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Use of wastes from the tea and coffee industries for the production of cellulases using fungi isolated from the Western Ghats of India


In this study, coffee pulp (Coffea arabica) and green tea (Camellia sinensis) residues were characterized for use as a substrate of solid-state fermentation for cellulases production. The invasion rate was evaluated, as well as cellulases production by strains of Aspergillus niger and Trichoderma asperellum from the western Ghats of India, on coffee pulp, green tea, and a mixture of both substrates (50:50). T. asperellum (AFP) strain was found to have the highest growth rate (0.409 ± 0.021 mm/h) using a mixture of both substrates. The production of cellulases by T. asperellum was unsatisfactory due to the presence of polyphenols in the supports to which A. nigger cellulases are more resistant. The production of cellulases by A. nigger was linked to the pH of the supports, favouring the use of T and TC. It was found that the extracts produced by A. niger (28A strain using a mixture substrate, 28A, and 20A strains using only green tea as a substrate) presented the highest cellulase activities when evaluated using a plate technique producing degradation halos of 2.3 ± 0.1 cm of diameter. Aspergillus 28A strain did not require mineral enrichment media for cellulase production using green tea residues as support of solid-state fermentation.


Coffee and green tea are crops used to make some of the world's most popular beverages, characterized by their unique flavour and contribution of antioxidants that can offer beneficial health effects [1]. Coffee is a plant native to Ethiopia that is currently grown in tropical and subtropical climates [2]. Although there are a large number of species, the most important from an economic point of view is the C. arabica and C. cenaphora, also known as Robusta [3].

In 2017, there was a global production of just over 9.2 million tons of coffee, with Brazil the world's largest producer (2.6 million tons) followed by Vietnam (1.5 million tons) [4]. Mexico is in 11th place (0.83 million tons) [5]. It has been estimated that 50% of the production of commercial green coffee is discarded as coffee pulp, representing 4.6 million tons [6].

Green tea is a plant native to Southeast Asia, currently associated with the C. sinensis plant, along with a less recognized variety called assamica [7, 8]. Commonly tea can be processed in three different ways, depending on the type of product desired. Drying it to prevent oxidation in the case of green tea, promoting oxidation in the case of black tea, and promoting semi-oxidation to produce oolong tea [9]. Global tea consumption and production have increased in recent years, with 6 million tons reported in 2017, of which 1.7 are green tea, driven mainly by China (1.2 million tons) [10]. It has been estimated that 75% of the production of commercial tea is discarded as residue, representing 3.8 million tons [11].

Coffee pulp residues are considered eco-toxic and anti-nutritional, because they have high polyphenolic content [12]. It has anti-physiological factors such as caffeine and condensed tannins that produce nutritional and physiological consequences through interaction with proteins and carbohydrates, compromising digestion, and preventing the absorption of metals [13]. And like tea, it has chlorogenic acid that reduce the diet and availability of amino acids by decreasing digestion in various insects [14].

One of the strategies that can be applied for the use of this class of waste is its use for the production of compounds of interest, such as enzymes, through solid-state fermentation processes (SSF) [15]. SSF defines it as an aerobic fermentation process developed in the absence of free water but with sufficient moisture for microbial growth [16]. It is a simple and economical process to implement, which allows the use of waste without the need for rigorous pretreated or complicated technologies [17].

Cellulases are some of the most used enzymes at the industrial level due to the wide range of activities in which they can be used, including the production of livestock feed, in the paper industry [18], detergent production [19], renewable energy generation [20], among others. These enzymes are commonly produced at an industrial level using filamentous fungi, because they produce the most complex enzyme pools and have a high secretion capacity [21].

These enzymes are catalogued based on the site of action in which they work to degrade cellulose. Endocellulases (EC fragment the internal part of the bio-polymer chain randomly, while exocellulases (EC degrade the fragments produced by the ends, generating cellobiose dimers, which are finally degraded to glucose monomers by the action of β-glucosidases (EC [22, 23].

Due to the limited information available using this type of substrates in the production of hydrolase enzymes, the present work is oriented to its use for the production of cellulase enzymes using strains of filamentous fungi endemic to the Western Ghats of India, one of the points with the highest biodiversity on the planet.


Microorganism used

Three fungal strains were used, A. niger 28A, A. niger 20A, T. asperellum AFP, all originating from the Western Ghats of India, provided by the Jawaharlal Nehru Tropical Botanic Garden and Research Institute (Palode, Thiruvananthapuram, Kerala, India).

Raw material

The research was carried out in the Bioprocesses laboratory of the Food Research Department at the Autonomous University of Coahuila, Saltillo, Mexico. Coffee pulp (C. arabica) from Xilitla San Luis Potosí; Mexico and green tea residues (C sinensis) generated by the preparation of infusions with green tea collected from the local market was used as substrates.


The characterization of the residues was based on the quantification of structural components, as well as the physicochemical parameters of the study substrates. Previously, the raw materials were dried at 60 °C and ground to a particle size of less than 1 mm.

Proximal analysis

The humidity was determined using a thermobalance (Ohaus MB 23, USA), 0.5 g of sample was evaluated at a temperature of 120 °C. Fiber, fat and ash contents were analysed using the procedures of the Association of Official Analytical Chemists (AOAC 1980). The ash content was analysed by evaluating the weight before and after incineration at 550 °C for 24 h, and the fat was determined by extraction using a Soxhlet apparatus. Soluble reducing sugars were determined from infusions made by mixing 1 g of the substrate with 19 ml of distilled water using the DNS technique [24]. Hemicellulose, cellulose, and lignin were determined using the methodology described by Sluiter et al. Acid hydrolysis of the substrates was carried out with 0.5 g of sample and 5 ml of 72% H2SO4 under constant stirring for 1 h, then they were diluted with distilled water and autoclaved at 121 °C for 1 h. Hydrolyzates were filtered using constant weight gooch filters with a vacuum pump system. The solids were weighed for the determination of the lignin percentage and a sample of the liquids was taken for analysis on HPLC to determine the content of hemicellulose and cellulose [25]. The analyses were carried out in triplicate and were expressed as a percentage.

Physicochemical characterization

pH determination

1 g of sample was homogenized in 10 ml of distilled water at a temperature of 20 °C; then, the pH was measured using a potentiometer (Ohaus starter 3100) following the guidelines established in the Mexican Standard [26].

Water absorption index

The water absorption index (WAI) was evaluated through the method described by Robledo et al. [27] with some modifications. 1.25 g of dry sample was weighed and mixed with 30 ml of distilled water at 60 °C; the samples were placed in a water bath at 60 °C for 30 min, stirring the suspension every 10 min after heating began. The samples were centrifuged at 4900 rpm for 30 min, to be then decanted, finally weighing the gel formed. WAI was calculated using formula (1)

$$WAI=\frac{\mathrm{g}\mathrm{e}\mathrm{l} \,\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\, (\mathrm{g})}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\, \mathrm{o}\mathrm{f} \,\mathrm{d}\mathrm{r}\mathrm{y} \,\mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\, (\mathrm{g})}.$$

Critical humidity point

0.5 g of the gel obtained from the quantification of the water absorption of each substrate was placed in a thermobalance (Ohaus MB 23, USA) at 120 °C, evaluating the percentage of moisture loss every minute. Defining the critical humidity point as the point at which the initial dehydration rate is altered [28].

Radial growth rate evaluation

The strains were reactivated on PDA agar at an incubation temperature of 30 °C for 5 days, using a 1% Tween 80 solution for spore recovery. The radial growth rate was evaluated on three substrates [100% coffee pulp (C), 100% green tea (T), and a mixture of both at 50% (TC)]. The tests were carried out in Petri dishes with 10 g of the substrate at 70% humidity. The center of the boxes was inoculated with 10 µl of spore solution at a concentration of 1 × 106 spores/ml following the technique used by Robledo et al. [27], leaving to incubate at 30 °C. The experiment was stopped when one of the experimental units reached the total invasion of the plate.

Recovery of enzymatic extracts

For the recovery of the enzymatic extracts, 3 ml of 0.05 M citrate buffer pH 4.8 were added to each of the experimental units, they were mixed manually and allowed to stand for 20 min, and finally, the extracts were recovered by filtration and mechanical compression using Whatman filter paper. #1 [29].

Evaluation of cellulase activity using the degradation halo technique

For the development of this test, a modification of the methodology reported by Meddeb-Mouelhi et al. [30] was used. Petri dishes with a mixture of carboxymethyl cellulose (CMC) at 500 ppm in agar were used. The Petri dishes were divided into five segments, three for the different repetitions, one for the blank (distilled water), and the other for positive control (Trichoderma reesei) cellulases (SIGMA), were applied 10 µl of each extracts as well as the blank and the control (which was previously diluted at a 1:40 ratio using citrate buffer pH 4.8 0.05 M) and allowed to incubate for 24 h at 30 °C. The Petri dishes were stained with 1% congo red solution for 15 min. They were washed with distilled water and finally with a 1 M NaCl solution for 15 min, measuring the degradation halos produced.

The following tests were carried out using only the A. nigger 28 A strain.

Evaluation of the use of mineral enrichment media in the production of cellulase enzymes using green tea residues as support

The strain was reactivated in PDA at 30 °C for a period of 5 days. Spore harvesting was performed with a 1% Tween 80 solution. The fermentations were carried out using 10 g of green tea residue at 70% humidity using Mandels mineral medium with the following composition (g/l): KH2PO4, 2; (NH4)SO4, 1.4; urea, 0.3; MgSO4·7H2O, 0.3; CaCl2, 0.3; FeSO4, 0.005; MnSO4, 0.0016; ZnSO4·7H2O, 0.0014; CoCl2·6H2O, 0.02; peptone, 1; yeast extract, 0.25; CuSO4·5H2O, 0.001 [31]. Czapek-Dox mineral medium with the following composition (g/l): NaNO3, 3; KH2PO4, 1; MgSO4, 0.05; KCL, 0.5; FeSO4, 0.01; peptone, 0.5 [32]. And finally a treatment using only distilled water without mineral enrichment media. It was inoculated with a concentration of 1 × 106 spores/ml and allowed to incubate at 30 °C for 96 h.

To recover the extracts, 10 ml of 0.05 M citrate buffer, pH 4.8, were added to each experimental unit and brought to 190 RPM for 10 min. Subsequently, the extracts were filtered using Whatman # 1 filter paper and stored frozen at − 4 °C until use.

Evaluation of total cellulase activity “filter paper units” (FPU)

For the assessment of total cellulase activity, the technique reported by Xiao et al. [33] was used. 40 µl of 0.05 M citrate buffer pH 4.8 was mixed with 20 µl of the diluted extracts, using 5.5 mm filter paper discs as substrate. The reaction mixture was incubated at 50 °C for 1 h. After, 120 µl of DNS were added and incubated at 95 °C for 5 min. Finally, 36 µl of each reaction were mixed with 160 µl of distilled water for analysis at 540 nm in a microplate.

Results and discussion

Proximal composition

The results of the proximal composition exhibited some variations with respect to the values reported in the literature; it is observed that both substrates had a high fiber content (Table 1). These are expected results, because the composition of the plants depends on the growing conditions, as well as on the variety and geographic location, among other factors involved in their development and processing [13]. The composition of these substrates shows potential for their use in the production of cellulases, due to the considerable content of cellulose, which is the main inducer of these enzymes [34].

Table 1 Proximal analysis of coffee and green tea pulp

The capacity of a support to absorb water is known as WAI, and CHP is the portion of water that cannot be used by the microorganism for the development of its metabolic reactions, because it is strongly bound to the substrate. Values similar to those obtained in the present study (Table 2) have been reported in materials used in solid-state fermentation processes as immobilization vehicles [35], for the production of polyphenolic compounds [28], ellagic acid [27], and β-fructofuranosidase [36]. The highest WAI was presented by green tea, which can be associated with low-fat content and a high amount of fiber [28].

Table 2 Physicochemical characterization of coffee pulp and green tea

Solid-state fermentation processes are triphasic systems with low humidity, substrates with low thermal conductivity, and a continuous gas phase. These produce a shortage in heat transfer and with it, an increase in localized temperature, decreasing humidity. Therefore, substrates with high WAI and low CHP are preferable for its development, allowing the incorporation of water without compromising the aeration of the system and its productivity [36, 37]. Therefore, these substrates are suitable to be used with SSF.

Radial growth rate

Figure 1 shows the growth kinetics of the different strains on the evaluated substrates. Table 3 details the growth rates of the different strains. It is observed that T. asperellum AFP with CT as support presented the highest radial growth rate 0.409 ± 0.021 mm/h, followed by A. niger 28 A and 20A on T with 0.224 ± 0.004 and 0.224 ± 0.030 mm/h, respectively.

Fig. 1
figure 1

Radial growth rate 20A TC (A. niger 20A, 50% mix), 20A T (A. niger 20A, 100% green tea), 20A C (A. niger 20A, 100% coffee pulp), 28A TC (A. niger 28A, 50% mix), 28A T (A. niger 28A, 100% green tea), 28A C (A. niger 28A, 100% coffee pulp), AFP TC (T. asperellum, 50% mix), AFP T (T. asperellum, 100% green tea), AFP C (T. asperellum, 100% coffee pulp)

Table 3 Radial growth rate

The A. strains showed very similar speeds to each other, depending on the substrate. Considering that both substrates have very similar values in soluble reducing sugars, the difference exhibited in radial growth rates is not associated with this factor. The growth rate of the Aspergillus strains is higher in T, followed by TC and finally in C.

This behaviour follows the expected trend, given by the WAI and CHP values. Because T has the highest WAI concerning its CHP, which favours the development of metabolic functions and with it the microbial growth [36]. Likewise, T and TC have the pH closest to 5, considered to be the optimum for the development of Aspergillus [28]. Also, C has some components with antifungal properties, such as ferulic acid [38] and caffeine [13, 39], that can hinder the development of the proven Aspergillus strains.

Trichoderma presented the highest growth using TC as a support, and its speed decreased when using C following the same pattern of the Aspergillus strains provided by the factors of WAI, PHC, and pH. However, he was inhibited using T as a support. This may be an indication that the valued Trichoderma strain shows sensitivity to some antifungal components of tea, such as EGCg (epigallocatechin-3-gallate) and phenyl lactic acid [40], which had a decreased concentration in TC.

Endocellulase activity plate assay

The degradation halos produced by the plate technique were analysed through a Tukey mean test (p > 0.05). The widest degradation halos were produced by the extracts obtained from treatments 28A TC, 28A T, 20A T. They did not show a significant difference concerning the control (Figs. 23).

Fig. 2
figure 2

Degradation haloes specified by the different extracts recovered on CMC plates stained with 1% Congo Red. 20A TC (A. niger 20A, 50% mix), 20A T (A. niger 20A, 100% green tea), 20A C (A. niger 20A, 100% coffee pulp), 28A TC (A. niger 28A, 50% mix), 28A T (A. niger 28A, 100% green tea), 28A C (A. niger 28A, 100% coffee pulp), AFP TC (T. asperellum, 50% mix), AFP T (T. asperellum, 100% green tea), AFP C (T. asperellum, 100% coffee pulp), B (blank, destilled water), C (positive control, Trichoderma reesei cellulases (SIGMA)

Fig. 3
figure 3

Degradation haloes specified by the different extracts recovered on CMC plates stained with 1% Congo Red. 20A TC (A. niger 20A, 50% mix), 20A T (A. niger 20A, 100% green tea), 20A C (A. niger 20A, 100% coffee pulp), 28A TC (A. niger 28A, 50% mix), 28A T (A. niger 28A, 100% green tea), 28A C (A. niger 28A, 100% coffee pulp), AFP TC (T. asperellum, 50% mix), AFP T (T. asperellum, 100% green tea), AFP C ( T. asperellum, 100% coffee pulp)

The highest degradation halos were generated by the extracts produced by the A. niger strains. This phenomenon is associated with what has been reported by Ximenes et al. [41], who indicate that Aspergillus cellulases are more resistant than Trichoderma cellulases to the inhibitory effect of polyphenols, which are found in abundance in both substrates [42, 43].

In the case of the Aspergillus strains, the highest activities were produced using T and TC as support. Both substrates have a pH quite close to 5.5, in which it is reported that the optimum point of production of a tannin-degrading protein complex is presented, inhibiting the antimicrobial effect produced by the tannins present in the worked substrates. Furthermore, they are within the range of pH 4.5–5 in which higher cellulase titers are usually produced [44], further highlighting that both T and TC have the highest WAI and CHP, which allows a better development of metabolic functions [36], and a lower caffeine content that also affects enzyme production [45].

The 28A T and 20A T treatments presented the highest cellulase activities by plate method and the highest growth rates, being significantly the same in both parameters. For this reason, the 28A T treatment was selected to continue the work.

Evaluation of the use of mineral enrichment media in the production of cellulase enzymes using green tea residues as support

The enzymatic activities obtained with each treatment were analysed through a Tukey mean test (p > 0.05). Production of 2.4 ± 0.3, 2.2 ± 0.42, and 2.5 ± 0.45 FPU/g was obtained using water, Mandel's medium, and Czapek-Dox, respectively. Enzymatic titers comparable to those obtained by other authors were obtained (Table 4). It was found that there is no significant difference in the production of FPU activity when using water or the Mandels and Czapek–Dox mineral enrichment media (Fig. 4).

Table 4 Comparison of FPU activity production with other fungal strains under SSF
Fig. 4
figure 4

Evaluation of the use of mineral enrichment media (Mandels, Czapek-Dox, and distilled water) in the production of total cellulase activity by A. niger 28A using green tea residues as support

The induction of cellulase induction is strongly influenced by carbon, nitrogen, and physical parameters such as pH, temperature, and incubation time [32]. The lack of an effect that drives the production of cellulase activity by the mineral media can be associated with a saturation of nutrients in the medium. High concentrations, some minerals like Mn, Co, Zn, and Fe, can cause a decrease in cellulase activity yields [31]. These results indicate that substrate T has sufficient nutrients intrinsically and does not require additional supplementation for the production of total cellulase activity by A. niger 28A.


The use of strains 28A on green tea residues and a mixture of substrates as support and 20A using green tea residues are the most viable options for the production of cellulases through solid fermentation with this type of material. Green tea residues turn out to be a suitable material for the production of cellulases through solid fermentation and do not require additional supplementation of mineral enrichment media.


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The authors thank the financial support given by the National Council of Science and Technology (CONACYT-Mexico) through the project FONCICYT-CONACYT-SRE-C0013-2015-03-266614, which was implemented within a framework of bilateral cooperation between Mexico and India. Author Salvador A. Saldaña Mendoza thanks CONACYT-Mexico as well as the Autonomous University of Coahuila for the financial support and the scholarship for the development of their master's studies.

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Saldaña-Mendoza, S.A., Ascacio-Valdés, J.A., Palacios-Ponce, A.S. et al. Use of wastes from the tea and coffee industries for the production of cellulases using fungi isolated from the Western Ghats of India. Syst Microbiol and Biomanuf 1, 33–41 (2021).

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  • Cellulases
  • Fungal strains
  • Solid-state fermentation
  • Agroindustrial wastes
  • Coffee pulp
  • Green tea