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Butanol production from lignocellulosics

Article in Biotechnology Letters · April 2012

DOI: 10.1007/s10529-012-0926-3 · Source: PubMed

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 Biotechnology Letters
Butanol production from lignocellulosics
--Manuscript Draft--

Manuscript Number: BILE3632R1

Full Title: Butanol production from lignocellulosics

Article Type: Review (invited by Dr. Archer)

Section/Category: Biofuels and Environmental Biotechnology

Keywords: butanol; Aceton-Butanol-Ethanol-fermentation; downstream; chemical catalysis;

lignocellulosics

Corresponding Author: Tom Granström, PhD

Aalto University
Espoo, FINLAND

Corresponding Author Secondary

Information:

Corresponding Author's Institution: Aalto University

Corresponding Author's Secondary

Institution:

First Author: German Jurgens

First Author Secondary Information:

Order of Authors: German Jurgens

Shrikant Survase

Oxana Berezina

Evangelos Sklavounos

Juha Linnekoski

Antti Kurkijärvi

Minna Väkevä

Adriaan van Heiningen, Professor

Tom Granström, PhD

Order of Authors Secondary Information:

Abstract: Various strains from genus Clostridium are producing n-butanol in a process called
ABE- fermentation. In terms of economical and sustainable industrial scale butanol
production a number of obstacles need to be addressed including choice of feedstock,
low product yield, toxicity to production strain, multiple end products and downstream
processing of alcohol mixtures. This review describes the use of lignocellulosic feed
stocks, bioprocess and metabolic engineering, downstream processing and catalytic
refining of n-butanol.

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Click here to download Manuscript: Butanol_prod_revised.docx

Title page
Section in which the paper is to be considered: Biofuels and Environmental Biotechnology

Manuscript title: Butanol production from lignocellulosics

The names of the authors plus email addresses of all authors:

1
German Jurgens, 1Shrikant Survase, 2Oxana Berezina, 3Evangelos Sklavounos, 1Juha Linnekoski,
1
Antti Kurkijärvi, 1Minna Väkevä, 3,4Adriaan van Heiningen, 1Tom Granström

german.jurgens@aalto.fi; shrikant.survase@aalto.fi; mashchenko@yandex.ru;

evangelos.sklavounos@aalto.fi; juha.linnekoski@aalto.fi; antti.kurkijärvi@aalto.fi;

minna.vakeva@aalto.fi; avanheiningen@umche.maine.edu; tom.granstrom@aalto.fi

The affiliations and addresses of the authors:

1Department of Biotechnology and Chemical Technology, Aalto University, FI-00076 AALTO

2 State Research Institute of Genetics and Selection of Industrial Microorganisms, 1-st Dorojniy

pr. 1, 117545, Moscow, Russian Federation

3Department of Forest Products Chemistry, Aalto University, FI-00076 AALTO

4Department of Chemical and Biological Engineering, University of Maine, 5737 Jenness Hall,

Orono, Maine USA

The email address, telephone and fax numbers of the corresponding author: Tom Granström
E.mail: tom.granstrom@aalto.fi ; Telephone: +358 9 4702 2560; Fax: +358 9 462 373
 2

Abstract
Various strains from genus Clostridium are producing n-butanol in a process called ABE-

fermentation. In terms of economical and sustainable industrial scale butanol production a

number of obstacles need to be addressed including choice of feedstock, low product yield,

toxicity to production strain, multiple end products and downstream processing of alcohol

mixtures. This review describes the use of lignocellulosic feed stocks, bioprocess and metabolic

engineering, downstream processing and catalytic refining of n-butanol.

Keywords: butanol, Acetone-Butanol-Ethanol-fermentation, downstream, chemical catalysis,

lignocellulosics

Introduction
The history of butanol has been covered thoroughly in the literature in a number of reviews

(Jones and Woods 1986; Dürre 1998; Rogers et al. 2006; Zverlov et al. 2006). The main conclusion

of Jones and Woods (1986) with regards to industrial ABE-fermentation is still highly relevant,

e.g. traditional fermentation is not cost effective and future metabolic engineering and process

technology are needed to increase its commercial viability. Butanol as a biofuel has been

reviewed recently by Jin et al. (2011) and Broustail et al. (2011), and the methods of metabolic

engineering by Lütke-Eversloh and Bahl (2011).

This review focuses on an industrially feasible alternative of butanol production from

lignocellulosics using Clostridium acetobutylicum or Clostridium beijerinckii. It covers various

aspects such as wood biomass pretreatment/fractionation, detoxification of wood hydrolysate

for fermentation, bioprocess and metabolic engineering of Clostridium, and downstream

processing of butanol from the fermentation broth.

 3
Lignocellulosic biomass
Plant biomass is the only sustainable source for production of liquid transportation fuels (Huber

et al. 2006). The key obstacle to take advantage of this feed stock is lack of a proven economic

technology, and the high capital cost relative to oil based transportation fuels due to the much

smaller scale of biomass processing (van Heiningen et al. 2011). The techno-economic potential

of woody biomass improves when integrated within an existing industrial forest products

complex since biomass may be collected simultaneously with tree harvesting to minimize

biomass feedstock cost and maximize supply. In such a lignocellulosic biorefinery the biomass

processing costs are also reduced due to integration with existing infrastructure such as

steam/power and water effluent treatment facilities (van Heiningen 2006).

Regarding the availability of biomass, a recent study of the US Department of Energy projects

that the available biomass in 2030 for industrial bioprocessing in the US would be between 1.1

and 1.6 billion tons. (U.S. Department of Energy 2011). Of this total the forest biomass represent

160 million dry tons at the lowest price ($40 per dry ton) to 664 million dry tons at $60 per dry

ton, while agricultural residues and wastes account for 404 million dry tons by 2030 at a

farmgate price of $60 per dry ton.

Fractionation/Pretreatment of lignocellulosics
In order to make the sugars in lignocellulosics available for fermentation the biomass must

either be fractionated into its principal constituents; cellulose, hemicellulose and lignin, or its

structure opened up by a so called pretreatment step. This is a difficult task because the

cellulose microfibrils are covered by a layer of hemicelluloses and are imbedded in a tight

composite structure of lignin and hemicelluloses bound to each other by covalent bonds (Fengel

and Wegener 1989). Fractionation of stem wood is traditionally used to release the cellulosic

fibers for pulp-based products by dissolution of lignin between the fibers as well as the lignin

and hemicelluloses between the cellulose microfibrils. However, when cheaper lignocellulosic

biomass is fractionated, the released lower quality fibers and dissolved hemicelluloses are still
 4
valuable as a sugar feedstock source for fermentation. Alternatively the cell wall structure of the

biomass may be opened up by pretreatment so that hydrolytic enzymes gain access to the sugar

polymers; cellulose and hemicellulose.

Pretreatment has been intensively studied because it is the most important step affecting the

production cost of lignocellulosic ethanol (Elander et al. 2009). While access to cellulose by

enzymes is the primary goal of pretreatment, clean separation of the major lignocellulosic

polymers is the key objective of fractionation (Bozell 2010). There is however a continuum

between the two processes; from steam explosion – dilute acid hydrolysis – SPORL (NaHSO3 and

H2SO4 treatment plus mechanical refining (Zhu et al. 2010) – Lignol (Ethanol-water with H2SO4

(Mabee et al. 2006) to AVAP (Ethanol-water with SO2 (Retsina and Pylkkänen 2007a).

Of all lignocellulosics, pretreated softwood biomass is most resistant to enzymatic hydrolysis

(Galbe and Zacchi 2007). Despite many pretreatment studies, it is still not possible to predict the

enzymatic hydrolysis behavior of pretreated biomass based on their chemical and physical

composition (Kumar et al. 2009). Examples of pre-treatment/fractionation methods include

steam explosion, dilute acid (0.1-3.0% sulfuric acid), SPORL (1-8% sulfite and 0.5-2% sulfuric

acid), AFEX (ammonia fibre explosion), ARP (ammonia recycled percolation, 10-15% ammonia),

and lime treatments. These methods increase the accessibility of cellulose by removing the

protective layers of either hemicelluloses (acidic processes) or lignin (alkaline processes) (Mosier

et al. 2005). Alkaline processes are considered less attractive due to the difficulty to recover

alkali/ammonia (Mosier et al. 2005; Wyman et al. 2005). Lime as an alkali source is cheap and

does not require recovery. However, lime pre-treatment suffers from extensive scaling (Zhu and

Pan 2010).

In acidic pre-treatments lignin is mostly preserved in the solid residue. However it is considerably

altered (melted, condensed, agglomerated) and this is seen as a reason for the enhanced

digestibility. Many of the acidic pretreatments are energy intensive as they operate at

temperatures higher than 150°C and sometimes even over 200°C with associated high pressures
 5
(Zhu et al. 2010). A significant water requirement related to the high required liquid-to-solids

ratios, the necessity to neutralise the acids prior to fermentation and associated salt (generally

gypsum) disposal problem, difficulty to treat softwoods, and corrosion problems are other

important drawbacks of acidic pre-treatment processes. Finally, an operating problem which has

mostly been overlooked for acidic pretreatment is formation and precipitation of sticky lignin on

reactor walls and piping (Leschinsky 2009). No economical solution to this problem is presently

available.

A qualitative comparison of different pre-treatment/fractionation processes is shown in Table 1.

(Place for Table 1 Qualitative comparison…)

The table includes sulphite pulping besides the previously discussed pre-treatment and

fractionation processes. Sulfite pulping is included because it is the only commercially operating

wood fractionation process which produces ethanol from the dissolved hemicelluloses. It also

shows that the SEW (SO2-EtOH-H2O) fractionation process used in the AVAP biorefinery process

is the only process which does not suffer from hemicellulose degradation, has a relatively low

energy requirement because of the low temperature (130-150°C) and liquid-to-wood ratio of 2-3

l/kg, does not create sticky lignin precipitates (Iakovlev 2011), can simultaneously process

softwoods and hardwoods biomass (Yamamoto et al. 2011), and only requires evaporation of

ethanol and SO2 for recovery of the chemicals because of the absence of a base (Mg or Na) in

the fractionation liquor. These characteristics make the SEW process uniquely suitable to release

sugars from lignocellulosic biomass for microbial fermentation.

Detoxification

Detoxification is the adequate removal of compounds that are inhibitory to fermentation. For

ABE fermentation the following compounds have been identified as inhibitors: formic acid,

dissolved lignin (Wang and Chen 2011), lignin and hemicellulose degradation products such as

syringaldehyde, ferulic and p-coumaric acids, and salts such as sulfate at relatively high
 6
concentration (13.6 g/l by Ezeji et al. 2007). Contrary to ethanologens, butanol producers such

as C. beijerinckii are not inhibited by furfural, HMF or acetic acid, rather they are stimulatory

(Pienkos and Zhang 2009). Formic acid, a common degradation product in lignocellulosic

hydrolysates, is a potent inhibitor for C. acetobutylicum at a level of 0.5 g/l (Sun et al. 2010). The

inhibition of Clostridium by formic acid may be related to the recent hypothesis (Wang et al.

2011) that formic acid triggers acid crash in ABE fermentation (Maddox et al. 2000). Wang et al.

(2011) found that formic acid at concentration of 1 mM or 46 ppm inside the cell wall is strongly

inhibitory. With ABE fermentation performed at about pH 5, and the pKa of formic acid being

3.8 this translates into a maximum total formic acid/formate concentration in the fermentation

broth of about 0.5 g/l. Detoxification methods include electrodialysis (Qureshi et al. 2008d),

liming (Qureshi et al. 2010b), treatment with cation and anion exchange materials (Qureshi et al.

2007) and activated carbon (Wang and Chen 2011).

SO2-Ethanol-Water (SEW) fractionation

In this review we focus on detoxification of SEW spent liquor from spruce before fermentation

by Clostridium. We found that ethanol and SO2 are inhibitors to Clostridium at concentration

levels of 10 g/L and 10-50ppm respectively (unpublished). Sklavounos et al. (2011) devised a

liquor conditioning scheme to detoxify SO2-ethanol-water (SEW) spent liquor from spruce (Fig.1)

to recover the fractionation chemicals (ethanol and SO2) and reduce the concentration of

ethanol, SO2, formic acid, furfural and HMF in the final conditioned hydrolysate to <0.1 g/l, 6

ppm, 0.2 g/l, non-detectable and 0.3 g/l. The final dissolved lignin concentration is 20 g/l. This

scheme also produces a hydrolysate with a hemicellulose monomeric sugar concentration of

around 100g/l.

(Place for Fig. 1. Conditioning scheme …)

In the above scheme the SEW spent liquor is produced by pulping of spruce (150°C, 3% w/w SO2

solution at a l/W ratio of 6:1 l/kg). Then the majority of ethanol and SO2 as well as volatile low

molecular acids and sugar degradation products are removed in the following vacuum

evaporation step (95°C for 90 min at 300mbar) to reduce the ethanol concentration to <10g/l.
 7
With a starting ethanol concentration of about 500 g/l this is equivalent to >98% wt. removal.

Mass balance calculations show that also about 90%wt. of SO2 and almost full removal of

furfural occurs during this step. The concentration of HMF decreases but the appearance of

some formic acid suggests that some HMF is formed due to C6 sugars degradation. In the

following atmospheric steam stripping step performed for 120 min the SO2 concentration

decreases to about 100ppm. Furfural, formic and HMF are practically fully removed from the

STR liquor. Retsina and Pylkkänen (2007b) describes steam stripping as a method to recover

ethanol and SO2 and remove inhibitors from spent liquor after SEW cooking. Steam stripping

also occurs during multiple effect evaporation of the spent liquor of sulfite and ASAMTM pulping

(Black, 1991). Liming is the third conditioning step with addition of Ca(OH)2 to the STR liquor

until pH=9.0. At this pH some residual SO2 precipitates as CaSO3 due to its low solubility.

Treatment with Ca(OH)2 also precipitates sulfate which is formed by oxidation of SO2 during SEW

fractionation (at 0.7 g/L) as gypsum. Some lignin is precipitated by forming lignin-CaSO3, lignin-

CaSO4 complexes. The sulfite and sulfate concentrations in the filtered LIME liquor are 33 ppm

and 3 g/l respectively.

Detoxification of dilute acid lignocellulosic hydrolysates by treatment with Ca(OH)2 before

fermentation to ethanol, is reported by many researchers (Horváth et al. 2005; Alriksson 2006,;

Mohageghi et al. 2006). Larsson et al. (1999) performed a comparison of 12 different

detoxification methods for treatment of dilute acid spruce hydrolysate prior to fermentation by

Saccharomyces cerevisiae, It is reported that Ca(OH)2 treatment at pH 10 is one of the best

methods with regard to improvement of ethanol productivity. Ranatunga et al. (2000) postulate

that if the cost of detoxification is considered then Ca(OH)2 liming seems to be a very economical

choice. Alriksson (2006) performed a study using dilute acid spruce hydrolysates to investigate

optimal conditions for Ca(OH)2 detoxification prior to ethanol fermentation. His conclusion is

that either high pH combined with moderate temperatures or low pH with high temperatures

gives the best balanced ethanol yield. The latter option is associated with the high heating costs

and therefore it is probable that the former option is more preferable. Mohagheghi et al. (2006)
 8
investigated liming with Ca(OH)2 in the range of pH 9-11 at a temperature of 50°C. The

conclusion is that liming to pH 10 is most appropriate to achieve detoxification with low sugar

loss and high ethanol yield at the same time.

The last conditioning step in Figure 1 is catalytic oxidation to further reduce the SO2 levels to

below 10ppm to allow fermentation by Clostridium. The catalytic oxidation step is performed by

bubbling air for 60 minutes through the solution at 60°C with addition of 20 mg/l FeSO4.7H2O as

catalyst. The latter concentration also brings the iron micronutrient level to that what is required

for the Clostridium. The residual sulfite anions are catalytically oxidized to sulfate (Linek and

Vacek 1981)

Fermentative production of biobutanol

In this section shall focus on recent developments on use of sustainable renewable substrates

for ABE production as well as use of immobilization materials in batch cultures. We shall also

discuss recent progress in continuous production of ABE solvents.

Batch fermentation
The use of sustainable feedstocks is one of the prerequisites for economical production of

biobutanol. Table 2 lists recent reports on different feedstocks, microorganisms used along with

maximum solvents, solvent yield and productivities achieved. Qureshi et al. (2008a) studied

different processes to produce ABE from wheat straw (WS) by C. beijerinckii P260 and found

simultaneous saccharification and fermentation with agitation by gas stripping efficient. Fed

batch operation with glucose supplementation was suggested to further improve solvent

production and yield. Lee et al. (2009) used various hydrolysates of rice bran and defatted rice

bran and observed highest butanol production (12.24 g/l) from the hydrolysate with acid and

enzyme treatment. Qureshi et al. (2010a) studied barley straw hydrolysate (BSH) in different

ways and overlimed BSH was reported to produce 26.64 g/l total solvents. Qureshi et al. (2010b)

performed similar experiments with corn stover and switchgrass hydrolysates and emphasized
 9
on the importance of dilution and overliming. Liu et al. (2010) used acid hydrolyzed wheat bran

and obtained an ABE production of 11.8 g/l with a yield of 0.32 in 72h. At optimum condition of

pH 6.7, sugar concentration of 42.2 g/l and agitation rate 48 rpm gave a maximum butanol yield

of 0.27 g/g sugar using maize stalk juice as substrate (Wang and Blaschek 2011). SO2-ethanol-

water (SEW) spent liquor obtained by fractionation of spruce chips was fermented after dilution

and supplementation with extra glucose (Survase et al. 2011a). The maximum ABE was found to

be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. Sun and Liu

(2011) subjected sugar maple wood to hot water extraction followed by membrane filtration

and hydrolysis with sulphuric acid. The hydrolysate was further treated with nanofiltration and

overliming to remove inhibitors (Sun and Liu 2011).

Using a hyper-butanol-producing C. acetobutylicum JB200A and highly concentrated cassava

bagasse hydrolysate containing mainly glucose 108.5 g/l ABE was produced in fed-batch

fermentation with simultaneous butanol recovery by gas stripping in 263 h. The integrated

fermentation process is claimed to be attractive because of a stable productivity and high

butanol yield for an extended period with periodical nutrients supplementation (Lu et al. 2011).

Qureshi et al. (2008b) demonstrated that simultaneous hydrolysis of wheat straw and

fermentation to butanol is possible if it is fed with extra sugar. They could achieve 100%

hydrolysis of WS to monomeric sugars and this also helped to improve the productivity by 16%

during a fed batch experiment of 533 h. Continuous lactic acid and glucose feeding produced

15.5 g/l butanol at a rate of 1.76 g/l/h in a fed batch culture with a pH-stat maintained at pH 5.5

(Sonomoto et al. 2010). They suggested that lactic acid, acetic acid and butyric acid can be used

as substrates for butanol production. Similar findings were reported by Tashiro et al. (2004).

(Place for Table 2 Different feedstocks and strains…)

Efremenko et al. (2011) have shown that immobilization of Clostridium cells using poly(vinyl

alcohol) cryogel changed the ratio of ABE solvents towards butanol. The ratio of ABE was 4:12:1

as compared to 3:6:1 with free cells of the same culture. Survase et al. (2011b) reported that
 10
addition of coconut fibers and wood pulp as support matrix helps in improving substrate

consumption and its conversion to solvents. Tripathi et al. (2010) suggested agarose-alginate

cryogel beads after comparing with other potential support matrices such as coconut fibers,

brick pieces and burnt coal. Shamsudin et al. (2006) reported that passive immobilization using

polyurethane foam could increase the solvent production by 215%.

Continuous fermentation

Continuous fermentation can be carried out with suspended cells, cell recycling or using

immobilized cell fermentation. The reactor configurations for continuous productions of

chemicals can be continuous stirred tank reactor (CSTR) with or without immobilization material,

packed bed reactor (PBR), fluidized bed reactor (FBR), airlift reactor, upflow anaerobic sludge

blanket reactor, or any other suitable configuration (Qureshi et al. 2005).

A continuous stirred tank reactor (CSTR) has been used in many studies for continuous

production of ABE (Ezeji et al. 2007; Lee et al. 2008; Survase et al. 2011c; Li et al. 2011). The use

of saccharified degermed corn in suspended cell continuous fermentation achieved a total ABE

concentration up to 14.16 g/l in 504 h. The average concentration of ABE was 10.12 g/l and

productivity 0.30 g/l/h (Ezeji et al. 2007). The continuous suspended cell fermentation could not

achieve high productivities because of its non-applicability at high dilution rates. The operation

at high dilution rate results in cell wash out (Lee et al. 2008; Survase et al. 2011c; Li et al. 2011).

When wood pulp was used as a cell holding material to prevent cell wash out, the solvent

(isopropanol plus butanol) productivity from glucose increased from 0.47 to 5.52 g/l/h with the

yield of 54% (Survase et al. 2011c). The average butanol concentrations with brick immobilized

cells and suspended cells were 8.07 and 4.56 g/l, respectively. The ABE productivity with

immobilized cells was 1.89 times that of suspended cells (Yen and Li 2011).

To overcome low cell density in traditional ABE fermentation, continuous fermentation processes

with cell recycling and retention have been successfully applied (Tashiro et al. 2005, Malaviya et
 11
al. 2011). A systematic comparison of continuous production of ABE using free cell cultivation

with and without cell recycling and cell recycling with cell bleeding was performed by Tashiro et

al. (2005). Continuous fermentation of C. pasteurianum MBEL_GLY2 with cell recycling was

carried out to get the ABE and butanol productivity of 8.3 and 7.8 g/l/h respectively, at a dilution

rate of 0.9 per h (Malaviya et al. 2011).

Packed bed reactors (PBRs) are packed with a suitable immobilization material on which a

biofilm is formed and used for continuous production of desired metabolites. The reaction rates

are much higher as compared to batch reactors. Recently studied immobilization materials in

PBRs are listed in Table 3. Qureshi et al. (2004) obtained ABE productivity of 16.13 g/l/h at

dilution rate of 2 per h with brick immobilized cells during reactor operation for 2302 h.

(Place for Table 3 Summary of recentcontinuous…)

To increase the sugar utilization Lienhardt et al. (2002) suggested to recycle the reactor effluent.

A maximum productivity of 12.14 g/l/h was achieved by Survase et al. (2011b) using a wood pulp

immobilized PBR and sugar mixture identical to that of a wood hydrolysate as feed. Wood pulp

as an immobilization material was also reported by Survase et al. (2011a) with SEW spent liquor

from spruce chips as feed. The highest productivity obtained was 4.86 g/l/h. Napoli et al. (2010)

used PBR reactor with Tygon rings as an immobilization material and lactose as feed to obtain a

maximum productivity of 5 g/l/h.

Metabolism of acetone-butanol-ethanol production

Clostridium is a genus of Gram-positive bacteria, belonging to the phylum Firmicutes. They are

obligate anaerobes capable of producing endospores. Due to their ubiquitous nature and as a

natural habitant of soil, Clostridium is capable of degrading a large range of carbohydrate

substances derived from plant, animal and microbial material sources. Clostridium

acetobutylicum is able to use all pentose and hexose sugars derived from wood biomass with a

preference for mannose and glucose, whereas the least preferred substrates are arabinose and
 12
galactose (Survase et al. 2011 a, b). Xylose uptake has been shown to be suppressed by glucose

in C.acetobutylicum batch cultures studies (Fond et al. 1986). Survase et al. (2011 a, b)

confirmed the sequential consumption of multiple substrates at different growth conditions.

C.acetobutylicum can grow on and produce solvents from a large variety of carbohydrate

sources such as starch (Rakkolainen et al. 2010), and sucrose (Tangney and Mitchell 2000).

Pentose sugars are metabolized through the pentose phosphate pathway resulting in fructose 6-

phosphate and glyceraldehyde 3-phosphate, which are further oxidized through the Embden-

Meyerhof pathway resulting in pyruvate. This is in turn oxidized to one of the central metabolites

acetyl-CoA by pyruvate:ferredoxin oxidoreductase. Clostridium can produce acetyl- CoA in

several ways, for example through pyruvate synthase or pyruvate-ferredoxin oxidoreductase (EC

1.2.7.1), or formate C-acetyltransferase (EC 2.3.1.54).

The first end product, acetate, is formed from acetyl-CoA by acetate kinase and ATP is generated.

Acetoacetyl-CoA is formed from two acetyl-CoA molecules, which are used to make 3-

hydroxybutyryl-CoA using one NADH. The second end product, acetone, is formed from

acetoacetyl-CoA by removing the CoA and decarboxylating acetoacetate into acetone. Butanol is

produced through crotonyl-CoA via two NADH reduction steps involving butyrul-CoA and

butyraldehyde (Fig. 2). Subsequently acetone can be reduced to isopropanol by chemical

reaction or by enzymatic conversion using isopropanol dehydrogenase (EC 1.1.1.80). The

metabolites of butyrate, acetate, acetyl-CoA, acetoacetyl-CoA and butyryl-CoA are common

substrates for CoA-transferase, acetate/butyrate kinase and phosphate acetyl/butyryl-

transferase resulting in the end products of acetone, butanol and ethanol.

(Place for Figure 2 Metabolic pathways in C. acetobutylicum…)

Metabolic engineering of Clostridium aiming at improving butanol

production and solvent tolerance
 13
Industrial fermentation of traditional Clostridium strains for large scale production of n-butanol

is not economically feasible in the current conditions because of low 1-butanol titer (<15 g/l),

two-phase metabolism hampering organization of continuous fermentation process, synthesis

of a significant amount of by-products, and expensive fermentation substrates. Multiple

attempts have already been made to improve Clostridium or to construct 1-butanol producing

strains by metabolic engineering of other microorganisms.

So far the best reported butanol-producing strain obtained by mutagenesis and selection was

the hyperamylolytic C. beijerinckii BA101 mutant generated with NTG together with selective

enrichment on a non-metabolizable glucose analog 2-deoxyglucose (Annous et al. 1991). In a

batch fermentation of semidefined P2 medium containing 6% glucose, C. beijerinckii BA101

consistently produced 18.6 g/l of butanol for 48.5 h versus 9.2 g/liter of butanol produced by the

parent NCIMB 8052 strain for 83.5 h. The yield of butanol (grams of butanol/ grams of glucose

utilized) was 32%; butanol was 71% of the total solvents (g/g). In comparison the parent strain,

the BA101 produced lower levels of butyric and acetic acids. This is consistent with an enhanced

capacity for uptake and recycling of these acids, more complete carbohydrate utilization, and

superior solvent production capacity during continuous culture fermentation in P2 medium

containing 6% glucose. Volumetric solvent yields of 0.78 and 1.74 g/l/h for BA101 and 0.34 and

1.17 g/l/h for NCIMB 8052 were obtained at dilution rates of 0.05 and 0.20 per hour,

respectively. No strain degeneration was observed for up to 200 h of fermentation (Formanek et

al. 1998).

Proper genetic tools are essential for successful metabolic engineering of clostridia. Shuttle

vectors, transformation and conjugation, circumvention of restriction barriers, transposon

mutagenesis, reporter genes, antisense RNA technology, and DNA microarrays have been

developed in most cases specifically for C. acetobutylicum ATCC 824 strain and its mutants.

Therefore these strains have become objects for genetic improvements (Davis et al. 2005 Lee et

al. 2008). The most promising technique in strain engineering - a reliable gene knock-out

technique for the genus Clostridium (ClosTron) - was invented and recently renewed by Heap et
 14
al. (2010). Same author published recently new method of larger DNA fragments integration into

clostridial chromosomes based on allele coupled exchange (Heap et al. 2012). Other methods

including deletion strategies and inducible promoter systems are currently under development

(Dürre 2011). Direct strategies for engineering strains with improved butanol formation include

inactivation of the genes required for the formation of other than butanol products such as

acetate (Kuit et al. 2012), acetoin, acetone, butyrate, ethanol, and lactate (Dürre 2007). The

highest titers of butanol, 16.7 g/l, were achieved by Harris et al. (2000) with a strain having

the gene for the butyrate kinase knocked out. Another approach aims to increase solvent

tolerance by overexpression of stress proteins like the heat shock protein groESL to produce 17

g/l of butanol (Tomas et al. 2003) or enzymes changing the lipid composition (Zhao et al. 2003),

to block sporulation process without affecting the solventogenesis (Jones et al. 2008) and to

increase aerotolerance of the C. acetobutylicum by single mutation (deletion of the perE gene,

Hillmann et al. 2008).

Understanding the way how regulatory elements are controlling the process of solventogenesis

could have a similar importance for achieving higher solvent production. It has been known for a

long time that the main regulator of sporulation, Spo0A also controls formation of acetone and

butanol (Ravagnani et al. 2000; Harris et al. 2002). However, only recently Dürre (2011)

suggested that many more regulators are involved in this process. AdcR and AdcS represent

novel transcription factors and regulators (Schiel et al. 2003), while CodY and CcpA (Standfest et

al. 2009) are known pleiotropic control proteins in Gram-positive bacteria. One of the best

genetic modifications of C. acetobutylicum ATCC824 is solR knockout mutant (Nair et al. 1999)

with simultaneous over-expression of the alcohol/aldehyde dehydrogenase gene aad (also

referred as adhE1). This resulted in 17.6 g/l of butanol (Harris et al. 2001).

Much more work is needed to understand how the Clostridium solventogenesis is organized,

functions and is connected to sporulation, carbon and nitrogen metabolism and other important

cell systems.
 15
Solvents and biofuels production by metabolically engineered Clostridium

A good example of using Clostridium metabolic engineering is modification of the

C.acetobutylicum type strain DSM792 that produces a mixture of IBE (isopropanol-butanol-

ethanol) solvents instead of “standard” ABE (acetone-butanol-ethanol). This mixture of solvents

can be sold as chemicals or used to replace gasoline in internal combustion engines. Such

genetically modified Clostridium strains could be used for industrial production of “green”

chemicals and biofuels from renewable and sustainable resources such as lignocellulosic

biomass.

Because C. acetobutylicum does not possess a secondary alcohol dehydrogenase, it is unable to

produce the secondary alcohol isopropanol from the ketone substrate acetone. In order to

modify the organism to produce isopropanol one could take adh gene for this alcohol

(isopropanol dehydrogenase) from C. beijerinckii strain NRRL B593 and insert it into C.

acetobutylicum strain DSM792. This has recently been done in several laboratories (Jurgens et

al. 2010; Heap et al. 2012; Lee et al. 2012). The resulting modified C. acetobutylicum DSM792-

pADH1 strain produced up to 14.3 g/l of IBE solvents from standard glucose media and 5 g/l

of IBE solvents from SO2–ethanol–water (SEW) spent liquor (Sklavounos et al. 2011) from spruce

chips. Strain C. acetobutylicum PJC4BK (pIPA3-Cm2) lacking in the butyrate kinase (buk) gene and

containing synthetic acetone operon (adc-ctfA-ctfB) produced 20.4 g/l of IBE solvents from CGM

media supplemented with 80 g/l glucose (Lee et al. 2012).

Subcloning of Clostridial genes required for butanol formation into a

heterologous host
Solventogenic genes (e.g. thiL/atoB, hbd, crt, bcd-etfB-etfA, adhE1, adhE2, and bdhB) derived

from ABE-producing clostridia or other microorganisms have been transferred for heterologous

expression in non ABE-producers such as Escherichia coli (Inui et al. 2008) to produce 1.2 g/l and

(Atsumi et al. 2008a) to produce 0.55g/l, Pseudomonas putida (Nielsen et al. 2009) to produce

0.11 g/l, Bacillus subtilis (Nielsen et al. 2009) to produce 0.024 g/l, and S. cerevisiae (Steen et al.
 16
2008) to produce 0.0025 g/l butanol.

Lactic acid bacteria are used for several biotechnological applications, and methods for their

manipulation have been well developed. Lactobacillus brevis naturally has the highest (3%)

tolerance against butanol among microorganisms (Knoshaug and Zhang 2009) and is able to

grow in plant-associated environments and ferment pentoses as well as hexoses (Lokman et al.

1991) therefore it was also subjected to metabolic engineering to produce butanol.

Transformation of crt, bcd, etfB, etfA, and bcd enabled Lactobacillus brevis to form 0.3 g/l

butanol on glucose media, using its own thiolase, aldehyde and alcohol dehydrogenase genes

(Berezina et al. 2010).

Shen et al. (2011) constructed a modified Clostridial 1-butanol pathway in E. coli to provide an

irreversible reaction catalyzed by transenoyl-CoA reductase (Ter). This created NADH and acetyl-

CoA driving forces to direct the flux. High titer (30 g/l) and high yield (70-88% of theoretical)

production of 1-butanol was achieved anaerobically, comparable to or exceeding the levels

demonstrated by native producers.

Alternative pathways for alcohols biosynthesis: Reversed fatty acid β-

oxidation pathway

Despite multiple attempts to develop 1-butanol producers by cloning heterologous genes in

various microbial hosts, the construction of effective butanol-producing strains is still a

challenge. Hence, the opportunity to create 1-butanol producers without using foreign genes is

also of interest. The reactions of the fatty acid β-oxidation pathway, which is native in many

organisms, and the main reactions of the clostridial 1-butanol biosynthesis pathway are

redox reaction sequences between metabolites with similar chemical structures (Fig. 3).

(Place for Fig. 3 Comparison of the widespread …)

The reactions of the fatty acid β -oxidation pathway, like most redox reactions, could be reversed.

The functional reversal of the beta-oxidation cycle can be used as a metabolic platform for the
 17
synthesis of alcohols, including 1-butanol, and carboxylic acids with various chain lengths and

functionalities (Seregina et al. 2009, Dellomonaco et al. 2011, Gulevich et al. 2011). The reversal

of the β-oxidation cycle has been engineered in E.coli (Dellomonaco et al. 2011, Gulevich et al.

2011) and used in combination with endogenous dehydrogenases and thioesterases to

synthesize n-alcohols, fatty acids and 3-hydroxy-3-keto- and trans-D2-carboxylic acids. The

superior nature of the engineered pathway was demonstrated by producing higher-chain linear

n-alcohols (C4) and extracellular long-chain fatty acids (C10) at higher efficiency (Dellomonaco et

al. 2011)

Alternative pathways for alcohols biosynthesis: use of valine biosynthetic

pathway to convert glucose into isobutanol

In recent years, the use of branched-chain amino acid (BCAA) biosynthesis pathways

togetherwith the heterologous Ehrlich pathway enzyme system (Hazelwood et al. 2008) has

been proposed by the Liao group as an alternative approach to aerobic production of higher

alcohols as new-generation biofuels (Atsumi et al. 2008b; Atsumi 2010; Cann and Liao 2008;

Connor and Liao 2008; Shen and Liao 2008; Yan and Liao 2009).

On the basis of these remarkable studies, the E. coli valine-producing strain H-81 was re-

engineered, which possess overexpressed ilvGMED operon, for the aerobic conversion of sugar

into isobutanol (Savrasova et al. 2011). To redirect valine biosynthesis to alcohol production the

enzymes of Ehrlich pathway were used. In particular, the following heterologous proteins were

exploited: branched-chain 2-keto acid decarboxylase (BCKAD) encoded by the kdcA gene from

Lactococcus lactis with rare codons substituted, and alcohol dehydrogenase (ADH) encoded by

the ADH2 gene from S. cerevisiae. The expression of both of these genes in the E. coli strain H-81

producing 12–13 g/l of valine from 60 g/l of glucose results in accumulation of isobutanol

instead of valine (Fig. 4). Expression of BCKAD alone also resulted in isobutanol accumulation in

culture broth, supporting the earlier obtained data (Atsumi et al. 2010) that native ADHs of E.

coli are also capable of isobutanol production.

 18

(Place for Fig. 4 Pathway of isobutanol biosynthesis…)

Catalytic upgrading of n-butanol

C. acetobutylicum produces n-butanol or 1-butanol [CH3(CH2)3OH]. Butanol molecule can be in

different isomeric forms like 1-butanol (n-butanol); isobutanol; 2-butanol (sec-butanol) and tert-

butanol depending on the methyl group placement in the carbon chain. From these isomeric

forms n-butanol has already existing markets and it is used in many different products such as

an ingredient in car waxes, cosmetics and industrial precursor for producing butyl acetate.

Isobutanol is an important industrial precursor having number of different applications. It can be

used as a biofuel or precursor for producing iso-octane (2,2,4 trimethylpentane), which is an

important component of gasoline (D’Amore et al. 2006).

The physical properties of n-butanol make it a potential component for fuels. Compared to

ethanol that is widely used in traffic fuels at the moment, many properties of butanol make it

more suitable for this purpose (Swana et al. 2011). n-Butanol has a higher energy content and

better miscibility with gasoline and diesel and is less corrosive than ethanol and thus better

compatible with existing fuel infrastructure (Bruno et al. 2009, Fortman et al. 2008). Without

any engine modifications, n-butanol can be mixed up to concentration of 30 % v/v (Patakova et

al. 2011). There are less phase separation problems with butanol compared to lower alcohols

(Bruno et al., 2009). n-Butanol is an oxygenate and the European Union allows 15 % of

oxygenates to be mixed in the gasoline as long as other specifications are fulfilled (Directive

2009/30/EC).

n-Butanol dehydration is an elimination reaction where one mole of alcohol produces an alkene

and a mole of water. This is done at elevated temperatures (150-400 °C) in atmospheric pressure

usually with heterogeneous catalysts that have acidic properties. Typical catalysts are zeolites

and pure and modified aluminum oxides (Shen et al. 1990; Macho et al. 2001; Makarova et al.

1990 and 1994, Berteau et al. 1985 and 1989; Zhang et al. 2010; Zotov et al. 2010). The reaction
 19
conditions and the choice of the catalyst have an effect on the product distribution. The main

product is 1-butene, but other butenes (2-butenes, isobutene) are also formed either through an

intermediate common in 1-butene formation or via 1-butene isomerization (Macho et al. 2001;

Berteau e al. 1985). There is a thermodynamical equilibrium between the different butene

isomers and at these temperatures isobutene is the most favored isomer (Domokos 1973).

One side reaction is the formation of dibutyl ether (DBE). It is favored at low temperatures, high

partial pressures and high density of active sites on the catalyst.

(Place for Fig. 5 1-Butanol dehydration reaction…)

Reaction between two butanol molecules has given name to a whole group of reactions. In this

Guerbet reaction two aliphatic alcohols react to form a dimer alcohol and simultaneously one

mole of water is lost. The Guerbet product is β-branched primary alcohol. The advantages of

Guerbet products are their unique branching pattern, oxidative stability and liquidity. Although

the chemistry is known for over a century, there are many relatively new applications for

Guerbet derivates (O’Lenick et al. 2011).

Hydrogen production from butanol via reforming has also been reported (Bimbela et al. 2009).

Hu et al. (2009) used alumina-supported nickel catalysts that are of low cost, active and very

selective (Bimbela et al. 2009) but tend to coke easily.

Butanol can be blended with gasoline as such but also after conversion to tertiary ether by

reaction with tertiary olefins using heterogeneous strong cation exchange resin catalyst

(Linnekoski et al. 1989). Tertiary ethers are used as oxygenates in gasoline to increase the

octane value of the gasoline and to reduce emission. The most common tertiary alkenes used

are isobutene and isoamylenes and alcohols methanol and ethanol. Methanol forms methyl

tert-butyl ether (MTBE) with isobutene and tert-amyl methyl ether (TAME) with isoamylenes.

Respectively ethanol forms ethyl tert-butyl ether (ETBE) and tert-amyl ethyl ether (TAEE) and

butanol butyl tert-butyl ether (BTBE) and tert-amyl butyl ether (TABE) with isobutene and

isoamylenes. Tertiary olefins are used in gasoline instead of alcohols, when enhanced fuel
 20
properties are needed. Tertiary ethers have many improved fuel properties compared to alcohols

(Krause et al. 2008). The typical reaction temperatures are 50-80°C to keep the equilibrium

conversion and reaction rate at reasonable levels. The most important side reactions are

alcohol-alcohol etherification and alkene-alkene dimerization. These occur in very small

amounts since the reaction temperature is quite low (Karinen et al. 2001).

Separation of ABE products from the fermentation medium

Traditionally butanol has been recovered from the ABE solution by energy intensive distillation

(Munday et al. 1980). In addition to distillation, literature covers a wide range of alternatives for

butanol recovery. These are for example extraction, gas stripping, membrane based methods,

flashing and adsorption-desorption. When comparing these different methods the most important

criterion is the energy consumption per unit of ABE products or butanol (Oudshoorn et al. 2009).

Despite that distillation consumes large amounts of energy it has an advantage over other

methods as it is the only method, which separates ABE fermentation products completely from the

broth. The other methods need recycling to avoid product loss and expensive waste water

treatment costs (Woodley et al. 2008). A forced phase separation has so far been considered to

be non-practical. Methods to achieve this are freeze crystallization, pressure change and salting

out with carbohydrates or salts (Oudshoorn et al. 2009; Aoki and Moriyoshi 1978; Oudshoorn et

al. 2011; Al-Sahhaf and Kapetanovic 1997; Li et al. 1995). So far these alternative methods for ABE

product removal have been only of academic interest. According to our knowledge all industrial

ABE processes are using conventional, energy intensive distillation.

Membrane methods
Membrane methods to separate ABE products from the fermentation broth are pervaporation,

perstraction, reverse osmosis and microfiltration. Of these pervaporation is the most widely

studied method. It is a process which uses a selective non-porous membrane. The liquids diffuse
 21
through the membrane leaving behind nutrients, sugar, and microbial cells (Matsumura et al.

1998; Oudshoorn et al. 2009; Fouad and Feng 2008; Ezeji et al. 2004). Selectivity and flux

through the membrane are functions of temperature, pressure, concentration differences across

the membrane, and the area and material of the membrane. Furthermore the flux is inversely

proportional to the selectivity of the membrane (Oudshoorn et al. 2009). Pervaporation has been

reported to be selective, simple to operate and low in terms of energy consumption. On the

other hand the achievable flux though the membrane is generally low and there is a high

possibility of clogging and fouling of the membrane (Dürre 1998; Fouad and Feng 2008).

To avoid the toxicity problem introduced by an extraction solvent, some investigators have used

a membrane to separate the solvent from the cell culture. Eckert and Schugerl (1987) used a

microfiltration unit to separate bacteria producing butanol from the extraction solvent (Grobben

et al. 1993; Ezeji et al. 2004). The use of a liquid pervaporation membrane, rather than a solid

membrane has been described by Matsumura et al. (1998). In this work liquid membrane was

supported on a flat sheet of microporous membrane. The membrane was not stable and a larger

scale usage would be very difficult (Ezeji et al. 2004).

Gas stripping and flash

In gas stripping an inert gas, usually nitrogen or the fermentation gases are bubbled through the

fermentation broth and the ABE products are condensed from the gas (Ezeji et al. 2004) The

equilibrium partitioning of the compounds between the gas and liquid phases can be estimated

using the Henry coefficients (Oudshoorn et al. 2009). Continuous flashing is a method which

relies on abrupt pressure reduction. This causes part of the liquid to evaporate and thus achieves

slight separation of components (Mariano et al. 2010; Mariano et al. 2008). In gas stripping and

continuous flashing there is no possibility of clogging or fouling, but they have a low

selectivity, poor removal efficiency and they are relatively energy intensive. However, these

methods are promising when used to enrich ABE products to reduce the energy cost of

subsequent separation operations (Dürre 1998).

 22

Adsorption

Adsorption is a method, where components are removed from a fluid phase by means of

selective adsorption of molecules on a solid surface (Oudshoorn et al. 2009). Most of the

materials used in adsorption are hydrophobic to minimize water adsorption. The adsorption

capacity is a function of temperature and aqueous butanol concentration. Regeneration,

(desorption) can be done with an auxiliary fluid extraction, or by using a temperature or a

pressure shift (Oudshoorn et al. 2009). Adsorption materials can be expensive, they have low

butanol capacity, relatively low selectivity and fouling is possible (Dürre 1998). However,

adsorption-desorption is a promising method when used in conjunction with other methods

such as gas stripping.

Extraction

Extraction is a good method to separate components from diluted solutions (Matsumura, 1988).

The efficiency of the extraction is often expressed in terms of the capacity of solvents to carry

ABE products, which is inversely proportional to the overall selectivity of the extraction (Sikkema

et al. 1995). This causes a dilemma, as only the low capacity solvents are selective enough. Also

the solvent should be non-toxic to the producing organism, which reduces the amount of

possible solvents even further. Luckily the most selective solvents are also the least toxic.

Furthermore the solvent should be non-toxic to humans and the environment, the fermentation

products should have high partition coefficients, the solvent should be non-emulsion forming

and have a small partition coefficient for water, the solvent should be inexpensive, easily

available, sterilizable and the fermentation products should be easily recoverable from the

solvent (Ezeji et al. 2004). Additional criteria in selecting extraction solvent are densities,

viscosities, volatility and flammability (Li et al. 2010; Groot et al. 1990; Oudshoorn et al. 2009).

Supercritical extraction with carbon dioxide is performed at 100 bar and 40°C. This means

expensive equipment and high operation costs. Furthermore as the butanol capacity of carbon
 23
dioxides is low, supercritical extraction has not attracted much attention (Oudshoorn et al. 2009;

Laitinen and Kaunisto 1999). Ionic liquids (ILs) are organic salts with low melting points (below

100°C). ILs consist typically of an organic or inorganic anion and a poorly coordinating, bulky,

organic cation. Distillation cannot easily be used to separate the ABE products and the ILs,

because at normal process operating conditions, ionic liquids essentially do not evaporate

(Simoni et al. 2010). Furthermore the price of ionic liquids is very high, and even the slightest

loss in operation would be expensive in industrial scale.

One approach to reduce toxicity of a solvent is to mix a toxic, high partition coefficient extractant

with a bio-compatible, low partition coefficient solvent. This results in a mixture, which has

a relatively high partition coefficient and low toxicity (Evans and Wang 1988; Ezeji et al. 2004).

However these solvents should be selected with care, because their regeneration by distillation

might require multiple columns or it could be completely hindered by azeotrope formation.

Another approach is to use biodiesel as the extractant in order to eliminate the need for

separating the butanol after extraction (Adhami et al. 2009). The problem with this method is

acetone, which is extracted together with other ABE products. It can be speculated that acetone

would have undesirable effects in an engine because of its high vapor pressure and its effect on

the engine’s rubber parts.

Conclusions

Butanol is produced by Clostridium in a process called ABE-fermentation. Historically oil-derived

n-butanol has been used in established industrial chemical markets and as an ingredient in

cosmetics. Future use will include sugar-derived biofuels due to its high energy content, low

miscibility with water and low corrosion properties. The limitations for large industrial scale

butanol production are low yield and productivity and cost effective downstream processing.

Furthermore, for production of butanol as a transportation fuel it should be produced from

lignocellulosic biomass. In order to use lignocellulosics as raw materials they need to be pre-
 24
treated. The most promising new development in this field is the SEW (SO2- EtOH-H2O)

treatment which does not degrade the sugars nor produce fermentation inhibitors to a large

extent. The resulting liquid has to further treat to remove the inhibitory substances for ABE-

fermentation predominantly SO2 and formic acid. This technology has been already developed.

The cost effective way to extract butanol from the dilute water solutions is the combinations of

already available downstream processing methods. Future production strains may be

metabolically engineered to produce different butanol molecules, but the prerequisite is that it

has to be able to utilize all hemicellulose sugars present in the wood biomass.

Acknowledgments

Authors would like to acknowledge and thank Professor Matti Leisola (Department of

Biotechnology and Chemical Technology, Aalto University, Finland) for the valuable comments,

suggestions and corrections of the manuscript.

 25
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Figures captions

Fig. 1 Conditioning scheme to detoxify SO2-ethanol-water (SEW) spent fractionation liquor

Fig. 2 Metabolic pathways of C. acetobutylicum (the open boxes indicate the end products) and

associated calculated in vivo fluxes. Enzymes (in open circles) are abbreviated as follows: AM

amylase; GAPDH glyceraldehyde 3-phosphate dehydrogenase, EC 1.2.1.12; PFO

pyruvate:ferredoxin oxidoreductase, EC 1.2.7.1; NFO NADH-ferredoxin oxidoreductase, EC

1.18.1.3; FNR NADPH-ferredoxin oxidoreductase, EC 1.18.1.2; H hydrogenase; PTA

phosphotransacetylase, EC 2.3.1.8; AK acetate kinase, EC 2.7.2.12; THL thiolase (acetyl-CoA

acetyltransferases), EC:2.3.1.9; BHBD 3-hydroxybutyryl-CoA dehydrogenase, EC 1.1.1.157; C

crotonase( 3-hydroxbutyryl-CoA dehydratase), EC 4.2.1.55; BCD butyryl-CoA dehydrogenase, EC

1.3.99.2; (CoAT) CoA transferase, EC 2.8.3.8; ADC acetoacetate decarboxylase, EC 4.1.1.4; BK

butyrate kinase, EC 2.7.2.7; PTB phophotransbutyrylase, EC 2.3.1.19; AAD alcohol/aldehyde

dehydrogenase, EC 1.1.1.1; BYDH butyraldehyde dehydrogenase. Note about AAD enzyme: it is

the primary enzyme for butanol and ethanol formation but additional genes (such as adhe2,

bdhA, bdhB, CAC3292 and CAP0059) that code for alcohol forming enzymes are exist

Fig. 3 Comparison of the widespread prokaryotic fatty acid β-oxidation pathway and 1-butanol

biosynthesis pathway in Clostridium acetobutylicum. Enzymes of E. coli and C. acetobutylicum

are indicated by their gene names: atoB acetyl-CoA C-acetyltransferase; fadA (fadI) acetyl-CoA

C-acyltransferase; fadB (fadJ) 3-hydroxyacyl-CoA dehydrogenase/enoyl-CoA hydratase; fade

(ydiO) acyl-CoA dehydrogenase; thlA, thlB thiolase (acetyl-CoA acetyltransferase); hbd 3-

hydroxybutyryl-CoA dehydrogenase; crt crotonase (3-hydroxybutyryl-CoA dehydratase); bcd

butyryl-CoA dehydrogenase (adapted from Gulevich et al. 2011)

Fig. 4 Pathway of isobutanol biosynthesis. IlvGM acetolactate synthase II, IlvC acetohydroxy acid
 36
isomeroreductase, IlvD dihydroxyacid dehydratase, IlvE branched-chain amino acid

aminotransferase, Kdc 2-keto acid decarboxylase, Adh alcohol dehydrogenase (adapted from

Savrasova et al. 2011)

Fig. 5 1-Butanol dehydration reaction and other butene isomers.

figure 1
Click here to download line figure: Fig1.pdf

Pulp Char Formed Precipitate

Spruce chips

SEW Cooking Evaporation Steam Stripping Liming Cat. oxidation Fermentation

SEW liquor EVAP liquor STR liquor LIME liquor CATOX liquor ABE solvents
figure 2
Click here to download line figure: Fig2.pdf

Starch
AM
Pentose Glucose
ATP

Embden-Meyererhof-Parnas pathway
ADP
Pentose phosphate Glucose 6-phosphate

Fructose 6-phosphate
ATP

ADP
(2) Glyceraldehyde 3-phosphate

GAPDH 2NAD+

4ADP 2NADH

4ATP
(2) Pyruvate
Fdox

CoA NADH NADPH

H2
PFO NFO FNR
2CO2 + H
NAD NADP+

Fdred

PTA AK AAD
(2) Acetate (2) Acetyl-CoA (2) Ethanol

ATP ADP THL 2NAD(P) 2NAD(P)+

H
Acetoacetate Acetoacetyl-CoA
NAD(P)H
ADC CoAT BHBD
CO2 NAD(P)+

Acetone 3-hydroxybutyryl-CoA
C H2O Butanol
Crotonyl-CoA NAD(P)+
NADH AAD
BCD
+ NAD(P)H
NAD
PTB BK BYDH
Butyrate Butyryl-CoA Butyraldehyde

+
ATP ADP NAD(P)H NAD(P)
figure 3
Click here to download line figure: Fig3.pdf

Escherichia,
Salmonella,
Bacillus, Clostridium acetobutylicum
Pseudomonas,
etc…

R = -(CH2)2n-CH3 R = CH3
0 O O
=

R S-CoA = S-CoA

atoB
fadA thlA
(fadI) thlB
CoA
O O
=
=

S-CoA
fatty acid -oxidation

R
NADH
fadB NADH NAD+
hbd
(fadJ) NAD+
OH O
1-butanol biosynthesis
=
_

R S-CoA

fadB H2O crt

(fadJ) H2O
O
=

R S-CoA

FADH2
fadE NADH bcd
FAD+
(ydiO) NAD +

O
=

R S-CoA

NADH
adhE NADH adhE
NAD+
NAD+
O
=

R H

NADH bdhA
adhE NADH bdhB
NAD+
NAD+ adhE
OH
_

R
figure 4
Click here to download line figure: Fig4.pdf

GLUCOSE
Glycolysis
pathway 2 NADH
PYRUVATE

IlvGM
CO2
2-ACETOLACTATE

IlvC
NADP+
2,3-DIHYDROXY-ISOVALERATE

IlvD
IlvE
2-KETOISOVALERATE L-VALINE

Kdc
CO2
ISOBUTANAL

Adh
NAD(P)+
ISOBUTANOL
figure 5
Click here to download line figure: Fig5.pdf
table 1
Click here to download table: Table 1.docx

Table 1 Qualitative comparison of pre-treatment/fractionation processes

Pretreatment or Full utilization of Low energy No sticky Omnivorous Simple

Fractionation hemicelluloses need lignin issue recovery
Alkaline treatment No Yes Yes No No
Steam explosion No No Intermediate No Yes
Autohydrolysis Intermediate Yes No No Yes
Acid hydrolysis Intermediate Intermediate No No No
Lignol (EtOH-H2O) Intermediate No Yes No Yes
SPORL Intermediate Intermediate Yes Intermediate No
SEW (SO2-EtOH-H2O) Yes Yes Yes Yes Intermediate
Sulfite pulping Intermediate Yes Yes Intermediate Intermediate
table 2
Click here to download table: Table 2.docx

Table 2 Different feedstocks and strains used along with maximum solvents and productivities achieved

Substrate Hydrolysis Strain used Yield (g/g) / Total Reference

method Productivity ABE
(g/l.h) (g/l)
Wheat straw H2SO4 + enzyme C. beijerinckii P260 0.60/0.42 25 Qureshi et al. 2007
Wheat straw H2SO4 + enzyme C. beijerinckii P260 0.41/0.31 21.42 Qureshi et al. 2008a
Corn fiber H2SO4 C. beijerinckii BA101 0.39/0.10 9.3 (Qureshi et al., 2008c)
Palm oil mill Enzyme C. saccharoperbutylacetonicum 0.40/0.10 14.38 Hipolito et al. 2008
effluent + sago N1-4
starch
Dried distillers’ Ammonium fiber C. beijerinckii BA101 0.34/0.14 10.4 Ezeji and Blaschek
grains and expansion + 2008
soluble (DDGS) enzyme
Rice bran and HCl + enzyme C. beijerinckii NCIMB 8052 0.31/0.26 16.42 Lee et al. 2009
defatted rice
bran
Barley straw H2SO4 + enzyme C. beijerinckii P260 0.43/0.39 26.64 Qureshi et al. 2010a
Corn stover H2SO4 + enzyme C. beijerinckii P260 0.44/0.31 26.27 Qureshi et al. 2010b
Switchgrass H2SO4 + enzyme C. beijerinckii P260 0.39/0.17 14.61 Qureshi et al. 2010b
Wheat bran H2SO4 C. beijerinckii ATCC 55025 0.32/0.16 11.8 Liu et al. 2010
SO2-ethanol- SO2-ethanol- C. acetobutylicum DSM 792 0.20/0.09 8.79 Survase et al. 2011a
water (SEW) water
spent liquor
Sugar maple Hot water C. acetobutylicum ATCC 824 0.22/0.15 11.0 Sun and Liu 2011
wood extraction +
sulfuric acid
Rice straw H2SO4 + enzyme C.acetobutylicum MTCC 481 1.04a/0.017 3.0 Ranjan and Moholkar
2011
Cassava bagasse Enzyme C. acetobutylicum JB200 0.39/0.62 33.87 Lu et al. 2011
a
Maize stalk juice --- C. beijerinckii NCIMB 8052 0.27 /0.30 11.5 Wang and Blaschek
2011
a
only butanol yield and productivity
table 3
Click here to download table: Table 3.docx

Table 3 Summary of recent continuous fermentation methods for ABE production along with solvent yield,
productivity and total solvents

Fermenta- Immobili- Strain Substrate Yield(g/g) / Total Reference

tion method zation Producti- ABE
material vity (g/l.h) (g/l)
Suspended cell
C. acetobutylicum BCRC 10 Glucose 0.11/0.37 6.85 Yen and Li
639 (2011)
C. acetobutylicum Glucose 0.23/0.89 8.85 Li et al. 2011
ATCC824
C. beijerinckii DSM 6423 Glucose 0.28/0.47 5.26 Survase et
al. 2011c
C. beijerinckii BA 101 Sachharified -/0.30 10.12 Ezeji et al.
degermed corn 2007
C. beijerinckii NCIMB 8052 Glucose + 0.24/0.22 7.1 Lee et al.
butyric acid Butanol Buta- 2008
nol
C. saccharoperbutyl- Glucose -/1.85 9.27 Tashiro et al.
acetonicum N1-4 2005
With cell recycle and bleeding
C. pasteurianum Glycerol -/8.3 9.2 Malaviya et
MBEL_GLY2 al. 2011
C. saccharoperbutyl- Glucose -/7.55 8.58 Tashiro et al.
acetonicum N1-4 2005
Immobilized cell reactor
Brick C. acetobutylicum BCRC 10 Glucose 0.19/1.21 11.28 Yen and Li
639 2011
Wood pulp C. beijerinckii DSM 6423 Glucose 0.29/5.52 9.2 Survase et
al. 2011c
Porous poly- C. beijerinckii NCIMB 8052 Glucose + 0.44/0.40 13.4 Lee et al.
vinyl alcohol butyric acid Butanol Buta- 2008
nol
Wood pulp C. acetobutylicum DSM SEW liquor + 0.27/4.86 7.59 Survase et
792 glucose al. 2011a
Wood pulp C. acetobutylicum DSM Sugar mixture 0.28/12.14 8.09 Survase et
792 al. 2011b
Corn stalk C. acetobutylicum ATCC Glucose 0.32/5.06 5.1 Zhang et al.
55025 2009
TygonTM C. acetobutylicum DSM Lactose 0.28/5.0 5.19 Napoli et al.
rings 792 2010
Brick C. beijerinckii BA101 Glucose 0.38/15.8 7.9 Qureshi et
al. 2000
Two stage chemostat
C. beijerinckii DSM 6423 Glucose 0.32/0.84 7.63 Survase et
al. 2011c
Sugarcane C. acetobutylicum B 5313 Glucose 0.35/2.47 12.37 Bankar et al.
baggase 2012

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