ee (yin 2aanioeysct og Copytight © 4095, Aner Sasol for Mievabiology 5 ‘ Vol oh, Nook Activily of Synchronized Cells of a Steady-State Biofilm Recirculated Reactor during Xeriobiotic Biodegradation MIGUEL A, ASCON-CADRERA,"* DANIEL THOMAS. ano JEAN-MICHEL LEBEAULT! Prvision des Poceis Bowe lunogiques, Département Genie Chinigus ard Labunauin de Technologie Cneymatiqne URA 1482 du Contre National de lo Recherche Setenifique, Lapuronen Cong Biologique™® Cente le Recherches dle Reyallcn, Universe Technologie dle Compiégne, 60206 Compityne Cés, Prince Received 5 Decensher 19% Accepted 1 Decembi 1994 slow fined-be reactor, as # consexetice of the 2 repre celh-physioloys phenomenon of steady-state hit) det % the bivdgrastatin hay Pacudomonas cells, wns detersined, Alter ell lesion Be ue nus formed bu n packed-bed recircuatel reactor, Alter tie stewlycetate f= 80 btn wie veachod theme of the interfacial cell deteimeit (at th FE Aleterivined. IL as established that (te hyde = (ean $0 to 807) ns the difution rateince ed cells daughter cells) rem rt 552) ly detaelved suspended cells shane ee Rewer Aletached cells grew synchro Iydrophobiciy dilterence of hi is probable that Hand at obilized sessile nud “pendent of shea s evowtl 4-Atser jnitial adhesion Rikia process of colo East evnutator pon nae ). In a continuous system with | s er than the maxinal.growth rate of the population), te pltguression of biofilm accumulation frequently takes the foray Jf sigmoidal curve, which ean be di Wo three phases: (i) {ial events (initial adhesion). Hor log acgurn x lation (sucface colonization), aud (i) plateau or steady-state S biofilm formation (9) ‘The steady-state biolilm is Uefined as a special ease in which te gains in biofiim mass (due to new cell growth) are ju ‘balanced by the loses in bfofilny mass (mainly from respira lecay and shearing to the liquid) (3, 22, 24). Thus, a steady. Slate biotin has a constant amount of ative biomass per unit of surface: area. ‘The overall aceunvukition of a stewdy-state biofilm in a reactor is determined by the average growth rave of Ue biofilm cells annus the rate of detache of cells fray the biofilm. ‘The biol can be remved by sloughing, abrasion, or predator grazing, but principally it is removed. by erosion, which is defined ais the removal of individual cells oF sna roups of cells from the susface of the biofilm as the result of ‘sheat forces exerted by moving fluid in contact with the biol Surface. (14, 20, 25). The varlables eonsidered in biofilm de ‘chntent include ateal biomass density, biol thickiess, i, wk! shear Stes, substrate wtliatic fat, yek eeicent, un ice followed by the 5 Ciena ata baie: Dene acts iscsi Gee Chine Ce ue Reka Se Koplen, ob, Vane oe hedwieg ae Caen Sinn Cones ls Tas {Trea wes: Depart of Nibley Wengen Agi ‘cultural University, 676: Wageningen, The Netheruadee a ie of the biofilm cells minus-the 1 the begimving af the eel suspended ielaehed ects peri EFe8S (ale 0 rec eaated How, the ssucheva fe the iain Factars pv which the overall ace ted the cell detachment ei in is detee fe of detuclustent of eels ren the Specific growth rate (8, 14, 20, 25), However, the last facta wich apparently best represents the plisiotoyieal state ol celle (6,15, 17), has been poorly studied inthe steady-state biohin W has been demonstrated that sarin iow fn the plsiological aetviy of cel causes cates nthe cell surfade bparcehne Lic which infuenees the iti elfadheson orceen te a, 17, 18, 28). Also, hydopobicy dilerences, i eal Sk d growth of sessile sand suspended cells of a sh observed (1 nized division of setivatedeshidge nvierootganisms hie beer, reported (12). However, these phenomena 6f cell synchron: zation and iydrophobicity have not been demonstrated in the "steady-state biofilm recisculited-texctors commonly used in biodegradation aud other biotechnological processes, probally because of the mistuse of cells atl levels of the teaciae a the reciseulation prot "s In fact, the ‘detichment process of a steady-state biotin could be contidered analogous to the dilation rate mostal; je, during steady’state, the qual to the average growth rate of the biofilm (6, 15, 11.22, 24). Ieis important 19 note that as.a result of detachnieut the + erage grovth rate ofthe biota cells s higher than might be infested froma direct observation of biotin accumutation ‘Thus, at high shear rates, detacluncut af cells from the bislilna surface can be aluiosi equivalent to the growth rat in. slow biothey accumulation rate (3,8, 20, 22) ‘The work reported here attempts to answer. wo. specific ‘questions: (i) isthe grow ofthe cells ofa steady-state Cisiia recirculated reactor synchronized and (i) are there hydicphio, bicity diflerences between sessile and. suspended cells of Seady-state biofilm recitculnied reactor during the depela tion of 24,6-trichloroplienol (2,4,6-TCP) at aillerent dotion che specilic detachinent rate is> Ah A995 z Yi FIG. 1. Schematic dain of continnys oe fate eftven ner engeonton, 0, B38 eceing hws SP, sung rales? Considerations of (i) hydrophobicity and synchron rowth as mechanisms of stendy-slate biotin mainies Gi) detachment and growth of the cells ns facta evaluation of a steady-state biol biodegradation are discussed, of a kinetic lm reactor during xenolviotie MATERIALS AND METHODS Siromenlons ae eiure med, Prtrimes cy tere tcctedom acheumba mie Mata 1 fou ste Antero sin ARS tedinanan sai SPL ‘a 2 preiony Key btch enh oo TAGTCD as tle sate energy source and aiferen atone Thus only ihe mistre of tt sans ws ale io eee depres fea ICP. Wen cuthntedseptey, stain SPI degra he ee kee 2EErmon{1S shin, whit sein SP2 del not epee heat [nip characteristic wet dieremate hese vats ms the paged (erence of claics alter eutute an mineral sale means mee place {sling the renobitic): After a8 ho elt. sialn SP here a es ame ole hig chi wile sirsin SP fumed tel eae io sble,shiny colonies. eer spre churns of thes ete ‘ioe saa eon were dace by Aeon Calvo or tence uf un open pore snr SIRAR, Noi, Germany (216) puted i 8 4 pce he an Fig white the phys ante peck veaio wat fea te ey SPI and sr2, 10 CEU af soppy Each [raponton (2), Then stbrae wow fe FeN a8 dtuiow rte ot nous ne Steady-state bai frat, The sno feeding medion (erveging “Tet ute) tothe so eeatlon tate aan 1 the rence cing the mere 1 pesmi cll elatzton we bt ht on na femed by contin: SIC fen mc ease co ed $title 3 Mee a a CELL ACTIVITY. OF A STEADY. Sse ed rene stem h « STATE BIOFILM REACTOR 924 TANLE 1 rameters , ed never 52 Mert Working va al reset, 20 ters Packing vol of rector, Las ters oil space va a ope Ha? lees No-of ting supyori 1s Ring support deny S117 Kyte cking dead wt ssl ANN Picking media rat a an Usable sirce anc 13 wy avo of support ate to etal val S404 i? Operational contin Subs 10 wd 60 mplner ong? he Mydraie reser tines Sih Recireatinn thn 20 nafnin pT ot reactor Th Tew wie fe the begining of ds biagkat pases fe whew = Deira a *Prcudoramer cls) "eshlihe dri he bikopet p ture} See lai and Be Ge Prudent co et has fr Tater ep ‘in. Calne comin ding at Nei orm ps fermeratnc, 0: ll 72h ant jsut igen ih cra a Tete hf womass tin dent na brome i {sho The lysate hin me cae ye the cies which emt nay fo ne itd ele "Ths hs han waevnmae ease bin fs torn ej! wagenaie bn (0; ata HFT) toc eaten 1 cso the dew npc, te ae a a cay bola was soni ty pte hile enna ‘hat cnt nny ned ghee cea Csi hyrepnabiey: For measurements of he ell hcogtotaah oa a ashrnicd promt the eee Gmmobitid ov equaled ee ae Tne es contin eee lug Now rect Lethe regnelation wasted gd ie eed fw 939 cn pn tion fate 08 rt sense the min of 200 pom. During the au § mi the eel aeee it (cu) poet the ees. he detache wih a sting tthe sages cre ne ced aft the fet Ween fra The ype or) hs, there coteced elle were nol ved sith le ode ‘Ces he we fo" he hydric we pint baer (ot 20) The eet Sentai at 208 Iatorlomleenane ‘Neier inns) Sita soncaton wren fan planktonic cette: The set were eh pain cocoon ee nl num pewre i uleson Wo droensbons (HATH ‘ecnqe (3) err ne wet ee vnlatangle mensurement and elecruphreie moti tecbagess G4) tie the BATH technique for hyophelncty mentoremens THe ss ‘he inntiied ses nd sper agin clots ste bass npated wit those withthe patie el calmed ee hea ements fel hep fh raters tnd sonsmteated (onened to drgerne Face seems hs koa {to the intensity ws des mat act the cel iy dronhebey mewereeny {an ots, ‘Te peor of each tan (5 Hdrgen prone to Aber eile peferentty mae opened cll were siete sere cenpemed hy 312) tthe bap ton the laces pies 66 409922 ASCON-GAIRERA EF AL Sa Jee We etertne by the ftw Soraonrdedr cee) omic, Ae in eae dete nate) ira oath of tends ln ects i cektted he ton af the pg en mer af eaters sue ae wer Met) The eakone woe mae nat MFC. Mte cvute stl el Wie 361 etn The proeron tied ite decries ‘oti fe seule Bain rnc: The sua concent i a comely miele ite Ff onsen eae Scribe bth ong mass comet ene ie FG 8)~ boas, = Sy =o 5,4 a fee cate oceans tthe tale 0d Km face, reper: thc ll ahare mabe ee Bik lactis mae wanes eociens onde a ek, f hi aad ttf es ‘amici Wedstenmine vee ‘he bance othe sate lation athe aia sc linedcerminey sees oe ” tate el, Vi ji bas een ae ameter he te pean betas ccerrallon} Aston state bob ee ecco biomass rate proprtonl W k Iw 4 Seateste hoi (eh ‘ustinam spcee pa te) the ‘woh detache ate 3) G1 0) at high ation tates > bliin growth tate Gn) cua weet 6 Sas, the Notte growth ene cam be expel bythe Folloruing o shite {ean e the nepended tnd biotin ataced Bons, especies stl ye ste aca for Watin scat aes A {nls dcrmined for ih ton rates cn be une fl ta han en {ace grout et of speed cel (ps eultues wih Low Sleen oc ig he folowing equate 1 im. Vhe bela solune ceupiog the teactor was ction i the mise the gai median athe ‘procedure. The tac ns stopped danny m, Aller the teacie wae empire tugeed anal collection of dropicc of kdl medion ote kee wate colecid dating the lt sep The mele a eel a fii LATO ya cated wit a matin dae eee hetsen fapicten Oy mei nue empsngprccede it ook ‘Samed itt the esd dnwant of bd medion woes er {alco nthe amount of water wth ine cette ihe heroes dg Votive io delenit i dry Loni of he Loti the weg of the exe bs srl hon ps support pais Bf dileveat ventas one) a ee the ape of elena supp pa ‘monte tomes ‘cone ia a enone the colegio ul we delecsined by record he UV serie ‘leure fetes (032 pm povesie Mer) Ie antic ene nena ly HAV tenes me, Arr. Es ne. shen Jne.ae ond are wears per Wer a lat vate es aie spetiontoioncice (Zeke, Ofetbochen fa a SH Cnc plete ccd or mem ae Feunay). Aw sboance ine AIF h eae wed os recy cs Ley Ute Fane) hp eh ey RESULTS Biotii formation. After adhesion was reached, the biosthn fonmation was followed by determination of wel Wot a Lime and billm dy weight, Fine 2 shows the evolutian uy biotin formation sluring a period of & ammuths: Tie ban brows daring the firs 60 days, alter Which, it beyan to stabilize, The stezdly-state biailn wag renched oh about 2 nwunths of eoatinuous eullure. The biohin wesonate Hoi rate duving the first pein (biotin formation) was stood 3.17 day". The maximal biomass reached aller 2 moa oe illu was 0.22 we of supprnt No sighiicontdillerenee (° U5) between biomass measiitements existed after this Yn Period, Steady-state biolilm. The Uniforan steady-state biofiIm was Aenionsirated by plate counts of viable cells of both the fay mobilized biofilm cells (sessile: cells) and. suspended cells (daughter cells). When the numbers in both populations were stable, the measurements were ablated on steady stele Dist film. ‘The cell numbers of the steadystate biotin were abviee 246 X 10? aud 2.3 X10" CEU of immobilized sesgie eels and Suspended dnugiter cells respectively, por ml. No signifiont tiflerence (P > 005) between the pepalation proputtion: of the Pseudomonas susins was established (determined by cok see Materials snd Methods), indicating. that ins hal similar geuseth cates atthe biofiln level, The average of suspended cells included about 852 of eels forage ‘garegates (which were considered 10 be the prodact of hace stress), ‘The oxygen concentration values rematved coneeaay when the biafi teached stability. Steady-state iohioe tare nisintained during 6 months of culture Mydrophobicity of steady-state biolitm cel. aeecrmine the hydrophobicity of steady-state bili cele ty tates of variation in pliysilogical activity the steady stay ighim was subjected to different dilution rates (0023 te 0.22 igure 3 shows the data for stain SPL (sintag date were Filablished for strain S12). Aluhough the hydropliouleay of (ath the immobilized (biotin sesite cells) and the planktowte (cultured in chemostat) cells increased as the dilution rate In order toL gis ee Phchcly28 acon of ton re. eee cel ee daugher ceo, Seow ease Me ae” wh Ditton rate (ty * etmsorm sendyste in reader Ge Macao Nee increased, the 80%) thaw th, the hydrophobicity of suspended biofiln celle W225 maintained alost constant at abort tas tion rates, A statistical a phobic Aasks), determi Browtls le cel ljlrophahiciy data dfered cheng from those of the to a sll hydrophobicity would bes the cell ete. The hyrophiostetnn {ould be these at the end of thecel ang Suspended daughter cells eould be hoe ste In order to demonstrate th was detern Psegaierensed asthe cell cycle progressed. The See, bi # ong fonden) toa Sincontzaton af {strepbibiciy of sessile cells was higher (50 to ite planktonic ells (30 to 60%). However (daughter celts} ‘luring all dil lysis Of these data showed that the ily (P< 0.08) ‘onic cells. These results suggest th of the pl hove hypothesis, cell hydro. {ime periods of 4) show that ty with regard tq different STepealseted Iminedistely (considered the beginalon Trang lycfle) had a hydeopiwbiety of about 48% even Masks) tee tll sce (aes 9 h of culture in Eaten: the bydrophobiet ine the uniform activity of steady-state biofin celle the (of suspended daughter cells was measured aver thie Sere ales. Figure 5 shows the increase of suspended viniig cells of strain SPI du three generations. The stepwise aug. ration demonst the culture Broa TF oe cts 53 fantom of et ely mer ain he as and Meth “iminobitized sessile cells grew synchremouey om lel plot of aspen ese Wn sll Cet ee ote Hot teacog sce Nirah and Nem 3 eee et im aye rom om Brew sith a high level of civiso significant difference (P > cell cele, However with the fourth ge hrony. There wis ad 0105) ete cell numer is the syachioniztion eg to deer tation (at not shown). Se abtished for stain SP Thede resale sagen nT thesendy tf ioiln recitculsted reactor. Ainetes of the steady-state bisa renctr. The kine rameters (Table 2) wers dstetnined eating Mate biolin. ‘The specific growth rate of the a film cells was obtsined from the eell how i diferent dilution rates: As shows concenteation of a mised euliare and the residual Sub remained constant during the ist f ni ates Cell concentration decreased aid the resi cutee eS feMtation Increased abruptly starting with scdlgting sake = O12 W". Therstore, this vive was commons Specific grovih rate of the stealy-stiie hey eo he maximum specific substrate uptake f biolta Gta) calculated at the bit sah eoncentratian (ie, aD 12 I ltd. of biomass. The volume ization rat biofilm reactor reached a maximusitwstue (Ore) of adeat oe ng al culture processes, the onygen concent I Was above 6.2 = U.S mpi st that the kinetics ofa steady-state {actor used for xenobiotic bode evaluated as a chemosta, using in Materials andl Meth ( owt rate fn st iform stextly- ch h predict the role played hy auy-state biofilm ivainienance), DIscussION, I hs always been thought that the steady-state bof i the bret of te sie bance of thee ee th tment aud detachinent on a sutlace, pialy due to () wears strate coneentralion, ate (8,20, 24,25). This few studies have nportance of celliphysiologicalelfects on he stendy-state biofilm (1,18). However, in ti 1 Pseudomonas mised culture ae to deste wwe show thik the cell hitante of « steasectars Dofiim eacine dues not depend:solely on the aboeaer tioned factors but mainly on Hattnsic cellular pthentonen se stress (6, 8, 10, 14), (Hl) limitation of & ne (i cell grow chading-() the steps in the cell ele (teats La) aa ai niyanogeo sae tt ei ih ur tesute show tht the steady-state Moin has two do/ A - soreness . Mrebiooe 98 ASCON-CABRERA ‘EE AL ‘CaP. 3913 ‘Arr Hinson, Mic gt am 151 = 10.0 mpliter 7 ¥ 11.25 = O02 mg of biomassimg of subitte ic residence time ory But peel pro mee Orit . 049 20103 iny’ofsubstrateining of biomass é 160 #42 aig of substrate aco [nhs 1 GF Uhl waa wo mg Co yi pe “Parmeter tt cid mani ml iy “ ie (Om O12 Wy. edie fon tod Cexgaton 3 " + tinue Ms DAS, 4) (exnation 2). portant aspects: (i) cell synchronized growth (Fig. 5) and (il) polymers segre the diteremt hydrophobicities of sessile (immobilized) aughter (suspended) cells (Fig. 3 and 4). On the one by blization of a xenobiotic compound agsole eathon and energy although we have only detérminted the sjuchronized prow source, snl (i) vaziation of cell physiological netivity at dillee Suspended caughiter cells, that sessile parent cells also grew ent lation rates (3, 13, 21); Caaten etal (Lo) have deg synchronously is j € eapacity of the immobilized * strated thal prolonged exposure of Pseudomonas cells te oe cells to develop synchronized growth hts already been dem- nobiatic eompourele cell niembrane ted! on the cell wall (1, 7), the preceding (i) pothesis would be favored by two imporiant piten rue onstrated (1, 12, 15). Is probable that synchronization is the ultasteuchare need oh lar inclusions, puoduct of a stable plysiblogical state of cells (je. simulta: Preusting et voustrated tthe eell protein neously oveurring e the cell eycle, such as cell division, nt Of Pariulomonas. cells inevewses by alt 50% (which DIA replication, structural changes esses [12)) indeed ry ‘hanes in nuttient flow, oxygen depleti ctabolic po: could Hot be excreted) when Ure cells prow quickly changes (x. (i... at high liltion rates) om rcubioties. Therelore, these ration [12, results indicate that independent of theshearstress ellects and. 15), Thus, while-the syncliron ined in immo: whether pure or mixed culture was used, the tenlyrciete pacity is lost inthe suspended daughter cells biofilm degrading xenobiotic connpounds could be the prodiet he other banc, the dillerence between the of eell synchronized prowth, whieh can be medioted teehee fharoplubicites of sessile and daughter cells could be ex dlfercnce: in hydropihabicies of both sesile (immobilized) plained by the vatiation in byclophobicity during the cell eyele und daughter (suspended celts, (1,615, 19), Cells atthe beginning of the cell eyele (daughter Our results suggest (ns shown in Fig."7) that a cell ole Cells) showed lower lyevaphobieity than cels at the end of the . containing two pliases, the free (suspensted) cell ple (os etl Gall Dele (sessile cells) (Fig. 3). Increased hydrophobicity in hydrophie piste) and the nahered eell phase (6r coll bene the sessile cells compared with the stable hyophobiciy ofthe _plubie phase), could le established inthe aenobiieegeat daughter cells as the dilution tereases suggesis.that the ing biofilm bacteria, ‘The five cell pltise Would contain (i) ; cell ndophoblety at the Leglnning of the cell cyele is iow even initially, free cells charactytized by the synchronised pee 3 high dilution rates, while the sell hydrophobicity at the end and (i) tinaly. free eells without snchuonized wowite tlhe Frere epee may be high al low diluion rates (1, 15}. adhered cel phase would contain (i) the adhered cells prowl Moreover as cell ydfophobiciy i the result f the general (colonizing « surface) nonsynclwonousy, and (i) thesiend, composition of the-cell wall and not solely of certain organiz state biofii. cells, growing, synclvonouly. “Therefore, i ie probable Ji similar phases could occur in eel cycles of othce g = . £ SUSPENDED CELLS. i jw § . sf symone 3] ee ee f CELL, i CYCLE ea "ed oly als ago oa A Sioa ‘ttutign rate (ety Sraclsentand biotite FIG 6, Kinetics ofthe sendy site bioflun reactor ducing 2.4,6-1CP degrara eee A. tO bee ahs coy, resid satstote concern steralsgd ee: epoerecaly by montriit day) Samper weve cashed hom the F* #1 PoahTBl cee of cele roving sn senobioc aroun ules in ‘onnilrm steadysite bili seater te Mitesh a ‘conteuoun syste, fie ells |. te cll"Saag — eee ye : ‘on 0461, 1555 CELL ACTIVITY OF A SIFADY-SEATE HOFMAP REACTOR: 36 Fee tenting ert Sh ey. gom around sues ese at, ar nina ads Hoth a constant dae centration i the reactor effluent up toa crilcal dilution ate 0 Count ua eerwest dynes pore am (0.12 hand thereafter the decrease of cell densly and the ns Mecha op aes og increase of substrate concentration (Fig. 6), suggest that ant 11 Weert. 17. Spica mon ape ne: Stace, ree, feRcoe his. strong snarky toa chemo.” masta htt mith ion Fae se {ua culture. Therefore, the critica flaw rate might correspond St ~Weachevemeh of maxinum specife growth rate or manimum —* {8.1 Rh imcchankont a Higa Mee Nea esa priate red eect! 7 Annis eh specie substrate utzon (Table 2, The samc eorekene ii et : ting ben peed by exer sutbors wh have used sendy.» Chin 0m Man measecn Manin we cern rence crm i aremenmt CD) ge Mk ae tt Som fectcaed teacir rable 2) could heebinnc ty ena (ip mrdcre ine Sbstae tllztion rte (eqation 3) and. Abia Chae ie, i ca Ca ea rnemt process of » steady-state biofin reactor" Cheb Ff Chane an ap Re 20a Sauivalent to the average growth rate ofthe biofilm (equations 11. ati et 3 and 4), Frsigict Padrnon sri ub rsh paone I ieee thet although iis ict to establish the ~ aug npc mc) hm. An. Envhon, Mii Sea netabicy andthe synchronized cell growth ina ree, SEA thine 9 Lmrrpsnad 8. Mec te Hn acacia Feactor (dve to the mixing of cells im the '* pact Az: Eycabna Jn ane “ice te biofilm reactor); %t is clear that in a steady-st film recir- ‘Tesponse 10 wutrient steess. Mier culaed reactor used for senohiotie hodseradalion tre ceig at a i ee al ah a grow synchronously and this phenomenon is mediated by the Grp te ate of eater ne bat Lilerenthydophobiciies of inmbilzed sessile cellgaid ome, q A619 degra acco ended daughter eels Thus, theve phenomena cout bese. ct al tend te inn ene te sponsible for the cell growth-cel detachment Galante ina sien a 17, Atal 1988, ates he nu spe erste Seagate (loin reactor. Therefore, the kinetics of m1: Mah tet Peers steady-state biofilm recirculated reactor could be evaluated fac Similatly to those of a chemostat Het Dae uly a A Migr tcl Now a1 6 ACKNOWLEDGMENTS urge. G-A LW: LC. Aches ML, Shale Everson and W. We thank J. M. Engtsser of the ENSAIA, tnsitt National Poty fa ance ston pwd oan Roe hie a technique ge karaine, Vandeusteles-Naney Cedex, France, aad Fonte 14; G Chae W929 til aa of he eet she stress the Lina itn ng HET ig J. Rlngnny and, Mel 191, Pysokoy at psjster, foxmation ol Fatomnes nine cnt ts dh nine Met Tec 17M 2 22 Rion Hy aa Pi AMeCarg 1980 Exelon Wf sey atten Copdevil ofthe URT, Institut National es Sciences Apples de Toulouse, Toulouse Cédes. France for helpful suggestions an forthe + | Gitcal reading of the manuscript. Ako, thant sre gett to. Wh Asc Reyes for her continuous interest mind enlightening eric 2 MhcouRhot this work neil support Was provided by Université de Technologie de SgupBencand Dg XI EVWACT Saunt Trom the Eoropean Com 2 man al arenas imisioe 2, semberg, Ml 198i Bae ulerence seta ec * sae or studng cll artic Iden TEATS Afraid te REFERENCES Boao Send "fiery tates mac, 1 See 24 Send CF mE fr gon ow en aie ent ator tamer wens hom bene tid Bre PON A A ct TEMS tcl tea Meith 2 Mama a " 2 AscbeCalnera, MAL 193. PHD. thai, Unie de Teehwogle de aT ‘Citgo vty of Psenenoae emia iP Seng teas i a 4 Bak, BL van J Diners 99), acini specie dominance within Aetiea is 26 Spat Ine 92, SSA, ceseak 824 son tf ont WG, Charachls 1984, 3p ns stent. Mkaechnol Seas Eas ae . Ons”