Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 1036–1043

Production of schizophyllan using Schizophyllum commune NRCM

Maushmi Kumari, Shrikant A. Survase *, Rekha S. Singhal

Food Engineering and Technology Department, Institute of Chemical Technology, University of Mumbai, Matunga, Mumbai 400 019, India

Received 23 November 2006; received in revised form 27 February 2007; accepted 28 February 2007Available online 18 April 2007

Abstract

In the present work, four strains were screened for schizophyllan production, of which Schizophyllum commune NRCM was selectedfor further work. The fermentation was carried out for 168 h at 28 ± 2 �C on an orbital shaker at 180 rpm. In the first step, one factor-at-a-time method was used to investigate the effect of media constituents such as carbon and nitrogen sources on schizophyllan production.Subsequently in the second step, concentration of the medium components was optimized using Response Surface Method (RSM). Theyield increased from 3.25 ± 0.72 g/l in the unoptimized media to 8.03 ± 1.12 g/l in the medium optimized by RSM.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Schizophyllan; Response surface method; Schizophyllum commune; Fermentation

1. Introduction

Schizophyllan is a non-ionic, water-soluble homopoly-saccharide consisting of a linear chain of b-D-(1-3)-gluco-pyranosyl groups and b-D-(1-6)-glucopyranosyl groupsproduced by fermentation from filamentous fungi Schizo-

phyllum commune ATCC 38548 (Rau et al., 1992). Thispolysaccharide has attracted attention in recent years inpharmaceutical industry as immunomodulatory, antineo-plastic and antiviral activities that are higher than otherglucans. It also finds applications in enhanced oil recovery,and in cosmetics (Rau and Brandt, 1994). The molecularweight ranges from 6 to 12 · 106 g mol�1 (Rau et al., 1990).

The optimization of fermentation conditions is an impor-tant step in the development of economically feasible biopro-cesses. A successful design of the fermentation process relieson producing a product to set specification (in terms of pur-ity, solubility, gel strength, pH of the solution). These goalscan be achieved by optimizing media composition, fermenta-tion conditions and fermentor design as well by developingsuperior strains by mutation (Margaritis and Pace, 1985).

0960-8524/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2007.02.029

* Corresponding author. Tel.: +91 022 24145616; fax: +91 02224145614.

E-mail address: shrikants.foodbio@udct.org (S.A. Survase).

Medium optimization by employing one factor-at-a-timemethod involves changing one independent variable (nutri-ent, pH, temperature, etc.) while fixing all the others at a cer-tain level. This single dimensional approach is laborious andtime consuming, especially for large number of variables,and frequently does not guarantee the determination of opti-mal conditions (Xu et al., 2003; Survase et al., 2006).

Response Surface Method (RSM) is a collection of sta-tistical techniques for designing experiments, buildingmodels, evaluating the effects of factors and searching forthe optimum conditions. RSM has been successfully usedin the optimization of bioprocesses (Kalil et al., 2000). InRSM, the experimental responses to design of experiments(DOE) are fitted to quadratic function. The number of suc-cessful applications of RSM suggests that second orderrelation can reasonably approximate many of the fermen-tation systems. Central composite design is the most widelyused response surface design. Rotatability is a desirableproperty of a central composite design. When there is a dif-ficulty in extending the star points beyond the experimentalregion defined by the upper and lower limits of each factor,a face-centered design can be used (Tsapatsaris andKotzekidou, 2004).

To the best of our knowledge the nutritional and envi-ronmental conditions for submerged culture of S. commune

M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043 1037

NRCM for schizophyllan production using RSM has notbeen demonstrated. Survase et al. (2006, 2007a,b) reportedoptimization of fermentation conditions for scleroglucan,another b-D-(1-3), (1-6) glucan. In the present work, isola-tion of S. commune and optimization of schizophyllan pro-duction using statistical approach is addressed. In the firststep, one factor at a time method was used to investigatethe effect of media constituents such as carbon and nitro-gen sources. Subsequently, in the second step concentrationof the medium components was optimized using RSM.

2. Methods

2.1. Materials

Media components such as glucose, sucrose, maltose,lactose, soluble starch, fructose, magnesium sulphate, yeastextract, peptone, casein digest, soybean meal, and beefextract, urea were purchased from Hi-Media Limited,Mumbai, India. Potassium di-hydrogen phosphate, sodiumnitrate, potassium nitrate were purchased from S.D. FineChemicals Ltd., Mumbai, India.

Strains of S. commune MTCC 1096, and S. communeCBS 103.96 were procured from Microbial Type CultureCollection (MTCC), Chandigarh, and Central Bureau voorSchimmelcultures (CBS), Netherlands, respectively. S.

commune NRCM was isolated at National Research Cen-tre for Mushroom (NRCM), Solan, Himachal Pradesh,India, while S. commune MSU was isolated at MaharajaSayaji Rao University of Badoda (MSU), Vadodara, Guj-arat, respectively.

2.2. Methods

2.2.1. Isolation of S. commune NRCM and S. commune

MSU

S. commune NRCM and S. commune MSU strains wereisolated from the wild growth on trees. The fruiting bodiesfrom the tamarind tree were used for the isolation. Dryfruiting body was washed in running tap water. They werestored in water for 6 h, treated with 70% alcohol, cut andagain soaked for 2 min in alcohol. These pieces were culti-vated on potato dextrose agar slants. They were grown for7 days at room temperature (28 ± 2 �C) and non-contami-nated slants were maintained and subcultured.

2.2.2. Maintenance of culture and seed culture preparationThe culture was grown on potato dextrose agar slants at

28 ± 2 �C for seven days. A 1 cm2 of mycelia along withagar from such slants were used to inoculate 50 ml of sterileseed culture medium in 250 ml conical flasks, which wasincubated at 28 ± 2 �C, 180 rpm for 3 days on rotaryshaker.

2.2.3. Selection of strainFour strains viz., S. commune NRCM, S. commune

MTCC 1096, S. commune MSU and S. commune CBS

103.96 were screened for schizophyllan production usingthe media reported by Rau et al. (1992) [in (g/l): Glucose– 30.0, yeast extract – 1.0, MgSO4 Æ7H2O – 0.5, KH2PO4

– 1.0] and Steiner et al. (1986) [in (g/100 ml): Avicel –4.0, bactopeptone – 3.5, CaNO3 – 0.5, KH2PO4 – 0.13,MgSO4 Æ7H2O – 0.05, trace element solution I, 0.1 ml{Stock Solution – in (g/100 ml) MnSO4 ÆH2O – 0.16,ZnSO4 Æ7H2O – 0.24 and CoCl2 Æ6H2O – 0.2} and trace ele-ment solution II, 0.1 ml {in g/100 ml FeSO4 Æ7H2O – 0.5}].Based on the initial screening, S. commune NRCM in med-ium of Rau et al. (1992) was selected for further studies.

2.3. Optimization of fermentation medium using one

factor-at-a-time method

Fifty milliliter of autoclaved medium (initial pH5.5 ± 0.2) was inoculated with 5 ml seed culture of S. com-

mune NRCM. Fermentation was carried out at 28 ± 2 �Cand 180 rpm for 168 h.

2.3.1. Effect of different carbon sources

In the production medium, glucose was substituted withfive different carbon sources viz., sucrose, fructose, solublestarch, maltose, dextrin and lactose. Initially all carbonsources were screened at 30 g/l, and after that sucroseand glucose were varied from 25 to 200 g/l. Fermentationwas carried out at 28 ± 2 �C and initial pH 5.5 ± 0.2 for168 h.

2.3.2. Effect of nitrogen sources

To study the effect of different nitrogen sources on schiz-ophyllan production, yeast extract was replaced with otherorganic nitrogen sources such as peptone, beef extract andinorganic nitrogen sources like sodium nitrate, ammoniumchloride, ammonium sulphate, ammonium nitrate and ureaat 3 g/l. Keeping total nitrogen source 3 g/l, the ratio oforganic to inorganic nitrogen source was varied from0:100 to 100:0. Fermentation was carried out at28 ± 2 �C with initial pH 5.5 ± 0.2 for 168 h.

2.3.3. Effect of initial pH

In order to study the effect of initial pH on schizophy-llan production, fermentation runs were carried out at dif-ferent initial pH values varying from 4.5 to 8.0. The effectof initial pH on biomass production and schizophyllanproduction was studied. Fermentation was carried out at28 ± 2 �C and 180 rpm for 168 h.

2.4. Optimization of medium components using RSM

RSM is an empirical statistical modeling techniqueemployed for multiple regression analysis using quantita-tive data obtained from properly designed experiments tosolve multivariable equations simultaneously (Rao et al.,2000). It was used to optimize nutrient concentrations forthe production of schizophyllan. A central composite rotat-able experimental design (CCRD) for four independent

1038 M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043

variables was used to obtain the combination of values thatoptimizes the response within the region of three dimen-sional observation spaces, which allows one to design aminimal number of experiments. The experiments weredesigned using the software, Design Expert Version 6.0.10trial version (State Ease, Minneapolis, MN). The mediumcomponents (independent variables) selected for the optimi-zation were sucrose, beef extract, potassium di-hydrogenphosphate and inoculum size. Regression analyses were per-formed on the data obtained from the design experiments.

Decoding of the variables was done according to the fol-lowing equation

xi ¼ ðX i � X cpÞ=DX i; i ¼ 1; 2; 3; . . . ; k; ð1Þ

where xi, dimensionless value of an independent variable;Xi, real value of an independent variable; Xcp, real valueof an independent variable at the center point; and DXi,step change of real value of the variable i correspondingto a variation of a unit for the dimensionless value of thevariable i.

Replicates at the centre of the domain in three blockspermit the checking of the absence of bias between severalsets of experiments. The relationship of the independentvariables and the response was calculated by the secondorder polynomial equation:

Y ¼ b0 þXk

i¼1

biX i þXk

i¼1

biiX iX j þX

i

Xj

i<j

bijX iX j; ð2Þ

Y is the predicted response; b0 a constant; bi the linear coef-ficient; bii the squared coefficient; and bij the cross-productcoefficient, k is number of factors. The second order poly-nomial coefficients were calculated using the software pack-age Design Expert Version 6.0.10 to estimate the responsesof the dependent variable. Response surface plots were alsoobtained using Design Expert Version 6.0.10.

2.5. Production profile for schizophyllan

Fourty two flasks, each containing 50 ml media wereinoculated with 10% inoculum. Three flasks containing50 ml fermentation broth were taken out from shaker every12 h during the course of 168 h fermentation. Broth (10 ml)was withdrawn from each of the flasks and analysed forbiomass, sugar utilization, phosphate utilization and schiz-ophyllan production.

2.6. Analytical determinations

2.6.1. Estimation of biomass

Three flasks containing 50 ml fermentation broth weretaken out from shaker. Broth (10 ml) from each flask wasdiluted 2–3 fold with distilled water, homogenized and thencentrifuged (10000g, 20 min). The pellet so obtained waswashed with distilled water and dried at 65 �C to constantweight and reported as dry cell weight (DCW). The superna-tant was used for estimation of schizophyllan production.

2.6.2. Estimation of schizophyllan production

Two volumes of 96% (v/v) isopropanol were added toprecipitate the schizophyllan from clear supernatant. Themixture was allowed to stand for 1 h at 4 �C for completeprecipitation. Schizophyllan was recovered and dried toconstant weight at 65 �C.

2.6.3. Sugar utilization during fermentation by S. commune

NRCM

Sucrose content of the cell suspension culture was esti-mated using DNSA (Di-nitrosalicylic acid) method asdeveloped by Miller (1959). Samples from the cell suspen-sion culture were withdrawn after every 12 h for 7 daysand estimated for the changes in the sucrose concentrationas follows. Cell suspension was centrifuged at 10 000g for20 min, and 10 ml of clear supernatant medium was takenand diluted with water to 50 ml which was then subjectedto hydrolysis using 6 ml of 6.34 N HCl at 60 �C for45 min. The solution was cooled and then neutralized using40% NaOH. The volume was then made up to 100 ml. To1 ml of this neutralized solution in a test tube, 1 ml ofDNSA reagent was added (1.6 g NaOH + 1 gDNSA + 30 g sodium potassium tartarate in 100 mlwater). The tubes were then placed in a boiling water bathfor 10 min, after which they were cooled and 10 ml of dis-tilled water was added to it to make up the volume to12 ml. The red coloured complex formed by the reactionof nitro group of DNSA with reducing sugars was readat 540 nm using Hitachi UV/Vis spectrophotometer(Dubois et al., 1956).

2.6.4. Phosphate utilization during fermentation by

S. commune NRCM

Phosphate content of the medium was estimated usingthe ascorbic acid method developed by Chen et al.(1956). Samples from the broth were withdrawn every12 h up to 7 days, and estimated for phosphate concentra-tion as follows. Cell suspension was centrifuged at 10000g

for 20 min, and 10 ml of clear supernatant medium wastaken and diluted with water 100 times. To 1 ml of dilutedsupernatant, 3 ml of distilled water and 4 ml of phosphatereagent (1 part 6 N H2SO4 + 2 parts of distilled water + 1part of 2.5% w/v ammonium molybdate + 1 part of 10%ascorbic acid) was added and incubated at 37 �C for a per-iod of 2 h. The colour formed as a result of the phospho-molybdate complex was estimated at 820 nm usingHitachi UV/Vis Spectrophotometer. The standard curvewas plotted with concentration against absorbance.KH2PO4 was used as standard in the range of 1–4 mg/ml.

3. Results and discussion

3.1. Isolation of S. commune NRCM

The cultures of the isolated fungi showed fruiting bodies(basidiocarps) and fast-growing cottony white mycelium.The fruiting bodies were characterized as pileus (cap of

M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043 1039

the mushroom) – 1–3 cm in diameter; surface – dark gray-ish brown and margin – lobed. S. commune was character-ized using scanning electron microscopy (SEM) as spores –white smooth, 2.8–4.6 · 1.25 lm; clamp connections andanastomoses close to hyphal tip. Clamp connection isdefined as a hyphal outgrowth, which, at cell division,makes a connection between the resulting two cells byfusion with the lower cell. S. fasciatum, S. palmatum, S.

umbrinum, S. brasiliense and S. commune show similarcharacteristics, and S. commune is the only species of thisgenus that has been recorded from India. Fruiting bodiesand clamp connections are distinctive features for S.

commune (Buzina et al., 2001; Kano et al., 2002).

4

/l)

3.2. Selection of microbial strain

Among the four strains, S. commune NRCM in mediareported by Rau et al. (1992) gave a maximum yield of1.62 ± 0.50 g/l schizophyllan. Therefore, this medium andS. commune NRCM was selected for further studies. Thiscould be due to its isolation from wild forest, which mayhave been responsible for good resistance towardsenvironment.

00.5

11.5

22.5

33.5

Yeast

Extrac

t

Beef E

xtrac

t

Pepton

e

Ammo. Chlo

ride

Ammo. Nitra

te

Sodium

Nitra

te

Ammo. Sulp

hate

Urea

Nitrogen Source

Schi

zoph

ylla

n (g

/l); D

CW

(g Yield (g/l) DCW(g/l)

Fig. 2. Effect of different nitrogen sources on schizophyllan and biomassproduction by S. commune NRCM. (Nitrogen source 3 g/l; DCW is dry

3.3. Optimization using one factor at-a-time

During the microbial fermentations, the carbon sourcenot only acts as a major constituent for building of cellularmaterial, but is also used in synthesis of polysaccharide,and as energy source (Dunn, 1985; Dube, 1983). Fig. 1shows the effect of different carbon sources on schizophy-llan production. The medium was supplemented with dif-ferent carbohydrates such as sucrose, glucose, fructose,lactose, soluble starch, dextrin and maltose as carbonsources, of which only glucose and sucrose were found tobe promising. Sucrose supported maximum production of3.20 g/l of schizophyllan, whereas glucose gave a yield of1.62 g/l of schizophyllan after 168 h of fermentation.

00.5

11.5

22.5

33.5

44.5

5

Glucos

e

Sucros

e

Maltos

e

Fruc

tose

Dextrin

Sol.Star

ch

Carbon Source

Schi

zoph

ylla

n (g

/l); D

CW

(g/l)

Schizophyllan (g/l) DCW (g/l)

Lacto

se

Fig. 1. Effect of different carbon sources on schizophyllan and biomassproduction by S. commune NRCM. (Carbon source 30 g/l; DCW is drycell weight.)

Sucrose gave maximum biomass (measured as dry cellweight, DCW), while dextrin gave minimum biomass.

Sucrose and glucose concentrations were varied from 25to 200 g/l. It was observed that increasing concentrationsof sucrose and glucose gave increasing schizophyllan pro-duction up to 150 g/l above which substrate inhibitionwas seen. The conversion rates of sucrose and glucose werefound to be low. Farina et al. (1998) has reported low con-version rates during the production of scleroglucan fromSclerotium rolfsii. They concluded that, high sucrose mighthave increased the osmotic pressure of the medium andthus influenced scleroglucan production and media con-taining high exopolysaccharide showed low sucroseconsumption.

Fig. 2 shows the effect of different organic and inorganicnitrogen sources on schizophyllan production. Among thethree selected organic nitrogen sources, beef extract gavemaximum yield of 2.62 g/l of schizophyllan. Among thefive inorganic nitrogen sources, ammonium nitrate gave amaximum yield of 1.2 g/l. Ammonium nitrate and beefextract gave maximum biomass production. Table 1 shows

cell weight.)

Table 1Effect of inorganic (ammonium nitrate) to organic nitrogen source (beefextract) ratio keep in total 3 g/l on schizophyllan and biomass productionby S. commune NRCM

Ratio Schizophyllana (g/l) DCWa (g/l)

0:100 2.62 ± 0.36 5.28 ± 1.1110:90 1.68 ± 0.45 4.39 ± 0.7920:80 1.32 ± 0.55 4.28 ± 0.7430:70 1.52 ± 0.23 4.13 ± 0.6640:60 1.40 ± 0.32 4.08 ± 0.7450:50 1.20 ± 0.56 3.92 ± 0.5860:40 1.20 ± 0.88 3.85 ± 0.4570:30 1.00 ± 0.45 2.45 ± 0.4780:20 0.82 ± 0.65 2.25 ± 0.8590:10 0.52 ± 0.74 2.13 ± 0.91100:0 0.49 ± 0.33 2.05 ± 0.97

DCW – Dry cell weight.a Results are mean ± SD of 3 determinations.

Table 3Analysis of variance (ANOVA) for the experimental results of the central-composite design (quadratic model)

Factora Coefficientestimate

Sum ofsquares

Standarderror

DFb F-value

Pc

Intercept 2.77 81.75 0.12 14 68.97 <0.0001X1 1.47 51.92 0.059 1 612.41 <0.0001

1040 M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043

effect of ratio of inorganic to organic nitrogen sources (at atotal concentration of nitrogen source as 3 g/l). Ratio of0:100 gave the maximum schizophyllan production of2.62 g/l.

The effect of different initial pH values on the schizophy-llan production was studied. An initial pH of 6.0 supportedmaximum production of 1.65 g/l of schizophyllan, whereasmaximum biomass production was obtained at a pH 6.5.Rau et al. (1992) reported a pH of 5.3 to be optimum forschizophyllan production. An inoculum size of 5 ml(10%) gave maximum yield of 1.62 g/l.

X2 �0.23 1.27 0.059 1 14.98 0.0015X3 �0.35 3.01 0.059 1 35.51 <0.0001X4 �0.16 0.58 0.059 1 6.80 0.0198X 2

1 0.25 1.76 0.056 1 20.80 0.0004X 2

2 0.29 2.34 0.056 1 27.64 <0.0001X 2

3 �0.45 5.44 0.056 1 64.13 <0.0001X 2

4 �0.13 0.48 0.056 1 5.70 0.0306X1 · X2 �0.60 5.71 0.073 1 67.38 <0.0001X1 · X3 �0.53 4.47 0.073 1 52.76 <0.0001X1 · X4 �0.40 2.53 0.073 1 29.82 <0.0001X2 · X3 0.19 0.57 0.073 1 6.72 0.0204X2 · X4 0.065 0.068 0.073 1 0.80 0.3860X3 · X4 �0.049 0.038 0.073 1 0.45 0.5132

a X1 = sucrose, X2 = beef extract, X3 = KH2PO4, X4 = inoculum size.b Degree of freedom.c *p < 0.05 are significant, R2 = 0.97.

3.4. Optimization using RSM

Four media components at four different levels wereselected for optimization. Table 2 depicted the media com-ponents selected and their concentrations. All experimentswere performed in duplicates. To examine the combinedeffect of four different medium components (independentvariables) on schizophyllan production, a central compos-ite factorial design of 24 = 16 plus 6 centre points and(2 · 4 = 8) star points leading to a total of 30 experimentswere performed. Eq. (3) represents the mathematical modelrelating the production of schizophyllan with the indepen-

Table 2Central composite rotatable design (CCRD) matrix of independent variables

Run Media components (g/l)b

Sucrose Beef extract KH2PO4

1 125 (0) 3.0 (0) 1.00 (0)2 100 (�1) 2.5 (�1) 1.25 (1)3 100 (�1) 2.5 (�1) 0.75 (�1)4 100 (�1) 3.5 (1) 0.75 (�1)5 150 (1) 3.5 (1) 1.00 (0)6 100 (�1) 3.5 (1) 1.00 (0)7 150 (1) 2.5 (�1) 0.75 (�1)8 125 (0) 2.0 (�2) 1.00 (0)9 150 (1) 2.5 (�1) 0.75 (�1)

10 125 (0) 3.0 (0) 0.50 (�2)11 125 (0) 3.0 (0) 1.00 (0)12 100 (�1) 3.5 (1) 0.75 (�1)13 75 (�2) 3.0 (0) 1.00 (0)14 150 (1) 2.5 (�1) 1.25 (1)15 125 (0) 3.0 (0) 1.00 (0)16 125 (0) 3.0 (0) 1.50 (2)17 125 (0) 4.0 (2) 1.00 (0)18 125 (0) 3.0 (0) 1.00 (0)19 125 (0) 3.0 (0) 1.00 (0)20 175 (2) 3.0 (0) 1.00 (0)21 125 (0) 3.0 (0) 1.00 (0)22 150 (1) 3.5 (1) 0.75 (�1)23 150 (1) 3.5 (1) 0.75 (�1)24 100 (�1) 2.5 (�1) 1.25 (1)25 150 (1) 3.5 (1) 1.25 (1)26 125 (0) 3.0 (0) 1.00 (0)27 150 (1) 2.5 (�1) 1.25 (1)28 100 (�1) 3.5 (1) 1.25 (1)29 125 (0) 3.0 (0) 1.00 (0)30 100 (�1) 2.5 (�1) 0.75 (�1)

a Readings are average of two determinations.b Values in parentheses are coded variables.

dent process variables, Xi and the second order polynomialcoefficient for each term of the equation determinedthrough multiple regression analysis using the Design

and the corresponding experimental and predicted yields of schizophyllan

Schizophyllan (g/l)

Inoculum size (ml) Predicted Experimentala

2.0 (�2) 2.55 2.304.0 (�1) 0.75 0.988.0 (1) 1.14 1.008.0 (1) 1.63 1.388.0 (1) 2.15 2.148.0 (1) 2.26 2.004.0 (�1) 6.68 6.926.0 (0) 3.48 3.958.0 (1) 5.54 5.756.0 (0) 1.70 1.836.0 (0) 2.77 2.784.0 (�1) 0.91 1.006.0 (0) 0.84 1.018.0 (1) 3.30 3.20

10.0 (2) 1.93 2.206.0 (0) 0.28 0.176.0 (0) 4.40 3.956.0 (0) 2.77 2.726.0 (0) 2.77 2.586.0 (0) 6.72 6.586.0 (0) 2.77 2.854.0 (�1) 4.51 4.308.0 (1) 3.63 3.408.0 (1) 1.01 1.214.0 (�1) 3.32 3.356.0 (0) 2.77 2.834.0 (�1) 4.63 4.864.0 (�1) 1.74 1.516.0 (0) 2.77 2.854.0 (�1) 0.69 0.68

Table 4Optimized medium composition for schizophyllan production by S.

commune NRCM

No Component concentration (g/l) Schizophyllane

(g/l)Sucrose Beefextract

KH2PO4 Inoculum size(ml/100 ml)

1a 30.0 3.00 1.00 5.0 3.25 ± 0.722b 125.0 3.00 1.00 6.0 6.25 ± 1.413c 170.5 2.04 1.08 8.3 8.03 ± 1.124d 170.5 2.04 1.08 8.3 8.06 ± 0.00

a The values before optimization.b The composition of center points.c The optimized values derived from RSM regression and schizophyllan

yield in this study.d The predicted optimum values and predicted maximum schizophyllan

yield derived from RSM regression in this study.e Results are mean ± SD of three determinations.

Yiel

d, g

/l

1.44 2.48 3.52

4.57 5.61

-1.0-0.5

0.0 0.5

1.0

-1.0

-0.5 0.0

0.5

1.0

B: Beef Extract

Yiel

d, g

/l

1.44

2.48

3.52

4.57

5.61

-1.0

-0.5

0.0

0.5

1.0

D: Inoculum Size

A: Sucrose

Fig. 3. Response surface plots for the yield of scleroglucan; changing componeinoculum size (c).

M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043 1041

Expert. The experimental and predicted values of yields ofschizophyllan are also given in Table 2. The coded valuesof independent variables are given in Table 2.

The results were analyzed using ANOVA, i.e., analysisof variance suitable for the experimental design used (Table3). The ANOVA of the quadratic regression model andmodel F-value indicates the model to be significant. ModelF-value is calculated as ratio of mean square regression andmean square residual. Model P-value (Prob > F) is verylow (0.0001). This again signifies that the model issignificant.

The P-values were used as a tool to check the signifi-cance of each of the coefficients, which, in turn, are neces-sary to understand the pattern of the mutual interactionsbetween the test variables. The t ratio and the correspond-ing P-values, along with the coefficient estimate, are given

Yiel

d, g

/l

1.44 2.48 3.52 4.57 5.61

-1.0-0.5

0.0 0.5

1.0

-1.0

-0.5

0.0

0.5

1.0

-1.0-0.5

0.0

0.5

1.0

C: KH2PO4 A: Sucrose

A: Sucrose

nts were sucrose and beef extract (a), sucrose and K2HPO4 (b), sucrose and

0

2

4

6

8

10

12

24 48 72 96 120144

168192

Time (h)

Schi

zoph

ylla

n (g

/l), D

CW

(g/l)

135

140

145

150

155

160

165

170

175

Res

idua

l Suc

rose

(g/l)

Schizophyllan (g/l) DCW (g/l) Res.sucrose (g/l)

Fig. 4. Production profile of schizophyllan by S. commune NRCM on themedia optimized by response surface method.

1042 M. Kumari et al. / Bioresource Technology 99 (2008) 1036–1043

in Table 3. The smaller the magnitude of the P, the moresignificant is the corresponding coefficient. Values of P lessthan 0.0500 indicate model terms are significant. The coef-ficient estimates and the corresponding P-values suggeststhat, among the test variables used in the study, X1

(sucrose), X2 (beef extract), X3 (KH2PO4), X4 (inoculumsize), X1 · X2 (sucrose · beef extract) and X3 · X4

(KH2PO4 · inoculum size) are significant model terms.Sucrose (P < 0.0001) has the largest effect on schizophyllanproduction, followed by KH2PO4 (P < 0.0001), inoculumsize (P < 0.0198) and beef extract (P < 0.0015). The mutualinteraction between sucrose and beef extract (P < 0.0001),sucrose and KH2PO4 (P < 0.0001), and sucrose and inocu-lum size (P < 0.0001) were also found to be important.Other interactions were found to be insignificant.

The corresponding second-order response model for Eq.(2) that was found after analysis for the regression was:

Yieldðg=lÞ ¼ 2:77þ ð1:47� sucroseÞ � ð0:23� beef extractÞ� ð0:35�KH2PO4Þ � ð0:16� inoculum sizeÞþ ð0:25� sucrose2Þ þ ð0:29� beef extract2Þ� ð0:45�KH2PO2

4Þ � ð0:13� inoculum size2Þ� ð0:60� sucrose� beef extractÞ� ð0:53� sucrose�KH2PO4Þ� ð0:4� sucrose� inoculum sizeÞþ ð0:19� beef extract�KH2PO4Þþ ð0:065� beef extract� inoculum sizeÞ� ð0:049�KH2PO4 � inoculum sizeÞ ð3Þ

The fit of the model was also expressed by the coefficientof determination R2, which was found to be 0.98, indicat-ing that 98.0% of the variability in the response could beexplained by the model.

By substituting the corresponding coded concentrationlevels of the factors into the regression equation, the maxi-mum predictable response for schizophyllan productionwas calculated and was experimentally verified. The maxi-mum production of schizophyllan obtained using the opti-mized medium was 8.03 g/l, which is in close agreementwith the predicted value of 8.06 g/l. However, Rau et al.(1992) described 10 g/l production of schizophyllan in a30 l batch cultivation of S. commune ATCC 38548 equippedwith three fan impellers at 100 rpm, at 27 �C, an initial pHof 5.3 and an aeration rate of 150 l/h. Table 4 documentsthe yields of schizophyllan before and after optimization.

Accordingly, three-dimensional graphs were generatedfor the pair-wise combination of the four factors, whilekeeping the other two at their center point levels. Graphsare given here to highlight the roles played by significantfactors (Fig. 3). From the central point of the contour plotor from the bump of the 3D plot the optimal compositionof medium components was identified. The optimal con-centrations for the four components as obtained from themaximum point of the model were calculated to be as170.5 g/l, 2.04 g/l, 1.08 g/l and 8.3 ml for sucrose, beef

extract, KH2PO4 and inoculum size, respectively. Fig. 4shows the production profile of schizophyllan on the mediaoptimized by RSM. The maximum yield was found at168 h, after which it decreased. Schizophyllan is reportedto be degraded by b-glucanases that are secreted by S. com-

mune when glucose is consumed as the carbon source (Rauet al., 1992).

4. Conclusion

It was possible to determine optimal operating condi-tions using one factor at a time method and RSM to max-imize production of schizophyllan by S. commune NRCMfrom an initial value of 1.06 g/l to 8.06 g/l.

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