You are on page 1of 145

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

All rights reserved

INFORMATION TO ALL USERS


The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a complete manuscript
and there are missing pages, these will be noted. Also, if material had to be removed,
a note will indicate the deletion.

UMI 3614766
Published by ProQuest LLC (2014). Copyright in the Dissertation held by the Author.
Microform Edition ProQuest LLC.
All rights reserved. This work is protected against
unauthorized copying under Title 17, United States Code

ProQuest LLC.
789 East Eisenhower Parkway
P.O. Box 1346
Ann Arbor, MI 48106 - 1346
2013
Copyright
John Paul Buyondo
All rights reserved
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 Results and discussion ........................................................................................... 67

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 Results and discussion ........................................................................................... 84

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

Figure 5.2: Sugar utilization and acids production profiles during fermentation of

med4: ........................................................................................................................... 68

Figure 5.3: Sugar utilization and acids production profiles during fermentation of

med1............................................................................................................................. 71

Figure 5.4: Sugar utilization and acids production profiles during fermentation of

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

Figure 6.3: Sugar utilization, lactic acid and acetic production profiles during batch

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
Rhamnose Mannose Galactose Glucose Arabinose Xylose

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
Rhamnose Mannose Galactose Glucose Arabinose Xylose

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 Results and discussion

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
Figure 5.3: Sugar utilization and acids production profiles during fermentation of

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)

Figure 5.4: Sugar utilization and acids production profiles during fermentation of

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 Results and discussion

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

Figure 6.1: Sugar utilization, lactic acid and acetic production profiles during batch
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

Figure 6.3: Sugar utilization, lactic acid and acetic production profiles during batch

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

sugars. Solid line kinetic model; Data points - Experiment

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

sugars. Solid line kinetic model; Data points - Experiment

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
References

Altok, D., Tokatl, F., & Harsa, S. (2006) Kinetic modelling of lactic acid production
from whey by lactobacilluscasei (NRRL B-441). Journal of Chemical
Technology and Biotechnology J Chem Technol Biotechnol. 81, 1190-1197.

Alvira, P., Toms-Pej, E., Ballesteros, M., & Negro, M. J. (2010) Pretreatment
technologies for an efficient bioethanol production process based on enzymatic
hydrolysis: A review. Bioresource technology. 101, 4851-4861.

Amidon, T. E., Wood, C. D., Shupe, A. M., Wang, Y., Graves, M., & Liu, S. (2008)
Biorefinery: Conversion of woody biomass to chemicals, energy and materials.
Journal of Biobased Materials and Bioenergy. 2, 100-120.

Amidon, T. E., & Liu, S. (2009) Water-based woody biorefinery. Biotechnology


Advances. 27, 542-550.

Bailey, J., & Ollis, D. (1986) Biochemical engineering fundamentals. McGraw Hill,
New York

Bajpai-Dikshit, J., Suresh, A. K., & Venkatesh, K. V. (2003) An optimal model for
representing the kinetics of growth and product formation by lactobacillus
rhamnosus on multiple substrates. Journal of Bioscience and Bioengineering. 96,
481-486.

Borrega, M., Nieminen, K., & Sixta, H. (2011) Degradation kinetics of the main
carbohydrates in birch wood during hot water extraction in a batch reactor at
elevated temperatures. Bioresource technology. 102, 10724-10732.

Bustos, G., Moldes, A. B., Cruz, J. M., & Dominguez, J. M. (2005) Influence of the
metabolism pathway on lactic acid production from hemicellulosic trimming vine
shoots hydrolyzates using lactobacillus pentosus. Biotechnology progress. 21,
793-798.

Bustos, G., de la Torre,N., Moldes, A. B., Cruz, J. M., & Dominguez,J. M. (2007)
Revalorization of hemicellulosic trimming vine shoots hydrolyzates trough
continuous production of lactic acid and biosurfactants by L. pentosus. Journal of
Food Engineering. 78, 405-412.

Buyondo, J. P., & Liu, S. (2012) Processes and bioreactor designs for butanol
production from lignocellulosic biomass Journal of Bioprocess Engineering and
Biorefinery. 1, 53-68.

114
Buyondo, J. P., & Liu, S. (2011) Lactic acid production by lactobacillus pentosus
from wood extract hydrolysate. Journal of Science & Technology for Forest
Products and Processes. 1, 38-47.

Datta, R., & Henry, M. (2006) Lactic acid: Recent advances in products, processes
and technologies - A review. Journal of Chemical Technology and
Biotechnology. 81, 1119-1129.

Dien, B. S., Nichols, N. N., & Bothast, R. J. (2002) Fermentation of sugar mixtures
using escherichia coli catabolite repression mutants engineered for production of
L-lactic acid. Journal of Industrial Microbiology and Biotechnology. 29, 221-
227.

Domann, M. U., Vogel, R. F., & Hammes, W. P. (1996) Mathematical description of


the growth of lactobacillus sake and lactobacillus pentosus under conditions
prevailing in fermented sausages. Applied Microbiology and Biotechnology. 46,
334-339.

Egli, T., Lendenmann, U., & Snozzi, M. (1993) Kinetics of microbial growth with
mixtures of carbon sources. Antonie van Leeuwenhoek, International Journal of
General and Molecular Microbiology. 63, 289-298.

Ek M., Gellerstedt G. & Henriksson G. Wood chemistry and wood biotechnology


(2009). Walter de Gruyter GmbH & Co. KG, Berlin

Ezeji, T. C., Qureshi, N., & Blaschek, H. P. (2004) Butanol fermentation research:
Upstream and downstream manipulations. Chemical Record. 4, 305-314.

Ezeji, T., Qureshi, N., & Blaschek, H. P. (2007) Butanol production from agricultural
residues: Impact of degradation products on Clostridium beijerinckii growth and
butanol fermentation. Biotechnology and bioengineering. 97, 1460-1469.

Fengel, D. and Wegner, G. (1989) Wood: chemistry, ultrastructure, reactions. Water


de Gruyter & Co., Berlin Ch 5 pp 125

Garde, A., Jonsson, G., Schmidt, A. S., & Ahring, B. K. (2002) Lactic acid production
from wheat straw hemicellulose hydrolysate by lactobacillus pentosus and
lactobacillus brevis. Bioresource technology. 81, 217-223.

Garlotta, D. (2001) A literature review of poly(lactic acid). Journal of Polymers and


the Environment. 9, 63-84.

Ghaly, A. E., & El-Taweel, A. A. (1997) Kinetic modelling of continuous production


of ethanol from cheese whey. Biomass and Bioenergy. 12, 461-472.

115
Hasan, A., Bujanovic, B., & Amidon, T. (2010) Strength properties of kraft pulp
produced from hot-water extracted woodchips within the biorefinery. Journal of
Biobased Materials and Bioenergy. 4, 46-52.

Hofvendahl, K., & Hahn-Hgerdal, B. (2000) Factors affecting the fermentative lactic
acid production from renewable resources. Enzyme and microbial technology. 26,
87-107.

http://www.plastemart.com. Accessed on December 02, 2012

Ishida, N., Saitoh, S., Tokuhiro, K., Nagamori, E., Matsuyama, T., Kitamoto, K., &
Takahashi, H. (2005) Efficient production of L-lactic acid by metabolically
engineered saccharomyces cerevisiae with a genome-integrated L-lactate
dehydrogenase gene. Applied and Environmental Microbiology. 71, 1964-1970.

Iyer, P. V., Thomas, S., & Lee, Y. Y. (2000) High-yield fermentation of pentoses into
lactic acid. Applied Biochemistry and Biotechnology - Part A Enzyme
Engineering and Biotechnology. 84-86, 665-677.

John, R. P., Nampoothiri, K. M., & Pandey, A. (2007) Fermentative production of


lactic acid from biomass: An overview on process developments and future
perspectives. Applied Microbiology and Biotechnology. 74, 524-534.

Koir, I. J., & Kidri, J. (2001) Identification of amino acids in wines by one-and two-
dimensional nuclear magnetic resonance spectroscopy. Journal of Agricultural
and Food Chemistry. 49, 50-56.

Krueger, K., & Peterson, W. H. (1948) Metabolism of biotin and oxybiotin by


lactobacillus pentosus 124-2. Journal of Bacteriology. 55, 693-703.

Kumar, M., & Gayen, K. (2011) Developments in biobutanol production: New


insights. Applied Energy. 88, 1999-2012.

Kumar, R., Singh, S., & Singh, O. V. (2008) Bioconversion of lignocellulosic


biomass: Biochemical and molecular perspectives. Journal of Industrial
Microbiology and Biotechnology. 35, 377-391.

Lampen, J. O., & Peterjohn, H. R. (1951) Studies on the specificity of the


fermentation of pentoses by lactobacillus pentosus. Journal of Bacteriology. 62,
281-292.

Laopaiboon, P., Thani, A., Leelavatcharamas, V., & Laopaiboon, L. (2010) Acid
hydrolysis of sugarcane bagasse for lactic acid production. Bioresource
technology. 101, 1036-1043.

116
Lendenmann, U., Snozzi, M., & Egli, T. (1996) Kinetics of the simultaneous
utilization of sugar mixtures by escherichia coli in continuous culture. Applied
and Environmental Microbiology. 62, 1493-1499

Lim, L. -., Auras, R., & Rubino, M. (2008) Processing technologies for poly(lactic
acid). Progress in Polymer Science (Oxford). 33, 820-852.

Litchfield, J. H. (2009) Lactic acid, microbially produced. In: Editor-in-


Chief: Moselio Schaechter (Ed.), Encyclopedia of Microbiology (Third Edition).
Academic Press, Oxford, pp. 362-372.

Liu, S., Lu, H., Hu, R., Shupe, A., Lin, L., & Liang, B. (2012) A sustainable woody
biomass biorefinery. Biotechnology Advances. 30, 785-810.

Liu, S. (2010) Woody biomass: Niche position as a source of sustainable renewable


chemicals and energy and kinetics of hot-water extraction/hydrolysis.
Biotechnology Advances. 28, 563-582.

Liu, S., Lu, H., Hu, R., Shupe, A., Lin, L., & Liang, B. (2012) A sustainable woody
biomass biorefinery. Biotechnology Advances. 30, 785-810.

Liu, T., Lin, L., Sun, Z., Hu, R., & Liu, S. (2010) Bioethanol fermentation by
recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood
extract hydrolyzate as substrate. Biotechnology Advances. 28, 602-608.

Lorca, G. L., Raya, R. R., Taranto, M. P., & De Valdez, G. F. (1998) Adaptive acid
tolerance response in lactobacillus acidophilus. Biotechnology Letters. 20, 239-
241.

Lpez-Rituerto, E., Cabredo, S., Lpez, M., Avenoza, A., Busto, J. H., & Peregrina, J.
M. (2009) A thorough study on the use of quantitative 1H NMR in rioja red wine
fermentation processes. Journal of Agricultural and Food Chemistry. 57, 2112-
2118.

Maas, R. H. W., Bakker, R. R., Jansen, M. L. A., Visser, D., De Jong, E., Eggink, G.,
& Weusthuis, R. A. (2008) Lactic acid production from lime-treated wheat straw
by Bacillus coagulans: Neutralization of acid by fed-batch addition of alkaline
substrate. Applied Microbiology and Biotechnology. 78, 751-758.

Madhavan Nampoothiri, K., Nair, N. R., & John, R. P. (2010) An overview of the
recent developments in polylactide (PLA) research. Bioresource technology. 101,
8493-8501.

117
Melzoch, K., Votruba, J., Hbov, V., & Rychtera, M. (1997) Lactic acid production
in a cell retention continuous culture using lignocellulosic hydrolysate as a
substrate. Journal of Biotechnology. 56, 25-31.

Mittal, A., Scott, G. M., Amidon, T. E., Kiemle, D. J., & Stipanovic, A. J. (2009)
Quantitative analysis of sugars in wood hydrolyzates with 1H NMR during the
autohydrolysis of hardwoods. Bioresource technology. 100, 6398-6406.

Monteagudo, J. M., Rodrguez, L., Rincn, J., & Fuertes, J. (1997) Kinetics of lactic
acid fermentation by Lactobacillus delbrueckii grown on beet molasses. Journal
of Chemical Technology and Biotechnology. 68, 271-276.

Nandasana, A. D., & Kumar, S. (2008) Kinetic modeling of lactic acid production
from molasses using Enterococcus faecalis RKY1. Biochemical engineering
journal. 38, 277-284.

Narayanan, N., Roychoudhury, P. K., & Srivastava, A. (2004) L (+) lactic acid
fermentation and its product polymerization. Electronic Journal of
Biotechnology. 7

Nord, L. I., Vaag, P., & Duus, J. . (2004) Quantification of organic and amino acids
in beer by 1H NMR spectroscopy. Analytical Chemistry. 76, 4790-4798.

Palmqvist, E., & Hahn-Hgerdal, B. (2000) Fermentation of lignocellulosic


hydrolysates. I: Inhibition and detoxification. Bioresource technology. 74, 17-24.

Pandey A., Soccol C., Nigam P., & Soccol V.T., (2000). Biotechnological potential
of agro-industrial residues. I: sugarcane bagasse. Bioresource Technology. 74,
69 - 80

Qureshi, N., & Ezeji, T. C. (2008) Butanol, 'a superior biofuel' production from
agricultural residues (renewable biomass): Recent progress in technology.
Biofuels, Bioproducts and Biorefining. 2, 319-330.

Qureshi, N., Saha, B. C., Hector, R. E., Dien, B., Hughes, S., Liu, S., Iten, L.,
Bowman, M. J., Sarath, G., & Cotta, M. A. (2010) Production of butanol (a
biofuel) from agricultural residues: Part II - use of corn stover and switchgrass
hydrolysates. Biomass and Bioenergy. 34, 566-571.

Saha, B. C. (2003) Hemicellulose bioconversion. Journal of Industrial Microbiology


and Biotechnology. 30, 279-291.

Schepers, A. W., Thibault, J., & Lacroix, C. (2006) Continuous lactic acid production
in whey permeate/yeast extract medium with immobilized lactobacillus

118
helveticus in a two-stage process: Model and experiments. Enzyme and microbial
technology. 38, 324-337.

Schmidt, S., & Padukone, N. (1997) Production of lactic acid from wastepaper as a
cellulosic feedstock. Journal of Industrial Microbiology and Biotechnology. 18,
10-14.

Shupe, A. M., Kiemle, D. J., & Liu, S. (2012) Quantitative 2D HSQC NMR analysis
of mixed wood sugars in hemicellulosic hydrolysate fermentation broth. Journal
of Bioprocess Engineering and Biorefinery. 1, 93-100.

Sjostrom, E. (1993) Wood chemistry: Fundamentals and applications. Academic Press


Inc, New York

Sjostrom, E and Westermark, U. (1998) Chemical composition of wood and pulps:


basic constituents and their distribution; in: Analytical Methods in Wood
Chemistry, Pulping, and Papermaking Springer-Verlag, Heidelberg, Germany,
pp. 1-19.

Subba Rao, C., Prakasham, R. S., Bhaskar Rao, A., & Yadav, J. S. (2008) Production
of L (+) lactic acid by lactobacillus delbrueckii immobilized in functionalized
alginate matrices. World Journal of Microbiology and Biotechnology. 24, 1411-
1415.

Sun, Z., & Liu, S. (2010) Production of n-butanol from concentrated sugar maple
hemicellulosic hydrolysate by Clostridium acetobutylicum ATCC824. Biomass
and Bioenergy. 24, 39 -47

Tanaka T., Hoshina M., Tanabe S., Sakai K., Ohtsubo S., & Taniguchi M. (2006)
Production of d-lactic acid from defatted rice bran by simultaneous
saccharification and fermentation. Bioresource Technology. 97, 211-217.

Tanaka, K., Komiyama, A., Sonomoto, K., Ishizaki, A., Hall, S., & Stanbury, P.
(2003) Two different pathways for D-xylose metabolism and the effect of xylose
concentration on the yield coefficient of L-lactate in mixed-acid fermentation by
the lactic acid bacterium Lactococcus lactis IO-1. Applied Microbiology and
Biotechnology. 60, 160-167.

Vazquez, J., & Murado, M. A. (2008) Unstructured mathematical model for biomass,
lactic acid and bacteriocin production by lactic acid bacteria in
batch fermentation. Journal of Chemical Technology and Biotechnology. 83, 91-
96.

Venus, J., & Richter, K. (2006) Production of lactic acid from barley: Strain selection,
phenotypic and medium optimization. Engineering in Life Sciences. 6, 492-500.

119
Walton, S. L., Bischoff, K. M., Van Heiningen, A. R. P., & Van Walsum, G. P.
(2010) Production of lactic acid from hemicellulose extracts by Bacillus
coagulans MXL-9. Journal of Industrial Microbiology and Biotechnology. 37,
823-830.

Wang, L., Zhao, B., Liu, B., Yu, B., Ma, C., Su, F., Hua, D., Li, Q., Ma, Y., & Xu, P.
(2010) Efficient production of l-lactic acid from corncob molasses, a waste by-
product in xylitol production, by a newly isolated xylose utilizing Bacillus sp.
strain. Bioresource technology. 101, 7908-7915.

Wee, Y. -., Kim, J. -., & Ryu, H. -. (2006) Biotechnological production of lactic acid
and its recent applications. Food Technology and Biotechnology. 44, 163-172.

Wee, Y. -., & Ryu, H. -. (2009) Lactic acid production by Lactobacillus sp. RKY2 in
a cell-recycle continuous fermentation using lignocellulosic hydrolyzates as
inexpensive raw materials. Bioresource technology. 100, 4262-4270.

Yang, X., & Tsao, G. T. (1994) Mathematical modeling of inhibition kinetics in


acetone-butanol fermentation by Clostridium acetobutylicum. Biotechnology
progress. 10, 532-538.

Yoon, H. H. (1998) Pretreatment of lignocellulosic biomass by autohydrolysis and


aqueous ammonia percolation. Korean Journal of Chemical Engineering. 15,
631-636.

Yun, J. -., & Ryu, H. -. (2001) Lactic acid production and carbon catabolite repression
from single and mixed sugars using Enterococcus faecalis RKY1. Process
Biochemistry. 37, 235-240.

Zanoni, P., Farrow, J., Phillips, B., & Collins, M. D. (1987) Lactobacillus pentosus
(fred, peterson, and anderson) sp. nov., norn, rev. International Journal of
systematic bacteriology. 37, 339-334.

Zhao, W., Sun, X., Wang, Q., Ma, H., & Teng, Y. (2009) Lactic acid recovery from
fermentation broth of kitchen garbage by esterification and hydrolysis method.
Biomass and Bioenergy. 33, 21-25.

Zhu, J. Y., & Pan, X. J. (2010) Woody biomass pretreatment for cellulosic ethanol
production: Technology and energy consumption evaluation. Bioresource
technology. 101, 4992-5002.

Zhu, Y., Lee, Y. Y., & Elander, R. T. (2007) Conversion of aqueous ammonia-treated
corn stover to lactic acid by simultaneous saccharification and cofermentation.
Applied Biochemistry and Biotechnology. 137-140, 721-738.

120
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