Influence of pig manure and its biochar on soil CO2 emissions and soil enzymes

Ecological Engineering 95 (2016) 19–24

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Influence of pig manure and its biochar on soil CO2 emissions and soil enzymes G. Gascó a,∗ , J. Paz-Ferreiro b , P. Cely a , C. Plaza c , A. Méndez d a

Departamento de Producción Agraria, E.T.S.I. Agrónomos, Universidad Politécnica de Madrid, Ciudad Universitaria, 28004 Madrid, Spain School of Engineering, RMIT University, GPO Box 2476, Melbourne 3001, VIC, Australia Instituto de Ciencias Agrarias, Consejo Superior de Investigaciones Científicas, Serrano 115 bis, 28006 Madrid, Spain d Departamento de Ingeniería Geológica y Minera, E.T.S.I. Minas y Energía, Universidad Politécnica de Madrid, C/Ríos Rosas n◦ 21, 28003 Madrid, Spain b c

a r t i c l e

i n f o

Article history: Received 6 June 2015 Received in revised form 27 November 2015 Accepted 15 June 2016 Keywords: Biochar Enzymatic activity CO2 emissions Manure wastes

a b s t r a c t Biochar production from manure wastes, including pig manure, could provide a valuable alternative to current waste management practices, while offering an opportunity to improve soil properties and to reduce the risk of contamination derived from the direct application of manure as a soil amendment. Two different biochar samples, produced from pig manure at 300 ◦ C (BPC300) and 500 ◦ C (BPC500) were used to evaluate the impact of biochar amendment on soil enzymatic activity and soil CO2 emissions. An incubation experiment was designed as follows: selected soil (S) was amended with pig manure (PC) and two pig manure biochars prepared at 300 ◦ C (BPC300) and 500 ◦ C (BPC500) at a rate of 8 wt%. All samples were incubated during 219 days. The results indicated that soil amendment with biochars decreased the carbon mineralization, in contrast to soil amended with the pig manure. Addition of pig manure increased dehydrogenase, phosphomonoesterase and phosphodiesterase activities, while B prepared at 300 ◦ C resulted on a positive effect on dehydrogenase activity. In contrast, B prepared at 500 ◦ C did not exhibit a positive effect on soil enzyme activity. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Previous studies have indicated that biochars can improve soil properties and also contribute to carbon sequestration when used as organic amendment. The amount of soil sequestered and the performance of biochar as a soil amendment depend on pyrolysis conditions and on the feedstock used for biochar production (Lehmann and Joseph, 2009; Paz-Ferreiro et al., 2012; Cely et al., 2015). However, biochars could also represent a risk due to their potential content of toxic compounds such as polycyclic aromatic hydrocarbons (PAHs) or heavy metals. There is an increasing body of literature providing evidence of biochar having a contrasting effect on different species of plants and microorganisms, depending on their sensitivity. Bastos et al. (2014) found the bioluminescent bacteria V. fischeri to be the most sensitive organism to a biochar produced from slow pyrolysis of mixed pine wood chips, however they did not observe any detrimental effect on the growth of the microalgae P. subcapitata. Besides, Cely et al. (2016) found

∗ Corresponding author. E-mail address: [email protected] (G. Gascó). http://dx.doi.org/10.1016/j.ecoleng.2016.06.039 0925-8574/© 2016 Elsevier B.V. All rights reserved.

that some plant species, including lettuce and tomato, seemed to be more sensitive to the presence of phytotoxic compounds in biochars, while all biochars used in the same experiment acted as phytostimulant for lentils. Enzymatic assays represent an important measurement in order to understand the metabolic activity in soils which underpins processes such as the mineralization and humification of organic matter. The latter in turn influences the biogeochemical cycles of elements including C, N, P and S (García and Hernández, 2003). Therefore, soil enzymes play an important role in organic matter decomposition and nutrient cycling (Walelign et al., 2014). Paz-Ferreiro et al. (2012) used the geometric mean of enzyme activities (GMea) as a soil quality index and reported a lower value of this index in soils amended with sewage sludge. On the contrary, sewage sludge biochar resulted in higher values of GMea than the control soil, indicating an improved soil quality. Biochar, as a soil amendment, can increase soil organic matter stocks and stimulate soil microbial activity (Lehmann and Joseph, 2009). Microorganisms are largely responsible for the decomposition of the organic matter via a variety of enzymes and hence, application of biochar is a method to improve soil quality and

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soil biological status, which usually implies an increase in enzyme activity. Soil organic matter content is a balance between inputs and decomposition rates and, as such, changes in agricultural practices can result in marked alterations in both the addition rates and decomposition rates of soil organic matter and therefore, nutrients (Walelign et al., 2014). The enhancement of C mineralization and microbial biomass, following biochar addition, indicates that the activity and mass of soil microorganisms have been promoted due to the presence of extra nutrients provided by the biochars (Zhao et al., 2015). Also, increasing application rates of biochar could enhance TOC and its labile fractions which improve the soil quality by increasing the microbial activities, aggregation and soil C sequestration. Cely et al. (2014) reported an increase in the amount of more humified or thermally stable organic matter after addition of diferent types of biochar. In addition, besides previous studies indicated that the CO2 evolved after adding biochar to the soil could be related to the labile carbon content in biochars (Méndez et al., 2013; Cely et al., 2014). The main purpose of this study was to evaluate the effects of pig manure and two biochars produced from pig manure at two different temperatures on soil carbon mineralization and soil microbial activities. 2. Methods 2.1. Soil samples selection and characterization The soil sample was taken from the northeast of Toledo-Spain (40◦ 7 6 N–4◦ 14 29 W—595 m). The soil was classified as a Cambisols, with a pH of 7.66 and a low content in CCr2O7 (%), N and P. Soil texture was sandy loam. Soil pH and electrical conductivity (EC) were determined with a soil: water ratio of 1:2.5 (g mL−1 ) using a Crison micro-pH 2000 (Thomas, 1996) and a Crison 222 conductivimeter (Rhoades, 1996) respectively. CEC was determined by NH4 OAc/HOAc at pH 7.0 (Sumner and Miller, 1996). Organic carbon oxidized with dichromate was determined by the Walkley-Black method (Nelson and Sommers, 1996). N was determined by Kjeldahl digestion (Bremmer and Mulvaney, 1982). P was determinated according to Watanabe and Olsen (Watanabe and Olsen, 1965). Total metal content was determined using a Perkin Elmer 2280 atomic absorption spectrophotometer after sample extraction by digestion with concentrated HCl/HNO3 following method 3051a (USEPA, 1997). Soil texture was determined following the methodology of Bouyoucos (1962). These analyses were performed in triplicate. 2.2. Pyrolysis of pig manure Biochars were prepared as by Gascó et al. (2012) as follows: 100 g of pig manure were placed inside a covered steel cup and introduced in an electric furnace where the temperature was increased to 300 ◦ C or 500 ◦ C at a rate of 10 ◦ C min−1 and the final temperature was maintained for 1 h. As a result two biochar samples were obtained: pig manure biochar prepared at a temperature of 300 ◦ C (BPC300) and pig manure biochar prepared at a temperature of 500 ◦ C (BPC500). 2.3. Biochar caracterization Biochar samples were produced from a pig manure (PC) taken from a farm in Avila (Spain). All samples were air-dried, crushed and sieved through a 2 mm mesh prior to analysis. The pH, CE, CIC, N Kjeldahl, total metal content and organic carbon oxidized were done as in section 2.1. The iodine number (mgI2 g−1 ), defined as the quantity of iodine adsorbed per gram of

activated carbon at an equilibrium concentration of 0.02 N was calculated according to D-4607 standard test method (ASTM, 1995). Proximate analysis was determined by thermogravimetry using a Labsys Setaram equipment. The sample was heated to a temperature of 600 ◦ C under N2 atmosphere and 30 ◦ C min−1 heating rate. Humidity was calculated as the weight loss from the initial temperature to 150 ◦ C. Volatile matter (VM) was determined as the weight loss from 150 ◦ C to 600 ◦ C under N2 atmosphere. At this temperature, air was introduced and fixed carbon (FC) was calculated as the weight produced when the final sample was burnt. The ashes were determined as the final weight of the samples (Cely et al., 2015; Gascó et al., 2012).

2.4. Treatments and soil respiration The selected soil (S) was amended with the two biochar samples and with the pig manure (PC) at 8 wt% and mixtures were incubated at constant temperature (28 ± 2 ◦ C) and humidity (60% water holding capacity) for 219 days. Treatments were named SPC (soil + pig slurry), SPC300 (soil + BPC300) and SPC500 (soil + BPC500). Soil basal respiration (mg C kg−1 ) was determined by static incubation as described previously (Paz-Ferreiro et al., 2012; Cely et al., 2014): each sample (100 g) was introduced into a 1 L airtight jar and the CO2 produced during incubation was collected in 50 mL of a 0.3 NaOH solution, which was then titrated using 0.3 NHCl after precipitation of carbonates following BaCl2 addition. As a further experiment, it was studied if the application of the different amendments had an additive or synergistic effect in the soil (priming effect). In this way each sample (PC, BPC300 and BPC500) was incubated individually using the same experimental conditions. All treatments were performed in triplicate.

2.5. Determination of soil enzymes Dehydrogenase, phosphomonoesterase, phosphodiesterase and ␤- glucosidase activities were determined following modifications of the original methods. While the amounts and concentrations of the substrates, buffers and other reagents were the same as in the original methods, different calibration curves were used for the control soil, soil amended with pig manure and soils amended with biochar. This correction has been described before (Paz-Ferreiro et al., 2009, 2012). Dehydrogenase activity was determined by a modification of ˜ et al. (1998). The results were the method as reported by Camina expressed as ␮mol iodonitrotetrazolium formazan (INTF) g−1 h−1 . Phosphomonoesterase, phosphodiesterase and ␤-glucosidase were determined after incubating soils with a substrate containing a p-nitrophenyl moiety and then measuring the amount of p-nitrophenol released during enzymatic hydrolysis, using a spectrophotomet er at a wavelenght of 400 nm (Paz-Ferreiro et al., 2012). The activity of each of these three enzymes was expressed as ␮mol p-nitrophenol g−1 h−1 . Adsorption of the enzyme reaction product or substrate can occur in the surface of biochar, resulting in an inaccurate measurement of soil enzyme activity. Both products of the enzyme reactions (INT and p-nitrophenol) differ in their ability to be adsorbed in soil and in soil amended with biochar as reported in previous studies (Paz-Ferreiro et al., 2012). Thus, a different calibration curve was used for each treatment in order to obtain an accurate measure of enzyme activity. We also ensured that the reaction was not substrate limited. Finally, the geometric mean (a general index to integrate information from variables that possess different units and range of

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Table 1 General properties of soil, pig manure and biochars.

pH (1:2.5) EC (1:2.5) (␮S cm−1 , 25 ◦ C) CEC (cmol(+) kg−1 ) NKjeldhal (%) P(g kg−1 ) CCr2O7 (%) Ca (g kg−1 ) Mg (g kg−1 ) Na (g kg−1 ) K (g kg−1 ) Zn (g kg−1 ) Ni (g kg−1 ) Cu (g kg−1 ) Sand (%) Silt (%) Clay (%) Iodine number (g Iodine 100 g−1 ) VM (wt%) FC (wt%) Ash (wt%) FC/(VM + FC)

PC

BPC300

BPC500

9.0 ± 0.1 124 ± 3 44.1 ± 0.3 2.51 ± 0.22 2.3 ± 0.2 13.5 ± 0.7 1.5 ± 0.2 0.7 ± 0.1 0.6 ± 0.1 0.7 ± 0.1 0.98 ± 0.10 0.011 ± 0.001 0.20 ± 0.06 – – – – 41.72 11.06 47.22 0.21

7.8 ± 0.1 102 ± 11 35.6 ± 0.1 2.24 ± 0.04 2.8 ± 0.2 7.9 ± 0.4 1.3 ± 0.2 0.8 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 1.2 ± 0.10 0.017 ± 0.001 0.34 ± 0.08 – – – 9.55 ± 1.04 31.25 18.50 50.25 0.37

8.2 ± 0.1 59 ± 3 32.7 ± 3.6 1.19 ± 0.14 1.2 ± 0.1 5.0 ± 0.3 0.8 ± 0.1 0.8 ± 0.1 0.6 ± 0.1 0.8 ± 0.1 1.3 ± 0.11 0.015 ± 0.001 0.31 ± 0.01 – – – 11.67 ± 1.49 6.50 19.62 73.88 0.75

Under detection level.

variation) of the assayed enzyme activities was calculated for each sample as: GMea = (DH × Glu × Phos × Phosdi)

1/4

where DH, Glu, Phos and Phosdi are dehydrogenase, ␤- glucosidase, phosphomonoesterase and phosphodiesterase activities, respectively. Paz-Ferreiro et al. (2012) have used this index as a quality index to condense a whole set of soil enzyme values in a single numerical value. 2.6. Statistical analysis The statistical analyses (calculation of means and standard deviations, differences of means) were performed using Statgraphics Centurion XVI. Differences of means were tested using an analysis of variance (ANOVA). Means were considered to be different when P < 0.05 for the Duncan test. 3. Results The general properties of soil, pig manure and two biochars are shown in Table 1. Biochars had pH values nearly 1 unit lower than the feedstock. EC values were reduced with pyrolysis temperature reaching a 52.4% of reduction in BPC500 with respect to the original pig slurry. CEC was slightly reduced (more than 19%) with respect to the feedstock. Both BPC300 and BPC500 presented very similar CEC. P content increased 22% at 300 ◦ C while was it reduced by 48% at the pyrolysis temperature of 500 ◦ C. Ca and Mg contents also diminished with temperature. N content was reduced by 52% with respect to PC at the pyrolysis temperature of 500 ◦ C. Also, the CCr2O7 (%) was drastically reduced by 41% (BPC300) and by 63% (BPC500) in the biochars with respect to the control. VM (%) content in biochar was reduced with temperature. The thermostabilty index, i.e., FC/(VM + FC) increased by 50% (BPC300) and by 257% (BPC500) with respect to pig manure. Finally, both biochars presented low and similar iodine numbers. C mineralization of the 3 materials followed the sequence: PC > BPC300 > BPC500 (Fig. 1). This implied a reduction on C mineralization of 34.5% (BPC300) and 49.8% (BPC500) with respect to the raw pig manure (Fig. 1). These mineralization rates correspond to 22.3%, 42.3% and 51% of the CCr2O7 content for PC, BPC300 and BPC500, respectively.

6000 C Mineralizaon (mg CO2 Kg-1 material)

a

Soil 7.66 69.65 15.71 0.15 0.14 ± 0.1 0.82 1.07 0.124 0.09 1.6 ua ua ua 77.78 17.78 4.44 – – – – –

5102a 5000

4000 3344b 3000

2557c

2000

1000

0 0

40

80

120 Time (d)

BPC300

BPC500

160

200

240

PC

Fig. 1. Cumulative C mineralization of PC,BPC300 and BPC500.

Basal respiration values for SPC500 treatment did not show any significant differences with respect to the control soil, while basal respiration achieved values of 229% and 90% of the value in the control soil for SPC and SPC300 respectively (Fig. 2). To quantify the priming effect of the pig manure and the two biochars, experimental data were compared with the addition of 92 g of soil with 8 g of raw material (Addition treatment). In this way, we were able to study the effect of the physical interaction between the soil and the raw material. Results showed that CO2 emissions calculated by addition were 576 mg CO2 kg−1 soil for the SPC treatment, a value slightly lower than the data obtained from the incubation of soil and pig manure together (Table 2). The addition data obtained for the SBPC300 and SBPC500 treatments were, respectively, 436 and 373 mg CO2 kg−1 soil. These values were higher than those obtained in the incubation experiment (Fig. 2). Soil dehydrogenase activity was higher in SPC and SPC300 than in the control soil, while no significant differences were found between SPC500 and the control. Soil dehydrogenase activity increased more than 70% following PC and BPC300 application. Phosphomonoesterase activity was higher in the soil amended with pig manure than in the control, SPC300 or SPC500. On the contrary, the application of biochar reduced phosphomonoesterase

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Table 2 CO2 emissions calculated by addition (mg C-CO2 kg−1 ) and enzyme activities of different soils. Enzyme activities are expressed as ␮mol product g dry soil−1 h−1 . For the same property different letters indicate statistically significant differences (P < 0.05).

S SPC SPC300 SPC500 1 2

CO2 emissions1

Dehydrogenase

Phosphomonoesterase

Phosphodiesterase

␤-glucosidase

Gmea

– 577 436 373

0.011a 0.019b 0.024b 0.008a

17.08a 46.07b 23.14a 3.01c

2.12a 7.02b 0.95a u2

9.41a 12.28a 12.30a 1.70b

1.38a 2.94b 1.57ab –

The addition of the experimental data has been made taking into account a dose of 8%. u: below detection limit.

700

C Mineralizaon (mg CO2 Kg-1 soil)

602a 600 500 400

348b

300 221c 200 183c 100 0 0

40

80

S

SPC

120 Time (d) SPCB300

160

200

240

SBPC500

Fig. 2. Cumulative C mineralization of soil after different treatments.

activity by 50% (SPC300) and by 93% (SPC500) with respect to SPC. Phosphodiesterase activity did not show differences among treatments, except for SPC500 whose value was under the detection limit. SPC500 treatment resulted in a decrement of 82% in ␤- glucosidase with respect to control soils, SPC and SPC300 treatments. Finally, GMea increased 113% in SPC soil compared to the control soil, while there was no difference between SPC300 and the control soil. 4. Discussion The decrease of pH after pyrolysis process was in agreement to Cely et al. (2014) and Hossain et al. (2011) who found a similar behaviour after pyrolysis of cattle manure and sewage sludges at lower temperatures (<400 ◦ C). With respect to EC values, Cely et al. (2014) found the same trend in the reduction of EC in poultry litter biochar prepared at the same temperatures than in this study. The low CEC values of BPC300 and BPC500 are in accordance to Song and Guo (2012) who found reductions of CEC following pyrolysis. The reduction of N content during pyrolysis is due to N being the most sensitive macronutrient to heating. Thus, the N content for the biochar prepared at 500 ◦ C was lower than in the other materials studied. This is in accordance to Cely et al. (2015) and Wang et al. (2012) who studied nitrogen availability in biochar produced from cattle manure and found that nitrogen in biochar became more stable as pyrolysis temperature increased. The reduction of carbon content by wet oxidation with potassium dichromate reflects the degree of biomass carbonisation and could therefore be used to estimate the labile fraction of C in biochar (Calvelo Pereira et al., 2011). This reduction is in accordance with the decreased in VM and the increment of FC with temperature. Indeed, this fact is in agreement with the values of thermostability index, which is a reliable parameter to evaluate the level of stability of organic matter in biochar

(Gascó et al., 2012) and increased with the temperature. A wide discussion about biochar properties, with respect to C mineralization and thermostability index, can be found on Cely et al. (2015). The reduction of C mineralization in samples with biochar with respect to pig manure is in accordance with the reduction of CCr2O7 (%), VM (%) and the increment of the FC (%) and thermostabilty index with temperature. This is similar to previous studies which showed a reduction of CO2 emissions in biochars when compared to feedstocks (Méndez et al., 2014; Cely et al., 2014). This fact is related to the decrement, with respect to the soil amended with pig manure, on soil basal respiration after biochar addition and the absence of significant differences with respect to the control soil when BPC300 is added. When experimental data (Fig. 2) and addition experiment data are compared (Table 2), a positive priming effect as a consequence of the addition of pig manure is detected. On the contrary, a negative priming effect was observed when biochars were added to soil. With respect to this last observation, Pignatello et al. (2006) has described the implications of adding biochar to soil. According to this author, soil humic substances can be absorbed to char surface reducing the sorption capacity of soil. Indeed, Pignatello et al. (2006) assumed that humic substances are restricted to the external surface of biochar where they act as pore blocking agents or competitive adsorbates. Our results are in agreement to Zimmerman et al. (2011) who concluded that C mineralization was generally less than expected for soils treated with biochar produced at high temperatures (500–600 ◦ C). Also, Paz-Ferreiro et al. (2012) reported similar results in soil amended with biochar at a rate of 8%. Nevertheless, Lu et al. (2014) and Zavalloni et al. (2011) did not observe any priming effect in soils amended with lower doses of biochars prepared from corn straw and coppiced woodlands at similar temperatures. Cely et al. (2015) concluded that there is not a unique factor involved in the alterations in C mineralization following biochar addition and it depends on C content, carbon aromaticity, volatile matter, fixed carbon, easily oxidised organic carbon, metal and phenolic substances content and surface biochar properties. In summary, our results have shown that preparation and addition of pig manure biochar to the soil can lead to a reduction of C emissions and therefore a reduction of the carbon footprint associated to pig farming. This can be strategic to countries possessing a sizeable pig farming industry such as Spain, Netherlands or Australia. Dehydrogenase activity is an important component of the enzymatic system of every microorganism, an indicator of soil redox status and participates in microbial respiration. It is considered a suitable indicator of soil quality and microbial activity as it does not accumulate (contrary to other enzymes) in soil humic and clay complexes. The improvement of dehydrogenase activities in SPC and SBPC300 can be attributed to the high content on volatile matter of the pig manure and the biochar prepared at 300 ◦ C with respect to the biochar prepared at 500 ◦ C. In fact, Serra-Wittling et al. (1996) related the enhancement of this enzyme with labile organic matter addition. Also, Ameloot et al. (2014) observed increases in dehydrogenase activity with biochar from swine manure prepared at

G. Gascó et al. / Ecological Engineering 95 (2016) 19–24

350 ◦ C and no differences on dehydrogenase values when biochar was prepared at high temperatures as in our results. Phosphatases catalyse the hydrolysis of both esters and anhydrides of phosphoric acid. Phosphomonoesterase and phosphodiesterase activities are inducible enzymes (Acosta-Martínez and Tabatabai, 2000) and therefore, excretion by plant roots and microorganisms are regulated by their requirement for orthophosphate, which is among other factors affected by soil pH. Phosphomonoesterase activity was lower in SBPC300 than in the control soil. This fact is consistent with previous studies by Paz-Ferreiro et al. (2014, 2012) and by Liang et al. (2014) who found an improvement in phosphomonoesterase after the application of biochar prepared from Miscanthus and poultry litter at temperatures between 400 and 450 ◦ C. However, the mechanism behind this observation has not been yet elucidated but could be related to the different mobility and availability to plants of the P contained in the control soil, the soil amended with manure and the soil amended with biochar. With respect to phosphodiesterase activity, the trend was similar to the one observed for phosphomonoesterase. The ratios of phosphomonoesterase to phosphodiesterase differed among the treatments and tended to increase in soils treated with biochar prepared at 300 ◦ C. This suggests major differences concerning how organic phosphate pools are accessed for the different treatments (Caldwell, 2005). Glucosidases play a key role in degrading organic compounds such as crop residues or animal manure in soils. In addition, ␤glucosidase has been used to monitor quick changes in soil organic matter caused by changes in soil management (Bandick and Dick, 1999) as ␤-glucosidase activity is strongly related to soil organic matter cycling. ␤-glucosidase activity is sensitive to alterations in soil pH. Biochar is generally an alkaline material which raises soil pH; however the response of ␤- glucosidase after biochar addition and the mechanisms involved in this process are not well established (PazFerreiro et al., 2014). In our case, there was no effect after the addition of PC or BPC300 to soil and there was a diminished activity in soil amended with PC500. A possible explanation for this result is an improved microbial efficiency as a consequence of the co-location of microorganisms and carbon on biochar surfaces. GMea is a quality index which has been used in the last years due to its usefulness to summarize under a unique value the response of different soil enzymes (Paz-Ferreiro et al., 2012, 2014) to the application of biochar, or other amendments, into the soil. In our study, the addition of SPC300 did not improve GMea values while the addition of pig manure improved this quality index. Overall, the results for the enzyme analysis show an enhanced diversity and microbial activity in soil amended with pig manure and with BPC300. However, this was untrue for BPC500. Our results highlight the importance of temperature selection for the production of biochars with an agronomic value. 5. Conclusions Pig manure is more stable after pyrolysis process; therefore soils amended with biochar exhibited a reduction in carbon mineralization. However, carbon mineralization is strongly dependent on pyrolysis temperature. In general, no enhancement of soil enzymatic activities was observed after biochar addition, except for dehydrogenase activity after the application of biochar prepared at 300 ◦ C. Our results highlight pyrolysis temperature as an important parameter in the production of biochar for agronomic purposes. References ASTM, 1995. D4607-94 Standard Test Method for Determination of Iodine Number of Activated Carbon. ASTM, Philadelphia.

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