Download Attached growth gains an advantage over suspended growth on

Attached growth gains an advantage over suspended growth on
enrichment of ANAMMOX bacteria
Yu Tao*, Dawen Gao, Haoyu Wang
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin
150090, China (E-mail: [email protected]; [email protected]; [email protected])
Abstract
Anaerobic ammonium oxidation is an energy saving biological nitrogen removal process limited to
slow growth rate of anammox bacteria during start-up period. Attached growth was reported to be
suitable to culture slow-growing populations like anammox bacteria. In this study, six bench-scale
reactors (suspended- and attached-growth processes) were compared to enrich anammox bacteria
from WWTP activated sludge, including SBR, external MBR (EMBR) and sequencing batch
biofilm reactor (SBBR). The results showed that SBBR is more suitable than the other reactors. It
took the least time (three months) for SBBR to successfully start anammox process, which also
gained highest anammox activity (at an average daily increase of 12.1 g-N·L-1·d-2). CLSM results
proved the dominance of anammox population in SBBR biofilm. Anammox cells were inlaid among
the flocs, and nanowires-like materials were also observed by SEM.
Keywords:
Anammox bacteria; Attached-growth; Enrichment
Introduction
Discharge of nitrogen-rich wastewater into aquatic systems nowadays is strictly regulated in many
countries, which requests WWTPs higher investment and operational cost. Anoxic ammonium
oxidation (anammox) is an energy saving biological nitrogen removal process and is an attractive
option for total nitrogen (TN) control. Bacteria responsible for anammox are related to phylum
Planctomycetes (Strous, Fuerst et al. 1999), and they can couple the oxidation of ammonium with the
reduction of nitrite to nitrogen gas (N2) as the terminal product (Vandegraaf, Mulder et al. 1995).
In a mixed culture system, since anammox bacteria grow at a slow rate (a common opinion is
doubling time 8-11 days) (Strous, Heijnen et al. 1998), their proliferation is challenged by other
populations and limited by some parameters (e.g. DO, organic matter and temperature) (Kartal,
Kuenen et al. 2010). Previous studies have found that several types of reactor were suitable to enrich
anammox bacteria, such as SBR (Strous, Heijnen et al. 1998), UASB (Schmidt, Batstone et al. 2004)
and EMBR (van der Star, Miclea et al. 2008). Attached growth is a widely applied biological process
for wastewater treatment because of its low energy consumption. Such process configuration can also
form two or more functionally separated spaces, providing a stable microenvironment in each
biofilm, which is suitable to slow-growing populations like anammox bacteria.
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In this study, suspended- and attached-growth processes were compared to enrich anammox
bacteria from WWTP activated sludge. Six bench-scale reactors were selected, including two SBRs
(suspended growth and not granules), two EMBRs (suspended growth) and two SBBRs (sequencing
batch biofilm reactor, attached growth). The results showed that enrichment efficiency of SBBRs is
much higher than the other reactors. Some advantages of attached growth were also analysed from a
microcosmic perspective.
Material and Methods
Six reactors were all made of plexiglass and the effective volumes were between 1.0~3.0 L (Figure
1). Synthetic medium (main source: NH4Cl and NaNO2) were fed as substrates. Six reactors were
inoculated with four different types of sludge (Table 1). Ammonium, nitrite, nitrate, MLVSS were
measured according to standard methods (APHA, 1998). Total organic carbon (TOC) and TN were
determined with a Shimadzu TOC-VCPN-6000 analyser. DO and pH were tested by Handheld
Multi-Parameter Instruments (pH/Oxi 340i, WTW, Germany).
Figure 1. Six reactors used for anammox bacteria enrichment.
A 100-mL serum bottle was inoculated with 0.76 g VSS biomass after washing with phosphate
buffer (pH 7.8) for three times, previously sparged with argon gas. The substrate had the same
content to the synthetic wastewater except that the concentrations of ammonium and nitrite were
fixed (5 mM for each). The bottles were incubated in an orbital shaker (rotating at 70 rpm) under
the same temperature of the source reactor (33±1 oC). All the tests were performed in triplicate.
Every cycle lasted 24 hours and liquid samples were taken for ammonium and nitrite analyses. The
anammox activity was calculated based on the concurrent depletion of ammonium and nitrite in a
strictly anaerobic and autotrophic environment.
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A sample (1.5 ml) was harvested and fixed in paraformaldehyde. The probe Amx 820
(S-*-Amx-0820-a-A-22, specific for Candidatus Brocadia anammoxidans and Candidatus
Kuenenia stuttgartiensis, purchased from TaKaRa, Dalian, China) was labelled with Cy3. The
hybridizations with fluorescent probes were performed according to a previously-described protocol
[22]. The samples were counterstained using mounting medium containing 4’,
6-diamidino-2-phenylindole (DAPI). A confocal laser-scanning microscope (CLSM, Carl Zeiss,
Oberkochen, Germany) equipped with an Ar ion laser (488 nm) and He-Ne laser (543 nm) was used
for observation. Scanning electron microscope (SEM, Hitachi S-4700) were used for microcosmic
morphologic observation, following the instructions previously reported (Dang, Chen et al. 2010;
Kim, Choi et al. 2010).
Table 1 Seeding sludge for six reactors
Reactor
Seeding sludge
Effective volume (L)
EMBR1
S1
1.0
EMBR2
50%S1+50%S2
0.8
SBR1
S1
3.0
SBR2
50%S1+50%S2
3.0
SBBR1
20%S1+80%S3
1.5
SBBR2
20%S1+80%S4
1.5
S1, seeding sludge 1, from a pilot UASB with low anammox activity;
S2, seeding sludge 2, from an anaerobic digestion reactor at Xiaohongmen
WWTP, Beijing
S3, seeding sludge 3, from a bench anammox reactor (Tao et al., 2012)
S4, seeding sludge 4, from conserved UAFB biofilms (Gao et al., 2011)
Five milliliters samples for molecular tests were collected and stored at -70°C until DNA extraction.
Nucleic acids were extracted using Aqua-SPIN Gel Extraction Mini Kit (WATSON
Biotechnologies, Inc., Shanghai, China) according to the manufacturer’s protocol. A forward primer
of Bact0009f (GGTTTGATCGTGGCTCAG) with the 5’ end labeled with dye
6-carboxyfluorescein, and a reverse primer of Bact1492r (ACGGYACCTTGTTACGACCTT) were
used for PCR analysis. PCR was performed using one denaturation step at 94°C for 5 min, followed
by 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 90 s
and a final extension at 72°C for 8 min. Fluorescently labeled PCR products were purified using a
QIAquick PCR purification kit (Qiagen Inc., Canada). One part of the purified PCR products were
sequenced by a commercial service (Sangon Biology Engineering Technology & Services Co. Ltd,
Shanghai, China) and submitted for comparison to GenBank database using BLAST algorithms.
Another part of the purified PCR products were digested with the restriction enzyme Rsa I at 37°C
overnight. Digested PCR products were precipitated with ethanol and re-suspended in 15 μL of
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Hi-Di formamide with 500LIZ standard (Applied Biosystems, Foster City, CA). Samples were
denatured at 95°C for 5 min, followed by rapid chilling on ice. The samples were run on an ABI
PRISM 3130 Avant Genetic Analyzer (Applied Biosystems, Foster City, CA) in the GeneScan
mode and analyzed with the GeneMapper program version 3.0 (Applied Biosystems, Foster City,
CA). Only fragment lengths in the range of 50–600 bps were considered for analysis to avoid the
detection of primers and uncertainties of size determination.
Results and Discussion
All the reactors have been operated for over 200 days (SBR#1 218 d; SBR#251 d; EMBR#1 420 d;
EMBR#2 242 d; SBBR#1 226 d; SBBR#2 252 d). Two SBBRs performed much better than the
other four reactors by following the specific anammox activity of each reactor (Table 2). Two SBRs
failed to enrich anammox bacteria even though the TN removal rate of each SBR reached to nearly
40 per cent during the final stage. Nitrite was almost completely depleted at the end of each period,
but ammonium was only partly removed in both SBRs. Nitrate accumulated obviously in SBR#1.
EMBRs did not perform anammox activity until half a year later (Table 2). EMBR#2 obtained
higher anammox activity than EMBR#1 due to the wash-out of biomass and withdrawal of the
liquid above the settled sediment at the beginning of start-up (day 1 to day 10). It took less than
three months for two SBBRs to successfully start anammox process, and their average daily
increase of specific anammox activities were almost ten times higher compared to EMBRs (Table
2).
Table 2 Enriching efficiency of anammox bacteria in each reactor
Reactor
Effective
volume (L)
Average daily increase of
specific anammox activity
a (g-N·L-1·d-2)
Start-up
period b
(days)
SBR#1
1.0
Failed
Failed
SBR#2
0.8
Failed
Failed
EMBR#1
3.0
0.7
212
EMBR#2
3.0
1.6
196
SBBR#1
1.5
10.8
90
SBBR#2
1.5
12.1
89
a, calculated only based on the data collected during stable anammox period, i.e.
simultaneous depleted of ammonium and nitrite without fluctuation;
b, the time from the beginning of start-up until the day that stable anammox began.
Two types of biofilm-attached carriers from SBBR#2 were taken out at different stages of the
experiment for morphology and microbial dynamic tests. Abundant and adhesive biomass closely
attached to the inner- (most) and outer-side of the carriers in dark orange colour (Figure 2 a). In
order to clearly observe the compactly-attached film, translucent carriers were also used in SBBR#2.
It can be obviously seen that the colour of back side of the carrier was fresh red, and the colour of
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face side (i.e. outer biofilm) was the same as Figure 2 a, which indicated that most anammox
bacteria closely attached to the carrier (inner-side of biofilm).
Figure 2 Biofilm attached onto the carriers (taken from SBBR#2).
(a) Polyethylene ring-shaped matrix pieces with anammox biofilm on it;
(b) Translucent plastic-piece carrier with anammox biofilm on it (face side);
(c) Translucent plastic-piece carrier with anammox biofilm on it (back side).
Figure 3 CLMS photos of the biofilm taken from SBBR#2. (a) anammox bacteria with the probe of
S-*-Amx-0820-a-A-22 (Amx820, 5’-AAAACCCCTCTACTTAGTGCCC-3’, ordered from
TaKaRa, Dalian, China); (b) all bacteria labelled with Cy5; (c) combined figures of a and b.
CLSM figures proved that anammox population covered most part among all bacteria, indicating a
dominant status (Figure 3). However, the concentrations of biomass in SBBRs were not high. The
value of MLVSS/MLSS for each SBBR was 0.29 - 0.32 even at stable stage. SEM figures showed
that anammox cells (with the length less than 1 um, Figure 4 a) were inlaid among the flocs (Figure
4 b), which were possibly the combination of organic (EPS and SMP, etc.) and inorganic matter
(metal-oxide and carbonate, etc.). It is interesting to find nanowires-like materials connecting
anammox cells with each other or connecting anammox cells and flocs (Figure 4 c-d). It is expected
that these wires played an important role in transferring electron, information or materials (Reguera
et al, 2005; Clauwaert, Rabaey et al. 2007).
The microbial dynamics of the biofilm taken from SBBR was closely investigated by using T-RFLP
(Figure 5). The abundance of anammox biomass can be seen accoding to the red bars. There was
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very little anammox bacteria in the seeding sludge (less than 4%), and the abundance increased
slowly to 12% after three month enrichment. However, the abundance became rapidly increasing
since then and the overall abundance increased to 40% at the end of the experiment. The formation
of stable and active biofilm in SBBR is believed to accelerate anammox due to its protective role as
a barrier to high dissolved oxgen concentration (Zekker et al., 2012).
Figure 4 SEM photos of the biofilm taken from SBBR#2.
Figure 5 T-RFLP results indicating a fast enrichment of anammox biomass (red bars) in the biofilm
taken from SBBR#2.
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