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Manuscript for review

Nitrification in fixed-bed reactors treating salinewastewater

Journal: Applied Microbiology and Biotechnology

Manuscript ID: AMB-09-18698.R1

Manuscript Category: Original Paper

Date Submitted by theAuthor:

07-Oct-2009

Complete List of Authors: Sudarno, Utomo; Universität Karlsruhe, Institut fürIngenieurbiologie und Biotechnologie des AbwassersBathe, Stephan; Universität Karlsruhe, Institut fürIngenieurbiologoe und Biotechnologie des Abwassers; UniversitätKarlsruhe, Institut für Ingenieurbiologie und Biotechnologie desAbwassersWinter, Josef; Universität Karlsruhe, Institut für Ingineurbiologieund Biotechnologie des AbwassersGallert, Claudia; Universität Karlsruhe, Institut für Ingenieurbiologieund Biotechnologie des Abwassers

Keyword:Halophilic nitrification; saline wastewater; Nitrosomonas;Nitrosospira; fixed-bed reactors, saline wastewater;, Nitrosomonas;, Nitrosospira; , Fixed-bed reactors

Applied Microbiology and Biotechnology


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Nitrification in fixed-bed reactors treating saline wastewater

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

Institute of Biology for Engineers and Biotechnology of Wastewater

University of Karlsruhe

76128 Karlsruhe

Am Fasanengarten

Corresponding author*:

PD. Dr. Claudia Gallert

Phone: +49-721-6082297

Fax: +49-721-6087704

[email protected]

Sudarno

Department of Environmental Engineering

University of Diponegoro UNDIP

Semarang, Indonesia

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


76128 Karlsruhe

Prof. Dr. Josef Winter

Institute of Biology for Engineers and Biotechnology of Wastewater


76128 Karlsruhe

age 1 of 42 Applied Microbiology and Biotechnology

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Abstract

Halophilic nitrifyers belonging to the genus Nitrosomonas and Nitrospira were

enriched from sea water and marine sediment samples of the North Sea. The

maximal ammonia oxidation rate (AOR) in batch enrichments with sea water was

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

Two fixed-bed reactors for continuous nitrification with either polyethylene/clay sinter

lamellas (FBR A) or porous ceramic rings (FBR B) were run at two different ammonia

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

velocity. A better overall nitrification without nitrite accumulation was observed in FBR

B. However, FBR A revealed a higher AOR and nitrite oxidation rate of 6 and 7 mg N

L-1 h-1, compared to FBR B with 5 and 5.9 mg N L -1 h-1, respectively. AORs in the

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

Whereas a shift within the ammonia oxidizing population in the genus Nitrosomonas

at the subspecies level occurred in FBR B with synthetic sea water at an increasing

ammonia loading rate (ALR) and a decreasing pH, the nitrite oxidizing Nitrospira

population apparently did not change.

Key words

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

reactors

Page 2 of 42Applied Microbiology and Biotechnology

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Introduction

Most biological wastewater treatment processes are running with polluted water

sources that contain either no or only little salt, whereas in coastal regions sea water

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

higher salt content. Thus, carbon removal, nitrification and denitrification in biological

wastewater treatment processes should function over a wide range of salt

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

bacteria must be present to cope with the salt content of a certain wastewater.

Saline wastewater containing high amounts of nitrogen and carbonaceous

compounds for instance is generated during aqua culturing of marine fish or shrimps

or by the fish canning industry. Nitrogen removal in real or artificial wastewater in the

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

(Campos et al. 2002, Fontenot et al. 2007, Huilinir et al. 2008). With increasing salt

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

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

et al. 1999, Uygur and Kargi 2004), whereas the highest nitrifying activity of a

halophilic bacterial population was obtained for an in-situ NaCl concentration of 28 g/l

(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

containing wastewater are designed for aerobic carbon removal and nitrification,

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


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The implementation of biological nitrification in a technical process can be

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

The activated sludge system for wastewater treatment works with sludge floc

enrichment. The effect of high inorganic salt concentrations on settling of sludge flocs

is mainly related to disintegration of flocculated sludge. The smaller flocs tend to float

as a result of a larger surface and the buoyancy force increases with increasing salt

contents due to a higher water density (Wu et al. 2008). Attached growth of bacteria

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

such as a long sludge retention time, prevention of wash-out of biomass from the

reactor and better process stability in terms of withstanding shock loadings or short-

term inhibitory effects (Fitch et al. 1998; Nogueira et al. 1998). For biofilm systems,

the selected support material as a “substratum” for attached growth has a great

influence on process stability and therefore a huge variety of different materials were

tested. Biofilm systems for wastewater treatment are well established but the

treatment of saline wastewater in biofilm reactors has not been studied extensively.

Nitrification of saline wastewater in biofilm reactors using PVC plastic tubes

(Gharasallah et al. 2002), wrinkled PVC plates (Rosa et al. 1998) or PVC discs

(Windey et al. 2005) was reported.

The aim of this study was i) to determine the original nitrifying activity in brine and

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

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

fixed-bed reactors (FBRs) for stable ammonia and nitrite removal rates with the most

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

enrichments.


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Materials and methods

Water and sediment sampling from different sites

Water and sediment samples A–D were collected from different costal locations at

the North Sea in January 2008 (Table 1). The water samples (10 L) and the sediment

samples (5 L) were filled into sterilized canisters and stored at 4 oC in a cool box

during transportation to the laboratory.

Batch assays for determination of the nitrifying activity

Water or sediment samples (250 mL each) from the different collection places at the

North Sea were filled into glass reactors of 500 mL total volume. A magnetic stirrer

was used to mix the samples in the reactors. The reactors were aerated with 2 L min -

1 air blown in via a syringe needle just above the stirrer. After addition of 50 mg N L -1

(190 mg L-1 NH4Cl) incubation at room temperature (20-23oC) was started. At

different time intervals, liquid samples were taken for analysis of dissolved oxygen

(DO), pH, ammonia, nitrite and nitrate. For pH correction, e.g. at day 42 and 52 (Fig.

1A,1C,1D), 1-3 mL 0.25 M KOH was added to the reactors until a pH > 8.0 was

reached. The DO in all samples was kept above 5 mg L-1.

Fixed bed reactors

Two cylindrical FBRs with 9 cm internal diameter and 45 cm height were used

(scheme see Fig. 1). The feed was pumped into the reactor at the bottom with a

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.

Polyethylene/clay sinter lamellas (PELIA, Herding, Amberg, Germany) with a specific

surface area of 440 m2 m-3, a fixed bed area of 0.46 m2 and a porosity of 59% were

used as support material in FBR A and porous ceramic rings (Poro Ring, Tropical)

with a specific surface area of 934 m2 m-3, a fixed bed area of 0.60 m2 and a porosity

of 38% in FBR B. Both FBRs were filled with sea water from Hafen Büsum (Table 1)

that contained 100 mg N L-1 (380 mg L-1 NH4Cl). During the first 40 days the FBRs

were operated batchwise until all ammonia and nitrite was oxidized to nitrate.

Afterwards, the FBR´s were operated continuously. The ammonia loading rate (ALR)

was increased by reducing the hydraulic retention time (HRT) or by increasing the

ammonia concentration. The reactors were aerated from the bottom with an air

diffuser at a flow rate of 2.5 L h-1, which was adjusted by a micro valve and controlled

by a TG 05 gas meter (Ritter, Bochum-Langendreer, Germany). This maintained an

oxygen concentration of ≥ 5 mg L-1 and a homogeneous distribution of the feeding.

To further improve liquid mixing, an external recirculation loop was installed from the

overflow to the inlet pipe for both FBRs. The reactor effluent was re-circulated at a

flow rate of 8 L h-1 into the inlet pipe of the reactor with a tube pump (model 604 U,

Watson-Marlow, Fallmouth, Cornwall, England). The pH was kept at 7.6 by a pH

titrator (Dulcometer D1C, Prominent, Heidelberg, Germany) and a dosing pump

(Prominent Gamma 74) with 0.25 M KOH solution. The reactors were run at room

temperature.

Synthetic seawater

The reactors were fed with a synthetic sea water containing in g L-1

: NaCl (28.13),

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),

H3BO3 (0.15), CuSO4 (0.03), KI (0.18), MnCl2 4.H2O (0.12), Na2MoO4

.2H2O (0.06),

ZnSO4.7H2O (0.12), CoCl2

.6H2O (0.15) and EDTA (10).

Sample preparation for analyses and physicochemical analyses

All samples were centrifuged at 10000 rpm for 5 min in a laboratory centrifuge (model

Z 233 M-2, Hermle, Gosheim, Germany). The clear supernatant was further

analyzed. Ammonia and nitrite were measured colorimetrically according to DEV

(1983), procedures E5 (DIN 38406) and D28 (DIN 38405), respectively. Nitrate was

analyzed spectrophotometrically at 320 nm according to Standard Methods (APHA

1998). The DO, pH, temperature and electrical conductivity (salinity) were measured

with respective standard probes attached to a multi meter (Inolab multi level 1, WTW

Weilheim, Germany). The alkalinity of the samples was calculated after titration to a

pH value of pH 4.5 with 0.02 M HCl and expressed as CaCO3 (mg L-1).

Nitritation and nitratation rates in the FBRs

To determine the ammonia oxidation rate (AOR, nitritation rate) and the nitrite

oxidation rate (NOR, nitratation rate), the continuously operated FBRs were run in

batch-mode for one day during a time period where the ammonia and nitrite removal

rates were high and no accumulation of ammonia or nitrite was observed. The

respective nitrogen sources, NH4Cl (60 mg N L-1) for nitritation or KNO2 (30 mg N L

-1)

for nitratation, were then added. Decreasing ammonia and nitrite concentrations were

monitored in the reactors. After complete removal of ammonia and nitrite, continuous

operation of the FBRs was re-established.


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Molecular biological analyses.

Five pieces of ceramic carrier material from FBR B were removed from the reactor

per sampling for analyses of the biofilm composition. They were placed in a 50 ml

Falcon tube, and 30 ml 0.85% KCl solution was added. The tube was vigorously

vortexed for 5 min to release the biofilm from the carrier material and the resulting

suspension was transferred into new tubes. Bacteria were sedimented by

centrifugation and the pellet was washed by re-suspension in 0.85% KCl and

repeated centrifugation. DNA from the pelleted bacteria was then extracted using an

SDS/CTAB detergent lysis protocol followed by phenol-chloroform extraction

(Ausubel et al. 1999).

PCR was conducted in a Biometra TGradient thermal cycler. Sequences of the

primers used in this study are listed in Table 2.

The following individual primer combinations were used in separate reactions to

target different organism groups: 8fm/1492r for Bacteria , βAMOf/NSO1225 for β-

subclass ammonia-oxidizing bacteria, 8fm/Ntspa662 for Nitrospira spp., 8fm/NIT3 for

Nitrobacter spp., and Arch-amoAF/Arch-amoAR for ammonia-oxidizing archaea. All

reactions were carried out in 25 µl volumes containing 0.5 units of polymerase

(Fermentas TrueStart Taq DNA polymerase) in 1x reaction buffer, 2.5 mM MgCl2, 0.2

mM of each dNTP, 0.2 µM of each primer, and approx. 10-50 ng of template DNA.

Thermal cycling was conducted with an initial denaturation of 5 min at 95 °C followed

by 35 cycles of 95 °C for 30 s, 54 °C for 1 min, and 72 °C for 2 min. The programme

was concluded by a final extension for 5 min at 72 °C.

The products of positive 16S rRNA gene targeted PCR reactions (Bacteria, AOB,

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 µl

of the first-round product was used in 25 µl reactions under the same concentrations

as listed above. The PCR program consisted of an initial denaturation at 95 °C for 5

min. followed by 20 cycles of 95 °C for 30 s, 58 °C for 30 s, and 72 °C for 1 min and

was concluded by a 20 min extension period at 72 °C.

DGGE was carried out in a CBS DGGE-2401 apparatus. Between 10 and 20 µl of

PCR product were separated in 10% poly acryl amide gels with a denaturant gradient

from 50 to 75% for 16.5 h at 60 V and a temperature of 60 °C. Gels were silver-

stained to visualize the DNA.

Products from AOB- and Nitrospira -specific first round PCR reactions were

additionally cloned using the GeneJet PCR cloning kit (Fermentas). The inserts of

individual clones were obtained by colony-PCR using primers pJET1f/pJET1r flanking

the MCS of the cloning vector pJET1. Reactions were carried out using a PCR

program consisting of 95 °C for 5 min. followed by 35 cycles of 95 °C for 30 s, 58 °C

for 30 s, and 72 °C for 2 min. Amplicons were screened by RFLP using RsaI, and the

PCR products of selected clones were then re-amplified using primers 341f-GC/518r

followed by DGGE analysis in order to identify clones co-migrating with dominant

bands in community profiles. The identified colony-PCR products were then custom-

sequenced (Seqlab Laboratories, Göttingen, Germany) using primer 341f.

Sequences were analysed using BLAST at NCBI and sequence alignments followed

by construction of phylogenetic trees were conducted using MEGA4 (Tamura et al.

2007). The evolutionary history was inferred using the Neighbor-Joining method. The

percentage of replicate trees in which the associated taxa clustered together in the

bootstrap test (1000 replicates) is shown next to the branches (Figures 6, 7 later on).

The evolutionary distances were computed using the Jukes-Cantor method and are

shown in the units of the number of base substitutions per site. All positions


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B also revealed the highest salinity and alkalinity, presumably caused by its mud

content. The influence of pollution by domestic waste water on the ammonia or

nitrate content can clearly be seen by comparing samples C and D from St. Peter-

Ording (Table 1). Wastewater discharge at the coast into brine water (sample C) led

to elevated ammonia and nitrate contents. At the point where the brine water flows

into the open sea (sample D) ammonia and nitrate concentrations were much lower

due to dilution with sea water.

To test the nitrifying capability of sea water or sea water/mud mixtures, samples A-

D (Table 1) were supplemented with 50 mg N L-1 ammonia. Portions of 250 ml were

filled into batch reactors for nitrification by the autochthonic bacteria. Ammonia

depletion, nitrite formation and utilization as well as nitrate formation were analysed.

After a lag-phase of approximately 10 d, ammonia oxidation began in all samples and

led to the formation of abundantly nitrite (samples A, B, D) or nitrate (sample C).

Ammonia conversion to nitrite or nitrate was incomplete in samples A, C and D

apparently due to the low alkalinity and a pH drop below or far below 7 (Fig. 2).

Ammonia was, however, completely converted to nitrite and then to nitrate in sea

water/mud-sample B, which had a high alkalinity and a stable pH with a minimal

value of 7.5 (Fig. 2B). When a second portion of ammonia was fed to this reactor at

day 42, ammonia was oxidized rapidly to nitrite. The nitrite was further oxidized to

nitrate within less than 10 d, indicating the successful enrichment of halophilic or at

least halotolerant nitrifyers. In the reactor with brine water sample C very little

ammonia was oxidized at the beginning due to the rapid drop of the pH (sample C

had the lowest alkalinity of all samples), but nitritation and nitratation proceeded

shortly after titrating the pH to 8.7 (Fig. 2C, day 42). The strict dependence of

ammonia oxidation on an alkaline pH can be seen in the three reactors where sea

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

oxidation started and nitrite was produced. When the pH was raised to around 8.5 by

addition of KOH at day 42 and 52, ammonia oxidation was completed and nitrite

conversion to nitrate started (Fig. 2A,C) or was finished (Fig. 2D).

The initial AOR of the enrichment culture obtained with sea water from Hafen

Büsum (sample source A) after the lag-phase and after pH correction was around

11.5 mg N L-1 d-1, that of enrichments from brine and sea water of St. Peter-Ording

(sample sources C and D) 7.3 and 6.6 mg N L-1 d-1, respectively. In the sea

water/mud-mixture (sample source B), the AOR during the first feeding cycle was

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

feeding cycle after day 42 (Fig. 2B).

Start-up of fixed-bed reactors with polyethylene/clay sinter lamellas (FBR A) and

porous ceramic rings (FBR B) for continuous nitrification

Sea water sample A from Hafen Büsum was used as an inoculation of FBR A and

FBR B. Sample A was preferred over sample B because it did not contain mud and

its AOR of 11.5 mg N d-1 was only slightly lower than that of sample B. Both FBRs

were filled with 2 L sea water sample A (Table 1). After addition of 104 mg N L-1

ammonia FBR A and FBR B were operated under batch conditions for 3 weeks to

acclimatize the nitrifying microorganisms.

After acclimatization a synthetic sea water medium was supplemented with NH4Cl

as indicated in Table 3 and both FBRs were run for more than 300 d. Four phases

were distinguished, concerning the HRT, ALR and installation of an external water

recirculation to improve the mixing intensity for better conversion rates (Table 3).


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In the initial phase a (day 0 - 56, Fig. 3a) in FBR A ammonia was oxidized

completely and more nitrite than nitrate was formed. Due to malfunction of the pH

titrator at day 12, causing an increase of the pH to 9.1, 60 mg N L-1 ammonia were

remaining in the reactor effluent. Restoration of the full AOR was obtained after

exchange of the pH probe within the next 10 d. From day 45 onward all ammonia

was oxidized to the expected stoichiometric amount of nitrate without intermediary

nitrite accumulation (Fig. 3a). An optimum pH of 8.0 – 8.3 was reported for Nitrospira

spec. (Blackburne et al. 2007).

In phase b (day 56 – 156, Fig. 3b) the HRT was reduced from 1.25 to 1 d and the

ALR was increased from 83 to 104 mg N L-1 d-1. Under steady state conditions

ammonia was still oxidized completely. Due to a failure of the pH probe at day 66 and

88 the pH fell below 6.5. Ammonia accumulation started immediately, but after

replacement of the pH probe degradation of ammonia resumed. From day 112 - 132

and 140 – 150 the aeration rate in FBR A increased drastically due to a leak of the

internal aeration system. The higher air flow rates caused higher shear forces and

this led to biofilm detachment from the carrier material and sedimentation/wash-out.

Ammonia was no longer completely oxidized and little nitrite accumulated at the

beginning of the disturbance (Fig. 3b, day 112).

In phase c (day 156 – 216, Fig. 3c) the ALR was further increased to 130 mg N L -1

d-1 by increasing the ammonia concentration in the medium. Ammonia was not

oxidized completely and some nitrite accumulated (Fig. 3c). Due to problems with the

stability of pH probes in the saline environment the titration system was switched off

and the medium was buffered by addition of NaHCO3 from day 186 - 194. Ammonia

oxidation to nitrate improved significantly, but precipitates in the sodium bicarbonate

buffered medium led to clogging of the inlet-tubes and thus to a disturbance of high

rate reactor operation. After re-installation of the pH titration system and the omission


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of sodium bicarbonate from day 194 onwards ammonia oxidation improved, leading

to around 20 mg N L-1 nitrite and up to 100 mg N L-1 nitrate, respectively (Fig. 3c).

In phase d of FBR A (conditions see Table 3), an effluent recirculation of 8 L h-1

(1.25 m h-1 upstream velocity) was installed. Immediately after starting recirculation of

the reactor content, ammonia was completely oxidized without intermediate formation

of nitrite (Fig. 3d, day 219 - 250). From day 250 to the end of reactor operation 20–40

mg N L-1 ammonia remained in the effluent, but no nitrite accumulated. The

decreasing AOR from day 250 onwards may have been due to a slow biofilm

detachment from the support material by effluent recirculation. The detached

biomass visibly settled in the conical bottom of the reactor.

Contrary to FBR A, the biofilm on the porous ceramic rings of FBR B oxidized all

ammonia to nitrate in phase a from the start of continuous operation (Fig. 4a). A

failure of the pH probe due to only short-term stability in saline medium caused a pH

decrease at day 8, 15 and 46, which led to short term incomplete oxidation of

ammonia until recalibration, but no significant amounts of nitrite accumulated. (Fig.

4a). An increase of the ALR to 104 mg N L -1 d-1 in phase b of FBR B led to an

increase of the ammonia and a decrease of the nitrate concentration from day 60–70

in the reactor effluent but no nitrite accumulated. All ammonia was oxidized to nitrate

from day 90 onwards (Fig. 4b). A further increase of the ALR to 130 mg N L -1 d-1 in

phase c did not affect the nitrifying performance of the reactor initially (Fig. 4c).

However, after day 180 ammonia was no longer oxidized completely, nitrite

accumulated and nitrate decreased. Replacing the titration unit by a bicarbonate

buffered medium (day 187-194) could not stop the decrease of the nitrate formation

(Fig. 4c, day 186–194). When the bicarbonate was omitted and the pH-titrator re-

installed, ammonia oxidation and nitrate formation was shortly improved but

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

to improve mixing. This led to a short recovery of ammonia and nitrite oxidation (Fig.

4d, day 219-240), but similar as in FBR A, the increased shear forces during

recirculation apparently led to a slow biofilm detachment, visible by the sludge, that

accumulated at the bottom of the reactor. The AOR decreased dramatically and at

first around 60, later on 80 mg N L-1 ammonia remained in the reactor effluent (Fig.

4d, day 248 and following).

Ammonia and nitrite oxidation rates in FBR A and FBR B

For determining the AORs and NORs in FBR A and FBR B, substrate supply was

stopped one day before rate analyses to obtain depletion of ammonia and nitrite. At

the respective days (Table 4) either NH4Cl or KNO2 was added to the reactor to

reach a final concentration of 60 or 30 mg N L-1, respectively. AORs and NORs were

determined from the slope of consumption curves and a surface area related

nitritation and nitratation rate for FBR A and FBR B was calculated (Table 4). The

NOR was always higher than the AOR, except for day 64 in FBR A and day 215 and

217 in FBR B, where nitrite accumulated due to an unknown disturbance (Fig. 4c,

4d). The surface area related AORs and NORs in FBR B with ceramic rings were

always lower than those in FBR A with polyethylene/clay sinter lamellas. The NOR,

especially in FBR B, was relatively stable, whereas the AOR showed a higher

fluctuation with time. After starting the external recirculation (phase d, Table 3), the

AOR in FBR A increased more than two-fold and the NOR more than 1.5-fold,

whereas the AOR and NOR in FBR B were not improved (Table 4. Day 217). There

was no positive short-term effect in FBR B as in FBR A, where the AOR at first

increased and then slowly decreased to the initial rates before recirculation, but the


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capacity for nitrite oxidation remained high. The NOR was not increased by a better

homogenization through recirculation in FBR B (Table 4).

Microbial population

Group-specific PCR analyses yielded

products with primer pairs specific for Bacteria , Nitrosomonas spp., and Nitrospira

spp. (Fig. 4). No products were obtained with primers specific for Nitrobacter spp.

and the archaeal amoA gene (not shown). Thus, the nitrifying population in the

reactors seemed to be dominated by Nitrosomonas spp. and Nitrospira spp..

The composition and dynamics of the nitrifyer populations was investigated using

PCR-DGGE followed by sequence analysis of cloned DGGE-bands. DGGE analyses

of samples from operational phases b and c (Fig. 5, Table 3: days 127, 160 and 210)

of FBR B showed in all cases relatively few bands for the bacterial profiles, indicating

that bacterial communities were represented by only a few phylo types. The most

dominant band was the same for all three sampling dates and migrated at the same

position as the single, dominant band (E) of Nitrospira -specific analyses (Fig. 5). The

ocurrence of only one band in the case of the Nitrospira -specific analyses indicated

that only one dominant organism was responsible for nitrite oxidation in the reactors,

which apparently did not change over time for different ALRs and pH-values. In

contrast, the AOB-specific profiles contained three bands each (A-C) for the first two

sampling dates, representing an ALR of 104 and 130 mg N L -1 d-1 at a pH of 7.5.

Band B disappeared and was replaced by a new band (D) in the third sample,

representing an ALR of 130 mg N L-1 d-1 in a bicarbonate buffered medium. Whereas

AOB-specific PCR products were not dominant or could not be seen in the DGGE

profiles for Bacteria of the first two samples, they were clearly recognizable in the


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third sample, indicating that AOB as well as Nitrospira spp. represented a significant

or even dominant part of the total bacterial population under these conditions (Fig. 5).

Elucidation of the sequences corresponding to dominant DGGE bands was done

by cloning of Bacteria -, AOB- and Nitrospira -specific PCR products and screening of

individual clones by RFLP and DGGE. Clones co-migrating with dominant DGGE

bands were sequenced. Figures 6 and 7 show the phylogenetic relatedness of the

obtained sequences to known sequences of AOB and Nitrospira spp., respectively.

The four detected AOB sequences (Fig. 5) were all related to salt-tolerant or salt-

requiring Nitrosomonas spp.: The DGGE-band A and B sequences were closely

related to Nitrosomonas aestuarii, which has been described as a common species in

marine and estuarine water with an obligate requirement for salt and an optimal

growth around 300 mM NaCl (1.7%) (Koops et al. 1991). The sequence related to

DGGE band D was more distantly related to N. aestuarii , but still clustered within the

N. aestuarii / N. marina clade of salt-requiring Nitrosomonas spp. The DGGE-band C

related sequence clustered within the Nitrosomonas Nm143 lineage, which contains

sequences found in coastal water and sediments with salinities above 1% (Purkhold

et al. 2003). The sequences of clones co-migrating with the Nitrospira -specific DGGE

band E were all closely related to the species N. marina , which was the first

obligately salt-requiring, marine nitrite oxidizer in this genus (Watson et al. 1986).

Discussion

Saline wastewater with a high load of nitrogen compounds is released by many

industries, such as sea food processing companies, tanneries, gelatin producers or


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even metal refining companies. An often reported but not always successful

approach for biological nitrification of saline wastewater is the stepwise adaption of

sweat water bacteria to halophilic conditions (e.g. Moussa et al. 2006). Adaption of a

sweat water nitrifying population to salt water conditions of up to 40 g/l NaCl seems

not to be possible (Moussa et al. 2006), whereas an enrichment of halophilic nitrifyers

from halophilic environmental sources would be a more promising approach, since

most halophilic nitrifyers are apparently ubiquitous in seawater environment (Francis

et al. 2005). Enrichment of halophilic nitrifying consortia from sea water or mud

samples of the coastal region of the North Sea in Germany with a salinity of about

3% NaCl (conductivity > 30 mS cm-1, Table 1) seemed thus to be most suitable to

establish halophilic nitrification in the laboratory. The applicability of marine

sediments as inocula for nitrification under saline conditions was also reported by

Antileo et al. (2002), who found ammonia oxidation after a lag-phase of 15 d. In our

first enrichments from different marine sources, AORs ranged between 4.9 and 15.1

mg N L-1d-1, which was in accordance with the work of Rejish Kumar et al. (2009),

who measured AORs of 4.6 and 12.2 mg N L-1d-1.

Villaverde et al. (1997) described a linear correlation between alkalinity (e.g. mg L -1

CaCO3) and pH with a stoichiometric coefficient of 7.1 mg CaCO3 consumed per mg

NH4+-N oxidized. If the alkalinity was not sufficient, the pH decreased during

oxidation of 50 mg N L-1 ammonia as it was observed in our samples A and C and to

a minor extent in sample D to values below pH 5 (Fig. 2). Sample B, a sea water/mud

mixture had the highest alkalinity and the pH was stable above 7.5 during the whole

experiment, including a second feeding with 50 mg N L -1 ammonia (Fig. 2b). Under

batch conditions, in samples A–D, a short lag-phase of approximately 10 d was

observed before ammonia oxidation started. An intermediate nitrite accumulation was


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observed in all samples (Fig. 2a-2d, 3a, 3c) independently of the pH. Whether nitrous

oxide was also formed in trace amounts under halophilic nitrification conditions, as

reported by Tsuneda et al. (2005) was not analysed.

In FBRs the support material seems to play a major role during the early stages of

a biofilm formed by fast-growing heterotrophic bacteria and during a much longer

time for biofilms with slow-growing autotrophic bacteria. Surface characteristics of the

support material are important, e.g. the roughness of surfaces and other surfaces

properties significantly influence bacterial colonization (Gjaltema et al. 1997, Verran

et al. 1991;). Crevices and pores act as niches for attachment of bacteria and protect

the biofilm from shear forces (Fox et al. 1990). In both of our FBRs, biomass

detachment was observed at increased shear forces after installation of liquid

recirculation. Concomitant with this phenomenon, AORs decreased notably, while the

NORs were apparently not influenced (Fig. 3d and 4d). The decrease was more

pronounced in FBR B with porous ceramic rings as a carrier material than in FBR A

with polyethylene-clay sinter lamellas. Turbulence in this reactor was visibly higher

than in FBR A due to more rugged surfaces through which the water must find it way

to the top. This might indicate, that the ammonia oxidizing nitrifyers were mainly

located in the outer layers of the biofilm on the support material and therefore may

have been sheared off to a higher extent by increased shear forces during liquid

recirculation. This would be in accordance with the report of Okabe et al. (1999), who

found a layering of AOB at the surface and of nitrite oxidizing bacteria in deeper

zones in nitrifying biofilms fed with domestic wastewater. A short-term effect

immediately after installation of the liquid recirculation was the increase of the AOR

and NOR in FBR A (Table 4, day 217) indicating that the better mixing, created by


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aeration and an increased up flow velocity (1.25 m h-1), apparently improved mass

transfer of oxygen and ammonia from the bulk liquid into the biofilm. Zhu and Chen

(2001) also observed an influence of turbulence on the AOR. The performance of

their nitrifying biofilters could be significantly improved by increasing the Reynolds

number in their biofilm reactor. In FBR B the aeration system was apparently

sufficient for an optimal supply of the nitrifyers in the biofilm with oxygen and

ammonia since the AOR and the NOR did not increase after starting the liquid

recirculation (Table 4, after day 215).

In FBR B, ceramic rings with a specific surface area of 934 m2 m-3 were used as

support material for biofilm formation, which was a twice as high specific surface area

than with polyethylene/clay sinter lamellas of FBR A. FBR B had a better nitrification

performance from the beginning of the continuous operation. Ammonia oxidation was

almost complete except when titration failed. Nitrite accumulation was only seen

during stage 3 (Table 3) after re-installation of the pH titrator (Fig. 4c). This was in

accordance with Krüner and Rosenthal (1983) who reported that the AOR was

proportional to the surface of the support material that was used for biofiltration. It

might be generalized, that the surface area of the support material significantly

influences the conversion rates when only a faint biofilm, as in the case of autotrophic

nitrifyers was formed, whereas the inner surfaces play only a minor role if a thick

biofilm was obtained as for carbon-rich industrial wastewater. Inner surfaces are then

blocked and diffusion of substrates into pores is highly limited.

The maximum AORs and NORs before installation of the liquid recirculation were 6

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

correlate the specific surface area of the support material with the maximum N-

removal rate, area related AORs and NORs were calculated. For FBR A a maximum


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surface area related AOR and NOR of 312 and 386 mg N m -2 d-1 and for FBR B of

199 and 236 mg N L-1 d-1, respectively, was determined (Table 4). Maximal and area

related AORs and NORs were better in FBR A than in FBR B, but the overall

performance (residual ammonia in reactor effluent, intermediate nitrite accumulation)

was better in FBR B. This may be due to a surface oriented and a more dense biofilm

in FBR A with higher rates but less efficient conversion. Nijhof and Bovendeur (1990)

determined an area related AOR under saline conditions at 24 oC of 280 mg N m-2 d-

1, whereas a much better AOR under fresh water conditions of 690 mg N m -2 d-1 was

obtained .

In continuously run FBRs for halophilic nitrification, that were inoculated with sea

water, organisms that were targeted specifically by a universal primer for Bacteria

were rather rare. DGGE revealed that the one dominant band, present at all sampling

times, represented Nitrospira spp.. The bands for AOB in the first two samples were

the same and were almost invisible with the primer for Bacteria. The nitrifyer

populations in FBR B were composed of salt-requiring ammonia- and nitrite-oxidizing

bacteria. The AOB population remained the same between the first two sampling

dates, consisting of three organisms related to Nitrosomonas aestuarii and

Nitrosomonas sp. Nm143. Between the second and the third sampling date, the

organism corresponding to DGGE band B became less dominant and was replaced

by the organism corresponding to DGGE band D. The nitrite oxidizer population

consisted of organisms closely related to Nitrospira marina , which yielded a single

strong DGGE band present in all three samples.

Acknowledgment


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This work was supported by a grant from the German Academic Exchange Service

DAAD to Mr. Sudarno.


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Windey K, Bo ID, Verstraete W (2005) Oxygen-limited autotrophic nitrification-

denitrification (OLAND) in a rotating biological contactor treating high-salinity

wastewater. Water Res 39:4512–4520

Wu G, Guan Y, Zhan X (2008) Effect of salinity on the activity, settling and microbial

community of activated sludge in sequencing batch reactors treating synthetic saline

wastewater. Water Sci Technol 58(2):351-358

Zhu S, Chen S (2001). Impacts of Reynolds number on nitrification biofilm kinetics.

Aquacult Eng 24:213-229


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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 Büsum (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.

Figure 3: Ammonia, nitrite and nitrate concentrations during continuous operation of

FBR A (support material: polyethylene/clay sinter lamellas) for conditions of Table 3.

Fig. 3a: day 0–55, phase a; Fig. 3b: day 55–155, phase b; Fig. 3c: day 155–215,

phase c; Fig. 3d: day 215–305, phase d. Symbols: ---- Ammonia, ---- Nitrite and --

-- Nitrate. The incubation temperature was 20-23 oC at an air flow rate of 2.5 L h -1.

Figure 4: Ammonia, nitrite and nitrate concentrations during continuous operation of

FBR B (support material: porous ceramic rings) for conditions of Table 3.

Fig. 4a: day 0–55, phase a; Fig. 4b: day 55–155, phase b; Fig. 4c: day 155–215,

phase c; Fig. 4d: day 215–305, phase d. Symbols: ---- Ammonia, ---- Nitrite and --

-- Nitrate. The incubation temperature was 20-23o

C at an air flow rate of 2.5 L h-1

.


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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.

Figure 6: Evolutionary relationships of ammonia-oxidizing bacteria.

Figure 7: Evolutionary relationship of Nitrospira spp.


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Table 1: Characterization of the used seawater/mud samples

___________________________________________________________________

Sample Conductivity Alkalinity NH4+-N NO2

--N NO3- -N

(mS cm-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1)as CaCO3

___________________________________________________________________A Sea water from 31.9 120 0 0.11 2.3Hafen Büsum

B Sea water-mud 37.5 400 0.2 0.32 10.3mixture from townNorden

C Brine water from 1.34 53 0.9 0.37 5.1

town St. Peter-Ording

D Sea water from 32.1 130 0.6 0.68 1.7town St. Peter-Ording ___________________________________________________________________


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Table 2. PCR primers used in this study.The indicated primers for Nitrospira and Nitrobacter spp. were used as reverseprimers in combination with 8fm as forward primer.

Primer

name

Sequence (5’-3’) Reference

8fm AGA GTT TGA TC(AC) TGGCTC AG

Lane 1991

1492r G(CT)T ACC TTG TTA CGA CTT Lane 1991βAMOf TGG GGR ATA ACG CAY CGA

AAGMcCaig et al. 1994

NSO1225 CGC CAT TGT ATT ACG TGTGA

Mobarry et al. 1996

Ntspa662 GGA ATT CCG CGC TCC TCT Daims et al. 2001NIT3 CCT GTG CTC CAT GCT CCG Wagner et al. 1996Arch-amoAF

(GC)TA ATG GTC TGG CTTAGA CG

Francis et al. 2005

Arch-amoAR

GCG GCC ATC CAT CTG TATGT

Francis et al. 2005

341f-GC CGC CCG CCG CGC GCG GCGGGC GGG GCG GGG GCACGG GGG GCC TAC GGG AGGCAG CAG

Muyzer et al. 1993

518r ATT ACC GCG GCT GCT GG Muyzer et al. 1993pJET1f GCC TGA ACA CCA TAT CCA

TCCPromega

pJET1r GCA GCT GAG AAT ATT GTAGGA GAT

Promega


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Table 3: Operational phases a-d of FBR A and FBR B.

___________________________________________________________________

Phase a b c d

___________________________________________________________________

Days 0-55 55-155 155-186 187-194 195-215 215-305

HRT (d) 1.25 1 1 1 1 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 83 104 130 130 130 130

External recirculation - - - - - 8 L h-1

___________________________________________________________________

HRT = Hydraulic Retention Time, ALR = Ammonia Loading Rate.


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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 B FBR A FBR B

__________________________________________________________________

48 274 230

64 306 156 65 230 226

84 176 51 85 386 236

113 145 156

173 312 199

215 207 197 216 363 182

217 502 197 218 547 181

257 295 112 258 484 210

284 272 149

295 218 82 296 525 214

___________________________________________________________________


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127 160 210127 160 210 127 160 210


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Nitrosospira spp.

AM295526

AM295525

AB212171

AM295527

AB239753

FN394308 DGGE band C

EF092247

AY123794 N. sp. Nm143

Nitrosomonas Nm143 lineage

AF272423 Nitrosomonas cryotolerans

AF272420 N. aestuarii

FN394309 DGGE band B

FN394310 DGGE band A

AF272424 N. sp. Nm51

AF272418 N. marina

M96400 N. sp. C-56

AJ621032 N. sp. Is343

AF386752 N. sp. R7c131

AF386751 N. sp. R7c155

EF092216

FN394311 DGGE band D

Nitrosomonas marina / aestuarii

Nitrosomonas ureae / oligotropha

Nitrosomonas nitrosa / communis

Nitrosococcus mobilis

Nitrosomonas halophila

Nitrosomonas eutropha / europaea

Outgroups

99

92

75

88

67

95

53

100

68

97

92

97

97

89

86

92

89

64

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81

91

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Lineage I

Lineage II

AY555798 AY555810

AY532586

EF018873

EU084879 Candidatus N. bockiana

EF626882

EU097147

Lineage III

AB015550

EU491612

EU491406

EF999362

AJ863251

EF157239

FN394314 DGGE band E-1


DQ058673

X82559 N. marina


AM295541

Lineage IV

Outgroups

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100

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66

68

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100 75

93

99

10028

96

67

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Documents

AMB Manuscript Sudarno Reviewer