Medium optimization for nuclease P1 production by Penicillium citrinum in solid-state fermentation...

ELSEVIER

Medium optimization for nuclease Pl production by Penicillium citrinum in solid-state fermentation using polyurethane foam as inert carrier Yang Zhu, Wieger Knol, Jan P. Smits, and Jan Bol

TN0 Nutrition and Food Research. Zeist. the Netherlands

A solid-state fermentation system, using polyurethune ,fi)um as an inert currier, was used for the production of nuclease PI by Penicillium citrinum. Optimization of nuclease PI production was carried out using a synthetic liquid medium. After a two-step medium optimization using a ,fractional jhctorial experimental design. nuclease PI productiviv and specific activity are greatly increased. Nuclease PI productivit?, and specific activity are ninefold and fourfold higher. respectively, than in solid-state fermentation of whear bran and 50Cfold and 20-fold higher, respectively. than in submerged fermentation. Additional advantages of the solid-state fermentation process using inert Currier are discussed.

Keywords: Solid-state fermentation; inert carrier; polyurethane foam: nuclrase PI: Pmicillium citrinum, medium optimization

Introduction

In recent years. advantages of solid-state fermentation (SSF) over submerged fermentations (SmF) have been in- creasingly recognized in various applications such as production of fungal metabolitesl and especially in the case of fungal enzyme production.’ However, one of the critical disadvantages of SSF processes which utilize agricultural materials such as wheat bran as substrate is the impurity of the product. These impurities result in difficult and compli- cated downstream processing’ when an end product of high purity is demanded.

One of the potential alternatives is to use an inert carrier such as polyurethane foam (PUF). Fujishima et al.’ used PUF as an inert carrier in a system containing a synthetic liquid medium for the production of fungal enzymes includ- ing nuclease Pl by Penicillium citrinum. However. enzyme activity expressed on the basis of the sugar content of the substrate was much lower in this process than in SSF of wheat bran and in SmF. Recently, Zhu et ~1.~ described a novel process using PUF as inert carrier in an SSF system

Address reprint requests to Dr. Yang Zhu. TN0 Nutrition and Food Rr- search, P.O. Box 360, 3700 AJ Zeist. Netherlands Received 7 September 1994: revised 16 March 1995: accepted 3 I March 1995

for the production of nuclease PI by P. citrinum. In this system, PIJF is impregnated with a liquid medium. The system simulates the chemical composition of wheat bran in which cellulose is replaced with PUF. Based on the substrate dry weight, the enzyme productivity in this PUF process is comparable with that in SSF of wheat bran.

This paper describes the results of research done on further development and optimization of the PUF process. A fractional factorial experimental design was used for medium optimization experiments to improve nuclease Pl productivity and specific activity. The PUF process with the optimized medium was compared with the PUF process before optimization, SSF of wheat bran, and SmF of a medium described by Fujishima.2

Materials and methods

Microorganism

Penicillium citrinum ATCC 14994 was used in the experiments. Potato dextrose agar slants were used for sporulation and storage. A suspension of I x 10s spores ml-’ was prepared in sterile 0.1% Tween 80 and stored at -80°C.

hrrt carrier

Polyurethane foam (PUF, type 528 I, AAIO, 45 pores in-.‘) was supplied by Declon Korby. Northants. England). The foam was

Enzyme and Microbial Technology 18:108-112, 1996 (6 1996 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0141.0229/96/$15.00 SSDI 0141-0229(95)00082-G

Table 1 Design of experimental runs in both optimization steps Table 2 Substrate constituents utilized in the first optimization one and two step

Constituent j

Run i (A) (B) (C) (D) (E) (F) (G) Constituent j

Low level k-) High level (+) (g constituent (g constituent 100 g-’ PUF) 100 g-’ PUF)

1 _ _ + + + _

2 + - - _ + + 3 _ + - + _ + 4 + + _ + _ _

5 - - + + - - + 6 + _ + - + - -

7 f + - + _

8 + + + + + + +

(A) Glucose (B) Starch (C) Peptone

y,’ y&W,

(F) CaClz4 (G) Vitamins

10.0 30.0 12.0 36.0

1 .oo 30.0 0.62 1.86 0.037 0.111 0.262 0.786 1xa 3Xb

+, high level; -, low level

sSame amount used by Zhu et a14 bThree times as much used by Zhu et aP

washed with warm water, dried, and cut into 5-mm cubes before use.

Submerged fermentation

The medium described by Fujishima* was used for SmF. It had the following composition (w/v): 5% glucose, 0.5% peptone, 0.05% KH,PO,, 0.05% K,HPO,, 0.04% MgSO,, and 0.04% CaCl,. All components were dissolved in distilled water; 50 ml of the medium was put into a 200-ml Erlenmeyer flask. After sterilizing at 12 1 “C for 20 min and cooling to room temperature, each flask was inoculated by adding 1 ml of spore suspension. Fermentation was performed by incubating the flasks on a shaker at 26’C and 200 rpm for 120 h. Samples were taken at 24 h intervals during the incubation period.

The concentrations of substrate constituents in medium optimization experiments and the final composition of the optimized medium used in comparison experiments are mentioned in RESULTS AND DISCUSSION. In all cases, soluble substrate constituents were dissolved in distilled water and the pH value was adjusted to 6.0.

Water activity measurement

Solid-state fermentation of wheat bran

An electric hydrometer (Type EK 84/3H/63T, Sensor type enBSK-4, Novasina, Pf&ffikon, Switzerland) was used to measure the water activity of impregnated PUF. Because different medium compositions were used throughout the optimization experiments, water activity varied with the medium composition. Therefore, water activity of each composition was measured in advance so that the water activity of all medium combinations could be set to a constant level of 0.97 by adjusting the water content of the impregnated PUF.

Ten g wheat bran was mixed with 6 ml tap water in a 300-ml Erlemneyer flask with a cotton plug. Flasks were sterilized at 121°C for 30 min. After cooling to room temperature, the wheat bran was inoculated (5 x lo7 spores g-i substrate) and cultivated in a climatic incubator (VEA-Instruments, Houten, Netherlands) with the temperature and relative humidity set at 26°C and 97%, respectively. Samples were taken every 24 h for 5 days by removing a single flask from the incubator.

Sample treatment

The PUF process

The contents of each Erlenmeyer flask were extracted for 30 s with distilled water in a laboratory blender (Stomacher LAB 400 Blender, Seward Medical, London, England) and filtered through a Whatman GF/C filter paper. Fermentation broth of SmF was filtered directly. Filtrates were used for the measurement of nuclease Pl activity and soluble protein. All results are the mean value of duplicate samples.

The substrate used for the PUF process based on the chemical composition of wheat bran has been described earlier.4 The liquid medium was added to a 300-ml Erlenmeyer flask with a cotton plug containing 1.5 g PUF and mixed thoroughly. Excess water was evaporated at 105°C until the final water activity was 0.97 while taking the effect of sterilization and inoculation on moisture content into account. The impregnated PUF was then sterilized at 121°C for 20 min. After cooling to room temperature, it was inoculated with spore suspension (5 x lo7 spores g-l substrate) and cultured in the same climatic incubator as for the SSF of wheat bran. Samples were taken every 24 h for 5 days by removing a single Erlenmeyer flask from the incubator.

Table 3 Biomass dry weight, nuclease PI activity, and specific activity in the first optimization step after 96 h of fermentation

Medium optimization for the PUF process

Medium optimization was carried out by using the fractional factorial experimental design described by De Meo5 and Christen.6 This method allows for improvement in the medium composition of seven substrate constituents utilizing only eight experiments.

Biomass Nuclease PI Specific activity dry weight (E,, ml-’ (E 260 mg-’

Run i (mg g-l PUF) extract) protein) PH

1 71.9 0.468 5.90 6.50 2 137.1 0.710 9.30 5.83 3 126.9 0.738 8.30 5.78 4 187.0 0.107 2.02 3.91 5 76.1 0.015 0.11 7.73 6 131.0 0.075 0.50 7.89 7 175.6 0.086 0.53 7.78 8 220.1 0.112 0.68 7.42 Average 131.7 0.112 3.41

Medium optimization for P. citrinum: Y. Z/w et al.

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Table 4 Coefficients for biomass dry weight CC,), nuclease PI activity (C,), and specific activity (C,) for each substrate constituent resulting from the first optimization step

Coefficient Glucose Starch Peptone (NH,),SO~ ZnSO, CaCI, Vitamins

Cb 28.1 36.7 9.99 -1.94 -3.24 10.5 -0.66 C, -0.038 -0.028 -0.22 -0.11 0.059 0.055 0.10 C, -0.29 -0.54 -2.96 -1.24 0.43 0.69 1.10

Biomass dry weight measurement in the PUF process

Biomass dry weight measurement in the PUF process was performed as described by Zhu et al.’

Protein assay

Soluble proteins in filtrates were measured using the BioRad protein assay based on Bradford’s dye-binding procedure.’

Nuclease PI assay

The activity of nuclease PI was measured using the method of Fujishima’ which was modified by replacing uranyl acetate with lead acetate. The absorbance at 260 nm (E& was used to express nuclease Pl activity. Specific activity of nuclease PI is expressed

as EzeO mg-’ protein.

Results and discussion

Optimization of medium

Two experimental steps were studied for optimization of the procedure. The purpose of the first step was to determine which substrate constituent has a significant effect on fungal biomass (expressed in this paper as biomass dry weight g-’ PUF), nuclease Pl activity, and specific activity. The goal of the second step was to further adjust the levels of the constituents. In both steps, seven substrate constituents were chosen for optimization. Each of these had two levels, so that one optimization step needs only eight experimental runs to estimate the influence of the constituent level (Table I). For evaluation of the effect of each constituent, the coefficients C, (biomass dry weight), Cp (nuclease Pl activ-

Table 5 Substrate constituents utilized in the second optimization step

Constituent j

Low level (-) (g constituent 100 g-’ PUF)

High level (+) (g constituent 100 g ’ PUF)

(A) Glucose 10.0 20.0 (B) Starch 10.0 20.0 (C) Peptone 7.50 15.0 (D) Phosphate 3.50 7.00 (E) ZnSO, 0.074 0.148 (F) CaCI, 0.525 1.05 (G) Vitamins 2x” 4Xb

“Twice as much used by Zhu et a/4 bFour times as much used by Zhu et a14

ity), and C, (specific activity) were calculated as follows. If B is the biomass dry weight (mg g-’ PUF). P is the nuclease activity (E,,, ml-’ extract), and S is the specific activity of nuclease Pl (E,,, mg-’ protein), the coefficients C,, C,, and C, relating to each of the seven given constituents are given by:

ChJ=VxX[$AjXBi] (1)

8 C,, = % x c Aj x Pi ;

[ 1 i=l

8

C,<] = l/x x c Aj x Si . I I i=l

(2)

(3)

Here Aj means either high (+) or low (-) level in experimental run i. If a calculated coefficient has a positive value, it means that this constituent has a positive effect at the high level and vice versa.

First optimization step

The setup of the first optimization step is shown in Tuble 2. The concentrations of other constituents, such as K,HPO,, KH,PO,, and FeSO, have been mentioned ear1ier.j The results of the first optimization step are given in Tab/e 3. The calculated results for C,. C,, and C, are shown in Ta- ble 4.

It can be seen from Table 4 that an increase in the level of glucose, starch, peptone, or CaCl, has a positive effect on the increase of biomass dry weight. An increase in the level

Table 6 Biomass dry weight, nuclease PI activity. and specific activity in the second optimization step after 96 h of fermentation

Biomass Nuclease PI Specific activity

dry weight (E,,, ml-’ (E 260 mg-’ Run i (mg 9-l PUF) extract) protein) PH

1 72.3 0.699 13.38 2 85.0 0.753 12.49 3 82.3 0.731 7.05 4 91.1 0.746 9.39 5 86.3 0.672 5.25 6 102.3 0.775 6.51 7 100.1 0.652 6.63 8 109.3 0.675 6.58 Average 91.3 0.713 8.41

6.16 5.93 6.05 5.78 6.68 6.74 6.78 6.44

110 Enzyme Microb. Technol., 1996, vol. 18, February

Medium optimization for P. citrinum: Y. Zhu et al.

Table 7 Coefficients for biomass dry weight CC,,), nuclease PI activity CC,), and specific activity CC,) for each substrate constituent resulting from the second optimization step

Coefficient

C,

Glucose Starch Peptone Phosphate ZnSO, CaCI, Vitamins

5.84 4.61 8.41 -1.34 0.463 0.588 -0.36 0.024 -0.012 -0.019 -0.015 0.007 1 -0.018 -0.0051

0.33 -0.99 -2.17 0.24 -0.030 1.36 -0.568

of (NH&S04, ZnSO,, or vitamins has a negative effect on biomass dry weight. A change in the amount of glucose and starch does not have a strong effect on nuclease Pl and specific activity. Peptone and (NH&SO, have a strong negative effect on nuclease Pl activity and specific activity while both ZnSO, and CaCl, have a positive effect on these two parameters, Vitamins have the most positive effect on specific activity. As for the influence of pH on nuclease Pl activity and specific activity, it can be seen from Table 3 that trials 4-8 have a pH which is either too low or too high. Nuclease Pl activity and specific activity are relatively low, which implies the importance of controlling the pH value of the medium during fermentation.

Second optimization step

In the second optimization step the dosages of constituents are adjusted based on the above results. The dosages of glucose and starch were set at an intermediate level. Peptone was adjusted to a level below the low level. Ammonium sulfate was omitted in this step. The dosages of both ZnSO, and CaCl, were further increased. In order to estimate the effect of buffer capacity of the medium on nuclease Pl activity and specific activity, phosphate was utilized to re- place ammonium sulfate as substrate constituent in this step.

The setup of the second optimization step is shown in Table 5. The FeSO, level remained the same in all eight runs as it was in the first optimization step. The calculation of coefficients was performed the same way as for the first optimization step.

The results of the second optimization step are shown in Table 6. The calculated coefficients C,, Cp, and C, are shown in Table 7.

In the second optimization step, nuclease Pl activity is largely increased. Compared with an average activity of 0.112 in the first optimization step, the value increases to 0.7 13. The mean value of specific activity, compared with a value of 3.41 for the first optimization step, increases to 8.41. From these data, it can be concluded that the concentrations of substrate constituents in the second optimization step are mostly optimized. The pH values in the second optimization step were all within the range of nuclease Pl stability. This indicates that the buffer capacity of the medium keeps the pH within the range of nuclease Pl stability.

From the coefficients given in Table 7, it can be concluded that glucose has a positive effect on biomass, nuclease Pl activity, and specific activity; consequently its dosage can remain at the high level for the following comparison experiment. Although starch shows a positive effect on biomass, it should be kept at a low level because of its negative effect on nuclease Pl and specific activity. Pep-

tone, although necessary as a nitrogen source for fungal growth, should be kept at a low level due to its negative effect on nuclease Pl activity and specific activity. Phos- phate and other constituents can be kept between the high and the low levels.

The results of the second optimization step were used to compose the medium for comparison experiments.

400 The PUF system

300

200

100

0

$ ,,i! 1 20

0.00 I, I I I

0 24 48 72 96 120

Fermentation time (h)

Figure 1 Nuclease Pl production in three fermentation systems

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Comparison experiments

TO compare the PUF process with the SSF of wheat bran and SmF, the medium used had the following composition (100 g-’ PUF): 25.0 g glucose, 10.0 g soluble starch, 7.40 g peptone, 3.70 g K,HPO,, 3.70 g KH,PO,, 0.618 g CaCl,, 29 mg FeSO,, 99 mg ZnSO,, 4.10 mg thiamin, 1.83 mg ribo- flavin, 136 mg nicotinic acid, 6.37 mg pyridoxine hydro- chloride, 7.26 mg a-tocopherol, 588 pg folic acid, 10.9 mg pantothenic acid, and 63.7 pg biotin. The water content of the impregnated PUF was adjusted to an amount satisfying a water activity of 0.97.

In comparing nuclease PI productivity in the PUF process with the SSF of wheat bran and SmF, enzyme productivity is expressed on the basis of substrate dry weight. The course of nuclease Pl production by Penicillium citrinum in the different fermentation systems is shown in Figure 1. Nuclease Pl productivity was largely increased after medium optimization compared with the productivity before optimization,4 in the SSF of wheat bran, and in SmF. The trend toward a decrease in nuclease PI activity disappears after medium optimization. In Table 8, nuclease Pl activity and specific activity after 120 h of fermentation are listed. The productivity of the PUF process using an optimized medium is about 9- and 500-fold higher than for SSF of wheat bran and SmF, respectively. Nuclease Pl activity and specific activity in the PUF process are approximately 4- and 20-fold higher than for SSF of wheat bran and SmF, respectively.

These results demonstrate that the PUF process utilizing an optimized medium results in both higher productivity and specific activity than the SSF of wheat bran and SmF. Compared with SmF, the PUF process produces relatively little waste because SmF requires a large quantity of fermentation broth and yields only a very low concentration of product. This can be a limiting parameter if downstream processing is the cost-determining factor. Compared with the SSF of wheat bran, the conversion of substrate to the desired product in the PUF process can be maximized because the medium is optimized. Consequently, a greater purity of the product can be obtained by only using the constituents necessary for fungal growth and product for- mation, while the composition of natural solid substrate such as wheat bran cannot be changed. Furthermore, in the

Table 8 Nuclease PI activity and specific activity after 120 h fermentation in the PUF process, SSF of wheat bran, and SmF

Process

Nuclease PI activity Specific activity

(E,,, 9-l E 260 mg-’ dry substrate) protein)

PUF process 430 58.9 SSF of wheat bran 49.2 13.9 SmF 0.818 2.92

case of the PUF process, the inert material (PUF) can be used repeatedly while the SSF of wheat bran will result in large quantities of unusable solid wastes.

In principle, in order to get high productivity and specific activity of certain valuable metabolites of fungi that are more suited for the SSF process than for the SmF process, the technique of using an inert carrier has potential. In ad- dition to the benefit of medium optimization which results in higher productivity and specific activity, the PUF process has many other advantages over the conventional SSF process. For instance, a much better environment for gas and heat transfer can be expected, easier filtration of extract with a low viscosity can be achieved, and a higher product purity and the possibility of a continuous or semi-continuous process is provided.

References

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Mitchell, D. A. and Lonsane, B. K. Definition, characteristics, and potential. In: Solid substrate cultivation (Doelle, H. W., Mitchell, D. A., and Rolz, C. E., Ed%). Elsevier, London, 1992, 1-16 Fujishima, T.. Uchida. K. and Yoshino, H. Enzyme production by molds in sponge culture. J. Ferment. Technoi. 1972, SO,726730 Meyrath, J. Production of amylase on vermiculite by Aspergillus rwyza~. J. Sci. Food Agrir. 1965. 16, 14-18 Zhu, Y.. Smits. J. P., Knol, W. and Bol, J. A. novel solid-state fermentation system using polyurethane foam as inert carrier. Biofech- rtol. I.&t. 1994. 16, 643-648 De Meo. M., Laget, M., Phan-Tan-Luu, R., Mathieu, D. and Dumknil. G. Application of experimental designs for optimization of medium and culture conditions in fermentation. Bio-Sci. 1985, 4,99-102 Christen, P. and Raimbault, M. Optimization of culture medium for aroma production by Ceratocystis jmbriuta. Biotechnol. Mt. I99 1. 13, 521-526 Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976. 72, 248-254

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