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Yunxin Zhu, YuJia Chen, Guangqi An, Cheng Zhang, Jingwei Yang, Rongyong Yang, [Guoping Chen](https://orcid.org/0000-0001-6753-3678), Yingnan Yang

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[Significance of homogeneous operation in light-assisted fixed-bed bioprocess under ammonia stress: Optimization, long-term operation and metagenomic analysis](https://mdr.nims.go.jp/datasets/2c556920-9397-47ae-916e-763ddfe7e4ff)

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Significance of homogeneous operation in light-assisted fixed-bed bioprocess under ammonia stress: Optimization, long-term operation and metagenomic analysis Yunxin Zhua, Yujia Chena, Guangqi Ana, Cheng Zhanga, Jingwei Yanga, Rongyong Yangb, Guoping Chenc, Yingnan Yanga* aGraduate School of Life and Environmental Science, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan bShanghai High Victory Science and Technology Co., Ltd., 4688 Jinshan Avenue, Shanghai 201512, China cResearch Center of Functional Materials, National Institute for Materials Science,1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan * Corresponding author:Tel/Fax: +81 29 8534650 E-mail address: yo.innan.fu@u.tsukuba.ac.jp (Y.Yang)Abstract 1 Activating microbes with light is a promising strategy for addressing ammonia-2 stressed anaerobic digestion (AD). However, as a critical in-process parameter, 3 homogenous operation, in light-assisted AD amended by bio-fixed bed has received 4 limited attention. This research endeavors to establish a uniform-illuminated biosystem 5 and assess its practical feasibility through a 90-day semi-continuous operation at pilot 6 scale under solar light illumination. The proposed system with an optimal stirring mode 7 (intermittent stirring for 3 minutes every 15 minutes) demonstrated a robust methane 8 yield of 914−971 mL/(g-DOCremoval∙d) across various organic loads, accounting for 9 88.7−94.3% of the theoretical one under ammonium-rich condition (3500 mg/L). The 10 metagenomic analysis revealed that the uniform illumination induced synergistic effects 11 of a diverse microbial consortium, the promotion of carbohydrate and methane 12 metabolism, and the formation of an electroactive bio-cluster for enhanced digestion. 13 With the implementation of solar light, this study offers an easy-operated approach for 14 sustainable waste-to-energy recovery in scale-up application. 15 Keywords: Light-assisted anaerobic digestion (AD), Ammonia inhibition, Homogenous 16 operation, Pilot-scale evaluation, Bioinformatics analysis 17 https://www2.cloud.editorialmanager.com/bite/viewRCResults.aspx?pdf=1&docID=184032&rev=0&fileID=3358081&msid=043d5ff2-fdd7-4c2d-997b-441158d3df2ehttps://www2.cloud.editorialmanager.com/bite/viewRCResults.aspx?pdf=1&docID=184032&rev=0&fileID=3358081&msid=043d5ff2-fdd7-4c2d-997b-441158d3df2e1. Introduction18 Anaerobic digestion (AD) stands out as a widely applied approach for waste 19 control and bioenergy production (i.e., H2, CH4), aligning with the goals of sustainable 20 development (Bose et al., 2021). While the ammonia inhibition that commonly occurs 21 in AD treating nitrogen-rich waste, impairs the treatment efficiency with the restrained 22 microbial productivity (Rajagopal et al., 2013). To address this challenge, the 23 combination of bio-zeolite fixed-bed system with light stimulation has been proposed to 24 enhance CH4 production (Zhang et al., 2016; Zheng et al., 2020; Zhu et al., 2021). The 25 bio-zeolite fixed-bed demonstrated notable capabilities in ammonia absorption and 26 microbial fixation, and the employment of light stimulation could effectively promote 27 the microbial activity and biomass growth, facilitating the biofilm formation to 28 counteract the ammonia toxicity. Moreover, natural sunlight could be adopted to further 29 highlight the easy-operability and sustainability of this system, making it highly 30 promising for practical applications. 31 Considering the scale-up impact of an illuminated biosystem, the light availability 32 need to be carefully controlled, as it generally influences the microbial metabolisms and 33 biomass growth (Rossi et al., 2020). However, the limitations of sludge sedimentation 34 and turbid mud commonly affect the light transmittance (Kariyama et al., 2018), 35 potentially hindering the transfer of light stimulus and photo-enhancement efficiency in 36 scaled-up reactors. Notably, in light-assisted system, Methanosarcina with high light 37 affinity (Qian et al., 2022; Tada et al., 2006; Zhu et al., 2022), typically resides at the 38 bottom of static reactors (Kariyama et al., 2018), which makes them less accessible to 39 light and thereby weakening the photo-stimulation efficiency. Therefore, achieving a 40 uniform light regime is essential for effective microbial stimulation and stable 41 performance of the developed system. As a commonly employed homogenization 42 technique, stirring, plays an influential role in influencing the mass transfer, the contact 43 between microbes and substrates, the stability of microbiome, the formation of biofilm, 44 the hydrolysis/fermentation rate, and so on  (Karim et al., 2005; Lindmark et al., 2014b). 45 It may also assist to avoid the sludge sedimentation and homogenize the distribution of 46 microbial community in lighted system, achieving a uniform environment for light 47 stimulation. Though traditionally, stirred tank digesters operated as continuous stirred 48 tank reactors (CSTRs), the high operational and maintenance cost of continuous stirring 49 (~54% of energy demand for system operation) restrained the energy production 50 efficiency (Lindmark et al., 2014a). Moreover, continuous stirring shear might lead to 51 the destruction of microbial flocs, the detachment of biomass, resulting in the long-term 52 upsets in large-scale application (Karim et al., 2005). Therefore, an appropriate 53 homogenization operation should be proposed for not only maximizing the energy 54 production but also minimizing the capital and operational costs. 55 Compared to no stirring and continuous stirring (CS), the economics of AD could 56 be improved by intermittent stirring (IS). Ideal IS could realize the homogenous 57 introduction of the fresh feed and intimate contact between the bacteria, meanwhile 58 reduce the shear impact and energy demand caused by CS  (Kariyama et al., 2018). 59 Suitable IS duration may also provide a uniform platform for the syntrophic association 60 among bacteria and methanogens with favorable extracellular polymeric substances 61 (EPS) production for resisting ammonia stress (Bose et al., 2021; Ong et al., 2002). 62 Moreover, IS may impact the community structure and formed microbial layer. 63 Methanogens with long-rod structure (i.e., acetoclastic Methanosaeta) are generally 64 sensitive to continuously stirring, while a good balance of Methanosaeta and 65 Methanosarcina could be achieved in the intermittent mixed digesters (Kariyama et al., 66 2018). Therefore, optimizing IS strategy is prerequisite for achieving a uniformly 67 illuminated consortia with efficient AD performance under ammonia stress. Until now, 68 existing studies mainly focused on the stirring effect in dark AD, leading to a lack of 69 experience regarding the homogenization of both the nutrients and light distribution in 70 illuminated bioreactor, especially under ammonia inhibition. Moreover, there have been 71 no report on the practical applicability of lighted AD system in terms of scale-up 72 implementation, long-term stability and solar assistance. Besides, the combined effect 73 of homogeneous operation and light stimulation on microbial communities and 74 metabolic pathways remains unclear. 75 Therefore, this research aimed to develop a homogenous-illuminated biosystem for 76 efficient ammonia-rich AD by optimizing stirring operation. The practical applicability 77 of proposed system was evaluated through a 3-month pilot-scale operation with 78 simulated solar light as light source. Finally, bioinformatics analysis delved into the 79 mechanisms underlying microbial communities, metabolic pathways, and biofilm 80 characteristics. This study provides a guide for establishing a sustainable waste 81 management system with efficient bioenergy recovery for scale-up application. 82 2. Materials and methods83 2.1. Seed sludge, feedstock and fixed-bed biosystem 84 The original seed sludge was regularly sourced from an anaerobic digester at a full-85 scale mesophilic wastewater treatment plant handling municipal wastewater (Ibaraki 86 prefecture, Japan). Subsequently, it was separated, sealed and refrigerated (4℃) before 87 pre-culturing. Following a two-week acclimatization at 55℃ with a synthetic medium 88 (glucose (2.5 g/L), sodium acetate (2.5 g/L), KH2PO4 (16 mg/L), yeast extract (200 89 mg/L), and a trace mineral solution (5% v/v), the cultured inoculum was prepared for 90 batch and semi-continuous assays. The chemical composition of synthetic medium and 91 the pre-culturing method were documented in previous study (Zhu et al., 2022). 92 Fermentation assays were conducted in a bio-zeolite fixed-bed reactor (Zhang et al., 93 2016), utilizing a 330 cm2/L porous nylon bag (H-[HN(CH2)XCO]–OH, T&T, Kainan, 94 Japan) as fixed material filled with 10 g/L zeolite A-3 (Wako Pure Chemical Industries, 95 Ltd.) for microbial immobilization and ammonia absorption (See Supplementary 96 Materials). 97 2.2. Exploration of optimal stirring condition for light-assisted anaerobic 98 bioreactor under ammonia stress  99 AD batch experiments were conducted in bio-zeolite fixed-bed bioreactors 100 (effective working volume of 200 mL, SIBATA) with synthetic medium and 101 acclimatized inoculum at a ratio of 4:1 (v/v) under thermophilic condition (55 ± 1℃). 102 NH4Cl was adopted in each reactor to simulate an ammonia-stressed environment (3000 103 mg NH4+-N/L). The initial pH of 7.0 ± 0.2 was adjusted using 1 M NaOH and 1 M HCl 104 before imparting the anaerobic condition. Daily incandescent lamp illumination 105 (400−800 nm, LW110V60W, Mitsubishi Ashram, Tokyo, Japan) was applied to lighted 106 reactors (marked as ‘L’) with the optimal light condition of photon number (NR) 107 =1.25×104 μmol/(day∙L) with 34 μmol/(m2∙s) for 90 min/day (Zhu et al., 2021). 108 To investigate the effect of homogenization on light-assisted system, a magnetic 109 stirrers (SW-M60, NISSIN) with a stirring speed of 100 rpm was applied based on 110 previous studies (Li et al., 2022; Liu et al., 2024). Preliminary experiments determined 111 suitable stirring modes for lighted reactors during 90-minute illumination (See 112 Supplementary Materials), including no-stirring (L), constant stirring for 90 mins (L-CS) 113 and intermittent stirring with 1 min stirring in every 15 min (L-IS1). Dark reactors with 114 the same stirring modes served as controls (D, D-CS, D-IS1, respectively). Enhanced 115 methanogenic productivity in the L-IS1 group indicated the advantage of intermittent 116 stirring over constant stirring and no stirring in the developed lighted reactor (See 117 Supplementary Materials). To optimize the IS condition, reactors were operated with 118 different stirring durations of 1, 3, 5, 7 min in every 15 min during 90-min illumination, 119 labeled as L-IS1, L-IS3, L-IS5, L-IS7, respectively. Each group was performed in 120 triplicate for 9 days. 121 2.3 Long-term effectiveness of homogenous-illuminated system at pilot scale 122 To assess the practical efficacy of the optimal stirring condition, a polit-scale semi-123 continuous AD digester (MDL-10L, B.E.Marubishi) was established (See 124 Supplementary Materials). The digester equipped with a double-layered six-blade 125 propeller device for stirring, and an automatic controlling system for real-time 126 monitoring (i.e., daily biogas volume, temperature (55 ± 1℃), pH and stirring operation). 127 Daily exchange of feedstock and fermentation broth was implemented by peristaltic 128 pump. Initially, a specified quantity of fixed materials and zeolite was suspended in the 129 digester (according to 8 L working volume). An ultraviolet-cut artificial solar light 130 (370−2000 nm, XC-100, 110 V1.8A, SERIC ltd., Tokyo, Japan) was applied as light 131 source to simulate solar illumination. 132 The 90-day semi-continuous operations were divided into three stages: Dark (day 133 0-20, dark without stirring), Light (day 21-50, light without stirring) and Light-S opt (day134 51-90, light with optimum stirring). Operations started with batch feeding for microbial135 acclimatization (10 days), in which the ammonia-rich feedstock (3500 ± 100 mg NH4+-136 N/L) and inoculum were initially fed to reactors on day 0. From day 11, the digester was 137 fed in semi-continuous mode with organic load rate (OLR) of 0.19 g-DOCadded/(L∙d) and138 hydraulic retention times (HRT) of 10 days. Specifically, 800 mL of ammonia-rich 139 synthetic medium was exchanged with the same volume of effluent. From day 21, 140 intermittent illumination was introduced to reactor using artificial solar light. From day 141 51, the proposed optimal stirring condition was applied to the illuminated digester 142 during daily illumination. After a 20-day stabilization period, the OLR was increased to 143 0.34 and 0.71 g-DOCadded/(L∙d) (during day 71-80 and day 81-90, respectively) with144 shorter HRT (5 days), to evaluate the capacity of proposed homogeneous-illuminated 145 system. 146 2.4 Analytical methods 147 Biogas was collected daily, and every other day, 10 mL of the sampled digestate 148 underwent centrifugation for further analysis. The biogas composition, total solid 149 content (TS), volatile solids content (VS), dissolved organic carbon (DOC), volatile 150 fatty acids (VFAs), ammonium nitrogen, pH during fermentation was measured 151 following a previous protocol (Zhu et al., 2021). The precipitate of the sludge sample 152 was utilized for assessing the following indicators. Adenosine triphosphate (ATP), 153 coenzyme F420 and sludge conductance were measured according to previous study 154 (Zheng et al., 2020). EPS was extracted from the sludge mixture using physical heat 155 method (He et al., 2021). Briefly, the sludge samples were centrifuged (4000 g for 10 156 min, 4ºC) and the obtained supernatants were filtered through 0.45 μm membrane filter, 157 serving as dissolved organic matters (DOM). The residue in the centrifuge tube was 158 then suspended to the original volume with distilled water, sheared by a vortex mixer 159 for 1 min, and loosely bound EPSs (LB-EPSs) were extracted using the same 160 centrifugation process. Finally, the residue was used to extract tightly bound EPSs (TB-161 EPSs) through heating (60℃, 30 min) in a water bath. The collected supernatants after 162 each centrifugation were filtered through a 0.45 μm membrane for total organic carbon 163 (TOC) analysis. To evaluate the long-term performance under different operation 164 conditions, the theoretical bio-CH4 yield (BMYthDOC) was calculated based on the 165 experimental DOC of the substrate (mL/g-DOCadded), according to previous study (Zhu 166 et al., 2021). 167 2.5 Microbial community analysis 168 The samples for DNA analysis were collected at the end of the characteristic 169 fermentation period of Dark, Light, and Light-S opt, and quick frozen at -80℃. Illumina 170 MiSeq paired-end sequencing (Illumina, USA) of 16S rRNA V4 regions (515F-806R) 171 was performed at Bioengineering Lab. Co., Ltd. (Kanagawa, Japan). Quality filtered 172 reads were assigned to operational taxonomic units (OTUs) using taxonomic 173 assignment against the EzBioCloud 16S database (https:// www. ezbiocloud. net/) (Yoon 174 et al., 2017). Indices of α-diversity for richness comparison (observed species and 175 Chao1 index) and diversity comparison (Shannon index and phylogenetic diversity (PD) 176 whole tree) were normalized using sequences (no less than 10000 sequences) obtained 177 among different samples. Functional prediction analysis was performed via PICRUSt2 178 (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), 179 to evaluate the expression of key metabolic pathways based on 16S rRNA data. The 180 KEGG (Kyoto Encyclopedia of Genes and Genomes) database was used to support 181 functional gene profiling (https://www.genome.jp/kegg/mapper/). 182 https://www.genome.jp/kegg/mapper/2.6 Kinetic and statistical analyses 183 The CH4 production data from the batch experiment were fitted to the modified 184 Gompertz model (Liu et al., 2022) using IBM SPSS statistics (Stats Guild Inc., Japan). 185 One way Analysis of Variance (ANOVA) followed by t-test with a threshold value of 186 0.05 on experimental data was performed to evaluate the statistical significance of the 187 results. Principal component analysis (PCA) and cluster analysis were conducted using 188 Origin 2021 software. 189 3. Result and discussion190 3.1 Optimal stirring condition for lighted system under ammonia stress 191 The performance of ammonia-rich AD under various intermittent stirring 192 conditions was illustrated in Fig. 1a. The cumulative CH4 productions of L (408 ± 15 193 mL/L), L-IS1 (447 ± 18 mL/L), L-IS3 (483 ± 19 mL/L), L-IS5 (383 ± 19 mL/L) were 194 1.44, 1.58, 1.71 and 1.35 folds of dark control (283 ± 21 mL/L), respectively. While L-195 IS7 exhibited a similar performance (295 ± 15 mL/L) to the dark group. This result 196 suggested that light stimulation coupled with proper homogeneous operation, might 197 synergistically enhance the methanogenic productivity under ammonia stress. Based on 198 experimental data, three major kinetic parameters, i.e., maximum cumulative CH4 199 production (Mmax), maximum CH4 production rate (Rmax), and lag-phase time (λ, d) 200 were further predicted from the Gompertz model with good fit (R2 = 0.983−0.995) (Fig. 201 1b). Besides, cumulative CH4 production in L-IS3 reached a plateau 1 day earlier than 202 the light control (Fig. 1a), indicating that intermittent string could shorten the start-up 203 period and digestion time under ammonia stress. As the stirring period increased to 3 204 min, Mmax (490 ± 8 mL/L) peaked in L-IS3 with the shortest λ (0.8 d) and fast Rmax (198 205 mL/(L·d)). But when the stirring time further ascending to 7 min, Mmax and Rmax sharply 206 decreased to 302 mL/L and 111 mL/(L·d), which were even lower than that of light 207 control. Consequently, 3-min intermittent stirring contributed to the highest CH4 yield 208 of 372 ± 21 mL/(g-DOCremoval∙d) in the homogeneous-illuminated system (L-IS3), 209 which was 1.57 folds and 1.25 folds of dark (191 ± 14 mL/(g-DOCremoval∙d)) and light 210 control (299 ± 15 mL/(g-DOCremoval∙d)), respectively (Fig. 1c). Whereas further 211 prolonging the stirring duration to 7 mins (L-IS7) could decrease the Mmax and CH4 212 yield by 28% and 34% as compared to that of light control. The significance of 213 optimized intermittent stirring for effective digestion demonstrated in this study, was in 214 aligned with previous studies (Bose et al., 2021), suggesting that intermittently minimal 215 stirring period could shorten the acclimatization period, accelerate the biogas generation 216 rate and maximize the digestion performance. 217 To further explore the potential synergy of optimal stirring and light stimulation, 218 ATP and coenzyme F420, as indicators of microbial activity and methanogenic 219 productivity, were analyzed (Fig. 1d). All illuminated reactors exhibited higher ATP 220 values than that of the dark control. The highest ATP concentration was obtained in L-221 IS3 (5.78 ± 0.31 μmol/L), leading to 20% and 67% increment compared to L (4.81 ± 222 0.17 μmol/L) and D (3.46 ± 0.29 μmol/L), respectively. ATP, as the intracellular energy 223 currency, drives the cellular processes and positively associates with bioprocess 224 efficiency (Zhu et al., 2022). The higher ATP value observed in L-IS3 indicated that 225 well-uniformed activation of ammonia-stressed anaerobes was achieved by proper 226 intermittent. Similar stimulating effects on methanogenic activity could also be found in 227 L-IS3, where the highest coenzyme F420 (0.70 ± 0.07 μmol/L) was obtained. Coenzyme228 F420, as a unique enzyme in methanogens, participates in the critical redox reaction of 229 the methanogenesis (Zheng et al., 2020). The promoted coenzyme F420 levels claimed 230 the enrichment of methanogens and stimulated methanogenic productivity in L-IS3. 231 However, longer stirring time in L-IS7 dramatically deteriorated the methanogenic 232 activity with deceased coenzyme F420 level to 0.41 ± 0.06 μmol/L, even lower than that 233 of the dark control (0.48 ± 0.05 μmol/L). This might be associated with the destabilized 234 methanogenic activity and structure under excessive stirring, as some filamentous 235 methanogen (i.e., Methanosaeta) would be vulnerable to harsh mixing (Kariyama et al., 236 2018). Moreover, the ammonia inhibitory effect on methanogenic consortia could be 237 further magnified with intensive stirring, as inappropriate mixing might obstruct the 238 bio-cluster formation and thus restrain methanogenic activity (Bose et al., 2021). 239 This would be further supported by the lower sludge conductivity obtained in L-240 IS7 (0.37 ± 0.07 μS/cm), which decreased by 52% and 33% compared to L-IS3 (0.77 ± 241 0.05 μS/cm) and L (0.55 ± 0.04 μS/cm), respectively (Fig. 1e). Sludge conductivity is an 242 index of efficiently electronical cell-cell association and biofilm formation for 243 syntrophic growth (Zheng et al., 2020). Previous study suggested that the immobilized 244 biomass in light-assisted fixed-bed system could promote electron (e-) transfer and 245 enhance methanogenic performance under ammonia stress, probably due to enriched 246 syntrophic partners and reduced interspecies distance for efficient contact (Zhu et al., 247 2022). However, this harmony in bio-cluster was disturbed by intensive stirring in L-IS7, 248 leading to the reduced electroactivity and lower microbial activity. In contrast, 249 homogenous illumination provided by L-IS3 could favor the microbial colonization for 250 electronic syntrophic growth and relieve ammonia stress on microbial activity, 251 contributing to efficient methanogenic productivity.  252 Therefore, intermittent stirring for 3 min/15 interval could be proposed as the 253 optimal stirring strategy for developing a homogenous-illuminated fixed-bed system 254 during ammonia-rich AD process. From a practical standpoint, the intermittently stirred 255 digester, producing the greatest amount of CH4 with minimized electrical energy 256 consumption, could make the process more economical for scaled-up operation. 257 3.2 Capacity of the proposed homogenous-illuminated system in long-term 258 operation at pilot scale 259 To validate the feasibility of proposed homogenous-illuminated system for long-260 term operation, a pilot-scale AD treating ammonia-rich feedstock was carried out with 261 different OLR conditions. Though under high ammonia stress, digester successfully 262 started up with steadily increased CH4 concentration during the first 10-day operation 263 (Fig. 2a). This could be ascribed to the advantages of fixed-bed system on microbial 264 fixation, ammonia absorption and buffer capacity (Zhang et al., 2016; Zheng et al., 265 2020), which was evidenced by increased biomass quantity from 2.17 to 2.64 g VS/L 266 (Fig. 2a) and reduced ammonium level from 3439 to 3042 mg/L during start-up period 267 (Fig. 2b). By amending with solar light stimulation (day 21−50), the digester could 268 maintain stable performance with a stable CH4 content (72.0 ± 2.2%) during Light stage 269 than that of Dark stage (68.8 ± 12.7%). When homogenous operation was further 270 introduced in Light-S opt stage (day 51-90), the average CH4 content increased from 73.5 271 ± 1.9% to 72.4 ± 2.5% and 73.4 ± 2.9% with elevated OLR of 0.19 to 0.34 and 0.71 g-272 DOC added/(L∙d), respectively (Fig. 2a). Though HRT sharply decreased from 10 to 5 273 days (day 71−90), stable biomass quantity (2.28−2.37 g VS/L) could be kept in 274 homogeneous-illuminated system without significant washout, which was ascribed to 275 the effective immobilization of biomass. Additionally, a reduced ammonium 276 concentration was observed in the Light-S opt stage (3159 ± 21 mg/L) than that of the 277 Light stage (3200 ± 50 mg/L), suggesting that more ammonium as essential nitrogen 278 source could be consumed by fixed anaerobes in homogenous-stimulated system. 279 Moreover, carbon conversion was also promoted by well-homogeneous light 280 stimulation, as evidenced by higher DOC removal rate of 85.3% than that of Dark 281 (80.6%) and Light (83.1%) (Table 1). With homogenous operation, organic carbon was 282 evenly distributed and consumed by well-illuminated anaerobes, resulting in boosted 283 CH4 yield in Light-S opt (914 ± 31 mL/(g-DOCremoval∙d)) compared to Dark (781 ± 26 284 mL/(g-DOCremoval∙d)) and Light (877 ± 36 mL/(g-DOCremoval∙d)) stages under the same 285 OLR (0.19 g-DOCadded/(L∙d)). Moreover, this well-homogenized system led to a steadily 286 increased CH4 yield to 971 ± 29 mL/(g-DOCremoval∙d) at OLR of 0.71 g-DOCadded/(L∙d), 287 covering 94.3% of theoretical CH4 yield (BMYthDOC = 1.03 L/g-DOCadded). Generally,288 the ammonia-stressed AD operated in practice often faces the so-called ‘inhibited 289 steady-state’ when treating degradable nitrogen-rich substrates with low C/N ratio (˂ 25) 290 (Zheng et al., 2021), where the reactor was characterized with a stable performance but 291 repressed CH4 yield (34−50% of the theoretical value at ammonium level of 3000−4000 292 mg/L) (Nielfa et al., 2015; Zhu et al., 2021). In this study, however, 88.7−94.3% 293 BMYthDOC was achieved under ammonium stress of 3500 mg/L with extremely lower 294 C/N ratio (0.31−0.56) in the proposed homogenous-illuminated system. This result 295 suggested the proposed system was effective to maximize the organic-to-CH4 296 conversion under ammonia stress at pilot scale for long-term application. 297 To further uncover the effect of homogenous-illumination on formed anaerobic 298 biofilm, indexes of microbial activity (ATP and coenzyme F420) and biofilm 299 characterizations (EPS variation and sludge conductivity) were analyzed (Fig. 2c−e). At 300 low OLR of 0.19 g-DOCadded/(L∙d), a higher ATP level was obtained in Light-S opt (18.1 301 ± 1.0 μmol/L) than that of Dark (11.9 ± 0.9 μmol/L) and Light (14.6 ± 0.8 μmol/L) (Fig. 302 2c), demonstrating a better microbial activity for biochemical conversion was achieved 303 under uniform illumination. The ATP level reached a peak (24.7 ± 1.2 μmol/L) in 304 homogenous-illuminated system when OLR increased to 0.71 g-DOCadded/(L∙d). This 305 could be ascribed to that sufficient carbon supply with homogenous operation might 306 evenly benefit the well-activated anaerobes. Similar tendency could be found in the 307 variation of coenzyme F420 (Fig. 2c), where relative higher levels appeared during the 308 Light-S opt stage (ranging from 0.42 ± 0.02 to 0.45 ± 0.02 μmol/L) than that of Dark 309 (0.36 ± 0.05 μmol/L) and Light (0.38 ± 0.03 μmol/L) stages. 310 Moreover, the optimized homogenization in the system provided an optimal 311 surrounding environment to microbes for electrically syntrophic metabolism, as 312 evidenced by doubled sludge conductance (17.6 ± 1.2 μmol/L) obtained in Light-S opt 313 (0.71) stage compared to that of the Dark (0.19) stage (8.9 ± 0.7 μmol/L) (Fig. 2d). 314 Electroactive microorganisms generally communicate with other cells or interact with 315 external environments via e- mediators (Dang et al., 2022). Those semiconductive 316 substances (i.e., polysaccharides, proteins, humic substances, etc.) that surrounded 317 microbial cells consisted the EPS matrix, possessing double-layer structure, including 318 inner-layered TB-EPS and outer-layered LB-EPS which enveloped by DOM and 319 (Babiak and Krzemińska, 2021). Outer-layered EPS absorbed organic compounds from 320 the surrounding environment, serving as carbon sources for biofilm. At low OLR, DOM 321 (352 ± 16 mg-DOC/g-VS) and LB-EPS (14 ± 1 mg-DOC/g-VS) in the Dark (0.19) stage 322 decreased to 259 ± 13 mg-DOC/g-VS and 11 ± 1 mg-DOC/g-VS when homogenous 323 illumination was induced, respectively (Fig. 2e). This indicated that more carbon source 324 could be consumed for biomass growth and biofilm formation, corresponding with a 325 higher DOC removal efficiency (Table 1) with promoted biomass activity (Fig. 2c). 326 Whereas TB-EPS quantity exhibited insignificantly change (14−15 mg-DOC/g-VS) 327 with or without homogenous illumination. Until OLR gradually increased to 0.71 g-328 DOCadded/(L∙d), peaked TB-EPS (22 ± 1 mg-DOC/g-VS) was observed in homogenous-329 illuminated stage. TB-EPS, storing the conducting polymers, plays a vital role in 330 extracellular e- transfer and promotes the aggregation of sludge cells. Besides, self-331 secreted coenzyme F420 and amino acids were mainly detected in the TB-EPS according 332 to (Wang et al., 2020). Accordingly, the highly secreted TB-EPS under homogenous 333 illumination aligned with higher coenzyme F420 bioactivity (Fig. 2c) and promoted 334 sludge conductivity (Fig. 2d), probably related to the existence of external e- accepter or 335 carriers. Moreover, TB-EPS quantity was found to be positively corelated with 336 ammonia level and organic load, as it directly coats on the cell surface as a natural shell 337 protecting cell or membrane-bound enzymes from toxics attacks (Yan et al., 2019). The 338 intermittent homogenous operation provided anaerobes with evenly and sufficient 339 chance to contact with substrate and each other, forming an effective biofilm for organic 340 removal. Therefore, light stimulation with proposed homogenous operation 341 synergistically promoted the biomass activity and quantity, methanogenic productivity 342 and syntrophic metabolism, contributing to a stable and outperformed CH4 conversion 343 from ammonia-rich feedstock. The 90-day superior efficiency obtained from pilot-scale 344 reactor provided a solid foundation for scaling-up application of developed system with 345 maximal bioenergy recovery from organic waste under solar illumination. 346 3.3 Variation of microbial communities  347 3.3.1 Effect of homogenous illumination on microbial diversity 348 In order to explore the synergy between light and homogenous operation on 349 microbial consortia, 16S rRNA genes of the microbial communities from Dark, Light 350 and Light-S opt were sequenced, resulting in a total of 41450 useful sequences per 351 sample. Fig. 3a−d depicted the alpha diversity based on effective sequences. Index of 352 observed species (Fig. 3a), representing the number of confirmed OTUs, increased from 353 675 (Dark) to 743 and 972 (in Light and Light-S opt stage, respectively). Moreover, a 354 higher Chao1 richness index of 1876 and 2095 was observed in Light and Light-S opt 355 stages, respectively (Fig. 3b), which were 1.17-fold and 1.31-fold of Dark (1605). Those 356 elevated indexes indicated the potential of light stimulation in increasing the microbial 357 richness and abundance, which were further strengthened by the optimized homogenous 358 operation. Moreover, PD whole tree and Shannon index under no-stirred darkness 359 drastically increased from 51 and 4.0 to 66 and 4.9 under homogeneous illumination 360 (Fig. 3c-d), respectively. PD whole tree indicates the systematic diversity by 361 summarizing the distances in a constructed phylogenetic tree, while Shannon calculates 362 the interspecies evenness based on the proportions of each species. The variations of 363 two indexes suggested that the community under darkness characterized by a poor 364 diversity could be regulated by homogenous illumination, which led to evenly 365 distributed populations with higher diversity in Light-S opt stage. The balanced and 366 diversified community structure formed under homogeneous illumination was further 367 supported by PCA results (Fig. 3e), where Methanosarcina and Methanosaeta closely 368 surrounded the Light-S opt and clustered with bacterial populations (Atribacteria OP9, 369 Actinobacteria, Proteobacteria, Chloroflexi, Synergistetes, Atribacteria OP8 and 370 Bacteroidetes). In contrast, Firmicutes (68% of bacterial populations) with 371 Methanothermobacter (85% of archaeal populations) overwhelmingly dominated during 372 the Dark stage (See Supplementary Materials). As biodiversity was crucial for the 373 longer-term resilience of ecosystem (Oliver et al., 2015), the homogenous-illuminated 374 system thereby achieved an outperformed and stable organic-to-CH4 conversion under 375 high ammonia stress. 376 3.3.2 Bacterial and archaeal structure under homogenous illumination 377 To delve into the diverse community, we represented the bacterial structure at the 378 family level in a heat map (Fig. 4a). In Light-S opt, a distinctive bacterial cluster 379 emerged, including OPB54 (Hydrogenispora), Rhodobacteraceae, Rhizobiales, 380 Solirubrobacterales, Actinomycetales, Atribacteria OP9_f, Anaerobaculaceae, 381 Thermotogaceae and Thermodesulfobiaceae. OPB54 (Hydrogenispora) is known for its 382 H2 productivity under ammonia stress (Fischer et al., 2019) and Rhodobacteraceae and 383 Rhizobiales are related to nitrogen fixation and ammonia utilization (Tian et al., 2018). 384 Their enrichment highlighted the microbial community's adaptability to the ammonia-385 rich condition. The filamentous Solirubrobacterales and Actinomycetales are linked to 386 organic hydrolysis and carbon removal, favoring the breakdown of complex organic 387 compounds (Haig et al., 2015). Moreover, Anaerobaculaceae specializes in VFAs 388 generation, while Atribacteria OP9_f is suggested as syntrophic propionate-oxidizing 389 bacteria that producing acetate (Dyksma et al., 2020). Thermotogaceae and 390 Thermodesulfobiaceae, as typical syntrophic acetate oxidizing bacteria (SAOB) 391 converts the metabolized acetate into H2/CO2 (Hattori, 2008). Notably, the presence of 392 Thermodesulfobiaceae is commonly associated with configurations using biofilm 393 support, corresponding to fixed-bed system used in this study (Lembo et al., 2020). In 394 Light-S opt, the bacterial cluster encompassed various bacterial families with specific 395 metabolic roles, contributing to a highly cooperative and specialized microbial network. 396 In contrast, only certain sugar fermenter (Bacillales), and H2 producing bacteria 397 (Ruminococcaceae. Clostridiaceae and Thermoanaerobacterales) were survived during 398 the non-stirred dark period under ammonia. 399 Corresponded to the regulated bacterial community, archaeal community was also 400 shifted to more balanced structure under homogenous illumination (Fig. 4b). The 401 dominance of Methanothermobacter in the Dark and Light stages reduced to 54% in 402 Light-S opt, when the abundance of Methanosarcina and Methanosaeta increased from 9% 403 to 35% and 3% to 7%, respectively. The former methanogen ubiquitously involves in 404 hydrogenotrophic methanogenesis (HM), while the latter two could produce CH4 via 405 acetoclastic methanogenesis (AM), a pathway that generally accounts 60-70% CH4 406 productivity. As reported, Methanosarcina spp. grow as cocci, usually shows high 407 resistance to stressful condition, due to their versatile metabolic ability in both HM and 408 AM. And the long-rod like Methanosaeta cells are sensitive to ammonia stress and 409 intensitive mixing, and only increase in abundance when there is no VFA accumulation 410 (Kowalczyk et al., 2013). Accordingly, homogenous illumination could effectively 411 recover the ammonia-sensitive Methanosaeta meanwhile enriched the versatile 412 Methanosarcina, contributing to a good balance of AM and HM for efficient CH4 413 production under ammonia stress. 414 3.4 Metabolic response to homogenous light stimulation 415 To unveil the impact of homogenous illumination on microbial metabolism, 416 bioinformatic analysis was performed based on categories in the KEGG databases. A 417 total of 19369550, 19924595 and 23605033 unique genes were predicted from the Dark, 418 Light and Light-S opt samples, respectively. Most detected functions were shared across 419 all the stages but were more abundant in Light-S opt (See Supplementary Materials), 420 indicating enhanced metabolic capabilities of the microbial community under 421 homogenous illumination. Within the Metabolism category, which governs the 422 bioconversion efficiency of AD, methane metabolism was the most abundant 423 classification, followed by energy production (Oxidative phosphorylation) as well as the 424 pathways related to carbohydrate conversion (Glycolysis and Pyruvate metabolism) and 425 organic acid degradation (Butanoate and Propanoate metabolism). The upregulated gene 426 expressions in these categories reflected the promoted metabolic activity for organics-427 to-CH4 in Light-S opt under ammonia stress (Fig. 5a). 428  Methanogens then consume the fermentative metabolites via AM and/or HM for 429 terminal CH4 production. The genes coding for critical methanogenic reactions 430 including EC:2.7.2.1 and EC:2.3.1.8 (for acetate consumption), EC:1.5.98.2 (for CO2 431 reduction) and EC:1.8.98.1 (for methanation) were more abundant in Light-S opt as 432 compared to Dark and Light stages (See Supplementary Materials), suggesting the 433 promoted dual methanogenic pathways under uniform light stimulation. Besides, genes 434 encoding carbamoyl phosphate synthase and glutamine synthetase, which function in 435 the protein synthesis using ammonia to constitute the microbes, were highly expressed 436 in the Light-S opt stage (Fig. 5a). This result indicated that the ammonia consumption 437 was promoted under uniformed light stimulation, aligning with the reduced ammonia 438 levels during the Light-S opt stage (Fig. 2b). Therefore, the activated and carbohydrate, 439 nitrogen and methane metabolism contributed to efficient organic-to-CH4 under 440 ammonia-rich condition. 441 Moreover, the successful bioconversion was fueled by intracellular energy of ATP 442 and e-, mainly producing from oxidative phosphorylation. The involved NADH: 443 quinone oxidoreductase (M00144) acted as critical precursors for e- generation, while 444 the F-type ATPase (M00157) and V/A-type ATPase (M00159) were responsible for ATP 445 synthesis in bacterial and archaeal cells, respectively (Zhao et al., 2020). Gene 446 abundances involved in the energy production were effectively upregulated in Light-S 447 opt as compared to other stages (Fig. 5a), supporting the higher ATP levels observed in 448 the Light-S opt stage (Fig. 2c). Furthermore, the yield of more e- flux and energy might 449 promote electroactive species to produce the biological e- transfer components of 450 electrically conductive pili (e-pili) and/or c-type cytochrome (Cyt C), which involved in 451 direct interspecies electron transfer (DIET) (Dang et al., 2022). Increased gene 452 abundances related to Cyt C and e-pili accessory proteins were detected in Light-S opt 453 compared to other stages (Fig. 5b). This suggested that homogeneously illuminated 454 anaerobes engaged in an energy-conserving syntrophic association through extracellular 455 e- exchange facilitated by Cyt C and e-pili. The higher conductivity observed in sludge456 exposed to homogeneous illumination (Fig. 2d) supported this evidence. 457 Additionally, the formation of DIET-dependent biofilm was reported to be highly 458 regulated by quorum sensing (QS) system (Dang et al., 2022). With more QS system-459 related genes observed during Light-S opt stage (Fig. 5a), anaerobes in biofilm might 460 produce more Cyt C and e-pili facilitating DIET-based syntrophic methanogenesis. 461 Additionally, QS signals were strongly correlated with the formation of compact 462 biofilm/aggregates accompanied with EPS production (Fig. 2e), leading to a structured 463 conductive bio-cluster for highly efficient organic conversion. 464 3.5 Proposed mechanism and significance of results 465 By integrating metagenomic analysis with digestion performance, the synergistic 466 effects of homogenous operation and light stimulation for enhanced AD performance 467 under ammonia stress were proposed (Fig. 5c). (1) Optimal stirring strategy ensured 468 uniform distribution of light photons in the bioreactor, and (2) facilitate efficient 469 contacts between anaerobes and nutrients. (3) The efficient light transmittance and 470 nutrients nourishment allowed for the even enrichment of increasing populations (Fig. 471 3), forming a diverse bio-cluster to resist ammonia stress. (4) Those light-activated 472 anaerobes triggered an efficient metabolic process and syntrophic association for 473 oragnics-to-CH4 conversion. Specifically, homogenous light stimulation upregulated the 474 carbohydrate metabolism, fatty acid degradation and ammonia consumption (Fig. 5a), 475 leading to an efficient DOC and ammonia removal during the long-term operation 476 (Table 1 and Fig. 2b). Moreover, the microbial activity and critical enzymes involved in 477 dual methanogenic pathways, particular the acetoclastic methanogenesis, were 478 significantly activated in proposed system (Fig. 5a), contributing to the boosted CH4 479 yield (Table 1). Additionally, an energy-saving and kinetically faster electronic 480 association for CH4 production via Cyt C and e-pili was established (Fig. 5b). This was 481 potentially ascribed to the light-enriched bio-cluster, including electroactive bacteria 482 (i.e., Anaerobaculaceae, Thermotogaceae and Thermodesulfobiaceae) and methanogens 483 (i.e., Methanosarcina and Methanosaeta) (Fig. 4). (5) With positive regulation of QS 484 signals (Fig. 5a), this close-knit cluster formed a conductive biofilm with well-485 structured EPS (Fig. 2d−e), which assisted to promote the ammonia resistance and 486 accelerated the syntrophic metabolism for CH4 production. Consequently, this 487 homogenous-illuminated community with favorable metabolic characteristics 488 contributed to outperformed and stable CH4 recovery in ammonia-stressed AD during 489 long-term operation. 490 4. Conclusion491 This study provided an innovative insight into the role of homogenous operation in 492 light-assisted anaerobic digester under ammonia inhibition. The synergy of intermittent 493 stirring and light stimulation induced remarkable shifts in microbial diversity, effective 494 activation on carbon and methane metabolism and the formation of electroactive bio-495 cluster. During 90-day polit-scale operation, the proposed homogenous-illuminated 496 system demonstrated the superior CH4 yield and carbon removal under high ammonia 497 stress. From an economic standpoint, the naturally viable solar illumination with 498 optimized intermittent mixing could minimize energy demand and maintenance cost in 499 full-scale application, presenting a sustainable and easy-operated treatment approach for 500 efficient waste-to-energy recovery. 501 Acknowledgements 502 This research was supported by Scientific Research (B) 22H03778 and Grant-in-503 Aid for Exploratory Research 21k19628 from Japan Society for the Promotion of 504 Science. The Figure 5c in the paper was drawn using Figdraw platform 505 (www.figdraw.com). 506 Appendix A. Supplementary data 507 E-supplementary data of this work can be found in online version of the paper.508 Reference 509 Babiak, W., Krzemińska, I., 2021. Extracellular polymeric substances (EPS) as 510 microalgal bioproducts: A review of factors affecting EPS synthesis and 511 application in flocculation processes. 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Manag. 238, 114155. 634 https://doi.org/10.1016/J.ENCONMAN.2021.114155 635 636 Table 1 Semi-continuous AD performance during different characteristic stages at pilot scale under ammonia stress Stage Parameter Dark Light Light-SoptPeriod Day 0-20 Day 21-50 Day 51-70 Day 71-80 Day 81-90 OLR (g-DOCadded/(L∙d)) 0.19 0.19 0.19 0.34 0.71 C/N 0.31 0.31 0.31 0.38 0.56 HRT (day) 10 10 10 5 5 DOC removal efficiency (%) 80.6 83.1 85.3 84.3 81.6 Average daily CH4 yield (mL/(g-DOCremoval∙d)) 781 ± 26 877 ± 36 914 ± 31 953 ± 35 971 ± 29 Table(Editable version) Click here to access/download;Table(Editable version);Tables.docxhttps://www2.cloud.editorialmanager.com/bite/download.aspx?id=3357945&guid=30186d6d-c597-4a2b-836d-0a6f6b4cd52d&scheme=1https://www2.cloud.editorialmanager.com/bite/download.aspx?id=3357945&guid=30186d6d-c597-4a2b-836d-0a6f6b4cd52d&scheme=1Fig. 1 Methane performance of (a) cumulative methane production, (b) maximal cumulative CH4 production, maximal CH4 production rate and lag-phase time fitted with Gompertz model, and (c) methane yield, and microbial activity indicators of (d) ATP and coenzyme F420, and (e) sludge conductivity in bioreactors under different stirring conditions during batch experiment. (Error bars designate standard deviations of triplicate experiments) Figure Click here to access/download;Figure;Figures1.docxhttps://www2.cloud.editorialmanager.com/bite/download.aspx?id=3358090&guid=a4bfbc60-3514-4225-8318-19feb179fbb7&scheme=1https://www2.cloud.editorialmanager.com/bite/download.aspx?id=3358090&guid=a4bfbc60-3514-4225-8318-19feb179fbb7&scheme=1Fig. 2 Long-term performance of pilot-scale bio-fixed bed digester under dark (Dark), illumination (Light) and homogenous illumination (Light-S opt) with different OLR. (a) Variations of CH4 concentration and biomass quantity, (b) variations of pH and NH4+-N, (c) average levels of ATP, coenzyme F420 and (d) sludge conductance, and (e) EPS components of sludge. (Error bars designate standard deviations of triplicate experiments)   Fig. 3 Boxplots for microbial richness and diversity with indexes of (a) observed species, (b) Chao1, (c) PD whole tree and (d) Shannon; and (e) PCA plot depicted dominant microbial populations (bacterial order and archaeal genera) during each stage in long-term operation.     Fig. 4 (a) Heat map with cluster analysis of dominant bacterial family, and (b) relative abundance of methanogenic genera from original inoculum, Dark stage, Light stage and Light-S opt stages. (a) (b) Fig. 5 (a) Gene copy numbers of key metabolic pathways from each stage during pilot-scale operation, and (b) heat map of genes abundance involved in cytochrome c and e-pili accessory proteins (pilA, pilB, pilC, pilE, pilF, pilM, pilN, pilO, pilP, pilQ, pilV, pilW, pilX pilY1 and pilZ), and (c) conceptual graph of mechanisms in homogenous-illuminated biosystem for enhanced ammonia-rich AD performance.