Production of humic substances from cotton stalks biochar by fungal treatment with Ceriporiopsis subvermispora

Sustainable Energy Technologies and Assessments 13 (2016) 31–37

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Original Research Article

Production of humic substances from cotton stalks biochar by fungal treatment with Ceriporiopsis subvermispora Jersson Placido a,⇑, Sergio Capareda a, Raghuparty Karthikeyan b a b

Bio-Energy Testing and Analysis Laboratory (BETA Lab), Biological and Agricultural Engineering Department, Texas A&M University, College Station, United States Water Quality Engineering Laboratory, Biological and Agricultural Engineering Department, Texas A&M University, College Station, TX, United States

a r t i c l e

i n f o

Article history: Received 18 February 2015 Revised 18 August 2015 Accepted 17 November 2015

Keywords: Biochar Biodepolymerization Ceriporiopsis subvermispora Ligninolytic enzymes Humic substances

a b s t r a c t This study evaluated the biodepolymerization of biochar from cotton stalks gasification using four fungal strains Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Postia placenta, and Bjerkandera adusta. After the biological treatment, the liquid and solid phases were analyzed using UV–Vis and FT-IR spectroscopy. The biochar’s largest depolymerization was obtained by C. subvermispora. The depolymerization kinetic of C. subvermispora showed the production of laccase and manganese peroxidase and a correlation between the depolymerization and the production of ligninolytic enzymes. The highest production of ligninolytic enzymes and depolymerization was observed after 24 days. The modifications in the UV–Vis and FT-IR spectra exhibited the presence of functional groups related with humic substances from cultures with C. subvermispora and cotton stalks biochar. Ó 2015 Elsevier Ltd. All rights reserved.

Introduction The biomass conversion to biofuels is done principally by biochemical and thermal conversions. In one hand, the biological conversion uses microorganisms or enzymes for the transformation of biomass in biofuels; in there, biogas and bioethanol are the two principal products. On the other hand, thermal conversion is defined as the biomass transformation in chemical compounds using high temperatures at different oxygen conditions. Gasification and pyrolysis are the two most important processes inside the thermal conversion. Gasification is performed at temperatures higher than 500 °C and small oxygen quantities. In contrast, pyrolysis is conducted at lower temperatures (400–600 °C) and with complete absence of oxygen. In both processes, the final products are synthesis gas (syngas), bio-oil and biochar. The syngas can be used directly to produce energy (combustion) or to generate liquid fuels using the Fischer–Tropsch process. The bio-oil can be upgraded to generate liquid biofuels as gasoline, diesel or jet fuel (JP-8). Different to the other two, biochar’s principal uses are soil amendment [1] and activated carbons [2]. The gasification of cotton gin trash and cotton plant trash has been studied at different scales and has been shown as a viable alternative for energy production [3,4]. The principal product from ⇑ Corresponding author at: 201 Scoates Hall, 2117 TAMU, College Station, TX 77843, United States. Tel./fax: +1 (979) 255 4341. E-mail address: [email protected] (J. Placido). http://dx.doi.org/10.1016/j.seta.2015.11.004 2213-1388/Ó 2015 Elsevier Ltd. All rights reserved.

cotton wastes gasification is syngas (80–90%); nevertheless, 10% to 20% of the biomass is converted to biochar. As described earlier, biochar is the final waste generated from thermal conversion, and there is not a process that can use this byproduct besides soil amendment or activated carbon. To increase the use of thermal conversion as a competitive energy production strategy; it is necessary to develop a process that allows the transformation of biochar in product. A new option for biochar utilization is the depolymerization. Depolymerization is the transformation of biochar’s complex structures in less recalcitrant compounds. The Biochar’s carbon content is around 20% to 40%; however, in that form, it cannot be employed in other process or be consumed by microbes or other organisms. To make this carbon available for consumption, it is necessary to depolymerize the biochar in less recalcitrant compounds. At this moment, the depolymerization of any type of biochar has not been evaluated; nevertheless, the depolymerization of low rank coal has been performed using physical, chemical and biological treatments [5–7]. Low rank coal can be related to biochar because both present similar chemical structure and composition [8]. The biological depolymerization use microorganisms or their enzymes to depolymerize coal using enzymatic and nonenzymatic mechanisms. On one hand, the non-enzymatic mechanisms include production of alkaline substances and chelators. Bacillus sp. Y7 was capable of solubilize around 40% of lignite in 12 days; this process was carried by the production of an alkaline

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substance that facilitate the coal transformation [9]. Quigley et al. [10] evaluated several bacterial and fungal strains, and found that Streptomyces setonii could depolymerize coal by generating an alkali medium. On the other hand, the enzymatic approach employs ligninolytic fungi or ligninolytic enzymes. These strains are able to solubilize low rank coal into different types of products such as humic substances using laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP). Selvi et al. [11] evaluated the depolymerization of three types of low rank coal (Lignite, Sub bituminous, Bituminous) and different fungal strains. They found Pleurotus djamor as the most effective strain to depolymerize lignite, and an inverse relation between the rank of coal and the depolymerization [11]. Phanerochaete chrysosporium transformed 85% of the coal in smaller molecules, this transformation was mediated by the production of LiP and MnP. Additionally, this study found a relation between the fungal depolymerization and the use of low nitrogen media [12]. Nematoloma frowardii b19 and Clitocybula dusenii b11 transformed coal humic substances, the reaction showed an 80% of discoloration and the formation of yellowish products [13]. The coal solubilization has been evaluated in different reactor configurations (stirred tank, fluidized bed and packed bed reactors). The best configuration was the stirred tank with a coal weight loss of 24.2% [14] . The research aim was to select which of these four strains P. chrysosporium, Ceriporiopsis subvermispora, Postia placenta, and Bjerkandera adusta can depolymerize cotton stalks biochar and to know the depolymerization kinetic of the selected strain.

design with four replicates for each fungus. The response variable was the biochar depolymerization, which was measured by the increment in the absorbance at 450 nm. This measurement has been used to correlate the level of transformation of coal into humic substances by different authors [5,11,15–17]. At the end of the experiment, the results were analyzed using the software SAS system 9.3. The depolymerization kinetic was performed to the fungi with the highest biochar depolymerization. The kinetic was executed during 35 days using the same conditions used in the past experiment. The variables evaluated in the depolymerization kinetic were the glucose content, depolymerization, and enzymatic activity.

Materials and methods

Depolymerisation products characterization

Biochar production

At the end of the experiment the solid and liquid phases were centrifuged for 10 min at 2500 rpm (Sigma 2–6, Sigma Laborzentrifugen GmbH). The liquid phase was analyzed immediately and maintained frozen; In contrast, the solid phase was dried at 105 °C for 24 h and maintained at room temperature until use [18].

The biochar was obtained after the gasification process of cotton stalks from J.G. Boswell, San Joaquin valley, California. The gasification process was done at 500–600 °C using the TAMU Fluidized Bed Gasifier. It is a 305 mm diameter skidmounted fluidized bed gasifier with a rating of 70 kg/h. The Biochar obtained from the gasifier was filtered using a mesh of 255 microns. Fungal strains The biological material involved in this research were P. chrysosporium ATCC 24725, C. subvermispora ATCC 90466, P. placenta ATCC 44394, and B. adusta ATCC 62023, all of them donated by Dr. Daniel Cullen from the USDA Forest Products Laboratory. These strains were selected because were originally isolated in the United States and they are the most studied strains in the USA. The strains were stored in potato dextrose agar (PDA) at 4 °C until use. Media and growing conditions The experiments used the Kirk’s culture medium: KH2PO4 (0.20 g/L), MgSO4
7H2O (0.05 g/L), CaCl2 (0.01 g/L), ammonium tartrate (0.22 g/L), glucose (10 g/L), thiamine (0.10 g/L), 10 ml/L of trace elements solutions. Trace elements solution composition: CuSO4 7H2O (80 mg/L), H2MoO4 (50 mg/L), MnSO4 (33 mg/L), ZnSO4
7H2O (43 mg/L) and Fe(SO4)3 (50 mg/L). The experiment employed 250 ml Erlenmeyer and 50 ml of Kirk’s medium. The solution was inoculated with four 10 mm plugs of active mycelium from a solid culture of the fungi (5 days); after that, the Erlenmeyers were agitated at 150 rpm and 30 °C. The biochar was added to the culture at day 10th to obtain a biochar solution of 0.5%. The biochar and the fungus were kept at the previous conditions for other 10 days. The experiment utilized a completely randomized

Enzymatic activities determination The laccase activity was calculated using the enzymatic oxidation of 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). The reaction was followed spectrophotometrically using the change in the absorbance at 420 nm and a molar extinction coefficient (e) of e420 = 36,000 M1 cm1. The LiP activity measured the formation of veratryl aldehyde from veratryl alcohol using the absorbance changes at 310 nm and a molar extinction coefficient of e310 = 9300 M1 cm1. The measurement of the MnP used the absorbance change at 469 nm produced by the oxidation of 2,6-dimethoxypenol (DMF) (e469 = 27,500 M1 cm1). All the enzymatic activities were reported in units (U), which are defined as the quantity of enzyme that catalyze 1 lmol of substrate per minute.

Solid phase analysis The treated and untreated solid samples were analyzed using the Fourier Transform InfraRed (FT-IR) spectroscopy. The FT-IR (Shimadzu, IR Affinity-1 with a MIRacle universal sampling accessory) was used to evaluate the structural properties of the biochar with and without pretreatments. The infrared spectra collected range from 4000 to 700 cm1 with a resolution of 4 cm1. Liquid phase analysis The liquid phase was analyzed using the FT-IR and the UV–Vis spectroscopy. The FT-IR (Shimadzu, IR Affinity-1 with a MIRacle universal sampling accessory) was used to evaluate the biochar depolymerization by following the production of new signals in the liquid phase’s spectrum compared with the controls. The UV–Vis (Perking Elmer) analyses were measurements at specific wavelengths that allowed the evaluation of the biochar’s depolymerization degree and some chemical properties of the compounds in the liquid. The degree of depolymerization was measured by the increment in the absorbance at 450 nm which is related with the production of humic substances [9,11]. The E270/400, E465/665, E250/365, E280/472, E280/664, E472/664, coefficients were calculated to identify some characteristics of the compounds found in the liquid phase [19]. The glucose content was evaluated using High performance liquid chromatography (Waters 2690, Separations Module, Waters Corporation, Milford, MA) equipped with auto sampler, Shodex SP 810 packed column and a Refractive Index (RI) detector. The samples were centrifuged and filter before

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in the level of depolymerization between C. subvermispora and the other strains was probably generated by the simultaneous production of laccase and MnP. In other substances, the synergic effect of both enzymes achieved a greater degradation than the strains that just have one type of enzyme [21]. Additionally, C. subvermispora could produce other enzymes or biochemical compounds which could produce biochar depolymerization. In other fungal strains the presence of chelators such as salicylic acid, triethanolamine, 1,10-phenanthroline have been associated with the biodepolymerization process [11]. The effect of other enzymes and other biochemical compounds was not evaluated in this research, however, the effect of them in biochar depolymerization should be evaluated in further research in this area. On the other hand, the depolymerization realized by P. placenta was leaded by the production of laccases, this fungus was the second largest producer of laccases (Table 1).The production of laccase from P. placenta has been reported in the depolymerization of lignocellulosic structures [22]. The presence of a like-laccase enzyme in P. placenta was reported by Alfredsen and Fossdal [23]; in this case, the laccase activity was only expressed when the wood was furfurylated (addition of furfuryl alcohol) [23]. The production of laccase by P. placenta can be associated with furfurals or other compounds attached into the biochar’s surface that could activate the laccase’s production. Additionally, it is important to describe that these activities were only observed under alkaline pH (8–9). The correlation between depolymerization and enzymatic activity showed the production of laccase and MnP as one of the possible factors involved in biochar depolymerization. The correlation between enzymes production and depolymerization has been described in low rank coal and humic substances [5,7,24]. An example of that is the strain RBSlb, it exhibited an association between the production of laccase and MnP and the depolymerization of low rank coal. However, the strain RBSlb obtained similar levels of laccase and MnP during the depolymerization [16]. The production of laccase has been associated with the

injection, each sample ran for 20 min at a flow rate of 1 ml/min, 60 °C using HPLC water as mobile phase. Statistical analysis The experiment utilized a completely randomized design with four replicates for each fungus. The response variable was the biochar depolymerization. At the end of the experiment, the results were analyzed using a one way ANOVA and the Duncan’s test to select the best treatment. The statistical analyses were performed in the software SAS system 9.3 using the proc anova. Results and discussion Biochar biodepolymerization The level of biochar biodepolymerization was measured by the absorbance at 450 nm, (Fig. 1), this measurement have been proved by different researches to correlate with the presences of humic substances [5,11,15–17]. The largest depolymerization was observed in C. subvermispora and P. placenta. These two treatments had absorbances greater than the control. In fact, C. subvermispora incremented the absorbance more than two fold compared with the control. In contrast, P. chrysosporium and B. adusta exhibited absorbances lower than the control; however, their differences were not representative. A one way Anova and the Duncan’s test were used to identify the differences in the experiment. C. subvermispora exhibited the largest production of laccase, the second of MnP (see Table 1), and the largest depolymerization. C. subvermispora generated greater quantities of laccase than MnP; however, this is an uncommon situation for C. subvermispora, which generally produce large quantities of MnP than laccase. A significant production of laccase than MnP was also found in cultures of C. subvermispora degrading wood particles [20]. One of the reasons for the difference

0.400 0.350

Absorrbace at 450nm

0.300 0.250 0.200 0.150 0.100 0.050 0.000 C. subvermispora

P. placenta

Control

P. chrysosporium

B. adusta

Fig. 1. Effect of selected fungal species on biodepolymerization of cotton stalks biochar. The bars indicate the statistical difference between the experiments.

Table 1 Ligninolytic activities observed in the depolymerization experiment. Enzymatic activity

P. placenta

B. adusta

C. subvermispora

P. chrysosporium

Laccase MnP LiP

28 U/L – –

– 3.4 U/L –

47 U/L 1.1 U/L –

– – –

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depolymerization of low rank coal [16]; however, the majority of researches reported a requirement of LiP or MnP to achieve depolymerization similar as the observed by C. subvermispora. The greatest production of MnP was achieved by B. adusta (Table 1); nevertheless, the production of MnP by B. adusta did not reflected a high increment in the absorbance at 450 nm. This result can be explained by the lack of production of laccase. P. chrysosporium did not exhibit a measurable production of enzymes. That lack of production leads to think in a negative effect created by biochar in the enzymatic production of P. chrysosporium. Similar to P. chrysosporium, biochar has been reported to reduce the growth and enzymatic production of different fungal strains [25]. This inhibition can be caused by substances in the biochar that reduced the enzymes production and are toxic to the microorganisms (polycyclic aromatic hydrocarbons (PAHs), volatile organic compounds (VOCs), heavy metals) [26,27]. In addition to the 450 nm absorbance; other significant UV–Vis signals were measured to analyze the depolymerized compounds (Table 2). B. adusta obtained the largest absorbances at 280 and 340 nm. These values evidenced the presence of aromatic compounds which were produced by biochar depolymerization. The low absorbance obtained by B. adusta in the other wavelengths indicates the absence of large aromatic compounds like humic substances. The relation between E280/472 describes the presence of aromatic groups in humic substances and in this case to describe the compounds obtained by the depolymerization [28]. Using this correlation, P. placenta and C. subvermispora generated molecules with more aromatic compounds in their structure; opposite to B. adusta, where the aromatic compounds are not located in a larger molecule [28]. A low absorbance at 436 nm indicates molecules with more aliphatic, carbohydrates or nitriles compounds; in contrast, high absorbance at 436 nm indicates carboxylic and phenolic groups [19]. The absorbances obtained by P. placenta and C. subvermispora suggested the presence of carboxylic and phenolic groups in the molecules formed. The coefficient E472/664 is associated with the level of condensation of the chain of aromatic carbons. P. placenta had a high value, which indicates more aliphatic structures and less condensed aromatic structures; in contrast, C. subvermispora obtained a low value, it represents a large degree of condensation in the chain of aromatic carbons [19].

FT-IR analysis The spectra of the liquid phase from the fungal strains exhibited a characteristic band or group of signals between 950 and 1200 cm1 (the FT-IR spectra are in the supplementary data); however, in that band each treatment exhibited peaks with different wavenumber and absorbance. The treatments that achieved low depolymerization (P. chrysosporium and B. adusta) exhibited similar signals than the controls; though, these signals were shorter than the controls. The spectra from the cultures of P. placenta and C. subvermispora exhibited a new band between 1300 and 1500 cm1; this band describes C–H bonds [29]. Additionally to this band, C. subvermispora generated other band between 1500

and 1700 cm1, signals at this wavenumbers have been associated with compounds with aromatic rings and double bonds. The FT-IR analysis of the solid phase detected some changes in the biochar’s structure after the biological treatment. The biochar control with the Kirk’s medium evidenced three significant signals 872, 900–1200, and 1200–1700 cm1. The 872 cm1 signal is associated with lone aromatic C–H wag [30]. The band between 900 and 1200 cm1 showed the maximum point at 1030 cm1 and is correlated with the Si–O stretching. Silica compounds make part of biochar because they were part of the original biomass. Finally, the band between 1200 and 1700 cm1 exhibited the highest peak at 1396 cm1. This peak is associated with the –COO symmetric stretching [31]. The solid phase generated after the culture of B. adusta exhibited the most significant modifications in the FT-IR spectrum. The biochar exhibited three new bands (two narrow peaks at 783 and 1311 cm1 and a broad band between 1500 and 1700 cm1) and the disappearance of two signals (872 cm1 and the 1200–1700 cm1). The band at 900–1200 cm1 did not showed significant modifications compared with the control. The strong difference between the control and this treatment is related with the large biochar adsorption in the biomass. This adsorption produced a solid phase with a mixture of biochar and biomass which created a substantial change in the FT-IR spectra. P. chrysosporium exhibited a peak profile with three signals 872, 900–1200, and 1300–1500 cm1. The first two signals were observed in the control and slightly lower than it. The greatest change was at 1300–1500 cm1 where a new band replaced the 1500–1700 cm1 band. This new band had a significant reduction in absorbance versus the control. P. placenta and C. subvermispora exhibited similar signals at 872, 900–1200, 1300–1500, and 1500–1700 cm1. The differences between both strains were the signal’s intensity. P. placenta had higher absorbances at 900–1200 and 1500–1700 cm1. In contrast, C. subvermispora exhibited stronger signals at 872 and 1300– 1500 cm1. The most substantial differences between both treatments and the control were observed at 872 cm1 (aromatic carbons [30]); in this signal, both strains significantly reduced their intensity. The reduction in these signals and the increment in the 450 nm absorbance can be associated with the biochar biodepolymerization because the enzymes found in P. placenta and C. subvermispora (laccase and MnP) catalyze reactions with aromatic compounds. Another important change was the transformation of the large band at 1200–1700 cm1 in two bands at 1300–1500 and 1500–1700 cm1. These two bands are associated with aromatic compounds and C–H bonds. Depolymerization kinetic The depolymerization kinetic evaluated the changes in the liquid phase of C. subvermispora cultures with cotton stalks biochar through time. The experiment employed a destructive sampling methodology for 35 days and followed the next variables, enzymatic activities, depolymerization at 450 nm, glucose concentration, UV–Vis spectra and FT-IR spectra. Fig. 2 shows the

Table 2 UV–Vis specific absorbances of the liquid phase from the depolymerization experiment. Fungal strains

P. placenta B. adusta C. subvermispora P. chrysosporium Kirk’s medium control

Wavelengths analyzed 280 nm

340 nm

360 nm

400 nm

436 nm

465 nm

472 nm

600 nm

664 nm

2.928 3.792 3.443 2.405 2.551

2.094 2.642 1.343 0.752 0.547

1.262 0.893 0.691 0.494 0.371

0.488 0.302 0.390 0.200 0.211

0.295 0.137 0.316 0.125 0.172

0.230 0.089 0.281 0.105 0.155

0.221 0.067 0.273 0.102 0.152

0.105 0.015 0.193 0.070 0.119

0.074 0.008 0.164 0.062 0.111

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0.45

90 Biochar Addion

0.4

70

0.35

60

0.3 0 3

50

0.25

40

0.2

30

0.15

20

0.1

10

0.05 0 05

Absorrbance

Enzyymac acvity a (U U/L)

80

0

0

0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

30

32

34

Time (Days) Laccase

MnP

Glucose [g/L]

450 nm

Fig. 2. Cotton stalks biochar depolymerization in relation to the production of ligninolytic enzymes by C. subvermispora.

6 5

Absorb bance

depolymerization kinetic of C. subvermispora. Similar to the previous observation, this fungus did not showed a significant increment in its biomass, this low quantity of biomass can be related with an inhibition by the agitation. Similar to the previous experiment, C. subvermispora produced laccase and MnP and did not produce LiP. Laccase was the enzyme greatest produced followed by MnP, Fig. 2 shows two production peaks, a small one during days 8 and 10 and a large peak during days 24–27. The initial increment in the laccase production dropped after the biochar’s addition. This drop is related with the presence of compounds such as PAHs, VOCs, or heavy metals, which could affect the production of the enzymes. This type of behavior has been found in other type of microorganisms when they are exposed to biochar. After that drop, the laccase production achieved its maximum at day 27 (87 U/L). The MnP production had the same production profile than laccase activity. Similarly to laccase, the maximum activity obtained by MnP was achieved at day 27 (12 U/L). These results evidence that the ligninolytic enzymes production by C. subvermispora was not associated with the secondary metabolism. In fact, C. subvermispora was capable to produce enzymes in every stage of the kinetic. This finding coincides with other studies with C. subvermispora; where laccase and MnP were secreted during the growing and stationary phase [32]. The biochar was added at day 10; however, the absorbance at 450 nm kept low values until day 12. After day 12, the absorbance constantly increased until day 27 when the absorbance achieved a stationary phase. The absorbance increment coincides with the production of the enzymatic activities; this association evidenced a relationship between the increment in the absorbance and the production of laccase and MnP. As mentioned before, the absorbance at 450 nm correlates with the presence of depolymerization compounds. Therefore, the relation between depolymerization and enzyme production was reported in the depolymerization of low rank coal by N. frowardii b19. This fungus showed large quantities of MnP which allowed the increment of the 450 nm absorbance by the production of humic substances [13]. However, is important to mention that the depolymerization could be affected by other enzymes or chemicals in the fermentation that were not evaluated in this research. The glucose consumption by C. subvermispora was small (Fig. 2). In fact, at the end of the kinetic (day 35) was possible to detect remaining glucose (4 g/L). Glucose concentration reduced following a linear pattern during the kinetic; however, this linear patter evidenced two different slopes one between days 0 and

4

Day 10 Day 6

3

Day 2 Day 21

2

Day 15

Day 24 Day 27 Day 35

1 Day 0

0 200

250

300

350

400

450

500

550

600

Wavelength (nm) Fig. 3. UV–Vis spectra of the liquid phase of the depolymerization kinetic of experiment C. subvermispora.

12, and other between day 15 and 35. The first one was around 0.11 g/d; whereas, the second was around 0.15 g/d. This augment in glucose consumption correlates with the days with the greatest enzymes production; for that reason, the increment in enzyme production increased the fungus’ glucose needs. The UV–Vis spectra (Fig. 3) exhibited the most considerable variations after day 15 and were detected between 450 nm and 300 nm. This band is associated with humic substances production [19]. Additionally, the spectrum evidenced an increment in this band from day 15 to 27; after day 27, the absorbance increments were smaller. This behavior agrees with the pattern observed in the 450 nm absorbance. The absorbance at 436 nm increased with the days, especially the last 10 days. The augment in this wavelength evidenced an increment in the compounds with carboxylic and phenolic groups [19]. The relation between E280/472 decreased principally when the ligninolytic enzymes were formed. This relation exhibited the increment in the number of aromatic compounds in their structures [28]. The increment in the UV–Vis signals at 450 nm and the variations obtained in the other wavelengths have been related with the production of humic substances from low rank coal [19]. This relationship indicates that humic substances were produced from the biochar. The FT-IR analysis exhibited three significant bands similar as the previous experiment (900–1200, 1300–1500, and 1500–1700 cm1). The largest modifications were observed at the 1300–1500 and 1500–1700 cm1 bands. In the initial days of the culture several

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signals at these wavenumbers were observed; however, after day 15, these signals forming a unique broad band. The broad band at 1300–1500 cm1 had its maximum at 1395 and 1390 cm1. The first signal is associated with acid groups, and the second corresponds to C–H bending vibrations. In contrast, 1500–1700 cm1 band exhibited its maximum at 1575 cm1; signal associated with aromatic rings and carboxylic acids. The depolymerization of biochar using C. subvermispora was explained by the production of laccase and MnP. On one hand, Laccases are principally associated with the catalysis of phenolic aromatics. However, these types of enzymes are known for their lack of specificity; which allows them to catalysis different type of molecules. On the other hand, MnP is associated with the cleavage of aromatic structures with or without phenols. This ability is associated with the production of Mn(III), a low size and high oxidative power ion, that ion attack molecules such phenols, some methoxylated aromatics, chloroaromatics, and organic acids [15]. The cotton stalks biochar can be described as a composite biochar [33], this type of biochar is composed by phenols, quinones, pyrroles and small polyaromatic units linked with graphitic crystallites. The ligninolytic enzymes can attack some of the amorphous structures and initiate the degradation of phenols, quinones, pyrroles and small polyaromatic molecules inside the biochar. Besides the structure, MnP and laccases can act synergistically to depolymerize the biochar structure. As mentioned before, the biodepolymerization of low rank coal is associated with the MnP catalysis and in minor level with the laccase activity. The increment of laccase activity in C. subvermispora can be related with the structural differences evidenced by biochar and the low rank coal. The biochar has more substituents in the aromatic structures which facilitate the laccase catalysis and production. Further studies need to evaluate the capacity of purified laccase and MnP to depolymerize biochar structures, a great number of strains, and the effect of other types of biochar in the products and levels of depolymerization. Conclusions Gasification biochar can be depolymerized when is treated with an adequate fungal strain. In this research, C. subvermispora was the most efficient strain to depolymerize the gasification biochar compared with the other fungus tested. The greatest depolymerization with C. subvermispora was obtained after 27 days of culture and an association between the increment in the depolymerization and the increment in the ligninolytic enzymes was found. The UV–Vis and FT-IR analyses exhibited the presence of chemical groups which correlates with the presence of humic substances from biochar. These results pave a way to increase the economic and environmental feasibility of biomass thermal conversion by the production of chemicals from biochar using ligninolytic fungus. Acknowledgements The Colombian government and the Fulbright organization are acknowledged for the financial support via the granting of FULBRIGHT-COLCIENCIAS for the PhD studies of Jersson Plácido in the USA. The BioEnergy Testing and Analysis Laboratory at the Biological and Agricultural Engineering Department and the Texas AgriLife Research of Texas A&M University for funding this research. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.seta.2015.11.004.

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