Polarforschung St; (2i3). 93-10J, /988

1.4 Preliminary Characterization and Identifica­tion of 1984/85 Continental Antarctic Soil

Microorganisms of Linnaeus Terrace(Altitude 1600 m; McMurdo Dry Valleys)

By Clauclia Gallikowski' and Peter Hirseh*

ataddcd nitroucn

onlya lC\\- strains.

11Ull1 13 Linnacus TCITi:lCC xoilxFungi ns wcll as phototropluc

thc bacrcria wcrc cocci. short rods.

144 bactcr!a tcstcd -vcrc Most isolutcs 12I"C\\

mincral saltsmcdium 'strmns oxidcs. but oxidation or was camcd out

IOq 2S(f· 01" thc strains cleavcd ..~:;~,:::::::r~~:,~~::~~,~'i::ea~l,:,~:,~::,:;i'~';l;:;;WiIS possib!c in man)' cascs. rr0111 mcrnbcrs of thc gcncra01' Brcvihactcnutnuvcvc wcrc also Bacillus nnd Except ror a fcw strains

0]" cvcn 3TC. rnost strains appcarcd to bc indigcnous to this cnvu'onmcnt.

Antarctic sununcr wc isclatcd 119 pure cuhurcx ofRange: South Victoria Land). AmoJH~ thcsc wcrc

yicklcd up 10 .16di!Tcr~nl morphorypcs. othcrs vvcrca

Zusammenfassung: Aus 13 die im Sudsommer 1984/85 auf Lumncus Torrace (1600 11l hoch Qcl<'QCIJ: ,'\sQard SÜd VictorinLand) ccsammch wurden, Reinkulturen oligotrophe! Darunter befanden sich heterotrophe 13'akt,ri"n.Herell.

Iädigc Pille und auch Grüunlgen. Während aus einigen Bodenproben bis IU 36 Morphotypen isoliert werden ;'::~~;~:I~II~!~I;;:;'C;;~I.;S:i;C;~handere als nahezu steril. Bei den Bakterien handelte es sich um Kokken. Kurzstäbchen. Stäbchen. Vibrionen. Sporenbildner.prosthckatc Bakteriell SOWlc Viele dieser Isolaie bildeten extrazelluläre Polvmcrc. Euva die Hälfte dieser Stämmegcn aller hinsichtlich in einem miniaturisierten Testsystem Bakterien erwiesen sichals Die der bei 4'C. und 7(Y;~ von ihnen waren bei bei .30"Wachstumzeigten. wuchsen oligocnrbophil auf Mincrnlsnlzmcdium 3.37 ohne zugesetzte 4Y{ wuchsen ohne zugegebene Stickstoffquelle.Unaefä!u die Hälfte der untersuchten Stämme konnte Eisenoxide oder nur bei lsolatcnnachgewiesen werden konnten. Ein Drittel der Kulturen oder tolerierte bis zu NaCL 25(>( der

spaltetcn Na-protocatechuat nach Induktion mit In vielen r-iillell war eine vorläufige möglich: nebenwie Micrococcus. oder Brcvihoctcriunt traten auch Bacillus und spp. auf.

und gutes Wachstum bei 30' -J7'C zeigten. schienen die meistenUmwelt zu gehören

I. INTRODUCTION

Linnaeus Terrace is locatecl in the continental Antaretic McMurclo Dry Valley region ("Ross Desert"). an areawhich is one of the harshest environments on earth (VISHNIAC & MAINZER 1973). This region is praetically

clevoicl of all hig her terrestrial life forms, except for oeeasional overflights of skuas (VISHNIAC & HEMPFLlNG1979), Some authors such as HOROWlTZ et a!. (1972) consie!erecl the Dry Valleys (espeeially the soils) to be

"abiotic". But eloser investigations of some valley soils in this region (BENOlT & HALL 1970, BOYD 1967,

CAMERON 1971) showee! that nearly all samplcs investigated there containecl numerous types of microorgan­isms, some of wh ich coule! be ie!entifiee!.

Taxonomie work has been performecl on Antarctic filamentous fungi and yeasts (ATLAS et al. 1978, SUGIYAMA1969, VISHNIAC & HEMPFLING 1979), on algae (TSCHERMAK-WOESS & FRIEDMANN 1984), and on

actinomycetcs (TSYGANOV et al. 1970). Among the baeteria the taxonomie work concentratecl mainly on

Bacillus spp. and eoryneform bacteria (JOHNSON & BELLINOFF 1978, MADDEN et al. 1978), In a cletailecl

stucly of stressecl Antarctic soils from several McMurclo Dry Valley sites, GALLIKOWSKI ( 1985) and GALLl­KOWSKI & HIRSCH (1989) observecl high microbial diversity in nine samples. It was possible to isolate 86

strains. ofthese there were 68 bacteria. 3 yeasts ancl15 filamentous fungi. The question neecls to be askecl ifthese

microorganisms were incligenous to Antarctic soils 01' contaminants that arrived recently from elsewhere. They

eoulc1have been transferrecl from weathering rocks, where a rich cryptocndolithic microbiota has been observecl

Clandia Gallikowsk i and Prof. Dr. Peter Hirsch. Institut für Allgemeine Mikrobiologie. Universität Kiel. Biclogiezcntrum. Olshnuscnstraße 40/60,0-2300 Kiel. Federn! Rcpublic of Genuany

93

(FRIEDMANN 1982). They could also be airborne "eontaminants" which have simply survived in these soils.UYDESS & VISHNIAC (1976) demonstrated in situ growth of bacteria in somc Antaretie valley soils whichindieated that at least some of the viable forms were indigenous. Similarconelusions were reaehed by VISHNIAC& HEMPFLING (1979) in their ecophysiologieal studies with Antarctic yeasts tCryptococrus spp., Sporobolo­mvces spp., and Tilletiopsis spp.). Reeently, more than 200 pure cultures of soil mieroorganisms were isolatedfrom Linnaeus Terrace 1984/85 sampies (HIRSCH et al. 1985). The purpose of this present ivestigation was tocharaeterize and possibly identify several of these recent isolates in order to eventually compare them to othersoil isolates from more favourable loeations and espeeially to organisms from the cryptoendolithic eeosystem. Itcould be expeeted, indeed, that several of the organisms from soil resembled those from rocks, which wauldindicate that there does exist an indigenous soil mieroflora in these exposed loeations of the MeMurdo Dry Valleys.

2. MATERIALS AND METHODS

Linnaeus Terrace is located in the Asgard Range (South Vietoria Land; 77'63'S, 1610 OSE; 1600----1650 maltitude). The Beaeon Sandstone boulders there are particularly rich ineryptoendolithie life (FRIEDMANN 1982).In Deeember of 1984 a total of 13 soil sampies were taken aseptieally (Table I, HIRSCH et al. 1985) andtransporred to Kiel over dry ice. The samples came from seemingly undisturbed areas, as weil as from a drysurface depression which was heavily eontaminated with human urine CKidney Pond"). The sampies' pH rangedfrom 5.2 to 6.7 (Table I). We employcd scveral enrichment methods (HIRSCH ct al. 1985) in order to obtain as

Sampie

845/201202203204205213224225226227228246247

Location

.Kidney Pond", site A. 0-2 cm2-5 cm

site B. O~2 cm2~5 cm

Strcam bed of SIlOW melt waterPowder insidc of sphereNorth of bouldcr INortheast of boulclerIUnder east end of boulder rSouth of boulder IUnder west end ofboulderIUnder bouldcr III. 0-5 cm

5-7 cm

plT'

5.96.26.56.76.66.76.46.36.46.36.35.25.6

Number ofpure culturesobservcd"

t8131121101229251736

41112

total: 229

Tab. 1: Pure cultures isolatcd from 1984/85 Linnaeus Terracc soil sam pies. 1) measured in l.0 N KCl für 2 hours. 2) Pure culturcs of a givcnsample were all morphologically different.

many different pure cultures as possible. For closer description and identification we selected 184 strains ofbacteria and yeasts showing good growth. We tested these strains for a great number of morphological andphysiological properties with the microtiter plate technique, a miniaturized test system (DOrr & THOFERN1980, MOALEDJ 1984). All experiments were carried out in triplicate. The standard medium was PYGV(STALEY 1968), which contained 0.25 g/l each of peptone, yeast extract, and glucose as well as mineral saltsand some vitamines. Incubation was normally at 9T in the dark for 4 weeks. For anaerobic cultivation we sealedthe wells of the microtiter plates with paraffin (1 part solid plus 5 parts liquid) and incubated in a nitrogenatrnosphere. The tests carried out were mostly done as described by MOALEDJ (1984), except that mediumPYGV was used as the basal medium. Growth on basal inorganic medium and the test for ring cleavage ofprotocatechuic acid were as described by GERHARDT et al. (1981). Manganese oxidation was tested foraecording to SCHWEISFURTH (1972). Base composition (Mol% G+C) was determined after MANDEL &MARMUR (1968). The strains were also tested for sialidase activity. This was done aecording to POTIER et al.(1979).

3. RESULTS

From the 13 soil samples we could isolate a total of 229 different pure eultures of heterotrophie bacteria, yeasts,

94

filamentous fungi, and green algae. But the numbers and diversity of mieraorganisms varied in these samples,While enriehments from one soil yielded only four different organisms, other soil sampIes with for example 36morphotypes showed a signifieantly higher diversity (Table I).

The eell morphology of the baeterial isolates ranged from eoeei to short rods, vibrios, and rod-shaped fonns (Figs.land 2). Appendaged 01' budding baeteria were rarely found among the isolates, but aetinomyeetes resemblingGeoderniatophilus spp. were quite eommon. Many strains had club-shaped eells and showed "snapping division"and thus were simi lar to eoryneforms. Twelve strains formed endospores; one of these baeilli had short rads 01'

eoeci (Fig, 2.1). Many baeteria produeed extraeellular polymers, some fonning large aggregations surrounded byslime eapsules (Fig. 2.4). 98 of 144 baeteria tested (68.1 %) were Gram-positive.

The effeet of temperature on grawth in PYGV was tested with 146 soil baeteria and yeasts (Table 2). Nearly altstrains (86.7%) grew weIl at 9'C; there were thiek pellets in the wells of these organisms. The remaining 13.3%showed only weak growth; there were small pellets 01' slight turbidity in the wells of the test plates. While themajority of isolates grew at 4'C, a few grew better at 20'C, 59% developed well at 30'C, and about 20% grewweil even at 3TC.

Growth rcponse 4'C 9'C 20'C 25°C 30'C 37'C 43'C

Good growth") 73.7 86.7 89.6 65.2 59.0 19.3 2.7Weak growth") 11.2 13.3 3.3 11.0 10.9 16.0 9.3No growth 15.1 0 7.1 23.8 30.1 64.7 88.0

Tab. 2: Effect of tcrnperuturc on growth of 146 Antarctic soil bactcria und ycats in medium PYGV. lncubation: 4 weeks. Data cxpresscd aspcrcentagc of Iota I numbcr of strains. I) distinct turbidity and/or pellet visible,~) turbidity and/or pellet burcly visible

Growth on five different media is shown for 146 strains in Table 3. As eould be expeeted, all strains grew onPYGV, the isolation medium. A basal inorganie medium (BIM) without any added organie earbon soureessupported growth of 56.9% of the strains, As this differenee to PYGV eould have been eaused by the missingsubstrates orby a speeific effect ofthe basal salts ofBIM, we also tested BIM with the addition ofPYGV substratesexcept for Hutner's basal salts. The result was that 9.3% of the organisms were inhibited by the BIM mineralsalts,

Growth PYGv') BIM') BPYGV') NTM') NTM')reponsc +0.1S(,(NH)2S0~

good growth 86.7 37.3 85.4 n.3 6Uweak growth 13.3 19.6 5.3 33.3 19.3no growth 0 43.1 9.3 53.3 19.3

Tab 3: Effccts of various media on rhe growth of 146 selected Antarctic soil bactena and yeasts. Dara expresscd as percernage of total nurnoer ofsrrains investigared.

I) PYGV (isolation medium): peptone (0.025%). yeast extract (0.025%). glucose (0.025%), vitanun solution. basal salts; 2) BIM: basal inorganic medium:3) BPYGV: basal inorganic medium + PYGV (without Hutncr's basal salts): 4) NTM: Nitrogen tcst medium (ROSSWALL & PERSSo.:-,; 1982).

To investigate the utilization of (NH412S04 as a nitrogen source, we compared N-free test medium with andwithout ammonium sulfate (Table 3). Surprisingly, many strains (46.6%) grew without a nitrogen source (i. e.oligonitrophilically). It was unlikely that the inoculum contained much nitrogen. Dinitrogen fixation by theseisolates still remains to be tested,

Table 4 shows some biochemical properties of the test strains. Nitrate reduction under aerobic conditions wascarried out by only 17 strains (forrnation ofnitrite). All isolates had been enriched for under aerobic conditions;therefore, the high percentage of catalase-positive strains could be expected, Only 7 cultures were cytochromeoxidase-positive. While manganese oxidation was carried out by only 5 out of 151 strains (in the presence oforganic carbon), iron precipitation was a more common property. Manganese reduction was also tested with 5 g/lof glucose and 0.75 g/l of meat extract as carbon, energy and nitrogen sources. One-fifth of all strains reduced

95

96

(uncontaminatcd}. J) "Mycococcus" (,;\1\-616) Irom

Fig.2: ofsclcctcd Anlarctic sci l isolntcs.1) Bacillus sp. !A ..\-65f)) frnm g,j5/.2!MiKldneyPol1C1.4) Pink. aggrcgating Irorn mtcron.

97

manganese oxide.

Decomposition of selecteel carbohyelrates was investigateel with 90 strains of bacteria anel yeasts uneler aerobicanelanaerobic conelitions (Table 5) accoreling to HUGH & LEIFSON (1953) as moelifieelby MOALEDJ (1984).Oxielative or fermentative elecomposition (formation of aciel) were evaluateel every 2-3 elays for 4 weeks for a

Charaktcristic

CatalascCytochrome oxidascNitrate reductionMangancsc oxidationManganese reducrionIren precipitation

Number ofstrainstcstcd

154148124151150148

Positive

1357

175

3672

Negetivc

1914110714611476

Tab. 4: Seme sclected biochemikal propcrties 01' Antarctic soil bactcria and ycasts.

color change ofthe aeleleel inelicator. Aerobically, glucose, mannose or galactose utilization leelto aciel formationin only a few strains. Anaerobically, the fennentation of glucose was more wielespreael among the test strains(Table 5). All other sugars were less weil metabolizecl.

Hyelrolysis of starch was testeel with 148 cultures, of which 32% hael extracellular amylase. Other extracellularenzymes active in some strains were DNase (present in 34% of the isolates) anel sialielase, which separatesneuraminic aciel from oligosaccharieles, glycolipiels or glycopeptieles. The latter enzyme was testeel far with 103strains accareling to POTIER et a1. (1979). Among the 14 sialidase-positive organisms were four isolates fromthe urine-contaminatcd "Kidney Pond" anel six yellow, rod-shaped bacteria from various other locations.

The ability to c1eave the aromatic ring of Na-protocatechuate was testeel with 107 strains after ineluction with0.1% Na-p-hydroxybenzoate. Ortho-cleavage was founel in 8 cases, anel c1eavage in the meta-position occurreelin 16 strains.

Substrate') Aerobically Anaerobicallyacid wcak acid 110 ucid ncid weak ncid

formation formation fortnation forrnation formation

Glucose 14 19 57 57 19Mannose 18 6 66 4 6Galactose 13 5 72 11 4Ribose 5 8 77 5 2

Maltose 4 7 79 3 3Saccharose 4 2 84 5 1

Fructose 0 1 89 0 4Ncacetyl-glucosaminc 0 1 89 0 0

no acidforrnation

1480758384848690

Tab. 5: Acid formation from selected sugars by 90 Antarctic soil bacteria and yeasts. Data expressed as number of strains in each case. I) sterile­filtcred sugar solutions were added to an 0.05% cesein hydrolysate medium to a final concentration of 0.1%

Growth rcsponse 0%NaCI addition to medium PYGV!% 5% 10%

good growthweak growthno growth

86.713.3o

80.78.0

11.3

66.721.312.0

21.316.762.0

Tab. 6: Effeet of sodiurn chloride on growth of 146 Anrarctic soil bactcria and yeasts. Data expressed as percentage of total number of strains inve­stigatcd.

The tolerance of 146 strains to increasing concentrations of NaCl was also investigateel with meelium PYGV at9T (Table 6). All strains grew without NaCl; the aelelition to PYGV of 5% NaClstill alloweel growth of 88% ofthe isolates testeel. Even 10% NaCl were tolerateel by mare than one-third of all cultures.

98

4. DISCUSSION

The miniaturized test system employed he re enabled us to study many physiological properties of a large number

of strains with a relatively sm all amount of material and within a short time. For most of the strains this method

worked well, Strains which grew extremely slowly (i. e. those which needed more than two months to form visible

colonies on agar) or cultures which grew faster and spread over a larger surface area (filamentous fungi, somebacilli) could not be tested together with other (normal) strains on the same microtiter plate. Generally the

necessity of having all strains to be tested in one plate (96 cultures) at the same state of activity, caused problems.

Therefore, we often had to exclude strains entirely because they did not grow in the positive contrals, i. e. on the

PYGV medium.

Another problem was that some bacteria with extensive slime production did not suspend in liquid media butinstead formed a viscous pellet. Also, onoculation by an atuomatic needle device was not always successful withthese strains so that often only one ofthe parallels grew up. Such tests had to then be repeated. The high percentage

of Gram-positive bacteria in Antarctic soils has been described by FLINT & STOUT (1960) and by GALLI­

KOWSKI (1985). Gram-postive strains seemed to be better adapted to the dry and cold environment of the

MeMurdo Dry Valleys. Survival in a periodieally dry soil would also be facilitated by the formation of copiousamounts of extraeellular polymer, which we so frequently observed in our cultures. The thick polymer layers

could even protect the cells form an aeidie environment. In enrichments it eould be observed that other organisms

(bacteria) grew within polymer capsules. Close proximity of cells inereases the possibility of exehanging excretion

products, of utilizing local dead cell material, or of taking advantage of partially degraded organie substrates. Theextraeellular polymer function could thus be looked upon as a me ans of furthering mutual relationships between

organisms which are strongly dependent on each other in this hostile environment.

The growth response to various temperatures was somewhat unexpected: most eultures grew equally weIl at 9°C.SIEBERT (1986) and SIEBERT & HIRSCH (1988) determined the temperature optimum for 15 Antarctic coeei:

it ranged from 100 to 21T. STRAKA & STOKES (1960) found for most of their Antarctie soil bacteria isolates

a temperature optimum of about 20T. If one follows the definition ofMORITA (1985), a psychrophilic organism

should have its optimum below IYC, which was not the ease with most of our strains. Rather, these should beclassified as "psychrotrophic" sinee their growth was possible near or slightly above 20T as well as around OT.

The observed growth of28 strains (19.3%) at 3TC indicated that possibly there were human contaminants among

our strains. A few ofthese 3TC eultures actually came from the "Kidney Pond" and others were spore-formingbacilli. It is possible that those strains wh ich did not grow at 4T (15.1 %) could have been such contaminants asweIl. Alternatively, the incubation time might have been too short for these organisms.

When we tested growth with different media, we included BIM, a mineral salts basal medium without addedcarbon sourees. More than half of the Antarctic soil organisms grew on this medium, which ean be taken as an

indieation for "oligocarbophily'' (BEIJERINCK & VAN DELDEN 1903). Most likely, the carbon and energy

sourees came from impurities of the laboratory air (HIRSCH 1958, 1965). As we also observed a capability for

"oligonitrophilic" growth, it eould be assumed that within the Antarctic soil there could occur a nutrient exchange

through volatile compounds released into the soil gas phase.

In the Ross Desert the frequently changing water regime often eauses the formation of salt aeeumulations and

crusts (BEHLING 1971, CAMERON et al. 1970). Soil mieroorganisms would have to develop a certain degreeof tolerance of higher salt coneentrations. We tested 146 soil isolates for growth in the presence of various

concentrations ofNaCI (Table 6), although it was not clear ifthe salt crusts we observed on Linnaeus terrace were

sulfates or ehlorides. A total of 88% of all test strains tolerated 5% NaCI, and 21.3% still grew well in 10% NaCl.

Halotolerant coeei seem to be quite eommon in the McMurdo Dry Valley soils; there are reports by MILLER &LESCHINE (1984) and the work by SIEBERT (1986) where halotolerance was mentioned.

The diversity of earbon sources in Antarctic soils is stilllargely unknown, exept for the presence of living or dead

microbial eells. CAMERON (1972) found a wide range of organic earbon eontents in Victoria Valley soils:0.01-0.21 weight%. This 20-fold variation expressed thevast differences between adjacent locations, differences

in microclimate, in microbial contens, and henee in mierobial produetion. Such differences also were observed

99

by us when we studicd the e!ensity anel morphotypes of microorganisms in soil samples by e!irect microscopy

methods (HIRSCH & GALLIKOWSKI, unpubl.). Thc main sources for organie carbon. rhercfore, would change

from one placc to another; the microbial soil population woule! have to be diverse with respeet to utilizing speeific

cornpounds.

Sevcral soil isolates were able to hydrolyze stareh 01' degrade DNA, ane! a varicty of sugars were utilized.

Moreover, the ability to clcave aromatic ring systcms was present in the populations wc investigared. This shows

that microhial matter ean be recycled to a considerable extent. Thc occasional presence of viable green algae in

Antaretic soil samples points to the possibility of a limited primary producnon. in soil, by photosynthesis. This

point necds closer investigation.

Acyl neurannnie acids (sialic acids) oeeur in blood serum of animals, in sputum, in all animal cells. but they have

rarely been found in microorganisms, Sialidases arc produccd by a fcw microorganisms. usually by those that

live in close proximity to man 01' higher animals. SCHAUER (1975) mentions sialidascs from Clostridiumperfringens, vibrio cholerac ancl a few other mieroorganisms. Sialidase activity has also been found in a few

actinomycetes (U. MEVS, personal communication). Biologically, the effect of acyl-neuraminic aciels lies in their

strongly negative charge which results in the bineling ofmany water molecules and gives a morc slimy consistency

to the polymer. One coule! spcculate that sialidases scrve thc purpose of elegrae!ing the protcctive polymer layers

which may cover many Antarctic soil organisms. An alternative explanation would have to postulate eontamina­

tion of thc Antarctic sampling sites with microorganisms derived from animals 01' man anel wh ich were also

sialidase-posi tive.

The properties so far observed in our soil isolates, ancl supporteel by the work of SIEBERT & HIRSCH (] 988)

allow us to place many of the coccal strains with the genera Micrococcus 01' Deinococcus. Micrococcaccae were

also found in Antarctic soils by BENOIT& HALL(1970), CAMERON et al. (]97]), ancl by CAMERON (1974).

The club-shaped, Gram-positive short roels eommon in soil sampies were often ie!entifiecl as Corvnebocterium,Arthrobactcr. 01' Brevibarteriunt spp. (JOHNSON & BELLINOFF ]978, SIEBERT 1986). Corynebactcrium spp.

wcre found to dominate in many soils (CAMERON 1974). While HOROWITZ er al. (1972) anel CAMERON et

al. (1976) assumed that Bacillus spp. were not ine!igenous to the Dry Valley soils, other authors (BOYD &ROTHENBERG 1968, UYDESS & VISHNIAC 1976. MADDEN et al. 1978) have isolatcd several bacilli from

Antarctie soils. NlILLER et al. (1982) foune! that Antaretie spore-formers were well aelapteel to cold conelitions

and increasee! salt eoncentrations. In the present stuely we inelueleel 12 strains of bacilli among our isolates.

Members of the genus Pselldomollas were represented in our colleetion of pure cultures by yellow-pigmenteel,

motile anel slene!erroels whieh were Gram-negative anel eata]ase- ancl eytochrome-oxielase-positive. Pseudomonas

spp. were also elescribee! from Dry Valley areas by CAMERON et al. (1976). Further ielentification of our strains

is in progress.

5. ACKNOWLEDGEMENTS

We gratefully aeknowlee!ge the he]p ane! stimulation by 01'. E. I. Frieelmann anel the members ofthe ACME group

(Antarctie Cryptoenelolithie Mierobia] Eeosystem research group) of the Polar Desert Research Center of Floriela

State University. Stimulating elisCllssions with T. Roggentin ancl the help ofK. Moaleelj were also appreciateel. P.

Roggentin supplieel information on the sialie!ase test. Skillful teehnical assistance was rene!ereel by M. Wunsch.

This work was supportee! by grants to E. I. Friee!mann from the US National Science Founelation (Division of

Polar Program; no DPP 83-14180) and by research anel travel grants to C. A. Gallikowski anel P.Hirsch from the

Deutsche Forschungsgemeinschaft.

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