High scleroglucan production by Sclerotium rolfsii: Influence of medium composition

High scleroglucan production bySclerotium rolfsii: Influence of mediumcompositionJ.I. Farina1*, F. Sineriz1,2, O.E. Molina1,3 and N.I. Perotti1,3

1 Planta Piloto de Procesos Industriales Microbiologicos (PROIMI), Tucuman, (4000) Argentina,fax: 154–81–344887, e-mail: [email protected];2 Catedra de Microbiologıa Superior, Facultad de Bioquımica, Química y Farmacia, UNT, Argentina;3 Catedra de Microbiologıa General e Industrial, Facultad de Cs. Exactas y Tecnologıa, UNT, Argentina

Scleroglucan production by Sclerotium rolfsii was markedly affected by the C-source concentration, showing a highestvalue with 150 g sucrose l21. Production was also influenced by the N-source, being considerably higher in mediacontaining NO3

2 than in those containing NH41, which had a clear inhibitory effect. Once defined the optimum culture

medium composition, the highest exopolysaccharide production (ca. 26 g scleroglucan l21) was achieved after 72 h offermentation at shake flask scale. High values of yield (Yp/c 5 0.49), productivity (Pr 5 0.365 g l21 h21) and specificproductivity (Pr/x 5 0.031 g (g biomass)21 h21) were observed, and productivity was 1.5 times further increased byscalling-up to fermenter scale. Addition of L-threonine, sunflower oil and ascorbic acid diminished exopolysaccharideproduction.

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IntroductionThe branched exopolysaccharide (EPS), b-1,3-b-1,6-glucan or scleroglucan, produced by Sclerotium rolfsii underappropriate culture conditions has attracted great interestbecause of its potential in chemically enhanced oil recoveryand its immune stimulatory, antineoplastic and antimicro-bial activities significantly higher than other b-glucans(Halleck, 1967; McNeil & Harvey, 1993).

Despite these interesting applications and the potentialdemand of scleroglucan, the study of the influence ofculture medium composition as a tool for improving theEPS production by S. rolfsii has received scant attention.Concerning this, it was observed that nitrate has beenusually preferred as N-source for the scleroglucan produc-tion by S. rolfsii (Griffith & Compere, 1978; Ziebolz et al.,1987; Pilz et al., 1991), but no reasons for this choice weregiven. With respect to the C-source, both glucose andsucrose have been used for scleroglucan production, andHalleck (1967) previously suggested initial concentrationsranging from 30 to 150 g l21.

In this context, we concentrated this work on the study ofthe influence of culture medium composition to examinesome physiological factors affecting EPS production insight of improving the kinetic parameters corresponding tothe scleroglucan production by S. rolfsii.

© 1998 Chapman & Hall

Materials and methodsMicroorganism and inocula preparationSclerotium rolfsii ATCC 201126 was preserved following theprotocol previously described (Farina et al., 1996a). Seedcultures were obtained by cultivating 2-day-old myceliumpreviously grown at 30°C on PM20 agar (see below Culturemedia). Five agar discs (5-mm diam. each) covered withmycelium were placed per 100 ml of PM20 in 500 mlErlenmeyer flasks and then incubated at 220 rpm, 30°C,for 48 h. Five-fold dilutions (with fresh PM20) of thesecultures were further incubated for 48 h at 30°C, 220 rpm.They were then blended in a CB-6 Waring blender and5 ml from this homogenate were used to inoculate at 10%(v/v) the different culture media to be evaluated.

Culture mediaBasal medium composition (PM20) was (in g l21): NaNO3,3; K2HPO4.3H20, 1.3; KCl, 0.5; MgSO4.7H2O, 0.5;FeSO4.7H2O, 0.05; yeast extract, 1; citric acid.H2O, 0.7;sucrose, 20 (initial pH, 4.5). N, P and C sources weremodified as detailed in Results.

Culture conditions for EPS productionExperiments were carried out in triplicate in 250 mlErlenmeyer flasks containing 50 ml of culture medium.Cultivations were performed at 220 rpm, 30°C, for 72 hunless stated otherwise.

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Figure 1 Influence of medium composition on the EPS production by S. rolfsii. a) Effect of NaNO3 as N-source (C:20 g sucrose l21, P: 1.3 g K2HPO4.3H2O l21). b) Effect of (NH4)2SO4 as N-source (C: 20 g sucrose l21, P: 1.3 gK2HPO4.3H2O l21). c) Effect of K2HPO4.3H2O as P-source (C: 20 g sucrose l21, N: 2.25 g NaNO3 l21). d) Effect ofsucrose as C-source (N: 2.25 g NaNO3 l21, P: 2 g K2HPO4.3H2O l21). Other medium components as in PM20. All resultsafter 72 h of cultivation.

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Figure 2 Effect of osmotic pressure on the EPS pro-duction by S. rolfsii. All results after 72 h of cultivation (N:2.25 g NaNO3 l21, P: 2 g K2HPO4.3H2O l21 and C-sourceas described in Results).

Optimized scleroglucan production by S. rolfsii

Scleroglucan production at fermenter scaleInocula were prepared according to the same protocoldescribed for shake flask scale. A 10 l stirred-tank reactorfitted with baffles and six-flat bladed Rushton turbineimpellers (Microferm, New Brunswick Scientific Co.) witha working volume of 8 l of optimized culture medium(MOPT, see Results) was inoculated at 10% (v/v). Thefollowing conditions were maintained throughout theexperiment: air flow rate, 0.5 vvm; stirrer speed, 400 rpm;temperature, 30°C.

Analytical determinationsNeutralized samples (with 1M NaOH or 1M HCl, asrequired) were 4-fold diluted with distilled water. Afterheating (80°C, 30 min), they were homogenized in aCB-6 Waring blender (low speed, 30 s) and centrifuged(27,500 g, 20 min, 10–15°C). For estimating biomass, thewashed pellet was dried at 105°C to constant weight. TheEPS from clear supernatant was precipitated by adding anequivalent volume of ethanol 96% (v/v). This mixture wasallowed to stand for 8 h at 4°C to complete EPS precipita-tion. Scleroglucan was recovered by filtration undervacuum through Schleicher & Schuell filter paper (589blue ribbon) and dried at 105°C to constant weight.

The following determinations were performed in super-natants after centrifugation: ammonium according to theBerthelot method (Srienc et al., 1984), nitrate by means ofa reduction method using Devarda’s alloy (APHA, AWWA& WPCF, 1975), sucrose with the 3,5-dinitro salicylic acidreaction (Miller, 1959) and phosphorous according to theLindberg & Ernster method (1955). In this last case,proteins were previously eliminated by addition of tri-chloroacetic acid to the supernatants (1:6, v/v) and sub-sequent centrifugation (8,400 g, 10 min).

ResultsInfluence of N-sourceScleroglucan production was influenced by both the natureand the concentration of the nitrogen source. The bestresults were reached when the fungus was growing onnitrate, when values higher than 0.12 g N l21 led to thehighest EPS yields (Fig. 1a), so that in subsequent experi-ments 0.37 g N l21 (; 2.25 g NaNO3 l21) was theconcentration used. On the other hand, even at very lowNH4

1 concentrations, EPS production was significantlylower than the one obtained with nitrate (Fig. 1b). More-over, the quality of EPS produced under ammoniumconditions was flaky and finely dispersed making its recov-ery very difficult.

Influence of phosphate concentrationAlthough the influence of the phosphate concentration onthe EPS production was not so marked as for the nitrogen

source, scleroglucan production was increased for valueshigher than 0.12 g P l21 (Fig. 1c), showing a highest EPSvalue when 0.28 g P l21 (; 2 g K2HPO4.3H2O l21) wasused. Thus, the latter was the concentration selected forfollowing experiments.

Influence of sucrose concentrationWhen increasing concentrations of sucrose were tested, themost significant improvement on EPS production wasobtained. The highest scleroglucan concentration wasreached with 150 g sucrose l21 (Fig. 1d), determiningabout a 4-fold increase with respect to the EPS productionreached in previous experiments (with 20 g sucrose l21).

The optimized culture medium (MOPT) was finallydefined containing: 2.25 g NaNO3 l21 as N-source, 2 gK2HPO4.3H2O l21 as P-source and 150 g sucrose l21 as C-source. The rest of components were (in g l21): KCl, 0.5;MgSO4.7H2O, 0.5; FeSO4.7H2O, 0.05; yeast extract, 1;citric acid.H2O, 0.7 (initial pH adjusted to 4.5).

Influence of osmotic pressureWhile studying the influence of the sucrose concentrationon the EPS production (Fig. 1d), it was noted that media

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Figure 3 Effect of ammonium addition on the EPS production by S. rolfsii after 72 h of cultivation in MOPT (N: 2.25 gNaNO3 l21, P: 2 g K2HPO4.3H2O l21, C: 150 g sucrose l21). a) Different initial (NH4)2SO4 concentrations. b) Differenttimes of addition of a 0.8 g N l21 (NH4)2SO4 pulse.

J.I. Farina et al.

containing high EPS concentrations showed relatively lowsucrose consumption. To try an explanation for this behav-iour, the possible influence of medium osmotic pressure onthe EPS production was studied. Osmosity values equiva-lent to media containing 125 or 150 g sucrose l21 wereachieved by addition of appropriate amounts of NaCl orKCl to media containing 50 or 75 g sucrose l21 (Wolf etal., 1986). Nitrogen and P-sources and the other compo-nents of culture medium were added at the same concen-tration as in MOPT, and C-source was modified as follows.50: 50 g sucrose l21; 75: 75 g sucrose l21; 125: 125 gsucrose l21 ; 50(125)Na: 50 g sucrose l21 1 8.3 g NaCll21 ; 75(125)Na: 75 g sucrose l21 1 5.6 g NaCl l21 ;50(125)K: 50 g sucrose l21 1 10.6 g KCl l21; 150: 150 gsucrose l21 ; 50(150)Na: 50 g sucrose l21 1 11.3 g NaCll21 ; 75(150)Na: 75 g sucrose l21 1 8.6 g NaCl l21

(where ; means osmosity equivalence).

Scleroglucan production was significantly increased whenNaCl or KCl were added to culture medium, though thehigh EPS concentration found when 150 g sucrose l21 wasthe C-source could not be attained (results from ANOVAand Dunnett multiple comparisons tests, 95% confidenceintervals). The effect of KCl was similar to that exerted byNaCl (Fig. 2).

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Effect of ammonium additionPolysaccharide production was markedly reduced when(NH4)2SO4 was added to MOPT (Fig. 3a), indicating thatscleroglucan production may be subjected to metabolicregulation by ammonium. Concentrations of (NH4)2SO4 aslow as 0.10 g N l21 caused a 44% decrease on the EPSproduction with respect to the control (without NH4

1)and, at 0.80 g N l21 the EPS concentration showed a 70%decrease.

When 0.80 g N l21 as (NH4)2SO4 was added in the earlierstages of cultivation (Fig. 3b), lower scleroglucan produc-tion was obtained. However, when ammonium was addedafter 24 h of cultivation, the consequent decrease on theEPS production was not so pronounced, being this effectprogressively slighter as the addition of (NH4)2SO4 wasdelayed.

Effect of different substances on the EPSproductionDiverse substances previously reported to affect b-glucanbiosynthesis or degradation in different organisms (Reese &Maguire, 1969; Kritzman et al., 1976; Sadana & Patil,1985; Kottutz & Rapp, 1990; Stasinopoulos & Seviour,1990; Pilz et al., 1991; Hamada & Watanabe, 1993) were

Table 1 Addition of diverse substances to theoptimized culture medium (MOPT#) and their effect onthe EPS production by S. rolfsiiƒ.

SubstanceBiomass

(g l21)EPS

(g l21)

None (Control) 10.2 6 0.4 20.6 6 0.110 mM cellobiose 9.4 6 0.5 19.7 6 0.30.5 ml (tween 80) l21 10.1 6 0.9 19.8 6 0.50.6mM EDTA 7.4 6 0.3 19.1 6 0.63 g sunflower oil l21 8.2 6 0.1 14.0 6 0.53 g olive oil l21 9.9 6 0.4 18.1 6 0.910 mM L-threonine 4.2 6 0.2 9.9 6 0.30.003 g ZnSO4.7H2O l21 8.4 6 0.2 18.7 6 0.93 g ascorbic acid l21 7.3 6 0.4 14.4 6 0.5

# MOPT: see composition above.ƒ Results at 72 h of cultivation. Values 6 S.E.M.

Figure 4 Effect of cultivation time on the EPS produc-tion by S. rolfsii cultured in MOPT (N: 2.25 g NaNO3 l21,P: 2 g K2HPO4.3H2O l21, C: 150 g sucrose l21).

Optimized scleroglucan production by S. rolfsii

evaluated. When sunflower oil, L-threonine and ascorbicacid were added, a statistically significant decrease in EPSproduction was observed (according to ANOVA andDunnett multiple comparisons tests, 95% confidenceintervals). Biomass was significantly reduced after theaddition of EDTA, L-threonine and ascorbic acid (Table1).

Influence of cultivation timeFor the first 40 h of cultivation in MOPT, scleroglucanproduction occurred simultaneously with growth (Fig. 4).Thereafter, biomass exhibited slight increases while EPScontinued significantly increasing until a maximum valueat 72 h. Residual sucrose remained high even after 120 hof cultivation, and the small increases after 72 h may berelated to the presence of b-glucanases (Rapp, 1989). Atthe same time, biomass, which seemed to have reached thestationary state, was slightly increased while the EPSshowed a significant decrease.

With respect to the fermentation kinetics, the parameterscalculated from the obtained values for EPS and biomasswere very promising as comparing with the literaturebackground (Table 2).

Table 2 Comparison of fermentation kinetic parameters cflask scale.

Microorganism ReferenceM

S. glucanicum McNeil & Harvey, 1993 datum S. glucanicum NRRL 3006 Taurhesia & McNeil, 1994S. rolfsii ATCC 201126 This workƒ

Yp/c (yield factor) 5 EPS production (g l21) / C-source consumption (g lPr (productivity) 5 EPS production (g l21) / fermentation time (h)Pr/x (specific productivity) 5 Pr (g l21 h21) / biomass concentration (g lƒ: results corresponding to EPS production in MOPT at 72 h of cultivat

Scalling-up to fermenter scaleUnder these conditions, the maximum EPS concentration(26 g l21) was achieved at 48 h of cultivation. In spite ofthe fact that production was almost the same as that oneobtained in shake flask experiments, a remarkable improve-ment in the productivity value (Pr 5 0.542 g l21 h21) wasfound. Working at fermenter scale, the cultivation timecould be 24-h shortened (Figure 5) as comparing withprevious shake flask experiments, this being obviouslyconvenient regarding the fermenter time usage.

DiscussionIn order to improve the EPS production by S. rolfsii, resultsconcerning to the influence of carbon source concentrationwere the most successful ones, allowing to reach a 4-fold

orresponding to the EPS production by S. rolfsii at shake

ax. EPS(g l21) Yp/c

Pr(g l21 h21)

Pr/x(g (g biomass)21 h21)

not available 0.3–0.5 0.08–0.12 0.01519 0.47 0.095 0.01626 0.49 0.365 0.031

21)

21)ion

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Figure 5 Scleroglucan production at fermenter scale(working volume: 8 liters) by S. rolfsii cultured in MOPT.For cultivation conditions see Materials and Methods.

J.I. Farina et al.

increase on the EPS concentration and a 2-fold increase onyield (Yp/c). This finding was very interesting since sucroseconcentrations higher than 45 g l21 have been previouslysuggested to inhibit growth and scleroglucan productionin S. glucanicum (Taurhesia & McNeil, 1994).

Although osmosity exhibited a significant influence on b-glucan production, the highest EPS values reached with150 g sucrose l21 could not be equaled just by increasingosmotic pressure in culture medium. For some yeast-likefungi it has been previously observed that osmotic stresscould stimulate exopolymer production (Breierova et al.,1996). In aqueous solutions, the OH-groups located out-side the chain of the EPS establish intermolecular hydro-gen bonds with the surrounding water molecules. Thisinteraction could account for the EPS protective role as anosmotic buffer and its increased production under high-osmotic pressure environments. As was noted in a previousreport (Fariña et al., 1996b), this strain of S. rolfsii hasshown to be relatively halotolerant and, it would bereasonable that the increase in scleroglucan productiongrowing under high-osmotic pressure could be a part of anosmoregulation mechanism.

The results concerning the N-source influence revealedthat nitrate led to significantly higher EPS production thanammonium, of which injurious effect on scleroglucanproduction could be clearly demonstrated. This inhibitory

830 Biotechnology Letters ⋅ Vol 20 ⋅ No 9 ⋅ 1998

effect may be direct or indirect and related to synthesisor activity of some enzyme/s involved in the carbonflux. Similar results were previously obtained for otherpolysaccharide-producing microorganisms (Souw &Demain, 1979; Seviour & Kristiansen, 1983; Stasinopoulos& Seviour, 1989; Badr-Eldin et al., 1994). Moreover, in thecase of S. rolfsii, ammonium seemed to exert its effect onthe biosynthetic machinery, since when the addition inculture medium was delayed a slighter incidence on EPSproduction has been observed.

Concluding, our results revealed that manipulation ofculture medium composition allowed to significantlyincrease the EPS production by S. rolfsii ATCC 201126 inassociation with very good kinetic parameters. The mainadvantages were reached regarding productivity (Pr) andspecific productivity (Pr/x), determining clear benefits inEPS recovery and costs. Scalling-up to fermenter scaleinvolved a significant shortening in fermentation time andallowed a considerable improvement in productivity, thisbeing of great economic importance considering that themost known drawbacks found when comparing to xanthangum are the fermentation time required and the sclero-glucan yields. Since scleroglucan is very attractive fordifferent industrial applications and as antitumor agent(Pretus et al., 1991), these findings are mainly valuable toface the potential demand of this EPS.

AcknowledgementsThe authors gratefully acknowledge to CONICET andCIUNT (Argentina) for financial support of this work.

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Received: 19 April 1998Revisions requested: 27 May 1998

Revisions received: 13 July 1998Accepted: 14 July 1998

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