Bio hydrogen bio butanol generation by bacteria isolated from spent mushroom substrate

BIO-HYDROGEN AND BIO-BUTANOL GENERATION
BY BACTERIA ISOLATED FROM
SPENT MUSHROOM SUBSTRATE

MARC-ANTOINE METAIS
(GRADUATE STUDENT OF LYCEE SAINTE GENEVIEVE, PARIS
DAUPHINE UNIVERSITY AND ECOLE CENTRALE PARIS)

A THESIS SUBMITTED FOR THE
DEGREE OF MASTER OF ENGINEERING
DEPARTMENT OF CIVIL AND ENVIRONMENTAL ENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE

2011


Acknowledgment

I would like to thank my supervisor, Professor He, who has helped me in this
research. I appreciate her time and willingness to share her ideas and opinions with
me.

I would also like to express my deep appreciation for the time, effort and energy
that my mentor, Xin Fengxue, invested in my research. He was always insightful,
supportive and understanding. His expertise and ability to guide, listen and provide
feedback contributed enormously to the quality of my research. Fengxue provided
invaluable advices and was always willing to provide me with needed assistance. I
feel extremely fortunate for having Xin Fengxue as my mentor.

I am also thankful for the support that my family, friends and labmates provided
me during my study: Yu Ran, Becky, Xiaomei, Constance, Chenxi, Shen Yujia,

Yan Yu, Si Yan, Cheng Dan, Dr Chua, Dr Ray, Dr Li, Qi Chao, Lam Yuen Sean,
Ding Chang, Wang Shan Quan, Lim Kah Bin, Thierry Desmarest, Kukik, Nur,
Alexandre and Victor. Unquestionably, without their support I would not have
been able to complete this study. Because of this, I would like to dedicate this
dissertation to all of you who made it possible.

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TABLE OF CONTENTS

Ackowledgment........................................................................................................i
Abstact......................................................................................................................v
List of figures.........................................................................................................vii
List of tables………….............................................................................................x
List of symbols………………………………………………….………………...xi

CHAPTER 1. Introduction and Literature Review.............................................1
1.1 Spent Mushroom Substrate............................................................................1
1.2 Value-added products.....................................................................................5
1.2.1 The potential of hydrogen....................................................................8
1.2.2 Butanol and its use.............................................................................11

CHAPTER 2. Methodology..................................................................................15
2.1 Medium Composition...................................................................................15
2.2 Medium Making Protocol............................................................................16
2.3 Inoculation....................................................................................................17
2.4 Biological Safety Cabinet………………………………………………….19
2.5 Analysis........................................................................................................20
2.5.1 Gas analysis…………………………………………………….…...20

2.5.2 Gas Chromatography – Thermal Conductivity Detector………..….21
2.5.3 Gas Chromatography – Flame Ionization Detector……………...….22
2.6 DNA Analysis – Strain Identification……………………………………..24

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CHAPTER 3. Isolation of bacteria from Spent Mushroom Substrate............27
3.1 Growth of mixed culture on Spent Mushroom Substrate.............................27
3.2 Growth of mixed culture on glucose............................................................31
3.3 Growth of mixed culture on different substrates..........................................36
3.4 Isolation of pure cultures using glucose as a carbon source.........................39
3.5 Identification of strains S4 and S11………………………….……………41

CHAPTER 4. Growth of bacteria using different kinds of carbon
sources....................................................................................................................47
4.1 Growth of pure cultures using glucose as a carbon source..........................47
4.2 Growth of pure cultures using xylose as carbon source……………….…..51
4.3 Growth of pure cultures using Spent Mushroom Substrate as carbon
source………………………………………………………………………...…...55
4.4 Pre-treatment of Spent Mushroom Substrate...............................................62
4.4.1 Enzymes production...........................................................................62
4.4.2 Enzymes purification..........................................................................63
4.4.3 Hydrolysis....................................... ..................................................70
4.4.4 Fermentation.......................................................................................71
4.5 Glucose concentration optimization.............................................................72
4.6 Upscaling of the laboratory-scale experiments............................................74
4.6.1 Protocol…………………………………………..…………………74
4.6.2 Results................................................................................................78

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CHAPTER 6. Conclusion.....................................................................................83
6.1 Major findings……………………………………………...……………...83
6.2 Recommendations and future studies…………………................………...84

References..............................................................................................................86

iv


Abstract

The world is now being overwhelmed by many kinds of waste. Agricultural waste
is a major issue, especially in emerging countries, where there is usually no
recycling facilities. This study focuses on Spent Mushroom Substrate, which is the
compost material remaining after a few cropping cycles of the mushroom industry.
The main objective of our study is to isolate and cultivate bacteria from this Spent
Mushroom Substrate. An anaerobic fermentation process producing bio-hydrogen
and bio-butanol is studied, using classic carbohydrates such as glucose, xylose,
cellulose and xylan, and then Spent Mushroom Substrate as carbon sources for the
micro-organisms. Few previous studies were done on this topic.

In this study, three strains of bacteria were isolated from Spent Mushroom
Substrate, able to generate value-added products from simple carbohydrates
(predominantly glucose). A full DNA analysis was conducted for the two best
strains. We further performed a thorough study of these bacteria growing on
several kinds of Spent Mushroom Substrate, in order to assess which kind of Spent
Mushroom Substrate could be used efficiently. Then, an optimization process of

the experiment conditions was conducted in order to improve the output of the
experiments. The substrate concentration was optimized, and a pre-treatment of
the Spent Mushroom Substrate, before the fermentation, was also performed. The
last part of the study is a pre-industrial simulation. We upscaled the laboratory
experiment in order to test whether the process could be done on-site, directly in a
mushroom farm.

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One strain was particularly good in generating bio-hydrogen and bio-butanol, and
was named S11. However, most of Spent Mushroom Substrates were not easily
degraded by S11. This is due to the molecular structure of Spent Mushroom
Substrate we tested: it can be made of very different kinds of materials (horse
manure, different kinds of wood sawdust…), come from very different places
(China, Malaysia…), and be stored in different conditions (temperature, humidity,
contamination…).

After optimization of the fermentation process of S11 on glucose, the molar
percentage of hydrogen was about 30%, and the butanol concentration was about
6.0 g/L, which are high concentrations. We noted that the pre-treatment process of
Spent Mushroom Substrate was not efficient enough, and therefore not costeffective for a mushroom farm. Finally, the pre-industrial simulation was
successful. We proved that upscaling the experiment did not cause any major
disturbance on the fermentation process: the results are very similar to those
obtained when using laboratory bottles. However, the process is slower than when
using laboratory bottles: this would be an issue on a practical viewpoint.

As a conclusion, we managed to isolate a good hydrogen–producing and butanolproducing strain, S11, from Spent Mushroom Substrate. The output of the
anaerobic fermentation is high concentrations of hydrogen and butanol, which are
highly-regarded value-added products nowadays. A pre-industrial simulation was

conducted and showed that the process we designed has potential to be
implemented on-site.

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List of figures

Figure 1 Top production of Mushroom and Truffles – Source: [1] ..........................1
Figure 2 Energy content of different chemicals. Source: [19] ..................................9
Figure 3 Biochemical Pathways in Clostridium acetobutylicum during acidogenic
phase - Source [47] .................................................................................................14
Figure 4 Orbital Shaker Incubator ..........................................................................18
Figure 5 Gas Chromatocgraphy Analyzer, Agilent Technologies ..........................22
Figure 6 Microscope view of Oyster Mushroom and Lentinula Edodes endogenic
mixed culture ..........................................................................................................28
Figure 7 Gas production of OM mixed culture growing on OM ............................29
Figure 8 Gas production of LE mixed culture growing on LE ..............................30
Figure 9 Total gas production of mixed cultures growing on glucose 30g/L .........32
Figure 10 Gas composition of OM mixed culture growing on glucose 30g/L .......33
Figure 11 Gas composition of LE mixed culture growing on glucose 30g/L .........33
Figure 12 Biosolvents production of OM mixed culture growing on glucose 30g/L
(mM) .......................................................................................................................34
Figure 13 Biosolvents production of LE mixed culture growing on glucose 30g/L
(mM) .......................................................................................................................35
Figure 14 Total gas production of mixed cultures growing on different carbon
sources.....................................................................................................................37
Figure 15 Biosolvents production of mixed cultures growing on different carbon
sources.....................................................................................................................38
Figure 16 Anaerobic Chamber ................................................................................40

Figure 17 Colonies on agar plate ............................................................................41
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Figure 18 Picture after DGGE (from left to right: S4, ladder, S11) .......................42
Figure 19 Extract of DNA Sequence for S4 Strain .................................................43
Figure 20 Extract of DNA Sequence for S11 Strain ...............................................43
Figure 21 Phylogenetic tree for S4 Strain ...............................................................44
Figure 22 Phylogenetic tree for S11 Strain .............................................................45
Figure 23 Cumulative gas production of pure cultures growing on glucose 30g/L 48
Figure 24 Biosolvents production of S4 pure culture growing on glucose 30g/L ..49
Figure 25 Biosolvents production of S11 pure culture growing on glucose 30g/L 50
Figure 26 Biosolvents production of ESE1 pure culture growing on glucose 30g/L
.................................................................................................................................50
Figure 27 Cumulative gas production of pure cultures growing on xylose 30g/L .52
Figure 28 Biosolvents production of S4 pure culture growing on xylose 30g/L ....53
Figure 29 Biosolvents production of S11 pure culture growing on xylose 30g/L ..53
Figure 30 Biosolvents production of ESE1 pure culture growing on xylose 30g/L
.................................................................................................................................54
Figure 31 Spent Mushroom Substrate : from the farm to dried powder .................56
Figure 32 Total gas production of S4 growing on SMS 30g/L...............................57
Figure 33 Total gas production of S11 growing on SMS 30g/L.............................58
Figure 34 Fermentation bottles ...............................................................................59
Figure 35 Biosolvents production of S4 growing on A 30g/L................................59
Figure 36 Biosolvents production of S4 growing on OMN 30g/L .........................60
Figure 37 Biosolvents production of S11 growing on A 30g/L..............................60
Figure 38 Biosolvents production of S11 growing on OMN 30g/L .......................61
Figure 40 Beaker with xylanase solution ................................................................64
Figure 41 Dialysis process ......................................................................................65

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Figure 42 Freeze-drying of the enzymes ................................................................66
Figure 43 Test tubes for optical density measurement ...........................................68
Figure 44 Spectrophotometer samples ....................................................................69
Figure 45 Standard curve for optical density of enzymes .......................................70
Figure 39 Gas composition of S11 pure culture growing on glucose at different
concentrations at pH=5.5 ........................................................................................73
Figure 46 Bio-fermenter and gas tank at the initial step .........................................75
Figure 47 Bio-fermenter and gas tank after 12 hours .............................................76
Figure 48 Total gas production of S11 pure culture growing on glucose 30 g/L on
pre-industrial scale ..................................................................................................78
Figure 49 Gas composition of S11 pure culture in Fermenter ................................79
Figure 50 Biosolvents production (mM) of S11 pure culture in Fermenter ...........80

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List of tables

Table 1 Components of Spent Mushroom Substrate. Source: [12]..……………….3
Table 2 PCR conditions …………………………………………………………..25
Table 3 Hydrogen yield of S4, S11 and ESE1 on Glucose 30 g/L ……………….49
Table 4 Standards composition …………………………………………………...67

x


List of symbols

SMS – Spent Mushroom Substrate
OM – Oyster Mushroom
LE – Lentinula Edodes
A – Abalone Mushroom
GC – Gas Chromatography
FID – Flame Ionization Detector
TCD – Thermal Conductivity Detector
PCR – Polymerase Chain Reaction
DGGE – Denaturing Gradient Gel Electrophoresis

xi


CHAPTER 1. Introduction and Literature review

1.1 Spent Mushroom Substrate

According to the Food and Agriculture Organization of the United Nations [1], the
global production of cultivated edible mushrooms has increased from 0.79 tons in
1970, to 2.07 million tons in 1990 and 6.53 million tons in 2009. It is expected that
this amount will increase in the future due to market demand. In 2009, Singapore
mushroom production was 18 tons. About 53% of cultivated edible mushrooms are
produced in Asian countries (China being the largest producer), followed by
European countries (32%) and the Americas (13%). In Asia, mushrooms are eaten
and appreciated for their flavor, and used medicinally for their healing properties.

Figure 1 Top production of Mushroom and Truffles – Source: [1]

1

After a few cropping cycles (usually 2 to 4), the mushroom productivity
diminishes, and the compost material is called “spent”, and it is then replaced by
fresh compost. Despite the evident benefits of mushrooms, the exponential
increase in their consumption worldwide is also generating a high volume of spent
mushroom substrate (SMS). Spent Mushroom Substrate is discarded and treated as
waste. It has been reported that about 5 kg of substrate are needed to produce 1 kg
of mushroom [2], and about 30 million tons of SMS are produced each year. SMS
has a storage problem because it is wet and putrefies quickly. Consequently, one of
the main problems faced by mushroom production companies is finding a way to
properly dispose of the SMS without contaminating the water and soil. In fact, the
lack of a sustainable waste management solution for SMS is the most significant
barrier to the future development of the mushroom industry [3].

Several studies have been carried out to demonstrate the benefits of SMS
application in mushroom re-cultivation, enrichment of soils, restoring areas that
have been destroyed through environmental contamination [4], cultivation of
vegetables, fruits and flowers in greenhouses and fields [5], use as animal feed [6]
and soil amendment and degradation of organopollutants [7]. The SMS can also be
used as a potential energy feedstock [8], and ethanol production [9].

Thus, environmental concerns have been escalating recently concerning its
effective disposal and recycling: in China, wastes are largely burnt by the farmers,
which causes air pollution issues. Mushroom producers use specially formulated
compost as growth medium to cultivate their mushrooms: the major ingredients
used to make the substrate are straw, hay, corncobs, horse manure, poultry litter,

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gypsum, lime. Among them, one of the most common sources is wood sawdust,
which is routinely used for the cultivation of king Oyster Mushroom (Pleurotus
eryngii) and Winter Mushroom (Flammulina velutipes).

Spent mushroom substrate is a nutrient-rich organic product. The chemical
composition of SMS determines its potential for reuse and environmental effects.
On average, fresh SMS is about 60% water and 40% dry material by mass [10],
whereas approximately 65% of the dry matter is organic and 35% is inorganic salts
[11]. Generally speaking, SMS contains high levels of organic, C, N, and inorganic
Ca+, Mg2+, Na+, K+, Cl-, SO42- .

Components

Content (g/kg)

Organic C

290 to 340

Organic N

17 to 26

P

5 to 7

S

50 to 60

K

21 to 26

Ca

83 to 97

Na

2 to 4

Mg

5 to 8

Cl

6 to 8

Table 1 Components of Spent Mushroom Substrate. Source: [12]

3


In this study, we use the Spent Mushroom Substrate from an organic mushroom
farm located in Johor, Malaysia. The SMS was used to harvest several species of
mushroom, mainly Pleurotus Ostreatus (OM, Oyster Mushroom), Lentinula

Edodes (LE, Shiitake), and Pleurotus cystidiosus (A, Abalone Mushroom). Oyster
mushroom is a mushroom species commonly found in Asia, is edible and is
believed to have medicinal virtues: it contains statins which work to reduce
cholesterol. Lentinula Edodes (Shiitake, LE) is also an edible mushroom native to
East Asia, and is used as a delicacy as well as a medicinal mushroom, in this
region. Abalone Mushroom is native to and still found growing wild in China. The
Abalone mushroom is named for the aquatic shellfish, abalone, whose shape the
mushroom resembles. It can be used for its bioremediation properties of
organopollutants.

The SMS from this mushroom farm was stored in anaerobic condition in a sealed
plastic bag. The time-duration of one mushroom culture cycle is about 4 months.
Then the anaerobic plastic bags were brought to our laboratory one month after the
harvest of the mushroom. Spent Mushroom Substrate was made of different
materials, depending on the mushroom to be grown on it. For OM, SMS was made
of oak tree sawdust, from China. For A, SMS was made of rubber tree sawdust,
from Malaysia. This mushroom farm is an organic mushroom farm, so the only
component of the SMS is wood sawdust: no chemical was added to the SMS.
Moreover, we can assume that the SMS is very clean, since, in order to grow the
mushroom mycelium, there has to be no contamination.

4


After harvest, mushroom have degraded part of the sawdust, producing
carbohydrates, constituting the major carbon source for the bacteria. Therefore,
there are two contradictory phenomena occurring at the same time. The more we
wait, the more wild bacteria will grow without any control and consume the
carbohydrates in the SMS. On the other hand, the more we wait, and the more the
mycelium will produce carbohydrates by degrading SMS. After a given period of

time, the former phenomenon will be predominant, because there is a fixed amount
of biomass that can be degraded by the mycelium.

A typical mushroom farm, such as the one we worked with, generates about 15
tons of SMS per month, which is 180 tons per year. This SMS has to be disposed
of, and most companies cannot do it by themselves, spending money on waste
management. The mushroom farm we worked with being an organic mushroom
farm, they convert most of their SMS into organic compost, to grow other on-site
agricultural products. Our aim is to provide a process which could essentially help
mushroom farms not to pay for SMS disposal, and turn this waste into value-added
products.

1.2 Value-added products

According to the second assessment report of the Intergovernmental Panel on
Climate Change (IPCC) [13], the Earth’s surface temperature has increased by
about 0.2ºC per decade since 1975. Furthermore, recognizing a number of
uncertainties, “the balance of evidence suggests that there is a discernible human
5


influence on global climate” as the result of activities that contribute to the
production of greenhouse gases. By preventing heat radiated from the sun-warmed
earth from escaping into space, the increased concentration of greenhouse gases in
the atmosphere contributes to climate change.

The gases that produce the greenhouse effect are carbon dioxide (CO2), methane
(CH4), nitrous oxide (N2O), and a host of engineered chemicals such as
hydrofluorocarbons (HFCs) and perflorocarbons (PFCs). About 90% of U.S.
greenhouse gas emissions from anthropogenic sources come from energy

production and use [14], and most (82%) of these emissions are a byproduct of the
combustion of fossil fuels. CO2 accounts for a majority of recent increases in the
heat-trapping capacity of the atmosphere, with worldwide atmospheric
concentrations of CO2 increasing at about 0.5% annually. Anthropogenic CO2 has
resulted in atmospheric CO2 concentrations that exceed preindustrial levels by
30%. Energy-efficient, renewable-energy, and other low-carbon technologies
reduce CO2 emissions by reducing the need for fossil fuel combustion.

Significant reductions in greenhouse gas emissions can be accomplished only
through an assemblage of actions ranging from more effective production,
distribution, and use of energy to a reliance on lower-carbon fuels. Given the
magnitude of carbon emission reductions needed to stabilize atmospheric CO2
concentrations, multiple approaches to carbon management will be needed. Such
changes have the potential to transform the world’s buildings, industries, vehicles,
and electricity production. Each of the three energy end-use sectors (buildings,
industry, and transportation) account for approximately one-third of CO2

6


emissions. This diversity of sources and uses of fossil energy means that no single
technological “fix” exists for reducing carbon dioxide emissions. Using the
framework of the 11-Lab study [15], there are three options for reducing
atmospheric carbon.

First, energy efficiency can decrease the energy intensity of the world economy,
thereby reducing carbon emissions. Energy-efficient technologies and products
such as more efficient cars, trucks, and household appliances provide the same
energy services using less fuel or electrical power and thereby emitting less
carbon. Similarly, energy requirements can be reduced through efficient system

designs, such as co-locating facilities that produce both electrical power and heat
with facilities that need them. A broad array of energy-efficiency options exists.

Second, the use of low-carbon technologies can decrease the carbon intensity of
the world’s energy economy, thereby reducing carbon emissions. These
technologies either increase the efficiency of energy production or use fuels that
emit less carbon such as renewable energy resources and nuclear power.
Electricity generation from natural gas is also a low-carbon technology when
compared to current coal-fired power plants; natural gas emits 13 MtC per quad of
energy used compared with 25 MtC per quad for coal [16]. Biomass feedstocks
offer an array of low-carbon options, including ethanol fuels, chemicals, materials,
and electricity. The carbon emissions from biomass combustion are largely offset
by CO2 absorption during plant growth.

7


Third, carbon sequestration technologies offer another suite of approaches to
reducing atmospheric concentrations of CO2. Carbon sequestration can include
various ways of removing CO2 from the atmosphere and storing it, or keeping
anthropogenic carbon emissions from reaching the atmosphere by capturing and
diverting them to secure storage [17]. Most approaches to carbon sequestration
will require considerable additional research to ensure their successful
development and acceptance. However, in the long-term, they could play
significant roles.

This thesis focuses on the second point: the use of low-carbon technologies. We
emphasize the need to generate value-added by-products from clean sources, such
as bacterial fermentation. We will highlight the importance of hydrogen and
butanol, both produced by either a mixed culture of bacteria or single-strain

colony, isolated from Spent Mushroom Substrate.

1.2.1 The potential of hydrogen

Hydrogen is considered to be one of the most promising fuels of the future [18]
due to its high energy content, 130 kJ/g, as compared to hydrocarbon fuels.

8


Figure 2 Energy content of different chemicals. Source: [19]

Hydrogen gas is also a clean fuel with no COx, SOx and NOx emissions from its
combustion. Besides, hydrogen is an important energy carrier and can be used in
fuel cells for generation of electricity [20].

However, hydrogen gas is not readily available in nature like fossil fuels and
natural gas, but can be produced from renewable materials such as biomass and
water [21]. Hydrogen gas production technologies have gained special attention
during the last fifty years due to the increasing energy demand, rapid consumption
of non-renewable fossil fuel reserves and hydrocarbon fuel based atmospheric
emissions. Users regularly purchasing pressurized hydrogen gas do it in heavy
steel cylinders containing about 0.6 kg of H2 per cylinder. The price of this
hydrogen has been reasonably stable at about $100/kg, plus cylinder rental [22].

9


Steam reforming of natural gas and water electrolysis are the most commonly used
processes for H2 gas production. Due to the energy intensive nature of those

processes, more energy efficient H2 production methods are searched for.
Hydrogen gas production from renewable resources such as biomass, and
carbohydrate rich waste materials by bioprocesses, offers distinct advantages over
energy intensive methods used. Major drawbacks in biohydrogen production are
low yields and productivities requiring large reactor volumes and long residence
times [23].

As mentioned beforehand, nowadays, hydrogen is usually produced by thermochemical processes, electro-chemical processes and bio-conversion processes.
Among them, bioconversion process is a promising method for two reasons:
utilization of renewable resources, and usually operated at ambient temperature
and atmospheric pressure.

Main bio-hydrogen production processes are direct or indirect bio-photolysis, dark
and photo-fermentations. Bio-photolysis of water under sunlight is considered as
the cleanest approach for bio-hydrogen production. However, low H2 gas
productivity, strict light requirement and oxygen inhibition are the main problems
in bio-photolysis of water [24].

Fermentative hydrogen gas production from carbohydrates is a much faster
process than bio-photolysis, with volatile fatty acids (VFAs) and H2 gas formation
[25]. However VFAs need to be fermented for further H2 gas production [26].
Major mechanisms for bio-hydrogen production by fermentation have been

10


elucidated. However, development of an effective bio-hydrogen production
process at industrial scale is still a challenge.

Up to now, the reported rates and yields of fermentative hydrogen gas production

were not high enough to make the process economically viable. The most suitable
raw materials, pre-treatment methods, bacterial cultures, operating conditions,
cultivation types, operating modes and processing schemes are yet to be
determined for an effective and economically viable fermentative hydrogen
production process.

1.2.2 Butanol and its use

The second byproduct we want to highlight is butanol C4H9OH. Though ethanol
C2H5OH and biodiesel are driving the world’s progress on the road of renewable
fuels, some attention is being generated by butanol, a fuel whose promoters
believe can become a real player in the world of energy alternatives.

Butanol is a 4-carbon alcohol: C4H9OH. Today, butanol is typically produced from
petroleum sources, but that has not always been the case. During the first half of
the 20th century, the production of butanol from biological sources was a
commercial reality.

According to the National Renewable Energy Laboratory (NREL), biobutanol had
previously been produced through a fermentation process known as “ABE” named
11


such because it produced Acetone, Butanol, and Ethanol in roughly 6:3:1 ratios.
Clostridium strains were the fermenting organisms to create the chemicals from
molasses-type feedstocks [27]. This ABE process nearly disappeared in the 1960s
because it could not compete on a cost basis with the economical creation of
solvents from cheap, plentiful petroleum.

The current market for butanol is largely industrial, for use as a plasticizer or

solvent. The world market for butanol is estimated at 350 million gallons per year,
with the U.S. accounting for about 220 million of that. The average cost for a
gallon of butanol is between three and four U.S. dollars [28]. Butanol has been
generating some attention as a potential alternative fuel, akin to ethanol but with
added benefits. One of those qualities is butanol’s low Reid vapor pressure (RVP),
a rating of 0.33 psi versus ethanol’s 2 psi and gasoline’s 4.5 psi, meaning butanol
has lower evaporative emissions [29].

Another property is its energy content. Butanol has a higher energy density (29.2
MJ/L) than ethanol (19.6 MJ/L), but lower than gasoline (32 MJ/L) [30]. So,
switching a gasoline engine over to butanol would in theory result in a fuel
consumption penalty of about 10%. However, using butanol instead of ethanol as
an alternative fuel would result in a consumption benefit of 48%. This figure
shows how efficient would butanol be compared to ethanol, if we were to switch
from gasoline engines to biofuel engines.

Butanol can be blended at higher concentrations in gasoline than existing biofuels
without modifications to automotive engines. Butanol is considered substantially

12


similar to gasoline for blending purposes and is certified by the U.S.
Environmental Protection Agency as a blending agent up to 11 percent [31].
Currently, no production vehicle is known to be approved by the manufacturer for
use with 100% butanol. As of 2009, only few vehicles are approved for even
using E85 fuel (i.e. 85% ethanol + 15% gasoline) in the USA. However, in Brazil
all vehicle manufacturers (Fiat, Ford, VW, GM, Toyota, Honda, Peugeot, Citroen)
produce flex fuel vehicles that can run on 100% ethanol or any mix of ethanol and
gasoline. These flex fuel cars represent 90% of the sales of personal vehicles in

Brazil, in 2009. BP and Dupont, engaged in a joint venture to produce and promote
butanol fuel, claim that "biobutanol can be blended up to 10%v/v in European
gasoline and 11.5%v/v in US gasoline" [31].

According to analysis done by the National Renewable Energy Lab [32], most of
the advantages of butanol come from its properties as a fuel, not from current
production technology. Traditionally, low yields - in the 15 to 25 percent range have plagued butanol production from bacterial reactions. Toxicity is also a
problem, with butanol generally being toxic to microbes at the rate of 20 g/L.

13