Protein Recovery from Secondary Paper Sludge and Its Potential Use as Wood Adhesive

123 Pages·2012·2.62 MB·English

Protein Recovery from Secondary Paper Sludge and Its
Potential Use as Wood Adhesive
by
Muhammad Pervaiz
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Forestry
University of Toronto
© Copyright by Muhammad Pervaiz 2012
Protein Recovery from Secondary Paper Sludge and Its Potential
Use as Wood Adhesive
Muhammad Pervaiz
Doctor of Philosophy
Graduate Department of Forestry
Abstract
Secondary sludge is an essential part of biosolids produced through the waste treatment
plant of paper mills. Globally paper mills generate around 3.0 million ton of biosolids and in the
absence of beneficial applications, the handling and disposal of this residual biomass poses a
serious environmental and economic proposition.
Secondary paper sludges were investigated in this work for recovery of proteins and their
use as wood adhesive. After identifying extracellular polymeric substances as adhesion pre-
cursors through analytical techniques, studies were carried out to optimize protein recovery from
SS and its comprehensive characterization.
A modified physicochemical protocol was developed to recover protein from secondary
sludge in substantial quantities. The combined effect of French press and sonication techniques
followed by alkali treatment resulted in significant improvement of 44% in the yield of
solubilized protein compared to chemical methods. The characterization studies confirmed the
presence of common amino acids in recovered sludge protein in significant quantities and heavy
metal concentration was reduced after recovery process. The sodium dodecyl sulfate
polyacrylamide gel electrophoresis analysis revealed the presence of both low and high
molecular weight protein fractions in recovered sludge protein.
After establishing the proof-of-concept in the use of recovered sludge protein as wood
adhesive, the bonding mechanism of protein adhesives with cellulose substrate was further
elucidated in a complementary protein-modification study involving soy protein isolate and its
glycinin fractions. The results of this study validated the prevailing bonding theories by proving
ii
that surface wetting, protein structure, and type of wood play important role in determining final
adhesive strength.
Recovered sludge protein was also investigated for its compatibility to formulate hybrid
adhesive blends with formaldehyde and bio-based polymers. Apart from chemical cross-linking,
the synergy of adhesive blends was evaluated through classical rule-of-mixture. The findings of
this study warrants further investigation concerning other potential uses of recovered sludge
protein, especially as food supplements and economic implications.

iii
Acknowledgements
I would like to express my sincere gratitude and appreciation to my supervisor Dr. Mohini M.
Sain for his guidance, and relentless support throughout this research thesis. I would also like to
acknowledge the valuable advice of advisory board members, Drs. Martin Hubbes, Ramin
Farnood and Sally Krigstin on this research work. I am also grateful to Dr. D.N. Roy, Dr. S.
Konar, Dr. R. Jeng, and Dr. V. Tiyagi for their valuable suggestions during entire research work.
My heartfelt thanks to all technical and secretarial staff members for their assistance in all
possible ways which helped in accomplishing this thesis in befitting manner. I also thank the
National Science and Engineering Research Council of Canada network industry partners for
their cash and in-kind support in carrying out this work.
Special thanks to Mr. Shiang Law for his uncompromising and dedicated support to everyone
around him and me.
I would also like to thank my wife, Nasreen Akhter, for her continuous and un-compromising
moral support and patience during my research work. Finally, special thanks to my daughter,
Maryam Shamin, for having lively discussions and helping in all possible ways to a successful
end of this project.
iv
Table of Contents
Abstract ……..…………………………………………………………………………………….ii
Acknowledgements…………………….……………………………………………...………….iv
Table of contents………..…………………………………………………………………………v
List of Tables……………………………………………………………………………………. ix
List of Figures ………..……………………………………………………………………….…. x
List of Appendices……………………………………………………………………………….xii
CHAPTER 1 – INTRODUCTION………………………………………………………… ……..1
CHAPTER 2 – LITERATURE REVIEW……………………………………………………..….3
2.1 Environmental and Economic Implication ……………………………………………...3
2.2 Paper Mill Residual Biosolids.…………….………...…………………………………. 4
2.3 Current Sludge Disposal Practices……………………………………………………….7
2.3.1 Landfilling……… …………………………………………………………………7
2.3.2 Incineration……..…………………………………………………………………. 8
2.3.3 Land Applications…………………………………………………………………10
2.4 Types of Sludge ……………………………………………………………………….. 12
2.4.1 Paper Mill Effluent Secondary Sludge…………………………………………... 13
2.4.1.1 Process of Generation and Significance………………………………….. 13
2.4.1.2 Current Applications of Wastewater Activated Sludge………………….. 16
2.4.1.3 Chemistry of Secondary Sludge…………………………………………. 16
2.4.1.4 Microbial Characteristics of Secondary Sludge…………………………. 19
2.4.1.5 Protein Recovery from Municipal Waste Effluents……………………… 19
2.5 Potential Role of Sludge Protein as Wood Adhesive………………………………….. 21
2.5.1 Significance of Bio-based Wood Adhesives…………………………………….. 21
2.5.2 Current Status of Sludge and Protein-based Adhesives……….………………… 22
2.5.3 Proposed Wood Adhesion Theories……………………………………………... 23
2.6 Problem Statement ……..……………………………….…………………………….. 27
2.7 Hypothesis and Objectives…………………………………………………………….. 29
2.7.1 Hypotheses………………………………………………………………………. 29
2.7.2 Objectives………………………………………………………………………... 29
v
2.8 Scope of Research Work……………………………………………………………….29

CHAPTER 3 – MATERIALS AND EXPERIMENTAL METHODOLOGY...…..………….....32
3.1 Materials ……………………………………………………………………………….32
3.2 General Characterization of Paper Mill Effluent Sludges ……………………………...33
3.2.1 Solids Contents Determination……………..……………………………………. 33
3.2.2 Ash Determination…………..……………………………………………………33
3.2.3 Total Nitrogen (TN) Determination…………...…………………………………..33
3.3 Extractives, Protein and Lignocellulose Analysis of Sludge…….……..…....………….34
3.3.1 Extractives Determination…………………………………………………………34
3.3.2 Analytical Protein Estimation……….……………………………………………..34
3.3.3 Lignocellulose Fractionation………………………………………………………35
3.3.4 Analytical Extraction of Cellular Biocomponents (EPS)…….….…………..…… 36
3.3.4.1 Biochemical Analysis………..………………………………………….... 37
3.3.4.2 FTIR Studies……….………………………………………………………37
3.3.4.3 Thermal Analysis - DSC Testing…………..………………………………38
3.4 Protein Recovery from Paper Mill Secondary Sludge…………………………………..38
3.4.1 Protein Recovery – General Scheme…..….……………………………………….39
3.4.2 Solubilization of Intracellular Materials…..…………..…………………………...39
3.4.3 Augmentation of Cell Disruption………..….………….…………………………. 40
3.4.4 Protein Precipitation……………………...……………….…………………….… 41
3.5 Characterization of Recovered Sludge Protein (RSP)……...…….…….……………… 41
3.5.1 Metal Toxicity……………………………………………….……………………41
3.5.2 Amino Acid Analysis…….…………………………………….…………………42
3.5.3 SDS-PAGE Analysis.……………………………………………….……...…….42
3.5.4 FTIR Analysis……………………………………………………….……………43
3.5.5 TGA Analysis….…………………………………………………………………43
3.6 Evaluation of Adhesive Character of RSP.…………….………………………………. 43
3.6.1 Native Adhesive Formulations………………………………………………….. 43
3.6.2 RSP Modifications and Adhesive Blends……………………………………….. 44
3.6.2.1 Denaturing of ProteinaceousAdhesives…………………………………..44
3.6.2.2 Cellulose Substrates and Contact Angle Measurements……………….... 45
3.6.2.3 RSP Adhesive blends with Synthetic and Bio-based Polymers…………..45
vi
3.6.3 Wood Composite ………………………..……………………………………….47
3.6.3.1 Lap-joint Preparation..………….…….…………………………………..47
3.6.3.2 Testing of Adhesion Strength….………………………………………....48
3.6.3.3 Surface and Internal Microscopy ..…..……….………………………….48

CHAPTER 4 - RESULTS AND DISCUSSION...………………………………………………49
4.1 Physiochemical and Proportional Sludge Composition…………………………………49
4.2 EPS Biochemical Analysis...……….…………………………..………………………..52
4.2.1 Functional Group Identification- FTIR Studies………..…………………………. 53
4.2.2 Thermal Analysis………..………………………………………………………... 54
4.3 High Yield Protein Recovery and Characterization……..…......………………………..56
4.3.1 Protein Solubilization and Cell Disruption from Raw Sludge…….……………….56
4.3.1.1 Effect of pH on Protein Solubilization.……..…………………………….. 56
4.3.1.2 Cell Disruption; French Press and Sonication….…………………………..57
4.3.2 Protein Recovery………………………………..………………………………….58
4.3.2.1 Effect of pH on Protein Precipitation……..………………………………..58
4.3.2.2 Effect of Precipitating Agents on Protein Recovery Yield…….…………..59
4.4 Recovered Sludge Protein (RSP) Characterization….….……………………………….60
4.4.1 Metal Toxicity……………………………………………………………………...60
4.4.2 Amino Acids……………………………………………………………………… 61
4.4.3 Molecular Weight Distribution…………………………………………………….63
4.4.4 FTIR Analysis ………………………….………………………………………… 64
4.4.5 Thermal Degradation of RSP………………………………………………………65
4.5 Use of Native RSP as Wood Adhesive; proof of concept……………………………….66
4.6 Protein Adhesion Mechanism on Cellulose Substrate……..………………………..…..67
4.6.1 Polyamide Chemistry of Recovered Soy and Sludge Proteins…..…………………68
4.6.2 Adhesion Mechanism of Protein-based Adhesives….………….………………….70
4.6.3 Validation of RSP vis-à-vis Soy Protein Bonding Mechanism……………………..72
4.6.3.1 Effect of Protein Denaturing ……...……...………………………………...73
4.6.3.2 Effect of Surface Wettability and Microstructure……….………………….74
4.6.3.3 Adhesion Strength…………………………………………………………..76
4.7 Synergy Evaluation of Chemically Modified RSP Adhesive Blends……..……………..77
4.7.1 Blend Compatibility through Rule of Adhesive Mixtures……..…………………..77
vii
4.7.2 Adhesion Synergy and Failure Mode Analysis……..……………………………..78
4.7.3 Statistical Significance and Discrepancy Analysis..………...……………………..80
4.7.4 Cross-linking Evidence………..……………………………………….…………. 81
CHAPTER 5- CONCLUSIONS, SIGNIFICANCE, AND RECOMMENDATIONS………......83
5.1 Conclusions……………………………………………………………………………...83
5.1.1 Distinct Nature of Secondary Activated Sludge…….……………………………..83
5.1.2 Protein Recovery from Secondary Paper Sludge………….……………………….84
5.1.3 Use of RSP as Wood Adhesive; adhesion mechanism and validation…..…………85
5.1.4 Chemical Modification of RSP and Adhesion Synergy…….……………………...85
5.2 Significance of Research Work………….……………………………………………….86
5.3 Future Work……….……………………………………………………………………...87
REFRENCES …………….……………………………………………………………………..88
APPENDICES……..…………………………………………………………………………...105

viii
List of Tables
Table 2.1 Solid waste generated by different pulping methods…………….……………………6
Table 2.2 Residue amount produced by different grades of paper……..………………………. 6
Table 2.3 Comparison of typical pulp and paper biomass and conventional fuels………...…….9
Table 2.4 Comparison of heavy metal concentration in paper mill sludges……………………11
Table 2.5 Comparison of SS characteristics of different types…………………………………17
Table 2.6 Protein contents of sewage sludge and associated bacteria………………………… 18
Table 2.7 Relative length scale of corresponding adhesion mechanisms………………………26
Table 3.1 Sources of sludge samples…………………………………………………………...32
Table 3.2 Summary of wood adhesives and their solid contents……………………………….43
Table 3.3 RSP/PF and RSP/SPI adhesive blends and glue line………………………………...46
Table 4.1 Comparison of secondary and other sludge characteristics………………………… 49
Table 4.2 Comparison of different protein fractions in sludge samples ……………………….50
Table 4.3 Extractives’ inventory of sludge samples……………………………………………51
Table 4.4 Gravimetric analysis and biochemical composition of extracted EPS……...……… 52
Table 4.5 Effect of different precipitating agents on protein recovery…………………………59
Table 4.6 Trace element concentration (%) in raw sludge, recovered proteins from
different biomass sources, and regulatory standards…………...………………...…….............. 61
Table 4.7 Comparison of amino acid % composition of recovered protein from paper
mill sludge and other sources……………………………………………………...……………..62
Table 4.8 Amino acid composition of RSP and soy proteins…………………………………..69
Table 4.9 Wettability data of RSP and SPI on hard and softwood substrates…………………. 74
ix
List of Figures
Figure 2.1 Simplified schematic of paper mill waste-water treatment plant……..……………...4
Figure 2.2 Sludge disposal practices by US pulp and paper industry..............…………………..7
Figure 2.3 Simplified schematic of paper mill secondary effluent treatment plant
showing aerobic and aerobic + anaerobic options……………………………………………….13
Figure 2.4 Geographical distribution of anaerobic integrated secondary treatment
plants in pulp and paper mills…………………………………………...……………………….15
Figure 2.5 Floc structure…………………………………………………………………….. ..15
Figure 2.6 Epistylis articulate; the most dominant protozoan in activated sludge……………. 18
Figure 2.7 Schematic of mechanism of bonding theories……………...……………………….25
Figure 2.8 Schematic showing equilibrium contact angle between adhesive drop
and substrate……………………………………………………………………………………...26
Figure 2.9 Phase-wise research approach of thesis work….…………………………………... 30
Figure 3.1 Dominant pulping methods of North America…………………………………… 32
Figure 3.2 EPS extraction; centrifuged sludge, chemical extraction/filtration, membrane
dialysis, and EPS sample……………...……………………………………………………...... 37
Figure 3.3 Schematic of protein recovery protocol………………….………………………… 39
Figure 3.4 Schematic diagram of French press and sonication optimization studies…………. 40
Figure 3.5 Wood composites showing glue-line and lap-joint specimen………………………47
Figure 3.6 Lap-joint veneer samples in hydraulic press with a pre-set process schedule……...47
Figure 4.1 Analytical protein determination of sludge samples………………………………. 50
Figure 4.2 Summative analysis of oven dry sludge samples………………………………….. 51
Figure 4.3 IR spectra of EPS extracted from SS by control and chemical methods…………. 54
Figure 4.4 DSC thermograms of SS, MS and extracted EPS………………………………… 55
Figure 4.5 Heat capacity curves of EPS and sludge samples from thermal analysis ……….. . 55
Figure 4.6 Effect of pH and reaction time on protein solubilisation………….……………….. 56
Figure 4.7 Amino acid % composition of recovered protein and other sources..…………...… 57
x