Methane emission from hypersaline microbial mats: Lack of aerobic methane oxidation activity

Methane emission from hypersaline microbial mats: Lack of aerobic methane oxidation activity

Abstract Methane emission was measured in intact cores of microbial mats taken from hypersalinc Solar Lake (Sinai) and from salterns of the city of Ei...

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Abstract Methane emission was measured in intact cores of microbial mats taken from hypersalinc Solar Lake (Sinai) and from salterns of the city of Eilat at salinities of 9% and 1396, respectively. The CH, emission rates were t1.4-2.2 nmol cm 2 h I irrespectively of the incubation conditions, i.e. incubation in the light versus dark, with air versus argon headspace. CH, emission rates did not increase under anaerobic conditions presence of potential

inhibitors

oxidation.

indicating

that the CH,

production

and resulted in zero emission. Aerobic

in uptake of CH,.

flux

of CH,

Attempts

was not affected

in the dark. The rate of CM, emission also did not increase in the

i.e. acetylene by CH,

( I H%), methyl fluoride ( t ..El%J), or dimcthyl elher ( 5 lil%)

oxidation.

incubation

However,

to obtain enrichment cultures of methanotrophic

failed. Measurement

of 0,

microprofiles

least 05 mm depth.

In the light, 0,

addition

of 20% acetylene

inhibited

CHJ

of mat pieces in the presence of 0.1 to 10% CH, did not result bacteria from the microbial

mats at 9% salinity

using ;1p&rugraphic 0, electrode showed that 0, was available in darkness to at

was produced

by oxygenic

depth, and penetrated to at least 2.5 mm depth. Measurement

photosynthesis,

of CH,

microprofiles

reached supersaturation

at about I.5 mm

using a gas diffusion

probe showed that

CH, concentrations increased linearly from the surface down to > 20 mm depth. The CH, flux calculated from the CH, gradient was the same as the ilux that was acrualiy measured. All these experiments indicate that hypersaiine micrubiai mats contain

no aerobic

CH,

oxidation

activity.

and probably

no methanotrophic

bacteria,

although

both 0,

and CH,

are

available. Kc~_ww~!.s:Solnr Lake; Methane

production:

Methane

emission;

1. Introduction Wetlands are important sources in the global methane cycle [I]. The source strerrgth of wetlands would be even higher, if part of the produced CH,

Corresponding rtuthor. Tel: (Oh321~ 2X 70.53; Fax ~OhK!l) lh117t.l; E-mail: crlnradlrl mailur.uni-marburp.Jc l

Inhibitor:

was

Methane gradient;

not oxidized

[z].

Oxygen gradient

the Cl-l, produced in the deeper layers of the wetland :+ediments are oxidized during its plassage through the oxic surface layers by methanotruphic bacteria. The mcthanotrophic bacteria require b,uth CM, and 0: for activity and growth [3,4]. Especially the avail&& ity of O,, e.g. produced by benthic algae, can in ence the magnitude of CH, oxidation and thus. control

the flux

of CH,

More

than 80%

of

inY0 the atmosphere

[5]. The

contribution of CH, oxidation in the oxic surface layers to the CH, flux has been quantified for various freshwater wetlands, e.g. deep lake sediments [6,7], Littoral sediments [B-l 11, and rice fields [ 14. Comparable measurements are lacking for marine and hypersaline environments. Hypersaline wetlands contain methanogenic bacteria and exhibit CH, production. Methane is usually formed from methylamines which are derived from glycine betaine, a common osmolyte in halophilic organisms [ 131, Hypersaline wetlands promote the development of microbial mats [14,1S], A wellstudied example is the microbial mat system in Solar Egypt), where the presence of Lake (Sinai, methanogenic bacteria [16] and CH, production (Geriing, Faber, Cohen, unpublished) have been demonstrated. Primary production in these mats is dominated by the cyanobacterium Microcolc~s cltehorrophsres which produces 0, during photosynthesis resulting in strong die1 fluctuations of the 0, concentration in the upper 3 mm layer of the mat [17,18]. Hence, we may assume that both CH, and 0, are available in the surface layers of the hypersaline microbial mats, which should be a suitable habitat for methanotrophic bacteria. Therefore, we measured rates of CH, emission from microbial mats of Solar Lake (Egypt), of an experimental pond (pond 2) at the Interuniversity Institute at Eilat (Israel) and of the municipal saltcrn of Eilat (Pond 202). and attempted to quantify the contribution of CH, oxidation to the CH, flux,

2. Mslerials and methods The following cyanobacterial mats were investigated: ( 1) Mats from pond 202 of the municipal saltern of EiIat having a salinity of 13.2%; the salterns are a system of ponds used for the production of sea salt and are flushed with sea water to establish increasing salinities in successive ponds. Cores of the mats were taken in April 1993, transported with about 5 cm brine on top and studied within 2 h at the biteruniversity Institute of Eilat. (2) Mats from the experimental pond 2 at the Interuniversity Institute of Eilat which had been initiated by introducing mat samples from Solar Lake

[19]; the salinity of pond 2 was 859.5%. Cores of were taken in April 1993 and studied immediately at the lnteruniversity Institute of Eilat. (3) Mats from the Solar Lake (Sinai, Egypt); large sheets of the mat (40 X 50 X 20 cm) were taken in November 1993, transported in brine-filled tanks to the Interuniversity Institute of Eilat (within 3 h) and stored in pond 2 (8.5% salinity). Within the next 5 days, cores were taken from these mats and studied immediately. Sets of cores were again taken from Solar Lake in March and May 1994, transported with brine on top to Germany (within 2-3 days) and studied in our laboratory during the next 3 days. Cores of about 10-15 cm length and 4-5 cm diameter were taken from the mats using plastic corers. The cores were closed with a rubber stopper at the bottom and covered with brine from the pond on the top for the transport. For measurement of the CH, flux, the brine was drained off leaving only a shallow (about 1 mm) deep water layer on top. Then, the core was closed with a rubber stopper and flushed with either ambient air (aerobic incubation) or argon (anaerobic incubation) using hypodermic needles. The accumulation of CH, in the headspace of the core was followed with time as described earlier [ 121. The cores were incubated under different conditions: (11 in the climatic room at a temperature of 35” C (April 1993) or 25-28” C (November 19931, either in darkness or at a light intensity of about (,O--80 pE m-’ s -- ’ using fluorescent light: (2) in pond 2 under ambient temperature and light conditions; or (3) in the laboratory at about 2528’ C illuminated with fiber light at intensities of 300-1800 PE mm’ s’. Emission rates were calculated from the linear increase of CH, in the headspace [ 121 and tested for statistical difference using the Lord test [21)]. This test is especially suitable for small numbers of replicates. Some flux experiments used mat pieces instead of cores. Cubic pieces were cut from the top 1 cm of the microbial mat and transfered into serum bottles (2.5 or 60 ml) to give a total fresh weig!lt of about X-10 g. After addition of about 3 mi brine the bottles were closed with black rubber stoppers and flushed with either air or argon to establish an aerobic or anaerobic headspace, respectively. The further processing was analogous to the cores. Some flux experiments were done in presence of the mats

potential inhibitors of aerobic C 1 oxidation. The gaseous inhibitors were injected into the headspace of the cores to give the desired concentration, followed by measurement of the CH, flux. Then additional amounts of the inhibitor were injected and the CH, flux measurement continued, and so on. Acetylene is reported to inhibit CH, oxidation at a concentration of about 1% [21,22], methyl fluoride at about 0.3% [23] and dimethylether at about 7% [23]. Vertical 0, microprofiles were measured using polarographic 0, electrodes as described earlier [ 181. The measurements were done either in Eilat or in Germany (see Results). Vertical CH, microprofiles were measured using gas diffusion probes described recently [24]. The meacurements were done in Gcrmany using a probe made of stainless steel (an improved version of the type F described by f2.5]) having a detection limit of about 1 PM CH, at a spatial resolution of about 0.6 mm. In laboratory experiments, photosynthetic active radiation (given as I_LEm-’ s ‘) at the mat surface was measured with a miniaturized quantum sensor (Type QS, Delta-T Devices Ltd, But-well, Cambridge, UK) which was mounted into the core so that it was exposed to the same light intensity as the microbial mat. Ambient light intensity (given as W m-’ ) in Eilat was measured by a solarimeter which was installed on top of the institute. 1 W m-’ is equivalent to about 4.6 ,uE m - so ’ [z]. Methane was analyzed in a gas chromatr:g:1aph (GC) with a flame ionization detector. In Ei!at, the GC was operated with a widebore capillary column (30 m length) at 40” C and had a detection limit of about 2 ppmv (using 200 ~1 gas samples). In Germany, the GC was operated with a packed column (Porapak Q, SO-100 mesh) at 40” C dnd had a detection limit of < 50 ppbv (using 1 ml gas samples). The linear slope of vertical CH, gradient (X/3x) in the microbial mat was used to calculate the CH, flux (J) by applying Fick’s law: J = D,&( AC/Ax) with 4 = porosity of the microbial mat; and Ds = diffusion coefficient of CH, in the microbial mat. The value of D, was approximated from the molecular diffusion coefficient (D) of CH, at 2s” C CD = 2 X 10-” cm2 s- ‘; [27]) using D, = [email protected]’ [2S]. The

porosity of the microbial mat was determined

from the bulk density and the water content of the top 1 cm layer, and was 4 = 0.79. The Bunsen solubility coefficient of cH, ( my) in the brine solution of pond 2 (8.5% salinity) at 25” c was experimentally determined. A glass syringe (50 ml) was filled with 20 ml of a known concentration j (between IO-50%) in air (m I ) and 20 ml of The gas phase was equilibrated with the water phase by heavy hand-shaking for 1 min. Then the gas phase was removed and replaced with air. The gas and aqueous phases were again equilibrated by shaking, followed by the analysis of the CH, mixing ratio (rn:) in the gas phase. The Bunsen solubility coefficient was calculated by cr = m?/m,, and resulted in CY= 0.0195 f 0.0015 (mean 2 S.D.; II = 3). Enrichment cultures for methanotrophic bacteria were set up analogously to the enumeration procedures in soil described previously [29]. However, the salinity of the medium was adjusted to 9% using NaCI.

The CH, flux (I.2 nmol cm -’ h _’‘) which was measured in a core of the microbial mat from the experimental pond 2 did not significantly increase when the incubation conditions were changed from aerobic-light to anaerobic-dark conditions indicating that CH, oxidation played no role for the emission of CH, (Fig. 1). Measurement of 0, microprofiles in the light (200-500 PE m-’ SC’) showed an increase of 0, from 250 PM at the surface to SO0 PM at 1 mm depth and a decrease to zero O2 at 3 mm depth. Microprofiles of 0, under dark conditions with aerobic headspace showed a decrease of 0, from 250 FM at the surface to zero 0, at about 0.5 mm depth. Therefore, 0, was not limiting for Cl-l, oxidation in the surface layers of the mat under aerobic conditions. By contrast, similar experiments which were conducted with sediment cores from freshwater lakes, wetlands and paddy fields. and which are described in the literature [7-121 gave completely different results, i.e. CH, fluxes which were much smaller under aerobic than under anaerobic conditions due to oxidation of most of the cH4 within the oxic surface layer of the sediments.

to sunlight showed no effects of light and exhibited

lime [h] Fig. I. Emission of CH, from an intxt cor1: trf the microhia? mat from the cxpcrimcntal pond 2 under acrtrhic light (80 PE ml’ s-

' )

itfld

;tnacrObic

at 35’ C. The

dark

ccxlditiclns llWilSllkXi

in ;I climatic room

arrow indicates the time when the incubation

conditions wcrc changed hy flushingthe hcAp;lcc

of the core

with argon.

The experiment was repeated with the microbial mat in the municipal saltern of Eilat (pond 2021, and gave similar results as that with the mats from pond 2. The CH, fluxes were in the narrow range of 0.4 to 0.7 nmol cm -? h ’ and exhibited no trend to higher emission rates when the cores were incubated under anaerobic-dark conditia.ms versus aerobic-light conditions. The same range of fluxes (0.4 to 0.7 nmol cm “’’ h ’ ) was ob served when two different cores were measured under the same incubation conditions. Repetition of the experiment with different cores which were either incubated under aerobic-light conditions (II = 3) or anaerobic-dark conditions (I? = 5) showed emission rates (mean f SD.1 of 1.15 + 0.93 nmol cm _’ h ’ and 0.75 + 0.30 nmol cm ’ h ‘, respectively. The results of the two incubation conditions were not significantly different (P < 0.01). Measurement of 0, microprofiles in the light (500- I (100 p E m ._’ s _-‘1 showed a maximum of 0, in 1.7 mm depth. Otherwise, the results were similar to those observed in the mat of pond 2. The experiment was once more repeated with the microbial mat from Solar Lake. Again, similar rates of CH, emission (about 0.3 nmol cm ’ h ’ ? were observed irrespectively of aerobic or anaerobic incubation conditions indicating that CH, oxidation played no role (Fig. 2). Also, cores of the Solar Lake mat which were incubated outdoors under ambient conditions and were either darkened or were exposed

no diurnal rhythm (Fig. 3a). The same result was obtained when pieces of the mat were incubated in flasks with outdoor sunlight conditions (Fig. 3b). Microprofiles of 0, were measured in the mat from Solar Lake both at low light intensities (SO I_LE -2 s- ‘1 typical fOF incubation in the climatic room Fig. 4a) and at high light intensities (1000 PE m-’ s-l ) typical for sunlight conditions (Fig. 4b). Both profiles showed an increase of the 0, concentration from the surface to a maximum at 1.25 mm depth demonstrating that 0, was not limiting for CH, oxidation in the surface layers of the mat. Microprofiles of both 0, and CH, concentrations were also measured in Solar Lake cores which were sampled and shipped to the German laboratory. The same cores were used for measurement of the CH, emission rate. The 0, profile which was measured in the light (300 PE rn-” s- ‘1 showed that 0, was available in the upper 1 mm layer of the mat. The variability of different CH, microprofiles (12 = 3) is shown in Fig. 5a. The data show a good agreement with a CH, macroprofile measured by extraction of CH 1 from 11) mm slices of the mat (Fig. 5a). The shape of the profiles indicates that CH, is produced at about 10-50 mm depth and is diffusing to the surface of the mat. Measurement of CH, profiles close to the surface of the mat showed that the concentration of CH, increased linearly with depth within the upper 20 mm mat layer (Fig. 5b). Nowever. the resolution of the individual CH, profiles

aemblc. darlr -e- allaeroelc. dark -O- aembic. light -O-&~~aemlx,hght

-B-

=E

6

0 5

E 6 P .-

4

E *

2

3

0

3

6

9

12

15

18

21

time [l-i] Fig.

2

microlkl

Emission

of CH,

from different

intact cores of

the

mat from Solar Lake. Sinai. mcasurcd under different

incubation conditions in :I climatic rrlom at 2X” C (light conditions X0 1c.E m --I s- ‘I.

dimethylether resulted in a significant increase in the emission rate, indicating t not involved in the fiux of C concentrations (20%) resulted in of CH, emission, indicating th was inhibited by this concentration [SO]. Attempts were made to isolate methanotrophic bacteria from mat samples from Solar Lake. Using media that proved to be suitable for enrichment of methanotrophic bacteria from various soils and sediments, however, a salinity of 9%. gave negative results. We also tried to measure potential CH, oxidation rates by incubating mat pieces from the experimental pond 2 under air containing 750 ppmv owever. CH, did not decrease during 3 days

1

-D-a--

12:oo

aerobic. aerobic,

18:00

light dark

24:00

06:OO

12:OO

lEt:OO

time [h] 100

r -K+

80

-

60

-

1200

,

aerobic,

light

40 20

I

0

o b 12:oo

I

18:OO

24:00

06:OO

12:OO

18:OO

time [h] Fig. 3. Emission of CH, the micrc&A ccmditicrns intact

hottlcs

Cows:

mat from hg incubation (bl

containing

givr-.

from intact Solar

Lakc.

in pond

rhc incrcasc

con5 Sinai.

Ia)

2: (al gives of C‘H,

i~nd picccs (h) ot

measured

under

the CI1,

flux

in ths hcadspacc

in situ from (,I the

the milt picccs.

ctia not allow us to decide whether the linear increase started immediately at the mat surface or at 1-2 mm depth. In the latter case, we would have to assume a zone of CH, consumption at 1-2 mm depth. However, CM, oxidation can be excluded. since the CH, flux calculated (0.3 nmol cm ’ h.- ’ ) from the linear increase of CH, with depth (average of II = 5 profiles is illustrated in Fig. 5h) was the same as the flux (0.4 nmol cm -’ h- ’ 1 that was actually measured at the mat surface (Fig. 6). Furthermore. the CH, emission did not increase upon addition of 1.5% methyl fluoride (Fig. 61, an inhibitor of CH, oxidation [z]. Additional potential inhibitors of CH J oxidation were checked for their influence on CH, emission using cores of microbial mats from the municipal saltern (acetylene; Fig. 7a) and from Solar Lake (dimethylether; Fig 7b). Neither acetlyene not

1000 ~JEm,2 s.’

b 0

Fig.

4. Vcrticlrl

mats from

0,

concentratkm

S~)l;lr I;kc sampled

1200

800

400

prclfilcs

in Novcmhcr

low (a) and high (b) light inlcnsities

(ZS’C).

1600

in cwcs of microhi:rl IV9.J and mc:awrcd

at

302

12 F-‘

7 E g t

8-

5

4-

A control

6-

2-

0”

0

10

20

30

40

50

60

time [min] Fig. 0. Emission

0 10 20 30 40 50 60 70 80 CH,

nf CH,

frc,m Solar LA.

IV4

frum intact cores trf the microbial mot

Sinai. sampled in March 19’1-1 and onulyzcd

without and with the air headspacccontaining I.% light intensity of IX00 FE

400,

300

0

5

10 CH,

15

20

1

.

m ” s

I

.

C’H qF at a

’ (25‘C).

I

.

I

.

I

.

,

8 2Qa

t

1

25

[PMI

Fig. 5. Vcrticnl C’li, conccntri;Iion prdilos

20

in C’NU of micrnhinl

30

40

50

trme [h]

matsfrom Scllilr Lake. Ski. .~,rmplcd in March 1YY-I and meam ” s ’ (25” 0. (a) The squaw give individ-

surcd at 300 PE WI

values

of

11 = 5

prdilcs

down to ;I depth of h0 mm. rcgrckrn.

unillyLcd with ;I gas diffusion

prohc

b

The curve shows the hcst fit hy

The hotchcd bars give the values ohtaincd by cxtritc-

150-

tion of slices of the mat. (b) Individual values of another WI of II = 9 profiles analyzed with ;I gas diffusion pmhc down to ;I depth

7

of 3) mm. The curve shows the host fit by lincar rcgrcssion.

k

4

loo-

t

E 50 -

of incubation. Using the Bunsen solubility coefficient determined for the brine solution of pond 2, a Cl-f, mixing ratio of 7%) ppmv is equivalent to it CH, concentration of 0.6 ,X-Mthat is typical for the CH, concentration in the upper 1-2 mm surfaze layer of the microbial mat (Fig. 51. Attempts to detect potential CH, oxidation in mar pieces from Solar Lake at initial CH, mixing ratios of 0.1, 1.O. and 1W CH, also failed (Fig. 8).

dimethylether

0’

0

20

40

.



60

.



80

*



100

-



120



time [h] Fig. 7. Emission

r)f c‘it,

from intact torch cJ lhc microbial math

from (a) the municipal pcrnd702 incuhatcd acrobirally in the light IHO pilE m- : s- ‘: 3.5”Cl with increasing

concentrations

of acoty-

Icnc: and (h) Solar Lake. Sinai. incubated ;uxohically in the light (till

PE

m --’ s-

dimcthvkthcr

‘-. _ W Cl with increasing conccntraticlns

of

We report emission rates of C 1 from two different hypersaline (8.5%-- 13.2% saiini the Gulf of Aquaba (Red Sea). The in the order of 1-5 mg m -’ d ~ ’ . Similar values were measured in hypersaline mats of Lake Sivash which exhibits ;n even higher salinity (30%) [31]. The values from the hypersaline environments are comparable to those measured in marine salt marshes at much lower salinities C!--Y%j, but are lower than those measured in brackish salt marshes at salinities < 2% [Xl. Obviously, halophilic methanogens were active in the hypersalinc microbial mats. This obscrvation is in agreement with reports of CH, production in sediments of hypersalinc lakes and marine basins [ 131. Our studies arc the first comprehensive expcriments demonstrating that the CH, flux from the hypersaline microbial mats was not affected by CM, oxidation in the oxic surface layers. Measurements of CH, fluxes under aerobic versus anaerobic incubation conditions, of CH, fluxes in the presence and absence of inhibitors of C J oxidation, and of vcrtical CH, microprofiles did not give any indication of CH, oxidation. Attempts to isolate aerobic methanatrophic bacteria from the hypersaline microbial mats failed. Therefore, we conclude that methanotrophic bacteria arc not present in the mats, although both

-O-

wlh

mat

pmcas

f 10 i T

I

L P

=

1

q-a-o-u-o

-u-0-0

U-U

O,l



0

I

20



40



60

I

80

o

fOO

time [h] Fig. X. Lack of pr~icnIisl C-H, c~xidation mcusurcd hy incubation of picccs from the microbial mat of Solar LAX. Sinai. acrt~hicail~ in rhc [email protected] tirws.

(C;O,uE WI

’ s ‘: 25” Cl 31diffcrcnt CA, conccntru-

CH, and 0, are available as substrates. lln the cyanobacterial mats of ersali9e Lake Sivash consumption activity tly was also abse;nt In fact, extremely lit methanotrophic bacteria have SO far not been described in the literature [41. The only halophilic species described so far were isolated from sea water and exhibit a salinity range of only 0.M to < 3.2% [33-353. In marine systems mcthanotrophic bacteria have indeed regularly been observed and counted [36-391. The role of aerobic CH, oxidation for the flux of CH, from marine sediments into the atmosphere is unclear. Experiments with the methqnotrophic inhibitor methylfluoridc were inconclusive [23]. One of the rexuns why aerobic CH, oxidation has so filr not been quantified in marine sys the observation that the zone of C only found in deeper (tens of centimeters) sediment layers below the zone of sulfate redluction and that the shape of the CH, profiles, as well as isotopic measurements. indicate that CM, is consumed within the anoxic zone of the sediment [39-41]. The CH, oxidation activity that was found in the oxic surface layers of a marine sediment were insignificant compared to those in the deeper anoxic zone [41]. Thcrcfore, the flux of CH, from marine sediments is prcscntly considered to b affected by anaerobic rather than by aerobic C I oxidation. However, more research is needed to learn about the relative role of aerobic versus aniicrobic CH, oxidation in marine sediments. here is evidence for the existence of anaerobic C j oxidation in the sediment and water of hypersaline alkaline lakes such as Big Soda Lake (Nevada) and Mono kake (California) 142,431. Aerobic CH, oxidation activity, on the other hand, was almost negligible, and inhibition studies were inconclusive [43], suggesting that aerobic methanatrophic bacteria were absent or inactive in these hypersaline lakes. This is similar to our observatrons in the hypersaline microbial mats. Hcwever, the vertical CH, profiles that we measured in the mats of Solar Lake (Fig. 5) also give no indication of anaerobic CH, oxidation. However, we do not want to completely rule out the existence of anaerdk CH, oxidation in hypersaline microbial mats, since our experiments were only designed fo test for uerobit CH, oxidalion.

Roscnbcrg. E..

Acknowledgements We thank Dr. M. Bender for conducting the enrichment cultures for aerobic methanotrophic bacteria and Dr. A. Fish for measuring some of the 0, microprofiles. We thank Professor K. Naguib, NRC, Cairo, for her help at the Solar Lake, Sinai. This research was sponsored in part by grants from the German BMFT and of the Israelian MOST.

Eds.). pp.

IXO-1%). American Society for

Micrahic~logy. Washington D.C. 1141Cohen. Y. and Rracnbcrg, E. (IYXY) Microbial Mats. Physiological Ecology nf Bcnthic Microbial Communities. Amaican Society for Microbiology, Washington D.C.

[ISI

Stal, L.J. and Caumettc, P. (1994) Microbial Mats. Structure, Dcvelopmcnt

and Environmental

Significance.

Springer,

Berlin.

[lfd Giani.

D.. Giani. L., Cohen. Y. and Krumhcin, W.E.

Mcthanogcncsis in Ihe hyporsalino S&r

t lYX4)

Lake (Sinai). FEMS

Microhicrl. Lctt. 2.5. 2 IY-224.

[I71 Jocrgcnscn. B.B.. Rcvhbcch, N.P.. Bhckburn, ‘T.H. and Cohen. Y.

( lY7Y) Diurnal cycle of oxygen and hulfidc microgru-

dicnts and microbial photosynthesis in a cyanc?hactcriidmat

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