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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.
i
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
ii
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
iii
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.
v
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.
vi
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
vii
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
viii
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
ix
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,
2
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
Figure 3 Biochemical Pathways in Clostridium acetobutylicum during acidogenic phase - Source [47]
14
CHAPTER 2. Methodology
All experiments were conducted under the same experimental conditions, unless
otherwise stated. All experiments were duplicated in order to have a higher
accuracy for the results.
2.1. Medium composition
The anaerobic bacteria used in all the following experiments were grown in a
liquid medium. This liquid medium is named modified DCB1, unless otherwise
stated. It provides pH-buffers and elements which are necessary to the growth of
our cultures [48], [49], [50].
In every cases, the medium used to grow the cultures of bacteria is the same. The
modifications are the following:
1- TES (pKa=7.55) is not used, but instead we use MES (pKa=6.15): 2-(Nmorpholino)ethanesulfonic acid: 10 mM. MES is more suitable because its
pH buffer range is relevant to the pH of the reactions required (around 6.0).
2- We add KH2PO4: 0.5 g/L
3- We add K2HPO4: 0.5 g/L
4- We add Yeast extract: 5 g/L
2.2 Medium Making Protocol
15
The protocol for making modified DCB1 medium is very strict, because the
anaerobic property must be strictly observed all along the making of the medium.
Following is the protocol used for the experiments in this paper. The volume of
medium prepared is 1800 mL, so all the amounts are given for 1800 mL of
modified DCB1 solution. Rinse rubber stoppers with deionized-H2O and dry them
with scientific cleaning wipes. Then, place them in clean beaker, cover with
aluminum foil and autoclave. This step is meant to prevent the rubber stoppers
from contamination.
The following step is to prepare defined mineral salt medium. Using a 2-L flask,
measure 1800 mL of deionized-H2O with 10 mL salt solution, 1 mL trace element
solution, 1 mL Se/W solution, and 0.25 mL resazurin. Put the flask onto an
electrothermal heating mantle and set up refrigerated bath circulator and
condenser. Bring to boil for 20 minutes under N2-purging at 1 psi.
After 20 minutes boiling, cool solution to room temperature under N2-purging at 2
psi in ice-water bath. Introduce new N2-source and switch off refrigerated bath
circulator and condenser. Measure buffering agents and reductants while solution
cools: MES 10 mM, 0.0242 g L-Cysteine, 0.048 g Na2S.9H2O, 0.0771 g DTT,
K2HPO4 0.5 g/L, KH2PO4 0.5 g/L, Yeast extract 5 g/L.
Add the buffering agents and reductants while stirring with a magnetic stir bar.
Insert pH-probe into solution and adjust pH to 7.0 by flushing with N2 and careful
addition of 10% H2SO4 or 20% NaOH. Flush 60-mL syringe with the headspace
16
gases 2 to 3 times and aliquot 35 mL of solution into the bottles, under N2-purging
for 1 to 2 minutes, stopper and seal bottle with crimper after 1 to 2 minutes. Ensure
all bottles are properly labeled, then autoclave for 20 minutes at 121°C. After
autoclave, all the bottles were stored at air temperature, and kept ready for addition
of carbon source and inoculation.
2.3 Inoculation
Inoculation was done inside of the anaerobic chamber. Anaerobic chamber
provides a strict anaerobic atmosphere of 0-5 parts per million using a palladium
catalyst and hydrogen gas mix of 5%. In order to use the chamber, one has to bring
in a baked catalyst, an anaerobic indicator (to test O2 level before starting the
experiment) and an empty beaker for condensed water every time entering the
chamber.
The condition to use the anaerobic chamber is to minimize the O2 input. If items
with sealed bags or caps (e.g., syringes larger than 3 mL, vials/tubes larger than 2
mL) are to be placed into the chamber, one has to slightly tear the bag open or
loosen a bit the caps of vials/tubes, in order to let O2 out during the autocycle.
Autocycle consists of a 3 times successive vacuum and N2 injections, inside of the
airlock, in order to make sure that the airlock is made anaerobic. After that, the
chamber itself, when bringing the items from the airlock to the chamber, can be
considered anaerobic.
17
The first step of inoculation is to remove the cap of the bottle, and scoop the
defined amount of carbon source (glucose or SMS most of the time, in this study)
into the 1st-generation autoclaved bottles. The second step is to inject the bacteria
into the bottle. Use sterile syringe and needle to inject the colony into the bottle.
Usually, in this study, and unless otherwise stated, the inoculation will be done at
5% (v/v). The last step is to stopper and seal the bottle with a crimper. Finally,
bring bottles out of the anaerobic chamber, use a syringe to release the gas (N2
flushed during medium preparation) contained in the headspace of the bottles, and
place in orbital shaker incubator at 35°C and 150 rpm.
Figure 5 Orbital Shaker Incubator
All the following experiments in this study were conducted with an additional
control bottle. This control bottle is not mentioned in the results to make this paper
18
more clear for the reader. However, for every experiment, a control bottle was
prepared with the same substrate as in the experiment, but without any inoculation
of bacteria. This control bottle was prepared in order to detect a possible
contamination. When gas or biosolvents were detected in the control bottle, the
experiment was re-conducted. This step is of paramount importance in order to
ensure that our results are as accurate as possible.
2.4 Biological Safety Cabinet
A Biological Safety Cabinet is an enclosed, ventilated laboratory workspace for
safely working with materials potentially contaminated with pathogens requiring a
defined biosafety level. The Biological Safety Cabinet used in our laboratory is a
ThermoScientific Herasafe KS, with a safety level of Class II. The US Centers for
Disease Control and Prevention defines Class II Biological Safety Cabinets as
hoods used to protect personnel, product and environment from bio-aerosols and
other particulates. These hoods offer personnel protection through engineered
airflow into the cabinet. To protect the product, the work area in the cabinet is
continuously bathed with ultra-clean air provided through the supply HEPA filter.
Approximately 70% of the air from each cycle is recirculated through this supply
HEPA filter [46].
Before and after using a Biosafety Cabinet, the working surface was disinfected
with a 70% alcohol solution. Materials for the experiment were placed inside the
cabinet prior to the start of the experiment. Overloading of material in the cabinet
19
should was avoided because materials may obstruct optimal airflow. We allowed
two minutes of purge time after placing materials in the cabinet. When performing
an experiment, materials were kept away from the front air intake and rear exhaust.
Transfer of viable materials were performed as deeply into the cabinet as possible.
The cabinet was never turned off while a flame is being used. Following
completion of the experiment, the cabinet was allowed to purge itself for about
two minutes.
2.5 Analysis
All samples were analysed on an everyday basis (unless otherwise stated). Three
different kinds of measures will be presented in this part. Unless otherwise stated,
all the graphs in this study are done until day 13. The reason is that 13 days after
inoculation, all the experiments had no gas production anymore, and there was no
more change in the biosolvents concentrations (including butanol).
2.5.1 Gas analysis
The first analysis that was automatically conducted is the gas release from the
bottles. The protocol for this analysis is very simple: apply ethanol on the stopper,
gently shake the bottle, insert needle and syringe, and allow the gas to fill the
syringe. Then, remove the syringe when the piston reaches equilibrium level,
dispense gas in the fume-hood. Reinsert the syringe into the bottle in case the
20
volume of gas produced is more than the syringe volume. Finally, the amount of
gas produced is noted down every day.
2.5.2 Gas Chromatography - Thermal Conductivity Detector
The second analysis is to measure the gas composition. We used a GC-TCD (Gas
Chromatography - Thermal Conductivity Detector), which is a non-specific and
non-destructive detector.
The GC-TCD is based on the principle of thermal conductivity which depends
upon the composition of the gas. The sample components in the carrier gas pass
into the measuring channel. A second channel serves as a reference channel where
only pure carrier gas flows. Electrically heated resistance wires are located in both
channels. The difference in thermal conductivity between the column effluent flow
(sample components in carrier gas) and the reference flow of carrier gas alone,
produces a voltage signal proportional to this difference. The signal is proportional
to the concentration of the sample components. Like all chromatographic
analytical processes, gas chromatography is a relative method: calibration with a
standard mixture is required, both to check linearity and as calibration for the
sample.
21
Figure 6 Gas Chromatocgraphy Analyzer, Agilent Technologies
The GC-TCD used was manufactured by Agilent Technologies. The protocol for
this analysis is to apply ethanol on the stopper, gently shake the bottle, insert
needle and small syringe, and extract a volume of gas of 100 L from the
fermentation bottle. Then, inject the gas onto the head of the chromatographic
column. The sample is transported through the column by the flow of inert,
gaseous mobile phase. The column was already calibrated to measure
concentrations of H2, N2 and CO2. CH4, which is also a good source of biofuel,
was also produced by the fermentation process, but this study focuses on H2 only.
2.5.3 Gas Chromatography - Flame Ionization Detector
The third analysis is to measure the organic compounds concentrations, using GCFID (Gas Chromatography – Flame Ionization Detector). In GC-FID, the FID
22
detects analytes by measuring an electrical current generated by electrons from
burning carbon particles in the sample. The FID is a non-selective detector, so
there is a potential for many non-target compounds present in samples to interfere
with this analysis and for poor resolution especially in complex samples. The FID
works by directing the gas phase output from the column into a hydrogen flame. A
voltage of 100-200V is applied between the flame and an electrode located away
from the flame. The increased current due to electrons emitted by burning carbon
particles is then measured. Although the signal current is very small (the ionization
efficiency is only 0.0015%) the noise level is also very small.
Except for a very few organic compounds (e.g. carbon monoxide) the FID detects
all carbon containing compounds. In our experiments, we focus on butanol, acetic
acid, and butyric acid. Most of the time, ethanol and acetone will also be
generated, and sometimes, propionic acid is produced in reasonable amount.
However, we do not mention it in this study since the main focus is butanol. All
the graphs shown below emphasize butanol, acetic acid and butyric acid only, I
order to make the graphs clearer for the reader.
The GC-FID used was manufactured by Agilent Technologies. To prepare the
samples, one need to use the Biological Safety Cabinet. The first step is to
dispense 25 L of 1 mol/L Sulfuric Acid H2SO4 in a 2 mL microfuge tube, and
then to add 0.475 mL of the sample to be tested. Then, vortex the sample and
centrifuge it at 10°C, 14,000 rpm for 20 minutes. Finally, aspirate 420 L of the
supernatant, dispense into a 2 mL screw cap vial, and tighten screw cap. Place the
tubes for reading by the GC-FID.
23
2.6 DNA Analysis – Strain Identification
The process for strain identification is in several steps. Cells (1 mL) used for DNA
extraction were withdrawn from cultures and then centrifuged immediately at
14,000 rpm for 15 minutes at 4°C. After removing the supernatant, the cell pellets
were stored at -20 ºC until further processing. The extraction of the genomic DNA
of the strains was done using DNeasy Blood and Tissue Kit (250), by Qiagen
GmbH, Germany, according to the manufacturer’s instructions.
Polymerase chain reactions (PCR) were carried out in an Eppendorf Master Cycler
ep gradient S thermocycler (Eppendorf AG, Hamburg, Germany). The extracted
DNA was used as a template for Polymerase Chain Reaction (PCR) amplification
of the 16S rRNA gene, which were amplified with a pair of universal bacterial
primers
8F
(Forward:
5'-AGAGTTTGATCCTGGCTCAG-3')
and
1392R
(Reverse: 5'-ACGGGCGGTGTGT-3'). The reaction mixture (50 µl) contained
5×PCR buffer, 2.5 mmol/L MgCl2, 0.13 mmol/L BSA, 0.25 mmol/L dNTP, 1.25 U
of Taq DNA polymerase, 0.05 µmol/L forward primer and 0.05 µmol/L reverse
primer, and 20 ng template DNA.
24
Process
Temperature °C
Duration
Initial Denaturation
95
2'10''
Denaturation
95
30''
55
45''
Extension
72
2'10''
Final Extension
72
6'
Annealing
30 cycles
Table 2 PCR conditions
The PCR-amplified 16S rRNA was purified and its size was verified by
low melting point agarose electrophoresis. TAE buffer was prepared by mixing 40
mL of TAE solution in 1960 mL of Milli-Q ultra-pure water. 1 gram of Agarose
powder (SeaKem LE Agarose, BioWhittaker Molecular Applications, USA) was
mixed with 100 mL of 1xTAE mixture in a conical flask. The Agarose powder was
thoroughly dissolved by heating up in microwave for about 90 seconds. Dissolved
Agarose was poured into a casting tray to allow solidification. The gel casting tray
was first leveled using a bubble level and its well comb placed securely before the
gel was poured in and allowed to harden. Trapped air bubbles in the hardening gel
were removed using a pipette tip so that they would not affect DNA migration
during electrophoresis. Comb was removed once gel is hardened.
Gel was then transferred, together with the tray, into the electrophoresis unit.
1xTAE buffer was poured into the tray to cover the gel completely. Thawed
extracted DNA samples were vortexed and centrifuge in micro-centrifuge tubes. 5
μL of 100 bp or 1-kb DNA ladder (Promega, Madison, US) was then loaded into
25
the first well. 1 μL of 6xblue/green loading dye (Promega, USA) was mixed with 5
μL of a DNA sample and then loaded into a well. Electrophoresis (Bio-Rad, U.S)
was done at 90V for 90 minutes when all DNA samples had been loaded. Gels
were removed from tray The bands in gel were visualised by UV excitation and
pictures were taken with a digital camera (Gel Doc, Bio-Rad, USA).
26
CHAPTER 3. Isolation of bacteria from Spent Mushroom
Substrate
The purpose of this study is to cultivate bacteria generating value-added products
for on-site production. So the emphasis is now put on finding, isolating and using a
particular strain, or consortium of strains, isolated from SMS and generating valueadded products (here, we focus on hydrogen and butanol).
Several steps were undertaken. The first one was to grow the mixed culture
directly from SMS on different carbon sources. The second one was to grow
isolated pure cultures from SMS on different carbon sources. The carbon sources
used in this paper are glucose, xylose, cellulose, xylan, and SMS itself (2 different
kinds). SMS should be particularly hard to degrade, according to its complex
composition.
3.1. Growth of mixed culture on Spent Mushroom Substrate
This first experiment was run in order to find out whether the bacteria (mixed
culture) growing on SMS can grow by themselves, using SMS as a carbon source,
to generate value-added products. No other carbon source was added in this
experiment, in order to be as accurate as possible, regarding the overall amount of
SMS consumed. The SMS was added fresh into the DCB1 medium, so the carbon
27
source is similar to the one found in the mushroom farm, after the mushroom are
grown and harvested. The SMS used is the one to grow the two mushroom strains
OM and LE, as previously stated.
A preliminary microscope screening shows the presence of a lot of different
bacterial strains in both bacterial sources (OM and LE). Several bacterial
morphologies can be observed, such as spheres (coccus), or round-ended cylinders
(bacillus). We can also notice different aggregation properties (bacteria density)
among these two samples. This diversity is a good indicator of the growth of
bacteria in the SMS after the harvest of mushroom. We will try to isolate
interesting strains from these samples, in order to generate value-added products.
Figure 7 Microscope view of Oyster Mushroom and Lentinula Edodes endogenic mixed culture
For this first experiment, the pH was controlled on a daily basis in order to
replicate the natural living conditions of the bacteria. The initial pH after
inoculation was 5.5; hence, everyday, the pH was adjusted to 5.5, using either
28
H2SO4 or NaOH solutions. Several SMS concentrations in the medium were used:
2.5 g/L, 5 g/L, 10 g/L, and 15 g/L, for both OM and LE substrates. The results of
the gas production for both OM and LE are presented here.
30
25
V
o 20
l
u
m 15
e
Day 13
Day 5
Day 4
Day 3
(
m 10
L
Day 1
)
5
0
OM-2.5g/L
OM-5g/L
OM-10g/L
OM-15g/L
Figure 8 Gas production of OM mixed culture growing on OM
29
30
25
Day 13
V
o 20
l
u
m 15
e
Day 5
Day 4
Day 3
(
m 10
L
)
Day 1
5
0
LE-2.5g/L
LE-5g/L
LE-10g/L
LE-15g/L
Figure 9 Gas production of LE mixed culture growing on LE
As can be seen on these graphs, the gas production is low (less than 30 mL after 13
days of fermentation). The hydrogen concentration was also low: about 0.5 mM for
all the samples. The conclusion is that mixed culture in the SMS cannot grow very
well by itself on the SMS in modified DCB1 medium.
Additional carbon source needs to be given, and pure cultures need to be isolated,
in order to improve the fermentation process.
30
3.2 Growth of mixed culture on Glucose
This second experiment was run in order to find out whether the bacteria (mixed
culture) growing on SMS can grow using glucose as a carbon source, to generate
value-added products. The pH was controlled on a daily basis in order to replicate
the natural living conditions of the bacteria. The initial pH was 5.5 after
inoculation; hence, everyday, the pH was set to 5.5, using either H2SO4 or NaOH
solutions.
The first step for this experiment was to prepare and isolate the mixed culture. A
5% (w/v) inoculation of fresh OM and LE SMS was done in a 30 mL bottle of
modified DCB1 medium with 30 g/L glucose. The incubation was done in a 35°C
shaker, 150 rpm. The gas produced by the bacteria was released every day, for 13
consecutive days. After 13 days, there was no more gas production, and the
concentration of glucose in the solution was close to 1.2 g/L, meaning almost all
the glucose was used by the bacteria. The colonies in fresh SMS can therefore
degrade glucose very well.
At the end of the previous experiment, 5% (v/v) of the previous bottle was
inoculated in another 30 mL bottle of DCB1 medium, with 30 g/L of glucose, for
both OM and LE mixed culture. This step is meant to cultivate the mixed culture,
and to constitute a stock-bottle of this mixed culture for future experiments.
The culture was incubated in the 35°C shaker, 150 rpm, for 13 days. At this stage,
the only carbon source was glucose 30 g/L: we can assume there was no leftover
31
from the previous culture because the glucose measured after 9 days was close to
0 g/L, and the new inoculation wass done at 5% (v/v). The daily gas production
was recorded for 13 days. The results are shown below: “LE” and “OM” being the
names given to the mixed cultures isolated from OM and LE SMS.
LE
OM
0
20
40
60
80
100
120
Volume (mL)
Figure 10 Total gas production of mixed cultures growing on glucose 30g/L
As can be seen on this graph, the results are very similar for the two different kinds
of SMS. The total gas production after 13 days is close to 120 mL. These results
are much more convincing than the previous study, where the only carbon source
was SMS itself. This track should be further studied.
32
25
(
C
o
20
n
c
e
15
n
m
t
M
r
10
a
t
5
i
o
n
0
H2
)
N2
CO2
1
5
9
13
Time (day)
Figure 11 Gas composition of OM mixed culture growing on glucose 30g/L
20
18
(
C
o
n
c
e
n
m
t
M
r
a
t
i
o
n
16
14
12
10
H2
8
N2
)
CO2
6
4
2
0
1
5
9
13
Time (day)
Figure 12 Gas composition of LE mixed culture growing on glucose 30g/L
33
As can be seen on these diagrams, the molar percentage of hydrogen increases
over time, and goes from 29% one day 1 to 36% on day 13 for OM; and from
12.5% on day 1 to 38% on day 13 for LE. So, the highest molar percentage of
hydrogen is found after 13 days of inoculation.
The amount of glucose remaining after 13 days was measured for OM and was
12.4 g/L. We can therefore calculate the yield of hydrogen production for this
reaction, which is:
H2 yield(mol hydrogen produced / mol glucose consumed) = 0.651
This yield, compared to existing papers, is relatively high [38].
Regarding the biosolvent production, the concentrations were measured 13 days
after inoculation. Acetic acid, butyric acid and butanol were detected for both OM
and LE mixed cultures.
23.6
42
Acetic Acid
Butyric Acid
Butanol
62
Figure 13 Biosolvents production of OM mixed culture growing on glucose 30g/L (mM)
34
40
52
Acetic Acid
Butyric Acid
Butanol
24.8
Figure 14 Biosolvents production of LE mixed culture growing on glucose 30g/L (mM)
As can be seen on the graphs above, butyric acid and butanol constitute the major
part of the biosolvents production. Butanol concentration is 3.11 g/L for OM
mixed culture and 3.85 g/L for LE mixed culture. The previous results imply that
some of the bacterial strains in OM and LE mixed cultures are potentially good
candidates to produce high amounts of hydrogen and butanol.
Up to now, we know that OM an LE mixed culture can grow well on glucose. In
order to use SMS as a carbon source, further studies should be done on whether
this mixed culture can degrade more complex carbohydrates, such as xylose,
cellulose and xylan.
35
3.3 Growth of mixed culture on different substrates
This third experiment was run in order to find out whether the mixed cultures
growing on spent OM substrate can grow using not only glucose as a carbon
source, but also xylose, cellulose and xylan, to generate value-added products. If
so, then we can be confident that these mixed cultures will be able to degrade
SMS, whose structure and composition are much more complex.
The OM mixed culture used in this experiment is the same as in the previous
experiment. A first inoculation was done at 5% (v/v) from the mixed culture stockbottle to a modified DCB1 bottle, in order to make sure that there was no glucose
leftover from the stock-bottle. Then, another 5% (v/v) inoculation was done to a
modified DCB1 bottle with only one carbon source, stated later for each particular
bottle.
Glucose and xylose were added at a concentration of 30 g/L (liquid phase) directly
into the modified DBB1 bottle. Cellulose and xylan from birchwood had to be
prepared in two steps, since the commercial products are in solid phase. The final
concentration in the modified DCB1 medium was 30 g/L.
The cultures were incubated in the 35°C shaker, 150 rpm, for 13 days. At this
stage, the only carbon source is the one stated for each bottle: glucose, xylose,
cellulose or xylan; there is no leftover from the stock bottle because two 5% (v/v)
inoculations were done successively in order to dilute the potential carried-over
glucose from the seed culture bottle. The pH was controlled on a daily basis in
36
order to replicate the natural living conditions of the bacteria. The initial pH after
inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H2SO4 or
NaOH solutions.
140
120
V 100
o
l
u 80
m
e
60
Day 13
(
Day 1
m
L
Day 9
Day 5
40
)
20
0
xylose
glucose
cellulose
xylan
Figure 15 Total gas production of mixed cultures growing on different carbon sources
This graph shows that the mixed cultures from OM SMS can grow well on
different substrates. The best results are for glucose 30 g/L, and cellulose is the
substrate which is the hardest to be degraded. The total gas production for all the
carbon sources is non negligeable: we thus can be confident that this mixed culture
can grow reasonably well on SMS as a carbon source. Regarding the gas
composition, hydrogen had a molar percentage from 10% to 30% for all carbon
37
sources, which is consistent with the previous experiments. Following are the
results for the biosolvents production after 13 days.
140
(
120
C
o
n
100
c
e
80
n
m
t
M
r
60
a
t
40
i
o
n
20
Butanol
Butyric Acid
)
Acetic Acid
0
xylose
glucose
cellulose
xylan
Figure 16 Biosolvents production of mixed cultures growing on different carbon sources
Again, glucose is the best carbon source for the mixed culture from OM SMS.
Xylose is also well degraded by the mixed culture, but in much lower quantities.
However, cellulose used as a carbon source doesn’t give any detectable amount of
butanol. Therefore, it is predictable that SMS could be degraded quite well by OM
mixed culture: the four carbon sources used here are already degraded by OM
mixed culture.
38
We now know that some of the strains both in OM and LE can produce hydrogen
and butanol in non-negligeable amounts. In order to get better results, we need to
isolate the pure cultures and to further experiment on these strains.
3.4 Isolation of pure cultures using glucose as a carbon source
The focus of this step is to isolate pure cultures from SMS. We need to isolate the
hydrogen and butanol producing strains, and then to improve their fermentative
efficiency, by optimizing the experiment conditions. All the following
manipulations were done in an anaerobic chamber. The previous bottles were kept
in order to store the mixed cultures from OM and LE SMS. Then, agar plates were
prepared, using glucose 30 g/L as carbon source.
Several dilution factors of the inoculate were tested in order to get the broadest
possible range: 1/10; 1/100; 1/1,000; 1/10,000; 1/100,000 and 1/1,000,000. 0.2 mL
to 0.5 mL liquid solution from OM and LE mixed cultures stock-bottles were
spread to different agar plates, with the stated dilution factors. Agar plates were
left in the anaerobic chamber for incubation. Single colonies started to appear
about 3 days later.
39
Figure 17 Anaerobic Chamber
The best dilution factor for our samples, both for OM and LE SMS was 1/100,000.
Then, 20 positive colonies were picked up for each of the two bacterial sources
(OM and LE), and transferred to modified DCB1 medium bottles, with glucose 30
g/L. These 40 new bottles were put in the shaker 35°C, 150 rpm, for 13 days. Gas
was released and tested every day; and biosolvent analysis was run at the end of
the 13 days, in order to discriminate the strains. Most of the strains couldn’t
produce any butanol, and their hydrogen concentration was not significant. The
full list of graphs for this experiment is not given here, since most of the strains
were not of any interest for our study.
40
Figure 18 Colonies on agar plate
Three strains were eventually selected among the 40, regarding their good
potential to produce high volume of gas, high hydrogen concentration, and
butanol. These three potentially good strains were named ESE1, S4 and S11. The
next step is to use these pure cultures on 30g/L glucose in order to test their
capability to degrade glucose and produce value-added products.
3.5 Identification of strains S4 and S11
As seen in the previous experiments, S4 and S11 are the two strains that seem to
have the best potential for our study. The following experiments were conducted
using strains S4 and S11: ESE1 was not used anymore. We therefore conducted
41
DNA analysis, according to the protocol detailed in 2.5, of these two strains in
order to identify them.
Figure 19 Picture after DGGE (from left to right: S4, ladder, S11)
The first part of the protocol is to carefully follow the instructions of 2.5. The final
step is to send our purified DNA samples (about 1,300 bp long) for sequencing to
Applied Biosystems Company, together with our 8-F primer. Following are the
results from this company.
42
Figure 20 Extract of DNA Sequence for S4 Strain
Figure 21 Extract of DNA Sequence for S11 Strain
43
Figure 22 Phylogenetic tree for S4 Strain
44
Figure 23 Phylogenetic tree for S11 Strain
45
The National Center for Biotechnology Information website [45] suggests that
Clostridium beijerinckii NCIMB 8052 is the closest strain to both S4 and S11.
However, the similarity percentage is 98% for S11 strain, and 99% for S4 strain.
We can therefore conclude that S4 and S11 are Clostridium sp. Further analysis
should be conducted in order to get a 100% accurateness regarding the DNA
sequence-matching with a known-microorganism.
The objective of this part was to conduct a DNA analysis of the two best strains
we have isolated. S4 and S11 are genetically similar. Although this is not the
purpose of this study, a genetically engineered microbe (based on S11 genome, for
instance), could be implemented in order to improve the performance of the
fermentation process.
46
CHAPTER 4. Growth of bacteria using different
kinds of carbon sources
This part is the core part of our study. In the previous chapter, we isolated three
strains (pure cultures) from Spent Mushroom Substrate in order to use them to
produce value-added products: bio-hydrogen and bio-butanol. This chapter will
focus on the degradation of several substrates by ESE1, S4 and S11.
4.1 Growth of pure cultures using glucose as a carbon source
This experiment was conducted with the same experiment conditions as the
previous ones. Every bottle was duplicated and the cultures put in the shaker 35°C,
150 rpm. Gas was released and tested every day; and a biosolvent analysis was run
in order to discriminate the strains. The pH was controlled on a daily basis in order
to replicate the natural living conditions of the bacteria. The initial pH after
inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H 2SO4 or
NaOH solutions.
47
400
350
V 300
o
l 250
u
m 200
e
S4
S11
(
150
ESE1
m
L 100
)
50
0
1
2
4
6
8
11
Time (day)
Figure 24 Cumulative gas production of pure cultures growing on glucose 30g/L
This graph clearly shows good results, considering the previous experiments done
in this study. The total gas volume goes from 297 mL for ESE1 strain to 375 mL
for S11 strain. Again, the hydrogen molar percentage was comprised between 10%
(for ESE1) and 30% (for S11). The amount of glucose remaining after 11 days was
measured for the three different strains. We can therefore calculate the yield of
hydrogen production (mol hydrogen produced / mol glucose consumed) for this
reaction.
48
H2 yield(mol hydrogen produced / mol glucose consumed)
S4
0.56
S11
0.68
ESE1
0.51
Table 3 Hydrogen yield of S4, S11 and ESE1 on Glucose 30 g/L
This yield, compared to existing papers, is relatively high [38]. In particular, S11
should be further studied. Then, the production of biosolvents was tested for every
strain. This step is crucial to assess the potential of these strains to degrade Spent
Mushroom Substrate.
120
(
C
o
100
n
c
80
e
n
m
60
t
M
r
a
40
t
i
20
o
n
0
Butanol
Acetic acid
)
Butyric Acid
0
1
4
Time (day)
6
11
Figure 25 Biosolvents production of S4 pure culture growing on glucose 30g/L
49
140
(
C
120
o
n
100
c
e
80
n
m
t
M
60
r
a
t
40
i
o
20
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 26 Biosolvents production of S11 pure culture growing on glucose 30g/L
100
C
o
n
c
e
n
t
r
a
t
i
o
n
90
80
70
60
Butanol
50
Acetic acid
40
Butyric Acid
30
20
(
m
M
10
)
0
0
1
4
6
11
Time (day)
Figure 27 Biosolvents production of ESE1 pure culture growing on glucose 30g/L
50
These three graphs demonstrate that the three strains generate butanol, which is
very promising for our SMS substrate. For S11, butanol concentration after 11
days is 6.0 g/L. For S4, butanol concentration after 11 days is 5.48 g/L. For ESE1,
butanol concentration after 11 days is 3.33 g/L These results are higher than the
butanol concentration measured when using the mixed cultures from SMS on the
same substrate (glucose 30 g/L). Nonetheless, further optimization will be done
using this strain in order to test its ability to produce more butanol.
Interestingly, butyric acid is generated in high quantity for S4, S11 and ESE1. Its
concentration is 9.3 g/L when the strain is S11: this result is particularly interesting
because the only carbon source here is xylose [44]. This is not the focus of our
study, but S11 could be further studied to improve the butyric acid production, by
optimizing the fermentation process.
4.2 Growth of pure cultures using xylose as a carbon source
This experiment was conducted with the same experiment conditions as the
previous one. Every bottle was duplicated and the cultures put in the shaker 35°C,
150 rpm. Gas was released and tested every day; and a biosolvent analysis was run
in order to discriminate the strains. The pH was controlled on a daily basis in order
to replicate the natural living conditions of the bacteria. The initial pH after
inoculation was 5.5; hence, everyday, the pH was set to 5.5, using either H 2SO4 or
NaOH solutions.
51
250
200
V
o
l
150
u
m
e
100
S11
S4
ESE1
(
m
L
)
50
0
1
2
4
6
8
11
Time (day)
Figure 28 Cumulative gas production of pure cultures growing on xylose 30g/L
Then, the production of biosolvents was tested for every strain. This step is crucial
to assess the potential of these strains to degrade Spent Mushroom Substrate.
Following are the results for the 3 pure cultures growing on xylose 30 g/L.
52
70
(
C
60
o
n
50
c
e
40
n
m
t
M
r
30
a
t
20
i
o
10
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 29 Biosolvents production of S4 pure culture growing on xylose 30g/L
80
(
C
70
o
60
n
c
50
e
n
m
40
t
M
r
30
a
t
20
i
o
10
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 30 Biosolvents production of S11 pure culture growing on xylose 30g/L
53
60
(
C
50
o
n
c
40
e
n
m
30
t
M
r
a
20
t
i
o
10
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 31 Biosolvents production of ESE1 pure culture growing on xylose 30g/L
These three graphs demonstrate that the three strains generate butanol, which is
promising for our SMS substrate. For S4, butanol concentration after 11 days is
4.0 g/L. For S11, butanol concentration after 11 days is 4.8 g/L. For ESE1, butanol
concentration after 11 days is 2.5 g/L. These concentrations are lower than when
using glucose as a carbon source, which is consistent. It has to be noted that the
concentration is still high, even if the molecular structure of xylose makes it harder
to degrade than glucose.
54
4.3 Growth of pure cultures using Spent Mushroom Substrate as
carbon source
This experiment is to test the degradation of SMS 30g/L, and the following
experiments are to change the parameters of the experiments in order to optimize
the value-added products generation. The carbon sources from Spent Mushroom
Substrates were used were named A (standing for Abalone Mushroom) and OMN
(standing for Oyster Mushroom New). The initial preparation process is important,
because, as seen in the previous chapter, fresh SMS is not a good substrate for
bacteria.
The first step of the initial preparation process is to dry the fresh SMS at 60°C in a
oven. The fresh SMS was manually shredded, spread on a tray, and this tray was
put in the 60°C oven for 3 days. After 3 days, the moisture of the SMS was
evaporated, the SMS can be called “dried”. This dried SMS was then blended
using a kitchen-mixer in order to make SMS powder, to get smaller dust particles,
which are easier to be degraded by the bacteria. This powder was then autoclaved
in the modified DCB1 medium bottles. Autoclave is of important since we want to
avoid bacterial contamination: the product generation should come exclusively
from our isolated strains, and not from any other unknown bacterial or fungal
source growing in the fresh SMS.
55
Figure 32 Spent Mushroom Substrate : from the farm to dried powder
This first experiment is meant to give a rough idea of what the potential of SMS as
carbon source is. This experiment was conducted with the same experiment
conditions as the previous ones (5% (v/v) inoculation). Every bottle was
duplicated and the cultures put in the shaker 35°C, 150 rpm. Gas was released and
tested every day; and biosolvent analysis was run in order to discriminate the
strains and SMS.
56
The pH was controlled on a daily basis in order to replicate the natural living
conditions of the bacteria. The initial pH after inoculation was 5.5; hence,
everyday, the pH was set to 5.5, using either H2SO4 or NaOH solutions. The SMS
concentration for this experiment was set to 30 g/L, to be consistent with the
previous experiments, where glucose and xylose concentrations were 30 g/L. The
main difference is that we do not know the composition of SMS. So it will not be
possible to assess our results regarding the yield of the experiment. The only
interpretable results of this experiment is its output: hydrogen production and
butanol production. This is the main concern of the mushroom farm: they do not
mind about the yield of the experiment, they want to generate value-added
products from their waste.
40
35
V 30
o
l 25
u
m 20
e
A
OMN
(
15
m
L 10
)
5
0
1
2
4
6
8
11
Time (day)
Figure 33 Total gas production of S4 growing on SMS 30g/L
57
35
30
V
25
o
l
u 20
m
e 15
A
OMN
(
m
10
L
)
5
0
1
2
4
6
8
11
Time (day)
Figure 34 Total gas production of S11 growing on SMS 30g/L
As can be seen on these graphs, the total gas production is lower than when the
carbon source is glucose or xylose 30 g/L. However, the total volume remains
reasonably high: between 28 mL and 35 mL. The best strain is S11, followed by
S4. This “ranking” of the strains is similar to when the carbon source was glucose
or xylose 30 g/L.
58
Figure 35 Fermentation bottles
16
(
C
14
o
n
12
c
e
10
n
m
8
t
M
r
6
a
t
4
i
o
2
n
0
Butanol
)
Acetic acid
Butyric Acid
0
1
4
6
11
Time (day)
Figure 36 Biosolvents production of S4 growing on A 30g/L
59
(
16
C
14
o
n
12
c
e
10
n
m
8
t
M
r
6
a
t
4
i
o
2
n
0
Butanol
)
Acetic acid
Butyric Acid
0
1
4
6
11
Time (day)
Figure 37 Biosolvents production of S4 growing on OMN 30g/L
16
(
14
C
o
12
n
c
10
e
n
m
8
t
M
r
6
a
t
4
i
o
2
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 38 Biosolvents production of S11 growing on A 30g/L
60
16
(
C
14
o
n
12
c
10
e
n
m
8
t
M
r
6
a
t
4
i
o
2
n
Butanol
)
Acetic acid
Butyric Acid
0
0
1
4
6
11
Time (day)
Figure 39 Biosolvents production of S11 growing on OMN 30g/L
According to these results, A and OMN SMS do not seem to have a very high
potential for butanol generation (the butanol concentration reached after 13 days is
about 0.1 g/L for these SMS species). We decide to continue all the following
experiments using S11 pure culture alone because S11 seems to be the most
promising strain to generate value-added products. More results regarding biohydrogen and bio-butanol productions by S11 will be shown further on.
These intermediate results are to be considered carefully. We need to take into
account that further optimization will be done: the use of enzymes to pre-treat
SMS (hydrolysis).
61
4.4 Pre-treatment of Spent Mushroom Substrate
This step is meant to pre-treat Spent Mushroom Substrate in order to generate
simple carbohydrates from SMS, which are easier to be degraded by strain S11.
We use a xylanase-producing strain isolated from SMS. Xylanase is an enzyme
able to degrade xylan, a complex sugar which is a major component of SMS. Four
main steps were undertaken: enzymes production, enzymes purification,
hydrolysis and fermentation.
4.4.1 Enzymes production
The first step is xylanase generation, using microbial anaerobic fermentation. In
order to produce enzymes able to degrade SMS, we use the same medium as usual,
modified DCB1. The carbon source for the bacteria is xylan birchwood 10 g/L.
The strain used to produce xylanase was isolated by my colleague Xin Fengxue,
from OM Spent Mushroom Substrate. After further study by my colleague,
genome identification and 16s rRNA analysis, this strain suitable for enzymes
production, is Kluyvera sp.
The fermentation process is the same as previously stated. Ten bottles with 50 mL
medium and xylan as carbon source were prepared anaerobically, then a 5% (v/v)
inoculation was done. Fermentation was done in the shaker 35°C, 150 rpm. Gas
produced by the culture was released every day and noted down. No analysis of
the gas composition or biosolvents concentrations were made, since this is not the
62
focus of this step. Nine days after inoculation, the fermentation process was
stopped: xylanase was fully generated by the fermentation process. Then, the
second step, enzymes purification could be undertaken.
4.4.2 Enzymes purification
This second step doesn’t need anaerobic conditions, because xylanase doesn’t need
anaerobic conditions. Thus, the ten 50 mL bottles containing the enzymes were
open and the 500 mL liquid was aliquoted into a beaker. Electronic charges
interactions are an issue for enzymes purification. The free charges in the medium
are an obstacle for the enzymes to act as catalyzers: in order to solve this issue,
ammonium sulfate H8N2O4S was added to the liquid, to annihilate the positive and
negative charges in the medium.
The beaker was installed in a 4°C cold room with a magnetic stirring bar. A 30%
(w/v) amount of solid ammonium sulphate, 150 g in our experiment, was added to
the 500 mL culture medium with constant stirring for 2 hours. The liquid was then
centrifuged down at 9,000 rpm for 15 minutes and the precipitate was discarded.
The supernatant was subsequently adjusted to 70% (w/v) saturation with addition
of 200 g of ammonium sulphate, with constant stirring for 12 hours at 4°C in the
cold room.
63
Figure 40 Beaker with xylanase solution
After 12 hours, the liquid medium was centrifuged down again, the supernatant
was discarded and the precipitate, which contains xylanase, was dissolved in a
small volume of 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0.
The xylanase solution was then subjected to dialysis for 24 hours at 4 °C against
0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0. Dialysis is a
diffusion process, which describes the property of substances in water which tend
to move from an area of high concentration (our liquid medium containing
enzymes) to an area of low concentration (the glycine buffer). Three intermittent
changes of the buffer were done, after 2 hours, 6 hours and 13 hours, in order to
improve the efficiency of the process, by lowering the concentration of dissolved
64
substances in the glycine buffer. After this step, the sample collected from the
dialysis is made of pure enzymes.
Figure 41 Dialysis process
In order to further improve the purification process, we also carried out a freezedrying of the sample, because it still contains a relatively high amount of water,
compared to the amount of (solid) enzymes isolated. Freeze-drying, or
65
lyophilization, is a dehydration process. It works by freezing the material, and then
reducing the surrounding pressure to allow the frozen water in the material to
sublime directly from the solid phase to the gas phase. In our case, the surrounding
pressure was reduced to 0.06 millibar, for 31 hours. After this step, there should
not remain any more water in the beaker, and the remaining solid phase is pure
xylanase.
Figure 42 Freeze-drying of the enzymes
66
Enzyme activity analysis was carried out from the dialyzed and freeze-dried
sample. Enzyme activity was measured using the optical density method (with a
spectrophotometer), and compared with the calibration curve obtained with
standard test tubes. In order to achieve this, 6 standard test tubes were prepared,
using the 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0. For every
standard test tube, 1.0 mL of 1% (w/v) xylan solution (in the 0.05 mol/L glycine
NH2CH2COOH buffer adjusted to pH=8.0) was added to the test tube. Then a
volume of xylose stock solution 10 g/L and glycine buffer were added to the test
tube in volumes stated in the following Table 2. The total volume of solution in a
standard test tube was therefore 1.5 mL. Total xylose concentration is the standard
which is going to be calibrated with the spectrophotometer, and compared with our
enzymes sample.
Xylose concentration (g/L)
St0
0
St1
Xylose stock (mL)
Glycine buffer (mL)
0
0.5
1.0
0.05
0.45
St2
2.0
0.10
0.4
St3
3.3
0.17
0.33
St4
5.0
0.25
0.25
St5
6.7
0.33
0.17
Table 4 Standards composition
67
The sample to be tested was prepared by mixing 1.0 mL of 1% (w/v) xylan
solution, in the 0.05 mol/L glycine NH2CH2COOH buffer adjusted to pH=8.0, with
0.5 mL of sample (containing the purified enzymes) in a test tube. Two test tubes
with our sample were prepared in order to duplicate the test, the total amount of
solution in each test tube being 1.5 mL.
The procedure for the test, similarly done for the standard test tubes and for our
samples, is the following. The first step is to incubate the 1.5 mL mixture at 50°C
for 10 minutes in a hot water-bath. Then the test tubes were placed on ice for 5
minutes. In every tubes, 3.0 mL of DiNitroSalicylic acid (DNS) solution was
added. Then, the test tubes were incubated in a hot water-bath at 90°C for 10
minutes. When removed from the hot water bath, they were placed on ice again,
for 20 minutes. Then, 20 mL of deionized water was poured in every test tube.
Figure 43 Test tubes for optical density measurement
68
At this stage, we can visually compare the standard tubes with our sample, in order
to have an idea of the concentration of reductive sugars in our sample. The darker
the solution, the higher the concentration of reductive sugars: DNS reacts with
reducing sugars to form 3-amino-5-nitrosalicylic acid, which absorbs light strongly
at 540 nm. For more accurateness, and in order to get the exact enzyme activity,
we use the spectrophotometer at 540 nm.
Figure 44 Spectrophotometer samples
The spectrophotometer gives the absorbance of every standard test tube at 540 nm.
This absorbance was noted down to get a standard calibration curve.
69
8
7
6
5
(
4
3
)
X
y
l
o
s
e
C
o
n
c
e
n g
t /
r L
a
t
i
o
n
2
1
0
0
0.172700003
0.241799995
0.309299991
0.362299994
0.476400003
Absorbance
Figure 45 Standard curve for optical density of enzymes
The mean absorbance for our sample is 0.25692. Reading on the standard curve for
our sample gives us a xylose concentration of 3.612 g/L. One unit (U) of enzyme
activity is defined as the amount of enzyme which liberate 1 μmol of product from
their respective substrate per minute. This calculation is fully detailed in [42]. We
can therefore calculate the U for our sample, which is 1.79/g substrate.
4.4.3 Hydrolysis
The first two steps of the pre-treatment process, enzymes production and enzymes
purification, are now completed. The third step is hydrolysis of Spent Mushroom
Substrate.
70
Enzymatic hydrolysis was carried out at a substrate loading of 2.0% (w/v) with 10
IU xylanase/g of substrate in 20 mL of DCB1 medium adjusted to pH=8.0. The
experiment was performed in duplicates at 55°C in a shaking hot-water bath for 72
hours. After hydrolysis, samples were centrifuged at 10,000 rpm for 10 minutes.
The supernatant, containing simple carbohydrates was then collected. This liquid
solution is to be the bacterial growth medium in the following fermentation
process.
4.4.4 Fermentation
The final step, after the pre-treatment process, is fermentation. The carbon source
is made of simpler carbohydrates, generated by the hydrolysis of Spent Mushroom
Substrate in the previous step. We pre-treated OM SMS, which did not give good
butanol concentration without pre-treatment. After pre-treatment, the results were
not significantly higher than when using OM SMS directly. One of the reasons
may be that OM SMS doesn’t contain much xylan, as previously expected, and
that the carbohydrates molecules consumed by strain S11 are the same when using
OM SMS or hydrolyzed SMS.
According to this remark, it can be concluded that SMS may not be a good
substrate to be degraded by S11 strain. The cost of implementing a fermentation
process using SMS as carbon source may be too high compared to the money that
can be made by selling hydrogen or other VFAs, such as butyric acid.
71
4.5 Glucose concentration optimization
This experiment is meant to evaluate which substrate concentration is optimal to
grow S11 on glucose. The purpose is to find out the highest hydrogen and butanol
concentrations, and at which glucose concentration these productions are achieved.
Regarding the results of the previous experiment, the pH was controlled every day
to pH=5.5, using H2SO4 or NaOH solutions. This was done in order to take full
advantage of the previous results. The experiment conditions were the same as
stated for the previous experiment: inoculation at 5% (v/v) of pure S11, every
bottle was duplicated, and the fermentation was done anaerobically in the shaker
35°C, 150 rpm.
The glucose concentrations tested in this experiment are the following: 2.5 g/L, 5
g/L, 10 g/L, 15 g/L, 20 g/L, 30 g/L and 60 g/L. The products concentrations were
measured 13 days after inoculation.
72
40
C
o
n
c
e
n
t
r
a
t
i
o
n
35
30
25
CO2
20
N2
H2
15
10
(
m
M
5
)
0
2.5g/L
5g/L
10g/L
15g/L
20g/L
30g/L
60 g/L
Figure 46 Gas composition of S11 pure culture growing on glucose at different concentrations at pH=5.5
Glucose 30 g/L show similar results to those obtained in the previous experiment
when using exactly the same experiment conditions. This shows a good stability of
S11 strain over time.
As can be seen, when glucose is used as a carbon source, the highest hydrogen
molar percentage is reached when glucose concentration is 60 g/L. However, it has
to be noted that the molar yield of the experiment drops dramatically from 0.7
when using glucose 30 g/L, to 0.5 when using glucose 60 g/L. It is much more
economically interesting to use glucose 30 g/L.
73
An interesting conclusion is that higher substrate concentration means higher
hydrogen production, but doesn’t mean higher molar yield. There is a limit above
which an increase of the substrate concentration doesn’t increase the hydrogen
molar yield.
These results have to be analysed on an industrial point of view. The main
objective of our study is to generate value-added products for the mushroom farm.
4.6 Upscaling of the laboratory-scale experiments
This part is meant to give a brief overview of what a farm-scale fermenter would
be. The results of the experiments shown before this part are from experiments
done on a laboratory-scale. We expect larger scale experiments to give the same
results, but we don’t know if bigger microbial colonies would interact with each
other, and disturb the fermentation process. We also don’t know how to deal with
the larger gas production, produced by a larger bio-fermenter.
4.6.1 Protocol
In a 4 Liter reactor, we are able to monitor the pH in real-time, which we cannot
conduct in a 30mL bottle. pH is of paramount importance in our experiments, this
is why it is crucial to control the pH as accurately and as regularly as possible.
The fermenter used is a 4L glass-tank, with a magnetic stirrer. Three of the top
openings were sealed using a rubber joint, and safely covered by a flexible plastic
74
paraffin film. The fourth opening is used to monitor the pH in real-time (by
introducing a pH-meter). The fifth opening is linked, through a rubber joint, to a
two-part flexible plastic tube (the two parts are linked with plastic paraffin film),
whose other extremity goes to a 2L graduated plastic cylinder. This cylinder is
reversed, filled with water, and stands using another cylinder to maintain the right
pressure inside of the first cylinder. When gas is produced by the fermenter, the
first cylinder goes up, pushed by the pressure difference between the gas and the
water level inside of the cylinder.
Figure 47 Bio-fermenter and gas tank at the initial step
75
Thus, the gas is continuously produced and stored in the cylinder. When the gas
volume exceeds 2L, we use a 50mL syringe to release the gas from the cylinder.
This syringe is inserted in the second part of the plastic tube which links the
fermenter to the cylinder. Gas is pulled-out and precisely measured with the
graduated 50mL syringe.
Figure 48 Bio-fermenter and gas tank after 12 hours
76
We tried the scale-up experiment using SMS as the carbon source. However, there
was no butanol produced: only Volatile Fatty Acids. Since our focus for this study
is butanol, we used glucose as the carbon source in order to have both hydrogen
and butanol productions: therefore we were able to analyze the results on a larger
scale than when using the 30mL bottles.
The methodology for the experiment is the following. We use 1.8L modified
DCB1 medium, prepared as usual (see protocol in Chapter II). The strain used is
S11 pure culture, inoculated at 5% (v/v). The results in the previous parts were
best when using glucose 30 g/L as a carbon source in this experiment. pH is set to
5.5 after inoculation, and controlled as often as possible to pH=5.5, which is the
best pH, as previously demonstrated. The fermenter was kept at air temperature
(about 20°C), and shaked using a magnetic stirrer. We use the same experiment
conditions as previous experiments, since we want to be able to compare the
results on the laboratory-scale and on the pre-industrial scale.
4.6.2 Results
As previously stated, the gas volume and composition were measured every day.
Gas was continuously produced for 16 days. After 16 days, no gas was released
anymore. So the total fermentation time is longer than when using the laboratory
bottles. The reason to this fact is probably that the fermenter is much bigger than
the laboratory bottles, so the bacteria growing in the fermenter have more
difficulties to encounter the carbon source in the medium. Thus, it takes longer for
77
the bacteria to find and degrade the substrate in the fermenter. In order to make it
on an industrial scale, an efficient stirring process should be implemented to
improve the efficiency and shorten the time taken by the fermentation process.
Following is the graph showing the cumulative gas production for 16 days.
18000
16000
14000
V
o 12000
l
u 10000
m
e 8000
(
m
L
6000
)
4000
2000
0
1
2
4
6
9
16
Time (day)
Figure 49 Total gas production of S11 pure culture growing on glucose 30 g/L on pre-industrial scale
As can be seen, the gas production is still very high on the pre-industrial scale. The
total cumulative gas production is 15,740 mL. The total volume of modified DCB1
is 60 times larger than when we use the laboratory bottles. Yet, the total gas
production using the fermenter is 41.4 times the gas volume produced by the
laboratory bottles. So the ratio is 0.69, meaning that there is a negative scaleeffect. If the mushroom farm were to implement our process, there would be a
78
slight difference in using a small volume or a high volume of medium: the scaleeffect is non-negligeable. We can even further guess than when using a larger
fermenter, the total gas production will be even lower than when using a smaller
volume. However, this is regarding the gas volume only. Bio-hydrogen and biobutanol can still be produced in large amounts, which is the focus of our study.
Regarding the gas composition, following are the time-course results in the
fermenter.
30
(
C
o
25
n
c
20
e
n
m
15
t
M
r
a
10
t
i
5
o
n
0
H2
)
N2
CO2
2
6
9
16
Time (day)
Figure 50 Gas composition of S11 pure culture in Fermenter
As can be noted, the gas composition is very similar to the one found when using
the small bottles. The molar percentage of hydrogen after 16 days is about 32%.
Then, the biosolvent analysis was conducted at the end of the 16 days. The
79
fermentation process was complete (no gas production anymore), and a sample
was taken from the fermenter to be analysed.
65.3
72.1
Acetic Acid
Butyric Acid
Butanol
112.7
Figure 51 Biosolvents production (mM) of S11 pure culture in Fermenter
The results are again similar to those found when using laboratory bottles. The
butanol concentration here is 4.84 g/L, which is slightly lower than when using the
laboratory bottles. However, butanol is still produced in reasonable quantities, and
this fact could be exploited on an industrial scale project.
Thus, using the same process and parameters (pH=5.5, substrate concentration set
to 30 g/L) on a pre-industrial scale gives similar results to those obtained when
using laboratory bottles. Hydrogen and butanol are generated by S11 strain in
almost the same concentrations, but the total gas volume is about 30% lower.
80
Nonetheless, it has to be noted that the process is slower than when doing the
experiment on a laboratory scale. If this process were to be implemented on an
industrial scale, a very efficient stirring process should be built. Moreover, a goodquality fermenter should be built, with perfectly fitted tubes to continuously
conduct gas out of the fermenter to a tank, cylinder, or membrane process; and
with all the safety precautions due to handling hydrogen in non-negligible
quantities.
81
CHAPTER 6. Conclusion
6.1 Major findings
With reference to the problem statement and the aims set out earlier in this report,
the objectives of this study have been met. Listed below are the central findings of
this study.
First, we disclose a fermentative pathway generating value-added products, using
bacteria isolated from Spent Mushroom Substrate. This process generates high
concentrations of bio-hydrogen and bio-butanol from glucose. The methodology
has been fully detailed earlier. It is an easily-implementable and reliable way for
mushroom farms to produce on-site bio-energy.
Second, the fermentative bacteria were isolated from Spent Mushroom Substrate
and several kinds of carbon sources were tested. The strain isolated and used for
the major part of the experiments in this paper is named S11. A full DNA analysis
showed that this strain is a Clostridium sp.
Third, we identified two main kinds of SMS, which were degraded by strain S11:
the one used to harvest Oyster Mushroom (OMN) and the one used to harvest
Abalone Mushroom (A). Other kinds of SMS were tested, but did not show could
results when being degraded by S11.
82
Fourth, we optimized the experiment conditions to obtain the highest amounts of
bio-hydrogen and bio-butanol. We found out that the optimal pH for the
fermentation process is 5.5. The optimal substrate concentration is 30 g/L when
using glucose as carbon source. This concentration does not give the highest
amounts of value-added products, but is the most economically relevant, because
the yield of the experiments drop when glucose concentration is higher. After a
study of the fermentation process of S11 on two different kinds of Spent
Mushroom Substrate, the conclusion is that butyric acid is produced in relatively
high concentration. This value-added product could be sold by mushroom farms,
who could produce butyric acid on-site.
Fifth, we also implemented a pre-industrial scale experiment, using glucose as
carbon source, which showed good products output. There seems to be a slight
negative scale-effect: further study should be done in order to improve this weak
point.
6.2 Recommendations and future studies
The outlook of this study seems promising. Obviously, the most efficient means to
prevent further waste generation of Spent Mushroom Subsrate lies in a better SMS
management. Farmers should try to reduce their SMS use. They tend to replace
their Spent Mushroom Substrate too often in order to increase the productivity of
their mushroom crops, which inevitably produces a very high quantity of waste.
They should also try to reuse it as a compost material, which is the simplest way,
83
today, of handling their SMS. Our process is a cost-effective and energy-efficient
way to generate value-added products using bacteria isolated from Spent
Mushroom Substrate.
It is also worthwhile to pay attention to the development and the extension of the
knowledge we currently possess in the field of genetic engineering of microbes.
With the combination of an in depth understanding of biochemistry and genetics,
and the applications of recombinant DNA techniques, it is possible to characterize
the appropriate genes and transfer them to construct engineered strains, derived
from our S11 strain, with enhanced capability for degradation of different
substrates. The combination of an optimized fermentation process with a
genetically engineered S11 strain would probably lead to very high outputs of
value-added products.
While we acknowledge and appreciate the contributions and works that have been
carried out in this field, there is so much more that needs to be accomplished in
this direction. Further study should be done in order to assess the total investments
needed to implement our on-site solution. It remains that on an environmentallyfriendly point of view, there is a strong point in implementing our process in
mushroom farms.
84
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[...]... 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... 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... 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... 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. .. 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... 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... 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. ..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 –... 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. .. 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 ... 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. .. 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, ... directly in a mushroom farm v 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