Cuban Journal of Agricultural Science, 50(3): 411-420, 2016, ISSN: 2079-3480

ORIGINAL ARTICLE

Isolation, selection and characterization of cellulolytic fungi from cocoa (Theobroma cacao L) hull

Aislamiento, selección y caracterización de hongos celulolíticos a partir de cáscara de cacao (Theobroma cacao L)

Ana L. Chafla,^I Zoraya Rodríguez,^II R. Boucourt,^II J. Espín,^I Lucia Silva,^III

^IUniversidad Estatal Amazónica, Puyo. Pastaza. Ecuador.
^IIInstituto de Ciencia Animal, Apartado Postal 24, San José de las Lajas, Mayabeque, Cuba.
^IIIEscuela Superior Politécnica de Chimborazo. Riobamba. Ecuador.

ABSTRACT

The objective of this study was to isolate, select and characterize fungi with cellulolytic capacity from fresh cocoa hulls, in order to use them as inoculum for solid fermentation of fiber residues. For taking the sampled fruits, the five points method was applied on cocoa crops (creole variety), belonging to the Center of Research and Postgraduate Courses (CIPCA, initials in Spanish) from the Universidad Estatal Amazónica, Ecuador. Samples were ground and exposed to three collection environments, with records of contamination due to microorganisms. For selecting isolates, growth in the medium, macroscopic characteristics, cellulolytic activity, measured by digestion halos and power index were considered. Out of 68 isolates, 21 strains were previously selected from the environment with the highest microbial culture diversity. Cluster analysis was applied to the characteristics of the selected colonies, as well as analysis of the obtained dendrogram, and four groups were formed. With help of taxonomical keys, nine strains of Aspergillus, seven of Trichoderma, four of Chrysosporium and one of Fusarium were identified. The 47.62% of the strains showed the highest degradation halo, which belonged to Aspergillus (5), Chrysosporium (3) and Trichoderma (2) genera. Strains A8 of Aspergillus and T1 of Trichoderma showed the highest power index (2.88and 2.45respectively), so they can be considered for their industrial use or as inocula in solid state fermentation, in order to obtain enzymes or animal feeding.

RESUMEN

El objetivo de este estudio fue aislar, seleccionar y caracterizar hongos con capacidad celulolítica a partir de cáscaras de cacao frescas, con el propósito de utilizarlos como inóculo en fermentación sólida de residuos fibrosos. Para la toma de los frutos muestreados, se aplicó el método cinco de oro en cultivos de cacao, variedad criollo, perteneciente al Centro de Investigación y Posgrado (CIPCA) de la Universidad Estatal Amazónica, Ecuador. Las muestras se molieron y expusieron a tres ambientes de colección, con historial de contaminación por microorganismos. En la selección de los aislados se consideró el crecimiento sobre el medio, características macroscópicas, microscópicas, actividad celulolítica medida por los halos de digestión y el índice de potencia. De 68 aislados, se preseleccionaron 21 cepas del ambiente con mayor diversidad de cultivos microbianos. A las características de las colonias seleccionadas se le aplicó análisis de conglomerados y del dendograma obtenido y se conformaron cuatro grupos. Con ayuda de claves taxonómicas se identificaron nueve cepas pertenecientes al género Aspergillus, siete de Trichoderma, cuatro de Chrysosporium y un Fusarium. El 47.62% de las cepas presentaron los mayores HD,  que se correspondieron con los géneros Aspergillus (5), Chrysosporium (3) Trichoderma (2). Las cepas A8 de Aspergillus y T1 de Trichoderma presentaron mayor índice de potencia (2.88 y 2.45 respectivamente), por lo que se podrían considerar para su empleo industrial o como inóculos en FES para la obtención de enzimas o alimento animal.

INTRODUCTION

Agriculture, agribusiness and industries of forestry, processing of pulp, paper and feed generate lots of residues, which are rich in lignin and cellulose and favor environmental pollution. These wastes represent losses due to the waste of matter that can be converted into several products with added values (Dashtban et al. 2010).

Ecuador is characterized by its great biodiversity, thanks to its geographical location and edaphoclimatic characteristics. Its economy is based on agriculture and livestock, mainly on cocoa production. In 2014, this production recorded an export of 235,000 metric tons (Castillo and Rosado 2016). Cocoa production experts have determined that this operation only uses 10 % of the weight of fresh fruit, because 90% are waste. Cocoa skin represents 75% of the total weight of harvested cobs (Barazarte et al. 2008).For this reason, it is necessary to optimize treatment systems for this type of waste (Arvanitoyannis and Varzakas 2008).

A use alternative with high potential is the biotechnological processing of cellulosic biomass by solid state fermentation (SSF), technology that allows to obtain human and animal feed (Chang 2007). Approximately 75-80 % of lignocellulosic residues can be degraded by the action of cellulases (Carvalho et al. 2013).However, hydrolytic activity of this enzyme complex is affected by physical and chemical factors, limiting the degradation of insoluble cellulose (Lee and Fan 1983).

For this reason, the search for microorganisms with better cellulolytic capabilities is very important because they could enable new biological sources for their use as products of high added value, such as enzymes for industrial purposes.

The objective of this research was to isolate, select and characterize native fungi strains from cocoa skin with high cellulolytic activity for their use as inoculum in SSF of fiber residues destined to the obtaining of enzymes or animal feed.

MATERIALS AND METHODS

Collection of cocoa fruit. Fruit collection was carried out at an experimental plot from the Center of Research and Postgraduate Courses (CIPCA, initials in Spanish), belonging to the Universidad Estatal Amazónica (UEA) in Ecuador. Fruits were selected through the five points method, proposed by Bautista et al. (2011). The minimum number of samples for an area of 5 ha was 12 fruits per each sampling point, with a total of 60 fruits. Mature and healthy fruits were selected, with mean weight of 478.94 g ± 95 per pod.

Processing of cocoa hulls. An amount of 20 cocoa fruit from the total obtained, was taken through a random sampling. These fruits were divided into the half with a vertical cut, seeds were removed and only the empty pods remained. The obtained subsamples were located for 15 d in three collection places: bromatology lab (BL), agribusiness lab (AL) and microbiology lab (ML). Average room temperature during the experiment was 23±3 ºC and 82±1.8 % of relative humidity. There parameters were daily registered with a digital thermometer with calibrated hygrometer (Thomas USA brand).

Preliminary selection and isolation of microorganisms. Out of every collection place, samples that presented fungal growth in cacao hull were taken. They were ground at particle size from 0.5 to 1 cm, in a feed grinder (Trapp-TRF brand 80/Brazil), with a sieve of 12 mm. An amount of 10 g of sample were dissolved in 20 mL of a peptone solution at 0.1% (p/v). The solution was filtered with a sterile gaze. A milliliter of the filtered was taken and added 9 mL of a saline solution at 0.09 % to achieve a dilution of 10^-6 (Stanier et al. 1996). Each microbial suspension to be evaluated was inoculated on filter paper circles (Whatman No.1) of 0.5 cm, located in the center of Petri dishes, containing solid medium of malt extract agar at 2% (p/v), supplemented with yeast extract 0.4% (p/v) at pH 6. For each dilution, a dish without inoculation was left as control. Dishes were located in an incubator (MEMMERT brand, model INB 400) at 30 ºC for eight days. For the selection of microorganisms, those with the highest number of isolates in the collection environment were considered. Isolates were purified by exhaustion in the same medium to which chloramphenicol at 1% was added to prevent bacterial growth. For conservation, they were cultivated in potato dextrose agar (PDA), in inclined tubes and kept at 4 °C. For their study, they were periodically cultivated and incubated at 28 °C.

Selection and characterization of colonies. From the areas that presented decomposition, repetitions were conducted in PDA, supplemented with carboxymethyl cellulose (CMC at 1% p/v) under the same conditions of the previous isolation. For selection, the morphotypes of higher size and frequency in the selective medium of each environment were considered. The characterization of fungi strains was performed by macroscopic and microscopic analysis (MOTIC stereoscope microscope) with digital camera and coupled micrometers, so the group of qualitative (growth, color and shape of the colony, medium pigmentation, reverse color, texture, exudate fluid, colony shape, conidia and mycelium shapes) and quantitative variables (lineage length, vesicle width, phialides and conidia) were determined.

Evaluation of cellulolytic ability of isolates. Petri dishes were prepared with synthetic medium, with carboxymethyl cellulose (CMC) (10 g CMC, 0.5 g NaNO₃, K₂HPO₄ 1.0 g, MgSO₄7H₂O 0.5 g, KCl 0.5 g, FeSO₄.7H₂O 0.001 g L^-1) as the only source of carbon (Waghmare et al. 2014). Three dishes per each isolation were cultivated. Dishes were incubated for three days and for the disclosure of degradation halos (DH), the superficial growth was covered with lactophenol blue solution of Merck. The evaluation of cellulolytic ability was categorized by DH intensity from 1 to 3, being 1 a less visible halo and 3 a very visible one (Pedroza et al. 2007). To establish the differences in the degradation activity, the analysis of comparison of proportions was used according to the COMPARPRO program (Font et al. 2007).

To determine the power index (PI), strains with DH equal to 3 were taken from each genera, and the relationship between degradation diameter and colony diameter was calculated (Valiño 1999). These diameters were measured with Vernier caliper (Suertek brand, sensitivity of ± 0.02 mm). A. niger was used as reference strain, from the UEA Microbiology laboratory. Strains with PI> 2 were selected.

Statistical analysis. Hierarchical cluster analysis was used for forming the groups with common characteristics, according to the linking method between groups with the measure of association cosine of vector angles (Torres 2015). The method of grouping from the Euclidian distance was used. To define groups, cut was made in 0.7. Statistical analyses for characterization were performed by IBM SPSS software (IBM Corporation 2013).

RESULTS AND DISCUSSION

Microorganisms are spread into the surrounding medium, so to select the isolation places, characteristics that should be preserved in isolates must be taken into account (Alcarraz et al. 2010). In general, the more demanding is the medium, the more restricted is life diversity (Valiño 1999). Sometimes, when the characteristics of microorganism are used since the selection, some steps are saved during isolation (Guzman et al. 2015). For primary isolation of cellulolytic fungi, Ferrer et al. (2011) used a culture medium with crystalline cellulose as the only carbon source, in order to increase the number of candidates. Therefore, this study conducted the isolation in the raw material that should be transformed, and thus, the guarantee that isolates counted on enzymes or enzymatic complexes adapted to the substrate itself.

From primary isolation, 53 morphotypes of LB, 11 morphotypes of LA and 4 morphotypes for LM were obtained, indicating that selected environments actually had potential for contamination. Considering the highest number of present morphotypes, the LB was chosen for purification and 21 fungi isolates were obtained, with distinct characteristics (tables 1 and 2).

Cluster analysis was applied to the group of qualitative variables and figure 1 shows the resulting dendrogram. It was possible to combine four groups and the obtained genera were identified using taxonomic keys: nine strains of Aspergillus genus (LB8, LB14, LB41, LB22, LB50, LB36, LB18, LB26, LB42) with 42.86 %; seven of Trichoderma (LB2, LB16, LB25, LB30, LB3, LB40, LB49), which represented 33.33%; four of Chrysosporium (LB9, LB11, LB24, LB44) that represented 19.05 % and one of Fusarium (LB37) with 4.76 %.

Around 56 species of cellulose-producer filamentous fungi have been identified (Valiño 1999). From the collected material, it was possible to have a diverse and representative sample of Aspergillus and Trichoderma genera, referred to in the literature as important producers of cellulase enzymes (Hill et al. 2009, Llacza 2012).

 Copetti et al. (2011), to study cocoa microbiota during drying and storage phase, observed fungal diversity in the collected samples. Among them, the most frequent were: Absidiacorymbiferanov, Penicillium and Aspergillus, the latter reported as the most frequent in this research.

With respect to cellulolytic activity, performed to 21 isolated strains by determination of degradation halo (HD), although there were no significant differences (P> 0.05) in the degrading activity, 10 strains (47.62 %) were obtained, which presented DH equals three. Out of these, Aspergillus genus showed the highest frequency (figure 2).

In the consulted literature, there were no results from degrading activity by this method in strains isolated from cocoa skin. However, Cruz et al. (2009) obtained 34.7% of the Aspergillus genus with DH equals three in isolates from agricultural residues. Ferrer et al. (2011) found only 10% of fungi, isolated from sugar cane bagasse, and showed good crystalline cellulose degradation, measured by methods as those used in this study. Several studies with fungi found that enzymatic activity depends heavily on the evaluated species and on factors like pH, temperature, nitrogen concentration and processes of inhibition by reaction products or the presence of proteases (Valiño 1999, Verlent et al. 2005).

Regarding PI values, table 3 shows that all evaluated strains with DH equal 3 presented PI values higher than those determined in the control strain. Out of them, T1 and A8 strains showed PI values higher than 2. This is an indicator of intense production of organic matter degrading cellulases, which colonize and hydrolyze these organic compounds very fast (Pérez et al. 2010).

Ferrer et al. (2011), after selecting five strains of fungi, isolated from sugar cane bagasse with degrading activity on crystalline cellulose, in which a similar rate to that of this study was calculated, found values inferior to the unit. However, the best diameters of cellulolytic activity found in this study were inferior to halos of 5 and 3 cm, which were produced by Trichoderma reesei and Aspergillus niger, respectively, reported by Gutiérrez et al. (2012).  They were also inferior to clearing zones of 7.5 cm for fungi from Trichoderma genus in studies of Ponce and Castillo (2011). However, due to the amount of factors that may affect the enzymatic activity, a better optimization of culture medium, supplemented with carbon and nitrogen sources and other surfactant substances, is required, besides adjusting the culture conditions to know the true cellulolytic potential of all the fungi found in all the different sampling sites.

An amount of 21 strains, with superior cellulolytic capacity to the reference strain, were isolated. A8 and T1 strains of Aspergillus and Trichoderma genera, respectively, showed power indexes superior to 2, indicating high cellulolytic capacity. These strains, native from cocoa skin, could be considered for its industrial use or as inocula in SSF to obtain enzymes or animal feed.

REFERENCES

Alcarraz, C. M., Flores, P. A. & Godoy, A. J. de D. 2010. “Producción de celulasas por inmovilización celular para el tratamiento de efluentes industriales lignocelulósicos”. Revista del Instituto de Investigación de la Facultad de Ingeniería Geológica, Minera, Metalurgica y Geográfica, 13(26): 97–102, ISSN: 1682-3087.

Arvanitoyannis, I. S. & Varzakas, T. H. 2008. “Vegetable Waste Treatment: Comparison and Critical Presentation of Methodologies”. Critical Reviews in Food Science and Nutrition, 48(3): 205–247, ISSN: 1040-8398, 1549-7852, DOI: 10.1080/10408390701279798.

Barazarte, H., Sangronis, E. & Unai, E. 2008. “La cáscara de cacao (Theobroma cacao L.): una posible fuente comercial de pectinas”. Archivos Latinoamericanos de Nutrición, 58(1): 64–68, ISSN: 0004-0622.

Bautista, F., Palacio, J. L. & Delfín, H. 2011. Técnicas de muestreo para manejadores de recursos naturales. 2nd ed., México: Universidad Nacional Autónoma de México, 670 p., ISBN: 978-607-02-21297-9, Available: <http://www.ciga.unam.mx/publicaciones/images/abook_file/tmuestreo.pdf>, [Consulted: November 10, 2016].

Carvalho, A. F. A., Neto, P. de O., da Silva, D. F. & Pastore, G. M. 2013. “Xylo-oligosaccharides from lignocellulosic materials: Chemical structure, health benefits and production by chemical and enzymatic hydrolysis”. Food Research International, 51(1): 75–85, ISSN: 0963-9969, DOI: 10.1016/j.foodres.2012.11.021.

Castillo, C. & Rosado, C. 2016. Análisis de la exportación de cacao y elaborados periodo 2004-2015 y evaluación de estrategias que contribuyan al crecimiento del sector considerando el cambio de la matriz productiva. Graduated Thesis, Universidad Católica Santiago de Guayaquil, Ecuador, 119 p., Available: <http://repositorio.ucsg.edu.ec/handle/3317/5226>, [Consulted: November 10, 2016].

Chang, S. T. 2007. “Mushroom cultivation using the ZERI principle: potential for application”. Micología Aplicada International, 19(2): 33–34, ISSN: 1534-2581.

Colina, A., Ferrer, A. & Urribarrí, L. 2009. “Cellulase production by Trichoderma reesei Rut C-30 from different cellulosic substrates”. Revista Técnica de la Facultad de Ingeniería. Universidad del Zulia, 32(2): 152–159, ISSN: 0254-0770.

Copetti, M. V., Iamanaka, B. T., Pereira, J. L., Fungaro, M. H. & Taniwaki, M. H. 2011. “Aflatoxigenic fungi and aflatoxin in cocoa”. International Journal of Food Microbiology, 148(2): 141–144, ISSN: 0168-1605, DOI: 10.1016/j.ijfoodmicro.2011.05.020.

Cruz, N., Castellanos, D. & Argüello, H. 2009. “Degradación de celulosa y xilano por microorganismos aislados de dos tipos de compost de residuos agrícolas en la Sabana de Bogotá”. Revista Colombiana de Ciencias Hortícolas, 3(2): 237–249, ISSN: 2011-2173, DOI: 10.17584/rcch.2009v3i2.1215.

Dashtban, M., Schraft, H., Syed, T. A. & Qin, W. 2010. “Fungal biodegradation and enzymatic modification of lignin”. International Journal of Biochemistry and Molecular Biology, 1(1): 36–50, ISSN: 2152-4114.

Ferrer, M. Y., León, R. M., Michelena, Á. G., Dustet, M. J. C., Duque, O. A., Ibañez, F. M. L. & Tortoló, C. K. 2011. “Selección de hongos aislados de bagazo de caña con actividad celulasa sobre celulosa cristalina para posibles aplicaciones industriales”. ICIDCA. Sobre los Derivados de la Caña de Azúcar, 45(1): 3–12, ISSN: 0138-620.

Font, H., Noda, A., Torres, V., Herrera, M., Lizazo, D., Sarduy, L. & Rodríguez, L. 2007. Comparpro. version 1.0, La Habana, Cuba: Instituto de Ciencia Animal, Departamento de Biomatemática.

Gutiérrez, R. L. A., Pérez, B. J. A. & Uribe, M. A. 2012. “Evaluación in vitro de celulasas producidas por cepas nativas de Trichoderma reesei, Cladosporium herbarum y Aspergillus niger”. Journal of Agriculture and Animal Sciences, 1(1): 7–15, ISSN: 2256-3342.

Guzmán, C. Á. M., Zambrano, P. D. E., Rivera, F. R. D., Rondón, A. J., Laurencio, S. M. & Pérez, Q. M. 2015. “Aislamiento y selección de bacterias autóctonas de Manabí-Ecuador con actividad celulolítica”. Cultivos Tropicales, 36(1): 7–16, ISSN: 0258-5936.

IBM Corporation. 2013. IBM SPSS Statistics. version 22, [Windows], U.S: IBM Corporation, Available: <http://www.ibm.com>.

Lee, Y. H. & Fan, L. T. 1983. “Kinetic studies of enzymatic hydrolysis of insoluble cellulose: (II). Analysis of extended hydrolysis times”. Biotechnology and Bioengineering, 25(4): 939–966, ISSN: 1097-0290, DOI: 10.1002/bit.260250406.

Llacza, H. 2012. Evaluación de la actividad celulolítica del complejo enzimático celulasa en cepas fúngicas de los departamentos de Cajamarca, Lima, Junín, Huánuco. Graduated Thesis, Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú, 68 p.

Pedroza, R. A. M., Matiz, V. A. & Quevedo, H. A. M. 2007. Manual de Laboratorio de Procesos Biotecnologicos. (ser. Apuntes), Bogotá, Colombia: Pontificia Universidad Javeriana, 9 p., ISBN: 97895876029, Available: <http://catalogo.udes.edu.co/cgi-bin/koha/opac-detail.pl?biblionumber=8486>, [Consulted: November 4, 2016].

Pérez, B. Y., Rebollido, R. R. & Martínez, S. J. 2010. “Aislamiento e identificación de hongos en compost elaborado a partir de residuos sólidos urbanos”. Agro Sur, 38(1): 1–7, ISSN: 0304-8802, DOI: 10.4206/agrosur.2010.v38n1-01.

Ponce, K. & Castillo, E. 2011. Aislamiento y caracterización morfológica y enzimo-funcional de hongos lignino-celulolíticos procedentes de la corteza de aliso (Alnus acuminata), Arrayán (Myrcian theshallii) y Pumamaqui (Oreopanax heterophyllus) presentes en las manchas del bosque nativo del Pasochoa, bajo condiciones de laboratorio. Graduated Thesis, ESPE, Sangolquí, Ecuador, 73 p.

Stanier, R. Y., Ingraham, J. L., Wheelis, M. L. & Painter, P. R. 1996. Microbiología. Villanueva, J. R. (ed.), Reverte, 776 p., ISBN: 978-84-291-1868-1, Available: <https://books.google.com.cu/books/about/Microbiolog%C3%ADa.html?id=2u-6Q2XCMDgC&redir_esc=y>, [Consulted: November 4, 2016].

Torres, V. 2015. “Aspectos estadísticos a considerar en el diseño, muestreo, procesamiento e interpretación de datos en la investigación de sistemas productivos agropecuarios”. In: Vargas, B. J. C., Benítez, J. D., Bravo, C., Leonard, I., Pérez, M., Torres, V., Ríos, S. & Torres, A., Retos y posibilidades para una ganadería sostenible en la provincia Pastaza de la Amazonía Ecuatoriana, Puyo, Ecuador: Universidad Estatal Amazónica, pp. 83–108, ISBN: 978-9942-932-16-7, Available: <https://www.researchgate.net/publication/295548677_Retos_y_posibilidades_para_una_ganaderia_sostenible_enla_provincia_de_Pastaza_de_la_Amazonia_Ecuatoriana>, [Consulted: November 4, 2016].

Valiño, E. 1999. Fermentación en estado sólido del bagazo de caña de azúcar por hongos conidiales productores de celulasas. Ph.D. Thesis, Instituto de Ciencia Animal, La Habana, Cuba.

Verlent, I., Smout, C., Duvetter, T., Hendrickx, M. E. & Van Loey, A. 2005. “Effect of temperature and pressure on the activity of purified tomato polygalacturonase in the presence of pectins with different patterns of methyl esterification”. Innovative Food Science & Emerging Technologies, 6(3): 293–303, ISSN: 1466-8564, DOI: 10.1016/j.ifset.2005.02.003.

Waghmare, P. R., Kshirsagar, S. D., Saratale, R. G., Govindwar, S. P. & Saratale, G. D. 2014. “Production and characterization of cellulolytic enzymes by isolated Klebsiella sp. PRW-1 using agricultural waste biomass”. Emirates Journal of Food and Agriculture, 26(1): 44–59, ISSN: 2079-052X, 2079-0538, DOI: 10.9755/ejfa.v26i1.15296.

Received: 08/2/2016
Accepted: 03/11/2016

Ana L. Chafla, Universidad Estatal Amazónica, Puyo. Pastaza. Ecuador. Email: achafla@uea.edu.ec