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Comparison of Andropogon gayanus (gamba grass) and corn straw compositions. Table 4

Table 4 Comparison of Andropogon gayanus (gamba grass) and corn straw compositions. Table 4

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YE: yeast extract; P: peptone; C: carbon source (gamba grass) concentration; M: mesh of the sieve. Uncoded values of independent variables for Plackett Burman design. Table 1
YE: yeast extract; P: peptone; C: carbon source (gamba grass) concentration; M: mesh of the sieve. Uncoded values of independent variables for Plackett Burman design. Table 1
Results of the Plackett Burman design for production of endoglucanase and cellobiase activities at 2, 4 and 7 days of cultivation. E: endoglucanase; C: cellobiase.
Results of the Plackett Burman design for production of endoglucanase and cellobiase activities at 2, 4 and 7 days of cultivation. E: endoglucanase; C: cellobiase.
The main effects observed in Plackett Burman design for independent variables on the enzymatic activities. Values represent coded units.
The main effects observed in Plackett Burman design for independent variables on the enzymatic activities. Values represent coded units.
Fig. 1. Endoglucanase production on different forage concentration: (@) 1%, (O) 2.5%, (W) 4%, (A) 5.5%, (HD 7%, (DZ) 8.5%, (#) 10%, in M1 (A) and Mz nedia.
Fig. 1. Endoglucanase production on different forage concentration: (@) 1%, (O) 2.5%, (W) 4%, (A) 5.5%, (HD 7%, (DZ) 8.5%, (#) 10%, in M1 (A) and Mz nedia.
Fig. 2. Cellobiase production on different carbon source concentration 1% (@), 2.5% (O), 4% (W), 5.5% (A), 7% (MD, 8.5% (1), 10% (@), in M1 (A) and M2 ( media. The kind of biomass interferes on the optimal concentration to produce each enzyme. Corn straw was slightly more efficient to induce endoglucanase, 8.28 U/mL (de Almeida et al., 2014), while 6.5 U/mL was obtained with gamba grass. However, M2, with gamba grass, showed to be ideal to induce cellobiase, allowing the achievement of 6.8 U/mL while in the tested medium using corn straw, maximum ac- tivity of 0.74 U/mL was observed (9-fold increase) (de Almeida et al., 2014). Gamba grass and corn straw are biomasses with different che- mical composition. From the available papers, it was possible to see that hemicellulose and lignin concentration on gamba grass can vary between cultivars from different countries. Gamba grasses from Brazil showed to have higher hemicellulose and lower lignin content than gamba grass form Nigeria and Senegal (Table 4). Comparing gamba grass and corn straw, it can be observed that, in general, gamba grass have a lower cellulose (31.8%) and lignin (6.7%) contents than corn straw (40.5% and 13.5% for cellulose and lignin, respectively) (Table 4). The higher cellulose and lignin contents in corn straw can be mainly inducing cellulases specialized to start the polymer degradation, like endoglucanase. Endoglucanases produces nicks in the cellulose chain exposing reducing and non-reducing ends, cellobiohydrolases acts upon these reducing and non-reducing ends to liberate cello-oli- gosaccharides and cellobiose units. Cellobiase acts in a final stage of cellulose degradation hydrolyzing cellobiose and, in minor extension, cellooligosaccharides (Lynd et al., 2002). Considering that gamba grass cellulose is more accessible than cellulose present in corn fiber (lower lignin content) it would be plausible suggest that higher levels of cel- lulose degradation would be observed during cultivation supplemented In previous study, when corn straw was used to optimize en- doglucanase and cellobiase production by Fusarium verticillioides, dif- ferent results were observed. For endoglucanase, higher corn straw concentration induces higher enzyme activity. For cellobiase produc- tion, corn straw concentration did not have a significant effect (de Almeida et al., 2014). The differences about endoglucanase and cello- biase production observed between M1 and M2 extract confirms that the medium composition interferes decisively over the enzymatic pro- file secreted by fungus Fusarium verticillioides. Furthermore, the results obtained in the present study and in the previous one (de Almeida et al., 2014) strongly suggest that Fusarium verticillioides presents distinct and antagonic inductions systems to regulate secretion of endoglucanase and cellobiase once it was observed that the improvement of en- doglucanase activity leads to a reduction of cellobiase secretion and
Fig. 2. Cellobiase production on different carbon source concentration 1% (@), 2.5% (O), 4% (W), 5.5% (A), 7% (MD, 8.5% (1), 10% (@), in M1 (A) and M2 ( media. The kind of biomass interferes on the optimal concentration to produce each enzyme. Corn straw was slightly more efficient to induce endoglucanase, 8.28 U/mL (de Almeida et al., 2014), while 6.5 U/mL was obtained with gamba grass. However, M2, with gamba grass, showed to be ideal to induce cellobiase, allowing the achievement of 6.8 U/mL while in the tested medium using corn straw, maximum ac- tivity of 0.74 U/mL was observed (9-fold increase) (de Almeida et al., 2014). Gamba grass and corn straw are biomasses with different che- mical composition. From the available papers, it was possible to see that hemicellulose and lignin concentration on gamba grass can vary between cultivars from different countries. Gamba grasses from Brazil showed to have higher hemicellulose and lower lignin content than gamba grass form Nigeria and Senegal (Table 4). Comparing gamba grass and corn straw, it can be observed that, in general, gamba grass have a lower cellulose (31.8%) and lignin (6.7%) contents than corn straw (40.5% and 13.5% for cellulose and lignin, respectively) (Table 4). The higher cellulose and lignin contents in corn straw can be mainly inducing cellulases specialized to start the polymer degradation, like endoglucanase. Endoglucanases produces nicks in the cellulose chain exposing reducing and non-reducing ends, cellobiohydrolases acts upon these reducing and non-reducing ends to liberate cello-oli- gosaccharides and cellobiose units. Cellobiase acts in a final stage of cellulose degradation hydrolyzing cellobiose and, in minor extension, cellooligosaccharides (Lynd et al., 2002). Considering that gamba grass cellulose is more accessible than cellulose present in corn fiber (lower lignin content) it would be plausible suggest that higher levels of cel- lulose degradation would be observed during cultivation supplemented In previous study, when corn straw was used to optimize en- doglucanase and cellobiase production by Fusarium verticillioides, dif- ferent results were observed. For endoglucanase, higher corn straw concentration induces higher enzyme activity. For cellobiase produc- tion, corn straw concentration did not have a significant effect (de Almeida et al., 2014). The differences about endoglucanase and cello- biase production observed between M1 and M2 extract confirms that the medium composition interferes decisively over the enzymatic pro- file secreted by fungus Fusarium verticillioides. Furthermore, the results obtained in the present study and in the previous one (de Almeida et al., 2014) strongly suggest that Fusarium verticillioides presents distinct and antagonic inductions systems to regulate secretion of endoglucanase and cellobiase once it was observed that the improvement of en- doglucanase activity leads to a reduction of cellobiase secretion and
Fig. 3. Stability of endoglucanase activity (@), FPase activity (O), cellobiase activity (W) and xylanase activity (AA) produced by fungus Fusarium verti- cillioides. The activity without pre-incubation was considered 100%. Thermal stability of the main cellulases and xylanases activities produced by fungus Fusarium verticillioides was carried out at 50 °C and pH 5 (Fig. 3). Endoglucanase and FPase activities were very stable re- taining 56 and 42% of the initial activity after 120 h of pre-incubation, respectively. This high stability showed that these enzymes suffered few thermal or pH inactivation along saccharification process. Purified en- doglucanases from the same fungus were reported to have an even higher thermostability than what was observed for crude extract,
Fig. 3. Stability of endoglucanase activity (@), FPase activity (O), cellobiase activity (W) and xylanase activity (AA) produced by fungus Fusarium verti- cillioides. The activity without pre-incubation was considered 100%. Thermal stability of the main cellulases and xylanases activities produced by fungus Fusarium verticillioides was carried out at 50 °C and pH 5 (Fig. 3). Endoglucanase and FPase activities were very stable re- taining 56 and 42% of the initial activity after 120 h of pre-incubation, respectively. This high stability showed that these enzymes suffered few thermal or pH inactivation along saccharification process. Purified en- doglucanases from the same fungus were reported to have an even higher thermostability than what was observed for crude extract,
Cellobiose accumulated in the beginning of the process in the assays with 100% M1 extract, but any amounts of M2 added helped to avoid this accumulation on the blends. Despite this initial difference, after 24h of saccharification, cellobiose was already consumed. In the end of the process, cellobiose concentration was similar in all tested blends (between 0.29 to 0.33 g/L). The ratio endoglucanase:cellobiase, 12:1, present on M1 was sufficient to avoid cellobiose inhibition. Among the enzyme activities analyzed, endoglucanase was the main difference between the blends and was probably responsible for the better per- formance of 100% M1 extract. Similar results were observed for su- garcane bagasse saccharification using enzymatic extract from Trichoderma and Aspergillus. It was reported that a cellobiase-rich (p- glucosidase) extract did not presents best results for saccharification if it do not contain an equilibrated cellulase composition (Gottschalk et al., 2010). In engineered Trichoderma reesei, a strain containing - glucosidase from Aspergillus aculeatus, an equilibrate composition was also achieved. The proportion enough to avoid cellobiose accumulation Fig. 4. Saccharification of sugarcane bagasse using different enzymatic blends between M1 and M2 extract produced by fungus Fusarium verticillioides. 100% M1 (A), 75% M1 — 25% M2 (B), 50% M1 - 50% M2 (C), 25% M1 — 75% M2 (D) and 100% M2 (E). Glucose (@), xylose (O), cellobiose (W), arabinose (A). sugarcane harvest for 2018/2019 was 635.51 million of ton (Conab, 2018) and sugarcane bagasse and straw are the main byproducts. Su- garcane bagasse was submitted to alkaline pretreatment before being hydrolyzed. Chemical composition of raw sugarcane bagasse sample used in the present study was as follows: glucan 45.4%, hemicellulose 26.8% (sum of xylans, galactan, mannan and arabinan), and lignin 22.0%. Proteins, extractives, and ash consisted the remaining 5.8%. After alkaline pretreatment the chemical composition of the pretreated biomass was: cellulose 66.1%, hemicellulose 25.8% and lignin 5.9%.
Cellobiose accumulated in the beginning of the process in the assays with 100% M1 extract, but any amounts of M2 added helped to avoid this accumulation on the blends. Despite this initial difference, after 24h of saccharification, cellobiose was already consumed. In the end of the process, cellobiose concentration was similar in all tested blends (between 0.29 to 0.33 g/L). The ratio endoglucanase:cellobiase, 12:1, present on M1 was sufficient to avoid cellobiose inhibition. Among the enzyme activities analyzed, endoglucanase was the main difference between the blends and was probably responsible for the better per- formance of 100% M1 extract. Similar results were observed for su- garcane bagasse saccharification using enzymatic extract from Trichoderma and Aspergillus. It was reported that a cellobiase-rich (p- glucosidase) extract did not presents best results for saccharification if it do not contain an equilibrated cellulase composition (Gottschalk et al., 2010). In engineered Trichoderma reesei, a strain containing - glucosidase from Aspergillus aculeatus, an equilibrate composition was also achieved. The proportion enough to avoid cellobiose accumulation Fig. 4. Saccharification of sugarcane bagasse using different enzymatic blends between M1 and M2 extract produced by fungus Fusarium verticillioides. 100% M1 (A), 75% M1 — 25% M2 (B), 50% M1 - 50% M2 (C), 25% M1 — 75% M2 (D) and 100% M2 (E). Glucose (@), xylose (O), cellobiose (W), arabinose (A). sugarcane harvest for 2018/2019 was 635.51 million of ton (Conab, 2018) and sugarcane bagasse and straw are the main byproducts. Su- garcane bagasse was submitted to alkaline pretreatment before being hydrolyzed. Chemical composition of raw sugarcane bagasse sample used in the present study was as follows: glucan 45.4%, hemicellulose 26.8% (sum of xylans, galactan, mannan and arabinan), and lignin 22.0%. Proteins, extractives, and ash consisted the remaining 5.8%. After alkaline pretreatment the chemical composition of the pretreated biomass was: cellulose 66.1%, hemicellulose 25.8% and lignin 5.9%.
Final activities on each cocktail blend used for saccharification assays. Endo: endoglucanase, Cello: cellobiase, Xyl: xylanase, B-Xyl: B-xylosidase, a- Arab: a-Arabinofuranosidase. Blends were defined by protein concentration (10 mg of protein for each blend). Table 5
Final activities on each cocktail blend used for saccharification assays. Endo: endoglucanase, Cello: cellobiase, Xyl: xylanase, B-Xyl: B-xylosidase, a- Arab: a-Arabinofuranosidase. Blends were defined by protein concentration (10 mg of protein for each blend). Table 5
Fig. 5. (A) Productivity of glucose ((]) and xylose (A) along the saccharification time for 100% M1 extract. (B): Determination of soluble protein and enzymatic activities along saccharification of sugarcane bagasse employing M1 extract produced by Fusarium verticillioides protein concentration (@), endoglucanase (OC), FPase (W), cellobiase (A\) and xylanase activities (§). The values equivalents to 100% were: protein 0.6 mg/mL, endoglucanase 4.2 U/mL, FPase 0.45 U/mL, cellobiase 0.16 U/mL and xylanase 51.9 U/mL.
Fig. 5. (A) Productivity of glucose ((]) and xylose (A) along the saccharification time for 100% M1 extract. (B): Determination of soluble protein and enzymatic activities along saccharification of sugarcane bagasse employing M1 extract produced by Fusarium verticillioides protein concentration (@), endoglucanase (OC), FPase (W), cellobiase (A\) and xylanase activities (§). The values equivalents to 100% were: protein 0.6 mg/mL, endoglucanase 4.2 U/mL, FPase 0.45 U/mL, cellobiase 0.16 U/mL and xylanase 51.9 U/mL.
Fig. 6. Saccharification using different doses and feed of enzymatic cocktail. Glucose concentration (A) and xylose concentration (B). 10 mg/g (@), 5 and 5 mg/g (0), 20 mg/g (W), 10 and 10 mg/g (A), 40 mg/g (MM), 20 and 20 mg/g ([)). The application of gamba grass as an inducer for cellulases pro- duction was tested for the first time in this study. Gamba grass is a plant commonly used as forage and that is widely available in farms, being an easy to find and costless carbon source. A statistical approach estab- lished the ideal composition for Fusarium verticillioides to produce two different enzymatic cocktails, one endoglucanase-rich (M1) and the other cellobiase-rich (M2). Studies with M1 and M2 blends for su- garcane bagasse saccharification revealed the best en- doglucanase:cellobiase ratio of 12:1. Among the non-productive factors on saccharification, enzyme adsorption was a bottleneck for en- doglucanase and xylanase activities while low thermostability was significant for cellobiase. Different protein dosages were tested, and the best biomass conversion was achieved using 40mg of protein/g of The higher glucose and xylose releasing were observed using 40 mg/g in unique dose, which is correspondent to 39 FPU/g. The fractioned enzyme feed produces slightly lower yield on product for- mation than when the enzymatic dose was entirely applied at the start reaction. Concerning the treatments carried out with no fractioned enzyme loading the glucan conversions were 29.6, 35.0 and 43.4% for treatments with 10, 20 and 40 mg of protein per gram of biomass, re- spectively. Xylan conversions were 41.2, 51.7 and 73.1% for 10, 20 and 40 mg/g, respectively. Some studies reported the stabilization of pro- duct formation using 10 FPU/g (Gottschalk et al., 2010) and 15 mg/g (49.5 FPU/g) (Zhang et al., 2010) for sugarcane bagasse hydrolysis. In the present study, this stabilization was not verified (Fig. 7). Similar result was obtained using cellulases from Trichoderma sp.. Stabilization was not observed when 10, 20, 30 and 40 FPU/g was used in sac- charification (Mkhize et al., 2016). The minimal enzyme dosage as well as the total biomass conversion is highly dependent not only on cocktail efficiency, but also on others factors. The biomass composition and
Fig. 6. Saccharification using different doses and feed of enzymatic cocktail. Glucose concentration (A) and xylose concentration (B). 10 mg/g (@), 5 and 5 mg/g (0), 20 mg/g (W), 10 and 10 mg/g (A), 40 mg/g (MM), 20 and 20 mg/g ([)). The application of gamba grass as an inducer for cellulases pro- duction was tested for the first time in this study. Gamba grass is a plant commonly used as forage and that is widely available in farms, being an easy to find and costless carbon source. A statistical approach estab- lished the ideal composition for Fusarium verticillioides to produce two different enzymatic cocktails, one endoglucanase-rich (M1) and the other cellobiase-rich (M2). Studies with M1 and M2 blends for su- garcane bagasse saccharification revealed the best en- doglucanase:cellobiase ratio of 12:1. Among the non-productive factors on saccharification, enzyme adsorption was a bottleneck for en- doglucanase and xylanase activities while low thermostability was significant for cellobiase. Different protein dosages were tested, and the best biomass conversion was achieved using 40mg of protein/g of The higher glucose and xylose releasing were observed using 40 mg/g in unique dose, which is correspondent to 39 FPU/g. The fractioned enzyme feed produces slightly lower yield on product for- mation than when the enzymatic dose was entirely applied at the start reaction. Concerning the treatments carried out with no fractioned enzyme loading the glucan conversions were 29.6, 35.0 and 43.4% for treatments with 10, 20 and 40 mg of protein per gram of biomass, re- spectively. Xylan conversions were 41.2, 51.7 and 73.1% for 10, 20 and 40 mg/g, respectively. Some studies reported the stabilization of pro- duct formation using 10 FPU/g (Gottschalk et al., 2010) and 15 mg/g (49.5 FPU/g) (Zhang et al., 2010) for sugarcane bagasse hydrolysis. In the present study, this stabilization was not verified (Fig. 7). Similar result was obtained using cellulases from Trichoderma sp.. Stabilization was not observed when 10, 20, 30 and 40 FPU/g was used in sac- charification (Mkhize et al., 2016). The minimal enzyme dosage as well as the total biomass conversion is highly dependent not only on cocktail efficiency, but also on others factors. The biomass composition and
Fig. 7. Cocktail dosage effect after 120h of saccharification. Glucose 10 mg/g (@) and xylose (©).
Fig. 7. Cocktail dosage effect after 120h of saccharification. Glucose 10 mg/g (@) and xylose (©).
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