Effect of moisture content in polyurethane foams as support

for solid-substrate fermentation of Lecanicillium lecanii

on the production profiles of chitinases

Marıa del Carmen Marin-Cervantes, Yoyi Matsumoto,Laura Ramırez-Coutino, Zaizy Rocha-Pino,

Gustavo Viniegra, Keiko Shirai *

Universidad Autonoma Metropolitana, Biotechnology Department, Laboratory of Biopolymers,

Avenue San Rafael Atlixco No. 186. Col. Vicentina, C.P. 09340, Mexico City, Mexico

Received 26 January 2007; received in revised form 31 August 2007; accepted 15 October 2007

www.elsevier.com/locate/procbio

Process Biochemistry 43 (2008) 24–32

Abstract

Finely minced (MPUF) and roughly cut (CPUF) polyurethane foam was used as inert support for the growth and chitinases production of

Lecanicillium lecanii by solid-substrate fermentation. L. lecanii growths on CPUF produced loose and disperse mycelia throughout the polymer

honeycomb, but MPUF resulted in dense aggregates at the ends of the polymer branches. Despite similar growth rates, m, and maximum biomass

concentration, Xmax, there were significant differences in the enzyme production. Highest enzyme titers (emax) without glucose supplementation

showed best results for>85% moisture content. emax, of exo-chitinase was 45-fold higher in CPUF than MPUF. Endochitinases, emax, were similar

for CPUF and MPUF. Catabolic repression of enzyme production depended on moisture level, being stronger for lower moisture contents for exo-

chitinases and milder or insignificant for endo-chitinases. Biomass yield coefficients of enzymes, Ye/x, were higher with MPUF than with CPUF for

endo-chitinases, but the reverse was found for exo-chitinases.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Lecanicillium lecanii; Polyurethane foam; Chitinases; Entomopathogenic fungi

1. Introduction

Verticillium are gathered mycoparasitic and entomopatho-

genic species that produce extracelullar enzymes, such as

chitinases. Several strains of Lecanicillium lecanii and

Verticillium fungicola were evaluated as chitinase producers,

and it is reported the activity to induce morphological changes

in phytopathogen fungi [1].

The strains of Verticillium fungicola USDA 4519 and those

of Lecanicillium lecanii USDA 974, USDA 2460, and ATCC

26854 showed the highest activities. Natural polymeric

substrates, such as chitin, have been used to support growth

of Lecanicillium lecanii as source of nutrients as well as inducer

[2]. Chitin is not degraded inside the cell due to its insolubility,

size, molecular complexity and heterogeneous composition but

* Corresponding author. Tel.: +52 5 5804 49 21; fax: +52 5 5804 47 12.

E-mail address: smk@xanum.uam.mx (K. Shirai).

1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2007.10.009

fungi secrete chitinases with different specificity, endochiti-

nases and exochitinases, which are able to transform or

hydrolyse chitin [3].

Verticillium lecanii (Lecanicillium lecanii) has been culti-

vated in solid substrate fermentation (SSF) using chitin as carbon

source. SSF produced a highly concentrated enzymatic extract

with very active chitinases [2]. The readily available sugar cane

bagasse was used as support for fungal growth in that study;

however, such SSF showed problems of substrate sterilization,

temperature and pH control, as well contamination of the system,

which was mainly detected at long process time. Besides, the

enzymatic extracts carried impureness from the organic support,

which involved a harder purification process.

The substrates in SSF, which are usually by products of agro-

industry, have some disadvantages, such as excessive thickness

of the substrate layer, low porosity, or inadequate internal

structures that disturbed the aeration, heat removal and

inefficient nutrient uptake. In most of these systems it is not

possible to separate residual solid substrate from biomass,

Table 1

Media composition of solid substrate fermentation of L. lecanii ATCC 26854 using polyurethane foam

Media Initial moisture

content (%)

PUF (%) Nutrients (%) PUF/nutrients H2O/solids Initial aw

Minced Cut

PGa 75 21.14 3.8 5.5 3 0.991 0.985

PGa 85 10.64 4.35 2.4 5.7 0.988 0.989

PGa 90 4.85 4.14 1.2 10.1 0.995 0.995

NGb 75 21.71 3.27 6.6 3 0.993 0.993

NGb 85 11.28 3.71 3.0 5.7 0.993 0.993

NGb 90 5.43 3.57 1.5 10.1 0.999 0.993

a PG Czapeck media with added colloidal chitin and glucose.b NG Czapeck media with added colloidal chitin.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–32 25

therefore, direct estimation of growth is not attainable, and

instead, that is usually measured by indirect methods based on

protein, ATP, glucosamine or CO2 concentrations.

Alternatively, SSF are also carried out on inert support

materials, such as ion exchanger resins (i.e. Amberlite) and

polyurethane foams (PUF) where the nutrients from a liquid

media are absorbed [4]. The use of inert supports allows direct

biomass determination, cleaner enzymatic extractions and

more homogeneous aeration [5]. PUF have been used as a

suitable support for SSF since it presents high porosity, low

density and relatively high water absorption. PUF have an

adequate pore size which provides a satisfactory environment

for fungal growth. Moreover, the biomass and support are easily

separated into the enzymatic extract with few impurities, which

facilitates further purification [5–7].

The research in this area has been focused in the past years

on how to improve chitinases productivity by the fungi,

however, there is no information, at least to the best of our

knowledge, on the relationships between size and shape of

foams as supports for chitinases production considering factors

such as moisture content and catabolite repression by glucose

addition in solid state fermentation.

2. Materials and methods

2.1. Organism and cultivation conditions

Lecanicillium lecanii (former Verticillium lecanii) ATCC 26854 (Amer-

ican Type Culture Collection, USA) was stored on potato dextrose agar slants

at 4 8C until needed. The spore suspension was obtained by mechanical

agitation with a solution of 0.1% (v/v) of tween 80 at a concentration of

107 spores/ml [2].

2.2. Chitin

Chitin was obtained from shrimp head waste silage that contained residual

proteins and ashes, 14 and 8% (dry weight basis), respectively [8]. For addition

into the media, colloidal chitin was prepared according to earlier reported

methodology [9].

2.3. Media

The composition of modified Czapeck medium was: NaNO3 3.73 g/l,

K2HPO4 3.0 g/l, MgSO4 0.5 g/l, FeSO4 0.096 g/l, KCl 0.5 g/l. Colloidal chitin

(36 g/l) was used as sole carbon source and this medium is referred as NG (non-

glucose). A second medium PG (plus glucose) was used. PG contained glucose

(15 g/l) and colloidal chitin (23.3 g/l). The pH of the media was maintained to 6

by addition of either HCl or NaOH (Table 1). The C/N, C/P, C/S molar ratios of

the media were 5.5, 32 and 110, respectively [10].

2.4. Polyurethane foam

PUF were used in two sizes: (i) minced (M) and (ii) cut into cubes (C). The

minced PUF (M) were prepared by using a Warring blender and meshed until a

0.3–0.5 cm particle size range, then washed and dried overnight at 70 8C. PUF

were also cut into cubes (C) with approximately 0.5 cm � 0.5 cm � 0.5 cm

cubic sizes, then washed and dried overnight at 70 8C.

2.4.1. PUF pore-size characterization

Digital image analysis was carried out to measure the interstitial spacing in

both minced and cut in cubes foams. A microscope (Olympus BX50, Japan) was

used to view the pore structure of the PUF in cross section of the foams. High

resolution photographs were taken using a cooled charge-coupled device

camera, and image analyses were performed using Image-Pro Plus software

(Media Cybernetics, Maryland, USA). Digital photographs were analyzed on

areas with dimensions of 8.95 mm � 7.21 mm by 4� magnification. The

photographs were taken randomly, from 120 to 140 measurements were carried

out for each type of PUF (minced and cut).

2.4.2. Porosity measurement

PUF in powder was prepared with a mincer (Thomas Scientific, model

3379-E) and meshed until a particle size of 1 mm. Foams minced or cut, as well

as powdered as a control sample, were placed in a measuring cylinder without

leaving any free spaces between the foams, and then weighed. Subsequently, the

void fraction (e) in the foam was measured by weighing PUF minced or cut

referred to powder foam. This procedure was repeated three times for MPUF

and CPUF samples.

The porosity (f) was calculated by means of the following Eq. (1):

f ¼ 1� e (1)

where e = (W/W0). W0 is the weight of PUF in powder, W is the weight of PUF,

minced or cut, and f is the porosity. Then when W �W0, the porosity (f) � 0.

2.4.3. Density and water storage capacity

The foams were dried to constant weight at 70 8C for 24 h and then cooled in

a dessicator and their masses were recorded. Each foam volume was calculated

considering height and diameter. PUF densities were calculated by dividing

mass by volume. Water storage capacity was calculated by dividing the

difference of wet and dry weights by the dry weight and multiplying by

100. Each determination was carried out by triplicate.

2.5. Solid substrate fermentation (SSF)

The culture media, PG and NG, as described above, and inoculum were

absorbed into polyurethane foams with two sizes: minced (MPUF) or cut

(CPUF). Three ratios of water:nutrients:support were evaluated, corresponding

to 75, 85 and 90% of moisture content. SSF was carried out in 250 ml flasks

closed with cotton plugs. Fermentation was carried out at 25 8C, during 7 days

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–3226

sampling by duplicates for each condition. Solid substrate cultures were

collected from each flask, mixed with their same weight in water and pressur-

ized to 1000 psi. Cultures were centrifuged at 14,000 � g at 4 8C for 15 min.

Spores and mycelia were separated by filtration and used as crude enzyme for

assays.

2.5.1. Determination of fungal biomass

L. lecanii growth was measured by dry weight determinations. A statistical

programme STATISTICA 6.0 (Stat Soft, Inc., USA) was used to estimating the

specific growth rate by fitting the biomass data, X, to the logistic Eq. (2):

X ¼ Xmax

1þ ððXmax=X0Þ � 1Þe�mmaxt(2)

where X was the biomass determined by dry weight after time (t); X0 was the

initial biomass, Xmax was the maximal biomass (t!1), and mmax was the

specific growth rate. The X0, Xmax, and mmax values were estimated using a non-

linear estimation programme. Ye/x is the yield coefficient for enzyme on total

biomass produced given by the Eq. (3):

Ye=x ¼½emax�Xmax

(3)

where emax is the maximum enzyme activity that was determined from the

experimental data [7].

2.5.2. Estimation of glucose consumption with Gompertz model

The glucose concentration in SSF was measured by the reducing sugar

method [11]. The Gompertz model was applied to glucose concentration data

during time and the constants Smax, b and k in the culture media with added

glucose were estimated for enzymes production by using the non-lineal

estimation program. In this model, S is the shift of glucose concentration as

function of time t during SSF and it was considered as S = S0 � S(t), where S0 is

the initial amount of glucose added to the media and S(t) is the glucose

consumed at time (t). S is obtained according to the following Eq. (4):

S ¼ Smax expð�b expð�ktÞÞ (4)

where Smax is the maximum glucose consumed (t!1), b is a constant related

to the initial conditions (when t = 0, then S = Smaxexp(�b)) and k is the

consumption rate.

2.6. b-N-Acetyl hexosaminidase activity

The presence of b-N-acetyl hexosaminidase activity was determined in the

extracts from SSF using p-nitrophenyl-b-N-acetyl glucosamine (Sigma Che-

mical Co., St. Louis, MO) as substrate. 200 ml of crude enzyme was added to

200 ml of a citrate-phosphate buffer (0.2 M, pH 5.6). The solution was mixed

with 200 ml of p-nitrophenyl-b-N-acetyl glucosamine (1 mg/ml) and then

incubated with agitation (180 rpm) at 37 8C for 1 h. The reaction was quenched

by addition of 1 ml of NaOH 0.02 M. The released p-nitrophenol was evaluated

by a UV–vis spectrophotometry (Jenway 6305, Essex UK) at 400 nm. The

enzymatic unit was defined as the amount of enzyme that releases 1 mmol of p-

nitrophenol per minute [12].

2.7. Endochitinase activity

The endochitinase activity was measured by reduction of turbidity of

suspensions of 1% (w/v) of colloidal chitin in phosphate buffer (50 mM)

and NaN3 (0.01 M) at pH 6.7. 500 ml of crude enzyme was added to 500 ml

Table 2

Characteristics of polyurethane foams used in this study

Type of PUF Porosity (f) Void fraction (e)

Minced PUF 0.912 � 0.010 0.0875 � 0.0098

Cut PUF 0.955 � 0.05 0.0453 � 0.0051

of colloidal chitin. The mixture was incubated at 30 8C and stirred continuously

at 180 rpm for 24 h. Then it was diluted with 5 ml of water and the absorbance

was determined at 510 nm. The enzymatic unit was defined as the amount of

enzyme that decreases 5% the turbidity of a colloidal chitin suspension [13].

2.8. Statistical analysis

A randomized design was carried out in quadruplicate for experiments with

different moisture content levels, PUF sizes and glucose addition in SSF. The

programme SPSS version 8.0 (SPSS, Inc., USA 1997) computed the analysis of

variance with enzyme activity and biomass as response variables. The means

were compared with Tukey Kramer multiple means comparison test (P � 0.05).

2.9. Scanning electron microscopy

SSF samples were prepared by immersion in 5% (v/v) glutaraldehyde for

24 h at 4 8C and post-treated with OsO4 1% (w/v) for 2 h at 4 8C. Then they

were dehydrated in a graded alcohol series and covered with carbon and gold

prior to examination in the Scanning Electron Microscope (JEOL JSM-5900 LV,

Tokyo).

3. Results and discussion

Minced (MPUF) and cut PUF (CPUF), approximated

density of 0.018 g/cm3, were evaluated on their properties to

retain the culture media due to their capillarity and porosity.

The water storage capacity of MPUF and CPUF were

determined as 19.8 and 21.4 ml/g, respectively (Table 2).

CPUF presented honeycomb of polygonal cells of the

polymer while MPUF, which cells of the polymer were

destroyed during the mince process, was observed as irregular

forms. The averages of the interstitial areas were different for

cut and for minced PUF; the former was of 73.77 mm2 and the

latter 99.48 mm2, while the porosity of the cut one was 0.955

and for the minced 0.912. According to these data, MPUF

presented a decrease in the capillarity due to the decreased

porosity that had significant effect on the absorption of the

culture media compared with the CPUF (Table 2). The CPUF

was subjected to less mechanical force causing minor

destruction of pores than MPUF and therefore, it presents

higher porosity than the MPUF (Fig. 1). The effect of surface

tension (capillarity) is to draw liquid back into the borders. Due

to the existence of highly curve borders in CPUF, the capillarity

pressure was higher than in the MPUF thin films (Fig. 1).

3.1. Lecanicillium lecanii growth using PUF in SSF

The direct measurement of the biomass is one of the

advantages of using PUF in SSF with regard to other supports

[5], however the use of colloidal chitin in the medium hinders

this determination for its insolubility and retention in this type

of supports. The chitin fraction absorbed in the PUF was

Maximum water

absorption (ml/g PUF)

Interstitial spacing (mm2)

19.8 99.48 � 6.83

21.4 73.77 � 4.59

Fig. 1. Stereomicroscope micrographs of polyurethane foams (PUF) (4�): (a and b) cut PUF; (c and d) minced PUF.

Fig. 2. Biomass production of Lecanicillium lecanii ATCC26854 in SSF using

minced (M) and cut (C) PUF at various moisture content: (a) culture media

without addition of glucose: (*) M 75%, (&) C 75%, (^) M 85%, (~) C 85%,

(—) M 90%, (+) C 90%. (b) Culture media with addition of glucose: (*) M

75%, (&) C 75%, (^) M 85%, (~) C 85%, (—) M 90%, (+) C 90%. The

experimental data are mean of four determinations.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–32 27

measured and considered for the biomass calculation. The

curves of growth of Lecanicillium lecanii in MPUF and CPUF

with different moisture contents and glucose addition are shown

in Fig. 2.

As it was expected, the supplementation of glucose to the

media as well as the increase of moisture content favored the

production of biomass. The biomass in chitin media (NG) was

significantly lower in both types of PUF and at all moisture

content levels tested (Fig. 2).

The growth parameters were estimated on the type of PUF,

moisture content and chitin media only or with added glucose.

The estimated kinetic parameters showed that for all the

experiments the production of maximum biomasses concen-

trations (Xmax) increased with the addition of glucose, which

was the main source of variation rather than the type of support.

The highest Xmax were determined with 90% of moisture

content in PG medium, 0.193 and 0.15, for MPUF and CPUF,

respectively. However, in PG media there is no correlation

between the type of support and the production of biomass,

whereas, Xmax were higher in CPUF than in MPUF for all the

moisture contents in NG media. The maximum specific growth

rate (mmax) was the highest with 75% of moisture content in

CPUF (Table 3). Although the interstitial spacing in MPUF was

significantly higher than in CPUF (Table 2); the growth was not

significantly different in both PUFs (Fig. 2). This is in

agreement with the Van de Lagemaat and Pyle [7] report, where

it was suggested that size of PUF did not have a significant

effect on biomass production of Penicillium glabrum, the

growth was mainly limited by the substrate rather than PUF

pore size.

Table 3

Estimated growth kinetic parameters of L. lecanii ATCC26854 and enzyme production in SSF using PUF (minced or cut) at 75, 85 and 90% of moisture contents in

media with or without addition of glucose

Moisture content (%, w/w) Polyurethane foam size Culture media Xmax (g/g PUF) X0 (g/g PUF) mmax (h�1) R2

75 Ca NGc 0.012 0.00009 0.32 0.94

75 Mb NGc 0.011 0.00006 0.19 0.94

75 Ca PGd 0.023 0.00026 0.22 0.96

75 Mb PGd 0.050 0.00445 0.08 0.92

85 Ca NGc 0.032 0.00075 0.18 0.99

85 Mb NGc 0.013 0.00014 0.27 0.90

85 Ca PGd 0.068 0.00680 0.05 0.90

85 Mb PGd 0.047 0.00059 0.20 0.90

90 Ca NGc 0.059 0.00057 0.20 0.97

90 Mb NGc 0.044 0.00202 0.14 0.96

90 Ca PGd 0.150 0.01471 0.07 0.90

90 Mb PGd 0.193 0.00575 0.06 0.92

a C cut PUF.b M minced PUF.c NG Czapeck media with added colloidal chitin.d PG Czapeck media with added colloidal chitin and glucose.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–3228

The distribution of the fungal cells in MPUF with NG media

was observed as loose and disperse, whereas in CPUF the

growth was observed in loosely packed hyphae as shown in

Fig. 3a–c. The fungal growths in CPUF covered the surface by

formation of a net of intertwinement hyphae in PG media while

in MPUF L. lecanii grew forming aggregates in the ends of the

branches made from the sectioned cells of the PUF (Fig. 3 b–d).

The formation of filamentous mycelia in PG media might be

due to the media composition, which contained glucose that

favors this fungal morphology. However it is remarkable that

mycelial aggregation was observed in MPUF maybe due to

losses of water during SSF.

Fig. 3. Scanning electron micrographs of the Lecanicillium lecanii ATCC 26854 g

addition of glucose; (b) with addition of glucose; and minced PUF: (c) without ad

3.1.1. Glucose consumption during SSF

The glucose consumption through the time of cultivation in

the SSF was determined at several moisture contents and the

results are shown in Fig. 4. The glucose consumption in SSF

with CPUF was faster during the first 24 h compared to MPUF.

The glucose was depleted at 72 h with CPUF at any moisture

content, while the sugar consumptions with MPUF were

achieved at 96 h with 85 and 95% of moisture content. The

experimental evidences point out that the PUF size and

moisture contents were significant for the rate of glucose

consumption. The maximum glucose consumed (Smax) was

lower with CPUF than MPUF. The highest Smax was determined

rowths with colloidal chitin at 85% moisture content on cut PUF: (a) without

dition of glucose; (d) with addition of glucose.

Fig. 4. Glucose concentration during SSF of Lecanicillium lecanii ATCC26854

in minced (M) and cut (C) PUF at various moisture content: symbols (*) M

75%, (&) C 75%, (^) M 85%, (~) C 85%, (—) M 90%, (+) C 90%.

Fig. 5. pH determination of Lecanicillium lecanii ATCC 26854 in SSF using

minced (M) and cut (C) PUF in media with different moisture contents: (a) culture

media without addition of glucose: (*) M 75%, (&) C 75%, (^) M 85%, (~) C

85%, (—) M 90%, (+) C 90%. (b) Culture media with addition of glucose: (*) M

75%, (&) C 75%, (^) M 85%, (~) C 85%, (—) M 90%, (+) C 90%.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–32 29

at 90% moisture content with MPUF as support. Smax also

increased with the moisture content; however, the consumption

rates (k) were similar in all the experiments (Table 4).

3.1.2. Effect of the pH on fungal growth

The pH is an important environmental factor for fungal

growth and chitinases production and it changes due to the acid

or basic compounds produced by microbial use of nitrogen

sources and the modification of the solid substrate [14]. In SSF,

it is not possible to control the pH by addition of acids or bases

to the medium since homogeneous distribution cannot be

achieved when the media is already impregnated in the support.

Agitation is not recommendable at this stage due to

contamination risk and possible destruction of mycelia;

therefore, buffered substrates are usually added into the media

for that purpose.

In this study, the media were adjusted to an initial pH of 6, and

after fungal growth, the NG medium was slightly alkaline, ca. 7,

due to the assimilation of nitrogen source from chitin or salts

(Fig. 5a). In the PG medium, glucose was readily consumed and

the chitin assimilation followed, then the pH increased (Fig. 4,

Fig. 5b). For instance, the pH variation in the PG medium at 85%

of moisture content in CPUF was different from the earlier since

it presented a pH decrease to ca. 5 (Fig. 5b). However, after 72 h,

the pH increased to ca. 8 (Fig. 5b).

The maintenance of pH on near values of 6 during culture

had also a positive effect on the fungal growth in NG media,

since mmax were higher than those found in PG media (Table 3).

Table 4

Estimated parameters of glucose consumption during SSF of Lecanicillium lecani

Moisture content (%, w/w) Polyurethane foam size Smax

75 Ca 0.053

75 Mb 0.054

85 Ca 0.120

85 Mb 0.123

90 Ca 0.281

90 Mb 0.301

a C cut PUF.b M minced PUF.

Neither moisture content nor the PUF sizes were significant,

thus the factor that affected significantly the pH was the

addition of glucose.

3.1.3. Effect of moisture content on fungal growth

The moisture content can be defined as the water contained in

the medium able to dissolve nutrients, products, and moisturize

spores thus it promotes germination [14]. Fungi can grow in a

wide range of aw, 0.61–0.98, in SSF they grow well at aw of 0.96–

0.98 [15]. In this study, the foams presented 21.4 ml/g PUF and

19.8 ml/g of PUF of water absorption for cut and minced,

respectively (Table 2). This high water retention of PUF allowed

aw among 0.985–0.999, which are in the range for optimal fungal

growth. However it is important to consider that high retention of

water in the support can also favors the contamination by bacteria

or yeasts, as well as oxygen diffusion.

i using polyurethane foam as inert support

(g/g PUF) b (g/g PUF) k (h�1) R2

4.65 0.054 0.98825

3.02 0.055 0.98634

5.92 0.050 0.97790

2.51 0.045 0.95280

3.67 0.030 0.96352

2.76 0.052 0.97404

Fig. 6. Production of b-N-acetyl hexosaminidases of Lecanicillium lecanii

ATCC 26854 in SSF using minced (M) and cut (C) PUF in media with different

moisture contents: (a) culture media without addition of glucose: (*) M 75%,

(&) C 75%, (^) M 85%, (~) C 85%, (—) M 90%, (+) C 90%. (b) Culture

media with addition of glucose: (*) M 75%, (&) C 75%, (^) M 85%, (~) C

85%, (—) M 90%, (+) C 90%.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–3230

The distribution of the fungal cells in the medium at the

highest moisture content might allow a better accessibility to

the substrate, without accumulation or precipitation of the

nutrients. Moisture content was significant as source of

variation for biomass production (Table 3).

3.2. Chitinases production using PUF in SSF

3.2.1. Effect of moisture content and glucose addition into

the media

In the reported SSF carried out with sugarcane bagasse as

support, L. lecanii was cultivated at several moisture contents,

65, 70, and 75%. The production of b-N-acetyl hexosaminidase

was 6.5-fold times higher when the humidity increased from 65

to 75%. At low moisture content the lag phase was long, for

instance with 70% it last 72 h, while at 75% the growth phase

began at 20 h [2].

In the present study, the yield coefficients for N-acetyl

hexosaminidase and endochitinase on total biomass produced

were the highest at 85% and maximal enzyme activities emax at

90% of moisture content (Table 5).

The addition of glucose improved the production of biomass

and the chitinases production was ruled by carbon catabolite

repression in PG media, as shown in Figs. 6 and 7.

In the SSF with chitin as sole carbon source, the N-acetyl

hexosaminidases production started later than biomass produc-

tion. The maximal values were reached after 96 h of culture. This

might be due to these enzymes are constitutive and they were

produced intracellular, subsequently, N-acetyl hexosaminidases

were induced and secreted. Endochitinases were produced

earlier (24 h), even with the presence of glucose into the media.

Chitin was pretreated with acid in order to increase solubility

and disrupt intraparticle barrier from diffusion, it is suggested

that this additional factor promoted the production of

chitinases. Ramirez-Coutino et al. [1] has found that alkaline

pretreatment of chitin changed the crystalline structure

facilitating enzymatic hydrolysis.

Table 5

Chitinases production of L. lecanii ATCC26854 in SSF using PUF (minced or cut) at 7

Moisture content

(%, w/w)

Polyurethane

foam size

Culture media N-Acetyl hexosa

emax (mU/g PUF

75 Ca NGc 1094.8 � 27.59

75 Mb NGc 443 � 4.64

75 Ca PGd 307.4 � 8.36

75 Mb PGd 488.8 � 1.40

85 Ca NGc 2703.6 � 184

85 Mb NGc 1912.1 � 7.48

85 Ca PGd 1262.3 � 36.04

85 Mb PGd 715.1 � 9.38

90 Ca NGc 5623 � 4.81

90 Mb NGc 123.7 � 3.55

90 Ca PGd 3202.5 � 12.66

90 Mb PGd 96.4 � 4.88

a C cut PUF.b M minced PUF.c NG Czapeck media with added colloidal chitin.d PG Czapeck media with added colloidal chitin and glucose.

The yield coefficients for N-acetyl hexosaminidase were

higher in NG media, however, the repression by glucose in PG

media substantially decreased with the increment of moisture

content. In the SSF at 90% with NG media, the YN-Hases/x were

about 5.6 times higher than SSF with PG media, while with

85% moisture content the difference was up to 10 times higher

in NG than in PG media (Table 5).

5, 85 and 90% of moisture contents in media with or without addition of glucose

minidase Endochitinases

) Ye/x (mU/g biomass) emax (U/g PUF) Ye/x (U/g biomass)

89742.4 187.1 � 15.08 15338.4

40581.6 197.6 � 14.98 18102.8

13385.7 77 � 11.80 3352.1

9690.7 331 � 15.61 6562.7

84410.4 717.3 � 7.34 22394.9

151814.6 481.1 � 9.07 38195.6

18428 422.6 � 53.59 6169.2

15068.1 562.1 � 12.31 11844.3

94817.8 925 � 20.77 15597.4

2798.3 1316.6 � 53.9 29788.3

21390.3 929.7 � 39.8 6210

500.1 1422.8 � 60.65 7381.4

Fig. 7. Production of endochitinases of Lecanicillium lecanii ATCC 26854 in

SSF using minced (M) and cut (C) PUF in media with different moisture

contents: (a) culture media without addition of glucose: (*) M 75%, (&) C

75%, (^) M 85%, (~) C 85%, (—) M 90%, (+) C 90%. (b) Culture media with

addition of glucose: (*) M 75%, (&) C 75%, (^) M 85%, (~) C 85%, (—) M

90%, (+) C 90%.

M.C. Marin-Cervantes et al. / Process Biochemistry 43 (2008) 24–32 31

This might be explained by the exhaustion of glucose in PG

media, which increased the production of N-acetyl hexosami-

nidases after 72 h as it is shown in Figs. 4–6.

3.2.2. Effect of pH and type of PUF

The effect of pH on the production of N-acetyl hexosami-

nidases was quite different from those reported in submerged

culture. L. lecanii, the highest activity of N-acetyl hexosami-

nidases was determined at pH 5 in submerged culture with

Czapeck medium supplemented with chitin from shrimp silage,

the pH range of the enzyme production was from 4 to 7 [16].

Herein, the highest emax were determined at the pH range of 7–8

(Table 5, Figs. 5 and 6). For instance, the N-acetyl

hexosaminidases reached their maximal enzyme activities,

emax, when pH shifted to basic (pH 8) in PG media (Figs. 5 and

6b). For endochitinases their maximal enzyme activities were

reached after 96 h, when pH was determined between 6.5 and

7.0 in both media (Figs. 5–7).

PUF size was significant source of variation on chitinases

production by multiple comparison tests, where it was shown

that the endochitinases production with MPUF was higher

than in CPUF whereas the opposite was found for N-acetyl

hexosaminidases production. The type of PUF was also

important for catabolic repression by glucose since with

CPUF the reduction in activity was lower than with MPUF

(Table 5).

The endochitinases underwent less catabolic repressed by

glucose than N-acetyl hexosaminidases. Diaz-Godinez et al. [6]

reported that Aspergillus niger was less sensitive to repression

of pectinases by sucrose when it was cultivated in SSF with

PUF as support than in submerged fermentation.

The highest yield coefficients of N-acetyl hexosaminidase

and endochitinase on biomass in MPUF were determined with

the SSF with 85% moisture content with the NG media, where

the state of micro-aggregation of mycelia was different to that

observed in CPUF (Table 2, Fig. 3). The experimental

evidences suggested that the different fungal morphologies

in both supports are responsible for these variations on the

yields in each type of enzyme, as it was found that Ye/x was

higher with CPUF than MPUF for N-acetyl hexosaminidases,

but it was just reverse for endochitinases.

4. Conclusions

Polyurethane foams employed in this study had similar

porosity, but the interstitial spacings and maximum water

absorption were different and displayed a remarkably different

effect to fungal morphology and chitinases production. This is, to

the best of our knowledge, the first report that related the changes

in the production of chitinases by entomopathogenic fungi based

on the different sizes and shapes of polyurethane foams.

Acknowledgements

The authors would like to thank to SEP-CONACYT (2004-

C01-46173) for research funding and scholarship grant to Miss

Marin. To the European Union for granting Miss Marin for

attendance to Biopolymers course through the project Alfa

Polylife. Dr. Jose Sepulveda is greatly acknowledged for his

invaluable assistance in the SEM studies.

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