Comparison and Optimization of Saccharification Conditions of Alkaline Pre-Treated Triticale Straw for Acid and Enzymatic Hydrol

energies

Article Comparison and Optimization of Sacchariﬁcation Conditions of Alkaline Pre-Treated Triticale Straw for Acid and Enzymatic Hydrolysis Followed by Ethanol Fermentation

Rafał Łukajtis *, Karolina Kucharska, Iwona Hołowacz, Piotr Rybarczyk, Katarzyna Wychodnik, Edyta Słupek, Paulina Nowak and Marian Kami ński Faculty of Chemistry, Department of Chemical and Process Engineering, GdańskUniversity of Technology, Narutowicza 11/12 Street, 80-233 Gdańsk,Poland; [email protected] (K.K.); [email protected] (I.H.); [email protected] (P.R.); [email protected] (K.W.); [email protected] (E.S.); [email protected] (P.N.); [email protected] (M.K.) * Correspondence: [email protected]; Tel.: +48-58-347-23-10

Received: 19 February 2018; Accepted: 8 March 2018; Published: 13 March 2018

Abstract: This paper concerns the comparison of the efﬁciency of two-stage hydrolysis processes, i.e., alkaline pre-treatment and acid hydrolysis, as well as alkaline pre-treatment followed by enzymatic hydrolysis, carried out in order to obtain reducing sugars from triticale straw. For each of the analyzed systems, the optimization of the processing conditions was carried out with respect to the glucose yield. For the alkaline pre-treatment, an optimal catalyst concentration was selected for constant values of temperature and pre-treatment time. For enzymatic hydrolysis, optimal process time and concentration of the enzyme preparation were determined. For the acidic hydrolysis, performed with 85% phosphoric acid, the optimum temperature and hydrolysis time were determined. In the hydrolysates obtained after the two-stage treatment, the concentration of reducing sugars was determined using HPLC. The obtained hydrolysates were subjected to ethanol fermentation. The concentrations of fermentation inhibitors are given and their effects on the alcoholic fermentation efﬁciency are discussed.

Keywords: lignocellulosic biomass; enzymatic hydrolysis; acid hydrolysis; alkaline pre-treatment; optimization; Box-Behnken design; bioethanol fermentation

1. Introduction Fermentation is an anaerobic respiration process carried out by many types of bacteria and fungi. Fermentation, by definition, is a disproportionation reaction. Ethanol fermentation is carried out by yeasts, including Saccharomyces cerevisiae [1–5]. Yeasts are able to ferment hexoses and some oligosaccharides, e.g., sucrose, maltose, or inulin, at specific conditions. A decreased pH value or the presence of inhibiting fermentation products, i.e., ethanol, influences the growthabilities of Saccharomyces cerevisiae. At ethanol concentrations up to 120 g/L, Saccharomyces are capable of performing fermentationand they areable to grow at ethanol concentrations up to 200 g/L [6–8]. The current global demand for biofuels is largely covered by the production of first-generation bioethanol from sucrose or starch contained in such raw materials as sugar cane, wheat, or corn. An interesting alternative is the production of the second-generation bioethanol as a result of fermentation processes from lignocellulosic waste. Lignocellulose, the most abundant renewable carbohydrate-rich biomass, is accumulated in large quantities from agricultural, forestry, or municipal activities and do not compete with food crops. It is also less expensive than conventional agricultural feedstocks. However, according to the complexity of the chemical structure, lignocellulosic biomass is

Energies 2018, 11, 639; doi:10.3390/en11030639 www.mdpi.com/journal/energies Energies 2018, 11, 639 2 of 24 resistant to biodegradation. Cellulose, hemicellulose, and lignin, main components of lignocellulosic complex, are strongly intermeshed and bonded trough covalent and non-covalent bonds. Lignin is the major barrier to make the carbohydrate components (cellulose and hemicellulose) suitable for bioconversion [9,10]. Therefore, the pre-treatment step has a large impact on subsequent enzymatic hydrolysis and fermentation steps [11]. It should improve the formation of sugars, be effective in the removal of lignin, and produce no, or limited amounts of, products that inhibit the action of the hydrolitic enzymes or the growth of fermentative microorganisms. Pre-treatment methods can be classsified as biological, physical, chemical, and physico-chemical processes. A combination of different pre-treatment methods have also been considered [12]. The main goal of alkaline treatment, one of the most common chemical pre-treatment methods, is to remove lignin from the biomass [13]. The action of alkaline reagents leads tothe degradation of side chains of esters and glycosides and then to the structural modification of lignin, cellulose swelling, cellulose decrystallization, and hemicellulose solvation. Ca(OH)2 also removes acetyl groups from hemicellulose, reducing steric hindrance and increasing the availability of the enzyme to the surface of hemicellulose and cellulose. These methods are more effective for cellulose digestibility and for lignin solubilization, exhibiting minor cellulose and hemicellulose dissolution than acid or hydrothermal processes [9]. The advantage of alkaline methods is their low cost, especially when using Ca(OH)2. The limitation is that alkali are converted into un-salted or introduced as biomass salts after the pre-treatment and, therefore, thewater consumption for salt removal from biomass is high. The lignin removal step is nessesary to avoid the formation of inhibitors (salts, furfural, phenolic acid, aldehydes). Abudi et al. [14] investigated the effects of alkaline and peroxide pre-treatment of rice straw (RS). The results indicate that NaOH and H2O2 pre-treatmentsaffected the lignocellulose composition differentlyand are particularly effective in terms of solubilization of RS hemicellulose more than of cellulose and lignin. NaOH pre-treatment led to approximately 11%, 32%, and 22% reductions in hemicellulose, cellulose, and lignin content, respectively. In the case of H2O2 pre-treatment, the corresponding reductions were 16%, 41%, and 7%, respectively. After the pre-treatment of lignocellulosic biomass, the hydrolysates are further processed by biochemical methods.During the enzymatic hydrolysis, crystalline cellulose and hemicellulose not decomposed during pretreatment, and are degraded to reducing sugars, fermentable by microorganisms. The conversion of cellulose and hemicellulose is catalysed by cellulases and hemicellulases, respectively.Cellulases are usually a mixture of several enzymes. The three predominant ones are: endo-1,4-β-glucanase, which hydrolyzes the inner β-1,4-glycosidic bonds; exo-1,4-β-glucanase, which removes mono- and dimers from the free chain-ends; and β-glucosidase (cellobiase), which hydrolyzes glucose dimers [15–17]. Cellulose hydrolysis starts with adsorption of cellulase enzymes onto the surface of cellulose. Afterwards, cellulose isbiodegraded to the fermentable sugars and cellulase is desorbed from the biomass surface.Cellulose microfibers are surrounded by hemicellulose polysaccharides. Therefore, auxiliary enzymes that attack hemicellulose are used. In the first step, endo-1,4-β-xylanase depolimerizes xylan into xylooligosaccharides.Further, xylanases, such as β-glucuronidase, α-arabinofuranosidase, and acetyl xylan esterase, cleave side chains and side groups of heteroxylan, while galactomannanase and glucomannanase hydrolyze glucomannan [18]. The optimal balanced combination of enzymes is needed to effectively modify the complex structure of lignocellulosic materials. In addition to the quality of the hydrolyzing enzymes, the digestibility of cellulose and hemicellulose is affected by pH, temperature, process time and porosity, degree of crystallization, and cellulose content [19]. Saccharification of lignocellulosic biomass with an acid catalyst is also possible. The main objective of acid treatment is the solubilization of hemicellulose and making cellulose accessible to enzymes. Acid hydrolysis is an alternative for enzymatic hydrolysis, as acid hydrolyzes biomass to fermentable sugars. However, intensive washing is required to remove the acid before fermentation is performed. The advantage of using acids is their low price and availability. Additionally, the use of organic acids (formic, acetic, maleic) makes the process environmentally friendly [20]. The disadvantage is the Energies 2018, 11, 639 3 of 24 need to neutralize the used acid by alkali, which generates a stream of reagents, or by vacuum distillation,Energies in order 2018, 11, tox FOR reuse PEER REVIEW the acid. Another disadvantage of using acid hydrolysisis3 of 25 is the possibilitydisadvantage of the formation is the need of to fermentation neutralize the used inhibitors, acid by alkali especially, which generates when higha stream acid of reagents, concentrations, high temperatures,or by vacuum and distillation long reaction, in order times to are reuse applied the acid [. 21Another,22]. During disadvantage acid of hydrolysis, using acid the acid catalyzes thehydrolysisis breakdown is the ofpossibility glycosidic of the bondsformation between of fermentation sugar inhibitors, residues, especially followed when by high the acid breakdown of releasedconcentrations, glucose to form high temperature hydroxymethylfurfurals, and long reaction (HMF), times formicare applied acid [21,22] (FA),. During and levulinic acid acid hydrolysis, the acid catalyzes the breakdown of glycosidic bonds between sugar residues, followed (LA) (Figureby1 the)[ breakdown23]. Compounds, of released glucose such to as form furfural, hydroxymethylfurfural 5-HMF, and (HMF phenolic), formic compoundsaffect acid (FA), and the microorganismlevulinic metabolism acid (LA) in(Figure the fermentation 1) [23]. Compoun step.ds, Dilutesuch as acid furfural, pre-treatment 5-HMF, and is morephenolic favourable for industrialcompoundsaffect applications the due microorganism to the reduction metabolism of the operational in the fermentation and maintenance step. Dilute costs(corrosion acid problems).pre Additionally,-treatment is more this kindfavourable of pre-treatment for industrial applications generates due fewerdegradation to the reduction of the compounds operational than the and maintenance costs(corrosion problems). Additionally, this kind of pre-treatment generates concentratedfewer aciddegradation pre-treatment compounds [18 ,than24,25 the]. concentrated acid pre-treatment [18,24,25].

Figure 1. FormationFigure 1. Formation of selected of selected fermentation fermentation inhibitors inhibitors from from glucose glucose during during acid hydrolysis acid hydrolysis..

The lignocellulosic hydrolysates are very attractive as a substrate for fermentative conversion The lignocellulosicprocesses to bioethanol. hydrolysates The results are of very the attractivelatest research as on a substrate the production for fermentative of bioethanol from conversion processes towaste bioethanol. lignocellulosic The biomass results are of presented the latest in researchTable 1. on the production of bioethanol from waste lignocellulosic biomass are presented in Table1. Table 1. The efficiency of bioethanol production related to the pre-treatment method and used microorganisms. Table 1. The efficiency of bioethanol production related to the pre-treatment method and used microorganisms. Type of Type of Ethanol Yield Lignocellulosic Used Microorganism Processes Involved References Pretreatment (g/g) Type ofMaterial Type of Used Ethanol Processes LignocellulosicCotton Hydrolysis and References Pretreatment Microorganism Yield (g/g) Involved Materialresidues,corn - Saccharomyces cerevisae 0.027 fermentation realized [21] stover separately HydrolysisSimultaneous and Cotton residues, SaccharomycesKluyveromyces Wheat straw Steam- explosion 00.027.27 saccharificationfermentation and [26] [21] corn stover marxianuscerevisae realizedfermentation separately SimultaneousSimultaneous Sugarcane Acid digestion, Kluyveromyces Wheat straw Steam explosion Pichia stipitis 0.0.2739 saccharificationsaccharification and and [27] [26] bagasse steam explosion marxianus fermentationfermentation Simultaneous Simultaneous Sugarcane Acid digestion, 0.29 saccharification and Sugarcane Pichia stipitis 0.39 saccharificationfermentation and [27] bagasse steam explosionMilling Pichia stipitis [28] bagasse Hydrolysisfermentation and 0.27 fermentation realized Simultaneous separately 0.29 saccharification and Hydrolysis and Sugarcane fermentation Rice husks AlkalineMilling digestion EscherichiaPichia stipitis coli 0.21 fermentation realized [29] [28] bagasse Hydrolysisseparately and 0.27 fermentation realized separately Hydrolysis and 0.21 fermentation realized separately Rice husks Alkaline digestion Escherichia coli [29] Simultaneous 0.20 saccharification and fermentation Energies 2018, 11, 639 4 of 24

Table 1. Cont.

Type of Type of Used Ethanol Processes Lignocellulosic References Pretreatment Microorganism Yield (g/g) Involved Material Acid digestion, Maize cob Zymomonas mobilis 0.42 Batch reactor [30] overliming Drying Clostridium 0.36 Acid digestionthermocellum; 0.40 Clostridium Alkaline digestionthermosaccharolyticum 0.42 Banana remnants Batch reactor [31] Drying 0.33 Clostridium Acid digestionthermocellum; 0.36 Clostridium ethanolicus Alkaline digestion 0.39

Currently, lignocellulosic biomass is regarded as the forward-looking, large-scale available non-fossil carbon source for biofuels production. Therefore, there is a strong motivation for research into industrially and economically viable processes for conversion of biomass to fuels, including bioethanol. However, successful and effective conversion of lignocellulosic biomass to monosaccharides in high yields is still a challenge. Acid or enzymatic hydrolysis of biomass polysaccharides after a pre-treatment stage are the main currently-investigated saccharification methods [32–36]. Therefore, the primary objective of the present study is the comparison of the efficiency of two-stage hydrolysis processes, i.e., alkaline pre-treatment followed by acid hydrolysis, as well as alkaline pre-treatment followed by enzymatic hydrolysis, carried out in order to obtain reducing sugars from triticale straw. The comparison is to concern not only results obtained for mono sugars, but for bioethanol as well. The aim of the work is also to examine the course and efficiency of the enzymatic hydrolysis using cellulolytic enzymes immobilized on diatomite. This procedure is intended to reduce process costs through the possibility of enzyme regeneration and reuse. The optimization of the conditions of the two-stage hydrolysis and the evaluation of the influence of process parameters on the glucose yield were performed according to the Box-Behnken design for the following variables: concentration of NaOH, duration time of acid hydrolysis, temperature of acid hydrolysis, the content of Viscozyme L, and the duration time of the enzymatic hydrolysis process. Finally, the parameters’ significance on the performance of sugars were optimized using the response surface methodology. The effect of acid hydrolysis conditions on the content of typical fermentation inhibitors, including 5-HMF, furfural, and the total phenolic compounds was investigated. Additionally, pre-treatment and hydrolysis effects were evaluated by performing bioethanol fermentation on previously-obtained hydrolysates.

2. Results and Discussion

2.1. Biomass Characterisation Milled and minced lignocellulosic biomass (triticale straw Triticum sp., originated from Wejherowo, Poland) was used in this study. The determined composition of the Triticum sp. was found to be: 43.6% cellulose, 23% hemicellulose, 21.5% lignin, 8.3% water extractives, 8.6% ethanol extractives, 1.2% ash, and a moisture content of 6.2% (on dry weight basis). Hemicellulose content was found as follows: xylose (18.1%), galactose (4.1%), and mannose with arabinose (0.8%).

2.2. Influence of Alkaline Pre-Treatment Conditions of the Chemical Composition of Triticale Straw The optimization of the two-stage hydrolysis conditions and the evaluation of the influence of process parameters on the glucose yield were made for two test systems. In the first system, alkaline pre-treatment and acid hydrolysis were carried out, while, in the second system, alkaline pre-treatment Energies 2018, 11, 639 5 of 24 followed by enzymatic hydrolysis. In both cases, the alkaline pre-treatment was carried out for six hours at a constant temperature of 65 ◦C. The treatment was carried out for the catalyst concentrations (NaOH) of 1%, 6%, and 11%. In the first stage of the research, the effect of alkaline pre-treatment on the change of triticale straw composition was determined. For this purpose, the composition of biomass (reducing sugars and lignin contents), as well as the degree of lignin removal, the degree of cellulose and hemicellulose recoveries were determined in the residue remaining after the alkaline treatment. The results (Table2) indicate that an increase in the concentration of NaOH causes an increase in the biomass loss of 37%, 49%, and 52%, respectively, for catalyst concentrations of 1%, 6%, and 11%. A similar dependence was obtained in [12,37,38]. The research results indicate that the use of 1% NaOH already significantly lowers the lignin content. A further increase in the catalyst concentration above 1% does not significantly affect the lignin removal. The alkaline pre-treatment of the material influences the change in the proportion of the sugar polymer content. The use of 1% NaOH allows the content of all reducing sugars to increase. For NaOH concentrations greater than 1%, a decrease in reducing sugars coming from hemicellulose with a simultaneous increase in glucose concentration is observed.

Table 2. Composition of raw biomass and obtained hydrolysates.

Glucose Xylose Galactose Mannose, Lignin Cellulose Hemicellulose Lignin Content Content Content Arabinose Content Content Recovery Recovery Removal (mg/gbiomass) (mg/gbiomass) (mg/gbiomass) (mg/gbiomass) (%) (%) (%) (%) Untreated 436 181 41 8 21.5 - - - 1% NaOH 548 230 58 13 9.5 79.3 82.4 72.1 6% NaOH 644 171 43 0 8.4 75.4 47.4 75.7 11% 701 141 22 0 8.4 77.2 33.9 75.2 NaOH

2.3. Optimisation ofAlkaline Pre-Treatment Followed by Acid Hydrolysis For the analysis of the inﬂuence of input variables on the result of two-stage hydrolysis, i.e., alkaline treatment and acid hydrolysis, experiments (Table3) were performed according to the Box-Behnken design for three variables. The following boundary values were assumed for the investigated variables: concentration of NaOH 1–11% at a constant temperature of 65 ◦C and alkaline pre-treatment time of 6h, duration of acid hydrolysis of 0.5–8 h, and the temperature of acid hydrolysis ◦ of 60–120 C. As an output parameter, glucose yield (Gy) was selected as a parameter describing the efﬁciency of the process. Additionally, Table3 shows the obtained values of the sum of reducing sugars.

Table 3. Box-Behnken design for alkaline pre-treatment and acid hydrolysis of Triticum sp.

◦ No. CNaOH (%) Temperature ( C) Time (h) Gy (mg/gbiomass) Sum of Sugars (mg/gbiomass) 1 1 90 0.5 334.5 397.3 2 11 90 0.5 293.9 360.8 3 1 90 8 104.6 122.3 4 11 90 8 106.4 118.5 5 1 60 4.25 229.6 314.6 6 11 60 4.25 204.9 285.0 7 1 120 4.25 7.3 13.2 8 11 120 4.25 7.6 8.2 9 6 60 0.5 56.4 147.9 10 6 60 8 301.7 369.3 11 6 120 0.5 271.4 325.3 12 6 120 8 14.3 14.3 13 6 90 4.25 265.6 314.8 14 6 90 4.25 260.7 312.8 15 6 90 4.25 269.8 326.1 Energies 2018, 11, 639 6 of 24

An inﬂuence of the process parameters (NaOH concentration, temperature and duration of acid hydrolysis) on the glucose yield was investigated. On the basis of multiple regression, the Equation (1) was proposed, describing the relationships occurring during the two-stage hydrolysis.

2 2 EnergiesY [%] 2018 =, 11−, 1426x FOR− PEER3.98X REVIEW1 + 34.45X 2 + 177.70X3 − 0.16X2 − 3.64X3 + 0.57X1·X3 − 1.97X2·X3 6 of (1)25

InIn Equation (1), (1), X1,,X X22, ,and and X X33 representrepresent sodium sodium hydroxide hydroxide concentration, concentration, temperature temperature,, and and timetime of of acid acid hydrolysis hydrolysis,, respectively respectively.. Y Y denotes glucose efficiency. efficiency. Equation (1) (1) enables calculat calculatinging thethe theoretical theoretical glucose glucose yield yield within within the the range range of of variables variables according according to to the the boundary boundary conditions. conditions. TheThe parameters parameters ofof Equation Equation (1) (1) are are given given in Table in Table4. In order 4. In to order determine to determine the effect of the the effect parameters of the parameterssignificance significance on the performance on the performance of sugars, of an sugars, analysis an ofanalysis variance of forvariance the surface for the ofsurface the model of the's model'sresponse response was made. was The made. form The of Equation form of Equation (1) is the result(1) is ofthe rejecting result of the rejecting least significant the least parameterssignificant parametersin the next iterativein the next steps. iterative This procedure steps. This showed procedure that showed the least that important the least parameters important in parameters such a system in suchis the a interaction system is the between interaction concentration between ofconcentration catalyst and temperatureof catalyst and (X 1temperature·X2). For this (X interaction,1·X2). For this the interaction,p-value was the much p-value higher was than much the higher assumed than significance the assumed level significanp < 0.1,ce which level isp why< 0.1, itwhich was rejected. is why itBased was onrejected. analysis Based of variance, on analysis the obtained of variance, value the of theobta determinationined value of coefficient the determination (R2 = 0.996, coefficient Figure2) (Rindicates2 = 0.996, a veryFigure good 2) indicates correlation a very between good the correlation efficiency between calculated the on efficiency the basis calculated ofEquation on (1) the and basis the ofactualEquation glucose (1) an efficiencyd the actual within glucose the investigated efficiency within range ofthe variables. investigated range of variables.

TableTable 4.4. Model parametersparameters forfor Box-BehnkenBox-Behnken designdesign for for alkaline alkaline pre-treatment pre-treatment and and acid acid hydrolysis hydrolysis of ofTriticum Triticumsp. sp. Parameter Value Std. Error t-Value p-Value ParameterIntercept Value−1426 Std.53.340 Error −t26.746-Value 1.81 ×p 10-Value−7 −7 InterceptX1 −1426−3.98 53.3401.234 −3.22726.746 0.0180 1.81 × 10 X −3.98 1.234 −3.227 0.0180 1 X2 34.45 1.118 30.824 7.74 × 10−8 × −8 X2 34.45 1.118 30.824 7.74 −7 10 X3 177.70 6.485 27.397 1.56 × 10 −7 X3 177.70 6.485 27.397 1.56 × 10 2 X22 −0.16 0.006 −28.087 1.35 × 10−7 −7 X2 −0.16 0.006 −28.087 1.35 × 10 2 2 −5 −5 X3 X3 −−3.643.64 0.376 −−9.6639.663 7.04 7.04 × 10× 10 X1·X3X1·X3 0.570.57 0.246 2.295 2.295 0.0615 0.0615 −8 X2·X3X2·X3 −−1.971.97 0.055 −−35.63135.631 3.26 3.26 × 10×−8 10

400

300

R2 = 0,996 Experimental 200 Gy, mg/mL

100

0 0 100 200 300 400

Model-predicted Gy, mg/mL

Figure 2. Analysis of variance for the theoretical (model-predicted) and experimental glucose yield for Figurealkaline 2. pre-treatment Analysis of variance and acid for hydrolysis. the theoretical (model-predicted) and experimental glucose yield for alkaline pre-treatment and acid hydrolysis. The attached ﬁgures show model surface responses and are the effect of the interactions between two selectedThe attached variable figures parameters, show while model maintaining surface response the thirds parameterand are the at a effect constant of thelevel. interactions The model betweenresponse two is the selected concentration variable of parameters, glucose (glucose while yield) maintaining after the the two-stage third parameter hydrolysis, at where a constant in the level. The model response is the concentration of glucose (glucose yield) after the two-stage hydrolysis, where in the second stage the obtained solid fraction after the alkaline pre-treatment was used. The analysis of the surface response was useful for the determination of the individual effects of each parameter and the impact of interactive effects of independent variables on the glucose yield. This approach allowed to determine the optimal conditions with respect to each investigated parameter. Figure 3 presents the effect of NaOH concentration and acid hydrolysis temperature on glucose yield (acid hydrolysis time 1h). The results indicate that the glucose yield reaches a Energies 2018, 11, 639 7 of 24 second stage the obtained solid fraction after the alkaline pre-treatment was used. The analysis of the surface response was useful for the determination of the individual effects of each parameter and the impact of interactive effects of independent variables on the glucose yield. This approach allowed to determine the optimal conditions with respect to each investigated parameter. EnergiesFigure 2018, 113 presents, x FOR PEER the REVIEW effect of NaOH concentration and acid hydrolysis temperature on glucose7 of 25 yield (acid hydrolysis time 1h). The results indicate that the glucose yield reaches a maximum at maximum at about 100 °C. Increasing the temperature of acid hydrolysis above 100 °C results in a about 100 ◦C. Increasing the temperature of acid hydrolysis above 100 ◦C results in a decrease of decrease of glucose yield due to the breakdown of simple sugars. The increase in NaOH glucose yield due to the breakdown of simple sugars. The increase in NaOH concentration during the concentration during the pre-treatment causes a decrease in a glucose yield. This is caused by a pre-treatment causes a decrease in a glucose yield. This is caused by a signiﬁcant loss of mass during significant loss of mass during alkaline pre-treatment. Biomass loss is related to the catalyst alkaline pre-treatment. Biomass loss is related to the catalyst concentration and it is equal to 38% for concentration and it is equal to 38% for the treatment of 1% NaOH and 52% when 11% NaOH is used. the treatment of 1% NaOH and 52% when 11% NaOH is used.

350 Gy, mg/g 300 300-350 250 250-300 200-250 200 150-200 150 11 100-150 100 9 7 50-100 50 0 5 0-50 60 CNaOH, % 70 3 80 90 Time = 1h o Temp, C 100 1 110 120 Figure 3.3. TheThe surface surface response response plots plots of of the effects of NaOH concentration and temperature acid hydrolysis on the glucose yield.yield.

Figure 44 showsshows thethe effecteffect ofof temperaturetemperature andand durationduration timetime ofof acidacid hydrolysishydrolysis onon thethe glucoseglucose yield for a constant sodium basebase concentrationconcentration (6%). Glucose efficiency efficiency increases with increasing temperature, however, however, when the optimal temperature (100 °C)◦C) is reached, a significant significant decrease in glucose efficiency efficiency is observed if the temperature is further increased.increased. For temperaturesintemperaturesin the range of 90 90–100–100 °C◦C,, and and for for a a short time time of of acid hydrolysis hydrolysis (0.5 (0.5 h), h), the the highest highest glucose glucose yields yields are are obtained. obtained. An increase increase in in time time over over 0.5 h 0.5 results h results in a decrease in a decrease in the hydrolysis in the hydrolysis efficiency. efficiency. Only for the Only temperature for the temperatureof 60 ◦C, anincrease of 60 °C, in glucose an increase efficiency in glucose along with efficiency the duration along time with of the acid duration hydrolysis time is observed. of acid hydrolysisFigure is5a,b observed. present an influence of the duration time of acid hydrolysis and NaOH concentrationFigure 5a (alkaline,b present treatment) an influence on the glucose of the yield duration at a constant time of temperature acid hydrolysis of acidic and hydrolysis. NaOH concentrationTwo possibilities (alkaline of interperation treatment) of two-stage on the glucose hydrolysis yield should at a be constant considered temperature here. In one of variant, acidic hydrolysis.when the acid Two hydrolysis possibilities temperature of interperation is 100 ◦ Cof (Figure two-stage5b), highhydrolysis yield ofshould reducing be considered sugars can onlyhere. beIn oneachieved variant, for whena short the duration acid hydrolysis of acidic hydrolysis temperature (0.5 is h). 100 The °C increase(Figure in 5b the), high duration yield of of treatment reducing sugarsresults canin a only decrease be achieved of the glucoseyield for a short duration due to the of degradation acidic hydrolysis of simple (0.5 h). sugars. The increase When a in lower the durationtemperature of treatment of 60 ◦C is results applied in (other a decrease variant, of Figurethe glucose5a), theyield highest due to yield the is degradation achieved after of simple 8 h of sugars.acidic hydrolysis. When a lower The choicetemperature of process of 60 temperature, °C is applied besides (other the variant, efficiency Figure of reducing5a), the highest sugars, yield should is achievedalso be supprted after 8 h by of the acidic concentration hydrolysis. of The fermentation choice of process inhibitors temperature, in the resulting besides hydrolysate. the efficiency of reducing sugars, should also be supprted by the concentration of fermentation inhibitors in the resulting hydrolysate. Energies 2018, 11, 639 8 of 24

Energies 2018, 11, x FOR PEER REVIEW 8 of 25

350 Gy, mg/g 300

300-350 250 250-300 200-250 200 150-200 150 100-150 100 50-100 0-50 50 0 CNaOH = 6% 0,5 2 3,5 5 120 6,5 100 Time, h 80 608 Temp, oC Figure 4. The surface response plots of the effects of temperature and time on the glucose yield. Figure 4. The surface response plots of the effects of temperature and time on the glucose yield.

Gy, mg/g

250-300 300 200-250 250

150-200 200 100-150 150 50-100 100 9 0-50 CNaOH, % 50 5 Temp= 60oC 0 1 0,5 1,5 2,5 3,5 4,5 5,5 6,5 7,5

Time, h (a)

Gy, mg/g 400

300-400 300 200-300 100-200 200 0-100 100 1

0 10 0,5 7 1,5 2,5 4 C , % Temp= 100oC 3,5 NaOH 4,5 5,5 6,5 1 Time, h 7,5 (b)

Figure 5. The surface response plots of the effects of NaOH concentration and time acid hydrolysis Figure 5. The surface response plots of the effects of NaOH concentration and time acid hydrolysis on on the glucose yield: (a) at a constant temperature 60 °C ; (b) at a constant temperature 100 °C . the glucose yield: (a) at a constant temperature 60 ◦C; (b) at a constant temperature 100 ◦C. Energies 2018, 11, 639 9 of 24

The analysis of the surface response areas (Figures3–5) allowed to determine the optimal conditions for a two-stage process involving alkaline pre-treatment (6% NaOH, 6 h, 65 ◦C) and ◦ acid hydrolysis (100 C, 0.5 h and 85% H3PO4). For these conditions, hydrolysis of the triticale straw was performed to obtain, from 1 g of raw material, 320 mg of glucose, 42.8 mg of xylose, 19 mg of galactose and 1.5 mg of arabinose, respectively. A similar result of the yield of sugars was obtained ◦ for acid hydrolysis with sulfuric acid (70% H2SO4, 30 C, 2 h) of Saccharum spontaneum, also after treatment with sodium base (7%, 48 h, 30 ◦C). For these conditions, a maximum yield of reducing sugars was obtained 369.6 mg/g [14]. The acid hydrolysis was carried out on straw which had not been subjected to the previous alkaline pre-treatment, obtaining, from 1 g of raw material, 420 mg of glucose, 150 mg of xylose, 38 mg of galactose and 4.7 mg of arabinose, respectively. It is clear that the efﬁciency of reducing sugars for acid hydrolysis with the omission of the alkaline pre-treatment is the highest.

2.4. Optimisation ofAlkaline Pre-Treatment Followed by Enzymatic Hydrolysis The system consists of 15 experiments planned according to the Box-Behnken design. Experiments include two-stage hydrolysis of biomass, i.e., alkaline pre-treatment followed by enzymatic hydrolysis. During the alkaline pre-treatment, NaOH concentration in the ﬁrst step of hydrolysis is optimized at a constant process temperature of 65 ◦C, pre-treatment time of 6h, and granulation of 0.75 mm. In the second step, enzymatic hydrolysis values of the time and ratio of cellulolytic enzymes immobilized on a solid support are optimized. As the boundary conditions, on the basis of previous experiments, NaOH concentrations of 1%, 6%, and 11% were assumed, and for enzymatic hydrolysis time 8, 16, and 24 h and the content of Viscozyme L in a mixture of 0.9, 0.95, and 1 were selected. Box-Behnken design estimation allowed the determination of the response surface based on the quadratic equation. As the initial parameter representing the efﬁciency of the process, the mass of the obtained glucose was selected (glucose yield, Gy). Table5 shows the obtained values of the glucose and the sum of reducing sugars.

Table 5. Box-Behnken design for alkaline pre-treatment and enzymatic hydrolysis of Triticum sp.

No. CNaOH (%) Viscozyme L Time (h) Gy (mg/gbiomass) Sum of Sugars (mg/gbiomass) 1 1 0.90 16 234.7 278.8 2 11 0.90 16 250.1 292.8 3 1 1.00 16 186.2 245.3 4 11 1.00 16 207.6 289.4 5 1 0.95 8 143.9 184.0 6 11 0.95 8 189.4 249.7 7 1 0.95 24 219.8 268.5 8 11 0.95 24 159.7 195.4 9 6 0.90 8 208.4 289.0 10 6 1.00 8 163.1 204.2 11 6 0.90 24 234.8 275.9 12 6 1.00 24 218.3 257.2 13 6 0.95 16 231.5 302.0 14 6 0.95 16 235.6 296.8 15 6 0.95 16 227.4 281.4

The inﬂuence of the input parameters, i.e., the NaOH concentration, the Viscozyme L content, and the duration time of the enzymatic hydrolysis process have been examined considering the glucose yield as the model output. The equation describing the relationships occurring during the two-stage hydrolysis was determined by multiple regression. The obtained regression equation describing the surface response takes the form:

2 2 Y [%] = 700.18 − 20.89X1 − 763.82X4 − 0,82 X1 − 0.52X5 − 0.66X1·X5 + 23.86X4·X5 (2) Energies 2018, 11, 639 10 of 24

EnergiesIn Equation 2018, 11, x FOR (2) XPEER1,X REVIEW4, and X 5 represent sodium hydroxide concentration, Viscozyme L10 content, of 25 and the time of enzymatic hydrolysis, respectively. Y denotes a glucose yield. Equation (2) enables thethe calculation calculation of of the the theoretical theoretical glucoseglucose yield within within the the range range of of variables variables according according to toboundary boundary conditions.conditions. In In order order to to obtain obtain Equation (2), (2), the the least least significant significant parameters parameters we werere rejected rejected in in the the consecutiveconsecutive iterative iterative steps. steps. It turned It turned out that out the that least the important least important parameters parameters are: NaOH are: concentration, NaOH durationconcentration, of enzymatic duration hydrolysis, of enzymatic and the hydrolysis ratio of enzymes, and the raised ratio to of the enzymes square. raised Equation to the 2 is square. described byEquation the model 2 parametersis described contained by the model in Table parameters6. Based conton theained analysis in Table of variance, 6. Based the on obtained the analysis value of of variance, the obtained value of the determination coefficient (R2 = 0.934) indicates a good correlation the determination coefficient (R2 = 0.934) indicates a good correlation of the results calculated from the of the results calculated from the equation with the experimental results of glucose efficiency equation with the experimental results of glucose efficiency (Figure6). (Figure 6).

TableTable 6. 6Model. Model parameters parameters for for thethe Box-BehnkenBox-Behnken design design for for alkaline alkaline pre pre-treatment-treatment and and enzymatic enzymatic hydrolysishydrolysis of ofTriticum Triticumsp. sp.

Parameter Value Std. Error t-Value p-Value Parameter Value Std. Error t-Value p-Value Intercept 700.177 75.480 9.276 1.48 × 10−5 −5 InterceptX1 700.17720.887 75.4803.463 6.031 9.276 0.0003121.48 × 10 X1 X4 20.887−763.822 3.46388.777 −8.604 6.031 2.58 × 0.000312 10−5 −5 2 − − × X4 X1 763.822−0.819 88.7770.220 −3.7258.604 0.0058302.58 10 2 − − X1 X52 0.819−0.523 0.2200.084 −6.2333.725 0.000250 0.005830 2 − − X5 X1·X5 0.523−0.656 0.0840.132 −4.9646.233 0.001102 0.000250 X ·X −0.656 0.132 −4.964 0.001102 1 X5 4·X5 23.864 2.975 8.022 4.281 × 10−5 −5 X4·X5 23.864 2.975 8.022 4.281 × 10

270

250

230 Experimental R2 = 0,934 210 Gy, mg/mL 190

170

150 150 200 250 300

Model-predicted Gy, mg/mL

Figure 6. Analysis of variance for the theoretical (model-predicted) and experimental glucose yield for Figure 6. Analysis of variance for the theoretical (model-predicted) and experimental glucose yield alkaline pre-treatment and enzymatic hydrolysis. for alkaline pre-treatment and enzymatic hydrolysis.

TheThe analysis analysis of reactionof reaction mixture mixture samples samples shows shows that thatthe main the main product product of lignocellulosic of lignocellulosic biomass hydrolysisbiomass hydrolysis is mainly glucose. is mainly Table glucose.5 presents Table the5 presents yield of the this yield monosugars of this monosugar in the hydrolysates.s in the Thehydrolysates obtained result. The was obtained compared result with was the compared sum of all with reducing the sum sugars. of all Physical reducingstudies sugars. have Physical shown lowstudies content have of cellobioseshown low in content hydrolysates, of cellobiose therefore, in hydrolysates it was crucial, therefore to use a small, it was addition crucial ofto βuse-glucosidase a small inaddition a mixture of with β-glucosidase Viscozyme in L. a Cellobiose mixture with is an Viscozy inhibitorme of L. cellulolytic Cellobiose enzymes is an inhibitor and its of presence cellulolytic lowers theenzymes efficiency and of itsenzymatic presence hydrolysis. lowers the Figure efficiency7 presents of enzymatic exemplary hydrolysis. changes in Figure glucose 7 presents efficiency forexemplary enzymatic changes hydrolysis in glucose conducted efficiency for 16hfor enzyma in the rangetic hydrolysis of variables conducted described for 16 inh Table in the5 .range Based of on thevariables shape of describe the responsed in Table surface 5. Based it can on be the stated shape that of forthe theresponse change surface in NaOH it can concentration, be stated that afor local maximumthe change around in NaOH 9% is concentration, observed, above a local and maximum below which around the 9% obtained is observed glucose, above yields andare below lower. Therefore,which the 9% obtained NaOH glucose concentration yields isare chosen lower. asTherefore the optimal, 9% value.NaOH Figureconcentration7 also shows is chosen that as there the is a linearoptimal relationship value. Figure between 7 also theshows yield that of there glucose is a and linear the relationship ratio of β-glucosidase between the in yield the mixtureof glucose with Viscozymeand the ratio L during of β-g thelucosidase enzymatic in the hydrolysis, mixture with where Viscozyme an increase L during in the ratiothe enzymatic of β-glucosidase hydrolysis, (or the where an increase in the ratio of β-glucosidase (or the decrease of Viscozyme L) corresponds with decrease of Viscozyme L) corresponds with an increase in the yield of glucose after enzymatic hydrolysis. an increase in the yield of glucose after enzymatic hydrolysis. The mentioned changes are small Energies 2018, 11, x FOR PEER REVIEW 11 of 25

EnergiesenoughEnergies 20182018 to, 11,decide11, x, 639FOR that PEER the REVIEW optimal addition of β-glucosidase chosen for further experiments 11is11 of0 of .2505 24 in proportion to Viscozyme L equal to 0.95. enough to decide that the optimal addition of β-glucosidase chosen for further experiments is 0.05 inThe proportio mentionedn to changesViscozyme are L small equal enough to 0.95. to decide that the optimal addition of β-glucosidase chosen for further experimentsGy, ismg/g 0.05 in proportion270 to Viscozyme L equal to 0.95.

G , mg/g 270 y 240-270 240

240-270 210-240 240210

210-240180-210 210180

180-210150-180 180150 11 0,9 9 150-180 0,92 7 Time = 16h 150 0,94 5 11 0,9 0,96 9 0,98 3 CNaOH, % Time = 16h Viscozyme0,92 L, - 7 0,94 1 5 0,96 3 CNaOH, % Viscozyme L, - 0,98 1 Figure 7. The surface response plots of the effects of NaOH concentration and Viscozyme L content onFigure the glucose 7. The surface yield after response 16 h of plots enzymatic of the effects hydrolysis of NaOH. concentration and Viscozyme L content on Figurethe glucose 7. The yield surface after response 16 h of enzymaticplots of the hydrolysis. effects of NaOH concentration and Viscozyme L content onFigure the glucose 8 presents yield afterchanges 16 h inof enzymaticglucose efficiency hydrolysis for. enzymatic hydrolysis carried for Viscozyme L contentFigure of8 0presents.95. Based changes on the incourse glucose of the efﬁciency response for surface enzymatic it can hydrolysis be stated carriedthat for for the Viscozyme change in Figure 8 presents changes in glucose efficiency for enzymatic hydrolysis carried for Viscozyme NaOHL content concentration, of 0.95. Based a local on themaximum course ofaround the response 7–9% is observed surface it. canObtained be stated results that confirm for the that change 9% L content of 0.95. Based on the course of the response surface it can be stated that for the change in NaOHin NaOH concentration concentration, is an a localadequa maximumte concentration around 7–9%of alkaline is observed. catalyst Obtained chosen as results the optimal conﬁrm value. that NaOH concentration, a local maximum around 7–9% is observed. Obtained results confirm that 9% A9% relationship NaOH concentration between time is an and adequate glucose concentration yield during of enzymatic alkaline catalyst hydrolysis chosen show as the that optimal an increase value. NaOH concentration is an adequate concentration of alkaline catalyst chosen as the optimal value. ofA relationshipthe hydrolysis between time above time and16 h glucoseis no longer yield reasonable. during enzymatic It was hydrolysisdecided that show the thatoptimal an increasetime of A relationship between time and glucose yield during enzymatic hydrolysis show that an increase enzyof thematic hydrolysis hydrolysis time is above equal 16 to h 16is noh for longer Viscozyme reasonable. L content It was of decided 0.95 and that 9% the concentration optimal time of of the hydrolysis time above 16 h is no longer reasonable. It was decided that the optimal time of NaOH.enzymatic hydrolysis is equal to 16 h for Viscozyme L content of 0.95 and 9% concentration of NaOH. enzymatic hydrolysis is equal to 16 h for Viscozyme L content of 0.95 and 9% concentration of NaOH.

Gy, mg/g 250

Gy,200-250 mg/g 250 200-250150-200 200 100-150 150-200 200 100-150 150 150 100 6 11 Viscozyme L = 0.95100 9 9 12 7 6 11 Viscozyme L = 0.95 9 15 5 9 12 18 3 Time, h CNaOH7 , % 15 21 1 5 18 3 Time, h 24 CNaOH, % 21 1 Figure 8. The surface response plots of the effects of NaOH concentration 24 and time on the glucose yield for enzyme ratio of 0.95.0.95. Figure 8. The surface response plots of the effects of NaOH concentration and time on the glucose yieldFigure for enzyme9 present ratios changesof 0.95. in response surface for glucose efficiency during the two-step Figure9 presents changes in response surface for glucose efﬁciency during the two-step alkaline alkaline and enzymatic hydrolysis conducted for NaOH concentration equal to 9%. A maximum in andFigure enzymatic 9 present hydrolysiss changes conducted in response for NaOH surface concentration for glucose equal efficiency to 9%. A maximum during the in twothe- rangestep β alkalineof about and 15–17 enzymatic h of the hydrolysis enzymatic conducted hydrolysis for time NaOH can beconcentration observed. Theequal ratio to 9%. of A-glucosidase maximum in to Energies 2018, 11, x FOR PEER REVIEW 12 of 25 Energies 2018, 11, 639 12 of 24 the range of about 15–17 h of the enzymatic hydrolysis time can be observed. The ratio of βViscozyme-glucosidase L to does Viscozyme not show L anydoes signiﬁcant not show changesany significant in the hydrolysischanges in course.the hydrolysis Yet, the course. addition Yet, of theβ-glucosidase addition of toβ-g Viscozymelucosidase Lto increasesViscozyme the L costincreases of enzymatic the cost hydrolysis,of enzymatic therefore, hydrolysis, the therefore content of, theViscozyme content of L ofViscozyme 0.95 is taken L of as 0.95 optimal is taken on as the optimal basis of on results the basis given of results in Figures given7 andin Figures9. 7 and 9.

Gy, mg/g 300

250 250-300 200-250 200 150-200 100-150 150

100 CNaOH = 9% 0,9 6 0,92 9 0,94 12 0,96 15 Viscozyme L, - 0,98 18 Time, h 1 21 24

Figure 9. The surface response plots of the effects of Viscozyme L content and hydrolysis time on the Figure 9. The surface response plots of the effects of Viscozyme L content and hydrolysis time on glucose yield for NaOH concentration of 9%. the glucose yield for NaOH concentration of 9%.

BasedBased on on statistical statistical analysis ofof experimentalexperimental data data and and the the analysis analysis of of response response surface surface course course and andchanges changes optimal optimal values values for furtherfor further experiments experiments have have been been determined. determined. It can It be can stated be stated that a that NaOH a NaOHconcentration concentration of 9%, anof 9%, enzyme an enzyme ratio equal ratio to equal 0.95, andto 0.95 a time, and of a 16 time h is of the 16 set h ofis the optimal set of conditions optimal conditionsfor two-step for alkaline-enzymatic two-step alkaline-enzymatic hydrolysis. hydrolysis.

2.5. Influence of the Pre-Treatment and Saccharification Conditions on the Formation of Fermentation Inhibitors 2.5. Influence of the Pre-Treatment and Saccharification Conditions on the Formation of Fermentation InhibitorsFor the hydrolysis experiments using phosphoric acid according to the Box-Benkhen design, theFor content the hydrolysis of typical fermentationexperiments using inhibitors phosphoric including acid 5-HMF, according furfural, to the andBox- theBenkhen total phenolicdesign, thecompounds content of was typical determined fermentation (Table 7 inhibitors). The application including of 5- acidHMF, hydrolysis furfural, involvesand the thetotal possibility phenolic compoundsof the transformation was determined of sugars (Table (including 7). The application pentoses) toof furfuralacid hydrolysis and levulinic involves acid, the andpossibility to 5-HMF of thein thetransformation case of glucose of sugars [23,39]. (including Partial hydrolysis pentoses) of to lignin furfural as a and result levulinic of which acid, phenolic and to compounds 5-HMF in thepass case into of the glucose hydrolysate [23,39] solution. Partial ishydrolysis also possible. of lignin Therefore, as a result the use of of which acid phenolicin the saccharification compounds passprocess into ofthe lignocellulosic hydrolysate solution biomass is may also be possible. associated Therefore, with the the formation use of acid of compoundsin the saccharification negatively processaffecting of thelignocellulosic metabolism biomass of microorganisms may be associated (so-called with inhibitors). the formation Depending of compounds on the concentration, negatively affectinginhibitors the may metabolism completely of block microorganisms the course of (so fermentation-called inhibitors). processes Depending or affect the on products the concentration, of microbial inhibitorsmetabolism may [40 completely,41]. Presence block of inhibitors the course in of the fermentation hydrolysate processes often involves or affect the the need products to perform of microthe detoxificationbial metabolism prior [40,41] to fermentation.. Presence of Ininhibitors this work, in the the hydrolysate effect of the often hydrolysis involves conditions the need with to perform85% phosphoric the detoxification acid solution prior on to the fermentation. content of fermentation In this work, inhibitors the effect wasof the investigated. hydrolysis conditions Analysis of withthe obtained 85% phosphoric results shows acid that solution temperature on the of content hydrolysis of fermen is the mosttation significant inhibitors parameter was investigated. influencing Analysisthe formation of the of obtained fermentation results inhibitors. shows that The temperature rate and efficiency of hydrolysis of catalytic is the conversion most significant of simple sugars to furfural and HMF is higher for higher temperatures of the hydrolysis. At temperatures parameter influencing◦ the formation of fermentation inhibitors. The rate and efficiency of catalytic conversionabove 100 ofC, simple degradation sugars processes to furfural occur and in which, HMF is apart higher from for the higher destruction temperature of sugars,s of there the hydrolysis.is also degradation At temperatures of furfural, above HMF, 100 as°C, well degradation as phenolic processes compounds. occur The in which, effects apart of degradation from the increase with the increase of the duration time of the hydrolysis process. This is why when the destruction of sugars, there is also degradation of furfural, HMF,◦ as well as phenolic compounds. Thehydrolysis effects of time degradation is 8 h and increase the hydrolysis with the temperature increase of isthe 120 durationC, the concentrationstime of the hydrolysis of simple process. sugars Thisand inhibitorsis why when are almost the hydrolysis zero. The performance time is 8 h of and the the hydrolysis hydrolysis at lower temperature temperature is 120 leads °C, to the the concentrationsformation of inhibitors, of simple however, sugars and their inhibitors concentration are almost is much zero. lower The than performance for the process of the carried hydrolysis out at athigher lower temperature temperature and leads for tothe the same formation time. Comparing of inhibitors, the processes however, carried their concentration out at temperatures is much of ◦ ◦ lower90 and than 60 forCfor the the process duration carried time out resulting at higher in similartemperature efficiency and offor reducing the same sugars time. (i.e.,Comparing 0.5 h, 90 theC Energies 2018, 11, 639 13 of 24 and 8 h, 60 ◦C), the concentration of selected inhibitors is similar (experiments 1 and 10, Table7). The concentration of total phenolic compounds is additionally affected by the alkaline pre-treatment. The use of alkaline treatment prior to acid hydrolysis (AlAcH) allowed to reduce the concentration of phenolic compounds by 29.2 mg/gbiomass, and furfural by 27.5 mg/gbiomass as compared to the process without alkaline treatment (AcH). However, the concentration of 5-HMF is almost doubled when alkaline pre-treatment is applied, mainly due to the higher content of glucan in the material after the alkaline pre-treatment. The higher the concentration of NaOH, the lower the concentrations of furfural and total phenolic compounds and the higher the concentration of NaOH, the higher the concentration of 5-HMF (compare the experiments i.e., 2 and 1, 4 and 3, etc., Table7). No vanillin, vanillic acid, and guaiacol was identified in the investigated samples of hydrolysates. In order to determine the effect of inhibitors on the bioethanol yield, detoxification of AlAcH and AcH hydrolysates was carried out. For this purpose, sorption on silica gel modified with octadecyl groups was applied. The use of this type of adsorbent allowed the almost complete removal of furfural and phenolic compounds and about a three- to five-fold decrease in 5-HMF concentration. The applied method of detoxification is effective and allows for the removal of phenolic compounds and furan derivatives from the hydrolysates, without the simultaneous loss of reducing sugars. Authors of [42] applied a XAD-4 sorbent polymer. As a result of detoxification with this method, 5-HMF was completely removed and the furfural concentration was reduced by about 6.5 times. In addition, acetic acid was also removed by means of electrodialysis. Interestingly, commonly-used activated carbon does not allow for complete removal of fermentation inhibitors. For example, the use of a two-stage detoxification (combination of hydrolysis treatment with calcium hydroxide and activated carbon) allows to remove 78.3% of the total phenolic and furan compounds from the solution after two-stage hydrolysis with sulfuric acid [43]. The use of electrodialysis allows for the complete removal of acetic acid and more than 50% of total phenolic compounds, however, it has no significant effect on reducing the concentration of furan derivatives [44]. After enzymatic hydrolysis, as in this study, no phenolic compounds, 5-HMF, and furfural were identified in the hydrolysate [45].

Table 7. Determined inhibitors of fermentation.

Experiment Total Phenolic Compounds (mg/g) Furfural (mg/g) 5-HMF (mg/g) 1 44.0 73.6 7.2 2 29.0 42.1 8.9 3 17.7 2.9 3.3 4 16.0 2.1 4.3 5 17.9 24.3 2.6 6 17.4 16.6 4.3 7 6.4 0.0 0.0 8 4.8 0.0 0.0 9 9.4 2.8 0.6 10 32.7 45.0 8.8 11 49.3 53.8 16.9 12 4.8 0.0 0.0 13 19.9 10.4 8.1 AcH 76.1 67.4 16.5 AlAcH 46.9 39.9 18.8 AcHD 1.5 0.0 4.9 AlAcHD 0.0 0.0 3.2

2.6. Inﬂuence of the Pre-Treatment on the Bioethanol Yield The industry uses various species and breeds of yeast belonging to the genus Saccharomyces. They are able to ferment simple sugars, i.e., glucose, fructose, mannose, maltose, and sucrose. During intensive fermentation, yeast cells are distributed throughout the entire volume of ﬂuid. The yeasts of the upper fermentation (Saccharomyces cerevisiae) conduct the process at a higher Energies 2018, 11, x FOR PEER REVIEW 14 of 25

2.6. Influence of the Pre-Treatment on the Bioethanol Yield The industry uses various species and breeds of yeast belonging to the genus Saccharomyces. They are able to ferment simple sugars, i.e., glucose, fructose, mannose, maltose, and sucrose. DuringEnergies 2018 intensive, 11, 639 fermentation, yeast cells are distributed throughout the entire volume of 14 fluid. of 24 The yeasts of the upper fermentation (Saccharomyces cerevisiae) conduct the process at a higher temperaturetemperature than than the the yeast yeast of the lower fermentationfermentation ((SaccharomycesSaccharomyces carlsbergensiscarlsbergensis),), the the process process itself itself is is more intensive with the release of a large amount of CO2. more intensive with the release of a large amount of CO2. InIn this this work, work, small small-scale-scale alcoholic alcoholic fermentation fermentation was was carried carried out. out. Hydroly Hydrolysatessates of of triticale triticale straw, straw, obtainedobtained as as a a result result of of alkaline alkaline pre pre-treatment-treatment and and acid acid or or enzymatic enzymatic hydrolysis, hydrolysis, carried carried out out under under previouslypreviously-optimized-optimized conditions, conditions, were were used used as the as processthe process feed. Acid feed. hydrolysis Acid hydrolysis with 85% phosphoricwith 85% phospacid (hydrolysishoric acid time(hydrolysis of 0.5 h time and temperatureof 0.5 h and oftemperature 100 ◦C) was of performed 100 °C) was for performed untreated straw for untreated (AcH), as strawwell as (AcH) for the, as alkaline well as pre-treated for the alkaline straw (AlAcH, pre-treated 6% strawNaOH, (AlAcH 6 h, 65, ◦ 6%C). The NaOH, enzymatic 6 h, 65 hydrolysis °C). The enzymaticunder optimal hydrolysis conditions under (EH,optimal hydrolysis conditions time (EH, of 16h,hydrolysis Viscozyme time of L 16h, content Viscozyme of 0.9, hydrolysisL content oftemperature 0.9, hydrolysis of 42 temperature◦C) was also of performed 42 °C) was for also the performed untreated material for the untreated and for the material alkaline and pre-treated for the alkalinematerial pre (AlEH).-treated In addition,material (AlEH). acidic hydrolysates In addition, were acidic detoxiﬁed hydrolysates (AcHD were and detoxified AlAcHD, respectively).(AcHD and AlAcHD,The results respectively). were compared The to results the results were of compared fermentation to the carried results out ofon fermen glucose,tation untreated carried straw, out and on glucose,alkaline untreatedpre-treated straw straw., and The alkaline course and pre- resultstreated obtained straw. The for coursethe different and results types of obtained treatment for were the differentevaluated. types Each of time,treatment the resultrefers were evaluated. to the Each amount time, of the sludge result orrefers hydrolysate to the amount obtained of from sludge 3 g or of hydrolysateraw material. obtained The differences from 3 g in of the raw amount material. of the The pre-treatment differences in method the amount on the of sugar the compositionpre-treatment of methodhydrolysates, on the assugar well composition as the composition of hydrolysates, of the broth as afterwell as the the fermentation composition processes of the broth are presentedafter the fermentationin Table8. processes are presented in Table 8. TheThe results results indicate indicate that that the the yeasts yeasts SaccharomycesSaccharomyces cerevisae cerevisae areare unable unable to to digest digest sugar sugar polymers polymers presentpresent in in the the plant plant material material if no if cellulolytic no cellulolytic enzymes enzymes are present are presentin the reaction in the mixture. reaction Figure mixture. 10 presentsFigure 10 the presents courses the of coursesthe fermentation of the fermentation processes. processes. The highest The ethanol highest yiel ethanold was yieldobtained was after obtained 9 h ofafter the 9fermentation h of the fermentation processes. processes.

14 Blank 12 Glucose AcH 10 AlAcH Bioethanol 8 yield, mg/mL AcHD 6 AlAcHD

4 EH AlEH 2

0 0 20 40 60 80 100 Time, h

Figure 10. Courses of performed fermentations. Figure 10. Courses of performed fermentations.

TThehe highest highest ethanol ethanol concentration concentration (8.8 (8.8 mg/ mg/mL),mL), corresponding corresponding to a to yield a yield of 0.29 of 0.29 g/gbiomass g/gbiomass, was , obtainedwas obtained for the for fermentationthe fermentation carried carried out out for for alkaline alkaline pre pre-treated-treated and and enzymatically enzymatically-hydrolyzed-hydrolyzed material.material. The The e ethanolthanol concentration concentration was was 3.7 3.7 mg mg/mL/mL higher higher than than the the concentration concentration obtained obtained for for the the hydrolysatehydrolysate subjectedsubjected to to enzymatic enzymatic hydrolysis hydrolysis only. only. This result This conﬁrms result confirms the desirability the desirability of conducting of conalkalineducting pre-treatment. alkaline pre-treatment.

Energies 2018, 11, 639 15 of 24

Table 8. Sugar content in hydrolysates and composition of fermentation broth.

Feed for Sugar Composition Before After Bioethanol Glycerin Succinic Acid Acetic Acid Methanol Fermentation (mg/mL) Fermentation Fermentation (mg/mL) (mg/mL) (mg/mL) (mg/mL) (mg/mL) Pure glucose Glucose 3 g 0 14.83 2.92 0.43 0.48 0 Glucose 10.1 0 Xylose 0.92 0 Mannose 0.11 0 EH 5.1 1.14 0.43 0.33 0.39 Arabinose 0.08 0.08 Cellobiose 82 0 Galactose 1.4 1.32 Glucose 15.06 0 Xylose 0.99 0 Mannose 0.03 0 AlEH 8.8 1.67 0.71 0.49 0.096 Arabinose 0.13 0.13 Cellobiose 0.77 0 Galactose 1.4 1.4 1.12 Glucose 13.4 13.4 0 Xylose 3.95 3.95 2.78 Mannose 0.45 0.45 0 AcH 6.4 2.35 1.9 1.34 0 Arabinose 0.21 0.21 0.21 Cellobiose 0.53 0.53 0.21 Galactose 0.8 0.8 0.76 Glucose 8.3 0 Xylose 0.89 0.32 Mannose 0.12 0 AlAcH 4.8 1.21 1.2 0.3 0 Arabinose 0 0 Cellobiose 0.35 0.03 Galactose 0.47 0.39 Glucose 13.3 0 Xylose 3.13 2.75 Mannose Arabinose 0.11 0 AcHD 6.7 2.04 1.4 1.18 0.99 Cellobiose 0.08 0.08 Galactose 0.53 0.33 0.7 0.7 Glucose 8.4 0 Xylose 0.95 0.65 Mannose 0.03 0 AlAcHD 4.9 1.1 1.08 0.33 0.15 Arabinose 0.13 0.13 Cellobiose 0.42 0.04 Galactose 0.44 0.44 Energies 2018, 11, 639 16 of 24

Energies 2018, 11, x FOR PEER REVIEW 17 of 25 The beneficial effect of alkaline treatment described above does not occur when the saccharification is carriedThe out bybeneficial means of effect acid of hydrolysis. alkaline treatment Higher ethanol described yield above 0.21 g/g doesbiomass not was occur obtained when forthe the processsaccharification where the untreated is carried materialout by means was hydrolyzedof acid hydrolysis. with acid Highe (AcH)r ethanol compared yield 0 to.21 the g/g fermentationbiomass was carriedobtained out on for the the materialprocess where after two-stagethe untreated treatment material (alkalinewas hydrolyzed pre-treatment with acid and (AcH) acid compared hydrolysis, to the fermentation carried out on the material after two-stage treatment (alkaline pre-treatment AlAcH), where the bioethanol yield was 0.16 g/gbiomass. The given low yield may be due to the large weightand loss acid (48%) hydrolysis, as a result AlAcH), of the where pre-alkaline the bioethanol treatment. yield Obtained was 0.16 results g/gbiomass of. The alcoholic given fermentation low yield provemay that be using due to the the alkaline large weight pre-treatment loss (48%) prioras a result to acid of hydrolysisthe pre-alkaline is pointless. treatment. Obtained results of alcoholic fermentation prove that using the alkaline pre-treatment prior to acid hydrolysis is pointless. In addition, fermentation was carried out on acidic hydrolysates after the removal of fermentation In addition, fermentation was carried out on acidic hydrolysates after the removal of inhibitors. Despite effective detoxification, no significant increase in ethanol yield was obtained. fermentation inhibitors. Despite effective detoxification, no significant increase in ethanol yield was For theobtained. fermentation For the fermentation of AcHD hydrolysate, of AcHD hydrolysate 0.3 mg/mL, 0.3 more mg/mL ethanol more ethano was obtainedl was obtained than for than AcH, whilefor for AcH, AlAcHD, while the for AlAcHD,increase in the ethanol increase was in even ethanol smaller was eve andn wassmaller only and 0.1 was mg/mL only 0.1more mg/ thanmL for the AlAcHmore than fermentation. for the AlAcH fermentation. In otherIn other studies, studies, using usingSaccharomyces Saccharomyces cerevisiae cerevisiae, partial, partial removal removal of inhibitors of inhibitors (vanillic (vani acid,llic furfural,acid, aceticfurfural, acid, feluric acetic acid, acid, feluric syringaldehyde) acid, syringaldehyde) allowedto allowed obtain to a concentration obtain a concentration of ethanol of ethanol higher by 0.27higher g/gbiomass by 0.27[8 ].g/g Thebiomass detoxification [8]. The detoxification by extraction by extraction with awith mixture a mixture of trialkylamine, of trialkylamine, octanol, octanol, and keroseneand kerosenesignificantly significantly improved improved the efficiency the efficiency of obtained of bioethanol obtained bioethanol (21.8 mg/mL), (21.8 mg/in comparisonmL), in withcomparison the ethanol with yield the obtained ethanol from yield the obtained hydrolysate from without the hydrolysate detoxification without (3.79 detoxification mg/mL) [ 46 ]. Despite(3.79 the mg/ poorermL) [46] results. Despite obtained the poorer for acid results compared obtained to for enzymatic acid compared hydrolysis, to enzymatic acid hydrolysis hydrolysis, may acid hydrolysis may be an attractive method of one-stage hydrolysis prior to alcoholic fermentation. be an attractive method of one-stage hydrolysis prior to alcoholic fermentation. It is mainly related to It is mainly related to the low cost of acid compared to the enzyme cost and short preparation time the low cost of acid compared to the enzyme cost and short preparation time of the fermentation feed of the fermentation feed material. Hydrolysates from waste lignocellulosic biomass, obtained by material.using Hydrolysates phosphoric acid from during waste hydrolysis, lignocellulosic mayenrich biomass, the obtained process feed by using for industrial phosphoric processes acid during of hydrolysis,alcoholic mayenrich fermentations the process of starch feed-rich for industrial substrates. processes This approach of alcoholic would fermentations increase the of share starch-rich of substrates.second- Thisgeneration approach biofuels. would In increase addition, the when share of neutralized second-generation with phosphoric biofuels. acid, In addition, e.g., with when neutralizedammonia, with a goodphosphoric medium acid, for e.g., yeast with is ammonia,obtained, which a good promotes medium a for reduction yeast is obtained, in the waste which promotesgeneration a reduction from the in use the of waste the acid generation during the from saccharification. the use of the acid during the saccharification. StudiesStudies on the on alcoholic the alcoholic fermentation fermentation of acid of acid hydrolysates hydrolysates [1] [1] were were also also carried carried out out using using dilute sulfuricdilute acid. sulfuric Obtained acid. hydrolysatesObtained hydrolysates were rich were mainly rich in mainly hemicellulose in hemicellulose hydrolysis hydrolysis products products (arabinose (arabinose and glucose), and the fermentation of these compounds allowed obtaining ethanol and glucose), and the fermentation of these compounds allowed obtaining ethanol (0.23g/gbiomass). (0.23g/gThe resultsbiomass of). fermentation on hydrolysates (Figure 11) indicate that glucose is completely digested The results of fermentation on hydrolysates (Figure 11) indicate that glucose is completely within the first 5 h of the process. During the fermentation of AlEH and EH hydrolysates, cellobiose digested within the first 5 h of the process. During the fermentation of AlEH and EH hydrolysates, is also digested, however, a significant loss of concentration of this disaccharide is observed only cellobiose is also digested, however, a significant loss of concentration of this disaccharide is after complete consumption of glucose. Additionally, xylose undergoes digestion only after the observed only after complete consumption of glucose. Additionally, xylose undergoes digestion completeonly after digestion the complete of glucose digestion by yeast. of glucose Total by xylose yeast. consumptionTotal xylose consumption was observed was afterobserved 24 hoursafter of fermentation.24 hours of The fermentation. order of sugar The digestion order of sugardescribed digestion above described is confirmed above by is other confirmed papers by indicating other thatpapers digestion indicating of xylose that by digestionSaccharomyces of xylose cerevisiae by Saccharomycesis possible cerevisiae only is after possible complete only after consumption complete of glucoseconsumption [3]. Interestingly, of glucose the yeast[3]. Interestingly,Saccharomyces the cerevisiae yeast D5ASaccharomyces, during the cerevisiae fermentation D5A, during of hydrolyzed the trunksfermentation of palm trees, of hydrolyzed was not able trunks to digestof palm xylose trees, was [6]. not able to digest xylose [6].

35 12 30 9 25 20 mg/mL mg/mL 6 15 10 3 5 0 0 0 50 100 0 50 100 Time, h Time, h

(a) (b)

Figure 11. Cont. Energies 2018, 11, 639 17 of 24 Energies 2018, 11, x FOR PEER REVIEW 18 of 25

16 16

12 12

mg/mL 8 mg/mL 8

4 4

0 0 0 20 40 60 80 100 0 20 40 60 80 100 Time, h Time, h

16 16

12 12

mg/mL 8 mg/mL 8

4 4

0 0 0 20 40 60 80 100 0 20 40 60 80 100 Time, h Time, h

(e) (f)

mg/mL 8

0 0 20 40 60 80 100 Time, h

(g)

Figure 11. Courses of bioethanol fermentation presenting bioethanol yield (blue) and consumption Figure 11. Courses of bioethanol fermentation presenting bioethanol yield (blue) and consumption of glucose (red) and xylose (green) in function of time from following substrates: (a) pure glucose; of glucose (red) and xylose (green) in function of time from following substrates: (a) pure glucose; (b) AcH; (c) AlAcH; (d) EH; (e) AlEH; (f) AcHD; and (g) AlAcHD. (b) AcH; (c) AlAcH; (d) EH; (e) AlEH; (f) AcHD; and (g) AlAcHD. The research results show that cellobiose and xylose were not completely digested by yeast duringThe research fermentation results onshow hydrolysates that cellobiose, both and after xylose one-step were acid not completely hydrolysis (AcH)digested and by after yeast acid during fermentationhydrolysis onpreceded hydrolysates, by alkaline both pre after-treatment one-step (AlAcH). acid hydrolysisOnly 30% and (AcH) 64% andof xylose, after acidrespectively, hydrolysis precededwere by converted alkaline into pre-treatment fermentation (AlAcH). products. Only This 30% result and 64% can of be xylose, explained respectively, by the werepresence converted of fermentation inhibitors in AcH and AlAcH hydrolysates, however, even after removal of inhibitors into fermentation products. This result can be explained by the presence of fermentation inhibitors (AcHD and AlAcHD), the fermentation of xylose does not proceed completely. It turns out that the in AcH and AlAcH hydrolysates, however, even after removal of inhibitors (AcHD and AlAcHD), presence of inhibitors affects the utilization of xylose in the alcoholic fermentation from Pichia the fermentation of xylose does not proceed completely. It turns out that the presence of inhibitors stipitis CBS 5776. The results presented in [46] indicate that the xylose from the detoxified affectshydrolys the utilizationate was digested of xylose completely in the alcoholic after 60 fermentation h, and during from the Pichia fermentation stipitis on CBS the 5776. hydrolysateThe results presentedwithout in detoxification [46] indicate only that 15% the xyloseof xylose from was the digested. detoxiﬁed The results hydrolysate of other was studies digested indicate completely that afterthe 60 digestibility h, and during of xylose the fermentation depends on onits initial the hydrolysate concentration without in the detoxiﬁcationhydrolysate and only on the 15% ratio of xyloseof wasglucose digested. to xylose. The results The highest of other conversion studies degree indicate of xylose that (83.2%) the digestibility to ethanol and of xylosexylitol was depends achieved on its initial concentration in the hydrolysate and on the ratio of glucose to xylose. The highest conversion degree of xylose (83.2%) to ethanol and xylitol was achieved when the xylose concentration was about Energies 2018, 11, 639 18 of 24

5.5 times higher than the glucose concentration. A much worse result (20%) was achieved when the fermentation broth contained three times more glucose [1]. Arabinose and galactose, regardless of the method and conditions of biomass pre-treatment and hydrolysis, are not consumed during the fermentation. The inability of S. cerevisiae to metabolize arabinose has been previously demonstrated in [1]. In addition, besides bioethanol, other typical byproducts were identiﬁed including glycerin, acetic acid, succinic acid, and methanol (Table8). Table9 presents a summary of the research results from this work compared to other published results regarding similar biomass types.

Table 9. Summary of investigations compared to other published results.

Type and Method Hydrolysis Biomass Ethanol Ethanol Microorganisms Ref. of Pre-Treatment Conditions Concentration % (w/v) Yield g/L Efﬁciency Hydrothermal180 ◦C, Saccharomyces cerevisiae 30 FPU/g, 45 ◦C 3% wheat straw 14.84 82.4% [2] 30 min 0.5% NaOH 121 ◦C, Kluyveromyces sp. 9 FPU/g substrate 10% wheat straw 18.29 79.64% [47] 30 min Steam explosion Cellulase 5 FPU/g Kluyveromycesmarxianus 100 ◦C, 2 min and 10% TS (w/v) wheat IB-glucosidase 12.7 67.1% [48] CECT 10875 laccase10 IU/g straw 5 IU/g 50 ◦C, 72 h 50 ◦C, 24 h Saccharomycescerevisiae 0.5% H SO 185 ◦C, 12 FPU/g, 50 ◦C, 2 4 10% SS wheat straw 30.3 83% [7] (T0936) 8 min 24 h 15 FPU,37 ◦C, 60 h Saccharomyces cerevisiae Steam explosion Spezyme® CP/g 15% (w/v) triticale 29.6 81.3% [49] Ethanol Red® as 200 ◦C, 7 min cellulose Without 85% H PO , 0.5 h, AcH 6.4 AcH 88.5% Saccharomyces cerevisae 3 4 3% This study pre-treatment 100 ◦C AcHD 6.7 AcHD 93.9% 85% H PO , 0.5 h, AlAcH 4.8 AlAcH 44.3% Saccharomyces cerevisae 6% NaOH, 6 h, 65 ◦C 3 4 3% This study 100 ◦C AlAcHD 4.9 AlAcHD 45.0% Immobilized Without Saccharomyces cerevisae enzymes 24 h, 3% 5.1 70.4% This study pre-treatment 42 ◦C Immobilized Saccharomyces cerevisae 6% NaOH, 6 h, 65 ◦C enzymes 24 h, 3% 8.8 80.5% This study 42 ◦C

Alcoholic fermentation of sugars in the presence of yeast belongs to the rapidly-growing industry and is used in brewing, viticulture, distilling, candling of fruit, yeast production, bread, as well as in environmental protection. This process involves the oxidation of carbohydrates by appropriate yeast strains, resulting in the formation of ethyl alcohol, used for food and industrial purposes. The raw materials used in the fermentation industry are divided into three groups. The first includes sugar products: molasses, fruit juices, sugar cane, the second: starch materials (rye, barley, and wheat), and the third: cellulose products (wood and sulphite waste liquids). Yeast contains enzymes that hydrolyze disaccharides to simple sugars. Only four hexoses are fermented under the influence of yeasts: D-glucose, D-mannose, D-fructose, and D-galactose. The last one is hardly fermented and not under the influence of all yeast species. Alcohol as the final fermentation product is isolated by distillation. The yield calculated on the ratio of fermenting sugars is about 90%.

3. Materials and Methods

3.1. Chemical Reagents and Enzymes • Chemical reagents (sodium hydroxide, phosphoric acid 85%, sulfuric acid 99.5%, hydrochloric acid, calcium oxide, glucose, and (NH4)2HPO4 are purity grade purchased from Chempur, (Piekary Sl´ ˛askie,Poland); • Chemical standards (cellobiose, glucose, mannose, arabinose, xylose, galactose, furfural, HMF, acetic acid, vaniline, succinic acid, lactic acid, and glycerin) are of analysis grade purity purchased from Sigma Aldrich, (Pozna´n,Poland); Energies 2018, 11, 639 19 of 24

• Demineralized water for HPLC analysis was filtered through a 0.45 µm filter; • Viscozyme L from the Aspergillus sp. Novozyme Corp. (Sigma Aldrich, Poznań,Poland) enzyme complex containsa great variety of carbohydrosides: arabinase, cellulase, β-glucanase, ◦ hemicellulase, and xylanase Topt 35–40 C, pH 5–6.5; • Glucosidase ≥ 750 U/g from Aspergillus sp. was acquired from Novozyme Corp. (Sigma Aldrich, ◦ Poznań,Poland), Topt 35–45 C, pH 4–6.5; • Cellulase ≥ 800 U/g from Aspergillusniger, was acquired from Novozyme Corp. (Sigma Aldrich, ◦ Poznań,Poland) Topt 30–40 C, pH 4–5.8; • Cellulase ≥ 700 U/g from Trichoderma Reesei ATCC 26921 was acquired from Novozyme Corp. (Sigma Aldrich, Poznań,Poland); • Saccharomyces cerevisae (Biomaxima, Gdańsk,Poland); • Saccharomyces carlsbergensis SA23 (ATCC, Manassas, VA, USA); • Diatomite (Chempur, Piekary Sl´ ˛askie,Poland);

• Phosphate buffer 0.1 M pH 5.7–8.0 (13.9 g NaH2PO4 + 35.85 g Na2HPO4 × 12H2O, 500 mL water); • McIlvainae buffer, pH 4.5 (mix 44.1 mL with 0.02 M Na2HPO4 with 55.9 mL 0.01 M citric acid and ﬁlled to 1000 mL with distilled water);

3.2. Biomass Preparation The air-dried lignocellulosic biomass was milled using a RETSCH Ultra Centrifugal Mill ZM 200 mill (Katowice, Poland). During the milling, a 0.75 mm diameter screen was used. The material after grinding was stored in sealed containers at room temperature. Before the alkalinepre-treatment, the material was dried in a laboratory dryer at 105 ◦C for 4 h and then stored in a desiccator with the drying substance (NaOH).

3.3. Alkaline Pre-Treatment The alkaline pre-treatment of lignocellulosic biomass was carried out using sodium hydroxide. The reactions were carried out in 250 mL sealed vessels, into which 4 g of milled and minced lignocellulosic biomass and 80 mL of catalyst solution were introduced. The dishes were placed in a shaking water bath. The pre-treatment was carried out at a constant temperature of 65 ◦C for 6 h for catalyst concentrations (NaOH) of 1%, 6%, and 11%. The reaction mixture was then neutralized with a hydrochloric acid solution, poured into centrifuge tubes, and centrifuged and ﬁltered through a Buchner funnel to separate the precipitate from the solution. The precipitate was then washed three times with water and then twice with acetone. The precipitate was dried for 24 h at 105 ◦C and placed in a desiccator with a drying substance.

3.4. Acid Hydrolysis The acid hydrolysis processes performed before and after the alkaline pre-treatment were carried out using 85% phosphoric acid. The reactions were carried out in 100 mL sealed vessels, into which 0.5 g of lignocellulosic biomass and 10 mL of catalyst solution were introduced. The vessels were placed in a thermostated shaker. The hydrolysis was carried out at the time and temperature given in Table4. For optimization purposes, the reaction mixture was diluted to a volume of 80 mL and neutralized with calcium carbonate. For fermentation, the mixture was diluted three times, then partially neutralized with phosphate acid with calcium carbonate. The calcium phosphate formed during the neutralization was ﬁltered off and the hydrolysate was subjected to fermentation.

3.5. Enzymatic Hydrolysis Milled and minced lignocellulosic biomass (0.2 g) after alkalinepre-treatment was added to the flasks and supplemented with a suspension of cellulolytic enzymes immobilized on diatomite to 10 mL. The reaction flasks were incubated in a thermostated shaker at 42 ◦C for 24 h. Subsequently, samples Energies 2018, 11, 639 20 of 24 were taken, the enzyme-containing bed was separated by centrifugation and filtration, and the contents of monosaccharides and glucose content were analyzed. Control experiments were carried without the addition of lignocellulosic material. To avoid non-linear changes of the process, buffers, such as McIlvainae or phosphate buffer, must be used. Immobilized enzyme preparation requires placing 20 mL of enzymatic solution (Viscozyme L, Glucosidase 0.9:0.1 m/m to 1:0) in a 50 mL beaker with 2.5 g dry diatomite and stirring the solution for 1 h at low speed (magnetic stirrer) at room temperature. The diatomite with the immobilized enzyme must be washed in a column with a small amount (about 10 mL) of McIlvaine buffer. After a few minutes the bed stabilizes. Next, the diluted diatomite must be stored under a layer of buffer (about 5 mm). To use the enzyme hydrolysis preparations, the bed should be shaken and then introduced to the solid residue of the lignocellulosic biomass solution after alkaline pre-treatment.

3.6. Detoxiﬁcation of Hydrolysates A SPE-C18 Polar Plus column (WITKO, Łód´z,Poland) with a bed length of 10 cm was used for detoxiﬁcation, attached to a receiver chamber connected to a vacuum pump. A hydrolysate solution was introduced into the column and passed through the bed under vacuum. The collected eluate was subjected to HPLC analysis in terms of the composition and content of sugars and inhibitors.

3.7. Ethanol Fermentation

A solution containing 0.04 g of (NH4)2HPO4, 0.5 g of glucose, and 7 g of mixture of Saccharomyces cerevisae and Saccharomyces carlbergensis SA23 in 200 mL of water was prepared. The mixture of given Saccharomyces strains is able to consume xylose [50]. The solution was incubated at 37 ◦C for 2 h. Twenty milliliters of grout was added to fermentation broths prepared as described in Table4 and ﬁlled to 100 mL with water. Fermentation was carried out at 37 ◦C for one week. Flasks were plugged with stoppers provided with fermentation tubes ﬁlled with water.

3.8. Methods • Total solids, ash, and extractives of raw triticale straw were determined according to the National Renewable Energy Laboratory (NREL) analytical procedures [51–54]. • The composition and analysis of sugar and lignin content in the native material and after alkaline hydrolysis were determined using the NREL procedure [52]. The content of cellulose and hemicellulose was determined by HPLC with a Rezex Pb2+ column (300 × 7.8 mm, 8 µm) (Phenomenex, Torrance, CA, USA) and RID detector (Knauer, Berlin, Germany). Water with a ﬂow rate of 0.6 mL/min was used as the eluent. • The presence and concentration of fermentation inhibitors (furfural, 5-HMF, levulinic acid) were determined by HPLC with a Shodex SH1011 column (300 × 7.8 mm, 8 µm) (Showa Denko, Toyama, Japan) and UV–VIS DAD (Merck-Hitachi, Düsseldorf, Germany) and RID detectors (Knauer, Berlin, Germany). The mobile phase was 5 mM H2SO4 solution.The composition of fermentation broths was determined by HPLC with the Shodex SH1011 column and UV–VIS DAD and RID detectors. The mobile phase was 5 mM H2SO4 solution. • The concentration of total phenolic compounds was determined by UV–VIS (HP, San Diego, CA, USA). The calibration curve was made for vanillin. The absorbance measurement was made at 280 nm. To obtain the sum of the phenolic compounds, the concentrations of furfural and 5-HMF (determined by HPLC) were subtracted from the concentrations determined by the UV–VIS method.

3.9. Design of Experiments and Data Analysis The experiments described in this paper were realized according to the general scheme presented in Figure 12. Symbols of variables are given in accordance to those used in Tables4 and7. Experiments regarding Energies 2018, 11, 639 21 of 24 the conditions of alkaline pre-treatment, as well as acid or enzymatic hydrolysis, were planned and realized according to Box-Behnken design for three factors. Table 10 presents the input variables for a givenEnergies coding 2018 level., 11, x FOR PEER REVIEW 22 of 25

Acid Optimization Hydrolysis Fermentation & Milling Biomass Alkaline Modelling Comparison & pre-treatment & mincing Conclusions Enzymatic Optimization Hydrolysis & Fermentation Modelling

Figure 12. Steps of lignocelulosic biomass processing towards bioethanol. Figure 12. Steps of lignocelulosic biomass processing towards bioethanol.

Table 10. Input variables for the Box-Behnken design. Table 10. Input variables for the Box-Behnken design. Coding Level Variable Symbol −1 Coding0 Level1 NaOHVariable concentration, % SymbolX1 1 6 11 Temperature of acid hydrolysis, °C X2 60 −190 0120 1 Time of acid hydrolysis, h X3 0.50 4.25 8.00 NaOH concentration, % X1 1 6 11 Viscozyme L content, % ◦ X4 0.90 0.95 1.00 Temperature of acid hydrolysis, CX2 60 90 120 Time of enzymatic hydrolysis, h X5 8 16 24 Time of acid hydrolysis, h X3 0.50 4.25 8.00 Viscozyme L content, % X4 0.90 0.95 1.00 Data analysisTime ofand enzymatic related hydrolysis,calculations h were performed X5 using8 R 16Studio Desktop 24 (version 1.0.143). Presentation of the results in the form of response surface as a function of investigated parameters was prepared using Microsoft Excel 2007. Data analysis and related calculations were performed using R Studio Desktop (version 1.0.143). Presentation3.10. Calculations of the results in the form of response surface as a function of investigated parameters was prepared using Microsoft Excel 2007. Biomass recovery was calculated from Equation (3): 3.10. CalculationsBR (%) = (Mass of pre-treated biomass (g)/Mass of feed biomass (g)) × 100% (3) BiomassGlucan recovery and xylan was recovery calculated was cal fromculated Equation as follows (3): :

Glucan/xylan recovery = BR (%) × (Cglukan/xylan pretreated biomass)/Cglucan/xylanorginal biomass) (4) BR (%) = (Mass of pre-treated biomass (g)/Mass of feed biomass (g)) × 100% (3) The delignification degree (i.e., lignin removal) was calculated from Equation (5):

Glucan and xylan recoveryLR (%) = was100% calculated − (BR (%) × as (C follows:lignin pretreated)/C lignin orginal biomass) (5) where C is lignin content (wt %). Glucan/xylan recovery = BR (%) × (C )/C ) (4) The theoretical ethanol yield was calculatedglukan/xylan according pretreated to biomass the NRELglucan/xylanorginal standard procedure biomass [55] from the following formula: The deligniﬁcation degree (i.e., lignin removal) was calculated from Equation (5): Theoretical EtOH yield % = (EtOH produced/(0.51 × f × Biomass × 1.111)) × 100% (6) where EtOH producedLR (%) = is 100% the ethanol− (BR concentration (%) × (Clignin at pretreated the end)/C of ligninthe fermentation orginal biomass (g/)L). The term (5) 0.51 × f × Biomass × 1.111 corresponds to the theoretical ethanol concentration, where Biomass is the wheredry C biomass is lignin weight content concentrati (wt %).on at the beginning of the fermentation (g/L); f is the cellulose fraction of dryThe biomass theoretical (g/g ethanol); 0.51 is yield the conversion was calculated factor according for glucoseto to the ethanol NREL based standard on the procedure stoichiometric [55] from the followingbiochemistry formula: of yeast, and 1.111 is the conversion factor for cellulose to equivalent glucose.

Acknowledgments:Theoretical EtOH This yieldwork was % = carried (EtOH out produced/(0.51 within the framework× f × ofBiomass the project× 1.111))“Studies× of100% alkaline (6) hydrolysis of lignocellulosic biomass and conversion conditions of hydrolyzed products to biogas”, supported wherefinancially EtOH producedby the National is the Science ethanol Center concentration through grant UMO at the-2014/13/B/ST8/04258. end of the fermentation We have received (g/L). funds The term 0.51 ×forf covering× Biomass the costs× 1.111to publish corresponds in open access. to the theoretical ethanol concentration, where Biomass is the dry biomassAuthor Contributions: weight concentration Rafał Łukajtis at and the Karolina beginning Kucharska of the conceived fermentation and designed (g/L); ftheis experiments; the cellulose Rafał fraction of dryŁukajtis, biomass Karolina (g/g ); Kucharska, 0.51 is the Edyta conversion Słupek, Katarzynafactor for Wychodnik glucose to, and ethanol Paulina based Nowak on theperformed stoichiometric the biochemistryexperiments; of yeast,Karolina and Kucharska 1.111 is and the Katarzynaconversion Wychodnik factor for performed cellulose the to equivalentethanol ferm glucose.entation, Piotr Energies 2018, 11, 639 22 of 24

Acknowledgments: This work was carried out within the framework of the project “Studies of alkaline hydrolysis of lignocellulosic biomass and conversion conditions of hydrolyzed products to biogas”, supported financially by the National Science Center through grant UMO-2014/13/B/ST8/04258. We have received funds for covering the costs to publish in open access. Author Contributions: Rafał Łukajtis and Karolina Kucharska conceived and designed the experiments; Rafał Łukajtis, Karolina Kucharska, Edyta Słupek, Katarzyna Wychodnik, and Paulina Nowak performed the experiments; Karolina Kucharska and Katarzyna Wychodnik performed the ethanol fermentation, Piotr Rybarczyk analyzed the data; Marian Kamińskiprovided reagents/materials/analysis tools; and Iwona Hołowacz, Rafał Łukajtis, Karolina Kucharska, and Piotr Rybarczyk wrote the paper. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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