Molecular characteristics of vermicompost and their relationship to preservation of inoculated nitrogen-fixing bacteria

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Molecular characteristics of vermicompost and their relationship to preservation of inoculated nitrogen-fixing bacteria Dariellys Martinez-Balmori a,b , Fábio Lopes Olivares a , Riccardo Spaccini c , Kamilla Pereira Aguiar a , Marcelo Francisco Araújo a , Natália Oliveira Aguiar a , Fernando Guridi b , Luciano Pasqualoto Canellas a,∗ a UENF – Universidade Estadual do Norte Fluminense Darcy Ribeiro, Núcleo de Desenvolvimento de Insumos Biológicos para a Agricultura (NUDIBA), Av. Alberto Lamego, 2000, Campos dos Goytacazes, 28013-602 Rio de Janeiro, Brazil b Departamento de Química, Universidad Agraria de La Habana, San José de las Lajas, Cuba c Dipartimento di Agraria, Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy

a r t i c l e

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Article history: Received 14 February 2013 Accepted 18 May 2013 Available online xxx Keywords: Thermochemolysis Pyrolysis off-line Structural–activity ratio Beneficial microorganisms Diazotrophs Microbial survival

a b s t r a c t The chemical nature of organic matter during the process of vermicomposting of cattle manure and filter cake from a sugar factory was characterized by thermochemolysis. The pyrolysates were mainly constituted of lignin moieties from propanoic acid units and short-chain (
1. Introduction A large number of organic wastes can be ingested by earthworms and excreted as vermicompost (VC), recycling nutrients and reducing environmental constraints [1]. This process accelerates organic residue stabilization, producing a greater proportion of hydrophobic organic matter, i.e., final stabilized organic products enriched with humic acids [2] having high biological activities [3,4]. VC is the final product of organic wastes processed by earthworms and can be used to alleviate soil contamination by heavy metals, PAHs and herbicides [5–7] due to its high chemical and biological activities. In another hand, there are a number of studies demonstrating the possibility of VC use as a vehicle for bioinoculants [8,9]. Microbiological processes such as biological nitrogen fixation, phosphate solubilization, and biostimulation can be

∗ Corresponding author. Tel.: +55 22 2739 7198. E-mail addresses: [email protected], [email protected] (L.P. Canellas).

improved with progressive knowledge about the relationship between the chemical nature of organic matter and the viability and activity of populations of natural or introduced beneficial microorganisms. Herbaspirillum seropedicae is an endophytic diazotrophic bacterium that colonizes mainly graminaceous plants such as sugarcane, rice, wheat, sorghum, and maize [10]. Canellas et al. [11] showed that inoculation of maize with H. seropedicae in the presence of humic acids isolated from VC increased the bacterial population associated with the plant root as well maize grain yield. In another study under a technological perspective stressing the importance of organic matter in driving a microbial process, Busato et al. [12] inoculated VC from cattle manure with a mixture bacteria able to solubilize phosphate and fix atmospheric nitrogen, resulting in increased phosphorus content for the agricultural substrate produced. However, in this study, the population of the introduced microorganisms decreased during VC maturation. A more effective plant growth medium using VC enriched with selected microorganisms depends on the maintenance of high levels of the selected

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population, since VC is a medium rich in microorganisms and microbial diversity. Detailed knowledge of the changes occurring in organic matter during vermicomposting can be useful for increasing the efficiency of VC use in many different processes, including VC enrichment with selected microorganisms or bioactive substances. Molecular characterization of organic matter can be improved using pyrolysis combined with methylation in the presence of tetramethylammonium hydroxide (TMAH) [13]. This method is efficient for transesterification of esters and methylation of fatty acids and lignin derivatives [14]. In fact, information obtained by preparative off-line pyrolysis (TMAH thermochemolysis) allows the use of large quantities of samples, allowing structural investigation of complex forms of natural organic compounds. Despite the importance of organic matter characterization to VC quality and the recognized efficiency of thermochemolysis in compost characterization [15,16], such approach using preparative off-line pyrolysis on VCs is still rare. The aim of this study was to monitor and compare the dynamics of organic matter transformation during vermicomposting of two raw materials (cattle manure and sugarcane filter cake) using thermochemolysis analysis. At the end of VC maturation, we assessed the size of the natural culturable diazotrophic population based on counts in semi-solid malate medium as well as by monitoring the survival of an introduced diazotrophic strain of H. seropedicae during storage using both VCs as inoculant carriers. A putative relationship between the molecular composition of a VC and its ability to preserve a microbial population was observed, opening a new trend for design of inoculant formulations based on the chemical composition of different VCs. This design should allow more effective application of microorganisms promoting plant growth for sustainable agriculture. 2. Materials and methods 2.1. VC preparation, sampling, and analysis of total C and N Two different VCs were prepared using cattle manure and filter cake from a sugarcane factory. Filter cake is the pulp resulting from the grinding of sugarcane. The sugarcane juice is treated with sulfur to clean it and with calcium to promote colloid flocculation. The resulting colorless cleaned juice is evaporated and the broth goes to vacuum filtration. The remaining solid stacked in the filter is called filter cake. Each cattle manure and filter cake were placed on a concrete cylinder (100 cm i.d.) with a 150-L capacity using two replicate (two cylinder per treatment) and the humidity was maintained at 65–70% with weekly addition of water followed by mixing. After approximately one month, the earthworm Eisenia foetida was introduced at a ratio of 5 kg of worms per m3 of organic residue. At the end of the transformation process (4 months after the distribution of the last organic residues), the worms were removed by placing a pile of fresh organic residue (cattle manure) in a corner of the container. The experiment were sampled at different time intervals, namely at 0, 30, 60, and 120 days after the introduction of the earthworms. Before sample collection, the content of the cylinder was vigorously mixed manually using a spade. The samples were air-dried, powdered by ball milling and finally sieved through a 500 ␮m mesh. Total organic carbon (C) and total nitrogen (N) contents were determined by dry combustion using an automatic CHN analyzer (Perkin-Elmer 2400 Series, Norwalk, CT, USA). 2.2. Off-line pyrolysis TMAH-GC–MS About 500 mg of dried VC was placed in a quartz boat and moistened with 1 mL of TMAH (25% in methanol) solution. After drying the mixture under a gentle stream of nitrogen for about 10 min, the

sample was introduced into a Pyrex tubular reactor (50 cm × 3.5 cm i.d.) and heated at 400 ◦ C for 30 min in a Barnstead Thermolyne 21100 furnace. The products released by thermochemolysis were continuously transferred by a flow of helium (20 mL/min) into two successive chloroform (50 mL) traps kept in ice/salt baths. The chloroform solutions were combined in a round flask and concentrated by rotoevaporation under reduced pressure. The residue was redissolved in 1 mL of chloroform and transferred to a glass vial for GC–MS analysis. Three thermochemolysis replicates were carried out for each compost sample. 2.3. Gas chromatography–mass spectrometry (GC–MS) The products of pyrolysis were analyzed by GC–MS. GC separations were carried out with a GCMS QP2010 Plus instrument (Shimadzu, Tokyo, Japan) equipped with an Rtx-5MS WCOT capillary column (Restek, 30 m × 0.25 mm; film thickness = 0.25 ␮m). Chromatographic separation was achieved with the following temperature program: 60 ◦ C for 1 min (isothermal), raised at 7 ◦ C min−1 to 100 ◦ C and then at 4 ◦ C min−1 to 320 ◦ C followed by 10 min at 320 ◦ C (isothermal). Helium was the carrier gas at 1.90 mL/min, the injector temperature was 250 ◦ C, and the split injection mode had a split flow at 30 mL/min. Mass spectra were obtained in EI mode (70 eV), scanning in the range of m/z 45–850 with a cycle time of 1 s. Compound identification was based on comparison of mass spectra with the NIST library database, published spectra, and real standards. For quantitative analysis, due to the large variety of detected compounds with different chromatographic responses, external calibration curves were built by mixing methyl esters and/or methyl ethers of the following molecular standards: tridecanoic acid, octadecanol, 16-hydroxyhexadecanoic acid, docosandioic acid, ␤-sitosterol, and cinnamic acid. Increasing amounts of standard mixtures were placed in a quartz boat and moistened with 0.5 mL of TMAH (25% in methanol) solution. The same thermochemolysis conditions as for compost samples were applied to the standards. 2.4. Quantification of natural culturable diazotrophic bacterial population associated with VC after maturation We took samples of VCs that had matured for 120 days after being prepared from cattle manure and sugarcane filter cake as described above. The samples were used to determine the population size of nitrogen-fixing bacteria capable of growth in semi-solid JNFb medium without nitrogen according to the following parameters: (a) Quantification of the basal population of diazotrophic bacteria (BP); (b) Quantification of the diazotrophic bacterial population after addition of a cocktail of soluble carbon sources (carbon induced basal population, CIBP); and (c) Quantification of diazotrophic bacterial population after sowing maize seeds in the vermicompost (rhizosphere induced basal population, RIBP). To count the bacteria, 10 g of both VCs were diluted in 90 mL saline solution (0.85% NaCl in 0.02% Tween 20) and incubated at 30 ◦ C under agitation at 150 rpm for 60 min. Thereafter, serial dilutions of 10−2 to 10−7 were made and aliquots (100 ␮L) of the respective dilutions were inoculated into 16 mL glass vials containing 5 mL of semi-solid JNFb medium, for which the composition of 1 L consists of: 5 g malic acid, 0.6 g K2 HPO4 , 1.8 g KH2 PO4 , 0.2 g MgSO4 ·7H2 O, 0.1 g NaCl, 0.02 g CaCl2 ·2H2 O, 4 mL Fe EDTA (1.64% sol), 2 mL bromothymol blue (0.5% solubilized in 0.2 M KOH), 2 mL of a culture medium for microelements, 1 mL vitamins for culture medium, adjusted to pH 5.8 with KOH and with 1.8 g agar added [17]. After inoculation, the flasks were incubated in an oven at 30 ◦ C for 7 days and evaluated for the presence of a thick white pellicle on the medium surface, which is an indication of the bacterium growth

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under N2 -fixing dependent. The population number was obtained using the McCrady table with 3 replicates per dilution [17]. The same procedure was adopted to quantify the diazotrophic bacterial population after carbon application (CIBP). Here, 10 g samples of both VCs were conditioned in sterilized plastic pots, incubated with 1 mL of an aqueous solution containing three carbon sources at final concentrations of 0.1% (glucose, mannitol, glycerol), to stimulate the basal population of microorganisms associated with VC. After 24 h of incubation at 32 ◦ C, aliquots were serially diluted and inoculated into semi-solid JNFb according to the procedure described above for BP quantification. For quantification of the rhizosphere induced basal population (RIBP), 100 g samples of both VCs were placed in different 300 mL plastic pot sowed with 10 seeds of maize per pot (Zea mays var. UENF 506/11). The seeds were surface-sterilized and heat-treated to eliminate seed-harboring microorganisms. At 7 days after germination, 1 g of VC under rhizosphere influence was obtained for quantification of diazotrophic bacteria as described above. All the data obtained represent the mean of three replicates. 2.5. Time-course quantification of introduced diazotrophic bacterial population after maturation of VC The population size of the diazotrophic bacterium H. seropedicae strain HRC 54, obtained from Laboratório de Biologia Celular e Tecidual culture collection (LBCT/UENF) was monitored over time after application in both mature VCs (cattle manure and sugarcane filter cake). The bacteria were grown in liquid DYGS medium [17] on a rotary shaker (120 rpm) at 30 ◦ C for 24 h. The bacterial suspension was adjusted to 109 viable cells per mL and the bacterial suspension (10 mL) was applied with a syringe into individual sealed plastic bags containing 100 g of different VCs that had matured for 120 days (the wet base was previously corrected to 40%). The assay was carried out under laboratory conditions at room temperature for 300 days after inoculation. Two individual bags (biological replicates) were collected monthly for each VC type and the population of H. seropedicae was determined based on two technical replicates per bag. Estimation of bacterial survival followed the same procedure described above. However, to selectively recover the introduced strain of H. seropedicae, we used semi-solid JNFb medium without nitrogen but containing nalidixic acid at 30 ␮g mL−1 as an antibiotic resistance marker. Uninoculated VCs were used as negative controls during the whole time course of the assay and showed no visible bacterial growth (data not shown). The estimated number was obtained for three replications per dilution by consulting the McCrady table. 3. Results During the vermicomposting process, the total organic carbon (TOC) content decreased (Fig. 1A) whereas the total nitrogen increased in the first 30 days and afterward remained stable in cattle manure and increases in filter cake VC. At the end of the incubation time, both VCs showed a higher N content than at the initial time, and this increase was higher in filter cake VC (+128%, Fig. 1B). As a consequence, the C/N ratio decreased, and at the end of the maturation period, the VC could be considered to be stabilized as pointed out by Jimenez and Garcia [18], who considered a C/N ratio lower than 15 a good indicator of the degree of maturity. The total ion chromatograms derived from the thermochemolysis products obtained during vermicomposting are shown in Figs. 2 and 3 for cattle manure and filter cake VC, respectively. The compounds identified in the VC samples are listed in Tables 1–3. Thermochemolysis of the VCs released more than 250 different molecules, which were identified as methyl ethers and esters of

Fig. 1. Evolution of total organic carbon (TOC) and nitrogen (TN) during vermicompost process of cattle manure and filter cake from sugar factory.

natural compounds. Most of these compounds originated from higher plant residues and microbial activity and were represented by lignin, waxes, and alkyl biopolymers and products of their transformation. Around 96 lignin components released by thermochemolysis from cattle manure and filter cake VC at the initial stage of maturation (Figs. 1 and 2) closely resembled those obtained for lignocellulose fractions of plant tissues and plant debris [19]. The different lignin derivatives (Table 1 summarizes the compounds shown in Figs. 1 and 2) are associated with current symbolism for basic lignin structures used in thermochemolysis analysis: P, p-hydroxyphenyl; G, guaiacyl (3-methoxy-4-hydroxyphenyl) and S, syringyl (3,5-dimethoxy-4-hydroxyphenyl) according to Spaccini and Piccolo [15]. As expected, the most representative lignin compound found among the pyrolyzed products, for both VCs, was a propenoic acid derivative (2-propenoic acid, 3-(4-methoxyphenyl)-methyl ester (P18), which is a basic component of lignified structures of herbaceous crops and grasses. The initial amount was 1.4-fold higher in VC from cattle manure (3259.42 mg kg−1 ), but at the end of composting, the final content was 1.6-fold lower than filter cake VC (619 mg kg−1 ) (Table 1). Various methylated guaiacyl derivatives were present at lower concentration with respect to propenoic acid derivatives while few syringyl units were found. The main G compounds were 3,4-dimethoxyphenol; 4-ethenyl-1,2-dimethoxy-; 1,2-dimethoxy4-(methoxymethyl)benzene; benzaldehyde, 3,4-dimethoxy-; cis/trans-1,2-dimethoxy-4-(2-methoxyethenyl)benzene; benzeneacetic acid, 3,4-dimethoxy-, methyl ester; cis/trans1,2-dimethoxy-4-(2-methoxyethenyl) benzene. The compound cis/trans-1,2-dimethoxy-4-(2-methoxyethenyl)benzene was found only in cattle manure VC at 120 days. A noticeable feature of the composting process was represented by the intense decomposition of lignin derivatives, which are often considered a refractory fraction of organic matter. Although a similar distribution of lignin monomers was found in both VCs at the initial maturation period (Table 1), an almost linear decrease in lignin components was

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Fig. 2. Total ionic current (TIC) obtained from off-line pyrolysis–GC–MS of cattle manure vermicompost at different maturation stage. (A) 0 days; (B) 30 days; (C) 60 days and (D) 120 days.

observed in the VC from cattle manure throughout the composting period, while an overall steady amount of lignin derivatives was found in the VC from filter cake at various stages of maturation. In fact, the initial content of lignin, obtained by the sum of all its derivatives, fell by 83% at the end of the cattle manure vermicomposting process. This decrease was lower in filter cake (17%), which showed 4297 mg kg−1 of lignin derivatives at the initial stage and 3546 mg kg−1 at 120 days. This was approximately the rate of decomposition found for the main lignin compound in both VCs (P18). The extent of lignin degradation may also be estimated by structural indices that are based on the relative amount of specific guaiacyl and syringyl thermochemolysis products [20]. These include both the aldehyde (G4,S4) and acid (G6,S6) derivatives, as well as the threo/erythro isomers of 1(3,4-dimethoxyphenyl)-1,2,3-trimethoxypropane (G14 and G15) and 1-(3,4,5-trimethoxyphenyl)-1,2,3-trimethoxypropane (S14 and S15) (Table 2). The aldehyde and acid forms of guaiacyl and syringyl structures result from the progressive oxidation of lignin monomers, while the corresponding homologs with a methoxylated side chain are indicative of unaltered lignin components, which retain intermolecular propyl ether linkages. The Ad/Al and  G indices are, respectively, the ratio of peak areas of acidic structures to the corresponding aldehydes (G6/G4, S6/S4) and to the sum of the peak areas for the threo/erythro isomers ( G ) G6/[G14 + G15];  S (S6/[S14 + S15]) [21]. These indices are considered to be good indicators of the biooxidative transformation

of lignin polymers [19] and are shown in Table 3. Both indices increased linearly with maturation stage for cattle manure VC while they increased for filter cake at 30 and 60 days and decreased at the end of maturation, confirming the high biological transformation of lignin components during vermicomposting. Lignin has been considered comparably resistant against microbial decomposition since only a limited group of fungi (white rot fungi) is capable of completely decomposing lignin to CO2 . However, our results showed that lignin is not a long-lived component of organic matter. Similar results have demonstrated this for soil organic matter [22]. Alkyl compounds were the most abundant thermochemolysis products released from VC samples of filter cake residues. The main alkyl compound in both VCs was short chain (
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Fig. 3. Total ionic current (TIC) obtained from off-line pyrolysis–GC–MS of filter cake vermicompost at different maturation stage. (A) 0 day; (B) 30 days; (C) 60 days and (D) 120 days.

which indicate organic matter input from plant material. Moreover, 12,13-epoxy-11-methoxy-9-octadecenoate is a plant component. The main FAMEs found in filter cake VC at all maturation stages were hexadecanoic and octadecenoic acids, typical of sugarcane fatty acid composition. As expected, due to a large content of sucrose in sugarcane, we found a large amount of long-chain alcohol units, mainly docosanol (C22), in methyl ether form (Table 3), while few and only small amounts of alcohols were detected in cattle manure VC. Alkenes and alkanes were present at relatively low amounts in both cattle manure and filter cake, and were observed to decrease during vermicomposting (Table 3). The compound 17-pentatriacontene was the only alkene found in cattle manure VC, while dodecene, pentadecene, nonadecene and tricosene were found in filter cake, although at low levels. A low amount of straight-chain alkanes was released from VC. Their exclusive presence as long-chain hydrocarbons (Table 3), the distinct predominance of odd versus even carbon numbers, and the high relative amount of eicosane or heneicosane and unidentified long-chain alkanes together with a low amount of long-chain alkenes suggested a prevalent origin of these alkyl compounds from the waxes of higher plants [23]. Tricyclic diterpenes and tetra- and pentacyclic triterpenes were distinctly identified, although in low amounts at the end of the vermicomposting period, among the thermochemolysis products

from VCs, especially filter cake VC (Table 3). At the end of the vermicomposting period, only a low content of squalene and fridoolean were found in cattle manure and only stigmasterol and ursane derivatives were found in filter cake. The tetracyclic triterpenes found in filter cake VC at relatively high (approximately 2800 mg kg−1 ) amounts were represented by methyl ethers and esters of methyl/ethyl cholesten-3-ol derivatives, whereas pentacyclic triterpenes were oleanane structures in cattle manure VC. Derivatives of plant stigmasterol (stigmasta-5,22-diene-3-ol acetate, Table 3) were present at high initial amounts in filter cake VC. Both sterol and triterpenol compounds decreased progressively with increasing maturity of VC samples, and at the end of incubation reached 25% of the initial content in cattle manure and 2% in filter cake VC. The high amount of terpenes and sterols in cattle manure VC was expected since these compounds are typically used to monitor fertilization using manure [24]. The presence of nitrogenated compounds in VC was notable during all stages of maturation. Derivative compounds from nitrogenated bases, especially uracil derivatives, were found in both VCs in line with very strong biological activity during vermicomposting, as demonstrated by high degradation and transformation of lignins and alkyl compounds. Moreover the contribution of microbial input is revealed by the identification of structural components of microbial cells represented by phospholipid fatty acids (PLFA) (Table 2), which are important microbial biomarkers of

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Table 1 Lignins derivatives found in different vermicomposting maturation stage (days) by Py–TMAH–GC–MS. Retention time (min)

10.46; 12.95 18.26 20.26 20.52 24.57 25.81 27.26 27.66 29.09 30.09 30.45 31.28 31.35 31.62 32.74; 32.99 34.30 34.79 35.36 35.74 36.63; 37.00; 39.75 38.83 38.84 38,.7 39.00 39.75 42.5 59.50–60.25

Lignins derivatives

Benzaldehyde, 4-methoxyPhenol, 3,4-dimethoxyBenzene, 4-ethenyl-1,2-dimethoxyBenzoic acid, 4-methoxy-, methyl ester 1,2-Dimethoxy-4-(methoxymethyl) benzene Benzaldehyde, 3,4-dimethoxycis/trans 1,2-Dimethoxy-4-(2-methoxyethenyl) benzene Benzenepropanoic acid, 4-methoxy-, methyl ester Ethanone, 1-(3,4-dimethoxyphenyl)Benzoic acid, 3,4-dimethoxy-, methyl ester Benzaldehyde, 3,4,5-trimethoxycis/trans 1,2-Dimethoxy-4-(2-methoxyethenyl) benzene Benzeneacetic acid, 3,4-dimethoxy-, methyl ester cis/trans 1,2-Dimethoxy-4-(2-methoxyethenyl) benzene 2-Propenoic acid, 3-(4-methoxyphenyl)-, methyl ester, Ethanone, 1-(3,4,5-trimethoxyphenyl)Benzoic acid, 3,4,5-trimethoxy-, methyl ester cis/trans 1,2-Dimethoxy-4-(3-methoxy-1-propenyl) benzene 1,2-Dimethoxy-4-(1,2-dimethoxyethyl) benzene 3-(3,4,5-Trimethoxyphenyl) propanoic acid threo/erythro 1,2-Dimethoxy-4-(1,2,3-trimethoxypropyl)benzene 2-Propenoic acid, 3-(3,4-dimethoxyphenyl)-, methyl ester 2-Propenoic acid, 3-(3,4-dimethoxyphenyl)-, methyl ester (trans) trans-3-Methoxy-1-(3,4-dimethoxyphenyl)-1-propene cis/trans 1,2-Dimethoxy-4-(1,3-dimethoxy-1-propenyl)benzene threo/erythro-1-(3,4,5-Trimethoxyphenyl)-1,2,3-trimethoxypropane cis-1-(3,4,5-Trimethoxyphenyl)-1,3-dimethoxyprop-1-ene 2-Propenoic acid, 3-(4-methoxyphenyl)-, methyl ester

Cattle manure (mg kg−1 )

Filter cake (mg kg−1 )

0-d

30-d

60-d

120-d

0-d

30-d

60-d

120-d

212 245 201 379 150 411 nd 170 78 350 304 269 47 237 3259 202 784 157 49 85 982 1074 52 45 151 nd nd 148

107 107 74 220 nd 189 nd 70 40 230 128 38 17 137 1735 96 580 78.76 25 53 425 588 nd nd 71 nd nd 116

65 85 73 195 52 306. nd 102 35 303 181 136 37 29 1618 74 379 54 53 54 334 542 nd nd 59 nd nd 34

25 14 20 72 nd 68 18 18 25 163 61 15 13 10 617 35 256 9 15 17 17 216 nd nd 13 nd nd 14

105 150 175 nd nd 61 nd 44 75 144 70 97 87 74 2236 108 108 nd nd nd nd 762 nd nd nd nd nd nd

82 27 117 nd nd 58 nd 28 22 82 120 59 21 21 1870 63 185 nd nd nd nd 657 nd nd nd nd nd nd

60 35 105 248 nd 162 nd 35 45 175 172 72 23 19 1438 52 262 nd nd nd nd 336 nd nd nd nd nd nd

140 40 61 235 81 185 nd 35 74 142 327 155 112 123 1049 51 457 nd nd nd nd 280 nd nd nd nd nd nd

Origin

P S G P18 G G G P G G S G G G P S S G G S G G P18 S13 G LG S14 G S16 P

nd = not detected. G, S and P represent guayacil, siryngil and p-hydroxiphenyl origins of lignins units.

natural organic matter found in soils, sediments, and recycled biomasses (15). The most abundant PLFA were identified as 13 and 12 methyltetradecanoic acids (iso and anteiso pentadecanoic acids), 14 and 15 methyl hexadecanoic acids (iso and anteiso heptadecanoic acids), and the cyclopropane-(2-hexyl)-octanoic acid. In addition, we found N compounds with different pyrrole units. These units are able to induce several physiological transformations in plants including lateral root induction by an auxin-like mechanism [24]. The main nitrogenous compound in cattle manure VC was a derivative of ␣-amino acid phenylalanine that is readily available for microbial mineralization despite its hydrophobic nature due to the presence of a benzyl group. Other amino acids in the form of purine, alanine, and methylbiobenzamide derivatives were found in both VCs. The content of nitrogenated compounds increased at the end of filter cake vermicomposting, as observed for total nitrogen (Fig. 1). In comparison with others compounds we found a relatively high content of carbohydrates derivatives in both VCs at the initial time and in filter cake VC at the end of incubation (Table 3). A lack of polysaccharides was previously noted after thermochemolysis of plant woody tissues and soil organic matter [15,25,26]. Our large samples and the high TOC may have favored the detection of this class of compounds. The main polysaccharide derivatives at the initial time of vermicomposting of cattle manure were furanone and furanose derivatives and methylated pentamethoxyheptanoic acid. These compounds are products of thermal rearrangement of sugars [27–29]. Some pyranoside structures from typical carbohydrate rings were detected in both VCs such as galacto-, gluco- and mannopyranoside derivatives. Methyl-2,3,5,-tetra-O-methyl ␣-d-glucopyranoside and methyl-2O-acetyl-3,4,6-tri-O-methyl-␣-d-mannopyranose and methyl-3,4di-O-acetyl-3,4,6-tri-O-methyl-␣-d-xylopyranoside were found only in cattle manure VC. The intense polysaccharide transformation was detectable in cattle manure VC since the initial content (around 4715 mg kg−1 ) decreased 10-fold at the end of the vermicomposting period, in contrast to filter cake, in which

carbohydrate compounds decreased around 60% in the first 30 days and reached the final stage of maturation with only 35% less carbohydrate derivatives than at the initial time. The main carbohydrate compounds at the start of filter cake vermicomposting were galactopyranoside and arabinofuranoside derivatives (Table 3). However, at the end of vermicomposting, methylated pentamethoxyheptanoic acids represented around 40% of the polysaccharides. Moreover, these polysaccharide-type materials may have been recalcitrant organic moieties in VC from filter cake residues. These could originate from glycolipid-like structures present in microbial cell wall biopolymers or cutinized hemicellulosic membranes since pyranoside structures from galacto, gluco, manno, xylo, and arabino sugars were found. In Table 3, we summarize the evolution of different classes of compounds during VC maturation. At the end of vermicomposting, mature filter cake VC showed 353% more FAMEs than cattle manure (Table 3). Other alkyl compounds were also increased in filter cake at 120 days compared to the same time for cattle manure, namely alcohols by 710%, alkenes by 1638%, and alkanes by 303%. The predominance of alkyl C in mature VC supports the view that aliphatic compounds are selectively preserved during biodegradation. However, these compounds are transformed by biological activity during vermicomposting. Nitrogenated compounds and carbohydrate derivatives were also present in greater amounts in filter cake. The only class of compounds higher in cattle manure VC was terpenes and steroids (Table 3). As a consequence, mature filter cake VC had organic matter with higher hydrophobicity than cattle manure. 4. Discussion Piccolo [30] postulated that recalcitrant hydrophobic components derived from plant degradation and microbial activities are able to randomly incorporate more polar molecules and hence protect them against degradation. This postulation is based on

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Table 2 Possible identities and yield (mg kg−1 ) of preparative off-line pyrolysis (TMAH thermochemolysis) products from vermicompost of two organic residues during different maturation stage (days). RT1 (min)

Fatty acids 27.5 33.5 36.1

Compound class

Cattle manure (mg kg−1 )

Filter cake (mg kg−1 )

0-d2

30-d

60-d

120-d

0-d

30-d

60-d

120-d

143 nd3 497

62 nd 235

112 nd 370

nd nd 56

70 nd 240

56 47 186

53 47 57

102 161 314

285

111

215

38

nd

34

112

155

252 831 64

180 751 63

137 734 30

27 131 7.69

nd 1.089 nd

99 1.010 nd

128 1052 nd

211 705 nd

145

111

78

nd

83

82

56

45

156 68 294 172 33 45 75 66 nd 152 595 253 179

98 48 310 98 214 39 37 52 nd 118 256 108 57

132 nd 321 145 272 nd 52 71 nd 71 316 68 85

23 nd 87 24 46 nd nd 17 nd 51 38 nd nd

214 43 400 45 237 184 60 41 nd nd 368 nd nd

119 53 406 49 147 212 55 41 nd nd 195 nd nd

87 32 407 55 135 148 48 35 nd nd 151 nd nd

173 45 474 101 188 247 38 60 106 nd 394 nd 269

75 168 99 2.089 58 189 637 102 1.126 106 647 123 193 56

85 106 102 1. 104 61 157 316 106 1.818 93 558 112 194 43

33 126 68 720 53 143 553 101 671 76 436 129 148 36

22 33 19 59 11 28 90 13 164 29 83 51 31 21

146 144 nd nd 180 335 nd 268 90 nd 2189 113 800 115

185 81 nd nd 90 177 nd 184 32 nd 1127 185 394 124

176 81 nd nd 50 174 nd 122 57 nd 710 147 275 96

132 61 nd nd 81 104 42 110 149 nd 520 96 151 35

52.1 53.4 54.0 54.5 55.4 57.2 57.8 60.8 61.3 63.0 64.1 64.6 67.3 70.2

Dodecanoic acid, ME Tetradecanoic acid ME Tetradecanoic acid, 13-methyl, ME (iso Pentadecanoic acid, ME) Tetradecanoic acid, 12-methyl, ME (ante-iso Pentadecanoic acid, ME) Pentadecanoic acid, ME Hexadecenoic acid, ME Hexadecanoic acid, ME Hexadecanoic acid, 15 methyl, ME (iso Heptadecanoic acid, ME) Hexadecanoic acid, 14 methyl, ME (ante-iso Heptadecanoic acid, ME) cy Heptadecanoic acid, ME Heptadecanoic acid, ME Octadecenoic acid, ME Octadecenoic acid, ME Octadecanoic acid, ME Hexadecanoic acid, 16 methoxy, ME Octadecanoic acid, 10-methyl, ME Cyclopropaneoctanoic acid, 2-hexyl-, ME Hexadecenoic acid, 9(10) 16 di methoxy, ME Hexadecanoic acid, 9(10)-16 di methoxy, ME Eicosanoic acid, ME Octadecenoic acid, 18 methoxy, ME Methyl 12,13 epoxy-11-methoxy-9-octadecenoic acid, ME Octadecenoic acid, 9(10)-18 di methoxy, ME Docosanoic acid, ME Nonadecanoic acid, 18-oxo-, ME Octadecanoic acid, 9,12,18 tri methoxy, ME Tricosanoic acid, ME Tetracosanoic acid, ME Docosanoic acid 22 methoxy ME Hexacosanoic acid, ME Tetracosanoic acid, 24 methoxy, ME Tricontanodienoic acid, DME Octacosanoic acid, ME Hexacosanoic acid, 26 methoxy, ME Triacontanoic acid, ME Dotriacontanoic acid, ME

Alcohols 43.74 48.48 52.67 56.51 58.34–68.65 62.45

n-Nonadecanol-1 10,11-Epoxy-n-undecan-1-ol n-Tetracosanol-1 1-Docosanol 1-Heptacosanol 1-Docosanol, methyl ether

nd 1065 141 nd 1.516 232

nd 515 nd nd 2.193 1.439

nd 712 60 nd 1.083 128

70 4 nd nd 132 39

nd nd 240 83 2.365 3.425

nd nd 58 40 1.159 1.876

nd nd 57 nd 783 1.052

nd nd 70 45 774 1.376

Alkenes 10.04 26.15 33.20; 36.18 43.95 50.60; 54.62 59.37

1-Dodecene 1-Pentadecene 1-Heptadecene 1-Tricosene 1-Nonadecene 17-Pentatriacontene

nd nd nd nd nd 233

nd nd nd nd nd 165

nd nd nd nd nd 204

nd nd nd nd nd 21

220 81 94 44 135 136

81 48 78 37 85 49

71 33 479 nd 33 33

146 76 38 nd 57 33

Alkanes 26.46 30.19 33.43 36.40; 39.10 46.39; 48.60; 50.72; 52.75; 54.71 56.60 58.40 63.50 65.09

Tetradecane Hexadecane Unidentified alkane Heptadecane Heneicosane Eicosane ou Heneicosane Nonacosane Unidentified alkane Unidentified alkane

nd nd 132 nd 131.01 32 36 120 nd

39 nd 78 nd 237 59 46 63 nd

115 75 93 nd 264 64 nd 102 nd

30 41 43 nd 31 9 14 45 nd

104 215 176 185 494 145 114 117 137

65 148 81 145 392 92 80 52 43

43 32 59 34 536 211 209 352 81

102 83 134 46 381 67 49 56 55

Terpenes and steroids 59.02 62.17

Squalene Cholest di-en

nd nd

157 nd

114 nd

222 nd

nd 180

nd nd

nd nd

nd nd

36.3 37.2 39.3/5 39.8 41.0 41.4 41.7 42.4 44.2 44.3 44.8 45.2 45.7 46.8 47.7 48.5 49.3 49.4 51.7

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8 Table 2 (Continued ) RT1 (min)

Compound class

Cattle manure (mg kg−1 ) 2

62.76 63.04; 65.4; 65.7; 67.4 63.18 63.36 63.70 64.94 65.10 65.26 65.46 65.71 66.38; 67.09; 68.5 66.40 67.87 68.51 69.41 Nitrogenous 10.05; 21.72 10.63; 11.04 11.47 11.68 13.76 17.85; 23.42 18.56; 33,75 19.52 19.80 20.70 31.90 33.85 34.40 35.96; 36.45

39.75; 40.06 45.99; 46.06 47.72 Carbohydrate derivatives 10.20 10.96 11.30 12.10; 26.00 12.19 12.27 12.45; 19.25 15.02; 16.38; 25.85; 27.07 17.38 20.55 21.29 22.42 41.27 43.04 47.78 48.16; 49.54 51.60 51.80 51.90 52.35

Filter cake (mg kg−1 )

0-d

30-d

60-d

120-d

0-d

30-d

60-d

120-d

Stigmasta-5,22-dien-3-MEt, Sterol Cholest-5-ene, 3-methoxy Cholest-4,6-dien-3-MEt ␤-Sitosterol, MEt Ergost-5-en-3-MEt 3,5-Dehidro 6-methoxy cholest-22-ene-21-ol Ergostan-3-ol, acetate Stigmaterol, acetate Cholesten 3-ol, MEt triterpenol Sterol 13,27 Cicloursan-3-en D:A-Friedooleanan-3-MEt Olean-12-en-28-acid ME

nd 90 68 nd nd nd 211 118 94 224 46 nd 58 221 nd

nd 56 35 nd nd nd 108 46 nd 84 nd nd 24 119 nd

nd 25 45 nd nd nd 165 79 51 124 nd nd nd 170 nd

nd 3 nd nd nd nd nd nd nd nd nd nd nd 30 nd

280 23 nd 103 286 888 nd nd 1.038 nd nd 1.824 nd nd 202

75 10 nd nd 49 nd nd nd 55 nd nd 76 nd nd nd

nd nd nd nd 138 50 nd nd 76 nd nd 155 nd nd nd

nd nd nd nd nd nd nd nd 24 nd nd 68 nd nd nd

1,3-Dimethyl uracil 3-Methyl hydrouracil 4-Hydroxy, 2-hydroxyaminopyrimidine N-Decanoylmorpholine 1-Methyl indole 1,3-Dimethyl-3,4,5,6-tetrahydro-2(1H)pyrimidinone 1-Ethyl-2-pyrrolidinecarboxamide 1-Methyl-hydrouracil 1,3-Dimethyl indole 4-Amino-2,6-dihydroxy-pyrimidine 1,3-Dimethyl hydrouracil N,N,9-Trimethyl purin-6-amine N-(p-vinylbenzoyl)-l-alanine 2-Methoxy-N-[2-(3,4dimethylphenoxy)ethyl]-4methylthiobenzamide N-Methoxycarbonyl-2,4-dimethyl phenylalanine 4,5,6,7-Tetrahydro-2-acethylaminobenzothiophene-3-carbohydrazide N,N-Dimethyl dacanamide

nd 287 148 nd 111 736

96 108 nd nd nd 266

150 157 nd nd 76 463

31 89 nd 67 39 72

69 nd nd nd 163 228

91 nd nd nd 67 160

80 nd nd nd 28 186

158 nd nd nd 183 512

606 nd 78.69 nd 171 264 161 680

194 nd nd nd 48 131 241 388

435 nd nd nd 70 120 1601 503

137 100.68 20.64 nd nd 30 211 90

187 nd 102.31 288 nd 123 nd 480

132 nd 58.51 82 nd 111 nd 245

146 nd nd 90 nd 83 nd 276

355 Nd 70 nd nd 175 981 6341

1513

556

670

27

829

109

37

927

nd

nd

nd

nd

nd

nd

nd

175

nd

50

nd

32

nd

nd

nd

nd

2-Methoxy 5-methyl phenol 2,3,4,6-tetra-O-methyl-d-galactopiranose 4-methoxy 2,5-dimethyl 3 (2H) furanone Methyl 2-(acetalamine)2-desoxy-4,6-di-Ometil-␣-d-galactopiranosídeo 3,4-dimethoxy toluene 3,4,6-Tri-O-methyl-d-glicose 3,5-Dimethoxy phenol 2,4,5,6,7-Pentamethoxy heptanoic acid, ME 2,3,5-Tri-O-methyl arabinofuranose 1,6-Anidro-␤-d-glucose, trimethyl ester 2,3,6-Tri-methyl-d-galactopiranose 1,3,5-Trimethoxy benzene Methyl 3,4-di-O-acethyl-2-O-methyl-␤glicero-d-glucoheptopiranosideo Methyl 3,4-di-O-acethyl-2-O-methyl-␣xylopiranosideo Methyl 2-O-acethyl-3,4,6-tri-O-methyl-␣manopiranoside Phenyl 2,3,4,6-tetra-O-methyl-␣-d-glucopiranosideo Methyl 4-O-acethyl-2,3,6-tri-O-methyl-␣-dgalactopiranoside Methyl 2,3,5,-tetra-O-methyl ␣-d-galactofuranoside Methyl 2,3,5,-tetra-O-methyl-␣-d-manofuranoside Methyl 2,3,5,-tetra-O-methyl-␣-d-glucopiranoside

69 179 849 nd

nd nd 309 nd

nd 64 390.92 nd

51 nd 193 nd

nd nd 567 nd

nd nd 303 40.44

nd nd 359 95.25

nd nd 399 137.70

nd nd 430 1095 199 nd 547 223 nd

nd nd 147 813 nd nd 380 88 nd

nd nd 89 513 nd nd 222 84 nd

45.02 nd 82 28 nd nd 41 nd nd

nd 77.74 359 298 nd 794 773 nd 102

83.93 nd 227 208 nd 400 278 nd nd

114.34 nd 172 190 nd nd 439 nd nd

106.46 nd 82 1.021 nd nd 373 nd nd

255

nd

106

nd

184

nd

nd

nd

104.88

nd

nd

nd

nd

nd

nd

nd

576

443

508

nd

123

nd

nd

450

nd

nd

nd

nd

590.87

82.97

80.08

nd

117

59

82

nd

170

nd

nd

98

nd

nd

nd

nd

75

nd

nd

nd

71.

70

59

nd

nd

nd

nd

nd

ME: methyl ester; Met: methyl ether. 1 Retention time. 2 Time of VC maturation. 3 Not detected.

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9

Table 3 Yields (␮g g−1 dry weight) and composition of main thermochemolysis products released from vermicompost at different maturities. Compounds

Lignins P G S G6/G4 S6/S4 G Alkyl-C (FAMES, alcohols, alkanes, alkenes) Terpenes and steroids Nitrogenous compounds Carbohydrates derivatives

Cattle manure (mg kg−1 )

Filter cake (mg kg−1 )

0-d

30-d

60-d

120-d

0-d

30-d

60-d

120-d

9946 4168 4157 1620 0.8 2.6 0.3 10,672 995 4909 4715

5123 2248 1912 963 1.2 4.5 0.5 9575 573 1949 2309

4789 2014 2012 772 1.0 2.1 0.91 9373 748 2873 2119

1712 748 602 383 2.4 4.2 9.6 2990 252 757 440

4297 2385 1475 436 2.4 1.5 – 14,751 4803 2470 4113

3415 1979 1037 399 1.4 1.5 – 8535 255 1120 1623

3242 1783 938 521 1.1 1.5 – 8027 420 989 1450

3546 1458 1212 874 0.8 1.4 – 8690 92 3473 2670

the concept that heterogeneous organic matter, such as humic substances, are present as supramolecular self-assembly with relatively loose binding of oligomers via hydrophobic interactions, ␲–␲ stacking, and hydrogen bonding [31–33]. It was previously demonstrated [34] that the organic compounds released in soils during mineralization of fresh maize residues were stabilized against microbial degradation by surrounding hydrophobic components. Based on such potential properties of preservation and considering the contrasting results of molecular characterization we observed for derivatives of cattle manure and sugarcane filter cake VCs, we decided to compare the influence of both mature VCs as complex media to support natural (Fig. 4A) or introduced culturable nitrogen-fixing bacteria (Fig. 4B). For the natural population, we proposed a new methodological approach to compare the ecological potential of both VCs for harboring natural diazotrophic bacteria. Such proposition has been justified by preliminary observations of low to undetectable populations of diazotrophic bacteria in mature VC, which results in low discriminative potential for different composting or vermicomposting materials suitable as microbial vehicles for agricultural bioinoculant formulations. The comparative diazotrophic basal population estimates are shown in Fig. 4A. Mature cattle manure VC exhibited no detectable diazotrophic bacteria, but surprisingly, filter cake VC was able to harbor 2.0 × 104 cells g−1 VC. This result may be due to the peculiar molecular characteristics of this VC conditioned by filter cake as a raw material and other aspects of the maturation process discussed later. Quantification of the diazotrophic bacteria population obtained after incubation with a cocktail of soluble carbon sources (CIBP) revealed an increase in the population size in filter cake VC (about 2log10 units) and the appearance of detectable populations in cattle manure VC (Fig. 4A, see CIBP column). These results were expected since the application of soluble carbon acts an extra energy source to support growth and diversity of microorganisms. A statistically significantly higher population size of N2 -fixing bacteria was observed for CIPB quantification of filter cake VC than for cattle manure VC (approximately 4log10 units), clearly highlighting the different carrying capacity of these materials. In addition, comparing the PB and CIPB estimation, we anticipate the potential of this proposed methodology for screening suitable materials as bioinoculant carriers. Both VCs were equally benefited based on quantification of diazotrophic bacteria population in the rhizosphere 7 days after sowing maize seeds, with no significant statistical differences in population size for rhizosphere induced basal population. Compared with CIBP quantification, plant root exudates were more diverse, and included complex compounds and biomacromolecules, such as amino acids, organic acids, carbohydrates, nucleic acid derivatives, vitamins and enzymes, which favor the

Fig. 4. (A) Estimation of natural culturable diazotrophic bacterial population associated with cattle manure and sugarcane filter cake mature vermicomposts (VC). The bacteria quantification was performed results were using JNFb semi-solid medium and the results were expressed in log n cells g−1 VC with 3 replicates per treatment. BP = quantification of the basal population of diazotrophic bacteria; CIPB = quantification carbon induced basal population; RIBP = quantification of rhizosphere induced basal population, RIBP (*) means population not detected; (B) population dynamics of introduced Herbaspirillum seropedicae strain HRC54 in both mature VCs (cattle manure and sugarcane filter cake) were expressed in log n cells g−1 VC with 3 replicates per treatment per time.

growth and multiplication of microorganisms and could explain the increased bacterial population relative to cattle manure VC (2log10 units). Otherwise, the noted reduction of the filter cake VC associated population in RIBP compared to CIBP could suggest that the type and concentration of carbon sources exudates from

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maize rhizosphere may be more limiting in supporting an increased population size. To evaluate the influence of the contrasting mature VC on microbial survival over the time, we introduced H. seropedicae strain HRC54 (naturally resistant to nalidixic acid at 30 ppm), a diazotrophic bacterium, and monitored its population for 10 months (Fig. 4B). The population dynamics revealed a decreasing trend in the microbial population in both VCs studied. However, the observed decrease was much more prominent in the cattle manure VC, especially between 30 and 60 days after inoculation. After this period, the population remained relatively constant until the end of the experiment. The bacterial population introduced to filter cake VC showed a more gradual decrease than in cattle manure VC, remaining above 106 cells g−1 , which represents a population of almost 2log10 units more bacterial cells than for cattle manure VC at 300 days after inoculation. It is worth mentioning that H. seropedicae has been ecologically characterized as an endophytic bacterium with low survival capacity in soil after artificial introduction, being undetectable in reisolation procedures after less than one month [10]. In our assay, both VC types were able to maintain high bacterial populations even after 10 months. Such findings allow us to point out that VC represents an excellent environment for long-term sustenance of the viability and activity of nitrogen-fixing bacterial cells. In addition, based on the population size of diazotrophic bacteria naturally associated with or introduced to VC, we found a higher microbial carrying capacity of sugarcane filter cake VC than cattle manure VC. Although many physicochemical or biological parameters of VC could be tentatively used to explain such differential preservation traits, our comparative data on molecular characterization obtained from thermochemolysis represent an important step in better understanding the relationship between the composition and quality of organic matter and its support of microbial density and activity. Even recognizing that it is not possible to point out specific compounds or classes of compounds directly involved with these differential microbial protective effects from such a complex plethora of chemical compounds identified by off-line pyrolysis TMAH, it appears that the more hydrophobic nature of filter cake contributes to the carrying capacity for microorganism. Such pursued knowledge is part of a new biofertilizer concept based on the combined use of stable organic matter and beneficial microorganisms, in which the selected bacterial strain benefits from the protective effect of stable organic matter followed by more consistent plant tissue colonization and effective plant growth promotion.

5. Conclusion The molecular changes during vermicomposting of cattle manure and sugarcane filter cake include intense transformation of lignin compounds. Alkyl C compounds were relatively well preserved, enhancing the hydrophobic nature of VC, especially from filter cake, which showed an increase in nitrogenated compounds and carbohydrate preservation, probably due to hydrophobic protection. Comparison of the natural diazotrophic bacterial population and monitoring the survival of an introduced strain of H. seropedicae on mature VC showed that sugarcane filter cake VC represents a more suitable material for preservation of the microbial population than cattle manure VC, again partially explained by the more hydrophobic nature of filter cake VC. The differential protective effect observed between VCs offers interesting possibilities in the direction of bioinoculant technological design, opening up the possibility of manipulating organic matter characteristics to enhance VC as a vehicle for inoculant delivery, allowing more

efficient plant inoculation and more effective plant growth promotion for sustainable agriculture.

Acknowledgements This work was supported by the following institutions: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundac¸ão de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenadoria de Aperfeic¸oamento de Pessoal de Nível Superior (CAPES), Instituto Nacional de Ciência e Tecnologia (INCT) para a Fixac¸ão Biológica de Nitrogênio, Internacional Foundation of Science (IFS) and OCWP.

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Please cite this article in press as: D. Martinez-Balmori, et al., Molecular characteristics of vermicompost and their relationship to preservation of inoculated nitrogen-fixing bacteria, J. Anal. Appl. Pyrol. (2013), http://dx.doi.org/10.1016/j.jaap.2013.05.015