Technoeconomic Study of Biobutanol AB Production. 1. Biomass

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Technoeconomic study of AB biobutanol production: Part 1: Biomass Pretreatment and Hydrolysis. Santiago Malmierca, Rebeca Díez-Antolínez, Ana Isabel Paniagua, and Mariano Martín Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.6b02943 • Publication Date (Web): 19 Jan 2017 Downloaded from http://pubs.acs.org on January 23, 2017

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Industrial & Engineering Chemistry Research

Technoeconomic study of AB biobutanol production. Part 1: Biomass Pretreatment and Hydrolysis Santiago Malmiercaa,b, Rebeca Díez-Antolínezb, Ana Isabel Paniaguab, Mariano Martína1 aDepartment

of Chemical Engineering. University of Salamanca. Plz. Caídos 1.5, 37008

Spain bCenter

of Biofuels and Bioproducts. Instituto Tecnológico Agrario de Castilla y León (ITACyL), Villarejo de Órbigo, León, Spain.

Abstract This paper presents a comprehensive study of different chemical pretreatments and enzymatic hydrolysis processes for the production of sugars from lignocellulosic biomass: switchgrass (Panicum virgatum L.) and corn stover. Experimental results were used to scale up the production process for biobutanol. With regards to pretreatments, acid, alkali, surfactants and organic solvents were tested to break down biomass structure. Acid pretreatment turned out to be the most efficient in terms of total released sugar. Besides, the optimal operating conditions for the enzymatic hydrolysis were determined experimentally. Comparing the degradation of biomass types, switchgrass showed high degradation when sulfuric acid treatment is used. However, corn stover showed the highest sugar production at complete degradation. Therefore, it will be the raw material and determines the operating conditions of choice for process scale up. Key Words: Biobutanol, Pretreatment, Corn stover, Switchgrass, Ultrasounds, Hydrolysis.

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Corresponding autor [email protected]

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1. Introduction Biobutanol is considered an advanced fuel because of its properties such as higher energy density and easy transportation, due to the lower vapor pressure, compared to ethanol. It is also miscible with gasoline and diesel and secures reduced GHG emissions.1 A number of petrochemical based processes already exist to produce butanol. Typically, it has been obtained from propylene, through propylene hydroformylation, oxo synthesis, Reppe synthesis or from crotonaldehyde hydrogenation. 2. Alternatively, butanol can be produced via biochemical processes, including fermentation of biomass wastes. Over the last decade, lignocellulosic biomass has been evaluated as raw material for the production of sugar monomers in the so-called second generation of biofuels production. The availability in different forms is proven, including energy crops, miscanthus, switchgrass or harvest residues.3 Among harvest crops, it is important to highlight corn, since it is the largest culture in the world, with an estimated production of 990.69 million tons in 2014/2015 according to the United States Department of Energy.3 United States, China, Brazil and European Union are the main producers. Therefore, the waste from the corn industry, the stover, is one of the most abundant raw materials. Since the composition of its structure has about 67% of fiber (11-15% of lignin, 18-22% of hemicellulose and 32-36% of cellulose), it represents an interesting source of fermentable sugars for biofuels production. As a result, it has attracted the attention of researches worldwide.4 Furthermore, energy crops such as switchgrass have also been evaluated as raw material for second generation biofuels and will also be considered in this work. There are many pretreatments that break down lignocellulose such as the use of chemicals, i.e. dilute acid, steam explosion, AFEX or the use of enzymes5,6 and the selection of the method depends on the biomass type. The use of sugars to produce butanol presents a number of challenges to be overcome at industrial scale. For instance, the fermentation yield is low. Together with butanol, ethanol and acetone are typically obtained. Furthermore, the butanol is toxic for the strain and the separation of the products is complex7 resulting in the fact that traditional fermentation is not cost effective. Advances in the processing technologies and genetically modified bacteria are needed.8 For instance, Clostridium bacteria are widely used to transform sugars into organic solvents alone, acetone and butanol.9 However, butanol inhibits the process, reducing the conversion of the sugars. Thus, the straight forward alternative is the use of methods for in-situ butanol recovery such as gas stripping,10 extraction,11 or pervaporation.12 2 ACS Paragon Plus Environment

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In this paper, a number of pretreatments combined with an enzymatic hydrolysis process are evaluated to determine the optimal operating conditions to produce sugars out of lignocellulosic biomass, corn stover and switchgrass. In a second paper, a flowsheet is proposed for the scaled-up process of the production of acetone and butanol where a complete technoeconomic evaluation is performed.13 The first step in the biochemical path to transform biomass into butanol consists of setting up and disrupting the raw material. Thus, the biomass is processed through a number of stages to break the bonds of the lignocellulosic structure. An ideal process would break down hemicelluloses and cellulose only, maintaining the lignin intact to avoid the formation of phenolic compounds, which are inhibitors for the subsequent fermentation. However, all pretreatments have an impact on the whole biomass structure, including lignin. The chosen process attempts to release as much hemicelluloses and cellulose as possible, trying not to affect the lignin. Thus, it is expected that the pretreatment breaks down the long chains and removes the amorphous part of the biomass structure so that it is prepared for the enzymatic hydrolysis, making cellulose – hemicelluloses – lignin structure much more accessible to the enzymes. The aim of the enzymatic hydrolysis is to release the glucose monomers resulting from cellulose. Once pretreatment has been carried out, the biomass has suffered a change in its cellulose – hemicellulose – lignin structure, and it can be digested using enzymes. Xilooligomers (XOS) and phenolic compounds have been found to be very important factors in the inhibition of cellulases and deactivation of βglucosidase,14,15 which are the main enzymes for biomass digestion. Hence, after the pretreatments discussed, the biomass structure is more accessible for enzymes involved in the process. The paper is organized as follows: Section 2 presents the description of the equipment and methods used to pretreat biomass and hydrolyze it. Section 3 shows the results comparing the various pretreatments and hydrolytic conditions. Finally, section 4 draws some conclusions.

2. Materials and methods 2.1. Materials 2.1.1. Substrate Switchgrass and corn stover biomass were recollected from test plot fields of Instituto Tecnológico Agrario de Castilla y León, ITACyL, located in Benavente (Zamora, Spain). 3 ACS Paragon Plus Environment


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Before pretreatment, lignocellulosic material is dried and reduced in size to improve the yield of the pretreatment. Biomass is dried at 45ºC for 48h in a hot air oven, and later milled to obtain small pieces, below 1 mm.16-18 An SM100 Comfort rotary mill (Retsch GmbH, Haan, Germany) is used. The particle size is doubled checked using a 1.0 mm screen.16

2.1.2. Chemical reagents The chemicals employed were: sulfuric acid (98% v/v), potassium hydroxide (85% w/w), ammonia hydroxide (25% v/v) and absolute ethanol (99.9% v/v), procured from Sigma Aldrich (Steinheim, Germany) and the surfactants (CTAB, Tween 80 and Tween 20), supplied by Merck (Darmstadt, Germany).

2.1.3. Enzymes The medium, where enzymes release sugars, is a buffer which consists of 50 mM of citrate with a pH of 4.8 and 0.04% (w/v) sodium azide. In addition, a cocktail of enzymes consists of NS50010 (βglucosidase) and NS50013 (cellulose complex) with an activity of 250 CBU/g and 70 FPU/g respectively, kindly provided by Novozymes (Bagsværd, Denmark). 2.2. Experimental procedure 2.2.1. Pretreatment 2.2.1.1. Dilute acid pretreatment The aim of this pretreatment is to disrupt the hemicellulose structure. Different concentrations of dilute sulfuric acid (AS) were tested to treat milled biomass with a solid loading of 10% (w/w) for switchgrass and 8.33% (w/w) for corn stover. The reason for reducing the solid loading for corn stover samples is its capacity to soak up a larger ammount of water. Treatment is performed in glass bottles of 500 mL that were introduced in an autoclave, MLS-3780 (Sanyo), heated up to 121ºC (1.034105 Pa) and maintained under these conditions for 90 min. After cooling down the mixture at room temperature, the samples were vacuum filtered, to separate the solid fraction from the liquid fraction. Next, the solid fraction is washed to recover the released sugars and to increase biomass pH in order to achieve the conditions required for enzymatic hydrolysis. Fermentable sugar concentrations of hydrosylate and wash water were measured by High Performance Liquid Chromatography (HPLC). 4 ACS Paragon Plus Environment

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2.2.1.2. Alkali and acid pretreatment Alkalis do not only make the structure accessible for further treatment, but they also act on the lignin. Hence, the process is expected to release phenolic compounds, although no sugars were found in the analysis. Thus, acid treatment is applied after the alkali based one to recover sugars free of furfural and HMF in solution. To evaluate the yield and competitiveness of the alkali based pretreatments, different alkalis and operating conditions are tested. We consider two alternative procedures by using different alkalies followed by a second stage that uses the same acid pretreatment described above. We test the alkali pretreatment on swichgrass aiming at larger yield to sugars than the one obtained for corn using acid pretreamtent alone as reference. The first procedure uses sole alkali treatments with KOH at 0.5, 1.0, 1.5 and 2.0 % (w/w) concentrations. Next, alkali treatments combined with AS at 0.5, 1.0, 1.5 and 2.0 % (w/w) are tested to assess their effects over sugar release yield. The pretreatment is performed in an autoclave at 121ºC for 90 min with a solid load of 10% (w/w) switchgrass. After that, biomass pH is neutralized by washing and dried at room temperature before pretreat it with AS, as it is described in section 2.1.3.1. A second alkali treatment is also considered. It consists of using a solution of NH4OH (15% v/v) to process a mixture of 14.28% w/w of solid loading. The solids are mixed with the medium in Erlenmeyer flasks in an Orbital Shaker (Ecotron, Infors, Bottmingen, Switzerland) at 60ºC for 24 h with a stirring of 100 rpm. After that, the sample is subjected to dilute acid process (Section 2.2.1.1). Fermentable sugar concentrations of pretreatment and wash water are measured by High Performance Liquid Chromatography (HPLC).

2.2.1.3. Ultrasound treatment The dilute acid pretreatment is also combined with sonication processes, trying to improve the yield to sugars. An ultrasound bath UR2 at 45 W with a frequency of 45 kHz (Retsch GmbH, Haan, Germany) is used. Biomass is previously treated with a solution of 1.5% AS at the same conditions described before in the diluted acid pretreatment. After that, different sonication times from 5 to 60 min are tested. The bath is 5 ACS Paragon Plus Environment


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filled with water as medium for ultrasound propagation. Fermentable sugar concentrations of hydrolysate and wash water are measured by High Performance Liquid Chromatography (HPLC).

2.2.1.4. Surfactant pretreatment Surfactants are supposed to enhance the digestibility of biomass because they reduce lignin and cellulose crystallinity.19 CTAB, Tween 80 and Tween 20 are used in the experiments based on previous work from the literature.18 A 3% (v/v) aqueous solution of surfactant (Tween 80 and Tween 20) is mixed with 10 % (w/w) of switchgrass in glass bottles of 500 mL, at 37.4 ºC for 60 min and 80 rpm in an Orbital Shaker (Ecotron, Infors). Then, the glass bottles are irradiated with ultrasonic waves for 60 s and finally, they are autoclaved for 60 min at 121 ºC. Solid recovered is washed and dried at atmosphere conditions. The effects of surfactant pretreatment alone and combined with AS pretreatment (Section 2.2.1.1) are assessed. The amount of sugars released is determined by HPLC. Fermentable sugar concentrations of hydrosylate and wash water are measured by High Performance Liquid Chromatography (HPLC). Based on the experimental results, see Table 4, Tween 80 is selected as surfactant of choice for further work. Biomass samples with a solid load of 13.83 % (w/w) are treated with a solution of 15% (v/v) NH4OH. Next, Tween 80 is added up to reach a concentration of 3% (w/w). After proper mixing in glass bottles, the samples are autoclaved for 90 min at 121 ºC. Once the solids are recovered, washed and dried, acid pretreatment is carried out.

2.2.1.5. Organic solvent treatment Similar to the alkali treatment, organic solvents such as ethanol favor biomass delignification. The effect of ethanol is also evaluated. The method consisted of mixing a solution of 35% v/v of ethanol, with a ratio of 14.28 ml of organic solution per gram of biomass in glass bottles of 500 mL. The sample is autoclaved for 10 min at 135 ºC. After that, the mixture is irradiated by ultrasounds for 15 min. Then, the mix is subjected to acid treatment. Fermentable sugar concentration of the pretreatment is measured by High Performance Liquid Chromatography (HPLC).


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2.2.2. Enzymatic Hydrolysis The pretreated method which provided a higher sugar release yield (mainly xylose) is selected as substrate for the enzymatic hydrolysis stage. A dosage of 440 and 400 µl of NS50010 (β-glucosidase) and NS50013 (cellulose complex) per gram of biomass is tested at a buffer hydrolysis broth with 2.5 % w/v of solid loading. The stirring speed, temperature and time are set to 150 rpm, 50ºC and 72h, respectively. The experimental set up is carried out in a thermomixer (Thermomixer Comfort, Eppendorf). The supernatant after centrifugation at 4000 rpm, are analyzed to quantify fermentable sugars recovered, mainly glucose.

2.3. Analytical methods Sugars concentration (glucose, xylose and arabinose) are determined using HPLC. This analysis is performed based on the NREL Analytical Procedures (NREL/TP-510-42623).20 A quantitative analysis is performed by Agilent 1200 HPLC (Agilent Technologies) with a 300 x 7.8 mm i.d. cation exchange column, Aminex HPX-87H (Biorad, Hercules, CA, USA), and a Refractive Index Detector (RID, Agilent Technologies, Santa Clara, CA, USA). The mobile phase is 5 mM H2SO4 at a flow rate of 0.6 mL/min and 60 °C. The injection volume is 20 µL. All samples are filtered through 0.22 µm filters prior to analysis.

3. Experimental results and discussion All experiments in this paper are carried out by duplicate. The quantity of recovered sugars showed in the following tables is the average of both runs and it represents the sum of sugar obtained from both, hydrolysate broth and wash water stream.

3.1. Pretreatment In Table 1, the results of the dilute acid pretreatment for both types of biomass, switchgrass and corn stover, are shown. For switchgrass, the production of C5 sugars increased with the concentration of acid until a concentration of 1%. The use of this pretreatment on the corn stover reaches its maximum yield for an acid concentration around 2%. However, as in the previous case, only marginal increment is found over 1%. Actually this maximum yield is for the total amount of sugars, since each analyzed sugar type, glucose, xylose and arabinose, has its maximum under different conditions. For instance, in the case of 7 ACS Paragon Plus Environment


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using corn stover, the maximum glucose obtained is for 2%, slightly superior that under other operating conditions, but xylose and arabinose production is larger at 1% v/v. Finally, note the improvement in the yield to sugars for both biomass types between 0.5 and 1.0% of acid concentration. We see that the yield for corn stover is larger than that obtained from switchgrass. Using this as a reference, we test alkali pretreatments on switchgrass to see whether it can become competitive with corn stover in terms of yield.

Table 1 Pretreatment of switchgrass and corn stover with dilute sulfuric acid in autoclave mg glucose/ mg xylose/ mg arabinose/ Treatment g biomass g biomass g biomass Switchgrass 1. AS (0.5% v/v) + ATCLV (90 min) 2. AS (1.0% v/v) + ATCLV (90 min) 3. AS (1.5% v/v) + ATCLV (90 min) 4. AS (2.0% v/v) + ATCLV (90 min) Corn stover 1. AS (0.5% v/v) + ATCLV (90 min) 2. AS (1.0% v/v) + ATCLV (90 min) 3. AS (1.5% v/v) + ATCLV (90 min) 4. AS (2.0% v/v) + ATCLV (90 min)

mg TS/ g biomass

26.77 35.05 35.60 35.45

168.21 196.10 196.02 189.54

25.30 31.56 31.87 31.17

220.28 262.71 263.49 256.16

17.43 26.42 28.67 30.55

182.48 211.57 210.97 210.12

42.71 46.17 45.75 45.89

242.61 284.16 285.39 286.56

We use of alkali pretreatments alone, with either KOH or NH4OH, on switchgrass samples. The analysis of the products show no release of hydrolysable sugars. Thus, alkali pretreatment was combined with dilute acid pretreatment in sequence. Table 2A shows the released sugar from switchgrass using KOH as alkali pretreatment combined with diluted acid pretreatment. For fixed concentration of KOH and operating conditions of the autoclave, the best yield to sugars is obtained using 1% of sulfuric acid. A similar analysis using NH4OH is presented in Table 2B. In this case, the best operating conditions require the use of a concentration of sulfuric acid of 1.5% v/v. Table 2A Pretreatment of switchgrass with different concentrations of KOH and dilute sulfuric acid in autoclave mg glucose/ mg xylose/ mg arabinose/ mg TS/ Treatment g biomass g biomass g biomass g biomass 1. KOH (0,5% v/v) + AS (0,5% v/v) 2. KOH (0,5% v/v) + AS (1,0% v/v) 3. KOH (0,5% v/v) + AS (1,5% v/v) 4. KOH (0,5% v/v) + AS (2,0% v/v)

8.60 16.40 13.00 15.90

121.90 132.80 89.40 97.70

24.60 27.80 19.70 22.10

155.00 176.90 122.10 135.70

Table 2B Pretreatment of switchgrass with NH4OH and dilute sulfuric acid in autoclave


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Treatment 1. NH4OH (15% v/v) + AS (0,5% v/v) + ATCLV (90 min) 2. NH4OH (15% v/v) + AS (1,0% v/v) + ATCLV (90 min) 3. NH4OH (15% v/v) + AS (1,5% v/v) + ATCLV (90 min)

mg glucose/ g biomass

mg xylose/ g biomass

mg arabinose/ g biomass

mg TS/ g biomass

9.53

116.79

29.42

155.74

18.54

156.38

32.06

206.98

22.50

175.19

34.02

231.71

If we compare the results of Table 1 with those in Tables 2A and 2B, the use of a sequence of alkali pretreatment followed by an acid treatment does not achieve any improvement to the total yield of sugars. Thus, the alkali treatment does not favor the pretreatment process. The comparison of the use of both alkalis shows that NH4OH yields better results than KOH. Note that the concentration of alkali is not the same for both. Finally, we cannot achieve competitive yields when using switchgrass compared to corn stover as raw material either. Table 3 shows the results for acid pretreatment coupled with sonication over switchgrass and corn stover samples. We fixed the dilute acid concentration to be 1.5% v/v since it showed the best results in terms of total sugars for switchgrass (Table 1). The optimal conditions for switchgrass are found after 15 min of sonication. It can be seen that sonication increases the production of sugars by 7%. In particular, the amount of xylose and arabinose is significantly larger compared to the results when dilute sulfuric acid is used alone. Therefore, we can conclude that sonication improves the pretreatment stage. Moreover, for corn stover we also fix the dilute acid concentration to be 1.5% v/v but ATCLV and US time varied. The highest yield to sugars is obtained by biomass autoclaving for 60 min and followed by 45 min of sonication. In the same way, the concentration of xylose and arabinose increases considerably in the juice. Direct comparison with the acid pretreatment and versus corn is not strictly possible since the autoclaving time is not maintained constant. But we see how autoclaving time has more influence than US time over biomass breaking down and only by using a larger autoclaving time the yield from switchgrass becomes as high as that from corn stover.



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Table 3 Pretreatment with dilute sulfuric acid in autoclave and sonication mg glucose/ mg xylose/ Treatment g biomass g biomass


mg TS/ g biomass

Switchgrass 1. AS (1,5% v/v) + ATCLV (90 min) + US (60min) 2. AS (1,5% v/v) + ATCLV (90 min) + US (45min) 3. AS (1,5% v/v) + ATCLV (90 min) + US (30min) 4. AS (1,5% v/v) + ATCLV (90 min) + US (15min) 5. AS (1,5% v/v) + ATCLV (90 min) + US (10min) 6. AS (1,5% v/v) + ATCLV (90 min) + US (5min) Corn stover 1. AS (1,5% v/v) + ATCLV (60 min) + US (60min) 2. AS (1,5% v/v) + ATCLV (60 min) + US (45min) 3. AS (1,5% v/v) + ATCLV (45 min) + US (45min) 4. AS (1,5% v/v) + ATCLV (45 min) + US (30min) 5. AS (1,5% v/v) + ATCLV (30 min) + US (15min) 6. AS (1,5% v/v) + ATCLV (30 min) + US (5min)

33.77 33.49 31.88 30.92 30.46 30.20

208.44 211.83 208.18 211.19 207.53 206.37

51.51 51.43 50.17 56.97 56.11 56.01

293.72 296.75 290.23 299.08 294.10 292.57

25.28 27.30 22.38 24.21 22.96 22.39

189.85 198.84 191.58 193.09 188.66 185.11

56.89 60.56 58.21 58.93 55.98 53.32

272.02 286.70 272.17 276.24 267.60 260.82

In Table 4 we compare the yield to sugars when using surfactants as pretreatment. It turned out that the use of surfactants does not increase the yield to sugars. It is the increase in the concentration of sulfuric acid which improves the yields. A part from evaluating the effect of an initial pretreatment based on the use of surfactants, we also added an initial stage using an alkali, NH4OH, runs 4-6 in Table 4. Although the yield is good, it is lower than the one obtained when no alkali step is used. Again, the increase in the concentration of sulfuric acid is the yield booster.

Table 4 Pretreatment of switchgrass soaked, sonication and surfactant and dilute sulfuric acid in autoclave mg glucose/ mg xylose/ mg arabinose/ Treatment g biomass g biomass g biomass 1. CTAB + US + ATCLV + AS (0,5% v/v) + 11.59 186.26 29.97 ATCLV 2. Tween 80 + US + ATCLV + AS (1,0% v/v) + 13.76 194.28 33.01 ATCLV 3. Tween 20 + US + ATCLV + AS (1,5% v/v) + 16.87 199.91 36.41 ATCLV 4. NH4OH + Tween 80 + ATCLV + AS (0,5% v/v) 10.21 143.92 37.62 + ATCLV 5. NH4OH + Tween 80 + ATCLV + AS (1,0% v/v) 17.92 175.88 40.65 + ATCLV 6. NH4OH + Tween 80 + ATCLV + AS (1,5% v/v) 19.58 183.40 39.41 + ATCLV

mg TS/ g biomass 227.82 241.06 253.19 191.74 234.45 242.39


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Finally, organic solvent is evaluated. Table 5 shows the results. The global yield to sugars is not competitive to the best ones. The total yield to sugar decreased up to 25%. Table 5 Pretreatment of switchgrass with organic solvent, sonication and dilute sulfuric acid in autoclave mg glucose/ mg xylose/ mg arabinose/ Treatment g biomass g biomass g biomass 1. Ethanol + ATCLV + US + AS (1,5 % v/v) + 16.07 163.24 40.54 ATCLV

mg TS/ g biomass 219.84

3.2. Enzymatic Hydrolysis The method described in section 2 is not straight forward, it has been developed based on screening several alternatives as presented in Table 6. The method is optimized to maximize the sugars yield by analyzing the effect of different variables. Hydrolysis targets glucose and arabinose.

Table 6a Enzymatic hydrolysis Solid loading (% w/v) 5 2.5 5 2.5 5 2.5

Enzymes/biomass (µl/g) N50013 N50010 200 220 200 220 300 330 300 330 400 440 400 440

mg glucose/ g biomass


mg TS/ g biomass

139.35 148.46 147.51 155.70 151.21 155.67

32.35 36.21 35.89 36.52 36.31 38.64

171.70 184.67 183.40 192.22 187.52 194.31

As it can be seen in Table 6a, the load of enzyme is the most important variable. The largest enzymatic loading, the highest amount of C6 sugars recovered. The same trend is found for the buffer ratio. An accumulation of sugars in the medium could inhibits or saturates the enzymes; hence, a high value of buffer ratio benefits the hydrolysis. It is also worth pointing out that the concentration of xylose is low and almost independent of the hydrolysis. The reason is that only a small part of hemicellulose remains after the pretreatment process. For further consideration we select a solid loading of 2.5% w/v and enzymes/biomass ratio of 400 and 440 µl/g of NS50013 and NS50010 respectively, as hydrolysis conditions. Once the enzymatic hydrolysis method is established, it is used over pretreated samples. Among the alternative pretreatments, we only focus at this point on the use of acid pretreatment. 11 ACS Paragon Plus Environment


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Before evaluating the results, a reference of enzymatic hydrolysis must be carried out with the selected conditions to characterize the process. The enzymatic cocktail consists of sugars which are measured at the hydrolysis wort but they do not come from biomass, so the amount of sugar, which is not released from biomass, must be determined. The results show that the sugar added by the enzymes is mainly arabinose. However, this sugar is included in the following tables because they are also included in the fermentation process described in part 2 “Process design”. The analysis of the results is divided between biomass types, switchgrass and corn stover, see Table 6b. Regarding switchgrass, hydrolysis releases a certain amount of glucose and arabinose while only a small amount of xylose is recovered. As discussed above, most of the hemicellulose is already disrupted into xylose in the pretreatment. Furthermore, there is not clear trend on the effect of the US processing time on the sugars yield. With regards to the hydrolysis of corn stover, there are also small yield differences with the sonication time. Among the experimental runs, the highest value is obtained for autoclaving the pretreated corn stover for 60 min and sonication for 45 min, recovering a total of 350 mg of sugars per gram of untreated biomass. The amount of recovered glucose is ten times larger than during the pretreatment and no xylose can be found in the juice. The pretreated structure is mostly made of cellulase while the hemicellulose has already been broken down in the pretreatment. Table 6b Enzymatic hydrolysis of pretreated biomass from Table 3 mg glucose/ Treatment g biomass Switchgrass 1. AS (1,5% v/v) + ATCLV (90 min) + US (60min) 2. AS (1,5% v/v) + ATCLV (90 min) + US (45min) 3. AS (1,5% v/v) + ATCLV (90 min) + US (30min) 4. AS (1,5% v/v) + ATCLV (90 min) + US (15min) 5. AS (1,5% v/v) + ATCLV (90 min) + US (10min) 6. AS (1,5% v/v) + ATCLV (90 min) + US (5min) Corn stover 1. AS (1,5% v/v) + ATCLV (60 min) + US (60min) 2. AS (1,5% v/v) + ATCLV (60 min) + US (45min) 3. AS (1,5% v/v) + ATCLV (45 min) + US (45min) 4. AS (1,5% v/v) + ATCLV (45 min) + US (30min) 5. AS (1,5% v/v) + ATCLV (30 min) + US (15min) 6. AS (1,5% v/v) + ATCLV (30 min) + US (5min)



mg TS/ g biomass

173,62 170,09 165,96 179,03 175,25 176,07

19,95 19,90 21,92 0,00 0,00 0,00

97,40 92,81 96,07 115,87 115,71 116,09

290,98 282,79 283,95 294,90 290,96 292,17

239,40 255,68 233,59 238,48 252,07 247,44

0,00 0,00 0,00 0,00 0,00 0,00

97,02 94,32 97,83 97,44 94,14 93,62

336,42 350,00 331,42 335,92 346,21 341,06


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Finally we evaluate the performance of hydrolysis on biomass samples where sonication was not used. The results are shown in Table 6c and highlights how dilute acids and moderate conditions in pretreatment, result in a higher amount of sugars recovered during the hydrolysis process. In run 1 for corn stover, the amount of every sugar is the largest found. It is due to the fact that pretreatment has not released as much sugars as other runs but it breaks down more efficiently. If we compare run 1 of both biomass types, the amount of xylose produced during pretreatment is higher in switchgrass which means pretreatment is less effective for this biomass, but the amount of glucose and arabinose reveals the large yield that enzymatic hydrolysis reaches when corn stover is processed. Best results are found when pretreated corn stover by sulfuric acid and without sonication is hydrolyzed, reaching a TS recovering of 434.38g. Table 6c Enzymatic hydrolysis of pretreated biomass from Table 1 mg glucose/ Treatment g biomass Switchgrass 1. AS (0,5% v/v) + ATCLV (90 min) 2. AS (1,5% v/v) + ATCLV (90 min) Corn stover 1. AS (0.5% v/v) + ATCLV (90 min) 2. AS (1.0% v/v) + ATCLV (90 min) 3. AS (1.5% v/v) + ATCLV (90 min) 4. AS (2.0% v/v) + ATCLV (90 min)



mg TS/ g biomass

155.67

38.64

112.93

307.24

148.07

36.78

109.09

293.95

260.96 210.53 197.53 189.15

29.44 19.78 15.74 13.86

143.99 100.30 95.67 89.95

434.38 330.61 308.94 292.96

Finally, it is important to measure the total amount of sugars obtained from the complete process involving both, pretreatment and enzymatic hydrolysis. Figure 2 shows the total sugar recovering from switchgrass and corn stover after pretreatment and hydrolysis. It can be seen that although switchgrass pretreatment shows better results than for corn stover, enzymatic hydrolysis for corn stover is very efficient. As a result, the yield of corn stover is larger than that of switchgrass.



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Figure 1. Total recovered sugars. A) Results from Tables 1 and 6c. B) Results from Tables 3 and 6b.

Figure 1 also shows the best operating conditions for processing biomass to sugars and the best raw material as a source for sugar. Corn stover reaches the highest yield, 677.00 mg of sugars by gram of biomass, for a pretreatment consisting of using a sulfuric acid solution 0.5% v/v combined with autoclaving during 90 min at 121 ºC and no sonication stage. The hydrolysis stage is set up at 50 ºC and 150 rpm with a solid loading of 2.5 % w/v and a ratio of enzymes/biomass of 400 and 440 µl/g of NS50013 and NS50010 respectively. To evaluate the actual performance of the pretreatment, and to compare with previous results in the literature, it is necessary to determine the amount of sugars coming from enzymatic cocktail. The measuring was performed by a blank of enzymatic hydrolysis with chosen conditions to characterize the process. The results show that applied sugars by enzymes are 3.24 g of glucose, 2.64 g of xylose and 103.80 g of arabinose per gram of untreated biomass. That means that TS from biomass add up to 567.32 mg per g of untreated corn stover. This result is close to the one obtained by Garlock et al.21 580 mg per g of untreated corn stover, or 80% of TS available. Other recent results such as the one from Vijay 14 ACS Paragon Plus Environment

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Sundaram et al22 show 92% of Glucose yield and 43% of Xylose yield when corn stover was pretreated using AFEX. In our case, we estimate corn stover has around 630 mg available sugars per gram of untreated biomass, so the TS represent 90% of available TS, 99% of Xylose yield and 86% of Glucose yield. These results show that our pretreatment is competitive with the ones in the literature in terms of yield. Therefore, we will be using these results for process scale up since they are produced for our local raw material.21

4. Conclusions In this study, the main stages of second generation biobutanol production, pretreatment, hydrolysis and butanol purification have been evaluated. In order to improve the release of sugars from lignocellulose biomass, a large number of experiments have been run for two types of biomass, switchgrass and corn stover. Biomass type is selected based on its yield to sugars after pretreatment and hydrolysis . The best operating conditions have been determined- Corn stover shows highest yield to sugars using dilute acid pretreatment followed by enzymatic hydrolysis with no further preprocessing stage. The procedure is competitive with the ones in the literature.

Nomenclature AS: sulfuric acid US: ultrasounds TS: total sugars ATCLV: autoclaving CTAB: Cetyltrimethyl ammonium bromide

Acknowledgements We are grateful for the funding from Instituto Tecnológico Agrario de Castilla y León in this research and thanks to the employees of the Center of Biofuels and Bioproduct for the work they have developed.

References [1] Lee, S.Y.; Park, J.H.; Jang, S.H.; Nielsen, L.K.; Kim, J.; Jung, K.S. Fermentative butanol production by clostridium. Biotechnol.Bioeng. 2008, 101, 209–228. [2] Hahn, H-D.; Dämbkes, G.; Rupprich, N.; Butanols. In Ullmann’s Encyclopaedia .Wiley – VCH. 2005. DOI: 10.1002/14356007.a04 463 15 ACS Paragon Plus Environment


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[3] U.S. Department of Energy. 2011. U.S. Billion-Ton Update: Biomass Supply for a Bioenergy and Bioproducts Industry. R.D. Perlack and B.J. Stokes (Leads), ORNL/TM-2011/224. Oak Ridge National Laboratory, Oak Ridge, TN. 227p [4] Weiss, N.D., Farmer, J.D:, Schell, D.J.. Impact of corn stover composition of hemicellulose conversion during dilute acid pretreatment and enzymatic cellulose digestibility of the pretreated solids. Bioresour Technol., 2010, 101, 674–678. [5] Keshwani, D.R.; Cheng, J.J. Switchgrass for bioethanol and other value-added applications: a review. Bioresour Technol. 2009, 100, 1515-1523. [6] Sun, Y.; Cheng J., Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour Technol. 2002, 83:1-11. [7] Kraemer, K.; Harwardt, A.; Bronneberg, R.; Marquardt, W. Separation of butanol from acetone-butanolethanol fermentation by hybrid extraction-distillation process. Comp. Chem. Eng. 2001, 35, 949-963 [8] Jurgens, G.; Survase, S.; Berecina, O.; Linnekoski, J.;, Kurkijärvi, A.;, Väkevä, M.;, Van Heiningen, A.; Granström T. Butanol production from lignocellulosics. Biotechnol Lett., 2012 Aug; 34(8):1415-34 [9] Raganati, F.; Curth, S.; Götz, P.; Olivieri, G.; Marzocchella, A. Butanol production from lignocellulosic – based hexoses and pentoses by fermentation of Clostridium Acetobutylicum, Chem. Eng. Trans. 2012, 27, 91-96 [10] Xue, C.; Zhao, J.; Lu, C.; Yang, S.-T.; Bai, F.; Tang, I.C. High-titer n-butanol production by C. acetobutylicum JB200 in fed-batch fermentation with intermittent gas stripping. Biotechnol.Bioeng. 2012, 109, 2746–2756. [11] Bankar, S.B.; Survase, S.A.; Singhal, R.S.; Granström, T. Continuous two stage acetone-butanolethanol fermentation with integrated solvent removal using C. acetobutylicum B 5313. Bioresour. Technol. 2012. 106, 110–116. [12] Wu, H.; Chen, X.-P.; Liu, G.-P.; Jiang, M.; Guo, T.; Jin, W.-Q.; Wei, P.; Zhu, D.-W. Acetone–butanol– ethanol (ABE) fermentation using C. acetobutylicum XY16 and in situ recovery by PDMS/ceramic composite membrane. Bioproc.Biosys. Eng. 2012, 1–9. [13] Marmierca, S.; Díez-Antolínez, R.; Paniagua, A.I. Technoeconomic study of AB biobutanol production. Part 2: Process design. Ind. Eng. Chem. Res. 2016 submitted. [14] Kong, R.; Kuraasin, M.; Teugjas, H.; Väljamäe, P. Strong cellulose inhibitors from the hydrothermal pretreatment of wheat straw. Biotechnol.Biofuels. 2013, 6, 135. [15] Kim, Y.; Ximenes, E.; Mosier, N.S.; Ldisch, M.R. Soluble inhibitiors/deactivators of cellulose enzymes from lignocellulosic biomass .Enzyme Microb. Technol. 2011, 48, 408 – 415 [16] Ruangmee, A.; Sangwichien, C. Response surface optimization of enzymatic hydrolysis of narrow – leaf cattail for bioethanol production. Energ. Conv. Manag. 2013, 73, 381-388. [17] Zang, D.S.; Yang, Q.; Zhu, J.Y.; Pan, X.J. Sulfite (SPORL) pretreatment of switchgrass for enzymatic saccharification. Biores. Technol. 2013, 129, 127 – 134. [18] Sindhu, R.; Kuttiraja, M.; Preeti, V.E.; Vani, S.; Sukumaran, R.K.; Binod, P. A novel surgactantassisted ultrasound pretreatment of sugarcane tops for improved enzymatic release of sugars. Biores. Technol. 2013, 135, 62 – 72.


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[19] Cao, S.; Aita, G.M. Enzymatic hydrolysis and ethanol yields of combined surfactant and dilute ammonia treated sugarcane bagasse. Bioresour. Technol. 2013, 131, 357 – 364 [20] Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D. Determination of Sugars, Byproducts, and Degradations Products in Liquid Fraction Process Samples. Technical Report NREL/TP510-42623 January 2008. [21] Garlock, R.J.; Bals, B:, Jasrotia, P.; Balan,V.; Dale, B.E. Influence of variable species composition on the saccharification of AFEX pretreated biomass from unmanaged fields in comparison to corn stover. Biomass Bioenerg. 2012, 37, 49-59. [22] Sundaram, V.; Muthukumarappan. K. Influence of AFEXTM pretreated corn stover and switchgrass blending on the compaction characteristics and sugar yields of the pellets. Industrial Crops and Products, 2016, 83, 537–544.



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Figure 1. Total recovered sugars. A) Results from Tables 1 and 6c. B) Results from Tables 3 and 6b. Figure 1 200x169mm (300 x 300 DPI)

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112x80mm (300 x 300 DPI)

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Technoeconomic Study of Biobutanol AB Production. 1. Biomass

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