links between morphology and physiology of ganoderma lucidum

Journal of Biotechnology 114 (2004) 153–164

Links between morphology and physiology ofGanoderma lucidumin submerged culture for the production of exopolysaccharide

Ricardo Wagnera, David Alexander Mitchella,∗, Guilherme Lanzi Sassakia,Maria Angela Lopes de Almeida Amazonasb

a Departamento de Bioqu´ımica e Biologia Molecular, Universidade Federal do Paraná, Caixa Postal 19046,Centro Politecnico, Curitiba 81530-990, Paraná, Brazil

b Centro Nacional de Pesquisa de Florestas, Embrapa Florestas, Cx.P. 319, Colombo 83411-000, Paraná, Brazil

Received 2 October 2003; received in revised form 26 April 2004; accepted 29 June 2004

Abstract

Ganoderma lucidumwas grown in submerged culture in shake flasks on a medium containing peptone, yeast extract andglucose. In pre-cultures, inoculated from an agar-grown culture, morphological and metabolic events were linked: the pelletsoriginally produced protuberances when glucose was present in the medium, although glucose was not consumed. The protu-berances were then liberated into the medium as second-generation pellets, at which time glucose consumption began and therate of exopolysaccharide (EPS) production increased. The synchrony between events was repeated in cultures fed with eitherglucose or peptone and yeast extract. In main cultures, inoculated from a 16-day-old pre-culture, the biomass concentrationi nces werep was addedt EPSc gicala tion.©

K

1

i

f

han

ricper-rte-ncer

unode

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ncreased linearly, while glucose consumption and EPS production were initially slow but then accelerated. Protuberaroduced and liberated similarly to the pre-culture, but there was less synchrony amongst the pellets. When glucose

o such a culture on day 10, an EPS concentration of 5.7 g L−1 was achieved on day 13, this being the highest reliableoncentration yet reported for submerged culture ofG. lucidum. We conclude that a greater understanding of the morpholond physiological events during the culture ofG. lucidumwill allow the proposal of culture strategies to improve EPS produc2004 Elsevier B.V. All rights reserved.

eywords: Ganoderma lucidum; Submerged culture; Exopolysaccharide

. Introduction

Ganoderma lucidum(Leyss.: Fr.) Karst, a basid-omycete belonging to the polyporaceae, has been used

∗ Corresponding author. Tel.: +55-41-361-1658;ax: +55-41-266-2042.

E-mail address:[email protected] (D.A. Mitchell).

as a traditional medicine in the orient for more t2000 years (Stamets, 1993). The basidiocarp ofG.lucidum is still used to treat conditions like gastulcer, chronic hepatitis, nephritis, hypertension, hylipemia, arthritis, insomnia, bronchitis, asthma, ariosclerosis, leukopenia, diabetes, anorexia and ca(Jong and Birmingham, 1992; Stamets, 1993; Mizet al., 1995). G. lucidum produces polysacchari

168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.jbiotec.2004.06.013

154 R. Wagner et al. / Journal of Biotechnology 114 (2004) 153–164

fractions containing�-d-(1 → 3) glucans, sometimesbranched at the C-6 position, which demonstrate highanti-tumor activity (Sone et al., 1985) and may be re-sponsible for some of the other biological activities at-tributed toG. lucidum. Although�-d-(1 → 3) glucansoccur both in the basidiocarp and cultured mycelium(Jong and Birmingham, 1992; Mizuno, 1999), mostcommercial products containing them are derived fromthe basidiocarp.

There has been recent interest in developing pro-cesses for the submerged culture ofG. lucidum, for thecommercial production of exopolysaccharide (EPS)fractions containing�-d-(1 → 3) glucans, promptedby the fact that wildG. lucidumis rarely found in na-ture and traditional mushroom cultivation takes months(Mizuno et al., 1995; Mayzumi et al., 1997). However,little is understood about howG. lucidumgrows in sub-merged culture, especially with regard to how morphol-ogy and physiology are linked (Wagner et al., 2003).The current work investigated these links, focussing onthe pre-culture step, which has received little attentionbut which can significantly affect the performance ofthe main culture. The insights gained from the stud-ies of the pre-culture step were applied to the mainculture, and enabled us to achieve the highest reliableEPS yields yet reported for the submerged culture ofG. lucidum.

2. Materials and methods

2i

aFb omB ofH asm in-cT erep d ont t-t1 oft thes ob-

tained was filtered through a stainless steel mesh (poresize 0.7 mm).

2.2. Medium and standard culture method

Cultures were done in replicate flasks, with threeflasks removed at each sampling time. To each500 mL flask were added 85 mL distilled water, 0.5 gyeast extract (Biobras), 0.5 g peptone (Biobras), 0.1 gNaH2PO4, 0.05 g MgSO4·7H2O and 0.005 g thiaminchlorohydrate (Vitamin B1). A 3.5 g of glucose wasdissolved in 10 mL water. After sterilization of both at121◦C for 15 min, the glucose was added aseptically tothe flask. The pre-culture was inoculated with 5 mL offiltered mycelial suspension. The main culture was in-oculated with 5 mL of a suspension obtained by adding50 mL distilled water to 100 mL of a 16-day-old pre-culture. All cultures were incubated in the dark at 29◦Cat 128 rpm on a rotary shaker.

2.3. Feeding of cultures

For pre-cultures fed with peptone and yeast extract,aliquots of 10 mL of aqueous solution were prepared,each aliquot containing 5.0 g of each of peptone andyeast extract. Each of several flasks received one ofthese 10 mL aliquots on day 13. For glucose feeding ofthe pre-culture, several flasks were initially preparedwith 10 mL water in the place of the glucose solution.After 17 days, aliquots of 10 mL of aqueous solutionw glu-c 0 mLa ul-t 0 mLa ning3

2d

t yt ),( alw thes lterpo ass.

.1. Culture maintenance and preparation ofnoculum for the pre-culture

G. lucidum (kindly provided to the Embraporests Culture Collection, Colombo, Parana, Brazily the Centre for International Services to Mushroiotechnology (CISMB) of the Chinese Universityong Kong, where it is stored as CMB 0246) waintained on potato dextrose agar (PDA). It was

ubated at 29◦C for 12 days and then stored at 4◦C.en 0.5 mm diameter disks of mycelium and agar wunched out from a maintenance culture, sprea

he surface of 80 mL of PDA within a 1 L Roux bole and incubated for 8 days at 29◦C in the dark. A00 mL of distilled water was then added to each

wo Roux bottles and the mycelium scraped fromurface with a scalpel. The mycelium suspension

ere prepared, each aliquot containing 38.5 g ofose. Each of several flasks received one of these 1liquots. Likewise, for glucose feeding of the main c

ure, after 10 days some of the flasks received a 1liquot of an aqueous solution, each aliquot contai8.5 g of glucose.

.4. Sampling, biomass fractionation and glucoseetermination

The biomass was separated into fractionsL (re-ained by a sieve with 2.0 mm pores),M (passed bhe 2.0 mm sieve but retained by a 0.8 mm sieveSpassed by both sieves) andA (adhered to the internall of the flask). Each fraction was washed fromieve with water, filtered through Whatman #1 fiaper, and dried to constant weight at 70◦C. The sumf all the fractions was denoted as the total biom

R. Wagner et al. / Journal of Biotechnology 114 (2004) 153–164 155

An aliquot of 0.5 mL of broth, removed prior to thebiomass fractionation, was centrifuged at 2200 rpm ina bench centrifuge and the supernatant stored at−18◦Cfor later determination of the glucose concentration bythe glucose oxidase method (DiaSys Diagnostic Sys-tems GMBH&Co.KG).

2.5. Recovery and quantification ofexopolysaccharide

The culture broth and the water used to wash thebiomass off the sieves were filtered through What-man #1 filter paper and evaporated to 50 mL underreduced pressure at 60–80◦C. This reduced volumewas added to 150 mL of ethanol, in order to pre-cipitate macromolecules, including polysaccharides.A glass rod was twirled within the solution. Theprecipitate, containing polysaccharide, was adsorbedonto the rod and removed from the solution. Glucosedoes not precipitate in a mixture of three volumes ofethanol per volume of water, but some soluble glucosemight be trapped within the polysaccharide precipitate,which was therefore washed with 96% ethanol. It wasthen re-suspended in distilled water, freeze-dried andweighed. Its carbohydrate content was determined bythe phenol–sulfuric method (Dubois et al., 1956) us-ing glucose as the standard, this carbohydrate beingreported as exopolysaccharide (EPS).

2.6. Characterization of the exopolysaccharide

iflu-o lso ysisw n( byG ol-u st o-g seo -t asiso ates byt on oft amo xide( R

in a Bruker Avance DRX 400 MHz spectrometer. Thechemical shifts were measured in ppm using tetram-ethylsilane (TMS) as standard.

2.7. Photography

Pellets in culture medium, or suspended in distilledwater, were photographed under either an optical mi-croscope (Zeiss Axiophot) or a magnifying glass (ZeissStemi 2000-C).

2.8. Statistical treatment

All experiments were repeated at least once. Withineach experiment triplicate flasks were used to generateeach data point. In the graphs, values are plotted asmean± S.E. If the error bars do not appear then theyare smaller than the size of the symbol. Student’st-test was used to determine whether differences werestatistically significant (P values are quoted).

3. Results

3.1. Characterization of the standard pre-culture

In a standard pre-culture ofG. lucidum, the glucoseconcentration was constant until day 13, at which timethe total biomass concentration had reached 6.4 g L−1

(Fig. 1a). On day 19, the biomass concentration was1 othh

atc lp pel-l pro-t y9 nces( andw day1 gantw up.T to ass ofilea . Onda

Several EPS samples were treated with 1 M trroacetic acid for 8 h at 100◦C. The acetate alditof the monosaccharides liberated by the hydrolere prepared as described byWolfrom and Thompso

1963), and the derivatized sugars were analyzedC-MS (Saturn 2000R-Varian), using a capillary cmn (DB-225 30 m× 0.25 mm i.d.) with helium a

he mobile phase (1.5 mL min−1). The temperature prram was 1 min at 50◦C, then a temperature increaf 40◦C min−1 until 220◦C, which was then main

ained until 25 min. Sugars were identified on the bf their retention times in relation to alditol acettandards (Sigma), with the identities confirmedhe mass spectrometer, and quantified by integratihe area under the GC elution curve. Thirty milligrf EPS was dissolved in deuterated dimethylsulfoDMSO) at 70◦C, and analyzed by carbon-13 NM

2.4 g L−1 and the glucose concentration in the brad fallen to 5.7 g L−1.G. lucidum grew with a pellet morphology th

hanged over time (Fig. 2). On day 7, the originaellets, hereafter referred to as first-generation

ets, were regular spheres with some long hyphaeruding from their surface (Fig. 2a and b). By da

the long hyphae had given rise to protuberaFig. 2c and d), which then increased in lengthidth, giving the pellet a starburst appearance by3 (Fig. 2e). On day 13 these protuberances be

o detach from the first-generation pellets (Fig. 2f),hich on day 17 had almost completely brokenhese detached protuberances, hereafter referredecond-generation pellets, were feather-like in prt the time of release, although they were not flatay 20 they were larger and ovoid in shape (Fig. 2gnd h).


Fig. 1. Culture profile for a standard pre-culture ofGanoderma lu-cidum: (a) total biomass (�) and glucose (�); (b) biomass fractionsL (�), M (�), S (�) andA (�); (c) EPS. The vertical dotted linerepresents day 13.

The pellet fragmentation affected the size distribu-tion of the biomass (Fig. 1b). FractionL increased untilpellet break-up began on day 13 and then decreased toalmost zero by day 17. FractionsM andSwere verylow until day 13, but then increased until day 19, dueto the liberation of the second-generation pellets, withthe increase in fractionM being more rapid. FractionA increased linearly from days 7 to 19. On day 9 thisfraction represented 65% of the total biomass, althoughby day 19 its proportion had fallen to 32%.

The EPS production rate was low during the first13 days, but then suddenly accelerated (Fig. 1c). Sincepellet break-up, the onset of glucose consumption andthe start of rapid EPS production all occurred aroundday 13, there appears to be a causal link between theseevents.

Fig. 2. Morphological development during growth ofGanodermalucidumin the standard pre-culture: (a) day 7, scale bar = 0.25 mm;(b) day 7, scale bar = 0.1 mm; (c) day 9, scale bar = 1.5 mm; (d) day9, scale bar = 1.0 mm; (e) day 13, scale bar = 1.5 mm; (f) day 13,scale bar = 0.8 mm; (g) day 20, scale bar = 1.5 mm, (h) day 20, scalebar = 0.3 mm.

3.2. Effects of peptone and yeast extract additionto the pre-culture

The previous experiment led us to hypothesize thata component of the yeast extract or peptone was pre-venting the uptake of glucose byG. lucidum. We there-fore added more peptone and yeast extract on day 13,just before the fungus had started utilizing glucosein the previous experiment (Fig. 1a). Our hypothesispredicted that glucose would not be consumed beforethe added nutrients were utilized. If there were a linkbetween the start of glucose consumption and pelletbreak-up, then pellet break-up would also be delayed.

Four days after feeding, the biomass concentra-tion in the fed culture was slightly higher than in a


standard pre-culture done as a control, but the max-imum biomass concentration, obtained on day 21,was similar, being 12.8 g L−1 for the fed culture and12.7 g L−1 for the standard pre-culture (Fig. 3a). Ofcourse, given that the fed culture contained an addi-tional 10% of volume, the total amount of biomass was10% larger in the fed culture.

Glucose consumption was not as predicted. In fact,the glucose concentration decreased slightly morerapidly in fed cultures than in the standard pre-cultureover days 13–17 (Fig. 3a). Furthermore, the feeding ac-celerated pellet break-up (Fig. 4). In the standard pre-culture, fractionL increased between the feeding at day13 and the removal of the next sample at day 17, rep-resenting enlargement of the first-generation pellets.

F ct ontasn

Fig. 4. Appearance, on day 17, of cultures ofG. lucidum: (a) a stan-dard pre-culture; (b) a culture fed with peptone and yeast extract onday 13. In both cases the scale bar represents 1.5 mm.

Note that, due to a slightly longer lag phase and thefact that a sample was not removed on day 15, the peakconcentration of fractionL in Fig. 3b is lower than the3.5 g L−1 obtained for this fraction on day 13 inFig. 1b.For the fed culture, fractionL decreased between days13 and 17, indicating that pellet break-up had begun.Due to their premature release, the second-generationpellets liberated in the fed culture were smaller thanthose liberated in the standard pre-culture.

Likewise, the feeding accelerated EPS production.On day 17, the concentration of EPS was significantlyhigher in the fed culture than in the standard pre-culture(P < 0.1), this probably being related to the earlierbreak-up of the first-generation pellets in the fed culture(Fig. 3c). The EPS concentration reached a maximum
ig. 3. Effect of feeding, on day 13, of peptone and yeast extra
he culture profile ofGanoderma lucidum: (a) total biomass (squares)nd glucose (triangles); (b) biomass fractionL: (c) EPS. In each grapholid symbols represent a standard pre-culture undertaken simulta-eously and hollow symbols represent the fed culture.

on day 21 for both the fed and the standard pre-cultures.The absolute amounts of EPS were not significantlydifferent: the 100 mL of the standard pre-culture, with


a concentration of 705 mg L−1, had 70.5 mg of EPS,while the 110 mL of the fed culture, with a concentra-tion of 686 mg L−1, had 75.5 mg of EPS. After day 21the EPS concentration fell rapidly in both cultures.

3.3. Effects of glucose on morphology andphysiology

We investigated the effects of glucose, based on theobservations that a significant amount of biomass wasobtained before the onset of glucose consumption andthat the onset of glucose consumption coincided withthe onset of pellet break-up.Fig. 5a shows the profileof growth and glucose concentration for three cultures:

F pre-c sG(G(

culture G35-0 was a standard pre-culture, with 35 g L−1

of glucose added at the beginning; culture G0-0 re-mained without glucose during the whole cultivation;culture G0-35 was initiated without glucose but glu-cose was added at day 17, giving a concentration of38.8 g L−1.

As observed previously, at day 13 the biomass con-centration in culture G35-0 had reached 7.4 g L−1,while the glucose concentration was still 35.0 g L−1,showing thatG. lucidumreproducibly produces a sig-nificant amount of biomass within the standard culturemedium before glucose consumption begins. In con-trast, the biomass concentration in culture G0-0 wasonly 1.3 g L−1 on day 13, even though it contained asmuch yeast extract and peptone as did culture G35-0.The maximum biomass concentration reached in cul-ture G0-0 was 2.7 g L−1 on day 17. In culture G0-35,the biomass concentration increased to 8.0 g L−1 at day21 after glucose was fed on day 17. However, despitethis increase of 5.3 g L−1 in biomass concentration, theglucose concentration fell by only 1.5 g L−1 during thisperiod.

The morphology was also affected by the addition ofglucose. Pellet break-up in culture G35-0 occurred be-tween days 13 and 17, as shown by the large decreasein fraction L (Fig. 5b). In culture G0-35, fractionLincreased only slowly in the absence of glucose, butincreased rapidly after glucose was added on day 17,peaking at day 21. This was followed by a drastic de-crease on subsequent days due to the liberation of thes ob-st noto el-a ucht f thep

0-0w 35-0a iumi entp tf gh-o
ig. 5. Effect of feeding of glucose on the culture profile of the
ulture stage ofGanoderma lucidum: (a) total biomass for culture
35-0 (�), G0-35 (�) and G0-0 (♦) and glucose for cultures G35-0�), G0-35 (�) and G0-0 (�); (b) biomass fractionL for cultures35-0 (�), G0-35 (�) and G0-0 (♦); (c) EPS for cultures G35-0�), G0-35 (�) and G0-0 (♦).
35-0 ex-p db in

econd-generation pellets. A different profile waserved in culture G0-0, in which fractionL remainedhe dominant fraction because pellet break-up didccur (Fig. 6). The pellets in culture G0-0 produced rtively few protuberances, which were very thin, s

hat the pellets did not have the starburst shape oellets in culture G35-0.

The biomass adhered to the flask in culture Gas softer than the adhered biomass of culture Gnd by day 17 this biomass had fallen into the med

n cultures G0-0 and G0-35, although for measuremurposes, it was still included in fractionA. Note tha

ractionA remained adhered in culture G35-0 throuut the entire culture period.

The maximum EPS concentration in culture Gwas similar to that obtained in the previous

eriments, remaining below 0.8 g L−1. EPS remaineelow 0.05 g L−1 in culture G0-0 throughout and


Fig. 6. Effect of glucose on the development of pellets in the pre-culture ofGanoderma lucidum. In all cases the scale bar represents 1.5 mm.(a–d) Culture G35-0; (e–h) culture G0-0; (i and j) culture G0-35. First column = day 9, second column = day 17, third column = day 21 andfourth column = day 25.

culture G0-35 prior to the addition of glucose. The ad-dition of glucose on day 17 to culture G0-35 stimulatedEPS production, which increased sharply after day 21,the day on which pellet break-up occurred, reaching1.3 g L−1 on day 25. This is 1.6 times that produced byculture G35-0, showing that biomass produced initiallyin the absence of glucose has a higher capacity for EPSproduction, once glucose is added, than that cultivatedin the presence of glucose from the beginning.

3.4. Characterization of the standard main culture

A cultivation was done, hereafter referred to as thestandard main culture, using a 16-day-old standardshake-flask pre-culture (i.e. type G35-0) as inoculum.The biomass concentration increased linearly, from thetime of inoculation to the tenth day, with a linear growthrate of 1.01 g L−1 day−1 (R2 = 0.996) (Fig. 7a). Thebiomass concentration on day 10, 10.3 g L−1, was themaximum obtained, the biomass concentration havingfallen slightly by day 13.

Glucose was consumed at a rate of only1.45 g L−1 day−1 over the first 4 days (Fig. 7a). It isunlikely thatG. lucidumconsumed only glucose dur-

ing this period, based on the production of 4.5 g L−1 ofbiomass for the consumption of 5.8 g L−1 of glucose. Inother words, a yield coefficient of 0.78 g-biomass g−1-glucose is unlikely, unless yeast extract and peptonewere also being consumed. Glucose consumption wasmore rapid between days 4 and 10, falling by 24.7 g L−1

over this period, representing a rate of 4.1 g L−1 day−1.The EPS profile behaved similarly (Fig. 7c). Some

EPS was brought into the culture with the inoculum.By day 4 it had increased, but only at an average rateof 0.06 g L−1 day−1. The EPS concentration then in-creased sharply, to its maximum value of 1.92 g L−1

at day 7, representing an average production rate of0.51 g L−1 day−1 between days 4 and 7.

The changes in pellet morphology and the distri-bution of the biomass between the different fractionsduring the main culture step had similarities to the pre-culture, but also some differences. At the time of in-oculation, the second-generation pellets were ovoid,but there were still a few first-generation pellets withprotuberances (Fig. 8a and b), which were then re-leased. The second-generation pellets also producedprotuberances (Fig. 8c and d). By day 7 some of thesehad been liberated into the medium as third-generation


Fig. 7. Culture profile for a standard main culture ofGanodermalucidum: (a) total biomass (�) and glucose (�); (b) biomass fractionsL (�), M (�), S(�) andA (�); (c) EPS.

pellets, however, many third-generation pellets be-gan to round-out while still attached to their second-generation mother pellet (Fig. 8e and f), only finallybeing liberated around day 10 (Fig. 8g and h), givinga predominance of third-generation pellets by day 13(Fig. 8i and j). In contrast, pellet break-up in the pre-culture occurred while the protuberances still had thefeather-like profile. Note also that in the main culturethe pellets did not reach the large pellet sizes of around10 mm that the first-generation pellets had attained inthe pre-culture.

Fig. 7b shows the distribution of the biomass frac-tions. At the time of inoculation, the biomass was dis-tributed between fractionsS andM. The increase inpellet size over the first 4 days caused fractionM to in-crease, with some of the pellets becoming large enoughto pass to fractionL, such that fractionL also increased.

Fig. 8. Morphological development during growth ofGanodermalucidumin the standard main culture: (a) day 0, scale bar = 1.25 mm;(b) day 0, scale bar = 0.8 mm; (c) day 4, scale bar = 1.25 mm; (d)day 4, scale bar = 0.6 mm; (e) day 7, scale bar = 1.5 mm; (f) day7, scale bar = 0.5 mm; (g) day 10, scale bar = 1.5 mm; (h) day 10,scale bar = 0.5 mm; (i) day 13, scale bar = 1.5 mm; (j) day 13, scalebar = 0.6 mm. In each case the photograph on the right is a highermagnification of a region of the photograph on the left.

Over days 4–7 fractionsM andL increased slightly, butthe main event was an increase in fractionS, associatedwith the release of third-generation pellets by some ofthe second-generation pellets. From days 7 to 10 a sig-nificant part of the large second-generation pellets withprotuberances broke up, causing fractionL to decrease.This caused fractionSto increase slightly, but a signifi-cant proportion of the liberated third-generation pellets


Fig. 9. Effect of feeding of glucose on the culture profile of a mainculture ofGanoderma lucidum. (a) Total biomass (�) and glucose(�) for a standard main culture and total biomass (�) and glucose(�) for a main culture started with glucose and fed with more glucoseon day 10. (b) EPS for a standard main culture (�) and for a mainculture started with glucose and fed with more glucose on day 10(�).

were already large enough to be classified in fractionM. Also, many of the fractionSpellets that had beenpresent on day 7 had grown sufficiently to enter intofractionM.

3.5. Effect of glucose feeding during the mainculture

In an attempt to increase biomass and EPS pro-duction in the main culture, a culture was undertakenthat was begun in the same manner as the standardmain culture but extra glucose was fed on day 10,increasing its concentration by 35 g L−1. A standard

Table 1Monosaccharide composition, in molar percentage, of EPS samples

Sample origin Glc Man a

Culture G35-0, day 17 (Fig. 5) 74.4 13.3Culture G0-35, day 25 (Fig. 5) 100.0 trStandard main culture, day 7 (Fig. 9) 82.5 8.2Fed culture, day 13 (Fig. 9) 76.0 9.9

Glc: glucose; Man: mannose; Gal: galactose; Ara: arabinose; Xyl: xyl

main culture was done as a control, in which, by day10, the biomass concentration had reached 11.7 g L−1

and the glucose concentration had fallen to 5.0 g L−1

(Fig. 9a). The biomass continued to increase in the cul-ture fed with glucose, although more slowly, reaching16.0 g L−1 on day 19. The glucose concentration beganto fall immediately upon feeding, maintaining the samerate of consumption that it had had before the feeding,and falling to 0.4 g L−1 by day 19. In comparison, thebiomass remained constant in the standard main culturebetween days 10 and 13 and then started to decrease(Fig. 9a).

In the fed culture, the EPS concentration was fallingwhen glucose was added on day 10. Feeding stimulatedthe production of more EPS, which was maintained forthree more days, at the same rate of production as hadbeen achieved between days 4 and 7 (Fig. 9b). Themaximum EPS concentration, of 5.7 g L−1, was ob-tained on day 13, after which the concentration beganto fall. The maximum EPS concentration in the fed cul-ture was significantly greater than that obtained in thestandard main culture (P< 0.01), which was 3.3 g L−1,obtained on day 7.

3.6. Characterization of the exopolysaccharide

When glucose was added on day 17 to a pre-cultureinitiated without glucose, the EPS contained only glu-cose (Table 1). This sample had an NMR spectrum typ-ical of a glucan. The presence of the�-configurationo -1s d1p i-c stst v-i -c ser s

removed from various cultures

Gal Ara Xyl Fuc Rh

7.6 3.6 1.1 tr trtr tr tr tr tr2.8 6.5 tr tr tr5.2 6.8 tr 1.3 tr

ose; Fuc: fucose; Rha: Rhamnose; tr: trace.

f d-glucopyranose was consistent with low field Cignals with chemical shifts (δ) of 102.7, 102.6 an02.5, and a high field H-1 signal atδ 4.55. Theresence of signals atδ 86.2, 86.0 and 85.7 indatedO-3 substitution in the glucan, which suggehat it was a�-d-(1 → 3) glucan. There was no edence of other types ofO-substitution of the gluopyranose in the13C NMR spectrum. Together, theesults suggest that a pure�-d-(1 → 3) glucan wa


produced by this culture. In all the other EPS sam-ples analyzed, the predominant monosaccharide wasglucose, but there were also mannose, galactose andarabinose, with traces of xylose, fucose and rham-nose.

4. Discussion

Submerged culture ofG. lucidum is poorly un-derstood (Wagner et al., 2003). The present workshows not only that the physiological and morpho-logical events are linked but also that the presenceof glucose affects growth, even when it is not be-ing consumed. Beyond this, the highest reliable EPSconcentration yet achieved was obtained in the mainculture.

4.1. Links between physiological andmorphological events in the pre-culture

The physiological and morphological changes thatoccur during the growth of pre-cultures ofG. lucidumhave not previously been reported: neither the pres-ence of protuberances on the pellets nor the mannerin which pellet break-up occurs (Wagner et al., 2003).This lack is due to the fact that previous workers haveconcentrated on the main culture step, and not the pre-culture, where the synchrony between events is mostevident.

entsd osec tionw tionp verale e fedw pre-c lesss er-a beings on-s rvedd atr s oft hyp re,h andm

4.2. The use of other substrates in preference toglucose and the stimulatory effect of glucose

The fact that significant growth occurred in the stan-dard pre-culture before the start of glucose consump-tion indicates that this strain uses yeast extract andpeptone preferentially over glucose after being trans-ferred from a solid culture grown on potato dextroseagar. Although the concentrations of the nutrients inthe yeast extract and peptone were not monitored, theysum to 10 g L−1, and, in order to produce biomassconcentrations of over 6 g L−1, it would have beennecessary forG. lucidumto have consumed a large pro-portion of these two carbon sources. The preferentialuse of substrates has not previously been reported forG. lucidum. Similar results were obtained byTang andZhong (2003), for a main culture ofG. lucidum, using amedium containing, per litre, 35 g lactose, 5 g peptoneand 2.5 g yeast extract. Over the first 2 days of culturethere was no significant consumption of lactose, whilethe biomass concentration reached 3.3 g L−1. However,the authors made no comment about this. In all othermain-culture experiments shown in the literature, thereis some sugar consumption over the first few days, aswas also obtained in the main culture in the currentwork.

The experiment done in which pre-cultures receivedglucose at different times showed that the presence ofglucose stimulates growth on peptone and yeast ex-tract, even during periods in which glucose is not be-i hadt vail-a ationo onooh ught ste nnotc ce ofg then lablef thato sep rsorsa smalla ducet ount

In the current work, the synchrony between evuring the pre-culture was striking: the onset of gluconsumption and the onset of rapid EPS producere linked to the liberation of the second-generaellets. This synchrony was consistent amongst sexperiments: the standard pre-culture, a pre-culturith yeast extract and peptone on day 13 and aulture fed with glucose on day 17. There wasynchrony in the main culture, the growth of protubnces and their release as third-generation pelletspread over a much wider time interval. Glucose cumption and EPS production were therefore obseuring the first days of the main culture, althoughelatively slow rates compared to the later stagehe culture. This lack of synchrony may explain wrevious workers, in their studies of the main cultuave not reported the links between morphologicaletabolic events.

ng consumed. Although cultures G0-0 and G35-0he same amount of peptone and yeast extract able, culture G35-0 reached a biomass concentrf 7.4 g L−1 on day 13, with negligible consumptif glucose, while culture G0-0 only reached 1.3 g L−1

n day 13, and a maximum of 2.7 g L−1 on day 17. Anypothesis consistent with this data is that, altho

his strain ofG. lucidumutilizes components of yeaxtract and peptone in preference to glucose, it caarry out gluconeogenesis. Therefore, in the absenlucose, growth is limited because only a fraction ofutrients in the yeast extract and peptone is avai

or the formation of those biosynthetic precursorsccur in the initial part of glycolysis and in the pentohosphate pathway. Once all the available precure used, growth stops. In the presence of glucose,mounts of glucose are consumed in order to pro

hese precursors, and therefore a much higher am


of growth is possible while consuming the yeast extractand peptone. However, this hypothesis does not explainwhy the addition of peptone and yeast extract on day 13to a standard pre-culture did not inhibit glucose uptake.Possibly an irreversible metabolic change had alreadybeen initiated.

4.3. The EPS levels obtained

In this work, we have obtained EPS levels higherthan any reliable values reported previously. In a mainculture started with glucose and fed with more glu-cose on day 10, we obtained an EPS concentration of5.7 g L−1 on day 13. In a survey of the literature under-taken byWagner et al. (2003), the best reliable max-imum EPS concentration was 1.71 g L−1, obtained byYang and Liau (1998). A value of 20 g L−1 has beenreported (Lee et al., 1999), but is unreliable becauseit represents the total weight of the ethanol precipi-tate obtained, not accounting for the presence of non-polysaccharide material (Wagner et al., 2003).

The addition of extra sugars to the main culture hasbeen tried previously.Tang and Zhong (2002)starteda culture with lactose and fed more lactose during theculture. In a similar study,Fang and Zhong (2002a)started a culture with glucose and added more glucoseon day 8. However, in both cases, although the max-imum EPS concentrations obtained were greater thanfor unfed cultures, the values were below 1.5 g L−1,much less than our value of 5.7 g L−1. The fact thati inedw rigi-n thatu aseE

4 kw

e, as askw ssi edb rred,o r in-c thig

et al. (2000)placed polyurethane sponges in a sub-merged culture, almost all the biomass grew adheredto the sponge. This characteristic could cause problemsin bioreactors since the biomass might foul impellersor sensors.

EPS is probably responsible for this adhesion. Theadhered biomass was covered with a gelatinous sub-stance on the side in contact with the flask; thissubstance reacting strongly with the phenol–sulfuricreagent, indicating the presence of polysaccharides.Further, for the culture undertaken in the absence ofglucose (G0-0), the biomass that grew adhered to theflask wall fell into the culture medium when the EPSconcentration fell to zero. This did not occur in any ofthe other cultures in which glucose was present and inwhich EPS levels did not fall to zero.

4.5. Nature of the exopolysaccharide produced

The EPS has a high glucose content, consistent withthe presence of glucans. The presence and relativequantities of monosaccharide units other than glucosedepended on the culture method. This agrees with theresults ofSone et al. (1985), who showed that, althoughglucose was always the dominant unit in EPS under arange of different culture conditions, the proportions ofglucose, mannose and galactose depended on the typeof sugar added to the culture medium.

Mizuno et al. (1995)separated the dried EPS ofG.lucidum into a water-insoluble fraction (which com-p tion(( ity,w cansc hichhpc usedt ive� har-a nderd

5

ofG e

n the pre-culture the highest EPS yield was obtahen glucose was added on day 17 to a culture oally cultivated in the absence of glucose suggestsse of this strategy in the main culture might increPS yields in the main culture still further.

.4. The presence of biomass attached to the flasall

During both the pre-culture and the main culturignificant quantity of biomass was attached to the flall. Fang and Zhong (2002b)separated the bioma

nto different fractions, but did not report attachiomass. Possibly no such attached growth occur it may have occurred and either been ignored oluded in the large pellet fraction. This type of grows not unexpected, since the mycelium ofGanodermarows in an attached state in nature and whenYang

rised 47% of the EPS) and a water-soluble frac53% of the EPS). The insoluble fraction contained�-1→ 3)-d-glucans and had a high anti-tumoral activhereas the soluble fraction contained heterogluomprised of glucose, mannose and galactose, wad no anti-tumoral activity. The fact thatG. lucidumroduced an apparently pure�-d-(1 → 3) glucan inulture G0-35 suggests that this strategy can beo select for the production of the biologically act-glucans. However, further study is required to ccterize the chemistry of the glucans produced uifferent conditions in greater depth.

. Conclusion

Our knowledge about the physiology of growth. lucidum in submerged culture is still limited. Th


current work has identified several key phenomena andopened avenues for future work, especially into the un-derlying mechanisms that link the pellet morphologywith metabolic activities. Such work will give us anunderstanding that will allow us to devise genetic andphysiological manipulations that will lead to higherEPS productivities. This potential is illustrated by thefact that we have already been able to obtain the highestreliable EPS concentration yet recorded for submergedculture byG. lucidum.

Acknowledgments

Ricardo Wagner, David Alexander Mitchell andGuilherme Lanzi Sassaki thank the Brazilian NationalCouncil for Scientific and Technological Develop-ment (CNPq, Conselho Nacional de DesenvolvimentoCientıfico e Tecnologico) for research scholarships.This work represents part of a project funded underthe PRODETAB scheme, which is a Brazilian Gov-ernment initiative, supported by the World Bank andadministered by EMBRAPA, funding research and de-velopment and technology transfer in the agricultural,forestry and agroindustrial sectors.

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links between morphology and physiology of ganoderma lucidum

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