Attenuation effects of ZVI/PDS pretreatment on propagation of antibiotic resistance genes in bioreactors: Driven by antibiotic residues and sulfate assimilation.

Sulfate radical-based advanced oxidation processes (AOPs) combined biological system was a promising technology for treating antibiotic wastewater. However, how pretreatment influence antibiotic resistance genes (ARGs) propagation remains largely elusive, especially the produced by-products (antibiotic residues and sulfate) are often ignored. Herein, we investigated the effects of zero valent iron/persulfate pretreatment on ARGs in bioreactors treating sulfadiazine wastewater. Results showed absolute and relative abundance of ARGs reduced by 59.8%- 81.9% and 9.1%- 52.9% after pretreatments. The effect of 90-min pretreatment was better than that of the 30-min. The ARGs reduction was due to decreased antibiotic residues and stimulated sulfate assimilation. Reduced antibiotic residues was a major factor in ARGs attenuation, which could suppress oxidative stress, inhibit mobile genetic elements emergence and resistant strains proliferation. The presence of sulfate in influent supplemented microbial sulfur sources and facilitated the in-situ synthesis of antioxidant cysteine through sulfate assimilation, which drove ARGs attenuation by alleviating oxidative stress. This is the first detailed analysis about the regulatory mechanism of how sulfate radical-based AOPs mediate in ARGs attenuation, which is expected to provide theoretical basis for solving concerns about by-products and developing practical methods to hinder ARGs propagation.

Synergistic analysis of performance, functional genes, and microbial community assembly in SNDPR process under Zn(II) stress.

One of the most prevalent heavy metals found in rural sewage is Zn(II), while its effect on simultaneous nitrification, denitrification and phosphorus removal (SNDPR) remains unclear. In this work, the responses of SNDPR performance to long-term Zn(II) stress were investigated in a cross-flow honeycomb bionic carrier biofilm system. The results indicated that Zn(II) stress at 1 and 5 mg L-1 could increase nitrogen removal. Maximum ammonia nitrogen, total nitrogen, and phosphorus removal efficiencies of up to 88.54%, 83.19%, and 83.65% were obtained at Zn(II) concentration of 5 mg L-1. The functional genes, such as archaeal amoA, bacterial amoA, NarG, NirS, NapA, and NirK, also reached the highest value at 5 mg L-1 Zn(II), with the absolute abundances of 7.73 × 105, 1.57 × 106, 6.68 × 108, 1.05 × 109, 1.79 × 108, and 2.09 × 108 copies·g-1 dry weight, respectively. The neutral community model demonstrated that deterministic selection was responsible for the system's microbial community assembly. Additionally, response regimes with extracellular polymeric substances and cooperation among microorganisms facilitated the stability of the reactor effluent. Overall, the findings of this paper contribute to improving the efficiency of wastewater treatment.

Simultaneous nitrification, denitrification and phosphorus removal: What have we done so far and how do we need to do in the future?

Nitrogen and phosphorus contamination in wastewater is a serious environmental concern and poses a global threat to sustainable development. In this paper, a comprehensive review of the studies on simultaneous nitrogen and phosphorus removal (SNPR) during 1986-2022 (538 publications) was conducted using bibliometrics, which showed that simultaneous nitrification, denitrification, and phosphorus removal (SNDPR) is the most promising process. To better understand SNDPR, the dissolved oxygen, carbon to nitrogen ratio, carbon source type, sludge retention time, Cu2+ and Fe3+, pH, salinity, electron acceptor type of denitrifying phosphorus-accumulating organisms (DPAOs), temperature, and other influencing factors were analyzed. Currently, SNDPR has been successfully implemented in activated sludge systems, aerobic granular sludge systems, biofilm systems, and constructed wetlands; sequential batch mode of operation is a common means to achieve this process. SNDPR exhibits a significant potential for phosphorus recovery. Future research needs to focus on: (1) balancing the competitiveness between denitrifying glycogen-accumulating organisms (DGAOs) and DPAOs, and countermeasures to deal with the effects of adverse conditions on SNDPR performance; (2) achieving SNDPR in continuous flow operation; and (3) maximizing the recovery of P during SNDPR to achieve resource sustainability. Overall, this study provides systematic and valuable information for deeper insights into SNDPR, which can help in further research.

A novel cross-flow honeycomb bionic carrier promotes simultaneous nitrification, denitrification and phosphorus removal in IFAS system: Performance, mechanism and keystone species.

Simultaneously achieving efficient nitrogen (N) and phosphorus (P) removal without adding external carbon source is vital for carbon-neutral wastewater treatment. In this study, a novel cross-flow honeycomb bionic microbial carrier (CF) was developed to improve the efficiency of simultaneous nitrification, denitrification, and P removal (SNDPR) in an integrated fixed-film activated sludge (IFAS) system. A parallel laboratory-scale sequencing batch reactor with the commercialized microbial carriers (CM) (CM-IFAS) was performed as the comparative system for over 233 d The results demonstrated that CF-IFAS exhibited a more consistent N removal efficiency and better performance than CM-IFAS. In the CF-IFAS, the highest N and P removal efficiencies were 95.40% and 100%, respectively. Typical cycle analysis revealed that nitrate was primarily removed by the denitrifying glycogen-accumulating organisms in the CF-IFAS and by denitrifying phosphate-accumulating organisms in the CM-IFAS. The neutral community model showed that the microbial community assembly in both the reactors was driven by deterministic selection rather than stochastic factors. Compared to those in CM-IFAS, the microorganisms in CF-IFAS were more closely related to each other and had more keystone species: norank_f_norank_o_norank_c_OM190, SM1A02, Defluviicoccus, norank_f_ Saprospiraceae, and norank_f_Rhodocyclaceae. The absolute contents of the genes associated with N removal (bacterial amoA, archaeal amoA, NarG, NapA, NirS, and NirK) were higher in CF-IFAS than in CM-IFAS; the N cycle activity was also stronger in the CF-IFAS. Overall, the microecological environment differed between both systems. This study provides novel insights into the potential of bionic carriers to improve SNDPR performance by shaping microbial communities, thereby providing scientific guidance for practical engineering.

Enhanced methane production in anaerobic digestion: A critical review on regulation based on electron transfer.

Anaerobic digestion (AD) is a potential bioprocess for waste biomass utilization and energy conservation. Various iron/carbon-based CMs (e.g., magnetite, biochar, granular activated carbon (GAC), graphite and zero valent iron (ZVI)) have been supplemented in anaerobic digestors to improve AD performance. Generally, the supplementation of CMs has shown to improve methane production, shorten lag phase and alleviate environmental stress because they could serve as electron conduits and promote direct interspecies electron transfer (DIET). However, the CMs dosage varied greatly in previous studies and CMs wash out remains a challenge for its application in full-scale plants. Future work is recommended to standardize the CMs dosage and recover/reuse the CMs. Moreover, additional evidence is required to verify the electrotrophs involved in DIET.

An influent responsive control strategy with machine learning: Q-learning based optimization method for a biological phosphorus removal system.

Biological phosphorus removal (BPR) is an economical and sustainable processes for the removal of phosphorus (P) from wastewater, achieved by recirculating activated sludge through anaerobic and aerobic (An/Ae) processes. However, few studies have systematically analyzed the optimal hydraulic retention times (HRTs) in anaerobic and aerobic reactions, or whether these are the most appropriate control strategies. In this study, a novel optimization methodology using an improved Q-learning (QL) algorithm was developed, to optimize An/Ae HRTs in a BPR system. A framework for QL-based BPR control strategies was established and the improved Q function, Qt+1(st,st+1)=Qt(st,st+1)+k·[R(st,st+1)+γ·maxatQt(st,st+1)-Qt(st,st+1)] was derived. Based on the improved Q function and the state transition matrices obtained under different HRT step-lengths, the optimum combinations of HRTs in An/Ae processes in any BPR system could be obtained, in terms of the ordered pair combinations of the . Model verification was performed by applying six different influent chemical oxygen demand (COD) concentrations, varying from 150 to 600 mg L-1 and influent P concentrations, varying from 12 to 30 mg L-1. Superior and stable effluent qualities were observed with the optimal control strategies. This indicates that the proposed novel QL-based BPR model performed properly and the derived Q functions successfully realized real-time modelling, with stable optimal control strategies under fluctuant influent loads during wastewater treatment processes.

Biological phosphorus removal in an extended ASM2 model: Roles of extracellular polymeric substances and kinetic modeling.

This paper presents the results of an extended ASM2 model for the modeling and calibration of the role of extracellular polymeric substances (EPS) in phosphorus (P) removal in an anaerobic-aerobic process. In this extended ASM2 model, two new components, the bound EPS (XEPS) and the soluble EPS (SEPS), are introduced. Compared with the ASM2, 7.71, 8.53, and 9.28% decreases in polyphosphate (polyP) were observed in the extended ASM2 in three sequencing batch reactors feeding with different COD/P ratios, indicating that 7.71-9.28% of P in the liquid was adsorbed by EPS. Sensitive analysis indicated that, five parameters were the significant influential parameters and had been chosen for further model calibration by using the least square method to simulate by MATLAB. This extended ASM2 has been successfully established to simulate the output variables and provides a useful reference for the mathematic simulations of the role of EPS in biological phosphorus removal process.

Enhancement of volatile fatty acid production by co-fermentation of food waste and excess sludge without pH control: The mechanism and microbial community analyses.

The study provided a cost-effective and high-efficiency volatile fatty acid (VFA) production strategy by co-fermentation of food waste (FW) and excess sludge (ES) without artificial pH control. VFA production of 867.42mg COD/g-VS was obtained under the optimized condition: FW/ES 5, solid retention time 7d, organic loading rate 9g VS/L-d and temperature 40°C. Mechanism exploration revealed that the holistic biodegradability of substrate was greatly enhanced, and proper pH range (5.2-6.4) was formed by the high buffering capacity of the co-fermentation system itself, which effectively enhanced hydrolysis yield (63.04%) and acidification yield (83.46%) and inhibited methanogenesis. Moreover, microbial community analysis manifested that co-fermentation raised the relative abundances of hydrolytic and acidogenic bacteria including Clostridium, Sporanaerobacter, Tissierella and Bacillus, but suppressed the methanogen Anaerolineae, which also facilitated high VFA production. These results were of great guiding significance aiming for VFA recovery from FW and ES in large-scale.

Economical evaluation of sludge reduction and characterization of effluent organic matter in an alternating aeration activated sludge system combining ozone/ultrasound pretreatment.

An ozone/ultrasound lysis-cryptic growth technology combining a continuous flow anaerobic-anoxic-microaerobic-aerobic (AAMA+O3/US) system was investigated. Techno-economic evaluation and sludge lyses return ratio (r) optimization of this AAMA+O3/US system were systematically and comprehensively discussed. Economic assessment demonstrated that this AAMA+O3/US system with r of 30% (AAMA+O3/US2# system) was more economically feasible that can give a 14.04% saving of costs. In addition to economic benefits, a 55.08% reduction in sludge production, and respective 21.17% and 5.45% increases in TN and TP removal efficiencies were observed in this AAMA+O3/US2# system. Considering the process performances and economic benefits, r of 30% in AAMA+O3/US2# system was recommended. Excitation-emission matrix and Fourier transform infrared spectra analyses also proved that less refractory soluble microbial products were generated from AAMA+O3/US2# system. Improvement in 2,3,5-triphenyltetrazolium chloride electron transport system (TTC-ETS) activity in AAMA+O3/US2# further indicated that a lower sludge lyses return ratio stimulated the microbial activity.

Role of carbon substrates in facilitating energy reduction and resource recovery in a traditional activated sludge process: investigation from a biokinetics modeling perspective.

Three activated sludge processes (ASPs) were modeled and driven by dissolved complex organics (F-ASP), propionic acid (P-ASP), and acetic acid (A-ASP), and various parameters were subsequently estimated. The energy depletion for carbon removal was 0.146, 0.120, and 0.119 kWh/m(3) of treated wastewater for F-ASP, P-ASP, and A-ASP, respectively, suggesting that acetic acid can forward energy conservation. The ratio of substrate storage to oxidation in F-ASP, P-ASP, and A-ASP was 0, 0.25, and 0.52, respectively, further demonstrating that substrate eliminations from P-ASP and A-ASP were both dominated by substrate storage for polymer production, not by total oxidation; thus, they exhibited lower energy-consuming levels than F-ASP. Quantification of bioenergy production and nutrient acquisition from the excess sludge of the three ASPs were conducted subsequently, and A-ASP was found to facilitate phosphorus capture.

Environmental profile of typical anaerobic/anoxic/oxic wastewater treatment systems meeting increasingly stringent treatment standards from a life cycle perspective.

Stringent new legislation for wastewater treatment plants (WWTPs) is currently motivating innovation and optimization of wastewater treatment technologies. Evaluating the environmental performance of a wastewater treatment system is a necessary precursor before proposing implementation of WWTPs designed to address the global requirements for reduced resource use, energy consumption and environmental emissions. However, developing overly-sophisticated treatment methods may lead to negative environmental effects. This study was conducted to employ a process modeling approach from a life cycle perspective to construct and evaluate six anaerobic/anoxic/oxic wastewater treatment systems that include a water line, sludge line and bioenergy recovery system and was designed to meet different treatment standards in China. The results revealed that improved treatments optimized for local receiving watercourses can be realized at the cost of higher resource consumption and greenhouse gas emissions. Optimal Scenarios were also identified from different positive perspectives.

[Evaluation and choice of PCR-DGGE primers in analyzing the microbial community structure of activated sludge in traditional Chinese medicine wastewater].

In the study of traditional Chinese medicine wastewater, it was discussed for the effect of different sets of 16S rDNA universal primers on DGGE fingerprinting and microbial community diversity of aerobic and anaerobic activated sludge from one traditional Chinese medicine wastewater treatment. The genome DNA of activated sludge was isolated, and eleven sets of primers were used to amplify the four variable regions of 16S rDNA, the resolution of DGGE fingerprinting and community diversity was analyzed. The results indicated that community diversity with different sets of universal primers by DGGE was obviously different. Separated patterns of the V3 and V6-V8 regions were better than of V1-V3 and V3-V5. In the DGGE profiles, bands and diversity from V3 were most, bands and diversity from V3-V5 and V6-V8 were a little worse than those of V3. According to the length of targeted sequence and the resolution of DGGE fingerprinting, V6-V8 (B968F/B1401R) are recommended to be used to do the DGGE analysis. Mix I341F/I534R and B341F/B534R PCR product equally to make DGGE analysis can get more community diversity information.

Effect of temperature on intracellular phosphorus absorption and extra-cellular phosphorus removal in EBPR process.

This study investigated the temperature influence on intracellular absorption and extra-cellular phosphorus removal by extra-cellular polymeric substance (EPS) in enhanced biological phosphorus removal (EBPR) process. Three sequencing batch reactors (SBRs) were operated in anaerobic/aerobic sequence at 5.0, 15.0 and 25.0 degrees C. Phosphorus removed by intracellular absorption was demonstrated as the dominant part (>80%) in total phosphorus removal operated under different temperatures and the highest total phosphorus removal rate of 95% was obtained due to the highest intracellular phosphorus absorption of 18.2mg P in a typical cycle at 15.0 degrees C. Phosphorus removed by EPS removal achieved the highest value at 5.0 degrees C (2.4 mg P/cycle), which resulted in a higher total phosphorus removal rate at 5.0 degrees C (90%) than that at 25.0 degrees C (83%). Low temperature was propitious to EPS phosphorus removal, accounting for 13% of total phosphorus removal at 5.0 degrees C, which could be mainly due to magnesium phosphate precipitation.

Back propagation neural network (BPNN) prediction model and control strategies of methanogen phase reactor treating traditional Chinese medicine wastewater (TCMW).

Based on the prototype experiment of treating herb wastewater by Up flow Anaerobic Sludge Bed and Anaerobic Filter reactor (UASBAF), an artificial neural network (ANN) model which adopts a back propagation algorithm with momentum and adaptive learning rate was established. And the effect of each parameter to the performance of the reactor was compared, using the method of partitioning connection weights (PCW). The result is pH values>influent of chemical oxygen demand (COD)>hydraulic retention time (HRT)>alkalinity. In addition, many strategies were proposed to optimize the working condition of the system. Adding some alkali to increase pH value when raising influent COD, was an effective way to avoid negative effect to system; low influent COD had a negative impact on the performance of the reactor; pH was suggested to be controlled more than 7.5 when the influent COD was increased over 6000mgL(-1). The best influent COD concentration was 6000-8000mgL(-1) when the conditions were that pH was 7.5, alkalinity was 2000mgL(-1) and HRT was 35-50h; HRT was suggested to be controlled more than 50h to maintain good performance of the reactor with high influent COD (8000-10,000mgL(-1)). These strategies provided an effective way of controlling UASBAF simply.

[Characterization of phosphate-accumulating organisms in starting-up EBPR by FISH analysis].

Enhanced biological phosphorus removal (EBPR) process was operated in a laboratory-scale sequencing batch reactor (SBR) for one-month fed with acetate as the carbon source. The characteristic and the microbial population structure and space distribution dynamics of phosphate-accumulating organisms (PAOs) of start-up period were analyzed by fluorescent in situ hybridization (FISH). The relationship between enrichment of PAOs and phosphorus removal was discussed. PAOs could be enriched by recirculation activated sludge containing heterotrophs through anaerobic aerobic conditions. Portion of PAOs in the sludge increase from 11.5% to 40.48%. Bacteria population competition lasted 34 days. It started from PAOs replacing heterotrophs which cost 5 days then followed by 19 days intra-specific competition of PAOs. The last step was re-increasing of PAOs predominance. Phosphorus uptake by the enriched microbial community was not observed immediately. An accumulating-phase was necessary for PHA and poly-P storage. A lag-stage of 4-8 days existed when taking the performance of the reactor into consideration. Phosphorus removal by the predominant PAOs through intra-specific competition was achieved after accumulating-phase too. The FISH picture indicated that in the quickly growing phase PAOs cells were small and community structure was loose. The latter "accumulating-phase" cells became larger and the community structure clustered densely. This stage presented by better reactor performance.

Behavior and characteristics of dissolved organic matter during column studies of soil aquifer treatment.

Soil column experiments were performed to investigate the behavior and characteristics of dissolved organic matter (DOM) during soil aquifer treatment (SAT), and to differentiate among the mechanisms responsible for the changes in the structural and functional properties of DOM during SAT. To determine the biological transformation of DOM, biodegradability tests using a biodegradation-column system were conducted. DOM was fractionated using XAD resins into 5 fractions: hydrophobic acid (HPO-A), hydrophobic neutral (HPO-N), transphilic acid (TPI-A), transphilic neutral (TPI-N) and hydrophilic fraction (HPI). Dissolved organic carbon (DOC) was removed by 70% during SAT, and the sorption and anaerobic biodegradation in SAT led to a DOC reduction of 27.4%. The significant changes in fluorescence properties of DOM were observed during SAT. However, the sorption and anaerobic biodegradation in SAT seemed to have no significant effect on the chemical structure of fluorescing constituents in DOM. The DOM fractions exhibited different changes in Fourier-transform infrared (FT-IR) spectra characteristics during SAT. Biodegradation resulted in the enrichment of aromatic structures and the decreased content of the oxygen-containing functional groups, such as CO and C-O, in DOM. On the other hand, the production of C-O and amide-2 functional groups occurred as a result of the sorption combined with anaerobic biodegradation in SAT.

[Novel phosphorus and nitrogen removal process for municipal sewage treatment: performance evaluation and design optimization].

A novel modified A2/O process (MMAO) was developed for nitrogen and phosphorus removal of municipal sewage. Bench-scale study was conducted to evaluate the performance of the MMAO process treating practical municipal sewage at normal temperature. Activated sludge model (ASM2D) was used to simulate the MMAO process and optimize its design and operation. It was found that the average treatment efficiency of COD, TN, NH4+-N and TP achieved by MMAO were up to 85.7%, 66.8%, 97.35% and 78.1%, respectively. When influent COD concentration of the system was more than 300 mg/L, a better nitrogen and phosphorus removal efficiency of 70% and 90% were achieved. After being calibrated and validated by the experimental results, the activated sludge model of MMAO could simulate the biological reactions occurred in the systems excellently. Optimization design and operational parameters could be accomplished by the mechanical activated sludge modeling. Furthermore, the model could also evaluate the process performance under peak load and low temperature and presented a whole scheme toward the unit combination and operation control. The effluent quality of MMAO process under stable operating could reach the first (B) standard of Municipal Sewage Treatment Plant Pollutants Discharge Standards (GB 18918-2002). The effluent of anaerobic unit was pumped directly into the anoxic unit to supply carbon source for denitrification instead of internal recirculation of mixture liquid, which would save operation cost significantly. The total hydraulic retention time of MMAO was lower than traditional biological organic removal system, so it was very suitable for the improvement of existing plant.

Effect of powdered activated carbon on Chinese traditional medicine wastewater treatment in submerged membrane bioreactor with electronic control backwashing.

Chinese traditional medicine wastewater, rich in macromolecule and easy to foam in aerobic biodegradation such as Glycosides, was treated by two identical bench-scale aerobic submerged membrane bioreactors (SMBRs) operated in parallel under the same feed, equipped with the same electronic control backwashing device. One was used as the control SMBR (CSMBR) while the other was dosed with powdered activated carbon (PAC) (PAC-amended SMBR, PSMBR). The backwashing interval was 5 min. One suction period was about 90 min by adjusting preestablished backwashing vacuum and pump frequency. The average flux of CSMBR during a steady periodic state of 24 d (576 h) was 5.87 L/h with average hydraulic residence time (HRT) of 5.97 h and that of PSMBR during a steady periodic state of 30 d (720 h) was 5.85 L/h with average HRT of 5.99 h. The average total chemical oxygen demand (COD) removal efficiency of CSMBR was 89.29% with average organic loading rate (OLR) at 4.16 kg COD/(m3 x d) while that of PSMBR was 89.79% with average OLR at 5.50 kg COD/(m3 x d). COD concentration in the effluent of both SMBRs achieved the second level of the general wastewater effluent standard GB8978-1996 for the raw medicine material industry (300 mg/L). Hence, SMBR with electronic control backwashing was a viable process for medium- strength Chinese traditional medicine wastewater treatment. Moreover, the increasing rates of preestablished backwashing vacuum, pump frequency, and vacuum and flux loss caused by mixed liquor in PSMBR all lagged compared to those in CSMBR; thus the actual operating time of the PSMBR system without membrane cleaning was extended by up to 1.25 times in contrast with the CSMBR system, and the average total COD removal efficiency of PSMBR was enhanced with higher average OLR.

Feasibility and simulation model of a pilot scale membrane bioreactor for wastewater treatment and reuse from Chinese traditional medicine.

The lack and pollution of water resource make wastewater reuse necessary. The pilot scale long-term tests for submerged membrane bioreactor were conducted to treat the effluents of anaerobic or aerobic treatment process for the high-strength Chinese traditional medicine wastewater. This article was focused on the feasibility of the wastewater treatment and reuse at shorter hydraulic retention time (HRT) of 5.0, 3.2 and 2.13 h. MLSS growth, membrane flux, vacuum values and chemical cleaning periods were also investigated. The experimental results of treating two-phase anaerobic treatment effluent demonstrated that the CODfilt was less than 100 mg/L when the influent COD was between 500-10000 mg/L at HRT of 5.0 h, which could satisfy the normal discharged standard in China. The experimental results to treat cross flow aerobic reactor effluent demonstrated that the average value of CODfilt was 17.28 mg/L when the average value of influent COD was 192.84 mg/L at HRT of 2.13 h during 106 d, which could completely meet the normal standard for water reuse. The maximum MLSS and MLVSS reached 24000 and 14500 mg/L at HRT of 3.2 h respectively. Membrane flux had maximal resume degrees of 94.7% at vacuum value of 0.02 MPa after cleaning. Chemical cleaning periods of membrane module were 150 d. A simulation model of operational parameters was also established based on the theory of back propagation neural network and linear regression of traditional mathematical model. The simulation model showed that the optimum operational parameters were suggested as follows: HRT was 5.0 h, SRT was 100 d, the range of COD loading rate was between 10.664-20.451 kg/(m3xd), the range of MLSS was between 7543-13694 mg/L.

[Anaerobic biodegradation pathway of the main ingredient of traditional Chinese medicine wastewater].

A laboratory-scale experiment was done. And the baicalin anaerobic biodegradation curve and pathway were discussed by analyzing the results of GC-MS. After analogue of status's trend and analysis of its regression, the biodegradation kinetic equation of baicalin was obtained. And the possible biodegradation pathway was "desugar --> debentures --> open benzene ring --> saturating --> disconnect links". It took at least 47 h to biodegrade baicalin. The main products of anaerobic biodegradation were micro-molecule oxide, such as alcohols, aldehydes, acids, esters, olefin, and hydrocarbon of methane series. This result demonstrated that the rate-limiting steps of bacalin biodegradation were the hydrolyzing and acidification phase. It was suitable to use two-phase anaerobic process and long HRT to biodegrade baicalin.