Bacteria concentration using a membrane type insulator-based

Journal of Environmental Sciences 21(2009) 568–574An innovative membrane bioreactor and packed-bed biofilm reactorcombined system for shortcut nitrification-denitrificationZHANG Yunxia, ZHOU Jiti ∗, ZHANG Jinsong, YUAN ShouzhiSchool of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian116024, China. E-mail: zyx9020@Received 21 May 2008; revised 04 July 2008; accepted 18 July 2008AbstractAn innovative shortcut biological nitrogen removal system, consisting of an aerobic submerged membrane bioreactor (MBR) and an anaerobic packed-bed biofilm reactor (PBBR), was evaluated for treating high strength ammonium-bearing wastewater. The system was seeded with enriched ammonia-oxidizing bacteria (AOB) and operated without sludge purge with a decreased hydraulic retention time (HRT) through three phases. MBR was successful in both maintaining nitrite ratio over 0.95 and nitrification efficiency higher than 98% at HRT of 24 h, and PBBR showed satisfactory denitrification efficiency with very low effluent nitrite and nitrate concentration (both below 3 mg/L). By examining the nitrification activity of microorganism, it was found that the specific ammonium oxidization rate (SAOR) increased from 0.17 to 0.51 g N/(g VSS ·d) and then decreased to 0.22 g N/(g VSS ·d) at the last phase, which resulted from the accumulation of extracellular polymers substances (EPS) and inert matters enwrapping around the zoogloea. In contrast, the average specific nitrite oxidization rate (SNOR) is 0.002 g N/(g VSS ·d), only 1% of SAOR. Because very little Nitrobactor has been detected by fluorescence in situ hybridization (FISH), it is confirmed that the stability of high nitrite accumulation in MBR is caused by a large amount of AOB.Key words: shortcut nitrification-denitrification; ammonia-oxidizing bacteria; nitrite accumulation; FISH DOI: 10.1016/S1001-0742(08)62309-8IntroductionBiological nitrification-denitrification is one of the most common processes for nitrogen removal from wastewa-ters. During nitrification step, ammonium is aerobically oxidized to nitrite by ammonia-oxidizing bacteria (AOB) and then nitrite is oxidized to nitrate by nitrite-oxidizing bacteria (NOB). During denitrification step, nitrate is re-duced to gaseous nitrogen by denitrifying microorganisms. Shortcut biological nitrogen removal (SBNR) is based on the fact that a partial nitrification to nitrite is performed which is followed by nitrite denitrification (Fig. 1). This new process has been gained great attention for its eco-nomic advantages in saving cost of aeration (25% less), organic carbons (40% less) and investment (Kim et al ., 2006). However, stability of nitrite accumulation, a key prerequisite for successful SBNR, is a great challenge for implementation of this process.In the last decade, research activities were mainly directed towards the achievement of consistent nitrite accumulation in a variety of reactor configurations by imposing specific process conditions to outcompete NOB, such as oxygen limitation and ammonia inhibition. Ruiz et al . (2003) determined the best condition for partial* Corresponding author. E-mail: jitizh@Fig. 1 Process of biological nitrogen removal by nitrification-denitrification.nitrification in an activated sludge reactor and achieved 65% nitrite accumulation at 0.7 mg /L of dissolved oxygen (DO), suggesting that pH was not a useful operation parameter to nitrite accumulation. Yun and Kim (2003) reported that inhibition of free ammonia on NOB was the major factor for nitrite accumulation, and nitrite ratio was maintained at around 95% with up to 2 kg NH 4+-N /(m 3·d). Kuai and Verstraete (1998) obtained average nitrite ratio of 80% with a 1000 mg NH 4+-N /L in a suspension sequencing batch reactor under oxygen limiting conditions. Canziani et al . (2006) achieved 90% nitrite ratio in a pure-oxygen membrane bioreactor with DO concentration range 0.2–0.5 mg /L and hydraulic retention time (HRT) higher than 45 d. Stable maintenance range partial nitrification is found to be very difficult in a long term operation because NOB , coexisting with AOB in mix nitrification system, can acclimatize the new growth environments and recover its activity, resulting in the failure of nitrite build-up. To solveNo. 5An innovative membrane bioreactor and packed-bed biofilm reactor combined system for shortcut nitrification-denitrification 569this problem, an enrichment culture of AOB is proposed here to achieve stable and high nitrite accumulation. Until now little is known about the feasibility of this novel ap-proach. Another problem is that nitrifiers with low growth rate can be washed out in conventional activated sludge process, resulting in reduction of nitrification performance.A submerged membrane bioreactor (MBR), an emerging new technology, is introduced for excellent nitrification efficiency because membrane filtration promises a com-plete solid-liquid separation and maintains a high number of slow-growing nitrifiers in the reactor (Yoon and Lee, 2005).The objective of this study was to evaluate the feasibility of a novel MBR-PBBR system for the treatment of high ammonium-bearing wastewater via nitrite pathway. En-riched AOB culture seeded in MBR as a new approach for stable nitrite accumulation was studied. The distribution of nitrifying bacteria was analyzed by fluorescence in situ hybridization (FISH), which has been successfully applied for phylogenetical identification and quantification in environmental and engineered systems (Amann, 1995; Koops and Prmmerening-Roser, 2001). These results can offer valuable perspectives for SBNR process.1 Materials and methods1.1 Experimental setupThe experimental system used for shortcut nitrification-denitrification is illustrated in Fig. 2. This system was composed of an aerobic MBR for partial nitrification and an anaerobic PBBR for nitrite denitrification, which were made of Plexiglas. A flat-sheet membrane module (PVDF, hydrophilic, pore size 0.22 μm, effective surface area 128 cm2, Millipore, USA) was installed in the MBR with a total volume of 2.4 L (working volume of 1.5 L). The constant flux operating mode was applied in this study using a suction pump (BT100-1J, Longer, China) to remove the permeate continuously from the MBR. An air diffuser, controlled by an air flow meter, was installed directly at the bottom of the membrane module to reduce membrane fouling and to supply oxygen to the microorganisms. Membrane module was removed for chemical cleaning once the transmembrane pressure (TMP) reached 25 kPa. The pH value in the MBR was automatically controlled at 7.8 ±0.2 by adding a mixture of alkaline solution (NaHCO3 5 g/L, NaOH 7 g/L, and Na2CO3 2 g/L) as pH buffer and carbon source for nitrifying bacteria.The permeate was pumped by a peristaltic pump to the PBBR with 2.0 L volume, in which 60% was packed with ceramic honeycombs as supports for biofilm growth. The external carbon source for denitrification was provided by dosing a 10 g/L trisodium citrate solution. The MBR-PBBR system was operated at 30 ±0.1°C constantly using a water bath after startup period. As shown in Table 1, this system was operated over a period of 105 d including four phases after startup. During the whole experimental period, no sludge was intentionally withdrawn from the MBR except for the measurement of suspended solids which was defined as complete sludge retention by Van Houten and Eikelboom (1997). Under the condition of complete sludge retention, the system could reach a steady state at a given sludge concentration because the biomass growth rate balanced the decay rate. The composition of synthetic wastewater in mineral medium used in this study was presented in Table 2. The AOB culture and denitrification microorganisms were obtained according to extinction dilution method (Aakra et al., 1999) and then enriched before been seeded in MBR and PBBR, respectively.Fig. 2 Schematic diagram of the combined membrane bioreactor (MBR) and anaerobic packed-bed biofilm reactor (PBBR) system.570ZHANG Yunxia et al.Vol. 21Table 1 Operating conditions of MBR-PBBR systemParameterStartupPhase IPhase IIPhase IIIPhase IVOperation time (d) 1–22 23–42 43–6364–84 85–105 HRT (h)2828242028Influent NH 4 +-N (mg/L)200–400 400 ± 20 400 ± 20 400 ± 20 500 ± 20 TSS in MBR (g/L) 1.58 ± 0.13 1.69 ± 0.12 1.78 ± 0.1 1.82 ± 0.08 1.88 ± 0.08 VSS in MBR (g/L)1.37 ± 0.1 1.45 ± 0.09 1.50 ± 0.07 1.51 ± 0.081.55 ± 0.07Temperature (°C) 28 ± 1.5 30 ± 0.1 30 ± 0.1 30 ± 0.1 30 ± 0.1HRT: hydraulic retention time; TSS: total suspended solids; VSS: volatile suspended solids. Data are expressed as mean ± SD (n = 10).Table 2 Composition of synthetic wastewater in mineral mediumSynthetic wastewaterTraces nutrient solutionCompoundConcentrationCompoundConcentration (g/L)(NH 4 )2 SO 4 (g/L)1.0–2.5EDTA50KH 2 PO 4 (g/L) 0.7 ZnSO 4 ·7H 2 O 2.2 MgSO 4 ·7H 2 O (g/L)0.05 CoCl 2 ·6H 2 O 1.61 NaCl (g/L)1.2 MnCl 2 ·4H 2 O 5.06 NaHCO 3 (g/L)1.0 CuSO 4 ·5H 2 O 1.57 Traces nutrient solution (mL/L) 2 (NH 4 )6 Mo 7 O 24 ·4H 2 O 1.1 CaCl 2 ·2H 2 O 5.54 FeSO 4 ·7H 2 O4.99KOHAdjust pH to 6.01.2 Analytical methodsAmmonia nitrogen (NH 4+-N), nitrite nitrogen (NO 2 −-N) and nitrate nitrogen (NO 3−-N) were analyzed by a UV /VS spectrophotometer (V-56, JASCO, Japan). Total suspended solids (TSS) and volatile suspended solids (VSS) were measured according to the standard methods (APHA, 1998). The total organic carbon (TOC) and total nitrogen (TN) were determined with a Total Organic Carbon Analyzer (TOC-VcpH, SHIMADZU, Japan). A phenol-sulphuric acid method (Dubois et al ., 1951) was used to qualify polysaccharide with glucose as the stan-dard. Protein content was determined by the colorimetric method with Coomassie Brilliant Blue G-250 (Bradford, 1976). The dissolved oxygen concentration and pH in re-actor was measured by a DO meter (Model 55, YSI, USA) and pH meter (PB-20, Sartorius, Germany), respectively. Sludge sample for every phase was taken from MBR to investigate the morphological changes of sludge floes with Scanning Electronic Microscopy (JEM-1200EX, JEOL, Japan).1.3 E nrichment of ammonia-oxidizing bacteria com-munityExtinction dilution method (Aakra et al ., 1999) was used for AOB isolation and cultivation, because many AOB do not form visible colonies on agar plates (Schmidt and Belser 1994). A 0.1-g nitrifying sludge taken from an airlift nitrifying bioreactor (Guo et al ., 2005) was suspended in the liquid mineral medium (Table 2) to a final concentration of 0.1 g /mL. A 100-μL sample was transferred to an Erlenmeyer flask with 100 mL liquid mineral medium which was sterilized by autoclave for 20 min at 121°C, and then was shaken in an incubator at 30°C, 150 r /min for two weeks. During the incubation, NH 4+-N, NO 2−-N and NO 3−-N were measured periodically. After two weeks, the above procedure was repeated again. Three months later, it was observed that nitrite was kept stable inmixture and nitrate was under detection limit. It is believed that the AOB community was successfully isolated from other bacteria especially nitrite-oxidizing bacteria. Then the mixture was enriched in twenty 1000-mL Erlenmeyer flasks with 500 mL medium and shaken in an incubator at 30°C in the dark for a month.1.4 Specific nitrification rate testThe specific nitrification rate including specific am-monium oxidization rate (SAOR) and specific nitrite oxidization rate (SNOR), were determined by batch ex-periments to evaluate the activities of AOB and NOB in MBR. One hundred milliliter mixture from MBR was centrifuged at 8000 r /min for 10 min and then washed with buffer medium to remove the background nitrogen. Batch experiment was performed in a 250-mL Erlenmeyer flask shaking in an incubator at 30°C with 100 mL mineral medium which had the same composition as synthetic wastewater except that 100 mg /L NH 4+-N and NO 2−-N were used here. Samples were taken at an interval of 30min to analyze the concentrations of NH 4+-N and NO 2−-N. The SAOR and SNOR were calculated by monitoring thedecreased rate of NH 4+-N concentration and NO 2−-N concentration versus time, respectively. Because the nitrifiers have a slow growth rate, it was considered that the biomass concentration was a constant during the batch experiments.1.5 Fluorescence in situ hybridization analysisThe seeded AOB culture and biomass sample taken from the MBR at day 105 were fixed in 4% freshly prepared paraformaldehyde solution for 2–3 h at 4°C, rinsed twice with phosphate buffered saline (PBS, pH 7.2) and then stored in the mixture of PBS and ethanol (1:1, V /V ) at –20°C. All hybridization experiments were performed according to the methods described by Amann (1995). The 16S rRNA-targeted oligonucleotide probes used in thisNo. 5An innovative membrane bioreactor and packed-bed biofilm reactor combined system for shortcut nitrification-denitrification571study were Nso190 (Mobarry et al ., 1996), Ntspa662 and Nit3 (Wagner et al ., 1996), which were synthesized and fluorescently labeled with a hydrophilic sulfoindocyanine dye (Cy3) or fluorescein isothiocyanate (FITC) at the 5 end. The cells were observed with a fluorescence micro-scope (BX-60, Olympus, Japan) equipped with a cooled charge-coupled device (CCD) camera system (PXL1400, Photometrics, USA).2 Results and discussion2.1 S table partial nitrification and nitrite accumulation in membrane bioreactorFigure 3 shows the nitrogen concentration of the feed and MBR effluent, nitrification efficiency and nitrite accu-mulation at each operational phase. At the startup period, the seeded sludge concentration in MBR was 1.58 g TSS /L (Table 1). It is arguable that the stability of VSS, as reflected by a standard deviation of less than 10% of the average at startup period and average 85% nitrification efficiency can clearly emphasize the attainment of steady state condition. The start-up period was operated for 23 d, demonstrating that this system is easy to manage and can startup rapidly. After startup, the MBR was monitored under decreased hydraulic retention time (HRT) (28, 24, 20 h) through three phases with the average influent ammonium nitrogen of 400 mg /L. As shown in Fig. 3, at phase I and II, 95% nitrification efficiency was detectedwith an average of effluent NH 4+-N of 20 mg /L and little nitrate was monitored. With the further decrease of HRTto 20 h (phase III), significant accumulation of NH 4+-N appeared (with the average of 138 mg /L), showing 30% reduction of nitrification efficiency. At phase IV, influentNH 4+-N was improved to 500 mg /L at the HRT of 28 h and only 60% nitrification efficiency was achieved. Interestingly, the concentrations of nitrite and nitrate in MBR were stable throughout the operation period: the nitrate nitrogen was very low (< 10 mg N /L) and the nitrite ratio was higher than 0.95. Up to now, the nitrite accumulation achieved over 0.95 is rarely for such a longtime period because NOB will recover its activity after being acclimated (Bernet et al ., 2005; Giudad et al ., 2004; Villaverde et al ., 1999). The high and stable nitrite accu-mulation achieved in this study might be due to the special AOB dominant microorganism in MBR. To confirm this assumption, batch biomass activity experiments and FISH analysis were followed.2.2 Performance of combined aerobic submergedmem-brane bioreactor and anaerobic packed-bed biofilm reactorFigure 4 shows overall nitrogen removal performanceof the MBR-PBBR system. A consistent NH 4+-N and TN removal efficiency of about 100% and 99% was detectedat phases I and II. At phases III and IV, both NH 4+-N and TN removal efficiencies were reduced by 20% due to lower nitrification efficiency in MBR. However, the e ffluentNO 2−-N and NO 3−-N concentration remained at low level (< 3 mg N /L) during the whole operation time, which contributed to satisfactory denitrification performance of anoxic PBBR. The carrier in PBBR provided good sur-roundings for the growth of dentrifiers, also helped to maintain high biomass concentration.2.3 Membrane foulingMembrane fouling is one of the main problems of MBR operation. In this work, two parameters, TMP and extracellular polymers substance (EPS), were introduced to reflect the membrane fouling of the aerobic MBR. It can be seen from Fig. 5 that a stable zero TMP was detected for almost 40 d and then climbed slowly in subsequent 10 d, which might be due to the fact that the permeate flux of 5.0 L /(m 2·h) is lower than the critical flux (Field et al ., 1995). Studies by Field et al . (1995) and Je fferson et al . (2000) demonstrated that when the MBR operated below the critical flux, the flux can be maintained constant over long period and the membrane fouling can be neglected. A rapid increase of TMP occurred in MBR from day 50 to about 25 kPa at day 67, demonstrating that membrane fouling became serious. Therefore, the membrane module was taken out from the bioreactor. The formation of a thickFig. 3 Variation of nitrogen concentrations and nitrification e fficiency in MBR.Fig. 4 Variation of nitrogen concentration and removal e fficiency in MBR-PBBR system.572ZHANG Yunxia et al.Vol. 21 Fig. 5 Variation of transmembrane pressure (TMP) over theoperating time.gel layer was observed on the membrane surface.Then, membrane was washed by sponge with tapwater followed by chemical cleaning with 0.5%NaOCl solution for 12 h (Shim et al., 2002). Afterthat, membrane permeability got recovered.It has been reported that some of the key factors in-cludingmicrobial floc size, soluble COD of mixed liquor, and theamount of EPS can affect the membrane fouling in MBR(Nagaoka et al., 1996; Pilly and Buckley, 1992). The totalamount of EPS has a significant positive effect on themembrane fouling resistance. Polysaccharides and proteinsare essential factors of EPS to influence microbial activity anddestruction (Lim et al., 2007). Figure 6 shows the variation ofsoluble EPS in the supernatant in MBR. The concentration ofpolysaccharides was found to increase gradually over theoperation time, while the proteins comparatively maintainedstable after phase II. The total soluble EPS (polysaccharidesplus proteins) was increased from 5 to 20 mg/L which wasresponsible for membrane fouling (Zhang et al., 2006). Inaddition, the accumulation of dead cells and EPS by theinterception of the membrane under a long sludge retentiontime (SRT) might affect the metabolism behavior ofammonium oxidizers.Fig. 6 Variation of soluble EPS, polysaccharides and proteins inMBR over the operation period.2.4 Specific ammonium/nitrite oxidization rateBatch experiments were carried out to clarify whether therelatively low population of NOB or free ammonia inhibition ofNOB caused nitrite accumulation in MBR. From Fig. 7, it canbe seen that MBR significantly enhanced the nitrificationperformance because the SAOR value was increased frominitial 0.17 to 0.49 g N/(g VSS·d) at phase I and thendecreased a little to 0.46 g N/(g VSS·d) at phase II. At phaseIII and IV, the SAOR decreased to 0.22 g N/(g VSS·d)gradually, which might be caused by the following reasons.First, a high free ammonia concentration selectively inhibitsAOB activity, because short HRT of 20 h in phase III and highinfluent NH4+-N concentration of phase IV (up to 500 mg/L)resulted in higher ammonia loads per cell which might exceedthe threshold concentration of AOB. Second, theaccumulation of EPS and inert matters caused byendogenous respiration under complete sludge retentionenwrapped around the zoogloea in MBR might impedetransfer rate of both DO and substrate (Han et al., 2005). Thishypothesis can be verified by the SEM image ofmicroorganism in MBR. On the other hand, very low SNOR(average of 0.002 g N/(g VSS·d)) was detected at phase I, II,and III, demonstrating that no NOB was present at thebeginning and little NOB went into the open MBR systemover the operation time.Fig. 7 Variation of specific ammonium oxidization rate (SAOR)and specific nitrite oxidization rate (SNOR) in MBR.2.5 Variation of surface morphology ofmicroorganisms in membrane bioreactorThe SEM observation of microorganism in MBR over theoperation time is shown in Fig. 8, which gives twoimplications. First, oval-shaped bacteria were found to bedominant in seeded bacteria and no other-shaped bacteriawas present (Fig. 8a). As operation over time, oval-shapedbacteria still remained dominant, however, a little bacilli wasobserved at day 105 (Fig. 8c). Second, with the extension ofoperational period, morphology of bacteria became more andmore unclear because cells were gradually enwrapped withsticky EPS. EPS with its sticky property and large molecularweight could not pass through membranes, and wastherefore retained in the MBR (Gao et al., 2004). Sticky EPSenwrapping bacteria had made a great resistance on thetransfer of substrate and DO to the surface of each cell,which might contribute toNo. 5An innovative membrane bioreactor and packed-bed biofilm reactor combined system for shortcut nitrification-denitrification573Fig. 8 SEM images of activated sludge in MBR. (a) seeded bacteria (×8000); (b) at day 42 (×8000), (c) at day 105 (×8000).Fig. 9 In situ identification of the distribution of nitrifiers in MBR at day 105 with total bacteria stained with DAPI (a), Cy3-labeled probe Nso190 (b), and FITC-labeled probe Nit3 (c).the decrease of ammonium oxidization activity in phase III and IV. 2.6 N itrifying community structure by fluorescence in situ hybridizationSince the activity of NOB was observed over operation time, biomass samples were taken from the MBR at day 105 and fixed immediately for FISH analysis. The purpose of FISH analysis was to verify the dominant microorgan-ism in MBR. The Cy3-labeled probes corresponding to AOB of β-Proteobacteria were applied for hybridization. The FITC-labeled probe specific for Nitrospira (Ntspa 662) and Nitrobacter (Nit3) was used for the hybridization of NOB. The total bacterial number was counted by DAPI staining. The fluorescence images of the biofilm samples from MBR at day 105 in Fig. 9 clearly showed that most of bacteria were consisted of Nso190-positive AOB and only a little Nitrobacter was detected. The seeded microorganism in MBR at the beginning was also analyzed by FISH, showing that it belonged to Nso190-positive AOB and no NOB (including Nitrobacter and Nitrospira ) was detected (data not shown). These results further confirm our hypothesis that the nitrite accumulation in MBR is not due to the inhibition of free ammonia on NOB activity, but due to the low population of NOB and high population of AOB. However, whether the appearance of NOB in MBR will influence the nitrite accumulation remains unknown. Further work is being carried out to evaluate the performance of this system over the long term.3 ConclusionsThis study focused on the feasibility and stability of shortcut nitrification-denitrification in a combined MBR-PBBR system using enriched AOB culture for nitrite accumulation in MBR. Particular emphasis was placed on nitrite accumulation characteristic, change of biomass activity and causes, and nitrifying community structure in MBR. The following conclusions are drawn:(1) The MBR-PBBR system was very effective for shortcut biological nitrogen removal. More than 99% TN removal efficiency was achieved at HRT of 28 and 24 h with a high influent ammonium nitrogen concentration of 400 mg /L. Satisfactory denitrification efficiency was observed with low effluent nitrite and nitrate nitrogen (< 3 mg /L) over the operation period.(2) Nitrification efficiency in MBR was significantly affected by the decrease of HRT, which was reduced from 95% (28 h) to 70% (20 h). However, the nitrite accumu-lation ratio could maintain higher than 0.95, and not be influenced by HRT or influent ammonium concentration.(3) The specific ammonium oxidization rate of biomass in MBR was increased from 0.17 to 0.49 g N /(g VSS·d) at the beginning and then decreased to 0.22 g N /(g VSS·d), which could be due to the accumulation of EPS and inert matters enwrapping around the zoogloea in MBR, and very low SNOR (average of 0.002 g N /(g VSS·d)) was detected.(4) The SEM and fluorescence images showed that AOB was dominated all the time, which was responsible for high and stable nitrite accumulation in MBR. 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2008年第3卷生态毒理学报Vol.3, 2008第3期, 307−312Asian Journal of Ecotoxicology No.3, 307−312应用流式细胞术测定淡水、海洋沉积物中异养细菌前处理条件的选择季倩，张经*华东师范大学河口海岸学国家重点实验室，上海 200062摘要：目前流式细胞术(FCM)已被广泛应用于水体异养细菌的检测，采用适当的前处理方法可将黏附在沉积物颗粒上的异养细菌提取到水相中进而利用FCM进行检测. 选择合适的前处理方法是将FCM应用于沉积物异养细菌检测的关键.论文对FCM测定沉积物中异养细菌的前处理方法进行了探讨，实验同步考虑了淡水及海洋沉积物. 结果表明，对于实验所用淡水、海洋沉积物，较优的前处理条件为：1mmol·L−1焦磷酸钠作为分散剂，暗处孵育10min，20w、40KHz水浴超声1min，并每30s人工振荡1次，2800r·min−1常温离心萃取3次.关键词：流式细胞术；异养细菌；沉积物；前处理文章编号：1673-5897(2008)3-307-06 中图分类号：Q93，X172文献标识码：AOptimization in Pretreatment Conditions for Determination of Heterotrophic Bacteria from Freshwater and Marine Sediments Using Flow CytometryJI Qian, ZHANG Jing*State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai 200062Received12 March 2008 accepted 6 May 2008Abstract:The Flow Cytometry (FCM) has been widely used to determine the heterotrophic bacteria in water environment presently. Also, it can be used to determine the heterotrophic bacteria in benthos environment after the appropriate pretreatment. The treatmentof extracting sediment-attached bacteria to water environment is the key point of applying the FCM to the study of benthos. This paper discussed the methods of pretreatment for the FCM to detect the heterotrophic bacteria both in freshwater and marine sediments. Results showed that the relatively preferable pretreatment conditions for sediments were: sodium pyrophosphate as the dispersant with the final concentration of 1mmol·L−1 and incubated 10min in dark; sonicate 1min in water bath at 20w, 40KHz power with a manual shake every 30s; and centrifugal extraction 3 times with 2800 r·min−1 at room temperature.Keywords: flow cytometry; heterotrophic bacteria; sediment; pretreatment收稿日期:2008-03-12 录用日期: 2008-05-06基金项目: 教育部“创新团队”项目(No. PCSIRT0427)；国家自然科学基金项目(No. 40476036)作者简介: 季倩(1982—)，女，硕士研究生，E-mail: jiqian22@；*通讯作者(Corresponding author)，E-mail: jzhang@sklec.308 生态毒理学报第3卷1引言(Introduction)流式细胞术(Flow cytometry)以其样品制备简单、可进行快速多参数数据采集、测定精确、不易受溶解有机物的干扰等优越性而成为水生和环境微生物学研究的重要工具(潘洛安等，2005). 上世纪80年代后，FCM被应用于海洋科学研究中，主要用于检测海水环境中的微型、微微型浮游生物，并已建立起一套相对完善的检测方法. 近年来，利用FCM 检测水体中异养细菌也屡有报道(焦念志等，1999；潘洛安等，2005). 异养细菌既是分解者又是生产者，底栖环境包括海洋沉积物、陆地土壤等均含有大量的异养细菌. 有毒物质在沉积物中的转化主要受到生物降解作用的影响，而底栖环境中异养细菌对生物降解、生态系统能量的传递等均具有十分重要的作用(张志南等，2003). 准确检测沉积物中的异养细菌是研究海洋沉积物生态毒理学、生物地球化学循环的重要前提之一.关于沉积物中异养细菌的检测，最开始采用直接培养法或荧光镜检的方法来计算其丰度(赵昌会等，2006；Dumahel et al., 2006；赵海萍等，2007)，过程繁琐，误差较大. 而FCM主要针对液相样品，因此对沉积物中异养细菌的检测存在一定的限制，需要将沉积物中的异养细菌转移到溶液中，而后根据已有的测定水样中异养细菌的方法进行检测. 为了有效地提取沉积物中的异养细菌，首先需要将沉积物和异养细菌分离. 目前，文献中提到的方法主要有添加分散剂、超声、离心萃取等(Maranger et al., 1996；Danovaro et al., 2001；2002；Dumahel et al., 2006). Danovaron等(2001；2002)讨论了FCM测定沉积物中的病毒的前处理方法，但是否可用于异养细菌的检测并没有详细分析；Dumahel等(2006)对FCM测定沉积物中的异养细菌进行了讨论，但其主要针对湖水沉积物，同时没有对磷酸盐分散剂种类及终浓度和孵育时间进行讨论. 国内关于利用FCM 检测沉积物异养细菌的前处理方法则未见报道. 本文以淡水、海洋沉积物为研究对象，对利用FCM测定沉积物中异养细菌的前处理条件进行了选择，初步确定了分散剂的种类、浓度、超声时间、萃取次数等，以期最大程度地将沉积物中的异养细菌萃取到上层液相样品中，从而运用FCM进行染色测定. 2 材料与方法(Materials and methods)2.1 沉积物样本实验中，海洋、淡水表层沉积物样品分别取自东海大陆架(125.01°E，28.27°N)及长江下游的徐六泾(121.03°E，31.77°N)，徐六泾沉积物样品由抓斗取得，东海大陆架沉积物样品由箱式采样器取得. 在实验中，为减少取样误差，先将沉积物样品搅拌均匀，后各取0.5mL沉积物作为平行分样，加入经0.02μm滤膜过滤的1%多聚甲醛4mL固定后，漩涡混合器振荡3min，暗处保存.2.2 主要的实验仪器及试剂FACScan型流式细胞仪(Becton Dickinson公司，美国)，SYBR Green I染料(SYBR-I，Molecular Probes 公司，美国)，0.02μm滤膜(Whatman公司，美国)，1.002μm荧光微球(Polysciences公司，美国)，微量移液枪，焦磷酸钠(Na4P2O7·10H2O，AR级)，六偏磷酸钠((NaPO3)6，CP级)，漩涡混合器，SCSF-1B 超声波仪，LXJ-II型离心沉淀机，高压灭菌锅. 实验中用新鲜制备的Milli-Q水配制所需试剂，并在0.1MPa、120℃条件下高压灭菌. 测定结果用Cell-Quest TM软件统计分析，t-检验用SPSS统计软件进行.2.3 实验方法参考文献报道数据，假定实验条件，然后对某一前处理条件进行优化选择. 考虑沉积物中异养细菌的可检测数量及相关的FCM分析的信号清晰程度，选择最优的前处理条件，以利于FCM的检测分析. 具体的假定实验条件见表1，所选择的前处理条件见表2.表1FCM测定沉积物中异养细菌实验的假定前处理条件Table 1 The assumed pretreatment conditions to detect the heterotrophic bacteria in the sediments using FCM假定实验条件沉积物0.5mL分散剂焦磷酸钠分散剂终浓度5mmol·L−1孵育时间10min超声时间1)3min离心萃取次数2)3次注：1): 采用SCSF-1B超声波仪(20w、40KHz)；2): 采用LXJ-II型离心沉淀机(650w、50Hz、2800r·min−1)第3期季倩等: 应用流式细胞术测定淡水、海洋沉积物中异养细菌前处理条件的选择309表2FCM测定沉积物中异养细菌实验所选择的前处理条件Table 2 The pretreatment conditions to detect theheterotrophic bacteria in the sediments using FCM所选择的实验条件分散剂焦磷酸钠、六偏磷酸钠分散剂终浓度0、1、2、3、4、5、10mmol·L−1孵育时间0、5、10、15、20min超声时间1)0、1、3、5、10、15min离心萃取次数2)1、2、3次注：1): 采用SCSF-1B超声波仪(20w、40KHz)；2): 采用LXJ-II型离心沉淀机(650w、50Hz、2800r·min−1)由于各个前处理条件的选择并不是一次完成，因此各次实验所用的沉积物可能略有不同，不同批次实验沉积物中的异养细菌数量可能会有一定变动.3结果与分析(Results and analysis)3.1 分散剂水体异养细菌常呈个体分散状态，但也可以因为水团扰动及细菌本身的生活习性而黏附在动植物排泄物和碎屑上(Tuner, 2002；Spiglazov et al., 2004)，集聚成菌团(Aggregates)(国家海洋局，1975)，从而沉降到水底，进入沉积物环境中. 因而需要添加分散剂或表面活性剂，使块状、粘状的沉积物分散为单个个体，解聚菌团，有利于微生物细胞与沉积物颗粒分离(徐永健等，2004).通常采用磷酸盐的缓冲液作为沉积物的分散剂，最常用的是六偏磷酸钠(Sodium hexameta- phosphate)，如在沉积物粒度分析、海洋地质调查中，均将六偏磷酸钠作为分散剂(国家海洋局，1975；孙有斌等，2001)，而在海洋底栖微生物的研究中，则常采用焦磷酸钠(Sodium pyrophosphate)作为分散剂(Bakken et al., 1985；Danovaro et al., 2001；Duhamel et al., 2006). 本实验选择焦磷酸钠和六偏磷酸钠进行对比研究，确定分散剂的终浓度、孵育时间、超声、离心萃取等前处理条件.3.1.1 分散剂终浓度Maranger等(1996)和Danovaro等(2001)指出，焦磷酸钠等分散剂终浓度越高，对微生物的检测噪音越大，因此需要选取最适宜的分散剂浓度. 图1为淡水沉积物、海洋沉积物添加不同浓度分散剂后异养细菌的测定结果. 对于淡水沉积物样品，选择焦磷酸钠所提取的异养细菌略高于六偏磷酸钠，但各浓度下差异不显著(p>0.01). 而对于海洋沉积物选择交磷酸钠所提取的异养细菌则远高于六偏磷酸钠，添加焦磷酸钠更有利于异养细菌的检测，这与Danovaro等(2001)、Duhamel等(2006)的研究结果基本相同. 由图1还可以看出，相比其他浓度，1mmol·L−1焦磷酸钠可以提取更多的异养细菌. 因此综合考虑淡水、海洋沉积物，实验选择焦磷酸钠为分散剂，并选用1mmol·L−1作为终浓度条件. 值得注意的是，对于淡水沉积物样品，添加分散剂后，样品中可检测得的异养细菌数量明显小于未添加分散剂的样品(p<0.01)，可能是由于未添加分散剂的沉图1不同终浓度分散剂对淡水沉积物(a)、海洋沉积物(b)异养细菌测定结果的影响(圆圈表示最终选择的实验条件；异养细菌单位为103·mL−1，以沉积物体积计)Fig.1 The heterotrophic bacterium abundance of freshwater sediment (a) and marine sediment (b) with different finalconcentrations of two dispersants(Circles indicate the selected experimental conditions; unit of bacteria: 103·mL−1, calculated with sediment volumes)310 生态毒理学报第3卷积物样品中，微生物颗粒之间以及非生物颗粒之间的相互粘连，不利于流式细胞仪染色荧光信号的检测，造成信号的重叠或假相增加.3.1.2 分散剂的孵育时间本实验以0~20min为界，讨论分散剂的孵育时间对沉积物中异养细菌的分散效果(图2). 由图2可见，分散剂对淡水及海洋沉积物的作用结果基本一致：随着孵育时间的增加，对异养细菌的提取效率呈先升高后降低趋势，当孵育时间为10min时，可以检测到相对较多的异养细菌. 同时，对于两种沉积物，焦磷酸钠作用效果均优于六偏磷酸钠. 因此，实验选择焦磷酸钠作为分散剂，孵育时间选择10min，以使沉积物获得最好的分散效果.3.2 超声时间通常在PBS缓冲溶液中对沉积物进行超声处理，可使细胞和颗粒进一步分散开，但超声是一种破坏性技术，破碎沉积物的同时也会造成微生物细胞的裂解(Ellery et al., 1984；Boenigk，2004；徐永健等，2004；Foladori et al., 2007)，因此需要选择适中的超声条件. Kepner等(1994)发现在50w条件下超声2.5min可以分离最大数量的吸附微生物，60w、冰浴条件下超声1min分离病毒的效果最好. Maranger和Bird(1996)选择室温超声45s，Danovaro 等(2001，2002)、Duhamel等(2006)则选择室温超声3min. Duhamel等(2006)指出，冰浴超声与室温超声对异养细菌的提取没有很大差别. 本研究根据SCSF-1B超声波仪具体的实验条件，选用20w、40KHz进行实验.超声时间对异养细菌提取的影响如图3所示. 对于淡水沉积物，不同超声时间下，选择焦磷酸钠所提取得异养细菌通常低于六偏磷酸钠，但在超声1min时，二者相差不大，同时超声1min时，焦磷酸钠的提取效果达到最佳. 对于海洋沉积物，在各超声时间下，焦磷酸钠提取效率通常高于六偏磷酸钠，且在超声1min时达到最高. 因此，实验在选用焦磷酸钠作为分散剂的前提下，室温下超声1min，每30s人工振荡1次，可以获得最好的分离效果.3.3 离心萃取次数采用LXJ-II型离心沉淀机(650w、50Hz)，2800r·min−1下离心15min可获得上清液. 通过计算每一次萃取得到的异养细菌浓度与各次萃取所得的异养细菌浓度总和的比值，来检测异养细菌的提取效率(Danovaro et al., 2001). 离心萃取次数对异养细菌提取效率的影响如图4所示. 以淡水沉积物为例，第一次离心萃取效率为69.1%，第二次萃取效率为23.5%，第三、第四次萃取效率分别为4.9%、2.5%. 可见萃取3次，基本上可将沉积物中的异养细菌完全提取.综合以上实验结果，同时考虑淡水、海洋沉积物，选择焦磷酸钠作为分散剂，并添加至1mmol·L−1终浓度，暗处孵育10min，室温下120w、40KHz水图2不同孵育时间对淡水沉积物(a)、海洋沉积物(b)异养细菌测定结果的影响(圆圈表示最终选择的实验条件；异养细菌单位为103·mL−1，以沉积物体积计)Fig.2 The heterotrophic bacterium abundance of freshwater sediment (a) and marine sediment (b) with different incubation time (Circles indicate the selected experimental conditions; unit of bacteria: 103·mL−1, calculated with sediment volumes)第3期季倩等: 应用流式细胞术测定淡水、海洋沉积物中异养细菌前处理条件的选择 311图3 不同超声时间对淡水沉积物(a)、海洋沉积物(b)异养细菌测定结果的影响(圆圈表示最终选择的实验条件；异养细菌单位为103·mL −1，以沉积物体积计)Fig.3 The heterotrophic bacterium abundance of freshwater sediment (a) and marine sediment (b) with different sonication time(Circles indicate the selected experimental conditions; unit of bacteria: 103·mL −1, calculated with sediment volumes)图4 离心萃取次数对淡水沉积物(a)、海洋沉积物(b)异养细菌测定结果的影响(异养细菌单位为103·mL −1，以沉积物体积计)Fig.4 The heterotrophic bacterium abundance of freshwater sediment (a) and marine sediment (b) withdifferent times of centrifuge extraction(unit of bacteria: 103·mL −1, calculated with sediment volumes)浴超声1min ，每30s 人工振荡1次，高速离心萃取3次，可获得较高的异养细菌提取效率.通讯作者简介：张经(1957—)，博士，中国科学院院士，华东师范大学教授，主要从事海洋生物地球化学研究.ReferencesBakken L R. 1985. 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Journal of Ocean University of Qingdao, 33: 375−383 (in Chinese)Zhao C H, Ye D Z, Wei W L. 2006. Research on deep-sea microbiology [J]. Microbiology, 33: 142−146 (in Chinese)Zhao H P, Li X Q, Tao J H. 2007. Methods of fluorescence enumeration of marine bacteria and application [J]. Journal of Hebei University of Engineering (Natural Science Edition), 24: 57−60 (in Chinese)中文参考文献国家海洋局. 1975. GB12763 海洋调查规范(第四分册：海洋地质调查)[S]. 北京: 国家海洋局潘洛安，张利华，张经. 2005. 应用流式细胞术测定水体异养细菌[J]. 海洋环境科学，24: 54−58孙有斌，高抒，鹿化煜. 2001. 前处理方法对北黄海沉积物粒度的影响[J]. 海洋与湖沼，32: 665−671徐永健，焦念志，钱鲁闽. 2004. 水体及沉积物中微生物的分离、检测与鉴定[J]. 微生物学通报，31: 151−155张志南，田胜艳. 2003. 异养细菌在海洋生态系统中的作用[J]. 青岛海洋大学学报，33: 375−383赵昌会，叶德赞，魏文铃. 2006. 深海微生物的研究进展[J]. 微生物学通报，33: 142−146赵海萍，李清雪，陶建华. 2007. 海洋细菌荧光显微计数法及其应用[J]. 河北工程大学学报(自然科学版)，24: 57−60 ◆。

A aAHLParts/signaling moleculeExplain:“N-Acyl homoserine lactones (AHLs or N-AHLs) are a class of signaling molecules involved in bacterial quorum sensing. Quorum sensing is a method of communication between bacteria that enables the coordination of group-based behavior based on population density. They signal changes in gene expression, such as switching between the flagella gene and the gene for pili for the development of a biofilm.”Reference:/wiki/Homoserine_LactoneRelated teams: Tokyo_Metropolitan(2011)Related works:1. Tokyo_Metropolitan(2011):In the BeE.coli design, the AHL producing bacteria was used as a model of the target. The CheZ is only expressed when AHL was receipted; therefore E.coli tend to move to the AHL concentrated area.Reference:/Team:Tokyo_Metropolitan/Project/TargetingAlkane chainsProductsExplain:“Alkanes (also known as paraffins or saturated hydrocarbons) are chemical compounds that consist only of hydrogen and carbon atoms and are bonded exclusively by single bonds (i.e., they are saturated compounds) without any cycles (or loops; i.e., cyclic structure)”Reference:/wiki/AlkaneRelated teams: Korea_U_Seoul(2011):Related works:1. Korea_U_Seoul(2011):The purpose of this team is to turn fatty acids into synthesize alkanes in the microorganisms.“Based on glycolysis, pyruvate oxidation, enzymes coded in luciferase genes (lux operon) and FAD from cyanobacteria, glucose is turned into alkane chain of about 13 carbon atoms in length. Synthesized fuel is functionally identical to natural petroleum and can be used as bioenergy. Produced alkane chain is part of a carbon circulation cycle as it is synthesized from glucose, in vivo. The fuel is relatively environment-friendly, unlikeordi prod the prod Refe http httpAs Part Exp “In e che L-as “Oth L-as met Refehttp Rela Rela 1. 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This e metabolism.”/Aspartate_a 2011):aspartase id and amm emoattracta will be overp UT-Tokyo/P EC 3.6.3.14synthesis of /ATP_syntha s (2011) hannel coup DP to ATP , th ase is used CO2 concen could be no glucose nev eoul/Project eoul/Project a-lyase (EC 4de aspartas enzyme pa ammonia-lya (AspA), wh onium ionant which wa produced in Project/Syste ) is an impo f adenosine aseled with ATP he energy m to provide ntration in th ot satisfied c vertheless w t/Designt/Abstract4.3.1.1) is a e, fumaric rticipates i aseich catalyze as chosen a the presenc emortant enzym triphosphate P synthesis,molecule of th the ATP w he atmosphe commercially will show ano n enzyme th aminase, L-n alanine es the react and used in ce of enoug me that prov e (ATP)”, uses up pr he cell”while consum ere. Though y, succeedin other metho hat catalyzes -aspartase, and aspa tion of aspa the project. gh AspA and vides energ roton gradie me the pote h the ng in od of s the and artate artate And d the y for ent to entialenergy build by ProteorhodopsinReference:/Team:IIT_Madras/Project/PrincipleB bBcrParts/geneExplain: A gene coding multidrug efflux pumpReference:/Team:HKUST-Hong_Kong/overview.htmlRelated teams: HKUST-Hong_Kong(2011)Related works:1. HKUST-Hong_Kong (2011)Used in the project so that the E.coli can have a “shield” to make themselves more vulnerable to antibiotics compared with the wild typeReference:/Team:HKUST-Hong_Kong/overview.htmlBeE.coliProjectExplain: The project of the team Tokyo_Metropolitan 2011Related teams: Tokyo_Metropolitan(2011)Related works:1. Tokyo_Metropolitan(2011)The project of the team Tokyo_Metropolitan 2011, as a mini injection system for pathogens killing, which is designed to be able to move fast and target pathogens concentrated area.“When bee attack enemy, they sting needle and inject toxin.” Similarly, the BeE.coli kills the bacteria using conjugation and sending the killing gene.Reference:/Team:Tokyo_Metropolitan/Team:Tokyo_Metropolitan/Project/KillingBiFCMethodExplain: bimolecular fluorescence complementation (BiFC) method, “based on the association between two nonfluorescent fragments of a fluorescent protein”. “When those fragments are brought in proximity to each other by an interaction between proteins, theywould fuse into one functional fluorescent protein (Tom K. Kerppola., 2008).” Reference:/Team:NYMU-Taipei/optomagnetic-designRelated teams: NYMU_Taipei(2011)Related works:1. NYMU_Taipei(2011):In the optomagnetic-design, the team mixed the BiFC method and BRET phenomenon into a BiFC-based BRET design in order to confirm the system can work like a switch, functioning in one of the two states -- “ON” and “OFF”.Reference:/Team:NYMU-Taipei/optomagnetic-designBiFC-based BRETDesigned SystemExplain: The designed system in the project of NYMU_Taipei(2011), which combined the BiFC method and BRET phenomenonRelated teams: NYMU_Taipei(2011)Related works:1. NYMU_Taipei(2011):In the optomagnetic-design, the team mixed the BiFC method and BRET phenomenon into a BiFC-based BRET design in order to confirm the system can work like a switch, functioning in one of the two states -- “ON” and “OFF”.Reference:/Team:NYMU-Taipei/optomagnetic-designBio-dosimeterProjectExplain: the project of the Osaka (2011)Reference:/Team:Osaka/ProjectRelated teams: Osaka (2011)Related works:1. Osaka (2011):The project of the Osaka (2011), which can evaluate the radio resistance with the heterologous genes, and response the radiation or DNA damage with visible outputs such as color productionReference:/Team:Osaka/ProjectThe BRET PhenomenonMethod/phenomenonExplain: Bioluminescence resonance energy transfer, called BRET for short, “based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor (Johan Bacart, et al., 2008)”In the optomagnetic-design, the team attempted to anchor the YFP on the N-terminus of Mms13 and r-Luciferase on the C-terminus, therefore the magnetic force will induce the fluorescent phenomenon.Related teams: NYMU_Taipei(2011)NYMU_Taipei(2011):Related works:1. NYMU_Taipei(2011):The BiFC-based BRET PhenomenonIt is known that transmembrane (TM) proteins usually have high propensities between the inter-helices.Reference:/Team:NYMU-Taipei/optomagnetic-designC cCarbon Stress BusterDesigned systemExplain: A device designed in the projectReference:/Team:IIT_Madras/Project/DeviceRelated teams: IIT_Madras (2011)Related works:1. IIT_Madras (2011):A design that “that rescues E.coli cells from carbohydrate starvation, and/or situations where Glycolytic pathway or components of the Electron Transport Chain is suppressed”, which has “green-light absorbing proteorhodopsin (GPR) generator”, ”Carbon stress induced promoter upstream of the PR generator”, and “A system for providing the Chromophore “Retinal” for light absorption activity of GPR”Reference:/Team:IIT_Madras/Project/DeviceCHAMP DesignMethodExplain: “The computed helical anti-membrane protein is designed by one of the computation and genetic methods available to engineer antibody-like molecules that target the water-soluble regions of transmemebrane (TM) proteins”Reference:/Team:NYMU-Taipei/optomagnetic-design#The_CHAMP_Design Related teams: NYMU_Taipei(2011)Related works:1. NYMU_Taipei(2011)In Optomagnetic design -- one project of NYMU-Taipei, Taiwan (2011) – CHAMP is used to design a peptide to inhibit the interaction inside the protein Mms13 to create the BiFC-based BRET system.Reference:/Team:NYMU-Taipei/optomagnetic-design#The_CHAMP_Design /Team:NYMU-Taipei/modelling-protein-structure-champ-design‘Charity’ by HR individualsMethod/phenomenonExplain: A kind of commensalism-like phenomenon in the community of the E. coli which is under the selection of the antibioticReference:/Team:HKUST-Hong_KongRelated teams: HKUST-Hong_Kong(2011)Related works:1. HKUST-Hong_Kong (2011)“Recent findings suggest that communities with a mixture of highly resistant (HR) and less resistant (LR) individuals are able to survive through ‘charity’ by HR individuals, which support LR individuals through indole signalling.”.Reference:/Team:HKUST-Hong_KongChemotaxisMethodExplain: Chemotaxis is the phenomenon whereby somatic cells, bacteria, and other single-cell or multicellular organisms direct their movements according to certain chemicals in their environment.Reference:/wiki/ChemotaxisRelated teams: UT-Tokyo(2011)Related works:1. UT-Tokyo(2011):A natural capability of the E.coliOne basic principle of the Substrate-induced Cell Assembling System design Reference:/Team:UT-Tokyo/Project/SystemChemoattractantsMethodExplain: Certain substances which can attracted the E.coliReference:/Team:UT-Tokyo/Project/SystemRelated teams: UT-Tokyo(2011)Related works:1. UT-Tokyo(2011):In the project of UT-Tokyo, SMART E.coli, the chemoattractant they have utilized is aspartate (L-Asp).Reference:/Team:UT-Tokyo/Project/SystemCheYParts/proteinExplain: One protein working in the mechanism of the motion of E.coliRelated teams: UT-Tokyo(2011), Tokyo_Metropolitan(2011)Related works:1. Tokyo_Metropolitan(2011)“Phosphorylated CheY is able to diffuse and bind to the flagellar motor, resulting in a change in motor rotation from counter-clockwise to clockwise. This causes a switch from smooth swimming to tumbling, allowing the bacteria to change direction.”Reference:/Team:Tokyo_Metropolitan/Project/TargetingCheZParts/proteinExplain: One protein working in the mechanism of the motion of E.coliRelated teams: Tokyo_Metropolitan(2011) ,UT-Tokyo(2011)Related works:1. Tokyo_Metropolitan(2011)One protein working in the mechanism of the chemotaxis in E.coli, which dephosphorylated CheY in the system; as a result, the team create a new chemotaxis by controlling the CheZ gene expression.Reference:/Team:Tokyo_Metropolitan/Project/Targeting2. UT-Tokyo(2011):In the design of Substrate-induced Cell Arrest System, the team constructed a system in which cheZ expression is repressed when the substrate is around the E.coli.Reference:/Team:UT-Tokyo/Project/SystemCheZ knockout strainStrainExplain: A strain of E.coli in which gene cheZ is knocked outRelated teams: UT-Tokyo(2011)Related works:1. UT-Tokyo(2011):In the design of Substrate-induced Cell Arrest System, a cheZ knockout strain is utilized to eliminate baseline expression. In this strain, the team construct a system in which cheZ expression is repressed when the substrate is around the E.coliReference:/Team:UT-Tokyo/Project/SystemchiA, chitinaseParts/gene + proteinExplain: “Chitinases are hydrolytic enzymes that break down glycosidic bonds in chitin. As chitin is a component of the cell walls of fungi and exoskeletal elements of some animals (including worms and arthropods), chitinases are generally found in organisms that either need to reshape their own chitin or dissolve and digest the chitin of fungi or animals.”Reference:/wiki/ChitinaseRelated teams: Kyoto (2011)Related works:1. Kyoto (2011):“Insect bodies are covered with hard integument mainly composed of chitin. To decompose the integument, we used ChiA gene, which encodes secreted chitinase.” Reference:/Team:Kyoto/DigestionCHOCOLATEProjectExplain: the project of the UNIST_Korea(2011)Reference:/Team:UNIST_KoreaRelated teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):The project of UNIST_Korea(2011), which aims to solve the problem of bio-safety by building a defense system in the E.coli – which can lead to the cell death and destruction of DNA inside the E.coli when they are in a “non-native environment” – to avoid horizontalgene transfer.Reference:/Team:UNIST_KoreacI inhibitorParts/inhibitorExplain: C1-inhibitor (C1-inh, C1 esterase inhibitor) is a protease inhibitor belonging to the serpin superfamily. Its main function is the inhibition of the complement system to prevent spontaneous activation.Reference:/wiki/C1_inhibitorRelated teams: UT-Tokyo(2011)Related works:1. UT-Tokyo(2011):The cI inhibitor is used in the system to construct a substrate-dependent manner to control the expression of cheZ“When the cell does not detect the substrate, cI inhibitor is not expressed and therefore cheZ is expressed and the product rescues motility of the cheZ- strain. Once the cell detects the substrate, the substrate-responsive promoter is activated and cI inhibitor is expressed. cI inhibitor then inhibits the activity of cI promotor and thereby represses cheZ expression, which leads to substrate-induced loss of motility.”Reference:/Team:UT-Tokyo/Project/SystemcI control systemDesigned systemExplain: the system in the work of UNIST_Korea(2011)Related teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):One design of the information processing module, which is the successful one as well “As expected, the cI system was able to provide an efficient control of gene expression with light despite a background expression. To reduce the background further we integrated the cI expressed from Pompc into the chromosome of E. coli. Chromosomally encoded cI reduced the background expression further”Reference:/Team:UNIST_Korea/project/modulesCOGSoftware/clusterExplain: Cluster of Orthologous Groups of proteinsReference:/Team:CBNU-Korea/Project_abstractRelated teams: CBNU-Korea (2011)Related works:1． CBNU-Korea (2011):The team attempted to re-group essential genes by COG distribution for construction of their databaseReference:/Team:CBNU-Korea/Project_abstractConjugationMethodExplain: The “sexual” activity between the bacteriaReference:/Team:Tokyo_Metropolitan/Project/KillingRelated teams: Tokyo_Metropolitan(2011)Related works:1. Tokyo_Metropolitan(2011)The “male” bacteria send the conjugation plasmid to the “female” in this phenomenon. The team design a killing system based on this mechanism: the killing gene on the plasmid will be passed during the conjugation, and expressed to induce the cell lysis of E.coli which is injected.Reference:/Team:Tokyo_Metropolitan/Project/KillingCph8Parts/protein/sensor/light sensorExplain: The light-sensitive protein Cph8 is a chimeric sensor kinase bearing the photoreceptor domain of the Synechocystis phytochrome Cph1 and the kinase domain of E.coli EnvZ (Levskaya et al., 2005).Reference:Jeffrey J. Tabor et.al, A Synthetic Genetic Edge Detection Program, Cell 137, 1272–1281, June 26, 2009Related teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):A kind of hybrid light sensor, which was chosen as an optical sensor and an osmolarity sensor of the sugar concentration in the project of UNIST_Korea, CHOCOLATE Reference:/Team:UNIST_Korea/project/modulesCrtE, CrtB, CrtIParts/cluster + proteinExplain: a lycopene biosynthetic gene clusterReference:/Team:Osaka/ProjectRelated teams: Osaka(2011)Related works:1. Osaka(2011):a lycopene biosynthetic gene cluster provided by 2009 Cambridge (BBa_K274100), whichis also used in the project of Osakaplease see more in the entry “Lycopene biosynthesis”Reference:/Team:Osaka/ProjectD dDEGSoftware/databaseExplain: Database of Essential Genes, where the information of essential genes will be obtained in the project of CBNU-KoreaReference:/Team:CBNU-Korea/Project_abstractRelated teams: CBNU-Korea (2011)Related works:1. CBNU-Korea (2011):Database of Essential Genes, where the information of essential genes will be obtained in the project of CBNU-KoreaReference:/Team:CBNU-Korea/Project_abstractDNS assayMethod/assayExplain: “3,5-Dinitrosalicylic acid (DNS or DNSA, IUPAC name 2-hydroxy-3,5-dinitrobenzoic acid) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly at 540 nm. It was first introduced as a method to detect reducing substancesin urine and has since been widely used, for example, for quantificating carbohydrates levels in blood. It is mainly used in assay of alpha-amylase. However, enzymatic methods are usually preferred to DNS due to their specificity.”Reference:/wiki/3,5-Dinitrosalicylic_acidRelated teams: Kyoto (2011)Related works:1. Kyoto(2011):“For characterizing the chitinase activity, we used the DNS assay, which is the method to determine the quantity of reducing sugar.”Reference:/Team:Kyoto/DigestionDpnIParts/protein/DNA damageExplain: One kind of Type IIM restriction endonucleases, which are able to recognize and cut methylated DNA.Reference:/wiki/Restriction_enzyme#Type_IIRelated teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):“We used two different lysis module to in order to compare the efficiency of lysis module to eradicate the genetically modified organisms: Holin mediated cell lysis and DpnI mediated DNA damage.Initially we had problems in cloning both the holin and Dpn genes under PL promoter as it is a constitutive promoter and lead to rapid cell death. We finally accomplished cloning the lytic cassette using ECNR2 strain which expresses cI constitutively. “Reference:/Team:UNIST_Korea/project/modulesD. radioduransStrainExplain:“The bacterium Deinococcus radiodurans shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.”Reference:/Team:Osaka/ProjectRelated teams: Osaka (2011)Related works:1. Osaka (2011):This strain of bacterium is studied in the project of bio-dosimeter because of its capabilities of radiation tolerance, and some of its genes related to the DNA damage resistance were transformed into the E.coli in the project so that the new strain might be able to resist the DNA damage.Reference:/Team:Osaka/ProjectDTOSoftware/objective resultsExplain: Distance to origin, which refer to the distance between replication origin and each essential geneReference:/Team:CBNU-Korea/Project_abstractRelated teams: CBNU-Korea (2011)Related works:1. CBNU-Korea (2011):In the project of CBNU-Korea, using this parameter, the team can easily confirm the distribution of the essential genes in each organismReference:/Team:CBNU-Korea/Project_abstractE eE. coli "guiders"StrainsExplain: A synthetic bacteria strain which works in the Substrate-induced Cell Assembling System, one project of UT-Tokyo(2011)Related teams: UT-Tokyo(2011)Related works:1. UT-Tokyo(2011):In the Substrate-induced Cell Assembling System, “E. coli "guiders" detect a substrate and attract other cells to themselves via chemoattraction”Reference:/Team:UT-Tokyo/Project/SystemE. coli K27StrainsExplain:An E. coli strain which is a FadD mutantRelated teams:Korea_U_Seoul(2011)Related works:1. Korea_U_Seoul(2011):A suitable host for the production of alkanes because it is a FadD mutant; therefore the E.coli accumlate fatty acids inside the cell and researchers can get more alkanes finally. Reference:/Team:Korea_U_Seoul/Project/DesignECNR2StrainsExplain: An E.coli strain which can express cI constitutivelyRelated teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):An E.coli strain which can express cI constitutively and hence was chosen for the construction of the lysis moduleReference:/Team:UNIST_Korea/project/modulesE. CRAFTStrainExplain: Escherichia coli Re-engineered for Antibiotics-Free Transformation, a strain made in the project E. Trojan of HKUST-Hong_Kong(2011)Reference:/Team:HKUST-Hong_Kong/overview.htmlRelated teams: HKUST-Hong_Kong(2011)Related works:1. HKUST-Hong_Kong (2011)“pre-E. trojan bacterial strain (E. CRAFT) that is capable of performing plasmid selection without antibiotics and consequently contains as few antibiotic resistance genes as possible”The team made the E. CRAFT as a pre E. Trojan so that the E. Trojan which will be introduced into the community will not “possess a wide spectrum of antibiotic resistance as this would give it an inherent selective advantage”Reference:/Team:HKUST-Hong_Kong/overview.htmlElectron Transport Chain (ETC)Method/phenomenonExplain: “An electron transport chain (ETC) couples electron transfer between an electron donor (such as NADH) and an electron acceptor (such as O2) with the transfer of H+ ions (protons) across a membrane.”Reference:/wiki/Electron_transport_chainRelated teams: IIT_Madras (2011)Related works:1. IIT_Madras (2011):The system which is responsible for providing the proton gradient generallyIn the project, ETC is always broken; therefore another system – the system built in thisproject, which consists of the Proteorhodopsin and ATP synthases – is needed. Reference:/Team:IIT_Madras/Project/PrincipleE. TrojanProjectExplain: The project of the HKUST, which “has its origins rooted in the Trojan Horse found in the tale of the Trojan War”Reference:/Team:HKUST-Hong_Kong/overview.htmlRelated teams: HKUST-Hong_Kong(2011)Related works:1. HKUST-Hong_Kong (2011)“Similar to how the Greeks destroyed the city of Troy from within using a wooden horse filled with soldiers, our E. trojan also works to destroy an E. coli population that is under antibiotic stress from within through indole quorum-sensing disruption.”Reference:/Team:HKUST-Hong_Kong/overview.htmlF ffim inversion systemDesigned systemExplain: One design of the information processing module, which is not introduced clearly and thought to be unsuccessful because of its difficulty on the basal fimE expression Related teams: UNIST_Korea(2011)Related works:1. UNIST_Korea(2011):“After sensing the environment through optical and physical sensor, Chop. coli should process the information. We introduced two different processor system: fim inversion system and cI control system.”Reference:/Team:UNIST_Korea/project/modulesG g“G & OR”Method/phenomenonExplain: A pattern of regulation of the replicationReference:/Team:HKUST-Hong_Kong/asm.htmlRelated teams: HKUST-Hong_Kong(2011)Related works:1. HKUST-Hong_Kong (2011)“Well studied examples of such origins include those of pSC101 and R6K plasmids, where the origins of replication (OR) appear together with a constitutive gene (G)” Reference:/Team:HKUST-Hong_Kong/asm.htmlGFPParts/proteinExplain: The green fluorescent protein (GFP) is a protein composed of 238 amino acid residues (26.9kDa) that exhibits bright green fluorescence when exposed to light in the blue to ultraviolet rangeReference:/wiki/Green_fluorescent_proteinRelated teams: HKU-Hong_Kong(2011), HKUST-Hong_Kong(2011)，Tokyo_Metropolitan(2011), UNIST_Korea(2011)Related works:1. HKU-Hong_Kong(2011):Used as reporter gene in the Super Silence – project of HKU(2011), to show whether the fusion protein work in a visible wayReference:/Team:HKU-Hong_Kong/Project2. HKUST-Hong_Kong(2011)：Please see the entry “sFP, sfGFP”3. Tokyo_Metropolitan(2011):In the BeE.coli design, the team used GFP twice. They used GFP instead of CheZ first to ensure that the idea of the design is workable, before testing Targeting Device system. And they build the Anti-Killer Device in the E.coli with a GFP generator to ensure that the Anti-Killer Device works as well.Reference:/Team:Tokyo_Metropolitan/Project/Targeting/Team:Tokyo_Metropolitan/Project/Killing2. UNIST_Korea(2011):In the project of UNIST_Korea, CHOCOLATE, the expression of GFP in either presence or absence of light was used to evaluate the efficiency of the optical sensor. Reference:/Team:UNIST_Korea/project/modulesgln AParts/geneExplain:glnA gene codes the glutamine synthetaseReference:/Team:Kyoto/HungerRelated teams: Kyoto (2011)Related works:1. Kyoto (2011)Please see entry “glutamine synthetase”Reference:/Team:Kyoto/Hungergln GParts/geneExplain:glnG gene codes the NtrC, a kind of nitrogen regulatory protein Reference:/Team:Kyoto/HungerRelated teams: Kyoto (2011)Related works:1. Kyoto (2011)Please see entry “NtrC”Reference:/Team:Kyoto/HungerglnLParts/geneExplain: glnL gene codes the NtrB, a kind of nitrogen regulatory protein Reference:/Team:Kyoto/HungerRelated teams: Kyoto (2011)Related works:1. Kyoto (2011)Please see entry “NtrC”Reference:/Team:Kyoto/HungerGlutamine synthetaseParts/proteinExplain:“Glutamine synthetase (GS) is an enzyme that plays an essential role in the metabolism of nitrogen by catalyzing the condensation of glutamate and ammonia to form glutamine” “Glutamine Synthetase uses ammonia produced by nitrate reduction, amino acid degradation, and photorespiration. The amide group of glutamate is a nitrogen source for the synthesis of glutamine pathway metabolites.”Reference:/wiki/Glutamine_synthetaseRelated teams: Kyoto (2011)Related works:1. Kyoto (2011)“When enteric bacteria are deprived of ammonia, they express glnA to produce glutamine synthetase (GS) under the σ54promoter. The transcription from σ54promoter is stimilated by phosphorylated form of NtrC (NtrC-P).”Reference:/Team:Kyoto/HungerGODSoftware/database + designerExplain: Genome Organization Database & DesignerReference:/Team:CBNU-Korea/Project_abstractRelated teams: CBNU-Korea (2011)Related works:1. CBNU-Korea (2011):Genome Organization Database & Designer, the software the team used in the project “We will re-group essential genes by COG distribution for construction of our database which is connected to software named GOD”Reference:/Team:CBNU-Korea/Project_abstractH hHNS protein (1)Parts/proteinExplain: An Escherichia coli nucleoid protein in E.coli genome, known only to function as a modulator of gene expression, “Gina M. Donato said that specific single amino acid substitutions in H-NS caused an approximately 50% increase in flagellum rotational speed (*1)”Reference:/Team:Tokyo_Metropolitan/Project/SpeedUp。

Concentration Calculations 11.Calculate the concentration of the following solutions:a.825 mL that contains 30.0 g of acetic acid, CH3OOHb.2500 mL that contains 49.0 g phosphoric acid, H3PO4c. 1.50 dm3 that contains 102 g potassium hydroxide, KOHd. 1.25 L that contains 8.50 g of sodium nitrate, NaNO32.How many cubic decimeters of solution can be made from each of the following?a. 2.00 M solution using 80.0 g NaOHb.0.500 M solution using 80.0 g NaOHc. 6.00 M solution using 126 g HNO3d.0.100 M solution using 170.0 g NaNO33.Calculate the mass of solute in the following solution:a.750.0 ml of a CaCl2 solution that is 0.500 Mb.3000.0 mL of a KOH solution that is 2.50 Mc.250.0 mL of a Na2SO4 solution that is 2.00 Md.50.0 mL of a NaNO3 solution that is 0.100 M4.Suppose that you need5.00 L of a 0.10 mol/L sodium nitrate solution. What massof sodium nitrate must you use. Describe how you would prepare this solution.5.Suppose that you want to make as large a volume of 5.00 mol/L sodium chloridesolution as possible. You have a bottle that contains 1.17 kg of this substance.What volume of solution can you make?6.If 8 g of sodium hydroxide, NaOH, is dissolved in 100 mL of solution, what is themolar concentration of this solution?7.If 20.8 g of barium chloride, BaCl2, is dissolved in 200 mL of solution, what is themolar concentration of this solution?8.You purchased a 25 mL container of a 15% m/V hydrogen peroxide solution.However, before the hydrogen peroxide can be used to disinfect a wound, it must be diluted with water to a concentration of 2.55 % m/V. What will the finalvolume of the solution if you dilute all 25 mL of the hydrogen peroxide that youpurchased?9.You need 100 mL of a 0.1 mol/L hydrochloric acid, HCl, and all you have instock is a bottle labeled 12.0 M HCl. How can you prepare the solution you need?10.If 0.75 L of a 5.00 M solution of HCl is diluted to a volume of 1.80 L by addingwater, what is the concentration of the resulting diluted solution?11.How many mL of water will be required to dilute 11 mL of a 0.45 H2SO4 to aconcentration of 0.12 M?12.It is desired to produce 1.00 L of 0.05 M nitric acid, HNO3, by diluting 14.00 Mnitric acid. Calculate the volume of the concentrated acid and the volume of water required for the dilution.13.A chemist adds water to a 120 mL of a 6.0 M solution of NaOH until the finalvolume is 2.0 L. What is the molarlity of the resulting solution?14.What concentration results when 150 mL of a 0.36 M solution of magnesiumsulphate, MgSO4, are added to a 750 mL of water?15.Ms. Pall needs 5.0 L of 5.0 M HCl for a class experiment and a supply of 14.6 Msolution of concentrated HCl is available. What volume of the concentrated acid should be measured out, by Ms. Pall to add to water to make up the final volume of 5.0 L?16.How much of 15.4 mol/L solution of HNO3 is needed so that the dilution resultsin 150 mL of 0.200 mol/L solution?17.Given a solution of ammonia, NH3, with a concentration of 15.0 mol/L, howwould you prepare 1.00 L of dilute ammonia with a concentration of 0.100 mol/L?18.If 10.0 mL of a 0.56 mol/L solution of aluminum nitrate, Al(NO3)3, is diluted to250.0 mL, what is…a.The concentration of aluminum nitrate in the new solution?b.The concentration of the nitrate ions in the new solution?19.Calculate the concentration of the solution formed if a 25.0 mL sample of HNO3,10.0 M is placed in a 500.0 mL volumetric flask and the flask is filled up to themark with distilled water.20.What is the total volume of 3.0 M H2SO4, that can be prepared from 5.0 L ofconcentrated sulfuric acid (18 M)?。

【收稿日期】2012-02-07【基金项目】国家自然基金（31160020）和云南省自然科学基金（2009ZC014X ）资助【作者简介】年洪娟，女，副教授，硕士生导师，从事微生物抗逆分子生物学研究，Email ：nianhongjuan@yahoo．com．cn文章编号：1005-376X （2012）08-0760-03【综述】不饱和脂肪酸在逆境胁迫中的作用年洪娟，陈丽梅（昆明理工大学生物工程技术研究中心，昆明650500）【摘要】生物膜是将细胞与环境分开的第一道屏障，是环境胁迫造成损伤的主要位点。

脂肪酸是生物膜的主要组成成分，不饱和脂肪酸在决定生物膜的生理特性中具有重要作用，增加脂肪酸的不饱和程度能增加膜脂的流动性。

近年来，很多研究发现，生物通过脂肪酸脱饱和维持膜的流动性来适应外界环境变化。

本文主要从不饱和脂肪酸在环境温度胁迫、盐胁迫、氧化胁迫、酸碱胁迫、干旱胁迫、乙醇胁迫及铝胁迫中的作用研究进展进行了综述。

【关键词】不饱和脂肪酸；胁迫【中图分类号】Q945；X173【文献标识码】AThe role of unsaturated fatty acids in various environmental stressesNIAN Hong-juan ，CHEN Li-mei（Biotechnology Research Center ，Kunming University of Science and Technology ，Kunming 650500，China ）【Abstract 】Biological membranes are the first barrier that separates cells from the environment and are a primary target for damage during environmental stress．Fatty acids are the major components of cell membranes．Unsaturated fatty acids play an important role in the biophysical characteristics of cell membranes．An increased degree of unsaturation increases fluidization of the membrane lipids．Recently ，many researches proved that organisms usually maintain the fluidization of membrane by de-saturation of the fatty acids to adapt to the environmental changes．This paper reviewed the recent progresses on the roles of un-saturated fatty acids in various stresses ，such as environmental temperature stress ，salt stress ，oxidative stress ，acid and alka-line pH tolerance ，drought tolerance ，ethanol tolerance and aluminum stress．【Key words 】Unsaturated fatty acids ；Stress生物将自身与环境分开的第一道屏障是生物膜，生物膜也是环境胁迫过程中发生损害的主要位点。

Internal environment:内环境refers to the liquid surrounding the cells in the body of multicellular animals, that is extracellular fluid.Homeostasis稳态: refers to a state of relatively constant of physical and chemical properties of internal environment, such as temperature, pH, osmotic pressure and all kinds of liquid ingredients in the body, and so on.positive feedback正反馈: A change in a condition leads to responses from the effectors which a mplifies that changenegative feedback负反馈: A change in a condition leads to responses from the effectors which counteracts that changefacilitated diffusion via carrier经载体易化扩散: Water soluble small molecules and ions under the carrier protein mediated, cross the plasma membrane follow concentration gradientfacilitated diffusion via ion channel经通道易化扩散: All kinds of charged ions under the channel proteins mediated, cross the plasma membrane follow concentration gradient and potential gradientPrimary active transport原发性主动转运:making direct use of energy derived from ATP to transport the ions across the cell membraneSecondary active transport次级主动转运:The ion gradients established by primary active transport permits the transport of other substances against their concentration gradientsresting potential静息电位: A potential difference across the membranes of inactive cells, with the inside of the cell negative relative to the outside of the cellaction potential动作电位: Some of the cells (excitable cells) are capable to rapidly reverse their resting membrane potential from negative resting values to slightly positive values. This transient and rapid change in membrane potential is called an action potential Excitation-contraction coupling兴奋收缩耦联: the mediation process of striated muscle cells generate action potentials and muscle filament contraction and relaxation. Hematocrit血细胞比容: The capacity percentage of the blood cells in the blood erythrocyte sedimentation rate红细胞沉降率: The distance that red blood cells settle in a tube of blood in one hourHemostasis止血: Small damaged blood vessels stop bleeding after a few minutes automaticallyBlood coagulation血凝固: blood change from sol to illiquid gel stateBlood group血型: The type of specific antigen on the surface of blood cellseffective refractory period:premature systole期前收缩: if ventricle is stimulated after ventricular muscle effective refractory period, before The next sinoatrial node excitement arrive, it will produce a contraction in advance.compensatory pause代偿间歇: A longer ventricular diastolic after premature systole. Atrioventricular delay房室延搁: Excitement slowly spread in atrioventricular junction and take longer time.Cardiac cycle心动周期: A cycle of heart contraction and relaxationStroke volume每搏输出量: One side of the ventricular injection volume of blood by a heart throbEjection fraction射血分数: The percentage stroke volume account for ventricularend-diastolic volumeCardiac output心输出量: The blood volume inject by one side of the ventricular per minuteCardiac index心指数: calculate cardiac output by unit surface areasystolic pressure收缩压: The highest blood pressure at mid ventricular systolic .diastolic pressure舒张压: The lowest blood pressure at ventricular end-diastolic.pulse pressure脉压: Systolic blood pressure minus diastolic blood pressurecentral venous pressure中心静脉压: The blood pressure in right atrium and chest cavity vena cavaMicrocirculation: Blood circulation between arteriole and micro veinEffective filtration pressure有效滤过压:the pressure difference between filtration and reabsorptionRespiration: The process of gas exchange between the body and its environmentvital capacity肺活量: After inhalation complete, the largest gas exhaled from the lungs forced vital capacity用力肺活量: After inhalation complete, exhale the largest gas from the lungs as fast as possibleforced expiratory volume用力呼气量: After inhalation complete, the gas exhaled from the lungs in a certain timeAlveolar Ventilation肺泡通气量: amount of inhaled the fresh air in the alveoli per minute. Pulmonary stretch reflex牵张反射: The reflection of inspiratory inhibit or inhale excited caused by pulmonary inflation and pulmonary deflation.Digestion消化: break down of food into small molecular components small enough to absorb.Mechanical digestion and chemical digestion.Absorption吸收: the small molecules that formed by digestion across the digestion tract mucosa go into blood and lymph.Small wave小波: the spontaneous rhythmic, subthreshold depolarization of the cell membrane of the gastrointestinal tract that characterizes the underlying electrical activity of the bowel.胃液主要成分1.HCl,parietal cell ,acid sterilization. Activation of pepsinogen, promotion of secretin secretion. Assisted effect of Fe and Ca absorption.2.Pepsinogen胃蛋白酶, chief cell, active in stomach, initially by H ions and then by active pepsin, autocatalytic activation. Pepsin an endopeptidase, which attacks peptide bonds in the interior of large protein molecules.3.Mucus粘液, neck cell and goblet cell, lubrication of the mucosal surface. Protection of the tissue from mechanical damage by food particle.4.intrinsic factor内因子, parietal cell. The intrinsic factor binds to vit B12 and facilitated its absorption.Stimulate gastric secretion刺激胃液分泌ACH gastrin histamine/ somatostatin Digestion phase gastric secretion消化期胃液分泌Cephalic gastric intestinal phase Regulation inhibitory gastric juice secretion胃液分泌抑制性调节1.HCl:inhibite G cell release gastrin;stimulate D cell release somatostatin;in the gastric antrum,inhibition of G cells,release of SST;in the duodenum ,release of secretin,bulbogastrone.2,fat:initiating release of enterogastrone.3,hepertonic solution:entero-gastric reflex.Receptive relaxation 容受性舒张:stimulation of receptor reflex muscle relax in the f undus and stomach body when chew and swallow.Pancreatic juice composition effect胰液成分作用:pancreatic amylase,pancreatic lip ase, trypsin,chymotrypsin,HCO3 bicarbonate balance the HCl in duodenum. Protect i ntestinal mucosa TPS and chymolase, pancreatic lipase, pancreatic amylase.Enteroh epatic circulation of bile salt.Bile salts were emptied into the small intestine with hepatic bile, about 95% is absorbed into the blood in the terminal ileum, and then synthesizing bile again after the hepatic vein to the liver, then empty into intestine.Physiological functions of bile胆汁生理作用1.Emulsifying or detergent function of bile salts.2.Help in the absorption of: fatly acid, lmonoglycerides, cholesterol, other lipids Peristalsis蠕动: the rhythmic waves of muscular relaxation and contraction are called peristalsis.Receptive relaxation 容受性舒张: stimulation of food on pharynx and esophagus produce relaxation of the lower esophageal sphincter and stomach.Gastric emptying胃排空: the process that the gastric contents are delivered to the duodenum.Thermal equivalent of food热价: calories liberated by 1g food oxidized in body. (kJ/g) Thermal equivalent of oxygen氧热价: heat production by consuming one liter of oxygen to oxidize a specific type of blood. (kJ/L)Respiratory quotient(RQ)呼吸商: in the process of oxidizing food, the ratio of CO2 produced to O2 used。

Membrane Bioreactors in Wastewater Treatment Wastewater treatment is a critical process that helps to protect public health and the environment. Membrane bioreactors (MBRs) are a type of wastewater treatment technology that has gained popularity in recent years due to its ability to produce high-quality effluent. In this response, we will explore the benefits and challenges of using MBRs for wastewater treatment.One of the primary advantages of MBRs is their ability to produce high-quality effluent. The use of membranes in the treatment process allows for the removal of suspended solids, bacteria, and other contaminants from the wastewater. This results in effluent that meets strict water quality standards and can be reused for a variety of purposes, including irrigation and industrial processes.Another benefit of MBRs is their compact size. Unlike traditional wastewater treatment plants, which can take up a significant amount of space, MBRs can be designed to fit into smaller areas. This makes them ideal for use in densely populated urban areas where space is at a premium.Despite these advantages, there are also some challenges associated with using MBRs for wastewater treatment. One of the primary challenges is the cost. MBRs can be more expensive to install and operate than traditional treatment methods. However, it is important to note that the long-term benefits of using MBRs, such as reduced maintenance costs and increased efficiency, can offset these initial costs.Another challenge associated with MBRs is the potential for membrane fouling. Membrane fouling occurs when the membranes become clogged with solids, bacteria, and other contaminants. This can reduce the efficiency of the treatment process and increase the frequency of membrane replacement. However, there are several strategies that can be used to minimize membrane fouling, including regular cleaning and the use of pretreatment processes.In addition to these challenges, there are also some environmental concerns associated with MBRs. One of the primary concerns is the energy required to operate the treatment process. MBRs require a significant amount of energy to power the pumps and other equipment used in the treatment process. However, there are several strategies that can be used to reduce energy consumption, such as using energy-efficient equipment and optimizing the treatment process.In conclusion, membrane bioreactors are a promising technology for wastewater treatment. They offer several advantages, including high-quality effluent and compact size. However, there are also some challenges associated with using MBRs, such as cost and membrane fouling. It is important to carefully consider these factors when deciding whether to use MBRs for wastewater treatment. With proper planning and management, MBRs can provide an effective and sustainable solution for treating wastewater.。

膜分离技术在工业废水治理作文## Membrane Separation Technology in Industrial Wastewater Treatment.Membrane separation technology has emerged as a promising and versatile solution for industrial wastewater treatment, offering numerous advantages in effectively removing contaminants and pollutants. This technology utilizes semipermeable membranes that selectively allow certain substances to pass through while retaining others, enabling the separation and purification of wastewater.One of the key advantages of membrane separation technology lies in its ability to remove a wide range of contaminants, including suspended solids, dissolved organic matter, heavy metals, and pathogens. This makes it particularly suitable for treating industrial wastewater streams that contain complex and diverse pollutants. Additionally, membrane filtration processes can be operated at varying pressures and temperatures, providingflexibility in adapting to different wastewater characteristics and treatment requirements.Moreover, membrane separation technology offers several benefits in terms of operational efficiency and sustainability. Membranes are durable and can withstand harsh conditions, allowing for continuous operation over extended periods without significant maintenance requirements. Furthermore, the energy consumption associated with membrane filtration processes is relatively low compared to other treatment methods, contributing to reduced operating costs.### Types of Membrane Separation Technologies.Various types of membrane separation technologies are employed in industrial wastewater treatment, each with its unique characteristics and applications:Microfiltration (MF): MF membranes have relatively large pores and are primarily used to remove suspended solids, bacteria, and other particulate matter.Ultrafiltration (UF): UF membranes possess smaller pores than MF membranes and can effectively remove larger organic molecules, colloids, and viruses.Nanofiltration (NF): NF membranes have even smaller pores than UF membranes and can selectively remove multivalent ions, organic matter, and color.Reverse Osmosis (RO): RO membranes have the smallest pores and are capable of removing virtually all dissolved solids and impurities, producing high-purity water.### Applications of Membrane Separation Technology in Industrial Wastewater Treatment.Membrane separation technology finds widespread applications in industrial wastewater treatment across various industries, including:Textile industry: Removal of dyes, chemicals, and heavy metals from wastewater.Pharmaceutical industry: Purification of wastewater containing active pharmaceutical ingredients and other contaminants.Food and beverage industry: Removal of organic matter, microorganisms, and other contaminants from wastewater.Metalworking industry: Treatment of wastewater containing heavy metals, oils, and suspended solids.### Advantages of Membrane Separation Technology.High efficiency and selectivity: Membranes can selectively remove specific contaminants while allowing other desired components to pass through.Compact and modular design: Membrane systems often require less space than conventional treatment methods and can be easily expanded or reconfigured as needed.Low energy consumption: Membrane filtration processestypically consume less energy compared to other treatment methods, such as distillation or evaporation.Environmental sustainability: Membranes can contribute to waste minimization by recovering valuable resources from wastewater and reducing the environmental impact of industrial discharge.### Limitations of Membrane Separation Technology.Membrane fouling: Membranes are susceptible to fouling by particles, organic matter, and other contaminants, which can reduce their efficiency and require regular cleaning or replacement.High capital investment: Membrane separation systems can involve significant capital investment, especially for large-scale applications.Operational challenges: Membrane systems require proper operation and maintenance to ensure optimal performance and longevity.### Conclusion.Membrane separation technology offers a powerful and versatile approach for industrial wastewater treatment, providing effective removal of contaminants, improved operational efficiency, and environmental sustainability.As research and development continue to advance membrane technology, its applications are expected to expand further, contributing to cleaner industrial wastewater streams and a more sustainable water management system.## 膜分离技术在工业废水治理中的应用。

Long-term operation of a partial nitritation pilot plant treating leachate with extremely high ammonium concentration prior to an anammox processRamon Ganiguéa,*,Jordi Gabarróa ,Alexandre Sànchez-Melsiób ,Maël Ruscalleda a ,Helio López a ,Xavier Vila b ,Jesús Colprim a ,M.Dolors Balaguer aaLaboratory of Chemical and Environmental Engineering (LEQUIA),Institute of the Environment,University of Girona,Campus Montilivi s/n,Facultat de Ciències,E-17071Girona,Catalonia,Spain bLaboratory of Molecular Microbial Ecology,Institute of Aquatic Ecology,University of Girona,Campus Montilivi s/n,E-17071Girona,Catalonia,Spaina r t i c l e i n f o Article history:Received 9March 2009Received in revised form 8June 2009Accepted 9June 2009Available online 3July 2009Keywords:Ammonium oxidising bacteria (AOB)Heterotrophic denitrification Nitrite oxidising bacteria (NOB)Operational strategy Partial nitritationa b s t r a c tThe goal of this work was to demonstrate the feasibility of treating leachate with high ammonium con-centrations using the SBR technology,as a preparative step for the treatment in an anammox reactor.The cycle was based on a step-feed strategy,alternating anoxic and aerobic conditions.Results of the studyverified the viability of this process,treating an influent with concentration up to 5000mg N–NH þ4LÀ1.An effluent with about 1500–2000mg N–NH þ4L À1and 2000–3000mg N–NO À2L À1was achieved,present-ing a nitrite to ammonium molar ratio close to the 1.32required by the anammox.Furthermore,taking advantage of the biodegradable organic matter,the operational strategy allowed denitrifying about200mg N–NO À2LÀ1.The extreme operational conditions during the long-term resulted on the selection of a sole AOB phylotype,identified by molecular techniques as Nitrosomonas sp.IWT514.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionLeachate generated from landfill sites is highly contaminated with a wide range of chemical contaminants.Among them,urban landfill leachate is usually characterised by high ammonium con-centrations and low biodegradable organic matter content (Kuli-kowska and Klimiuk,2008).Because of this,treating leachate through conventional nitrification–denitrification processes is eco-nomically expensive due to the high oxygen demand and the requirement of a supplemental external carbon source.Treatments based on anaerobic ammonium oxidation (anam-mox)metabolism may pose a more sustainable alternative to the treatment of such wastewater,due to the reduced aeration requirements and lower dosage of external organic carbon (Liang and Liu,2008).Eq.(1)presents the anammox stoichiometry (Strous et al.,1998):NH þ4þ1:32NO À2þ0:066HCO À3þ0:13Hþ!1:02N 2þ0:26NO À3þ2:03H 2O þ0:066CH 2O 0:5N 0:15ð1ÞPrior to the anammox process,the ammonium present inwastewater must be partially oxidised to nitrite (Eq.(2))by ammo-nium oxidising bacteria (AOB).Further nitrification to nitrate (Eq.(3)),carried out by nitrite oxidising bacteria (NOB),must beavoided in order to allow optimal N-removal by the anammox bac-teria (Yamamoto et al.,2008).In addition,biodegradable organic matter should be removed to avoid its negative effects on the sub-sequent anammox process (Chamchoi et al.,2008;Ruscalleda et al.,2008)NH þ4þ2HCO À3þ1:5O 2!NO À2þ3H 2O þ2CO 2ð2ÞNO À2þ0:5O 2!NO À3ð3ÞAccording to the literature,nitrite build-up can be achieved suc-cessfully by oxygen limitation (Aslan et al.,2009),as well as by high temperatures coupled with low sludge residence times (Fux et al.,2002;Hellinga et al.,1998;van Dongen et al.,2001).It can also be accomplished by inhibiting NOB with free ammonia (FA)and/or free nitrous acid (FNA)(Ganiguéet al.,2007;Lai et al.,2004).However,several studies (Fux et al.,2004;Villaverde et al.,2000)have reported problems in maintaining nitrite build-up over the long-term in such systems when the NOB becomes acclimatised to high concentrations of these inhibitory compounds.The aim of a partial nitritation system is to oxidise about half the influent ammonium to nitrite.In the particular case of highly ammonium-loaded wastewater as the landfill leachate,the ammo-nium and nitrite concentrations inside a partial nitritation reactor could be very high.This overcomes an operational problem,since AOB can be inhibited by the unionised forms of their substrate0960-8524/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2009.06.023*Corresponding author.Tel.:+34972183249;fax:+34972418150.E-mail address:ramon@lequia.udg.cat (R.Ganigué).Bioresource Technology 100(2009)5624–5632Contents lists available at ScienceDirectBioresource Technologyjournal homepage:www.elsevier.c o m /l o c a t e /b i o r t echand product,NH3and HNO2(widely described by Anthonisen et al.,1976;Ganiguéet al.,2007;Vadivelu et al.,2007;Van Hulle et al., 2007).Partial nitritation systems have been used extensively to treat sludge digester supernatants(Fux et al.,2002;Gut et al., 2006;Vázquez-Padín et al.,2009;among others),which present ammonium concentrations between500and1500mg N–NHþ4LÀ1.Nevertheless,inhibition can be a critical issue when deal-ing with landfill leachate,which can present concentrations up to6000mg N–NHþ4LÀ1(Kurniawan et al.,2006).In this light,any reduction in total nitrogen during the partial nitritation reactor must be observed as an opportunity to reduce the inhibition fac-tors and thus lower operational costs.Therefore,despite the low levels of biodegradable organic matter available in the leachate, the inclusion of anoxic phases during the feeding events may help reduce the inhibition of AOB by heterotrophic denitrification via nitrite.The primary aim of this paper is to demonstrate the feasibility of treating urban landfill leachate with extremely high ammoniumconcentrations(up to5000mg N–NHþ4LÀ1)by a partial nitritation-sequencing batch reactor(PN-SBR),as a step prior to an anammox reactor.Specifically,this study seeks to achieve the stable produc-tion of a suitable mixture of ammonium and nitrite,as well as to demonstrate the viability of long-term nitrite build-up in a bio-mass retention system.This study also focuses on harnessing the low levels of available biodegradable organic matter for denitrifica-tion purposes,and assesses the role of bicarbonate on the nitrita-tion process.Furthermore,the microbial populations involved in the aerobic processes of N-compound oxidation(AOB and NOB) have been discerned by DNA-based molecular techniques to better understand the partial nitritation process under these extreme conditions.2.Methods2.1.PN-SBR pilot plantThis study was conducted in a PN-SBR pilot plant,located at the LEQUIA facilities of the University of Girona.The pilot plant was comprised of a250L pilot-scale SBR operated at a minimum vol-ume of111L.The reactor was water jacketed,allowing tempera-ture control by means of a thermostated water plete mixture was achieved by means of a mechanical stirrer,and aera-tion was carried out using air diffusers(Magnum,from OTT System GmbH&Co.)located at the bottom of the reactor.Raw leachate was stored in a300L storage tank prior to treatment.The pilot plant was also equipped with a monitoring and control system. On-line data provided by pH,ORP,DO,and temperature probes (CPF81,CPF82and OXYMAX-W COS-41,from Endress-Hauser) were acquired by means of interface cards(PCI-1711and PCLD-8710from Advantech)and by our own software,which was devel-oped using Lab-ViewÒ.Program commands were transmitted to the pilot plant through another interface card(PCI-885from Advantech)and a relays output board,which controlled the switch on/off of all electrical devices and thus allowed the repetition of a previously defined operational cycle.2.2.Operational conditionsTemperature was maintained at36±1°C,and dissolved oxygen (DO)was controlled at a set-point concentration of2mg LÀ1during the aerobic reaction phases.The pH was kept below a maximum set-point value of8through the addition of hydrochloric acid (1M).The reactor was operated according to an anoxic–aerobic step-feed strategy.The cycle,with a total length of24h,consisted of14feeding events under anoxic conditions,followed each by an aero-bic reaction phase of85min.Total inflow was equally distributed among all feeding events.Therefore,the cycle could be divided into 14identical sub-cycles of100min,each consisting on15min of anoxic phase(feeding between4and14min)followed by85min of aerobic reaction.The cycle ended with a20min settling phase followed by20min of draw.Due to the high amount of suspended solids removed from the reactor by the outflow(393.7±179.4mg TSS LÀ1),no purge phase was carried out.Despite this,the average concentration of mixed liquor suspended solids(MLSS)during the study was 666.0±240.6mg TSS LÀ1with a volatile fraction of about66–75%.On the other hand,due to significant oscillations in the raw leachate ammonium concentration,influentflow had to be ad-justed to keep the nitrogen loading rate(NLR)within a suitable range of values.This caused significant hydraulic retention time (HRT)fluctuations,ranging from3to6days.Under these condi-tions the sludge retention time(SRT)was not a controlled system parameter,but was calculated considering reactor MLSS and efflu-ent suspended solids concentrations;its average value was 6.44±2.34days(ranging from3.1to12days).2.3.Analytical methodsTotal suspended solids(TSS;APHA-2540D),volatile suspended solids(VSS;APHA-2540E),chemical oxygen demand(COD; APHA-5220B),total organic carbon(TOC;APHA-S310),inorganic carbon(IC;APHA-S310),ammonium(N–NHþ4;APHA-4500-NH3.B-C5220B),total kjeldahl nitrogen(TKN;APHA-4500-Norg.B),nitrites(N-NOÀ2;APHA-4110B),and nitrates(N–NOÀ2;APHA-4110B)were all measured according to Standard Methods(APHA,2005).Biolog-ical oxygen demand(BOD)was measured using OxiTopÒsystem (from WTW),based also on Standard Methods(APHA-5210D).To-tal nitrogen was calculated as the sum of N–TKN,nitrite,and ni-trate concentrations as mg N–TN LÀ1.Conductivity was measured by a conductimeter(EC-Meter Basic30+from Crison).Concentrations of Free Ammonia(FA)and Free Nitrous Acid (FNA)were calculated as a function of pH,temperature,and Total Ammonium as Nitrogen(TAN)for FA,or Total Nitrite(TNO2)for FNA,according to Anthonisen et al.(1976).The specific oxygen uptake rate(OUR,mg O2g VSSÀ1hÀ1)was calculated according to Puig et al.(2005)from on-line measure-ment of the drop in dissolved oxygen when no airflow was supplied.2.4.Molecular analysis of AOB and NOBDNA isolation.DNA was isolated from the collected samples using DNeasy Blood&Tissue commercial kit(Qiagen,Venlo,The Netherlands),in accordance with the manufacturer’s instructions for Gram-negative microorganisms.The DNA isolation efficiency was verified in a0.8%(w/v)electrophoresis gel.Polymerase chain reaction(PCR).PCR analyses were carried out with a GeneAmp PCR system2700thermocycler(Applied Biosys-tems,Perkin–Elmer,CA,USA),using the PCR programs as described in the respective references of the primer sets.AOB populations were detected through amplification of the16S rDNA operon with the CTO primer set:CTO654R(Kowalchuk et al.,1997)coupled to a CTO189F mix,which is a2:1mixture of CTO189F A/B and CTO189F C.Detection of Nitrobacter and Nitrospira was also achieved by amplification of the16S rDNA operon.The primer set FGPS872R coupled with FGPS1269F(Degrange and Bardin,1995)was used for Nitrobacter species,while Nitrospira species were amplified with NSR1113F in combination with NSR1264R(Dionisi et al., 2002).R.Ganiguéet al./Bioresource Technology100(2009)5624–56325625Cloning,sequencing and identification.For cloning procedures, PCR products were ligated to pGEM Teasy vectors(Promega)and transformed into Top10Escherichia coli cells following manufac-turer instructions.The vectors were then isolated from E.coli colo-nies growing in LB+Ampicillin medium using the Ultraclean 6minute Mini Plasmid Prep Kit(MoBio).Cloning was only neces-sary to achieve DNA sequences from amplifications performed with CTO primers.PCR products obtained with Nitrobacter and Nitrospira primer sets were sequenced directly,since only one phylotype was detected in each product.16S rDNA fragments were sequenced by the Macrogen Service(Macrogen Ltd.,Seoul Korea, )and the partial sequences were compared with the National Center for Biotechnology Information(NCBI) database using the BLASTn algorithm tool(Altschul et al.,1990) to identify the phylotypes.The sequences were also submitted to the Greengenes()via the Bellerophon software to check for the presence of chimeras.2.5.Raw leachateThe raw leachate treated in this study,which came from the Corsa urban landfill site(41°602800N,1°70400E;Reus,Spain),pre-sented a highly variable composition.The concentration range and mean values of the principal chemical compounds,along with the electrical conductivity(EC)and pH,are summarised in Table1.2.6.Experimental procedureThe SBR was inoculated with a mixture of nitrifying sludge from the Sils-Vidreres municipal WWTP(41°4705800N,2°450700E;Cata-lonia,Spain)and the Orís urban landfill leachate treatment plant (42°0302800N,2°1401500E;Catalonia,Spain).After a brief start-up,the PN-SBR was operated under an anoxic–aerobic step-feed strategy.From the process operation point of view,the450-day study can be divided into three periods.First,the reactor was operated without any bicarbonate adjustment(Period I).Once the low per-centage of ammonium being oxidised to nitrite was observed, bicarbonate dosage(NaHCO30.5M)was implemented on day59 to increase the conversion(Period II).This addition was made when pH decreased below a set-point value of7.2.Due to the low reliability of this control,on day220,solid bicarbonate began to be dosed at the influent.The amount of bicarbonate added was calculated based on the stoichiometrical requirements for achiev-ing a suitable effluent to feed an anammox reactor.During Period III,the minimum pH was also controlled by the bicarbonate solu-tion dosage.3.Results and discussionA suitable nitrite to ammonium ratio in the influent of around1.32is crucial for proper operation of an anammox reactor.With this objective,partial nitritation was conducted in an industrial-scale PN-SBR to achieve the desired conversion,as well as to high-light the keys to this proper operation.In a previous study(Ganig-uéet al.,2008),the step-feed strategy was proven to be a good cycle design for achieving stable partial nitritation,since the distri-bution of the inflow across different feeding events avoided load-ing shocks and subsequent important pH variations in the reactor.Nevertheless,because of the high nitrogen concentrations in this study,anoxic phases were included during feeding events to promote heterotrophic denitrification.3.1.PN-SBR performanceThe reactor was successfully operated for450days treating raw urban landfill leachate,according to an anoxic–aerobic step-feed strategy.Fig.1presents the evolution of influent ammonium con-centration and nitrogen loading rate(NLR)(Fig.1a)and the con-centration of effluent nitrogen compounds(Fig.1b)and bicarbonate(Fig.1c)along the whole study.Fig.1a clearly shows the notable oscillations in influent ammo-nium concentration throughout the study,ranging from2000to 5000mg N–NHþ4LÀ1.These variations were due to the raw leach-ate supplied by the landfill site.The initial NLR was around 0.85kg N mÀ3dÀ1,ultimately reaching a value of1.2kg N mÀ3dÀ1 by the end of the study.Fig.1b reveals that partial nitritation of influent ammonium was achieved over the450day period,preventing further nitriteoxidation to nitrate(NOÀ3concentration throughout the studywas always below25mg N–NOÀ2LÀ1).A more in-depth analysis shows that during Period I(no exter-nal bicarbonate addition)the concentration of effluent ammonium was much higher than nitrite,with conversions around25%.In this period,as presented in Fig.1c,the available inorganic carbon in the raw leachate was much lower than the theoretical stoichiometric amount required for achieving the desired effluent composition. To overcome this,bicarbonate was added during Period II to en-hance the oxidation of ammonium to nitrite.Thus,nitrite concen-tration substantially increased,achieving an effluent with about1500mg N–NOÀ2LÀ1and1200mg N–NHþ4LÀ1.Nevertheless,reac-tor conversion was quite unstable(ranging from22%to78%)due to the imprecise control of the bicarbonate dosing,and the nitrite to ammonium ratiofluctuated considerably.Furthermore,at the end of Period II(195th day)ammonium started to accumulate since the control system was unable to supply enough bicarbonate, reaching at the end of this period ammonium concentrations high-er than3000mg N–NHþ4LÀ1.The problem was solved by the addi-tion of solid bicarbonate at the pre-treatment tank(Period III) resulting in an enhanced and stabilised nitrite production.This method resulted in the production of a suitable influent to feed an anammox reactor during Period III,with ammonium and nitriteconcentrations reaching about1800mg N–NHþ4LÀ1and2600mg N–NOÀ2LÀ1.Finally,it should also be mentioned that all bicarbonate sup-plied to the reactor was removed from the system,as presented in Fig.1c,mainly by the AOB activity.All biodegradable organic matter should be removed in the par-tial nitritation step.Fig.2presents the organic matter evolution ofTable1Leachate characteristics throughout the study.Compound Units Range Mean±rAmmonium,NHþ4mg N–NHþ4LÀ12237–49383772±956Nitrite,NOÀ2mg N–NOÀ2LÀ10.0–1.20.2±0.5Nitrate,NOÀ3mg N–NOÀ3LÀ10.0–8.0 1.4±3.2Alkalinity mg HCOÀ3LÀ12059–11,2238638±3314Total Kjeldahl nitrogen,TKN mg N LÀ12494–55404058±987Chemical oxygen demand,COD mg O2LÀ12480–70404357±692Biological oxygen demand,BOD5mg O2LÀ1230–1025810±278Total organic carbon,TOC mg C LÀ11509–24201946±457Total carbon,TC mg C LÀ12977–38123541±385Inorganic carbon,IC mg C LÀ11336–19041571±296Conductivity,EC l S cmÀ160,600–70,50068,065±1863pH–7.48–8.568.11±0.205626R.Ganiguéet al./Bioresource Technology100(2009)5624–5632both influent and effluent in terms of TOC values and removal percentage.The raw leachate presented influent TOC concentrations rang-ing from1500to3000mg C LÀ1.The biodegradable fraction of this organic matter was removed in the PN-SBR process(either aerobi-cally or anoxically).Nevertheless,except for the periods with oper-ational problems,TOC concentrations at the effluent were higher than1000mg C LÀ1throughout the study.The low TOC removal percentage(less than50%)was related to the high refractory or-ganic matter fraction in the raw leachate.This was corroborated by a mean BOD u to COD ratio of0.32in the raw leachate and a sol-uble BOD u of zero in the effluent.3.2.Heterotrophic denitritationThe nitrogen balance and organic matter removal over a period of stable operation were assessed to estimate the nitrogen removal by denitritation.Based on this stability requirement the assess-ment was done between days145th and335th(Periods II and III),neglecting all data biased due to influent composition changes. The theoretical amount of COD necessary for denitrification was then calculated and plotted,based on Tchobanoglous et al. (2003),obtaining a theoretical ratio of1.97g COD per g N–NOÀ2. Results are depicted in Fig.3.The average amount of nitrogen eliminated in the system was only about200–250mg N LÀ1.As can be observed in Fig.3a,15% to20%of the influent nitrogen was removed by denitritation be-tween days145and225(Period II),declining to5%over the next 110days(Period III).Fig.3b presents the amount of COD elimi-nated in respect to the total organic matter at the influent.The COD removed from the system over this190-day period was about 25–30%.During Period II,more than half of the biodegradable or-ganic matter was used for denitrification purposes,and this value plummeted to less than10%in Period III.It is important to high-R.Ganiguéet al./Bioresource Technology100(2009)5624–56325627light that denitrification performance declined when solid bicar-bonate began to be dosed to the influent(day225).There is no lit-erature regarding any direct harmful effect of NaHCO3on heterotrophic bacteria.However,indirect effects as the high in-crease in conductivity may have negatively affected heterotrophic bacteria.3.3.Assessment of influent and effluent molar ratiosFig.4shows the evolution of HCOÀ3:NHþ4influent molar ratio(Fig.4a)and the NOÀ2to NHþ4effluent molar ratio(Fig.4b).Notethat bicarbonate supplied by the pH control in Period II has alsobeen taken-up in the influent.During Period I,the influent presented HCOÀ3:NHþ4molar ratiosaround0.6that lead to effluent molar ratios between0.18and0.41,far from the stoichiometric requirements of the further anammoxprocess(1.32mol of NOÀ2per mole of NHþ4).The external NaHCO3dosage during Period II resulted in an increase in HCOÀ3:NHþ4influ-ent molar ratio,which manifested in an increased nitrite to ammo-nium effluent molar ratio.Nevertheless,the dosage strategyinduced significantfluctuations,with values ranging from0.3to3.7.Preconditioning the influent with solid NaHCO3addition(Per-5628R.Ganiguéet al./Bioresource Technology100(2009)5624–5632iod III)provided a more stable HCOÀ3:NHþ4influent molar ratio;thiskept the effluent molar ratio within a suitable range over the230days,between1and1.5mol NOÀ2per mole of NHþ4with peaksup to2.With the aim of further studying the relationship betweeninfluent HCOÀ3:NHþ4molar ratio and effluent NOÀ2:NHþ4molar ratio,a data subset was selected wherein the reactor operated under sta-ble conditions.Fig.5shows the experimental nitrite to ammonium effluent molar ratio versus influent bicarbonate to ammonium mo-lar ratio.In addition the theoretical effluent NOÀ2:NHþ4molar ratiohas been calculated based on the AOB stoichiometry(Eq.(2)).As can be seen,experimental resultsfit quite well with the stoi-chiometric curve,validating bicarbonate as the key to control the conversion of ammonium to nitrite.Experimental points deviating from the theoretical behaviour gave information about the process performance and the ongoing phenomena.When the effluent mo-lar ratio was lower than the theoretical,this could be attributed to a bias linked to the heterotrophic denitrification process and/or a bicarbonate loss by CO2stripping.On the other hand,a higher than theoretical effluent ratio could be related to ammonium removal from the system due to NH3stripping,and/or to additional CO2 coming from the organic matter elimination,which may allow a higher conversion.3.4.Determination of AOB and NOB populationsOne of the aims of this study was to evaluate the initial AOB and NOB populations and analyze their evolution over the course of aR.Ganiguéet al./Bioresource Technology100(2009)5624–56325629long-term operation.Given the high ammonium and nitrite con-centrations in the bulk media (both higher than 1000mg N L À1),the elevated salinity (always above 60,000l S cm À1)and high tem-perature (36°C),identifying the AOB capable of resisting such ex-treme conditions would represent an important microbiological feature with potential environmental implications.The microbial community analysis also intended to determine whether or not NOB organisms were present in the community after long-term system operation.With these purposes,five samples were col-lected and the genomic DNA was isolated and processed.R0was an aliquot from the initial sample from the mixture of nitrifying sludges used to inoculate the reactor,whereas R192,R288,R415and R450were obtained from the PN-SBR on days 192nd,288th,415th and 450th,respectively.All DNA isolations were screened by PCR using different combi-nations of primers,each of them specific for a bacterial group.Po-sitive PCR amplifications with the CTO primer sets confirmed thepresence of AOB during the entire working period.Based on these results,only R0and R450were cloned since no changes were de-tected in the PCR products between days 192and 450(data not shown).16S rDNA sequences created from the cloning procedure showed a high homology with known uncultured bacteria phylo-types,all of them related to Nitrosomonas -like species.Phylotypes detected in R0were grouped into five Organism Taxonomic Units (OTU),while all R450sequences clustered together,indicating that only one of the OTUs present in the inoculum became dominant in the reactor (Table 2).This OTU showed a high similarity (98–99%)with Nitrosomonas sp.IWT514,which was therefore positively se-lected by the severe operational conditions in the reactor.Despite the stable nitrite build-up over the long-term,positive PCR amplifications were also obtained for NOB in all the DNA iso-lations using FGPS and NSR primer sets,which were chosen to search for the main NOB groups in wastewater treatment plants,respectively Nitrobacter and Nitrospira .All the sequences from each amplification belonged to the same phylotype,and no changes were detected between the inoculum and the reactor samples.Se-quences for Nitrobacter showed high homology with Nitrobacter winogradskyi (99%),while Nitrospira sequences perfectly matched (100%homology)with Candidatus Nitrospira defluvii.It was ini-tially expected that such extreme conditions would completely re-move NOB from the reactor.However,results proved that both Nitrobacter and Nitrospira were still present in the system and coexisted after 450days of operation.Therefore,despite being strongly inhibited,changes in environmental conditions may lead to the development of NOB populations and the expression of ni-trite oxidation activity.3.5.Cycle analysis:on-line parametersOn-line parameters,such as pH,DO and specific OUR,provide information about the biological activity and the process perfor-mance.Thus,in order to clearly understand the behaviour of the system a specific presentation of an aerobic–anoxic sub-cycle (100min of the whole 1440min cycle)is depicted in Fig.6.The plot is comprised of three sections:aerobic reaction (white area),anoxic reaction (stripped area)and feeding in anoxic conditions (gray stripped area).Table 2OTUs obtained from the successfully identified AOB phylotypes throughout PCR with CTO primers.OTUClosest BLASTn phylotype NCBI accession number%phylotypes R01Uncultured bacterium clone IIIEA1-rp-O2nit gi|161367780|gb|EU267435.1|272Nitrosomonas sp.Is32gi|40994846|emb|AJ621027.1|273Uncultured bacterium clone S_1gi|121592404|gb|EF175894.1|204Uncultured bacterium clone 58gi|89348071|gb|DQ413117.1|105Nitrosomonas sp.IWT514gi|13958147|gb|AF363293.1|AF36329310R4505Nitrosomonas sp.IWT514gi|13958147|gb|AF363293.1|AF3632931005630R.Ganiguéet al./Bioresource Technology 100(2009)5624–5632。