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Acta Scientiarum 

 

http://www.uem.br/acta 
ISSN printed: 1679-9283 
ISSN on-line: 1807-863X 
Doi: 10.4025/actascibiolsci.v39i3.32384 

 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

Production of cellulases by Penicillium oxalicum through solid state 

fermentation using agroindustrial substrates  

Fabrícia Vieira Silva Bomtempo

1*

, Franciane Maria Mantovani Santin

1

, Raphael Sanzio 

Pimenta

2

, Deyla Paula de Oliveira

3

 and Emerson Adriano Guarda

1

 

1

Laboratório de Pesquisa em Química Ambiental e de Biocombustíveis, Universidade Federal do Tocantins, Avenida Ns 15, Al C 14, s/n,  

77015-016, Palmas, Tocantins, Brazil. 

2

Laboratório de Microbiologia Ambiental e Biotecnologia, Universidade Federal do Tocantins, Palmas, 

Tocantins, Brazil. 

3

Pró-Reitoria de Pesquisa e Pós-Graduação, Universidade Estadual do Tocantins, Palmas, Tocantins, Brasil. *Author for 

correspondence. E-mail: fabriciavs@gmail.com  

ABSTRACT. The purpose of this study was to define the factors that influence the production of 
cellulases by Penicillium oxalicum, a strain obtained from a leaf-cutting ant colony and identified based on 
the ITS gene. The experimental design included solid state fermentation using sugarcane bagasse and 
lignocellulosic sorghum as the lignocellulosic substrate. The variables were analyzed using a 2

5-1 

fractional 

factorial design, with three replicates on the central point. All the variables analyzed influenced the 
production of at least one of the three cellulose types analyzed. The highest values observed were: FPase 
4.2 U g

-1

, CMCase 9.2 U g

-1 

and Avicelase 8.4 U g

-1 

using lignocellulosic sorghum as the substrate. The 

best conditions for enzyme production were: incubation temperature at 40ºC, initial moisture of 60%, pH 
of 4.0 and a growth period of four days using lignocellulosic sorghum as the substrate.

 

Keywords: sugarcane bagasse; lignocellulosic sorghum; cellulolytic enzymes. 

Produção de celulases por Penicillium oxalicum em fermentação em estado sólido usando 
diferentes substratos agroindustriais 

RESUMO. O estudo teve como foco a determinação de fatores que influenciam a produção de celulases 

por uma cepa isolada de ninho de formigas cortadeiras e identificada por meio do gene ITS como 
Penicillium oxalicum. O processo produtivo foi  Fermentação em Estado Sólido utilizando como substrato 
lignocelulósico bagaço de cana-de-açúcar e sorgo lignocelulósico. As variáveis foram analisadas através de 
um planejamento fatorial fracionário 2

5-1

, com três repetições no ponto central. Todas as variáveis analisadas 

influenciaram a produção de pelo menos um dos três tipos de celulases analisados. As maiores atividades 
observadas foram: FPase 4,2 U g

-1

; CMCases 9,2 U g

-1 

e avicelase 8,4 U g

-1

, utilizando sorgo 

lignocelulósico como substrato. As melhores condições para produção foram: temperatura de incubação a 
40

o

C, umidade inicial 60%, pH 4,0, tempo de cultivo de quatro dias, utilizando como substrato sorgo 

lignocelulósico. 

Palavras-chave: Bagaço de cana-de-açucar, sorgo lignocelulósico, enzimas celulolíticas. 

Introduction 

Lignocellulose is the most abundant organic 

compound in the biosphere; however, only a small 

amount produced in agriculture or forestry is used, 

the rest being considered waste, causing consequent 

deterioration of the environment (Sánchez, 2009). 

Much is being done to reduce losses of this resource 

and to diminish the resulting environmental 

degradation (Ballesteros, 2001) through the 

generation of a series of high-value products and by-

products such as cellulolytic enzymes and cellulosic 

ethanol (Isikgor & Becer, 2015). 

Despite the potential of cellulose, it is highly 

crystalline and compact (Aristidou & Penttila, 2000; 
Gray, Zhao, & Emptage, 2006; Pérez, Muñoz-

Dorado, de la Rubia, & Martínez, 2002). 
Fortunately, some microorganisms, like Penicillium 
oxalicum
 are able to degrade this lignocellulosic 
material using hydrolytic and oxidative enzymes 
naturally produced by the microorganisms 
themselves (Xian, Wang, Yin, & Feng, 2016). These 
enzymes include cellulases, the main recruitable 
resource for the bioconversion of cellulosics to 
useful products, and usually the most costly part of 
the production process. In ethanol production, for 
example, the cost of cellulases is about 40% of the 
total cost of production.  

Commercially, the production of cellulases is 

carried out on purified cellulose such as avicel and 

solka floc, which is quite expensive. Therefore, it is 

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322 

Bomtempo et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

possible to produce large quantities of cellulase 
using materials like lignocellulosic residues, which 

have much lower costs and would allow for large 

economically viable bioconversions (Chandra, Kalra, 

Sharma, Kumar, & Sangwan, 2010) 

The strain P. oxalicum F-3380, was selected for 

identification and analysis in the present study 

because it showed the highest productivity among 

the isolates from leafcutter ant species’ nests 

(Acromyrmex balzani) previously tested. These ants 

cultivate symbiotic fungus, prune, clean the garden, 

and take the substrate rejected by the fungus to a 

disposal site located outside the nest (Caldato, 

Andrade, Forti, Barbieri, & Lopes, 2010). The close 

relationship of lignocellulose with microorganisms 

that exist in the nest reinforces the idea that these 

organisms have a high ability to produce a series of 

catalytic enzymes and degrade polymers, cellulose, 

hemicellulose and lignin, existing on the substrate 

on which they grow.  

The production of substrates for fermentation of 

these microorganisms and the consequent 

production of these lignocellulolytic enzymes are 

also a rational alternative for the use of 

agroindustrial lignocellulosic residues (Pandey, 

Soccol, Nigam, & Soccol, 2000). Solid State 

Fermentation (SSF) has emerged as a great resource 

for enzyme production by microorganisms; 

according to Hölker, Höfer, and Lenz (2004) the use 

of SSF has been shown to be particularly 

advantageous for the growth of filamentous fungi 
and the consequent production of enzymes, since it 

simulates their natural habitat and thus provides 

higher productivity when compared to the process 

of submerged fermentation. In addition, the 

enzymes produced by SSF are less susceptible to 

substrate inhibition problems and also have greater 

stability in temperature and pH variations.  

However, it is known that the variables that 

affect the SSF process are numerous and need to be 

analyzed, because the success of the enzymatic 

production depends on the type of microorganism 

used and the setting of the most significant 

operating variables of the production process. Thus, 

based on the biotechnological potential of P. 

oxalicum F-3380 (Xian et al., 2016) with the evidence 

of high enzymatic activity in the leafcutter ant nests 

(Caldato et al. 2010), in the face of the abundance of 

residual lignocellulosic material available in nature 

(Sánchez, 2009), and based on the need to obtain 

ideal conditions for the SSF process using the strain, 

the study aims to define the most influential factors 

on cellulase production from Penicillium oxalicum F-

3380, isolated from a leaf-cutting ant nest belonging 

to the species Acromyrmex balzani, using sugarcane 

bagasse SB and lignocellulosic sorghum LS as 
substrates. 

Material and methods 

Microorganism 

The filamentous fungus F 3380 was obtained 

from an Acromyrmex Balzani colony (Hymenoptera: 

Formicidae), which is a leaf-cutting ant. The strain 

belongs to the microorganism culture collection: 

Coleção de Culturas Carlos Rosa at Laboratório de 

Microbiologia Ambiental e Biotecnologia from 

Universidade Federal do Tocantins. It was chosen due to 

present high cellulolytic activity according to 

previous research. The microorganism was 

revitalized in Petri dishes containing PDA for 7 days 
at 25° C and, with the addition of distilled water, 

spore suspensions were obtained.

 

Molecular identification 

Genomic DNA was extracted from the strains 

following the protocol of Rosa, Vaz, Caligiorne, 

Campolina, and Rosa (2009). The ITS region was 

amplified and sequenced using the primers ITS1 

and ITS4 (White, Bruns, Lee, & Taylor, 1990). 

Amplification was performed in a Mastercycler

®

 

nexus thermocycler (Eppendorf) using the GoTaq® 

DNA Polymerase kit (Promega Corp., Madison, 

WI, USA) in a final reaction volume of 25 μL 
containing 13.375 μL of ultrapure water; 2.5 μL of 

25 mM MgCl

2

; 1.0 μL of 10 mM dNTPs; 5.0 μL of 

10 X buffer (100 mM Tris-HCl, pH 8.5, 500 mM 

KCl); 1.0 μL of the primer ITS1 (2 mM); 1.0 μL of 
the primer ITS4; 0.125 μL of 5 U of the enzyme 

Taq polymerase, 1.0 μL of DNA (50 ng μL

-1

). For 

the amplification cycles, an initial denaturation was 

performed at 95° C for 2 minutes, followed by 39 

denaturation cycles at 95° C for 1 minute; after that, 

the annealing of primers was performed at 52° C for 

2 minutes and extension at 72° C for 2 minutes. A 
final extension was performed at 72° C for 10 

minutes. 

After amplification, agarose gel electrophoresis 

was executed at 1% (w/v) containing GelRed

®

 

(Biotium Inc. California, USA), and 1 X TBE buffer 
(2 mM EDTA, 1 M Tris-HCl, 0.1 M boric acid, pH 
8.0 with the PCR product to see if amplification had 
occurred. It was visualized under ultraviolet light in 
a LPIX-EX imaging system (Loccus Biotechnology). 
The 1 Kb DNA Ladder (Promega Corp., Madison, 
WI, USA) was used as a molecular marker. 

The amplified product was purified with a 

solution of Exonuclease I and Shrimp Alkaline 

Phosphatase (ExoSAP-it

®

) per the manufacturer's 

instructions. After purification, the amplified 

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Production of cellulases by Penicillium oxalicum 323 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

fragment was sequenced in both directions using the 
same PCR primers in an ABI 3500 XL automated 

sequencer (Life Technologies) according to the 

dideoxy  or chain-termination method (Sanger, 

Nicklen, & Coulson, 1977) and using the BigDye 

Terminator kit v3.1 (Life Technologies). This step 

was carried out at the Laboratório de Polimorfismo de 

DNA of the Universidade Federal do Pará (UFPA). 

After automatic sequencing, the nucleotide 

sequences of the microorganism were imported into 

the Geneious 6.1.8 program (Kearse et al., 2012) to 

be analyzed, aligned and edited. The consensus 

sequence, which was generated from the forward 

and reverse sequences, was manually corrected and 

aligned. This sequence was exported into a FASTA 

file extension (.fasta*/*. Fas) for further analysis, to 

search for and compare sequence identities in the 

GenBank database (www.ncbi.nlm.nih.gov/blast/) 

and sequences in the CBS Database 

(http://www.cbs.knaw.nl/Collections/). 

Substrates (SB) (LS) and their pretreatment 

Sugarcane bagasse (SB) was obtained from the 

company Bunge (Pedro Afonso, Tocantins, Brazil) 

and lignocellulosic sorghum (LS) was obtained from 

the company EMBRAPA Milho e Sorgo (Sete Lagoas, 

Minas Gerais, Brazil). SB and LS were passed 

through a sieve with a 20 mesh particle size and 

treated with 4% sodium hydroxide in an autoclave at 

1.1 kg cm

-2

 of pressure for 15 minutes. After that, 

the pH was adjusted using phosphoric acid (pH 2 

was maintained for 30 minutes) and the liquid 

portion was discarded; then, the solids were 

autoclaved with distilled water 1:1 (w/s) and dried at 

65° C to a constant weight. 

Enzyme production 

All enzyme production was conducted in 250 mL 

Erlenmeyer flasks with each flask containing 5 g of 

substrate and the mineral salt solution described by 

Mandels and Weber (1969). The flasks were 

sterilized in an autoclave for 15 minutes, then 

cooled, and after that, inoculated with fungal spore 

suspension and incubated under constant stirring at 

150 rpm. The variables were adjusted according to 

the experimental design. Enzyme extraction was 
done by adding citrate buffer (0.05 M, pH 4.8) 

followed by agitation for 1h, simple filtration and 

finally storage at -20° C for later analysis (Aguiar & 

Lucena, 2011). 

Enzyme assay 

Endoglucanase activity (CMCase) was 

determined according to the method described by 

Ghose (1987) using a reaction mixture containing 

0.5 mL of the enzyme solution with 0.5 mL of 2% 
carboxymethylcellulose solution in citrate buffer 

(0.05M, pH 4.8), which was incubated at 50°C for 

30 minutes. Exoglucanase (Avicelase) activity was 

determined according to that described for 

endoglucanase activity, but the incubation was 

carried out with 0.5 mL of 1% avicel suspension 

instead of carboxymethylcellulose. The FPase 

activity (filter paper activity) was determined by the 

Ghose (1987) method using 0.5 mL of the enzyme 

solution and a whatman n. 1 filter paper strip (1 x 6 

cm; 50 mg) immersed in 1.0 mL of 0.05 M citrate 

buffer at a pH of 4.8, with incubation at 50° C for 30 

minutes. 

The reducing sugar released was estimated using 

the DNS method described by Miller (1959). One 

unit (U) of enzyme activity in each case was defined 

by the amount of enzyme that produces one μmol of 

glucose from the substrate per minute of the 

reaction. The activities were expressed in U g

-1

 of 

dry substrate used in the process (U g

-1

 s).  

Experimental design and Statistical Analysis 

A 2

5-1 

fractional factorial design was performed with 

16 observations and 3 replications in the central point, 

according to Rodrigues and Iemma (2005). The tested 

variables were: incubation temperature (TPT), 

moisture content (M), pH of the mixture (pH), 

incubation time (T), and inoculum concentration (IC); 

the responses were filter paper activity (FPase), 

endoglucanase activity (CMCase) and exoglucanase 

activity (Avicelase). All the results were analyzed using 

the software Statistica

®

 10.0 (Statsoft Inc., Tulsa, OK, 

USA), with a significance level of 95%. 

Results and discussion 

Fungal strain identification 

This study molecularly identified the F33805 

strain from the Coleção de Culturas Carlos Rosa, 

Universidade Federal do Tocantins. The sequence, 

which was generated using the ITS gene, shows 98% 

similarity with the strain Penicillium oxalicum 

(GenBank Accession LT558936.1) (Guevara-Suarez 

et al., 2016). For this reason, it was identified as 

Penicillium oxalicum and its identity was confirmed in 

the CBS Database http://www.westerdijkinstitute.nl/ 

collections/), where it presented 99% similarity 

(IHEM Accession 2931).  

Species from the genus Penicillium  have been 

described as potential producers for commercial 

cellulases (Saini et al., 2015). Zhang et al. (2014) 

described fungi with cellulolytic activity, which were 

isolated from a subtropical and tropical forest in 

China and mostly from the genus Penicillium. 

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324 

Bomtempo et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

Difference between the evaluated substrates (SB) (LS) 

The mean the of values obtained from the 

enzymatic activity with sorghum was higher than 

the ones using sugarcane bagasse (Figure 1). The 

activity using sugarcane bagasse varied between 3.39 

and 0.32 U g

-1

 for FPase activity; 3.52 - 0.17 U g

-1

 

for CMCases and 1.85 - 0.32 U g

-1

 for Avicelase 

activity. The cellulolytic activities obtained using LS 

varied between 4.2 and 2.2 U g

-1

 for FPase activity; 

9.2 - 2.2 U g

-1

 for CMCases and 8.4 - 1.8 U g

-1

 for 

Avicelase activity. 

Rocha, Barros, Fischer, Coutinho-Filho, and 

Cardoso (2013), upon analysis of different 

substrates’ ability to induct the production of 

cellulolytic enzymes by Aspergillus niger, found that 

the highest values were obtained using sorghum, 

approximately 38 U g

-1

 compared to 5 U g

-1

 with 

sugarcane bagasse. 

 

 

Figure 1. Mean of cellulolytic activity obtained using sugarcane 
bagasse and sweet sorghum as carbon sources. 

Latifian, Hamidi-Esfahani, and Barzegar (2007) 

evaluated the effect of the following variables: initial 

pH, fermentation medium content and temperature, 

using rice straw as a substrate and a genetically 

modified strain of Trichoderma reesei; as a result, they 

obtained a maximum FPase activity of 1.1635 U g

-1

 

under the following conditions: pH 5.0, medium 

containing 70% rice straw and incubation 

temperature was 30° C. However, Scheufele, Butzke, 

Marra, Hasan, and Fiorese (2012), in analyzing the 

effects of the following variables: time of hydrolysis, 

enzymatic dilution, concentration of Tween 80 and 

the solid:liquid ratio in SSF using Trichoderma sp

obtained a maximum enzymatic activity of 2.778 U g

-1

Basso, Gallo, and Basso (2010) used sugarcane 

bagasse as a substrate and obtained a maximum 

FPase activity of 2.3 U g

-1

 on the fifteenth day of the 

experiment. Singhania, Sukumaran, and Pandey 

(2007) obtained total cellulase activity of 0.17 U g

-1

 

after 5 days of culture, using T. reesei RUT C30 in a 

wheat bran substrate. The current study observed 

greater values than those described above, with 

relatively less incubation time, which indicates that 

the filamentous fungus used has cellulolytic capacity 

and that sugarcane bagasse and sorghum are good 

substrates for SSF using this microorganism.  

Fractional factorial design 

The results of cellulolytic activity (FPases, 

CMCases, Avicelases) obtained from the 2

5-1 

fractional factorial design using sugarcane bagasse 

and Lignocellulosic sorghum as substrates are shown 

in Table 1. 

Table 1. Fractional factorial design 2

5-1 

obtained during SSF 

using sugarcane bagasse and lignocellulosic sorghum as 
substrates.

 

Tests 

Factors 

Substrate Yield (U g

-1

TPT 

(X1)

(X2)

pH

(X3)

(X4)

Ci 

(X5) 

SB LS 

1* 2* 3* 1* 2* 3* 

1 30 

60

10

8

  0.5 0.9 0.3 2.5 4.5 4.8

2 40 

60

10

6

  0.3 0.6 0.3 4.2 9.2 8.4

3 30 

80

10

6

  3.3 1.3 1.6 2.5 5.3 4.5

4 40 

80

10

8

  2.0 1.3 0.2 3.1 5.3 4.6

5 30 

60

10

6

  1.4 1.3 1.8 2.8 4.4 6.4

6 40 

60

10

8

  2.5 0.9 0.3 3.3 4.1 3.7

7 30 

80

10

8

  1.8 2.8 0.6 2.9 4.6 2.2

8 40 

80

10

6

  1.3 1.2 0.2 2.3 5.4 4.5

9 30 

60

12

10

6

  0.8 0.6 0.1 2.4 4.0 4.7

10 40 

60

12

10

8

  0.4 0.1 0.2 3.5 5.4 4.2

11 30 

80

12

10

8

  2.7 1.4 1.4 2.3 3.2 2.7

12 40 

80

12

10

6

  1.6 0.8 0.4 2.3 3.0 2.2

13 30 

60

12

10

8

  0.7 0.1 0.4 3.1 2.3 2.8

14 40 

60

12

10

6

  1.6 0.8 1.3 2.5 4.6 5.6

15 30 

80

12

10

6

  2.7 3.5 0.3 2.3 4.2 3.4

16 40 

80

12

10

8

  1.3 1.0 1.5 2.2 3.1 6.5

17 35 

70

5x10

7

  1.3 0.9 0.9 2.9 2.8 1.8

18 35 

70

5x10

7

  1.4 0.7 1.0 3.3 2.2 2.3

19 35 

70

5x10

7

1.3 1.0 0.8 2.8 4.6 3.5

TPT - Incubation temperature (

o

C); M- initial moisture content (wt%); pH- initial pH 

of the mixture; T- incubation time; Ci - inoculum concentration; SB - sugarcane 
bagasse; LS - Lignocellulosic sorghum; 1* - FPase; 2* - CMCase; 3* - Avicelase. 

The data indicates that there was a large variation 

in cellulase activity in the different assays carried 
out; this variation increases the importance of the 
experimental design used in this study because it 
may contribute to achieving higher enzyme 
production by adjusting the variables involved in 
SSF. 

Another way to confirm the importance of 

orienting experimental design to improve SSF 

conditions is to observe the values of enzymatic 

activity presented in the Central Point (CP) of the 

experiment (tests 17, 18 and 19). CP values were 

established, based mostly on the best results of 

similar cultivations performed in previous studies 

such as Kang, Park, Lee, Hong, and Kim (2004); 

Gao, et al. (2008) and Zhang and Sang (2012). From 

these values, ranges of the variables were expanded. 
CP tests resulted in an average activity of 1.1 U g

-1

 

of SB and 2.9 U g

-1

 of LS; this shows that the 

experimental design performed promoted an average 

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Production of cellulases by Penicillium oxalicum 325 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

increase in enzymatic activity of about 70% in 
sugarcane and 20% in sorghum, considering the 

highest values obtained in the experiment.  

It is possible to observe in Table 1 that, for the 

assays using sugarcane bagasse as their substrate, the 

highest FPase activity (3.3 U g

-1

) was in test 3, which 

was performed at a temperature of 30º C; initial 

moisture of 80%; pH of 4.0; over four days and an 

inoculum concentration of 10

spores mL

-1

. In 

comparison, the highest CMCase activity (3.52 U g

-1

was in test 15, which was performed with an initial 

incubation temperature of 30º C; initial moisture of 

80%; pH of 8.0; over twelve days and an inoculum 

concentration of 10

spores mL

-1

. Also, the highest 

activity of avicelase (1.85 U g

-1

) was seen in test 05, 

which was performed at an incubation temperature 

of 30º C; initial moisture of 60%; pH of 8.0; over 

four days and an inoculum concentration of 10

spores mL

-1

.  

In contrast, the results of fermentation using 

sorghum showed that the highest activities for FPase 

(4.2 U g

-1

), CMCase (9.2 U g

-1

) and Avicelase (8.4 

U g

-1

) were provided by combining test 2 levels, 

with a temperature of 40º C; 60% initial moisture; 

pH  of  4.0;  four  days  of  incubation  and  an  initial 

inoculum concentration of 10

8

 spores mL

-1

Comparisons of cellulase activity produced by 

different fungal strains can be seen in Table 2. 

Effect of the analyzed variables 

Table 3 shows the significant effects of the 

factors and their interactions on FPase activity. 

Initial moisture influences the activity of FPase in 

both SB and LS substrates and this influence is 

positive for the SB substrate (1.07 U g

-1

), which 

means  that  due  to  a  rise  in  moisture  from  60%  to 

80%, there was a gain in activity of 1.07 U g

-1

According to Martins, Kalil, and Costa (2008), low 

moisture reduces the solubility of the solid 

substrate, the swelling index and produces high 

superficial tension; this can explain the low yield 

presented in the tests with low moisture. However, 

the LS substrate’s high values for initial moisture 

negatively affected FPase activity (-0.55 U g

-1

), 

which means that due to the increase in initial 

moisture from 60% to 80%, there was a reduction of 

0.55 U g

-1

 in enzymatic activity. According to 

Hölker et al. (2004), this fact can be related to 

fungus inhibition due to extrapolation of the water 

level. 

The divergence of effects on the SB and LS 

substrates can be explained by natural and structural 

differences between the materials utilized, the 

chemical composition of the substrate itself and the 
distribution of its main components, such as lignin, 

hemicellulose and cellulose (Aguiar & Ferraz, 2011). 
According to Hölker et al. (2004), the appropriate 

level of moisture in solid state fermentation is 

variable, depending not only on the needs of the 

microorganism and the expression of desired 

metabolites, but also on the nature of the substrate 

used. This difference is a factor that may indicate an 

advantage for using sorghum as a substrate, in the 

sense that one problem with this type of enzyme 

production on a large scale is maintaining the same 

moisture levels even with a natural increase in 

temperature during SSF. Due to the fact that the 

studied microorganism is able to produce more in a 

sorghum substrate with low moisture, the 

maintenance of fermentation would be facilitated.  

It was also observed (Table 3) that combinations 

of higher temperatures caused a reduction in 

enzymatic activity in SB (-0.39 U g-1), and 

increased production in SSF using LS. This 

enzymatic production capacity at high temperatures 

indicates that sorghum can maintain an ideal 

environment for fermentation by the filamentous 

fungus. This is a very promising result since heat 

removal is one of the greatest difficulties in SSF 

processes, due to the low thermal conductivity of 

the fermented material, a problem that increases for 

large-scale SSF (Durand, 2003). In the present 

study, it is noticeable that the sorghum substrate 

would be a better option than sugarcane bagasse for 

use on a large scale, due to its ease of temperature 

maintenance and considering the higher enzymatic 
activities obtained at high temperatures using the 

same fungal strain in fermentation. 

The significant effects of the factors and their 

interactions on CMCase activity (Table 3) involve 

mainly M, TPT and pH; TPT alone presented a 

negative influence (-0.63 U g

-1

) for SSF using SB. 

On the other hand, for SSF with the LS substrate, 

the T variable alone presented significance, though 

it was negative; when the time was increased from 4 

to 12 days, CMCase activity was reduced to 1.8 U g

-1

The reduction or stabilization of the amount of 

enzymatic activity due to the increase in 

fermentation time may possibly be attributed to the 

exhaustion of nutrients or the accumulation of 

products that inhibit enzymatic synthesis or cell 

growth (Sanchez & Demain, 2008). 

Biazus, Souza, Curvelo-Santana, and 

Tambourgi (2006), working with maize malt, 

observed that enzyme production, in principle, is 

slow, accelerating until it reaches its maximum 

value. Thenceforth, the concentration of 

products generated by the enzymes causes that 

part of them to become inhibited and their 

activity to be reduced to a constant value. 

 

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326 

Bomtempo et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

 

Table 2. Comparison of cellulase activities produced by cellulolytic fungi strains 

Strains Substrate 

Cellulases U g

-1

 

References 

1* 2* 

3* 

P. oxalicum 

LS 4.2 

9.2 

8.4 

Current 

study 

P. oxalicum 

SB 3.3 

3.5 

1.8 

Current 

study 

T. reesei 

RB 

1.2 

 

 

Latifian et al. (2007) 

Penicillium decumbens 

SB 

3.97 

 

 

Long et al. (2009) 

Trichoderma reesei 

SB 

 

5.4 

 

Basso et al. (2010) 

Aspergillus fumigatus 

SB 

0.14 

 

 

Soni, Nazir, and Chadha (2010) 

A. fumigatus 

RB 

 

 

1.7 

Sherief, El-Tanash, and Atia (2010) 

Aspergillus niger 

SM and SB 

 

2.2 

 

Rodrigues-Zúñiga, Farinas, Bertucci Neto, Couri, and Crestana (2011) 

Trichoderma sp 

SB 

2.7 

 

 

Marra et al. (2015) 

LS - Lignocellulosic sorghum; SB - Sugarcane bagasse; RB - Rice bran; SM - Soybean meal; 1* - FPase; 2* - CMCase; 3* - Avicelase. 

Table 3. Estimate of significant effects (p <0.05) for FPase activity in SB and LS substrates. 

 

Substrate 

Effect U g

-1

 

Standard Deviation

T (3) 

P value 

Lim. Conf. 

-95% 

Lim. Conf. 

+95% 

1* 

SB 

 

Mean/Interc

1.59

 

0.06 24.34 0.000152 1.38 1.80 

M 1.07

 

0.13 7.52 0.004867 0.62 1.52 

M x pH 

-0.82

 

0.13 -5.80 0.010200 -1.28 

-0.37 

TPT x M 

-0.73

 

0.13 -5.10 0.014564 -1.18 

-0.27 

TPT x Ci 

0.51 

0.13 

3.60 

0.036673 

0.06 

0.97 

T -0.39 0.13 -3.03 

0.012697 

-0.68 

-0.10 

TPT x pH 

0.37 

0.13 

2.87 

0.016688 

0.08 

0.66 

 

 

 

 

 

 

 

 

LS 

 

Mean/Interc. 2.80 

0.07 

39.77  0.000035 

2.58 

3.02 

M -0.55 0.12 -3.58 

0.037164 

-104 

-0.06 

TPT x pH 

-0.53 

0.12 

-3.42 

0.041791 

-1.01 

-0.04 

T -0.38 0.12 -3.09 

0.020262 

-0.65 

-0.09 

TPT x M 

-0.35 

0.12 

-2.89 

0.002505 

-0.63 

-0.07 

TPT 0.33  0.12  2.68 0.027853 0.05 0.60 

2* 

SB 

Mean/Interc

1.17 0.08 13.99 

0.000790 0.91 1.44 

M 0.97 0.18 -3.46 

0.040713 

-1.21 

-0.05 

TPT -0.63  0.18  5.33 0.012924 0.39 156 

pH 0.58 0.18 2.94 

0.012455 

0.15 101 

T x Ci 

-0.57 

0.18 

-2.89 

0.013481 

-1.00 

-0.14 

TPT x M 

-0.54 

0.18 

-2.76 

0.017209 

-0.97 

-0.11 

 

 

 

 

 

 

 

 

LS 

Mean/Interc. 4.26 

0.26 

39.77  0.000001 

3.65  4.877 

T -1.78 0.57 -3.58 

0.016638 

-3.11 

-0.43 

3* 

SB 

Mean/Interc. 0.77 

0.05 

15.96  0.000535 

0.61 

0.92 

TPT x T 

0.57 0.10 5.43 0.012266 0.23 0.90 

M x pH 

-0.49 

0.10 

-4.72 

0.018011 

-0.83 

-0.16 

M x Ci 

0.45 

0.10 

4.33 

0.022676 

0.12 

0.78 

T x Ci 

0.48 

0.10 

4.61 

0.019194 

0.15 

0.82 

 

 

 

 

 

 

 

 

LS 

No significant variables 

TPT - Incubation temperature (

o

C); M- Initial moisture content (wt%); pH- Initial pH of the mixture; T- Incubation time; Ci - Inoculum concentration; 1* - FPase; 2* - CMCase; 3* 

- Avicelase. 

 

Thus, the optimal incubation time for maximum 

enzyme production depends on the type of material, 
microorganism growth rate, carbon source and its 
standard production. For the production of 
Avicelases it is possible to observe that the variables 
were significant only when studied relating one 
factor to another, while for SSF using LS, no 
variable or interaction was significant with a 95% 
confidence interval.  

The coefficient of determination (R-squared) 

values were observed at 0.97726 for FPase, 0.94624 

for CMCase and 0.93391 for avicelase, which 

validates the use of equations 1, 2 and 3 and 

indicates that they can be used to predict enzymatic 

production tendencies. ANOVA analysis showed, 

through the F-test, that CMCase and Avicelase 

activities in LS were not significant. Thus, the 

models generated cannot be used. 

 

1

2

1

2

1

3

1

5

2

3

1.59 0.20

0.54

0.36

0.19

0.26

0.41

production

X

X

X X

X X

X X

X X

FPase

=

+

+

+

(Equation 1) 

 

where: X

1

 represents the incubation temperature; X

= is the initial moisture content; X

= is the initial 

pH of the mixture; X

= is the incubation time; X

is the inoculum concentration. 

 

1

2

3

1

2

4

5

.17 – 0.31

0.49

0.29

– 0.27

– 0.28

1

production

X

X

X

X X

X X

CMCase

+

+

=

 

(Equation 2) 

 

where: X

1

 represents the incubation temperature; X

= is the initial moisture content; X

= is the initial 

pH of the mixture; X

= is the incubation time; X

is the inoculum concentration. 

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Production of cellulases by Penicillium oxalicum 327 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

1

4

2

3

2

5

4

5

0.77 0.28

– 0.25

0.23

0.24

production

X X

X X

X X

X X

Avicelase

+

+

=

+

 

(Equation 3) 

 

where: X

1

 represents the incubation temperature; X

= is the initial moisture content; X

= is the initial 

pH of the mixture; X

= is the incubation time; X

is the inoculum concentration. 

Figure 2 displays the response surface generated 

from FPase production in SB. The best production 
region is in the extensions: Temperature (≤30 to  
35

C), Moisture (70 to ≥80 %). 

 

     

 

Figure 2. Response Surface of FPase activity obtained based on 
the SSF process using sugarcane bagasse as the substrate, 
considering Moisture (M) and Temperature (TPT) as factors. 

In Figures 3 and 4 there is a region of best 

CMCase production in sugarcane bagasse in the 

following extensions: temperature (<30 to 35 

o

C), 

Moisture (75 to > 80 %), pH (4 to <6). 

 

 

 

Figure 3. Response Surface of CMCase activity obtained based 

on the SSF process using sugarcane bagasse as the substrate, 
considering Moisture (M) and Temperature (TPT) as factors. 

In Figures 5 and 6 it is possible to observe the 

existence of a region of best FPase production in 
sorghum. This region is in the following extensions: 
temperature (≥37 to ≥40

C), Moisture (≤60 to 70 

%), pH (6 to <4). 

    

 

Figure 4. Response Surface of CMCase activity obtained based 
on the SSF process using sugarcane bagasse as the substrate, 
considering Moisture (U) and pH as factors. 

    

 

Figure 5. Response Surface of FPase activity obtained based on 

the SSF process using sorghum as the substrate, considering pH 
(pH) and Temperature (TPT) as factors 

    

 

Figure 6. Response Surface of FPase activity obtained based on 

the SSF process using sorghum as the substrate, considering 
Moisture (M) and Temperature (TPT) as factors.

 

Conclusion 

Cellulase production by Solid State 

Fermentation, using Penicillium oxalicum F 3380 and 

biomass LS and SB has shown to be promising due 

to high values of enzymatic activity; notably, the best 

performance was with LS. The huge fluctuation of 

enzymatic activity production in the tests performed 

and the differences in the influence of the variables 

in both substrates reinforce the importance of 

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328 

Bomtempo et al. 

Acta Scientiarum. Biological Sciences 

Maringá, v. 39, n. 3, p. 321-329, July-Sept., 2017 

detailed adjustments based on SSF parameters to 
obtain greater enzymatic production. The initial 

analysis in this study indicates the path for future 

optimization of SSF by Penicillium oxalicum using SB 

or LS as a substrate. 

Acknowledgements 

The authors thank Dr. Marcelo Mendes Pedroza 

and João Pedro Kappes Marques for their help with 

statistical analyses and the CAPES Foundation for 

the financial support through a doctoral scholarship. 

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Received on June 21, 2016. 
Accepted on April 10, 2017. 

 

 

License information: This is an open-access article distributed under the terms of the 
Creative Commons Attribution License, which permits unrestricted use, distribution, 
and reproduction in any medium, provided the original work is properly cited. 

 

 

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