protein recovery from secondary paper sludge and its ......protein recovery from secondary paper...
TRANSCRIPT
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
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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
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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.
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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.
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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
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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
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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
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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
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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
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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
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Figure 4.8 Combined effect of French pressing and sonication on protein solubilisation…….. 58
Figure 4.9 Effect of pH on protein precipitation and recovery yield….………………………. 59
Figure 4.10 SDS PAGE analysis of proteins from activated secondary sludge………………...63
Figure 4.11 FTIR spectra of RSP, secondary sludge and residual mass ……….……………….65
Figure 4.12 TGA analysis of proteins from different sources………………………………… .66
Figure 4.13 Bond shear strength of wood composites bonded with different adhesives…….… 67
Figure 4.14 Failure study of ruptured joint surfaces…………….……………………………... 67
Figure 4.15 Polyamide backbone and reactive sites of RSP and soy protein….…….………….69
Figure 4.16 Availability of three hydrogen bonding sites for each anhydroglucose unit….…....71
Figure 4.17 Schematic of proposed adhesion mechanism of protein adhesives with cellulose substrate………………………………………………………………………… 71
Figure 4.18 FTIR spectra of unmodified and Urea modified protein adhesives…….….……….73
Figure 4.19 Transformation of urea’s keto linkage into enol alkene after hydrogen bonding…..74
Figure 4.20 Initial contact angles of (A) unmodified and (B) modified RSP adhesives……… 75
Figure 4.21 Microstructure of poplar and pine veneers in a composite lap joint glued with RSP adhesives……………………………………………………………………………. 75
Figure 4.22 Adhesion strength of protein adhesives on hard and softwood substrates………. 76
Figure 4.23 Shear strength of adhesive blends; experiment vs model………….………………79
Figure 4.24 Typical failure modes during lap-shear adhesion test and examples…………… 80
Figure 4.25 FTIR spectra of unmodified and modified RSP with PF………...………………. 82
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List of Appendices
Appendix –A. Typical press-schedule of lap-joint hot-pressing………..…………………… 105
Appendix – B. Effect of alkali treatment on suspended solids and protein content of secondary sludge………………………………………………………………………….….. 106
Appendix – C. Comparison of protein yield and adhesion strength at different pH levels…... 107
Appendix – D. Amino acids; Calibration data……….……………………………………… 108
Appendix – E. Amino acid determination; Sample information and analysis…….………… 109
Appendix – F. ‘Goodness of Fit’ Analysis: Shear Strength of RSP/PF blend…….………… 110
Appendix – F. ‘Goodness of Fit’ Analysis: Shear Strength of RSP/SPI blend...…………… 111
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CHAPTER 1 - INTRODUCTION
The pulp and paper manufacturing facilities generate a large quantity of sludge from the
wastewater treatment processes. Generally on average basis, approximately 45 kg of waste
sludge per ton of product is produced from a typical paper mill (Edalatmanesh et al. 2010). The
bulk of this residual material consists of wastewater sludge accompanied by lesser quantities of
woodyard waste, causticizing wastes (Kraft mills), mill trash, and ash from boilers (Amini et al.,
2012; Thacker, 2007). After recovering energy from some of these residues, the disposal of
remainder material has always been an economical and ecological burden for the pulp and paper
industry.
Depending on the disposal method, sludge pollutes soil, air, and water inevitably. Recent public
awareness in the last two decades, strict environmental legislation, and outspoken NGOs have
been pressurizing the pulp and paper industry and researchers to reduce sludge amounts going to
landfills by finding alternate recycling uses of residual biosolids (Chase, 2000; Environment
Canada, 2003; Gorrie, 2005; Kay, 2003; McNair et al., 2003; Suriyanarayanan et al., 2010;
Van Dongen, 2006; Webb, 2000; Yamashita et al., 2008)
Apart from environmental and ecological concerns, the North American pulp and paper industry
has been passing through difficult financial circumstances for the last decade due to continuous
downturn in forest-based industries and low returns on investment (NRC, 2011). Coupled with
the fact that the capital and operating costs incurred to dispose of mixed sludge are estimated to
be in the tune of 50% of the total cost of the wastewater treatment plant (Krigstin, 2008) and
scarcity of disposal lands, it has become imperative to develop new technologies in finding novel
uses of this residual biomass which is rich in valuable components.
Secondary sludge (SS), generated through biological treatment of effluent, typically consists of
polysaccharides, nucleic acids, enzymes, and proteins (Jung et al. 2002) and accounts for 33 to
60 % of total sludge disposed by a paper mill (Rashid et al., 2006). The potential of reutilization
of SS as a resource is possible either by directly recovering useful biomaterials or modifying SS
into value-added materials through biological or physicochemical techniques. Recovery of
valuable biomaterials from activated secondary sludge has gained much attention recently
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(Garcia-Becerra et al., 2010, Edalatmanesh et al. 2010) and the potential of raw SS as a co-
adhesive or an extender has also been demonstrated (Geng et al., 2007a).
Since the bacterial cells in the activated sludge are believed to contain about 50%
proteins (Shier and Purwono 1994), therefore availability of this large amount of residual
biomass from paper mills also gives an alternative opportunity to be explored as a source of
proteinaceous substances. The recovery of protein from municipal sewage treatment plants
(Hwang et al., 2008; Jung et al., 2001 and 2002; Lerch et al., 1993a and 1993b) has been
practiced for analytical purposes. The main focus of this study is to explore the recovery of
proteins from paper secondary sludge in economical and substantial quantities. Further, a
comprehensive characterization of recovered sludge protein (RSP) is also warranted to
understand its chemical composition, thermal behaviour and molecular structure for its potential
use in value-added applications. Investigating the interaction of RSP with cellulose substrates in
a bio-based adhesive system is another unique and scientific feature of this study which can
benefit Canada’s wood composite industry by providing formaldehyde –free binders as a
strategic marketing tool.
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CHAPTER 2 – LITERATURE REVIEW
2.1 Environmental and Economic Implication
About four decades ago the concept of environmentally sound management of industrial
and municipal waste became a matured topic due to emergence of some contentious issues.
Major looming challenges, especially regarding solid waste disposal, identified at that time were
reduced capacity of existing landfill sites, difficulty in opening new site due to strict legislation,
leaching problem of toxic materials, and a growing public awareness about unwanted greenhouse
gas emissions (Krigstin, 2008).
In a rapidly urbanizing global society, the issue of solid waste management has become
even more challenging in recent times. Reducing waste at source and recycling has become an
integral part of waste management and governments of the industrialized world have taken a
number of initiatives in this regard. European governments’ legislation has been successful to
reduce their waste generation per capita owing to pressures of land scarcity and increasing
population densities (Riebel, 2001). According to an EU directive adopted in 1999 only one third
of municipal waste intended for landfilling could be of compostable nature. Among Nordic
countries Sweden has altogether banned dumping of combustible organic waste, whereas its
neighbors have imposed harsh taxes to reduce landfilling (Soderman, 2003).
Canada, being a major forest-based economy, produces around 31 million ton of pulp
annually. Though it exports one third of its pulp production which was worth $23 billion in 2004
(Rashid et al., 2006), the pollutants generated during production process pollutes our lakes, air
and soil. The Canadian industry rightly defends itself by citing its investment of six billion
dollars towards environmental upgrades since 1990 (FPAC, 1999), but there is ample evidence
that the pulp and paper industry remains the third largest industrial polluter in Canada (McNair et
al., 2003). One of the current approaches followed by Canadian waste management think tanks is
to increase the recycling of post-consumer paper products to reduce the intensity of virgin fibre
utilization. Although paper recycling is perceived as the number one priority to transform paper
manufacturing industry around the world, this strategy has its own pitfalls. The recycled paper
manufacturing generates almost ten times the amount of solid residues compared to using wood
as a raw material (Krigstin, 2008). Further, the de-inked sludge (DS) has much higher ash
4
content and lower heating values thereby requiring extra treatments to utilize or dispose of this
material. (Mjoberg et al., 1993). In these scenarios it becomes imperative to develop
technologies to reduce and recycle paper sludge from virgin mills in substantial quantities
2.2 Paper Mill Residual Biosolids
Conventionally the term ‘sludge’ is used for all residues that results from pulp and paper
manufacturing process, however, more specifically it is the solid residue recovered from the
wastewater treatment plants of the pulping and papermaking process as shown schematically in
Figure 2.1.
Figure 2.1 Simplified schematic of paper mill waste-water treatment plant. PS: Primary sludge, SS: Secondary sludge, MS: Mixed sludge, DS: De-inked sludge
The first stage of the processing at the primary clarifier removes primary sludge through
sedimentation or by dissolved air flotation. The sludge thus removed from the bottom of the
tank, usually fibrous material, contains 1.5 to 6.5% solids depending on the material
characteristics. The overflow containing considerable proportion of nitrogenous materials is
passed on to the activated secondary treatment, a biological process in which micro-organisms
convert the waste to carbon dioxide and water while consuming oxygen (Lau, 1981). The
5
activated sludge process generates a large amount of excess sludge due to consumption of
organic pollutants in the wastewater and the associated microbial growth (Jung et al., 2001). The
resulting solids are removed as secondary sludge through clarification and then mixed with the
primary sludge prior to dewatering and disposal as mixed sludge. The ratio of primary to
secondary sludges in mixed sludge varies from mill to mill and is usually maintained at 50:50;
40:60 or 67:33 levels (Rashid et al., 2006).
The characteristics of sludges, or biosolids, and their quantity recovered from pulp and paper
mills are variable and directly related to the technology used to process the wood into pulp and
manufacture the paper and to the type of effluent treatment employed (Vance, 2000). The sludge
generated by the European pulp and paper industry accounts for 4.3% of final production
whereas in Quebec, Canada, this figure is 4.8% (Koubaa et al., 2010). Given the size of the pulp
and paper industry in Canada, large amounts of paper mill sludge originating from primary and
secondary waste water treatment processes are generated which goes mostly to landfills, 41%,
burned, 54%, or applied to land, 5% (Marche et al., 2003; Rashid et al. 2006).
The statistics about the waste generated by pulp and paper industry is limited and most of
the information available is sourced from North American studies carried out in the 1990s to
assess the amount of sludge from pulp mills. However, some studies on USA and Canadian mills
in recent years have generated useful data in this regard. Globally, around 5.8 million ton (Mt) of
dry residues from the entire pulp and paper industry was reported in 1995 (Das and Jain, 2001).
USA, the largest producer of paper products, has been reported recently as generating 15 Mt of
all kinds of waste biosolids from pulp and paper manufacturing industry which includes about
5.5 Mt of sludge from effluent treatment plants (Thacker, 2007). The Canadian figure for solid
wastes from the pulp and paper industry is reported as 7.1 Mt out of which 12% is mentioned as
deinked paper solids (Reid, 1998). In another study, the pulp and paper mill industry in Canada
is reported producing 1.5 million ton of paper mill sludge annually, originating from primary and
secondary waste water treatment processes (Marche et al., 2003).
Different amounts of sludge are produced by pulp mills depending largely on the nature
of raw materials used, manufacturing process, and final product (Bajpai, 2011), Table 2.1.
6
Similarly, the amount of sludge residue produced for each grade of paper is quite different from
each other and depends, in part, on the source of the raw material, Table 2.2.
Table 2.1 Solid waste generated by different pulping methods Pulping process Sludge
(kg/ton) Non-sludge waste
(kg/ton) Kraft mill 58 - Sulphite mill 102 - Deinking mill 234 - Bale wrappers - 23 Sorting rejects - 9 Metal wires/others - 6
Source: Bajpai, 2011
The use of recycled fiber as raw material have more waste to dispose of than mills that
use wood as raw material largely due to fillers in the paper which are mostly not recovered. This
is more pronounced in the tissue grades where office waste, highly filled stock is used to produce
a low-filler content product, Table 2.2.
Table 2.2 Residue amount produced by different grades of paper
Paper grade Wood only (Residue-kg/ton)
Recycled paper (Residue-kg/ton)
Mixed (Residue-kg/ton)
Tissue 33 406 382 Newsprint 57 164 69 Printing writing 62 187 165 Speciality 45 12 11
Source: Abubakr et al. 1995
Similarly in case of printing writing grade, when office waste is used as a raw material,
both the product and the raw material are highly loaded, but the filler material (clay or calcium
carbonate) may be difficult to recover, contaminated, or its characteristics may have changed
making it unusable in the recycled sheet.
Typically, Kraft pulp mill sludge tends to have higher sulfur content, while deinking mill
sludge has more ash content. Between pulp mill and paper mill sludges, the later tends to be
lower in sulfur compounds but it is high in fibrous content (Board, 1986; Ferguson, 1993).
The main objectives of the paper mill effluent treatment plants are to remove enough
suspended solids and BOD (biological oxygen demand) from the effluent to pre-describer limits
dictated by local regulations. Raw effluent flows are reported in a range from 30-70 m3/AD ton
of finished product containing on average 224 kg/ton of total suspended solids (TSS) and 64
kg/ton of BOD5 (Badar, 1994; Krigstin, 2008). Though recycling mills have lower flows of
7
effluent, 10-25 m3/ton of product, recycling also increases the BOD; it is reported that BOD is
increased by 0.57 kg/ton for each percent increase in the use of non-deinked recovered fibre
(Badar et al., 1995; Mabee, 2001)
2.3 Current Sludge Disposal Practices
The most practiced options of sludge disposal have always been landfilling, incineration
and land applications (Badar, 1994; Bellamy, 1994; Reid, 1998), however with the passage of
time their intensity has changed drastically. The current strategies to dispose of pulp and paper
mill sludges in USA are shown in Figure 2.2 which clearly indicates the first choice being
landfilling (Bird and Talberth, 2008; Thacker, 2007). However, compared to 1980’s the
landfilling figure is reduced drastically when up to 85% of pulp and paper mill sludge was
landfilled (Amberg 1988). During the early 1990’s, individual companies privately owned about
50% of landfills used in the pulp and paper industry, however this figure is declining due to
increasingly stringent legislations and construction costs (Glowacki 1994).
Figure 2.2 Sludge disposal practices by US pulp and paper industry Source: Bird and Talberth, 2008; Thacker, 2007
Incineration requires huge capital investment to install sophisticated equipment to burn the wet
and heterogeneous material effectively, whereas land applications have been under strict scrutiny
over the last two decades due to long-term toxicity implications of sludge materials.
2.3.1 Landfilling
Landfill, the most commonly used disposal technique for sludge (Ham et al., 2009), has
been made safer but very expensive due to increased legislative requirements. Recently, concern
has risen over the amount and quality of future landfill space due to cost of construction, more
8
stringent regulations, diminishing land availability, and public opposition. In 1978, the United
States had about 14,000 landfills, in 1988 there were 5,500, and by the year 2000, the number of
landfills were expected to drop to 2,200 (Ingram, 1993).
The most important criteria that can limit this route for disposal is the presence of trace
chemicals, such as PCB which may leach into the water table beneath the site. Modern landfills
are designed with the objective to minimize these risks and very few problems have been noted
with leachates or gas production in landfills and these landfill programs also have compensation
programs for nearby residents (Heeney and Trott, 1991; Miner and Gellman, 1988).
In Canada, especially, Ontario, the landfill construction cost and other charges have
increased significantly. Tipping fees in and around Southern Ontario have increased over the past
several decades due to limited landfill capacity and the urging of environmentalists to promote
ecofriendly waste management practices. Transporting the waste material to the landfill site must
also be considered in terms of both environmental and monetary costs. The recent cost of`
transporting Toronto municipal waste to Michigan for disposal in landfills was $58/ton plus
$65/ton tipping fee (American Recycler, 2006). Costs for landfilling in the B.C, Canada are even
higher ; expected in the range of $130 to $150/ ton by Jan, 2012 in the region of Hartland (CRD,
2009).
Different construction techniques have been used to avoid sub-soil or air contamination
through leachates or gas emissions. One technique is the lining of landfills with dense clays or
with a synthetic material such as high-density polyethylene, reduces the threat of leaching
(Maule et al. 1993).
Pulp and paper sludge has also been used as an effective barrier layer in landfill closures.
In a recent full-scale pilot plant study, Abitibi Bowater Inc., Canada, has successfully closed a
1.4 hectare (ha) ash landfill using compacted pulp sludge and the barrier layer appears to be
performing in a manner equal to that of the conventional barrier systems (Ham et al., 2009).
2.3.2 Incineration
Incineration of sludge serves two purposes; it disposes of the material, and it provides
heat energy that can be used in the papermaking process. Quite recently, the rising cost and
uncertainty of traditional energy sources has shifted the energy focus to renewable sources.
National energy policies in many parts of the world are being updated or initiated for the first
time with priority on renewable energy, and in North America and Europe the incineration of
9
pulp and paper mill sludge for energy generation is currently a predominant strategy for its
disposal. It is widely practiced in Europe and gaining broader acceptance globally as newer and
less polluting technologies are developed. In 2004 the bio-energy accounted for 638.8 PJ (15.3
Mtoe-million tonnes of oil equivalent) in the pulp and paper industry in Europe, which represents
50% of the total energy consumption in the industry. The pulp and paper industry accounts on
average 23% of total bioenergy use in EU countries whereas renewable energy share in the pulp
and paper industry has increased from 45.5 % (1996) to 54.5 % (2006), (CEPI, 2007).
The option to use paper sludge as a biowaste fuel for energy production has found
renewed interest in North America and Europe. The organic fraction in paper sludge is
renewable, and therefore it does not contribute to net CO2 emissions. A few mills incinerate
paper sludge in their boilers as “hog” fuel but this practice is not widespread, because the heating
value is very low (Table 2.4) and the high moisture of the sludge affects its ability to burn
efficiently. To enhance the heating value, the sludge is mixed with dryer waste materials such as
wood residue (Gavrilescu, 2008). Sludge ash concentrates heavy metals, however if their
concentration surpasses hazardous levels, the ash requires special handling, (Shin et al., 2005).
Table 2.3 Comparison of typical pulp and paper biomass and conventional fuels.
Analysis (%) Biomass
Solids Ash C H S N O
Heating Value1 (kJ/kg)
Mixed sludge2 37 20 33.7 4.4 0.3 0.7 41.2 4200 DIP sludge3 42 50 19.0 2.4 0.05 1.0 27.4 2800 Bark 40 3 50.6 5.9 - 0.5 40.2 5900 Peat 50 5 57.1 6.2 0.2 1.9 29.6 9200 Coal 88 14 71.6 4.9 0.6 1.9 7.0 24000
Source: Gavrilescu, 2008 1. as received basis 2. Mixed sludge (primary and secondary paper mill sludge) 3. De-inked paper mill sludge
The heating value of deink sludge depends on the ash content and is 4.7–8.6 GJ/t of dry
substance, (Hamm, 2006). Halogenated organic compounds such as polychlorinated biphenyls
(PCB), polychlorinated dibenzodioxins (PCDD), and polychlorinated dibenzofurans (PCDF) are
found in the flue gas. All types of sludge contain toxic trace metals (Pb, Cd, Cu, Zn, Hg) in
various quantities. The behavior of toxic trace metals in de-inking and biological sludge, has
been found to be strongly affected by metal interactions with the solid substrate in the furnace
10
(mostly with the Al/Si structure) which acted as a limit to their vaporization. Paper sludge is also
characterized by high chlorine content, which will be partly retained in the fly ash as condensed
alkali chlorides, the rest forming HCl to a high extent. Kaolin enhances HCl release in the flue
gas, while calcium carbonate enhances chlorine capture on the coarse fly ash fraction (Coda,
2004).
2.3.3 Land Applications
The earliest studies on paper sludge utilization in agriculture (Dolar et al., 1972;
Hermann, 1982; NCASI, 1984; Thiel, 1984) have shown benefits like increased plant growth and
yield, improved soil moisture and nutrient retention. Adverse effects, limited to high salt levels
(ammonium, sodium and sulfate) and deficiencies of available N in some types of sludge, have
been minimized by controlling sludge application rates and by delaying planting for some period
after treatment, and/or by applying fertilizer to soil or sludge (Simpson et., 1983).
Several pilot plant studies have been conducted during 1990’s to evaluate the
effectiveness of different types of sludge and any associated drawbacks and ecological concerns.
One study examining yields of Bermuda grass on mine soils treated with primary sludge at rates
of 56 000, 112 000, and 224 000 kg/ha found the downward trend in yield of Bermuda grass
especially at higher rates. It was suggested to combine the sludge treatments with fertilizer and a
yield increase of tenfold was achieved after this modified treatment (Feagley et al., 1994).
When the primary and secondary sludge streams are separated and applied individually to
soil, it was found that secondary sludges, due to higher nitrogen and phosphorus content, are
actually more suitable for application. Values of nutrients vary, but one reported range of
secondary sludge values are 3% N, 1.5% P, and 0% K (Pickell and Wunderlich 1995).
In a recent study (Quayea et al., 2011) the effect of paper sludge, dairy manure, and urea
fertilizer on biomass production of shrub willow (Salix dasyclados SV1) was studied and it was
found that both fertilizer and paper sludge behaved in a similar fashion.
Amini et al. (2012) have found increase in wheat yield through a research which
diagnosed nutrients that affect crop production in a high specific surface soil with illite as the
dominant mineral in clay fraction and incorporation of paper mill sludge as mulch.
A potential limitation for the use of raw deinking sludge as a soil amendment is the
possibility of N and P deficiencies for adequate plant nutrition and growth. However, when N
and P fertilizers are supplemented, de-inking paper mill biosolids can be an adequate soil
11
amendment (Norrie and Gosselin, 1996; Krigstin, 2008). Fierro et al. (1997) evaluated de-inking
paper mill biosolids as a soil amendment supplemented with N and P in a greenhouse study on
different species of grass and found that the best response in sand-sludge mixtures was obtained
with the mixture containing 30% biosolids. De-inking paper mill biosolids are suitable for use as
a soil amendment in re-vegetation studies on degraded light soils when supplemented with P and
N ingredients.
The potential for heavy metal contamination of water and plants is one of the major
public concerns over the use of sludge on agricultural land (Gendebien, 2010). Generally the
contents of heavy metals and organic toxic compounds in paper mill sludges are low, Table 2.4,
and comparable to those found in livestock manure (Bellamy et al., 1995; Cabral et al., 1998;
Demeyer and Verloo, 1999; Rashid et al., 2006). Although concentrations of heavy metals in soil
amended with paper mill biosolids have usually been below established standards
(Baziramakenga and Simard 2001), there are some exceptions , especially in the case of deink
biosolids when copper levels in soil estimated after crop harvest, 135Mg ha-1, were found
exceeding the permissible limits (Goss and Rashid, 2004).
Table 2.4 Comparison of heavy metal concentration (mg/kg) in paper mill sludges
Metal Primary sludge1 Secondary sludge1 Mixed sludge2 CMCS3
Arsenic 0.09-1.7 0.2-0.98 <1 20 Cadmium 0.25-2.5 0.25-2.5 <1 3 Chromium 17-29 31-46 5.2-12 750 Cobalt 1-5 <1 1.5-2.5 40 Copper 15-26 12-30 250-310 150 Lead 1-10 4-10 8.3-10 375 Mercury 0.02-0.2 0.14-0.25 0.57-0.87 0.8 Molybdenum 4-20 <4 2.5-3.8 5 Nickel 7-10 2-9 2.9-5.6 150 Selenium 0.5-2.0 0.5-2.0 <1 - Zinc 26-38 30-79 130-250 600
Source: Rashid et al., 2006 1. Powell River pulp and paper mill, B.C, Canada 2. Abitibi Bowater, Canada 3. CMCS limits = Criteria for Managing Contaminated Sites Limits in British Columbia
By the mid-nineties Canada was recycling 7% of all available pulp and paper mill
biosolids through land application. Twenty mills made use of land application in 2001 and half
of these mills applied more than 95% of their biosolids production (Krigstin, 2008). The majority
12
of the biosolids are applied to agricultural land (70%) whereas 4% to silvicultural land and the
remainder for land rehabilitation (Velema, 1999). In southern Ontario, the Ontario Ministry of
Environment has been responsible for managing the spread of paper mill biosolids on agriculture
lands, however some controversies have been reported as recently as 2006 when the ministry had
to issue an order requiring the owner of a sludge pile to address contamination created by runoff
from a biosolids stockpile near St. Catherines (Van, 2006).
The option of composting mixed sludges and then applying them to the ground is another
alternative that has been pursued by several mills in the USA. In Canada this practice has been
facing some perception related issues. For example in July 2006, an Ontario Ministry of the
Environment order was issued to Abitibi to clean up a pile of sludge in Pelham, Ontario
(Grittenden, 2006). Though the scientific data indicated no health related hazards, strong
emotional issues were behind this incident.
The federal and provincial governments in Canada are now developing new legislation to
regulate the application of residual biosolids on agriculture lands. As of January 1, 2011, Ontario
will stop issuing “Organic Soil Conditioning Sites” Certificates of Approval for the application
of non-agricultural source materials (NASM), which includes biosolids, and will instead issue a
new type of approval called a NASM Plan, based on nutrient and toxin elements of source
material (CCME, 2010).
2.4 Types of Sludge
Broadly speaking, sludge can be separated into two major categories, primary and
secondary sludge. Primary sludge is a fibre-rich material formed from mechanical cleaning of the
wastewater stream and consists primarily of woody fibres and fines, and as such is the easiest
material to characterize and reuse. In a virgin pulp mill, this material is fairly clean containing
only woody waste material and impurities washed out of the raw material. In a recycling plant,
the primary sludge also consists of synthetic materials such as plastics and stickies, as well as
traces of glass or metal. After processing, primary sludge can vary in moisture content from 30 to
70%, depending on the drying technology and the targeted final moisture content (Coburn and
Dolan 1995). Primary treatments are effective at removing suspended solids, but do not reduce
BOD levels to any great extent (O'Connor et al. 2000) .
13
Secondary sludge is produced through activated wastewater treatments and is composed
of a combination of organic and inorganic detritus that includes fibres, fines, and bacterial cells.
A more detailed description of this sludge is mentioned in the following sections.
Deink sludge is by definition a primary sludge produced through mechanical treatment of
deinking effluent of a recycle mill. Mostly it is processed through the same mechanical cleaners
and filters as regular primary sludges but it is much dirtier and more problematic to deal with.
Due to its very different characteristics, it is kept separate from other sludges.
2.4.1 Paper Mill Effluent Secondary Sludge
2.4.1.1 Process of Generation and Significance
The secondary paper sludge is generated through activated wastewater treatments in a
similar way to the treatment of municipal sludge via either aerobic and/or anaerobic bacteria.
Aerobic treatment of sludge is facilitated through use of an aerated stabilization basin (ASB) or
lagoons where the bio-organisms catalyze the conversion of organic substances in wastewater to
carbon dioxide, water, and microbial biomass, or secondary sludge. The semi-solid sludge is
Figure 2.3 Simplified schematic of paper mill secondary effluent treatment plant showing aerobic and aerobic + anaerobic options (IC Reactor: Internal circulation reactor)
14
dredged out or drained from the basin into a clarifier as shown in the schematic of Figure 2.3.
The supply of excess air and nutrients facilitate the rapid growth of bacteria which consume
organic material available in the waste stream (Kroiss, 2004; Maybee, 2001; Wood et al., 2009).
Although much of the toxicity of the effluent is substantially reduced with the ASB
process, certain chemicals, such as pentachlorophenol, tend to resist this treatment at toxically
high levels (Cowan et al., 1995). Certain hard to biodegrade compounds in pulp mill effluents,
such as EDTA (ethylenediaminetetraacetic acid) (Ridge, 2005) which is used in TCF (total
chlorine free) bleaching process to scavenge metal ions, can be effectively controlled by
secondary effluent treatment. The ASB system can reduce the concentration of EDTA in the
range of 50 - 80% under alkaline conditions (Ginkel and Geerts, 2005; Virtapohja and Alén,
1998).
In a modified version of the aerobic treatment process, tanks are used instead of lagoons
to hold highly aerated wastewater during biological activity and solid precipitates are removed in
a sedimentation tank known as a secondary clarifier. Though this system works in a shorter
process time needing less space, it is expensive in terms of hardware requirements and difficult
to control higher microbial concentrations. (Johnson and Chatterjee, 1995).
Aerobic activated systems are found to be effective in reducing BOD loading of the
effluent stream; generally up to 90% reduction is achieved. However, in case of deink sludge,
this effectiveness is somewhat reduced (NCASI 1991).
Most commonly used versions of secondary effluent treatment usually include anaerobic
or hybrid aerobic-anaerobic pretreatment, cultivating different bacteria in the absence of oxygen.
However, aerobic activation is mostly an integral part of the activated treatment system because
the anaerobic method alone is not found sufficient to detoxify the effluent sludge. Though
anaerobic treatment has a number of advantages like lower energy, less chemicals, small space
requirements, and potential of methane production as a byproduct (Driessen and Vereijken,
2003; Karlsson et al., 2011) there exists a large discrepancy between Europe and North
America in the adoption of this technology as evident from Figure 2.4 ( Lerner et al., 2007). The
main reasons for North America lagging behind in adopting anaerobic technology could be lack
of incentives and regulatory policies. Further, this system has low tolerance to toxic shock and
the inhibitory compounds and is also responsible for producing many of manure-like smells
15
Figure 2.4. Geographical distribution of anaerobic integrated secondary treatment plants in pulp and paper mills. Source: Lerner et al., 2007
accustomed with secondary treatment plants (Lerner et al., 2007; O'Connor et al. 2000).
Sometimes it is advantageous to use coagulants to facilitate the precipitation of
suspended material in the sludge suspension. For this purpose certain chemicals including alum,
Al2(SO4)3, and polyacrylamide polymers are used especially in zero-effluent mills, where it is
important to precipitate all materials. In these cases, these polymers can be used on a large scale,
with up to four ton per day of alum and 0.5 ton per day of polyacrylamide per day.
Since the generation of activated sludge largely depends on microorganisms, a delicate
process control through microscopic studies is an essential part of an optimized waste water
treatment plant (WWTP). The monitoring of sizes and shapes of floc particles, Figure 2.5, which
largely depends on retention times, air flow, metal toxicity, and sludge flow rates is one of the
key parameters in ensuring desired reductions in BOD of discharged effluents.
Figure 2.5 Floc structure. (left) compact floc larger than 500 µm and (right) floc with open structure and interflocular filamentous bacteria Source: Arregui et al., 2010
16
The solid content of treated secondary sludge are fairly low, around 0.5 to 3%, and due to
the high water content in the sludge even after dewatering, secondary sludge disposal can
account for as much as 60% of total wastewater treatment costs (Wood et al., 2009). Generally,
to assist dewatering, secondary sludge is mixed with primary sludge before final pressing. The
ratio of primary to secondary sludges in mixed sludge varies from mill to mill and is usually
maintained at 50:50; 40:60 or 67:33 levels (Rashid et al., 2006). .
2.4.1.2 Current Applications of Wastewater Activated Sludge
Although wastewater activated sludges have been used as landspread and fertilizer, their
utility in this regard is limited due to metal toxicity if biosolids are sourced from municipal
treatment plants (Dewil et al., 2006). Currently, the other major value-added use of activated
sludge is the production of biofuels. Some wastewater treatment facilities have integrated
cogeneration systems where activated sludge is further processed in anaerobic digesters to
produce methane and hydrogen (Ghosh et al., 1975). A Canadian paper mill in B.C has recently
installed a high pressure cell disrupter named MicroSludge to breakdown bacteria’s tough cell
walls of activated sludge followed by anerobic reactor to produce synthetic biogas (Caulfield,
2012). Similarly, in a recent pilot plant study, pulp mill SS was used in an anaerobic
bioconversion process to generate biogas at high rates of production (Wood et al., 2009). In fact,
biogas synthesis is the only economical viable method to produce biofuels from activated
sludges. Other biofuel related applications involve pyrolysis to transform waste biomass of SS
into liquid fuels (Kim & Parker, 2008; Konar et al.,1994) and using various fluid extraction
techniques to recover the lipid fraction and converting them into biofuels (Dufreche et al., 2007).
Manufacturing of bioplastics such as poly hydroxyl alkanoates (PHAs), is another value-added
application, however, the economics of the production of PHAs is an issue as it includes costly
processes such as cell culture isolation and complex product recovery (Wallen & Rohwedder,
1974; Yan et al., 2006). Development of nylon-enabled composites with SS as reinforcing-filler
is another research area which has been explored recently (Edalatmanesh, 2012)
2.4.1.3 Chemistry of Secondary Sludge
The characteristics of secondary sludge compared to other common sludge types are very
distinct in terms of carbohydrate, protein, ash, lignin and lipid contents (Barzelatto, 1995; Deng
et al., 2007 Geng et al., 2007a).Secondary sludge (SS), generated through biological treatment of
17
effluent, typically consists of polysaccharides, nucleic acids, enzymes, and proteins (Jung et al.
2002). Most of the analytical and pilot plant studies have been focused on the secondary sludge
of municipal waste treatment plants to recover protein and very little data is available on pulp
and paper activated sludge. Unfortunately the composition of activated sludge is never likely to
be constant even from a single plant and variations between plants may be enormous. Table 2.5
gives an overview of different activated secondary sludges characterised for different properties.
Table 2.5 Comparison of secondary sludge (SS) characteristics of different types
Characteristics PP-SS1a PP-SS1b MW-SS2c
(n=5)
MW-SS2 ST-SS3 DY-SS3
TS (mg/ml) 11.1 24.4 - - 20.0 20.0 TSS (mg/ml) 8.7 17.9 - - 19.2 19.6 VS (mg/ml) 7.9 13.9 - - - - Ash (%) - - 27.1 25.1 - - Fat (%) - - 16.0 1.73 - - C/N ratio 7.3 11.4 - - - - COD (mg/ml) 11.7 27.0 - - 8.6 0.23 Protein % 35.1 18.9 13.0 37.8 73.4 5.2 Carbohydrate (%) 7.2 8.1 4.1 9.8 - -
Sludge sources from waste treatment plants of; PP: pulp and paper manufacturing, MW: municipal waste, ST: starch industry, DY: dairy industry
1 Wood et al., 2009 (a: sulphite pulp mill, b: kraft pulp mill) 2 Stafford et al., 1979 (c: average of 5 municipal waste plants) 3 Yan et al., 2006
The protein content of sewage sludge has been reported as direct function of quality and
number of microorganisms available. The activated sludge system facilitates the aggregation of
microorganisms that are embedded within or in the surface of a complex heterogeneous floc
structure which are freely suspended in the mixed liquor. Biological communities within the
activated sludge plants are mainly composed by microorganisms of which 90-95% biomass
pertains to bacteria found free or forming part of the floc (isolated, grouped or in a filamentous
form). These bacterial populations are responsible of the biodegradation of organic material and
the removal of toxic contaminants (Bitten, 2005; DNR, 2011).
Protists, involved in the predation of bacterial populations and in the flocculation process and
other microorganisms represent 5-10% of the biological total biomass of activated sludge. These
microorganisms, especially the dominant protozoan, Epistylis articulate (Figure 2.6) and
associated bacteria are substantially proteinacous in nature as found in analytical studies on
18
activated sludge and shown in Table 2.6. In the qualitative analysis of this study, it was found
that glutelin protein was the highest fraction, whereas, the percentage yields of albumin and
globulin fractions were almost the same in all the materials (Arregui et al., 2010; Sridhar and
Pillai 1973).
Figure 2.6 Epistylis articulate, the most dominant protozoan in activated sludge Source: Arregui et al., 2010
Table 2.6 Protein contents of sewage sludge and associated bacteria
Material Total Protein (%)
Raw sewage sludge 11.4 Septic tank sludge 17.2 Activated sludge 43.1 Epistylis articulata 59.5 Bacteria from activated sludge 72.6 Bacteria from Epistylis articulata 77.8
Unfortunately, not many references are available in literature pertaining to the composition
of pulp and paper secondary sludge in terms of lignocellulosic materials which are inherently
present in substantial quantities in such activated effluents. It has been reported in a recent work,
(Edalatmanesh et al., 2010), that the ratios of cellulose, hemicellulose and lignin are somewhat
similar in the secondary sludge compared to typical wood fibers while the high amount of ash in
the sludge was attributed to additives, especially clay, which are incorporated as a part of raw
material to manufacture paper products to improve their opacity, printing and other properties.
Although it is mentioned in the same study that selected sludge apparently had substantial
amount of protein, but it seems fractionation has been performed without extracting this material.
19
2.4.1.4 Microbial Characteristics of Secondary Sludge
Extracellular polymeric substances (EPS) of bacterial cells are reported as major
component of activated sludge originating from metabolism/lysis of microorganisms and the
wastewater itself (Garcia-Becerra et al., 2010; Wingender et al., 1999). The EPS are believed to
be the building blocks of activated sludge flocs and biofilms and represent the main constituents
of the organic fraction for these microbial aggregates (Sutherland, 2001; Vaningelgem et al.,
2004).
Due to unique and significant role in biofilm formation and bio-adhesion qualities, a
number of investigative studies are available in literature dedicated in exploring the polymeric
nature of EPS (Garnier et al., 2005, 2006; Goh et al., 2005; Sheng et al., 2005; Weinbreck et al.,
2003). In activated sludge EPS acts like a polymeric matrix that holds the microbial biomass
together. Proteins and polysaccharides are estimated as main constituents of EPS whereas humic
substances, uronic acid and deoxyribonucleic acids (DNA) have been detected as well (Garnier
et al., 2005; Liu & Fang 2002).
The molecular masses of EPS proteins and polysaccharides range from a few thousands
to several million Daltons and their components contain a large number of negatively charged
functional groups including carboxyl, amino, sulphate and phosphate (Garnier et al., 2005).
Consequently, proteins and exopolysaccharides play a significant role in adhesion phenomena
through formation of expolymeric networks as the supporting matrix of microbial aggregates
(Görner et al., 2003; Wilen et al., 2003).The average amount of extracted EPS from various
municipal and industrial waste secondary sludges has been reported as 20% of the total organic
content with a wide range of 10 to 42%. Carbohydrates represent 11% while proteins makeup
36% of the extracted fractions (Garcia-Becerra et al., 2010). A study on the EPS extracted from
pulp and paper secondary sludge using Formaldehyde/NaOH process has been reported as 15%
(Edalatmanesh et al., 2010)
2.4.1.5 Protein Recovery from Municipal Waste Effluents
A considerable proportion of the nitrogenous materials present in the sewage or paper
mill effluents is settled out during treatment process and is incorporated into the primary sludge
through the sedimentation process. Alternatively, it may be removed from the sewage effluent
and be converted into the biomass of micro-organisms through the biological oxidation and
coagulation processes known as activated secondary treatment process (Lau, 1981). The
20
activated sludge process generates a large amount of excess sludge due to consumption of
organic pollutants in the wastewater and the associated microbial growth (Jung et al., 2001).
Typical municipal wastewater activated sludge consists mainly of microorganisms with
concentrations of up to 1011 cells/ml, surrounded by large macromolecules, along with varying
proportions of dissolved organic compounds and heavy metals (Wagner, 2005; Watson and
Pletschke, 2006 ). In terms of microbiological nature of activated sludge, it is composed of
approximately 95% bacteria which have protein as a major fraction of their biomass (Shier and
Purwono, 1994; Sridhar and Pillai, 1973).
Due to industrial significance a considerable interest has been shown by researchers to
extract and characterize the proteins from municipal sludge (Hwang et al., 2008; Jung et al.,
2001 and 2002; Lau, 1981; Lerch, 1991; Lerch et al., 1993a and 1993b), wastewaters of fish
processing facilities (Stine et al. 2012), cheese manufacturing, and poultry industries (Potter et
al. 1974). Most of these studies are analytical in nature and Hwang et al. (2008) has explored the
potential use of recovered protein as poultry feed.
Though literature is not very specific about the true optimum conditions necessary to
obtain maximum protein extraction, various protein recovery protocols based on
physicochemical techniques have been reportedly used, which essentially comprise the
solubilization of intracellular contents of municipal sludge into the aqueous phase by disrupting
the floc structure (Jung et al. 2001; Onyeche et al. 2002; Navia et al. 2002;). A significant
increase in the soluble protein and decrease in total suspended solids (TSS) was observed after
sludge disintegration (Zhang et al. 2007; Weemaes et al. 2000). Lau (1981) has reported protein
recovery from both primary and secondary sludge of three different sewage plants by using a
pre-concentration anionic ion-exchange process followed by the use of 2.5% sodium chloride
solution to facilitate protein regeneration. Finally, sodium lignosulphonate was used as
precipitating agent. The efficiency of protein extraction into solution from the sludge is reported
around l0%, while the protein recovery from the solution by the use of sodium lignosulphonate
was approx. 38%. The recovered sludge protein had considerable protein content: as high as 71%
for the secondary sludge.
Sodium hydroxide has been found the most common and effective alkali used by researchers
for protein solubilisation from municipal sewage sludge. Lerch et al. (1993a) used a number of
21
reagents and found that 1M NaOH solution was significantly more effective than Triton (a non-
ionic detergent) and water as solubilizing agent. Similarly Chishti et al., (1992) has reported up
to 89% protein solubilization by using NaOH compared to only 10% solubilization while using
NaCl at optimum conditions.
Characterization studies on recovered proteins from sewage sludge indicate wide
variation in results, however, generally it is reported that recovery process reduces the amount of
heavy metals compared to raw sludge (Hwang et al., 2008). Lau (1981) and Chishti et al. (1992)
have reported the presence of all essential amino acids whereas Lerch et al. (1993b) have
reported both high and low MW fractions of proteins extracted from activated sludge samples of
different sewage treatment plants. Goodwin and Forster (1989) have reported the presence of
predominantly low MW proteins, < 10 kDa, in an activated sludge while using membrane
filtration.
2.5 Potential Role of Sludge Protein as Wood Adhesive
2.5.1 Significance of Bio-based Wood adhesives
The recorded history of use of bonded wood products goes back to at least 3,000 years in the past
to the Egyptians (Skeist and Miron, 1990; River, 1994), and multipurpose adhesive bonding goes
back to even earlier times (Keimel, 2003). Although many of the early civilizations had skills to
make adhesives from plants and animals, however advent of petrochemicals led to wide spread
use of formaldehyde-based adhesives in the last century.
Although known for good performance in a variety of applications, a growing concern regarding
health related issues has been emerging against mass scale use of formaldehyde-based adhesives
over a period of time. In 1995, the International Agency for Cancer Research determined that
formaldehyde should be listed as a suspect carcinogen. Further testing was conducted and in
2004 formaldehyde was reclassified to a known carcinogen, specifically for nasal cancer (IARC,
2004). A major related issue emerged in July, 2007, when FEMA testified before the Committee
on Oversight and Government Reform in the U.S. House of Representatives that trailers built for
Katrina hurricane victims had 400 times the level of formaldehyde emissions permitted by law.
These and other initiatives have prompted several jurisdictions to adopt strict legislation against
the use of formaldehyde based resins. The State of California, for example, is introducing phase
22
II of its legislation in 2011-12 to control formaldehyde emissions which is believed to be hard to
comply with current technology of producing wood panel composites (Orr, 2007).
Apart from health related issues, recent climate change and environmental concerns have further
focused the bio-based wood adhesives in the spotlight which are derived from animal, plant, or
marine kingdoms. The most common of bio-based adhesives are protein-based sourced from
animal bones and hides, milk (casein), blood, fish skins, and soybeans.
2.5.2 Current Status of Sludge and Protein-based Adhesives
In the area of wood adhesives, paper sludge had been suggested as organic filler in the
past (Geng et al., 2007; Robertson and Robertson, 1977) and as a reinforcing agent in
thermoplastic composites (Krigstin and Sain, 2006). Prototypes of medium density fibreboard
(MDF) have been also developed from sludge sourced from different pulp mills (Koubaa et al.,
2010) and deinked paper mills (Davis et al., 2003).
A growing interest has been shown over a period of time to isolate and find the potential
applications of biopolymers from secondary sludge of municipal sewage plants. Most of these
studies are analytical in nature and have characterized the material in detail. Garcia-Becera et al.
(2010) have extracted surface active agents from returned activated sludge of an urban sewage
plant to explore their utility as detergent and also suggested the potential application of these
organic materials as wood adhesive after chemical crosslinking. In other analytical studies on
the recovery and characterization of protein from municipal waste sludge (Chishti et al., 1992;
Hwang et al., 2008; Lerch et al., 1993b), poultry food supplement has been emphasized as the
potential application area of recovered proteinacous biomass.
Apart from traditional animal kingdom sources like blood and hide, non-conventional
resources have been also investigated to develop protein-based glues. Graham et al. (2005) have
investigated “frog glue” secreted from Notaden bennetti , Australian frogs, and found that this
mostly protein-based biopolymer acts as a promiscuous pressure-sensitive adhesive that
functions even in wet conditions. Gary et al. (2010) have reported the discovery of a novel,
electrically mediated protein-based adhesive formed from silkworm silk through a process
termed electrogelation which offers biomimetic features when used in conjunction with devices.
Similarly protein-based adhesives derived from mussels, an aquatic bio organism, are known to
23
be extremely strong and water resistant (Zhang et al., 2010), however, all these kind of glues are
very un-economical to produce even at pilot scale levels.
Today, soy flour is the most abundantly available and commercially used raw material for
protein-based wood adhesives (Orr, 2007) which was first introduced in 1923 when a patent was
issued to manufacture soy meal-based glue (Johnson et al., 1984). Due to renewed interest in
developing bio-based adhesives, various studies have been carried out in recent past to enhance
the effectiveness of soy protein adhesives through chemical modifications, enzymatic treatments
, and denaturing techniques ( Kumar et al., 2004; Liu and Li, 2007; Qi and Sun, 2011; Zhang
and Hua, 2007).
Phenol formaldehyde (PF) induced hybrid bio-adhesives have been discussed in
literature. The invention of cold-setting adhesive for finger-jointing lumber containing equal
parts of soy protein isolate and phenol–resorcinol–formaldehyde resin is available in prior art
(Steele et al., 1998). A US patent (Riebel et al., 1997) illustrates the methods for preparing a
soybean-based molding compound by cross-linking soy flour with polymeric 4, 4-
diphenylmethane diisocyanate. A PF-cross-linked light-coloured soy resin containing 70% soy
flour and 30% PF has been also developed which can be used as a liquid resin for exterior
plywood or as a powder resin for molded products, but it lacked the ability to be used as spray
resin (Kuo et al., 2001).
Since the sludge protein extracted from municipal sewage plants are reported to have all
primary amino acids available in soy protein, it seems a logical extension of this research study
to explore bonding mechanism of wood adhesives in pursuit to explore potential applications of
paper sludge proteins for same purpose.
2.5.3 Proposed Wood Adhesion Theories
An adhesive, by definition, is a substance capable of holding materials together by a
commonly observed phenomenon of surface attachment also known as adhesion. Adhesion itself
is a complicated phenomenon involving adhesive penetration, mechanical anchoring, chemical
bonding and physical interactions. The actual adhesion mechanism has never been clearly
24
defined and universally agreed upon, although some have proposed a unifying adhesion theory
on the subatomic level (Nevolin et al., 1990).
A number of adhesion theories have been presented over period of time; each being
subjected to critique and controversy. However, these theories do provide concepts and useful
information to understand basic requirement of a good adhesive bond and even predict
qualitative realization of adhesion strength (Petrie, 2000).
As mentioned earlier, at present no practical single theory describes all wood adhesive
bonds. However, as per more recent prior art, six adhesion theories have been proposed by
researchers in this area (Pocius, 2002; Schultz and Nardin, 1994)
- Mechanical interlocking
- Electronic or electrostatic
- Adsorption / or wetting theory
- Diffusion theory
- Covalent bonding
- Weak boundary layers and interphases
Depending on the particular conditions, these mechanisms are not mutually exclusive and
several may be interacting at the same time in a given adhesion system. Mechanical interlocking,
a purely physical friction that prevents the adhesive from slipping off the porous substrate under
stress, is considered as important component to the strength of wood adhesion (Cheng, 2004;
Gardner et al., 2005). This theory emphasizes on the spreading of adhesive, wetting of substrate
surface, and subsequently curing and hardening of adhesive in cavity like an anchor, Figure 2.7
(A). The chemical bonding theory has long been a focus of study to understand durable wood
bonding with thermosetting adhesives. It is possible to have covalent bonds through sharing of
electrons across the interface between the adhesive and substrate as illustrated in Figure 2.7 (B).
This kind of bonding is believed to be strongest and durable where mutually reactive chemical
groups tightly bond the substrate surface and adhesive film; such as hydroxyl and carboxyl
groups of adhesive coatings tend to react chemically with similar reactive groups of substrate
surface (Gollob and Wellons, 1990).
The electrostatic theory is more relevant to wood in finishing and coating operations
whereas studies pertaining to adsorption or wetting theory have been mostly focused on wood
adhesive systems (Gray 1962; Shi and Gardner 2001).
25
Figure 2.7 Schematic of mechanism of mechanical (A) and chemical (B) bonding theories
The theory of weak boundary layers is mostly pertinent to the impact of mechanical
damage on preparing wood surfaces for bonding and the impact of surface aging (Christiansen,
1990; Stehr, 1999). The bonding mechanism of thermoplastic matrices used in wood plastic
composites has been generally explained through diffusion theory which presents the concept of
tentacles of adhesive penetrating into the substrate from the macro scale to the molecular level
(Wool, 2002).
The thermodynamic model of adhesion is another widely used approach in explaining the
adhesion science (Schultz and Nardin, 1999). In this mechanism, the adhesive is adhered to the
substance as a result of the interatomic and intermolecular forces at the interface through
intimate contact. The magnitudes of interfacial forces, van der Waals and Lewis acid-base
interactions, responsible for establishing intimate contact are generally related to the fundamental
thermodynamic quantities such as surface free energies of both the adhesive and the adherent.
The perfect interface is supposed to materialize when the liquid adhesive completely “wets” the
substrate surface. Usually, this “spread ability” or “wettability” is expressed and measured in
terms of “equilibrium contact angle”, θ, formed by the drop of adhesive on the substrate surface
as shown in Figure 2.8.
The wetting equilibrium in a solid-liquid system is defined from the profile of a drop on a
solid surface and expressed through Young’s equation;
γSG = γSL + γLG cos θ
This equation defines the balance of forces caused by an adhesive wet drop on a dry surface by
relating the surface tension (γ) of materials at the three-phase contact point to the equilibrium
26
contact angle θ where subscripts S, L, and G stand for, respectively, solid phase of substrate,
liquid phase of the droplet and gas (vapor phase) of the ambient.
Figure 2.8 Schematic showing equilibrium contact angle between adhesive drop and substrate
Length scale and bond energies; Adhesion interactions
The knowledge of length scale over which the adhesion interactions transpire has been
described as a useful tool to categorize bonding theories (Gardner et al., 2005). Similarly, bond
energies associated with chemical interactions play an important role in understanding and
interpreting adhesion effectiveness of various polymers (Charles, 2005). A comparison of length
scale to relative adhesion interactions is presented in Table 2.7 which demonstrates that
interlocking or entanglement (mechanical/diffusion) adhesion interactions can transpire over
larger length scales compared to charge interactions. The influence of diffusion interactions
(entanglement) takes place mainly from nano to several millimeters length scale depending on
the size of the bond interphase, whereas charge interactions occur on the molecular level or
nano-length scale
Table 2.7 Relative length scale of corresponding adhesion mechanisms.
Interaction type Mechanism Length scale Mechanical 0.01 to 2000 µm Interlocking/Entanglement
Diffusion 10 nm to 2 mm
Electrostatic 0.1 to 1.0 µm
Covalent 0.1 to 0.2 nm
Acid-base interactions 0.1 to 0.4 nm
Charge
Van Der walls 0.5 to 1.0 nm
Data source: Gardner et al., 2005
27
As far as the bio-adhesion of protein with cellulose substrate is concerned, researchers
have deduced from their experimental work that bonding mechanism in this case is mostly
governed by three type of interactions; mechanical bonding, physical adsorption, and chemical
bonding. Apart from surface characteristics of cellulose substrate and degree of wettability, the
primary structure of protein adhesive, dictated by linear sequence of amino acid residues or
polypeptides, is also a contributing factor for adhesion efficiency in these systems (Sun, 2005).
Since the protein amino acids’ side groups are characterized with both negative and positive
charges, the polarity of these functional groups plays a major role in the adhesion by establishing
strong bonds with wood surface (Richard, 2001).
The adsorption linkage in a protein-cellulose adhesion relies on any physical or
electrostatic attraction between protein polymers and wood surfaces through hydrogen bonding
and van der Waals forces. In case of wood, the cellulose material has abundant amount of polar
hydroxyl groups which can develop hydrogen bonds effectively with oxygen and nitrogen atoms
of protein adhesive (Xu et al., 2011). The mechanical interaction is believed to occur through
penetration of protein adhesive molecules into the fiber cells through crevices via capillary paths
followed by anchoring due to in-place curing of adhesive (Sun, 2005). A more thorough
explanation on this subject is available in the discussion part of section “Protein Adhesion
Mechanism on Cellulose Substrate”
2.6 Problem Statement
The management of large quantity of residual biosolids generated from pulp and paper
mills is a source of economic liability for an industry going through financial hardships for better
part of last decade, especially in Canada (Edalatmanesh, 2012; Gorrie, 2005; Koubaa, 2010;
NRC, 2011; Webb, 2000). At the same time the widely practiced sludge disposal options, land
filling and incineration, are generally associated with ecological concerns as well (Environment
Canada, 2003; Krigstin, 2008, Mahmood and Elliott, 2006; Kay, 2003; McNair et al., 2003;
Van Dongen, 2006; Yamashita et al., 2008). Land spreading, the only recycling option
accounting for a small share of total sludge produced, has its own environmental and logistic
issues and regulatory bodies have been drafting stricter legislation to control the use of sludge on
agriculture lands (CCME, 2010; Gendebien, 2010; Grittenden, 2006; Van, 2006). This research
28
project is intended to explore the bio-adhesion characteristics of secondary paper sludge and its
value-added utilization as wood adhesive.
Several analytical studies on the secondary sludge from municipal waste treatment plants
have confirmed the presence of biopolymers, especially protein, in significant quantities and
suggested its use as poultry feed (Hwang et al., 2008; Jung et al., 2001 and 2002; Lau, 1981;
Lerch, 1991; Lerch et al., 1993a and 1993b). Paper mill secondary sludge, accounting for almost
one half of total mixed sludge, is believed to be similar to municipal effluent activated sludge in
terms of microbial chemistry and contains 30 -40% proteins on dry weight basis (Edalatmanesh,
2012; Jung et el., 2002). Although microbial and cellular studies have suggested use of
secondary sludge as potential surface active agents and adhesives( Garcia-Becerra et al., 2010,
Edalatmanesh et al. 2010), but current body of knowledge is devoid of any reference pertaining
to protein recovery from paper sludge and its characterization . This work, first of its kind, is
related to protein recovery from paper secondary sludge through a modified protocol in
substantial quantities and its comprehensive characterization.
The potential use of recovered sludge protein as wood adhesive is also explored in this
research work. Soy flour, a proteianacous rich conventional bio-based wood adhesive, is
available in commercial quantities. However, use of food crops for adhesives and fuel/plastic has
detrimental effect on food prices which has been a major global concern during the last few years
(Daynard, 2011; UN-WFP, 2008). Though raw secondary sludge as a co-adhesive or an extender
has been demonstrated (Geng et al., 2007a), but no background information on adhesion
mechanism is elucidated in this area. Derived from fundamental knowledge available on wood
bonding theories (Sun, 2005) and denaturing dynamics of soy protein (Van et al., 2000), a
comprehensive model on sludge protein-cellulose adhesion mechanism is proposed and
substantiated through experimental work in this study.
Several methods are discussed in literature to enhance the effectiveness of protein
adhesives through chemical modifications, enzymatic treatments, and denaturing techniques
(Kumar et al., 2004; Liu and Li, 2007; Qi and Sun, 2011; Zhang and Hua, 2007). Soy protein
adhesives have been effectively modified with phenol and urea formaldehyde (Kuo et al., 2001;
Qi and Sun, 2011; Wescott et al., 2006). This study investigates the modification of sludge
protein with PF over a wide range of formulation as well as adhesive blends with soy protein are
also studied in detail which includes synergy evaluation through mathematical model.
29
2.7 Hypothesis and Objectives
The main goal of this study was to investigate technical viable and economically sound strategies
to isolate protein from raw secondary paper sludge in substantial quantity for reuse in value-
added applications through comprehensive characterization and testing.
2.7.1 Hypotheses
Two hypotheses are addressed in this work.
- The inherent physicochemical and microbial composition of secondary sludge warrants
the possibility of exploiting this residual biomass as a source of protein in substantial
quantities.
- The recovered sludge protein and its modified versions may have better adhesion
properties than SS due to high profile protein-cellulose interactions.
2.7.2 Objectives
Specific objectives related to above hypotheses were;
i. Evaluating physical and chemical characteristics of different type of paper mill
sludges.
ii. Characterizing bio-chemical composition of secondary sludge and quantifying extra
cellular polymeric substances through analytical studies.
iii. Comprehensive characterization of recovered sludge protein.
iv. Understanding physico-chemical mechanism underlying protein adhesion to woody
component.
2.8 Scope of Research Work
The objectives were achieved through collection and pre-treatment of appropriate sludge samples
and experimental design based on four phases as shown in Figure 2.9. Brief overview of each
phase is mentioned below.
30
Preliminary Characterisation
The preliminary phase of characterization was accomplished through physical and bio-
chemical characterization of selected samples of SS from different sources. This task was
facilitated through use of various standard and modified methods of testing available for woody
and pulp/paper product analysis. The interpretation was focused on comparing the results of
analysis of different sourced sludge materials which gave information about the variability of
important properties and their suitability for intended applications.
Figure 2.9 Phase-wise research approach of thesis work
The characteristics evaluated in this phase included; ratio of organic to inorganic material,
woody polymeric and extractives quantification, carbohydrate, protein and lignin contents,
and thermal degradation through TGA.
Extra cellular polymeric substances were extracted through both physical and chemical
methods from SS and MS and yield was reported in terms of gravimetric analysis of EPS and
associated protein concentration.
31
Protein Recovery from SS
This phase was further arranged in three sequential processes by using experimental designs
planned for each optimizing study.
Protein solubilisation and cell rupture in alkaline media was performed over a wide pH range
of 8 to 12 for two different time intervals. After finding optimum conditions of this step, further
yield improvement in protein solubilisation was achieved by employing physical techniques of
French press and sonication both as standalone and in-series mode. Final step of protein
precipitation was optimized by using three different acidic media.
The RSP was characterized for molecular weight distribution (SDS-PAGE), amino acid
analysis (revers-phase HPLC), heavy metal analysis (ICP AES), functional group determination
(FTIR), and thermal degradation behaviour (TGA). The RSP was tested for adhesion properties
by testing shear strength of ruptured lap-joints of poplar veneer bonded with RSP adhesive.
Evaluation of Protein-adhesion Mechanism on Cellulosic Substrate.
The complementary nature of chemistry of proteins isolated from soy and secondary
paper sludge and other relevant evidence regarding well-established bonding theories helped in
deducing RSP-wood adhesion mechanism.
An experimental design was chalked out to further validate bonding mechanism of RSP –
cellulose system which is inspired from a literature work involving denaturing of soy globulin
protein and its effect on adhesion strength with hard and soft wood substrates.
Synergy Evaluation of Adhesion Blends; Sludge protein adhesive blends with synthetic and
bio-resourced materials
Adhesive blends of RSP with phenol formaldehyde (PF) and soy protein isolate (SPI)
were formulated over a wide range of concentrations and tested for adhesion strengths. Apart
from chemical modifications, the trend of adhesion performance of wood composites was
validated through a classical adhesive-mixture law.
32
CHAPTER 3 – MATERIALS AND EXPERIMENTAL METHDOLOGY
3.1 Materials Sludge samples for this study were selected from two Canadian pulp and paper mills
representing most dominant pulping processes used globally to manufacture a variety of paper
products. Chemical pulping ranks first in manufacturing bulk of market pulp; out of total world
market pulp capacity of 57.9 Mt (PPPC, 2009), about 92 % belongs to chemical pulping. In
North America and rest of the world, traditionally Kraft pulping has been the most dominant
method to produce both soft and hardwood pulps on large scale, Figure 3.1 (Smook, 1997)
Figure 3.1. Dominant pulping methods of North America
The secondary sludge samples were arranged from Abitibi Consolidated Inc. Amos, Quebec, and
Abitibi Bowater, Thunder Bay, Ontario. For comparative studies, a sample of mixed sludge (MS)
was also arranged from first mill, however the ratio of SS to PS mixed in this sludge was
unknown. The details of operations of these mills are mentioned in Table 3.1.
Table 3.1 Sources of sludge samples
Pulp/paper Mill
Location Process & Furnish Capacity
(Tonne/year)
Sample Label
Abitibi Consolidated Inc
Amos, Quebec
TMP: (100% SW)
Newsprint: (99.5% TMP, 0.5% DIP)
209,000 SS1
MS
Abitibi Bowater
Thunder Bay, Ontario
Kraft Bl. (ECF) (95% SW, 5%HW)
W/printing
579,000 SS2
33
It may be noted that Abitibi Bowater Thunder Bay is now owned by Resolute Forest Products.
The sludge samples having consistency of around 1.5% were arranged in polyethylene bags
placed in buckets. Upon arrival at laboratories, these samples were immediately stored at 4oC till
further testing and use.
For basic characterization, the sludge suspensions were dried at 60oC till constant weight, and
converted to powder of 40mesh through Wiley mill grinding. For determining suspended solids,
volatile solids, and extracting protein contents, the raw sludge was used in as received
suspension form.
3.2 General Characterization of Paper Mill Effluent Sludges
3.2.1 Solid Contents Determination
The solid contents, total solids (TS), total suspended solids (TSS) and volatile suspended
solids (VSS), of secondary sludge samples were determined by using standard methods 2540 B ,
2540 D, and 2540 E respectively. These methods have been devised by American Public Health
Association, APHA (2005).
The moisture content of dried sludge samples and recovered protein was measured
through TAPPI Standard T 412 om-94 (TAPPI 1996) and accounted for on an oven-dry basis to
correcting compositions and reporting yields where applicable.
3.2.2 Ash Determination
Ash content of sludge samples was determined by igniting samples at 525°C for four
hours. This procedure follows TAPPI Standard Method T 211 om-93 (TAPPI 1996). The
material remaining after incineration is completely inorganic, as all carbon complexes within the
sample are combusted.
3.2.3 Total Nitrogen (TN) Determination
Invariably always the average protein content of activated sewage sludge has been
reported in terms of TN determined through Kjeldahl method (Chishti, et al., 1992; Helrich,
1990; Kyllönen et al., 1988; Peres, 1992; Wood et al., 2009). Because the Kjeldahl method does
not measure the protein content directly a conversion factor of 6.25 (equivalent to 0.16 g
nitrogen per gram of protein) is used for many applications (Hattori and Mukai, 1986). The
Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and
34
titration. The principle behind the method is that when biomass is digested with a strong acid, it
releases nitrogen which can be determined by a titration technique. For this study, these tests
were done in Department of Chemical Engineering, University of Toronto.
3.3 Extractives, Protein and Lignocellulose Analysis of Sludge
3.3.1 Extractives Determination
In case of the secondary sludge, the extractives are mainly nonstructural wood
constituents (e.g., fats and waxes, phenolic compounds, etc.) along with the low molecular
weight polysaccharides and lipids associated with the microorganisms. The TAPPI Standard T
264 om-88 (TAPPI 1996) was applied by substituting benzene, a carcinogenic solvent, with
toluene due to their close solubility values (Barton, 1975).
Three extraction stages were employed to remove the full range of extraneous substances
from the material, leaving behind major wood polymers and protein contained within the sludge
matrix. The first extraction is carried out with a 1:2 mixture of 95% ethanol and analytical grade
toluene. As a non-polar solvent, this mixture removes substances from the sludge that include
waxes, fats, resins, salts, wood gums, phytosterols, and non-volatile hydrocarbons. The second
extraction utilizes 95% ethanol, and serves as a rinse, removing any of these materials that the
first cycle left behind. The third extraction with boiling distilled water is effective for dissolving
colouring matter, gums, tannins and sugars.
All the extraction tests were done by wrapping the samples in ‘tea-bags’ made out of kimwipe
tissue papers, a cellulose tissue product.
3.3.2 Analytical Protein Estimation
Fractionation of recycled paper sludge has been practiced without extracting protein since
these materials have very small amounts of nitrogen (Krigstin, 2008). However, since activated
sludge being rich in bio-organisms therefore it is essential to extract protein contents from sludge
before attempting cellulose and lignin extractions.
Unfortunately, there is no single standard method available to extract protein completely
from complex biomass like activated sludge. The most commonly used method is the alkali
solubilization of protein, using IM NaOH solution as the extraction agent (Barzelatto, 1995;
35
Chishti, et al., 1992; Lerch, et al., 1993a). A more detailed description of this technique, which is
part of a protein extraction optimization study, is mentioned in Section 3.4.2.
Extractive-free sludge sample, 5.0 g each time, was used to solubilize the protein with
alkali at 25oC for 12 hour constant stirring at pH 12. After treatment the protein-solubilized
solution was filtered and the residue was used for cellulose and lignin estimations.
The solubilized protein was quantified through Bio-Rad® protein assay, a standard
colorimetric method for measuring total protein concentration based on the Bradford dye-binding
method (Bradford, 1976). This assay is very reproducible and rapid with the dye binding process
and there is virtually no interference from cations such as sodium and potassium or from
carbohydrates as reported in other standard protein quantification methods such as Lowry
procedure (Bradford, 1976). In this method a standard curve is generated using 0.2–1.5 mg/ml of
bovine gamma globulin standard protein solution and diluted dye reagent after absorbance
measurement at 595nm with spectrophotometer, Smart Spec TM Plus.
Lipids solubility in alkali solutions and inherent existence of water-soluble proteins in
secondary sludges are the two major issues which can potentially interfere with the fractionation
estimates (Peres et al., 1992). Since the protein estimation was done after removing extractives,
therefore, any erroneous estimation due to solubility of lipids in alkali has been completely ruled
out. However to rule out any interference in the final stage of extractives removal with hot
water, a parallel test was arranged on raw sludge to estimate water-soluble proteins through
Bradford assay method.
3.3.3 Lignocellulose Fractionation
Apart from extractives, lignin and cellulose are major components of woody biomass
which are commonly found in appreciable quantities in primary and mixed sludge. Although
activated sludge systems harbour a number of complex microorganism which might change the
composition and quantity of lignocellulose components in secondary sludge, but still this
residual biomass contain significant amounts of these materials. Lignocellulose composition of
woody materials is determined through standard methods devised for wood materials and same
methods have been extensively used by researchers investigating paper mill sludges of all sorts
(Barzelatto, 1995; Edalatmanesh, 2012; Mabee, 2001; Krigstin, 2008).
36
Holocellulose represents the entire cellulosic portion of wood material and includes
alpha, beta and gamma cellulose. Quantitative isolation of the holocellulose component from
woody material is accomplished through delignification with acidified chlorite solutions, a
method developed by Browning (1967). In this method extractive-free sludge is treated
repeatedly with glacial acetic acid and sodium chlorite followed by washing with water and
acetone.
Alpha-cellulose is distinguished from other wood polysaccharides by its insolubility in
aqueous alkali solutions. The alphacellulose fraction was determined by further reducing the
holocellulose fraction of the sludge through multiple treatments with 17.5% NaOH to dissolve
and remove polyoses. Finally, the isolated alpha-cellulose was washed with acetic acid, dried and
weighed.
Lignin from extractive-free sludge was isolated as a residue by removing the
polysaccharide portion of extractive-free sludge by hydrolysis with strong acid. This method is
based on the assumption that lignin does not degrade significantly in acidic mediums. The solid
residue was measured as Klason lignin content as per ASTM Method D5896-96 (ASTM, 2005).
3.3.4 Analytical Extraction of Cellular Biocomponents (EPS)
Extracellular Polymeric Substances (EPS) are considered as one of the major component
of the secondary sludge which mostly contains proteins and carbohydrates. Extraction of EPS is
routinely performed for analytical purposes and several such studies (Cetin and Erdincler, 2004;
Garnier et al., 2005; Goh et al. 2005; Dungi et al., 1998) have investigated the polymeric features
of the EPS in activated sludges due to their significant role in biofilm formation. No standard
method to extract EPS is reported in literature but researchers have been using a number of
physical and chemical techniques to extract EPS from sludge of waste water treatment plants
(Liu and Fang, 2003). Though the amount and composition of extracted EPS largely depends on
extraction method, type of wastewater, and the operating conditions of the treatment plant (Liu
and Fang, 2002; Sponza, 2003), Guibaud (2003) has reported maximum yield of extracted EPS
by using the alkaline-formaldehyde chemical technique. Same method has been used in this work
described below.
In this work, two sludge samples, SS1 and MS were used to isolate EPS through both
chemical (alkaline-formaldehyde) and physical techniques. In chemical method, centrifuged
37
sludge was treated with formaldehyde (36.5%) for one hour at 4oC followed by NaOH 1M
treatment at same temperature, stirred for three hours while maintaining the pH level at 11.
Separation of EPS from treated sludge was achieved by ultra centrifugations at 20,000g for 20
minutes followed by 10,000g for 15 minutes at 4oC. For further removal of microbial cells, the
sample was dialyzed using a 6500 D membrane suspended in distilled water at 4oC. The freeze-
dried EPS was kept at -18oC till further use. An overview of the EPS extraction procedure is
elaborated in Figure 3.2.
Figure 3.2. EPS extraction (left to right); centrifuged sludge, chemical extraction/filtration, membrane dialysis, and EPS sample
EPS extraction was also done through a physical control method (Liu and Fang, 2003) in which
sludge is ultra-centrifuged at 4000g for 20 min, at 4 ◦C without using any chemicals or extended
dialysis.
3.3.4.1 Biochemical Analysis
Analytical analysis involved measurements of protein and carbohydrate contents of
extracted EPS. Protein was determined using Bradford assay method, already mentioned in
earlier sections.
Carbohydrates were quantified through Anthrone method (Raunkjaer et al., 1994) using
glucose as a standard reagent to generate a standard curve after absorbance measured at 625 nm.
3.3.4.2 FTIR Studies
Infra-red (IR) spectrometry was used to determine the main functional groups in extracted
EPS samples. Fourier transform infrared (FT-IR) spectra were acquired on a Bruker Tensor-27
spectrometer over a range of 400–4000 cm-1 at a resolution of 4 cm-1 with 200 scans.
38
3.3.4.3 Thermal Analysis – DSC Testing
Calorimetric measurements of sludge and extracted EPS were obtained using differential
scanning calorimeter (DSC) DSC- Q1000, TA Instruments Inc, New Castle, DE, USA. The
samples, 2-3 mg, were weighed into standard aluminium pans and sealed hermetically with lids.
A similarly sealed empty aluminium pan was used as the reference. The DSC cell was
pressurized with Nitrogen gas at 22 psi throughout the experiment and thermograms were
recorded at a standard ramp mode of 10oC/min.
3.4 Protein Recovery from Paper Mill Secondary Sludge
The activated sludge of pulp and paper mill effluents consists of microbial biomass which
is typically composed of polysaccharides, nucleic acids, enzymes, and proteins (Jung et al.
2002). Unfortunately, as per author’s information, no effort has been made so far to recover
protein from paper mill activated sludge.
In this work a multi-step physicochemical recovery process was used to extract protein
from secondary sludge of Kraft paper mill effluent plant. Briefly, a combination of French cell
press and sonication techniques was used to augment the disruption of the floc structure of
sludge solids and optimize the release of the intracellular proteins into the aqueous phase after
the initial alkali treatment. These simple and inexpensive physical methods have been used
successfully by researchers for cell lysis (Abram et al. 2009; Benov and Al-Ibraheem 2002).
The precipitation of soluble protein in the current study has been studied through use of
sulphuric acid, hydrochloric acid, and ammonium sulphate. The extracted crude protein was
characterized for quantitation of protein, determinations of molecular weight of extracted
proteins, evaluation of amino acids, and assessment of heavy metal toxicity. The main results of
RSP characterization are compared with available data pertaining to sewage sludge protein
leading to conclusive discussion in exploring the potential utilization of paper mill secondary
sludge.
Finally, in this part of study, RSP was used to develop lap-jointed veneer composites to
validate the concept-of-proof in the application of sludge protein as wood adhesive. The
39
adhesion properties of RSP for wood bonding was accessed through lap-joint shear testing and
results compared with commonly used synthetic and bio-based wood adhesives.
3.4.1 Protein Recovery – General Scheme A general scheme involving a physicochemical protocol to recover protein from SS has
been illustrated in the schematic of Figure 3.3. Each step is further elaborated below.
Figure 3.3 Schematic of protein recovery protocol
3.4.2 Solubilization of Intracellular Materials
The initial solubilization of intercellular contents from sludge into aqueous phase was
facilitated through alkali (NaOH) treatment by using liquid SS as starting material. Since
sufficient evidence is available in literature in recommending NaOH as appropriate protein
solubilizing agent (Chishti et al. 1992; Lerch et al. 1993a; Hwang 2008), therefore, the same
alkali was used to disintegrate the sludge and release the intracellular materials into aqueous
phase. Various pH values ranging from 8.0 to 12.5 were maintained by treating with alkali at
25oC followed by continuous stirring for two different intervals; 2 and 24 hours. Finally, the
40
protein concentration was estimated in alkali treated sludge solutions to calculate solubilization
yield at each pH level.
3.4.3 Augmentation of Cell Disruption
In order to augment the protein yield after alkali treatment, optimization studies were
carried out by using the French press and sonication methods individually as well as in a
combination sequence in which French pressing was followed by sonication. The methodology
of these treatments is shown in Figure 3.4
Figure 3.4 Schematic diagram of French press and sonication optimization studies French pressing was done on a Spectronics Instruments, SLM-AMINCO, by adjusting
the pressure at 20,000 psi. A quantity of 30ml of alkali treated (12 pH, 24 hour treatment) sludge
was poured in minicell for each pressing and six replicates were carried out for each test.
Sonication was performed on a Branson Sonifier 450 equipped with a micro tip. French
pressed sludge samples were intermittently sonicated on ice for 30 s with 30 s allowed for
cooling. The total sonication time was 1, 3, or 5 min. and six replicates were processed for each
cycle.
Total number of passes through French press and duration of sonication were varied in
this study to investigate the maximum release of proteins from alkali treated sludge. The
41
disrupted floc mass containing mostly soluble protein was separated as supernatant by
centrifugation of disintegrated sludge at 7000 rpm for 30 minutes at 4 oC.
3.4.4 Protein Precipitation
The solubilized protein of highest concentration obtained after alkali treatment was
further precipitated out by lowering the pH of supernatant with 2.0M H2SO4. Four different pH
levels, 1.5, 3.0, 4.5, and 5.5 were investigated to optimize the protein recovery. Similarly 2.0 M
solution of hydrochloric acid was also used to precipitate the solubilized protein at pre-
determined pH level of 3.0. Finally the precipitates were centrifuged at 7000 rpm for 30 minutes
at 4oC to obtain the recovered sludge protein (RSP) in the pellet form.
Ammonium sulphate was the third precipitating agent to recover soluble protein. In this
method a 40% saturated solution of this salt was used to precipitate out protein from highest
concentrated solution of solubilized protein. To remove excess salt from recovered protein,
dialysis was performed using regenerated cellulose dialysis tubes (Fisherbrand #21-152-5).
The final pellet of RSP in each case was dried at 60oC overnight to estimate yield and
adhesion strength. Selected sample of RSP of highest yield was used for various characterization
studies.
3.5 Characterization of Recovered Sludge Protein (RSP)
Total and soluble protein was measured as per methods already mentioned in earlier sections. 3.5.1 Metal Toxicity
The concentrations of heavy metals in SS and RSP were determined using an inductively
coupled plasma mass spectrometer (Perkin Elmer Model Optima 7300DV ICP AEOS-
PerkinElmer Inc. USA).
The dried and powdered samples were acid digested, diluted volumetrically with 18 Mohm water
and assayed by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP AES) directly.
The detected concentration of metals has been certified by spectrometric analysis against an
independent source, which is traceable to National Institute of Standards and Technology.
42
3.5.2 Amino Acid Analysis
Protein precipitated out by use of H2SO4 was analysed to determine amino acid
composition of RSP. The method involved the hydrolysis and pre-column derivatization of the
hydrolyzates using PITC followed by reverse phase HPLC.
The amino acid analysis was performed by Advanced Protein Technology Centre, Dept.
of Molecular Structure & Function, Hospital for Sick Children, Toronto, Canada. Briefly, the
samples were dried in pyrolyzed borosilicate tubes in a vacuum centrifugal concentrator and
subjected to vapour phase hydrolysis by 6N HCl with 1% phenol at 110°C for 24 hours under
pre-purified nitrogen atmosphere. After hydrolysis, excess HCl was removed by vacuum,
hydrolyzates washed with redrying solution and derivatized with phenyisothiocyanate (PITC) to
produce phenylthiocarbamyl (PTC) amino acids. The derivatized amino acids were re-dissolved
in phosphate buffer and transferred to injection vials which were loaded into the auto-sampler for
automatic injection. Quantitation of amino acids was done through Waters Acquity UPLC
detection at 256nm.
3.5.3 SDS-PAGE Analysis
The lysis of the samples for extraction of protein from sludge slurry was performed by
treatment with alkali, liquid nitrogen with or without detergents (NP-40, Triton X-100, or a
combination of these methods. To prevent endogenous protease activity, protease inhibitor
cocktails were also added in the sample. Unless stated otherwise, samples were boiled for 5 min
in sample buffer (final concentration 0.25 M Tris-HCl, pH 6.81, 30% (v/v) glycerol, 8% (w/v)
SDS) supplemented with 10% (v/v) 2-mercaptoethanol, bromphenol blue indicator dye (Sigma
chemical) and centrifuged to remove any un-dissolved material. The loaded sample volume for
each well of gel was 20 µL, whereas for marker, a pre-stained SDS-PAGE standard broad range
ladder (Bio-Rad: 161-0318) was used.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was routinely
done using 4–20% precast linear gradient polyacrylamide gel, 10-well, 30 µl, 8.6 x 6.8 cm
(W x L), while using Mini-Protein II Bio-Rad electrophoresis system working on an electrical
potential of 120 V and 60mA. Proteins were stained using brilliant blue G solution Sigma
B8522-1EA containing 0.1% w/v brilliant blue, 25% v/v Methanol, and 5%v/v acetic acid.
43
3.5.4 Fourier Transform Infrared (FT-IR) Analysis
The study on the functional groups of secondary sludge, recovered protein, and residual
mass was performed on a Bruker Tensor-27 spectrometer. All spectra were captured over a range
of 400–4000 cm-1 at a resolution of 4 cm-1 with 200 scans.
3.5.5 Thermogravimetric (TGA) Analysis
Thermogravimetric analysis was performed by using TA instrument Q500 with a heating
rate of 10 oC/min. The samples were heated from 40 to 900 oC to determine the complete thermal
degradation of RSP in nitrogen atmosphere using a flowing rate of 60 mL/min.
3.6 Evaluation of Adhesive Character of RSP
In the area of wood adhesives, paper sludge had been suggested as organic filler in the
past (Geng et al., 2007; Robertson and Robertson, 1977). In this work, RSP has been utilized as
wood adhesive to manufacture veneer lap-joints and evaluate its adhesion strength. Phenol
formaldehyde, a commonly used synthetic resin, and soy protein have been also used as wood
adhesive for comparative purpose.
3.6.1 Native Adhesive Formulations
Soybean protein isolate (SPI) powder, PRO-FAM® 974, arranged from ADM-USA and
containing 90% protein was suspended in distilled water and stirred for 2 hours to make
unmodified soy wood adhesive of 10% solid contents (Wang et al., 2006).
Table 3.2 Summary of wood adhesives and their solid contents W o o d a d h e s i v e s
PF SPI SS RSP
Solids (%) 49.5 10.0 15.0 8.1
Glue Line 5mg/cm2
Phenol formaldehyde (PF) liquid resin, 2220-109, arranged from Arclin Canada Inc. was
used as received whereas secondary sludge (SS) was concentrated before use as adhesive. The
solid contents of different adhesives used are mentioned in Table 3.2.
44
The selection of right quantity of glue-line for this kind of study is always quite a
challenge since each adhesive has very different texture and spreadability at any given solid
contents and especially raw sludge is difficult to apply evenly on wood ply (Geng et al. 2007a).
A quantity of 5 mg/cm2 of glue-line, fairly close to similar studies (Liu et al. 2005; Wang et al.
2006), was found appropriate to ensure uniform spreading of all tested adhesives at their
designated solid contents. The same glue-line was used in all the lap-joint preparation tests for
both unmodified and modified adhesive systems.
3.6.2 RSP Modifications and Adhesive Blends
3.6.2.1 Denaturing of Proteinaceous Adhesives
Protein-based adhesive must consist of relatively large, flexible and interwoven polymer
chains for better adhesion with wood substrate. Chemical modifications have been used for this
purpose to break the internal bonds and facilitate unfolding of the protein molecules (Van et al.,
2000). Soy protein isolates (SPI) treated with urea, alkali, and sodium dodecyl sulfate (SDS)
have shown improved adhesion strength and water resistance (Huang and Sun, 2000a; Huang
and Sun, 2000b; Sun and Bian, 1999).
Urea has hydrogen, nitrogen, and negatively charged oxygen atoms that would interact
with hydroxyl groups of the proteins, which could break down the hydrogen bonding in the
protein body and, consequently, unfold the protein complex. At lower urea concentrations, urea
can destabilize globular protein by forming strong hydrogen bonds with water molecules
surrounding the proteins, resulting in partially unfolded protein structures desirable for improved
wet adhesion strength (Sun, 2005).
In this study RSP was modified with urea to elucidate the bonding mechanism of sludge
protein with cellulose substrate in conjunction with similar parallel modification of soy protein
and testing both adhesive systems for their wettability and adhesion strength.
Recovered sludge protein (RSP) and soy protein Isolate (SPI) were modified with 1.0 M
urea solution separately as per method mentioned by Zhang and Hua (2007). In this method ten
grams of protein adhesives on dry basis were suspended in 100 g of urea solution, stirred, and
allowed to react for 6 h at room temperature. The final solid content of urea modified SPI and
RSP adhesives was maintained at 10 and 8% respectively, whereas the glue line for lap-joints
was controlled at 5mg/cm2.
45
3.6.2.2 Cellulose Substrate and Contact Angle Measurements
Poplar (hardwood) and pine (softwood) veneers, used as wood substrate to prepare lap-joints,
were arranged locally from forest laboratory of Faculty of Forestry, University of Toronto. In
case of urea modified protein formulations, both hard and softwood veneers were used to prepare
test specimens.
Contact angle measurement
Contact angles measurement between adhesive and wood were measured with a goniometer
Mitutoys 519-109. Three readings for each adhesive for each position (immediate and
equilibrium) were taken to calculate average values of contact angle drop percentage as follows;
η = (θi - θe) *100/ θi
Where
η: contact angle drop percentage
θi: immediate contact angle
θe: equilibrium contact angle.
Unmodified RSP and SPI were used as control adhesives.
3.6.2.3 RSP Adhesive Blends with Synthetic and Bio-based Polymers
Enhancing adhesion properties of proteinaceous adhesives through chemical modifications or
crosslinking has been studied extensively in the past and both synthetic and bio materials have
been used as co-polymers for this purpose. PF induced hybrid bio-adhesives have been widely
discussed in prior art (Kuo et al., 2001; Qi and Sun, 2011; Wescott et al., 2006).
In this work, recovered sludge protein was modified with different concentrations of phenol
formaldehyde and soy protein separately. The formulated adhesive blends of RSP:PF and
RSP:SPI were investigated as wood adhesive and trend of adhesion performance was validated
through a classical adhesive-mixture law.
In order to investigate any cross-linking of the RSP with PF and SPI, the selected
adhesive blends 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 sample. The powdered samples were mixed with
KBr, pressed into a disc, and the IR spectra were collected in the range 4000 − 400 cm−1 using
46
TENSOR 27 spectrometer with a resolution of 4 cm−1. Spectral outputs were recorded in the
absorbance mode as a function of wave number.
RSP/PF Adhesive Blends
PF-cross-linked protein resins were formulated by reacting the recovered sludge protein
(RSP) hydrolyzates with phenol-formaldehyde (PF) in various solid-to-solid ratios as shown in
Table 3.3. The entire modification process involved essentially denaturization and
copolymerization steps to develop PF-protein cross-linked adhesive.
After adjusting the pH of RSP to 8.0, the RSP solution was treated with about 50% PF
solution which was prepared separately according to method available in literature (Yang et al.,
2006). In short a mixture of 1 mol phenol, 2.4 mol formaldehyde, and 0.1mol NaOH with a
required amount of water was heated at 65°C for 1.5h, and then regularly heated at 95°C for 1h.
The characteristics of the PF resin were 50% solids, and pH 9.5 at 25°C. PF-protein formulations
were prepared by heating RSP hydrolyzates to 50°C, followed by slow addition of PF resin with
vigorous stirring for 20 min so that the resulting resins contained desired concentrations of both
components. Commercial resin and RSP were used as control resins.
RSP/SPI Adhesive Blends
Soybean protein isolate (SPI), PRO-FAM® 974-ADM USA, containing 90% (dry basis)
protein was suspended in distilled water and stirred for 2 hours to make wood adhesive of 10%
solid contents, pH adjusted to 10.0 with NaOH (Liu and Lie, 2007). Adhesive blends, RSP/SPI,
of various ratios were prepared at room temperature as per concentrations shown in Table 3.3.
Table 3.3 RSP/PF and RSP/SPI adhesive blends and glue line.
RSP/PF and RSP/SPI adhesive formulation Blend 1 2 3* 4 5* 6* 7* 8* 9 RSP (wt.%) 100 90 80 70 60 50 40 20 0 PF or SPI (wt.%) 0 10 20 30 40 50 60 80 100 Glue line 32 mg/in2 Samples Lap-joint: 101.6x25.4x3.0 mm; minimum 5 samples per test
* Formulations for experimental adhesive blends
47
3.6.3 Wood Composite
3.6.3.1 Lap-joint Preparation
Single-lap specimens are the most widely used specimens for development, evaluation,
and comparative studies involving adhesives and bonded products, including manufacturing and
quality control (ASTM, 2008; Cetin and Ozmen, 2002; Desai et al., 2003; Li et al., 2004). .
Poplar or pine veneer strips having dimensions of 25.4mmx101.6mmx3mm were used to
evaluate the bonding ability of selected adhesives. The adhesive preparations were applied on an
area of 6.95 cm2 (1.0 in2) to one side and one end of a poplar veneer strip (Figure 3.5) in such a
way that glue line was maintained at 5 mg/cm2 on a dry content basis. Adhesive coated pairs of a
series of strips were stacked together and hot-pressed at 130°C for 3 min to a final combined
thickness of 4.5 mm of bonded veneer pairs which corresponded to 200 PSI pressure.
Figure 3.5 Wood composites showing glue-line and lap-joint specimen
The hydraulic press, PressMAN - Dieffenbacher North American Inc., Figure 3.6, having an
automatic process control system was set on a pre-determined final thickness, pressing time, and
temperature parameters. A typical pressing schedule is exhibited in Appendix -A. After pressing,
the samples were equilibrated at ambient conditions for 24 hours before further processing
Figure 3.6 Lap-joint veneer samples placed in hydraulic press with a pre-set process schedule
48
3.6.3.2 Testing of Adhesion Strength
The pressed specimens were tested for the lap-shear strength with a Zwick-z100 Testing
Machine equipped with a load cell of 10kN according to ASTM D906 standard method while
maintaining the crosshead speed at 1 mm/min. The bond strength was reported as the maximum
shear strength at failure of lap-joint
3.6.3.3 Surface and internal microscopy
Surface study of wood substrate and adhesive spreading was performed by using
microscope Zoom Stereo SZ and images analyzed through software AmScope MT-3.0.0.1.
Internal microstructure of cellulose substrate in association with glue-line was scanned by using
X-Ray tomography through SkyScan 1172 Micro-CT at 80kV and 140 µA. The resolution used
was from 2.3 to 34.1 µm.
49
CHAPTER 4 - RESULTS AND DISCUSSION
4.1 Physiochemical and Proportional Sludge Composition
The results of physiochemical analysis are shown in Table 4.1. The total solid contents
and suspended solids in secondary sludges, which are inherently composed of high water
content, are used to estimate the availability of total biomass, and yield calculation for
microcellular polymers and protein amounts. Total solids and volatile solids are a common
measure of the mass extractable from wastewater sludge.
Though highly dependent on individual waste treatment plant conditions, the solid
contents of sludge samples used in this study are within range of similar sludges reported in
literature. The ratio of volatile to total solids, around 1:1.5, in both secondary sludges of this
study also corresponds to usual values available in literature. The total nitrogen content,
depending on raw materials, process, aerobic bacteria and internal recycling ratio of SS in the
effluent treatment plant, was found consistent among Kraft mill secondary sludges, and
significantly lower for MS which obviously lacks bio-organisms. Interestingly, primary sludge of
municipal sewage plant shows substantial value of nitrogen which relates to significantly higher
protein contents in these systems due to domestic and commercial food-related effluents.
Table 4.1 Comparison of secondary and other sludge characteristics.
Literature Parameter SS1 SS2 MS
SSa-1 SSb-1 PS2
pH 6.8 6.7 7.1 - - 6.3
Solids (%) - - 16.8 - - 69.2 TS (mg/ml) 7.8 (0.2) 19.7 (0.3) - 11.1 24.4 -
TSS (mg/ml) 5.5 (0.1) 16.7 ( 0.2) - 8.7 17.9 - VSS (mg/ml) 5.2 (0.1) 11.5 (0.2) - 7.3 13.9 - TN (wt. %) 6.3 4.4 1.9 6 4 3.5
TS: Total solids, TSS: Total suspended solids, VSS: Volatile suspended solids
1. Wood et al., 2009 (SSa : Sulphite pulp mill SS, SSb : Kraft mill SS)
2. Chishti et al., 1992 (PS : primary sludge of sewage treatment plant)
(Values in bracket represent standard deviation (+) based on three replicates)
The protein estimation based on TN and extracted through alkali and water solubilisation
techniques is shown in Figure 4.1.
50
0
2
4
6
SS1 SS2
Pro
tein
(m
g/m
l)AP(KN) ASP WSP
0
5
10
15
20
25
MS
pro
tein
(m
g/m
l)
Figure 4.1 Analytical protein determinations of sludge samples.
AP(KN): available protein based on Kjeldahl nitrogen, ASP: alkali soluble protein, WSP: water soluble protein (measured through Bradford assay method)
Unfortunately, prior art has no reference on the chemical protein solubilization sourced
from paper mill secondary sludge. For sewage activated sludges, the range of protein solubility
in alkali is quite wide and ranges between 30 to 90% of available protein depending largely on
the source and bacterial treatment (Chishti et al., 1992; Lerch et. al, 1993). Similarly, the water
extractable protein from sewage sludges has been reported from 1.3 to 7% (Lerch et al., 1993a;
Peres et al., 1992). The total extractable protein (TEP) and respective alkali and water-soluble
protein quantities of this study are summarized in Table 4.2. The low quantity of protein content
in MS is attributed to presence of primary sludge in this material which is obviously low in
micro-organism activity.
Table 4.2 Comparison of different protein fractions in sludge samples
Sludge type AP (%)
(based on o.d. sludge)
ASP (%)
(based on AP)
WSP (%)
(based on AP)
TEP (%)
(based on o.d. sludge)
SS1 37.5 (1.7) 81.6 (3.1) 3.0 (0.1) 31.7
SS2 27.5 (0.9) 77.6 (2.2) 3.2 (0.1) 22.3
MS 11.9 (0.5) 75.2 (2.0) 4.8 (0.2) 9.5
AP: available protein, ASP: alkali soluble protein WSP: water soluble protein (quantification done on raw sludge) TEP: total extractable protein (Values in bracket show standard deviation (+) based on four replicates)
51
The extractives determined in different sludges and corrected for water-soluble protein
are summarized in Table 4.3. This study has shown that the water-extractable protein represents
a significant part of water-soluble extractives pertaining to especially secondary sludges. For
SS1(TMP), water-induced extractives represented up to 85% protein content, whereas for
SS2(Kraft), this figure was 50%. Statistically, for non-polar extractable materials isolated
through ethanol/toluene mixture, no significant difference was found between SS2 and MS
sludges (p<0.001).
Table 4.3 Extractives’ inventory (%age) of sludge samples
Solvent SS1 (TMP) SS2(Kraft) MS
Toulene:Ethanol(2:1) + Ethanol rinse
8.2 (0.60) 11.0 (0.14) 11.8 (0.20)
Water 1.3 (0.03) 1.8 (0.02) 1.6 (0.06)
WSP (% of od. Sludge) 1.1 (0.02) 0.9 (0.02) 0.6 (0.01)
Total Extractives 8.4 11.9 12.8
(Values in bracket show standard deviation (+) based on four replicates)
Although it is very difficult to have a proportional material balance accounting for each
component of any kind of sludge due to interference/interaction of inorganic materials (Krigstin,
2008), a representative summative analysis after corrections for water-soluble protein is shown
in Figure 4.2.
10 15 168
12 1310
914
2320
2617
21
2232
239
0%
20%
40%
60%
80%
100%
SS1 SS2 MS
Protein
Lignin
Alpha-Cel
Hemi-Cel
Extractives
Ash
Figure 4.2 Summative analysis of oven dry sludge samples
The macro characteristic analysis, Figure 4.2, has confirmed that protein makes up a
significant part of secondary sludges followed by lignocellulose materials. This typical
52
characteristic of higher protein fractions make secondary sludge distinct from mixed and
especially recycled sludge of paper mill which have high ash contents , up to 40%, and devoid of
any proteinacous substance (Krigstin, 2008). Since mixed sludge of virgin fibre mill contain
usually at least one third of secondary sludge, its protein content can still make a considerable
contribution to overall composition as evident from this study.
Regarding lignocellulose composition, it is hard to find difference among virgin fibre
mill sludges. For example, Mabee (2001) has reported no statistical difference among TMP and
Kraft mills’ mixed sludges although their effluent treatment were also using different processes.
In this work, no statistical difference is found in hemicellulose content of secondary sludges,
however a same trend in ratio of hemicellulose to alphacellulose was observed as reported in
other works pertaining to virgin fibre mixed sludges (Mabee, 2001; Krigstin, 2008). On the
other hand Barzelatto (1995) has reported a significantly high ratio of hemicellulose to
alphacellulose in a study conducted on sewage sludge. The amount of total lignin is within range
as reported by Edalatmanesh (2010) in a study on similar secondary sludge.
4.2 Extracellular Polymeric Substances (EPS) Biochemical Analysis
The summarized values of EPS yields by control and chemical methods from SS1 and
MS are shown in Table 4.4. For both types of sludge, the chemical method
(formaldehyde - NaOH) has yielded significantly high quantities of EPS compared to physical
control method. In case of SS, the chemical method has extracted up to 3.5 times the amount of
Table 4.4 Gravimetric analysis and biochemical composition of extracted EPS
Extraction Method Sludge
Type
Gravimetric Analysis
Control Chemical
Protein
(mg/g) of
EPS
Carbohydrate
(mg/g) of EPS
EPS extracted1 (mg/L) 401 (19) 1390 (77) SS
EPS yield2 (%) 5.1 17.8 320 (12) 109(6)
EPS extracted1 (mg/L) 192 (12) 881 (53) MS
EPS yield2 (%) 1.1 5.2 290 (8) 116(4)
1. EPS extracted in mg per litre of sludge 2. Yield of extracted EPS based on TS
53
EPS compared to control whereas from MS, the EPS yield is almost 4.5 times higher than
control.
The overall yield of EPS by chemical method is in accordance with literature findings
where average amount of extracted EPS from various municipal and industrial waste secondary
sludges has been reported as 20% of the total organic content with a wide range of 10 to 42%.
Carbohydrates represent 11% while proteins makeup 36% of the extracted fractions (Garcia-
Becerra et al., 2010; Liu and Fang, 2003).
The high extraction efficiency of the chemical method may be attributed to floc
dispersion mechanism in which the ability of NaOH and formaldehyde reagents to create floc
dispersion facilitates EPS extraction process. While formaldehyde fixes itself on the cell,
resisting cell lysis by engaging amino, hydroxyl, and carbonyl groups of proteins and nucleic
acids of the cell membrane, the presence of NaOH increases the pH which facilitates the
dissociation of acidic groups in EPS and also increases the EPS solubility in water resulting in
even more extraction yield (Alcamo, 1997; Wingender, et al., 1999).
The lower quantity and yield of EPS from MS is attributed to the nature of sludge.
Primary sludge, the major portion of MS along with SS, contains mostly suspended solids in the
shape of cellulose fibres and is devoid of any biological activity. On the other hand SS, obtained
from activated treatment plant, consists of bacteria and organic materials on which the bacteria
feed and believed to be rich in bacterial cells and microbial extracellular polysaccharides (Cetin
and Erdincler, 2004; Geng et al., 2007b).
4.2.1 Functional Group Identification- FTIR Studies
Since the IR spectra of extracted EPS from both kinds of sludge were more or less same,
only SS spectra of extracted EPS are shown in Figure 4.3. Apart from asymmetric (2919-2945
cm-1) and symmetric (2854 cm-1) stretching vibrations of CH2 , the major characteristic bands of
control and chemical spectra can be attributed to polymeric compounds’ OH stretching
vibrations (3300 – 3420 cm-1), amide I and II bands of protein (1550 – 1660 cm-1 ) and
polysaccharide (1040-1080 cm-1) functional groups. The IR spectrum obtained for EPS extracted
by the NaOH + formaldehyde method shows some particular bands not visible in IR spectrum of
EPS extracted by control method. These extra bands around 800, 1400 and 1750 cm−1 could be
54
interpreted as specific bands for formaldehyde and/or the result of a formaldehyde and EPS
reaction. Further, less intense band at 1240 cm-1 suggest the presence of uronic acids. The
findings of this IR study agree with the Guibaud’s work (2003).
Figure 4.3. IR spectra of EPS extracted from SS by control and chemical methods
4.2.2 Thermal Analysis
The thermograms for SS, MS and extracted EPS from secondary sludge are shown in
Figure 4.4. The peaks pertaining to SS and MS (around 94-98 oC) are obviously dehydration
ones as raw sludge contains significant amount of hygroscopic materials. Although EPS contains
diverse biopolymers such as proteins and polysaccharides, a broad endotherm peak confirms its
homogenous nature. This is supported by the fact that a miscible blend of polymers is known to
form a homogeneous mixture and to present a single glass transition or melting temperature
(Meireles et al., 2007). The polymeric structure of EPS is further supported by its temperature of
melting transition of 110 oC. Unfortunately, no thermal analysis on EPS is available in literature;
however Parlouer (1988) has reported a melting transition temperature of a common polymer,
polyethylene, as 110.6 oC.
The lower heat values observed for raw MS and SS may be attributed to higher ash
content and other impurities in these sludge samples, whereas significantly high heat capacity of
EPS, Figure 4.5, pertains to relatively higher molecular weight components like protein and
carbohydrates.
55
Figure 4.4. DSC thermograms of SS, MS and extracted EPS.
Figure 4.5 Heat capacity curves of EPS and sludge samples from thermal analysis.
56
4.3 High Yield Protein Recovery and Characterization
4.3.1 Protein Solubilization and Cell Disruption from Raw Sludge 4.3.1.1 Effect of pH on Protein Solubilization
Since most of the protein present in activated sludge is not in solution form, therefore it
cannot be readily separated from other complex organic and inorganic materials. It is reported
(Sridhar and Pillai 1973; Christiansen and Mitchell 1978) that chemical treatment involving
alkalis is a suitable solubilization method whereby encrusting substances (cellulose
hemicellulose and lignin) are effectively removed.
The effect of sodium hydroxide on paper mill secondary sludge was studied at different
pH levels for two different time intervals. A rapid and linear increase in protein recovery was
observed with increase of hydroxyl ions for both 2 and 24 hour intervals as shown in Figure 4.6.
Figure 4.6 Effect of pH and reaction time on protein solubilization
The maximum protein recovery for both time intervals was possible at pH 12, beyond
which no further improvement in solubilization was observed. However, after pH 10 the increase
in recovery rate was steeper for 24 hour treatment compared to 2 hour period of reaction time.
The maximum recovery of available protein was achieved at pH 12.0 (24 hour treatment),
which was also about 32% more compared to 2 hour treatment for same pH. A significant high
quantity of alkali was required to increase the pH from 12 to 12.5 and a 3 – 4% drop in protein
recovery was observed for the same incremental shift in pH level.
57
The effect of alkali treatment on suspended solids vis-à-vis protein content of treated
sludge is shown in Appendix B. An eighteen fold decrease in suspended solids was observed
corresponding to about six times increase in soluble protein. This result is in consistent with
literature finding (Zhang et al. 2007; Weemaes et al. 2000) in which a significant increase in the
soluble protein and decrease in total suspended solids (TSS) was observed after sludge
disintegration
4.3.1.2 Cell Disruption (French press and sonication)
In order to augment the protein yield after alkali treatment, optimization studies were carried out
by using French press and sonication methods individually as well as in a combination sequence
in which French pressing was followed by sonication.
4.64.8
55.2
5.45.6
5.8
0 1 2 3Number of passes (French press)
Pro
tein
con
c, (
mg/
ml)
(a)
4.6
5.1
5.6
6.1
6.6
0 30 60 90 120
Sonication duration (seconds)
Pro
tein
con
c, (
mg/
ml)
(b)
Figure 4.7 Protein yield optimization; effect of number of passes (French press) and sonication time on the solubilisation of alkali treated sludge protein. In standalone mode, alkali treated sludge was used as starting material individually for both
French press and sonication studies, The effect of number of passes in French press and duration
of sonication is shown in Figure 4.7. After two passes of French press, a maximum cell
disruption was observed which showed about 15% increase in protein solubilisation compared to
alkali treated control sample. After two passes, no further improvement was noticed which is the
usual case as mentioned by others (Benov and Al. Ibraheem 2002; Thermo scientific 2011). The
sonication protocol proved to be better choice by yielding 23% more solubilised protein
compared to alkali treated control sample. The maximum yield was realized in about 3 to 4
sonication cycles of 30 second each, beyond which no further solubilisation was observed. To
investigate the combined effect of French press and sonication, the samples expelled from
58
French press having maximum solubilised protein were further processed through sonicator for
total of four cycles.
012345678
C FP SN FP-SN
Pro
tein
con
c. (
mg/
ml)
Figure 4.8 Combined effect of French pressing and sonication on protein solubilisation. (C: control; alkali treated sludge, FP: French pressing, SN: sonication, FP-SN: French pressing followed by sonication)
Although statistically no significant difference was found among French press and
sonication treatments individually, however the results of in –series experiments confirmed,
Figure 4.8, that an extra protein solubilisation of about 1mg/ml can be achieved in a combined
mode; thereby enhancing the overall protein yield by about 44% compared to alkali treated
secondary sludge.
4.3.2 Protein recovery
4.3.2.1 Effect of pH on Protein Precipitation
The effect of pH on the precipitation of alkali disrupted sludge is shown in Figure 4.9.
The dotted column on far left indicates the highest concentration of solubilized protein after
alkali disruption at 12 pH followed by French press/sonication treatments. Several specimens
from alkali disrupted sludge were treated with sulphuric acid at different pH levels to precipitate
out the pellet of protein. The vertical-bar columns in Figure 4.9 denote the protein concentration
of supernatant after protein recovery.
Though more than 80% protein recovery was observed at pH 1.5, but maximum process
efficiency was achieved at pH value of 3.0 where 90% of soluble protein was precipitated out. A
peak protein recovery of about 82% for municipal sludge has been reported by Hwang et al.
59
(2008) at pH value of 3.3. In another work (Chishti et al., 1992), a maximum protein recovery of
83% is reported by using sulphuric acid at pH 3.0.
010002000
3000400050006000
70008000
So
lub
le P
rote
in (
mg
/L)
0
20
40
60
80
100
Pro
tein
Yie
ld (
%)
pH 1.5 pH 3.0 pH 4.5 pH 5.5
Disrupted sludge pH adjusted sludge Protein recovery
Figure 4.9. Effect of pH on protein precipitation and recovery yield
4.3.2.2 Effect of Precipitating Agents on Protein Recovery Yield
Procedures involving sufficient amounts of acids, inorganic salts or organic solvents have
been used in the past to extract protein from solubilized protein solutions of different origins
(Florkin and Stoz 1963; Sastry and Virupaksha 1967; Hwang et al. 2008). In this study instead
of using organic solvents as precipitating agents, commonly available dilute acids and inorganic
salt is used to recover protein from solution of solubilized protein processed from secondary
sludge of paper mill. The commonly used precipitating agents mentioned in literature are
sulphuric acid, hydrochloric acid, and ammonium sulphate (West et al. 1966; Knorr et al., 1977;
Christiansen and Mitchell 1978; Chishti et al., 1992)
Maximum recovery of solubilized protein, around 90%, was achieved by using sulphuric
acid at pH 3.0 followed by hydrochloric acid and ammonium sulphate, Table 4.5. Though protein
Table 4.5 Effect of different precipitating agents on protein recovery
1. Quantity of precipitating agent used to bring down the pH of protein solution from 12 to 3.0 2. 40% saturated solution of ammonium sulphate. (Values in bracket represent standard deviation (+) based on three replicates)
Reagent Quantity Used1
Dry wt. of precipitates
(g/l)
Protein content (%)
Protein Recovery (%)
Sulphuric acid 8.0 ml/l (0.32) 12.7 (0.49) 49.0 (2.30) 90.1 Hydrochloric acid 31.4 ml/l (1.63) 11.2 (0.50) 44.6 (2.20) 72.4 Ammonium sulphate2
40% sat. soln. 8.9 (0.43) 46.3 (2.31) 59.7
60
content of ammonium sulphate treated precipitates was higher than HCl treated precipitates,
however the protein recovery was less due to low amount of centrifuged mass. The results of this
part of study clearly indicate that most of the protein from solution can be extracted using these
precipitating agents. The most cost-effective method proved to be treatment with sulphuric acid
in terms of yield and ease of operation. The treatment with ammonium sulphate required much
more time due to added steps of membrane dialysis to remove extra salt from recovered protein
precipitates. This extra filtration step was detrimental in reducing process efficiency and yield
due to inherent material losses.
Protein precipitated out by using sulphuric acid was used for all the characterization
studies of this work.
In a separate set of experiments, the effect of H2SO4 and HCl was also studied on protein
recovery from alkali treated sludge at pH 8.0. The recovered proteins were tested for adhesion
strength and results are reported in Appendix -C.
4.4 Recovered sludge Protein (RSP) Characterization 4.4.1 Metal toxicity
The measured concentrations of hazardous trace elements including heavy metals in the
original activated sludge and the recovered protein pellet are listed in Table 4.6 with some
regulatory standards. For comparative purposes, similar results from literature pertaining to de-
inked paper sludge and municipal sewage sludge are also inserted in this comprehensive chart.
The starting sludge contained a variety of metals which largely depend on the type of raw
materials and numerous additives used for pulp and paper manufacturing. Although primary and
secondary wastewater treatment processes are mainly responsible for concentrating heavy metals
into effluent sludge, these hazardous materials can be further absorbed by biomass, precipitated
by some anions such as sulfide, and accumulated in excess sludge (Stephenson and Lester, 1987;
Ito et al., 2000).
A significant amount of metals were reduced during protein recovery process as seen in
Table 4.6. Regarding heavy metals, the concentration were reduced either to very low and below
detection limits (Cd, Ni, Pb) or 4 - 6 times of original values (Cu, Fe, Zn). The only exception
was Na metal showing a substantial increase after protein recovery which can be attributed to use
61
of alkali (NaOH) for solubilization purpose. The low concentration of metals in DIP sludge
relates to use of cleaner furnish, recycled paper, compared to virgin wood in terms of metal
toxicity.
Table 4.6 Trace element concentrations (%age) in raw sludge, recovered proteins from different biomass sources, and regulatory standards.
Biomass Type Muncipal Sewage
Element SS1
(Paper) Rec,
protein (SS1)
DIP sludge2 PS3 SS4 Rec.
protein (SS4)
Canadian compost
standard2. (class B)
Poultry feed legal
limit4
Al 1.190 0.311 0.776 0.464 As BDL BDL <0.010 0.075 2x10-4 B 0.004 0.001 <0.001 Ca 0.774 0.164 0.173 0.091 Cd 0.001 BDL <1x10-4 0.002 0.002 1x10-4 Cr 0.003 0.002 0.106 0.01 Cu 0.048 0.008 0.011 0.094 0.278 0.025 0.076 Fe 0.571 0.111 0.043 0.504 0.288 K 0.137 0.073 Mg 0.339 0.022 Mn 0.023 0.006 0.002 0.016 Mo BDL BDL 0.002 Na 0.950 1.940 Ni 0.003 0.001 <0.001 0.015 0.005 0.001 0.018 Pb 0.056 BDL <0.002 0.053 0.002 0.000 0.050 0.001 Ti 0.015 0.003 Zn 0.080 0.024 0.004 1.38 0.058 0.021 0.185
SS: Secondary sludge, PS: Primary sludge, DIP: De-inked pulp sludge, BDL: Below detection limit 1. Current study 2. Beauchamp et al. 2002 3. Chishti et al 1992 4. Hwang et al. 2008
Overall, it is interesting to note that paper secondary sludge has less metal toxicity
compared to municipal sludge except in concentration level of Ca which pertains to excessive
use of clays in paper manufacturing. Though recovered protein from sewage sludge is also
shown (Hwang et al. 2008) as lower in metal concentration compared to its original sludge,
however the recovery process does not seem to be as effective to reduce metal toxicity as already
demonstrated in the current study. Compared to composting and poultry feed standards, the
recovered protein from paper sludge is definitely safer to be considered for supplementary
animal feeds and other value-added utilizations such as wood adhesive.
4.4.2 Amino Acids
A detailed analysis of the amino acid composition of protein extracted from paper
secondary sludge was carried out and results are shown in Table 4.7, which also includes similar
62
Table 4.7 Amino acid % composition of recovered protein and other sources
Recovered Protein Protein products Amino Acid
Paper
mill
sludge1
Municipal
sewage
sludge2
Municipal
sewage
sludge3
Soybean
flour4
Wheat
flour2
FAO5
Asparagine 12.1 - 2.36 11.3 -
Glutamine 10.8 - 3.37 17.2 - Leucine* 8.7 6.9 6.28 6.5 7.0 4.8
Alanine 7.5 - 2.3 4.0 - Valine* 7.2 5.4 3.3 4.6 4.1 4.2
Arginine 7.0 - 1.6 7.0 -
Phenylalanine* 5.9 4.2 2.2 4.7 5.5 2.8 Glycine 5.8 - 2.2 4.0 - Threonine* 5.6 5.4 1.6 4.3 2.7 2.8 Tyrosine* 5.5 3.1 1.3 3.4 - 1.4 Isoleucine* 5.5 - - 4.8 4.2 4.2
Proline 4.7 - 1.4 4.7 -
Lysine* 4.5 9.0 2.4 5.7 1.9 4.2 Serine 4.2 - 0.8 5.0 - Histidine 2.4 - 1.6 2.6 - Methionine* 2.4 4.6 0.5 1.3 1.5 2.2
Cysteine* 0.1 1.0 - 1.5 1.9 2.0 1. Recovered protein from secondary sludge of paper mill effluent; current study 2. Lau 1981. Recovered protein from secondary sludge of municipal sewage. 3. Chishti et al. 1992. Recovered protein from primary sludge of municipal sewage 4. Cheng 2004. 5. Provisional amino acid requirements for food products, recommended by Food and Agricultural
Organization (Lau 1981) * Essential amino acids
data of other protein products for comparison purpose. This quantitative analysis explicitly
indicates the value of sludge protein in terms of amino acid contents, especially the essential
amino acids whose levels exceeded the amino acid requirements provisionally recommended by
the Food and Agricultural Organization (FAO), making the paper sludge as an attractive
candidate to be explored for food supplements
The results further indicate that protein recovered from paper mill secondary sludge is
compatible to proteins recovered from sludge of municipal sewage treatment plants in terms of
63
amino acid contents. The other important finding is the close similarity between soy flour and
paper sludge proteins in terms of amino acid composition. Since soy flour is the most common
raw material to produce soy protein wood adhesives and except in glutamine composition, the
protein recovered from paper mill sludge is equally compatible with this resource. Hence, these
results strongly suggest a possibility of replacing food crops with abundantly available biomass
from paper mill sludge to develop bio-based wood adhesives. For reference purpose, calibration
and sample data of amino acid analysis is given in Appendices D and E.
4.4.3 Molecular Weight Distribution
The composition of sludge protein was analyzed by SDS-PAGE and results are shown in
Figure 4.10 along with literature values. The most distinct patterns were obtained by using a
Figure 4.10 SDS PAGE analysis of proteins from activated secondary sludge. M. Marker RSP(a). Liquid Nitrogen+ detergent RSP(b). Liquid Nitrogen+ detergent+protease inhibitor cocktail Literature. Lerch et al., 1993. Extracted protein from municipal sewage secondary sludge
combination of liquid nitrogen and detergent or liquid nitrogen, detergent, and protease inhibitor
(Sample A and B). The SDS-PAGE clearly indicates that activated secondary sludge contained
proteins in two distinct molecular weight (MW) ranges; the higher MW proteins were found
concentrated in the range of 30 kDa to 70 kDa. The other band of proteins in paper sludge had
quite low MW that was mostly concentrated around 7 kDA. Though there is no literature
available on SDS-PAGE analysis of paper SS, however Lerch et al (1993b) have reported high
64
MW ( 29 – 66 kDa) and low MW fractions (< 17 kDa) of proteins, Figure 4.10 (literature
values), extracted from activated sludge samples of different sewage treatment plants by using
water or alkali as solubilizing agents.
Goodwin and Forster (1989) have reported the presence of predominantly low MW
proteins, < 10 kDa, in an activated sludge while using membrane filtration. However,
Karapanagiotis et a1. (1989) have mentioned that the major MW fraction of protein and
nonprotein components from activated sludge separated by SDS-PAGE chromatography is
greater than 5 kDa. Alkali extraction is most likely responsible for hydrolysis of peptide bonds,
generating lower MW components, which is supported by earlier studies (Zubay 1983; Lerch
1991)
4.4.4 FTIR Analysis
The FTIR spectra of three main process streams of protein recovery procedure are shown
in Figure 4.11. It may be noted that residual (RS) stream represents the semi-solid mass settled
out after removing solubilized protein solution.
The FTIR analysis based on the identification of bands related to the functional groups
present in activated sludge and extractable proteinaceous components is supported by literature
(Hong et al. 1995; Garnier et al. 2005; Edalatmanesh et al. 2010). The main characteristics
absorption bands of recovered sludge protein are related to C=O stretching at 1656 cm-1 (primary
amino group- amide I), angular deformation of N–H at 1545 cm-1 (secondary amines-amide II)
and C–H deformation at 1455 cm-1 which might be contributed by the secondary amines due to
CH2 groups of aliphatic chains. A slight shoulder peak around 1430 cm-1 could be attributed to
lignin extracted with protein and inherently present in sludges of wood pulping processes
(Pandey, 1999). The band near 1405 cm-1 is believed to result from the N-C-H deformation in the
protein. The band in the proximity of 1250 cm-1 originates from the asymmetric stretching
vibration of C-O-C ester in the fat and C-O-C ether in the cellulose. The intense band near 1100
cm-1 is attributed to C-N stretching vibrations of both primary and secondary amines. Further, a
sharp band near 670 cm-1 belongs to unsaturated C=C bonds.
65
Figure 4.11. FTIR spectra of recovered sludge protein (RSP), secondary sludge (SS), and residual mass (RS) of sludge. A broad band, between 3600 and 3000 cm-1, observed in all three materials is attributed to free
and bound O–H and N–H groups. In may be noted that secondary sludge is less intense in
showing amide groups compared to RSP. Moreover the residual mass after protein recovery is
devoid of any significant presence of amide II and C-O-C ester groups related to fats and lipids.
4.4.5 Thermal Degradation of RSP
The TGA analysis of RSP extracted from secondary paper sludge is shown in Figure
4.12. Unfortunately, no reference is available on thermal degradation study of protein recovered
from paper mill or municipal secondary sludge; however, two relevant citations from literature
pertaining to thermal analysis of soy protein isolate (SPI) ((Nanda et al., 2007) and BSA protein
(Rico et al., 2005) have been used in this work to facilitate in the interpretation TGA data.
Apart from the first degradation for SPI at 105oC owing to its high purity, a comparison
can be drawn for rest of thermal behaviour for both RSP and SPI materials. In case of RSP, the
first thermal degradation takes place at 211 oC having mass loss of about 5%. In the initial phase
the degradation is slow due to entrapped moisture in the polymer matrix. In the next phase, the
degradation is rapid and pertains to quicker breakage of hydrogen bonds, electrostatic and other
weak bonds. At 306 oC, RSP shows mass loss of up to 24% whereas SPI is reported to have mass
66
Figure 4.12 Thermal degradation of recovered sludge protein (RSP)
loss of 20% and 40% at degradation temperatures of 250 oC and 353 oC respectively (Nanda et
al., 2007). The leaner degradation observed after 400oC is probably due to hard to break peptide
and disulphide bonds, and due to presence of lignin in RSP whose thermal degradation is from
400 oC to 500 oC (Martin-Sampedro et al., 2011). Strangely, a sharp phase change at 700oC for
RSP is not available in SPI, however, the thermogram of BSA, blood protein, shows this
degradation. Most probably this is due to high molecular weight polymerization of these distinct
proteins.
4.5 Use of Native RSP as wood Adhesive; proof of concept
Shear strength of wood composites bonded with PF, SPI, RSP and SS are shown in
Figure 4.13.
As anticipated, the PF (phenol formaldehyde) bonded wood composites yielded highest
strength whereas SPI (soy protein isolate) showed better results among two bio-based adhesives.
The better performance of SPI is understandable as it had much purified and high concentration
of protein (90%) compared to RSP (49%). The ratio of shear strength of SPI to PF and their
values are consistent with literature values (Cetin and Ozmen, 2002; Sun and Bian, 1999).
67
Figure 4.13 Bond shear strength of wood composites bonded with different adhesives.
The RSP has shown significant improvement in bonding efficiency by yielding twice the amount
of shear strength compared to raw sludge. Compared to PF and SPI, the strength of RSP adhesive
is about 40% and 48% respectively.
The other significant observation regarding RSP’s bonding efficiency was the study of
failure mode of tested lap-joints. Though PF and SPI showed a dominant phenomenon of wood-
failure, RSP also showed a mixed failure mode of both wood and adhesive failure whereas in
case of raw sludge, no wood failure was observed as shown in Figure 4.14.
Figure 4.14. Failure study of ruptured joint surfaces: (a) PF: total wood failure, (b) RSP: partial wood/adhesive failure, (c) SS: total adhesive failure.
4.6 Protein Adhesion Mechanism on Cellulose Substrate
Recovered sludge protein (RSP) from residual biomass gives an excellent opportunity to
be explored for bio-based wood adhesives. Preliminary work on characterization of RSP has
68
already shown the similarities between soy protein and RSP in terms of amino acid composition
and molecular weights. Although wood and paper bonding are the largest applications for
adhesives, some of the fundamental aspects are not fully understood. Better understanding of the
critical aspects in wood adhesion should lead to improved composites.
.
In this section the adhesion mechanism of RSP vis-à-vis soy protein has been elaborated
with examples from prevailing bonding theories. Based on an experimental design, the bonding
mechanism of RSP –cellulose system is validated which is inspired from a literature work
involving denaturing of soy globulin protein and its effect on adhesion strength with hard and
soft wood substrates.
4.6.1 Polyamide Chemistry of Recovered Soy and Sludge Proteins
Most of the soybean varieties contain 35-40% protein, 18-20% oil, 5% carbohydrates,
and about 5% ash on dry basis. A pure form of soy protein, soy protein isolate (SPI), is recovered
from soy flour by removing carbohydrates and other residues through an aqueous extraction
process followed by isoelectric precipitation, washing, neutralization and drying . The resulting
SPI contains about 90% protein (Cheng, 2004).
The primary structure of protein is dictated by linear sequence of amino acid residues or
polypeptides. Based on their sedimentation coefficients, the protein content of defatted soy meal
is further classified into four fractions namely, 2S, 7S, 11S, and 15S. The 7S (β-conglycinin)
and 11S (glycinin) fractions account for majority of the proteins, about 68% of total available
protein (Liu, 1997; Silvana and Anon, 1995; Thanh and Shibaski, 1976). These fractions are also
believed to play major role in the adhesion of cellulosic substrates and have been studied to
understand bonding mechanism of protein-based adhesives (Richard, 2001; Zhang and Hua,
2007). The amino acid composition of various soy proteins and recovered sludge protein (RSP)
is given in Table 4.8.
The robust structure of polyamides of RSP and soy proteins is characterized by hydrogen
bonding between the amide backbone and amino acid side group. Further, soy protein, and
essentially RSP, also contains many chemically active sites such as amine, carbolic, sulfhydryl,
69
and aliphatic aromatic hydroxyl groups. Polyamide backbone structure and reactive site groups
of soy and RSP protein are shown in Figure 4.15.
Table 4.8 Amino acid composition of RSP and Soy proteins
Amino acid RSP1 Soy
flour2
7S
(β-Conglycinin)2
11S
(Glycinin)2
Asparagine 12.1 11.3 14.1 11.8 Glutamine 10.8 17.2 20.5 18.8 Leucine* 8.7 6.5 10.2 7.2 Alanine 7.5 4.0 3.7 6.7 Valine* 7.2 4.6 5.1 5.6 Arginine 7.0 7.0 8.8 5.9 Phenylalanine* 5.9 4.7 7.4 3.9 Glycine 5.8 4.0 2.8 7.8 Threonine* 5.6 4.3 2.8 4.2 Tyrosine* 5.5 3.4 3.6 2.5 Isoleucine* 5.5 4.8 6.4 4.6
Proline 4.7 4.7 4.3 6.3
Lysine* 4.5 5.7 7.0 4.1 Serine 4.2 5.0 6.8 6.6 Histidine 2.4 2.6 1.7 1.8 Methionine* 2.4 1.3 0.2 1.0 Cysteine* 0.1 1.5 0.3 1.1
1. Recovered sludge protein – current work 2. Cheng, 2004
Figure 4.15 Polyamide backbone and reactive sites of RSP and soy protein.
The polarity of functional groups plays a major role in the adhesion by establishing
strong bonds with wood surface and it has been shown in literature that soy protein amino acids
side groups are characterized with both negative and positive charges (Richard, 2001).
70
4.6.2 Adhesion Mechanism of Protein-based Adhesives The adhesion between adhesive and substrate at the interface is a complicated phenomenon and
no single theory is available to justify various interactions responsible for a durable bonding.
However, in the case of protein adhesion with wood substrate, at least there is some degree of
consensus among researchers to explain the adhesion mechanism based on experimental studies.
(Cheng, 2004; Sun, 2005). Adhesion mechanism between protein polymers and cellulose
substrate is attributed to three main type of interactions; mechanical bonding, physical
adsorption, and chemical bonding.
The mechanical interaction is explained in two parts; first the protein adhesives spread
and wet the surface of the substrate and then adhesive molecules penetrate into the fiber cells
through crevices via capillary paths. Eventually this interaction develops into mechanical
anchoring due to in-place curing of adhesive. Both interlocking and penetration are two major
contributors to mechanical bonding theory. The degree of mechanical interlocking or resistance
to the shearing load is largely depends on two major variables; surface roughness of cellulose
substrate and flow behavior of protein adhesive. Penetration mechanism relies on an appropriate
penetration depth in to the wood surface which is reported as about 2-6 fiber depths (Gollob and
Wellons, 1990). Since protein polymers contain a certain molecular weights distribution, a
fraction of smaller molecules could easily penetrate into cell walls of surface cellulose fibres.
The adsorption theory relies on any physical or electrostatic attraction between protein polymers
and wood surfaces through hydrogen bonding and van der Waals forces (Xu et al., 2011). The
hydrogen bonding strength is achieved by the attraction between positive hydrogen atoms and
negative oxygen or nitrogen atoms. In case of wood, the main material is cellulose which has
abundant amount of polar hydroxyl groups. Hydrogen bonds can be formed between these polar
groups and oxygen and nitrogen atoms of protein adhesive. As shown in Figure 4.16, for each
anhydroglucose unit of wood cellulose, there are three possible (C2, C3, C6) sites where both
intra and intermolecular hydrogen bonding is possible. Upon application of pressure, protein
molecules make intimate molecular contact with substrate surface due to shortened distance
which results in hydrogen bonding as well as Van der Waals forces.
71
Figure 4.16 Availability of three hydrogen bonding sites for each anhydroglucose unit.
A three dimensional model is shown in Figure 4.17 as a schematic to illustrate proposed
Figure 4.17 Schematic of proposed adhesion mechanism of protein adhesives with cellulose substrate.
72
adhesion mechanism of protein adhesive with wood substrate. As illustrated in Figure 4.17, the
smaller molecules of protein adhesive eventually become part of the fibre component after
getting cured within the fibre cell to a hard mass. In this case the interactions between smaller
and bigger molecules contribute to the bonding strength and the interface transforms into a three-
dimensional network of interactions due to an effective penetration depth. The three-dimensional
network then becomes entangled with the substrate to form a continuous mass which, in
combination with hydrogen bonding and adsorption interactions, contributes to the overall
bonding mechanism.
4.6.3 Validation of RSP vis-à-vis Soy Protein Bonding Mechanism Apart from literature and theoretical manifestation of the subject matter, this section of current
study is an effort to further substantiate the adhesion mechanism of sludge protein through
experimental work and correlate the findings with relevant prior art.
Protein-based adhesive must consist of relatively large, flexible and interwoven polymer chains
for better adhesion with wood substrate. Chemical modifications have been used for this purpose
to break the internal bonds and facilitate unfolding of the protein molecules (Van et al., 2000).
Soy protein isolates (SPI) treated with urea, alkali, and sodium dodecyl sulfate (SDS) have
shown improved adhesion strength and water resistance (Huang and Sun, 2000a; Huang and Sun,
2000b; Sun and Bian, 1999). Zhang and Hua (2007) have studied 7S and 11S globulins, the
dominant storage proteins in soybean, under the influence of urea to investigate adhesion
mechanism of soy protein with wood surfaces.
Urea has hydrogen, nitrogen, and negatively charged oxygen atoms that would interact
with hydroxyl groups of the proteins, which could break down the hydrogen bonding in the
protein body and, consequently, unfold the protein complex. At lower urea concentrations, urea
can destabilize globular protein by forming strong hydrogen bonds with water molecules
surrounding the proteins, resulting in partially unfolded protein structures desirable for improved
wet adhesion strength (Sun, 2005).
In the current study we have modified recovered sludge protein (RSP) with 1.0 M urea
solution and tested for its wettability and adhesion strength for both soft and hardwoods.
73
4.6.3.1 Effect of Protein Denaturing
Chemicals containing amino groups can interact with protein polymers. The compact
protein structure unfolds or cross-links during denaturation due to breaking and reforming of
inter and intra-molecular interactions (Careri et al., 1979). Urea, an amino-based compound, has
been widely used to modify soy proteins to improve its adhesion characteristic and water
resistance as a wood adhesive.
Figure 4.18 FTIR spectra of unmodified and Urea-modified protein adhesives
(RSP: recovered sludge protein, SPI: soy protein isolate)
Urea can efficiently form hydrogen bonds with the protein backbone or with the charged
hydrophilic residues in the same way as it does with water. This high ability of hydrogen bond
formation of urea is attributed to its similarity with protein backbone and hydrogen bonding thus
formed have been reported to be of longer lifetimes (Das and Mukhopadhyay, 2008).
Although in literature no significant compositional changes have been reported in protein
after urea modification (Das et al., 2011), however, in this work few prominent changes were
observed during FTIR study in the modified RSP and SPI at exactly same wave numbers as
shown in Figure 4.18. One additional peak is due to hydrogen bonding which has transformed
74
the keto linkage of urea into enol , Figure 4.19, forming an amide functional group identified at
3390 cm-1 due to NH stretching (Dyer, 1965). The other changes are the slight shift in OH
stretching (3280 cm-1), C=O stretching of protonated –COOH groups (Laberge et al., 1997), and
shift in NH and CN bending of protein amide groups to 1550 cm-1 . A new shoulder at 1610 cm-1
could be attributed to deformational vibrations of the NH2 groups of urea (Kulakov et al., 1987).
Figure 4.19 Transformation of urea’s keto linkage into enol after hydrogen bonding.
4.6.3.2 Effect of Surface Wettability and Microstructure
A better adherence of adhesive with substrate is largely affected by surface wettability
dynamics and a contact angle less than 90o is considered adequate for reasonable wetting
between adhesive and substrate (Wang, 1987). The contact angle drop percentage, a measure of
difference between immediate and equilibrium contact angles, is an indicator of wettability
which dictates that a lower equilibrium contact angle corresponds to better wettability of
adhesive on a given substrate (Zhang and Hua, 2007). The wettability of unmodified (control)
and urea modified RSP and SPI adhesives on hard and softwood was evaluated by contact angles
Table 4.9 Wettability data of RSP and SPI on hard and softwood substrates
Adhesive Wood θi1 θe
2 η3
RSP-C4 Poplar 84 64 23.8
Pine 88 70 20.5
RSP-M5 poplar 75 38 50.6
Pine 78 46 41.0
SPI-C poplar 85 54 36.5
Pine 87 62 28.7
SPI-M poplar 71 24 66.2
Pine 70 32 54.3
1. Initial contact angle 2. Equilibrium contact angle 3. % drop in contact angle 4. Control 5. Modified
75
and contact angle drop percentages. Since the entire contact angle data were less than 90o, Table
4.9, it means these adhesives can wet both kind of wood samples sufficiently. However, both
adhesives had varying degree of wettability on substrate samples for their un-modified and
modified states.
The data in Table 4.9 confirms that protein modification with urea has significantly
enhanced the wettability of RSP and SPI adhesives. However, since RSP contains lower amount
of protein originally, its drop in contact angle in unmodified state is much less compared to SPI.
The drop in initial contact angle for modified RSP is further illustrated in Figure 4.20.
Figure 4.20 Initial contact angles of (A) unmodified and (B) modified RSP adhesive
The effect of wood surface structure is also somewhat evident from contact angle data,
however, it may be noted that wettability is not the only mechanism responsible for overall
bonding strength. The microstructure of substrate surface also plays important role in mechanical
Figure 4.21 Microstructure of poplar and pine veneers in a composite lap-joint glued with RSP adhesive (central horizontal area represents glue-line joining two substrates)
76
and finger-joint interlocking effects. Generally, the gluing strength with hardwoods like poplar,
walnut, and cherry have been found higher compared to pine (Sun and Bian, 1999). The main
contributing factor to this behavior is attributed to presence of pores in hardwoods compared to
softwoods as shown in micrographs of Figure 4.21. The protein molecules penetrate into the
pores from the wood surface, forming a complex matrix upon curing. The rough surface under
pressure can form random micro “finger joints” effects enhancing adhesion strength (Sun, 2005).
4.6.3.3 Adhesion Strength
Shear strength of hard and softwood composites bonded with unmodified (control) and
modified recovered sludge protein (RSP), soy protein isolate (SPI), and 11S (glycinin) fraction
of soy protein are shown in Figure 4.22.
Figure 4.22 Shear adhesion strength of protein adhesives on hard and softwood substrates. c: Control m: Urea modified protein adhesives 1. Literature results: Huang and Sun, 2000 2. Literature results: Zhang and Hua, 2007 The highlights of shear strength results are;
- The bonding strength of protein-based adhesives is a direct function of amount of actual
protein content; RSP and SPI used in current study contained 49% and 90% protein content
respectively, whereas, SPI used in literature study (Huang and Sun, 2000) had 88.3% protein
and 11S (glycinin) fraction contained 96.6% protein (Zhang and Hua, 2007).
- Microstructure of wood effects adhesion strength significantly, but not consistently. Though
hardwood contains more micro-pores, i.e, wider lumen and capillary path, which enables
smaller protein molecules to penetrate through crevices and get established into finger-joints
77
after curing, however, there are some other wood attributes which contribute to wettability
of adhesive. Apart from surface component of wood (Hemingway, 1969), seasonal growth
of wood also has importance in ultimate bond strength; for example spring wood has better
wettability than autumn wood (Hse, 1971; 1972). Similarly sapwood has better adhesive
spreading ability than heartwood (Kajita, 1992).
- Protein denaturation significantly increases the functionality of protein-based adhesives.
Compared with unmodified RSP and soy protein adhesives, the urea-modified adhesives
have shown improved adhesion strengths on both poplar and pine. The secondary structure
is considered desirable for protein adhesion and proteins modified at relatively lower urea
concentrations may have been partly unfolded and have a certain amount of secondary
structure, resulting in better shear strengths (Huang and Sun, 2000).
4.7 Synergy Evaluation of Chemically Modified RSP Adhesive Blends
Chemical modifications or crosslinking of protein-based native adhesives with synthetic
and biopolymers has been practiced in the past to enhance their adhesion properties. PF induced
hybrid bio-adhesives have been studied by researchers (Qi and Sun, 2011; Wescott et al., 2006).
Similarly, soy protein is reported to be modified with biopolymers in the development of
analytical grade adhesives and membrane composites (Liu and Li, 2004; Zhu, 2012).
Based on the analytical results of current work which has confirmed the presence of
adhesion extenders in sludge protein, an experimental design was envisaged to formulate a broad
range of RSP:PF and RSP:SPI adhesive blends and trend of adhesion performance was validated
through a classical adhesive-mixture law.
4.7.1 Blend Compatibility through Rule of Adhesive Mixtures
One of the most important factors influencing the final adhesion properties of a adhesive
blend is the mutual miscibility of the components and any possible chemical cross-linking.
The log-additive rule, equation (1), has been mentioned and utilized as an appropriate
mathematical model to investigate synergy and compatibility of adhesive blends (Gedde, 1995;
Crawford, 1998; Khonakdar et al., 2006)
78
log E = ∑ ωi log Ei (1)
In this model equation E is one of the properties of blend and Ei is properties of the ith
component, respectively, and ωi is the weight fraction of the ith component. Now for two
component system, A and B, the equation (1) can be written for adhesive blend’s property of
shear strength, ST, as follows
log STblend= ωA log STA + ωB log STB (2)
Since the shear strength of pure components determined through control experiments were; RSP:
2.3 MPa, PF: 5.2 MPa, and SPI: 4.0 MPa, therefore equation (2) can be further modified for both
blends as below
log STblend= ωRSP log 2.3 + ωPF log 5.2 (3)
log STblend= ωRSP log 2.3 + ωSPI log 4.0 (4)
Equations 3 and 4 were used to generate curves for correlation studies to investigate deviation of
experimentally determined shear strength of adhesive blend compared to mathematical model
derived through log-additive rule of mixing.
4.7.2 Adhesion Synergy and Failure Mode Analysis
Shear strength results of wood composites bonded with various blends of RSP/PF and
RSP/SPI are shown in Figure 4.23 (a) and (b).
Though both blends have shown negative deviation from rule of mixture, however, a
significantly less distinctive deviation was observed in the case of RSP/PF blend. Wood adhesion
mechanism is largely affected by interfacial wettability when adhesive is applied on the opposing
faces of a substrate. A marked difference in surface wetting was observed in this comparative
study; RSP/PF blend being more easily spreadable compared to RSP/SPI formulation.
At 50% blend ratio, RSP has shown 61 and 75 % bond strength compared to neat PF and
SPI adhesives respectively. In literature (Yang et al., 2006), 50% Soy/PF blend is reported to
have around 68% bond strength compared to neat PF.
79
(a)
0
1
2
3
4
5
6
0 20 40 60 80 100
PF conc. (%)
She
ar S
tren
gth
(MP
a)
Model
Experiment
(b)
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
0 20 40 60 80 100
SPI conc. (%)
She
ar S
tren
gth
(M
Pa
)
Model
Experiment
Figure 4.23 Shear strength of adhesive blends; experiment vs model. (a) RSP/PF, (b) RSP/SPI (b)
ANOVA study on raw data for both type of blends has confirmed the statistical
significance in the improvement of adhesion property of RSP by modifying it with PF and SPI
adhesives (ANOVA (4-20), P<0.05).
Since the lap-shear test determines the level of stress needed to exceed the cohesive
strength of the adhesive, the shear strength of the substrate, or the adhesive strength of the bond
(Strong, 1996), a study on mode of joint failure is possible by visual surface inspection of broken
joints. Obviously, some combination of these failure mechanisms is always observed in these
kinds of tests. Typical failure modes during lap shear tests (Cherian and Lehman, 2005) and
observed phenomenon for this study are shown in Figure 4.24. No adhesive failure was observed
80
for any blend of adhesives whereas lap-joints bonded by pure PF, SPI adhesives usually
experienced substrate failure.
Figure 4.24 Typical failure modes during lap-shear adhesion test and examples.
In case of RSP and its blends a combination of cohesive and substrate failure was
observed quite often. The morphology of this mode consisted of patches of cohesively failed
adhesive surrounded by sheared substrate.
Compared to literature findings on similar studies involving soy protein and whey
protein, the results of this study are encouraging. Even though the RSP contains only 40-50%
protein, its blends with PF has shown significant improvements in the adhesion strength of RSP.
For example, RSP’s modification with 30% PF has increased its shear strength by 25% which is
very close to adhesion strength of whey protein when it was modified with same amount of PF
(Wang et al., 2011). Similarly, compared to soy protein (Yang et al., 2006), RSP has also shown
competitive performance by yielding up to 60% strength of pure PF in a 50:50 blend. In case of
formulated adhesive blends of RSP and SPI, a bonding synergy has been observed with
significant improvements in adhesion strength of RSP
4.7.3 Statistical Significance and Discrepancy Analysis:
Model versus Experimental Values
The performance of prediction models can be assessed using a variety of methods and the
customary statistical approach is to quantify how close predictions are to the actual experimental
values. The method of computing r2, a measure of “Goodness of Fit” of linear regression, is a
widely practised technique to explore the statistical significance among predictive and
experimental results (Motulsky, 2007; Zar. 1984.). In this method, the statistical significance of
81
matching trends is determined by comparing slopes and intercepts of regression lines at 95%
confidence level. First a P value is calculated to test the null hypothesis that the slopes are
identical. A second null hypothesis that intercepts are the same (lines are parallel) is tested by not
rejecting the first one for P values greater than 0.05. For this study GraphPad Software,
GarphPad Prism® - Version 5.04 (www.graphpad.com), was used for “Goodness of Fit” analysis
to determine statistical significance of protein adhesive blends against their predictive model.
In case of RSP/PF blends, the ‘Goodness of Fit Test’ difference between the slopes and
elevations was not significant; thus high degree of agreement between modelled and
experimental values was confirmed (Appendix F -Goodness of Fit Test, RSP/PF blend).
Although successive RSP/SPI blends have demonstrated significant improvement in
adhesion strength compared to neat RSP, however “Goodness of Fit” analysis showed a
moderate agreement between model and experimental values. In this case difference between the
slopes was not significant but elevations differed significantly (Appendix F -Goodness of Fit
Test, RSP/SPI blend)
The discrepancy among model and experimental values can be traced back to two
fundamental issues; inherent limitations in the model structure and complex phenomenon of
interface/substrate interactions in a binary adhesive system. The log-additive model incorporates
only weight fractions of individual resins and their respective shear strength in native state. The
chemical cross-linking and other thermodynamic changes on molecular level also play a major
role in the final adhesion performance of polymers and their blends (Charles, 2005; Zhu, 2012).
The better agreement among model and experimental values for RSP/PF compared to RSP/SPI
can be attributed to chemical bonding in the former blend as mentioned in following section.
4.7.4 Cross-linking Evidence
The cross-linking mechanism between denatured soy protein and PF has been proposed to
proceed in two possible modes (Wescott et al., 2006) which have been used here to explain the
RSP-PF interactions.
The first possible reaction of cross-linking is the propagation of a methyl-induced
network through self-condensation step of methylolated protein groups as depicted below.
82
RSP – CH2OH + RSP – CH2OH → RSP – CH2 - RSP
The other possible reaction is the ability of denatured or modified RSP to cross-link with PF and
form a thermoset network as shown below
RSP – CH2OH or RSP+PF → RSP – CH2 – PF.
This kind of mechanism involving soy protein and PF has been also endorsed by others (Tome
and Naulet, 1981). A crosslinking mechanism was also observed during FTIR study of RSP/PF
Fig. 4.25 FTIR spectra of unmodified and modified RSP with PF
blend. FTIR spectra, Figure 4.25, confirm that the characteristic RSP IR bands shifted on cross-
linking with PF.
The peaks at 3421 and 3425 cm-1 correspond to N–H stretching of RSP band. The
characteristic peak position at 1632 and 1636 corresponds to C=O stretching of amide groups. In
case of PF modified RSP the band at 1525 cm-1 is shifted slightly when compared to unmodified
RSP sample which indicates that the cross-linking was effected by the NH2 group of RSP and
OH groups of PF by hydrogen bonding. Similar findings have been reported by Reddy and
Rajulu (2009) while studying cross-linking of soy protein with Phenol Resorcinol Formaldehyde.
In case of RSP/SPI blends, a significant increase in the intensity of amide I and amide II peaks of
RSP was observed during FTIR study suggesting a simple impregnation of two protein
compounds rather a chemical bonding.
83
CHAPTER 5- CONCLUSIONS, SIGNIFICANCE, AND
RECOMMENDATIONS
5.1 Conclusions
In this study, an attempt has been made to understand the physicochemical and microbial
characteristics of secondary paper mill sludge in exploring the recycling opportunities of this
abundantly available residual biomass from pulp and paper industry. The main objective of this
thesis, recovery of protein from secondary sludge of paper mill and its use as wood adhesive,
was realized through results of a series of studies and main conclusions are;
5.1.1 Distinct Nature of Secondary Activated Sludge
No study is available in prior art where protein estimation could have accounted for in
chemical compositional analysis of secondary sludge. Only lignocellulose analysis has been
reported, whereby protein is estimated through total nitrogen determination. In this study, for the
first time, both regent-soluble and water-soluble protein content of secondary and mixed sludge
are analytically estimated.
This study has shown that the water-extractable protein represents a significant part of
water-soluble extractives pertaining to especially secondary sludges. For secondary sludge of
TMP origin, water-induced extractives represented up to 85% protein content, whereas for
secondary sludge from Kraft mill, this figure was 50%. Alkali-soluble protein content is the
major component of total available protein in secondary (78-82 %) as well as of mixed sludge
(75%).
Regarding microbial characteristics, this study has conclusive evidence to support results
from literature on municipal sewage activated sludge. Paper secondary sludge in this study is
found to contain up to 18% extracellular polymeric substances (EPS) of which protein and
carbohydrate are the major fractions. The mixed sludge, as anticipated, contained significantly
low, up to 37% less, quantity of EPS compared to secondary sludge. Same study also resulted in
a conclusion that chemical recovery method is 5-6 time more yield efficient than physical
technique to isolate EPS from bacterial sludge.
84
Secondary activated sludge sourced from pulp and paper mill has a distinct nature
compared to other common type of virgin fibre and recycle paper mill sludges. The main
distinctive characteristic of activated sludge is the presence of extractable protein in significant
quantities, up to 32%, which is inherently available in this biomass due to aerobic and anaerobic
activity of bacterial organisms. Consequently, overall composition of these sludge types show
lower concentrations of woody components compared to mixed sludge. Regarding
lignocellulose composition, it is hard to find difference among virgin fibre mill sludges. In this
work, no statistical difference is found in hemicellulose content of secondary sludges from
different sources; however a same trend in ratio of hemicellulose to alphacellulose was observed
as reported in other works pertaining to virgin fibre mixed sludges.
5.1.2 Protein Recovery from Secondary Paper Sludge
For the first time this study confirms the technical feasibility to recover proteinaceous
materials from paper mill sludge in appreciable quantities. Highlighted conclusions are;
- The reaction kinetics of alkali-induced protein solubilisation is highly pH dependent; pH
of 12 being optimum to release protein from encrusting substances, i.e., cellulose and
lignin.
- Augmented cell disruption by using sonication and French press is proved highly
productive in releasing intracellular materials into aqueous phase.
- Statistically, stand-alone treatments of sonication and French press had no yield
difference, however their combined effect resulted in 44% more solubilized protein
compared to only alkali treatment.
- Recovery of solubilized protein is best achieved by using sulphuric acid as precipitating
agent at pH 3.
- Overall, up to 90% of extractable protein can be recovered from secondary paper mill
sludge by using commonly available reagents and equipment.
- Though lower in total protein content, recovered protein from paper sludge is
qualitatively more superior to protein extracted from sewage sludge in terms of higher
amino acid contents and lower in metal toxicity.
- The hybrid physiochemical protocol of protein recovery used in this study was able to
reduce the metal toxicity up to 4 to 6 times compared to raw sludge.
85
- In terms of essential amino acids’ concentration, RSP is compatible with soy protein and
other similar products.
- RSP contains both lower and high molecular weight proteins in the range of 5 – 70 k Da.
5.1.3 Use of RSP as Wood Adhesive; adhesion mechanism and validation
As the compact protein structure unfolds during denaturation due to breaking and
reforming of intermolecular and intramolecular interactions, same approach applied to RSP with
urea modification has yielded in conclusive evidence of increased adhesion strength by 21%. It is
believed that urea-induced denaturation results in partially disturbing hydrogen bonds of primary
helical structure of protein thereby increasing the surface area of adhesive leading to improved
gluing strength. Further, denaturation enables high degree of entanglements and cross-links
during curing facilitated through availability of various amino residual groups, such as amino,
carboxyl, and hydroxyl in RSP and soy protein adhesives. Although microstructure of wood also
effects adhesion strength significantly, but this relationship is not very consistent statistically as
other wood attributes like seasonal growth patterns play an important role as well in the ultimate
wettability of substrate surface and mechanical anchoring of adhesive upon curing.
Deduced from prevailing adhesion theories and extensive experimental setup, it is
concluded that RSP follows essentially same functionality traits as observed in well-established
protein adhesives; especially soy protein isolates and its pure globulin fraction. The extensive
and relevant literature findings and observations arising from case studies give ample evidence to
support the conclusion that adhesion mechanism of RSP with cellulose substrate is a complex
phenomenon which depends largely on mechanical, chemical, and adsorption theories for better
bonding strength.
5.1.4 Chemical Modification of RSP and Adhesion Synergy
It is technically feasible to modify RSP with synthetic and bio-based resins to improve
the functionality of wood adhesive and findings are relevant to similar literature studies
involving soy protein and whey protein formulations. RSP’s modification with 30% PF has
increased its shear strength by 25% which is very close to adhesion strength of whey protein
when it was modified with same amount of PF. Similarly, compared to soy protein RSP has also
shown competitive performance by yielding up to 60% strength of pure PF in a 50:50 blend.
86
Cross-linking evidence of RSP with PF is evident in FTIR analytical spectra and
supported through two-step reaction mechanism starting with possible propagation of a methyl-
induced network in a self-condensation step of methylolated protein groups followed by cross-
linking of modified RSP with PF to form a thermoset network. In case of formulated adhesive
blends of RSP and SPI, it is concluded to have a significant bonding synergy; however no proof
of chemical cross-linking was established through analytical study.
The validation of adhesion performance of RSP modified with PF and soy protein
through a classical model warrants RSP’s blending compatibility with synthetic and bio-based
adhesives used widely in wood composite industry. The significant rise in adhesion strength of
RSP modified with PF is also supported through literature findings based on soy and whey
proteins.
Both the hypothesis of this research thesis, potential use of secondary sludge biomass as a
source of protein and its application as wood adhesive, are positively proved through results and
conclusions mentioned above.
5.2 Significance of Research Work
Current options for management and disposal of paper mill sludge are associated with
serious financial and ecological concerns which have been the driving force to compel industry
owners in finding alternate strategies. Since the final mixed sludge ready for disposal from paper
mills contain a substantial quantity of secondary sludge, ranging from 33 to 60% (Rashid et al.,
2006), the outcome of this theses work in terms of alternate uses can lead to a significant
reduction in landfilling, incineration and landfilling of this important residual biomass.
Consequently, re-use of any quantity of secondary sludge will ultimately lead to reduced
GHG emissions and extend the life cycle of the carbon source. The availability and potential use
of protein-based wood adhesives sourced from secondary sludge is probably the most important
benefit which can be derived from the analytical and practical approach of this research work.
There is an urgent need in Canadian wood composite industry to develop formaldehyde-
free adhesives from bio-renewable resources in anticipation of new stringent environmental
legislation and growing public awareness. Validity of adhesion mechanism of sludge protein
with cellulose substrate and proven compatibility of RSP with both synthetic and bio-based
adhesives can open new commercial opportunities for wood adhesive manufacturers. Therefore,
87
this study has a strategic importance for both paper and wood products industry of Canada by
contributing significantly to economic sustainability and environmental health of these important
job sectors through a biorefinery approach.
Regarding protein recovery from activated sludge, prior art has mostly dealt with
municipal sewage sludge plants. The outcomes of this thesis, first of its kind on paper mill
secondary sludge, will serve as a significant contribution to the body of knowledge. The
technical feasibility to recover protein from secondary paper sludge in substantial quantities and
significantly low metal toxicity in RSP compared to sewage sludge protein provides another
dimension in the potential value-aided utilization of abundantly available residual biomass as
proteinacous food supplements for poultry and other livestock.
5.3 Future Work
The following areas of research can further enhance the understanding and practical
applications of recovered protein from secondary sludge of paper mills.
1. Fractionation of RSP into more pure fractions through ion-exchange chromatography or
other methods. The main foreseeable challenge in this area is the presence of complex-
natured impurities in the RSP which might interfere with buffers or separating columns.
2. Selective enzymatic or fungal modification of RSP or raw sludge to improve adhesion
functionality of proteinacous components.
3. Investigating the effect of water absorption on adhesion between protein-based adhesives
and cellulosic materials. This research area is vital to investigate the application of RSP
wood adhesives in outdoor or humid environments.
88
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APPENDIX - A
Typical press-schedule of lap-joint hot-pressing
106
APPENDIX - B
Effect of alkali treatment on suspended solids and protein content of secondary sludge
0
5000
10000
15000
20000
Raw sludge Treated sludge
TS
S, V
SS
(m
g/L)
05001000150020002500300035004000
Sol
uble
pro
tein
(m
g/L)
TSS VSS Protein
Sludge sample: SS2
Reaction time: 2 hours at pH 12.0
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APPENDIX - C
Comparison of protein yield and adhesion strength at different pH levels.
Protein solubilzation at pH level of 8 and 12
Reaction time: 24 hours
Precipitation agents: H2SO4 and HCl (at pH 3.0)
Statistical analysis:
Protein yield from 8.0 pH alkali treated sludge; no significance observed (P>0.05)
Protein yield from 12.0 pH alkali treated sludge; significance observed (P<0.05)
No significance in shear strength for protein recovered from 8 pH sludge; (P>0.05)
Statistical significance observed for shear strength of protein recovered from 12 pH sludge; (P<0.05)
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APPENDIX – D
Amino acid; Calibration data
109
APPENDIX – E
Amino acid; Sample information and analysis
110
APPENDIX – F
111
APPENDIX – F