fermentative production of butanol. the industrial perspective

COBIOT-862; NO. OF PAGES 7

Available online at www.sciencedirect.com

Fermentative production of butanol—the industrial perspectiveEdward M Green

A sustainable bacterial fermentation route to produce

biobutanol is poised for re-commercialization. Today,

biobutanol can compete with synthetic butanol in the

chemical market. Biobutanol is also a superior biofuel and, in

longer term, can make an important contribution towards the

demand for next generation biofuels. There is scope to

improve the conventional fermentation process with

solventogenic clostridia and drive down the production cost

of 1-butanol by deploying recent advances in biotechnology

and engineering. This review describes re-commercialization

efforts and highlights developments in feedstock utilization,

microbial strain development and fermentation process

development, all of which significantly impact production

costs.

Address

Green Biologics Ltd., 45A Milton Park, Abingdon, Oxfordshire OX14

4RU, United Kingdom

Corresponding author: Green, Edward M ([email protected])

Current Opinion in Biotechnology 2011, 22:1–7

This review comes from a themed issue on

Energy biotechnology

Edited by Peter Duerre and Tom Richard

0958-1669/$ – see front matter

# 2011 Elsevier Ltd. All rights reserved.

DOI 10.1016/j.copbio.2011.02.004

Introduction1-Butanol (butyl alcohol or n-butanol) is a four carbon

straight chained alcohol with a molecular formula of

C4H9OH (MW 74.12) and boiling point of 118 8C. 1-

Butanol is an important chemical precursor for paints,

polymers and plastics. In 2008, the global market for 1-

butanol was 2.8 million t [1], estimated to be worth

approximately $5 billion. The average growth is expected

to be 3.2% pa with demand concentrated in North Amer-

ica (28%), Western Europe (23%) and North East Asia

(35%).

Most 1-butanol produced today is synthetic and derived

from a petrochemical route based on propylene oxo

synthesis in which aldehydes from propylene hydrofor-

mylation are hydrogenated to yield 1-butanol. Synthetic

butanol production costs are linked to the propylene

market and are extremely sensitive to the price of crude

oil (Figure 1).

Please cite this article in press as: Green EM. Fermentative production of butanol—the industria

www.sciencedirect.com

Renewable 1-butanol is produced from the fermentation

of carbohydrates in a process often referred to as the ABE

fermentation, after its major chemical products: acetone,

butanol and ethanol. The ABE fermentation is a proven

industrial process that uses solventogenic clostridia to

convert sugars or starches into solvents [2]. The fermen-

tation occurs in two stages; the first is a growth stage in

which acetic and butyric acids are produced and the

second stage is characterized by acid re-assimilation into

ABE solvents. During this stage, growth slows, the cells

accumulate granulose and form endospores. The fermen-

tation also produces carbon dioxide and hydrogen.

Biobutanol is an attractive renewable liquid transportation

biofuel. The superior properties have been well documen-

ted (http://www.butamax.com/_assets/pdf/butamax_

advanced_biofuels_llc_fact_sheet.pdf). Bio-butanol fits

the existing fuel infrastructure; it has a better energy

density and performance than ethanol and can be made

from more sustainable feedstocks than bio-diesel. There-

fore, bio-butanol has the potential to substitute for both

ethanol and bio-diesel in the biofuel market estimated to

be worth $247 billion by 2020 (http://www.pikeresearch.

com/research/biofuels-markets-and-technologies).

Commercial productionThe ABE fermentation process was first developed in the

UK in 1912 and commercial production quickly spread

around the globe during the first and second world wars;

first to produce acetone for ammunitions and then later to

produce butanol for paint lacquers. The fermentation

process fell out of favour in the US and Europe in the

1950s when renewable solvents could no longer compete

with their synthetic equivalents on price. Some pro-

duction via fermentation remained in China, Russia

and South Africa until the early 1980s [3–5].

Commercial solvent titres peak at about 20 g/L from 55 to

60 g/L of substrate giving solvent yields of around 0.35 g/

g sugar [4]. The butanol:solvent molar ratio is typically 0.6

with an A:B:E ratio of 3:6:1 [2]. Butanol is the preferred

solvent since it attracts the highest price in the chemical

market.

China leads efforts to re-commercialize the ABE fermen-

tation process. Over $200 million has recently been

invested in China to install 0.21 million t pa of solvent

capacity with plans to expand to 1 million t pa. There are

six major plants that produce about 30 000 t butanol pa

from corn starch [6�]. Most plants operate in a semi-

continuous fashion with each fermentation lasting up

to 21 days. The plants typically house several trains of

l perspective, Curr Opin Biotechnol (2011), doi:10.1016/j.copbio.2011.02.004


http://dx.doi.org/10.1016/j.copbio.2011.02.004

mailto:[email protected]


http://www.butamax.com/_assets/pdf/butamax_advanced_biofuels_llc_fact_sheet.pdf

http://www.butamax.com/_assets/pdf/butamax_advanced_biofuels_llc_fact_sheet.pdf

http://www.pikeresearch.com/research/biofuels-markets-and-technologies

http://www.pikeresearch.com/research/biofuels-markets-and-technologies

2 Energy biotechnology


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Current Opinion in Biotechnology

The relationship between crude oil ($/barrel) and the synthetic butanol price in China during 2010.

up to eight fermentation tanks (300–400 m3 volumes)

linked together in series. Fresh feedstock, together with

periodic additions of seed culture, cascades through the

fermentors in a process that provides sufficient residence

time for re-assimilation of acids to solvents. Conventional

distillation is then used to recover acetone, butanol and

ethanol. Most plants are located next to ethanol plants to

reduce utility and operating costs. Co-located operations

tend to share effluent treatment facilities based on

anaerobic digestion (AD). Biogas, produced from the

AD process, is used to generate heat and power. Although

not widely practiced, additional value can be gained from

the recovery of hydrogen from the fermentation exhaust

gas (typically 1/10th of mass of butanol produced).

A relatively new plant has been built in Brazil and

operated by HC Sucroquimica. This plant produces

8000 t solvent pa from sugar cane juice and is located


Table 1

The challenges and solutions for ABE fermentation

Challenge

High feedstock cost significantly increase operating costs.

Low butanol titres increase recovery costs. Low titres also

reduce sugar loadings and increase water usage.

Low butanol yield increases feedstock costs.

Low volumetric solvent productivities increase capital

and operating costs.

Solvent recovery using conventional distillation is energy

intensive and relatively expensive.

High water usage is not sustainable and increases

the cost of effluent treatment.


next to an ethanol distillery and sugar mill (GBL market

data).

The challenges for ABE fermentationThe technical and related commercial challenges for the

conventional ABE fermentation have been extensively

reviewed [7–9] and are summarized in Table 1. In gen-

eral, there is a need for cheaper feedstocks, improved

fermentation performance and more sustainable process

operations for solvent recovery and water recycle. Feed-

stocks contribute most to production cost. On a conven-

tional plant, corn starch accounts for up to 79% of the

overall solvent production cost while energy for oper-

ations including distillation, contributes 14% to the over-

all cost [10]. Today, the production cost (and profitability)

of the plant largely depends upon the price of feedstock

and is extremely sensitive to any price fluctuation. There-

fore, transition towards cheaper (non-edible) feedstocks


Solutions

Transition towards cheaper (and more sustainable) feedstocks

such as wastes and agricultural residues.

Develop improved microbes with improved solvent titres and/or

develop methods for in situ product removal to alleviate end

product tolerance.

Develop improved microbes with higher butanol yields and/or

develop microbes with higher butanol: solvent ratios.

Develop continuous fermentation processes that reduce down

time and increase volumetric productivity.

Develop low energy methods for solvent recovery and purification.

Recovery can also be improved by improving the solvent titre.

Recycle process water back through the fermentation.



Fermentative production of butanol—the industrial perspective Green 3


offers the biggest opportunity for cost reduction and

improved sustainability.

Fermentation plant profitability is also sensitive to the

butanol price which in turn is linked to the cost of oil

(Figure 1). For example, in 2008 the oil price crashed and

the butanol price fell from $2400/t to $1000/t between

June and November [6�]. All plants in China ceased

production. By the end of 2010, the butanol price recov-

ered to around $2000/t prompting several plants to restart

(Figure 1; GBL market data).

Butanol titre and yieldThe butanol titre and yield that can be achieved are

largely a function of the microbe. Performance can be

improved using chemical mutagenesis, specific genetic

manipulation or a combination of both techniques. To

date, Clostridium acetobutylicum ATCC 824 remains the

best studied and manipulated strain although this species

group is quite distinct, both genetically and physiologi-

cally, from the three other main solvent producing

species: C. saccharobutylicum, C. beijerinckii and C. sacchar-operbutylacetonicum [5]. Proven commercial strains include

C. saccharobutylicum P262 and C. beijerinckii P260, which

were used in South Africa in the early 1980s [11]. Rather

than focus on just a single one strain Green Biologics Ltd

(GBL) (http://www.greenbiologics.com) has built a large

collection of strains representing the four main species

groups. Strains are selected based on performance against

specific substrates and developed for growth and fermen-

tation on specific feedstocks.

Following the publication of the genome sequence of C.acetobutylicum ATCC 824 in 2001 [12], significant progress

has been made using specific gene integration and anti-

sense RNA technology to knockout or down regulate

gene expression and better understand gene function.

Advances in genomics, transcriptomics and genetic

manipulation have been extensively reviewed by Durre

[13], Lee et al. [14�] and Papoutsakis [15�]. More recently,

proteomic reference maps have been developed for acido-


Table 2

Summary of chemically mutated and genetically engineered solvento

Strain Host strain Solvent titre (g/L) Butano

PJC4BK C. acetobutylicum

ATCC 824

23.7 16.7

SolRH (ptAAD) C. acetobutylicum

ATCC 824

27.9 17.6

BA101 C. beijerinckii

NCIMB 8052

24.2–26.1 15.8–19

EA2018 C. acetobutylicum

(strain 4)

21 14.4


genic and solventogenic cultures [16] together with insilico genome models [17–19]. Genetic advances in other

strains have lagged. For example, the genome sequence

of C. beijerinckii NCIMB 8052 was only completed in 2007

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&

cmd=search&term=NC_00961721) and tools for genetic

engineering this strain have not been fully developed.

For C. acetobutylicum, significant advances have been

made to methods for gene integration. Methods based

on a group II intron system for gene knockout have been

described [20,21�]. More recently an improved method,

based on allele coupled exchange (ACE), has been

described for stable integration of larger DNA fragments

[22��]. It is now possible to construct multi-step biosyn-

thetic pathways paving the way for new synthetic clos-

tridia.

Table 2 summarizes the best chemically mutated or

genetically engineered solventogenic clostridia strains

recorded in the literature. These include C. beijerinckiiBA101, a hyper-butanol producer, developed in 1991

using chemical mutagenesis [23]. TetraVitae Bioscience

(http://www.tetravitae.com) has licensed this strain from

the University of Illinois for commercial use. Strain

EA2018 was also developed using chemical mutagenesis

of C. acetobutylicum and found to produce higher buta-

nol:solvent ratios (0.7) than the parental strain (0.6) [24].

This strain has been licensed to several commercial

producers in China (GBL market data). The acetone

pathway has also been knocked out in this strain resulting

in higher butanol:solvent ratios (0.8) but no overall

increase in higher butanol titre was observed [25].

Superior performance has also been demonstrated from

genetically engineered derivatives of C. acetobutylicumATCC 824 [26,27].

Synthetic biology has recently been used to introduce

biosynthetic capacity for butanol into non-natural hosts.

The choice between using or engineering natural func-

tion versus importing biosynthetic function has been


genic clostridia displaying improved solvent titre

l titre (g/L) Details Reference

The buk gene encoding butyrate

kinase has been inactivated.

[27]

The solR regulatory gene

has been inactivated and the gene

encoding aldehyde/alcohol

dehydrogenase has been

overexpressed.

[26]

.6 Chemical mutagenesis with

N-methyl-N9-nitro-N-nitrosoguanidine.

[23]

Chemical mutagenesis. This strain

produces an A:B:E ratio of 2:7:1.

[24]


http://www.greenbiologics.com/

http://www.ncbi.nlm.nih.gov/sites/entrez%3Fdb=genome%26cmd=search%26term=NC_00961721

http://www.ncbi.nlm.nih.gov/sites/entrez%3Fdb=genome%26cmd=search%26term=NC_00961721

http://www.tetravitae.com/




reviewed [28�]. Commonly used host strains include

Escherichia coli and Saccharomyces cerevisiae that are rela-

tively easy to genetically manipulate but do not tolerate

more than 2% 1-butanol [29]. In addition, these strains do

not display broad substrate ranges and cannot compete

with natural or engineered clostridia for the production of

1-butanol from a broad range substrates including pentose

sugars and sugars derived from cellulosic feedstocks.

Isobutanol is a more promising product because it is less

toxic than 1-butanol. It is an attractive biofuel but cannot

substitute for 1-butanol in the chemical market. One

synthetic approach involves the introduction of genes

encoding enzymes that convert either acetyl-CoA or

pyruvate to butanol. Alternatively, genes encoding

enzymes that convert 2-keto acids intermediates (from

amino acid synthesis) into isobutanol and branched-chain

alcohols; 2-methyl-1-butanol and 3-methyl-1-butanol can

be introduced [30�,31,32]. Several companies are cur-

rently involved in scale-up and demonstration. Gevo

Inc. (http://www.gevo.com) has engineered E. coli to

produce isobutanol [33] and recently acquired a com-

mercial-scale ethanol plant in Minnesota for retrofit to

produce isobutanol. The company has also received

Environmental Protection Agency certification to blend

isobutanol in fossil fuels. DuPont has also engineered

several biocatalysts for isobutanol [34] and assigned the

technology to ButamaxTM Advanced Biofuels (http://

www.butamax.com), a joint venture between BP and

Dupont. ButamaxTM is collaborating with Kingston

Research Limited, another BP–Dupont joint venture,

to build a demonstration plant in the UK.

FeedstocksToday, the ABE fermentation process is economic on

starch and sugar based feedstocks if 1-butanol is sold at a

premium into the chemical market (GBL model data).

Significant cost reduction can be achieved using cheaper

agricultural residues or wastes such as corn cobs, corn

stover, sugar cane bagasse, wheat straw and municipal

solid waste (MSW) that should enable 1-butanol to com-

pete, on price, with ethanol for the biofuel market. Also,

use of cellulosic and waste material is more sustainable

offering a lower carbon footprint and reduced green house

gas (GHG) emissions.

Solventogenic clostridia are particularly well suited for

fermenting sugars derived from cellulosic feedstocks.

They have a broad substrate range including pentose

sugars [2] and tolerate many known growth inhibitors

formed during the pre-treatment and hydrolysis. A recent

review provides comprehensive information on both

feedstock and end product inhibition in a variety of

fermentative microbes together with strategies to

improve strain tolerance [35��]. Interestingly, furans

(formed from sugar degradation) and lactic acid were

found to stimulate butanol production in clostridia [9,36].



Recent work with cellulosic feedstocks has focused on C.saccharobutylicum P262, C. beijerinckii P260 and C. beijer-inckii BA101. Good fermentation performance data have

been demonstrated on corn fibre [37], wheat straw [38]

and dried distillers grains and solubles [39]. Other reports

describe methods for desalting and detoxifying wheat

straw and corn stover hydrolysates that result in improved

fermentation performance [40,41]. Further improve-

ments have been demonstrated by combining hydrolysis

and fermentation in a consolidated bioprocess and by

integrating a method for in situ solvent recovery, based on

gas stripping [42,43].

Solventogenic clostridia produce good solvent yields and

titres from a wide range of cellulosic material [44�] but

demonstration of the technical and economic feasibility

of cellulosic butanol at scale is required. In particular, the

cost of enzymatic hydrolysis of cellulose is still proble-

matical although there is considerable scope to optimize

the enzyme cocktail specifically for clostridia thereby

reducing the cost. An attractive option is to use the

pentose sugar rich hemi-cellulose streams resulting from

agricultural, paper pulp and wood processing plants.

Typically, this fraction does not require hydrolysis but

may still require some detoxification to reduce growth

inhibitors. This approach for ABE production has been

demonstrated in China by Songyuan Laihe Chemicals. A

600 t pa pilot plant has been built to ferment sugars

contained within the hemi-cellulose fraction from pre-

treated corn stover (http://english.ipe.cas.cn/ns/Events/

200911/t20091125_47623.html). Plans are now underway

to demonstrate this process at commercial scale.

Volumetric productivityVolumetric solvent productivity (g solvent/L fermenta-

tion broth/h) has a big impact on capital cost. For

example, a two-fold increase in productivity reduces

capital expenditure by approximately 20% together with

significant reductions in operating costs (unpublished

GBL model data).

The Chinese semi-continuous fermentation process

offers 40% higher solvent productivity than a convention-

al batch process [6�]. Continuous culture, over a pro-

longed period, offers even greater improvements in

productivity but this is difficult to achieve because sol-

vent production in strains such as C. acetobutylicum tends

to degenerate over time [45]. Also the fermentation is

biphasic and maintenance of the culture in just the

solventogenic phase is difficult. A new method, based

on flow cytometry, provides some insight into the

relationship between morphology and solvent production

and could help monitor and control solventogenic cell

populations for stable continuous culture [46�].

Recently, a continuous culture system, fed with lactic

acid and glucose and using a pH-stat to control dilution



http://www.gevo.com/

http://www.butamax.com/

http://www.butamax.com/

http://english.ipe.cas.cn/ns/Events/200911/t20091125_47623.html

http://english.ipe.cas.cn/ns/Events/200911/t20091125_47623.html




rate, produced 5.5 g/L butanol at a productivity rate of

1.76 g/L/h [36]. Significant improvements have also been

demonstrated using high dilution rates and immobiliz-

ation to retain the microbe in the vessel [47–49]. A biofilm

bioreactor offering higher productivity is under develop-

ment by Cobalt Technologies (http://www.cobalttech.

com). However, immobilized systems tend to produce

low solvent titres that are expensive to recover and they

tend to suffer from operational problems with high solid

feedstock loadings. An alternative approach involving a

two-stage continuous fermentation process that produces

butyric acid and then 1-butanol with a different bacterial

strain in each vessel has been developed by Butylfuel

(http://www.butanol.com) [50].

Solvent recoverySolvent recovery using conventional distillation is robust

and proven but energy intensive. For every 1 t of solvent,

approximately 12 t of steam is required [6�]. Improve-

ments can be made to conventional distillation but non-

conventional methods are required to significantly reduce

energy and the associated cost.

Integrating solvent recovery with fermentation is an

attractive process option. Gas stripping was found to

alleviate end product inhibition and improve both solvent

titre and productivity [51]. Other methods for solvent

recovery include liquid–liquid extraction, adsorption,

pervaporation, reverse osmosis and aqueous two phase

separation. These methods have been extensively

reviewed [52,53�] but few have been proven at scale or

commercialized to date.

For isobutanol, the use of oleyl alcohol extractive tech-

nology coupled with gas stripping has been patented by

Dupont [54]. An alternative GIFTTM process using flash

distillation followed by phase separation has been devel-

oped by Gevo Inc. [33]. However, continuous operation

using flash distillation may be difficult to control due to

fluctuations in substrate and product concentration and

the need to maintain a thermodynamic equilibrium in the

flash tank [55].

ConclusionsThe clostridial ABE fermentation is an old, but proven,

industrial fermentation process that has recently been

re-established in China. Newly installed production

capacity can be optimized and expanded with further

improvements to the microbe and refinements to the

fermentation process. Over time, it should be possible

to convert plants to use cheaper cellulosic feedstocks.

Also, the clostridial ABE fermentation process is rela-

tively simple and can be performed in existing sugar or

starch ethanol plants with little modification. This

retrofit model provides an attractive option to rapidly

expand renewable 1-butanol production in the US and

Brazil.



The chemical market for 1-butanol provides an excellent

entry point because the value is approximately three-fold

higher than the value, likely to be achieved, in the biofuel

market.

The chemical demand is approximately 3 million t pa. If

biobutanol completely substitutes for synthetic butanol

in this market this would require at least fifty world scale

ABE plants.

In order to penetrate the larger biofuel market, bio-

butanol needs to compete on cost (priced on an energy

basis) with ethanol despite its superior fuel properties.

Reduction in feedstock cost offers the best opportunity

especially since clostridia are well suited for sugars

derived from cellulosic material. Clostridia have broad

substrate ranges (including pentose sugars) and display

superior tolerance to typical feedstock inhibitors.

An alternative approach for the biofuel market resides

with isobutanol using a synthetic microbe. However, it is

still unclear how robust and economic these processes are

at commercial scale and whether they can accommodate

cellulosic feedstocks. Further advances for both 1-butanol

and isobutanol are likely to come from the deployment of

continuous culture, especially when coupled with in situmethods for solvent extraction and recovery.

The application of advances in biotechnology and engin-

eering to the clostridia ABE fermentation process will

drive down the cost of 1-butanol production. Several

technology companies have become established to capi-

talize on this opportunity. The choice of strain is crucial

since it determines fermentation performance and heav-

ily influences methods for feedstock pre-treatment/

hydrolysis and solvent recovery. Microbial strain perform-

ance can be improved using advances in genetic manip-

ulation together with improved genome sequence

information and systems-based tools but the work does

need to focus on commercially relevant and robust strains.

Synthetic biology of non-butanol producing hosts is an

exciting longer term prospect but advances also require

robust host strains capable of tolerating high solvent

concentrations and feedstock inhibitors.

AcknowledgementsThe author would like to acknowledge his colleagues at Green BiologicsLimited: Rosa Dominguez, Elizabeth Jenkinson and Preben Krabben forhelp preparing this manuscript.

References and recommended readingPapers of particular interest, published within the period of review,have been highlighted as:

� of special interest

�� of outstanding interest

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http://www.cobalttech.com/

http://www.cobalttech.com/

http://www.butanol.com/




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Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM,Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridiumrefined and streamlined. J Microbiol Methods 2010, 80:49-55.

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22.��

Heap JT, Minton NP: Methods. International Patent Application2009, PCT/GB2009/000380.

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This review highlights factors that influence the decision to engineernatural function or import synthetic pathways. The authors argue thattransfer of new biological function to a basic recombinant microorgan-ism is more promising than upgrading the properties of a native micro-organism.

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A comprehensive review with information on both feedstock and endproduct inhibition in a variety of fermentative microbes together withstrategies to improve strain tolerance and performance.

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A summary of fermentation performance with several solventogenicclostridial strains on a variety of cellulosic residues. The review demon-strates the versatility and potential of using solventogenic clostridia withlow cost cellulosic feedstocks.

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This extensive review describes the advantages and disadvantagesassociated with the many methods for integrated product recovery.Opportunities do exist to challenge distillation for low alcohol concentra-tions but new methods need to be proven at scale.

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http://dx.doi.org/10.1002/bbb.108

http://dx.doi.org/10.2202/1934-2659.1226


fermentative production of butanol. the industrial perspective

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