Bioconversion of cassava bagasse and sugarcane bagasse using cheap home-made enzymatic cocktails


 The agricultural industries generate lignocellulosic wastes that can be modified by fungi to generate high value-added products. The aim of this work was to analyze the efficiency of the bioconversion of sugarcane bagasse and cassava bagasse using two cheap home-made enzymatic cocktails from Aspergillus niger LBM 134 (produced also from agroindustrial wastes) and compare the hydrolysis yield with that obtained from the bioconversion using commercial enzymes. Sugarcane bagasse and cassava bagasse were pretreated with a soft alkaline solution before the hydrolysis carried out with home-made enzymatic cocktails of A. niger LBM 134 and with commercial enzymes to compare their performances. Mono and polysaccharides were analyzed before and after the bioconversion of both bagasses as well as their microscopic structure. The maximal yield was the 80% of total glucans saccharified from cassava bagasse. The bioconversion of both bagasses were better when we used the home-made enzymatic cocktails than commercial enzymes. We obtained high added-value products from agroindustrial wastes, home-made enzymatic cocktails and hydrolysates rich in fermentable sugars. The importance of this work lays in the higher performance of the cheap home-made enzymatic cocktails over the hydrolytic performance of commercial enzymes due to the cost of producing the home-made enzymatic cocktails were more than 500 times lower than commercial enzymes.


Introduction 33
Biomass is the core of the bioeconomy concept where the efficient and sustainable 34 use of this renewable resource constitutes the basis of bioeconomy development [1]. In 35 this context, biorefineries are a key pillar in the development of a future bioeconomy-36 based society based on the development of biorefineries to produce biofuels and 37 bioproducts from renewable biomass sources and efficient bioprocesses to achieve 38 sustainable production [1]. Renewable feedstocks can be obtained from primary 39 biomass sources or wastes derived from household, industrial and agricultural activities. 40 Using wastes from agricultural activities adds value to the whole chain and those from 41 worldwide crops are an interesting resource. 42 Cassava (Manihot esculenta Cranz) and sugarcane (Saccharum sp.) are two of the 43 major tropical and subtropical agricultural crops [2]. The root of cassava is processed 44 to isolate the starch or to sell cassava as a pre-cooked meal [3]. The industry of cassava 45 4 generates CB as one of the solid by-products; this waste is a problem due to its high 46 percentage of water, which makes more expensive drying and transporting operations 47 9 β-D-glucobioside (PNPG) as substrate; and BXL activity was determined according to 131 Ghose and Bisaria [17] using ρ-nitrophenyl-β-D-xylobioside (PNPX) as substrate, 132 through the quantification of ρ-nitrophenol method. Absorbance was measured at 410 133 nm. BGL and BXL activities were expressed as U, defined as the amount of enzyme 134 releasing 1 μmol of ρ-nitrophenol per min at 50 °C. 135

Bioconversion of SCB and CB 136
SCB and CB were pretreated with an alkaline solution of NaOH 0.85% (w/v) to 137 remove lignin and avoid the holocellulose hydrolysis. For that, 10 g of bagasse was 138 mixed with 200 mL of the alkaline solution for a consistence of 5% (w/v) at 121 °C 139 during 30 min. Then, the bagasses were washed with water and 0.5 M sodium acetate 140 buffer pH 5.0 at 80 rpm, 25 °C for 12 h; bagasses were dried at 45 °C during 24 h. The 141 enzymatic hydrolysis of both agroindustrial wastes were carried out by the home-made 142 enzymatic cocktails of A. niger LBM 134 and by commercial enzymes for comparing 143 their performance. Also, two controls of these enzymatic hydrolysis were carried out: 144 1) incubation of bagasses without enzymes for determining the reducing sugars 145 previous the hydrolysis; 2) incubation of the home-made enzymatic cocktails without 146 the bagasses enzymes for determining the reducing sugars of the cocktails. The home-147 10 made enzymatic cocktail for carrying out the hydrolysis of SCB was obtained from A. 148 niger LBM 134 grown on SCB and in the same way, the home-made enzymatic cocktail 149 for the CB hydrolysis was obtained from the fungus grown on CB. For that, 1 g of 150 pretreated bagasse was incubated with 25 mL of reaction solution consisting of 0.05 M 151 sodium acetate buffer pH 5.0, 30 °C and the corresponding enzymatic cocktail 152 containing (in Ug -1 of biomass): EX 300, FPU 10 and BGL 20. The commercial 153 enzymes used were EX of Xylanase (Sigma-Aldrich, USA) 300 U g -1 , FPU of 154 Celluclast (Sigma-Aldrich, USA) 10 U g -1 and BGL of Viscozyme (Sigma-Aldrich, 155 USA) 20 U g -1 . All the enzymatic hydrolysis and the control assays were carried out at 156 30 °C, pH 5.0, 200 rpm during 24 h without the addition of any antibiotic for no 157 increasing the cost of the bioprocess. After this period, the assays were vacuum filtered 158 and centrifugated at 12,000 g during 20 min. The resulting supernatants were used to 159 quantify reducing sugars with the DNS method [18] and to identify and quantify 160 monomeric sugars by HPLC analysis. 161 The values were presented as the means of the triplicates ± the standard deviation.  where, FC corresponds to the conversion factor, that is 1.11 for glucose, 1.05 for 174 cellobiose, and 1.13 for xylose. 175

Electron microscopic structure of SCB and CB before and after the hydrolysis 176
Bagasses were observed by scanning electron microscopy (SEM) to evaluate the 177 changes in their microscopic structure during each step of the bioprocess: before and 178 after of the alkaline pretreatment and after the hydrolysis with the home-made 179 enzymatic cocktails and with the commercial enzymes. For that, 0.01 g of bagasses 180 were fixed in each evaluated step with formaldehyde:alcohol:acid (FAA, 10:50:5). proposed. These models were validated with experimental data from the work of 194 Kamoldeen et al. [20]. These models were used to stablish a simulation process of 195 bioethanol production. 196

Statistical analysis 197
The experimental and theorical results were analyzed and graphed with the software 198

Characterization of SCB and CB 201
The bioprocesses carried out in this study as a strategy to convert both SCB and CB 202 into enzymatic cocktails and fermentable sugars offered the possibility of obtaining 203 these high added-value products from agroindustrial wastes. Firstly, to know the 204 chemical composition of both SCB and CB for comparing then with monomeric sugars 205 after the enzymatic hydrolysis, the main components of the raw bagasses were 206 identified according to NREL analytical procedure (Table 1). SCB presented more 207 quantities of extractives (fat, proteins, wax), hemicelluloses and lignin than CB. 208 Conversely, CB had more glucans than SCB. 209

Characterization of the home-made enzymatic cocktails of A. niger LBM 134 211
The pH and thermostability of the key enzymes involved in the hydrolysis of 212 lignocellulosic biomass was studied (FPase, BGL, EX and BXL) in the home-made 213 enzymatic cocktails of A. niger LBM 134 due to the pH and the temperature are two 214 main factors affecting the stability of the enzyme activity. The enzymes of both home-215 made cocktails showed considerable stability, making them promising to be used in the 216 bioconversion of SCB and CB. 217 The polysaccharide hydrolytic activities, FPU, BGL, EX and BXL, of the home-218 made enzymatic cocktails of A. niger LBM 134 were measured (Table 2) and the 219 enzymatic levels demonstrated that these cocktails were suitable for carrying out the 220 bioconversion of SCB and CB. Also, the effect of temperature (30 °C) and pH (5.0) on 221 the stability of the enzyme activities were studied due to the importance of the 222 enzymatic stability of in any bioprocess (Figure 1). Thermostability of enzymes was 223 above 50% after 24 h (Figure 1a-b) and pH stability was above 50% after 24 h ( Figure  224 1c-d). Therefore, the hydrolysis assays were carried out under these conditions: 30 °C 225 and pH 5.0 for 24 h. 226

Bioconversion of SCB and CB 229
Also, raw materials, SCB and CB, were extensively characterized hence the correct 230 choice of any pretreatment strategy depends on knowing the fundamental biochemistry 231 of the biomass and the desired products [21]. For that reasons, we employed a soft 232 15 alkaline pretreatment on SCB and CB guarantying a specific lignin removal and 233 preserving the polysaccharides into the sold fraction, a fundamental feature required 234 for the hydrolysis [22]. 235 In addition to this effective pretreatment, we used the crude (home-made) carbohydrates were formed. The lignin was removed and discarded with the liquid 256 fraction 88.39 ± 5.83% for SCB and 73.20 ± 0.23% for CB, from the total lignin content. 257 The lignin removed was also evidenced by the change of colour of the solid fraction; 258 SCB and CB were initially brown before the alkaline treatment and after that, SCB 259 changed to light brown and CB, to yellow cream (data not shown). In addition, there 260 was no polysaccharides loss after the pretreatment of both bagasses due to there were 261 no sugars detected in the liquid fraction by the DNS assay. 262 The enzymatic hydrolysis of the pretreated bagasses was carried out with the home-263 made enzymatic cocktails of A. niger LBM 134. The reducing and monomeric sugars 264 from both hydrolysates and controls were shown in Table 3. The main products of the 265 hydrolysis of SCB were in (mg mL -1 ) 4.51 ± 1.14 glucose and 3.66 ± 1.06 xylose, 266 achieving a 28% of conversion to glucose and 42% to xylose, respectively. These 267 conversion percentages were similar to that obtained from the hydrolysis of pretreated 268 SCB using commercial enzymes: 23% conversion to glucose and 42% to xylose. The 269 hydrolysates from CB were rich in glucose, 5.12 ± 0.89 mg mL -1 ; reaching a 16.5% of 270 conversion, three times higher than that obtained using commercial enzymes. Reducing 271 sugars were also determined to estimate the saccharification yield; hydrolyzed 272 pretreated CB with the home-made enzymatic cocktails of A. niger LBM 134 showed 273 the maximal saccharification yield, 80%. 274

TABLE 3 275
Changes in the structure of SCB and CB were analyzed through SEM (Figure 2). 276 Electronic microscopic photographs were taken of typical features of both bagasses 277 before any treatment; the SCB fibers were covered by lignin material (Figure 2a  The cost of having a more or less complete commercial cocktail of cellulases and 292 xylanases is at least almost $900, more than 500 times the cost of producing the home-  where, G is glucose; X is xylose; t is time. These models were validated with 322 experimental data and no statistical difference was found for P <0.05 (Supplementary 323 Table 2). The validated conversion models and the kG and kX constants were used to 324 established the bioethanol production models (Supplementary Table 3): 325 where, EG is ethanol production from glucose; G0, glucose concentration at time 0; EX, 327 ethanol production from xylose; X0, xylose concentration at time 0; t, time. 328 The validation of the models was carried out applying them to experimental and 329 theorical data and comparing with the experimentally produced bioethanol. The model 330 fitted well with the experimental data, there was no significance difference for P < 0.05 331 (Supplementary Table 4). Once the ethanol production model was validated, the curve-332 fitting was employed for simulating the bioethanol yield from experimental data of the 333 saccharification of SCB and CB, achieving 4.16 mg mL -1 and 2.57 mg mL -1 , 334 21 respectively (Figure 3e). 335

FIGURE 3 336
The successful bioconversion of both SCB and CB occurred due to the home-made 337 enzymatic cocktails were produced using the respective bagasse as substrate for the 338 fungus [8]. Moreover, as the hydrolysis was carried out using fungal enzymes, there 339 was no need to detoxify the hydrolysates since there were no formation of inhibitors 340 that can negatively influence on the fermenting microorganism [10]. 341 Regarding the fermentation step, we used two yeasts enabled to simulate the 342 metabolization of hexoses such as glucose and pentoses as xylose for a more complete 343 utilization of all the sugars released during the hydrolysis of SCB [10]. On the other 344 hand, the fermentation of the hydrolysates of CB was simulated only using the glucose-345 metabolizing yeast, S. cerevisiae because CB hydrolysates were mainly rich in glucose. 346 From the bioethanol model simulation, the SCB hydrolysates would reach a higher 347 bioethanol yield than the CB hydrolysates; this behavior can be explained by xylose 348 sugars present in the SCB hydrolysates. The importance of the xylose as a fermentable 349 sugar for obtaining bioethanol in higher quantities is relevant since it has been identified 350 that non or poor utilization of the xylose components of biomass is a principal factor 351