production of succinic acid from glycerol and
TRANSCRIPT
PRODUCTION OF SUCCINIC ACID FROM GLYCEROL AND GLYCEROL
WASTE
NOR HIDAWATI BINTI HJ ELIAS
Thesis submitted for the fulfillment of the requirements
For the award of the degree of
Master of Engineering in Bioprocess
Faculty of Chemical and Natural Resources Engineering
UNIVERSITI MALAYSIA PAHANG
JANUARY 2012
vi
ABSTRACT
Succinic acid is extensively used globally for manufacture of synthetic resin, biodegradable
polymer, pharmaceutical product and food flavoring. Traditionally, succinic acid produced
through petrochemical route. The process is non-renewable and will affected pollution in
the environment. Therefore, more attention has been focused towards a friendly
environment processes for succinic acid production by way of fermentation process; which
is highly recommended nowadays. In this research, studies been done to produce succinic
acid through anaerobic fermentation of glycerol waste using Escherichia coli and to
investigate the effect of each factors that influence succinic acid production. The most
influential parameter for succinic acid production obtained via preliminary study of
parameter range selection were glycerol concentration, tryptone concentration, sodium
carbonate (Na2CO3) concentration , inoculums density, pH and incubation period. This
experiment has produced 0.973g/l succinic acid using One-factor-at-a-time (OFAT)
approach from commercial glycerol sample. These six variables screened to select the most
significant factor affected succinic acid production using fractional factorial design (FFD)
based on first order model term. Four factors selected while, two factors eliminated for the
next stage of experiment. Subsequently, full factor central composite designs (CCD) falling
under RSM employed to develop a mathematical correlation model between the significant
factors for physio-chemical variables of succinic acid production. The experiment has
produced 7.55g/l succinic acid from commercial glycerol sample. Besides, a second order
polynomial regression fitted and the model was adequate by R-squared 0.9864. Meanwhile,
under the optimum level, it is about 7.34g/l succinic acid being produced from commercial
glycerol and 7.32g/l of succinic acid produced from recovered glycerol. Overall, this
research shows glycerine pitch can be used safely and effective as a carbon source for
Escherichia coli to produce succinic acid. In addition, this research provides an exciting
opportunity to transform low-grade glycerine pitch into highly demanded chemical such
succinic acid.
vii
ABSTRAK
Asid suksinik digunakan secara meluas di seluruh dunia dalam pembuatan resin sintetik,
polimer terbiodegradasikan, produk farmaseutikal dan perasa makanan. Secara tradisinya,
asid suksinik dihasilkan melalui proses petrokimia. Proses ini tidak boleh diperbaharui dan
mencemarkan alam sekitar. Oleh itu, perhatian yang lebih telah difokuskan pada proses
pengeluaran asid suksinik yang lebih mesra alam melalui proses penapaian; yang amat
digalakkan pada masa kini. Dalam kajian ini, kajian telah dilakukan untuk menghasilkan
asid suksinik melalui penapaian anaerobik sisa gliserol dengan menggunakan Escherichia
coli untuk menyiasat kesan bagi setiap faktor yang mempengaruhi pengeluaran asid
suksinik. Parameter yang paling berpengaruh untuk pengeluaran asid suksinik yang
diperolehi daripada kajian awal pemilihan julat parameter adalah kepekatan gliserol,
kepekatan tripton , kepekatan natrium karbonat (Na2CO3), ketumpatan inokulum, pH dan
jangka masa inkubasi (pengeraman). Kajian ini telah menghasilkan asid suksinik 0.973g/l
menggunakan kaedah Satu faktor dalam satu masa (OFAT) daripada sampel komersial
gliserol. Enam pembolehubah telah disaring untuk memilih faktor yang paling penting yang
mempengaruhi pengeluaran asid suksinik menggunakan bentuk faktorial pecahan (FFD)
berdasarkan hukum model pertama. Empat faktor telah dipilih manakala dua faktor telah
dihapuskan untuk percubaan ke peringkat seterusnya. Selepas itu, faktor keseluruhan pusat
reka bentuk komposit (CCD) telah digunakan untuk membangunkan model korelasi
matematik di antara faktor-faktor yang signifikan bagi pembolehubah fisio-kimia
penghasilan asid suksinik. Ujikaji ini telah menghasilkan asid suksinik sebanyak 7.55g/l
menggunakan sampel komersial gliserol. Regresi polinomial peringkat kedua telah
digunakan dan model yang terhasil yang telah menepati R-kuasa dua 0.9864. Manakala
diperingkat optimum, sebanyak 7.43g/l asid suksinik terhasil daripada gliserol komersial
dan 7.32g/l asid suksinik terhasil daripada sampel gliserol terpulih. Secara keseluruhannya,
kajian ini telah menunjukkan bahawa sisa gliserol boleh digunakan dengan selamat dan
berkesan sebagai sumber karbon untuk Escherichia coli dalam penghasilan asid suksinik.
Di samping itu, kajian ini menyediakan peluang yang menarik untuk mengubah sisa
gliserol bergred rendah kepada bahan kimia permintaan tinggi seperti asid suksinik.
viii
TABLE OF CONTENTS
Page
SUPERVISOR’S DECLARATION ii
STUDENT’S DECLARATION iii
DEDICATION iv
ACKNOWLEDGEMENTS v
ABSTRACT vi
ABSTRAK xii
TABLE OF CONTENTS vii
LIST OF TABLES xii
LIST OF FIGURES xiv
LIST OF SYMBOLS xvii
LIST OF ABBREVIATIONS xvii
CHAPTER 1 INTRODUCTION
1.1 Research Background 1
1.2 Problem Statement 2
1.3 Objectives of the Study 3
1.4 Scopes of Study 3
1.5 Rationale and Significant 4
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction 6
2.2 Fermentation Process 7
2.2.1 Type of Fermentation Process 8
ix
2.2.2 Nutritional and Physiological Requirement in Fermentation 10
Processes
2.3 Factors Affecting Succinic Acid through Fermentation Process 12
2.3.1 Carbon and Energy Source for Fermentative Processes 12
2.3.2 Glycerol as a Substrate in Succinic Acid Production 15
2.3.3 Waste Glycerol from Biodiesel Manufacturing 18
2.4 Bio-based succinic acid producing bacteria 30
2.4.1 Anaerobiospirillum succiniciproducens 31
2.4.2 Actinobacillus succinogenes 33
2.4.3 Mannheimia succiniciproducens 35
2.4.4 Bacteroides fragilis 36
2.4.5 Escherichia coli K-12 MG1655 as a Succinic Acid Producer 36
2.5 Succinic Acid as a Product from Glycerol Fermentation Process 38
2.5.1 Physical and Chemical Properties of Succinic Acid 38
2.5.2 The Importance of Succinic Acid 41
2.5.3 Natural Source of Succinic Acid 44
2.5.4 Succinic Acid Production 45
2.6 Optimization of Succinic Acid Production using Response 50
Surface Methodology (RSM)
CHAPTER 3 METHODOLOGY
3.1 Operational Framework 52
3.1.1 Phase 1: Treatment of Glycerine Pitch 54
3.1.2 Phase 2: Characterization of Materials 54
3.1.3 Phase 3: Anaerobic Fermentation in Batch System 55
3.2 Pre-treatment of Glycerine Pitch 55
3.2.1 Material 55
3.2.2 Experimental Procedure 55
3.3 Characterization of Material 56
3.3.1 Glycerol 56
3.3.2 Succinic Acid 59
3.3.3 Escherichia coli K-12 MG1655 60
x
3.4 Microorganism, Medium, Inoculums Prepaation and Product
Estimation 61
3.4.1 Microorganism and Medium 61
3.4.2 Inoculums Preparation 61
3.4.3 Succinic Acid and Cell Growth Estimation 62
3.5 Anaerobic Fermentation in Batch System 63
3.5.1 Preliminary Study of Factor Range Selection using
OFAT Approach 63
3.5.2 Screening Experiment using Fraction Factorial Design (FFD) 64
3.5.3 Optimization Experiment using Response Surface
Methodology (RSM) 68
3.5.4 Confirmation and Validation of Model 71
3.6 Chapter Summary 72
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Introduction 73
4.2 Glycerol Recovery from Glycerine Pitch (Biodiesel Waste) 75
4.2.1 Glycerine Pitch Pre-treatment 75
4.3 Characterization of Materials 77
4.3.1 Glycerol 77
4.3.2 Kinetic Profile Growth of Escherichia coli K-12 Strain MG1655 86
4.3.3 Succinic Acid 88
4.4 Glycerol Fermentation for Succinic Acid Production 92
4.4.1 A Preliminary Study of the Parameter Range Selection on
Succinic Acid Production from Glycerol Fermentation using
Escherichia coli K-12 MG1655 through OFAT Approach. 92
4.4.2 Screening the Factors Using Fractional Factorial Design (FFD) 103
xi
4.4.3 Optimization of Succinic Acid Production by Response Surface
Methodology (RSM) 116
CHAPTER 5 GENERAL CONCLUSIONS AND RECOMMENDATIONS
FOR FUTURE WORK
5.1 General Conclusions 133
5.2 Recommendations for Future Work 135
REFERENCES 136
APPENDICES
A Standard Calibration Curves 164
B Pictures of Experimental Set Up and Result 169
C Experimental Data 175
D Diagnostics Cases of Fractional Factorial Design (FFD) and
Response Surface Methodology (RSM) 185
E List of Publications 190
xii
LIST OF TABLES
Table No. Title Pages
2.1 Bio-based production of chemical and fuels via glycerol 7
fermentation
2.2 Physiological .function of principle element for cell growth 11
2.3 Global sources of glycerol production 16
2.4 Physical properties of glycerol 17
2.5 Summary of product and process derived from crude glycerol 22
2.6 Analysis result of macro element in crude glycerol 26
2.7 The summary of glycerol waste treatment procedure 27
2.8 The summary of bio-based succinic acid production 31
2.9 The comparison of batch fermentation in making succinic acid
production using various Escherichia coli strain 37
2.10 Physical properties of succinic acid 40
2.11 Application of succinic acid-based production and its derivatives 43
3.1 Experimental range of six independent variables in preliminary
Study factors range selection 64
3.2 Experimental range and level of the six influential variables used
for FFD in terms of actual and coded values 65
3.3 Experimental design of 26–1
fractional factorial designs 66
3.4 The central composite design (CCD) of the significant variables
was expressed in coded levels. 69
3.5 Independence variable and concentration levels for through CCD 70
xiii
4.1 Characterization of glycerine pitch, recovered glycerol and comparison
with commercial glycerol 77
4.2 Comparison of FTIR spectra of glycerin pitch, recovered glycerol and
commercial grade glycerol 80
4.3 Summary of preliminary study of parameters range selection 102
4.4 Experimental layout and result of 26-1
fractional factorial design 103
4.5 ANOVA for 26-1
fractional factorial design (FFD); Response:
Succinic acid concentration (g/l) 107
4.6 Experimental layout and result of 24 full factorial Central Composite
Design (CCD) 117
4.7 Analysis of variance for complete quadratic model 121
4.8 Result of operating condition with experimental design in confirmation
experiment using commercial glycerol 130
4.9 Result of operating condition with experimental design in validation
experiment using recovered glycerol 131
4.10 Summary of reaction before and after optimization for succinic acid
production from commercial glycerol and recovered glycerol as a
substrate 132
xiv
LIST OF FIGURES
Figure No. Title Page
2.1 Structural formula of glycerol and 3D view 16
2.2 Transesterification reaction to produce biodiesel 20
2.3 Comparison of crude glycerol with pure glycerol 25
2.4 Schematic diagram of the chemical and physical treatment
to recover crude glycerol 28
2.5 The spiral shaped gram-negative anaerobic rods of
A. succiniciproducens 32
2.6 Actinobacillus succinogenes strain 130Z (ATCC 55618) 34
2.7 Chemical structure and physical appearance of succinic acid 39
2.8 Various chemical and product can be synthesized from
Succinic acid 42
2.9 Reaction of Kreb cycles. 45
2.10 The catalytic cycle on hydrogenation of maliec anhydride 47
2.11 The pathway of MA reduction to succinic acid 48
3.1 The operational framework of research 53
3.2 High Performance Liquid Chromatography (HPLC) systems 57
3.3 Brookfield Rotational Digital Viscometer 58
4.1 Bar chart shows the effect of recovered glycerol concentration
on pH. 76
4.2 The changes in colour of glycerol samples 79
4.3 FTIR spectra for glycerine pitch 82
xv
4.4 FTIR spectra for recovered glycerol 83
4.5 Representative of HPLC spectra of glycerol samples 85
4.6 Time course of cell growth and production of succinic acids in batch
fermentation from glycerol by Escherichia coli K-12 MG1655 87
4.7 Succinic acid structure 88
4.8 FTIR spectrum on fermentation product containing succinic acid 90
4.9 HPLC chromatogram of succinic acid 91
4.10 Effects of glycerol concentration 94
4.11 Effects of tryptone concentration 95
4.12 The effect of incubation period 97
4.13 Effects of sodium sarbonate concentration 98
4.14 The effects of pH on succinic acid production 100
4.15 Effects of inoculum density on succinic acid production 101
4.16 The plot of (a) half normal plot for 26-1
FFD, (b) normal probability
plot of residuals for succinic acid production, (c) residual against
predicted response in succinic acid production and (d) outlier-t plot 109
4.17 Plot of 3D surface response graph (a) and interaction effect graph
(b) between glycerol concentration and tryptone concentration on
succinic acid production (g/l) 111
4.18 Plot of 3D surface response graph (a) and interaction effect
Graph (b) between tryptone concentration and Na2CO3
concentration on succinic acid production (g/l) 112
4.19 Plot of 3D surface response graph (a) and interaction effect graph
(b) between tryptone concentration and pH on succinic acid
production (g/l) 113
4.20 Plot of 3D surface response graph (a) and interaction effect graph
(b) between Na2CO3 concentration and incubation period on
succinic acid production (g/l) 114
xvi
4.21 Normal probability plot of residual for succinic acid production.
The residual point follow a straight line which indicates the errors
distributed normally 122
4.22 Plot of residual against predicted response in succinic acid
production random scatter implies the models proposed are
adequately free from any violation of independence or constant
variance assumption 123
4.23 Parity plot showing the distribution of experimental versus
predicted values of succinic acid production 123
4.24 Perturbation plot representing succinic acid concentration.
Maximum succinic acid concentration was obtained at coded
value +1 124
4.25 (a) Contour plot, (b) interaction graph and (c) 3D surface of
succinic acid production from model equation: effect of glycerol
(A) and tryptone (B) concentration 126
4.26 (a) Interaction graph, (b) contour plot and (c) 3D surface of
succinic acid production from model equation: effect of glycerol
concentration (A) and pH (C) 127
4.27 (a) Interaction graph, (b) contour plot and (c) 3D surface of
succinic acid production from model equation: effect of glycerol
concentration (A) and incubation period (D) 128
xvii
LIST OF SYMBOLS
°C Degree Celsius
g gram
g/l Concentration (gram per liter)
mm Millimeter
3-D Three-dimensional
% Percentage
gl/h Productivity (gram liter per hour)
cm-1
A reciprocal centimeter (or wave number)
cSt centistokes
Rt Retention time
OD550 Optical density at wavelength 550nm
Yx/s Growth yield
µM Maximum specific growth
n Variable quantity
w/w Mass fraction (mass per mass)
v/v Volume concentration (volume per volume)
w/v Mass concentration (mass per volume)
rpm rotation per minute
xviii
LIST OF ABBREVIATIONS
NaOH Sodium Hydroxide
RSM Response Surface Method
FTIR Fourier Transform Infrared Spectroscopy
SEM Scanning electron Microscope
ANOVA Analysis of Variance
CCD Centre Composite Design
HPLC High Performance Liquid Chromatography
FFD Fractional Factorial Design
Na2CO3 Sodium Carbonate
H2SO4 Sulfuric Acid
MONG Matter-non-glycerol
KOH Potassium hydroxide
SD Standard deviation
CHAPTER 1
INTRODUCTION
1.1 RESEARCH BACKGROUND
Succinic acid is a colourless crystalline dicarboxylic acid which has the
molecular formula of C4H6O4 (Song and Lee, 2006). This chemical has a potential
precursor in the chemicals industry and has a high market volume as a versatile
building-block chemical, suitable for many uses (Sauer et al., 2007). It has the potential
to become a chemical platform for the production of various value added derivatives
including surfactant, electroplating, and flavour enhancer in food products, antibiotics,
amino acid, pharmaceuticals and vitamins (Zeikus et al., 1999; Lin et al., 2008). It is
also widely used to produce well known industrial chemicals such as butanediol (BDO),
tetrahydrofuran (THF) and pyrrolidone (Zeikus et al., 1999).
Succinic acid can be synthesized using both chemical and biological processes.
Conventionally, succinic acid is being produced from butane through maliec anhydride
in petrochemical routes (Zeikus et al., 1999). Nowadays, technology has created green
succinic acid from glucose, generated from cane molasses (Lin et al., 2008), wood
hyrolysate (Kim et al., 2004) and straw hydrolysate (Zheng et al., 2009).Although there
were many researches being done using glucose, an alternative non-food material such
as glycerine pitch which originated from biodiesel production plant could also be used
as raw material in succinic acid production. Besides, application of this material as
carbon source in succinic acid production is relatively new and there is limited
information on the process behaviour. This includes factors affecting on the production
as well as the optimum condition in producing the succinic acid. Thus, an understanding
of anaerobic fermentation process with the application of batch system could be
2
obtained via research and experimentation in order to determine the optimum reaction
condition for succinic acid production.
1.2 PROBLEM STATEMENT
Generally, glycerol waste is an alternative substrate being used for succinic acid
production because it has become inexpensive and is abundant in the world market.
Silva et al. (2009) had reported that an excess glycerol generated might cause
environmental problems since it cannot be disposed into the environment. However, to
produce succinic acid from glycerol waste directly without performing treatment, is
quite difficult as it contains high impurities such fatty acid (soap) and salts (Yong et al.,
2001) which might complicate further utilization of glycerol (Hajek and Skopal, 2010).
Besides, it is important to gain a concentrated glycerol with the least amount of other
chemical substances to get the highest purity of glycerol that could be used as a
substrate in the production of succinic acid. Thus, alternative methods of glycerine pitch
treatment are being introduced to overcome these problems as proposed by Kongjao et
al. (2010) by the ways of treating glycerine pitch using chemical and physical treatment
methods. Nevertheless, not many studies had been carried out to optimize glycerol
waste fermentation by Escherichia coli in experimental design. Prior to the succinic
acid production, preliminary works of parameter range selection were first generated, as
this part was essential to produce high concentration of succinic acid. Inappropriate
selection of parameters range would attribute to low succinic acid production and
crucial problem would occur when it is being introduced to the optimization of
fermentation condition. The reaction parameters which mainly contributed to succinic
acid production were glycerol concentration as carbon sources/substrate, nitrogen
sources concentration, pH level, metal ion dosage, reaction time, temperature, agitation
and inoculums density. Improper employment of reaction time, pH level and metal ion
dosage are being attributed to microbial defective and thus would reduce succinic acid
production. However, unsuitable amount of glycerol concentration would contribute to
the inhibition of Escherichia coli growth and subsequently decrease succinic acid
production. The present study focuses in the treatment process of glycerine pitch as well
3
as determining the optimum condition of anaerobic fermentation of glycerol using
Escherichia coli in producing succinic acid.
1.3 OBJECTIVES OF STUDY
The objectives of this study are:
(i) To treat and to recover glycerol content from glycerine pitch.
(ii) To study the significant variables in succinic acid production
(iii)To optimize glycerol fermentation process in succcinic acid production.
1.4 SCOPES OF STUDY
There are mainly four scopes in this research:
(i) The glycerine pitch was purchased from a local biodiesel plant. Prior to the
experiment, the glycerine pitch was being recovered using chemical and
physical treatment methods in order to remove impurities and gain the
glycerol content. Besides, study effect of pH value on glycerol recovery is
also being presented throughout this study.
(ii) The characterization of raw material (glycerol), microbe (Escherichia coli)
and product were being characterized along the way of this research being
conducted. Three glycerol samples (glycerine pitch, recovered glycerol,
commercial glycerol) was being characterized by its functional groups
(Fourier Transform Infra Red - FTIR), quantitative analysis (High
Performance Liquid Chromatography - HPLC), physical observation (color
changes) and chemical analyses (water, pH, ash and MONG). The
Escherichia coli was being characterized based on kinetic profile growth and
cell concentration (optical density; OD550nm) while, the succinic acid were
being characterized by its functional group (FTIR) and quantitative analysis
using High Performance Liquid Chromatography (HPLC).
(iii)The commercial strain of Escherichia coli (wild strain, K-12 MG1655;
ATCC 700926TM
) was being fed into a formulated medium which contained
commercial glycerol to produce the succinic acid. The glycerol
4
concentration, tryptone concentration, inoculums density, pH value,
incubation period and sodium carbonate (Na2CO3) concentration were being
studied using one factor at one time approach (OFAT). Followed by
screening significant parameter using Fractional Factorial Design (FFD).
Finally, the optimum condition of glycerol fermentation was obtained by
using Centre Composite Design (CCD) falling under Response Surface
Methodology (RSM).
(iv) The generated mathematical model from RSM being confirmed and
validated using commercial glycerol and recovered glycerol as carbon source
in fermentation process. This stage is purposely done to investigate
developed model can also applied using recovered glycerol sample in
producing succinic acid. Finally, the optimum condition of succinic acid
production using both commercial glycerol and recovered glycerol are
presented and being compared to un-optimized fermentation condition.
1.5 RATIONALE AND SIGNIFICANCE
Succinic acid production through the fermentation process is widely discussed
and accepted as one of the most recent and advance techniques in biotechnology field
over conventional method of petrochemical route. None in the previous journals had
reported the employing of experimental design in optimizing succinic acid production
using low-grade biodiesel glycerine pitch as a raw material. As glycerine pitch contains
high impurities such as ash and salts, it would produce high concentration of succinic
acid after specific waste treatment being performed on glycerine pitch sample.
Besides, this research had utilized Escherichia coli as a microbe which is a very
amendable to industrial application since it could help to overcome pathogenicity
problems, strict anaerobic conditions, unavailability of genetics tools and lack of
physiological knowledge that could be found in other microbes (Murarka et al.,
2008).Thus, the process would be more helpful when the industrial organism like
Escherichia coli is being used as biocatalysts (Murarka et al., 2008). Furthermore,
anaerobic fermentation under batch system that been employed in this work could be
used as a stand point for succinic acid production from renewable resources towards
5
producing commercials succinic acid. In addition, the process being employed is more
reliable to scale up into a pilot scale or industrial scale based on mathematical modelling
obtained from the experiment.
CHAPTER 2
LITERATURE REVIEW
2.1 INTRODUCTION
Annually, industrial residues such as glycerol are produced in large amounts in
both oleochemical and biodiesel industries worldwide. This product is normally
classified as waste and can be processed in the production of highly demanded product
as succinic acid. Succinic acid plays an important role as a precursor in many industrial
chemical productions. It has been found in a wide range of applications such as in food
industries, pharmaceutical and other important chemical productions such as BDO,
THF, and adipic acid. Recent developments in biotechnology have created an impact on
succinic acid production. The increase of awareness in environmental issues and the
depletion of mineral oil reserves have led to the search for alternatives in making
greener succinic acid. One of the prominent solutions in producing this greener succinic
acid is through the fermentation process using renewable source that exists in nature
such as glycerol. The word fermentation in succinic acid production is described as the
process of succinic acid production which involves interaction between mass culture of
microorganism and substrate, used under specific condition; either aerobic or anaerobic
condition. In this chapter, basic knowledge on succinic acid production through
fermentation process would be presented as well as earlier works that are related to this
current research.
2.2 FERMENTATION PROCESS
Fermentation process is a biological process that strictly requires sterility during
the process, use of cellular enzymatic reactions instead of chemical reactions aided by
inanimate catalysts and sometimes operating at higher temperature and pressure.
7
Fermentation is not necessarily carried out in an anaerobic environment. It can also be
run in an aerobic way. In modern fermentation technology, aerobic conditions are
maintained in a closed fermenter with submerged cultures. The culture contents are
agitated with an impeller and aerated by forcing sterilized air. Table 2.1 presents earlier
works on glycerol fermentation which also included the conditions being used.
Table 2.1: Bio-based production of chemical and fuels via glycerol fermentation
Product
(concentration,
yields*)
Substrate Strains Culture References
1,3-Propanediol
(30g/l, Y=0.61)
Crude
glycerol
Clostridium
acetobutylicum
DG1 (psPD5)
Anaerobic
(N2),
continuous
Gonzalez-
Pajuelo et al.
(2005)
Hydrogen
(60mM) Ethanol
(90mM, Y=0.69)
Crude
glycerol
Enterobacter
aerogenes HH-101
Anaerobic
(N2), Batch/
continuous
Ito et al.
(2005)
Citric acid
(77.4g/l, Y=0.69)
Crude
glycerol
Yarrowia lypolitica
NCIM 3589
Aerobic,
batch
Imandi et al.
(2007)
Succinic acid
(4.9g/l, Y=0.59)
Pure glycerol Anaerobiospirillum
succiniciproducens
ATCC 29305
Anaerobic
(CO2), batch
Lee et al.
(2001a)
Butanol (17g/l,
Y=0.39)
Pure glycerol Clostrodium
pasteurianum
DSM 525
(ATCC6013)
Anaerobic
(N2), batch
Biebl et al.
(2001)
*yield (mol product per mol glycerol)
8
2.2.1 Type of Fermentation
Industrial fermentation processes can be divided into two main types. They are
batch fermentations and continuous fermentations.
i) Batch fermentation process
Batch fermentation is considered as ‘closed system’. In the course of an entire
fermentation, no nutrient is being added into the fermenter except for oxygen, anti-foam
agent and pH controller, either in base or acidic form. In general, batch fermentation is
being used to produce biomass and primary metabolite. In order to maximize biomass
production, optimization of cultural condition for both physical and chemical nutrients
is highly recommended. There are a few advantages of batch fermentation such as low
contamination risk, able to run different successive phase in the same vessel and close
control of genetic stability of microorganism(s). In batch fermentation mode, the
systems are normally controlled culture pH, dissolved oxygen tension and temperature.
There are many researches which had applied this batch processing system to produce
new products. Lee et al. (2008) had successfully produced succinic acid by
Anerobiosprillum succiniciproducens in batch fermentation of mixed sugar of galactose,
glucose and lactose. Furthermore, Jiang et al. (2010) had employed batch fermentation
system in producing succinic acid from enzymatic hydrolysate of brewer’s yeast (EBY)
by A. succinogenes NJ113 with the addition of vitamins to replace commercial yeast
extract. After 48h of cultivation under batch mode, it has resulted in 46.8 g/L succinic
acid concentration and yield of 68.8% being produced.
ii) Continuous fermentation process
Continuous fermentation process is a system where the mixed culture is
continuously being supplied with fresh nutrients, and volume of culture is being kept
maintained by continuous removal of liquid culture at the same flow rate as the feeding
9
rate of fresh nutrients. Hence, this method offers a continuation of growth for a long
period of cell cultivation. With this feature, the designated medium could overcome the
problem of limitation of substrate concentration, besides the exponential growth phase
is being prolonged until an extra substrate is being exhausted on the cell. The
uniqueness of this fermentation style is it operates under steady state conditions, where
cell growth occurs at constant growth rate and stable environmental conditions. Factors
such as pH of the culture, specific growth rate, nutrient and cell concentration,
metabolites concentration and dissolved oxygen; are all keep maintained in the
continuous culture. Using this kind of fermentation method, continuous system
promises important potential advantages in reducing operating and raw material costs.
Brethauer and Wyman (2009) mentioned that some continuous fermentation is now
being employed for commercial ethanol production from cane sugar and corn. This is to
take advantage of higher volumetric productivity, reduced labour costs, and reduced
vessel down time for cleaning and filling. In contrast, these systems are more
susceptible to microbial contamination and need more advanced operations.
Continuous operation in succinic acid production gives more advantages over
batch operation mode. Since the system is fully equipped with continuous media
sterilization and aseptic technique operation, the contamination problem to continuous
system could also be eliminated (Shuler and Kargi, 2002). Many scientific papers and
patents published today deal with the continuous fermentation system on succinic acid
production. Research done by Lee et al. (2003b) had performed anaerobic continuous
culture of Mannheimia succiniciproducens MBEL55E in whey based medium
containing Corn Steep Liquor (CSL) which had resulted in succinic acid yield of 69%
and productivity as high as 3.9gL/h and Kim et al. (2004) had attempted to use
continuous culture of Mannheimia succiniciproducens MBEL55E in wood hydrolysate
based medium and had produced succinic acid yield of 56% and the productivity being
achieved at 1.17gL/h. Besides, another research had shown another type of microbe
which posses the capabilities to produce succinic acid through the continuous system.
Kim et al. (2009) had successfully produced succinic acid by continuous fermentation
of Actinobacillus succinogenes sp. 130Z in an external membrane cell recycle reactor.
This work had resulted in 16.4g/L production and productivity at 6.63gL/h was
achieved. In addition, Oh et al. (2008) had reported anaerobic continuous fermentation
10
of Mannheimia succiniciproducens LPK7 yielded 0.38mol/mol of succinic acid and
productivity at 1.77gL/h was successfully being attained.
2.2.2 Nutritional and Physiological Requirement in Fermentation Processes
Normally, bacterial growths in fermentation processes require specific
nutritional needs which would suit to its type and its physical conditions. The nutrient
includes chemicals and elements that used for bacterial growth. These refer to
nutritional requirements for energy generation, cell maintenance, reproduction and
biosynthesis of cellular. Essentially, the key components for bacterial growth consist of
carbon, hydrogen, oxygen, nitrogen, sulfur, phosphorus, magnesium, iron, calcium,
manganese and traces elements. The common physiological functions of the elements
are summarized as in Table 2.2. The microbial cells grow under a huge range of
physical conditions such as dissolved oxygen, agitation, hydrogen ion concentration
(pH) and temperature. For example, a thermophile grows at high temperatures; an
acidophile grows at low pH; and osmophile grows at high solute concentration. Water
availability in culture medium also becomes a crucial factor in microbial growth.
Table 2.2: Physiological function of principle element for cell growth (Kampen, 1997)
Element Symbol (atomic
number)
Physiological function
Carbon C (6) Constituents of organic cellular material
Oxygen O (8) Constituent of cellular water and organic
materials, as O2 electron acceptor in respiration of
aerobes