Biogas and phosphorus recovery from waste activated sludge with protocatechuic acid enhanced Fenton pretreatment, anaerobic digestion and microbial electrolysis cell

Journal Pre-proofs Biogas and Phosphorus Recovery from Waste Activated Sludge with Protocatechuic Acid Enhanced Fenton Pretreatment, Anaerobic Digestion and Microbial Electrolysis Cell Huijie Hou, Zhen Li, Bingchuan Liu, Sha Liang, Keke Xiao, Qian Zhu, Shaogang Hu, Jiakuan Yang, Jingping Hu PII: DOI: Reference:

S0048-9697(19)35266-0 https://doi.org/10.1016/j.scitotenv.2019.135274 STOTEN 135274

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Science of the Total Environment

Received Date: Revised Date: Accepted Date:

29 August 2019 16 October 2019 27 October 2019

Please cite this article as: H. Hou, Z. Li, B. Liu, S. Liang, K. Xiao, Q. Zhu, S. Hu, J. Yang, J. Hu, Biogas and Phosphorus Recovery from Waste Activated Sludge with Protocatechuic Acid Enhanced Fenton Pretreatment, Anaerobic Digestion and Microbial Electrolysis Cell, Science of the Total Environment (2019), doi: https://doi.org/ 10.1016/j.scitotenv.2019.135274

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Biogas and Phosphorus Recovery from Waste Activated Sludge with Protocatechuic Acid Enhanced Fenton Pretreatment, Anaerobic Digestion and Microbial Electrolysis Cell Huijie Hou a,b,Zhen Li a,b, Bingchuan Liu a,b, Sha Liang a,b, Keke Xiao a,b, Qian Zhu a,b,

Shaogang Hu a,b, Jiakuan Yang a,b,c, Jingping Hu a,b,c,* a

School of Environmental Science and Engineering, Huazhong University of

Science and Technology (HUST), Wuhan, 430074, P. R. China b

Hubei Provincial Engineering Laboratory of Solid Waste Treatment, Disposal and

Recycling, Wuhan, 430074, P. R China c

State Key Laboratory of Coal Combustion, Huazhong University of Science and

Technology (HUST), Wuhan, 430074, P. R. China *Corresponding

author:

Jingping

Hu,

+86-27-87793948.

1

E-mail:

[email protected],

Tel:

Abstract Biogas and phosphorus recovery from waste activated sludge (WAS) with sequential homogeneous protocatechuic acid (PCA) enhanced Fenton pretreatment, anaerobic digestion (AD) and microbial electrolysis cell (MEC) were investigated. The accumulation of biogas production of WAS-Fenton-AD was 330.4 mL/g VS, which were 2.05-fold of the control without pretreatment (WAS-AD) during anaerobic digestion. Biogas production of 178 mL/L/d from WAS-Fenton-AD-MEC was achieved, which was 5.23-fold of the WAS-MEC, 2.28-fold of WAS-Fenton-MEC and 1.46-fold of WAS-AD-MEC, respectively. Enhanced phosphorus recovery in form of struvite reached 1.72 g/g TS (18.03% of total P) with a purity of 74.4%. Microbial community richness and diversity analysis revealed that the pretreatment process under circumneutral condition improved the diversity of microbial community, which was consisted of Bacteroidetes (33.90%), Proteobacteria (33.14%), and Chloroflexi (10.14%), compared to a majority of Firmicutes (70.81%) in WAS-AD. This study provides a feasible strategy for the recovery of biogas combined with phosphorus from WAS. Keywords: Waste activated sludge, Anaerobic digestion, Microbial electrolysis cell, Struvite, Homogeneous Fenton process.

2

1. Introduction The ever increasing of population and industrial evolution inevitably generate a large amount of contaminated wastewater that requires proper treatment before final discharge to the surface water bodies (Lu et al., 2012). Accompanied with the wide application of the activated sludge process due to their cost-effective benefits and easy operation, massive amount of waste activated sludge (WAS) is produced and requires proper treatment (Yang et al., 2014). Meanwhile, WAS is a promising source of renewable organic matters and phosphorus. Anaerobic sludge digestion is one the most efficient and feasible sludge treatment process for methane recovery, particularly due to its capabilities of reducing the amount of final sludge solids for disposal and decreasing the health risk of pathogens (Appels et al., 2010). However, the complex structures of extracellular polymeric substances (EPS) in sludge, mainly characterized with high levels of proteins, polysaccharides and lipids, lead to long hydraulic retention time and limit its digestion efficiency. Furthermore, phosphate would remain unclaimed in the digested supernatant that may induce eutrophication in natural water if not properly managed. Pretreatment with Fenton processes has been intensively investigated for the rapid increase of sludge biodegradability (Pilli et al., 2015) to enhance the hydrolysis step in the anaerobic digestion process. The in situ generation of hydroxyl radicals (•OH) could efficiently disintegrate EPS structures and promote EPS solubilization (Oturan & Aaron, 2014). However, effective Fenton reactions proceed within a limited pH range of 3-4 (Wang et al., 2016), which is deviated from that of the anaerobic digestion process (pH 6.5-7.5) (Dhar et al., 2011). Furthermore, the accumulation of Fe3+ would form precipitates and decrease the pretreatment efficiency with prolonged reaction time. To 3

address this issue, a homogeneous Fenton-like process with a chelate agent that would form stable and soluble complexes with ferrous or ferric ions was proposed, enabling high reactivity at neutral pH conditions (Romero et al., 2016). Typical chelate agents are carboxylic acid linked to a number of tertiary nitrogen atoms (De Luca et al., 2014), such as

ethylenediaminetetraacetic

acid

(EDTA)

(Hu

et

al.,

2018),

ethylenediamine-N,N′-disuccinic acid (EDDS) (Soriano et al., 2018), nitrilotriacetic acid (NTA) (Zhang et al., 2017b), and citrate (Ruales L et al., 2016). The formed Fe(III) complexes could prevent iron precipitation and improve the degradation efficiency of organics (Wang et al., 2017). However, either the recalcitrant nature, poor biodegradability, or the adverse effects on Fe2+ regeneration by the commonly used chelates limit their practical applications (He et al., 2017). Protocatechuic acid (PCA) has recently been proposed as an environmentally-friendly iron chelate, and the Fe(III)/PCA/H2O2 system was able to degrade alachlor at near-neutral pH with PCA concentration as low as 0.1 mmol/L (Qin et al., 2015). However, its performance for sludge treatment to enhance anaerobic digestion has not been explored. Anaerobic digestion of sludge (Pepè Sciarria et al., 2019) would generate sludge fermentation liquid (SFL) rich with organic matters (Zhen et al., 2016) and phosphorus (Yang et al., 2014; Zhou et al., 2018). Phosphorus precipitates from AD generally in forms of calcite (CaCO3), hydroxyapatite (Ca5(PO4)3OH) (Cusick et al., 2014), strengite (FePO4•2H2O), vivianite (Fe3(PO4)2•8H2O) (Happe et al., 2016) or Al (oxy)hydroxides (Shepherd et al., 2017), which have limited plant uptake (Latif et al., 2018). Microbial electrolysis cell (MEC) is proposed as an effective strategy for simultaneous biogas production (Liu et al., 2005) and phosphorus recovery in forms of struvite, a slow-release fertilizer (Cusick & Logan, 2012; Happe et al., 2016). In addition, phosphorus recovery 4

via struvite crystallization could be enhanced at the cathode of the MEC (Cusick & Logan, 2012), where the localized alkaline condition near the cathode (Yuan & Kim, 2017) would be created due to the hydrogen evolution reaction (Cusick et al., 2014). Phosphate recovery in a scaled-up triple-chambered MEC reached 67% in 26 h (Happe et al., 2016). However, solid component of sludge interfered with the struvite crystallization process (Pepè Sciarria et al., 2019), and the current density (Ki et al., 2015) and chemical oxygen demand (COD) removal were also lower compared to those with SFL as substrate in MEC reactors (Ullery & Logan, 2015) . Therefore, the integration of anaerobic digestion with MECs (AD-MEC) would promote biogas production as well as phosphate recovery from SFL after AD process. In this study, we proposed an efficient strategy for simultaneous biogas production and struvite recovery from WAS via a three-step process: WAS pretreatment with Fe(III)/PCA/H2O2 at circumneutral pH to accelerate EPS disintegration, a subsequent AD process for biogas production and the release of phosphorus, and a final process for further biogas and phosphorus recovery from SFL within an MEC system. The concentrations of dissolved organic matters (soluble protein, soluble polysaccharide) and ortho-phosphorus in supernatant were studied to evaluate the effect of WAS pretreatment. The yields of biogas and ortho-phosphorus in the supernatant were evaluated for the AD process. Biogas production and the purity of struvite precipitation products were also studied. This study provides a feasible coupled strategy to maximize biogas production and phosphorus recovery from WAS as sustainable renewable energy.

2. Materials and Methods 2.1. Materials 5

Waste activated sludge was collected from the secondary sedimentation tank of Shahu municipal wastewater treatment plant (WWTP) in Wuhan, China, where the anaerobic/anoxic/oxic (A2O) activated sludge process was utilized. The collected WAS was stored at 4 C before use. The main characteristics of the collected WAS, including total solids (TS), volatile solids (VS), total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD) and total phosphorus (TP), are presented in Table 1.

2.2. Process design and operation procedure The effects and mechanisms of resource recovery from waste activated sludge with the proposed three-step process were explored as illustrated in the schematic diagram (Fig. S1). Step 1: WAS was pretreated with Fe (III)/PCA/H2O2 at circumneutral pH (WAS-Fenton), and WAS without pretreatment was used as control (WAS). Soluble EPS and phosphorus release were characterized for the pretreatment effect. Step 2: WAS-AD and WAS-Fenton-AD were systems with raw WAS and WAS pretreated with Fe(III)/PCA/H2O2 respectively as the influent for the anaerobic digestion process, where soluble EPS, VFAs, SCOD, biogas products, phosphorous release, dry solid, and microbial community were analyzed for both efficiency and mechanisms of resource recovery. Step 3: the effluents of anaerobic digestion (WAS-AD and WAS-Fenton-AD) were injected into MEC reactors for biogas production and phosphorus recovery (named as WAS-AD-MEC and WAS-Fenton-AD-MEC). Raw WAS and WAS with Fenton pretreatment without the AD process were also inoculated in MEC reactors as controls (WAS-MEC and WAS-Fenton-MEC). Both biogas production and phosphorus recovery were analyzed.

6

2.2.1 WAS pretreatment Waste activated sludge with a volume of 0.5 L was pretreated with Fe(III)/PCA/H2O2 without pH adjustment (pH = 6.87) as follows: ferric chloride (10 mM) of 4.75 mL and PCA (10 mM) of 7.125 mL were added into the waste activated sludge solution and stirred at 240 rpm for 30 seconds, then 30% H2O2 of 0.51 mL was added dropwise to initiate the Fenton reaction and operated for 2 hours. The mass of H2O2 was 0.2 g/g TS (Yu et al., 2017), the molar ratio of Fe3+ : PCA : H2O2 was optimized to be 1 : 1.5 : 200, based on maximized capillary suction time (CST) (data not shown). Sludge pretreatment were conducted in duplicates. Analyses were sampled every 30 min.

2.2.2 Anaerobic digestion A serum bottle of 0.55 L was used as the anaerobic digestion batch reactor with a working volume 0.5 L. Anaerobic digestion reactors were inoculated by anaerobic granular sludge with an inoculum ratio of 20% by volume and a solid concentration of 5 wt%. Both WAS and WAS-Fenton samples were subjected to the anaerobic digestion process, WAS-AD and WAS-Fenton-AD were systems with raw WAS and WAS pretreated with Fe(III)/PCA/H2O2 respectively. The AD experiments terminated the anaerobic digestion process at end of hydrolysis, which typically finishes in 1-3 days (Asztalos & Kim, 2015), and conducted at a constant temperature of 25  1 C for 3 days with continuous stirring at 200 rpm.

2.2.3 Microbial electrolysis cell A single-chamber microbial electrolysis cell with a volume of 550 mL was utilized after 7

anaerobic digestion. Carbon brush (CTN0310, Kangsidun technology Co., Ltd. Shenzhen China) was used as the anode electrode, and a piece of stainless steel mesh (50 mesh, Mingwan mesh, Guangzhou Co., Ltd., China) with a projective area of 78 cm2 (13 cm × 6 cm) was used as the cathode electrode. During the startup, MECs were inoculated with the effluent of microbial fuel cells and fed with 250 mL 1.0 g/L NaAc (CH3COONa) mixed with 250 mL 100 mM phosphate buffer solution (PBS) (Ren et al., 2014), 10 mL minerals solution and 10 mL vitamin solution as the electrolyte (Ahn & Logan, 2012). All MECs were purged with nitrogen (99.99%) for 15 minutes to eliminate the presence of oxygen inside the reactors. All reactors were operated with an applied voltage of 1.0 V (IT6302, ITECH electronics Co., Ltd., USA) with a 20 Ω resistor connected in series. Voltage across the resistor was recorded every 10 minutes by a data acquisition instrument (model 2750, Keithley Instrument, USA). The reactor was considered acclimated up with stable voltage output over three continuous cycles. The supernatant of the effluents of WAS, WAS-Fenton, WAS-AD, and WAS-Fenton-AD was collected with a high-speed centrifuge (G16, YingTai Instrument Co., Ltd., China) at 8000 rpm for 10 minutes, and 200 mL of each collected supernatant was mixed with 300 mL 100 mM PBS and inoculated in the MEC reactor. Each MEC reactor was stirred at 200 rpm and operated under a constant temperature of 25  1 C. At the end of each cycle, a mixed solution of 3 mol/L NH4Cl and 3 mol/L MgCl2 with a total volume of 30 mL was injected to each MEC to initate struvite precipitation. Struvite crystals were collected via a filter paper and introduced to the freeze drying (FD-1A-50, Boyikang Technology Co., Ltd., China) for 48 hours before characterization.

2.3 Analytical methods

8

2.3.1 Physiochemical analysis TS and VS were characterized in accordance with the Standard Methods for the Examination of Water and Wastewater (Walter & William. G., 2012). Supernatant samples were filtered via 0.45 μm polyethersulfone filter before performing the chemical analysis. pH of samples was also measured with a pH meter (FE28, Mettler Toledo Co., Ltd., China). SCOD and NH4+-N in the influent and effluent were determined by a spectrophotometer (DR3900, HACH Colorado, USA). The concentrations of soluble proteins and polysaccharides were measured using the Lowry–Folin (Lowry, 1951) and phenol–sulfuric method (Herbert et al., 1971). Moreover, TP and TFe (Total Fe element) in sludge samples were detected by adding 0.1 g solid sample into 10 mL HNO3 and H2O2 solution (HNO3 : H2O2 = 5 : 1, v/v) and the mixture was digested in a microwave digestion system (MDS-6G, Sineo Technology Co., Ltd., China) at 180 C for 60 min. Dissolved Fe and orthophosphate (OrthoP) in supernatant were filtered via 0.22 μm polyethersulfone filter before analyzed with an inductively coupled plasma optical emission spectrometer (ICP–OES) (Optima 8300, PerkinElmer, USA). The yield of volatile fatty acids (VFAs) was regularly characterized with gas chromatography (GC-2030, Shimazu, Japan) that equipped with a flame ionization detector (FID) and a DB-FFAP capillary column (30 m × 0.25 mm × 0.25 μm). The supernatant sample was acidified by 3% formic acid (sample : formic acid = 1 : 1, v/v) before measured and with an injection port temperature of 230 C, injection volume of 1 μL, and a split ratio of 1 : 50. The column temperature was maintained at 100 C for 5 min, and then increased to 210 C with an elevating speed of 15 C min−1 and remained for 3 min. The detector temperature was 230 C. Nitrogen was used as the carrier gas at a flow rate of 3 mL min−1. 9

2.3.2 Gas analysis Gas production during AD and MEC were measured using gas chromatography (GC-2030, Shimazu, Japan) that equipped with a thermal conductivity detector (TCD) and a capillary column carboxen 1010 (30 m × 0.53 mm × 30 μm). Argon was used as the carrier gas at a flow rate of 60 cm s−1. GC was operated with an injection port temperature of 200 C, a column temperature of 150 C, a detector temperature of 230C, an injection volume of 10 μL, and a split ratio of 1 : 1.

2.3.3 Struvite crystal analysis The morphology of the accumulated struvite was analyzed by scanning electron microscope-energy dispersive X-Ray spectroscopy (SEM-EDX) (Germini SEM300, Carl Zeiss, Germany). The crystal phases of struvite products were examined by X-ray diffraction (XRD 7000, Shimazu, Japan). Quantitative analysis of XRD spectra was by Jade (version 6.5) and results were retrieved from the Joint of Committee on Powder Diffraction Standards (JCPDS) database. Fourier transform infrared spectroscopy (FTIR, VERTEX 80, Bruker, Germany) was used to analyze the functional groups of the struvite products. Radicals produced during the Fe(III)/PCA/H2O2 Fenton-like reaction were measured by electron paramagnetic resonance spectroscopy (EPR, EMX nano, Bruker, Germany). The purity of struvite was determined following the previous study (Qian et al., 2016) and calculated using Eq. (1) (Ye et al., 2016): Eq. (1)

where nN (mol) is the mole number of NH4-N released from struvite after hydrochloric acid treatment, Mstruvite (g/mol) is the molar mass of struvite and mproduct (g) is the mass of 10

the product.

2.3.4 Microbial community analysis Microbial communities of WAS-AD and WAS-Fenton-AD after the completion of anaerobic digestion stage were analyzed using Illumina MiSeq sequencing. The purity and concentration of extracted DNA were measured by NanoDrop2000 (NanoDrop, USA). Bacteria barcode primer sets were 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’), and archaeal barcode primer set were

524F10extF

(5’-TGYCAGCCGCCGCGGTAA-3’)

and

arch958RmodR

(5’-YCCGGCGTTGAVTCCAATT-3’). Polymerase chain reaction (PCR) amplification was conducted with GeneAmp (9700, ABI, USA) and quantitatively analyzed by QuantiFluor-ST blue fluorescence quantitative system (Promega, USA). Miseq pyrosequencing analysis was conducted by TruSeqTM DNA Sample Prep Kit.

2.3.5 Statistics analysis The data of physiochemical analysis and gas analysis were calculated based on triplicate experiments and the standard deviations were also examined. The confidence of different groups of data was examined with a two-tailed t-test (5% significance threshold). Statistics analysis of microbial richness and diversity of anaerobic digestion process was performed using the free online platform of Majorbio I-Sanger Cloud Platform (www.majorbio.com). Raw fastq files were filtered by Trimmomatic and merged by FLASH. Operational taxonomic units (OTUs) with 97% cutoff were clustered using UPARSE (version 7.1), and chimeric sequences were identified and removed. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier (http://rdp.cme.msu.edu/) against the 16S rRNA database (Silva SSU128) using 11

confidence threshold of 0.7. Microbial Alpha diversity of Sobs and Shannon index analysis was conducted with Mothur software (version 1.30.1) under OTU with 97% similarity.

3. Results and discussion 3.1. EPS solubilization and phosphorus release during pretreatment To investigate the effect of WAS pretreatment on sludge solubilization and phosphorus release, soluble protein, soluble polysaccharide, soluble orthoP and dissolved iron concentrations in supernatant were characterized, as shown in Fig. 1. A dramatic increase in soluble protein from 8.23  0.85 mg/L to 50.54  2.03 mg/L and soluble polysaccharide from 5.82  0.41 mg/L to 20.02  0.45 mg/L were observed after pretreatment. WAS is composed of protein (35-61% of TCOD), carbohydrates (7-11% of TCOD) and other component (<21% of TCOD) (Wang et al., 2014). The produced •OH radicals during the pretreatment could enhanced sludge biodegradability in two ways. Hydroxyl radicals break the microbial cell membrane, leading to the oxidative decomposition of cell walls, and in the release of organic substances into the liquid phase (Pilli et al., 2015). Furthermore, hydroxyl radicals would partially oxidize the macromolecules of EPS compounds into more biodegradable forms (Şahinkaya et al., 2015). In addition, PCA successfully prevent the release of free Fe2+ and Fe3+ ions into the supernatant, therefore, avoids the precipitation of iron species as inactive oxyhydroxide iron under neutral pH and promotes the cyclic reactions of Eqs. (4) - (6) for continuous •OH generation. Soluble protein and soluble polysaccharide are the main carbon resources that could be utilized by microbial organisms during the AD process, the enhanced release of these compounds would facilitate the AD process for methane production. In addition, EPS solubilization facilitates the release of soluble orthoP to the 12

supernatant, from 30.64  0.87 to 42.03  0.35 mg/L (Fig. 1). XRD pattern of the dried raw sludge sample showed that inorganic phosphorus was mainly insoluble AlPO4 in the solid phase of raw WAS (Fig. S2, Table S1). The pretreatment process facilitates the breakage of long chain organic polymeric phosphorus (polyP) and pyrophosphate (pyroP) rich in the EPS structure to orthophosphate (Zhang et al., 2013), accompanied by the conversion of insoluble phosphorus to soluble phosphorus via •OH oxidation. Furthermore, a slight increase of dissolved iron in supernatant was observed (from 0.36  0.04 mg/L to 0.51  0.07 mg/L) within the pretreatment process. It is worth to note that the original does of iron was 5.32 mg/L, much higher than the measured soluble Fe (0.51 0.07 mg/L), indicating that the added Fe3+ ions were successfully chelated with PCA and formed stable Fe-PCA complexes. Furthermore, the low soluble iron prevented the formation of Fe-P precipitates (strengite and phosphates-vivianite) during the next step AD process, enhancing the transfer of P element into the liquid phase to facilitate phosphorus recovery.

3.2. Anaerobic digestion 3.2.1 Variations of SCOD and VFAs during the AD process The effluent characteristics of anaerobic digestion were also evaluated in terms of SCOD and volatile fatty acids (VFAs) in both WAS-Fenton-AD and WAS-AD. As shown in Fig. S3, SCOD concentrations increased in both reactors, in which the untreated reactor (WAS-AD) increased from 91  8 mg/L to 1580  210 mg/L, and the WAS-Fenton-AD increased from 140  7 mg/L to 720  16 mg/L. The increases of SCOD in both samples were mainly due to the decomposition of organic substrate through fermentative microorganisms, resulting in the release of soluble EPS into the effluent in forms of low 13

molecular weight organic matter. However, the effluent SCOD in WAS-Fenton-AD was lower than that in WAS-AD, suggesting the increased consumption rate of SCOD in WAS-Fenton-AD, which was confirmed with the enhanced methane production in WAS-Fenton-AD. VFAs are the major intermediate compounds that are formed from the decomposition of macromolecule organics in the hydrolysis and acidogenesis phases, and they are subsequently converted to biogas in the following acetogenesis and methanogenesis anaerobic phases. As shown in Fig. 2a, VFAs concentrations in both reactors (WAS-AD and WAS-Fenton-AD) were characterized, in which VFAs in WAS-Fenton-AD were far below the WAS-AD control, with only acetic acid and iso-butyrate acid observed at the 2nd day in WAS-Fenton-AD. This might be ascribed to the utilization of VFAs by acetoclastic methanogens for methane production, resulting in decreased concentration of VFAs. VFAs accumulation is favorable for methane production, however, the excessive level of VFAs may lead to the inhibition of the whole AD process (Yu et al., 2015). The reduced accumulation of VFAs in WAS-Fenton-AD indicated that the pretreatment of WAS with Fe(III)/PCA/H2O2 at circumneutral pH could promote the consumption of VFAs and enhance the progress of the AD process. Similar result was obtained in previous research that the addition of rusty scrap decreased the accumulation of VFAs and improved the rate of methane production for WAS fermentation (Zhang et al., 2014).

3.2.2 Biogas production and composition during the AD process Biogas, mainly composed of carbon dioxide and methane, are considered as a key indicator for successful sludge solubilization and system efficiency. Cumulative biogas 14

production and cumulative methane production, as shown in Fig. 2b, demonstrate the biogas production with the pretreated sludge (WAS-Fenton-AD) reached the maximum biogas at 140 mL/L/d in the 2nd day of AD, much higher than WAS-AD (20 mL/L/d at the 2nd day). Furthermore, the yield of methane exhibited a continuous growth and reached 123 mL/L/d in the 4th day. In addition, biogas production calculated based on VS also reveals the advantages of the pretreatment, where biogas production of 330.4 mL/g VS in WAS-Fenton-AD was higher than that of the control (WAS-AD, 160.7 mL/g VS). Moreover, the compositions of daily biogas are shown in Fig. 2c, in which the percentages of hydrogen and methane were significantly enhanced with the Fe (III)/PCA/H2O2 pretreatment process. Enhanced release of soluble EPS accelerates the hydrolysis of organics to produce hydrogen, where the amount of H2 reached the maximum amount at the 2nd day in WAS-Fenton-AD, one day earlier than that of WAS-AD. Furthermore, the generation of H2 was also enhanced in WAS-Fenton-AD (36.69% in 2nd day) than that of WAS-AD (24.29% of the 3rd day), consistent with higher methane production in the WAS-Fenton-AD system.

3.2.3 Phosphorous release during AD process Phosphorus in both WAS-AD and WAS-Fenton-AD reactors was also released to the supernatant during the anaerobic digestion process, as shown in Fig. 2d. This was ascribed to the effect of hydrolysis and acidogenesis steps in AD, and the release of orthoP into the supernatant (Li et al., 2017; Ohlinger et al., 1998). While the WAS-AD showed a rapid increase from 28.47  0.54 mg/L to 37.05  0.58 mg/L in two days and then decreased at day 4 (34  0.56 mg/L). The presence of Ca, Mg, Fe, and Al in the sludge would form inorganic phosphorus precipitates and reduce the P concentration in supernatant (Table S2). Quantitive XRD analysis of WAS-AD showed that 42.3 wt% 15

aluminum phosphate was present in the dry sludge, which was much higher than WAS (9.2 wt%), WAS-Fenton (6.8 wt%), and WAS-Fenton-AD (7.8 wt%) (Fig. S2, Table S1). PCA as a chelate successfully prevent the release of free Fe2+ and Fe3+ ions into the supernatant, with a final Fe of 2.83  0.05 mg/L (data not shown). Thus, the formation of Fe-P or Al-P compounds was prevented and the amount of P in the supernatant was sustained in the WAS-Fenton-AD system.

3.2.4 Microbial community richness and diversity analysis Alpha diversity estimators for bacterial and archaeal community richness and diversity in AD digesters were analyzed by Sobs richness and Shannon diversity index (Fig. 3a). The WAS-Fenton-AD digesters had higher richness and diversity of bacterial and archaeal than WAS-AD. The Sobs richness index of bacterial and archaeal were 385 and 45 at the OUT level in WAS-Fenton-AD digesters, respectively, higher than 160 and 23 in WAS-AD. Moreover, the rarefaction curve of Shannon diversity at WAS-Fenton-AD digesters demonstrated higher diversity of bacterial and archaeal than WAS-AD. The higher richness and diversity of microorganism are benefited from the more biodegradable effluent substrates rich in soluble protein and soluble polysaccharides after the Fe(III)/PCA/H2O2 pretreatment at circumneutral pH. The taxonomic distribution of bacterial at phylum level and archaeal at genus level involved in the anaerobic digestion process with correlation coefficients of p < 0.01 is shown in Fig. 3b. For the bacterial community at phylum level, Firmicutes (70.81%), Bacteroidetes (15.31%), and Proteobacteria (10.85%) were the three majorities bacterial phyla in the WAS-AD. However, the major bacteria were Bacteroidetes (33.90%), Proteobacteria (33.14%), and Chloroflexi (10.14%) in WAS-Fenton-AD. Firmicutes, Proteobacteria, Bacteroidetes and Chloroflexi were the dominate phyla in the bacterial 16

community under anaerobic conditions (Niu et al., 2015; Yang et al., 2014). Proteobacteria and Bacteroidetes in WAS-Fenton-AD instead Firmicutes in WAS-AD accelerated the utilization of polysaccharides (Yang et al., 2014) and protein (Riviere et al., 2009) and greatly shortened the processes of hydrolysis and acetogenesis. The pretreatment enhances the conversion of complex organics to soluble organic matter, resulting in the change of bacterial community diversity in WAS-Fenton-AD. Archaeal that utilizes organic intermediates to produce methane are the core functional microbial community in the AD process (Zhang et al., 2017a). Investigation of the archaeal at genus level (Fig. 3b) revealed that the most dominant microbial genus was Methanobrevibacter (89.05%), a type of hydrogenotrophic methanogen, in WAS-AD. However, it was worthy to note that the relative abundance of acetoclastic methanogens (Methanosaeta 37.25%, Methanosarcina 9.67 and Methanomethylovorans 9.37%) increased dramatically to 56.29% in WAS-Fenton-AD, eliminating the inhibition of methane production at high hydrogen pressure. Furthermore, the appearance of acetoclastic methanogens consume VFAs rapidly (Fig. 2b) and improve AD performance in WAS-Fenton-AD.

3.3 Biogas production and struvite precipitation in MEC 3.3.1 Current and biogas generation in MEC reactors WAS-AD-MEC and WAS-Fenton-AD-MEC demonstrated obvious higher currents than those of WAS-MEC and WAS-Fenton-MEC (Fig. 4a). This is consistent with the previous report that the pre-fermentation ahead of a MEC could enhance the conversion of particulate organics to simple organic acids favorable for the electrogenic biofilm (Ki et al., 2015). MEC with the supernatant of the effluent of the WAS-Fenton-AD-MEC 17

reactor showed the highest current up to 24 mA, even though a lower influent SCOD (385.3 ± 60 mg/L) was detected compared to that of WAS-AD-MEC (637.7 ± 90 mg/L). This is probably due to the substrate composition in the WAS-Fenton-AD-MEC system with pretreatment, which is more favorable for electrogenic biofilm. It was consistent with the highest SCOD removal in WAS-Fenton-AD-MEC (26.12%) (Fig. 4b). The increase in SCOD in the WAS-Fenton-MEC might be due to the release of small molecules into the solution through further MEC degradation of macromolecules, which was not measurable for influent samples due to the filtration process before SCOD measurement. The cumulative gas production and gas composition of different systems are shown in Fig. 4c. The highest gas production was achieved with Fenton pretreatment, anaerobic digestion (WAS-Fenton-AD-MEC), with a total volume of 178 mL/L/d after 6 days of operation, while the direct use of WAS supernatant as the influent of MEC (WAS-MEC) demonstrated the lowest gas production of 34 mL/L/d within the same time frame. This was consistent with its highest SCOD removal in WAS-Fenton-AD-MEC (26.12%). The composition of gas collected from the reactor at the 4th day revealed that it was mainly consisted of methane due to the hydrogenotrophic methanogenesis and acetoclastic methanogenesis processes (Cusick et al., 2014) that converted SCOD and hydrogen into methane. Microbial community analysis on the anode carbon bush (Fig. S4) showed that Methanobacterium (91.93%) was dominated in MECs. The richness of hydrogenotrophic methanogens facilitates the conversion of H2 to methane in MEC reactors.

3.3.2 Struvite crystal identification and purity analysis Irregular shaped struvite crystals were observed and orthorhombic structures mainly existed in WAS-MEC, WAS-Fenton-MEC, WAS-AD-MEC, while the rodlike structure 18

was observed mainly in WAS-Fenton-AD-MEC (Fig. 5a-5d). The yield of struvite formed at the end of the cycle was 0.0065 g/g TS (WAS-MEC), 0.058 g/g TS (WAS-Fenton-MEC),

1.09

g/g

TS

(WAS-AD-MEC)

and

1.72

g/g

TS

(WAS-Fenton-AD-MEC), respectively. EDX analysis (Fig. S5) demonstrated that the main elements in struvite precipitation products were oxygen, phosphorus, magnesium and nitrogen. The functional groups and crystal structures of all precipitation products were measured by FTIR (Fig. 5e). A set of phosphate group bands that have strong antisymmetric stretching vibration band appear at 1006 cm–1 (Wei et al., 2017), and a sharp antisymmetric bending vibrations band presented at 572 cm–1 could be assigned to P–O bond (Shiba & Ntuli, 2017). Furthermore, the absorption band at 467 cm–1 is attributable to the symmetric bending vibration of PO43–. Two strong absorption bands presented at 1640 cm–1 and 1435 cm–1 are associated with antisymmetric bending vibrations assigned to N–H of NH4+ (Frost et al., 2004), and the characteristic bands at 1435 cm–1 was ascribed to the ammonium group in struvite (Huang et al., 2017) . The FTIR spectra manifested that the main components in all precipitation products were struvite, which was further confirmed by XRD analysis (Fig. 5f). The purity of struvite precipitates was analyzed by dissolving these products in 0.5% hydrochloric acid solution (Qian et al., 2016) and measuring the ammonium concentration (Eq. (1) and Fig. S6). The results

manifested

that

the

highest

purity

of

struvite

is

74.4

wt%

in

WAS-Fenton-AD-MEC products compared to the control (WAS) at 44.64 wt%. The high purity of struvite in WAS-Fenton-AD-MEC sample was mainly induced by the increase of orthophosphate in the supernatant during the pretreatment and AD process. Phosphorus

of

90.24

wt%

in

SFL

was

successfully

recovered

in

the

WAS-Fenton-AD-MEC system, which was higher than WAS-MEC (1.26 wt%) and WAS-AD-MEC (44.10 wt%) (Fig. 4d). 19

3.4 Mechanisms of coupled process for biogas production and phosphorus recovery A three-step coupled process was investigated for enhanced biogas production and struvite recovery from waste activated sludge. The pretreatment of WAS at circumneutral pH with Fe(III)-PCA complexes enhances the disintegration of EPS structures and accelerates the speed of hydrolysis process during anaerobic digestion, resulting in enhanced biogas production and further release of phosphorus, the final MEC process achieves further biogas production with high purity of struvite recovery. The mechanisms of this research was proposed as a coupled three-steps process: first, Fe(III)-PCA complexes are formed by the bonding of two chelate ligands per metal ion (Rekik et al., 2017). The formed Fe(III)-PCA complexes react with H2O2 to generated Fe(II)-PCA complexes and •OH, as confirmed by EPR spectrum (Eqs.(3) - (6), Fig. S7). The Fe(III)/PCA/H2O2 Fenton pretreatment of WAS generates highly reactive hydroxyl radicals •OH that could disintegrate the large molecules of EPS structures into small molecule organics, represented by increased SCOD, which is easier to be degraded further during anaerobic digestion (Fig. 6). PCA+ Fe(III)  Fe(III)-PCA complexes

Eq. (3)

Fe(III)-PCA complexes + H2O Fe(II)-PCA complexes+HO2•+H+

Eq. (4)

Fe(II)-PCA complexes + H2O2 Fe(III)-PCA complexes+•OH+OH−

Eq. (5)

Fe(III)-PCA complexes + HO2• Fe(II)-PCA complexes+O2+H+

Eq. (6)

Secondly, during the AD process, the pretreated WAS flocs are further disintegrated and promotes the release of protein, polysaccharides and phosphorus into the liquid phase. The increase of SEPS greatly promotes the hydrolysis process that benefits the acidogenic process for biogas production and released phosphorus at anaerobic condition. 20

This is accompanied by the release of protein and VFA compounds into the supernatant. Thirdly, the organic compounds in liquid phase continuously support the growth of the electrogenic biofilm in MECs for simultaneous biogas production and phosphorus recovery as struvite.

4. Conclusions A coupled three-step process was investigated for enhanced biogas production and struvite recovery from waste activated sludge. The pretreatment process improved the bacterial community richness and diversity which enabled the high utilization efficiency of VFAs during AD. The accumulation of biogas was 330.4 mL/g VS, which was 2.05-fold of the control (WAS-AD) during anaerobic digestion. Biogas production of 178 mL/L/d from WAS-Fenton-AD-MEC was achieved, which was 5.23-fold of the WAS-MEC. Meanwhile struvite with a purity of 74.4% and yield of 1.72 g/g TS was obtained. This research provides a feasible strategy for much enhanced biogas and phosphorus

recovery

from

waste

21

activated

sludge.

Acknowledgements This research was supported by Natural Science Foundation of China (51878309, 51608217). The authors also thanks to the Analytical and Testing Center of Huazhong University of Science and Technology for providing the facilities to conduct the characterization work.

22

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25

Figures

Fig. 1. Soluble compounds (polysaccharide, protein and orthophosphate) and iron concentrations during the pretreatment process.

26

(a)

(b)

(c)

(d)

Fig. 2. Variations of (a) VFA production and composition during the anaerobic digestion process (ND: not detectable), (b) cumulative biogas production and cumulative methane production in WAS-AD and WAS-Fenton-AD systems, (c) biogas composition, and (d) soluble phosphorus after anaerobic digestion with (WAS-Fenton-AD) and without (WAS-AD) pretreatment.

27

(a)

(b)

Fig. 3. Microbial community richness and community diversity analysis (p  0.01): (a) Sobs richness and Shannon diversity index and (b) bacterial at class level and archaeal at Genus level.

28

(a)

(b)

(c)

(d)

Fig. 4. Performances of different MEC systems: (a) current outputs, (b) influent and effluent concentrations of SCOD and removal efficiencies, (c) cumulate gas productions and compositions (at 4th day), and (d) influent and effluent concentrations of orthoP and removal efficiencies.

29

(a)

(b)

(c)

(d)

(e)

(f)

Fig. 5. SEM images of struvite precipitation products: (a) WAS-MEC, (b) WAS-Fenton-MEC, (c) WAS-AD-MEC, (d) WAS-Fenton-AD-MEC (scale bar was 100 μm and enlarge plot scale bar was 10 μm ), (e) FTIR and (f) XRD analysis of the struvite products from MEC reactors with different inoculum.

30

H2 O

CH4

HO2 •

CH4

Struvite

Acetate, H2 , CO2 H2 , CO2

Fe(III)-PCA Fe(II)-PCA

glucose

•OH

H2 O2

Amino acid SCOD

Polysaccharide Protein

Mg2+, NH4 +, PO4 3−

0

Biogas production

EPS disintegration Waste Activated Sludge

Pretreatment

SCOD released

Cell lysis P release

Anaerobic P release Anaerobic digestion

Biogas production

SCOD removal

Struvite precipitation Microbial electrolysis cell

Fig. 6. Mechanism of the proposed three-step process for simultaneous biogas production and phosphorus recovery from waste activated sludge.

31

Tables

Table 1 Characteristics of the collected WAS (n = 3). Sol. TCOD Parameters

TS (g/L)

VS (g/L)

SCOD

Sol. protein

pH

TP

Total Fe

(mg/L)

(mg/L)

polysaccharide (mg/L)

(mg/L)

(mg/L) (mg/L)

Values

5.790.04

2.810.03

6.920.01

4253297

262

32

6.06  0.84

7.370.43

23327 9.750.13

Conflict of Interest Statement No conflict of interest exits in the submission of this manuscript, and the manuscript is approved by all authors for publication. I would like to declare on behalf of my coauthors that the work described was an original research that has not been published previously, and not under consideration for publication elsewhere, in whole or in part. All authors listed have approved the manuscript that is enclosed.

33

Graphical Abstract H2 O

CH4

HO2 •

CH4

Struvite

Acetate, H2 , CO2 H2 , CO2

Fe(III)-PCA Fe(II)-PCA

glucose

•OH

H2 O2

Amino acid SCOD

Polysaccharide Protein

Mg2+, NH4 +, PO4 3−

0

Biogas production

EPS disintegration Waste Activated Sludge

Pretreatment

SCOD released

Cell lysis P release

Anaerobic P release Anaerobic digestion

34

Biogas production

SCOD removal

Struvite precipitation Microbial electrolysis cell

Highlights 

A three-step process for biogas production and struvite recovery from WAS.



WAS pretreatment with Fe(III)/PCA/H2O2 at neutral pH enabled EPS disintegration.



AD process achieved efficient biogas production and release of orthophosphate.



MEC process achieve further biogas production with recovery of high purity of struvite.

35