Chemosphere 186 (2017) 453e458

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Preservation and reactivation of Candidatus Jettenia asiatica and

Anammoxoglobus propionicus using different preservative agents
A. Viancelli a, *, M.C. Pra b, L.A. Scussiato c, M. Canta
~o b, A.M.G. Ibelli b, A. Kunz b, c
a rdia, SC, Brazil
Complexo de Desenvolvimento Científico - Universidade do Contestado, 89700-000, Conco
b
Embrapa Suínos e Aves, 89700-000, Concordia, SC, Brazil
c
PGEAGRI/CCET, UNIOESTE, Cascavel, PR, Brazil

h i g h l i g h t s

Only anammox biomass preserved using glycerol or skimmed cow milk recovered activity.

Preservation using glycerol was successful for Candidatus Jettenia asiatica.

skimmed cow milk favored the selection of Candidatus Anammoxoglobus propionicus.

a r t i c l e i n f o a b s t r a c t

Article history: Anaerobic ammonium oxidation (anammox) bacteria have peculiar characteristics that make them
Received 24 April 2017 difficult to cultivate. The conservation of these microorganisms in culture collections or laboratories
Received in revised form requires successful preservation and reactivation techniques. Furthermore, studies have shown that
10 July 2017
successful reactivation may be preservative dependent. Considering this, the present study aimed to
Accepted 11 July 2017
Available online 12 July 2017
evaluate the preservation and reactivation of anammox consortia enriched from swine manure treat-
ment lagoons, by using different preservative agents at different temperatures: KNO3 (at 4  C), glycerol
Handling Editor: Y Liu (20  C, 80  C), and skimmed cow milk (20  C, 80  C, 200  C). After 4 months, the biomass was
thawed (except for KNO3), and the reestablishment of anammox activity was evaluated by stoichiometric
Keywords: coefficients. Microbial community transformation during the reactivation process was also studied by
Deammonification 16S rDNA sequence analysis. The results showed that the anammox biomass preserved with glycerol or
Preservative agents skimmed cow milk at 80  C recovered activity, while the biomass preserved with other methodologies
Sludge did not reestablish activity during the studied time (90 days). The bacterial community from the biomass
with anammox activity was characterized and showed the presence of Candidatus Brocadia anammox-
idans, Candidatus Jettenia asiatica, and Candidatus Anammoxoglobus propionicus. Preservation with
skimmed cow milk at 80  C favored the selection of Candidatus Anammoxoglobus propionicus, while
preservation with glycerol at 80  C was successful for Candidatus Jettenia asiatica. The present study
was effective on anammox sludge preservation and reactivation using low-cost processes for anammox
cultures preservation, which is important for biomass transport and deammonification reactor start up.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction responsible for the process. Candidatus Kuenenia, Candidatus Bro-

The anaerobic ammonium oxidation (anammox) process was
discovered in the early 1990s, and it became a promising bioprocess
for nitrogen removal from wastewaters (Strous et al., 1998). Until
now, nineteen species and five genera have been identified as
* Corresponding author.
E-mail address: alineviancelli@unc.br.
cadia, Candidatus Anammoxoglobus, and Candidatus Jettenia have
been enriched from activated sludge. The fifth genus, Candidatus
Scalindua, has been enriched from natural habitats, especially from
marine sediments and oxygen minimum zones (van Niftrik and
Jetten, 2012).
Because of the long doubling time (between 1.2 and 11 days) of
the bacteria, microbial biomass cultures are difficult to initiate, and
they require controlled reactor conditions (Ibrahim et al., 2016;
Zhang et al., 2017). Today, these microorganisms are kept viable

http://dx.doi.org/10.1016/j.chemosphere.2017.07.053
0045-6535/© 2017 Elsevier Ltd. All rights reserved.
454 A. Viancelli et al. / Chemosphere 186 (2017) 453e458
through continuous culturing (Heylen et al., 2012), which is time
consuming and expensive and makes it difficult to transport the
biomass to different places. To solve this problem, freezing meth-
odologies have been studied (Vlaeminck et al., 2007; Rothrock et al.,
2011; Heylen et al., 2012; Ali et al., 2014).
Several techniques have been used for the preservation of mi-
crobial cultures, including freezing (20  C to 200  C), lyophili-
zation, or a combination of these processes (Ali et al., 2014).
Cryoprotective additives (CPAs), especially glycerol and skimmed
milk, have also been used. Glycerol can slowly penetrate the cell
wall and the cytoplasm; however, the efficiency of the process
depends on the temperature and cell characteristics (Hubalek,
2003). Skimmed milk is the preferred CPA for bacteria. Cody et al.
(2008) reported that the use of a skimmed milk freezing solution
(10%) provides cell protection against loss of viability if a freezer
fails and may increase the long-term survival of bacterial stocks
when maintained at 80  C.
Although these techniques have been successfully used to pre-
serve the cultures of various microorganisms, research on the
storage of enriched cultures, consortium, or other nonpure cultures
remains in its infancy, and few studies have been conducted on
preserving anammox bacteria (Ali et al., 2014). Rothrock et al.
(2011) reported that prefreezing with liquid nitrogen (200  C)
was required for the long-term preservation of anammox bacteria
by lyophilization. Magri et al. (2012) preserved an anammox
biomass immobilized in polyvinyl alcohol gel at 8  C for 17 h, but
the anammox activity was severely damaged. Heylen et al. (2012)
introduced a protocol for long-term cryopreservation of a single-
cell anammox biomass using dimethyl sulfoxide (DMSO) as a
CPA; however, DMSO was highly toxic to the cells when thawed.
Wang et al. (2016) reported the anammox storage at 35  C for a 6-
month period; however, it was necessary to add nutrient medium
every week to retain the bacterial activity.
Recent studies have shown that successful anammox reac-
tivation (activity recovery after thawing) could be species-
preservative dependent (Heylen et al., 2012; Ali and Okabe, 2015;
Xing et al., 2016). Candidatus Kuenenia stuttgartiensis was reac-
tivated after being stored at 4  C with glycerol (Vlaeminck et al.,
2007), and Candidatus Brocadia caroliniensis was reactivated after
freezing in liquid nitrogen and subsequent lyophilization in skim-
med milk medium (Rothrock et al., 2011). However, storage at 4  C
was not successful for single-cell cultures of the marine anammox
species Candidatus Scalindua (Heylen et al., 2012).
Viancelli et al. (2011) showed the presence of Candidatus Jettenia
asiatica and Candidatus Anammoxoglobus propionicus species in a
consortium enriched from swine manure lagoons. This consortium
was kept viable through continuous culturing and was used as a
biomass provider for various studies (Viancelli et al., 2011; Casagrande
et al., 2013; Chini et al., 2016; De Pra et al., 2016). Considering this, the
present study was performed to evaluate reactivation using different
preservative agents (KNO3, glycerol, and skimmed cow milk) at
different temperatures (4  C, 20  C, 80  C, and 200  C) after
biomass freezing. Because of the correlation between anammox
species and the preservative agent, the bacterial community compo-
sition was evaluated immediately after the thaw and after achieving
anammox activity. The 16S rDNA pyrosequencing method was used.
2. Materials and methods
2.1. Biomass harvesting
A biomass (15 g for each preservation method in duplicate) with
established anammox activity (deposited at Embrapa Swine and
Poultry microorganism bank BRMSA 0323) was harvested from a
laboratory bioreactor operated at 35 ± 1  C, with a hydraulic
retention time of 2 h, dissolved oxygen of 0.2 mg L1, and nitrogen
loading rate of 1.2 gN L1.d1 feed with synthetic culture medium
containing 100 mgN L1 (50 mgNH3-N L1 and 50 mgNO-2-N L1)
(Casagrande et al., 2013).
2.2. Preservation methodologies
The harvested biomass was washed in deionized water and
sieved five times to remove any residual nutrients prior to preser-
vation. The biomass was conditioned in 50-mL centrifuge tubes
(duplicate for each method), and the respective preservative was
immediately added at the concentration as listed in Table 1. Treat-
ment 1 was performed in KNO3 media at a refrigeration tempera-
ture of 4  C, as described by Rothrock et al. (2011). Treatment 2 was
performed by adding glycerol to the biomass to a final concentration
of 25% (v v1). Treatment 3 was performed using skimmed cow
milk, where a solution of 20% of skimmed cow milk was prepared
with deionized water, and this solution was added to anammox
biomass to a final concentration of 20% (v v1). Treatments 4e6
were performed with the addition of preservatives as described
above, prefreezing at 20  C, followed of lyophilization at <1 mm
Hg, temperature from 40  C to 20  C, for 6 days, elevating 10  C per
24 h. Each tube was stored at the noted temperature. All samples
were preserved for 4 months.
2.3. Biomass thaw
After 4 months, the frozen biomass was thawed to room tem-
perature. Once thawed, the liquid fraction was separated from the
sludge, and the biomass was washed five times with synthetic
culture medium (Casagrande et al., 2013) to remove all preservative
agent residues.
2.4. Anammox reactivation process
Each biomass (duplicates for each method) that was previously
submitted to different preservative conditions was transferred to a
100-mL continuous upflow bioreactor, which was filled to 30% of
the reactor useful volume. The system was continuously fed with
synthetic medium (Casagrande et al., 2013) at the same operational
conditions described in 2.1.
2.5. Analytical methods
Total ammoniacal nitrogen (as NH3-N), nitrite (NO- 2 N), nitrate
(NO3 -N), and total alkalinity were monitored twice a week in the
influent and effluent samples (APHA, 2012). The NH3-N and NO 2N
consumption and NO 3 N production values were compared to the
anammox process stoichiometry (Strous et al., 1998; Kunz et al.,
2007; Ibrahim et al., 2016).
2.6. Anammox sludge without preservation (ASWP)
Biomass from a laboratory reactor, which works at the same
operational conditions as the preservation samples, was harvested
immediately before the reactivation experiment and used as a
positive control in the reactivation experiments.
2.7. Bacterial structure characterization
Biomass samples were collected immediately after thawing and
after anammox activity establishment. The samples underwent
DNA extraction and pyrosequencing of the 16S rDNA. The genomic
material was extracted using the MoBioPowerSoil® kit (Carlsbad,
CA, USA) according to the manufacturer's protocol. 16S rDNA
 A. Viancelli et al. / Chemosphere 186 (2017) 453e458 455

Table 1
Different conditions (preservative agent, concentration, and temperature) used to preserve the anammox biomass for 4 months.

Treatment Preservation medium Concentration (% (v v1))a Storage temperature ( C)

1 KNO3 80 4
2 Glycerol 25 20
80
3 skimmed cow milk 20 20
80
4 Lyophilization þ glycerol 25 20
80
5 Lyophilization þ skimmed cow milk 20 20
80
6 Lyophilization þ skimmed cow milk 20 200
a
Except for KNO3 (mgN L1).

sequence library was constructed by a limited cycle polymerase
chain reaction amplifying the V3 and V4 regions of the 16S rDNA
gene according to the manual from Illumina® (ILLUMINA, 2016):
Forward Primer 16S_341F 50 -tcgtcggcagcgtcagatgtgtataagaga-
cagcctacgggnggcwgcag-30 and Reverse Primer 16S_805R 50 -
gtctcgtgggctcggagatgtgtataagagacaggactachvgggtatctaatcc-3’.
3. Results and discussion
3.1. Anammox activity reestablishment after thawing
The different preservative agents used to reactive anammox
sludge had different behaviors on biomass activity. To understand
whether the biomass inoculated in the upflow reactors underwent
nitrogen removal by the anammox process, the nitrogen species
concentrations (NH3-N, NO  Kuenenia stuttgartiensis and the marine anammox species Candi-
2 N, and NO3 -N) in the influent and
effluent were tested. At each time point, the concentration differ-
ences of each species were converted to molar ratios (compared to
NH3-N) and compared to the literature coefficients (Strous et al.,
1998).
The stoichiometric coefficients obtained from each biomass are
presented in Tables 2 and 3. Anammox sludge without preservation
(ASWP) required 60 days to achieve stoichiometric coefficients
(Table 2), and this period could be related to the lag phase described
in other studies (Vlaeminck et al., 2007; Tang et al., 2009). This
phase is characterized by duplicating bacterial cell preparation.
Vlaeminck et al. (2007) showed that a longer preservation time led
to a longer lag phase.
All tests using lyophilization failed to recover anammox activity
(Table 3). Stoichiometric coefficients higher than 1 for N0-2-N and
NO
3 -N, the light brown biomass color, and the fluffy flocs format
(data not shown) suggest a strong contribution of nitrification ac-
tivity (Crites and Techobanoglous, 1998; Lo pez-Palau et al., 2011;
Chini et al., 2016). In a reactor, nitrifying bacteria
(mmaxnitritation ¼ 0.77 d1 and mmaxnitratation ¼ 1.08 d1) can grow
very fast when compared to anammox bacteria (mmax ¼ 0.06e0.10
d1) because nitrifying bacteria are favored when the anammox pro-
cess is inhibited or inactivated (Wiesmann, 1994; Kunz et al., 2012;
Awata et al., 2013).
Lyophilization is a very complex procedure, and Heylen et al.
(2012) highlighted that lyophilization is not recommended for
fastidious microorganisms because of the potentially damaging
drying process. This damage occurs in the case of anammox bac-
teria, which have a peculiar composition of cell membranes and
compartments (such as the anammoxosome) (van Niftrik et al.,
2004). However, the complete composition of the anammox cell
is presently unknown (van Teeseling et al., 2014), and thus, it is
difficult to know which characteristics are responsible for the
failure.
The biomass preserved using KNO3 at 4  C did not reactivate, as
was previously observed with single-cell cultures of Candidatus
datus Scalindua (Heylen et al., 2012). The cells lysed within days,
which may be due to the lack of the protective polymeric matrix
present in aggregated cultures. However, other studies have
described successful activity recovery after 2 months using the
preservation method of storage without cryoprotectant at 4  C (Ji
and Jin, 2014).
The best results were obtained when the bacteria consortium
was preserved using skimmed cow milk or glycerol and stored
at 80  C. However, when using the same preservative at 20  C,
only skimmed milk showed a tendency toward anammox stoichi-
ometry (Table 2).
The results from the reactivation with different preservation
methodologies used in the present study showed that 90 days were
necessary until the biomass had anammox activity. The establish-
ment of anammox activity was achieved when the bacterial con-
sortia reached stoichiometric coefficients similar to those described
in the literature, with the NO þ
2 N/NH4 -N ratio ranging from 1.14 to
 þ
1.32 and the NO3 /NH4 ratio from 0.13 to 0.26 (Strous et al., 1998;
Kunz et al., 2007; Ibrahim et al., 2016).

Table 2
Stoichiometric coefficients of ASWP and anammox sludge þ preservative agents after thawing and during reactivation time. All coefficients are related to NH3-N.

Reactivation time (d) ASWP skimmed cow milk skimmed cow milk Glycerol (80  C) Glycerol (20  C) KNO3 (4  C)
(80  C) (20  C)

N-NO-2 N-NO-3 N-NO-2 N-NO-3 N-NO-2 N-NO-3 N-NO-2 N-NO-3 N-NO-2 N-NO-3 N-NO-2 N-NO-3

0e28 0.47 0.08 ND ND ND ND ND ND ND ND ND ND

29e45 0.48 0.08 ND ND ND ND ND ND ND ND ND ND
46e65 0.75 0.22 0.36 0.39 ND 0.18 0.13 0.30 ND 0.28 ND 0.07
66e90 1.15 0.36 1.25 0.37 0.70 0.49 1.17 0.58 0.63 0.36 ND ND

ND ¼ not detected.
ASWP ¼ anammox sludge without preservation.
456 A. Viancelli et al. / Chemosphere 186 (2017) 453e458

3.2. Bacterial community structure

N-NO-3

0.32
1.54
1.95
2.12
skimmed cow milk
Reactors that had anammox activity underwent bacterial
community characterization after process re-establishment. A

Lyophilized þ
total of 107,425 high-quality sequence reads were obtained from

(200  C)
bacterial 16S rDNA. It was possible to observe that the community

N-NO-2
structure changed (Fig. 1) when the species present on the first

0.11
1.05
1.47
1.58
operation day (T0) were compared to those on the 90th day (T90).
The biomass preserved using glycerol at 80  C was mainly
composed of Candidatus Jettenia asiatica, Pseudomonas, Thermo-
nonas, Diaphorobacter, Nitrosomonas, and Nitrospira immediately
N-NO-3
Lyophilized þ Glycerol

0.49
2.46
1.56
2.54
after thawing. The same biomass was characterized after 90 days
of operation, when it had a good amount of anammox activity, and
Stoichiometric coefficients of ASWP and anammox sludge þ preservative agents after thawing and lyophilization and during reactivation time. All coefficients are related to NH3-N.

the relative abundance of this bacterial group changed with an

increase in the number of Thermononas and a decrease in the
(20  C)

N-NO-2

number of Candidatus Jettenia asiatica. Additionally, the number

0.42
2.25
1.14
1.86

of Pseudomonas, Nitrosomonas, and Nitrospira decreased, showing

bacterial community improvement and the establishment of the
anammox process.
All the bacteria identified have been identified during the
N-NO-3
Lyophilized þ Glycerol

nitrification or denitrification processes. The presence of different

0.37
2.79
1.79
1.99

species is frequently observed in anammox reactors. Candidatus

Jettenia asiatica has been identified in wastewater treatment
systems (Quan et al., 2008) and in a laboratory reactor inoculated
(80  C)

with an abandoned lagoon treating swine wastewater (Viancelli

N-NO-2

et al., 2011). Pseudomonas are denitrifying bacteria that, under

0.54
1.59
1.42
1.53

anoxic conditions, use nitrate as a final electron acceptor, and they

have been found on SBR reactors converting NO3 to N2 (Merzouki
et al., 1999). Thermononas (belonging to g-Proteobacteria) is a
genus of denitrifiers that was isolated from treatment systems
N-NO-3

0.55
3.40
2.55
1.51

(Mergaert et al., 2003; Karanasios et al., 2010) by performing

skimmed cow milk

denitrification at an optimum temperature between 28  C and

35  C (Mergaert et al., 2003). Nitrosomonas and Nitrospira partic-
Lyophilized þ

ipate in the nitrification process by converting nitrate or nitrite

(20  C)

through ammonium oxidation. These bacteria have been detected

N-NO-2

0.76
2.77
2.31
1.27

in anammox granular sludge (Quan et al., 2008), suggesting that a

small oxygen gradient in the granular sludge increases the
amount of nitrite available for anammox bacteria (Hu et al., 2012).
The occurrence of partial nitrification and the anammox process
N-NO-3

in the same reactor has been studied (Abma et al., 2010; Chini
0.43
1.23
0.98
1.16
skimmed cow milk

et al., 2016), with the advantage that single reactors are compact
because of high granule concentration. However, the disadvantage
Lyophilized þ

is that the reactors require high control to achieve stable simul-

(80  C)

taneous nitrification and anammox processes (Capodaglio et al.,

N-NO-2

2016).
0.12
0.90
0.56
1.22

The biomass that was preserved using skimmed cow milk

at 80  C was composed of Candidatus Jettenia asiatica, Thermo-
nonas, Diaphorobacter (capable of performing simultaneous
nitrification and denitrification under aerobic conditions), and a
N-NO-3

low number of Candidatus Anammoxoglobus propionicus imme-

0.08
0.08
0.22
0.36

diately after thawing. After 90 days, the dominant bacterium in

ASWP ¼ anammox sludge without preservation.

the reactor was Candidatus Anammoxoglobus propionicus.

Candidatus Anammoxoglobus propionicus was described by
Kartal et al. (2007) in an enriched culture medium, by oxidizing
N-NO-2
ASWP

0.47
0.48
0.75
1.15

propionate, acetate, and formate with nitrate as a final electron

accepter. These bacteria were the first anammox bacteria with a
defined niche. The oxidation of propionate and ammonium in a
mixed culture leads to competitive behavior with anammox and
Reactivation time (d)

other denitrifying heterotrophic bacteria by the oxidation of

propionate in the presence of ammonium, nitrite, and nitrate.
ND ¼ not detected.

After 4 months of Candidatus Anammoxoglobus propionicus cul-

ture, according to Kartal et al. (2007), other anammox species
65e106

were eliminated from the reactor, as was observed in the present

29e45
46e65
Table 3

0e28

study.
In the reactor with active anammox sludge, we observed the
 A. Viancelli et al. / Chemosphere 186 (2017) 453e458
Fig. 1. Relative abundance of bacterial 16S rDNA genes found in the biomass from different bioreactors. ASWP ¼ Anammox sludge without preservation; SCM ¼ skimmed cow milk.
presence of Diaphobacter, Pseudomonas, and Candidatus Jettenia
asiatica, and after 90 days, we observed only anammox-related
microorganisms, including Candidatus Jettenia asiatica, Plancto-
mycete, and Candidatus Brocadia anammoxidans, with the latter as
the predominant species. These bacteria were the first anammox
bacteria described (Strous et al., 1999) in wastewater treatment
systems.
3.3. Anammox bacteria community structure
In the biomass that was preserved with glycerol preservation
at 80  C, we observed Candidatus Jettenia asiatica predominance,
with less than 10% Candidatus Anammoxoglobus propionicus
(Fig. 2). However, in the community preserved using skimmed cow
milk at 80  C, we observed an inversion in the relative abundance
of two anammox species. Immediately after thawing, Candidatus
Jettenia asiatica was predominant; however, after 90 days the pre-
dominant species was Candidatus Anammoxoglobus propionicus.
This result could be explained on the basis of studies by Heylen
et al. (2012) and Ali and Okabe (2015), who affirmed that preser-
vation efficacy (activity recovery after thawing) could be species
dependent. Considering this, the preservation with glycerol was
successful for Candidatus Jettenia asiatica. However, skimmed cow
milk favored the selection of Candidatus Anammoxoglobus
propionicus.
Changes observed in the anammox sludge could be due to the
species Candidatus Brocadia anammoxidans, which can eliminate
other anammox species when in co-culture (Kartal et al., 2007).
Fig. 2. Relative abundance of anammox-related bacterial 16S rDNA genes found in the biomass from the different bioreactors. ASWP ¼ Anammox sludge without preservation;
SCM ¼ skimmed cow milk.
457
Rothrock et al. (2011) found that it was possible to successfully
reactivate anammox activity and populations after a storage period
of 4 months at an ultra-low freezing temperature of 200  C in
liquid nitrogen followed by lyophilization. Li and Gu (2013) re-
ported that the regular addition of nutrients is desirable for the
long-term storage of anammox sludge at a room temperature of
15 ± 2  C as opposed to starvation.
The present study corroborates all studies that describe anam-
mox preservation and highlights that before deciding to preserve
anammox biomass, it is essential to know the species present in the
reactor because some species can naturally eliminate others and
because different species may be favored by the preservative agent
used. If an inappropriate preservative is chosen, the entire com-
munity could be lost.
4. Conclusion
The present study described the successful preservation and
reactivation of bacteria with anammox activity, namely Candidatus
Anammoxoglobus propionicus and Candidatus Jettenia asiatica. The
success of anammox reactivation is strain dependent, and a con-
sortia contaning Candidatus Anammoxoglobus propionicus was
best reactivated when preserved using skimmed cow milk and
stored at 80  C. A consortia contaning Candidatus Jettenia asiatica
was best reactivated when preserved using glycerol and stored
at 80  C. All the results obtained can be used as a guide for the
future preservation of anammox species in culture collections,
where it is strongly recommended to characterize the bacterial
458 A. Viancelli et al. / Chemosphere 186 (2017) 453e458

community before choosing an appropriate preservation method. Kartal, B., Rattray, J., van Niftrik, L.A., van de Vossenberg, J., Schmid, M.C., Webb, R.I.,
Strous, M., 2007. Candidatus “Anammoxoglobus propionicus” a new propionate
oxidizing species of anaerobic ammonium oxidizing bacteria. Syst. Appl.
Notes Microbiol. 30 (1), 39e49.
Kunz, A., Vanotti, M., Szogi, A., Garcia, M.C., Neto, G.F.S., Soares, H.M., 2007.
The authors declare no competing financial or personal Development of Anammox Process for Animal Waste Treatment: Experiences in
Brazil.
interests. Kunz, A., Steinmetz, R., Damasceno, S., Coldebela, A., 2012. Nitrogen removal from
swine wastewater by combining treated effluent with raw manure. Sci. Agric.
Acknowledgments 69 (6), 352e356.
Li, M., Gu, J.D., 2013. Community structure and transcript responses of anammox
bacteria, AOA, and AOB in mangrove sediment microcosms amended with
sFert Network
This work was financially supported by the Bioga ammonium and nitrite. Appl. Microbiol. Biotechnol. 97 (22), 9859e9874.

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