ASIAN J. EXP. BIOL. SCI.

, Vol 1 (2)2010: 352-359

Society of Applied Sciences

ORIGINAL ARTICLE

Effect of Co-Fungal Treatment on Biodegradation of Coffee Pulp Waste in Solid state Fermentation
K.Parani* and M. Eyini1
Research and Development, Multiplex Biotech Pvt. Ltd, Bangalore 560058, Karnataka, India 1 Reader, Research Centre in Botany, Thiagarajar College (Autonomous), Madurai 625009, Tamilnadu, India. Corresponding author : parani_k@yahoo.co.in ABSTRACT The ability of seven fungal strains viz., Phanerochaete chrysosporium, Pleurotus eous, Pleurotus flabellatus, Ganoderma lucidum, Fomes badius, Chaetomium globosum, Aspergillus terreus to decrease the cellulose, hemicellulose content and to enhance in the highest amount of reducing sugars using coffee pulp substrate was assessed. In monoculture experiments, viz., Phanerochaete chrysosporium, Pleurotus eous, Ganoderma lucidum and Chaetomium globosum showed a cellulose loss of more than 50% of the original content in 30 days, while Pleurotus eous and Chaetomium globosum monocultures degraded 53.7% of hemicellulose during the experimental period of 40 days. In coculture experiments, Pleurotus flabellatus with Pleurotus eous and Phanerochaete chrysosporium combinations resulted in the maximum cellulose loss of 84 % and 81%; hemicellulose loss of 62.5 to 68.7 % respectively in 40 days of degradation and simultaneously these cocultures showed an enhancement in the reducing sugar content (9.18 % and 9.64% dry wt. from the initial value of 3.2% dry wt.) on the 30th day of degradation as compared to the monoculture experiments, indicating their higher coffee pulp degradation potential. In contrast, Pleurotus eous with Fomes badius coculture caused 56.6% cellulose loss and 47.5% hemicellulose loss within the same period of degradation. Keywords: White rot fungus, Brown rot fungus, Soft rot fungus, Coffee pulp waste, Cellulose, Hemicellulose, Reducing sugars, Biodegradation
*

INTRODUCTION Coffee pulp is one of the most abundantly available agro-industrial wastes, produced during the pulping operation of the coffee cherries to obtain coffee beans in many coffee-producing areas of the tropics.Thus, for every 2 tonnes coffee cherries processed, nearly one ton pulp is generated [1]. Only the coffee bean has a real commercial value. It represents 55.4% of the fruit on dry weight basis and the rest is considered to be the byproducts or residues. At different stages from harvesting to the processing and consumption, several residues viz., coffee pulp or husk, leaves and spent-ground are generated in more than two million tonnes quantity yearly [2]. Among these byproducts, coffee pulp is the most important. It is formed from the epicarp and part of the mesocarp of the fruit and it represents about 28.7% on a dry weight basis when it is obtained by the wet coffee processing method [3]. Millions of tonnes of coffee pulp are produced all over the world every year and due to its difficult and improper handling, it causes many water pollution problems in the rivers and insalubrious conditions in the land areas near cities or towns where it is discarded. Due to the presence of these compounds (caffeine, tannins and polyphenols), these organic solid residues show toxic nature and thus have not been utilized beneficially. This has also led to the problem of environmental pollution [2]. In spite of the toxic components, coffee husk and pulp are very rich in organic components and could be used as substrates in bioprocesses to produce enzymes, aroma compounds, plant hormones, edible mushrooms and feeds [4]. The most efficient degraders are the white rot fungi [5]. In nature, the selective removal of cellulose from wood, leaving lignin, is the work of the brown rot fungi, the brown residue being essentially lignin [6]. Experiments on biological treatment of agro-residues have been successfully done by SSF (solid state fermentation) with various microorganisms like Candida utilis, Aspergillus pullulans, Cellulomonas sp, Alcaligenes fecalis, the soft rot fungus Chaetomium cellulolyticum and white rot fungi, Phlebia

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tremellosus, Ganoderma applanutum, Pleurotus sp and Dichomitus squalens. Environmental conditions such as temperature, humidity, microclimate, nitrogen content of the substrate and compartmentalization may also govern the selectivity of lignin biodegradation in vivo [7]. Solid state fermentation of lignocellulosics leading to the production of animal feed, human food and spent fermented residues as the compost for soil-remediation, is economical and can be practiced worldwide in all countries, where these substrates are abundantly available [2]. Hence, the present investigation was designed to study the biodegradation potential of selected microbial strains of mushroom / white rot, brown rot and soft rot fungi in co cultures. MATERIALS AND METHODS Fungal Strains and Medium Fungi A) Pleurotus eous (Berk.) Sacc, B) Pleurotus flabellatus (Berk and Br.) Sacc were obtained from Tamilnadu Agricultural College, Coimbatore, India. Pure cultures of Phanerochaete chrysosporium Burdsall (NCIM, 1197) was procured from National Chemical Laboratory, Pune, India. All the cultures were maintained on Potato Dextrose Agar (PDA) slants and stored at 4 C and the slants were subcultured once a month. Sporophores of the brown rot fungi i.e., Ganoderma luciderm and Fomes badius growing on wood logs were collected from the coffee estate. Actively growing mycelia were used to inoculate malt extract agar slants to get pure cultures of the respective brown rot fungus. The slants were stored at 4C and they were retrieved and subcultured once in every 15 days. Chaetomium globosum a known cellulolytic fungus was laboratories isolate and had been maintained on PDA slants. It was earlier recovered from paper mill effluent by primary selection through enrichment culture method. Aspergillus terreus was isolated from a sample of coffee pulp -dumped soil by serial dilution and pour plate technique. The pure cultures were made on PDA plates and they were identified by their morphological and colony characteristics [8]. The organisms were maintained on PDA slants at 4C and were sub cultured once a month. Substrate and Degradation Coffee pulp, the solid waste of coffee industry, processing the coffee beans by wet processing method was used as the substrate for biodegradation studies. Fresh coffee pulp was collected from Kardana Estate, Chinnamanur, Theni district, Tamil Nadu, India. It was sun dried, coarsely ground to uniform size (2mm) and was stored in gunny bags. The material was used within three months after procurement. Biodegradation of coffee pulp was studied in solid state in Erlenmeyer flasks (250 ml) using the selected mushroom fungi and their fungal associations. Ten grams of coffee pulp containing 60 % moisture was taken in individual Erlenmeyer flasks (250 ml). The flasks were plugged with cotton and autoclaved at 121C for 15 mins. Single mycelial agar block (8 mm) from seven days old cultures of the selected fungi was used as inoculum for monoculture experiments. For coculture studies, two agar blocks of the test white rot fungus and its respective fungal association were used as inocula. The conical flasks were incubated at 28 2 C for a period of 40 days in the culture room. Separate flasks were maintained for studying the compositional changes in coffee pulp. Treatment of Samples At each 10 days interval of study, the entire content of each flask was withdrawn, dried at 60 C overnight and was used in the analyses for measuring cellulose [9]; hemicellulose [10] and total reducing sugars [11]. Experimental Design All the experiments were carried out in triplicates and were replicated twice. RESULTS AND DISCUSSION Effect of Fungal pretreatment on cellulose content Coffee pulp contained cellulose as the major carbohydrate component (27. 25 % by dry wt.). It contained 16.02 % of hemicellulose and 3.24 % reducing sugars on a dry wt. basis. The highest 64.3% cellulose degradation in coffee pulp was found to be caused by G. lucidum monoculture. Cellulose degradation potential of C. globosum was on par with that of P. chrysosporium (62.5% and 62.1% cellulose degradation respectively). P.eous and P.flabellatus monoculture treatments decreased the coffee pulp cellulose content by 61% and 54.4 % respectively (Table1). The cellulolytic potential of Chaetomium globosum had been well documented and it had been recommended as the choice organism for cellulose bioconversion processes [12]. Several authors suggested that the reduction in
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cellulose could be attributed to the activity of the hydrolytic enzymes secreted by the fungus for its growth [13, 14] and reported 17% reduction in cellulose of paddy straw by P.sajor-caju in 30 days [15]. Pleurotus sp. are reported to be efficient colonizers and degraders of lignocellulosics [16-19]. The differential degradation of cellulose and lignin by different species of Pleurotus has been discussed [20]. The results of cellulose degradation by the monocultures were substantiated by the observations made on corresponding cellulase plate assays.
Table 1.Percent degradation of cellulose in coffee pulp during solid state biodegradation of selected fungal monocultures Cellulose Percent degradation S.No. Organisms Degradation (Days) 10 1. 2. 3. 4. 5. 6. 7. P. chrysosporium P. eous P. flabellatus F. badius G. lucidum A. terreus C. globosum LSD (0.05) = 1.153 25.0 18.7 12.8 13.6 16.5 10.6 18.7 20 37.5 32.7 28.6 33.4 38.6 36.3 43.0 30 56.6 54.4 43.0 46.3 52.9 50.3 55.8 40 62.1 61.0 54.4 61.3 64.3 51.1 62.5

Initial cellulose content: 27.25 % dry wt All the cocultures except that of P.chrysosporium+P.flabellatus, P.chrysosporium + P.eous caused a 50% cellulose loss within 30days (Table 2 ). Mixed cultures of fungi reportedly degraded 74% of cellulose of paddy straw within 22 days compared with 46% of cellulose degradation in the untreated control [21]. Our results on the higher efficiency of cocultures over monocultures in coffee pulp cellulose degradation are in agreement with this report. Cellulose degradation by P.eous + F.badius coculture (56.2% of cellulose loss in 40 days) was significantly less than that caused by the individual monocultures. Antagonism among members of a coculture had been observed that to have inhibited lignocellulosic degradation [22]. Effect of Fungal pretreatment on Hemi cellulose content The highest 53.7% hemicellulose degradation in coffee pulp was found to be caused by P.eous followed by 46.8% hemicellulose decrease in P.flabellatus monoculture treatments, during the experimental period of 40 days. Some of the workers recorded that a 37.5% reduction in hemicellulose on paddy strawcoir waste mixtures colonised by P.citrinopileatus in 45 days [23, 24, 25, 18] in several species of Pleurotus. Our results showed that both the soft rot fungi C.globosum and A.terreus showed a high efficiency (52-54.0% loss in hemicellulose) in degrading coffee pulp hemicellulose which was on par with that caused by P.eous and the brown rot fungi (Table 3). These results conform to those of who reported a greater loss of hemicellulose in rice straw treated with Chaetomium sp. and Trichoderma viride [26]. Lower utilisation of hemicellulose resulting in higher digestibility of the substrate is preferred in bioconversion studies for feed production [27]. In the present study, in dual cultures involving P.chrysosporium with the brown rot or soft rot fungi and those of P.chrysosporium + P.eous and P.eous + A.terreus, hemicellulose degradation could not be detected after 30days (Table 4). Selection of such cocultures, might be suitable for these type of bioconversion studies provided these cocultures

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also showed high efficiency for the removal of toxic compounds in coffee pulp and similar findings with the cocultures of the white rot fungi (P.chrysosporium, C.fimetarius) and the bacterium A.chroococcum [14]. In this study, cocultures involving P.flabellatus had a longer duration and showed higher (>60% decrease) hemicellulose degradation. P.ostreatus with a longer duration degraded a larger quantity of lignin, cellulose and hemicellulose in 32 days as compared to P.djamor was observed [28]. The fungus with a longer crop duration and higher BE continued to degrade various components with greater efficiency during the study period and finally ended up with achieving higher mineralization of the substrate. Thus the choice of organisms in a coculture depended on the specific purpose of SSF i.e., mineralization of the substrate or conversion of the substrate into cattle feed or other products [28].
Table 2 Percent degradation of cellulose in coffee pulp during solid state biodegradation of white rot and their fungal associations S. No I White rot + White rot 1 P. chrysosporium + P. eous 2 3 II P. chrysosporium + P. flabellatus P. eous + P. flabellatus White rot + Brown rot 27.9 P. chrysosporium + F. badius 2. 3 4 5 6 III 1 2 3 4 5 6 P. chrysosporium + A. terreus P. eous + A.. terreus P. flabellatus + A. terreus P. chrysosporium + C. globosum P. eous + C. globosum P. flabellatus + C. globosum LSD (0.05) =2.4 P. P. P. P. P. eous + F. badius flabellatus + F. badius chrysosporium + G. lucidum eous + G. lucidum flabellatus + G. lucidum White rot + Soft rot 27.5 25.0 17.2 32.7 31.6 31.6 43.0 45.2 51.0 47.0 46.3 59.5 56.6 61.3 69.1 59.5 62.8 73.1 57.7 63.2 77.2 63.6 74.2 75.7 19.4 23.5 34.5 31.6 29.7 38.2 46.3 45.9 56.6 61.7 50.7 71.3 59.9 73.8 76.1 56.6 76.4 61.3 74.6 79.4 41.9 56.9 59.1 32.7 27.5 50.3 52.2 73.1 74.2 80.8 83.8 34.1 52.5 67.2 68.3 Organisms Degradation (days) 20 30

10

40

Effect of co culture on total reducing sugars accumulation The substrate reducing sugars content increased to 6.9% dry wt (2.1 fold over the initial value) on the 30th day and the sugar content was nearly maintained at the same level during the course of biodegradation in P. flabellatus, F. badius and G. lucidum monocultures (Table 5) and a similar pattern of reducing sugars content of sago hampas colonized by P. sajor- caju was noted [29] . The different fungi produced different amounts of reducing sugars played an important role inducing, promoting and regulating the biosynthesis of cellulase enzymes by the cellulolytic fungi [30]. In lignocellulolytic organisms, lignin decomposition was found to be accompanied by the culture growth and release of sugars in Coprinus fimetarius fermented bagasse [31]. One f the author [32] opined that high FPA activity positively correlated with high sugar production but it also depended on the particular substrate and the fungus. Our results showing different patterns of reducing sugar accumulation and utilization for the different types of fungal associations on coffee pulp conform to
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this observation. A similar progressive increase in soluble reducing sugars throughout in Rhizopus colonized cassava peel. Similar results were reported [33] in wheat bran using A.niger.
Table 3 Percent degradation of hemicellulose in coffee pulp during solid state biodegradation of selected fungal monocultures Hemicellulose Percent degradation S.No Organisms 10 1 2 3 4 5 6 7 P. chrysosporium P. eous P. flabellatus F. badius G. lucidum 11.8 10.0 8.1 9.37 12.5 Degradation (Days) 20 21.2 26.2 18.7 23.7 27.5 30 35.6 42.5 31.2 36.2 38.7 40 48.7 53.7 46.8 51.2 51.2

A. terreus 15.6 36.8 50.6 51.8 C. globosum 16.8 31.8 51.8 53.7 LSD (0.05) = 0.528 Initial hemicellulose content: 16.02 % dry wt. Table 4 Percent degradation of hemicellulose in coffee pulp during solid state biodegradation of white rot and their fungal associations. S.No I 1 2 3 II 1 P. chrysosporium + F. badius 2 3 4 5 6 III 1 P. chrysosporium + A. terreus 2 3 4 5 6 P. eous + A.. terreus P. flabellatus + A. terreus P. chrysosporium + C. globosum P. eous + C. globosum P. flabellatus + C. globosum LSD (0.05) = 0.764 16.8 17.5 18.7 20.6 20.0 34.3 31.2 40.0 42.5 39.3 54.3 51.2 58.1 55.6 54.3 56.2 62.5 60.0 59.3 59.3 P. eous + F. badius P. flabellatus + F. badius P. chrysosporium + G. lucidum P. eous + G. lucidum P. flabellatus + G. lucidum White rot + Soft rot 16.8 31.2 48.7 49.3 16.2 17.5 36.8 40.6 53.7 61.2 62.5 64.3 10.6 11.8 21.8 23.7 29.3 39.3 35.6 50.0 55.0 47.5 63.1 56.8 Organisms White rot + White rot 21.8 P. chrysosporium + P. eous P. chrysosporium + P. flabellatus P. eous + P. flabellatus White rot + Brown rot 15.6 36.8 45.6 48.1 21.2 19.8 35.6 38.7 51.2 53.1 65.0 68.7 41.8 59.3 62.5 10 Degradation (days) 20 30 40

Initial hemicellulose content: 16.02 % dry wt.

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Table : 5. Percent increase or decrease in reducing sugars content in coffee pulp during solid state biodegradation of selected fungal monocultures. Reducing sugars Percent increase or decrease S.No Organisms 10 1 2 3 4 5 6 7 P. chrysosporium P. eous P. flabellatus F. badius G. lucidum A. terreus C. globosum LSD (0.05) =10.527 27.8 43.7 31.2 27.8 40.6 60.6 70.6 Degradation (Days) 20 30 71.8 100.9 65.6 53.1 86.8 86.8 86.8 79.6 106.8 109.3 115.6 110.0 24.3 56.8 40 9.4 71.8 110.9 116.5 110.0 24.3 - 6.25

Initial reducing sugars: 3.2 % dry wt

Table: 6. Percent increase or decrease in reducing sugars content in coffee pulp during solid state biodegradation of white rot and their fungal associations. S.No I 1 2 3 II 1 2 3 4 5 6 III. 1 P. chrysosporium + A. terreus 2 3 4 5 6 P. eous + A. terreus P. flabellatus + A. terreus P. chrysosporium + C. globosum P. eous + C. globosum P. flabellatus + C. globosum LSD (0.05) = 8.105 93.1 72.0 102.8 116.2 119.6 96.5 172.0 122.0 33.4 159.4 56.2 59.3 - 40.6 50.0 6.25 - 10.6 Organisms White rot + White rot P. chrysosporium + P. eous P. chrysosporium + P. flabellatus P. eous + P. flabellatus White rot + Brown rot P. chrysosporium + F. badius P. eous + F. badius P. flabellatus + F. badius P. chrysosporium + G. lucidum P. eous + G. lucidum P. flabellatus + G. lucidum White rot + Soft rot 81.2 112.5 31.2 25.0 65.6 68.7 112.5 78.1 112.5 103.0 150.0 112.5 156.2 93.1 156.2 119.6 31.2 50.0 90.3 3.1 90.3 40.6 12.5 - 22.0 50.0 - 3.1 50.0 - 4.68 89.6 86.2 93.1 129.6 172.5 171.2 12.5 201.2 186.8 - 28.1 100.0 50.0 Degradation (days) 10 20 30 40

Initial reducing sugars: 3.2 % dry wt.

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P. flabellatus + P.eous coculture, which showed the highest biomass protein caused 2.86 fold increase in reducing sugar content followed by P.flabellatus + A. terreus coculture which showed the highest 2.6 fold increase in reducing sugar content on 30th day of biodegradation (Table 6). Similarly, the reducing sugar content of coffee pulp substrate positively correlated with the biomass or substrate protein content in coculture treatments. The change in reducing sugar content seemed to correspond with the duration of coculture in SSF flask and the nature of the fungal association involved in the coculture. Asiegbu et al. (1996) who worked with monocultures, cocultures and mixed cultures accumulated higher amounts of soluble sugar compared with monocultures and cocultures. Results on coculture treatments which showed significant accumulation of reducing sugars over the monocultures conform to this report. Solid state fermentation has scored high among other fermentation types used in large scale biodegradation and bioconversion processes, due to its economically viable and practically acceptable design [2, 34, 35,]. One of the current approaches in improving the efficiency of biodegradation and bioconversion of agricultural or agroindustrial residues in SSF is the use of cocultures or mixed cultures of lignocellulolytic microorganisms. Many of these cocultures or mixed cultures were reportedly more efficient in lignocellulolytic biodegradation in producing high activity enzymes due to their synergistic action [36-40]. Hence, the work was directed towards identifying the best synergistic coculture which could successfully colonize and degrade coffee pulp in solid state fermentation. REFERENCES
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