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Applied Microbiology and Biotechnology
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Nitrification in fixed-bed reactors treating saline

wastewater
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Original Paper
07-Oct-2009
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Date Submitted by the

Author:
AMB-09-18698.R1
er
Sudarno, Utomo; Universitt Karlsruhe, Institut fr

Ingenieurbiologie und Biotechnologie des Abwassers
Bathe, Stephan; Universitt Karlsruhe, Institut fr
Ingenieurbiologoe und Biotechnologie des Abwassers; Universitt
Karlsruhe, Institut fr Ingenieurbiologie und Biotechnologie des
Abwassers
Winter, Josef; Universitt Karlsruhe, Institut fr Ingineurbiologie
und Biotechnologie des Abwassers
Gallert, Claudia; Universitt Karlsruhe, Institut fr Ingenieurbiologie
und Biotechnologie des Abwassers
Re
Halophilic nitrification; saline wastewater; Nitrosomonas;

Nitrosospira; fixed-bed reactors, saline wastewater;, Nitrosomonas;
, Nitrosospira; , Fixed-bed reactors
ew
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Keyword:
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Nitrification in fixed-bed reactors treating saline wastewater
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Sudarno, S. Bathe, J. Winter, C. Gallert *
Institute of Biology for Engineers and Biotechnology of Wastewater
University of Karlsruhe
76128 Karlsruhe
Am Fasanengarten
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Corresponding author*:
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PD. Dr. Claudia Gallert
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Phone: +49-721-6082297
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Fax: +49-721-6087704
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Claudia.gallert@iba.uka.de
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Sudarno
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Department of Environmental Engineering
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University of Diponegoro UNDIP
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Semarang, Indonesia
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Present address: Institute of Biology for Engineers and Biotechnology of Wastewater
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76128 Karlsruhe
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Prof. Dr. Josef Winter
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Institute of Biology for Engineers and Biotechnology of Wastewater
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76128 Karlsruhe
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Abstract
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Halophilic nitrifyers belonging to the genus Nitrosomonas and Nitrospira were
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enriched from sea water and marine sediment samples of the North Sea. The
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maximal ammonia oxidation rate (AOR) in batch enrichments with sea water was
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15.1 mg N L-1 d-1. An intermediate nitrite accumulation was observed.
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Two fixed-bed reactors for continuous nitrification with either polyethylene/clay sinter
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lamellas (FBR A) or porous ceramic rings (FBR B) were run at two different ammonia
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concentrations, three different ALRs, pH adjustment and at an increased upflow
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velocity. A better overall nitrification without nitrite accumulation was observed in FBR
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B. However, FBR A revealed a higher AOR and nitrite oxidation rate of 6 and 7 mg N
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L-1 h-1, compared to FBR B with 5 and 5.9 mg N L-1 h-1, respectively. AORs in the
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FBRs were at least 10 times higher than in suspended enrichment cultures.
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Whereas a shift within the ammonia oxidizing population in the genus Nitrosomonas
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at the subspecies level occurred in FBR B with synthetic sea water at an increasing
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ammonia loading rate (ALR) and a decreasing pH, the nitrite oxidizing Nitrospira
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population apparently did not change.
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Key words
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Halophilic nitrification; saline wastewater; Nitrosomonas; Nitrosospira; fixed-bed
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reactors
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Introduction
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Most biological wastewater treatment processes are running with polluted water
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sources that contain either no or only little salt, whereas in coastal regions sea water
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dominated domestic wastewater may be generated (Wu et al. 2008). In addition
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several sources of industrial wastewater may contain 3 % sodium chloride or an even
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higher salt content. Thus, carbon removal, nitrification and denitrification in biological
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wastewater treatment processes should function over a wide range of salt
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concentrations to meet wastewater discharge criteria. Halo-tolerant or halophilic
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bacteria must be present to cope with the salt content of a certain wastewater.
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Saline wastewater containing high amounts of nitrogen and carbonaceous
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compounds for instance is generated during aqua culturing of marine fish or shrimps
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or by the fish canning industry. Nitrogen removal in real or artificial wastewater in the
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presence of 0 6 % NaCl in lab- or full-scale SBR systems has been investigated
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(Campos et al. 2002, Fontenot et al. 2007, Huilinir et al. 2008). With increasing salt
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concentrations up to 6 % removal efficiencies decreased drastically in the lab-scale
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SBRs inoculated with salt-adapted, but non-halophilic microorganism (Intrasungkha
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et al. 1999, Uygur and Kargi 2004), whereas the highest nitrifying activity of a
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halophilic bacterial population was obtained for an in-situ NaCl concentration of 28 g/l
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(Fontenot et al. 2007).
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Removal of carbonaceous and nitrogenous pollutants before discharging the
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wastewater into a water body is essential to avoid oxygen depletion and
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eutrophication. Conventional nitrogen removal processes for protein or ammonia
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containing wastewater are designed for aerobic carbon removal and nitrification,
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followed by anoxic denitrification with addition of an external carbon source, e.g. in
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sequencing batch reactors (Fontenot et al. 2007). Alternatively, new approaches like
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the completely autotrophic nitrogen removal over nitrite (CANON process) with
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nitritation of ammonia and anaerobic oxidation of ammonia with nitrite to gaseous
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nitrogen (Anammox) could be applied for treatment of saline wastewater in rotating
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biological contactor reactors (Liu et al. 2008). Chemolithoautotrophic nitrification
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proceeds in two steps, catalysed by phylogenetically different bacteria. Ammonia is
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oxidized to nitrite (nitritation) by ammonia oxidizing bacteria (AOB) and the nitrite is
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further oxidized to nitrate (nitratation) by nitrite oxidizing bacteria (NOB).
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Osmotic stress in the presence of high salt concentrations in wastewater reduces
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bacterial metabolic activities (Omil et al. 1995). The enrichment of halophilic
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organisms to treat wastewater with high concentrations of NaCl may help to
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overcome treatment problems (Dincer and Kargi 2001). The utilization of halophilic
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microbial consortia or even of enrichments from a conventional manure flora, that
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were adapted to saline conditions (selection of halotolerant bacteria), reduces the
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effect of salt stress on bacterial metabolism (Antileo et al. 2002, Mariangel et al.
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2008). Salt adapted microorganisms were also used in an anaerobic/anoxic/aerobic
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system for an improved organic matter and nitrogen removal (Panswad and Anan
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1999). Nitrite oxidizers were apparently more sensitive to high salt concentrations
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than ammonium oxidizers (Schenk and Hegemann 1995, Dincer and Kargi 2001). It
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has been demonstrated that in nitrifying seawater, nitrite was accumulating even
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when marine genera, adapted to high saline concentrations, were present (Catalan et
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al. 1997). Another effect of salinity on the nitrification process was an increased
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emission of the greenhouse gas N2O due to inhibition of N2O reductase (Tsuneda et
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al. 2005).
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The implementation of biological nitrification in a technical process can be
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attempted with suspended or immobilized bacteria in different types of bioreactors.
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The activated sludge system for wastewater treatment works with sludge floc
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enrichment. The effect of high inorganic salt concentrations on settling of sludge flocs
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is mainly related to disintegration of flocculated sludge. The smaller flocs tend to float
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as a result of a larger surface and the buoyancy force increases with increasing salt
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contents due to a higher water density (Wu et al. 2008). Attached growth of bacteria
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in biofilm systems offers several advantages as compared to suspended growth,
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such as a long sludge retention time, prevention of wash-out of biomass from the
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reactor and better process stability in terms of withstanding shock loadings or short-
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term inhibitory effects (Fitch et al. 1998; Nogueira et al. 1998). For biofilm systems,
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the selected support material as a substratum for attached growth has a great
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influence on process stability and therefore a huge variety of different materials were
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tested. Biofilm systems for wastewater treatment are well established but the
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treatment of saline wastewater in biofilm reactors has not been studied extensively.
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Nitrification of saline wastewater in biofilm reactors using PVC plastic tubes
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(Gharasallah et al. 2002), wrinkled PVC plates (Rosa et al. 1998) or PVC discs
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(Windey et al. 2005) was reported.
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The aim of this study was i) to determine the original nitrifying activity in brine and
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seawater samples to obtain a suitable inoculum for nitrification of an ammonia-
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containing wastewater with 3% NaCl content, ii) to establish continuously operated
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fixed-bed reactors (FBRs) for stable ammonia and nitrite removal rates with the most
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suitable enrichment and iii) to characterize the nitrifying population in these
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enrichments.
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Materials and methods
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Water and sediment sampling from different sites
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Water and sediment samples AD were collected from different costal locations at
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the North Sea in January 2008 (Table 1). The water samples (10 L) and the sediment
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samples (5 L) were filled into sterilized canisters and stored at 4 oC in a cool box
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during transportation to the laboratory.
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Batch assays for determination of the nitrifying activity
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Water or sediment samples (250 mL each) from the different collection places at the
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North Sea were filled into glass reactors of 500 mL total volume. A magnetic stirrer
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was used to mix the samples in the reactors. The reactors were aerated with 2 L min-
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(190 mg L-1 NH4Cl) incubation at room temperature (20-23 oC) was started. At
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different time intervals, liquid samples were taken for analysis of dissolved oxygen
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(DO), pH, ammonia, nitrite and nitrate. For pH correction, e.g. at day 42 and 52 (Fig.
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1A,1C,1D), 1-3 mL 0.25 M KOH was added to the reactors until a pH > 8.0 was
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reached. The DO in all samples was kept above 5 mg L-1.
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Fixed bed reactors
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Two cylindrical FBRs with 9 cm internal diameter and 45 cm height were used
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(scheme see Fig. 1). The feed was pumped into the reactor at the bottom with a
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Gilson Minipuls 3 pump (Abimed, Dreieich) and effluent was flowing out at the top of
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the reactor through an overflow pipe, maintaining a working volume of 2.1 L.
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Polyethylene/clay sinter lamellas (PELIA, Herding, Amberg, Germany) with a specific
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surface area of 440 m2 m-3, a fixed bed area of 0.46 m2 and a porosity of 59% were
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used as support material in FBR A and porous ceramic rings (Poro Ring, Tropical)
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with a specific surface area of 934 m2 m-3, a fixed bed area of 0.60 m2 and a porosity
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of 38% in FBR B. Both FBRs were filled with sea water from Hafen Bsum (Table 1)
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that contained 100 mg N L-1 (380 mg L-1 NH4Cl). During the first 40 days the FBRs
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were operated batchwise until all ammonia and nitrite was oxidized to nitrate.
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Afterwards, the FBRs were operated continuously. The ammonia loading rate (ALR)
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was increased by reducing the hydraulic retention time (HRT) or by increasing the
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ammonia concentration. The reactors were aerated from the bottom with an air
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diffuser at a flow rate of 2.5 L h-1, which was adjusted by a micro valve and controlled
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by a TG 05 gas meter (Ritter, Bochum-Langendreer, Germany). This maintained an
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oxygen concentration of 5 mg L-1 and a homogeneous distribution of the feeding.
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To further improve liquid mixing, an external recirculation loop was installed from the
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overflow to the inlet pipe for both FBRs. The reactor effluent was re-circulated at a
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flow rate of 8 L h-1 into the inlet pipe of the reactor with a tube pump (model 604 U,
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Watson-Marlow, Fallmouth, Cornwall, England). The pH was kept at 7.6 by a pH
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titrator (Dulcometer D1C, Prominent, Heidelberg, Germany) and a dosing pump
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(Prominent Gamma 74) with 0.25 M KOH solution. The reactors were run at room
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temperature.
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Synthetic seawater
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The reactors were fed with a synthetic sea water containing in g L-1: NaCl (28.13),
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KCl (0.77), CaCl2 .2H2O (1.6), MgCl2 .6H2O (4.8), NaHCO3 (0.11), MgSO4 .7H2O (3.5)
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and 0.3 mL L-1 nutrient solution of the following composition (g L-1): FeCl3 .6H2O (1.5),
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H3BO3 (0.15), CuSO4 (0.03), KI (0.18), MnCl2 4.H2O (0.12), Na2MoO4 .2H2O (0.06),
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ZnSO4 .7H2O (0.12), CoCl2 .6H2O (0.15) and EDTA (10).
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Sample preparation for analyses and physicochemical analyses
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All samples were centrifuged at 10000 rpm for 5 min in a laboratory centrifuge (model
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Z 233 M-2, Hermle, Gosheim, Germany). The clear supernatant was further
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analyzed. Ammonia and nitrite were measured colorimetrically according to DEV
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(1983), procedures E5 (DIN 38406) and D28 (DIN 38405), respectively. Nitrate was
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analyzed spectrophotometrically at 320 nm according to Standard Methods (APHA
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1998). The DO, pH, temperature and electrical conductivity (salinity) were measured
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with respective standard probes attached to a multi meter (Inolab multi level 1, WTW
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Weilheim, Germany). The alkalinity of the samples was calculated after titration to a
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pH value of pH 4.5 with 0.02 M HCl and expressed as CaCO3 (mg L-1).
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Nitritation and nitratation rates in the FBRs
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To determine the ammonia oxidation rate (AOR, nitritation rate) and the nitrite
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oxidation rate (NOR, nitratation rate), the continuously operated FBRs were run in
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batch-mode for one day during a time period where the ammonia and nitrite removal
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rates were high and no accumulation of ammonia or nitrite was observed. The
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respective nitrogen sources, NH4Cl (60 mg N L-1) for nitritation or KNO2 (30 mg N L-1)
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for nitratation, were then added. Decreasing ammonia and nitrite concentrations were
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monitored in the reactors. After complete removal of ammonia and nitrite, continuous
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operation of the FBRs was re-established.

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Molecular biological analyses.
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Five pieces of ceramic carrier material from FBR B were removed from the reactor
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per sampling for analyses of the biofilm composition. They were placed in a 50 ml
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Falcon tube, and 30 ml 0.85% KCl solution was added. The tube was vigorously
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vortexed for 5 min to release the biofilm from the carrier material and the resulting
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suspension was transferred into new tubes. Bacteria were sedimented by
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centrifugation and the pellet was washed by re-suspension in 0.85% KCl and
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repeated centrifugation. DNA from the pelleted bacteria was then extracted using an
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SDS/CTAB detergent lysis protocol followed by phenol-chloroform extraction
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(Ausubel et al. 1999).
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PCR was conducted in a Biometra TGradient thermal cycler. Sequences of the
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primers used in this study are listed in Table 2.
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The following individual primer combinations were used in separate reactions to
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target different organism groups: 8fm/1492r for Bacteria, AMOf/NSO1225 for -
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subclass ammonia-oxidizing bacteria, 8fm/Ntspa662 for Nitrospira spp., 8fm/NIT3 for
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Nitrobacter spp., and Arch-amoAF/Arch-amoAR for ammonia-oxidizing archaea. All
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reactions were carried out in 25 l volumes containing 0.5 units of polymerase
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(Fermentas TrueStart Taq DNA polymerase) in 1x reaction buffer, 2.5 mM MgCl2, 0.2
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mM of each dNTP, 0.2 M of each primer, and approx. 10-50 ng of template DNA.
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Thermal cycling was conducted with an initial denaturation of 5 min at 95 C followed
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by 35 cycles of 95 C for 30 s, 54 C for 1 min, and 72 C for 2 min. The programme
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was concluded by a final extension for 5 min at 72 C.
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The products of positive 16S rRNA gene targeted PCR reactions (Bacteria, AOB,
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Nitrospira) were then used as templates in a second round of PCR (nested PCR) with
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primers 341f-GC/518r to generate amplicons suitable for analysis by DGGE. One l
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of the first-round product was used in 25 l reactions under the same concentrations
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as listed above. The PCR program consisted of an initial denaturation at 95 C for 5
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min. followed by 20 cycles of 95 C for 30 s, 58 C for 30 s, and 72 C for 1 min and
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was concluded by a 20 min extension period at 72 C.
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DGGE was carried out in a CBS DGGE-2401 apparatus. Between 10 and 20 l of
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PCR product were separated in 10% poly acryl amide gels with a denaturant gradient
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from 50 to 75% for 16.5 h at 60 V and a temperature of 60 C. Gels were silver-
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stained to visualize the DNA.
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Products from AOB- and Nitrospira-specific first round PCR reactions were
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additionally cloned using the GeneJet PCR cloning kit (Fermentas). The inserts of
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individual clones were obtained by colony-PCR using primers pJET1f/pJET1r flanking
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the MCS of the cloning vector pJET1. Reactions were carried out using a PCR
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program consisting of 95 C for 5 min. followed by 35 cycles of 95 C for 30 s, 58 C
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for 30 s, and 72 C for 2 min. Amplicons were screened by RFLP using RsaI, and the
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PCR products of selected clones were then re-amplified using primers 341f-GC/518r
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followed by DGGE analysis in order to identify clones co-migrating with dominant
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bands in community profiles. The identified colony-PCR products were then custom-
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sequenced
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Sequences were analysed using BLAST at NCBI and sequence alignments followed
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by construction of phylogenetic trees were conducted using MEGA4 (Tamura et al.
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2007). The evolutionary history was inferred using the Neighbor-Joining method. The
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percentage of replicate trees in which the associated taxa clustered together in the
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bootstrap test (1000 replicates) is shown next to the branches (Figures 6, 7 later on).
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The evolutionary distances were computed using the Jukes-Cantor method and are
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shown in the units of the number of base substitutions per site. All positions
(Seqlab
Laboratories,
Gttingen,
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Page 10 of 42
Germany)
using
primer
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containing gaps and missing data were eliminated from the dataset (Complete
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deletion option). There were a total of 614 (Figure 6) and 722 (Figure 7) nucleotide
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positions in the datasets for ammonia-oxidizing bacteria and Nitrospira spp.,
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respectively
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The DNA sequences obtained in this study have been deposited in the EMBL
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nucleotide
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FN394308-FN394314.
database
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accession
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(http://www.ebi.ac.uk/embl/)
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Chemicals
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All chemicals used were of analytical grade and were purchased from VWR/Merck
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(Darmstadt), Fluka (Taufkirchen) or Roth (Karlsruhe, Germany).
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Results
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Characterization of marine water and sediment samples as natural sources of
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halophilic nitrifiers
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Sea water and sediment samples from different coastal regions at the North Sea
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were characterized for parameters such as salinity, alkalinity, ammonia, nitrite and
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nitrate concentrations (Table 1). The ammonia concentration in all four samples was
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less than 1 mg L-1. In sea water/mud-sample B from a brine water pond that
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remained in the mud during low tide and that was fed with treated sewage effluent at
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the harbor of the city of Norden the nitrate concentration was highest with 10.3 mg N
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L-1 (Table 1), most probably due to incoming nitrified domestic waste water. Sample
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B also revealed the highest salinity and alkalinity, presumably caused by its mud
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content. The influence of pollution by domestic waste water on the ammonia or
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nitrate content can clearly be seen by comparing samples C and D from St. Peter-
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Ording (Table 1). Wastewater discharge at the coast into brine water (sample C) led
284
to elevated ammonia and nitrate contents. At the point where the brine water flows
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into the open sea (sample D) ammonia and nitrate concentrations were much lower
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due to dilution with sea water.
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To test the nitrifying capability of sea water or sea water/mud mixtures, samples A-
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D (Table 1) were supplemented with 50 mg N L-1 ammonia. Portions of 250 ml were
289
filled into batch reactors for nitrification by the autochthonic bacteria. Ammonia
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depletion, nitrite formation and utilization as well as nitrate formation were analysed.
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After a lag-phase of approximately 10 d, ammonia oxidation began in all samples and
292
led to the formation of abundantly nitrite (samples A, B, D) or nitrate (sample C).
293
Ammonia conversion to nitrite or nitrate was incomplete in samples A, C and D
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apparently due to the low alkalinity and a pH drop below or far below 7 (Fig. 2).
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Ammonia was, however, completely converted to nitrite and then to nitrate in sea
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water/mud-sample B, which had a high alkalinity and a stable pH with a minimal
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value of 7.5 (Fig. 2B). When a second portion of ammonia was fed to this reactor at
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day 42, ammonia was oxidized rapidly to nitrite. The nitrite was further oxidized to
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nitrate within less than 10 d, indicating the successful enrichment of halophilic or at
300
least halotolerant nitrifyers. In the reactor with brine water sample C very little
301
ammonia was oxidized at the beginning due to the rapid drop of the pH (sample C
302
had the lowest alkalinity of all samples), but nitritation and nitratation proceeded
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shortly after titrating the pH to 8.7 (Fig. 2C, day 42). The strict dependence of
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ammonia oxidation on an alkaline pH can be seen in the three reactors where sea
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water samples with a low alkalinity were incubated for the establishment of halophilic
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nitrification (Fig. 2A,C,D). In these reactors the pH dropped rapidly when ammonia
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oxidation started and nitrite was produced. When the pH was raised to around 8.5 by
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addition of KOH at day 42 and 52, ammonia oxidation was completed and nitrite
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conversion to nitrate started (Fig. 2A,C) or was finished (Fig. 2D).
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The initial AOR of the enrichment culture obtained with sea water from Hafen
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Bsum (sample source A) after the lag-phase and after pH correction was around
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11.5 mg N L-1 d-1, that of enrichments from brine and sea water of St. Peter-Ording
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(sample sources C and D) 7.3 and 6.6 mg N L-1 d-1, respectively. In the sea
314
water/mud-mixture (sample source B), the AOR during the first feeding cycle was
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only 4.9 mg N L-1 d-1. It increased three-fold to 15.1 mg N L-1 d-1 during the second
316
feeding cycle after day 42 (Fig. 2B).
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Start-up of fixed-bed reactors with polyethylene/clay sinter lamellas (FBR A) and
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porous ceramic rings (FBR B) for continuous nitrification
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Sea water sample A from Hafen Bsum was used as an inoculation of FBR A and
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FBR B. Sample A was preferred over sample B because it did not contain mud and
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its AOR of 11.5 mg N d-1 was only slightly lower than that of sample B. Both FBRs
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were filled with 2 L sea water sample A (Table 1). After addition of 104 mg N L-1
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ammonia FBR A and FBR B were operated under batch conditions for 3 weeks to
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acclimatize the nitrifying microorganisms.
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After acclimatization a synthetic sea water medium was supplemented with NH4Cl
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as indicated in Table 3 and both FBRs were run for more than 300 d. Four phases
329
were distinguished, concerning the HRT, ALR and installation of an external water
330
recirculation to improve the mixing intensity for better conversion rates (Table 3).
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331
In the initial phase a (day 0 - 56, Fig. 3a) in FBR A ammonia was oxidized
332
completely and more nitrite than nitrate was formed. Due to malfunction of the pH
333
titrator at day 12, causing an increase of the pH to 9.1, 60 mg N L-1 ammonia were
334
remaining in the reactor effluent. Restoration of the full AOR was obtained after
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exchange of the pH probe within the next 10 d. From day 45 onward all ammonia
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was oxidized to the expected stoichiometric amount of nitrate without intermediary
337
nitrite accumulation (Fig. 3a). An optimum pH of 8.0 8.3 was reported for Nitrospira
338
spec. (Blackburne et al. 2007).
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In phase b (day 56 156, Fig. 3b) the HRT was reduced from 1.25 to 1 d and the
340
ALR was increased from 83 to 104 mg N L-1 d-1. Under steady state conditions
341
ammonia was still oxidized completely. Due to a failure of the pH probe at day 66 and
342
88 the pH fell below 6.5. Ammonia accumulation started immediately, but after
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replacement of the pH probe degradation of ammonia resumed. From day 112 - 132
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and 140 150 the aeration rate in FBR A increased drastically due to a leak of the
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internal aeration system. The higher air flow rates caused higher shear forces and
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this led to biofilm detachment from the carrier material and sedimentation/wash-out.
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Ammonia was no longer completely oxidized and little nitrite accumulated at the
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beginning of the disturbance (Fig. 3b, day 112).
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349
In phase c (day 156 216, Fig. 3c) the ALR was further increased to 130 mg N L-1
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d-1 by increasing the ammonia concentration in the medium. Ammonia was not
351
oxidized completely and some nitrite accumulated (Fig. 3c). Due to problems with the
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stability of pH probes in the saline environment the titration system was switched off
353
and the medium was buffered by addition of NaHCO3 from day 186 - 194. Ammonia
354
oxidation to nitrate improved significantly, but precipitates in the sodium bicarbonate
355
buffered medium led to clogging of the inlet-tubes and thus to a disturbance of high
356
rate reactor operation. After re-installation of the pH titration system and the omission
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Page 15 of 42
357
of sodium bicarbonate from day 194 onwards ammonia oxidation improved, leading
358
to around 20 mg N L-1 nitrite and up to 100 mg N L-1 nitrate, respectively (Fig. 3c).
359
In phase d of FBR A (conditions see Table 3), an effluent recirculation of 8 L h-1
360
(1.25 m h-1 upstream velocity) was installed. Immediately after starting recirculation of
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the reactor content, ammonia was completely oxidized without intermediate formation
362
of nitrite (Fig. 3d, day 219 - 250). From day 250 to the end of reactor operation 2040
363
mg N L-1 ammonia remained in the effluent, but no nitrite accumulated. The
364
decreasing AOR from day 250 onwards may have been due to a slow biofilm
365
detachment from the support material by effluent recirculation. The detached
366
biomass visibly settled in the conical bottom of the reactor.
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Contrary to FBR A, the biofilm on the porous ceramic rings of FBR B oxidized all
368
ammonia to nitrate in phase a from the start of continuous operation (Fig. 4a). A
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failure of the pH probe due to only short-term stability in saline medium caused a pH
370
decrease at day 8, 15 and 46, which led to short term incomplete oxidation of
371
ammonia until recalibration, but no significant amounts of nitrite accumulated. (Fig.
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4a). An increase of the ALR to 104 mg N L-1 d-1 in phase b of FBR B led to an
373
increase of the ammonia and a decrease of the nitrate concentration from day 6070
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in the reactor effluent but no nitrite accumulated. All ammonia was oxidized to nitrate
375
from day 90 onwards (Fig. 4b). A further increase of the ALR to 130 mg N L-1 d-1 in
376
phase c did not affect the nitrifying performance of the reactor initially (Fig. 4c).
377
However, after day 180 ammonia was no longer oxidized completely, nitrite
378
accumulated and nitrate decreased. Replacing the titration unit by a bicarbonate
379
buffered medium (day 187-194) could not stop the decrease of the nitrate formation
380
(Fig. 4c, day 186194). When the bicarbonate was omitted and the pH-titrator re-
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installed, ammonia oxidation and nitrate formation was shortly improved but
382
accumulating nitrite led to a reversal of the improvement (Fig. 4c). To overcome this
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decreasing efficiency, an external recirculation was installed in phase d after day 216
384
to improve mixing. This led to a short recovery of ammonia and nitrite oxidation (Fig.
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4d, day 219-240), but similar as in FBR A, the increased shear forces during
386
recirculation apparently led to a slow biofilm detachment, visible by the sludge, that
387
accumulated at the bottom of the reactor. The AOR decreased dramatically and at
388
first around 60, later on 80 mg N L-1 ammonia remained in the reactor effluent (Fig.
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4d, day 248 and following).
390
391
Ammonia and nitrite oxidation rates in FBR A and FBR B
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For determining the AORs and NORs in FBR A and FBR B, substrate supply was
394
stopped one day before rate analyses to obtain depletion of ammonia and nitrite. At
395
the respective days (Table 4) either NH4Cl or KNO2 was added to the reactor to
396
reach a final concentration of 60 or 30 mg N L-1, respectively. AORs and NORs were
397
determined from the slope of consumption curves and a surface area related
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nitritation and nitratation rate for FBR A and FBR B was calculated (Table 4). The
399
NOR was always higher than the AOR, except for day 64 in FBR A and day 215 and
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217 in FBR B, where nitrite accumulated due to an unknown disturbance (Fig. 4c,
401
4d). The surface area related AORs and NORs in FBR B with ceramic rings were
402
always lower than those in FBR A with polyethylene/clay sinter lamellas. The NOR,
403
especially in FBR B, was relatively stable, whereas the AOR showed a higher
404
fluctuation with time. After starting the external recirculation (phase d, Table 3), the
405
AOR in FBR A increased more than two-fold and the NOR more than 1.5-fold,
406
whereas the AOR and NOR in FBR B were not improved (Table 4. Day 217). There
407
was no positive short-term effect in FBR B as in FBR A, where the AOR at first
408
increased and then slowly decreased to the initial rates before recirculation, but the
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Page 16 of 42
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Page 17 of 42
409
capacity for nitrite oxidation remained high. The NOR was not increased by a better
410
homogenization through recirculation in FBR B (Table 4).
411
412
Microbial population
413
414
Group-specific PCR analyses yielded
415
products with primer pairs specific for Bacteria, Nitrosomonas spp., and Nitrospira
416
spp. (Fig. 4). No products were obtained with primers specific for Nitrobacter spp.
417
and the archaeal amoA gene (not shown). Thus, the nitrifying population in the
418
reactors seemed to be dominated by Nitrosomonas spp. and Nitrospira spp..
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The composition and dynamics of the nitrifyer populations was investigated using
420
PCR-DGGE followed by sequence analysis of cloned DGGE-bands. DGGE analyses
421
of samples from operational phases b and c (Fig. 5, Table 3: days 127, 160 and 210)
422
of FBR B showed in all cases relatively few bands for the bacterial profiles, indicating
423
that bacterial communities were represented by only a few phylo types. The most
424
dominant band was the same for all three sampling dates and migrated at the same
425
position as the single, dominant band (E) of Nitrospira-specific analyses (Fig. 5). The
426
ocurrence of only one band in the case of the Nitrospira-specific analyses indicated
427
that only one dominant organism was responsible for nitrite oxidation in the reactors,
428
which apparently did not change over time for different ALRs and pH-values. In
429
contrast, the AOB-specific profiles contained three bands each (A-C) for the first two
430
sampling dates, representing an ALR of 104 and 130 mg N L-1 d-1 at a pH of 7.5.
431
Band B disappeared and was replaced by a new band (D) in the third sample,
432
representing an ALR of 130 mg N L-1 d-1 in a bicarbonate buffered medium. Whereas
433
AOB-specific PCR products were not dominant or could not be seen in the DGGE
434
profiles for Bacteria of the first two samples, they were clearly recognizable in the
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435
third sample, indicating that AOB as well as Nitrospira spp. represented a significant
436
or even dominant part of the total bacterial population under these conditions (Fig. 5).
437
Elucidation of the sequences corresponding to dominant DGGE bands was done
438
by cloning of Bacteria-, AOB- and Nitrospira-specific PCR products and screening of
439
individual clones by RFLP and DGGE. Clones co-migrating with dominant DGGE
440
bands were sequenced. Figures 6 and 7 show the phylogenetic relatedness of the
441
obtained sequences to known sequences of AOB and Nitrospira spp., respectively.
442
The four detected AOB sequences (Fig. 5) were all related to salt-tolerant or salt-
443
requiring Nitrosomonas spp.: The DGGE-band A and B sequences were closely
444
related to Nitrosomonas aestuarii, which has been described as a common species in
445
marine and estuarine water with an obligate requirement for salt and an optimal
446
growth around 300 mM NaCl (1.7%) (Koops et al. 1991). The sequence related to
447
DGGE band D was more distantly related to N. aestuarii, but still clustered within the
448
N. aestuarii / N. marina clade of salt-requiring Nitrosomonas spp. The DGGE-band C
449
related sequence clustered within the Nitrosomonas Nm143 lineage, which contains
450
sequences found in coastal water and sediments with salinities above 1% (Purkhold
451
et al. 2003). The sequences of clones co-migrating with the Nitrospira-specific DGGE
452
band E were all closely related to the species N. marina, which was the first
453
obligately salt-requiring, marine nitrite oxidizer in this genus (Watson et al. 1986).
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Page 18 of 42
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Discussion
457
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Saline wastewater with a high load of nitrogen compounds is released by many
459
industries, such as sea food processing companies, tanneries, gelatin producers or

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460
even metal refining companies. An often reported but not always successful
461
approach for biological nitrification of saline wastewater is the stepwise adaption of
462
sweat water bacteria to halophilic conditions (e.g. Moussa et al. 2006). Adaption of a
463
sweat water nitrifying population to salt water conditions of up to 40 g/l NaCl seems
464
not to be possible (Moussa et al. 2006), whereas an enrichment of halophilic nitrifyers
465
from halophilic environmental sources would be a more promising approach, since
466
most halophilic nitrifyers are apparently ubiquitous in seawater environment (Francis
467
et al. 2005). Enrichment of halophilic nitrifying consortia from sea water or mud
468
samples of the coastal region of the North Sea in Germany with a salinity of about
469
3% NaCl (conductivity > 30 mS cm-1, Table 1) seemed thus to be most suitable to
470
establish halophilic nitrification in the laboratory. The applicability of marine
471
sediments as inocula for nitrification under saline conditions was also reported by
472
Antileo et al. (2002), who found ammonia oxidation after a lag-phase of 15 d. In our
473
first enrichments from different marine sources, AORs ranged between 4.9 and 15.1
474
mg N L-1d-1, which was in accordance with the work of Rejish Kumar et al. (2009),
475
who measured AORs of 4.6 and 12.2 mg N L-1d-1.
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Villaverde et al. (1997) described a linear correlation between alkalinity (e.g. mg L-1
477
CaCO3) and pH with a stoichiometric coefficient of 7.1 mg CaCO3 consumed per mg
478
NH4+-N oxidized. If the alkalinity was not sufficient, the pH decreased during
479
oxidation of 50 mg N L-1 ammonia as it was observed in our samples A and C and to
480
a minor extent in sample D to values below pH 5 (Fig. 2). Sample B, a sea water/mud
481
mixture had the highest alkalinity and the pH was stable above 7.5 during the whole
482
experiment, including a second feeding with 50 mg N L-1 ammonia (Fig. 2b). Under
483
batch conditions, in samples AD, a short lag-phase of approximately 10 d was
484
observed before ammonia oxidation started. An intermediate nitrite accumulation was

19
485
observed in all samples (Fig. 2a-2d, 3a, 3c) independently of the pH. Whether nitrous
486
oxide was also formed in trace amounts under halophilic nitrification conditions, as
487
reported by Tsuneda et al. (2005) was not analysed.
488
489
In FBRs the support material seems to play a major role during the early stages of
490
a biofilm formed by fast-growing heterotrophic bacteria and during a much longer
491
time for biofilms with slow-growing autotrophic bacteria. Surface characteristics of the
492
support material are important, e.g. the roughness of surfaces and other surfaces
493
properties significantly influence bacterial colonization (Gjaltema et al. 1997, Verran
494
et al. 1991;). Crevices and pores act as niches for attachment of bacteria and protect
495
the biofilm from shear forces (Fox et al. 1990). In both of our FBRs, biomass
496
detachment was observed at increased shear forces after installation of liquid
497
recirculation. Concomitant with this phenomenon, AORs decreased notably, while the
498
NORs were apparently not influenced (Fig. 3d and 4d). The decrease was more
499
pronounced in FBR B with porous ceramic rings as a carrier material than in FBR A
500
with polyethylene-clay sinter lamellas. Turbulence in this reactor was visibly higher
501
than in FBR A due to more rugged surfaces through which the water must find it way
502
to the top. This might indicate, that the ammonia oxidizing nitrifyers were mainly
503
located in the outer layers of the biofilm on the support material and therefore may
504
have been sheared off to a higher extent by increased shear forces during liquid
505
recirculation. This would be in accordance with the report of Okabe et al. (1999), who
506
found a layering of AOB at the surface and of nitrite oxidizing bacteria in deeper
507
zones in nitrifying biofilms fed with domestic wastewater. A short-term effect
508
immediately after installation of the liquid recirculation was the increase of the AOR
509
and NOR in FBR A (Table 4, day 217) indicating that the better mixing, created by
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510
aeration and an increased up flow velocity (1.25 m h-1), apparently improved mass
511
transfer of oxygen and ammonia from the bulk liquid into the biofilm. Zhu and Chen
512
(2001) also observed an influence of turbulence on the AOR. The performance of
513
their nitrifying biofilters could be significantly improved by increasing the Reynolds
514
number in their biofilm reactor. In FBR B the aeration system was apparently
515
sufficient for an optimal supply of the nitrifyers in the biofilm with oxygen and
516
ammonia since the AOR and the NOR did not increase after starting the liquid
517
recirculation (Table 4, after day 215).
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518
In FBR B, ceramic rings with a specific surface area of 934 m2 m-3 were used as
519
support material for biofilm formation, which was a twice as high specific surface area
520
than with polyethylene/clay sinter lamellas of FBR A. FBR B had a better nitrification
521
performance from the beginning of the continuous operation. Ammonia oxidation was
522
almost complete except when titration failed. Nitrite accumulation was only seen
523
during stage 3 (Table 3) after re-installation of the pH titrator (Fig. 4c). This was in
524
accordance with Krner and Rosenthal (1983) who reported that the AOR was
525
proportional to the surface of the support material that was used for biofiltration. It
526
might be generalized, that the surface area of the support material significantly
527
influences the conversion rates when only a faint biofilm, as in the case of autotrophic
528
nitrifyers was formed, whereas the inner surfaces play only a minor role if a thick
529
biofilm was obtained as for carbon-rich industrial wastewater. Inner surfaces are then
530
blocked and diffusion of substrates into pores is highly limited.
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531
The maximum AORs and NORs before installation of the liquid recirculation were 6
532
and 7 mg N L-1 h-1, respectively in FBR A, and 5 and 5.9 mg N L-1 h-1 in FBR B. To
533
correlate the specific surface area of the support material with the maximum N-
534
removal rate, area related AORs and NORs were calculated. For FBR A a maximum
21
535
surface area related AOR and NOR of 312 and 386 mg N m-2 d-1 and for FBR B of
536
199 and 236 mg N L-1 d-1, respectively, was determined (Table 4). Maximal and area
537
related AORs and NORs were better in FBR A than in FBR B, but the overall
538
performance (residual ammonia in reactor effluent, intermediate nitrite accumulation)
539
was better in FBR B. This may be due to a surface oriented and a more dense biofilm
540
in FBR A with higher rates but less efficient conversion. Nijhof and Bovendeur (1990)
541
determined an area related AOR under saline conditions at 24 oC of 280 mg N m-2 d-
542
543
obtained .
, whereas a much better AOR under fresh water conditions of 690 mg N m-2 d-1 was
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In continuously run FBRs for halophilic nitrification, that were inoculated with sea
545
water, organisms that were targeted specifically by a universal primer for Bacteria
546
were rather rare. DGGE revealed that the one dominant band, present at all sampling
547
times, represented Nitrospira spp.. The bands for AOB in the first two samples were
548
the same and were almost invisible with the primer for Bacteria. The nitrifyer
549
populations in FBR B were composed of salt-requiring ammonia- and nitrite-oxidizing
550
bacteria. The AOB population remained the same between the first two sampling
551
dates, consisting of three organisms related to Nitrosomonas aestuarii and
552
Nitrosomonas sp. Nm143. Between the second and the third sampling date, the
553
organism corresponding to DGGE band B became less dominant and was replaced
554
by the organism corresponding to DGGE band D. The nitrite oxidizer population
555
consisted of organisms closely related to Nitrospira marina, which yielded a single
556
strong DGGE band present in all three samples.
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Acknowledgment
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Page 23 of 42
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This work was supported by a grant from the German Academic Exchange Service
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DAAD to Mr. Sudarno.
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References
563
APHA (1995) Standard Methods for the Examination of Water and Wastewater.
564
American Public Health Association, Washington, DC.
565
Antileo C, Aspe E, Urrutia H, Zaror C, Roeckel M (2002).Nitrifying biomass
566
acclimation to high ammonia concentration. J Environ Eng 128, 367-375
567
Ausubel FM, Brent R, Kingson RE, Moore DD, Seidman JG, Smith JA, Struhl K
568
(1999) Short Protocols in Molecular Biology. Wiley and Sons, New York, USA
569
Campos JL, Mosquera-Collal A, Snchez M, Mndez R, Lema JM (2002) Nitrification
570
in saline wastewater with high ammonia concentration in an activated sludge unit.
571
Water Res 36, 25552560
572
Blackburne R, Vadivelu VM, Yuan Z, Keller J (2007) Kinetic characterization of an
573
enriched Nitrospira culture with comparison to Nitrobacter. Water Res 41(14):3033-
574
3042
575
Catalan MAB, Wang PC, Matsumura M (1997) Nitrification performance of marine
576
nitrifyers immobilized in polyester and macroporous cellulose carriers. J Ferment
577
Bioeng 84, 563571
578
Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M (2001). In situ
579
characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater
580
treatment plants. Appl Environ Microbiol 67: 5273-5284
581
DEV
582
Schlammuntersuchung. Verlag Chemie, Weinheim
583
Dincer AR, Kargi F (2001) Performance of rotating biological disc system treating
584
saline wastewater. Process Biochem 36, 901906
(1983)
Deutsche
iew
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 24 of 42
Einheitsverfahren
24
zur
Wasser-,
Abwasser-
und
Page 25 of 42
585
Fitch MW, Pearson N, Richard, G, Burken JG (1998) Biological Fixed-film System.
586
Water Environ Res 70, 495-518
587
Fontenot Q, Bonvillain C, Kilgen M, Boopathy R (2007) Effects of temperature,
588
salinity, and carbon: nitrogen ratio on sequencing batch reactor treating shrimp
589
aquaculture wastewater. Bioresource Technol 98:17001703
590
Fox P, Suidan MT, Bandy JT (1990) A comparison of media types in acetate fed
591
expanded-bed anaerobic reactor. Water Res 24:827-835
592
Francis CA, Roberts KJ, Beman J M, Santoro A E, Oakley BB (2005) Ubiquity and
593
diversity of ammonia-oxidizing archaea in water columns and sediments of the
594
ocean. Proc Natl Acad Sci USA 102:14683-14688
595
Gharsallah N, Khannous L, Souissi N, Nasri M (2002) Biological treatment of saline
596
wastewaters from marine-products processing factories by a fixed-bed reactor. J
597
Chem Techno. Biol 77:865-870
598
Gjaltema A, van der Marel N, van Loosdrecht MCM, Heijnen JJ (1997) Adhesion and
599
biofilm development on suspended carriers in airlift reactor: Hydrodynamic conditions
600
versus surface characteristics. Biotechnol Bioeng 55:880-889
601
Huilinir M, Cristina Marti M, Aspe E, Roecke M (2008) Organic and nitrogenous
602
matter effects on the denitrification of saline protein-rich effluents. Environ Technol
603
29:881-890
604
Intrasungkha N, Keller J, Blackall LL (1999) Biological nutrient removal efficiency in
605
treatment of saline wastewater. Water Sci Technol 39(6):183-190
606
Koops HP, Bttcher B, Mller UC, Pommerening-Rser A, Stehr G (1991)
607
Classification of eight new species of ammonia-oxidizing bacteria: Nitrosomonas
608
communis sp. nov., Nitrosomonas ureae sp. nov., Nitrosomonas aestuarii sp. nov.,
iew
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
25
609
Nitrosomonas marina sp. nov., Nitrosomonas nitrosa sp. nov., Nitrosomonas
610
eutropha sp. nov., Nitrosomonas oligotropha sp. nov., and Nitrosomonas halophila
611
sp. nov. J Gen Microbiol 137:1689-1699
612
Krner G, Rosenthal H (1983) Efficiency of nitrification in trickling filters using
613
different substrates. Aquacult Eng 2:49-67
614
Liu S, Yang F, Gong Z, Su Z (2008) Assessment of the positive effect of salinity on
615
the nitrogen removal performance and microbial composition during the start-up of
616
CANON process. Appl Microbiol Biotechnol 80:339348
617
Lane DJ (1991) 16S/23S rRNA sequencing. In Nucleic Acids Techniques in Bacterial
618
Systematics. Stackebrandt, E., and Goodfellow, M. . Chichester, UK: John Wiley &
619
Sons, pp. 115175
620
Mariangel L, Aspe E, Cristina Marti M, Roeckel M, 2008. The effect of sodium
621
chloride on the denitrification of saline fishery wastewater. Environ Technol 29:871-
622
879
623
McCaig AE, Embley TM, Prosser JI (1994) Molecular analysis of enrichment cultures
624
of marine ammonia oxidisers. FEMS Microbiol Lett 120(3):363368
625
Mobarry BK, Wagner M, Urbain V, Rittmann BE, Stahl DA (1996). Phylogenetic
626
probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl
627
Environ Microbiol 62: 2156-2162
628
Moussa MS, Sumanasekera DU, Ibrahim SH, Lubberding HJ, Hooijmans CM, Gijzen
629
HJ, van Loosdrecht MCM (2006) Long term effects of salt on the activity, population
630
structure and floc characteristics in enriched bacterial cultures for nitrifiers. Water
631
Res 40:1377-1388
iew
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Page 26 of 42
26
Page 27 of 42
632
Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial
633
populations by denaturing gradient gel electrophoresis analysis of polymerase chain
634
reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 59:695-700
635
636
637
Nijhof M, Bovendeur J (1990) Fixed film nitrification characteristic in sea water
638
recirculation fish culture systems. Aquaculture 87:133-134
639
Nogueira R, Lazarova V, Manem J, Melo LF (1998) Influence of dissolved oxygen on
640
the nitrification kinetics in a circulating bed biofilm reactor. Bioprocess. Eng 19: 441-
641
449
642
Okabe S, Satoh H, Watanabe Y (1999) In situ analysis of nitrifying biofilms as
643
determined by in situ hybridization and the use of microelectrodes. Appl Environ
644
Microbiol 65:3182-3191
645
Omil F, Mendez R, Lema JM (1995) Anaerobic treatment of saline wastewaters
646
under high sulphide and ammonia content. Bioresource Technol 54:269278
647
Panswad T, Anan C (1999) Specific oxygen, ammonia, and nitrate uptake rates of a
648
biological nutrient removal process treating elevated salinity wastewater. Bioresource
649
Technol 70:237-243
650
Purkhold U, Wagner M, Timmermann G, Pommerening-Rser A, Koops HP (2003)
651
16S rRNA and amoA-based phylogeny of 12 novel betaproteo bacterial ammonia-
652
oxidizing isolates: extension of the dataset and proposal of a new lineage within the
653
nitrosomonads. Int J Syst Evol Microbiol 53:1485-94
654
Rejish Kumar VJ, Achuthan C, Manju NJ, Philip R, Bright Singh IS (2009). Mass
655
production of nitrifying bacterial consortia for the rapid establishment of nitrification in
656
saline recirculating aquaculture systems. World J Microb Biot 25:407-414
iew
ev
rR
ee
rP
Fo
1
2
3
4
5
6
7
8
9
10
11
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43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
27
657
Rosa MF, Furtado AAL, Albuquerque RT, Leite SGF, Medronho RA (1998) Biofilm
658
development and ammonia removal in the nitrification of a saline wastewater.
659
Bioresource Technol 65:135-138
660
Schenk H, Hegemann W (1995) Nitrification inhibition by high salinity concentrations
661
in the aerobic biological treatment of tannery wastewater. Wasser/Abwasser
662
136:465470
663
Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary
664
Genetics Analysis (MEGA) software version 4.0. Molecular Biology and Evolution
665
24:1596-1599
666
Tsuneda S, Mikami M, Kimochi Y, Hirata A (2005) Effect of salinity on nitrous oxide
667
emission in the biological nitrogen removal process for industrial wastewater. J
668
Hazard Mater.119: 9398
669
Uygur A, Kargi F (2004) Salt inhibition on biological nutrient removal from saline
670
wastewater in a sequencing batch reactor. Enzyme Microb Tech 34:313-318
671
Verran J, Lees G, Shakespeare AP (1991) The effect of surface roughness on the
672
adhesion of Candida albicans to acrylic. Biofouling 3:183-192
673
Villaverde S, Garcia-Encina.A, Polanco (1997) Influence of pH over nitrifying biofilm
674
activity in submerged biofilters. Water Res 31:1180-1186
675
Wagner M, Rath G, Koops HP, Flood J, Amann R (1996). In situ analysis of nitrifying
676
bacteria in sewage treatment plants. WatSciTechn 34: 237-244
677
Watson SW, Bock E, Valois FW, Waterbury JB, Schlosser U (1986)
678
marina gen. nov. sp. nov.: a chemolithotrophic nitrite-oxidizing bacterium. Arch
679
Microbiol 144:1-7
iew
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Page 28 of 42
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Page 29 of 42
680
Windey K, Bo ID, Verstraete W (2005) Oxygen-limited autotrophic nitrification-
681
denitrification (OLAND) in a rotating biological contactor treating high-salinity
682
wastewater. Water Res 39:45124520
683
Wu G, Guan Y, Zhan X (2008) Effect of salinity on the activity, settling and microbial
684
community of activated sludge in sequencing batch reactors treating synthetic saline
685
wastewater. Water Sci Technol 58(2):351-358
686
Zhu S, Chen S (2001). Impacts of Reynolds number on nitrification biofilm kinetics.
687
Aquacult Eng 24:213-229
688
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Legends of Figures
Figure 1: Arrangement of the fixed-bed reactors used for nitrification experiments
Figure 2: Initiation of nitrification of ammonia (50 mg N L-1) by autochthonic nitrifyers
from different sea water and mud samples
Sample sources: A: Hafen Bsum (sea water), B: Town Norden (sea water), C: Town
St. Peter-Ording (brine water), D: Town St. Peter-Ording (sea water). At day 42 and
52 the pH was raised with KOH. In assay B ammonia was added a second time at
day 42. Symbols: ---- Ammonia, ---- Nitrite, ---- Nitrate, pH. The incubation
temperature was 20-23 oC.
rR
ee
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Fo
12
Figure 3: Ammonia, nitrite and nitrate concentrations during continuous operation of
13
FBR A (support material: polyethylene/clay sinter lamellas) for conditions of Table 3.
14
Fig. 3a: day 055, phase a; Fig. 3b: day 55155, phase b; Fig. 3c: day 155215,
15
phase c; Fig. 3d: day 215305, phase d. Symbols: ---- Ammonia, ---- Nitrite and --
16
-- Nitrate. The incubation temperature was 20-23 oC at an air flow rate of 2.5 L h-1.
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Page 30 of 42
17
18
Figure 4: Ammonia, nitrite and nitrate concentrations during continuous operation of
19
FBR B (support material: porous ceramic rings) for conditions of Table 3.
20
Fig. 4a: day 055, phase a; Fig. 4b: day 55155, phase b; Fig. 4c: day 155215,
21
phase c; Fig. 4d: day 215305, phase d. Symbols: ---- Ammonia, ---- Nitrite and --
22
-- Nitrate. The incubation temperature was 20-23 oC at an air flow rate of 2.5 L h-1.
30
Page 31 of 42
Figure 5: DGGE analysis of samples from FBR B.
The image shows DGGE profiles specific for Bacteria, ammonia-oxidizing bacteria,
and Nitrospira spp.. Samples were taken at day 127, 160 and 210 from FBR B.
5
6
Figure 6: Evolutionary relationships of ammonia-oxidizing bacteria.
7
8
9
11
Figure 7: Evolutionary relationship of Nitrospira spp.
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Table 1: Characterization of the used seawater/mud samples
___________________________________________________________________
3
4
5
6
7
8
9
10
11
12
13
14
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16
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18
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20
21
22
Conductivity Alkalinity
NH4+-N NO2--N NO3- -N
-1
-1
(mS cm )
(mg L )
(mg L-1) (mg L-1) (mg L-1)
as CaCO3
___________________________________________________________________
A Sea water from
31.9
120
0
0.11
2.3
Hafen Bsum
Sample
B Sea water-mud
mixture from town
Norden
37.5
Fo
C Brine water from

town St. PeterOrding
1.34
400
0.2
0.32
10.3
53
0.9
0.37
5.1
rP
D Sea water from

32.1
130
0.6
0.68
1.7
town St. PeterOrding
___________________________________________________________________
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Page 32 of 42
Page 33 of 42
Table 2. PCR primers used in this study.

The indicated primers for Nitrospira and Nitrobacter spp. were used as reverse
primers in combination with 8fm as forward primer.
Primer
name
8fm
1492r
AMOf
NSO1225
Lane 1991
Lane 1991
McCaig et al. 1994
Mobarry et al. 1996
Daims et al. 2001
Wagner et al. 1996
Francis et al. 2005
Francis et al. 2005
Muyzer et al. 1993
Muyzer et al. 1993

Promega
Promega
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rR
pJET1r
AGA GTT TGA TC(AC) TGG

CTC AG
G(CT)T ACC TTG TTA CGA CTT
TGG GGR ATA ACG CAY CGA
AAG
CGC CAT TGT ATT ACG TGT
GA
GGA ATT CCG CGC TCC TCT
CCT GTG CTC CAT GCT CCG
(GC)TA ATG GTC TGG CTT
AGA CG
GCG GCC ATC CAT CTG TAT
GT
CGC CCG CCG CGC GCG GCG
GGC GGG GCG GGG GCA
CGG GGG GCC TAC GGG AGG
CAG CAG
ATT ACC GCG GCT GCT GG
GCC TGA ACA CCA TAT CCA
TCC
GCA GCT GAG AAT ATT GTA
GGA GAT
ee
518r
pJET1f
Reference
rP
Ntspa662
NIT3
ArchamoAF
ArchamoAR
341f-GC
Sequence (5-3)
Fo
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8
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51
52
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58
59
60
Table 3: Operational phases a-d of FBR A and FBR B.
___________________________________________________________________
Phase
___________________________________________________________________
Days
0-55 55-155
155-186 187-194 195-215
215-305
HRT (d)
1.25 1
pH adjustment with
KOH KOH
KOH
NaHCO3 KOH
KOH
NH4+-Nin (mg L-1)
104
104
130
130
130
130
ALR mg NH4+-N L-1 d-1
104
130
130
130
130
8 L h-1
Fo
83
10
External recirculation
11
___________________________________________________________________
12
HRT = Hydraulic Retention Time, ALR = Ammonia Loading Rate.
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Page 34 of 42
Page 35 of 42
Table 4: Surface area specific ammonia and nitrite oxidation rates in FBR A and FBR
B.
________________________________________________________________
Days
AOR
Days
NOR
(mg N m-2 d-1)
(mg N m-2 d-1)
FBR A
FBR A
FBR B
FBR B
__________________________________________________________________
48
274
230
156
65
230
226
51
85
386
236
216
ee
363
182
218
547
181
484
210
Fo
64
306
10
84
176
11
113
145
12
173
312
13
215
207
197
14
217
502
197
15
257
295
112
16
284
272
149
17
295
218
82
18
___________________________________________________________________
rP
156
199
258
296
ev
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214
Figure 1:

pH controller
0.25 M KOH
Effluent
pH probe
Support material
External recycle
Air diffuser
rP
Fo
Air
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Influent
NaCl 2.8%
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Figure 5
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Figure 6
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Nitrosospira spp.
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AM295526
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AF272423 Nitrosomonas cryotolerans
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M96400 N. sp. C-56
Nitrosomonas marina / aestuarii
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AF386752 N. sp. R7c131
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AF386751 N. sp. R7c155
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Nitrosomonas nitrosa / communis

Nitrosococcus mobilis
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Nitrosomonas halophila
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Nitrosomonas eutropha / europaea

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Outgroups
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Figure 7
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X82559 N. marina
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Lineage IV

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Applied Microbiology and Biotechnology

Manuscript for review

Nitrification in fixed-bed reactors treating saline

Applied Microbiology and Biotechnology

Complete List of Authors:

Date Submitted by the

Sudarno, Utomo; Universitt Karlsruhe, Institut fr

Halophilic nitrification; saline wastewater; Nitrosomonas;

Nitrification in fixed-bed reactors treating saline wastewater

Sudarno, S. Bathe, J. Winter, C. Gallert *

Institute of Biology for Engineers and Biotechnology of Wastewater

PD. Dr. Claudia Gallert

Department of Environmental Engineering

University of Diponegoro UNDIP

Present address: Institute of Biology for Engineers and Biotechnology of Wastewater

Applied Microbiology and Biotechnology

Prof. Dr. Josef Winter

Institute of Biology for Engineers and Biotechnology of Wastewater

Applied Microbiology and Biotechnology

Halophilic nitrifyers belonging to the genus Nitrosomonas and Nitrospira were

15.1 mg N L-1 d-1. An intermediate nitrite accumulation was observed.

concentrations, three different ALRs, pH adjustment and at an increased upflow

FBRs were at least 10 times higher than in suspended enrichment cultures.

population apparently did not change.

Halophilic nitrification; saline wastewater; Nitrosomonas; Nitrosospira; fixed-bed

dominated domestic wastewater may be generated (Wu et al. 2008). In addition

several sources of industrial wastewater may contain 3 % sodium chloride or an even

wastewater treatment processes should function over a wide range of salt

concentrations to meet wastewater discharge criteria. Halo-tolerant or halophilic

Saline wastewater containing high amounts of nitrogen and carbonaceous

presence of 0 6 % NaCl in lab- or full-scale SBR systems has been investigated

concentrations up to 6 % removal efficiencies decreased drastically in the lab-scale

SBRs inoculated with salt-adapted, but non-halophilic microorganism (Intrasungkha

(Fontenot et al. 2007).

Removal of carbonaceous and nitrogenous pollutants before discharging the

wastewater into a water body is essential to avoid oxygen depletion and

eutrophication. Conventional nitrogen removal processes for protein or ammonia

followed by anoxic denitrification with addition of an external carbon source, e.g. in

Applied Microbiology and Biotechnology

Applied Microbiology and Biotechnology

nitritation of ammonia and anaerobic oxidation of ammonia with nitrite to gaseous

nitrogen (Anammox) could be applied for treatment of saline wastewater in rotating

biological contactor reactors (Liu et al. 2008). Chemolithoautotrophic nitrification

proceeds in two steps, catalysed by phylogenetically different bacteria. Ammonia is

further oxidized to nitrate (nitratation) by nitrite oxidizing bacteria (NOB).

Osmotic stress in the presence of high salt concentrations in wastewater reduces

bacterial metabolic activities (Omil et al. 1995). The enrichment of halophilic

organisms to treat wastewater with high concentrations of NaCl may help to

microbial consortia or even of enrichments from a conventional manure flora, that

were adapted to saline conditions (selection of halotolerant bacteria), reduces the

2008). Salt adapted microorganisms were also used in an anaerobic/anoxic/aerobic

The implementation of biological nitrification in a technical process can be

attempted with suspended or immobilized bacteria in different types of bioreactors.

in biofilm systems offers several advantages as compared to suspended growth,

Nitrification of saline wastewater in biofilm reactors using PVC plastic tubes

(Windey et al. 2005) was reported.

Applied Microbiology and Biotechnology

seawater samples to obtain a suitable inoculum for nitrification of an ammonia-

containing wastewater with 3% NaCl content, ii) to establish continuously operated

suitable enrichment and iii) to characterize the nitrifying population in these

Applied Microbiology and Biotechnology

Materials and methods

Water and sediment sampling from different sites