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 Metabolic Regulation and
Metabolic Engineering for Biofuel
and Biochemical Production
 Metabolic Regulation and
Metabolic Engineering for Biofuel
and Biochemical Production

Kazuyuki Shimizu
Institute for Advanced Biosciences
Keio University, Yamagata
Japan

p,
p,
A SCIENCE PUBLISHERS BOOK
A SCIENCE PUBLISHERS BOOK
CRC Press
Taylor & Francis Group
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Boca Raton, FL 33487-2742
© 2017 by Taylor & Francis Group, LLC
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No claim to original U.S. Government works

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Version Date: 20170223

International Standard Book Number-13: 978-1-4987-6837-5 (Hardback)

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 Preface

The increasing economic growth and the prosperity has accelerated

worldwide with the increasing demand on the energy, mostly generated
from fossil fuels since the beginning of the industrial revolution in the mid
eighteenth century. This has brought about the rapidly increasing global
warming, caused by the emission of the green-house gas such as CO2,
resulting in disastrous climate change, where this problem will become
crucial even to the level of human survival by the end of this century.
It is, therefore, the time to take action to protect further global warming
by employing the alternative clean renewable sources of energy. Among
the renewable energy sources such as nuclear, solar, wind energies, etc.,
bioenergy seems to be the most promising alternative source of renewable
energy in the long range future.
This book intends to explain the current status and future perspectives
for biofuel and biochemical production from biomass, and motivates the
innovation for future energy problem. Moreover, attention is focused on
the production of biofuels and biochemicals directly from atmospheric CO2
by photosynthetic microorganisms. In particular, it is important to create
new approaches that bring innovation or revolution for bio-based energy
generation. The center for this is the metabolism of organisms. Although
many papers have been published on the production of biofuels and
biochemicals by metabolic engineering or synthetic biological approaches,
less attention is focused on the metabolic regulation. Understanding the
metabolism or metabolic regulation mechanisms in response to pathway
modification or genetic modulation is critical for the innovative design of
the cell metabolism for efficient biofuel production providing a solution to
the future energy problems. Thus the present book intends to explain the
metabolic regulation mechanisms including catabolic regulation prior to
the metabolic engineering practices. The metabolic engineering practices
are subdivided into two categories: one, using heterotrophic bacteria, yeast,
and fungi, and second, the photoautotrophic autotrophic microorganisms
such as algae and cyanobacteria. Finally, brief explanation is given for the
systems biology approach and modeling.
 Contents

Preface v
Notations xiii

1. Background 1
1. Current Status of Global Warming and Action Plan 2
2. Attempts to Reduce Energy Consumption 5
3. Alternative to Petroleum-based Fuels 6
3.1 Nuclear power energy 6
3.2 Wind energy 7
3.3 Solar energy 8
3.4 Other sources of renewable energy 8
3.5 Grid energy storage and future perspectives of 9
renewable energy sources
4. Bio-based Energy Generation for the Reduced CO2 Emission 9
5. Biofuel and Biochemical Production from Biomass 11
6. Brief Summary and the Outlook of the Book 12
References 14
2. Pretreatment of Biomass 16
1. Introduction 17
2. Various Pretreatments 20
2.1 Physical pretreatment 22
2.2 Biological pretreatment 22
2.3 Chemical pretreatment 24
2.4 Physiochemical pretreatment 26
3. Simultaneous Saccharification and Fermentation (SSF) 28
4. Consolidated Biomass Processing (CBP) 30
5. Concluding Remarks 32
References 32
3. Transport of Nutrients and Carbon Catabolite Repression for 38
the Selective Carbon Sources
1. Introduction 39
2. Variety of Regulation Mechanisms 40
viii Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

3. Porin Proteins in the Outer Membrane and their Regulation 42

4. Transport of Carbohydrates and PTS 46
5. Carbohydrate Uptake by Various PTSs and without PTS 50
6. Nitrogen PTS 52
7. Carbon Catabolite Repression for the Selective Carbon 53
Source Uptake
8. CCR in other Bacteria than E. coli 58
9. Concluding Remarks 61
References 61
4. Catabolite Regulation of the Main Metabolism 70
1. Introduction 70
2. Regulation of the Glycolytic Flux 72
3. Enzyme Level Regulation of the Glycolysis 74
4. Regulation of Pyruvate Kinase 79
4.1 Regulation of Pyk expression by isozymes 79
4.2 Effects of Pyk mutation on the metabolism 79
5. Transcriptional Regulation of the Glycolysis 84
6. Overflow Metabolism and the Oxidative Stress Regulation 86
7. Constraint on ATP Production by Respiration 90
8. Respiratory Pathways and the Competition with 91
Catabolic Transport
9. Coordination of the Metabolism by cAMP-Crp at Higher 94
Catabolic Rate
10. Carbon Catabolite Repression 96
10.1 Carbon catabolite repression in E. coli 97
10.2 Catabolite regulation in Corynebacteria 99
10.3 Catabolite regulation in Baccili 100
10.4 Catabolite regulation in Clostridia 100
11. Heteroginity of the Cell Population and CCR 101
12. Carbon Storage Regulation 103
12.1 Carbon storage regulation in E. coli 104
12.2 Carbon storage regulation in a variety of organisms 106
13. Concluding Remarks 109
References 110
5. Metabolic Regulation in Response to Growth Environment 126
1. Introduction 127
2. Nitrogen Regulation 127
3. Sulfur Regulation 131
4. Phosphate Regulation 132
5. Metal Ion Regulation and Oxidative Stress Regulation 133
6. Redox State Regulation 135
7. Acid Shock Response 137
 Contents ix

8. Heat Shock Stress Response 139

9. Cold Shock Response 140
10. Solvent Stress Regulation 141
11. Osmoregulation 142
12. Biofilm, Motility by Flagella, and Quorum Sensing 143
13. Concluding Remarks 145
References 147
6. Metabolic Engineering for the production of a Variety of 155
Biofuels and Biochemicals
1. Introduction 156
2. Typical Fermentation 159
2.1 Pyruvic acid fermentation 161
2.2 Lactic acid fermentation 162
2.3 Acetic acid fermentation 186
3. Production of TCA Cycle Intermediates 187
3.1 Citric acid production 189
3.2 Malic acid production 192
3.3 Fumaric acid fermentation 200
3.4 Succinic acid production 206
4. Diol Fermentation 207
4.1 Propanediol production 207
4.1.1 1,3-propanediol production 208
4.1.2 1,2-propanediol production 208
4.2 Butandiol production 212
4.2.1 2,3-butandiol production 212
4.2.2 1,4-Butandiol production 215
4.2.3 1,3-Butandiol production 215
5. Other Organic Acid Fermentation 216
5.1 Itaconic acid fermentation 217
5.2 Glucaric acid production 219
5.3 Muconic acid production 220
5.4 Adipic acid production 222
6. Amino Acids and Related Fermentation 222
6.1 Shikimic acid fermentation 223
6.2 Phenylalanin and biofuels production by 225
modulating Csr
6.3 Production of L-Tyrosine and its derived compounds 225
7. Isoprenoid, Polyketide, and Alkanoid Production 225
8. Biofuel Production 226
8.1 Hydrogen production 227
8.2 Ethanol fermentation 228
8.3 Higher alcohol production 239
8.4 Fatty acid fermentation 241
x Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

9. Tolerance to Solvent Stresses 242

10. Concluding Remarks 244
References 245
7. Biofuel and Biochemical Production by Photosynthetic 276
Organisms
1. Introduction 277
2. Candidate Photosynthetic Micro-organisms for Biochemical 278
and Biofuel Production
3. Metabolism of Photosynthetic Microorganisms 280
3.1 Brief overview of the metabolism of photosynthetic 280
organisms
3.2 Metabolism of algae 281
3.3 Metabolism of cyanobacteria 283
3.4 Nitrogen regulation and carbon storage regulation in 288
cyanobacteria
4. Metabolic Engineering of Photosynthetic Microorganisms 290
4.1 Metabolic engineering of algae 290
4.2 Metabolic engineering of cyanobacteria 291
4.2.1 Hydrogen production 292
4.2.2 Ethylene production 293
4.2.3 Ethanol production 296
4.2.4 Isopropanol production 297
4.2.5 Lactic acid production 297
4.2.6 Acetone production 300
4.2.7 Butanol production 301
4.2.8 Fatty acid and fatty alcohol production 302
4.2.9 Isoprene production 306
4.2.10 Diols production 308
(a) 1,2-propanediol 308
(b) 1,3-propanediol 309
(c) 2,3-butanediol 310
4.2.11 PHB production 311
4.2.12 3-hydroxybutyrate production 314
4.2.13 Other metabolite production 315
4.3 Efficient CO2 fixation 317
5. Systems Biology Approach and Modeling of the Metabolism 318
6. Cultivation Methods 320
7. Harvesting of Algal Biomass 320
8. Downstream Processing 321
9. Concluding Remarks 323
References 324
 Contents xi

8. Systems Biology Approach and Modeling for the 341

Design of Microbial Cell Factories
1. Introduction 342
2. Flux Balance Analysis and its Extensions 343
3. Kinetic Modeling and Incorporation of Metabolic Regulation 346
4. Modeling of the main Metabolism for Catabolite Regulation 348
5. Importance of the Modeling for the main Metabolic Pathways 350
6. Metabolic Regulation Mechanisms to be Incorporated in the 352
Kinetic Model
7. Modeling for the Metabolism under Oxygen Limitation 361
8. Concluding Remarks 363
References 364
Appendix A 373
Index 375
About the Author 387
 Notations

(General)
ABC transporter ATP-binding cassette transporter
CBB cycle Calvin-Benson-Bassham cycle
CBP consolidated bioprocess
CCR carbon catabolite repression
ETC electron transport chain
FFA free fatty acid
PP pathway pentose phosphate pathway
TCA cycle tri-carboxylic acid
PS photo system
PTS phosphotransferase system
ROS reactive oxygen species
SHF separate hydrolysis and fermentation
SSF simultaneous saccharification and
fermentation
(Metabolite)
AA amino acid
AcAcCoA acetoacetyl coenzyme A
ACC 1-aminocyclopropane 1-carboxylic
acid
AcCoA acetyl coenzyme A
AcP acetyl phosphate
ADP adenosine diphosphate
ADPG adenosine 5’-diphospho glucose
ALA amino-levulinic acid
Alg alginine
xiv Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

AMP adenosine monophosphate

Asp aspartic acid
ATP adenosine triphosphate
BDO butanediol
1,3BPG 1,3-bisphosphoglycerate
CHA chorismic acid
CIT citrate
DAHP 3-deoxy-D-arabinoheptulosonate
7-phosphate
DHA dihydroxyacetone
DHAP dihydroxyacetone phosphate
DHQ 3-dehydroqunic acid
DHS 5-dehydro shikimic acid
DMAPP demethylalyl diphosphate
E4P erythrose 4-phosphate
EPSP 5-enolpyruvate-shikimate-3-
phosphate
F6P fructose 6-phosphate
FBP fructose 1,6-bisphosphate
Fru fructose
FUM fumarate
G1P glucose 1-phosphate
G6P glucose 6-phosphate
GABA ɤ-amino butyric acid
GAP glyceraldehyde 3-phosphate
GL3P glycerol 3-phosphate
Glc glucose
Glu glutamic acid
Gln glutamine
GOX glyoxylate
GSH glutathinon
GTP guanosine triphosphate
HA hydroxyalkanoate
 Notations xv

HB hydroxybutyric acid
IA itaconic acid
ICI isocitrate
KDPG 2-keto-3-deoxy-6-phosphogluconate
αKG α-ketoglutaric acid
KMBA 2-keto-4-methyl-thiobutyric acid
Lys lysine
MA muconic acid
MAL malate
2MB 2-methyl-1-butanol
MEP 2-C-methyl-D-erythritol 4-phosphate
MG methylglyoxal
MQ menaquinone
MVA mevalonic acid
OAA oxaloacetate
OSP oseltamivir phosphate
PDO propanediol
2PG 2-phosphoglycerate
3PG 3-phosphoglycerate
PEP phosphoenol pyruvate
PHA polyhydroxy alkanoate
PHB polyhydroxybutyrate
PLA polylactic acid
PMA plymalic acid
6PG 6-phosphogluconate
PGA poly-β-1,6-N-acetyl-D-glucosamine
PPY phenylpyruvate
PQ plastoquinone
PQQ pyrroloquinone
PUFA polyunsaturated fatty acid
PYR pyruvate
R5P ribose 5-phosphate
R5PI ribose phosphate isomerase
xvi Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

Ru5P ribulose 5-phosphate

RuBP ribulose 1,5-bisphosphate
SA shikimic acid
S7P sedoheptulose 7-phosphate
Ser serine
SUC succinate
TAG triacylglycerol
Trp tryptophane
Tyr tyrosine
UQ ubiquinone
UQOH ubiquinol
X5P xylulose 5-phosphate
XOS xylo-oligosaccharide

(Protein)
ACC acyl-acyl carrier protein
Ack acetate kinase
ACP acyl carrier protein
Acs acetyl coenzyme A synthetase
ADC (Adc) acetoacetate decarboxylase
ADH alcohol dehydrogenase
Adk adenylate kinase
ADSL adenylosuccinate lyase
AHL acyl-homoserine lactone
AI arabinose isomerase
Ald aldolase
ALDC 2-acetolactate decarboxylase
ALDH aldehyde dehydrogenase
ALS acetolactate synthase
AOR aldehyde oxidoreductase
ASL alginino-succinate lyase
AspP adenosine diphosphate sugar
pyrophosphatase
 Notations xvii

AtoB acetyl transferase

BDH butanediol dehydrogenase
CA carboxylic anhydrase
cAMP cyclic adenosine monophosphate
CcpA catabolite control protein A
CimA citramalate synthase
Cra catabolite repressor/ acticvator
Crp cAMP receptor protein
CS citrate synthase
Csr carbon storage regulator
Ctf CoA transferase
Cya adenylate cyclase
DXS 1-deoxy-D-xylulose 5-phosphate
synthase
EI enzyme I
EII enzyme II
EFE ethylene forming enzyme
Eno enolase
F1PK D-fructose-1-phosphate kinase
FAA fumarylacetoacetase
FAS fatty acid synthase
FHL formate hydrogenlyase
Fnr fumarate nitrate reductase
Fum fumarase
G6PDH glucose 6-phosphate dehydrogenase
GADC glutamate decarboxylase
GAPDH glyceraldehydes 3-phosphate
dehydrogenase
GDH glutamate dehydrogenase
GlgA glycogen synthase
GlgB glycogen branching enzyme
GlgC ADPG phosphorylase
GlgP glycogen phosphorylase
xviii Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

Glk glucokinase
Glx glyoxalase
GlgX glycogen debranching enzyme
GlpK glycerol kinase
GlyDH glycerol dehydrogenase
GOGAT Glutamate synthase (glutamine
oxoglutarate aminotransferase)
GS glutamine synthetase
Hbd 3-hydoxybutyryl-CoA
dehydrogenase
HPr histidine-phosphorylatable protein
HPrK HPr kinase
Hxk hexokinase
Hyd hydrogenase
ICDH isocitrate dehydrogenase
Icl isocitrate lyase
IspS isoprene synthase
KDC ketoacid decarboxylase
Kivd 2-keto-acid-decarboxylase
LADH L-arabitol 4-dehydrogenase
LALDH lactaldehyde dehydrogenase
LDH lactate dehydrogenase
Lrp leusine responsive regulatory protein
LXR L-xylulose reductase
MarR multiple antibiotic resistant regulator
MDH malate dehydrogenase
Mez(Mae) malic enzyme
Mgs methylglyoxal synthase
MIOX myo-inositol oxygenase
MS malate synthase
Nac nitrogen assimilation control protein
NR nitrogen regulator
Pck phosphoenolpyruvate caroxykinase
 Notations xix

PDC pyruvate decarboxylase

PDH pyruvate dehydrogenase
PDOX propanediol oxidoreductase
Pfk phosphofructokinase
PGDH 6-phosphogluconate dehydrogenase
Pgi phosphoglucose isomerase
Pgm phosphoglucomutase
Pgk phosphoglycerate kinase
PhaA β-ketothiolase
PhsB acetoacetyl-CoA reductase
PhaC polyhydroxy alkanoate (PHA)
synthase
PI3K phosphoinositide 3-kinase
Ppc phosphoenolpyruvate carboxylase
Pps phosphoenolpyruvate synthase
PRK phosphoribulokinase
Pta phosphotransacethylase
Pyc pyruvate carboxylase
Pyk pyruvate kinase
RhaD rhamnose dehydrogenase
RK ribulokinase
Rpe ribulose 5-phosphate 4-epimerase
Rpi ribulose 5-phosphate isomerase
RubisCo ribulose 1,5-bisphosphate
carboxylase
SADH secondary alcohol dehydrogenase
SDH succinate dehydrogenase
SOD superoxide dismutase
Tal transaldolase
TE thioesterase
Ter trans-2-enoyl-CoA reductase
Thl thiolase
Tkt transketolase
xx Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

Udh urinate dehydrogenase

XDH xylitol dehydrogenase
XI xylulose isomerase
XK xylulokinase
XR xylose reductase
 1
Background

ABSTRACT
The increasing economic growth and the prosperity has been accelerated
worldwide with the increasing demand on the energy mostly generated
from fossil fuels since the beginning of the industrial revolution in the mid
eighteenth century. This has brought about rapid global warming caused
by the emission of the greenhouse gases such as CO2, resulting in the
disastrous climate change, and this problem will become endangering even
to the level of human survival by the end of this century. It is, therefore,
the time to take action to prevent further global warming by employing
the alternative clean renewable sources of energy. Among the renewable
energy sources such as nuclear, solar, wind energies, etc., bioenergy seems
to be the most promising alternative source of renewable energy in the
long range future.
The so-called 1st generation biofuels have been produced from corn starch
and sugarcane in USA and Brazil. However, this causes a problem of the
so-called “food and energy issues” as the production scale increases. The
2nd generation biofuels production from lignocellulosic biomass has thus
been paid more attention recently. However, it requires energy intensive
pre-treatment for the degradation of lignocellulosic biomass. The 3rd
generation biofuels production from photosynthetic organism such as
cyanobacteria and algae has also come under attention, but the cell growth
rate and thus the productivity of the fuels is significantly low. The typical
processes for biofuel and biochemical production from biomass include
pre-treatment of biomass, saccharification, fermentation, and separation
of the dilute fermentation broth followed by purification.
Keywords
Global warming, renewable energy, pretreatment, lignocellulosic biomass,
biofuel, biochemical, 1st generation biofuel, 2nd generation biofuel, 3rd
generation biofuel, low carbon society
2 Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

1. Current Status of Global Warming and Action Plan

The increasing economic growth and the prosperity have been accelerated
worldwide with the increasing demand on the energy generated from
mostly fossil fuels since the beginning of the industrial revolution in the mid-
eighteenth century. This has been promoted by the steam powered trains
and ships, and then the internal combustion engines significantly changed
the human’s life style and production system in relation to transportation
and industrial production. Nowadays, many people live comfortable
lives spending much electricity for air conditioning and freezing of food,
etc. Many people use cars and airplanes to move across the countries and
the continents spending more and more fossil fuels as sources of energy.
Unlike the beginning of the industrial revolution, where the population
of the world was 700 million, the current population is 7 billion and
is estimated to grow to 9 billion by 2,050, and even about 10 billion by
2,100 (Lee 2011). Unlike the developed countries, the population growth
is more eminent in the developing countries like Asia and Africa, where
the economic growth is accelerating year by year, caused by promotion
of industrialization to catch up to the standard of posh and comfortable
living of the developed countries. The International Energy Agency (IEA)
has projected that the world’s energy demand will increase from about
12 billion ton oil equivalents (t.o.e.) in 2,009 to either 18 billion t.o.e. or 17
billion t.o.e. by 2,035 under the current policies or new policies scenarios,
respectively (International Energy Agency 2011). In association with this,
carbon dioxide (CO2) emissions are expected to increase from 29 giga tons
(Gt) per year to 43 Gt/year or 36 Gt/year under the current policy and new
policies, respectively. This creates significant climate-change risks in either
policies, and we are now facing a risk to even human survival by the end of
this century. Namely, we need another industrial revolution for the energy
sources to be affordable, accessible, and sustainable. Energy efficiency and
conservation, as well as decarbonizing the energy sources are essential to
the revolution (Chu and Majumdar 2012).
International Panel on Climate Change (IPCC) keeps warning the
global society on global warming caused by greenhouse gases (GHGs) such
as CO2 based on the accumulating data and the reliable prediction model.
IPCC asks world societies to make decisions to take actions and to invest
in the reduction of CO2 emissions caused mostly by human activities. This
may be also considered from the point of view of future cost caused by the
severe climate change due to global warming. Namely, the global warming
may cause serious local climate change as well as the rise in the sea level,
which will severely damage the societies worldwide. In fact, every year
we are experiencing disastrous climate change, and it seems to grow more
and more severe. We have recently experienced disastrous phenomena as
 Background 3

extreme heat waves, droughts due to segregated rain falls, wild fires in
the broad forest, melting glaciers, etc. These phenomena may have been
caused largely by the global warming most likely due to the accumulation
of GHGs in the atmosphere, mainly CO2 from the burning of fossil fuels. In
2013, a big news was broadcasted throughout the world warning that the
CO2 level in the earth’s atmosphere had passed over 400 ppm (parts per
million) for the first time in several million years. As shown in Fig. 1, the
CO2 level periodically changed in accordance with the repetition of warming
and cooling of the earth probably due to the effect of black body irradiation
from the surface of the sun since 400,000 years ago. However, the sharp
increase in the CO2 level caused by the human activities after industrial
revolution becomes eminent, and this is becoming more and more severe.
The international society has recognized the importance of this
problem since the late 20th century. The international political activities
against climate change began at the Rio Earth summit in 1992, where Rio
convention included the adoption of the United Nations Framework
Convention on Climate Change (UNFCCC). This convention attempted
to take action for stabilizing atmospheric concentrations of GHGs to avoid
“dangerous anthropogenic interference with climate system.” The UNFCCC
has currently a near universal membership of 195 parties including both
developed and developing countries.

Figure 1. Global change in CO2 concentration and the temperature on earth (https://
stevengoddard.wordpress.com/2014/09/26/climate-is-not-difficult/, http://cdiac.ornl.gov/
trends/co2/recent_mauna_loa_co2.html).
4 Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

The annual conference of parties (COP) began in Berlin in 1995,

followed by several conferences such as COP3 in Kyoto, where the Kyoto
Protocol was adopted, COP11 in Montreal, where the action plan was
determined, COP17 in Durban, where the Green Climate Fund was created.
Unfortunately, some large CO2 producing countries denied signing the
agreement of Kyoto Protocol, and the efficient action against climate change
has been unsuccessful. As mentioned above, however, the global warming
steadily proceeds, where the glaciers and the iceberg in the north-pole
are melting, causing sea level to rise, and causing drastic climatic change
worldwide, and the atmosphere is becoming more and more humid
due to vaporization of sea water at an unprecedented rate caused by the
temperature rise. The international societies strongly recognize the current
and future critical situations caused by CO2 emission from fossil fuels.
In 2015, COP21 (also known as the 2015 Paris Climate Conference)
aimed to achieve a legally binding and universal agreement on climate,
where the conference was attracted about 50,000 participants including
25,000 official delegates from government, intergovernmental organizations,
united nation (UN) agencies, non-governmental organizations (NGOs) and
civil societies. Then a historic agreement was signed to combat climate
change and unleash actions and investment towards a low carbon, resilient
and sustainable future. Each county must set individual target and must
attain it, where the action result will be checked every 5 years, but this is
rather effort target without legal constraint, even though this is a big one
step towards preventing further global warming. The aim of the universal
agreement is to keep the global temperature rise well below 2 degrees
Celsius (2ºC) during this century, and take strong action to limit the
temperature rise even further to 1.5ºC above pre-industrial level.
Now the developed countries face the problem of promoting
industrialization without or much less emission of CO2 with efficient
transportation and production systems, or may have to change the life
styles drastically, while the developing countries may have to keep
industrialization without or less amount of emission of CO2 with the aid
of technology transfer and investment from the developed countries.
Namely, green based innovation and revolution is necessary to attain such
target and to realize a low carbon society. Now is the time to pay attention
to innovations that provide an entirely new energy system including
transportation and stationary systems together with energy generation
systems. It is also of importance to investigate the ways of energy efficiency
and the integration of energy sources with electricity transmission,
distribution and storage (Chu and Majumdar 2012).
Currently, petroleum-oriented liquid fuels are the main source of energy
in the transportation infrastructure throughout the world. The geographical
distribution of petroleum resources changes as the new resources such as
 Background 5

shale oil field are found and accessed by new technologies for discovery
and production. The distribution of petroleum production does not coincide
with the place of the demand. This means that fair amount of fuels must
be transported spending energy for this purpose. For example, about 2.690
billion tons of oil were consumed, where 1.895 billion tons of crude oil and
0.791 billion tons of refined products crossed national borders (BP Statistical
review of the world 2012). This also brings along with itself political problem
of national-security for energy, where many countries are forced to import
oils from the limited oil producing countries.
The overall loss caused by the increase in the number of heat waves,
floods, wildfires, droughts, and storms may be estimated to be over $150
billion per year (Munich Re 2012), where these phenomena may possibly be
caused by the climate change (Cambridge University Press 2012). The cost
of renewable energy is rapidly becoming competitive with other sources
of energy.

2. Attempts to Reduce Energy Consumption

Improvements in energy efficiency can contribute for the reduction of fuel
usage. One idea is to use light-weight materials for vehicles. Some attempts
have been made for ultra-high tensile strength steels, carbon-fiber reinforced
composite materials, aluminium and magnesium alloys, and polymers
(Gibbs et al. 2012). The potential of reducing the weight of vehicles such as
cars and airplanes has already been shown without sacrificing safety, and
it is expected to reduce more about 20–40% (Powers 2000). It is estimated
that for every 10% weight reduction of the vehicle, 6–8% of improvement
is expected in fuel consumption (Holmberg et al. 2012). Moreover, the
reduction of the friction loss in vehicles may also contribute for the efficient
energy usage, where several attempts have been made for the development
of cost effective technologies such as tyres, braking and waste-heat energy
recovery, etc. (Holmberg et al. 2012), where Rankin cycle may be utilized
to convert waste heat to work by low-cost and high efficiency solid state
thermoelectric systems (Yang and Caillat 2006).
Plug-in hybrid electric vehicles (PHEVs) or electric vehicles (EVs)
have the opportunity to displace a significant amount of liquid fuel use
in transportation, and reduce GHG emission. Such vehicles are becoming
competitive, a mass-market car with the internal combustion engine (ICE)
using liquid transportation fuel. The high efficiency of fuel-cell-powered
electric vehicles may become a potential option in the near future, where
there are inherent volumetric energy density issues for hydrogen-gas
storage. The supply infrastructure and a low-carbon source of hydrogen
are challenged in several countries. The technology advances in shale-gas
production may give impact on the transportation system. In addition to
6 Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

the direct use of natural gas as a fuel, low-cost natural gas may give local
reforming or hydrogen filling stations in the near future, where delivery
to local filling stations may be made through high-pressure gaseous tube
trailers in practice. Due to the low cost of natural gas as compared to
petrol, natural-gas vehicles may become more widely used, where liquefied
natural gas (LNG)-powered trucks and trains may be considered for the
long distance transportation (Chu and Majumdar 2012).

3. Alternative to Petroleum-based Fuels

The time-scale and the mix of the current energy sources is shown in Fig. 2,
where the ratios have been significantly changed after industrial revolution
in mid 1800s.

Figure 2. Change in energy usage after industrial revolution. World energy consumption by
source, based on Vaclav Smil estimates from Energy Transitions: History, Requirements and
Prospects together with BP Statistical Data for 1965 and subsequent. https://ourfiniteworld.
com/2012/03/12/world-energy-consumption-since-1820-in-charts/.

3.1 Nuclear power energy

Nuclear power may play an important role in decarbonizing the energy

production, where the percentage of nuclear energy usage is about 14% of
the total electric power generated in 2009 (International Energy Agency
 Background 7

2010), although this percentage dropped to about 12% in 2011, mainly

caused by the less usage in Japan due to Fukushima disaster and Germany
(BP 2012, Ruhl 2011). After the Fukushima nuclear disaster in 2011, all the
nuclear power plants were eventually shut down in Japan. This disaster
affected the energy policies of various countries, where some countries keep
running the nuclear power plants with careful caution; some have opted
to reverse their decision on nuclear energy, or accelerating planned-phase-
outs (Chu and Majumdar 2011). In some developing countries, however,
the electricity shortage is critical, which prevents the comfortable life and
industrialization, and thus the nuclear energy is among the top priority
for such countries with the risk of safety. The most significant problem in
using nuclear energy is treating the spent fuel before disposal, which is still
contaminated with highly radioactive particles. Moreover, the numbers of
old nuclear power plants (say more than about 40 years old) are increasing,
and the cost of amortizing plants is not low. Hence the humans are yet to
learn to control the nuclear energy.

3.2 Wind energy

Significant improvement has been made in turbines, blades and gearboxes,

and the height of the wind towers to reduce the cost. As the height and
size of the wind turbines increase, more and more mechanical stresses are
imposed on the gearbox, blades and tower. Direct-drive wind turbines can
convert the slow rotary motion directly into alternating current electrical
power using electromagnetic generators, which consist of permanent
magnets composed of rare-earth metals. The dependence of the materials
on the rare-earth gives a problem of unforeseen future availability, since
more than about 90% of the rare-earth metals are located in China (US
Department of Energy: DOE 2012).
Offshore wind farms are attractive in the sense that they can be placed
in near-proximity to cities, and yet far enough away to mitigate local
opposition. However, it is not easy to construct a marine structure that
can withstand the harsh marine environment for many years, and thus
the cost remains high, which prevents its application in practice (Chu and
Majumdar 2012).
As the scales and the number of wind turbines increase, the effect of
such towers with fans on the aerodynamics, and the effect of high frequency
sound on the human health must be carefully analyzed. Such problems
could be avoided by installing the wind towers far from the population
area, but another problem of energy loss during the long-distance transfer
of energy may become eminent.
8 Metabolic Regulation and Metabolic Engineering for Biofuel and Biochemical Production

3.3 Solar energy

Although the installation cost for solar panels are becoming lower due to
mass production, the apparent limitations are the locations and the areas
to be used, and the dependence on the sunny weather. The costs may be
further decreased by increasing the efficiency of the solar cells. The ultimate
efficiency limit in the conversion of sunlight energy to electric energy is
determined by a Carnot heat engine, where the limit is estimated to be
about 94% based on the temperature of the sun’s black body at 5,800 degree
Kelvin (K) and the cell temperature of about 350 K. The Shockley-Queisser
limit is a well-known efficiency limit of single-junction solar cells, which
suffer from spectrum losses, recombination and black-body radiation, where
the limit of the silicon is about 37%, but may actually be low at around
29% in practice (Swanson 2005). This efficiency may be more improved by
considering the above theoretical upper bound.
The solar thin films are made of crystalline substances, and
polycrystalline-silicon photovoltaics, direct-gap semi-conductors such
as cadmium telluride and copper-indium gallium-selenide (CIGS).
Cadmium-telluride thin films are used, but there is yet significant room
for improvement. Improvement of solar-module efficiency may also come
from multi-function cells that capture a large fraction of the solar spectrum,
multi-exciton generation, multi-photon absorption or photon up-conversion
and light concentration (Chu and Majumdar 2012).
Solar flat panels may be also replaced by the concentrated solar
photovoltaics, where the main cost of the system is the mirrors, lenses, and
their tracking system rather than solar cell at the focal point of the system
(Angel and Olbert 2011).
In order to convert direct current to alternating current electricity and
to integrate solar power into the grid, power electronics is crucial (US
Department of Energy 2012).

3.4 Other sources of renewable energy

Hydrolytic power energy has long been employed in particular at

mountainous regions, where water fall is necessary to catch the energy from
the river. Although this may be important for the protection of flooding
by the dam capacity during strong heavy rain, etc., this is limited to the
mountainous locations.
Geothermal energy is another source of energy associated with
volcanos, but the installation of the power plant may dry up the hot springs,
and may disturb the geothermal environment.
Exploring the Variety of Random
Documents with Different Content
 "Mrs. Varney laughed, too; but she knew by this time
Miss Allen would be expecting her scholar; so she said,
'Come, I'll show you the way.'"

CHAPTER VII.

BILLY'S CLEAN FACE.

I CAN'T tell you how surprised Miss Allen was, when she
found this was really Billy. She seated him among the
scholars, by her side, and then gave him his second lesson.

"This was the story of the Saviour's love to little

children. Billy sat with parted lips, and listened as if for his
life.

"But I must hasten to tell you how the poor boy, after
school was dismissed, found himself near his own home. He
lived in what is called a tenement house, and in the one
entry where he lodged there were, only think of it, ninety
persons!

"On the Sabbath the men, and women too, were mostly
out of doors, lounging in the dirty court. Now, as Billy
entered it, the boys and girls stared at him in some
surprise, thinking, of course, that he was a stranger.

"'Hollo!' at last shouted one fellow, 'it's our Bill! Look at

him. What a prig! Oh! Oh!' And he laughed boisterously.
 "Billy smiled quietly to himself, and, making his way
through the door, started to run upstairs, when a woman
called out,—

"'Who are ye after, if I may be so bold?'

"'It's Billy Foster, no mistake,' said another, 'but I can't

tell what he's been doing to himself.'

"Billy's mother started to her feet when he entered. She

stared at him with open mouth, not daring to speak until he
said, laughing,—

"'Don't you know me, mother?'

"'And what has happened ye, Bill? Sure it's like a

miracle ye are.'

"The boy eagerly related what had occurred, adding

that he had promised to put aside his new jacket for
Sunday wear.

"'Wait till I see will yer father know his own son,' she
said; eagerly; 'sure I can't believe my own eyes.'

"Mr. Foster agreed with his wife, that it was little short
of a miracle; but the next day the mother said,—

"'Feth, if soap and wather can do it for Billy, it can for

Jack.' So she set to work, and washed and scrubbed all her
children, greatly to the astonishment of the neighbors.

"'Sure and washing is a good thing,' she said to her

husband, 'I'll be trying it on myself;' which she did. Then
she argued that the room was not clean enough for such
tidy looking folks; and when Mr. Foster came home at night
from his wood-sawing, he saw her scouring the floor on her
knees, the chairs being piled up on the bed.

"First her neighbor on the right, and then the neighbor

on her left, imitated her example, until to the astonishment
of the landlord, who came weekly for his rent, there was a
gradual but thorough renovation of the place.

"It was astonishing how much more these poor people

respected themselves, now that their persons and clothes
and rooms were clean. Soon they began to ask themselves
whether they, too, could not go to the mission school where
Billy had been treated so kindly.

"All this was not done at once. It required a good deal

of arguing on the part of Mrs. Foster to induce some of her
lazy neighbors to commence a reformation, and Billy was
called in from his play a great many times to describe the
bath-room, the good nature of Mrs. Varney, and the
kindness of the pretty lady in the school, before they would
consent to follow so good an example.

"Miss Allen had thought often of Billy, and resolved to

visit him in his home, not dreaming of all the changes
wrought there by the sight of his clean, fresh face.

"She set out early Sunday morning, and was delighted

to find Billy's two brothers and one sister ready to
accompany her to the school.

"I cannot stop to tell you all the particulars," said

Cousin Stuart, smiling, "for Lily is growing very sleepy; but
I will say that Billy's clean face was the means of leading
thirty persons, men, women, and children, to the mission
school, where they learned to love the Saviour."
 "That's a nice story," exclaimed Helen, "I wish mamma
had heard it."

"I think so, too," said grandma. "I should like to see
Billy."

CHAPTER VIII.

THE AUGUST TREE.

WHEN the buds began to swell, the crocuses to push

their bright-colored blossoms out of the ground, and the
golden robins to sing, mamma said it was time for the little
ones to be in the country. The Rector did not say nay,
though he must stay a good many weeks longer to take
care of his parish.

The children began to dance and clap their hands at the

idea of going to the farm where they always had such a
happy time, where there was plenty of nice milk and cream,
and, in the summer, fruit and berries in abundance.

Mamma and Sarah were often now in earnest

consultation about the packing. There were calls to make,
too, and shopping for the summer; so the month of May
was a busy time at the rectory, you may be sure.

Early in June, when the roses were in bloom, and the

peach trees were garlanded with their beautiful blossoms,
the transit was made, and one evening after a very tedious
journey of over two hundred miles, our travellers found
themselves in their summer home by the sea.

And now the happy days commenced, and life seemed

one long dream of innocent enjoyment.

Aunt Josephine, with Ida, Rose, Forrest, and baby, came

a few days later, then grandma, Russell, and Aunt Mary, so
that there was a large company to be merry with. And then
there was the pleasure of expecting the two papas, who
came for a day or two now and then.

On the lawn in front of the spacious mansion, there was

a game of croquet, which in fine weather occupied both old
and young for many an hour; while for the little ones a
long, unfinished hall over the shed was the scene of daily
frolics.

Russell, who had declared himself a candidate for

orders, built a pulpit at one end, which was really quite an
ingenious affair. In this, he officiated as minister nearly
every day in the week. Here, too, they kept store, played
"come and see," or rolled hoop in rainy weather.

Out of doors, the chickens, the lambs, and the pony

were unfailing sources of delight. Star, indeed, was a very
important character. He was of a light sorrel color, with a
white spot in his forehead, from which he received his
name. The pony was the especial property of Master
Forrest, a lad of twelve, the oldest of all the cousins.

Uncle George, his papa, thought him too old to spend

all his summer in play, so he bought a working-cart for Star,
and sent the boy out occasionally to labor with the hired
men.
 There was also a pony carriage, in which four of the
cousins rode regularly to church.

I suppose some little girls and boys will inquire whether

Helen, Ida, and Forrest studied any in those bright days. To
be sure they did, though the lessons were short. When the
trunks were packed, Sarah put in Helen's books, "just so
that she sha'n't forget all she knows," mamma said.

In the winter she had been studying very hard, and was
getting on finely. Now she had a short lesson in geography,
history, and French; and Ida recited with her.

Forrest recited, too, to his mamma; but his delight was

to get a new book, when he would seat himself in the midst
of the children and devour it. They might talk, and laugh,
and sing, and shout; but he heard nothing.

Forrest was a manly boy, rather grave and reserved,

quite unlike his Cousin Russell, who was always ready for
any kind of fun.
 THE AUGUST TREE.

I wish my little readers could see Lily and Rose, and

how merrily the days passed with them; or that I had time
to tell you more of Russell and Berty, at their boyish plays in
the hall. But I must hasten to give you an account of an
August tree, and the celebration that accompanied it.

The little folks—there were nine in all—had been very

good through the long warm days, and the two mammas
were quite willing to afford them any reasonable pleasure.
 So, when, one morning, Russell said, "Oh, I wish it was
Christmas!"

And Helen answered, "We can have an August tree, as

well as a Christmas tree."

A ready consent was given to her earnest request.

The plan was at last arranged; a beautiful spruce tree

brought from the woods; and then Aunt Josey wrote tiny
notes inviting the guests.

There were the farmer and his wife and family, the
carpenter and the painter, with their families, all
dependents of the great house; each must come to the
August tree, have his or her present, and stay to the big
supper.

What a busy time that was, and how earnestly the

children consulted in regard to suitable gifts, and the
decorations of their tree! In the kitchen what a clatter of
dishes and of tongues, beating of eggs and sugar! Russell,
who seemed to be everywhere, ran at last to announce the
fact that the cake had come out of the oven and smelled
real good.

The day so long and eagerly anticipated came at last,

and at an early hour the children's voices rung through the
house.

The guests had all signified their pleasure at the

invitation, and their resolution to be "on hand." The
celebration was to be in the afternoon; but the little folks
had enough to do to keep themselves busy till about eleven.
After this, the hours lagged terribly. Russell thought the
clock had stopped, and Helen said, earnestly,—
 "I do wish two o'clock would come."

Frank Follen, the farmer's boy, came first, dressed in his

Sunday suit, with his face scrubbed till it shone. Then Mr.
Pratt, the carpenter, made his appearance, followed by his
entire family; and after this, not ten minutes elapsed before
every guest had been ushered into his or her seat.

The tree having been greatly admired, the company

were requested to go downstairs and stand under the
window.

This made a good deal of laugh; even the Rector was

observed to grow quite red in the face; but, under Master
Russell's excellent management, the request was carried
out.

Ida and Helen then took the gift from the tree, called
out the name of the one to receive it, and began to let it
down by a cord tied to a basket.

This part of the entertainment consumed nearly an

hour, but was enjoyed amazingly.

Mr. Follen handled the little necktie, which fell to him, as

daintily as if it had been china, saying with a laugh,—

"I shall think a heap of this ere, Miss Helen."

When the presentations were over, the guests were

invited to the dining-room, where the children waited on
them.

Everybody had a good time, and were loud in their

praises of the August tree.
 At an early hour the children went to bed, weary, it is
true, but happy in the consciousness that they had made
others so; while the two papas and mammas rejoiced that
their children so early showed a disposition to contribute to
the comfort of those about them, and especially that in all
their plays they remembered the apostle's injunction, "Be
kindly affectioned one to another."
 *** END OF THE PROJECT GUTENBERG EBOOK LILY'S BIRTHDAY
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