微生物英文文献及翻译—原文

m batch reactor Zhang Bin a,b, Chen Zhe a,b, Q Li Junwen a,b, Wang Xuan c,*, tal Med sment r Memb 0, PR tion. Accordingly, biodegradation and elimination of ammonia in wastewater are the primary functions of the NOB). Aerobic ammonia-oxidation is often the first, rate- limiting step of nitrification; however, it is essential for the * Corresponding author. ** Corresponding author. Institute of Hygiene and Environmental Medicine, Academy of Military Medical Sciences, Tianjin 300050, PR China. Tel.: +86 22 84655498; fax: +86 22 23328809. wang@hotmail.com (W. Jingfeng). Available online at www.sciencedirect.com .e ls wat e r r e s e a r c h x x x ( 2 0 1 1 ) 1e1 0 E-mail addresses: wangxuan0116@163.com (W. Xuan), jingfeng 1. Introduction At sufficiently high levels, ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica- wastewater treatment process. Nitrification, the conversion of ammonia to nitrate via nitrite, is an important way to remove ammonia nitrogen. It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and Keywords: Ammonia-oxidizing bacteria Granular sludge Community development Granule size Nitrifying bacteria distribution Phylogenetic diversity process conditions. Denaturing gradient gel electrophoresis (DGGE) and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settlingewashing out action. Furthermore, DGGE analysis revealed that larger granules benefit more AOB species surviving in the reactor. In the SBR were various size granules coexisted, granule diameter affected the distribution range of AOB and NOB. Small and medium granules (d< 0.6 mm) cannot restrict oxygen mass transfer in all spaces of the sludge. Larger granules (d> 0.9 mm) can result in smaller aerobic volume fraction and inhibition of NOB growth. All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems. ª 2011 Elsevier Ltd. All rights reserved. a Institute of Hygiene and Environmen bTianjin Key Laboratory of Risk Asses cTianjin Key Laboratory of Hollow Fibe Polytechnical University, Tianjin 30016 a r t i c l e i n f o Article history: Received 30 June 2011 Received in revised form 10 September 2011 Accepted 10 September 2011 Available online xxx Please cite this article in press as: Bin, Z., sludge granulation in an anaerobiceaero 0043-1354/$ e see front matter ª 2011 Elsev doi:10.1016/j.watres.2011.09.026 iu Zhigang a,b, Jin Min a,b, Chen Zhiqiang a,b, Chen Zhaoli a,b, Wang Jingfeng a,b,** icine, Academy of Military Medical Sciences, Tianjin 300050, PR China and Control for Environment and Food Safety, Tianjin 300050, PR China rane Material and Membrane Process, Institute of Biological and Chemical Engineering, Tianjin China a b s t r a c t The structure dynamic of ammonia-oxidizing bacteria (AOB) community and the distribution of AOB and nitrite-oxidizing bacteria (NOB) in granular sludge from an anaerobiceaerobic sequencing batch reactor (SBR) were investigated. A combination of process studies, molecular biotechniques and microscale techniques were employed to identify and characterize these organisms. The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge. Along with granules formation, the AOB diversity declined due to the selection pressure imposed by anaerobiceaerobic sequencing communities during sludge granulation in an Dynamic and distribution of a journal homepage: www et al., Dynamic and dis bic sequencing batch re ier Ltd. All rights reserved monia-oxidizing bacteria evier .com/locate/watres tribution of ammonia-oxidizing bacteria communities during actor, Water Research (2011), doi:10.1016/j.watres.2011.09.026 . four stainless steel sieves of 5 cm diameter having respective wat e r r e s e a r c h x x x ( 2 0 1 1 ) 1e1 02 removal of ammonia from the wastewater (Prosser and Nicol, 2008). Comparative analyses of 16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti- cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen, 2001). However, a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages, and that environmental factors such as process parameter, dis- solved oxygen, salinity, pH, and concentrations of free ammonia can impact certain species of AOB (Erguder et al., 2008; Kim et al., 2006; Koops and Pommerening-Ro¨ser, 2001; Kowalchuk and Stephen, 2001; Shi et al., 2010). Therefore, the physiological activity and abundance of AOB in waste- water processing is critical in the design and operation of waste treatment systems. For this reason, a better under- standing of the ecology and microbiology of AOB in waste- water treatment systems is necessary to enhance treatment performance. Recently, several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems (Li et al., 2008; Yin and Xu, 2009). Currently, the application of molec- ular biotechniques can provide clarification of the ammonia- oxidizing community in detail (Haseborg et al., 2010; Tawan et al., 2005; Vlaeminck et al., 2010). In recent years, the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy (de Bruin et al., 2004; Liu et al., 2009; Schwarzenbeck et al., 2005; Schwarzenbeck et al., 2004a,b; Xavier et al., 2007). Granules have a more tightly compact structure (Li et al., 2008; Liu and Tay, 2008; Wang et al., 2004) and rapid settling velocity (Kong et al., 2009; Lemaire et al., 2008). Therefore, granular sludge systems have a higher mixed liquid suspended sludge (MLSS) concentration and longer solid retention times (SRT) than conventional activated sludge systems. Longer SRT can provide enough time for the growth of organisms that require a long generation time (e.g., AOB). Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al., 2010; Tsuneda et al., 2003).Other researchers reported that larger granules have been developed with the synthetic organicwastewater insequencingbatchreactors (SBRs) (Lietal., 2008; Liu and Tay, 2008). The diverse populations of microor- ganisms that coexist in granules remove the chemical oxygen demand (COD), nitrogen and phosphate (de Kreuk et al., 2005). However, for larger granules with a particle diameter greater than0.6mm, anouter aerobic shell andan inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core. These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth. Some research has shown that particle size and density induced the different distribution and dominance of AOB, NOB and anam- mox (Winkler et al., 2011b). Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems, the information on the dynamics, distribution, and quantification ofAOB communities during sludge granulation is still limited up to now. To address these concerns, the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seeding flocs Please cite this article in press as: Bin, Z., et al., Dynamic and dis sludge granulation in an anaerobiceaerobic sequencing batch re mesh openings of 0.9, 0.6, 0.45, and 0.2 mm. A 100 mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the 0.9 mm mesh sieve. The sample was subsequently washed with distilled water and particles less than 0.9 mm in diameter passed through this sieve to the sieves with smaller openings. The washing procedure was repeated several times to separate the gran- ules. The granules collected on the different screens were recovered by backwashing with distilled water. Each fraction was collected in a different beaker and filtered on quantitative filter paper to determine the total suspended solid (TSS). Once the amount of total suspended solid (TSS) retained on each sieve was acquired, it was reasonable to determine for each into granules, and the distribution of AOB and NOB in different size granules from an anaerobiceaerobic SBR. A combination of process studies, molecular biotechniques and microscale techniques were employed to identify and char- acterize these organisms. Based on these approaches, we demonstrate the differences in both AOB community evolu- tion and composition of the flocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size. It is ex- pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems. 2. Material and methods 2.1. Reactor set-up and operation Thegranuleswere cultivated ina lab-scaleSBRwithaneffective volume of 4 L. The effective diameter and height of the reactor was10cmand51cm, respectively.Thehydraulic retention time was set at 8 h. Activated sludge from a full-scale sewage treat- ment plant (Jizhuangzi Sewage Treatment Works, Tianjin, China) was used as the seed sludge for the reactor at an initial sludge concentration of 3876 mg L�1 in MLSS. The reactor was operated on 6-h cycles, consisting of 2-min influent feeding, 90- min anaerobic phase (mixing), 240-min aeration phase and 5- min effluent discharge periods. The sludge settling time was reducedgradually from10to5minafter 80SBRcycles in20days, and only particles with a settling velocity higher than 4.5m h�1 were retained in the reactor. The composition of the influent media were NaAc (450 mg L�1), NH4Cl (100 mg L �1), (NH4)2SO4 (10 mg L�1), KH2PO4 (20 mg L �1), MgSO4$7H2O (50 mg L �1), KCl (20mgL�1), CaCl2 (20mgL �1), FeSO4$7H2O (1mgL �1), pH7.0e7.5, and 0.1 mL L�1 trace element solution (Li et al., 2007). Analytical methods-The total organic carbon (TOC), NHþ4eN, NO�2eN, NO � 3eN, total nitrogen (TN), total phosphate (TP) concentration, mixed liquid suspended solids (MLSS) concentration, and sludge volume index at 10min (SVI10) were measured regularly according to the standard methods (APHA-AWWA-WEF, 2005). Sludge size distribution was determined by the sieving method (Laguna et al., 1999). Screening was performed with class of size (<0.2, [0.2e0.45], [0.45e0.6], [0.6e0.9], >0.9 mm) the percentage of the total weight that they represent. tribution of ammonia-oxidizing bacteria communities during actor, Water Research (2011), doi:10.1016/j.watres.2011.09.026 2.2. DNA extraction and nested PCReDGGE The sludge from approximately 8 mg of MLSS was transferred into a 1.5-mL Eppendorf tube and then centrifuged at 14,000 g for 10 min. The supernatant was removed, and the pellet was added to 1 mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules. Genomic DNA was extracted from the pellets using E.Z.N.A.� Soil DNA kit (D5625-01, Omega Bio-tek Inc., USA). To amplify ammonia-oxidizer specific 16S rRNA for dena- 2.3. Distribution of nitrifying bacteria Three classes of size ([0.2e0.45], [0.45e0.6], >0.9 mm) were chosen on day 180 for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules. 2 mg sludge samples were fixed in 4% para- formaldehyde solution for 16e24 h at 4 �C and then washed twice with sodium phosphate buffer; the samples were dehydrated in 50%, 80% and 100% ethanol for 10 min each. Ethanol in the granules was then completely replaced by in t si 16 16 16 wat e r r e s e a r c h x x x ( 2 0 1 1 ) 1e1 0 3 turing gradient gel electrophoresis (DGGE), a nested PCR approachwas performed as described previously (Zhang et al., 2010). 30 ml of nested PCR amplicons (with 5 ml 6 � loading buffer) were loaded and separated by DGGE on polyacrylamide gels (8%, 37.5:1 acrylamideebisacrylamide) with a linear gradient of 35%e55% denaturant (100% denaturant ¼ 7 M urea plus 40% formamide). The gel was run for 6.5 h at 140 V in 1 � TAE buffer (40 mM Tris-acetate, 20 mM sodium acetate, 1 mM Na2EDTA, pH 7.4) maintained at 60 �C (DCode� Universal Mutation Detection System, Bio-Rad, Hercules, CA, USA). After electrophoresis, silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994). These were followed by air-drying and scanning with a gel imaging analysis system (Image Quant350, GE Inc., USA). The gel images were analyzed with the software Quantity One, version 4.31(Bio-rad). Dice index (Cs) of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes (LaPara et al., 2002). This index ranges from 0% (no common band) to 100% (identical band patterns) with the assistance of Quantity One. The Shannon diversity index (H ) was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population. H was calculated using the followingequation:H ¼ �P � ni N � log � ni N � ,whereni/N is the proportion of community made up by species i (bright- ness of the band i/total brightness of all bands in the lane). Dendrograms relating band pattern similarities were automatically calculated without band weighting (consider- ation of band density) by the unweighted pair group method with arithmetic mean (UPGMA) algorithms in the Quantity One software. Prominent DGGE bandswere excised and dissolved in 30 mL Milli-Q water overnight, at 4 �C. DNA was recovered from the gel by freezeethawing thrice. Cloning and sequencing of the target DNA fragments were conducted following the estab- lished method (Zhang et al., 2010). Table 1 e Oligonucleotide probes used for ecology analysis Probe Probe sequence (50e30) Targe NSO190 CGATCCCCTGCTTTTCTCC NIT3d CCTGTGCTCCATGCTCCG NSR1156 CCCGTTCTCCTGGGCAGT a Escherichia coli numbering. b Percentage of formamide in the hybridization buffer. c Millimolar concentration of sodium chloride in washing buffer. d Used with an equimolar amount of unlabeled competitor oligonucleot Please cite this article in press as: Bin, Z., et al., Dynamic and dis sludge granulation in an anaerobiceaerobic sequencing batch re xylene by serial immersion in ethanol-xylene solutions of 3:1, 1:1, and 1:3 by volume and finally in 100% xylene, for 10 min periods at room temperature. Subsequently, the granules were embedded in paraffin (m.p. 56e58 �C) by serial immer- sion in 1: 1 xylene-paraffin for 30 min at 60 �C, followed by 100% paraffin. After solidification in paraffin, 8-mm-thick sections were prepared and placed on gelatin-coated micro- scopic slides. Paraffin was removed by immersing the slide in xylene and ethanol for 30 min each, followed by air-drying of the slides. The three oligonucleotide probes were used for hybridiza- tion (Downing and Nerenberg, 2008): FITC-labeled Nso190, which targets themajority of AOB; TRITC-labeled NIT3, which targets Nitrobacter sp.; TRITC-labeled NSR1156, which targets Nitrospira sp. All probe sequences, their hybridization condi- tions, and washing conditions are given in Table 1. Oligonu- cleotides were synthesized and fluorescently labeled with fluorochomes by Takara, Inc. (Dalian, China). Hybridizations were performed at 46 �C for 2 h with a hybridization buffer (0.9 M NaCl, formamide at the percentage shown in Table 1, 20 mM Tris/HCl, pH 8.0, 0.01% SDS) containing each labeled probe (5 ng mL�1). After hybrid- ization, unbound oligonucleotides were removed by a strin- gent washing step at 48 �C for 15 min in washing buffer containing the same components as the hybridization buffer except for the probes. For detection of all DNA, 4, 6-diamidino-2-phenylindole (DAPI) was diluted with methanol to a final concentration of 1 ng mL�1. Cover the slides with DAPIemethanol and incubate for 15 min at 37 �C. The slides were subsequently washed once with methanol, rinsed briefly with ddH2O and immediately air-dried. Vectashield (Vector Laboratories) was used to prevent photo bleaching. The hybridization images were captured using a confocal laser scanning microscope (CLSM, Zeiss 710). A total of 10 images were captured for each probe at each class of size. The representative images were selected and final image evaluation was done in Adobe PhotoShop. different size granules. tea (rRNA positions) %FAb NaCl (mM)c S (190e208) 55 20 S (1156e1173) 40 122 S (1035e1048) 30 56 ide cNIT3. tribution of ammonia-oxidizing bacteria communities during actor, Water Research (2011), doi:10.1016/j.watres.2011.09.026 3. Results gradual process of sludge granulation, i.e., from flocculent granular sludge. Biodiversity based on the DGGE patterns was 0 20 40 60 80 100 120 140 1 25 41 59 73 84 94 104 115 125 135 147 160 172 188 Time (d) SV I10 (m L. g-1 ) 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 10000 M LS S (m g. L-1 ) SVI10 MLSS Fig. 1 e Change in biomass content and SVI10 during whole operation. SVI, sludge volume index; MLSS, mixed liquid suspended solids. wat e r r e s e a r c h x x x ( 2 0 1 1 ) 1e1 04 3.1. SBR performance and granule characteristics During the startup period, the reactor removed TOC and NH4 þ- N efficiently. 98% of NH4 þ-N and 100% of TOC were removed from the influent by day 3 and day 5 respectively (Figs. S2, S3, Supporting information). Removal of TN and TP were lower during this period (Figs. S3, S4, Supporting information), though the removal of TP gradually improved to 100% removal by day 33 (Fig. S4, Supporting information). To determine the sludge volume index of granular sludge, a settling time of 10 min was chosen instead of 30 min, because granular sludge has a similar SVI after 60 min and after 5 min of settling (Schwarzenbeck et al., 2004b). The SVI10 of the inoculating sludge was 108.2 mL g�1. The changing patterns of MLSS and SVI10 in the continuous operation of the SBR are illustrated in Fig. 1. The sludge settleability increased markedly during the set-up period. Fig. 2 reflects the slow and Fig. 2 e Variation in granule size distribution in the sludge dur solids. Please cite this article in press as: Bin, Z., et al., Dynamic and dis sludge granulation in an anaerobiceaerobic sequencing batch re analyzed by calculating the Shannon diversity index H as sludge to granules. 3.2. DGGE analysis: AOB communities structure changes during sludge granulation The results of nested PCR were shown in Fig. S1. The well- resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig. 3). The community structure at the end of experiment was different from that of the initial pattern of the seed sludge. The AOB communities on day 1 showed 40% similarity only to that at the end of the GSBR operation (Table S1, Supporting information), indicating the considerable difference of AOB communities structures between inoculated sludge and ing operation. d, particle diameter; TSS, total suspended tribution of ammonia-oxidizing bacteria communities during actor, Water Research (2011), doi:10.1016/j.watres.2011.09.026 ring r). wat e r r e s e a r c h x x x (