Variations of organic matters and extracellular enzyme activities during biodrying of dewatered sludge with different bulking agents

Biochemical Engineering Journal 147 (2019) 126–135

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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Regular article

Variations of organic matters and extracellular enzyme activities during biodrying of dewatered sludge with different bulking agents Zongdi Haoa, Deokjin Jahngb, a b

T

⁎

School of Chemistry and Chemical Engineering, Shanghai University of Engineering Science, 333 Longteng Road, Shanghai, 201620, People's Republic of China Department of Environmental Engineering & Energy, Myongji University, 116 Myongjiro, Cheoingu, Yonginshi, Gyeonggido, 17058, Republic of Korea

H I GH L IG H T S

was biodried with various bulking agents. • DS achieved the highest water removal in the shortest biodrying period. • SCG readily biodegradable fractions in SCG accelerated DS biodrying. • Various promoted VS degradation into DOM and then humic- and fulvic-like substances. • SCG • Mannanase were significantly active during DS biodrying with SCG.

A R T I C LE I N FO

A B S T R A C T

Keywords: Biodrying Bulking agent Dewatered sludge Extracellular enzyme activity Organic matter degradation Spent coffee ground

Biodrying of dewatered sludge (DS) mixed with air-dried sludge (AS, Trial A), spent coffee ground (SCG, Trial B) and sawdust (SD, Trial C) was investigated by analyzing general physico-chemical parameters, organic matters degradation and extracellular enzyme activities. Results showed that SCG accelerated biodrying in terms of the highest water removal (80.50%) in the shortest period (8 days) in Trial B. Compared to Trial B, Trial A showed a shorter high-temperature period, and Trial C reached a lower temperature peak and biodrying period was long. During the high temperature period in Trial B, hemicellulose, lipids and proteins successively served as main sunbstrates for producing metabolic heat, and their degradation accounted for 28.9%, 34.7% and 10.9% of the consumed VS, respectively. Mean while, proteins and cellulose, representing 65.6% and 32.1% of the consumed VS were the main energy sources in Trials A and C, respectively. During high-temperature period in Trial B, SCG accelerated the conversion of VS to dissolved organic matters (DOM), and DOM to humic- and fulvic-like substances. The foremost induced enzyme was mannanase with activity of 3103 μg min−1 g−1 TS (total solids) on d 2, followed by protease, xylanase and cellulase with activities of 183, 1356 and 648 μg min−1 g−1 TS on d 6 in Trial B. Trial C showed evolution of xylanase and cellulase while Trial A slowly induced protease. These findings indicated that bulking agents with different compositions influenced the DS biodrying by stimulating different extracellular enzymes and thereby affecting the sequence and extent of organic matters degradation.

1. Introduction Biodrying has been demonstrated to be an effective pretreatment method for producing solid recovered fuel (SRF) from dewatered sludge (DS) [1]. In biodrying, water is removed from wet organic wastes by metabolic heat generated from the aerobic decomposition of biodegradable volatile solids (BVS) contained in the wastes [2]. Since biogenerated heat is only produced from biodegradable fractions of the waste [3], biodrying processes are strongly affected by BVS content. Although it is known to be composed mainly of excess biomass

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generated from the aerobic and/or anaerobic digestion of the organic matters contained in municipal sewage, DS is not readily biodegradable, and thus BVS available for heat production is limited [4]. It is also known that a bulking agent is needed for DS biodrying to provide structural support and to adjust the initial moisture content (MC) into optimal ranges [5]. Thus a bulking agent with a high biodegradation potential could serve as an extra BVS source for DS biodrying. Many reports have indeed showed that bulking agents not only enhance the void volume of a biodrying feedstock but also increase the amounts of energy sources for biodrying [6,7]. For example, Cai et al.

Corresponding author. E-mail address: [email protected] (D. Jahng).

https://doi.org/10.1016/j.bej.2019.04.001 Received 1 December 2018; Received in revised form 9 March 2019; Accepted 2 April 2019 Available online 03 April 2019 1369-703X/ © 2019 Published by Elsevier B.V.

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feedstock and distributing air. Dehumidified ambient air was supplied to the bottom of the reactor. The outlet gas exhausted from the top of the reactor passed through a condenser to remove the water vapor carried by the air stream prior to reaching a gas analyzer (Multi-Master, Sensoronic Co., Korea). Every 30 min, the CO2 concentrations were monitored by the gas analyzer. A 250 mm-long thermocouple was installed in the middle of the biodrying pile (200 mm above the bottom and 150 mm apart from the reactor wall), and the temperature was recorded every 1 h onto a data recorder [11].

[8] confirmed that hydrolyzable compounds were dominant fractions for bio-heat production during DS biodrying with sawdust (SD) as a bulking agent. For enhancing water removal from DS biodrying, various materials with high biodegradation potentials including straw [6], airdried sludge (AS) [9], wheat residues [10] and spent coffee ground (SCG) [11] have been used as bulking agents. It has also been reported that uses of different bulking agents result in different profiles of organic matters during biodrying because organic matter compositions in these bulking agents are different [6]. Similarly, differences in sequence and extent of organic matter biodegradation are also observed in composting [7] and municipal solid waste (MSW) biodrying [12]. During biodrying, solid organic matters need to be solubilized to be biodegradable because dissolved organic matters (DOM) are the direct substrates for the microorganisms. As a result, DOM greatly affected the microbial activity and biodrying efficiency [13]. DS biodrying performances were influenced by aeration strategy [13], bulking agents [11], and sludge type [10] because they all influenced DOM evolution. In order to form DOM from insoluble polymeric nutrients, proteins, amylums, hemicellulose, cellulose, etc., need to be decomposed first by extracellular enzymes. Then degradation products with appropriate molecular sizes are transported into microbial cells [14]. Since extracellular enzymes are produced by microorganisms, the degradation of organic matters and microbial community are deeply inter-connected in biodrying as Cai et al. [8] reported. Other researchers also characterized both composting and biodrying by investigating extracellular enzyme activities [15,16] and changes of microbial communities [11]. Recently, Zhang et al. [17] reported that lignocellulose-hydrolyzing enzymes, especially xylanase, cellulase and lignin peroxidase, were induced during DS biodrying with SD. It was also reported that different profiles of extracellular enzyme activities were found in different MSW biodrying processes. Among various enzymes, amylase was the most prominent because the main composition of the MSW was kitchen waste [18]. However, to the best of our knowledge, little is known how extracellular enzyme activities are associated with organic matter degradation during DS biodrying with various bulking agents. In this study, three bulking agents, AS, SCG and SD, were used for biodrying DS, during which temperature profiles and CO2 evolution, water removal, and volatile solids (VS) degradation were monitored. At the same time, degradation of organic matters and chemical characteristics of DOM were also investigated, and extracellular enzyme activities were analyzed. From these results, the reasons for the excellent performances of SCG as a bulking agent of DS biodrying were revealed.

2.3. Experimental set-up and operation Three trials were set up for biodrying DS with bulking agents AS (Trial A), SCG (Trial B) and SD (Trial C). For preparing the biodrying feedstock, DS and each bulking agent were thoroughly mixed manually with the mixing ratio of 3:1 (w/w, wet basis) for 60 min. The particle sizes of the mixtures were lower than 10 mm. Biodrying was conducted in the described reactor filled with 10 kg of feedstock. Free air space (FAS), an important parameter for evaluating the porosity of biodrying pile, was calculated by Eq.(1) [5,6]. The FAS of Trials A, B and C were 51.23%, 53.10% and 67.59%, respectively. FAS = 1 – BD·[(1-DM)/dw + DM·VS/dVS + DM·(1 – VS)/dASH] Eq. (1) Here, BD was the total bulk density on a wet basis (kg m−3). DM and VS were the contents of dry matter (wet weight basis) and VS (dry weight basis) of the bulk, respectively. dW, dVS and dASH were the densities of water, VS and ash (inorganic fraction), respectively. Values of dVS and dASH were 2.5 × 103 and 1.6 × 103 kg m−3, respectively [5]. During biodrying, the airflow rate was kept at 3 L min−1 [0.056 m3 −1 kg total solids (TS) h−1]. The biodrying pile was manually turned every two days, and the samples were taken after turning for analyzing MC, VS, organic matters, DOM and enzyme activities. 2.4. Organic matter analysis For measuring contents of organic matters, sludge mixtures under biodrying were sampled from reactors, air dried for 48 h at 60 °C (SOFW155; SciLab, Korea), and pulverized by using a grinder (HR2084, Xin Sheng Electrical Co., China). Proteins were estimated by multiplying the organic nitrogen value (total Kjeldahl nitrogen subtracted by total ammonia nitrogen) by 6.25 [19]. Lipids were gravimetrically measured after extracting 1 g of a sample with the mixture of chloroform and methanol (2:1, v/v) [20]. For the determination of amylums, the lipids and soluble sugars in 2 g samples were removed with 50 mL diethyl ether and 100 mL 85% (v/v) ethanol, respectively. The residual ethanol in the samples was removed by air-drying for 2 h at 60 °C. Then the samples were suspended in 25 mL deionized water and kept in a boiling water bath for 30 min for gelatinization. Subsequently, the samples were transferred to 55 °C water bath and added with 5 mL 1% (w/v) αamylase from Aspergillus oryzae (Sigma, St Louis, MO, USA). The reaction terminus was judged by using iodine solution (containing 0.02 M KI and 0.01 M I2) [21]. Then the mixture was centrifuged at 3000 rpm for 15 min, and the reducing sugar content in the supernatant was determined by using 3,5-dinitrosalicylic acid (DNS) reagent composed of 6.3 g L−1 DNS, 182 g L−1 potassium sodium tartrate, 5 g L−1 NaKC4H4O6·4H2O [potassium sodium L(+)-tartrate tetrahydrate], 5 g L−1 phenol, 5 g L−1 NaOH[22]. The amylums content was calculated based on multiplying the amount of reducing sugar by 0.9 [23]. The fiber components (hemicellulose, cellulose and lignin) were analyzed by following a proposed method [6,24] with slight modification. Briefly, 2 g air-dried sample was loaded in a test tube, and soluble fractions were removed by washing the sample with deionized water. Then the hemicellulose and cellulose were sequentially extracted by using aciddetergent solution (ADS) containing 0.5 mol L−1 H2SO4 and 20 g L−1 cetyl trimethylammonium bromide (CTAB) and 72% (w/w) H2SO4. The soluble reducing sugars dissolved in ADS and 72% H2SO4 were

2. Materials and methods 2.1. Characteristics of raw materials The DS was obtained from a belt press facility at a wastewater treatment plant in Yongin, Korea. SD was purchased from a traditional market in Yongin, Korea and used without any further treatment. SCG was collected from local coffee shops and dried at 105 °C until the MC ≤ 10 wt%. AS was prepared by spreading the DS under the ambient atmosphere and dried for a couple of weeks until the MC became constant [5]. Particle sizes of bulking agents were 0.05–10 mm in diameter. The characteristics of raw materials used in this study are shown in Table 1. 2.2. Biodrying reactor A laboratory biodrying reactor used in this study was an air-tight 28.3 L cylindrical vessel (inner diameter of 300 mm, height of 450 mm) made of polymethyl methacrylate (PMMA) insulated with 100 mmthick cotton. Headspace of the reactor was filled with cotton for preventing heat loss and absorbing condensed water. A perforated plate was located at the bottom of the reactor for supporting the biodrying 127

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Table 1 Characteristics of raw materials used in this study. DS MC (wt%) VS (wt%, dry basis) Organic components (wt%, dry basis) Lipids Proteins Amylums Hemicellulose Cellulose Lignin Dissolved organic matter (DOM) contents (mg g−1 TS) Dissolved organic carbon (DOC) Proteins Polysaccharides Reducing sugars Others Electric conductivity (EC) pH Dissolved chemical oxygen demand (DCOD) (mg L−1) NH3-N (mg g−1 TS) SR (S275-300/S350-400) a b

AS

SCG

SD

83.11 ± 0.12 69.85 ± 0.04

13.68 ± 0.01 74.27 ± 0.01

3.75 ± 0.18 97.94 ± 0.03

8.88 ± 0.07 97.06 ± 0.03

9.72 ± 0.18 35.33 ± 0.18 4.37 ± 0.16 5.41 ± 0.24 0.88 ± 0.07 23.05 ± 0.72

4.77 ± 0.25 39.38 ± 0.99 4.07 ± 0.25 8.05 ± 0.51 1.10 ± 0.16 25.88 ± 1.61

19.79 ± 0.77 15.06 ± 0.31 5.21 ± 0.62 25.76 ± 1.21 20.27 ± 1.62 23.74 ± 4.86

2.48 ± 0.34 0.87 ± 0.06 6.69 ± 0.34 12.81 ± 0.87 42.72 ± 1.22 26.60 ± 2.93

6.69 ± 0.32 5.63 ± 0.02 1.88 ± 0.16 NDb

19.49 ± 0.87 4.30 ± 0.14 9.16 ± 0.05 2.50 ± 0.23

35.86 ± 0.12 0.52 ± 0.05 19.60 ± 0.31 10.25 ± 0.18

14.18 ± 0.38 0.96 ± 0.01 11.18 ± 0.02 10.20 ± 0.20

409.5 ± 0.5 7.69 363 ± 13 4.76 ± 0.04 2.55

887.0 ± 3.6 7.48 2789 ± 62 2.34 ± 0.02 5.08

620.3 ± 0.9 5.36 4279 ± 48 0.14 ± 0.01 1.16

194.3 ± 0.3 5.77 1431 ± 96 ＜0.01 1.08

a

Mean ± standard deviation for triplicate analyses. ND: Not detected.

were separately pre-warmed at the each reaction temperature for 10 min. Enzyme activities of amylase, cellulase, xylanase and mannanase were measured based on generation rates of reducing sugars from the specific substrates under respective incubation conditions (Table 2). Briefly, the carbohydrate-hydrolyzing reactions were terminated by adding 2 mL of the DNS reagent followed by boiling in a water bath for 10 min. After cooling down the test tube, deionized water was added to make the volume to 15 mL, and absorbance at 540 nm was read by a spectrometer (Genesys 20, Thermo Fisher Scientific, Waltham, MA, USA). The control was run simultaneously with inactivated enzyme which was prepared by adding the DNS reagent prior to the substrates at the same conditions for each enzyme activity. The protease activity was assayed by determining the amount of amino acids released after incubation with casein as a substrate for 15 min at 40 °C as described by Zhang et al. [18] and Castaldi et al. [28]. All the enzyme activities were reported on basis of a gram TS of the sludge sample.

considered as hemicellulose and cellulose. Then, the ADS- and 72% H2SO4-extracted residual were combusted at 550 °C for 2 h, and the volatile fraction was considered as lignin [6]. 2.5. DOM extraction and analysis DOM was extracted from the fresh samples (2 g) using deionized water (solid to water mass ratio of 1:20) for 2 h on a shaking incubator (150 rpm) at 20 °C [25]. The suspensions were then centrifuged at 4000 rpm for 10 min and filtered through a 0.45-μm polytetrafluoroethylene membrane [10]. The pH and electric conductivity (EC) of the filtrates were measured using a pH meter (Orion star A211, Thermo Fisher Scientific, Waltham, MA, USA) and an EC meter (Orion star A222, Thermo Fisher Scientific, Waltham, MA, USA), respectively. NH3-N concentrations were analyzed by using the Ammonia Reagent Set (Hach, Loveland, CO, USA) following the Hach method 10,031. Chemical oxygen demand (COD) of the filtrates, which was defined as dissolved COD (DCOD) was measured using the high range COD digestion vials following Hach method 8000. Dissolved organic carbon (DOC) concentrations of the filtrates were determined by TOC-VCPN analyzer (Shimadzu, Japan). Ultraviolet and visible (UV) spectra were determined by a UV-1800 spectrometer (Shimadzu, Japan). Molecular weights of the DOM were estimated by calculating the spectral ratio (SR) of the 275–295 nm slope to 350–400 nm slope (S275-295/S350-400) [10,26]. Before measuring synchronous fluorescence spectra (SFS), the DOM extracts were diluted with HPLC-grade water (Samchun, Korea), and the final DOC was kept below 20 mg L−1. The SFS were measured using a FluoroMate FS-2 fluorescence spectrometer (Scinco, Korea) by ranging the excitation wavelengths from 250 to 550 nm with a constant off-set (Δλ) of 60 nm [27].

2.7. Statistical analysis All the experiments were conducted for three parallel samples. The relationships between pairs of variables were analyzed by the Pearson correlation coefficient using Statistical Product and Service Solutions (SPSS v22.0, IBM, USA). The significance threshold was assigned as P < 0.05. 3. Results and discussion 3.1. Variations of temperature and CO2 concentration As shown in Fig. 1a, the biodrying pile temperatures of all trials increased rapidly without lag phase and reached the high-temperature phase (≥ 45 °C) within 0.5 day. This rapid increase of temperature indicated that the abundant microorganisms in DS utilized energy sources contained in both DS and bulking agents. Turning decreased the pile temperature as biodrying pile was exposed to ambient air but did not influence metabolic heat production because the temperature rebounded rapidly. However, temperature profiles of three trials showed obvious differences after the first turning on d 2. In Trial A with AS, the temperature rebounded to 67.5 °C after the first turning on d 2, which was close to the first temperature peak (68.4 °C) on d 1.5. After the second peak, temperature of Trial A decreased and maintained at

2.6. Assay of extracellular enzyme activities For obtaining extracellular enzymes, 2 g of the fresh sample taken from the biodrying reactor were immersed in 10 mL phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4) and vortexed for 15 s. Then the suspension was centrifuged at 4000 rpm for 10 min. This aqueous extraction was repeated twice, and the combined supernatant was defined as the enzyme extract (EE) [18]. For measuring enzyme activities, EE and the respective substrate 128

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[28] Folin-phenol method, A660 Addition of 0.4 M trichloroacetic acid solution 15

Tyrosine-like amino acids

[48] DNS method, A540 Addition of DNS reagent 15

Reducing sugars

[47] DNS method, A540 Addition of DNS reagent 30

Reducing sugars

[46] Addition of DNS reagent 30

Reducing sugars

DNS method, Absorbance at 540 nm (A540) DNS method, A540 Reducing sugars Addition of DNS reagent 15

40

55

Fig. 1. (a) Temperature of the biodrying pile, and (b) CO2 concentration in the exit gas from the biodrying reactor. Vertical arrow (↓) indicates turning and sampling.

around 30 °C (5–10 °C higher than the ambient temperature). In Trial B with SCG, however, the temperature rebounded to 70.8 °C after the first turning, which was higher than the first peak (65.1 °C) on d 1. Meanwhile, the temperature of Trial B was maintained very high during d 2 to d 4 (≥ 65 °C). Even after the second turning on d 4, temperature increased to 67 °C and then decreased rapidly this time. The third turning on d 6 exerted almost no effect on the temperature, indicating that biodrying of Trial B was over. When DS was biodried with SD as the bulking agent (Trial C), the temperature profile was different from the other two trials in that temperature peak after each turning was lower than the previous one, i.e., the peak temperatures were 67.2 °C on d 0.9, 58.2 °C on d 2.6 and 47.5 °C on d 6. But, after d 6, the temperature rebounded to 49.1 °C, slightly higher than the previous one. In addition, temperature of Trial C maintained higher than Trials A and B after the third turning on d 6. These different temperature profiles of three trials suggested that bulking agents supplied extra substrates for microorganisms in biodrying piles and influenced the microbial metabolic heat production. Profiles of CO2 concentrations, another microbial activity indicator in exit gases, from biodrying reactors are shown in Fig. 1b. For all trials, the CO2 concentrations showed synchronous behaviors with the temperature elevation rate throughout the entire biodrying periods. After d 4, Trial A showed a moderate CO2 concentration (1–2 vol%) and

Protease

1% Locust bean gum in 0.05 M sodium phosphate buffer (pH 7) 1% Casein in 0.1 M sodium phosphate buffer (pH 7) Mannanase

50 Xylanase

Cellulase

1% Soluble starch in 0.1 M sodium phosphate buffer (pH 6.9) 0.8% Sodium carboxymethyl cellulose in 0.1 M sodium acetate buffer (pH 5.5) 1% Xylan in 0.1 M sodium acetate buffer (pH 5.5) Amylase

50

50

0.5 mL EE + 1.5 mL substrate 0.2 mL EE + 1.8 mL substrate 1 mL EE + 1 mL substrate 1 mL EE + 1 mL substrate 1 mL EE + 1 mL substrate

Termination Reaction time (min) Temperature(oC) Enzymolysis system

Substrate Enzymes

Table 2 Methods used for enzyme assays.

Incubation conditions

Assay method

Product

Measurement

[45]

References

Z. Hao and D. Jahng

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and 82.45 wt% with VS degradation ratios of 32.98%, 28.12% and 34.78% for Trials A, B and C, respectively. Due to the lower VS content of AS (Table 1), the VS of Trial A decreased more rapidly than Trials B and C, especially in the early stage of biodrying. There was no obvious VS decrease after d 8 in all trials. The mass of biodried products were 4.15, 3.64 and 4.03 kg with mass reduction of 58.5%, 63.6% and 59.7% for Trials A, B and C, respectively. Biodrying index (BI), which was defined as the mass ratio of removed water to degraded VS [2,4], was calculated to compare biodrying performances of three trials. The BI of Trials A, B and C were 6.53, 6.26 and 4.64, respectively. Although Trial B showed a slightly lower BI compared to Trial A, the abundant biodegradable organic matters in SCG prolonged high-temperature period and enhanced water removal. Li et al. [29] also found that the BI of DS biodrying with wheat residues as a bulking agent was slightly lower than that with straw or SD [30] albeit that BVS content and water removal were higher for wheat residues. Meanwhile, compared to the SD, which is the most frequently-used bulking agent [8], SCG achieved both higher water removal efficiency and BI. Thus it was necessary to investigate how organic matters contained in the mixture of DS and a bulking agent changed during biodrying.

3.3. Biodegradation of organic matters In order to investigate VS consumption in details, organic matters including lipids, proteins, amylums, hemicellulose, cellulose and lignin contained in the biodrying piles were analyzed (Fig. 3). Proteins was the main organic matter in Trial A, occupying 39.24 wt% (dry basis) of the feedstock. As a result, proteins decreased much in initial two days and were continuously degraded until d 12. After 14-day biodrying, the degraded proteins (0.57 kg) accounted for 65.6% of the consumed VS (0.87 kg). Other organic matters did not show evident changes except lipids, which were degraded slowly during the entire biodrying period. In the case of Trial B, only proteins and hemicellulose decreased slightly during the initial two days. Considering rapid increases of temperature and CO2 concentration in exit gas (Fig. 1), it was thought that the abundant dissolved polysaccharides (Table 1) supplied by SCG were firstly dissimilated by the microorganisms in the initial two days. Then hemicellulose and lipids decreased from 0.63 kg and 0.53 kg to 0.51 kg and 0.40 kg, respectively, from d 2 until d 4. Subsequently, proteins and lipids served as the main substrates and decreased from 0.77 kg and 0.40 kg to 0.67 kg and 0.19 kg, respectively, during d 4 to d 6. After d 6, there was no obvious change in VS components of Trial B except that cellulose was gradually biodegraded, because the cellulosedegrading microorganisms were thought to be developed slowly. Finally, consumed lipids (0.34 kg), proteins (0.11 kg), hemicellulose

Fig. 2. Variations of (a) MC and (b) VS/TS ratio during DS biodrying with different bulking agents.

temperature (Fig. 1a), indicating that biodegradation rates of organic matters were low. Trial B showed very low CO2 concentration (≤ 0.2 vol%) after d 6, which might be due to the complete exhaustion of rapidly-biodegradable substrates. For Trial C, the CO2 concentration and temperature were higher than Trials A and B after d 4, perhaps because organic matter degradation, although slow, still continued. The distinct CO2 emission profiles of three trials also suggested that bulking agents affected microbial respiration. 3.2. Water removal and VS degradation In biodrying, water in the waste is removed by metabolic heat produced at the expense of BVS. As more BVS are degraded, temperature increases higher and longer, which, in turn, removes more water. The water removal (and VS degradation) was calculated based on the differences between the initial and final water (and VS) masses of the biodrying mixture [11]. Since a high-temperature period lasted longer (Fig. 1a), Trial B achieved the lowest MC among three trials in a short time (MC of 31.20 wt% on d 8) with water removal as high as 80.50% (Fig. 2a). In Trials A and C, the water removal during the initial 8 days were only 65.04% and 59.90%, respectively. Although Trial C was kept warm even after d 6 until d 14, its water removal was 74.77% on d 14, which was lower than that of Trial B achieved by 8-day biodrying. Thus it was obvious that SCG accelerated DS biodrying, saving both time and energy (with less aeration). As Fig. 2b shows, VS contents decreased from 72.26 wt%, 88.67 wt% and 86.53 wt% to 65.78 wt%, 85.51 wt%

Fig. 3. Weight changes of organic matters contained in DS biodrying piles with different bulking agents. 130

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Trials A and B were much higher than that of Trial C. It was likely that the polymers of lignocellulose in SD were either insoluble or blocked by the insoluble matrix [6], resulting in low DOC in biodrying pile. The NH3-N concentration in DOM extract was determined and shown in Fig. 4e. The extremely high NH3-N concentration in Trial A certified that the main energy sources for metabolic heat production were proteins or other nitrogen-containing organic matters. Such high NH3-N concentration in biodrying pile might be toxic to microorganisms, resulting in a short high temperature period [34]. After d 2, NH3N concentration dropped, because NH3 was discharged from the biodrying pile by the forced aeration [35]. Besides, the sharp decline of NH3-N concentration in Trial C from d 6 to d 8 also demonstrated that the pH decrease was mainly due to the volatilization of ammonia. SR, which is considered to be inversely related to the molecular weights of the DOM [26], is presented in Fig. 4f. In all trials, it was obvious that the DOM molecular weights increased during the initial 2 days of biodrying, because high temperature facilitated the release of DOM from the organic solids. Afterwards, SR of Trials A and C decreased while that of Trial B increased slightly, indicating that the molecular weights in Trial B decreased. From these results, it was thought that organic matters were more actively decomposed in Trial B with SCG than other two trials. Fig. 5 presents the SFS of DOM during DS biodrying with various bulking agents. There were three distinctive regions with the excitation wavelength ranges of 250–300, 300–380, and 380–550 nm, which are known to be protein-like, fulvic-like, and humic-like DOM, respectively [27]. Thus it was clear that the protein-like DOM decreased in all three trials while fulvic-like and humic-like substances increased. The most conspicuous changes were observed in Trial B (Fig. 5b), as the fluorescence intensities of humic-like and fulvic-like substances greatly increased during d 2 to d 4 and maintained at high levels until the end of biodrying. In Trial C (Fig. 5c), although the fluorescence intensity indicated that the protein-like substances were consumed rapidly at the beginning of the biodrying, the production rates of humic- and fulviclike substances were lower than Trial B. In summary, the consumption rates of DOM in Trial B during biodrying were obviously higher than those of Trials A and C so that SCG accelerated biodrying with efficient water removal by providing a long and intense high-temperature period.

(0.28 kg) and cellulose (0.19 kg) accounted for 34.7%, 10.9%, 28.9% and 19.3% of the consumed VS (0.97 kg), respectively. When SD was supplied as a bulking agent (Trial C), lipids and proteins firstly decreased from 0.37 kg and 0.48 kg to 0.16 kg and 0.40 kg, respectively, during the first two days. After the first turning, the hemicellulose decreased from 0.43 kg to 0.34 kg, indicating that the high temperature was achieved mainly by consuming hemicellulose during d 2 to d 4. Thereafter, cellulose started to be hydrolyzed and became the main substrate during d 6 to d 10. Lipids decreased again during d 10 (0.13 kg) to d 12 (0.09 kg), and cellulose was the main substrate for last two days and decreased from 0.53 kg to 0.49 kg. Interestingly, amylums did not show obvious changes during the entire biodrying period, perhaps because its concentration was too low (< 5 wt%) to be the main substrate in the biodrying pile. At the beginning of biodrying, proteins and lipids, which were abundant in DS (Table 1), were consumed because they might be more readily-biodegradable than the main component of SD (lignocellulose). Unlike Trial B, when cellulose was the main energy source for biodrying, the temperature of Trial C increased to a moderate level (50 °C). After the second turning on d 4, there appeared a lag period of around 1 day, during which cellulose-degrading microorganisms might be developed. The lag period was also observed by Zhao et al. [6] and Cai et al. [8], who biodried DS with SD as a bulking agent, and they speculated that the cellulose-degrading microorganisms slowly dominated. Hao et al. [11] confirmed that the cellulase-producing phyla, mainly Actinobacteira, were enriched during the lag phase before temperature rebound. The degradation proportions of lipids, proteins, hemicellulose and cellulose in consumed VS (0.96 kg) of Trial C were 29.7% (0.28 kg), 12.7% (0.12 kg), 16.7% (0.16 kg) and 32.1% (0.31 kg), respectively. From these results, it was inferred that the different organic matters in three bulking agents resulted in different VS consumption (0.87 kg, 0.97 kg and 0.96 kg for Trials A, B and C), and different patterns of organic matters degradation. With abundant easily-biodegradable organic matters such as lipids, hemicellulose and proteins in SCG, Trial B showed higher heat and CO2 production than Trial C with SD mainly composed of cellulose. Although it seemed that proteins in AS were also easily-biodegradable, low VS content was the main restriction for heat and CO2 production in Trial A. Contents of lipids and hemicellulose in SCG were higher than other two bulking agents. Meanwhile, proteins content of SCG was also higher compared to SD. Higher contents of these organic matters in SCG seemed to be beneficial for inducing various thermophilic bacteria.

3.5. Variations of enzyme activities It is well known that the extracellular enzymes are responsible for de-polymerizing high molecular-weight organic matters during biological degradation [36]. The addition of different bulking agents might induce the different expression of these hydrolyzing enzymes during biodrying. As shown in Fig. 6a, due to low amylums contents in DS and three bulking agents (Table 1), the amylase activity markedly decreased after d 2. In case of Trial A, cellulase (Fig. 6b), xylanase (Fig. 6c) and mannanase (Fig. 6d) activities were low during the entire biodrying period, probably because contents of substrates for these enzymes were low in DS and AS (Table 1). Although proteins, the most abundant organic substances in DS and AS, were considered as the main energy sources for biodrying (Fig. 3), protease activity in the early stages of Trial A was not as high as expected (Fig. 6e). Instead, the protease activity increased gradually until biodrying was finished. For Trial A, biodrying feedstock was prepared by mixing DS and AS, both were excess biomass consisted of bacteria, protozoa and small amount of fungus [37]. Thus the biodrying occurred by cryptic growth of the microorganisms, which meant that the energy sources for biological heat production were lysed cells [38]. It is also well known that nitrogen content of bacterial cell is 7–12 wt% (dry basis) [39]. In this study, the protein content was determined by using the traditional Kjeldahl method, in which the proteins, amino acids, peptides, peptidoglycans, and other organic substances containing amidogen were degraded to produce ammonia. Thus in Trial A, various nitrogen-

3.4. Chemical characteristics of DOM In order to understand microbial degradation of organic matters, biodrying piles were extracted with water, and the obtained DOM was analyzed. Since different organic matters were degraded differently during biodrying, it was likely that the behavior of DOM would also be different among three trials. Fig. 4 shows changes of DOM parameters during biodrying. The high EC of DOM of Trial A seemed to be coincident with protein content in Fig. 3. As proteins were degraded (Fig. 3), a large amount of NH4+ was released (Fig. 4e), which increased EC [10]. The EC of Trial B increased gradually while that of Trial C increased until d 4 and then decreased. This might be due to that the mineral salts released from the mineralization of the organic matters were assimilated by the thermophilic microorganisms [31]. Meanwhile, the gaseous metabolites such as CO2 and NH3 could be firstly captured in moisture contained in the biodrying pile and then released together by forced aeration. For all trials, pH increased and then decreased. This decrease of pH might be caused by the volatilization of ammonia and/or formation of organic acids [32,33]. The pH of Trial C decreased more rapidly (pH 6.2 on d 8) than others, perhaps due to that more organic acids were produced from metabolism of cellulose and hemicellulose. In three trials, DOC and DCOD increased first and then became stable. The DOC and DCOD concentrations of 131

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Fig. 4. Time courses of characteristic parameters of DOM water-extracted from biodrying piles. (a) EC; (b) pH; (c) DOC; (d) DCOD; (e) NH3-N; (f) SR.

rapidly increased on d 6 (Fig. 6e) and proteins were mainly biodegraded during d 4 to d 6, indicating that the proteins were also important for biodrying in Trial B. The biodegradation sequence of organic matters in Trial B was hemicellulose, proteins and then cellulose, indicating that hemicellulose was more biodegradable than proteins and cellulose. In Trial C, it was suspected that the hemicellulose was mainly xylan because xylanase activity was very high after d 4 (Fig. 6c). It also seemed that the microorganisms hydrolyzed hemicellulose prior to cellulose since the activity of xylanase (3578 μg min−1 g−1 TS on d 6) was much higher than cellulase (1136 μg min−1 g−1 TS on d 6) (Fig. 6b–c). Both xylanase and cellulase activities reached peak values of 9364 and 2500 μg min−1 g−1 TS on d 10. These variations of enzyme activities well agreed with patterns of organic matter degradation shown in Fig. 3. For example, the biodegradation of cellulose mainly

containing organic matters, peptides, amino acids, nucleic acids and peptidoglycan (the main component of bacterial cell wall) in sludge might be the main energy sources rather than proteins [40]. During DS biodrying with SCG as a bulking agent (Trial B), activities of amylase (Fig. 6a), cellulase (Fig. 6b) and xylanase (Fig. 6c) were moderate. On the contrary, the mannanase activity boomed (3103 and 4295 μg min−1 g−1 TS on d 2 and d 4, respectively) and maintained higher than Trials A and C until biodrying was finished (Fig. 6d). Correspondingly, obvious decrease of hemicellulose was observed during d 2 to d 4 in Trial B (Fig. 3). It is well known that the main hemicelluloses in SCG are mannan (more than 50% of the total hemicellulose) and galactan (35–45% of the total hemicellulose) [41]. Thus it seemed that the high mannanase activity was induced by the high content of mannan and played an important role for accelerating biodrying of Trial B. It was also noticed that protease activity in Trial B 132

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which might be transported across the bacterial cells without depolymerization [43]. Secondly, most lipases in the biodrying pile might be membrane-bounded or intracellular [44] so that the EE did not contain the lipases. Table 3 shows the correlation coefficients among enzymes. In Trial A, xylanase and cellulase activities were positively correlated with each other significantly (P < 0.05), while protease activity was negatively correlated with other enzymes. Mannanase also showed positive correlation with xylanase and cellulase. Since AS with low contents of amylums, cellulose and hemicellulose (Table 1) was used as a bulking agent in Trial A, activities of amylase, cellulase, xylanase and mannanase kept decreasing during the whole biodrying process, while protease activity gradually increased. As a result, protease activity was negatively correlated with activities of other enzymes. When SCG was used as the bulking agent (Trial B), however, synergistic effects were observed among lignocellulose-degrading enzymes (xylanase, mannanase and cellulase) because they were almost positively correlated with each other significantly (P < 0.05). Furthermore, protease activity was positively related to lignocellulose-degrading enzyme activities, particularly xylanase with P < 0.01. These results suggested that when SCG was added into the biodrying feedstock, the abundant BVS induced various extracellular enzymes, causing synergistic effects on various organic matters biodegradation, especially proteins and hemicellulose. Previous study showed that biodrying with SCG as a bulking agent enriched thermophilic microorganisms [11], indicating that the simultaneous biodegradation of various organic matters was contributed by the thrived thermophilic genera, which were reported to be able to secret various heat-stable extracellular enzymes [8]. In Trial C, lignocellulose-degrading enzyme activities were all positively correlated to each other significantly (P < 0.01), while negatively related to protease. Other than Trial B, the biodegradation of proteins and lignocellulose was not smoothly linked during biodrying with SD as a bulking agent. This was because the main constituent of SD was lignocellulose, while proteins were mainly from DS. Also the development of lignocellulose-degrading microorganisms, which were important for enzyme secretion, was slow in Trial C due to that cellulose was the main component of SD (Table 1). These results suggested that various nutrients supplied by bulking agents induced extracellular enzymes in different levels, resulting in different biodrying performance. 4. Conclusions Bulking agents with different compositions of organic matters affected degradation of organic matters and enzyme activities so that heat production, CO2 emissions and water removal were varied. Biodrying with AS with a high content of lysed microorganisms and SD containing abundant cellulose resulted in short high-temperature period and long biodrying period, respectively, while SCG with various readily-biodegradable organic matters accelerated biodrying, achieving the highest water removal (80.50%) with the shortest biodrying period (8 days). Although biodrying with AS and SCG showed higher DOC, the shortage of readily biodegradable organic matters in AS resulted in lower water removal compared to SCG. In addition, SCG promoted the conversion of VS to DOM and the degradation of high molecular weight DOM to humic- and fulvic-like substances. SCG showed high activities of various enzymes including mannanase, xylanase and protease during biodrying, while AS and SD induced mainly protease and lignocellulose-related enzymes, respectively. It was concluded that bulking agents not only functioned as structural supports but also affected DS biodrying because of their different organic compositions and biodegradation potentials.

Fig. 5. SFS of DOM in biodrying piles of (a) Trial A (AS as a bulking agent), (b) Trial B (SCG as a bulking agent), and (c) Trial C (SD as a bulking agent). The sample temperature when collected was shown in the legend.

occurred after d 6 (Fig. 3) when the cellulase activity became high. At the beginning of Trial C, proteins served as the main energy source (Fig. 3), and the protease activity increased during d 2 to d 4. The high mannanase activity detected in Trial C (especially after d 4) might be due to that SD contained mannan, or, other lignocellulose-degrading enzymes in Trial C were able to degrade the substrate used for mannanase assay [42]. Interestingly, although lipids were also degraded in Trials B and C (Fig. 3), no lipase activity was detected in the extracellular enzyme extract (data not shown). The reason might be the following. Firstly, lipids in biodrying piles were mainly free fatty acids or phospholipids,

Acknowledgements This study was supported by the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2015R1D1A1A01059073). Z. Hao is also grateful for the 133

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Fig. 6. Extracellular enzyme activities of (a) amylase (b) cellulase (c) xylanase (d) mannanase and (e) protease during biodrying. Table 3 Correlation coefficients of enzyme activities in biodrying samples (n = 8). Trial A (AS as a bulking agent)

Amylase Cellulase Xylanase Mannanase a b

Cellulase 0.830a

Xylanase 0.830a 1.000a

Mannanase 0.434 0.247 0.247

Trial B (SCG as a bulking agent) Protease −0.245 −0.433 −0.433 −0.212

Cellulase −0.719a

Xylanase −0.511 0.842a

Correlations were significant at the 0.05 level (2-tailed). Correlations were significant at the 0.01 level (2-tailed).

134

Mannanase −0.974b 0.740a 0.548

Trial C (SD as a bulking agent) Protease −0.580 0.818a 0.938b 0.594

Cellulase −0.754a

Xylanase −0.684 0.990b

Mannanase −0.625 0.935b 0.940b

Protease −0.296 −0.269 −0.379 −0.318

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support of the China Scholarship Council (No. 201307565002). [26]

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