Autotrophic NHD
4 removal has been extensively researched, but few studies have investigated alternative electron
D
acceptors (for example, SO2L
4 ) in NH4 oxidation. In this study, sulfate-reducing anaerobic ammonium oxidation (SRAO)
and conventional Anammox were started up in upow anaerobic sludge blanket reactors (UASBRs) at 36 (0.5) C and 20
2L
(0.5) C respectively, using reject water as a source of NHD
or NOL
2 , respectively, were applied as electron ac4 . SO4
ceptors. It was assumed that higher temperature could promote the SRAO, partly compensating its thermodynamic
disadvantage comparing with the conventional Anammox to achieve comparable total nitrogen (TN) removal rate.
3
Average volumetric NHD
4 LN removal rate in the sulfate-reducing UASBR1 was however 5e6 times less (0.03 kg-N/(m
day)) than in the UASBR2 performing conventional nitrite-dependent autotrophic nitrogen removal (0.17 kg-N/(m3 day)).
However, the stoichiometric ratio of NHD
4 removal in UASBR1 was signicantly higher than could be expected from the
extent of SO2L
4 reduction, possibly due to interactions between the N- and S-compounds and organic matter of the reject
water. Injections of N2H4 and NH2OH accelerated the SRAO. Similar effect was observed in batch tests with anthraquinone-2,6-disulfonate (AQDS). For detection of key microorganisms PCR-DGGE was used. From both UASBRs, uncultured
bacterium clone ATB-KS-1929 belonging to the order Verrucomicrobiales, Anammox bacteria (uncultured Planctomycete
clone Pla_PO55-9) and aerobic ammonium-oxidizing bacteria (uncultured sludge bacterium clone ASB08 Nitrosomonas) were detected. Nevertheless the SRAO process was shown to be less effective for the treatment of reject water,
compared to the conventional Anammox.
2014, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Sulfate-reducing ammonium oxidation; Upow anaerobic sludge blanket reactor; Humic matter; Anammox intermediates; Autotrophic
NH
4 removal]
* Corresponding author. Tel.: 372 5691 2374; fax: 372 737 5264.
E-mail addresses: ergo.rikmann@ut.ee, rikmannster@gmail.com (E. Rikmann).
2
8NH
4 3SO4 /4N2 3HS 12H2 O 5H
22kJ=mol
(1)
D G0
(2)
1389-1723/$ e see front matter 2014, The Society for Biotechnology, Japan. All rights reserved.
http://dx.doi.org/10.1016/j.jbiosc.2014.03.012
Seeding of UASBRs
Both UASBRs were seeded with anaerobic sludge containing Anammox bacteria, obtained from the facility treating wastewater of the
427
FIG. 1. Scheme of the UASBR1 (performing SRAO) and the UASBR2 (performing conventional Anammox process).
428
RIKMANN ET AL.
J. BIOSCI. BIOENG.,
UASBR1 SRAO
UASBR2 Anammox
300
0,50
Average
0.43
0.5
0.23
1.10
20
7.5
250
Stdev
0.3
1
0.5
1
17
0.09
0.02
85
70
71
64
19.1/1
0.2
9
0.03
0.01
28
23
6
9
1
75.4
0.22
0.21
80.5
42
0.2
21
0.04
0.06
14.23
39.5
1
18
0.15
0.02
134
119
82
78
26.8/1
0.2
15
0.01
0.02
24
33
7
5
1
70
0.37
0.25
80.42
1.15
1
25
0.17
0.04
158
125
123
110
8.2/1
0.3
11
0.04
0.02
46
42
32
31
1
70
0.43
0.25
123.5
9.8
2
30
0.11
0.04
221
155
193
154
6.7/1
0.4
8
0.01
0.01
22
18
16
26
2
69
0.40
0.28
178.23
4.6
0.4
2.9
0.11
0.077
47.00
3.4
24
0.13
0.03
155
119
123
106
13.3/1
10
0.03
0.01
32
30
18
20
70
0.37
0.25
120
11
14.8
0.1
0.08
50
nd
0,40
200
0,30
150
0,20
100
0,10
50
0,00
0
0
0.2
18.3
0.04
0.04
13.22
0.41
0.3
20
0.09
0.07
38.63
5.4
250
50
100
150
200
Days of operation
250
300
350
250
300
350
200
150
100
50
0
0
TN loading rate after day 86 for the UASBR1 was lower due to
diminished NH
4 values in reject water. Efuent TN values of both
reactors were unstable during this period (Figs. 2a and b), but the
TN removal rate remained low only in the UASBR1. After day 239
(period IV), stable and efcient TN removal was achieved in the
UASBR2; also the performance of the UASBR1 was more stable than
in earlier periods.
After day 239 in the UASBR1 and day 259 in the UASBR2, the
HRT was set to 2 days to facilitate a better process performance. For
the same purpose, Anammox intermediates were injected into the
reactors. Finally, a more stable performance of the UASBR1 with
fewer uctuations in the TN removal efciencies and rates than in
the UASBR2 was achieved (Table 1, Figs. 2 and 3).
Nevertheless, the process efciency in the UASBR1 remained
somewhat lower compared to other researches performed with
synthetic wastewaters. NH
4 removal efciencies of 40e45% have
been achieved (9,17e19), while Fdz-Polanco et al. (8) has reported a
Stdev
Sulfate/Ammonium conc,
mg N/L, mg S/L
1.87
36
8.11
Average
VSS of seeding sludge, g/L
Temperature, C
pH
Period I, start-up (days 0e40)
HRT, days
TN removal efciency, %
TN loading rate, kg-N/(m3 day)
TN removal rate, kg-N/(m3 day)
NH
4 N (inuent), mg N/L
NH
4 N (efuent), mg N/L
SO2
4 S (inuent), mg/L
SO2
4 S (efuent), mg/L
2
NH
4 Nconsumed =SO Sconsumed
Period II, days 41e100
HRT, days
TN removal efciency, %
TN loading rate, kg-N/(m3 day)
TN removal rate, kg-N/(m3 day)
NH
4 N (inuent), mg N/L
NH
4 N (efuent), mg N/L
2
SO4 S (inuent), mg/L
2
SO4 S (efuent), mg/L
2
NH
4 Nconsumed =SO4 Sconsumed
Period III, days 101e239
HRT, days
TN removal efciency, %
TN loading rate, kg-N/(m3 day)
TN removal rate, kg-N/(m3 day)
NH
4 N (inuent), mg N/L
NH
4 N (efuent), mg N/L
SO2
4 S (inuent), mg/L
SO2
4 S (efuent), mg/L
2
NH
4 Nconsumed =SO4 Sconsumed
Period IV, from day 239 onwards
HRT
TN removal efciency, %
TN loading rate, kg-N/(m3 day)
TN removal rate, kg-N/(m3 day)
NH
4 N (inuent), mg N/L
NH
4 N (efuent), mg N/L
SO2
4 S (inuent), mg/L
2
SO4 S (efuent), mg/L
2
NH
4 Nconsumed =SO4 Sconsumed
Entire experimental period
TN removal efciency, %
TN loading rate, kg-N/(m3 day)
TN removal rate, kg-N/(m3 day)
NH
4 N (inuent), mg N/L
NH
4 N (efuent), mg N/L
2
SO4 S (inuent), mg/L
2
SO4 S (efuent), mg/L
2
NH
4 Nconsumed =SO4 Sconsumed
50
100
150
200
Days of operation
FIG. 2. Process performance of the UASBR1 (SRAO) and the UASBR2 (conventional
Anammox) reactors. (a) Dynamics of ammonium-N and sulfate-S in the UASBR1.
Symbols: open triangles, inuent ammonium-N; closed triangles, efuent ammoniumN; open diamonds, inuent sulfate-S; closed diamonds, efuent sulfate-S; open circles,
inuent sulde-S; closed circles, efuent sulde-S. (b) Changes in the inuent and
efuent NH
4 N and NO2 N, NO3 N concentrations in the UASBR2. Symbols:
open triangles, inuent ammonium-N; closed triangles, efuent ammonium-N; open
circles, inuent nitrite-N; closed circles, efuent nitrite-N; closed diamonds, efuent
nitrate-N.
(3)
a 100
80
300
70
250
200
60
150
100
50
429
NO2-N control
NO2-N with humic substance
NH4-N control
NH4-N with humic substance
NO3-N with humic substance
NO3-N control
50
40
90
TN conc, mg N/L
500
TN control
450
TN with humic substance
Hydrazine with humic substance 400
Hydrazine control
350
N conc mg N/L
30
20
10
50
40
0
2
4
Time (h)
4
Time (h)
d
Sulfate control
500
270
Ammonium-N control
265
400
Hydrazine control
300
450
800
Sulfate with humic
substance
780
SO42- conc, mg/L
260
760
350
250
255
740
200
250
720
245
700
240
150
100
50
10
15
20
25
0
0
10
15
20
25
Time (h)
Time (h)
UASB1: crosses, NO
2 in control; open diamonds, NO2 with humic substance; pluses, NH4 in control; closed triangles, NH4 with humic substance; open squares, NO3 with humic
2
mechanism of O2 generation in anoxic medium by oxygenic bacteria was described by Ettwig et al. (25), although the presence of
anoxic oxygenic bacteria in the engineered systems has not been
reported yet. As sulde concentrations in the efuent of UASBR1
were very low (not exceeding 0.1 mgS/L) it indicated to a rapid
sulde oxidation into S0 or SO2
4 .
Interactions between HM present in the reject water, nitrous
and sulfurous compounds can also contribute to the above2
mentioned effects on NH
4 oxidation. Lower SO4 reduction than
assumed could be due to partial re-oxidation of S0 or HS into SO2
4
taking place via sulfur-utilizing denitrication/denitritation. This
D 2
alters the nal balance DNH
4 consumed = SO4 consumed vratio,
increasing it. Presence of denitrifying sulfur-oxidizing Sulfurimonas
denitricans DSM 1251 in the seeding sludge as well as in the
reactor provides clear evidence in favor of this denitrication
mechanism. Sulfur-utilizing denitrication/denitritation reactions
include (26):
2
2
3
4
S0 6 5 NO
3 5 H2 O/SO4 5 N2 5 H
=
(4)
2
7
4
4
HS 8 5 NO
3 5 H /SO4 5 N2 5 H2 O
(5)
(6)
DG0 739kJ=mol
2
7
4
4
HS 8 3 NO
2 3 H /SO4 3 N2 3 H2 O
(7)
430
RIKMANN ET AL.
J. BIOSCI. BIOENG.,
TABLE 2. Bacterial strains identied in the seeding sludge and in the reactors.
Name of species
Seeding sludge
Uncultured bacterium clone Inoculum14
(Porphyromonadaceae), GenBank: HM008286
Unclassied Planctomycetaceae uncultured
bacterium JJB347, GenBank: GQ143799
Uncultured Verrucomicrobia bacterium
Sulfurimonas denitricans DSM 1251
Methylobacterium spp. (strains EI189, EI187,
AKB-2008-OT7, ED323, SEMIA 6407)
UASBR1 SRAO
ATB-KS-1929 (order Verrucomicrobiales),
GenBank: EF686989
Uncultured Verrucomicrobiales bacterium clone
De2102, GenBank: HQ183974
Uncultured Planctomycete clone Pla_PO55-9,
GenBank: GQ356109
Uncultured bacterium clone Dok23,
GenBank: FJ710742.1
Uncultured bacterium clone A1-F6_M13R Thiobacillus,
GenBank: GU083403.1
Uncultured sludge bacterium clone ASB08 Nitrosomonas,
GenBank: FJ947122.1
Uncultured bacterium clone 081203-OL-PVP22:1-9
Desulfobulbaceae, GenBank: FJ823212.1
UASBR2 Anammox
ATB-KS-1929 (order Verrucomicrobiales),
GenBank: EF686989
Uncultured Verrucomicrobiales bacterium clone
De2102 16S ribosomal RNA gene, partial sequence,
GenBank: HQ183974
Uncultured Planctomycete clone Pla_PO55-9 16S
ribosomal RNA gene, partial sequence,
GenBank: GQ356109
Uncultured Planctomycetales bacterium clone P4
16S ribosomal RNA gene, GenBank: DQ304521.2
Uncultured sludge bacterium clone ASB08
Nitrosomonas, GenBank: FJ947122.1
Uncultured bacterium clone KIST-JJY016 Nitrospira,
GenBank: EF594049.1
Uncultured bacterium clone B17_926R Thiobacillus,
GenBank: HQ141362.1
Primer
Determination method
Reference
GC-BacV3f/907r
PCR-DGGE
32
GC-BacV3f/907r
PCR-DGGE
32
Eub27f/Eub1492rPla46F-GC/Amx368R
8F/357R with 454 specic sequencing
primer parts
8F/357R with 454 specic sequencing
primer parts
PCR-DGGE
Pyrosequencing
33e35
454 platform manufacturer
(Roche) protocol
454 platform manufacturer
(Roche) protocol
GC-BacV3f/907r
PCR-DGGE
33e35
Pla46F-GC/Amx368R
PCR-DGGE
32
PCR-DGGE
32
GC-BacV3f/907r
PCR-DGGE
32
Pyrosequencing
Pyrosequencing
Pyrosequencing
Pyrosequencing
GC-BacV3f/907r
PCR-DGGE
33e35
Pla46F-GC/Amx368R
PCR-DGGE
32
PCR-DGGE
32
PCR-DGGE
32
Pyrosequencing
Pyrosequencing
Pyrosequencing
40 % of total NH
4 oxidation, while 60 % of NH4 N oxidation was
achieved in Anammox pathway. At the same time the concentra
tions of NH
4 and NO2 in the efuent were very low, obviously due
to the joint activity of the AOB and the nitrite oxidizing bacteria
(NOB).
NO
3 was formed in the UASBR2 either as a by-product of the
Anammox reaction or a product of oxidation of NO
2 performed by
NOBs. The calculated extent of direct NO
2 oxidation into NO3 can
be estimated from the difference between inuent NO
N
and
2
efuent NO
2 N by subtracting from it the amount of NO2 utilized
in the Anammox process and in the heterotrophic denitrication,
and adding the amount of NO
2 formed in the aerobic oxidation of
NH
4 performed by AOB-s. The calculated amount of NO3 formed
431
FIG. 4. Phylogenetic neighbor-joining tree, reecting the relationships between identied sequences [uncultured Planctomycetaceae bacterium clone JJB347 (GenBank: GQ143799)
and uncultured bacterium clone ATB-KS-1929 (GenBank: EF686989)] and 16S rDNA sequences of other known bacteria belonging to the phyla Planctomycetes and Verrucomicrobia.
432
RIKMANN ET AL.
J. BIOSCI. BIOENG.,
ASB08 Nitrosomonas) were detected. Our results indicate that
Verrucomicrobiales may be involved in some nitrogen removal
process. In the UASBR1, sulfur cycle microorganisms were dominant, while the UASBR2 was dominated by nitrogen-cycle
microorganisms.
ACKNOWLEDGMENTS
The research was supported by the Estonian target-nanced
research project Processes in macro- and microheterogeneous and
nanoscale systems and related technological applications
(SF0180135s08) and by the Estonian Science Foundation research
project Alternative ways of anaerobic ammonium oxidation process and the ways of its usage (ETF 9370). BiotaP LLC, Estonia and
Madis Metsis, Triin Lillsaar and Jaak Simm from the Integrated
Systems Biology Centre, Tallinn University of Technology are
acknowledged for the pyrosequencing results. Salutaguse Yeast
Factory (subsidiary of Lallemand Inc.) is acknowledged for fruitful
cooperation.
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