Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s00253-014-5835-z
ENVIRONMENTAL BIOTECHNOLOGY
Abstract Nitrite-dependent anaerobic methane oxidation (ndamo) is mediated by bacteria that anaerobically oxidize
methane coupled with nitrite reduction and is a potential
bioprocess for wastewater treatment. In this work, the effect
of reactor configuration on n-damo bacterial cultivation was
investigated. A magnetically stirred gas lift reactor (MSGLR),
a sequencing batch reactor (SBR), and a continuously stirred
tank reactor (CSTR) were selected to cultivate the bacteria.
Microbial community was monitored by using quantitative
PCR, 16S rRNA gene sequencing, pmoA gene sequencing,
and fluorescence in situ hybridization (FISH). The effects of
substrate inhibition, methane mass transfer, and biomass
washout in the three reactors were focused on. The results
indicated that the MSGLR had the best performance among
the three reactor systems, with the highest total and specific ndamo activities. Its maximum volumetric nitrogen removal
rate was up to 76.9 mg N L1 day1, which was higher than
previously reported values (5.137.8 mg N L1 d1).
Introduction
Nitrite-dependent anaerobic methane-oxidizing (n-damo) bacteria were first discovered and confirmed in a laboratory
enrichment culture (Raghoebarsing et al. 2006) and were
subsequently found in natural habitats (Deutzmann and
Schink 2011; Kojima et al. 2012; Shen et al. 2013; Hu et al.
2014). N-damo bacteria can mediate the bioprocess of
B. Hu : Z. He : S. Geng : C. Cai : L. Lou : P. Zheng : X. Xu (*)
Department of Environmental Engineering, Zhejiang University,
Hangzhou 310058, China
e-mail: xuxinhua@zju.edu.cn
Schematic diagrams of the three reactors with different configurations (SBR, CSTR, and MSGLR) are shown in Fig. 1.
Each reactor has a total volume of approximately 1.3 L, with a
working volume of 1.0 L. The CSTR and MSGLR had an
additional 0.1 L for the gas-liquid-solid separator, but their
headspaces were slightly smaller than those of the SBR.
The SBR was operated in successive 24-h cycles consisting
of 0.1 h of medium feeding, 22 h of aeration and stirring, 1.8 h
of settling, and 0.1 h of effluent discharge. The volume exchange ratio for the SBR was 0.5. The CSTR and MSGLR
were both continuous flow reactor systems with hydraulic
retention times (HRTs) of 48 h. Methane (99.99 %) was
supplied at 10 mL min1 into the three reactors, and gas was
recirculated in the MSGLR at 1,000 mL min1. All three
reactors were magnetically stirred at 150 rpm, and the temperature was controlled at 300.5 C. The feeding tank and
the effluent collection tanks were flushed with pure Ar
(99.999 %) for 15 min and subsequently sealed with argon
gas bags. Each day, 2 mL of influent and effluent were
sampled and centrifuged (5 min, 7,440g) to measure nitrite
and nitrate concentrations, and 2 mL of sludge was sampled
for molecular analyses on day 75. Moreover, suspensions in
the reactors and the effluent were collected to determine the
VSS level on days 0 and 75 and day 75, respectively.
The inoculum for the three reactors was taken from a previous
SBR enrichment cultivated after 650 days. The inoculum was
completely mixed and divided into three portions before the
reactors were seeded. The volatile suspended solid (VSS)
concentration of the seeding sludge was 5.30.3 g VSS L1,
and the inoculum volume was 30 % of the working volume for
each reactor.
The medium was modified from previous protocols (Ettwig
et al. 2009). The KHCO3 concentration was reduced from 1.0
to 0.25 g L1 to avoid the generation of CaCO3 and MgCO3
deposits. Here, the modified medium contained (per liter)
0.25 g KHCO3, 0.05 g KH2PO4, 0.3 g CaCl2 2H2O, 0.2 g
8
15
9
15
15
7
11
11
3
13
12
12
14
12
10
preincubation, 1 mL of the medium was sampled and centrifuged (5 min, 7,440g) every 1.5 h to measure the nitrite and
nitrate concentrations, and 30 L of gas was extracted to
measure the 13CH4 and 13CO2 concentrations in triplicate.
To precisely determine the n-damo activity, two control
groups were established. Control group (I), without methane
in the headspace, was designed to eliminate the disturbances
of denitrification by heterotrophic denitrifier, and control
group (II), without nitrite in the medium, could present the
methane consumption of other methane oxidation processes.
Both of the two control groups were studied simultaneously
with the experimental group.
Analytical methods
The concentrations of VSS, nitrite, and nitrate were measured
according to the APHA standard methods (APHA 2005). The
13
CH4 and 13CO2 concentrations in the headspace were measured by a mass spectrometer (Agilent 7890/5975C inert
MSD; Agilent, USA) as previously described (Ettwig et al.
2009). The pH was measured with a Mettler-Toledo FE20 pH
meter (Mettler-Toledo Instruments Co., Ltd., Shanghai).
Molecular analyses
The inoculum and cultures of the three reactors on day 75
were collected (2 mL) for molecular analysis. M. oxyfera-like
bacteria were identified by 16S rRNA and pmoA gene sequencing and FISH microscopic analysis. The quantities of
M. oxyfera-like bacteria in the biomass were represented by
the abundance of the 16S rRNA genes of NC10 bacteria
(copies g1 VSS) determined by qPCR. The densities of
M. oxyfera-like bacteria in the reactors were characterized by
the copy numbers of the 16S rRNA genes of NC10 bacteria
(copies L1) that were calculated by multiplying the 16S
rRNA gene abundance (copies g1 VSS) times VSS concentrations (g VSS L1).
The total DNA was isolated with a PowerSoil DNA
Isolation Kit (Mo Bio Laboratories, Carlsbad, CA, USA)
according to the manufacturers instructions. Nested PCR
was employed to amplify the 16S rRNA and pmoA genes
from the DNA samples. The primers and the amplification
conditions were the same as previously reported (Luesken
et al. 2011a, b). The PCR products were examined by agarose
gel (1.0 %) electrophoresis. Cloning of the PCR products was
performed using the pMD19-T vector (TaKaRa Bio Inc.,
Shiga, Japan) according to the manufacturers instructions.
The vectors were transformed into competent cells. They were
incubated in SOC medium for 1 h and in LB medium containing ampicillin, X-gal, and IPTG for 16 h at 37 C. Using
blue-white screen technique, the white colonies were picked
out. About twenty positive clones were randomly selected
from each of the 16S rRNA and pmoA gene clone libraries
and were sequenced by BGI-Hangzhou, China.
Results
Volumetric nitrogen removal rates
Figure 2 presents the volumetric nitrogen removal rate (NRR)
of the three reactors. The 75-day operating period can be
divided into three phases (marked by dashed lines in Fig. 2)
according to the nitrite loading rate (NLR). The NLR was
approximately 30 mg N L1 day1 in phase I,
50 mg N L1 day1 in phase II, and 70 mg N L1 day1 in
phase III (the NLRs of the SBR and CSTR decreased to
20 mg N L1 day1 after day 50 due to substrate inhibition).
In phase I (114 days), the NRRs in all three reactors
increased over time. The performances of the SBR and CSTR
were similar, and the MSGLR performed better than the other
two. The NRR in the MSGLR rapidly increased to the maximum (approximately 30 mg N L1 day1) for this phase during
the first 5 days and was then stable near the maximum for the
remaining time. In phase II (1545 days), the NRRs in the
CSTR and MSGLR continuously increased, but the SBR exhibited a stagnation period. The nitrite concentrations in the
reactors, the SBR, the CSTR, and the MSGLR, were 4.15
5.87, 2.753.31, and 0.021.42 mmol L1, respectively. In
phase III (4675 days), the NRR in the MSGLR continuously
increased after a short stagnation period, but those in the SBR
and CSTR were rapidly falling when the NLR increased to
70 mg N L1 day1 for 5 days, and the nitrite concentrations in
the SBR and the CSTR sharply increased to 9.17 and
8.71 mmol L1 on the fifth day after NLR increasing.
Moreover, the nitrite concentration in the MSGLR increased
to 3.80 mmol L1 on the fifth day after NLR increasing but
decreased gradually in the remaining time. After the NLR was
reduced, the NRR in the SBR and CSTR partially recovered,
but it was difficult to resume to the previous best level (in phase
II). Finally, the MSGLR achieved the highest NRR,
76.9 mg N L1 day1, which is almost twice as high as the
previously reported highest values of 37.8 mg N L1 day1
(Kampman et al. 2012) (see Table 1).
Biomass washout
The copy numbers of the 16S rRNA genes of the NC10
bacteria in all three reactors (copies L1) increased over the
75-day operation period, and the copy number for the
MSGLR was the highest (see Table 2), which agrees with
the NRR results. A comparison of the SBR and CSTR showed
that the SBR maintained more biomass (higher biomass concentration) but the abundance of the 16S rRNA genes of
NC10 bacteria in the SBR was slightly lower than that in the
CSTR. The MSGLR owned both the highest biomass concentration and 16S rRNA gene abundance among the three reactors, which indicated that the MSGLR could be the optimal
choice to cultivate n-damo bacteria.
To further investigate the sludge retention performances of
the three reactors, the concentrations of biomass in the effluent
of the SBR, CSTR, and MSGLR were determined on day 75,
which were 0.039, 0.038, and 0.050 g VSS L1, respectively.
The percentages of biomass washed out with the effluent of
the SBR, CSTR, and MSGLR were estimated to be 1.63, 2.00,
and 1.85 %, respectively, as calculated from the concentration
of biomass in the effluent divided by that in the reactor. The
ratios of washed-out biomass to produced biomass in the
SBR, CSTR, and MSGLR were estimated to be 57.2, 77.0,
and 55.3 %, respectively, by assuming that the percentages of
washed-out biomass were constant during the period of
operation.
Batch activities by stable isotope tests
The consumption of nitrite and 13CH4 occurred simultaneously, and 13CO2 was detected in the headspace after 3.0 h. The
generation of 13CO2 occurred later than 13CH4 consumption
(Fig. 3), which may be explained by the fact that 13CO2 was
dissolved into the liquid. The specific activities of n-damo
Phase II
Phase I
Phase III
70
60
50
MSGLR
CSTR
SBR
40
30
20
10
0
0
10
20
30
40
50
60
70
80
time (d)
bacteria were used to compare the performances of the different cultures and are represented by the generation rates of
13
CO2 and the conversion rates of nitrite and 13CH4 (Fig. 4).
The specific activities of the cultures from the SBR, CSTR,
and MSGLR after 75 days increased at varied levels. The
specific activity was the highest in the MSGLR, followed by
the CSTR and lowest in the SBR. Moreover, almost all of the
specific activities increased during the 75 days of operation
(except for the 13CO2 generation rate in the SBR). The results
of the specific activities support the qPCR findings (the abundance of the 16S rRNA genes of the NC10 bacteria in
Table 2).
According to the stable isotope batch tests, the molar ratios
of the 13CO2, 13CH4, and nitrite generation/conversion rates
for the inoculum and the cultures from the SBR, CSTR, and
MSGLR were 2.6:3:7.1, 2.3:3:7.5, 2.8:3:6.6, and 2.8:3:8.5
Table 1 Overview of the maximum nitrogen removal rates for the n-damo process
Configuration
Temperature
(C)
HRT
(day)
Maximum NRR
(mg N L1 day1)
Reference
SBRa
SBRa
25
30
15.3b
29.3b
SBRa
SBR
SBR
CSTR with an external settler
CSTR
2023
30
30
30
30
5.5b
1330.3b (initial)
4.3b (after 6 months)
1550b
2.0
2.0
16.2565b
2.0
5.1b
17.9b
11.4
9.6b
26.4
30
30
4.855.0b
2.0
37.8
76.9
These reactors are SFBRs actually, although they were called as SBRs in the references
Calculated value
Inoculum
SBR
CSTR
MSGLR
1.60.1
9.32.01011
1.50.31012
2.40.1
9.61.31011
2.30.31012
1.90.2
1.30.41012
2.50.81012
2.70.1
1.30.51012
3.51.41012
Discussion
Substrate inhibition and methane mass transfer
0.05
0.10
0.04
0.09
0.03
0.08
0.02
0.07
0.01
13
0.11
13
High concentrations of nitrite may inhibit the n-damo activities in the reactors, as is well known that nitrite is a substrate
inhibitor. The stagnation period of the SBR in phase II may be
resulted from the substrate inhibition of nitrite for its nitrite
concentration was slightly higher than the reported nitrite
inhibition constant for n-damo bacteria (K I NO2 , 4.1
0.06
0.00
0
time (h)
0.05
13
CO2
13
13
CH4
0.04
13
nitrite
0.03
0.02
0.01
0.00
Inoculum
CSTR
MSALR
the MSGLR makes the flocs small (Biggs and Lant 2000;
Jarvis et al. 2005), which reduces the solid-liquid mass transfer resistance and improves the apparent activity of the ndamo bacteria.
SBR
28
40
59
49
group A
12
25
83
98
0.02
group B
24
69
74
32
31
95
36
60
0.05
Microbiological characteristics
Fig. 7 FISH of the biomass from the three reactors after 75 days. The
cells were hybridized with probe S-*-DBACT-1027-a-A-18 (Cy3, red)
specific for NC10 bacteria and a mixture of probes EUB I-III and EUB
V (dark blue), which detects nearly all eubacteria. The NC10 bacteria
appear purple due to double hybridization with the specific (red) and
general (blue) probes. Scale bar = 5 m
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