774 Chiang Mai J. Sci. 2017; 44(3)

Chiang Mai J. Sci. 2017; 44(3) : 774-787http://epg.science.cmu.ac.th/ejournal/Contributed Paper

Optimization of Cellulase Production by Pycnoporussanguineus in 5 L Stirred Tank Bioreactor andEnhanced Fermentation by Employing External LoopYi Peng Teoh [a,b], Mashitah Mat Don [a] and Kamaluddin Fadzilah [a]

[a] School of Chemical Engineering, Universiti Sains Malaysia, 14300 Nibong Tebal, Seberang Perai South,

Penang, Malaysia.

[b] Department of Chemical Engineering Technology, Faculty of Engineering Technology,

Universiti Malaysia Perlis, P.O. Box 77, D/A Pejabat Pos Besar, 01000 Kangar, Perlis, Malaysia.

*Author for correspondence; e-mail: teoh.yipeng@gmail.com

Received: 27 August 2015

Accepted: 3 March 2016

ABSTRACT

Palm oil mill effluent (POME) and oil palm frond (OPF) could be used as an adequatesource of cellulose for the production of cellulolytic enzyme. Batch culture of Pycnoporussanguineus in conventional stirred tank bioreactor (CSTB) showed that biomass and cellulaseproduction were influenced by POME concentration, aeration rate, and agitation speed.Optimization with central composite design (CCD) indicated an optimum condition at 70%(v/v) POME concentration, 350 rpm agitation speed and 1.0 vvm aeration rate with maximumenzyme activities at 16.073, 10.012, 2.348 and 12.186 IU/ml for CMCase, FPase, BG, andlaccase, respectively. These optimized parameters were then adapted in the studies using stirredtank bioreactor with external loop (STBEL), in which OPF served as a supporting matrix.Enzyme productions were shown to improve with higher maximum biomass (52.75 g/L)and enzyme activities (CMCase 18.221 IU/mL, FPase 13.406 IU/mL, BG 3.370 IU/mL,and laccase, 17.481 IU/mL) compared to CSTB.

Keywords: palm oil mill effluent (POME), Pycnoporus sanguineus, conventional stirred tankbioreactor (CSTB), external loop

1. INTRODUCTION

Palm oil industry plays an importantrole in the economic development ofMalaysia and in enhancing the economicwelfare of the population. Despite theobvious benefits, this industry also significantlycontributed to environmental degradation.Palm oil manufacturing processes generatedlarge quantities of wastewater commonly

known as palm oil mill effluent (POME),solid waste (fiber, kernel, shell, trunk, andfronds) and air pollution (smoke and dust)[1]. The environmental issues of the crudepalm oil industries were primarily relatedto water pollution due to indiscriminatedischarged of untreated or partially treatedpalm oil mill effluents into public watercourse.

Chiang Mai J. Sci. 2017; 44(3) 775

In fact, improper interim storage of solidwaste materials including boiler and incineratorash, decanter ash, spent bleaching earth andsludge separator residues, improper landapplication practices for liquid/solid wastes,odour emission from poor managed effluentsystem, and some noise from the millingprocess were also reported by Ibarahim et al.[2].

Oil palm fronds (OPF) are a by-productof the cultivation of oil palm trees (Elaeisguineensis Jacq.). They are used to be burnedbut environmental concerns led to banningthe practice in the 1990s. Nowadays, they areusually left on the ground to rot and fertilizethe soil. A considerable amount of researchhas been dedicated to demonstrate thenutritional value and economic viabilityof OPF for ruminants in order to improveself-sufficiency in dairy and meat production.In fact, OPF are a low-protein, high-fibrematerial that has been shown to be palatableand to have a good feeding potential formany classes of herbivore livestock, includingcattle, buffaloes, sheep, goats, deers andrabbits [3]. The discharge of untreatedPOME though created adverse impact to theenvironment, the notion of nurturing POMEand its derivatives as valuable resources shouldnot be dismissed. This was because POMEcontained high concentrations of protein,carbohydrates, nitrogenous compounds,lipids, and minerals that may be convertedinto useful materials using microbialprocesses [4]. Thus, POME and OPF aresome of the plentiful oil palm residues thatwould be used as an adequate source ofcellulose for the production of cellulolyticenzyme.

Enzymatic hydrolysis of cellulose is agood use of waste cellulose. During cellulasehydrolysis process, a single enzyme cannotaccomplish the task of extensive cellulosedegradation; multiple enzymes were required.

Consequently, microorganisms that grewsuccessfully on cellulose as substrates werecapable of doing so by producing a cellulasecomplex that which consists of at least threeextracellular enzyme activities: endoglucanase[EC 3.2.1.4], exoglucanase [EC 3.2.1.91] andβ-glucosidase [EC 3.2.1.21] [5]. As reportedby Mathew et al. [6], fungal cellulases haveproved to be a better candidate than othermicrobial cellulases with their secreted freecellulase comprising all three components ofcellulase. Pycnoporus sanguineus, a white-rotfungus are so far unique in their ability tocompletely degrade all components oflignocellulosic materials [7,8]; recentlyhad shown their production of xylanaseand β-glucosidase when grown in a suitableculture medium [9].

During fermentation in conventionalstirred tank bioreactor (CSTB), mixing is animportant aspect in the microbial synthesisof enzymes and can be imparted by meansof aeration and agitation. In order to achievemaximum yield, it is necessary to establishoptimum combination of airflow andagitation [10]. Both parameters are especiallyimportant in cellulase fermentation due tothe non-Newtonian nature of the broth, asthe presence of fungal cells and solidsubstrates results in a viscous mixture thatis not conducive for oxygen transfer [11].

Unfortunately, Kamaluddin [12] revealedthat submerged cultivations of P. sanguineusresulted in operational problems caused byextension of the fungal biomass andpropensity of the filamentous fungi to growon solid surfaces. Among others, foaming,wrapping of fungal mycelium aroundimpeller, gas sparger and baffles, andgrowth on vessel wall were apparent. In turn,such phenomena resulted in reduction ofmass transfer and mixing efficiency. In orderto overcome operational problems associatedwith filamentous growth, several authors

776 Chiang Mai J. Sci. 2017; 44(3)

have studied the immobilization of cellseither by entrapment or attachment on asolid support [13,14]. Collectively, thesestudies were aimed at replicating theconditions under which the fungus grows innature through the provision of a solidmatrix to support fungal growth. In essence,this approach is similar to the solid statefermentation (SSF) which was shown to beparticularly suitable for the production ofenzymes by filamentous fungi [6,15,16].Thus, the addition of a solid support systemin this study is expected to improve growthcharacteristics and enzyme production ofP. sanguineus. In this study, OPF is a potentialcandidate that can serve simultaneously as asupport matrix for fungal growth and anatural inducer for both ligninolytic andcellulolytic enzymes production due to itslignocellulosic content. The fibrous nature ofOPF offers high surface area withoutincreasing the mass of the support and canbe easily incorporated into the external vessel.

In view of such potential, this studywas carried out by investigating the effect ofPOME concentration, agitation, and aerationrate to the cellulase production in CSTBusing statistical optimization tool. Then,the study followed by enhancement thecellulase production by P. sanguineus in a stirredtank bioreactor with external loop (STBEL)using OPF as a supporting matrix.

2. MATERIALS AND METHODS

2.1 MicroorganismP. sanguineus was obtained from the Forest

Research Institute of Malaysia (FRIM).It was grown on potato dextrose agar at30°C for 7-10 d and stored at roomtemperature until further use. Stock cultureswere sub-cultured monthly.

2.2 Fermentation MediumPOME was collected from the United

Oil Palm (M) Sdn. Bhd., Penang, Malaysia.Raw POME was subjected to ultrasonictreatment (40 kHz, 50°C) for 1 h andsubsequently stored at 4°C until furtheruse. Pretreated POME was diluted withdistilled water to obtain concentration of70% (v/v). Sigmacell cellulose Type 101(10 g/L) and peptone (5 g/L) were added tothe media as co-substrate/cellulase inducerand nitrogen source, respectively.

2.3 Pretreatment of Oil Palm Frond(OPF)

OPF was collected from the UnitedOil Palm (M) Sdn. Bhd., Penang, Malaysia,and used for studies in bioreactor with anexternal vessel. The OPF were sun-dried for48 h to remove moisture so as to avoidmicrobial growth during storage. The driedOPF were treated with nitric acid followingthe findings of Umikalsom et al. [17]. Thefronds were soaked in 0.5% (v/v) of nitricacid at 30°C for 4 h. The chemically-treatedOPF were subsequently autoclaved togetherwith the chemical solutions at 121°C for5 min. The treated OPF were then filteredand washed with distilled water until notrace of acids could be detected, and thendried in oven at 95°C for 48 h.

2.4 Cell Suspension PreparationCell suspension was prepared by

suspending cell discs taken from 14 d-oldP. sanguineus culture plate in standard bottlecontaining sterilized distilled water and afew drops of Tween 80. Using a 5 mm (o.d.)cork borer, the discs are taken by count of10 discs per 100 mL of distilled water.

2.5 Inoculum PreparationSeed culture or inoculum was prepared

by inoculating 15 mL of cell suspension intoa 500 mL Erlenmeyer flask containing135 mL of fermentation medium. The flask

Chiang Mai J. Sci. 2017; 44(3) 777

was incubated in a rotary shaker at 30°C,250 rpm for 48 h prior inoculate intobioreactor.

2.6 Fermentation Condition2.6.1 Fermentation using conventionalstirred tank bioreactor (CSTB)

Batch fermentations with CSTB werecarried out in a 5 L CSTB (Minifors, InforsHT, Switzerland) with a working volume of3.5 L. The bioreactor is equipped withdigitally-controlled pH electrode, temperatureprobe, polarographic dissolved oxygenelectrode (pO

2), and two six-blade Rushton

turbine impellers fixed on the agitator shaft.The pH electrode was calibrated at pH 4 and7 using buffer solutions (ChemAR, USA)prior to sterilization at 121°C for 30 min.Two-point calibration of pO

2 probe was

done after sterilization by sparging purenitrogen for a 0% reading followed byair until 100% saturation was achieved.Silicone-based antifoam (Witeg GmbH,Germany) was used to control formation offoam. The pH of the culture media duringfermentation was maintained at pH 7±0.1using 1 M NaOH and 1 M HCl. In thisCSTB study, three different parameters(e.g., POME concentration, agitation, andaeration rate) were investigated using statisticaloptimization method. Fermentation wasconducted for 7 d at 30°C. Samples werewithdrawn at 12 h intervals, and followedby centrifuged (4500× g) for 30 min to

separate the cells and residual solids fromthe supernatant. The collected supernatantwas analysed for cellulase activity andreducing sugar estimation based on DNSmethod [18]. Sediments were used todetermine dry cell weight and residualcellulose based on a shortened procedure ofUpdegraff [19]. Each analysis experimentwas carried out in triplicate.

2.7 Experimental Design using ResponseSurface Methodology (RSM)

RSM is a collection of mathematicaland statistical techniques that were usefulfor the modelling and analysis of problemsin which a response of interest is influencedby several variables and the objectives wereto optimize this response [1]. The RSM usedin this present study was Central Compositedesign (CCD) experimental plan involvingthree key factors as listed in Table 1. Theresults were then analysed using Analysisof Variance (ANOVA) by Design Expert6.0.6 software (Stat Ease Inc., USA). Threedimensional plots and their respectivecontour plots were obtained based on theeffect of the levels of the three factors.From these three-dimensional plots, thesimultaneous interactions of the threefactors on the responses were studied.The optimum region was also identifiedbased on the main parameters in theoverlay plot.

Table 1. Coded and actual values of independent variables.

Factor

POME concentrationAgitation rateAeration rate

Unit

% (v/v)rpmvvm

Factor code

ABC

Level (coded)-1501000.5

0653001.0

1805001.5

778 Chiang Mai J. Sci. 2017; 44(3)

2.8 Fermentation using Stirred TankBioreactor with External Loop (STBEL)

Batch fermentation with STBEL wascarried out in a 5 L stirred tank bioreactorlinked to a custom-made 5 L external vessel(Infors HT, Switzerland) using siliconetubing that were connected to two peristaltic

Figure 1. Experimental setup of stirred tank bioreactor with external vessel (STBEL).

In the first phase of this experiment,the fermentation process was carried out at30°C, pH 7 for 7 d in the stirred tankbioreactor (working volume of 3.5 L) withoutcirculation into external vessel. Fermentationparameters were adapted from the resultsobtained in above section. Samples werewithdrawn at 12 h intervals, and followed bycentrifuged (4500× g) for 30 min to separatethe cells and residual solids from thesupernatant. The collected supernatant wasanalysed for cellulase activity and reducingsugar estimation based on DNS method [18].Sediments were used to determine dry cellweight and residual cellulose based on ashortened procedure of Updegraff [19].

Next, the external loop was commencedonce the growth rate and overall enzymeactivities in the main bioreactor reachedstationary phase. Flow rate for the inletand outlet stream of the main bioreactorwere initially set at 5 L/min. This flow ratewere monitored and adjusted to achieve a

continuous flow while maintaining thevolume of the culture media in the mainbioreactor so as not to be below � from theoriginal volume. Fermentation was continueduntil analysis of samples showed no furtherincrease in enzyme activities.

3. RESULTS AND DISCUSSION

3.1 Cellulase Production by P. sanguineususing Conventional Stirred TankBioreactor (CSTB)3.1.1 Model development and statisticalanalysis

In order to assess the interactive effectof cellulase production in CSTB, a CCDwas applied at three levels (low, high andcentral) with regard to POME concentration,agitation and aeration rate (Table 1).The complete design matrix consisting of20 experimental runs was shown in Table 2with the respective responses. Six replicateruns at the centre point were included toevaluate the reproducibility of data at

pumps (Integra Bioscience, Switzerland) foreach inlet and outlet stream, as portrayed inFigure 1. The external vessel consisted of twoperforated stainless steel plates at the top andbottom, and four connection ports. The spacebetween the plates was packed with thepretreated OPF as mentioned in above.

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1.321-2.877, and 5.114-12.186 IU/mL,respectively. The observed responses werefurther analysed using regression equationsobtained from ANOVA to determine thesuitability of the model.

the following conditions: 65% (v/v) ofPOME concentration, 300 rpm agitation,and 1.0 vvm aeration. Responses forCMCase, FPase, BG and laccase were foundto range between 9.641-16.073, 6.113-10.012,

Regression analysis as given by ANOVAshowed that the interactions between theindependent variables and all responsesfitted well with the quadratic model. Thequadratic regression models in termsof coded factors are presented in Eq. (1) to(4). The plus and minus notations of therespective terms in the regression model isan indication of the type of effect of eachterm on the responses, i.e. positive andnegative signs respectively posed positive and

negative effect. Eq. (1) for CMCase activityshowed that the first order (A, B, and C) andsecond order (A2, B2, and C2) terms imposedpositive and negative effect on CMCase,respectively, whereas positive interaction wasindicated only by POME concentration-agitation (AB). Accordingly, the terms A, B,C, B2, and AC in Eq. (2) indicated positiveeffect towards FPase activity. Three of themodel terms for BG exhibited positivecoefficients (A, B, and BC), with the remaining

Table 2. Experimental design matrix and responses for batch fermentation in conventionalstirred tank bioreactor (CSTB)

Std.order

1234567891011121314151617181920

Independent variables(coded)

A-11-11-11-11-110000000000

B-1-111-1-11100-1100000000

C-1-1-1-111110000-11000000

Responses (IU/mL)

CMCase9.64112.0519.74713.12711.24612.05411.47513.50812.94515.23414.22815.10613.71214.82615.90215.88416.07315.96316.01415.855

FPase6.1136.8846.7517.2137.4318.6537.5278.4768.8149.5929.96510.0127.4138.8729.7159.9139.7319.8149.8219.785

BG1.3081.6151.6251.7121.3141.4181.6521.7882.0362.1511.9152.0712.2562.1872.3472.3432.3422.3502.3452.348

Laccase5.5217.8254.1148.9619.2079.2327.27111.3708.73512.1868.2438.50810.03511.34111.92111.31411.96511.11211.88711.923

780 Chiang Mai J. Sci. 2017; 44(3)

terms giving negative connotations.Conversely, all model terms for laccaseshowed positive effect except for four terms(A2, B2, C2, and AC).

Y1 = 15.88 + 1.09A + 0.37B + 0.48C - 1.69A2

- 1.11B2 - 1.51C2 + 0.27AB - 0.37AC +0.063BC (1)

Y2 = 9.85 + 0.42A + 0.093B + 0.66C -

0.72A2 + 0.61B2 - 1.78C2 - 0.073AB +0.12AC - 0.13BC (2)

Y3 = 2.35 + 0.075A + 0.13B - 0.016C - 0.27A2

- 0.38B2 - 0.15C2 - 0.024AB - 0.019AC +0.037BC (3)

Y4 = 11.43 + 1.47A + 0.02B + 1.20C - 0.58A2

- 2.66B2 - 0.35C2 + 0.83AB - 0.38AC +0.059BC (4)

where A, B, C were the coded values forPOME concentration, agitation, and aerationrate, while Y

1, Y

2, Y

3 and Y

4 were responses

for CMCase, FPase, BG, and laccase,respectively.

ANOVA for all responses were

summarized in Table 3 and 4. As shown inTable 3, all models were significantwith Prob>F values less than 0.0001 for a99% level of confidence, while lack of fitwere found to be insignificant relative to thepure error. For all models, R2 values wereclose to unity (CMCase, 0.9980; FPase,0.9966; BG, 0.9934; laccase, 0.9667),indicating satisfactory adjustment of thequadratic model to the experimental data.The high value of adjusted R2 indicatedthat the models were highly significant.Thus, the models were deemed adequateand could represent the actual relationshipbetween the response and the significantvariables. Model terms and their respectiveProb>F values for each response weresummarized in Table 4. All terms in theregression model developed for CMCasewere found to be significant (Prob>F < 0.005)except for agitation-aeration interaction (BC).The second order term of agitation (B2)and POME concentration-agitationinteraction (AB) were found to be insignificanton FPase activity. On the other hand, allmodel terms were deemed significant forthe regression models of both BG and laccase.

Table 3. ANOVA for the regression models.

Response

i) CMCaseModelResidualLack of fitPure error

ii) FPaseModelResidualLack of fitPure error

Sum ofsquares

85.490.170.140.035

31.870.110.0840.026

Degree offreedom

91055

91055

Mean square

9.500.0170.028

7.013×10-4

3.540.0110.017

5.109×10-3

F-value

546.73

3.95

324.38

3.27

Prob>F

<0.0001

0.0788

<0.0001

0.1095

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Table 3. Continued.

i) R2: 0.9980, Adj. R2: 0.9961; ii)R2: 0.9966, Adj. R2: 0.9935; iii)R2: 0.9983, Adj. R2: 0.9968; iv)R2:0.9967, Adj. R2: 0.9937

Positive interaction of POMEconcentration and agitation rate onCMCase production was evident from thesurface plot in Figure 2(a), which showedCMCase activity converging into a peakas POME concentration and agitationwere increased. This indicated that theoptimum conditions were within theselected ranges. Maximum CMCase activity(16.073 IU/mL) was obtained at 65% (v/v)POME concentration and 300 rpm of

agitation. Beyond these points, CMCasenoticeably decreased. Similar pattern wasalso observed with BG, as maximum BGactivity (2.35 IU/mL) was obtained at 65%(v/v) and 300 rpm of POME concentrationand agitation rate, respectively. Optimalcellulase activities at this condition werealso reported in other studies [20, 21]. On theother hand, FPase activity was negativelyaffected through the interaction of POMEconcentration and agitation rate, as was

Table 4. F values and Prob>F for model terms of all responses.

* Superscript denotes insignificant term

Response

iii) BGModelResidualLack of fitPure error

iv) LaccaseModelResidualLack of fitPure error

Sum ofsquares

2.620.018

3.495×10-3

4.683×10-5

96.323.322.620.70

Degree offreedom

91055

91055

Mean square

0.291.752×10-3

3.495×10-3

9.367×10-6

10.700.330.520.14

F-value

166.44

2.31

32.28

3.75

Prob>F

<0.0001

0.1899

<0.0001

0.0865

Modelterm

ABCA2

B2

C2

ABACBC

F valueCMCase686.3280.63134.32449.55194.24358.8934.6662.571.81

FPase160.207.97

397.19132.190.94

802.513.8810.0712.58

BG32.0293.221.41

118.70221.3133.922.521.696.17

Laccase65.410.01243.182.7658.761.0116.513.450.084

Prob>FCMCase<0.0001<0.0001<0.0001<0.0001<0.0001<0.00010.0002

<0.00010.2087a

FPase<0.00010.0180

<0.0001<0.00010.3551b

<0.00010.0772c

0.00990.0053

BG0.0002

<0.00010.2630d

<0.0001<0.00010.00020.1434e

0.2225f

0.0324

Laccase<0.00010.9146g

<0.00010.1278h

<0.00010.3386i

0.00230.0927j

0.7774k

782 Chiang Mai J. Sci. 2017; 44(3)

suggested by the negative regressioncoefficient of AB in Eq. (2). From Figure 2(b),agitation was shown to impose a lineareffect during interaction with POMEconcentration, whereas increasing POMEconcentration up to 65% (v/v) increases FPase

activity. The lack of effect of mixing rateon FPase production observed in this studywas also reported by Lee et al. [22], whereasthe maximum level of FPase was achievedafter 5 d regardless of the different mixingrates used during the fermentation.

Figure 2. Surface plots of the effect of POME concentration and agitation rate on: a) CMCase,b) FPase, c) BG, and d) laccase activity.

Response surface plots for the interactionbetween POME concentration and aerationrate were illustrated in Figure 3. All cellulasecomponents showed converging surfaceplots with prominent peaks, indicating thatthe selected range of POME concentrationand aeration was preferable for cellulaseproduction and that optimum conditionswere within the studied ranges. Optimumyield of CMCase (16.073 IU/mL), FPase(10.012 IU/mL) and BG (2.35 IU/mL) wasobtained at 65% (v/v) and 1.0 vvm ofPOME concentration and aeration rate,respectively.

Meanwhile, response surface plotsdepicting interactions between agitation and

aeration were illustrated in Figure 4. Theirinteractive effect were analysed by keepingPOME concentration constant at its optimumlevel (65% v/v) and varying agitation andaeration within the experimental range.Both factors were found to favour CMCaseand BG production (Figure 4(a) and (b)).Agitation showed a linear effect on FPasewhile aeration produced a quadratic curve,which peaked at 1.0 vvm. Conversely, aerationwas found to impose an almost linear effecton laccase (Figure 4(d)) while agitation gavequadratic impact. It was noted that higheraeration range may be more favourable forlaccase production, as shown by the ascendingcurve in Figure 4(d).

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Figure 3. Surface plots of the effect of POME concentration and aeration rate on: (a) CMCase,(b) FPase, (c) BG, and (d) laccase activities.

Figure 4. Surface plots of the effect of agitation and aeration rate on: (a) CMCase, (b) FPase,(c) BG, and (d) laccase activity.

784 Chiang Mai J. Sci. 2017; 44(3)

3.2 Optimization and Model ValidationIn order to validate the developed model,

the fermentation was carried out using theoptimal culture conditions as predicted byRSM. The experiment was carried out intriplicate and results were given as average.Experimental and predicted responses werefound to be in close agreement, with errorranging from 0.35 to 0.77%, as portrayed inFigure 5. The small error margin (<1%)indicated that the generated modeladequately predicted batch cellulaseproduction in standard stirred tankbioreactor. Thus the optimum conditions formaximum cellulase production at 70% (v/v)POME concentration, 350 rpm agitationspeed, and 1.0 vvm aeration rate weresuccessfully developed by RSM.

3.3 Cellulase Production by P. sanguineususing Stirred Tank Bioreactor withExternal Loop (STBEL)3.3.1 Growth and cellulase production inSTBEL

In this study, batch fermentation in the5 L CSTB was supplemented with an externalloop to enable recycling of cells andsimultaneously overcome cellulase inhibition

by glucose. The external loop consisted ofpretreated OPF which served as a naturalsupport for adhesion of the fungal cells.Fermentation in this modified bioreactorsetting was carried out in triplicate atoptimum conditions (e.g., 70% (v/v) POMEconcentration, 350 rpm, and 1.0 vvm)obtained from the optimization experimentsof CSTB in above section. Followinginoculation, the CSTB was operatedindependently without the external loopup to mid-exponential phase. This wasdone to ensure active growth and adequatecell concentrations.

Figure 6(a) shows the fermentationprofiles of P. sanguineus in the STBEL.Prominent exponential growth observedbetween 0-24 h could be attributed tothe optimized conditions at which thefermentations were conducted. Based onabove findings, recycle stream to the externalloop were started at 60 h (mid-exponentialphase). Initiation of the external loop sawcell concentrations decreasing as partof the cell mass were transferred intothe external vessel. Afterwards, dry cellweight decreased steadily due to lowercell concentration in the inlet recycle streamas cells became attached to the OPF matrixin the external vessel. Consequently, oxygenuptake in the main vessel was reducedas evident from increasing dissolvedoxygen concentrations after 60 h (Figure 6(a)).Foaming was also alleviated when theexternal streams were activated, which couldbe attributed to the foam-breaking effectof the inlet recycle stream into the mainvessel. The findings in this study differedslightly from the study by Karim and Annuar[14], in which no freely suspended myceliawere observed in the liquid medium whenP. sanguineus was immobilized using coconuthusk as support matrix in a bubble columnreactor. This variation could be attributed to

Figure 5. Comparison of experimental andpredicted value of cellulase components usingoptimal conditions given by RSM underoptimal condition: POME concentration70%, agitation speed 350 rpm, and aerationrate 1.0 vvm.

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the use of different type of reactor and themuch smaller working volume (100 mL)applied in their study.

The time course for the production ofenzymes by the modified system (Figure 6(b))revealed a visible lag phase followingthe activation of the recycle stream.This observation was expected as cellsneeded to adapt to the new environment.Similar finding was reported by Hui et al.[16] using Aspegillus niger immobilized onnylon pads. They attributed the lag phaseto diffusional constraints of oxygen andsubstrate movements, and slow diffusionof enzyme from the immobilized cellmatrix to the bulk liquid. However,diffusional constraints can be ruled out inthis study given that the OPF supportmatrix were highly porous, as demonstratedby uninterrupted flow of culture mediainto and out of the external vessel.

Figure 6. Fermentation profiles in stirred tankbioreactor with external vessel (STBEL). Timecourses are shown for: (a) dry cell weight anddissolved oxygen concentration, and (b)CMCase, FPase, BG, and laccase activities.

Table 5. Comparison of cellulase productions in CSTB and STBEL.

Parameter

1) CMCaseMaximum activity (IU/mL)Yield, Y

P/X (g/g)

Volumetric rate of production (IU/mL.h)

2) FPaseMaximum activity (IU/mL)Yield, Y

P/X (g/g)

Volumetric rate of production (IU/mL.h)

3) BGMaximum activity (IU/mL)Yield, Y

P/X (g/g)

Volumetric rate of production (IU/mL.h)

4) LaccaseMaximum activity (IU/mL)Yield, Y

P/X (g/g)

Volumetric rate of production (IU/mL.h)

Conventional stirredtank bioreactor

(CSTB)16.0730.5040.096

10.0120.3140.060

2.3500.0740.014

12.1860.3820.073

Stirred tank bioreactorwith external loop

(STBEL)18.2210.3450.108

13.4060.2540.080

3.3700.0640.020

17.4810.3310.104

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3.4 Comparison Study between CSTB andSTBEL

In order to evaluate the performance inSTBEL, the data were compared withoptimum results in CSTB. The results inTable 5 shows slightly higher enzymeactivities as a result of the STBEL setting,whereby CMCase, FPase, BG and laccaseactivity of P. sanguineus increased 13.4, 33.9,43.4, and 43.5%, respectively.

The improved enzyme productionobserved in STBEL could be attributed totwo main factors: a) simultaneous recyclingand retention of cells in the OPF supportmatrix, and b) lower repression by glucoseas a result of continuous-like operation.As stated by Shuler and Kargi [23], cell recycleoperation is the most common approach toincrease conversion rate and productivity.Furthermore, recycling of cells also increasesthe stability of some systems by minimizingthe effects of process perturbations.

Subsequently, continuous flow of theculture medium in and out of the main vesselmay have alleviated the repression caused byaccumulation of glucose and other reducingsugars. As stated by Sukumaran et al. [24],cellulase synthesis can be halted by glucoserepression when glucose generation is fasterthan consumption. In addition, the presenceof PPF, an insoluble lignocellulose, may havetriggered an increase in cellulase synthesis.In their review, Zhang and Lynd [25] pointedthat cellulase synthesis in batch cultures wasnine-fold greater with Avicel compared tocellobiose, whereby the former is insolublecellulose. In addition, increases in enzymeactivities were most likely contributed inlarge part by the role of P. sanguineus as a lignin-degrading fungus, whereby depolymerizationof lignin by laccase from P. sanguineus hasbeen widely reported [26,27]. In turn,breakdown of lignin improved enzyme accessto cellulose, thus allowing more cellulose to

be hydrolysed.

4. CONCLUSION

Cultivation of P. sanguineus in CSTB washighly affected by POME concentration,aeration rate, and agitation. The optimumconditions predicted by RSM (i.e. 70% (v/v)POME concentration, 350 rpm and 1.0 vvm)resulted in 16.073, 10.012, 2.348 and 12.186IU/mL of CMCase, FPase, BG and laccase,respectively. On the other hand, the overallenzyme activities were improved as a resultof introducing external loop to the stirredtank bioreactor. Despite a lower maximumcell concentration (27.82 g/L), enzymeactivities in STBEL were higher comparedto those obtained in CSTB (CMCase 18.221IU/mL, FPase 13.406 IU/mL, BG 3.370IU/mL, and laccase, 17.481 IU/mL).

ACKNOWLEDGEMENT

The authors are grateful to the ResearchUniversity (RU) team grant (no. account:1001/PKIMIA/854002) provided byUniversiti Sains Malaysia to support thiswork. They would also like to extend theirgratitude to Dr. Salmiah Ujang of the ForestResearch Institute of Malaysia (FRIM) forsupplying the stock culture of fungus.

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