Cell survival and death programmes

Accepted Manuscript Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of the potential role of microorganisms from a metagenomic analysis Lu Cai, Tong-Bin Chen, Sheng-Wei Zheng, Hong-Tao Liu, Guo-Di Zheng PII:

S0045-6535(18)30389-8

DOI:

10.1016/j.chemosphere.2018.02.177

Reference:

CHEM 20935

To appear in:

ECSN

Received Date: 12 August 2017 Revised Date:

26 February 2018

Accepted Date: 27 February 2018

Please cite this article as: Cai, L., Chen, T.-B., Zheng, S.-W., Liu, H.-T., Zheng, G.-D., Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of the potential role of microorganisms from a metagenomic analysis, Chemosphere (2018), doi: 10.1016/ j.chemosphere.2018.02.177. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ACCEPTED MANUSCRIPT

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

Decomposition of lignocellulose and readily degradable carbohydrates during

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sewage sludge biodrying, insights of the potential role of microorganisms from a

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metagenomic analysis

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Lu Cai1,*, Tong-Bin Chen2, Sheng-Wei Zheng1, Hong-Tao Liu2,3, Guo-Di Zheng2,3

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1

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Ningbo 315211, China.

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2

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Natural Resources Research, Chinese Academy of Sciences, Beijing 100101, China.

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3

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Faculty of Architectural, Civil Engineering and Environment, Ningbo University,

Center for Environmental Remediation, Institute of Geographic Sciences and

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College of Resources and Environment, University of Chinese Academy of

Sciences, Beijing 100049, China.

11 *Corresponding author.

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*Lu (given name) Cai (family name):

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Postal address: Faculty of Architectural, Civil Engineering and Environment,

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Ningbo University, 818 Fenghua Road, Ningbo, 315211, China

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Email: [email protected]

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Tel: +8618867862220

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Tong-Bin (given name) Chen (family name)

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Postal address: Institute of Geographic Sciences and Natural Resources Research,

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Chinese Academy of Sciences, 11A Datun Road, Beijing, 100101, China

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Email: [email protected]

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Sheng-Wei (given name) Zheng (family name)

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Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

Postal address: Faculty of Architectural, Civil Engineering and Environment,

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Ningbo University, 818 Fenghua Road, Ningbo, 315211, China

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Hong-Tao (given name) Liu (family name):

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Postal address: Institute of Geographic Sciences and Natural Resources Research,

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Chinese Academy of Sciences, 11A Datun Road, Beijing, 100101, China

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Email: [email protected]

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Guo-Di (given name) Zheng (family name):

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Postal address: Institute of Geographic Sciences and Natural Resources Research,

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Chinese Academy of Sciences, 11A Datun Road, Beijing, 100101, China

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Email: [email protected]

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Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

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ABSTRACT Sewage sludge biodrying is a waste treatment method that uses bio-heat

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generated from organic degradation to remove moisture from sewage sludge.

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Lignocellulose and carbohydrate decomposition is important when assessing

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biodrying performance. This study investigated lignocellulose and carbohydrate

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decomposition, and the potential microbial functions during biodrying. We

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determined the lignocellulose and carbohydrate contents, assayed related enzyme

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activity, performed a complete metagenomic study on sewage sludge biodrying

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material during the thermophilic phase, annotated potential genetic function

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involved in the decomposition, and summarized the key metabolic pathways. The

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results indicated that lignocellulose, readily degradable carbohydrates, and starch,

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significantly decomposed after biodrying. During the thermophilic phase, the

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majority of lignocellulase and carbohydrate-related enzymes showed significantly

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higher activity, and glycoside hydrolases and glycosyl transferases showed higher

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gene counts and reads. Moreover, the top five microorganisms enriched with

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carbohydrate-active enzyme genes, i.e., Bacillus, Intrasporangium, Tetrasphaera,

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Rhodobacter, and Streptomyces, were also among the top ten ecologically dominant

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genera. These findings highlight the crucial phases for biodrying process, reveal the

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ecologically functional diversity of biodrying-originated microbial consortia, and

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suggest potential candidates for optimizing biodrying decomposition.

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Keywords: enzyme activity; genetic function; lignocellulose decomposition; 3

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

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microbial consortia; sewage sludge biodrying

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1. Introduction Biodrying has been used as an organic waste treatment method worldwide in

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recent decades, sustaining a vast microbial consortia specializing in organic

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decomposition, during which bio-heat is generated and the sludge moisture is

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reduced. Biodrying can be used to pretreat compost, coupled with a maturing period

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after biodrying, or it can be used as a treatment to generate biofuels or contaminated

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soil conditioners (Winkler et al., 2013; Rada and Ragazzi, 2014; Lü et al., 2017).

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Previous studies indicate the simplified potential of sewage sludge biodrying.

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Some biostabilisation processes of municipal solid wastes occur, generally, only for

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2–4 weeks to obtain a product with medium level of biological stability (Scaglia et

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al., 2013). Based on the significant decomposition and sterilization during the

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thermophilic phase (Huiliñir and Villegas, 2015; Zhang et al., 2017), and the

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challenge of increasing sludge generation, it is desirable to shorten the biodrying

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duration without deteriorating the biodrying product.

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The efficiency of organic decomposition is of concern if biodrying is shortened.

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The biodrying product can be used as biofuel or land conditioner, but when intended

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for land use, insufficient organic decomposition would negatively affect the

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utilization (Lü et al., 2017; Syamala et al., 2017). Consequently, the decomposition

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of lignocellulose and carbohydrates during the shortened biodrying merits

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investigation. 4

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

Various enzymes and microorganisms are required for decomposition of

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lignocellulose and carbohydrates (Feng et al., 2011; Eichlerová et al., 2012; Xu et al.,

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2014; Wang et al., 2016). Studies have reported the presence of several enzymes and

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microbial diversity during biodrying (Zhang et al., 2015; Zhang et al., 2017).

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However, in sewage sludge biodrying, to the best of our knowledge, the association

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between this diversity and biodrying-related functions and the actual decomposition

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remains obscure. The development of metagenomics allows the identification of

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functional microorganisms and genes participating in related activities, which can be

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used to infer the mechanism of the biodegradation process (Wang et al., 2016).

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Accordingly, searching for the potential function of microbial communities to

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characterize

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carbohydrate decomposition in the biodrying ecosystem seems promising.

biotechnology

of

lignocellulose

and

readily

degradable

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the

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This study investigated lignocellulose and carbohydrate decomposition, and the

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functions of the corresponding microbial consortia during simplified biodrying. The

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duration of biodrying process was shortened to 15 days, based on the application of

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the biodrying product (biofuel, soil conditioner, or landfill cover), as well as the time

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required for treatment engineering. First, the lignocellulose and carbohydrate

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contents and enzyme activity were determined. Next, a complete metagenomic study

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on sewage sludge biodrying material (SSBM) during the thermophilic phase was

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performed. Finally, annotations against gene databases and analyses of microbial

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function were conducted. This provided information about the critical microbial

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Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

consortia and genetic functions related to biodrying that may improve understanding

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about the control of biodrying.

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2. Materials and methods

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2.1. Materials

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Mechanically dewatered sewage sludge cake and biodrying product were

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collected from a sewage sludge treatment plant in Shanghai, China, as described by

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Cai et al. (2017). Wood shavings with particle size 5–10 mm were collected from a

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timber mill. Biodrying product and wood shavings were used as the combined

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bulking agent. Sewage sludge, biodrying product, and wood shavings were

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mechanically mixed at a mass ratio of 6:3:1. The moisture contents of sewage sludge

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and initial SSBM were 83.49% and 66.28%, respectively (Supplementary

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Information Table S1 showing the characteristics of the raw materials).

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2.2. Biodrying procedure

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The initial SSBM was loaded into a tank, with dimensions 15 m long, 5 m wide,

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and

m

high.

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acrylonitrile-butadiene-styrene

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(Supplementary Information Figure S1 showing the structure). The pile was

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subjected to an auto control technology for biodrying (CTB, GreenTech

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Environmental Engineering Co., Beijing, China). Details of the biodrying reactor

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were described by Cai et al. (2017). During the 15-day bio-drying period, each pile

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was aerated intermittently. The total forced air volume over the 15 days was 900 m3

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The

tank

was

divided

copolymer

panels

6

into to

three

enable

units

using

triplicate

tests

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

ton-1 SSBM. The aeration period for each trial was 40 min, including 10-min

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aeration time and 30-min no-aeration time. Each pile was mechanically turned two

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times (on days 9 and 12).

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2.3. Sample collection

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To compare the decomposition of lignocellulose and readily degradable

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carbohydrates in different phases, biodrying samples on the beginning days of the

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mesophilic phase, the initial thermophilic phase, and the second thermophilic phase

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were collected for composition and enzyme analysis, including: a sample on day 0

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(21 °C), samples during the initial thermophilic phase on day 3 (66 °C) and the

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second thermophilic phase on day 9 (57 °C), and a sample of the biodrying product

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on day 15 (41 °C). A previous study indicated that the initial thermophilic phase

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contributed greatly to the biodrying (Cai et al., 2016); accordingly, a sample

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collected at the steady stage during the initial thermophilic phase (on day 5, 67 °C)

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was selected for metagenomic sequencing. Each sample consisted of approximately

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1.0 kg in wet weight, and was stored at −80 °C until analysis.

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2.4. Composition determination

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The moisture contents of samples were analysed using the gravimetric method,

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measured as the mass loss in biodrying samples following heating at 105 °C. Total

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carbohydrates were extracted and then determined using the 3,5-dinitrosalicylic acid

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method, as described by Teixeira et al. (2012). Lignin, cellulose, and hemicellulose,

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were processed using sequential extraction with neutral and acidic detergents, 7

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followed by a strong acid extraction, contents of which were analyzed according to

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the method described by Yan et al. (2015). Starch content was determined using

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perchloric acid method (Fernandes et al., 2012). Total readily degradable

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carbohydrate content was calculated by subtracting the cellulose, hemicellulose, and

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starch contents from the total carbohydrate content. The compositions were analysed

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in triplicate, on dry matter of SSBM.

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2.5. Enzyme assay

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The activities of polyphenol oxidase (EC 1.10.3.1), laccase (EC 1.10.3.2),

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manganese peroxidase (EC 1.11.1.13), lignin peroxidase (EC 1.11.1.14),

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β-glucosidase (EC 3.2.1.21), endo-1,4-β-glucanase (EC 3.2.1.4), exo-β-1,4-glucan

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cellobiohydrolase (EC 3.2.1.91), β-xylosidase (EC 3.2.1.37),

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α-L-arabinofuranosidase (EC 3.2.1.55), xylanase (EC 3.2.1.8), α-amylase (EC

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3.2.1.1), and urease (EC 3.5.1.5), were spectrophotometrically determined as

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described previously. Table S2 can be found online, showing the apparatus and

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references involved in enzyme assay in the study.

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Due to the difference between the enzyme assays, in the assay of polyphenol

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oxidase (EC 1.10.3.1), a change in absorbance value of 0.005 was defined as one

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unit of enzyme activity, expressed as U g-1 (Tang and Newton, 2004; Kiewning et al.,

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2013), whereas in the assays of the other enzymes the unit of enzyme activity was

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defined as the amount of enzyme forming 1 nmol of reaction product per min and

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was expressed as nmol min-1 g-1. The enzyme activity was analysed in triplicate, on 8

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

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dry matter of SSBM. For statistical analysis, one-way analysis of variance (One-way ANOVA) was

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performed using IBM SPSS Statistics for Windows, Version 24.0 (IBM Corp.,

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Armonk, USA). Figures were plotted using Origin 9.0 (OriginLab Corp.,

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Northampton, USA).

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2.6. DNA extraction

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DNA for metagenomics was extracted from the biodrying sample (0.5 g) by

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using the E.Z.N.A. Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) according

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to the manufacturer’s protocols. The DNA concentration and purity were quantified

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with a TBS-380 mini-fluorometer (Turner BioSystems, Sunnyvale, CA, USA) and

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NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA,

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USA), respectively. DNA quality was examined by 1% agarose gel electrophoresis.

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2.7. Metagenomic sequencing

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2.7.1. Library construction and metagenomics sequencing

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DNA was fragmented to an average size of approximately 300 bp using Covaris

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M220 (Gene Company Limited, China) for paired-end library construction. The

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paired-end library was prepared by using a TruSeq DNA Sample Prep Kit (Illumina,

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San Diego, CA, USA). Adapters containing the full complement of sequencing

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primer hybridization sites were ligated to the blunt-end fragments. Paired-end

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sequencing was performed on an Illumina HiSeq 4000 platform (Illumina Inc., San

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Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China)

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using a HiSeq 3000/4000 PE Cluster Kit and HiSeq 3000/4000 SBS Kits according

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to the manufacturer’s instructions (www.illumina.com). The sequence data

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generated in this study are publicly available in the NCBI Sequence Read Achieve

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(SRA) database with an accession number SRP096475.

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2.7.2. Quality control of Illumina Hiseq reads

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Illumina raw reads were subject to the following treatments: the 3′-end and

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5′-end of reads were trimmed to the first high-quality base using Seqprep

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(https://github.com/jstjohn/SeqPrep); raw reads were trimmed to remove low quality

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reads that contained ambiguous N bases or had a Phred score lower than 20 using

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Sickle (https://github.com/najoshi/sickle).

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2.7.3. De novo assembly of Illumina high quality reads

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SOAPdenovo (http://soap.genomics.org.cn/, version 1.06) was used to

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assemble short reads. The parameter k-mer varied from 39 to 47. The resulting

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scaffolds were cut into contigs and only contigs longer than 500 bp were saved for

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gene prediction.

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2.7.4. Gene prediction and catalogue

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MetaGene (http://metagene.cb.k.u-tokyo.ac.jp/, version 3.25) was used for open

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reading frame (ORF) prediction. Removing redundancy was done with CD-HIT

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(http://www.bioinformatics.org/cd-hit/) using bacterial and fungal redundant coding

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sequence catalogues. Sequences with ≥ 95% identity and 90% coverage were

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considered redundant (Temu et al., 2016). Taxonomy identification was performed 10

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by BLASTP search (http://blast.ncbi.nlm.nih.gov/Blast.cgi, BLAST version 2.2.28+)

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with E-value < 1 × 10−5 against the non-redundant (NR) database.

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2.7.5. COG, KEGG, and CAZyme annotations The catalogue, comprised of non-redundant coding sequence-correspondent

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protein sequences, was annotated to achieve the clusters of orthologous groups of

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proteins (COG), based on the evolutionary genealogy of genes: Non-supervised

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Orthologous Groups (eggNOG), using BLASTP (BLAST version 2.2.28+) with

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E-value < 1 × 10−5. This catalogue was also annotated to inform the metabolic

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pathway based on the Kyoto Encyclopedia of Genes and Genomes (KEGG), using

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BLASTP (BLAST version 2.2.28+) with E-value < 1 × 10−5. Protein coding genes

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identified in the metagenome of microbial consortia in SSBM were searched in the

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carbohydrate-active

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classification in categories and families to annotate the CAZyme family (Shinkai et

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al., 2016).

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2.7.6. Function-microorganism collinear relation diagram

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which

offers

functional

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The CAZyme-encoding genes in the SSBM microorganisms were annotated

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using hmmscan (http://hmmer.org/). The data were visualised using Circos software

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(http://circos.ca/) (Wang et al., 2016).

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3. Results

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3.1. Pile temperature profile

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The moisture content significantly decreased from 66.28% to 50.23% after 15 11

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

days of biodrying (P < 0.01), indicating a satisfactory biodrying result. Pile

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temperature, indicating the biodrying progress, can affect the water removal and

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biodegradation of biodrying (Mohammed et al., 2017; Yu et al., 2017). The pile

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experienced a mesophilic phase (days 0-2), an initial thermophilic phase (pile

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temperature higher than 50 °C; days 3-8), a second thermophilic phase (after the

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turning on day 9, the pile temperature dropped and then rebounded above 50 °C;

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days 9-12), and a cooling phase (days 13-15) (Supplementary Information Figure S2

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showing the pile temperature variation of triplicate piles). Once entering the initial

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thermophilic phase, the pile temperature increased from 50°C (morning on day 3) to

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higher than 60 °C (evening on day 3). The pile temperature during the initial

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thermophilic phase was mostly higher than 60 °C, with a peak value of 67 °C on day

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5, and that during the second thermophilic phase was lower than 60 °C. The

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temperature of the biodrying product was 39 °C when it was loaded out onto the

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lorry on day 16, and it finally cooled to ambient temperature due to shoveling and

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loading procedures.

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3.2. Carbohydrate decomposition

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Cellulose and readily degradable carbohydrates were the most abundant,

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comprising more than 95%, of the total carbohydrates (Fig. 1). Cellulose,

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hemicellulose, starch, and readily degradable carbohydrate contents before

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biodrying accounted for 59.30, 3.37, 0.70, and 36.63% in the total carbohydrates,

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respectively; whereas these contents after biodrying were 49.73, 1.86, 0.96, and 12

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47.45%, respectively. Lignin, cellulose, hemicellulose, starch, and readily degradable carbohydrates,

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were all significantly degraded after biodrying (P < 0.01). Their decomposition

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proportions were 23.40%, 58.20%, 72.44%, 31.90%, and 35.41%, respectively.

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The total carbohydrates and readily degradable carbohydrates

were

significantly decomposed in each phase (P < 0.01). Cellulose and hemicellulose

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were significantly degraded in each phase (P < 0.05); however, their decomposition

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during the second thermophilic phase was not significant at the 0.01 level,

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suggesting that their decomposition before the second thermophilic phase was more

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robust. Lignin degraded significantly during the mesophilic phase and the second

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thermophilic phase (P < 0.01), but the degradation was not significant during the

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initial thermophilic phase (P > 0.05). Starch, accounting for less than 1% of the total

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carbohydrates, decomposed gradually, but it decomposed significantly during the

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second thermophilic phase (P < 0.05).

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3.3. Enzyme activity

As shown in Table 1, the enzyme activities related to lignocellulose

decomposition were significantly different during different phases (P < 0.05).

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< Table 1 >

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During the initial thermophilic phase, the activities of laccase, manganese

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peroxidase, lignin peroxidase, exo-β-1,4-glucan cellobiohydrolase, β-xylosidase, 13

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α-amylase, and urease all showed significant peaks (P < 0.05), with maxima of

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61.15, 87.48, 49.62, 485.88, 977.72, 22566.74, and 1339.34 nmol min-1 g-1,

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respectively. During the second thermophilic phase, enzyme activities of polyphenol oxidase,

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α-L-arabinofuranosidase,

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significant peaks (P < 0.05), with the values of 67.11 U g-1, 28.50, 173.85, and 29.35

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nmol min-1 g-1, respectively.

and

β-glucosidase

showed

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endo-1,4-β-glucanase,

Some enzymes were negatively sensitive to extremely high temperature in the

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initial thermophilic phase. The activities of polyphenol oxidase, β-glucosidase, and

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α-L-arabinofuranosidase were significantly lower in the initial thermophilic phase

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than in other phases (P < 0.05). The activity of xylanase peaked on day 0, and then

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decreased continuously.

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Significant lignin decomposition was observed during the second thermophilic

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phase. Activity of the ligninolytic enzyme polyphenol oxidase significantly peaked

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(67.11 U g-1) during the second thermophilic phase, facilitating lignin decomposition.

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However, the activities of other ligninolytic enzymes, i.e., laccase, manganese

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peroxidase, and lignin peroxidase, peaked on day 3.

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Significant decomposition of cellulose and hemicellulose was observed in each

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phase (P < 0.05), and the decomposition before the second thermophilic phase was

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more robust (P < 0.01). This was probably due to the significantly higher activity of

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exo-β-1,4-glucan cellobiohydrolase for cellulose decomposition and β-xylosidase 14

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and xylanase for hemicellulose decomposition, in the initial thermophilic phase

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rather than the second thermophilic phase. Starch decomposed gradually, but it significantly decomposed during the

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second thermophilic phase (P < 0.05); this profile did not reflect the extremely high

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activity of α-amylase in the mesophilic and initial thermophilic phases, probably as a

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result of its chemical structure and its low content in SSBM, only accounting for less

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than 1% of the total carbohydrates.

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3.4. Genome properties

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Metagenomic sequencing of the biodrying sample yielded 73,308,756 total raw

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reads, with a read length of 151 bp and 11.0 Gb of raw bases. The percentage of

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clean reads among raw reads was 96.90% after quality control. The number of

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contigs was 149,899 after genome assembly, and 266,722 open reading frames

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(ORFs) were predicted, with an average length of 573 bp.

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3.5. COG annotation reveals functions related to carbohydrate decomposition

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The function of energy production and conversion, and that of carbohydrate

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transport and metabolism were 7.5% and 4.7% in the COG function classification,

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respectively. To determine the potential roles of microbial communities in

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carbohydrate decomposition, the specific COGs involved in related transport and

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metabolism and those abundantly persisted during the thermophilic phase were

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analysed. The total items of related COGs were 506, among which, the top 20

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abundant COGs are shown in Table S3.

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A carbohydrate-related function enriched in the SSBM microorganisms was the

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major facilitator Superfamily COG0477 (31206 reads), which can catalyse the

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transport of various substrates, including ions, carbohydrates, lipids, amino acids

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and peptides, nucleosides, and other small molecules (Madej et al., 2014). It couples

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the movement of substrates to the proton motive force generated across the cell

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membrane, rather than ATP hydrolysis and is, therefore, referred to as a secondary

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active transporter (Chaudhary et al., 2016). COG1109 (24040 reads), known as a

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phosphomannomutase gene, potentially encodes enzymes that catalyze the

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conversion of glucosamine-6-phosphate to glucosamine-1-phosphate (Rashid et al.,

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2004). COG0366 (23452 reads) potentially encodes α-amylase that hydrolyses alpha

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bonds of large, alpha-linked polysaccharides, such as starch and glycogen, yielding

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glucose and maltose (Janeček and Gabriško, 2016). COG0726 (23278 reads)

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potentially encodes polysaccharide deacetylase. The extracellular solute-binding

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protein family COG1653 (15638 reads), and binding-protein-dependent transport

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systems inner membrane component COG0395 (8996 reads), are involved in the

321

uptake of carbohydrate (Wang et al., 2016).

SC

M AN U

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EP

AC C

322

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306

Altogether, the wide diversity of gene functions in carbohydrate metabolism

323

and transport reveal a high potential of the SSBM microorganisms for glycan

324

degradation and sugar uptake and transport in the biodrying ecosystem.

16

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

325

3.6. KEGG annotation reveals key enzymes and pathways for lignocellulose

326

metabolism The KEGG annotations of functional enzymes contributing to lignocellulose

328

degradation during biodrying are elucidated here.

329

3.6.1. Functional enzymes and their presentations in KEGG

RI PT

327

For lignin: Pathways associated with laccase (EC 1.10.3.2), manganese

331

peroxidase (EC 1.11.1.13), and lignin peroxidase (EC 1.11.1.14) are not reported in

332

KEGG, but similar functional enzymes such as polyphenol oxidase (EC 1.10.3.1)

333

and peroxiredoxin (EC 1.11.1.15, reads 10744) exist in the KEGG pathways.

334

However, the gene encoding polyphenol oxidase (EC 1.10.3.1) was not detected

335

using metagenomic sequencing during the initial thermophilic phase, correlating

336

with the significantly lower activity of polyphenol oxidase (EC 1.10.3.1) during this

337

phase (Table 1), probably because of extremely high temperatures.

M AN U

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For cellulose: Pathways associated with cellulases such as β-glucosidase (EC

EP

338

SC

330

3.2.1.21, reads 9956), endo-1,4-β-glucanase (EC 3.2.1.4, reads 3546), and

340

exo-β-1,4-glucan cellobiohydrolase (EC 3.2.1.91, reads 156) exist in KEGG,

341

whereas those associated with endo-1,3-β-glucanase (EC 3.2.1.6, reads 3004) are not

342

reported in KEGG. β-glucosidase (EC 3.2.1.21) participates in starch and sucrose

343

metabolism (ko00500, reads 88442).

344 345

AC C

339

For hemicellulose: Pathways associated with β-xylosidase (EC 3.2.1.37, reads 692) and α-L-arabinofuranosidase (EC 3.2.1.55, reads 874) exist in KEGG, whereas 17

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

those with xylanase (EC 3.2.1.8, reads 850) are not reported in KEGG. Both

347

β-xylosidase (3.2.1.37) and α-L-arabinofuranosidase (3.2.1.55) participate in amino

348

sugar and nucleotide sugar metabolism (ko00520, reads 166684). Table S4, showing

349

the enzymes, the reads of corresponding genes, and the serial number of the

350

pathways, can be found online.

351

3.6.2. Pathway illustrations of lignocellulose metabolism To

illustrate

the

critical

metabolic

SC

352

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346

pathways

during

lignocellulose

decomposition, marker genes that were abundant during the thermophilic phase

354

were annotated using the KEGG pathway (Fig. 2).

M AN U

353



356

Glycolysis is the metabolic pathway that converts glucose into pyruvate. The

357

free energy released in this process is used to generate biological energy during

358

biodrying. Fig. 2a illustrates the glycolysis in the thermophilic phase. D-glucose

359

could be phosphorylated into D-glucose 6-phosphate, which plays a key role in

360

carbohydrate metabolism, and D-glucose 6-phosphate is connected to the pentose

361

phosphate pathway. D-glucose 6-phosphate could be metabolized into pyruvate, and

362

under aerobic fermentation, pyruvate could be oxidized into acetyl-CoA, which

363

enters the citrate cycle.

AC C

EP

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355

364

Sugar monomers in hemicellulose include glucose, xylose, mannose, galactose,

365

rhamnose, and arabinose. The pathway analysis showed that amino and nucleotide

366

sugars are metabolized during the thermophilic phase (Fig. 2b). D-glucose from the 18

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

glycolytic pathway was connected either to starch and sucrose metabolism or to

368

ascorbate and aldarate metabolism. 1,4-β-D-xylan could be hydrolysed into

369

D-xylose by β-xylosidase (EC 3.2.1.37), which entered the pentose and glucuronate

370

interconversions. Mannose could be metabolized via fructose and mannose

371

metabolic pathways. α-D-galactose obtained from galactose metabolism could be

372

transferred and isomerized, finally entering ascorbate and aldarate metabolism.

373

Arabinan could be degraded into L-arabinose by α-L-arabinofuranosidase (EC

374

3.2.1.55).

M AN U

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367

Fig. 2c illustrates that the starch and sucrose metabolic pathways occurred in

376

the thermophilic phase. Readily degradable carbohydrates could be significantly

377

decomposed during this phase (P < 0.01). Cellulose in the SSBM could be

378

hydrolysed into cellodextrin with endo-1,4-β-glucanase (EC 3.2.1.4), followed by

379

hydrolysis into cellobiose with endo-1,4-β-glucanase (EC 3.2.1.4) and cellulose

380

1,4-β-cellobiosidase (EC 3.2.1.91). Subsequently, cellobiose could be glycosylated

381

into D-glucose with β-glucosidase (EC 3.2.1.21). Alternatively, cellodextrin could be

382

directly glycosylated into D-glucose with EC 3.2.1.21. In addition, cellulose could

383

be directly degraded into cellobiose with EC 3.2.1.91, and then glycosylated into

384

D-glucose with EC 3.2.1.21.

AC C

EP

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375

19

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

385

3.7. CAZyme annotation reveals relationships between enzymes and

386

microorganisms

387

3.7.1. Families of CAZyme-encoding genes To better understand carbohydrate decomposition in the biodrying ecosystem,

389

where microorganisms synergistically degrade and utilize the ready and recalcitrant

390

organic

391

carbohydrate-active enzymes. The SSBM microorganisms contained 5,902

392

CAZyme-encoding genes distributed unequally among glycoside hydrolases (GHs),

393

glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases

394

(CEs), carbohydrate binding modules (CBMs), and auxiliary activities (AAs), and

395

their gene count abundances were 26.52%, 36.28%, 1.29%, 18.59%, 12.69%, and

396

4.64%, respectively (Fig. 3a). The total reads of these CAZyme-encoding genes

397

were 454,316, and the read abundances of GHs, GTs, PLs, CEs, CBMs, and AAs

398

were 25.91%, 38.76%, 1.22%, 11.85%, 15.54%, and 6.73%, respectively (Fig. 3b).

399

GTs and GHs had higher percentages both in gene counts and reads. Enzymes of the

400

GH and GT families play key roles in the cleavage of polymeric substrates (Roth et

401

al., 2017).

403

screened

our

metagenome

for

genes

encoding

EP

TE D

M AN U

SC

we

AC C

402

matter,

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388



3.7.2. Genera enriched with CAZyme genes

404

The overall distribution of CAZyme-encoding genes in the SSBM

405

microorganisms was analysed to associate biodrying microbial community 20

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

composition

407

CAZyme-encoding genes varied across the bacterial phyla. As shown in Fig. 4, most

408

of these genes in the SSBM microorganisms were from Bacillus (phylum Firmicutes,

409

9.6% of all microorganisms in the analysed sample), followed by Intrasporangium

410

(phylum Actinobacteria, 2.6%), Tetrasphaera (phylum Actinobacteria, 3.1%),

411

Rhodobacter

412

Actinobacteria, 2.3%), which were present in the thermophilic phase and were

413

among the top ten ecologically dominant genera.

415

Members

(phylum

of

decomposition.

Proteobacteria,

3.5%),

and

The

Actinobacteria

and

abundance

of

Streptomyces

(phylum

SC

lignocellulose

M AN U

414

with

RI PT

406

Firmicutes

were

dominant

in

carbohydrate-active modules of GHs, GTs, CEs and CBMs, whereas those from

417

Proteobacteria were dominant in PLs (Table S5).

TE D

416

There are several implications of this observation: First, the composition of

419

functional members corresponds well to the structure of the ecologically dominant

420

species in SSBM microorganisms. A potential link exists between microbial taxa

421

and their functional traits. This suggests that polysaccharides within the sewage

422

sludge biodrying habitat are hydrolysed by the predominant Actinobacteria,

423

Firmicutes, and Proteobacteria. Furthermore, although lignocellulose-degrading

424

activity was previously identified in Bacillus, Rhodobacter, and Streptomyces

425

(Singh et al., 2015; Zhang et al., 2016; Gong et al., 2017), our data provide genetic

426

evidence for high cellulolytic and transglycosylatic abilities of these genera, which

AC C

EP

418

21

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

harbour abundant genes encoding GHs and GTs.

428

4. Discussion

429

4.1. Satisfactory biodrying performance in lignocellulose and carbohydrate

430

decomposition

RI PT

427

After 15 days of biodrying with wood shavings (particle size between 5–10

432

mm), total carbohydrates, lignin, cellulose, hemicellulose, starch, and readily

433

degradable carbohydrates were significantly decomposed, with decomposition

434

proportions of 50.14%, 23.40%, 58.20%, 72.44%, 31.90%, and 35.41%, respectively.

435

Although starch was structurally simple and rapidly biodegradable (Adams and

436

Umapathy, 2011), its decomposition was not as rapid as that of other carbohydrates

437

in this study.

M AN U

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431

The modified and simplified biodrying process in this study did not impede

439

lignocellulose decomposition when compared to 20 days of biodrying with sawdust

440

(particle size ≤2 mm) (Zhang et al., 2017). The decomposition proportions of the

441

latter were 48.4%, 38.8%, and 45.0%, for lignin, cellulose, and hemicellulose,

442

respectively, and lignocellulases were more active after day 10. In this study,

443

lignocellulases were significantly active before day 10. The differences in

444

decomposition between the two biodrying processes may have resulted from

445

discrepancies in bulking agents and operation procedures. It appears that

446

lignocellulose was decomposed efficiently when the biodrying period was shortened

447

to 15 days and the mechanical turning frequency was halved.

AC C

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438

22

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

The carbohydrate decomposition observed in this study was also compared

449

with that in composting, showing satisfactory performance. During composting with

450

wheat straw, 57.63% of the total carbohydrates were decomposed (Jurak et al., 2015);

451

in studies of composting with straw, green trimmings, or pomace (Haddadin et al.,

452

2009; Lashermes et al., 2012; Yan et al., 2015), lignin, cellulose and hemicellulose

453

were decomposed by 12–38%, 18–64%, and 7–68%, respectively, whereas starch

454

was decomposed by approximately 50% when composting with cassava leaves

455

(Syamala et al., 2017). Although aerobic degradation is the working principle of

456

both composting and biodrying, the composting period is normally longer than

457

biodrying due to differences in their objectives. Therefore, carbohydrate

458

decomposition in biodrying competes with that in composting.

459

4.2. Microorganisms involved in lignocellulose and carbohydrate decomposition

460

The action of multiple microbial members during biodrying contributed to

461

lignocellulose decomposition. Based on the annotations of COG, KEGG, and

462

CAZyme, 124, 101, 95, 83, 65, and 60 genera were associated with COG0477,

463

COG1109, COG0366, COG0726, COG1653, and COG0395, respectively. These

464

COGs belonged to the function classification of carbohydrate transport and

465

metabolism as illustrated in Section 3.3. There were 264, 322, 24, 193, 112, and 88

466

identifiable genera responsible for secreting carbohydrate-active enzymes such as

467

GHs, GTs, PLs, CEs, CBMs, and AAs, respectively. The involved genera included,

468

but were not limited to, Acinetobacter, Aureobasidium (domain Eukaryota, phylum

AC C

EP

TE D

M AN U

SC

RI PT

448

23

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

469

Ascomycota),

Bacillus,

Batrachochytrium

470

Chytridiomycota)

471

Ilumatobacter, Intrasporangium, Methanosphaerula (domain Archaea, phylum

472

Euryarchaeota), Tetrasphaera, Thermomonas, Thermococcus (domain Archaea,

473

Euryarchaeota) Rhodobacter, and Streptomyces, which belong to Archaeabacteria,

474

Eubacteria, and Eukaryota; moreover, some of these are ecologically dominant

475

microorganisms in SSBM. Clearly, the functional microorganisms were remarkably

476

diverse.

Caldibacillus,

Eukaryota,

Cellulomonas,

phylum

Glycomyces,

M AN U

SC

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Brevibacillus,

(domain

Notably, the dominant lignocellulolytic bacteria such as Rhodobacter, Bacillus,

478

Tetrasphaera, Streptomyces, Ilumatobacter, and Brevibacillus were also the

479

dominant functional genera for tryptophan and tyrosine decomposition during

480

thermophilic biodrying (Cai et al., 2017).

TE D

477

Rhodobacter has been reported to be capable of steroid hormone degradation

482

(Yang et al., 2011) and is a prospective candidate for biohydrogenation (Sargsyan et

483

al., 2016). Bacillus and Streptomyces are noted for lignocellulose degradation (de

484

Gannes et al., 2013; Zhang et al., 2016; Ventorino et al., 2016). Tetrasphaera has

485

been reported to degrade benzene and amino acids and remove phosphorus

486

(Kristiansen et al., 2013; Yoshikawa et al., 2016; Marques et al., 2017). Type species

487

of Ilumatobacter usually inhabit saline environments such as estuarine sediments,

488

coastal beaches, and marine sponges (Matsumoto et al., 2013; Fang et al., 2015).

489

The presence of Ilumatobacter in SSBM is probably because of its geographical

AC C

EP

481

24

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

source, Shanghai, a coastal metropolis. Brevibacillus has been detected during

491

composting (Joshua et al., 2013), and is capable of degrading polymers (Ye et al.,

492

2013). This study provides genetic evidence of microbial function in the biodrying

493

ecosystem, showing that the biodrying microbiome contained a variety of both

494

well-known and less-studied members potentially capable of decomposing

495

lignocellulose and carbohydrates.

SC

RI PT

490

This study focused on the potential microbial function in the initial

497

thermophilic phase; however, whether the decomposition dynamics of lignocellulose

498

and readily degradable carbohydrates could be attributed to the microbial succession

499

or the result of changes in gene expression is still unclear. Therefore, microbial

500

function in different phases, as well as the relationship between the actual

501

decomposition and the bioinformatics of SSBM, merit further research.

502

4.3. Implications

TE D

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496

In recent years, dewatering and stabilization in a short run has been the target of

504

sewage sludge treatment and disposal engineering to meet the challenges of the

505

dramatically increasing sludge production. The 15-day biodrying that is

506

characterized by efficient dewatering, high-temperature sterilization, and effective

507

decomposition, with the product used as biofuel, contaminated site conditioner, or

508

landfill cover, can cater to the sludge treatment demand.

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503

509

This study reveals the critical decomposition phases and key microorganisms

510

involved in biodrying and stabilization, underlining the ecologically functional 25

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

diversity of biodrying-originated microbial consortia in lignocellulose and

512

carbohydrate decomposition. From an applied point of view, it would be practical to

513

inoculate the dominant functional microorganisms at the beginning of biodrying or

514

further optimize the temperature control via aeration and turning to improve the

515

incubation of the functional microbial consortia. Furthermore, this study observed

516

ecologically minority but functionally similar microorganisms that were beneficial

517

for biodrying, which could also provide a guideline for other biodrying or

518

biodegradation processes.

519

5. Conclusions

M AN U

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511

520

The modified and simplified biodrying, with 15-day running and two

521

mechanical turnings, was characterized by efficient dewatering, sufficient

522

thermophilic lasting,

523

degradable carbohydrate, and starch significantly decomposed after biodrying.

524

Metagenomic analysis revealed that carbohydrate transport and metabolism occurred

525

in 4.7% cases of the COG function classification. The diverse repertoire of CAZyme

526

genes in the biodrying-originated microbial consortia was primarily distributed

527

within the genera of Bacillus, Intrasporangium, Tetrasphaera, Rhodobacter, and

528

Streptomyces, which were also among the top ten ecologically dominant genera. The

529

actual decomposition of lignocellulose and readily degradable carbohydrates, the

530

assay of related enzyme activities, and the genetic function of biodrying-originated

531

microbial consortia illustrate the importance of microbial degradation. This work

Lignocellulose,

readily

AC C

EP

TE D

and effective decomposition.

26

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

reveals potentially important microorganisms, and highlights typical phases and

533

pathways for biodrying process. The relationship between the variation of microbial

534

function and the decomposition dynamics throughout biodrying merits further

535

research.

AC C

EP

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M AN U

SC

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532

27

Decomposition of lignocellulose and readily degradable carbohydrates during sewage sludge biodrying, insights of ACCEPTED MANUSCRIPT the potential role of microorganisms from a metagenomic analysis

536

Acknowledgments This work was supported by the National Natural Science Foundation of China

538

(Grant No. 41401538), and the Ningbo Natural Science Foundation of China (Grant

539

No. 2017A610303), and it was sponsored by the K. C. Wong Magna Fund at Ningbo

540

University.

541

Appendix A. Supplementary data

SC

542

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537

Supplementary data related to this article can be found online. References

544

1.

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543

Adams, J.D.W., Umapathy, D., 2011. Investigating microbial activities during a

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starch-amended co-composting process at mesophilic and thermophilic

546

temperatures. Environ. Technol. 33, 1817–1823. 2.

Cai, L., Gao, D., Hong, N., 2015. The effects of different mechanical turning

TE D

547

regimes on heat changes and evaporation during sewage sludge biodrying. Dry.

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Technol. 33, 1151–1158. 3.

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Cai, L., Chen, T.B., Gao, D., Yu, J., 2016. Bacterial communities and their

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association with the bio-drying of sewage sludge. Water Res. 90, 44–51.

4.

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Cai, L., Krafft,T., Chen, T.B., Lv, W.Z., Gao, D., Zhang, H.Y., 2017. New

insights into biodrying mechanism associated with tryptophan and tyrosine

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scale census of major facilitator superfamily transporters in trichoderma reesei

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using protein sequence and structure based classification enhanced ranking.

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Eichlerová, I., Šnajdr, J., Baldrian, P., 2012. Laccase activity in soils: considerations for the measurement of enzyme activity. Chemosphere 88,

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Microbiol. 63, 3404–3408.

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27. Roth, C., Weizenmann, N., Bexten, N., Saenger, W., Zimmermann, W., Maier, T.,

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Sträter, N., 2017. Amylose recognition and ring-size determination of

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ACCEPTED MANUSCRIPT

Name (Unit)

EC number

D0

Polyphenol oxidase (U g-1)

1.10.3.1

24.44±0.67c

Laccase (nmol min-1 g-1)

1.10.3.2

41.62±4.18b

Manganese peroxidase (nmol min-1 g-1)

1.11.1.13

51.42±1.80b

Lignin peroxidase (nmol min-1 g-1)

1.11.1.14

β-Glucosidase (nmol min-1 g-1)

3.2.1.21

Endo-1,4-β-glucanase (nmol min-1 g-1)

3.2.1.4

Exo-β-1,4-glucan cellobiohydrolase (nmol min-1 g-1)

3.2.1.91

β-Xylosidase (nmol min-1 g-1)

3.2.1.37

α-L-Arabinofuranosidase (nmol min-1 g-1)

3.2.1.55

Urease (nmol min-1 g-1)

D9

D15

20.14±2.13c

67.11±5.37a

49.77±2.23b

61.15 ±3.34a

28.38 ±1.30c

26.39±1.02c

87.48±3.09a

28.25±2.10c

21.62±2.58d

30.94±1.69b

49.62±2.21a

27.59±1.24c

18.13±0.66d

24.62±0.43b

14.61±0.36c

29.35±2.40a

27.13±1.14ab

136.36±1.96c

156.00±0.09b

173.85±3.25a

159.60±2.46b

303.88±1.89c

485.88±2.05a

313.28±0.19b

125.06±0.80d

535.91±16.08b

977.72±15.28a

319.93±4.70c

179.56±11.74d

29.47±0.33a

11.38±0.66c

28.50±0.70a

13.63±0.36b

1373.03±51.95a

898.71±11.30b

591.71±49.35c

236.42±11.41d

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α-Amylase (nmol min-1 g-1)

3.2.1.8

3.2.1.1

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Xylanase (nmol min-1 g-1)

3.5.1.5

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Table 1 Enzyme activities in different phases during sewage sludge biodrying*

19317.87±396.47b 22566.74±323.32a 226.10±23.21d

1339.34±15.79a

17916.38±137.76c 16934.70±161.83d 852.42±50.45b

560.43±59.72c

*: Values followed by different lowercase letters mean significant differences at 5% level. EC number means the enzyme commission number, a numerical classification scheme for enzymes. D0, D3, D9, and D15 mean the samples collected on day 0, 3, 9, and 15, respectively.

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Fig. 1. Contents of total carbohydrates, lignocellulose, starch, and readily degradable

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carbohydrates. The samples were collected on days 0, 3, 9, and 15. Different lowercase and uppercase letters indicate significant differences at the 5% level and 1% level, respectively.

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Fig. 2. Summarized Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways occurring in the initial thermophilic phase of sewage sludge biodrying. (a) Metabolic pathway of glycolysis. (b)

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Metabolic pathway of amino sugar and nucleotide sugar. (c) Metabolic pathway of starch and sucrose. Substrates and products are annotated with red circles; enzymes abundant in biodrying

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material are annotated using enzyme commission number with blue rectangles. Other connected pathways are annotated with green chamfered rectangles. The blue solid lines indicate only one reaction on the pathway; the dotted lines indicate more than one reaction.

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Fig. 3. Distributions of carbohydrate-active enzyme families in the initial thermophilic phase of

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sewage sludge biodrying: glycoside hydrolases (GHs), glycosyl transferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), carbohydrate binding modules (CBMs), and auxiliary activities (AAs). (a) Gene count distributions; (b) read distributions.

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Fig. 4. Distribution of genera enriched with carbohydrate-active enzyme (CAZyme) genes during the initial thermophilic phase of sewage sludge biodrying. The left semicircle shows the CAZyme families, including glycosyl transferases (GTs), glycoside hydrolases (GHs), carbohydrate binding modules (CBMs), carbohydrate esterases (CEs), auxiliary activities (AAs), and polysaccharide lyases (PLs). The right semicircle shows the top genera enriched with the CAZyme families. For the left semicircle: each single colour in the inner semicircle represents a CAZyme family,

ACCEPTED MANUSCRIPT and the bar width of the single colour indicates the relative abundance of the CAZyme family; the diverse colours in the outer semicircle indicate the distribution of different genera enriched with related genes, and the bar widths of the diverse colours indicate the percentages of corresponding genera. For the right semicircle: each single colour in the inner semicircle represents a genus, and

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the bar width of the single colour indicates the relative abundance of the genus; the diverse colours in the outer semicircle indicate the distribution of different CAZyme families detected in the genus,

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and the bar widths of the diverse colours indicate the percentages of corresponding CAZyme

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ACCEPTED MANUSCRIPT Highlights Lignocellulose and carbohydrates significantly degraded after biodrying.




The majority of related enzymes were significantly active in thermophilic phase.




GHs and GTs had higher gene counts and reads in the thermophilic phase.




Top five genera enriched with CAZyme genes were ecologically dominant.




Summarized

key

pathways

illustrated

lignocellulose

and

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decomposition.

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carbohydrate