FODDER RADISH CAKE ( Raphanus sativus L . ) AS AN ALTERNATIVE BIOMASS FOR THE PRODUCTION OF CELLULASES AND XYLANASES IN SOLID-STATE CULTIVATION

Fodder radish (FR) is an oilseed crop with a high potential for biodiesel production due to its high productivity and the quality of its seed oil. FR oil extraction results in a residue that is rich in protein and fiber. In this study, FR cake (FRC) was evaluated as carbon and nitrogen source for the production of cellulases and xylanases using Penicillium echinulatum S1M29 during solid-state cultivation. It was determined that it is possible to partially replace wheat bran (WB) by FRC, resulting in 24.22 ± 0.25U/g Filter Paper Activity (144 hours), 210.5 ± 5.8U/g endoglucanase activity (144 hours), 22.62 ± 0.01U/g β-glucosidase activity (96 hours) and 784.7 ± 70.19U/g xylanase activity (120 hours). These values are equal or higher than the enzymatic activity obtained using WB. These results may contribute to the reduction of the cost of enzymes used in the production of cellulosic ethanol or other biotechnological applications.


INTRODUCTION
In recent decades, the enzymatic hydrolysis of cellulose from lignocellulosic residues has been widely studied because cellulosic biomass is a possible resource for alternative fuel production.Additionally, this process has a great potential for reducing carbon dioxide emissions, thereby contributing to reducing global warming.However, to convert lignocellulosic biomass into biofuel, specifically ethanol, it is necessary to perform hydrolysis on the biomass, which requires enzymes, particularly cellulases and xylanases (Basso et al., 2014;Mabee et al., 2011;Reis et al., 2014).
Both cellulases and xylanases can be produced in solidstate cultivation (SSC) or submerged cultivation (SC).Commercially, most cellulases and xylanases are produced by SC because the production factors are easier to control.However, the SC method can be complex because it involves mixing, aeration, and control and monitoring of temperature, pH, dissolved oxygen and other factors (dos Reis et al., 2014;Reis et al., 2015), and the SC method requires specific equipment.Using SSC for enzyme production has attracted interest because it is a lower cost technology and has a relatively high enzyme production capacity (Camassola and Dillon, 2007;Camassola and Dillon, 2016;Macedo et al., 2013;Pirota et al., 2013;Yoon et al., 2014).
Among the microorganisms that can potentially produce cellulases that generate second-generation ethanol, Penicillium echinulatum mutants are notable because they have high enzymatic titers (Dillon et al., 2011) and produce appropriate proportions of FPA and β-glucosidase (Martins et al., 2008).
A major advantage of solid-state cultivation is the use of agricultural and agro-industrial residues (Camassola and Dillon, 2010).Agro-industrial residues include residues from the production of biodiesel, like fodder radish.Fodder radish has a rapid growth and high capacity to recycle nutrients, particularly nitrogen and phosphorus, developing reasonably in poor soils with acidity problems.It is important for crop rotation and produces great amounts of dry weight and is excellent for the practice of direct planting.Fodder radish enters the crop rotation system, minimizing soil compaction, producing green mass and reducing weed infestation during the fallow season of agricultural areas (Sluszz and Machado, 2006).Fodder radish produces 20 to 35 t / ha green mass, 3.5 to 8 t / ha of dry matter and 0.5 to 1.5 t / ha grains.The oil productivity is around 150 to 550 kg / ha (Ávila and Sodre, 2012).Seeds of fodder radish (Raphanus sativus L.) derive 40-54% of their mass from oil (Domingos et al., 2008) and the postextraction residue of oil for biodiesel production contains approximately 39% (w/w) protein and 5% (w/w) minerals (Ávila and Sodré, 2012).
Additionally, fodder radish cake (FRC) is a promising residue that can be used in combination with lignocellulose for enzyme production.This study evaluated the potential of using FRC, alone and in combination with wheat bran (WB), as a substrate for the production of cellulase and xylanase during solid-state cultivation with P. echinulatum.

Microorganism
The strain S1M29 of P. echinulatum was obtained from the strain 9A02S1 after several steps of hydrogen peroxide mutagenesis and selection in medium containing 2-deoxyglucose (Dillon et al., 2006).This strain was stored in the cultivation collection of the Enzymes and Biomass Laboratory, Institute of Biotechnology, Caxias do Sul, Rio Grande do Sul, Brazil.This strain was grown on C-agar slants (Dillon et al., 2011) for up to 7 days at 28°C until conidia formed and then was stored at 4°C until further use.

Enzyme production
WB and FRC were used as the support and primary carbon and nitrogen sources.The cultivation media consisted of a mixture of various ratios of WB and FRC, as specified in Table 1.The controls were WB and FRC alone.
The SSC was performed as described in Camassola andDillon (2007, 2010) (Camassola and Dillon, 2010;Camassola and Dillon, 2007).The cultivation flasks were incubated at 28 °C and 90% humidity for 6 days.Four replicate experiments containing 2 g of dry mass biomass were performed for the same strains and each incubation time.The extraction of enzymes was done according to Camassola and Dillon (2007), the contents of each flask were separately added to a 125 mL-Erlenmeyer flask containing 10 mL distilled water, and the pH was measured.Next, 17 mL of 0.05 M citrate buffer (pH 4.8) was added, mixed, incubated under agitation for 30 min at 4 °C and then centrifuged at 3220 × g for 20 min.Enzyme assays were performed on the broth samples as described below.

Enzyme assay
The enzymatic activity was analyzed on filter paper (Filter Paper Activity -FPA) according to Camassola and Dillon (2012).The β-glucosidase activity was determined using p-nitrophenyl-β-D-glucopyranoside as the substrate (Daroit et al., 2008).The endoglucanase activity was determined according to the method outlined by Ghose (1987) using 2% (w/v) carboxymethyl cellulose solution in citrate buffer.The xylanase activity was determined using the same method as the endoglucanase activity assay, except that 1% (w/v) xylan from oat spelt solution was used as the substrate in place of carboxymethylcellulose.
The reducing sugar was estimated as either the xylose or glucose equivalent using the dinitrosalicylic acid (DNS) method (Miller, 1959).
One unit (U) of enzyme activity was defined as the amount of enzyme required to liberate 1 µmol of reducing sugar from the appropriate substrates per minute under assay conditions.The enzymatic activities are expressed as units per gram of dry medium (U/g).

Mycelial Mass Determination
Growth determinations were performed by determining the amount of N-acetyl-D-glucosamine present in the mycelium and converted fungal biomass according to the method described by Novello et al. (2014).

Statistical tests
The results were statistically analyzed using analysis of variance (ANOVA) and Tukey post-test using the Prism GraphPad program (GraphPad Software, Inc., USA).A p-value < 0.05 was considered statistically significant.
For β-glucosidase (Fig. 1B), the highest activity was observed at 96 hours (22.62 U/g) in the 75% WB + 25% FRC medium.At 120 hours, there was an increase in β-glucosidase activity in the 50% WB + 50% FRC and 100% FRC media; however, the cultures were not significantly different.At 144 hours, the β-glucosidase enzyme activity was statistically indistinguishable for all media, with the exception of the FRC only medium (FRC 100%), which had decreased enzyme activity compared with 120 hours.
The FPA of the 75% WB + 25% FRC media was superior to the control (100% WB) at 120 hours.Although the average of the 75% WB + 25% FRC was higher at 144 hours (24.22 ± 0.25 U/g), the results were not significantly different from the 100% WB and 50% WB + 50% FRC treatments.The lowest FPA was observed using 100% FRC and 25% WB + 75% FRC (Figure 1C).
The highest xylanase enzyme activity (Figure 1D) was obtained with the 100% WB medium after 144 hours of cultivation (1137.59± 4.76 U/g).All cultures containing FRC had lower activities, with increasing FRC concentrations resulting in lower xylanase production.

Evaluation of fungal mass and pH of the cultures
The mycelial masses in various SSCs using the fungus P. echinulatum S1M29 are shown in Figure 2. The mycelial mass was estimated indirectly by analyzing the production of N-acetyl-D-glucosamine from the enzymatic hydrolysis of chitin in the fungal cell wall after 120 hours of cultivation.
The mycelial mass is proportional to the ratio of WB present in the medium; as the WB concentration increased, the mycelial mass increased.It was determined that that there was no direct relationship between enzymatic activities and mycelial mass for the evaluated enzymes (Figure 2).
The pH profile was measured in the experimental cultures (Figure 3).It was determined that for 48 hours there was no pH change; however, at 96 hours, there was acidification in all cultures.During this period, there was potentially carbon source consumption in the medium that resulted in acidification, with the pH reaching values near 4.0.After 96 hours, when there was increased enzymatic activity, the pH values increased to between 6.0 and 7.0.These data indicate that there is a correlation between pH and growth; these findings are according to Sternberg and Dorval (1979).They interpreted the pH variation in Trichoderma reesei cultures as a period of growth and NH 3 uptake, which released H + into the medium, while the pH increase at the end of the cultivation was due to NH 3 secretion by the fungus.This hypothesis is potentially corroborated by the results of the present work because the 100% WB cultures had the lowest pH and had the highest growth (Figure 2).There were lower pH values in the WB only medium after 48 hours of cultivation.These results are contrary to the results observed by Camassola and Dillon (2007); however, in that study, the WB was complexed with cane sugar bagasse.
Identical results can be observed when comparing enzymatic production using A. niger NRRL 567 (Dhillon et al., 2012); however, the FPA values were very high compared with other studies.A mixed culture of T. reesei and A. niger (Dhillon et al., 2011) had relatively similar enzyme concentrations compared with P. echinulatum S1M29 in 75% WB + 25% FRC medium, demonstrating the potential use of FRC for the production of enzymes that mediate lignocellulose hydrolysis (Table 2).The results indicated that FRC alone did not produce adequate cellulase and xylanase levels during solid-state cultivation.However, the replacement of 25% of the WB with FRC may be favorable for the production of cellulases because higher values were observed for FPA activity and β-glucosidases in cultures grown in this media.These experiments are relevant because they indicate that WB could be replaced by up to 25% for cellulase production in solid-state cultivation.

CONCLUSIONS
The present study has demonstrated that fodder radish cake can be employed as a component in media for cellulase production during P. echinulatum solid-state cultivation.Specifically, this study determined that up to 25% WB, a traditional substrate for cellulase production, may be substituted and is advantageous for cellulase production.Therefore, using FRC as an alternative carbon source to partially replace WB may lower the costs of producing enzyme complexes, which may in turn reduce the production costs of cellulosic ethanol.

Figure 1 .
Figure 1.Activities of endoglucanases (A), β-glucosidases (B) FPA (C) and xylanase (D) in solid-state cultivation with Penicillium echinulatumS1M29 in various culture media using fodder radish cake and wheat bran as a substrate.The treatment means that have the same letters for the same day are not significantly different when evaluated using Tukey's test (p> 0.05).WB -wheat bran; FRC -fodder radish cake.

Figure 2 .
Figure 2. Mycelial mass concentrations in solid-state cultivation using Penicillium echinulatum S1M29 after 120 hours of cultivation in media with different formulations.WB -wheat bran; FRC -fodder radish cake.

Figure 3 .
Figure 3. Variations of pH at different time points during the solidstate cultivation using Penicillium echinulatum S1M29.

Table 1 .
Formulations used in the solid-state cultivation to produce xylanases and cellulases from Penicillium echinulatum S1M29.

Table 2 .
Comparisons of enzyme activities for different microorganisms grown on various lignocellulosic substrates.