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Nitrogen loss in a nitrifying biofilm system
e>
Pergamon
War. Sci Tech. Vol. 39, No.7, pp. 13-21, 1999 <01999 IAWQ Published by Elsevier Science Ltd
Pnnted In Great Bnlam. All rights reserved
PH: 80273-1223(99)00145-6
0273-1223/99 $20.00 + 0.00
NITROGEN LOSS IN A NITRIFYING BIOFILM SYSTEM C. Helmer*, S. Kunst*, S. Juretschko**, M. C. Schmid**, K-H. Schleifer** and M. Wagner** • Institute ofSanitary Engineering and Waste Management, University ofHannover, Welfengar/en J, D-30J67 Hannover, Germany •• Technische Universitlit Miinchen, LehrstuhlfUr Mikrobiologie, Arcisstr. 16, D-80190 Miinchen, Germany
ABSTRACT In a biological contaclor that is part of the biological pretreatment of landfIll leachate in Mcchemich (Gennany) nitrogen elimlJlation of 60% or more was observed under low dissolved oxygen (DO) conditions. Ammonia was converted without aceumulalJon of nitrite and with only little nitrate production. Interestingly, due to limited supply with organic substrate 10 the system, this observation cannot simply be explamed by a combination of conventional autotrophic nitnfication and heterotrophic denitrificatIOn. In situ hybridization with 16S rRNA-targeted probes revealed the pteSencc of large microcolonies of at least three different types of ammonia-oxidizing bacteria 10 those biofilm regions where extremely high nitrogen losses occurred. These results were confll111ed by comparative sequence analysis of biofilm-derived amoA (encoding the active-site polypeptide of anunonia-monooxygenase) clones for molecular fme-scale analysIS of the anunorua-oxldizing population. In batch tests inoculated with biofilm material nitrogen loss occurred without dosage of organic substrate at a DO concentration of I mg/l. The simultaneous presence of arnmODJa and nitrite 10 the reactor induced the process of complete nitrogen elimination. N z was idenllfied to be the gaseous end product of the reaction. These results indicate that under low DO concentrations autotrophic ammonia-oxidizers might be the causative agents of the observed nitrogen los. by performing aerobic/anoxic denitrification with nitrite as electron acceptor and ammonia (or pcrhaps hydroxylamin) as electron donor. C 1999 lAWQ Published by ElseVier Science Ltd. All rights reserved .
KEYWORDS Biological contactor; nitrogen loss; nitrification/aerobic denitrification; aerobic/anoxic deammonification; biofilm. INTRODUCTION During recent years microbiologists have shown that nitrifiers as well as denitrifiers are of larger physiological variety than previously expected (e.g. Jetten et al., 1997). Three novel principles in the microbial conversion of nitrogen compounds have been recognized. (I) In addition to the classical autotrophic ammonia- and nitrite-oxdizing bacteria, some heterotrophic bacteria are also capable of nitrification. Many of these heterotrophic nitrifiers are able to denitrify aerobically as well as anaerobically (Castignetti and Hollocher, 1984; Robertson, 1988). The best investigated heterotrophic nitrifier-aerobic denitrifier is Paracoccus denitrificans DSM 2944 (formerly Thiosphaera pantotropha) (Robertson et al., 1995; Arts et al., 1995; Stouthamer et al., 1997). The autotrophic ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosomonas eutropha are also able to denitrify aerobically under low oxygen
(m
13
14
C. HELMER et al.
concentrations (Bock et al., 1995; Zart et al.. 1995). (III) During ANaerobic AMMonium Oxidation ANAMMOX (Mulder et al., 1995; Graaf et al., 1997) ammonium is used by as yet unidentified prokaryotes as electron donor with nitrite as electron acceptor. The end product of this reaction is Nz. A better understanding of the phylogeny, physiology and ecology of the prokaryotes responsible for these (and other) unconventional principles of nitrogen elimination might allow in the future the design of novel single-stage treatment plants for nttrogen removal. The biological pretreatment of landfill leachate at Mechernich (near Cologne, Germany) consists of a nitrification step in a biofilm system (biological contactor (BC» and a denitrification step in an activated sludge system with a topped transition aeration and a settling tank. The plant was laid out by Seyfried (1987). Detailed design characteristics of the leachate treatment plant are presented together with an overview of the main operating results in Hippen et al. (1997, 1998). Surprisingly, the BC unit of this plant accomplishes not only complete nitrification of high ammonia concentrations but also high nitrogen losses of more than 60% (referred to the sum of soluble organic and inorganic N compounds) without producing nitrite or significant amounts of nitrate. Due to the very low COD removal in the BC unit (ratio of CODel,fNeh.tnorg is 1,3), heterotrophic denitrification in anoxic biofilm-microniches is unlikely to be the main reason for this phenomenon. Nitrogen loss in the absence of added organic substrate was confirmed in recent aerobic batch tests with biofilm material from the BC unit (DO concentration at 1 mgtl). Furthermore, the amount of lost nitrogen was even higher after homogenizing the biofilm mechanically to destroy possible anoxic microzones (Helmer and Kunst, 1998). Therefore we decided to use the term "aerobic deammonification" for the direct biological conversion from ammonia to nitrogen gas under (micro)aerobic conditions (Hippen et al., 1998). However, we cannot yet definitely exclude a denitrification under anoxic conditions being jointly responsible for nitrogen loss in the Be unit or in batch tests, as it is still to be proved if anoxic microniches are present or not, even after homogenization. So we use here, a little more carefully, the term "aerobic/anoxic deammonification". A process design that allows for aerobic/anoxic deammonification with low demand of oxygen and organic carbon might be of great interest for the treatment of nitrogen rich waste water (e.g. industrial waste water or sludge liquor). Therefore, we have started to investigate the complete nitrogen removal in the BC more closely by combining precise balancing of nitrogen conversion processes with an in situ identification of dominant bacterial groups in the biofilm. MATERIALS AND METHODS One street of the BC unit consisting of three chambers was sampled at nine points following the flow direction of waste water (Figure I). For detailed documentation of nitrogen losses the concentrations of NH 4 -N, NOz-N and NOJ-N were determined. In addition, temperature, DO concentrations and pH-values were measured. Batch tests Biofilm was taken from the disks at the same sampling points. After dilution with water aerobic nitrification/denitrification rates, TKN and gaseous end products were measured in a gasproofbatch reactor in order to balance nitrogen conversion. The reactor was operated at a DO concentration of I mgtl (comparable to the BC unit). At the beginning of the experiments ~·N was added to obtain a concentration of 300 mgtl in the reactor. No organic substrate was added. Additional batch tests were performed at different DO concentrations and with an additional dose of 60 mgtl NOz-N at the beginning.
Nitrogen loss in a nitrifying biofilm system
IS
I ·9· ••""lmS pomLt
Figure 1. Investigated Be unit with nine sampling points following the flow direction of waste water.
In situ analysis ofammonia-oxidizin~ bacteria
Biofilm samples were fixed for in situ hybridization for 3h with 4% paraformaldehyde. The following 16S rRNA-targeted oligonucleotides were used: (i) NEU, complementary to a signature region of most halophilic and halotoleraot ammonia-oxidizers (Wagner e/ aJ., 1995); (ii) Nso 190, specific for most ammonia-oxidizers in the beta-subclass of Proteobacteria (Mobarry et aJ., 1996); (iii) Nso1225, specific for the ammonia• oxidizers in the beta-subclass of Proteobacteria (Mobarry et aJ., 1996); (iv) Nsv443, specific for the Nrtrosospira-cluster (Mobarry et aJ., 1996); (v) NmV, specific for the Ni/rosococcus mobilis lineage (Juretschko et aJ., in press); and (vi) S---Nse-1472-a-A-18, targeted against all members of the Nitrosomonas europaea lineage (Juretschko et aJ., in press). Cy3 and Cy5-labelled derivatives of the oligonucleotides were used for in situ hybridization. All hybridizations were performed at a temperature of 46°C. Subsequently, a stringent wash step was performed for 10 min at 48°C. Simultaneous hybridization with probes requiring different stringency was realized by a successive hybridization procedure (Wagner e/ aJ., 1994). A Zeiss LSM 510 scanning confocal microscope (Carl Zeiss, Germany) equipped with an UV• laser (351nm + 364nm), an Ar-ion laser (450-514nm), and two HeNe-Lasers (543nm + 633nm) was used to record optical sections. Image processing was performed with the standard software package delivered with the instrument (version 1.5). Reconstructed and processed images were printed by using the software package Microsoft Power Point (version 7.0, Microsoft, USA) in combination with a Kodak printer 8650 PS. Comparative sCQuence analysis QfamoA For PCR amplification of the amoA gene fragment the primers published by Rotthauwe et aJ. (1997) were applied. Cloning, sequencing and phylogenetic analysis of the biofilm-derived amoA fragments was performed as previously described (Juretschko et aJ., in press) RESULTS AND DISCUSSION For documentation of nitrogen loss in the biofilm of the BC unit, Figure 2 shows the concentrations of NH4 -N, N02-N and NOJ-N at the different sampling points. The loss of inorganic nitrogen amounted to 78%. In the first chamber of the BC unit (points 1-3) the conversion of ammonia went slowly compared to the second chamber (points 4-6). This was probably due to the high concentrations of free ammonia at the beginning of the BC unit with a maximum of 36,5 mg/I NHJ-N at sampling point 1. Till sampling point 5 ammonia was converted without any production of nitrite or nitrate. Nitrate production could not be observed before the NHJ-N concentrations were below a threshold of 10 mgll. The waste water flowed
C. HELMER el al
16
through the BC umt at a temperature of about 28°C and at a pH-value of 8.2. DO concentratIOns up to 1.2 mg/I were reached. 350
sum of inorganic N ~NH4-H
300
N02-N _ _ N03-N
_ _ (>
_
250
~ 200 Z
150 100 50 3
2
4
5
6
7
8
9
sampling points
fIgure 2. Profiles ofNH,-N. NO,-N and NO,-N along with the samphng pomts
10
the BC urnt
Batch tests Ammoma conversion rates and nitrite and nitrate accumulation rates were measured in batch tests wtth suspended biofilm taken from the disks at the nine samphng points. The results in Ftgure 3 vtsualize that maximal ammonia conversion rates between -20 and -23 mg N/(gYSS*h) were measured from the middle of the second chamber (point 5) to the middle of the third chamber (point 7). From sampling pomt I to sampling point 7 ammoma conversIon rates significantly exceeded the sum of the nitnte and nitrate accumulation rates. Consequently. nitrogen losses can also be confirmed in batch, even after homogenizing the samples wtth an ultraturrax blender (data not shown).
o ammonia conversion rates • nitrite accumulation rates o nitrate accumulation rates
.c
en
en
>Ol ~ Ol
oS
/
5
/
6
sampling points
9
hgure 3 Anlmoma conversIOn rates and nItnte/mtrate accumulation rates measured
In
batch with blofilm from the
nme samplmg pomts.
Comparable to the concentration profiles measured 10 the BC unit. the maximum nitrogen loss could be observed with biofilm from the middle of the second chamber (sampling polOt 5). In batch 82.7 mgN/gYSS disappeared. Figure 4 shows the values of lost nitrogen, measured in batch tests with biofilm from all nine samphng points. To study the fate of the lost N-compounds, gaseous end products were measured. The complete nitrogen balance for sampling point 5 displayed 10 Figure 5 demonstrates that the lost mtrogen was converted to the gaseous end product N 2 .
Nitrogen loss In a nitnfying blOfilrn system
5
17
6
sa~ngJlO''''''
Figure 4. Nitrogen lost in batch test (5 hours). 900
800
ON2
._N
700
.ir'O'gan.N
800 500
f
400
z
300 200 100 0
90
300
TImo(........../
Figure 5. Fate ofmtrogen
a batch test With biofilrn from the nuddle of the second chamber (sampling point 5).
In
Sl.ITlollr»rgaricN
80
~"""'-N
..... N02-N
__ ~
70. 60 50
~
Z
40
JO 20 1O
0 0
JO
60 TIme Imlt\Ml)
90
120
Figure 6. Nitrogen conversion rates at a DO concentration of6 mgll (MLSS-content = 4,7 gil). _~o'lr'Of'ga~CN
80
-o--NH4-N ..0- .NQ2-N
_ _ N03-N
70
60.
tz
50
40 JO 20 10 o_~_-,
o
JO
60 Tome lml1Ues)
90
120
Figure 7. Nitrogen conversion rates at a DO concentration of I mgll (MLSS-content = 3,3 gil).
As mentioned before, recent studies in parallel batch tests with biofilm not homogenized and biofilm mechanically homogenized have shown that nitrogen loss cannot be simply explained by conventional
C HELMER et al.
18
demtnfication in anoxic microzones (Helmer and Kunst, 1998). However, results of parallel batch tests at DO concentrations of I mg/l and 6 mg/I demonstrated that the process of aerobic/anoxic deammomfication was also dependent on the concentration of DO. At a DO concentration of 6 mg/l ammonia was oxidized in the conventional way to nitrite (Figure 6), whereas at a DO concentration of I mg/I a combination of ammonia-oxidation and aerobic/anoxic deammonification took place (Figure 7). While comparable ammonia conversion rates were observed in both assays (-7,5 mg N~-N/(gMLSS'h)at a DO concentratIOn of 6 mg/I and -7,2 mg NH4-N/(g MLSS'h) at a DO concentration of I mg/I) inorganic nitrogen loss of almost 60% appeared exclUSively at a DO concentration of I mg/1. Interestingly, loss of nitrogen-compounds did not start immediately so that conventional ammonia-OXidatIOn to nitnte without any aerobic/anoxIc deammonification took place during the first 30 minutes (Figure 7). The delay of the aerobic/anoxIc deammonification process could be avoided by addition of nitrite at the beginning of the batch expenment at a DO concentratIOn of Img/I (Figure 8). The presence of nitrite m the absence of ammonia at the beginnmg of a batch test led to conventional nitrite-oxidalion to nitrate while reestablishment of the aerobic/anoxic deammonification process required the additIOn of ammonia (Fig 9). sum of inorganic N ~ NH4-N -.<> - N02·N _
140
&-
N03-N
120 100 80
"" ,[
60
z
.......
-
40. 20. ----
0 0
15
30
45
TIme (minutes]
Figure 8. NItrogen converston rates after an Imtlal dosage ofmtrlle
~NH4-N
120
~ NH,-N dosage
•• <> - N02-N
- _ N03-N - - sum of ,norganoc N
100 80
'a,
60
oS z
40 20 0 0
50
100
150
200
Time [minlies]
Figure
l)
Starling of aerobic/anOXIc deammomficallOn by addmg ammoma to mtrlle and mtrale
MlcroblOl°i:Y Thc ammonia-oxidizer population in the biofilm samples was analyzed by whole cell fluorescent In situ hybndizatlon with an encompassmg 16S rRNA-targeted probe set and by comparaltve sequence analysis of amoA. Initial In .\·Uu hybndizatlon experiments were performed with probe NEU (targeting halophilic and
Nitrogen loss in a mtnfying blOfilm system
19
halotolerant members of the genus Nitrosomonas) to study in situ the abundance of ammonia-OXidizers in the biofilm at the nine sampling points and to compare microbiological results with data from the batch tests. Confocal laser scanning microscopy revealed that the ammonia-oxidizers form typical clusters in the biofilm (Figure 10). Their number remarkably increased at the end of the first chamber (sampling point 3) and reached a maximum in the middle of the second chamber (sampling point 5) reflecting the measured ammonia conversion rates. Interestingly, a correlation between the size of the ammonia-oxidizers clusters and the nitrogen loss at the various sampling points could be observed. Formation of very large aggregates by the ammonia-oxidizers (>50 J.1lIl in diameter) was exclusively detected in biofilm samples from sampling points characterized by a high nitrogen loss (e.g. middle of chamber 2).
Sampling point I
Sampling point 2
Sampling point 3
Sampling point 4
Sampling point 5
Sampling point 6
Sampling point 7
Sampling point 8
Sampling point 9
FIgure IO./n SItu Identification ofammoma-oxldizers with probe NEV.
For detailed ammonia-oxidizer population structure analysis additional hybridizat.ion experiments were fi rmed on a biofilm sample from sampling point 5 using probes Nso190 (targetmg most beta-subclass :~onia-oxidizers), Nso 1225 (targeting the beta-subclass am~onia-oxidizers), .~sv443 (t:rgeting the Nltrososplra-cluster), NmV (complementary to a target region of Nitrosococcus moblils) an~ S- -Nse-1472• a-A-18 (targeting the N. europaea-Iineage). These analyses demonstrated that at least three different types of
20
C. HELMER el al.
ammonia-oxidizers were present in the biofilm. Approximately 50% of the cells identified in situ as ammonia-oxidizers hybridized with probes Nso1225, Nso190, NEU and S-*-Nse-1472-a-A-18 _ a hybridization pattern which is indicative of N. halophila. N. europaea and N. eutropha. Forty percent of the detected ammonia-oxidizers in the biofilm hybridized exclusively with the general probe Nso1225 while a third population of cells with a low in situ abundance (approximately 10% of the ammonia-oxidizers) showed an unexpected hybridization pattern indicative of a novel, as yet uncultured (or if cultured, then previously not characterized on the 16S rRNA level) ammonia-oxidizer. This population hybridized with probes NEU, Nso1225 and S-*-Nse-I472-a-A-18 but did not hybridize with the probe Nso190. No hybndization signals could be obtained with probes Nsv443 and NmV indicating the absence of Nitrosococcus mobilis and members ofthe Nitrosospira-cluster in the biofilm. Comparative sequence analysis ofbiofilm derived amoA clones independently confirmed the presence of at least three types of ammonia-oxidizers in the biofilm at sampling point 5 (Figure II). Six of the eleven analyzed amoA clones were highly similar to the amoA sequence of N. europaea. The other five clones formed two well separated clusters within the Nitrosomonas europaea.lineage indicating the presence of additional yet undiscovered diversity within this lineage. Methytococcul ClpsuiatUi Bath
M.e".m!e" clOIl•• G13 Mle".m!e" elOIlI. GI6
Meclzernicll clone, G8 M.e".mie" elOIl', mDIO Mechunich clone ~ G18 Mechernich clolle, mD2 Nltrosomonas europau NmSO
M.c".m!c" elell', mD4 M.c".rn!c" elOIl', /liDS Meclzernich
C[Ollt,
mD6
Nilrosomona. eUlropha Nm57 amoA 1 Nltrosomonu eulropha Nm57 amoA2
Mee".m!c" CIOIl', mD3
' - - - - - - Mechernich clolfe, mD7
L_--G~~==::::::::;:~-Nltroaospira
Nllrosocoecus mobJbs Nc2 dUller
010
Figure II. Phylogenetic tree reflecting the relationships of amrnoma-oxldizmg bacteria based on amoA sequences.
CONCLUSIONS In the investigated biofilm of the biological contactor high nitrogen losses were observed which, due to the lack of organic substrate, cannot be explained by conventional heterotrophic denitrifying activity within anoxic biofilm microniches. In batch tests nitrogen loss occurred without any dosage of organic substrate (even after biofilm homogenization) at dissolved oxygen concentrations of I mg/1. N2 was the gaseous end product. Beside a reduced DO concentration, ammonia and nitrite but no organic substrate had to be present in the reactors to induce aerobic/anoxic deammonification. These results suggest, that aerobic/anoxic denitrification catalyzed by autotrophic nitrifiers under low DO concentrations is the main reaction in the process of aerobic/anoxic deammonification. The high in situ abundance of conspicuous large microcolonies of at least three populations of ammonia-oxidizers in biofilm regions where extremely high nitrogen losses were reported is an additional hint in this direction. Keeping in mind the results of the batch experiments, the process of aerobic/anoxic deammonification obviously uses ammonia (or perhaps hydroxylamin) as electron donor and nitrite as electron acceptor with N2 as gaseous cnd product. This hypothesis is supported by recent analyses of an enrichment culture from the biofilm of the BC unit under autotrophic and micro-aerobic conditions showing a consumption of ammonia and nitrite in a ratio of III to 'I. (Hippen et al., 1998). However, the nitrogen balance presented in Figure 5 demonstrates that nitrogen lost in the batch test did not only originate from inorganic but also from organic nitrogen including nitrogen from biomass. Consequently, we cannot yet exclude that apart from
Nitrogen loss in a nitrifying biofilm system
21
nitrogen. ~OD may have been released via decay of biomass which might serve heterotrophic mlcroorgarusms able to nitrifY and denitrify aerobically as substrate. The presence of bacteria with these physi~logical. prope~ies wi~in the biofilm of the BC unit was recently confirmed by the isolation of a bactenal s~am affilIated WIth the genus Thauera (Lukow and Diekmann, 1997; Hippen et al.• 1998). However, . In our batch tests COD release via biomass decay resulted in ~light increase of COD concentratIOns. whereas no COD removal could be measured throughout the test duration. These results indicate that heterotrophic processes play only a minor role in the process of aerobic/anoxic deammonification in the investigated BC unit.
REFERENCES Arts, P. A. M., Robertson, L. A. and Kuenen, lG. (1995): Nitrification and denitrification by Thiosphaera pantotropha m aerobic chemostat cultures. FEMS Microbiol. Ecol., 18,305-316. Bock, E., Schrrudt, I., Stilven, R. and Zart, D. (1995): Nitrogen loss caused by denitrifytng Nitrosomonas cells using ammonia or hydrogen as electron donors and Dltnte as electron acceptor. Arch. Microbiol., 163, 16-20. Castignetti, D. and Hollocher, T.C. (1984): Heterotrophic nitrification among denitnfiers. Appl. Env. Microbiol., 47, 620-623. Helmer, C. and Kunst, S. (1998): Simultaneous mtnficationldemtrification in an aerobic biofilm system. Wat. &1 Tech., 37(4-5), 183-187. Hippen, ~., Baumgarten, G., Rosenwinkel, K.-H. and Seyfried, C. F. (1997). Aerobic deammonification - A new experience in the treatment of wastewaters. Wat Sci. Tech., 35(10), 111-120. Hippen, A., Scholten, E., Helmer, c., Kunst, S., Seyfried, C. F., Rosenwinkel, K.·B. and Diekmann, H. (1998). Aerobic deammonification in high nitrogen loaded wastewaters. "New Advances in Biological Nitrogen and Phosphorus Removal for Municipal or Industrial Wastewaters". Conference held in Narbonne, France, 12-14 October, 1998. Jellen, M. S. M., Logemann, S. M., Muyzer, G., de Vries, S., van Loosdrecht, M. C. M., Robertson, L. A. and Kuenen, I. G. (1997). Novel prinCiples in the microbial convefSlon of nitrogen compounds. Anthonie van Leeuwenhoelc, 71, 75-93. luretschko, S., Timmermann, G., Schmid, M. C., Schleifer, K.-H., Pommerening-Rllser, A., Koops, H.-P. and Wagner, M. (in press). Combined molecular and conventional analyses of nitrifying bacterial diversity in activated sludge: N1trosococcus mobilis and Nitrospira-like bacteria as donunant populations. Appl. Environ. Microblol. Lukow, T. and Diekmann, H. (1997). Aerobic denitrification by a newly isolated heterotrophic bacterium strain 11.1. Biotechnol. Lett., 19, 1157-1159. Mobarry, B. K., Wagner, M., Urbain, V., Rittrrumn, B. and D.A. Stahl. (1996): Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ. Microbiol., 6, 2156-2162. Mulder, A., van de Graaf, A. A., Robertson, L. A. and Kuenen, I. G. (1995). Anaerobic ammonium oxidation discovered in a dcnitnfying flUidized bed reactor. FEMS Microbiol. Ecol., 16, 177-184. Robertson, L. A. (1988). Aerobic demtrification and heterotrophic nitrification in Thiosphaera pantotropha and other bacteria. PhD thesIS, University of Technology, Delft, The Netherlands. Robertson, L. A., Dalsgaard, T., Revsbach, N.-P. and Kuenen, I. G. (1995). Confirmation of aerobic deDltrification in batch cultures using gas chromatography and N mass spectrometry. FEMS Microbiol. Ecol., 18, 113-120. Rotthauwe, J.-H., Witzel, K.-P. and Liesack, W. (1997). The ammonia monooxygenase struclUral gene amoA as a functional marker: molecular fme-scale analysis of nalUral ammonia-oxidizing populations. Appl Environ. Microbiol., 63. 4704• 4712. Seyfried, C. F. (1981). Studie zur Sickerwasserreinigung der Deponie Mechemich durch ein kombiniertes biologischphysikahsches Verfabren, Hannover (unpublished). Stouthamer, A. H., de Boer, A. P. N., van der Oost, J. and van Spanning, R. J. M. (1997). Emerging principles of inorganic nItrogen metabolism in Paracoccus denitrificans and related bacteria. Antonie van Leuwenhoek, 71, 33-41. . Van de Graaf, A. A., de Bruljn, P., Robertson, L. A., Jellen, M. S. M. and Kuenen, 1. G. (1997). Metabohc pathway of anaerobiC ammonium oxidation on the basis of l~ studies in a flUidized bed reactor. Microbiology, 143,2415-2421. Wagner, M., Amann, R., Klmpfer, P., AJlmus, B., H~nn, A., Hutzler~ ~., Springer, N. and Schleifer K.•~. (1.994). Identification and in situ detection of gram-negatIve filamentous bactena m acnvated sludge. System. Appl. Mlcrobrol. I, 405-417. Wagner. M., Ratb, G., Amann, R., Koops, H.-P. and Schleifer, K.-H. (1995). In situ identification of ammonia oxidizing bacteria. System. Appl. MicroblOI., 18, 251-264. Zart, D., Schmidt, I. and Bock, E. (1996). Neue Wege vom Ammonium zorn Stickstoff: In: Oleologie der Abwasserorganismen, Lemmer, H.• Griebe, T. and Flemmmg, H.-G. (eds), Springer.Verlag, Berlm, Heidelberg, 183-191.
Pergamon
War. Sci Tech. Vol. 39, No.7, pp. 13-21, 1999 <01999 IAWQ Published by Elsevier Science Ltd
Pnnted In Great Bnlam. All rights reserved
PH: 80273-1223(99)00145-6
0273-1223/99 $20.00 + 0.00
NITROGEN LOSS IN A NITRIFYING BIOFILM SYSTEM C. Helmer*, S. Kunst*, S. Juretschko**, M. C. Schmid**, K-H. Schleifer** and M. Wagner** • Institute ofSanitary Engineering and Waste Management, University ofHannover, Welfengar/en J, D-30J67 Hannover, Germany •• Technische Universitlit Miinchen, LehrstuhlfUr Mikrobiologie, Arcisstr. 16, D-80190 Miinchen, Germany
ABSTRACT In a biological contaclor that is part of the biological pretreatment of landfIll leachate in Mcchemich (Gennany) nitrogen elimlJlation of 60% or more was observed under low dissolved oxygen (DO) conditions. Ammonia was converted without aceumulalJon of nitrite and with only little nitrate production. Interestingly, due to limited supply with organic substrate 10 the system, this observation cannot simply be explamed by a combination of conventional autotrophic nitnfication and heterotrophic denitrificatIOn. In situ hybridization with 16S rRNA-targeted probes revealed the pteSencc of large microcolonies of at least three different types of ammonia-oxidizing bacteria 10 those biofilm regions where extremely high nitrogen losses occurred. These results were confll111ed by comparative sequence analysis of biofilm-derived amoA (encoding the active-site polypeptide of anunonia-monooxygenase) clones for molecular fme-scale analysIS of the anunorua-oxldizing population. In batch tests inoculated with biofilm material nitrogen loss occurred without dosage of organic substrate at a DO concentration of I mg/l. The simultaneous presence of arnmODJa and nitrite 10 the reactor induced the process of complete nitrogen elimination. N z was idenllfied to be the gaseous end product of the reaction. These results indicate that under low DO concentrations autotrophic ammonia-oxidizers might be the causative agents of the observed nitrogen los. by performing aerobic/anoxic denitrification with nitrite as electron acceptor and ammonia (or pcrhaps hydroxylamin) as electron donor. C 1999 lAWQ Published by ElseVier Science Ltd. All rights reserved .
KEYWORDS Biological contactor; nitrogen loss; nitrification/aerobic denitrification; aerobic/anoxic deammonification; biofilm. INTRODUCTION During recent years microbiologists have shown that nitrifiers as well as denitrifiers are of larger physiological variety than previously expected (e.g. Jetten et al., 1997). Three novel principles in the microbial conversion of nitrogen compounds have been recognized. (I) In addition to the classical autotrophic ammonia- and nitrite-oxdizing bacteria, some heterotrophic bacteria are also capable of nitrification. Many of these heterotrophic nitrifiers are able to denitrify aerobically as well as anaerobically (Castignetti and Hollocher, 1984; Robertson, 1988). The best investigated heterotrophic nitrifier-aerobic denitrifier is Paracoccus denitrificans DSM 2944 (formerly Thiosphaera pantotropha) (Robertson et al., 1995; Arts et al., 1995; Stouthamer et al., 1997). The autotrophic ammonia-oxidizing bacteria Nitrosomonas europaea and Nitrosomonas eutropha are also able to denitrify aerobically under low oxygen
(m
13
14
C. HELMER et al.
concentrations (Bock et al., 1995; Zart et al.. 1995). (III) During ANaerobic AMMonium Oxidation ANAMMOX (Mulder et al., 1995; Graaf et al., 1997) ammonium is used by as yet unidentified prokaryotes as electron donor with nitrite as electron acceptor. The end product of this reaction is Nz. A better understanding of the phylogeny, physiology and ecology of the prokaryotes responsible for these (and other) unconventional principles of nitrogen elimination might allow in the future the design of novel single-stage treatment plants for nttrogen removal. The biological pretreatment of landfill leachate at Mechernich (near Cologne, Germany) consists of a nitrification step in a biofilm system (biological contactor (BC» and a denitrification step in an activated sludge system with a topped transition aeration and a settling tank. The plant was laid out by Seyfried (1987). Detailed design characteristics of the leachate treatment plant are presented together with an overview of the main operating results in Hippen et al. (1997, 1998). Surprisingly, the BC unit of this plant accomplishes not only complete nitrification of high ammonia concentrations but also high nitrogen losses of more than 60% (referred to the sum of soluble organic and inorganic N compounds) without producing nitrite or significant amounts of nitrate. Due to the very low COD removal in the BC unit (ratio of CODel,fNeh.tnorg is 1,3), heterotrophic denitrification in anoxic biofilm-microniches is unlikely to be the main reason for this phenomenon. Nitrogen loss in the absence of added organic substrate was confirmed in recent aerobic batch tests with biofilm material from the BC unit (DO concentration at 1 mgtl). Furthermore, the amount of lost nitrogen was even higher after homogenizing the biofilm mechanically to destroy possible anoxic microzones (Helmer and Kunst, 1998). Therefore we decided to use the term "aerobic deammonification" for the direct biological conversion from ammonia to nitrogen gas under (micro)aerobic conditions (Hippen et al., 1998). However, we cannot yet definitely exclude a denitrification under anoxic conditions being jointly responsible for nitrogen loss in the Be unit or in batch tests, as it is still to be proved if anoxic microniches are present or not, even after homogenization. So we use here, a little more carefully, the term "aerobic/anoxic deammonification". A process design that allows for aerobic/anoxic deammonification with low demand of oxygen and organic carbon might be of great interest for the treatment of nitrogen rich waste water (e.g. industrial waste water or sludge liquor). Therefore, we have started to investigate the complete nitrogen removal in the BC more closely by combining precise balancing of nitrogen conversion processes with an in situ identification of dominant bacterial groups in the biofilm. MATERIALS AND METHODS One street of the BC unit consisting of three chambers was sampled at nine points following the flow direction of waste water (Figure I). For detailed documentation of nitrogen losses the concentrations of NH 4 -N, NOz-N and NOJ-N were determined. In addition, temperature, DO concentrations and pH-values were measured. Batch tests Biofilm was taken from the disks at the same sampling points. After dilution with water aerobic nitrification/denitrification rates, TKN and gaseous end products were measured in a gasproofbatch reactor in order to balance nitrogen conversion. The reactor was operated at a DO concentration of I mgtl (comparable to the BC unit). At the beginning of the experiments ~·N was added to obtain a concentration of 300 mgtl in the reactor. No organic substrate was added. Additional batch tests were performed at different DO concentrations and with an additional dose of 60 mgtl NOz-N at the beginning.
Nitrogen loss in a nitrifying biofilm system
IS
I ·9· ••""lmS pomLt
Figure 1. Investigated Be unit with nine sampling points following the flow direction of waste water.
In situ analysis ofammonia-oxidizin~ bacteria
Biofilm samples were fixed for in situ hybridization for 3h with 4% paraformaldehyde. The following 16S rRNA-targeted oligonucleotides were used: (i) NEU, complementary to a signature region of most halophilic and halotoleraot ammonia-oxidizers (Wagner e/ aJ., 1995); (ii) Nso 190, specific for most ammonia-oxidizers in the beta-subclass of Proteobacteria (Mobarry et aJ., 1996); (iii) Nso1225, specific for the ammonia• oxidizers in the beta-subclass of Proteobacteria (Mobarry et aJ., 1996); (iv) Nsv443, specific for the Nrtrosospira-cluster (Mobarry et aJ., 1996); (v) NmV, specific for the Ni/rosococcus mobilis lineage (Juretschko et aJ., in press); and (vi) S---Nse-1472-a-A-18, targeted against all members of the Nitrosomonas europaea lineage (Juretschko et aJ., in press). Cy3 and Cy5-labelled derivatives of the oligonucleotides were used for in situ hybridization. All hybridizations were performed at a temperature of 46°C. Subsequently, a stringent wash step was performed for 10 min at 48°C. Simultaneous hybridization with probes requiring different stringency was realized by a successive hybridization procedure (Wagner e/ aJ., 1994). A Zeiss LSM 510 scanning confocal microscope (Carl Zeiss, Germany) equipped with an UV• laser (351nm + 364nm), an Ar-ion laser (450-514nm), and two HeNe-Lasers (543nm + 633nm) was used to record optical sections. Image processing was performed with the standard software package delivered with the instrument (version 1.5). Reconstructed and processed images were printed by using the software package Microsoft Power Point (version 7.0, Microsoft, USA) in combination with a Kodak printer 8650 PS. Comparative sCQuence analysis QfamoA For PCR amplification of the amoA gene fragment the primers published by Rotthauwe et aJ. (1997) were applied. Cloning, sequencing and phylogenetic analysis of the biofilm-derived amoA fragments was performed as previously described (Juretschko et aJ., in press) RESULTS AND DISCUSSION For documentation of nitrogen loss in the biofilm of the BC unit, Figure 2 shows the concentrations of NH4 -N, N02-N and NOJ-N at the different sampling points. The loss of inorganic nitrogen amounted to 78%. In the first chamber of the BC unit (points 1-3) the conversion of ammonia went slowly compared to the second chamber (points 4-6). This was probably due to the high concentrations of free ammonia at the beginning of the BC unit with a maximum of 36,5 mg/I NHJ-N at sampling point 1. Till sampling point 5 ammonia was converted without any production of nitrite or nitrate. Nitrate production could not be observed before the NHJ-N concentrations were below a threshold of 10 mgll. The waste water flowed
C. HELMER el al
16
through the BC umt at a temperature of about 28°C and at a pH-value of 8.2. DO concentratIOns up to 1.2 mg/I were reached. 350
sum of inorganic N ~NH4-H
300
N02-N _ _ N03-N
_ _ (>
_
250
~ 200 Z
150 100 50 3
2
4
5
6
7
8
9
sampling points
fIgure 2. Profiles ofNH,-N. NO,-N and NO,-N along with the samphng pomts
10
the BC urnt
Batch tests Ammoma conversion rates and nitrite and nitrate accumulation rates were measured in batch tests wtth suspended biofilm taken from the disks at the nine samphng points. The results in Ftgure 3 vtsualize that maximal ammonia conversion rates between -20 and -23 mg N/(gYSS*h) were measured from the middle of the second chamber (point 5) to the middle of the third chamber (point 7). From sampling pomt I to sampling point 7 ammoma conversIon rates significantly exceeded the sum of the nitnte and nitrate accumulation rates. Consequently. nitrogen losses can also be confirmed in batch, even after homogenizing the samples wtth an ultraturrax blender (data not shown).
o ammonia conversion rates • nitrite accumulation rates o nitrate accumulation rates
.c
en
en
>Ol ~ Ol
oS
/
5
/
6
sampling points
9
hgure 3 Anlmoma conversIOn rates and nItnte/mtrate accumulation rates measured
In
batch with blofilm from the
nme samplmg pomts.
Comparable to the concentration profiles measured 10 the BC unit. the maximum nitrogen loss could be observed with biofilm from the middle of the second chamber (sampling polOt 5). In batch 82.7 mgN/gYSS disappeared. Figure 4 shows the values of lost nitrogen, measured in batch tests with biofilm from all nine samphng points. To study the fate of the lost N-compounds, gaseous end products were measured. The complete nitrogen balance for sampling point 5 displayed 10 Figure 5 demonstrates that the lost mtrogen was converted to the gaseous end product N 2 .
Nitrogen loss In a nitnfying blOfilrn system
5
17
6
sa~ngJlO''''''
Figure 4. Nitrogen lost in batch test (5 hours). 900
800
ON2
._N
700
.ir'O'gan.N
800 500
f
400
z
300 200 100 0
90
300
TImo(........../
Figure 5. Fate ofmtrogen
a batch test With biofilrn from the nuddle of the second chamber (sampling point 5).
In
Sl.ITlollr»rgaricN
80
~"""'-N
..... N02-N
__ ~
70. 60 50
~
Z
40
JO 20 1O
0 0
JO
60 TIme Imlt\Ml)
90
120
Figure 6. Nitrogen conversion rates at a DO concentration of6 mgll (MLSS-content = 4,7 gil). _~o'lr'Of'ga~CN
80
-o--NH4-N ..0- .NQ2-N
_ _ N03-N
70
60.
tz
50
40 JO 20 10 o_~_-,
o
JO
60 Tome lml1Ues)
90
120
Figure 7. Nitrogen conversion rates at a DO concentration of I mgll (MLSS-content = 3,3 gil).
As mentioned before, recent studies in parallel batch tests with biofilm not homogenized and biofilm mechanically homogenized have shown that nitrogen loss cannot be simply explained by conventional
C HELMER et al.
18
demtnfication in anoxic microzones (Helmer and Kunst, 1998). However, results of parallel batch tests at DO concentrations of I mg/l and 6 mg/I demonstrated that the process of aerobic/anoxic deammomfication was also dependent on the concentration of DO. At a DO concentration of 6 mg/l ammonia was oxidized in the conventional way to nitrite (Figure 6), whereas at a DO concentration of I mg/I a combination of ammonia-oxidation and aerobic/anoxic deammonification took place (Figure 7). While comparable ammonia conversion rates were observed in both assays (-7,5 mg N~-N/(gMLSS'h)at a DO concentratIOn of 6 mg/I and -7,2 mg NH4-N/(g MLSS'h) at a DO concentration of I mg/I) inorganic nitrogen loss of almost 60% appeared exclUSively at a DO concentration of I mg/1. Interestingly, loss of nitrogen-compounds did not start immediately so that conventional ammonia-OXidatIOn to nitnte without any aerobic/anoxIc deammonification took place during the first 30 minutes (Figure 7). The delay of the aerobic/anoxIc deammonification process could be avoided by addition of nitrite at the beginning of the batch expenment at a DO concentratIOn of Img/I (Figure 8). The presence of nitrite m the absence of ammonia at the beginnmg of a batch test led to conventional nitrite-oxidalion to nitrate while reestablishment of the aerobic/anoxic deammonification process required the additIOn of ammonia (Fig 9). sum of inorganic N ~ NH4-N -.<> - N02·N _
140
&-
N03-N
120 100 80
"" ,[
60
z
.......
-
40. 20. ----
0 0
15
30
45
TIme (minutes]
Figure 8. NItrogen converston rates after an Imtlal dosage ofmtrlle
~NH4-N
120
~ NH,-N dosage
•• <> - N02-N
- _ N03-N - - sum of ,norganoc N
100 80
'a,
60
oS z
40 20 0 0
50
100
150
200
Time [minlies]
Figure
l)
Starling of aerobic/anOXIc deammomficallOn by addmg ammoma to mtrlle and mtrale
MlcroblOl°i:Y Thc ammonia-oxidizer population in the biofilm samples was analyzed by whole cell fluorescent In situ hybndizatlon with an encompassmg 16S rRNA-targeted probe set and by comparaltve sequence analysis of amoA. Initial In .\·Uu hybndizatlon experiments were performed with probe NEU (targeting halophilic and
Nitrogen loss in a mtnfying blOfilm system
19
halotolerant members of the genus Nitrosomonas) to study in situ the abundance of ammonia-OXidizers in the biofilm at the nine sampling points and to compare microbiological results with data from the batch tests. Confocal laser scanning microscopy revealed that the ammonia-oxidizers form typical clusters in the biofilm (Figure 10). Their number remarkably increased at the end of the first chamber (sampling point 3) and reached a maximum in the middle of the second chamber (sampling point 5) reflecting the measured ammonia conversion rates. Interestingly, a correlation between the size of the ammonia-oxidizers clusters and the nitrogen loss at the various sampling points could be observed. Formation of very large aggregates by the ammonia-oxidizers (>50 J.1lIl in diameter) was exclusively detected in biofilm samples from sampling points characterized by a high nitrogen loss (e.g. middle of chamber 2).
Sampling point I
Sampling point 2
Sampling point 3
Sampling point 4
Sampling point 5
Sampling point 6
Sampling point 7
Sampling point 8
Sampling point 9
FIgure IO./n SItu Identification ofammoma-oxldizers with probe NEV.
For detailed ammonia-oxidizer population structure analysis additional hybridizat.ion experiments were fi rmed on a biofilm sample from sampling point 5 using probes Nso190 (targetmg most beta-subclass :~onia-oxidizers), Nso 1225 (targeting the beta-subclass am~onia-oxidizers), .~sv443 (t:rgeting the Nltrososplra-cluster), NmV (complementary to a target region of Nitrosococcus moblils) an~ S- -Nse-1472• a-A-18 (targeting the N. europaea-Iineage). These analyses demonstrated that at least three different types of
20
C. HELMER el al.
ammonia-oxidizers were present in the biofilm. Approximately 50% of the cells identified in situ as ammonia-oxidizers hybridized with probes Nso1225, Nso190, NEU and S-*-Nse-1472-a-A-18 _ a hybridization pattern which is indicative of N. halophila. N. europaea and N. eutropha. Forty percent of the detected ammonia-oxidizers in the biofilm hybridized exclusively with the general probe Nso1225 while a third population of cells with a low in situ abundance (approximately 10% of the ammonia-oxidizers) showed an unexpected hybridization pattern indicative of a novel, as yet uncultured (or if cultured, then previously not characterized on the 16S rRNA level) ammonia-oxidizer. This population hybridized with probes NEU, Nso1225 and S-*-Nse-I472-a-A-18 but did not hybridize with the probe Nso190. No hybndization signals could be obtained with probes Nsv443 and NmV indicating the absence of Nitrosococcus mobilis and members ofthe Nitrosospira-cluster in the biofilm. Comparative sequence analysis ofbiofilm derived amoA clones independently confirmed the presence of at least three types of ammonia-oxidizers in the biofilm at sampling point 5 (Figure II). Six of the eleven analyzed amoA clones were highly similar to the amoA sequence of N. europaea. The other five clones formed two well separated clusters within the Nitrosomonas europaea.lineage indicating the presence of additional yet undiscovered diversity within this lineage. Methytococcul ClpsuiatUi Bath
M.e".m!e" clOIl•• G13 Mle".m!e" elOIlI. GI6
Meclzernicll clone, G8 M.e".mie" elOIl', mDIO Mechunich clone ~ G18 Mechernich clolle, mD2 Nltrosomonas europau NmSO
M.c".m!c" elell', mD4 M.c".rn!c" elOIl', /liDS Meclzernich
C[Ollt,
mD6
Nilrosomona. eUlropha Nm57 amoA 1 Nltrosomonu eulropha Nm57 amoA2
Mee".m!c" CIOIl', mD3
' - - - - - - Mechernich clolfe, mD7
L_--G~~==::::::::;:~-Nltroaospira
Nllrosocoecus mobJbs Nc2 dUller
010
Figure II. Phylogenetic tree reflecting the relationships of amrnoma-oxldizmg bacteria based on amoA sequences.
CONCLUSIONS In the investigated biofilm of the biological contactor high nitrogen losses were observed which, due to the lack of organic substrate, cannot be explained by conventional heterotrophic denitrifying activity within anoxic biofilm microniches. In batch tests nitrogen loss occurred without any dosage of organic substrate (even after biofilm homogenization) at dissolved oxygen concentrations of I mg/1. N2 was the gaseous end product. Beside a reduced DO concentration, ammonia and nitrite but no organic substrate had to be present in the reactors to induce aerobic/anoxic deammonification. These results suggest, that aerobic/anoxic denitrification catalyzed by autotrophic nitrifiers under low DO concentrations is the main reaction in the process of aerobic/anoxic deammonification. The high in situ abundance of conspicuous large microcolonies of at least three populations of ammonia-oxidizers in biofilm regions where extremely high nitrogen losses were reported is an additional hint in this direction. Keeping in mind the results of the batch experiments, the process of aerobic/anoxic deammonification obviously uses ammonia (or perhaps hydroxylamin) as electron donor and nitrite as electron acceptor with N2 as gaseous cnd product. This hypothesis is supported by recent analyses of an enrichment culture from the biofilm of the BC unit under autotrophic and micro-aerobic conditions showing a consumption of ammonia and nitrite in a ratio of III to 'I. (Hippen et al., 1998). However, the nitrogen balance presented in Figure 5 demonstrates that nitrogen lost in the batch test did not only originate from inorganic but also from organic nitrogen including nitrogen from biomass. Consequently, we cannot yet exclude that apart from
Nitrogen loss in a nitrifying biofilm system
21
nitrogen. ~OD may have been released via decay of biomass which might serve heterotrophic mlcroorgarusms able to nitrifY and denitrify aerobically as substrate. The presence of bacteria with these physi~logical. prope~ies wi~in the biofilm of the BC unit was recently confirmed by the isolation of a bactenal s~am affilIated WIth the genus Thauera (Lukow and Diekmann, 1997; Hippen et al.• 1998). However, . In our batch tests COD release via biomass decay resulted in ~light increase of COD concentratIOns. whereas no COD removal could be measured throughout the test duration. These results indicate that heterotrophic processes play only a minor role in the process of aerobic/anoxic deammonification in the investigated BC unit.
REFERENCES Arts, P. A. M., Robertson, L. A. and Kuenen, lG. (1995): Nitrification and denitrification by Thiosphaera pantotropha m aerobic chemostat cultures. FEMS Microbiol. Ecol., 18,305-316. Bock, E., Schrrudt, I., Stilven, R. and Zart, D. (1995): Nitrogen loss caused by denitrifytng Nitrosomonas cells using ammonia or hydrogen as electron donors and Dltnte as electron acceptor. Arch. Microbiol., 163, 16-20. Castignetti, D. and Hollocher, T.C. (1984): Heterotrophic nitrification among denitnfiers. Appl. Env. Microbiol., 47, 620-623. Helmer, C. and Kunst, S. (1998): Simultaneous mtnficationldemtrification in an aerobic biofilm system. Wat. &1 Tech., 37(4-5), 183-187. Hippen, ~., Baumgarten, G., Rosenwinkel, K.-H. and Seyfried, C. F. (1997). Aerobic deammonification - A new experience in the treatment of wastewaters. Wat Sci. Tech., 35(10), 111-120. Hippen, A., Scholten, E., Helmer, c., Kunst, S., Seyfried, C. F., Rosenwinkel, K.·B. and Diekmann, H. (1998). Aerobic deammonification in high nitrogen loaded wastewaters. "New Advances in Biological Nitrogen and Phosphorus Removal for Municipal or Industrial Wastewaters". Conference held in Narbonne, France, 12-14 October, 1998. Jellen, M. S. M., Logemann, S. M., Muyzer, G., de Vries, S., van Loosdrecht, M. C. M., Robertson, L. A. and Kuenen, I. G. (1997). Novel prinCiples in the microbial convefSlon of nitrogen compounds. Anthonie van Leeuwenhoelc, 71, 75-93. luretschko, S., Timmermann, G., Schmid, M. C., Schleifer, K.-H., Pommerening-Rllser, A., Koops, H.-P. and Wagner, M. (in press). Combined molecular and conventional analyses of nitrifying bacterial diversity in activated sludge: N1trosococcus mobilis and Nitrospira-like bacteria as donunant populations. Appl. Environ. Microblol. Lukow, T. and Diekmann, H. (1997). Aerobic denitrification by a newly isolated heterotrophic bacterium strain 11.1. Biotechnol. Lett., 19, 1157-1159. Mobarry, B. K., Wagner, M., Urbain, V., Rittrrumn, B. and D.A. Stahl. (1996): Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ. Microbiol., 6, 2156-2162. Mulder, A., van de Graaf, A. A., Robertson, L. A. and Kuenen, I. G. (1995). Anaerobic ammonium oxidation discovered in a dcnitnfying flUidized bed reactor. FEMS Microbiol. Ecol., 16, 177-184. Robertson, L. A. (1988). Aerobic demtrification and heterotrophic nitrification in Thiosphaera pantotropha and other bacteria. PhD thesIS, University of Technology, Delft, The Netherlands. Robertson, L. A., Dalsgaard, T., Revsbach, N.-P. and Kuenen, I. G. (1995). Confirmation of aerobic deDltrification in batch cultures using gas chromatography and N mass spectrometry. FEMS Microbiol. Ecol., 18, 113-120. Rotthauwe, J.-H., Witzel, K.-P. and Liesack, W. (1997). The ammonia monooxygenase struclUral gene amoA as a functional marker: molecular fme-scale analysis of nalUral ammonia-oxidizing populations. Appl Environ. Microbiol., 63. 4704• 4712. Seyfried, C. F. (1981). Studie zur Sickerwasserreinigung der Deponie Mechemich durch ein kombiniertes biologischphysikahsches Verfabren, Hannover (unpublished). Stouthamer, A. H., de Boer, A. P. N., van der Oost, J. and van Spanning, R. J. M. (1997). Emerging principles of inorganic nItrogen metabolism in Paracoccus denitrificans and related bacteria. Antonie van Leuwenhoek, 71, 33-41. . Van de Graaf, A. A., de Bruljn, P., Robertson, L. A., Jellen, M. S. M. and Kuenen, 1. G. (1997). Metabohc pathway of anaerobiC ammonium oxidation on the basis of l~ studies in a flUidized bed reactor. Microbiology, 143,2415-2421. Wagner, M., Amann, R., Klmpfer, P., AJlmus, B., H~nn, A., Hutzler~ ~., Springer, N. and Schleifer K.•~. (1.994). Identification and in situ detection of gram-negatIve filamentous bactena m acnvated sludge. System. Appl. Mlcrobrol. I, 405-417. Wagner. M., Ratb, G., Amann, R., Koops, H.-P. and Schleifer, K.-H. (1995). In situ identification of ammonia oxidizing bacteria. System. Appl. MicroblOI., 18, 251-264. Zart, D., Schmidt, I. and Bock, E. (1996). Neue Wege vom Ammonium zorn Stickstoff: In: Oleologie der Abwasserorganismen, Lemmer, H.• Griebe, T. and Flemmmg, H.-G. (eds), Springer.Verlag, Berlm, Heidelberg, 183-191.