowrate regulated by a needle valve coupled to a flow meter (AalborgTM FT10201SAVN, Aalborg, Denmark), located before each slurry container ( Figure 1). The outlet air from the 12 plastic containers was exhausted out of the climatic room by a fume hood. The concentrations of NH3, N2O, CO2 and CH4 were measured in the exhaust air with a photoacoustic multigas monitor (INNOVA 1412i-5, Lumasense Technologies, Ballerup, Denmark) and air samples collected, in sequence (120 s intervals), through one sampling point (Teflon tube with 3 mm internal diameter) per container, by a multipoint sampler (INNOVA 1409-12, Lumasense Technologies, Ballerup, Denmark) provided with PTFE-filters (0.001 mm pore size, Whatman, Ome, Japan) ( Figure 1). The photoacoustic multigas monitor was equipped with an optical filter for water vapour (filter type SB0527) and the detection limits for NH3 (filter type UA0973), N2O (filter type UA0985), CO2 (filter type UA0982) and CH4 (filter type UA0969) were, respectively, 0.1521, 0.0589, The concentrations of NH 3 , N 2 O, CO 2 and CH 4 were measured in the exhaust air with a photoacoustic multigas monitor (INNOVA 1412i-5, Lumasense Technologies, Ballerup, Denmark) and air samples collected, in sequence (120 s intervals), through one sampling point (Teflon tube with 3 mm internal diameter) per container, by a multipoint sampler (INNOVA 1409-12, Lumasense Technologies, Ballerup, Denmark) provided with PTFE-filters (0.001 mm pore size, Whatman, Ome, Japan) ( Figure 1). The photoacoustic multigas monitor was equipped with an optical filter for water vapour (filter type SB0527) and the detection limits for NH 3 (filter type UA0973), N 2 O (filter type UA0985), CO 2 (filter type UA0982) and CH 4 (filter type UA0969) were, respectively, 0.1521, 0.0589, 2.9471 and 0.2864 mg m −3 .

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The photoacoustic multigas monitor was calibrated by the manufacturer before the beginning of the experiment, being operated in a mode for compensation of water interference and cross-interference.

Data Analysis
The gas concentrations of NH 3 , N 2 O, CO 2 and CH 4 from the outlet sampling points were used to calculate means per hour and day. The gas (NH 3 , N 2 O, CO 2 or CH 4 ) emissions from each outlet sampling point were determined (per hour) by a mass balance, using Equation (1).
where, EMISSION was the gas emission (mg h −1 ), GOUTLET was the outlet gas concentration (mg m −3 ), GINLET was the inlet gas concentration (mg m −3 ) using the following background coefficients: 0.00266 mg m −3 for NH 3 , 0.58942 mg m −3 for N 2 O, 628.71429 mg m −3 for CO 2 and 1.07411 mg m −3 for CH 4 , and FLOW was the air flowrate in the plastic container (m 3 h −1 ).
The cumulative values of NH 3 , N 2 O, CO 2 and CH 4 were determined by averaging the flux between two sampling occasions and multiplying by the time interval between the measurements [19]. The global warming potential (GWP) for each plastic container was determined using the global warming potential coefficients for direct greenhouse gas emissions (265 for N 2 O, 1 for CO 2 and 28 for CH 4 ) and indirect N 2 O emissions (1% of NH 3 -N volatilised for N 2 O-N) [2].
All data obtained were analysed by two-way analysis of variance (ANOVA) to test the effects of each treatment and time on slurry composition and gaseous emissions, followed by Tukey's honestly significant difference test comparisons of means tests (p < 0.05), using the statistical software package STATISTIX 10 (Analytical Software, Tallahassee, FL, USA).

Composition of the Slurries
The initial (0 days) and final (85 days) composition of the Control and amended treatments (Biochar, Clinoptilolite and Biochar + Clinoptilolite) are presented in Tables 1 and 2. The initial pH values did not differ significantly (p > 0.05) among treatments Control and Clinoptilolite (pH = 8.5), being significantly higher (p < 0.05) in treatments Biochar and Biochar + Clinoptilolite (pH = 9.0) ( Table 1). Compared to the beginning of the experiment, the pH values of treatments Biochar and Biochar + Clinoptilolite increased significantly (p < 0.05) in the end of experiment ( Table 1). The initial dry matter content increased significantly (p < 0.05) in amended treatments (Biochar, Clinoptilolite and Biochar + Clinoptilolite) when compared with treatment Control, with higher values for treatments Biochar and Biochar + Clinoptilolite (2.1% for Biochar against 2.7% for Clinoptilolite). However, the dry matter content did not differ significantly (p > 0.05) between the beginning and the end of the experiment, although being observed a difference of about 30% (2.7 vs. 1.9) for treatment Biochar + Clinoptilolite (Table 1).
At the beginning of the experiment, the treatments Biochar and Biochar + Clinoptilolite increased significantly (p < 0.05) by about 100% the total C, as well as the C/N ratio when compared with the treatments Control and Clinoptilolite (C/N = 22 for Biochar treatments against C/N = 11 for non-Biochar treatments). In all treatments, the initial values of total C were reduced significantly (p < 0.05) at the end of the experiment ( Table 1). The initial values of total N, NH 4 + -N, NO 3 − -N, NH 4 + /total N ratio, biological oxygen demand (BOD) and volume of slurry were not significantly different (p > 0.05) among treatments (Tables 1 and 2). These same parameters decreased in the end of the experiment. However, the final values of total N were significantly higher (p < 0.05) in treatments Clinoptilolite and Biochar + Clinoptilolite (1.5 g total N kg −1 for Clinoptilolite-alone against <0.5 g total N kg −1 for Biochar treatments) ( Table 1). In addition, the final BOD values of amended treatments decreased significantly (p < 0.05) by the following order: Biochar + Clinoptilolite < Biochar < Clinoptilolite (Table 2).
At the beginning of the experiment, the number of colonies of E. coli were significantly higher (p < 0.05) in treatment Biochar, but significantly lower (p < 0.05) in treatment Clinoptilolite, when compared with treatment Control or Biochar + Clinoptilolite (Table 2). At the end of the experiment, the values the number of colonies of E. coli were not significantly different (p > 0.05) among all treatments and there was no evidence of the presence of E. coli (1.0 colony-forming units (CFU) mL −1 ) ( Table 2).
Results of this study (Tables 1 and 2) are in line with previous studies [3,[15][16][17][18][19][20][21][22][23] who reported that pre-treating animal slurries with biochar could modify the physicochemical properties, like as increasing pH, C/N ratio and cation-exchange capacity, and microbial activities. The explanation is that biochar was a porous carbonaceous material largely containing C jointly with the inorganic components of the biomass utilized, such as alkali and alkaline earth metals [21]. On other hand, the addition of biochar at a rate of 2.5% appear not reduce E. coli during the 85 days of experiment (Table 2), agreeing with Soares et al. [18] who reported a marked decrease in CFU mL −1 for survival of E. coli by the storage period (90 days) and not by the addition of biochar at a rate of 4.5% to cattle slurry. However, recent studies [24,25] suggest that the presence of biochar influences the removal of E. coli and minimizes the impact on bacterial viability, which requires further research.
The addition of clinoptilolite reduced NH 3 losses from slurry due the great affinity for NH 4 + and consequently preserved N in amended treatments (Table 1). Such an effect was because clinoptilolite are crystalline, hydrated aluminosilicates of alkali and alkaline earth cations with high porosity, ion exchange and adsorption capacity for NH 4 + retention [19,26]. At the beginning of the experiment, clinoptilolite decreased the prevalence of E. coli in amended treatments ( Table 2) which might be explained by the antibacterial properties, already documented in a previous study [27].

Nitrogen Emissions
The daily fluxes of NH 3 and N 2 O from treatments during the experiment are shown in Figure 2A,B. Comparatively to amended treatments, the daily NH 3 fluxes of treatment Control were significantly higher (p < 0.05) in the first 19 days, with a great peak in the first 6 days (20 to 65 mg NH 3 m −2 h −1 ) followed by a progressive decrease (65 to 35 mg NH 3 m −2 h −1 ) until day 19 of experiment. The daily NH 3 fluxes of amended treatments increased (15 to 35 mg NH 3 m −2 h −1 ) in the first 19 days, followed by a progressive decrease (35 to 2 mg NH 3 m −2 h −1 ) until the end of the experiment including in the treatment Control (Figure 2A). Compared to the treatment Control, the amended treatments reduced significantly (p < 0.05) the daily NH 3 fluxes in the first 19 days (30-35% lower), followed by an significant increase (p < 0.05) (10-38% higher) until day 30 and a significant decrease (p < 0.05) until day 60 (34-52% higher), and finally reduction for the Control level until the end of the experiment (Figure 2A). It is noteworthy that the daily NH 3 fluxes from treatments Clinoptilolite and Biochar + Clinoptilolite were lower in about 15% when compared with treatment Biochar whereas, in most measurement days, were observed quite similar NH 3 fluxes between treatments Biochar and Biochar + Clinoptilolite (Figure 2A). The cumulative NH 3 emissions (expressed in g m −2 or as % of applied N) did not differed significantly (p > 0.05) among amended treatments, being significantly lower (p < 0.05) by about 26% than in treatment Control ( Table 3). The cumulative NH 3 losses in treatments Clinoptilolite and Biochar + Clinoptilolite were lower in about 10% when compared with treatment Biochar, despite these differences were not statistically significant (p > 0.05) ( Table 3).
As can be seen in Table 3, the addition of biochar and clinoptilolite alone or in combination decreased NH 3 emissions due the high specific surface area and the high cation exchange capacity of these additives, that enhance the NH 4 + and NH 3 binding [20]. Kalus et al. [23] reported that the addition of biochar (1-12% w/w) to animal manure could reduce NH 3 emissions from 12 to 77%, being comparable with the value observed in the present study (26% of reduction). Maurer et al. [22] reported up to 23% of NH 3 reduction for pig slurry amended with pinewood biochar (1.14-4.56 kg m −2 ) and Brennan et al. [28] found 77% NH 3 reduction for dairy cattle slurry treated with wood shavings biochar (12% v/v). In this study, the reduction of NH 3 emissions (26% reduction) by the addition of clinoptilolite, was lower than emissions reported in other studies [29,30] for cattle and pig slurries (50-70% reduction), which could be related with the higher rate of clinoptilolite used in referred studies (0.40-6.25 w/w). In addition, no advantages were gained from the combination of biochar with clinoptilolite.
Appl. Sci. 2020, 10, x FOR PEER REVIEW 7 of 14 pig slurries (50-70% reduction), which could be related with the higher rate of clinoptilolite used in referred studies (0.40-6.25 w/w). In addition, no advantages were gained from the combination of biochar with clinoptilolite.    As can be seen in Table 3, the addition of biochar and clinoptilolite alone or in combination decreased NH 3 emissions due the high specific surface area and the high cation exchange capacity of these additives, that enhance the NH 4 + and NH 3 binding [20]. Kalus et al. [23] reported that the addition of biochar (1-12% w/w) to animal manure could reduce NH 3 emissions by from 12% to 77%, being comparable with the value observed in the present study (26% of reduction). Maurer et al. [22] reported up to 23% of NH 3 reduction for pig slurry amended with pinewood biochar (1.14-4.56 kg m −2 ) and Brennan et al. [28] found 77% NH 3 reduction for dairy cattle slurry treated with wood shavings biochar (12% v/v). At this study, the reduction of NH 3 emissions (26% reduction) by the addition of clinoptilolite, was lower than emissions reported in other studies [29,30] for cattle and pig slurries (50-70% reduction), which could be related with the higher rate of clinoptilolite used in referred studies (0.40-6.25 w/w). In addition, no advantages were gained from the combination of biochar with clinoptilolite. The daily N 2 O fluxes follow the same trend, independently of the treatment, with a progressive increase (0.5 to 2 mg N 2 O m −2 h −1 ) in the first 50 days followed by a strong increase (2 to 10 mg N 2 O m −2 h −1 ) until the end of the experiment ( Figure 2B). Compared to the treatment Control, the amended treatments reduced significantly (p < 0.05) the daily N 2 O fluxes in the first 50 days (14-18% lower) followed by a significant increase (p < 0.05) until the end of the experiment (32-67% higher) ( Figure 2B). On most measurement days, significantly higher (p < 0.05) N 2 O fluxes from day 50 until the end of the experiment were observed according to the following order: Clinoptilolite > Biochar + Clinoptilolite > Biochar > Control ( Figure 2B). In the 85 days of experiment, the daily N 2 O fluxes of treatments Clinoptilolite and Biochar + Clinoptilolite were higher in about 11% relative to treatment Biochar ( Figure 2B). The cumulative N 2 O emissions (expressed in g m −2 or as % of applied N) were not significantly different (p > 0.05) between amended treatments; and only treatment Clinoptilolite was significantly higher (p < 0.05), by about 30%, than treatment Control ( Table 2). The cumulative N 2 O loss in treatment Clinoptilolite was higher in about 21% relative to treatment Biochar, but not statistically different (p > 0.05) ( Table 3).
The N 2 O losses originated in the nitrification and denitrification processes, which occurs when both aerobic and anaerobic conditions coexist in the slurries [31]. The N 2 O emissions observed in the present study appears to be mainly emitted by the nitrification process, because the aerobic condition created by the continuous air exchange and low depth of the slurry containers. The addition of biochar and clinoptilolite alone or combined led to NH 4 + retention, decreasing the N 2 O losses until day 50 of experiment. From this day until the end of the experiment an increase of N 2 O emissions was observed, which may be related with the saturation of the capacity of NH 4 + adsorption of the additives [19,20].
In addition, no further reduction in N 2 O emissions was observed by combining the two additives. Excluding differences on rates and composition of additives, the results of this study are lower than previous studies, where Brennan et al. [28] reported that cattle slurry amended with pinewood biochar (12% v/v) reduced N 2 O loss by 63% and Pereira et al. [19] referred that poultry manure amended with clinoptilolite (2.344 kg m −2 ) reduced N 2 O loss by 34% and Wang et al. [32] found that pig manure amended with biochar (10% w/w) mixed with clinoptilolite reduced N 2 O loss in about 80%. The N (NH 3 + N 2 O) emissions, expressed as g m −2 or as % of applied N, were not significantly different (p > 0.05) among amended treatments, being significantly lower (p < 0.05) in about 21% than in treatment Control ( Table 3). The cumulative N losses in treatments Clinoptilolite and Biochar + Clinoptilolite were reduced in about 8% relative to treatment Biochar but not statistically significant (p > 0.05) ( Table 3).

Carbon Emissions
The CO 2 and CH 4 daily fluxes from treatments along the experiment are shown in Figure 3A,B. Higher CO 2 fluxes were observed over the first 20 days of the experiment (630 to 1670 mg CO 2 m −2 h −1 ), followed by a small reduction (640 to 510 mg CO 2 m −2 h −1 ) until the end of the experiment ( Figure 3A). Compared to treatment Control, the amended treatments reduced significantly (p < 0.05) the daily CO 2 fluxes, in about 19%, during the first 13 days of experiment. After this day until the end of the experiment, no significant differences (p > 0.05) were observed between all treatments including Control ( Figure 3A). The cumulative CO 2 emissions, expressed in g m −2 , from amended treatments were significantly lower (p < 0.05) in about 5% when compared with treatment Control. When expressed as % of applied C, the CO 2 emissions from treatments Biochar and Biochar + Clinoptilolite were significantly lower (p < 0.05) in about 50% than from treatments Control or Clinoptilolite ( Table 3).
The CO 2 losses coming from the microbial degradation of the organic matter and the hydrolysis of the urea [19]. Biochar decreased the CO 2 emissions in 50% (expressed in % of applied C) whereas clinoptilolite appears had no effect on these losses (Table 3). In agreement with our results, previous studies reported that CO 2 emission from animal manure was reduced by 34-63% by adding biochar (10% w/w) [28,32] whereas the application of clinoptilolite (2.344 kg m −2 ) appears have no effect on CO 2 emission [19]. Excluding differences among biochar's like feedstock, method and temperature pyrolysis, the reduction of CO 2 emissions by adding biochar could be related with either sorption of onto the biochar or a reduction in the labile C availability [23,28].
In all amended treatments, the CH 4 daily fluxes peaked (7 to 25 mg CH 4 m −2 h −1 ) on the first day of the experiment, followed by a progressive decrease (7 to 0 mg CH 4 m −2 h −1 ) between day 2 and 50 and then increased (0.5 to 8 mg CH 4 m −2 h −1 ) from day 51 until the end of the experiment ( Figure 3B). Compared to treatment Control, in the first 17 days of the experiment the daily CH 4 fluxes were significantly lower (p < 0.05), by about 30%, in treatments Biochar and Biochar + Clinoptilolite but significantly higher (p < 0.05), by about 26%, in treatment Clinoptilolite ( Figure 3B). Then, on most measurement dates, the daily CH 4 fluxes from amended treatments were significantly reduced (p < 0.05) by 9-20% between days 2 and 50 of the experiment ( Figure 3B). Up to day 51 until the end of the experiment, the daily CH 4 fluxes in amended treatments were significantly higher (p < 0.05) than in the treatment Control with increases between 240 and 380% ( Figure 3B). The cumulative CH 4 emissions, expressed in g m −2 , were not significantly different (p > 0.05) among treatments (Table 3). When expressed as a % of applied C, the CH 4 cumulative emissions from treatments Biochar and Biochar + Clinoptilolite were significantly lower (p < 0.05) in about 55% relative to treatments Control or Clinoptilolite (Table 3).
the end of the experiment, no significant differences (p > 0.05) were observed between all treatments including Control ( Figure 3A). The cumulative CO2 emissions, expressed in g m −2 , from amended treatments were significantly lower (p < 0.05) in about 5% when compared with treatment Control. When expressed as % of applied C, the CO2 emissions from treatments Biochar and Biochar + Clinoptilolite were significantly lower (p < 0.05) in about 50% than from treatments Control or Clinoptilolite (Table 3).  Methane is produced mainly by microbial decomposition of organic matter under anaerobic conditions [33,34]. Biochar reduced the CH 4 emission by 55% while clinoptilolite seems had no effect on such loss (Table 3). Previous studies [32,35,36] reported that the emission of CH 4 from animal manures could be reduced by 50-95% by adding biochar and clinoptilolite alone or in combination (5-10% w/w of each additive), this being explained by the adsorption ability of the additives. However, in this study, the addition of clinoptilolite to pig slurry did not cause an increase or a reduction in CH 4 emissions because the experiment has been done on the liquid fraction of the slurry.
The C (CO 2 + CH 4 ) emissions (in g m −2 ) were not significantly different (p > 0.05) among amended treatments (Table 3). However, the cumulative C losses, as % of applied C, from the treatments Biochar and Biochar + Clinoptilolite were significantly lower (p < 0.05) in about 50% relative to treatments Control or Clinoptilolite (Table 3). The cumulative emissions expressed as GWP did not differed significantly (p > 0.05) between treatments (Table 3). Thus, the addition of biochar or clinoptilolite to pig slurry did not cause an increase or a reduction in GWP in this study.

Benefits of Storage Additives
The results of this laboratory study showed the potential of adding biochar and clinoptilolite in order to reduce NH 3 emissions in the storage of pig slurry, without increasing the GWP, and avoiding "pollution swapping" between NH 3 and N 2 O emissions. However, no advantages were gained from the combination of these two additives on gaseous emissions. In addition to the gains on the reduction of environmental impacts to the atmosphere during the storage stage, the treatment of slurry either by biochar or clinoptilolite will increase the N availability for crops or consequently increase yields. Moreover, biochar will improve the soil biological activities, nutrient retention, water-retention capacity, increase of pH value and amount of soil organic matter [21], whereas clinoptilolite will increase moisture retention in the soil due to increased soil surface area and cation exchange capacity [37].
Although caution must be exercised when extrapolating laboratory studies to farm-scale conditions, the inclusion of the additives with the slurry at the storage stage and prior to soil application represent an efficient method because of reduced environmental impacts from these two stages of manure management (storage and soil application). Therefore, integrated studies are necessary to evaluate the impact of biochar and clinoptilolite on all stages of animal manure management, namely feeding, housing, storage and soil application.

Conclusions
The results indicated that the addition of biochar could modify the physicochemical properties of the liquid fraction. The addition of biochar did not reduce the E. coli during the experiment while clinoptilolite decreased its prevalence. The addition of biochar or clinoptilolite reduced significantly the NH 3 emission during the storage of pig slurry, but no advantages were gained with their combination. The addition of biochar significantly reduced the CO 2 and CH 4 emission relative to clinoptilolite, when expressed as % of total C applied, however N 2 O emission and global warming potential did not differ among the additives. Hence, biochar and clinoptilolite are recommended as a mitigation measure to reduce gaseous emissions and preserve the fertiliser value of slurry, and do not require modification of the storage structure.

Conflicts of Interest:
The authors declare no conflict of interest.