Chemosphere 48 (2002) 445451 www.elsevier.

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A comparison of organic and inorganic carbon controls over biological denitrication in aquaria
S. Vidal *, C. Rocha, H. Galv~o a
CIMACenter for Marine and Environmental Research, Faculdade de Ci^ncias Marinhas e Ambientais (FCMA), e University of Algarve, Campus de Gambelas, PT-8000-062 Faro, Portugal Received 23 January 2001; received in revised form 6 December 2001; accepted 30 January 2002

Abstract In aquaria and rearing tanks, nitrate accumulation as a result of organic matter degradation is inevitable and has two major negative side eects: direct toxicity to organisms, specially invertebrates, and the introduction of a reducing environment by oxygen consumption. The aim of this study was to compare two alternate methods of removing nitrogen compounds from closed systems, autotrophic columnar denitrication (ACD) and heterotrophic columnar denitrication (HCD) by following end product concentrations as reaction progressed. A pilot plant consisting of two series of 50 dm3 recirculating ow systems (each in triplicate) was used to test both methods. Absence of pH control was also useful in autotrophic denitrication systems in order to follow eects over reaction rates and pathways. Concentrations of NO , NO and NH were followed throughout the experiment, as well as pH, temperature and salinity. Under dierent 3 2 4 ow conditions results show that higher nitrate reduction rates were possible in the autotrophic systems (35:1 4:7 lM/ day without pH control until reversal of the process and 20:6 7:3 lM/day after reestablishment of pH control) in comparison with heterotrophic (9:9 1:3 lM/day). However, pH control through calcium bicarbonate addition was found to be crucial in maintaining constant levels of total denitrication in ACD systems, just as it was necessary to closely maintain organic carbon addition to HCD systems. 2002 Elsevier Science Ltd. All rights reserved.
Keywords: Nitrate; Remediation; Toxicity; Denitrication; Autotrophic columnar denitrication (ACD); Heterotrophic columnar denitrication (HCD)

1. Introduction In closed systems, such as aquaria and rearing tanks, the progressive accumulation of nitrate can be detrimental to specimens sensitive to captivity, like the majority of invertebrates (Hirayama, 1974; Spotte, 1979; Landau, 1992). Even at mid-range concentrations (700 1400 lM), nitrate will interfere with normal tissue development, inducing continuous stress on captive animals, leading to serious debilitation or even death (Dakin, 1992). Besides the compound toxicity, the main

Corresponding author. Fax: +351-289-818-353. E-mail address: svidal@ualg.pt (S. Vidal).

disadvantage of nitrate accumulation is its eect on alkalinity and pH. Nitrate production during nitrication process consumes alkalinity and produces protons consequently reducing pH (Delbeek and Sprung, 1994). The reversibility of this process is, however, understudied. Many small aquarium facilities control nitrate levels by performing regular water exchange. However, this strategy becomes impractical in very large aquaria, because the size makes it operationally dicult to replace a signicant fraction of water. In addition, the expense of preparing large volumes of articial saline water in locations with no available natural seawater can be prohibitive (Grguric et al., 2000). As a result, biological denitrication has been selected as a method of choice for controlling NO concentrations in closed systems. 3

0045-6535/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 5 - 6 5 3 5 ( 0 2 ) 0 0 0 7 3 - 5

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metabolism of heterotrophic bacteria (mostly Pseudomonas). Methanol and glucose are the most extensively studied model compounds for organic matter in the development of stoichiometric relationships for the process (Balderston and Sieburth, 1976). In biolters, methanol is the most extensively used organic additive, in which case the reaction would progress according to (EPA, 1993): 6NO 5CH3 OH ! 3N2 5CO2 7H2 O 6OH 3 1
Fig. 1. Microbial nitrate reduction steps (adapted from Koike and Sorensen (1988)).

Eectiveness of this method has been established in the treatment of aquaculture ponds (Balderston and Sieburth, 1976), ground water (Wang, 1998), surface water (Flere and Zhang, 1998; Zhang and Lampe, 1999) and sewage (Smith et al., 1972), but studies comparing the reaction progress of alternate processes in aquaria are rare. Denitrication (Fig. 1) is a process carried out by an environmentally ubiquitous group of bacteria that sequentially reduce NO to NO and other gaseous forms 3 2 (N2 O and N2 ) under suboxic conditions (EPA, 1993). Rates of NO3 reduction in aquatic systems are consequently dependent on temperature and pH, which inuence oxygen availability, and substrate concentrations including NO and organic matter (Delwiche and 3 Bryan, 1976; Koike and Sorensen, 1988). The application of biological denitrication for the purpose of nitrate removal from articial systems involves a decision on which of the main natural pathways should be used. More specically, whether heterotrophic or chemoautotrophic nitrate reduction should be implemented as the main biological removal process. The two dier on the type of substrate used as bacterial support and consequentially on the species that will develop, but also on the electron donor driving nitrate reduction. In both cases it is essential to provide an anaerobic environment within the biolter in order to allow the reduction process to start. For the practical application to closed systems two major types of denitrication lters may therefore be considered: heterotrophic columns (heterotrophic columnar denitrication, HCD), where the electron donor would be organic matter of some sort, and autotrophic columns (autotrophic columnar denitrication, ACD), where the electron donor would be elemental sulphur. Heterotrophic denitrication in natural systems progresses through oxidation of labile organic matter using NO as the electron acceptor. The availability of a 3 carbon source is thus crucial for reaction progress, and has to be added periodically to promote growth and

In contrast, the chemoautotrophic bacteria Thiobacillus denitricans oxidize sulphur and sulphur compounds while reducing NO to free dinitrogen gas (N2 ). 3 In this case, the metabolic reaction would progress according to (Santanna et al., 1996): 11S 10NO 4:1HCO 0:5CO2 1:71NH 4 3 3 2:5H2 O ! 0:92C5 H7 NO2 biomass 11SO2 4 5:4N2 9:62H 2 In this study, two alternate methods of columnar denitrication to remove nitrogen compounds from aquaria were compared, emphasising the major controls over reaction progress on both processes, which would be organic matter supply and pH control, and whether reversibility of reaction pathways could be observed and followed stoichiometrically. The experiment was conducted from April to June 1998 in the aquariology department laboratories of Zoomarine, a thematic park situated in Albufeira (Southern Portugal). Microbial metabolic pathways known from environmental research were compared with experimental data, in order to infer stoichiometric relationships of the various possible reaction pathways, both chemoautotrophic and heterotrophic, thus allowing relevant reaction kinetics to be assessed.

2. Methods 2.1. Experimental set-up In order to insure reliable comparison of nitrate reduction rates between the two dierent pathways of denitrication, three pilot plants (see Fig. 2) were used to test each method. The pilot plants had a total working volume of 50 dm3 with a circulating ow system between the glass holding tanks (80 26 26 cm3 ) and the denitrication unit. Each system consisted of a denitrication stage (PVC cylinder) with an internal diameter of 80 mm, packed with specic media for conditioned bacteria xation to a height of 400 mm.

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2.2. Experimental program, sampling and analytical methods Articial seawater purposefully contaminated with nitrate was used in the pilot study. An articial seawater solution (salinity 36) was prepared with synthetic salt (Coral Reef Red Sea Salt) diluted into freshwater. Before the experiment began, an aerobic lter colonized with nitrifying bacteria (Bac-300FTM ; Coralife ) was installed and periodic additions of ammonium chloride (NH4 Cl) were made until sucient nitrate concentration evolved. When adequate nitrate concentration was present (after the nitrication process) bicarbonates were added to raise pH to 8.4 and create a suitable environment for denitrication to progress. In order to follow reaction pathways under alkalinity deciency, and study the eect of reaction progress on pH, no additional inorganic carbon (IC) was added. The experiment started with ammonia and nitrate concentrations respectively 0.10 and 630 lM. Nitrite concentration was below detection limit. Water temperature was thermostatically controlled so that temperature oscillation did not range more than 2 C, with values kept between 23 and 26 C in all systems. In order to compensate water loss by evaporation, distilled water was added daily. To follow progress of both reactions throughout the experiment, water samples were taken on a daily basis to produce a time series for nitrate, nitrite and ammonium concentration. Samples for nutrient determination were ltered through 0.45 lm MSITM Acetate PlusTM lters, xed with 50 ll of a saturated HgCl2 solution (Oremland and Capone, 1988) and stored at 4 C until analysis. Ammonium (NH ), nitrate and nitrite-nitrogen 4 (NO NO ) were quantied using the methodology 2 3 described in Grassho (1976). Corrections allowing for periodic water addition were made on the resulting concentrations. pH (WTW pH 330/SET 1 with WTW SEN TIX 97T pH probe), dissolved oxygen (WTW Oxi 320/SET with WTW Cell Ox 325 oxygen probe), temperature and salinity were followed in all settings. pH values were measured 1 cm above the media of the denitrication units (Fig. 2). Missing values between 28th and 60th day were due to a malfunction of the pH meter.

Fig. 2. Experimental pilot plant (arrows indicate the direction of the ow). (A) Outow from the denitrication reactor to the aquarium; (B) denitrication column; (C) locus of glucose injection (only on HCD systems); (D) water pump (power-head 201 of 220 v/50 HzAquaclearTM ); (E) locus of ow regulation; (F) aeration system (water mixing mechanism).

Dimensions of the reactor were tailored to accommodate a reactor surface area to water volume ratio of 1=100, allowing comparison with marine environments with an average near shore water column depth of 10 m to 1 dm2 surface area. Upward circulation of water was chosen to ease the evacuation of gaseous nitrogen via the open top end of the column. In this experiment no direct bacterial inoculation was performed. The intention was that xation of bacteria would be induced naturally, by use of the type of substrate better suited for attachment of each kind of denitrier. Heterotrophic columnar denitrication (HCD): Porous volcanic media with 7 mm particle size was used. Fixation of heterotrophic bacteria was induced by periodical addition of an organic carbon source (1 ml of 20% glucose aqueous solution) prepared from analytical grade glucose. The water ow was regulated and maintained at 1.5 dm3 /h throughout the experiment due to the low oxygen tolerance of this bacteria species as reported by Seitzinger (1988). Autotrophic columnar denitrication (ACD): Elemental sulphur (commercial powder) with 24 mm particle size was used as bacterial xating media. Water ow was kept low (1.5 dm3 /h) in the beginning to accelerate the creation of an anaerobic environment and after nitrate reduction started the ow was raised (4 dm3 /h) due to the higher oxygen tolerance of this bacteria species as reported by Le Cloirec and Martin (1990). In both systems, the experiment began when very low concentrations of oxygen, as well as reduction of nitrate concentrations, were measured in the euent of the denitrication units (the day after the start of both runs).

3. Results and discussion The experimental run lasted for 103 days and was given as concluded when all studied nitrogen compounds (NH , NO and NO ) where removed from all 4 2 3 aquaria. Nitrate was removed successfully in both systems, albeit at dierent time scales. Decrease of total NO was a continuous process in 3 HCD systems (Fig. 3). Levels of NH and NO were 4 2

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S. Vidal et al. / Chemosphere 48 (2002) 445451 Table 1 Relevant parameters derived from HCD systems (nitrate reduction rates were calculated from the maximum slope of NO 3 decay prole) Column 1 Nitrate reduction (lM/day) Aquarium volume (dm3 ) Denitrication volume (dm3 ) Flow rate (dm3 /h) Volume turnover time (days) 8.4 50 2 1.2 1.7 Column 2 10.6 50 2 1.2 1.7 Column 3 10.8 50 2 1.3 1.6

Fig. 3. Evolution prole of measured concentrations of NH , 4 NO and NO in HCD systems, columns 1, 2 and 3 from left to 2 3 right.

very close to zero except in column 3 (Fig. 3c) where a small accumulation of NO was observed. Removal of 2 NO by heterotrophic denitrication progressed at an 3 average rate of 9.9 (1.4) lM/day (calculated from Table 1). The rate of heterotrophic denitrication depends both on the availability and nature of the organic matter undergoing degradation and on the concentration of NO . Most environmental researchers agree that deni3 trication can be perceived and modelled as a zeroth order reaction with respect to NO down to very low 3 levels (Ros, 1995). In fact, notwithstanding assumptions for packed bed reactors (Harremoes, 1976), NO con3

centration time series in systems 1 and 2 follow a typical zeroth order reaction trend down to low dissolved N levels. However, concentrations in system 3 evidence a deceleration in the transformation rate, which is more characteristic of a rst order reaction. This could be due to the higher ow rate through the denitrication column (Table 1). The presence of NO in the euent also 2 indicates that denitrication was incomplete, suggesting that NO reduction was the rate-limiting step (Koenig 2 and Liu, 1999). Zhang and Lampe (1999) have shown that higher ow rates result in increased oxygen ux to the lter, contributing to nitrite accumulation. In this case, although NO removal eciency can be formally 3 higher, it leads to NO accumulation, which is detri2 mental to captive organisms. Another possible explanation for nitrite accumulation would be carbon limitation in the system, which can lead to an incomplete nitrate reduction (Sauthier et al., 1998). According to several authors (Balderston and Sieburth, 1976; Ingersoll and Baker, 1998; Sauthier et al., 1998) the higher the C:N ratio, the quicker the nitrate removal rate. However, even if carbon excess furthers the denitrication rate, it could bring about a quick decrease of oxidationreduction potential, which enhances sulphate reduction when its Eh is negative, releasing toxic sulphides in an environmental setting. An identical pattern of change in nitrogen compound concentrations can be identied in all ACD systems, corresponding to ve distinct phases. First, a decrease of NO is observed concomitantly with an increase of NO 3 2 concentration and appearance of small quantities of NH , which suggested the start of nitrate reduction 4 (phase I). However, reaction progress in this direction stops at day 13 and takes the reverse direction: decrease of NO and reappearance of NO (phase II). A third 2 3 phase is characterized by small irregular variations on nitrate concentrations and no apparent removal. A decrease of total NO is observed again concomitantly 3 with increase of NO concentrations (phase IV), which 2 corresponded to active pH control through calcium bi-

S. Vidal et al. / Chemosphere 48 (2002) 445451 Table 2 Relevant parameters derived from ACD systems (nitrate reduction rates were calculated from the maximum slope of NO 3 decay prole, except in phase V, where the maximum slope of NO2 decay was used) Nitrate reduction (lM/day) Phase I Phase IV Phase V Aquarium volume (dm3 ) Denitrication volume (dm3 ) Flow rate (dm3 /h) Volume turnover time (days) Column 4 35.7 20.7 10.7 50 2 4.0 0.5 Column 5 30.1 27.8 5.2 50 2 4.0 0.5 Column 6 39.4 13.1 6.6 50 2 3.6 0.6

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carbonate addition from day 60 (Fig. 5) and nally the levels of all nitrogen compounds decrease until complete removal (phase V) (Table 2). Oxygen concentrations above 0.3 and 1.5 mg/dm3 , inhibit enzymatic activity from heterotroc denitrifying bacteria (EPA, 1993; Joye and Paerl, 1994). In the ACD systems an anoxic environment inside the denitrication units evolved rapidly (after four days), values oscillated between 5 and 0.02 mg/dm3 (data not shown). In the HCD systems oxygen concentrations inside denitrication units ranged between 0.2 and 3.8 (values obtained after seventh day). Outside the lter unit oxygen concentrations ranged between 5.4 and 7 mg/dm3 on both systems (data not shown). Rapid initial decrease of NO in ACD systems, 3 promoted the emergence of higher NO concentrations 2 than those measured in HCD systems. In view of the fact that both auto- and heterotrophic denitrication involve a common step, the respiration of nitrate to nitrite (Fig. 1), this observation could be related to the higher advective ux of oxygen into ACD columns, due to higher ow rates, which ranged from 3.6 to 4 dm3 /h, compared to 1.21.3 dm3 /h in HCD columns. As with any biological lter, the eciency of the reactive process is dependent on the advective ux of reactants and products through the lter media. The magnitude of the advective ux of compounds through the permeable column controls the availability of both reaction enhancers (in this case, substrates) but also rate-limiting compounds for anaerobic reactions (oxygen). In the particular case of nitrate removal, the eciency of the process can be controlled by two dierent approaches: either maintaining the ow rate and enlarging the section area, which eectively reduces advective ux of reactants to the lter, therefore improving anaerobic conditions, or, on the other hand, maintaining the lter sectional area

and reducing the ow rate, which also reduces the O2 advective ux. In practice, however, the rst option is preferable because it allows a higher turnover rate of the aquarium water. In accordance, Zhang and Lampe (1999) suggest that development of methods to further reduce the dissolved oxygen in ACD recirculating systems might be cost eective for achieving activated denitrication and higher removal eciencies. In this experiment it was not possible to measure dissolved oxygen on a regular basis. However, it was possible to observe that euents from ACD systems attained lower OD values faster than HCD systems. OD apparently had a smaller inuence in autotrophic denitrication compared to heterotrophic denitrication, which needs that OD concentrations stay below 0.20.4 mg/dm3 (Seitzinger, 1988). NH is not an expected end product of biological 4 denitrication (see Eqs. (1) and (2)), but evidence from marine environments suggests that dissimilatory NO 3 reduction to NH can occur under conditions required 4 for denitrication (Koike and Sorensen, 1988). Nitrate ammonication in seawater was found to occur simultaneously with denitrication in a recent study, accounting for 1233% of the total NO reduced (Omnes 3 et al., 1996). Our results showed mild accumulation of ammonia at the start of ACD denitrication (Fig. 4ac), corresponding to approximately 17% of the total reduced nitrate (phase I). This was observed even in large-scale systems (Grguric et al., 2000). Once low concentrations of ammonia were present in the aquarium water at startup, this result suggests that the nitrate reduction process starts by nitrate respiration followed by nitrate ammonication, and only when sucient ammonia is present autotrophic denitrication will progress according to reaction 2.

3.1. Eect of IC deciency and pH control in ACD lters pH time series in ACD systems (Fig. 5) showed that NO reduction was accompanied by the decrease of pH 3 values until 28th day (phase I). In fact, the presence of low pH values coincided with the inversion of the process (phase II) and pause of all reactions (phase III). Karlsen (1938) in Delwiche and Bryan (1976) showed that a great number of denitriers are capable of denitrication between pH 5.8 and 9.2 with an optimum between 7 and 8.2. Baalsrud and Baalsrud (1954) in Driscoll and Bisogni (1978) suggest a minimum of 6.2 for T. denitricans. In concordance with these studies, interruption of autotrophic denitrication was veried in our experiments between pH 5.9 and 6.9. Our results support the observation that pH variation is a clear indicator of denitrication progress, in agreement with results reported by Glass and Silverstein (1998). During autotrophic denitrication, relevant

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Fig. 4. Evolution prole of measured and calculated concentrations of NH , NO and NO in ACD systems, columns 4, 5 and 6 from 4 2 3 left to right.

similar to natural seawater, and reactions progressed accordingly, illustrating perfectly the eect of alkalinity on NO transformation. After the 60th day, pH was 3 gradually increased by addition of an IC source (calcium bicarbonate) and the denitrication process restarted as a consequence, showing almost perfect reversibility of the reaction. In contrast, the nature of products in heterotrophic denitrication process (see Eq. (1)) maintained alkalinity levels and did not allow pH oscillation.

3.2. ACD versus HCD systems Comparing the performance of ACD and HCD systems (see Table 3) we conclude that in terms of formal chemical eciency, both methods were not signicantly dierent. However, in terms of practical application in ow chamber systems the results suggest that the turnover time seemed to determine the availability of reactants and consequently the performance and overall eciency in nitrate removal.

Fig. 5. Prole of pH average values throughout experiment in ACD () and HCD () systems.

bacteria use IC as a source of cellular carbon. The presence of bicarbonate and carbonate compensates metabolic addition of hydrogen ions maintaining a constant pH level (Driscoll and Bisogni, 1978). This way, the amount of IC necessary for biomass synthesis is 1.5 mg C (in form of CaCO3 ) and alkalinity consumed is 4.38 mg per mg of NO reduced. Buer capacity of the 3 system, which was low in the rst place (see methodology) was rapidly surpassed dropping pH below physiologic needs before limiting conditions for bacterial growth are installed. In the meantime, reversal of the denitrication process was observed following stoichiometric evaluation of the direct reaction. Addition of a certain quantity of IC is then essential to insure buering of the system. This eect is especially visible in these experiments since no eort was done to insure that articial seawater used at the beginning had alkalinity

Table 3 Comparison of removal eciencies of ACD and HCD systems ACD systems Nitrate reduction (lM/day) Volume turnover time (days) NO residence 3 time (days) Phase I Phase IV Phase I Phase IV 19:0 3:6 11:0 3:4 0:54 0:04 17.9 30.5 1:69 0:08 63.1 HCD systems 16:8 2:0

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4. Conclusions The autotrophic denitrication showed double the nitrate reduction rates in comparison with heterotrophic denitrication, albeit, in terms of overall system performances no substantial dierence was observed. Due to the constraints imposed by experimental design, the results were more useful to compare biological denitrication pathways and stoichiometric in relation to carbon source than to compare eciency of removal based on reactor design. In this way, we were able to compare two major reaction pathways for biological denitrication. Dierences between the two were mainly due to the microbiological group mediating the nitrogen transformation. Thus, we were able to show through time series of NO , NH and NO , that in heterotrophic systems, 4 3 2 organic carbon is the chief control over reaction progress, while IC exerted this role in autotrophic systems. Another important role of organic carbon in the HCD systems is to allow the establishment of an anaerobic environment in the reactor column, which leads to the use of nitrate as an oxidant by heterotrophic bacteria when O2 is consumed (depending on ow) and consequently remove it from the system. Acknowledgements The authors would like to thank Dr. Daniel Andrade for his suggestions and to Zoomarine for technical and nancial support.

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