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  • ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184

    ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/

    WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en

  • GICOM (Composting Research Group)

    DEPARTAMENT D' ENGINYERIA QUIMICA, BIOLÒGICA I AMBIENTAL

    Escola d'Enginyeria

    SUSTAINABLE CARBOHYDRASE PRODUCTION USING ORGANIC WASTES THROUGH

    SOLID-STATE FERMENTATION: OPERATIONAL STRATEGIES AND MICROBIAL

    COMMUNITIES ASSESSMENT

    PhD Thesis

    Alejandra Patricia Cerda Llanos

    Supervised by:

    Antoni Sánchez Ferrer

    Teresa Gea Leiva

    2017

  • Antoni Sánchez Ferrer i Teresa Gea Leiva, professors titulars del Departament d' Enginyeria Química,

    Biològica i Ambiental de la Universitat Autònoma de Barcelona,

    Certifican:

    Que la enginyera bioquímica i màster en estudis ambientals Alejandra Patricia Cerda Llanos ha

    realitzat sota la nostra direcció el treball amb títol "Sustainable carbohydrase production using organic

    wastes through solid-state fermentation: Operational strategies and microbial communities

    assessment", que es presenta en aquesta memòria i que constitueix la seva tesi per optar al Grau de

    Doctor per la Universitat Autònoma de Barcelona.

    I perquè en prengueu coneixement i consti els efectes oportuns, es presenta a l'Escola de Enginyeria

    de la Universitat Autònoma de Barcelona l'esmentada tesi, signant el present certificat.

    Dr. Antoni Sánchez Ferrer Dra Teresa Gea Leiva

    Bellaterra, Febrer 2017.

  • To my mother.

  • "Real education must ultimately be limited to men/women who insist on knowing, the rest is mere

    sheep-herding"- Ezra Pound.

    "Sólo con el corazón se puede ver bien, lo esencial es invisible para los ojos"- Antoine de Saint-

    Exupèry.

  • Acknowledgements

    This thesis was carried out thanks to the financial support of the Spanish Ministry of Economy and

    Competitiveness for the "Waste to product" CTM2012-33663 and BIOPRO project, the European

    commission for the "Decisive" CTM2015-69513-R project and to the Universitat Autònoma de

    Barcelona for the predoctoral fellowship granted (PIF-UAB).

    In first place, i have to thank both of mi supervisors: Tere and Toni. Thanks for providing me with the

    great opportunity to join the big GICOM family and for all of the support, trust and patience (above

    all patience!!!). You two are an endless source of inspiration for me in both, personal and professional

    aspects. I have to specially acknowledge the coolest, craziest and nicest supervisor i could ever ask

    for: Tereeee!!!. More than a supervisor, a great person and friend and most definitely one of the

    persons that i will remember with a smile whenever i remember my stayed at UAB.

    To all the GICOM group, thanks for all the shared moments even those including crying, stress,

    sweat, sludge and terrible odours!!. To my only (PhD) padawan: Pedro, keep sharing love, energy and

    happiness to the world, you made every minute spent in the "caseta" unforgettable, jajaja! I wish you

    the best for the bright future ahead!. Ahmad, Nora, Maria (let's not forget the last name

    Compostatge"), Cindy, Óscar, Eva, Dani, the grad padawans Laura and Aida (you don't have to

    google it anymore)...and everyone who passed for the composting lab thanks for being there.

    To my mexican BFF's: Gaby and CChan!. I love you both. We will definitely be seeing you guys!

    even if we have to go to Cancún to stay for two month in your house CChan, ajjajaajaj don't forget!.

    Gabriela, in spite of the toughest time you had the last couple of years you still keep your smile and

    positiveness. I admire your will to carry on even when the odds do not favour you.

    To my dearest office mates: Jordi and Xavi. Thanks for making our office the coolest place to work

    (among others), to always put a smile of my face, to have the perfect taste in music ((each one on our

    style but no matter what other people say, it is very important!) and for being there and for sharing a

    piece of your lives with me. I will miss you both.

    Last but not least, thanks to my dear family!!!! Without all of you we (Carlos, Vicente and me)

    probably wouldn't have made it through all this time in Spain. To my parents: Vivi and Nelson, thank

    you for shaping me the way i am. I'm deeply grateful for all you have given to me, especially my

    beautiful sisters: Vivi, Coté and Gabi! I love you all forever and ever!. To "los suegros": Carlos and

    Maggi, i couldn't ask for a better family!!! I love you and thanks for all of the love, support and trust!.

    To Matias (Makikis) and Manuel (papum): I always wanted brothers and now i have two!!! I'm so

    lucky!!!. We miss you all always, everyday.

  • To the loves of my life: Carlos and Vicente. I can't imagine my life without you both. Carlos, you are

    the partner i always imagined and more. Thanks for holding my hand during this (sometimes difficult)

    road, i love you!!. Vicentin!!, with a smile melts my heart and at the same time makes me strong.

    With you i feel i can accomplish anything.

    As the late Gustavo Cerati would say: Gracias Totales!!

  • Summary

    According to the solid-state fermentation (SSF) research line of the composting research group

    GICOM, the main goal of this thesis is to develop a sustainable process for the production of

    carbohydrases using organic wastes in a representative scale. To achieve this goal, several organic

    wastes were screened on its potential to produce cellulase at laboratory scale. Also, compost was

    added as a complex mixture of biomass in order to provide cellulose-degrading microorganisms. After

    the selection of the two best substrates: orange peels and coffee husk using 0.5 L reactors, the scale up

    was assessed in 10 and 50 L reactors. All SSF were undertaken in near-adiabatic conditions using

    thermally isolated reactors with a continuous monitoring of the biological activity reflected as specific

    oxygen uptake rate (sOUR) and temperature.

    The main challenges to overcome in SSF using organic wastes is on one hand the possibility to work

    in a continuous configuration and, on the other hand, the reproducibility of the proposed process.

    These two aspects were assessed in this thesis.

    A first approach towards a continuous process was carried out for the production of amylase using

    bread waste and soy fiber as substrate and adding Thermomyces sp as inoculum. Three different

    strategies were developed in a sequential batch operation by removing part of the substrate in three

    diffeerent stages of the process: during maximum sOUR, maximum amylase activity and maximum

    amylase activity with a posterior enzymatic extraction. An increase of above 200% in amylase activity

    production was obtained in all the assessed strategies.

    Due to the success of the amylase production in a continuous configuration, the next step was to use

    the operational conditions obtained at laboratory scale for cellulase production and develop a

    sequential batch operation in 4.5L reactors. An initial compost addition as inoculum was considered

    only at the beginning of the fermentation. After reaching the maximum cellulase production the

    system started working in cycles, removing one part of the substrate and replacing it for fresh

    substrate. Two substrate exchange ratios were evaluated: 90% and 50%. Using this configuration, a

    continuous process was carried out, with a continuous production of cellulase and valorization of

    organic wastes and eliminating the requirement for fresh inoculum in each cycle. The sequential batch

    operation resulted in the successful production of cellulase with sustained values around 8-9 filter

    paper units per grams of dry matter in both configurations. In addition. the microbial communities

    present at the end of this operation was characterized, identifying several cellulose, lignin and

    hemicellulose degraders. This fermented solid was described as a specialized inoculum able to

    colonize the solid matrix of the reactor and provides the opportunity to develop a reproducible

    process.

  • Finally, the reproducibility and consistency of the process was assessed in triplicates in a 50 L reactor

    using the specialized inoculum and coffee husk as the substrate. In addition the gaseous emissions

    were measured in order to fully understand the process and to determi