142
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
of great interest in the field of biological nitro-
gen removal (Ahn, 2006; Jetten
et al
., 2005). The
prevailing view holds that the main functional
bacteria in this process are ammonia oxidizing
bacteria (AOB), nitrite oxidizing bacteria (NOB)
and anaerobic ammonium oxidizing bacteria
(ANAMMOX) (Dong & Tollner, 2003; Sliekers
et al
., 2002; Third, Sliekers, Kuenen, & Jetten,
2001). Ammonium is partially oxidized by AOB
to nitrite, which is subsequently consumed by
ANAMMOX together with the remaining am-
monium. However, AOB and NOB are aerobic
bacteria while ANAMMOX are obligate anaer-
obes, which do not tolerate dissolved oxygen
(DO) (Strousm, Vanger, Kuenen, & Jetten, 1997).
Therefore, the key to one-step autotrophic de-
nitrification in a single reactor is to balance the
supply and demand of dissolved oxygen (Qin,
Guo, Fang, & Yang, 2009; Hippen, Rosenwin-
kel, Baumgarten, & Seyfried, 1997; Galluzzo,
Ducato, Bartolozzi, & Picciotto, 2001) and how
to make them coexist in one reactor.
In this study, autotrophic nitrogen removal
was conducted in an SBBR (Guo, Qin, Fang, &
Yang, 2008). Previous research has shown that
autotrophic nitrogen removal process has been
set up and microbial community structure ex-
erts significant influence on the performance of
this system (Fang, Qin, Guo, Jia, & Yang, 2010).
Microbes are at the core of the reactor, and the
denitrification effect of activated sludge and bio-
film is directly determined by the composition
of the microbial community. This is a compara-
tive study of activated sludge and biofilm in an
SBBR, with quantitative analysis of functional
bacterial populations as well as nitrogen transfer
pathways. This study is to analyze the different
roles between activated sludge and biofilm in
autotrophic nitrogen process.
Materials and methods
Experimental apparatus
A schematic view of the SBBR autotrophic
denitrification reactor is shown in figure 1. The
reactor was filled with hollow balls fillers and
soft combination packing. Under the condi-
tion of 30 ℃ and pH of 8.0, artificial synthetic
ammonia nitrogen wastewater was added in.
The concentration of NH
4
HCO
3
was 160 mg/l
without organic carbon sources. According
to the optimal operation condition about 2h:
2h aeration/non-aeration ratio and DO 2.0
(aeration) mg/l /0.4 (non-aeration) mg/l, after
half year domestication, autotrophic nitrogen
removal system was set up successfully. TN
removal rate reached to 80% while ammonia
conversion rate reached to 100%.
Nitrosation and anaerobic ammonium
oxidation were tested separately in both acti-
vated sludge and biofilm samples in the SBBR.
Other tests were done in conical flasks (figure
2), which were sealed with rubber plugs to
maintain a stable DO concentration. Artificial
wastewaters with different NH
4
+
-N or NO
2
-
-N
concentrations were added.
Experimental apparatus
(1) Quantitative analysis of functional bacteria
DNAwas extracted using a Shanghai Biocolors
BioScience and Technology Company K717
environment genomic DNA extraction kit.
Quantitative real-time PCR was carried out
using SYBR Green Real-time PCR Master Mix
Kit (Bio-Rad) in an iCycler IQ® Multiplex Real-
Time PCR DNA System (Bio-Rad). A melting
curve analysis was performed for each sample
after PCR amplification to ensure that a single
product with the expected melting curve char-
acteristics had been obtained. The recombinant
plasmid vectors were constructed by standard
procedures.
(2) Nitrosation rate
Activated sludge and biofilm samples were
taken from an SBBR which was performing ef-
fective and stable autotrophic nitrogen removal.
The samples were diluted with distilled water
and then inoculated into two 250 ml flasks,
into which had already been added 100 ml of
artificial wastewater. The initial concentration of
NH
4
HCO
3
was 70 mg/l, and pH was adjusted to
8.0 (± 0.2) by the addition of NaHCO
3
. The nutri-
ent solution with trace elements such as Fe, Mg
