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Enhancemen o hizocompe ence in pa hogenic bac e ia emo al o
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cons uc ed we land sys em
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Ma wa BEN SAAD1, My iam BEN SAID1, Isabel SANZ SÁEZ2, Olga SANCHEZ3, Jo di
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MORATO4, La i a BOUSSELMI1 and Ahmed GHRABI1
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1Wa e Resea ches and Technologies Cen e , CERTE, BP 273 - 8020 Soliman Tunisia
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2Ins i u de Ciències del Ma (ICM-CSIC), Pg. Ma í im de la Ba celone a,08003 Ba celona, Spain
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3Depa men o Gene ics and Mic obiology Facul y o Biosciences, UAB · 08193 Bella e a Ba celona Spain
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4UNESCO Chai in Sus ainabili y, Poly echnic Uni e si y o Ca alunya, C1 Te assa, 08222, Ba celona, Spain
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*ma [email protected]
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Abs ac
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The main goal o he p esen s udy was o enhance he hizobac e ium po en ial in ho izon al
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subsu ace low cons uc ed we land sys em plan ed by Ph agmi es aus alis h ough
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en i onmen ally iendly biological app oaches. The bioinocula ion o an agonis bac e ia has
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been used o p omo e highe hizosphe e compe ence in pa hogenic bac e ia emo al. The
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expe ience was pe o med wi h once and wi h sequen ial bio-inocula ion. The esul s show
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ha he s ain PFH1 played an ac i e ole in pa hogenic bac e ia emo al. In ac , he
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indi idual bioinocula ed imp o es ema kably he inac i a ion kine ics o a pa hogenic es ed
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bac e ia; S. yphi in plan izosphe e by, 0.8 U-Log10 wi h once bio-injec ion and
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app oxima ely, 2.5 U-Log10 wi h sequen ial bio-injec ions.These esul s sugges ed ha his
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s ain ep esen s a p omising candida e o imp o e he wa e pu i ica ion by cons uc ed
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we land.
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Key wo ds: An agonism, Bioinocula ion, Cons uc ed We land, Rhizosphe e.
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In oduc ion
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Cons uc ed we lands (CWs) ha e been used as a g een echnology o ea a ious
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was ewa e s o se e al decades. They o e a land-in ensi e, low-ene gy, and less
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ope a ional- equi emen s al e na i e o con en ional ea men sys ems, especially o small
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communi ies and emo e loca ions(Gh abi e al. 2011; Shen e al. 2015; Tee e al. 2016)
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(Gh abi e al. 2011; S. Wu e al. 2015) These enginee ed sys ems a e designed o ea
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con aminan s in su ace wa e , g oundwa e o was e s eams by using na u al unc ions o
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we land ege a ion, soils and hei mic obial popula ions (Vymazal 2014). They ha e a g ea
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po en ial o he ea men o was ewa e o di e en o igin (Wang e al. 2015; Zay se e al.
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2011) such as domes ic sewage, ag icul u al was ewa e , indus ial e luen , mine d ainage,
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land ill leacha e, u ban uno , and pollu ed i e wa e (Liu e al. 2015). CWs ha e been
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success ully used o mi iga e en i onmen al pollu ion by emo ing o a wide a ie y o
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pollu an s om was ewa e such as o ganic compounds, suspended solids, pa hogens, me als,
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and nu ien s (Zhang e al. 2014). Du ing was ewa e ea men in CWs, pollu an s a e
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emo ed h ough an in eg a ed combina ion o biological, physical and chemical in e ac ions
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be ween he plan s, he subs a e and he inhe en mic obial communi y (Wang e al. 2015) .
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CWs a e ypically classi ied in o wo ypes acco ding o he we land hyd ology: ee wa e
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su ace (FWS) CWs and subsu ace low (SSF) CWs. FWS sys ems a e simila o na u al
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we lands, wi h a shallow low o was ewa e o e a sa u a ed subs a e. On SSF sys ems,
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was ewa e lows h ough he subs a e which suppo s he g ow h o plan s, and based on he
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low di ec ion, SSF CWs could be u he di ided in o e ical low (VF) and ho izon al low
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(HF) CWs. A hyb id CW, a combina ion o a ious we land sys ems, was also in oduced o
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he ea men o was ewa e (Wu e al. 2015). Fo he pu pose o his pape , only a subsu ace
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low cons uc ed we land and especially he ho izon al subsu ace low cons uc ed we land
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plan ed by Ph agmi es aus alis is conside ed.
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Mic oo ganisms play a i al ole in deg ada ion o mul iple pollu an s in CWs. I has been
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ecognized ha he emo al o mos pollu an s in CWs is due p ima ily o mic obial ac i i y
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(Meng e al. 2014). Remo al o a pa icula pollu an is ypically associa ed wi h a speci ic
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mic obial unc ional g oup, he e o e he employmen o design and ope a ional
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me hodologies ha enhance he ac i i y o ha g oup will be e op imize pe o mance
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(Faulwe e e al. 2009).
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I has long been enowned ha many na u ally occu ing hizosphe e bac e ia and ungi may
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o e a iable subs i u e o he use o chemicals and a e an agonis ic owa ds c op pa hogens.
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Thus, Plan g ow h p omo ing hizobac e ia (PGPR) has been shown bene icial o plan
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g ow h and heal h by emancipa ing hei ac i i y on ni ogen ixa ion, he p oduc ion o
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phy oho mones and an i ungal compounds, and induced sys emic esis ance (Sindhu,
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Rakshiya, and Sahu 2009).
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Based on he impo ance o hizosphe e compe ence o oo coloniza ion in bene icial plan –
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mic obe in e ac ions (Ben Saad e al. 2016), he main goal o he p esen s udy was o enhance
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he inac i a ion o pa hogenic bac e ia a es in ho izon al subsu ace low cons uc ed we land
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sys em plan ed by Ph agmi es aus alis using an agonis ic bac e ia. This wo k aimed o
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demons a e he bene icial applica ion o bio echnology o con e highe hizosphe e
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compe ence in he emo al o pa hogenic bac e ia; S. yphi ATCC 560 by en i onmen
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iendly biological app oaches.
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Me hods
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1. Sampling and isola ion o bac e ial s ains om di e en en i onmen s
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Bac e ial s ains we e isola ed om di e en ecological niches: was ewa e , soil, Ph agmi es
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aus alis oo s and shee s om he Technical Demons a ion Cen e (TDC) ha ea s sewage
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om he uni e si y home loca ed a he Ag onomic Ins i u e o Tunis (INAT). Rhizosphe e
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samples we e collec ed om each we land a he en ance, middle, and exi a a dep h o
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app oxima ely 30 cm unde he g a el su ace. All samples we e p ocessed in he labo a o y.
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To isola e bac e ia om he hizosphe e, he oo s we e ini ially sepa a ed om he hizomes,
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and hen small pieces o oo s we e imme sed in s e ile saline solu ion (0.85 g/L NaCl) and
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o exes 15min in o de o elease he bac e ia a ached o oo s in o he solu ion.
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The same p o ocol was ollowed o isola e bac e ia om he se s o eeds. Conce ning he
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was ewa e samples, hese samples unde wen decimal dilu ions in s e ile saline solu ion and
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sp ead ou o e selec i e medium.
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2. Iden i ica ion o he s ain and de ec ion o side opho es p oduc ion
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The iden i ica ion o selec ed bac e ia was based on he pheno ypical aspec o colonies, he
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mic oscopic examina ion s anda d mic obiological and biochemical es s. Side opho e was
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de ec ed by he me hod o Jalal and Vande Helm (1990) using a spec opho ome ic assay
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whe e a peak a 495 nm on he addi ion o 2% aqueous solu ion o FeCl3 o 1 mL o
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supe na an indica ed he p esence o side opho es.
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3. An agonism es be ween isola ed bac e ial s ains and agains pa hogenic bac e ia
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S. yphi ATCC 560
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An agonism es had been pe o med be ween isola ed bac e ial o a oid nega i e in e ac ion
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be ween hem a e hei bioinocula ion. The Pe i dish su ace was seeded by an indica i e
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s ain and hen he blank discs deposi ed on he cul u e medium had been d enched wi h 50µL
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o il e ed supe na an o a pu a i e an agonis s ain, collec ed a e cen i uga ion a 4000
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.p.m o 15 min. The di usion o he an imic obial agen s was enhanced by incuba ion a
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37°C o 24h . An agonis ac i i y was e ealed by he appea ance o inhibi ion zone a ound
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he discs.
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4. S udy o mo ili y o isola ed bac e ia and bio ilm p oduc ion.
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The di e en ypes o mobili y (swimming, swa ming and wi ching) we e de e mined by he
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me hod o Reimmann e al. (2002). The bio ilm p oduc ion o bac e ial isola es was de ec ed
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by wo me hods: he i s desc ibed by F eeman and al. (1989), consis ed o pla ing he es
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s ains on solid medium con ained b ain hea in usion b o h 37 g/L, suc ose 50 g/L, aga 10
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g/L and Congo Red indica o 8 g/L (Suja ha N. 2013). A e incuba ion a 30°C o 24h ,
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black colonies indica e bio ilm p oduc ion. A e his quali a i e s udy, we p oceeded in a
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quan i a i e s udy desc ibes by O’Toole (1998). This me hod uses he dye c ys al iole (CV).
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bio ilm p oduc ion was es ima ed by spec opho ome ic measu emen o he OD 600nm.
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5. Molecula iden i ica ion o he selec ed s ain
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Bac e ial DNA was ex ac ed and pu i ied using he -DNA eagen (GenIUL) acco ding o
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he manu ac u e ’s ins uc ions. The concen a ion o he ex ac ed DNA was measu ed using
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a spec opho ome e a 260nm. DNA pu i y was es ima ed om he A260/A280 a io. The
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comple e 16S RNA gene was ampli ied using bac e ial p ime s 27F (5’-TAC GGY TAC
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CTT GTT AYG ACT T-3’) and 1492Rmod (5’-AGR GTT TGA TCM TGG CTC AG-3’).
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Each PCR eac ion wi h a inal olume o 25 µl con ained: 2µl o empla e DNA, 0.5 µl o
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each deoxynucleoside iphospha e a a concen a ion o 10 µM, 0.75 µl o MgCl2 1.5 mM,
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0.5 µl o each p ime a a concen a ion o 10 µM, 0.125 µl o Taq DNA polyme ase
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(In i ogen), 2.5 µl o PCR bu e supplied by he manu ac u e (In i ogen, Paisley, UK) and
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Milli-Q wa e up o he inal olume. Reac ions we e ca ied ou in a Bio ad he mocycle
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using he ollowing p og am: ini ial dena u a ion a 94ºC o 5 min, ollowed by 30 cycles o
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1 min a 94ºC, 1 min a 55 ºC and 2 min a 72 ºC, and a inal ex ension s ep o 10 min a 72ºC.
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PCR p oduc s we e e i ied and quan i ied by aga ose gel elec opho esis wi h s anda d low
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DNA mass ladde (In i ogen). Pu i ica ion and One Sho Sange sequencing o 16S RNA
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gene p oduc s was pe o med by Genosc een (Lille, F ance) wi h p ime s 27F and
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1492Rmod.
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6. Concep ion and cons uc ion o he pilo -scale sys ems
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The expe imen al sys em designed o he bio-inocula ion, included wo small pa allel
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iden ical ho izon al subsu ace low cons uc ed we lands. Bo h basins we e illed wi h g a el
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and plan ed wi h eed. The i s one se ed as a con ol and he second se ed o he di e en
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bio-assays. The size o each cons uc ed we land bed was 0.3 x 0.44 x 0.28. The ea men
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a ea was packed wi h 8-12mm diame e pea g a el while bigge and la ge g a el o 30-
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80mm diame e was used a he inle and ou le a eas in o de o p e en clogging o he il e
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media. The ela i e po osi y has been calcula ed in 0.26 (n = V /V whe e V is he oid
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olume and V is he o al olume (Meng e al. 2014)). The pilo e cons uc ed we lands had
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bo om slope o 1% o acila e he low o wa e by g a i y. The plan s we e allowed o g ow
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and mul iply o e h ee mon hs. The e was a pe idioc applica ion o was ewa e o se e as a
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sou ce o nu ien s o he plan s. The main cha ac e is ics o he expe imen al sys em a e as
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ollows: Su ace a ea: 13.2dm2, Hyd aulic Residence Time HRT ( heo e ical): 0.385, G a el
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dep h: 2dm, A e age s a ing eed heigh s: 57cm.The igu es 1 and 2 show he concep ion o
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ho izon al subsu ace low cons uc ed we land sys ems adop ed in his s udy.
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Figu e1. The ep esen a i e igu e o he pilo -scale o subsu ace low cons uc ed we land
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7. Moni o ing o bac e ia emo al
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To op imize he expe ience, he kine ic g ow h o he selec ed s ain was de e mined using a
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spec opho ome e o es ima e abso bance o cell suspensions (DO600). Se ies es s we e
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conduc ed o de e mine he lag ime ha made he selec ed bac e ia o adap o new
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condi ions.
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The expe ience in si u was done as ollows: Phase I s a om he sowing o in e es bac e ia
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in o he izosphe e en i onmen . The main e en s a e ac i a ion o he an agonis inoculum
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and es ablishmen o an an agonis popula ion in he plan izosphe e. Phase II is he p ocess
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o he in oduced an agonis and na i e oo -associa ed mic obes o es ablish a popula ion
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densi y and pe sis in he hizoplane, hizosphe e o inside he oo .
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The e o e, he an agonis bac e ium was inocula ed in o he hizosphe e o he pilo -scale (F).
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A e an adap a ion pe iod (depends on g ow h kine ic pa ame e s), a con amina ed e luen
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by 104 UFC/ml o an indica o bac e ia; S. yphi ATCC 560 was added o bo h pilo - scales (F
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and T).
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Based on g ow h kine ic pa ame e s o in e es bac e ia (PFH1), sequen ial bio-injec ions we e
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pe o med in pilo scale (F) o es o he spa io- empo al dynamics and mic obial ecological
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p ocesses o oo coloniza ion by an an agonis and o explo e he impac o accumula ion
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e ec o sequen ial bioinocula ion o an agonis bac e ia o p omo e inhibi ion o pa hogenic
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bac e ia.
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The moni o ing o pa hogenic bac e ia emo al a e bioinocula ion was de e mined by
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cul u e on selec i e medium (SS: Selmonella Shigella aga ).
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Resul s and discussion
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1. Isola ion and Sc eening o bac e ial s ains
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A e sampling, isola ion and pu i ica ion s ages, 19 bac e ial s ains we e isola ed om
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di e en ecological niches om he Technical Demons a ion Cen e (TDC) ha ea s
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sewage om uni e si y home loca ed a he Ag onomic Ins i u e o Tunis (INAT). The isola s
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s ains we e selec ed and sc eened o gene al unc ional p ope ies o plan p omo ing
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hizobac e ia; namely, side opho e p oduc ion and an agonis ac i i y agains pa hogenic
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bac e ia in addi ion o he bac e ial mo ili y (swimming, swa ming and wi ching mo ili ies)
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and bio ilm p oduc ion.
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Bac e ial bio ilm o ma ion is impo an o oo coloniza ion. Indeed, oo -associa ed bac e ia
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ha e been s udied ex ensi ely, and many o hese p omo e he g ow h o hos plan s o a e
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used as biocon ol agen s (Dekke s e al. 1998). The plan -g ow h-p omo ing bac e ia ha e
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been epo ed o discon inuously colonize he oo su ace, de eloping as small bio ilms along
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epide mal issu es.
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Among he isola es, we we e sc eened PFH1 s ain o apply as an in e es o be inocula ed in o
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he izosphe ic zone o enhance he educ ion o pa hogenic bac e ia. Indeed, he an agonism
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es had e ealed he s ain PFH1 as he mos an agonis bac e ia agains S. yphi. Indeed, The
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abili y o mic obes o p oduce a wide ange o an imic obial compounds, including ly ic
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agen s, an ibio ics, bac e iocins, p o ein exo oxins and o he seconda y me aboli es, is c i ical
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o hei success in an agonis ic ac i i ies (W.-Y. Liu e al. 2013).
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2. Molecula iden i ica ion o he selec ed s ain
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The 16S RNA gene sequences o he selec ed bac e ia we e de e mined. The sequence
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analysis e ealed ha his bac e ia has 99% o simila i y wi h En e obac e cloaceae.
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En e obac e cloacae a e a g am-nega i e P o eobac e ium belonging o he
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En e obac e iaceae amily. Wi hin his amily, En e obac e is mos closely ela ed o, and is
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g ouped in a sub-clade wi h, Klebsiella. The E. cloacae species comp ises an ex emely
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di e se g oup o bac e ia ha has been ound in di e se en i onmen s, anging om plan s o
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soil o humans (Liu e al., 2013).
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En e obac e species ha e been epo ed as bo h plan pa hogens and human oppo unis ic
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pa hogens (Nishijima e al., 2007), and also as impo an enginee ing and plan g ow h-
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p omo ing bac e ia (Nie e al., 2002). Some En e obac e s ains may play impo an oles in
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plan –mic obe in e ac ions and hence in biocon ol mechanisms. In his sense, we a e used he
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selec ed bac e ia o con ol he pa hogenic densi y and i s applica ion o imp o e he wa e
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ea men by cons uc ed we land.
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3. Applica ion bioinocula ion o selec ed bac e ia.
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To op imize he expe ience and be o e inocula ion in o he hizosphe e, he kine ic g ow h o
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PFH1 s ain was in es iga ed in saline wa e and au ocla ed was ewa e a oom empe a u e
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in o de o de e mine he speci ic g ow h cha ac e is ics o PFH1 namely he lag ime, (ʎ ) he
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maximum speci ic g ow h a e (µmax), he bac e ium gene a ion ime ( G) (Figu e 2).
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8
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Figu e 2. Kine ics g ow h o PFH1 in di e en g ow h empe a u e condi ions.
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Da a a e a e ages o h ee expe imen s
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3.1. Remo al o pa hogenic bac e ia wi h single bioinocula ion
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The igu e 3 shows he kine ic o bac e ia emo al wi h an ini ial concen a ion o indic o
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bac e ia (S. yphi) equal o 106 UFC/ml in p esence o PFH1.
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0
0,2
0,4
0,6
0,8
1
1,2
0
15
30
45
60
75
90
105
120
135
OD600
Time (min)
Saline Wa e
Au ocla ed was ewa e
Saline Wa e
µmax = 0. 25 min-1
G= 2.77 min
ʎ = 60 min
Au ocla ed WW
µmax = 0.31 min-1
G= 2.23 min
ʎ =75 min
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de eloping a comme cial bioinoculan o be applied in biological wa e ea men p ocess o
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imp o e he ea ed wa e quali y.
2
3
CONCLUSION
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F om he p esen esea ch, we can conclude ha applica ion o bioinocula ion has a po en ial
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o enhance he pa hogenic bac e ia emo al p ocess. Indeed, he p elimina y esul s show he
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bene icial e ec o he bioinocula ed s ain (PFH1) in he hizosphe e o inc ease ema kably
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he e iciency o he wa e ea men sys em o he educ ion o pa hogenic bac e ia wi h a
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educ ion in con ac ime.
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This s udy has con ibu ed wi h an eco- iendly s a egy o imp o e wa e ea men p ocess
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by cons uc ed we land, and highligh ed he ac ha be e yields can be ob ained hough
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bio-inocula ion.
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As a pe spec i e o his s udy, he applica ion o his s a egy in ield condi ions wi h mul i-
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inocula ion o an agonis s subs ances p o ec ed by na u al polyme s o inac i a e pa hogenic
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bac e ia in ea ed wa e wi hou chemical addi ion, ex ension in he e en ion ime o addi ion
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o a complemen wa e ea men s ages.
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Acknowledgmen
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This wo k is suppo ed by CERTE con ac p og ams unded by he Minis y o Highe
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Educa ion and Scien i ic Resea ch o Tunisia.
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