Artisanal and controlled pyrolysis-based biochars differ in biochemical composition, thermal recalcitrance, and biodegradability in soil

Biomass and Bioenergy 84 (2016) 1e11

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Research paper

Artisanal and controlled pyrolysis-based biochars differ in biochemical composition, thermal recalcitrance, and biodegradability in soil K. Jegajeevagan a, L. Mabilde a, M.T. Gebremikael a, N. Ameloot a, S. De Neve a, P. Leinweber b, S. Sleutel a, * a b

Department of Soil Management (Ghent University), Coupure Links 653, 9000 Gent, Belgium Soil Science (University of Rostock), Justus von Liebig Weg 6, 18059 Rostock, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 May 2015 Received in revised form 27 October 2015 Accepted 28 October 2015 Available online xxx

Biochar composition and stability is under intense research. Yet the question remains to what extent the current state-of-the-art applies to artisanally charred biomass in tropical regions. We compared kiln and drum based biochars with their counterpart controlled (at 400
C) slow pyrolysis biochars from coconut shells, rice husks and Palmyra nutshell for their biochemical composition, thermal stability and biodegradability in soil. Thermal behavior of individual organic constituents was quantified by pyrolysis-field ionization mass spectroscopy (Py-FIMS). Comparison of the mass spectra demonstrated higher abundances of either phenols, lignin and carbohydrate monomers or of lipids in the artisanally produced biochars. Hence, relatively more untransformed plant matter was preserved by artisanal charring and also the thermal stability of carbohydrates, alkylaromatics and N-containing compounds was lower for all three feedstocks. This indicates lower prevailing temperatures compared to controlled pyrolysis biochar, at least in parts of the biomass charring in the kilns or drum. Nine-weeks biochar derived C mineralization upon soil incorporation revealed a relatively lower biological stability of the controlled pyrolysis biochars. The proportion of detected ion intensity from thermolabile lower mass signals (<400
C, m/z < 250) was negatively correlated to the net-biochar derived C mineralization. We hypothesize this fraction to be composite and act both as a C-substrate and at the same time to hold unidentified substances inhibiting microbial activity. Compared to controlled pyrolysis biochar, traditionally charred biomass, i.e. the ‘biochar’ most likely to be actually applied to soil in developing countries, has a heterogeneous thermal and biochemical composition and unpredictable biological stability. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Biochar Py-FIMS Thermal stability Biochar production method Pyrolysis Borassus flabellifer Cocos nucifera L. Oryza sativa L

1. Introduction Farming practices in the subtropics often result in depletion of soil organic matter (SOM) [1,2], which not only jeopardizes soil fertility but also contributes to global warming. Because of rapid microbially-mediated decomposition, options to maintain or lift SOM levels in tropical sandy soils are limited. Over the past decade, the beneficial effects of field application of carbonized biomass [3], so-called biochar, on agro-ecosystem functioning has been showcased by different researches [4,5]. Biochar additions have often been shown to improve plant growth by supplying and, crucially in a tropical context, retaining nutrients in the soil profile and by improving soil physical, chemical and biological properties [4]. For

* Corresponding author. E-mail address: [email protected] (S. Sleutel). http://dx.doi.org/10.1016/j.biombioe.2015.10.025 0961-9534/© 2015 Elsevier Ltd. All rights reserved.

instance, Lehmann et al. [6] demonstrated that the ability of biochar to retain applied nutrients against leaching resulted in increased fertilizer use efficiency because of the higher surface charged area of biochar. The Jaffna Peninsula, Sri Lanka is situated in a climatic region characterized by dry tropical conditions, and the soils are predominantly sandy and calcic in nature. The cation exchange capacity is as low as 0.04 mol kg1 [7,8] and soils mostly have a very limited soil organic carbon (SOC) content (often less than 8 g C kg1). Common problems with soil fertility in the sandy soils in Sri Lanka have prompted the question if biochar production by farmers from traditional feedstocks would be a viable option. While a large body of research up to now has concerned laboratory produced biochar, very little is known on the differences between these biochars and traditionally produced biochars. If biochar is going to be produced in developing countries, it will most probable

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occur by means of relatively uncontrolled pyrolysis or charring of traditional nutrient-poor feedstocks. These conditions may yield very different biochars in terms of biochemical composition and biological stability when compared to abundantly studied lab slow pyrolysis ‘biochar’, limiting the practical representativity of current literature on biochar. Our aim was to compare artisanally produced and controlled pyrolysis biochars for key parameters characterizing their biochemical composition and biological stability. Agricultural byproducts such as rice husks (Oryza sativa L.), Coconut shells (Cocos nucifera L.) and Palmyra nut shells (Borassus flabellifer L.) are available as waste material in plentiful amounts in Sri Lanka and were therefore selected as feedstock. We produced biochar by traditional charcoal making methods and in a controlled pyrolysis system in at the University of Ghent, Belgium. The biochars were compared by proximate analysis and physico-chemical characterizations. Secondly, we used pyrolysis field ionization mass spectroscopy (Py-FIMS) to assess the biochemical quality and thermal stability of the biochars. The Py-FIMS specifically provides information on the biochemical composition of the organic matter (OM), while the volatilization temperature of molecular markers is related to their binding strength to organic or mineral constituents [9]. This method has frequently been used to assess the molecularlevel biochemical composition of bio-wastes [10,11], wood [12], rhizo-deposits [13,14], dissolved organic matter [15] and SOM [16,17]. Lastly, we assessed short-term C mineralization of soil amended with the three artisanally produced biochars and their counterpart controlled (at 400
C) pyrolysis biochars and we compared the effect of biochars soil amendment on C evolution from SOM mineralization. At the end of the incubation experiment, we also assessed differences in pH and biological assays among the biochar amended mesocosms. 2. Material and methods Six different biochars were produced from three different types of biomass, namely Rice husks, Palmyra nutshells and Coconut shells (Table 1) by two different methods. Halved coconut shells (Cocos nucifera L.) and rice husks (Oryza sativa) were collected as waste from local households (Chulipuram, Jafna, Sri Lanka, 9
450 50.000 N 79
560 39.200 E) on 20th of June 2011. One to two years old entire Palmyra nuts were collected from the ground near 30e40 years old native Borassus flabellifer trees (9
420 28.300 N 79
570 28.200 E on 5th of May 2011) after surrounding pulp was decomposed or eaten by roaming cattle and endosperm had already sprouted. Biomass was left to dry in the sun for two days and was then stored in nylon bags before biochar production. Two techniques were used: (1) slow pyrolysis with a cylindrical furnace system in a laboratory environment and (2) by artisanal charcoal production methods. About 1 kg of each biomass was shipped in sealed PE plastic bags to Ghent University and were then oven dried at a temperature of 60
C for 24 h before controlled pyrolysis. 2.1. Biochar production under controlled laboratory conditions The biomass were slowly pyrolysed at 400
C in the laboratory at the department of Biosystems Engineering (Ghent University) on

9th of September 2011, yielding three types of slow pyrolysed biochar, viz. Coconut shell biochar (CC), Palmyra nutshell biochar (PC) and Rice husks biochar (RC). The slow pyrolysis unit consisted of a cylindrical furnace in which a 30 cm high vertical stainless steel reactor is covered with an inner diameter of 3.6 cm. The biomass was ground to particles of about 4e8 mm and was then added into the reactor tube to a height of about 25 cm and flushed with N2 at a gas flow rate of 13.3 cm3 s1. After a total residence time of 10 min at the selected temperature of 400
C following a heating phase, the reactor tube was cooled and the produced biochar was collected, weighed and stored in polypropylene containers at temperatures of about 18
C. 2.2. Biochar production by using traditional charring methods Following traditional charring practices, artisanal Coconut shell biochar (CA), artisanal Palmyra nutshell biochar (PA) and artisanal Rice husks biochar (RA) were produced Nutshells were not further ground or dried. We produced biochar from coconut or Palmyra nutshells in Jaffna by piling biomass in ground pits (approximately 1  1 m2 opening and 0.7 m depth) on an initial pre-combusting bio-fire source. The progress of piling was continuously adapted to prevent excess or limitation of oxygen supply, as assessed only from smoke formation and color of the smoke (see Appendix 1). Finally, after several hours of piling, the well smoked pits were covered air tightly by a steel lid covered by earth. These setups were let for three to four days. Finally the lids were opened and the well charred nutshells were removed from the pit and further spread on a leveled metal sheet on adjacent ground for further cooling. The RA biochar on the other hand was produced on the ground surface with a ‘drum method’. In this method, the heating source was placed under a steel drum perforated with tiny holes over its entire surface. Once the outer surface of the drum was heated, rice husks were heaped all over the drum. From time to time the rice husk heap was mixed by shovel and heated air flow was maintained through drum's pores until the whole heap turned into black in color (Biochar). Then the biochar was spread immediately on metal sheets and water was spring on the biochar for cooling down. Finally, the biochar was sundried for several days and packed in nylon bags and further ground and sieved. A further description of the artisanal charring is given in Appendix 1. 2.3. General biochar properties All biochar samples were analyzed for their proximal composition at the Biosystems-engineering lab of Ghent University. Moisture content, volatile matter and ash content were determined by the ASTM D1762-84 standard testing method [18]. The pHKCl of the biochar samples was measured by a pH glass electrode in suspensions of 5 g soil and 0.05 L of 1 mol L1 KCl. The cation exchange capacity (CEC) was measured according to the Chapman method [19]. Five grams of 2 mm-sieved biochar material were well mixed with acid-washed sand. After saturation with ammonium acetate (pH 7) solution the mixtures were subsequently leached with 90% pure ethanol. Finally, sand-biochar mixture was extracted with 1 mol L1 KCl and mineral N (NHþ 4 -N) content was measured

Table 1 Chemical properties of three tropical feedstocks. Feedstock

Ash content (g kg1)a

C (g kg1)a

N (g kg1)a

pHKCl ()

pHH2O ()

ECb (mS)

Coconut shells Palmyra nutshells Rice husks

8±0 13 ± 2 146 ± 6

472 ± 0 469 ± 0 369 ± 0

0.7 ± 0.2 2.2 ± 0.1 4.0 ± 0.2

5.16 4.89 5.46

6.24 5.80 6.01

719 263 1506

a b

Means ± standard deviations, n ¼ 3. Electrical Conductivity determined in a suspension of 5 g biochar and 25 cm3 deionized water.

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colorimetrically with a ‘continuous flow auto-analyzer’ (Chemlab System 4, Skalar, the Netherlands). CEC was calculated in equivalent of hydrogen per kg dry biochar (mol kg1). The total C and N contents of the biochar samples were measured with a Variomax CNSanalyzer (Elementar Analysensyteme, Germany). 2.4. Pyrolysis-field ionization mass spectroscopy (Py-FIMS) Temperature-resolved Py-FIMS of the biochars was carried out at the Mass Spectrometry Laboratory of the Chair of Soil Science, University of Rostock. About 2e5 mg biochar material was thermally degraded in the ion-source of a modified Finnigan MAT 95 high-performance mass spectrometer. The samples were heated in three replicates under a high vacuum from ambient temperature to 700
C at a heating rate of 10 K per magnetic scan (~1.7 K s1). After about 20 min, in total 60 magnetic scans were recorded for the mass range 16e1000 Da (single spectra). The single scan spectra were integrated to obtain one summed spectrum. In general, the summed spectra of three replicates were averaged to give the final survey spectrum. For each of the 60 single scans, the ion intensities of these marker signals were calculated. All samples were weighted before and after Py-FIMS to normalize ion intensities per mg sample. Detailed descriptions of the Py-FIMS methodology and statistical evaluations of sample weight and residue and total ion intensities are given by Sorge et al. [20]. We furthermore calculated difference mass spectra between the corresponding biochars produced traditionally and in the controlled UGhent lab pyrolysis unit. 2.5. C mineralization experiment Soil mesocosms with 256 g of air dried soil were prepared in 6.8 cm diameter PVC tubes. Each mesocosm was amended with 2.14 g biochar, equivalent to a rate of 20 Mg fresh biochar 10 000 m2 or biochar to dry soil ratio of 1:119. An Eutrustox (7th approximation of USDA) with sandy loam soil texture (clay < 2 mm: 150 g kg1, silt 2e50 mm: 40 g kg1 and sand 50e2000 mm: 810 g kg1), representative for intensive agriculture in Jaffna peninsula, was collected to a depth of 20 cm from an field in the Regional Agriculture research station, Jaffna, Sri Lanka. The soil had a SOC content of 6.0 ± 0.8 g kg1, a total N content of 0.6 ± 0.1 g kg1, pHKCl 7.5, pHH2O 8.1 and CEC 0.079 ± 0.008 mol kg1 soil. Soil was thoroughly mixed with the biochar, filled in the tubes, and slightly compacted to obtain a bulk density of 1400 kg m3. There were three replicates per biochar treatment and one unamended control treatment. Deionized water was added to the soils to achieve a fixed moisture content of 50 water filled pore space. Soil microcosms were placed in closed glass jars and kept in an incubator at 27
C. The emitted CO2 was trapped in 15 cm3 of 1 mol L1 NaOH. At day 1, 3, 4, 6, 13, 20, 26, 36, 48, and 60 the vials with NaOH were removed and titrated with HCl in the presence of BaCl2. The water content of the microcosms was adjusted weekly in order to maintain the set out soil moisture level. At the end of the incubation (after 120 days) soil microbial biomass carbon (MBC), soil mineral nitrogen content (1 mol L1 KCl extracts) and beglucosidase and dehydrogenase enzyme activity [21] were determined. Microbial biomass carbon was determined using the fumigation-extraction technique [22]. Both fumigated soil and unfumigated controls (25 g) were extracted in duplicate with 50 cm3 of 0.5 mol L1 K2SO4. Organic carbon contents of the extracts were determined with a TOC analyser (TOC-VCPN, Shimadzu Corp., Kyoto, Japan). Dehydrogenase is an intracellular enzyme participating in the processes of oxidative phosphorylation of microorganisms and is thus linked with microbial respiratory processes. It is often used as an overall measure for soil microbial activity. The procedure for dehydrogenase activity [22] relies on the reduction of a colorless, water

3

soluble substrate (TTC) added to 5 g of fresh soil by dehydrogenases present in a buffered soil environment. In this process an insoluble product with red color (triphenylformazan-TPF) is formed. TPF was quantified colorimetrically at the range of visible light (485 nm) with a Varian, Cary 50 spectrophotometer. b-glucosidase is an enzyme involved in the C cycle that catalyses the conversion of disaccharides into glucose. b-glucosidase activity was determined on 1 g of fresh soil using mM p-nitrophenyl-b-D-glucoside as substrate by incubating in a pH 6 modified buffer at 37
C. The pnitrophenol produced was measured colorimetrically at 400 nm with a Varian, Cary 50 spectrophotometer. All measurements were carried out in triplicate with one blank. The cumulative C mineralization, C(t) was plotted against the time t and a parallel zero and first order kinetic model was fitted to the data:

  CðtÞ ¼ t$ks þ Cf $ 1  ekf$t , with Cf the size of an easily-mineralizable C pool, that is mineralized according to first-order kinetics at rate kf; and ks, the zeroorder mineralization rate of a slowly decomposing C pool. The net C mineralization (Cmin net) derived from degradation of the biochar was calculated by subtracting the calculated cumulative 60 days C mineralization from the biochar amended minus the unamended control soil 2.6. Statistical analysis All statistical analyses were carried out with IBM SPSS Statistics 22 (SPSS inc., Chicago, USA). Kinetic model fitting to cumulative Cmineralization data was performed with SPSS's non-linear regression function. Differences between biochar properties, net 9-week cumulative C mineralization (i.e. biochar treated e untreated soil C mineralization), C-mineralization kinetic model parameters, bglucosidase and dehydrogenase activity and MBC were assessed by one-way ANOVA and Tukey's post-hoc test. Pearson correlation coefficients were calculated between these variables and between summed ion intensities of specific Py-FIMS derived mass signals. Principal component analysis on the Py-FIMS summed 0e700
C ion intensity-data sets (m/z 15e900) was used to identify molecular marker mass peaks which most strongly explained differences in biochemical composition over all biochars. 3. Results and discussion 3.1. General biochar characterization Controlled pyrolysis produced uniformous powdery to mainly granular (4e8 mm) black char with a strong tar-like smell for all three feedstocks. Artisanal charring produced clearly recognizable crispy charred rice husks (RA), with lower odor, and sometimes discrete still brown colored undercooked husks. The CA and PA chars instead consisted of 0.01e0.15 m deep black shell pieces with no odor. Ash and white patches were seen on the artisanal chars, representing ashed portions, especially in case of the RA biochar. The yield percentages of pyrolysis biochar production and proximate analyses of the six biochars are summarized in Table 2. Controlled pyrolysis at Ghent University resulted mass recovery of 410 ± 6.6 g kg1 on average. The highest biochar percentage yields were obtained for the rice husks feedstock, regardless of production method. The controlled pyrolysis biochars consistently had higher volatile matter contents than the artisanally produced biochars, with a divergent very high content in the PC biochar. In addition, the H:C-ratio was consistently lower in the traditionally produced

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Table 2 Char yield, proximate analysis and chemical properties of six biochars (means ± standard deviations, n ¼ 3). Biochara

Char yield (g kg1)

Volatile matter (g kg1)

CC CA PC PA RC RA

350 ± 11 -b 406 ± 41 -b 481 ± 47 -b

286 209 442 177 263 60

± ± ± ± ± ±

8 50 20 20 16 2

Ash (g kg1) 14 66 23 391 295 523

± ± ± ± ± ±

1 45 2 101 2 262

C (g kg1) 705 715 746 488 490 216

± ± ± ± ± ±

59 11 12 7 23 8

N (g kg1) 1.9 1.5 4.4 9.3 5.5 1.3

± ± ± ± ± ±

0.3 0.1 0.3 1.9 0.6 0.3

H:Cc ()

C:Nd (kg kg1)

CEC (mol kg1)

pHKCl ()

0.63 0.44 0.64 0.37 0.73 0.17

371 470 170 53 89 166

0.015 0.039 0.038 0.080 0.1115 0.114

7.61 7.59 6.53 7.58 7.45 8.15

± ± ± ± ± ±

0.14 0.06 0.03 0.11 0.13 0.02

a Coconut shell (C), Palmyra nut shells (P) and Rice husk (R) chars, suffixed by C when the biochar production was under controlled conditions or A when artisanally produced. b Yield not determined during local charring. c Atomic ratio. d Mass ratio.

biochars when compared to their UGhent counterparts (Table 2). Consequently, pyrolysis system had a considerable control on biochar volatile matter percentage and H:C ratio. Cross and Sohi [23] found sugarcane bagasse and trash derived biochars to hold lower contents of volatile matter when obtained with increasing pyrolysis temperature. It is furthermore well known that during pyrolysis hydrogen and oxygen are lost as bio-oil or bio-gas, while C accumulates in the biochar and therefore C content increases with increasing pyrolysis temperature [24]. While we have no temperature recordings of the uncontrolled thermal conversion during traditional charring, both a lower volatile matter content and H:Cratio (Table 2) suggest higher prevailing temperatures than the 400
C, that was maintained in the controlled biochar production at UGhent. The highly variable ash content of the artisanally produced biochars (Table 2) suggests heterogeneity in their composition, indicating larger variation in production conditions compared to the probably more homogeneous controlled pyrolysis biochars. The C:N ratios of traditionally produced CA and PA biochars were lower compared to controlled pyrolysis counterparts (Table 2). Lang et al. [25] reported that relatively half or more of the N is lost during biochar production. Specifically, these high pyrolysis temperature (700e950
C) biochars display low total N content because amines, amino acids, and amino-sugars are volatilized and any remaining N will occur in recalcitrant heterocyclic compounds [26]. The RA biochars' N mass fraction was lower than the RC biochar's and this may be due to the high temperature and air (oxygen) mixing during production of RA biochars under the steel drum method' open air conditions. It is unlikely, however, that pyrolysis temperatures approached 700e950
C in the CA and PA kilns as in Refs. [25], because a partial combustion reaction or external heat source would be required to maintain such temperatures. Still, establishment of kiln temperatures >400
C seem realistic. The measurements of pHKCl, pHH2O, and CEC among different six biochars showed the trend that traditionally produced biochars tended to have higher pH, EC and lower CEC values compare to respective UGent counterpart biochars. Bagreev et al. [27] stated that during the high temperature pyrolysis, the organic N present as amine functionality transformed in to pyridine-like compounds due to dehyroxylation reactions which largely responsible for increasing the basicity and increasing the pH of biochars' surface. The pH and CEC of biochar have often been found to be positively linked to each other [28]. However, this correlation of CEC and pH was found (pHKCl, R2 ¼ 0.91; pHH2O, R2 ¼ 0.58) only among control pyrolysis biochars and for the traditionally produced biochar the correlation was very low. 3.2. Pyrolysis-field ionization mass spectroscopy: total ion intensity and thermograms The total ion intensity (TII) counted during Py-FIMS of the CC

(89.3106 mg1), PC (116.0106 mg1) and RC (79.7106 mg1) biochars were comparable, which is logical as they were all prepared in the same pyrolysis unit and with equal process conditions. Ameloot [29] found comparable TII around 60e150106 mg1 for biochars produced in the same UGhent pyrolysis unit at 400
C. The TII of the RA (0.5106 mg1) and CA (2.3106 mg1) biochars produced traditionally in kilns was much lower. Sorge et al. [20] concluded that TII depends not only on the organic C content of samples, but also on structural features of the C. It appears logical that these are affected by biochar production conditions (temperature, duration, oxygen availability). For instance, it is known that at higher pyrolysis temperature the amorphous character of biochars lessens [30], although establishment of graphite-like crystalline structures seems unlikely. Ameloot [29] found a much lower TII for Py-FIMS analysis of biochars that were produced at 500
C as compared to 400
C. Likewise, the high resistance of the RA and CA biochars against volatilization in the Py-FIMS ion source may have resulted from a higher pyrolysis temperature in the coconut husks kiln (CA) and in the steel drum (RA) charring. In stark contrast, a higher TII in the PA (130.2106 mg1) compared to the PC biochar suggests temperatures exceeding 400
C to have been infrequent in the Palmyra nutshell kiln. A further insight in thermal behavior of the biochars is derived from the temperature resolved detection of the TII of the mass spectra. Most of the thermal degradation during Py-FIMS results from free radical reactions initiated by bond breaking (large molecules will break apart and rearrange in a characteristic way) and depends on the relative strengths of the bonds that hold the molecules together [31]. Thus the temperature course of the detected ion intensity of individual mass signals, as derived from Py-FIMS, gives an indication of the thermal energy required for the volatilizing of individual detected molecular compounds from the biochars [20,11]. The thermal evolution of m/z signals can be readily observed by plotting the ion intensity measured for these signals per temperature interval of 10
C in so-called ‘thermograms’ (Fig. 1). Volatilization started at 200
C for all three controlled pyrolysis biochar (CC, PC and RC) and evolved to a slightly skewed broad volatilization peak around 500
C. The traditionally produced biochars' thermograms instead showed very noisy patterns with earlier (from 50
C) volatilization. The PA biochar's thermogram was less noisy with a single peak at 400
C, approaching the controlled pyrolysis biochars. In the PA and RA spectra, 45% and 47% of TII was recorded at ionization chamber temperatures below 400
C while this was only 5% and 10% of TII for their PC and RC counterparts (Fig. 1). This demonstrates much more thermolabile material in the PA and RA biochars, in line with the reasoning that temperatures remained below 400
C at least in some parts of the traditional kilns. Considering the low recorded TII in case of RA, it cannot be excluded that thermolabile components were preferentially volatilized and detected though. There was only a smaller

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Fig. 1. Thermograms of the total ion intensities (upper right) and summed, averaged pyrolysis-field ionization mass spectra of six biochars produced out of three feedstocks (Palmyra Nutshell (P), Coconut shell (C), Rice Husks (R)) by either artisanal charring (suffix A) or controlled pyrolysis at Ghent University Bioscience Engineering lab (suffix C).

difference in <400
C volatilization between the CA (27%) and CC (18%) biochars. Instead the CA thermogram revealed substantial volatilization of molecular ions at high (>600
C) temperatures, i.e. originating from very thermostable matter. In general, the thermogram peak width of the traditionally produced biochars was broader than that of the controlled pyrolysis counterparts, regardless of ample (PA) or limited (CA, RA) recorded ion intensity. Hence, it is clear that traditional kiln or drum-based charring generates biochar that is a composite of matter varying in thermal stability. This diversity in thermal behavior of the biochar is likely due to the limited control over the traditional charring process with presumably substantial spatial and temporal heterogeneity in temperature, gas phase composition and pressure within the kiln or steel drum. 3.3. Chemometric analysis of the pyrolysis-field ionization mass spectroscopy datasets 3.3.1. General composition of the biochars Py-FIMS offered a more detailed insight into the molecular

chemical composition of the biochars. All mass spectra were characterized by intense peaks ranging from m/z 50 to 400 (Fig. 1). Important contributors of the detected TII were cfr [16]. identified marker peaks for carbohydrates (e.g. m/z 96, 110, 132, 162) (3e7% of TII) and lignin monomers and dimers and phenols (e.g. m/z 94, 108, 124, 168, 196, 212, 284, 342) (8e34% of TII). This shows that structural plant constituents like hemicellulose and lignin survived controlled pyrolysis and traditional charring. Using Py-FIMS, Ameloot [29] also found clear indications that slow pyrolysis of pine and willow wood partly leaves plant matter chemically untransformed. Identified marker m/z peaks for alkylaromatics (substituted benzenes and pentrenes, e.g. m/z 162, 198, 234, 246) and N-containing compounds (e.g. m/z 120, 195, 229) amounted 9e20% of TII and 4e16% of TII, respectively. Since alkylaromatics are not abundantly present in plant matter, these compounds must have formed during biochar production. Dong & Ouchi [32] postulated that catalytic thermal reactions at 120e300
C between phenols in lignin, and fatty acids or alcohols in lipids leads to the genesis of alkylaromatics in coal. Hence, most alkylaromatics in biochar likely originate from lignin and other phenols. Most N was

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Fig. 2. Score plots of the first two p.c.’s (p.c. 1 versus p.c. 2) calculated from the set of pyrolysis-field ionization mass spectra of six biochars produced out of three feedstocks (Palmyra Nutshell (P), Coconut shell (C), Rice Husks (R)) by either artisanal charring (suffix A) or controlled pyrolysis at Ghent University Biosystems Engineering lab (suffix C). Individual points of repeated Py-FIMS measurements per biochar are enclosed by a line.

detected in heterocyclic N-containing compounds, likely produced during charring of proteinaceous N, although heterocyclic N is also part of plant matter (e.g. in DNA, RNA, chlorophyll, pyridine). It should also be commented that Py-FIMS analysis of the artisanally produced biochars was hampered by their low thermal depolymerization and hence volatilization. Indeed, a large proportion of the TII could not be specifically assigned to organic compound classes (22e50% of TII) and is likely part of the aromatic ring cluster structure. Such selective detection of thermally untransformed C in biochar is indicated by the discrepancy between the average summed ion intensity of phenols, lignin monomers, lignin dimers and alkylaromatics (41% of TII) as opposed to generally reported high aromaticity levels. For example, the solid-state 13 C NMR-derived aromaticity of a kiln-produced wheat straw biochar was 85% [33] and similar to that of switchgrass biochar produced under controlled slow pyrolysis conditions at 500
C (82e93%).

3.3.2. Influence of biochar production method A principal component analysis was conducted on the Py-FIMS datasets. The principal component (p.c.) scores of individual measurements on the first versus the second component are shown in Fig. 2. The first two p.c.'s accounted for 71.0% of the total variance. Clearly, all three controlled pyrolysis biochars showed quite similar mass spectra when compared to the dissimilarity with and between artisanally prepared biochars. The 30 m/z signals having the highest absolute loadings on p.c. 1 and p.c. 2 are listed in Table 3. P.c. 2 separated the latter from the controlled pyrolysis biochars (Fig. 2). Dominant loadings derived from m/z indicative of N-containing compounds, alkylaromatics and lipids. But biochar production method in fact resulted in differences in content of nearly any detected organic building blocks. These differences in the biochemical composition are very clearly visualized by difference Py-FIMS mass spectra (controlled pyrolysis spectrum minus traditionally produced biochar spectrum (Fig. 3)). For the rice husks and coconut shell feedstocks, there were higher relative TII abundances of lower mass peaks and lower abundances of higher mass peaks in the artisanally compared to the controlled environment produced biochars. Both RC-RA and CC-CA difference spectra actually revealed an ‘infliction point’ around m/z 200e210. Compared to their controlled pyrolysis counterparts, the CA and RA biochars had higher abundances of marker m/z signals for phenols (m/z 94 phenol, 108 cresol, 120 vinylphenol), lignin monomers (m/z 154 syringic acid, 168 c1-syringic acid, 178 coniferyl aldehyde, 180 confery alcohol) and carbohydrates (m/z 96 c2-furan, 128, 132 anhydropentose, 142). These differences show that plant matter transformation appears to have been less in artisanal charring of coconut shells and rice husks. It should be borne in mind though that Py-FIMS analysis cannot discern between originally present carbohydrate and lignin fragments and recondensed or readsorbed moieties. Yet, given the much lower volatile matter content of the artisanal compared to the controlled pyrolysis biochars (Table 2), it is unlikely that the higher contribution to TII from m/z indicative of untransformed plant matter in case of CA and RA would have been due to extra re-adsorption from the vapor phase relative to CC and RC. For all three feedstocks, biochar production method also strongly affected abundance of tentatively assigned alkylaromatics. The TII of alkylaromatics was higher in case of the RA and CA compared to RC and CC biochars, logically following trends in phenols and lignin monomers, from which alkylaromatics are derived during pyrolysis. For example, the CC-CA difference peaks for m/z 78 benzene, 92 methyl-benzene, 106 xylene, 134 benzofuranone, 156 c2-alkylnaphthalene, 170 c3-alkylnaphthalene were negative (Fig. 3). In case of the Palmyra nutshells, selective conservation of

Table 3 Py-FIMS signals which load the first two principal components (p.c.) with loadings > j0.075j; m/z which dominantly load the p.c.'s are given in bold. p.c.1 m/z Positive loadings N-containing compounds: 257, 281, 285, 363 Sterols: 394, 412, 414, 416, 426 Lipids: 282, 378, 380, 422 Free fatty acids: 368, 382 Not specified: 283, 339, 341, 351, 353, 355, 357, 361, 365, 373, 375, 381, 391, 404, 443 m/z Negative loadings e

a

Partial overlap with lipids marker m/z signal.

p.c.2 N-containing compounds: 227 Lipids: 226, 238, 252, 254, 258, 266, 272 Alkylaromatics: 234, 236, 248, 262, 288 Free fatty acids: 242 Lignin Dimers: 300 Not specified: 211, 213, 225 N-containing compounds: 104, 129 Alkylaromatics: 78, 118, 142, 156a Phenols and lignin monomers: 166, 168 Carbohydrate pentose or hexose subunits: 96, 128a Not specified: 58, 169, 227

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7

Fig. 3. Difference pyrolysis-field ionization mass spectra of biochars produced by three different feedstocks (P: Palmyra Nutshell, C: Coconut Shell, R: Rice Husks): spectra of biochar produced by controlled pyrolysis minus spectra from counterpart biochars produced traditionally by kiln or drum charring. (Positive values indicate larger relative ion intensities in the Controlled pyrolysis biochars; negative values indicate larger relative ion intensities in traditionally produced biochars).

relatively untransformed plant matter by artisanal charring was also suggested, but by a very high abundance of marker m/z for long-chained lipids and sterols in the PA biochar compared to the PC biochar. Lipid marker m/z also dominantly loaded p.c.1 (Fig. 2), clearly discriminating the PA biochar's biochemical composition from all others. Surprisingly, the PA biochar's lower content of lignin and carbohydrate m/z markers compared to the PC biochar does not follow these indications of relatively preservation of original plant matter upon artisanal charring. Nevertheless, the summed abundance of m/z markers for aromatic building blocks (phenols and lignin monomers and dimers and alkylaromatics) of the PA biochar (17% of TII) was much lower than that of any other biochar (36e54% of TII). As was already indicated by the PA thermogram (Fig. 1), this once more suggests that lower temperatures prevailed in the PA kiln, because it is well known that aromaticity generally increases with biochar production temperature [34]. In

sum, from these data we cannot identify the specific plant constituents preserved by artisanal charring over controlled pyrolysis. While in the PA kiln, lipids were relatively retained, carbohydrates and lignin monomers were preserved in the CA kiln and RA rice drum. Understanding this discrepancy would require close tracking of pyrolysis conditions during artisanal charring. But this is certainly not a trivial task, given the expected large spatial and temporal heterogeneity in temperature and oxygen availability. 3.4. C mineralization experiment The first- and zero order kinetic model was able to describe the cumulative course of C mineralized very well in all cases (R2 always >0.99) (Fig. 4). Over the 60-days incubation 4.0% of the native soil organic matter was mineralized. In comparison, less than 0.2% of the biochar's C was mineralized, indicating its relative stability over

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SOM and to previous reports, e.g. by Bruun et al. [35], who found that wheat straw biochar produced between 500 and 575
C lost <5% of its C when incubated in soil for 100 days. While the mineralization rates kf and ks were equal in biochar and unamended soil, parameter Cf was significantly higher (P < 0.01) in the PC and RC treatments compared to the unamended control (Table 4), indicating the presence of a substantial biologically available C pool in these biochars. In sum, biochar amendment induced only limited differences in soil C mineralization (Fig. 4). Nevertheless, the predicted 9-weeks cumulative amount of C mineralized, i.e. at the end of the incubations, was significantly (P < 0.01) higher in biochar amended compared to the control soil, except for RA. Hence, part of the biochar appeared to be biodegradable over this time-frame. Biochar production method had a significant (P < 0.01) effect on the soil C mineralization, with a higher mean C mineralization upon soil amendment of controlled pyrolysis (325 ± 42 mg kg1 of dry soil) than artisanally produced biochars (280 ± 28 mg kg1 of dry soil). Biochar feedstock as well affected the cumulative C mineralization (P < 0.05), in the order: Palmyra nut shells (323 ± 24 mg kg1 of dry soil) > rice husks (303 ± 75 mg kg1 of dry soil) > coconut shells (280 ± 4 mg kg1 of dry soil). Soil dehydrogenase and b-glucosidase activity, soil mineral nitrogen content and soil pHH2O were all significantly (P < 0.05) different among the treatments, with lower values in the RA treatment and higher net biochar-derived C mineralization and dehydrogenase activity in the RC treatment. In all cases, biochar addition lowered the b-glucosidase activity relative to the control soil (P < 0.01). From these bulk analyses it already appears that biodegradability differs between biochars produced under traditional or in advanced pilot facilities and from different feedstocks. The net 9-weeks C mineralization and ks were positively correlated with the dehydrogenase activity (respectively r ¼ 0.91; P < 0.05 and r ¼ 0.97; P < 0.01), while they were negatively correlated with the biochar's pHKCl (respectively r ¼ 0.98, P < 0.01 and r ¼ 0.89; P < 0.05) and the Cf parameter (r ¼ 0.95; P < 0.01). Consequently, it appears that additional soil CO2 emission caused by biochar application stemmed from microbial activity and not from degradation of carbonates in the biochars. The 9-weeks cumulative C mineralization and Cf were positively correlated to the biochar's volatile matter content (respectively, r ¼ 0.81; P < 0.05 and r ¼ 0.87; P < 0.05). This relation between biochar biochemical composition and soil microbial activity was further investigated by correlation and regression analysis of the Py-FIMS thermometric and chemometric datasets and the kinetic C mineralization model parameters. 3.5. Relation between C mineralization and biochar composition

Fig. 4. Nine-week cumulative amount of C mineralized during aerobic incubation of soil amended with biochar or left unamended (control), with a first-order kinetic model fitted (solid lines) to the data. Bars represent ±1 standard deviations.

Although traditionally and controlled pyrolysis biochars could be distinguished from each other by p.c. 2 or by their different biodegradability, the p.c. 2 scores were not correlated to the fitted zero- and first order C mineralization model parameters. Neither did we find such relations between the dominant loading masses of p.c. 2 on the one hand and the TII on the other. This may imply that not the biochar biochemical composition per se, but rather the organization of its molecular-level constituents determines the size of the microbially degradable biochar C pool. Since bonding strength is reflected in Py-FIMS volatilization temperature, it instead is plausible to hypothesize that biochars with a larger share of thermolabile matter would be more biodegradable. However, there existed only weak (P < 0.1) negative correlations between the %TII <400
C and the net C-mineralization after 9 weeks and Cf. Nevertheless, %TII <400
C of lower mass peaks (from m/z 15 to 250) was significantly and negatively correlated with the net Cmineralization after 9 weeks (r ¼ 0.83; P ¼ 0.04) (Fig. 5), CA

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9

Table 4 Parameters of a parallel first and zero order kinetic model fitted to cumulative C mineralization data, Net biochar derived C mineralization, microbial biomass C and soil enzyme activities after 9 weeks of incubation (means ± standard errors, n ¼ 3). Cf is the size of the easily-mineralizable C pool, ks and kf are the mineralization rates of the slow and fast C pools, respectively. Biochara

Fitted kinetic model parameters Cf ()

CC CA PC PA RC RA Soil

7.42 6.12 9.00 6.41 10.06 6.92 6.37

kf (day ± ± ± ± ± ± ±

0.85ab 0.40a 0.57c 0.41ab 0.52bc 0.37a 0.55a

0.22 0.20 0.36 0.18 0.19 0.19 0.22

± ± ± ± ± ± ±

1

ks (g of C kg of dry soil C day1)

0.04a 0.01a 0.09a 0.08a 0.03a 0.01a 0.04a

3.2 3.5 4.0 3.8 4.1 2.9 3.2

)

1

± ± ± ± ± ± ±

0.6a 0.4a 0.6a 0.9a 0.2a 0.6a 0.3a

Net C mineralization (mg kg1 of dry soil)

12.2 18.2 75.0 34.7 91.6 15.5 e

± ± ± ± ± ±

28.9 ab 26.5ab 30.3ab 40.3ab 23.2b 28.8a

MBC (mg kg1 of dry soil)

80.6 116.7 102.6 92.9 82.2 66.4 97.1

± ± ± ± ± ± ±

4.9a 47.7a 32.0a 20.3a 8.8a 18.7a 24.1a

Dehydrogenase activity (mg of TPF kg1 of moist soil) 10.6 10.5 18.0 15.7 24.5 4.6 12.5

± ± ± ± ± ± ±

1.3ab 2.3ab 2.2bc 8.3abc 5.6c 1.3a 0.6ab

b-glucolidase activity

(mg of PNP kg1 of moist soil)

32.2 32.0 30.1 28.4 32.8 23.4 36.9

± ± ± ± ± ± ±

0.5bc 0.9bc 2.7abc 2.7ab 4.2bc 1.4a 3.5c

a Coconut shell (C), Palmyra nut shells (P) and Rice husk (R) chars, suffixed by C when the biochar production was under controlled conditions or A when artisanally produced. b Means followed by different lowercase letters are significantly different according to ANOVA and Tukey's HSD post-hoc test.

(r ¼ 0.82; P ¼ 0.05) and dehydrogenase activity (r ¼ 0.83; P ¼ 0.04). In other words, a higher content of low-molecular-weight thermolabile organic building blocks, i.e. the likely most bioavailable part of biochar, surprisingly seemed to inhibit microbial activity. From Py-FIMS analysis it is, however, very clear that a wide range of molecules constitute this thermolabile low molecular weight OM pool and it would be simplistic to term this fraction as either entirely a microbial substrate or as toxic to the soil microbial community. Based on previous extensive Py-FIMS investigations of soils, leachates, rhizodeposits, manure and plant matter Py-FIMS marker peaks m/z can be assigned to specific compound classes of which summed ion intensities may be calculated. Peak assignment is tentative for biochar, and with care we calculated summed ion intensities of carbohydrates, carbohydrates with pentose and hexose subunits (CHYDR), phenols and lignin monomers (PHLM), lignin dimers (LDIM), lipids, alkanes, alkenes, bound and free fatty acids and alkylmonoesters (LIPID), alkylaromatics (ALKY), peptides and other mainly heterocyclic N-containing compounds (NCOMP). By plotting thermograms for these compound classes (Fig. 6) it is

clear that higher proportions of thermolabile carbohydrates, heterocyclic N-compounds and alkylaromatics (and lipids in the case of the PC-PA pair) account for the higher %TII of <400
C matter of the biologically more resistant artisanally produced biochars (Fig. 1). All these abundantly present thermolabile components are obvious potential C-substrates, including even the n-alkylbenzenes, which have been shown to be quite susceptible to biodegradation in seawater [36]. Ion intensities of marker peaks for these OM classes were all negatively correlated to the cumulative C-mineralization. Although some amino acids are known to be strong reducing agents and have been correlated to soil anti-oxidant capacity, thus limiting oxidation of native soil OM [37], we rather hypothesize that the seemingly unlogical negative relation between proportion of thermolabile material and the cumulative C mineralization is indirect. I.e., a higher proportion of thermolabile material may imply a larger part of the biochar to be bioavailable, with however, also a higher availability of potentially inhibitory substances. Py-FIMS analysis did not allow the specific identification of toxic substances so a plethora of candidate substances remains. Graber et al. [38] identified compounds in biochar that are known to adversely affect microbial growth and survival at high concentrations. These included ethylene glycol and propylene glycol, hydroxy-propionic and butyric acids, benzoic acid and ocresol, the quinones (recorsinol and hydroquinone), and 2phenoxyethanol. Also volatile organic compounds absorbed to fresh biochar, like phenol and 3-methyl-phenol were found to inhibit growth of microbes like Bacillus mucilaginosus [39]. 4. Conclusions

Fig. 5. Relation between C mineralized from 6 biochars during 9-week lab incubation and the Py-FIMS quantified relative fraction of thermolabile low-molecular weight organic matter constituents.

Controlled pyrolysis and artisanal charring both partly preserve structural plant constituents like hemicellulose and lignin. A larger share of these constituents was found to survive the artisanal charring, especially thermolabile material (i.e. with Py-FIMS volatilization <400
C). This selective preservation may be due to lower prevailing temperature in parts of the charring biomass, inherent to weakly controlled artisanal charring. Principal component analysis on the Py-FIMS data indicated that lab-based pyrolysis, when compared to artisanal charring, generates more similar biochars from different feedstocks. Biochar biodegradability in soil was found to depend on production method with a higher soil microbial activity in case of controlled pyrolysis biochar amendment. The biochar's biochemical composition on its own, however, failed to sufficiently explain variation in biodegradability. Yet, net biocharderived C mineralization correlated unexpectedly inversely to the proportion of thermally labile low-molecular weight (m/z < 250) constituents. We therefore hypothesize this fraction to comprise

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Fig. 6. Thermograms of detected ion intensities of marker peaks for carbohydrates (CHYD), alkylaromatics (ALKY) and nitrogen-containing compounds (N-COMP) for six biochars, produced out of three feedstocks.

unidentified inhibiting constituents. Given the abundance of carbohydrates and N-compounds in this thermolabile biochar fraction as well, it should be considered as composite and may not be termed exclusively toxic nor a microbial substrate. Overall, we conclude that the overwhelming literature on biochemistry and biodegradability of controlled pyrolysis produced biochar does not apply to artisanally charred biomass, i.e. the ‘biochar’ to be most likely applied to soil in developing countries.

researcher for (12N3315N).

the

Research

Foundation

Flanders

(FWO)

Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biombioe.2015.10.025. References

Acknowledgments This research was carried out with financial support by FWOproject G.0426.13N.N. K. Jegajeevagan is funded by a VLIR-ICPPhD scholarship. N. Ameloot is working as a post-doctoral

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