Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses

146 Pages·2014·3.2 MB·English

by  Turkia Heidi

S C IENCE
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VI Dissertation O
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GILHGIHHCRA E S E R
Capillary electrophoresis
for monitoring carboxylic,
phenolic and amino acids
in bioprocesses
Heidi Turkia

VTT SCIENCE 47
Capillary electrophoresis
for monitoring carboxylic,
phenolic and amino acids
in bioprocesses
Heidi Turkia
VTT Technical Research Centre of Finland
Lappeenranta University of Technology, Faculty of Technology,
Department of Chemistry, Process and Environmental Analytics
Thesis for the degree of Doctor of Philosophy to be presented with due
permission for public examination and criticism in Auditorium 1381, at
the Lappeenranta University of Technology, on the 24.1.2014 at 10:00.
ISBN 978-951-38-8107-8 (Soft back ed.)
ISBN 978-951-38-8108-5 (URL: http://www.vtt.fi/publications/index.jsp)
VTT Science 47
ISSN-L 2242-119X
ISSN 2242-119X (Print)
ISSN 2242-1203 (Online)
Copyright © VTT 2013
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Capillary electrophoresis for monitoring of carboxylic, phenolic and
amino acids in bioprocesses
Kapillaarielektrofeesin käyttö karboksyyli-, fenoli- ja aminohappojen monitorointiin
bioprosesseissa.Heidi Turkia.Espoo 2013. VTT Science 47. 103 p. + app. 36 p.
Abstract
Bioprocess technology is a multidisciplinary industry that combines knowledge of
biology and chemistry with process engineering. It is a growing industry because
its applications have an important role in the food, pharmaceutical, diagnostics
and chemical industries. In addition, the current pressure to decrease our depend-
ence on fossil fuels motivates new, innovative research in the replacement of
petrochemical products. Bioprocesses are processes that utilize cells and/or their
components in the production of desired products. Bioprocesses are already used
to produce fuels and chemicals, especially ethanol and building-block chemicals
such as carboxylic acids. In order to enable more efficient, sustainable and eco-
nomically feasible bioprocesses, the raw materials must be cheap and the biopro-
cesses must be operated at optimal conditions. It is essential to measure different
parameters that provide information about the process conditions and the main
critical process parameters including cell density, substrate concentrations and
products. In addition to offline analysis methods, online monitoring tools are be-
coming increasingly important in the optimization of bioprocesses.
Capillary electrophoresis (CE) is a versatile analysis technique with no limita-
tions concerning polar solvents, analytes or samples. Its resolution and efficiency
are high in optimized methods creating a great potential for rapid detection and
quantification. This work demonstrates the potential and possibilities of CE as a
versatile bioprocess monitoring tool. As a part of this study a commercial CE de-
vice was modified for use as an online analysis tool for automated monitoring. The
work describes three offline CE analysis methods for the determination of carbox-
ylic, phenolic and amino acids that are present in bioprocesses, and an online CE
analysis method for the monitoring of carboxylic acid production during biopro-
cesses. The detection methods were indirect and direct UV, and laser-induced
frescence. The results of this work can be used for the optimization of bioprocess
conditions, for the development of more robust and tolerant microorganisms, and
to study the dynamics of bioprocesses.
Keywords bioprocess monitoring, capillary electrophoresis, online capillary electro-
phoresis, carboxylic acids, phenolic acids, amino acids
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Kapillaarielektroforeesin käyttö karboksyyli-, fenoli- ja
bioprosesseissa
Capillary electrophoresis for monitoring of carboxylic, phenolic and amino acids in bioprocesses.
Heidi Turkia.Espoo 2013. VTT Science 47. 103 s. + liitt. 36 s.
Tiivistelmä
Bioprosessitekniikka on monitieteellinen teollisuudenala, joka yhdistää biologian ja
kemian tietämyksen kemiantekniikkaan. Se on kasvava teollisuudenala, koska sen
sovellukset ovat hyvin tärkeitä ruoka-, farmasia-, diagnostiikka- ja kemianteolli-
suudelle. Tämän lisäksi riippuvuutta öljystä tulee vähentää merkittävästi, mikä
mahdollistaa uuden ja innovatiivisen tutkimuksen petrokemikaalituotteiden kor-
vaamiseksi. Bioprosessit ovat prosesseja, jotka käyttävät soluja ja/tai niiden osia
tuottamaan haluttuja tuotteita. Bioprosesseja on jo käytetty polttoaineiden ja kemi-
kaalien tuottamiseen, erityisesti etanolin ja ns. building-block kemikaalien, kuten
karboksyylihappojen, tuottamiseen. Jotta bioprosesseista saadaan tehokkaampia
ja taloudellisesti kannattavampia, raaka-aineiden on oltava halpoja ja bioprosessien
on toimittava optimaalisissa olosuhteissa. Erilaisten parametrien mittaaminen on
välttämätöntä, sillä ne antavat tietoja prosessin tilasta ja kriittisistä prosessipara-
metreista, joita ovat biomassa, lähtöaineet ja tuotteet. Offline-mittausten lisäksi
online-määritykset kasvattavat merkitystään bioprosessien optimoinnissa.
Kapillaarielektroforeesi (CE) on monipuolinen analyysitekniikka, jolla ei ole ra-
joituksia polaaristen liuottimien, analyyttien tai näytteiden kanssa. Optimoitujen
menetelmien korkea resoluutio ja tehokkuus mahdollistavat nopean detektoinnin ja
kvantifioinnin. Tämä työ osoittaa CE:n potentiaalin monipuolisena bioprosessi-
monitorointityökaluna. Osana tätä tutkimusta kaupallinen CE-laitteisto muutettiin
toimimaan online-analysointilaitteistona prosessien automaattista monitorointia
varten. Tämä työ kuvaa kolme offline-CE analyysimenetelmää karboksyyli-, fenoli-
ja aminohappojen analysointiin sekä yhden online-CE analyysimenetelmän kar-
boksyylihappojen monitorointiin bioprosessien aikana. Käytetyt detektointimene-
telmät olivat epäsuora ja suora UV sekä laser-indusoitu fluoresenssi. Työn tuloksia
voidaan käyttää bioprosessiolosuhteiden optimoinnissa, elinvoimaisempien ja
kestävämpien mikro-organismien kehittämisessä sekä bioprosessien dynamiikan
tutkimisessa.
Avainsanat bioprocess monitoring, capillary electrophoresis, online capillary electro-
phoresis, carboxylic acids, phenolic acids, amino acids
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Preface
This work was carried out at VTT Technical Research Centre of Finland during the
period 2008–2013. Financial support from the Finnish Centre of Excellence in
White Biotechnology – Green Chemistry Research granted by the Academy of
Finland (grant number 118573), the EU-project Nanobe which received funding
from the European Community’s Seventh Framework Programme (FP7/2007–
2013) under grant agreement no. 227243, Laboratory of Chemistry at the
Lappeenranta University of Technology and Graduate School for Chemical
Sensors and Microanalytical Systems (CHEMSEM) is gratefully acknowledged.
I express my deepest gratitude to my supervisors Professor Heli Sirén and Dr.
Sci. (tech.) Juha-Pekka Pitkänen. Your help, support and encouragement during
the work was invaluable. The fruitful conversations and brainstorming were of
great importance for the success of this study. In addition, your knowledge and
enthusiasm has been inspirational for me. Vice President of Bio and Process
Technology Dr. Anu Kaukovirta-Norja, Technology manager Dr. Kirsi-Marja Oks-
man-Caldentey, her predecessor Dr. Tiina Nakari-Setälä, and team leader Dr.
Jaana Uusitalo are acknowledged for providing excellent working facilities and
research equipment.
I thank Professor Mihkel Kaljurand and Professor Krist V. Gernaey for their ex-
cellent review of the thesis. Kathleen Ahonen and Michael Bailey are thanked for
the language revision of the articles, and Michael Bailey for the language revision
of the thesis. Your work upgraded the scientific value of my writings.
I thank my co-authors Sami Holmström, Toni Paasikallio, Merja Penttilä, Juha-
Pekka Pitkänen, Heli Sirén and Marilyn Wiebe for your contribution. Working with
you has been a privilege. All the supervisors and students at the CHEMSEM
graduate school are thanked for the interesting and fruitful annual meetings and
courses.
I would also like to thank all of my colleagues, especially Sami, Toni, Jouni, Su-
sanne, Dorothee and everyone else in my team. Working with you has been a
pleasure and I sure do hope to work with you in the future. I have had so many
interesting conversations with the people in the morning coffee table of all imagi-
nable subjects, thank you for making starts of mornings so much fun.
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Finally I would like to thank my friends and family for their love and support.
Especially my husband Ville is acknowledged for his never ending support and
understanding. I love you so much.
Espoo, December 2013
Heidi
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Academic dissertation
Supervisors Professor Heli Sirén
Department of Chemistry
Faculty of Technology
Lappeenranta University of Technology
Lappeenranta, Finland
D.Sc. (tech.), Principal Scientist Juha-Pekka Pitkänen
VTT Technical Research Centre of Finland
Espoo, Finland
Reviewers Professor Mihkel Kaljurand
Department of Chemistry
Tallinn University of Technology
Tallinn, Estonia
Professor Krist V. Gernaey
Department of Chemical and Biochemical Engineering
Technical University of Denmark
Lyngby, Denmark
Opponent Professor Mihkel Kaljurand
Department of Chemistry
Tallinn University of Technology
Tallinn, Estonia
Custos Professor Heli Sirén
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List of publications
This thesis is based on the following original publications which are referred to in
the text as I–IV. The publications are reproduced with kind permission from the
publishers.
I Turkia, H., Sirén, H., Pitkänen, J.-P., Wiebe, M., Penttilä, M., 2010.
Capillary electrophoresis for the monitoring of carboxylic acid production by
Gluconobacter oxydans. Journal of Chromatography A 1217, 1537–1542.
II Turkia, H., Sirén, H., Penttilä, M., Pitkänen, J.-P., 2013. Capillary
electrophoresis for the monitoring of phenolic compounds in bioprocesses.
Journal of Chromatography A 1278, 175–180.
III Turkia, H., Holmström, S., Paasikallio, T., Sirén, H., Penttilä, M., Pitkänen, J.-P.,
2013. Online capillary electrophoresis for monitoring carboxylic acid
production by yeast during bioreactor cultivations. Analytical Chemistry 85,
9705–9712.
IV Turkia H., Sirén, H., Penttilä, M., Pitkänen, J.-P., 2013. Capillary
electrophoresis with laser-induced fluorescence detection for studying
amino acid uptake of yeast during beer fermentation. Submitted to Journal
of Chromatography B.
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