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5?Recovery Techniques Integrated with Fermentation Process

5?Recovery Techniques Integrated with Fermentation Process

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M. Kumar and K. Gayen

Table 7.3 Integrated systems for enhancing the production of ABE fermentation


Type of

Max. titer of ABE

Max. titer of ABE (with References



(without online recovery) online recovery) in g/l

in g/l

Gas stripping Batch








Pervaporation Fed-batch











































[75, 76]













20–30% increment in the fluidity of cell membrane [71, 72]. C. acetobutylicum

was found sensitive to the higher concentration of butanol than 12–13 g l-1 [43,

73]. Various attempts are being made at the organism and process level for

reducing the butanol inhibition. One attractive development in process is as an

integrated system of fermentation and recovery processes, which allows simultaneous production and selective removal of solvents illustrated very significant

results at laboratory scale studies. The common butanol recovery techniques are

adsorption, liquid–liquid extraction, perstraction, reverse osmosis, pervaporation,

and gas stripping, which can be integrated with ABE fermentation for online

removal of products (Table 7.3) [74].

Gas stripping (Fig. 7.3) comprises the most advantageous characteristics such

as simple and economical process (no need of expensive equipments), does not

harm the culture, does not remove the nutrients and reaction intermediates, and

reduces butanol inhibition effectively [39, 40]. At laboratory scale, gas stripping

showed the significant results as integrated with various kinds of fermentation

processes, batch [39, 40, 53], fed-batch [53, 75], and continuous [40]. While

nitrogen [40] and gases produced in fermentation (hydrogen and carbon dioxide)

[39, 53, 75] are used for stripping purpose, whereas utilization of nitrogen gas

reflected more effective recovery than other gases.

7 Biobutanol: The Future Biofuel


Fig. 7.3 Schematic diagrams

of gas stripping recovery

process integrated with

fermentation process for

online removal of solvents.

Along with external gases

(e.g. nitrogen), gases

(hydrogen and carbon

dioxide) produced in

fermentation can also be used

for stripping

7.6 Economic Aspects

Fermentation processes are exothermic as its products contain less energy than

substrates. Theoretically, mass and energy yield of ABE fermentation is 37 and 94%,

respectively calculated on the basis of energy of combustion and products ratio in the

fermentation. During the study, it was suggested that yield of ABE fermentation

might not be possible to meet its 100%, whereas product yield less than 25% can

cause the economical unfeasibility even with any process development [50]. In the

account of above fact, strain improvement may be a necessary step to enhance

the theoretical yield. In this direction, many endeavors have been made to engineer

the strain or transfer the gene in a heterogeneous host organism. But, to time none of

genetically engineered strain produced higher yield than native organism [4].

However, the most valuable strain, C. beijerinckii BA101, has been developed

through chemical mutagenesis from native organism, C. beijerinckii N-CIMB 8052

[80, 81]. C. beijerinckii BA101 can generate 19–20 g l-1 solvent, which is much

higher than the native and other organisms [25, 40]. Through recent endeavors in

process development for butanol production using C. beijerinckii BA101, improved

solvent concentration (20–30 g l-1), solvent yield (0.30–0.50 g g-1), and reactor

productivity (0.30–1.74 g l-1) have been achieved [64]. A high productivity of

15.8 g l-1 h-1 has also been achieved in immobilized reactor. Due to the high

concentration of solvents, this organism leads ABE fermentation to be economical.

An economic evaluation of ABE fermentation from corn using C. beijerinckii BA101

reported butanol cost of US$0.25 lb-1. The improvement in yield from 0.42 to

0.45 g g-1 resulted in lesser butanol cost of US$0.20 lb-1 [49].

Apart from the yield, other vital factor is feedstock in economics of ABE

fermentation, it almost contributes to 60% of the total production cost of butanol

[82, 83]. Utilization of none of starch and sugar-containing crops can make this

fermentation economically feasible in the present scenario. Moreover the


M. Kumar and K. Gayen

continuous use of these food materials can cause the food shortage. On the basis of

recent studies, cheaper agriculture biomass (lignocellulosic materials) and industrial wastes were found suitable for sustainable production of butanol. Still, efforts

are being made for scaling-up the process for economical industrial production

using lignocellulosic biomass.

7.7 Prospective

Significant activity of clostridia toward consuming lignocellulosic biomass

uncovers the space of cheaper feedstock for ABE fermentation. However, efficient

techniques for removing the inhibitors, generated during hydrolysis of lignocellulosic materials, can make it a more effective feedstock. From the economic point

of view, the integrated system of hydrolysis, fermentation, and recovery process

also opens vital ways to reduce the capital and operational cost of butanol synthesis. More developments in the recovery techniques such as gas stripping will

boost up to this integrated fermentation process for improving the productivity.

Additionally, completion of genome sequencing of two clostridial species provided the crucial opportunity to genetic engineers to engineer the genome of

butanol-producing species to improve its capability toward high yield and butanol



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Appl Energy 86:108–117

Chapter 8

Molecular Genetic Strategies

for Enhancing Plant Biomass

for Cellulosic Ethanol Production

Rengasamy Ramamoorthy and Prakash P. Kumar

8.1 Introduction

Biofuels are renewable and sustainable sources of energy that can be in the solid,

liquid or gas forms. A major source of biofuels is the biomass of plants rendered as

bioethanol, biodiesel and biogas. Biofuels are the natural alternative sources to

fossil fuels and are environmentally friendly. The concept of biofuels is not new,

with firewood as the most primitive form of solid biofuel used ever since the

discovery of fire. In fact, wood is still being used for cooking food and to generate

heat during winter in many parts of the world. The liquid form of biofuels is either

vegetable oils or ethanol derived by fermentation of plant materials. The biogas

produced by anaerobic digestion of animal manure and organic household wastes

into gas (methane) used for cooking is also a biofuel. Biodiesel is obtained from

the vegetable oils produced from several plant species including, oil palm, canola,

soybean that are also used as food oils, and more recently, from non-food sources

such as Jatropha seed oil. The liquid forms of biofuels are preferred over other

forms due to the ease of storage and transportation; and in many cases these can

directly replace petroleum fuels. Thus, the so-called ‘‘flex fuel vehicles’’ on the

road today can use gasoline blended with 15–85% of bioethanol.

The world bioethanol production in 2010 was about 86 billion liters (Renewable

Fuel Association: http://www.ethanolrfa.org/news/entry/global-ethanol-productionto-reach-85.9-billion-litres-22.7-billion-ga/). Bioethanol is currently produced

mainly from corn starch in the USA and from sugarcane in Brazil. The use of food

crops for fuel production affects the food chain and has the potential to lead to

serious socioeconomic issues as reflected in escalating food price. Therefore,

R. Ramamoorthy Á P. P. Kumar (&)

Department of Biological Sciences and Temasek Life Sciences Laboratory, National

University of Singapore, 10 Science Drive 4, Singapore 117543, Singapore

e-mail: dbskumar@nus.edu.sg

C. Baskar et al. (eds.), Biomass Conversion,

DOI: 10.1007/978-3-642-28418-2_8, Ó Springer-Verlag Berlin Heidelberg 2012



R. Ramamoorthy and P. P. Kumar

cellulosic ethanol is becoming a viable alternative for corn starch and sugarcane as

the feedstock. Because cellulosic ethanol is produced from plant biomass such as

crop residues (straw), forestry and wood waste it does not disturb the food chain.

The use of bioethanol can greatly reduce the greenhouse gas (GHG) emission,

which can reach up to 94% lower than gasoline GHG emission [1, 2]. Therefore, it

is hoped that the use of more bioethanol in the coming decades can help to achieve

the significant displacement of petroleum use mandated by the advanced energy

initiative (AEI) in the USA [3, 4]. The AEI requires 30% reduction from the levels

of 2005 petroleum use in the transportation sector to be replaced by domestically

produced renewable bioethanol. Accordingly, numerous cellulosic ethanol production facilities are being opened or the existing facilities are expanding their

capacities in the USA (Renewable Fuel Association).

Biomasses such as corn stover (stalk ? leaves), rice straw and wheat straw are

produced in large-scale as the by-products of food production and a large portion

of it is going waste by getting burnt in the field and leading to more GHG

emission. In 2009–2010, the world production of corn was about 890 million tons

(mt) and at the proportion of 1:1 the corn stover produced will also be about

890 mt [5]. Similarly, around 730 mt of rice straw was reportedly produced in

Africa, Asia, Europe and America, out which around 678 mt comes from Asia [6].

Also, the current global production of wheat is about 675 mt and the wheat grain

to straw yield ratio is estimated at around 1:1.6 [7]. The yield of ethanol from corn

grain is in the range of 400–500 liters/ton, and the yield of cellulosic ethanol from

digestion of dried cellulosic biomass is (380 liters/ton) in the same range.

Therefore, by not using the plant biomass from the major grain crops we are

discarding an excellent renewable source of fuel. Nevertheless, it should be noted

that even if the entire global non-grain biomass from the three main cereal crops

(corn, wheat, rice) is used for ethanol fermentation, it can only yield about 25% of

the annual use of petroleum in the world. Hence, we need to develop additional

sources of lignocellulosic feedstock to generate higher amounts of bioethanol.

In addition to the agricultural by-products, fast growing grasses such as

switchgrass (Panicum virgatum L.), Miscanthus X giganteus, reed canary and trees

such as willows and hybrid poplar have been identified as dedicated biofuel crops.

Of these, switchgrass and Miscanthus are the most favored candidates due to their

low input needs and high yield that can be harvested with existing agricultural

methods [8, 9]. There are varieties suitable for different ecosystems [10] with

estimated net energy yield of over 60 GJ/hectare/year [1]. Similarly, Miscanthus

has been shown to yield harvestable biomass between 30 and 60 t/hectare/year [4].

At the 30 t/hectare yield, it was estimated that 12 million hectares of US cropland

can yield adequate volumes of ethanol (133 9 109 l) corresponding to about 20%

of the annual gasoline used in the USA, and in comparison, corn starch grown in a

similar land area would yield only about 49 9 109 liters of ethanol with much

higher fertilizer needs and other inputs accounting for significantly higher GHG

emission [4]. Hence, it is clear that the net GHG release will be highly reduced by

using switchgrass and Miscanthus as feedstock for bioethanol.

8 Molecular Genetic Strategies


To get sustainable amount of biomass for the future biofuel production needs it

is important to enhance the biomass yield of these dedicated biofuel crops. In this

chapter we will discuss some of the possible molecular and genetic strategies to

enhance plant biomass.

8.2 Strategies for Enhancement of Biomass

The second-generation bioethanol production facilities depend on lignocellulosic

biomass, unlike the first-generation bioethanol plants that use corn starch or sugar.

Demands on agricultural land for food production are expected to increase significantly in the coming decades and hence use of marginal land to grow and

harvest the highest possible levels of biomass using plants such as switchgrass and

Miscanthus will contribute significantly to ensure sustainable production of

renewable fuel in the future. In order to enhance their productivity, these grasses

have to be targeted for intensive research aimed at improving the biomass yield

and other attempts to change the characteristics of the chemical contents

(e.g., lignin, hemicellulose, cellulose).

Expanding the industry to use biomass feedstock from the agricultural and

forestry waste materials and enhanced plant biomass from biofuel crops from

marginal lands might be the best ways to get more bioethanol and reduce net

emission of GHG. Hence, it is important to develop strategies to increase the yield

of plant biomass in a unit area of marginal land, and save the arable land for food

production. In this context, the following strategies can be employed to enhance

biomass production and ensure a sustainable and constant supply of lignocellulosic

biomass for bioethanol production.

8.2.1 Genetic Basis of Plant Architecture

Plant architecture is one of the important points to be considered for biomass

enhancement. It is clear that different plant species grow to different heights, sizes

and shapes. The final size and shape are determined by genetic and environmental

factors. Thus, it would be appropriate to conclude that plant architecture is

determined and influenced by the genetic information and the environmental

factors, respectively. The final shape of a mature plant is established by postembryonic growth of the shoot apical meristem (SAM) and root apical meristem

(RAM). SAM activity involves development of lateral organs such as leaves,

flowers and branches as well as maintenance of the meristem identity in a pool of

stem cells within the meristem. Recent data show that SAM is controlled by

several genes such as SHOOTMERISTEMLESS, CLAVATA and WUSCHEL in

dicotyledonous plants (e.g., Arabidopsis) and OSH1 and MOC1 in monocotyledonous plants (e.g., rice) (see [11] for detailed review). The involvement of


R. Ramamoorthy and P. P. Kumar

Table 8.1 Some of the mutants with demonstrated changes in branching phenotype (based on


Mutants with increased branching

Mutants with decreased branching




auxin insensitive1

branched1 and 2

more axillary branching1, 2, 3 and 4


ramosus1, 2, 3, 4 and 5


decreased apical dominance1


Maize (corn), wheat, sorghum

teosinte branched1


fine culm1 (OsTB1)

high tillering dwarf

dwarf3 and dwarf10


many noded dwarf





regulator of axillary meristems1, 2

and 3


lateral suppressor


lateral suppressor



tiller inhibition number3




low number of tillers1

uniculm2, uniculm4

absent lower laterals

semi-brachytic (uzu)

intermedium spike-b

various phytohormones such as cytokinin, gibberellin, auxin and abscisic acid in

regulating shoot development has been well recognized by plant physiologists and

developmental biologists. Therefore, it is interesting to note that besides the genes

listed above, several key regulatory genes that influence shoot development have

been identified, among which are phytohormone signaling intermediates such as

ARR5, ARR6 and ARR7 [11].

A number of other genes are known to be involved in regulating branching.

Table 8.1 lists some of the known mutants with increased or decreased branching

(for review see [12, 13]). The process of branching could be viewed as a multipronged developmental event, because it will involve establishment of axillary

meristem, development of axillary bud, promotion of the outgrowth of the branch

by overcoming the apical dominance [13]. Therefore, one can expect to find genes

regulating the various steps in this developmental program, and they can be the

targets of genetic modification of branching.

Manipulation of selected genes that are involved in plant growth and development may lead to the increase in the biomass. For example, mutation in a

cytochrome P450 gene called SUPERSHOOT resulted in significantly increased

axillary bud growth and led to profuse branching and significant increase in biomass [14].

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