Scholarly article on topic 'Consolidated briefing of biochemical ethanol production from lignocellulosic biomass'

Consolidated briefing of biochemical ethanol production from lignocellulosic biomass Academic research paper on "Agricultural biotechnology"

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Electronic Journal of Biotechnology
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{Bioconversion / Bioethanol / Fermentation / Hydrolysis / "Renewable fuels"}

Abstract of research paper on Agricultural biotechnology, author of scientific article — Spyridon Achinas, Gerrit Jan Willem Euverink

Abstract Bioethanol production is one pathway for crude oil reduction and environmental compliance. Bioethanol can be used as fuel with significant characteristics like high octane number, low cetane number and high heat of vaporization. Its main drawbacks are the corrosiveness, low flame luminosity, lower vapor pressure, miscibility with water, and toxicity to ecosystems. One crucial problem with bioethanol fuel is the availability of raw materials. The supply of feedstocks for bioethanol production can vary season to season and depends on geographic locations. Lignocellulosic biomass, such as forest-based woody materials, agricultural residues and municipal waste, is prominent feedstock for bioethanol cause of its high availability and low cost, even though the commercial production has still not been established. In addition, the supply and the attentive use of microbes render the bioethanol production process highly peculiar. Many conversion technologies and techniques for biomass-based ethanol production are under development and expected to be demonstrated. In this work a technological analysis of the biochemical method that can be used to produce bioethanol is carried out and a review of current trends and issues is conducted.

Academic research paper on topic "Consolidated briefing of biochemical ethanol production from lignocellulosic biomass"


EJBT-00183; No of Pages 10

Electronic Journal of Biotechnology xxx (2016) xxx-xxx

Contents lists available at ScienceDirect

Electronic Journal of Biotechnology


Consolidated briefing of biochemical ethanol production from lignocellulosic biomass

Spyridon Achinas *, Gerrit Jan Willem Euverink

Faculty of Mathematics and Natural Sciences, University of Groningen, Nijenborgh 4, 9747 AG Groningen, Netherlands ARTICLE INFO ABSTRACT

Article history: Received 11 February 2016 Accepted 1 April 2016 Available online xxxx




Crude oil reduction


Environmental compliance



Fossil fuels



Renewable fuels

Technological progress

Bioethanol production is one pathway for crude oil reduction and environmental compliance. Bioethanol can be 16 used as fuel with significant characteristics like high octane number, low cetane number and high heat of 17 vaporization. Its main drawbacks are the corrosiveness, low flame luminosity, lower vapor pressure, miscibility 18 with water, and toxicity to ecosystems. One crucial problem with bioethanol fuel is the availability of raw 19 materials. The supply of feedstocks for bioethanol production can vary season to season and depends on 20 geographic locations. Lignocellulosic biomass, such as forest-based woody materials, agricultural residues and 21 municipal waste, is prominent feedstock for bioethanol cause of its high availability and low cost, even though 22 the commercial production has still not been established. In addition, the supply and the attentive use of 23 microbes render the bioethanol production process highly peculiar. Many conversion technologies and 24 techniques for biomass-based ethanol production are under development and expected to be demonstrated. In 25 this work a technological analysis of the biochemical method that can be used to produce bioethanol is carried 26 out and a review of current trends and issues is conducted. Q2

© 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. 29 This is an open access article under the CC BY-NC-ND license (


1. Introduction..............................................................................................................................0

2. Current status............................................................................................................................0

3. Lignocellulosic sources and composition....................................................................................................0

3.1. Raw materials and characteristics....................................................................................................0

3.1.1. Forest woody sources......................................................................................................0

3.1.2. Agricultural and municipal solid wastes (MSW)..............................................................................0

3.1.3. Marine algae..............................................................................................................0

3.2. Lignocellulosic molecular components................................................................................................0

3.2.1. Hemicellulose..............................................................................................................0

3.2.2. Cellulose..................................................................................................................0

3.2.3. Lignin....................................................................................................................0

4. Processing routes to bioethanol............................................................................................................0

4.1. Pretreatment......................................................................................................................0

4.2. Hydrolysis........................................................................................................................0

4.3. Fermentation......................................................................................................................0

5. Recent issues in bioethanol production......................................................................................................0

5.1. Is recalcitrance of biomass a barrier? ................................................................................................0

5.2. Sustainable balance of water-biofuels................................................................................................0

5.3. Gap between biotech research and commercialization..................................................................................0

5.4. Bioethanol-based economy..........................................................................................................0

* Corresponding author. E-mail address: (S. Achinas).

Peer review under responsibility of Pontificia Universidad Católica de Valparaíso.

http: //

0717-3458/© 2016 Pontificia Universidad Católica de Valparaíso. Production and hosting by Elsevier B.V. All rights reserved. This is an open access article under the CC BY-NC-ND license (


S. Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2016) xxx-xxx

6. Conclusion................................................................................................................................0

Uncited reference..............................................................................................................................0


1. Introduction

Nowadays, the depletion of fossil fuels and the environmental compliance regarding the greenhouse gases has attracted the interest in non-conventional fuel from bioresources [1,2,3,4,5]. For the past few years, the biomass-based ethanol has caught the attention of global industry. According to the Renewable Fuels Association [6], United States (U.S.) and Brazil are the pioneer countries in global bioethanol production with a percentage of approximately 90%. The involvement of several countries has already begun in new pathway development for biogasoline from biomass [7]. Wheals et al. [8] refer that in North America, bioethanol is primarily provided from starch sources (corn starch) while in South America is mostly extracted from sugars (sugarcane juice) and molasses [8,9]. (See Tables 1-3.)

On the other side, the European countries focus on biodiesel and biogasoline production which exceeds 50% of the global production cause of engines development and feedstocks supply costs [10,11,12, 13,14]. Despite the fact that most of the countries in the world, China, India and Japan continue to invest in technologies from agricultural residues and appear as future producers [15,16,17,18,19]. Although bioethanol based on corn and sugar is an encouraging replacement to gasoline in transportation sector, the amount produced is insufficient with respect to the annual consuming amount worldwide. There is no black-and-white answer to the question of what constitutes the most suitable feedstock for the bio-based economy. Generally, sugars, oils and proteins can be used in many applications. The concern for the food security has globally increased the interest of researchers to focus on alternative feedstocks [20,21,22].

The nova Institute of Germany claims that lignocellulosic resources are favorable in terms of environmental sustainability and food security as they do not antagonize food crops and animal feed as renewable substrate for bioethanol production [23,24]. Moreover, the availability of lignocellulosic materials in industrial-scale basis is increased cause of the exploitation of industrial wastes and agricultural residues [25,26,27]. Lignocellulosic wastes are a promising feedstock considering its availability and low cost. The utilization of corn stover, rice, wheat and sugarcane bagasse is gaining significant importance worldwide [28,29,30,31].

Nonetheless, the recalcitrant structure of lignocellulose requires high capital cost processing. Therefore, these technologies are not economically achievable [32,33]. During the decomposition of lignocellulosic material, it must be considered that D-xylose is the second important sugar which has to be broken down as is found in high portion in the feedstock [34]. The conversion of biomass to ethanol has 4 main steps: pretreatment, hydrolysis, fermentation and distillation. During the last decades genetic engineering and enzymatic processing have provided significant improvements in all of the four steps of ethanol production and making capable to ferment different sugars concurrently [35,36,37]. Even though there is a wide range of

Table 1

Top five bioethanol producers (billion gallons) [45]

Country 2008 2010 2012 2014

US 9.31 13.30 13.22 14.34

Brazil 6.47 5.57 5.57 6.19

Europe 0.73 1.21 1.14 1.45

China 0.50 0.54 0.56 0.64

Canada 0.24 0.36 0.45 0.51

bacteria, they cannot all be adapted to saccharification process 123 conditions and several bacteria produce low ethanol yields. For this 124 reason, subtle improvements are sometimes required [38]. 125

The microbial contamination is a crucial problem in bioethanol 126 production process. Bacterial infections occur during bioethanol 127 fermentation which consume nutrients necessary for the fermentation 128 itself and it is possible to produce toxic products too. Both of these 129 situations can negatively affect the bioethanol yield [39,40]. The 130 formation of inhibitory by-products during the biofuel production must 131 be taken into account. Pienkos and Zhang [41] refer that pretreatment 132 and conditioning processes release toxic compounds into the 133 hydrolysate which inhibit the bacteria growth and decrease the ethanol 134 yield. The mechanism/methodology applied for biomass pretreatment 135 influences the relevant toxicity rate [41,42]. This review examines 136 recent technologies and trends that are used in lignocellulosic 137 bioethanol production. It also provides a summary of the current 138 problems and barriers concerning the different pathways and analyses 139 potential issues and trends of biotechnological conversion performance. 140

2. Current status 141

In 2014, the global production ofbioethanol reached 24.5 billion gal, 142 up from 23.4 billion gal in 2013 which shows the international 143 bioethanol market is at a very dynamic stage [43]. More than half 144 (about 60%) of global bioethanol production is based on sugar cane 145 conversion and the rest (40%) comes from other crops [44]. United 146 States and Brazil are the global producers as they produce more than 147 70% of the global bioethanol production. 148

Even the main source for bioethanol production is considered to be 149 the corn from US and sugar cane from Brazil, any country with 150 agro-industrial economy can be involved in bioethanol fermentation. 151 This is feasible cause of the current progress in bioconversion of 152 non-food crops in large scale production [46]. 153

In Europe the biochemical pathways show a crucial potential 154 for research development in conjunction with the progress in 155 biorefineries. It is important to clarify that several technologies are 156 under development such as the SSCF technology which gains space in 157 biotechnology research area. Research requires effort to solve problems 158 concerning process improvement and confront challenges regarding 159 the overall efficiency of a biorefinery [47]. It was also reported in 2009 160 that notwithstanding the global economic-constraints, bioethanol 161 production continues to increase and to support significantly to the 162 global development [48]. 163

3. Lignocellulosic sources and composition 164

3.1. Raw materials and characteristics 165

Sustainable biofuel production in Europe can be met with 166 lignocellulosic biomass usage [49]. There is a wide variety of raw 167 materials that are discerned by their makeup, structure and 168 process-ability. In North America most cultivated land comprises. 169

The land cultivation is mainly based on forestland (around 35%), 170 grazed land (27%) as well as crop lands (19%) which constitute 171 approximately 9.0 million km2 [51,52,53]. Forest sources include 172 woody biomass consisting mainly of residues or by-products from 173 manufacturing processes, biomass plantations, agricultural residues 174 (trees and branches) [54,55]. Cellulose materials can also be collected 175


S. Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2016) xxx-xxx

t2.l Table 2

t2.2 Total energy potential from different feedstocks (KTOE). [50].

t2.3 Different biomass sources availability 2004-2010 2020

t2.4 Netherlands EU-27 Netherlands EU-27

t2.5 Biomass from agricultural land and by-products Woody residues of fruit trees, nuts and berry plantations, olives, 16 9362 13 10105

citrus and vineyards

t2.6 Straw 39 22936 195 49285

t2.7 Manure 3916 56817 4574 46724

t2.8 Grassland cutting 38 1097 40 1143

t2.9 Biomass from forestry Primary forestry residues 22 20285 71.5 41186

t2.10 Round wood 148.4 56735 137.8 56115

t2.11 Sawmill by-products (excluding saw-dust) 21 9072 31 10093

t2.12 Saw-dust 10 4496 - 4984

t2.13 Other industrial wood residue - 4637 - 5461

176 from municipal and industrial wastes which include food residues and

177 pulping sludge [56,57].

178 3.1.1. Forest woody sources

179 According the taxonomical division of woody materials, there are

180 two species: softwoods and hardwoods. Softwoods are gymnosperms

181 and originate from coniferous trees including pines, spruces and firs.

182 Hardwoods are angiosperms and originate from deciduous trees

183 including oaks, maples and birches [59].

184 Fig. 2 shows the type of forest biomass that can be supplied globally.

185 Forest biomass represents a valuable feedstock cause of its composition

186 (more lignin and less ash content than agricultural residues). Forestry

187 wastes like wood chips, branches, and sawdusts have also been used Q6 Q5 as bioethanol feedstocks [60]. (See Fig. 1.) (See Figs. 3-5.)

189 3.1.2. Agricultural and municipal solid wastes (MSW)

190 Agricultural residues are a widespread lignocellulosic biomass

191 source available in many countries. The available amount of

192 agro-residues is estimated to be 1010 Mt. globally, which corresponds

193 to an energy value of 47 EJ [61]. Crops residues consist of an extensive

194 variety of types. They are mostly comprised of agricultural wastes

195 such as corn stover, corn stalks, rice and wheat straws as well as

196 sugarcane bagasse [62]. Crop residues contain more hemicellulosic

197 material than woody biomass (approximately 25-35%) [63]. Besides

198 from environmental point of view, agricultural residues help to avoid

199 non-sustainable cutting trees decreasing the phenomenon of Q7 deforestation [30].

201 Moreover, municipal and industrial solid wastes are also a

202 prospective pathway for biofuels production [64]. Li [65] studied that

203 integrated bioconversion of cellulose-enriched municipal solid waste

204 offers promising alternatives but the processing cost is still high.

205 However, their utilization associated with the disposal of garbage,

organic waste and household by-products has to be considered in case 206 of environmental effects [66]. Even though intensifying crop 207 management is applied to improve yields, the high cost of biomass 208 still remains a crucial constraint [67]. According to this study the 209 available amount of biomass for 30% petroleum-based gasoline 210 displacement will almost meet the target of 2030 [68]. 211

3.13. Marine algae 212

Since the 1970s special interest has existed in marine algae as third 213 generation biofuel feedstock but the research was discontinued when 214 funding stopped. Particularly the research has focused on examination 215 of its production efficiency per acre including water consumption and 216 estimation of by-products during ethanol production [62]. Even 217 though exists progress in algae development commercial applications 218 are still limited during the 20th century. Currently, algae conversion is Q8 regaining interest as future biofuel feedstock in order to replace 220 energy crops and cover any limitations in supply. 221

Marine algae are a suitable raw material for several chemical 222 processes especially due to biorefineries expansion that aims at the 223 production of different substances such as biofuels (i.e. bioethanol, 224 biodiesel, biogasoline etc.) and other value-added chemicals [69]. 225 Rodolfi et al. [70] state that algae feedstock can provide 60 times more 226 alcohol than soybeans per acre of land. According to the study of 227 Ferrel and Sarisky-Reed [71] algae can provide ten-fold the amount of 228 ethanol than corn per growing area. Harel [72] refers that algae are 229 consuming high amounts of CO2 during their growth which make 230 them very attractive to use as an environmental friendly feedstock. 231

3.2. Lignocellulosic molecular components

The main components of lignocellulosic biomass are cellulose 233 (30-35%), hemicellulose (25-30%) and lignin (10-20%) In addition, 234

t3.l Table 3

t3.2 Pros-and-cons of potential microorganisms for bioethanol fermantation [140].

t3.3 Species

t3.4 Saccharomyces cerevisiae

t3.5 Z. mobilis

t3.6 Escherichia coli

t3.7 Thermophilic species:

t3.9 t3.10

• Thermoanaerobacter

• Clostridium

-Alcohol yield up to 90%

-High tolerance to chemical inhibitors and to ethanol (10% v/v)

-Naturally adapted to ethanol


-Complaisance to genetic modifications -Bioethanol yield up to 97% -High ethanol tolerance (up to 14% v/v) -Does not require additional oxygen -Complaisance to genetic modification -Ability to use both pentose and hexose sugars -Amenability for genetic modifications

-Resistance to high temperature of 70°C. -Suitable for consolidated bioprocessing -Ferment a variety of sugars -Amenability to genetic modification

-Not able to ferment xylose and arabinose sugars -Not able to survive at high temperature of hydrolysis

-Not able to ferment xylose sugars -Low tolerance to inhibitors

-Low tolerance to inhibitors and ethanol -Narrow pH and temperature growth range -Production of organic acids -Low tolerance to ethanol


4 S. Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2016) xxx-xxx

Simultaneous sacchariftcation and fermentation (SSF)

Fig. 1. Stages of bioethanol fuel production.

235 lignocellulose contains protein, lipids, water and other items [73,74,75,

236 76]. Cellulosic and hemicellulosic polymers constitute approximately

237 70% of the entire biomass and are connected to the lignin component

238 through a variety of covalent bonds that give the lignocellulosic

239 biomass significant robustness and resistance to (bio-)chemical or

240 physical treatment [77,78].

that for the conversion of cellulosic crystalline to an amorphous 256 structure, a temperature of 320°C and a pressure of 25 MPa is 257 required. Cellulose is the rifest organic polymer on earth and make up 258 30% of plant biomass. However, cotton consists of almost 100% 259 cellulose [84]. 260

241 3.2.1. Hemicellulose

242 Hemicellulose has a vague and changeable structure of

243 heteropolymers including hexoses (glucose, galactose, mannose),

244 pentoses (xylose, arabinose) as well as sugar/uronic acids (glucuronic,

245 galacturonic, methylgalacturonic) [79]. The hemicellulosic chain

246 consists of xylose (90%) and arabinose (10%). Xylan is the primary

247 component of hemicellulose and its composition varies in each

248 feedstock. For this reason, hemicellulose stands in need of wide variety

249 of enzymes to be completely hydrolyzed into free monomers [80,81,82].

250 3.2.2. Cellulose

251 Cellulose is a linear polymer which contains several thousand of

252 1,4-b-glucosidic bonds connecting thousands of glucose units. The

253 structure is crystallic because of the hydrogen bridges between the

254 polymers. This large amount of hydrogen linkages provides toughness

255 and compactness to the cellulose molecule. Deguchi et al. [83] refer

3.2.3. Lignin 261

Lignin is a complex polymer coupled via covalent bonds to xylans 262 rendering massiveness and stability to the plant cell wall. It contains 263 three main monomers, coumaryl alcohol, coniferyl alcohol, and 264 sinapyl alcohol [75]. Lignin is a copious natural polymer and a 265 dominant constituent of wood (30-60% for softwoods and 30-55% for 266 hardwoods), while agricultural residues and grasses contain 3-15% and 267 10-30% respectively [63]. Contrarily, crop residues like corn stover, rice 268 and wheat straws contain particularly hemicellulose. Heretofore, lignin 269 effects on hydrolysis have partially been investigated, even though in 270 recent studies it is reported that lignin characteristics, such as structure 271 and composition, can positively contribute to the whole hydrolysis 272 process [85]. Chen et al. [86] pointed out that lignin modification via 273 genetic engineering techniques could increase the bioethanol yield and 274 furthermore to be a potential source to give biorefineries financial 275 solvency [86]. 276

Forest biomass

Primary residues

2. Secondary residues

3. Tertiary residues

Forest residues:

- Logging residues

- Residues from first and intermediate residues

- Stumps

Forest manufacturing residues:

- Bark

- Chips and slabs

- Sawdust from kei f

- Shavings

- Endings and cross cut ends

- Black liquor

Used wood residues from:

- Construction

- Demolition

- Wooden packaging

Fig. 2. Different types of forest biomass. Adopted from the source [58].


S.Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2016) xxx-xxx

Bioma» Haodhig

Вютаи Pretreatment

Enzyme Production




Glucose Fermentation

Pentose „ FtrmenUîxxi

Ethanol Recovery



Fig. 3. Schematic of a biochemical cellulosic ethanol production process. Adopted from the source [95].

277 4. Processing routes to bioethanol

278 There are two different approaches (i.e. biochemical and

279 thermochemical conversion) for bioethanol production from biomass

280 [87]. Both pathways conclude into fragments of lignin, hemicellulose

281 and cellulose via degradation of lignocellulose. Polysaccharides are

282 hydrolyzed into sugars and subsequently are converted into

283 bioethanol [88,89]. However, these conversion technologies are not

284 similar techniques. Mu et al. [90] state that the thermochemical route

285 includes feedstock gasification at 800°C with a catalytic reaction to

286 ensue. This technology requires high level of heat and results into a

287 synthesis gas (syngas) such as CO, H2 and CO2. Syngas can be

288 chemically converted into a mixture of alcohols at 300°C using MoS2

289 as the catalyst. Ethanol is separated from the mixture via distillation

290 [91]. Alternatively, syngas can also be further processed into ethanol

291 using the microorganism Clostridium Ijungdahlii, Saccharomyces

292 cerevisiae or Zymomonas mobilis [92,93,94].

293 In contrast to the thermochemical pathway towards syngas, the

294 biochemical route includes mild physical and/or thermochemical

295 pre-treatment, and biological pretreatment using hydrolytic enzymes

296 to degrade cellulose and hemicellulose. The physical and/or

297 thermochemical pretreatment is mainly used to overwhelm

298 contumacious substances and boost cellulose availability/accessibility

299 to cellulases and hemicellulases in the biological pretreatment to

300 produce the monomeric sugars. [96,98].

301 The upstream process includes hydrolysis of cellulose and breakdown

302 of hemicellulose into soluble sugars. Afterwards the sugars are converted

303 into bioethanol via fermentation and pure ethanol is produced via

304 distillation [88,97]. Contemporaneously, the recalcitrant by-product,

305 lignin, can be combusted and converted into power and heat [89].

In general, biochemical conversion consists of four unit operations 306

i.e. pretreatment, hydrolysis, fermentation and distillation [99,100]. 307

Nowadays, the biochemical approach is the most commonly used 308

process [101]. 309

4.1. Pretreatment 310

Hydrolysis and downstream processing can be optimized by effective 311

pretreatment. The basic treatment methods include physical and 312

thermochemical processes which disrupt the recalcitrant materials and 313

enable the cellulose be undergone hydrolysis with higher efficiency Q9

and lower energy consumption [102]. The pretreatment process 315

required for each feedstock was chosen according to its characteristics. Q10

Zhu and Pan [103] reported that agricultural biomass treatment differs 317

from woody biomass because of its physical properties and chemical 318

composition. Unlike agricultural biomass, woody biomass requires high 319

content of energy to reach size reduction for further enzymatic 320

saccharification. 321

Toxic compounds have also to be considered for evaluating the 322

pretreatment cost. Different substances may act as inhibitors of 323

microorganisms that are used in the ethanol fermentation. These 324

inhibitors include phenolic compounds, furans (furfurals and 5-HMF), Q11

aliphatic acids and inorganics compounds (iron, chromium or nickel). 326

Several alternative measures can be taken to avoid problems caused 327

by inhibitors [104]. The detoxification process is an important step 328

which can affect the pretreatment performance [103,105,106]. General 329

feedstock versatility and toxic inhibitors produced have to be 330

considered on the pretreatment efficiency in order to reach optimal 331

conditions [107]. 332

-O HO-

0H Cellulose



Fig. 4. Mechanism of acid-catalyzed cellulose hydrolysis to glucose. HMF = hydroxymethylfurfural, LA = levulinic acid, FA = formic acid [113,114]


Fig. 5. Mechanism of the enzyme catalyzed hydrolysis of cellulose into glucose [116]

333 42. Hydrolysis

334 The performance of the hydrolysis is highly associated to the

335 pretreatment process [80]. During this reaction, cellulose and

336 hemicellulose are hydrolysed into simplistic and soluble compounds

337 available for further conversion (fermentation) to ethanol [88]. There

338 are two different types of hydrolysis processes that involve either

339 acidic (sulfuric acid) or enzymatic reactions. The acidic reaction can be

340 divided into dilute or concentrated acid hydrolysis. Dilute hydrolysis

341 requires a high temperature of 200-240°C to disrupt cellulose crystals

342 [108]. On the other side, concentrated acid hydrolysis is a more

343 effective method as it produces higher amount of free sugars (80%)

344 and lower concentrations of inhibitors. Despite this process requires Q12 high quantity of acid which makes it usage less attractive [109,110].

346 When acids are used in the hydrolysis, the phenomenon of chemical

347 dehydration occurs on monosaccharides resulting in the appearance of

348 other compounds like aldehydes [20]. This specific issue has driven the

349 researcher to focus on enzymatic hydrolysis. Compelling pretreatment

350 is fundamental to an efficient enzymatic hydrolysis [111]. Eggeman

351 and Elander [112] have demonstrated that Trichoderma reesei is a

352 very efficient fungus to produce industrial grade cellulolytic enzymes.

353 Recent studies proved that lignin is a source of sustainable energy

354 and added-value compounds. The application of metal components

355 like Ca(II) and Mg(II) could intensify the enzymatic hydrolysis

356 [112,115].

Sewalt et al. [117] have reported that the unfavorable influence of 357 lignin on cellulases activities can be surpassed by ammonium and 358 N-based components. Spindler et al. [118] report that the enzymatic 359 pretreatment can be attained in simultaneous way with the 360 co-fermentation (known as simultaneous saccharification and 361 fermentation (SSF)) process in order to produce ethanol from woody 362 biomass. In SSF process the concentration of saccharides is kept low 363 and cellulose inhibition is deterred. In a separate hydrolysis and 364 fermentation (SHF) process cellulases (hydrolytic enzymes) are 365 inhibited by glucose and cellobiose (saccharide products) resulting in 366 a slower process and a lower yield of fermentable sugars [119]. 367

4.3. Fermentation 368

Fermentation is the following step and requires the presence of 369 microorganisms to degrade sugars into alcohols and other end 370 products. The previously described processes are fundamental for the 371 fermentation process [88,89]. Typically S. cerevisiae converts the 372 sugars into ethanol under anaerobic conditions at a temperature of 373 30°C. In this pathway other by-products are also generated in the 374 form of CO2 and N-based compounds. S. cerevisiae is a prevalent 375 microorganism and provides a high yield of ethanol (12.0-17.0% w/v; 376 90% of the theoretical yield) from sugars [119]. 377

The SHF is the traditional method for bioethanol production. Several 378 studies have reported the weakness of S. cerevisiae to ferment only 379


S.Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2016) xxx-xxx

380 hexose sugars and the interest for versatile-acting microorganisms

381 increased [121]. To date, extensive research has been conducted to

382 develop microorganisms which enable to i) ferment pentose and

383 hexose sugars synchronously available from the hemicellulose fraction

384 and ii) endure under inhibitory conditions. Recently, research

385 attention focuses on efficient techniques like SSF in order to establish

386 a consolidated bioprocessing so that hydrolysis and fermentation

387 occur in a single reactor. This leads to a reduction in costs and

388 avoidance of high amount of inhibitory compounds. While there is a

389 wide variety of microorganisms which are able to convert sugars to

390 ethanol as well as the use of one microorganism seems promising for

391 efficient fermentation, their limitation from the standpoint of ethanol

392 yield, tolerance to chemical inhibitors and temperature is still obvious

393 in many demonstrated projects [85].

394 The end-product from fermentation process is a mixture of

395 ethanol-water and requires further separation through a distillation

396 process. Fractional distillation is a very common process to separate

397 ethanol from water based on their different volatilities. The distillation

398 column is heated and on the top of the column the distillate

399 (bioethanol) is collected as it has lower boiling point (78.3°C) whereas

400 water's boiling point is (100°C). However, the concentration of the

401 ethanol distillate is about 92% and further dehydration is required to

402 obtain 99% ethanol [25].

403 5. Recent issues in bioethanol production

404 5.1.1s recalcitrance ofbiomass a barrier?

Q13 Despite lignocellulosic biomass is a promising feedstock for

406 biorefineries, its recalcitrant structure and complexity make up an

407 economic and technical constraint to lignocellulosic-based biofuel

408 production. The three constituents ofbiomass (cellulose, hemicellulose

409 and lignin) enhance its compactness and strength. There are strong

410 linkages between molecules resulting in a complex structure of

411 lignocellulosic material. As a consequence, it is necessary to use specific

412 enzymes as a pre-treatment for fermentation [122].

413 Moreover, there are other materials which are inhibitory, such as

414 xylose, and must be removed in order to prevent any negative

415 influence to enzymatic hydrolysis [123,124]. Recent studies have

416 indicated that bioconversion efficiency is related to the pretreatment

417 performance [103]. For instance, the recent SPORL treatment

418 technology is of great interest for its broad on acting in different types Q14 of woody materials [126,127]. Zhu et al. [128] reported that SPORL

420 technology is effective for softwoods (e.g., spruce and red pine) and

421 capable to solve problems concerning their poor digestibility in

422 enzymatic saccharification. The SPORL was effective even when it was

423 applied to directly pretreat wood chips without chip impregnation.

424 Generally, each feedstock has different characteristics and for this

425 reason the pretreatment process has to be chosen carefully [125].

426 The recalcitrance issue still remains a technical constraint that has to

427 be eliminated. This problem is not current but is concerned to the

428 evolution mechanism of natural plants which have developed those

429 mechanisms to resist and avoid the attack of insects on theirs sugars.

430 In general this 'natural' recalcitrance of plants makes up an

431 impediment for the transformation of lignocellulosic biomass into

432 fermentable sugars. For this reason, research development has been

433 focused on sugars capture by re-engineering (genetic techniques

434 applied in cell wall structure) in order to increase the sugar yields

435 following by enzymatic hydrolysis. The use of such approaches may

436 promote and accelerate the future use of lignocellulosic feedstocks for

437 the bioethanol industry [129].

438 5.2. Sustainable balance ofwater-biofuels

439 Water consumption in sustainable biorefineries is a crucial issue

440 considering the industrial and agricultural practices implemented to

date [130]. Although water resources are not constraint for countries 441 such United States, Canada and Brazil, for other countries like China 442 and India water availability is a crucial issue which project 443 investments have to be encountered [131,132]. In United States, the 444 production of energy feedstocks and fuels requires substantial water 445 input. So far, bioethanol from lignocellulosic resources is produced in 446 laboratory and pilot scale. 447

The Argonne National Laboratory refers that the water requirements 448 for lignocellulosic ethanol production vary with technology and invokes 449 that nearly 35 l of water required to produce biochemically 3.5 l of 450 cellulosic ethanol [133,134]. The U.S. National Academy of Science 451 (NAS) has reported that the overuse of water via the expansion of 452 energy crops makes up serious problem. Even the biorefineries 453 consume a specific amount of water, the main problem is concerned 454 with the water used for cultivation [135,136]. Huffaker [137] states 455 that significant steps are required and must include best available 456 practices (BATs) (for instance recycling) for sustainable use of water. Q15

5.3. Gap between biotech research and commercialization 458

Bioethanol production from lignocellulosic biomass at large scale has 459 not yet been demonstrated as an economically feasible option. Research 460 efforts have to focus on second generation (cellulose-based) bioethanol 461 because it has potential to be improved. A wide variety of technical 462 problems occur in the different steps of bioethanol processing from 463 pretreatment to the final separation of the ethanol-water mixture. 464 Further development has to be carried out in order to mature and 465 consequently to industrialize the second-generation-based production 466 technologies. However, the comprehension of the interconnection 467 between science and applied technology is crucial to identify the voids 468 and rifts of research-industry system, so that through an overall 469 analysis the socio-economical, technical and environmental aspects can 470 be determined [138]. 471

However, in order to reduce the cost of bioethanol production, it is 472 necessary to clarify the important technological steps (i.e. enzyme 473 development: activity, stability and production costs). Many companies 474 are developing enzymes to increase the range of applications and the 475 performance of the enzymatic hydrolysis of cellulose and hemicellulose. 476 The hydrolysis n may involve the application of micro-organisms (fungi, 477 yeast, bacteria) and/or enzymes. The choice of micro-organisms and/or 478 catalysts has to be made in terms of type and quantity as this has an 479 impact on conversion rates and process stability. However, the use of 480 enzymes and microorganisms increases the production cost of 481 lignocellulosic ethanol. Further research has to be conducted in the area 482 of microorganisms and enzymes to increase the conversion efficiencies, 483 decrease the cost of microorganisms and enzymes to positively 484 contribute to profitable lignocellulosic-based ethanol production plants 485 [139]. 486

5.4. Bioethanol-based economy 487

Bioethanol economy is based on different factors like feedstock 488 availability, bioprocessing technology efficiency, and end-products 489 characteristics. There is a wide variety of sources (corn starch, sugar 490 cane lignocellulosic biomass, etc.) with low cost and high availability 491 which can be used for bioethanol. Research & Development 492 communities have to focus on the development of cheap and efficient 493 bioconversion technology of solid cellulosic materials into bioethanol 494 as a feasible industrialized technology in order to be considered 495 economically attractive. 496

Furthermore, significant initiatives like the registration of cellulosic 497 bioethanol for sale and use under the RFS eliminate the gap between 498 research and commercialization. Blenders and refiners of 499 transportation fuels are obligated under the RFS to include certain 500 percentages of renewable fuels in their total fuel sales. Industry 501 ensures that since cellulosic bioethanol technology is ready for 502


S. Achinas, G.J.W. Euverink / Electronic Journal of Biotechnology xxx (2QÍ6) xxx-xxx

503 commercialization. The production of bioethanol could reach the

504 required levels to be economically viable from the demand caused by

505 the RFS. Both lawmakers and industry expect that the creation of a

506 guaranteed market as federal programs such as grants, loans, and tax Q16 incentives boost the market introduction of this fuel [140,141].

508 However, the lignocellulosic-base ethanol is not yet widely

509 demonstrated because of its high costs [142]. In addition, efforts have

510 to be continued and studies to be carried out to optimize the

511 efficiency of the existing process technology from the pretreatment to

512 the dehydration [143]. There are margins for further development and

513 combination (i.e. consolidated bioprocessing) of these pilot

514 technologies in order to achieve higher bioethanol yields. Especially

515 processes based on enzyme technology have high cost and for this

516 reason have to be improved [144]. Bioethanol production plays a key

517 role on bio-based economy as there are strategic perspectives for

518 global producers, mainly US and Europe, especially when the price of

519 oil is reduced.

520 6. Conclusion

521 In the next decades, biomass will be the most meaningful renewable

522 energy source as an alternative to fossil fuels. Lignocellulosic bioethanol

523 is a potential pathway for the global producers which provide renewable

524 fuels. Bioethanol production will be probably the most successful biofuel

525 because it has plenty of usable forms (heat, power, electricity or vehicle

526 fuel). Different feedstocks can be used in bioethanol production and

527 studies have focused on their characteristics. The benefits anticipated

528 from mandated use of cellulosic biofuels include energy security

529 through domestic production of transportation fuel and environmental

530 improvement through the reduction of greenhouse gas and other

531 particulate emissions associated with fossil fuel combustion. Additional

532 benefits include creating new markets for agricultural products,

533 keeping productive farmland in use, and improving trade balances.

534 The main steps leading to the end-user product (bioethanol) are

535 pretreatment, hydrolysis, fermentation and separation/distillation. High

536 attention has to be given for all four major steps so that the

537 bioconversion will be optimized and the ethanol yield increased. In the

538 USA and Europe, previous and planned research initiatives and efforts

539 are still funded by federal sources. Also significant research funding

540 exists through various companies which are making investments in

541 applied research that addresses topics concerning the genetics of

542 energy crops, the production of stable and active hydrolytic enzymes,

543 the further development of yeast and bacterial ethanol fermentation

544 systems. Even though technological advances and research efforts are

545 still progressing, multiple configurations of systems and techniques are

546 developed in order to design efficient, sustainable and economically

547 feasible bioethanol production technologies and confront issues Q17 concerning the feedstocks and operations costs.

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