4.Production of Fungal Antibiotics Using Polymeric Solid Supports in Solid State and Liquid Fermentation

ORIGINAL PAPER

Ramunas Bigelis Haiyin He Hui Y. YangLi-Ping Chang Michael Greenstein

Production of fungal antibiotics using polymeric solid supportsin solid-state and liquid fermentation

Received: 1 December 2005 / Accepted: 2 March 2006 / Published online: 6 May 2006 Society for Industrial Microbiology 2006

Abstract The use of inert absorbent polymeric supportsfor cellular attachment in solid-state fungal fermenta-tion inuenced growth, morphology, and production ofbioactive secondary metabolites. Two lamentous fungiexemplied the utility of this approach to facilitate thediscovery of new antimicrobial compounds. Cylindro-carpon sp. LL-Cyan426 produced pyrrocidines A and Band Acremonium sp. LL-Cyan416 produced acremoni-dins AE when grown on agar bearing moist polyestercellulose paper and generated distinctly dierentmetabolite proles than the conventional shaken orstationary liquid fermentations. Dierences were alsoapparent when tenfold concentrated methanol extractsfrom these fermentations were tested against antibiotic-susceptible and antibiotic-resistant Gram-positive bac-teria, and zones of inhibition were compared. Shakenbroth cultures of Acremonium sp. or Cylindrocarpon sp.showed complex HPLC patterns, lower levels of targetcompounds, and high levels of unwanted compoundsand medium components, while agar/solid supportcultures showed signicantly increased yields of pyr-rocidines A and B and acremonidins AE, respectively.This method, mixed-phase fermentation (fermentationwith an inert solid support bearing liquid medium),exploited the increase in surface area available forfungal growth on the supports and the tendency ofsome microorganisms to adhere to solid surfaces, pos-sibly mimicking their natural growth habits. The pro-duction of dimeric anthraquinones by Penicillium sp.LL-WF159 was investigated in liquid fermentationusing various inert polymeric immobilization supportscomposed of polypropylene, polypropylene cellulose,

polyestercellulose, or polyurethane. This culture pro-duced rugulosin, skyrin, avomannin, and a new bis-anthracene, WF159-A, after fermentation in thepresence and absence of polymeric supports for myce-lial attachment. The physical nature of the dierentsupport systems inuenced culture morphology andrelative metabolite yields, as determined by HPLCanalysis and measurement of antimicrobial activity.The application of such immobilized-cell fermentationmethods under solid and liquid conditions facilitatedthe discovery of new antibiotic compounds, and oersnew approaches to fungal fermentation for naturalproduct discovery.

Keywords Fermentation Antibiotics Naturalproducts Pyrrocidines Acremonidins

Introduction

New fermentation methods that inuence the growthand metabolism of microorganisms enhance their va-lue as sources of natural products and potentialtherapeutic compounds [20, 21]. Solid-state fermenta-tion draws on traditional fungal fermentation tech-nology [1012], even ancient food fermentationmethods [9], yet still oers opportunities for thedevelopment of new bioprocesses for natural products.Such alternative bioprocesses include the use of poly-meric supports for mycelial attachment and growthduring fermentation.

Both solid-substrate and solid-state fermentation[1, 6, 16, 30, 35, 41, 42, 46, 4952, 55] employ a naturalsubstrate as a carbon/energy source in the presence oflittle or no free water, but the broader designation,solid-state fermentation, includes the possible applica-tion of an inert substrate as a solid support in a suit-able medium. Both approaches avoid free liquid anddier from liquid or submerged fermentations, whichare performed in dilute solutions or slurries [4, 39, 53,

R. Bigelis (&) H. He H. Y. Yang L.-P. Chang M. GreensteinNatural Products Research, Chemical and Screening Sciences,Wyeth Research, 401 N. Middletown Road, Building 205,Room 407, Pearl River, NY 10965, USAE-mail: [email protected].: +1-845-6023994Fax: +1-845-6025687

J Ind Microbiol Biotechnol (2006) 33: 815826DOI 10.1007/s10295-006-0126-z

58]. Solid supports in solid-state fermentation mayconsist of natural materials, a variety of syntheticmaterials especially polymeric substances, or homoge-nous combinations of natural and synthetic substances,such as woven bers, incubated in the presence ofnutrients. Membrane lters used in microbiology aretypically thin porous sheet structures made of celluloseesters or similar polymeric materials, and may beconsidered inert if they are resistant to degradation orare metabolized poorly [44]. Solid support systemsapplied to solid-state fermentation provide new ways tomanipulate variables that inuence growth and physi-ology [6], and, thus, to exploit fungal dierentiationand developmental processes that are often linked topathways of secondary metabolism that produce com-plex compounds [7, 15]. Similarly, solid supports ap-plied to liquid fermentation permit the manipulation ofthese variables in new ways, as documented by anextensive literature on microbial cell immobilization byattachment, entrapment, aggregation, or containmentand by a long history of its application to commercialprocesses [25, 26, 36, 47, 49, 54, 62, 66, 67, 73].

Solid-state fermentation has a record of successfulapplication to the production of secondary metabo-lites, as the mycelial state is associated with produc-tion of many industrial compounds of this class and iswell suited for growth on solid substrates [35]. Forexample, antibiotic production under such conditionsis often associated with higher yields in shorter timeperiods compared to the alternative submergedfermentation approach, even oering processingadvantages [55]. Solid-state systems, their denitionsand advantages, and the physico-chemical andenvironmental factors that aect them have beenrecently reviewed by Krishna [35], as well as others[5, 33, 55, 59].

We are interested in alternate approaches to theproduction of bioactive microbial metabolites, and therapid and ecient evaluation of fungi for secondarymetabolite production. We have investigated variationsof solid-state and liquid fermentation, as well as mixed-phase fermentation, a fermentation method withabsorbent inert materials that bridges solid-state andliquid fermentation. Here we report the production oftwo antibiotic groups, pyrrocidines and acremonidins,by growing two fungi on an easily harvested solid sup-port composed of polyestercellulose bers able to ab-sorb a liquid nutrient medium and allow cellularattachment. Also, we report the production of four otherantibiotics, two known anthraquinones, avomannin,and a new bisanthracene, by growing another mycelialfungus in the presence of various solid supports agitatedin liquid medium.

We have reported the chemical elucidation of thepyrrocidines [28] and acremonidins [29] in earlier work.Wicklow et al. [69] have reported the production ofpyrrocidines by Acremonium zeae and have broughtattention to the biocontrol potential of this organism foragricultural applications.

Materials and methods

Sources of fungal cultures

Fungal cultures Cylindrocarpon sp. LL-Cyan426 andAcremonium sp. LL-Cyan416 were obtained from theWyeth collection. Both fungi are isolates from a mixedDouglas Fir-Hardwood forest, Crane Island Preserve,San Juan County, Washington State. Penicillium sp. LL-WF159 (subgenus Furcatum) was also obtained from theWyeth Culture Collection.

Fermentation and processing of cultures

All fungal fermentations were performed at 22C usingDifco medium ingredients (Becton, Dickinson andCompany, Franklin Lakes, NJ), except where indicatedotherwise. D-glucose was obtained from Sigma-Aldrich,St. Louis, MO.

Fungal cultures Cylindrocarpon sp. LL-Cyan-426 orAcremonium sp. LL-Cyan-416 were plated on Bennettsagar medium (10 g/l D-glucose, 1 g/l beef extract, 1 g/lyeast extract, 2 g/l NZ amine A, 20 g/l agar) from afrozen 25% glycerol storage vial. A small agar slicebearing mycelial growth was used to inoculate 50 ml ofseed culture in potato dextrose broth (PDB) in a 250-mlErlenmeyer ask shaken at 200 rpm for 1 week. Pro-duction medium (1 l) for use with a solid support ofpolyestercellulose consisted of malt extract medium(ME) with agar (25 g malt extract, 5 g peptone, 0.5 gyeast extract, 20 g agar) that had been sterilized andpoured into a sterile 30 20 13 cm polypropylene traycovered with aluminum foil. The solidied agar was thenoverlaid with a sterile 28 46 cm sheet of nongauzemilk-lter paper (KenAG Animal Care Group, Ashland,OH) that had been sterilized separately. The productionmedium was inoculated by pipeting 50 ml of seed cultureuid onto the sheet of polyestercellulose. The inocu-lated tray culture was incubated stationary at 22C.Fungal mycelia were primarily associated with thepolyestercellulose bers and this growth pattern facili-tated rapid and convenient harvest of the biomass withminimum carryover of agar and associated mediumcomponents. After 2 weeks of incubation, the milk-lterpaper bearing prolic mycelial growth was easily re-moved from the surface of the agar, lyophilized for5 days, and then extracted with 1 l of methanol. Liquidmedium fermentations were performed in 2.8-l Fernbachasks containing 1 l of ME medium shaken for 2 weeksat 200 rpm.

The extraction of cultures of Cylindrocarpon sp. LL-Cyan-426 and Acremonium sp. LL-Cyan-416 was per-formed by agitation with Diaion HP20 resin (MitsubishiChemical Co., Tokyo, Japan) for at least 2 h. The resinand biomass were recovered by centrifugation, and thismaterial was lyophilized and then extracted with meth-anol prior to analysis. Culture broth was also lyophilized

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without resin treatment, extracted with methanol, andanalyzed for comparison. The resin was found to be anecient means of concentrating natural products withexcellent recovery of metabolites and antimicrobialactivity. When desired, the methanol extracts wereconcentrated tenfold with a Thermo Electron SpeedVacConcentrator (Thermo Electron Corp., Waltham, MA)equipped with a cold-trap and vacuum.

Seed cultures of Penicillium sp. LL-WF159 wereprepared as indicated above. Experiments were per-formed in 50-ml Erlenmeyer asks (Pyrex No. 4442)containing 15 ml of PDB shaken at 200 rpm, condi-tions that favored secondary metabolite production.Nonaerated, static cultures were not productive, norwere cultures grown in Czapek-Dox Broth or Sabou-raud Maltose Broth. Initial experiments with solidsupports employed 50-ml shake-asks bearing cylindersof milk-lter paper (4.5 10 cm, KenAG) that hadbeen creased gently along their length and then insertedwith extension into the ask stem. The vertical cylin-ders absorbed PDB medium added afterwards andwere able to withstand both autoclaving and agitation.In subsequent experiments, thin polymeric discs 4.2 cmin diameter composed of polyurethane (HT4201 foamwipe, Wilshire Technologies, Carlsbad, CA), polypro-pylene (Spectrawipe 6, Baxter Healthcare, Deereld, ILor MicroFirst wipe, Berkshire/Dupont, Great Barring-ton, MA/Wilmington, DE), polypropylene cellulose(Fabwipe TX3009, Texwipe Co., Kernersville, NC), orpolyestercellulose (KenAG milk-lter paper or Su-rex805 wipe, Berkshire, Great Barrington, MA) wereemployed in shake-asks containing PDB. Cultureswere either shaken at 200 rpm for a given period orwere rst maintained static, then shaken at 200 rpm.Both protocols generated distinctly yellow-orange cul-tures. After an incubation period, the cultures werelyophilized, extracted with methanol, and the methanolextracts were concentrated tenfold with a ThermoElectron SpeedVac Concentrator. Since antimicrobialactivity was primarily associated with fungal biomass,immobilized mycelia could, if desired, be selectivelyremoved from shake-asks, and thus easily separatedfrom medium components for harvesting and extrac-tionor even reinoculation into fresh media understerile conditions. None of the polymeric supports

employed in these studies contained antimicrobialactivity after methanol extraction of uninoculatedmaterial, tenfold concentration, and bioassay, nor wereany prominent peaks apparent after HPLC analysis ofsuch preparations.

Analytical procedures

HPLC analysis was performed using a C18 column(YMC Co., Kyoto, Japan), YMC ODS-A, 5 lm, 120 A,4.6 150 mm, and employed a linear gradient: 20100% acetonitrile in water in 23 min and 100% aceto-nitrile 1 min.

Measurement of antibacterial activity

The antimicrobial activities of tenfold concentratedfungal extracts were determined by the standard agardiusion method using selected antibiotic-sensitive andantibiotic-resistant microorganisms [17]. A volume of20 ll of extract was dispensed into wells of an agar assayplate and the zones of inhibition were measured after1618 h of incubation at 37C using a handhelddigital caliper. The test microorganisms includedmethicillin-susceptible Staphylococcus aureus SA375,methicillin-resistant S. aureus SA310 (MR), and vanco-mycin-resistant Enterococcus faecium (VR).

Results

Production of pyrrocidines A and B by Cylindrocarponsp. LL-Cyan426

Cylindrocarpon sp. LL-Cyan426 was reported earlier toproduce the antibiotics pyrrocidines A and B (Fig. 1),natural products containing rare 13-membered macro-cycles [28]. Solid-state fermentation with a moistenedpolyester-cellulosic support residing on agar, i.e., mixed-phase fermentation, was indispensable to the discoveryof these compounds. The two pyrrocidines were detect-able under these special growth conditions in a malt ex-tract medium, but not in a liquid version of this medium.

O NHOH

O

OH

H

H HO NHOH

O

OH

H

H

Pyrrocidine A Pyrrocidine B

Fig. 1 Structures ofpyrrocidines A and B

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Cylindrocarpon sp. LL-Cyan426 was grown on MEagar medium bearing a polyestercellulose support, asillustrated in Fig. 2. The fungus was also grown in liquidME medium by conventional shake-ask fermentation.Production of pyrrocidines A and B under these twoconditions was compared by HPLC analysis afterextraction and concentration. In addition, antimicrobialactivities associated with tenfold concentrated methanolextracts from such fermentations were monitored by theagar diusion method using antibiotic-resistant andantibiotic-susceptible test organisms. The production ofantimicrobial activity was favored by growth of strainLL-Cyan426 on an inert support able to absorb waterand nutrients from agar medium, (Table 1). Extracts ofcultures with the solid support generated larger zones ofgrowth inhibition against strains of S. aureus andE. faecium than did comparable concentrated extracts ofconventional shake-ask cultures. A concentrated (10 )extract of residual ME agar (post-harvest) containedonly traces of antimicrobial activity. Stationary culturesin liquid ME medium also showed trace or no activity.Concentrated extracts of lyophilized whole agar culturesfrom petri plates bearing a lawn of growth producedsmall, hazy zones of antimicrobial activity.

HPLC analysis of tenfold concentrated methanolextracts revealed dierences between the metaboliteproles of 2-week fungal cultures grown on agar med-ium under mixed-phase conditions and cultures grown

by conventional liquid fermentation. Pyrrocidines A andB were produced in measurable amounts only underconditions of mixed-phase fermentation. HPLC analysisof methanol extracts of Cylindrocarpon sp. LL-Cyan426immobilized on the solid support resolved pyrrocidine Aas peak 23.9 and pyrrocidine B as a shoulder of thispeak, as shown in Fig. 3a. The yield of pyrrocidine Awas 21.8 mg/l, and the yield of pyrrocidine B was3.1 mg/l. Cylindrocarpon sp. LL-Cyan426 grown in li-quid culture did not produce pyrrocidine A, the morepotent antibiotic of the two species, and pyrrocidine Bwas present at the threshold of detection, as shown inFig. 3b. Cylindrocarpon sp. LL-Cyan426 also produceda number of illicicolins (designated I in Fig. 3a, b),known fungal metabolites with antibiotic activity, underboth growth conditions.

The levels of polar material, consisting mainly ofresidual medium components, were signicantly reducedin extracts of fungal growth harvested on solid supportsfrom the agar surface, and this characteristic greatlyfacilitated purication of the two antibiotics. Extracts ofliquid cultures contained signicantly more componentsfrom the malt extract medium as represented by theearly peaks in the HPLC prole of Fig. 3b (retentiontimes of 015 min).

Pyrrocidines A and B have been puried and chemi-cally characterized after fermentation by these methodsand then tested against selected microorganisms [28].Pyrrocidine A exhibited potent antibiotic activityagainst most Gram-positive bacteria, including drug-resistant strains, but showed only moderate activityagainst S. pneumoniae. Pyrrodicine B was less activeagainst these test organisms.

Production of acremonidins AE by Acremonium sp.LL-Cyan416

Acremonium sp. LL-Cyan416 was reported in earlierstudies to produce the antibiotics acremonidins AE, allnatural products of polyketide origin [29]. Solid-statefermentation with a polyester-cellulosic support on MEagar was important in the discovery of these compoundssince these unique growth conditions signicantly ele-vated yields. The chemical structures of these ve nat-ural products are shown in Fig. 4.

As was the strain of Cylindrocarpon sp., Acremoniumsp. LL-Cyan416 was grown both on ME agar mediumbearing a polyestercellulose support (Fig. 2) and byconventional shake-ask fermentation in liquid MEmedium. The production of the ve acremonidins wascompared by HPLC after extraction and concentration.In addition, antimicrobial activities associated withtenfold concentrated methanol extracts from such fer-mentations were monitored by the agar diusion meth-od using antibiotic-resistant and antibiotic-susceptibletest organisms. The production of antimicrobial activityby strain LL-Cyan426 was favored by mixed-phase fer-mentation on moistened brous sheets.

Fungal growth

Polyester-cellulosicsupport

Petri plateAgar medium

Fig. 2 Fungal growth on polyester-cellulosic support on agarsurface

Table 1 Eect of growth conditions on the production of antimi-crobial activity by Cylindrocarpon sp. LL-Cyan426

Fermentation conditions Antibacterial activitiesa

(zone of inhibition in mm)

S. aureus S. aureus(MR)

E. faecium(VR)

Solid support from ME agar 22 mm 19 mm 16 mmResidual ME agar afterremoval of solid support

0 0 0

ME broth shaken 9 12 10ME broth stationary 0 0 0

MR methicillin-resistant, VR vancomycin-resistantaActivity determined by the standard agar diusion method, datafor 20 ll/well of the 10 extract

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Preparations from both types of fermentations pos-sessed antimicrobial activity against methicillin-resistantS. aureus SA310, methicillin-susceptible S. aureusSA375, and vancomycin-resistant E. faecium EC379.However, extracts of fungal biomass harvested on thepolyestercellulose support were more active and gen-erated larger zones of growth inhibition, as shown inTable 2. An extract of residual ME agar (post-harvest)showed low levels of antimicrobial activity. Concen-trated extracts of lyophilized whole agar cultures frompetri plates bearing a lawn of growth revealed smallerzones of antimicrobial activity, than did extracts ofcultures grown on polyestercellulose supports. Sta-tionary cultures in liquid ME medium showed noactivity.

HPLC analysis of tenfold concentrated methanolextracts revealed dierences between 2-week cultures ofAcremonium sp. LL-Cyan416 grown under mixed-phaseconditions (Fig. 5a) and those by conventional liquidfermentation (Fig. 5b). HPLC analysis of extracts ofmixed-phase fermentations revealed acremonidins AE(peaks 19.014, 15.261, 16.378, 13.347, and 12.576,respectively). The yields of these antibiotics were: A130 mg/l, B 4.5 mg/l, C 4.2 mg/l, D 3.1 mg/l, and E21 mg/l. Liquid fermentation produced signicantlylower levels of acremonidins A, B, D, and E and lackedacremonidin C completely, as indicated in the HPLC

chromatograms shown in Fig. 5b employing a threefoldexpanded mAU scale for the liquid fermentation. Bothtypes of fermentation extracts also contained knownquinones (designated Q in Fig. 5).

Acremonidins AE were puried from the methanolextracts of Acremonium sp. LL-Cyan416 grown by bothfermentation methods. Extracts of liquid fermentationsgenerated signicantly more polar material (Fig. 5b),mostly medium components that were eluted earlyduring HPLC (retention times of 010 min), whereasextracts obtained after mixed-phase fermentation hadsignicantly lower levels of such components (Fig. 5a).

Production of anthraquinones and avomanninby Penicillium sp. LL-WF159 in liquid fermentation

Initial experiments in liquid fermentation examined theinuence of a polyestercellulose support (KenAGmilk-lter paper) on secondary metabolism with Peni-cillium sp. LL-WF159 according to two dierent shake-ask protocols (Table 3). In the rst procedure, theorganism was preincubated without agitation for3 days, promoting mycelial attachment to a moistenedcylinder and immobilization, and then shaken for4 days. In the second procedure, a parallel fermenta-tion lacking such a polyestercellulose support was

Fig. 3 a, b HPLC analysis of extracts of Cylindrocarpon sp. LL-Cyan426 showing peaks corresponding to pyrrocidines A, B, and variousillicicolins (I)

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agitated without interruption for 7 days. After 7 daysof fermentation, all of the mycelial growth was asso-ciated with the cellulosic cylinder and the culture uidwas clear, while conventional fermentation broth con-tained dispersed mycelia. All fermentation broths(including supports when present) were lyophilized,extracted with methanol, concentrated tenfold, and

then analyzed for antimicrobial activity. Extracts ofculture LL-WF159 grown in the presence of a polyes-tercellulose support were more active in antimicrobialassays (Table 3). The zones of inhibition againstmethicillin-resistant S. aureus SA310, methicillin-susceptible S. aureus SA375, and vancomycin-resistantE. faecium EC379 were larger than zones obtained withstandard shaken cultures (without discs). Mycelialadhesion of Penicillium sp. LL-WF159 to the solidsupport had an apparent eect on the production ofbioactive compounds.

Shake-ask fermentations of Penicillium sp. LL-WF159 in PDB were extracted with methanol, analyzed,and subjected to bioassay-guided fractionation. All fer-mentation broths were typically a deep yellow. Fouraromatic polyketides were isolated, and a spectroscopicanalysis led to the determination of the structures ofthese compounds. Penicillium sp. LL-WF159 was foundto produce rugulosin and skyrin [18], two anthraqui-nones known to possess antimicrobial activity, as well asavomannin and new bisanthracene WF159-A (Fig. 4),in fermentations with and without a polyestercellulosesupport. Puried preparations of all four compounds

O

OHHO

COOCH3OH

OH

O OH

HO

OH

OH

O

COOCH3

HO

HO

OH

O OH

HO

OH

O

O

COOCH3

HO

HO

O

OH

O O

HO

OH

O

O

COOCH3

HO

HO

O

OH

OH

Acremonidin A

OOH

OHO OH

OH

HO

OHO

OH

O

O O

OHHO

COOCH3OH

OHOH

HO

H3COOC

OH

HO

Acremonidin B

Acremonidin C Acremonidin E

Acremonidin D WF159-A

Fig. 4 Structures of acremonidins AE and compound WF159-A

Table 2 Eect of growth conditions on the production of antimi-crobial activity by Acremonium sp. LL-Cyan416

Fermentation conditions Antibacterial activitiesa

(zone of inhibition in mm)

S. aureus S. aureus(MR)

E. faecium(VR)

Solid support from ME agar 17 mm 17 mm 14 mmResidual agar after removalof solid support

7 0 0

ME broth shaken 12 12 11ME broth stationary 0 0 0

MR methicillin-resistant, VR vancomycin-resistantaActivity determined by the standard agar diusion method, datafor 20 ll/well of the 10 extract

820

inhibited at least one of three Mur enzymes, (UDP-N-acetylglucosamine enolpyruvyl transferase), MurB(UDP-N-acetylenolpyruvoyl glucosamine reductase), orMurC (UDP-N-acetylmuramate: L-alanine ligase), threecytoplasmic steps involved in the biosynthesis of pepti-doglycan precursor [23].

The growth of strain LL-WF159 was then investi-gated in shake-asks containing a polymeric disc thatwas free to agitate in the growth vessel. The organismwas grown in PDB with and without discs composed of

polyurethane, polypropylene (two types), polypropylenecellulose, or polyestercellulose. Two dierent fermen-tation protocols were employed, and the relative yieldsof the four antibiotics were determined. All shake-askswere either agitated for 2 weeks, or they were main-tained static for 1 week followed by a period of shakingfor 1 week. The support matrices, all of which permittedmycelial attachment and growth, not only inuencedculture morphology and allowed cell immobilization(Table 4), but also elevated relative metabolite yields asdetermined by HPLC analysis (Table 5). Production ofall four compounds in fermentations without an addedsupport was enhanced by a static preincubation periodof 1 week that allowed a mycelial mat to form on themedium surface, followed by another weeklong periodof agitation. The thin white mycelial aggregate was stillintact after 2 weeks, though the medium was turbid withsome dispersed growth. The levels of the four antibioticscould be further elevated by the addition of a polymericdisc and the formation of an articial mycelial matthat was free to agitate in the growth vessel (Fig. 6). Thephysical nature of the support system inuenced therelative yields of the two bisanthraquinones and avo-mannin to varying degrees (Table 5). In general, therelative yields were increased, though in a few cases, thepolymeric disc lowered metabolite production owing topossible interference with mixing and aeration.

Fig. 5 a, b HPLC analysis of extracts of Acremonium sp. LL-Cyan416 showing peaks corresponding to acremonidins AE, and alsoseveral quinones (Q)

Table 3 Eect of mycelial adhesion of Penicillium sp. LL-WF159to a polyestercellulose support on antimicrobial zones of inhibi-tion

Fermentation conditions Antibacterial activitiesa

(zone of inhibition in mm)

S. aureus S. aureus(MR)

E. faecium(VR)

PDB shaken 7 days 12:15RC mm 9:16RC mm 8H mmPDB with support, static3 days, shaken 4 days

15 16 10

MR methicillin-resistant, VR vancomycin-resistant, RC zone withresistant colonies, H hazy zoneaActivity determined by the standard agar diusion method using10 ll/well of 10 extract

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Considering the two dierent fermentation protocols(Table 5), avomannin production was signicantly en-hanced by stationary preincubation that permittedmycelial mat formation on the surface of the medium ora comparable period of growth during which the moldinltrated the supplemented disc. For example, theproduction of avomannin was enhanced tenfold bystatic preincubation of the mycelial mat before agitation,and even more when a disc was employed for mycelialimmobilization. The extent of the enhancement was re-lated to the makeup of the disc. A polyurethane discemployed in a 14-day static/shaken fermentation wasespecially eective in elevating avomannin production,and the level of this compound was 22-fold greater thanthat attained after a standard 14-day fermentationlacking any supplement. The 14-day shaken fermenta-tion with disc elevated avomannin levels ninefold. Thetwo fermentation protocols incorporating a polymericsupport typically resulted in similar relative yields ofrugulosin, skyrin, and the new bisanthracene compound.However, both types of fermentations (shaken or static/shaken) that incorporated a disc support consistentlyoutperformed standard shake-ask fermentations thatlacked any polymeric disc, resulting in higher relativeyields of these three compounds (Table 5).

Morphological and physiological changes have beenobserved with a number of other mycelial fungi grownby shake-ask fermentation with a PUF support inour laboratory. Mycelial attachment to the support iscommon in such agitated cultures, and changes in

developmental patterns and secondary metabolism,pigment production, for example, are sometimes evi-dent (R. Bigelis and H. He, unpublished observations).

Discussion

Fungi are a remarkably diverse group of microorgan-isms that include an estimated 1.5 million species [27,34]. The manipulation of fungal growth conditions innew ways magnies their chemical diversity and in-creases their potential as sources of useful secondarymetabolites, and possibly new therapeutic compounds[2022]. Alternative approaches to fungal growth andsecondary metabolism in the fermentation laboratorycomplement bioprospecting for natural products withunique microbial isolates from unexplored or extremeenvironments [14]. While the developmental or ecologi-cal role fungal secondary metabolites may be complexand poorly understood [15, 22], the regulatory networksthat modulate natural product production can bemanipulated during fermentation to generate new anti-microbial compounds. As reported here, alternativeapproaches using solid supports in solid-state or liquidfermentation facilitated the discovery of two new classesof natural products, pyrrocidines and acremonidins[28, 29], as well as a new bisanthracene, by revealing newcompounds, elevating their yields, and simplifying har-vesting/processing steps. Thus, fermentation under solidconditions with a support bearing liquid medium, and

Table 4 Culture characteristics of Penicillium sp. LL-WF159 grown in liquid medium with dierent polymeric discs

Disc compositiona Disc source Culture appearanceb

No added disc None Turbid culture uidPolyurethane Wilshire Fungal biomass attached to supportPolypropylene 1 Baxter Fungal biomass attached to supportPolypropylene 2 Berkshire/Dupont Fungal biomass attached to supportPolypropylene cellulose Texwipe Fungal biomass attached to supportPolyestercellulose Berkshire Fungal biomass attached to support

aDiscs were 4.2 cm in diameter and resided on the bottom of 50-ml Erlenmeyer shake-asks containing 15-ml PDBbAll cultures were either shaken at 200 rpm at 22C for 2 weeks or were maintained static for 1 week then shaken for 1 week at 200 rpm at22C. All cultures were shades of yellow-orange after 2 weeks

Table 5 Relative yields of four metabolites in shaken and stationary/shaken fermentations with Penicillium sp. LL-WF159

Disc added to PDB Flavomannin Compound WF159-A Rugulosin Skyrin

Shakea Stat/shakeb Shakea Stat/shakeb Shakea Stat/shakeb Shakea Stat/shakeb

PDB only 418 4,205 184 460 1,955 4,586 245 1,068Polyurethane 3,784 9,384 779 758 8,784 6,778 984 1,246Polypropylene 1 140 9,049 1,167 1,048 5,918 7,578 942 1,759Polypropylene 2 89 9,683 606 889 6,287 7,563 1,448 1,352Polypropylene cellulose 316 6,197 1,003 758 5,407 7,852 829 1,573Polyestercellulose 157 4,585 778 533 4,020 6,329 397 1,037

All productive cultures were dark yellow to yellow-orange revealing colors characteristic of anthraquinones. Static 2-week culturescontained pale white, nonpigmented myceliaaShaken 14 days at 200 rpmbStatic 7 days, then shaken 7 days at 200 rpm

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fermentation in liquid medium bearing solid supportswith immobilized mycelia enhanced the discovery pro-cess for new natural products. The solid fermentationapproach reected growth conditions more commonlyencountered in nature, typically environments populatedby terrestrial fungi. These growth conditions are dis-cussed below together with applications to naturalproducts discovery.

The culturing of fungi on static supports in abundantliquid medium, i.e., mixed-phase fermentation with solidand liquid environments, exploits various environmentalstimuli that inuence metabolism, morphogenesis, anddierentiationand consequently natural product pro-les. The physicochemical characteristics of the supportsystem and its interaction with available water, oxygen,and medium components are primary variables that caninuence growth and metabolism under these condi-tions. Various other factors, some subtle, may also playa role in mediating the response of the colonizingorganisms to the support system. These factors includewater activity, water absorbency and retention, avail-ability of the gaseous phase in interstitial spaces andaeration, porosity, accessible surface area, surfacetopographies and architecture, mediators of adhesionand anchorage (such as adhesins as agents in biolmformation), and air-interface-induced development andaerial stimulation, as well as nutrient and waste productabsorbance and diusion [26, 52, 60, 61, 65, 70]. Somecharacteristics of the support system may change sig-nicantly during growth stages of the culture and mayeven generate gradients of environmental conditionsthat produce nutrient limitations simulating the vari-ability found in nature. The support environment itselfmay also reect subtle characteristics of natural fungalhabitats, ranging from inorganic native locales to livinghosts. Some supports such as cellulosic or syntheticpapers, cellulosic pulps, fabrics, membranes, plasticfoams, sponges, porous inorganic particulates, andespecially agricultural products may closely mimic nat-ural substrates. Such conditions may elicit growth habitsrelated to proliferation, cellular attachment, and dier-entiation.

Fungal interactions with native solid substrates areknown to inuence growth and physiology. Theseinteractions may initiate a cascade of events that inu-ence dierentiation. Articial membranes and etchedsurfaces have been used as tools to mimic leaf topog-raphy and induce the development of fungal infection

structures in vitro [24, 31, 32, 40, 71]. Obligate plantpathogens possess a unique and precise capability fortopographical perception and thigmotropic response toplant microarchitecture [31, 64, 72]. For example, thedierentiation program of the rust fungus Uromycesappendiculatus is responsive to leaf surface topographyof the bean Phaseolus vulgaris. The ability of U. ap-pendiculatus to infect the host plant and mobilize adevelopmental sequence involving gene expression,mitosis, and specic morphological adaptation is trig-gered by such surface signals [31, 72]. The topographicalsignal can orient growth, promote infection structureformation, and induce cell dierentiation along withnuclear division [2, 3, 31]. The rst infection structureforms directly over the stomata, through which it pen-etrates and forms other structures. U. appendiculatus iseven responsive to the microfabricated ridges on siliconwafers or plastic membrane replicas of the leaf surfacethat simulate stomatal guard cells [3, 31]. This organ-isms exquisite sensitivity to substrate elevation isremarkable: the induction of appressorium formationoccurs at ridge heights of 0.5 lm, while those less than0.25 lm or greater than 1.0 lm are signicantly lessinductive.

Though the mechanisms of surface recognition thatcan trigger fungal development are poorly understood,the hydrophobicity of the substratum is known to be animportant variable in the dierentiation processes ofsome fungi [24, 45]. Some pathogenic fungi undergospecic developmental processes that are inuenced bysuch interactions with surfaces. The rm attachment ofplant pathogenic fungi to plant surfaces is believed to beessential for prepenetration development and successfulinfection, followed by processes of dierentiation. Theformation of the appressorium by the rice blast fungusMagnaporthe grisea is primarily determined by thehydrophobicity of the contact surface [38]. Similarly,U. appendiculatus spore and germling adhesion andinduction of appressoria are closely related to the degreeof hydrophobicity of substrata [63]. A role for hydro-phobins, hydrophobic proteins located on the cell sur-face, has been proposed as mediators of these types ofresponses to surfaces [60, 61, 68]. Other fungi, however,can attach to a variety of surfaces, including articialsurfaces, and their behavior does not reveal a linkagebetween adhesion and surface hydrophobicity. Surfaceproperties such as charge, texture, and hardness mayplay a role in these cases. The corn leaf pathogenCochliobolus heterostrophus is an example of a plantpathogenic fungus that is insensitive to surface hydro-phobicity and diers from M. grisea, U. appendiculatus,Botrytis cinerea, Colletotrichum sp., Candida albicans,Nomuraea rileyi, Metarhizium anisopliae and a numberof other fungi that have been shown to adhere withgreater tenacity to more hydrophobic surfaces [13, 63].Moreover, fungal surface hydrophobicity can varyamong species, as well as within one species, dependingon the age of the fungus and the composition of thegrowth medium [57].

Mycelial mat

Polymeric disc with immobilized mycelia

Stationary culture, no disc Shaken culture with disc

Free mycelia

Fig. 6 Growth of Penicillium sp. LL-WF159 as a mycelialaggregate in stationary and shaken asks

823

Environmental stimuli associated with solid-statefermentation using polymeric materials may createadvantages over conventional liquid fermentation forproduction of certain fungal metabolites, especiallywhen such stimuli favor cellular dierentiation thatoccurs in the natural state. Investigations with lichenfungi by Culberson and Armaleo [19] parallel ndingsreported here with Cylindrocarpon sp. LL-Cyan426and Acremonium sp. LL-Cyan416. Their experimentswith the mycosymbiont Cladonia grayi suggest that thecharacteristic of secondary metabolism of natural li-chen is linked to their aerial habit of growth. Quan-titative analysis of polyketide metabolite yield coupledwith culturing on nylon microlters [48] placed onagar medium revealed that secondary metabolismcould be induced under restrictive conditions. Disag-gregated fungal mycelia were seeded by suction onnylon lters forming a mat before placement on solidmedium. Filters bearing the attached fungal mat wereremoved at set time points, harvested, and analyzedfor the production of a depside and two depsidonesfound in the natural lichen. The three natural productsappeared a few days after transfer of mycelia fromliquid to solid medium. Induction of the pathway wasenhanced on drier substrates, and was correlated withgrowth of aerial mycelia. Decreased water activity wascritical for this lichen mycobiont pathway, along withexposure to air. Specialized growth conditions areimportant for the study of this group of organisms,and their requirements provide insights into factorsthat may mediate secondary metabolism in otherclasses of fungi.

The relationship between morphological dierentia-tion, such as sporulation, and secondary metabolismsuggests independent processes regulated by a commonmechanism [8, 22]. Most solid-state fermentation pro-tocols are associated with environmental conditionssuitable for sporulation or conidiation [49], cellulardevelopment programs that can inuence secondarymetabolism and thus natural product proles [4]. And,solid-state fermentations involving a polymeric supportsystem are suitable for the induction of such fungaldierentiation patterns. The sporulation exhibited bydiverse lamentous fungi, especially species of Penicil-lium and Aspergillus, is stimulated primarily by ofexposure of hyphae to air. Additional stimuli associatedwith solid substrates and added supports, such as sur-face eects, dessication, osmotic stress, and nutrientlimitation, also inuence developmental processes ableto alter growth and physiology, as well as the productionof natural products [6, 24, 37, 43, 56, 73].

We have described the production of antimicrobialmetabolites by three fungi in media containing dierentinert solid supports composed of polymeric materials.These approaches not only improved yields of somepolyketide natural products, but also facilitated theexperimental process from fermentation to extractionto analysis, raising the question of other applications.It is conceivable that such immobilized-cell fermenta-

tion methods could be extended to miniaturized for-mats with small, absorbent brous discs amenable torapid automated screening. Alternative methods of thisnature could simplify and even condense some of themultiple steps necessary for microbial natural productdiscovery.

Acknowledgments We acknowledge the assistance Je Janso withantimicrobial assays and Eric Solum with fermentation work. Weare grateful to Dr Maya P. Singh for providing antimicrobial testorganisms and protocols.

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Production of fungal antibiotics using polymeric solid supportsin solid-state and liquid fermentationAbstractIntroductionMaterials and methodsSources of fungal culturesFermentation and processing of culturesAnalytical proceduresMeasurement of antibacterial activityResultsProduction of pyrrocidines A and B by Cylindrocarpon sp. LL-Cyan426Fig1Production of acremonidins A ndash E by Acremonium sp. LL-Cyan416Fig2Tab1Production of anthraquinones and flavomannin by Penicillium sp. LL-WF159 in liquid fermentationFig3Fig4Tab2Fig5Tab3DiscussionTab4Tab5Fig6AcknowledgmentsReferencesCR1CR2CR3CR4CR5CR6CR7CR8CR9CR10CR11CR12CR13CR14CR15CR16CR17CR18CR19CR20CR21CR22CR23CR24CR25CR26CR27CR28CR29CR30CR31CR32CR33CR34CR35CR36CR37CR38CR39CR40CR41CR42CR43CR44CR45CR46CR47CR48CR49CR50CR51CR52CR53CR54CR55CR56CR57CR58CR59CR60CR61CR62CR63CR64CR65CR66CR67CR68CR69CR70CR71CR72CR73

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