microbial degradation of banana waste under

Process Biochemistry 40 (2005) 445451

Microbial degradation of banana waste under solid state bioprocessingusing two lignocellulolytic fungi (Phylosticta spp. MPS-001 and

Aspergillus spp. MPS-002)Maulin P. Shah a, G.V. Reddy b, R. Banerjee a, P. Ravindra Babu c, I.L. Kothari a,

a Post Graduation Deptartment of Biosciences, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, Indiab School of Life Sciences, University of Hyderabad, Hyderabad 500046, India

c Department of Environmental Sciences, Nagarjuna University, Nagarjunanagar, Guntur 522510, IndiaReceived 22 September 2003; received in revised form 11 December 2003; accepted 18 January 2004

Abstract

Phylosticta spp. MPS-001 and Aspergillus spp. MPS-002 were investigated for their ability to produce various lignolytic and cellulolyticenzymes such as laccase, lignin peroxidase, xylanase, endo-1,4--d-glucanase (CMCase) and exo-1,4--d-glucanase (filter paper activity (FPactivity)) on banana agricultural waste (leaf and pseudostem biomass) biomass under solid state fermentation (SSF) condition. The productionpattern of these enzymes were studied during the growth on the organisms for a period of 40 days. Very low levels of cellulolytic enzymeactivities were observed compared to lignin degrading enzymes by both the organisms. Maximum specific activities of studied enzymes wereobtained at 20 days of culture growth. 2004 Elsevier Ltd. All rights reserved.

Keywords: Phylosticta; Aspergillus; Solid substrate fermentation; Banana waste

1. Introduction

Banana is one the most consumed fruits in the worldand India is one of the largest producing countries ofthis fruit, which is cultivated in 4.796 105 ha yielding16.37 106 t of banana [1]. Each hectare of banana cropgenerates nearly 220 t of plant residual waste that consistsmainly of lignocellulose material. Most of the residualwaste produced due to banana cultivation is discardedby farmers into nearby rivers, lakes and on roads, whichcauses a serious environmental concern. In recent yearsthere has been a significant interest in efficient use ofagro-industrial residues [24]. Several processes have beendeveloped based on these materials as substrates in bio-process for production of single cell protein, organic acids,ethanol, mushrooms, enzymes and biologically importantsecondary metabolites [2,5]. Solid state fermentation (SSF)

Corresponding author. Tel.: +91-2692-2235157;fax: +91-2692-236475.

E-mail address: [email protected] (I.L. Kothari).

has important industrial application including manufactureof selected high value microbial products. SSF is advan-tageous in obtaining concentrated metabolites and sub-sequent purification procedures are therefore economical[6].

The main residual wastes of the banana crop are leavesand pseudostem, both containing high levels of ligno-cellulose [7]. These lignocellulose materials are efficientsubstrates for some fungi, which produce lignolytic andcellulolytic enzymes that have numerous application inindustrial processes for food, drug, textile and dye use[811]. In the present study two fungal species, Phylostictaspp. MPS-001 and Aspergillus MPS-002 were assessedfor their ability to produce lignolytic and cellulolytic en-zymes such as laccase (EC 1.10.3.2), lignin peroxidase (EC1.11.1.14), xylanase (EC 3.2.1.8), endo-1,4--d-glucanase(carboxy methyl cellulase, CMCase EC 3.2.1.4) and exo-1,4--d-glucanase (FP activity EC 3.2.1.91) by solid sub-strate fermentation on banana residual waste. The dynamicsof these extracellular enzymes were studied during thegrowth of these organisms on leaf and pseudostem residualwaste.

0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved.doi:10.1016/j.procbio.2004.01.020

446 M.P. Shah et al. / Process Biochemistry 40 (2005) 445451

2. Materials and methods

2.1. Fungal strain isolation and inoculum development

The fungal strains (Phylosticta spp. and Aspergillus spp.)were isolated from degraded banana waste samples collectedfrom local cultivated fields by an enrichment culture methodusing 1% (w/v) carboxy methyl cellulase. The culture weremaintained on potato dextrose agar slants at 24 2 C bysub-culturing at every 15-day interval. The inocula for boththeses cultures were produced on boiled wheat grains sup-plemented with 0.5% calcium carbonate. Cultures were in-cubated at 30 C for 10 days and these grains with myceliumwere used as inocula.

2.2. SSF substrate preparation, inoculation and cultureconditions

Agricultural wastes of banana plants were collected, driedand divided into pseudostem and the leaf portion, each ofwhich were cut into 2 cm pieces. The SSF substrate wasprepared by following a published method [12]. Twenty-fivegrams of each portion was placed in 1000 ml conical flasksand moistened with 75 ml of distilled water. The flasks wereautoclaved for 2 h at 121 C and they were inoculated sep-arately with 3 g of the wheat grain-based inocula of Phy-losticta and Aspergillus spp. Cultures were incubated at32 2 C in a BOD incubator and samples were collectedat every 5-day interval until the 40th day.

2.3. Sampling, extraction and analytical methods

Enzymes were extracted from 5 g of sample with 20 mlof cold 0.05 M acetate buffer (pH 6.5). The homogenate

Fig. 1. Production patterns of lignolytic and cellulolytic enzymes on pseudostem biomass of banana waste by Phylosticta spp. MPS-001.

was filtered through nylon cloth of 200 mesh and the filtratewas centrifuged at 6000 g at 4 C for 20 min. The su-pernatant was analysed for activities of laccase [13], ligninperoxidase [14], xylanase [15], carboxy methyl cellulase(CMCase) [15] and filter paper activity (FP activity) [15].Laccase unit activity was defined as the amount catalyzing0.1 absorbance change in guaicol per minute. The amountof the enzyme catalyzing a change of 1.0 absorbency unitin o-dianisidine per minute was defined as activity unit ofliginin peroxidase. One activity unit of FP activity, carboxymethyl cellulase (CMCase) and xylanase was expressedas 1mol of glucose or xylose equivalents liberated perminute, respectively. Specific activities of these enzymeswere estimated by finding the total protein contents inthe enzyme extract expressed as units per milligrams ofprotein.

The amount of reducing sugars were estimated by thedinitro salicylic acid (DNS) method [16] and protein concen-trations were estimated by following the method of Lowryet al. [17].

The cellulose, hemicellulose and lignin contents in de-garded and undegraded samples of banana waste were esti-mation by following published methods [7].

3. Results

The banana waste was divided into leaf biomass and pseu-dostem biomass to assess the feasibility of both the lig-nolytic and cellulolytic activity. Enzymes namely laccase,lignin peroxidase, xylanase, CMCase and FP activity wereassayed to determine the presence of lignolytic and cellu-lolytic enzymes.

M.P. Shah et al. / Process Biochemistry 40 (2005) 445451 447

3.1. Phylosticta spp. MPS-001 on pseudostem biomass

Production patterns of lignolytic and cellulolytic enzymeson pseudostem biomass of banana waste by Phylostictaspp. MPS-001 are shown in Fig. 1. Increase in specific ac-tivity of laccase from 5th day (0.8507 units mg1) to 20thday (2.4925 units mg1), then gradual decrease in its activityon 25th day has been observed. Lignin peroxidase specificactivity is 0.5558 units mg1 on 5th day which increasedon 20th day (2.2231 units mg1), and then declined on 40thday (0.246 units mg1). Xylanase specific activity has beenfound to be maximal at 20th day (1.6767 units mg1). Very

Fig. 2. Production patterns of lignolytic and cellulolytic enzymes on leaf biomass of banana waste by Phylosticta spp. MPS-001.

Fig. 3. Production patterns of lignolytic and cellulolytic enzymes on pseudostem biomass of banana waste by Aspergillus spp. MPS-002.

low levels of CMCase and FP activity could be detected(Fig. 1).

3.2. Phylosticta spp. MPS-001 on leaf biomass

Maximal specific activities for all the above mentionedenzymes were observed on the 20th day, afterwards up tothe 40th day a gradual decrease is observed. The highestspecific activity of laccase was observed on the 20th day ofthe culture by giving 2.726 units mg1 protein, followed bya gradual decrease. The specific activity of lignin peroxi-dase increased from day 15 (1.3076 units mg1) to day 20


Fig. 4. Production patterns of lignolytic and cellulolytic enzymes on leaf biomass of banana waste by Aspergillus spp. MPS-002.

(2.3018 units mg1), and thereafter the low levels could alsobe detected. Maximal xylanase-specific activity was foundto be 1.6863 on the 20th day. Low levels of CMCase andFP activity could be detected (Fig. 2).

3.3. Aspergillus spp. MPS-002 on pseudostem biomass

Laccase activity was quite distinct from the 5thday (0.8826 units mg1) onwards, peaking at day 20(2.8309 units mg1). The highest specific activities of ligninperoxidase and xylanase were 1.8796 and 2.7895 units mg1on day 20, respectively. Low levels of CMCase and FPactivity could also be detected (Fig. 3).

3.4. Aspergillus spp. MPS-002 on leaf biomas

The maximal specific activities of laccase and ligninperoxidase were recorded on day 20 and were 2.9567and2.6293 units mg1, respectively. In the case of xylanase,maximum specific activity was 1.9515 units mg1 was ob-tained on the 20th day. CMCase for endo glucanase andFP activity for exo glucanase were very low throughout theculture period (Fig. 4).

4. Discussion

Banana crop cultivation has been on rise at the globallevel, consequently generating a huge amount of rich resid-ual wastes [1,2]. There is enormous potential for exploitationof this substrate. The present investigation indicates that mi-

crobial biotransformed banana substrate can be a rich sourceof organics [7]. These organics are produced by the dynamickinetics of the enzymes, which are produced by two fungalstrains. It is clearly seen that the Aspergillus spp. is moreactive than that of the Phylosticta spp., both, in growth rate(data not shown) as well as enzyme activity. However, thelaccase activity was found to be quite high. The extracel-lular protein contents of Aspergillus spp. are higher thanthat of Phylosticta spp. Laccase enzyme production showeda very close correlation with the growth phase of the fun-gal mycelium. Both organisms under investigation exhibitvery good laccase activity. Reddy et al. [12] also reported asimilar pattern of laccase activity in two Pleurotus species.Increase in laccase activity during the vegetative phase hasbeen reported in Schizophylum commune [18], Agaricus bis-porus [19], L. edodes [20] and Coprinus congregatus [21].A rapid decrease in the laccase production from the day20th onwards, visibly indicates maximal mycelial growth.Laccase could be used as a morphogenetic indicator, itsrapid decrease indicates that maximal mycelium growth wasachieved. The present study clearly indicates that maximalmycelial growth occurred within 20 days on both leaf andpseudostem biomass. Again the results are very similar tothat of growth of Pleurotus sajor-caju and Pleurotus ostrea-tus in which maximal enzyme activity is observed on day20 [12].

The present study reveals that banana waste can be used asan alternative substrate to other agricultural/agro-industrialwaste, wheat bran/straw, sawdust and bagasse, which arealready in use for the production of lignino and cellulolyticenzyme production. The maximal production of laccasereported on rubber tree sawdust by P. sajor-caju was


27.4 units mg1 protein [22]. In the present study with theAspergillus and Phylosticta production of laccase on leafbiomass was 2.5 units mg1 protein and for pseudostem wasroughly 2.3 units mg1 protein by Phylosticta spp., whilefor Aspergillus it was 3 units mg1 protein, for both thepseudostem as well as leaf biomass. Similar yields of lac-case and lignin peroxidase were obtained in a SSF systemusing a culture of Polyporus versicolor [23]. Laccases ofsome white-rot fungi have been purified and characterisedbiochemically [23,24]. However, not many systematic andcomparative studies are available on quantitative laccaseproduction.

The importance of laccase in various biotechnological ar-eas underlines the need of expanding the spectrum of laccase

Fig. 5. Cellulose, hemicellulose and lignin contents (dry weight basis) in degraded and undegraded banana pseudostem biomass.

Fig. 6. Cellulose, hemicellulose and lignin contents (dry weight basis) in degraded and undegraded banana leaf biomass.

producing organisms and enhancing the potential of theirlaccase producing ability [25]. The present study revealsthat banana waste can be used as an alternative substrate toother agricultural/agro-industrial waste, wheat bran/straw,sawdust and bagasse which are already in use for the pro-duction of ligninolytic and cellulolytic enzyme production[26]. The present study shows maximum laccase productionon day 20th (to be 2.9 and 2.8 units mg1 on leaf biomassand pseudostem biomass, respectively by Aspergillus spp.)This production does not reach commercial productionstandards although improvement of culture conditionsthrough changes in mineral supplements and strain im-provement will lead towards the production of the enzymescommercially.


Lignin peroxidase enzymes had maximal activity onthe day 20th for both Phylosticta and Aspergillus forboth the substrates, pseudostem and leaf biomass rangingfrom 22.5 units mg1 protein, but significantly Aspergillusgrowing on pseudostem biomass had a much reducedperoxidase activity of roughly 1.5 units mg1 protein.Lignin peroxidase plays a central role in the biodegrada-tion of the plant cell wall constituent lignin. The ligno-cellulosic components of plant cells are lignin, celluloseand hemicellulose. To attack these complex polymers,white-rot fungi produce an array of enzyme families in-cluding lignin peroxidase, Mn peroxidase and cellulases[27].

Lignin peroxidase is an extracellular enzyme produced byfungi during secondary metabolism. The role in vivo of thisprotein is the degradation of lignin contained in the woodas it has been observed that LiP can catalyse degradationreactions of recalcitrant aromatic compounds. Its use hasbeen proposed in waste waster treatment and soil detoxifica-tion [28]. Lignin peroxide can be used for bioremediation ofpentochlorophenols and other toxic chlorinated compounds.[11,29].

Application of xylanase with other bioleaching agentssuch as oxygen and hydrogen peroxide in the pulp industryhas been extensively investigated and projection of a totallychlorine free pulp technology suggested [30]. Xylanase pro-duction, though low in the present study, could be enhancedby adjusting culture condition. However, very low levels ofCMCase and FP activities were observed. These results arein agreement with the reported results [7,31].

Production pattern of lignolytic and cellulolytic enzymeson pseudostem biomass of banana waste by Phylostictaspp. MPS-001 and and Aspergillus spp. shows the more util-isation of lignin contents then hemicellulose and cellulose.This is supported by reduction of lignin contents when com-pared with hemicellulose and cellulose contents in degradedsamples (Figs. 5 and 6).

The present study gives an insight into the dynamics of theextracellular enzyme production by the two potential isolatesPhylosticta spp. MPS-001 and Aspergillus spp. MPS-002on banana pseudostem and leaf biomass. The SSF conditionreveals a cheaper protocol for the production of these ex-tracellular enzymes. In the context of present environmen-tal production problems this paper gives a insight into theexploitation of a recalcitrant material for the production ofsome industrial enzymes.

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Microbial degradation of banana waste under solid state bioprocessing using two lignocellulolytic fungi (Phylosticta spp. MPS-001 and Aspergillus spp. MPS-002)IntroductionMaterials and methodsFungal strain isolation and inoculum developmentSSF substrate preparation, inoculation and culture conditionsSampling, extraction and analytical methods

ResultsPhylosticta spp. MPS-001 on pseudostem biomassPhylosticta spp. MPS-001 on leaf biomassAspergillus spp. MPS-002 on pseudostem biomassAspergillus spp. MPS-002 on leaf biomas

DiscussionReferences

microbial degradation of banana waste under

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