JOURNAL OF BIOSCIENCE AND BIOENGINEERING

Vol. 93, No. 1,25-30.2002

Effect of Seed Culture on Solid-State Bioconversion of Wheat Straw by Phanerochaete chrysosporium

for Animal Feed Production SHUBHAYU BASU,’ RAJNEESH GAUR,’ JAMES GOME&‘*

T. R. SREEKRISHNAN,’ AND VIRENDRA S. BISARIA’

Department of Biochemical Engineering & Biotechnology, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi-110 016, India’

Received 28 May 2001 /Accepted 17 October 200 1

The solid-state bioconversion of wheat straw by Phanerochaete chtysosporium the produc- of animal was studied. study was based on central composite

mental design. conditions of seed culture suitable for induction of lignino- lytic of the when the culture is used for bioconver- sion wheat straw, determined. When seed culture an initial of 5.8 grown under conditions at rpm in flasks at it was to give deg- radation 19.5% and degradation of A time study of solid-state bio-

of wheat indicated that highest lignin lowest cellulose levels occurred the sixth of cultivation. desirability coeff%zient this process passed through maximum of on the day.

[Key animal feed, degradation, Phanerochaete pellet morphology]

of the of the bioconversion (SSB) lignocellulosic residues white rot is to

these renewable to more feed for nants. Lignin in these is interspersed hemicelluloses forming matrix that the orderly

microfibrils and their digestibility. removal of from the residues using

chemical or treatment enhances di- gestibility these materials. treatment is able over methods because is specific lignin, produces harmful by-products has low requirements.

In study, Phanerochaete ATCC 24725 employed for bioconversion of straw. p

has been studied and known to high rates lignin degradation This white

fungus produces major enzyme lignin peroxi- (Lips), manganese-dependent (MnPs)

and all of are responsible the biodeg- of lignin 3). In chrysosporium, lignin

degraded only secondary (idiophasic) which is by limiting concentrations be-

2 mM Addition of to cultures prior to expression of activity delayed induction of

degradation, suggesting NH,+ interfered the synthesis the enzyme (4). It evident, therefore,

the lignin of lignocellulosic by l? might be if the culture used

Corresponding author. gomes@dbeb.iitd.emet.in +91-11-659 6134, l-659 6145 +91-l l-686

25

for has already through the growth phase.

a study the effect the growth white rot on wheat by solid-state Moyson and

(5) found digestibility improved in- creasing degradation. They that the bility correlated with lignin Using Pleu-

sajor-caju, a in lignin from 12.6% 5.6% improved digestibility of straw from

to 59.2% a period 12 weeks. p sajor-caju hemicellulose and behind most the cellulose an energy for the

For large-scale of animal it is to achieve lignin degradation minimum cel-

utilization (6). achieve this it is neces- sary determine the of inoculum prepa- ration submerged cultivation which the cul- ture p chyosporium the most induction of activity when to the substrate. Furthermore, is very that the culture is the form individual and pellets as

to mycelia, uniform inoculation the moist straw substrate. the effect the condi-

of submerged on the number and cell weight the pellets I? chrysosporium stud-

ied. effect of characteristics, which turn de- on the of seed preparation by

merged cultivation, the SSB wheat straw terms of and cellulose and crude content

was The results a series statistically de- experiments to the integrated of the

26 BASU ET AL.

culture (prepared by submerged cultivation) and the SSB of wheat straw are presented in this paper.

J. BIOSCI. BIOENG.,

MATERIALS AND METHODS

Methodology and experimental design Considering process and economic requirements of the large-scale bioconversion of lignocellulosic residues, an integrated procedure for experiments was adopted to determine the optimum conditions. The spores of P chrysosporium ATCC 24725 were grown under specified sub- merged conditions and the harvested pellets were used to inoculate wheat straw for SSB. Clearly, the residual carbon and nitrogen concentrations in the medium as well as the diameter, number and dry cell weight (dew) of the fungus at the time of harvest would affect the degradation of lignin in wheat straw during SSB. To study this lignin degradation capability, a series of experiments based on the central composite method were designed and per- formed. Each experiment consisted of two stages. In the first stage, l? chrysosporium was grown according to the procedure discussed in the section on preparation of seed culture. In the second stage, the culture (pellet form) was harvested and used for inoculating the wheat straw. The details of the procedure are described in the sec- tion on the SSB of wheat straw. In total, the experimental design comprised 20 experiments that were carried out to obtain the sur- face response. Three variables, agitation speed (rpm), temperature (“C) and pH, were examined at three different levels resulting in 6 star points (a= 1.682) and the center point was repeated 6 times. For the submerged culture, five responses were measured to char- acterize the culture, namely, pellet diameter (mm), pellet number (qualitative), dry cell weight (g/l), residual glucose (g/Z) and re- sidual nitrogen (g/r). To characterize the bioconversion of wheat straw, lignin degradation (%), cellulose degradation (%) and crude protein content (%) were the responses measured. The responses for the submerged cultivation (stage 1) were measured only once at the end of 62 h, whereas, for the bioconversion of wheat straw, the three responses were measured on day 1 and day 3 to day 8. The day-2 responses were not measured because it was observed that in general the changes in the responses between day 1 and day 2 were insignificant.

The models selected for describing the surface response were based on the regression coeffkient R2 and ANOVA (analysis of variance). In most cases a partial cubic model gave the best tit in which some of the cubic terms of the general equation

were not used to ensure that all the parameters could be estimated independently and that only those cubic terms were included which improved the degree of fit.

Since the number of variables is three and their effect on the SSB is interactive, it is necessary to perform the analysis in two steps. A predictive model for the response of a set of experiments was first obtained by performing an analysis of variance and then a desirability index for each response was evaluated based on the prediction using a statistical software (Stat-Ease Inc., USA). The individual desirability indices were then used to construct the combined objective function called the desirability function 6, which is the geometric mean of all the transformed responses and is given by (7)

S=(d,xd,x xdj where 4’s are values obtained by transforming the measured response based on the desired goal. Hence, when the goal is to maximize a certain response, the di values are defined as

where wi is the weight index and yy and y?” are the maximum and minimum values of the responses used for calculating 6. When the goal is to minimize a response, the di values are defined as

Since the di values are in the range O<d$ 1, the desirability co- efficient is also in the range 0165 1. The index n equals Ci wi. The desirability coefficients reported here are based on the di values computed for lignin degradation (d,), cellulose degradation (d,) and crude protein content (d,). The goals considered were maxi- mizing lignin degradation and crude protein content and minimiz- ing cellulose utilization. The weights wi have values of 5, 4, and 3 for 4, d2 and d3, respectively, indicating their relative degree of importance. Clearly, n equals 12. Using this procedure, d, is 0.2887, d, is 0.4745 and d3 is 0.110 for the sixth day data and it re- sults in a value 6 of 0.705 shown in Fig. 1.

Preparation of seed culture Fifty milliliters of medium con- sisting of 10 g/Z dextrose, 3 8/Z yeast extract, 0.5 g/Z (NH,),SO,, 0.02 g/Z KH,PO,, 0.02 g/Z K,HPO, and 0.01 g/Z MgSO, were pre- pared in baffled 250-ml Erlenmeyer flasks in duplicate. Each flask was inoculated with 4 x 1 O5 spores taken from a stock suspension containing 1.8 x 1 O6 spores per ml and incubated in a rotary shaker. After 62 h, the content of one flask was used to analyze the re- sponses for submerged cultivation while the other was used to in- oculate the wheat straw for SSB. The details of optimization of the medium constituents, initial spore count and the incubation time are presented elsewhere (Basu, S., M., Tech. Thesis, IIT Delhi, India, 2000).

Solid-state bioconversion (SSB) of wheat straw Seventy five grams of wheat straw cut into pieces, l-2 cm long, were sterilized for 40 min at 12 1 “C in polypropylene trays (290 mm x 240 mm x 50 mm). After sterilization, the wheat straw was aseptically mixed with 50 ml of seed culture. The final moisture content in the trays was 80% (w/w). A cover consisting of a thin uniform layer of cot- ton sandwiched between two layers of cheesecloth was used to cover the trays. This cover maintained the sterility while allowing the diffusion of gases from the tray. Samples were taken from the tray at the start of the experiment and every day starting from the second day up to the eighth day. These samples were then dried, ground and sieved through a mesh of 53 pm average pore size. The sample passing through this mesh size was used for analysis of crude protein, lignin and cellulose.

0.8

0.3 L I

2 3 4 5 6 7 8

Time(d)

FIG. 1. Variation of the desirability coefficient 6 with the duration of SSB of wheat straw.

VOL. 93,2002 BIOCONVERSION OF WHEAT STRAW TO ANIMAL FEED 27

TABLE 1. Details of central composite experimental design for solid-state bioconversion of wheat straw using P chryso.sporium

Expt. Agitation speed Temperature Expt. Agitation speed Temperature no. @pm) (“Cl PH no. @pm) (“Cl PH

1 102 30 5.80 11 121 34 5.30 2 153 34 5.30 12 121 34 5.30 3 121 34 5.30 13 102 30 4.80 4 140 30 4.80 14 102 38 4.80 5 121 34 5.30 15 121 34 5.30 6 89 34 5.30 16 140 30 5.80 7 121 27 5.30 17 121 34 4.50 8 140 38 4.80 18 121 41 5.30 9 121 34 6.10 19 102 38 5.80

10 140 38 5.80 20 121 34 5.30

The conditions for submerged seed culture preparation and subsequent bioconversion of straw were identical.

Analytical methods Lignin was measured by the acetyl bro- mide soluble lignin assay (8) while cellulose was measured by the anthrone calorimetric method (9). Nitrogen content was estimated by Kjeldahl’s method and crude protein was assumed to be equal to 6.25 times the nitrogen content (10).

RESULTS AND DISCUSSIONS

In Table 1, we present the details of the experimental design. It shows the values of the variables in each of the experiments based on the central composite design (11). Examples of other methods that may also be used for ob- taining a surface response are a factorial design (12), the Box-Wilson design (13) and the Box-Behnken design (14). In this study, the high and low values for each variable were chosen based on the results of several preliminary experi- ments. The responses of the experiments were determined in terms of the five parameters, namely, pellet diameter, pel- let number, dry cell weight, and residual glucose and nitro- gen concentrations at 62 h. The response of the second stage of the experiment, consisting of the SSB of wheat straw, was measured in terms of the degradation of lignin and cel- lulose, and crude protein content. These measurements were then used to determine the performance of bioconversion in terms of the desirability coefficient 6.

For the first phase of the experiments, a high value of the coefficient 6 implies that the seed culture prepared by sub- merged cultivation had a high dry cell weight value and rel- atively small pellets. It also indicates that the residual nitro- gen and glucose concentrations were low. For the second phase of the experiments, a high value of the coefficient 6 indicates high lignin degradation accompanied by low cel- lulose degradation and a high yield of crude protein. These values are bounded within the range defined by the actual maximum and minimum values achieved for each of the re- sponses. One may infer from the definition of the desirabil- ity coefficient 6 given in the Materials and Methods section and the requirements for the SSB of wheat straw (5) that a high value of 6 not only means high digestibility but also in- dicates the overall suitability of the wheat straw as animal feed. The results obtained are summarized in Figs. 2 and 3.

For the seed culture preparation, a single 6 value was ob- tained for the entire set of experiments. However, for the SSB of wheat straw carried out in trays (second phase of the experiments), it was possible to compute the coefficient F

for all the days when samples were taken. When the desir- ability coefficient for each day was calculated, it was ob- served that the highest value of 6 was obtained for the sixth day (Fig. 1).

A polynomial expression was used for describing the sur- face response obtained for the parameters. All the data had an adequate degree of precision statistically. A cubic model was required for describing the lignin data whereas a qua- dratic model was found suitable for the cellulose and crude protein data. The overall level of confidence in the data was better than 90%. This is reasonable considering the prob- lems usually faced with sampling. The predicted responses using these models are used to determine the conditions for growing the seed culture and subsequent SSB, which results in the most desirable bioconversion. For the seed culture preparation, the surface responses for pellet diameter, resid- ual nitrogen and glucose concentrations, cell dry weight and the desirability coefficient 6 are presented in Fig. 2. Simi- larly, for the SSB of wheat straw, the lignin and cellulose degradation, crude protein content and the desirability co- efficient 6 are presented in Fig. 3.

Metz and Kossen (15) identified agitation, pH and oxy- gen tension as important parameters, which, among others, influenced the formation of fungal pellets. They reported a decrease in pellet diameter with increasing agitation inten- sity. Our results are in agreement with these findings. In the submerged cultivation of P chrysosporium carried out in shake flasks, we observed the influence of temperature, agi- tation speed and pH not only on pellet diameter but also on pellet number, dry cell weight, and residual glucose and ni- trogen concentrations. Since we also studied the bioconver- sion capability of cultures grown under various conditions, it was desirable to obtain a large number of small diameter pellets so that it would be easy to uniformly distribute the culture over the mass of wheat straw. Clearly, a higher dry cell weight was also preferable as it increased the density of the fungus in the wheat straw. Furthermore, since low resid- ual glucose and nitrogen concentrations have been associ- ated with high ligninase and cellulase activity, obtaining a culture with low concentrations of these constituents at the time of harvesting was desirable.

The surface response results obtained from the experi- mental data of the central composite design are presented in Figs. 2a to 2e. The predicted condition most suitable for growing the seed culture of P chrysosporium was an agita-

28 BASU ET AL. J. BIOSCI. BIOENG.,

00

FIG. 2. Effect of variation of speed culture conditions (agitation speed and temperature) at a constant pH of 4.8 on (a) desirability coeffkient, (b) dry cell weight, (c) pellet diameter, (d) residual nitrogen concentration and (e) residual glucose concentration.

tion speed of 130 rpm, incubation temperature of 37°C and pH of 4.8. Submerged cultivation under these conditions meets the requirement of small pellet diameter, high dry cell weight and minimum residual glucose and nitrogen concen- trations to a satisfactory degree. The desirability coefficient 6 for this prediction was 0.943 and indicates that the char- acteristics of the submerged seed culture come close to the

pre-specified requirements. These observations are in good agreement with the temperature of 37°C and pH of 5.0 previously reported by Abdullah et al. (lo), and a tempera- ture of 39°C and pH of 4.5 reported by Kirk et al. (16) and by Faison and Kirk (17) for R chrysosporium. If the fungus is grown under the optimal conditions determined in this study, the expected pellet diameter is 1.05 mm, dry cell

VOL. 93,2002 BIOCONVERSION OF WHEAT STRAW TO ANIMAL FEED 29

(c) 00 FIG. 3. Effect of variation of speed culture conditions (agitation speed and temperature) at a constant pH of 5.8 on (a) desirability coefficient,

(b) cellulose degradation, (c) lignin degradation and (d) crude protein content when used for the SSB of wheat straw.

weight 4.5 gfl, residual glucose concentration 0.15 gll and residual nitrogen concentration 2.13 mgll. However, if the wheat straw is inoculated with a seed culture grown under these conditions, only 10.85% lignin is degraded. Simulta- neously, about 36% cellulose is consumed accompanied by a crude protein production of 4.45% on a dry weight basis. Consequently, the desirability coefficient 6 of the process is only 0.5.

Bioconversion of lignocellulosic residues for animal feed needs to satisfy the condition that the highest lignin degra- dation level is achieved with minimum utilization of cellu- lose. If excess cellulose is utilized, the energy content of the lignocellulosic residues decreases significantly which is not desirable. One of the main purposes of bioconversion is to make the residues susceptible to further degradation, so that the energy contained in cellulose is made available. In its native form, this energy is not easily available due to the presence of lignin. The optimum was determined with this objective in mind. High lignin degradation and crude pro- tein content are usually accompanied by high cellulose deg- radation. The surface response shows that if the wheat straw is inoculated with seed grown under conditions of 130 rpm

agitation speed, 38°C incubation temperature and initial pH of 5.8, the predicted lignin degradation is as high as 19.5% and cellulose degradation is only 17.8%. This prediction is associated with a desirability coefficient 6 of 0.705, which is acceptable. Under these conditions, it is predicted that the seed culture would produce pellets of 1.43 mm diameter, a final cell dry weight of 3.35 g/l, a residual glucose concen- tration of 0.78 gll and a residual nitrogen concentration of 1.67 mg/Z. Although we compromised on the seed culture characteristics when we grew the fingus at a higher pH value of 5.8, these were within acceptable margins.

Figure 2(c) shows a linear relationship between pellet di- ameter, agitation and temperature. Since the maximum dry cell weight was achieved at about 125 rpm and 37”C, this indicates that although at higher agitation speeds smaller pellets were obtained, the shear was detrimental to growth. Perhaps the attrition between individual particles becomes significant close to 135 rpm. In observations independent of pellet diameter and dry weight, we noted that under the same conditions of temperature and agitation, increasing the pH from 4.8 to 5.8 results in the production of more fluffy pellets. In addition, an increasing tendency of mycelial

30 BASU ET AL. J. BIOSCI. BIOENG.,

growth of the fungus was observed as the pH approached 5.8. The pellet number was recorded only qualitatively. The most desirable situation in this study, namely, high pellet number and minimum diameter, was obtained at a tempera- ture of 38°C an agitation speed of 140 rpm and a pH value of 4.8.

5.

6. Another important consideration in SSB for the produc-

tion of animal feed is to determine the time of harvesting so that cellulose utilization by fungi is within acceptable limits. The desirability coefficient for each day, starting from the second to the eighth day, was ascertained and is plotted in Fig. 1. The highest desirability coefftcient was obtained for the sixth day. Therefore, wheat straw harvested after 6 d of SSB using R chrysosporium is suitable for use as animal feed.

7.

zyme system of Phanerochaete chrysosporium: synthesized in the absence of lignin in response to nitrogen starvation. J. Bacterial., 135,79&797 (1978). Moyson, E. and Verachtert, IL: Growth of higher fungi on wheat straw and their impact on the digestibility of the sub- strate. Appl. Microbial. Biotechnol., 36,421424 (1991). Jung, H. G. and Vogel, K. P.: Influence of lignin on the di- gestibility of forage cell wall material. J. Animal Sci., 62, 1703-1707 (1986). Derringer, G. and Suich, R.: Simultaneous optimization of several response variables. J. Qualitv Technol., 12, 214-219

8. (1980). -

. _

Morrison, I. M.: A semi-micro method for the determination of lignin and its use in predicting the digestibility of forage

Lignin degradation is a strongly oxidative process and aeration and oxygen transfer are essential for lignin degra- dation (18). Bar-Lev and Kirk (19) have shown that in SSB, increased oxygen concentration stimulates the synthesis of the lignin degrading enzymes in I? chrysosporium. Our SSB experiments using wheat straw were performed in trays where there was no forced air circulation. The only mode of aeration was diffusion through the porous cover. Thus, it is very probable that higher lignin degradation will be achieved under the same conditions in reactors where aera- tion is provided. Development of suitable large-scale (200 I) reactors with aeration and other control features for the bio- conversion of wheat straw is in progress in our laboratory.

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ACKNOWLEDGMENT

The authors gratefully acknowledge the financial support pro- vided by the Department of Biotechnology, Ministry of Science & Technology, Government of India.

15.

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