Brewer’s spent grain as a source of renewable fuel through optimized dilute acid pretreatment

Journal Pre-proof Brewer’s Spent Grain As A Source Of Renewable Fuel Through Optimized Dilute Acid Pretreatment

José A. Rojas-Chamorro, Inmaculada Romero, Juan C. López-Linares, Eulogio Castro PII:

S0960-1481(19)31902-0

DOI:

https://doi.org/10.1016/j.renene.2019.12.030

Reference:

RENE 12739

To appear in:

Renewable Energy

Received Date:

05 May 2019

Accepted Date:

05 December 2019

Please cite this article as: José A. Rojas-Chamorro, Inmaculada Romero, Juan C. López-Linares, Eulogio Castro, Brewer’s Spent Grain As A Source Of Renewable Fuel Through Optimized Dilute Acid Pretreatment, Renewable Energy (2019), https://doi.org/10.1016/j.renene.2019.12.030

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Journal Pre-proof

BREWER’S SPENT GRAIN AS A SOURCE OF RENEWABLE FUEL THROUGH OPTIMIZED DILUTE ACID PRETREATMENT

José A. Rojas-Chamorro1, Inmaculada Romero1,2, Juan C. López-Linares1, Eulogio Castro1,2* 1Dept.

of Chemical, Environmental and Materials Engineering, Universidad de Jaén,

2Center

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

Abstract In this work, a dilute sulfuric acid pretreatment at mild conditions was employed to fractionate brewer’s spent grain (BSG) with the aim of achieving high carbohydrate recovery. A Box-Behnken experimental design was used with temperature (110– 130ºC), acid concentration (1–3% w/v) and time (10–40 min) as independent factors, the objective being to determine the best conditions for the release of glucose (following enzymatic hydrolysis of the pretreated solids) along with that of hemicellulosic sugars in the liquors. The optimal pretreatment conditions were found to be 130ºC, 1% H2SO4 and 26 min, which allowed the recovery of 94% of the sugars in raw BSG. Next, the effect of substrate concentration on the simultaneous saccharification and fermentation (SSF) process of the pretreated solid was studied. Likewise, the fermentability of the resulting prehydrolysate was evaluated with two xylose fermenting microorganisms, Scheffersomyces stipitis and Escherichia coli. The overall proposed BSG bioconversion process yielded 22.9 L bioethanol from 100 kg of dry biomass. Keywords Brewer’s spent grain; Acid pretreatment; response surface methodology; high solid loading; co-fermentation 1

Journal Pre-proof 1. Introduction Biomass represents the only renewable source capable of replacing fossil fuels for transportation. Advanced biofuels based on residual biomass constitute the most interesting alternative to conventional biofuels based on food feedstocks [1] and will play a crucial role in the transition from fossil fuels to sustainable resources. The use of lignocellulosic materials to produce biofuels and green chemicals is currently widely studied because lignocellulose is the most abundant renewable carbon source in nature. Among biomass resources, brewer`s spent grain (BSG) constitutes a prominent example of an agroindustrial, lignocellulosic residue with low-value applications (primarily animal feed) that has been proposed as a feedstock for fuel ethanol production [2]. To overcome the recalcitrant structure of lignocellulose, materials must be subjected to a pretreatment step that fractionates the biomass and facilitates the carbohydrate release which has been proved to be essential for producing fuels and chemicals from lignocellulosic materials [3]. Dilute acid pretreatment solubilizes hemicelluloses and opens the structure of the lignocellulosic biomass. Moreover, this technology has the advantage of not requiring special construction materials, thus improving industrial application possibilities [4]. After pretreatment, the conversion process also includes the hydrolysis of the pretreated materials by means of enzymes and fermentation of the resulting glucose to ethanol by selected microorganisms. Ethanol production can be increased by also transforming the sugars released during pretreatment; these sugars come from the hemicellulosic fraction of the lignocellulosic material that enters the liquid phase that is usually separated by filtration of the pretreated solids. Recent studies have revealed several different bioproducts that can be obtained from BSG. For example, BSG is a source of antioxidant compounds that have been extracted using either conventional solvent extraction [5] or ultrasound or microwave assisted 2

Journal Pre-proof systems [6]. Ionic liquids have been used on BSG as a delignification method with the goal of obtaining sugars that may, in turn, be used for the production of other valueadded products [7]. One of the main applications is the fractionation of protein from BSG; Rommi et al. [8] reported that the use of an enzyme (protease) was required for effective extraction of proteins from hydrothermally pretreated BSG. Qin et al. [9] compared several pretreatment options and concluded that hydrothermal pretreatment at 60ºC is an advantageous technology for protein extraction when both cost and environmental issues are taken into account. Dávila et al. [10] reported on the application of the biorefinery concept based on BSG; they concluded that the production of ethanol alone is not an economically feasible option, but heat integration could make the joint production of xylitol and polyhydroxybutyrate, along with ethanol, a viable alternative that would also benefit from the environmental point of view. The environmental impacts of a BSG-based biorefinery producing xylooligosaccharides and bioethanol are also discussed in a recent work by González-García et al. [11], who identified steam and enzyme production as environmental hotspots in the proposed biorefinery. From the literature review, ethanol is still one of the products to be obtained from BSG given the high sugar content of this material. In this work, an experimental design considering the main operational variables was applied to optimize the pretreatment conditions of BSG to produce cellulose-enriched solids and liquids where hemicellulosic sugars and other compounds are included. This work addresses the integral conversion of all the sugars present in BSG, including those from the hemicellulosic and starch fractions and the glucose coming from enzyme hydrolysis of the cellulose contained in the pretreated solids. An ethanologenic Escherichia coli strain and a pentose-fermenting yeast, Scheffersomyces stipitis, were used to ferment starchy3

Journal Pre-proof glucose and hemicellulosic sugars, while Saccharomyces cerevisiae fermented the glucose derived from cellulose. 2. Materials and methods 2.1. Raw material Brewers’ spent grain (BSG), which was kindly donated by the Cruzcampo-Jaén brewery (Heineken España, S.A., Spain), was washed with distilled water in order to attain a neutral pH, dried at 50°C and stored at 4°C until use. The chemical composition of BSG is (dry weight): cellulose 15.2 ± 0.5, hemicellulose 25.1 ± 0.7 (xylan 16.9 ± 0.7, galactan 1.3 ± 0.0, arabinan 6.6 ± 0.3, mannan 0.3 ± 0.1), starch 5.3 ± 0.2, acid-soluble lignin 5.5 ± 0.3, protein 21.2 ± 0.2, ash 2.3 ± 0.1, acid-insoluble material (lignin 7.0 ± 0.5, protein 1.9 ± 0.5, ash 1.2 ± 0.1), and extractives 18.5 ± 1.0 [12]. For biomass samples containing protein, the acid-insoluble material includes acid-insoluble protein besides of acid-insoluble lignin and ash [13]. 2.2. Sulfuric acid pretreatment and experimental design BSG and the sulfuric acid solution were mixed at a liquid/solid ratio of 8:1 (w/w) and put into a 1 L Parr reactor (Parr Instr. Co., IL, USA). Once the temperature was set, pretreatment was initiated with a heating rate of 5ºC/min and an agitation speed of 350 rpm. The conditions at which the raw material was pretreated were determined following a Box-Behnken experimental design, resulting in 17 experiments, including five points at the centre of the experimental domain. Three major process variables, temperature (110–130ºC), sulfuric acid concentration (1–3% w/v) and reaction time (10–40 min), were chosen as factors. The real values of these independent factors are reported in Table 1. Analysis of the experimental data was

4

Journal Pre-proof carried out using Design-Expert 8.0.7.1 statistical software, Stat-Ease Inc., Minneapolis, USA. Table 1 Experimental conditions for sulfuric acid pretreatment of BSG and experimental and predicted values for hemicellulosic sugar recovery in liquids (HSR) and enzymatic hydrolysis yield (YEH). Responses

Experimental conditions

HSR, %

YEH, %

Run

T, ºC

CA , %

t, min

Experimental

Predicted

Experimental

Predicted

1

110

1

25

82.0

80.9

69.1

69.1

2

120

3

10

93.6

92.7

81.1

82.4

3

120

3

40

92.4

92.7

77.4

76.0

4

110

3

25

90.2

91.6

80.3

80.4

5

120

2

25

85.5

83.7

71.7

73.0

6

110

2

10

73.3

74.7

64.1

64.1

7

130

1

25

98.0

97.7

82.7

82.6

8

120

2

25

83.3

83.7

80.0

73.0

9

120

2

25

83.0

83.7

82.4

73.0

10

130

2

40

88.0

86.6

80.3

80.3

11

130

2

10

85.6

86.6

78.4

78.4

12

110

2

40

80.1

80.8

70.8

71.0

13

120

1

40

87.4

89.2

54.2

55.5

14

120

2

25

84.3

83.7

73.1

73.0

15

130

3

25

89.7

90.2

65.3

73.0

16

120

2

25

82.3

83.7

67.5

73.0

17

120

1

10

83.3

84.5

63.3

62.0

HSR: hemicellulosic sugar recovery in liquids; YEH: enzymatic hydrolysis yield.

After treatment, the reactor was cooled down to about 40ºC. Then, the pretreated material or water-insoluble solids (WIS) and the liquid fraction (prehydrolysate) were separated by vacuum filtration. The WIS were water washed, dried at 35ºC, and analysed to determine their chemical composition. In addition, the WIS were used as substrates in enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) tests. The content of sugars and inhibitors, such as formic acid, acetic acid,

5

Journal Pre-proof furfural, hydroxymethylfurfural (HMF) and phenolic compounds, was measured in the prehydrolysates. The efficiency of the sulfuric acid pretreatment was assessed by determining the concentration of hemicellulosic sugars in the liquors (HSR). 2.3. Enzymatic hydrolysis Pretreated BSG was enzymatically hydrolysed using Cellic CTec3, a cellulolytic commercial enzyme complex (Novozymes A/S, Denmark). To avoid possible inhibition attributed to cellobiose, the Cellic CTec3 enzyme preparation was supplemented with fungal β-glucosidase (Novozym 188, Novozymes A/S). Cellulase and β-glucosidase enzyme loadings used were 15 filter paper units (FPU)/g WIS and 15 international units (IU)/g WIS, respectively. The substrate concentration used in the enzymatic assays was 5% (w/v) using 100 mL Erlenmeyer flasks with a working volume of 25 mL. The pH of enzymatic solutions was 4.8, which was achieved using 0.05 M sodium citrate buffer. Triplicate tests were performed in an orbital shaker (Certomat-R, B-Braun, Germany) at 50ºC and 150 rpm for 24 h. Samples were withdrawn every 6–8 h, centrifuged at 11,500 rpm (Sigma 1-14 Centrifuge) for 10 min and analysed by high-performance liquid chromatography (HPLC) for glucose concentration. Moreover, as commercial enzyme preparation are known to contain glucose, this was taken into account for calculations through enzyme blanks. The enzymatic hydrolysis yields were calculated by dividing the amount of glucose from enzymatic hydrolysis by the glucose in the WIS (enzymatic digestibility) or by the glucose in raw BSG (enzymatic hydrolysis yield, YEH). 2.4. Microorganisms and inocula 6

Journal Pre-proof Saccharomyces cerevisiae (Fermentis Ethanol Red, Cedex, France) was used for SSF tests. Yeast was kept on a solid culture medium consisting of (g/L): glucose, 20; yeast extract, 5; peptone, 5; NH4Cl, 2 g/L; KH2PO4, 1; MgSO4.7H2O, 0,5; and agar 20, at 4ºC. To make the cells, solid media-cultured yeasts were transferred to the growth medium, using 100 mL Erlenmeyer flasks and a working volume of 25 mL. The growth medium composition was (g/L): yeast extract, 5; ClNH4, 2; KH2PO4, 1; MgSO4.7H2O, 0.3; and glucose, 30. The inoculum was grown in a rotatory shaker (Certomat-R, B-Braun, Germany) at 150 rpm and 30ºC for 16 h. Escherichia coli strain SL100 and Scheffersomyces stipitis strain CBS6054 were used for the fermentation of the liquor from pretreatment at optimal conditions. E. coli was preserved in 40% glycerol stocks at -80°C. To grow the inoculum, 250 mL Sterilized Erlenmeyer flasks were used with 100 mL of AM1 culture medium [14]. The cells were incubated at 37ºC and 200 rpm in a shaking incubator for 24 h, centrifuged (3,500 rpm, 10 min) and washed. S. stipitis was maintained in agar slants. The growth of inoculum was carried out in 250 mL Erlenmeyer flasks employing 75 mL of sterilized culture medium, and whose composition was (g/L): yeast extract, 5; NH4Cl, 1; KH2PO4, 1; MgSO4.7H2O, 0.5; and xylose, 40. The inoculum was grown at 30°C and 180 rpm for 24 h and manipulated in the same way as for E. coli. 2.5. Simultaneous saccharification and fermentation (SSF) SSF tests using BSG pretreated at optimal conditions as the substrate were carried out at different biomass loadings, 5, 15, 20 and 25% w/v. For SSF assays, 25 mL of citrate buffer was used in 100 mL Erlenmeyer flasks containing the previously mentioned nutrients for S. cerevisiae growth (except for glucose) at microaerobic conditions. The inoculum represented 4% v/v, equivalent to 0.25 g cell/L.

7

Journal Pre-proof Cellic CTec3 and β-glucosidase enzymes were used for SSF tests with the same enzyme loading that was used in the enzymatic hydrolysis assays. In addition, commercial xylanase enzyme (Cellic HTec2, Novozymes A/S) with an enzyme loading of 15 FPU/g WIS was added. The SSF tests were done in triplicate at 37ºC and 150 rpm for 72 h. Samples were withdrawn every 24 h and the ethanol produced and glucose consumed were analysed. SSF results were expressed as a percentage of the theoretical yield (0.51 g ethanol/g glucose). 2.6. Fermentation of BSG prehydrolysate The acid prehydrolysate of BSG obtained at optimal pretreatment conditions was fermented with E. coli SL100 and S. stipitis CBS6054 without prior detoxification. Fermentation with E. coli was carried out by supplementing the hydrolysate with AM1 growth medium salts as reported in Section 2.5 for this strain, without glucose and xylose. In order to decrease the toxicity of the hydrolysate, sodium metabisulfite was also added [15]. The concentration of metabisulfite (1.5 mM, equivalent to 43.20 mg in each fermentor) was added after the salts and just before the inoculum. Then, the hydrolysate was sterilized by filtration (0.22 mm, Millipore, Ireland). Fermentation tests were carried out at 37°C, 300 rpm and 6.5 pH in 300 mL glass flasks with a total volume of 150 mL. During all the fermentation tests, 2 M KOH was used to adjust the pH. The initial cell concentration was 0.5 g/L, which was determined using absorbance data at 620 nm. For S. stipitis fermentation, the medium described above in Section 2.5 for this microorganism (except xylose) was added to the acid prehydrolysate. The hydrolysate sterilization process used was the same as described for E. coli. The initial biomass concentration was 1 g/L. Fermentation experiments with 100 mL working volume were

8

Journal Pre-proof carried out in a rotary shaker at 30°C, 150 rpm, and 5 pH for 60 h, in 250 mL Erlenmeyer flasks. All fermentation experiments were achieved by duplicate. Samples were withdrawn at planned times, centrifuged at 11,500 rpm for 10 min and analysed by HPLC for cell, sugar and ethanol composition. Ethanol yields (grams of ethanol/grams of consumed sugars) were calculated to assess the effectiveness of the optimum prehydrolysate fermentation process. The ethanol yield as a percentage of the theoretical one (0.51 g of ethanol/g of sugar) was also reported. 2.7. Analytical methods The National Renewable Energy Laboratory’s analytical methodology [13] was used to determine the composition of the pretreated solids. Elemental analysis (EA1112 Thermo Finnigan elemental analyser) was used to determine their nitrogen content, which was later converted to protein using a conversion factor (N × 6.25). According to NREL methodology, the presence of protein in the biomass interferes in the determination of acid-insoluble lignin since it is partly accounted in the acid-insoluble material and therefore, protein and ash content in the acid-insoluble material should be determined and subtracted to estimate the acid-insoluble lignin in the biomass [13]. In this work, the acid-insoluble material was determined after a two-step acid hydrolysis for all pretreated solids although only in the case of BSG from the pretreatment under optimal conditions, the protein content was determined in the acid-insoluble material and, together with the acid-insoluble ash, were taken into account for estimating its acidinsoluble lignin content. The determination of biomass concentration, sugar content in liquors, including oligomeric sugars, inhibitor concentration, ethanol produced and total phenolic concentration were done as described elsewhere [12].

9

Journal Pre-proof All analytical determinations were performed in triplicate. Average results, with standard deviations lower than 5%, are reported. 3. Results and discussion 3.1. Effect of dilute acid pretreatment on BSG Dilute sulfuric acid pretreatment of BSG achieved the partial solubilization of the biomass; the solid recovery after pretreatment ranged from 36.9% (run 15, 130ºC, 3% H2SO4, 25 min) to 57.1% (run 6, 110ºC, 2% H2SO4, 10 min) depending on the pretreatment harshness (Table 2). This fact is related to the hydrolysis of hemicellulose and starch fractions and the solubilization of the extractives during pretreatment. Consequently, cellulose was enriched in the pretreated solids. Considering that the raw BSG used in this work contained 15.2% cellulose, the pretreatment yielded celluloseenriched solids and, in most of the assayed conditions, the cellulose content after pretreatment was almost twice higher than in the raw BSG (Table 2). Likewise, dilute acid pretreatment of BSG resulted in pretreated solids free of hemicellulose or with a low hemicellulose content, less than 6%. Hydrolysis of the hemicellulose during pretreatment favours biomass fractionation and contributes to making the cellulose chains more accessible to enzymatic attack.

10

Journal Pre-proof Table 2 Solid recovery and characterization of the solid fractions after pretreatment (%). Run

Solid Acid-insoluble Cellulose Hemicellulose recovery material

Acid-soluble lignin

Protein

Ash

1

54.5

25.3

4.0

34.6

6.3

29.8

2.6

2

46.5

27.1

0.8

41.1

5.0

19.0

2.6

3

42.1

27.8

0.6

44.3

4.5

18.5

3.0

4

50.0

30.6

2.2

40.0

5.6

19.7

2.8

5

50.0

26.5

2.7

37.3

5.0

21.0

2.6

6

57.1

25.0

5.8

28.5

5.8

27.4

2.3

7

43.2

29.2

0.2

44.4

3.5

18.1

2.9

8

50.4

28.3

1.7

40.6

4.4

21.4

2.7

9

46.0

29.0

1.9

37.5

5.4

23.8

2.8

10

44.2

30.4

0.5

45.6

3.5

23.0

2.8

11

46.0

29.9

1.4

39.9

4.4

19.4

2.8

12

48.6

30.1

2.5

37.0

4.7

22.8

2.6

13

45.7

33.3

0.0

47.7

2.2

12.0

2.9

14

47.2

26.2

1.8

37.5

4.7

23.1

2.9

15

36.9

31.3

0.0

53.1

1.5

11.2

2.9

16

50.4

23.5

1.9

37.3

4.7

26.5

2.4

17

47.2

27.3

3.5

34.3

4.8

22.3

2.8

Average of three determinations with standard deviations lower than 5%. Acid-insoluble material includes lignin, protein and ash.

The protein was also partly solubilized; the pretreated solids resulted to have a protein contain between 29.8% (run 1; 110ºC, 1% H2SO4, 25 min) and 11.2% (run 15; 130ºC, 3% H2SO4, 25 min). This means that the acid pretreatment solubilized from 23% (run 1) up to 80% (run 15) of the protein, considering that raw BSG contains 21.2% protein. Pretreated solids with about 20% protein were obtained from the same raw material pretreated by steam explosion at 180ºC [16] or with phosphoric acid at 140ºC [2]. It can be noted that the values of acid-insoluble material depicted in Table 2 for the pretreated solids include acid-insoluble fractions of lignin, ash and protein. Taking into account that raw BSG accounted for 10% acid-insoluble material [12], the pretreatment 11

Journal Pre-proof meant an important increase in the acid-insoluble material content. Kemppainen et al. [16] determined also higher values of acid-insoluble material after steam explosion pretreatment of the same feedstock, even at the softest conditions. They attributed this fact to the enrichment of lignin in solids by the formation of pseudolignin structures resulting from reactions between lignin and other compounds like sugars and sugar degradation products. In addition to cellulose-enriched solids, the pretreatment yielded sugar solutions with concentrations greater than 40 g/L, mainly containing hemicellulosic sugars as monomers (Table 3). The main sugar in all prehydrolysates was the xylose from hemicellulose, which accounted for almost 50% of total sugars in these liquids. Arabinose in the acid prehydrolysates of BSG was also noticeable, about 25% of total sugars, due to the high arabinan content in this biomass, more than 6%, compared to other lignocellulosic materials. Overall, the sulfuric acid pretreatment of BSG reached hemicellulosic sugar recoveries greater than 80% in all tested conditions (Table 1), with a maximum value of 98% (run 7; 130ºC, 1% H2SO4, 25 min). The experimental and predicted values for HSR were very close for all conditions assayed. Compared to other pretreatments, low pH systems such as dilute acid pretreatment tend to achieve higher hemicellulosic sugar recovery [17]. Glucose was present in all prehydrolysates, with concentrations varying between 4.5 g/L (run 9) and 11 g/L (run 15) (Table 3). This glucose mainly originated from the starch fraction in raw BSG (5.25 g/100 g dry BSG) because starch is hydrolysed to glucose under milder conditions than cellulose. In addition to sugars, inhibitor compounds were detected in the acid liquors from BSG, although in low concentrations. In fact, the concentrations of acetic acid, furfural and phenols in these liquids were lower than 1.2, 0.9 and 2.2 g/L, respectively. HMF and formic acid were not detected in any prehydrolysate (Table 3).

12

Journal Pre-proof Table 3 Composition (g/L) of pretreatment liquors obtained after pretreatment. Sugars Inhibitors Run Glucose Xylose Galactose Arabinose Acetic acid Furfural

Phenols*

1

7.05

17.85

2.18

10.38

0.88

0.05

1.68

2

7.26

22.06

2.16

11.31

1.21

0.18

1.46

3

9.70

21.66

2.59

11.40

1.23

0.91

1.64

4

5.74

20.88

2.24

11.11

1.18

0.13

2.17

5

9.45

18.57

2.37

10.91

1.03

0.13

1.79

6

6.81

14.78

1.82

9.89

0.55

0.04

1.15

7

9.44

21.68

2.31

11.08

1.17

0.31

2.08

8

7.90

17.25

2.09

9.89

1.11

0.14

1.56

9

4.48

18.20

1.65

10.01

1.05

0.10

1.45

10

8.88

20.09

2.07

10.47

1.20

0.38

1.87

11

9.13

18.15

2.08

10.05

1.09

0.11

1.91

12

7.26

17.03

1.88

9.67

1.06

0.08

1.49

13

6.75

19.87

1.9

9.92

1.14

0.31

1.45

14

9.01

18.35

2.08

10.29

1.06

0.10

1.49

15

11.12

20.67

2.50

11.31

1.00

1.11

2.09

16

8.48

17.79

2.05

9.96

1.05

0.12

1.36

17

8.56

18.16

2.07

10.69

0.93

0.03

1.37

* Expressed as gallic acid equivalents Average of three determinations with standard deviations lower than 5%

3.2. Enzymatic saccharification as a function of pretreatment conditions The effect of acid pretreatment on the enzymatic digestibility of BSG was assessed through enzymatic hydrolysis, which was performed at 5% (w/v) substrate concentration. In general, enzymatic digestibilities higher than 70% were obtained after 24 h, even achieving complete cellulose hydrolysis when the pretreatment was carried out at 130ºC with 1% H2SO4 for 25 min (run 7) (data not shown). Compared with other raw materials, the enzymatic saccharification of BSG after sulfuric acid pretreatment was shorter and the hydrolytic process had finished after only 24 h in all cases. Other researchers have also reported low durations for enzymatic

13

Journal Pre-proof hydrolysis of this feedstock after being pretreated with HCl or NaOH [18] or by steam explosion [17]. The enzymatic hydrolysis yield (YEH) is an interesting parameter for evaluating the performance of both pretreatment and enzymatic hydrolysis. The experimental and predicted YEH values after 24 h of hydrolysis are showed in Table 1, ranging from 54.2% (run 13) up to 82.7% (run 7). The highest value was obtained at the same pretreatment conditions that yielded the maximum value of enzymatic digestibility (run 7, 130ºC, 1% H2SO4, 25 min). 3.3. Optimization of the sulfuric acid pretreatment Three independent variables (temperature, acid concentration and pretreatment time) were varied and their effect on the overall sugar recovery from BSG was studied. The recovery of hemicellulosic sugars in the liquors (HSR) and the enzymatic hydrolysis yield (YEH) were chosen as responses, allowing the determination of the recovery of sugars from hemicellulose and cellulose fractions, respectively. From the experimental results of both dependent variables (Table 1), second-order regression models with interaction between the factors were obtained as Eq. (1) and (2): HSR (%) = 83.66 + 2.91T +1.78 CA - 5.39 T CA + 7.27 C2A R 2 = 0.9541 R 2 adjust = 0.9337

(1)

YEH (%) = 70.78 + 11.34 T -3.22 t + 10.23 CA + 4.18T t + 4.58T CA + 7.16 T 2 - 9.94 t 2 + 8.14 C2A R 2 = 0.9723 R 2 adjust = 0.9170

(2)

where T (ºC) is the pretreatment temperature, t (min) is the reaction time and CA (% w/v) is the sulfuric acid concentration. Eq. 1 can be used to estimate the percentage of sugars released to the pretreatment liquor, while Eq. (2) predicts the enzymatic saccharification yield of pretreated BSG. As can be deduced from the coefficients in both equations, the two factors (temperature and acid concentration) exerted a positive influence on HSR even though these 14

Journal Pre-proof influences were less significant than the interaction between both factors, with a clear negative effect on this response. The influence of the reaction time on HSR was not significant (Eq. 1). As far as YEH is concerned, the effect of the two factors was also positive, whereas the pretreatment time showed a negative effect. Likewise, interactions between the temperature and the other two factors, Tt and TC, also showed a positive effect on YEH (Eq. 2). Applying Eq. (1) to determine HSR obtains the 3D response surface plots versus C and T shown in Fig. 1. As can be seen, the maximum HSR is achieved at the highest level of temperature and lowest level of acid concentration. It can be noted that at higher acid concentration, the temperature had almost no effect on HSR. Regarding YEH, Fig. 2 shows the interactive effect of the pretreatment temperature and time on this response. As shown, the maximum yield is achieved at the highest level of temperature and intermediate time level. Therefore, an increase or decrease of the pretreatment time means a drop of the enzymatic hydrolysis yield.

15

Journal Pre-proof

98.0

93.0

HSR (%)

RAHL

88.0

83.0

78.0 130 125 3.0

120

A: T

2.5 2.0

115 1.5

C: CA

1.0 110

Fig. 1. Recovery of hemicellulosic sugar as a function of pretreatment conditions (acid concentration and temperature for 25 min pretreatment time).

16

Journal Pre-proof

90.0

YYEH EH (%)

79.8

69.5

59.3

49.0

40

130 33

125 25

B: t

120 18

115 10

110

A: T

Fig. 2. Influence of (a) temperature and reaction time at 2% sulfuric acid, and (b) sulfuric acid concentration and temperature (25 min pretreatment time) on the enzymatic hydrolysis yield (YEH). The model predicted the optimal pretreatment conditions for BSG as 130ºC, 1% (w/v) sulfuric acid concentration and 26 min. BSG was pretreated according to these conditions, yielding a cellulose-enriched WIS (more than 28%) with less than 2% hemicellulose and a sugar solution with 43.4 g sugars/L. This means that 94% of sugars from the hemicellulose fraction of raw BSG were recovered in the liquor at these pretreatment conditions. Additionally, the resulting pretreated solid was enzymatically hydrolysed, yielding 82.7 g glucose/100 g glucose in raw BSG. Comparing experimental and predicted values for the two responses differences lower than 5% were found. This validation suggested the high reliability of the optimization experiment in this work.

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Journal Pre-proof In addition to hemicellulosic sugars, 34% of the glucose in raw BSG was released during the pretreatment, mainly the starchy-glucose. Therefore, considering all sugars solubilized during the pretreatment (glucose plus hemicellulosic sugars) and the cellulosic-glucose solubilized by enzymatic hydrolysis, a total of 47.7 g sugars/100 g BSG were obtained at the optimized pretreatment conditions, which corresponds to a sugar recovery of 94%. This overall sugar yield is greater than that obtained with the same material pretreated at 155ºC, and 2% H3PO4 [2]. 3.4. Ethanol production from BSG pretreated at optimal conditions 3.4.1. Cellulosic ethanol from SSF of pretreated BSG The potential of cellulosic ethanol production from BSG was evaluated by SSF at varying solid concentrations (5, 10, 15 and 25% w/v). BSG pretreated under optimal conditions (130ºC, 1% H2SO4, 26 min) was simultaneously saccharified and fermented and the effect of the solid loading on the performance of this biomass conversion process was investigated after 72 h. As can be expected, the increase of dry matter resulted in more concentrated ethanolic solutions, reaching a maximum value of 27 g/L ethanol at 25% solids loading (Fig. 3). From an economic point of view, high dry matter in the SSF process is required to reduce the energy demand of the subsequent distillation step, which is crucial to ensure a cost-competitive biomass conversion process [19]. However, the use of high substrate concentration can cause difficulties in mixing, lack of homogeneity in the bioreactor and, consequently, mass transfer limitations [20]. For this reason, in general, the simultaneous saccharification and fermentation of very concentrated slurries imply lower ethanol yields. The drop in the ethanol yields because of increasing the substrate concentration in the SSF process has been widely reported [20,21]. In this work, the ethanol yield did not decrease noticeably with the increase of solid loading in the SSF process (72% at 5% solids vs 68% at 25% 18

Journal Pre-proof solids) (Fig. 3). This fact can be considered very advantageous because, in addition to producing very concentrated ethanolic solutions, high yields are also required to increase the viability of the process. Overall, these results improve those obtained during the SSF of phosphoric acid pretreated BSG using lower solid loadings [2].

Fig. 3. Ethanol concentration (g/L) and ethanol yield (%) obtained by SSF of BSG pretreated at different solid loadings under optimal conditions. Ethanol yield is expressed as a percentage of the theoretical ethanol yield (0.51 g ethanol/g glucose).

In addition, solids resulting from the SSF process were characterized in order to determine their residual cellulose content, which ranged between 10% and 13% for SSF tests performed at 5% and 25% solids, respectively (data not shown). These results are in agreement with the slight drop in ethanol yields by increasing the substrate concentration (Fig. 3) due to part of the cellulose not being hydrolysed by enzymes. The use of fed-batch strategies in the SSF process to achieve the complete conversion of the cellulose fraction deserves further attention.

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Journal Pre-proof 3.4.2. Fermentation of the acid prehydrolysate with S. stipitis and E. coli The bioconversion of the sugars released during the pretreatment of biomass can be an attractive option to the valorization of these sugars, although the presence of acetic acid or some sugar degradation products can limit their use [22]. The sulfuric acid pretreatment of BSG at optimal conditions produced a prehydrolysate containing (g/L): glucose, 9.4; xylose, 21.7; galactose, 2.3; and arabinose, 10.0. All these sugars were found in the monomeric form. Mannose was not detected in the liquor. In addition, phenolic compounds accounted for 1.2 g/L, acetic acid 0.3 g/L and furfural 2.1 g/L, but formic acid and HMF were not detected in the liquor. The fermentability of the acid prehydrolysate from BSG was tested with both S. stipitis and E. coli without previous detoxification. The high sensitivity of S. stipitis to acetic acid, released from the hydrolysis of the hemicelluloses, which is typical in lignocellulosic hydrolysates, has been previously reported [23]. In addition, phenolic compounds are the main inhibitors in the fermentation of xylose to ethanol [24] and, together with acetic acid, inhibit the microbial metabolism of S. stipitis and reduce the ethanol production [25]. In this work, low concentrations of these inhibitor compounds in the BSG prehydrolysate allowed S. stipitis to metabolize all sugars available in the fermentation medium after 41 h (Fig. 4a). Nevertheless, yeasts such as S. stipitis, capable of naturally fermenting both hexoses and pentoses, prefer glucose over xylose and other sugars and, therefore, the fermentation of xylose starts when the concentration of glucose in the medium is below a critical level [26]. As can be observed in Fig. 4a, the yeast started to metabolize glucose and then the remaining sugars. Arabinose consumption started once glucose was exhausted, although this did not result in an increase of ethanol concentration in the medium. In this context, Nigam et al. [27]

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Journal Pre-proof showed that S. stipitis can assimilate arabinose for cell growth but not for ethanol production Fig. 4b shows the variations in sugar and ethanol concentrations during the fermentation of the BSG prehydrolysate with E. coli. Compared with S. stipitis, E. coli has been reported to be more resistant to the presence of inhibitors, mainly acetic acid. As can be observed, E. coli assimilated glucose faster than S. stipitis (8 h vs 30 h) but the remaining sugars had been completely metabolized by both microorganisms after 41 h (Fig. 4a and 4b). However, unlike with S. stipitis, E. coli began to assimilate all the sugars simultaneously from the beginning. This behaviour of E. coli SL100 is in agreement with the results obtained using hydrolysates from other lignocellulosic materials such as sweet sorghum, sugarcane [14] and olive tree biomass [28].

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a)

b) Fig. 4. Time course of sugar consumption, ethanol production and cell growth of (a) S. stipitis and (b) E. coli in the fermentation of the acid prehydrolysate from BSG without detoxification.

Comparing the fermentation of the liquor with both microorganisms, E. coli reached greater ethanol production, 17 g/L at 41 h, corresponding to 76% of the theoretical 22

Journal Pre-proof yield, whereas S. stipitis reached a maximum ethanol concentration of 11.4 g/L (53% ethanol yield) at the same fermentation time. In addition, the ethanol productivity achieved by E. coli at 41 h was also greater, 0.41 g/L/h versus 0.28 g/L/h. Nevertheless, the results obtained in this work with S. stipitis compare favourably with those reported in the fermentation of hemicellulosic hydrolysates from spent coffee grounds [24], palm press fibre [29] or sugarcane bagasse [30] with the same yeast. Concerning the fermentation of the BSG prehydrolysate with E. coli, similar ethanol production has been reported with the same microorganism in the fermentation of hydrolysates from BSG pretreated by phosphoric acid [12] or sulphuric acid pretreated rapeseed straw, although in this case a previous detoxification step was required [31]. 3.5. Overall mass balance A mass balance is crucial for assessing the biomass conversion process for advanced bioethanol production. Fig. 5 shows the material balance of the complete process for BSG, considering the optimized acid pretreatment, SSF of the pretreated solid at 25% solids and the co-fermentation of the prehydrolysate with E. coli SL100. Enzymatic solution 165.5 g Inoculum (4%) 6.70 mL

H2S04 ( 96 %) 1.04 g H20 799.43 g 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: 12.5 g Protein : 21.2 g

SSF

SULFURIC ACID PRETREATMENT 12.5% solids, 1% H2SO4 130ºC, 26 min

LIQUID Glucose: 7.5 g Xylose: 17.3 g Galactose: 1.4 g Arabinose: 7.6 g Phenols: 1.6 g Formic acid: 0.1 g Acetic acid: 0.9 g Furfural: 0.2 g

SOLID 43.1 g Glucose: 13.8 g Xylose: 0.1 g Lignin: 15.5 g Protein : 7.8 g

25% solids, 40 ºC, 72 h, 150 rpm Cellic CTec 3 15 FPU/g β-glucosidase 15 IU/g S. cerevisiae 4 % (v/v)

SOLID: 19.6 g

LIQUID Ethanol: 4.6 g

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

LIQUID Ethanol: 13.5 g

Fig 5. Mass balance of ethanol production from brewer’s spent grain pretreated by sulfuric acid at optimal conditions (130ºC, 1% H2SO4, 26 min).

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Journal Pre-proof Dilute-acid pretreatment allowed the recovery of 95% of the glucose in raw BSG, mainly in the pretreated solids although 34% was solubilized and recovered in the pretreatment liquor. This high recovery of glucose in the prehydrolysate can be justified by the fact that 26% of the total glucose in raw BSG is starchy-glucose, which is easy to hydrolyse. In addition, the pretreatment of BSG solubilized 93% of the hemicellulosic sugar content in raw BSG. This means that sulfuric acid pretreatment at optimized conditions achieved a relevant fractionation of this biomass with a carbohydrate recovery as high as 94%. Concerning lignin content, a noticeable increase after acid pretreatment can be observed. As previously mentioned, this fact can be related to the formation of insoluble complexes known as pseudolignin [8]. After pretreatment, the solid fraction was simultaneously saccharified and fermented at high dry matter (25% solids) yielding 4.6 g ethanol/100 g raw BSG (67% of the maximum potential yield). Regarding the co-fermentation of the sugars solubilized during the pretreatment, E. coli assimilated all sugars in the prehydrolysate with an ethanol production of 13.5 g ethanol/100 g raw BSG, which corresponds to 78% of the theoretical ethanol yield. All in all, 70% of the total sugars of BSG can be converted into ethanol following the proposed process configuration, yielding 18 g ethanol/100 g BSG. Wilkinson et al. [32] reported an ethanol yield of 9.4 kg/100 g dry BSG using fungal consolidated bioprocessing.

Conclusions The acid pretreatment at optimized conditions (130ºC, 1% sulfuric acid, 26 min) efficiently fractionated brewer’s spent grain, with total recovery of starch and 94% of hemicellulosic sugars in the pretreatment liquor as well as 90% cellulose in the pretreated solid. The behavior of two microorganisms able to ferment both pentoses and

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Journal Pre-proof hexoses have been compared in this work. Overall, 100 kg of dried BSG yielded 18.1 kg ethanol (4.6 kg from cellulose by SSF at 25% solids and 13.5 kg by co-fermentation of hemicellulosic sugars and glucose with E. coli without previous detoxification). The results reported here can be used as a basis for the development of a biorefinery scheme based on BSG, including also the valorization of other fractions, such as protein or lignin, to make an integral utilization of this feedstock, which will be the focus of future works. Aknowledgments Technical support from the Agrifood Campus of International Excellence (ceiA3) is gratefully acknowledged. References [1]

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[2]

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Duque, A., Manzanares, P., Ballesteros M., 2017. Extrusion as a pretreatment for lignocellulosic biomass: Fundamentals and applications. Renew. Energy 114, 1427-1441.

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Singh, R., Shukla, A., Tiwari, S., Srivastava, M., 2014. A review on delignification of lignocellulosic biomass for enhancement of ethanol production potential. Renew. Sust. Energ. Rev. 32, 713-728.

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Socaci, S.A., Farcas, A.C., Diaconeasa, Z.M., Vodnar, D.C., Rusu, B., Tofan, M., 2018. Influence of the extraction solvent on phenolic content,

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Journal Pre-proof antioxidant, antimicrobial and antimutagenic activities of brewers’ spent grain. J. Cereal Sci. 80, 180–187. [6]

Carciochi, R.A., Sologubik, C.A., Fernández, M.B., Manrique, G.D., D’Alessandro, L.G., 2018. Extraction of antioxidant phenolic compounds from brewer’s spent grain: optimization and kinetics modeling. Antioxidants 7, 45.

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Outeiriño, D., Costa-Trigo, I., Paz, A., Deive, F.J., Rodríguez, A., Domínguez, J.M., 2019. Biorefining brewery spent grain polysaccharides through biotuning of ionic liquids. Carbohydr. Polym. 203, 265-274.

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Rommi, K., Niemi, P., Kemppainen, K., Kruus, K., 2018. Impact of thermochemical pre-treatment and carbohydrate and protein hydrolyzing enzyme treatment on fractionation of protein and lignin from brewer’s spent grain. J. Cereal Sci. 79, 168-173.

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Qin, F., Johansen, A.Z., Mussatto, S.I., 2018. Evaluation of different pretreatment strategies for protein extraction from brewer’s spent grains. Ind. Crop. Prod. 125, 443–453.

[10] Dávila, J.A, Rosenberg, M., Cardona, C.A., 2016. A biorefinery approach for the production of xylitol, ethanol and polyhydroxybutyrate from brewer’s spent grain. AIMS Agriculture and Food 1(1), 52-66. [11] González-García, S., Morales, P.C., Gullón, B., 2018. Estimating the environmental impacts of a brewery waste–based biorefinery: Bio-ethanol and xylooligosaccharides joint production case study. Ind. Crop. Prod. 123, 331340. [12] Rojas-Chamorro, J.A., Cara, C., Romero, I., Ruiz, E., Romero-García, J.M., Mussatto, S.I., Castro, E., 2018. Ethanol production from brewers’ spent grain pretreated by dilute phosphoric acid. Energy Fuels 32, 5226−5233.

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Journal Pre-proof [13] Sluiter, A., Hames, B., Ruiz, R., Scralata, C., Sluiter, J., Templeton, D., Crocker, D., 2012. Determination of structural carbohydrates and lignin in biomass. Golden, Colorado: National Renewable Energy Laboratory. August, Report No. TP-51042618. [14] Martínez, A., Grabar, T.B., Shanmugam, K.T., Yomano, L.P., York, S.W., Ingram, L.O., 2007. Low salt medium for lactate and ethanol production by recombinant Escherichia coli B. Biotechnol. Lett. 29(3), 397–404. [15] Geddes, C.C., Mullinnix, M.T., Nieves, I.U., Hoffman, R.W., Sagues, W.J., York, S.W., Shanmugam, K.T., Erickson, J.E., Vermerris, W.E., Ingram, L.O., 2013. Seed train development for the fermentation of bagasse from sweet sorghum and sugarcane using a simplified fermentation process. Bioresour. Technol. 128, 716-724. [16] Kemppainen, K., Rommi, K., Holopainen, U., Kruus, K., 2016. Steam explosion of Brewer’s spent grain improves enzymatic digestibility of carbohydrates and affects solubility and stability of proteins. Appl. Biochem. Biotechnol. 180, 94-108. [17] Yang, B., Wyman, C.E., 2008. Pretreatment: the key to unlocking low-cost cellulosic ethanol. Biofuels, Bioprod. Bioref. 2, 26–40. [18] Wilkinson, S., Smart, K.A., Cook, D.J., 2014. A comparison of dilute acid- and alkali-catalyzed hydrothermal pretreatments for bioethanol production from brewers’ spent grains. J. Am. Soc. Brew. Chem. 72(2), 143-153. [19] Liu, Z-H., Qin, L., Zhu, J-Q., Li, B-Z., Yuan, Y-J., 2014. Simultaneous saccharification and fermentation of steam-exploded corn stover at high glucan loading and high temperature. Biotechnol. Biofuels 7, 167.

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Journal Pre-proof [20] Qiu, J., Tian, D., Shen, F., Hu, J., Zeng, Y., Yang, G., Zhang, Y., Deng, S., Zhang, J., 2018. Bioethanol production from wheat straw by phosphoric acid plus hydrogenperoxide (PHP) pretreatment via simultaneous saccharification and fermentation (SSF) at high solid loadings. Bioresour. Technol. 268, 355362. [21] Hoyer, K., Galbe, M., Zacchi, G., 2013. The effect of prehydrolysis and improved mixing on high-solids batch simultaneous saccharification and fermentation of spruce to ethanol. Process Biochem. 48(2), 289-293. [22] Chaabane, F.B., Marchal, R., 2013. Upgrading the hemicellulosic fraction of biomass into biofuel. Oil Gas Sci. Technol. – Rev. IFP Energies nouvelles 68(4), 663-680. [23] van Zyl, C., Prior, B.A., du Preez, J.C., 1991. Acetic acid inhibition of Dxylose fermentation by Pichia stipitis. Enzyme Microb. Technol., 13, 82-86. [24] Mussatto, S.I., Machado, E.M.S., Carneiro, L.M., Teixeira, J.A., 2012. Sugars metabolism and ethanol production by different yeast strains from coffee industry wastes hydrolysates. Appl. Energy 92, 763–768. [25] Dussán, K.J., Silva, D.D.V., Perez, V.H., da Silva, S.S., 2016. Evaluation of oxygen availability on ethanol production from sugarcane bagasse hydrolysate in a batch bioreactor using two strains of xylose-fermenting yeast. Renew. Energy 87, 703-710. [26] Wang, L., York, S.W., Ingram, L.O., Shanmugam, K.T., 2019. Simultaneous fermentation of biomass-derived sugars to ethanol by a coculture of an engineered Escherichia coli and Saccharomyces cerevisiae. Bioresour. Technol. 273, 269-276.

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Journal Pre-proof [27] Nigam, J.N., 2001. Ethanol production from wheat straw hemicellulose hydrolysate by Pichia stipitis. J. Biotechnol. 87, 17–27. [28] Martínez-Patiño, J.C., Romero, I., Ruiz, E., Cara, C., Romero-García, J.M., Castro, E., 2017. Design and optimization of sulfuric acid pretreatment of extracted olive tree biomass using response surface methodology. BioResources 12(1), 1779-1797. [29] Brito, P.L., Ferreira, C.M.A., Silva, A.F.F., Pantoja, L.A., Nelson, D.L., dos Santos, A.S., 2018. Hydrolysis, detoxification and alcoholic fermentation of hemicellulose fraction from palm press fiber. Waste Biomass Valor. 9, 957968. [30] Terán-Hilares, R., Reséndiz, A.L., Martínez, R.T., Silva, S.S., Santos, J.C., 2016. Successive pretreatment and enzymatic saccharification of sugarcane bagasse in a packed bed flow-through column reactor aiming to support biorefineries. Bioresour. Technol. 203, 42–49. [31] López-Linares, J.C., Romero, I., Cara, C., Castro, E., 2016. Bioconversion of rapeseed straw: enzymatic hydrolysis of whole slurry and cofermentation by an ethanologenic Escherichia coli. Energy Fuels 30, 9532–9539. [32] Wilkinson, S., Smart, K. A., James, S., Cook, D. J., 2017. Bioethanol Production from brewers spent grains using a fungal consolidated bioprocessing (CBP) approach. Bioenergy Res., 10, 146-157.

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Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Journal Pre-proof Highlights  H2SO4 pretreatment of BSG for bioethanol production is optimized for the first time  94% of sugars in raw BSG were recovered at optimized pretreatment conditions  E. coli co-fermented sugars from hemicellulose and starch with 76% ethanol yield  The proposed bioconversion process yielded 230 L bioethanol per ton of dry BSG