144
Qin
et al
.,
Comparison on nitrosation and anaerobic ammonium oxidation between activated sludge and biofilm from an autotrophic...
Tecnología y Ciencias del Agua
, vol. VIII, núm. 2, marzo-abril de 2017, pp. 141-149
•
ISSN 2007-2422
was also added (EDTA 5.0 g/l, CoCl
2
·6H
2
O 1.6
g/l, ZnSO
4
·7H
2
O 2.2 g/l, MnCl
2
·4H
2
O 5.1 g/l,
CuSO
4
·5H
2
O 1.6 g/l (NH
4
)
6
Mo
7
O
24
·4H
2
O 1.1 g/l,
CaCl
2
·2H
2
O 5.5 g/l, FeSO
4
·7H
2
O 5.0 g/l). The
flasks were maintained at a temperature of 30 °C
using a heated magnetic stirrer. DO concentra-
tion was controlled to 2.0 mg/l by aerating with
a mixture of O
2
and Ar. The flasks were placed
in a constant temperature shaking reactor at 30
°C and shaken at 150 revs/min.
Concentrations of NH
4
+
, NO
2
-
, NO
3
-
and TN
were measured at intervals during each 48 h test
to obtain concentration curves. The maximum
reaction rate Vmax was calculated from the
maximum slope of each concentration curve. At
the end of each test, activated sludge or biofilm
samples were taken out of the flask by brushing
with a sterile brush and rinsing with distilled
water, and volatile suspended solids (VSS) were
measured:
V
max
=
k
max
mgN/
d
(
)
VSS mgVSS
(
)
(3) Anaerobic ammonium oxidation rate
The experimental procedure and analysis was
similar to the study of nitrosation rate, except
for the components of artificial wastewater
and DO concentration. NH
4
HCO
3
and NaNO
2
concentrations in the artificial liquid were both
70 mg/l. The liquid was sparged with Ar gas
for 30 min to remove DO to below the detection
limit (0.01 mg/l) of an LDO
TM
HQ10 dissolved
oxygen meter.
Results and discussion
Identification of recombinant plasmid
The recombinant plasmids sequences were
analyzed using a GenBank BLAST search. As
shown in table 2, conservative regions of 16S
rDNA of functional bacteria were obtained.
These recombinant plasmids were suitable
for quantitative analysis of AOB, NOB, and
ANAMMOX in real-time PCR experiments.
Quantitative analysis of AOB, NOB, and
ANAMMOX
Quantitative real-time PCR results are shown
in table 3. The populations of the three main
functional bacteria (ammonia oxidizing bacteria
AOB, nitrite oxidizing bacteria NOB and anaero-
bic ammonium oxidizing bacteria ANAMMOX)
in activated sludge and biofilm samples were
Table 1. Primers and parameters used in real-time PCR.
Primers
Reaction parameters
Reference
AOB
5’-GGAGRAAAGCAGGGGATGG-3’
5’-CGTCCTCTCAGACCARCTACTG-3’
95 ℃ × 5 min; 95 ℃ × 15 s, 60 ℃ × 45 s;
45 cycles
Li, Liu, Zhang,
Du, & Chen, 2007
NOB
5’-CCTGCTTTCAGTTGCTACCG-3’
5’-GTTTGCAGCGCTTTGTACCG-3’
95 ℃ × 5 min; 95 ℃ × 1 min; 60 ℃ × 1
min; 72 ℃ × 2 min;
35 cycles
Dionisi
et al
., 2002
ANAMMOX 5’-ATGGGCACTMRGTAGAGGGGTTT-3’
5’-AACGTCTCACGACACGAGCTG-3’
50 ℃ × 2 min; 94 ℃ × 10 min; 94 ℃ × 15 s;
60 ℃ × 1 min; 40 cycles
Ikuo, Tomonori,
& Satoshi, 2007
Table 2. A partial result of recombinant sequence alignment with genes in GenBank.
Sample
Length
Number
Similarity
Blast result
I
117bp
FJ445022
CP000450 (98%)
Nitrosomonas
II
151bp
FJ490152
AJ224046 (100%)
Nitrospira
III
275bp
FJ490144
AF375995 (99%)
Candidatus kuenenia stuttgartiensis
