Pretreatment of Hemp Fibers to Enhance Enzymatic Accessibility for Hemp Fibers Biological ...

129 Pages·2015·3.81 MB·English

Pretreatment of Hemp Fibers to Enhance Enzymatic
Accessibility for Hemp Fibers

Diogo Alexandre Santos Silva

Thesis to obtain the Master of Science Degree in
Biological Engineering
Supervisors: Prof. Dr. Anne Strunge Meyer
Prof. Dr. Pedro Carlos de Barros Fernandes

Examination Committee
Chairperson: Prof. Dr. Helena Maria Rodrigues Vasconcelos Pinheiro
Supervisor: Prof. Dr. Pedro Carlos de Barros Fernandes
Members of the Committee: Dr. Maria Teresa Ferreira Cesário Smolders

October, 2015

Author: Diogo Alexandre Santos Silva
2015
Title: Pretreatment of hemp fibers to enhance
enzymatic accessibility for hemp fibers
Institute: Center for Bioprocess Engineering Project Period:
(BioEng), Department of Chemical and Biochemical
Engineering, Technical University of Denmark (DTU) 16.02.2015 – 16.07.2015
ECTS:
30

Education:

Master of Science

Main Supervisors:

Professor Anne S. Meyer, DTU, Chemical
Engineering
Researcher/Assistant Professor Pedro Carlos de
Barros Fernandes, IST-DBE

Co-supervisors:

Anders Thygesen, PhD, DTU Chemical Engineering

Ming Liu, MSc, DTU Chemical Engineering

Comments:

This report is submitted as partial fulfillment of the
requirements for graduation in the above education

at Technical University of Lisbon

Pages: 111
Tables: 30

Figures: 52
References: 84

Technical University of Lisbon
DBE - Department of Bioengineering
Torre Sul, piso 0
Avenida Rovisco Pais, N1
1049-001, Lisbon

Preface
The current thesis is submitted as a partial fulfilment of the requirements for obtaining the Master of
Science degree in Biological Engineering at Technical University of Lisbon (IST), Portugal. The studies
were conducted from the 16th of February 2015 to the 16th of July 2015, at the Center of Bioprocess
Engineering (BioEng), Department of Chemical and Biochemical Engineering, Technical University of
Denmark (DTU), Denmark.
The carried out investigation was part of the plan of studies of PhD student Ming Liu and it is integrated in
CelfiMat project, with the aim of using high quality cellulosic fibers as reinforcements in the production of
strong biocomposite materials. The project counts with the partnership of the Departments of Wind
Energy, Chemistry and Chemical Engineering of the Technical University of Denmark, the Department of
Forest Products of the Swedish Agricultural University, the German company Bafa Neu GmbH and the
French company Planète Chanvre.
The master thesis has been supervised by:
Anne S. Meyer, Professor, DTU Chemical Engineering Main supervisor
Pedro Fernandes, Assistant Professor, IST Department of Bioengineering Main supervisor
Anders Thygesen, PhD and Project Leader, DTU Chemical Engineering Co-supervisor
Ming Liu, PhD Student, DTU Chemical Engineering Co-supervisor

I would like to acknowledge my supervisor Anne S. Meyer, first for accepting me at BioEng as a guest
student, providing all the fundamental conditions to develop my investigation. Then, for all the confidence,
enthusiastic encouragement and invaluable feedback in our frequent meetings throughout my period of
studies. Furthermore I would like to extend my thanks to my local supervisor Pedro Fernandes for his
availability, spent time, advices and nearness to my project.
A special acknowledgment goes to Ming Liu, with whom I share my project results. Most of all, it was an
honor and a privilege to have the opportunity to work with Ming, to whom I deeply thank for the insight into
this project and the constant guidance, inspiration, friendship and gaiety.
Annette Eva Jensen, Mikkel Justesen, Rikke Nielsen, Jonas Kreutzfeldt Heininge and Jan Sjølin are
acknowledged for technical support and also Michael K. Nielsen, in the condition of Project Manager at
BioEng.
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I have to highlight the importance of my parents Natália and Fernando and my brother Fábio, during these
6 months abroad, for undoubted support and encouragement to embrace this crusade. Then, because
“behind a great man there is always a great woman”, I deeply thank Laura for unconditional love and
support in the most difficult times.
Lastly I would like to express my gratitude to some people, who somehow contributed to my academic
path, which is now ending, by writing their names: Alberto Santos, Maria Eduarda Teles, Fernanda
Marques, Isabel Marques, Mário Estevens, Dulce Simão, Joaquim Simão.

Diogo Alexandre Santos Silva
Lisbon, Portugal
October, 2015

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Abstract
Hemp (Cannabis sativa) fibers have been considered as a sustainable biomaterial to replace man-made
fibers in composite applications, due to their low cost and density, good mechanical properties and
biodegradability. However, for high-grade composites, the cementing materials from middle lamella (ML)
regions must be degraded to obtain individual fibers or small fiber bundles, in order to create a strong
interface and reduce void space between fibers (or fiber and matrix). In this context, an enzymatic
treatment is one of the most promising and ecological methods, but the process is not efficient because
enzymes are too large to penetrate the well lignified ML region, and thus requires an auxiliary
pretreatment.
Among the conducted experiments, a fungal pretreatment at controlled conditions for half a week, a
hydrothermal pretreatment in an autoclave at 1 bar (121 ºC) for 30 min and a chemical pretreatment with
NaOH at 60ºC for 4 h were shown to be the best solutions, by allowing partial degradation of pectin
(decrease of 55, 41 and 67% in galacturonic acid content, respectively) and subsequently enhancing the
accessibility of pectinases for said substrate, indicated by the final low content of GalA in final treated
fibers. The direct combination of EDTA-2Na (0.5%) with endopolygalacturonase was also demonstrated
as a time saving option.
However, significant negative effects (p<0.05) in the mechanical performance of pretreated hemp fibers
were noted, except for hydrothermally pretreated, for which only strain significantly (p<0.05) decreased by
40% and no apparent effects on stiffness and Ultimate Tensile Strength were recorded.
Keywords: Hemp fibers; Enzyme; Hydrothermal pretreatment; Biological pretreatment; Chemical
Pretreatment; Mechanical Properties

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Resumo
As fibras de cânhamo (Cannabis sativa) têm sido reconhecidas como uma alternativa sustentável às
fibras sintéticas para aplicações com materiais compósitos, devido ao seu baixo custo e densidade, boas
propriedades mecânicas e biodegradabilidade. No entanto, para fabricação de compósitos de alta
qualidade, os materiais de cimentação da lamela média (LM) devem ser degradados a fim de se obter
fibras individuais ou pequenos feixes de fibras, criando uma interface forte e reduzindo o espaço vazio
entre as fibras (ou entre fibras e matriz). Neste contexto, um tratamento enzimático é um dos métodos
mais promissores e ecológicos, mas o processo não é eficiente porque as enzimas são demasiado
grandes para penetrar a bem lignificada LM, e, portanto, exige-se um pré-tratamento auxiliar.
Entre as experiências realizadas, um pré-tratamento fúngico em condições controladas durante meia
semana, um pré-tratamento hidrotérmico numa autoclave a 1 bar (121 ºC) durante 30 min e um pré-
tratamento químico com NaOH a 60 °C durante 4 h, mostraram ser as melhores soluções, permitindo a
degradação parcial de pectina (diminuição de 55, 41 e 67% do teor de ácido galacturónico,
respectivamente) e aumentando posteriormente a acessibilidade de pectinases para o referido substrato,
resultando num baixo teor de ácido galacturónico nas fibras finais. A combinação direta de EDTA-2Na
(0,5%) com endopoligalacturonase demonstrou ser também uma alternativa rápida.
No entanto, foram registados efeitos negativos significativos (p<0.05) no desempenho mecânico das
fibras de cânhamo pré-tratadas, excepto quando pré-tratadas hidrotermicamente, para as quais apenas a
deformação diminui significativamente (p< 0,05) em 40% e não foram registados efeitos aparentes sobre
a rigidez e limite de resistência à tração.
Palavras-chave: Fibras de Cânhamo; Enzimas; Pretratamento Hidrotérmico; Pretratmento Biológico;
Pretratamento Químico; Propriedades mecânicas.

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Table of Contents
PREFACE................................................................................................................................................ I
ABSTRACT ........................................................................................................................................... III
RESUMO ............................................................................................................................................. IV
TABLE OF CONTENTS ............................................................................................................................... V
INDEX OF FIGURES ................................................................................................................................ VII
INDEX OF TABLES ................................................................................................................................... XI
INDEX OF ABBREVIATIONS ...................................................................................................................... XIII
1. INTRODUCTION ........................................................................................................................... 1
1.1 NATURAL FIBERS ............................................................................................................................ 1
1.1.1 HEMP FIBERS ........................................................................................................................................ 3
1.2 HEMP FIBERS STRUCTURE ................................................................................................................. 7
1.3 CHEMICAL COMPOSITION OF HEMP FIBERS ............................................................................................ 8
1.3.1 CELLULOSE ............................................................................................................................................ 9
1.3.2 HEMICELLULOSE .................................................................................................................................. 10
1.3.3 LIGNIN ............................................................................................................................................... 11
1.3.4 PECTIN ............................................................................................................................................... 12
1.3.5 WAXES AND ASHES............................................................................................................................... 14
1.4 HEMP FIBERS APPLICATIONS ............................................................................................................ 15
1.4.1 HEMP FIBERS AS REINFORCEMENTS FOR COMPOSITES ................................................................................. 15
1.5 PROCESSING OF HEMP FIBERS .......................................................................................................... 17
1.5.1 FIELD RETTING ..................................................................................................................................... 17
1.5.2 WATER RETTING .................................................................................................................................. 18
1.5.3 FUNGAL TREATMENT ............................................................................................................................ 18
1.5.4 CHEMICAL TREATMENT ......................................................................................................................... 19
1.5.5 ENZYMATIC TREATMENT........................................................................................................................ 22
2. AIM OF STUDIES ........................................................................................................................ 25
3. MATERIALS AND METHODS........................................................................................................ 26
3.1 MATERIALS ................................................................................................................................. 26
3.2 METHODS .................................................................................................................................. 26
3.2.1 FUNGAL PRETREATMENT ....................................................................................................................... 27
3.2.2 HYDROTHERMAL PRETREATMENT ........................................................................................................... 28
3.2.3 CHEMICAL PRETREATMENT .................................................................................................................... 28
3.2.4 ENZYMATIC TREATMENT........................................................................................................................ 29
3.2.5 CHEMICAL COMPOSITION ANALYSIS ......................................................................................................... 30
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3.2.6 TENSILE STRENGTH TESTING OF FIBER BUNDLES ......................................................................................... 35
3.2.7 WATER RETENTION DETERMINATION ....................................................................................................... 38
3.2.8 TOTAL PROTEIN CONCENTRATION DETERMINATION .................................................................................... 39
3.2.9 STATISTICAL ANALYSIS ........................................................................................................................... 40
4. PRETREATMENT OF HEMP FIBERS TO ENHANCE ENZYME ACCESSIBILITY FOR HEMP FIBERS ......... 41
4.1 BIOLOGICAL PRETREATMENT OF HEMP FIBERS TO ENHANCE ACCESSIBILITY OF PECTINASES .............................. 41
4.1.1 EXPERIMENTAL DESIGN ......................................................................................................................... 41
4.1.1 RESULTS AND DISCUSSION ..................................................................................................................... 42
4.2 HYDROTHERMAL PRETREATMENT OF HEMP FIBERS TO ENHANCE ACCESSIBILITY OF PECTINASES ........................ 52
4.2.1 EXPERIMENTAL DESIGN ......................................................................................................................... 53
4.2.2 RESULTS AND DISCUSSION ..................................................................................................................... 54
4.3 CHEMICAL PRETREATMENT OF HEMP FIBERS TO ENHANCE ACCESSIBILITY OF ENZYMES .................................... 64
4.3.1 EXPERIMENTAL DESIGN ......................................................................................................................... 65
4.3.2 RESULTS AND DISCUSSION ..................................................................................................................... 66
4.4 A COMPARISON OF DIFFERENT PRETREATMENTS .................................................................................. 75
5. CONCLUSIONS AND FUTURE PROSPECTS .................................................................................... 78
6. BIBLIOGRAPHY .......................................................................................................................... 81
7. APPENDIXES .............................................................................................................................. 88
7.1 EFFECT OF DIFFERENT CHEMICAL AGENTS ON HEMP FIBERS ...................................................................... 88
7.1.1 EFFECT OF EDTA ON HEMP FIBERS .......................................................................................................... 88
7.1.2 EFFECT OF ACID-CHLORITE ON HEMP FIBERS ............................................................................................. 93
7.1.3 EFFECT OF SODIUM HYDROXIDE ON HEMP FIBERS ...................................................................................... 97
7.1.4 CONCLUSION ..................................................................................................................................... 105
7.2 ENZYMATIC ACTIVITY ASSAYS ........................................................................................................ 106
7.2.1 ENDOPOLYGALACTURONASE ACTIVITY ASSAY .......................................................................................... 106
7.2.2 PECTIN LYASE ACTIVITY ASSAY .............................................................................................................. 107
7.2.3 XYLANASE ACTIVITY ASSAY ................................................................................................................... 108
7.2.4 XYLOGLUCANASE ACTIVITY ASSAY .......................................................................................................... 109
7.2.5 LACCASE ACTIVITY ASSAY ..................................................................................................................... 109
7.3 EXAMPLE OF HPLC CHROMATOGRAPH FOR STANDARD MOTHER SOLUTION .............................................. 111

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