Out

KINETIC STUDIES OF LACTIC ACID PRODUCTION FROM WOOD
EXTRACT HYDROLYSATE VIA BATCH AND CONTINUOUS

FERMENTATION PROCESSES
by
John Paul Buyondo
A dissertation
submitted in partial fulfillment
of the requirements for the degree of
Doctor of Philosophy (Bioprocess Engineering)
State University of New York
College of Environmental Science and Forestry
Syracuse, New York
September, 2013
Approved: Department of Paper and Bioprocess Engineering
___________________________ _____________________________
Shijie Liu, Major Professor William Powell, Chair Examining
Committee
______________________________ _____________________________
Gary M. Scott, Department Chair and S. Scott Shannon, Dean
Director Division of Engineering The Graduate School
UMI Number: 3614766
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Acknowledgements
This dissertation could not have been written without valuable contributions
from several individuals. First and most importantly, I would like to thank my major
Advisor, Dr. Shijie Liu, who continuously supported, challenged and encouraged me
to aim higher and strive for excellence throughout my academic program. I am
grateful also to my committee members, Dr. Thomas Amidon, Dr. Gary Scott, Dr.
Bandaru Ramarao and Dr. Biljana Bujanovic who have made valuable comments and
suggestions to me during the dissertation process. I am very thankful for their input.
Many thanks go to my group members: Alan Shupe, Karin Arens, Yang
Wang, Jipeng and Micheal Garver for their valuable input and help. Thanks are also
extended to Mr. Dave Kiemle for his support with NMR analytical work and Mr.
Christopher Wood for his support with production of hemicelluloses experimental
work.
My deepest gratitude goes to my parents, Mr. and Mrs. Yiga together with my
brothers and sisters without whom my accomplishments in life could not have been
achieved. Their unconditional love and unending support of my personal endeavors
has been a major factor in giving me the strength of mind to complete my Ph.D.
program. I will never be able to express enough gratitude to my parents. My greatest
thanks go to my dear wife, Mary Louise Nakitende, who has been my friend, partner
and supporter throughout my Ph.D. program. I am grateful for all of her love, help,
and understanding, and thank her for giving me such a beautiful life. Our baby
iii
daughter, Gabriella, who is my angel, brings a new light to our family and gives me
tremendous happiness that I could never imagine.
J.P.B
iv
Table of Contents
List of Tables ................................................................................................................. x
List of Figures ............................................................................................................... xi
List of Abbreviations .................................................................................................. xiv
Abstract ......................................................................................................................xvii
CHAPTER 1: INTRODUCTION .................................................................................. 1
1.1 Introduction .............................................................................................................. 1
1.2 Objectives of the study............................................................................................. 3
1.3 Dissertation outline .................................................................................................. 4
CHAPTER 2: LITERATURE REVIEW ....................................................................... 6
2.1 Introduction .............................................................................................................. 6
2.2 Lactic Acid Applications ......................................................................................... 7
2.2.1 Lactic Acid Applications in Food Industry ........................................................... 7
2.2.3 Lactic Acid Applications in Chemical Industry.................................................... 9
2.2.4 Lactic Acid Applications in Poly (lactic acid) Synthesis ...................................... 9
2.3 Why biorefinery? ................................................................................................... 11
2.4 Wood Chemical Composition and Structure ......................................................... 13
2.4.1 Cellulose ............................................................................................................. 14
2.4.2 Hemicelluloses .................................................................................................... 14
2.4.2.1 Glucomannan ................................................................................................... 15
2.4.2.1 Xylan ................................................................................................................ 16
2.4.3 Lignin .................................................................................................................. 17
2.4.4 Extractives and Inorganics .................................................................................. 18
v
2.5 Extraction of Hemicelluloses from Woody Biomass............................................. 19
2.5.1 Hot water extraction of woody biomass ............................................................. 20
2.5.1.1 Advantages of Hot Water Extraction ............................................................... 21
2.5.1.2 Disadvantages of Hot Water Extraction .......................................................... 22
2.5.2 Organosolv Pretreatment of Woody Biomass..................................................... 22
2.5.3 Acid pretreatment................................................................................................ 24
2.5.4 Steam Explosion Pretreatment ............................................................................ 25
2.6 Advantages of using Wood Hemicelluloses as Substrate for Lactic Acid

Production .................................................................................................................... 26
2.6.1 Abundant/Readily Available ............................................................................... 26
2.6.2 Current cost comparison ..................................................................................... 27
2.6.3 Non food competitive ......................................................................................... 27
2.6.4 Renewable and environmentally friendly ........................................................... 28
2.6.5 Contain vital salts................................................................................................ 28
2.7 Challenges Involved in using Wood Hemicelluloses for Lactic Acid Production 29
2.7.1 Microbial growth Inhibitory compounds ............................................................ 29
2.7.2 Lack of Microorganisms which can utilize all Hemicellulosic Sugars ............... 30
2.8 Lactobacillus pentosus ........................................................................................... 30
2.8.1 The metabolic pathway for lactic acid production by Lactobacillus pentosus ... 31
2.8.1.1 Embden-Meyerhoff-Parnas Pathway ............................................................... 31
2.8.1.2 Pentose Phosphate Pathway ............................................................................. 33
2.8.2 Nutrient requirements for Lactobacillus pentosus .............................................. 34
2.9 Using NMR to Quantify Fermentation Substrates and Metabolites ...................... 35
CHAPTER 3: MATERIALS AND METHODS ......................................................... 39
3.3 Acid hydrolysis of membrane separated sugar maple wood extract ...................... 40
vi
3.4 Nanofiltration membrane fractionation of sugar maple wood extract ................... 41
3.5 Microorganisms and seed culture preparation ....................................................... 42
3.5.1 Adaptation of Lactobacillus pentosus cells to concentrated WEH ..................... 43
3.6 Batch fermentation of WEH to produce lactic acid ............................................... 44
3.7 Continuous Fermentation ....................................................................................... 45
3.8 Sterilization of media components......................................................................... 46
3.9 Sample preparation for NMR analysis ................................................................... 47
3.10 NMR Spectroscopy .............................................................................................. 47
3.11 Quantitative NMR Analysis. ................................................................................ 48
CHAPTER 4: PRODUCTION OF HEMICELLULOSIC SUGARS FROM SUGAR

MAPLE WOODCHIPS ............................................................................................... 50
4.1 Introduction ............................................................................................................ 50
4.2 Results and discussion ........................................................................................... 51
4.2.1 Hot water extraction of sugar maple woodchips................................................. 51
4.2.3 Dilute acid hydrolysis of membrane separated sugar maple wood extract ......... 56
4.2.4 Nanofiltration of sugar maple wood extract and wood extract hydrolysate ....... 60
4.3 Conclusion ............................................................................................................. 63
CHAPTER 5: BATCH PRODUCTION OF LACTIC ACID FROM WOOD

EXTRACT HYDROLYSATE BY LACTOBACILLUS PENTOSUS ....................... 64
5.1 Introduction ............................................................................................................ 64
5.2.1 Batch Lactic Acid Fermentation ......................................................................... 67
5.2.2 Effect of concentrated WEH loading on production of lactic acid ..................... 73
5.2.3 Adaptation of L pentosus to WEH for Lactic Acid Production .......................... 76
5.3 Discussion and Conclusion .................................................................................... 79
vii
CHAPTER 6: A KINETIC STUDY OF BATCH AND CONTINUOUS
FERMENTATION OF LACTIC ACID FROM SUGAR MAPLE WOOD EXTRACT
HYDROLYSATE ........................................................................................................ 81
6.1 Introduction ............................................................................................................ 81
6.2.1 Batch fermentation of sugar maple WEH ........................................................... 84
6.2.2 Continuous production of lactic acid from WEH ............................................... 88
6.2.3 Effect of dilution rate on lactic acid and acetic acid production......................... 89
6.3. Discussion and conclusion .................................................................................... 92
CHAPTER 7: UNSTRUCTURED KINETIC MODELING OF BATCH

PRODUCTION OF LACTIC ACID FROM HEMICELLULOSIC SUGARS ........... 94
7.1 Introduction ............................................................................................................ 94
7.2 Mathematical model............................................................................................... 94
7.3 Results and Discussion .......................................................................................... 99
7.3.1 Estimation of kinetic parameters ....................................................................... 99
7.3.2 Comparison of kinetic parameter values........................................................... 102
7.3.3 Experimental data fitting................................................................................... 103
7.4 Discussion and conclusion ................................................................................... 107
CHAPTER 8: CONCLUSION AND RECOMMENDATION ................................. 109
8.1 Conclusion ........................................................................................................... 109
8.1.1 Hot water extraction of hemicelluloses from sugar maple woodchips ............. 109
8.1.2 Batch production of lactic acid from wood extract hydrolysate by lactobacillus
pentosus...................................................................................................................... 110
8.1.3 A kinetic study of batch and continuous production of lactic acid ................... 111
8.1.4 Unstructured kinetic modeling of batch production of lactic acid .................... 112
8.2 Recommendations for future work ...................................................................... 113
viii
References .................................................................................................................. 114
Appendices ................................................................................................................. 121
Appendix A: Standard curve for Lactobacillus pentosus biomass concentration ..... 121
Appendix B: Continuous fermentation calibration curve for influent and effluent

pumps ......................................................................................................................... 121
Appendix C: Sugar concentration calibration curves ................................................ 122
RESUME ................................................................................................................... 125
ix
List of Tables
Table 2.1: Chemical and structure composition of wood ............................................ 13
Table 4.1: Concentrations of different inhibitor compounds in WEH before and after
membrane nanofiltration .............................................................................................. 55
Table 4.2: Hemicelluloses concentrations at different stages of production ............... 60
Table 5.1: Hemicelluloses sugar composition of prepared fermentation .................... 74
Table 5.2: Fermentation of concentrated WEH using native and adapted Lactobacillus
pentosus strain .............................................................................................................. 77
Table 6.1: Influence of dilution rate on the production of lactic acid, acetic acid and
residual sugars at steady state ...................................................................................... 90
Table 7.1: Optimum kinetic parameter values for kinetic model of LA production
from WEH by Lactobacillus pentosus ....................................................................... 101
Table 7.2: Kinetic parameters of lactic acid fermentation reported in literature ....... 103
Table 7.3: Squared correlation coefficients for biomass growth, substrate utilization
and product formation ................................................................................................ 107
x
List of Figures
Figure 2.1: The L-and D- enantiomers of lactic acid .................................................... 6
Figure 2.2: Structure of cellulose ................................................................................. 14
Figure 2.3 Structure of softwood glucomannan (Galactoglucomannans) ................... 15
Figure 2.4: Structure of hardwood glucomannan . ...................................................... 16
Figure 2.5:Structure of softwood xylan ....................................................................... 16
Figure 2.6: Structure of hardwood xylan ..................................................................... 17
Figure 2.7: Monomers forming lignin polymer ........................................................... 18
Figure 2.8: Biochemical steps during the homolactic fermentation of glucose ......... 32
Figure 2.9: Biochemical steps during the heterolactic fermentation of pentoses ........ 33
Figure 3.1: The digester used for hot water extraction of Sugar maple woodchips. .. 40
Figure 3.2: Schematic diagram of nanofiltration process ........................................... 42
Figure 3.3: Schematic diagram of continuous fermentation system. ........................... 46
Figure 4.1: 2D NMR spectrum for hemicelluloses contained in hot water extracts of
sugar maple woodchips. ............................................................................................... 54
Figure 4.2: Hemicelluloses of hot water extraction of sugar maple woodchips .......... 55
Figure 4.3: Temperature profile for dilute acid hydrolysis of wood extract ................ 57
Figure 4.4: 2D NMR of hemicellulosic sugars contained in sugar maple wood extract
hydrolysate. .................................................................................................................. 59
Figure 4.5: Concentration of hemicelluloses after acid hydrolysis and neutralization of
sugar maple wood extracts. .......................................................................................... 59
xi
Figure 4.6: Concentration of hemicelluloses after membrane filtration of wood extract
hydrolysate. .................................................................................................................. 62
Figure 5.1: Sugar utilization and acids production profiles during fermentation of
med2:............................................................................................................................ 68
med4: ........................................................................................................................... 68
med1............................................................................................................................. 71
med3............................................................................................................................. 72
Figure 5.5: Effect of initial sugar concentrations in wood hydrolysate-based medium
on lactic acid production by L. pentosus. ..................................................................... 75
Figure 6.1: Sugar utilization, lactic acid and acetic production profiles during batch
fermentation of sugar maple WEH. ............................................................................. 85
Figure 6.2: 1D NMR for quantification of lactic acid and acetic by using glucosamine
as the internal standard................................................................................................. 85
fermentation of sugar maple WEH before continuous process was initiated. ............. 89
Figure 6.4: The effect of dilution rate on residual xylose, lactic acid and acetic acid
production during steady state continuous fermentation of sugar maple WEH. ......... 92
Figure 7.1: Curve fitting of the predicted data to experimental data for biomass
growth ................................................................................................................... 104
xii
Figure 7.2: Curve fitting of the predicted data to experimental data for product
formation .................................................................................................................... 105
Figure 7.3: Curve fitting of the predicted data to experimental data for substrate
utilization ................................................................................................................... 106
xiii
List of Abbreviations
ID One dimensional
2D Two dimensional
AA Acetic Acid
ADP Adenosine diphosphate
Arab Arabinose
ATCC American Type Culture Collection
ATP Adenosine triphosphate
D Dilution rate
EMP Embden-Meyerhof Parnas
F Flow rate
FDA Food and Drug Authority
Gala Galactose
GAP Glyceraldehyde-3-Phosphate
GC Gas chromatography
Gluc Glucose
GlucN Glucosamine
GRAS Generally recognized as safe
h hour
HDPE Higher density polypropylene
HMF Hydroxylmethyl furfural
HPLC High performance liquid chromatography
HSQC Heteronuclear single quantum correlation
LA Lactic acid
LAB Lactic acid bacteria
xiv
Mann mannose
min Minute
N/A Not applicable
NADH Reduced Nicotinamide adenine dinucleotide
ND Not detected
NMR Nuclear magnetic resonance
OD Optical density
ODE Ordinary differential equation
PET Polypropylene terephthalate
PK-P Phosploketolase pathway
PLA Polylactic acid
PP Polypropylene
PP-P Phosphate pentose pathway
PS Polysterene
qNMR Quantitative Nuclear magnetic resources
Qp Productivity
RPM Rounds per minute
Rham Rhamnose
S Substrate
State University of New York, College of Environmental Science and
SUNY- ESF Forestry
TMA Trimethylamine
TSP Trimethylsilyl
US United States
V Distilled water volume
xv
v/v Volume by volume
VBA Visual basic application
WEH Wood extract hydrolysate
Xylo Xylose
xvi
Abstract
J. P. Buyondo. Kinetic studies of lactic acid production from wood extract

hydrolysate via batch and continuous fermentation processes. 144 Pages, 9 Tables, 30
Figures. December, 2013.
The research work presented in this dissertation describes kinetic studies of

batch and continuous fermentation of hemicelluloses to lactic acid. Sugar maple wood
chips were subjected to hot water to extract hemicelluloses, predominantly as
oligomers. Hemicelluloses oligomers were hydrolyzed to fermentable monomeric
sugars by dilute acid hydrolysis and concentrated by nanofiltration process. The
concentrated wood extract hydrolysate contained 138.7 g/L xylose, 22.2 g/L mannose,
18.7 g/L glucose, 10.7 g/L galactose, 4.6 g/L arabinose, and 5.2 g/L rhamnose. The
effect of initial sugar loading was investigated by diluting the concentrated wood
extract hydrolysate to obtain desired total sugar concentrations. In the batch
fermentation process lower total sugar concentration led to the highest lactic acid
yield of 0.83 g/g using Lactobacillus pentosus ATCC 8404 cells. Acetic acid was
produced as the byproduct. Adaptation of Lactobacillus pentosus strain to
concentrated wood extract hydrolysate led to 10 h reduction in batch fermentation
time and 15.5% increase in lactic acid production.
Adapted Lactobacilus pentosus cells were used to study the kinetics of lactic
acid production via batch and continuous fermentation processes. The continuous
fermentation process led to higher lactic acid productivity and lower acetic acid to
lactic acid ratios ranging between 0.27 and 0.60 as compared to the batch
fermentation process which had 0.62 acetic acid to lactic acid ratio. For both batch
and continuous fermentation processes all wood hemicellulosic sugars were utilized
with glucose being the preferred sugar whereas the rest of sugars were simultaneously
utilized.
A kinetic model for batch lactic acid fermentation from hemicellulosic sugars was
developed. Kinetic parameters were determined by ODEXLIMS routine to solve a set
of ordinary differential equations for biomass growth rate, product formation rate and
substrate utilization rate while minimizing the variance between experimental and
predicted values using Excel solver. The model performed satisfactorily for
predicting the transient responses of biomass growth, product formation and substrate
utilization with squared Pearson correlation coefficient (R2) ranging between 0.97 and
0.99 for the initial substrate (total hemicellulosic sugar) concentrations of 40.0 g/L
and 55.0 g/L.
xvii
Key words: Lactic acid, batch, continuous, kinetic modeling, Lactobacillus pentosus,
wood extract hydrolysate.
J. P. Buyondo
Candidate for the degree of Doctor of Philosophy, September 2013
Shijie Liu, Ph.D.
Department of Paper and Bioprocess engineering
State University of New York College of Environmental Science and Forestry,
Syracuse, New York
Shijie Liu, Ph.D. _________________________________
xviii
CHAPTER 1: INTRODUCTION
1.1 Introduction
Traditionally, lactic acid is mainly utilized in food and beverage industries where
it plays an important role in food preservation, shelf life and flavor
enhancement/control. In personal care industry it is used in cosmetics for deep skin
treatments. More specialized lactic acid end-uses include dialysis media and
pharmaceutical intermediates. However, in the past decade lactic acid has become one
of the most valuable chemicals because of its use as a monomer in the production of
polylactic acid (PLA). The scale of lactic acid production has been rising significantly
in order to supply feedstock for the production of PLA. The global lactic acid and
PLA market is growing at a compound annual growth rate of 18.7% and is expected
to reach US$3831.3 million by 2016, as per Markets and Markets
(http://www.plastemart.com). Public concern regarding the environment, increasing
desire of manufacturing companies to develop more sustainable products, limited
available fossil fuel resources, and climate change are important reasons for
governments and companies to find substitutes to crude oil-based products. Bio-based
plastics, with PLA a leading example, present a huge potential to reduce the
dependence on fossil fuels and the related environmental impacts.
In 2010, the global lactic acid market was dominated by North America,
accounting for 35.8% of the overall market. Europe and Asia-Pacific were the second
and third largest markets for lactic acid; accounting for 29.9% and 29.2% of the
overall market, respectively. In the last few years, industrial applications have
1
surpassed food and beverages as a leading application for the consumption of lactic
acid. This has primarily been a result of strong growth in the PLA and solvent markets
for which lactic acid is the primary raw material. The U.S. currently leads in
consumption of lactic acid, while Europe is projected to be the fastest growing
regional market for lactic acid, with an average annual growth rate of more than 8.0%
(http://www.plastemart.com).
Traditionally lactic acid is produced by fermentation of carbohydrates such as
glucose, starch and molasses. Low cost lactic acid is required for the successful
commercial development of cost competitive lactate esters as substitutes to those
derived from petrochemical sources. To produce low cost lactic acid requires
utilization of cheap and readily available substrates. Hemicelluloses from woody
biomass offer one of the most economically favorable substrates alternatives for
biotechnological production of optically pure lactic acid. For instance, in the pulp
industry much of the wood hemicelluloses together with removed lignin are burned to
generate energy which in turn is used for heating purposes. However, compared to
lignin, hemicelluloses have low heating value. The biorefinery concept at Department
of Paper and Bioprocess Engineering, SUNY ESF involves removal of
hemicelluloses and extractives from woodchips prior to pulping or wood pellets
making. The separation of hemicelluloses from woodchips prior to pulping and
utilization of the extracted hemicelluloses for the production of valuable chemicals
like lactic acid would enable the paper and pulp industry to become more profitable.
Economical production of lactic acid from wood hemicelluloses requires a
2
microorganism which can efficiently utilize both pentoses (xylose and arabinose) and
hexoses sugars (glucose, galactose and mannose) to produce high lactic acid yields
with no or minimum by product. In addition, knowledge of the microorganisms
kinetics for cell growth, hemicellulosic sugar utilization and product formation would
be vital for the fermentation process development, optimization and bioreactor design.
This dissertation presents the investigation of kinetics for biotechnological
production of lactic acid from hemicelluloses extracted from sugar maple woodchips
via batch and continuous fermentation processes.
1.2 Objectives of the study
The objectives of this study are:
1. To extract hemicelluloses from sugar maple woodchips and acid hydrolyze the
hemicelluloses oligomers to obtain fermentable monomeric sugars.
2. To adapt Lactobacillus pentosus ATCC 8041 to concentrated wood extract
hydrolysate in order to produce high lactic acid yields via batch and continuous
fermentation processes.
3. To study the kinetics of utilization of hemicelluloses by the adapted Lactobacillus
pentosus-WEH cells to produce lactic acid via batch and continuous fermentation
processes.
4. To develop unstructured kinetic model to simulate the cell growth of adapted
Lactobacillus pentosus, utilization of hemicellulosic sugars and lactic acid
production from wood extract hydrolysate.
3
1.3 Dissertation Outline
Chapter 2 of this dissertation presents the literature review related to lactic acid
production and applications, utilization of lignocellulosic materials for biorefinery
industry and challenges involved.
Chapter 3 of this dissertation explains the experimental methods employed in the
ensuing research. A summary of the sugar maple woodchips preparation,
hemicelluloses extraction equipment and operating conditions are described. Methods
for extract processing, including concentration, hydrolysis and neutralization, are
detailed. The microorganism adaptation techniques, batch and continuous
fermentation process setup are also included, along with procedures for chemical
analysis.
In Chapter 4, the complete compositional determination of hemicelluloses
extracted from sugar maple woodchips is presented plus ultra-filtered wood extract
and its hydrolysates.
In Chapter 5, batch production of lactic acid from wood extract hydrolysate by
native and adapted Lactobacillus pentosus cells is examined. This bacterial strain was
found to utilize all hemicellulosic sugars found in sugar maple wood extract
hydrolysate (WEH) to produce high lactic acid yield. The effect of concentrated WEH
loading and adaptation of Lactobacillus pentosus to concentrated WEH were also
studied.
Chapter 6 details the kinetic study of batch and continuous production of lactic
acid from concentrated WEH by adapted Lactobacillus pentosus ATCC 8041 with
4
focus on the effect of glucose in the fermentation broth to the production of acetic
acid as a by-product via batch and continuous fermentation processes.
Chapter 7 provides the unstructured mathematical kinetic modeling of
Lactobacillus pentosus cell growth rate, product formation rate and hemicellulosic
sugar utilization rate via batch fermentation process.
This dissertation concludes with Chapter 8, which summarizes the findings of the
previous chapters. Chapters 5, 6 and 7 were prepared as manuscripts for publication.
Chapter 5 was published in the Journal of Science and Technology for Forest
Products and Processes. A modified version of Chapter 6 was submitted to the
Journal of Biobased Materials and Bioenergy and Chapter 7 was published in the
Journal of Bioprocess Engineering and Biorefinery.
5
CHAPTER 2: LITERATURE REVIEW
2.1 Introduction
Lactic acid (2-hydroxy propionic acid) is a non-toxic, naturally occurring
(Narayanan et al., 2004; Dien et al., 2002) and renewable raw material. Lactic acid is
the simplest hydroxy acid with an asymmetric carbon atom. It exists in two optically
active configurations; D- and L-enantiomers (Garlotta, 2002) (Figure 2.1).
Figure 2.1: The L-and D- enantiomers of lactic acid
L-lactic acid rotates polarized light to the left, whereas D-lactic acid rotates
polarized light to the right. The L-isomer is an intermediate of carbohydrate
metabolism in humans and other mammals whereas both the D- and L-enantiomers
are produced in bacterial systems (Garlotta, 2001). In general, lactic acid can be
obtained either by chemical synthesis or microbial fermentation of carbohydrate based
substrates, although the fermentation route predominates. The fermentation route
generates pure L (+) or D (-) lactic acid whereas chemical synthesis route generates
racemic mixture of L (+) and D (-) lactic acid isomers referred to as DL lactic acid
(Subba Rao et al., 2008). The chemical synthesis route was established in the early
1960s purposely to synthesize heat-stable lactic acid to be used in baking industry
(Wee et al., 2006). DL-lactic acid is optically inactive. By 2006, the worldwide annual
demand of lactic acid was estimated to be 130,000 150,000 (metric) tones, and the
6
commercial prices of food-grade lactic acid ranged between 1.38 US$/kg (for 50%
purity) and 1.54 US$/kg (for 88% purity). Technical-grade lactic acid with 88% purity
has been priced as much as 1.59 US$/kg (John et al., 2007). Most of the worlds
commercially produced lactic acid is made by the bacterial fermentation of
carbohydrates (Dien et al., 2002), using homolactic organisms such as various
optimized or modified strains of the genus Lactobacilli, which exclusively form lactic
acid (Garlotta, 2001).
2.2 Lactic Acid Applications
Lactic acid is an industrially important product with a large market due to its
attractive properties. Lactic acid and its derivatives have a wide range of applications
in many industries such as food industry, cosmetics and pharmaceutical industry,
chemical industry and poly (lactic acid) synthesis.
2.2.1 Lactic Acid Applications in Food Industry
Lactic acid is considered as generally recognized as safe (GRAS) for use as a
food additive by the regulatory agencies like FDA in USA (Litchfield, 2009). Food-
related applications are the major use of lactic acid in the United States and account
for about 85% of the commercially produced product (Garlotta, 2001). The acid and
salts are preferred to other acids in the food industry because they do not dominate
other flavors and also act as preservers (Monteagudo et al., 1997). Lactic acid has
been used as a preservative and acidulant in the food and beverage sector for several
decades. For instance, calcium lactate is a good dough conditioner whereas sodium
7
lactate acts both as conditioner and as emulsifier (John et al., 2007; Litchfield, 2009).
Lactic acid is used as acidulant, flavoring, pH buffering agent or inhibitor of bacterial
spoilage in a wide variety of processed foods, such as candy, breads and bakery
products, soft drinks, soups, sherbets, dairy products, beer, jams and jellies,
mayonnaise, and processed eggs (John et al., 2007). Lactic acid or its salts are also
used in the disinfection and packaging of carcasses, particularly those of poultry and
fish, where the addition of their aqueous solutions during processing increased shelf
life and reduced microbial spoilage by Clostridium botulinum (John et al., 2007).
2.2.2 Lactic Acid Applications in Cosmetics and Pharmaceutical Industry
The natural occurrence of lactic acid in the human body makes it very useful
as an active ingredient in cosmetics and its water-retaining capacity makes lactic acid
suitable for use as moisturizer in cosmetic formulations (John et al., 2007). In skin
care products, lactic acid is incorporated in formulations as an alpha-hydroxy acid for
improving skin texture and appearance (Litchfield, 2009). The ability of lactic acid to
suppress the formation of tyrosinase is responsible for its effects like skin lightening
and rejuvenation. As humectants, the lactates are often superior to natural products
and more effective than polyols and lactic acid esters with fatty acids as emulsifiers,
builders, and stabilizers in toiletries and personal care products (Litchfield, 2009). For
instance, ethyl lactate is the active ingredient in many anti-acne preparations. Lactic
acid has long been used in pharmaceutical formulations, mainly in topical ointments,
lotions, and parenteral solutions (Wee et al., 2006). Sodium lactate is used in
parenteral and kidney dialysis solutions, while calcium and magnesium lactates can be
8
used in treating mineral deficiencies. In addition, optically pure methyl, ethyl, and
isopropyl lactate esters are used for chiral molecules as pharmaceutical intermediates
(Litchfield, 2009).
2.2.3 Lactic Acid Applications in Chemical Industry
Technical-grade lactic acid is extensively used in the leather tanning industries
as an acidulant for deliming hides and in vegetable tanning. Lactic acid is used as
descaling agent, solvent, cleaning agent, slow acid-releasing agent, and humectants in
a variety of technical processes (John et al., 2007). Owing to their lower toxicities,
ethyl and butyl lactates have industrial applications as alternatives to glycol ethers. In
particular, ethyl lactate is a suitable replacement for chlorinated hydrocarbon solvents
for precision metal cleaning in the aerospace, electronics, and semiconductor
industries (Litchfield, 2009; Datta and Henry, 2006).
2.2.4 Lactic Acid Applications in Poly (lactic acid) Synthesis
Currently, the most important application of lactic acid may be as the raw material
for the production of the biodegradable polymer, poly(lactic acid) (PLA). The
physical properties of PLA polymers depend on the isomeric composition of the lactic
acid used. Therefore, it has become much more important to produce optically pure
lactic acid to be used for the synthesis of high-grade polymers (Hofvendahl and Hahn-
Hagerdal, 2000). PLA is one of the most promising biodegradable polymers owing to
its mechanical property profile, thermoplastic processibility and biological properties,
such as biocompatibility and biodegradability. PLA has several advantages: it is
9
biocompatible and biodegradable, and can be readily broken down thermally by
hydrolysis (Madhavan Nampoothiri et al., 2010). Because PLA is compostable and
derived from renewable sources, it has been considered as one of the solutions to
alleviate solid waste disposal problems and to lessen the dependence on petroleum-
based plastics for packaging materials (Lim et al, 2008). Worldwide production of
high volume consumer plastics continues to be dominated by non degradable
petroleum-based polymers (Madhavan Nampoothiri et al., 2010). While some
petroleum-based plastics are being recycled and reused, the majority are disposed in
landfills due to end-use contaminations. For instance in 2005, plastics were recovered
at a rate lower than 10% in the USA (Lim et al., 2008). PLA is a sustainable
alternative to petrochemical-derived products, since the lactides from which it is
ultimately produced can be produced on a mass scale by the microbial fermentation of
agricultural product and by-products mainly the carbohydrate rich substances (John et
al., 2007).
PLA has wide applications not only as a biodegradable plastic, but also as a
biomedical material because of its excellent mechanical properties and its adjustable
hydrolyzability. These polymers have potential markets in commodity packaging,
fabrication of prosthetic devices, and controlled delivery of drugs in humans (Schmidt
and Padukone, 1997; Garlotta, 2001). It has also been demonstrated that PLA can be
used to enhance the strength properties of paper sheets made from bleached or
unbleached kraft pulp produced from hot-water-extracted and unextracted sugar
maple woodchips (Hasan et al., 2010). PLA has reasonably good optical, physical,
10
mechanical, and barrier properties compared to existing petroleum-based polymers.
For instance, the permeability coefficients of CO2, O2, N2, and H2O for PLA are lower
than that for polystyrene (PS) but higher than that for poly(ethylene terephthalate)
(PET) (Lim et al, 2008). Mechanically, unoriented PLA is quite brittle, but possesses
good strength and stiffness. However, oriented PLA provides better performance than
oriented PS, but comparable to PET. Tensile and flexural moduli of PLA are higher
than high density polyethylene (HDPE), polypropylene (PP) and PS, but the Izod
impact strength and elongation at break values are smaller than those for these
polymers. Overall, PLA possesses the required mechanical and barrier properties
desirable for a number of applications to compete with existing petroleum-based
thermoplastics (Lim et al, 2008).
2.3 Why biorefinery?
Naturally, plant materials (referred to as biomass in this work) are energy storage
units which humans use in their livelihoods. Plants absorb energy from the sun and
use it to make carbohydrates/food via a biological process known as photosynthesis.
For all of mankind existence, humans have utilized biomass as a source of food and
energy. However, the industrial revolution of 1750 to 1850 led to discovery of fossil
fuels and the establishment of routes to generate energy and chemicals from fossil
sources more efficiently than biomass. The discovery and utilization of fossil fuels
has tremendously improved the lifestyle of humans across the globe. However, in
recent years there has been steady increase in the price of petroleum reaching a record
high of $140/barrel in 2009. Because of the continuous increase in world population
11
and a high demand for petroleum products, it is projected that the current worldwide
petroleum deposits will shrink and those fossil fuels will become more expensive as
they become less available (Liu et al., 2012). In addition, research has shown that
continued use of fossil fuels has led to the current global warming and climate change
due to the emission of greenhouse gases in the atmosphere. As a result many
researchers across the world have dedicated much work to searching for alternative
chemical and energy generation routes which are more sustainable and renewable.
Woody biomass has been identified as a potential source of renewable energy and
chemicals. Woody biomass is an important renewable resource that offers an
environmentally attractive alternative to petroleum in the production of fuels,
chemicals and polymeric materials. Harvesting and utilization of woody biomass does
not alter the environment when performed in a sustainable manner (Liu, 2010). In
addition, biomass can be regenerated at a rate as desired by human needs. Therefore,
biomass as a chemical and energy source is sustainable over a time scale longer than
its recycle time (1 80 years) and does not contribute to any net change in the carbon
dioxide in the atmosphere (Liu, 2010). About 370 million oven dry tons of woody
biomass, accounting for 30% of the total biomass projected to be available for biofuel,
can be sustainably produced annually in the United States (Zhu and Pan, 2010).
12
2.4 Wood Chemical Composition and Structure
Woody biomass is composed of three main components: cellulose,
hemicellulose and lignin (Table 2.1). Cellulose provides the structure and strength,
while hemicellulose and lignin provide bonding to the cellulose structure.
Table 2.1: Chemical and structure composition of wood
Compound Composition Structure Building Unit (s)
(%)
Cellulose 40 - 45 linear homo- Glucose
polysaccharide
(cellobiose)
Hemicelluloses 20 - 30 branched hetero- Xylose
polysaccharide
Arabinose
Mannose
Glucose
Galactose
Lignin 20 25 hardwood Three dimensional Guaiacyl
molecule
Syringyl
26 32 softwood Hydroxyphenyl
13
2.4.1 Cellulose
Cellulose is the most abundant carbohydrate on earth. Cellulose is a highly
crystallized linear homopolysaccharide of -D-glucopyranose linked together by -
(14) glycosidic bonds (Figure 2.2). Cellulose occupies some specific properties
including high tensile strength, insolubility to most solvents, and relatively high
resistance to degradation (Amidon et al., 2008; Sjostrom, 1993). Because of the strong
tendency for intra and intermolecular hydrogen bonding, bundles of cellulose
molecules aggregate to microfibrils, which form either highly ordered (crystalline) or
less ordered (amorphous) regions. The tight fiber structure created by hydrogen bonds
results in the typical material properties of cellulose (Sjstrm and Westermark,
1998).
Figure 2.2: Structure of cellulose
2.4.2 Hemicelluloses
Hemicelluloses are the second most abundant carbohydrate source on earth and in
most wood species their content is 20 30% of the dry wood weight (Sjstrm and
Westermark, 1998). Hemicelluloses are heterogeneous polymers made of pentoses
(D-xylose, D-arabinose) and hexoses (D-mannose, D-glucose, D-galactose) (Liu,
2010; Kumar et al., 2008). In addition to the principal pentoses and hexoses building
14
units, uronic acid residues are present, of which 4-O-methyl-D-glucuronic acid
predominates (Kumar et al., 2008; Sjstrm and Westermark, 1998). Main
hemicelluloses constituents in wood are classified as glucomannan and xylan.
2.4.2.1 Glucomannan
There are two types of glucomannan; galactoglucomannans which are primarily
found in softwood (Figure 2.3) and glucomannans (Figure 2.4) which are constituents
of hardwood (Saha, 2003; Sjstrm and Westermark, 1998). Glucomannans and
galactoglucomannans are mainly composed of a linear backbone of (1 4)-linked and
partially acetylated -D-glucopyranose and -D-mannopyranose units, which are
substituted at C-6 with a variable number of a single -D-galactopyranose unit.
Glucomannan has a higher glucose to mannose than galactoglucomannan ratio
(Sjstrm and Westermark, 1998).
Figure 2.3: Structure of softwood glucomannan (Galactoglucomannans)
15
Figure 2.4: Structure of hardwood glucomannan (Ek, 2009, ch 4 - 5).
2.4.2.1 Xylan
There are two types of xylan; arabinoglucuronoxylan found in softwood (Figure
2.5) and glucuronoxylan found in hardwood (Figure 2.6). Xylan consists of a linear
framework of (1 4)-linked -D-xylopyranose units with branches of both 4-O-
methyl- -glucuronic acid and -L-arabinofuranose. Softwood xylan contains no
acetyl groups whereas hardwood xylan contains no arabinose units and the xylose
residues are partially acetylated. A notable detail in both types of xylans is the
presence of a dimeric segment consisting of an -L-rhamnopyranose unit linked
(1 2) to an -D-galacturonic acid residue, which is attached to the 4-position of the
reducing xylose end group (Saha, 2003; Sjstrm and Westermark, 1998).
Figure 2.5: Structure of softwood xylan
16
Figure 2.6: Structure of hardwood xylan
2.4.3 Lignin
Lignin is an amorphous polymer, composed of hydroxylated phenyl propane units,
which are linked together mainly with carbon-oxygen (ether) bonds but also with
carbon-carbon bonds (Sjstrm and Westermark, 1998). Structurally, lignin is a three
dimensional macromolecule made of three building units: hydroxyphenyl, guaiacyl
and syringyl (Liu et al., 2012) (Figure 2.7). Lignin is hydrophobic and covalently
links to hemicellulose, cross linking different plant polysaccharides to confer
mechanical strength to the cell wall. The -O-4 inter-unit linkages are the most
abundant in lignin, estimated to be as high as 50% in softwoods and almost 60% in
hardwoods (Sjostrom, 1993).
17
Figure 2.7: Monomers forming lignin polymer
2.4.4 Extractives and Inorganics
Woody biomass consists of extractives which are extractable compounds that can
be readily dissolved with organic solvents or water at room temperature and under
atmospheric conditions. The main types of extractives are: terpenoids and steroids,
fats (fatty acid glycerol esters), waxes (fatty acid esters of fatty alcohols, terpene
alcohols, and sterols), and phenolic constituents including stilbenes, lignans, tannins,
and flavonoids. Most of the compounds in these groups are lipophilic and soluble in
organic solvents. Various hydrophilic (water soluble) constituents, such as certain
carbohydrates, and inorganic components, are also present in wood (Sjstrm and
Westermark, 1998).
Although there are over 70 metals, earth elements and inorganic compounds in
woody biomass (Amidon and Liu, 2009), the content of inorganic components in
wood is normally low, seldom exceeding 1% of the dry wood weight (determined as
ash) (Sjstrm and Westermark, 1998). The cations potassium, and magnesium, are
present mainly as bound to the acid groups in wood (Amidon and Liu, 2009; Sjstrm
18
and Westermark, 1998). Calcium comprises 0.08 0.2% of dry stem wood mass and
0.85 3.05% stem bark mass. Magnesium comprises 0.02 0.04% of dry hard wood
stem mass and 0.07 0.11% dry hardwood stem bark mass (Amidon and Liu, 2009).
Iron and manganese occur in much smaller amounts and among the other transition
metals, copper and cobalt are present only as traces (Sjstrm and Westermark,
1998).
2.5 Extraction of Hemicelluloses from Woody Biomass
Cellulose and hemicellulose components of woody biomass have been
demonstrated to be potential substrates for many fermentations including lactic acid
(Bustos et al., 2005; Garde et al., 2002; Schmidt and Padukone, 1997; Tanakaa et al.,
2006; Walton et al., 2010; Zhu et al., 2007). To obtain fermentable sugars woody
biomass has to undergo pretreatment. The pretreatment of woody biomass results in
enlargement of the inner surface area of substrate particles, accomplished by partial
solubilization of hemicellulose and lignin. This leads to the fractionation of the three
components and opening of cellulose structure (Pandey et al, 2000).
Therefore, to be utilized as substrate in fermentation process, hemicelluloses need
to be separated / fractionated from other wood components. Although there are
several methods which have been investigated to effectively remove hemicelluloses
from woody biomass, the most commonly used methods are; hot water extraction,
organosolv process, steam explosion, and acid treatment.
19
2.5.1 Hot water extraction of woody biomass
Hot water extraction is a hydrothermal treatment which does not require rapid
decompression and does not employ any catalyst or chemicals (Alvira et al., 2010).
Hot water extraction of woody biomass involves addition of water to woody material
and heating the contents to the desired temperature. The temperatures used usually
range between 120C and 240C. The particle size of the raw material varies from
industrial-sized chips to fine wood meal. Due to mass and heat transfer limitations,
the larger the particle size, the lower is the yield of extracted products. The extraction
efficiency may also depend on the liquid-to-wood ratio of the process, owing to
solubility limitations (Amidon et al., 2008; Borrega et al., 2011). Under high
temperatures, acetyl groups in woody biomass hydrate and dissociate, causing the
extraction liquor pH to drop and create acidic conditions. The formed protons further
catalyze the de-acetylation and hydrolysis. The acidic conditions then generated in the
water extract promote the hydrolysis of wood components (Liu, 2010; Borrega et al.,
2011). The extraction reactions are thus called autocatalytic (Liu, 2010). The acetyl
groups bound to the hemicelluloses are cleaved with the consequent formation of
acetic acid. The acids formed in autohydrolysis are weak acids (such as formic acid,
acetic acid, and glucuronic acid) and are not strong enough to hydrolyze cellulose;
therefore, autohydrolysis can selectively extract hemicellulose from the biomass (Liu,
2010; Yoon, 1998). In the case of extraction/dissolution, proton and water molecules
need to be adsorbed onto the solid surface at the (14)-glycosidic bond sites for the
reaction to begin. The reaction occurs on the solid (biomass) surface, whereas
20
hydrolysis occurs in the liquid phase. The resulting liquor contains a host of
compounds such as polysaccharide or oligosaccharides, formic acid and acetic acid.At
a given operation temperature, the monosaccharide concentration increases as the
residence time increases, with polysaccharides showing a maximum during the
extraction / dissolution process (Liu, 2010).
2.5.1.1 Advantages of Hot Water Extraction
The extraction of the readily hydrolysable carbohydrates with hot water in the
absence of added mineral acids or bases renders the hot water extraction process to be
environmentally friendly. In addition, the heating values of the residual chips
recovered after extraction increases (as more sugars than lignin is removed), there is
no reduction in value due to NaOH or metal hydroxides added to the chips, and no
increase in corrosion due to mineral acids. Furthermore, adverse environmental and
recovery effects are avoided because no NaOH or Na is added to the process streams
and no by-products from mineral acid neutralization are produced from the extraction
process (Liu, 2010).
A hot water extraction above 180C appears to quantitatively remove the
hemicelluloses and a large fraction of the lignin, but only a small portion of the
cellulose. Therefore, hot water extractions at elevated temperatures may be utilized as
a pretreatment to manufacture highly purified dissolving-grade pulps (Borrega et al.,
2011).
21
Liquid hot water pretreatments are attractive from a cost-savings potential: no
catalyst is required and relatively low-cost reactor construction is possible due to
modest corrosion potential. Hot water extraction also has the major advantage that the
solubilized hemicelluloses and lignin products are present in lower concentration, due
to higher water input, and subsequently concentration of degradation products is
reduced. High pentosan recovery and low formation of inhibitors are obtained.
The wood residue after hot water extraction, composed mostly of cellulose and
lignin, can be further subjected to pulping, using less chemicals and shorter pulping
times than in the case of untreated wood (Hasan et al., 2010;Borrega et al., 2011). The
residue wood can also be used for making wood pellets. Wood pellets from hot water
extracted chips are of better quality than pellets made from non-extracted woody
biomass (Amidon et al., 2008).
2.5.1.2 Disadvantages of Hot Water Extraction
The limitation of hot water extraction is that pulps produced from water-
extracted wood generally show lower yield on a wood basis and reduced strength
properties than pulps produced from untreated wood, due to the removal of the
hemicelluloses (Borrega et al., 2011).
2.5.2 Organosolv Pretreatment of Woody Biomass
In the organosolv process, woody biomass is treated with organic solvents at
elevated temperature which leads to separation of hemicelluloses from biomass.
Numerous organic or aqueous solvent mixtures, including methanol, ethanol, acetone,
22
ethylene glycol and tetrahydrofurfuryl alcohol have been used in the organosolv
pretreatment of lignocellulosic biomass (Zhao et al, 2009; Alvira et al., 2010). In
some studies these mixtures are combined with acid catalysts (HCl, H2SO4, oxalic or
salicylic) to break hemicellulose bonds. A high yield of xylose can usually be
obtained with the addition of acid. However, this acid addition can be avoided for a
satisfactory delignification by increasing process temperature (above 185C). Typical
organosolv pretreatment conditions for woody biomass are temperatures about 160
190 C, pretreatment time of 30 60 min, and ethanol concentration of 40 60%
(Zhu and Pan, 2010). An advantage of employing ethanol is because of its low boiling
point, ease of recovery by simple distillation with concomitant modest energy
requirement. Compared to methanol another low boiling point alcohol, ethanol is
safer to handle, relatively affordable and also fully miscible with water.
After organosolv pretreatment of woody biomass, the organic fraction is
drained from the reactor, evaporated, and condensed and the solvent is recycled to the
reactor. The condensed black liquor is diluted with water to precipitate lignin. The
main products from pretreatment are the following:
1. Cellulosic fibers, which contain the original cellulose component and varying
amounts of hemicellulose and residual lignin.
2. Solid lignin, obtained after removal of the volatile solvent from the black
liquor by distillation.
3. An aqueous solution of the hemicellulose sugars, which consists mainly of
xylose in the case of hardwoods or agricultural residues (Zhao et la, 2009).
23
Compared to other chemical pretreatments of lignocellulosic materials the main
advantage of the organosolv process is the recovery of relatively pure lignin as a by-
product (Alvira et al., 2010). On the other hand, the limitations of organosolv process
include generation of high concentrations of inhibitory compounds like furfurals and
soluble polyphenols due to high operation temperatures. Thus the hemicellulose
sugars dissolved in the water-soluble stream are not fermentable without extensive
detoxification. Furthermore, complete solvent recovery is a critical issue for the
process to be economic (Zhu and Pan, 2010).
2.5.3 Acid pretreatment
The main objective of the acid pretreatments is to solubilize the hemicellulosic
fraction of the woody biomass. This type of pretreatment can be performed with
concentrated or diluted acid but utilization of concentrated acid is less attractive for
fermentation process due to the formation of microbial inhibiting compounds,
equipment corrosion problems and acid recovery. Dilute acid pretreatment can be
performed at high temperature (e.g. 180C) for a short period of retention time; or at
lower temperature (e.g. 120C) for longer retention time (30 90 min) (Alvira et al.,
2010). Dilute acid pretreatment presents the advantage of solubilizing hemicellulose,
mainly xylan, but also converting solubilized hemicellulose to fermentable sugars.
Nevertheless, depending on the process temperature, some sugar degradation
compounds such as furfural and hydroxylmethylfurfural (HMF) and aromatic lignin
degradation compounds are detected, and affect the microorganism metabolism in the
fermentation step (Alvira et al., 2010).
24
2.5.4 Steam Explosion Pretreatment
Steam explosion is a hydrothermal pretreatment in which the biomass is subjected
to pressurized steam for a period of time ranging from seconds to several minutes, and
then suddenly depressurized. This pretreatment combines mechanical forces and
chemical effects due to the hydrolysis (autohydrolysis) of acetyl groups present in
hemicellulose. Autohydrolysis takes place when high temperatures promote the
formation of acetic acid from acetyl groups; furthermore, water can also act as an acid
at high temperatures. During steam explosion process the sudden reduction in
pressure leads to explosive decompression of woody biomass and separation of fibers.
In combination with the partial hemicellulose hydrolysis and solubilization, the lignin
is redistributed and to some extent removed from the material (Alvira et al., 2010). .
With steam explosion pretreatment good success in sugar recovery from hardwoods
has been achieved with total monomer sugar recovery ranging from 65% to 80% from
poplar species (Zhu and Pan, 2010).
In some steam explosion processes acid is added as catalyst to increase the
process yield and efficiency. In such a process wood chips or chip-sized wood
materials are first impregnated with acid catalyst either in the gas phase with sulfur
dioxide or in aqueous phase with sulfuric acid before steam pretreatment. Acid-
catalyzed steam pretreatment is actually another form of dilute acid pretreatment in
which the pretreatment is carried out in vapor phase rather in aqueous phase. Typical
acid or sulfur dioxide charge on oven dried (OD) wood varies between 0% and 5%,
25
and temperature ranges from 190 to 210C for hardwoods and 200 to 220C for
softwoods. Pretreatment time varies from 1 to 10 minute (s) (Zhu and Pan, 2010).
The main technical barriers for the steam explosion process include its scalability
for commercialization and ineffectiveness with softwood species (Zhu and Pan,
2010). Another limitation of steam explosion pretreatment is the partial hemicellulose
degradation and the generation of microbial toxic compounds that could affect the
following hydrolysis and fermentation steps. The toxic compounds generated and
their amounts depend on the raw material and the harshness of the pretreatment
(Alvira et al., 2010).
2.6 Advantages of using Wood Hemicelluloses as Substrate for Lactic Acid
Production
Wood hemicelluloses offer an alternative as substrate for biotechnological
production of lactic acid because they are abundant, cheap, do not compete with
human food and are renewable.
2.6.1 Abundant/Readily Available
Hemicelluloses are the second most abundant polysaccharides in nature
representing about 20 - 35% of lignocellulosic biomass. Hemicelluloses are often
underutilized or even an undesirable byproduct in certain industries and agricultural
processes (Sun and Liu, 2010). For instance, in the paper and pulp industry black
liquor containing hemicelluloses and lignin is usually burned to generate energy.
However, compared to lignin, hemicelluloses have a lower heating value. Therefore,
26
utilization of such hemicelluloses in the production of valuable chemicals like lactic
acid would help the pulp and paper industry to become more profitable.
2.6.2 Current cost comparison
Compared to agricultural residues woodchips are at cost ranging from 25 $
tonne-1 to 50 $ tonne-1 depending on sources (Buyondo and Liu, 2012) whereas wheat
straw is 26.46 $ tonne-1, barley straw is 28.66 $ tonne-1, pea straw is 48.50 $ tonne-1,
corn stover is 55.12 $ tonne-1, grass hay is 55.12 $ tonne-1, and switch grass is 66.14 $
tonne-1 (Qureshi et al., 2010). Wood is competitively priced.
2.6.3 Non food competitive
On the basis of utilization of feedstocks, biochemicals and biofuels are
classified into first generation and second generation. In the first generation
biochemicals and biofuels, raw materials are sugarcane and cereal grains while in the
second generation biochemicals and biofuels, lignocellulosic materials (e.g.
agriculture and forest residues) are used as feedstocks. It should be noted that the raw
materials used for first generation biochemicals and biofuels is food competitive
while second generation biofuels are produced from non-edible biomass (Kumar and
Gayen, 2011). Because wood hemicelluloses are not used for human consumption,
their utilization in lactic acid production does not compete with food meant for human
consumption nor does it interfere with human food chain.
27
2.6.4 Renewable and environmentally friendly
Harvesting and utilization of woody biomass to produce lactic acid does not alter
the environment when performed in a sustainable manner. Trees and forests are
available during all weather seasons of the year and thus offer better alternative to
agricultural crops as a reliable biomass for biochemical and bioenergy production.
Therefore, there is a niche position for the woody biomass as a sustainable renewable
chemical and energy source when used together at a facility (Liu, 2010). In addition,
unlike fossils which take over 100 million years to be regenerated, biomass can be
regenerated at a rate as desired by human needs. Therefore, biomass as a chemical and
energy source is sustainable and over a time scale longer than its recycle time (1 80
years), biomass does not contribute to any net change in the carbon dioxide in the
atmosphere (Liu, 2010).
2.6.5 Contain vital salts
In the process of hot water extraction of hemicelluloses from wood, inorganic
compounds are extracted as well. Extracted salt ions such as Ca2+, Mg2+, K+ and Mn2+
are vital for microbial growth of LAB and lactic acid production. The presence of
inorganic compounds makes wood extract based fermentation medium to be cheaper
than refined sugar based medium because the former would require less
supplementation with inorganic salts to achieve optimum nutrient requirements for a
particular lactic acid producing microorganism.
28
2.7 Challenges Involved in using Wood Hemicelluloses for Lactic Acid
Production
There are two main challenges involved in using wood hemicelluloses for
biotechnological production of lactic acid. Namely: microbial growth inhibitory
compounds and lack of microorganisms which can utilize both hexose and pentose
monosaccharides.
2.7.1 Microbial growth Inhibitory compounds
During the pretreatment process of woody biomass to generate hemicelluloses
there are occurrences of partial degradation of hemicelluloses and generation of toxic
compounds that could be inhibitory to the growth of LAB and lactic acid yield in the
fermentation stage. For instance, previous studies have shown that most
microorganisms demonstrate relatively poor pentose sugar fermentation performance
on dilute acid hydrolysates compared to laboratory formulations containing pure
sugars. The lower conversion yield and productivity from dilute-acid hydrolysates
have been attributed to the presence of inhibitory compounds such as acetic acid,
furfurals and assorted phenolics (Garde et al., 2002; Liu et al., 2010; Sun and Liu,
2010). Phenolic acids like ferulic and p-coumaric acids are phenolic compounds
which have long been known to be toxic to bacterial cells. It is likely that lethal
concentrations of ferulic and p-coumaric acids may inhibit bacterial growth by
damaging the hydrophobic sites of the bacterial cells. This is because phenolic
29
compounds cause increase in membrane fluidity, a property known to affect
membrane permeability causing leakage of cellular contents (Ezeji et al., 2007).
2.7.2 Lack of Microorganisms which can utilize all Hemicellulosic Sugars
Wood hemicelluloses is composed of both pentose sugars; xylose and
arabinose and hexose sugars; glucose, galactose and mannose. Theoretically, lactic
acid producing microorganisms would be able to ferment both pentose and hexose
sugars to produce high lactic acid yields. However, while several LAB can efficiently
utilize glucose or hexose sugars, few LAB can efficiently utilize pentose sugars. Thus
fermentation of pentoses (arabinose and xylose) remains a primary obstacle to
economical woody biomass conversion. Economic conversion of hemicelluloses to
lactic acid requires microbial strains capable of fermenting sugar mixtures containing
both hexoses and pentoses. Naturally occurring LAB such as Lactobacillus pentosus,
and Bacillus coagulans have been selected to produce high lactic acid yields from
hemicelluloses under optimized fermentation conditions. On the other hand
recombinant microbial strains of bacteria (Dien et al., 2002) and yeast (Ishida et al.,
2005) have been genetically modified to selectively produce lactic acid from mixtures
of pentose and hexose sugars.
2.8 Lactobacillus pentosus
Lactobacillus pentosus is one of the few lactic acid bacteria cells which have
been demonstrated to produce high lactic acid yields from both pentose and hexose
sugars. Lactobacillus pentosus is a very sturdy organism that readily ferments such
30
unfavorable sugar-containing materials as acid hydrolyzates of wood, sawdust, and
sulfite waste liquor. Zanoni and coworkers (Zanoni et al., 1987) characterized
Lactobacillus pentosus cells to be rod shaped (1 to 1.2 m by 2 to 5 m) with rounded
ends, straight, which may occur singly, in pairs or short chains. In addition,
Lactobacillus pentosus cells are gram positive, nonmotile and facultatively anaerobic,
which grow at temperatures between 10 and 40C. Lactobacillus pentosus cells are
facultatively heterofermentative producing D- and L-lactic acid from a wide variety of
substrates which include amygdalin, L-arabinose, arbutin, cellobiose, D-fructose,
galactose, -gentiobiose, gluconate, D-glucose, glycerol, N-acetylglucosamine,
lactose, D-mannose, mannitol, maltose, melibiose, raffinose, ribose, salicin, sorbitol,
sucrose, trehalose, and D-Xylose (Zanoni et la 1987). Lactobacillus pentosus cells
metabolize glucose and hexoses to produce almost exclusively lactic acid. It also
ferments pentoses to produce lactic acid with acetic as the main byproduct (Krueger
and Peterson, 1948).
2.8.1 The metabolic pathway for lactic acid production by Lactobacillus pentosus
Lactobacillus pentosus is a heterofermentatative microorganism which utilizes
glucose or hexose sugars via Embden-Meyerhoff-Parnas (EMP) pathway and xylose
or pentose sugars via pentose phosphate pathway.
2.8.1.1 Embden-Meyerhoff-Parnas Pathway
During anaerobic growth of obligatory homofermentative LAB in the presence of
excess substrate, one mole of glucose yields two moles of pyruvate via the Embden-
31
Meyerhoff-Parnas pathway (EMP-P), and the pyruvate is further metabolized to lactic
acid (Bustos et al., 2005;Bailey and Ollis, 1986). The homolactic fermentation
follows the biochemical steps indicated in the scheme of Figure 2.8. Intracellular
redox balance is maintained through the oxidation of nicotinamide adenine
dinucleotide (NADH), concomitant with pyruvate reduction to lactic acid. This
process yields two moles of adenosine triphosphate (ATP) per glucose consumed.
Although 100% yield can be predicted from the glucose to lactic acid conversion, a
small part of the sugar is employed for biomass maintenance and generation.
Figure 2.8: Biochemical steps during the homolactic fermentation of glucose

(Redrawn based on Bustos et al., 2005)
32
2.8.1.2 Pentose Phosphate Pathway
Heterofermentative LAB including Lactobacillus pentosus metabolize sugars
via pentose phosphate pathway (PP-P), alternatively referred to as the pentose
phosphoketolase (PK) pathway (Figure 2.9). In PK pathway one mole of xylose
undergoes oxidative reactions to yield xylulose-5-phosphate. The resulting xylulose-
5-phosphate is cleaved into one mole glyceraldehyde phosphate (GAP) and one mole
acetyl phosphate. GAP is further metabolized to lactate as in homofermentation, with
the acetyl phosphate reduced to ethanol via acetyl-CoA and acetaldehyde
intermediates. In theory, end-products (including ATP) are produced in equimolar
quantities from the catabolism of one mole of glucose.
Figure 2.9: Biochemical steps during the heterolactic fermentation of pentoses

(Buyondo and Liu, 2011)
33
2.8.2 Nutrient requirements for Lactobacillus pentosus
Like many LAB, Lactobacillus pentosus have complex nutrient requirements, due
to their limited ability to synthesize B vitamins and amino acids (Hofvendahl and
Hahn-Hgerdal, 2000). In addition to carbon and nitrogen sources, LAB requires
amino acids, vitamins, and minerals for optimal growth. There are several growth-
stimulation factors that have a considerable effect on the production rate of lactic acid.
The mixture of amino acids, peptides, and amino acid amides usually stimulates the
growth of LAB, and the resulting growth rates are much higher than those obtained
with free amino acids (Wee et al., 2006). For LAB growth, the nutrients found in
commercial complex media (like yeast extract, proteose petone, beef extract etc) is
usually sufficient.
Because of high amino acids and vitamins required for LAB during fermentation
of hydrolyzed lignocellulosic sugars, the medium is supplemented with nitrogen,
vitamins and amino acids sources. Meat extract, peptone, yeast extract and corn steep
liquor are used as sources of nitrogen, vitamins and amino acids (Hofvendahl and
Hahn-Hgerdal, 2000). Ammonium ions cannot serve as the sole nitrogen source, but
they seem to have some influence on the metabolism of certain amino acids.
Pantothenic acid, nicotinic acid, and biotin plus the amino acids valine, leucine,
isoleucine, cysteine, and glutamic acid were reported to be essential vitamins for
lactic acid production and growth of Lactobacillus pentosus (Hofvendahl and Hahn-
Hgerdal, 2000: Garlotta, 2001).
34
2.9 Using NMR to Quantify Fermentation Substrates and Metabolites
The ability to accurately quantify fermentation substrates and metabolites in
fermentation broth is an important parameter in determining the efficacy of the
fermentation process and estimating the potential yield of the total sugars that can be
derived from a specific feedstock. The increased use of lignocellulosic materials as
substrates for the fermentation of biochemicals and biofuels requires an analytical
technique which is fast, accurate and able to detect and quantify all hemicellulosic
sugars and metabolites in fermentation broth. Traditionally, fermentation products
like lactic acid and sugars in the wood extract hydrolyzates have been quantified
chromatographically using high performance liquid chromatography (HPLC) or a
related technique. Although chromatographic methods are more sensitive than high-
resolution nuclear magnetic resonance (NMR) spectroscopy, their disadvantage lies in
the time consuming preparation of samples before measurements. On the contrary,
sample preparation for NMR spectroscopy is simpler and less time-consuming (Koir
and Kidri, 2001).
Traditionally NMR spectroscopy was mainly used for identification of chemical
structures for different compounds. However, recent research has proven that NMR
technique can as well be used to accurately quantify many compounds including
carbohydrates, organic acids, amino acids, vitamins and aromatics (Mittal et al.,
2009). The NMRs ability to detect analytes in both one dimension (proton NMR) and
two dimensional (proton carbon NMR) makes it an outstanding technique to detect
most of the fermentation substrates and fermentation products in one prepared sample
35
compared to chromatography methods like HPLC and gas chromatography (GC). The
speed and accuracy of NMR makes it an attractive tool for quantification but also for
explorative analyses and monitoring (Lpez-Rituerto et al., 2009). Modern NMR
spectroscopy permits analysis of low concentrations of analytes and it is a
nondestructive technique that selectively can simultaneously detect a large number of
compounds in fermentation broth (Nord et al., 2004; Koir and Kidri, 2001).
The term qHNMR was recently introduced as an abbreviation for proton-specic
quantitative NMR and highlights the enormous potential of qHNMR in the
identication, characterization, and quantification of metabolites and natural products
(Lpez-Rituerto et al., 2009). Complex chemical analysis of products of fermentation
and utilization of lignocellulosic biomass is becoming of great importance due to the
general endeavor of achieving high product yields, which would lead to an
establishment of economically competitive biorefinery industry. Two dimensional (2
D) NMR spectroscopy has also been used to quantify hemicelluloses and metabolites
in fermentation broth (Buyondo and Liu, 2011; Shupe et al., 2012). NMR
quantification of fermentation metabolites and substrates is made either by integration
of NMR chemical shifts together with the use of calibration references or by partial
least-squares regression (Nord et al., 2004; Shupe et al., 2012; Mittal et al., 2009).
The 1H NMR signals could be characterized as partially overlapped or overlapped
(Nord et al., 2004; Mittal et al., 2009) depending on the degree of how signals
interfere with each other. In aqueous solution at equilibrium monosaccharides mainly
exist in the form of alpha () and beta () monomeric compounds.
36
In a study to quantify monosaccharides in aqueous solution, the proton
resonances associated with the C1- and C1- anomeric protons are integrated and
summed for each sugar providing an estimate of its relative molar concentration in a
mixture of sugars. For those sugars where the individual C1- and C1- proton
resonances are not well resolved, the integrated intensity of either the C1- or C1-
proton is used to approximate the intensity of the other anomeric proton (Mittal et al.,
2009). An integration method could be correctly used with non-overlapped signals
and with careful manual integration (Nord et al., 2004; Lpez-Rituerto et al., 2009;
Mittal et al., 2009). To avoid problems of overlapping signals, two dimensional 1H-
13
C Heteronuclear Single Quantum Correlation (HSQC) NMR technique was
proposed to accurately quantify individual monosaccharides contained in the wood
13
extract hyrolysate (Shupe et al., 2012). In this NMR method the addition of C
second dimension to spectrum results in almost no overlap of the peaks between
various sugars as it is the case with 1H NMR spectrum. For instance, the peaks of -
D-glucopyranose and -D-arabinopyranose have similar chemical shifts at 5.25 ppm
in the single 1H NMR dimension. The addition of a second 13

C dimension leads to
clear distinction of the two peaks where -D-glucopyranose and -D-arabinopyranose
have chemical shifts at 94.93 ppm and 95.42 ppm, respectively. A similar distinction
is made between -D-xylopyranose and -D-mannopyranose whose chemical shifts
occur at 5.21ppm in single 1H NMR dimension whereas in the second 13C dimension
-D-xylopyranose and -D-mannopyranose chemical shifts occur at 95.08 ppm and
96.88 ppm, respectively.
37
In qNMR a reference standard of known molar concentration is used to
determine the concentration of the analyte. The reference standard should be a pure
compound which is structurally unrelated to the analyte with a resonance that does not
overlap that of the analyte. The requirement for lack of overlap means that most
standards have simple NMR spectra, often producing only singlet resonances.
Additional requirements for standards to be used for quantitative NMR analysis
include: chemically inert, low volatility and similar solubility characteristics as the
analyte. There is a wide choice of standard compounds which can be used for qNMR
analysis and a single standard can be used to quantify many components of the same
solution. The direct proportionality of the analytical response and molar concentration
is a major advantage of NMR over other spectroscopic measurements for quantitative
analysis.
38
CHAPTER 3: MATERIALS AND METHODS
This chapter describes the materials and methods used in this study for all
experimental work, including equipment descriptions and analytical procedures. Some
methods such as NMR analysis are common to all chapters, while others are more
specific to a given experiment.
3. 1 Woodchips
Sugar maple wood logs were obtained from the State University of New York,
College of Environmental Science and Forestry (SUNY-ESF) forest properties and
were debarked and chipped in a Carthage chipper. The wet woodchips were air-dried
to a moisture content of 9 12 % and screened using a vibratory screen to 2.5 x 2.0 x
0.5 cm, a size normally used in industry. The screened chips were stored at room
temperature in air tight barrels in a single lot to avoid variation in the experiments.
3.2 Hot-water extraction of sugar maple woodchips
Air-dried sugar maple woodchips were transferred to a 1840.5 L digester (Figure
3.1) and an appropriate amount of water added. For efficient extraction of
hemicelluloses from woodchips, the wood to water ratio was maintained at 1:4. Steam
was used to indirectly heat up the digester and contents to 160C and maintained at
this temperature for 2 h. After hot-water extraction the reactor was cooled down to -
40C by running cold tap water in the reactor jacket. The wood extracts (liquor) and
residual woodchips were removed from the reactor and stored at 8C in the cold room
until further processing. The Wood extract was subjected to nanofiltration processing
39
(described in section 3.4) whereas the residual woodchips were used for pulping and
paper making or wood pellet making. Pulping/paper making and wood pellets making
were beyond the scope of this work.
Figure 3.10: The digester used for hot water extraction of sugar maple woodchips
located in the papermaking pilot plant at department of Paper and Bioprocess
Engineering, SUNY-ESF.
3.3 Acid hydrolysis of membrane separated sugar maple wood extract
After nanofiltration concentration, the concentrated sugar maple wood extract
was transferred to a reactor and 1.5 % wt concentrated sulfuric acid (Acros organics)
was added. High pressure steam at 689.4 kPa and 170C was used to indirectly heat
up the reactor and contents to 135C reaction temperature. The acid hydrolysis
reaction was held at that temperature for 40 min. At the completion of acid hydrolysis
reaction, the reactor and contents were cooled down to 40C indirectly with cold tap
40
water and transferred to a clean and dry barrel. The wood extract hydrolysate (WEH)
was kept at 8C in cold room for about 18 - 24 hr to allow the acid insoluble lignin to
precipitate out of the liquor and settle down at the bottom of the barrel. Acid-insoluble
lignin plus any other solid material were separated from the wood extract hydrolysate
by centrifugation (CEPA High speed centrifuge Z81G, cylinder speed 16,000 rpm,
cylinder diameter 125 mm, New Brunswick Scientific, NJ 08818-4005, USA).
The centrifuged WEH was transferred to a clean barrel where free sulfuric acid
contained in WEH was stoichiometrically neutralized (based on the initial added
amount of acid) by the addition of calcium hydroxide powder (Fisher Scientific,
Pittsburgh, PA, USA). Calcium hydroxide powder was gently added to WEH and the
contents immediately and vigorously mixed to avoid formation of calcium hydroxide
lumps. The pH of WEH before and after neutralization was measured and recorded.
The neutralized WEH was kept at 8C in cold room overnight to allow further
neutralization reaction and calcium sulfate precipitation to occur. The neutralized
WEH was then centrifuged to remove lignin, calcium sulfate crystals plus any other
solid material before it was subjected to nanofiltration processing
3.4 Nanofiltration membrane fractionation of sugar maple wood extract
The obtained wood extract and wood extract hydrolysate were subjected to ultra
filtration process to separate hemicelluloses and oligomers from other wood extract
components. Figure 3.2 shows the schematic diagram of the nanofiltration process
employed in this study. The wood extract or wood extract hydrolysate to be processed
41
was pumped from the feed tank into the nano-size membrane where separation of
hemicelluloses and oligomers from other components occurred. Components were
separated on the principle of the difference of molecular weight. High molecular
weight components like hemicelluloses were retained in the concentrate stream
whereas low molecular weight components were passed through the membrane and
sent to permeate stream. The wood extract and wood extract hydrolysate were
concentrated about 10-fold by recycling the concentrate stream. The concentrated
wood extract hydrolysate was kept in cold room at 8C for further use.
Figure 3.11: Schematic diagram of the design of nanofiltration process at department
of Paper and Bioprocess Engineering, SUNY-ESF.
3.5 Microorganisms and seed culture preparation
The bacterial strain Lactobacillus pentosus ATCC 8041 used was obtained from
The American Type Culture Collection (ATCC). The strain was maintained on MRS
agar medium slant and stored at 4 C. The strain was transferred every 3 - 4 weeks to a
fresh medium. The MRS medium (Difco, Maryland, USA) contained: 10.0 g/L
proteose peptone 3, 10.0 g/L beef extract, 5.0 g/L yeast extract, 20.0 g/L dextrose, 1.0
42
ml/L Tween 80, 2.0 g/L ammonium citrate, 0.1 g/L MgSO4, 0.05 g/L MnSO4, 2.0 g/L
K2HPO4 and 5.0 g/L CH3COONa. The MRS medium was supplemented with 20 g/L
agar to make slant. The seed culture was prepared by picking 1 - 2 big colonies from
the slant and inoculating them into 50 mL MRS (55 g/L) medium contained in 125
mL screw capped plastic flasks (NALGENE, Rochester, NY, USA). The seed culture
was incubated at 37 C for 15 - 24 h on a rotary shaker (GYROMAXTM 747R,
Amerex Instruments, Lafayette, CA, USA), operating at 150 rpm.
3.5.1 Adaptation of Lactobacillus pentosus cells to concentrated WEH
The effect of adapting the strain to concentrated wood extract hydrolysate (WEH)
prior to fermentation was also studied. MRS-WEH solid medium contained 10% (v/v)
WEH, 55 g/L MRS and 20 g/L agar dissolved in distilled water. MRS-WEH solid
medium was inoculated with 2 - 3 big colonies picked from MRS medium slants and
incubated at 37 C for 24 hr in Incumax IC 150 incubator (Amerex Instruments, Inc.
Lafayette, CA, USA). The seed culture of adapted strain was prepared by picking 1 -
2 big colonies from MRS-WEH medium plate and inoculated into 50 mL MRS
medium supplemented with 20% (v/v) WEH. The seed culture was incubated at 37 C
for 15 - 24 h on a rotary shaker (GYROMAXTM 747R, Amerex Instruments,
Lafayette, CA, USA), operating at 150 rpm. The adapted Lactobacillus pentosus
strain was named Lactobacillus pentosus-WEH.
43
3.6 Batch fermentation of WEH to produce lactic acid
Batch fermentation experiments were conducted in a 1.0 L New Brunswick
Bioreactor (BIOFLO 110; New Brunswick Scientific Co., Edison, NJ, USA) with 800
mL working volume. The desired wood extract hydrolysate concentration was
obtained by diluting the concentrated wood extract hydrolysate with the appropriate
volume, V, of distilled water. The fermentation medium contained wood extract
hydrolysate supplemented with 10 g/L yeast extract, 1 g/L K2HPO4, 1 g/L KH2PO4
and 0.5% (v/v) Tween 80. All medium components were purchased from Fisher
Scientific, Pittsburgh, PA, USA. After cooling to room temperature, an appropriate
amount (800 - V) mL of filter sterilized concentrated WEH was added to the
bioreactor. Prior to inoculation the pH of medium in the bioreactor was adjusted to 6.0
by addition of 5 mol/L NaOH. The bioreactor was inoculated with 40 mL of actively
growing 15 24 h-old seed culture and incubated at 37C. Agitation speed was set at
150 rounds per minute (rpm) and air flow rate set at 25 mL/min. During batch process
the fermentation broth pH was maintained at 6.0 by automatic addition of 5 mol/L
NaOH. Two parallel fermentation experiments were performed; one with native seed
culture and the other with adapted seed culture. Samples (2 mL) were taken at given
fermentation times and centrifuged at 4000 rpm for 5 min. The supernatants were
stored at -10 C for sugars, lactic acid and acetic acid analyses. Experimental data
were obtained in triplicate, where the values reported represent sample means.
44
3.7 Continuous Fermentation
Continuous fermentation experiments were conducted in a 1.0 L New Brunswick
Bioreactor (BIOFLO 110; New Brunswick Scientific Co., New Brunswick, NJ, USA)
with 500 mL working volume. The desired wood extract hydrolysate concentration
(40% v/v) was obtained by diluting the concentrated wood extract hydrolysate with
the appropriate volume of distilled water. The fermentation medium contained wood
extract hydrolysate supplemented with 10 g/L yeast extract, 2 g/L K2HPO4, 2 g/L
KH2PO4 and 0.5% (v/v) Tween 80. All medium components were purchased from
Fisher Scientific, Pittsburgh, PA, USA. Wood extract hydrolysate was sterilized by
filtration through a 0.22 m sterile filter (nitrocellulose membrane, Millipore) which
was held on a filter holder with a receiver (1000 mL, NALGENE, Rochester, NY,
USA). The fermentation medium nutrient supplements were dissolved in the desired
amount of distilled water and autoclaved at 121C for 20 min. After cooling to room
temperature, an appropriate amount of filter sterilized concentrated wood extract
hydrolysate was added to the bioreactor and pH adjusted to 6.0 prior to inoculation.
The bioreactor was inoculated with 40 mL of actively growing 15 24 h old seed
culture and incubated at 37C. Agitation speed was set at 150 rpm and air flow rate
was set as 25 mL/min. The pH was maintained at 6.0 by dosing with a 5 mol/L NaOH
solution. The sterile medium was fed at the desired dilution rate by a peristaltic pump
and the effluent was received by another pump at the same speed (Figure 3.3).
Initially the fermentation was carried out in batch operation mode for 16 h after which
continuous fermentation was started. The steady state in the continuous culture was
45
considered to reach after being replaced by at least five residence times and when
three successive measurements of lactic acid and substrate concentrations were
similar with a maximum deviation of 10%. Residence time was defined as reactor
volume/ feed flow rate whereas dilution rate was defined as the ratio of the feed flow
rate to culture volume.
Figure 3.12: Schematic diagram of continuous fermentation system: F flow rate, V
bioreactor volume.
3.8 Sterilization of media components
In order to avoid degradation of hemicelluloses and generation of inhibitory by
products, WEH was sterilized by filtration through a 0.22 m sterile filter
(nitrocellulose membrane, Millipore) which was held on a filter holder with a receiver
(1000 mL, NALGENE, Rochester, NY, USA). Other medium nutrient supplements
were dissolved in the desired amount of distilled water and sterilized by autoclaving
(HIRAYAMA HICLAVETM HV-110, Amerex Instruments, Lafayette, CA, USA) at
46
121 C for 20 min. After cooling to room temperature, an appropriate amount of filter
sterilized concentrated WEH was added to the bioreactor and pH adjusted to 6.0 prior
to inoculation.
3.9 Sample preparation for NMR analysis
To 0.1 mL of thawed sample was added 0.1 mL of a solution of internal standard
plus 800 L deuterium oxide (Acros organics). Each sample was prepared and
analyzed from a 5-mm-o.d. NMR tube (Corning, NY, USA). Sample contents were
thoroughly mixed and the tube cleaned of any particulate matter by wiping the surface
with a soft tissue. The internal standard consisted of 95.6 % wt deuterium oxide, 4.2
% wt glucosamine, 0.2 % wt trimethylamine (TMA) and 0.1% wt trimethylsilyl
propionate (TSP).
3.10 NMR Spectroscopy
NMR spectra were recorded on a Bruker DRX 600 spectrometer (proton
frequency 600.13 MHz) equipped with a 5-mm triple-resonance inverse probe.
Acquisition of spectra was carried out with TOPSPIN software (version 1.2). The
spectrometer was locked on D2O, and all spectra were acquired at 30C. The 1H NMR
spectra were recorded with the standard pulse sequence for presaturation of the water
signal at 1875 Hz (zgpr with pl9 at 60 dB and ip angle 90). The spectral window
was 10 ppm, and data were collected into 64k data points after 128 scans plus 4
dummy scans. The acquisition time was 2.7 s, and the relaxation delay was 1.0 s. All
experiments were carried out with a xed receiver gain in 2300 which was estimate to
47
be adequate through several tests (not automatic receiver gain function was carried
out). The experiments were carried out with automatic tuning and matching (ATM)
and with GRADSHIM tools and using the NMR CASE as a NMR sample changer
allows the automatic analysis of several samples. For 2D NMR spectroscopy, the 1H
13
C HSQC experiment focused on the C1/H1-monosaccharides spectrum in the
chemical shift range of 4.1 to 6.0 ppm for 1H axis and 91 to 106 ppm for the 13C axis.
The two dimensional NMR spectroscopy required approximately 1 h to run one
sample.
3.11 Quantitative NMR Analysis.
Standard solutions of lactic acid, acetic acid, xylose, mannose, glucose,
arabinose, rhamnose and galactose with known ranges of concentrations were
subjected to NMR spectroscopy and respective calibration curves of normalized peak
area versus concentration were generated (Appendix C). Standard solution samples
were prepared by mixing 0.1 mL sample, 0.1 mL internal standard and 0.8 mL
deuterium oxide. For both 1HNMR and 1H 13

C HSCQ NMR, the -glucosamine
signal peak area was used as the reference for quantification of analytes whereas TSP
was set as the reference point at 0 ppm chemical shift for 1HNMR spectroscopy.
Lactic acid and acetic acid methyl group signals located at 1.30 1.35 ppm and 1.95
2.01 ppm, respectively were used for quantification of analytes. For hemicelluloses,
the peaks corresponding to C1- and C1- anomeric sugars were integrated using
MestReNova software and normalized to C1- anomeric peak for glucosamine.
Concentrations of individual analytes were determined by comparing the obtained
48
peak area to the respective calibration curve. The concentration of individual sugars
was determined as the average of and -anomeric sugars concentrations. Where
necessary, the concentration of a particular monosaccharide was determined from
either C1- or C1- anomeric peak alone as it was the case for galactose.
3.12 Determination of cell biomass concentration
Cell biomass concentration was determined by measuring the optical density
(OD) of the sample at 600 nm wavelength using a spectrophotometer (Mapada
Instruments Inc). The OD readings were then converted into cell mass dry weights
using a predetermined correlation standard curve between OD and cell mass dry
weight. The spectrophotometer was calibrated to 100% transmission with fresh
fermentation medium as a reference before the actual OD readings. Between 0.10 and
0.80 OD unit a linear relationship existed between OD and cell mass dry weight.
Samples were thus diluted in such a way that OD readings were in the range of 0.10 -
0.80 OD unit. The cell mass of the respective samples was determined by a filtration
method using pre-weighed 0.22 m Millipore filter paper, followed by air drying in
an oven at 80oC for 24 h. Cell mass was defined as the difference in the measured
weights of the filter paper before and after drying whereas cell dry weight was defined
as dry weight of bacteria per unit volume. The standard curve was a plot of OD
against cell dry weight (Appendix A).
49
CHAPTER 4: PRODUCTION OF HEMICELLULOSIC SUGARS FROM
SUGAR MAPLE WOODCHIPS
4.1 Introduction
In the past decades, substrates such as glucose, starch, molasses and whey
permeate have been used to produce lactic acid. However, such substrates are
expensive whereas lactic acid is a relatively cheap commodity. Consequently, there
has been a steady increase in search for affordable feedstock for lactic acid
production. Lignocellulosic biomass offers a favorable alternative as a feedstock for
the biological production of lactic acid because it is readily available and is less
expensive than either cornstarch or sugars. The main components of lignocellulosic
biomass are: cellulose, hemicellulose, lignin, ash, and extractives. Hemicelluloses are
the second most abundant carbohydrate source on earth and account for 25 35% of
lignocellulosic material (Liu, 2010; Kumar et al., 2008). Woody biomass
hemicelluloses are made up of both five and six carbon sugars attached by glycosidic
bonds. Hemicellulosic sugars include: xylose, glucose, mannose, galactose, rhamnose
and arabinose.
In order to be an effective fermentation substrate, hemicelluloses need to be
extracted from woody biomass and hydrolyzed to fermentable sugars
(monosaccharides). One way of extracting hemicelluloses without damaging or
degrading the cellulosic components is through hot-water extraction. Hot water
extraction of sugars from woody biomass is one exciting developmental-stage
technology that could significantly reduce upstream processing costs associated with
50
pretreatment of lignocellulosic biomass. This concept has been proposed at SUNY-
ESF resulting in a goal for efficient separation of biomass components. This method
requires relatively modest capital and operational expenditures. The wood extracts
from hot water extraction consist of monosaccharides, polysaccharides, acetic acid,
degraded lignin and other low molecular weight extractable compounds (Liu, 2010).
Hot water extraction of hemicelluloses from wood is a preferred method because it
requires less capital, is environmentally friendly because no chemicals are used and
mild operation conditions like temperature and pH, and leads to minimum degradation
of cellulose.
Xylan is the main polymeric molecule in hardwood hemicelluloses. Xylan is a
polymer made of -xylopyranose units linked through -(14)-glycosidic bonds,
where arabinose, acetyl groups, and uronic acids are also present as lateral chains.
Hydrolysis of hemicelluloses by breaking the interconnecting -(14)-glycosidic
bonds as well as the side chain inter-residue bonds leads to generation of pentose
sugars (mainly xylose) and hexose sugars (mainly glucose).
4.2 Results and discussion
4.2.1 Hot water extraction of sugar maple woodchips
Hot water extraction of sugar maple woodchips was carried out in a digester at
160C for 2 hr at water to solid ratio of 1:4. During the hot water extraction process,
the liquor pH becomes low because of the presence of acidic compounds in the hot-
water extractable portion of the woody biomass. The acetyl groups from
51
hemicellulose, for example, contribute to acetic acid formation in the extraction liquor
or the wood extracts. The dissolution of acidic components in water causes the liquor
pH to drop and effectively generate acid as catalyst for the extraction (Borrega et al.,
2011Amidon et al., 2008). Our results showed that the pH level of the hot-water
sugar maple woodchips extracts when cooled and measured at room temperature was
approximately 3.5. The acidic conditions catalyze extraction and hydrolysis reactions
of sugar maple woodchips. Therefore, hot-water extraction is frequently referred to as
autocatalytic and sometimes as an autohydrolysis process (Liu, 2010; Amidon et al.,
2008). At the completion of hot water extraction cellulose (glucan) and lignin (klason
lignin) were mostly retained by the residual woodchips whereas the other wood
components were mostly found in the extraction liquor.
Figure 4.1 shows the 2D NMR spectrum of hemicelluloses extracted from sugar
maple woodchips. It can be observed that the extracts contain mainly xylo-oligomers.
This is not surprising because sugar maple is a hardwood and since xylan is the main
component of hardwood hemicelluloses, xylooligomers and xylose are the main
products obtained in hot water extracts of sugar maple woodchips. In addition,
hemicelluloses is the easiest component to separate among the three major
components of hardwood: cellulose (glucan), hemicellulose (xylan), and lignin. The
hemicellulose is extracted from woodchips in the form of xylo-oligomers with various
degree of polymerization and thus the amount of xylose monomer in the extract is low
(Amidon et al., 2008).
52
Figure 4.2 shows the concentrations of hemicelluloses extracted from sugar
maple woodchips. It can be observed (from Figure 4.2) that xylose was the main sugar
component of sugar maple wood extracts contributing about 70% of total
hemicelluloses in hot water wood extract. Other hemicelluloses extracted from sugar
maple woodchips included mannose, glucose, galactose, arabinose and rhamnose. The
main source of xylose in sugar maple is glucuronoxylan. Glucomannan
hemicelluloses is the main source of glucose and mannose (Liu, 2010). In sugar maple
hardwood rhamnoarabinogalactan is the main source of galactose, arabinose and
rhamnose and occur in the ratio of 1.7:1:0.2, respectively (Fengel and Wegener,
1989).
Other components detected in the hot water extracts of sugar maple woodchips
included; acetic acid, formic acid, furfural and HMF (Table 4.1). Acetic acid is a
principal by-product of the hemicelluloses hot water extraction process. Acetyl groups
are liberated from the hemicelluloses in the form of acetate when the pH is above the
pKa of acetic acid (4.8). At lower pH the acetyl groups lead to the formation of acetic
acid. In the extract, the amount of acetyl groups removed from the hemicelluloses and
the split between the acetate and the acid formed will depend upon pH of the
extraction liquor (Walton et al., 2010; Alvira et al., 2010). Formic acid is derived
from further degradation of furfural and HMF. Furfural is a dehydration product of
pentose sugars: xylose and arabinose whereas HMF is a dehydration product of
glucose or hexose sugars. In addition, wide ranges of phenolic compounds are
53
generated due to lignin breakdown varying widely between different raw materials
(Alvira et al., 2010).
Figure 4.1: 2D NMR spectrum for hemicelluloses contained in hot water extracts of
sugar maple woodchips using deuterium oxide as the solvent.
54
Table 4.1: Concentrations of different inhibitor compounds in WEH before and after
membrane nanofiltration
Inhibitor Concentration, g/L
compound
Before After
Acetic acid 38.86 2.17
Formic acid N/A N/A
HMF 0.27 N/A
Furfural 3.12 N/A
8 40
6 30 Xylose Conc., g/L

Conc., g/L
4 20
2 10
0 0
Rhamnose Mannose Galactose Glucose Arabinose Xylose
Figure 4.2: Hemicelluloses of hot water extraction of sugar maple woodchips
55
4.2.3 Dilute acid hydrolysis of membrane separated sugar maple wood extract
As observed in Figure 4.1 and Table 4.2 sugar maple wood extract mainly
contained oligomers and a less amount of monomers. However, in lactic acid
fermentation oligomers cannot be utilized by Lactobacillus pentosus cells. Therefore,
oligomers contained in wood extract had to be hydrolyzed to fermentable sugars. In
this study sulfuric acid was used to hydrolyze oligomers contained in sugar maple
wood extract to monomers. The nanofiltrated and concentrated hot water extract from
sugar maple woodchips was hydrolyzed by 1.5 % wt concentrated sulfuric acid at 130
C for 40 min to obtain fermentable hemicellulosic sugars. The reactor was charged
with 154.22 kg wood extract plus 1.5 wt% (1310 mL) concentrated sulfuric acid. It
can be observed from the reactor temperature profile in Figure 4.3 that it required
about 35 min for the charged reactor to reach the reaction temperature of 130C and
.the reaction temperature was well maintained at 130C for 40 min reaction time.
However, the reactor and contents required 70 min to cool down to 40C, the
temperature that was regarded to be safe for unloading the reactor.
56
140
120
100
Temp, C
80
o
60
40
20
0
0 20 40 60 80 100 120 140
Time, min
Figure 4.3: Temperature profile for dilute acid hydrolysis of wood extract
It should be noted that operation temperature and time for acid hydrolysis
reaction was lower than that for hot water extraction, as a lower operation temperature
led to minimal formation of furfurals. Formation of furfural compounds leads to low
hemicelluloses yield due to degradation of sugars. In addition, furfurals are inhibitory
to microbial growth. In acid hydrolysis reaction, hydrogen ions or acidic conditions
act as a catalyst to hydrolyze the glycosidic bonds and induce their depolymerization.
The measured pH of wood extract hydrolysate at room temperature was 0.61.The
glycosidic bond breakage is enhanced when the breakaway oligomers are attracted
away from the solid surface. High temperature and high electrolyte content facilitate
the oligomers to be in the liquor phase (Liu, 2010).
Figure 4.4 shows the 2D NMR spectrum for the obtained mixture of
hemicellulosic sugars in the sugar maple wood extract hydrolysate (WEH). Looking
at two Figures 4.1 and 4.4, it can clearly be observed that wood extract hydrolysate
57
(Figure 4.4) contained far less oligomers and more monomers than wood extract
(Figure 4.1). The C1- and C1- anomeric sugar components were located between
5.0 - 5.3 ppm and 4.4 - 4.95 ppm chemical shifts, respectively. This shows that the
acid hydrolysis reaction led to break down of oligomeric sugars to monomeric sugars.
As a result in Figure 4.5 and Table 4.2 one can observe that the total concentrations of
hemicellulosic monomeric sugars contained in wood extract hydrolysate generally
increased by more than 3 times the total concentrations of monomeric sugars
contained in wood extract.
Calcium hydroxide was used to neutralize the free sulfuric acid contained in
WEH. Calcium hydroxide reacts with sulfuric acid according to the equation below;
Ca(OH)2 (s) + H2SO4 (aq) CaSO4 (s) + H2O (l) (4.1)
The measured pH of neutralized WEH was 3.5, which was nearly equal to the
initial pH 3.3 of wood extract before acid hydrolysis reaction.
58
Figure 4.4: 2D NMR of hemicellulosic sugars contained in sugar maple wood extract
hydrolysate using deuterium oxide as the solvent.
20 200
18 180
16 160
Minor Sugars Conc., g/L
14 140
Xylose Conc., g/L
12 120
10 100
8 80
6 60
4 40
2 20
0 0
Figure 4.5: Concentration of hemicelluloses after acid hydrolysis and neutralization of
sugar maple wood extracts. Minor sugars include: rhamnose, mannose, galactose,
glucose and arabinose.
59
Table 4.2: Hemicelluloses concentrations at different stages of production
Concentration, g/L
Hot water Wood After Acid Hydrolysis and After membrane
Hemicelluloses Extract Neutralization nanofiltration
Galactose 4.0 8.6 10.7
Arabinose 7.0 4.1 4.6
Glucose 5.6 15.2 18.7
Xylose 36.9 175.1 138.7
Mannose 3.8 17.3 22.2
Rhamnose 2.8 3.0 5.2
Total 60.2 223.3 200.1
4.2.4 Nanofiltration of sugar maple wood extract and wood extract hydrolysate
Wood extracts after hot-water extraction and acid hydrolysis consisted of
monomeric and oligomeric sugars (hexoses and pentoses), acetic acid, degraded lignin
(or aromatics), furfurals as well as minor extractives like formic acid. Nanofiltration
membrane system was used to separate desired monomeric and/or oligomeric sugars
from other components and as well concentrate the desired sugars. The fractionation
of hemicellulosic sugars and byproducts from wood extract is a key step in the
biorefinery process because byproducts like furfural, hydroxylmethylfurfural (HMF),
acetic acid, formic acid, methanol and aromatics have been identified as potential
60
inhibitors of many fermentation processes (Liu et al., 2010; Ezeji et al., 2007)
including biotechnological production of lactic acid (Hofvendahl and Hahn-Hgerdal,
2000; Buyondo and Liu, 2011).
Nanofiltration membrane separation was adapted to fractionate wood extracts
and/or hydrolysates into desired chemical components. The membrane used in this
study had a molecular weight cut-off of about 100 g/gmol. However, filtration was
not entirely governed by molecular weight, as molecular shape and hydrophobicity
can also affect penetration. Sugars have a ring structure and thus have a relatively
large diameter. Thus, so long as the pore size is small enough to prevent the sugar ring
structure from permeating through, all the sugars are likely to be retained in the
concentrate stream. Using a membrane with a molecular weight cut-off of 100
g/gmol, almost all the sugars were retained on the retentate or concentrate side. Free
acetate and small molecules such as formic acid and furfural passed through the
membrane almost as readily as water. Membrane technology can therefore be utilized
to purify sugars, as well as to separate other chemicals (Liu et al., 2012).
Table 4.2 shows the different concentration of hemicellulosic sugars obtained at
different stages of wood extract processing. It can be observed that apart from xylose,
the concentration of other hemicellulosic sugars remained about the same at all stages
of production. The decrease in xylose concentration after neutralization could be
attributed to occurrence of sugar losses during precipitation of calcium sulfate and
lignin from wood extract hydrolysate. At completion of the final stage of membrane
separation/purification, the concentration of fermentable sugars in sugar maple wood
61
extract hydrolysate were: 138.7 g/L xylose, 22.2 g/L mannose, 18.7 g/L glucose, 10.7
g/L galactose, 4.6 g/L arabinose, and 5.2 g/L rhamnose (Table 4.2 and Figure 4.6).
Here, xylose, a five carbon sugar, is the dominant component in sugar maple wood
extract hydrolysate. Arabinose is another five carbon sugar. Mannose, glucose and
galactose are six carbon sugars, while rhamnose is a six carbon sugar derivative
(deoxy L-rhamnose). The main source of xylose in sugar maple is glucuronoxylan.
The ratio of glucose to mannose is near one which suggests that their main origin was
glucomannan (Amidon et al., 2008). There was no formic acid, furfural and HMF
detected in the membrane separated/purified wood extract hydrolysate and the
concentration of acetic acid was significantly reduced (Table 4.1)
25 160
140
20
120
Minor Sugars Conc., g/L
100
Xylose Conc., g/L
15
80
10
60
40
5
20
0 0
Figure 4.6: Concentration of hemicelluloses after membrane filtration of wood extract
hydrolysate.
62
4.3 Conclusion
Hemicelluloses form lignocellulosic materials like sugar maple are potential
source of sugars for production of a wide range of biological valuable products such
as lactic acid. Prior to their utilization hemicelluloses must be fractionated from other
wood components. In this study the hot water extraction technique was demonstrated
to successfully extract hemicelluloses from sugar maple woodchips. At mild
extraction conditions, with temperatures at 160C and 2 hr extraction period favored
the recovery of high-molar mass xylooligomers in the water extract. Increasing the
severity of the extraction process would lead to the degradation of sugars to microbial
inhibitory compounds like furfural and HMF. Sugar oligomers in hot water extract
were hydrolysed by concentrated sulfuric acid to generate fermentable monomeric
sugars. The total monomeric sugars increased from 60.2 g/L before acid hydrolysis to
222.3 g/L after acid hydrolysis. The wood extract hydrolysate was subjected to
nanofiltration membrane system to fractionate the desired sugars from other
components like acetic acid and furfurals. Upon nanofiltration membrane processing,
water and other low molecular weight compounds, (such as acetic acid, formic acid
and furfurals), were preferentially sent to the permeate stream, while sugar monomers
and oligomers along with high molecular weight compounds were concentrated in the
concentrate stream. Xylose was the main sugar component contributing about 70% of
the total sugars in the WEH.
63
CHAPTER 5: BATCH PRODUCTION OF LACTIC ACID FROM WOOD
EXTRACT HYDROLYSATE BY LACTOBACILLUS PENTOSUS
5.1 Introduction
In commercial processes, glucose and corn starches have been widely used as
substrates for biological lactic acid production. However, this is economically
unfavorable, since pure sugars have a higher economic value than the produced lactic
acid (Hofvendahl and Hahn-Hgerdal, 2000). In addition, the recent increase in
refined sugar and corn prices due to high demand for its conversion to fuel ethanol,
climate change impact and increase in world population have led to increase in cost
for refined sugars and starches and yet lactic acid market price has remained relatively
low. In order for the biotechnological production of lactic acid to be feasible, low cost
raw materials are necessary to meet the demand of PLA polymer producers and other
industrial users at a relatively low cost. Therefore, for the biotechnological production
of lactic acid to be economically feasible and competitive, the process requires
utilization of inexpensive and abundant raw materials and efficient conversion of
these materials to lactic acid. As a result, enormous research work has focused on
screening alternative potential substrates for biotechnological lactic acid production.
Materials screened include: (1) Industrial and household wastes such as molasses
(Monteagudo et al., 1997), waste paper (Schmidt and Padukone, 1997), sugarcane
baggasse (Laopaiboon et al., 2010), whey (Schepers et al., 2006), and kitchen waste
(Zhao et al., 2009). (2) Agricultural residues such as wheat straw (Garde et al., 2002;
Maas et al., 2008), Corn stover (Zhu et al., 2007), rice bran ( Tanakaa et al., 2006).
Barley straw (Venus and Richter, 2006), Corncob (Wang et al., 2010), vine trimmings
64
(Bustos et al., 2007) and (3) Woody biomass such as cellulose and hemicelluloses
(Buyondo and Liu, 2011; Walton et al., 2010; Wee and Ryu, 2009).
Lignocellulosic biomass offers a favorable alternative as a feedstock for the
biological production of lactic acid because it is readily available, has no competing
food value, and is less expensive than either cornstarch or refined sugars. The main
challenges of using lignocellulosic biomass for lactic acid production include;
presence of microbial inhibitory compounds and there is not currently identified a
LAB which can efficiently utilize all hemicellulosic sugars to produce high lactic acid
yields. Microbial inhibitory compounds are formed as by-products during
pretreatment of lignocellulosic biomass to obtain fermentable sugars and this
challenge must also be overcome.
Although systematic research on inhibition mechanisms of wood extract
hydrolysate (WEH) in lactic acid fermentation have rarely been reported, weak acids,
furfural, and 5- hydroxymethyl furfural (HMF) were reported to be the main
inhibitors of WEH fermentations in ethanol production (Palmqvist and Hahn-
Hgerdal, 2000). Weak acids inhibit cell proliferation due to uncoupling and
intracellular anion accumulation, and furfural or HMF inactivates cell replication by
inhibiting specific enzymes related with glycolysis or inhibitor reduction. To reduce
the impact of microbial inhibitory compounds on lactic acid production yields, several
techniques have been applied which include adaptation of LAB to fermentation
medium containing inhibitory compounds (Lorca et al., 1998; Wee et al., 2006) and
65
detoxification of inhibitory compounds contained in wood hydrolysate (Sun and Liu,
2012).
Because of the heterogeneity of sugar composition in wood hydrolyzates,
complete utilization of all sugars is difficult (Abdel-Rahman et al., 2011). For
profitable biotechnological production of lactic acid from lignocellulosic biomass, a
LAB strain capable of fermenting hemicellulosic sugar mixtures of hexoses and
pentoses is needed. Although much research has been focused on genetically
engineering strains that can efficiently utilize both 5- carbon and 6-carbon sugars
(Dien et al., 2002), the recombinant cells have a tendency to be genetically unstable
on repeated application. All LAB are well known to metabolize glucose and other
hexoses, but only a few of LAB such as Lactococcus lactis, Lactobacillus casei,
Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus pentosus and Bacillus
coagulans have the ability to ferment pentoses (Doran-Peterson et al., 2008; Buyondo
and Liu, 2011, Walton et al., 2010). The versatility of Lactobacillus pentosus makes it
a potential alternative to genetically modified strains. L. pentosus ferments hexoses
via the EMP (Embden-Meyerhoff-Parnas) pathway (Figure 2.8) and pentoses via the
phosphoketolase pathway (Figure 2.9).
In this work, a bacterial strain Lactobacillus pentosus ATCC 8401 was challenged
by different levels of concentrated sugar maple wood extract hydrolysate (WEH) to
produce lactic acid via a batch fermentation process. The effect of adaptation of
Lactobacillus pentosus cells to concentrated WEH was also examined.
66
5.2.1 Batch Lactic Acid Fermentation
Concentrated WEH was diluted with distilled water to obtain total sugar
concentration of 61.47 g/L for medium med2 and 129.50 g/L for medium med4. L.
pentosus seed culture (40 mL) was inoculated into the prepared fermentation medium
and the batch fermentation reaction set to run for 55 hours. Unlike many other
Lactobacillus strains, L. pentosus is capable of metabolizing both 5-carbon and 6-
carbon sugars. Therefore, all hemicellulosic sugars in fermentation media were
potential carbon sources for microorganisms growth and lactic acid production.
Acetic acid was produced as the main by-product. As shown in Figure 5.1, during
fermentation of med2, arabinose, glucose, rhamnose and galactose were utilized
within the first 8 hours while utilization of mannose and xylose started after 8 hours.
Mannose was consumed in the next 12 hours of fermentation reaction, whereas xylose
was slowly utilized.
67
Figure.5.1: Sugar utilization and acids production profiles during fermentation of
med2.
Figure.5.2: Sugar utilization and acids production profiles during fermentation of
med4.
68
In the fermentation of med4 (Figure 5.2), arabinose, glucose, rhamnose, and
galactose were utilized within the first 24 hours while utilization of mannose and
xylose started after 10 hours. In this case, mannose was consumed in the next 14
hours and xylose was slowly utilized. Within 55 hours of fermentation, all sugars
contained in med2 were completely consumed while for med4 118.11 g/L total sugars
had been consumed and 10.39 g/L xylose remained unconsumed.
The lactic acid concentration obtained was 43.66 g/L after 55 hours of
fermentation of medium med2, with a productivity of 0.99 g/(L h) and product yield
(YP/S) of 0.72 g lactic acid /g consumed total sugar. Acetic acid (21.60 g/L) was
produced as the main by-product. Figures 5.1 and 5.2 show that the production of
acetic acid was pronounced after glucose had been exhausted. In the fermentation of
medium med4 (Figure 5.2) 59.62 g/L lactic acid was produced after 55 hours of
fermentation, with a productivity of 1.36 g/(L h) and a product yield (YP/S) of 0.50 g-
lactic acid /g-consumed total sugar. The concentration of produced acetic acid was
21.13 g/L. During fermentation of media med2 and med4 both pentoses and hexoses
sugars were simultaneously consumed, though xylose and mannose consumption was
delayed, by L. pentosus. These results agree with those obtained by Bustos and
coworkers (Bustos et al., 2005) who reported that L. pentosus is a heterofermentative
bacteria, which utilizes glucose (a hexose sugar) via EMP pathway and in the
absence of glucose, xylose (a pentose sugar) is utilized via PK pathway. Conversion
of hexoses and pentoses by L. pentosus has been projected to follow the following
overall stoichiometric equations (Lampen and Peterjohn, 1951):
69
C6H12O6 2C3H6O3 (5.1)
C5H10O5 C3H6O3 + CH3COOH (5.2)
During this process carbon dioxide, lactate, and acetate or ethanol are produced in
variable proportions depending on several factors such as aeration, initial sugar
concentration or even the presence of other proton and electron acceptors (Bustos et
al., 2005). L. pentosus catabolizes one mole of glucose (or hexose sugar) to yield two
moles of pyruvate via the EMP pathway (Bustos et al., 2005). The terminal electron
acceptor in this pathway is pyruvate, which reduces to lactic acid. In the PK pathway,
the pentose sugar undergoes oxidative reactions to generate one mole of each of
Glyceraldehyde 3-Phosphate (GAP) and acetyl phosphate. GAP is metabolized into
lactic acid by following the same pathway as in the EMP pathway (Bailey and Ollis,
1986). The acetylphosphate has two possible destinations depending on
environmental conditions. In one condition, this molecule successively reduces to
ethanol via acetyl-CoA and acetaldehyde intermediates. In another condition, the
acetyl-phosphate can produce acetate (acetic acid) through the enzyme acetate kinase.
However, in this study no ethanol was detected.
70
med1.
The fermentation profiles in Figure 5.1 indicate that acetic acid production started
after all the arabinose, galactose, rhamnose and glucose were depleted, indicating that
the acetic acid coproduction came only from the xylose and, perhaps, mannose
consumption. Fermentation of media med1 containing 54.08 g/L total sugars (Figure
5.3) and med3 containing 94.10 g/L total sugars (Figure 5.4) followed the same trend.
The obtained results are in agreement with that reported by (Bustos et al., 2005),
working with same bacterial strain (L. pentosus) but different raw material (trimming
vine shoots hydrolysate). Although Bustos and coworkers (2005) did not measure
mannose, rhamnose and galactose sugars, lactic acid and acetic acid production
followed the same trend. When glucose is present during the initial stage of
fermentation, the sole product is lactic acid while 5-carbon (arabinose), 6-carbon
71
(glucose and galactose), and deoxy 6-carbon (rhamnose) sugars were catabolyzed.
Therefore, when glucose is present, one may infer that;
3CH2O C3H6O3 (5.3)
2C6H12O5 + O2 4C3H6O3 (5.4)
med3.
Because of apparent acetic acid production and absence of ethanol production
from the mixture of mannose and xylose after the early stage of fermentation, one
may infer that the 6-carbon sugar follow EMP pathway while 5-carbon sugars follow
PK pathways when glucose is not present. In both fermentation media med2 and
med4, L. pentosus preferred to first utilize glucose (6-carbon), galactose (6-carbon)
arabinose (5-carbon), and rhamnose (6-carbon deoxy) as compared to mannose (6-
72
carbon) and xylose (5-carbon). For med2, mannose and xylose utilization started after
or near the complete utilization of other sugars, while in med4 mannose and xylose
utilizations started after 10 hours of fermentation. Fermentation of med4 with higher
initial glucose concentration (12.53 g/L) had a higher average rate of sugar utilization
(2.68 g L-1 h-1) than medium med2 with lower initial glucose concentration (6.33 g/L),
which had sugar utilization rate of 1.37 g L-1 h-1. Compared to glucose, the conversion
of xylose requires additional enzymatic reaction steps. Some enzymes are inducible;
therefore theres a lag time before the enzymes required for assimilation appear when
cells are exposed to xylose (Tanaka et al., 2003). It has also been proposed that an
increase in xylose concentration will result in increased intracellular concentrations of
the intermediates, such as fructose 1,6-diphosphate, which inhibits enzyme activity
during xylose metabolism (Wang et al., 2010). Nevertheless, the ability of L. pentosus
to utilize 5-carbon and 6-carbon sugar mixtures makes it a potentially useful
microorganism to be used in industrial fermentation of wood extract hydrolysate.
5.2.2 Effect of concentrated WEH loading on production of lactic acid
To investigate the possible effect of wood extract hydrolysate (WEH)
concentration on the production of lactic acid, concentrated WEH was diluted with
distilled water. Fermentation media were prepared by adding different amounts of
distilled water to the known volume of concentrated WEH (mL) to obtain different
sugar concentrations (Table 5.1). The main sugar component of fermentation media
was xylose, contributing about 68% of total sugar concentration. This agrees with the
73
results presented in literature by Liu et al., (2010) and Amidon et al., (2008) that
xylose is the main component of sugar maple wood extract hydrolysate
Table 5.1: Hemicelluloses sugar composition of prepared fermentation
WEH, V, Xylose, Glucose, Galactose, Arabinose, Mannose, Rhamnose, Total Sugars,
Medium mL mL g/L g/L g/L g/L g/L g/L g/L
med1 300 500 36.90 5.75 2.04 1.73 6.22 1.44 54.08
med2 400 400 41.22 6.33 3.24 1.86 6.91 1.91 61.47
med3 500 300 64.15 9.62 3.83 2.54 10.47 3.48 94.10
med4 600 200 86.34 12.53 6.98 4.14 15.54 3.98 129.50
To study the effect of sugar loading on the production of lactic acid fermentation
media containing total sugar concentrations of 54.08 g/L (med1), 61.47 g/L (med2),
94.10 g/L (med3) and 129.50 g/L (med4) were used. As shown in Figure 5.5, the final
lactic acid concentration increased with an increase in total sugar concentration
contained in the fermentation medium. The maximum lactic acid concentration of 65
g/L was obtained after 106 h of fermentation of medium med4. However, the yield
was higher with fermentation media containing lower concentration of WEH
74
Figure 5.5: Effect of initial sugar concentrations in wood hydrolyzate-based medium
on lactic acid production by L. pentosus.
Medium med1 had the highest product yield of 0.83 g-lactic acid/g-sugar followed
by med2 (0.72 g/g), med3 (0.65 g/g), and med4 (having 0.50 g/g). In addition, the
time required for complete fermentation (or complete substrate utilization) generally
increased with an increase in hydrolysate loading. Fermentation media (med1 and
med2) which contained lower WEH concentrations required shorter fermentation time
to attain maximum lactic acid production as compared to media (med3 and med4).
The low lactic acid yields at higher WEH loading could be largely attributed to
product inhibition since known microbial inhibitor compounds; furfural, HMF and
acetic acid had been removed from WEH during the nanofiltration process. This result
is in good agreement with literature reports (Bustos et al., 2005; Iyer et al., 2000) that
product inhibition leads to a decrease in fermentation rate and microbial growth rate.
75
In all fermentations acetic acid was produced as the main byproduct. Acetic acid
production followed the same trend as lactic acid production. Although there was no
significant difference in the produced acetic acid for different levels of WEH
containing media, generally acetic acid concentration increased with increase in sugar
concentration in fermentation medium. Fermentation medium med4 produced the
highest acetic acid concentration of 23.90 g/L, followed by medium med3 (22.61
g/L), medium med2 (21.61 g/L) and medium med1 (18.47 g/L). In all fermentations,
acetic acid was mainly produced when mannose and xylose were the only remaining
fermentable sugars. This observation indicates that in the presence of glucose L.
pentosus metabolizes both 5-and 6-carbon sugars to exclusively produce lactic acid
whereas in the absence of glucose it metabolizes 5-carbon sugars via the PK pathway
to produce lactic acid and acetic acid.
5.2.3 Adaptation of L pentosus to WEH for Lactic Acid Production
Because wood extract hydrolysate contains different levels of inhibitory
compounds like lignin and furfural, adaptation of the fermentation microorganisms to
the hydrolysate containing medium can lead to achievement of higher production of
lactic acid. In this study 40 mL of actively growing 15 24 h old WEH adapted
Lactobacillus pentosus cells named Lactobacillus pentosus-WEH was inoculated into
bioreactors containing fermentation medium med2 or med4 and set at 37 C, 150 rpm
agitation speed and pH 6.0. The fermentation reaction was allowed to run till
maximum lactic acid concentration was obtained. Generally fermentation of the
adapted L. pentosus strain on WEH containing media showed higher lactic acid yields
76
and productivity compared to WEH fermentations performed with native L. pentosus
strain (Table 5.2). The adapted L. pentosus strain also achieved maximum lactic acid
production in a shorter time as compared to fermentation with the native strain.
Table 5.2: Fermentation of concentrated WEH using native and adapted Lactobacillus
pentosus strain
Fermentation medium
Parameter Strain
med2 med4
Native 43.66 62.45

Maximum lactic acid Concentration ,g/L
Adapted 50.45 63.96
Time required to attain maximum lactic Native 55 75
acid production, h Adapted 45 52
Native 0.72 0.50

Lactic acid yield, g/g
Adapted 0.78 0.54
Native 0.99 1.04

Lactic acid productivity, g/(L h)
Adapted 1.15 1.53
With the adapted L. pentosus strain, batch fermentation of medium med2
(containing 50%WEH (v/v) led to 15.5% increase in lactic acid production as
77
compared to 2.0% increase in lactic acid production in med4 (containing 75%WEH
(v/v) loading). These results show that even with the WEH adapted L. pentosus strain,
fermentation of lactic acid from medium containing high levels of WEH is affected by
product inhibition, which is in agreement with the previous observations made during
this study. Wee and coworkers (2006) reported that adaptation of E. faecalis RKY1 to
wood hydrolysate, cell growth, lactic acid productivity, and lactic acid yield were
increased by 32%, 19%, and 4%, respectively. This implies that E. faecalis RKY1
grown on WEH-based medium might be partially tolerant to inhibitory compounds in
wood hydrolysate. In another study Lorca and coworkers (1998) reported that cellular
activity of Lactobacillus acidophilus could be enhanced by its adaptation to acidic
environments by induction of acid-tolerance. However, the exact nature of the genetic
and biochemical changes occurring during these "adaptations" is not known (Lampen
and Peterjohn, 1951). Such biochemical changes could lead to gene mutation of the
microorganism which enhances the ability of the adapted cells to utilize WEH.
However, there is no evidence at present to indicate whether all the cells can gain the
ability to ferment a given sugar, or only a fraction of the original population is
selected by these procedures.
78
5.3 Discussion and Conclusion
Although wood extract hydrolysate contains high concentrations of mixed sugars,
including xylose, mannose, glucose, arabinose and rhamnose, little research has been
published where wood hemicelluloses was used as carbon source for lactic acid
production. In our study, batch fermentation of different WEH containing media led
to high lactic acid production levels ranging between 43.2 g/L and 65.02 g/L
representing 0.83 g/g and 0.54 g/g product yield, respectively. The obtained lactic
acid production/yield is higher than most values reported in literature. For example,
Walton and co-workers (Walton et al., 2010) using bacteria strain Bacillus coagulans
MXL-9 obtained 33 g/L lactic acid and yield of 0.73 g/g from wood hemicellulose
extracts, Bustos and coworkers reported 46.0 g/L lactic acid concentration and yield
of 0.78 g/g from vine trimming hydrolysate using L. pentosus strain, Iyer et al., (2000)
obtained 0.82 g/g lactic acid yield from acid-treated softwood hydrolysates using L.
casei subsp. Rhamnous strain, Garde et al., (2002) obtained 9 g/L lactic acid
concentration and yield of 0.90 g/g using L. pentosus and 10 g/L lactic acid
concentration and yield of 0.61 g/g using L. brevis from wheat straw hemicellulose
hydrolysates.
In this study we demonstrated that batch fermentation of sugar maple wood
extract hydrolysate based media using L. pentosus yields high productivity of lactic
acid. The strain utilized each component of the sugar mixture, though xylose and
mannose more slowly, of hexoses and pentoses in the fermentation media. In the first
12 h of fermentation, the strain preferably utilized glucose, arabinose, rhamnose and
79
galactose while utilization of mannose and xylose started after 12 h with mannose
being consumed in the next 12 h. However, L. pentosus growth rate and product yield
appeared inhibited by high concentration of produced lactic acid during fermentation
and some residual xylose remained at the highest starting concentration of sugars.
Although L. pentosus strain adaptation to concentrated WEH led to significant
increase in lactic acid productivity and yield for fermentation media containing lower
concentrations of WEH, there was no significant effect on product yields with
fermentation media containing high WEH concentration. The low lactic acid yield
was largely attributed to product inhibition since the known microbial inhibitors
contained in WEH had been removed by the nanofiltration process. The main by-
product, acetic acid, was produced after glucose had been depleted from fermentation
broth.
80
CHAPTER 6: A KINETIC STUDY OF BATCH AND CONTINUOUS
FERMENTATION OF LACTIC ACID FROM SUGAR MAPLE WOOD
EXTRACT HYDROLYSATE
6.1 Introduction
Although the batch fermentation process is a simple and the most commonly
studied method for lactic acid production, lactic acid productivity in batch bioreactors
is often low due to downtime, a long lag phase, and end-product inhibition. Therefore,
a continuous fermentation process may be used to reduce the lactic acid inhibitory
effect and also improve lactic acid productivity. In a continuous fermentation system,
the reactor is initiated in a batch mode and cell growth is allowed until the cells are in
the exponential phase. While the cells are in the exponential phase, the reactor is fed
continuously with the medium and a product stream is withdrawn at the same flow
rate as the feed, thus keeping a constant volume in the reactor (Ezeji et al., 2004).
When referring to continuous culture systems, the terms lag phase is usually
eliminated. In this set-up, one expects the retaining of the exponential growth phase
(Liu et al., 2012). Continuous free cell systems offer comparatively higher
productivities because of the elimination of down time faced in a batch fermentation
process (Qureshi and Ezeji, 2008). In addition, cells can be maintained at a constant
physiological state and growth rate (Buyondo and Liu, 2012; Wee and Ryu, 2009).
Ideally, the growth can be identified as only in exponential phase or in the stationary
phase for a useful chemostat (Liu et al., 2012).
Microbes in nature usually grow under carbon-limited conditions in the
presence of complex mixtures of potential substrates, all of which are usually present
81
at concentrations of only nanograms to micrograms per liter. In batch cultures
containing a mixture of two carbon substrates at high initial concentrations,
microorganisms frequently exhibit diauxic growth. As a rule, the substrate that
supports the highest growth rate is utilized first, while the consumption of other
substrate is repressed (Lendenmann et al., 1996). In batch culture simultaneous
utilization of 'diauxic' carbon substrates occurs when initial substrate concentrations
are low (Egli et al., 1993). This suggests that under natural conditions a heterotrophic
microbe is unlikely to rely on a single carbon compound for growth but that it will
make use of as many as possible of the different substrates available in its
environment (Egli et al., 1993).
Chapter 5 reported batch lactic acid production from wood extract hydrolysate
where Lactobacills pentosus utilized both hexose and pentose hemicellulosic sugars in
each fermentation. It was observed that during batch fermentation Lactobacillus
pentosus first utilized glucose, followed by utilization of the remaining hemicellulosic
sugars galactose, arabinose, rhamnose, mannose and xylose. After the other sugars are
used up, xylose and mannose were consumed with xylose being the most slowly
utilized (Buyondo and Liu, 2011). Several authors have reported similar observations
about batch lactic acid production from hemicellulosic sugars (Walton et al., 2010;
Bustos et al., 2005). A critical look at these studies shows that lactic acid bacteria
simultaneously utilized all of the hemicellulosic sugars to produce lactic acid. Walton
et al. (2010) reported that Bacillus coagulans MXL-9 grown on medium of green
liquor-extracted hardwood consumed all hemicellulosic sugars within 24 h of batch
82
fermentation to obtain 0.94 g/g lactic acid yield. Similar observations were reported
by Bustos et al., (2005) working with Lactobacillus pentosus achieved lactic acid
yield of 0.61 g/g within 24 h of batch fermentation of vine shoots hydrolyzates
medium containing xylose, glucose and arabinose.
Similarly, diauxic growth kinetics has been observed with a chemostat culture
grown on mixed refined sugars. In these studies it was shown that in carbon-limited
chemostat cultures in which the steady-state substrate concentrations are very low,
mixtures of diauxic carbon substrates are utilized simultaneously. This behavior is
referred to as mixed-substrate growth (Lendenmann et al., 1996). Furthermore, in
carbon-limited chemostat cultures the simultaneous utilization of mixtures of carbon
sources results in lower steady-state concentrations of individual substrates than the
concentrations observed during growth with a single substrate (Lendenmann et al.,
1996).
Although the kinetics of single substrate and diauxic growth are well known
for batch fermentation process, little is known about the kinetics of growth of lactic
acid producing microorganisms on the medium containing a mixture of hemicellulosic
sugars in a chemostat culture. In this study we report kinetics of the continuous
production of lactic acid from a mixture of hemicellulosic sugars extracted from sugar
maple woodchips. The effect of bioreactor dilution rate on lactic acid productivity,
residual sugar, biomass production and acetic acid to lactic acid ratio are also
reported. The batch growth kinetics were also studied and are compared to chemostat
culture.
83
6.2.1 Batch fermentation of sugar maple WEH
The WEH adapted L. pentosus-WEH bacterial strain was utilized in the kinetic
study experiments of batch and continuous fermentation of WEH. From Figure 6.1, it
can be observed that of all hemicellulosic sugars contained in sugar maple wood
extract hydrolsate, L. pentosus-WEH preferred glucose and it was depleted in the first
6 hours of batch fermentation. In the same period of the first 6 h of fermentation,
xylose and mannose were also consumed but at a slower rate. At this time L.
pentosus-WEH solely produced lactic acid with no acetic acid detected in the
fermentation broth. L. pentosus-WEH ferments hexose sugars via the Embden-
Meyerhoff-Parnas (EMP) pathway and pentose sugars via the phosphoketolase
pathway (PK). In the EMP pathway one mole of glucose (or hexose sugar) is
catabolyzed to yield two moles of pyruvate, which finally generates two moles of
lactic acid (Bustos et al., 2005; Bailey and Ollis, 1986). In the PK pathway, the
pentose sugar undergoes oxidative reactions to generate one mole of each of
Glyceraldehyde 3-Phosphate (GAP) and acetyl phosphate. GAP is metabolized into
lactic acid by following the same pathway as in the EMP pathway (Bailey and Ollis,
1986). The acetylphosphate has two possible destinations depending on
environmental conditions. In one condition, this molecule successively reduces to
ethanol via acetyl-CoA and acetaldehyde intermediates. In another condition, the
acetyl-phosphate can produce acetate (acetic acid) through the enzyme acetate kinase.
In this type of mixed acid fermentation, the yield coefficient of L-lactate per
84
consumed xylose (pentose sugar) is less than 1.0 mol/mol and the molar ratio of
lactate to acetate is also less than 1.0 (Tanaka et al., 2003). However, in our study
there was no ethanol detected. Figure 6.2 shows the 1D NMR spectrum for lactic acid
and acetic acid contained in WEH fermentation broth.
Gluc
6 30 Gala
Gluc, Mann, Gala, Rham, Arab Conc., g/L
Mann
Arab
5 Rham
Xylo, LA, AA Conc., g/L

Xylo
LA
4 20 AA
2 10
0 0
0 10 20 30
Time, hr
fermentation of sugar maple WEH.
Figure 6.2: 1D NMR for quantification of lactic acid and acetic by using glucosamine
as the internal standard
85
Conversion of hexose and pentose sugars by L. pentosus follows the following
overall stoichiometric equations;
C6H12O6 2C3H6O3 (6.1)
C5H10O5 C3H6O3 + CH3COOH (6.2)
It is worthy to note that the adapted L. pentosus (L. pentosus-WEH) cells
utilized hemicellulosic sugars in somewhat different trend compared to the native
cells. Figure 6.1 shows that by 8 h, mannose had been depleted and still there was no
acetic acid detected in fermentation broth. Acetic acid production was first observed
at 10 h of batch fermentation and by this time there was no arabinose and galactose
detected in the fermentation broth. According to equations (6.1) and (6.2), 1 mole of
hexose sugar generates two moles of lactic acid whereas one mole of pentose sugar
generates 1 mole of lactic acid and 1 mole of acetic acid. By 8 h of batch process
17.85 g/L total sugars (0.038 moles hexoses and 0.028 moles pentoses) had been
consumed in the bioflo 110 reactor with a working volume of 600 mL.
Stoichiometrically we would expect to obtain 0.104 moles LA and 0.028 moles AA.
However, by 8 hr of batch fermentation of sugar maple WEH, L. pentosus had
produced 10.55 g/L (0.07 mol) lactic acid and no acetic acid. Of course, some sugars
were used for cell growth and development. The production of LA as the sole product
could be attributed to the presence of glucose and/or mannose in fermentation broth
during the early stage of batch fermentation of WEH. Under these conditions L.
pentosus may simultaneously catabolyze pentose sugars (arabinose and xylose),
86
hexose sugars (glucose, mannose and galactose), and deoxy 6-carbon (rhamnose)
sugars to produce lactic acid. Therefore, one may infer that in the presence of glucose
and/or mannose both hexose and pentose sugars are catabolyzed to lactic acid
according the following stoichiometry;
C6H12O6 2C3H6O3 (6.3)
3C5H10O5 5C3H6O3 (6.4)
The tendency of microorganisms to simultaneously utilize mixed sugars to
produce lactic acid was previously observed in fermentation of a mixture of refined
sugars. (Yun and Ryu, 2001) reported that Enterococcus faecalis RKY1 grown on a
mixture of glucose and fructose simultaneously consumed both sugars, and the cell
growth and average lactic acid volumetric productivity were higher than when grown
on the individual sugars. However, one of the limitations of mixed sugar cultures is
the tendency to suffer from a catabolite repression effect. Carbon catabolite repression
is a regulatory mechanism in which the expression of genes for the utilization of other
carbon sources is prevented by the presence of a preferred substrate (Yun and Ryu,
2001). Glucose is well known to be a global metabolic regulator in many
microorganisms where it controls the expression of a large number of genes involved
in sugar utilization, gluconeogenesis, mitochondrial biogenesis, and cell cycle
regulation (Yun and Ryu, 2001). Although in our experiment glucose never
completely suppressed the utilization of other hemicellulosic sugars, glucose
87
depletion led to an increase in the rate of utilization of the remaining sugars (Figure
6.1).
Of all the WEH hemicellulosic sugars, xylose and rhamnose were slowly
utilized by L. pentosus- WEH with the latter being depleted by 12 h of fermentation
process whereas xylose utilization required 34 h with residual amount of 6.73 g/L at
the end of batch fermentation. Nonetheless, at the end of batch fermentation the final
lactic acid concentration was 32.52 g/L representing 0.67 g/g LA yield and 0.96 g L-
1 -1
h productivity. The final acetic acid concentration was 20.39 g/L representing 0.42
g/g AA yield, which led to total product yield of 1.08 g/g-total sugars consumed and
AA/LA ratio was 0.62.
6.2.2 Continuous production of lactic acid from WEH
Although traditionally batch fermentation process has been used to produce
lactic acid from refined sugars, starch and some lignocellulosic biomass, it is desirable
to study kinetics of lactic acid production from lignocellulosic biomass via continuous
fermentation process in the ongoing search for a more economic process. In this study
before continuous fermentation was started, the bioreactor was operated in batch
mode for 16 h. Figure 6.3 shows the profiles for production of lactic acid, acetic acid
and sugar utilization during the 16 hr period of batch fermentation operation. From
Figure 6.3 it can be observed that L. pentosus-WEH preferably utilized glucose,
mannose, galactose and arabinose. Xylose and rhamnose were utilized at a slower
rate. By the time continuous fermentation was initiated (at 16 hr), all hemicellulosic
88
sugars had been depleted except for xylose and a very small amount of rhamnose. At
that time the WEH fermentation broth contained 14.82 g/L xylose, 20.72 g/L lactic
acid (LA) and 8.03 g/L acetic acid (AA) and the ratio of AA to LA was 0.39. These
results for sugar utilization, LA and AA production profile confirm the observations
reported in previous section (6.2.1) in batch fermentation of sugar maple WEH.
6 35
Gluc
30
Gluc, Mann, Gala, Rham, Arab Conc., g/L
5 Gala
Mann
25 Arab
Xylo, LA, AA Conc., g/L

4 Rham
Xylo
20 LA
3 AA
15
2
10
1
5
0 0
0 2 4 6 8 10 12 14 16
Time, hr
fermentation of sugar maple WEH before continuous process was initiated.
6.2.3 Effect of dilution rate on lactic acid and acetic acid production
The continuous fermentation process was initiated and allowed to reach steady
state, which was attained after the culture had been replaced by at least five residence
times. During continuous fermentation of sugar maple WEH, hemicellulosic sugars
were simultaneously utilized by L. pentosus-WEH to produce lactic acid as the main
89
product and acetic acid as the by-product. Operating the chemostat under the lowest
dilution rates considered in this work, the prolonged residence times resulted not only
in practically higher xylose consumption but also in higher lactic acid and acetic acid
production. Conversely, the higher dilution rates resulted in short residence times,
increasing the amounts of unconsumed sugars and consequently reducing both yields
and productivities for lactic acid and acetic acid (Table 6.1). Figure 6.4 shows the
results of lactic acid and acetic acid production obtained for steady states achieved
under flow rates of 10, 25, 40 and 50 mL/hr, corresponding to dilution rates of 0.02,
0.05, 0.08 and 0.10 h-1, respectively. Lactic acid production reached a maximum of
27.56 g/L at dilution rate of 0.02 h-1.
Table 6.1: Influence of dilution rate on the production of lactic acid, acetic acid and
residual sugars at steady state
F (mL/h) D (h-1) LA (g/L) QP (g/L h) AA (g/L) AA/LA S (g/L)
10 0.02 27.56 0.55 16.65 0.60 6.02
25 0.05 24.39 1.22 9.92 0.41 19.92
40 0.08 21.17 1.69 7.86 0.37 19.71
50 0.10 23.58 2.36 6.42 0.27 24.34
F: feed flow rate, D: bioreactor dilution rate, LA: lactic acid concentration, AA: acetic
acid concentration, S: residual xylose, QP; volumetric LA productivity.
90
As bioreactor dilution rate was increased, lactic acid production decreased
with a simultaneous increase in the residual xylose content in the effluent stream
(Table 6.1). For all dilution rates investigated, xylose was the only residual sugar and
its content in the bioreactor effluent stream increased with increase of the dilution
rate. In addition, lactic acid and acetic acid productivities increased with an increase
in dilution rate (Figure 6.4). From Figure 6.4 and Table 6.1, it can be observed that
acetic acid production and ratio of produced lactic acid to acetic acid (AA/LA)
decreased with increase in the bioreactor dilution rate. The lower AA/LA ratio is
particularly important during LA purification from fermentation broth. Low AA
concentration would lead to lower LA purification costs.
30
LA
AA
25 Xylo
20
Conc., g/L
15
10
0
0.02 0.04 0.06 0.08 0.10
D, h-1
91
Figure 6.4: The effect of dilution rate (D) on residual xylose (xylo), lactic acid (LA)
and acetic acid (AA) production during steady state continuous fermentation of sugar
maple WEH.
6.3. Discussion and conclusion
In recent years lactic acid has become one of the worlds most valuable
commodities mainly because of its use as a monomer in the production of PLA, a
biodegradable plastic. Traditionally refined sugars and starch have been used as
substrates for biotechnological production of lactic acid but such materials are
expensive and some compete with human food. Lignocellulosic biomass such as sugar
maple woodchips have been identified as potential feedstock for lactic acid
production mainly because lignocellulosic materials are abundant and relatively
cheap. However, for lactic acid to be an economically feasible biorefinery product all
hemicellulosic sugars should be utilized by the chosen production microorganisms to
exclusively produce lactic acid with no or with minimal byproduct.
Although lactic acid bacteria (LAB) are well known to utilize glucose and/or
6C sugars to exclusively produce lactic acid, few LAB can utilize pentose sugars like
xylose to solely produce lactic acid. Most LAB which utilize pentose sugars tend to
produce lactic acid and acetic acid as the main by-product. In this study Lactobacillus
pentosus-WEH simultaneously utilized pentose and hexose sugars via batch and
continuous fermentation processes to produce lactic acid with acetic acid as the main
byproduct. However, in presence of glucose and/or mannose L. pentosus-WEH
92
utilized both 5C and 6C sugars via batch fermentation process to exclusively produce
lactic acid with no acetic acid detected in the fermentation broth. Generally batch
fermentation of sugar maple WEH led to higher AA/LA ratio (0.62) as compared to
continuous fermentation process which ranged between 0.27 and 0.60 depending on
bioreactor dilution rate. The lower AA/LA ratio in the continuous fermentation of
sugar maple WEH can be attributed to the continued supply of WEH feed containing
glucose and/or mannose to the L. pentosus cells in the bioreactor. This phenomenon
can be further supported by the fact that at higher dilution rate/flow rate the AA/LA
ratio was lower than at lower dilution rate/flow rate. Therefore, it can be noted that
glucose presence acts as an inducer of lactic acid production by L. pentosus-WEH via
both batch and continuous fermentation processes. The lower AA/LA ratio offers
great advantage in the purification of lactic acid from the fermentation broth of WEH,
as pure lactic acid is required for the production of PLA.
93
CHAPTER 7: UNSTRUCTURED KINETIC MODELING OF BATCH
PRODUCTION OF LACTIC ACID FROM HEMICELLULOSIC SUGARS
7.1 Introduction
The increase in use of lignocellulosic materials as substrates for biotechnological
production of lactic acid, calls for the development of mathematical kinetic models to
describe the fermentation process of lignocellulosic biomass. Kinetic models provide
a general understanding of the metabolic processes involved in lactic acid production
as well as the basis for the development of better strategies for the optimization and
control of the fermentation process to ensure economic viability. Mathematical
models have been used to predict the influence of fermentation operating parameters
on cell growth rate, cell concentration, substrate utilization rate and lactic acid
production rate (Nandasana and Kumar, 2008). Such models can be used to cautiously
predict the growth of organisms under conditions for which no experiment was
performed (Domann et al., 1996). Various structured (Bajpai-Dikshit et al., 2003)
and unstructured (Nandasana and Kumar, 2008) kinetic models describing
fermentative production of lactic acid by lactic acid bacteria have been reported.
Compared to structured models, unstructured models are much easier to use, and have
proven to accurately describe lactic acid fermentation in a wide range of experimental
conditions and media (Nandasana and Kumar, 2008). In unstructured models, all
cellular components are pooled into a single biomass represented by the total biomass
concentration X (grams of dry weight per liter of medium). Furthermore, in these
models the fermentation process is normally modeled by one reaction only the black
box model. Modeling of the process involves specification of specific growth rate, ,
94
as a function of extracellular concentrations of S (substrate), P (product) and X
(biomass), i.e f S , P, X (Bailey and Ollis, 1986).
When microorganisms are grown on medium containing multiple substrates, such
as the case in nature, different growth phenomena are observed. These include growth
due to (i) sequential utilization of substrates, (ii) simultaneous consumption of
substrates, and (iii) co-metabolism of substrates (Bajpai-Dikshit et al., 2003). When
faced with a choice to take up multiple substrates, microorganisms tend to regulate the
uptake of the less-preferred substrate. This regulation is achieved, either by control at
the metabolic level (i.e., at the level of enzymatic activity) or at the genetic level (i.e.,
at the level of enzyme synthesis) (Bajpai-Dikshit et al., 2003). In our previous studies
we reported successful production of high lactic acid yields from fermentation of
sugar maple wood extract hydrolysate using Lactobacillus pentosus ATCC 8401
bacterial strain (Buyondo and Liu, 2011). Lactobacillus pentosus cells nearly
simultaneously utilized both 5C and 6C hemicellulosic sugars, though at different
rates, of sugar maple wood extract hydrolysate contained in fermentation broth.
During the LA fermentation process, experimental data is collected over a
particular time series. Basically such data describes the substrate utilization rate, cell
growth rate and product formation rate. To solve such rate equations and also obtain
best fitting kinetics parameters the variance of experimental data around the
regression model predictions is minimized. A viable approach is to use a general
differential equation solver (or integrator) to compute a variable for a particular time
series and then minimize the variance associated with predicted and experimental
95
values (Liu, 2013). Excel software is an easy to use but powerful program which
easily performs regression analysis on spreadsheet data. Spreadsheets have built-in
optimization routines, allow interface of user-defined visual basic for applications
(VBA) routines and also have graphical capabilities to make the regression analysis
visually at every step of the way. In this study a VBA routine ODEXLIMS was
employed to solve a set of ordinary differential equations for substrate utilization rate,
biomass growth rate and product formation rate. ODEXLIMS function solves a set of
first order ordinary differential equations using linearly implicit midpoint method with
smoothing and extrapolation (Liu, 2013). The solver program in Excel was used to
minimize the variance associated with predicted and experimental values and at the
same time obtain the best fitting kinetic parameters for Lactobacillus pentosus
substrate utilization rate, biomass growth rate and product formation rate.
Although there are several lactic acid production kinetic models reported in
literature, most of these models used refined sugars or starch as fermentation
substrate. In this study we report development of unstructured mathematical model
capable of predicting cell growth, substrate utilization and lactic acid production rates
during the batch fermentation of sugar maple wood extract hydrolysate and to test the
validity of the model using experimental data.
7.2 Mathematical Model
In developing unstructured mathematical model, the following assumptions were
made:
96
1. Lactobacillus pentosus cells simultaneously utilize sugar maple wood extract
hemicellulosic sugars, though at different rates, contained in fermentation
broth.
2. Total product concentration is the sum of lactic acid (LA) and acetic acid
(AA) and is represented as single product, P .
3. End product inhibition is due to both LA and AA.
The most widely used unstructured models to describe cell growth are the Monod
kinetic model for a known limiting substrate and the logistic equation. In certain
cases, microbial processes do not follow the classical kinetic model of substrate-
limited biomass growth and product formation proposed by Monod in 1949. When
limiting substrate is unknown, the logistic equation, a substrate-independent model, is
used as an alternative empirical function. A generalized cell growth model can be
written as:
n a
X
rX m X 1 (7.1)
Xm
where m is the maximum specific growth rate (h1), X m is the stationary population
size, or upper biomass concentration, above which bacteria do not grow, n and a are
parameters. When n a 1 equation 7.1 reduces to a logistic growth model
(Equation 7.2). Logistic equation well describes biomass growth for both exponential
and stationary phases,
X
rX m X 1 (7.2)
Xm
97
The rate of total extracellular product formation was based on modified
Michaelis-Menten or the Monod equation. Monod model, however, fails to reflect the
effects of product inhibition and environmental conditions. A good model should
account for such factors in order to derive a proper representation of the culture
kinetics. In this study the Michaelis-Menten equation for rate of product formation
was modified to account for end product inhibition of lactic acid and acetic acid
fermentation.
SX
Pm ax
rP X (7.3)
P
KP S
K PI
where pmax is the maximum specific rate of product formation (h-1), S is the
concentration of the limiting substrate for microbial growth (g/L), KP (g/L) and KPI
are saturation constant and product inhibition constant on substrate utilization and
lactic acid production, respectively, whereas (h-1) is a kinetic parameter
During batch fermentation process microbial cells utilize sugars mainly for
biomass growth and product formation. Therefore, the uptake rate of hemicellulosic
sugars from batch bioreactor by Lactobacillus pentosus-WEH is defined as;
rX rP
rS (7.4)
YX / S YP / S
Where rX is the biomass growth rate, rP is the product formation rate, YX/S and YP/S
are yield coefficients for biomass and product on substrate (g/g).
98
2.6 Parametric Estimation
To estimate kinetic parameters, it was required to search for values of these
parameters for which predicted values of X , S and P are closely equal to the
experimental values, X exp , S exp , and Pexp within acceptable tolerance at all times
during fermentation process. Kinetic parameters were determined by simultaneously
solving equations 7.2, 7.3 and 7.4 using ODEXLIMS function with Excel (Liu,
2013). ODEXLIMS function solves a set of first order ordinary differential equations
using linearly implicit midpoint method with smoothing and extrapolation. Best
fitting parameters were determined using Excel solver by minimizing the variance
between the predicted and the experimental values.
7.3 Results and Discussion
7.3.1 Estimation of Kinetic Parameters
Batch production of lactic acid was performed at two different levels of
concentrated WEH with total hemicellulosic sugar concentration of 40.0 g/L and 55.0
g/L. The experimental data obtained from batch fermentation of WEH were used for
the determination of kinetic parameters. The theoretical response for each of the
parameters was predicted by solving ODE equations 7.2, 7.3 and 7.4 for the
dependent variables X , S and P using ODEXLIMS function developed in Excel. The
kinetic parameter values, which resulted in the minimum variance between the
experimental and predicted data were determined and are listed in Table 7.1.
99
From Table 7.1 it can be observed that the estimated parameters were the same for
both initial substrate concentrations of 40.0 g/L and 55.0 g/L apart from maximum
specific product formation rate ( pmax) and product yield coefficient (YP/S). This
implies that the maximum specific product formation rate and product yield of
Lactobacillus pentosus-WEH depend on the initial total hemicellulosic sugars in
fermentation broth. The maximum specific product formation rate, pmax, was
estimated to be 33.9 h-1 and 25.9 h-1 for initial substrate concentration of 40.0 g/L and
55.0 g/L, respectively. Estimated product yield was 1.24 g/g and 1.34 g/g for initial
substrate concentration of 40.0 g/L and 55.0 g/L, respectively. The estimated
maximum specific growth rate, max and maximum biomass concentration, X m were
found to be 0.55 h-1 and 2.59 g/L. Maximum specific growth rate ( max ) is an
empirical coefficient to the Logistic equation. It differs between microbial species and
based on the ambient environmental conditions.
100
Table 7.1: Optimum kinetic parameter values for kinetic model of LA production
from WEH by Lactobacillus pentosus
Kinetic Parameter Initial substrate concentration
40.0 g/L 55.0 g/L
max (h-1) 0.55 0.55
X m (g/L) 2.59 2.59
pmax (h-1) 33.9 25.9
K P (g/L) 176.0 176
K PI 0.04 0.04
Yx /s (g/g) 0.46 0.46
Yp /s (g/g) 1.24 1.34
(h-1) 0.178 0.178
The values of product inhibition constants on substrate utilization K P and lactic
acid production K PI were found to be 176.0 g/L and 0.04, respectively. Lactic acid
fermentation process suffers from end product Inhibition which limits not only
microbial metabolic rates but also final product concentrations (Buyondo and Liu,
2011; Datta and Henry, 2006). Therefore, a good model for LA production must take
into account the effects of end product inhibition and, substrate limitation as well as
growth conditions. The drawbacks of the end product inhibition include: (1) slow
101
metabolic rate which leads to requirement of a large reactor size and/or a long time to
process a given quantity of substrate; (2) the inhibition also causes a dilute final
product concentration, which raises the energy costs for product separation and
recovery (Yang and Tsao, 1994). In the fermentation processes product inhibition is
caused by both undissociated and dissociated lactic acid and acetic acid. In our study
of batch fermentation of sugar maple WEH the fermentation broth pH was maintained
at 6.0 by automatic addition of 5M NaOH. Therefore, product inhibition was mainly
due to the electrolytes such as lactate and acetate ions. Biomass yield coefficient
(YX/S) was independent of the initial total sugar concentration and was estimated to
be0.46 g/g.
7.3.2 Comparison of kinetic parameter values
Kinetic parameter values obtained in this study were compared with the kinetic
parameter values reported for lactic acid production from different substrates using
different lactic acid bacteria (Table 7.2). From Table 7.2, it can be observed that the
kinetic parameters obtained in this study are within the range of values reported in the
literature. The difference in the kinetic model parameters obtained between these
studies may be attributed to the use of different substrates, different bacteria strains,
different fermentation conditions and use of different kinetic models. Although at the
time of this study there was no publication about kinetic modeling of lactic acid
production from wood hemicellulosic sugars, the model developed in this study
simulated well lactic acid and acetic acid productions from hemicellulosic sugars.
102
Table 7.2: Kinetic parameters of lactic acid fermentation reported in literature
Microorganism Substrate and max pmax KP Yp/s Yx/s Reference
initial
(h-1) (h-1) (g/L) (g/g) (g/g)
concentration
Lactobacillus Wood extract 0.55 33.9 176 1.24 0.46 This work
pentosus hydrolysate,
55 g/L
Lactococcus Glucose, 21 g/L 0.687 - - 1.601 0.139 Vazquez
lactis and Murado,
2008
Lactobacillus Whey lactose 0.265 - - 0.682 - Altok et al.,
casei 2006
7.3.3 Experimental data fitting
The developed VBA program was used to solve ODE equations 7.1 7.3 and
simulate the prediction of substrate utilization rate, rS , cell growth rate, rX and
product formation rate, rP during the batch fermentation process of sugar maple
wood extract hydrolysate. Figures 7.1, 7.2 and 7.3 respectively present the curve fitted
prediction and experimental data for cell growth rate, substrate utilization rate and
product formation rate at different initial substrate concentrations. From Figures 7.1,
7.2 and 7.3 it can be observed that the developed model predicts well the substrate
utilization rate, cell growth rate and product formation rate to yield good agreement
103
between the model predictions and the experimental data.
3.5
40 g/L Model
40 g/L Expt
3.0 55 g/L Model
55 g/L Expt
2.5
Conc., g/L
2.0
1.5
1.0
0.5
0.0
0 5 10 15 20 25 30
Time, hr
Figure 7.1: Curve fitting of the predicted data to experimental data for biomass
growth, for initial substrate concentrations of 40 g/L and 55 g/L total hemicellulosic
sugars. Solid line kinetic model; Data points - Experiment
104
60
40 g/L Model
40 g/L Expt
50 55 g/L Model
55 g/L Expt
40
Conc., g/L
30
20
10
0
0 5 10 15 20 25 30
Time, hr
Figure 7.2: Curve fitting of the predicted data to experimental data for product
formation for initial substrate concentrations of 40 g/L and 55 g/L total hemicellulosic
105
60
40 g/L Model
40 g/L Expt
50 55 g/L Model
55 g/L Expt
40
Conc., g/L
30
20
10
0 5 10 15 20 25 30
Time, hr
Figure 7.3: Curve fitting of the predicted data to experimental data for substrate
utilization for initial substrate concentrations of 40 g/L and 55 g/L total hemicellulosic
106
The closeness between experimental and predicted data was further demonstrated
by computing the squared Pearson correlation coefficient, R 2 (Table 7.3).
Table 7.3: Squared Pearson correlation coefficients for biomass growth, substrate
utilization and product formation
Initial substrate R2
concentration, g/L
Biomass Substrate Product
40.0 0.99 0.97 0.99
55.0 0.98 0.99 0.98
From Table 7.3, it can be observed that the squared Pearson correlation
coefficients ranged between 0.97 and 0.99, which indicated that the developed model
was able to predict the experimental results with a high degree of accuracy.
7.4 Discussion and conclusion
The growth of microorganisms can be best imagined as the increase of cell
material expressed in terms of mass (or cell numbers). Microbial cell growth is the
result of a highly coordinated series of enzymatically catalyzed biological steps.
Optimal expression of growth kinetics depends on optimal maintenance of the
transport of the necessary nutrients in the medium to the cell surface, rate of mass
transfer from the medium into the cells and environmental parameters (such as
temperature and pH) (Ghaly and El-Taweel, 1997). The simultaneous utilization of
107
pentose and hexose sugars by Lactobacillus pentosus-WEH represents how
microorganisms survive in natural environment where pentose and hexose sugars do
exist in small or micro amounts. Therefore, in this study substrate limitation was due
to the total concentration of all sugars contained in the sugar maple wood extract
hydrolysate.
In conclusion, the study presented in this chapter a kinetic model describing batch
fermentation of wood extract hydrolysate by Lactobacillus pentosus-WEH to produce
lactic acid was developed by kinetic equations of biomass growth, product formation
and substrate utilization rates. Biomass growth rate was modeled by logistic equation
whereas total product formation kinetic equation was based on modified Michaelis-
Menten and Monod models. Substrate utilization rate equation described substrate
utilized for cell growth and product formation. The model performed satisfactorily in
predicting the biomass growth rate, product formation rate and substrate utilization
rate with high degree of accuracy (R2 = 0.97 - 0.99).
108
CHAPTER 8: CONCLUSION AND RECOMMENDATION
8.1 Conclusion
In this study use of sugar maple woodchips hemicelluloses as an alternative
feedstock for lactic acid production was investigated. The major innovations of this
research included: (1) kinetic study of utilization of pentose and hexose sugar maple
hemicellulosic sugars for lactic acid production by Lactobacillus pentosus via batch
and continuous fermentation processes and (2) development of unstructured kinetic
model to describe batch lactic acid production from sugar maple wood
hemicelluloses. The main findings obtained from this research are summarized below:
8.1.1 Hot water extraction of hemicelluloses from sugar maple woodchips
Hot water extraction technique was demonstrated to successfully extract
hemicelluloses from sugar maple woodchips. At mild extraction conditions, with
temperatures at 160C and 2 hr extraction period favored the recovery of high-molar
mass xylooligomers in the water extract. Increasing the severity of the extraction
process would lead to the degradation of sugars to microbial inhibitory compounds
like furfural and HMF, which could as well reduce the sugars yield. Hot water
extracted sugar oligomers were hydrolyzed with 1.5% wt concentrated sulfuric acid to
generate fermentable monomeric sugars. The total monomeric sugars increased from
60.2 g/L before acid hydrolysis to 222.3 g/L after acid hydrolysis. The wood extract
hydrolysate was subjected to nanofiltration membrane system to fraction the desired
sugars from other components like acetic acid and furfurals. Upon nanofiltration
membrane processing, water and other low molecular weight compounds, (such as
109
acetic acid, formic acid and furfurals), were preferentially sent to the permeate stream,
whereas sugar monomers and oligomers along with high molecular weight
compounds were concentrated in the concentrate stream. Xylose was the main sugar
component contributing about 70% of the total sugars in the sugar maple wood extract
hydrolysate.
8.1.2 Batch production of lactic acid from wood extract hydrolysate by

lactobacillus pentosus
Concentrated wood extract hydrolysate was diluted with deionized water to
obtain four different sugar concentrations: 54.08 g/L, 61.47 g/L, 94.10 g/L and 129.50
g/L, labeled media med1, med2, med3 and med4, respectively. After 55 h of
fermentation in a 1 L bioreactor at 37 C and pH 6.0, medium med1 had the highest
lactic acid yield (Yp/s) of 0.83 g-lactic acid/g-sugar, representing about 97.3% of
theoretical yield. In the first 12 h of batch fermentation, the strain preferably utilized
glucose, arabinose, rhamnose and galactose while utilization of mannose and xylose
started after 12 h with mannose being consumed in the next 12 h. Acetic acid was
produced as the main by product up to 49% of the obtained lactic acid concentration.
It was observed that acetic acid production started after depletion of glucose.
Lactobacillus pentosus cells suffered from end product inhibition. Adaptation of L.
pentosus strain to concentrated wood extract hydrolysate led to 10 h reduction in
fermentation time and 15.5% increase in lactic acid production for fermentation media
containing lower concentrations of WEH, there was no significant effect on product
yields with fermentation media containing high WEH concentration. The low lactic
110
acid yield was largely attributed to product inhibition since known microbial
inhibitors contained in WEH had been removed by nano-filtration process
8.1.3 A kinetic study of batch and continuous production of lactic acid
A kinetic study was carried out to compare lactic acid production from sugar
maple wood extract hydrolysate via continuous and batch fermentation processes
using WEH adapted Lactobacillus pentosus-WEH cells. Operating a one stage
continuous fermentation process high lactic acid (LA) productivity of 2.36 g L-1h-1
was obtained at high dilution rate of 0.1 h-1. At lower dilution rate of 0.02 h-1, the LA
productivity was lower (0.55 g L-1h-1) but the steady state lactic acid concentration
(27.56 g/L) was higher than at high dilution rate (23.68 g/L). Compared to continuous
fermentation process, at the end of 34 h batch fermentation process the final LA
concentration was 32.52 g/L representing 0.67 g/g LA yield and 1.59 g L-1h-1
productivity. In the presence of glucose and/or mannose L. pentosus-WEH cells
exclusively produced lactic acid from sugar maple WE H. The final acetic acid (AA)
concentration in batch process was 20.39 g/L representing 0.42 g/g AA yield, which
led to total product yield of 1.08 g/g-total sugars consumed. However, continuous
fermentation process led to lower AA/LA ratios ranging between 0.27 and 0.60 as
compared to batch fermentation process which had 0.62 AA/LA ratio. For both batch
and continuous fermentation processes all sugar maple wood hemicellulosic sugars
were utilized with glucose being the preferred sugar whereas the rest of sugars were
simultaneously utilized. In both batch and continuous fermentation processes xylose
was the only detected residual sugar. For continuous fermentation process residual
111
xylose concentrations ranged between 24.34 g/L and 19.02 g/L depending on the
dilution rate whereas for batch process there was minimal residual xylose at 6.73 g/L
concentration.
8.1.4 Unstructured kinetic modeling of batch production of lactic acid
An unstructured kinetic model was developed to describe batch lactic acid
production from hemicellulosic sugars using L. pentosus-WEH. The model accounted
for cell growth rate, total hemicellulosic sugars utilization rate, product (lactic acid
and acetic acid) formation rate and end product inhibition. Kinetic parameters were
determined by a Visual Basic Application (VBA) routine ODEXLIMS to solve a set
of ordinary differential equations for biomass growth rate, product formation rate and
substrate utilization rate and minimizing the variance between experimental and
predicted values using Excel solver. Kinetic parameters max, KP, KPI, YX/S and
were found to be independent of initial total sugar concentration and estimated as=
0.55 h-1,176.0 g/L, 0.04, 0.46 g/g, and 0.178 h-1. Kinetic parameters pmax and Yp/s
depended on the initial total sugar concentration. For initial total sugar concentration
of 40.0 g/L, pmax and Yp/s were estimated to be 33.9 g/L and 1.24 g/g, respectively.
For initial total sugar concentration of 55.0 g/L, pmax and Yp/s were estimated to be
25.9 g/L and 1.34 g/g, respectively. The developed model performed satisfactorily for
predicting the transient responses of biomass growth, product formation and substrate
utilization with squared Pearson correlation coefficient (R2) ranging between 0.97 and
0.99 for the initial substrate (total hemicellulosic sugar) concentrations of 40 g/L and
55 g/L.
112
8.2 Recommendations for future work
To further economically investigate the feasibility of utilization of wood
hemicelluloses as an alternative feedstock for lactic acid production, it would be
necessary to study the methods of separation and purification of lactic acid from
fermentation broth. Potential methods would include ion exchange, esterification and
membrane technologies.
Lactic acid kinetic model could further be improved by studying the effect of
individual sugars, pH and temperature on kinetic parameters. It is also recommended
to develop a structured model for batch lactic acid production from wood extract
hydrolysate and compare the obtained kinetic parameters to unstructured kinetic
model.
113
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Appendices
Appendix A: Standard curve for Lactobacillus pentosus biomass concentration
4.00
y = 0.2338x - 0.183
3.50 R = 0.9987
3.00
2.50
Conc., g/L2.00
1.50
1.00
0.50
0.00
0 5 10 15 20
Absorbance, 600 nm
Appendix B: Continuous fermentation calibration curve for influent and effluent
pumps
30
y = 0.5x
25 R = 1
20
Pump set
15
point, %
10
0
0 10 20 30 40 50 60
Flow rate, mL/hr
121
Appendix C: Sugar concentration calibration curves
500 y = 28.478x
450 -Arabinose R = 0.9978
400
350
300
Peak
250
Area
200
150
100
50
0
0 2 4 6 8 10 12 14 16
Conc., g/L
250
-Galactose y = 12.542x
200 R = 0.9985
150
Peak
Area
100
50
0
0 5 10 15 20
Conc., g/L
122
120
y = 6.3518x
-Glucose R = 0.9976
100
80
Peak
Area 60
40
20
0
0 5 10 15
Conc., g/L
450
-Rhamnose y = 27.931x
400
R = 0.9981
350
300
Peak 250
Area 200
150
100
50
0
0 2 4 6 8 10 12 14 16
Conc., g/L
123
160
-Mannose y = 9.4683x
140 R = 0.9988
120
100
Peak
80
Area
60
40
20
0
0 2 4 6 8 10 12 14 16
Conc., g/L
160
-Xylose y = 9.595x
140 R = 0.9988
120
100
Peak 80
Area
60
40
20
0
0 2 4 6 8 10 12 14 16
Conc., g/L
124
RESUME
John Paul Buyondo
1256 North McLean Blvd, Memphis, TN, 38018,
(315) 420-7074 jpbuyondo@buckman.com.
Skills
Fermentation of lignocellulosic and non-lignocellulosic biomass to lactic acid,
ethanol and butanolkinetics of enzymatic and acid hydrolysis of biomass. ,.
Fungi, yeast and bacteria cell growth kinetics, aerobic and anaerobic
fermentation processes, bioreactor design and operation.
Instrumentation NMR, HPLC, GC, and UV-VIS Spectrophotometer.
Education
PhD. Bioprocess Engineering .. Aug. 2009 Dec. 2013
State University of New York, College of Environmental Science and Forestry
MS. Chemical Engineering Sept. 2007 July 2009
Xiamen University, Xiamen, China
Work Experience
Biotechnology Research Specialist Dec. 2012 present
Buckman International Laboratories, Memphis, TN
Design and execution of a set of in-situ 5 L 20 L batch fermentation

experiments for production of enzymes using fungi and yeast cells.
Development of enzyme assays.
Formulation of enzyme based products to be used in water, leather and pulp
and paper industries.
Work with the global marketing team to address customers concerns about
enzyme based products.
Process Engineer Intern May July 2012

Sunoco Ethanol Plant, Fulton, NY
Designed and executed a set of in-situ process experiments for optimization of
alpha amylase enzyme dosage and liquefaction pH and process temperature for
efficient starch conversion yield from corn mash.
Determined the daily steam production efficiency of boilers and steam
requirements for upstream and downstream processes.
Designed an excel macro spreadsheet that tracks the daily utility and
consumable material costs in the ethanol plant.
Worked with the plant engineer to investigate an economical feasibility to
establish a profitable process to make biodiesel onsite and also evaluated the
return on investment.
Academic Experience
Teaching Assistant Aug. 2009 May 2012
Department of Paper and Bioprocess Engineering, SUNY - ESF
Teaching assistant for the classes; bioprocess engineering laboratory (PBE
440), computing methods for engineers and physical scientists (GNE 106),
principles of mass and energy balances (PSE 370).
125
Conducted students review sessions for the topics covered in class.
Graded students assignments and advised them on their performances.
Mentored students to solve engineering problems by using mathcad, matlab
and excel visual basic computing software.
Graduate Research
Hot water extraction and NMR quantification of hemicelluloses from woody
biomass.
Developed and optimized wood hydrolysate based medium for bacterial lactic
acid production in shake flasks and laboratory scale bioreactors of capacity 1
10 L.
Extracted the -1,3-glucanase DNA gene from yeast cells, inserted it into a
plasmid and expressed the protein in E. coli (Masters Thesis).
Accomplishments
Award
Renata Marton award for outstanding graduate research student at Department
of Paper and Bioprocess Engineering, SUNY-ESF. Nov. 2012.
Peer reviewed journal articles
Buyondo J.P and Liu S. Unstructured Kinetic Modelling of Batch
Production of lactic acid from hemicellulosic sugars. Final acceptance
granted on March 23, 2013 for publication in Journal of Bioprocess
Engineering and Biorefinery,
Buyondo, J.P and Liu, S. Processes and bioreactor designs for butanol
production from lignocellulosic biomass. Journal of Bioprocess Engineering
and Biorefinery, 2012; 1:53 - 68
Buyondo, J.P and Liu, S. Lactic acid production by Lactobacillus pentosus
from wood extract hydrolysates. Journal of Science and Technology for
Forest Products and Processes, 2011; 1: 38 - 40.
Book Chapter
Liu S., Wang YG., Buyondo J.P, Wang YN, and Garver M. Biochemical
conversion. In Stuart P.R and El-Halwagi M. Integrated Biorefineries:
Design, Analysis and Optimization, 2013 (PP 591 650). Taylor & Francis,
Boca Raton, FL, USA.
126

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KINETIC STUDIES OF LACTIC ACID PRODUCTION FROM WOOD

EXTRACT HYDROLYSATE VIA BATCH AND CONTINUOUS

John Paul Buyondo

Approved: Department of Paper and Bioprocess Engineering

All rights reserved

INFORMATION TO ALL USERS

to aim higher and strive for excellence throughout my academic program. I am

Many thanks go to my group members: Alan Shupe, Karin Arens, Yang

Christopher Wood for his support with production of hemicelluloses experimental

achieved. Their unconditional love and unending support of my personal endeavors

program. I will never be able to express enough gratitude to my parents. My greatest

tremendous happiness that I could never imagine.

List of Tables ................................................................................................................. x

List of Figures ............................................................................................................... xi

List of Abbreviations .................................................................................................. xiv

CHAPTER 1: INTRODUCTION .................................................................................. 1

1.1 Introduction .............................................................................................................. 1

1.2 Objectives of the study............................................................................................. 3

1.3 Dissertation outline .................................................................................................. 4

CHAPTER 2: LITERATURE REVIEW ....................................................................... 6

2.1 Introduction .............................................................................................................. 6

2.2 Lactic Acid Applications ......................................................................................... 7

2.2.1 Lactic Acid Applications in Food Industry ........................................................... 7

2.2.3 Lactic Acid Applications in Chemical Industry.................................................... 9

2.2.4 Lactic Acid Applications in Poly (lactic acid) Synthesis ...................................... 9

2.3 Why biorefinery? ................................................................................................... 11

2.4 Wood Chemical Composition and Structure ......................................................... 13

2.4.1 Cellulose ............................................................................................................. 14

2.4.2 Hemicelluloses .................................................................................................... 14

2.4.2.1 Glucomannan ................................................................................................... 15

2.4.2.1 Xylan ................................................................................................................ 16

2.4.3 Lignin .................................................................................................................. 17

2.4.4 Extractives and Inorganics .................................................................................. 18

2.5.1 Hot water extraction of woody biomass ............................................................. 20

2.5.1.1 Advantages of Hot Water Extraction ............................................................... 21

2.5.1.2 Disadvantages of Hot Water Extraction .......................................................... 22

2.5.2 Organosolv Pretreatment of Woody Biomass..................................................... 22

2.5.3 Acid pretreatment................................................................................................ 24

2.5.4 Steam Explosion Pretreatment ............................................................................ 25

2.6 Advantages of using Wood Hemicelluloses as Substrate for Lactic Acid

2.6.1 Abundant/Readily Available ............................................................................... 26

2.6.2 Current cost comparison ..................................................................................... 27

2.6.3 Non food competitive ......................................................................................... 27

2.6.4 Renewable and environmentally friendly ........................................................... 28

2.6.5 Contain vital salts................................................................................................ 28

2.7.1 Microbial growth Inhibitory compounds ............................................................ 29

2.8 Lactobacillus pentosus ........................................................................................... 30

2.8.1.1 Embden-Meyerhoff-Parnas Pathway ............................................................... 31

2.8.1.2 Pentose Phosphate Pathway ............................................................................. 33

2.8.2 Nutrient requirements for Lactobacillus pentosus .............................................. 34

2.9 Using NMR to Quantify Fermentation Substrates and Metabolites ...................... 35

CHAPTER 3: MATERIALS AND METHODS ......................................................... 39

3.5 Microorganisms and seed culture preparation ....................................................... 42

3.5.1 Adaptation of Lactobacillus pentosus cells to concentrated WEH ..................... 43

3.6 Batch fermentation of WEH to produce lactic acid ............................................... 44

3.7 Continuous Fermentation ....................................................................................... 45

3.8 Sterilization of media components......................................................................... 46

3.9 Sample preparation for NMR analysis ................................................................... 47

3.10 NMR Spectroscopy .............................................................................................. 47

3.11 Quantitative NMR Analysis. ................................................................................ 48

CHAPTER 4: PRODUCTION OF HEMICELLULOSIC SUGARS FROM SUGAR

4.1 Introduction ............................................................................................................ 50

4.2 Results and discussion ........................................................................................... 51

4.2.1 Hot water extraction of sugar maple woodchips................................................. 51