A global approach to obtain biobutanol from corn stover

Journal Pre-proof A global approach to obtain biobutanol from corn stover María Hijosa-Valsero, Jerson Garita-Cambronero, Ana I. Paniagua-García, Rebeca Díez-Antolínez PII:

S0960-1481(19)31898-1

DOI:

https://doi.org/10.1016/j.renene.2019.12.026

Reference:

RENE 12735

To appear in:

Renewable Energy

Received Date: 5 February 2019 Revised Date:

20 November 2019

Accepted Date: 5 December 2019

Please cite this article as: Hijosa-Valsero Marí, Garita-Cambronero J, Paniagua-García AI, DíezAntolínez R, A global approach to obtain biobutanol from corn stover, Renewable Energy (2020), doi: https://doi.org/10.1016/j.renene.2019.12.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

CREDIT AUTHORS STATEMENT María Hijosa-Valsero: Writing – Original Draft, Investigation, Formal analysis. Jerson Garita-Cambronero: Writing – Original Draft, Investigation. Ana I. Paniagua-García: Methodology, Investigation, Writing – Review and Editing. Rebeca Díez-Antolínez: Writing – Review and Editing, Project administration.

A global approach to obtain biobutanol from corn stover

María Hijosa-Valseroa,*, Jerson Garita-Cambroneroa, Ana I. Paniagua-Garcíaa,b, Rebeca Díez-Antolíneza,b

a

Centro de Biocombustibles y Bioproductos, Instituto Tecnológico Agrario de Castilla

y León (ITACyL), Villarejo de Órbigo, 24358 León, Spain b

Instituto de Recursos Naturales (IRENA), Universidad de León, Avenida de Portugal

42, 24071 León, Spain

*Corresponding Author: María Hijosa-Valsero E-mail: [email protected] Telephone: +34987374554

Other authors’ e-mail addresses: Jerson Garita-Cambronero: [email protected] Ana I. Paniagua-García: [email protected] Rebeca Díez-Antolínez: [email protected]

Declarations of interest: none.

Pretreatment with very dilute acid Lignocellulosic biomass

BIOBUTANOL

Gas stripping

Strain selection and fermentation

Detoxification with adsorption resins

1

Abstract

2

The aim of this research was to subject corn stover to a complete biorefinery process at

3

laboratory-scale in order to assess the production of biobutanol. The research was

4

conducted to focus on process simplification, reduction of reagents and optimization of

5

acetone-butanol-ethanol (ABE) fermentation. The main recommendations include the

6

use of low acid concentrations during the physicochemical pretreatment, the selection of

7

adequate Clostridium strains, detoxification of the hydrolysates with reusable

8

adsorption resins and the possibility of performing gas stripping offline to recover ABE

9

solvents. Various pretreatment conditions, fifteen bacterial strains and three polymeric

10

adsorption resins were assessed. The proposed method consisted of a physicochemical

11

pretreatment with 0.89% H2SO4 (w/w) at 160 °C during 5 min, followed by an

12

enzymatic hydrolysis, which released 75% of the sugars contained in corn stover. The

13

hydrolysate was detoxified with the resin Dowex® Optipore® SD-2 and fermented by

14

C. saccharobutylicum DSM 13864, producing 4.75 ± 0.25 g/L acetone, 9.02 ± 0.11 g/L

15

butanol and 0.39 ± 0.01 g/L ethanol in 72 h, with a sugar consumption of 97.3 ± 0.27%.

16

A two-stage gas stripping was applied to the fermentation broth, obtaining butanol-rich

17

condensates (418-425 g/L in the organic phase) in a total time of 6 h.

18 19

Keywords

20

Lignocellulosic biomass; biobutanol; corn stover; pretreatment; detoxification;

21

Clostridium.

22

1

23

1. Introduction

24

The development of a sustainable industry based on the use of renewable resources to

25

produce fuels, chemicals and environmentally friendly materials has been recognised as

26

a key issue of importance [1]. One alternative that is gathering promising scientific

27

efforts is the study of new fuels obtained from different lignocellulosic biomasses, such

28

as butanol production through acetone-butanol-ethanol (ABE) fermentative process [2].

29

Lignocellulosic wastes from agricultural industry have attracted much more attention as

30

a sustainable alternative as they avoid the “food vs fuel” issues that arise by using

31

agricultural land to produce biomass for fuels. Biobutanol production by ABE

32

fermentation has already been successfully achieved using several agricultural wastes

33

[3-6] including corn stover [7]. Due to its immediate availability at the required

34

industrial scale [8], corn stover is currently seen as one of the most important feedstocks

35

for bioethanol production in industrialized countries. In addition, corn stover also

36

represents an interesting feedstock for biobutanol production due to its fibrous structure

37

with a high carbohydrate content (about 18-22% hemicellulose and 32-36% cellulose).

38

Both characteristics have attracted the attention of researchers for using corn stover to

39

produce second generation biobutanol [9].

40

Aside from the selection of an appropriate lignocellulosic feedstock, biobutanol

41

production requires the improvement of several technical procedures before

42

fermentation, such as biomass pretreatment and hydrolysis of structural carbohydrates,

43

selection of a suitable bacterial strain and optimization of the physicochemical

44

conditions in the fermentation broth [10]. In addition, the pretreatment processes –

45

because of sugars and lignin degradation– produce chemical by-products (phenolic

46

compounds, furan derivatives and weak organic acids) which cause inhibition on

47

solventogenic strains of Clostridium affecting biobutanol production [11]. The

2

48

production of these inhibitors is usually enhanced with dilute acid pretreatments,

49

particularly when using sulfuric acid [12]. To address this problem several

50

detoxification methods for corn stover hydrolysates prior to bacterial fermentation have

51

been proposed, including inhibitors extraction, adsorption, evaporation, electrodialysis,

52

overliming, neutralization, steam stripping, as well as enzymatic and microbial

53

treatments [11]. There is a clear need for further research to better understand and assess

54

the potential of industrial scale biobutanol production from corn stover. For example,

55

using alternatives such as a simple and efficient biomass pretreatment or testing

56

alternative detoxification techniques such as reusable resins, could decrease the

57

economic and energetic requirements associated with the fermentative process [13].

58

From an economic and energetic point of view, downstream processes such as solvent

59

recovery and purification from fermentation broths are extremely important in

60

biorefineries. Recently, gas stripping has emerged as a very attractive alternative, since

61

its energy requirements (including a final step of distillation) could be lower than

62

previously calculated [14-17] and due to configuration innovations like two-stage gas

63

stripping [15].

64

This study explores different strategies for addressing the major barriers that are

65

currently present at each step of the ABE fermentative process when using corn stover

66

as lignocellulosic feedstock. In particular, acid pretreatment conditions will be refined

67

in order to obtain suitable sugar and inhibitor concentrations in the hydrolysate,

68

followed by a clostridial strain selection and the optimization of certain fermentation

69

conditions (pH, T, CaCO3 and nutrient supplementation). The necessity of a

70

detoxification process to remove inhibitors by inert adsorption resins prior to

71

fermentation is also evaluated. Finally, the ABE solvents contained in the fermentation

72

broth are recovered by two-stage gas stripping. This study presents for the first time a

3

73

complete ABE biorefinery process based on corn stover, including a detoxification step

74

with reusable resins.

75 76

2. Material and methods

77 78

2.1. Chemicals and reagents

79

All chemicals used were of analytical grade. The enzyme Cellic CTec2 (enzymatic

80

activity 105 FPU/mL) was provided by Novozymes (Tianjin, China). The polymeric

81

resin Amberlite® XAD-4 was bought from Acros Organics (Geel, Belgium), Dowex®

82

Optipore® L-493 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and

83

Dowex® Optipore® SD-2 was obtained from Supelco (Bellefonte, PA, USA).

84 85

2.2. Biomass description

86

Corn stover samples were obtained in November 2017 from experimental plots

87

(ITACyL, Finca Zamadueñas, Valladolid, Spain). These agricultural by-products were

88

dried in an oven at 45 °C until constant weight, ground in a rotary mill SM100 Comfort

89

(Retsch GmbH, Haan, Germany) and sieved to a size of 0.5-1.0 mm. Moisture, ash,

90

structural carbohydrates (cellulose and hemicellulose), Klason lignin, fats, proteins and

91

total phenolic compounds were analyzed as reported elsewhere [4]. Corn stover

92

composition is shown in Table 1.

93 94

2.3. Physicochemical pretreatment

95

Corn stover was subjected to a dilute-acid physicochemical pretreatment. It was

96

observed that autohydrolysis was not an efficient pretreatment for corn stover (data not

97

shown). In preliminary tests, nitric acid and sulfuric acid were assessed under similar

4

98

conditions [pretreatment at 0.89% (w/w) acid, 125 °C, 5 min; followed by enzymatic

99

hydrolysis], and finally sulfuric acid was chosen for the subsequent experiments

100

because its hydrolysates were more easily fermentable by bacteria (Figure SM1).

101

Physicochemical pretreatments were carried out with a 2-L high-pressure reactor made

102

of alloy Carpenter-20 (Parr Instrument Company, Moline, IL, USA). Corn stover was

103

immersed in a solution of sulfuric acid, with a solid-to-solvent ratio of 10% (w/w).

104

Operation details of the reactor are described elsewhere [5]. After the thermal

105

pretreatment, an enzymatic hydrolysis was performed on the solid/liquid mixture

106

obtained in the reactor according to the procedure described in section 2.4.

107 108

2.3.1. Pretreatment optimization

109

The physicochemical pretreatment was optimized in order to obtain a fermentable broth

110

with the maximum concentration of simple sugars (glucose, xylose, etc.) and the

111

minimum concentration of inhibitors [formic acid, acetic acid, levulinic acid, furfural,

112

5-hydroxymethyl furfural (5-HMF) and phenolic compounds]. In particular, the

113

variables to be optimized were H2SO4 concentration (0.89, 1.13, 1.37, 1.60 and 1.84 %

114

w/w) and temperature (125, 135, 145 and 160 °C). The treatment time in the reactor was

115

experimentally set at 5 min, after observing that longer treatment times did not improve

116

sugar release. The analysis of sugars and inhibitors was performed after the combined

117

and subsequent steps of physicochemical pretreatment and enzymatic hydrolysis.

118 119

2.4. Enzymatic hydrolysis

120

Upon completion of the thermal pretreatment, an enzymatic hydrolysis with Cellic

121

CTec 2 was performed on the biomass solid/liquid mixture obtained in the reactor,

5

122

following the method described by [4]. The employed dose of 36 µL/g biomass is

123

equivalent to 3.78 FPU/g biomass.

124 125

2.5. Strain cultivation

126

The strains Clostridium acetobutylicum DSM 792, DSM 1732, DSM 6228, C.

127

beijerinckii DSM 51, DSM 552, DSM 791, DSM 1820, DSM 6422, DSM 6423, C.

128

pasteurianum

129

saccharoperbutylacetonicum DSM 2152 and DSM 14923 were purchased from DSMZ

130

(Braunschweig, Germany), whereas the strain C. acetobutylicum NRRL B-530 was

131

obtained from NRRL (Peoria, IL, USA) and the strain C. beijerinckii CECT 508 was

132

supplied by CECT (Paterna, Spain). Spores from all the strains (except DSM 6228)

133

were prepared and stored as explained in [18]. For the asporogenic strain DSM 6228,

134

lyophilised cells were resuspended in 10 mL of sterile Reinforced Clostridial Medium -

135

RCM (Oxoid, Basingstoke, UK) supplemented with 10 g/L glucose, and incubated 24 h

136

at 35 °C under anaerobic conditions. Then, 1.5 mL were mixed with 0.4 mL glycerol

137

(80% v/v) and stored at -80 °C until used. Cellular reactivation and inocula preparation

138

were performed in liquid RCM or in a potato based medium (in the case of DSM 2152

139

and DSM 792) as detailed in [6]. Bacterial cultures were incubated for 20-48 h at 35 ºC

140

until obtaining a density of 5·108 cells/mL as determined by counting in a Bürker

141

chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany).

DSM

526,

C.

saccharobutylicum

DSM

13864,

C.

142 143

2.6. Strain selection for the fermentation of corn stover hydrolysates

144

Corn stover hydrolysates were obtained after a pretreatment with H2SO4 (125 °C, 5 min,

145

0.89% w/w acid) and a subsequent enzymatic hydrolysis. For fermentation tests, non-

146

detoxified corn stover hydrolysates were filtered through filter paper (No. 1305, 73

6

147

g/m2, Filtros Anoia SA, Barcelona, Spain) and supplemented with the standard nutrients

148

(S) listed in Table 2. Inoculation and medium preparation were performed as described

149

in [18]. Fermentations were conducted at 35 °C, 100 rpm and 96 h in an Infors HT

150

Minitron orbital shaker (Infors AG, Bottmingen, Switzerland). These experiments were

151

performed in triplicate with the fifteen bacterial strains listed in section 2.5.

152

Fermentation controls were prepared with aqueous solutions containing glucose and

153

xylose mixtures at similar concentrations to those of corn stover hydrolysates and

154

supplemented with the abovementioned nutrients and salts. The most appropriate strain

155

was selected for the next experiments.

156 157

2.7. Optimization of fermentation conditions

158

Once the most efficient strain had been selected, the most adequate nutrients and

159

fermentation conditions for that strain were determined. Firstly, a Plackett-Burman

160

experimental design was proposed to select the necessary nutrients and their

161

concentrations. Corn stover was pretreated with H2SO4 (125 °C, 5 min, 0.89% acid

162

w/w) and a subsequent enzymatic hydrolysis. The Plackett-Burman design consisted of

163

twelve experimental runs combining the maximum and minimum values established for

164

the ten independent variables (yeast extract, KH2PO4, K2HPO4, NH4Cl, MgSO4·7H2O,

165

FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O, cysteine and CaCO3). The hydrolysates were

166

fermented as described in Section 2.6 and butanol was measured as the response

167

variable. The characteristics of this experimental design are shown in Table SM1. The

168

nutrients and their concentration ranges were selected according to literature data [19-

169

22].

170

In the second place, after having established the essential nutrients for the selected

171

strain, fermentation conditions (temperature, initial pH and CaCO3 concentration for pH

7

172

control) were optimized via response surface methodology (RSM) with a Box-Behnken

173

experimental design consisting of 3 factors, 1 replicate, 15 runs, 1 block and 3 central

174

points (Table SM4). Corn stover was pretreated with H2SO4 (125 °C, 5 min, 0.89% acid

175

w/w) and a subsequent enzymatic hydrolysis, and then it was fermented using the

176

nutrients and conditions listed in Table SM4.

177 178

2.8. Detoxification

179

The polymeric adsorption resins Amberlite® XAD-4, Dowex® Optipore® L-493 and

180

Dowex® Optipore® SD-2 were evaluated for the removal of inhibitors from corn stover

181

hydrolysates. About 58 g of each polymeric resin were placed inside a 120-mL glass

182

column (chromatography column with soldered porous plate No. 0, model 340634, 400

183

mm x 20 mm i.d.; Pobel, Madrid, Spain) where they were soaked with distilled water,

184

reaching a total volume of 90 mL. The resins were conditioned following a modification

185

of [23]. In brief, the resin Amberlite® XAD-4 was washed three times with 90 mL

186

methanol at 4 mL/min and then was left in methanol overnight. Afterwards it was

187

washed with 900 mL distilled water at 10 mL/min. The resins Dowex® Optipore® L-

188

493 and Dowex® Optipore® SD-2 were washed three times with 90 mL NaOH 1 M at

189

4 mL/min and then with 900 mL distilled water at 10 mL/min.

190

For the detoxification step, corn stover (10% solid biomass) was pretreated with a

191

0.89% H2SO4 w/w solution for 5 min. Two distinct pretreatment temperatures (125 °C

192

or 160 °C) had to be tested in order to generate different sugar concentrations in the

193

hydrolysate (see section 3.4). A subsequent enzymatic hydrolysis was then applied. The

194

hydrolysate was passed through filter paper (No. 1305), and its pH was adjusted to 5.42

195

(optimal for fermentation). Then, the hydrolysate was fed into the column at a rate of 2

196

mL/min using a peristaltic pump (10 min residence time). The detoxified hydrolysate

8

197

was collected and fermented with C. saccharobutylicum DSM 13864 under the optimal

198

conditions (O) (Table 2). The use of other strains for detoxified hydrolysates was

199

experimentally discarded (Supplementary Material and Figure SM2).

200 201

2.9. Gas stripping

202

In order to recover solvents (acetone, butanol and ethanol) from the fermentation broths,

203

a two-stage gas stripping was applied as described in [24]. Corn stover hydrolysate was

204

obtained by treating 10% solid biomass in a solution of 0.89% H2SO4 (w/w) at 160°C

205

for 5 min, followed by enzymatic hydrolysis and detoxification with the resin Dowex®

206

Optipore® SD-2. The hydrolysate was fermented by C. saccharobutylicum DSM 13864

207

during 72 h using the optimized combination (O) (Table 2).

208

About 3.2 L of fermentation broth containing 5.02 g/L acetone, 9.02 g/L butanol, 0.42

209

g/L ethanol, 1.61 g/L acetic acid, 0.95 g/L butyric acid and 1.19 g/L total sugars, were

210

subjected to gas stripping over 5 h under the following conditions: Tfeed = 60 °C,

211

Trefrigeration = 5 °C and gas flow = 1.34 L/min. Five millilitres of Antifoam A (Fluka

212

Analytical, Sigma-Aldrich, Steinheim, Germany) were added at the beginning of the

213

stripping to avoid foam formation. The condensate was collected and separated

214

spontaneously in two phases (organic and aqueous). The aqueous phase was further

215

subjected to another gas stripping process for 1 h after which its condensate was

216

collected.

217

The performance of the gas stripping process was calculated based on solvent recovery

218

(η) and selectivity (α), according to Equations 1 and 2 [25-26]:

219

=

(1)

220

=

(2)

9

221

where αi is the selectivity for compound i (for instance, butanol; αB), xi is the mass ratio

222

of metabolite i in the feed solution, yi is the mass ratio of metabolite i in the condensate,

223

ηi is the percentage recovery efficiency for metabolite i (in this case, butanol; ηB), mic is

224

the mass of metabolite i in the condensate (expressed in g) and miF is the mass of

225

metabolite i in the feed solution. Gas stripping rates [g/(L·h)] were calculated as

226

proposed by [27].

227 228

2.10. Chemical analyses

229

The sugars cellobiose, glucose, xylose, rhamnose and arabinose, and the potential

230

inhibitors formic acid, acetic acid, levulinic acid, 5-HMF and furfural were analyzed by

231

HPLC-RID; phenolic compounds were analyzed by Folin-Denis’ assay; whereas

232

fermentation metabolites, like acetone, butanol, ethanol, isopropanol, acetic acid and

233

butyric acid were determined by GC-FID, according to [4].

234

Fermentation yields (Yi/S, g/g), metabolite productivity rates [Wi, g/(L·h)] and sugar

235

recovery or sugar conversion efficiency (%) were calculated as explained elsewhere [6].

236 237

2.11. Statistical analyses

238

Samples were compared with a one-way ANOVA and Tukey’s HSD test using the

239

software Statistica 7 (StatSoft Inc., Tulsa, OK, USA). Plackett-Burman experimental

240

designs and Box-Behnken RSM experimental designs were made with Minitab 16

241

(Minitab Inc., State College, PA, USA).

242 243

3. Results

244 245

3.1. Composition of corn stover hydrolysates

10

246

The effects of temperature and H2SO4 concentration on the corn stover pretreatment

247

were assessed in order to increase sugar release and minimize the generation of

248

fermentation inhibitors (Table 3). Temperature had a clear impact on sugar release and

249

inhibitor formation when acid concentration was kept constant at 0.89% w/w (Table 3).

250

On the contrary, for a fixed temperature of 125°C, an increase in H2SO4 concentration

251

did not improve sugar release, but favoured the generation of inhibitors, especially

252

formic acid, acetic acid, furfural and phenolic compounds (Table 3). These data suggest

253

that a sulfuric acid concentration of 0.89% w/w (which is equivalent to ~0.54% v/v)

254

could be sufficient for corn stover degradation. In fact, Gao and Rehman [28] pretreated

255

Phragmites australis with increasing concentrations of H2SO4 (0.5-2.0% v/v) and they

256

observed that the use of higher acid concentrations did not release more sugars, but only

257

more inhibitors. In addition, they reported that H2SO4 concentration influenced the type

258

of phenolic compounds generated.

259

The amount of total sugars released from corn stover ranged between 34.7 and 50.4 g/L,

260

depending on the physicochemical pretreatment used (Table 3), which implies a sugar

261

recovery efficiency of 48-75%. According to a review published by [12], the sugar

262

yields obtained from corn stover employing different pretreatment methods vary

263

between 42 and 66 g of fermentable sugars per 100 g of dry corn stover. In the present

264

study, as corn stover contained 57.23 % carbohydrates (Table 1), that yield would reach

265

~43 g of fermentable sugars per 100 g of dry corn stover in the best case.

266

In order to avoid inhibitory problems, it was decided to select the pretreatment

267

conditions of 0.89% H2SO4 w/w, 125 °C and 5 min, because they offered acceptable

268

sugar concentrations and low inhibitor concentrations. The total sugar concentration

269

obtained (~40 g/L), moderate as it may seem, might guarantee successful ABE

270

fermentations, as observed in previous works with cheese whey [29] or coffee silverskin

11

271

[6], where initial sugar concentrations as low as 30-34 g/L enabled the generation of

272

7.0-8.5 g/L biobutanol.

273 274

3.2. Strain comparison for corn stover hydrolysates

275

Different bacterial strains were compared for the fermentation of corn stover

276

hydrolysates after pretreating this biomass at 125 °C, during 5 min with 0.89% w/w

277

H2SO4 and subjecting the sample to enzymatic hydrolysis (Figure 1).

278

The best fermentations were performed by the strains DSM 13864 (5.95 ± 0.06 g/L

279

butanol, 94 ± 0.3 % total sugar consumption), DSM 6423 (5.49 ± 0.20 g/L butanol, 78 ±

280

2.4 % total sugar consumption) and DSM 2152 (4.97 ± 0.33 g/L butanol, 80 ± 2.1 %

281

total sugar consumption). It must be noted that the broths fermented by C.

282

saccharobutylicum DSM 13864 acquired a gelatinous appearance, probably due to the

283

formation of a polysaccharide, a fact that could explain the low concentration of total

284

free sugars at the end of the fermentation with this strain. According to these results and

285

taking into account both ABE concentrations and sugar consumption, it was decided to

286

select the strain DSM 13864 for the optimization experiments. In fact, the species C.

287

saccharobutylicum has been reported to cope with mixed agricultural and waste-based

288

substrates [30]. Moreover, C. saccharobutylicum DSM 13864 had been previously used

289

by [31] to ferment corn stover hydrolysates obtained after a pretreatment with deep

290

eutectic solvents.

291 292

3.3. Optimization of fermentation conditions for non-detoxified corn stover

293

hydrolysates

294

The most adequate nutrients and fermentation conditions for C. saccharobutylicum

295

DSM 13864 were evaluated as described in the Supplementary Material (Tables SM1-

12

296

SM7). The optimal working conditions, as well as the nutrient concentrations have been

297

summarised in Table 2 (optimized conditions - O). The calculated optimum answer

298

established that it would be possible to produce 7.56 g/L butanol at 28 °C, pH 5.42 and

299

8.0 g/L CaCO3. In order to validate the model, this optimal point was tested by

300

fermenting non-detoxified corn stover hydrolysates. The results of model validation

301

yielded a butanol concentration of 5.38 ± 0.21 g/L, which is far from the estimated

302

value (7.56 g/L). In addition, other fermentation experiments were performed on

303

different days under similar circumstances obtaining variable butanol concentrations in

304

the range of 4.49-6.38 g/L. This lack of repeatability might be related to the inhospitable

305

nature of corn stover acid hydrolysate for bacteria [7], which could cause a selective

306

pressure on microorganisms via toxic effects that would lead to the survival of those

307

organisms with a higher resistance to inhibitors, which may not be the best butanol

308

producers. Even if the same spores batch of a bacterial strain is used in different

309

experiments, not all the individuals are identical and, in addition, can suffer genetic

310

changes during their development and reproduction causing a large amount of

311

phenotypical variability inside this environment. This potential selective pressure

312

generated by the toxic compounds could be faced by applying a detoxification step for

313

this corn stover hydrolysate. In any case, under other circumstances (detoxified

314

hydrolysates; Figure SM2) the optimized conditions (O) were proved to be superior to

315

the standard conditions (S) for C. saccharobutylicum DSM 13864 and therefore they

316

were maintained for further experiments.

317

Table 4 shows fermentation performances of non-detoxified corn stover hydrolysates

318

found in literature. The results from the present work are in agreement with other

319

comparable studies employing batch fermentations, where butanol concentrations of 0-

320

6.93 g/L were attained. However, these studies were carried out with hydrolysates

13

321

containing clearly higher initial sugar concentrations (52-75 g/L total sugars), which

322

highlights the good performance of the proposed pretreatment/fermentation process.

323

This satisfactory result could be due to the selection of the most appropriate strain for

324

this specific hydrolysate (DSM 13864). In addition, the registered sugar consumption of

325

83% is notably high, and it is near the values observed in semicontinuous reactors with

326

in-situ butanol recovery [32]. This fact could be related to the relatively low initial sugar

327

concentration in the broth and to the hypothetical formation of an extracellular

328

polysaccharide by C. saccharobutylicum DSM 13864; although other non-

329

polysaccharide forming strains, such as DSM 6423 and DSM 2152, had also shown

330

sugar consumption values above 70% (Figure 1).

331 332

3.4. Detoxification of corn stover hydrolysates

333

Due to the presence of inhibitors, corn stover hydrolysate was not readily fermentable,

334

and butanol production did not exceed the threshold of 4-6 g/L in spite of strain

335

screening, nutrient optimization and adjustment of fermentation conditions. Therefore,

336

after physicochemical pretreatment (0.89% H2SO4 w/w, 125°C, 5 min) and enzymatic

337

hydrolysis, corn stover hydrolysate was detoxified with three adsorption resins (Table

338

5). Most inhibitors were removed from the hydrolysates (especially 5-HMF and

339

phenolic compounds), but sugars were also retained by the resins (14.7-17.8%), which

340

implies an important loss of sugars for the subsequent fermentation process. These

341

detoxified hydrolysates were fermented during 96 h with C. saccharobutylicum DSM

342

13864. According to Figure 2a, the detoxification process did not clearly enhance

343

bacterial activity. In fact, butanol production in the best case (5.62 ± 0.02 g/L, with

344

Amberlite® XAD-4) was similar to that of the non-detoxified hydrolysate (p > 0.05). In

345

spite of the successful removal of inhibitors, ABE concentrations were not improved in

14

346

a significant way, probably because of the decrease in sugar concentrations caused by

347

the resin treatment (33.8-35.1 g/L initial sugars).

348

As a consequence, the ability of detoxified hydrolysate with higher sugar concentrations

349

to produce greater ABE yields was tested. For this experiment, a hydrolysate containing

350

about 50 g/L total sugars, obtained with an aqueous solution of 0.89% H2SO4 (w/w) at

351

160°C during 5 min (Table 3), was chosen and detoxified with the three resins. Inhibitor

352

concentrations were reduced by the resins, while sugar concentrations decreased by

353

11.9-13.2% (Table 5). Therefore, fermentations commenced with initial sugar

354

concentrations of 43.7-44.4 g/L, using the optimized nutrients and fermentation

355

conditions (O) (Table 2). The fermentation was finished after 72 h. The non-detoxified

356

hydrolysate was not fermentable, yielding barely 0.12 ± 0.04 g/L butanol (Figure 2b).

357

On the contrary, detoxified samples obtained butanol concentrations of 8.71 ± 0.03 g/L,

358

8.39 ± 0.23 g/L and 9.02 ± 0.11 g/L for resins Amberlite® XAD-4, Dowex® Optipore®

359

L-493 and Dowex® Optipore® SD-2, respectively. In addition, butanol concentrations

360

recorded for the sample detoxified with Dowex® Optipore® SD-2 were not

361

significantly different (p > 0.05) from those of the control, which indicates that any

362

potential inhibitory effects had been eliminated during detoxification. Therefore, the

363

pretreatment at 160 °C aiming at releasing more sugars was successful for ABE

364

fermentation, provided that a detoxification step is performed. This process implied a

365

clear improvement in butanol production (from 5.38 ± 0.21 g/L to 9.02 ± 0.11 g/L

366

butanol) and fermentation time (from 96 h to 72 h) in comparison to the previous values

367

obtained before detoxification (section 3.3). The fermentation of the hydrolysate

368

detoxified with resin Dowex® Optipore® SD-2 obtained 4.75 ± 0.25 g/L acetone, 9.02

369

± 0.11 g/L butanol, 0.39 ± 0.01 g/L ethanol, 2.17 ± 0.07 g/L acetic acid and 1.26 ± 0.07

15

370

g/L butyric acid, with a sugar consumption of 97.3 ± 0.27%, a butanol yield YB/S of

371

0.222 ± 0.003 g/g and a butanol productivity WB of 0.125 ± 0.002 g/(L·h).

372

The three adsorption resins used in the present study had been previously assessed for

373

the adsorption of phenolic compounds [33], for the in situ recovery of butanol from

374

fermentation broths [34] and for the detoxification of various lignocellulosic

375

hydrolysates, but they had never been used for the detoxification of corn stover

376

hydrolysates. Gao and Rehman [28] used the resin Dowex Optipore L-493 to

377

simultaneously adsorb inhibitors and butanol during the fermentation of an acid

378

hydrolysate of P. australis. The resin adsorbed 2.98% glucose, 4.85% xylose, 7.84%

379

acetic acid, 33.3% 5-HMF, 77.8% furfural and 95.1% total phenolic compounds.

380

Shukor et al. [35] detoxified the acid hydrolysate of palm kernel cake with Amberlite

381

XAD-4 and observed a reduction of 50% furfural and 77% 5-HMF, without losses of

382

glucose or mannose. On the other hand, Ezeji et al. [36] compared overliming and

383

Amberlite XAD-4 as detoxification methods for corn fiber hydrolysates and concluded

384

that overliming was superior, although the resin removed 60-80% furfural, 5-HMF and

385

ferulic acid. Sugar losses were 2.99% for overliming and 10.72% for the resin. Weil et

386

al. [37] subjected a corn fiber hydrolysate to detoxification with resins Amberlite XAD-

387

4 and XAD-7 and were able to remove 96% furfural. Our results are in agreement with

388

the reported removal ranges in the case of furfural, 5-HMF and phenolic compounds,

389

and are slightly higher in the case of acetic acid. However, sugar losses in the present

390

study (12-18%) are clearly higher than those mentioned in literature (0-11%).

391

Table 6 presents a literature summary of fermentation performances using detoxified

392

corn stover hydrolysates. Butanol production in the present work (9.02 g/L) is similar or

393

higher than that of other papers (0.36-14.5 g/L) and it is in the range of those

394

experiments where fermentations were started with sugar concentrations below 45 g/L

16

395

(7.1-11.5 g/L butanol). The most common detoxification techniques for this substrate

396

are dilution with water, overliming, washing of the solid biomass after physicochemical

397

pretreatment, alkaline peroxide and adsorption onto activated charcoal (Table 6). To the

398

best of the authors’ knowledge, this is the first time that polymeric resins are employed

399

for the detoxification of corn stover hydrolysate.

400 401

3.5. Two-stage gas stripping of fermentation broths

402

The detoxified hydrolysate was fermented, and the fermentation broth was subjected to

403

a two-stage gas stripping process. During the first stage, butanol concentration in the

404

fermentation broth decreased from 9.02 g/L to 2.07 g/L in 5 h, and a condensate

405

containing 169.26 g/L butanol was collected (Table 7). This implies solvent recoveries

406

(η) of 33.22% for acetone, 67.70% for butanol and 38.98% for ethanol, and selectivity

407

factors (α) of 9.61 for acetone, 22.38 for butanol and 10.85 for ethanol. Gas stripping

408

rates were 0.58 g/(L·h) for acetone, 1.39 g/(L·h) for butanol, 0.03 g/(L·h) for ethanol

409

and 2.00 g/(L·h) for ABE. These results are in the upper range of previously reported

410

values for gas stripping. Typical condensates of integrated fed-batch gas stripping

411

processes contain 20-120 g/L acetone, 17-113 g/L butanol and 7-21 g/L ethanol [17,

412

38]; with selectivity values of 0.45-30.5 for butanol and 4-30.5 for ABE; and gas

413

stripping ABE rates of 0.02-1.34 g/(L·h) [17, 39-40]. The gas stripping process was

414

successful despite the presence of an antifoaming agent, which is known to reduce

415

system performance [41]. The relatively high butanol concentration in this condensate,

416

notably above butanol solubility in water (7.4 g/100g at 298 K) [42], resulted in the

417

spontaneous separation of an aqueous and an organic phase. The organic phase had a

418

butanol concentration of 425.35 g/L. The aqueous phase, with a butanol concentration

419

of 88.70 g/L, was collected and subjected to a second gas stripping stage.

17

420

The second stage caused a decrease in butanol concentration in the feed solution from

421

88.70 g/L to 1.60 g/L in 1 h. In this case, a condensate containing 263.29 g/L butanol

422

was collected (Table 8). The recorded solvent recoveries (η) were 45.81% for acetone,

423

73.38% for butanol and 70.01% for ethanol. In this second gas stripping step, selectivity

424

factors (α) were lower than in the first stage, since solvent concentrations in the initial

425

feed solution were much higher at the beginning of the second stripping. Thus,

426

selectivity values (α) of 1.94 for acetone, 3.67 for butanol and 2.86 for ethanol were

427

attained. Gas stripping rates during this second stage were 46.9 g/(L·h) for acetone, 87.1

428

g/(L·h) for butanol, 3.79 g/(L·h) for ethanol and 27.6 g/(L·h) for ABE. It has been

429

reported that condensates of two-stage gas stripping processes contain 119-198 g/L

430

acetone, 337-451 g/L butanol and 21-23 g/L ethanol [38]. Once more, the condensate

431

spontaneously separated in two phases. The aqueous phase contained 129.67 g/L

432

butanol, a value which is above its solubility in water, but this fact could be explained

433

by the presence of 99.54 g/L acetone in this aqueous phase, which could have favoured

434

butanol solubility [15]. On the other hand, the organic phase contained 417.71 g/L

435

butanol, which is a high value similar to that obtained in the organic phase of the first

436

stripping stage. Figure 3 summarises butanol fate during the steps of the gas stripping

437

process. Total mass recoveries (sum of organic phase 1, organic phase 2 and aqueous

438

phase 2) reached 19.91% acetone, 60.28% butanol and 30.37% ethanol.

439 440

3.6. Implications of the proposed process for biorefineries

441

Previous studies on biobutanol production from corn stover have explored the main

442

technical issues related to ABE processes (pretreatment, hydrolysis, toxicity, bacterial

443

performance, fermentation conditions) through the use of different pretreatment

444

reagents and conditions [13, 43-45]; hydrolysate detoxification by various methods [7,

18

445

10, 19, 21, 43, 45-46] and the selection of mutant and wild type solventogenic

446

Clostridium strains [7, 10, 19, 43, 45-46].

447

Steam explosion, dilute sulfuric acid, dilute NaOH, organosolv and deep eutectic

448

solvents have been employed as physicochemical pretreatments for corn stover

449

hydrolysis (Table 4 and Table 6). Although dilute sulfuric acid pretreatment is frequent

450

at an industrial scale [12], the concentration of H2SO4 used in the present work (0.89%

451

w/w or ~0.54% v/v) is clearly below the values of 1-2% v/v reported in literature (Table

452

4 and Table 6), which constitutes an advantage from both an economic and

453

environmental perspective. In addition, the short pretreatment time (5 min) proposed

454

would require less energy consumption.

455

Current detoxification methods include overliming, evaporation, adsorption (onto resins

456

or activated charcoal) or biological methods (use of peroxidases and laccases) [12].

457

Among the adsorption methods, the use of polymeric resins has been determined as an

458

efficient method to detoxify biomassic hydrolysates, as well as a technique to remove

459

alcohols from the fermentation broth [28, 47]. In addition, polymeric resins potentiate

460

the reduction of the overall production cost due to their reusability without affecting the

461

process efficiency and in some cases without altering the sugar concentration contained

462

in the hydrolysate [28]. On the other hand, adsorption resins are regarded as an

463

expensive detoxification method [48]. However, the possibility of immobilising resins

464

inside columns to perform continuous processes and the recyclability of these materials

465

by regeneration make them attractive for industrial applications. Other detoxification

466

methods employing alkali addition or activated carbon can reduce acetic acid, furfural

467

and phenolic compounds concentration, but they can also induce salt formation, which

468

can be detrimental for Clostridium species [12]. Furthermore, detoxification techniques

469

by washing the solid biomass before the enzymatic hydroysis imply the loss of the

19

470

pentoses and other hemicellulosic sugars released during the physicochemical

471

pretreatment. In comparison to detoxification by washing (Table 4, Table 6), resins

472

could entail water savings. Sainio et al. [49] proved that the resin Amberlite XAD-16

473

(similar to XAD-4) could be regenerated with a 50% ethanol aqueous solution in order

474

to remove fermentation inhibitors such as furfural. Weil et al. [37] confirmed the

475

regenerability of the resin Amberlite XAD-4 with ethanol. Therefore, if adsorption

476

resins were used for inhibitor removal at industrial scale in ABE biorefineries, they

477

could be regenerated by washing them with the same solvents recovered from the

478

fermentation broth (acetone or ethanol), thus obtaining a solution rich in furans and

479

phenolic compounds which could also have commercial value.

480

Corn stover hydrolysates have been subjected to ABE fermentation employing several

481

wild strains [7, 10, 19, 21, 31, 32, 44, 46], or modified strains [43, 45, 50]. However,

482

their correct performance is frequently related to high initial sugar concentrations in the

483

fermentation broth or to the application of detoxification methods involving the use of

484

non-recyclable reagents (Table 4 and Table 6). The screening of an adequate strain,

485

which are able to survive in a specific hydrolysate, can improve ABE fermentation in a

486

substantial way.

487

Regarding solvent recovery, the application of integrated processes (which remove

488

acetone, butanol and ethanol as they are being produced in the fermentation broth), like

489

gas stripping, liquid-liquid extraction, vacuum fermentation, adsorption, reverse

490

osmosis, pervaporation and perstraction, has been extensively reviewed [40, 51-54].

491

Gas stripping has been usually tested as an integrated (in situ) recovery technique; but

492

these operating conditions can increase the whole process energy requirements [51]. It

493

has been observed that gas stripping is also successful when applied upon completion of

494

the fermentation, which requires much shorter stripping times [55]. In the present work,

20

495

a total stripping time of 6 h has been proven efficient for solvent recovery. Therefore,

496

off-line recovery techniques could be attractive alternatives in order to reduce energetic

497

costs.

498 499

4. Conclusions

500

It is possible to pretreat corn stover by employing low H2SO4 concentrations (0.89%

501

w/w) and yet release about 75% of its constitutive carbohydrates after enzymatic

502

hydrolysis. However, high temperatures (160 °C) are necessary to guarantee an efficient

503

pretreatment. Due to the presence of fermentation inhibitors in the hydrolysate,

504

selecting appropriate strains and detoxification steps are essential to carry out a

505

successful ABE fermentation. Despite the fact that in this study one of the highest-ever

506

butanol values has been obtained without the necessity of using genetically improved

507

bacterial strains, it would be useful to test the whole butanol production workflow

508

proposed in this paper with some hyerproducing solventogenic strain, for instance C.

509

beijerinckii P260. The use of polymeric adsorption resins immobilised in columns for

510

the detoxification is an attractive alternative, since they may be regenerated and reused

511

by washing them with organic solvents (ethanol, acetone); this could imply the

512

simultaneous recovery of retained phenolic compounds and furans. The present method

513

(including pretreatment, detoxification and fermentation) enabled the production of 43.9

514

g acetone/kg corn stover, 83.4 g butanol/kg corn stover and 3.61 g ethanol/kg corn

515

stover. These solvents could be recovered from the fermentation broth by two-stage gas

516

stripping, producing condensates with 46-91 g/L acetone, 169-263 g/L butanol and 4.5-

517

13.2 g/L ethanol, which separated spontaneously into an aqueous and an organic phase,

518

the latter containing 38-99 g/L acetone, 418-425 g/L butanol and 4.2-13.9 g/L ethanol.

519

21

520

Acknowledgements

521

The authors thank Novozymes China for kindly providing the enzymes. Authors thank

522

R. Antón del Río, N. del Castillo Ferreras and G. Sarmiento Martínez for their technical

523

help.

524 525

Funding

526

The present work has been performed as part of the H2020-WASTE-2015-two-stage

527

Agrocycle project (Sustainable techno-economic solutions for the agricultural value

528

chain. GA - 690142), funded by the European Union’s Horizon 2020 Research and

529

Innovation Programme. MH-V is supported by a postdoctoral contract (DOC-INIA,

530

grant number DOC 2013-010) funded by the Spanish National Institute for Agricultural

531

and Food Research and Technology (INIA) and the European Social Fund.

532 533

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30

TABLES Table 1. Chemical composition of corn stover. Note: Xylan comprises xylan, galactan and mannan.

Total carbohydrates (%) Soluble sugars (%) Glucan/Cellulose (%) Hemicellulose (%) - Xylan (%) - Arabinan (%) Klason lignin (%) Protein (%) Fat (%) Moisture (%) Ashes (%) Phenolic compounds (mg/g)

Corn stover 57.2 2.00 31.9 17.4 15.5 1.90 15.2 3.29 0.57 12.5 5.36 6.10

Table 2. Different nutrient supplementation and fermentation conditions applied to corn stover hydrolysates. Standard (S) Optimized (O) Nutrients Yeast extract (g/L) 5.0 KH2PO4 (g/L) 1.0 K2HPO4 (g/L) 0 NH4Cl (g/L) 2.1 MgSO4·7H2O (g/L) 0.2 FeSO4·7H2O (g/L) 0.01 MnSO4·7H2O (g/L) 0 Cysteine (g/L) 0.5 CaCO3 (g/L) 5.0 Fermentation conditions Temperature (°C) 35 Initial pH 6.00 Shaking (rpm) 100

5.0 1.0 1.0 2.1 0.2 0.01 0.01 0 8.0 28 5.42 100

Table 3. Composition of corn stover hydrolysates after H2SO4 pretreatment and enzymatic hydrolysis under various temperatures and acid concentrations in the physicochemical pretreatment. (*) Note: Numbers between brackets indicate the percentage of sugar recovery considering the total carbohydrate content of corn stover. Physicochemical pretreatment (0.89% H2SO4, 5 min) + Enzymatic hydrolysis

Physicochemical pretreatment (125°C, 5 min) + Enzymatic hydrolysis

125 °C

135 °C

145 °C

160 °C

0.89% H2SO4

1.13% H2SO4

1.37% H2SO4

1.60% H2SO4

1.84% H2SO4

Cellobiose

1.36

1.59

1.87

1.18

1.36

1.38

1.17

1.33

0.21

Glucose

21.7

20.8

23.3

29.8

21.7

20.7

19.1

21.1

17.9

Xylose

15.7

15.5

17.7

17.8

15.7

15.1

14.3

15.7

14.6

Rhamnose

< 0.05

< 0.05

< 0.05

< 0.05

< 0.05

< 0.05

< 0.05

< 0.05

0.14

Arabinose

1.69

1.82

1.76

1.67

1.69

1.77

1.73

1.8

1.85

40.5 (61%)

39.7 (59%)

44.6 (66%)

50.4 (75%)

40.5 (61%)

39.0 (56%)

36.3 (54%)

40.0 (59%)

34.7 (48%)

Formic acid

0.08

0.10

0.14

0.17

0.08

0.09

0.09

0.12

0.12

Acetic acid

2.47

2.6

2.93

3.25

2.47

2.52

2.44

2.73

2.95

< 0.02

< 0.02

0.04

0.09

< 0.02

0.03

0.03

0.04

0.05

5-HMF

0.16

0.12

0.23

0.43

0.16

0.08

0.09

0.11

0.09

Furfural

0.05

0.07

0.22

0.64

0.05

0.08

0.08

0.10

0.12

Phenolic compounds

1.06

1.09

1.12

1.24

1.06

1.15

1.19

1.23

1.38

Concentration (g/L) Sugars

Total sugars* Inhibitors

Levulinic acid

Table 4. Comparison of ABE fermentations from non-detoxified corn stover hydrolysates. Note: In all cases, an enzymatic hydrolysis was performed after physicochemical pretreatment.

Initial sugars (g/L)

A (g/L)

B (g/L)

E (g/L)

Sugar consumption (%)

t (h)

Reference

n.a.

15.2

n.a.

93

24

[32]

C. beijerinckii P260

0

0

0

0

n.a.

[7]

75.0

C. beijerinckii NCIMB 8052

2.30

3.70

n.a.

20

48

[19]

Batch.

52.0

C. acetobutylicum zzu-02

n.a.

6.93

n.a.

n.a.

n.a.

[10]

Batch.

52.0

C. beijerinckii zzu-01

n.a.

5.24

n.a.

n.a.

n.a.

[10]

Batch.

40.5

C. saccharobutylicum DSM 13864

2.70

5.38

0.27

83

96

This work

Batch.

47.2

C. saccharobutylicum DSM 13864

<0.05

0.12

<0.05

3.6

72

This work

Pretreatment

Other

Steam, 3% SO2. Water:solid 2:1. 160°C, 30 min.

Semicontinuous. In situ recovery (liquid-liquid).

81.0

C. acetobutylicum P262

H2SO4 1%, v/v. 160°C, 20 min. ~8% solid.

Batch.

60.0

H2SO4 2%, v/v. Steam explosion 0.7 MPa, 150°C, 15 min.

Batch.

Steam explosion 1.5 MPa.

Steam explosion 1.5 MPa. H2SO4 0.89%, w/w (~0.54% v/v). 125°C, 5 min. 10% solid. H2SO4 0.89%, w/w (~0.54% v/v). 160°C, 5 min. 10% solid.

n.a.: Not available.

Strain

Table 5. Concentrations of total sugars and inhibitors in corn stover hydrolysates after detoxification with three adsorption resins. Percentage changes are shown between brackets. Total sugars (g/L)

Formic acid (g/L)

Acetic acid (g/L)

Levulinic acid (g/L)

5-HMF (g/L)

Furfural (g/L)

Phenolic compounds (g/L)

Pretreatment at 125 °C, 0.89% H2SO4 w/w, 5 min Undetoxified

41.2

0.07

2.57

≤ 0.02

0.10

0.03

1.17

Amberlite® XAD-4 Dowex® Optipore® L-493 Dowex® Optipore® SD-2

35.1 (-14.7%)

0.07 (0%)

2.10 (-18.3%)

≤ 0.02 (0%)

0.02 (-80.0%)

≤ 0.02 (-33.3%)

0.19 (-83.8%)

33.8 (-17.8%)

0.06 (-14.3%)

1.78 (-30.7%)

≤ 0.02 (0%)

0.02 (-80.0%)

≤ 0.02 (-33.3%)

0.05 (-95.7%)

34.0 (-17.4%)

0.07 (0%)

2.06 (-19.8%)

≤ 0.02 (0%)

0.02 (-80.0%)

≤ 0.02 (-33.3%)

0.01 (-99.2%)

Pretreatment at 160 °C, 0.89% H2SO4 w/w, 5 min Undetoxified

50.4

0.10

3.61

0.08

0.36

0.78

1.43

Amberlite® XAD-4 Dowex® Optipore® L-493 Dowex® Optipore® SD-2

43.7 (-13.2%)

0.10 (0%)

2.95 (-18.3%)

≤ 0.02 (-75.0%)

≤ 0.02 (-94.4%)

≤ 0.02 (-97.0%)

0.19 (-86.7%)

44.4 (-11.9%)

0.11 (10.0%)

2.57 (-28.8%)

≤ 0.02 (-75.0%)

≤ 0.02 (-94.4%)

≤ 0.02 (-97.0%)

0.10 (-93.0%)

44.1 (-12.5%)

0.11 (10.0%)

2.93 (-18.8%)

≤ 0.02 (-75.0%)

≤ 0.02 (-94.4%)

≤ 0.02 (-97.0%)

0.06 (-95.8%)

Table 6. Comparison of ABE fermentations from corn stover hydrolysates detoxified with several methods. Note: In all cases, an enzymatic hydrolysis was performed after physicochemical pretreatment.

Pretreatment

Detoxification

Initial sugars (g/L)

Other

Strain

A (g/L)

B (g/L)

E (g/L)

Sugar consump. (%)

t (h)

Reference

Dilution with water 1:1

Batch

60.0

C. beijerinckii P260

4.70

10.4

0.90

62

96

[7]

Dilution with wheat straw hydrolysate 1:1

Batch

59.3

C. beijerinckii P260

5.10

12.3

0.64

70

84

[7]

Overliming

Batch

60.3

C. beijerinckii P260

8.00

14.5

3.77

99

85

[7]

Overliming

Batch

39.0

C. beijerinckii P260

5.55

6.04

0.89

89

96

[21]

Overliming

SSF

39.0

C. beijerinckii P260

4.82

8.98

0.40

87

73

[21]

Overliming

SSF and recovery

39.0

C. beijerinckii P260

8.10

11.6

1.11

100

60

[21]

Overliming

Batch

75.0

C. beijerinckii NCIMB 8052

7.80

10.4

0.41

71

48

[19]

Washing

Batch

53.5

C. acetobutylicum ATCC 824

1.15

0.36

2.20

52

72

[46]

Alkaline peroxide, 4% H2O2, 1% NaOH, 24 h. 10% solid biomass

Batch

45.0

C. acetobutylicum ATCC 824

̴2.70

̴8.30

̴1.20

96

72

[46]

Activated charcoal 7.5% (w/v), 30°C, 150 rpm, 12 h

Batch

49.0

C. acetobutylicum ATCC 824

̴2.50

̴8.40

̴1.50

91

72

[46]

Steam explosion 1.5 MPa.

Washing

Batch

57.5

C. acetobutylicum zzu-02

4.14

9.88

1.80

98

70

[10]

NaOH 2%, 121°C, 30 min.

Washing

Batch

71.3

C. beijerinckii CC101 (adaptative mutant)

7.50

11.2

1.10

68

57

[45]

H2SO4 1%, v/v. 160°C, 20 min. ~8% solid.

H2SO4 2%, v/v.Steam explosion 0.7 MPa, 150°C, 15 min.

Steam explosion, 1.1 MPa, 4 min.

Pretreatment

Detoxification

Other

Initial sugars (g/L)

Strain

A (g/L)

B (g/L)

E (g/L)

Sugar consump. (%)

t (h)

Reference

Deep eutectic solvents, 130°C, 2 h.

Washing

Batch

48.2

C. saccharobutylicum DSM 13864

̴1.20

5.63

̴0.30

71

48

[31]

Twin-screw extrusion, NaOH 8%, 99°C, 1 h. 33% solid.

Washing

Batch

42.4

C. acetobutylicum ATCC 824

̴2.70

7.10

̴1.20

93

72

[44]

Organosolv (60% ethanol, 4% NaOH), 110°C, 90 min.

Washing

Batch

30.0

C. beijerinckii NCIMB 4110 (mutant)

-

9.90

-

̴95

72

[43]

H2SO4 0.89%, w/w (~0.54% v/v). 125°C, 5 min. 10% solid.

Resin Dowex Optipore® SD-2

Batch

31.7

C. saccharobutylicum DSM 13864

3.70

5.58

0.28

96

96

This work

H2SO4 0.89%, w/w (~0.54% v/v). 160°C, 5 min. 10% solid.

Resin Dowex Optipore® SD-2

Batch

41.7

C. saccharobutylicum DSM 13864

4.75

9.02

0.39

97

72

This work

Table 7. Performance indicators of the first gas stripping stage for the fermented detoxified corn stover hydrolysate.

Fermentation broth Condensate

Volume (mL)

Acetone (g/L)

Butanol (g/L)

Ethanol (g/L) 0.42

Acetic acid (g/L) 1.61

Butyric acid (g/L) 0.95

Initial

3243

5.02

9.02

Final

3126

2.11

2.07

0.28

1.10

0.75

Mixture

117

46.26

169.26

4.53

< 0.05

< 0.05

Aqueous phase Organic phase

89

48.87

88.70

4.65

< 0.05

< 0.05

28

37.96

425.35

4.17

< 0.05

< 0.05

Table 8. Performance indicators of the second gas stripping stage applied to the aqueous phase from the first gas stripping stage.

Aqueous phase from the 1st stripping Condensate

Volume (ml)

Acetone (g/L)

Butanol (g/L)

Ethanol (g/L) 4.65

Acetic acid (g/L) < 0.05

Butyric acid (g/L) < 0.05

Initial

89

48.87

88.70

Final

67

2.01

1.60

0.86

< 0.05

< 0.05

Mixture

22

90.57

263.29

13.17

< 0.05

< 0.05

Aqueous phase Organic phase

12

99.54

129.67

13.19

< 0.05

< 0.05

10

98.59

417.71

13.88

< 0.05

< 0.05

FIGURES

Fermentation metabolites (g/L)

8 7

100 90 80

6 70 5

60

4

50 40

3

30 2 20 1

Sugar consumption (%)

Acetone Butanol Ethanol Acetate Butyrate Isopropanol Total sugars

10

0

0 M DS

86 13

4 M DS

64

23

6 2 3 2 1 8 0 52 20 22 28 51 32 92 52 79 55 79 50 -53 21 18 64 62 M 17 14 CT M M M M M SM SM SM SM LB DS M E S S S S S D D D D R C D D D D D DS NR

Figure 1. Sugar consumption and ABE parameters for a 96-h fermentation of nondetoxified corn stover hydrolysates (pretreatment 125 °C, 5 min, 0.89% w/w H2SO4) by several Clostridium strains.

100

8 7

b

95

b

6

b b

5

90

4 85

3 2

80

1 0

75 ol nt r Co

a

10

d Un

e xifi eto

d

Am

4 -2 93 DSD L-4 XA ore ore ptip ptip O O x x we we Do Do

100

a cd

9

c

ad

8

80

7 6

60

5 4

40

3 2

20

1

0 n Co

b

Acetone Butanol Ethanol Acetate Butyrate Total sugars

b

0

e rlit be

Sugar consumption (%)

105 a

Sugar consumption (%)

9

Fermentation metabolites (g/L)

Fermentation metabolites (g/L)

10

l tro d Un

eto

xif

ied

Am

4 -2 93 DSD L-4 XA ore ore ptip ptip O O x ex we ow Do

e rlit be D

Figure 2. Fermentation of corn stover hydrolysates before and after detoxification with three adsorption resins with C. saccharobutylicum DSM 13864. Different letters above butanol bars represent statistical differences among samples (p < 0.05). a) Pretreatment at 125°C, 0.89% H2SO4 w/w, 5 min. The control consisted of an aqueous solution containing 22 g/L glucose, 16 g/L xylose and nutrients. b) Pretreatment at 160°C, 0.89% H2SO4 w/w, 5 min. The control consisted of an aqueous solution containing 31 g/L glucose, 22 g/L xylose and nutrients. Note: More fermentation parameters are given in Table SM8.

Figure 3. Butanol recovery during the two-stage gas stripping process. Note: α, butanol selectivity factor; η, butanol recovery.

Highlights • A complete workflow for butanol production from corn stover has been developed. • Corn stover is efficiently pretreated with very dilute H2SO4 (0.89% w/w). • Corn stover hydrolysates can be detoxified with reusable polymeric resins. • The use of appropriate strains enables proper ABE production in this complex matrix. • Offline gas stripping is efficient for butanol recovery from fermentation broths.

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: