ethanol production from brewers' spent grain pretreated by dilute

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Biofuels and Biomass

ETHANOL PRODUCTION FROM BREWERS’ SPENT GRAIN PRETREATED BY DILUTE PHOSPHORIC ACID José A. Rojas-Chamorro, Cristobal Cara, Inmaculada Romero, Encarnación Ruiz, Juan M. Romero-García, Solange I. Mussatto, and Eulogio Castro Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.8b00343 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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ETHANOL PRODUCTION FROM BREWERS’ SPENT GRAIN PRETREATED BY DILUTE PHOSPHORIC ACID José A. Rojas-Chamorro1, Cristóbal Cara1,2, Inmaculada Romero1,2*, Encarnación Ruiz1,2, Juan M. Romero-García1, Solange I. Mussatto3, Eulogio Castro1,2 1

Dept. of Chemical, Environmental and Materials Engineering, Universidad de Jaén, 2

Center for Advanced Studies in Energy and Environment, Universidad de Jaén, Campus Las Lagunillas, 23071 Jaén, Spain

3

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220, 2800, Kongens Lyngby, Denmark

ABSTRACT This paper deals with the characterization of brewer’s spent grain (BSG) and the optimization of the phosphoric acid pretreatment for this feedstock. The influence of temperature and acid concentration on BSG was studied and the optimal conditions were found to be 155 ºC and 2% H3PO4. The use of both pretreatment and enzymatic hydrolysis together recovered 92% of total sugars in BSG, mainly solubilized in the prehydrolysate (63%). Escherichia coli SL100 fermented this mixed sugar solution containing hemicellulosic sugars and starchy glucose without previous detoxification with an ethanol yield of 0.40 g/g. Considering also the glucose released from the cellulosic structure and converted to ethanol by a simultaneous saccharification and fermentation process, an overall ethanol yield of 17.9 g of ethanol per 100 g of raw BSG was achieved. Thereby, the process configuration proposed in this work allowed 69% of the total sugars in the BSG to be converted to ethanol. 1. INTRODUCTION Brewers’ spent grain (BSG) constitutes a by-product of the beer-making process that isgenerated in large amounts and lacks economically feasible applications. The production of beer from barley was 37.4 million tonnes in the European Union and

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more than 180 million tons in the world in 2014.1 According to Mussatto2, BSG is the main residue generated in the brewery process (85% of solid by-products) and it is estimated that 20 kg of wet BSG is produced per 100 L of beer produced.3,4 Nowadays, BSG is disposed of directly in landfills or used as animal feed. However, the high moisture content of this material (about 80%) makes its transportation expensive and limits its exploitation because microbial growth occurs easily.3,5,6 Different applications of BSG have been reported in the literature. The potential of this feedstock as a source of arabinoxylans with prebiotic activity,7-9 in food applications with associated health benefits,3 or for biogas10 and biobutanol production11 has been recently reported. Likewise, the combustion of BSG to produce thermal heat and electric power has been proposed with the aim of reducing the demand for energy in breweries, although there are problems associated with its high moisture12 and nitrogen content.13 Researches concerning the bioconversion of the carbohydrates in BSG have been traditionally directed to xylitol production due to its noticeable xylose content.14-16 The utilization of lignocellulosic materials as feedstocks in biorefineries requires a pretreatment that alters their crystalline structure and allows their fractionation. Pretreatment is an essential stage in the bioconversion of lignocellulosic biomass to fuels and chemicals.17 Different works have been recently reported with the aim of producing ethanol from BSG. Physicochemical pretreatments such as steam explosion,18 microwave,19 liquid hot water,20 acid hydrolysis,21-23 and alkaline treatments24 have been used for the BSG bioconversion. However, while sulfuric acid is the most widely used acid, the utilization of phosphoric acid in the pretreatment as an alternative to sulfuric acid shows advantages such as a lower formation of toxic compounds and lower corrosiveness.25 2 ACS Paragon Plus Environment

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The main objective of this work was to solubilize the hemicellulosic fraction of BSG by dilute phosphoric acid pretreatment and to achieve high enzymatic digestibility of the cellulose fraction. Ethanol production from the cellulosic and hemicellulosic sugars in BSG was evaluated. For this purpose, the liquid fraction from the phosphoric acid pretreatment under optimized conditions was fermented, without previous detoxification, with Escherichia coli SL100, an ethanologenic microorganism capable of assimilating both hexoses and pentoses. Likewise, the resulting cellulose-rich solid was enzymatically hydrolysed, yielding a glucose solution to be converted into bioethanol. 2. MATERIALS AND METHODS 2.1 Raw material BSG with 22% total solids was kindly supplied by a local brewery, Cruzcampo-Jaén (Heineken España, S.A., Spain). The BSG was washed in water until a neutral pH was achieved and then oven dried at 50 ºC until it reached a total solids content of around 90%. This feedstock was stored at 4 ºC until use. 2.2 Phosphoric acid pretreatment In this work, BSG was pretreated by dilute phosphoric acid according to a rotatable central composite design (α = 1.414) to evaluate the effect of pretreatment temperature (140–180 ºC) and phosphoric acid concentration (2–6% w/v). Once the experimental conditions had been reached according to the experimental design, the reactor was cooled down. The coded and uncoded values of both factors in the design are shown in Table 1. The experimental design included one point and four replicates at the centre of the domain selected for each factor with a total of 13 experiments to determine the best conditions for phosphoric acid pretreatment of BSG. 3 ACS Paragon Plus Environment

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Pretreatment of BSG was carried out in a 1-L reactor (Parr Instr. Co., IL, USA). The liquid/solid ratio in the reactor was maintained at 8:1 (w/w). After pretreatment, the liquid and solid fractions were separated by vacuum filtration. The liquid fraction (prehydrolysate) was characterized with regard to its composition of sugars and inhibitory compounds such as acetic acid, furfural, hydroxymethylfurfural (HMF), formic acid, and total phenols. The solid fractions were washed with distilled water to eliminate acid solution and dried at 35 ºC. Then, the solid recovery was determined in each case and these pretreated solids were characterized. 2.3 Enzymatic hydrolysis Commercial Cellic® CTec3 (Novozymes A/S, Denmark) was used for enzymatic hydrolysis. The enzyme loading was 15 Filter Paper Units (FPU)/g substrate of Cellic® CTec3 supplemented by fungal β-glucosidase (Novozyme 50010, Novozymes A/S, Denmark) with 15 International Units (IU)/g substrate. Enzymatic saccharification tests of acid-pretreated BSG were performed at 5% solids with 0.05-M sodium citrate buffer (pH 4.8) in 100-mL Erlenmeyer flasks. The flasks were incubated at 50 ºC in an orbital shaker (Certomat-R, B-Braun, Germany) at 150 rpm for 48 h. Samples were taken every 24 h for glucose concentration measurements. The concentrations of initial glucose in the commercial enzyme mixtures were also quantified by HPLC and subtracted from the final glucose concentrations by enzymatic hydrolysis. All experiments were carried out in triplicate, the average results and standard deviations are shown. For the purpose of comparison, raw BSG was also saccharified by enzymes under the same conditions. 2.4 Assessment of results Commercial software (Design Expert 7.0.0, Stat-Ease Inc., Minneapolis USA) was used to analyse the results and optimize the pretreatment conditions. The response surface 4 ACS Paragon Plus Environment

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methodology was applied for assessment of the results. Hemicellulosic sugar recovery (HSR) in the liquid fractions, expressed as grams of hemicellulosic sugars in the prehydrolysate per 100 g of hemicellulosic sugars in raw BSG, and the enzymatic hydrolysis yield (YEH), expressed as grams of glucose obtained by enzymatic hydrolysis per 100 g of cellulosic-glucose in raw BSG, were chosen as the responses. The influence of both temperature and phosphoric acid concentration on the responses was evaluated. The optimal conditions for the acid pretreatment considering the maximization of both responses simultaneously were estimated. 2.5. Microorganism E. coli SL100 (kindly provided by Dr. Ingram from the University of Florida, USA) was maintained in 40% glycerol tubes at –80 ºC and transferred before inoculation to an AM1 culture medium26 with a sugar concentration similar to that of prehydrolysate, which was previously sterilized by filtration through 0.2 µm membranes. Finally, the inoculum was grown at 37 ºC in an orbital incubator at 180 rpm for 24 h, centrifuged (3500 rpm, 10 min), washed, and added to the fermenter. 2.6 Fermentation of acid prehydrolysate Fermentation tests were carried out with 150 mL of fermentation liquor in 300 mL glass flasks equipped with a pH probe and magnetic agitation. The temperature control was assured by using a water bath, and pH was monitored and automatically corrected by the addition of 2 M KOH (for more details, see Romero-García et al.27). The experimental conditions were 37 °C, 400 rpm, pH 6.5, and an initial cell concentration of about 0.5 g/L. The prehydrolysates were supplemented with salts of AM1 culture medium. At different times during 92 h, samples were taken and centrifuged (11,500 rpm, 10 min) to determine cell growth, sugar consumption, and ethanol production. 5 ACS Paragon Plus Environment

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Duplicate experiments were performed, and average values and standard deviations are shown. 2.7 Analytical methods Untreated and acid-pretreated BSG were characterized using standard laboratory analytical procedures for biomass analysis provided by the National Renewable Energy Laboratory.28 The total starch content was measured in raw BSG using the Total Starch Assay Kit (Megazyme, Ireland) method. The nitrogen content in BSG was determined by elemental analysis (EA1112 Thermo Finnigan Elemental Analyser) and converted into protein using the N × 6.25 conversion factor. Liquids (prehydrolysates obtained from pretreatment and enzymatic hydrolysates) were centrifuged and filtered through 0.45-µm membranes (Gelman Sciences, Inc., Michigan, USA) and analysed by HPLC for quantitative carbohydrate analysis. The HPLC system (Waters, Milford, USA) was equipped with a refractive index detector (model 2414). A CARBOSep CHO-782 Pb (Transgenomic, Inc., Omaha, USA) carbohydrate analysis column operating at 70 °C with ultrapure water as a mobile phase (0.6 mL/min) was used for the monomeric sugars (arabinose, galactose, glucose, mannose, and xylose) determinations. Oligomeric sugars were determined as the difference between total free sugars in the liquor before and after post-hydrolysis (120 °C, 3% w/v H2SO4, 20 min). The content of phenolic compounds was determined using Folin-Ciocalteu’s reagent method.29 A 0.2 mL aliquot of each prehydrolysate was mixed with 1.5 mL of water, 0.1 mL of Folin-Ciocalteu’s reagent (1:1 v/v), and 0.2 mL of Na2CO3 (10 %w/v). After one hour, the absorbance of the mixtures was measured at 765 nm and a calibration curve for gallic acid was constructed. The concentration of the total phenolic


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compounds in the prehydrolysates was expressed as grams of gallic acid equivalent (GAE). 3. RESULTS AND DISCUSSION 3.1 Feedstock composition BSG is mainly constituted by carbohydrates, protein, and lignin, although its composition can vary depending of the barley variety, the harvesting time, and different brewery process parameters.3,30 Table 2 shows the composition of BSG determined in this work. According to the NREL analytical methods, a two-stage extraction (water + ethanol) is necessary to remove the extractives from biomass prior to determining its carbohydrate content. Thus, 18.5% of dried BSG was determined to be extractives, of which 25% was water soluble materials and 75% ethanol soluble materials. Besides, 8.6% lipids were determined in raw BSG after 6 h of extraction with hexane. A similar concentration of lipids in BSG has been previously reported.8,18 As shown in Table 2, structural sugars represent as much as 51% of the dry weight of BSG, mainly glucose and xylose. Glucose in BSG is mainly in the form of cellulose, although a minor fraction, about 26%, is like starchy glucose, accounting for 5.25% of the BSG composition. The starch content in BSG can vary depending on the brewery technology utilized.5 Other lignocellulosic materials such as banana pseudostem31 or wheat bran32 have also been reported to have comparable starch contents. Additionally, it is worth noting the high arabinose content determined in BSG, representing about 25% of the total hemicellulosic sugars. This is in agreement with the content previously reported in other researches on this raw material.33,34 The arabinose-to-xylose ratio determined in this work (0.4) is typical for this lignocellulosic material.5 The BSG


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carbohydrate content indicates that this by-product has excellent potential for bioethanol production if both pentoses and hexoses in BGS can be utilized. Regarding the lignin content, BSG can be considered as a material with a relatively low lignin content (12.5%) compared to other lignocellulosic materials such as barley straw,35 corn stover,36 or sweet sorghum.37 This is advantageous because lignin acts as a barrier that hinders the enzymatic access to cellulose chains.38 BSG is also rich in proteins, with a content of 21.2% (based on total nitrogen, N × 6.25). According to the NREL procedures, 2% of protein content was quantified in the gravimetric determination of acid insoluble material to take account of the interferences of the protein in the analytical methods. This means that 9% of the protein in raw BSG was acid insoluble. However, the content of protein in BSG was also determined after two-stage extraction (water + ethanol) and no solubilization was observed. This result agrees with the findings of Xiros and Christakopoulos5, who reported that the protein in BSG was highly insoluble. In addition to protein, acid insoluble material in BSG (10.13%) includes mainly lignin as well as ash (Table 2). These results are in agreement with those of Kemppainem et al.18, who determined an acid insoluble material content of 14.7 % in BSG, which also included a minor fraction of protein. 3.2 Influence of H3PO4 pretreatment on BSG The effectiveness of the phosphoric acid pretreatment on BSG was evaluated by the hemicellulosic sugar solubilization and the enzymatic digestibility of the pretreated solids. The experimental results of the sugar composition of the liquid fractions from the acid pretreatment of BSG under different conditions are shown in Table 3. A main objective of the biomass pretreatment is the hydrolysis of its hemicellulose fraction, which has been evaluated by determining the HSR in the prehydrolysate. In general, 8 ACS Paragon Plus Environment

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high HSR values of above 70% were determined, reaching a maximum of 89.4% (run 13, 140 ºC, 6% H3PO4). However, when the temperature was higher than 180 ºC (runs 2, 3, and 5), HSR decreased noticeably due to sugar degradation. As shown, sugar solutions with concentrations ranging between 9 and 43.4 g/L resulted from the pretreatment. Besides hemicellulosic sugars, mainly xylose and arabinose, noticeable glucose concentrations were also measured in the liquid fractions. As can be observed, regardless of the pretreatment conditions, glucose was partially solubilized and recovered in the prehydrolysate. This can be attributed to the fact that BSG contains non-cellulosic glucose that is easy to hydrolyse. Thus, glucose recovery in the prehydrolysates ranged between 15 and 36% and is likely to have mainly originated from the starch fraction, which accounted for 26% of the total glucose determined in raw BSG. In most of the prehydrolysates, all sugars were found in monomeric form. Thus, the presence of gluco-oligomers and xylo-oligomers in the prehydrolysates was only detected at the lowest level of phosphoric acid concentration (runs 10 and 12). As can be expected, these prehydrolysates also contained acetic acid from the hydrolysis of acetyl groups of hemicellulose and sugar degradation compounds such as formic acid, furfural, and HMF. Likewise, phenolic compounds from the extractives and lignin degradation were originated during the pretreatment (Table 3). All of them have been identified as compounds that inhibit the cell growth of the ethanologenic microorganisms. Therefore, it is desirable to maintain low concentrations of these toxic compounds in these sugar solutions to avoid hindering their subsequent fermentation. Overall, low inhibitor compound concentrations were measured in the phosphoric acid prehydrolysates. Thus, acetic acid concentrations lower than 1.5 g/L were determined in the prehydrolysates, whilst the presence of formic acid was negligible. However, phenols and furfural achieved concentrations as high as 3.6 and 4.4 g/L, respectively. 9 ACS Paragon Plus Environment

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The toxic effect of these individual compounds and their synergetic action on the ethanologenic microorganisms have been previously reported.39 High concentrations of inhibitory compounds in the prehydrolysates could be related to low sugar recovery and vice versa. Thus, the poorest prehydrolysate in sugars (9 g/L) corresponded to the pretreatment conducted at the highest temperature (188.28 ºC, run 3). This indicates that sugar degradation occurred due to the high severity of the pretreatment, as confirmed by the presence of 1.7 g/L HMF from the degradation of hexoses (Table 4). However, the highest sugar recovery in the liquid fraction was achieved with run 13 (140 ºC, 6% H3PO4), corresponding with low concentrations of toxic compounds. The determination of solid recovery after each pretreatment made it possible to find the level of material solubilization achieved under the different pretreatment conditions assayed. Table 5 shows the solid recovery and the composition of pretreated BSG under different conditions. As shown, more than 65% of BSG was solubilized in all assayed conditions. This high level of solubilization is related to the presence of easily hydrolysable fractions such as extractives, hemicellulose, starch, or protein. Overall, hemicellulose was almost completely solubilized under most of the assayed conditions. It can be noted that the low residual hemicellulose content determined in the pretreated solids is attributed to the exclusive presence of xylan, since arabinan has been reported to be more hydrolysable than xylan.34 As far as the protein is concerned, the phosphoric acid pretreatment of BSG resulted in solids with protein content ranging between 9 and 20%. It means that a noticeable solubilization occurred during the pretreatment, above 60% in all assayed conditions. Up to 80% of the original protein was released when the pretreatment was carried out at maximum acid concentration, 6.83% (run 6). In this


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context, Kemppainen et al.18 determined lower protein solubilization, between 2% and 12% during steam explosion pretreatment of BSG without acid catalyst. The high BSG solubilization during the pretreatment yielded solids that were highly enriched in cellulose. Overall, the cellulose content in BSG was at least doubled after the pretreatment by phosphoric acid. Therefore, the cellulose content in the pretreated solids ranged between 29% (run 3) and 43% (run 2), while raw BSG had a cellulose content of 15.2%. BSG pretreatment with different inorganic acids under mild conditions (1% acid concentration, 121 ºC, 30 min) including phosphoric acid was reported by Wilkinson et al.23. Only 45% of material was solubilized with that acid and a cellulose-enriched solid remained but contained 37% xylan, probably due to the soft pretreatment conditions used in that research. 3.3 Enzymatic saccharification of pretreated BSG Solids pretreated by phosphoric acid under different conditions were enzymatically hydrolysed to find the effect of the pretreatment on the enzymatic digestibility of these solids. In addition, a test of enzymatic hydrolysis using non-pretreated BSG as substrate was conducted as a control with a glucose production of 4.0 ± 0.11 g/L after 48 h. Besides removing hemicellulose, the aim of the pretreatment is to reduce the crystallinity of the cellulose recalcitrant structure. All pretreated solids were hydrolysed under standard conditions (5% substrate loading, 50 ºC, pH 4.8) with Cellic® CTec3 during 48 h. Glucose concentration in the enzymatic hydrolysates varied in a narrow range, from 16 g/L (run 3) to 21 g/L (run 7). In both cases, complete conversion of the cellulose was achieved. Meanwhile, enzymatic digestibility (grams of glucose obtained by enzymatic hydrolysis per 100 g of glucose in pretreated BSG) of above 80% was achieved in all 11 ACS Paragon Plus Environment

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assayed conditions. On comparing these results with the enzymatic digestibility of raw BSG, it could be seen that the phosphoric acid pretreatment led to an improvement of 55%. This noticeable increase in the enzymatic hydrolysis yield indicates that the pretreatment was successful in opening up the cellulose structure, and this increased the enzymatic access to the cellulose chains. Similar cellulose conversion to glucose has been reported from BSG pretreated by liquid hot water at 190 ºC20 or by 1% (w/v) ClH or 3% (w/v) NaOH.23 Likewise, enzymatic hydrolysis yields (YEH), expressed as grams of glucose obtained by enzymatic hydrolysis per 100 g of cellulosic-glucose in raw BSG, were determined in all cases. Starchy glucose in the raw BSG was not considered to calculate these yields since starch is easy to hydrolyse,40 and therefore this starchy glucose had already been solubilized during the pretreatment and recovered in the liquid fractions (Table 3). Thereby, the pretreatment of BSG made it possible to recover more than 70% cellulosicglucose by enzymatic hydrolysis in all cases with a maximum value of YEH of 94.5% (run 6; 160 ºC, 6.83% H3PO4). These yields compare well with those reported for BSG pretreated with sulfuric acid under mild conditions.11 To assess the efficiency of the phosphoric acid pretreatment, the overall amount of sugars in BSG recovered by both the pretreatment and the subsequent enzymatic hydrolysis of the resulting solids was determined. This parameter was called the overall sugar yield and was calculated as the sum of sugars recovered in liquid and the glucose recovered by enzymatic hydrolysis from 100 g of raw BSG. As can be seen in Table 6, overall sugar yields of above 40 g per 100g were determined except in the experiments carried out at pretreatment temperatures higher than 180 ºC, regardless of the acid concentration (runs 2, 3, and 5). As previously indicated, these experiments resulted in low sugar recoveries in the prehydrolysates (Table 3) as well as a low enzymatic 12 ACS Paragon Plus Environment

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hydrolysis yield in spite of their complete cellulose conversion in runs 3 and 5 (Table 5). This is attributed to a noticeable sugar degradation that resulted in high concentrations of inhibitory compounds (Table 4). Phosphoric acid pretreatment made it possible to obtain more than 40 g of fermentable sugars from 100 g of BSG (78% of the potential sugars). Caetano et al.22 reached an overall sugar yield of 72% with the same feedstock in a configuration process that included the acid pretreatment (HCl plus HNO3) simultaneously with the enzymatic hydrolysis. 3.4 Optimization of phosphoric acid pretreatment The aim of this research was the production of fermentable sugars from both cellulose and hemicellulose in BSG. Therefore, the enzymatic hydrolysis yield (YEH, grams of glucose obtained by enzymatic hydrolysis per 100 g of cellulosic glucose in raw BSG) and the HSR in the liquid fraction (HSRL, grams of hemicellulosic sugars in the prehydrolysate per 100 g of hemicellulosic sugars in raw BSG) were chosen as responses. It can be noted that glucose in the prehydrolysates was not included in the determination of HSRL since this sugar is not originated from the hemicellulose fraction. Overall, the solubilization of cellulosic glucose during the pretreatment is not desirable since the cellulose should be preferentialy hydrolysed in the subsequent enzymatic hydrolysis stage. However, in the case of the BSG, the presence of starchy glucose (5.25%), which is easy to hydrolyse, meant a noticeable recovery of glucose in the prehydrolysates (Table 3). Temperature (T) and phosphoric acid concentration (CA) were chosen as factors to predict the optimal pretreatment conditions based on a multiple-criteria optimization 13 ACS Paragon Plus Environment

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that maximized both responses, HSRL and YEH. The relationships of the independent variables and the individual responses were calculated by the second-order polynomial equations. The regression equations in terms of normalized values are shown in Eqs. (1) and (2):

HSR (%) = 77.60- 21.17T −1.38CA - 4.92T CA -14.70T2 R 2 = 0.9685 R 2 adjust = 0.9527

YEH (%) = 86.54-1.03T - 7.11T2 R 2 = 0.9399 R 2 adjust = 0.9227

(1)

(2)

According to R2 and adjusted R2, the models were highly predictive for both HSR (Eq. 1) and enzymatic hydrolysis yield (Eq. 2). As regards the HSR, both temperature and phosphoric acid concentration showed a negative effect on this response, although the influence of the temperature was more significant (Eq. 1). However, in the case of the enzymatic hydrolysis yield, only the temperature was significant, with a slight negative influence, whereas the acid concentration did not affect this response (Eq. 2). It can be noted that the interaction between the two factors was not detected in either response. Figure 1 shows the influence of both temperature and acid concentration on the response HSRL. As can be appreciated, the HSR in the prehydrolysate decreases when the pretreatment temperature increases. However, the phosphoric acid concentration only shows a slight positive influence at the lowest temperature, whereas at 180 ºC this influence was not detected. As for YEH, the lack of influence of the acid concentration on this response is presented in Fig. 2. However, the temperature showed a significant effect on YEH with highest values at the intermediate assay temperatures, while higher and lower temperatures resulted in decreases in YEH. 14 ACS Paragon Plus Environment

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Once the influence of both factors—temperature and phosphoric acid concentration—on both responses—HSR and YEH—had been analysed, the software was able to determine the optimal conditions for the phosphoric acid pretreatment of BSG with a solid/liquid ratio of 1:8 (w/v). According to the statistical model, when maximizing both responses simultaneously, the optimal conditions were found to be 155 ºC and 2% H3PO4. Then, these optimal conditions for the phosphoric acid pretreatment of BSG were experimentally reproduced and 80% hemicellulosic sugars were recovered in the prehydrolysate. Moreover, the resulting BSG pretreated under these conditions was enzymatically hydrolysed and the complete conversion of cellulose was achieved, yielding 84 g of glucose per 100 g of glucose in raw BSG. Therefore, considering that the model predicted an HSR of 84% and an enzymatic hydrolysis yield of 86%, it can be assumed that a notable adjustment was achieved. This result compares favorably with those obtained by Wilkinson et al.23 for the same raw material pretreated with different inorganic acids (121 ºC, 1% w/v acid concentration, 30 min), also including phosphoric acid. In addition, taking into account that 36% glucose was also recovered in the prehydrolysate, an overall sugar yield of 46.4 g per 100 g of raw BSG was experimentally determined under optimized conditions (155 ºC, 2% H3PO4). Therefore, since 100 g of raw BSG contains 51 g of sugars (Table 2), 91% of the potential sugars in BSG were experimentally recovered by phosphoric acid pretreatment under optimized conditions and subsequent enzymatic hydrolysis. 3.5 Fermentation of acid prehydrolysate obtained under optimal conditions The bioconversion of all carbohydrates in the BSG is crucial to carry out the integral utilization of this feedstock. Therefore, 71% of the total sugars in BSG was solubilized


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during the phosphoric acid pretreatment under optimal conditions and recovered in the prehydrolysate, including glucose (20% of the total sugars in the liquor). This high solubilization can be explained by the fact that 67% of the sugars in the BSG forms structures such as hemicellulose and starch, which are easier to hydrolyse than cellulose. This indicates an important difference between BSG and other lignocellulosic materials such as barley straw, corn stover, eucalyptus, and rice straw,41 whose sugars are mainly in cellulose form. For this reason, in the case of BSG, it is essential to valorize the stream of solubilized sugars that contains both pentoses and hexoses. This proposal suggests the use of an ethanologenic microorganism that is able to convert xylose and arabinose, in addition to glucose, into ethanol whilst having a high inhibitor tolerance. The prehydrolysate resulting from the phosphoric acid pretreatment of BSG with more than 40 g/L sugars also contained inhibitory compounds. As can be seen in Table 7, organic acids and HMF were present in the prehydrolysate at low concentrations. However, the levels of furfural and phenols were higher, although they were not limiting for the cell growth. Thereby, the liquor showed good fermentability, E. coli was able to assimilate all sugars in the prehydrolysate, and a detoxification step was not necessary. Martínez-Patiño et al.42 fermented liquors from olive tree biomass that was also pretreated with phosphoric acid, but E. coli needed a previous detoxification step by overliming. It is worth noting that the high protein content determined in the BSG (21%) was partly solubilized during the pretreatment and could be utilized by the microorganism as a nitrogen source, boosting the fermentability of the prehydrolysate.16 Figure 3 shows the sugar uptake and ethanol production by E. coli from raw BSG prehydrolysate. As shown, E. coli consumed glucose at a higher rate than the rest of the


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sugars and it was depleted after only 7 h whereas the rest of sugars were assimilated in a more gradual way. Thus, all of them had been assimilated by the microorganism after 45 h of fermentation with a maximal ethanol production of 16 g/L at 55 h. This corresponds to a yield of 0.40 g of ethanol per gram of sugars in the medium (78% of the theoretical ethanol yield, 0.51 g of ethanol per gram of sugar). This result compares favourably with that reported by Wilkinson et al.23 in the fermentation of prehydrolysate from BSG pretreated by HCl with S. cerevisae, which yielded an ethanol production of 5.4 g/L and 69% of the theoretical yield. 3.6 Overall process material balance BSG fractionation by phosphoric acid pretreatment plus the enzymatic hydrolysis of the pretreated solid yielded two sugar solutions from the hemicellulosic, starch, and cellulosic fractions in this feedstock, leaving a final solid enriched in lignin and protein with the possibility to be exploited. The phosphoric acid pretreatment of BSG under optimal conditions made it possible to recover 80% of the hemicellulosic sugar content in this feedstock. Besides, 35.5% of total glucose in BSG was also recovered in the liquid fraction. All these sugars were converted to bioethanol by E. coli after 55 h of fermentation, as described in Section 3.5. Likewise, the pretreatment of BSG yielded a highly cellulose-enriched solid (36% cellulose). The simultaneous saccharification and fermentation (SSF) of this solid pretreated using Saccharomyces cerevisiae has been recently reported. This simultaneous process at 15% solids yielded an ethanolic solution of 21 g/L (74% of the theoretical yield) after 30 h, corresponding to 5.1 g of ethanol per 100 g of raw BSG.43 The mass balance of the whole process, including the phosphoric acid pretreatment of BSG under optimal conditions, fermentation of the resulting prehydrolysate, and SSF of 17 ACS Paragon Plus Environment

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the pretreated solid, was determined to assess the performance of the BSG conversion process (Fig. 4). All in all, the optimal conditions of phosphoric acid pretreatment led to an ethanol production of 17.9 g of ethanol per 100 g of raw BSG (12.9 g from hemicellulose and starchy glucose plus 5.1 g from cellulose). Considering that 51 g of fermentable sugars was determined in 100 g of raw BSG (Table 2), the process configuration studied in this research allowed 69% of these sugars to be converted to ethanol. On this basis, 1 t of BSG (dry weight) would yield 179 kg of ethanol. This overall process yield compares favorably with those reported with the same feedstock using fungal consolidated bioprocessing44 or using the mesophilic fungus Neurospora crassawith ethanol productions (per ton of BSG) of 94 and 74 kg, respectively. 24 4. CONCLUSION BSG is an interesting feedstock for producing easily hydrolysable fermentable sugars that can be converted to ethanol. Phosphoric acid pretreatment of BSG with a solid/liquid ratio of 1:8 (g/g) under optimized conditions (155 ºC, 2% H3PO4) and subsequent enzymatic hydrolysis made it possible to recover 91% of total sugars in BSG, mainly solubilized in the prehydrolysate (63%). E. coli fermented this sugar solution containing hemicellulosic sugars and starchy glucose, and no detoxification step was necessary. E. coli assimilated both pentoses and hexoses with an ethanol yield of 0.40 g/g. The process configuration proposed in this work, including phosphoric acid pretreatment of BSG under optimal conditions, fermentation of the resulting prehydrolysate, and SSF of the pretreated solid, gave an overall yield of 227 L of ethanol per dried ton of BSG. Further research should focus on the processing of the complete slurry in order to achieve higher ethanol concentrations.


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AUTHOR INFORMATION Corresponding Author Phone: +34 953213644, Fax: +34 953212779, E-mail: [email protected] ORCID Inmaculada Romero: 0000-0002-4152-8034 ACKNOWLEDGEMENTS Financial support from Universidad de Jaén (project UJA2015/07/21).

REFERENCES (1) FAOSTAT Statistics Division. Food and Agriculture Organization of the United Nations (FAO). Rome, Italy. http://faostat3.fao.org/browse/Q/QC/S (accessed November 27, 2017). (2) Mussatto, S. I. Biotechnological potential of brewing industry by-products. In Biotechnology for Agro-Industrial Residues Utilisation: Utilisation of Agro-Residues; 2009; pp 313-326. (3) Lynch, K. M.; Steffen, E. J.; Arendt, E. K. J. Inst. Brew. 2016, 122, 553-568. (4) Kunze, W. Technology Brewing and Malting-International Edition; VLB: Berlin, 1996; , pp 726. (5) Xiros, C.; Christakopoulos, P. Waste Biomass Valoris. 2012, 3, 213-232. (6) Aliyu, S.; Bala, M. Afr. J. Biotechnol. 2011, 10, 324-331. (7) Kabel, M. A.; Carvalheiro, F.; Garrote, G.; Avgerinos, E.; Koukios, E.; Parajó, J. C.; Gı́rio, F. M.; Schols, H. A.; Voragen, A. G. J. Carbohydrate Polymers 2002, 50, 47-56.


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(8) Vieira, E.; Rocha, M. A. M.; Coelho, E.; Pinho, O.; Saraiva, J. A.; Ferreira, I. M. P. L. V. O.; Coimbra, M. A. Ind. Crops Prod. 2014, 52, 136-143. (9) Reis, S. F.; Gullón, B.; Gullón, P.; Ferreira, S.; Maia, C. J.; Alonso, J. L.; Domingues, F. C.; Abu-Ghannam, N. Appl. Microbiol. Biotechnol. 2014, 98, 93659373. (10) Cater, M.; Fanedl, L.; Malovrh, Š; Marinšek Logar, R. Bioresour. Technol. 2015, 186, 261-269. (11) Plaza, P. E.; Gallego-Morales, L. J.; Peñuela-Vásquez, M.; Lucas, S.; GarcíaCubero, M. T.; Coca, M. Bioresour. Technol. 2017, 244, 166-174. (12) Olajire, A. A. J. Clean. Prod. 2012. (13) Johnson, P.; Paliwal, J.; Cenkowski, S. Stewart Postharvest Rev. 2010, 6, 1-8. (14) Carvalheiro, F.; Duarte, L. C.; Medeiros, R.; Gírio, F. M. Appl. Biochem. Biotechnol. Part A Enzyme Eng. Biotechnol. 2004, 115, 1059-1072. (15) Duarte, L. C.; Carvalheiro, F.; Lopes, S.; Marques, S.; Parajó, J. C.; Gírio, F. M. Appl. Biochem. Biotechnol. Part A Enzyme Eng. Biotechnol. 2004, 115, 1041-1058. (16) Mussatto, S. I.; Roberto, I. C. J. Sci. Food Agric. 2005, 85, 2453-2460. (17) Dávila, I.; Gullón, P.; Andrés, M. A.; Labidi, J. Bioresour. Technol. 2017, 244, 328-337. (18) Kemppainen, K.; Rommi, K.; Holopainen, U.; Kruus, K. Appl. Biochem. Biotechnol. 2016, 180, 94-108.


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(19) Wilkinson, S.; Smart, K. A.; Cook, D. J. Journal of Fuels 2015, 2015, 13. (20) Michelin, M.; Teixeira, J. A. Bioresour. Technol. 2016, 216, 862-869. (21) White, J. S.; Yohannan, B. K.; Walker, G. M. FEMS Yeast Res. 2008, 8, 11751184. (22) Caetano, N. S.; Moura, R. F.; Meireles, S.; Mendes, A. M.; Mata, T. M. Chem. Eng. Trans. 2013, 35, 1021-1026. (23) Wilkinson, S.; Smart, K. A.; Cook, D. J. J. Am. Soc. Brew. Chem. 2014, 72, 143153. (24) Xiros, C.; Topakas, E.; Katapodis, P.; Christakopoulos, P. Bioresour. Technol. 2008, 99, 5427-5435. (25) Geddes, C. C.; Peterson, J. J.; Roslander, C.; Zacchi, G.; Mullinnix, M. T.; Shanmugam, K. T.; Ingram, L. O. Bioresour. Technol. 2010, 101, 1851-1857. (26) Martinez, A.; Grabar, T. B.; Shanmugam, K. T.; Yomano, L. P.; York, S. W.; Ingram, L. O. Biotechnol. Lett. 2007, 29, 397-404. (27) Romero-García, J. M.; Martínez-Patiño, C.; Ruiz, E.; Romero, I.; Castro, E. Bioethanol 2016, 2, 51-65. (28) Sluiter, J. B.; Ruiz, R. O.; Scarlata, C. J.; Sluiter, A. D.; Templeton, D. W. J. Agric. Food Chem. 2010, 58, 9043-9053. (29) Singleton, V. L.; Rossi, J. A. Am. J. Enol. Vitic. 1965, 16, 144-158. (30) Mussatto, S. I. J. Sci. Food Agric. 2014, 94, 1264-1275. 21 ACS Paragon Plus Environment

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(31) Guerrero, A. B.; Ballesteros, I.; Ballesteros, M. J. Chem. Technol. Biotechnol. 2017, 92, 2351-2359. (32) Gullón, B.; Gullón, P.; Tavaria, F.; Pintado, M.; Gomes, A. M.; Alonso, J. L.; Parajó, J. C. J. Funct. Foods 2014, 6, 438-449. (33) Carvalheiro, F.; Esteves, M. P.; Parajó, J. C.; Pereira, H.; Gı́rio, F. M. Bioresour. Technol. 2004, 91, 93-100. (34) Mussatto, S. I.; Roberto, I. C. J. Chem. Technol. Biotechnol. 2006, 81, 268-274. (35) Sáez, F.; Ballesteros, M.; Ballesteros, I.; Manzanares, P.; Oliva, J. M.; Negro, M. J. J. Chem. Technol. Biotechnol. 2013, 88, 937-941. (36) Buruiana, C. -.; Vizireanu, C.; Garrote, G.; Parajó, J. C. Ind. Crops Prod. 2014, 54, 32-39. (37) Castro, E.; Nieves, I. U.; Rondón, V.; Sagues, W. J.; Fernández-Sandoval, M. T.; Yomano, L. P.; York, S. W.; Erickson, J.; Vermerris, W. Ind. Crops Prod. 2017, 109, 367-373. (38) Kim, J. S.; Lee, Y. Y.; Kim, T. H. Bioresour. Technol. 2016, 199, 42-48. (39) Zaldivar, J.; Martinez, A.; Ingram, L. O. Biotechnol. Bioeng. 1999, 65, 24-33. (40) Karimi, K.; Taherzadeh, M. J. Bioresour. Technol. 2016, 200, 1008-1018. (41) Dahadha, S.; Amin, Z.; Bazyar Lakeh, A. A.; Elbeshbishy, E. Energy Fuels 2017, 31, 10335-10347.


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(42) Martínez-Patiño, J. C.; Romero-García, J. M.; Ruiz, E.; Oliva, J. M.; Álvarez, C.; Romero, I.; Negro, M. J.; Castro, E. Energy Fuels 2015, 29, 1735-1742. (43) Rojas-Chamorro, J. A.; Romero, I.; Ruiz, E.; Cara, C.; Castro, E. Chem. Eng. Trans. 2017, 61, 637-642. (44) Wilkinson, S.; Smart, K. A.; James, S.; Cook, D. J. Bioenergy Res. 2017, 10, 146157.


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Figure captions Figure 1. Response surface and contour plot of the combined effects of phosphoric acid concentration and pretreatment temperature on the hemicellulosic sugar recovery in the prehydrolysate (HSRL). Figure 2. Response surface and contour plot of the combined effects of phosphoric acid concentration and pretreatment temperature on the enzymatic hydrolysis yield (YEH). Figure 3. Time course during E. coli SL 100 fermentation of raw phosphoric acid BSG prehydrolysate obtained at optimal conditions (155ºC, 2% H3PO4). Figure 4. Material balance flow scheme of the overall process for ethanol production from BSG after pretreatment with 2% (w/v) H3PO4 at 155 °C.


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Table 1. Experimental design for H3PO4-pretreatment of BSG

Run

Temperature (ºC)

H3PO4 conc. (%)

Coded

Real

Coded

Real

1

0

160

0

4

2

+1

180

-1

2

3

+1.41

188.28

0

4

4

0

160

0

4

5

+1

180

+1

6

6

0

160

+1.41

6.83

7

0

160

0

4

8

0

160

0

4

9

-1.41

131.72

0

4

10

-1

140

-1

2

11

0

160

0

4

12

0

160

-1.41

1.17

13

-1

140

+1

6


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Table 2. Composition (% of dry matter) of the brewery spent grains. Component Cellulose Starch Hemicellulose Xylose Galactose Arabinose Mannose Acid Insoluble Material Acid Insoluble lignin Acid Insoluble ash Acid Insoluble protein Acid soluble lignin Acetyl groups Extractives Fat Protein Ash

% of dry matter 15.24±0.48 5.25±0.16 25.14±0.72 19.13±0.67 1.38±0.03 7.50±0.26 0.34±0.08 10.13±0.73 7.00±0.53 1.23±0.14 1.90±0.48 5.50±0.32 0.22±0.02 18.47±0.97 8.61±0.47 21.2±0.22 2.28±0.05

Data are the mean values and standard deviation of six measurements Fat: determined as hexane extractives after 6 h


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Table 3. Sugar composition and sugar recovery in the acid prehydrolysates

1

Glucose (g/L) 8.80

2

6.31

7.16

1.31

4.7

0.50

38.3

22.4

1.81

3

4.31

1.63

0.83

2.03

0.18

13.2

15.3

1.46

4

7.95

18.90

1.78

8.44

0.60

81.8

28.2

1.45

5

6.34

4.13

1.19

3.53

0.40

26.0

22.5

1.27

6

9.48

15.63

1.97

8.61

0.47

74.5

33.7

1.02

7

7.93

18.73

1.88

9.21

0.55

84.6

28.1

1.48

8

8.61

17.79

2.01

9.16

0.43

82.0

30.5

1.46

9

8.79

14.83

2.03

8.04

0.61

70.6

31.2

1.30

10

8.54

17.81

1.83

10.05

0.47

82.0

30.3

1.55

11

8.07

17.17

1.82

8.7

0.50

79.1

28.6

1.46

12

10.11

17.05

1.95

8.89

0.27

78.8

35.9

1.93

13

10.06

20.40

2.07

10.71

0.19

89.4

35.7

1.03

Run

Xylose Galactose Arabinose Mannose (g/L) (g/L) (g/L) (g/L) 14.83 2.03 8.04 0.62

HSR (%) 70.6

GR (%) 31.2

1.50

pH

HSR: Hemicellulosic sugar recovery (g hemicellulosic sugars in the prehydrolysate/100 g hemicellulosic sugars in raw BSG) GR: Glucose recovery (g glucose in the prehydrolysate/100 g glucose in raw BSG)


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Table 4. Inhibitor composition of phosphoric acid prehydrolysates from BSG. Run

Formic acid Acetic acid (g/L) (g/L)

1 0.04 2 0.07 3 0.16 4 0.01 5 0.13 6 nd 7 0.03 8 0.03 9 nd 10 nd 11 0.05 12 nd 13 nd nd: not detected

1.24 1.24 1.54 1.25 1.42 1.49 1.23 1.26 0.89 0.41 1.31 0.63 0.90

HMF (g/L)

Furfural (g/L)

0.42 0.92 1.70 0.24 1.23 0.52 0.23 0.28 0.01 nd 0.35 0.10 0.03

3.44 nd nd 2.67 nd 4.44 2.42 2.75 0.46 0.12 3.35 1.48 0.50

Phenolic compounds (g/L) 3.28 3.11 2.31 3.70 2.57 2.66 2.48 3.60 1.72 1.75 2.86 1.89 1.64


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Table 5. Solid recovery and pretreated solids composition Run

Composition (%) Solid recovery (%) Cellulose Hemicellulose AIL

1

36.1

36.7

1.4

48.9

2 3

33.6 37.8

42.9 28.7

2.4 1.0

47.8 45.0

4 5

36.0 32.2

40.2 33.6

3.6 0.2

38.0 54.1

6 7 8

38.2 34.9 35.0

39.6 38.8 35.5

0.2 1.8 0.0

41.0 43.2 45.7

9 10 11 12 13

35.3 39.4 32.4 36.7 42.7

33.8 32.3 41.5 36.2 34.9

2.5 9.0 2.0 2.8 6.1

36.6 31.3 38.1 36.6 36.5

Protein 11.3 14.7 12.1 16.7 10.2 9.3 9.9 10.0 17.6 20.6 11.5 14.2 14.7

AIL: Acid insoluble lignin


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Table 6. Results of enzymatic hydrolysis of pretreated solids and sugar yields obtained after pretreatment and enzymatic hydrolysis Run

Glucose concentration (g/L)

Enzymatic digestibility (%)

EH yield (%)

Overall yield (g/100 g)

1

19.72 ± 0.61

97.6

85

41.30

2

19.51 ± 0.55

82.6

78.2

29.02

3

15.86 ± 0.17

100

71.6

19.19

4

21.31± 0.40

96.4

91.5

44.96

5

20.27 ± 1.49

100

77.9

25.53

6

20.74 ± 0.16

95.2

94.5

45.19

7

21.31 ± 1.26

99.9

88.7

45.21

8

20.32 ± 0.85

100

84.8

44.35

9

17.14 ± 1.09

92.3

72.1

39.15

10

17.17 ± 0.50

96.7

80.8

43.63

11

20.06 ± 0.17

87.9

77.6

42.04

12

19.81 ± 0.70

99.7

86.7

44.97

13

16.25 ± 0.31

84.7

82.7

47.26

EH yield: g glucose by EH/100 g cellulosic-glucose in raw BSG Overall yield: sum of glucose by EH + sugars in the prehydrolysate/100 g raw BSG


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Table 7. Sugar and inhibitor composition of the prehydrolysate obtained at optimal pretreatment conditions (155ºC, 2% H3PO4).

Sugars (g/L) Glucose

Xylose

Galactose

Arabinose

Mannose

7.75 ± 0.54

21.04 ± 0.76

1.86 ± 0.35

9.23 ± 0.70

0.36± 0.10

Inhibitors (g/L) Formic acid

Acetic acid

HMF

Furfural

Phenols

0.19± 0.05

0.81± 0.12

0.10± 0.03

1.36± 0.14

3.31± 0.22

HMF: Hydroxymethylfurfural


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94

74

54

HSR (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 35

33

13

2

140 3

150 4

B: C_fosfórico

160 5

170

A: T

6 180

Figure 1.


Page 33 of 35

89

85

80

YEH (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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76

71

6

1 80 5

170 4

B: C_fosfórico

160 3

150

A: T

2 140

Figure 2.


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25 Glucose 20 Concentration (g/L)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 35

XGM 15

Ethanol

10

Arabinose Cell

5

0 0

20

40 Time( h )

60

Figure. 3


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H3P04 (85 %) 18.8 g H20 781.2 g

Enzymatic solution 242.6 g Inoculum (4%) 9.70 mL

BREWERY SPENT GRAIN 100 g dry weight Glucose: 22.5 g Xylose: 19.1 g Galactose: 1.4 g Arabinose: 7.5 g Mannose: 0.3 g Lignin: 10.1 g Protein : 21.2 g

SSF PHOSPHORIC ACID PRETREATMENT 155 ºC, 0 min, 2 % w/w

SOLID 36.4 g Glucose: 14.2 g Xylose: 0.7 g Lignin: 15.5 g Protein: 4.9 g

72 h, 150 rpm 40 ºC Cellic CTec 3 15 FPU/g β-glucosidase 15 IU/g Substrate loading: 15 % (w/v) S.cerevisiae 4 % (v/v)

Glucose: 1.9 g Lignin: 10.3 g Protein: 3.2 g

LIQUID

LIQUID Glucose: 6.2 g Xylose: 16.8 g Galactose: 1.4 g Arabinose: 7.4 g Mannose: 0.30 g Phenols: 2.6 g Formic acid: 0.1 g Acetic acid: 0.6 g HMF: 0.1 g Furfural: 1.1 g

SOLID : 15,7 g

Ethanol: 5 g

FERMENTATION E.coli SL100 pH 6.5, 37º C, 300 rpm

LIQUID Ethanol: 12.8 g

Figure 4.


ethanol production from brewers' spent grain pretreated by dilute

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