BASE Biotechnol. Agron. Soc. Environ.201519(4),430-442 Focus on:

Bioreactordesignandimplementationstrategiesforthecultivationoffilamentousfungiandtheproductionoffungalmetabolites:fromtraditionalmethodstoengineeredsystemsMichelMusoni(1,3),JacquelineDestain(1),PhilippeThonart(1,2),Jean-BaptisteBahama(3),FrankDelvigne(1)(1)UniversitédeLiège-GemblouxAgro-BioTech.CentreWallondeBiologieIndustrielle.PassagedesDéportés,2.BE-5030Gembloux(Belgique).E-mail:Michael.Musoni@gmail.com;F.Delvigne@ulg.ac.be(2)UniversitédeLiège.ServicedeTechnologiemicrobienne.BoulevardduRectorat,29.BE-4000Liège(Belgique).(3)UniversitéduBurundi.FacultédesSciencesagronomiques.LaboratoiredelaProtectiondesVégétaux.BP2940.Bujumbura(Burundi).

ReceivedonNovember28,2013;acceptedonJuly27,2015.

Theproductionoffungalmetabolitesandconidiaatanindustrialscalerequiresanadequateyieldatrelativelylowcost.Tothisend,manyfactorsareexaminedandthedesignofthebioreactortobeusedfortheselectedproducttakesapredominantplaceintheanalysis.Oneapproachtoaddressingtheissueistointegratethescaling-upprocedureaccordingtothebiologicalcharacteristics of the microorganism considered, i.e. in our case filamentous fungi. Indeed, the scaling-up procedure isconsideredasoneofthemajorbottlenecksinfermentationtechnology,mainlyduetothenearimpossibilityofreproducingtheidealconditionsobtainedinsmallreactorsdesignedforresearchpurposeswhentransposingthemtoamuchlargerproductionscale.Thepresentreviewseekstomakethepointregardingthebioreactordesignanditsimplementationforcultivationoffilamentousfungiandtheproductionoffungalmetabolitesaccordingtodifferentdevelopmentalstagesoffungiofindustrialinterest.Solid-state(semi-solid),submerged,fermentationandbiofilmreactorsareanalyzed.Thedifferentbioreactordesignsusedforthesethreeprocessesarealsodescribedatthetechnologicallevel.Keywords.Bioreactors,fungi,secondarymetabolites,conidia,solidstatefermentation.

Conception de bioréacteurs et mise en œuvre de stratégies pour la culture de champignons filamenteux et la production de métabolites d’origine fongique : des méthodes traditionnelles aux technologies actuellesLaproductiondemétabolitesd’origine fongiqueetdeconidiesà l’échelle industrielle requiertunprocédéadéquatàcoutrelativementbas.Àceteffet,beaucoupdefacteurssontanalysésetlaconfigurationdubioréacteuràutiliserpourleproduitretenuoccupeuneplaceprépondérantedansl’analyse.Uneapprocheàsuivrepourrésoudreleproblèmerelatifàlaproductiondemétabolitesetdeconidiesparleschampignonsconsisteàmaitriserlamontéeenéchelleduprocédédefermentationaveclerespectdecaractéristiquesbiologiquesdemicro-organismes,telsqueleschampignonsfilamenteuxpourlecasquiconcernenotreétude.Eneffet,cettemontéeenéchelleestconsidéréecommel’undesprincipauxpointsd’achoppemententechnologiedefermentation:cecienraisondelaquasi impossibilitédereproduireexactement lesmêmesconditionsidéalesobtenuesenutilisantlespetitsréacteursderechercheaumomentdelestransposeràlaproductionàgrandeéchelle.Laprésenterevueviseàfairelepointsurlaconceptiondebioréacteursetleurmiseenœuvrepourlaculturedechampignonsfilamenteuxetlaproductiondemétabolitesen fonctiondesdifférents stadesdedéveloppementdechampignonsd’intérêt industriel.Lesculturessolide(semi-solide),submergéeetenbiofilmontétéprisesencomptedanslesdifférentesétudes.Lesdifférentstypesdebioréacteursutiliséspourlestroisprocédéssontaussidécritsdupointdevuetechnologique.Mots-clés.Bioréacteur,champignon,métabolitesecondaire,conidie,fermentationàl’étatsolide.

Designoffungalbioreactors 431

1. INTRODUCTION

Threefungalgeneraarewidelyusedforbiotechnologicalapplications:Aspergillussp.,Penicilliumsp.,andtoalesserextent,Trichodermasp.Filamentousfungihavethecapabilitytoproduceextremelylargequantitiesofhomologousproteins, includingseveralenzymes thatcanbeusedasindustrialbiocatalysts.Severalvolatilemetabolites(MVOC)canalsobeproduced.However,theproductionofthesemetabolitesreliesontheculturemode at both the quantitative and qualitative levels.Indeed,thesefungiareabletogrowindifferentforms:from free mycelium and pellet in liquid phase, topelletandmyceliumtoconidiaatasolid-airinterface.Basedonthis,differentbioreactorstrategieshavebeendeveloped in order to promote a given developmentstage.Inthiscontext,threemodesofcultivationscanbeconsidered:–submerged fermentation where two kinds ofmorphologies are most often depicted, i.e. freemycelium (with different degrees of ramification)andpellets;

–semi-solid fermentation which is reflected by thedevelopmentofmyceliumonthesurfaceandattheinterior of a solid substrate in the absence of freewater;

–biofilm formation, represented by the developmentofmycelium on an inert support, such as stainlesssteel,glassorTeflonmaterialintegratedinanadaptedreactor.

Among these three strategies, submergedfermentation is the most widely used mode offermentation.However,thelasttwostrategiesleadtotheenhancementofthedevelopmentofaerialhyphaewiththeproductionofconidiaonconidiophorepositionoffilamentsdependingonthedevelopmentstageofthemicroorganism.Theywill be able togenerate fungalproductsofbiotechnologicalinterest,sincetheexcretioncapacityisincreasedwhenfungalbiomassisattachedonagivensupport.Barrios-Gonzalez(2012)reportedthat the solid-state fermentation is considered as analternativeculturemethodthathasgainedresearchersattentionoverthepast20years,andcredibilityamongmany industrial corporations. The present review isaimed at understanding the influence of the culturalconditionsonthedevelopmentalstageofthefungi,aswell as on the excretionof secondarymetabolitesofindustrial interest,whichhighlight theorientationforbioreactordesignneeded.It is focusedonbioreactor,fermentation, strategies for secondary metabolitesenhancement and some applications cultivation ofTrichoderma, Penicillium and Aspergillus. In theconclusion, a new range of biofilm reactors will beproposed for the improvement of the production offungalmetabolites.

2. TRADITIONAL METHODS AND ENGINEERED SYSTEMS APPLIED FOR THE CULTIVATION OF FILAMENTOUS FUNGI IN ORDER TO PRODUCE THE METABOLITES AND CONIDIA

2.1. Basic bioreactor design: from submerged fermentation to biofilm reactor

A bioreactor can be defined as mechanically stirredvessel in which organisms are cultivated in acontrolled manner and/or materials are convertedor transformed through specific reactions. Indeed,traditionalmethodsforsolidstatefermentationincludetray,drumandpackedbedbioreactorswithproblemsregarding the control of different parameters; while,for submerged fermentation they include continuousstirred-bioreactors, continuous flow stirred-tankreactors,plug-flowreactor,andfluidized-bedreactors.For biofilm processes, the material supporting themicroorganism can be used in reactors applied forsubmergedfermentationandadaptedforusedinordertocultivateimmobilizedcellsorbiofilmswithrespectto the production of value-addedmolecules (Muffleretal.,2014).

2.2. Factors affecting bioreactor design

Manyfactorscontributetothedesignofabioreactor.Durand (2003) reported that compared to submergedfermentation, the solid media used in solid-statefermentation contain less water but an importantgas phase existed between the particles.This featureis of great importance because of the poor thermalconductivityofaircompared towater.Anotherpointis the wide variety of matrices used in solid-statefermentation,whichvaryintermsofcomposition,size,mechanical resistance, porosity and water-holdingcapacity.Allthesefactorscanaffectthereactordesignandthestrategytocontrolthekeyparameters.Indeed,with submerged fermentation, it can be consideredan approximation that all the media are made upessentiallyofwater.Inthisenvironment,temperatureandpHregulationsaretrivialanddonotposeproblemsduringthescaling-upofaprocess.

Withsubmergedfermentation,thedifficultymainlyencountered is related to limitations at the level ofoxygentransfercapacity,whichdependsupontheshapeandsizeofthereactorandtheagitation/aerationsystemused. To characterize this transfer, a parameter, KLa(oxygentransfercoefficient),hasbeendefined.Itcanbeconsideredasa“similarityinvariant”,whichmeansthatitsvalueexpressesthecapacityoftheequipmentto transfer oxygen independent of the volumeof thereactorand,assuch,constitutesanimportantparameterused in scale-up studies in submerged fermentation.

432 Biotechnol. Agron. Soc. Environ. 201519(4),430-442 MusoniM.,DestainJ.,ThonartPh.etal.

Withsolid-statefermentation,besidesoxygentransfer,whichcanbealimitingfactorforsomedesigns, theproblemsaremorecomplexandaffectthecontrolofthree importantparameters, i.e. temperature,pHandwater content of the solid medium. There are alsootherfactorsaffectingthebioreactordesign,namely:–themorphologyof thefungus(thepresenceornotofaseptumin thehyphae)and, related to this, itsresistancetomechanicalagitation,

–thenecessityornotofhavingasterileprocess.

Concerning fungal morphology, as reportedby Papagianni (2004), filamentous fungi aremorphologicallycomplexmicroorganisms,exhibitingdifferentstructuralformsthroughouttheirlifecycles.Thebasic vegetative structure of growth consists oftubularfilamentsknownashyphaethatoriginatefromthe germination of a single reproductive conidiumor a piece ofmycelium.As the hyphae continue togrow, theybranchfrequentlyandrepeatedlytoformamassofhyphalfilamentsreferredtoasamycelium.Whengrowninsubmergedculture,thesefungiexhibitdifferentmorphologicalforms,rangingfromdispersedmycelial filaments to densely interwoven mycelialmassesreferredtoaspellets,whereasconidiation,asthe end point of the fungi’s developmental cycle, israrelyachievedduringsubmergedcultivation,partiallyduetorelativelygoodnutrientavailabilityandpartlyto the physical nature of the hyphal wall. Differentmetabolites are produced by these different formsin submerged culture, so the successful productionof fungal metabolites requires detailed knowledgeof the growth characteristics and physiology of thefungus considered.Not only does the production ofdifferent metabolites require different physiologicalconditions, but each fungal species is also uniquein its anatomical, morphological and physiologicaldevelopment. Thus, for fungal fermentation, theprecise physiological conditions and correct stageof development must be established to achievethe maximal product formation. In other words,controlling the form of these microorganisms is arealissuethatneedsgreatattentioninordertomakeoptimal use of their potential production capacitiesduringtheirdevelopmentaccordingtothestructureofbioreactordesigned.

2.3. Solid-state fermentation processes

Manyexamplesofsolid-statefermentationhavebeenreportedbyresearchers,someofwhicharedepictedin a figure presented by Durand (2003) and otherswheretheyrepresentedtraybioreactors,rotatingdrumbioreactors,packedbedbioreactorsandothersmoresophisticated.Regardingexamplesofsubmergedandbiofilm fermentations, the different developmental

stagesobserveddependontheprocessesusedtoobtainthe final product desired at the end of the process.Some examples are outlined in figures 1A, 2A and3A.Thefiguresconfirmed that thebiomass thatcanbeaccumulatedonthesupportwashigherinbiofilmreactor(Figure 3B)thanitsequivalentinsubmergedreactor, pointing out a possible way of bioprocessintensification (Figure 1A). The same conclusionhas been outlined by Seye et al. (2014) concerningimmobilisationofmicroorganismsonsupportusedinfungalbiofilmreactorforproductionofconidiausingAspergillus clavatus.The choice of a bioreactor fora given fermentation is based on the production orproductivity yields and the regulation of the systemrequiredforthefilamentousfungi’sgrowth.Regardingbioreactorsforsolid-statefermentationatthepilotandindustriallevels,afewcategoriescanbedistinguishedbased on the aeration and mixing strategies. Theseare:forcedorunforcedaeration;static,pulsedmixed,orcontinuouslymixed.

Among cultivation systems, while solid-statefermentation exhibits a higher productivity ofsecondarymetabolites, Kumar et al. (1987) as wellas Hölker et al. (2004) compared submerged withliquid-surface fermentations and pointed to someinherentdisadvantages,suchaspoorheatdissipation.Slowdiffusionratesofnutrients,products,water,andoxygeninpackedbedshavealsobeenreported(Viccinietal.,2001;Bellon-Maureletal.,2003;Durand,2003;Mitchelletal.,2003;Moetal.,2004).Muffleretal.(2014)pointedoutthattherelevanceoftheemergingfield of biofilm research in biotechnology becomesparticularly obvious if one considers the number ofpublications per year for the terms ‘‘biofilm’’ and‘‘biotechnology’’collectedbytheWebofKnowledge(ThomsonReuters) in June2013.Even thoughonlya small fraction of the number of papers is directlyfocusedonproductivebiofilms,theillustrationshowstheprosperousfieldofthisresearcharea.

2.4. Strategies for enhancing the production of fungal metabolites

Byconsideringthefungalbiologyfoundwhenbiomassisattachedontoasupport, someresearchers tried todevelop a process by which secondary metabolitescould be enhanced according to the needs of themicroorganisms.Nevertheless,generallymuchhigheryieldswereobtainedinsolid-statefermentation.Theyused,forexample,submergedculturesinflasksand/orastirrertank(Gasparetal.,1997;Gasparetal.,1998;Vavilova et al., 2003; Seyis et al., 2005; Ahamedet al., 2008; Meshram et al., 2008), or solid-statefermentation(Bakrietal.,2003;Sandhyaetal.,2005;Assamoietal.,2008a;Assamoietal.,2008b;Mitchelletal.,2010)toproducethecompoundsunderstudy.

Designoffungalbioreactors 433

Figure 1. Scheme of a bioreactor used in submerged fermentation and illustration ofwall growth fungi—Schéma d’un bioréacteur utilisé en culture submergée et illustration de la croissance du micro-organisme sur les parois de la cuve.A:classicalstirredtankbioreactorinwhichtwoorthreeidenticalturbinesweremountedonthecentralshaft—bioréacteur agité classique équipé d’un axe comprenant deux à trois mobiles d’agitation identiques;B:wallgrowthoffungiinsubmergedbioreactor—biomasse sur les parois en culture submergée.

Figure 2.Schemeofabioreactorusedinbiofilmfermentationandillustrationof thebiomassaccumulatedonthesupportused—Schéma d’un bioréacteur utilisé en culture à biofilm et la biomasse accumulée sur les plateaux perforés utilisés.A:perforatedplatebioreactoradaptedfromamechanicallystirredtankinwhichtenperforatedstainlesssteelplatesweremountedataregulardistanceandaflowingmediumsystemadopted—bioréacteur à plateaux perforés avec système d’aspersion;B:biomassaccumulatedontheplatesusedinaperforatedplatebioreactor— illustration de la biomasse accumulée sur les plateaux perforés utilisés.

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434 Biotechnol. Agron. Soc. Environ. 201519(4),430-442 MusoniM.,DestainJ.,ThonartPh.etal.

Hölker et al. (2004) reported that solid-statefermentation is currently the best method forobtaining fungal conidia from aerial hyphae. Theproperties of conidia produced by solid-statefermentation differ distinctly from those obtainedbysubmergedfermentation.Fungalconidia,usedasbiocontrolagentsagainstfungalplantpathogens,arepreferentially produced in solid-state fermentation;theconidiaobtainedmanifestahigherquality.Theyaremoreresistanttodesiccationandmorestableinthe dry state. To obtain a high number of conidia,a combination of submerged fermentation (forbiomassproduction in thefirst step)andsolid-statefermentation (for subsequent conidia production)proved to be successful. However, a directcomparison between solid-state fermentation andsubmerged fermentation is very difficult due to thedifferentconsistenciesofthefungalculturesusedinthetwotechnologies.Inthisregard,biofilmculturesshouldrepresentagoodobjectiveduetothefactthataerialgrowthwithaflowingmedium,whichseemstobesimilartotheirnaturaldevelopment,canresultincontinuoushighdensityproductionofsecondarymetabolitesandconidia.

3. APPLICATIONS INVOLVING TRICHODERMA, PENICILLIUM AND ASPERGILLUS

According to their capability of producing differentcompounds, the three genera, Trichoderma,PenicilliumandAspergillusareusedinmanyindustrialapplications.Thesemycofactoriescanbeusedfortwoclasses of applications i.e. either the production ofsecondarymetabolites,ortheproductionofconidiaforbiocontrolapplications.

3.1. Cultivation of Trichoderma for the production of fungal metabolites

The filamentous fungus Trichoderma has beencultivated using submerged, solid-state and biofilmfermentationinordertoproducesecondarymetabolitesas well as conidia. The molecules which have beentaken into account were cyclosporin, 6-pentyl-α-pyrone,cellulase,hemicellulaseandamylase.

Vinale et al. (2008) showed that MVOCs ofthe fungus Trichoderma act antibiotically againstpathogenicplantmouldsandcanconferplantgrowth

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A:Biofilmbioreactorwithinertsupportadaptedformamechanicallystirredtanktowhichaninertsupportcanbeaddedandmediumcirculatedinsystemduringfermentation—bioréacteur à biofilm avec système de circulation du milieu aspergé sur le garnissage métallique structuré; B:biomassaccumulatedonaninertsupportandanemptyone—incrustation de biomasse dans le garnissage métallique structuré et un autre sans dépôt.

Designoffungalbioreactors 435

promoting effects as well as systemic resistance toplants,thusrenderingplantslessvulnerabletofungalpathogens(Harmanetal.,2004).

A number of studies have been conducted toevaluate theeffectofnutritional factors (Yonget al.,1986;Serrano-Carreonetal.,1992)onsolidandliquidfermentation methods (Kalyani et al., 2000; Sarhy-Bagnonetal.,2000),on6-pentyl-α-pyroneproduction.Particularattentionhasbeenaccordedtoenzymeswithhigh xylanolytic activity produced by Trichodermaspp.(Wongetal.,1992).Variousfermentationsystemshave been used with Trichoderma harzianum Rifai,TrichodermaviridePersoonandTrichodermakoningiiOudemans to produce these compounds, such assubmerged (Cutler et al., 1986; Simon et al., 1988;Evidenteetal.,2003),aqueoustwo-liquid-phase(Rito-Palomares et al., 2000;Rito-Palomares et al., 2001),organic-aqueous two-liquid-phase (Serrano-Carreonetal.,2002;Rocha-Valadezetal.,2006),liquid-surface(Kalyani et al., 2000), and solid-state cultivationsystems(Cooneyetal.,1997a;Cooneyetal.,1997b).

Shinobu et al. (2009) developed an extractivefermentationsystemwithcharacteristicsofbothaliquidsurface immobilization (LSI) system and a liquid-liquid interface bioreactor (L–LIBR). The systemis tentatively named by the authors an “extractiveliquid surface immobilization” (Ext-LSI) system.In the Ext-LSI system, the fungus-microsphere matproduces hydrophobic secondary metabolites usingnutrientscontainedinaliquidmedium.Itisexpectedthat the hydrophobic metabolites are spontaneouslyextracted from the fungalcells intoanorganicphasewheretheyaccumulatetohighconcentrationsbecausefeedback inhibition and cellular toxicity of themetabolitesmaybeeffectivelyalleviated.Thereportedmaximum accumulations of 6-pentyl-α-pyrone(6PP) in submerged, liquid surface, and solid-statefermentationswere474mg.l-1(Serrano-Carreonetal.,2004), 455mg.l-1 (Kalyani et al., 2000) and 3g.kg-1(deAraujo et al., 2002), respectively. Shinobu etal.(2009) also tried to apply theExt-LSI systemwithafungus to produce an aromatic coconut compound.Following optimization of the conditions to producethis volatile, the accumulation of the metabolite intheorganicphasereached7.1g.l-1duringafourweekcultivation.Theseexamplesprovedthattheproductionof secondary metabolites can be enhanced by usingsolid-statefermentation.

Looking at the relationship between secondarymetabolites and conidiation, Calvo et al. (2002)suggestedthreekindsofrelationsthatcanbeconsidered:–metabolites that activate conidiation (for example,the linoleic acid derived compounds produced byAspergillus nidulans);

–pigments required for conidiation structures (forexample, melanins required for the formation or

integrity of both sexual and asexual conidia andoverwinteringbodies);

–toxicmetabolitessecretedbygrowingcoloniesattheapproximate time of conidiation (for example, thebiosynthesis of some deleterious natural products,suchasmycotoxins).

Table 1 summarizes someexamplesof secondarymetabolites produced by microorganisms withcommercialimportance,likeantibiotics,organicacidsandenzymes.

The term ‘‘secondary metabolite’’ includes aheterogeneous group of chemically distinct naturalcompoundspossibly related to survival functions fortheproducingorganism such as competition (againstothermicro- andmacroorganisms), symbiosis,metaltransport, differentiation, etc. (Demain et al., 2000).Includedinthisgroupareantibiotics,whicharenaturalproductsthatcaninhibitmicrobialgrowth.Antibioticproduction is often well correlated with biocontrolability, and theapplicationofpurifiedantibioticshasbeenfoundtoshowpositiveeffectsonthehostpathogensimilartothoseobtainedusingthecorrespondinglivingmicroorganism.Ghisalbertietal.(1990)demonstratedthatthebiocontrolefficacyofTrichoderma harzianumRifai isolates against Gaeumannomyces graminisvar.‘tritici’isrelatedtotheproductionofpyronelikeantibiotics.

Trichodermaspp.producesaplethoraofsecondarymetaboliteswithbiologicalactivity(Ghisalbertietal.,1991;Sivasithamparamet al., 1998).Theproductionof secondary metabolites by Trichoderma spp. isstrain dependent and includes antifungal substancesbelonging to a variety of chemical classes thatwereclassified by Ghisalberti et al. (1991) into threecategories:–volatile antibiotics, for example 6-pentyl-α-pyroneandmostoftheisocyanidederivates;

–water-soluble compounds, like heptelidic acid orkoningicacid;

–peptaibols, which are linear oligopeptides of12–22amino acids rich in α-aminoisobutyric acid,N-acetylated at the N-terminus and containing anamino alcohol (Pheol or Trpol) at the C-terminus(LeDoanetal.,1986;Rebuffatetal.,1989).

Theresultsobtainedfromexperimentsconductedbysomeresearcherssuggestthatmicroorganismsproducesecondary metabolites under different bioprocesses(Table 2).Bothmutantandwildstrainswereused.Insomecases,amutantgaveahigherproductionthanthewildstrain.Theparticularbioprocessusedalsohadanimpactonenhancingtheproduction.

Polizeli et al. (2005) reported that in eithersubmerged or solid-state fermentation could be usedto generate xylanase production by microorganisms.

436 Biotechnol. Agron. Soc. Environ. 201519(4),430-442 MusoniM.,DestainJ.,ThonartPh.etal.

Table 1.Somebiocompounds(antibiotics,organicacidsandenzymes)producedbyfilamentousfungiappreciatedfortheircommercial value—Quelques composés (antibiotiques, acides organiques et enzymes) produits par les champignons filamenteux appréciés pour leur importance commerciale.N° Fungal biocompounds Filamentous fungi in usedA Antibiotic Fungal strains1 Cyclosporins Trichoderma polysporumGams2 EchinocandinB Aspergillus nidulans Winter,Aspergillus rugulosusThomandRaper.3 Griseofulvin Penicillium aurantiogriseumDierckx,Penicillium griseofulvumDierckx,

Penicillium italicum Wehmer4 Patulin Penicillium urticaeBainier5 Penicillins Aspergillus flavus Link,Aspergillus giganteusWehmer,Aspergillus nidulansWinter,

Aspergillus nigervanTieghem,Aspergillus oryzae (Ahlburg)Cohn,Aspergillus parasiticusSpaere,Aspergillussp.,Penicillium baculatum Westling,Penicillium chrysogenum Thom,Penicillium turbatumWestling

B Organic acid Fungal strains6 Citricacid Aspergillus citricus(Wehmer)Mosseray,Aspergillus clavatusDesm.,Aspergillus niger

vanTieghem,Aspergillus phoenicisWisconsin,Penicillium decumbensThom,Penicillium isariiformeStolkandMeyer,Trichoderma viride Persoon

7 Gluconicacid Aspergillus carbonarius(Bainier)Thom,Aspergillus niger vanTieghem,Aspergillus oryzae (Ahlburg)Cohn,Aspergillus wentiiWehmer,Penicillium chrysogenumThom,Penicillium luteumZukal,Penicillium simplicissimum(Oudemans)Thom

8 Itaconicacid Aspergillus itaconicusKinoshita,Aspergillus terreus Thom9 Kojicacid Aspergillus candidusLink,Aspergillus flavusLink,Aspergillus oryzae(Ahlburg)Cohn,

Aspergillus parasiticus Spaere,Aspergillus tamariiKita,Penicillium jensenii Zalessky10 L-Malicacid Aspergillus atroviolaceusMosseray,Aspergillus citricus(Wehmer)Mosseray,

Aspergillus flavus Link,Aspergillus nigervanTieghem,Aspergillus ochraceusWilhelm,Aspergillus oryzae(Ahlburg)Cohn,Aspergillus wentiiWehmer

11 D-Araboascorbicacid Penicillium chrysogenum Thom12 Erythorbicacid Penicillium griseoroseumDierckxC Enzyme Fungal strains13 α-Amylase Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Aspergillus awamori

Nakazawa,Trichoderma viridePersoon14 Amyloglucosidase Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Aspergillus awamori

Nakazawa,Aspergillus phoenicis Wisconsin15 Catalase Aspergillus nigervanTieghem,Penicillium vitalePidoplichkoandBilai16 Cellulase Aspergillus nigervanTieghem,Aspergillus soyaeSakagawaandK.Yamada,

Aspergillus terreusTohm,Penicillium citricum Thom,Penicillium funiculosum Thom,Trichoderma longibrachiatumRifai,Trichoderma reeseiRifai,Trichoderma viridePersoon

17 Dextranase Aspergillus carneus(vanTieghem)Blockwitz,Penicillium funiculosum Thom,Penicillium lilacinum Thom,Penicillium pinophilumHedgeock

18 α-Galactosidase Aspergillus awamori Nakazawa,Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Penicillium dupontii GriffonandMaubl.

19 β-Galactosidase Aspergillus awamori Nakazawa, Aspergillus nigervanTieghem,Aspergillus nidulans Winter,Aspergillus oryzae(Ahlburg)Cohn,Penicillium funiculosum Thom

20 β-Glucanase Aspergillus niger vanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Trichoderma viridePersoon,Trichoderma reeseiRifai

21 Glucoamylase Aspergillus nigervanTieghem,Aspergillus awamori Nakazawa,Aspergillus oryzae(Ahlburg)Cohn

./..

Designoffungalbioreactors 437

For example, submerged fermentation was used toproducexylanasefromT. reesei.Inthatexperiment,theoptimalpHandtemperaturewere4.5and40°C.This enzyme is usually used in cellulose pulpbleachingandanimalfeed.

Dashtban et al. (2011) performed experimentsrelating to theeffectofdifferentcarbonsourcesoncelluloseproductionbyHypocrea jecorina(T. reesei)strainsusingT. reeseiQM6a,T. reeseiQM9414,andT. reeseiRUT-C30.Timecourseexperimentsshowedthat maximum cellulose activity with QM6a andQM9414 strains occurred at 120h for themajorityof the tested carbon sources, while RUT-C30had its greatest cellulose activity at around 72h.Maximum cellulase productionwas observed to be0.035,0.42and0.33μmolglucoseequivalentsusingmicrocrystallinecellulosesforQM6a,QM9414,andRUTC-30, respectively; while the maximum dryweightforallthreeT. reeseistrainswasobtainedatthe highest carbon concentration level (2%). This

suggests that an increase in enzyme production isdirectlycausedbyan increase inmyceliumdensityandmaybearesultofcomplexinducingfactorsinarelationshipwiththebioreactorconfigurationused.

In the course of experiments carried out in ourlaboratory,itwasobservedthatduringculture,wallgrowth of the fungi is amplified by the speed ofagitation,whichsplashessomefragmentsofmyceliaon the wall vessel (Figure 1B). This phenomenonreducesthebiomassinthattypeofculture.Inordertoperformtheproductionofbiomassandthevolatile,a biofilm bioreactor has been adapted, modulatingplate bioreactor from the mechanical one with tenperforated stainless steel plates (Figure 2B) andalsousingan inertmetallic supportnamedpacking(Figure 3B)onwhichbiomass canbeaccumulatedwithoutgrowthonthevesselwall.Withtheseresults,itcanbesuggestedthatthewallgrowthinsubmergedculture will be moderated using the strategiesadopted.

Table 1 (continued). Some biocompounds (antibiotics, organic acids and enzymes) produced by filamentous fungiappreciatedfortheircommercialvalue—Quelques composés (antibiotiques, acides organiques et enzymes) produits par les champignons filamenteux appréciés pour leur importance commerciale.N° Fungal biocompounds Filamentous fungi in usedC Enzyme Fungal strains22 Glucose

aerohydrogenaseAspergillus nigervanTieghem

23 Glucoseoxidase Aspergillus niger vanTieghem,Penicillium amagasakienseKusai,Penicillium simplicissimum(Ouedmans)Thom,Penicillium vermiculatumDangeard

24 α-Glucosidase Aspergillus awamori Nakazawa,Aspergillus flavusLink,Aspergillus fumigatusFresenius,Aspergillus nigervanTieghem

25 α-D-Glucosidase Aspergillus nigervanTieghem26 β-Glucosidase Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Trichoderma reesei

Rifai27 Hemicellulase Aspergillus nigervanTieghem,Aspergillus oryzae (Ahlburg)E.Cohn,Aspergillus

phoenicisWisconsin,Trichoderma longibrachiatumRifai,Trichoderma viridePersoon28 Hesperidase Aspergillus nigervanTieghem

29 Invertase Aspergillus awamoriNakazawa,Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn

30 Lipase Aspergillus nigervanTieghem,Aspergillus oryzae(Ahlburg)Cohn,Penicillium roquefortiThom

31 Pectinase Aspergillus alliaceusThomandChurch.,Aspergillus nigervanTieghem32 Phytase Aspergillus nigervanTieghem,Aspergillus ficuum(Reichardt)Henn.33 Protease Aspergillus nigervanTieghem,Aspergillus melleusYukawa,Aspergillus oryzae (Ahlburg)

Cohn,Aspergillus saitoi,Penicillium dupontiiGriffonandMaubl.34 Tannase Aspergillus fumigatusFresenius,Aspergillus versicolor,Aspergillus flavusLink,Aspergillus

niger van Tieghem, Aspergillus tamarii Kita, Aspergillus japonicus Saito, Aspergillus parasiticus Spaere, Aspergillus oryzae (Ahlburg) Cohn, Penicillium charlesii Thom,Penicillium crustosumThom,Penicillium restrictumGilmanandAbbott

35 Xylanase Trichoderma reesei Rifai

438 Biotechnol. Agron. Soc. Environ. 201519(4),430-442 MusoniM.,DestainJ.,ThonartPh.etal.

ThesimilarsystemhasbeenusedbyKhalesietal.(2014)fortheproductionof hydrophobin HFBII by T. reesei.They noted that the use of a biofilmreactorhasledtoasignificantincreaseofHFBIIproductioninshortertimebycomparisonwithaclassicalsubmergedbioreactor.

3.2. Use of Penicillium for the production of fungal metabolites and conidia

The main molecules produced byPenicilliumweregriseofluvin,penicillin,citric acid, koji acid, erythorbic acid,catalase, dextranase and tamase.Chávezetal.(2005)reviewedthegenusPenicillium, particularly the speciesproducingxylanase.Theyreportedthatthe Penicilliaweremostly saprophyticin nature, and numerous species areof particular value for humans. Thebest known arePenicillium canescens10-10c and Penicillium notatumWestling, producing xylanase and theantibiotic penicillin, respectively, aswellasPenicilliumroquefortiThomandPenicillium camemberti Thom, whichareimportantinthefoodindustry.Thelatterareassociatedwiththeproductionof particular types of cheese. A lotof Penicillia are soil fungi, and growin a variety of organic substances,particularly dead plant material. Theyproduce extracellular hydrolases suchas pectic enzymes, lipases, proteases,cellulases and xylanases. The latterenzymes are known for a numberof biotechnological applications.One important use in cellulose pulpbiobleaching (Polizeli etal., 2005) isto eliminate lignin residues from kraftpulp.Theyalsoincreasethedigestibilityoffeedbyloweringtheviscosityintheintestinaltract,thusimprovingnutrientuptake(Twomeyetal.,2003).Inbaking,they are added to increase the specificvolume and in this way improve finalflavour(Maatetal.,1992).Inbeerandjuiceprocessing,theyareusedtoreducehaze formation by solubilising longchain arabinoxylans (Dervilly etal.,2002).

In studies carried out withP. canescens 10-10c, it was concludedTa

ble

2. Productionofxylanaseand6-pentyl-α-pyronebysom

efilam

entousfungiusingdifferentbioprocesses—

Pro

duct

ion

de x

ylan

ase

et d

e 6-

pent

yl-α

-pyr

one

par

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ham

pign

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lam

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poun

dPr

oduc

tion

Mic

roor

gani

smFe

rmen

tatio

n ap

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efer

ence

Xylanase

630IU. ml-1

Tric

hode

rma

rees

eiRutC-30

SbmF

Flask

Xiongetal.,2004

1,350IU. ml-1

Tric

hode

rma

rees

eiRutC-30

SbmF(Fed-batch)

Bioreactor(2l)

Xiongetal.,2004

14,790IU

. ml-1. h

-1As

perg

illus

nig

erKK2

SSFwheatraw

Flask

Parketal.,2002

844IU. ml-1

Peni

cilli

um c

anes

cens10-10c

SbmF

Shakeflask

Gaparetal.,1997

7,448IU. ml-1

Peni

cilli

um c

anes

cens10-10c

SbmF

Shakeflask

Bakrietal.,2003

9,632IU. g

-1Pe

nici

llium

can

esce

ns 10-10c

SSF

Flask

Bakrietal.,2003

9,300IU. g

-1Pe

nici

llium

can

esce

ns 10-10c

SSF

Plasticgags

Assam

oietal.,2008a

10,200IU

. g-1

Peni

cilli

um c

anes

cens10-10c

SSF

Multi-layer

Assam

oietal.,2009

18,895IU

. g-1

Peni

cilli

um c

anes

cens10-10c

SSF

Flask

Assam

oietal.,2009

6-pentyl-α-pyrone

455mg.l-1

Tric

hode

rma

harz

ianu

mRifai

LSC

Flask(500ml)

Kalyanietal.,2000

474mg.l-1

Tric

hode

rma

harz

ianu

mSbmF

Flask

Serrano-Carreonetal.,2004

3,000mg.kg

-1Tr

icho

derm

a ha

rzia

num

SSF

Flask

deAroujoetal.,2002

7,100mg.l-1

Tric

hode

rma

atro

viri

de AG2755-5NM398

SbmFExt-L

SIPlate(50ml)

Shinobuetal.,2009

230mg.l-1

Tric

hode

rma

harz

ianu

m IM

I206040

SbmF

Shakeflask(500ml)

Rocha-Valadezetal.,2006

230mg.l-1

Tric

hode

rma

harz

ianu

mIM

I206040

SbmF

Stirredtank(10l)

Rocha-Valadezetal.,2006

5,000mg.kg

-1Tr

icho

derm

a ha

rzia

num4040

SSF

Flask(250ml)

Ram

osetal.,2008

SbmF:SubmergedFermentation;SSF:SolidSubstrateFermentation;Ext-LSI:E

xtractiveLiquid-SurfaceIm

mobilisation.

Designoffungalbioreactors 439

that the production of xylanase is influenced by theprocess used in its production, whether submergedor solid-state fermentation. The researchers whoconducted the experiments in different types offermentation noted the influence of the solid-statefermentation (9,632IU.g-1) (Gaspar et al., 1997;Bakri etal.,2003),or solid-state fermentationwithamulti-layer system (10,200UI.g-1) (Assamoi et al.,2008a;Assamoi etal., 2008b;Assamoi et al., 2009).In order to improve oxygen transfer inP. canescens10-10c culture, Gaspar et al. (1997) studied theinfluenceoftheagitationandaerationratesonKLaina mono-agitated reactor; the authors concluded thata reduction in specific power is more profitable toxylanase production than an increase inKLa beyonda critical value.Too littleKLa is harmful to biomassgrowth.Thiswasverifiedwhenproductionat200rpmwithDT4turbine(P=102W.m-3,KLaatabout20h

-1)reached only 370IU.ml-1.An optimumwas found at300rpmwithFBT8mobile(P=about232W·m-3,KLaatabout62h-1),whereproductionreached844IU.ml-1.Bakri et al. (2003) performed experiments in orderto evaluate the influence of carbon sources, nitrogensources,andmoisturecontentonxylanaseproductionby P. canescens 10-10c in solid-state fermentation.The experiments started in submergedculturesusingErlenmeyer flasks (250ml) containing 50ml of themedium; other experiments were conducted usingsolid-stateculture.Themaximumenzymeproductionobtained was 7,448IU.g-1 in submerged culture and9,632IU.g-1 in solid-state culture after 12days. Theyields of xylanase productivity from P. canescens10-10c observedwere approximately 1.5 fold higherthanoptimumproductivitiesreportedintheliteraturefor some microorganisms grown in solid-statefermentation.According to the results fromAssamoietal. (2008a),Assamoi et al. (2008b), andAssamoietal. (2009),xylanasecanbeproducedinsolid-stateculture. The experiments were carried out eitherin Erlenmeyer flasks or reactors with P. canescens10-10c.Inflasks,itwasobservedthatproductionwasmaximal after seven days, 18,895IU.g-1. Thereafter,theauthorscarriedoutascale-upinplasticbagsandamulti-layerreactor.Theresultsaftersevendayswere9,300IU.g-1 in plastic bags and 10,200IU.g-1 in themulti-layer reactor. These results demonstrated thatmanyproblemsoccurinsolid-statefermentationasthescaleof theprocess increases; inparticular, transportphenomena are strongly affected by the scale of theprocess.Inthiscase,oxygentransferinsidetheplasticbagsisexpectedtobeverylow.Inordertoimprovetheoxygentransfereffectively,itwouldbeinteresting,forexample,tosupplementtheplasticbagswithasoftenedforcedaeration(lowandintermittenthumidifiedairflowrate).Polizelietal.(2005)summarizeddataonvariouscommercial xylanases produced in submerged and

solid-statefermentationculturesbymicroorganisms.Itshouldbenotedthatabout80-90%ofallxylanasesareproduced in submerged culture.Wheat bran and riceareregardedasinducers.

3.3. Secondary metabolites produced by Aspergillus in different bioreactor designs

The fungal metabolites produced by Aspergilluswere penicillin, citric acid, koji acid, L_malic acid,amylase,catalase,cellulase,galactosidase,glucanase,glucosidase, hemicellulase, lipase, pectinase andprotease.deVriesetal.(2001)reportedthatwithinthegenusAspergillus,comprisingagroupoffilamentousfungiwithalargenumberofspecies,themostimportantfor industrial applications are some members of thegroupofblackAspergilli(AspergillusnigervanTieghemand Aspergillus tubingensis [Schöber] Mosseray).Reclassification using molecular and biochemicaltechniques resulted in clear distinctions being madebetween eight groups of black Aspergilli: A. niger,A. tubingensis,Aspergillusfoetidus(Nakazawa)ThomandRapper,Aspergilluscarbonarius(Bainier)Thom,Aspergillus japonicus Saito, Aspergillus aculeatusIizuka,Aspergillus heteromorphus Batista andMaia,andAspergillusellipticusRaperandFennell.Productsof several of these species have attained a generallyrecognized as safe (GRAS) status, which allowsthemtobeusedinfoodandfeedapplications.BlackAspergillihaveanumberofcharacteristicsthatmakethemidealorganismsforindustrialapplications,suchas good fermentation capabilities and high levels inproteinsecretion.Theresultsobtainedinacomparativestudy on protease yield by solid-state fermentationor submerged fermentation indicated that among thevariousagro-industrialresiduesused,wheatbranwasthemost suitable substrate for protease synthesis byA. oryzeaNRRL1808insubmergedcultureaswellassolid-state fermentation.The solid-state fermentationshowed its superiority for enzyme production oversubmerged culture, since a comparative evaluationof protease yields by the two fermentation systemsshowed3.5-foldgreaterenzymeproductionusingthesolid-state format (Sandhya et al., 2005).A mixtureof rice bran, rice husk and gram hull in 5:3:2 ratiowas found to be themost suitable,wheremaximumenzyme production occurred after 72h and 96hof incubation under submerged and solid-statefermentation conditions, respectively (Nehra et al.,2002). Production of xylanase by the A. niger KK2mutantinsolid-statefermentationreached14,790IU.l-1.h-1(Parketal.,2002);theauthorsremarkedduringexperimentationthatthehighestproductionofxylanase(2,596IU.gds-1)wasobtainedat40°Caftersixdays;athighertemperatures,itsproductiondecreasedsharply,suggesting that the end point of fermentation should

440 Biotechnol. Agron. Soc. Environ. 201519(4),430-442 MusoniM.,DestainJ.,ThonartPh.etal.

be carefully controlled because synthesized xylanasecouldbedegradedbynon-specificproteasessecretedbythefungus;themaximumvalueofxylanaseactivityachievedwas5,071IU.g-1ofricestraw,whichissimilartothe5,484IU.g-1ofricestrawpredictedbythemodelproposed.Wang et al. (2005) reported that proteasecouldbeproducedfromAspergillusoryzea(Ahlburg)Cohnbyeithersolid-stateorsubmergedfermentation.However, problems exist in both processes. Besidesincomplete nutrient utilization and frequent bacterialcontamination, solid-state fermentation also suffersfrom difficulties in establishing a constant moisturecontent, evenly distributed aeration and effectiveremovalofmetabolicheat.Insubmergedfermentation,filamentous fungi exhibit various morphologies,ranging from dispersed mycelia to pellets. In somecases, dispersed growth benefits enzyme production,but in a medium of low viscosity; it is difficult toachieveabsolutelydispersedmycelia,althoughfungalmorphologyvarieswithbrothpH, inoculumsizeandquality, dissolved oxygen tension, mixing intensity,polymer additives and surfactant addition. For theproductionofxylanase,Begetal.(2001)reportedthatthe advantages of solid-state fermentation processesover liquid-batch fermentation include smallervolumesofliquidrequiredforproductrecovery,cheapsubstrate, low cultivation cost for fermentation, andlowriskofcontaminationinadditiontohighenzymeyield.Seyeetal.(2014)carriedoutexperimentswitha fungal biofilm reactor combining the technologicaladvantages of submerged fermentation (i.e., freewater facilitating the control of culture parameters)withthebiologicalcharacteristicsfoundinsolid-statefermentationinordertoestablisheffectofinsecticidalactivity of conidia and metabolites secreted by anentomopathogenic A. clavatus strain in this culturesystem against Culex quinquefasciatus larvae andadults.Theyconcludedthattheprocessallowedfacilityinrecuperationandpurificationofconidia(confinedonthesolidsubstrate)andmetabolites (contained in theliquidmedium).Theseresultsconfirmtheusefulofthesystem,which can be used in similar production forotherfungi.Submergedfermentationisusedmoreoftenthansolid-statefermentationeventhissolid-stategiveshighproductionofbiomoleculesandconidia.Anotherbioreactortypethatcanenhanceproductionoffungalmetabolites and conidia in comparison with thoseproduced in submerged culture is biofilm bioreactorwhich simulates solid-state. Gutiérrez-Correa et al.(2012) discussed the main biological mechanismsthat support the development of filamentous fungalbiofilms and the recent advances on their use inindustrial processeswith an emphasis onAspergillusbiofilms. They concluded that cell adhesion appearsto be the trigger for biofilm formation and its mainindustrial features such as increased production of

metabolitesandenzymes.Inthissense,agreatdealofinformationhasbeengainedrelatingtotheindustrialusesofbiofilmsfromAspergillusandotherfilamentousfungi, which were originally considered as derivedfromcell immobilization systems.Efforts to seek anagreement are required to differentiate filamentousfungalbiofilmsfromthosecellimmobilizationsystemsbasedon“natural”or“passive”forces,sincethiswouldencouragedeepresearchtounderstandgeneregulationsystems and behaviour in such a way to achieve itsoptimization,scale-up,andindustrialproduction.

4. CONCLUSIONS

Theaimofthispaperwastohighlighttheprocessusedforoptimizingtheproductionofbiomoleculessecretedby filamentous fungi. Traditional methods used fortheproductionofenzymesandsecondarymetaboliteshavebeenfocusedonsolid-statefermentationwithoutcontrollingfactorsessentialformicroorganismgrowth.However, submerged fermentation is more widelyused,considering thepossibility to implement robustcontrol loops allowing to ensure the reproducibilityof the process. The cost of secondary metabolite isone of the main factors determining the economicsof process. In some cases, solid-state fermentationoffers advantages over submerged fermentation, butin general submerged cultures are used most oftensince theparameterscanbecontrolledefficiently.Asfilamentous fungi are naturally adapted to grow onsurfaces,underwhichconditionstheyshowaparticularphysiological behaviour different from that observedinsubmergedfermentation,anotherbioreactorprocessnamed “biofilm” that can enhance the production offungal metabolites using a synthetic support withina liquid environment should also be used in orderto enhance the production of biomolecules needed.Thisbiofilmreactorcanbeconsideredasapromisingtechnology combining the advantages of submergedfermentation in terms of process control and theadvantages of solid-state fermentation in terms offungalbiologyanddevelopment.

Acknowledgements

MichelMusoniistherecipientofagrantfromtheBelgianTechnicalCooperation(BTC/CTB).

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