Engineering microbial communities using thermodynamic principles and electrical interfaces

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Original citation:

Zerfass, Christian, Chen, Jing and Soyer, Orkun S.. (2018) Engineering microbial communities

using thermodynamic principles and electrical interfaces. Current Opinion in Biotechnology,

50 . pp. 121-127.

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Engineering

microbial

communities

using

thermodynamic

principles

and

electrical

interfaces

Christian

Zerfa

ß

,

Jing

Chen

and

Orkun

S

Soyer

Microbialcommunitiespresentthenextresearchfrontier.We arguehere thatunderstanding andengineeringmicrobial communitiesrequiresaholisticviewthatconsidersnotonly species–species,butalsospecies–environmentinteractions, andfeedbacks betweenecologicalandevolutionary dynamics(eco-evofeedbacks).Duethismulti-levelnatureof interactions, wepredictthatapproachesaimedsoley at alteringspecificspeciespopulationsinacommunity (through strainenrichmentorinhibition),wouldonlyhavea transient impact,andspecies–environmentandeco-evo feedbackswouldeventuallydrivethemicrobialcommunityto its originalstate. Wepropose ahigher-levelengineering approach thatis basedon thermodynamicsofmicrobial growth,andthat considersspecifically microbialredox biochemistry. Withinthisapproach,theemphasisison enforcing specificenvironmentalconditionsontothe community.These areexpectedto generatehigher-level thermodynamic boundsonto thesystem,whichthe community structureandfunctioncanthen adaptto. We believethatthe resultingend-statecanbeecologicallyand evolutionarilystable,mimickingthenaturalstatesofcomplex communities.Towarddesigningthe exactnatureof the environmentalenforcement,thermodynamicsandredox biochemistrycanactascoarse-grainedprinciples,whilethe useofelectrodes—aselectronprovidingoracceptingredox agents—canprovideimplementationwithspatiotemporal control.

Addresses

1_{WarwickIntegrativeSyntheticBiologyCenter(WISB),Universityof} Warwick,UnitedKingdom

2

SchoolofLifeSciences,UniversityofWarwick,UnitedKingdom

Correspondingauthor:Soyer,OrkunS([email protected])

CurrentOpinioninBiotechnology2018,50:121–127 ThisreviewcomesfromathemedissueonEnvironmental biotechnology

EditedbyMikeJettenandIreneSa´nchezAndrea

https://doi.org/10.1016/j.copbio.2017.12.004

0958-1669/_ã2017TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense( http://creative-commons.org/licenses/by-nc-nd/4.0/).

Introduction

Microbialcommunitiesperformkeybiochemical

trans-formations of organic and inorganic matter,

underpin-ning the biogeochemical cycles on Earth [1,2] and

playing a crucial part in the nutrition and health of

higherorganismsincludinghumans,animals,andplants

[3,4].Thus, it is notsurprisingthat there isincreasing

interest in understanding and engineering microbial

communities forenvironmental, medical, and

biotech-nological applications [5,6–9,10,11]. Engineering of

microbial communities has been proposed both as a

top-down approach, controlling metabolic processes

for stabilizing complex, natural communities [5,6,7]

and as a bottom-up approach, for designing defined,

synthetic communities with desired functionality

[8,9,10,11]. In the former direction, most focus has

been on gut communities for impacting human and

animal health [6,7], and on anaerobic digestion (AD)

communitiesforimprovingindustrialmethane

produc-tion from organic wastes [11]. In the latter direction,

earlystudiesfocusedonimplementingdefined

commu-nities for degradation oforganic matter using existing

species(e.g.[12–14]),whilemorerecentstudiesfocused

on creating synthetic communities with defined (and

sometimes synthetically engineered) interactions that

giverisetospecificbiotechnologicalapplications,

popu-lation dynamics, and community control (e.g. [15–

17,18,19]). In the future, these top-down and

bot-tom-upapproachescouldmerge,withdefined,synthetic

communities being used to impact and engineer the

behaviorofcomplex,natural communities.

Irrespectiveoftheirspecificaimsandleveloffocus,any

engineeringapproachtomicrobialcommunitiesrequires

predictiveprinciplesfordescribingcommunitystructure

andfunctionrelationships,andpracticaltoolsforshaping

these. A simplistic view(that could be considered as a

guidingprincipleintheengineeringsense)istoconsider

complex microbial communities as being composed of

different functional groups performing key tasks. This

viewpointsuggeststhattheoverallbehaviorofacomplex

community canthenbemodulatedin adesired wayby

includingthenecessaryfunctionalgroups orbyaltering

thepopulation fractionsof suchgroups(Figure 1a). We

believethatthis simplisticviewis,however,unlikelyto

befruitfulasanengineeringapproachtomicrobial

com-munities, as it ignores secondary interactions between

speciesandtheenvironment,andtheensuingfeedback

Species–species and species–environment interactions,

aswellasevolutionarydynamicspresentsignificant

chal-lengestocomplexcommunityengineering.Toillustrate

the above point, consider for example, increasing the

populationfractionof aspeciesinvolvedin the

fermen-tationof aparticular organiccompound. Such an

inter-ventionisexpectedtoimpactotherspeciesina

commu-nitydirectlythroughcreationofsubstrate-competition(e.

g.forcarbohydrates),butalsoindirectlythrough

environ-mentalpH-changes(e.g.acidificationthroughformation

oforganicacids)[20]andemergenceofnewcross-feeding

interactions (e.g. through organic acids acting as new

substrates) [21]. Thus, altering the population of one

functional group might present unexpected impacts, or

alternatively no impact at all. Indeed, several recent

studiesfindthat functional improvementsto a

commu-nityemergefromlarge-scalecommunity‘implantation’or

mixingofmultiplecommunities[22,23],supportingthe

notionthatcommunityfunctionistheresultofa

commu-nity as a whole,inclusive of its myriad species–species

andspecies–environment interactions.

Giventheshortgenerationtimesofsomemicrobes,itis

alsopossiblethatlong-termspecies–speciesinteractions

canresultintheevolutionofadditionalgenetic

interac-tions. Such evolutionary adaptation is implicated for

examplebyfindingsofabundantauxotrophicinteractions

(emergingfromtheinabilityofonespeciestosynthesizea

compound required for its growth) in communities

enriched for degradation of specific compounds [24].

Evolutionary dynamics can also be driven by species–

environment interactions resulting in so-called eco-evo

feedbacks[25].Thesefeedbacksareshowntoimpactthe

populationdynamicsofcooperativetraitsinapopulation

[26,27,28],andareproposedasapotentialdrivingforce

beyond physiological specialization [29,30]. The latter

possibility has been demonstrated theoretically in the

context of monocultures of Escherhichia coli, where it is

shownthatmetabolicactivitiesalteringtheenvironment

can result in a feedback that drives the evolution of

differentmetabolic strategieswithinthis organism[21].

Todevelop applications of microbial communities,

engi-neering approaches hence need to deal not only with

species–species,butalsowith species–environment

inter-actionsandwiththeensuingeco-evofeedbacks(Figure1b).

Bottom-up

engineering

needs

to

consider

species–environment

and

eco-evo

feedbacks

For differentspecies to co-exist and achievea common

functionalgoal,theirenvironmentneedstobedesignedin

a way to support (or even enforce) their growth and

interactions.Thishas been achievedfor synthetic

auxo-trophic interactions within one species [17], and

cross-feedingandsyntrophicinteractionsamongdifferent

spe-cies [15,16,31]. A key example in the latter direction

involves amethanogen anda sulfatereducer, which

co-existinanenvironmentthatlackssulfate(sulfatereducers’

natural choiceas an electronacceptor) [31]. Thismodel

system isachieved byenforcing aspecificenvironment,

122 Environmentalbiotechnology

Figure1

(a) (b)

SO42- SO4

2-H₂S H₂S

H₂O O₂

pH E0

Current Opinion in Biotechnology

namelythe lackof sulfate,thatthennaturallydrivesthe

emergence of the syntrophic interaction. Indeed, it is

interestingto notethatthe syntrophicinteraction inthis

model system is impacted by evolutionary dynamics; a

maintainedpolymorphisminthesulfatereducerisfound

to berequiredfortheinitiationofsyntrophy[32],anda

rangeofpotentiallystabilizingmutationsarefoundtoarise

overlong-termco-culturing[33,34].Similarly,microbial

communitiesenrichedfordegradationofspecificorganic

compoundsarefoundtodisplaymultiplesyntrophic

inter-actions,aswellas secondarydependenciessuchas

auxo-trophic interactions [24]. These examples suggest that

microbialcommunitiescannaturallyadapttheirstructure

and specificmetabolicinteractionsto the given

environ-mental conditionsandtoachievestabilityand

productiv-ity. In other words, it might be possible that enforcing

specific environmental constraints can directly facilitate

the engineering of community structure and function

towardanevolutionarilystablestate.

Thermodynamics

and

redox

processes

as

‘design

principles’

for

community

engineering

In the above example of syntrophic interactions, the

enforcementoftheenvironment(throughsulfate

deple-tion) relates directly to the thermodynamic basis of

microbial growth.Microbial(catabolic) metabolismcan,

ata coarselevel,beunderstood as acollection of

path-ways, eachimplementingadifferentredoxreaction

uti-lizing a different terminal electron acceptor (TEA)

(Figure 2) [35,36,37,38,39]. Shortageof strongTEAs

is common in many micro- and macro-environments,

includingsoil,ADreactors,gut,andlakes,whileoxygen

Figure2

(a)

CS

ES

TEA

TEA

ES

Microbial

Biomass

ATP

[H]

(b)

pH=7 acetate/pyruvate

lactate+CO₂/glucose CO2/formate

CO2/lactate

CO2/butyrate

CO2/methane pyruvate/lactate

fumarate/succinate

glyoxylate/glycolate

nitrite/NO

nitrate/nitrite acetate/methane

sulfur/HS–

sulfite/HS–

H+_/H 2

MnO2/Mn2+

O2/H2O

Fe3+_/Fe2+

H+_/H

2 pH=0

800

600

400

200

reduction poteential E

0

(mV)

0

–200

–400

–600

Current Opinion in Biotechnology

Microbialmetabolismcanbeseenascomposedofintra-molecularandinter-molecularredoxreactionswithassociatedenergyharvesting.(a) Microbescoupleoxidationofreducedenergysources(ESred)withthereductionofterminalelectronacceptors(TEAs),therebyharvesting reductiveenergy([H]).Thisenergyisinvestedinthebuildingofbiomassbyreducingexternalcarbonsources(CS),orintheproductionofother energyequivalents(e.g.ATP).NotethatCSandEScanbethesamecompound,andthatinfermentationESessentiallyequalsTEA,butafter intramolecularredoxconversion.Furtherbiomasscomponents(e.g.nitrogen,phosphorous)areignoredinthisschemeforsimplicity.(b)Standard reductionpotentialsatbiologicalconditions(i.e.allsoluteconcentrations=1M(exceptfor[H+_]=10-7_{),pressure=1bar,temperature=25C)of}

biologicallyrelevantorganicandinorganiccompounds(notethatprotonreductionpotentialat[H+_{]=1M,i.e.pH=0,isshownseparately,} correspondingtostandardhydrogenelectrodepotential).Eachgraydotindicatesareductionreaction,withthesubstrateandproductshownin thelabels.Microbialgrowthinvolvescombiningonesuchreductionreactionwithanotherthatisruninthereverse(i.e.oxidationdirection),soto formaredoxcouple.Twoexamplesforsuchmicrobialgrowth-supportingredoxcouplesarehighlighted;theoverallprocessinmethanogenesis, whereH2oxidation(toH+)iscoupledtoreductionofCO2(tomethane);andakeypartofthesulfatereduction,wherelactateoxidation(to pyruvate)iscoupledtoreductionofsulfite(fromactivatedsulfate).Forthesecouplings,theoxidationandreductionreactionsareshowninblack andredrespectively(adaptingthecathodeandanodecolor-coding).Notethatindividualreactions’reductionpotentialswouldshiftas

depletioncanevenhappeninsealedculturesorbiofilms

offast-growingfacultativeorganisms[40,41].Underthe

absenceofstrongTEAs,thecommunity-membersneed

toadapttoredoxprocessesthroughweakTEAs(suchas

H+,CO2,andSO42 ),andfermentativepathwayswhich

mitigateintracellularreductiveenergyoverflow.This,in

turn,leadstothepossibilityofthermodynamicinhibition,

wherebymicrobescease growthdueto accumulationof

theirownmetabolic end-products[42].

Avoiding thermodynamic inhibition is only possible by

switchingtoalternativeredoxprocesseswithnew

chemi-calproducts or actively engaging in syntrophic

interac-tions[38,42].Indeed,thermodynamicinhibitionis

sug-gestedtoleadtomaintenanceofmicrobialandmetabolic

diversity[38],andtostronglyaffectcommunitystructure

anddynamics[1,2].Inanaerobiccommunities,for

exam-ple,depletionof strong TEAsleads to accumulation of

acetateandhydrogen,whichcanbeconsumedby

metha-nogens.Atthesametime,however,acetateaccumulation

candecreaseenvironmentalpHandinhibitmethanogens

[43].The resulting delicate balance can spiral out to a

feedbackdynamic,withincreasedacidificationleadingto

moreinhibitionofmethanogenesis,andthereforetomore

acidification,andfinallytowhole-communityinhibition.

DifferentialTEAavailabilitycanalsocausemore direct

alterationsin communitystructure.Adrasticexampleis

thefindingthatguthost cellresponsesto inflammation

canleadtoformationofnitrate,whichcanactasTEAfor

selectivegroupsofmicrobesandtherebygiveriseto

host-mediated changes in community dynamics [44].

Simi-larly, severalredoxactive compoundssuchas Azodyes

andhumic substances(i.e.polyaromatic

lignin-degrada-tionfragments)arefoundtoalteroverallmethanogensis

ratesinAD communities[45–47,48].

Electrical

interferences

as

dynamic

and

controllable

means

for

community

engineering

The above findings highlight TEA availability as a key

driver of both environmental conditions and microbial

interactions,givingrisetothepossibilitythatcommunity

structure and function could be manipulated through

TEAs provision or removal, guided by thermodynamic

considerationsofdifferentmicrobialrespirationprocesses.

This approach has already been used successfully to

achieveenrichment of specificmicrobial processes,

pre-dictedbythermodynamics(e.g.[49,50]),andissuggested

as a route to design anaerobic production strategies in

biotechnology[36,51,52].TheTEA-basedredox

inter-ventionscanbeachievedbothatcellular-and

community-levels,andtodatewere successfullyimplemented using

differentapproachesincludingtheprovisionofgases(i.e.

oxygen, hydrogen), chemical supplementation of media

(chemical electrondonors/acceptors) [45,47,48],genetic

modification(rate-controlonNADH-synthesis/depletion)

(e.g. [53]), and electrode-based intervention [54,55,56].

Chemicalinterventionsareestablishedparticularlyinthe

contextofAD,however,theydonotreadilyallowtemporal

control. Similarly, genetic interventions cannot be

trig-gered in a temporal fashion and only target a specific

species, which might not be stable in the context of a

community.Electrode-basedintervention,whereelectrons

canbesuppliedorretrievedfromthesystematset

reduc-tionpotentials,provideamoredirectandtemporalcontrol

overredoxprocesses,providedthatsomekeycommunity

membersareabletointerfacewiththeelectrodebydirect

orindirectelectronexchange.

Promising results of electrode-based intervention with

communitydynamics arealready beingobtained. In the

contextof AD, electrons were successfuly suppliedto a

complex anaerobic community via electrodes poised at

differentpotentialsandcurrents[57].Thisstudyfound

thatatconstantpotential,ahighcurrentfavored

acetogen-esiswithsomemethaneproductionthroughacetotrophic

methanogens. This suggests that the rate of electron

supplycantosomeextentcontroltheelectrons’ destiny,

highlightingtheinterlinkedeffectofthermodynamicand

kineticreactioncontrolincellmetabolism[36,38,59].A

similaruseofelectrodestocontrolcommunitydynamicsin

bottom-upengineeringisstilltobeattempted,butseveral

studieshaveshownthatmanydifferentbacteriaincluding

Clostridiaspecies[59]andmethanogens[60,61]are

capa-bleofelectrontransferfromandtoelectrodes,openingthe

route to implement separate redox reactions such as

organicsdegradationandmethanogenesisacross

compart-mentscoupledwithelectrodes.

Future

outlook

Weargued herefor amicrobial communityengineering

approachthattakesaholisticviewandthatconsidersnot

onlyspecies–species,butalsospecies–environment

inter-actions and eco–evo feedbacks. Shifting the emphasis

awayfromindividualspecies(orfunctionalgroups)tothe

system as a whole, this approach is similar to those

advocated for modeling connected biotic and abiotic

geochemical processes [62], where thermodynamics

andconsiderationsofentropymaximizationarebrought

to the fore as fundamental guiding principles. Holistic

engineering of microbial communities with such

high-levelguidingprinciples will requireusto better

under-standthethermodynamicbasisof microbialgrowth,and

in particularthe energeticsand dynamics of respiratory

and fermentative metabolic pathways. A crucial gap in

thisunderstanding, amongstothers, istheenergetics of

cellulargrowthwithinmicro-environments,suchas

bio-filmsandmicrobialgranules.Besidesimproved

measure-menttechniquesofmicro-andmacro-environments,the

fillingof this gap willalso requireincreased interaction

betweenresearchcommunitiesfromgeochemistry,

phys-ics,electrochemistry,andbiology.Increased

understand-ingatthemicrolevelcansubsequentlyallowbetteruseof

thermodynamic principles at the microbial community

level,andtodesignspecificelectrode-basedintervention

strategies. Inthis direction, more research is neededto

betterunderstandelectrode-microbeinteractions,aswell

as to develop cost effective electrical manipulations.

Together,thesedevelopmentscanallowelectrical

engi-neeringof complexmicrobialcommunitiesfoundinthe

soil, gut, and theanaerobic digestion reactors,and

bot-tom-up designof functional,definedcommunities.

Acknowledgements

We’dliketothankMunehiroAsallyandChristopherQuinceforcomments on an earlier version of this manuscript. We would like to acknowledge fundingfromBiotechnologicalandBiologicalResearchCouncil(BBSRC)of theUK,throughresearchgrants:BB/K003240/2(toOSS),andBB/M017982/ 1(totheWarwickIntegrativeSyntheticBiologyCentre,WISB).

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