by Maryam Edalat Manesh A thesis submitted in conformity ...€¦ · Rodrigues, Dr. Suhbash Mojumdar, Mr. Muhammed Pervaiz and Mr. Dan Matters who provided helpful technical collaboration

Utilization of Pulp and Paper Mill Sludge as Filler inNylon Biocomposite Production

by

Maryam Edalat Manesh

A thesis submitted in conformity with the requirementsfor the degree of Doctor of Philosophy

Graduate Department of Chemical Engineering and Applied ChemistryUniversity of Toronto

Copyright c⃝ 2012 by Maryam Edalat Manesh

Abstract

Utilization of Pulp and Paper Mill Sludge as Filler in Nylon Biocomposite Production

Maryam Edalat Manesh

Doctor of Philosophy

Graduate Department of Chemical Engineering and Applied Chemistry

University of Toronto

2012

The biological treatment of pulp and paper mills effluents results in the production of

waste secondary sludge which is hard and costly to dewater and dispose. Secondary

sludge, which is structurally comparable to the municipal sewage sludge, is composed of

microbial cells, organic woody materials, and ash. In this work, the use of this waste

biosolid as renewable and cost-cutting filler in the composite industry is proposed. More-

over, the effect of enzymatic treatment of the waste biosolid on the final properties of

the manufactured biocomposite is studied. The high protein content of the secondary

sludge (35± 5%) and the surface thermodynamics measured by Inverse Gas chromatog-

raphy (IGC) led us to choose Nylon 11 as the main polymeric matrix. The biocomposite

samples produced by compounding and injection molding of different mixtures of dried

secondary sludge and Nylon were tested. The results of mechanical strength tests showed

that a 10% sludge content does not lead to any significant deterioration of either tensile

or flexural strengths. Therefore, it is concluded that the secondary sludge may be used

as filler to reduce the cost while maintaining the mechanical properties of Nylon. En-

zymatic modification of the waste biosolid to advance its application from cheapening

filler to reinforcing filler has also been proposed in this work. Lipase and laccase utilized

for the modification of the sludge in order to reduce the hydrophobicity and increase the

molecular weight, respectively. Lipase application did not lead to any significant changes

in either tensile or flexural strengths. This is attributed to the rather low content of lipids

ii

in the sludge. On the other hand, enzymatic modification of the sludge by laccase which

increases the molecular weight of the existing lignins, resulted in significant improvement

of the flexural strength of the manufactured biocomposite.

iii

Acknowledgements

I would like to thank my advisors, Professor M. Sain and Professor S. N. Liss for their

supervision and support. They have inspired me with their valuable qualities as profes-

sionals and scientists. I am also very much grateful to my committee members: Professor

D. N. Roy and Professor R. Farnood from the University of Toronto, for their scientific

and constructive input, criticisms, advice, and time. I also appreciate the time and

valuable feedbacks of Professors G. Allen, D. W. Kirk and E. Master.

I genuinely appreciate the financial support of Natural Science and Engineering Re-

search Council of Canada.

I would like to thank Mr. Shiang Law, Dr. Tony Ung, Dr. Robert Jeng, Dr. Arturo

Rodrigues, Dr. Suhbash Mojumdar, Mr. Muhammed Pervaiz and Mr. Dan Matters

who provided helpful technical collaboration and training in equipment operation in this

study. I am deeply thankful to Dr. Tayebeh Behzad, Professor Sally Krigstin, Dr. Suhara

Panthapulakkal and Dr. Mojgan Nezhad for their valuable inputs and time.

My deepest appreciation goes to Mr. Ian Kennedy and Mr. John McCarron from

the faculty of Forestry, and Ms. Pauline Martini from the Department of Chemical

Engineering for making life much easier on graduate students.

I would like to thank my parents, Minoo and Jamshid, for their constant encourage-

ment and valuable guidance throughout my life. I am very much thankful to my dearest

friends, Hamideh, Azadeh and Hajiha family for their invaluable friendship and support.

Finally, words alone cannot express the thanks I owe to Amir, my husband, for ten

years of love and support. He was not only willing to put up with four years of long-

distance relationship, but also supported me unconditionally through the ups-and-downs

of my doctoral studies.

iv

Contents

1 Introduction 1

1.1 Background and Motivation . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.3 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4 Thesis Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2 Literature Review 5

2.1 Pulp and Paper Wastewater Treatment Residues . . . . . . . . . . . . . . 5

2.1.1 Sludge Composition . . . . . . . . . . . . . . . . . . . . . . . . . . 7

2.1.2 Biorefining of the Biomass . . . . . . . . . . . . . . . . . . . . . . 7

2.1.3 Paper Sludge Composites . . . . . . . . . . . . . . . . . . . . . . 9

2.2 Separation and Quantification of the Constituents: Solubility and the

Thermodynamic Approach . . . . . . . . . . . . . . . . . . . . . . . . . . 10

2.3 Composites and Role of Surface Energy in Composite Properties . . . . . 14

2.3.1 Fiber Surface Characteristics and Modification . . . . . . . . . . . 18

2.4 Enzyme Catalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.4.1 Lignin Structure and Biosynthesis . . . . . . . . . . . . . . . . . . 24

2.4.2 Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

2.4.3 Laccase Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . 26

2.4.4 Lignification by laccase . . . . . . . . . . . . . . . . . . . . . . . . 27

v

2.4.5 Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2.5 Problem Statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

3 Molecular Structure and Cellular Biopolymers 32

3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3.3 Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

3.3.1 Secondary Sludge Sample . . . . . . . . . . . . . . . . . . . . . . 37

3.3.2 Moisture and Ash Content . . . . . . . . . . . . . . . . . . . . . . 37

3.3.3 Extractive Content Determination . . . . . . . . . . . . . . . . . . 38

3.3.4 Isolation and Quantification of Lignocellulosic Materials . . . . . 38

3.3.5 Extraction of Cellular Polymers . . . . . . . . . . . . . . . . . . . 39

3.3.6 Biochemical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 40

3.3.7 Chromatographic Method (HPSEC analysis of cellular biopolymers) 40

3.3.8 Fourier Transformed Infrared Spectroscopy (FTIR) . . . . . . . . 41

3.4 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

3.4.1 FTIR Spectra of the Secondary Sludge . . . . . . . . . . . . . . . 41

3.4.2 Biomass Fractionation: Extraction Method . . . . . . . . . . . . . 43

3.4.3 The Dynamics of the Extraction Process . . . . . . . . . . . . . . 46

3.4.4 Quantities of Cellular Polymeric Substances Extracted from the

Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

3.4.5 FTIR Spectra of Extracted Cellular Biopolymer Samples . . . . . 50

3.4.6 Chromatographic Separation . . . . . . . . . . . . . . . . . . . . . 53

3.4.7 Choices of Working Wavelength . . . . . . . . . . . . . . . . . . . 54

3.4.8 Mobile Phase and the Flow Rate . . . . . . . . . . . . . . . . . . 54

3.4.9 Molecular Size and Weight Distribution . . . . . . . . . . . . . . . 55

3.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

vi

4 Surface Thermodynamics and Thermal Behavior 58

4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59


4.3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

4.3.2 Inverse Gas Chromatography . . . . . . . . . . . . . . . . . . . . 62

4.3.3 Thermogravimetric Analysis . . . . . . . . . . . . . . . . . . . . . 63

4.4 Results and Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

4.4.1 Dispersive Component of the Surface Free Energy . . . . . . . . . 63

4.4.2 Acid-Base Interactions . . . . . . . . . . . . . . . . . . . . . . . . 66

4.4.3 Thermogravimetric Behavior . . . . . . . . . . . . . . . . . . . . . 70

4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

5 Enzymatic Modification of Sludge 73

5.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

5.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74


5.3.1 Secondary Sludge Sample . . . . . . . . . . . . . . . . . . . . . . 76

5.3.2 Lipase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

5.3.3 Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

5.3.4 Assay of Laccase Activity and Kinetic Studies . . . . . . . . . . . 76

5.3.5 Lipid Measurement . . . . . . . . . . . . . . . . . . . . . . . . . . 77

5.3.6 Klason Lignin Determination . . . . . . . . . . . . . . . . . . . . 77

5.3.7 Alkali Extraction of Lignin . . . . . . . . . . . . . . . . . . . . . . 78

5.3.8 Enzymatic Treatment . . . . . . . . . . . . . . . . . . . . . . . . . 78

5.3.9 Fourier Transformed Infrared Spectroscopy (FTIR) . . . . . . . . 79

5.3.10 Chromatographic Method . . . . . . . . . . . . . . . . . . . . . . 79

5.4 Results and Discussions . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

vii

5.4.1 Lipid Content of the Sludge . . . . . . . . . . . . . . . . . . . . . 79

5.4.2 Lipase Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

5.4.3 Laccase Characterization . . . . . . . . . . . . . . . . . . . . . . . 82

5.4.4 Laccase Modification of the Sludge . . . . . . . . . . . . . . . . . 84

5.4.5 Lignin Determination and Alkali Extraction of Lignin from the Sludge 86

5.4.6 Modification of the Molecular Weight Distribution . . . . . . . . . 87

5.5 Conslusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

6 Nylon/Sludge Biocomposite 91

6.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91

6.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92


6.3.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

6.3.2 Composite Preparation . . . . . . . . . . . . . . . . . . . . . . . . 95

6.3.3 Mechanical Properties Measurements . . . . . . . . . . . . . . . . 95

6.3.4 Statistics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

6.4 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

6.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104

7 Conclusions and Future Work 105

7.1 Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

7.2 Significance of Research Work . . . . . . . . . . . . . . . . . . . . . . . . 106

7.3 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

7.4 Publications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

Appendices 110

A Dispersive Component of the Surface Energy 110

B Calculated Graphs for the Bowater Sludge 112

viii

C Calculated Graphs for the Tembec Sludge 115

Bibliography 115

ix

List of Tables

2.1 Molecular composition of typical municipal sludge . . . . . . . . . . . . . 8

2.2 Molar volumes and solubility parameters of solvents . . . . . . . . . . . . 12

2.3 Hansen solubility parameters of representative polymers and biopolymers 12

2.4 Surface tension components and parameters of different wood fibers and

some polymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.5 Representation of polymers synthesized/modified by enzymes (Gubitz) . 23

3.1 Wood-associated biopolymers quantified in sludge samples B and T . . . 46

3.2 Polysaccharide and protein content of sludge samples and extracellular

polymeric substances extracted by NaOH and CER methods . . . . . . . 50

4.1 Characteristics of the nonpolar probes used in the analysis . . . . . . . . 65

4.2 Surface characteristics (determined by IGC) of the sludges . . . . . . . . 66

4.3 Characteristics of the polar probes used in the analysis . . . . . . . . . . 68

4.4 Surface energies of a few commonly used polymeric resins . . . . . . . . . 70

5.1 Experimentally observed reaction rates for sludge with different initial

concentration of dissolved oxygen . . . . . . . . . . . . . . . . . . . . . . 84

6.1 Mechanical properties of Nylon composites . . . . . . . . . . . . . . . . . 97

6.2 Mean tensile and flexural strengths of pure Nylon and 25% sludge-filled

Nylon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

x

6.3 Mean tensile and flexural strengths of pure Nylon and 10% sludge-filled

Nylon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

6.4 Mean tensile and flexural strengths of 10% sludge-filled Nylon with and

without laccase modification . . . . . . . . . . . . . . . . . . . . . . . . . 101





xi

List of Figures

2.1 A liquid drop on a solid surface at equilibrium. . . . . . . . . . . . . . . . 15

2.2 Reaction coordinate diagram. . . . . . . . . . . . . . . . . . . . . . . . . 22

2.3 Laccase redox centers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

3.1 FTIR spectra of sludge samples (B and T) . . . . . . . . . . . . . . . . . 42

3.2 Comparing the obtained results for the secondary sludge constituents with

typical wood fibers (Pettersen, 1984) constituents . . . . . . . . . . . . . 45

3.3 Effect of extraction time on absorbance at 300 nm, extraction by ethanol-

toluene (1:2 v/v) and 290 nm, extraction by ethanol at boiling temperature. 47

3.4 Kinetic of the extraction processes (logarithmic scale) . . . . . . . . . . . 48

3.5 FTIR spectra of EPS samples extracted by NaOH . . . . . . . . . . . . . 53

3.6 FTIR spectra of EPS samples extracted by CER . . . . . . . . . . . . . . 53

3.7 HPSEC chromatograms obtained for CER extracted samples at flow rate

of 1 ml/min, Signal at 280 nm, injection volume of 25 µL . . . . . . . . . 55

3.8 HPSEC chromatogram obtained for CER extracted samples at flow rate

of 0.5 ml/min, Signal at 280 nm, injection volume of 25 µL. . . . . . . . 56

4.1 Determination of acid-base contribution to the free energy of adsorption:

IGC data of n-alkanes polar probes for B-sludge sample . . . . . . . . . . 66

xii

4.2 Determination of the specific component of the enthalpy of adsorption and

the entropy of adsorption for each of the polar probes for secondary sludge

(B) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

4.3 Determination of Ka and Kb for secondary sludge (B) . . . . . . . . . . . 69

4.4 The DSC thermogram for the sludge sample . . . . . . . . . . . . . . . . 71

5.1 FTIR spectra of the toluene-extracted lipids from the secondary sludge. . 80

5.2 FTIR spectra of the untreated and lipase treated sludge for 4, 8, and 24

hours (E1 to E3, respectively). . . . . . . . . . . . . . . . . . . . . . . . . 81

5.3 Typical changes in dissolved oxygen during laccase-sludge reaction in a

batch system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

5.4 FTIR spectra of the untreated and laccase treated sludge . . . . . . . . . 85

5.5 HPSEC chromatogram obtained for the laccase treated alkali extracts with

signals at 210 and 280 nm (flow rate of 1 ml/min, and injection volume of

25 µL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

5.6 HPSEC chromatogram obtained for the alkali extracted sample before and

after laccase treatment (flow rate of 1ml/min, Signal at 280nm, injection

volume of 25 µL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

6.1 Flexural properties of the sludge-filled Nylon composites . . . . . . . . . 99

6.2 Tensile properties of the sludge-filled Nylon composites . . . . . . . . . . 99

6.3 Reaction scheme of maleic anhydride with amide end groups of Nylon at

high temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

6.4 Flexural properties of the sludge-filled Nylon composites . . . . . . . . . 102

6.5 Tensile properties of the sludge-filled Nylon composites . . . . . . . . . . 102

A.1 Determination of acid-base contribution to the free energy of adsorption . 111

B.1 Calculation of γDS for Bowater sludge from Inverse Gas Chromatography 112

xiii



B.4 Free energy of desorption ∆GAB for the Bowater sludge . . . . . . . . . . 114

B.5 Plot of ∆HAB/AN versus DN/AN for the Bowater sludge . . . . . . . . 114

C.1 Calculation of γDS for Tembec sludge from Inverse Gas Chromatography . 115




C.5 Free energy of desorption ∆GAB for the Tembec sludge . . . . . . . . . . 117

C.6 Plot of ∆HAB/AN versus DN/AN for the Tembec sludge . . . . . . . . 118

xiv

Chapter 1

Introduction

1.1 Background and Motivation

The by-products of biological treatment of organic wastes, commonly referred to as

biosolids, are produced in large amounts in wastewater treatment plants on a daily basis.

Biosolid management can cost as much as 60% of the total operating costs of a wastewa-

ter treatment plant (Canales et al., 1999), thereby rendering waste management a crucial

task in any industry with a high biosolid production. Pulp and paper industry, in par-

ticular, generates approximately 45 kg of waste biosolid per ton of pulp (Campbell et al.,

1991). The mechanical and biological treatments of waste effluents from pulp and paper

mills result in the production of primary and secondary sludges, respectively. Secondary

sludge, which is structurally comparable to municipal sewage sludge, is mainly composed

of microbial cells, organic woody materials, and ash. Secondary sludge, due to its compo-

sition, is harder to dewater and dispose than the primary sludge. Thus far, landfilling and

incineration have been the common methods of dealing with the huge amount of waste

sludge. However, shortage of landfill space, environmental consequences of landfilling,

as well as the rising environmental concerns over emissions during incineration, call for

new alternatives. Among different possibilities, material reuse/recycling such as using

1

Chapter 1. Introduction 2

the biosolid as a soil amendment or as a feed to composite production (Krigstin and Sain,

2006) has received increasingly more attention. One alternative is to use this biosolid

as renewable and cost-cutting filler in composite industry. Unless the addition of this

biosolid results in the deterioration of the composite’s properties, their main advantage

will be reducing the costs. This is due to the fact that this cheap biosolid replaces a

part of the expensive polymeric resin. It may also enhance the material stiffness which

should be examined and proved. Since paper products form the largest recyclable com-

ponent of the solid waste generated in Canada (Krigstin and Sain, 2006), production of

biocomposites from pulp and paper biosolid is the main focus of this thesis.

1.2 Hypotheses

The main hypotheses of this work are:

• Secondary sludge of pulp and paper mills has satisfactory physico-chemical char-

acteristics to fill Nylon without deteriorating its mechanical properties.

• Selective enzymatic modification of the sludge enhances the mechanical properties

of the manufactured biocomposite by reducing the hydrophobicity and increasing

the components’ molecular weights.

1.3 Objectives

The objectives of this work are:

• Determination and quantification of the major secondary sludge constituents and

identification of the potential reinforcing components for biocomposite production;

• Understanding of the surface characteristics and thermogravimetric behavior of the

sludge biopolymers;


• Enzymatic modification of the secondary sludge and application of the sludge as

filler coupled with appropriate polymeric matrix;

• Providing knowledge on the relationships between sludge constituents and their

effects on the properties of the manufactured biocomposite;

1.4 Thesis Outline

This thesis is divided into seven chapters: a general introduction, a general literature

review, four chapters that include the main findings of this research, and a final chapter

containing the main conclusions and recommendations.

Chapter 2 offers a general literature review and background on the scientific concepts

discussed in the context of this thesis. It reviews the components commonly present in

the waste activated sludge in order to identify the ones contributing to the composite’s

final properties. The thermodynamic approach to solubility is presented since it is the

main scientific concept behind the isolation and quantification of the major constituents.

Also, to understand the mechanism of adhesion, the most popular theories and equations

explaining and quantifying the adhesion are reviewed. Moreover, the lignin structure

and synthesis as well as the role of laccase enzyme and its mechanism are explored in

order to give a better understanding of the pathway taking place during the enzymatic

modification of the sludge. Chapter 2 is basically providing the theoretical basis of this

work as well as studying the potential of secondary sludge to be used as filler in composite

manufacturing. More in-depth literature review is also provided in Chapters 3 to 6 as

necessary.

Chapter 3 is based on a manuscript published in the Journal of Water Science and

Technology (vol. 62, no. 12, 2010). It presents the results obtained from the isolation

and quantification of the sludge’s major constituents. Moreover, this chapter includes

details on the CER and alkaline extraction of the cellular biopolymers and molecular


weight determination of the resulting extracts. Further characterizations of the secondary

sludge in terms of the thermogravimetric behavior and surface energy are described in

Chapter 4. The results presented in this chapter are published as a paper in the Journal of

International Review of Chemical engineering (vol. 2, no. 1, 2010). Chapter 5 discusses

the enzymatic modification of the secondary sludge by lipase and laccase and the changes

made in the molecular structure and molecular weight of the sludge. The results of the

works presented in Chapters 3 and 4 led to the selection of the main polymeric matrix.

Chapter 6 details the process of pairing both the unmodified and the enzymatically

modified secondary sludge with the appropriate polymeric matrix in order to make new

biocomposite material. Finally, Chapter 7 reviews the main conclusions of this thesis,

and offers recommendations for improvement of the biocomposite’s final properties. The

results of chapters 5 and 6 are submitted to the Journal of Reinforced Plastics and

Composites in the form of two journal papers; with one being already accepted while the

other one is under review.

Chapter 2

Literature Review

2.1 Pulp and Paper Wastewater Treatment Residues

The primary and secondary wastewater treatments in pulp and paper mills result in

the production of waste residues. Primary treatment is the mechanical cleaning of the

wastewater stream to finish the incomplete solid/liquid separations at various stages of

pulp and paper production. It removes solid particles by gravity, settling/clarification,

and filtration. The residue collected through screens and filters is known as primary

sludge which mainly consists of wood fibers and fillers (Mahmood and Ellikot, 2006).

The primary sludge of recycling plants also contains synthetic materials such as plastics

and stickies, as well as traces of glass or metal (Mabee, 2001).

Primary wastewater treatment eliminates the suspended solids and reduces the amount

of heavy metals; however, it does not reduce the BOD (Biological Oxygen Demand) to

satisfactory levels. Therefore, the primary treatment is followed by a biological treatment

similar to the municipal wastewater treatment. The latter process is often an activated

sludge treatment in which the wastewater’s organic matter is broken down by means of

biodegradation. The by-product of this process is the secondary sludge which is struc-

turally similar to the municipal sewage sludge. It is composed of microbial cells, organic

5

Chapter 2. Literature Review 6

woody materials, and ash.

The principal component of both primary and secondary sludges is water, thereby

requiring the sludges to undergo dewatering operations. Dewatering must be carried out

before biosolids are composted, landfilled, or incinerated. The most common dewatering

techniques are air drying, vacuum filtration, plate-and-frame filtration and centrifugation.

Dewatering reduces the moisture content of the liquid sludge from 95-97% to about 50%

(wet weight basis) (Krigstin and Sain, 2006).

Primary sludge is fairly easy to dewater because of its high proportion of woody

materials. On the other hand, due to the highly colloidal nature of the small particles in

suspension (Mabee, 2001), the secondary sludge is far more difficult to dewater compared

to the primary sludge. Moreover, the secondary sludge has a high moisture content (97-

99.5%) (Kenny et al., 1995).

Therefore, primary and secondary sludges are usually combined to improve the dewa-

tering potential. The combined sludge from primary and secondary treatment consists

of a muddy mass of microorganisms, fibrous material, lignin, mineral components (lime-

stone and phosphorous), clay, inert solids rejected during the recovery process, ash, and

water (Navaee, Bertrand and Stuart, 2006). In general, as the primary to secondary

(P/S) ratio decreases, so does the ease of dewatering (Mahmood and Ellikot, 2006).

The primary sludge generation rate decreases with the increasing efficiency of the

fiber recovery system installed at the mill. Higher secondary sludge production and the

increased fiber conservation will worsen the secondary sludge dewatering potential. Thus,

from the dewatering standpoint, decreasing the secondary sludge production should be

a goal for the future (Mahmood and Ellikot, 2006). Producing less secondary sludge will

help improving or at least maintaining the current P/S ratio. Moreover, a decreased

amount of produced sludge will require less polymer addition to aid the dewatering.

Savings towards polymer costs can be substantial since conditioning chemicals constitute

the largest part of a wastewater treatment plant’s operating budget (Kenny et al., 1995).


2.1.1 Sludge Composition

Activated sludge from a typical municipal wastewater treatment plant consists mainly of

microorganisms with concentrations of up to 1011 cells/ml (Wagner, 2005), surrounded

by large macromolecules (Watson and Pletschke, 2006) along with heavy metals and also

dissolved organic compounds. The typical microbiology of activated sludge consists of

approximately 95% bacteria and 5% higher organisms such as protozoa and rotifers. The

activated sludge from pulp and paper mills also consists mainly of microbial biomass.

However, its main difference with the municipal sludge is the higher content of cellulosic

fibers (Chen et al., 2002). Cellulose fibers were also reported as a constituent of municipal

sludge and their existence has been attributed to the discharge of toilet paper (Honda,

Miyata and Iwahori, 2002).

From a chemical standpoint, activated sludge is mainly composed of biochemical

compounds that are characteristic of all living matter (proteins, carbohydrates, lipids,

and inorganics) (Rozich and Gaudy, 1992). Table 2.1 shows the molecular composition

of a typical municipal sludge as reported in the literature.

2.1.2 Biorefining of the Biomass

Biomass is any abundant carbon-neutral renewable resource for the production of sus-

tainable biopower and bioproducts. The challenge is to develop a new manufacturing

concept for converting renewable biosolid into bioenergy and biomaterials, referred to as

the “biorefining” (Ragauskas et al., 2006). Biorefining contributes to sustainability by

using sustainable bioresources, and recycling the waste. It can also be an effective way

of reducing the greenhouse gas emissions.

Biosolids (historically known as sewage sludge) are the by-products of the biological

treatment of organic wastes. They are mainly composed of organic materials, plant nu-

trients, and other elements which reflect the origin of the waste. They also include heavy


Table 2.1: Molecular composition of typical municipal sludge

Sludge Components % (Dry Weight)

Protein23-36 (Dignac et al., 2000)

30 (Kelly, Miller and Namazian, 2001)

Carbohydrate and lignin 8-15 (Dignac et al., 2000)

(Dignac et al., 2000)20 (Kelly, Miller and Namazian, 2001; Parmar, Singh

and Ward, 2001)

Lipids 6-30 (Kelly, Miller and Namazian, 2001)

Inorganics17-28 (Kelly, Miller and Namazian, 2001)

20 (Poulsen and Hansen, 2003)

metals which occur naturally or come from the industry. Biosolids undergo additional

treatment on site prior to being used or disposed in order to meet the regulatory require-

ments, facilitate handling and reduce costs. The two most common types of biosolids

treatment processes are stabilization and dewatering. To date, the most common solution

for the huge amount of waste biosolids has been landfilling. The problems associated with

landfilling include the high cost of the process, emissions of odor and gas, the percolating

water, and the growing shortage of suitable landfill sites. Moreover, the mixed sludge

from pulp and paper industry, when landfilled, is an important source of greenhouse gases

(Navaee, Bertrand and Stuart, 2006).

Anaerobic digestion, composting and combustion with energy recovery are some of

the methods that are used in addition to landfilling (Sorum, Gronli and Hustad, 2001).

However, material reuse/recycling such as using the biosolid as a soil amendment or as

a feed to composite production (Krigstin and Sain, 2006) are desired the most. Due to

the significant content of nitrogen and phosphorus, residues of the municipal wastewater


treatment can be applied to land as fertilizer and soil conditioner (Werthera and Ogadab,

1999). This method has the advantage of returning the organic materials into the bio-

cycle and it provides a good alternative to artificial fertilizers whose production consumes

a lot of energy (Werthera and Ogadab, 1999). However, concerns over the risk of heavy

metals and organic contaminants in the sludge, and caution over the addition of nitrogen-

and phosphorus-rich manure to the land, are the limiting factors in the use of sludge as

fertilizer (Werthera and Ogadab, 1999).

Incineration of the sludge can reduce the waste volume up to 90%. This treatment,

producing energy, generates solid residues containing variable amounts of heavy metals,

chlorides and sulfates salts originally present in the sludge, which can be released into

the environment (Bruder-Hubscher et al., 2002).

However, the best alternative is to use the biosolid in a value-added manner. For

instance, the waste wood fibers separated from the paper sludge, can be employed as

fillers in manufacturing thermoplastic polymer composites. Producing the environmental-

friendly biocomposites from the biomass is not only considered as a solution to the

growing environmental threat but also as a solution to the uncertainty of the petroleum

supply. Moreover, this method maintains the carbon dioxide neutrality and solves the

problem of persistent plastics in the environment (Mohanty, Misra and Drzal, 2002).

2.1.3 Paper Sludge Composites

Primary sludge, also known as paper sludge, is considered a cellulosic material (Jang and

Lee, 2001) since it is mainly composed of cellulosic fibers and inorganic materials. Due

to its composition it has the potential to substitute the conventional fillers such as talc,

calcium carbonate, and clay (Son, Yang and Kim, 2004).

Paper sludge is reported not to be a major threat in terms of heavy metals (Boni,

D’April and Casa, 2004) and can be introduced to phenolic resins (Jang et al., 2000)

as well as low melting-point thermoplastics such as polypropelene (Jang and Lee, 2001).


Generally, cellulosic materials are extensively used in phenolic and other thermosetting

resins since they reduce the shrinkage during molding, thereby resulting in improved

impact strength of the finished products (Simitzis, Karagiannis and Zoumpoulakis, 1996).

For instance, paper sludge/ polypropylene composites can be applied as construction

materials in home furnishing, domestic and industrial buildings. The drawback of such

applications is their flammability which can be improved by adding flame retardant

materials (Jang and Lee, 2001).

2.2 Separation and Quantification of the Constituents:

Solubility and the Thermodynamic Approach

Extraction is a separation technique based on the solubility of a substance in a liquid

which is being used to isolate that substance from a mixture. The solubility parameter

is a numerical value indicating the relative solvency behavior of a specific solvent. It

is derived from the cohesive energy density of the solvent. The cohesive energy density

is defined as the amount of energy required for complete removal of the unit volume of

molecules from their neighbors to infinite separation. It can be derived from the heat of

vaporization (cal/cm3) by the following equation:

c =∆H −RT

Vm

(2.1)

where c is the cohesive energy density, ∆H is the heat of vaporization, and Vm is the

molar volume.

In the case of solubility, when two liquids are mixed, the molecules of each liquid are

physically separated by the molecules of the other one. This is similar to the separation

that happens during vaporization where the same intermolecular van der Waals forces

must be overcome. Therefore, the correlation between vaporization and van der Waals

forces can also be translated into a correlation between vaporization and solubility be-


havior. Hildebrand (Hildebrand, 1936) proposed the square root of the cohesive energy

density as a numerical value indicating the solvency behavior of a specific solvent:

δ =√c =

√∆H −RT

Vm

(2.2)

The proposed Hildebrand solubility parameter, δ, provides a numerical estimate of

the degree of interaction between particularly nonpolar materials. Since the solubility

of two materials is possible only when their intermolecular attractive forces are similar,

materials with similar values of δ are expected to be miscible. However, Hildebrand solu-

bility parameter is only useful for predicting the solubility of nonpolar and slightly polar

systems without hydrogen bonding. Therefore, most researchers have relied on more com-

plicated, three-dimensional solubility parameters for predicting the solvency behavior of

polar molecules. The most widely accepted three component system is the one proposed

by Hansen (Hansen, 1967). The three Hansen parameters include: the dispersion force

component (δd), the hydrogen bonding component(δh), and the polar component (δp).

The values of all three components are related through the total Hildebrand value (δt):

δ2t = δ2d + δ2p + δ2h (2.3)

A three-dimensional model was applied by Hansen to plot polymer solubilities. Hansen

solubility parameters of few solvents and polymers are presented in Tables 2.2 and 2.3.

Hansen realized that by doubling the dispersion parameter axis, an approximately

spherical volume of solubility would be formed for each polymer. The radius of this

sphere is called the interaction radius (R). A polymer is probably soluble in a solvent (or

solvent blend) if the Hansen parameters for the solvent lie within the solubility sphere of

the polymer. In order to determine this (without building a model) it must be calculated

whether the distance of the solvent from the center of the polymer solubility sphere is

less than the radius of interaction for the polymer:


Table 2.2: Molar volumes and solubility parameters of solvents

SolventsMolar volume

(cm3mol−1)

δ(MPa1/2)

δP δh δd

Water (Ham, Choi and

Chung, 2005)18.0 16.0 42.3 15.6

Ethanol (Ham, Choi and

Chung, 2005)58.5 8.8 19.4 15.8

Benzene (Ham, Choi and

Chung, 2005; Barton, 1975)89.4 0.0 2.0 18.4

Toluene (Barton, 1975) 106.8 1.4 2.0 18

Acetic Acid (Barton, 1975) 57.1 8.0 13.5 14.5

Table 2.3: Hansen solubility parameters of representative polymers and biopolymers

Polymerδ(MPa1/2)

δP δh δd

Cellulose acetate (Hansen, 1971) 12.73 11.01 18.6

Cellulose nitrate (Hansen, 1971) 14.73 8.84 15.41

Amorphous Cellulose (Hansen, 2000) 19.9 22.5 24.3

Lignin (Hansen, 2000) 14.1 16.9 21.9


D =[4 (δdS − δdP )

2 + (δpS − δpP )2 + (δhS − δhP )

2]1/2

(2.4)

where D is the distance between solvent and center of solubility sphere.

In conclusion, a solvent with solvent parameters whose values are close to that of the

solute will win over a solvent whose solvent parameters differs significantly.

The experimental solubility data can help choosing the appropriate solvents for sep-

aration and purification of solid solutes. However, in the case of polymers with high

molecular masses (about 2 million) even the best match in solubility parameters between

solvent and solute will not lead to a true solution (Horvath, 2006). This is due to the

fact that in addition to the solubility parameter, several other factors affect the solubility,

including molar volume, molecular surface area, polarizability, polarity, and H-bonding

strength. Moreover, it is common for polymers to have higher Hansen solubility parame-

ters than the monomers from which they are made(Hansen, 2000). Since biomass consists

of a mixture of different large molecules with intramolecular forces, isolated constituents

do not represent their natural state in the biomass structure. Nonetheless, they can

give indications on possible conformation (Horvath, 2006). Wood fiber is a mixture of

40-50% cellulose, 20-35% hemicellulose (polysaccharides), and 15-30% lignin along with

some extractives (3-5%) and minerals (1-2%). Unfortunately, due to the large number of

variables involved, the thermodynamic principles of the solubility of such a structurally

complicated material in various liquids (e.g., solvents, oil, etc.) have not been adequately

investigated (Horvath, 2006).

In the biomass, large molecules of wood fiber including cellulose, hemicellulose, and

lignin are bound together where hemicellulose acts as a surfactant bounding to both cel-

lulose and lignin. This is due to the fact that hemicellulose has backbone and side groups

favoring energetically to cellulose environment and lignin environment, respectively. Also

biopolymers associated with the microorganisms including proteins and polysaccharides

are present in the biomass. The considerable interactions of these polymers results in the


crucial role of the solvent diffusion in their solubilities.

Cellulose is distinguished analytically from the extractives by its insolubility in water

and organic solvents. These extractives are the nonstructural wood constituents (e.g., fats

and waxes, phenolic compounds, etc.) along with the low molecular weight polysaccha-

rides and lipids associated with the microorganisms. Moreover, cellulose is distinguished

from the proteins and hemicelluloses by its insolubility is aqueous alkaline solutions, and

from lignin by its relative resistance to oxidizing agents and susceptibility to hydrolysis

by acids (Horvath, 2006). The presence of one primary (C6) and two secondary (C2, C3)

hydroxyl groups in an anhydroglucose unit of cellulose predetermines the occurrence of a

system of inter- and intramolecular hydrogen bonds in the polymer. This strongly con-

fines the application of one component solvents suitable for practical use (Bochek, 2003).

On the other hand, hemicelluloses are easily hydrolyzed by acids to their monomeric

compounds and lignin has a very low solubility in most solvents.

In conclusion, there are several standard methods recommended for isolation and

quantification of each of these compounds considering their molecular structure and

interactions with each other. Extraction techniques adapted in this work in order to

quantify each of the biomass constituents are based on these standard methods.

2.3 Composites and Role of Surface Energy in Com-

posite Properties

One significant factor which determines the composite’s performance is the quality of

interface between the fiber/filler and the polymeric matrix. The interface quality is,

mainly governed by the adhesion of these phases to each other. It has been shown in

the literature that the work of adhesion, the ultimate interfacial shear strength, and

consequently, the strength of the composites correlate with each other (Pisanova and

Mader, 2000).


S

L

V

SLγ SV

γ

LVγ

θ

Figure 2.1: A liquid drop on a solid surface at equilibrium.

The term adhesion stands for both the establishment of interfacial bonds as well as

the mechanical load required to break an assembly. So far, different theories have been

proposed explaining the scientific concept of adhesion including: mechanical interlocking,

electronic theory, theory of weak boundary layers, and the thermodynamic theory. These

theories have been validated based on a wide range of related experimental observations.

Nonetheless, each of them has many weaknesses and none of them, by itself, can explain

all of the adhesion related experimental observations (Schultz and Nardin, 1999).

The thermodynamic model of adhesion is currently the most widely used approach in

the adhesion science (Schultz and Nardin, 1999). According to this theory, the adhesive

will adhere to the substance as a result of the interatomic and intermolecular forces at

the interface, provided that the intimate contact is achieved. The interfacial forces result

from the van der Waals and Lewis acid-base interactions. The magnitudes of these forces

are generally related to the fundamental thermodynamic quantities such as surface free

energies of both the adhesive and the adherent. Generally, the adhesion goes through a

liquid-solid contact step and therefore, the criteria for good adhesion become basically

the criteria for good wetting. It should be noted, however, that this is a necessary but

not sufficient condition.

Based on this theory, therefore, the perfect fiber-matrix interface happens when the

liquid resin completely “wets” or “spreads on” the fiber surface. The measure of this

“wettability” is expressed by the observed equilibrium “contact angle” formed by the

drop of liquid on the surface, θ (Figure 2.1) at a thermodynamic equilibrium.


In a solid-liquid system, wetting equilibrium is defined from the profile of a drop on

a solid surface. Young’s equation (Young, 1805) defines the balance of forces caused

by a wet drop on a dry surface. It relates the surface tension (γ) of materials at the

three-phase contact point to the equilibrium contact angle θ via the following equation:

γSV = γSL + γLV cos θ (2.5)

where subscripts S, L, and V stand for, respectively, solid phase of substrate, liquid

phase of the droplet and vapor phase of the ambient such that a combination of two of

these subscripts corresponds to the solid-vapor interface.

Moreover, the reversible work of adhesion, WA, is defined as the work required to

separate two incompatible substances (A and B) with the result of creating two new

surfaces. WA is defined in terms of surface tension:

WA = γA + γB − γAB (2.6)

The work of adhesion is related to the intermolecular forces which operate at the

interface between two materials. However, there is also energy loss due to irreversible

deformation processes within the adhesive which should be taken into account. Fowkes

(Fowkes, 1964) suggested that the work of adhesion and surface free energy of a given

body could be represented by the sum of the contributions of different types of interac-

tions, such as dispersion forces (D), dipole interactions (P ), hydrogen bonding (H), and

metallic bonds (M):

WA = WD +W P +WH +WM . . . (2.7)

and,

γA = γD + γP + γH + γM . . . (2.8)


Moreover, Fowkes (Fowkes, 1964) proposed that the dispersive part of the interactions

between solids 1 and 2 can be quantified by twice the geometric mean of the dispersive

components of the surface tension of the two entities. Therefore, for the type of interac-

tions where only dispersion forces are involved, W12 is given by:

W12 = 2√γD1 γ

D2 (2.9)

Van Oss et. al (van Oss, Good and Chaudhury, 1988, 1987) suggested to combine

the contribution of polar (Keesom and Debye) and dispersion forces which is denoted by

LW, i.e. Lifschitz-van der Waals. Additionally there is a short-range interaction which

is caused by acid-base interactions and is denoted by AB. Therefore, we have:

γTOTs = γLW

s + γABs (2.10)

WLW12 can be expressed by the geometric mean, same as in Equation 2.5. However,

this is not the case for WAB12 since the basic components of the surface only interact

with the acidic components of the liquid, and vice versa. Thus, the following equation is

proposed by van Oss et. al (van Oss, Good and Chaudhury, 1988):

WAB12 = 2

√γ+1 γ

−2 + 2

√γ−1 γ

+2 (2.11)

where γ+i represents the acidic part and γ−

i the basic part. In terms of having one or both

of the acidic and basic groups, substances are either monopolar or bipolar, respectively.

Therefore, the wettability of a material depends on its surface tension, the surface

tension of the liquid which it is in contact with, and the effective surface in contact with

the liquid. Generally, wetting requires the surface tension of the adherent (reinforcement)

to be at least equal to or greater than that of the adhesive (matrix) (Rudd, 1997). Thus,

predicting the wetting level requires extensive knowledge of the surface characteristics

of both the solid and the liquid in contact with it. Thermodynamically, a high-energy


surface is the most conducive to good wetting, particularly if adhesive contains polar

functional group.

In order to modify the interaction across fiber/matrix interface, information about

the biofiber surface is required. There are several methods available for characterizing the

fiber surface and fiber/matrix interface properties. Inverse Gas Chromatography (IGC) is

a well established technique for semi-quantitative expression of the dispersive component

of the surface tension and acid-base characteristics of the polymers and reinforcing fibers

(Gulati and Sain, 2006; Hildebrand, 1936). It is reported to be a more reliable technique

compared to wetting techniques (Heng et al., 2007). The obtained data via IGC is

significant since it helps predicting the degree of interaction, and hence adhesion, between

the fiber/filler and the polymeric matrix. This experiment is carried out via injections

of very small amounts of volatile probes (mobile phase) with known properties, through

a column packed with the unknown solid (stationary phase). The probes are carried

through the column by an inert carrier gas. The retention time of each probe is related

to their interaction with the stationary phase. Since the theory and principles of IGC

have been well explained in other articles (Riedl and Mauana, 2006) only the critical

equations and assumptions are mentioned in Chapter 4.

2.3.1 Fiber Surface Characteristics and Modification

Wetting of wood fiber surfaces by resins is an intensively investigated phenomenon in

biocomposites since knowledge on the interactions taking place at the fiber-matrix in-

terface may lead to enhance the poor interfacial adhesion. Table 2.4 shows the surface

free energy components of some polymeric matrices and wood fibers of different species

reported in the literature.

Since wetting requires the surface tension of the fibers to be greater than that of the

resin, surface energies of some of these fibers need to be raised in order to make a proper

choice for the reinforcing phase. Compatibilization is any operation performed on the


fiber and polymer that increases the wetting within the blend (Holbery and Houston,

2006). The common methods for effective adhesion enhancement across the interface of

the two dissimilar phases include physical/chemical treatment of fiber, and the use of

coupling agent (Saheb and Jog, 1999).

Table 2.4: Surface tension components and parameters of different wood fibers and some

polymers

SpeciesδTOTs

(mJ/m2)

δLWs

(mJ/m2)

δABs

(mJ/m2)

δ+s

(mJ/m2)

δ−s

(mJ/m2)

Cherry (Gardner, 1996) 54.3 47.46 6.84 0.42 28.00

Maple (Gardner, 1996) 53.3 45.48 7.85 0.46 33.19

Red oak (Gardner, 1996) 47.97 39.67 8.30 0.46 37.74

White oak (Gardner, 1996) 40.0 34.02 5.98 0.39 22.80

Walnut (Gardner, 1996) 42.55 37.92 4.63 0.09 58.93

PVC (Qin and Chang, 1996) 42.5 41.8 0.7 0.0 5.1

PS (Qin and Chang, 1996) 43.5 43.2 0.2 0.0 12.2

PMMA (Qin and Chang,

1996)43.6 42.1 1.6 0.2 2.6

PET (Oss, 1994) 44.6 44.1 0.6 0.0 7.4

Cellulose (Wu, Giese and van

Oss, 1995)56.1 44.9 11.2 2.2 14.3

Physical treatments of fiber surfaces do not change the chemical composition of fibers,

but tends to modify the structural and surface properties of fibers rendering stronger

mechanical bonding with the matrix. Examples of physical treatment methods are low


temperature plasma and corona discharge. Low temperature plasma improves the surface

characteristics of the fiber surface by using particles such as electron, ion, radical and

excited molecules produced by electrical discharge. On the other hand, sputter etching

brings about mainly physical changes such as surface roughness. It should be considered

that the modification may have adverse effects on the individual properties of fibers, so

the optimum treatment time is to be verified.

Chemical methods, basically, modify the chemical structure and moieties present at

the fiber surface in order to facilitate the occurrence of chemical bonding between the fiber

surface and the matrix. This, in turn, improves the stress transfer at the fiber/matrix

interface. Examples of chemical modification techniques include: silane treatment, alka-

nization, isocyanate treatment, and graft copolymerization. Treatments which lead to

eliminate waxes and lignin from the fiber surface are also promising techniques; since the

only component which contributes to the surface energy is cellulose. Coupling agents are

also popular chemical substances for improving adhesion. They improve the interfacial

properties by interacting with both the fiber and the matrix, thus forming a bridge of

chemical bonds between the components.

Chemical modification of fibers, although effective, can be a threat to the environment

and sometimes costly. On the other hand, biological modification of surface is a new

environmentally safe method of treating natural fibers surfaces. Modification of fiber

surface by fungal enzymatic activity is a novel method suggested in the literature. Gulati

and Sain (Gulati and Sain, 2006) treated hemp fibers with a fungus: Ophiostoma ulma,

obtained from elm tree infected with Dutch elm disease. The treated fibers showed

improve acid-base characteristics which, in turn, enhanced the interfacial adhesion.


2.4 Enzyme Catalysis

Catalysts are molecules reducing the magnitude of the energy barrier required for a chem-

ical reaction to take place. Enzymes, the natural catalysts, can perform efficiently under

mild conditions while preserving the functionality and characteristics of the biological

systems. Biocatalysis refers to the use of enzymes for catalysis reactions under artificial

conditions (i.e. in vitro), where they will be transformed into process catalysts able to

catalyze industrial processes under rough conditions.

Enzyme catalysis acts by changing the energetics of the reaction. Chemical thermo-

dynamic is able to explain the energy changes due to the equilibrium composition of the

system. Free energy controls the extent and direction of a chemical reaction which can

be quantitatively described by the equilibrium constant, K:

∆G = −RT lnK (2.12)

where R and T are the gas constant and absolute temperature, respectively.

Enzyme catalysis can be explained by the “transition state theory”. Based on this the-

ory, an intermediate is formed between the substrates and products (i.e. the transition-

state intermediate) in an enzyme catalyzed reaction. This intermediate has the configu-

ration of atoms in the maximum free energy, where the bonds of reactants are breaking

and new bonds are forming. The energy which is needed to form the intermediate is the

activation energy, ∆G∗. Enzymes, or more generally catalysts, accelerate the reaction by

reducing this activation energy (see Figure 2.2). However, Gibbs free energy is a state

function and therefore, enzymes are not changing the equilibrium constant of the overall

reaction. They can only accelerate the attainment of equilibrium.

Enzymes have been used as biocatalysts in textile industry due to their low envi-

ronmental impact and product quality enhancement (Araujo, Casal and Cavaco-Paulo,

2008). As in Table 2.5, enzymes can be used to synthesize/modify both natural and syn-


G

Reaction coordinate

Reactants

Products

Non-catalyzed path

Catalyzed path

∆G

∆G*

Figure 2.2: Reaction coordinate diagram.

thetic polymers. Hydrolytic and oxidative enzymes are being utilized for surface modifi-

cation of most widely used synthetic fibers in textile industry: Polyethylene terephtalate

(PET) (Fischer-Colbrie et al., 2004), polyamide (PA), and polyacrylonitrile (PAN) (Bat-

tistel, Morra and Marinetti, 2001). The surface modification is performed in order to

improve wettability, increase hydrophilicity, and also surface functionalization for easy

coupling with the finishing agents (e.g., flame retardants, dyes, etc.) (Fischer-Colbrie,

Heumann and Guebitz, 2006). Enzymatic surface modification has several advantages

over the conventional chemical ones, including milder treatment conditions and specific

non-destructive reactions limited to surfaces. Moreover, when using chemicals, their dis-

charge is causing environmental concerns (Araujo, Casal and Cavaco-Paulo, 2008). Thus,

for specific and non-destructive functionalization of fiber surfaces and also increasing their

surface energy, enzymes (from commercial sources) can be promising candidates.

To the knowledge of the author, the only work on the biological surface modification of

reinforcing fibers for biocomposite production is the one by Gulati and Sain (Gulati and

Sain, 2006). They employed a white rot fungus to increase the acid-base characteristics


Tab

le2.5:

Representation

ofpolymerssynthesized/m

odified

byenzymes

(Gubitz)

Modification

Synthesis

Synthetic

Polymers

•Polyethyleneterephthalate

•Polyamides

•Polyacrylonitrile

•Thesynthesisof

polyesterswithlipases

•Thesynthesisof

vinylsugars

withproteases

•Thesynthesis

ofphenolic

andacrylicpolymerswithoxi-

doreductases

Natural

Polymers

•Polysaccharides

•Lignocellulose

materials

•lign

in

•Thesynthesisof

cellulose

andXylanbyendoglucanases

•Thesynthesisof

chitin

bychitinase


of hemp fibers and consequently improve adhesion with acidic and basic resins. White-

rot fungi secrete one or more of three extracellular enzymes (Lignin peroxidase (LiP,

EC.1.11.1.14), manganese peroxidase (MnP, EC.1.11.1.13), and laccase (EC.1.10.3.2))

that are essential for lignin degradation and mineralization (Pointing, 2001).

2.4.1 Lignin Structure and Biosynthesis

Lignin is a statistically amorphous biopolymer and the second most abundant biopoly-

mer in nature. It is formed in plant cell walls by radical coupling of its phenylpropanoid

precursors (i.e. coniferyl alcohol, sinapyl alcohol, and p-hydroxycinnamyl alcohol) which

leads to the formation of an amorphous, polyphenolic material without a consistent re-

peating structure. The precursors are oxidized by one electron to resonance-stabilized

phenoxy radicals in a reaction which is catalyzed by laccase or peroxidases (Dean and

Eriksson, 1994). These reactions result in the complex 3-D structure of lignin with the

monomeric units bonded by carbon-carbon and ether linkages in different bonding pat-

terns. The reaction of these radicals with the oxygen of carbohydrates forms a stable

ether bond and links the aromatic moiety of the lignocellulose complex to the carbohy-

drate part which brings mechanical strength to the cell wall.

Due to the cross-linking of lignin with hemicellulose and cellulose, lignin is not di-

gestible by animal enzymes. However, some fungi and bacteria have the appropriate

enzymes to degrade lignin. The lignin-degrading fungi are the wood-decaying fungi in-

cluding brown rot, soft rot or white rot. White rot fungi secrete three classes of extra-

cellular lignin-degrading enzymes, manganese peroxidase (MnP), lignin peroxidase (LiP)

and laccase (Hatakka, 1994). The ligninolytic enzymes have to be much less specific than

the typical enzymes, since lignin consists of a mixture of stereo-irregular units (Hammel,

1997).


2.4.2 Laccase

Laccase is an oxidoreductase which contains four copper atoms bound to three oxidative

sites. Laccases can be found in many plants, fungi, and microorganisms, but fungi are

known to be the main producers of laccase. Laccase is known to cause polymerization as

well as depolymerization of lignin compounds through free-radical reactions (Felby et al.,

1997).

Free radicals formed by the enzymatic oxidation lead to depolymerization of polymeric

substances like lignin (Hammel, 1997) and a range of pollutants. These free radicals might

also cross-link and polymerize. Each of these reaction pathways is favorable for a specific

application. Laccases, due to their broad substrate specificity, are proposed for oxidation

of industrial pollutants including textile industry (Fu and Viraraghavan, 2001) and soil

pollutants such as chlorinated phenols (Baldrian et al., 2006).

In pulp and paper industry laccase has been applied commercially for pulp delignifi-

cation (biobleaching) (Bourbonnais and Paice, 1990) and also proposed as adhesive for

fiberboard production. In the former application, bonding of wood fibers through activa-

tion of surface lignin by laccase-catalyzed oxidation instead of toxic synthetic adhesives

occurs (Felby, Pedersen and Nielsen, 1997). This adhesion is linked to the formation

of free radicals in the lignin matrix of the fibers which further react with each other

to form covalent bonds in the manufacturing of lignocellulose-based composite materials

such as fiberboards (Couto and Herrera, 2006). Moreover, lignocellulosic fibers can be

functionalized by laccases in order to improve the chemical or physical properties of the

fiber products (Lund and Ragauskas, 2001). This application, i.e. grafting of different

functional groups on the fiber surfaces, may result in fiber materials with completely

novel properties such as hydrophobicity or charge (Couto and Herrera, 2006).


Figure 2.3: Laccase redox centers.

2.4.3 Laccase Mechanism

Laccase catalyzes the oxidation of various phenolic compounds. These proteins contain

4 atoms of copper, on three redox sites and are designated as Type 1, 2, and 3 copper

catalytic centers (Figure 2.3).

These copper atoms include one mononuclear site with a type-1 Cu (blue Cu) and a

trinuclear copper site containing one type-2 Cu (normal Cu) and two type -3 Cu (coupled

binuclear Cu) (Hakulinen et al., 2002). Substrates are oxidized near type-1 Cu, while

the electrons are transferred to type-2 and type-3 centers, where the molecular oxygen is

reduced. The overall outcome of the catalytic cycle is the one-electron oxidation of the

substrate with the simultaneous four-electron reduction of molecular oxygen to water.

The laccase catalysis can be summarized in 3 steps:

1. Oxidation of the substrate at the mononuclear center

2. Electron transfer from the mononuclear center to the trinuclear center

3. Reduction of oxygen molecule to water at the trinuclear center


However, neither the electron transfer mechanism nor the oxygen reduction to water is

fully understood (Hakulinen et al., 2002). The direct oxidation of the substrate molecules

to radicals by interaction with the copper centers is not always the case. In some cases,

the large size of substrate makes the contact with the laccase active site hard. This

problem can be overcome by the use of “chemical mediators”. These compounds act as

intermediate substrates which are able to penetrate and interact with the bulky or high

redox-potential substrate targets.

The laccase-catalyzed reactions might have two opposite fates: they can lead to poly-

merization by the cross linking of monomers or depolymerization of the existing polymers

(Claus, 2004). The destination is governed by the direction of the nonenzymatic reac-

tions. However, it is reported that the fate of the reaction depends also on the type of

laccase catalyzing the reaction as well as the immediate microenvironment of the reaction

(Hakulinen et al., 2002). In terms of different crystal structures of laccases, some laccases

are reported to have the structure which might trap the oxygen molecule. This reduces

the speed of the oxygen reduction to water, and giving a chance for the oxidized free

radicals in the surrounding environment to polymerize as opposed to structures in which

the rapid exchange of oxygen and water does not allow for the build-up of free radicals

in the microenvironment thus avoiding polymerization (Arora and Sharma, 2010).

2.4.4 Lignification by laccase

Enzymatic polymerization is defined as chemical polymer synthesis in vitro via non-

metabolic pathways catalyzed by an isolated enzyme (Kobayashi, Uyama and Kimura,

2001). Utilizing enzymes for organic synthesis has several advantages including the reac-

tion being carried out under mild conditions (in terms of catalysis temperature, pressure,

and pH), high selectivity, and being environmentally-friendly due to the nontoxic nature

of the catalyst.

It is known that the white-rot fungi are able to degrade lignin employing a cocktail


of oxidative enzymes, including laccases (Riva, 2006). However, further studies showed

that the treatment of pulp with laccase alone does not degrade lignin but instead leads to

structural changes and repolymerization (Haars and Huttermann, 1980; Bajpai, 1999).

Bajpai et al. concluded that even though laccases play an important role in degrading

the lignin in vivo, in vitro oxidation reactions catalyzed by the enzyme result in further

polymerization of the lignin. It is currently believed that phenoxy radicals are formed

by single-electron transfers from the lignin, and these radicals react further to form the

polymerized lignin (Huttermann et al., 2000). Moreover, the addition of phenols to the

reaction mixture, results in their reaction with the lignin molecule and consequently the

formation of copolymers (Milstein et al., 1993).

Huttermann et al. (Huttermann et al., 2000), showed that the incubation of a 20%

(w/w) aqueous solution of lignin with rather high concentrations of laccase and intensive

aeration leads eventually to a sudden increase in the reactivity of lignin with cellulose

followed by a sudden increase in the molecular weight of the incubated lignin. They

found that after about 6 hours of incubation with laccase and oxygen, more than 70% of

the lignin was bound to cellulose covalently. Their report is the first and only one so far

in the literature where an enzymatically catalyzed reaction of lignin with cellulose takes

place. They succeeded in enhancing the lignin polymerization by adding small amounts

of activated lignin to normal, non-activated lignin. They concluded that during the in-

cubation with laccase under aeration, stable phenoxy radicals are formed and acts as a

cross-linking agent for the added lignin and thus dramatically enhances the polymeriza-

tion of the untreated lignin with laccase. These phenoxy radicals are so active that they

are also able to react with nucleophiles such as cellulose or starch (Huttermann et al.,

2000).

In another work Huttermann et al. (Huttermann, Mai and Kharazipour, 2001) studied

the enzymatic copolymerization of lignin and alkenes to produce defined lignin-acrylate

copolymers while controlling the molecular weights of the products. Their work was


an attempt to enzymatically induce grafting of polymeric side chains onto the lignin

backbone, which might lead to the use of lignin as part of new engineering materials.

2.4.5 Lipase

The term lipid includes a great variety of compounds: mainly triglycerides(> 95%),

phospholipids, waxes, fatty acids, cholesterols, etc. In terms of their chemical structure,

lipids are esters of long-chain fatty acids and alcohols. In fixed oils and fats, the alcohol

is glycerol, while in waxes, the alcohol has a higher molecular weight, e.g., cetyl alcohol.

The most common lipids, triglycerides, are made from two kinds of molecules: glycerol

(a type of alcohol with a hydroxyl group on each of its three carbons) and three fatty

acids joined by dehydration synthesis. The “tail” of a fatty acid is a long hydrocarbon

chain, making it hydrophobic. The “head” of the molecule is a carboxyl group which

is hydrophilic. Moreover, when the head end is attached to glycerol to form a fat,

that whole molecule is hydrophobic. The lipid content of the secondary sludge, being

hydrophobic, is not contributing to the surface interactions between the sludge and the

hydrophilic Nylon resin. Therefore, reducing the hydrophobicity by breaking down the

lipid molecules might enhance the surface adhesion between the filler (sludge) and the

polymeric resin.

Lipases (EC 3.1.1.3) are a group of enzymes which catalyzes the hydrolysis of tria-

cylglycerols to fatty acids and glycerol. A number of lipases are available commercially

which are used for the production of fatty acids for chemical industry from triacylglyc-

erols, olive oil, soybean oil, coconut oil, palm oil, etc.

Lipase from Candida rugosa, a yeast, is a relatively cheap (Yu, Wu and Ching, 2004)

commercial enzyme. Lipase from Candida rugosa owes its popularity mainly to its high

activity in hydrolysis (Hernaiz, Sanchez-Montero and Sinisterra, 1994) of tryglycerols

regardless of the glycerol position (Macrae and Hammond, 1985). Lipase from Candida

rugosa lacks the stereospecificity of triglycerides and can catalyze the complete hydrolysis


of several oils to free fatty acids (Han and Rhee, 1985). Due to the non-consistent nature

of the sludge, the low selectivity of this lipase is advantageous.

2.5 Problem Statement

Currently the disposal of the secondary sludge is expensive and considered to be a threat

to the environment (Bruder-Hubscher et al., 2002; Kenny et al., 1995; Krigstin and Sain,

2006; Mahmood and Ellikot, 2006; Mabee, 2001; Sorum, Gronli and Hustad, 2001). The

practical way of using sludge in a value-added manner is as land-fertilizer (Werthera and

Ogadab, 1999; Krigstin and Sain, 2006) which is limited due to the concerns over the

risks of heavy metals and organic contaminants of the sludge (Werthera and Ogadab,

1999). This work aims to study the potential of the waste sludge to be used as filler in

composite industry.

Owing to its industrial significance, there have been many studies on the sewage

sludge characterization and composition (Wagner, 2005; Watson and Pletschke, 2006;

Rozich and Gaudy, 1992; Parmar, Singh and Ward, 2001; Poulsen and Hansen, 2003;

Kelly, Miller and Namazian, 2001; Honda, Miyata and Iwahori, 2002; Chen et al., 2002;

Dignac et al., 2000). However, literature on the pulp and paper sludge characterization

and composition is scarce (Jang et al., 2000). Determination of the contents of wood-

associated biopolymers in the sludge which contributes to the reinforcing potential of the

sludge as composite’s filler, is performed in this work.

Other than the secondary sludge composition, the thermogravimetric behavior of the

sludge and its surface thermodynamics are further explored in this work. These qualities

of the sludge which have not been looked at in the literature are essential for further

verification of the compatible polymeric matrices and appropriate processing conditions.

Surface modification of the filler/fiber to increase the compatibility of the composite’s

phases is known to enhance the adhesion and consequently the composite’s properties


(Mohanty, Misra and Drzal, 2002; Sain et al., 2005). Among different modification meth-

ods, enzymatic modification is mostly preferred mainly due to the selective, environment-

friendly and mild reaction conditions (Araujo, Casal and Cavaco-Paulo, 2008). However,

to the knowledge of the author, the only work on the biological surface modification of

reinforcing fibers for biocomposite production is the one by Gulati and Sain (Gulati and

Sain, 2006) which employed a white rot fungus to increase the acid-base characteristics

of hemp fibers. This work investigates the possibility of enhancing the sludge properties

as the reinforcing phase by enzymatic modification.

Laccase is an oxidoreductase which causes polymerization and depolymerizatoin of

lignin compounds through free radial reactions (Felby et al., 1997; Hammel, 1997; Haku-

linen et al., 2002; Claus, 2004; Riva, 2006; Arora and Sharma, 2010). Laccase has been

studied to be used in various industries (Fu and Viraraghavan, 2001; Baldrian et al.,

2006; Bourbonnais and Paice, 1990; Couto and Herrera, 2006; Lund and Ragauskas,

2001; Kobayashi, Uyama and Kimura, 2001). The repolymerization of lignin compounds

have been specifically studied (Huttermann et al., 2000; Haars and Huttermann, 1980;

Bajpai, 1999) and a few applications have been proposed. The use of laccase to increase

the molecular weight of the secondary sludge is suggested and explored in this work.

Chapter 3

Characterization of the Secondary

Sludge: Molecular Structure and

Cellular Biopolymers

3.1 Abstract

Secondary sludge produced by pulp and paper mills is the by-product of the biological

treatment of the waste stream and its disposal is a significant cost imposed on the treat-

ment plants. In order to identify the potential applications of this waste biosolid as part

of a new value-added biomaterial, qualitative and quantitative determination of its char-

acteristics are essential. In this chapter, FTIR spectroscopic technique was applied and

showed the existence of major functional groups of lignocellulosic materials and cellular

biopolymers. Further extraction techniques were employed to isolate and quantify the

wood-associated and cellular biopolymers. Soxhlet extraction with polar and nonpolar

solvents was performed to extract the low-molecular-weight substances where most of

the target substances (> 15% w/w) were extracted in 2 hours. The results of further

analyses were indicative of the presence of a considerable amount (> 30% w/w) of wood

32

Chapter 3. Molecular Structure and Cellular Biopolymers 33

biopolymers in the secondary sludge. Extraction of intra- and extracellular polymeric

substances from the secondary sludge was carried out by cation exchange resin (CER) and

alkali (NaOH). FTIR characterizations of the extracted cellular biopolymers revealed the

functional groups of lignin in the NaOH-extracted samples, which suggests this method

to be inappropriate for isolation of the cellular biopolymers from the pulp and paper mills

residues. High pressure size exclusion chromatography (HPSEC) was utilized to explore

the molecular weight distributions of the CER extracted cellular biopolymers. These

analyses confirmed that the secondary sludge consists mainly of wood biopolymers and a

considerable amount of proteins (> 30% w/w). Moreover, the majority of the extracted

proteins (> 80%) have molecular masses in the order of 104 Da.

3.2 Introduction

Waste sludge produced in the wastewater treatment plants is the by-product of the

mechanical/biological treatment of organic wastes. The mechanical and biological treat-

ments of the waste stream in a pulp and paper mill result in the production of primary

and secondary sludges, respectively. Primary sludge mainly consists of wood fibers and

fillers (Mahmood and Ellikot, 2006) and the one originating from the recycling plants

also contains synthetic materials such as plastics and stickies, as well as traces of glass or

metal (Mabee, 2001). Primary sludge has been proposed in literature to be used as filler

in thermoplastic composites (Jang et al., 2000; Jang and Lee, 2001; Park and Balatinecz,

1996; Son, Kim and Lee, 2001; Son, Yang and Kim, 2004). However, there has been no

such interest in the secondary sludge. Secondary sludge, typically the by-product of an

activated sludge process, is structurally very similar to the municipal sewage sludge since

it contains microbial cells and ash. However, unlike municipal sludge, it includes wood

biopolymers. In order to identify the potential applications of the secondary sludge in

the production of new biomaterials, characterization of the secondary sludge in terms of


their chemical and biochemical composition is essential.

The isolation and quantification of major wood-associated and cellular biopolymers

are the primary objectives of this chapter. For isolation and quantification of each com-

ponent in a mixture, and extraction technique is employed based on different solubilities

of the components in various solvents. Experimental solubility data can help choosing

the appropriate solvents for separation and purification of solid solutes. However, in the

case of polymers with high molecular masses (about 2 million) even the best match in

solubility parameters between solvent and solute will not lead to a true solution (Horvath,

2006). This is due to the fact that in addition to the solubility parameter, several other

factors affect the solubility, including molar volume, molecular surface area, polarity,

and H-bonding strength. Since secondary sludge consists of a mixture of different large

molecules with intramolecular forces, isolated constituents do not represent their natural

state in the biomass structure. Nonetheless, they can give indications on possible confor-

mation (Horvath, 2006). There are several standard methods recommended for isolation

and quantification of each of the wood-associated components considering their molec-

ular structure and interactions with each other. Extraction techniques adapted in this

work in order to quantify each of the biomass constituents are based on these standard

methods.

One major component of the secondary sludge is the cellular biopolymers. The sec-

ondary sludge contains both Intracellular (within the cellular membrane) and Extracel-

lular (within the polymeric network) Polymeric Substances (EPS). Several researchers

(Goh, Hemar and Singh, 2005; Garnier et al., 2005, 2006; Tuinier et al., 1999; Vaningel-

gem et al., 2004; Weinbreck et al., 2003) have studied the polymeric features of the EPS

in sludges due to their significant role in biofilm formation and holding the microbial mass

together. The EPS are complex mixtures of highly charged organic macromolecules which

are located at or outside of the cell surface independent of their origin. The extracel-

lular localization of EPS and their composition may be the result of different processes


including active secretion, shedding of cell surface material, cell lysis, and adsorption

from the environment (Wingender, Neu and Flemming, 1999). The EPS are composed

of a selection of organic substances. Proteins and polysaccharides are identified as its

predominant constituents, while humic substances (Liu and Fang, 2002), uronic acid and

deoxyribonucleic acids (DNA) have been detected in the EPS as well.

A couple of studies have been conducted on the evaluation of different EPS extrac-

tion methods (Comte, Guibaud and Baudu, 2006; Liu and Fang, 2002) and the relative

contents of different components of EPS. However, to the knowledge of the author, the

only study on the EPS extracted from pulp and paper mill sludge was done by Garnier et

al. (Garnier et al., 2005) which showed no major difference with the municipal sludge’s

EPS. According to various studies, the extracted EPS typically represents up to approx-

imately 15% of the sludge’s suspended solid (SS) (Frølund, Keiding and Nielsen, 1994;

Urbain, Block and Manem, 1993). However, quantification of EPS depends strongly on

the extraction method while there is no universal extraction procedure established so far

(Wingender, Neu and Flemming, 1999). The main forces involved in the binding of poly-

mers in the EPS matrix are the van der Waals forces, electrostatic interactions, hydrogen

bonds, hydrophobic interactions, and in some cases covalent bonds such as the disulfide

bonds in glycoproteins(McSwain et al., 2005). The extraction method is based on the

type of interactions keeping the EPS components together in the matrix. The extraction

methods used in this work are cation exchange resin (CER) and the alkali process. Both

of these methods are reported to have higher efficiencies compared to other extraction

methods (Frølund et al., 1996; Comte, Guibaud and Baudu, 2006).

These intra- and extracellular biopolymers, due to their high molecular weight and

functional groups, are expected to affect the miscibility and viscoelastic properties of

the secondary sludge as part of a biocomposite and consequently the final properties

of the manufactured composite. Therefore, the biochemical composition and molecular

weight distribution of these cellular biopolymers helps identifying the sludge-compatible


polymeric resins.

The specific physicochemical properties of EPS and its molecular weight distribution

have been of major interest due to the ongoing debates about the role of large biopolymers

in contributing to good sludge settalibility (Comte, Guibaud and Baudu, 2007; Frølund

and Keiding, 1994; Grner et al., 2003). During the past decade, HPSEC (Comte, Guibaud

and Baudu, 2007; Frølund and Keiding, 1994; Grner et al., 2003; Garnier et al., 2005) ,

and before that low-pressure size exclusion chromatography (SEC) (Goodwin and Forster,

1985) have been used to determine the molecular weight of the extracted EPS samples.

However, the HPSEC is established to separate the EPS into more distinct groups (i.e.

peaks) (Comte, Guibaud and Baudu, 2007). Garnier et al. (Garnier et al., 2005) looked

at the molecular weight and nature of EPS from various activated sludges. They reported

the molecular weight of proteins to be varying from small (10 kDa) to large (600 kDa)

numbers, while all polysaccharides were smaller than 1 kDa.

Quantification of proteins and polysaccharides of the sludge, and the extracted cel-

lular biopolymers due to the high molecular weight of proteins and high functionality of

polysaccharides is also of major significance. The amount of EPS extracted, and thus

the protein and carbohydrates contents are strongly dependant on the extraction method

used (Liu and Fang, 2002). The conventional quantitative chemical analysis methods are

based on extraction, purification and photospectrometric analysis. In this chapter, the

total polysaccharides of the extracted cellular biopolymers were determined using An-

throne method and the total proteins were measured according to Lowry method, and

also Kjeldahl test.


3.3 Materials and Methods

3.3.1 Secondary Sludge Sample

The aerobic activated sludge samples were taken from two different pulp and paper

mills: AbitibiBowater newsprint and kraft pulp mill, Thunder Bay, Ontario and Tembec

paperboard and kraft pulp mill, Temiscaming, Quebec. The AbitibiBowater and Tembec

sludge samples are noted as B and T in this chapter, respectively. The samples were kept

at 4◦C to minimize the bacterial activity and were not stored for more than one month.

The samples were dried in the oven at 102◦C to constant weight. In order to disrupt

the microorganisms’ cell walls in the characterization process, mechanical method utiliz-

ing the rotating blades was selected. The sample then passed through a mesh (0.3 mm)

in order to produce a fine homogeneous powder for further characterization experiments.

3.3.2 Moisture and Ash Content

The moisture content of the sample was measured through the testing procedure de-

scribed in the TAPPI (Technical Association of the Pulp and Paper Industry) Standard

T 412 om-94 (TAPPI 1996). In this method, samples from each batch were weighed

and placed in the oven at 103◦C (±3◦C) for 48 hours. Then the samples were removed

from the oven and placed in a desiccator immediately to cool down without re-absorbing

moisture. The samples were weighed again and the moisture contents were calculated as

a fraction of the oven-dried weight of the specimen.

The ash content of the secondary sludge was determined based on the procedure

presented in the TAPPI Standard T 211 om-93 (TAPPI 1996). The ash contents of

the samples were calculated gravimetrically after complete combustion of 2 g samples at

525◦C in a furnace and expressed in relation to the dry weight of the original samples

specimen.


3.3.3 Extractive Content Determination

The testing methods chosen to determine the extractive content were based on the pro-

cedure presented in the TAPPI Standard T 264 om-88 (TAPPI 1996). However, due to

the carcinogenic effects of benzene, in the case of benzene/ethanol extraction, benzene

was substituted by toluene. Two soxhlet extraction stages employing a 1:2 mixture of

95% ethanol and analytical grade toluene, and by 95% ethanol were followed by pure

water digestion each for 4 hours. The residuals were used to determine the percentage

of cellulose, hemicellulose and lignin constituents in the following steps.

In order to monitor the amount of the extracted substances, the absorbance of the

solvent was recorded as a function of time. Absorbance was measured with Shimadzu

spectrophotometer (model UV-160, Tokyo, Japan) at 300 and 290 nm for the ethanol-

toluene and ethanol extractions, respectively. These wavelengths were the ones with the

highest absorbance for each solvent. Since the extracts may not be in soluble form at

ambient temperature, the absorbance was measured immediately after taking the sample

out of the extraction system.

3.3.4 Isolation and Quantification of Lignocellulosic Materials

The cellulose and hemicellulose contents of the extractive-free biosolid were determined

using the procedure adapted from Zobel and McElwee (Zobel and McElwee, 1958). This

method uses acid, sodium hydroxide, and chlorite reduction in order to decompose lignin

and protein fractions of the sludge, leaving behind the resistant holocellulose. The residue

is further reduced in order to distinguish the cellulose and hemicellulose fractions.

In this method, extractive-free samples (0.70 ± 0.01 g) were weighed and placed in

tared Erlenmeyer flasks. Then, 10 ml of the acid solution (60 ml glacial acetic acid and 20

g NaOH per liter of distilled water) was added to each flask. 1 ml of a chlorite solution

was added and stirred into each flask, before placing them in a 70◦C hot water bath


for 4 hours. Chlorite solution was added to the flasks after 45, 90, and 150 minutes.

Upon removal from the hot water bath, the content of each flask was transferred to an

oven-dried, tared coarse crucible and suction was applied to remove the liquid part. The

remaining solids were washed with acetic acid and a final rinse with acetone to remove

the moisture was carried out. The dried crucibles were weighted to determine the final

percentage.

Klason lignin determination is based on what was originally proposed by Hagglund

(Hagglund, 1951). In this method, Klason lignin determination is based on the acidic

digestion of polysaccharides of the sample. This method is based on the assumption that

lignin does not degrade significantly in acidic mediums. This method has been codified

in TAPPI standard T 222 om-88 (TAPPI 1996).

All tests were run in triplicates and averaged.

3.3.5 Extraction of Cellular Polymers

In order to disrupt the cell walls and extract the mixture of intra and extracellular

polymeric substances, freezing and thawing of the sludge samples 3 times prior to the

extraction process were carried out. Cellular biopolymers from the two sludge samples

were extracted by cation exchange resin and alkali extraction. Before the extractions,

the activated sludge samples were harvested by centrifugation (15000× g, 20 min, 4◦C),

then washed by resuspension in double distilled water and centrifuged again (15000× g,

20 min, 4◦C).

In CER extraction procedure, DOWEX 50×8, 20-50 mesh in the sodium form (Fluka)

was used based on what proposed by Frølund et al. (Frølund et al., 1996). The CER

was washed for 1 hour in the extraction buffer, then filtered and used for the extraction.

Extraction time was 4 hours due to the best extraction yield (Garnier et al., 2005). In

the alkali extraction procedure the pellets were resuspended to the original volume in

NaOH (Caledon, 97%) at pH 11, for 4 hours and 4◦C.


After the extraction, the samples were centrifuged at 15000 × g to remove the cells.

In order to completely remove the microbial cells filtration by a 0.5 µm filter (millipore

FH) was performed. For further biochemical analysis, part of the samples was frozen at

−20◦C and finally freeze-dried hours under vacuum conditions.

3.3.6 Biochemical Analysis

The freeze-dried extracted cellular biopolymers were dissolved in double distilled water

before analyzed for total polysaccharide and proteins. The total polysaccharides in the

EPS were determined using Anthrone method as described by Gaudy (Gaudy, 1962)

where glucose used as the standard in the range 0-100 mg/L. The total proteins were

measured according to Lowry method with bovine serum albumin (BSA) as the stan-

dard in the range 0-60 mg/L. Absorbances were measured with a Beckman UV-Visible

spectrophotometer (USA) at 750 nm and 578 nm for protein and polysaccharide mea-

surements, respectively. Also, Kjeldahl test was employed to measure the total nitrogen

content, then using the conversion factor of 6.25 the protein content was estimated.

3.3.7 Chromatographic Method (HPSEC analysis of cellular

biopolymers)

The separation of the extracted cellular biopolymers was carried out with a DIONEX

DX600 chromatograph equipped with an AD25 absorbance detector. A Nucleogel GFC

1000-8 column (Macherey-Nagel, Duren, Germany) of 300× 7.7 mm, with 8 mm particle

size and a size separation range of 1 × 104 to 1 × 107, was employed for the analytical

separation. The detection was carried out at room temperature (25◦C) at the wavelength

of 280 nm. The mobile phase used consisted of 9mM NaCl and 0.9 mM Na2HPO4. All

samples were filtered with 0.2 µm filters (Millipore) before injection (25 µL).

Five protein standards (Cytochrome C: 12.4 kDa, Carbonic Anhydrase: 29 kDa,


Albumin: 66 kDa, Alcohol Dhydrogenase: 150 kDa, -Amylase: 200 kDa) were used for

calibration. The reproducibility of the chromatograms was checked and the influence of

samples storage time was also tested.

3.3.8 Fourier Transformed Infrared Spectroscopy (FTIR)

The sludge and cellular biopolymer samples were characterized by Fourier Transformed

Infrared (FTIR) spectroscopy. A Perkin Elmer spectrum 1000 (Perkin Elmer Life and

Analytical Sciences Inc., Waltham, MA, USA) was used to obtain the spectra of each sam-

ple. The powdered samples were mixed with KBr (sample/KBr ratio: 1/100) and pressed

into a disc of 1mm thick. The IR spectra were collected in the range 4000− 500cm−1

using TENSOR 27 spectrometer with a resolution of 4cm−1. The spectral outputs were

recorded in the absorbance mode as a function of wavenumber. The obtained spectra

were subtracted from the background daily measures on dried KBr pellet standard.

3.4 Results and Discussion

3.4.1 FTIR Spectra of the Secondary Sludge

Spectroscopy experiments were performed for preliminary characterization of the biosolid

to identify the major functional groups. The FTIR spectra of the sludge samples (T and

B) are presented in Figure 3.1. The similarity of the obtained spectra illustrates the close

chemical structure of the various sludge samples; the obtained spectra also demonstrate

the existence of lignocellulosic compounds, proteins, lipids and polysaccharides. How-

ever, distinguishing these chemicals by the FTIR spectra is not easy due to their similar

functionalities.

As illustrated in Figure 3.1, a broad band at 3550− 3100cm−1 is observed which

corresponds to the H-bonded OH groups of alcohols, phenols and organic acids, as well as

the H bonded N-H groups. The intensive absorption bands at 2850, 2920, and 2960cm−1


Figure 3.1: FTIR spectra of sludge samples (B and T)

corresponding to the respective symmetrical and asymmetrical -CH- vibrations (Schmitt

and Flemming, 1998), belong to the cellular lipids (Sheng, Yu and Yue, 2006). The

shoulder at 1728cm−1 is corresponding to C=O stretching of protonated -COOH groups

(Laberge et al., 1997). Wide and intensive absorption bands at frequencies of 1630

and 1531cm−1 could be identified, corresponding to the characteristic vibrations of the

-CONH- group (Schmitt and Flemming, 1998) of Amide I and Amide II in proteins

(Comte, Guibaud and Baudu, 2006). The small shoulder at 1510cm−1 is originated

from lignin and lignocellulosic structure (Smidt and Meissl, 2007). The less intense

bands between 1450 and 1380cm−1 corresponds to several chemical groups such as CH3,

OH of phenols, COO- and/or ortho-disubstituted aromatic rings (Amir et al., 2004).

In the 1255cm−1 region, a typical band of phosphate group absorption was observed,

which can be attributed to nucleic acids (Sheng, Yu and Yue, 2006). Wide and intensive

carbohydrate bands between 900 and 1250cm−1 belong to C-O stretching of carbohydrate

and alcohol functions which reflect the occurrence of polysaccharides or cellulose (Comte,

Guibaud and Baudu, 2006). The fingerprint zones (below 1000cm−1) of the two spectra

are almost identical which confirms their similar chemical structure.

The experiments provide evidence of qualitative comparability between the sludge

and the natural lignocellulosic material components namely extractives, cellulose, and


lignin. However, distinguishing these chemicals by the FTIR spectra is not easy due to

their similar functionalities (e.g., functional groups of polysaccharides originated from

microorganisms and wood fibers).

3.4.2 Biomass Fractionation: Extraction Method

For an exclusive extraction, the solubility of a substance is a vital piece of information.

Even if the molecular structure of a substance is not known, its solubility characteristics

can be of great help in predicting its behavior in a solution. In the secondary sludge,

large molecules of wood fiber including cellulose, hemicellulose, and lignin are bound

together where hemicellulose acts as a surfactant bonded to both cellulose and lignin.

This is due to the fact that hemicellulose has backbone and side groups favoring energet-

ically to cellulose environment and lignin environment, respectively. Also biopolymers

associated with the microorganisms including proteins and polysaccharides are present

in the biomass. The considerable interactions of these polymers results in the crucial role

of the solvent diffusion in their solubilities. Unfortunately, due to the large number of

variables involved, the thermodynamic principles of the solubility of such a structurally

complicated material in various liquids (e.g., solvents, oil, etc.) have not been adequately

investigated (Horvath, 2006). Therefore, the standard methods recommended for isola-

tion and quantification of each of these components have been adapted while considering

the specific molecular structures and their interactions with each other.

The first step in the isolation of the wood-associated biopolymers is to extract out

the low-molecular-weight substances. In case of the secondary sludge, these extractives

are the nonstructural wood constituents (e.g., fats and waxes, phenolic compounds, etc.)

along with the low molecular weight polysaccharides and lipids associated with the mi-

croorganisms. The TAPPI Standard T 264 om-88 (TAPPI 1996) was applied, however,

benzene was substituted by toluene. Considering their close solubility values (Barton,

1975), it is believed that this substitution does not affect the experiment. The extractions


with nonpolar solvents (1:2 mixture of 95% ethanol and toluene, ethanol) are supposed to

remove a number of substances from the sludge including waxes, fats, resins, salts, wood

gums, phytosterols, and non-volatile hydrocarbons. Distilled water as a polar solvent

was used to remove substances unaffected by the ethanol or ethanol-toluene extractions,

including tannins, gums, sugars, and coloring matter that may be present in the sludge.

The residue left after the extraction is supposed to consist of wood biopolymers (lignin,

cellulose and hemicellulose) and proteins. Cellulose is distinguished from proteins and

hemicelluloses by its insolubility in aqueous alkaline solutions, and from lignin by its

relative resistance to oxidizing agents and susceptibility to hydrolysis by acids (Horvath,

2006). The presence of one primary (C6) and two secondary (C2, C3) hydroxyl groups

in an anhydroglucose unit of cellulose predetermines the occurrence of a system of inter-

and intramolecular hydrogen bonds in the polymer. This strongly confines the applica-

tion of one component solvents suitable for practical use (Bochek, 2003). On the other

hand, hemicelluloses are easily hydrolyzed by acids to their monomeric compounds and

lignin has a very low solubility in most solvents. Thus, after extraction of the low-

molecular-weight substances acidic digestion isolates lignin and acid chlorite reduction

isolates holocelluloses. Further reduction by basic solution will separate cellulose and

hemicellulose.

The moisture content of both samples was 97.5± 0.5%. All numbers presented here-

after are dry-basis percentages (w/w). The total amounts of substances extracted from

the two secondary sludge samples were 17±1.3% and 16.5±0.5%. The extraction residue

should be mainly composed of the three wood polymers as well as proteins. The content

of cellulose, hemicellulose and lignin were obtained to be 21±3, 8±4, and 15±3 percent

of the dry mass, for sludge B and 19 ± 3, 9 ± 2, and 16 ± 3 for sludge T, respectively

(ash-free content). The ash content of the secondary sludge samples were measured to

be 17± 0.5% and 16.5± 1% for sludge samples B and T, respectively. The results of the

isolation and quantification of the extractives, ash and wood-associated biopolymers of


0

10

20

30

40

50

60

Extractives Cellulose Hemicellulose Lignin Ash

Dry mass (%)

Secondary sludge (B)

Secondary sludge (T)

Wood fiber

Figure 3.2: Comparing the obtained results for the secondary sludge constituents with

typical wood fibers (Pettersen, 1984) constituents

the sludge samples along with the constituents of typical wood fibers (Pettersen, 1984)

are presented in Figure 3.2. Typical wood fiber is a mixture of 40-50% cellulose, 20-35%

hemicellulose (polysaccharides), and 15-30% lignin along with some extractives (3-5%)

and minerals (1-2%) (Pettersen, 1984).

It can be readily observed from Figure 3.2 that the extractive and ash content of the

sludge samples are extremely high compared to typical wood fibers. The high amount

of minerals can be explained by the chemicals added during the pulp and paper making

process as well as the metals present in the recycled papers. Moreover, clay is often added

to the pulp as filler in order to improve the characteristics of the paper for printing or

writing. Clay minerals are also expected in the sludge. Moreover, a considerable portion

of the extraneous substances is expected to be lipids, polysaccharides and other low-

molecular-weight substances associated with microorganisms. The extractives, lignin,

cellulose, hemicellulose, and ash contents obtained for the sludge samples B and T are

not statistically different at P = 0.1 level (Table 3.1). Thus, in spite of the heterogeneity

of the sludge samples, they are similar in terms of their main constituents.


Table 3.1: Wood-associated biopolymers quantified in sludge samples B and T

B Sludge Sample

(percentage w/w)

T Sludge Sample

(percentage w/w)

t-ValueSignificance

(df=3, P=0.1)

Lignin 15± 3 16± 3 0.52 No

Cellulose 21± 3 19± 3 1.05 No

Hemicellulose 8± 4 9± 2 0.5 No

Extractives 17± 1.3 16.5± 0.5 0.80 No

Ash 17± 0.5 16.5± 1 1 No

3.4.3 The Dynamics of the Extraction Process

The objective of the soxhlet experiment is to extract the low-molecular-weight substances

from the sludge, leaving behind the biopolymers including proteins, cellulose, hemicel-

lulose and lignin. Studying the dynamics of this process can be helpful for finding the

optimum extraction time, operating parameters, and for future industrial implementa-

tion.

Here, due to the numerous known and unknown low-molecular-weight substances

extracted from the sludge, quantification of the extracted substances is a very difficult

task. Thus, in order to study the dynamics of the extraction process, spectrophotometry

was selected as the analysis method for determining the concentration of the extracted

substances. Beer-Lambert law or Beer-Lambert-Bouguer law states that the fraction of

light absorbed by a system does not depend on the incident spectral radiant power, and

the absorbance is proportional to the number of the constituent molecules absorbing the

radiation. It can be concluded that there is a linear relationship between the absorbance

and the concentration of the absorbing species. Thus, here, absorbance is a qualitative

measure of the concentration of the extracted substances.


0

0.5

1

1.5

2

2.5

3

0 100 200 300

Extraction time (min)

Absorbance at 300 nm

0

0.1

0.2

0.3

Absorbance at 290 nm

300 nm - ethanol-

toluene (1:2 v/v)

290 nm - ethanol

Figure 3.3: Effect of extraction time on absorbance at 300 nm, extraction by ethanol-

toluene (1:2 v/v) and 290 nm, extraction by ethanol at boiling temperature.

Figure 3.3 shows the evolution of absorbance of the extracts for the two soxhlet

extractions with ethanol-toluene and ethanol, respectively. It can be observed that after

about 3 hours of extraction there will be no significant change in terms of the extractives

concentration.

Figure 3.4 shows the initial kinetics of the two extraction processes in logarithmic

scale. Based on the straight lines varying in slope for each solvent, the extraction process

follows first-order kinetics. Considering the Beer-Lambert law, for the calculation of k0 in

each case, absorbance can be used instead of the concentration. The calculated k’s can be

used for comparing the reaction rates under different conditions. The fact that the slopes

are of the same order here, shows that both solvents are of the same significance in terms

of the extraction kinetics. However, the final concentrations of the extracted substances

are influenced by the composition of solvents. In conclusion, a first-order reaction can

represent the global kinetics of extraction. This approach can also be used in order to

find the optimum temperature and solvent concentration for maximum extraction yield.


y = 0.003x - 0.1394

R2 = 0.9247

y = 0.0054x - 1.3506

R2 = 0.9762

-1.6

-1.2

-0.8

-0.4

0

0.4

0.8

0 50 100 150 200

Extraction time (min)

log (A)

Ethanol: Toluene

Ethanol

Figure 3.4: Kinetic of the extraction processes (logarithmic scale)

3.4.4 Quantities of Cellular Polymeric Substances Extracted

from the Sludge

The extraction method significantly affects the yield and structure of the extracted cel-

lular biopolymers (Wingender, Neu and Flemming, 1999). The extraction efficiency

of different methods is the common debate in EPS extraction studies. Frølund et al.

(Frølund et al., 1996) compared different extraction procedures and found the most ef-

fective method to be the CER extraction. While results obtained by Liu and Fang (Liu

and Fang, 2002) showed the base extraction to be the most efficient one. Both of these

methods have been applied in this work. The extraction methods employed here, CER

and NaOH, have been able to extract 16±2.5%, and 24±2% (dry weight basis) of the

sludge sample B and 17±3%, and 26±1.5% from the sludge sample T. However, since

this work mainly focuses on the qualitative characterization of the extracted cellular

biopolymers, the effect of different process variables on the extraction yield have not

been studied.

To extract EPS from the bacterial sample, the different linkages in the EPS matrix

should break in order to facilitate the release of EPS into the water. Addition of NaOH


results in the ionization of several charged groups, since the isoelectric points in proteins

are generally below pH 4-6. Consequently, ionization of proteins causes a strong repul-

sion between the cellular biopolymers which results in a higher water solubility of the

compounds (McSwain et al., 2005). While in CER extraction, the divalent ions of the

cellular polymeric matrix (mostly Ca2+ and Mg2+) are attracted by the active reagent

and replaced with monovalent cations. Similar to the alkali extraction, the created repul-

sion between cellular biopolymers results in a higher water solubility of the components

leading to the extraction to take place. On the other hand, for extraction of the cellular

biopolymers, disrupting the cell walls is necessary. After cell lysis, intracellular biopoly-

mers can be extracted along with the extracellular polymeric substances. Since EPS is

partly originated from autolysis, it can be assumed that its polymeric features are not

that different from the intracellular polymeric substances.

Unfortunately, there are no standardized methods established for analysis of proteins

and polysaccharides of the waste sludge. In order to measure the amount of proteins and

polysaccharides in the extracted EPS different methods and reagents have been employed

in the literature including Anthrone and phenol-sulphuric for polysaccharide determina-

tion and Lowry, Bradford, and Kjeldahl tests for protein measurements. The methods

employed in this work are Anthrone for polysaccharide measurement and both Lowry

and Kjeldahl methods for protein determination. Since different components of the cel-

lular biopolymers show different accessibility for extraction, all extractions in this work

were done for the same duration of time. The results are presented in Table 3.2 and are

within the range of the values reported in literature (Dignac et al., 1998; Liu and Fang,

2002; Leppard et al., 2003; Sheng, Yu and Yue, 2006; Dogsa et al., 2005). As expected

(Comte, Guibaud and Baudu, 2006), not only the yield of extraction, but also the con-

stituents vary for different extraction methods. It can be observed that CER method is

capable of extracting more proteins which agrees with literature results (Comte, Guibaud

and Baudu, 2006). These results also indicate that proteins, the high molecular weight


Table 3.2: Polysaccharide and protein content of sludge samples and extracellular poly-

meric substances extracted by NaOH and CER methods

SampleAmount (mg g−1 suspended solids)

Polysaccharide (Anthrone) Protein (Lowry) Protein (Kjeldahl)

Sludge (B) 270± 5 360± 7 346.8± 0.4

Sludge (T) 245± 6 331± 2 307.2± 0.7

CER- B 37.5± 3 257± 4 280.1± 1.0

CER- T 29± 2 201± 3 220.7± 2.1

NaOH- B 67± 1 231± 3 201.8± 1.0

NaOH- T 71± 5 233± 1 190.01± 0.4

portion of the cellular biopolymers, make up for more than 30% of the sludge samples.

Clearly, not all the proteins in the sludge can be extracted by these extraction methods.

However, since all the proteins are originated from the microorganisms, it can be assumed

they have similar properties. Therefore, the results obtained for characterization of the

CER extracted proteins by HPSEC can be attributed to the total protein content.

3.4.5 FTIR Spectra of Extracted Cellular Biopolymer Samples

The Fourier transform infrared (FTIR) spectroscopy has been employed in this study

as an effective and easy technique to compare and characterize the main components of

the extracted cellular biopolymer samples. The spectra of the secondary sludge samples

from two different mills and cellular biopolymer extracted by two different methods were

obtained. These experiments provide verification of comparability between the sludge

samples and the cellular biopolymer samples extracted by different methods.

Comparing the spectra obtained for EPS samples extracted by different methods

(CER and NaOH) and from different sludge sources (Figure 3.5 and Figure 3.6), it can


be observed that samples extracted by similar extraction methods show similar band

positions and band shapes. Moreover, cellular biopolymer samples extracted by different

methods, exhibit very distinct fingerprints (wavelengths < 1000cm−1). Nonetheless, all

samples have similar spectra from 3500 to 1600cm−1. Broad bands at 3500 to 3300cm−1

in all the obtained spectra, corresponding to the stretching vibrations of OH groups, are

indicative of proteins and polysaccharides. All cellular biopolymer samples, extracted

by NaOH or CER methods, have absorption bands at 2850, 2920, and 2950cm−1, corre-

sponding to the respective symmetrical and asymmetrical -CH- vibrations due to cellu-

lar lipids (Schmitt and Flemming, 1998). Additionally, all biopolymer samples exhibit

peaks around 1630cm−1 which is attributed to stretching C=O of proteins (Garnier et al.,

2005). For the samples extracted by NaOH method, the absorption bands at frequencies

of 1630 and 1570cm−1 are attributed to the characteristic vibrations of the -CONH- group

(Schmitt and Flemming, 1998) of Amide I and Amide II in proteins (Comte, Guibaud

and Baudu, 2006). However, for the samples extracted by CER, the absorption bands of

Amide I are much stronger than Amide II, which is almost completely masked. The amide

bands can provide information on the structural properties of the cellular proteins. Here

the bands at 1637− 1624cm−1 have been assigned to β-sheet or β-turns conformation

(Omoike and Chorover, 2004).

A band appearing at 1400cm−1 in the EPS samples extracted by CER method is

due to the symmetric stretching of carboxylate (-COO-) anions of proteins (Omoike and

Chorover, 2004). Carboxylate ions are also expected to exist in the EPS samples extracted

by NaOH which are most probably masked by the very intense peak at 1430cm−1. Both

EPS samples extracted by NaOH, show distinct, sharp peaks around 1430cm−1 which

is absent in the spectra belong to CER extracted samples. The band at 1430cm−1

corresponding to COH in-plane deformation with aromatic ring stretching, is almost

certainly (Pandey, 1999) due to lignin extracted along with the EPS.

The band observed around 1150cm−1 has been allocated to exocyclic vibrations of


the C-O-C bond or the glycosidic bridge (Kaurkov et al., 2000). The band appear-

ing at 1080cm−1 is assigned to the complex vibrations involving the stretching of the

C6-O6 bond and the deformational vibrations of the C4-C5 bond (Shingel, 2002). Cellu-

lar biopolymers extracted by alkali have the same spectral shape but with considerably

diminished bands at 1080cm−1 and 1150cm−1 compared to the samples extracted by

CER method. This can be explained by the glycosidic cleavage reaction due to alkaline

hydrolysis during the extraction by NaOH. In this process, glycosidic linkages in car-

bohydrates are cleaved through hydrolysis which also results in the reduction of overall

molecular weight. Diminishing of the bands at 1080cm−1 and 1150cm−1 establishes the

polysaccharides hydrolysis during the 4 hours of alkaline extraction. The fingerprint area,

wavelengths less than 1000cm−1, is specific for each extraction method which can be at-

tributed to the specific interactions of each extracting agent with the cellular biopolymer

samples.

In spite of the similar peak positions, the differences in absorption ratios suggest

differences in the contributions of particular functional groups in the extracted samples.

It can be observed that the absorption ratio for the protein peaks (1600− 1700cm−1) and

the polysaccharides (3500− 3300cm−1) are not similar for different samples and different

extraction methods, suggesting that the relative contents of proteins and carbohydrates

are different.

One major conclusion from the FTIR spectra is the presence of lignin in the alkali

extracted biopolymers. It is known that, unlike municipal sludge, the secondary sludge

contains lignin and it has been extracted along with the cellular biopolymers in the case

of alkali extraction. Therefore, for further molecular weight-determination experiments

the alkali extracted samples have been excluded.


Figure 3.5: FTIR spectra of EPS samples extracted by NaOH

Figure 3.6: FTIR spectra of EPS samples extracted by CER

3.4.6 Chromatographic Separation

HPSEC method is known to be the most precise and successful method for molecular

weight determination of EPS samples (Comte, Guibaud and Baudu, 2006). The molecular

weight distribution of the sludge proteins is very significant since it affects the viscosity

and miscibility with other polymers and consequently the biocomposite’s final properties.

In this work, HPSEC has been applied to determine the molecular weight distribution of

the cellular polymeric samples and also as a means for comparing the samples extracted


from different sludge sources.

3.4.7 Choices of Working Wavelength

In literature, the choices of working wavelengths for the HPSEC chromatogram have

been 280, 260, and 210 nm (Comte, Guibaud and Baudu, 2007; Frølund and Keiding,

1994; Garnier et al., 2005; Grner et al., 2003). The results showed no significant difference

between chromatograms of 280 and 260 nm with other parameters kept constant (Comte,

Guibaud and Baudu, 2007; Frølund and Keiding, 1994). However, for the wavelength

of 210 nm no absorbance was recorded (Comte, Guibaud and Baudu, 2007). Based on

the results on literature the wavelengths of 260 and 280 nm are chosen for the HPSEC

procedure where, again, no significant difference between the obtained chromatograms

was observed.

3.4.8 Mobile Phase and the Flow Rate

Frølund and Kieding (Frølund and Keiding, 1994) studied the effect of mobile phase ionic

strength and mobile phase pH. They reported that using a mobile phase of pH 7 results

in the separation of EPS into the maximal number of peaks. This pH also does not

cause irreversible column absorption. Based on their results pH 7 is selected in this work

for the mobile phase. The mobile phase in this study consisted of 9mM NaCl and 0.9

mM Na2HPO4; since it is known to give the highest number of separated species for the

extracted EPS in similar studies on sludge (Comte, Guibaud and Baudu, 2007; Frølund

and Keiding, 1994; Grner et al., 2003).

Flow rates of 1.0 mL/min and 0.5 mL/min were chosen in this study for the mobile

phase and the results are shown in Figure 3.7 and Figure 3.8 where the lower flow rate

revealed two more peaks. Thus, lower flow rate increases the sensitivity, i.e. separa-

tion efficiency. However, further reducing the flow rate did not increase the separation

efficiency.


Figure 3.7: HPSEC chromatograms obtained for CER extracted samples at flow rate of

1 ml/min, Signal at 280 nm, injection volume of 25 µL

3.4.9 Molecular Size and Weight Distribution

Calibration of molar mass to retention time was achieved by linear regression of standards

of nominal molecular masses of 12.4 kDa, 29 kDa, 66 kDa, 150 kDa, 200 kDa.

Chromatograms of the extracted biopolymer samples extracted from two different

sludge sources (B and T) showed similar fingerprints. It can be concluded that the

cellular biopolymers of the two different sludge sources have similar chemical composition.

Nonetheless, it should be noted that the two different sludges in this work are coming

from the same industrial origin (i.e. pulp and paper secondary sludge). This phenomenon

which has been observed previously (Wloka et al., 2006) can be attributed to two possible

causes: 1) The extracted biopolymers might have gone through chemical alterations by

the extracting reagents, or 2) each extraction method, extracts specific proteins and

polysaccharides, selectively. However, since the mechanism of biopolymer’s extraction is

not completely known, the contribution of each cause will remain unclear. Moreover, the

majority of the extracted biopolymers are in the same range of molecular mass: based on


Figure 3.8: HPSEC chromatogram obtained for CER extracted samples at flow rate of

0.5 ml/min, Signal at 280 nm, injection volume of 25 µL.

the calibration data around 80% of each chromatogram corresponds to molecular mass

of 50× 103 Da.

Garnier et al. (Garnier et al., 2005) compared the chromatograms of EPS samples ex-

tracted from municipal sludge with EPS extracted from industrial sludge. They observed

different profiles where industrial EPS samples exhibited fewer peaks. In this study, also,

not more than 4 peaks are detected, whereas EPS from municipal sludge has been sep-

arated into 7 peaks by HPSEC (Garnier et al., 2005; Grner et al., 2003; Frølund and

Keiding, 1994). The fewer peaks observed in the EPS samples extracted from pulp and

paper secondary sludge can be attributed to the different microbial cultures characteristic

for each type of sludge at industrial plants.

The chromatograms, each show 4 peaks detected for biopolymers extracted by CER

method from the two different sludge samples (Figure 3.8). Comparing the two chro-

matograms, the peak retention volumes are almost identical. The calibration of the

column by protein standards, resulted in the determination of the molecular weights

associated with chromatograms peaks assuming that the extracted proteins and the pro-


tein standards have the same mechanism interacting with the column. In case of the

biopolymers extracted by CER method, the main contribution was attributed to the

molecular mass of 50 × 103 Da which is about 80% of the chromatogram area. Other

peaks were attributed to molecular masses of more than 2× 106 Da (outside of the cali-

bration range), 12× 103 Da, and 1 kDa, where they contributed only a minor portion of

the chromatogram area.

3.5 Conclusions

Pulp and paper sludge identification in terms of the content of extractives, wood-associated

and cellular biopolymers, their molecular weight distribution, and functionality, is the key

to finding new applications for this solid waste in biocomposite industry. To gain further

insight into the molecular structure of these biopolymers both FTIR spectroscopy and

HPSEC were employed in this chapter. The FTIR spectroscopy allowed identification of

the major functional groups in both sludge and the extracted cellular biopolymers. The

FTIR experiments provided evidence of qualitative comparability between the sludge

and the natural lignocellulosic materials. The detection of lignin in the spectra of the

NaOH extracted cellular biopolymers proves this extraction method to be inappropriate

for pulp and paper mills secondary sludge. Proteins were determined to be a major

component of the secondary sludge (>30% dry weight). In order to study the molecu-

lar weight distribution of this major component, HPSEC was applied to the extracted

cellular biopolymers.

Based on the results of this chapter, the secondary sludge has the potential to be used

as binder material in compatible polymeric matrices due to the dominant portion of high

molecular-weight proteins. Further characterization of the secondary sludge in terms of

its thermogravimetric behavior and surface energy is presented in the next chapter in

order to help find the appropriate polymer matrices compatible with this waste biosolid.

Chapter 4

Secondary Sludge Characterization:

Surface Thermodynamics and

Thermal Behavior

4.1 Abstract

Secondary sludge from pulp and paper mills can be considered as potential filler for

composite industry. This chapter studies the potential of secondary sludge from pulp

and paper mills in terms of its surface thermodynamics and thermogravimetric behavior

to be used as filler for composite production. To this end, a secondary sludge sample

was characterized through different techniques including Inverse Gas Chromatography

(IGC) and differential scanning calorimetry (DSC). The dispersive component of surface

energy for the secondary sludge samples was obtained to range from 60 to 42 mJ/m2

measured at 313− 373◦K with dominant acidic nature. Thus, the biosolid has a higher

dispersive component compared to Nylon and the opposite polarity which may result

in an acceptable Nylon-sludge adhesion. The thermal analysis showed that the sludge

samples, a heterogenous mixture of biopolymers, act as a homogeneous blend. Moreover,

58

Chapter 4. Surface Thermodynamics and Thermal Behavior 59

the processing temperature should not exceed 200◦C due to the decomposition of the

biosolid.

4.2 Introduction

Biocomposites are defined as biopolymers or synthetic polymers reinforced with natural

fibers which are alternatives to artificial fibers such as carbon, aramid, and glass fibers

(Krigstin and Sain, 2006). During the past two decades, there has been a growing urgency

to develop new bio-based products from renewable agricultural and forestry feed stocks

in order to unhook the widespread dependence on fossil fuels and alleviate the major

cause of environmental pollution. Biocomposites are widely used for building, automo-

tive (Mohanty, Misra and Drzal, 2002), and packaging applications and are extending

their horizon even to aerospace applications. Natural fibers have several advantages

over synthetic fibers including low cost, low density, acceptable strength properties, ease

of separation, carbon dioxide sequestration, and biodegradability (Mohanty, Misra and

Drzal, 2002; Han et al., 2005). Composite’s mechanical properties strongly depend on

the effectiveness of stress transfer between the fibers/fillers and the matrix. All plant-

based natural fibers are lignocellulosic in nature with cellulose, a hydrophilic linear glucan

polymer with alcoholic hydroxyl groups, as their main constituents. Although the chem-

ical structures of cellulosic components of different natural fibers are the same, they

have different mechanical properties due to the different degrees of polymerization (DP).

Different polymeric resins have diverse affinities towards the fibers/fillers owing to the dif-

ference in their chemical structure and surface energies. Due to the significance of stress

transmission from the matrix to the fiber, interfacial adhesion between these two phases

plays an important role in composite’s physical properties and performance (Holbery and

Houston, 2006). Therefore, the compatibility of the two phases is necessary. In order

to assess the compatibility of the biosolid with common polymeric matrices, the surface


thermodynamics (i.e. surface energy) of the raw and modified sludge samples should be

determined. Adhesion theory and the thermodynamic approach can help explaining and

quantifying the degree of compatibility between the phases of a biocomposite.

So far, different theories have been proposed explaining the scientific concept of ad-

hesion including: mechanical interlocking, electronic theory, theory of weak boundary

layers, and the thermodynamic theory. The thermodynamic model of adhesion which

is currently the most widely used approach in the adhesion science (Shultz, Laville and

Martin, 1987), states that adhesion takes place mainly due to weak, short-range, polar

and non-polar intermolecular interactions at the interface, provided that the intimate

contact is achieved. Fowkes (Fowkes, 1964) suggested that the surface free energy (γ)

of a given body could be represented by the sum of the contributions of different types

of interactions, such as dispersion forces (D), dipole interactions (P ), hydrogen bonding

(H), and metallic bonds (M):

γ = γD + γP + γH + γM . . . (4.1)

Based on this theory, the perfect fiber-matrix adhesion happens when the liquid

resin completely “wets” or “spreads on” the fiber surface. The wetting requires the

surface tension of the fibers/fillers to be greater than that of the resin. Thus, in order

to predict the interactions across filler/matrix interface, information about the filler’s

surface is required. There are several methods available for characterizing the filler surface

and filler/matrix interface properties. Inverse Gas Chromatography is known to be a

convenient yet reliable tool to get semi-quantitative estimate of the dispersive component

of the surface tension and acid-base characteristics of the polymers and reinforcing fibers

(Hildebrand, 1936; Gulati and Sain, 2006). It is reported to be a more reliable technique

compared to wetting techniques (Heng et al., 2007). The obtained data via IGC is

significant since it helps predicting the degree of interaction, and hence adhesion, between

the filler and the selected polymeric matrix in advance.


The thermal behavior of polymers and biopolymers should be known in order to

employ the appropriate manufacturing techniques. To this end, Differential Scanning

Calorimetry has been employed in this work. The DSC measures the heat flow, dH/dt,

transferred to or from the sample as the temperature of the sample holder, T, is changed

at a constant rate, dT/dt = β (Brown, 2001) rendering the heat capacity of the system,

dH/dT :

dH/dt = dH/dT × dT/dt (4.2)

For an absolutely pure compound with zero melting range, dH/dt would become

infinite at the melting point, T0. For an impure compound, dH/dT is finite and is a

function of T. The DSC measures the specific heat capacity, heat of transition, and the

temperature of phase changes and melting points

Moreover, cellular biopolymers and biofibers are complex mixtures of organic materi-

als and thus, thermal treatment leads to physical and chemical changes. The prolonged

exposure to high temperature may result in discoloration, volatile release, poor interfa-

cial adhesion, or embrittlement of the natural fibers. As such, only thermoset polymers

with low curing temperatures and thermoplastic matrices with lower melting points can

be applied in natural fiber biocomposites. Therefore, the processing temperature which

is to be applied for biopolymers and natural fibers should be determined. In this work,

Thermal Gravimetric Analysis (TGA) technique was chosen to verify the decomposition

temperature. The TGAmeasures the mass loss of the sample as a function of temperature

and hence presenting the temperatures of phase changes, reactions or decomposition.

In summary, the primary goal of this chapter is to assess the feasibility of utilizing pulp

and paper mill secondary sludge as functional and/or cheapening filler in biocomposite

manufacturing by determining the surface energy and thermogravimetric behavior.



4.3.1 Materials

The aerobic activated sludge samples were collected from two different pulp and paper

mills: AbitibiBowater newsprint and kraft pulp mill, Thunder Bay, Ontario and Tembec

paperboard and kraft pulp mill, Temiscaming, Quebec. The AbitibiBowater and Tembec

sludge samples are noted as B and T, in this chapter, respectively. The samples were

kept at 4◦C to minimize the bacterial activity and were not stored for more than one

month. The sludge samples were centrifuged at 15000× g for 15 minutes and the pellets

were dried in the oven (102◦C) over night before the IGC test.

4.3.2 Inverse Gas Chromatography

Inverse Gas Chromatography data were obtained utilizing a Perkin-Elmer 8500 gas chro-

matograph equipped with a flame ionization detector (FID) system. For the preparation

of IGC columns, the dried samples were crushed and sieved (< 0.3 mm) and packed into

a copper column using a vibrator. All columns used, were 33 cm in length and 14in in

diameter. Helium was used as the carrier gas. The corrected flow rate of helium ranged

from 10 to 36 ml/min. Small quantities of volatile probe (less than 10 µL) of known

properties injected into the columns using Hamilton microsyringes. Methane was used

as the non-interacting probe (i.e. Marker). The experiments were performed at 40, 60,

80, and 100◦C for the sludge samples.

Multiple injections of probes into the columns (at least 3 times) were performed in

order to confirm the peaks reproducibility. No significant change in the residence time

or the shape of the peaks was observed. The reproducibility of the peaks rules out the

permanent sorption of the probes into the sample (Swaminathan, Cobb and Saracovan,

2006).


4.3.3 Thermogravimetric Analysis

TGA analysis has been carried out by TGA analyzer by TA Instrument (Q500) at a heat-

ing rate of 10◦C/min in a nitrogen atmosphere. DSC measurement has been performed

with DSC analyzer by TA Instrument (Q1000) at a heating rate of 10◦C/min in nitrogen

environment, over a temperature range of 40 to 160◦C.

4.4 Results and Discussions

4.4.1 Dispersive Component of the Surface Free Energy

IGC experiment is carried out via injections of small (µL) amounts of volatile probes

(mobile phase) with known properties, through a column packed with the solid of un-

known properties (stationary phase). The probes are carried through the column by an

inert carrier gas. The retention time of each probe is related to their interaction with

the stationary phase. Since the theory and principles of IGC have been well explained

in other articles (Riedl and Mauana, 2006) only the critical equations and assumptions

are mentioned here. Since the volume of the injected solutes (i.e., probes) are extremely

small, in the order of 10µL or less, the interactions between solid and the particular probe

are measured by the net retention volume (Vn). Vn is the volume of carrier gas required

to elute the probe from the column and it is defined by the following equation:

Vn = Q0J(tr − t0) (4.3)

where tr and t0 are the retention times of the probe and of a non-interacting probe

(methane here), respectively. Q0 is the measured flow rate (ml/min), and J (James-

Martin compressibility factor) is the correction factor due to the pressure drop along the

chromatographic column which is determined via the following equation:


J =3

2

1−(Pi

Po

)21−

(Pi

Po

)3 (4.4)

where Pi and Po are the inlet and outlet pressures of the carrier gas, respectively.

From the measured specific net retention volume, the free energy of adsorption can

be calculated as follows:

−∆Gd = ∆Ga = RTc lnVn + C (4.5)

where ∆Gd and ∆Ga are changes of the standard free energy for isothermal desorption

and adsorption of each probe from standard gaseous state to a standard adsorption

state on the surface (Riedl and Mauana, 2006), respectively. C is usually treated as a

constant for a given chromatographic column depending on the reference states (Riedl

and Mauana, 2006).

The free energy of adsorption, ∆Gd, is related to the work of adhesion, WA, between

the probe molecule and the stationary phase, per unit surface area of the solid by:

∆Ga = RTc lnVn + C = N.a.WA (4.6)

where, N is the Avogadro’s number and a is the surface area of an absorbed probe

molecule.

The Fowkes (Fowkes, 1964) approach can be used to determine the free energy of

adsorption and the dispersive component of the stationary phase when only dispersion

interactions are being exchanged (i.e., when nonpolar probes are used). The energy of

adhesion between two species interacting only by dispersion, can be described by the

geometric mean of the free surface energy (Fowkes, 1964) and is given by:

WA = 2√γDS γ

DL (4.7)


Table 4.1: Characteristics of the nonpolar probes used in the analysis

Probe Area (A2) γDL (mJ/m2)

n-Hexane 51.5 18.4

n-Heptane 57 20.3

n-Octane 62.8 21.3

n-Nonane 68.9 22.7

where γDS and γD

L are dispersive components of surface energies of the stationary phase

and the probe, respectively. The two equations can now be written as:

RTC lnVn = 2N√γDS a

√γDL + C (4.8)

Based on the above equation, RT lnVn is a linear function of the quantity a√γDS .

Values of a and γDL of the probes used in this work are presented in Table 4.1.

Based on the Fowkes approach, the dispersive components of the surface free energies

for four different temperatures are determined. For each temperature and each substrate

of study, the calculated values of RT lnVn were plotted against a√γDL as seen in Figure

4.1 for one of the sludge samples. This figure also includes the data obtained from the

studies of polar probes, which will be discussed later in this chapter. The good correlation

coefficients obtained in each of these plots for n-alkane lines, show a linear relationship

illustrating that IGC technique is a good choice for the studied substrates. At each

temperature, the slope of this linear fit gives the dispersive component of the surface

free energy (γDS ) as listed in Table 4.2. More details on the calculations and graphs

are provided in Appendices A-C. It can be observed that, as the temperature increases,

the dispersive part of the surface free energy is decreasing. Fagelman (Fagelman and

Guthrie, 2005) attributed this to the trend that the weaker the dispersive interactions,

the easier it would be to remove the molecules from the surface. Since there was no


y = 0.0843x - 16.389

R2 = 0.9996

0

2

4

6

8

10

12

170 220 270 320 370

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol] Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

353ºK

Figure 4.1: Determination of acid-base contribution to the free energy of adsorption:

IGC data of n-alkanes polar probes for B-sludge sample

Table 4.2: Surface characteristics (determined by IGC) of the sludges

TypeγDS (mJ/m2)

Ka Kb

40◦C 60◦C 80◦C 100◦C

Bowater Sludge 52 50 49 42 0.113 0.0397

Tembec Sludge 60 58 55 51 0.1196 0.0138

significant variation within the temperature range tested, the change in the chemical

nature of each substrate can be ruled out. Hence, the variations are most likely due to

the increase in the molecules kinetic energies. Since wetting requires the surface energy

of the fibers/fillers to be greater than that of the resin, these values being higher than the

conventional plastics, in theory, means that the polymeric resins can “wet” the biosolid.

4.4.2 Acid-Base Interactions

Polar probes have both dispersive and acid-base interactions with the stationary phase.

Therefore, the total free energy of adsorption, ∆Gtota , can be calculated by adding the


dispersive, ∆GDa , and acid-base, ∆GAB

a , contributions:

∆Gtota = ∆GD

a +∆GABa (4.9)

In order to determine the contribution of the acid-base properties of the stationary

phase, polar probes must be injected into the column. Based on Gutmann’s acid-base

concept (Guttmann, 1983) the enthalpy of adsorption can be calculated from the variation

of the free energy of adsorption with temperature:

∆GABa = ∆HAB − T∆SAB (4.10)

where ∆SAB is the entropy of adsorption. The plot of ∆GAB vs. Temperature for each

probe results in a linear line with ∆HAB as the intercept.

According to Papirer’s approach solid surfaces can be characterized by the numbers

describing their acidic (Ka) and basic (Kb) characteristics:

∆HAB = KaDN +KbAN (4.11)

where DN (kcal/mol) and AN (dimensionless) are electron donor (base) and electron

acceptor (acid) numbers of the probes, respectively (Guttmann, 1983). DN and AN of

the probes used in this work are presented in Table 4.3.

Based on Equation (4.11), a plot of ∆HAB/AN vs. DN/AN should result in a straight

line and Ka and Kb can be determined from the slope and intercept at the origin of this

straight line, respectively.

The error of the measurement of retention time was less than 3%. Since the surface

energy is derived through a series of calculations based on the retention time data, the

final error for the surface energy can be calculated through “error propagation”. The

error propagation is a mathematical procedure which allows calculating the influence of

errors of one measurement in another value based on them. As such, here, the errors are


Table 4.3: Characteristics of the polar probes used in the analysis

ProbeArea

(A2)

γDL

(mJ/m2)

DN

(kcal/mol)

AN

(dimensionless)

Character

Chloroform 44 25.9 0 23.1 Acidic

Ethyl Acetate 48 19.6 17.1 9.3 Amphoteric

Ethyl Ether 47 15 19.2 3.9 Basic

Tetrahydrofuran 45 22.5 20 8 Basic

Acetone 42.5 16.5 17 12.5 Amphoteric

caused by the experimental measurements of the retention times by the IGC device. In

theory, the influence of these errors on the final surface energy values can be calculated

through error propagation. However, the error estimates for non-linear functions are

biased (Taylor, 1997). Specifically, here, the bias on the error calculated for lnVn increases

as Vn increases since the expansion to 1 + Vn is a good approximation only when Vn is

small. Moreover, the error propagation in a long sequence of steps such as IGC does not

give reliable final errors (Loukopoulos et al., 2004; Gavril et al., 2005; Atta et al., 2004).

Therefore, the errors of the calculated surface energies are not reported.

A range of polar probes (Table 4.3) are used in this work to determine the acid-base

interactions. Similarly as for the n-alkanes, the calculated values of RT lnVn were plotted

against a√γDL (Figure 4.1). The calculated ∆GAB was then plotted versus temperature

which resulted in a straight line with intercept equal to ∆HAB (Figure 4.2).

Based on the obtained intercepts, a plot of ∆HAB/AN versus DN/AN was generated

(Figure 4.3). According to the slope and intercept of the obtained linear plot, Ka and

Kb were determined, respectively. These values are summarized in Table 4.2.

As shown by the obtained results in Table 4.2, both secondary sludge samples are


0

1

2

3

4

5

6

7

8

300 320 340 360 380 400

Temperature (K)

∆GAB(kJ/mol) Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

Figure 4.2: Determination of the specific component of the enthalpy of adsorption and

the entropy of adsorption for each of the polar probes for secondary sludge (B)

y = 0.113x - 0.0397

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25

DN/AN

∆H/AN

Ethyl Ether

Acetone

THF

Ethyl Acetate

Chloroform

Figure 4.3: Determination of Ka and Kb for secondary sludge (B)

showing higher values of Ka than Kb. This dominant acidic nature can be related to

the electron acceptor character of the hydroxyl group’s protons in cellulose. Table 4.4

shows the surface free energy components of a few polymeric matrices and wood fibers

of different species reported in the literature. As presented in literature (Huang et al.,

2006), Nylon has a lower dispersive component compared to the secondary sludge and the

opposite polarity (Table 4.4) which should result in an acceptable Nylon-sludge adhesion.

Wood fibers are mainly composed of cellulose and hemicellulose and the high polarity

of cellulose and hemicellulose causes strong attraction to polyamide chains by dipole-

dipole interactions or hydrogen bonding. Moreover, both Nylon and sludge proteins


Table 4.4: Surface energies of a few commonly used polymeric resins

Polymer

material

Ka Kb γDS (mJ/m2) Temperature (K) Reference

PES 0.086 1.523 32.09-27.2 333.2-363.2 (Zhang et al., 2007)

PC 0.09 0.48 33.7-29.3 353-393(Santos, Fagelman and

Guthrie, 2002)

PE 0.328 0.189 27.92-19.35 353-383 (Zhao et al., 2008)

PS 0.085 0.339 37.2 333(Felix, Gatenholm and

Schreiber, 1993)

PP 0.012 0.004 32.8 333(Felix, Gatenholm and

Schreiber, 1993)

Nylon 66 2.03 7.17 37.4-47.5 343.2-373.2 (Huang et al., 2006)

have amine-amid functional groups which enhances the adhesion. These interactions are

expected to provide a reinforcing effect which probably leads to a biocomposite material

with acceptable strength and stiffness.

4.4.3 Thermogravimetric Behavior

In order to examine the thermal stability of the extracted cellular polymeric substances,

the samples were tested by TGA technique. The TGA was performed in a TGA-50,

Shimadzu. The samples were heated from room temperature to 600◦C at a rate of

10◦C/min under nitrogen atmosphere. The result showed that the thermal decomposition

of the sample occurs around 200◦C. Based on the TGA result, DSC experiment was

performed below this temperature in a nitrogen atmosphere and at a heating rate of

10◦C/min.


Figure 4.4: The DSC thermogram for the sludge sample

A miscible blend of polymers in known to form a homogeneous mixture and to present

a single glass transition or melting temperature (Meireles et al., 2007). Therefore, pres-

ence of a single peak in the thermogram (Figure 4.4) demonstrates the compatibility of

the different constituents of the sample with each other (Wong and Lam, 2002). It should

be noted that the occurrence of a miscible polymer blend requires specific interactions

between the polymers at the molecular level. On the other hand, an immiscible blend of

polymers results in multiple Tgs, similar to the isolate materials Tgs. Thus, from the first

scan (Figure 4.4) it can be concluded that even though the extracted cellular biopolymers

consist of different biopolymers such as proteins, polysaccharides, lipids, etc. they act as

a homogenous blend of polymers when it comes to their thermal behavior. This counts

as a significant advantage for the sludge biopolymers at the processing stage.

4.5 Conclusions

In order to utilize the secondary sludge as filler to produce a composite with acceptable

mechanical strength and good adhesion of the phases, an appropriate polymeric resin

should be selected. Therefore, characterization of the sludge in terms of the surface

energy and thermogravimetric behavior was carried out as the first step. To achieve


good adhesion, the surface energy of the fiber/filler should be greater than that of the

resin. In this chapter, the surface dispersive and acid-base energies of the secondary

sludge samples from two different pulp and paper mills were characterized by inverse gas

chromatography. The dispersive component of surface energy for the secondary sludge

samples was found to range from 60 to 42 mJ/m2 measured at 313− 373◦K. The surface

energies being greater than those of most common polymeric resins indicates those resins

are capable of “wetting” the biosolid. In the previous chapter, Nylon was proposed

as the appropriate polymeric resin due to its similar functional groups with proteins.

Considering the surface energy results presented in this chapter, Nylon has a higher

dispersive component (Huang et al., 2006) compared to the secondary sludge and the

opposite polarity which, in theory, should result in an acceptable Nylon-sludge adhesion.

Moreover, in this chapter the thermal behavior of the secondary sludge was studied

by the TGA and DSC techniques. Based on the TGA results, the biosolids were found to

decompose beyond 200◦C. For future composite production, the processing temperature

should be chosen based on these findings. In addition, the DSC thermograms showed

that even though the sludge biopolymers are structurally different and have a rather

wide molecular weight distribution, they act like a homogeneous blend. In conclusion,

the results of this chapter support the idea of using secondary sludge as filler in polyamide

composites.

Chapter 5

Enzymatic Modification of Sludge

5.1 Abstract

Enzymatic modifications of waste secondary sludge from pulp and paper mills to reduce

the hydrophobicity and increase the molecular weight were carried out by lipase and lac-

case, respectively. The enzymatic modification was performed to enhance the reinforcing

capability of the secondary sludge for further composite production. The lipid content of

the secondary sludge, which was measured to be 6±0.5%, was hydrolyzed by lipase from

Candida rugosa and the structural changes were followed by fourier transform infrared

(FTIR) spectroscopy.

Laccase from Trametes versicolor was tested for its activity and reaction rate in

the secondary sludge and the alkali-extracted lignin. Characterization of the sludge

before and after the laccase treatment was carried out by FTIR spectroscopy. High-

pressure size exclusion chromatography (HPSEC) was applied to determine the molecular

weight distribution of the lignin samples and also as a means for comparing modified and

unmodified samples. Biokinetic parameters for the Michaelis-Menten kinetic model as

a function of dissolved oxygen concentrations were determined and the Km and the

Vmax values were calculated for the sludge and the alkali extract. The FTIR results on

73

Chapter 5. Enzymatic Modification of Sludge 74

the laccase treated secondary sludge showed clear changes in the molecular structure

which was mainly attributed to the crosslinking reactions and generation of new bonds.

Moreover, the HPSEC results revealed that laccase modifies the sludge by increasing the

molecular weight.

5.2 Introduction

Secondary sludge, which is proposed to be used as filler in composite industry, contains

cellular and lignoncellulosic biopolymers. These biopolymers, due to their high molecu-

lar weight and functionality, can potentially contribute to the thermodynamic compat-

ibility and final properties of the manufactured composite. Therefore, modification of

the sludge in terms of increasing the biopolymers’ molecular weights or eliminating the

non-contributing elements can increase the viability of utilizing this biosolid as filler in

composite industry.

The lipid content of the secondary sludge, being hydrophobic, is not contributing to

the surface interactions between the sludge and the hydrophilic Nylon resin. Therefore,

reducing the hydrophobicity by breaking down the lipid molecules might enhance the sur-

face adhesion between the filler (sludge) and the polymeric resin. Lipase from Candida

rugosa, is chosen here to catalyze the hydrolysis of triglycerides to fatty acids and glyc-

erols, thus, to reduce the hydrophobicity of the sludge. Due to the non-consistent nature

of the sludge, the low selectivity of this lipase is advantageous (Macrae and Hammond,

1985).

The high content of lignin in the secondary sludge can be to the advantage of a variety

of applications. Enzymatic modification of this waste biosolid in terms of increasing the

molecular weight of the lignin component is expected to contribute to the final properties

of the manufactured biomaterials.

Lignin is a statistically amorphous biopolymer without a consistent repeating struc-


ture which is formed in plant cell walls by radical coupling of its phenylpropanoid precur-

sors (i.e. coniferyl alcohol, sinapyl alcohol, and p-hydroxycinnamyl alcohol). The precur-

sors are oxidized by one electron to resonance-stabilized phenoxy radicals in a reaction

which is catalyzed by laccase or peroxidases (Dean and Eriksson, 1994). These reactions

result in the complex 3-D structure of lignin with the monomeric units bonded by carbon-

carbon and ether linkages in different bonding patterns. The reaction of these radicals

with the oxygen of carbohydrates forms a stable ether bond and links the aromatic moiety

of the lignocellulose complex to the carbohydrate part which brings mechanical strength

to the cell wall (Hatakka, 1994).

Laccase is an oxidoreductase which can be found in many plants, fungi, and microor-

ganisms, but fungi are known to be the main producers of laccase. It is known that

white-rot fungi are able to degrade lignin employing a cocktail of oxidative enzymes,

including laccases (Riva, 2006). However, further studies showed that the treatment of

pulp with laccase alone does not degrade lignin but instead leads to structural changes

and repolymerization (Haars and Huttermann, 1980). Bajpai et al. (Bajpai, 1999) con-

cluded that even though laccases play an important role in degrading the lignin in vivo,

in vitro oxidation reactions catalyzed by the enzyme result in further polymerization of

the lignin. It is currently believed that phenoxy radicals are formed by single-electron

transfers from lignin, and these radicals react further to form the polymerized lignin

(Huttermann et al., 2000).

This chapter describes the enzymatic modification processes for the pulp and paper

mill secondary sludge with considerable lignin and lipid content. The objective of this

chapter is to study the effectiveness of lipase treatment on the secondary sludge in terms of

the chemical changes. Moreover, the effects of laccase application on the sludge in terms

of the reaction rates, molecular weight, and the chemical structure were determined.



5.3.1 Secondary Sludge Sample

The aerobic activated sludge samples were taken from AbitibiBowater newsprint and

kraft pulp mill, Thunder Bay, Ontario. The samples were kept at 4◦C to minimize the

bacterial activity and were not stored for more than one month.

5.3.2 Lipase

Lipase from Candida rugosa (E.C.3.1.1.3, Type VII) was purchased from Sigma-Aldrich

Chemical (Oakville, ON). The lipase is a crude preparation with a nominal specific

activity of 835 U/mg. 1 U corresponds to the amount of enzyme which liberates 1 µmol

oleic acid per minute at pH 8.0 and 40◦C.

5.3.3 Laccase

Laccase (EC 1.10.3.2) from Trametes versicolor was purchased from Sigma-Aldrich Chem-

icals (Oakville, ON) with an activity of > 20 U/mg. The actual activity of the enzyme

was determined in this work.

5.3.4 Assay of Laccase Activity and Kinetic Studies

Laccase activity was determined by the oxidation of ABTS (Jonas et al., 1998). The

assay mixture contained 18µM ABTS in 0.10 M sodium acetate buffer (pH 5.0), and

laccase. The linear absorbance increase due to the oxidation of ABTS was measured at

420 nm in 1 cm cuvettes and at an incubation temperature of 25◦C. One unit of laccase

activity was defined as the amount of enzyme required to oxidize 1µmol of ABTS per

minute. Assays were replicated three times.

For reaction rate determination, the experiments were carried out in 50 ml Pyrex


flasks containing 50 ml of the sludge sample/alkali extract and 5 ml of the sodium acetate

buffer (pH=5) in which laccase was dissolved (0.017 U ml−1). The dissolved oxygen probe

was immersed in the liquid using a clamp while not touching the flask walls. The flask

was sealed to prevent the dissolution of more oxygen in the liquid during the reaction.

When required and prior to reaction, the dissolved oxygen concentration were increased

by sparging air through the sludge/alkali extract solution while controlling the level with

the DO meter. Oxygen consumption during the laccase reaction was measured with a

YSI 52CE dissolved oxygen meter at room temperature (25◦C) and under atmospheric

pressure which was calibrated after each reaction. A magnetic stirrer at a 100 rpm was

used to mix the reaction medium. The reaction was started with the addition of the

laccase solution and the dissolved oxygen concentration was continuously monitored.

5.3.5 Lipid Measurement

Lipids were measured by toluene extraction in the Soxhlet apparatus. The dried sludge

samples were crushed and sieved (0.3 mm mesh) and then transferred into a cellulose

extraction thimble. The thimble was then inserted into a Soxhlet apparatus and extracted

for 6 hours (5-6 cycles/h). The crude extracts were carefully evaporated by rotary vacuum

evaporator at 60◦C and the rest of solvent was removed in the oven before the FTIR

spectroscopy.

5.3.6 Klason Lignin Determination

Klason lignin determination is based on what was originally proposed by Hagglund (Hag-

glund, 1951). Klason lignin determination is based on the acidic digestion of polysac-

charides of the sample. This method has been codified in TAPPI standard T 222 om-88

(TAPPI 1996).


5.3.7 Alkali Extraction of Lignin

Lignin from the sludge samples were extracted by 1N NaOH solutions. Before the ex-

tractions, the activated sludge samples were harvested by centrifugation (15000 × g, 20

min, 4◦C), then washed by resuspension in double distilled water and centrifuged again

(15000×g, 20 min, 4◦C). The extraction took place for 4 hours at 4◦C. The samples were

then centrifuged at 15000×g to remove the cells. The solution was also filtered to remove

the remaining microbial cells. The solution was then dialyzed against sodium acetate to

reduce the pH. These extracts went through further modification by the enzyme before

the molecular weight determination.

5.3.8 Enzymatic Treatment

The lipase from Candida rugosa exhibits an optimal activity at 37◦C and at pH 7.5 (Khor,

Tan and Chua, 1986) and hence, the reactions were carried out at this condition. Due to

the buffer capacity of the sludge, no pH adjustment was made. The reactors will be loaded

with 100 ml (1 g TS) of secondary sludge and 10 mg of enzyme product. The hydrolysis

was carried out for different incubation times (4-24 h). To stop the hydrolysis, 50 ml

of 50:50 (v/v) mixtures of acetone in ethanol were added to the samples to denature

the enzyme (Mendes, Pereira and Castro, 2006). The samples were then centrifuged

(at 10000 g, for 15 min) and dried in the oven overnight before further analysis. All

experiments were carried out in triplicate.

Laccase (approximately 500 U) was added to a 100 ml of sludge/alkali extract, which

was subsequently incubated for 4 h at room temperature (25◦C) on an orbital shaker.

The treated sludge was then centrifuged and the pellets were oven-dried for further

FTIR analysis. On the other hand, to follow the changes in molecular weight, the alkali

extracted solutions were used. Before analysis by HPSEC chromatography, the extracts

were diluted approximately 10 times and the chromatographic separation was performed


right after the enzymatic treatment.

5.3.9 Fourier Transformed Infrared Spectroscopy (FTIR)

The extracted and the modified samples were characterized by Fourier Transformed In-

frared (FTIR) spectroscopy. A Perkin Elmer spectrum 1000 (Perkin Elmer Life and Ana-

lytical Sciences Inc., Waltham, MA, USA) was used to obtain the spectra of each sample.

The powdered samples were mixed with KBr (sample/KBr ratio: 1/100) and pressed into

a disc of 1 mm thick. The IR spectra were collected in the range 4000− 500 cm−1 using

TENSOR 27 spectrometer with a resolution of 4 cm−1. Spectral outputs were recorded

in the absorbance mode as a function of wave number.

5.3.10 Chromatographic Method

High pressure size-exclusion chromatography (HPSEC) of the treated and untreated alkali

extracts were carried out with a DIONEX DX600 chromatograph equipped with an AD25

absorbance detector and a PSS MCX column (1000A, 300 × 8 mm). 0.5 M NaOH was

used as the mobile phase and at a flow rate of 1 ml/minute. The UV detection was

carried out at room temperature (25◦C) at the wavelength of 280 nm. All samples were

filtered with 0.2 µm filters (Millipore) before injection (25 µL). Five protein standards

from Sigma Aldrich were used for calibration. The reproducibility of the chromatograms

was checked by three replicate experiments.

5.4 Results and Discussions

5.4.1 Lipid Content of the Sludge

Microorganisms of the secondary sludge are responsible for the pollution removal from

the waste stream. The main constituent of the microorganisms are proteins, polysac-


Figure 5.1: FTIR spectra of the toluene-extracted lipids from the secondary sludge.

charides, humic substances and lipids. Lipids are the hydrophobic components of the

sludge which are expected to affect the composite’s properties adversely. Quantification

of lipids depends on the extraction method where no universal extraction procedure is

established so far. The toluene extraction method which was employed in this work, was

able to extract 6± 0.5%, (dry weight basis) of the sludge samples.

The FTIR spectra of the extracted lipids are presented in Figure 5.1. It shows nine

distinct absorption bands over the wave number range 3500-800 cm−1. The bands at 2924

and 2852 cm−1 are originating from stretch vibrations of C-H groups of the hydrophobic

chains (fatty acids) of lipids. The very sharp and distinct band at 1730 cm−1 is assigned

to stretching vibrations of the -C=O group of fatty esters. The -C=O vibrations of fatty

acid should be observed at 1710-1715 cm−1 which is most probably masked by the band

at 1730 cm−1. The C-H deformation vibrations which can be seen via the bands at 1460,

and 713 cm−1 are originated from the saturated fatty acids. The band at 1379 cm−1

belongs to the C-O stretching of COO- groups of the lipids. The bands observed at 1257,

1168, and 1089 cm−1 are attributed to asymmetric and symmetric stretch vibrations of

phosphodioxy groups, respectively. Moreover, the vibrations at 1020 and 800 are specific

to cholesterol (Dreissig et al., 2009). Based on the FTIR results, the toluene has extracted

lipids and phospholipids and not the other compounds.


Figure 5.2: FTIR spectra of the untreated and lipase treated sludge for 4, 8, and 24 hours

(E1 to E3, respectively).

5.4.2 Lipase Treatment

Lipases (EC 3.1.1.3) are enzymes catalyzing the hydrolysis of triacylglycerols to fatty

acids and glycerol. Lipase application is expected to digest lipids, the hydrophobic com-

ponents of the sludge, in order to reduce their hyrophobicity and enhance the sludge

surface energy. Lipase from Candida rugosa was applied on the secondary sludge for

different durations of time. The FTIR spectra of the lipase-treated sludge samples (E1

to E3: 10 mg/g of sludge for 4, 8, and 24 hours, respectively) are compared with the non-

treated sludge in Figure 5.2. When the spectra are compared to the untreated sludge,

they are surprisingly similar to each other. Clearly, the sensitivity of the method was not

high enough to detect changes in the structure of different sludge samples after lipase

treatments. Lipase application is supposed to cause changes in the carbonyl absorption

of the triglyceride ester linkage at 1744 cm−1 (van de Voort et al., 1994). However,

these changes are only reflecting in the E3 spectra which belongs to sludge that has been

exposed to lipase for 24 hours.


5.4.3 Laccase Characterization

Laccase activity is mostly determined by spectrophotometry monitoring of the colored ox-

idation products of phenolic/non-phenolic substrates. 2,2’-azinobis-(3-ethylbenzyl thiozoline-

6-sulphonate) (ABTS) is an electron-rich, non-phenolic substrate with an oxidation po-

tential that is not pH-dependent within the range 2-11, producing colored radical cation

(ABTS+•) (Johannes and Majcherczyk, 2000). The reaction took place at 0.10 M sodium

acetate buffer (pH 5.0), since the enzyme is reported to have maximum activity at this

pH (Leonowicz and Grzywnowicz, 1981). The absorbance of the oxidation product is

measured at 420 nm with an extinction coefficient of ϵ420 = 3.6× 104 M−1cm−1 (Bour-

bonnais and Paice, 1990). The enzyme activity is expressed in U which is the µmol

of ABTS oxidized per minutes and calculated to be 17.3 U per mg for the purchased

enzyme.

The activities of laccase both on the sludge and the alkali extracted lignin from the

sludge were studied. There have been few works in literature on the estimation of kinetic

parameters of laccase-catalyzed polymerization of phenolic compounds (Akta, Kibarer

and Tanyola, 2000; Akta et al., 2003; Soegiaman, 2006). Studying the kinetics of the

laccase catalytic system is essential in understanding the reaction route and eventually for

designing a suitable reactor system. In the sludge system, it also serves the key purpose

of proving that laccase is actively reducing the sludge and producing free radicals.

Since the oxidation of any substrate by laccase is paired with the reduction of oxygen

to water, the enzyme’s activity can be monitored with a dissolved oxygen meter. The

Michaelis-Menten kinetic parameters were determined by the experimental observations

of the oxygen consumption over time at a variety of initial oxygen concentrations.

V =Vmax[S]

KM + [S](5.1)

The rates were estimated by linear regression of initial dissolved oxygen concentrations

data (Figure 5.3). The individual data points of dissolved oxygen concentration in the


R2 = 0.9864

0

1

2

3

4

5

6

0 500 1000 1500 2000 2500

Reaction time (sec)

Dissolved O

xygen Concentration

(g m

-3)

Figure 5.3: Typical changes in dissolved oxygen during laccase-sludge reaction in a batch

system.

linear regression analysis were included to the point that gave the correlation coefficient

not less than 0.98. The initial oxygen consumption rates were calculated for different

experiment runs and the results are presented in Table 5.1.

Employing the Michaelis-Menten kinetic model as a function of dissolved oxygen

concentrations, the corresponding biokinetic parameters for sludge bioreactions and the

alkali extracts are slightly different. The Km values calculated to be 3.491 and 2.318 g

m−3 for sludge and the alkali extract, respectively. It can be concluded the enzyme has a

lower affinity for the sludge which might be due to the inhibitory effect of one or few of its

numerous components. The values calculated for Vmax were 0.023 and 0.014 g m−3min−1,

respectively. The estimated rate constants are in accordance with the previously reported

values for laccase-catalyzed reactions of different monomers in terms of their orders of

magnitude (Akta, Kibarer and Tanyola, 2000; Akta et al., 2003).

Taking the rate of dissolved oxygen consumption in the flask as a measure of the


Table 5.1: Experimentally observed reaction rates for sludge with different initial con-

centration of dissolved oxygen

Runs

Initial dissolved

oxygen concentration

(g m−3)

Initial reaction rate

(g DO m−3min−1)

1 7.9 0.0157

2 7 0.0153

3 5.9 0.0147

4 5.4 0.0138

5 4.6 0.0130

6 4 0.0122

reaction rate, the rate can be increased by maximizing the dissolved oxygen concentration.

Therefore, the laccase modification of sludge is carried out by full aeration of the reactor

in this work.

5.4.4 Laccase Modification of the Sludge

In the pulp and paper industry laccase has been applied commercially as adhesive for

fiberboard production through activation of surface lignin by laccase-catalyzed oxidation

and bonding of wood fibers (Felby, Pedersen and Nielsen, 1997). Moreover, the reported

redox potentials of laccases are lower than those of non-phenolic compounds (Couto and

Herrera, 2006), so these enzymes cannot oxidize such substances and hence, they are

not degrading the other components. Laccase application on the waste sludge which

contains lignin might result in further polymerization of lignin and even bonding to

carbohydrates. In this work, the lignin content of the secondary sludge was measured to


0

0.3

5001000150020002500300035004000

Wavenumber (cm-1)

Absorbance

Sludge

Laccase

treated

Figure 5.4: FTIR spectra of the untreated and laccase treated sludge

be 15± 3% (w/w). The laccase modification will significantly affect the final properties

of the value-added materials produced from this waste biosolid. Enzymatic modification

has several advantages compared to other chemical methods including the reaction being

carried out under mild conditions (in terms of catalysis temperature, pressure, and pH),

high selectivity, and being environmentally-friendly due to the nontoxic nature of the

catalyst.

Modification of the Molecular Structure

The changes in the structural properties of laccase-treated sludge are evaluated with

corresponding FTIR spectra and presented in Figure 5.4. The FTIR spectra give evi-

dence of differences in the frequency domains of 3500− 3200 cm−1 and elimination of the

absorption band at 1640 cm−1 after laccase treatment.

The band at 1425 cm−1 is assigned to the bending vibrations of CH2 groups of the

aromatic ring of lignocelluloses (Carrillo et al., 2004; Gastaldi et al., 1998; Pavan et al.,

2008). The sudden decrease of the intensity of CH2 bending band is due to the structural

transformation during laccase treatment. The CH2 groups have bonded with the free


radicals generated by laccase activity.

Different FTIR analysis in literature on lignin degradation by fungal activity shows

the formation (Vivekanand et al., 2008) or increase in the intensity (Sealey and Ra-

gauskas, 1998) of the absorption bands at 1719− 1720 cm−1. It has been attributed

to the increase of free carbonyl groups resulted from the release of ketones and alde-

hydes. The absence of such a peak in the laccase-treated sludge samples suggests this

reaction has gone a different path. It can be concluded that instead of lignin degrada-

tion, cross-linking and polymerization has occurred. The differences in the frequency

domains of 3500− 3200 cm−1 supports this assumption: In the sludge spectra, broad

doublet peaks at 3404 and 3269 cm−1 belong to characteristic hydrogen bonded O-H vi-

bration bands of lignin and polysaccharides. Thus, the changes of characteristics in the

region 3500− 3200 cm−1 show the transformed nature of the hydrogen bonding of the

OH groups due to polymerization.

5.4.5 Lignin Determination and Alkali Extraction of Lignin from

the Sludge

Lignin, due to its molecular structure and covalent bonds with holocelluloses, cannot be

isolated without damaging its structure (Reale et al., 2004). Isolation methods based

on solvent-extraction only release parts of the relatively unchanged lignin (Bjrkman,

1956). On the other hand, the common and well-known method of acid hydrolysis of

carbohydrates with 72% sulfuric acid give structurally modified lignin and is only useful

for quantitative determination of lignin. Using the latter method here, the lignin content

of the secondary sludge was determined to be 15± 3% (w/w). This considerable amount

of lignin present in the sludge is proposed to be enzymatically modified to increase the

molecular weight for new applications.

In order to follow the changes in the molecular weight by size-exclusion chromatog-

raphy (SEC), a non-destructive method of lignin extraction should be applied. For this


reason, the alkaline extracted lignin has been enzymatically treated and the changes in

the molecular weight were monitored. The alkaline extraction of lignin is not causing

further oxidative damage to phenolic lignin unlike other extraction methods (Chakar and

Ragauskas, 2004). Issues about the possible degradation of the macromolecules during

isolation (Reale et al., 2004) can be overlooked here, since the main purpose is to follow

the changes of the extracted lignin.

5.4.6 Modification of the Molecular Weight Distribution

Molar mass distribution of lignins affects their reactivity and physiochemical proper-

ties and is also involved in assessing lignin potential for new uses (Baumberger et al.,

2007). High-pressure size-exclusion chromatography (HPSEC) was applied to determine

the molecular weight distribution of the lignin samples and also as a means for comparing

modified and unmodified samples. Employing HPSEC for molecular weight determina-

tion of lignins offers advantages including availability, short analysis time, and low sample

demand (Baumberger et al., 2007).

Alkali solution, also used for lignin isolation, is widely used as an eluent (from 2.5

mM to 1 M NaOH) by size exclusion chromatography (Bikova et al., 2004; Wong and

de Jong, 1996; Pellinen and Salkinoja-Salonen, 1985; Hortling, Turunen and Kokkonen,

2004). Alkali is a convenient eluent since it can dissolve almost all lignins and the sample

derivatization for analysis is not needed (Wong and de Jong, 1996). Thus, the 0.5 M

NaOH with a flow rate of 1 ml/min was used as the eluent in this work.

In literature, the choices of working wavelength for the HPSEC chromatogram of

lignin have been mostly 280 and 210 nm (Baumberger et al., 2007; Bikova et al., 2004;

Gosselink et al., 2004; Pellinen and Salkinoja-Salonen, 1985) which are the wavelengths

reflecting the concentration of the methoxylated phenol ring (Bikova et al., 2004). Based

on the results on literature the wavelengths of 210 and 280 nm were chosen for the HPSEC

procedure. The results showed no significant difference between chromatograms of 280


0 1 2 3 4 5 6 7 8

Retention time (min)

Absorbance

Absorption at 210 nm

Absorption at 280 nm

Figure 5.5: HPSEC chromatogram obtained for the laccase treated alkali extracts with

signals at 210 and 280 nm (flow rate of 1 ml/min, and injection volume of 25 µL).

and 210 nm with other parameters kept constant (Figure 5.5).

The interactions of alkaline eluent with the packing material might cause changes

in the performance of the gel filtration medium over time (Sun et al., 2004). All the

experiments in this work have been performed with a brand new column and in less than

two weeks.

The HPSEC chromatograms of the alkaline extracted lignin from the secondary sludge

before and after laccase modification are shown in Figure 5.6. It can be observed that

laccase treatment has changed the chromatogram and shifted it towards higher molecular

weights.

Calibration of molar mass to retention time was achieved by linear regression of

standards of nominal molecular masses. The values for weight (Mw) and number (Mn)

average molecular mass are calculated based on the following equations:

Mw =

∑ni=1 hiMi∑ni=1 hi

(5.2)


Figure 5.6: HPSEC chromatogram obtained for the alkali extracted sample before and

after laccase treatment (flow rate of 1ml/min, Signal at 280nm, injection volume of 25

µL).

Mn =

∑ni=1 hi∑n

i=1 hi/Mi

(5.3)

where hi and Mi are the height and molecular mass, respectively.

Using the above formula and the data provided by the HPSEC experiments, the size-

and weight-average molecular weights of the alkali extracted lignins were determined to

be 4 and 30 kDa, respectively. The low polydispersity is probably due to the extrac-

tion of low-molecular weight polysaccharides along with lignins. The sample after the

laccase treatment had the size- and weight-average molecular weights 20 and 80 kDa,

respectively. These values show the increase in the molecular weight after the enzyme

treatment. The hydrocarbons probably have reacted with the free radicals forming an

ether bond and thus, increased the molecular weight. The lignins molecular weights,

however, cannot be compared to literature due to the lack of uniform values. This lack of

uniform values is attributed to lignins different origins (e.g., kraft, alkali, enzyme, milled-

wood) (Baumberger et al., 2007), different isolation methods and also the polydisperse

character of the isolated lignins (Reale et al., 2004).


5.5 Conslusions

Waste secondary sludge from a pulp and paper mill was enzymatically modified by lipase

and laccase enzymes. The lipid content of the sludge was shown to be hydrolyzed by lipase

after twenty four hours. The lignin content of this waste biosolid was measured to be

15±3% (w/w). Enzymatic modification of the sludge by laccase from Trametes versicolor

was performed in a batch system while being aerated. The laccase activity assay was

determined using previously published methods. In order to study the reaction rate of

laccase in the sludge, the uptake of oxygen in the samples was monitored after adding

the laccase. The consumption of oxygen primarily showed that the laccase activity was

not inhibited completely by the diverse components of the sludge. However, the higher

reaction rate of laccase in the alkali-extracted samples indicated some inhibitory activity

of the sludge. Moreover, the biokinetic parameters for the Michaelis-Menten kinetic

model as a function of dissolved oxygen concentrations were determined. The Km values

were found to be 3.491 and 2.318 g m−3 for sludge and the alkali extract, respectively

while the corresponding Vmax values were 0.023 and 0.014 g m−3 min−1, respectively.

Finally, the FTIR and HPSEC results showed that laccase structurally modifies the

sludge and increases the molecular weight of its components via cross-linking reaction.

This modification is expected to enhance the properties of the waste secondary sludge

as filler in composites. Utilizing the enzymatically modified sludge as filler in Nylon

composite is discussed in the following chapter.

Chapter 6

Nylon/Sludge Biocomposite

6.1 Abstract

Secondary sludge from pulp and paper mills can be considered as potential filler for com-

posite industry. Based on the characterization results, i.e. surface thermodynamics and

the chemical structure, presented in chapters 3 and 4, polyamides were suggested to be a

compatible polymeric resin with the biosolid. The processing compatibility and material

characteristics of the biocomposites filled by secondary sludge was studied in this chapter.

The manufactured Nylon/sludge composites showed acceptable, yet not improved, me-

chanical strength. 10% of the dried sludge as filler proved to be an effective amount which

is sufficient to fill but not degrade the tensile and flexural strengths of the composite.

Sludge-filled composites compounded by a twin screw extruder exhibited considerably

better tensile properties than those compounded by the K-mixer. Maleated polyolefins

coupling agent also improved the composite’s mechanical properties significantly. The

lipase treated sludge/Nylon composite showed deteriorated mechanical strength (both

tensile and flexural). However, the laccase-treated sludge/Nylon composite showed en-

hanced mechanical strength which is due to the increase in the components molecular

weight.

91

Chapter 6. Nylon/Sludge Biocomposite 92

6.2 Introduction

Secondary sludge, which is structurally comparable to municipal sewage sludge, is com-

posed of microbial cells, organic woody materials, and ash. One alternative to the costly

disposal methods which are also threats to the environment is to use this biosolid as re-

newable and cost-cutting filler in composite industry. Unless the addition of this biosolid

results in the deterioration of the composite’s properties, the main advantage would be

reducing the costs since this cheap biosolid replaces a part of the expensive polymeric

resin. It may also enhance the material stiffness which should be examined and proved.

The surface characteristics of fillers can be tailored by physical, chemical, or enzymatic

modifications in order to obtain good adhesion with the polymeric resin. Enzymes, the

natural catalysts, can perform efficiently under mild conditions and unlike chemicals, have

no harmful discharge causing environmental concerns (Araujo, Casal and Cavaco-Paulo,

2008). Thus, for specific and non-destructive functionalization of fiber surfaces and also

increasing their surface energy, enzymes (from commercial sources) have been employed.

Lipase has been applied in order to reduce the hydrophobicity of the sludge by breaking

down the lipid molecules. Moreover, the enzyme laccase was applied to cause further

polymerization and increase the lignins molecular weights. The effectiveness of these

enzymes can be established by the results of the mechanical strengths of the produced

biocomposites.

One significant factor which determines the composite’s performance is the quality

of interface between the fiber/filler and the polymeric matrix. An acceptable interface

quality requires the surface tension of the fibers/fillers to be greater than that of the resin.

Inverse gas chromatography results showed the surface energy of the sludge to be greater

than that of the most conventional plastics. Moreover, the filler should be compatible

with the matrix in terms of their chemical structure. A major solid component of the

secondary sludge is the bacterial biomass which develops during the sewage digestion

process. These cellular biopolymers consist mainly of proteins, which, when coupled


with polyamides, might act as a binder. For this reason polyamides can be considered

as an appropriate choice for the polymeric resin. In terms of the surface energy, Nylon

has a lower dispersive energy and the opposite polarity compared to the sludge samples

which may result in acceptable adhesion of these phases.

Nylon is a thermoplastic polyamide with peptide bonds (-NH-CO-) linking the repeat-

ing units. It is a condensation copolymer formed by reacting equal parts of a diamine and

a dicarboxylic acid (forming peptide bonds). Nylon can be utilized as the matrix phase

in composites; Nylon composites are mostly glass-filled for higher structural and impact

strength and rigidity. The primary drawback of using natural fibers as the reinforcing

phase in Nylon composites is the thermal degradation of natural fibers at processing

temperatures beyond 200◦C. However, long-chain polyamides including Nylon 11 have

relatively lower melting points compared with other Nylons. Nylon 11 is an important

commercial polyamide with excellent mechanical properties which is used in a large range

of industrial fields from automotive to offshore applications (Liu et al., 2003).

The main concerns for selection of a suitable technology for processing the natural-

fiber composite are the final desired product form, performance attributes, cost, and

ease of manufacturing (Holbery and Houston, 2006). The methods used to manufac-

ture biocomposites are mainly based on the existing techniques for processing plastics

or composite materials including press molding, hand lay-up, filament winding, pultru-

sion, extrusion, injection molding, compression molding, resin transfer molding and sheet

molding compounding (Fowler, Hughes and Elias, 2006).

Generally, processing of the natural fiber thermoplastic composites consists of the

extrusion of the ingredients at melting temperature and the shaping operations such as

injection molding and thermoforming (Saheb and Jog, 1999). However, their manufac-

turing is limited by two essential physical factors: the upper temperature at which the

fiber can be processed and the significant difference between the surface energies of the

lignocellulosic fibers and the polymeric matrix (Holbery and Houston, 2006). Thus, most


of the work reported so far deals with polymers with lower process temperatures such

as polyethylene, polypropylene, polystyrene, and polyvinylchloride (Sain et al., 2005)

to avoid thermal degradation of natural fibers. Press molding, injection molding, and

compounding/extrusion are the most commonly used processing techniques for natural-

fiber-reinforced thermoplastic composites (Panthapulakkal, Sain and Law, 2005).

Throughout compounding, the thermoplastic polymer is heated to melt. In this

state, natural fiber, usually in the form of flour, is added along with other additives

such as surfactants and coupling agents. Once the components are thoroughly mixed,

the compound can be either extruded directly in the final product, or be pelletized and

packed for further extrusion or injection molding processes. Compounding processes are

gaining wide acceptance due to the high degree of consistency feasible in the pellet form

(Holbery and Houston, 2006).

The primary goal of this chapter is to assess the feasibility of utilizing pulp and paper

mill secondary sludge as cheapening and/or functional filler in biocomposite manufactur-

ing. For this reason, the dried biosolid along with the selected resin will be used in man-

ufacturing biocomposites by compounding, pelletizing, and injection molding. Moreover,

two types of compounding, batch and continuous, were carried out in order to establish

the best choice of process. These processes have been executed at the Centre for Bio-

composites and Biomaterials Processing, Faculty of Forestry, University of Toronto. In

order to determine the advantages and limitations of utilizing secondary sludge as filler

material, the mechanical properties of the manufactured biocomposite will be studied

subsequently.


6.3.1 Materials

Nylon 11 (BMNO commercial grade, gift from Arkema) was used as the polymeric resin.


6.3.2 Composite Preparation

Blending of the sludge and Nylon was carried out by two methods. Once, the dried

sludge (25% by weight) was added to molten Nylon in a laboratory K-mixer (C.W.

Brabender) at 190◦C. After the sample was well-mixed it was removed from the K-

mixer and the compounds were allowed to cool to room temperature and granulated in

a Brabender granulator (Model S-10-9). Compounding was also performed using a twin-

screw extruder with a screw nominal diameter of 25 mm, screw centre distance 21.2 mm

and L/D of 40. The compounded Nylon/sludge was then cooled down and pelletized. The

pellets compounded by two different methods, were pre-dried in an air circulated oven

at 105◦C for 1 hour and then injection molded with an Engel ES-28 machine equipped

with a standard ASTM test specimen mold. The injection molding was carried out at

the injection temperature of 200◦C, injection pressure of 4.8 MPa, and clamp pressure

of 11.7 MPa. The injection time, cooling time, and mold opening time were 9.5 s, 25 s,

and 2 s, respectively.

6.3.3 Mechanical Properties Measurements

Tensile and flexural tests were performed according to ASTM D 638 and ASTM D 790,

respectively. These mechanical characteristics of Nylon and the sludge-filled Nylon sam-

ples were tested by an Instron 5860 (Grove City, PA) in tensile and flexural modes with

a local cell of 2 kN and 30 kN, respectively. Tensile tests were performed at a crosshead

speed of 12.5 mm/min. The values reported in this work are the results of averaging over

at least 5 measurements.

6.3.4 Statistics

Statistical analyses were performed with Student’s t-test to assess statistical significance

within the data. T-test was used to establish any reinforcing effects of the sludge on


composite’s properties.

6.4 Results and Discussion

In general, Nylon has been shown to be improved in terms of its mechanical properties

when filled with typical reinforcing fillers (Caulfield et al., 2001). However, Klason et al.

(Klason, Kubat and Strmvall, 1984), showed severe degradation of cellulosic fibers when

used with Nylon 6. They concluded that for higher melting temperature thermoplastics,

e.g., Nylon 6, cellulosic fibers cannot increase the stiffness and strength of the composite

despite their obvious potential (Klason, Kubat and Strmvall, 1984). Bio-based Nylon 11

is chosen in this work for its lower melting temperature compared to Nylon 6. Differential

scanning calorimetry was used to determine the melting point of the employed Nylon and

it was found to be 190◦C±0.0.

Table 6.1 shows some of the literature results on the tensile and flexural strength of

Nylon composites filled with mineral and natural fibers. It can be seen that mineral fillers

increase the composite’s strength considerably. Glass fibers are known to be the most

effective fillers so far (Caulfield et al., 2001). On the other hand, natural fibers usually

degrade the mechanical properties of the filled Nylon in most cases (Santos et al., 2007;

Xu, 2008). However, enhanced mechanical properties for the wood-fiber-filled Nylon is

also reported (Caulfield et al., 2001). Moreover, different manufacturing methods did not

change the final properties to a considerable extent (Xu, 2008).

From Table 6.1, the plastic composites of polyamide (PA6) and Curaua fiber have

mechanical properties comparable to those of mineral filled composites. Curaua is a plant

from bromeliad family which has a higher mechanical strength than other natural fibers

such as sisal, jute and flax (Santos et al., 2007). On the other hand, the mechanical

strengths of the sludge-filled Nylon are at best similar to the unfilled Nylon (Figure 6.1

and Figure 6.2). This is due to the numerous components of the sludge that are not


Tab

le6.1:

Mechan

ical

properties

ofNyloncomposites

Sou

rce

Method

Sam

ple

Tensile

test

Flexuraltest

Max

(MPa)

E(G

Pa)

Max

(MPa)

E(G

Pa)

SABIC

Innovative

Plasticsa

Extrusion

and

Injection

molding

Nylon6unfilled

631.4

95

+20%

Curaua

835.5

115

+20%

Glass

101

6.5

160

+20%

Talc

736.5

115

San

toset

al.,2007

Extrusion

and

Injection

molding

Nylon6unfilled

44±

30.9±

0.3

+20%

shortvegetalfiber

34±

101.9±

0.1

+30%

shortvegetalfiber

34±

62.1±

0.1

+40%

shortvegetalfiber

30±

32.4±

0.2

+20%

longvegetalfiber

30±

31.8±

0.1

+30%

longvegetalfiber

32±

82.1±

0.2

+40%

longvegetalfiber

26±

32.8±

0.1

Xu,2008

Extrusion

and

Com

pression

molding

+10%

Cellulose

Fiber

53.7

3.01

103

2.72

+20%

Cellulose

Fiber

54.3

4.16

103

3.69

+30%

Cellulose

Fiber

48.9

4.58

93.3

3.59

Nylon

44.2

1.92

69.8

1.37

Extrusion

and

Injection

molding

+10%

Cellulose

Fiber

47.9

3.12

71.6

1.57

+20%

Cellulose

Fiber

54.2

4.32

85.6

2.00

+30%

Cellulose

Fiber

53.3

5.15

95.7

2.6

Cau

lfieldet

al.,2001

Injection

molding

Nylon6

60.2

2.75

64.2

2.38

+30%

Wallastarite

62.7

6.51

105.7

2.38

+33%

Glass

fiber

111.2

8.02

146.7

7.55

+33%

HW

fiber

86.5

5.71

121.6

5.88

+33%

SW

fiber

81.9

5.35

113.9

5.45

ahttp://w

ww.ptonline.com/a

rticles/kuw/34

715.htm

l


necessarily all compatible with each other and Nylon. Moreover, the fiber content of

the sludge can be considered similar to recycled fibers, with considerable strength loss.

The mean lengths of these fibers have been shortened during various processes. This

strength loss is attributed to the loss in flexibility and/or changes in surface condition of

the recycled fibers (Cao, Tschirner and Ramaswamy, 1999).

In this work, Nylon/sludge composites have been tested for their mechanical proper-

ties and compared with unfilled Nylon (Figure 6.1 and Figure 6.2). The tensile strength

of the Nylon/sludge composite which was compounded by the K-mixer is measured to be

considerably lower than the other samples. In other words, the compounding method can

greatly affect the composite’s final properties. K-mixer is a batch-style machine which

consists of two rotors and a heated chamber where the compounded materials should be

removed manually from. Consequently, the longer exposure to heat while compounding

with the K-mixer, due to the time-consuming sample removal, might result in the infe-

rior mechanical properties of the biocomposite. In this work, removing the Nylon/sludge

blend from the K-mixer took about 30 minutes, while the time that the mixture spent in

the twin-screw did not exceed 10 minutes. Since cellular biopolymers and natural fibers

are complex mixtures of organic materials, heat exposure leads to physical and chemi-

cal changes (Holbery and Houston, 2006). The prolonged exposure to high temperature

resulted in poor interfacial adhesion and embrittlement of the biocomposite.

The tensile and flexural mean strengths for unfilled and sludge-filled Nylon samples

are summarized in Table 6.2. Pure Nylon and the 25% sludge-Nylon mixture compounded

by twin-screw extruder were injection molded into test specimens. The decrease in the

tensile strength of the samples is proved to be significant at the P ≤ 0.001 level. However,

the flexural strength has not deteriorated. On the other hand, if the sludge content be

lowered to 10% (Table 6.3), there will be no significant deterioration of either tensile or

flexural strengths. Therefore, even without further modification, sludge can be utilized as

cheapening filler for the appropriate applications. It should be noted that the mechanical


0

10

20

30

40

50

60

70

80

Nylon Sludge-filled Nylon

(25%) (Brabender)

Sludge-filled Nylon

(25%) (Twin screw)

Sludge-filled Nylon

(70-5-25%) (Twin

screw-CA)

Flexural Strength (MPa)

0

0.4

0.8

1.2

1.6

2

Flexural Modulus (GPa)Strength Modulus

Figure 6.1: Flexural properties of the sludge-filled Nylon composites

0

10

20

30

40

50


(25%) (Brabender)

Sludge-filled Nylon

(25%) (Twin screw)

Sludge-filled Nylon (70-

5-25%) (Twin screw-

CA)

Tensile Strength (MPa)

0

0.4

0.8

1.2

1.6

2

Tensile Modulus (GPa)

Strength Modulus

Figure 6.2: Tensile properties of the sludge-filled Nylon composites

strengths of the sludge-filled Nylon are at best similar to the unfilled Nylon (Figure 6.1

and Figure 6.2).

The potential role of coupling agent in composite properties was also tested (Table

6.4). Addition of 5% maleated olefinic coupling agent resulted in a statistically signif-

icant increase of flexural and tensile strengths at the P ≤ 0.05 level. Maleated poly-

olefins are usually PE or PP with maleic anhydride functional groups grafted onto the

polymer backbones. These coupling agents, in theory, when melted with polymers and

cooled, crystallize into the main polymers and the maleic anhydride groups react with

the hydroxyl groups of cellulosic fibers forming strong covalent ester linkages, thereby,

linking the polymer phase to the reinforcing fibers. Maleated polyolefins have been exten-


Table 6.2: Mean tensile and flexural strengths of pure Nylon and 25% sludge-filled Nylon

NylonSludge-filled Nylon

(25%)

Significance

(P ≤ 0.001)

t-Value

Tensile (MPa) 38.598± 0.750 32.436± 0.794 Yes 12.61532

Flexural (MPa) 55.55± 1.42 55.4± 0.98 No 0.19442

Table 6.3: Mean tensile and flexural strengths of pure Nylon and 10% sludge-filled Nylon

NylonSludge-filled Nylon

(10%)

Significance

(P ≤ 0.001)

t-Value

Tensile (MPa) 38.598± 0.750 35.476± 1.530 No 4.09698

Flexural (MPa) 55.55± 1.42 53.67± 2.11 No 1.6528

sively used for natural fiber composites, and reported to increase the tensile and flexural

strength by 80% (Keener, Stuart and Brown, 2004; Panthapulakkal, Sain and Law, 2005).

Here, adding the coupling agent increased the tensile and flexural strength about 10%.

This improved adhesion at the interface is due to the possible reactions given in Figure

6.3 (Groeninck, 1999).

Therefore, utilizing the secondary sludge as cost-cutting filler does not degrade the

mechanical properties of Nylon. In other words, 10% of dried sludge as filler is an

effective amount which was sufficient to fill but not degrade the tensile and flexural

strengths of the composite. Sludge-filled composites compounded using a twin screw

extruder exhibited considerably better tensile properties than those compounded by the

K-mixer. Maleated polyolefins coupling agent also improved the composite’s mechanical

properties significantly.

In addition, the enzymatic treated sludge was compounded with Nylon 11 and in-


Table 6.4: Mean tensile and flexural strengths of 10% sludge-filled Nylon with and with-

out laccase modification

Sludge-filled Nylon

(70-5-25%)

Sludge-filled Nylon

(25%)

Significance

(P ≤ 0.005)

t-Value

Tensile (MPa) 35.705± 0.538 32.436± 0.794 Yes 7.62139

Flexural (MPa) 61.07± 1.35 55.40± 0.98 Yes 7.6001

Figure 6.3: Reaction scheme of maleic anhydride with amide end groups of Nylon at high

temperature

jection molded into test specimens. The manufactured composites were tested for their

mechanical properties and compared with unfilled Nylon and Nylon filled with untreated

sludge (Figure 6.4 and Figure 6.5).

It can be observed that the lipase treated sludge when used as filler for Nylon com-

posite, slightly degrades the mechanical properties (both tensile and flexural). Therefore,

it can be concluded that the 5-6% lipid content of the sludge is acting as a plasticizer and

in fact is helping with the final composite’s strength. Moreover, the reduced molecular

weight caused by lipase treatment has a stronger effect on the composite’s properties

than the lowered hydrophobicity.

However, laccase treatment of the sludge resulted in a statistically significant increase

of flexural strength at the P ≤ 0.001 level for both 10% and 25% sludge-Nylon biocom-

posites (Table 6.5 and Table 6.6). The tensile strength also showed an increased value

which is not statistically significant. This can be attributed to the cross linking reaction


0

10

20

30

40

50

60

70

80


(10%)

Laccase treated

Sludge-filled

Nylon(10%)

Lipase treated

Sludge-filled Nylon

(10%)

Flexural Strength (MPa)

0

0.4

0.8

1.2

1.6

2

Flexural Modulus (GPa)Strength Modulus

Figure 6.4: Flexural properties of the sludge-filled Nylon composites

0

10

20

30

40

50


(10%)

Laccase treated

Sludge-filled

Nylon(10%)

Lipase treated

Sludge-filled Nylon

(10%)

Tensile Strength (MPa)

0

0.4

0.8

1.2

1.6

2

Tensile Modulus (GPa)

Strength Modulus

Figure 6.5: Tensile properties of the sludge-filled Nylon composites

taking place in the presence of laccase which causes an increase in the molecular weight

and consequently a better reinforcement to the biocomposite.

Based on the results of this chapter, the secondary sludge can be considered a cheapen-

ing filler for Nylon biocomposite without degrading Nylon’s mechanical strengths. More-

over, enzymatic treatment of the secondary sludge resulted in a statistically significant

enhancement of the flexural properties. It can be concluded that the biosolid not only

serves as cheapening filler, but also has the potential to be a reinforcing phase in Nylon

biocomposites.




Sludge-filled Nylon

(10%)

Laccase-treated

Sludge-filled Nylon

(10%)

Significance

(P ≤ 0.001)

t-Value

Tensile (MPa) 35.476± 1.530 37.057± 2.104 No 1.359

Flexural (MPa) 53.67± 2.11 60.52± 1.68 Yes 5.675



Sludge-filled Nylon

(25%)

Laccase-treated

Sludge-filled Nylon

(25%)

Significance

(P ≤ 0.001)

t-Value

Tensile (MPa) 32.436± 0.794 32.791± 4.660 No 0.167

Flexural (MPa) 55.40± 0.98 61.05± 1.25 Yes 7.951


6.5 Conclusions

Current disposal practices of waste sludge are expensive and, in most case, are detri-

mental to the environment. Utilizing secondary sludge of pulp and paper mills as filler

in composite industry has been proposed in this work. Based on the chemical com-

patibility and surface thermodynamic data, Nylon was selected as the main polymeric

matrix. Mixtures of 10% and 25% dried secondary sludge and Nylon were compounded

by k-mixer and twin-screw extruder and then injection-molded into test specimens. The

results of mechanical strength tests showed that composites compounded using a twin

screw extruder exhibit considerably better tensile properties than those compounded by

the k-mixer. This was attributed to the long exposure of the mixture to heat in the

k-mixer. Filling Nylon with 25% of sludge showed statistically significant decrease in the

tensile strength at the P ≤ 0.001 level. With lower sludge content (10%), however, there

was no significant deterioration of either tensile or flexural strengths. Therefore, even

without further modification, sludge can be utilized as cheapening filler for the appro-

priate applications. Moreover, using the lipase treated sludge as filler slightly degraded

the mechanical properties (both tensile and flexural) of Nylon composite. This might

be due to the plasticizing effect of lipids. On other hand, it might be indicating that

reduced molecular weight has a greater effect on the composite’s properties than the

reduced hydrophobicity. In contrast, enzymatic treatment of the secondary sludge with

laccase improved the composite’s mechanical strength. This is attributed to the increased

molecular weight of the sludge components caused by the cross-linking reactions.

Chapter 7

Conclusions and Future Work

7.1 Concluding Remarks

In this work the potential of the secondary sludge from pulp and paper mills for applica-

tion as filler in composite production was explored. The main hypotheses were verified

by the results and the main conclusions are as follows:

1. The studied waste biosolid has satisfactory qualities to fill Nylon without

degrading its mechanical properties. The thorough characterization of the

secondary sludge showed proteins to be a major component of the sludge (> 30%

dry weight). More than 80% of these biopolymers have molecular weight of more

than 50,000 Da. The thermal behaviour study showed that the secondary sludge

acts as a homogeneous blend of biopolymers which decomposes at temperatures

above 200◦C. The dispersive component of surface energy was found to be greater

than most common polymeric resins. The IGC results also revealed the dominant

acidic nature of the biosolid thereby rendering Nylon, as a basic resin, a proper

polymeric matrix to pair with it.

The mixture of 25% dried sludge and Nylon, when compounded with twin-screw

and injection molded into test specimens, caused slight degradation of the strength.

105

Chapter 7. Conclusions and Future Work 106

On the other hand, the 10% sludge-filled nylon biocomposite showed no degradation

of either tensile or flexural strengths.

2. Selective enzymatic modification of the sludge enhances the mechani-

cal properties of the manufactured biocomposite due to the increased

molecular weight of the components. Enzymatic treatment of the secondary

sludge with laccase is shown to increase the components’ molecular weight. The bio-

composites made with laccase-modified sludge show enhanced mechanical strength

compared to those filled with unmodified-sludge. On the other hand, use of lipase-

treated sludge as filler for Nylon composite results in slightly degraded mechanical

properties which is attributed to the reduced molecular weight of the sludge com-

ponents.

7.2 Significance of Research Work

Municipal and industrial wastewater treatments result in the production of significant

amount of waste sludge. The disposal of this waste biosolid is costly and environmentally

harmful. Currently, the common disposal practices are landfilling, incineration, and land-

spreading. The environmental concerns and processing difficulties associated with each

and every one of these practices have motivated research to seek alternative strategies.

This research shows that biorefining of the waste sludge can decrease the environmental

impacts of the current disposal practices significantly. This new waste reuse approach

helps the industries by assisting the facilities in meeting the environmental regulatory

requirements at a lower cost. Utilizing the waste sludge as filler in biocomposites, as

proposed in this work, not only helps reducing the greenhouse gas emissions, but also

addresses the problem of persistent plastics in the environment. This approach also helps

preserving the precious resources including petroleum, minerals and forests by replacing

them in the biocomposite. The focus of this study was the secondary sludge from the


pulp and paper industry. Due to the similarity of the pulp and paper secondary sludge

and the municipal sludge in terms of their high protein content, this biorefining approach

may also be extendable to the latter case.

Prior studies on sludge characterization are mainly related to the settling charac-

teristics. This work attempts to offer information relevant to sludge constituents and

properties for identifying the best reuse options. Moreover, this study provides an un-

derstanding of the surface characteristics and thermogravimetric behavior of the waste

sludge in order to investigate the suitability of the sludge for new applications.

In order to enhance the properties of the biocomposites, physical and chemical surface

modifications are being applied extensively. In this work the environmentally friendly

enzymatic modification of the sludge to improve its compatibility with polymeric matrices

is performed. Close monitoring of the laccase application on the sludge proved its activity

towards increasing the molecular weight of the lignin component of the sludge. The

application of enzymes to alter the chemical structure of the waste sludge opens new

avenues in recycling of the biosolid.

The heterogeneity and inconsistent nature of the sample sources may be argued as

a weakness of this study. Here, sludge samples from two different pulp and paper mills

were taken over 3 years did not exhibit any significant difference in terms of their major

constituents and the physico-chemical properties tested. This is not surprising since the

samples were taken from mills processing similar furnish and treatment plants (both

kraft/recycle pulp and paper mills). However, feed and operational parameters may

impact the quality and properties of the sludge (Kelly, Miller and Namazian, 2001).

The inherent inconsistency of raw material, depending on its source, is similar for other

industrial processes relying on raw materials (e.g. petrochemical), and must be considered

when developing engineering solutions for scale-up and production, as well as through

research.


7.3 Future Work

The following research topics can help in producing composites with better qualities and

for a wider application range:

1. Developing new dewatering techniques with less energy consumption to provide an

economically viable feed stock.

2. Investigating other waste sludges’ suitability for application as filler in composite

industry.

3. Further research on enzymatic copolymerization of lignin to produce new engineered

materials. The enzymatically induced grafting of polymeric side chains onto the

lignin backbone opens new possibilities for compatibilization of the sludge with

other polymeric matrices.

7.4 Publications

JOURNAL ARTICLES:

• M. Edalatmanesh, M. Sain and S. Liss, Enzymatic Modification of Secondary

Sludge by Lipase and Laccase to Improve the Nylon/sludge Composite Properties,

Journal of Reinforced Plastics and Composites, In Press.

• M. Edalatmanesh, M. Sain and S. Liss, Utilization of Secondary Sludge as Filler

in Composites: Surface Energy and Final Mechanical Properties, Journal of Rein-

forced Plastics and Composites, vol. 30, no.10, pp. 864-874, May 2011.

• M. Edalatmanesh, M. Sain and S. Liss, Cellular Biopolymers and Molecular Struc-

ture of a Secondary Pulp and Paper Mill Sludge Verified by Spectroscopy and Chem-

ical Extraction Techniques, Water Science and Technology, vol. 62, pp. 2846-2853,

2010.


• M. Edalatmanesh, M. Sain and S. Liss, Usage of Pulp and Paper Secondary Sludge

for Biocomposite Production: FTIR and DSC Analysis, International Review of

Chemical Engineering, vol. 2, no. 1 , pp. 25-30, January 2010.

Appendix A

Determination of the Dispersive

Component of the Surface Energy

Calculations for Hexane probe at 313◦K are shown below:

• Average retention time, tr, of hexane: 1.42 min

• Methane retention time, t0: 0.45 min

• Column outlet pressure, Po, (atmospheric pressure in the laboratory): 14.4 psi

• Column inlet pressure, pi: 15.3 psi

• Measured flow rate of carrier gas, Q0: 10.6 ml/min

• Surface area of hexane: 51.5 A2

• Surface free energy of hexane: 18.4 mJ/m2

The net retention time for pentane is: tn = tr − t0 = 0.97 min

The correction factor, J , is obtained from Equation 4.4, as follows: J = 0.971

The retention volume can then be calculated from Equation 4.3: V n = 9.983 ml

110

Appendix A. Dispersive Component of the Surface Energy 111

y = 0.0867x - 13.275

R2 = 0.9991

0

2

4

6

8

10

12

14

16

18

170 190 210 230 250 270 290 310 330 350

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol] Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

313ºK

Figure A.1: Determination of acid-base contribution to the free energy of adsorption

Retention volume for the same probe is calculated similarly for each temperature (313,

333, 353, and 373◦ K) and the resulting values of RT lnVn are plotted against a√γdi .

The dispersive component of the surface free energy for each temperature can be

determined from the slope of the linear fit:

At 313◦K: γDS = 52 mJ/m2

The acid-base characteristics are calculated based on the polar probe retention times;

For each temperature and each substrate of study, the ∆GAB is plotted against T

(Kelvin), where the intercept is the ∆HAB for that probe. Plotting ∆HAB/AN vs.

DN/AN gives a straight line with Ka and Kb as the slope and intercept.

Appendix B

Calculated Graphs for the Bowater

Sludge Sample from the IGC Results

y = 0.0854x - 14.825

R2 = 0.9996

0

2

4

6

8

10

12

14

170 190 210 230 250 270 290 310 330 350

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol]

Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

333ºK

Figure B.1: Calculation of γDS for Bowater sludge from Inverse Gas Chromatography

112

Appendix B. Calculated Graphs for the Bowater Sludge 113

y = 0.0843x - 16.389

R2 = 0.9996

0

2

4

6

8

10

12

170 220 270 320 370

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol] Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

353ºK


y = 0.0786x - 16.509

R2 = 0.9953

0

1

2

3

4

5

6

7

8

9

10

170 220 270 320 370

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol] Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

373ºK


Appendix B. Calculated Graphs for the Bowater Sludge 114

0

1

2

3

4

5

6

7

8

300 320 340 360 380 400

Temperature (K)

∆GAB(kJ/mol) Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

Figure B.4: Free energy of desorption ∆GAB for the Bowater sludge

y = 0.113x - 0.0397

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25

DN/AN

∆H/AN

Ethyl Ether

Acetone

THF

Ethyl Acetate

Chloroform

Figure B.5: Plot of ∆HAB/AN versus DN/AN for the Bowater sludge

Appendix C

Calculated Graphs for the Tembec

Sludge Sample from the IGC Results

y = 0.0933x - 19.096

R2 = 0.9977

-2

0

2

4

6

8

10

12

14

170 220 270 320 370

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol]

Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

313ºK

Figure C.1: Calculation of γDS for Tembec sludge from Inverse Gas Chromatography

115

Appendix C. Calculated Graphs for the Tembec Sludge 116

y = 0.0919x - 20.89

R2 = 0.9978

-4

-2

0

2

4

6

8

10

170 220 270 320 370

a(γL)0.5[A

º2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol]

Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7C8

333ºK


y = 0.0896x - 22.529

R2 = 0.9964

-6

-4

-2

0

2

4

6

8

170 220 270 320 370

a(γL)0.5[Aº

2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol]

Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

353ºK



y = 0.0787x - 16.936

R2 = 0.9981

-8

-6

-4

-2

0

2

4

6

8

170 220 270 320 370

a(γL)0.5[Aº

2(mJ/m

2)0.5]

RTln(V

N) [kJ/mol]

Alkanes

Chloroform

Ethyl Acetate

Ethyl Ether

THF

Acetone

C9

C6

C7

C8

373ºK


-3

-1

1

3

5

7

300 320 340 360 380 400

Temperature (K)

∆GAB (kJ/mol) Chloroform

Ethyl Acetate

THF

Acetone

Figure C.5: Free energy of desorption ∆GAB for the Tembec sludge


y = 0.1196x - 0.0138

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25

DN/AN

∆H/AN

Ethyl Ether

Acetone

THF

Ethyl Acetate

Chloroform

Figure C.6: Plot of ∆HAB/AN versus DN/AN for the Tembec sludge

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