Substrates)for)the)sustainable)production)of ...· !ii!! Abstract) This!work!aims!the!sustainable!production!of!biosurfactants,!mannosylerythritol!lipids!(MEL),!

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Substrates for the sustainable production of mannosylerythritol lipids: biological oils vs nanofiltrated

lignocellulosic hydrolysates

Aldo Robles Arvalo

Thesis to obtain the Master of Science Degree in

Biotechnology

Supervisors: Prof. Dr. Frederico Castelo Alves Ferreira.

Prof. Dr. Csar Simes da Fonseca.

Examination Committee

Chairperson: Prof. Dr. Leonilde de Ftima Morais Moreira.

Supervisor: Prof. Dr. Frederico Castelo Alves Ferreira

Members of the Committee: Prof. Dr. Pedro Carlos de Barros Fernandes.

October 2015

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Acknowledgments On first place, I would like to thank to my advisor Prof. Frederico Ferreira for the continuous

support during my research work, for his patience, motivation, enthusiasm, and immense knowledge.

His assistance helped me to perform my lab work and writing of this thesis.

Besides my advisor, I would like to thank my co-advisor Prof. Csar Fonseca, for their insightful

and accurate comments. Adding to that, for the opportunity to collaborate at LNEG and for introducing

me to lignocellulose biomass.

I would like to thank for the funding which allowed the development of the work. This work was

co-funded by Fundao para Cincia e Tecnologiaby National with the Exploratory Project "TAKE-OFF"

through FEDER funds Programa Operacional Factores de Competitividade COMPETE (FCOMP-

01-0124-FEDER-041191) and national (FCT) funds (EXPL/AAG-TEC/0610/2013).

My sincere gratitude also goes to Dr. Nuno Faria for his invaluable guidance. Also for the hard

questions which incepted me to widen my research from various perspectives.

Additionally, I am grateful to Marisa Santos who gave access to the laboratory and research

facilities at IBB. Also to Dr. Teresa Esteves for her support and enlightening comments.

This project couldnt have been completed without the collaboration of Dr. Cristina Oliveira for

her assistance to acquire the oils and their characterisation. Also to Dr. Cristina Matos for providing the

environmental analysis of my samples and for her invaluable guidance in this topic.

I thank my fellow lab mates for their support and comprehensiveness, and for all the fun we

have had in the last two years. Specially to Sara Matias and Clara Lopes. Also I thank my class mates

who welcomed me and made me feel at home.

I take this chance to express my gratitude to all of the IBB members for their help and support.

I also recognize my parents for the unceasing encouragement. Specially to my Portuguese parents

Florindo Santos and Leticia Santos who opened the doors of their home and gave me his invaluable

support to conclude this dream. I am also grateful to my Portuguese and Mexican friends who supported

me through out this venture.

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Abstract This work aims the sustainable production of biosurfactants, mannosylerythritol lipids (MEL),

exploring the use of different carbon sources by non-conventional yeasts, Pseudozyma spp. Two main

sources were assessed: 1) biological oils (poultry oil, waste frying oil, crude soybean oil, and crude

rapeseed oil) and 2) lignocellulosic biomass (wheat straw). This thesis demonstrates the capacity of

Pseudozyma spp. in producing MEL from biological oils and from sugars in the presence hemicellulosic

hydrolysates, inhere evaluated for the first time. The highest MEL titres were achieved with oils as

substrate after 14 days of bioconversion: refined soybean oil (18.3 gMEL/l), crude soybean oil (13.7

gMEL/l), poultry oil (12.9 gMEL/l), crude rapeseed oil (11.5 gMEL/l), and waste frying oils (8.3 gMEL/l).

The best MEL titre using hemicellulosic hydrolysates supplemented with glucose (1.47 gMEL/l) was

achieved, after detoxification by nanofiltration. The nanofiltration with a polyamide membrane (NF-PA2)

removed 80% of 2-furfuraldehyde, 56% of formic acid, and 43% of acetic acid were removed with a low

monosaccharides loss, (e.g. 17% of D-xylose). Formic and acetic acids revealed inhibitory effect on

biomass yield and MEL titres, which is reduced up to approx. 50% when formic acid is present at 2 g/l.

MEL recovery was achieved by, liquid-liquid extraction with ethyl acetate attaining nearly 80% purity

and 82% yield.

KEYWORDS: Biosurfactants, Mannosylerythritol Lipids, Nanofiltration technologies,

Lignocellulosic biomass, Detoxification.

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Resumo Este trabalho visa produo sustentvel de biossurfatantes, manosileritritolpidos (MEL), pela

levedura no convencional Pseudozyma sp. estudando o uso de diferentes fontes de carbono. Duas

principais fontes de carbono foram avaliadas: 1) leos biolgicos (leo de galinha, leo residual de

frituras, leo de soja e leo de colza no refinados) e 2) biomassa lignocelulsica (palha de trigo). Esta

tese demonstra a capacidade de produo de MEL pela levedura Pseudozyma sp. a partir de leos

biolgicos e de acares na presena de hidrolisados de hemicelulose. As concentraes de MEL mais

elevadas foram atingidas aps 14 dias de bioconverso usando leos como substrato: leo de soja

refinado (18.3 gMEL/l), leo de soja no refinado (13.7 gMEL/l), leo de galinha (12.9 gMEL/l), leo de

colza no refinado (11.5 gMEL/l), e leos residuais alimentares (8.3 gMEL/l). A melhor concentrao

de MEL atingida a partir de hidrolisados de hemicelulose, suplementados com glucose (1.47 gMEL/l),

foi obtida aps destoxificao por nanofiltrao. A nanofiltrao com uma membrana de poliamida (NF-

PA2) removeu 80% de 2-Furfuraldeido, 56% de cido frmico e 42% de cido actico com baixas

perdas de monossacridos (ex. 17% de D-Xilose). Os cidos frmico e actico demostraram ter um

efeito inibitrio no rendimento de biomassa e de MEL, efeito que reduz em aproximadamente 50%

quando o cido frmico est presente em 2 g/l. O MEL foi recuperado por extrao lquido lquido

com acetato de etilo atingindo cerca de 80% de pureza e 82% de rendimento.

PALAVRAS CHAVE: Biossurfatantes, manosileritritolpidos, nanofiltrao, biomassa

lignocelulsica, destoxificao.

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Table of Contents

Acknowledgments .................................................................................................................................... i

Abstract ................................................................................................................................................... ii

Resumo .................................................................................................................................................. iii

Table index ............................................................................................................................................ vii

Figure index ........................................................................................................................................... vii

Abbreviations .......................................................................................................................................... x

Chapter 1 - Introduction .......................................................................................................................... 1

1.1 Overview ................................................................................................................................. 1

1.2 Objectives and challenges ...................................................................................................... 1

1.3 Research questions ................................................................................................................ 2

1.4 Research strategy ................................................................................................................... 2

Chapter 2 Theoretical background and State-of-the-art ........................................................................ 4

2.1 - Biosurfactants .......................................................................................................................... 4

2.1.1 Characteristics .................................................................................................................. 4

2.1.2 Surfactants Market ........................................................................................................... 7

2.1.3 Industrial Perspectives ................................................................................................... 10

2.1.4 Production ...................................................................................................................... 11

2.1.5 New generation of biosurfactants ................................................................................... 12

2.2 Process Intensification .......................................................................................................... 15

2.2.1 Principles ........................................................................................................................ 15

2.2.2 Process design trends: Process intensification (PI) ....................................................... 17

2.3 - Mannosylerythritol Lipids ....................................................................................................... 19

2.3.1 Properties and differentiation among other molecules ................................................... 19

2.3.2 Significance of MEL and