Enzyme and Microbial Technology 39 (2006) 743–749

Improvement of exopolysaccharide production by macro-fungusAuricularia auricula in submerged culture

Jing Wu, Zhong-Yang Ding, Ke-Chang Zhang ∗The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology,

Southern Yangtze University, 170 Huihe Road, Wuxi 214036, PR China

Received 8 August 2005; received in revised form 16 December 2005; accepted 20 December 2005

Abstract

The effect of culture conditions and pH on the performance of exopolysaccharide (EPS) production by Auricularia auricula, an edible andmedical macro-fungus, was investigated in submerged culture. First, the optimal cultivation condition was determined in shake flasks by thesingle-factor tests. Second, the effect of pH on EPS production was investigated in batch submerged fermentation, a highest EPS concentration(7.5 ± 0.14 g/l), a highest mycelial growth rate but a lowest residual glucose concentration (3.9 ± 0.04 g/l) were achieved at the pH 5.5, 5.0 and 4.5,respectively. Based on kinetic parameters analysis, a two-stage pH-control strategy was proposed, in which pH was controlled at 5.0 in the first4pwtc©

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8 h and then switched to 5.5. The effectiveness of the strategy was verified successfully by experiments. Higher concentration (8.7 ± 0.08 g/l) androductivity (0.091 g/l/h) of EPS were achieved by applying this strategy. The productivity increased 93.3, 110.4 and 45%, respectively, comparedith that of the non-pH control, and controlling pH at 5.0 and 5.5. Those results demonstrated that EPS production by A. auricula was sensitive

o the pH change in submerged culture. In addition, the idea of changing environmental conditions at different culture stages to suit the metabolicharacteristics of cell, could be applied to the other bioactive metabolites production from macro-fungi in submerged culture.

2006 Elsevier Inc. All rights reserved.

eywords: Auricularia auricula; Exopolysaccharide (EPS); Submerged culture; pH-control strategy

. Introduction

Auricularia auricula, a precious macro-fungus, has beensed as food and drug in China for a long time [1]. Itas recently acquired interests due to its attractive potentialpplication in pharmaceutical industries [2]. It was reportedhat the immunomodulation activities, such as anti-tumour,nti-inflammatory, anti-coagulant, hypocholesterolemic, hypo-lycemic and ameliorating, have been observed in A. auricula1,3–8]. In addition, its ethanolic extracts have been proveno possess the antioxidant and nitric oxide synthase activationroperties [9]. Those medical applications would result in anxpansion of the commercial demand in the near future.

Up to now, polysaccharides are mainly extracted from theruiting body of macro-fungi, which grow on the solid cul-ure. However, when macro-fungi grow on solid culture, the

∗ Corresponding author. Tel.: +86 510 5864675; fax: +86 510 5809237.E-mail addresses: wujing@sytu.edu.cn (J. Wu), zhang3806@yahoo.com

K.-C. Zhang).

time to complete the fruiting body is too long, and the productquality control is difficult. Submerged culture is an alternativeapproach to produce a large amount of macro-fungi polysac-charides. There are several advantages of the submerged cultureover solid culture on polysaccharides (ESP) production: low fer-mentation period, low costs, availability of convenient control,high product concentration and easy downstream processing[10,11]. For macro-fungi submerged culture, it was succeeded inAgaricus campestris in 1949 [12]. Subsequently, a large amountof studies have been focusing on the production of bioactivecompounds with submerged culture [10,11,13]. However, fewstudies have been reported on bioactive polysaccharides produc-tion by A. auricula through submerged culture [14].

For macro-fungus A. auricula batch submerged fermentation,the optimal conditions for mycelial growth and EPS formationmight be quite different. The mycelial growth rate, EPS produc-tion rate, EPS productivity might vary with medium compositionand environmental conditions, including carbon source, nitro-gen source, pH, temperature, etc. [11]. In order to achieve ahigh specific mycelial growth rate, high EPS production rate andhigh EPS productivity, it is necessary to optimize the nutritional

141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2005.12.012

744 J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749

and environmental conditions for mycelial growth and EPS pro-duction in submerged culture [10,13,15]. Previous studies havedemonstrated that pH plays a central role on the mycelial growthand EPS production [16–18]. In addition, kinetic informationabout macro-fungi in submerged culture is very important fordeveloping a better EPS fermentation process using an optimalcontrol strategy [18]. However, few studies have been reportedon the effect of pH on the EPS production by A. auricula insubmerged culture.

In this work, the optimal nutritional and environmental con-ditions for EPS production by A. auricula were determined inshake flasks. Subsequently, a two-stage pH control strategy, aim-ing at achieving a high concentration and high productivity ofEPS by A. auricula, was carried out based on the kinetic analysisof batch processes with pH control at different levels, in a 7-lstirred fermentor. The effectiveness of the control strategy wasverified experimentally.

2. Materials and methods

2.1. Microorganism and media

The strain of A. auricula AA-2 was purchased from Strain Research Centerof Huazhong Agricultural University of China. It was maintained on potato dex-trose agar (PDA) slants. The slant was inoculated and incubated at 25 ◦C for 7days, then stored at 4 ◦C for about a month. The seed medium consisted of (g/l):glucose 40, soybean powder 7, KH2PO4 4, MgSO4·7H2O 2. The basal fermen-tKaoa

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2

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2

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For EPS preparation, the supernatant was firstly filtered through a 0.45 �mmembrane filter (Millipore Co., Bedford, MA, USA), then 95% (v/v) ethanol wasadded under vigorous agitation condition until the absolute ethanol concentra-tion reached 30%, and then left overnight at 4 ◦C. The mixture was centrifugedfor 20 min at 4000 rpm, the resulting sediment was discarded and the super-natant was treated with 95% (v/v) ethanol with the same procedure mentionedabove with the exception that the absolute ethanol concentration reached 75%.Consequently, the crude EPS was precipitated by centrifugation at 3000 rpmfor 20 min. The polysaccharide was determined by phenol–sulfuric acid method[19]. Residual glucose was measured according to the method described byMiller [20].

3. Results and discussion

3.1. Effect of carbon and nitrogen source on mycelialgrowth and EPS production

For selection of the best carbon source for mycelial growthand EPS production, different carbon sources (glucose, sucrose,lactose, maltose, xylose, soluble starch and corn powder), wereadded to the basal medium individually at 20 g/l. When themycelial grew on the glucose medium, both mycelial biomassand EPS concentration reached the highest levels (Fig. 1A). Theoptimum glucose concentration for both mycelial growth andEPS production was determined as 40 g/l (Fig. 1C).

To investigate the effect of nitrogen sources on mycelialgrowth and EPS production, eight kinds of nitrogen sources(tbpFcis

3g

wtofhw

dTdbw(

3

a

ation medium in shake flasks was as follows (g/l): glucose 20, yeast extract 2,H2PO4 4, MgSO4·7H2O 2. To optimize medium composition, different kinds

nd levels of carbon and nitrogen sources were employed to compare with theriginal medium in this study. Soybean and corn powder were purchased fromlocal market and then filtrated with the 60 screen mesh.

.2. Inoculum and shake flask culture

For the seed culture, three pieces of agar culture in pea size from a slantulture were inoculated into a 250 ml flask with 80 ml seed medium, and thenultivated for 6 days on a reciprocal shaker at 150 rpm. The pre-culture was thennoculated either into a 250-ml flask containing 80 ml fermentation medium ornto a 7-l stirred fermentor containing 4 l fermentation medium with an inoculumize of 10% (v/v).

To find the optimum temperature for A. auricula fermentation, the inoculatedasks were incubated at 22, 25, 28, 30 and 34 ◦C, respectively. To determine theptimum initial pH, medium was adjusted to the required pH after sterilizationy addition of sterile 0.5 mol/l HCl or 0.5 mol/l NaOH before inoculation.

.3. Batch fermentation in the 7-l stirred fermentor at different pH

Fermentations were carried out in a fermentor (KF-7 l, Korea Fermentor Co.,nchon, Korea) with 4 l fermentation medium. The agitation speed and aerationate were controlled at 200 rpm and 4 vvm, respectively. pH was controlled atfixed level in the range of 3.0–7.0 with addition of either 1 mol/l NaOH ormol/l HCl at 28 ◦C. For each pH control experiment, about eight samples were

aken and analyzed at least twice. All experiments were done in triple.

.4. Analytical methods

The mycelial biomass of A. auricula was determined by weighting the dryell amount. Samples collected at different time were centrifuged for 20 min at000 rpm, and then the resulting precipitate was washed repeatedly with distilledater and dried at 105 ◦C until a constant weight was achieved to get the dry

ell weight (DCW) measurement. At the same time, the supernatant was usedor EPS preparation and measurement.

peptone, yeast extract, sulfate ammonium, urea, glycin, glu-amic acid, soybean powder and bran) were examined in theasal medium with 40 g/l glucose (Fig. 1B). Maximum EPSroduction was achieved when soybean powder (7 g/l, seeig. 1D) was used. Higher mycelial biomass and EPS con-entration were observed when using organic nitrogen sources,n comparison with the cases of using inorganic nitrogenources.

.2. Effect of initial pH and temperature on mycelialrowth and EPS production

Effect of initial pH on mycelial growth and EPS productionas investigated in the range of 3.0–8.0. Medium was adjusted to

he required pH level after sterilization by addition of sterile HClr NaOH before inoculation. No mycelial growth was observedor the initial pH 3.0 or 8.0 cases. As illustrated in Fig. 2, theighest mycelial biomass and EPS concentration were achievedhen initial pH was 5.4.The effect of temperature on mycelial growth and EPS pro-

uction was also studied at different temperatures (22–34 ◦C).he optimal temperature for both mycelial growth and EPS pro-uction was 28 ◦C. As shown in Fig. 2, the maximum mycelialiomass (7.2 ± 0.13 g/l) and EPS concentration (2.3 ± 0.03 g/l)ere obtained under the condition of optimal temperature

28 ◦C).

.3. Time course of EPS fermentation at natural pH

By combinational consideration of the optimum nutritionalnd environmental conditions in shake flasks, EPS produc-

J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749 745

Fig. 1. Effects of carbon sources (A), nitrogen sources (B), glucose concentration (C) and soybean powder concentration (D) on mycelial growth and EPS productionby A. auricula. EPS (black bar and �) and DCW (blank bar and �).

Fig. 2. Effects of initial pH and temperature on mycelial growth and EPS production by A. auricula. EPS (�) and DCW (�).

tion by A. auricula at natural pH (the initial pH 5.4, pH wasnot controlled in EPS fermentation) was performed in a 7-l stirred fermentor, the results are presented in Fig. 3. Theresults demonstrated that the EPS production was stronglyassociated with mycelial growth. The maximum concentra-tion of EPS (4.5 ± 0.12 g/l) was achieved after 96 h. The con-centration of glucose in culture broth was also detected. Theresults showed that the glucose consumption rate decreased after96 h, and high residual glucose concentration (16.1 ± 0.24 g/l)was detected in culture broth at the end of fermentation. Thedecrease rate of pH in the culture broth was slow at the earlyperiod (0–48 h), and then became fast during 48–96 h (pHdropped to 3.96 from 5.4). At the final phase, pH remainedunchanged at 3.83. Fig. 3 indicated that the favourite pHlevel for EPS production by A. auricula was higher than 4.0.These results also suggested that, it is important to investigatethe effect of pH on the mycelial growth and EPS production

Fig. 3. Time courses of EPS production by A. auricula at natural pH. pH (�),EPS (�), DCW (�) and glucose (�).

746 J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749

at different fermentation phase for enhancement of the EPSproduction.

3.4. Time courses of EPS fermentation at different pH

The effects of pH (ranging from 3.0 to 7.0) on the mycelialgrowth, glucose consumption and EPS production were studied.Time courses of EPS concentration at different pH are shownin Fig. 4. Almost no mycelial growth was observed when pHwas lower than 4.0 or higher than 6.0, as a result, the relevanttime courses of EPS concentration are not depicted in Fig. 4.As shown in Fig. 4, the maximum EPS concentration and themaximum DCW did not achieved at the same pH. For mycelialbiomass, the maximum value (14.5 ± 0.48 g/l) was achieved atpH 5.0 after 144 h (Fig. 4A) with the shortest lag time. As forthe EPS production, the maximum concentration (7.5 ± 0.14 g/l)was obtained at pH 5.5 (Fig. 4C). However, the lowest residualglucose concentration (3.9 ± 0.13 g/l) was found for the caseof pH 4.5 (Fig. 4B). These results indicated that, in order toenhance the EPS concentration and productivity, using thosekinetic parameters such as specific growth rate, etc. to analyzethe fermentation process systematically might be a good choice.

3.5. Kinetics analysis of EPS production at different pH

As illustrated in Fig. 4D–F, the kinetic parameters, such as thespecific cell growth rate (µ), the specific glucose consumptionrate (qs), and the specific EPS production rate (qp), were obtainedfrom the following definitions:

qs = − 1

X

dS

dt= − 1

Xlim

�t→0

�S

�tand µ = 1

X

dX

dt= 1

Xlim

�t→0

�X

�t

qp = 1

X

dP

dt= 1

Xlim

�t→0

�P

�t

The smoothly fitted cell density curve with regards to fermenta-tion time was firstly regressed, the differential of the fitted curve(versus time) was calculated and divided by the fitted cell densityat the same instant to get the value of the specific cell growth rate(µ). The calculations were implemented with Microsoft Excelprogram. Similar method was applied to calculate qs and qp.Furthermore, the time courses of the cell yield on glucose (YX/S)and the EPS yield on glucose (YP/S) at different pH are shownin Fig. 5. They were determined with the following definitions

F

ig. 4. Time courses of EPS production by A. auricula at different pH. pH 4.0 (� and 1), pH 4.5 (� and 2), pH 5.0 (� and 3), pH 5.5 (� and 4) and pH 6.0 (� and 5).

J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749 747

Fig. 5. The change of DCW and EPS yields on glucose at different pH. pH 4.0 (1), pH 4.5 (2), pH 5.0 (3), pH 5.5 (4) and pH 6.0 (5).

using the calculated µ, qs and qp:

YX/S = −dX

dS= µ

qsYP/S = −dP

dS= qp

qs

Figs. 4 and 5 showed the time courses of kinetic parameters ofEPS production at different pH values. The patterns of mycelialgrowth, glucose consumption, and EPS production under dif-ferent pH control levels were significantly different. First, thepH values did not affect the mycelial growth rate at the initialstage (0–24 h). However, the maximum specific cell growth rate(µm) was obtained at pH 5.0 after 24 h. In addition, the highestcell yield on glucose (0.426 g/g) was achieved when control-ling pH at 5.0 (Fig. 5A). Second, high pH was advantageous toincrease glucose consumption rate. The maximum specific glu-cose consumption rate (qs) was obtained at pH 5.0. However,the lowest concentration of residual glucose was found at pH4.5 (Fig. 4B and E). Third, both the EPS concentration and theEPS yield on glucose increased with the increase of pH in cul-ture broth. In addition, a high EPS concentration, a high specificEPS production rate (qp) and a high EPS yield on glucose (YP/S)were observed at pH 5.5 (Figs. 4C and 5B). The concentrationsof EPS at pH 5.5 and 5.0 increased 68.3% (7.5 ± 0.14 g/l) and38.9% (6.2 ± 0.12 g/l), respectively, compared with that of thenatural pH. However, the time when the maximum EPS concen-tration reached at pH 5.5 and 5.0 (144 h) were longer than thatop

itcahaci

3d

mi

could increase EPS production and glucose consumption. Toincrease the concentration and the productivity of EPS further-more, and to decrease the residual glucose concentration in theculture broth as well, it is a good choice to use a two-stage pHcontrol strategy instead of the constant pH control. An optimaltwo-stage pH strategy for EPS production was proposed as fol-lows: pH was controlled at 5.0 during 0–48 h and then switchedto 5.5 after 48 h.

The time course of pH-shift strategy is shown in Fig. 6, and theresults of pH-shift strategy and the different pH experiments aresummarized in Table 1. With the two-stage control strategy, themaximum DCW (12.9 ± 0.14 g/l) was achieved at 96 h. In addi-tion, the average mycelial growth rate was 102, 33.0 and 72.2%higher than that of natural pH and constant pH control (5.0 and5.5) cases. At 96 h, the concentration of EPS was achieved themaximum of 8.7 ± 0.08 g/l. The concentration of EPS increased93.3, 40.3 and 16.0%, respectively, compared with that of naturalpH and constant pH control (5.0 and 5.5) cases. Furthermore, theproductivity of EPS of using the pH-shift control strategy was93.3, 110.4 and 45% higher than that of natural pH and constantpH control (5.0 and 5.5) cases, respectively. On the other hand,with the pH-shift control strategy, the residual glucose concen-tration of the culture broth at the time when the maximum EPSappeared was 10.91 ± 0.22 g/l, which was about 67.8 and 84.8%decrease of that in natural pH (16.1 ± 0.24 g/l) and constant (pH5.5) pH cases (12.9 ± 0.18 g/l). Those results demonstrated that

Fs

f the natural pH (96 h), which resulted in the decrease in theroductivities of EPS.

It is concluded that, at early phase of EPS production, its appropriate to control pH at a lower level (e.g. pH 5.0)o maximize the mycelial growth rate and accelerate the glu-ose consumption, while at mid- and later-fermentation phase,higher pH (e.g. pH 5.5) was favourable for achievement of

igher EPS yield on glucose and EPS production rate. The resultslso suggested that there is some relationship between glucoseoncentration and EPS production [21], which will be studiedn other studies.

.6. A two-stage pH strategy and the experimentalemonstration

The above results demonstrated that a lower pH at early fer-entation stage was advantageous to cell growth, while a proper

ncrease in pH of the submerged culture at mid-and later-stage

ig. 6. Time courses of EPS production by A. auricula under the two-stage pHtrategy. EPS (�), DCW (�) and glucose (�).

748 J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749

Table 1Comparison of parameters of EPS fermentation at different pH

Parameters Natural pH pH 4.0 pH 4.5 pH 5.0 pH 5.5 pH 6.0 Two-stagepH strategy

DCW (g/l) 11.1 ± 0.17 6.0 ± 0.09 12.2 ± 0.24 14.5 ± 0.48 11.2 ± 0.21 7.0 ± 0.11 12.9 ± 0.14The time of maximumEPS achieved (h)

96 168 144 144 144 168 96

The concentration ofmaximum EPS (g/l)

4.5 ± 0.12 1.2 ± 0.05 4.6 ± 0.15 6.2 ± 0.12 7.5 ± 0.14 2.8 ± 0.21 8.7 ± 0.08

The concentration ofresidual glucose (g/l)

16.1 ± 0.24 20.9 ± 0.18 3.9 ± 0.13 6.6 ± 0.08 12.9 ± 0.18 22.2 ± 0.16 10.9 ± 0.22

YP/S (g/g) 0.20 0.07 0.16 0.20 0.30 0.18 0.32YX/S (g/g) 0.50 0.35 0.36 0.46 0.45 0.44 0.36The EPS productivity(g/l/h)

0.046 0.0071 0.032 0.043 0.063 0.017 0.091

the proposed two-stage pH strategy could efficiently improvethe mycelial growth, EPS production, as well as glucose con-sumption in A. auricula fermentation.

4. Conclusions

The optimal medium composition for EPS production by A.auricula determined by the single-factor tests was as follows(g/l): glucose 40, soybean powder 7, KH2PO4 4, MgSO4·7H2O2. Time course of fermentation in a 7-l stirred fermentor withthe natural pH revealed that pH plays a critical role in affectingthe mycelial growth, glucose consumption and EPS production.A higher mycelial growth rate could be obtained at a lower pHlevel (pH 5.0), but higher EPS production rate, EPS concentra-tion and EPS productivity were achieved at a higher pH level (pH5.5). This study has verified that the two-stage pH strategy (pHswitching from 5.0 to 5.5) was superior to the constant pH andthe natural pH controls, in the term of EPS production enhance-ment. Furthermore, the proposed two-stage pH control strategycould supply an alternative optimization way for the bioactivecompounds production by other macro-fungi in submerged cul-ture.

Acknowledgement

t

R

[4] Ukai S, Kiho T, Hara C, Kuruma I, Tanaka Y. Polysaccharides in fungiXIV. Anti-inflammatory effect of the polysaccharides from the fruit bod-ies of several fungi. J Pharmacobiodyn 1983;6:983–90.

[5] Yuan Z, He P, Cui J, Takeuchi H. Hypoglycemic effect of water-solublepolysaccharide from Auricularia auricula-judae Quel.on geneticallydiabetic KK-Ay mice. Biosci Biotechnol Biochem 1998;62:1898–903.

[6] Yuan Z, He P, Takeuchi H. Ameliorating effects of water-solublepolysaccharides from woody ear (Auricularia auricula-judae Quel.)in genetically diabetic KK-Ay mice. J Nutr Sci Vitaminol (Tokyo)1998;44:829–40.

[7] Yoon SJ, Yu MA, Pyun YR, Hwang JK, Chu DC, Juneja LR, et al.The nontoxic mushroom Auricularia auricula contains a polysaccha-ride with anticoagulant activity mediated by antithrombin. Thromb Res2003;112:151–8.

[8] Takeujchi H, He P, Mooi L. Reductive effect of hot-water extracts fromwoody ear (Auricularia auricula-judae Quel.) on food intake and bloodglucose concentration in genetically diabetic KK-Ay mice. J Nutr SciVitaminol (Tokyo) 2004;50:300–4.

[9] Acharya K, Samui K, Rai M, Dutta B, Acharya R. Antioxidant and nitricoxide synthase activation properties of Auricularia auricula. Indian JExp Biol 2004;42:538–40.

[10] Lee BC, Bae JT, Pyo HB, Choe TB, Kim SW, Hwang HJ. Sub-merged fermentation conditions for the production of mycelial biomassand exopolysaccharides by the edible Basidiomycete Grifola frondosa.Enzyme Microb Technol 2004;35:369–76.

[11] Papagianni M. Fungal morphology and metabolite productionin submerged mycelial processes. Biotechnol Adv 2004;22:189–259.

[12] Humfeld H, Sugihara TF. Mushroom mycelium production by sub-merged propagation. Food Technol 1949;3:355–6.

[

[

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The authors acknowledge Prof. Z.P. Shi for critically readinghe manuscript.

eferences

[1] Cheung P. The hypocholesterolemic effect of two edible mushrooms:Auricularia auricula (tree-ear) and Tremella Fuciformis(white jelly-leaf)in hypercholesterolemic rats. Nutr Res 1996;16:1721–5.

[2] Wasser SP. Medicinal mushrooms as a source of antitumor andimmunomodulating polysaccharides. Appl Microbiol Biotechnol 2002;60:258–74.

[3] Misaki A, Kakuta M, Sasaki T, Tanaka M, Miyaji H. Studies oninterrelation of structure and antitumor effects of polysaccharides: anti-tumor action of periodate-modified, branched (1 goes to 3)-beta-d-glucan of Auricularia auricula-judae, and other polysaccharides con-taining (1 goes to 3)-glycosidic linkages. Carbohydr Res 1981;92:115–29.

13] Kim SW, Hwang HJ, Xu CP, Sung JM, Choi JW, Yun JW. Optimiza-tion of submerged fermentation process for the production of mycelialbiomass and exo-polysaccharides by Cordyceps militaris C738. J ApplMicrobiol 2003;94:120–6.

14] Jeong SC, Cho SP, Yang BK, Gu YA, Jang JH, Huh TL, et al. Pro-duction of an anti-complement exo-polymer produced by Auriculariaauricula-judae in submerged fermentation. Biotechnol Lett 2004;26:923–7.

15] Lee H, Song M, Yu Y, Hwang S. Production of Ganoderma lucidummycelium using cheese whey as an alternative substrate: response surfaceanalysis and biokinetics. Biochem Eng J 2003;15:93–9.

16] Fang QH, Zhong JJ. Effect of initial pH on production of ganoderic acidand polysaccharide by submerged fermentation of Ganoderma lucidum.Process Biochem 2002;37:769–74.

17] Elmahdi I, Baganz F, Dixon K, Harrop T, Sugden D, Lye GJ. pH con-trol in microwell fermentations of S. erythraea CA340: influence onbiomass growth kinetics and erythromycin biosynthesis. Biochem EngJ 2003;16:299–310.

J. Wu et al. / Enzyme and Microbial Technology 39 (2006) 743–749 749

[18] Lee KM, Lee SY, Lee HY. Bistage control of pH for improvingexopolysaccharide production from mycelia of Ganoderma Zucidum inan air-lift fermentor. J Biosci Bioeng 1999;88:646–50.

[19] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetricmethod for determination of sugars and related substances. Anal Chem1956;28:350–6.

[20] Miller GL. Use of di-nitrosalicylic acid reagent for determination ofreducing sugars. Anal Chem 1959;31:426–8.

[21] Baruque-Ramos J, Hiss H, Arauz LJ, Mota RL, Ricci-Silva ME, Paz MF,et al. Polysaccharide production of Neisseria meningitidis (SerogroupC) in batch and fed-batch cultivations. Biochem Engine J 2005;23:231–40.