Bioresource Technology 142 (2013) 52–62

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Bioresource Technology

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Characterization of bacterial communities in hybrid upflow anaerobic sludge blanket (UASB)–membrane bioreactor (MBR) process for berberine antibiotic wastewater treatment ⇑ Guanglei Qiu a,b,c,d, Yong-hui Song a,b,d, , Ping Zeng a,b, Liang Duan a,b, Shuhu Xiao a,b

a State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Dayangfang 8, Anwai Beiyuan, Beijing 100012, China b Department of Urban Water Environmental Research, Chinese Research Academy of Environmental Sciences, Dayangfang 8, Anwai Beiyuan, Beijing 100012, China c Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576, Singapore d College of Water Science, Beijing Normal University, Xinjiekou Wai Street 19, Beijing 100875, China

highlights

High berberine removal was achieved in hybrid UASB–MBR process. UASB was dominated by Firmicutes and Bacteroidetes, while MBRs by . Clostridium, Eubacterium and Synergistes were functional species in UASB. Functional species in MBRs were mainly Alpha-, Beta-, and Gammaprotebacteria members. Discrepant community compositions in two MBRs however with analogous functions.

article info abstract

Article history: Biodegradation of berberine antibiotic was investigated in upflow anaerobic sludge blanket (UASB)– Received 11 March 2013 membrane bioreactor (MBR) process. After 118 days of operation, 99.0%, 98.0% and 98.0% overall remo- Received in revised form 19 April 2013 vals of berberine, COD and NHþ–N were achieved, respectively. The detailed composition of the estab- Accepted 19 April 2013 4 lished bacterial communities was studied by using 16S rDNA clone library. Totally, 400 clones were Available online 28 April 2013 retrieved and grouped into 186 operational taxonomic units (OTUs). UASB was dominated by Firmicutes and Bacteroidetes, while Proteobacteria, especially Alpha- and Beta-proteobacteria were prevalent in the Keywords: MBRs. Clostridium, Eubacterium and Synergistes in the UASB, as well as Hydrogenophaga, Azoarcus, Sphingo- 16S rDNA clone library monas, Stenotrophomonas, Shinella and Alcaligenes in the MBRs were identified as potential functional spe- Bacterial community structure Berberine wastewater cies in biodegradation of berberine and/or its metabolites. The bacterial community compositions in two Upflow anaerobic sludge blanket (UASB) MBRs were significantly discrepant. However, the identical functions of the functional species ensured Membrane bioreactor (MBR) the comparable pollutant removal performances in two bioreactors. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction (Martinez, 2008). Extensive research has been conducted to inves- tigate the degradation profile of antibiotics in domestic and indus- The occurrence of antibiotics in environment has received con- trial wastewater treatment systems, and the results showed that siderable public concerns in recent years (Göbel et al., 2005; Joss many of them are not well removed when they present in nano- et al., 2006). They were considered to be emerging pollutants scale concentrations (Radjenovic´ et al., 2009). Some studies also due to their bioactivity, polarity and persistence which may cause suggested that dilution of wastewater is expected to retard the adverse effects on aquatic life and humans. Additionally, antibiot- biological removal of antibiotics, which highlight the significance ics and their transformation products were also regarded as impor- of treatment at the source if dilution can be avoided (Joss et al., tant inducements of long term development/maintenance/ 2006). transfer/spread of antibiotics resistant and genes Berberine (5,6-dihydro-9,10-dimethoxybenzo[g]-1,3-benzodi- oxolo [5,6a] quinolizinium; 7,8,13,13a-tetradehydro-9, 10-

dimethoxy-2,3-(methylenedioxy) berbinium, C20H18NO4)isa ⇑ Corresponding author at: State Key Laboratory of Environmental Criteria and quaternary ammonium salt from the protoberberine group of Risk Assessment, Chinese Research Academy of Environmental Sciences, Dayangf- isoquinoline alkaloids, originally isolated from Berberidaceae fam- ang 8, Anwai Beiyuan, Beijing 100012, China. Tel./fax: +86 10 84928380. ily plants, which has been extensively used as a broad-spectrum E-mail address: [email protected] (Y.-h. Song).

0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.04.077 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 53 antibiotic medicine against a wide variety of microorganisms facilitate the design of signature oligonucleotides that are comple- including Gram-positive and Gram-negative bacteria, fungi, proto- mentary to target groups, as well as the utilization of genetic engi- zoa, trypanosomes and plasmodia (Cˇernˇáková and Koštˇálová, neering technique to improve the biodegradation of certain 2002). However, the production of berberine either by plant refractory compounds. extraction or by chemical synthesis resulted in the generation of In this study, berberine was treated in UASB coupled with two a large amount of wastewater containing up to 1000 mg/L of resid- novel MBRs (aerobic granular sludge membrane bioreactor ual berberine, which requires proper treatment before discharge. (AGMBR) and moving bed membrane bioreactor (MBMBR)) in par- Studies showed that, the IC50, minimum inhibitory concentration allel. The composition of the established berberine degradation (MIC) and minimum microbicidal concentration (MMC) values of functional bacterial communities was studied by using 16S rDNA berberine against resistant and Escherichia clone library. And the berberine degradation functional consortia coli are 99.2, 240, 250 mg/L and 87.0, 469, 500 mg/L, respectively were further identified based on the clone library results. This (Cˇernˇáková and Koštˇálová, 2002). High concentrations of berberine study was planning to provide some fundamental information on in the wastewaters make it recalcitrant and toxic to the biological the bacterial community composition in the biological berberine cultures. Most recently, some physico-chemical technologies were treatment process, and which is expected to lay a foundation for proposed to treat wastewaters containing berberine, and the further studies on the berberine biodegradation functional groups promising results have shown that, the berberine removal could and potential berberine resistant groups. reach up to 90%. However, a great deal of residual berberine (up to 150 mg/L) remains untreated in the effluent, which need further disposal (Ren et al., 2011). Unfortunately, few related studies up to 2. Methods date focus on biological removal of berberine from wastewater. The application of combined anaerobic–aerobic treatment sys- 2.1. Hybrid UASB–MBR system tem has proven to be quite effective for a wide range of wastewa- ters containing antibiotics and pharmaceutical effluents. And The UASB was a cylinder with 400 mm internal diameter and among all the anaerobic processes, upflow anaerobic sludge blan- 1500 mm height providing a working volume of 120 L (Fig. 1). A ket (UASB) was much efficient due to the advantage of their gran- 3-phase separator was installed upside the reactor, angled at 60° ular sludge, which could protect susceptive microorganisms from and placed 300 mm below the effluent ports, to prevent floating toxic substrate, and make the reactor highly resistant to antibiotics granules from washing out with the effluent. The reactor was (Oktem et al., 2008). As for the aerobic processes, membrane biore- equipped with sampling ports at 200 mm intervals (the lowest actor (MBR) was found to be a promising alternative in eliminating being 100 mm from the bottom) that allowed biological solids recalcitrant and toxic antibiotics (Göbel et al., 2007; Radjenovic´ and liquid samples to be withdrawn from the sludge bed. The reac- et al., 2009). In MBR, the entire retention of activated sludge ex- tor walls were wrapped with a tubular heater, to maintain the tends the sludge–substrate contacting time, thus improving the reactor temperature at 37 ± 1 °C. A peristaltic pump (Longer- removals of low biodegradable pollutants. Additionally, the mem- BT100, Baoding, China) was used to control the influent rate to brane separation process could efficiently retain slow-growing and the UASB. dispersed non-flocculating bacteria, which could facilitate the An AGMBR and a MBMBR were operated in parallel with the development of much abundant microbial community and enrich feed wastewater delivered from the UASB (Fig. 1). Each MBR was the target contaminants biodegradation functional species/groups a cubic column with 400 mm length, 300 mm weight, and (Göbel et al., 2007). And more importantly, via membrane separa- 600 mm height, and the effective volume was 60 L. Perforated baf- tion, MBR technology could effectively stop potential pathogenic fles with pore size of 20 mm diameter and 20 mm pore spaces microorganisms with increased antibiotic resistance in activated were placed at 300 mm length location in the MBRs, which divided sludge from flowing through the treatment system, which prevents the bioreactor into reaction compartments (effective volume 45 L) the release and dispersion of the resultant antibiotic resistant bac- and membrane separation compartments (effective volume 15 L). teria and genes during antibiotic wastewater treatment (Xia et al., For the MBMBR, The reaction compartment was filled with Kaldnes 2012). Currently, anaerobic–aerobic combinations have been K3 carriers (Kaldnes Miljøteknologi A/S, Tønsberg, Norway) pos- extensively used for treatment of antibiotic containing wastewa- sessing an estimated available surface area for biomass growth of ter, but berberine containing wastewaters still have not yet been 500 m2/m3 at a 0.6 filling ratio. And for the AGMBR, no carriers tried. were added in the reaction compartment, and aerobic granular In the biological wastewater treatment processes, the charac- sludge was formed naturally in the reactor by using berberine tox- terization of microbial assemblages is essential for better under- icity as a driving force. Aerobic granulation process was observed standings of the effects and contributions of microbial activities in the MBR in operation days of 67–70, and from then on, the per- on target contaminants removal. However, traditional cultiva- centage of aerobic granular sludge in mixed liquor suspended sol- tion-dependent methods in general provide with insufficient in- ids in the MBR kept above 80%. The granulation process in MBR sight into the in situ structures of microbial assemblages. would be discussed elsewhere. The membrane separation com- Although recent developments in molecular techniques have pro- partments of both MBRs were equipped with hollow fiber microfil- vided many novel approaches that enable a better investigation tration membrane modules made of polyvinylidene fluoride that of complex microbial communities, 16S rDNA clone library method had a total surface area of 0.3 m2 and a nominal pore size of with high degrees of precision and specificity was still widely used 0.4 lm (Originwater, Beijing, China). In the MBRs, aeration was for microbial community studies in wastewater treatment pro- continuously carried out at flow rates of 160 L/h in the reaction cesses (Deng et al., 2012). Compare to other molecular techniques, compartment and 600 L/h in the membrane separation compart- 16S rDNA clone library method could provide much detailed and ment, respectively. Filtration was carried out in a constant flow precise information on the full-length 16S rDNA sequences, which rate of 0.2 m3/m2/d driven by peristaltic pump (Longer-BT100, accurately reflect the phylogenetic position of the corresponding Baoding, China). No excess sludge was extracted from the hybrid 16S rDNA sequences, thereby help future research on a better UASB–MBR system throughout the operation period. understanding of biotreatment of target contaminants. The utiliza- Inoculums for the starting up of UASB were obtained from the tion of 16S rDNA clone library method may also be able to identify wastewater treatment facilities (hydrolysis/acidification tank) of some new and valuable bacterial resources, therefore further a chemical synthetic pharmacy company located at Shenyang, 54 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62

Biogas Vacuum gauge AGMBR AGMBR Pump influent

Membrane module Aeration UASB effluent Flowmeter AGMBR

Air diffuser effluent UASB Vacuum gauge UASB Influent MBMBR MBMBR Pump

influent Carriers Flowmeter Regulating Membrane module MBMBR Feed reservoir Flowmeter efflent tank

Pump Efflent Air diffuser reservoir Pump Pump Sludge

Fig. 1. Schematic structure of UASB–MBR system.

Table 1 Operation conditions of the hybrid UASB–MBR system.

Stage 1 2 3 4 5 6 7 Time (day) 1–17 18–47 48–72 73–81 82–97 98–111 112–118 HRT (h) UASB 24 MBRs 24 Mean berberine loading rates (g/m3/d) UASB 72.1 99.5 118.7 187.2 260.2 375.1 66.4 MBRs 0.7 1.2 1.9 7.3 12.4 82.5 26.8 Mean COD loading rates (kg/m3/d) UASB 1.97 3.55 3.32 3.20 3.51 3.26 2.30 MBRs 0.52 0.84 1.08 1.45 1.85 2.34 2.00

þ 3 Mean NH4 –N loading rates (g/m /d) UASB 98.9 133.6 116.0 115.9 122.6 118.7 110.7 MBRs 107.4 138.5 122.3 123.8 130.2 118.8 107.5

China. An aerobic activated sludge, collected from the aeration Agilent HB-C8 column (150 4.6 mm, 5 lm) maintained at 30 °C tank of the same wastewater treatment facilities, was used as inoc- temperature. Signals were detected with UV detector at 345 nm, ulum for the MBRs. The initial concentrations of the inoculum were and the LOQ is 0.05 mg/L. approximately 9560 mg MLSS/L in the UASB, 3570 and 3530 mg MLSS/L in AGMBR and MBMBR, respectively. Other operation con- 2.3. Sampling and DNA extraction ditions of the combined UASB–MBR system were summarized in Table 1. The biomass samples taken after 90 days of system operation The UASB–MBR system was fed with synthetic wastewater con- were used to build the clone libraries. In MBMBR, floc sludge and sisting of 2000.0–3000.0 mg/L glucose, 75.0–375.0 mg/L berberine, biofilm biomass were sampled separately, the biofilm was re- 535.0 mg/L NH4Cl, 104.6 mg/L KH2PO4, 71.0 mg/L MgSO4 7H2O, moved from the carriers by using sonication and suspended in 19.3 mg/L CaCl2 2H2O, 17.4 mg/L FeSO4 7H2O, 0.07 mg/L CuCl2 sterile water. Then all the samples were centrifuged for 5 min at 2H2O, 0.13 mg/L MnCl2 4H2O, 0.13 mg/L ZnSO4 7H2O, 0.03 mg/L 1000g, and the supernatant was decanted and the pellet was resus- Na2MoO4 2H2O, 0.025 mg/L H3BO3 and 0.033 mg/L KI. The opera- pended in Tris–EDTA buffer (10.0 mM Tris–HCl, 1.0 mM EDTA, pH tion of the system was divided into 7 stages according to different 8.0). All samples were immediately frozen and stored at 80 °C proportions of glucose and berberine added as carbon sources, as after resuspention until DNA extraction. shown in Table 1. DNA was extracted from the samples with a QIAamp DNA Mini Kit (Qiagen, Valencia, CA, USA) as described in the manufacturer’s 2.2. Analyses of wastewater quality instructions. To minimize variations in DNA extraction, templates used for PCR amplification were prepared by mixing the DNA ex- The water sample was collected daily form the reactor. The COD tracted in triplicate for each sample. þ and NH4 –N were analyzed following standard methods. Berberine was determined using high performance liquid chromatography 2.4. PCR amplification and 16S rDNA cloning with autosampler (Agilent 1100, Santa Clara, CA, USA). Acetoni- trile/0.05 mol/L KH3PO4 (30/70, v/v) was chose as mobile phase. The 16S rDNA genes were amplified from the DNA extracts The flow-rate was 1.0 ml/min. After filtered by 0.4 lm polytetraflu- using universal primers 27F and 1492R (Duan et al., 2009). PCR oroethene microfiltration membrane, 20 ll of the wastewater sam- amplifications were carried out in a total volume of 50 llin ple was inject automatically. Analytes were separated using 200 ll tubes using a DNA thermocycler (Bio-Rad, Richmond, CA, G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 55

þ USA). The PCR mixture contained 1.25 U of Taq polymerase (Pro- fluctuation of COD and NH4 –N in the inflow wastewater. Table 2 mega, Madison, WI, USA), 1 PCR buffer, 2 mM MgCl2, 0.5 lmol summarizes the treatment performance of the system during the of each primer, each deoxynucleoside triphosphate at a concentra- biomass sampling campaign (days 82–97). A major proportion of tion of 200 lM, and 40 ng of template DNA. The temperature berberine (95.2 ± 0.6%) and about half of the total COD þ cycling conditions were as follows: pre-incubation at 95 °C for (47.3 ± 3.0%) removals were achieved in UASB, whereas, NH4 –N þ 2 min, 25 cycles of 95 °C for 1 min, 62 °C for 1.5 min, and 72 °C was mainly removed by MBRs. It is also noted that, the NH4 –N for 1 min, finally 72 °C for 10 min. contents increased slightly after UASB treatment, which suggested The PCR products were purified with the Qiaquick PCR cleanup the transformation of organic nitrogen in berberine molecule to þ kit (Qiagen, Valencia, CA, USA) and ligated into a PCR 2.1-TOPO NH4 –N. In general, the treatment performance of AGMBR and vector and transformed into TOP 10 E. coli competent cells follow- MBMBR was comparable. Both reactor achieved COD, berberine þ ing the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). and NH4 –N removals of about 97.5%, 93% and 98%, leaving an efflu- þ Ampicillin and x-gal were used to screen for colonies with plas- ent COD, berberine and NH4 –N less than 50.0 mg/L, 1.0 mg/L and mids. For each sample, approximately 100 clones were picked 3.0 mg/L, respectively. These results suggested that, the hybrid out randomly for analysis. Positive clones were identified by PCR UASB–MBR system was efficient in berberine reduction as well as þ amplification with the primer pairs M13 using the same program COD and NH4 –N removal. Moreover, these results also suggested as for 16S rDNA amplification. All the positive clones were that a functional stable bacterial community for berberine degra- subjected to sequencing analysis with an ABI 3730 automated dation have already established. So as to better understand the sequencer (Invitrogen, Carlsbad, CA, USA). bacterial community composition and to identify the key func- tional bacterial species/groups in the berberine biodegradation 2.5. Phylogenetic and statistical analysis process, the biomass samples in UASB and both AGMBR and MBMBR reactors were subjected to 16S rDNA clone library The sequences sharing 97% or great similarity were grouped analysis. into the same operational taxonomic units (OTUs). Representative sequence of each OTU was compared with similar sequences of the 3.2. Bacterial community structure reference microorganisms by performing a BLAST search in the NCBI database. The most similar reference sequence was retrieved Four clone libraries were individually constructed for UASB and aligned with clone sequence using ClustalX. Phylogenetic trees anaerobic sludge, AGMBR aerobic granular sludge, MBMBR floc were constructed by the neighbor-joining method with the MEGA, sludge and MBMBR biofilm. Totally 400 sequences were success- version 5.0 software. UniFrac computational analysis was per- fully retrieved and grouped into 186 OTUs, which were expected formed to compare 4 clone libraries of the UASB–MBR system. to representing most of the berberine antibiotic resistant and ber- Then 4 clone libraries were clustered by the application of the UP- berine degradation functional bacteria in the hybrid UASB–MBR GMA method to the UniFrac metric matrix to compare the differ- system. Fig. 2 shows the taxonomic breakdown at the bacterial ence among community structures. phyla level for the four clone libraries. Shannon–Wiener diversity index (H0), Equitability index (E) and For the UASB, 104 clones were retrieved and grouped into 52 Richness index (R) were calculated to evaluate the structural diver- OTUs. As shown in Fig. 2a, 104 clones and 52 OTUs fell into 12 phy- sity of the bacterial communities by the following equations la, Firmicutes (34.6% both clones and OTUs) and Bacteroidetes (Gafan et al., 2005): X (28.8% clones and 17.3% OTUs) were dominant in the UASB, fol- 0 H ¼ pi lnpi ð1Þ lowed by Chloroflexi (9.6% clones and 15.4% OTUs). The next fre- quently detected phyla were Actinobacteria, Synergistetes, E ¼ H0=ðlognÞð2Þ Spirochaetes, each accounting for 5.7% of total clones and OTUs. Some minor phyla and candidate divisions such as Chlorobi,TM7, R ¼ðn 1Þ=lnN ð3Þ OP10 and OD1 were also found in the UASB community. Proteobac- teria was obviously less favored in the UASB. The phylum Firmi- where pi is the ratio of i-th OTU to total community, n is the total cutes, all of which were Gram-positive low GC bacteria, were number of OTUs in the sample, and N is the total number of clones. generally found to be major bacterial constituents in other anaer- obic bioreactors (Narihiro et al., 2009). 3. Results and discussion Fig. 2b presents the community structure of the AGMBR com- munity. Sequencing analysis of 94 clones from the AGMBR com- 3.1. Treatment performance munity indicated 8 distinct phyla. The relative abundance of Proteobacteria, especially (31.9% clones and The hybrid UASB–MBR system was operated for 118 days, 25.0% OTUs) and (41.5% clones and 33.3% OTUs) which was divided into 7 stages mainly with respect to different were remarkably high. And the second dominant phylum was Bac- influent berberine concentrations (72.1 ± 11.3 to 375.1 ± 10.5 mg/ teroidetes, which occupied about 16.0% clones and 22.2% OTUs. The L) (Table 1). Generally, in each stage, high and stable overall re- rest phyla all accounted for less than 5.0% of the whole community, þ moval rates of berberine, COD and NH4 –N were achieved, regard- including (4.3%), Deltaproteobacteria (2.1%), less of the elevation of the berberine concentration and the Nitrospirae (1.1%), Actinobacteria (1.1%) and Chlorobi (1.1%).

Table 2 Average influent and effluent pollutant contents and removal efficiencies (mean ± SD) in the hybrid UASB–MBR system during the sampling campaign.

Influent UASB effluent AGMBR effluent MBMBR effluent UASB removal AGMBR removal MBMBR removal Overall removal (mg/L) (mg/L) (mg/L) (mg/L) rate (%) rate (%) rate (%) rate (%) Berberine 260.2 ± 11.9 12.4 ± 1.7 0.90 ± 0.10 0.86 ± 0.22 95.2 ± 0.6 92.6 ± 1.5 93.0 ± 1.9 99.7 ± 0.1 COD 3509 ± 125 1845 ± 65 45.4 ± 7.9 46.2 ± 9.1 47.3 ± 3.0 97.5 ± 0.5 97.5 ± 0.5 98.7 ± 0.2 þ NH4 –N 122.6 ± 12.0 130.2 ± 9.9 1.80 ± 0.40 2.9 ± 1.4 -6.6 ± 7.2 98.6 ± 0.4 97.8 ± 0.9 98.1 ± 0.7

SD = standard deviation obtained from daily analysis. 56 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62

40.0% Table 3 Bacterial community richness (R), equitability (E), simpson domination (D) and Shannon–Wiener’s diversity (H0) indexes of the UASB and MBR communities. 30.0% Clones OTUs H0 ER D

20.0% UASB 3.69 0.80 10.98 0.033 AGMBR 3.17 0.70 7.70 0.063

Proportion , Proportion % MBMBR biofilm 3.35 0.72 9.06 0.049 10.0% MBMBR Floc sludge 3.57 0.78 11.75 0.030

0.0% Nitrospirae was also identified in the AGMBR community, which þ were responsible for the NH4 –N removal in the reactor. Xia and co-workers suggested that Betaproteobacteria and Gammaproteo- bacteria members played an important role in wastewater treatment that contained antibiotics (Xia et al., 2012). a Phyla As for the MBMBR (Fig. 2c and d), both biofilm sample and floc 50.0% sludge was subjected to the 16S rDNA clone library analysis. Sequencing analysis of 103 clones from MBMBR biofilm revealed 40.0% Clones OTUs a total of 43 OTUs belonging to 9 different phyla. Similar to the AGMBR, Proteobicteria was dominant in dominate the MBMBR 30.0% biofilm, in which Alphaproteobacteria and Betaproteobacteria accounted for 35.0% and 48.5% of total clones, and 44.2% and 20.0% 20.9% of total OTUs, respectively, followed by Gammaproteobacteria Proportion , Proportion % 10.0% (9.3% clones and 5.8% OTUs). However, it is distinct from the AGMBR that, Bacteroidetes was much rare, which only occupied 0.0% 3.9% of the community. Members belonging to Nitrospirae were also detected in the MBMBR biofilm. Other minor lineages detected include Firmicutes, Chlorobi, Acidobacteria and Actinobacteria, each accounting for 0.97% of the total community. While for MBMBR sludge, 99 positive clones were retrieved and grouped into 55 b Phyla OTUs, showing a higher diversity and equitability than the biofilm (Table 3). Moreover, the composition of MBMBR sludge community 50.0% also exhibited significant difference from the biofilm. Although Proteobicteria was still the most abundant group in the MBMBR 40.0% Clones OTUs sludge, the dominant status was less significant, Beta- and Gamma- proteobacteria were obviously less favored, while the proportion of 30.0% Alphaproteobacteria was comparable with that in the biofilm. Addi- 20.0% tionally, Firmicutes members were found to be the second most

Proportion , Proportion % abundant bacterial group which comprised of 28.3% of the total 10.0% clones and 21.8% of OTUs. The next frequently detected phylum was Bacteroidetes, accounting for 12.1% of the clones and 16.4% of 0.0% the OTUs. In particular, Actinobacteria was highly abundant, and and Candidate division TM7 members were also detected in the MBMBR sludge community, all of which did not appear in the biofilm. The similarities of OTUs among 4 libraries were compared by c Phyla using UniFrac computational analysis. Totally, 8 OTUs were shared by the AGMBR and MBMBR biofilm libraries, 6 OTUs were shared 40.0% by the AGMBR and MBMBR floc sludge libraries and 7 OTUs were Clones OTUs shared by the MBMBR biofilm and floc sludge libraries. Four OTUs 30.0% were furthermore shared across 3 aerobic libraries. And no OTU was found to be shared among UASB library and the three MBR 20.0% libraries, suggesting the obvious difference between the anaerobic community and the aerobic community. However, cluster analysis Proportion , Proportion % 10.0% (Fig. 3) showed that the MBMBR floc sludge community was closer to the UASB community than to these of the AGMBR and MBMBR 0.0% biofilm, indicating a high difference between floc sludge and the biofilm communities even in the same reactor. However, since aer- obic granular sludge was a kind of self-immobilized biofilm (Ren et al., 2013), it was reasonable that the bacterial compositions of AGMBR and MBMBR biofilm were similar. The MBMBR floc sludge d Phyla community and UASB community were clustered together because that Firmicutes all dominated in two communities. However, no Fig. 2. Percentage of the bacterial groups in (a) UASB, (b) AGMBR, (c) MBMBR more confirmative relations could be drawn from two bacterial biofilm and (d) MBMBR sludge. groups, since the Firmicutes membranes in the two communities G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 57

aromatic compounds biodegradation (Gorny and Schink, 1994). Additionally, some other species was also reported to be able to completely transform a series of nitrogen containing analogues of quinoline and isoquinoline (Johansená et al., 1997). Since berberine was a pyridine ring containing isoquinoline alkaloids, it is expected that OTU 13 and OTU 23 were related to the cleavage of pyridine ring and/or aromatic ring in berberine molecule and in its Fig. 3. Jackknife cluster analysis of four bacterial clone libraries of hybrid UASB– metabolites. MBR system. The scale bar shows the distance between clusters in UniFrac units: a distance of 0 means that two environments are identical; and a distance of 1 means Since most of UASB OTUs were affiliated with uncultured bacte- that two environments contain mutually exclusive lineages. ria, and many OTUs showed <97% sequence similarity with the clo- sely related bacterial 16S rDNA gene sequences already known in were further classified into different genera: Clostridium, Rumino- the GenBank, which implies that a large population of the UASB coccus, Acidaminococcus, Eubacterium and Erysipelotrichaceae for community are novel species, and some new bacterial resources UASB Firmicutes members, while Catenibacterium, Lactococcus and may embedded in them. Selenomonas for MBMBR floc sludge. As for the MBR communities, the most abundant species in AGMBR community, OTU 12 (14 clones, 14.9% of TPo), was closely 3.3. Potential functional species related to Hydrogenophaga bisanensis (NR044268.1), which was isolated from textile wastewater (Yoon et al., 2008). Additionally, In the UASB library, only 6 out of 52 total OTUs were related to the three most abundant species in the MBMBR biofilm commu- cultured species (Fig. 4). The most abundant species in the UASB nity: OTU 1 (11 clones, 10.7% of TPo, related to uncultured Hydrog- community were OTU 52 and OTU 44. Both of them have 8 clones, enophaga sp. clone W5S29 (GU560177.1) identified from biofilms accounting for 7.7% of the total population (TPo), and were closely for pharmaceutical wastewater treatment), OTU 5 (9 clones, 8.8% related to two uncultured bacteria: SHA-5 (AJ306736.1) obtained of TPo, related to Hydrogenophaga atypica (NR029023.1) isolated from an anaerobic dechlorinating bioreactor and 054A03 B DI from activated sludge) and OTU 4 (8 clones, 7.9% of TPo, related P58 (CR933243.1) from an anaerobic sludge digester, respectively. to Hydrogenophaga sp. GPTSA100-30 (DQ854974.1)) were all affil- Both of them are clustered into the Bacteroidetes phylum. The third iated to Hydrogenophaga genus of Betaproteobacteria. Furthermore, most abundant population was OTU 28 (7 clones, 6.7% of TPo), Hydrogenophaga species (OTU 7, 2 clones, 2.1% of TPo, also related which is closely related to another uncultured bacterium clone to Hydrogenophaga sp. GPTSA100-30 (DQ854974.1)) were also 24h04 (EF515475.1), identified from a microbial fuel cell, belong- identified in the MBMBR floc sludge library. Since many species ing to Erysipelotrichi class of Firmicutes. The roles of these three of Hydrogenophaga were reported to code the genes for a protoca- top species were still unclear. The relative abundance of the rest techuate 3,4-dioxygenase, a key enzyme in aromatic ring ortho- OTUs were all less than 3.8%. cleavage (Contzen et al., 2001), it is expected that Hydrogenophaga As for non-dominant species, OTU 8 (1 clone, 0.9% of TPo) is clo- were potential functional species in cleavage of aromatic rings in sely related to Clostridium sardiniense (AB161368.1), a phospholi- berberine molecules and/or the degradation of its aromatic pase C producing strain of genus Clostridium. Some Clostridium metabolites. strain was reported to be able to utilize the methyl group of aro- The second abundant species in AGMBR, OTU 1 (11 clones, matic methyl ethers as a carbon source via O-demethylation reac- 11.7% of TPo), was closely related to Azonexus hydrophilus tion (Kasmi et al., 1994). Some other stain was also reported to be IMCC1716 (DQ664239.1), a nifH gene-harboring bacterium iso- capable of cleaving aromatic rings (Winter et al., 1991). Therefore, lated from freshwater, which belongs to genus Azonexus within OTU 8 was probably a functional species in the cleavage of aro- the Betaproteobacteria. Azonexus, which formerly included in genus matic ring and/or the degradation of methoxyl groups in berberine Azoarcus, are very similar in physiology and ecology to Azoarcus molecule as well as in its metabolites. OTU 10 (2 clones, 1.9% of species. Studies recognized that, many strains of Azoarcus contrib- TPo) was closely related to two Eubacterium sp. (AM884910.1 ute significantly to the biodegradation of aromatic and other and DQ337532.1), which was clustered into the Eubacterium genus, refractory compounds via a meta-cleavage pathway of the aromatic belonging to the same class, Clostridia, as OTU8. Studies showed rings (Rabus et al., 2005). Additionally, Azoarcus species (MBMBR that, some Eubacterium can demethoxylate O-methoxylated aro- biofilm OTU 6, 2 clones, 1.9% of TPo, related to Azoarcus sp. mXyN1 matic acids to produce mixed volatile fatty acids (Mountfort (X83533.1), a new aromatic compounds degrader (Rabus and Wid- et al., 1988). Additionally, some Eubacterium was also reported to del, 1995)) was also identified in the MBMBR biofilm library. It is be able to metabolize various oxygen heterocyclic aromatic com- expected that Azonexus and Azoarcus were also functional species pounds, e.g. Eubacterium callanderi which can cleave the oxygen in the degradation of berberine and its anaerobic metabolites. heterocyclic ring and realize p-dehydroxylation in the aromatic Sphingomonas, a genus within Alphaproteobacteria, was identi- ring (Wang et al., 2001). Since two methoxyl groups and an oxygen fied in all three MBR communities: AGMBR OTU 24 (5 clones, heterocyclic ring were contained in the berberine molecule. OTU 5.3% of TPo), MBMBR biofilm OTU 18 (2 clones, 1.9% of TPo) and 10 was probably involved in the demethoxylation and/or oxygen MBMBR sludge OTU 15 (1 clone, 1.0% of TPo) were all related to heterocyclic ring cleavage of berberine molecules as well as its Sphingomonas sp. IJ1 (HQ260905.1), a carbofuran-degrading bacte- metabolites. rium. Additionally, AGMBR OTU 21 was found to be closely related For OTU 13 (2 clone, 1.9% of TPo), the closest relative of which is to Sphingomonas sp. IC075 (AB196249.1), which carried the carba- uncultured Synergistes sp. (JF736632.1), belonging to the obligate zole-catabolic car genes encoding carbazole 1,9a-dioxygenase, anaerobic bacteria genus of Synergistes. Previously, 4 strains of Syn- meta-cleavage enzyme, and meta-cleavage compound hydrolase ergistes were identified to be able to degrade a class of pyridine and altogether realized the cleavage of nitrogen heterocyclic ring derivatives via pyridine ring reduction enzymatically (Rincon and the aromatic rings (Inoue et al., 2005). As a group, Sphingomon- et al., 1998). While for OTU 23 (2 clone, 1.9% of TPo), it was closely as have broad catabolic capabilities and therefore high potential for related to Desulfovibrio marrakechensis (NR042704.1), a mesophilic bioremediation and waste treatment. The range of contaminants 1,4-tyrosol degrading bacterium isolated from olive mill wastewa- that various Sphingomonas can degrade is extensive and includes ter. Actually, many sulfate-reducing bacteria were recognized as a class of nitrogen heterocyclic, oxygen heterocyclic and aromatic functional species in anaerobic aromatic ring cleavage and compounds (Fredrickson et al., 1995). Some carbazole-degrading 58 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62

OTU 1 (23 67 68 79) Uncultured Firmicutes bacterium QEDN5DE07 (CU927131.1) OTU 2 (43) Uncultured bacterium clone RRH_aaa01h03(EU474867.1) OTU 3 (5 20) Uncultured bacterium clone WD11_aak21b02(EU509791.1) OTU 4 (110) Uncultured bacterium clone B21.29(GU559825.1) OTU 7 (64 71) Uncultured bacterium clone RL246_aai74h06 (DQ793639.1) OTU 5 (6 75) Uncultured bacterium clone BARB_aaa01d10(EU475641.1) Eubacterium sp. ADS17 (AM884910.1) Eubacterium sp. BBDP67(DQ337532.1) OTU 10 (93 103) OTU 6 (8) Uncultured Pelospora sp. clone De3154 (HQ183798.1) OTU 9 (95) Uncultured bacterium clone 1103200824977(EU842505.1) OTU 8 (81) Uncultured bacterium clone WA_aaa03c07(EU473501.1) Clostridium sardiniense strain DSM 2632(AB161368.1) OTU 14 (25) Anaeromusa acidaminophila strain DSM 3853 (NR_024921.1) Uncultured Veillonellaceae bacterium clone MFC-B162-A03(FJ393062.1) OTU 15 (89) OTU 17 (72) Uncultured Unclassified bacterium clone QEDV2BD01(CU919999.1) OTU 16 (18) Uncultured bacterium clone 054B07_B_DI_P58(CR933236.1) OTU 18 (74 77 88 92) Uncultured bacterium clone MRA2011(FN428757.1) OTU 26 (50 82 101) Uncultured bacterium clone 23a07(EF515334.1) OTU 28 (12 17 33 36 40 51 58) Uncultured bacterium clone 24h04 (EF515475.1) OTU 27 (91) OTU 13 (49 108) Uncultured Synergistetes bacterium clone QEDN10AF01(CU926983.1) OTU 12 (97) Uncultured Aminanaerobia bacterium clone QEDT3AB07(CU920428.1) OTU 11 (26 53 55) Uncultured low G+C Gram-positive bacterium clone PD-UASB-13(AY261810.1) OTU 42 (21 85 99) Uncultured Olsenella sp. clone J27 (DQ168843.1) OTU 43 (105) Uncultured bacterium clone gir_aah95c09 (EU775234.1) OTU 41 (15 28) Uncultured bacterium clone R1Cb30 (EF063633.1) OTU 29 (9) Uncultured OP10 bacterium clone QEEA3DA03(CU918792.1) OTU 30 (27) Uncultured bacterium clone 655076(DQ404736.1) OTU 31 (61) Uncultured bacterium (AB195911.1) OTU 32 (3) Uncultured bacterium clone BacIV_clone38 (FN870312.1) OTU 33 (66) OTU 34 (69) Uncultured Chloroflexi bacterium clone QEDN4BA01(CU925970.1) OTU 35 (22) uncultured bacterium SHD-245(AJ278174.1) OTU 36 (84) Leptolinea tardivitalis (AB109438.1) OTU 37 (70) Longilinea arvoryzae (AB243673.1) OTU 38 (19) uncultured bacterium C1-28 (AJ387901.1) OTU 39 (29 57) Uncultured Chloroflexi bacterium clone QEDN1DF06 (CU925778.1) OTU 40 (56 65) uncultured bacterium SJA-131(AJ009492.1) Desulfovibrio marrakechensis strain DSM 19337 (NR_042704.1) Desulfovibrio sp. S14 PV-2008(AM946979.1) OTU 23 (4) OTU 24 (86) OTU 25 (96 100) Uncultured bacterium clone C55(EU234257.1) OTU 19 (7 16 62 109) Uncultured bacterium clone ambient_uncontrolled-45(GU454906.1) OTU 20 (107) Uncultured bacterium clone 613_I10_PCE_column_outflow(FM178812.1) OTU 21 (87) Bacterium enrichment culture clone GD-A-1(HQ122957.1) OTU 22 (38 39) Uncultured bacterium clone TSSUR001_A14 (AB487884.1) OTU 44 (2 13 24 31 46 54 73 78) Uncultured bacterium clone SHA-5 (AJ306736.1) OTU 45 (11) OTU 46 (59) Uncultured bacterium clone ambient_uncontrolled-118(GU454979.1) OTU 47 (32 42) Uncultured bacterium clone EUB57(AY693825.1) OTU 48 (35 41 44 106) OTU 49 (1 30 80) Uncultured bacterium clone T2WK15D90 (HQ716474.1) Uncultured bacterium clone VWP_aaa02h04(EU475061.1) OTU 51 (47 48) Uncultured bacterium clone BT5_15 (GQ458220.1) OTU 50 (63) Uncultured bacterium clone DC53 (HM107047.1) OTU 52 (14 37 45 52 63 90 94 98 102) Uncultured bacterium clone 054A03_B_DI_P58(CR933243.1) a 0.02

Fig. 4. Phylogenetic relationships of bacterial 16S rDNA gene sequences in the (a) UASB, (b) AGMBR, (c) MBMBR biofilm and (d) MBMBR floc sludge libraries. G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 59

OTU 5 (32 110) OTU 8 (70 86) OTU 4 (83) OTU 1(1 5 31 34 44 46 55 57 69 85 94) Azonexus hydrophilus IMCC1716(DQ664239.1)-AGMBR OTU 2 (82) OTU 3 (12 90) OTU 6 (98) Uncultured Rhodocyclus sp. clone R15-23 (JF808915.1) OTU 7 (88) Methyloversatilis universalis (AY436796.2) OTU 9 (73) Uncultured bacterium clone ambient_alkaline-94 (GU455077.1) OTU 10 (68) Variovorax sp. S24561 (D84645.2) OTU 11 (80 81) Uncultured Hydrogenophaga sp. clone W5S29 (GU560177.1) OTU 12 (19 48 107 10 71 4 6 1 62 72 11 13 76 99 100) Hydrogenophaga bisanensis strain K102 (NR_044268.1) OTU 16 (104) Pseudoxanthomonas sp. R-24339 (AM231052.1) OTU 17 (51 52 54) Stenotrophomonas sp. Toyama-1 (AB180662.1) OTU 21 (18) OTU 22 (6 43) Uncultured sludge bacterium A3 (AF234730.1) Uncultured Acetobacteraceae bacterium clone AMNE3 (AM934744.1) Sphingomonas sp. IJ1 (HQ260905.1) OTU 29 (7 64 78 101 105) OTU 27 (89) OTU 26 (40 87) Sphingosinicella sp. OC5S (AB429069.1) OTU 18 (3 26 27 30 35 38 84 96 102 103) Uncultured bacterium clone HP1B78 (AF502220.1) OTU 20 (24 50) OTU 25 (49 93) Nordella sp. P-63 (AM411927.1) OTU 23 (2 17 29) Oligotropha carboxidovorans S28 (AB099660.1) OTU 24 (97 56 106) Uncultured Alphaproteobacteria bacterium clone QEDN4DF03 (CU927408.1) OTU 32 (53 59) Uncultured Nannocystaceae bacterium clone X369 (JF815509.1) OTU 30 (9) Uncultured Olsenella sp. clone J27 (DQ168843.1) OTU 31 (66) Candidatus Nitrospira defluvii (DQ059545.1) OTU 33 (67) Unidentified Cytophagales/green sulfur bacterium clone MFC-EB28 (AJ630296.1) OTU 34 (95) Uncultured Bacteroidetes bacterium clone OTU10 (JN802702.1) OTU 35 (92) Chryseobacterium sp. CHNTR56 (DQ337588.1) OTU 36 (74) Flavobacterium sp. I-111-12 (FJ786049.1) Flavobacterium sp. PR6-5 (FJ889628.1) OTU 37 (22 37 65) OTU 38 (21 63 91) Uncultured Flavobacterium sp. clone 2g11 (JF979360.1) OTU 39 (8) Uncultured bacterium clone M0111_08 (EU104012.1) OTU 40 (23) OTU 41 (20 58 60 79) Flavobacterium sp. 3A5 (AF368756.1) Uncultured Flavobacterium sp. clone GS15 (JF736634.1) b 0.02

Fig. 4. (continued) 60 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62

OTU 18 (3 30) Sphingomonas sp. IJ1(HQ260905.1) OTU 21 (7 54) Sphingomonas sp. D12 (AB105809.1) OTU 20 (14 68 81) Sphingosinicella microcystinivorans strain MDB3 (AB219941.1) OTU 19 (95) Uncultured bacterium DSSF5 (AY328628.1) OTU 23 (9) Uncultured Sphingomonadales bacterium clone CMJC5 (AM935865.1) OTU 13 (52) Uncultured alphaproteobacterium clone delph2G4 (FM209141.1) OTU 12 (16) Alphaproteobacterium BAC233 (EU180520.1) OTU 15 (44 57 93) Uncultured Stellasp. clone AMLG5 (AM934982.1) OTU 14 (92) Uncultured Rhodospirillales bacterium clone blastula_6 (HQ111145.1) OTU 16 (66 90) Uncultured bacterium clone F57 (FJ230929.1) OTU 17 (79) OTU 22 (107) Brevundimonas aveniformis strain EMB102 (NR_043770.1) Uncultured Phenylobacterium sp. clone 5.17h2 (JN679157.1) OTU 26 (102) OTU 11 (100) OTU 24 (10) OTU 25 (105) Aminobacter sp. Sokolova (FJ907162.1) OTU 9 (84) Shinella sp. HZN1 (HM535627.1) OTU 28 (65 73 88 89 96 113 35 55 109) OTU 27 (22 29) Agrobacterium sp. HY-35 (EU580738.1) Uncultured Dokdonella sp. clone S70 (JN217047.1) OTU 32 (58 46) Pseudoxanthomonas sp. P2-3(EU276093.1) OTU 10 (12) Uncultured bacterium clone A62 (FJ660561.1) OTU 31 (72) OTU 30 (41 97) OTU 29 (37 64) Uncultured bacterium clone B175 (HQ640630.1) Azoarcus sp. mXyN1 (X83533.1) Uncultured bacterium clone ambient_uncontrolled-24 (GU454885.1) OTU 6 (87 98) OTU 8 (21 51 50 61 99 104) Uncultured bacterium clone ZBAF3-24(HQ682050.1) OTU 7 (78) Sterolibacterium sp. TKU1 (AM990454.1) OTU 2 (114 76 4 69 94) OTU 3 (20 47 70 71 85 86) Acidovorax sp. OS-6(AB076844.1) Uncultured bacterium clone BF3-8 (HM584338.1) OTU 1 (1 2 60 53 110 17 23 63 67 19 62) Uncultured Hydrogenophaga sp. clone W5S29(GU560177.1) OTU 4 (40 80 39 34 56 13) Hydrogenophaga atypica strain BSB 41.8(NR_029023.1) OTU 5 (27 103 24 42 45 82 49 48) Hydrogenophaga sp. GPTSA100-30(DQ854974.1) OTU 33 (101) Uncultured bacterium clone 31c11 (EF515637.1) OTU 39 (31) Agrococcus sp. DNPRC1034 (DQ232611.2) OTU 35 (33) Uncultured bacterium clone FFCH13501 (EU132213.1) OTU 36 (43) OTU 37 (6) Nitrospirasp. clone b30 (AJ224041.1) Candidatus Nitrospira defluvii (DQ059545.1) OTU 38 (5) OTU 34 (108) Unidentified Cytophagales/green sulfur bacterium OPB56(AJ630296.1) OTU 40 (18) Uncultured bacterium clone BF4-3(HM584346.1) Uncultured bacterium clone A19(FJ660546.1) OTU 41 (112) OTU 42 (75) Uncultured bacterium clone CNTE12 (HQ728224.1) OTU 43 (91) Uncultured bacterium clone M0509_10(EU104097.1)

c 0.02

Fig. 4. (continued) G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 61

OTU 30 (51 29 82 7 66) key enzymes for the aerobic degradation of aromatic compounds. Uncultured bacterium clone L57 (EU834772.1) Sinorhizobium sp. J1 (DQ294628.1) Some of these multicomponent enzyme systems carry out a spe- OTU 28 (56) OTU 29 (69) cific regioselective angular dioxygenation, which is necessary for Uncultured Mesorhizobium sp. clone AMKD6 (AM935031.1) OTU 49 (42 71) the mineralization of a class of oxygen and nitrogen heterocyclic Sphingomonas sp. IJ1(HQ260905.1) aromatic compounds (Pieper et al., 2004). Studies also suggest that OTU 19 (17) OTU 24 (96) some Sphingomonas strains could degrade oxygen heterocyclic aro- Uncultured bacterium clone L65(EU834774.1) OTU 25 (68 79) matic compounds by hydrolysis at the oxygen heterocyclic ring Uncultured Hyphomicrobium sp. clone ENR15 (FJ536922.1) OTU 20 (2 102) (Seonkim et al., 2004). Some strain was also reported to be capable Caulobacter sp. C6a1 (AB552896.1) OTU 21 (86) of mineralizing both the oxygen heterocyclic group and the aro- Uncultured Phenylobacterium sp. clone X-2 (HQ132473.1) matic ring of a heterocyclic compound, carbofuran (Feng et al., OTU 22 (52) Uncultured bacterium clone 92-ORF10 (DQ376579.1) 1997). Consequently, Sphingomonas were expected to be functional OTU 23 (61) Uncultured bacterium clone M3B31 (FJ439862.1) species in the cleavage of nitrogen heterocyclic, oxygen heterocy- OTU 26 (50 64) Uncultured bacterium clone C24.47 (GU559802.1) clic and/or aromatic rings in berberine molecule, as well as in the OTU 27 (78) Uncultured bacterium clone June05-pIV-H11 (HQ592567.1) degradation of its nitrogen heterocyclic, oxygen heterocyclic and/ OTU 39 (22 100) or aromatic intermediates. Uncultured alphaproteobacterium clone lhap15 (DQ648974.1) OTU 15 (34) Additionally, some species only identified in single library such OTU 17 (65) Uncultured bacterium clone 1013-28-CG3 (AY532560.1) as AGMBR OTU 14 (3 clone, 3.2% of TPo) was found to be closely OTU 16 (108) OTU 14 (33) related to Stenotrophomonas sp. Toyama-1 (AB180662.1), a dib- Drinkingwater bacterium MB12 (AY328843.1) OTU 9 (4 97) enzoflan-degrading species within the Gammaproteobacteria. Some yabuuchiae strain: OA1-4 (AB233212.1) Stenotrophomonas strain was reported to be capable of inducing OTU 10 (35) Uncultured Gammaproteobacteria clone QEDN4BE08 (CU927839.1) and synthesizing various dioxygenases (protocatechuate 3,4-diox- OTU 13 (37 58 32 90 98) Uncultured bacterium clone A62 (FJ660539.1) ygenase and catechol 2,3-dioxygenase) depending on different aro- Dokdonella sp. LM 2-5 (FJ455531.1) Bacterium enrichmentculture clone heteroA75_4W (GU731285.1) matic substrate, which involved in either intradiol cleavage or OTU 11 (24) OTU 18 (109) extradiol cleavage of the aromatic ring (Wojcieszynska et al., OTU 3 (21 31) 2011). MBMBR biofilm OTU 28 (9 clones, 8.8% of TPo) was closely Alcaligenes sp. YcX-20 (AY628412.1) Uncultured bacterium clone OD-14 (HM584301.1) related to Shinella sp. HZN1 (HM535627.1), which belonging to Shi- OTU 2 (70 84) OTU 5 (6 53) nella genus of Alphaproteobacteria. A new strain of Shinella was re- Methylibium petroleiphilum PM1 (AF176594.1) OTU 7 (23 15) cently isolated from the activated sludge of a coking wastewater Hydrogenophaga sp. GPTSA100-30(DQ854974.1) treatment plant, which could cleave the pyridine ring between C OTU 8 (105) Uncultured bacterium clone MACA-RR15 (GQ500750.1) and N and convert pyridine-N directly into ammonium (Bai et al., OTU 35 (11 27) Uncultured bacterium clone V201-88 (HQ114103.1) 2009). Alcaligenes was identified only in MBMBR sludge, OTU 2 (3 OTU 36 (62 76) Uncultured bacterium clone E51 (HQ827955.1) clones, 3.2%), related to Alcaligenes sp. YcX-20, a methyl parathione OTU 33 (9 74) Uncultured bacterium SHA-105 (AJ249113.1) degrading bacterium. Aromatic amine dehydrogenase has been OTU 34 (73) identified in some Alcaligenes species, which catalyze the oxidative Uncultured bacterium clone R3F (EU499455.1) OTU 32 (101 93 87) deamination of various aromatic amines (Sukumar et al., 2006). Uncultured Olsenella sp. clone J27 (DQ168843.1) OTU 40 (3 106 81 99 95 91 14 92 67) Additionally, some Alcaligenes species was also found to be able Uncultured bacterium clone 31c11 (EF515637.1) OTU 42 (72) to dissimilate aromatic compounds via both b-ketoadipate and Uncultured bacterium clone 23a07 (EF515334.1) OTU 41 (18) meta-cleavage pathways (Johnson and Stanier, 1971). Therefore, Uncultured bacterium clone tios26a(AM950252.1) these species were also expected to be responsible for the degrada- OTU 43 (39) Uncultured bacterium clone 42-9BM (HQ143303.1) tion of berberine and its metabolites via the degradation of differ- OTU 48 (8 28) Bacterium enrichment culture clone DPF04 (GQ377124.1) ent target groups (pyridine ring and aromatic ring) in their OTU 51 (38 10 94 12 54) Uncultured bacterium clone SP2-0 (GQ167189.1) molecules. Uncultured bacterium clone RL180_aao75g10 1(DQ808370.1) OTU 50 (48) Furthermore, Nitrospira were successfully identified in both OTU 45 (60) AGMBR and MBMBR, i.e. AGMBR OTU 26 (1 clone, 1.1% of TPo) Uncultured bacterium clone TC76 (EF644509.1) Uncultured bacterium clone MM3 (GQ871731.1) and MBMBR biofilm OTU 38 (1 clone, 1.0% of TPo), both of them OTU 44 (16) OTU 46 (19) were related to Candidatus Nitrospira defluvii (DQ059545.1) identi- Uncultured bacterium clone 3-4E11 (FJ677949.1) OTU 47 (30) fied from activated sludge. These species must be responsible for Uncultured bacterium clone p-3024-SwA5 (AF371827.1) þ OTU 37 (36) the good removal of NH4 -N in both MBRs. The occurrence of Uncultured TM7 bacterium clone QEDN2CH07 (CU925068.1) Nitrospira under high berberine environment implies that some OTU 38 (45) Arcobacter butzleri strain ED-1 (FJ968634.1) Nitrospira could acquire a certain degree of resistance via acclima- OTU 58 (80) Uncultured bacterium clone BF-50 (HQ609652.1) tion to the berberine exposure. Whereas, no species affiliated to Uncultured bacterium clone NBDTU19 (FJ529939.1) OTU 56 (77) Nitrospira was found in the MBMBR floc sludge, which might sug- OTU 59 (55 110) Uncultured Bacteroides sp. clone J3 (DQ168847.1) gest that the loose structure of planktonic floc sludge could not OTU 60 (89 107) provide sufficient protection of Nitrospira from the biotoxic envi- Bacteroides sp. W7 (FJ862827.1) Uncultured bacterium clone M0509_27(EU104113.1) ronment in comparison with aerobic granular sludge and biofilm OTU 61 (41) OTU 53 (63) which possess much compact structures. Uncultured bacterium clone D46 (GQ389176.1) OTU 55 (49) In general, the compositions of three MBR communities were Uncultured bacterium clone BF-48 (HQ609650.1) OTU 54 (103) significantly discrepant. However, analogous functional species OTU 52 (5 88) could be found in all the communities correlating to the degrada- Uncultured bacterium clone A19 (FJ660546.1) tion of diverse structure groups in the berberine molecule (aro- d 0.02 matic groups, oxygen heterocyclic groups, nitrogen heterocyclic groups and methoxyl groups). The analogous function of the com- Fig. 4. (continued) munities in the AGMBR and MBMBR ensures the identical pollu- tant removal performance of two MBRs. Sphingomonas strains were also observed harboring genes coding It still should be noted that, the dominant species in the UASB rieske-type non-heme iron oxygenases (Habe et al., 2002), the and in the MBMBR sludge community were all related to 62 G. Qiu et al. / Bioresource Technology 142 (2013) 52–62 uncultured species. The implications of these species in berberine Habe, H., Ashikawa, Y., Saiki, Y., Yoshida, T., Nojiri, H., Omori, T., 2002. Sphingomonas removal process still require further study. Additionally, Bacteroi- sp. strain KA1, carrying a carbazole dioxygenase gene homologue, degrades chlorinated dibenzo-p-dioxins in soil. FEMS Microbiol. Lett. 211 (1), 43–49. detes was found to be prevalent in all the clone libraries, but the Inoue, K., Habe, H., Yamane, H., Omori, T., Nojiri, H., 2005. 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Anaerobic degradation of catechol by Desulfobacterium Microbiol. 58 (2), 393–397. sp. strain Cat2 proceeds via carboxylationto protocatechuate. Appl. Environ. Microbiol. 60 (9), 3396–3400. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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