Original Article




In search of native cellulase-producing microorganisms, collected from lignocellulosic waste in Ecuatorian Andes, Amazon region, and in Antarctica, 17 sporulating strains were found. These strains and the cocktails obtained from them were used in assays to measure glucose concentration produced from carboxymethylcellulose, crystalline cellulose and filter paper units (FPU). The obtained values were that only the cocktail from Bacillus sp. collected at a “panela” artisan factory produced glucose concentrations that might be of interest, after the cellulases degraded all substrates: carboxymethylcellulose, crystalline cellulose and filter paper. The amount of glucose liquor obtained in the best assay was measured in HPLC, producing 3.45 mg/ml of glucose after the action of endo-beta-glucanase and 1.8 mg/ml of glucose in presence of exo-glucanase. The amount of filter paper units produced in vitro were 0.0073 FPU. However, the study found that, since the Payback Period for the investment was 6 years, it would not be recommendable to invest in a production plant with these cocktails. Due to this, is recommended to continue the research with this strain of Bacillus sp. to improve the dynamic indicators.

Key words: 

Bacillus; bagasse; cellulases; enzymatic; cocktail; hydrolysis.


En la búsqueda de microorganismos nativos productores de celulasa, recolectados en desechos lignocelulósicos en los Andes, Amazonia y Antártida, se encontraron 17 cepas esporulantes. Estas cepas y los cocteles provenientes de éstas, fueron empleados en ensayos para medir la concentración de glucosa a partir de carboximetilcelulosa; celulosa cristalina y papel filtro (UPF) producidos por la acción de los microorganismos y de los cocteles enzimáticos. Se obtuvo como resultado que solamente el coctel de Bacillus sp., proveniente de una fábrica de panela, produjo concentraciones de glucosa que pueden ser interesantes, después de que las celulasas degradaran todos los sustratos: carboximetilcelulosa, celulosa cristalina y papel filtro. La cantidad de glucosa de licor obtenida en el mejor ensayo se midió en HPLC, produciendo 3.45 mg/ml de glucosa después de la acción de la endobetaglucanasa y 1,8 mg/ml de glucosa en presencia de la exoglucanasa. La cantidad de unidades de papel filtro producidas in vitro ha sido de 0,0073 UPF. Sin embargo, el estudio logró identificar que siendo el Período de Recuperación de la Inversión (PRI) de 6 años no sería recomendable invertir en una planta de producción de estos cocteles. Por estas razones se recomienda continuar las investigaciones con esta cepa de Bacillus para mejorar los indicadores dinámicos.

Palabras clave: 

Bacillus; bagazo; celulasas; enzimática; coctel; hidrólisis.


Bioprospecting or biodiversity prospection is defined as the exploration of biodiversity for new resources of social and commercial value (Beattie et al., 2011). Historically, humans have been bioprospecting for all their existence on Earth. Societies have been and still are committed to obtain as many useful things as possible from the nature around. In modern times, the frontiers have been pushed from animals and plants to bacteria, fungus, virus, etc. regarding where the commercially valuable biochemical and genetic resources can come from. This is possible due to the new applications and developments that both demand and enable the discovery of new products. One example is the recent improvements in metagenomics (Madhavan et al., 2017. These efforts can be channeled for better results, such as the case of Colombia, which has been developing with a broad perspective of providing greater benefits for the nation (Malgarejo, 2013). This is an activity of nature exploration, non-destructive, which, through scientific research, aims to obtain useful information derived from the collection of small quantities of biological material for application in medicine, agriculture and industry (Setzer et al., 2003).

Enzymes are biological catalysts and most are of microbial origin (Prabha et al., 2007, Hairchar, 2007). Microbial cellulolytic enzymes are essential for the degradation of cell walls of plants. However, there are limitations for their industrial use, such as the vast diversity of microorganisms in the environment and the complexity to culture them (Gomashe and Bezalwar, 2013). Fungi and bacteria are groups that present extracellular enzymes secreted in the medium in which they are cultured. The mechanism of degradation of cellulose and how microbial communities carry out this work is not yet clear. There are two types of enzymes: extracellular and intracellular. The waste of plant biomass on which they catalyze is composed of lignin, cellulose and hemicellulose (de Gonzalo et al., 2016). Enzymatic hydrolysis of cellulose by microorganisms is a key step in the global carbon cycle, which explains both the fossil fuels deposits and the bioavailability for current living beings. Cellulose degradation by microorganisms is not unique, there being at least five ways to do it with the indispensable presence of cellulases (Wilson, 2011). These microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. One of the biggest industrial interests is bioethanol production, which has experienced considerable impulse in the recent years, due to which many lines of research have been followed (Acharya and Chaudhary, 2012). Some of their recent developments involve metagenomics oriented towards the improvement of processes in biorefineries through identifying new cellulases (Tiwari et al., 2017). One of the agro-industrial products of interest for bioethanol, due to its large amounts of cellulose, is bagasse from sugarcane (Pandey et al., 2000).

The objective of the study was to find microorganisms native to Ecuador, if not better suited or more productive regarding their cellulolytic activity, at least capable of locally and inexpensively produce regular-performing cellulolytic enzymes that can be scaled up to an industrial setting for the degradation of bagasse and perform the analysis of the cost of investment and total production.


2.2. Qualitative and quantitative screening of cellulolytic enzymes

The isolated sporulating strains were cultured in CMC medium (Carboxymethylcellulose), following the protocol by Shi et al., (2011). For the quantitative assays, an approximate concentration of 5.67 * 109 CFU/g of each purified strain was inoculated into medium specific for endo-, exo-glucanase and filter paper (Lin et al., 2009). In addition, the filter paper activity was determined and calculated according to the methodology established by Adney and Baker (1996) evidenced with dinitrosalicylic acid (Miller, 1959). To evaluate the differences, the 95% confidence intervals for average degradation were established.

2.6. Economic technical analysis

A production of 50 000 l of ethanol per day was considered. The conditions were the same ones that were maintained in this investigation in the pre-fermenter and fermenter stages. From these data, a scale-up was performed for a production of 2 320 l of bacteria per day, with an enzymatic cocktail production of 1.72 tons of cocktails in 15 hours. The dynamic indicators Net Present Value (NPV), Internal Rate of Return (IRR) and Payback Period (PP) were also calculated.


3.1. Isolation of sporulating microorganisms

Hundreds of bacteria and fungi were isolated, being the Balzapamba (Andes) sample the most diverse with a figure of 2.85 CFU. Of these, only those that formed spores were studied (shown in Table 1). The strains with the highest production of cellulases belong to the bacterial species Bacillus sp and the fungus Scopulariopsis sp., according to morphological and biochemical analysis. Literature shows that this fungus belongs to a group that causes fungal tinea.

Table 1. 

Amount of sporulating microorganisms (Tolerate industrial stressful conditions)

RegionAmount of microorganismsSporulating organisms
Andes2.8 E52.2 E5
Antarctica1.3 E50.8 E3
Amazonia1.3 E41.2 E4

Source: Own construction (2018) for all figures and tables, except when stated otherwise

3.2. Screening of cellulase producing strains

17 sporulating strains producing interesting degradation halos were isolated, as shown in Figure 2. Of these, the fungus produces 6.5 mm of halo, this is the greatest degradation. The bacterial strains Y3 and BAL3 show heterogeneous results due to their variability (Figure 3). Species and cocktails from Antarctica show a low degradation of less than 1 mm.


Figure 2. 

Average degradation (mm) of the strains and confidence intervals at 95%

3.3. Quantitative assays for cellulase production

It was found that the enzymatic cocktails have a higher endoglucanase, exoglucanase and filter paper activity, unlike microorganisms. The results obtained for FPU are: 0.0073 FPU for the Bal 3 bacteria; 0.0041 FPU for the fungus, and 0.0012 FPU for Ant 12. The glucose concentration produced in the endoglucanase assays are: 0.0001 mg/ml for Bal 3; 0.0007 mg/ml for Fungus, and 0.0001 mg/ml for Ant12. The reducing sugar concentration produced in the exoglucanase assays are: 2.7 mg/ml for Bal 3; 3.4 mg/ml for Fungus, and 8.7 mg/ml for Ant12.


Figure 3. 

Average strain degradation (Abs) and 95% confidence intervals

The glucose concentration values, obtained from enzymatic cocktails using mathematical models with an R2 of 0.99, for the endobetaglucanase activity are: 2.9 mg/ml, 0.0 mg/ml and 0.8 mg/ml for Fungus, Ant12 and Bal 3, respectively.

In relation to exobetaglucanase activity, the glucose concentration values were 1.0 mg/ml; 5.9 mg/ml and 1.8 mg/ml for fungus, Ant12 and Bal 3. The results obtained for FPU are: 0.013 FPU for the Bal 3 bacteria; 0.0095 FPU for the fungus, and 0.0012 FPU for Ant 12.

3.4. Economic Technical Analysis of Bacillus sp.

For the scale-up, the conditions produced by the Bacillus sp. species (Bal 3) were used. The cost estimates of the investments were made by the methods recommended by (Peter and Timmerhaus, 1991).

Salvador et al., (2018) presented a chart showing the projected equipment expenses increase through the years for industrial purposes. This chart was taken as a reference to calculate the production costs.

Table 2. 

Calculation of the investment value for the production of the enzymatic cocktail from the bacteria Bacillus sp

NConceptEstimated USDAmount USD
IDirect Costs (DC)A+B+C+D453 629
A. Equipment + othersS (1 a 5)402 944
1. Equipment purchase costs (EC)571 776253 424
2. Installation including isolation and painting.35 % EC88 698
3. Installation of instruments and control6 % EC15 205
4.Pipes installation10 % EC25 342
5. Electric installations8 % EC20 274
B. Buildings10 % EC25 342
C. Facilities and services10 % CE25 342
D. Land0%0
IIIndirect Costs (IC)A+B+C81 176
A. Engineering and supervision5 % DC22 681
B. Construction and hiring expenses7 % DC31 754
C. Contingency5 % IFC26 740
IIIInvested Fixed Capital (IFC)I + II534 805
IVWorking Capital10 % TWC59 423
VTotal Working Capital (TWC)III + IV594 227

Table 3. 

Total Cost of producing bacterial enzymes, taking into account raw materials

Total Production CostPrice $/UMAmountUMCost M$/year
1. Manufacturing Expenses 444 289
Direct costs 296 789
1.Raw Materials 175 414
Nutritious broth5003062.4Kg/a165 000
Water0.0011 405 800L/a1500
2. Operational labor10 %TPC 85 000
3. Direct supervision 10 % operational labor10 % of 2 8500
4.Utilities and services
Water0.00116 000L/a240
5.Maintenance and reparation % de IFC 1058
6. Supplies % of 5 52.9
7. Laboratory charges 8500
8. Patents % CTP 8500
1. Depreciation 10 % IFC 52 910
2. Local fees 1-4% IFC 52 911
3. Taxes 0,4-1% IFC 2116.4
II. General Expenses 403 289
A.Marketing and sales 17 000
B. Management 17 000
C. Research and Development 17 000
D. Financial Interests 5291.0
III. Total Production Cost 827 578

It can be noted in Table 3 that the production cost is $ 827 578 per year. This, divided by 50 000 liters of ethanol per day and by 330 days, gives the cost of enzyme production per liter of ethanol as being $ 0.051. In relation to the commercial enzyme whose cost is USD 0.059 per liter of ethanol (Salvador et al., 2018), the cost reduction is $ 0.0075 per liter of ethanol produced. This cost reduction means a total of $ 123 750 per year, as shown in table 4.

Table 4. 

Cost reduction in enzymatic expenses when using the enzymatic cocktail

EnzymeCost per liter
Cocktail Bacillus sp$ 0.0515
Commercial enzyme$ 0.0590
Cost reduction$ 0.0075

In addition, cost reductions are obtained in investment of foreign currency expenditures due to unnecessary purchases, since the necessary supplies are provided in Ecuador for an amount of $ 517 325.12 (Table 5, Table 6). This in turn has a stimulating effect in domestic economy.

Table 5. 

Investment cost reductions

Production investmentCost ($)
Total Investment587 896
Expenses70 571
Expenses reduction517 325

There is a cost reduction in raw materials since locally produced supplies are used for an amount reflected in Table 6.

Table 6. 

Raw materials cost reduction

Raw materialCost ($)
Bagasse7 260
Bacteria1 654
Water (raw material)1 500
Water (general use)240
Whey and sugar cane bagasse77 220
Total87 874

In total, a cost reduction in annual imports of $691 649 is achieved, which allows determining investment recovery in relation to expenses abroad (Table 7).

Table 7. 

Amounts for production investments

SuppliesPricePercentage (%)
Total$827 578100
Imported$158 35119.13
National$669 22780.87

What is observed in Table 7 are the cost reductions in imports, since 80,87% of the expenses that will be made for the production of the enzymatic cocktail, use a large number of raw materials locally produced and easily accessible, and the engineering design and manpower will be trained Ecuadorian professionals, encouraging the implementation of new technologies, and innovating production processes. This has a direct impact on the country's technological development.

The following results were obtained: NPV (Net Present Value), $ 198 386.2; IRR (Internal Rate of Return), 19% and PP (Payback Period) of 6 years, Figure 5.


Figure 5. 

Payback period

In addition, as shown in Figure 5, the payback period of 6 years starts after the full operation of the production plant of cellulolytic enzymes from Bacillus sp. This time may well serve as a reason to determine that it is not convenient to put it into practice, since the time of money immobilization is maybe too long. A recommendable payback time should be three years or less.


  1. In order to obtain the degradation of lignocellulosic residues in the enzymatic hydrolysis stage, extra cellular enzymatic cocktails are better, unlike the use of microorganisms in the substrate in which the culture medium hydrolysis takes place.

  2. Of the 17 microorganisms analyzed, bacteria of the genus Bacillus was isolated and exhibited activity on crystalline cellulose substrate. This shows that it is feasible to investigate microorganisms that produce a cocktail of autochthonous enzymes.

  3. Biodiversity is a sustainable source that generates value-added products that can provide technological independence.

  4. Through technical economic analysis, it was determined that in the production process of native Bacillus sp. enzymes, cost reductions of ethanol production and investment are achieved, which can have a positive impact on the national economy of Ecuador.


Acharya, S. and Chaudhary, A., Bioprospecting thermophiles for cellulase production: a review., Brazilian Journal of Microbiology, Vol. 43, No.3, 2012, pp. 844-856.

Adney, B. y Baker, J., Measurement of Cellulase Activities., Chemical Analysis and Testing Task Laboratory Analytical Procedure., National Renewable Energy Laboratory, Golden, Colorado, USA, 1996, pp. 4-30.

Beattie, A.; Hay, M; Magnusson, B; De Nys, R; Smeathers, J; Vincent, J; Ecology and bioprospecting; Austral Ecol. Vol.36, No. 3,2011,pp. 341-356.

González, E. y Castro, E., Aspectos Técnico-económicos de los estudios previos inversionistas para la producción de etanol de caña de azúcar. Editorial Gráficas La Paz, Torredonjimeno, Jaén, España, 1era Edición, 2012, pp. 34-54.

Gomashe, P.A. and Bezalwar, P.M., Isolation and Screening of Cellulose Degrading Microbes from Nagpur Region Soil., Int. J. of Life Sciences, Vol. 1, No. 4, 2013, pp. 291-293.

De Gonzalo, G., Colpa, D., Habib, M., Fraaije, M., Bacterial enzymes involved in lignin degradation., Journal of Biotechnology, Vol. 236, 2016, pp. 110-119.

Hairchar, F., Identification of cellulolytic bacteria in soil by stable isotope probing. Environ Microbiol, Vol. 9, No. 3, 2007, pp. 625-34.

Lauer, A., Simon, M.A., Banning, J.L., Andre, E., Duncan, K., Harris, R.N., Common cutaneous bacteria from the eastern redbacked salamander can inhibit pathogenic fungi., Copeia, Vol. 3, 2007, pp. 630-640.

Lin, L., Meng, X., Liu, P., Hong, Y., Wu, G., Huang, X., Li, C., Dong, J., Xiao, L,. Liu, Z., Improved catalytic efficiency of Endo-β-1,4-glucanase from Bacillus subtilis BME-15 by directed evolution., Applied Microbiology and Biotechnology, Vol. 82, No. 4, 2009, pp. 671-679.

Madhavan, R., Sindhu, R., Parameswaran, B., Sukumaran, R., Pandey, A., Metagenome Analysis: a Powerful Tool for Enzyme Bioprospecting., Applied Biochemistry and Biotechnology, Vol.183, No.2, 2017, pp. 636-65.

Malgarejo L., Bioprospecting as a possible development mechanism for Colombia., Acta. Biol. Colombia, Vol. 18, No. 1, 2013, pp. 19-30.

Miller, G. L., Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar., Analytical Chemistry, Vol. 31, No. 3, 1959, pp. 426-428.

Pandey, A., Soccol, C., Singh-Nigam, P., Soccol, V., Biotechnological potential of agro-industrial residues. I: sugarcane bagasse., Bioresource Technology, Vol. 74, No. 1, 2000, pp. 69-80.

Peter, M., Timmerhaus, K., West, R., Instructions for file “Equipment costs” accompanying Plant Design and Economics for Chemical Engineers, 5th edition, McGraw-Hill, USA, 1991, pp. 209-211.

Prabha, M., Jayaraaj, I. and Jeyaraaj, R., Comparative studies on the digestive enzymes in the gut of earthworms, Eudriluseugeniae and Eiseniafoetida., Indian Journal of Biotechnology , Vol. 6, No. 4, 2007, pp. 567-569.

Salvador, C., Mesa, A., Batallas, F., Villavicencio, J., Mesa, L., González, E., Assessment of the best operating conditions in the enzymatic hydrolysis of pretreated bagasse for bagasse ethanol., International Conference Series on Multidisciplinary Sciences, MOL2NET, Puyo, Ecuador, Diciembre, 2018, pp. 1-15. Disponible URL en: http://sciforum.net/conference/mol2net-03.

Setzer, M., Moriarity, D., Lawton, Ro., Setzer, W., Gentry, G., Haber, W., Phytomedical potential of tropical cloud fores tplant from Monteverde, Costa Rica., Rev. Biol.Trop, Vol. 51, No. 3-4, 2003, pp. 647-674.

Shi, Q.Q., Sun, J, Yu, H.L., Li, C.X., Bao, J., and Xu, J.H., Catalytic performance of corn stoverhydrolysis by a new isolate Penicillium sp. ECU0913 producing both cellulase and xylanase., Applied Biochemistry and Biotechnology , Vol. 164, No. 6, 2011, pp. 819-830.

Sidiras, D., and Salapa, I., Organosolv pretreatment as a major step of lignocellulosic biomass refining., Conferences international Biorefinery I: Chemicals and Materials From Termical Chemical Biomass conversion and related processes, Crete, Grecia, 2015, pp. 17-37.

Tiwari R., Nain L., Labrou N., Shukla P., Bioprospecting of functional cellulases from metagenome for second generation biofuel production: a review., Critical Reviews in Microbiology, Vol. 44, No. 2, 2017, pp.1-14.

Wilson, D.B., Microbial diversity of cellulose hydrolysis., Current Opinion in Microbiology, Vol. 14, No. 3, 2011, pp. 1-5.





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