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Biochar insights from laboratory incubations monitoring O2 consumption and CO2 production

  • Risely Ferraz AlmeidaEmail author
  • Kurt A. Spokas
  • Daniel de Bortoli Teixeira
  • Newton La Scala Júnior
Original Article


Biochar has been touted as a long-term carbon sequestration tool. However, there are no studies evaluating biochar’s effect on oxygen (O2) consumption as a measure of the microbial respiration response to biochar. To gain insight into this aspect, we evaluated O2 consumption rates to test the hypothesis that biochar is an efficient agent for carbon dioxide (CO2) sequestration in soils. Four different biochar types and one activated charcoal were incubated alone and associated with three different soils for approximately 2 months in laboratory incubations. Headspace concentration of CO2 and O2 was periodically quantified. The data presented here confirm that the CO2 production following biochar’s addition to soils results in a process that is correlated to oxygen consumption. However, this overall stimulation is not clearly related to biochar type. Activated carbon resulted in the highest statistically significant stimulation of activity, despite it possessing the lowest quantity of volatile carbon and mineral nutrient sources. Taking into consideration our results, we conclude that using biochar does achieve total carbon sequestration. However, the amount of available soil organic carbon following soil incorporation appears to be reduced following biochar addition and its long-term implication on this mineralizable soil organic carbon pool does deserve more research attention.


Biochar activation Charcoal CO2 sequestration CO2 sorption 

1 Introduction

The carbon (C) stock in the soil is the net balance between C inputs into soil and the outputs from the soil [i.e., carbon dioxide (CO2) respiration; dissolved CO2 leaching; particulate C transport] (Davidson and Janssens 2006). This balance is influenced by both the quality and quantity of the C inputs and magnitudes of the outputs (mainly carbon dioxide emission—CO2) (Fearnside and Barbosa 1998). Thereby, the terrestrial biosphere can be a C source or a sink (Guo and Gifford 2002), depending on whether the inputs or outputs are greater. This balance can also vary temporally.

Soil CO2 emission directly results from plant roots and microorganism respiration by biochemical processes (Melillo et al. 2002; Davidson and Janssens 2006), consuming oxygen (O2) and producing CO2 emissions through organic matter decomposition (Chen et al. 2011). Soil temperature, moisture, microbial abundance, and substrate quality and availability are major properties that control soil CO2 emission (Almeida et al. 2015). The net CO2 emission from the soil is also controlled by overall soil use and tillage management decisions that also directly influence the processes of CO2 sequestration and emission (i.e., Figueroa et al. 2008; Huang et al. 2012; ESRL 2016). According to FAO (2010), the adoption of agricultural practices that sequester C is one means to mitigate climate change.

One such methodology of sequestering C is the use of biochar that presents the ability to sequester atmospheric CO2 in a more stable form (Lehmann 2007). Biochar has received increasing attention due to its high C content, cation exchange capacity, large specific surface area and stable chemical structure (Wang and Wang 2019). The properties of biochar are a function of the original biomass materials used (e.g., animal manures, lignocellulosic feedstocks) (Novak et al. 2013), production conditions (e.g., heating rates, pyrolysis systems) (Laird et al. 2009), and any post-production weathering (i.e., surface, oxidation, cooling conditions) (Spokas and Reicosky 2009; Spokas 2013). Thereby, this variability in biochar chemistry hampers our ability to extract mechanistic level information across different studies.

There does appear to be the possibility of similar behavior across different biochar types. For instance, biochar as a short-term mitigation tool to reduce nitrous oxide and ammonia emissions has been more consistently reported, for example, by Fidel et al. (2019) and Fungo et al. (2019). However, biochar’s effect on CO2 emission is less consistent. Increase in CO2 emission from soil amended by biochar (bamboo, rice husk, and sawdust biochar) was shown by Oo et al. (2018). In contrast, Fidel et al. (2019) demonstrated no effect on CO2 emission using a mixture of hardwood and softwood biochars (primarily Quercus, Ulmus, and Carya spp. woodchips). Interestingly, there were no studies available that evaluated biochar’s effect on O2 consumption as an additional means to assess the microbial response. Almeida et al. (2018) and Kyaw Tha Paw et al. (2006) noticed a negative relation between CO2 production and O2 consumption from biological processes in soil. However, other factors can influence the balance of CO2 and O2 in the soil. For example, desorption of CO2 from the solid phase (Smagin et al. 2016), moisture content (Linn and Doran 1984), as well as soil temperature (Angert et al. 2015), all impact the relationship between O2 consumption and CO2 production. To gain insight into biochar’s effect on O2 consumption as a surrogate for microbial activity, we evaluated the use of O2 consumption and CO2 production rates to produce an improved insight into the effect of biochar on the soil microbial population.

2 Materials and methods

2.1 Soils and biochar sampling

Laboratory incubation experiments were performed at the University of Minnesota (St. Paul, MN). The experiment was carried out using three soil types: agricultural soil (AG; Rosemount, MN); commercial potting soil (PS; Sunshine); and urban soil (US; St. Paul, MN) (Table 1). There were five biochars evaluated: pine chip biochar—B1; oak hardwood lump charcoal—B2; slow pyrolysis bamboo biochar—B3; fast pyrolysis macadamia nut biochar—B4, as well as an activated coconut shell charcoal (Accurel)—AC (Table 2). Control incubations were also conducted without biochar addition. Both soil and biochar characteristics are summarized in Tables 1 and 2, respectively. The AG (Waukegan silt loam, collected in Rosemount, MN) and UM soil (Chetek sandy loam, collected in St. Paul, MN) were collected (at 0–5 cm) and air dried until the start of the incubations. A synthetic potting soil mixture (PS; Brand #1; Sun Gro Horticulture Distribution Inc, Agawam, MA) was also used in this experiment.
Table 1

Properties of the soils used in this experiment


Soil series


OM (%)


Commercial potting soil




Agricultural soil




Urban soil



pH in H2O

OM organic matter

Table 2

Properties of biochars [pine chip biochar—B1; hardwood lump charcoal—royal oak—B2; slow pyrolysis bamboo-based biochar—B3; fast pyrolysis macadamia nut biochar—B4, as well as one activated charcoal (Accurel)—AC] used in this experiment


Tempa (°C)

Timeb (h)











% dry weight basis




50:50 of Pinus ponderosa: Pinus banksiana













Equal proportions of: Quercus robur; Acer saccharum; Fraxinus americana













Mixed source of Phyllostachys aureosulcata; Phyllostachys rubromarginata; Phyllostachys bissetii; Phyllostachys aurea












< 0.2

Macadamia integrifolia (nut shell)













Cocos nucifera (nut shell)c










C carbon, N nitrogen, O oxygen, H hydrogen, S sulphur, VM volatile matter

aPyrolysis temperature (°C)

bPyrolysis time (hour and minutes)

cActivation process: post-production 1100 °C with steam

2.2 Soils and biochar incubations

The first experimental design was set up using a completely randomized block to assess the impact of the biochar solely. Four replicates of 1 g of biochar along with 0.5 mL of water were placed into 125 mL serum bottles (125 mL), which were then sealed with butyl rubber septa. A set of blank control incubation (no additions) was also conducted to assess if there was any sorption of oxygen or carbon dioxide resulting from the incubation setup. No alteration in the headspace concentration of CO2 and O2 was detected during the 60-day incubation (data not shown). The serum vials (total of 20 vials total) were incubated in a laboratory environment with a controlled temperature of 25 °C for 22-day incubation. The CO2 and O2 concentrations were measured periodically to assess CO2 production and O2 consumption rates of the biochar alone. Consumption or production of CO2 and O2 (in micromoles CO2/O2 g−1 biochar day−1) was determined by linear regression of the headspace concentration with time. A positive value indicates production and a negative value indicates consumption of the respective gas during the incubation.

The second experimental design was developed to evaluate the CO2 and O2 from incubation of soil and biochar. The factorial was represented by three soils (AG, US, and PS), five biochars (B1, B2, B3, B4, and AC) as well as soil control (no biochar), which were conducted with three replications. Soil (5 g), biochar (0.1 g) and deionized water (0.5 mL) were added to each serum bottle (125 mL), sealed with butyl rubber septa at a controlled temperature of 25 °C for 57 days. The CO2 and O2 concentrations were measured periodically over approximately 60-day incubation (sampled on 1, 5, 8, 13, 15, 20, 27, 37, 52 and 57 days after sealing).

To conduct the gas sampling in both studies, 5 mL of laboratory air (known composition) was injected using a syringe. The syringe was then flushed repeatedly without removing the needle from the serum bottle to flush the headspace and mix the sample adequately for sampling (3 × flushing with the syringe). A 5 mL sample of the headspace was then collected and injected in a 10 mL headspace vial, previously flushed with helium. CO2 and O2 headspace concentrations were measured using gas chromatography on a system previously described (Spokas and Reicosky 2009).

2.3 Data processing

The gas concentration data were initially assessed for outliers by the Grubb’s test. The assumptions of data normality and homogeneity of variance were evaluated using the Shapiro–Wilk (SigmaPlot Inc., USA) and Bartlett tests (SPSS Inc., USA), respectively. Concentrations of CO2 from the second experimental design were fitted to a logistic growth model (package = growth rates; Petzoldt 2018) in R (R Core Team 2019) and the oxygen consumption rates were estimated from the linear fit of the headspace concentration versus time. Effects of biochar were subject to analysis of variance (ANOVA) based on the F test for statistical significance. When there were significant differences (p ≤ 0.05), the means of the treatments were compared by the Tukey test (p ≤ 0.05). Pearson’s correlation was further used to explain relationships between property variables (significant at p ≤ 0.05).

3 Results

3.1 Biochar alone

Incubations of biochar alone indicated that biochar’s potential to produce CO2 was highly variable, whereas the detected O2 consumption was more uniform across all types of biochar. AC removed all the headspace CO2, resulting in a negative CO2 production rate (Fig. 1a). There was a significant difference between the biochar types with B3 and B1 possessing a positive production rate of 0.767 and 0.518 mmol CO2 g−1 day−1, respectively, over the 22-day incubation. This rate represents less than 0.22% of the original carbon present in the biochar for the 22-day incubation. Rates of CO2 production were significantly lower for B2 (0.08 mmol CO2 g−1 day−1) and B4 (0.02 mmol CO2 g−1 day−1). The AC possessed a negative CO2 production potential of − 0.059 mol of CO2 g−1 day−1. However, this sorption capacity for CO2 could be significantly larger, since AC removed all detectable CO2 from the sealed incubation (Fig. 1a).
Fig. 1

a CO2 and b O2 production from the biochar alone incubations [pine chip biochar—B1; hardwood lump charcoal—royal oak—B2; slow pyrolysis bamboo-based biochar—B3; fast pyrolysis macadamia nut biochar—B4, as well as an activated charcoal (Accurel)—AC]. The letters indicate which treatments are statistically equal when compared by the Tukey test (p ≤0.05)

On the other hand, despite the differences observed in the CO2 production, there were no statistically significant differences between the biochars in the observed O2 consumption rates (Fig. 1b). All biochars removed O2 from the headspace with an overall average of − 8.06 mmol of O2 g−1 day−1. Additionally, there was no statistically significant relationship between CO2 and O2 production rates (R2 = 0.169, p = 0.07; Fig. 2), with significantly more O2 removed than the corresponding observed CO2 production. This also suggests that the biochar is a scavenger for gaseous O2.
Fig. 2

Comparison of the CO2 and O2 production rates from the biochar alone incubations

3.2 Biochar + soil

Results of the model fit of the CO2 production rates are shown in Table 3 and the cumulative CO2 production curves are shown in Figure S1. Overall, there is no consistent behavior of any biochar across all three soil types evaluated here in the CO2 production rates. Despite the appearance of differences in the measured rates, none of these differences are statistically significant for each soil groupings (p ≥ 0.05). However, there is a statistically significant difference in the CO2 production as a function of soil type, with the PS possessing a slightly lower production rate (0.15 day−1 for PS) compared to 3.0 day−1 and 2.14 day−1 for the AG and US soils, respectively. This agrees with the similarities in curve shapes seen in Figure S1. However, there are a few significant differences when we examine the total CO2 produced as a function of soil × treatment, as shown in Fig. 3.
Table 3

Results of model fits of the CO2 and O2 production rates for biochar [pine chip biochar—B1; hardwood lump charcoal—royal oak—B2; slow pyrolysis bamboo-based biochar—B3; fast pyrolysis macadamia nut biochar—B4, as well as an activated charcoal (Accurel)—AC] and soils (PS—commercial potting soil; AG—agricultural soil; US—urban soil)



CO2 rates

O2 rates (mg O2 g−1 day−1)

μmax (day−1)

K (mg CO2)

R 2






− 0.04





− 0.05





− 0.04





− 0.07





− 0.05





− 0.05






− 0.08





− 0.07





− 0.07





− 0.09





− 0.06





− 0.04






− 0.04





− 0.06





− 0.04





− 0.05





− 0.01





− 0.04

Fig. 3

Differences in the total C–CO2 production during the laboratory incubation period for the three soils (PS—commercial potting soil; AG—agricultural soil; US—urban soil) and biochars [pine chip biochar—B1; hardwood lump charcoal—royal oak—B2; slow pyrolysis bamboo-based biochar—B3; fast pyrolysis macadamia nut biochar—B4, as well as activated charcoal (Accurel)—AC]. Significant differences between the biochar treatments and the control group are shown. Note: “ns” designates no significant difference between the treatment and control

All the biochar treatments increased cumulative CO2 production, although not all were statistically significant. AC was the only amendment which was statistically significant across all soil types, and B1 was significant only in the AG soil. Results were similar for the O2 consumption rates in biochar-amended soils (Table 3). Except for the AC, there was no clear effect of biochar type across all three soils in this experiment.

Despite the lack of individual differences, soil amended by biochar presented a strong negative correlation between CO2 and O2 production rates across all soils and treatments with a slope of − 0.83 (Fig. 4; R2 = 0.70; p = 4.377 × 10−15). This value is within 20% to the theoretical value of − 1 for this relationship, assuming for 1 mol of CO2 it takes 1 mol of O2.
Fig. 4

Relationship between the calculated linear CO2 and O2 production rates for all soils and treatments in this study

4 Discussion

4.1 Biochar alone

Biochar offers a means of CO2 sequestration, as demonstrated in the incubation without soil. The production of CO2 was highly variable as a function of the kind of biochar. AC presented a higher capacity for CO2 sequestration, resulting in a negative CO2 production rate. This value could be significantly larger since the AC removed all detectable CO2 from the sealed incubation. Biochar as an alternative for CO2 sequestration also has been presented by Creamer et al. (2014) using biochar produced from sugarcane bagasse and hickory wood; Guo et al. (2018) using peanut shell pyrolyzed; and Rashidi and Yusup (2017) using a synthesized palm kernel shell-based activated carbon. Activated biochar possesses a high capacity for sorption of other substances, such as nitrate, fungicides, tricyclazole, heavy metals and soil organic pollutants, as demonstrated by several studies published by Dempster et al. (2012), García-Jaramilloa et al. (2015), and Yu et al. (2010). On the other hand, B3 and B1 possessed positive CO2 production rates which were not directly correlated to the consumption of O2 (Fig. 2). However, the overall production of CO2 is well below 1% of the original C contained in the biochar. The lack of correlation between CO2 and O2 also suggests the lack of microbial pathways for this observed CO2 production.

The CO2 sorption by AC is explained by the activation process (post-production 1100 °C with steam); this capacity appears to be directly linked to the pyrolysis temperature, with the highest capacity to sorb CO2 in activated charcoal which underwent a high-temperature post-thermal activation process. An “activation-like” process could be obtained during cooling using water or exposing hot biochar to atmospheric oxygen altering the biochar surface chemistry (Nuithitikul et al. 2010; Cheng et al. 2006). Gupta and Kua (2017) described that biochar’s capability for adsorption depends on factors such as pyrolysis conditions. Pyrolysis temperature, heating rate, and pressure all increased the capacity for CO2 adsorption (Guo et al. 2018). Creamer et al. (2014) using biochar produced from sugarcane bagasse and hickory wood hypothesized that the adsorption of CO2 on biochar is mainly controlled by physisorption, which is a weak interaction arising from intermolecular forces (van der Waals forces). The presence of nitrogenous functional groups on biochar could be responsible for increase in the physisorption of CO2 on biochar surfaces, due to the strong interaction between the acidic CO2 and basic nitrogenous surface functional groups (Zhang et al. 2013, 2017). This effect was explained by Zhang et al. (2017) who demonstrated that after the adsorption of CO2, the CO2 can be converted to C=O groups, nitrogen atom-containing heterocyclic groups, and N–O groups on the surface of biochar. Surprisingly, there was no clear evidence of the role of the C/N ratio in the observed trends in CO2 production among biochar (Table 2).

Production of CO2 that was observed in the biochar alone incubation most likely did not principally result from microbial activity. This is supported by the lack of a clear relationship between O2 and CO2 production rates (Fig. 2). More likely, this CO2 gas is due to degassing from isolated pores (or pockets) within the biochar and/or CO2 gas reversibly held by surface moieties. It is particularly noteworthy since the O2 consumption rates exceeded the theoretical 1 mol of O2 consumed for each mmol of CO2 produced. The chemical stability of biochar against microbial degradation might be associated with low O/C molar ratios (O/C < 0.20; Spokas 2010), high C distribution (Keiluweit et al. 2010), and low amounts of oxygen-containing functional groups, such as: hydroxyl (–OH) and carboxylate (–COOH) (Yuan et al. 2011). Oxygen-containing moieties confer a net negative charge on biochar surfaces with the dissociation of oxygen-containing functional groups (Inyang et al. 2010; Yuan et al. 2011).

The presence of micropores smaller than 0.5 nm appears to promote additional biochar–chemical interactions (e.g., chemical sorption and desorption) (Weldon et al. 2019; Yargicoglu et al. 2015). Serafin et al. (2017) noticed that micropores in the range of 0.3–0.6 nm were effective for CO2 adsorption and the highest adsorbed amount of CO2 was equal to 1.25 mmol CO2/g. Studies of Plaza et al. (2014) demonstrated that the optimum production of micropores in biochar occurs at low oxygen concentrations (3–5%) and temperatures ranging from 550 to 650 °C. We did not evaluate the nano- and micro-pore volume in this study.

4.2 Biochar + soil

Our results indicated that soil amended by biochar significantly affected the net CO2 production. The hypothesis that biochar can be a source for CO2 emission in soil was accepted and is corroborated by the study of Sigua et al. (2014), who observed a linear increase in CO2 emission from two ultisols amended with biochar incubated for 50-day incubation. Additionally, Shen et al. (2017) noticed an initial increase of CO2 emission from an incubation of biochar in soil (total of 30 and 70 t ha−1 of corn stover biochar), followed by a decreasing trend in CO2 emission after 10 days of incubation (total of 46-day incubation). Our results support these trends demonstrating a positive CO2 emission followed by a consecutive decreasing production trend (e.g., first-order kinetics) (Figure S1). On the other hand, in a 3-year field study, Lentz et al. (2014) verified lower CO2 emissions from irrigated calcareous soils with a single application of fast pyrolyzed hardwood biochar (22.4 t ha−1 in 0- to 15-cm soil). While no significant differences in the flux of CO2 were observed by Karhu et al. (2011) comparing biochar amended (total of 9 t ha−1 of Charcoal Finland biochar) and control plots in a short-term field study, and Scheer et al. (2011) comparing biochar incorporation (total of 10 t ha−1 of cattle feedlot biochar) into soil from an intensively managed subtropical pasture. An explanation for contradictory results is postulated due to differing biochar properties (biomass raw material and process of pyrolysis) that influence the sorption and production of CO2, as we observed in the incubation of the different types of biochar alone.

In the short term, an increase of CO2 emission in soil with biochar addition is explained by the increment of soil microbiological and enzymatic activities in soil with the addition of biochar. This result is confirmed in our study by a negative relation between CO2 and O2 due to the biological respiration of soil microorganisms. According to Lehmann et al. (2011) application of biochar in soil is responsible for increasing the abundance and activity of soil biota due to the release of a variety of organic molecules from the biochar. However, this trend was not evident in our study, since the lowest temperature biochar did not universally increase observed CO2 production following incorporation. Biochar has been hypothesized to provide a direct habitat for soil microbes offering favorable microsites for the microbes and shelter against soil faunal predators (Pietikainen et al. 2000). These actions could promote a better condition for soil biological activities with time (Agegnehu et al. 2015; Lentz et al. 2014; Lehmann et al. 2011) but would not provide a direct explanation for the immediate increase observed. Applications of biochar can create additional external soil porosity, which could increase the availability of oxygen and water holding capacity in soil (Lim et al. 2016). Both conditions promote the biological activity in soil (Almeida et al. 2018). Han et al. (2017) demonstrated an increase in the bacterial community with higher abundance of Sphingomonas sp. and Pseudomonas sp. in the biochar-treated soils with continuous cropping systems. However, these favorable conditions in soil porosity alterations can be lost by biochar fragmentation from microbial activities (Sigua et al. 2014) or natural biochar fragmentation (Spokas et al. 2014). Biochar can also affect indirect soil properties that can improve the biological activity in soil, mainly higher availability of nitrogen, phosphorus, potassium, magnesium, and calcium following biochar application (Laird et al. 2010; Lentz et al. 2014; Agegnehu et al. 2015). In this study, the AC amendment universally bolstered CO2 production across all soils, despite being the C source that was the most resistant to microbial degradation as suggested by the chemical properties (low O2 content, high fixed C; Table 2), lowest volatile C source, and the lowest in ash content (mineral fraction).

If we assume that this stimulation of CO2 production is resulting from the soil organic carbon (SOC) pool, these biochar additions on average resulted in a 1.59-fold increase in C losses from the soil, or an additional 2.2 mg C loss over the 60-day incubation period. Assuming that this C was from the available SOC pool, this would represent a decrease of 2–5% in the SOC during this period. If these rates are maintained, the 64–93 mg addition of C from the biochar addition could be offset by this increased CO2 respiration activity with time. Additionally, even though this might not be noticeable in total C analysis, this is a critical component to the overall microbial health of the soil system. Since microbes can only utilize soil organic C and not biochar structural C sources.

5 Conclusion

The data presented here confirm that the CO2 production following biochar addition in the soil is due to a process that is correlated to oxygen consumption. However, this overall stimulation is not clearly related to biochar type in our study. Activated carbon resulted in the highest statistically significant stimulation of activity, despite it possessing the lowest quantity of volatile C sources. This is contrary to the current hypothesis of volatile C sources on biochar stimulating initial microbial activity. Utilizing O2 consumption rates along with CO2 production allows one to assess the potential source mechanisms. The addition of biochar does stimulate the native microbial population increasing the loss of soil organic carbon (a decrease of 2–5% in the available SOC pools in 60 days). The exact mechanism behind this stimulation is not known and does not appear to be directly correlated to volatile C sources on the biochar. We conclude that using biochar does achieve total C sequestration. However, the amount of available SOC following soil incorporation appears to be reduced with biochar addition and its long-term implication on the active SOC pool does deserve more research attention.


Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this article.

Supplementary material

42773_2019_21_MOESM1_ESM.docx (117 kb)
Supplementary material 1 (DOCX 116 kb)


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Copyright information

© Shenyang Agricultural University 2019

Authors and Affiliations

  1. 1.University of São Paulo-USPPiracicabaBrazil
  2. 2.United States Department of Agriculture-Agricultural Research ServiceSt. PaulUSA
  3. 3.University of Marília-UnimarMaríliaBrazil
  4. 4.Universidade Estadual Paulista-UNESPJaboticabalBrazil

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