ࡱ>      5@ bjbj22 4XX2222222F & & & &t~'LF(777789; E=yyyyyyy$Ry2/X88/X/Xy2277ananan/X.2727yan/Xyan,ann22n7( @  &]gnr0nKnnFF2222n 2r(M>AFanK{PM>M>M>yyFF$ &KnFF &World Journal of Microbiology and BiotechnologySpringer Science+Business Media B.V.200710.1007/s11274-007-9405-8Original Paper Nitrate removal with bacterial cells attached to quartz sand and zeolite from salty wastewaters LucijaFoglar1 HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "ContactOfAuthor1#ContactOfAuthor1"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/contact.gif" \* MERGEFORMATINET , LaszloSipos2 and NenadBolf3 (1)Division of Industrial Ecology, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruliev trg 19, HR-10000Zagreb, Croatia (2)Division of General and Inorganic Chemistry, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruliev trg 19, HR-10000Zagreb, Croatia (3)Division of Measurement and Process Control, Faculty of Chemical Engineering and Technology, University of Zagreb, Maruliev trg 19, HR-10000Zagreb, Croatia  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/contact.gif" \* MERGEFORMATINET LucijaFoglar Email:  HYPERLINK "mailto:lfoglar@pierre.fkit.hr" lfoglar@fkit.hrReceived: 5September2006Accepted: 6April2007Published online: 3June2007 AbstractA mixed bacterial culture was acclimated to the removal of high nitrate-N concentrations (100750mg NO3 "-NL"1) from salty wastewaters. The experiments were carried out under anoxic conditions in the presence of 0.5, 1.5 and 3% (w/v) NaCl at different temperatures. The acclimated mixed bacterial culture was attached to quartz sand and zeolite. Denitrification was monitored in a continuous-flow bioreactor at different hydraulic retention times (HRT). Nitrate removal with cells attached to quartz sand and zeolite was completed at HRT of 167h and 25h respectively. Then brine denitrification with bacterial cells attached to zeolite was monitored for 85days. Under the increased nitrate loading rate, nitrate removal was above 90%. Furthermore, during denitrification, not more than 0.5mg NO2 "-NL"1 could be produced. It can be concluded that nitrate removal with the cells attached to zeolite is economically and operationally a promising solution to denitrification of brine wastewaters. KeywordsDenitrification-Spent brine-Mixed bacterial culture-Anoxic conditions- Quartz sand- Zeolite  Introduction Nitrate-contamination of groundwater used for drinking is a health hazard due to harmful effects of nitrate (e.g. asphyxia and methemoglobinemia of infants) including the increased risk of cancer development (Shuval and Gruener  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR32#CR32" 1977; Weisenburger etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR35#CR35" 1991; Crespi etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR4#CR4" 1991). Although nitrate content in pristine groundwater is usually insignificant a recent survey of wells revealed its detectable levels in more than one half of them, whereas in 2.4% of the rural ones it exceeded the highest contaminant level of 10mg NO3 "-NL"1 (Briskin etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR3#CR3" 1991). This is so all over the world where human activities, particularly agriculture, are continuously increasing contamination with nitrates. Ion exchange, biological denitrification, and membrane desalting by reverse osmosis, as well as hyper filtration or electrodialysis are common methods for removal of nitrates from water supplies (Shrimali and Singh  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR31#CR31" 2001; Wisniewski etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR36#CR36" 2001). Although technically and economically efficient, nitrate removal by ion exchange is linked with significant problems. The biggest one is disposal of spent regenerant or waste brine from regeneration of exhaust resins. Brine waste contains high concentrations of nitrate-N and NaCl, which are usually within 100900mg NO3 "-NL"1 and 3 6% (w/v) respectively (Clifford etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR5#CR5" 1993; Okeke etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR24#CR24" 2002; Chang etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR6#CR6" 2004). With regard to the ever increasing environmental concerns and relevant legislation, its handling requires appropriate methods. Consequently, nitrate removal from brine wastewaters is in the focus of the research. Most of the reactors used for brine denitrification in the previous studies were the sequencing batch reactor, upflow sludge blanket reactor (van der Hoek  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR11#CR11" 1987, Clifford etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR5#CR5" 1993; Okeke etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR24#CR24" 2002; Chang etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR6#CR6" 2004) or a different type of the reactor used to trap the microorganisms in the polymer carrier (Yang etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR37#CR37" 1995) and immobilized or attached by physical procedures such as adsorption on the surface of an insoluble porous material e.g. sand, plastic or ceramic (Mateju etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR21#CR21" 1992). Immobilization or attaching of microorganisms to a solid is very attractive because of its simplicity. In this context, the use of zeolitic materials is of great interest. Zeolites and the related materials have interesting and potentially useful properties, such as large surface area (between 200 and <"1000m2/g), they can be hydrophobic or hydrophilic create electrostatic interactions, have different ion-exchanged forms, and exert mechanical and chemical resistance (Nikolaeva etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR23#CR23" 2002; Jung etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR15#CR15" 2004). The additional advantages are their ease of water dispersion/recuperation and high water uptake capacity. Therefore, compositional and structural varieties of molecular sieves provide a powerful tool for tuning up carriers properties. Nitrate is efficiently removed with the attached bacterial cultures when the external organic carbon source is added. Most studies in biological denitrification suggest the use of methanol, ethanol or acetic acid as the organic carbon source (van der Hoek  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR11#CR11" 1987; Mohseni-Bandpi and Elliott  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR22#CR22" 1998; Bae etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR2#CR2" 2002; Nikolaeva etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR23#CR23" 2002; van Rijn etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR28#CR28" 2006). Methanol is the least expensive and the most efficient carbon source used for denitrification. Present work has studied biological denitrification of synthetic wastewater with high concentrations of nitrate and sodium chloride. It investigated the potentials of the used mixed bacterial culture, which utilizes methanol in nitrate removal. The acclimated mixed bacterial culture was attached to quartz sand and zeolite. Removal of nitrate in the continuous-flow bioreactor by bacterial cells attached to the quartz sand and zeolite was investigated at different HRT and at different nitrate loading rates. The aim was to determine the reactors stability and denitrification potentials.  Materials and methods Organisms and culture media The microorganisms originated from the mixed liquid from aerobic municipal sewage treatment plant in Velika Gorica, Croatia, and active sludge of the two-stage anaerobic-aerobic wastewater treatment plant Anamet in Savski Marof, Croatia. The mixed liquid and active sludge (from 50ml samples) were mixed and centrifuged at 12500rpm (12557נg) and 5C for 10min. The obtained biomass was washed twice, diluted with mineral medium, refrigerated at 4C and stored for further use. The mixed culture was adjusted to nitrate ions (up to 750mg NO3 "-NL"1) and NaCl (up to 3% w/v) at pH=6.8 and 37C under anoxic conditions for 30days before being used under anoxic batch denitrification conditions. Term anoxic means that medium (synthetic wastewater) was not flushed to remove present dissolved O2 and during the tests dissolved O2 present in medium was only source of oxygen. After every experiment the biomass suspension was prepared as described and used as the inoculum in the next experiment. Composition of the medium was slightly different from the originally proposed one by Wang etal. ( HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR34#CR34" 1995). The mineral medium contained (gL"1): K2HPO4 2.5; KH2PO4 1; MgSO47H2O 0.1; NaCl 5 and deionised water up to 1L. It was used to prepare synthetic wastewater (SW) and synthetic brine wastewaters (BW1 and BW2). During preparation of BW1 and BW2 in the mineral medium, additional 10gL"1 NaCl and 25gL"1 NaCl were added. Consequently, brine wastewaters contained 1.5 and 3.0% (w/v) of NaCl respectively. The solutions were autoclaved and allowed to cool to room temperature before adding NaNO3 and CH3OH. For each experiment nitrate-N from 100 750mg NO3 "-NL"1 (the stock solution was aqueous solution of NaNO3 containing nitrate-N 10gL"1) and 2moles of methanol per mole of nitrate were added separately. The excess methanol was used to avoid carbon-limited conditions. Phosphate salts in the mineral medium were used as buffer. This provided unchanged pH of the prepared wastewaters throughout the experiments. Feed solution used during the tests in the continuous-flow bioreactor was BW2 containing 100700mg NO3 "-NL"1 and 2 moles of methanol per mole of nitrate. The carriers of the microorganisms and cell attachment The acclimated mixed bacterial culture was collected by centrifugation (Sigma 3K15, 12500g, 10min, 5C) and resuspended in the mineral medium (3% (w/v) NaCl). Quartz sand and zeolite (35mm), used as carriers of microorganisms, were washed with HCl (pH=2) and then with deionised water to achieve neutral pH. The bioreactor was filled with 200g of quartz sand or zeolite. Natural zeolite (Clinoptilolite) was provided from the large sedimentary deposit in Donje Jesenje, Croatia, and quartz sand from the plant Tovarna duika Rue, Puconci, Slovenia. To attach the bacterial cells to the carrier, the mineral medium with the mixed bacterial culture was pumped and recirculated with a peristaltic pump through the bioreactor filled with carrier over 48h. The carriers were then washed with sterile mineral medium to remove excess bacterial cells. Washing was discontinued when microscopic examination (at 1000) of eluate showed that the bacterial cells were brought to zero. Denitrification tests started at that point. Experimental set-up The experiments in the first run were performed in 0.5dm3 closed, sterile serum bottles. Each bottle was filled with 0.3L of synthetic wastewater (SW, BW1 or BW2) and 0.1dm3 of biomass suspension. The initial inoculum concentration was measured and controlled photometrically by monitoring of optical density at OD540 in suspension samples. The stopper was equipped with a thermometer and two disposable syringes with needles, one for measuring the produced gas and the other for sampling. During denitrification tests, the bottled inoculum as a static culture was placed in an air thermostat at selected temperature. All experiments were performed under anoxic conditions. Headspace gas and dissolved oxygen in the medium were not flushed to remove O2. Thus, at the beginning, the mixed bacterial culture consumed the initially present O2. The first part of the experiment examined the effect of various nitrate concentrations (100750mg NO3 "-NL"1), of NaCl concentrations (0.5, 1.5, and 3.0%), and of temperature on biological denitrification. For that purpose the bottles with SW, BW1 and BW2 (pH=6.8) were incubated at 20C, 28C and 37C. Previous to the second run, nitrate adsorption on quartz sand or zeolite was investigated. For that purpose, the tests were performed in sterile 0.5L Erlenmeyer flasks filled with 1g of carrier and 0.1L of synthetic wastewater BW2 (100 750mg NO3 "-NL"1) and closed with rubber stoppers. For sampling purposes the stoppers were equipped with a disposable syringe supplied with a needle. The tests were carried out at 25C on the rotary shaker at 80rpm over 24h. The samples were taken at beginning of the experiment, at 5, 15, 30 and 60min intervals and then in regular 4h intervals over 24h. In the second run, brine wastewater denitrification was carried out in the bioreactor filled with quartz sand or zeolite. The used apparatus was similar to the previously described one (Foglar and Briki  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR9#CR9" 2003). In the 0.3dm3 bioreactor (diameter 54mm and height 200mm) the barrier-grid was placed above the magnetic mixer. Then the bioreactor was filled with 200g of quartz sand or zeolite to the respective carrier volume and void volume of 144and 156mL. Each test began with addition of BW2 (156mL) as a batch test. The initial nitrate concentration was 100mg NO3 "-NL"1. Continuous flow of feed started after complete removal of nitrate. For continuous cultivation, synthetic brine wastewater was pumped with a peristaltic pump at different flow rates into the bottom of the reactor to give different hydraulic retention times (HRT). Incubation was conducted at 252C, pH 6.8, and the agitation speed of 400rpm (only for feed solution mixing) under anoxic conditions. Nitrate removal from BW2 with bacterial cells attached to zeolite (the initial CFU value was 4נ107CFUg"1) was then continuously monitored under same incubation conditions over 85days. During that denitrification process nitrate concentration in the feed was gradually increased from 100mg NO3 "-NL"1 (first 30days) to 700mg NO3 "-NL"1. All the experiments (except long-term denitrification of BW2) were performed in duplicate and the data reported here represent their average values. The nitrate, nitrite and the dissolved oxygen measured during duplicate measurements differed by 0.01 0.5mgNL"1 and 0.1mg O2L"1 respectively. The confidence intervals are computed based on the sample mean and sample standard deviation. The number of cells determined by plate count differed by less than 5%. Analytical methods During nitrate removal from wastewaters, the microbial growth was monitored and their growth kinetics was established. To study the kinetics of nitrate removal from the medium, the bottle and the reactor contents were sampled at the preset time and processed immediately. Concentration of the dissolved oxygen (corrections for the atmospheric local pressure was not calculated) and pH of wastewaters were monitored by the oxygen-meter MA 5485 and pH-meter MA 5750 (Metrel, Horjul, Slovenia). Liquid samples were filtered through the 0.20m sterile syringe filters immediately after sampling and used for nitrate and nitrite analysis. Nitrate concentration in wastewater during the experiment was monitored spectrophotometrically on Hach DR/2400 (Hach Company, Loveland, Colorado, USA) by chromotropic acid method at =410nm (Standard Methods  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR33#CR33" 1989). Nitrite (Hll  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR12#CR12" 1979) was determined by the absorbance measurements at =500nm on the photometer (MA 9510 Iskra, Kranj, Slovenia). Chemical oxygen demand (COD) and biomass dry weight were determined according to the Standard Methods ( HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR33#CR33" 1989) for expressing methanol and biomass concentrations. During determination of the biomass dry weight liquid samples (20mL) were filtered through the 0.45m sterile filters and washed with 0.5L sterile water. The washed biomass was carefully scraped off and transferred with 1mL of sterile deionised water (in sterile conditions) to the previously dried and weighted small Petry dish, and dried at 1041C. The numbers of bacterial colonies (CFU) in wastewater samples and the number of cells attached to the carriers was determined by plate count on the standard nutrient agar and synthetic wastewater (SW, BW1 or BW2) solidified with the addition of 1.5% (w/v) agar-agar, after repeated dilution with NaCl (m/V ratio=9gL"1). The carriers were weighed (1g-wet weight) in the sterile Erlenmeyer flasks and 100mL of sterile 0.9% NaCl solution was added. The flask was agitated vigorously with vortex over 30min on the magnetic stirrer in order to remove the bacteria from quartz sand or zeolite particles. The resulting suspension was serially diluted in the sterile 0.9% NaCl solution and triplicate aliquots were plated on the standard nutrient agar and synthetic wastewater agar. After incubation over two days at 37C, all plates containing 30150 discrete colonies were selected for determination of the initial cell number by plate count. Different denitrifiers in the mixed bacterial culture were distinguished according to their colony forms and by optical microscopy after Gram staining. Bacterial species isolated as pure cultures were identified by API 20 E and API 20 NE systems and according to Bergeys Manual of Determinative Bacteriology (Holt etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR13#CR13" 1994). According to literature data (Casey etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR7#CR7" 1997; Li etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR20#CR20" 2001; Sarioglu and Horan  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR30#CR30" 2001) and obtained results, specific constant rate is determined by the widely applied Monod equation. It was employed for calculating kinetic constants using data from batch experiments:  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/11274_2007_9405_Article_Equ1.gif" \* MERGEFORMATINET (1)where r D is the rate of nitrate utilization (mg NO3 "-NL"1h"1), C N ( nitrate concentration (mg NO3 "-NL"1), K s  the half-saturation constant (mg NO3 "-NL"1), and k D is the maximum rate of nitrate utilization (NO3 "-NL"1h-1), which include the influence of microbial concentration. The kinetic parameters of the Monod equation were determined using the Nelder-Mead simplex method of non-linear parameter search incorporated in Matlab program. The initial guess of the kinetic parameter is entered into the program. Using this set of parameters the response curves are generated by the Runge-Kutta (IV) numerical integration method. Once the optimal kinetic parameters were established, the final optimal theoretical curve was compared with the experimental data plot. During denitrification in the continuous-flow stirred reactor, the NO3 "-N removal rate, COD removal rate, volumetric loading rate, and volumetric denitrification rate were calculated as follows:  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/11274_2007_9405_Article_Equa.gif" \* MERGEFORMATINET where (Cnitrate-N)infl. and (Cnitrate-N)effl represented the influent and effluent nitrate nitrogen concentrations (mg NO3 "-NL"1). The dilution rate, D (h"1) and hydraulic retention time, HRT (h) were calculated from the influent wastewater flow rate value, R (mLh"1) and the void (working) reactor volume V (mL). For determination of initial value of D (0.006h"1) was used previously determined value of  max (maximum specific growth rate), obtained during the conducted batch tests.  Results and discussion Acclimation of mixed bacterial culture for nitrate removal from brine wastewaters The initial nitrate concentration (100, 500 and 750mg NO3 "-NL"1), in the presence of 2moles of methanol per mole of nitrate, under static conditions at 37C was removed from SW during 3, 4 and 11days respectively. Nitrite content in the SW samples generated during the first 48h was 0.1, 0.13 and 0.15mg NO2 "-NL"1 respectively. By the end of the tests it was completely removed. The microbial biomass concentration (expressed as biomass dry weight) increased from initial 100mgL"1 to 300mgL"1 and showed that the mixed bacterial culture was able to utilize methanol and remove nitrate from SW. The pH of SW was regularly monitored. Throughout all the experiments its value had been maintained at 6.8 with phosphate salts, K2HPO4 and KH2PO4, acting as a buffer system. Next run was performed with BW1 and BW2 at different temperatures. At 37, 28 and 20C, 750mg NO3 ""NL"1 was removed from BW1 within 8, 13 and 35days respectively. Similar was recorded during denitrification of BW2. At 37C and 28C nitrate was removed from BW2 within 11 and 15days respectively (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1A). At lower temperature, nitrate removal was slower taking 40-days time. Obviously, the temperature of 20C was too low for satisfactory removal of nitrate. The experimental and model results of the nitrate nitrogen concentration during denitrification were plotted in Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1A. The batch kinetic analysis shows a close agreement between the experimental data and the predicted values. Obtained k D and K s values for denitrification at 37C were 0.0844mg NO3 --NL"1h"1 and 5.1810"5mg NO3 --NL"1 . Since K s value was very small in comparison to nitrate concentration (C N ), denitrification approaches a zero order reaction with respect to nitrate concentration, so Eq. 1 becomes r D =k D . This observation is similar to other published data (Wang etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR34#CR34" 1995; Casey etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR7#CR7" 1997; Li etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR20#CR20" 2001; Sarioglu and Horan  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR30#CR30" 2001). The comparison of experimental results, obtained during denitrification of BW1 and BW2 (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1), indicated that at higher NaCl concentrations denitrification was not significantly slower and that the volume of the generated nitrite was not considerably increased. That might have been due to the acclimation of microbial culture, used after every experiment as the inoculums in the next experiment.  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig1_HTML.gif" \* MERGEFORMATINET  Fig.1Removal of 750mg NO3 "-NL"1 during the denitrification at pH=6.8 and in the presence of 3% NaCl, at different incubation temperatures, 37C (%), 27C ( INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figa_HTML.gif" \* MERGEFORMATINET ) and 20C (%). (A) Experimental (symbols) and modelled Eq. 1 (lines) nitrate-N concentrations, (B) biomass production, (C) nitrite-N and (D) dissolved O2 concentrations  Generation of nitrite is known to be one of the main problems of direct biological denitrification (Almeida etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR1#CR1" 1995). Therefore, its levels had been regularly checked (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1C). It was lower or near the proposed maximum targeted contaminant level of 1.0mg NO2 "-NL"1 (Mateju etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR21#CR21" 1992) for denitrification processes conducted at 37C and 28C. At lower temperature (20C) it increased up to 1.7mg NO2 "-NL"1 by day 25, after which its ions had been gradually removed over 40days. The kinetic of biomass production was monitored as well (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1B) and the fastest microbial growth was observed at 37C. The comparison of CFU, grown on the standard nutrient agar and on the solidified wastewater solution, during denitrification tests showed that almost equal CFU values was assigned. Consequently, it was concluded that the mixed bacterial culture contained denitrifying bacteria. Microscopic examinations indicated that only five different types of bacteria were dominant in wastewaters. Three types were rod-shaped, and one was a short coccobacillus. Gram staining showed that they were Gram-negative. The mixed bacterial culture contained one Gram-positive long rod-shaped bacterium, too. The identified Pseudomonas sp. and Paracoccus sp. were the only true denitrifiers (Zellner etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR38#CR38" 1995; Glass and Silverstein  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR10#CR10" 1998; Foglar and Briki  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR9#CR9" 2003). Paracoccus and Pseudomonas bacterial genera being such, their presence in mixed culture obviously accomplished complete nitrate removal with minimum nitrite accumulation. The analysis of dissolved oxygen (DO) from the samples, performed during the denitrification tests showed that it gradually dropped to approximately 0.3mg O2L"1 and remained at that level (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig1#Fig1" 1D). During these experiments, almost 50% nitrate-N was removed at DO concentration above 2mg O2L"1. Therefore, it can be assumed that the initially present dissolved oxygen did not inhibit the studied denitrification process. The obtained results were in agreement with previously published studies (Patureau etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR26#CR26" 1997; Huang and Tseng  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR14#CR14" 2001; Foglar etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR8#CR8" 2005) performed with some bacteria capable of aerobic denitrification. Also, in agreement with that observation, Lemmer etal. ( HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR19#CR19" 1997) discuss that Paracoccus bacteria use simultaneously oxygen and nitrate as a terminal electron acceptor by which denitrification is possible even under partially aerobic conditions. The denitrification with bacterial cells attached on quartz sand and zeolite At the beginning of this part of investigation, adsorption of nitrate from brine wastewater on quartz sand and zeolite was monitored. For that purpose, 1g of the carrier and 100mL of BW2 (100 750mg NO3 "-NL"1) were placed in 0.5L Erlenmeyer flasks. The test was performed at 25C on the rotary shaker at 80rpm over 24h. Nitrate concentrations determined at the beginning and during the tests were almost unchanged. The difference between the obtained values was 0.2mg NO3 "-NL"1. The results clearly indicate that quartz sand and zeolite did not adsorb nitrate from BW2. Continuous denitrification of BW2 by the attached mixed bacterial cells was investigated subsequently. Denitrification in the bioreactor (containing the carrier with attached cells) was monitored in the first part of the experiments at different dilution rates in order to achieve the optimal dilution rate. Hydraulic retention times were within the range of 16725h (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig2#Fig2" 2). Each test started as a batch and when nitrate was completely reduced, continuous flow of feed (BW2 containing 100mg NO3 "-NL"1 and 2moles of methanol per mole of nitrate) was started. The feed was prepared daily and checked for nitrate-N, nitrite-N and methanol concentrations (methanol concentration was expressed as COD value). Nitrite-N and the DO concentrations in the feed were usually in the range of 0.01 0.03mg NO2 "-NL"1 and 5.40 6.20mg O2L"1 respectively. The initial number of bacterial cells attached to quartz sand and zeolite was 2107 4107CFUg"1. The values obtained were similar to those from the recent denitrification experiments conducted with pure culture of Paracoccus denitrificans (Kim etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR17#CR17" 2004).  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig2_HTML.gif" \* MERGEFORMATINET  Fig.2Effluent concentration values at steady state during denitrification with bacterial cells attached to quartz sand: nitrate-N (%) and nitrite-N (%) concentrations, and zeolite: nitrate-N (  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figb_HTML.gif" \* MERGEFORMATINET ) and nitrite-N ( INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figa_HTML.gif" \* MERGEFORMATINET ) concentrations at different HRT values at 25C and pH=6.8  In the first test set with bacterial cells attached to quartz sand at the dilution rate of 0.006h"1 (HRT=167h) nitrate ions in the bioreactor were completely reduced. At lower HRT nitrate and nitrite concentrations in the effluent at the steady state were up to 7.20.2mg NO3 "-NL"1 and 3.20.05mg NO2 "-NL"1 respectively (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig2#Fig2" 2). Bacterial count at the end of the tests increased to 6.9107CFUg"1 at the top of the bioreactor and to 9.2108CFUg"1 at its bottom. The difference between these two values might be due to the fact that the feed solution was introduced at the bottom of the bioreactor (optimal growth conditions) and to the presence of bacteria that were aerobic heterotrophs or a consequence of bioreactor properties and fact that the bioreactor was filled with 200g of zeolite. The initial DO concentration dropped from 6.35mg O2L"1 to 1.60mg O2L"1 and after some fluctuations it went down to 1.4mg O2L"1. That unexpected decrease and increase of DO during the first 6 days might be attributed to fluctuations in the number of living cells and consumption of O2 by bacterial cells. In the second test set denitrification started at the dilution rate of 0.006h"1, since the previous test had shown (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig2#Fig2" 2) that the effluent at the dilution rate of 0.012h"1 contained nitrate. During 7days of continuous flow there was no trace of nitrate, and so over further 7days HRT was lowered to 83h and finally to 48h (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig3#Fig3" 3). Nitrate concentration in the effluent increased to 5mg NO3 "-NL"1. At the same time, during denitrification with bacterial cells attached to zeolite, at HRT=48h nitrate ions were completely reduced in the bioreactor. Furthermore, even at HRT=25h there were only 2mg NO3 "-NL"1 in the effluent. Additionally, the comparison of nitrate-N and nitrite-N concentrations at the steady state during the experiments with bacterial cells attached to quartz sand and zeolite, (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig2#Fig2" 2) showed that in our work zeolite was the preferred carrier of microorganisms. This observation may be explained with structure and properties of zeolite, obviously interactions of bacterial cells with zeolite influence and improve denitrification process. Thus, further study of denitrification process was performed with the mixed bacterial culture attached to zeolite.  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig3_HTML.gif" \* MERGEFORMATINET  Fig.3Time course of a continuous denitrification of BW2 with bacterial cells attached to quartz sand operated at 25C and pH=6.8. Effluent nitrate-N ( % ), effluent nitrite-N ( INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figa_HTML.gif" \* MERGEFORMATINET ) and DO effluent concentrations ( % )  Nitrate removal with bacterial cells attached to zeolite Prolonged denitrification with bacterial cells attached to zeolite was carried out. Initial concentration of nitrate-N and nitrite-N in the feed was 100mg NO3 "-NL"1 and 0.01mg NO2 "-NL"1 respectively (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4). The process started at HRT 25h and was conducted at 252C. The operating temperature was selected for economical reasons and from the previous batch results. The DO concentration and chemical oxygen demand (COD) in the feed were from 5.4 to 6.2mg O2L"1 and 900 1000mg O2L"1 respectively (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig5#Fig5" 5). The initial number of bacterial cells attached to zeolite was 4107CFUg"1. After 10days of continuous operation, the system reached the steady state and the process was monitored over further 20days. After some fluctuations during 10days, nitrate-N and nitrite-N concentration in the effluent were between 0.1 0.3mg NO3 "-NL"1 and 0.01 0.04mg NO2 "-NL"1 respectively (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4). The reactor was opened to sample the carrier. The number of bacterial cells attached to zeolite at the top of the bioreactor increased during the first 30days to 5נ108CFUg"1. Due to opening the reactor nitrate-N concentration increased to 2mg NO3 "-NL"1 on day 31, but the system slowly reached the steady state at 0.5mg NO3 "-NL"1. Then, nitrate-N in the influent gradually increased to 500mg NO3 "-NL"1 (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4). The effluent nitrate and COD concentrations having reached the steady state were below 2mg NO3 "-NL"1 and 100mg O2L"1 respectively. Moreover, nitrite-N was below 0.5mg NO2 "-NL"1, accordingly, nitrate and COD removal was still above 99 and 90% respectively (Figs. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4 and  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig5#Fig5" 5). Gas production was also measured, as described in our previous papers (Foglar and Briki  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR9#CR9" 2003; Foglar etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR8#CR8" 2005). At the nitrate loading rate of 11.76mg NO3 "-NL"1 the generated nitrogen gas volume accounted for 9.7mLN2L"1h"1 which was in agreement with the previously published data (Clifford and Liu  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR5#CR5" 1993).  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig4_HTML.gif" \* MERGEFORMATINET  Fig.4Continuous-flow denitrification with bacterial cells attached to zeolite at 25C and pH=6.8: Inlet nitrate-N (%), effluent nitrate-N ( % ) and effluent nitrite-N ( INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figa_HTML.gif" \* MERGEFORMATINET ) concentrations   INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig5_HTML.gif" \* MERGEFORMATINET  Fig.5Continuous-flow denitrification with bacterial cells attached to zeolite at 25C and pH=6.8. COD values ( INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Figc_HTML.gif" \* MERGEFORMATINET ) in the feed, in the effluent (+) and COD removal values (נ)  During the experiment the dissolved O2 was regularly checked. The influent value was in the range of 5.4 to 6.2mg O2L-1 and the level of the residual DO in the effluent was 2.30.4mg O2L"1 (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig6#Fig6" 6). Kesser etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR16#CR16" 2003 observed similar effluent values during denitrification in the continuous-flow bioreactor containing immobilized Pseudomonas butanovora cells. According to them and the literature data (Lemmer etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR19#CR19" 1997; Patureau etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR26#CR26" 1997; Huang and Tseng  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR14#CR14" 2001; Foglar etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR8#CR8" 2005), it could be concluded that brine denitrification was possible even at partially aerobic conditions. The temperature was also monitored and it was always close to 25C, except on day 34 and within the period from days 63 and 65 when it was 22C (Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig6#Fig6" 6). The volumetric denitrification rates were calculated from results obtained during the tests. During first 30 days it was 3.960.48mg NO3 "-NL"1h"1 and until day 42 it raised to 7.610.25mg NO3 "-NL"1h"1. On day 43, the inlet nitrate concentration went up to 300mg NO3 "-NL"1. Consequently, volumetric denitrification rate was raised to 11.790.15mg NO3 "-NL"1h"1. Having reached steady state on day 55 the approximate inlet nitrate concentration was 500mg NO3 "-NL"1 and volumetric denitrification rates was 19.360.90mg NO3 "-NL"1h"1. Finally, on day 66 the nitrate concentrations in feed were raised again to 700mg NO3 "-NL"1 and therefore denitrification rates increased to 28.430.35mg NO3 "-NL"1h"1. Comparison of the obtained volumetric denitrification rates and the temperature indicated that lower ambient temperature caused reduction of denitrification rates. Observed increase of the volumetric denitrification rate with the increase in the influent nitrate concentration was supported by the fact that denitrification rates are dependent on nitrate concentration. That was demonstrated by the presented results, which were in a range between 3.960.48mg NO3 "-NL"1h"1 and 28.430.35mg NO3 "-NL"1h"1. The obtained denitrification rates were compared with literature data (Table HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Tab1#Tab1" 1). Those recorded after day 66 compare well with the references (Roennefahrt etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR29#CR29" 1986; Kesser etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR16#CR16" 2003) as shown in Table HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Tab1#Tab1" 1. Conversely, Park etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR25#CR25" 2001 used immobilized freshwater denitrifier consortium for salinity acclimation. They achieved NO3 "-N removal rate of 0.33 0.75mg NO3 "-NL"1h"1 at HRT=12h. Since their values were significantly lower than ours, it could be assumed that the selected mixed culture was advantageous for brine denitrification.  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/MediaObjects/11274_2007_9405_Fig6_HTML.gif" \* MERGEFORMATINET  Fig.6A continuous nitrate removal from BW2 with bacterial cells attached to zeolite at 25C and pH=6.8 under anoxic conditions. Denitrification rates (%), temperature values () and DO concentrations (%)  Table1Comparison of denitrification rates obtained during the denitrification processes with immobilized or attached bacterial cells SystemCarrierElectron donorDenitrification rate (mgNL"1h"1) ReferenceBiologicalNa-alginate (cell entrapped)Ethanol22.536.67Kesser etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR16#CR16" 2003 Biological Buoyant carriersEthanol31.25Roennefahrt etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR29#CR29" 1986 Biological/ion exchange Na-alginateAcetate16.67Richard etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR27#CR27" 1989 Biological Macroporous celluloseSoluble starch19.16Kim etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR18#CR18" 2002 Biological Polyvinil alcohol gel beadsGlucose0.731.42Park etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR25#CR25" 2001 Biological ZeoliteMethanol428.5This studyThe proposed system was obviously a stable process. Therefore, nitrate-N concentration in the influent was further increased to 700mg NO3 "-NL"1 on day 66, as shown in Fig. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4. On day 71, the attached biomass was found on the wall of the bioreactor opened for scratching of the attached biomass. Its presence there had been recorded during some previous investigations and might have been due to the excess organic carbon and optimal conditions for the microbial growth. The bacterial cells attached to zeolite at the top of the bioreactor increased to 7.5נ108CFUg"1, but in the effluent they amounted to 2.1107CFUmL"1. Previously determined free bacterial cells in the effluent never exceeded 1.0102CFUmL"1. The obtained values were similar to the previous ones from denitrification study (Kim etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR17#CR17" 2004). However, the presence of free bacterial cells in the effluent suggested that the results of the denitrification were boosted with the presence of free cells rather than being fully attributable to the attached bacterial cells. Furthermore, it was assumed that the increase of nitrate-N to 5.57mg NO3 "-NL"1 on day 72 occurred because the reactor was opened. 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"*026!ν h=h=>*B*mH phsH jh=h=UmH sH h=h=H*mH sH h=h=H*mH sH h=h=mH sH C!"rstu -.yjTh=h=UmH sH h=h=H*mH sH h=h=H*mH sH  h=h=>*B*mH phsH jh=h=UmH sH h=h=mH sH &h=h=6>*B*]mH phsH h=h=6]mH sH #jh=h=6U]mH sH *lnp XY_`degh  UVdeijlm稚稚稚稚稚&h=h=6>*B*]mH phsH h=h=6]mH sH #jh=h=6U]mH sH h=h=CJaJmH sH h=h=5H*\mH sH jPh=h=UmH sH h=h=mH sH h=h=5\mH sH 4  kd$$If4rp& 20634abp26>I8kd:$$Ifrp&0634abdd$If[$\$gd=gh8kd$$Ifrp&0634abdd$If[$\$gd=h8kd$$Ifrp&0634abdd$If[$\$gd=5DJ8kd$$Ifrp&0634abdd$If[$\$gd=lm8kd$$Ifrp&0634abdd$If[$\$gd=my8kd^$$Ifrp&0634abdd$If[$\$gd=mLNPRZ^XZ\^12bdrv  . 2   X Y ] ^           & * 8 :   z&h=h=6>*B*]mH phsH h=h=6]mH sH #jh=h=6U]mH sH  h=h=>*B*mH phsH jh=h=UmH sH h=h=H*mH sH h=h=H*mH sH h=h=CJaJmH sH h=h=mH sH 0E oooovvv6y7y9y8kd$$If&634a $Ifgd= dd@&[$\$gd=gd= dd[$\$gd=   hhhhhhh6i8i:i*B*mH phsH 6). Surprisingly, nitrite-N concentration at that time was still below 1mg NO2 "-NL"1, but from day 77, the effluent nitrite-N slowly increased to 2mg NO2 "-NL"1. The effluent COD values, from day 66 on increased and the organic removal rate was lowered to 80% (Figs. HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig4#Fig4" 4 and  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "Fig5#Fig5" 5). Peristaltic pump hosepipe had been released gradually during that period. It broke down on day 80 and was immediately replaced with a new one. Probably this caused the fluctuations of nitrate and nitrite concentrations. The steady state was reached on the day82. Nitrate and nitrite concentrations and COD removal were 6.3mg NO3 "-NL"1, 2mg NO2 "-NL"1 and 75% respectively. Therefore, on day 85, denitrification was discontinued. At the end of the experiment the number of bacterial cells attached to zeolite at the top and at the bottom of the bioreactor was 1.8נ109CFUg-1and 3.1נ109CFUg-1 respectively. The CFUg-1 resembled that obtained during denitrification with immobilized cells (Shrimali etal.  HYPERLINK "http://www.springerlink.com/content/e8l55473521p3035/fulltext.html" \l "CR20#CR20" 2001). Irrespective of the breakthrough after day70, this study has shown that the mixed culture attached to zeolite is an advantageous system for brine denitrification.  Conclusions Biodenitrification with immobilized or attached bacterial cells is an effective method for the removal of nitrates from brine wastewaters. Furthermore, proper selection of the carrier for bacterial immobilization can improve the process. The study of synthetic brine wastewater denitrification was aimed at investigating stability and denitrification of the bioreactor with bacterial cells attached to quartz sand and zeolite. According to obtained results denitrification was more efficient in presence of zeolite as a carrier. The results indicate that during continuous brine denitrification (at up to 500mg NO3 "-NL"1 in the influent) with the mixed bacterial cells attached to zeolite, nitrate and COD removal exceeded 99 and 90% respectively. Complete nitrate removal with the mixed bacterial culture containing dominant Pseudomonas sp. and Paracoccus sp. species was achieved in the presence of DO concentration of 2mg O2L"1. Moreover, during that period the effluent nitrite-N concentration was below 0.5mg NO2 "-NL"1. The obtained results suggests that biological denitrification with the mixed bacterial cells attached to zeolite, is an operationally promising solution to denitrification of the spent regenerant.  References Almeida JS, Reis MAM, Carrondo MJT (1995) Competition between nitrate and nitrite reduction in denitrification by Pseudomonas fluorescens. 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Desalination 139:199205  HYPERLINK "http://dx.doi.org/10.1016/S0011-9164(01)00311-3" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/crossref_link.gif" \* MERGEFORMATINET  HYPERLINK "http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=springer&version=1.0&coi=1%3ACAS%3A528%3ADC%252BD3MXnt1Gmsrs%253D&md5=d93f55867f5bf991d58424f0090b7510" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/chemport_link.gif" \* MERGEFORMATINET  o ooooooVrXrZr\rdrhrtt,t@tttttuuuuuuvvvvw+wJwϨϜϐϐςςϜϐϜϐϐs[ς.h=h=5CJ OJQJ\^JaJ mH sH jh=h=UmH sH h=h=6]mH sH h=h=H*mH sH h=h=H*mH sH .h=h=5CJ&OJQJ\^JaJ&mH sH j2h=h=UmH sH h=h=mH sH #jh=h=6U]mH sH &h=h=6>*B*]mH phsH !JwKwwwwwwwwwxxx1y2y3y4y5y6y7y9y:yyyzz zuzvzwzxzzzB{C{D{{{{{Ѯњwc&jh=h=B*UmH phsH &jJh=h=B*UmH phsH h=h=CJaJmH sH &j'h=h=B*UmH phsH &jjh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH h=h=mH sH jh=h=UmH sH &9y:y{{{{ |K8kd$$If&634a8kd$$If&634a $Ifgd=8kd$$If&634a{{{{{{ | |||;|<|||||~}}}}}5~6~7~8~:~~~lmnopqrtuYZ[ǀȀ𴥴𴥴}𴥴&jmh=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH h=h=6]mH sH h=h=CJaJmH sH h=h=mH sH jh=h=UmH sH , | ||||||K8kd$$If&634a8kd$$If&634a $Ifgd=8kdB$$If&634a||qrtùK8kd5$$If&634a8kd$$If&634a $Ifgd=8kdG$$If&634aȀɀʀˀ̀̀πЀ=>@24ȃʃ̃RSTUW`abΆφɾɾؠ،ɾؠxɾɾؠ&j h=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH h=h=CJaJmH sH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &jh=h=B*UmH phsH (̀̀πЀ=>@K8kd $$If&634a8kd$$If&634a $Ifgd=8kd[$$If&634a@AK8kdJ$$If&634a8kd$$If&634a $Ifgd=8kd`$$If&634aφІцӆ     mnopr678^_ɾد؛ɾɾدxɾدdɾ&jj(h=h=B*UmH phsH &j!h=h=B*UmH phsH h=h=CJaJmH sH &jrh=h=B*UmH phsH h=h=B*mH phsH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &jh=h=B*UmH phsH 'K8kd/$$If&634a8kdB!$$If&634a $Ifgd=8kd $$If&634aYK8kd5$$If&634a8kdc5$$If&634a $Ifgd=8kd20$$If&634a_   YZ\]/Cčōƍ23457mnopqrү~j&jO<h=h=B*UmH phsH &js7h=h=B*UmH phsH h=h=6]mH sH h=h=CJaJmH sH &j0h=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=mH sH )YZ\]K8kd6$$If&634a8kdh6$$If&634a $Ifgd=8kd6$$If&634arsuv1K8kdD$$If&634a8kdC$$If&634a $Ifgd=8kd7$$If&634arsuv:;,-./01245В:;<ôàôÌ~ôj&jpSh=h=B*UmH phsH h=h=6]mH sH &jEKh=h=B*UmH phsH &jzDh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=mH sH h=h=CJaJmH sH )1245K8kd=Z$$If&634a8kdS$$If&634a $Ifgd=8kdR$$If&634aefhi*K8kdi$$If&634a8kd/i$$If&634a $Ifgd=8kdZ$$If&634a$<pq%&'(*`abcdefhi=>|}~%&ĵġĵč~~ĵjĵ&jih=h=B*UmH phsH h=h=CJaJmH sH &jah=h=B*UmH phsH &jZh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=6]mH sH h=h=mH sH '&'()*+-.FGH}~МќҜ>?@ACɾɾؠ،ɾؠxɾɾؠd&jh=h=B*UmH phsH &jh=h=B*UmH phsH &jxh=h=B*UmH phsH h=h=B*mH phsH h=h=CJaJmH sH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &jph=h=B*UmH phsH '*+-.K8kd$$If&634a8kdfx$$If&634a $Ifgd=8kdx$$If&634a~K8kdN$$If&634a8kd$$If&634a $Ifgd=8kdV$$If&634aC   yz{|}~KLMrs*+үҌx&jth=h=B*UmH phsH &jh=h=B*UmH phsH h=h=CJaJmH sH &j~h=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=mH sH )jkmK8kd߳$$If&634a8kd<$$If&634a $Ifgd=8kd$$If&634a+,-/efghijkmnMN ҥӥԥ@ABCDEFHIΦϦɾد؛ɾɾدxɾدdɾ&j\h=h=B*UmH phsH &jh=h=B*UmH phsH h=h=CJaJmH sH &jdh=h=B*UmH phsH h=h=B*mH phsH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &jh=h=B*UmH phsH 'mnEFHIƨK8kd,$$If&634a8kd$$If&634a $Ifgd=8kd4$$If&634aϦSTU¨èĨŨƨǨɨʨIJ  ɪʪүқxjh=h=6]mH sH &jh=h=B*UmH phsH h=h=CJaJmH sH &jTh=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=mH sH )ƨǨɨʨ K8kdL$$If&634a8kd$$$If&634a $Ifgd=8kd$$If&634a  LK8kd$$If&634a8kd<$$If&634a $Ifgd=8kd$$If&634aJKLSTٮڮۮGHIJKLMOܹܚܹwܹcܹ&jh=h=B*UmH phsH &jh=h=B*UmH phsH h=h=CJaJmH sH &jh=h=B*UmH phsH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH &LMOPK8kd$$If&634a8kd$$If&634a $Ifgd=8kd6$$If&634aOPѯү#$&'yz{PQ    մִ״CôàôÌôxô&j>h=h=B*UmH phsH &jh=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH &jh=h=B*UmH phsH jh=h=UmH sH h=h=mH sH h=h=CJaJmH sH /#$&'K8kd$$If&634a8kdc$$If&634a $Ifgd=8kd$$If&634aHIKK8kd$$If&634a8kd$$If&634a $Ifgd=8kd$$If&634aCDEFGHIKLڵ۵    %&ghiոָ׸ظڸɾɾɾ~ɾjɾ&jh=h=B*UmH phsH h=h=6]mH sH &j6h=h=B*UmH phsH h=h=B*mH phsH h=h=CJaJmH sH h=h=mH sH jh=h=UmH sH &j h=h=B*UmH phsH &jh=h=B*UmH phsH )KL   K8kd$$If&634a8kd$$If&634a $Ifgd=8kd$$If&634aK8kd).$$If&634a8kd$$If&634a $Ifgd=8kdI$$If&634a  +,-fghԽսֽ׽ؽٽڽܽݽfgܹ܋ܹwܹuU&jV6h=h=B*UmH phsH &j/h=h=B*UmH phsH h=h=CJaJmH sH h=h=mH sH jh=h=UmH sH &j&h=h=B*UmH phsH &jh=h=B*UmH phsH h=h=B*mH phsH +ٽK8kd./$$If&634a8kd.$$If&634a $Ifgd=8kd~.$$If&634aٽڽܽݽK8kdRG$$If&634a8kd&>$$If&634a $Ifgd=8kd=$$If&634aYang PY, Nitisoravut S, Wu JYS (1995) Nitrate removal using a mixed-culture entrapped microbial cell immobilization process under high salt conditions. Water Res 29:15251532  HYPERLINK "http://dx.doi.org/10.1016/0043-1354(94)00296-J" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/crossref_link.gif" \* MERGEFORMATINET  HYPERLINK "http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=springer&version=1.0&coi=1%3ACAS%3A528%3ADyaK2MXlsVGhtr0%253D&md5=f1a6aa17764af5a46f1e4b1f16f48345" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/chemport_link.gif" \* MERGEFORMATINET Zellner G, Feuerhake E, Jordening HJ, Macario AJL, Conway de Macario E (1995) Denitrifying and methanogenic bacteria in the biofilm of a fixed-film reactor operated with methanol/nitrate demonstrated by immunofluorescence and microscopy. App Microbiol Biotechnol 43:566571  HYPERLINK "http://dx.doi.org/10.1007/s002530050452" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/springer_link.gif" \* MERGEFORMATINET  HYPERLINK "http://chemport.cas.org/cgi-bin/sdcgi?APP=ftslink&action=reflink&origin=springer&version=1.0&coi=1%3ACAS%3A528%3ADyaK2MXntlaqsLY%253D&md5=0d7c461d4b030ba554585145743dd657" \t "_blank"  INCLUDEPICTURE "http://www.springerlink.com/content/e8l55473521p3035/chemport_link.gif" \* MERGEFORMATINET  ghik/01hijkm123ɾد؛ɾɾدxɾدdɾ&jLh=h=B*UmH phsH &jHh=h=B*UmH phsH h=h=CJaJmH sH &j?h=h=B*UmH phsH h=h=B*mH phsH h=h=mH sH jh=h=UmH sH &jh=h=B*UmH phsH &j>h=h=B*UmH phsH %KI8kdO$$If&634a8kd4O$$If&634a $Ifgd=8kdG$$If&634ah[7]21h:p[7]/ =!"n#$% $$If!vh5#v:V   06,5/ 34p $$If!vh5#v:V 06,5/ 34$$If!vh5#v:V 06,5/ 34Dd  s \A(contactContact InformationyK  yK http://www.springerlink.com/content/e8l55473521p3035/fulltext.html"ContactOfAuthor1#ContactOfAuthor1bбUQI7SnбUQIPNG  IHDR vrPLTE }~IJWbKGDH cmPPJCmp0712OmBIDATcH0gHRRRRugHPA@Z)HIU D*hP%'wD';C 3Bd&IENDB`a$$If!vh55$#v#v$:V 6534a$$If!vh55$#v#v$:V 6534a$$If!vh55$#v#v$:V 6534Ddz  S VA(contactContact InformationbбUQI5SnбUQIPNG  IHDR vrPLTE }~IJWbKGDH cmPPJCmp0712OmBIDATcH0gHRRRRugHPA@Z)HIU D*hP%'wD';C 3Bd&IENDB`g$$If!vh55#v#v:V 6,534Dd%<P  3 3"((Dd%<P  3 3"((>Dd I  s A:0$$11274_2007_9405_Article_Equ1 $$ r_{D} = \frac{{_{{{\text{d}}C_{N}}} }} {{{\text{d }}t}} = \frac{{k_{D} \cdot C_{N} }} {{{\text{(}}K_{s} + {\text{ }}C_{N} {\text{)}}}}{\text{ }} $$b2299|OXux Sn299|OXuxPNG  IHDR'`hsBITw0PLTEwwwUUU333fffDDD"""t tRNSbKGDH cmPPJCmp0712Om4IDATHǵOh`cAŵtnN2e&Ãlp.peΝ"(<̋=(((XR$ڤIOM3q2KvvT@P!1Ztة`fka+ ezcPPyS?R,%KS3JנKC B;mo'<^-hgR! a%tq(f9&Ѡ? ' i1 e10N9_±'㟏=NCzU"sN*k) rP$1Mb#}5֗Fi1XXCkJ86u V E4R;Δ@#Xn{N lD bU6ޮωGuE%s#]:68"NC&!6t׫ B`-E@HaCNrW.Of) 'k`ﺜ/U(4j&c=. bv4*ٞK{RT-hzS+5cBU#2 ƲF9{rlaA RxIm{[IE/z@-2bXmeN?u vǜ؁a'* "zI6iCm0VZ!WR=S+LХ|/> Lh@-k'ʶnNy;RQIENDB`g$$If!vh5"52#v"#v2:V 6,534N(Dd&`    s A:À $$11274_2007_9405_Article_Equa $$ \begin{aligned}{} & {\text{NO}}_{{\text{3}}} ^{ - } {\text{ - N removal rate}} = \frac{{{\text{(C}}_{{{\text{nitrate - N}}}} {\text{)}}_{{{\text{infl}}{\text{.}}}} {\text{ }} - {\text{ (C}}_{{{\text{nitrate - N}}}} {\text{)}}_{{{\text{effl}}{\text{.}}}} {\text{ }}}} {{{\text{(C}}_{{{\text{nitrate - N}}}} {\text{)}}_{{{\text{infl}}{\text{.}}}} }} \times 100(\% ) \\ & {\text{COD removal rate}} = \frac{{{\text{(COD)}}_{{{\text{infl}}{\text{.}}}} - {\text{ (COD)}}_{{{\text{effl}}{\text{.}}}} {\text{ }}}} {{{\text{(COD)}}_{{{\text{infl}}{\text{.}}}} }} \times 100\;(\% ) \\ & {\text{Organic load}} = {\text{(COD)}}_{{{\text{infl}}{\text{.}}}} \times {\text{D (mg COD}}\;{\text{L}}^{{ - {\text{1}}}} \;{\text{h}}^{{ - {\text{1}}}} {\text{)}} \\ & {\text{Nitrate - N load}} = {\text{(C}}_{{{\text{nitrate - N}}}} {\text{)}}_{{{\text{infl}}{\text{.}}}} \times {\text{D (mg NO}}_{{\text{3}}} ^{ - } {\text{ - N}}\;{\text{L}}^{{ - {\text{1}}\;}} {\text{h}}^{{ - {\text{1}}}} {\text{)}} \\ & {\text{Volumetric denitrification rate}} = {\text{ [(C}}_{{{\text{nitrate - N}}}} )_{{{\text{infl}}{\text{.}}}} - {\text{ (C}}_{{{\text{nitrate - N}}}} {\text{)}}_{{{\text{effl}}{\text{.}}}} {\text{]}} \times {\text{D (mg NO}}_{{\text{3}}} ^{ - } {\text{ - N}}\;{\text{L}}^{{ - {\text{1}}}} \;{\text{h}}^{{ - {\text{1}}}} {\text{)}} \\ \end{aligned} $$bDlXLSnDlXPNG  IHDRp,`PLTEwwwUUU333pppfffXXXDDD888222"""dtRNSEbKGDH cmPPJCmp0712HsIDATx^]cnU]45RL{aB盪Y1@5:VPҲ&`^Ou8`hPD\!2'V_|+7\*kPڢ/>W2wH K4ER _S!|US|E]ViP<&qlSEYQT(ԠHEZ2hQ~5(*j2;J \5G2),QrgX惦6S(E5 )  I[eXc=*\ 2l a i'ɽ<5kNV `d*D!h;orT TUyY]w!SF<~3[s W(!L{yi^^7nE`aa]5V"q{@zhg(6d6V7Yn >V ,K <*5U ls\3}>/ݼϘU~A >'1Ih(&,V9F^׷zV0~z50KU5E^8ˍHQ&iЪҀUԳ4hS5W5~"2ߔ Û%>uXʈW^r6YȪY ʫ]~j() Grpt\ DhѠ I 8nPl}ۭ+^d|}jӍ#~ׯ,7L C O PrS5秕|*!l$(A,Y==Wߋ~^໅L )(S@D!s` dH5hZR:V2z)ik}K[b+U2!@ƭ8T˷;ݡ'4-//<o}%k}ѹnM 3GP0N",5BK \\9 xKu\GTo"*_l_x`:wx-[̶4usV%.C'Af^ oZ5AFRg|lu(6Whu:$Xg  S[ =F au&V7WW5+Uzd#0oT7Ad :ia`o$ʸ_iARQP+rYY$sw˧uSrlSahqyzkך`k_jgt2f9ܮߛb0:1-hX3էZ3>֙Xzl&xjNm:[܎'u0_Ãe@6!K$׺GxIT/(heX?{4M](ʧߎLpnoY~eL4UO*oI%`LWn7fɿ?<2ii]kk6ݽ)?<gF{41 R0xL=6]k q>JzmIҰQN厄f}warAg(ٵ'Kd`a3DPkObfwL@C{,w3ʓ'2rySW'2%f07vIcxxecc S8BDdn7,Md@&qX027oܞX2oLxc{DXogALxL fl(#ělo*3i7|Ȳ_ˈu]g.-&Td->dQ (<6P[DyZ*2̈́R=xmyKq-F)~Q?賀oI,~H;OKE&ejGf)1O|2 ][Lo47Z|ȪeLfq CͶ-~XHLxNLd86xKzX*2&=SoI s KEfx,Sⷯ/ސΔ{KieFwuQ@ҙ V'=(yRVxS>u?fM1LǦtZ0׶|a# J=?8[N `Ѝ(~IufO|OQw]ݖO2hc 0ByצӒB!"bt#*hǦcFkΎ^r.^M{Pxt)1bIΣpk%<6P"\9YLe\34 ӥp[aĢ U; k9=+#oN* 9cq/&g^LLם.i5}OXy.kNP "⑗<:y7۸.yG'OԟܚOݩL04']#r+y׻TFyGzSw:dfM#hx5=*G^]_ۖO7Lr*!3smZ5N!U<.yذG^ן.eu/*ʍԝjGkӭM%L咗=\RqD=%O;]*ĭԝ2~Y4u2cRT* XkXjs aA4ZkRk9 `[>uG4%j3Ȕw-6SwLØ6L1ӱZpx8F,dJF򐙵OO-5dm)| @OA֐;+c23g46LgkK9 ǵ, ljяgi5j²5H͔:'Ay5jg 6cAXXVIs Vd^V@ 3.gcUyMPGHot# Ya(+QjKeo -YK2=^bQL"se?udב(iy seȜaf=LXfՈuԄ;ZjV&7!>(IB Ez5(ئ",0K6iz,2>p_<4ݝ';j{Ƕ ,brGkB yK| C.`B۳wx9a)>kj_\k/J͢_gS…%@"-T+.:z/q,Oy [QFs|b.,ʥvk|1_5672G$,pE}\0Mn,5!L>`PL4`Æ]|~Ͷ\O.A*01-F$,pUꓒrUδF>=WP0F}6,bf \F+LEŎE x@85u >z*?tj/rv]@PyLmPl;Z5pb4Bv cSL÷A0@ϣ)q lrt՚.5>7suo Rb>P_k–̬T!kN"s]`:g} vag-/Y#vDunuQ-dR8U}` Tjo5W%׸8B;kdtiۣrC m)pEy3]`U0b)pEVCGd~W@~& $pxLO rt)9$Iʫ^Y "ycalW'OW+ &eu8zF_ >^Bi6"aCεrJ SR"3+5AϯG::́t߰Xv+0c8OjҜ:ր'Tuw@bvg(aԤ9ۇ:*B&qiXrJk8aA}ʜ9F: Bf27MFP/I؟iH aA/sm(ґ!SYeu2g3P/ e#l89:S^@f7U. i^-5 bL2+>(EdN? 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