Biochar (BC) application to soil suppresses emission of nitrous- (N2O) and nitric oxide (NO), but the mechanisms are unclear. One of the most prominent features of BC is its alkalizing effect in soils, which may affect denitrification and its product stoichiometry directly or indirectly. We conducted laboratory experiments with anoxic slurries of acid Acrisols from Indonesia and Zambia and two contrasting BCs produced locally from rice husk and cacao shell. Dose-dependent responses of denitrification and gaseous products (NO, N2O and N2) were assessed by high-resolution gas kinetics and related to the alkalizing effect of the BCs. To delineate the pH effect from other BC effects, we removed part of the alkalinity by leaching the BCs with water and acid prior to incubation. Uncharred cacao shell and sodium hydroxide (NaOH) were also included in the study. The untreated BCs suppressed N2O and NO and increased N2 production during denitrification, irrespective of the effect on denitrification rate. The extent of N2O and NO suppression was dose-dependent and increased with the alkalizing effect of the two BC types, which was strongest for cacao shell BC. Acid leaching of BC, which decreased its alkalizing effect, reduced or eliminated the ability of BC to suppress N2O and NO net production. Just like untreated BCs, NaOH reduced net production of N2O and NO while increasing that of N2. This confirms the importance of altered soil pH for denitrification product stoichiometry. Addition of uncharred cacao shell stimulated denitrification strongly due to availability of labile carbon but only minor effects on the product stoichiometry of denitrification were found, in accordance with its modest effect on soil pH. Our study indicates that stimulation of denitrification was mainly due to increases in labile carbon whereas change in product stoichiometry was mainly due to a change in soil pH.
Citation: Obia A, Cornelissen G, Mulder J, Dörsch P (2015) Effect of Soil pH Increase by Biochar on NO, N2O and N2 Production during Denitrification in Acid Soils. PLoS ONE 10(9): e0138781. https://doi.org/10.1371/journal.pone.0138781
Editor: R. Michael Lehman, USDA-ARS, UNITED STATES
Received: May 7, 2015; Accepted: September 3, 2015; Published: September 23, 2015
Copyright: © 2015 Obia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Data Availability: Data have been deposited to Dryad (doi:10.5061/dryad.m8q78).
Funding: This work was funded by Norwegian Research Council project FRIMUF No. 204112 awarded to JM and Norwegian University of Life Sciences PhD internal financing to AO, with co-funding from Norwegian Research Council FriPro project No. 217918 awarded to GC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Denitrification, the microbially mediated, stepwise reduction of nitrogen oxides to N2 via nitric oxide (NO) and nitrous oxide (N2O) , is the dominant pathway returning reactive nitrogen to the atmosphere. NO and N2O are gaseous intermediates of denitrification which, once escaped to the atmosphere, have adverse effects on plant and animal health , stratospheric ozone  and the radiative balance of the Earth . About 45% of the total terrestrial N2O emissions can be attributed to nitrogen (N) cycling in agriculture , making denitrification a primary target for greenhouse gas abatement .
Numerous studies have shown that biochar (BC), a biomass pyrolysis product originally devised for carbon (C) sequestration and soil amelioration [7–10] suppresses N2O emissions ( and references therein) alongside with increasing crop production [12–14]. Only few studies have reported that BC leads to increased N2O emissions [15, 16]. Thus, BC appears to be a promising agent to mitigate N2O emissions from agroecosystems, but the mechanisms mediating the suppression are unresolved. Various mechanisms have been proposed, such as increased N2O reductase activity at raised soil pH , increased electron flow to N2O through BC-mediated electron shuttling , reduced rates of denitrification through competition for electrons , direct sorption of N2O , improved soil aeration  and immobilization of ammonium or nitrate through adsorption or enhanced soil cation/anion exchange [15, 21, 22]. Other proposed mechanisms are ethylene production by BC resulting in temporary inhibition of nitrification  and microbial N immobilization due to the presence of labile organic carbon in BC . Increased N2O emission after BC application has been attributed to high N content in certain BC such as that made from poultry manure [16, 22].
Most BCs are alkaline owing to their ash content, causing release of base cations, and alkaline properties of organic functional groups . Biochar addition to soils neutralizes soil acidity and may increase the cation exchange capacity (CEC) and base saturation, depending on the intrinsic properties of the soil and the BC [26, 27].
Soil pH strongly controls the N2O/(N2O+N2) product ratio of denitrification. This has been demonstrated for pure cultures of denitrifying bacteria  and for soil denitrifying communities [29–33]. The likely reason is that low pH prevents the assembly of functional N2O reductase (N2OR), the enzyme reducing N2O to N2 in denitrification [29, 34]. Since BC is generally alkaline, increased N2OR activity due to pH rise could be one of the major mechanisms behind the observed suppression of N2O emission in BC treated acid soils. If so, N2O suppression by BC would be mainly a “liming effect”.
The objectives of the present study were to evaluate the role of BC-induced pH change on NO and N2O net production in soil denitrification. Although, NO is an important regulator in many biological processes including denitrification [35, 36], only few BC studies have addressed NO . We carried out ex situ denitrification experiments in closed bottles with two acidic agricultural soils from Indonesia and Zambia. We applied increasing doses of two types of BC strongly differing in amount and type of alkalinity and studied the responses of soil pH, overall denitrification rate and gaseous reaction products (NO, N2O, N2). To shed light on the role of soil pH, we removed alkalinity from the BCs through leaching with water and acid prior to incubation in a second experiment. In a third experiment, sodium hydroxide (NaOH) was used as an alkali analogue to study the effect of pH per se in the absence of BC. Furthermore, the NO and N2O suppressing effect of BC was compared to that of uncharred feedstock. The denitrification kinetics were studied in stirred soil slurries in helium (He) atmosphere, using a high-throughput incubation robot which monitors oxygen (O2), carbon dioxide (CO2), NO, N2O and N2 at high temporal resolution . Stirring ensured homogeneous soil slurries and equilibrium of gases between headspace and liquid phase. Unlike previous studies, our investigations were carried out under fully anoxic conditions, preventing confounding effects on denitrification NO and N2O production by other N-cycling processes.
Materials and Methods
Soils and biochars
Acidic, sandy loam Acrisols were sampled at Lampung (Sumatra, Indonesia; 05°00.406' S; 105°29.405' E) and Mkushi (Zambia; 13°36.264′ S; 29°29.768′ E) in October 2012 and April 2013, respectively. The soils were sampled from private lands with permission of the owners during the dry season and stored air-dried. Selected soil and BC properties are presented in Table 1. Different N-forms in soils and BCs were not considered. The NH4+ content was deemed irrelevant because our main experiments were under anaerobic conditions ruling out nitrification. The added ample amount of NO3- would override any sorption effect and denitrification and its product stoichiometry, are not sensitive to small differences in NO3- availability .
The BCs were prepared from rice husk and cacao shell, two common agricultural wastes in Lampung, Indonesia. The two BCs differed in extent and type of alkalinity (Table 1); cacao shell BC had a lower ash content but a ~10 times higher CEC than rice husk BC. The exchangeable cations of cacao shell BC were dominated by potassium (K). Overall, cacao shell BC had a ~5 times higher acid neutralizing capacity (ANC) than rice husk BC (217 vs 45 cmolc kg-1) .
The BC pyrolysis conditions, taken from Hale, Alling , can be found in Description A in S1 File. Since the BCs were not produced in the laboratory, thermogravimetric analyses (TGA) was used to estimate the pyrolysis temperature, indicating that this was between 400 and 500°C. In short, during the TGA, the temperature was stepwise increased up to 900°C, and weight loss was monitored. The weight loss profile was then compared to three temperature series of laboratory-generated BCs where pyrolysis had taken place at an exactly measured temperature. Weight loss and high to low temperature weight loss ratios of our BC samples both showed pyrolysis temperature of 400–500°C.
The BCs used in this experiment were either untreated or leached with water or acid. Leaching of the BCs to partly remove their alkalizing effect before use in the experiments was done on the size fraction ≤ 2 mm. For leaching, columns of 5 cm diameter and 30 cm length were filled with BC. The columns were fitted with tubing at the inlet and outlet and filter paper (0.45μm) was placed on both ends of the column. Biochars were first leached with demineralized water at a 1:50 (BC:water w/w) ratio with a flow rate of 70–80 ml hr-1 for 4 days to produce “water-leached” BC. After removing part of the BC from the column (water-leached), leaching continued with 0.05 M HCl at a 1:10 (BC:acid w/w) ratio with a flow rate of 20–30 ml hr-1 for 1 day to produce “acid-leached” BC. During the leaching, water and subsequently HCl were pumped through the vertical columns from the bottom upwards. Pumping stopped temporarily when leachate appeared on the top of the column and resumed after 2 days (in the case of water) or 1 day (in the case of HCl). A peristaltic pump was used to control the flow rate. Both water- and acid-leached BCs were oven-dried at 40°C for 3 days resulting to a moisture content of 13 and 6%w/w, respectively. Prior to mixing with the soil, the BCs (both untreated and leached) were ground and passed through a 0.5 mm sieve. Despite possible release of fresh materials after grinding of leached BCs to ≤ 0.5 mm, the pH measured in soil-leached BC slurries before incubation (Table 2) was lower than in slurries with untreated BC, hence satisfying the purpose of reducing or removing alkalizing effect of BC. Cations, anions and dissolved organic C removed by leaching with water and acid, respectively, can be found in S2 File.
Air-dried soils were saturated with water and equilibrated to 5 kPa suction in a sand box (Eijkelkamp Agrisearch Equipment, Giesbeek, The Netherlands) over a 5 days period. Controlled pre-wetting was done to accommodate for the flush of microbial activity commonly observed upon rewetting of dry soil . For the incubation assays, approx. 10 g sand box equilibrated soil was placed in 120 ml serum bottles together with a magnetic stirring bar. Treatments included BCs (untreated and leached) and uncharred cacao shell, the latter to assess the effect of the feedstock alone (in Lampung soil only), at doses of 0, 1, 2, 5 and 10% (dry weight basis). Weight losses during leaching were implicitly corrected for since the same weights of the treated chars were used. To investigate if the effect of BC on denitrification and its gaseous reaction products was merely a pH effect, another set of experiments was run with Lampung soil in which soil pH was manipulated by adding 0.35, 1.25 and 1.80 ml of 0.1M NaOH prior to anoxic incubation. Dose of NaOH was decided based on the alkalizing effect of BC, e.g. 1.8 ml 0.1M NaOH was equivalent to 10% untreated cacao shell BC in Lampung soil. All treatments were prepared in triplicate. In preparation of soil slurries, 30 ml of a 2 mM KNO3 solution were added to the bottles thereby providing ample NO3- for denitrification. After the amendment, the effective pH values in the soil slurries were measured by a pH meter (Orion 2 Star, Thermo Fisher Scientific, Fort Collins, CO, USA) after 0.5 hour of oxic stirring. Thereafter, bottles were tightly closed with rubber septa and aluminum crimp seals and flushed with He (99.999%, AGA Industrial Gasses, Oslo, Norway) by alternately evacuating and He-filling the bottles 5 times using an automated manifold. This was done under constant stirring to achieve close to fully anoxic conditions. Measurements of pH in the slurries were repeated at the end of the incubation. An oxic incubation was carried out independently to check for BC-induced toxicity or stimulation of microbial activity (measured as O2 consumption) (Figure A in S3 File).
Incubation and data collection
All incubations were carried out in a water bath at 20°C (which is within optimal range for microbial activities ) under constant stirring to maintain equilibrium of gases between the soil slurry and the bottle headspace. We used a robotized incubation system similar to that described by Molstad, Dörsch  to monitor the kinetics of O2 depletion, CO2 production and N-gas accumulation (NO, N2O, N2) during denitrification. The system consists of a water bath connected to a cryostat, placed under the robotic arm of an autosampler (Combi Pal, CTC, Switzerland). The water bath can accommodate up to 30 stirred bottles which are pierced repeatedly (here five-hourly) by the hypodermic needle of the autosampler which is connected to a peristaltic pump transporting the gas sample to a gas chromatograph equipped with various detectors and further to an NO-chemiluminescence analyzer. Details of the incubation system and gas analysis can be found in Description B in S1 File.
Rates of gas production and consumption were corrected for sampling loss and dilution as described by Molstad, Dörsch . Maximum induced denitrification rate was calculated as the slope of the steepest part of the accumulation curve given by the sum of all N-gas products. The N2O/(N2O+N2) product ratio was calculated as the area under the curve of N2O divided by the area under the curve of NO+N2O+N2 . As a cut off, the maximum accumulation of N2 was used, usually coinciding with the complete exhaustion of N-oxides in the bottles. In the instances where N-oxides were not exhausted, the accumulation curves were integrated over the entire experimental period. As a measure of NO net production in denitrification, maximum dissolved NO (nM) was calculated from headspace concentrations, using Henry’s law.
Statistical analysis was carried out using the R software . Progression of denitrification was inspected by plotting cumulative N-gas and CO2 production as well as depletion of residual O2 over time. Maximum induced denitrification rates for each of the amendment type across its doses were subjected to one-way ANOVA and mean values of the doses were separated using Tukey’s Test at 5% significance level to establish statistically significant differences between BC doses.
To identify the possible factors explaining the effect of the amendments on maximum induced denitrification rate, N2O/(N2O+N2) ratio and maximum NO production, analyses of covariance (ANCOVA) were carried out. Firstly, ANCOVA was used to assess the effect of different types of untreated BC and doses, which was then followed by inclusion of BC leaching (untreated, water-leached and acid-leached) and effective pH in the statistical model as explanatory variables. Secondly, ANCOVA was used to separate the effect of labile C and other factors in BC on rate, N2O/(N2O+N2) ratio and maximum NO production by comparing charred and uncharred cacao shell. Furthermore, the effect of labile C and pH increase after adding BC on rate, N2O/(N2O+N2) ratio and maximum NO production were separated by comparing uncharred cacao shell and NaOH treatments using ANCOVA. pH, being an important explanatory variable for BC effect on N2O/(N2O+N2) ratio and maximum NO production, its values at the beginning and end of incubation are also presented.
Effect of biochar on soil pH before and after anoxic incubation
The addition of BC increased the pH of both soils (Table 2). The dose-dependent pH rise was more pronounced in Mkushi soil than in Lampung soil, reflecting the weaker buffer capacity (lower CEC) of the former (Table 1). Biochar from cacao shell and rice husk differed vastly in alkalinity and thus in its alkalizing effect on soil. For instance, addition of 1% (w/w) cacao shell BC to Lampung soil increased the soil pH by 2.3 units, whereas adding the same amount of rice husk BC resulted in only 0.2 units pH increase. Carbonate contributed a large part to the alkalizing effect of cacao shell BC as shown by high CO2 concentrations immediately following mixing the BC with acid soils (Fig 1).
Shown are averages of three incubations; error bars denote SE. Approximately 6.1 μmol NO3--N g-1 was added to 9.8 g soil in the bottles. Note the differences in the scale of y-axis.
Water leaching removed 159 cmolc of base cations kg-1 (S2 File) from cacao shell BC and reduced its pH(H2O) from 9.8 to 9.6. Additional leaching with acid removed another 61 cmolc of base cations and reduced its pH(H2O) to 8.0. For rice husk BC, water leaching removed 15cmolc kg-1 base cations and reduced the pH from 8.4 to 8.2. Acid leaching removed an additional 19 cmolc kg-1 and effectively acidified the BC (pH 2.5). In terms of mass, leaching with water and acid removed materials of approx. 65 and 14 mg g-1, respectively, of cacao shell BC and 7 and 5 mg g-1, respectively, of rice husk BC, and increased the surface area of BC (Table 1). For both BCs, base cations, in particular K+, removed by sequential water and acid leaching exceeded ammonium acetate exchangeable amounts (Table 1). The leaching treatment removed a significant part of the alkalizing effect of both BCs in soil (Table 2) and it may have changed other properties of BC. The cacao shell feedstock increased soil pH only modestly compared to its BC, if applied at an equivalent dose of mass (Table 2).
Anoxic incubation of soil slurries caused an increase in soil pH from initial values between 4.0 and 9.8 to final values between 5.4 and 9.9 (Table 2). In control soils and acidic soil-BC slurries, the pH increased more strongly than in alkaline slurries. This increase in pH can be attributed to denitrification (an alkalizing process), continuous release of cations from the BCs and exchange reactions during stirring.
Kinetics of denitrification
Fig 1 and Figure B in S3 File show the kinetics of N-gas production and consumption together with the depletion of residual O2 (after He-flushing) and cumulative CO2 production (total inorganic carbon) in response to addition of untreated BC to Lampung and Mkushi soil, respectively. Controls (no BC addition) showed transient NO accumulation, instantaneous N2O net production and measurable N2 production after ~100 hours of incubation. Maximum NO accumulation was one order of magnitude greater in the Lampung soil (0.3–0.5 μM, Fig 1) than in Mkushi soil (0.05 μM, Figure B in S3 File).
Both BCs suppressed the net production of NO and N2O and increased N2 production, but cacao shell BC (Fig 1; lower panel) stimulated overall denitrification (measured as total N2 accumulation) more than rice husk BC (Fig 1; upper panel). With cacao shell BC doses > 2%, N2 production reached a plateau after slightly more than 100 hours incubation, indicating that all N-oxides were exhausted. In this case, cumulative N2 production roughly balanced the sum of initially present total soil N and added NO3-. Biochar also shortened the time needed to detect measurable N2 production (except in the 10% cacao shell BC addition to Mkushi soil), indicating earlier induction of N2O reductase (N2OR) activity in the presence of BC. In Lampung soil, the suppression of NO and N2O and stimulation of N2 as well as CO2 production was dose-dependent irrespective of BC type. In Mkushi soil, 2% cacao shell BC addition stimulated complete denitrification resulting in high production rates of N2 and practically eliminated N2O accumulation (Figure B in S3 File). However, with further increases in the dose of cacao shell BC, slurry pH increased up to pH 9 in this weakly buffered soil and maximum NO accumulation and N2 production decreased, indicating inhibition of denitrification at high pH. N2O suppression with concomitant increase in N2 production was also seen in the NaOH treatments of Lampung soil (Figure C in S3 File) and in incubation of BC in 2 mM KNO3 without soil (Figure D in S3 File). In contrast, uncharred cacao shell stimulated overall denitrification strongly, while suppression of N2O was small (Figure C in S3 File).
Water-leached rice husk BC caused only a modest decline in pH and resulted in denitrification kinetics similar to those with untreated BC in Lampung soil (compare Fig 1 and Figure E in S3 File). By contrast, addition of acid-leached rice husk BC reduced soil pH, but left the net production of N2O and overall N-gas largely unchanged when compared with the control soil (Figure F in S3 File). Unlike acid-leached rice husk BC, acid-leached cacao shell BC retained some of its N2O suppressing effect in Lampung soil (Figure F in S3 File) in line with its remaining alkalizing effect. However, the N2O suppressing effect of water or acid-leached cacao shell BC was non-linear with maximum suppression already reached at 2% BC. At higher doses of leached cacao shell BC, no further N2O suppression occurred and we observed biphasic kinetics in particular of NO accumulation showing two peaks during incubation (Figures E and F in S3 File).
Table 3 shows maximum induced denitrification rates for Lampung and Mkushi soil amended with rice husk and cacao shell BC, uncharred cacao shell and NaOH. In Lampung soil, addition of more than 2% untreated cacao shell BC significantly increased denitrification rates compared to the control (P˂0.05), whereas rice husk BC did not. Water- and acid-leaching of the cacao shell BC removed most of the stimulating effect. Higher doses of acid-leached rice husk BC caused a small but significant decrease in denitrification rate in Lampung soil (P˂0.05). In Mkushi soil, only 2% untreated cacao shell BC stimulated denitrification whereas leached BC did not. This contrasts findings from aerobic incubations, which showed clear stimulation of respiration by all doses of untreated BCs in both soils (Figure A in S3 File). NaOH also stimulated denitrification (Table 3) but to a much lesser extent compared to untreated cacao shell BC and uncharred cacao shell despite similar increases in soil pH (Table 2).
Possible factors contributing to the BC effect on net N2O and NO production and denitrification rate
Linear model ANCOVA showed differences in the response of denitrification product ratio (N2O/(N2O+N2)), maximum NO accumulation and denitrification rate to BC type (rice husk or cacao shell) and dose, total C content (at onset of the experiment) and pH of the slurry (Table 4). In particular, BC type was a very important factor (p = 0.000). Doses were also important (p = 0.000 for denitrification product ratio and maximum NO accumulation; p = 0.01 for denitrification rate). Upon incorporation of BC leaching (untreated, water- and acid-leached BC) and pH as factors in addition to BC type and doses in the analysis, N2O/(N2O+N2) ratio, maximum NO accumulation and denitrification rates were significantly affected by all the factors at p = 0.000 (except the effect of BC dose on denitrification rate, which was at p = 0.003). Several interaction terms between factors were also significant (p<0.05).
ANCOVA also showed that total organic C (either as cacao shell or as its BC) added to the system was important in determining denitrification rate (p = 0.006) and maximum NO accumulation (p = 0.000) but not N2O/(N2O+N2) ratio (p = 0.41). In addition, a comparison of treatments with uncharred cacao shell, providing significant amounts of labile C, and NaOH, without addition of labile C, showed the strong influence of labile C on denitrification rate (p = 0.000) but not on N2O/(N2O+N2) ratio (p = 0.06). In this comparison, pH significantly affected both denitrification rate and N2O/(N2O+N2) (p = 0.000).
NO accumulation and N2O/(N2O+N2) product ratios
Increasing doses of both untreated rice husk and cacao shell BC, as well as NaOH, caused maximum NO accumulation to decrease (Fig 2 upper panel). Corresponding doses of leached BC reduced suppression of maximum NO accumulation. Acid leaching of rice husk BC entirely eliminated the suppression of NO accumulation. Uncharred cacao shell had weaker effect on suppression of NO accumulation than corresponding doses of cacao shell BC whether leached or not. Maximum NO accumulation decreased asymptotically with increasing pH to trace levels at pH > 6.5 (Fig 2 lower panel). The NO accumulation rate was greatest at the beginning of the incubation reaching maximum values within 72 hours (Fig 1 and Figures C, E and F in S3 File), except in Mkushi soil with > 5% cacao shell BC (Figure B in S3 File). Here NO accumulation gradually increased throughout the incubation period.
The N2O/(N2O+N2) product ratio decreased with increasing doses of untreated BC (Fig 3 upper panel). Rice husk BC addition to Lampung soil resulted in a decrease of the N2O/(N2O+N2) ratio with increase in dose, reaching values below 0.1 at 10% BC addition (Fig 3A1). Adding the same amounts of cacao shell BC to Lampung soil suppressed the denitrification product ratio much more strongly; reaching low product ratios already with 1% addition and increasing the doses did not have additional benefit in suppressing N2O. Cacao shell BC with its strong alkalizing effect was more effective in suppressing N2O than its feedstock at equivalent doses of mass (Fig 3B1). Thus, the strong effect of cacao shell BC compared to that of rice husk BC on the N2O product ratio could be linked to its strong alkalizing effect, resulting in greater soil pH increase at equivalent doses (Table 2, Fig 3). Due to its strong alkalizing effect, no N2O/(N2O+N2) data are available for cacao shell BC-amended Lampung soils in the pH range 4.8–6.6 (Fig 3B2). Therefore, our data do not allow a direct comparison of pH-related effects of the two BCs. In Mkushi soil, the N2O/(N2O+N2) ratio was reduced to zero even at the lowest dose (here 2%, which increased soil pH to 8.3; Fig 3C). A 10% cacao shell BC addition to Mkushi caused high, but uncertain values of product ratio probably due to suppression of overall denitrification activity (Figure B in S3 File). Thus, our data for BC-amended soils indicate that the N2O/(N2O+N2) ratio decreased from close to 1 at pH < 4 (no induction of N2OR activity) to close to zero at pH > 6 (sufficient induction of N2OR to prevent significant net production of N2O).
Addition of NaOH also decreased net N2O production (Fig 3D). In the pH range 4 to 7, the relationship between pH and N2O/(N2O+N2) product ratio had a significantly smaller slope for NaOH-amended- than for BC-amended soils but similar to that of uncharred cacao shell (Table 5).
Applying water-leached rice husk BC to Lampung soil resulted in a similar relationship between N2O/(N2O+N2) ratio and dose (or pH) as observed in soils with untreated rice husk BC (Fig 3A). Addition of acid-leached rice husk BC, which had lost all its alkalizing effect, resulted in large N2O/(N2O+N2) product ratios independent of BC dose (Fig 3A). Water and acid leached cacao shell BC decreased the N2O/(N2O+N2) ratio at low dose (2%), albeit less than untreated BC. At higher doses of leached BC, the ratios were relatively high compared to those of in response to untreated cacao shell BC additions at similar doses (Fig 3B).
N2O reduction to N2, which requires functional N2OR, only occurred after dissolved NO concentrations decreased to values ≤ 100 nM (Fig 1). In addition, N2O reduction only occurred at soil pH ≥ 5. At pH ≥ 5, e.g. after the amendment of rice husk BC at 5%, N2OR activity started immediately at the beginning of incubation (Fig 1). For soils or soil-BC mixtures with initial pH < 5 (e.g. treatments with 1–2% rice husk BC), denitrification driven alkalization, increasing pH to ~5 had to take place before induction of N2OR activity was observed. Initial delay in N2OR activity caused high accumulation of N2O in acidic soil or soil-BC mixtures.
Effect of biochar on NO, N2O and N2 production and denitrification rate
Addition of untreated BCs to the two acidic Acrisols in this study suppressed the net production of both NO and N2O during anoxic incubation (Fig 1), which is in line with previously reported studies ( and references therein). Here we show that this suppression went along with increase in N2 production, suggesting increase in the activity of N2OR  due to alkalization .
Leaching of the BCs except for water-leached rice husk BC reduced or eliminated the effect of NO and N2O suppression (Figs 2 and 3), indicating that some of the BC constituents removed by leaching (S2 File) contributed to the suppression. Base cations and carbonates (shown by the high amount of CO2 released upon mixing of acidic soil with BC—Fig 1) were the major constituents removed by leaching, thus causing a decrease in alkalizing effect. Suppression of NO and N2O production in response to the addition of NaOH indicated that pH is an important factor contributing to the suppression. A recent study reported loss of alkalizing effect together with a loss in N2O suppression due to field aging of BC , suggesting that N2O suppression by BC might be a transient effect connected to the transiency of its alkalizing effect.
The N2O/(N2O+N2) product ratio decreased when the initial soil pH increased from pH 4 to 6 in response to the addition of BC (Fig 3). The rise in pH through addition of BC or NaOH removed the impairment of N2OR, typically seen at low pH [29, 33, 49]. The relief of this impairment through pH increase is similar to what has been reported for denitrifying pure cultures and for soils from long-term liming experiments in which raised pH stimulated N2OR and reduced N2O production or emissions [28, 29, 31, 33]. This direct effect of pH was attributed to a threshold pH above which functional N2OR is assembled [29, 31]. In this study, we found a threshold of pH ≈ 5 for the induction of N2OR based on the timing of N2 production onset (Fig 1), amount of accumulated denitrification intermediates (Fig 1) and pH at the beginning and end of incubation (Table 2). This threshold pH is close to threshold pH values for N2OR induction around pH 6, observed through detection of measurable N2 in earlier anoxic studies [29, 32]. The greater decrease of the N2O/(N2O+N2) ratio with increasing pH in rice husk BC-amended soil compared to that of previously published data [29, 32] and results from the NaOH-amended soil (Fig 3D and Table 5) suggest that BC has a somewhat stronger effect on the suppression of N2O than explained by pH alone. However, the effectiveness of N2O suppression seems to depend on the timing of induction of N2OR, which is controlled by the alkalizing effect of BC. Denitrification-driven alkalization contributed to induction of N2OR if the threshold pH for N2OR induction was not achieved by the BC alkalizing effect alone. Recently, Harter, Krause  reported an increased relative abundance of nosZ genes encoding for N2OR during 80 days of incubation after BC addition to soil, which is in line with the increased activity of N2OR observed in this study.
Only few recent studies have reported BC effects on NO production. Recently, Nelissen, Saha  reported a decrease in NO production similar to this study. The driver behind NO suppression by BC appears to be similar to that underlying N2O suppression because the two gases decreased with increasing doses of untreated BCs in a similar fashion (Fig 1). The concentration of the two gases increased initially and reached a peak before decreasing, although in all cases, NO reached the peak earlier than N2O. Low NO concentrations in BC- or NaOH-amended soils (Fig 1 and Figure C in S3 File) were likely due to the pH-increasing effect (Table 4), which prevents chemical decomposition of NO2- to NO [51, 52], leaving only enzymatically produced NO to accumulate. Higher NO production in Lampung compared to Mkushi soil was probably due to higher microbial activities producing nitrite, part of which was decomposed chemically to NO at low pH. Our data also suggest that induction of N2OR is linked to low NO concentration, as N2OR activity was not initiated before NO concentration dropped to values below 100 nM. NO has been proposed to play an important role in the regulation of denitrification enzyme regulation , but little is known how reactive gaseous N species like NO react with BC.
In general, both aerobic and anaerobic respiration were stimulated by BC addition to soil (Fig 1 and Figure A in S3 File). Suppression of anaerobic respiration was only found at high doses of cacao shell BC added to Mkushi soil resulting in soil pH values > 9 (Figure B in S3 File). Anoxic incubation of untreated BC in 2 mM KNO3 solution without soil revealed that BC themselves carried out some denitrification activity which was expressed when residual O2 was fully exhausted (Figure D in S3 File). Interestingly, no N2O accumulated, suggesting full N2OR induction at high pH. Denitrification activity was clearly greater with rice husk (pH 8.4) than cacao shell BC (pH 9.8). This might reflect the inability of the denitrifier community to thrive when too much BC is added driving soil pH to high values at which NO2- may accumulate to toxic levels . Additionally, the osmotic effect of salts due to high dose (10%) BC in poorly buffered Mkushi soil may have inhibited microbial activity. Other than at high dose, our BC did not have any direct inhibitory effect on microbial activities such as shown for BC-mediated ethylene production .
BC is a complex material, which may alter many soil variables besides pH. In particular, BC increased bioavailable carbon (C) (Figure D in S3 File; residual O2 was consumed and CO2 was produced during incubation of BC without soil) [55, 56] and nutrients (S2 File) which could stimulate microbial growth  and affect the regulation of denitrification. Addition of bioavailable C clearly affected denitrification rate as seen after adding uncharred cacao shell (Tables 3 and 4), but it did not affect the product ratio (Table 4). The decrease in product ratio with increase in BC dose applied was better explained by pH increase than by C-addition in our ANCOVA. The contribution of bioavailable organic C and/or nutrients of cacao shell BC to increased denitrification rates is clearly seen when comparing cacao shell BC treatments with NaOH treatments at similar soil pH.
Leaching of BC, which mimics field aging, affected both its alkalinity and surface chemistry (Table 1 and S2 File). Changes to BC surface chemistry may occur through alterations of surface functional organic groups. The leaching experiments showed that certain BC types such as cacao shell BC may be more resistant to aging presumably through release of base cations and secondary carbonation, which would explain the relatively minor effect of acid leaching on cacao shell BC’s alkalinizing effect (Table 1 and S2 File). Denitrification experiments with leached cacao shell BC did not show ordinary dose response. Instead, higher doses of leached cacao shell BC resulted in conspicuous biphasic NO kinetics with two peaks in Lampung soil, a delayed peak of N2O production as well as delayed production of N2 by either enzymatic or chemical pathways (Figures E and F in S3 File) . This went along with higher N2O/(N2O+N2) ratios at high doses as compared with untreated BC (Fig 3B). This may point at some chemical interaction of newly exposed BC surfaces with denitrification intermediates. Initially, leached cacao shell BC may have acted as electron sink [11, 18], competing with denitrification reductases for electrons. However, there was no indication of chemical reaction such as sorption and desorption between BC and N-compounds in an anoxic incubation of BC (untreated or leached) without soil (Figure D in S3 File).
Factors determining NO and N2O suppression by biochar
In this study, we found that the pH effect of BC in acid soil played a major role in the suppression of both NO and N2O under anoxic conditions. However, any extrapolation of our data beyond acidic soils needs to be done with caution. Cayuela, Sánchez-Monedero  also observed reduced N2O/(N2O+N2) ratios during N2O peak emission in wet soils amended with brush BC but a direct pH effect was not clearly captured probably because of the small pH increase (0.1 pH units). Instead, Cayuela, Sánchez-Monedero  could show that the observed reduction in N2O/(N2O+N2) ratios were positively correlated to the buffer capacities of the added BC. Earlier, Yanai, Toyota  had concluded that suppression of N2O emissions (which they believed originated from denitrification) by BC was not the result of changes in soil chemical properties. Cayuela, Sánchez-Monedero  and the present study clearly show that BC can affect the soil chemical properties with consequences for the product stoichiometry of denitrification. In this study, we used controlled anoxia with direct quantification of N2 production to study the effect of BC on denitrification stoichiometry. Yanai, Toyota  did not separate N cycling processes and their study could have been confounded by nitrification, an acidifying process, as suggested by the decrease in pH at the end of their incubations. We did not account for dissimilatory nitrate reduction to ammonium (DNRA) in this study; however, it is unlikely that this process played a major role as we recovered the added nitrate quantitatively as N2.
The steeper slopes of N2O/(N2O+N2) versus pH in BC treatments compared to NaOH and uncharred cacao shell treatments (Table 5) indicate that some other factors may have contributed to the suppression of N2O in addition to the pH effect. The similarity of the slopes for uncharred cacao shell and NaOH suggests that stronger suppression of N2O by BC was not due to cacao shell itself or to labile C but to some other BC property. Biochar redox behavior (electron shuttling), where the electron-conductance of BC serves as a catalyst in denitrification as suggested by Cayuela, Sánchez-Monedero  could be one of these factors. The reduction or elimination of BC suppression of N2O after leaching of BC in this study raises questions about how leaching affects electron shuttling and how important electron shuttling is, in suppressing N2O.
This study is the first of its kind assessing BC effects under full denitrification conditions, simultaneously quantifying NO, N2O and N2 production at high temporal resolution. We found compelling evidence that BC strongly suppresses relative NO and N2O net production from denitrification in two acid soils, resulting in a reduced propensity for NO and N2O emissions. Increase of soil pH by BC addition was identified as a major factor mediating this suppression. NO suppression was linked to less chemical decomposition of NO2- to NO due to pH increase. N2O suppression on the other hand was in accordance with the notion that raising pH in acid soils greatly stimulates N2OR activity resulting in more complete denitrification with N2 as the dominating end product. Other factor(s) contributing causally to the observed increase in N2OR activity cannot be excluded and need further testing.
S1 File. Description of biochar production, incubation system operation and gas chromatograph detectors.
Biochar production (Description A). Incubation system operation and gas chromatograph detectors (Description B).
S2 File. Constituents removed from BC through leaching.
Constituents removed from BC through leaching with water and strong acid (HCl) (Table A).
S3 File. Mean oxygen consumption during oxic incubations and kinetics of gas production (N2, N2O, NO, CO2) and consumption (O2) during anoxic incubations.
Mean oxygen consumption in BC amended soils during oxic incubations (Figure A). Denitrification kinetics and CO2 and O2 concentrations in incubations of Mkushi soil amended with untreated cacao shell BC (Figure B). Denitrification kinetics and CO2 and O2 concentrations in incubations of Lampung soil amended with uncharred cacao shell (upper 2 panels) and 0.1M NaOH (lower 2 panels) (Figure C). Denitrification kinetics and CO2 and O2 concentrations in anoxic incubations of 2.36 g BC without soil in 30 ml 2mM KNO3 (Figure D). Denitrification kinetics and CO2 and O2 concentrations in incubations of Lampung soil amended with water-leached rice husk BC (upper 2 panels) and cacao shell BC (lower 2 panels) (Figure E). Denitrification kinetics and CO2 and O2 concentrations in incubations of Lampung soil amended with acid-leached rice husk BC (upper 2 panels) and cacao shell BC (lower 2 panels) (Figure F).
We thank Dr. Sarah Hale, NGI, for the support during leaching of the BCs and Dr. Shahid Nadeem, NMBU, for his technical support during the incubation experiments. David Rutherford (US Geological Survey, Denver, CO, USA) is gratefully acknowledged for performing the TGA analyses. We also thank Jeremy Selby at Mkushi, Zambia and the Indonesian Soil Research Institute (ISRI) at Bogor, Indonesia for making soil samples available for the project.
Conceived and designed the experiments: AO GC JM PD. Performed the experiments: AO PD. Analyzed the data: AO PD. Contributed reagents/materials/analysis tools: AO GC JM PD. Wrote the paper: AO GC JM PD.
- 1. Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol R. 1997;61(4):533–616.
- 2. Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, Schindler DW, et al. Human alteration of the global nitrogen cycle: Sources and consequences. Ecol Appl. 1997;7(3):737–750.
- 3. Ravishankara AR, Daniel JS, Portmann RW. Nitrous Oxide (N2O): The Dominant Ozone-Depleting Substance Emitted in the 21st Century. Science. 2009;326(5949):123–125. pmid:19713491
- 4. IPCC. Climate change 2007: The physical science basis. Agenda. 2007;6(07):333.
- 5. Reay DS, Davidson EA, Smith KA, Smith P, Melillo JM, Dentener F, et al. Global agriculture and nitrous oxide emissions. Nature Clim Change. 2012;2(6):410–416.
- 6. Montzka SA, Dlugokencky EJ, Butler JH. Non-CO2 greenhouse gases and climate change. Nature. 2011;476(7358):43–50. pmid:21814274
- 7. Spokas KA, Koskinen WC, Baker JM, Reicosky DC. Impacts of woodchip biochar additions on greenhouse gas production and sorption/degradation of two herbicides in a Minnesota soil. Chemosphere. 2009;77(4):574–581. pmid:19647284
- 8. Lehmann J. A handful of carbon. Nature. 2007;447.
- 9. Lehmann J, Gaunt J, Rondon M. Bio-char Sequestration in Terrestrial Ecosystems—A Review. Mitig Adapt Strategies Glob Chang. 2006;11(2):395–419.
- 10. Lehmann J. Bio-energy in the black. Front Ecol Environ. 2007;5(7):381–7.
- 11. Cayuela ML, van Zwieten L, Singh BP, Jeffery S, Roig A, Sánchez-Monedero MA. Biochar's role in mitigating soil nitrous oxide emissions: A review and meta-analysis. Agr Ecosyst Environ. 2014;191:5–16.
- 12. Zhang A, Cui L, Pan G, Li L, Hussain Q, Zhang X, et al. Effect of biochar amendment on yield and methane and nitrous oxide emissions from a rice paddy from Tai Lake plain, China. Agr Ecosyst Environ. 2010;139(4):469–475.
- 13. Cornelissen G, Martinsen V, Shitumbanuma V, Alling V, Breedveld G, Rutherford D, et al. Biochar Effect on Maize Yield and Soil Characteristics in Five Conservation Farming Sites in Zambia. Agronomy. 2013;3(2):256–274.
- 14. Jeffery S, Verheijen FGA, van der Velde M, Bastos AC. A quantitative review of the effects of biochar application to soils on crop productivity using meta-analysis. Agr Ecosyst Environ. 2011;144(1):175–187.
- 15. Clough TJ, Bertram JE, Ray JL, Condron LM, O'Callaghan M, Sherlock RR, et al. Unweathered Wood Biochar Impact on Nitrous Oxide Emissions from a Bovine-Urine-Amended Pasture Soil. Soil Sci Soc Am J. 2010;74(3):852–860.
- 16. Singh BP, Hatton BJ, Singh B, Cowie AL, Kathuria A. Influence of Biochars on Nitrous Oxide Emission and Nitrogen Leaching from Two Contrasting Soils. J Environ Qual. 2010;39(4):1224–1235. pmid:20830910
- 17. Cayuela ML, Sánchez-Monedero MA, Roig A, Hanley K, Enders A, Lehmann J. Biochar and denitrification in soils: when, how much and why does biochar reduce N2O emissions? Sci Rep. 2013;3:1732. pmid:23615819
- 18. Joseph S, Camps-Arbestain M, Lin Y, Munroe P, Chia C, Hook J, et al. An investigation into the reactions of biochar in soil. Soil Res. 2010;48(7):501–515.
- 19. Cornelissen G, Rutherford DW, Arp HP, Dörsch P, Kelly CN, Rostad CE. Sorption of pure N2O to biochars and other organic and inorganic materials under anhydrous conditions. Environ Sci Technol. 2013;47(14):7704–7712. pmid:23758057
- 20. Yanai Y, Toyota K, Okazaki M. Effects of charcoal addition on N2O emissions from soil resulting from rewetting air-dried soil in short-term laboratory experiments. Soil Sci Plant Nutr. 2007;53(2):181–188.
- 21. Clough TJ, Condron LM. Biochar and the Nitrogen Cycle: Introduction. J Environ Qual. 2010;39(4):1218–1223. pmid:20830909
- 22. Clough T, Condron L, Kammann C, Müller C. A Review of Biochar and Soil Nitrogen Dynamics. Agronomy. 2013;3(2):275–293.
- 23. Spokas KA, Baker JM, Reicosky DC. Ethylene: potential key for biochar amendment impacts. Plant Soil. 2010;333(1–2):443–452.
- 24. Bruun EW, Ambus P, Egsgaard H, Hauggaard-Nielsen H. Effects of slow and fast pyrolysis biochar on soil C and N turnover dynamics. Soil Biol Biochem. 2012;46(0):73–79.
- 25. Yuan J-H, Xu R-K. Effects of biochars generated from crop residues on chemical properties of acid soils from tropical and subtropical China. Soil Res. 2012;50(7):570–578. http://dx.doi.org/10.1071/SR12118.
- 26. Biederman LA, Harpole WS. Biochar and its effects on plant productivity and nutrient cycling: a meta-analysis. GCB Bioenergy. 2013;5(2):202–214.
- 27. Verheijen FGA, Jeffery S, Bastos AC, van der Velde M, Diafas I. Biochar Application to Soils: A Critical Scientific Review of Effects on Soil Properties, Processes and Functions. EUR 24099 EN, Office for the Official Publications of the European Communities, Luxembourg, 149pp. 2009.
- 28. Bergaust L, Mao Y, Bakken LR, Frostegard A. Denitrification response patterns during the transition to anoxic respiration and posttranscriptional effects of suboptimal pH on nitrous oxide reductase in Paracoccus denitrificans. Appl Environ Microb. 2010;76(19):6387–6396.
- 29. Liu B, Morkved PT, Frostegard A, Bakken LR. Denitrification gene pools, transcription and kinetics of NO, N2O and N2 production as affected by soil pH. FEMS Microbiol Ecol. 2010;72(3):407–417. pmid:20370831
- 30. Dörsch P, Braker G, Bakken LR. Community-specific pH response of denitrification: experiments with cells extracted from organic soils. FEMS Microbiol Ecol. 2012;79(2):530–541. pmid:22093000
- 31. Liu B, Frostegard A, Bakken LR. Impaired reduction of N2O to N2 in acid soils is due to a posttranscriptional interference with the expression of nosZ. mBio. 2014;5(3):e01383–14. pmid:24961695
- 32. Qu Z, Wang J, Almøy T, Bakken LR. Excessive use of nitrogen in Chinese agriculture results in high N2O/(N2O+N2) product ratio of denitrification, primarily due to acidification of the soils. Glob Change Biol. 2014;20(5):1685–1698.
- 33. Raut N, Dörsch P, Sitaula BK, Bakken LR. Soil acidification by intensified crop production in South Asia results in higher N2O/(N2 + N2O) product ratios of denitrification. Soil Biol Biochem. 2012;55:104–112.
- 34. Bakken LR, Bergaust L, Liu B, Frostegard A. Regulation of denitrification at the cellular level: a clue to the understanding of N2O emissions from soils. Philos T Roy Soc B. 2012;367(1593):1226–1234.
- 35. Nadeem S, Dörsch P, Bakken LR. Autoxidation and acetylene-accelerated oxidation of NO in a 2-phase system: Implications for the expression of denitrification in ex situ experiments. Soil Biol Biochem. 2013;57:606–614.
- 36. Nadeem S, Dörsch P, Bakken LR. The significance of early accumulation of nanomolar concentrations of NO as an inducer of denitrification. FEMS Microbiol Ecol. 2013;83(3):672–684. pmid:23035849
- 37. Nelissen V, Saha BK, Ruysschaert G, Boeckx P. Effect of different biochar and fertilizer types on N2O and NO emissions. Soil Biol Biochem. 2014;70:244–255.
- 38. Molstad L, Dörsch P, Bakken LR. Robotized incubation system for monitoring gases (O2, NO, N2O N2) in denitrifying cultures. J Microbiol Meth. 2007;71(3):202–211.
- 39. Wang R, Feng Q, Liao T, Zheng X, Butterbach-Bahl K, Zhang W, et al. Effects of nitrate concentration on the denitrification potential of a calcic cambisol and its fractions of N2, N2O and NO. Plant Soil. 2012;363(1–2):175–189.
- 40. Smebye A. The effect of biochar on dissolved organic matter in soil. Master Thesis. University of Oslo. 2014. Available: https://www.duo.uio.no/handle/10852/42266?show=full
- 41. Martinsen V, Alling V, Nurida NL, Mulder J, Hale SE, Ritz C, et al. pH effects of the addition of three biochars to acidic Indonesian mineral soils. Soil Sci Plant Nutr. 2015:1–14.
- 42. Smebye A, Alling V, Vogt RD, Gadmar TC, Mulder J, Cornelissen G, et al. Biochar amendment to soil changes dissolved organic matter content and composition. Chemosphere. 2015.
- 43. Hale SE, Alling V, Martinsen V, Mulder J, Breedveld GD, Cornelissen G. The sorption and desorption of phosphate-P, ammonium-N and nitrate-N in cacao shell and corn cob biochars. Chemosphere. 2013;91(11):1612–1619. pmid:23369636
- 44. Kieft TL, Soroker E, Firestone MK. Microbial biomass response to a rapid increase in water potential when dry soil is wetted. Soil Biol Biochem. 1987;19(2):119–126.
- 45. Saad OALO, Conrad R. Temperature dependence of nitrification, denitrification, and turnover of nitric oxide in different soils. Biol Fert Soils. 1993;15(1):21–27.
- 46. R Core Team. R: A language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2014.
- 47. Holtan-Hartwig L, Dörsch P, Bakken LR. Comparison of denitrifying communities in organic soils: kinetics of NO3- and N2O reduction. Soil Biol Biochem. 2000;32(6):833–843.
- 48. Spokas KA. Impact of biochar field aging on laboratory greenhouse gas production potentials. GCB Bioenergy. 2013;5(2):165–176.
- 49. Simek M, Cooper JE. The influence of soil pH on denitrification: progress towards the understanding of this interaction over the last 50 years. Eur J Soil Sci. 2002;53:345–354.
- 50. Harter J, Krause HM, Schuettler S, Ruser R, Fromme M, Scholten T, et al. Linking N2O emissions from biochar-amended soil to the structure and function of the N-cycling microbial community. ISME J. 2013:1–15.
- 51. Islam A, Chen D, White RE, Weatherley AJ. Chemical decomposition and fixation of nitrite in acidic pasture soils and implications for measurement of nitrification. Soil Biol Biochem. 2008;40(1):262–265.
- 52. Braida W, Ong SK. Decomposition of nitrite under various pH and aeration conditions. Water Air Soil Poll. 2000;118(1–2):13–26.
- 53. Spiro S. Regulators of bacterial responses to nitric oxide. FEMS Microbiol Rev. 2007;31(2):193–211. pmid:17313521
- 54. Bollag J-M, Henninger NM. Effects of nitrite toxicity on soil bacteria under aerobic and anaerobic conditions. Soil Biol Biochem. 1978;10(5):377–381.
- 55. Bruun EW, Hauggaard-Nielsen H, Ibrahim N, Egsgaard H, Ambus P, Jensen PA, et al. Influence of fast pyrolysis temperature on biochar labile fraction and short-term carbon loss in a loamy soil. Biomass Bioenergy. 2011;35(3):1182–1189.
- 56. Luo Y, Durenkamp M, De Nobili M, Lin Q, Brookes PC. Short term soil priming effects and the mineralisation of biochar following its incorporation to soils of different pH. Soil Biol Biochem. 2011;43(11):2304–2314.
- 57. Dail DB, Davidson EA, Chorover J. Rapid abiotic transformation of nitrate in an acid forest soil. Biogeochemistry. 2001;54(2):131–146.