Operating conditions of a 200 l staged vertical reactor for bioconversion of wheat straw by...

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Bioresource Technology 99 (2008) 6917–6927

Operating conditions of a 200 l staged vertical reactor forbioconversion of wheat straw by Phanerochaete chrysosporium

Ankur Bhatnagar a, Sanjay Kumar b, James Gomes b,*

a Biocon Ltd., 20th KM, Hosur Road, Electronics City, Bangalore 560 100, Indiab Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110 016, India

Received 2 November 2007; received in revised form 14 January 2008; accepted 16 January 2008Available online 7 March 2008

Abstract

Bioconversion of wheat straw using Phanerochaete chrysosporium was carried out in a 200 l staged vertical reactor. The bioconversionprocess was characterized by measuring the percentage degradation of lignin and cellulose, and increment in crude protein content. Theeffect of airflow rate, inoculum amount and wheat straw loading on bioconversion was investigated using a statistical experimentaldesign. An analysis of variance was performed to determine response surfaces. The quality of bioconversion indicated by an optimizationindex called the desirability coefficient had the highest value of 0.75 for the fifth day of cultivation. This corresponded to an operatingcondition of 1.5 kg wheat straw per stage using an inoculum amount of 0.38 g (100 g dry wheat straw)�1 and an airflow rate of15 l min�1. The lignin and cellulose degradation achieved at this operating condition was 27% and 29%, respectively. A ratio of 3 forthe weight of wheat straw to inoculum amount gave the highest crude protein of 5.9% on dry weight basis. Among the variables inves-tigated, the airflow rate exhibited a significant effect on the quality of bioconversion. Our results indicate that the quality of bioconver-sion may be controlled by implementing a predetermined airflow rate schedule.� 2008 Elsevier Ltd. All rights reserved.

Keywords: Staged vertical reactor; Airflow rate; Lignin degradation; Cellulose degradation; Desirability coefficient

1. Introduction

Lignocellulosic compounds are the most abundantrenewable organic matter and a key ingredient in the devel-opment of sustainable processes and products. Crop resi-dues constitute about 123 � 106 tons per year of thisrenewable resource, containing approximately 60% cellu-lose and hemicellulose, and 30% lignin on a dry mass basis.Lignin is essentially a three dimensional phenylpropanepolymer having extensive cross-linked ether and carbon–carbon double bonds. The presence of lignin limits the uti-lization of crop residues in bioconversion processes, such asproduction of alcohol as a renewable energy source andproduction of animal feed (Tengerdy and Szakacs, 2003;

0960-8524/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.biortech.2008.01.031

* Corresponding author. Tel.: +91 1126591013; fax: +91 1126582282.E-mail address: [email protected] (J. Gomes).

Wyman, 2007). The production of animal feed from cropresidues finally translates into the generation of food man-ufactured from milk and meat, and is consequently animportant industry. Therefore, the development of pro-cesses to improve the nutritional quality and digestibilityof lignocellulosic residues are important (Robinson andNigam, 2003; Villas-Boas et al., 2002; Leoniwicz et al.,1999; Zadrazil and Isikhuemhem, 1997).

The digestibility of lignocellulosic residues may beimproved by employing physical, chemical and biologicalmethods of removing lignin. Since many of the physicaland chemical methods are energy intensive and utilizechemicals that contribute to environmental hazards, micro-bial methods have an advantage where these issues are ofserious concern. Phanerochaete chrysosporium is a rapidlignin degrader and has been widely studied for its abilityto degrade lignin. This fungus, when deprived of essentialnutrients such as carbon, nitrogen or sulphur (Keyser

mailto:[email protected]

Nomenclature

A operating point on the inoculum versus wheatstraw loading plane

B operating point on the inoculum versus wheatstraw loading plane

C operating point on the inoculum versus wheatstraw loading plane

d1 transformed lignin degradation for computingdesirability coefficient

d2 transformed cellulose degradation for comput-ing desirability coefficient

d3 transformed increment in crude protein for com-puting desirability coefficient

di values obtained by transforming the measuredresponse based on the desired goal

LiP lignin peroxidases

LOM loss of organic matterMnP manganese peroxidasesrpm revolutions per minutewi weight indexx1 wheat straw per stagex2 inoculum amountx3 airflow rateymax

i maximum value of the responses used for calcu-lating d

ymini minimum value of the responses used for calcu-

lating d

Greek symbol

d desirability coefficient

6918 A. Bhatnagar et al. / Bioresource Technology 99 (2008) 6917–6927

et al., 1978), secretes lignin peroxidases (LiP) and manga-nese peroxidases (MnP) (Martin et al., 1999). These seriesof isoenzymes are central to the lignin-degrading systemof P. chrysosporium (Arora et al., 2002; Malherbe and Clo-ete, 2002; Mansur et al., 1997). Although structurally sim-ilar, these two heme-containing peroxidases exhibitdifferent substrate specificity. The secreted ligninases serveto break down the lignin sheath and expose the cellulosicstructures within. P. chrysosporium also secretes cellulolyticenzymes. The combined action of these enzymes results inthe utilization of agro residues as carbon and energysource. Hence, lignin removal by white rot fungi is alwaysaccompanied with some loss of cellulose.

The ligninolytic system of P. chrysosporium is non-spe-cific. The degree of lignin removal depends greatly on thespecies, the substrate and on several culture parameterssuch as nitrogen source and concentration, temperature,pH, moisture content, carbon dioxide and oxygen level,and mixing (Zadrazil and Brunnert, 1981). It is reportedthat CO2 is inhibitory while oxygen stimulates lignin degra-dation (Kamara and Zadrazil, 1988). Similarly, poor ther-mal conductivity of lignocellulosic substrates reduces therate of metabolic heat exchange; this in turn causes localhot spots, which affects lignin degradation. Consequently,the rate of aeration, mixing, substrate loading and amountof inoculum culture influences the process of lignin degra-dation (Breen and Singleton, 1999; Lopez et al., 2002).

Lignocellulose substrate has been used for the produc-tion of enzymes, secondary metabolites, biopulp, biofuelsand animal fodder (Tengerdy and Szakacs, 2003; Sabuet al., 2006; Roopesh et al., 2006). In each of these pro-cesses both lignin and cellulose is utilized by the fungusfor growth and energy requirements. Reactors in whichthese processes are carried out are three phase reactorswhere the kinetics and transport of various constituentsoccur across solid, liquid and gaseous phases. These pro-cesses have been modeled mathematically to describe the

underlying phenomenon and for predicting the behaviorunder existing constraints (Mitchell et al., 2004; Hamidi-Esfahani et al., 2004). However, problems related to theoperation and control of the solid-state fermentation hasnot been addressed fully. There also exists a wide scopefor improvement in instrumentation of solid-state fermen-tation processes (Bellon-Maurel et al., 2003; Khanahmadiet al., 2006). A recent review on laccase production showsthe volume of the reactors used range from 300 ml to 20 l(Couto and Toca-Herrera, 2007). The scaling of these pro-cesses to larger volumes is required to determine if the pro-cesses are economically viable. Data even at the pilot scalethat essentially states the conditions of operations of thesereactors is lacking.

In the case of animal feed production, there is concernfor minimizing the consumption of cellulose by the micro-organism so that the energy value of the lignocellulose isretained as far as possible. The process needs to be simple,either semi-aseptic or non-aseptic and easy to adopt byunskilled farm hands. Hence, what is really required is arelation between operating (manipulated) variables, suchas the airflow rate and weight of raw material, and thequality of the final product.

In this paper, the determination of operating conditionsbest suited for the bioconversion of wheat straw to animalfeed carried out in a 200 l staged vertical reactor is pre-sented. The optimum humidity and temperature for thisbioconversion was previously determined (Basu et al.,2002) and, in this study, these variables are controlled atthese values using digital controllers. Experiments wereperformed based on a statistical design to study the effectof wheat straw loading, inoculum amount and airflow rate,on the bioconversion of wheat straw. The bioconversionwas quantified in terms of the lignin and cellulose degrada-tion, and increment in the crude protein content. The timefor batch processing was determined from the time profilesof degradation where lignin degradation was the highest

A. Bhatnagar et al. / Bioresource Technology 99 (2008) 6917–6927 6919

and cellulose degradation was the lowest. A discussion ofthe results is presented.

2. Methods

2.1. Microorganism

P. chrysosporium (ATCC 24725) was used in the presentstudy. The culture was maintained on glucose agar slant(1% glucose, 0.3% yeast extract), stored at 4 �C and trans-ferred to fresh slants every month.

2.2. Seed culture preparation

A suspension of spores of P. chrysosporium was preparedin 2.0% glycerol solution and its concentration was adjustedto 4 � 105 spores ml�1. One milliliter of this suspension wasused for every 50 ml of the seed culture. Using this sporeconcentration, we obtained about 4 mg DW ml�1 of pelletbiomass at the end of 62 h of cultivation (Basu et al.,2002). For each bioconversion experiment, 8 l of culturemedium was prepared in a 15 l reactor (B-Braun BiostatC, Germany). The culture medium had the following com-position: 10 g l�1 dextrose, 3 g l�1 yeast extract, 3 g l�1

(NH4)2SO4, 0.02 g l�1 KH2PO4, 0.02 g l�1 K2HPO4, and

AirSupply

WaterSupply

Humidity and Temperature

Controller

Condensers

HT

HA

LA

LT

PI

Fig. 1. A schematic diagram (not to scale) of the 200 l staged vertical reactor dpasses through a pressure reducing station, is humidified and supplied throucontrolled by a microprocessor based on signals from the hold up tank, temperfrom the steam generator are use for sterilization of the wheat straw. - - - signatransmitter; HT, humidity transmitter; TT, temperature transmitter; PT, press

0.01 g l�1 MgSO4. The pH of the medium was adjusted to5.8 before sterilization. The reactor was inoculated with160 ml of spore suspension and the seed culture was grownat 38 �C, 600 rpm and 8 l min�1 airflow rate in the reactorfor 62 h.

2.3. Solid sate cultivation in 200 l reactor

A schematic diagram of the reactor is presented inFig. 1. Each stage of the solid-state reactor consists of aperforated plate provided with an independent air supplyline for aeration. A branch line from the main air supplyheader is shunted through a humidifier so that the relativehumidity of the air supplied to the reactor is maintainedabove 90%. Heat exchange in the reactor takes placemainly by convection and evaporative cooling. The humid-ity in the head space was controlled between 80% and 90%and average temperature on each stage was controlled at38 �C by a cascade PID control module (Decibel, India).

A measured amount of wheat straw of particle sizeapproximately 1 to 2 cm was taken in sterilizable polythenebags and mixed with a known amount of distilled water tobring the moisture level of wheat straw to approximately50%. The polythene bags containing wheat straw were auto-claved for 20 min at 121 �C. After cooling, the sterilized

200 Liter Reactor

TT

TT

TT

PI

PI

Air Exit

SteamGenerator

PI

esigned for bioconversion of wheat straw. The air from a supply header isgh a condenser manifold. The humidity of the air entering the reactor isature of each stage and humidity of air leaving the reactor. The steam linesl lines; — flow lines; HA, high level alarm; LA, low level alarm; LT, levelure transmitter; PI, pressure indicator.

Table 1Relation between coded and actual values of the variables used forevaluating reactor performance

Response variable Coded level of variable

�a �1 0 +1 +a

Wheat straw per stage (kg) x1 1.08 1.25 1.50 1.75 1.92Inoculum amount (g/100 g) x2 0.16 0.25 0.375 0.50 0.59Airflow rate (l min�1) x3 6.59 10.0 15.0 20.0 23.41


wheat straw was aseptically mixed with appropriate amountof the pellets of P. chrysosporium. The moisture content ofwheat straw was adjusted to 60% (wet basis) by adding dis-tilled water and loaded into the 200 l reactor. The bed heightof the wheat straw on each stage varied with the loading foran experiment. The airflow rate was maintained as per sche-dule of the experimental design. The reactor was operated at38 �C for 7 days and samples were collected through thesampling ports for analysis. At the end of the experiment,the content of each stage of the reactor was harvested.About 1.0 kg of treated wheat straw was weighed accuratelyand dried overnight in an oven at 85 �C. Its dry weight wasmeasured and loss of organic matter (LOM) determined.After each experiment, the reactor was cleaned with deter-gent and fumigated with formalin.

2.4. Analytical method

The treated wheat straw samples passing through 53 lmmesh was used for analysis of cellulose, lignin, hemicellu-lose, protein and ash content. Lignin was estimated byacetyl-bromide method given by Morrision (1972), cellu-lose was estimated by calorimetric method of anthrone

Table 2Analysis of various components of wheat straw after three days of solid-state

Expt.no.

Wt. of wheat straw perstage (kg)

Inoculum (g/100 g W. S.)

Airflow rate(l min�1)

Lignindegrad(%)

1 1.75 0.50 10.00 6.292 1.50 0.16 15.00 7.953 1.25 0.50 20.00 12.614 1.50 0.38 15.00 8.915 1.50 0.59 15.00 15.476 1.50 0.38 15.00 18.587 1.75 0.25 10.00 6.188 1.50 0.38 15.00 8.319 1.50 0.38 6.59 6.87

10 1.25 0.25 10.00 22.2811 1.75 0.25 20.00 4.7412 1.25 0.25 20.00 11.8813 1.08 0.38 15.00 13.3314 1.75 0.50 20.00 8.8615 1.50 0.38 15.00 13.1816 1.25 0.50 10.00 10.3817 1.92 0.38 15.00 9.3918 1.50 0.38 15.00 12.2519 1.50 0.38 15.00 7.9620 1.50 0.38 23.41 10.67

given by Updegraff (1969). The lignin and cellulose degra-dation was determined as described by Abdullah et al.(1985). Ash content was determined according to themethod (ID No. 942.05) of the AOAC (2000). Crude pro-tein was determined from Kjeldahl nitrogen determinationof whole unwashed samples of substrate and product. Pro-tein content was assumed to be 6.25 times of the nitrogencontent (Abdullah et al., 1985). Hemicellulose was deter-mined by the difference between the measurements of neu-tral detergent fibre (NDF) and acid detergent fibre (ADF)(Van Soest and Jones, 1968).

2.5. Experimental design

To determine conditions suitable for bioconversion ofwheat straw, the responses measured were lignin and cellu-lose degradation, and increment in protein content. Theother variables also measured were the total hemicelluloseand ash content. The degradation of lignin is directlyrelated to the digestibility of lignocellulosic residues (Guptaet al., 2008; Bach Knudsen, 2001; Haddadin et al., 2002),while cellulose, hemicellulose and protein content of thetreated material determines its nutritional quality. Themanipulated variables (factors) were the weight of wheatstraw per stage, the amount of inoculum per unit weightof the wheat straw and the airflow rate. These variableswere selected because they influence overall economics ofthe process.

To reduce the total number of experiments required todetermine suitable operating conditions for the reactor, acentral composite design was constructed with 5 repetitionsof the centre point. The design with actual factor values hasbeen presented with the results in Tables 2–4. The low and

bioconversion using Phanerochaete chrysosporium

ationCellulosedegradation (%)

Hemicellulose(%)

Crudeprotein (%)

Ashcontent(%)

4.33 23.89 4.64 13.417.30 23.86 5.83 15.336.89 25.55 4.92 16.98

17.86 28.29 5.12 15.8115.21 23.36 5.41 11.9317.49 28.85 4.96 11.4910.85 25.31 1.50 10.257.30 24.01 5.58 12.859.20 30.43 5.43 13.48

23.93 23.68 4.93 11.8110.19 24.50 4.32 16.958.22 25.66 4.82 4.089.25 28.32 5.18 10.947.96 25.48 5.26 15.43

10.32 29.11 5.56 14.2611.18 24.06 4.76 12.317.13 29.98 5.46 11.83

11.66 29.86 5.93 11.3815.51 29.23 5.58 17.439.65 27.11 5.46 12.20

Table 3Analysis of various components of wheat straw after five days of solid-state bioconversion using Phanerochaete chrysosporium

Expt.no.




Lignindegradation(%)

Cellulosedegradation (%)

Hemicellulose(%)

Crudeprotein (%)

Ashcontent(%)

1 1.75 0.50 10.00 14.86 19.25 22.58 5.63 13.482 1.50 0.16 15.00 15.11 16.28 22.43 5.61 13.493 1.25 0.50 20.00 15.64 24.91 23.11 4.89 15.324 1.50 0.38 15.00 27.91 28.98 26.59 4.58 12.305 1.50 0.59 15.00 21.21 22.29 21.96 5.81 11.326 1.50 0.38 15.00 29.25 31.71 26.18 5.48 17.327 1.75 0.25 10.00 9.45 18.95 24.11 1.15 10.868 1.50 0.38 15.00 16.67 19.71 22.86 5.72 16.679 1.50 0.38 6.59 14.04 15.86 28.16 5.13 11.19

10 1.25 0.25 10.00 28.94 31.85 23.63 4.68 16.4811 1.75 0.25 20.00 10.82 21.65 24.01 4.63 12.8612 1.25 0.25 20.00 17.23 22.91 23.42 4.01 11.9813 1.08 0.38 15.00 30.90 24.99 27.19 4.93 11.2314 1.75 0.50 20.00 19.80 42.77 27.37 4.32 12.8115 1.50 0.38 15.00 26.81 27.98 28.27 5.43 12.4816 1.25 0.50 10.00 13.13 23.59 22.23 4.87 13.9817 1.92 0.38 15.00 22.59 25.33 28.47 5.19 10.9418 1.50 0.38 15.00 28.83 30.62 28.44 5.69 12.4019 1.50 0.38 15.00 32.30 35.17 27.38 5.36 16.1120 1.50 0.38 23.41 23.60 30.01 25.18 5.71 10.46

Table 4Analysis of various components of wheat straw after seven days of solid-state bioconversion using Phanerochaete chrysosporium

Expt.no.




Lignindegradation(%)

Cellulosedegradation (%)

Hemicellulose(%)

Crudeprotein (%)

Ashcontent(%)

1 1.75 0.50 10.00 16.98 25.56 21.32 4.84 14.582 1.50 0.16 15.00 22.60 28.92 21.08 5.52 13.673 1.25 0.50 20.00 18.64 35.14 23.19 4.86 11.924 1.50 0.38 15.00 35.16 51.12 24.36 5.31 12.595 1.50 0.59 15.00 28.88 34.46 21.32 5.52 10.446 1.50 0.38 15.00 37.22 43.18 22.15 4.83 15.567 1.75 0.25 10.00 15.67 32.17 23.01 1.32 11.018 1.50 0.38 15.00 26.30 36.60 21.11 5.42 16.469 1.50 0.38 6.59 22.26 28.51 27.09 5.26 12.23

10 1.25 0.25 10.00 31.60 49.93 21.92 4.53 12.4611 1.75 0.25 20.00 13.17 30.19 22.00 4.93 12.8612 1.25 0.25 20.00 20.24 34.54 22.16 3.98 13.4213 1.08 0.38 15.00 35.13 41.46 26.23 5.69 10.4814 1.75 0.50 20.00 25.33 48.98 26.44 5.61 12.1015 1.50 0.38 15.00 34.07 38.10 27.46 4.98 11.0316 1.25 0.50 10.00 21.28 30.79 20.58 4.28 14.6617 1.92 0.38 15.00 26.67 36.06 27.19 5.39 11.4918 1.50 0.38 15.00 31.76 38.41 27.83 5.84 13.2919 1.50 0.38 15.00 34.81 50.36 25.81 5.68 14.3220 1.50 0.38 23.41 34.22 46.55 21.81 5.34 14.20


high value for each factor was decided based on the resultsfrom preliminary experiments. The low and high value forthe weight of wheat straw per stage was 1.25 kg and1.75 kg, for the amount of inoculum added per 100 g ofdry wheat straw it was 0.25 g and 0.50 g, and for airflowrate, it was 10 and 20 l min�1. The relation between codedand actual values is presented in Table 1. Quadratic andcubic models were used to describe the response surfaces.A complete analysis of variance was performed to validatethe empirical models that describe the response surfaces.

2.6. Determination of desirability coefficient

Primary variables measured to evaluate reactor perfor-mance were lignin and cellulose degradation and incrementin crude protein content because these variables are impor-tant to animal feed production processes. Hence, the com-bined effects of these three performance parameters wereconsidered in determining the quality of bioconversion.The analysis was performed in two steps. A predictivemodel for the response of performance parameters in the


bioconversion experiments was first obtained by an analy-sis of variance and then a desirability index for eachresponse was evaluated using statistical software (Stat-EaseInc., USA). The individual desirability indices were thenused to construct the combined objective function calledthe desirability coefficient d, which is the geometric meanof all the transformed responses and is given by (Derringerand Suich, 1980)

d ¼ ðd1 � d2 � � � � � dnÞ1n ð1Þ

where di’s are values obtained by transforming the mea-sured response based on the desired goal. Hence, whenthe goal was to maximize a certain response, the di valueswere defined as

di ¼yi � ymin

i

ymaxi � ymin

i

� �wi

ð2Þ

where wi is the weight index and ymaxi and ymin

i are the max-imum and minimum values of the responses used for calcu-lating d. When the goal was to minimize a response, the di

values were defined as

di ¼ymax

i � yi

ymaxi � ymin

i

� �wi

ð3Þ

One may look upon the di as the value of the responseon a new scale between zero and unity. The exponent(weighting factor) defines curvature of the interpolationequation. For example, when wi = 1, the interpolation islinear. Since the di values are in the range 0 6 di 6 1, thedesirability coefficient is also in the range 0 6 d 6 1. Theindex n equals

Piwi. The contour plot of desirability coef-

ficients reported here are based on the di values computedfor three variables, namely, for lignin degradation (d1), cel-lulose degradation (d2) and crude protein content (d3). Thegoals considered were maximizing lignin degradation andcrude protein content and minimizing cellulose utilization.The weights wi have values of 1, 4.5 and 3 for d1, d2 and d3,respectively. Hence, the desirability coefficient d is the geo-metric mean of the di’s with index n equal to 8.5. Theweight d1 gives a uniform importance to all lignin degrada-tion data. The weights d2 and d3 gives less importance todata that does not minimize cellulose degradation or max-imize increment in crude protein. The desirability coeffi-cient d was computed in this fashion and the contours ofequal d values were plotted.

3. Results and discussion

The production of animal feed in reactors through bio-conversion of lignocellulosic residues should be translat-able to low-end technology. The operation of the reactormust be made as simple as possible so that no difficulty isfaced in its execution by farm hands. Therefore, a clearrelationship between the operating condition and qualityof end product must be established. We have performeda series of experiments in a 200 l staged vertical reactor,

where the wheat straw loading, inoculum amount and air-flow rate was varied and the quality of bioconversion wasdetermined. A schematic diagram of the reactor showingthe control loops, instrumentation and signal lines, acces-sory utilities layout and piping is presented in Fig. 1. Thehumidity in the reactor was controlled between 80% and90% and average temperature on each stage was controlledat 38 �C (±1.0 �C). Samples were taken on day 3, 5 and 7for each of the experiments. The best results were obtainedon day 5 of the reactor operation. From an analysis of theresults, the operating condition for the most desirable bio-conversion was obtained. It was determined that a wheatstraw loading of 1.50 kg per stage, inoculum amount of0.38 g per 100 g of wheat straw and an airflow rate of15 l min�1 was the best operating condition for the reactor.The corresponding lignin and cellulose degradation was27% and 29%, respectively. The quality of bioconversionachieved was quantified in terms of the desirability coeffi-cient (Section 2.6). For this operating condition, the high-est value of 0.75 was obtained for the desirabilitycoefficient.

The results of the experiments carried out on day 3, day5 and day 7 are presented in Tables 2–4, respectively. Theseexperiments were designed using the central compositemethod (Box et al., 1978). The effects of three variables,namely, weight of wheat straw per stage (kg), inoculumamount (g (100 g dry wheat straw)�1) and airflow rate(l min�1), on the bioconversion of wheat straw were stud-ied. The high and low values for each variable were chosenbased on several preliminary experiments. Degradation oflignin and cellulose was measured; in addition the hemicel-lulose, protein and ash content were also determined.

The surface response was developed separately for day3, 5 and 7 and for each of the three responses. Reducedquadratic and cubic models were developed using statisticalanalysis for describing the response surface. The desirabil-ity coefficient d for each of the days was computed usingwheat straw loading and the inoculum amount as the inde-pendent variables. The variation in the contours of desir-ability coefficient d was then examined for different valuesof the airflow rate. From among these many cases studied,the condition giving the highest d value was selected.

3.1. Lignin and cellulose degradation

The lignin and cellulose degradation achieved throughbioconversion of wheat straw needs to be examinedtogether for the production of animal feed. The objectivehere is to obtain the highest lignin degradation with mini-mum utilization of cellulose so that the energy content ofthe product is not compromised (Kamara and Zadrazil,1988; Zadrazil et al., 1983; Villas-Boas et al., 2002). Theresponse surface of lignin and cellulose degradation forday 5 is presented in Figs. 2 and 3 for an airflow rate of15 l min�1. All the points on the surface of these figuresare candidates for the operating condition. These surfaceschange as the airflow rate changes.

15.017.520.022.525.027.530.0

Lign

in D

egra

datio

n (%

)

1.251.33

1.421.50

1.581.67

1.75

0.25

0.30

0.35

0.40

0.45

0.50

Wheat Straw (kg)

Inoculum(g/100 g)

A

B

C

Fig. 2. Lignin degradation response to variations in the amount of theinoculum and wheat straw loading in the reactor. The points A, B and Care the operating conditions at which the effect of airflow rate on ligninand cellulose degradation have been studied in detail.

182124273033

Cel

lulo

se D

egra

datio

n (%

)

1.251.33

1.421.50

1.581.67

1.75

0.25

0.30

0.35

0.40

0.45

0.50

Wheat Straw (kg)

Inoculum(g/100 g)

Fig. 3. Cellulose degradation response to variations in the amount of theinoculum and wheat straw loading in the reactor.


Consider the data for day 5 samples. We observed thatfor an airflow rate of 15 l min�1, the maximum lignin deg-radation of 30% occurs at point (0.33,1.25) in Fig. 2 andthe maximum cellulose degradation of 31% occurs at point(0.48, 1.75) in Fig. 3. These values are consistent with pre-viously reported literature (Nigam, 1990). Since the rates oflignin and cellulose consumption are different and becausetheir maxima conditions do not coincide, the time profiles

of degradation does not result in a unique solution. A sim-ilar analysis at the same airflow rate was performed for day3 and day 7 samples (Table 2 and 4). For day 3, the highestlignin and cellulose degradation obtained from theresponse analysis was 18.1% and 16.8%, respectively. Forday 7, the highest lignin and cellulose degradation obtainedwas 34% and 44%, respectively. In these cases also the max-ima do not coincide. Further, the magnitude of degrada-tion shows that for day 3, although lignin degradationhad begun, it was not substantial. On the other hand theresult for day 7 shows that a significant amount of cellulosepresent in the wheat straw had been utilized by the fungusand lower energy content of the treated material would notbe acceptable. Therefore, intermediate points were exam-ined to find the operating point.

The following procedure was adopted to narrow downthe search region. Since it is required that the lignin degra-dation is as high as possible, the line AC in Fig. 2 is takenas the basis for subsequent analysis and the airflow rate isscanned from 10 to 20 l min�1 to determine the operatingpoint. Over this region of the response surface, the desir-ability coefficient d values were scanned. The point havingthe highest d was selected as the operating point.

3.2. Increment of crude protein

The crude protein has been estimated indirectly (Section2.4). The surface response of crude protein was also exam-ined for a fixed airflow rate of 15 l min�1. The maximumvalue obtained for all three days was approximately 5.9%on dry weight basis. However, the position of the maxi-mum was different for each day. It was (1.38, 0.45) forday 3, (1.57, 0.49) for day 5 and (1.70, 0.5) for day 7, forvariation in weight of wheat straw and inoculum amounttaken in sequence within the parenthesis. The incrementin crude protein in response to variations of the wheatstraw loading, inoculum and airflow rate for day 5 of reac-tor operation has been presented in Fig. 4. It appears thatthe ratio of the weight of wheat straw to inoculum amountequal to about 3.0 gives the highest crude protein. The rea-son for this could be that for the conditions within the reac-tor, the best distribution of nutrients occurs at this ratio.

3.3. Desirability coefficient d

A high value of d (0 6 d 6 1) indicates that the operat-ing point can produce acceptable bioconversion results.The highest value of d for day 3, 5 and 7 was 0.11, 0.75and 0.66, at corresponding airflow rates of 12, 15 and10 l min�1, respectively. Contours of increasing desirabilitycoefficients with respect to changes in the inoculum amountand wheat straw loading for day 5 are shown in Fig. 5. Thevalue d = 0.75 was obtained for an inoculum amount of0.38 g (100 g dry wheat straw)�1, a wheat straw loadingof 1.5 kg per stage and an airflow rate of 15 l min�1. Thecorresponding lignin and cellulose degradation achievedwas 27% and 29%, respectively.

3.84.24.65.05.45.86.2

Cru

de P

rote

in (%

)

1.251.33

1.421.50

1.581.67

1.75

0.25

0.30

0.35

0.40

0.45

0.50

Wheat Straw (kg)

Inoculum(g/100 g)

Fig. 4. Increment of crude protein content in response to variations in theamount of the inoculum and wheat straw loading in the reactor.

Wheat Straw (kg)

Inoc

ulum

(g/1

00 g

)

1.25 1.35 1.45 1.55 1.65 1.750.25

0.30

0.35

0.40

0.45

0.50

0.520.60

0.63

0.68

0.71

0.730.75

Fig. 5. Contours of the desirability coefficient as a function of theinoculum amount and wheat straw loading in the reactor. The airflow ratewas 15 l min�1.

Table 5The analysis of variance for the response surfaces obtained for lignindegradation, cellulose degradation and protein content

Measure Lignindegradation

Cellulosedegradation

Crude proteincontent

F-value 3.44 3.59 2.53P > Fa 0.031 0.030 0.082Degrees of freedom

of model8 9 9

RMSE 5.076 4.519 0.784R2 0.71 0.76 0.69Coefficient of

variance24.2 17.56 15.87

Adequate precisionb 5.99 7.91 8.08

a Values indicate that confidence level for lignin and cellulose is >96%and for crude protein >91%.

b Value >4 indicates adequate precision in the model.


A low value of the coefficient for day 3 samples indicatesthat the bioconversion was minimal even after three days.This is related to the time required by the fungus to grow,to express enzymes necessary for degradation of lignin andcellulose, and penetrate into the solid substrate (Fujianet al., 2001). We also observe that for day 3, a wheat strawloading of 1.25 kg and inoculum amount of 0.25 g (100 gdry wheat straw)�1 gives satisfactory results. At this condi-tion, the highest amount of resource is available per unitweight of fungus. However, since the desirability coefficient

depends on the criteria of maximizing lignin degradationand simultaneously minimizing cellulose consumption,the value of d obtained is low. Lignin is utilized at the high-est rate during the initial phase of colonization. In the laterphase, cellulose is the preferred substrate. Consequently, asthe bioconversion process progresses, d passes through amaximum.

3.4. Statistical analysis

A complete analysis of variance (ANOVA) was per-formed to validate the models describing the response sur-faces (Table 5). The best results were obtained using areduced cubic model for lignin and crude protein and areduced quadratic model for cellulose. The empirical mod-els describing lignin, cellulose and crude protein are givenby the following equations:

R1 ¼ 26:24� 2:49x1 þ 0:96x3 � 3:99x22 � 3:76x2

3

þ 3:97x1x2 þ 1:94x2x3 þ 2:22x3x1 � 1:33x1x2x3

R2 ¼ 28:90� 0:0049x1 þ 1:85x2 þ 3:10x3 � 0:59x21 � 2:67x2

2

� 1:38x23 þ 3:46x1x2 þ 4:23x2x3 þ 3:892:22x3x1

R3 ¼ 5:31� 0:17x1 þ 0:98x3 � 0:34x21 � 0:21x2

3 þ 0:39x1x2

� 0:51x2x3 þ 0:35x3x1 � 0:33x32 � 0:66x1x2x3;

ð4Þ

where R1, R2 and R3 are, respectively, the responses lignindegradation, cellulose degradation and increment in crudeprotein; x1, x2 and x3 are the factors, namely, the wheatstraw loading, inoculum amount and airflow rate. The F

statistic and the corresponding P > F values show that thelevels of confidence for lignin and cellulose degradation ishigher than 96%, and for increment in crude protein, it ishigher than 91%. There is adequate precision in the models.The regression coefficients are 0.71, 0.76 and 0.69 for theseresponses. An examination of the normal plot of residualsfor each response showed a linear trend. In addition, thestudentized residuals also exhibited a random scatter. These

15

20

25

30

35

40

45

10 12 14 16 18 20Airflow Rate (L/min)

Cel

lulo

se D

egra

datio

n (%

)

A

B

C

Fig. 7. The variation of cellulose degradation for the operating conditionsA (0.33,1.25), B (0.38,1.5) and C (0.48,1.75) for the fifth day data ofbioconversion. The first value in the parenthesis is the inoculum amountand the second value is the wheat straw loading.


response models were used to investigate the effect of air-flow rate variation on lignin and cellulose degradation.

3.5. Airflow rate

Varying the airflow rate from 10 l min�1 to 20 l min�1

between the points (0.33, 1.25) and (0.48, 1.75) in Fig 2,we observed that lignin and cellulose degradation is sensi-tive to process variables along this line. The response interms of lignin degradation varies marginally between22.5% and 27.1% as the airflow rate varies from 10 l min�1

to 20 l min�1. At the lower airflow rate of 10 l min�1, thehighest lignin degradation of 26.7% was observed for aninoculum amount of 0.30 g/100 g of dry wheat straw and1.25 kg wheat straw loading. At the higher end of20 l min�1, the highest lignin degradation was 25.4% foran inoculum amount of 0.48 g/100 g of dry wheat strawand 1.75 kg wheat straw loading (Fig. 6). The correspond-ing cellulose degradation at these extreme airflow rateswere 29.9% and 40.6%, respectively (Fig. 7).

The lignin degradation passes through a maximum forcurves A and B (curves are named after the points to whichthey correspond) as the airflow rate varies from 10 l min�1

to 20 l min�1; however, for curve C the lignin degradationstabilizes at about 18 l min�1. The maximum for curve Boccurs at 15 l min�1 (Fig. 6). On the other hand, for cellu-lose degradation the behavior is different. In Fig. 7, weobserve that for curve A, cellulose degradation is higherfor lower values of airflow rates and it decreases as the air-flow rate increases. We also observe that at low airflowrates, the cellulose degradation is higher than it is forcurves B and C. Although the curves B and C start at lowervalues of cellulose degradation, these curves show increas-ing cellulose degradation as the airflow rate increases.Beyond the pivotal point near 14 l min�1, both curves B

10

15

20

25

30

10 12 14 16 18 20

Airflow Rate (L/min)

Lign

in D

egra

datio

n (%

)

A

B

C

Fig. 6. The variation of lignin degradation for the operating conditions A(0.33,1.25), B (0.38,1.5) and C (0.48,1.75) for the fifth day data ofbioconversion. The first value in the parenthesis is the inoculum amountand the second value is the wheat straw loading.

and C have higher cellulose degradation than curve A.However, since the wheat straw loading is low for pointA, it is not a favorable choice for process operation. Inaddition, the slope of curve C near the pivotal point is morethan three times the slope of curve B. The penalty in termsof cellulose consumed is higher for point C. Hence, thepoint of operating point B is most favorable for bioconver-sion. The airflow rate of 15 l min�1 gives the maximum lig-nin degradation. However, even at point B, a higher airflowrate results in undesirable cellulose consumption.

Lignin degradation is an oxidative process. Barlev andKirk (1981) had reported that oxygen is instrumental instimulating the transcription of the lignin-degradingenzymes as well as in actual lignin degradation. It wouldbe expected that higher airflow rates would increase the lig-nin degradation achieved. However, an intermediate airflowrate of 15 l min�1 gives the highest lignin degradation. Thisis related to the actual mass transfer process at the microlevel. Oostra et al. (2001) have shown that the oxygen limi-tation resides in the aqueous film of the fungal mat thatforms on the solid substrate. However, Rahardjo et al.(2002) presented an alternative explanation where it wasshown that about 75% of oxygen requirement is obtainedthrough aerial mycelia. A recent review on modeling bio-conversion and transport discusses various issues relatedto oxygen limitation in solid-state fermentation (Rahardjoet al., 2006). Our results show that airflow rate can exert onlyan indirect influence. Airflow rate affects the film thicknesslocally in the substrate interstices, thereby affecting the oxy-gen mass transfer and consequently the degree of lignin deg-radation. Degradation of the lignin sheath then exposes thecellulose to attack by cellulases.

The maximum value of the desirability coefficient doccurs for different conditions on each of the days ofsampling. For day 3, the condition (1.25,0.31, 12.0) andfor day 7, the condition (1.64,0.30,10.0) gave the best


results. These results show that if the airflow rate was grad-ually increased from the start of the experiment reaching amaximum of 15 l min�1 on day 5, and thereafter reduced to10 l min�1 on day 7, the highest lignin degradation withminimum cellulose degradation could be achieved. Thisapproach may be applicable to similar bioconversion pro-cesses and it should be possible to schedule the airflow pro-gram automatically.

4. Conclusion

The bioconversion of wheat straw was carried out in a200 l staged vertical bioreactor. Samples were collectedon days 3, 5 and 7 to determine the extent of lignin and cel-lulose degradation, and increment of crude protein content.Although, lignin degradation stabilizes by day 7, cellulosedegradation continues to increase at a steady rate. A com-plete analysis of variance was performed to determine theresponse surfaces and the influence of airflow rate on thesevariables was determined using the desirability coefficient.It has been shown that the highest lignin degradation withcommensurate minimum cellulose degradation wasachieved by using the operating condition of 1.5 kg wheatstraw per stage, inoculum amount of 0.38 g (100 g drywheat straw)�1 and an airflow rate of 15 l min�1. On theother hand, the increment in crude protein depended onthe ratio of the weight of wheat straw per stage and inocu-lum amount. The maximum increment observed was 5.9%on a dry weight basis.

Among the process inputs, the airflow rate exerts a sig-nificant influence on the degree of bioconversion. The air-flow rate affects the heat transfer in the substrate bed andthe aqueous film thickness on individual solid substrateparticles. Only the macroscopic relation between the oper-ating conditions and quality of bioconversion has beenshown. Our results show that the airflow rate requirementvaries during the course of bioconversion. Initially, theairflow rate requirement is low and reaches a maximumon day 5. This is followed by a reduced requirement onday 7. The exact requirement can be computed from theexperiment data indirectly from the response surfaces.Therefore, by manipulating the airflow rate, we can con-trol the preferential uptake of lignin or cellulose by thefungus and improve profitability. Other aspects of palat-ability of feed formulations and nutrition would also playa role in the process economics. A complete study thatincludes the implementation of operational conditionsfor the best bioconversion along with other importantrequirements of feed formulation may be considered infuture work.

Acknowledgement

The funding received from DBT to carry out this work isgratefully acknowledged.

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