Enhanced anaerobic digestion performance by two artificially constructed microbial consortia capable of woody biomass degradation and chlorophenols detoxification

Journal Pre-proof Enhanced anaerobic digestion performance by two artificially constructed microbial consortia capable of woody biomass degradation and chlorophenols detoxification Sameh S. Ali (Conceptualization) (Methodology) (Writing - original draft) (Formal analysis) (Writing - review and editing), Michael Kornaros (Conceptualization) (Writing - review and editing), Alessandro Manni (Methodology) (Software), Jianzhong Sun (Funding acquisition)Writing- review and editing), Abd El-Raheem R. El-Shanshoury (Investigation) (Conceptualization), El-Refaie Kenawy (Visualization) (Validation), Maha A. Khalil (Writing - review and editing)

PII:

S0304-3894(20)30062-5

DOI:

https://doi.org/10.1016/j.jhazmat.2020.122076

Reference:

HAZMAT 122076

To appear in:

Journal of Hazardous Materials

Received Date:

28 September 2019

Revised Date:

10 January 2020

Accepted Date:

10 January 2020

Please cite this article as: Ali SS, Kornaros M, Manni A, Sun J, El-Shanshoury AE-RaheemR, Kenawy E-Refaie, Khalil MA, Enhanced anaerobic digestion performance by two artificially constructed microbial consortia capable of woody biomass degradation and chlorophenols detoxification, Journal of Hazardous Materials (2020), doi: https://doi.org/10.1016/j.jhazmat.2020.122076

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier.

Enhanced anaerobic digestion performance by two artificially constructed microbial consortia capable of woody biomass degradation and chlorophenols detoxification Sameh S. Alia,b,*, Michael Kornarosc, Alessandro Mannid, Jianzhong Suna,*, Abd ElRaheem R. El-Shanshouryb, El-Refaie Kenawye, Maha A. Khalilb,f a

Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Zhenjiang, 212013, China b

Botany Department, Faculty of Science, Tanta University, Tanta, 31527, Egypt

c

d

ro of

Laboratory of Biochemical Engineering & Environmental Technology (LBEET), Department of Chemical Engineering, University of Patras, 1 Karatheodori Str., University Campus, 26504 Patras, Greece Department of Industrial Engineering, University of Rome Tor Vergata, Italy

e

-p

Polymer Research Group, Department of Chemistry, Faculty of Science, Tanta University, Tanta 31527, Egypt f

lP

re

Biology Department, Faculty of Science, Taif University, KSA

*Corresponding authors at: Biofuels Institute, School of the Environment and Safety Engineering, Jiangsu University, Xuefu Rd. 301, 212013, Zhenjiang, China. [email protected]

(J.

Sun);

ur na

E-mail:

[email protected]

Jo

Graphical abstract

1

[email protected],

(S.S.

Ali),

Highlights 1. CS-5 and BC-4 constructed for lignocellulose deconstruction and chlorophenols detoxification. 2. The achieved weight loss of the CSW induced by CS-5 and BC-4 reached 69.2 and 56.3%, respectively. 3. CS-5 and BC-4 would potentially serve as a useful agent for detoxifying CPs. 4. The synergistic action of CS-5 and BC-4 increased CH4 production by 113.7% at the AD peak phase. 5. Methanosataceae represented 45.1% of the methanogenic Archaea.

ro of

Abstract Catalpa sawdust (CSW) is a promising biomass-based biofuel. However, the complex

lignocellulosic structure limits its efficient utilization in biorefinery applications. It is even more so when chlorophenols (CPs), highly toxic organic substances widely used as wood preservatives,

are present. Hence, it is crucial to develop effective and eco-friendly approaches to attain

-p

deconstruction of lignocellulose and chlorophenols simultaneously as well as to improve methane (CH4) production efficiently. This study might be the first to explore the performance of the novel

re

constructed microbial consortia CS-5 and BC-4 on woody biomass degradation and CPs detoxification simultaneously with CH4 production. After the degradation of CSW and CPs for 15 days by C5-5 or BC-4, significant reduction in lignocellulosic components and CPs mixture

lP

was realized with a total weight loss of 69.2 and 56.3% and CPs degradation of 89 and 95%, respectively. The toxicity of individual or mixed CPs after 15 days of degradation was reduced by approximately 90%. The synergistic action of CS-5 and BC-4 enhanced biogas and CH4 yields

ur na

over 76 and 64% respectively, higher than control. Furthermore, CH4 production increased by 113.7% at the peak phase of AD process. Methanosataceae represented 45.1% of the

Jo

methanogenic Archaea in digester G-III.

Keywords: Chlorophenols; woody biomass; microbial consortium; biodegradation and detoxification; anaerobic digestion; methanogenic Archaea.

2

1. Introduction

ro of

The extensive use of fossil fuels makes human beings face two major crises: that of energy

shortage and global warming. Therefore, great attention has been focused, in recent years, to explore alternative and renewable energy resources. Lignocellulosic biomass, which can be considered as an abundant carbon-based resource for producing valuable products, including

biofuels and bio-based materials, has the potential to fulfil the renewable fuels demand in near

-p

future. Although current research and development attention has been focused mainly on agricultural biomass, wood has great potential in biomass-based biorefinery (Ali et al., 2020; Ali

re

et al., 2017). Wood, such as catalpa sawdust (CSW), is one of the most valuable natural resources of outdoor applications and building interiors. CSW is an organic material composed of

lP

lignocellulosic components (mainly cellulose, hemicellulose and lignin), which are prone to biological and chemical degradation by both biotic and abiotic agents (Ali et al., 2019a; Kim et al., 2020). Chlorophenols (CPs), simultaneously, are representative highly toxic aromatic

ur na

hydrocarbons that has been used extensively over several decades for wood protection, leading to severe ground water contamination (Fang et al., 2020; Liang et al., 2018). In fact, the coexistence of CPs with woody biomass contributes in the release of hazardous materials both for the environment and public health. Anaerobic digestion (AD), on the other hand, is a promising approach for waste exploitation. It has been used for decades as a potential technology for the treatment of a broad spectrum of organic wastes. However, a wide range of exogenous pollutants

Jo

(e.g., aromatic hydrocarbons, heavy metals and phenolic compounds) that co-exist with biomass wastes and their influence on AD performance have been neglected (Genethliou et al., 2020; Luo et al., 2020; Tsigkou et al., 2020). Therefore, it is crucial to develop effective and eco-friendly approaches to enhance woody biomass degradation simultaneously with CPs detoxification as well as to improve methane (CH4) production efficiently. The major obstacle in the production of CH4 from CSW is the inefficient deconstruction of its recalcitrant lignocellulosic network structure toward microbial enzymatic attack, which

3

results in low digestion rates and long digestion periods (Jin et al., 2016). Therefore, pretreatment is required to unlock the complex structure of CSW via its delignification, cellulose crystallinity reduction and sugars solubilization thereby promoting both its enzymatic attack and CH4 production (Ali et al., 2019b). Various pretreatment approaches, including physical (adsorption, steam explosion, microfiltration, hydrothermolysis or irradiation), chemical (ozonation, oxidation, acid, alkali, sedimentation or coagulation), and combined procedures have been extensively studied for various lignocellulosic substrates prior to AD processing (Ali and Sun, 2015; Ali and Sun, 2019; Hu et al., 2020; Jin et al., 2016; Zhu et al., 2020). However, these methods are in general cost ineffective and some pretreatments, especially under acidic conditions, often release phenol, furfural, and 5-hydroxymethylfurfural, with inhibitory and/or

ro of

toxic effects on anaerobic microorganisms during AD process. Such hazardous compounds are

the degradation products of C6 and C5 sugars as well as lignin, or acetic acid derived from the cleavage of acetyl groups of the hemicellulosic components (Tao et al., 2020). Therefore, it is imperative to develop a cost-effective, eco-friendly and efficient pretreatment method for the

-p

bioremediation of CSW.

Biological methods are competitive and promising alternatives when compared with physical and chemical methods, exploiting the metabolic potential of microorganisms,

re

individually or in a consortium, to clean up the environment. The performance of monocultures on the degradation of complex lignocellulose structure is generally poor, as individual strains

lP

typically can degrade only simple substrates like cellulose (Ali et al., 2019c; García-Mancha et al., 2017). However, multi-species consortia have more balanced enzymatic complements and tolerance to extreme environments than individual species (Ali et al., 2017; Ali et al., 2019a; Ali

ur na

et al., 2019b). During pretreatment, the lignocellulosic carbohydrates are required at certain levels by aerobic microorganisms, which increase the competition on carbohydrates in pretreatment stages and subsequent biogas production (Li et al., 2018). In addition, inoculation of anaerobic microbial consortia could be a cost-effective option for AD enhancement. Thus, recent biological strategies have started using microbial consortia for improved biomass degradation simultaneously with the enhancement of CH4 production during AD process.

Jo

CPs are a group of organochlorides of phenol containing one or more covalently bonded

chlorine atoms. Typically, CPs are classified into five groups, including monochlorophenol (e.g., 2-CP, 3-CP and 4-CP), dichlorophenol (DCP), trichlorophenol (TrCP), tetrachlorophenol (TeCP) and pentachlorophenol (PCP) (Ali et al., 2020). These compounds are persistent in the environment, having poor biodegradability together with toxic and carcinogenic characteristics (Liang et al., 2018). It has been reported that CPs are toxic at concentrations of a few µg/L. Even

4

at very low concentrations (0.005-20 µg/L) of CPs in surface water bodies, it exhibited chronic effects on human beings (Ge et al., 2017). Hence, CPs are considered priority hazardous pollutants by the Agency for Toxic Substances and Disease Registry (ATSDR, 1999). Woody biomass co-existing with CPs makes complex effluents with high biological oxygen demand and chemical oxygen demand (Liang et al., 2015). In an effort to accelerate the simultaneous degradation of CSW and CPs xenobiotics, developing highly effective degrading microbial consortia are required to overcome such challenges. As per our earlier report, the pretreatment of sawdust by the constructed microbial consortium LCDC exhibited significant reduction in cellulose (37.5%), hemicelluloses (39.6%) and lignin (56.7%) within 10 days when 2 g/L milled sawdust was supplemented into the culture medium (Ali et al., 2017). The

ro of

constructed microbial consortium OEM2 successfully removed 75% CPs mixture (concentration of 5% w/v each CP) after 12 days’ culture (Liang et al., 2018). As far as it is known, very few studies are conducted for developing a microbial consortium capable of degrading lignocellulose

and CPs simultaneously. To date, to the authors’ knowledge, no report has been published on

-p

simultaneous biodegradation of CSW and CPs detoxification for enhancing CH4 production. Therefore, screening and construction of novel microbial consortia with an impressive performance on degradation and detoxification of hazardous pollutants are meaningful. In this

re

study, screening and enrichment experiments led to the construction of two novel microbial consortia, the aerobic microbial consortium CS-5 and the anaerobic microbial consortium BC-4.

lP

The performance of the developed consortia was evaluated in terms of their ability and efficiency to degrade of CSW and CPs pollutants simultaneously. The synergistic effect of both microbial consortia on the mixed pollutant hydrolysate deserved more attention in light of AD performance,

ur na

involving CH4 production and microbial community structure. A proposed mechanism of CPs and lignocellulose biodegradation with the assistance of methanogenic Archaea during AD process was also investigated. 2. Material and methods

2.1 Experimental materials

CSW was used as a lignocellulosic substrate in this study. The woody chips were firstly

Jo

air-dried, milled to give a particle size of 2-3 mm thickness using a laboratory hammer mill, then screened with a portable sieve shaker and stored at 4 °C. The main properties of raw CSW were determined. Cellulose was identified as its main component, representing 45.9%, while 18.1 and 21.7% were recorded for hemicellulose and lignin content of CSW, respectively. Holocellulose, which represents the main carbon source in AD process, was 64% of CSW total dry weight. In addition, the raw CSW contained 923 g of total solids (TS) per Kg, while its contents in ash, total

5

organic carbon (TOC) and organic matter (OM) were 6.1, 33.1 and 59.6% of TS, respectively. The Whatman No. 1 filter paper, which consists of 100% cellulose, was used in the experiments of microbial screening and enrichment. Seven representatives of CPs, namely 2-CP; 3-CP; 4-CP; 2,4-DCP; 2,4,6-TrCP; 2,3,4,6-TeCP and PCP were used in this study for the screening of microbial consortia with efficient capability on degradation and detoxification of phenolic xenobiotics. A stock solution of each CP was prepared aseptically using 0.1 N NaOH. Then, a stock solution mixture (concentration of 5% w/v each CP) was prepared and stored in a brown screw cap vial at room temperature. Sawdust waste and water hyacinth compost samples were used as isolation sources for screening and construction of microbial consortia with high degradation capability on

ro of

lignocellulose and CPs simultaneously. Samples of non-sterilized milled sawdust, collected from

local carpentry workshops, were kept under humid conditions up to one month before the screening process. Samples from a running anaerobic batch digester, which used pretreated water

hyacinth co-digested with cow dung for enhancing CH4 production (Ali et al., 2019c), were also

-p

used in this study during microbial screening and enrichment experiments. Anaerobic sludge sample, obtained from thermophilic anaerobic digester of waste-activated sludge, was also used

as inoculum seed in all anaerobic batch digesters during AD process. The sludge contained 96.1

2.2 Culture media and conditions

re

g/kg of TS, while its contents in ash, TOC and OM were 45.5, 33.2 and 54.2% of TS, respectively.

lP

Peptone Cellulose Solution (PCS) medium and Mineral Salt (MS) medium were used for the screening process and biodegradation performance assessment. The PCS medium contained (per liter of distilled water): 5.0 g peptone, 5.0 g NaCl, 1.0 g yeast extract, 1.5 g CaCO3 and

ur na

supplemented with 5.0 g of cellulosic substrate. The MS medium contained (per liter of distilled water): 0.3 g (NH4)2SO4, 0.008 g CaCl2.2H2O, 0.3 g K2HPO4, 0.3 g KH2PO4, 0.002 g FeSO4, 0.61 g NaCl, 0.13 g MgSO4.7H2O, 2.0 g trypticase and 2 g yeast extract. Then, a volume of 1 mL trace element solution (with initial pH 6.8) was mixed with the above MS medium ingredients. The trace element solution contained (per liter of distilled water): 0.1 g CuSO4.5H2O, 0.2 CaCl2, 0.11 g ZnCl2, 0.03 Na2MoO4.2H2O, 0.09 g MnCl2.4H2O, 0.16 g CoCl2.6H2O, 0.02 g H3BO4 and 0.06

Jo

NiCl2.6H2O.

The medium was supplemented with 0.5% (w/v) CSW, while it was replaced by 1% (w/v)

filter paper when used in the experiment. For aerobic cultivation, the cultures were incubated at 50 °C with an agitation of 130 rpm, while under anaerobic conditions, 1 mg resazurin was added to enrichment medium-containing serum bottles to wipe off the dissolved oxygen. The serum

6

bottles were then boiled under the steam of O2-free N2 gas in order to maintain anaerobic conditions. 2.3 Screening and enrichment of woody biomass- and CPs-degrading microbial community In order to construct stable microbial consortia capable of degrading lignocellulosic biomass and CPs simultaneously, the enrichment culture technique was carried out in two stages as depicted in the experimental set up (Fig. 1). Firstly, 1% (w/v) filter paper was added separately into 100 mL of MS and PCS media supplemented with 50 µL CPs stock solution and 5 g of the isolation source. The cultures were then incubated under aerobic or anaerobic conditions. Once the filter paper decomposed into megascopic fiber fragments, 10% (v/v) of the culture fluid was transferred to the new enrichment medium. Successive culture transfer for more than 20

ro of

generations led to two stable microbial consortia with high degradation performance. At the second stage, the procedures were repeated as in the first stage to improve the performance of

microbial consortia on the simultaneous degradation of CSW (woody biomass) and CPs, but filter

paper was replaced by 0.5% w/v of CSW. Successive transfers were continued until the CSW

-p

was softened. As a result, two novel and stable microbial consortia with the efficient capability of lignocellulosic biomass and CPs degradation were constructed and named as aerobic microbial consortium CS-5 and anaerobic microbial consortium BC-4 (Fig. 1). The individual strains of

re

each microbial consortium were mixed in equal proportions (500 µl, OD 0.2) to maintain the same cell count in multi-species consortia as well as in monocultures. Stocks of the constructed

lP

microbial consortia CS-5 and BC-4 were stored at -80 °C. 2.4 16S rRNA gene sequencing

The identities of the pure strains were identified based on 16S rRNA gene sequencing. The

ur na

total genomic DNA of bacteria was extracted using the commercial Takara kit according to the manufacturer's instructions. For the amplification of 16S rDNA, the extracted DNA was used as a

template

together

with

a

pair

of

universal

bacterial

primers

27F

(5'-

AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCTACCTTGTT ACGACTT-3'). The PCR process and amplification conditions were performed as previously described (Ali et al., 2019b). The sequences of 16S rRNA gene were then aligned and compared with the sequences

Jo

available in the GeneBank database (NCBI). The neighbor-joining method was used to construct the phylogenetic relationship in terms of 16S rRNA gene sequencing using MEGA 7.0. 2.5 Analytical methods The potential performances of the aerobic microbial consortium CS-5 and the anaerobic microbial consortium BC-4 on the degradation of CSW and CPs were evaluated for 15 days. The preserved consortia CS-5 and BC-4 were activated on the enrichment media for one generation

7

prior to biodegradation process, which was performed in 100 mL PCS medium supplemented with CSW (0.5% w/v), 50 µL of CPs stock and an inoculum volume (10%) of CS-5 or BC-4. The flasks with the aerobic microbial consortium CS-5 were incubated at 50 °C and at an agitation level of 130 rpm for 15 days. However, this experiment was performed in an anaerobic incubator at 50 °C to ensure the maintenance of an anaerobic environment for the growth of BC-4 (Fig. 1). For weight loss experiments, the hydrolysate (fermentation broth containing CPs solution and residual CSW) was centrifuged at 5000 rpm for 15 min. The residual solids were then washed for 2-3 times with acetic acid/nitric acid reagent, followed by sterilized deionized water to remove non-lignocellulosic materials and dried at 105 °C for 2 h and the weight loss of residual CSW was determined (Ali et al., 2019b). The components of the dried solid residues of CSW were analyzed

ro of

based on Goering and Van Soest’s method (Goering and Van Soest, 1970).

The degradation efficiency of CSW was calculated using the following equation: X(L) =

[S0(L) – S(L)]/S0(L), where X(L) is the efficiency of CSW degradation in terms of lignocellulosic

components (cellulose, hemicellulose and lignin). S0(L) is the mass of the lignocellulosic

-p

component before biodegradation. S(L) is the mass of the lignocellulosic component at a time

within the biodegradation process. The degradation efficiency of CP was calculated using the following equation: R(cp) = [CA(cp) – CMC(cp)]/CA(cp), where R(cp) is the efficiency of CP degradation

re

that varies from one CP type to another one in the CPs stock solution. CA(cp) is the concentration of CP on days 5, 10 and 15 of cultures devoid of CS-5 and BC-4. CMC(cp) is the concentration of

lP

CP after its microbial degradation on days 5, 10 and 15. The concentration of CP was measured by HPLC following the method reported earlier (Jianping et al., 2006). The routine parameters including TS, VS, pH and VFAs were determined according to the

ur na

Standard Methods (APHA, 2012). The concentration of VFAs was determined using GC (PerkinElmer, Model Clarus-680, USA) with a sample volume of 1 µL and an injector temperature of 180 °C. During the biodegradation process, samples of CSW hydrolysate were withdrawn at different time intervals for determining their weight loss together with Chemical Oxygen Demand (COD) (Kong et al., 2018; Yuan et al., 2012). The CODloss was calculated from the following equation: CODloss = (CODs + CODm) – (CODr + sCODh), where CODs is the

Jo

substrate COD before its biodegradation, CODr is the substrate residual COD during its biodegradation, CODm is the COD of uninoculated PSC medium, and (sCODh) is the hydrolysate COD.

Scanning Electron Microscopy (SEM) was also investigated for a better understanding of the structural alterations of CSW made by the microbial consortia CS-5 and BC-4 in the biodegradation process. The CSW residues were washed for 2-3 times with sterilized deionized

8

water to remove non-lignocellulosic materials. The samples were centrifuged at 5000 rpm for 15 min before drying at 105 °C for 2 h and then examined by SEM (JEM-100SX, JEOL, Japan). Biogas compositions were analyzed by a GC (Agilent HP5972) equipped with a thermal conductivity detector and a packed column. Argon was used as the carrier gas with a flow rate of 14 mL/min. The injection volume of the sample was 1 mL. The operational temperature of the oven was adjusted at 40 °C for 6 min. The injector and detector temperatures were controlled at 120 and 100 °C, respectively. 2.6 Enzyme assay The degradative lignocellulolytic enzyme activities in the crude hydrolysates made by CS5 and BC-4 were assessed in samples collected at time intervals during degradation experiments

ro of

which were then centrifuged at 5000 rpm for 15 min. The 3,5-dinitrosalicylic acid (DNS) method (Miller, 1959) was used to determine xylanase, polygalacturonase and endoglucanase activities using beechwood xylan, polygalacturonic acid and carboxymethylcellulose (CMC), respectively

as the corresponding substrates. In the reaction mixture, 150 µL of the corresponding substrate

-p

(1% w/v) in sodium acetate buffer (50 mM, pH 5) was mixed with 50 µL of filtered supernatant

before incubation at 50 °C for 10 min. Activities were calculated based on xylose, galacturonic acid and glucose as standards. One unit of enzyme activity was defined as the amount of enzyme

re

equivalent to the release of reducing sugar (1 μmol) per minute under assay conditions. In order to determine the enzymatic activities of β-glucosidase, β-xylosidase and

lP

cellobiohydrolase, the corresponding substrates namely, p-nitrophenyl glucopyranoside (p-NPG), p-nitrophenyl-β-D-xylopyranoside (p-NPX), and cellobioside p-nitrophenyl-β-D-cellobioside (pNPC) were used, respectively. The reaction mixture contained 50 µL of filtered supernatant mixed

ur na

with 100 µL of 5 mM corresponding substrate solution and 50 µL of 200 mM sodium acetate buffer (pH 5). The reaction mixture was maintained at 50 °C for 10 min. One unit of enzyme activity was defined as the amount of enzyme equivalent to the release of 1 μmol of the standard (p-nitrophenol) per minute under assay conditions. Ligninolytic enzyme activities were determined in terms of manganese peroxidase, lignin peroxidase, and laccase using 2,6-dimethoxyphenol (2,6-DMP), veratryl alcohol and 2,2-azonodi-

Jo

3-ethylbenzothiazoline-6-sulfuric acid (ABTS), respectively as the corresponding substrates. In a total volume of 1 mL reaction mixture used for measuring manganese peroxidase activity, the crude enzyme was mixed with H2O2 (10 mM), sodium tartrate (50 mM, pH 5) and 1 mM MnSO4 at room temperature. In a total volume of 1 mL reaction mixture used for assaying lignin peroxidase activity, the crude enzyme and 10 mM H2O2 were mixed with 40 mM veratryl alcohol substrate in sodium tartrate (100 mM, pH 3.5) at room temperature. Laccase activity was

9

measured in a reaction mixture volume of 1 mL containing ABTS substrate (1 mM) in sodium acetate buffer (100 mM, pH 5) mixed with the crude enzyme. One unit of enzyme activity was defined as the amount of enzyme catalyzing the release of corresponding products (1 μmol) per 1 min. 2.7 Toxicity test Acute toxicity of the reaction solution (CPs mixture) was evaluated during CPs degradation by CS-5 or BC-4 using three toxicity tests. Vibrio fischeri, Daphnia magna and Lactuca sativa were used as targeting species belonging to three different trophic levels in the Microtox ®, Daphnia and seed germination toxicity tests, respectively. Samples were withdrawn at the beginning and at the end of the biodegradation process for evaluations. The toxicity rating was

ro of

performed based on acute toxicity classification (Supplementary data, Table S1). 2.7.1 Microtox® test

It depends on measuring the inhibition in the luminescence of marine luminescent bacteria,

V. fischeri after 15 min of exposure to the reaction solution, which is due to disruption of the

-p

respiratory process of V. fischeri. The test was performed following the manufacturer's protocol

ISO 11348-3 (1998). Phenol was used as a reference toxic compound. Toxicity was expressed as an effective concentration 50 (EC50), which is identified as the concentration causing a 50%

re

decrease in the bacterial luminescence after 15 min of incubation at 15 °C. 2.7.2 Daphnia test

lP

It depends on measuring the inhibition of D. magna mobility after 48 h of exposure at 20 °C following the manufacturer's protocol ISO 6341(1996). Potassium dichromate was used as a reference toxic compound. EC50 was identified as the concentration causing immobility in 50%

ur na

of D. magna after 24 h or 48 h exposure at 20 °C. 2.7.3 Seed test

The toxicity test was evaluated by measuring a reduction in root elongation of Lactuca sativa following the manufacturer's protocol EPA/600/3-88 (EPA, 1989). Ten seeds of Lactuca sativa were soaked with a 5 mL solution of the reaction solution and incubated for 120 h at room temperature. EC50 was identified as the concentration which provokes a 50% inhibition of Lactuca

Jo

sativa elongation after 120 h of incubation at room temperature. 2.8 Pretreatment and AD process The biological pretreatment stage of CSW prior to AD process and the experimental set up

of AD process are depicted in Fig. 1. The main objective of the biological pretreatment was to make cellulose and hemicellulose components more readily available and digestible for further AD processing. The results of biodegradation process revealed that the optimal length of time for

10

pretreatment of CSW by CS-5 was 7 days. For the pretreatment stage, CS-5 from the frozen stock was firstly activated for one generation into a PCS medium containing 1% (w/v) filter paper and the cultures were incubated at 50 °C for 5 days. In 300 mL PCS medium supplemented with 150 µL of CPs stock solution, 1.5 g CSW was added. Afterward, 30 mL of 5-day-old CS-5 culture was used as inoculum. The mixture was incubated at 50 °C and an agitation level of 130 rpm for 7 days. The control preparation was also prepared as illustrated in Fig. 1, using sterilized CS-5 as inoculum. To assess the influence of biological pretreatment on digester performance and CH4 production, AD experiment was performed using three groups of anaerobic batch digesters, namely G-I, G-II and G-III as summarized in Fig. 1. Each digester had a volume of 5 L, with a

ro of

working volume of 80% of the total digester volume. The hydrolysate of untreated CSW was fed into the control digester G-I. After 7-days’ pretreatment of CSW by CS-5 consortium in PCS medium supplemented with CPs stock solution, the hydrolysate was fed into digester G-II. In the batch digester G-III, the synergistic effect of the 7-days’ pretreated hydrolysate mixed with the

-p

anaerobic microbial consortium BC-4 deserved more attention in light of their influence on

digester performance. All digesters were first seeded with a 7-day old anaerobic sludge (2% w/v). All digesters were supplemented with a mixture of N2/CO2 (80/20 v/v) for securing anaerobic

re

conditions before sealing the digesters with rubber stoppers. The sealed anaerobic digesters were incubated at 50 °C for 40 days. The volume of biogas produced was daily determined based on

lP

the water displacement method. Hence, the corresponding cumulative biogas volume was calculated. CH4 content was analyzed daily by gas chromatography (GC) using a biogas analyzer. When no biogas was obtained over three successive days, the AD process was terminated. The

ur na

variations in the operational parameters of pH, VFAs and sCOD were determined at time intervals during pretreatment and AD experiments. The alterations in lignocellulosic compositions and residual weights were also analyzed.

2.9 Microbial diversity analysis with specific identification and quantification of methanogenic Archaea

An anaerobic reactor sample stabilized (fixed) was analyzed regarding its microbial

Jo

populations using VIT® gene probe technology. The culture-dependent approach for isolation, identification and enumeration of microbial communities was carried out following the methods described previously (Ali and Sun, 2019). The VIT® gene probe technology was used for the identification and relative quantification of the culture-independent microbial community (Bacteria and Archaea)-based fluorescence in situ hybridization (FISH) as described in details (Ali et al., 2019a; Ali and Sun, 2019). Domain Bacteria were targeted using the probe sequence

11

EUB338, while Archaea and remaining Bacteria were targeted by ARC915 and EUB338+ probe sequences, respectively. Genomic DNA was quantified by qPCR-targeting 16S rDNA with a pair of primers 27F and 1510R (Ali et al., 2019a). Primer pairs of MetF-MetR and BAC1055FBAC1392R were used for detecting mcrA gene of methanogenic Archaea. This gene encodes methyl co-enzyme M reductase (Ali and Sun, 2019). The viability of Bacteria and Archaea was examined by DAPI-based fluorescence microscopy as described previously (Ali et al., 2019a). Viable cells with integrated membranes were fluorescent green, while dead cells were of red damaged membranes. 2.10 Statistical analysis The data were visualized and analyzed statistically using Minitab 17.1.0.0 software

ro of

(Minitab Inc., Pennsylvania, USA) and SigmaPlot 12.5.0.38 software (SigmaPlot, Systat

Software Inc., UK). The two-ways hierarchical clustering analysis was performed by the PCORD for windows (ver.5) using Sorensen methods for distance and beta (-0.025) for group linkage. The normality of data was analyzed using Shapiro-Wilk test. Comparisons between two

-p

or more groups were performed using one-way ANOVA test for variance with multiple

comparisons using Tukey test. Pearson’s correlation coefficient test was used to examine the

3. Results and discussion 3.1 Screening and enrichment process

re

correlation between different numerical data. P value < 0.05 was considered significant.

lP

Screening and enrichment procedure, which aimed at looking for stable microorganisms with great potential on lignocellulose and CPs degradation, is depicted in Fig. 1. In the screening process, the filter paper was significantly degraded when isolation sources of microorganisms

ur na

(rotten sawdust and Eichhornia crassipes compost) were inoculated into the enrichment media within 48 h. It could be probably due to that the substrates assisted the development of an abundant lignocellulose-degrading microbial community of high enzymatic degradation activity, which could accelerate the simultaneous degradation of lignocellulose and CPs. Hence, the resultant microbial community was suitable for a new generation transfer. In fact, successive transfers were associated with the enhancement of microbial degradation performances. At the

Jo

early stage of the screening process, the microorganisms may undergo a fierce competition stage, leading to the elimination of non-adapted microbes and only microorganisms, which have great potential in processes related to the simultaneous degradation of lignocellulose and CPs, were dominant. After enrichment for 23 generations, two microbial communities, one aerobic and another one anaerobic, with stable and high degradation performance, were obtained and inoculated into the new culture media for the second enrichment stage. Meanwhile, the CSW

12

substrate replaced the filter paper in the enrichment media to increase the lignin load as well as the concentration of CPs (50%). During this stage, biodegradation efficiencies were improved. The characteristics of CSW biodegradation revealed that 17.3 and 13.8% of the CSW were degraded with the assistance of aerobic and anaerobic microbial communities, respectively during the fourth generation of the enrichment process. The degradability performance of CSW by both microbial communities improved rapidly during the enrichment process and reached the stability trend through twelve generations with a CSW weight loss of 58.1 and 49.6%, respectively. After successive enrichment, the CSW weight loss achieved 70.6 and 59.7% within 5 days' incubation by aerobic and anaerobic microbial communities, respectively in the sixteenth generation. Clearly, both microbial communities showed relatively high potential in woody biomass

ro of

degradation at aerobic and anaerobic conditions, which was probably due to their capability to produce various enzymes that may potentially synergize in the degradation processing of CSW.

Seven representatives of CPs, namely 2-CP, 3-CP, 4-CP, 2,4-DCP, 2,4,6-TrCP, 2,3,4,6-

TeCP and PCP were investigated to evaluate the efficiency of microbial communities on CPs

-p

degradation during the second enrichment stage. Clearly, mono- and di-chlorophenols (2-CP, 3CP, 4-CP and 2,4-DCP) were degraded completely by both aerobic and anaerobic microbial

communities within 5 days' incubation of generation twelve and generation sixteen. However, 2-

re

CP, 3-CP, 4-CP and 2,4-DCP were degraded by 91.8, 94.2, 90.6 and 69.1%, respectively by the aerobic microbial community during the fourth generation of the enrichment process. All tested

lP

mono- and di-chlorophenols were degraded completely by the anaerobic microbial community of the fourth generation. In addition, 2,4,6-TrCP was degraded completely by both aerobic and anaerobic microbial communities within 5 days' incubation of generation sixteen, while it

ur na

achieved degradation percentages of 49.5 and 68.0% by the aerobic microbial community of generation twelve and generation sixteen, respectively. Anaerobic microbial community of generation twelve and generation sixteen degraded 67.3 and 89.7% of 2,4,6-TrCP, respectively. The 2,3,4,6-TeCP compound showed no degradation by both microbial communities of the fourth generation, while the aerobic microbial community degraded 10.3 and 19.4% of 2,3,4,6-TeCP in generation twelve and generation sixteen, respectively. The anaerobic microbial community of

Jo

generation twelve and generation sixteen degraded 18.5 and 30.4% of 2,3,4,6-TeCP, respectively. Both microbial communities showed no efficiency in PCP degradation within 5 days' incubation of the fourth generation. In generation twelve and generation sixteen, PCP was degraded by the aerobic microbial community (5.04 and 9.2%) and anaerobic microbial community (12.04 and 18.2%), respectively. Therefore, it could be concluded that microbial communities obtained

13

through the enrichment process exhibited great potential for the simultaneous degradation of woody biomass and CPs. 3.2 Clustering analyses and construction of microbial consortia CS-5 and BC-4 After the enrichment procedure for 23 generations in culture media supplemented with CPs and CSW, 21 stable strains, denoted as SR-1 to SR-21, were obtained. It was impressive to determine the performance of these strains in terms of degradation of aromatic dyes, aromatic compounds and toxic related phenolic compounds, tolerance to high temperatures and production of lignocellulolytic enzymes. As a result, the characteristic profile of each strain was recorded as depicted in Fig. 2. Clustering analyses revealed that nine bacterial strains with the highest and efficient performances were shifted together into one cluster designated as cluster X. In this

ro of

cluster, five bacterial strain codes (green color) were aerobic and denoted as SR1, SR3, SR5,

SR14 and SR16, while the other four-strain codes (red color) were anaerobic and denoted as SR4, SR7, SR8 and SR13 (Fig. 2). The nine strains were capable of degrading biophenyl (BPL),

tolerating high temperatures up to 50 °C (except for SR-4 strain) as well as producing cellulolytic

-p

(CEL), xylanolytic (XYL) and ligninolytic (LGN) enzyme activities. Degradation of veratryl alcohol (VEA) and 4-methyl-2,6-dimethoxyphenol (MMP) was observed in all strains of cluster

X except the SR-16 strain. However, the degradation of vanillyl alcohol (VAA) and benzoic acid

re

(BZA) was recorded in all strains except SR-4 and SR-5, respectively. Azure B (AZB) dye was successfully decolorized by SR-7, SR-4, SR-13, SR-3 and SR-16. However, methylene blue

lP

(MEB) dye was decolorized by all strains except SR-13 and SR-3 strains. Both strains SR-8 and SR-14 failed to degrade p-cresol (CRS), while SR-5 and SR-1 showed inability to degrade vanillin (VA). As depicted in Fig. 2, six strains (SR-7, SR-4, SR-5, SR-1, SR-8 and SR-14) showed a

ur na

capability to degrade 3-chlorobenzoic acid (CBA) successfully. Clearly, the bacterial strains of cluster X present impressive functions to deconstruct lignocellulose, CPs and some other hazardous materials simultaneously with their lignocellulolytic activities and tolerance to high temperature, which implies that they may be valued to work on the bioremediation for various xenobiotics under extreme conditions of high temperature. Therefore, two novel aerobic and anaerobic microbial consortia, designated as CS-5 and BC-4, respectively were constructed.

Jo

Identification and a phylogenetic tree of the components of the constructed microbial

consortia CS-5 and BC-4 were performed based on 16S rRNA gene sequencing. The results revealed that the individual strains (SR1, SR3, SR5, SR14 and SR16) consisting CS-5 consortium were belonging to the following genera Micrococcus, Citrobacter, Exiguobacterium, Acidisoma and Dyella, respectively. SR1 and SR5 strains showed 97% identity to Micrococcus luteus strain KL3 (KT901825) and Exiguobacterium acetylicum strain Lmb007 (KT986080), while SR3 and

14

SR16 strains showed 98% identity to Citrobacter freundii strain FC18565 (MK561018) and Dyella sp. LD14149 (JX630093), respectively. SR14 strain showed 96% identity to Acidisoma sibirica (KF241161). On the other hand, the individual strains (SR4, SR7, SR8 and SR13) of the microbial consortium BC-4 were belonging to the following genera Thermoanaerobacterium, Ruminococcus, Caproiciproducens and Methanobrevibacter, respectively. SR4 strain showed 99% identity to Thermoanaerobacterium aciditolerans strain 761-119 (NR_042856). SR8 strain showed 98% identity to Caproiciproducens galactitolivorans strain BS-1 (NR_145929), while SR7 and SR13 strains showed 97% identity to Ruminococcus flavefaciens strain C94T (AM915269) and Methanobrevibacter thaueri strain CW (NR_044787), respectively. 3.3 Performance of constructed microbial consortia on CSW degradation

ro of

3.3.1 Variations in CSW chemical composition

Cellulose was the main component of the raw CSW, representing 45.9% of the dried

biomass weight, while 18.1% and 21.7% for hemicellulose and lignin, respectively. Fig. 3 depicts the degradation efficiency of CSW components (cellulose, hemicellulose and lignin) by the

-p

constructed microbial consortia CS-5 and BC-4 over an incubation period of 15 days. CSW was

degraded more strongly during the first 5 days, at which cellulose, hemicellulose and lignin realized the fastest weight loss of respectively, 52.7, 54.5 and 33.5% by CS-5, while exhibited

re

48.4, 52.4 and 35.5 % by BC-4. Cultivation of CSW with the microbial consortium CS-5 resulted in lignin, cellulose and hemicellulose loss, which reached above 39, 56 and 60%, respectively at

lP

the end of the cultivation period (Fig. 3 A). In comparison, BC-4 incubated with CSW resulted in lignin, cellulose and hemicellulose loss of above 40, 47 and 58%, respectively after 15 days' cultivation (Fig. 3 B). Clearly, hemicellulose was evidenced as the primary component of lost

ur na

mass during the degradation of CSW by CS-5 or BC-4. It was degraded at a greater extent than other lignocellulosic components during the entire degradation time under the experimental conditions, demonstrating that the individual strains of constructed consortia CS-5 or BC-4 can co-exist synergistically for extracellular hemicellulases production and lignocellulose degradation. Hemicellulose forms a protective physical barrier to decomposition. It is a priority degradation target by the microbial consortia to support their microbial growth and metabolism

Jo

(Liado et al., 2015). Comparisons of biodegradation of lignocellulosic components with previous studies are given in Table 1. The performed weight loss of CSW induced by CS-5 and BC-4 was 69.2 and 56.3%, respectively within 15 days. The pretreatment of wheat straw, rice straw and corn straw by the microbial consortium XDC-2, resulted in a loss of their weights by 25.2, 39 and 17.6%, respectively (Hui et al., 2013). The loss of Napier grass induced by the microbial consortia MC1, WSD-5 and XDC-2 was 25, 29 and 27%, respectively within 21 days (Wen et al., 2015),

15

while the microbial consortium NSC-7 induced a total weight loss of rice straw by 73.6% within 14 days (Liu, 2008). A total weight loss of 41.5 and 45.7% was achieved when rice straw and wheat straw were degraded by OEM2 and TC-5 consortia for 12 days (Kong et al., 2018; Liang et al., 2018). 3.3.2 Structural variations of CSW The SEM analysis was applied to investigate the structural characteristics of degraded CSW after inoculation with CS-5 or BC-4 (Fig. 4). The raw CSW showed a non-decayed, smooth, compact and intact surface structure (Fig. 4 A&B). The 7-day CSW samples inoculated with the microbial consortium CS-5 or BC-4 exhibited partial degradation with distorted and collapsed surfaces (Fig. 4 C&D). In 15-day CSW inoculated with CS-5 or BC-4, the samples appeared with

ro of

extensive disruption and decay, suggesting the degradation of the main CSW components (Fig. 4

E&F). These results have generally corresponded to the variations of cellulose, hemicellulose and lignin levels as depicted in Fig. 3. In addition, the presence of pores in the biodegraded CSW was probably associated with higher lignin degradation, increasing the exposed surface area that

-p

could effectively increase the accessibility of cellulases and hemicellulases to lignocellulosic biomass, thereby making it easier for subsequent AD processing (Dong et al., 2019). 3.3.3 Lignocellulolytic enzyme activities

re

The activities of lignocellulolytic enzymes produced by CS-5 and BC-4 during 15 days of CSW degradation were determined (Fig. 5). Xylanase and β-xylosidase activities were used for

lP

evaluating hemicellulose degradation (Fig. 5 A). Among hemicellulases, xylanase enzyme produced by CS-5 achieved its maximum specific activity (7.11 U/mg) on day five, after which it decreased to 2.91 ± 0.04 U/mg at the end of the degradation process. For BC-4, the specific

ur na

activity of xylanase increased to its highest value on day five (5.67 U/mg), then decreased to 1.55 U/mg on day 15. As for xylanase, the maximum specific activities of β-xylosidase (3.09 and 2.86 U/mg) of CS-5 and BC-4 were achieved, respectively on day five. Then it decreased gradually until the end of the process.

β-glucosidase, endoglucanase and cellobiohydrolase activities were determined in order to evaluate the activities of cellulases produced by the microbial consortia during the degradation

Jo

process (Fig. 5 B). In both CS-5 and CB-4, the β-glucosidase specific activity increased gradually to reach its highest level of 0.47 and 0.36 U/mg, respectively on day 10. For comparison, the specific activity of endoglucanase remained at a low level for both microbial consortia during the entire incubation period. In CS-5, the cellobiohydrolase achieved its maximum specific activity on day five (0.49 U/mg), while the cellobiohydrolase specific activity reached its highest level of 0.44 U/mg on day 10. The polygalacturonase specific activity in CS-5 fluctuated and reached its

16

highest level of 0.24 U/mg at the end of the degradation process. In contrast, the specific activity of polygalacturonase in BC-4 increased gradually until day 15 (0.20 U/mg). In terms of ligninolytic enzymes produced by CS-5 and BC-4 during the degradation of CSW, the activities of manganese peroxidase, lignin peroxidase and laccase were determined (Fig. 5 C). Manganese peroxidase produced by CS-5 and BC-4 showed a high specific activity of 2.71 and 0.22 U/mg on days five and 10, respectively and declined by the end of the degradation process. In contrast, the specific activity of lignin peroxidase produced by CS-5 and BC-4 increased gradually to reach its highest level of 0.29 and 0.19 U/mg, respectively on day 15. Laccase activity was detected only in CS-5 and the peak specific activity was 1.58 U/mg on day five, while laccase activity was below detectable level in the microbial consortium BC-4. Overall,

ro of

the highest degradation rate of hemicellulose by both microbial consortia CS-5 and BC-4 over other lignocellulosic components of CSW suggested that hemicellulases-producing microbial consortia played a major role in reaching and degrading hemicellulose.

3.3.4 Variations in routine parameters during CSW biodegradation

-p

Variations in sCOD, pH and the predominant VFAs (acetic acid, butyric acid and propionic

acid) during CSW degradation by the constructed microbial consortia CS-5 and BC-4 were studied (Fig. 6). In general, as the biodegradation time of CSW prolonged, the concentration of

re

VFAs increased gradually, then decreased by the end of the degradation process. On day 7, acetic acid reached its peak value of 1730 and 1450 mg/L at pH 5.9 and 7 during the degradation of

lP

CSW by CS-5 and BC-4, respectively (Fig. 6 A&B). However, the peak values of propionic acid were achieved at 560 and 450 mg/L and pH values of 5.9 and 6.9 when CS-5 and BC-4 performed CSW degradation on days 7 and 9, respectively (Fig. 6 C&D). The concentration of butyric acid

ur na

reached its peak value of 1970 and 1900 mg/L at pH 7.5 and 7.2 when the degradation of CSW by CS-5 and BC-4 occurred on day 11 and 13, respectively (Fig. 5 E&F). In fact, pH is one of the main parameters affecting the efficiency and composition of VFAs production (Chen et al., 2020). The optimal pH values for VFAs production found between a range of 5.3 and 11 (Lee et al., 2014). However, the specific pH range depends on the type of waste used. In this study, the pH values for VFAs production were ranged between 5.9 and 8.4 when CSW was degraded by

Jo

CS-5, while the pH range was between 6.5 and 8.7 when VFAs were produced from CSW inoculated with BC-4. Although acidic pH affected positively the production of VFAs as well as microbial growth as reported previously (Wang et al., 2020), our findings are in agreement with Atasoy et al. (2020) and Jankowska et al. (2017) who reported that alkaline pH enhanced the production of VFAs. On the other hand, acetic and propionic acids are important organic acids that can be used for CH4 fermentation (Yang et al., 2008). In addition, acetic and butyric acids

17

play key intermediate roles in AD process as well as in enhancing lignocellulose degradation (Hui et al., 2013). The level of sCOD in the hydrolysate increased rapidly during the early stage of CSW degradation by CS-5 and BC-4. The peak values of sCOD concentrations with CS-5 and BC-4 during the degradation of CSW occurred at days 7 and 9, reaching 15132 and 1321.4 mg/L, respectively, followed by a decrease of sCOD until the end of biodegradation process. Fig. 7 depicts the performance of CS-5 and BC-4 on the sCOD and VFAs during the degradation of CSW. Clearly, as the concentration of VFAs (acetic acid and propionic acid) increased, the level of sCOD also increased up to day 7, followed by a decrease of VFAs and sCOD concentrations until day 15 during the degradation of CSW by CS-5 (Fig. 7 A&C). However, the peak value of

ro of

VFAs (butyric acid) obtained on day 11 showed a low level of sCOD (5648.7 mg/L) when CSW was degraded by CS-5 (Fig. 7 E). Both VFAs (acetic acid, butyric acid and propionic acid) and

sCOD concentrations increased simultaneously up to day 7 after CSW degradation by BC-4. The peak value of sCOD, which was achieved on day 9, was attributed to the maximum increase in

-p

propionic acid concentration (450 mg/L) (Fig. 7 D) with a relative decrease in acetic acid

concentration (1410 mg/L) (Fig. 6 B). However, at the peak value of sCOD, the butyric acid was behind its peak value, which achieved 1900 mg/L on day 13. (Fig. 6 F). Our results are in

re

agreement with Yuan et al. (2014) who found that the VFAs and sCOD concentrations increased rapidly up to day 4 of corn stalk degradation using the microbial consortium MC1. The increase

lP

of VFAs and sCOD concentrations during the biodegradation of CSW was attributed to the deconstruction of insoluble macromolecular organic compounds of CSW (Poszytek et al., 2016). The subsequent decrease of VFAs and sCOD concentrations was probably due to the generation

ur na

of soluble organic products by hydrolytic individual strains within the constructed microbial consortia, followed by the consumption of the soluble metabolites by other microbial groups (fermentative strains) (Wen et al., 2015). The degradation of lignocellulose into soluble products can be regarded as a rate-limiting phase during AD process. In addition, the optimum pretreatment time should be equal to the time at which both VFAs and sCOD concentrations reach their peak values (Poszytek et al., 2016). As

Jo

VFAs (acetic and propionic acids) and sCOD simultaneously reached their peak values on day 7 of the degradation of CSW by CS-5 consortium, the pretreatment time was set up at 7 days. The pretreatment time was also chosen based on the weight loss of lignocellulosic components, which rapidly increased during the early stage of CSW degradation (i.e., first 7 days). The CODloss during CSW biodegradation rapidly increased from 3055 to 10095 mg/L at days 7 and 15, respectively, which was probably due to gas production during the degradation process. The findings obtained

18

from the evaluation of the performance of CS-5 and BC-4 on CSW degradation (VFAs, sCOD, pH, SEM and weight loss) were proof of high hydrolytic activity of these consortia including their hemicellulolytic activities. The cooperation and synergistic effect of the individual strains constituting the CS-5 and BC-4 consortia support their abilities in terms of degradation performance. The performance of microorganisms in a mixture is more efficient than using individual microorganisms, especially when conditions change during the hydrolytic process. Microbial consortia are usually better adapted to extreme conditions as well as their higher tolerance to xenobiotics, such as toxic organic compounds and heavy metals (Giovanella et al., 2020; Wahla et al., 2019). 3.4 Performance of the constructed microbial consortia on CPS degradation

ro of

CPs are aromatic ring compounds containing at least one chlorine atom at the benzene

rings. Conventional chemical and physical methods used for the degradation of CPs are not ecofriendly due to the formation of hazardous materials as by-products (Olaniran and Igbinosa,

2011). Conversely, the removal of CPs from the environment via the biodegradation process has

-p

gained great attention, in recent, due to the complete mineralization of CPs using microorganisms

in the environment. Therefore, the performance of CS-5 (under aerobic conditions) and BC-4 (under anaerobic conditions) on CSW degradation over an incubation period of 15 days was

re

determined.

Monochlorophenols, which are the simplest form of CPs, include 2-CP, 3-CP and 4-CP. In

lP

this study, the aerobic microbial consortium CS-5 showed complete degradation of 3-CP on day 5 and until the end of the degradation process, while CS-5 showed a high degradation potential of 2-CP and 4-CP by 90.3 and 88.4% on day 5 and increased subsequently, reaching 98.6 and

ur na

94.8% on day 15, respectively. Both of them showed complete degradation on day 15. In the case of 2,4-DCP, the degradation increased from 56.8% on day 5 to reach its highest level of 77.1 and 91.5% on day 10 and 15, respectively. The 2,4,6-TrCP was also degraded by CS-5 and showed different levels during the degradation process, in which 37.9, 55.2 and 72.1% were obtained on days 5, 10 and 15, respectively. The degradation of PCP by CS-5 reached its highest level (70.8%) on day 15, while it was 33.4 and 58.6% on days 5 and 10, respectively. These results indicate that

Jo

CS-5 has great potential in the aerobic degradation of CPs. Aerobically, many bacteria have the ability to utilize CPs as a sole source of carbon and energy source using hydroxylation mechanism (monooxygenases), resulting in ortho-, meta- or para-cleavage of CP rings (Arora and Bae, 2014). Pseudomonas pickettii LD1, Rhodococcus opacus 1G, Alcaligenes xylosoxidans JH1and Alcaligenes sp. A7–2 strains have been reported previously for their abilities to utilize monochlorophenols as their sole carbon and energy sources (Fava et al., 1995). The bacterial

19

degradation of 2,4-DCP and 2,4,6-TrCP was initiated through the action of 2,4dichlorophenoxyacetate-α-ketoglutarate dioxygenase and FADH2-dependent-2,4,5-TrCP-4monooxygenase, respectively (Fava et al., 1995). The bacterial degradation of PCP is initiated via the action of either PCP-4-monooxygenase or cytochrome P-450 type enzyme (Fukumori and Hausinger, 1993). The performance of BC-4 to degrade CPs under anaerobic conditions was also evaluated. BC-4 showed complete degradation of 4-CP on day 5 and until the end of the degradation process. Similarly, BC-4 exhibited a high degradation potential on 2-CP and 3-CP over the degradation time and both reached 100% degradation on day 15. In the case of 2-CP, the degradation increased from 89.7% (day 5) to 95.6% (day 10), while the degradation of 3-CP achieved 91.4 and 100%

ro of

on days 5 and 10, respectively. The efficiency of BC-4 on the degradation of 2,4-DCP and 2,4,6TrCP increased over the incubation time and reached the maximum degradation of 100 and

88.6%, respectively on day 15. On day 5, the degradation levels of 2,4-DCP and 2,4,6-TrCP were 45.7 and 51.6%, while levels increased to 61.2 and 74.8% on day 10, respectively when 2,4-DCP

the degradation of CPs under anaerobic conditions.

-p

and 2,4,6-TrCP were degraded by BC-4. These results indicate that BC-4 has great potential in

The mechanism of CPs degradation by bacteria or enriched cultures depends on the

re

reductive dehalogenation step (chlorine atoms are replaced by hydrogen atoms), which is a crucial step especially for polychlorophenols degradation under anaerobic conditions (Field and Sierra-

lP

Alvarez, 2008). Several polychlorophenolic compounds are recalcitrant towards bacterial degradation under aerobic conditions but can be reductively dehydrogenated into lesser CPs, which subsequent mineralized easily (Fava et al., 1995). For example, PCP was dehydrogenated

ur na

reductively by anaerobic bacteria to 2,3,4,5-TeCP, followed by 3,4,5-TrCP, then to 3,5-DCP and 3-CP, which subsequently degraded to phenol and finally CH4 was obtained (Londry and Fedorak, 1992). In order to favor potential anaerobic degradation and dechlorination of CPs, several conditions should be taken into consideration such as methanogenic, denitrification as well as sulfate and iron reduction (Arora and Bae, 2014; Londry and Fedorak, 1992). Anaerobic degradation of CPs (monochlorophenols and polychlorophenols) is initiated by their reductive

Jo

dehalogenation to form phenol, which is transformed into benzoic acid, then mineralized to CO2 and produce CH4 under anaerobic conditions. Although the efficiency of aerobic and anaerobic biodegradation of monochlorophenols and polychlorophenols was variant, more than 89 and 95% degradation of the total CPs consortium was obtained by CS-5 and BC-4, respectively at the end of biodegradation process. Overall, the newly constructed microbial consortia CS-5 and BC-4

20

could be promising for the simultaneous degradation of recalcitrant pollutants, such as woody biomass and CPs. 3.5 Detoxification of CPs The toxicology assessment of CPs after a degradation processing by CS-5 and BC-4 was finally conducted using standardized toxicity bioassays. As a design, the evaluations of the Microtox test, Daphnia test and seed test were performed. The toxicity of CPs (individually and in a mixture) was calculated at the beginning and at the end of the biodegradation process based on EC50 values (Table 2). The toxicity of 15 days' degradation of an individual or mixed CPs was reduced to around 90%. The remaining toxicity portions could be associated with the nondegraded CPs, which remained inside the reaction solution after the biodegradation assay.

ro of

Differences in the toxicity level of CPs at the beginning and at the end of degradation by CS-5 and BC-4 provided a piece of evidence for the capability of the constructed microbial consortia CS-5 and BC-4 to detoxify the highly toxic hazardous compounds, such as CPs after aerobic as

well as anaerobic biodegradation process. The biodegradation of CPs mixture (2-CP, 2,4-DCP,

-p

2,4,6-TrCP and PCP) by the laccase-producing Trametes pubescens allowed 90% reduction in mixture toxicity (Gaitan et al., 2011). Despite the high levels of toxicity obtained at the beginning of 2,4,6-TrCP by an indigenous bacterial community, more than 90% reduction in the toxicity

re

level was estimated at the end of the biodegradation process using Microtox test, Pseudokirchneriella test and Daphnia test (Gallego et al., 2009). Liang et al. (2018) constructed

lP

a new microbial consortium OEM2 with a high potential to detoxify monochlorophenols, 2,4DCP and 2,4,6-TrCP. Clearly, our findings in this safety evaluation, as well as a comparison with previous investigations, suggested that the constructed microbial consortia CS-5 and BC-4 would

ur na

potentially serve as a useful agent for detoxifying CPs, which can be safely implemented in bioremediation processing and biomass-based biorefinery. 3.6 Influence of biological pretreatment on digester performance 3.6.1 Biogas production

The experimental set up of the biodegradation process, pretreatment stage and AD process is summarized in Fig. 1. The results of the biodegradation process for 15 days revealed that the

Jo

optimal length of time for pretreatment of CSW by CS-5 is 7 days, in which VFAs and sCOD concentrations of the hydrolysate were the highest. Some macromolecules hydrolyzed efficiently during the pretreatment stage, and thus, more produced substances were available for subsequent AD processing. Therefore, more produced substances were available for subsequent AD processing. The biogas and CH4 yields obtained during the entire AD retention time are shown in Fig. 8. The results of daily biogas and cumulative biogas yielded by G-I, G-II and G-III revealed

21

that no obvious lag period in biogas production was detected, since G-I, G-II and G-III increased to their peak values of daily biogas production on the second day before subsequent declining (Fig. 8 A). The cumulative biogas yielded by G-III (204 L/Kg VSadded) was significantly higher than that of G-I and G-II, which yielded 116.4 and 157 L/Kg VSadded, respectively. In the stage of most biogas production, it was found that the volume of biogas production using G-III, increased over 76 and 28% for G-I and G-II, respectively. Clearly, the synergistic effect and cooperation of both microbial consortia CS-5 and BC-4 on the mixed pollutant hydrolysate deserved more attention in terms of AD performance as proofed from increased biogas yield in G-III if compared with other groups. The performance of biological pretreatment in enhancing biogas production has also been reported in references. The biogas production from pretreated sawdust by the

ro of

microbial consortia LCDC and SSA-9 was significantly increased, achieving cumulative biogas

yield of 312 L/Kg VS (25.6%) and 55 L/Kg VS (86.4%) over the untreated substrates, respectively (Ali et al., 2017; Ali et al., 2019a). For lignocellulose of maize silage and corn stalks, the biogas

yield was also significantly increased by 38% (393 L/Kg VS) and 68.3% (2450 mL) compared

-p

with the untreated substrates, respectively (Poszytek et al., 2016; Xufeng et al., 2011). In this

study, the biogas yield obtained from the pretreated sawdust hydrolysate containing CPs was comparatively lower than that reported earlier by Ali et al., 2017, Ali et al., 2019a, Poszytek et

re

al., 2016 and Xufeng et al., 2011, which was probably due to the structure of this woody substrate together with the presence of non-degraded CPs associated with CSW hydrolysates in the

lP

digesters. Hence, these factors may affect negatively the performance of microbial community of the AD process and consequently the biogas/CH4 yields. Compared with chemical pretreatment methods used for enhancing biogas production, the biogas yields of corn stover treated with

ur na

ammonia, acetic acid and phosphoric acid were increased by 26.7% (427.1 mL/g VS), 34.1% (71.7 mL/g TS) and 40.7% (75.3 mL/g TS), respectively than the untreated samples (Li et al., 2015; Tian et al., 2016). 3.6.2 Methane yield

The daily and cumulative CH4 yields were also determined (Fig. 8 B). All groups reached their peak values of daily CH4 yields on the second day. The results showed that the daily CH4

Jo

yield in G-III was always significantly higher than that in G-I and G-II during the stage of most biogas production. In addition, a significant difference in cumulative CH4 yields between the treated and untreated groups was observed. The cumulative CH4 yield of G-III was increased by 64.7% over the control digester. The CH4 production was increased by 113.7% at the peak phase of AD process compared to the corresponding control. The cumulative CH4 yield was increased by 36.6% (314 L/Kg VS) when wheat straw was treated by the microbial consortium TC-5 (Kong

22

et al., 2018). The maximum cumulative CH4 yield of sawdust treated with the microbial consortium LCDC was increased by 72.6% (155.2 L/KgVS) over the control (Ali et al., 2017). In fact, the cumulative CH4 yield in this study was lower than those reported by Ali et al. (2017) and Kong et al. (2018), which was probably due to the complex structure of CSW, the presence of non-degradable CPs as well high dry mass loss. Alexandropoulou et al. (2017) reported that the lower CH4 yield values after the fungal pretreatment of willow sawdust was attributed to the high loss of dry mass, in which a high portion of the holocellulose which could be converted to CH4 during AD process was transformed to carbon dioxide upon fungal pretreatment. The daily CH4 content in biogas was also measured during AD process of untreated and pretreated CSW. The peak value of CH4 content (up to 66%) was noted between day 6 and day

ro of

20 of the total biogas production in all anaerobic digesters. Clustering analysis of CH4 content in the biogas of G-I, G-II and G-III showed a significant increase in CH4 content of G-III with

respect to other digesters through 40 days retention time of AD process (Supplementary data, Fig. S1). The peak value of CH4 content for the XDC-treated corn stalk hydrolysate was above

-p

63% on day 5, compared with 58.2% for the untreated substrate, which was achieved on day 8

(Xufeng et al., 2011). Clearly, the cumulative biogas and the CH4 content in the produced biogas during AD process revealed that the constructed microbial consortia CS-5 and BC-4 significantly

re

increased the efficiency of methanization of the woody biomass substrate (CSW). The correlation between the AD retention time and biogas or CH4 yield was significant (Supplementary data,

lP

Fig. S2).

3.6.3 Lignocellulose degradation and digestion time In order to evaluate the effect of CS-5 on the destruction of the lignocellulosic substrate

ur na

CSW during pretreatment stage and its susceptibility to AD process for biogas/CH4 production, the changes in the main components of CSW at the end of 7-day pretreatment stage and 40-day AD process were determined (Table 3). After 7 days’ pretreatment of CSW by CS-5 at 50 °C and an agitation level of 130 rpm, cellulose, hemicellulose and lignin were significantly reduced by 49.2 ± 2.2, 54.3 ± 1.7 and 30.8 ± 1.1%, respectively, with respect to the control. The highest degradation rate of hemicellulose during the pretreatment stage implied that hemicellulase

Jo

enzymes made by CS-5 were effective to reach and degrade hemicellulose prior to AD process. Reduction of cellulose, hemicellulose and lignin contents of anaerobic digesters of untreated and pretreated CSW after AD process was also determined. The reduction of cellulose and hemicellulose content of pretreated CSW of digester G-III was significantly higher than that of the control digester G-I. Despite the reduction of lignin of pretreated CSW lignin in digester GIII was lower than that of cellulose and hemicellulose, it was higher than that of other untreated

23

(G-I) and pretreated digester (G-II). The increased lignin degradation of CSW pretreated with the aerobic microbial consortium CS-5 in the digester G-III was probably due to the anaerobic microbial consortium BC-4 in this digester. Hence, the inoculation of BC-4 into G-III digester was effective not only for its delignification capacity but also higher CH4 yield in this digester. In order to evaluate the effect of biological pretreatment and substrate degradability on the performance of digester during AD process, the technical digestion time 80 (DT 80) was determined. Palmowski and Müller (2000) identified DT80 as the time needed to produce 80% of the maximal digester gas production. In this study, the retention time of AD process was lasted up to 40 days until the biogas production neared zero. The cumulative biogas production was considered as the total produced digester gas. Hence, it was used to calculate DT80 (Zheng et al.,

ro of

2009). G-III showed a DT80 value of (24.3%) after 12 days over the control digester. The reduction of digestion time further indicated that the lignocellulosic substrate CSW had become more

accessible and readily available for degradation after microbial consortium CS-5 pretreatment,

thus a shorter digestion time can be taken (Yuan et al., 2016). This could increase the pretreatment

-p

capacity as well as the production efficiency of an existing digester by using a shorter digestion time (Zheng et al., 2009).

3.6.4 Correlation between process parameters and digester performance

re

The performance of digester during AD process was also determined by physical and chemical parameters. Of these, pH, VFAs and sCOD were determined in this study (Fig. 9). In

lP

general, the trend of pH values during AD was opposite to that of VFAs and sCOD concentrations (Fig. 9 A), which had similar trends (Fig. 9 B&C). The pH values had a similar trend of increasing with the increased AD retention time and then kept stable with less fluctuation. The initial pH

ur na

value in G-I was 5.6, which increased by the end of AD process to 8.7. The pH values of the pretreated digesters G-II and G-III increased from 5.4 and 5.0 to 8.3 and 8.0, respectively after 40 days. The pH values in this experiment were in the range between 5.0 and 8.7, which was appropriate for the microbial community in AD. Hwang et al. (2004) reported that the fermentative microorganisms have the capability to function within the pH range of 4.0-8.5. In addition, the increased pH in the digesters with increasing AD retention times was attributed to

Jo

the consumption of VFAs by methanogenic bacteria. Both VFAs (r = - 0.9, p < 0.001) and sCOD (r = - 0.8, p < 0.001) showed significant negative correlation with pH changes during AD process (Fig. 9 B). The less fluctuation in pH values was probably attributed to VFA and sCOD consumption and production in the digesters. In addition, sCOD was significantly positive correlated with VFAs (r = 0.8, p < 0.001) during AD process (Fig. 9 C).

24

On the other hand, both sCOD and VFAs showed their peak concentrations at the beginning while a decreasing trend in all digesters during AD process was then observed. The decrease of sCOD and VFAs concentration during AD is given in Fig. 9 D. The sCOD concentration was greatly declined from 15864 and 12885 mg/L, reaching 6256 and 7463 mg/L after 40 days by GIII and G-II, respectively than in the control digester G-I (from 9787 to 5672 mg/L), which indicated that large amounts of soluble substrates in the hydrolysates were available for AD process (Poszytek et al., 2016). The initial high concentrations (2278 and 1867 mg/L) of VFAs in the pretreated digesters G-III and G-II rapidly decreased and reached 266 and 211 mg/L, respectively by the end of the digestion process. Therefore, the formed VFAs can play a crucial role in enhancing the hydrolysis of the lignocellulosic substrate. The increased concentration of

ro of

VFAs is due to the degradation of organic materials by acidogenic bacteria, while the decreasing

of VFAs concentration to the minimum level is due to the production of CH4 by methanogenic bacteria (Lin et al., 2017). The VFAs concentration of the control digester G-I was lower than

that of the pretreated digesters and it decreased from 1392 to 155 mg/L, which was probably due

-p

to the rapid conversion of glucose to VFAs, and then conversion of VFAs to other intermediates

during AD process (Poszytek et al., 2016). On the other hand, the formed VFAs in the pretreated digesters can play a crucial role in enhancing the hydrolysis of a lignocellulosic substrate (Yu et

re

al., 2004). The high biogas production in pretreated digesters was probably due to the high hydrolytic activity occurred during AD process, which suggests that the pretreatment stage is

lP

crucial for enhancing biomass-based biogas productivity (Wongwilaiwalin et al., 2010). The inoculation of CSW by the novel constructed microbial consortia CS-5 and BC-4, which were constructed from naturally hydrolytic bacteria for the simultaneous degradation of CSW and CPs,

ur na

significantly enhanced biogas and CH4 yields by 74.6 and 64.6%, respectively higher than the control at the end of AD process. The synergistic action of CS-5 and BC-4 increased CH4 production by 113.7% at the peak phase of AD process compared to the corresponding control. 3.7 Microbial community structure and AD performance: A proposed mechanism Based on our investigation and those findings of CS-5 and BC-4 performances on simultaneous degradation of lignocellulose and CPs as mentioned above, a possible pathway was

Jo

proposed to understand the synergistic effect and cooperation of both novel constructed microbial consortia CS-5 and BC-4 together with the digester microbial community on AD performance in terms of CH4 production (Fig. 10-12). 3.7.1 Microbial distribution Molecular characterization of the bacterial and methanogenic Archaea community was performed based on real-time PCR in order to present variations in the quantitative composition

25

of community profiles in the anaerobic digesters containing untreated and pretreated hydrolysates as well as in anaerobic sludge. Taxonomic profile of microbial communities of digesters G-I, GII and G-III at the phyla and families’ level was determined (Fig. 10). Euryachaeota, Bacteroidetes, Firmicutes, Proteobacteria, Chlotoflexi, Actinobacteria and Planctomycetes were the seven dominant phyla of the microbial community in all digests but with different relative abundance percentages (Fig. 10). The bacterial diversity is beneficial for the improvement of biogas production performance (Cai et al., 2017). Clearly, Bacteroidetes phylum dominated the bacterial communities of G-III over that of other digesters, representing 19.3% of the microbial community and Flavobacteriaceae was the predominant family of the G-III Bacteroidetes (Fig. 10 A). In digesters G-II and GIII, Proteobacteria dominated the bacterial phyla of digester

ro of

microbial communities, achieving 13.7 and 21.4%, respectively. Pseudomonadaceae, Hyphomonadaceae and Rhodobacteraceae were the most predominant families of G-II

Proteobacteria, while Comamonadaceae, Sphingomonadaceae, Pseudomonadaceae and

Nitrosomomonadaceae dominated the bacterial families of G-I Proteobacteria (Fig. 10 B&C).

-p

On the other hand, Planctomycetes, Chloroflexi and Actinobacteria were determined in minor

abundances of less than 15% in all digesters. The G-III digester sample contained not only more viable cells but also the activity of detected cells seemed to be higher than other digesters due to

re

the brighter fluorescence signals obtained (data not shown). The sample contained (per mL) a total cell count and a total amount of viable cells of 3.2 x 1010 and 1.1x 1010, respectively with a

lP

percental share of viable cells of 38%. Pseudomonadaceae, Flavobacteriaceae, Clostridiaceae and Thermoanaerobacteraceae showed the highest relative abundance of bacterial families of GIII digester (Fig. 10 A). Within the anaerobic sludge, Firmicutes, Chloroflexi and Bacteroidetes

ur na

were the most predominant bacterial phyla (83.6%). Thermotogaceae (12.3%) was the most abundant bacterial family in the sludge sample. The efficiency of bacterial members of Thermotogaceae on utilizing various organic compounds and producing H2 gas as a by-product has been previously reported (Cai et al., 2017). It has also been reported that the members of the two acid-forming bacterial phyla Bacteroidetes and Firmicutes showed potential on cellulose degradation in order to produce organic acids as a metabolic endpoint (Lee et al., 2017).

Jo

The major groups of Euryarchaeota were also analyzed. Euryarchaeota is the only group

of methanogenic Archaeal population that can use CH4 as a final metabolic product. The relative abundance of G-III Euryarchaeota was 38% over that of G-I and G-II, representing 27.8 and 32.7%, respectively. Gene probe analysis revealed that Methanosataceae (Methanosaeta) was the most abundant family of Archaeal methanogens. The dominant methanogenic family (Methanosataceae) represented 45.1% of the methanogenic population in the digester G-III. In

26

addition, G-III Methanosataceae was 63.5% more than that of the control digester G-I. Methanomicrobiales, Methanobacteriales and Methanosarcinaceae represented the rest of the major methanogenic Archaeal population in G-III (Fig. 11 B). On the other hand, the analysis of the methanogenic community showed that Methanosataceae (42.3%) was the most predominant methanogenic family within the sludge, while Methanosarcinaceae was the least family, representing 3.2%. Methanobacteriales and Methanomicrobiales together represented more than 54% of the dominant methanogenic families of the sludge (Fig. 11 B). Boone et al. (1993) reported that Methanosaeta use only acetate as a substrate, while H2/CO2 was the substrate of most Methanosarcinaceae. The predominant Methanosataceae hence supports the fact that acetoclastic methanogens favor a high level of the organic acid environment (Zhang et al., 2011).

ro of

The results of molecular characterization in this study were consistent with the obtained results of biogas and CH4 yields.

3.7.2 A pathway proposed for CH4 production from biodegraded lignocellulose and CPs hydrolysate

-p

Conversion of woody biomass into CH4, as the final product of AD process, is one of the major challenges due to the complexity of lignocellulosic biomass structure. It is even more so when the toxic wood preservatives CPs are present. Lignocellulose is naturally degraded via the

re

cooperation of various microorganisms in order to avoid the problems that are posed by using monocultures. Several studies have demonstrated the construction of microbial consortia, aiming

lP

to hydrolyze lignocellulose successfully in the pretreatment stage prior to AD process for efficient CH4 production. However, to our knowledge, such microbial consortia have not been used directly in the pretreatment of woody biomass (CSW) with simultaneous detoxification of CPs.

ur na

The constructed microbial consortia CS-5 and BC-4 were proved to be efficient in improving the degradation of CSW prior to and within the AD process cooperated efficiently and results revealed that the CH4 production yield was significantly increased over the control. AD can be integrated into a biorefinery as an effective biological pretreatment that facilitates the subsequent breakdown of lignocellulose into its simple sugars (e.g., glucose, xylose and galactose) and/or short fatty acids (acetic acid, propionic acid and butyric acid) via hydrolytic enzymes. Then

Jo

solubilized components can be used as a precursor in the production of CH4 (Fig. 11 A). The effect of biological pretreatment on AD process was associated with the increased methanogenic efficiency (Zhao et al., 2019). The sensitivity of AD process to potential toxicants is one of the considerable challenges that interfere with CH4 production. CPs are toxic compounds and their presence can negatively affect the microbial community in the AD process. Chen et al. (2014) reported that CPs were

27

toxic to the syntrophic methanogens. As a result, the CH4 production of wastewater treatment plants was significantly reduced. In this study, the results based on microbial community structure revealed that methanogenic Archaea were dominant during the peak of CH4 production, which could be an important explanation for achieving CH4 in the presence of non-degradable CPs in the digester during AD process. Based on the performance of the constructed microbial consortia on the degradation and detoxification of CPs mentioned above, it was proposed that the degraded polychlorophenols (during the pretreatment stage), as well as monochlorophenols, were converted into phenols via reductive dehalogenation. Under methanogenic conditions, the mineralization of phenols can be achieved through two routes (Levén et al., 2012) and the produced caproic acid or 4-hydroxy benzoate can then be converted into acetic acid (Fig. 11 A).

5 pretreated hydrolysate, inside the digester during AD process?

ro of

However, what about the non-degradable CPs and CSW residues, which remained after the CS-

It was obvious that Methanosataceae was dominant (45.1%) at the peak of methanogenesis. The population profile of Methanosataceae during AD process was obtained (Fig. 12). At the

-p

genera level, Methanosaeta, Methanosarcina, Methanofollis, Methanoplanus, Methanosphaera

and Methanoculleus were the identified methanogens. Of these Archaeal genera, Methanosaeta and Methanosarcina together were dominant, representing over 50% of the total genera.

re

Methanosaeta is one of the few Archaeal genera that can efficiently metabolize acetic acid to produce CH4, since it has an arsenal of enzymes and genes required for converting carbon to CH4,

lP

which characterize the CH4-producing Archaea (Zhao et al., 2019). As obtained in the pretreatment stage (Fig. 6), the concentration of acetic acid was significantly increased, which can explain the foundation of active Methanosaeta cells and their involvement in CH4 production

ur na

as well as their tolerance to non-degradable CPs and CSW. In addition, Methanosarcina is another strong tolerant genus of Archaeal methanogens, which can also use acetic acid for methanogenic metabolism as well as its capability of producing CH4 from lignocellulose (Vrieze et al., 2012). As a result, the tolerance of Methanosaeta and Methanosarcina probably provided an answer for the production of CH4 under the stress conditions of non-degradable CPs and CSW. Hence, Methanosaeta and Methanosarcina can become key functional Archaeal methanogens when non-

Jo

degraded lignocellulose (CSW) and/or CPs are present or when lignocellulose hydrolyzes slowly. Finally, acetic acid was metabolized by the acetoclastic methanogens, forming CH4 as the final product (Fig. 11). Four enzymes namely, tetrahydromethanopterin S-methyltransferase (Mtr), methyl-coenzyme M reductase (Mcr), acetyl-CoA synthetase (Acs) and formylmethanofuran dehydrogenase (Fmd) have been reported to be involved in the acetoclastic pathway in AD process (Ali et al., 2019a; Zhao et al., 2019). The expression of Mtr, which is responsible for the

28

transfer of CH3 groups to HS-CoM, influences CH4 production. Mcr is the unique enzyme for methanogenic archaea, which catalyzes the last step of CH4 production. In addition, the activation of Mcr is followed by that of Acs, which is responsible for the capture and activation of acetic acid by Methanosaeta (Li et al., 2015). The reduction of CO2 into formylmethanofuran is the key function of the Fmd enzyme (Cornelia and Uwe, 2014). Overall, the Archaeal community, especially the dominant Methanosataceae (Methanosaeta) methanogens, successfully improved CH4 yield during simultaneous biodegradation of CSW and CPs. 4. Conclusion The coexistence of CPs with woody biomass contributes in the release of hazardous materials both for the environment and public health. In this study, two novel microbial consortia

ro of

CS-5 and BC-4 were first constructed for the efficient decomposition of CSW and CPs simultaneously with enhancing CH4 production. Clearly, hemicellulose was evidenced as the

primary component of lost mass during the degradation of CSW by CS-5 or BC-4. The achieved

weight loss of the CSW induced by CS-5 and BC-4 reached 69.2 and 56.3%, respectively within

-p

15 days. The highest degradation rate of hemicellulose by both microbial consortia CS-5 and BC-

4 over other lignocellulosic components of CSW suggested that hemicellulases-producing microbial consortia played a major role in reaching and degrading hemicellulose. The findings

re

obtained from the evaluation of the performance of CS-5 and BC-4 on CSW degradation (VFAs, sCOD, pH, SEM and weight loss) were proof of high hydrolytic activity of these consortia

lP

including their hemicellulolytic activities. Clearly, our findings in the safety evaluation, as well as in comparison with previous investigations, suggested that the constructed microbial consortia CS-5 and BC-4 would potentially serve as a useful agent for detoxifying CPs, which can be safely

ur na

implemented in bioremediation processing and biomass-based biorefinery. The synergistic action of CS-5 and BC-4 increased CH4 production by 113.7% at the peak phase of AD process compared to the corresponding control. On the other hand, Euryachaeota, Bacteroidetes, Firmicutes, Proteobacteria, Chlotoflexi, Actinobacteria and Planctomycetes were the seven dominant phyla of the microbial community in all digesters but with different relative abundance percentages. Gene probe analysis revealed that Methanosataceae (Methanosaeta) was the most family

Jo

abundant

of

Archaeal

methanogens.

The

dominant

methanogenic

family

(Methanosataceae) represented 45.1% of the methanogenic population in the digester G-III. Overall, the newly constructed microbial consortia CS-5 and BC-4 could be promising for the degradation of lignocellulosic substrates such as woody biomass simultaneously with recalcitrant pollutants like CPs, with high performance in CH4 production.

CRediT authorship contribution statement

29

Sameh Ali: Conceptualization, Methodology, Writing-original draft, Formal analysis, Writing- review & editing. Michael Kornaros: Conceptualization, Writing- review & editing. Alessandro Manni: Methodology, Software. Jianzhong Sun: Funding acquisition, Writing- review. Abd El-Raheem R. El-Shanshoury: Investigation, Conceptualization. El-Refaie Kenawy: Visualization, Validation. Maha Khalil: Writing- review & editing. Declaration of competing interest

The authors declare that they have no known competing financial interests or personal

ro of

relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key R&D Program of China

(2018YFE0107100), National Natural Science Foundation of China (31772529), Egyptian

-p

Ministry of Higher Education & Scientific Research (MHESR), Support of Excellent Students Projects (SESP), and the Project funded by the Priority of Academic Program Development of

Appendix A. Supplementary data

re

Jiangsu Higher Education Institutions (PAPD 4013000011).

References

lP

E-supplementary data for this work can be found in e-version of this paper online.

Agency for Toxic Substances and Disease Registry (ATSDR), 1999. Toxicological Profile for Chlorophenols. Department of Health and Human Services, Public Health Service,

ur na

Atlanta.

Alexandropoulou, M., Antonopoulou, G., Fragkou, E., Ntaikou, I., Lyberatos, G., 2017. Fungal pretreatment of willow sawdust and its combination with alkaline treatment for enhancing biogas production. J. Environ. Manage. 203, 704-713. Ali, S.S., Abomohra, A., Sun, J., 2017. Effective bio-pretreatment of sawdust waste with a novel

Jo

microbial consortium for enhanced biomethanation. Bioresour. Technol. 238, 425-32.

Ali, S.S., Al-Tohamy, R., Manni A., Luz, F.C., Elsamahy, T., Sun, J., 2019a. Enhanced digestion of bio-pretreated sawdust using a novel bacterial consortium: Microbial community structure and methane-producing pathways. Fuel. 254, 115604.

Ali, S.S., Al-Tohamy, Sun, J., Wu, J., Huizi, L., 2019b. Screening and construction of a novel microbial consortium SSA-6 enriched from the gut symbionts of wood-feeding termite, Coptotermes formosanus and its biomass-based biorefineries. Fuel. 236,1128-1145.

30

Ali, S.S., Mustafa, A.M., Kornaros, M., Manni A., Sun, J., Khalil, M.A., 2020. Construction of novel microbial consortia CS-5 and BC-4 valued for the degradation of catalpa sawdust and chlorophenols simultaneously with enhancing methane production. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2019.122720 Ali, S.S., Nessem, A.A., Sun, J., Li, X., 2019c. The effects of water hyacinth pretreated digestate on Lupinus termis L. seedlings under salinity stress: A complementary study. J. Environ. Chem. Eng. 7, 103159. Ali, S.S., Sun, J., 2015. Physico-chemical pretreatment and fungal biotreatment for park wastes and cattle dung for biogas production. SpringerPlus. 4, 712. Ali, S.S., Sun, J., 2019. Effective thermal pretreatment of water hyacinth (Eichhornia crassipes)

ro of

for the enhancement of biomethanation: VIT® gene probe technology for microbial

community analysis with special reference to methanogenic Archaea. J. Environ. Chem. Eng. 7, 102853.

APHA, Standard Methods for the Examination of Water and Wastewater, 2012. 22nd ed.,

-p

American Public Health Association, Washington, DC.

Arora, P.K., Bae, H., 2014. Bacterial degradation of chlorophenols and their derivatives. Microb. Cell Fact. 13:31.

re

Atasoy, M., Eyice, Ö., Cetecioglu, Z., 2020. Volatile fatty acid production from semi-synthetic milk processing wastewater under alkali pH: The pearls and pitfalls of microbial culture.

lP

Bioresour. Technol. 297, 122415.

Boone, D.R., Whitman, W.B., Rouviere, P., 1993. Diversity and taxonomy of methanogens. In: Methanogenesis - Ecology, Physiology, Biochemistry and Genetics (Ferry, J.G., Ed.), pp.

ur na

35-80. Chapman and Hall, New York.

Cai, Y., Hua, B., Gao, L., Hu, Y., Yuan, X., Cui, Z., Zhu, W., Wang, X., 2017. Effects of adding trace elements on rice straw anaerobic mono-digestion: Focus on changes in microbial communities using high-throughput sequencing, Bioresour. Technol. 239, 454–463. Chen, J.L., Ortiz, R., Steele, T.W., Stuckey, D.C., 2014. Toxicants inhibiting anaerobic digestion: a review. Biotechnol. Adv. 32, 1523–34.

Jo

Chen, Y., Yang, Z., Ren, N., Ho, S., 2020. Optimizing the production of short and medium chain fatty acids (SCFAs and MCFAs) from waste activated sludge using different alkyl polyglucose surfactants, through bacterial metabolic analysis. J. Hazard Materi. 384, 121384. Cornelia, W., Uwe, D., 2014. Bioenergetics and anaerobic respiratory chains of aceticlastic methanogens. Biochim. Biophys. Acta. 1837 (7), 1130–1147.

31

Dong, M., Wang, S., Xu, F., Wang, J., Yang, N., Li, Q., Chen, J., Li, W., 2019. Pretreatment of sweet sorghum straw and its enzymatic digestion: insight into the structural changes and visualization of hydrolysis process. Biotechnol. Biofuels. 12, 276. Environmental Protection Agency (EPA), 1989. Protocols for short term toxicity screening of hazardous waste sites. A.8.7. Lettuce root elongation (Lactuca sativa). EPA 600/388/029. Fang, F., Wang, S-N., Li, K-Y., Dong, J-Y., Xu, R-Z., Zhang, L., Xie, W., Cao, J-S., 2020. Formation of microbial products by activated sludge in the presence of a metabolic uncoupler o-chlorophenol in long-term operated sequencing batch reactors. J. Hazard. Materi. 384, 121311.

ro of

Fava, F., Armenante, P.M., Kafkewitz, D., 1995. Aerobic degradation and dechlorination of 2chlorophenol, 3-chlorophenol and 4-chlorophenol by a Pseudomonas pickettii strain. Lett. Appl. Microbiol. 21(5), 307–312.

Field, J.A., Sierra-Alvarez, R., 2008. Microbial degradation of chlorinated phenols. Rev. Environ.

Fukumori,

F.,

Hausinger,

R.P.,

-p

Sci. Biotechnol. 7, 211–241. 1993.

Purification

and

characterization

of

2,4-

dichlorophenoxyacetate/alpha-ketoglutarate dioxygenase. J. Biol. Chem. 268(32),

re

24311–243117.

Gaitan, I.J., Medina, S.C., González, J.C., Rodríguez, A., Espejo, Á.J., Osma, J.F., Sarria, V.,

lP

Alméciga-Díaz, C.J., Sánchez, O.F., 2011. Evaluation of toxicity and degradation of a chlorophenol mixture by the laccase produced by Trametes pubescens. Bioresour. Technol. 102, 3632–3635.

ur na

Gallego, A., Gemini, V., Rossi, S., Fortunato, M., Planes, E., Gomez, C., Korol. S., 2009. Detoxification of 2,4,6-trichlorophenol by an indiogenous bacterial community. Int. Biodeterior. Biodegrad. 63, 1073-1078. García-Mancha, N., Monsalvo, V.M., Puyol, D., Rodriguez, J.J., Mohedano, A.F., 2017. Enhanced anaerobic degradability of highly polluted pesticides-bearing wastewater under thermophilic conditions. J. Hazard. Materi. 339, 320–329.

Jo

Ge, T., Han, J., Qi, Y., Gu, X., Ma, L., Zhang, C., Naeem, S., Huang, D., 2017. The toxic effects of chlorophenols and associated mechanisms in fish. Aquat. Toxicol. 184, 78–93.

Genethliou, G., Kornaros, M., Dailianis, S., 2020. Biodegradation of olive mill wastewater phenolic compounds in a thermophilic anaerobic upflow packed bed reactor and assessment of their toxicity in digested effluents. J. Environ. Manage. 255, 109882.

32

Giovanella, P., Vieiralgor, G.A.L., Pais, E., Otero, I.V.R., Pellizzer, E.P., Fontes, B.J., Sette, L.D., 2020. Metal and organic pollutants bioremediation by extremophile microorganisms. J. Hazard. Materi. 382, 121024. Goering, H.k., Van Soest, P.J., 1970. Forage Fiber Analysis (Apparatus, Reagents, Procedures and Some Applications). USDA Agriculture Handbooks No. 379. U.S. Department of Agriculture, Washington, DC. Hu, Y., Wang, J., Shen, Y., 2020. Enhanced performance of anaerobic digestion of cephalosporin C fermentation residues by gamma irradiation-induced pretreatment. J. Hazard. Materi. 384,121335. Hui, W., Jiajia, L., Yucai, L., Peng, G., Xiaofen, W., Kazuhiro, M., Zongjun, C., 2013.

ro of

Bioconversion of un-pretreated lignocellulosic materials by a microbial consortium XDC-2. Bioresour. Technol. 136, 481–487.

Hwang, M.H., Jang, N.J., Hyum, S.H., Kim, I.S., 2004. Anaerobic biohydrogen production from ethanol fermentation: the role of pH, J. Biotechnol. 111, 297-309.

-p

ISO 11348-3, 1998. Water quality. Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test).

(Cladocera, Crustaceae).

re

ISO 6341, 1996. Water quality. Determination of the inhibition of the mobility of D. magna Straus

Jankowska, E., Chwialkowska, J., Stodolny, M., Oleskowicz-Popiel, P., 2017. Volatile fatty acids

lP

production during mixed culture fermentation – The impact of substrate complexity and pH. Chem. Eng. J. 326, 901-910.

Jianping, W., Hongmei, L., Jing, B., Yan, J., 2006. Biodegradation of 4-chlorophenol by Candida

ur na

albicans PDY-07 under anaerobic conditions. Chinese J. Chem. Eng. 14(6), 790-795. Jin, S., Zhang, G., Zhang, P., Li, F., Wang, S., Fan, S., Zhou, S., 2016. Microwave assisted alkaline pretreatment to enhance enzymatic saccharification of catalpa sawdust. Bioresour. Technol. 221, 26–30. Kim, J-Y., Oh, S., Park, Y-K., 2020. Overview of biochar productionfrom preservative treated wood with detailed analysis of biochar characteristics, heavy metalbehavior, and their

Jo

ecotoxicity. J. Hazard. Materi. 384, 121356.

Kong, X., Du, J., Ye, X., Xi, Y., Jin, H., Zhang, M., Guo, D., 2018. Enhanced methane production from wheat straw with the assistance of lignocellulolytic microbial consortium TC-5. Bioresour Technol. 263, 33-9.

33

Lee, J., Kim, J.R., Jeong, S., Cho, J., Kim, J.Y., 2017. Long-term performance of anaerobic digestion for crop residues containing heavy metals and response of microbial communities, Waste Manag. 59, 498–507. Lee, W.S., Chua, A.S.M., Yeoh, H.K., Ngoh, G.C., 2014. A review of the production and applications of waste-derived volatile fatty acids. Chem. Eng. J. 235, 83-99. Levén, L., Nyberg, K., Schnürer, A., 2012. Conversion of phenols during anaerobic digestion of organic solid waste –A review of important microorganisms and impact of temperature. J. Environ Manage. 95, S99-S103. Li, L., Zheng, M., Ma, H., Gong, S., Ai, G., Liu, X., Li, J., Wang, K., Dong, X., 2015. Significant performance enhancement of a UASB reactor by using acyl homoserine lactones to

Biotechnol. 99 (15), 6471–6480.

ro of

facilitate the long filaments of Methanosaeta harundinacea 6Ac. Appl. Microbiol.

Li, W., Khalid, H., Zhu, Z., Zhang, R., Liu, G., Chen, C., Thorin, E., 2018. Methane production through anaerobic digestion: Participation and digestion characteristics of cellulose,

-p

hemicellulose and lignin. Appl. Energ. 226, 1219-28.

Li, X.J., Feng, D., Zhang, Y.T., Zou, D.X., Yuan, H.R., 2015. Anaerobic digestion performance and mechanism of ammoniation pretreatment of corn stover. Bioresources. 10, 5777-

re

5790.

Liado, S., Covino, S., Solanas, A.M., Petruccioli, M., D’annibale, A., Vinas, M., 2015.

lP

Pyrosequencing reveals the effect of mobilizing agents and lignocellulosic substrate amendment on microbial community composition in a real industrial PAH-polluted soil. J. Hazard. Materi. 383, 35-43.

ur na

Liang, J., Fang, X., Lin, Y., Wang, D., 2018. A new screened microbial consortium OEM2 for lignocellulosic biomass deconstruction and chlorophenols detoxification. J. Hazard. Materi. 347, 341-8.

Liang, J., Peng, X., Yin, D., Li, B., Wang, D., Lin, Y., 2015. Screening of a microbial consortium for highly simultaneous degradation of lignocellulose and chlorophenols. Bioresour. Technol. 190, 381-387.

Jo

Lin, Y., Liang, J., Zeng, C., Wang, D., Lin, H., 2017. Anaerobic digestion of pulp and paper mill sludge pretreated by microbial consortium OEM1 with simultaneous degradation of lignocellulose and chlorophenols. Renew. Energy. 108, 108-115.

Liu, C.L., 2008. The WANG, cultural characteristics and stability of the dual-functional bacteria community NSC-7. Microbiol. 35, 725-730.

34

Londry, K.L., Fedorak, P.M., 1992. Benzoic-acid intermediates in the anaerobic biodegradation of phenols. Can. J. Microbiol. 38, 1-11. Luo, J. , Zhang, Q., Zhao, J., Wu, Y., Wu, L., Li, H., Tang, M., Sun, Y., Guo, W., Feng, Q., Cao, J., Wang, D., 2020. Potential influences of exogenous pollutants occurred in waste activated sludge on anaerobic digestion: A review. J. Hazard. Materi. 383, 121176. Miller, G.L., 1959. Use of dinitrosalicyclic reagent for determination of reducing sugar. Anal. Chem. 31,426-8. Olaniran, A.O., Igbinosa, E.O., 2011. Chlorophenols and other related derivatives of environmental concern: properties, distribution and microbial degradation processes. Chemosphere. 83, 1297–1306.

anaerobic digestion. Water Sci. Technol. 41, 155-162.

ro of

Palmowski, L.M., Müller, J.A., 2000. Influence of the size reduction of organic waste on their

Poszytek, K., Ciezkowska, M., Sklodowska, A., Drewniak, L., 2016. Microbial Consortium with

High Cellulolytic Activity (MCHCA) for Enhanced Biogas Production. Front. Microbiol.

-p

7, 324.

Tao, Z., Wang, D., Yao, F., Huang, X., Wu, Y., Du, M., Xhen, Z., An, H., Li, X., Yang, Q., 2020. The effects of thiosulfinates on methane production from anaerobic co-digestion of

re

waste activated sludge and food waste and mitigate method. J. Hazard. Materi. 384, 121363.

lP

Tian, Y.L., Zhang, H.Y., Mi, X.Y., Wang, L.J., Zhang, L.Y., Ai. Y.J., 2016. Research on anaerobic digestion of corn stover enhanced by dilute acid pretreatment: mechanism study and potential utilization in practical application. J. Renew. Sustain. Energy. 8, 8179.

ur na

Tsigkou, K., Tsafrakidou, P., Kopsahelis, A., Zagklis, D., Zafiri, C., Kornaros, M., 2020. Used disposable nappies and expired food products valorisation through one- & two-stage anaerobic co-digestion. Renew. Energy, 147, 610-619. Vrieze, J.D., Hennebel, T., Boon, N., Verstraete, W., 2012. Methanosarcina: the rediscovered methanogen for heavy duty biomethanation. Bioresour. Technol. 112 (5), 1–9. Wahla, Q.A., Iqbal, S., Mueller, J., Anwar, S., Arsla, A., 2019. Immobilization of metribuzin

Jo

degrading bacterial consortium MB3R on biochar enhances bioremediation of potato vegetated soil and restores bacterial community structure. J. Hazard. Materi. 121493.https://doi.org/10.1016/j.jhazmat.2019.121493

Wang, T., Li, C., Wang, L., Zhou, M., Ning, J., Pan, X., Zhu, G., 2020. Anaerobic digestion of sludge filtrate assisted by symbionts of short chain fatty acid-oxidation syntrophs and

35

exoelectrogens: Process performance, methane yield and microbial community. J. Hazard. Materi. 384, 121222. Wen, B., Xufeng, Y., Li, Q.X., Liu, J., Ren, J., Wang, X., Cui, Z., 2015. Comparison and evaluation of concurrent saccharification and anaerobic digestion of Napier grass after pretreatment by three microbial consortia. Bioresour. Technol. 175, 102-111. Wongwilaiwalin, S., Rattanachomsri, U., Laothanachareon, T., Eurwilaichitr, L., Igarashi, Y., Champreda, V., 2010. Analysis of a thermophilic lignocellulose degrading microbial consortium and multi-species lignocellulolytic enzyme system. Enzyme Microb. Technol. 47, 283–290. Xufeng, Y., Li, P., Wang, H., Wang, X., Cheng, X., Cui, Z., 2011. Enhancing the anaerobic

ro of

digestion of corn stalks using composite microbial pretreatment. J. Microbiol. Biotechnol. 21(7), 746-752.

Yang, Y., Tsukahara, K., Sawayama, S., 2008. Biodegradation and methane production from glycerol-containing synthetic wastes with fixed-bed bioreactor under mesophilic and

-p

thermophilic anaerobic conditions. Process Biochem. 43, 362–367.

Yu, Y., Park, B., Hwang, S., 2004. Co-digestion of lignocellulosics with glucose using thermophic acidogens. Biochem. Eng. J. 18, 225–229.

re

Yuan, X., Cao, Y., Li, J., Wen, B., Zhu, W., Wang, X., Cui, Z., 2012. Effect of pretreatment by a microbial consortium on methane production of waste paper and cardboard. Bioresour.

lP

Technol. 118, 281-8.

Yuan, X., Ma, L., Wen, B., Zhou, D., Kuang, M., Yang, W., Cui, Z., 2016. Enhancing anaerobic digestion of cotton stalk by pretreatment with a microbial consortium (MC1). Bioresour.

ur na

Technol. 207, 293-301.

Yuan, X., Wen, B., Ma, X., Zhu, W., Wang, X., Chen, S., Cui, Z., 2014. Enhancing the anaerobic digestion of lignocellulose of municipal solid waste using a microbial pretreatment method. Bioresour. Technol. 154, 1–9. Zhang, Q., He, J., Tian, M., Mao, Z., Tang, L., Zhang, J., 2011. Enhancement of methane production from cassava residues by biological pretreatment using a constructed

Jo

microbial consortium. Bioresour. Technol. 102, 8899-8906.

Zhao, Y., Xu, C., Ai, S., Wang, H., Gao, Y., Yan, L., Mei, Z., Wang, W., 2019. Biological pretreatment enhances the activity of functional microorganisms and the ability of methanogenesis during anaerobic digestion. Bioresour. Technol. 290, 121660. Zheng, M., Li, X., Li, L., Yang, X., He, Y., 2009. Enhancing anaerobic digestion of corn stover through wet state NaOH pretreatment. Bioresour. Technol. 100, 5140-5145.

36

Zhu, N., Wang, L., Li, X., Deng, Y., Zhang, W.,

2020. Activation or sequestration of heavy

metals during hydrothermal process of swine manure: Interactions among metal species

Jo

ur na

lP

re

-p

ro of

and particulates. J. Hazard. Materi. 385, 121549.

37

of ro -p re al P ur n

Jo

Fig. 1. Experimental set up for screening and construction of novel aerobic and anaerobic microbial consortia valued for degrading woody biomass (CSW) and chlorophenols (CPs) simultaneously, and their influence with Bacterial population and Archaeal methanogens on the AD digester performance and methane production.

38

of ro -p re al P ur n

Jo

Fig. 2. Clustering analysis and characteristic profile of the stable strains obtained after the enrichment process. SR; code of each individual strain used in this cluster (SR-1 to SR-21), AZB; Azure B, CRS; p-cresol, MEB; methylene blue, CBA; 3-chlorobenzoic acid, VA; vanillin, BPL; biphenyl, XYL; xylanolytic, LGN; ligninolytic, CEL; cellulolytic, BZA; benzoic acid, VAA; vanillyl alcohol, VEA; veratryl alcohol and MMP; 4-methyl-2,6-dimethoxyphenol.

39

ro of -p re

Jo

ur na

lP

Fig. 3. The performance of CS-5 (A) and BC-4 (B) microbial consortia on the fractionation of CSW, in terms of cellulose, hemicellulose and lignin during 15 days of cultivation.

40

Jo

ur na

lP

re

-p

ro of

Fig. 4. Scanning electron microscopy (SEM) of CSW. Control (A&B), after 7 days of incubation with CS-5 (C) and BC-4 (D) and after 15 days of incubation with CS5 (E) and BC-4 (F) microbial consortia.

Fig. 5 Lignocellulolytic enzymes activities of biodegradation hydrolysates made by CS5 and BC-4, in terms of hemicellulases (A), cellulases (B) and ligninases (C).

41

ro of

Jo

ur na

lP

re

-p

Fig. 6. VFAs and pH contour plot of CS-5 (A,C&E) and BC-4 (B,D&F), illustrating variations in acetic acid (A&B), propionic acid (C&D) and butyric acid (E& F) concentrations with pH changes during the biodegradation process.

Fig. 7. sCOD and VFAs contour plot of CS-5 (A,C&E) and BC-4 (B,D&F), illustrating variations in acetic acid (A&B), propionic acid (C&D) and butyric acid (E& F) concentrations with the sCOD concentration change during biodegradation process. 42

ro of -p re lP ur na

Jo

Fig. 8. Digester performance. Biogas (A) and methane (B) yields during 40 days of AD process. G-I; hydrolysate of untreated CSW. G-II; hydrolysate of 7-days’ pretreatment of CSW by CS-5 consortium in PCS medium supplemented with CPs stock solution. The synergistic effect of the 7-days’ pretreated hydrolysate mixed with the anaerobic microbial consortium BC-4 was observed in the digester G-III.

43

of ro -p re al P ur n Jo

Fig. 9. Correlations between process parameters in terms of VFAs, sCOD and with the digester performance during the AD process.

44

ro of -p re lP

Jo

ur na

Fig. 10. Microbial community composition of digesters G-III (A) and G-II (B) compared to the control digester G-I (C) at phylum and family levels.

45

Jo

ur na

lP

re

-p

ro of

Fig. 11. A proposed mechanism of CH4 production from the hydrolysate of simultaneously CPs and woody biomass biodegradation (A) based on the digester Bacterial and Archaeal community structure, with special reference to the Archaeal methanogens (B).

46

of ro -p re al P ur n

Jo

Fig. 12. Gene probe analysis of Methanosataceae (Methanosaeta) of digester G-III on days 1 (A), 5 (B), 15 (C), 25 (D) and 40 (E) of AD process. All viable Bacterial and Archaeal population (I). Specific detection of Methanosataceae (Methanosaeta) using vermicon VIT® test of methanogenic Archaea (II). Total images (1000x).

47

of

Table 1

-p

XDC-2 XDC-2 XDC-2 WSD-5 OEM2 TC-5 LCDC CS-5 BC-4

Jo

ur n

Wheat straw Rice straw Corn stalk Napier grass Rice straw Wheat straw Sawdust Catalpa sawdust Catalpa sawdust

Cellulose 16.1 9.7 10.4 22.0 76.0 30.1 37.5 58.1 47.5

Weight loss (%) Hemicellulose 37.2 84.4 16.5 40.0 85.4 65.4 39.5 60.51 58.7

re

Microbial consortium

al P

Raw material

ro

Comparisons of the biodegradation of lignocellulose components with previous studies.

48

lignin 40.8 30.2 9.6 35.5 18.9 48.6 56.7 39.8 40.3

Degradation time (day)

Reference

6 6 6 21 12 12 10 15 15

Hui et al. (2013) Hui et al. (2013) Hui et al. (2013) Wen et al. (2015) Liang et al. (2018) Kong et al. (2018) Ali et al. (2017) This study This study

of ro

Table 2

Performance of the constructed microbial consortia CS-5 and BC-4 on chlorophenols detoxification.

> 90 (99.1) > 90 (99.0) > 90 (99.0) > 90 (99.1) > 90 (97.2) > 90 (98.0) 94.6 concentration

Jo

ur n

2-CP 84.7 3-CP 87.2 4-CP 90.3 2,4-DCP 83.6 2,4,6-TrCP 60.9* PCP 72.5* Mixture 77.8 *Toxic, EC50; effective pentachlorophenol.

Final > 90 (98.0) > 90 (99.2) > 90 (98.6) > 90 (96.1) > 90 (92.4) > 90 (91.0) 91.2

al P

Initial 86.3 90.1 83.2 69.1* 54.0* 47.8* 65.6

re

EC50 (% v/v) 2-CP 3-CP 4-CP 2,4-DCP 2,4,6-TrCP PCP Mixture

D. magna CS-5 consortium Initial Final 85.7 > 90 (98) 87.3 > 90 (99.0) 75.7 > 90 (99.1) 60.3* > 90 (96.2) 47.2* > 90 (95.8) 40.8* > 90 (93.0) 67.4 90.5 BC-4 consortium 85.1 > 90 (99.2) 85.4 > 90 (98.1) 87.4 > 90 (99.4) 84.3 > 90 (99.0) 61.4* > 90 (97.2) 70.8* > 90 (97.0) 79.1 95.3 CP; chlorophenol, DCP;

-p

V. fischeri

50,

49

Lactuca sativa Initial 88.2 94.8 84.7 74.3* 62.3* 55.3* 64.9 85.9 86.5 90.0 84.7 62.4* 72.2* 77.5 dichlorophenol,

Final > 90 (99.5) > 90 (99.2) > 90 (99.1) > 90 (96.0) > 90 (95.4) > 90 (94.5) 90.6 > 90 (99.2) > 90 (99.0) > 90 (99.0) > 90 (99.1) > 90 (97.0) > 90 (98.3) 94.7 TrCP; trichlorophenol,

PCP;

of ro

Table 3

Component

Before

-p

Changes in the main components of CSW at the end of 7-day pretreatment stage and 40-day AD. pretreatment 7-day pretreatment

re

(%)

G-I

G-II

G-III

45.8

49.2 ± 2.2

8.34 ± 0.5

57.5 ± 2.0

69.3 ± 1.7

Hemicellulos

18.11

54.3 ± 1.7

17.85 ± 1.4

62.4 ± 2.1

78.3 ± 2.6

2.43 ± 0.1

32.1 ± 1.3

45.3 ± 1.8

e Lignin

21.7

al P

Cellulose

30.8 ± 1.1

Jo

ur n

G-I; hydrolysate of untreated CSW, G-II; hydrolysate of 7-days’ pretreatment of CSW by CS-5 consortium in PCS medium supplemented with CPs stock solution and G-III; the 7-days’ pretreated hydrolysate mixed with the anaerobic microbial consortium BC4.

50