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Removal(of(Ni((II)(from(water(using(
recombinant(Escherichia(coli(
Master!thesis!at!Division!of!Bioprocess!Technology,!KTH!
Marie!Berg!
2012=12=04(
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SUPERVISOR(AND(EXAMINER:(GEN(LARSSON(
! ! !EXECUTIVE(SUMMARY
!!Heavy! metals,! nickel! being! one! of! them,! are! natural! constituents! in! soil! and! rocks,! and! can! be! released!into!waters!due!to!weathering.!Other!large!sources!for!pollution!are!manBbased!activities,! such!as!farming,!mining!and!other!industries. (Ochieng!et!al.!2007)!These!metals!accumulate!in!the! nature!and!can!cause!health!problems!to!humans!and!the!environment.!The!methods!for!removing! heavy!metals!from!wastewater!available!today!are!too!expensive!to!be!implemented!in!large!scale,! both!in!the!western!world!and!in!developing!countries!(Kumar!2006).!!
Nickel! is! one! of! thirtyBthree! substances,! prioritized! by! the! EU,! to! reduce! emission! of! (EUROPAPARLAMENTETS! OCH! RÅDETS! DIREKTIV! 2000/60/EG! 2000).! The! aim! of! this! study! was! to! present!the!nickel!issues!in!Sweden!and!Vietnam,!as!well!ass!comparing!the!use!of!a!recombinant!
Escherichia) coli! strain! for! removal! of! nickel! from! water! with! already! available! methods,! such! as!
reverse!osmosis,!nanofiltration,!and!ion!exchange.!
The!recombinant!cell!used!in!this!study!was!an!OmpTBnegative!Escherichia)coli!strain,!overexpressing! the! phytochelatin! analogue! EC4BGly! on! its! surface.! The! expression! system! used! was! an! autotransporter,!native!to!E.!coli,!called!pAIDA1.!
The! experiments! showed! that! the! recombinant! cell! expresses! the! peptide! satisfactory,! both! with! IPTG!and!lactose!as!inducer!of!the!lacUV5!operon,!and!that!it!can!adsorb!nickel.!The!cell!also!shows! some! biofilm! formation! potential,! however,! not! as! much! as! desired.! The! nickel! removal! efficiency! reached! 77%! in! an! experiment! where! the! cells! had! been! let! form! a! biofilm! on! Cytoline! 1! beads.! However,!a!large!part!of!the!nickel!ions!seem!to!adsorb!to!the!bead!itself,!and!not!just!the!cell.! The!conclusion!reached,!was!that!this!method!is!not!implementable!now,!but!with!further!research! and!optimization,!it!can!be.!
SAMMANFATTNING(
Tungmetaller,! nickel! en! av! dem,! är! naturliga! beståndsdelar! i! jord! och! berg,! och! kan! komma! ut! i! vattnet! på! grund! av! vittring.! Andra! stora! källor! för! förorening! av! tungmetaller! är! mänskliga! aktiviteter,! såsom! jordbruk,! gruvdrift! och! andra! industrier.! (Ochieng! et! al.! 2007)! Dessa! metaller! ackumuleras!i!naturen!och!kan!orsaka!hälsoproblem!för!människor!och!miljö.!Dagens!metoder!för!att! avlägsna!tungmetaller!från!avloppsvatten!är!för!dyra!för!att!användas!i!stor!skala,!både!i!västvärlden! och!i!utvecklingsländer!(Kumar!2006).!
Nickel! är! ett! av! trettiotre! ämnen,! prioriterade! av! EU,! för! att! minska! utsläpp! av! (EUROPAPARLAMENTETS!OCH!RÅDETS!DIREKTIV!2000/60/EG!2000).!Syftet!med!denna!studie!var!att! presentera! nickelproblematiken! i! Sverige! och! i! Vietnam,! och! att! jämföra! användningen! av! en! rekombinant! Escherichia) coliBstam! för! avlägsnande! av! nickel! från! vatten! med! redan! tillgängliga! metoder,!såsom!omvänd!osmos,!nanofiltrering,!och!jonbyte.!
Den!rekombinanta!cellen!som!används!i!denna!studie!var!en!OmpTBnegativ!Escherichia)coliBstam!som! överuttrycker! phytochelatinBanalogen! EC4BGly! på! dess! yta.! Uttryckssystemet! som! användes! var! en! autotransporter,!nativ!för!E.)coli,!som!kallas!pAIDA1.!
Experimenten! visade! att! den! rekombinanta! cellen! uttrycker! peptiden! tillfredsställande,! både! med! IPTG!och!laktos!som!inducerare!av!lacUV5Boperonet,!och!att!den!kan!adsorbera!nickel.!Cellen!visar! också!viss!biofilmbildningspotential,!dock!inte!så!mycket!som!önskas.!Effektiviteten!nådde!77%!i!ett! experiment! där! cellerna! hade! låtits! bilda! en! biofilm! på! Cytoline! 1Bkulor.! Emellertid,! en! stor! del! av! nickeljoner!tycks!adsorbera!till!kulan!själv,!och!inte!bara!till!cellen.!
Slutsatsen!var!att!denna!metod!inte!kan!implementeras!i!nuläget,!men!med!ytterligare!forskning!och! optimering,!kan!den!kanske!det.!
ACKNOWLEDGEMENTS
!I!would!like!to!show!appreciation!to!everyone!who!has!made!this!project!possible.!First,!I!would!like! to!thank!Sida!for!providing!me!with!the!MFSBscholarchip.!I!would!also!like!to!thank!Professor!Gen! Larsson! for! introducing! us! to! this! project,! Professor! Hai! for! welcoming! us! to! IBT,! and! Huyen! for! guiding! us! during! our! stay! in! Hanoi,! both! scientifically! as! well! as! in! everyday! life.! Also! thanks! to! Hoang!Ahn,!Ngoc,!Robin,!and!Loi!for!all!their!help.! I!would!like!to!thank!Emelie!for!enduring!me!twentyBfour!hours!a!day!for!two!and!a!half!months!(still! friends!fortunately).!I!would!also!like!to!thank!Martin!Gustafsson,!Johan!Norén!and!Johan!Jarmander! for!invaluable!guidance!when!back!in!Sweden.! And,!of!course,!thank!you!Johan!Lindholm!for!always!supporting!me!!
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TABLE(OF(CONTENTS(
1!
INTRODUCTION*...*7!
1.1!
PROBLEM!...!7!
1.2!
HYPOTHESIS!...!7!
1.3!
AIM!...!7!
1.4!
SOLUTION!STRATEGY!...!7!
2!
FUNDAMENTALS*...*8!
2.1!
NICKEL!...!8!
2.2!
WASTEWATER!TREATMENT!...!8!
2.2.1!
Biofilm*in*wastewater*treatment*...*9!
2.3
!
WASTEWATER!TREATMENT!IN!VIETNAM!...!9!
2.4
!
CURRENT!METHODS!FOR!REMOVING!NICKEL!...!10!
2.4.1
!
REVERSE*OSMOSIS*...*10!
2.4.2
!
NANOFILTRATION*...*10!
2.4.3
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ION*EXCHANGE*...*10!
2.5
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BIOCHELATORS!...!11!
2.6
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EXPRESSION!SYSTEM!AND!GENE!CONSTRUCT!...!12!
3
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MATERIALS*AND*METHODS*...*13!
3.1
!
EXTENT!OF!NICKEL!POLLUTION!IN!VIETNAM!...!13!
3.1.1
!
WATER*SAMPLING*IN*NORTHERN*VIETNAM*...*13!
3.2!
PEPTIDE!EXPRESSION!...!13!
3.2.1!
PRIMARY*ADSORPTION*TEST*...*13!
3.2.1.1!
Cell!cultivation!...!13!
3.2.1.2!
Adsorption!test!...!13!
3.2.1.3!
EXPRESSION!OF!PEPTIDES!...!14!
3.2.2!
SURFACE*EXPRESSION*USING*LACTOSE*AS*INDUCER*AND*SOLE*CARBON*SOURCE*....*15!
3.2.2.1!
Cultivation!...!15!
3.2.2.2!
FACS!analysis!...!15!
3.3!
BIOFILM!FORMATION!...!15!
3.3.1!
BIOFILM*FORMATION*WITH*LACTOSE*AS*CARBON*SOURCE*...*15!
3.3.1.1!
BiofilmUcultivation!...!15!
3.3.2!
BIOFILM*FORMATION*ON*CYTOLINE*1*BEADS*...*16!
3.3.2.1!
Washing!procedure!...!16!
3.3.2.2!
Wet!weight!and!dry!weight!measurements!...!16!
3.3.2.3!
Biofilm!cultivation!for!adsorption!test!...!17!
3.3.2.4!
FluorescenceUmicroscopy!...!17!
3.3.3!
BIOFILM*FORMATION*ON*BIOFILMCHIP*M*CARRIERS*...*17!
3.3.4!
ADSORPTION*TEST*ON*BIOFILM*...*17!
4!
RESULTS*...*19!
4.1
!
EXTENT!OF!NICKEL!POLLUTION!IN!VIETNAM!...!19!
4.1.1
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WATER*SAMPLING*IN*NORTHERN*VIETNAM*...*19!
4.2!
PEPTIDE!EXPRESSION!...!19!
4.2.1!
PRIMARY*ADSORPTION*TEST*...*20!
4.2.1.1!
Expression!of!peptides!...!20!
4.2.2!
SURFACE*EXPRESSION*USING*LACTOSE*AS*INDUCER*AND*SOLE*CARBON*SOURCE*....*21!
4.2.2.1!
Cultivation!...!21!
4.2.2.2!
FACS!analysis!...!22!
4.3
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BIOFILM!FORMATION!...!23!
4.3.1!
BIOFILM*FORMATION*WITH*LACTOSE*AS*CARBON*SOURCE*...*23!
4.3.2!
BIOFILM*FORMATION*ON*CYTOLINE*1*BEADS*...*24!
4.3.2.1!
FluorescenceUmicroscopy!...!27!
4.3.3!
BIOFILM*FORMATION*ON*BIOFILMCHIP*M*CARRIERS*...*27!
4.3.4!
ADSORPTION*TEST*ON*BIOFILM*...*28!
5!
DISCUSSION*...*29!
5.1!
IMPLEMENTATION!ISSUES!...!30!
6!
CONCLUSION*...*31!
7!
LITERATURE*...*32!
8!
APPENDICES*...*35!
8.1!
APPENDIX!1:!BIOFILM!FORMATION!WITH!LACTOSE!AS!CARBON!SOURCE!...!35!
8.2
!
APPENDIX!2:!RESULTS!FROM!ADSORPTION!TEST!...!35!
!1 INTRODUCTION(
1.1 PROBLEM(
Heavy!metals!can!be!found!everywhere.!They!are!natural!constituents!in!soil!and!rocks,!and!can!be! released! into! waters! due! to! weathering.! ManBbased! activities,! such! as! farming,! mining! and! other! industries,!are!also!a!large!source!for!pollution. (Ochieng!et!al.!2007)!These!metals!accumulate!in!the! nature!and!can!cause!health!problems!to!humans!and!the!environment!(Kumar!2006),!which!is!why!it! is!important!to!reduce!the!release!of!heavy!metals.!
Nickel! is! a! metal! that! is! hard! to! remove,! while! chemical! compounds! used! for! precipitation! within! wastewater!treatment!plants!today!often!contain!nickel!(Björlenius,!Personal!communication!2012).! Therefore,!the!water!coming!out!of!the!plant!has!almost!the!same!concentration!of!nickel,!as!do!the! wastewater!coming!in!(André!et!al.!2006).! The!methods!for!removing!heavy!metals!from!wastewater!available!today!are!too!expensive!to!be! implemented!in!large!scale,!both!in!the!western!world!and!definitely!in!developing!countries!(Kumar! 2006).!What!is!needed!is!a!method!that!is!as!effective!as!the!methods!available!today,!but!much!less! expensive.! The!method!used!in!this!work!has!the!potential!of,!being!cheaper,!both!in!regard!to!initial!cost!and! the! and! required,! and! simpler! than! conventional! methods! (Kumar! 2006).! The! idea! is! to! use! biochelators,!expressed!on!the!surface!of!Escherichia)coli!bacteria,!for!biosorption!of!the!metals.!The! cells!can,!after!adsorption,!be!regenerated!and!used!again.!
1.2 HYPOTHESIS(
The!hypothesis!is!that!E.)coli!overexpressing!biochelating!peptide!EC4BGly!on!its!surface!can!adsorb! nickel! in! 1:1! stoichiometry,! that! is! to! say! five! nickel! ions! per! peptide,! and! that! the! E.) coli! cell! can! express!approximately!100!000!peptides!per!cell.!It!is!also!that!this!cell!can!form!a!biofilm.!Further,! the! hypothesis! is! that! the! efficiency! of! this! method,! when! used! for! removal! of! nickel! from! water,! increases!with!increased!number!of!peptides!on!the!cell!surface!and!increased!number!of!cells.!!
1.3 AIM(
The! aim! of! this! study! is! to! compare! the! efficiency! of! this! method! with! the! efficiency! of! already! available! methods! for! removing! nickel! from! water.! Further,! it! is! to! present! the! nickel! issues! in! Sweden!and!in!Vietnam.!
1.4 SOLUTION(STRATEGY(
The!solution!strategy!is!to!investigate!the!two!variables!that!can!increase!efficiency!of!this!method,! the!expression!of!peptides!on!cell!surface!and!increased!cell!numbers!in!biofilm,!and!thus!the!total! amount!of!peptides.!It!is!also!to!test!Vietnamese!waters!for!pollution.!
Investigation! of! the! extension! of! the! nickel! pollution! was! carried! out! through! literature! studies! as! well!as!by!some!water!sampling.!The!investigation!of!expression,!biofilm!formation!and!adsorption! tests!were!set!up!according!to!literature!findings!and!the!implementation!discussion!was!based!on! literature!as!well!as!experimental!findings.!!
2 FUNDAMENTALS(
2.1 NICKEL(
Heavy!metals!have!a!tendency!to!accumulate!in!nature!and!several!of!them!are!quite!toxic,!nickel! being! one! of! them! (Kumar! 2006).! Nickel! has! an! important! role! in! the! homeostasis! of! the! human! organism.!It!has!been!one!of!the!most!used!metals!in!billons,!silver!substitutes,!and!stainless!steel,! ever!since!it!was!first!discovered.!(Darlenski!et!al.!2012)!
Nickel! is! suspected! to! be! an! embryotoxin! and! teratogen! (Chen! et! al.! 1998),! while! higher! concentrations! of! nickel! causes! effects! of! poisoning.! These! effects! can! for! example! be! headaches,! nausea,!vomiting,!and!extreme!weakness!(Pandey!et!al.!2007).!Nickel!is!also!a!possible!carcinogen,! present!in!the!effluent!water!of!many!industries!(Pandey!et!al.!2007).!The!most!commonly!known! effect!of!nickel!exposure,!however,!is!probably!nickel!allergy,!which!is!the!most!common!cause!of! contact!dermatitis!(Darlenski!et!al.!2012).! The!guideline!value!of!nickel!for!drinking!water!is,!according!to!WHO,!70!μg/l!(WHO!2011).!This!is! considerably!more!than!the!effluent!water!from!for!example!Henriksdalsverket!in!Stockholm,!which! is! 7.16! μg/l! (André! et! al.! 2006).! However,! nickel! is! one! of! the! thirtyBthree! by! EU! prioritized! substances.! These! are! substances! that! are! considered! hazardous! and! that! should! be! reduced! in! effluent! waters.! (EUROPAPARLAMENTETS! OCH! RÅDETS! DIREKTIV! 2000/60/EG! 2000)! These! are! all! good!reasons!for!investigating!different!options!that!could!improve!nickel!removal!at!a!reasonable! cost.!
2.2 WASTEWATER(TREATMENT(
Even!though!the!wastewater!treatment!methods!are!getting!better!and!better,!some!metals,!nickel! one! of! them,! are! still! hard! to! remove! from! the! water! at! reasonable! cost! (Björlenius,! Personal! communication! 2012).! At! municipal! wastewater! treatment! plants! there! are! usually! no! treatment! step! designed! for! this.! For! example,! in! 2005,! the! nickel! concentration! in! at! Henriksdalsverket,! in! Stockholm,!was!7.8!μg/l!and!the!nickel!concentration!out!was!7.16!μg/l.!At!Brommaverket,!also!in! Stockholm,!the!concentrations!were!6.02!μg/l!in!and!6.18!μg/l!out.!This!can!be!compared!with!the! concentrations! of! cadmium,! which! are! 0.21! μg/l! in,! and! <0.009! μg/l! out! at! Henriksdalsverket,! and! 0.13! μg/l! in,! and! <0.008! μg/l! out! at! Brommaverket.! (André! et! al.! 2006)! This! shows! a! much! higher! removal!rate!for!cadmium!than!for!nickel.! A!typical!design!of!a!municipal!wastewater!treatment!plant!can!be:! I. Sieving!–!removal!of!large!particulate!contaminants.! II. Filtering!–!often!a!sandbed!removing!smaller!particulate!contaminants.! III. Biological!treatment!–!Aerobic!or!anaerobic!treatment!removing!organic!compounds,!as!well! as!biological!nitrogen!reduction.! IV. Chemical!precipitation!–!usually!for!removing!phosphorus!from!the!water.! Where,!sedimentation!tanks!are!situated!before!and!after!the!aerobic!treatment!as!well!as!after!the! chemical!precipitation!as!a!last!step.!The!chemical!precipitation!can!also!be!placed!before!the!first! sedimentation!tank!or!together!with!the!biological!treatment.!(P.!O.!Persson!2005)!
2.2.1 Biofilm(in(wastewater(treatment(
Biofilms! create! an! advantage! for! bacteria! in! nature! by! sheltering! the! bacteria! from! harmful! substances,! as! well! as! through! metabolic! cooperation! and! facilitating! gene! transfer (Davey! et! al.! 2000).!Further,!biofilm!can!be!used!to!increase!the!cell!density.!These!advantages!can!also!be!applied! to!biofilm!in!wastewater!treatment.!
There! are! also! other! advantages! of! using! biofilm! systems! instead! of! suspended! cell! systems! in! wastewater!treatment,!high!biomass!concentration,!less!space!demand,!and!less!sludge!production! being!a!few!of!them.!However,!there!are!also!some!disadvantages!and!limitations!in!using!biofilm.! Due!to!the!fact!that!the!substrate!must!cross!the!solidBliquid!interface!and!then!reach!the!cells!in! lower! layers,! there! will! be! a! concentration! gradient! that! may! cause! only! part! of! the! biofilm! to! actually!take!part!in!the!metabolism.!(Wilderer!et!al.!2004)!In!wastewater!treatment!biofilm!can!be! used! in! both! fixed,! for! example! trickling! filter,! and! mobile! designs,! for! example! fluidized! beds (Persson!et!al.!2005).!
Biofilms! can! form! on! almost! every! surface! in! a! moist! environment.! The! biofilm! formation! can! be! divided!into!a!few!steps:!
I. A!film!of!inorganic!and!organic!compounds!is!formed!at!the!surface.!
II. The! bacteria! move! by! chemotaxis! or! Brownian! movement! towards! the! surface! and! attach! reversibly.!
III. Production!of!extracellular!polymeric!substances!(EPS)!starts!and!the!bacteria!attach!to!the! surface!by!so!called!irreversible!attachment.!
IV. The! biofilm! starts! to! mature! by! cell! division! and! further! …(recruitment)! of! planktonic! bacteria.!
V. The! final! step! is! detachment,! and! is! when! bacteria! detach! from! the! biofilm! community! to! search!for!a!new!environment.!(Andersson!2009)!
It!usually!takes!several!days!for!a!biofilm!to!reach!maturity!(Andersson!2009),!while!the!initial!shift! from!reversible!to!irreversible!attachment!can!be!quite!fast!(Palmer!et!al.!2007).!
The!production!of!EPS!is!often!considered!to!be!essential!in!the!biofilm!formation!process.!However,! a!study!from!2008!showed!that!Acinetobacter)calcoaceticus!did!not!produce!detectable!amounts!of! EPS,! but! formed! a! strong! biofilm.! This! was! probably! due! to! the! hydrophobicity! of! the! cell,! which! made!the!biofilm!hold!together!by!hydrophobic!or!electrostatic!forces.!(Andersson!et!al.!2008)!The! normal!composition!of!EPS!is!40B95%!polysaccharides,!1B60%!proteins,!1B10%!nucleic!acids,!and!1B 40%!lipids!(Andersson!2009).!!
2.3 WASTEWATER(TREATMENT(IN(VIETNAM(
As!in!most!developing!countries,!the!wastewater!treatment!in!Vietnam!has!not!been!sufficient.!It!has! been! estimated! that! only! 60! percent! of! the! Vietnamese! population! has! access! to! potable! water,! which!makes!the!shortage!of!clean!water!one!of!Vietnams!most!urgent!problems!(Anh!2008).!At!the! moment,!only!approximately!10!percent!of!the!wastewater!in!the!capital!Hanoi!is!treated!before!it!is! discharged!into!rivers!and!lakes!(Anh!2008).!!
The! To! Lich! and! Kim! Nguu! rivers! are! two! rivers! floating! through! Hanoi.! They! are! the! principal! irrigation! water! source! for! fish! farming! and! agriculture! in! the! Hanoi! area.! These! two! rivers! were! examined! by! Japanese! scientists,! and! found! to! be! polluted.! The! pH! was,! at! three! of! the! eight!
sampling!sites,!higher!than!the!Vietnamese!standards!for!surface!water.!The!dissolved!oxygen!was!at! all!sites!much!lower!than!the!Vietnamese!standards,!and!the!chemical!oxygen!demand!and!the!total! suspended!solids!widely!exceeded!the!allowed!values.!The!total!nickel!concentration!exceeded!the! WHO! standards! (1989)! for! irrigation! water,! 0.2! mg/l,! at! all! sites,! while! it! was! well! within! the! Vietnamese!standards!of!1!mg/l.!(Huong!et!al.!2008)!
The!wastewater!treatment!in!Vietnam!is,!however,!slowly!improving.!In!Hanoi,!the!Yen!So!sewage! treatment!plant,!with!a!capacity!of!200,000!m3!per!day,!is!to!be!able!to!treat!half!of!the!wastewater! in!Hanoi.!In!the!original!plans,!it!was!to!be!in!operation!by!the!end!of!2011,!but!seems!not!to!have! been!launched!yet.!(Biwater)!
It! has! also! been! reported! that! the! Vietnamese! government! now! takes! pollution! violations! by! industries!more!seriously!than!previously,!putting!these!manufacturers!on!environment!black!lists.! This!has!resulted!in!boycotts!of!these!manufacturers,!from!banks!as!well!as!the!public. (Anh!2008)!! Despite!the!improvements!shown!in!Vietnam,!there!are!still!a!long!way!to!go!before!the!wastewater! treatment!in!the!country!is!adequate.!
2.4 CURRENT(METHODS(FOR(REMOVING(NICKEL(
Today,! there! is! no! treatment! in! wastewater! treatment! plants! specialized! in! removing! nickel.! However,!there!are!a!few!options!that!could!be!suitable,!was!it!not!for!the!high!cost!of!using!these! methods.!Reverse!osmosis,!nanofiltration!and!ion!exchange!are!three!of!those!options.!
2.4.1 REVERSE(OSMOSIS(
In! reverse! osmosis! (RO),! a! semiBpermeable! membrane! rejects! pollutants,! large! molecules! and! charged! ions,! while! letting! the! liquid! to! be! decontaminated! to! pass! through,! thus! overcoming! the! osmotic!pressure!(Fu!et!al.!2011).!!
Suggested! advantages! of! RO! are! that! organic! and! inorganic! contaminants! can! be! removed! at! the! same! time,! the! design! is! simple,! and! the! maintenance! requirements! are! low! (Williams! 2003).! The! removal!efficiency!has!shown!to!be!99.3%!for!Ni2+Bconcentrations!of!44B169!mg/l!(Ipek!2005).!There!
is!however!a!major!drawback!of!this!method,!the!energy!needed!for!the!pumping!pressures!and!the! restoration!of!the!membranes,!makes!this!a!quite!expensive!process.!(Fu!et!al.!2011)!
2.4.2 NANOFILTRATION(
Nanofiltration!(NF)!shows!great!promise!for!heavy!metal!removal!from!wastewater.!The!simplicity!of! the! method,! the! reliability! and! the! fairly! low! energy! consumption! are! a! few! advantages! (Fu! et! al.! 2011).!The!method!has!shown!a!maximum!nickel!removal!of!98.94%!at!an!initial!nickel!concentration! of!5!mg/l,!when!using!a!commercial!membrane!(Murthy!et!al.!2009).!
The! membrane! used! in! NF! has! pores! of! approximately! 1! nm! and! is! charged.! The! separation! is! therefore! both! based! on! electrical! effects! and! size! exclusion.! These! effects! make! it! possible! to! separate!bivalent!ions!from!both!water!as!well!as!solutions!of!monovalent!ions.!The!pressure!needed! for!this!separation!is!lower!than!the!pressure!needed!for!reverse!osmosis.!(Chilyumova!et!al.!2008)!
2.4.3 ION(EXCHANGE(
!Ion! exchange! (IE)! is! based! on! the! fact! that! the! resin! can! exchange! cations! with! the! metals! in! the! wastewater,! and! thereby! binding! the! metal.! Fast! kinetics! and! high! removal! efficiency! are! two!
advantages!of!ion!exchange.!(Fu!et!al.!2011)!When!using!clinoptilolite,!a!form!of!zeolite,!(Argun!2008)! was!able!to!reach!a!removal!efficiency!of!93.6%!at!an!initial!nickel!concentration!of!25!mg/l.!!
2.5 BIOCHELATORS(
In! nature,! organisms! have! developed! certain! metal! chelating! peptides! to! protect! themselves! from! the!metals!they!are!exposed!to.!Phytochelatins!(PCs),!a!family!of!short!and!cysteine!rich!peptides,!are! such!metal!chelating!peptides.!The!general!structure!of!PCs!is!(γGluBCys)nBGly,!where!n!is!a!number!
between!2!and!11,!see!Figure!1 (Bae!et!al.!1997).!PCs!regulate!the!metal!concentration!by!binding! metal!ions!with!its’!evenly!distributed!cysteine!thiolate!groups.!The!theory!that!PCs!are!essential!in! heavy!metal!detoxification!is!supported!by!the!fact!that!they!are!induced!by!exposure!to!for!example! Cd2+,!Pb2+,!Zn2+,!Sb2+,!Ag1+,!Ni2+,!Hg2+,!but!not!by!exposure!to!Ca2+,!Al3+,!and!Mg2+!(Cheng!et!al.!2005).!!
! ! ! ! ! ! ! The!EC4Bgly!(αGluBCys)4BGly)!metal!chelator!used!in!this!study!is!a!phytochelatin!analogue!which!has! been!shown!to!bind!several!heavy!metal!ions,!among!them!Ni2+,!in!1:1!stoichiometry!(Cheng!et!al.! 2005).!The!γBlinkage!of!the!PCs!cannot!be!encoded!by!a!gene,!but!is!synthesized!enzymatically!by!the! PC!synthase,!why!the!analogue!has!an!αBlinkage!instead,!see!Figure!2!(Bae!et!al.!1997).! !
Figure( 1.( The( molecular( structure( of( phytochelatin( (PC),( drawn( from((Bae(et(al.(1997)(
Figure( 2.( The( molecular( structure( of( ECs,( drawn( from((Bae(et(al.(1997)(
Biosorption!of!heavy!metals!have!been!tried!several!times!before.!For!example,!(Tahir!et!al.!2009)! used!immobilized!Bacillus)species!with!shown!biosorption!of!nickel,!and!estimated!the!nickel!removal! to! be! 97.4%.! Algae! have! also! been! used! as! biosorbent.! (Tamilselvan! et! al.! 2012)! investigated! the! biosorption!potential!of!Sargassum)wightii!(brown)!and!Caulerpa)racemosa!(green)!for!Cr6+,!Cr3+,!Pb2+!
and!Cd2+,!and!showed!78%!to!85%!biosorption.!The!adsorption!efficiency!may!be!dependent!on!pH!
and! temperature! (Kao! et! al.! 2009).! Also,! time! can! be! an! important,! why! these! three! factors! are! important!to!take!into!account!when!optimizing!adsorption.!
One! suggested! solution! to! the! problem! with! heavy! metal! pollution! has! been! to! introduce! PC! synthase!into!common!plants!that!grow!fast!and!produce!large!quantities!of!biomass,!and!use!these! plants!in!phytoremediation!(Pal!et!al.!2010).!This!solution!does,!however,!come!with!a!large!problem;! How!are!the!plants!to!be!disposed?!! This!problem!is!circumvented!when!using!a!cell!that!adsorbs!the!metal!on!its!surface!instead!of!inside! the!cell,!and!thereby!making!regeneration!a!possibility.!
2.6 EXPRESSION(SYSTEM(AND(GENE(CONSTRUCT(
Used!in!this!study!was!an!Escherichia)coli!strain!K12!overexpressing!biochelators!on!its!surface.!The! strain!is!OmpTBnegative!(0:17ΔOmpT)!since!this!decreases!the!possibility!of!the!expressed!protein!to! be!cleaved!by!this!outer!membrane!protease!(Mangel!et!al.!1994).! The!vector!pAIDABEC4Bgly!was!used!for!overexpression!of!biochelators!on!the!surface.!AIDA!(Adhesin! Involved!in!Diffuse!Adherence)!is!an!autotransporter!with!the!wildBtype!purpose!of!enabling!E.)coli!to! adhere!to!cells!in!the!intestine (Benz!et!al.!1989).!The!reason!AIDA!is!considered!more!advantageous! than!other!autotransporters!is!that!it!is!a!native!protein!to!E.)coli!(Jose!2006).!!The! AIDA! vector! consists! of! the! NBterminal! AIDA! signal! peptide,! a! linker! region! and! the! AIDAc!
(membraneBspanning! βBbarrel! in! the! CBterminal! domain).! The! passenger! (EC4Bgly! sequence)! is! situated!between!the!signal!peptide!and!the!linker,!see!Figure!3.!The!EC4Bgly!construct!consists!of!a! His6Btag,!a!four!times!repetition!of!cysteine!and!glutamate,!ending!with!a!glycine,!and!a!MycBepitope! tag.!The!vector!has!a!regulated!promoter,!LacUV5,!which!can!be!induced!by!lactose!or!IPTG.! ! !
(
(
(
!(
Figure(3.(Illustration(of(the(AIDA(vector.(In(this(case,(the(passenger(consists(of(the(EC4=Gly( sequence.(3 MATERIALS(AND(METHODS(
3.1 EXTENT(OF(NICKEL(POLLUTION(IN(VIETNAM(
3.1.1 WATER(SAMPLING(IN(NORTHERN(VIETNAM(
At!all!locations,!500!ml!bottles!were!used!for!water!sampling.!Previously!to!sampling!the!bottles!was! treated!with!H2SO4!overnight,!to!ensure!that!the!metals!do!not!absorb!to!the!bottle.!The!bottles!were! then!rinsed!in!the!sampling!water,!before!the!final!sample!was!taken.!! There!were!also!several!attempts!to!take!samples!from!wastewater!treatment!plants!in!Hanoi,!to!get! an! idea! of! the! pollution! levels! before! and! after! treatment.! However,! this! was! not! possible! due! to! regulations.!The! samples! were! analyzed! with! Atomic! Absorption! Spectroscopy! (AAS),! at! the! Institute! of! Environmental!Technology,!Vietnam!Academy!of!Science!and!Technology,!Hanoi,!Vietnam.!
3.2 PEPTIDE(EXPRESSION(
3.2.1 PRIMARY(ADSORPTION(TEST(
3.2.1.1 Cell(cultivation(
The!EC4BGly!cells!were!cultivated!on!minimal!medium!with!10!g/l!glucose!as!carbon!source,!in!25!ml! shake!flasks!overnight.!The!minimal!medium!consisted!of!7!g!(NH4)2SO4,!1.6!g!KH2PO4,!6.6!g!Na2HPO4!
×2H2O!(or!16.7!g!Na2HPO4!×12H2O)!and!0.5!g!(NH4)BHBcitrate!per!liter.!1!ml/l!trace!elements!and!1!
ml/l!MgSO4!(1!M)!was!added!to!the!medium.!The!composition!of!the!trace!element!solution!was:!0.5!
g!CaCl2!×2H2O,!16.7!g!FeCl3!×6H2O,!0.18!g!ZnSO4!×7H2O,!0.16!g!Cu!SO4!×5H2O,!0.15!g!MnSO4!×4H2O,!
0.18!g!CoCl2!×6H2O,!20.1!g!NaBEDTA,!per!liter.!25!μg/ml!Chloramphenicol!was!used!as!antibiotic.! In!the!morning!the!cells!were!reBinoculated!to!a!final!OD600!of!approximately!0.1,!in!several!100!ml! shake!flasks!with!the!same!content.!When!the!cell!density!had!reached!OD600!=!0.3B0.5,!the!cells! were!induced!by!IPTG!at!a!final!concentration!of!0.2!mM.!The!cells!were!grown!until!they!reached! stationary!phase,!at!which!point!they!were!harvested!by!centrifugation,!3500!rpm!for!12!minutes!at! 4°C.!The!cells!were!dissolved!in!PBS!with!20%!glycerol!and!stored!at!B80°C.! The!0:17!cells!were!cultivated!in!the!same!way!as!the!EC4BGy!cells,!apart!from!that!they!were!never! induced!and!Chloramphenicol!was!not!included!in!the!minimal!medium.! 3.2.1.2 Adsorption(test(
The! cells! were! taken! from! the! B80°C! freezer! and! thawed! on! ice.! The! thawed! cells! were! then! centrifuged!for!12!minutes!at!3500!rpm,!washed!in!PBS!pH!6,!and!centrifuged!again.!When!washed,! the!cells!were!dissolved!in!PBS!pH!6.!!
The! samples! were! prepared! by! adding! one! half! of! cells! in! PBS! pH! 6,! OD=112! and! OD=200! respectively,!and!one!half!PBS!pH!6!with!approximately!20!mg/l!Ni2+!to!a!centrifugation!tube,!to!get!a!
final!Ni2+Bconcentration!of!10!mg/l.!
The! reference! was! prepared! by! adding! one! half! of! PBS! pH! 6,! and! one! half! PBS! pH! 6! with! approximately!20!mg/l!Ni2+.!!
After!incubation,!the!samples!were!centrifuged!twice!at!5000!rpm!for!15!minutes.!The!samples!were! analyzed!by!AAS.! 3.2.1.3 EXPRESSION(OF(PEPTIDES( 3.2.1.3.1 SDSBPAGE!with!Coomassie!Brilliant!Blue!staining! Forty!μl!sample!(OD=20)!and!5!μl!treatment!buffer!was!mixed!well!by!vortexing.!The!samples!were! then!treated!in!100°C!for!10!min.!Before!loading!onto!the!gel!(12%!bisacrylamide!separating!gel),!the! samples!were!centrifuged!at!13000!rpm!for!10!minutes!to!remove!cell!debris.!15!μl!of!sample!was! loaded!to!each!well!on!the!gel!according!to!the!following!scheme!(EF:!cells!cultivated!in!Erlenmeyer! flask,!SF:!cells!cultivated!in!shake!flask).!Marker!was!added!as!1!and!2!μl!respectively!for!unstained! and!preBstained!marker.! Unstained! marker! 0:17! EC4BGly! EF! EC4BGly! SF! X! PreBstained! marker! 0:17! EC4BGly! EF! EC4BGly! SF!
An! electric! current! of! 12! mA! was! applied! until! the! samples! had! passed! the! stacking! gel! into! the! separating!gel.!At!that!time!the!current!was!increased!to!24!mA.! The!run!was!stopped!when!the!dye!had!reached!the!end!of!the!gel.!The!gel!was!split!into!two,!where! the!part!with!the!unstained!marker!was!stained!with!Coomassie!brilliant!blue.! Coomassie(brilliant(blue(staining:( • The!gel!was!destained!in!destaining!buffer!containing!methanol!for!10!min.! • After!that,!the!gel!was!stained!with!Coomassie!Brilliant!Blue!R250!for!30!min.! • Another!destaining!with!destaining!buffer!containing!methanol!was!performed!for!10!min.! • Destaining! buffer! containing! ethanol! was! used! for! 20! minutes! to! destain! the! gel.! This! step!
was!repeated!until!the!gel!was!clear.! • Finally!the!gel!was!washed!in!deionized!H2O,!and!then!kept!in!this.! 3.2.1.3.2 Western!blot! Western!blot!was!used!to!visualize!only!the!expressed!peptides.! • A!PVDF!membrane!was!soaked!in!methanol!for!10!min!and!two!filter!papers!were!soaked!in! blotting!buffer!for!5!minutes.! • PVDF!membrane!was!applied!to!one!filter!paper,!then!gel!and!last!the!second!filter!paper,!in! the! transfer! apparatus.! The! filters,! gel! and! membrane! were! gently! rolled! between! each! application!to!make!sure!no!air!was!left.!
• The!proteins!were!transferred!at!15!V,!198!mA,!for!15!min.!
• The! membrane! was! washed! twice! in! TBST,! and! then! blocking! buffer! was! applied! and! incubated!for!1!h.!
• The! membrane! was! washed! 3x3! min! with! TBST.! And! the! primary! antibody! (antiBHis6)! was! diluted!in!skimmed!milk!(2!μl!to!10!mL!skimmed!milk).! • The!membrane!was!incubated!with!primary!antibody!for!1!h.! • The!membrane!was!washed!3x3!min!with!TBST!and!incubated!with!secondary!antibody!(2!μl! in!10!mL!skimmed!milk)!for!1!h.! • It!was!washed!3x3!min!with!TBST.! • 2!mL!Tetramethylbenzidine!was!added!and!incubated!until!color!appeared.!
• The!membrane!was!washed!with!dH2O!to!stop!reaction.!
3.2.2 SURFACE(EXPRESSION(USING(LACTOSE(AS(INDUCER(AND(SOLE(CARBON(SOURCE(
3.2.2.1 Cultivation(
The! cells! were! cultivated! overnight! in! 25! mL! minimal! medium,! described! elsewhere,! with! chloramphenicol!and!either!10!g/L!glucose!or!lactose!as!carbon!source.!When!reaching!exponential! phase! and! approximately! OD600=1,! 150! mL! minimal! medium! was! inoculated! with! the! overnight!
culture! to! a! final! OD600! of! approximately! 0.1.! The! glucose! cultivation! was! induced! with! IPTG,! final!
concentration!0.2!mM,!when! reaching!OD600≈0.7,!while!the!lactose!cultivation!was!not!induced!by!
IPTG.!
Samples!from!the!cultures!where!taken!every!30!to!60!minutes,!starting!at!OD600≈2.!If!the!sample!had!
an!OD600>2,!it!was!diluted!to!OD600=2!in!Saline!(NaCl!0.9%).!500!μl!glycerol!(50%)!was!added!to!500!μl!
sample,!giving!a!final!OD600!of!1,!and!mixed.!The!samples!where!then!stored!in!B80°C!until!analysis.!! 3.2.2.2 FACS(analysis( The!surface!expression!was!measured!by!FluorescenceBactivated!cell!sorting!(FACS).!First,!the!cells! needed!to!be!labeled!with!a!Mouse!monoclonal!antibody!(MsMAb!to!cBMyc!Surelight!Allophycyanin),! binding!to!the!MycBtag!on!the!expressed!peptide.!The!lactose!and!glucose!cultures!where!compared! to!a!negative!control,!the!0:17!cell!not!carrying!the!pAIDA!plasmid!and!thus!not!expressing!peptides.! The!protocol!was!as!follows:!
• A! volume! corresponding! to! OD*V! =! 50! was! added! to! an! eppendorfBtube! containing! 800! μl! PBS!and!mixed.!
• The!sample!was!centrifuged!at!4500!rpm,!for!10!minutes!at!4°C,!after!which!the!supernatant! was!pipetted!off.!
• The! cell! pellet! was! reBsuspended! in! 100! μl! antibody! solution! (diluted! 10! times! in! PBS),! and! incubated!for!60!minutes!in!room!temperature.!
• After!incubation,!the!samples!were!centrifuged!in!the!same!manner!as!before,!and!washed! with!100!μl!PBS.!They!were!then!centrifuged!again.!
• When! supernatant! had! been! removed,! the! cell! pellet! was! reBsuspended! in! 500! μl! ice! cold! PBS,!and!kept!on!ice!until!FACS!analysis.!
Right!before!FACS!analysis,!the!samples!were!vortexed!and!then!moved!to!a!flow!cytometer!tube!and! inserted! into! the! FACSBmachine! (Gallios! Flow! Cytometer,! Beckman! Coulter).! The! intensity! of! the! samples!was!analyzed!at!660!nm,!after!samples!had!been!excited!at!638!nm.!
3.3 BIOFILM(FORMATION(
3.3.1 BIOFILM(FORMATION(WITH(LACTOSE(AS(CARBON(SOURCE(
3.3.1.1 BiofilmEcultivation((
25Bml! cultures! with! either! lactoseB! or! glucoseBminimal! medium! were! cultivated! overnight! at! 37°C.! The!cells!were!inoculated!into!fresh!medium!when!they!had!reached!exponential!phase!and!added!to! I!column!(eight!wells)!of!a!96Bwell!polystyrene!microtiter!plate,!100!μl!in!each!well.!The!cultures!were! then!incubated!for!24!and!48!hours,!at!37°C!and!180!rpm,!after!which!the!biofilm!formation!of!the!to! media!were!compared!using!crystal!violet.!The!staining!of!the!cells!were!performed!as!follows:!
• The!medium!was!removed,!and!the!wells!were!washed!from!loosely!attached!cells,!five!times! with!200!μl!sterilized!deionized!H2O.!
• The!plates!were!then!set!to!airBdry!for!45!minutes.!
• 150! μl! of! Crystal! Violet! (1%)! was! added! to! each! well,! and! they! were! incubated! for! 45! minutes.! • Crystal!violet!was!removed!and!the!wells!were!washed!in!the!same!manner!as!before.! • 200!μl!of!ethanol!(95%)!was!added!to!each!well!to!destain!the!biofilm.! • 100!μl!liquid!was!moved!to!new!wells!and!analyzed!in!a!plate!reader!at!595!nm.!
3.3.2 (BIOFILM(FORMATION(ON(CYTOLINE(1(BEADS(
3.3.2.1 Washing(procedure( Before!use,!the!beads!needed!to!be!washed!according!to!instructions!provided!by!GE!Healthcare.! • First,! the! beads! were! put! in! an! Erlenmeyer! flask! and! twice! the! volume! of! deionized! water!was!added.!The!beads!were!then!autoclaved!at!121 °C!and!1!bar!for!10!minutes.!
• After! autoclaving,! the! beads! were! sieved! to! remove! the! water,! put! back! in! the! flask! and! twice! the! volume! of! deionized! water! was! once! again! added.! The! flask! was! stirred! for! 10! minutes.!This!step!was!repeated!two!times.!
• An! equal! volume! of! NaOH! 0.1! M! was! added! to! the! microcarriers! and! this! mixture! was! incubated!overnight.!
• The!beads!were!washed,!in!the!same!manner!as!before,!five!times!until!the!NaOH!was!totally! removed.!
• The!beads!were!then!once!again!autoclaved,!this!time!at!121!°C,!1!bar!for!20!minutes.! 3.3.2.2 Wet(weight(and(dry(weight(measurements(
The! autoclaved! beads! were! sieved! for! ten! minutes! after! which! 13! aliquots! of! approximately! 1.3! grams! were! weighed! in.! According! to! GE! Healthcare! the! density! of! Cytoline! 1! is! 1.32! g/cm2,! and!
therefore!1.3!g!should!correspond!to!1!ml!beads!(GEHealthcare!2006).! The!beads!were!after!weighing!put!in!preBweighed!DWBtubes!and!put!in!a!105°C!oven!overnight.!The! following!morning!the!tubes!were!weighed!and!the!beads!were!put!in!100!ml!Erlenmeyer!flasks.!10! ml!minimal!medium!was!added!to!each!flask!and!they!were!autoclaved!at!121°C!and!1!bar!for!20! minutes.! One!of!the!aliquots!were!sieved!for!10!minutes!and!weighed!after!autoclaving.!The!beads!were!then! put!in!a!DWBtube!and!dried!overnight!in!a!105°C!oven,!after!which!they!were!weighed!again.!This!was! to!see!whether!the!weight!of!the!beads!is!affected!by!autoclaving.!
Lactose! of! a! final! concentration! of! 10! g/l! was! added! to! six! 100! ml! flasks,! while! glycerol! of! a! final! concentration!of!10!g/l!was!added!to!the!other!six.!To!all!flasks!Chloramphenicol,!MgSO4!and!trace!
elements! at! a! final! concentration! of! 10! mg/l,! 1! mM! and! 1! mM! respectively,! were! added.! To! the! glycerol! flasks,! IPTG! at! a! final! concentration! of! 0.2! mM! was! added! for! induction! of! the! lacUV5! operon.!
Three!flasks!with!lactose!and!three!flasks!with!glycerol!were!inoculated!with!inoculum!in!exponential! phase! (grown! on! lactose! and! glycerol! respectively)! to! a! final! OD600!=! 0.1.! All! twelve! flasks! were!
The!incubation!was!stopped!after!seven!days.!At!this!time,!the!beads!were!washed!with!deionized! water!and!sieved!for!10!minutes,!after!which!wet!weight!was!measured.!When!this!was!done,!the! beads! were! put! in! preBweighed! DWBtubes! and! put! in! 105°C! overnight.! The! following! morning! the! beads!were!weighed!in!the!tubes,!for!dry!weight!measurements.!
3.3.2.3 Biofilm(cultivation(for(adsorption(test(
The,! after! washing! procedure,! autoclaved! beads! were! sieved! and! four! aliquots! of! approximately! 3! grams!of!beads!were!weighed.!The!beads!were!put!in!500!ml!Erlenmeyer!flasks!and!50!ml!minimal! medium! was! added.! The! flasks! containing! the! beads! were! autoclaved! at! 121°C! and! 1! bar! for! 20! minutes.!
Lactose!of!a!final!concentration!of!10!g/l!was!added!to!two!500!ml!flasks,!while!glycerol!of!a!final! concentration! of! 10! g/l! was! added! to! the! other! two! 500! ml! flasks.! To! all! flasks! Chloramphenicol,! MgSO4!and!trace!elements!at!a!final!concentration!of!10!mg/l,!1!mM!and!1!mM!respectively,!were!
added.!To!the!glycerol!flasks,!IPTG!at!a!final!concentration!of!0.2!mM!was!added!for!induction!of!the! lacUV5!operon.!
One! flask! with! lactose! and! one! flask! with! glycerol! were! inoculated! with! inoculum! in! exponential! phase! (grown! on! lactose! and! glycerol! respectively)! to! a! final! OD600!=! 0.1.! All! four! flasks! were!
incubated!at!37°C!and!100!rpm.!Every!three!days!approximately!half!the!medium!was!taken!out!and! fresh!medium!was!added.! The!incubation!was!stopped!after!eleven!days.! 3.3.2.4 FluorescenceEmicroscopy( In!order!to!visualize!the!bacteria!on!the!carrier,!they!were!labeled!in!the!same!manner!as!previously! described!for!FACSBanalysis,!but!with!two!labels!instead!of!one.!One!binding!to!the!MycBtag!and!one! binding!to!the!His6Btag.!Two!samples!were!prepared,!one!with!beads!incubated!with!cells!and!lactose! as!carbon!source,!and!one!with!lactose!as!carbon!source!but!no!cells,!for!11!days.! A!fluorescence!microscope!was!used!to!visualize!the!cells,!along!with!phase!contrast!to!visualize!the! bead!surface.!A!carrier!bead!not!treated!in!any!way!was!used!as!reference!to!investigate!whether!the! carrier!itself!fluoresces.!
3.3.3 BIOFILM(FORMATION(ON(BIOFILMCHIP(M(CARRIERS(
Four!aliquots!of!three!Biofilmchip!M!was!dried!in!105°C!overnight.!The!following!morning!the!chips! were! weighed.! The! aliquots! were! put! in! flasks! and! 50! ml! minimal! medium! was! added.! The! biofilmchips!were!autoclaved!at!121°C!and!1!bar!for!20!minutes,!together!with!the!medium.!Glycerol,!Chloramphenicol,!MgSO4,!trace!elements!and!IPTG!were!added!at!the!final!concentrations!
stated!above.!Two!flasks!were!inoculated!with!inoculum!in!exponential!phase!to!a!final!OD600!=!0.1.!
All!four!flasks!were!incubated!at!37°C!and!100!rpm.!
After! four! days! approximately! half! the! medium! was! taken! out! and! fresh! medium! was! added.! The! incubation! was! stopped! after! seven! days! and! the! chips! were! washed! with! deionized! water! and! sieved.!The!chips!were!then!dried!in!80°C!overnight,!after!which!the!chips!were!weighed.!
3.3.4 ADSORPTION(TEST(ON(BIOFILM(
The!wet!weight!of!the!beads!was!measured!and!the!beads!were!put!in!50!ml!Erlenmeyer!flasks.!9900! μl!PBS!pH!7.5!and!100!μl!NiSO !(1!g/l)!was!added!to!each!aliquot.!The!samples!were!incubated!at!
22°C! and! 180! rpm! for! one! hour,! after! which! 5! ml! sample! was! pipetted! off! from! each! aliquot! and! stored!at!4°C!until!analysis.!
The!samples!were!analyzed!by!ICPBOES!at!Applied!Physical!Chemistry!at!KTH.!
4 RESULTS(
4.1 EXTENT(OF(NICKEL(POLLUTION(IN(VIETNAM(
4.1.1 WATER(SAMPLING(IN(NORTHERN(VIETNAM(
In!order!to!investigate!the!nickel!pollution,!three!locations!in!Hanoi,!S1,!S2!and!S3,!were!chosen!for! water!sampling.!The!locations!chosen!had!in!a!previous!study!showed!high!concentrations!of!nickel! due!to!pollution!from!industries!in!the!area!(Huong!et!al.!2008).!The!chosen!locations!can!be!seen!in! Figure!4.! Two!locations!in!Hai!Phong,!S4:!dam,!S5:!lake,!and!three!locations!in!Thai!Nguyen,!S6:!swamp,!S7:! dam,!and!S8:!creek,!all!close!to!industrial!zones,!were!also!tested!for!nickel!pollution.! As!can!be!seen!in!Table!1!the!nickel!concentrations!detected!at!the!sampling!sites!in!Vietnam!are!not! as!high!as!have!been!detected!previously!(Huong!et!al.!2008).!This!can!be!due!to!the!fact!that!the! removal! of! nickel! from! industrial! water! has! improved! since! previous! study! was! performed.! Or! perhaps!the!nickel!pollution!varies!depending!on!day!or!time!of!the!day.! Table(1.(The(nickel(concentrations(at(the(eight(different(sampling(sites(in(Vietnam.( Sampling(site( S1( S2( S3( S4( S5( S6( S7( S8( Concentration(mg/l( 0.005! 0.006! 0.008! 0,028! 0,013! <0.005! <0.005! <0.005! !4.2 PEPTIDE(EXPRESSION(
This!section!investigates!the!first!variable!connected!to!efficiency!of!the!method,!peptide!expression.! Previous! work! has! shown! that! this! cell! expresses! the! EC4BGly! peptide! when! cultivated! on! glucose! minimal!medium!and!induced!by!IPTG.!Expression!may!be!different!with!different!inducers!as!well!as! different! times! of! induction.! The! growth! phase,! which! the! cell! is! in,! may! also! influence! the! expression.!The!strategy!of!this!part!was!therefore!to!show!that!the!cell!expresses!the!peptide!and!then! compare! the! expression! using! different! carbon! sources,! as! well! as! compare! expression! at! different!growth!phases.!
4.2.1 PRIMARY(ADSORPTION(TEST(
A!primary!adsorption!test!was!performed!to!see!whether!or!not!the!peptides!actually!adsorb!nickel! ions.!Adsorption!to!EC4BGly!was!tested!at!two!different!optical!densities!(OD),!56!and!100.!The!wild! type!0:17!was!used!as!reference.!The!metal!concentration!in!the!reference!without!cells!was!measured!to!7.65!mg/l.!In!Table!2!the! concentrations!after!adsorption!are!shown.! Table(2.(The(remaining( concentration(of(Ni2+(after( adsorption(test(in(mg/l.( ( 0:17( EC4=Gly( OD(56( 2.30! 0.98! OD(100( 1.66! 1.83! Considering!the!first!experiment!where!OD600!=!56,!and!assuming!that!each!EC4BGly!can!bind!four! metals,!that!the!His6Btag!can!bind!one!metal!ion!and!that!each!cell!expresses!100!000!peptides!on!its!
surface,! one! milliliter! with! OD600! =! 56! has! the! potential! of! binding! 3.3×1016! metal! ions.! In! fact,!
6.8×1016! metal! ions! have! been! adsorbed.! This! probably! means! that! the! cell! surface! itself! adsorbs!
metal!ions.!This!can!also!be!seen!in!Table!2.!!
The!0:17!cells,!not!expression!peptides,!have!adsorbed!5.5×1016!metal!ions!per!milliliter.!Considering!
this!it!seems!that!the!peptides!have!adsorbed!1.3×1016!metal!ions,!meaning!that!less!than!half!of!the!
peptides!are!occupied.!
However,!the!second!experiment,!with!OD600!=!100,!where!these!cells!actually!adsorb!less!than!they!
did! at! OD=55.! The! result! is! therfore! not! conclusive.! This! was! further! investigated! by! Holmström,! 2012.!
4.2.1.1 Expression(of(peptides(
To! make! sure! that! the! cells! used! in! the! above! experiment! actually! express! the! peptides! on! its! surface,! SDSBPAGE! and! Western! Blot! was! performed.! By! this! method,! one! cannot! know! if! the! peptides!are!expressed!on!the!surface!of!the!cell,!or!intracellular.!However,!this!was!the!only!method! available!at!the!university!in!Hanoi.!
! Figure( 5.( The( results( of( the( SDS=PAGE( and( Western( blot( done( to(
!
The! EC4BGly! expression! was! compared! to! that! of! pAIDA! and! 0:17.! For! Western! blot,! an! antiBHis6!
antibody! was! used,! which! means! that! there! should! be! an! expression! for! both! pAIDA! and! EC4BGly.! One! gel! was! used! for! both! SDSBPAGE! and! Western! blot.! For! SDSBPAGE,! an! unstained! marker! was! used,!and!for!Western!blot,!a!preBstained!marker!was!used.! Figure!5!shows!the!Coomassie!brilliant!blue!stained!SDSBPAGE!gel!and!the!Western!blot!membrane.! On!the!Western!blot!membrane,!shown!to!the!right,!one!can!see!that!the!two!lines!where!expression! was!expected!actually!show!two!clear!lines,!at!approximately!60!kDa.!Because!of!uneven!lines!it!is! not!possible!to!find!the!bands!in!the!SDSBPAGE!that!corresponds!to!the!band!in!the!Western!blot.!It! can!hereby!be!deduced!that!the!cells!express!the!His6Btag,!and!thus!most!probably!also!the!EC4BGly! peptide.!This!was!also!reinforced!by!results!from!FACS,!see!4.2.2.2.!
4.2.2 SURFACE(EXPRESSION(USING(LACTOSE(AS(INDUCER(AND(SOLE(CARBON(SOURCE(
Previous! work! has! shown! that! the! E.! coli! cell! expresses! the! wanted! peptides! on! its! surface! when! cultivated!on!glucose!and!induced!by!IPTG,!a!lactose!analogue!(Tan!2011).!However,!IPTG!is!a!quite! expensive!chemical,!as!well!as!more!toxic!than!lactose.!It!was!therefore!interesting!to!see!if!the!E.)
coli) would! express! the! peptides! as! well! if! cultivated! on! lactose! as! sole! carbon! source,! and! thus!
inducing!the!lacUV5Boperon!continuously.! 4.2.2.1 Cultivation(
In! the! two! graphs! in! Figure! 6! the! two! curves,! one! for! cells! growing! on! glucose,! and! one! for! cells! growing!on!lactose!are!almost!identical.!Both!cultivations!shows!a!μmax!of!0.87.!Therefore,!one!can!
see!that!there!is!no!difference!between!growing!the!cells!on!glucose!or!lactose.! ! 0! 0,5! 1! 1,5! 2! 2,5! 3! 3,5! 0! 100! 200! OD 600 ( Time((min)( Glucose! Lactose! 0! 1! 2! 3! 4! 5! 0! 100! 200! 300! 400! OD 600 ( Time((min)( Glucose! Lactose! Figure(6.(To(the(right(the(growth(curve(of(pAIDA(EC4=Gly(cultivated(on(glucose(and(lactose(are(compared.(To(the(left(are(the( same(two(curves,(however,(here(the(exponential(phase(is(zoomed(in.(
!
4.2.2.2 FACS(analysis(
Four! samples! were! chosen! for! FACSBanalysis,! two! from! the! lactose! cultivation! and! two! from! the! glucose!cultivation.!To!be!comparable,!sample!with!similar!OD600,!and!hence!probably!in!the!same!
phase! were! chosen.! The! OD600! of! the! samples! analyzed! are! shown! in! Table! 3.! One! sample! from!
exponential! phase,! and! one! sample! from! when! the! cells! just! had! reached! stationary! phase,! were! taken!from!each!cultivation!flask.!The!reason!for!taking!samples!from!two!different!growth!phases!is! that!the!cell!may!express!differently!at!different!growth!rates.!Also,!peptides!may!start!to!be!cleaved! off!when!the!cells!enter!stationary!phase.! ! Table(3.(The(mean(fluorescent(values(of(the(four(samples(analyzed.(The(value(within(brackets(is(the(ungated(value.( ( Exponential(phase( Early(stationary(phase( ( Glucose! Lactose! Glucose! Lactose!
OD600( 3.35! 3.48! 4.30! 4.28! Mean( Fluorescence( 288! 206!(171)! 209! 208! ! In!Figure!7,!the!flow!cytometry!results!from!0:17!without!plasmid,!0:17!pAIDABEC4BGly!cultivated!on! lactose,!and!0:17!pAIDABEC4BGly!cultivated!on!glucose!are!visualized!in!the!same!image.!These!are! the!results!from!the!samples!taken!during!the!exponential!growth!phase.!As!can!be!seen!from!the! picture,!the!cells!grown!on!glucose!and!induced!by!IPTG!shows!a!slightly!higher!mean!fluorescence! compared!to!the!cells!grown!on!lactose,!288!compared!to!206!(gated).!! ! !
Figure( 7.( Visualization( of( expression( at( exponential( phase.( The( results( to( the( left( shows( the( visualization( of( the( fluorescence( ungated.(Here,(one(can(see(there(is(a(number(of(cells(cultivated(on(lactose(that(does(not(express(the(Myc=tag,(or(at(least(no(anti( Myc(antibodyhas(bound(to(them.(The(figure(to(the(right(shows(a(gated(version(of(the(same(results.(The(mean(fluorescence(value( of(the(cells(cultivated(on(lactose(is(171(for(the(ungated(and(206(for(the(gated(version.(
Looking!at!Figure!8,!where!results!from!early!stationary!growth!phase!are!shown,!one!can!see!that! mean!fluorescence!values!are!the!same!208!and!209.!From!these!two!results,!it!was!concluded!that! the!difference!in!peptide!expression,!if!any,!was!small!enough!to!be!disregarded.!Therefore,!lactose! can!be!used!as!inducer!as!well!as!carbon!source!with!regards!to!surface!expression!and!maximum! growth!rate.! ! ! !
4.3 BIOFILM(FORMATION(
This!section!investigates!the!second!variable!connected!to!the!efficiency!of!the!method,!total!amount! of!peptides.!When!the!cells!express!the!peptide!satisfactory,!the!next!step!to!improving!the!efficiency! of!the!method!is!to!increase!the!concentration!of!peptides!by!increasing!the!cell!numbers.!This!can! be!done!by!letting!the!cells!form!biofilm,!and!thereby!increasing!the!cell!mass!in!a!certain!volume.! The! strategy! of! this! part! was! therefore! to! investigate! the! biofilm! formation! potential! of! the! cell! expressing!the!peptide!and!also!to!compare!the!biofilm!formation!potential!of!the!cell!using!different! carbon!sources.!Finally,!it!was!to!try!the!efficiency!of!this!method.!4.3.1 BIOFILM(FORMATION(WITH(LACTOSE(AS(CARBON(SOURCE(
A!previous!master!thesis!work!has!shown!that!the!strain!used!in!this!study!can!form!a!biofilm.!The! work! then! showed! that! the! cells! formed! most! biofilm! if! cultivated! in! glycerol! minimal! medium! (compared! to! glucose! minimal! medium),! at! 37°C,! and! the! inoculation! was! performed! during! the! exponential! phase.! (Nobili! 2010)! However,! the! plasmid! then! used! for! surface! expression! of! an! enzyme!had!a!constitutive!promoter,!instead!of!a!regulated!promoter!used!in!this!work.!This!meant! that! the! cells! in! the! biofilm! never! needed! to! be! induced! to! express! the! desired! enzyme.! This! induction!can!cause!problems!when!one!does!not!know!the!optimal!time!for!induction.!Therefore!it! was!decided!to!investigate!whether!this!strain!forms!biofilm!when!cultivated!in!lactose!medium.! The! results! from! the! biofilm! cultivation! in! the! 96Bwell! polystyrene! microtiter! plates! are! shown! in! Table!4!and!visualized!in!Figure!9.!
Table(4.(Absorbance(after(24(and(48(hours(cultivation(in(polystyrene(microtiter(plates,(with(glycerol(and(lactose(as( carbon(source.( Glycerol Lactose 24 h 48 h 24 h 48 h Absorbance 0,14 0,43 0,15 0,25 Standard deviation 0,03 0,12 0,02 0,10 ! As!can!be!seen!there!is!no!difference!between!glycerol!and!lactose!as!carbon!source!after!24!hours,! but! there! is! a! difference! after! 48! hours.! However,! after! 48! hours! a! large! part! of! the! medium! had! evaporated,!which!is!why!it!is!uncertain!whether!this!large!increase!in!absorbance!for!glycerol!is!due! to!biofilm!formation!or!drying!of!cells!to!the!surface.!In!previous!work,!the!absorbance!had!actually! decreased!after!48!hours,!which!would!mean!degradation!of!the!biofilm!(Nobili!2010).! ! ! The!absorbance!in!glycerol!minimal!medium!after!24!hours!is!considerably!less!than!what!was!shown! in!previous!work,!which!may!be!due!to!high!shaking,!180!rpm!compared!to!100!rpm!or!no!shaking,! commonly! used! in! this! sort! of! experiment! (Andersson! et! al.! 2008;! Pratt! et! al.1998;! Danese! et! al.! 2000).!This!theory!is!supported!by!the!fact!that,!cell!mass!could!be!seen!as!little!granules!in!the!wells! after!24!and!48!hours!of!cultivation.!!
Due! to! lack! of! time! this! experiment! was! not! repeated! with! the! same! conditions! used! in! pervious! work,!and!the!reason!for!this!low!absorbance!is!not!conclusive.!
4.3.2 BIOFILM(FORMATION(ON(CYTOLINE(1(BEADS(
Cytoline! 1,! a! macroporous! microcarrier,! see! Figure! 10,! from! GE! Healthcare! was! chosen! as! carrier! since! previous! work! has! shown! that! this!organism!can!form!a!biofilm!on!this!carrier!(Nobili 2010).!To!see!if! there! was! a! difference! in! the! formation! and! when! doing! adsorption! tests,!two!sets!of!biofilm!cultivations!were!set!up,!one!with!lactose!and! one! with! glycerol! as! carbon! source.! All! biofilm! cultivations! were! performed!in!Erlenmeyer!flasks,!and!not!shake!flasks,!see!Figure!11.!This! was!partly!due!to!limitations!in!materials,!but!also!because!of!fear!that! this!would!lead!to!shearing!of!the!biofilm.!The!downside!of!not!having! baffles!is!the!even!more!limited!oxygen!transfer.!!
Different! methods! for! detection! of! biofilm! formation! were! considered.! Crystal! violet! staining,! dry! 0! 0,1! 0,2! 0,3! 0,4! 0,5! 0,6! Glycerol! Lactose! Ab so rb an ce ( 24!h! 48!h! Figure(10.(Picture(of(a(Cytoline(1( bead.( Figure(9.(Visualization(of(the(difference(in(absorbance(after( 24(and(48(hours(cultivation(in(polystyrene(microtiter(plates,( with(glycerol(and(lactose(as(carbon(source.(
violet! bound! to! the! beads! themselves! and! is! therefore! not! a! good! method.! Dry! weight! was! not! successful!either,!since!the!weight!of!the!cells!in!the!biofilm!was!so!much!less!than!the!weight!of!the! beads!and!DWBtube.!While,!wet!weight!measurements!indicated!biofilm!formation.!(Nobili!2010)!! FluorescenceBmicroscopy! where! the! MycB! and! His6Btag! are! labeled! with! fluorescent! probes,! and!
elemental!analysis!for!CHN!were!also!considered.!Since!elemental!analysis!is!quite!expensive,!it!was! decided!that!dry!weight,!wet!weight!and!fluorescence!microscopy!as!methods!of!analysis.! ! ! ! ! ! ! When!comparing!wet!weight!measurements!before!and!after!7!days!of!cultivation,!the!wet!weight! had! actually! decreased! for! all! aliquots.! This! is! probably! due! to! that! the! beads! were! drier! when! weighed!after!cultivation.!
However,!Table!5!and!Figure!12!shows!the!average!weight!increase!for!the!aliquots!from!dry!weight! before! biofilm! cultivation! to! wet! weight! after! cultivation.! Since! the! bead! aliquots! that! were! incubated!with!cells!have!increased!more!than!those!incubated!without!cells,!for!both!lactose!and! glycerol!as!carbon!source,!this!is!an!indication!that!a!biofilm!has!been!formed.! ! 0! 10! 20! 30! 40! 50! 60! 70! Lactose! Glycerol! W ei gh t(i nc re as e( in (%( No!cells! EC4!
Figure( 11.( Picture( of( cultivation( flasks( for( biofilm( formation( on( Cytoline(1(beads.(
Figure(12.(Visualization(of(weight(increase(from(dry(weight(of(beads( to(wet(weight(of(beads(with(cells.(
Table(5.(Weight(increase(from(dry(weight(of(beads(to(wet(weight(of(beads(with(cells.(
( Lactose( Glycerol(
( No!cells! EC4BGly! No!cells! EC4BGly!
Weight(increase(in(percent( 8.75±1.09! 45.5±3.21! 12.5±5.95! 47.7±12.9! However,!when!comparing!dry!weight!of!the!beads!before!and!after!biofilm!cultivation,!there!was!a! weight!increase!for!both!the!aliquots!incubated!with!and!without!cells.!Since!the!aliquots!incubated! without!cells!should!not!have!a!weight!increase,!this!is!a!mystery.!Not!properly!dried!DWBtubes!or! beads!could!be!an!explanation.! In!Table!6!and!Figure!13!the!dry!weight!increases!are!shown.!Even!though!there!was!an!increase!both! with! and! without! cells,! for! the! cultivation! with! lactose! as! carbon! source,! the! increase! with! cells! is! significantly!larger!than!the!increase!without!cells.!This!is,!however,!not!the!case!for!the!cultivation! with!glycerol!as!carbon!source.!
Table(6.(Increase(in(dry(weight(after(cultivation.(
( Lactose( Glycerol(
( No!cells! EC4BGly! No!cells! EC4BGly!
Weight(increase(in(percent( 0.23±0.038! 0.55±0.046! 0.31±0.23! 0.97±0.51!
!
Previous! work! has! shown! that! this! cell! forms! biofilm! better! with! glycerol! than! glucose! as! carbon! source!(Nobili!2010).!In!this!study,!no!difference!was!seen!after!24Bhour!incubation!in!polystyrene! microtiter!plates.!However,!the!reason!for!the!large!standard!deviation!seen!when!using!glycerol!as! carbon! source! on! Cytoline! 1! beads,! could! be! that! the! biofilm! is! not! very! stable! and! varies! a! lot,! compared!to!when!lactose!is!used!as!carbon!source.!
When!assessing!biofilm!formation!of!a!number!of!bacterial!strains!found!in!wastewater!treatment! systems,!(Andersson!et!al.!2008),!found!that!E.)coli)KB12!did!not!produce!detectable!quantities!of!EPS,! which!is!considered!essential!in!biofilm!formation.!This!could!be!the!reason!why!no!larger!quantities! of!biofilm!were!seen.!Also,!several!weeks!or!even!months,!are!often!used!for!biofilm!formation!and! stabilization! (Björlenius,! Personal! communication! 2012).! This! was! a! quite! short! time! for! biofilm! formation,!so!there!is!no!saying!if!this!strain!can!produce!a!more!stable!and!robust!biofilm.! 0! 0,2! 0,4! 0,6! 0,8! 1! 1,2! 1,4! 1,6! Lactose! Glycerol! W ei gh t(i nc re as e( in (%( No!cells! EC4! Figure(13.(Visualization(of(increase(in(dry(weight(after(cultivation.(
The! reason! for! forming! a! biofilm! was! to! increase! the! number! of! cells,! and! thereby! peptides,! in! a! certain!volume.!The!increase!in!dry!weight!for!the!beads!cultivated!in!lactose!medium!was!in!average! 0.0039!grams,!which!then!would!be!the!dry!weight!of!the!cells.!In!the!primary!adsorption!test!the!cell! concentration! used! was! OD600! =! 100,! which! roughly! corresponds! to! 30! g/l.! To! get! that! same!
concentration!using!the!Cytoline!1!beads!with!biofilm,!the!volume!would!have!to!be!as!little!as!130!μl! to! approximately! 1! ml! beads.! This! is! of! course! impossible;! hence,! even! though! the! cells! do! form! biofilm,!they!had!not!been!concentrated!when!forming!biofilm.! 4.3.2.1 FluorescenceEmicroscopy( Fluorescence!microscopy!was!used!in!order!to!visualize!the!bacteria!on!the!carrier.! The!fluorescenceBmicroscopy!visualization!of!cells!was!not!successful!since!the!surface!of!the!carrier! also!showed!fluorescence,!why!there!was!no!way!to!see!the!bacteria.!This!is!probably!due!to!that!the! labels!also!bound!to!the!carrier!surface!and!not!just!the!cells.!A!more!vigorous!washing!may!have! helped,!however,!a!too!vigorous!washing!may!also!wash!away!the!cells!from!the!surface.!
When! looking! at! the! carrier! bead! that! had! not! been! treated! with! any! of! the! labels,! a! slight! fluorescence!was!seen,!but!It!was!much!less!than!for!the!treated!bead.!
4.3.3 BIOFILM(FORMATION(ON(BIOFILMCHIP(M(CARRIERS(
Although! Cytoline! 1! has! shown! positive! results! for! biofilm! formation! previously,! it! would! not! be! feasible!to!use!them!in!large!scale!in!wastewater!treatment!plants.!Due!to!the!fact!that!Cytoline!1! beads!are!developed!for!cultivation!of!animal!cells,!the!price!is!much!too!high.!Biofilmchip!M,!Figure! 14,! from! Anoxkaldnes! is! however! a! much! cheaper.! Biofilmchip! M! is! the! carrier! from! Anoxkaldnes! with!the!largest!protected!surface!area,!1200!m2/m3!(Christensson!2009).!!
!
Only!biofilm!formation,!and!not!adsorption!by!that!biofilm!was!investigated!on!Biofilmchip!M.!The! biofilm!formation!was!investigated!using!dry!weight!since!that!seem!to!have!been!successful!for!the! biofilm!formation!on!Cytoline!1!beads.!
The! dry! weight! measurements! of! the! Biofilmchip! M! showed! increase! in! weight! for! both! the! chips! incubated!with!and!without!cells.!There!was!no!significant!distinction!between!the!weight!increase,! why!there!seem!not!to!have!been!any!detectable!biofilm!formation.!Of!course,!there!may!have!been! a!biofilm!formation!that!was!too!small!in!mass!to!be!detected!by!dry!weight.!!
This! E.) coli) strain! has! shown! biofilm! formation! in! polystyrene! microtiter! plates! and! indications! of! biofilm! formation! on! Cytoline! 1! beads! coated! with! silica.! The! Biofilmchip! M! is! made! out! of! polyethylene!or!polypropylene!(AnoxKaldnes!2009),!two!hydrophobic!plastics,!just!as!polystyrene.!It!
looks!as!the!cell!should!be!able!to!form!biofilm!just!as!good!on!either!of!the!materials,!however,!of! course!there!could!be!some!difference.!
A! possible! way! to! have! detected! any! biofilm! formation! could! have! been! crystal! violet! staining.! However,!that!was!not!performed.!
4.3.4 ADSORPTION(TEST(ON(BIOFILM(
An!adsorption!test!was!performed!to!investigate!the!efficiency!of!the!method!at!this!stage.!Although! the!differences!were!small,!a!study!running!parallel!to!this!one!showed!that!the!EC4BGly!adsorbed! nickel!best!at!pH!7.5!and!22°C.!It!also!showed!that!oneBhour!incubation!was!adequate.!(Holmström,! Personal! communication! 2012)! The! nickel! concentration! used! was! 10! mg/l,! which! is! much! higher! than!concentrations!seen!in!Swedish!municipal!wastewater!treatment!plants.!The!reason!for!this!is! the! detection! limit! of! the! instrument! used! for! analysis,! ICPBOES! with! approximately! 0.1! mg/l! in! detection!limit.!
The!wet!weight!measurements!showed!an!approximately!5%!increase!for!both!lactose!and!glycerol! cultivated! biofilms.! However,! the! Cytoline! beads! without! cells! in! glycerol! media! showed! an! approximately! 2%! increase,! which! should! not! be! possible.! This! was! probably! due! to! inadequate! sieving! of! those! beads,! and! this! further! emphasizes! the! room! for! error! involved! in! wet! weight! measurements.!
! !
The!diagram!in!Figure!15!shows!a!quite!extensive!adsorption!of!Ni2+!to!the!Cytoline!bead!itself!even!
though!a!significant!difference!between!beads!with!and!without!cells!can!be!seen.!!
The! starting! concentration! of! Ni2+! was! 10! mg/l.! The! beads! with! cells! cultivated! on! lactose! had!
adsorbed! 7.7! mg/l! and! the! beads! with! cells! cultivated! on! glycerol! had! adsorbed! 7.5! mg/l,! corresponding!to!790×1015!and!770×1015!Ni!ions!respectively.!Disregarding!what!the!beads!appear!to!
have! adsorbed,! this! indicates! that! the! cells! have! adsorbed! 135×1015! and! 120×1015! Ni! ions!
respectively.! Given! that! this! adsorption! test! has! only! been! performed! once! for! each! growth! condition,!and!thus!does!not!show!a!statistically!significant!difference,!this!difference!in!adsorption! cannot!be!determined.! 0! 10! 20! 30! 40! 50! 60! 70! 80! 90! Lactose! Glycerol! %( N i 2+(ad so rb ed ( Inoculated! Not!inoculated!
Figure( 15.( Visualization( of( the( adsorption( of( nickel( ions( to( Cytoline( 1( beads( with( and( without(biofilm,(cultivated(on(lactose(or(glycerol.(
5 DISCUSSION(
According!to!GE!Healthcare!specifications,!Cytoline!1!has!a!surface!area!of!>0.3!m2/g!(GEHealthcare! 2006).!Approximating!the!cells!to!circular!spheres!and!approximating!the!cell!diameter!to!0.8!μm,!the! number!of!cells!that!could!fit!on!the!surface!of!3!g!beads!in!a!single!layer!would!be!920×109!cells.! Assuming!that!100!000!peptides!are!expressed!on!the!cell!surface,!and!that!each!peptide!adsorbs!five! ions,!these!cells!should!be!able!to!bind!460×1015!ions.!The! number! of! ions! that! have! been! adsorbed! to! the! cells! is! only! about! 25%! of! the! hypothetical! potential.!Further,!it!has!previously!been!shown!that!the!cell!surface!itself!is!quite!good!at!adsorbing! nickel!ions.!
Either;!
• The! potential! of! the! peptides! is! lower! than! expected.! There! could! be! a! number! of! explanations!to!this!phenomenon;!the!cells!may!not!express!as!many!peptides!as!expected,! some! of! the! peptides! may! not! be! available! for! adsorption! due! to! cell! adhesion! to! bead! surface,!or!the!pH!of!the!nickel!buffer!may!make!the!amino!acids!in!the!peptides!neutrally! charged.!
• Or,!the!biofilm!formation!is!not!as!good!as!hoped.!!
The!typical!pKa!of!Cysteine!is!8.3,!while!the!typical!pKa!value!of!Glutamic!acid!is!4.1,!although!this!is!
dependent! on! temperature! and! ionic! strength! (Berg! et! al.! 2007).! Thus,! at! pH! 7.5! used! in! this! experiment!Cysteine!should!be!neutrally!charged,!while!Glutamic!acid!is!negatively!charged.!Histidine! has!a!typical!pKa!of!6.0,!which!makes!it!neutrally!charged!with!a!free!electron!pair!at!pH!7.5.!This!
means!that!the!EC4Bpeptide!may!only!bind!two!instead!of!four,!nickel!ions,!and!the!His6Btag!binding!
one.!
What! does! the! removal! efficiency! shown! here! translate! into! in! real! life?! Suppose! one! wants! to! implement!this!method!at!Henriksdalsverket!in!Stockholm.!Henriksdalsverkat!has!an!average!flow!of! 252!000!m3!per!day (Stockholm!Vatten!AB),!and!the!average!nickel!concentration!coming!into!the! plant!is!7.8!μg/l!(André!et!al.!2006).!This!means!that!every!day,!1966!g!nickel!reaches!the!plant.!With! the!adsorption!potential!shown,!using!the!same!beads!to!liquid!ratio,!a!15!m3!basin,!containing!4540! kg!Cytoline!1!beads,!would!be!needed.!However,!this!only!takes!into!account!the!maximal!adsorption! and!not!that!that!adsorption!is!an!equilibrium!or!the!retention!time!in!the!basin.!! In!the!example!above,!the!retention!time!would!only!be!5!seconds,!considering!one!hour!a!day!is! used! for! regeneration! of! the! cells,! and! probably! not! enough! for! optimal! adsorption.! For! one! hour! retention! time! in! the! basin,! the! volume! would! have! to! be!10960! m3,! with! 3300! tonnes! Cytoline! 1!
beads,!which!is!an!enormous!amount.!
In!a!study!running!in!parallel!with!this!one,!the!optimal!adsorption!conditions!for!this!recombinant! cell!was!investigated.!These!experiments!were!performed!as!batch!with!suspended!cells.!The!highest! removal! efficiency! was! reached! at! 16! C,! at! pH! 6.5! with! an! incubation! time! of! 2! h.! This! removal! efficiency! was! 89.6%.! However,! the! reference! cell,! the! wild! type! 0:17,! had! a! maximum! removal! efficiency! of! 90.3%! at! 22! C! and! pH! 7.5.! (Holmström! 2012)! That! such! large! part! of! the! nickel! ions! adsorb!to!the!cell!surface!itself!could!become!a!problem!when!regenerating!the!cells.!
None!of!the!experiment!performed!in!this!study!or!in!the!study!done!by!Holmström,!2012,!shows!as! high!removal!efficiency!as!do!RO,!NF,!and!IE,!which!is!99.3%,!98.94%,!and!93.6%!respectively!(Ipek!
2005;!Murthy!et!al.!2009;!Argun!2008).!However,!the!methods!with!highest!nickel!removal!efficiency! are! the! methods! operating! at! very! high! pressures.! The! pressures! used! in! the! experiments! where! these! numbers! are! derived! from! operated! at! 1100! kPa! for! RO! (Ipek! 2005)! and! 2000! kPa! for! NF (Murthy!et!al.!2009),!this!can!be!compared!with!the!pressure!in!a!car!tire,!which!is!approximately!200! kPa.!
A! few! measures! that! have! been! suggested! for! improving! the! adsorption! is! longer! linkers! or! more! repeats!of!Glutamic!acid!and!Cysteine.!Longer!linker!would!provide!the!benefit!of!making!the!EC4! more! available,! since! it! may! be! hidden! at! the! rough! cell! surface! using! a! too! short! linker.! A! longer! linker!may,!however,!have!its!own!disadvantages;!a!longer!linker!may!fold!itself,!and!thus!not!making! the!EC4!more!available!and!maybe!even!less!available,!through!for!example!HistidineBlinkages.!!
5.1 IMPLEMENTATION(ISSUES(
The!hope!is!to!eventually!be!able!to!implement!this!method!in!large!scale.!However,!there!is!still!a! long!way!to!go!before!this!can!happen.! When!looking!at!where!to!implement!this!nickelBremoving!step!there!are!two!options,!before!or!after! the!biological!treatment.!Implementing!nickel!removal!before!has!the!advantage!of!removing!metals! before!they!can!end!up!in!the!sludge.!The!sludge!produced!in!wastewater!treatment!plants!may!be! used! as! fertilizer! on! farmlands! or! as! substrate! for! biogas! production.! In! both! cases,! it! is! crucial! to! have!as!little!metals!as!possible!in!the!sludge.!However,!before!biological!treatment,!there!may!be!a! lot!of!compounds!in!the!water!that!are!either!harmful!to!the!cells,!or!prohibit!adsorption.!Implementing! the! nickelBreducing! step! after! biological! treatment! has! the! advantage! of! the! cells! working!in!a!cleaner!and!more!controlled!environment.!However,!large!parts!of!the!incoming!nickel! may!already!have!ended!up!in!the!sludge.!
Where! to! implement! this! method! is! not! the! only! issue! at! hand.! Many! studies! using! cells! for! biosorption!uses!already!dead!cells,!maybe!this!is!a!better!option!than!using!live!cells,!as!are!used! here.!However,!that!will!mean!that!other!issues!come!up;!where!are!the!cells!to!be!cultivated!in!the! first! place,! how! are! they! to! be! transported! to! the! location! for! adsorption,! will! this! effect! the! expressed!peptides?!
In!wastewater!treatment!plants!today,!the!organisms!in!each!biological!step!are!selected!by!using! optimal!conditions!for!the!organism!needed.!This!means!that!the!cells!are!growing!all!the!time,!and! some! are! always! discarded! as! sludge.! If! this! were! the! case! for! these! recombinant! organisms,! measures!would!have!to!be!taken!to!make!sure!that!no!cells!are!released!into!recipient!waters!since! there!are!many!regulations!regarding!such!organisms.!Also,!in!this!study,!a!resistance!to!antibiotics!is! used!for!selection;!this!could!not!be!used!in!large!scale,!in!case!of!cells!escaping.!!
Another! thing! that! would! have! to! be! investigated! is! whether! the! cells! should! be! suspended! or! cultivated! as! biofilm.! The! question! is! what! gives! highest! efficiency,! and! if! the! efficiency! is! in! fact! increased!by!increased!cell!mass.!In!this!study,!Cytoline!1!beads!and!Biofilmchip!M!were!tested!as! support! materials! for! biofilm! formation,! the! beads! being! far! too! expensive! for! large! scale! and! the! Biofilmchip! M! being! a! more! reasonable! material.! An! even! cheaper! material! that! could! be! used! is! sand.!If!a!sandBbed!were!to!be!used,!it!would!probably!have!to!be!placed!after!biological!treatment! when!the!water!is!clean!and!the!risk!of!clogging!is!minimized.!
The! biofilm! formation! on! Biofilmchip! M! was! not! detectable! in! the! experiments! performed! in! this! study.!However,!the!briefness!of!the!tests!may!explain!lack!of!biofilm.!But!if!one!were!to!implement! this!on!a!large!scale,!and!do!not!want!to!use!suspended!cells,!one!would!need!a!cell!that!forms!strong! biofilm.!Either,!the!expression!system!can!be!tried!in!a!biofilm!forming!cell!strain!that!also!grows!on! minimal!medium,!or!the!E.)coli!strain!could!be!modified!to!form!biofilm,!for!example!by!producing! EPS.! Despite!the!issues!of!implementing!this!method!it!has!advantages!that!makes!it!worth!the!effort!of! further! research.! Using! peptides! for! adsorption! enables! specificity! for! certain! metals! not! possible! with! other! methods,! whether! that! metal! is! nickel,! cadmium! or! any! other! problematic! metal.! And! using!such!an!easily!cultivated!and!wellBknown!cell!as!E.)coli!gives!a!great!advantage.!
6 CONCLUSION(
The!method!of!using!E.)coli!expressing!EC4BGly!peptides!on!its!surface!does!not!show!the!same!nickel! removal!efficiency!as,!for!example,!reversed!osmosis!or!nanofiltration.!Examples!of!how!to!improve! the!efficiency!could!be!to!modify!the!expressed!peptide!to!better!adsorb!nickel,!or!to!modify!the!cell! to! produce! EPS! and! form! stronger! biofilm.! This! method! is! not! implementable! at! the! moment,! but! with!further!research,!it!could!be!in!the!future.!
