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ISBN 978-952-62-0882-4 (Paperback)ISBN 978-952-62-0883-1 (PDF)ISSN 0355-3213 (Print)ISSN 1796-2226 (Online)
U N I V E R S I TAT I S O U L U E N S I SACTAC
TECHNICA
U N I V E R S I TAT I S O U L U E N S I SACTAC
TECHNICA
OULU 2015
C 540
Johanna Panula-Perälä
DEVELOPMENT AND APPLICATION OF ENZYMATIC SUBSTRATE FEEDING STRATEGIES FOR SMALL-SCALE MICROBIAL CULTIVATIONSAPPLIED FOR ESCHERICHIA COLI, PICHIA PASTORIS, AND LACTOBACILLUS SALIVARIUS CULTIVATIONS
UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU, FACULTY OF TECHNOLOGY
C 540
ACTA
Johanna Panula-Perälä
C540_etukansi.fm Page 1 Monday, June 22, 2015 3:49 PM
A C T A U N I V E R S I T A T I S O U L U E N S I SC Te c h n i c a 5 4 0
JOHANNA PANULA-PERÄLÄ
DEVELOPMENT AND APPLICATION OF ENZYMATIC SUBSTRATE FEEDING STRATEGIES FOR SMALL-SCALE MICROBIAL CULTIVATIONSApplied for Escherichia coli, Pichia pastoris, and Lactobacillus salivarius cultivations
Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Technology andNatural Sciences of the University of Oulu for publicdefence in Kuusamonsali (YB210), Linnanmaa, on 14August 2015, at 12 noon
UNIVERSITY OF OULU, OULU 2015
Copyright © 2015Acta Univ. Oul. C 540, 2015
Supervised byProfessor Heikki OjamoDoctor Antti VasalaProfessor Peter Neubauer
Reviewed byDocent Markku SaloheimoDoctor Kristiina Kiviharju
ISBN 978-952-62-0882-4 (Paperback)ISBN 978-952-62-0883-1 (PDF)
ISSN 0355-3213 (Printed)ISSN 1796-2226 (Online)
Cover DesignRaimo Ahonen
JUVENES PRINTTAMPERE 2015
OpponentDoctor Juha-Pekka Pitkänen
Panula-Perälä, Johanna, Development and application of enzymatic substratefeeding strategies for small-scale microbial cultivations. Applied for Escherichiacoli, Pichia pastoris, and Lactobacillus salivarius cultivationsUniversity of Oulu Graduate School; University of Oulu, Faculty of TechnologyActa Univ. Oul. C 540, 2015University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland
Abstract
Small-scale cultivation methods are a necessity for the development of new biotechnologicalprocesses. The most common method for submerged microbial cultivation is a shake flask usedwith a batch operation protocol. Well plate cultivation formats have also increased theirimportance, due to the need to utilize high-throughput cultivations for efficient productdevelopment. However, batch cultivation is often not the optimal method for obtaining high celldensities and good product quality, due to unlimited microbial growth.
The aim of this dissertation was to improve small-scale microbial cultivations for microbialgrowth and product formation. Hydrolytic enzymes were utilized to relieve nutrient limitation byhydrolysis of proteins in lactic acid bacteria cultures to improve lactic acid production from dairyside products. Hydrolytic enzymes were also utilized in the enzymatic release of glucose fromstarch to create a fed-batch-like cultivation system applicable on small scale. The wireless sensorsystem developed was applied in shake flask cultivations to monitor oxygen and pH levels.
Enzymatic polymer processing was applicable for small-scale cultivations. Lactic acidproduction by Lactobacillus salivarius ssp. salicinius was enhanced four-fold when the proteinswere hydrolyzed either by proteases or by proteolytic microbes. The fed-batch-mimickingcontrolled glucose feeding and growth control were obtained by means of the simultaneousenzymatic hydrolysis of starch-polymer during cultivation. Controlled growth, higher celldensities, decreased side product formation and increased amount of soluble protein product wereobtained in Escherichia coli cultivations. When this method was applied to the cultivation andrecombinant protein production of the methylotrophic yeast Pichia pastoris, higher cell densitiesand higher amounts of active protein were obtained. The glucose concentration remained lowenough to avoid the substrate repression of the alcohol oxidase promoter.
The fed-batch method is suitable for high-throughput cultivations since the method can beutilized in well plate formats without external feeding devices. The method can be utilized in thedevelopment of new biotechnological products, especially when the production system is sensitiveto growth conditions, and growth control is preferred.
Keywords: cultivation conditions, fed-batch, high cell density, high-throughput,recombinant protein, shake flask, well plate
Panula-Perälä, Johanna, Pienen mittakaavan mikrobikasvatuksiin soveltuvanentsymaattisen ravinnesyötön kehittäminen ja soveltaminen. SovelluskohteinaEscherichia coli, Pichia pastoris ja Lactobacillus salivarius -mikrobikasvatuksetOulun yliopiston tutkijakoulu; Oulun yliopisto, Teknillinen tiedekuntaActa Univ. Oul. C 540, 2015Oulun yliopisto, PL 8000, 90014 Oulun yliopisto
Tiivistelmä
Pienen mittakaavan mikrobikasvatusmenetelmiä tarvitaan kehitettäessä uusia bioteknologisiaprosesseja. Tavallisin menetelmä mikrobien liuoksessa tapahtuvaan kasvatukseen on panostyyp-pisesti tehtävä sekoituspullokasvatus. Kuoppalevykasvatukset ovat myös tulleet entistä tärkeäm-miksi, koska tuotekehityksen tehostamiseksi on tarvetta käyttää high-throughput-menetelmiä.Tavoiteltaessa korkeita mikrobisolutiheyksiä ja tuotteen hyvää laatua, panostyyppinen kasvatusei ole usein paras vaihtoehto, johtuen mikrobien rajoittamattomasta kasvusta.
Tämän työn tarkoituksena oli parantaa mikrobien kasvua ja tuotteen muodostusta pienen mit-takaavan kasvatuksissa. Meijeriteollisuuden sivutuotteiden proteiineja pilkottiin entsyymienavulla, jotta maitohappobakteerit pystyivät hyödyntämään proteiinit tehokkaammin ja tuotta-maan enemmän maitohappoa. Hydrolyyttisiä entsyymejä hyödynnettiin myös glukoosin vapaut-tamiseen tärkkelyksestä, jolloin saatiin luotua pieneen mittakaavaan sopiva panossyöttötyyppi-nen kasvatusmenetelmä. Työn aikana kehitettyä langatonta mittausjärjestelmää hyödynnettiinsekoituspullokasvatuksissa happipitoisuuden ja pH:n seurantaan.
Entsymaattinen polymeerien käsittely oli soveltuva menetelmä pienen mittakaavan kasvatuk-siin. Maitohapon tuotto Lactobacillus salivarius ssp. salicinius -mikrobilla nelinkertaistui, kunravinneproteiinit pilkottiin joko proteaasien tai proteolyyttisten mikrobien avulla. Panossyöttö-menetelmää muistuttava hallittu glukoosin syöttö ja mikrobin kasvun hallinta saavutettiin pilk-komalla tärkkelystä glukoosiksi kasvatuksen aikana. Escherichia coli kasvatuksissa saavutettiinhallittu solumäärän kasvu, korkeammat solutiheydet, vähentynyt sivutuotteiden muodostus jasuurempi liukoisen tuoteproteiinin määrä. Tätä menetelmää sovellettiin myös vierasproteiinintuottoon metylotrofisella Pichia pastoris -hiivalla, jolloin saavutettiin korkeammat solutiheydetja suurempi aktiivisen tuoteproteiinin määrä. Glukoosin määrä kasvatusliuoksessa pysyi riittä-vän alhaisena, jotta se ei repressoinut hiivan alkoholioksidaasi-promoottoria.
Panossyöttömenetelmä on sopiva high-throughput-mikrobikasvatuksiin, koska sitä voidaankäyttää kuoppalevyillä ilman syöttölaitteita. Menetelmää voidaan hyödyntää uusien bioteknis-ten tuotteiden kehittämisessä erityisesti silloin, kun tuottoisäntä on herkkä kasvuolosuhteidensuhteen ja mikrobin kasvua halutaan hallita.
Asiasanat: kasvatusolosuhteet, korkea solutiheys, kuoppalevy, panossyöttömenetelmä,sekoituspullo, vierasproteiini
7
Acknowledgements
This doctoral dissertation was started in the Bioprocess Engineering Laboratory
and finalized in the Chemical Process Engineering research group of the Faculty of
Technology at the University of Oulu. Funding from Finnish Academy, Finnish
Funding Agency for Technology and Innovation, University of Oulu Graduate
School, Finnish Foundation for Technology Promotion, and Tauno Tönning
Research Foundation are gratefully acknowledged. The Biocenter Oulu Doctoral
Programme and the follow-up group chaired by Doctor Petri Kursula are
acknowledged for the scientific support. I also thank the industrial partner BioSilta
Ltd. for the scientific and material support for this work. All these partners made
the work possible. I am very grateful for Docent Markku Saloheimo and Doctor
Kristiina Kiviharju for the careful pre-examination of this thesis, and their valuable
comments for the scientific content and writing.
I wish to thank especially the people who have been around me during this
process, whether they have been abroad, at work, or at home, since they have been
“the biocatalysts” for all the scientific (and not so scientific) ideas and thoughts. I
wish to warmly thank Professor Peter Neubauer who gave me an opportunity to
work at Bioprocess Engineering Laboratory. He is a true innovator and gives a great
inspiration to achieve more. Doctor Antti Vasala is greatly and warmly
acknowledged for acting as an advisor already at my undergraduate years in a
laboratory course (which eventually led to the first scientific article that is part of
this thesis), throughout the M.Sc. thesis to the finalization of this dissertation. Your
support has been priceless! Professor Heikki Ojamo is also warmly thanked as he
supervised my thesis to the finals! I wish to thank you for all the advices and
arrangements. The co-authors of the research articles of this thesis are warmly
acknowledged for their valuable scientific input. Doctor Anu Mursula I wish to
thank also for all the support! I also thank my previous colleagues at BPEL, it was
fun, and unforgettable!
Completely new page in my work was turned as I was warmly welcomed to
the Chemical Process Engineering research group. I wish to express my gratitude
to Professor Juha Tanskanen for welcoming me to the group, giving an opportunity
to work in such an inspiring environment! Special thanks also to Doctor Juha Ahola
for all the advices, and Doctor Sanna Taskila for all the discussions we had! My
dear colleagues at CPE group deserves special thanks: it has really not been just a
job, is has been an adventure, as promised!
8
I wish to thank my family, the blood-related and the marriage-related, and my
other friends. You have reminded me that there is life outside of this thesis, and you
are very valuable! My parents Sisko and Juhani, and my brother Janne, you have
always given your solid support! Finally, I wish to thank my husband Juho. You
have always been there for me, with your love and your support, and there is no-
one like you.
Oulu, 17th of June,
Johanna Panula-Perälä
9
Abbreviations
AOX alcohol oxidase enzyme
AOX1 alcohol oxidase 1 promoter
AOX1 alcohol oxidase 1 gene
AOX2 alcohol oxidase 2 gene
API analytical profile index
ATP adenosine triphosphate
BMEB buffered minimal EnBase medium
B. megaterium Bacillus megaterium
BMG buffered minimal medium
BMM buffered methanol medium
E. coli Escherichia coli
FMN flavin mononucleotide
H. polymorpha Hansenula polymorpha
HPLC high performance liquid chromatography
Ks saturation constant [g l-1]
λem emission wavelength [nm]
LB Luria-Bertani broth
L. salivarius Lactobacillus salivarius
M molarity [mol l-1]
MRS De Man-Rogosa-Sharpe broth
mRNA messenger ribonucleic acid
MSM mineral salt medium
Mut- methanol utilization minus
Mut+ methanol utilization plus
MutS methanol utilization slow
OD optical density
OD600 optical density at 600 nm
PAGE polyacrylamide gel electrophoresis
P. pastoris Pichia pastoris
Pphxt1 Pichia pastoris hexose transporter I
Pphxt2 Pichia pastoris hexose transporter II
PVDF polyvinylidene difluoride
ROL Rhizopus oryzae lipase
rpm rotations per minute
SDS sodium dodecyl sulfate
10
sp. species
ssp. subspecies
TCA tricarboxylic acid cycle
Tb Trypanosoma brucei brucei
TIM triosephosphate isomerase
U enzyme activity unit
YDP yeast peptone dextrose medium
Yx/s yield coefficient for glucose
Yx/m yield coefficient for methanol
11
List of original publications
This dissertation is based on the following publications which are referred to in the text by their Roman numerals.
I Vasala A, Panula J, Neubauer P (2005) Efficient lactic acid production from high salt containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre-treatment by proteolytic microorganisms. J Biotechnol 117: 421–431.
II Vasala A, Panula J, Bollók M, Illmann L, Hälsig C, Neubauer P (2006) A new wireless system for decentralised measurement of physiological parameters from shake flasks. Microb Cell Fact 5: 8.
III Panula-Perälä J, Šiurkus J, Vasala A, Wilmanowski R, Casteleijn M, Neubauer P (2008) Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb Cell Fact 7: 31.
IV Panula-Perälä J, Vasala A, Karhunen J, Ojamo H, Neubauer P, Mursula A (2014) Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations. Bioproc Biosyst Eng 37: 1261–1269.
The author’s contributions in the original publications:
I: The author, J. Panula-Perälä (nee Panula), participated in the planning and
implementation of the experiments related to the treatment of dairy side products
with enzymes or B. megaterium and participated in writing the manuscript. II: The
author participated in planning the experiments, carried out the Escherichia coli
cultivations, assisted in all the experiments that were monitored with the wireless
monitoring system by setting up and supervising measurements and data analysis,
and participated in writing the manuscript. III: The author was responsible for
planning most of the experiments, carried out most of the experiments, drafted the
manuscript, and wrote the manuscript in co-operation with the other authors. IV:
The author was responsible for writing the manuscript and planning the
experiments, and carried out most of the experiments.
12
13
Contents
Abstract
Tiivistelmä
Acknowledgements 7
Abbreviations 9
List of original publications 11
Contents 13
1 Introduction 15
1.1 Background ............................................................................................. 15
1.2 Objectives and scope of research ............................................................ 16
2 Literature review 17
2.1 Development of new bioprocesses begins on small scale ....................... 17
2.2 Batch and fed-batch cultivation modes ................................................... 18
2.2.1 Batch operation principle ............................................................. 18
2.2.2 Fed-batch operation principle ....................................................... 20
2.3 Effect of cultivation conditions on metabolism ...................................... 22
2.3.1 Responses of Escherichia coli to cultivation conditions .............. 23
2.3.2 Responses of the methylotrophic yeast Pichia pastoris to
cultivation conditions ................................................................... 27
2.3.3 Lactobacillus salivarius ssp. salicinius, a fastidious lactic
acid bacterium .............................................................................. 30
2.4 Development of small-scale cultivation methods ................................... 31
2.4.1 Problems in small-scale cultivations: low oxygen transfer
and batch-like nature of the culture .............................................. 33
2.4.2 Problems in small-scale cultivations: online measurement
and control .................................................................................... 40
2.5 Enzymatic polymer processing ............................................................... 42
3 Materials and methods 45
3.1 Microbial strains ..................................................................................... 45
3.2 Enzymes .................................................................................................. 45
3.3 Preparation of nutrient storage gels ......................................................... 45
3.4 Microbial cultivations ............................................................................. 46
3.5 Analysis methods .................................................................................... 48
4 Results and discussion 51
4.1 Utilization of enzymatic nutrient release in production of lactic acid by
Lactobacillus salivarius ssp. salicinius (I) .............................................. 51
14
4.2 Cultivation conditions in shake flask cultures of Escherichia coli (II) ... 53
4.3 Development of small-scale fed-batch system (III) ................................ 55
4.3.1 Studies on glucose storing in agar gel .......................................... 56
4.3.2 Starch as a glucose source ............................................................ 57
4.4 Benefits obtained with small-scale fed-batch (III, IV) ............................ 67
4.4.1 Production of recombinant TIM ................................................... 67
4.4.2 Use of enzymatic glucose feeding in the cultivation of
methylotrophic Pichia pastoris producing heterologous
lipase ............................................................................................. 71
4.4.3 Applications benefitting from enzymatically controlled
glucose feeding ............................................................................. 75
5 Conclusions and future perspectives 79
References 81
Original publications 99
15
1 Introduction
1.1 Background
Small-scale bacterial cultivations, such as well plate and shake flask cultivations,
are commonly used in industrial and scientific laboratories in research and
development (Büchs 2001). The methods are used for example in the optimization
of cultivation media and conditions, scale-up, preparation of inoculum cultures, and
production of recombinant proteins. As Büchs (2001) concluded, shake flask
cultivation has several rewards such as simplicity and low cost, and they have been
used in laboratories for several decades (see e.g. Smith & Johnson (1954)). Flasks,
however, require a relatively large amount of hands-on time, and space in
incubators, especially when several parallel cultivations are being done. Well plate
cultivations are attractive because of the capacity to perform several parallel
cultivations in a small area with small medium volumes. When processes like
screening of production hosts for new enzymes or secondary metabolites, a lot of
resources like working time, space, chemicals, and equipment costs, can be saved
if experiments can be done in parallel in a small space.
Small-scale cultivation methods are usually performed as batch cultivation,
where all medium components are added to the medium at the same time (reviewed
e.g. by Scheidle et al. (2010)). Due to the batch-method, small-scale applications
may be limited by low cell densities, oxygen transfer rate, and pH changes (see
Kensy et al. (2005) and Weuster-Botz et al. (2001)). Excess substrate, especially
glucose, induces overflow metabolism and fast oxygen depletion due to a non-
controlled substrate consumption rate and fast respiration (Xu et al. 1999b). During
overflow metabolism or fermentative metabolism, harmful metabolites are formed
and acidification of the medium occurs. Therefore, the cell densities remain low
and the potential of microbes for successful product formation is poor. Furthermore,
false negative results or suboptimal strain selection may be obtained if the
screening is made in a different cultivation mode than the mode used in the
production phase (Graslund et al. 2008, Lattermann & Büchs 2015, Siurkus et al.
2010). If cultivation conditions can be controlled and conditions can be made ideal
for growth, the benefits could be remarkable.
In large-scale controlled bioreactors, fed-batch operation mode based on
substrate limitation is used to avoid the inhibitory conditions related to substrate
and oxygen levels (Johnston et al. 2002, Xu et al. 1999a). This approach requires
16
tools for monitoring (pH, oxygen) and control (feeding pumps). Applying the
substrate-limited fed-batch for small-scale cultures is challenging, due to the small
feeding volumes required (e.g. feeding to 300 µl in the well plate). However, small-
scale methods with the quality of large-scale cultivations would speed up the
development of bioprocesses considerably.
1.2 Objectives and scope of research
The objectives for this research were
– To improve growth and product formation on small scale by improving
cultivation conditions.
– To develop, characterize and apply the fed-batch method to shake flask and
micro plate cultivations in order to reach higher cell densities and improved
product concentrations.
– To control the metabolism of microbes preventing accumulation of harmful
side products.
– To apply the developed method with the industrially and scientifically
important organisms Escherichia coli and Pichia pastoris.
The following research questions are covered in this work. It must be pointed out
that not all the data presented in the research articles are included in the dissertation:
Research article 1: Is in situ enzymatic hydrolysis of proteins a suitable technique
to improve the utilization of dairy side products by a lactic acid bacterium?
Research articles 2 and 3: Why do small-scale cultivations often result in low
product concentrations? What are the cultivation conditions in shake flasks and can
they be monitored easily and efficiently? Can the information received be used in
cultivation design?
Research articles 3 and 4: How can fed-batch system be effectively implemented
in small-scale cultivations? What are the benefits obtained?
Research article 4: Can the problem of starvation between methanol pulses with
methanol utilizing Pichia pastoris be solved with small-scale fed-batch?
17
2 Literature review
2.1 Development of new bioprocesses begins on small scale
The development of a new biological product utilizes small-scale microbial
cultivations. For example, development of a new industrial enzyme can start with
target gene modification by a suitable technique (e.g. random mutagenesis),
continuing to screening and selection of possible improved candidates in small-
scale cultivations. After a suitable construct is found, the production process is
scaled up to the final production process (for reference see e.g. Bhamhure et al.
(2011)). Traditionally, shake flask cultivations have been utilized in the screening
of potential candidates. However, this approach is rather inefficient when several
hundreds of variants have to be cultivated. Therefore, high-throughput cultivations
in the well plate format should be, and are applied. (For review, see e.g. Long et al.
(2014))
Process development from strain modification to final product may take years
to complete. Bareither et al. (2011) presented a process development scheme for
monoclonal antibody production in Pichia. After strain construction, the first
screening steps were done in 96- and 24-well plates, which were then eventually
scaled up to 12 m3. The development process was evaluated as taking from 1.5 to
3 years. As they stated, efficient and comprehensive research would benefit from
high-throughput experimentation. Parallel cultivations enable implementation of
the statistical design of experiments and the production of statistically meaningful
data. As Bareither et al. (2011) emphasized in their review article, process
development, and strain selection and evaluation would benefit from the
quantitative scale-up of growth kinetics and product formation done by parallel
small-scale cultivations that mimic large-scale process conditions. Neubauer et al.
(2013) concluded that large-scale conditions should be utilized already in the early
developmental phase, since growth conditions, growth mode, and the growth
history of the cell has an impact on cell behavior. Consequently, the conventional
batch cultivation protocols, although still widely used, are not optimal methods for
early phase process development.
Various approaches have been used in developing small-scale cultivation
techniques (see chapter 2.4). The miniaturization of batch and fed-batch bioreactors,
development of shake flask cultivation systems, as well as milliliter-scale and
microliter-scale systems have been applied (for a review, see e.g. Lattermann &
18
Büchs (2015)). The need for better cultivation control in shaken cultivations has
been widely recognized. However, improved systems easily lose some of the
simplicity of the standard shaking cultivations.
2.2 Batch and fed-batch cultivation modes
The amount of biomass will increase exponentially unless cell growth is limited.
However, unlimited exponential growth is possible only for a short period of time.
Eventually, factors like nutrient concentration, products, side products, or oxygen
start to limit the cultures and growth rate, and often also the product formation
ceases. The cell metabolism and product formation can be affected by selection of
the operation mode. For example, the oxygen consumption rate and side product
formation can be controlled by controlling the specific growth rate of the cells. This
control can be achieved by adjusting substrate feeding according to the desired
growth rate (see e.g. El-Mansi (2004) and Korz et al. (1995)).
There are several operation modes for microbial cultivations including batch,
fed-batch, and continuous operation modes. The mode is selected depending on the
application and the equipment available. The first two will be discussed in more
detail in sections 2.2.1 and 2.2.2. The continuous operation mode is utilized for
example in the production of single cell protein, in wastewater treatment, and in
the brewing industry (although batch fermentation is the prevalent method in beer
brewing) (Macauley-Patrick & Finn 2008). In continuous cultivation, fresh
cultivation medium flows constantly through the reactor, and the culture volume is
kept constant. As a steady state can be achieved, this mode is also used for studying
cell physiology. This operation mode, however, is not in the scope of this thesis and
therefore it is not described further. The principles of operation modes are well
presented in several books, including Liu (2013) and Doran (2012).
2.2.1 Batch operation principle
The batch cultivation method is a regularly used operation mode in large-scale
cultivations as well as in small-scale. A batch process is a closed system where all
medium components are added at the beginning of the process and the product is
collected at the end. Antifoam and pH controlling agents and gases may, however,
be added during cultivations. The process is typically continued until product
formation slows down or ceases. (Macauley-Patrick & Finn 2008)
19
The batch operation mode is used a lot since it is quite reliable and repeatable
on a large scale (Macauley-Patrick & Finn 2008). The method is easier to control
than fed-batch and continuous processes, and it is well known since it has been
scientifically studied for several decades. On a small scale, the method is easy and
affordable and is therefore attractive to use in research and development (Kumar et
al. 2004). However, due to its simplicity, the batch process has also drawbacks
related to cultivation conditions in the process.
In aerobic processes, the highest oxygen demand occurs usually during the
exponential growth phase (Garcia-Ochoa et al. 2010). The increased oxygen
demand can be compensated by increasing the oxygen available in bioreactors. If
the oxygen transfer capacity is not high enough, the oxygen consumption rate
exceeds the transfer rate, resulting in oxygen depletion (Maier & Büchs 2001).
Oxygen limitation in aerobic cultures causes stress responses, which affect growth
and product formation, as will be discussed in chapter 2.3.
High substrate concentrations should not be used in batch cultivations, due to
their osmotic or toxic effects. High osmotic pressure causes for example cell
shrinking when water leaks out of the cells. The deformation of the cytoplasmic
membrane causes conformational changes in the membrane-related proteins,
resulting in inhibition of the electron transfer chain and transport systems (Houssin
et al. 1991). The concentration of solutes and macromolecules increase in the cell,
causing disruption in cytoplasmic molecular interactions (Cayley & Record 2003).
As a result, growth is inhibited. For example, a glucose concentration of 50 g l-1 is
growth-inhibiting for Escherichia coli (Lee 1996). Methanol is an example of a
toxic substrate. Methanol concentrations higher than 30 g l-1 are inhibitory for
Pichia pastoris (Katakura et al. 1998, Zhang et al. 2000). Reactive oxygen species
are formed during the metabolism of the substrate causing cell damage, as will be
discussed in section 2.3.2. Theoretical cell dry weights (xt) obtained with these
inhibitive substrate concentrations (ci) are 24.0 g l-1 and 16.77 g l-1 (when xt =ci Yx/s,
Yx/g = 0.48 g g-1; Yx/m = 0.559 g g-1, yield values for non-inhibitive substrate
concentrations, Xu et al. (1999a), Maurer et al. (2006), respectively). However, in
practice, the obtained cell dry weights are lower since the yield coefficients
decrease when the maintenance requirement increases, due to the increased osmotic
stress. When product concentration is dependent on cell density, low product
amounts are observed due to the low cell concentrations. High substrate
concentration may also result in unwanted metabolic effects, as discussed in
chapter 2.3.
20
More than 90% of the cultivations done in laboratories are performed in shake
flasks as batch cultivations (Büchs 2001). The simplicity and low cost of the
method makes it attractive for research and development. Controlled feeding to
small-scale cultivations with external pumps requires special equipment, as
concluded by Funke et al. (2010). The small volume restricts the feeding amounts,
and the precise addition of highly concentrated feeding solution to a small volume
is challenging. The external feeding devices also increase the risk of contamination
and raise the operating costs (Long et al. 2014). All these reasons explain the
popularity of the batch operation mode in small-scale work, despite the
disadvantages.
2.2.2 Fed-batch operation principle
The fed-batch operation mode is the technology generally applied to achieve high
cell densities and high product concentrations (Shiloach & Fass 2005). This
operation mode is also utilized when control over the growth rate is required. In
one of the most common methods, control is obtained by keeping one substrate
component at the limiting level (Korz et al. 1995). Consequently, control over
oxygen consumption rate and, at some level, over side product formation (see
chapter 2.4) can be obtained. The limiting substrate is added continuously or
intermittently to the reactor by feeding pumps. However, the fed-batch strategy can
also be utilized with several other control strategies. For example, the substrate
feeding can be set to keep the substrate concentration at a non-limiting level (e.g.
Minning et al. 2001), or to keep the dissolved oxygen concentration at a certain
level (e.g. Hu et al. 2008). The control strategy is selected based on the application
and available instrumentation.
The concentration of the feeding solution should be high to avoid dilution of
the reactor content. For E. coli, the most common substrate is glucose, and it is fed
as a solution with concentrations up to as high as 80% (Korz et al. 1995). For P.
pastoris, typical substrates also include glycerol and methanol.
The fed-batch process usually begins with a batch phase that continues until
the carbon source is exhausted from the growth medium (e.g. Kim et al. 2004, Korz
et al. 1995, Riesenberg et al. 1990). Substrate feed is started and the feed rate is
kept proportional to biomass growth to maintain the desired growth rate and/or to
avoid oxygen becoming the limiting factor (Shiloach & Fass 2005). The growth
rate is adjusted according to the oxygen transfer capacity of the reactor to keep the
21
metabolism respirative, and preferably at the level known to be optimal for product
formation (Illanes 2008a).
Fed-batch operation mode enables high cell density cultures due to the control
over growth (Shiloach & Fass 2005). High cell density cultivation techniques are
economically important, since volumetric productivity increases together with cell
mass concentration. The highest cell density that it is possible to obtain in E. coli
fed-batch cultivation is estimated to be about 220 g l-1 of cell dry weight (Lee 1996).
For E. coli, a cell dry weight of 190 g l-1 has been obtained with fed-batch operation
mode, although it was combined with dialysis (Fuchs et al. 2002, Nakano et al.
1997). For the yeast Pichia pastoris, a cell dry weight of 218 g l-1 has been reported
(Heyland et al. 2010). As concluded by Shiloach & Fass (2005), the highest cell
densities are eventually limited by the oxygen transfer rate, the nutrients available
for the cells, and toxic side product formation.
Mixed feeding strategies
The yeast Pichia pastoris is widely applied in recombinant protein production. The
methylotrophic strains utilize methanol as the carbon source as well as an inducer
for recombinant protein production. In most methylotrophic P. pastoris fed-batch
fermentations, methanol is fed as the only carbon source during induction after
glycerol feeding phases. However, a transition phase with simultaneous feeding of
methanol and glycerol is also used (e.g. Minning et al. 2001, Zhang et al. 2000).
The transition phase allows faster induction and also conditions the cell metabolism
to the new substrate (Jungo et al. 2007). The use of methanol as the only carbon
source is problematic, since methanol and its metabolites are toxic. For this reason,
several studies have been made where mixed feeding was continued during the
induction phase to improve cell and/or product yields. Mixed feeding fermentations
with non-repressing substrates, like sorbitol, alanine, mannitol, trehalose, and yeast
extract, have been performed with success (Celik et al. 2010, Gao et al. 2012,
Guerrero-Olazaran et al. 2009, Inan & Meagher 2001, Niu et al. 2013, Thorpe et al.
1999, Zhu et al. 2013). However, the two most common substrates for P. pastoris,
glycerol and glucose, repress the AOX promoters responsible for methanol usage
even at relatively low concentrations. Nevertheless, several studies indicate that the
derepression of the AOX promoter does not require the complete depletion of
glucose (Boettner et al. 2002) or glycerol (Abad et al. 2010, Holmes et al. 2009,
Jungo et al. 2007). Thus, small amounts of these substrates can be present.
22
2.3 Effect of cultivation conditions on metabolism
Non-optimal growth conditions together with recombinant protein expression
trigger several metabolic responses that may be unfavorable for the product yield
of the process (reviewed by Shiloach & Fass (2005) and Carneiro et al. (2013)).
The capability of the host strain to tolerate these conditions can be altered by
genetic modifications and host strain selection (see e.g. Marisch et al. (2013)), but
it is not always the most suitable or even required option. As Porro et al. (2005)
concluded for yeasts, among some other factors, the designing of a process for
efficient protein production requires the recognition of “the physiological
determinants that maximize the potential of the genetic determinants.” The
selection of operation mode has an effect on metabolism, growth, and product
formation. In general, improved control of the process increases the control over
the metabolism of the microbes (see e.g. Riesenberg et al. (1991)). On the other
hand, a more complicated process will increase the production costs. The selected
operation mode has an effect on the growth behaviour of the microbe and the
growth conditions (e.g. Hewitt et al. (1999) and Xu et al. (1999b)). Therefore,
different metabolic effects are observed with different operation modes depending
on how the operation conditions are controlled.
A wide range of microbes including bacteria (e.g. Escherichia coli, Bacillus
subtilis, Lactobacilli, Lactococcus lactis, Streptomyces spp.), yeasts (e.g. Pichia
pastoris and Saccharomyces cerevisiae), and filamentous fungi (e.g. Penicillium
spp., Trichoderma spp. and Aspergillus spp.) have been applied for scientific and
industrial use. The metabolic and genetic properties as well as the ease of genetic
modification dictate which organism is selected for use in a bioprocess. E. coli, P.
pastoris, and L. salivarius ssp. salicinius were studied in this thesis. E. coli
(prokaryote) and P. pastoris (eukaryote) are widely used hosts for recombinant
protein production and can be grown for high cell densities in production-scale
fermentations using fed-batch operation mode. E. coli has problems related to the
overflow metabolism, which can be minimized by utilizing an appropriate
cultivation mode (see e.g. Xu et al. (1999b)). Recombinant protein production in
methylotrophic P. pastoris is repressed by glucose and glycerol and therefore the
inducer and carbon source, methanol, is fed during the production phase (reviewed
e.g. by Cereghino (2002)). Both of these microbes have specific problems with
batch operation mode when grown on a small scale, as will be reviewed in sections
2.3.1 and 2.3.2. L. salivarius ssp. salicinius is a homofermentative lactic acid
bacterium that tolerates high salt concentrations (Research article I). This makes it
23
a suitable organism for the production of lactic acid from the concentrated by-
products of cheese manufacturing. Since lactic acid is an inexpensive bulk chemical,
its production costs, especially the use of medium supplements, should be
minimized. Production of lactic acid is typically done in batch or fed-batch
cultivations (reviewed by Datta et al. (2006) and Ghaffar et al. (2014)), where all
the necessary amino acids should be available. The need for amino acids is one
cost-increasing factor in the production process and is reviewed in section 2.3.3.
The metabolic responses of the selected microbes, E. coli, P. pastoris, and L.
salivarius ssp. salicinius, to cultivation conditions are discussed in the following
sections.
2.3.1 Responses of Escherichia coli to cultivation conditions
Escherichia coli is the most widely used prokaryote host, especially in the
production of biopharmaceuticals. This microbe is relatively easy to grow in
inexpensive mineral salt-based media, and its genome is well characterized
(Blattner et al. 1997, Hayashi et al. 2006, Jeong et al. 2009). A large number of
mutant strains and cloning vectors are available. E. coli can be grown to high cell
densities by utilizing the fed-batch operation mode, for example. However, the
microbe has limited capability for post-translational modifications and therefore
heterologous protein production is limited to non-glycosylated proteins, although
research into genetically engineered E. coli capable of glycosylation has been
increasing, as reviewed by Jaffé et al. (2014). Additionally, E. coli has a poor
capacity to produce proteins having disulfide bonds, as the cytoplasm of the
bacterium is a reducing environment (reviewed by Messens & Collet (2006),
Saaranen & Ruddock (2013)). Such proteins have to be either targeted to the
periplasm, or alternatively host strains deficient in reductive pathways (Bessette et
al. 1999, Lobstein et al. 2012) or host strains that simultaneously co-express
enzymes for disulfide bond formation (Hatahet et al. 2010, Nguyen et al. 2011)
have to be used.
Overflow metabolism, also known as the bacterial Crabtree effect, occurs
during aerobic growth of E. coli with excess glucose. The glucose flows through
glycolysis until it yields acetyl-CoA. Due to the limited capacity of the citric acid
cycle, not all the carbon continues to the citric acid cycle but part of the flow is
transferred to alternative metabolic routes that produce overflow metabolites –
especially acetate (Fig. 1) (Han et al. 1992, Hollywood & Doelle 1976, Wolfe 2005,
Xu et al. 1999b) as well as to central metabolic intermediates like pyruvate,
24
glucose-6-phosphate, and α-ketoglutarate (Fig. 1), which are excreted to the growth
medium, as shown by Paczia et al. (2012). In general, glucose assimilation and its
utilization for biosynthesis and energy production are not in balance (el-Mansi &
Holms 1989). The phenomenon occurs with excess glucose amounts in the growth
medium, as for instance at the beginning of the batch process or in chemostat
cultures operated at a high dilution rate. Acetate formation increases with high
growth rates (el-Mansi & Holms 1989, Holms 1996, Kayser et al. 2005, Majewski
& Domach 1990) and therefore high acetate production occurs during the
exponential growth phase. When glucose is exhausted, acetate is re-assimilated by
the cells (Andersen & von Meyenburg 1980, Varma & Palsson 1994). Overflow
metabolism can be reduced by running the cultivation with glucose limitation, thus
limiting the growth rate below the threshold value, as reviewed by Eiteman and
Altman (2006). The fed-batch operation mode is suitable for this purpose (Lee 1996,
Luli & Strohl 1990). Acetate accumulation is higher with E. coli K strains than with
E. coli B strains, probably due to the higher capacity for re-assimilation of acetate
by B strains (Phue et al. 2005, Shiloach et al. 1996, van de Walle & Shiloach 1998).
Acetate formation has also been decreased through genetic modification of the
strains. Such approaches include directing the carbon flow from glucose to a
product other than acetate, and reducing the glucose uptake rate. For example, Veit
et al. (2007) increased the specific activities of succinate dehydrogenase, α-
ketoglutarate dehydrogenase, and succinyl-CoA synthetase enzymes in the citric
acid cycle to direct the carbon flux to carbon dioxide instead of acetate and De
Anda et al. (2006) modified the glucose phosphotransferase transport system to
decrease the glucose intake rate.
25
Fig. 1. Schematic pathways of glucose to overflow metabolism end products (yellow
background), mixed acid fermentation end products (blue background), and to the citric
acid cycle (green background). Two molecules of phosphoenolpyruvate are formed
from one glucose molecule. Consequently, two molecules are also further metabolized
(indicated with 2 x). The figure is combined from Paczia et al. (2012), Wolfe (2005), Xu et
al. (1999b), Böck & Sawers (1996), and Cronan & Laporte (1996).
Escherichia coli is a facultative anaerobe. When the culture is limited by oxygen,
energy is produced with mixed acid fermentation (Xu et al. 1999a). Instead of the
citric acid cycle, phosphoenolpyruvate is converted to succinate or pyruvate, and
pyruvate is converted to acetate, lactate, ethanol, formate and H2, for ATP
generation and NAD+/NADH recycling, as presented in Fig. 1 (reviewed by Böck
& Sawers (1996)). Consequently, biosynthesis decreases since the production of
TCA intermediates decreases, including α-ketoglutarate and oxaloacetate, the
precursors of several amino acids. The net energy obtained through fermentative
metabolism is considerably lower compared to aerobic metabolism. Growth of the
cells slows down. Mixed acid fermentation may also occur in aerated culture when
26
the oxygen transfer rate in the growth vessel is not high enough (Xu et al. 1999a).
In such case, cells consume oxygen faster than it can be dissolved into the growth
medium. Anaerobic zones may occur due to inefficient mixing, and consequently
part of the bacterial population utilizes anaerobic metabolism (Xu et al. 1999a).
Overflow metabolism and mixed acid fermentation result in growth medium
acidification due to the organic acids produced. As concluded by Bearson et al.
(1997), weak organic acids permeate the cell membrane in their protonated form
by diffusion. The higher the acidity of the medium, the higher the amount of organic
acids in their protonated form. Decreased internal and external pH causes growth
inhibition and decreased product formation, as reviewed by Eiteman & Altman
(2006). ATP is consumed during proton extrusion. Decreased ion gradients over the
cell wall and increased anion concentrations inside the cell are observed (Zaldivar
& Ingram 1999). Transport through the cell wall and ATP generation are hampered.
Formation of organic acid decreases the product yield since part of the carbon flow
goes to side products. Product formation is also inhibited (Jensen & Carlsen 1990,
Shimizu et al. 1988). However, not all inhibitive effects relate to the decreased pH
since they also occur in pH-controlled cultivations (Jensen & Carlsen 1990, Luli &
Strohl 1990, Nakano et al. 1997).
A non-optimal growth environment and/or protein overexpression cause stress
responses in E. coli. A general stress response is triggered by various different
environmental stresses while specific stress responses are triggered by a specific
condition or smaller variety of conditions. For example, too low or too high
temperature or pH, hyperosmolarity, oxidative conditions, and starvation are
known to cause stress responses, as reviewed by for example Chung et al. (2006)
and Hengge-Aronis (2002). Recombinant protein production causes a high
metabolic load on cells, as concluded in a review by Carneiro et al. (2013) and also
triggers stress responses (Hoffmann & Rinas 2004, Schweder et al. 2002). A heat
shock response is caused not only by temperature up-shift but also by recombinant
protein expression. A stringent response is triggered by the starvation of amino
acids or energy. The purpose of the stress responses is to protect the cell and restore
cell viability and functionality. The general stress response increases the resistance
of the cells against the stress, while specific responses are more reparative
responses, as Hengge-Aronis (2002) concluded. Stress responses cause changes in
cell metabolism, as well as changes in cellular physiology and morphology.
Changes in cell membrane compositions and envelope structure, in DNA
supercoiling and packing are possible (Hengge-Aronis (2002)). The growth rate is
27
reduced or cells enter the stationary phase, recombinant product accumulation
slows down, and the maintenance energy requirement increases.
2.3.2 Responses of the methylotrophic yeast Pichia pastoris to cultivation conditions
The methylotrophic yeast Pichia pastoris is a widely used organism for
heterologous protein production. The yeast has advantages over the other widely
used organism, E. coli, due to its eukaryotic expression system including post-
translational modification systems, such as glycosylation and disulfide bond
formation. Furthermore, Pichia can secrete proteins into the cultivation medium.
This property has been further enhanced by introducing a Saccharomyces secretion
signal for Pichia expression vectors (reviewed by Damasceno et al. (2012)). The
yeast itself secretes only small amounts of endogenous proteins. The genome
sequences of P. pastoris strains CBS7435, GS115, and DSMZ 70382 have been
published (De Schutter et al. 2009, Küberl et al. 2011, Mattanovich et al. 2009,
respectively). The P. pastoris cultivations are robust, and the yeast can be grown in
defined or in complex media (for a review see Cereghino & Cregg (2000)).
P. pastoris is a Crabtree-negative yeast, thus incapable of aerobically
fermenting excess glucose into ethanol as Saccharomyces cerevisiae (De Deken
1966, van Urk et al. 1989). As stated by Mattanovich et al. (2009), the glucose
uptake of Crabtree-negative yeasts is limited as they contain only a few hexose
transporter genes. Consequently, they do not exhibit severe overflow metabolism.
Cell yields achieved with Pichia are much higher than with Crabtree-positive S.
cerevisiae.
P. pastoris is an obligate aerobe. There are, however, cases where oxygen-
limited fed-batch cultivation with unlimited methanol has resulted in good product
quality and productivity compared to methanol-limited fully aerobic cultivations
(Charoenrat et al. 2005, Trentmann et al. 2004). However, improved results are not
always obtained with this technique when compared to fully aerobic culture, as
shown in the case of recombinant mouse endostatin (Trinh et al. 2003). The total
production was at the same level with both techniques, although the specific
production per methanol was higher with unlimited predefined methanol feed.
The methanol-inducible alcohol oxidase promoter of the AOX1 gene is the
most commonly used promoter for recombinant protein expression in P. pastoris.
Alcohol oxidase enzymes are encoded by two genes, AOX1 and AOX2, of which
AOX1 is responsible for more than 90% of the enzyme in the cell, as reviewed by
28
Cos et al. (2006). The capability of the P. pastoris host strain to utilize methanol
depends on whether it is the methanol utilization plus (Mut+), methanol utilization
slow (MutS), or methanol utilization minus (Mut-) phenotype, as reviewed by
Macauley-Patrick et al. (2005). Deletion of the AOX1 gene in the MutS type results
in slower methanol utilization and slower growth. The Mut- type is unable to grow
on methanol due to deletion of both the AOX1 and AOX2 genes.
Alcohol oxidase is the first enzyme in the methanol utilization pathway, and it
can represent up to 30% of the total soluble protein in cells growing on methanol
(Couderc & Baratti 1980). The strong AOX1 promoter is repressed by glucose and
glycerol, and the repression is relieved upon glucose/glycerol limitation or
starvation. The promoter is fully induced when methanol is utilized as the only
carbon source. Consequently, the growth medium is exchanged for a glucose- and
glycerol-free medium prior to induction in shaken cultures (Boettner et al. 2002,
Invitrogen 2010). The change of the growth substrate causes stress since cells have
to adapt their metabolism to the new substrate. In shaken cultures, methanol is
usually delivered manually by adding 0.5% final concentration once or twice per
day (Boettner et al. 2002). In bioreactors, methanol-induced stress can be alleviated
by applying a transfer phase in order to accustom the cells gradually to the more
toxic methanol substrate, as reviewed in section 2.2.2. This is not easy to perform
in shaken cultures without external feeding devices. However, complete
glucose/glycerol starvation is not required for derepression of the AOX1 promoter,
and mixed feeding with glucose and glycerol has been utilized, as reviewed in
section 2.2.2. Mixed feeding often results in increased cell densities and increased
protein yields for both Muts and Mut+ strains (for a review see Cos et al. (2006)).
For example, the glycerol/methanol mixed feeding strategy has been successful in
the production of human β2-glycoprotein I domain V (Katakura et al. 1998) and
trimeric CD40 ligand (Mcgrew et al. 1997) in P. pastoris GS115, where higher
specific growth rates and production rates were observed. However, the effect of
mixed feeding is clearly dependent on the application. Abad et al. (2010) obtained
40% higher biomass yield with glucose-methanol and glycerol-methanol mixed
feeding strategies compared to pure methanol feed in P. pastoris Tv1_mc
cultivations but the volumetric yield [U l-1] of the produced recombinant protein
was higher with a pure methanol feed.
The mechanisms of catabolite repression in P. pastoris are not yet well known.
As concluded by Weinhandl et al. (2014), only two hexose transporters in P.
pastoris are known, Pphxt1 and Pphxt2. Zhang et al. (2010) proposed that the
Pphxt1 transport system is directly involved in AOX1 repression. The glucose
29
repression of AOX1 did not occur in the strain where Pphxt1 gene was deleted. The
induction of the transporters in the wild-type strain was dependent on the glucose
concentrations. While Pphxt2 mRNA was fully induced in cells growing on less
than 1 g l-1 of glucose in the growth medium, Pphxt1 was induced only to a level
of one third compared to cultures growing on 5 g l-1 of glucose. Therefore, it was
suggested that different hexose transporters are expressed depending on the glucose
concentrations. Pphxt1 is expressed at higher and Pphxt2 at lower glucose
concentrations.
Methanol as an inducer and carbon source poses its own challenges for
cultivation. Methanol induces cell lysis and, consequently, increased proteolytic
activities and decreased product yields are obtained (Jahic et al. 2003). Methanol
is growth-inhibiting in concentrations higher than 3% (Katakura et al. 1998),
although Zhang et al. (2000) reported negative effects on growth even for
0.365% methanol. Curvers et al. (2001) observed that the productivity of the cells
falls immediately after cells are exposed to toxic levels of methanol, and the cells
revert to the wild-type growth characteristics. In shaken cultures, methanol is
usually added as pulses at 12 h or 24 h intervals (Boettner et al. 2002). This,
however, results in starvation phases between pulses, as observed in Research
article II and later confirmed by Ruottinen et al. (2008), since only a limited amount
of methanol can be added to the cultivation due to its toxic effects. The effects of
carbon starvation of P. pastoris at the metabolic level have not yet been studied.
However, Ruottinen et al. (2008) observed increased product yields in shake flasks
by avoiding methanol starvation through applying continuous methanol feeding.
Methanol utilization by methylotrophic yeasts, as well as disulfide bond
formation during protein folding, causes oxidative stress by creating reactive
oxygen species. Disulfide bond formation is an oxidative folding process, creating
reactive oxygen species that may eventually cause cellular damage or cell death
(Haynes et al. 2004, reviewed by Kincaid & Cooper (2007)). Hydrogen peroxide is
formed during methanol utilization in peroxisomes. Alcohol oxidase (AOX)
oxidize methanol to toxic formaldehyde and simultaneously reduce oxygen to
hydrogen peroxide (Couderc & Baratti 1980). Formaldehyde is dissimilated to
carbon dioxide or assimilated in cell metabolism (reviewed by Hartner & Glieder
(2006)). Hydrogen peroxide is further oxidized to oxygen and water by catalase.
However, the oxygen radicals in hydrogen peroxide react with the peroxisomal
membrane, resulting in alkyl hydroperoxide formation with further oxidation by
glutathione peroxidase to alkyl alcohol (Horiguchi et al. 2001). Yano et al. (2009)
showed severe defects in P. pastoris growth due to oxidative stress by altering the
30
genes related to the detoxification of oxygen radicals and formaldehyde. Growth
on methanol increases the metabolic burden of the cells.
2.3.3 Lactobacillus salivarius ssp. salicinius, a fastidious lactic acid bacterium
Unlike E. coli or P. pastoris, Lactobacilli are anaerobic or aerotolerant organisms
that obtain energy by fermentation. Overflow metabolism is thus not an issue for
these microbes. Lactobacillus salivarius ssp. salicinius is a homofermentative
lactic acid bacterium producing lactic acid as the main fermentation product (Li et
al. 2006, Rogosa et al. 1953). This probiotic microbe produces bacteriocins
(Arihara et al. 1996) that have potential applications in the food industry
(Messaoudi et al. 2013). Lactic acid is a natural end product of the energy
metabolism of the microbe. The organic acid is secreted to the environment. As
concluded by Panesar et al. (2007), the produced lactic acid has to be neutralized
to avoid a major pH decrease, which would inhibit the growth of the microbe. The
optimal fermentation pH depends on the strain, varying from 5.5 to 6.5, and
fermentation is strongly inhibited below pH 4.5. Many lactic acid bacteria respond
to an external pH decrease by decreasing the intracellular pH to maintain a constant
pH gradient (Hutkins & Nannen 1993, Shabala et al. 2006, Siegumfeldt et al. 2000),
whereas e.g. E. coli maintains near neutral pH in cytosol (Slonczewski & Foster
1996). The mechanism of lactic acid bacteria reduces the energy needed for proton
translocation from cytosol. However, when the concentration of lactate increases,
or the pH of the medium decreases, the concentration of protonated lactic acid also
increases. Protonated lactic acid diffuses through the cell membrane and dissociates
in more neutral cytosol (Bearson et al. 1997). After the intracellular buffering
capacity is exceeded, a decrease in the intracellular pH results in failures in cellular
functions (Hutkins & Nannen 1993). In addition, as reviewed by Carpenter &
Broadbent (2009), the accumulation of acid anions may inhibit cell growth to a
higher extent than proton accumulation.
Lactic acid bacteria are demanding organisms in relation to the growth medium,
and it is therefore technically challenging to prepare the fermentation broth. While
E. coli can synthesize all essential amino acids, lactic acid bacteria have to obtain
the amino acids from the growth medium. (Pritchard & Coolbear 1993). Lactic acid
bacteria have complex proteolytic systems for processing proteins into smaller
fragments that can be transported through the cell membrane (Kunji et al. 1996).
31
The bacteria have a transport system for oligopeptides containing up to eight amino
acid residues as well as transport systems for amino acids (Tynkkynen et al. 1993).
The proteolytic system of several lactic acid bacteria is unfortunately
inefficient. Therefore, several studies have been done to improve the use of whey
in lactic acid production, as reviewed by Panesar et al. (2007). Whey is a side
product from the cheese manufacturing process. It contains high concentrations of
lactose as well as proteins, lipids, and salts. The two most abundant proteins in cow
milk whey are β-lactoglobulin and α-lactalbumin (Smithers et al. 1996). Different
Lactobacillus species have different capabilities for hydrolyzing these two proteins.
For example, in the research of Tzvetkova et al. (2007) and Pescuma et al. (2008),
α-lactalbumin was hydrolyzed more efficiently than β-lactoglobulin. Generally, the
proteolytic mechanism of most lactic acid bacteria is not efficient enough to support
fast growth and lactic acid formation. The growth and production of lactic acid can
be improved by adding an easily assimilated amino acid source like yeast extract
to whey (Aeschlimann & Stockar 1990, González et al. 2007). Protein lysates as
well as enzymatically hydrolyzed whey proteins have also increased the yield
(Amrane & Prigent 1993). The use of such additives however increases the
production costs.
2.4 Development of small-scale cultivation methods
Development of small-scale cultivation methods has continued ever since
microbial cultivations were started in laboratories in the 19th century. For example,
heat sterilization by Pasteur, the use of cotton for closing flasks to prevent medium
contamination by Schröder and von Dusch (Block 2001), and the development of
agar containing solid cultivation media by Hesse (Madigan et al. 2012) eventually
enabled the use of pure cultures in biotechnology laboratories. In the 20th century,
knowledge related to cultivation conditions, especially oxygen demand, was further
increased. The effect of baffles and flask closure on oxygen transfer and microbial
growth was studied in the 1960s (McDaniel et al. 1965, McDaniel & Bailey 1969,
Schultz 1964). The problems related to small-scale cultivation have been fully
recognized by a large group of scientists during the last two decades. Recently, the
development has been intensive and progress has been made in developing
measurement systems, in improving the controllability of small-scale cultivation,
as well as in developing the cultivation methods (for reviews see Betts & Baganz
(2006) and Lattermann & Büchs (2015)).
32
The interest shown towards high-throughput process development in
biotechnology is increasing. High-throughput methods include a rapid process
development environment, use of robotics, data processing and control software,
liquid handling devices, and sensitive detectors. Miniaturization, automation, and
parallelization are required in the design of a high-throughput process. (For reviews
see Bhambure et al. (2011) and Long et al. (2014)) Miniaturization saves the space
and materials required. Parallelization of the experiments for gene cloning,
screening of expression strains, and media optimization enhances process
development and improves the reliability of the experimental data. Automation and
computation are needed for handling the large amount of experimental data
gathered from miniaturized parallel experiments. However, small scale sets
additional demands on cultivation methods. Therefore, several protocols to
improve cell yields and/or controllability in small-scale cultivation have been
implemented (Funke et al. 2010, Huber et al. 2009a, Jeude et al. 2006, Krause et
al. 2010, Ruottinen et al. 2008, Sanil et al. 2014). These methods have been
developed to overcome the known problems in nutrient feed, pH variation, and
scale-up.
Well plate cultivation formats, from microwell plates to deepwell plates
(volumes generally from scales of 10 µl to 10 ml), have increased their importance
in research and development together with improved robotics, development in
bioinformatics, and improved methods for high-throughput screening and strain
modification (concluded from Bhambure et al. (2011), Bornscheuer et al. (2012),
Huber et al. (2009a) and Long et al. (2014)). The well plate formats are attractive
due to their high capacity for parallel cultivations. If parallel cultivations can be
made with good repeatability and cultivation conditions comparable to large-scale
cultivations, the benefits in research and development may be considerable.
Cell densities and product concentrations tend to remain relatively low in
traditional shake flask cultivations, including Erlenmeyer’s and baffled shake
flasks (volumes generally from scales of 10 ml to 2 l). Several problems related to
the small scale have been recognized (see e.g. Büchs (2001), Losen et al. (2004)
and Kunze et al. (2014)):
– Problems related to oxygen transfer
– Batch-like nature of the cultures
– Problems related to online measurements and control
Several solutions are presented in the literature to solve or avoid these problems
(reviewed in sections 2.4.1 and 2.4.2). Most of the effort has been focused on
33
solving the problems related to oxygen transfer. Since the 1950s, oxygen transfer
in shaken scales and/or the effect of oxygen limitation have been studied
excessively (Büchs 2001, Corman 1957, Duetz & Witholt 2004, Giese et al. 2013,
Gupta & Rao 2003, Hermann et al. 2003, Kensy et al. 2005, Maier & Büchs 2001,
McDaniel et al. 1965, McDaniel & Bailey 1969, Smith & Johnson 1954, Ukkonen
et al. 2011, Ukkonen et al. 2013b).
2.4.1 Problems in small-scale cultivations: low oxygen transfer and batch-like nature of the culture
Oxygen transfer in shake flasks and well plate formats differs greatly from oxygen
transfer in fermenters. In shaken cultures, oxygen transfer to the medium is
provided by surface aeration powered by the shaker, while in bioreactors oxygen is
sparged to the reactor vessel. As early as the 1970s, Van Suijdam et al. (1978)
concluded that oxygen transfer across the gas-liquid interface can be a bottleneck
in shake flasks. Flask and well geometry as well as filling volume, surface tension
in the well, shaking speed, and shaking diameter have a high impact on the oxygen
transfer rates (Anderlei et al. 2007, Corman 1957, Duetz & Witholt 2001, Duetz &
Witholt 2004, Giese et al. 2013, Hermann et al. 2003, McDaniel et al. 1965,
McDaniel & Bailey 1969, Smith & Johnson 1954, Van Suijdam et al. 1978). Even
though these parameters are known, the oxygen transfer capacity of the vessel is
not always sufficient and cannot be altered to maintain the aerobic growth
conditions in batch cultures. The batch-like nature of shaken cultivations causes
exponential growth, resulting in oxygen depletion when the oxygen transfer
capacity is not sufficient (see e.g. Ferreira-Torres et al. (2005), Ge & Rao (2012)
and Kensy et al. (2005)). In practice, this problem often remains unrecognized, as
the oxygen level is not commonly monitored on small scale. Researchers tend to
further impair aeration by wrapping the flask closure tightly with aluminum foil.
The high nutrient concentrations used may also cause osmotic stress and metabolic
effects like overflow metabolism and/or the Crabtree effect, as reviewed in chapter
2.3.
Controlled substrate feeding allows control over the growth rate and thereby
over the oxygen consumption rate. Additionally, the high substrate concentration
present in batch cultures can be avoided. Several applications have been developed
for shaken cultures where the batch operation mode have been replaced by more
controlled substrate feeding and/or increased aeration (Table 1). Such methods can
be implemented with or without external feeding devices. An intermittent feeding
34
system with pH control using external feeding devices was developed for shake
flasks by Weuster-Botz et al. (2001). This system supported a maximum of 16
parallel shake flasks at the same time. Individual feeding of pH controlling agent
and substrate to each flask was facilitated by means of miniature pinch valves and
a piston pump. A process computer was needed to control the intermittent feeding.
Higher cell concentrations were obtained when compared to non-controlled batch
cultures. Miniaturized bioreactors have also been developed for milliliter and
microliter scales (Maharbiz et al. 2004, Puskeiler et al. 2005, Szita et al. 2005).
Puskeiler et al. (2005) developed milliliter scale reactors for fed-batch operation
having up to 48 parallel cultivations. The system was equipped with automated
substrate feeding, gas inducing impellers, and automated at-line pH and optical cell
density monitoring. With this system, a cell dry weight of 20.5 g l-1 of E. coli K-12
was reached in 5 ml bioreactors in 16 h of cultivation time. Maharbiz et al. (2004)
developed microbioreactors containing eight 250 μl reactors including continuous
monitoring of optical density by scattered light measurement, temperature control,
and electrodes for electrolytic oxygen generation for gas dosing. However, this
system was operated as a batch. Bähr et al. (2012) developed a dialysis shake flask
for fed-batch cultivations without external feeding devices. Glucose feeding was
diffusion-driven from a specially designed feeding reservoir attached to the flask.
Catabolite repression of product formation was avoided, and reduced medium
acidification and overflow metabolism were obtained.
External feeding devices, customized flasks, and miniaturized bioreactors are
interesting and useful devices in research, but they are laborious to use and
expensive when applied to hundreds of parallel cultivations. Wilming et al. (2014)
developed microwell plates with internal feeding chambers for high-throughput
screening. The plates allow 44 parallel fed-batch cultivations and can be used with
common well plate shakers. This method, however, still requires specially prepared
plates and is therefore not available for researchers without suitable manufacturing
devices. There have been several interesting attempts for more controlled
cultivations with simpler solutions, mainly utilizing in situ storage systems. In the
method of Jeude et al. (2006), glucose was continuously released to the growth
medium from glucose-containing silicone discs added in the growth medium in
shake flasks. Improved biomass yield and green fluorescent protein expression by
the yeast Hansenula polymorpha pC10-FMD was obtained compared to batch
cultivation. Later, Huber et al. (2009b) implemented the same principle for well
plate formats by pouring a glucose-containing silicone layer to the bottom of the
wells. Scheidle et al. (2011) used silicone discs for storing alkaline sodium
35
carbonate for pH control. Sodium carbonate was gradually released from the discs
to compensate the pH decrease caused by microbial activity in the cultivation. Sanil
et al. (2014) had a pH-responsive base release in E. coli cultivations to control
medium acidification. They added magnesium hydroxide-loaded hydrogel discs to
cultivations in a shake flask. As the solubility of magnesium hydroxide increases
with decreasing pH, more of the base was released as a response to the pH decrease
in the cultivation. During E. coli K-12 cultivation in an LB-glucose medium, the
pH levels were maintained at between 6 and 8. Without control, the pH decreased
to 5. They demonstrated the system in plasmid production by E. coli in which they
also utilized the glucose-releasing hydrogels presented in a previous study for
mammalian cell cultivations (Hegde et al. 2012). Plasmid production increased 4-
fold compared to a system without pH control. Lübbe et al. (1985) used ethylene-
vinyl acetate copolymer beads for the controlled release of ammonia for
Streptomyces clavuligerus fermentation in shake flasks and obtained improved
cephalosporin production. The biphasic approach for nutrient delivery had already
been used in 1950s, when nutrients were concentrated in a separate phase to
increase cell numbers in shake flask cultivations. Gorelick et al. (1951) used a
cellophane sack to separate the culture medium from the nutrient reservoir and
obtained higher cell numbers than purely liquid medium based cultivations. Tyrrell
et al. (1958) used a layer of solid nutrient medium overlaid with a small volume of
nutrient broth for microbial cultivations. They obtained 2-30 times higher cell
numbers compared to broth only. However, their purpose was not to control the
growth but to combine agar-plate cultivations (diffusional access to nutrients) and
submerged cultivations (homogenous cells in cultures) to increase cell yields.
A purely liquid fed-batch-like cultivation method without external feeding
devices has been presented by Krause et al. (2010). They used a controlled release
of glucose to the growth medium from a glucose polymer. Glucose was released
enzymatically from the polymer, and the release rate was controlled by the amount
of the enzyme. The complex additives in the growth medium were also metabolized,
resulting in intrinsic pH control due to the ammonia released to the medium. The
yield of correctly folded recombinant proteins (R-alcohol dehydrogenase from
Lactobacillus and multifunctional enzyme type 2 from Drosophila) produced in E.
coli was improved in comparison to cultivations in LB, Terrific Broth, or a mineral
salt medium. Improved pH control and 2-5 times higher optical cell densities were
also obtained.
36
Ta
ble
1. S
olu
tio
ns
fo
r im
pro
ved
an
d m
ore
co
ntr
olled
cu
ltiv
ati
on
co
nd
itio
ns
fo
r sm
all
-sc
ale
mic
rob
ial c
ult
iva
tio
ns
.
Applic
atio
n
Resu
lt P
ros
Cons
Refe
rence
Inte
rmitt
ent
feedin
g
and p
H c
ontr
ol i
n E
.
coli
sha
ke fla
sk
culti
vatio
ns.
Inte
rmitt
ent fe
edin
g thro
ugh s
yrin
ge p
um
p to the fla
sk w
as
imple
mente
d. In
crease
d a
ero
bic
cell
conce
ntr
atio
ns
com
pare
d to
batc
h w
ere
obta
ined. A
vera
ge C
DW
of 5.1
g l-1
wa
s re
ach
ed a
fter
12 h
of cu
ltiva
tion.
Up to 1
6 p
ara
llels
poss
ible
.
Indiv
idua
l feedin
g
pro
files
for
each
flask
. C
ontr
olle
d
gro
wth
poss
ible
.
Requires
ext
ern
al f
eedin
g
syst
em
. N
o s
imulta
neous
feed
ing
to fla
sks.
Inte
rmitt
ent fe
edin
g
cause
s osc
illatio
ns
in o
xygen
tensi
on.
Weust
er-
Bo
tz e
t al.
(20
01
)
Fe
d-b
atc
h o
pe
rate
d
ml-sc
ale
aera
ted
bio
rea
cto
rs f
or
E.
coli
culti
vatio
ns
with
pH
and O
D m
on
itorin
g.
Inte
rmitt
ent fe
edin
g o
f glu
cose
was
imple
mente
d b
y au
tom
ate
d
pip
ettin
g. In
crease
d s
urf
ace
aera
tion w
as
imple
mente
d w
ith fre
e-
floatin
g m
agn
etic
impelle
r. A
uto
mate
d a
t-lin
e m
easu
rem
ent
of
pH
and O
D w
as
util
ize
d. C
DW
of 20.5
g l-1
was
obta
ined in
16 h
of
culti
vatio
n. U
p to 4
8 p
ara
llels
poss
ible
.
Auto
mate
d
sam
plin
g a
nd
feedin
g. C
ontr
olle
d
gro
wth
poss
ible
.
Requires
speci
fic r
eact
ion b
lock
and s
epara
te li
quid
handlin
g
syst
em
for
sam
plin
g a
nd feedin
g.
Inte
rmitt
ent fe
edin
g m
ay
cause
osc
illatio
ns
in o
xyg
en tensi
on.
Pu
ske
iler
et
al.
(20
05
)
Ba
tch
op
era
ted
µl-
scale
bio
rea
ctors
for
E. co
li cu
ltiva
tions
with
pH
and O
D
monito
ring,
tem
pe
ratu
re c
on
tro
l,
and e
lect
roly
tic o
xygen
genera
tion.
Oxy
gen, ge
nera
ted in
ele
ctro
lyte
cha
mbers
set belo
w e
ight para
llel
250 µ
l mic
robio
rea
ctors
, w
as
added t
o the r
eact
ors
thro
ugh a
gas-
perm
eable
sili
cone
mem
bra
ne in
sert
ed b
etw
een th
e e
lect
roly
te
cham
ber
and the m
icro
react
ors
. O
ptic
al d
ensi
ty, te
mpe
ratu
re,
oxy
gen in
put, a
nd p
H w
ere
follo
we
d.
Oxy
gen tra
nsf
er
of
40 m
mol O
2 h
-1l-1
was
obta
ined.
Gas
dosa
ge
can b
e
independently
contr
olle
d. M
ulti
ple
gase
s ca
n b
e
ge
ne
rate
d.
Additi
onal s
enso
r
can b
e a
dded t
o th
e
syst
em
.
Requires
a s
peci
al s
yste
m to
opera
te. T
he s
ilico
ne m
em
bra
ne
bulg
es
due t
o g
as
flow
decr
easi
ng th
e c
ulti
vatio
n
volu
me. B
ubble
s co
ale
sce a
t th
e
mem
bra
ne s
urf
ace
unle
ss the
react
or
is s
tirre
d.
No c
arb
on
subst
rate
feedin
g.
Maharb
iz
et
al.
(20
04
)
37
Applic
atio
n
Resu
lt P
ros
Cons
Refe
rence
Dia
lysi
s sh
ake
fla
sk for
fed-b
atc
h c
ulti
vatio
ns
of E
scherich
ia c
oli
and
Hanse
nu
la
poly
morp
ha
The s
hake
fla
sk w
as
equip
pe
d w
ith a
feedin
g r
ese
rvo
ir. T
he
rese
rvoir in
clu
ded a
rota
ting feedin
g t
ip w
ith a
n u
ltrafil
tra
tion
me
mb
ran
e f
or
con
tinu
ou
s g
luco
se d
iffu
sio
n f
rom
th
e r
ese
rvo
ir t
o
the g
row
th m
ediu
m. C
ata
bolit
e r
epre
ssio
n o
f th
e p
rod
uct
form
atio
n
pre
sent in
the r
efe
rence
batc
h c
ulti
vatio
n w
as
avo
ided w
ith b
oth
mic
roorg
anis
ms.
Reduce
d m
ediu
m a
cidifi
catio
n a
nd o
verf
low
meta
bolis
m w
as
obta
ined. E
. co
li C
DW
of ~
8 g
l-1 a
fte
r 2
2 h
of
glu
cose
fed-b
atc
h c
ulti
vatio
n w
as
rep
ort
ed.
Equip
ment
can b
e
use
d w
ith c
om
mon
shaki
ng in
cubato
rs.
The m
eth
od c
an b
e
use
d w
ith s
eve
ral
solu
ble
nutr
ients
.
Counte
r diff
usi
on o
f w
ate
r to
feedin
g r
ese
rvo
ir.
A s
peci
al s
hake
flask
and fe
edin
g r
ese
rvoir
needed. T
he s
ize o
f th
e d
iffusi
on
mem
bra
ne t
ip h
as
to b
e a
dju
ste
d
acc
ord
ing to th
e s
haki
ng s
pe
ed.
Bä
hr
et
al.
(20
12
)
Modifi
ed m
icro
well
pla
tes
for
fed
-ba
tch
culti
vatio
ns
of E
. co
li
and H
. poly
morp
ha.
A m
odifi
ed m
icro
well
pla
te w
as
de
sig
ned for
44 p
ara
llel f
ed-b
atc
h
culti
vatio
ns.
A c
ha
nnel w
as
mill
ed b
etw
een the r
ese
rvoir
and
culti
vatio
n w
ell,
an
d fill
ed w
ith h
ydro
gel t
o a
llow
diff
usi
onal f
eedin
g
of su
bst
rate
to the c
ulti
vatio
n w
ell.
Th
e feedin
g r
ate
was
adju
sted
by
changin
g the fe
edin
g s
olu
tion c
on
centr
atio
n, hyd
rog
el,
and/o
r
geom
etr
y of
the c
hannel.
Four-
fold
hig
her
pro
duct
form
atio
n w
as
obse
rved in
E.
coli
culti
vatio
ns
com
pare
d to b
atc
h. In
H.
poly
morp
ha
cu
ltiva
tions,
cata
bolit
e r
epre
ssio
n w
as
relie
ved a
nd
pro
duct
was
form
ed in
fed-b
atc
h c
ulti
vatio
n.
Su
itab
le f
or
seve
ral
diff
ere
nt
nutr
ients
.
Ea
sy t
o u
se a
fte
r
pla
te h
as
be
en
pre
pare
d.
Can b
e
use
d w
ith c
om
mon
mic
row
ell
pla
te
shake
rs.
Half
of th
e w
ells
in the p
late
s are
rese
rve
d f
or
nu
trie
nts
. P
late
manufa
cturing r
eq
uires
speci
al
equip
ment. A
dju
stm
ent
of
the
feedin
g r
ate
is c
om
plic
ate
d w
hen
it has
to b
e d
one b
y ch
angin
g t
he
pla
te c
hara
cteri
stic
s. W
ate
r
cou
nte
r d
iffu
sio
n t
o t
he
re
serv
oir
well
reduce
s cu
ltiva
tion v
olu
me.
Wilm
ing
et
al.
(20
14
)
Glu
cose
-rele
asi
ng
silic
on
e d
iscs
for
H.
poly
mo
rph
a f
ed-b
atc
h
culti
vatio
ns
in s
ha
ke
flask
s.
Glu
cose
cry
stals
were
sto
red in
sili
cone e
last
om
er
dis
cs a
s
nutr
ient su
pply
. G
luco
se r
ele
ase
was
contr
olle
d b
y ch
angin
g the
thic
kness
of th
e d
isc
and the n
um
ber
of dis
cs.
Reduce
d o
verf
low
meta
bolis
m d
ue to
decr
ease
d g
luco
se c
once
ntr
atio
ns
was
obse
rved. G
reen f
luore
scent pro
tein
yie
lds
were
incr
ea
sed fro
m 3
5
to 4
20 tim
es
com
pare
d to b
atc
h.
No e
xtern
al f
eedin
g
devi
ces
needed.
Larg
e w
idth
of th
e d
iscs
com
pare
d t
o g
luco
se s
tora
ge
am
ou
nt.
Th
e a
dju
stm
en
t o
f
feedin
g r
ate
is r
est
rict
ed b
y th
e
diff
usi
ona
l rele
ase
chara
cterist
ics
of th
e d
iscs
.
Jeude e
t
al.
(20
06
)
38
Applic
atio
n
Resu
lt P
ros
Cons
Refe
rence
So
diu
m c
arb
on
ate
rele
asi
ng p
oly
mer
dis
cs for
pH
-contr
olle
d
E. co
li cu
ltiva
tions
in
shake
fla
sk.
Sodiu
m c
arb
onate
was
store
d in
poly
mer
dis
cs for
pH
contr
ol.
Conse
que
ntly
, th
e b
uffer
conce
ntr
atio
ns
in the g
row
th m
ediu
m
were
reduce
d t
o h
alf
or
zero
depend
ing o
n t
he c
arb
on s
ourc
e
use
d. T
he p
H w
as
kept at phys
iolo
gic
al l
eve
l.
No e
xtern
al f
eedin
g
devi
ces
needed.
Buffer
usa
ge w
as
reduce
d.
Non-c
ontr
olle
d g
row
th. D
iscs
cannot be a
uto
cla
ved. T
he
adju
stm
ent
of
the b
uffer
feedin
g
rate
is r
est
rict
ed b
y th
e d
iffusi
onal
rele
ase
chara
cteri
stic
s of th
e
dis
cs.
Sch
eid
le
et
al.
(20
11
)
Magnesi
um
hyd
roxi
de
rele
asi
ng h
ydro
gel
dis
cs for
pH
-contr
olle
d
E. co
li cu
ltiva
tions.
Magnesi
um
hyd
roxi
de w
as
store
d in
poly
mer
dis
cs for
pH
contr
ol.
The s
olu
bili
ty o
f M
g(O
H) 2
incr
ease
d a
s th
e p
H in
the c
ulti
vatio
n
decr
ease
d, allo
win
g p
H r
esp
on
sive
contr
ol.
The m
eth
od w
as
test
ed in
E.
coli
TO
P10 c
ulti
vatio
n f
or
pla
smid
pro
du
ctio
n r
esu
lting
in a
four-
fold
incr
ease
in v
olu
metr
ic p
lasm
id y
ield
. In
E.
coli
K-1
2
culti
vatio
n in
LB
-glu
cose
mediu
m, pH
was
main
tain
ed b
etw
een 6
-
8, as
in r
efe
rence
pH
decr
ease
d to 5
. A
n O
D (
550 n
m)
of 14 w
as
report
ed fro
m this
culti
vatio
n.
No e
xtern
al f
eedin
g
devi
ces
needed.
pH
-contr
olli
ng a
ge
nt
rele
ase
d a
ccord
ing
to the d
ecr
easi
ng
pH
.
Sta
ggere
d a
dditi
on o
f dis
cs
needed to a
void
to
o h
igh b
ase
rele
ase
at th
e b
egin
nin
g. T
oo
hig
h p
H in
crease
ove
rall
poss
ible
.
Sanil
et
al.
(20
14
)
Po
lym
er
be
ad
s fo
r
am
moniu
m s
tora
ge
and feedin
g in
Str
ep
tom
yce
s
clavu
ligeru
s
culti
vatio
ns
in s
ha
ke
flask
s.
Am
moniu
m s
tore
d in
poly
mer
bea
ds
as
nitr
ogen s
ourc
e. Im
pro
ved
cephalo
sporin p
roduct
ion w
as
obta
ined c
om
pare
d to b
atc
h
culti
vatio
n w
ith fre
e a
mm
oniu
m.
No e
xtern
al f
eedin
g
devi
ces.
Am
ount
of
am
moniu
m a
dded
to the c
ulti
vatio
n
can b
e in
crea
sed
with
out in
hib
itive
effect
s co
mpare
d t
o
traditi
onal b
atc
h.
Beads
cannot be a
uto
clave
d.
Lübbe e
t
al.
(19
85
)
39
Applic
atio
n
Resu
lt P
ros
Cons
Refe
rence
Cello
ph
ane s
ack
as
a
nutr
ient
mediu
m
stora
ge, and a
ctiv
ate
d
carb
on in
sha
ke fla
sks
for
impro
ved m
icro
bia
l
cell
con
centr
atio
ns.
Nutr
ient bro
th w
as
seale
d in
a c
ello
phane s
ack
and a
dded to a
shake
fla
sk c
onta
inin
g s
alin
e, m
icro
org
anis
ms,
and c
ha
rcoal.
Up to
4 tim
es
hig
her
Bru
cella
su
is a
nd E
. co
li ce
ll co
nce
ntr
atio
ns
were
obta
ined c
om
pare
d to c
ultu
res
with
out th
e c
ello
phane s
ack
.
No e
xtern
al f
eedin
g
devi
ces
needed.
Applic
able
for
various
mic
roorg
anis
ms.
Nutr
ient diff
usi
on n
ot co
ntr
olle
d.
Act
ivate
d c
arb
on m
ay
inte
rfere
with
optic
al m
easu
rem
ents
.
Gore
lick
et
al.
(19
51
)
So
lid n
utr
ien
t m
ed
ium
ove
rlaid
with
nutr
ient
bro
th f
or
impro
ved
cell
conce
ntr
atio
ns.
Solid
nutr
ient
mediu
m w
as
poure
d o
nto
the b
ottom
of
the
culti
vatio
n m
ediu
m a
nd o
verlaid
with
nutr
ient bro
th. A
ga
r/bro
th
ratio
s fr
om
1 to 1
0 w
ere
test
ed. 2-3
0 t
imes
hig
her
cell
conce
ntr
atio
ns
we
re o
bta
ined, depe
ndin
g o
n the b
act
eri
um
speci
es,
com
pare
d to a
n e
qual v
olu
me o
f liq
uid
culti
vatio
n (
volu
me
equal t
o c
om
bin
ed
agar
and b
roth
volu
mes
in a
bip
hasi
c sy
stem
).
Sim
ple
me
tho
d.
Applic
able
for
various
mic
roorg
anis
ms
and
nu
trie
nt
bro
ths.
Non-c
ontr
olle
d g
row
th. R
ela
tively
low
am
ount
of liq
uid
mediu
m
com
pare
d to the s
hake
fla
sk s
ize.
Tyr
rell
et
al.
(19
58
)
Enzy
matic
glu
cose
rele
ase
fro
m s
olu
ble
glu
cose
po
lym
er
for
contr
olle
d r
ele
ase
of
glu
cose
to t
he g
row
th
mediu
m.
Glu
cose
fe
edin
g to
the g
row
th m
ediu
m w
as
contr
olle
d b
y ch
angin
g
the e
nzy
me a
mo
unt. M
ediu
m w
as
als
o “
boost
ed”
with
com
ple
x
additi
ves
for
impro
ved r
eco
mbin
ant p
rote
in e
xpre
ssio
n a
nd in
trin
sic
pH
contr
ol.
Impro
ved c
ell
and p
roduct
yie
lds
an
d p
H c
ontr
ol w
as
obta
ined c
om
pare
d t
o L
B,
TB
, and m
inera
l mediu
m c
ulti
vatio
ns.
Max
OD
60
0 o
f 51.3
in 2
4-d
eepw
ell
pla
tes
was
report
ed.
Sim
ple
me
tho
d.
Contr
olle
d g
row
th
befo
re in
duct
ion a
nd
intr
insi
c p
H c
ontr
ol.
Su
itab
le f
or
we
ll
pla
te f
orm
ats
.
Imp
rove
d p
rod
uct
yield
s.
After
boost
ing w
ith c
om
ple
x
additi
ves,
som
e o
f th
e
contr
olla
bili
ty o
f th
e s
yste
m is
lost
. P
rop
rie
tary
pa
ten
ted
me
diu
m
com
posi
tion.
Kra
use
et
al.
(20
10
)
40
2.4.2 Problems in small-scale cultivations: online measurement and control
Traditionally, sensors and analytics methods have been developed for larger
bioprocess scales and larger samples. Small scales pose a challenge for
measurement and control. Small volumes limit the size of on-line electrodes,
sample sizes, and the addition of nutrients or pH-controlling agent. Several studies
have been conducted to miniaturize bioreactors (Funke et al. 2010, Szita et al.
2005), and to develop sensors for shake flasks (Anderlei & Buchs 2001, Ge & Rao
2012, Gupta & Rao 2003, Heyland et al. 2009, Schneider et al. 2010, Tolosa et al.
2002, Wittmann et al. 2003) and well plate formats (John et al. 2003a, John et al.
2003b, Kensy et al. 2005, Samorski et al. 2005).
The sensors developed for shake flasks include sensor devices for off-gas
measurements and luminescence. Anderlei & Büchs (2001) presented a device for
the intermittent measurement of the oxygen transfer rate in a shake flask. The
change in partial pressure of oxygen in the headspace of a maximum of 12 flasks
was measured by oxygen gas sensors. Prior to the measurement, a gas with a
calculated composition was sent through the flask head-space for calibration.
Change in the partial pressure was measured after the gas flow was shut down, and
the oxygen transfer rate was calculated based on a model. These cycles were
repeated during cultivation to follow the oxygen transfer rate during cultivation.
Later, the authors included a differential pressure sensor to evaluate the carbon
dioxide transfer rate (Anderlei et al. 2007), and improved the model used for data
calculations (Hansen et al. 2012). Heyland et al. (2009) used a different set-up for
the measurement of carbon dioxide concentration. They measured carbon dioxide
and ethanol continuously from the shake flask off-gas using infrared sensors. They
obtained the molar amounts of the gases by means of pressure monitoring in the
headspace of the flask.
Tolosa et al. (2002) immobilized a luminescing sensor spot at the bottom of
the shake flask and measured dissolved oxygen concentrations during shaking
based on the reaction between oxygen and the luminescent dye. The flask was
placed on the top of a detector device containing light emitting diodes and detectors,
and accurate analysis up to a relative concentration of 60% dissolved oxygen could
be obtained. Gupta & Rao (2003) utilized a similar sensor in oxygen transfer studies,
though they were limited by the sensor to oxygen concentrations below 60%.
Wittmann et al. (2003) presented a more stable luminescence-based measurement
system that is also applicable for higher dissolved oxygen concentrations in shake
41
flasks. Schneider et al. (2010) applied fluorescence technology for the wireless
measurement of dissolved oxygen concentration and pH in shake flasks. They
measured the parameters wirelessly by adding a RF transmitter to a measurement
tray containing nine measurement positions. Ge & Rao (2012) presented
fluorescence-based disposable optical sensor patches for the measurement of
oxygen, pH, and carbon dioxide in shake flasks. Carbon dioxide and oxygen could
also be measured from the flask headspace.
Well plate formats are even more challenging regarding online monitoring than
shake flasks or microreactors stirred with a stirrer bar. The microliter scale
combined with orbital shaking, and the number of wells placed in a small area,
place their own requirements on measuring devices. The methods developed are
mainly based on optical measurements. Such technologies apply fluorescing optical
sensor spots that are monitored through the bottom of the well. John et al. (2003a)
measured pH from dairy starter cultures, and dissolved oxygen from
Corynebacterium glutamicum cultures (John et al. 2003b) with immobilized
fluorophores using off-line measurements with a fluorescence reader. Samorski et
al. (2005) measured biomass by utilizing scattered light and NADH fluorescence
online without interfering shaking. Kensy et al. (2005) inserted an optode with
immobilized fluorophores in the bottom of the well and utilized LEDs and
photodiodes for online measurements of pH and OD. Later, Kensy et al. (2009)
improved the online monitoring for the technology presented by Samorski et al.
(2005) and were able to monitor the pH online using a soluble fluorescent pH
indicator. Many of the developed methods for shake flasks and well plates rely on
fluorescence, although they have accuracy problems. Kunze et al. (2014) showed
that the fluorescing products of the cells, like FMN-binding fluorescent protein
(emission wavelength λem = 492 nm), green fluorescent protein (λem = 520 nm) and
yellow fluorescent protein (λem = 532 nm), may interfere with the fluorescence
measured with pH and DOT sensor spots, causing errors in the data. The
excitation/emission wavelengths used in the sensors should be outside the emission
range of such a fluorescing product.
The incorporation of microfluidic devices in microwell plates brings shaken
cultures close to miniaturized bioreactors. Funke et al. (2010) replaced the bottom
of the microtiter plate with a microfluidic chip for feeding nanoliter volumes of pH-
controlling agents or growth substrates. Dissolved oxygen tension and pH were
measured online using fluorescence optodes and biomass using scattered light and
NADH fluorescence. The mixing of the system still relied on shaking.
42
Fluorescence sensors are also utilized in miniaturized bioreactors. Szita et al.
(2005) presented a multiplexed micro-scale bioreactor with a working volume of
150 μl, stirred with a magnetic spin bar and having the possibility to monitor pH,
OD600, and dissolved oxygen in situ and in real time. Fluorescence lifetime sensors
were embedded in the bottom of the reactor chambers to monitor the dissolved
oxygen tension and pH in the cultures. Optical density was monitored by a
transmittance measurement through the reaction chamber. Eventually, good
comparability with bench-scale fermentations as well as good reproducibility were
obtained with the developed system.
2.5 Enzymatic polymer processing
Hydrolytic enzymes catalyze the hydrolysis of various bonds. For example,
proteases (E.C.3.4) hydrolyze peptide bonds and glycosylases (E.C.3.2) glycosyl
compounds (e.g. starch). Enzymes can be highly specific to certain substrate
structures or they can be non-specific, hydrolyzing several bond types or polymer
structures. Highly specific enzymes can be utilized to cut the polymer structures at
a specific site. (Illanes 2008b)
Proteins are important structural and functional macromolecules. Protein is a
polypeptide where amino acid monomers are linked to each other with peptide
bonds. Efficient hydrolysis of proteins is obtained for example by using protease
mixtures such as mixtures of endopeptidase and exopeptidase (Kofoed et al. 2000,
Pommer 1995). Endopeptidases hydrolyze internal peptide bonds releasing
oligopeptides. Exopeptidases hydrolyze peptide bonds from the C- or N-terminal
residues of the polypeptide and release amino acids. Consequently, exopeptidases
are capable of completely hydrolyzing the protein molecule to amino acids.
Proteases are generally utilized for example in the food and chemical industries,
especially in the dairy and detergent industries (Kirk et al. 2002). They are used,
for example, in producing different flavors during cheese ripening or used as
additives in detergents for improved stain removal. The selection of proteases
affects the properties of the product. Proteases can be selected for instance to
remove terminal hydrophopic amino acids from peptides to remove bitterness
(Izawa et al. 1997). Highly specific proteases can be applied in medicine (Craik et
al. 2011). A therapeutic protease may either activate or inactivate its target protein
by cleavage. For example, microplasmin is utilized in detaching vitreous from the
retina in the eye (Gandorfer et al. 2004, Stalmans et al. 2010). The serine protease
hydrolyzes laminin and fibronectin, the glycoproteins present at the vitreoretinal
43
interface, releasing vitreous. As the protease has no activity towards collagen IV,
which is a major component of the basement membranes and the inner limiting
membrane in the eye, the membranes are preserved.
Starch, which is one of the most abundant biopolymers in nature, can be
processed with amylases. Starch consists of two glucose-containing
polysaccharides, linear amylose (Fig. 2A) and branched amylopectin (Fig. 2B).
Glucose units are joined with (14) glycosidic bonds in the linear molecule. In
amylopectin, branching occurs with an (16) bond at about every 20-25
glucosidic units, since 4 - 5% of the glucosidic bonds are (16) linkages (Preiss
2009, Shannon et al. 2009). The properties of the different plant origin starches
depend on the botanical source and plant growth environment, as reviewed by
Singh et al. (2003). For example, the ratio between amylopectin and amylose and
the branching of amylopectin varies between starches of different plant species but
also within the same species.
Native starches can be modified to generate new functional properties. For
example, solubility, heat tolerance, adhesion, and texture can be modified to be
suitable for specific applications. Chemical, physical, and enzymatic methods or
combinations of them are applied. Physical methods are mainly applied to change
the starch granule structure, to make native starch cold-water soluble, or to modify
the crystallite structure of starch. Chemical methods are utilized in adding
functional groups into the starch molecule. Enzymatic methods include mainly
hydrolysis reactions. (For a review, see Ashogbon & Akintayo (2014) and Kaur et
al. (2012)).
Native starch is insoluble in water at room temperature. When the temperature
of a starch suspension increases above the gelatinization temperature, starch
granules start to absorb water and swell. Consequently, solubility to water increases,
and especially amylose dissolves. When the hot starch dispersion is cooled down,
the dissolved amylose realigns and a gel is formed. The solubility of the starch at
room temperature can be increased by partial hydrolysis of the starch, for example.
This partial hydrolysis can be done by using enzymes or acids. (Biliaderis 2009,
Kusunoki et al. 1982)
Several different enzymes can hydrolyze starch. For example, pullulanase
cleaves (16) bonds releasing straight-chain maltodextrins, -amylase cleaves
(14) bonds releasing -dextrin and oligosaccharides, and β-amylase cleaves
(14) bonds releasing maltose (a disaccharide). Glucoamylase cleaves both type
of bonds, although it cleaves (14) bonds at a higher rate (Hiromi et al. 1966,
Meagher et al. 1989). Consequently, glucoamylase can convert the starch molecule
44
completely to glucose. Glucoamylase closes the non-reducing end of the starch
chain to a pocket including the active site (Aleshin et al. 1992, Sevcik et al. 1998).
Due this pocket-like structure, the starch molecule is released after cleavage of the
glycosidic bond to allow the product to leave the active site (Robyt 2009).
Some microbes (e.g. many bacilli and filamentous fungi) have the ability to
produce starch-degrading enzymes, which gives them the capability to grow on
starch. However, microbes lacking such enzymes cannot consume starch unless it
is hydrolyzed by other means. Therefore, E. coli or P. pastoris cannot grow on
starch as the sole source of carbon. One interesting application of the use of
hydrolytic enzymes was presented by Rheinwald & Green (1974). They cultivated
mammalian cells in a medium containing amylases provided by fetal calf serum
and a low amount of starch or maltose. They found that mammalian cells growing
with slow glucose liberation produced less acid than cultures growing on free
glucose with the same growth rate. After five days they obtained glucose-limited
growth, indicating that the cells consumed all the glucose at the same rate as it was
liberated. They were able to affect the rate of glucose release by inactivating the
enzymes by heat treatment of the serum. At a smaller glucose-liberating rate they
could prolong the cell cultivations compared to conventional culture. However,
they did not present any accurate relationships between enzyme dosing and glucose
production or biomass accumulation.
Fig. 2. Amylose (A) and amylopectin (B) in starch.
45
3 Materials and methods
3.1 Microbial strains
The following microbes were used in the research work:
Lactobacillus salivarius ssp. salicinius ATCC 11742
Bacillus megaterium CCM 2037
Escherichia coli K-12 RV308 ATCC 31608
Escherichia coli BL21(DE3)
Escherichia coli BL21(DE3) pET3a pLysS
Pichia pastoris X33 pPICZA-ROL
Pichia pastoris X33
3.2 Enzymes
The polymer-processing enzymes used in the research work are presented in Table
2.
Table 2. The hydrolytic enzymes utilized in this work.
Enzyme Application Original article,
or this work
Alcalase (subtilisin endopeptidase), Novo
Nordisk
For treatment of whey permeate and
lactose mother liquor
I
Flavourzyme (Aspergillus oryzae
endopeptidase/ exopeptidase complex),
Novo Nordisk
For treatment of whey permeate and
lactose mother liquor
I
Amylase AG300L (glucoamylase),
Novozymes.
For enzymatic glucose release III, this work
Enz I’m (amylase mixture), BioSilta Oy For enzymatic glucose release IV
3.3 Preparation of nutrient storage gels
Preparation of glucose-agar gel to the shake flask. Glucose solution (20%, 40% or
60%, w/v) in distilled water was sterilized by autoclave at 121 C for 20 min. Agar
(BD, Bacto Agar, Franklin Lakes, USA), 3% or 5% (w/v), was mixed into the
glucose solution (at room temperature) and 100 ml of the glucose-agar mixture was
poured into the bottom of a 1000 ml shake flask. The flask was autoclaved at
121 C for 20 min for sterilization.
46
Preparation of starch-agar gel for shake flasks. Powdered soluble potato starch
(S2004, Sigma-Aldrich, St. Luis, USA) was dispersed into a small amount of cold
water to create a slurry. The slurry was diluted to 12% or 20% (w/v) by mixing into
boiling water. Mixing was continued until the starch was evenly dispersed in the
water. The solution was autoclaved at 121 C for 20 min. To ensure the removal of
bacterial spores the solution was left overnight at 37 C. For gel preparation, the
solidified starch was melted in a microwave oven and 5% (w/v) of agar (BD, Bacto
Agar, Franklin Lakes, USA) was stirred into the solution. The solution was
autoclaved at 121 C for 20 min. 100 ml of sterile agar-starch solution was poured
aseptically into the sterile shake flask and was left to solidify at room temperature.
Preparation of EnBase gel for shake flasks and microwell plates. The
preparation protocol for the two-layer gel system is presented in the methods
section of research article III.
3.4 Microbial cultivations
The cultivation media used in the research work are listed in Table 3. More detailed
information of the methods, and the medium recipes is described in the published
articles.
L. salivarius ssp. salicinius and B. megaterium cultivations. L. salivarius ssp.
salicinius and B. megaterium were cultivated in 250 ml glass minifermenters
(Glasgerätebau Ochs GmbH, Germany) equipped with automated pH control in
cheese-whey based cultivation media as presented in the research article I.
SenBit flasks. The baffled 1000 ml shake flasks (Glasgerätebau Ochs GmbH,
Germany) including a sample needle, standard electrochemical pH sensors
(EGV150, Sensortechnik Meinsberg, Germany) and polarographich Clark-
electrodes for dissolved oxygen measurements (Medorex, Nörten-Hardenberg,
Germany), contained three 25 mm diameter side necks for positioning the sample
needle and the electrodes (Research article II). Wireless SenBit transmitters
connected to the electrodes were attached to the flask. The digitalized data from
transmitter was received by a receiver connected to the computer with SenBit
control program for data collecting and visualization.
E. coli batch cultivations. The methods and cultivation conditions for shake
flask batch cultivations of E. coli RV308 in mineral salt medium, referred as MSM,
were carried out as presented in the research article II. E. coli RV308 batch
cultivation in 200 ml Luria-Bertani broth (Research article I), referred as LB, was
carried out in SenBit flasks. Incubation temperature was 37 C and shaking rate
47
116 rpm (Finepcr SH 30 orbital shaker, 10 cm radius). Preculture in 10 ml LB at
37 C, 116 rpm, was prepared from frozen glycerol stocks and carried out in 100
ml Erlenmeyer flask.
E. coli glucose-gel cultivation. E. coli RV308 cultivation in 150 ml MSM and
100 ml 20% glucose – 3% agar-gel was performed in SenBit flask. Incubation
temperature was 37 C and shaking rate 180 rpm (Finepcr SH 30 orbital shaker, 10
cm radius). Preculture in 10 ml MSM at 37 C, 116 rpm, was prepared from frozen
glycerol stocks and carried out in 100 ml Erlenmeyer flask.
E. coli starch-agar-gel cultivations. E. coli RV308 cultivations with starch-agar
gel were carried out in SenBit flasks. Preculture in 10 ml MSM at 37 C, 116 rpm,
was prepared from frozen glycerol stocks and carried out in a 100 ml Erlenmeyer
flask.
E. coli EnBase cultivations. The methods and cultivation conditions for
EnBase shake flask cultivations of E. coli RV308, EnBase microwell plate
cultivations of E. coli BL21(DE3), and E. coli BL21(DE3) pET3a pLysS were
implemented as presented in research article III.
P. pastoris cultivations. The methods and cultivation conditions for deepwell
plate cultivations of P. pastoris X33 pPICZA-ROL and X33 were implemented
as presented in research article IV.
48
Table 3. The cultivation media used in this research. Suitable antibiotics used to
maintain the selective pressure for plasmids are listed in the original research articles.
Medium Application Original article for
recipe
Whey permeate (JK
JuustoKaira Oy)
B. megaterium CCM 2037 cultures for pretreatment,
L. salivarius ssp. salicinius ATCC 11742 cultures for
lactic acid production (I)
I
Lactose mother liquor (JK
JuustoKaira Oy)
B. megaterium CCM 2037 cultures for pretreatment of
the medium (I),
L. salivarius ssp. salicinius ATCC 11742 cultures for
lactic acid production (I)
I
MRS (Difco) L. salivarius ssp. salicinius ATCC 11742 cultures for
inoculation for lactic acid production (I)
I
Luria-Bertani broth B. megaterium CCM 2037 cultures for inoculation (I),
E. coli RV308 cultivations in shake flask (this work)
I
Mineral salt medium with
added glucose solution 5 g l-1
E. coli RV308 cultivation (II), and preculture (this work,
III),
E. coli BL21(DE3) preculture (III),
E. coli BL21(DE3) pET3a pLysS preculture (III)
III
Mineral salt medium without
added glucose solution
E. coli K-12 RV308 cultivation with starch-agar gel and
enzymatic glucose release (this work),
E. coli K-12 RV308 cultivation with EnBase (III),
E. coli BL21(DE3) cultivation with EnBase (III),
E. coli BL21(DE3) pET3a pLysS cultivation with
EnBase for production of TbTIM (III)
III
M9ZB E. coli BL21(DE3) pET3a pLysS cultivation for
production of TbTIM (III)
Buffered minimal medium
(Invitrogen)
P. pastoris X33 pPICZA-ROL and X33 cultures for
inoculation, and cultivations before induction (IV)
IV
Buffered methanol medium
(Invitrogen)
P. pastoris X33 pPICZA-ROL and X33 cultivations
during induction (IV)
IV
Buffered minimal EnBase
(Invitrogen and BioSilta)
P. pastoris X33 pPICZA-ROL cultivations for
recombinant ROL production, and X33 cultivations (IV)
IV
3.5 Analysis methods
The main analysis methods used in the research are listed in Table 4. More detailed
protocols are available in the research articles referred to if utilized in the published
results.
49
Table 4. The main analysis methods utilized in this research.
Analysis Description Research article
for protocol
Optical density from shake
flasks
Optical density was measured at 600 nm in cuvettes with
appropriate dilutions made with a sterile cultivation
medium.
III
Optical density from
multiwell plates
Optical density was measured at 490 nm in 96 microwell
plates with appropriate dilutions made with a sterile
cultivation medium. The OD490 values were converted to
corresponding OD600 values in cuvette. For E. coli, OD490
of 1 corresponded to OD600 of 0.73 (research article III).
For P. pastoris, OD490 of 1 corresponded to OD600 of 0.12
(research article IV)
III, IV
Cell number by plating The E. coli cell numbers obtained by plating were
calculated as averages by counting two LB-agar plates
containing 30 – 300 colonies. The LB-agar spread plates,
prepared by adding 15 g l-1 Bacto agar (Becton Dickinson
and Company) to LB broth (Research article I) were
incubated for 24 h at 37 C.
-
Glucose analysis Samples were centrifuged for cell removal prior to
analysis. Glucose was analyzed from the clear
supernatant diluted to appropriate concentrations, with a
YSI 2007 Select bioanalyzer (YSI Inc., USA) using an
enzymatic test.
III, IV
Starch analysis Samples were centrifuged for cell removal prior to
analysis. Starch was analyzed indirectly by hydrolyzing
starch with acid and measuring the obtained glucose
residues with a YSI 2007 Select bioanalyzer (YSI Inc.,
USA) using an enzymatic test.
III
Lactate analysis by HPLC
in L. salivarius ssp.
salicinius cultivations
Samples were centrifuged and filtered for cell removal
prior to analysis. The chemical compounds in the sample
were separated by reversed phase HPLC (Merck–Hitachi,
Model D-7000), and lactate was quantified with a UV-vis
detector.
I
Acetate analysis by HPLC
in E. coli cultivations
Samples were centrifuged and filtered for cell removal
prior to analysis. Compounds were separated by reversed
phase HPLC (Merck–Hitachi, Model D-6000), and acetate
was quantified with a UV-vis detector.
III
50
Analysis Description Research article
for protocol
Analysis of organic acids
by HPLC in P. pastoris
cultivations
Samples were centrifuged and filtered for cell removal
prior to analysis. Compounds were separated by reversed
phase HPLC (Agilent Technologies, 1200 series), and
organic acids were quantified with a diode array detector
(succinate and lactate) and refractive index detector
(formate, acetate and ethanol).
IV
Trypanosoma brucei
triosephosphate isomerase
analysis by SDS-PAGE
Samples were centrifuged to remove the culture media
prior to analysis. A cell pellet was suspended into lysis
buffer for cell disruption. Insoluble and soluble debris
were separated by centrifugation and the suspensions
were normalized by dilution. Insoluble debris was washed
and resuspended in urea. Insoluble and soluble samples,
and pure TbTIM as reference, were applied to SDS-
PAGE. The lane intensities were quantified using
ImageQuant 5.2 software (GE Healthcare, UK).
III
Rhizopus oryzae lipase
analysis by Western
blotting
Samples were centrifuged for cell removal prior to
analysis. The proteins were separated by SDS-PAGE,
and transferred to a PVDF membrane for Western
blotting. The blocked membrane was incubated with a
primary antibody that was recognized by horseradish
peroxidase conjugated goat anti-Rabbit IgG. Enhanced
chemiluminescence was utilized for more sensitive
detection by applying a detection kit (GE Healthcare).
IV
Rhizopus oryzae lipase
activities by assay
Samples were centrifuged for cell removal prior to
analysis. ROL activity was obtained by utilizing a Lipase
colorimetric assay kit (Roche) and monitoring the
enzymatic reaction with a spectrophotometer for
5 minutes. The activity was calculated utilizing the Beer-
Lambert law.
IV
51
4 Results and discussion
The focus area of the thesis was the improvement of biomass and product formation
in microbial cultures by improving the cultivation conditions with an enzymatic
nutrient delivery system. Publication I describes the in situ enzymatic approach
where proteolytic enzymes were applied during the cultivation to relieve growth
limitation by peptides and amino acids during the cultivation. Publication II
describes studies where a wireless measurement system was developed and applied
to identify the problems in shake flask cultivations. Publications III and IV describe
the development and use of the enzymatic glucose release system.
4.1 Utilization of enzymatic nutrient release in production of lactic acid by Lactobacillus salivarius ssp. salicinius (I)
The use of enzymes or proteolytic microbes in the pretreatment of whey permeate
and lactose mother liquor for cultivation with Lactobacillus salivarius ssp.
salicinius was studied in research article I. The cultivations were implemented in
250 ml glass minifermenters with pH control utilizing standard pH sensors
connected to a control computer. The in situ hydrolysis of proteins for amino acid
supply was utilized for improved growth and product formation.
Whey permeate and lactose mother liquor, the side products from cheese
manufacturing, have high concentrations of lactose, proteins and other nutrients,
and are therefore potential growth media for microbes for lactic acid production.
The whey permeate, obtained by the ultrafiltration of cheese whey, contained 50 g l-
1 of lactose as a carbon source, and 1.5 g l-1 of proteins. The lactose mother liquor,
a byproduct from lactose recovery, contained 90 g l-1 of lactose, and 9 g l-1 of
proteins. However, these side products also had high salt concentrations that the
microbes have to tolerate. L. salivarius ssp. salicinius was taken as a production
organism, since it was known to tolerate such salt concentrations, as concluded by
other researchers in research article I. It has, however, an inefficient proteolytic
system and therefore, despite the relatively high protein content, whey permeate or
lactose mother liquor could not be directly used as such as growth media.
The whey permeate supplemented with 3 g l-1 proteins, or lactose mother liquor,
was treated with a protease mixture after heat sterilization, since it became obvious
that strong coagulation of proteins occurs if treatment is done prior to sterilization.
Protease treatment of the medium increased the amount of lactic acid produced
about four-fold in both media when compared to non-treated media after 60 h of
52
cultivation (Fig. 4 in research article I). Since similar enhancement in lactic acid
production could be achieved by supplementing the medium with yeast extract, it
was concluded that the low availability of amino acids or easily assimilable small
peptides was limiting lactic acid production.
As an interesting alternative for medium modification, treatment with the
proteolytic microbe Bacillus megaterium was applied. B. megaterium is a widely
used strain in the industry for enzyme production. It secretes proteases, and has
GRAS status. The strain did not utilize lactose, as verified with the API test
(Biomerieux, France), although some contradictory observations for the strain
occur (Obruca et al. 2011). However, even if the result from the API test was
incorrect, its use of lactose was insignificant based on the lactic acid concentration
obtained in L. salivarius ssp. salicinius fermentation. The lactose mother liquor and
whey permeate were pretreated by cultivation overnight with proteolytic B.
megaterium. After a temperature shift to 40 °C, L. salivarius ssp. salicinius was
added to the pretreated medium for the lactic acid production phase in
minifermenters. An almost four-fold increase in lactic acid concentration was
observed compared to a non-pretreated medium. The final concentrations of lactic
acid were a few g l-1 higher in the lactose mother liquor medium than with
enzymatic treatment, and at the same level in both cultures made in whey permeate
(Fig. 4 in research article I).
Interestingly, a pH increase of ~1.5 units was observed during the pretreatment
of whey permeate and lactose mother liquor by B. megaterium (Fig. 5 in research
article I), possibly due to oxidative deamination during the utilization of proteins
by the microbe. This phenomenon was later also observed in E. coli cultivations
(see chapter 4.2). The oxidation of amino acids releases ammonia, which increases
the pH in the growth medium. Consequently, the consumption of external pH-
adjusting agents may be reduced when the ammonia-releasing catabolic reactions
occur.
The in situ enzymatic treatment of lactose mother liquor and whey permeate
was clearly a competitive method to improve lactic acid production in salt-
containing dairy side products. Further, almost equal amounts of lactic acid were
obtained when the mixed fermentation of B. megaterium and L. salivarius ssp.
salicinius was implemented. The inefficiency of the proteolytic system of lactic
acid bacteria could be compensated by utilizing proteolytic enzymes or microbes,
thus allowing the use of the proteins present in dairy side products. Simultaneous
hydrolysis of proteins during fermentation seemed to be a promising technique, and
the principle was later applied in the in situ hydrolysis of starch in E. coli
53
cultivations as will be discussed in section 4.3.2. Deamination was later found to
be a useful principal mechanism for maintaining the pH level in glucose-limited
cultivations, as utilized by Krause et al. (2010). The online measurement of pH was
useful for gathering continuous information from small-scale fermenters. The
small-scale reactors were run is parallel, but the capacity was limited to four
fermenters. This was one motivating factor for the development of wireless sensor
devices for the less expensive shake flasks discussed in the next chapter.
4.2 Cultivation conditions in shake flask cultures of Escherichia
coli (II)
Shake flask cultivations are commonly used in biotechnology laboratories and they
are usually operated as batch cultivations. Due to the batch method, the growth is
exponential until some factor starts to limit the cultivation. The measured product
concentrations often remain much lower than in bioreactor cultivations. Monitoring
of the process conditions during (or even after) the cultivation is often neglected.
The flexible online monitoring system SenBit®, for measurement of these
parameters, was presented in research article II.
Online measurements of pH and pO2 from conventional Escherichia coli
cultivations with the SenBit system showed that oxygen transfer and medium
buffering capacity were not high enough in baffled shake flasks (Fig. 3). Aerobic
growth conditions and steady pH could not be maintained throughout the
cultivation.
The pH was seen to change with different patterns in the mineral salt medium
and in the rich cultivation medium Luria-Bertani broth. A pH decrease in the
mineral salt medium occurs through the cultivation (Fig. 3a), while in Luria-Bertani
broth the pH started to increase after three hours (Fig. 3b). E. coli produces organic
acids via the overflow metabolism when growing on excess glucose (Hollywood &
Doelle 1976). In fermentative metabolism, organic acids are produced via mixed
acid fermentation (reviewed by Böck & Sawers 1996, Xu et al. 1999a).
Consequently, the organic acids acidify the medium. E. coli also consumes the
ammonium sources, ammonium sulfate and ammonium chloride, in a mineral
medium resulting in a pH decrease. In complex Luria-Bertani broth, after a certain
time point, the pH started to increase again (Fig. 3b). A pH increase was also
observed in a complex medium in Bacillus megaterium cultivations (research
article I) suggesting that it is a typical phenomenon in microbial cultures grown in
54
a rich cultivation broth. The phenomenon has also been recognized in other studies
(e.g. Losen et al. 2004, Sezonov et al. 2007).
Fig. 3. Escherichia coli RV308 batch cultivations at 37 °C, baffled shake flask and 200 ml
cultivation volume a) in a mineral salt medium with 180 rpm shaking and 0.1 ml l-1
antifoam (Sigma) (Research article II, reprinted with permission of BioMed Central), and
b) in Luria-Bertani broth with 116 rpm shaking (modified from Panula (2006)).
The Clark-type pO2 electrode was selected for the system for several reasons. This
technology was known to be suitable for microbial cultivations. In addition,
autoclavable small-size Clark sensors were available. The batteries implemented to
the transmitter were supporting enough input voltage for the sensor to operate for
several days. As reviewed by Suresh et al. (2009), operation of the oxygen electrode
is based on the electrochemical cell, where the polarization voltage is supplied by
an external source. The Clark sensor creates an output current based on the oxygen
partial pressure in the system measured. The use of Clark-type electrodes in shake
flasks has, however, been criticized, since the electrode shaft affects the
hydrodynamics of the culture (Hansen et al. 2011, Tolosa et al. 2002), and therefore
the results obtained do not represent typical shake flasks. Hansen et al. (2011)
showed that the oxygen transfer rate is higher in flasks containing such invasive
sensors. A shake flask with 30 ml filling volume in a 250 ml Erlenmeyer flask had
a maximum oxygen transfer capacity of 12 mmol l-1 h-1 with an invasive sensor,
and without the invasive sensor the oxygen transfer capacity was 10 mmol l-1 h-1.
Oxygen transfer in shake flasks is more efficient when the oxygen transfer area is
increased by increasing the shaking speed (Maier & Büchs 2001), or using baffled
shake flasks (McDaniel & Bailey 1969), for example. The invasive oxygen and pH
sensors used in SenBit, with diameters of 6 mm and 12 mm, respectively, increase
the oxygen transfer rate in the cultivation. The sensors increase the liquid mixing
Time [h]0 5 10 15 20
pO
2 [
%]
0
20
40
60
80
100
OD
600
0
1
2
3
4
5
6
7
pH
3
4
5
6
7
8
Time [h]
0 5 10 15 20
pO
2 [
%]
0
20
40
60
80
100
OD
600
0
1
2
3
4
5
6
7
pH
3
4
5
6
7
8
a b
55
and the liquid surface area by breaking the smooth liquid flow together with baffles
(see sensor positions in Fig. 4). Therefore, the obtained dissolved oxygen graphs
are not completely representative of baffled flasks but show results from an even
better mixed system. However, the results give a good indication of the conditions
in the flask. When no oxygen is measured from the cultures, it can be assumed that
the situation is the same or even worse in flasks without sensor shafts and their
effect on oxygen transfer. However, if the oxygen flow through flask closure is the
limiting step in oxygen transfer, the increased mixing does not increase oxygen
transfer to the medium.
The advantage of the SenBit system is its flexibility. The small transmitters can
be added to normal-sized shake flasks and operated in an incubation chamber
already present in the laboratory. No new incubators, shaking vessels, or gas
analyzers are needed, although flasks containing side necks for electrode positions,
as seen in Fig. 4, were used in order to facilitate the use of several sensors and to
ensure sufficient aeration. However, if exact oxygen transfer rates are required, off-
gas measurements are needed.
The SenBit system has been used for the development of optimal cultivation
protocols for microbial cultivations. By following the oxygen levels in Pichia
pastoris cultivations, methanol addition could be done right after the substrate had
been consumed, which was indicated by an increase in oxygen level (Fig. 3 in
research article II). Ruottinen et al. (2008) used the system to study further the
effect of conventional pulse feeding of methanol to the shake flask. Later, the
SenBit system was utilized in screening the optimal substrate feeding to shake
flasks in E. coli cultivations (Fig. 9). The effect of changing the glucose feed could
be observed in pO2 and pH, as well as in off-line measured acetate and glucose
levels. These parameters will be further discussed in chapter 4.3.
4.3 Development of small-scale fed-batch system (III)
The problems with oxygen transfer became visible in the results in research article
II. The batch-like nature of small-scale cultivations was a clear reason for the low
cell densities obtained. Therefore, it was decided to implement the fed-batch
method to shaken cultures, as it is a common protocol for controlling the oxygen
consumption in larger-scale bioreactors in the production of recombinant proteins.
However, the premise for the system was to remove the need for external feeding
devices in different cultivations. Such a system is presented in research article III.
Controlled growth and improved product concentrations were obtained, as was
56
demonstrated in research article III with Escherichia coli and in research article IV
with Pichia pastoris. However, the foundation for the fed-batch method was
already laid in the application in research article I, where enzymes (proteases) were
applied for the in situ hydrolysis of dairy side products for lactic acid production.
4.3.1 Studies on glucose storing in agar gel
It was necessary to control the amount of glucose available in shake flasks to avoid
the negative effect of high substrate concentrations, while supporting enough
glucose for high cell densities. Therefore, glucose-agar gels were prepared to study
whether controlled glucose feeding to the shake flask could be applied from the
storage gel poured into the bottom of the shake flask (Fig. 4). However, this
approach was not successful since glucose diffusion was not restricted enough (Fig.
5). According to Lee (1996), the growth-inhibiting glucose concentration for E. coli
is approximately 50 g l-1. The glucose level in the sterile medium in flasks
containing 60% and 40% glucose-gels reached a growth-inhibiting level after only
half an hour from the start (Fig. 5a). For the 20% glucose-gel this limit was reached
after 1.5 h. After 6 h, the glucose concentration in the medium was in equilibrium
with the gel. The E. coli cultivation implemented with 20% glucose-gel became
oxygen-limited due to non-controlled growth. Consequently, cell density was low,
and a pH decrease down to 4.0 was observed (Fig. 5b).
Fig. 4. E. coli RV308 cultivations in baffled shake flasks with glucose-agar gel. 1: Growth
medium, 2: Glucose-agar gel, 3: pH sensor, 4: pO2 sensor. The flask is tilted to make the
gel more visible.
57
Fig. 5. Glucose-containing storage gel as a glucose source for E. coli cultivation. a)
Glucose diffusion studies without bacterial cells. The amount of glucose in storage gel
was 20% (two parallels), 40% or 60% (three parallels) and the agar concentration was 3%
or 5%. b) E. coli RV308 cultivation with 20% glucose - 3% agar-gel. The experiments
were carried out in baffled shake flasks at 180 rpm, 150 ml MSM, 37 °C and 100 ml
storage gel. Figures modified from Panula (2006).
Tyrrell et al. (1958) used a layer of solid nutrient medium overlaid with a small
volume of nutrient broth for microbial cultivations. They obtained 2-30 times
higher cell numbers compared to broth only. However, their purpose was not to
control the growth but to combine the agar-plate cultivations (diffusional access to
nutrients) and submerged cultivations (homogenous cells in cultures) to increase
cell yields. They also used a complex medium without the risk of inhibiting
concentrations of glucose. They compared the results against a control broth with
the same combined volumes of solid nutrient medium and overlaid nutrient broth.
This setup suggests differences for oxygen transfer that could have affected the cell
numbers obtained, since the medium volume has an effect on oxygen transfer
(Maier & Büchs 2001). In the glucose-agar method tested in this work, the gel acted
as storage for one growth-supporting component whereas Tyrrell et al. (1958) had
nutrient agar gels containing all the medium components. Nutrient agar gels can
counteract substrate inhibition, since nutrients are not highly concentrated.
However, with this system, growth control in the sense of fed-batch cannot be
obtained.
4.3.2 Starch as a glucose source
It was obvious that the agar-gel could not sufficiently limit glucose diffusion to the
growth medium. The successful in situ proteolytic degradation implemented in
Time [h]
0 2 4 6 8
Rel
ease
d gl
ucos
e [g
l-1]
0
50
100
150
200
250
5 % agar5 % agar5 % agar3 % agar
Time [h]0 10 20 30 40
pH
2
3
4
5
6
7
8
pO2
0
20
40
60
80
100
120
140
160
OD
600
0
2
4
6
8pH pO2
OD600
20
Glucose in agar [%]
40
60
a b
58
research article I (as discussed in chapter 4.1) encouraged us to study a similar
approach for the in situ hydrolysis of starch for glucose production. Starch is a
biopolymer consisting of glucose monomers and it can be enzymatically
hydrolyzed to glucose. The hypothesis was that the growth rate of the cells could
be controlled by regulating the amount of glucose-releasing enzyme.
The method using soluble starch dispersed in the growth medium as a glucose
source was not applicable for high cell density E. coli cultures. The amount of
starch that remained soluble in the growth medium was not high enough to support
high final cell densities. In addition, the growth medium was cloudy due to the
dispersed starch, which complicated the analysis made from the cultivation. Even
when solubilized by heating, starch quickly loses its solubility due to the tendency
for retrogradation. A similar kind of approach to utilize soluble starch as a glucose
source was used in mammalian cell cultures by Rheinwald & Green (1974), as
reviewed in chapter 2.5. Their system was not suitable, however, for controlled
high cell density bacterial or yeast cultures due to the use of a complex medium,
the very slow glucose release rate, and the small amount of starch used (5 g l-1).
This amount was too low to reach theoretically higher than 2.5 g l-1 of E. coli cell
dry weight ~ OD600 of 7.5 (if 100% of starch is converted to glucose and Yx/g = 0.5).
Furthermore, only about 50% of the added starch could be utilized for glucose
production in their system.
The amount of glucose stock available to the cells should be significantly
higher to provide high cell densities. Therefore, starch was stored in starch-agar gel
that was poured onto the bottom of the shake flask. Cultivation remained
submerged as the liquid growth medium, including the starch-degrading
glucoamylase, was on top of the gel. In this case, the glucose release was too high,
resulting in exponential growth, consequent oxygen depletion, and strong medium
acidification (Fig. 6a). However, the obtained final optical density was several OD
units higher than that obtained with the same strain in conventional MSM
cultivation (Fig. 3a), despite the observed oxygen limitation.
When the starting cell density was increased to OD600 of 2, and the amount of
enzyme was kept the same, the glucose concentration remained below 1 g l-1 also
at the beginning of the cultivation (Fig. 6b). Consequently, the exponential growth
phase was much shorter since excess glucose was consumed faster from the growth
medium. The oxygen depletion was still quite strong and oxygen was at zero level
for a short time at five hours from induction. Manual pH control with 4 M KOH
was utilized to reduce the effect of the pH decrease. The consumption of 4 M KOH
during 22 h of cultivation was 2.8 ml (Fig. 6b), which indicate acid formation.
59
To obtain fully aerobic cultivations, the glucoamylase concentration was
reduced to half (Fig. 6c). This strategy was successful. The cultivation remained
fully aerobic, although medium acidification was still observed, and 2.4 ml of 4 M
KOH was added during 22 h of cultivation. Obviously, the volume and quality of
the inoculum has to be considered when adjusting the glucoamylase dosage. Good
quality inoculum harvested in the exponential growth phase has a higher substrate
consumption rate than inoculum growing in the stationary phase. Therefore, with
the same glucoamylase concentration and starting cell density, glucose may
accumulate at a different rate depending on how fast the cells grow and consume
the released glucose. A too high glucose accumulation at the beginning will
eventually lead to oxygen depletion when the oxygen consumption rate of the
exponentially growing cells exceeds the oxygen transfer capacity of the flask.
60
Fig. 6. Enzymatic glucose feeding in shake flask cultivations of E. coli RV308.
Cultivation in 180 ml MSM, shaking speed 180 rpm, 37 C. a) Glucoamylase 167 AGU l-1,
OD600 0.1 at inoculation, 100 ml starch (12%) - agar (5%) gel as a glucose source b)
Glucoamylase 167 AGU l-1, OD600 2 at inoculation, manual pH control with 4 M KOH, total
2.4 ml added during 22 h of cultivation. 100 ml starch (20%) - agar (5%) gel as a glucose
source. c) Glucoamylase 83 AGU l-1, OD600 2 at inoculation, manual pH control with 4 M
KOH, total 2.4 ml added during 22 h of cultivation. 100 ml starch (20%) - agar (5%) gel
as a glucose source. Figures modified from Panula (2006).
Time [h]
0 5 10 15 20 25 30
pO
2 [%
]
0
20
40
60
80
100
pH
6.0
6.2
6.4
6.6
6.8
7.0
OD
600
0
2
4
6
8
10
12
14
Glu
cose
[g
l-1]
0
1
2
3
4
pO
2 [%
]
0
20
40
60
80
100
pH
6.0
6.2
6.4
6.6
6.8
7.0
OD
600
0
2
4
6
8
10
12
14
Glu
cose
[g
l-1]
0
1
2
3
4
pO
2 [%
]
0
20
40
60
80
100
pH
4
5
6
7
8
9
10
Glu
cose
[g
l-1]
0
2
4
6
8
10
12
14
16
18
OD
600
0
2
4
6
8
10
12
14a
b
c
Glucoamylase 167 AGU l-1
Glucoamylase 167 AGU l-1
Glucoamylase 83 AGU l-1
61
Applying regulating gel for better control
Even though the starch-gel based glucose feeding worked, several problems were
observed with the gel. Small starch-agar gel pieces detached from the gel during
cultivation and sometimes the gel broke down during vigorous shaking. Moreover,
the soluble starch accumulated in the growth medium all too quickly, impairing the
oxygen transfer rate by increasing the medium viscosity. A regulating agar-gel was
poured on top of the storage gel to control these phenomena. The regulating gel
retarded the starch accumulation (Fig. 7) and prevented the gel breakage during
cultivation (data not shown).
Fig. 7. The effect of regulation gel and its composition on the accumulation of starch in
the culture medium. Starch diffusion from the storage gel through the regulating gel,
and a control without regulating gel was investigated. (Research article III, reprinted
with permission of BioMed Central)
Different glucose release rates were obtained by varying the amount of the glucose-
releasing enzyme (Fig. 8a and c), indicating the possibility to adjust cell growth
through the amount of glucoamylase. This was clearly observed in the microwell
plate cultivations of E. coli BL21(DE3) (Fig. 4 in research article III). The residual
starch measured from the medium depended on the glucoamylase concentration
(Fig. 8b). The higher the enzyme concentration, the less starch was accumulated in
the medium. The starch diffusion from the gel to the medium was slow enough to
see the clear effect of the varied enzyme amount on starch accumulation. However,
accumulation of glucose in the medium increased the risk of product inhibition,
which may have had an effect on the glucose release rates. Especially with 30 and
12 AGU l-1, the glucose release rate decreased in time while the residual starch
Time [h]5 15 25 350 10 20 30
Sta
rch
[g l-1
]
0
10
20
30
40
No regulating gel1.5 %2.5 %3.25 %5 %
Agar concentration in regulating gel
62
concentrations remained above zero. Therefore, the starch amount should not be
the limiting factor for the release rate. Glucose has been recognized as a
competitive inhibitor for glucoamylase (Cepeda et al. 2001, Hiromi et al. 1973). In
competitive inhibition, the inhibitor prevents the substrate from binding to the
active site. When the amount of glucose increases, inhibition also increases.
Therefore, the saturation of the curves observed especially for the higher
glucoamylase amounts may be a result of product inhibition. However, significant
product inhibition should not occur in cultivations since bacterial cells consume the
released glucose from the growth medium.
63
Fig. 8. Optimization of the glucose delivery system. a) Glucose accumulation from a
two-phase gel into a sterile liquid medium with different amounts of glucoamylase (0.15-
30 AGU l-1). b) Residual starch in the growth medium measured utilizing acid hydrolysis.
c) Glucose release rate calculated based on measured glucose in a). Microwell plates
were incubated at 37 °C, with a shaking speed of 750 rpm. Whole wells were harvested
for each analysis. (a) and c) are modified from III and reprinted with permission of
BioMed Central)
Time [h]
0 20 40 60 80 100 120 140
Glu
cose
rele
ase
rate
[g l-1
h-1
]
0.0
0.2
0.4
0.6
0.8
1.0
1.2
3012 6 3 1.2
Time [h]0 20 40 60 80 100 120 140
Glu
cose
[g l-1
]
0
5
10
15
20
25
30
35
AGU l-1
c
a
b
Time [h]0 20 40 60 80 100 120 140
Res
idua
l sta
rch
[g l-1
]
0
10
20
30
40
0.60.30.150
3012 6
3 1.2
AGU l-1
64
The reaction mechanism of glucoamylase was deduced to be suitable for
controlled glucose release. Glucoamylase has no endo-mechanism and it acts only
on the reducing end of the molecule (Meagher et al. 1989, Phillips & Caldwell 1951,
Robyt 2009). Therefore, the released product is glucose, not dextrin or higher chain
length glucose polymer. The amount of available polymer molecules for
glucoamylase does not increase during the cultivation by release of glucose
polymer branches from starch molecules (Kusunoki et al. 1982), but it depends on
the diffusion of starch from the gel. When the amount of soluble starch is not liming
and no inhibition occurs, glucose release remains approximately linear. However,
if necessary the solubility of the starch substrate can be improved by treatment with
α-amylases.
The control of the growth by controlled glucose release was obtained also in
shake flask cultivations of E. coli RV308 (Fig. 9). Optical density, glucose
concentration, pH, pO2 and acetate levels were monitored. The amount of the
enzyme had to be adjusted according to the desired growth rate, but also to keep
the cultivation aerobic. At the beginning of the cultivation the amount of cells was
not high enough to consume all glucose released by the enzyme, leading to the
accumulation of glucose into the medium. Therefore, a batch phase, the typical
phase in fed-batch cultures in bioreactors, was observed at the beginning of the
cultivation (Fig. 9). Depending on the amount of the released glucose, unlimited
growth was observed until the amount of the cells was high enough to consume the
released glucose immediately. However, such batch phase cannot be avoided if
glucoamylase is added only at the beginning of the cultivation. In that case, the
amount of glucoamylase cannot be adjusted according to the cell mass at the
beginning: the amount would be too small to release enough glucose later for higher
cell concentrations. The two phase glucose releasing enzyme addition was utilized
by Krause et al. (2010) for increased need of glucose for higher cell concentrations,
as will be discussed later.
Acetate is well-known side product from E. coli metabolism when excess
glucose is present (Hollywood & Doelle 1976). When over optimal amount of
glucose releasing enzyme was present in the cultivation, acetate was produced
during the whole cultivation (Fig. 9a). By decreasing the amount of the enzyme,
the amount of glucose at the beginning was decreased and consequently also the
acetate accumulation could be reduced (Fig. 9b and c). In cultivations with small
enzyme amounts, acetate was consumed immediately after cultivations shifted to
glucose limitation. This influenced the pH level, which could even temporally raise
due to acetate consumption (Fig. 9b).
65
Control over growth was also indicated by the pO2 levels of the cultivations
(Fig. 9). While with highest glucoamylase concentrations metabolism was
fermentative short time, with smallest glucoamylase concentration cultivation was
fully aerobic. Consequently, highest cell densities were obtained when oxygen
depletion could be completely avoided.
Fig. 9. Shake flask cultivations of E. coli RV308 at 37 °C with the EnBase system with
different amounts of glucoamylase (a: 60; b: 30; c: 12 AGU l-1). pO2 and pH values were
registered in 12 sec intervals with the SenBit system. The spikes in the pO2 curves
reflect intermediate stops of the shaker for sampling. (Research article III, reprinted with
permission of BioMed Central)
The principle of the developed method (known as EnBase®) is comparable to fed-
batch cultivation where glucose is fed at a constant rate to the fermentor (Fig. 10 a
and b). In the developed method, glucose is released continuously to the growth
medium by glucoamylase from starch stored in the bottom gel in the cultivation
vessel. The regulating gel reduced starch accumulation and also protected the
bottom gel. The growth rate in the system decreases as the cell mass increases and
consumes the added glucose. This phenomenon also occurs in large-scale fed-batch
cultivations where the cooling capacity or oxygen transfer rate of the reactor does
not allow exponential feed after a certain cell concentration. Exponential feeding,
often utilized in fed-batch cultivations having none of the above-mentioned reactor-
limiting issues, is not easy to obtain in EnBase after the batch phase. Even the pH
decrease observed in the cultivation does not enhance the enzyme activity
sufficiently to provide exponential growth. Therefore, the growth rate decreases
Glu
cose
[g
l-1]
0
1
2
3
4
Ace
tate
[g l-1
]
0.0
0.5
1.0
1.5
2.0
2.5
3.0
OD
600
0
5
10
15
20
25
30
Time [h]
0 5 10 15 20 25
pO2
[%]
0
20
40
60
80
100
Time [h]
0 5 10 15 20 25
Time [h]
0 5 10 15 20 25
pH
4.0
4.5
5.0
5.5
6.0
6.5
7.0
a b c
66
during cultivation. To increase the growth rate, more enzymes can be added.
However, over six times higher cell densities and a smaller decrease in pH were
obtained with the enzymatically controlled glucose release (Fig. 9) compared to a
conventional cultivation in a mineral salt medium (Fig. 3a). Also, it was possible
to control the oxygen consumption rate and acetate production by adjusting the
amount of glucoamylase.
Fig. 10. Principal concepts of high cell density cultivation by substrate-limited fed-batch
cultivation in a bioreactor a), and by the enzyme-controlled substrate delivery system
b). Designations: (1) substrate reservoir, (2) control system for supply of the substrate
with a pump in the standard bioreactor or with a specific concentration of an enzyme in
the substrate delivery system, (3) liquid cell culture medium. A typical fed-batch
process often starts with a batch phase, which is characterized by a high initial glucose
concentration that steadily decreases (a). In contrast, in enzymatic glucose feeding (b)
the glucose level increases in the first phase due to the glucoamylase function and low
consumption by the small number of cells. In both cases, a) and b), the glucose
concentration is very low after the initial batch phase. The biomass increase over time
is controlled either by the pump (a), or by the enzyme concentration (b). (Research
article III, reprinted with permission of BioMed Central)
67
The downside of the method was the necessity to prepare a gel structure that
had to be pipetted to the bottom of the well plate or poured into the flask. Especially
in the 96-well microwell plate, the gel may take 50% of the working volume
consequently decreasing the volume available for cultivation. Also, on rare
occasions, the gel could still break during shaking and release particles to the liquid
phase. The developed EnBase method was further developed for a totally soluble
form by Krause et al. (2010). The method was based on the same working principle.
Glucose was released enzymatically from glucose polymer, and the release rate was
controlled by the amount of the enzyme. However, they used soluble
polysaccharide instead of starch, which allowed completely soluble cultivation.
They used a semi-defined cultivation medium and had also complex additives
added at the time of induction to “boost” recombinant protein production and to
control pH via the deamination reaction. An additional dose of the glucose-
releasing enzyme was also added at the time of induction to increase the glucose-
releasing rate for higher biomass concentrations. The developed method was
successful, although some of the controllability of the growth was lost due to the
use of complex additives.
4.4 Benefits obtained with small-scale fed-batch (III, IV)
The results from E. coli cultivations were promising since fully aerobic cultivations
could be achieved by controlled glucose release in a starch-based system. However,
the method was also tested in the production of recombinant protein with E. coli as
well as with methylotrophic Pichia pastoris.
4.4.1 Production of recombinant TIM
The developed small-scale fed-batch method was applied in E. coli BL21(DE3)
pET3a pLysS cultivations for the production of Trypanosoma brucei brucei
thiosephosphate isomerase (TbTIM) (Borchert et al. 1993). The protein was an
example of highly expressible proteins, which have a tendency to form inclusion
bodies in E. coli. Also, the cultivation conditions were known to have an effect on
the aggregation (Borchert et al. 1993, Casteleijn 2009). The TIM enzyme catalyzes
the reversible isomerization of dihydroxyacetone phosphate to D-glyceraldehyde
3-phosphate, a reaction occurring in glycolysis. Deficiency in the enzyme in
humans is lethal and causes for example congenital hemolytic anemia and
progressive neuromuscular dysfunction, as reviewed by Orosz et al. (2009). The
68
enzyme structure and function have been extensively studied, however; no effective
therapy is available.
Previous experiments in producing TbTIM in shake flasks had shown that a
more soluble protein was obtained with enzymatic glucose release compared to
conventional cultivation, induced at the same optical density (Panula 2006).
Aerobic growth conditions, monitored by the SenBit system, were maintained
throughout the cultivation whereas oxygen depletion occurred in a conventional
batch cultivation. The induction cell density has an effect on the yield of
recombinant proteins (Graslund et al. 2008), and the culture should be in the middle
or end of the exponential phase when induced. The cell density should be high
enough to yield a sufficient amount of protein, but the cells should still be vital and
not entering the stationary phase. The typical optical density during induction is
therefore between an OD600 of 0.6 and 1.2 (Sivashanmugam et al. 2009). Since
previous experiments showed improved protein production with the glucose release
system induced at the same optical density (Panula 2006), the effect of induction
cell density on the amount of product was studied.
The microwell plate cultivations were implemented in 300 µL 96-well plates
with 150 µL cultivation volumes. The glucose-releasing method was compared
with the conventional batch cultivation in an M9ZB medium, so far the best
conventional production medium, in similar microwell plates. In microwell plate
cultivations the overall product amount obtained, and especially the soluble protein
amount, was 8-10 times higher with fed-batch than with batch (Fig. 11). The
induction cell density in the conventional cultivation was relatively low, at OD600
of 0.3. Although the total product per cell was higher with the conventional
cultivation method, the soluble protein amount per cell was at the same level as in
EnBase. Furthermore, the lower product yield per cell in EnBase was well
compensated by the higher cell densities obtained.
The production of recombinant protein causes relatively high metabolic stress
to the cells (Bentley et al. 1990). A mineral medium does not contain all the
necessary building blocks for growth and therefore biosynthesis of these
components is required (Tao et al. 1999). When protein synthesis is induced, the
demand for amino acids for example is further increased. This leads to a decreased
growth rate but also slower product synthesis compared to the complex medium
containing amino acids (Tao et al. 1999). The amount of ribosomes is also higher
in fast-growing cells, increasing the product formation rate compared to a slower
growth rate (Sanden et al. 2003). As Rosano & Ceccarelli (2014) have concluded,
a slower production rate decreases the amount of cellular proteins, but also gives
69
the recombinant protein more time to fold properly. Sanden et al. (2005) obtained
more active protein and a higher total amount of protein by decreasing the growth
rate in a minimal medium. They suggested that the result was due to less proteolysis
occurring at smaller feeding rates. In our recombinant TIM case, the specific
productivity was higher with a rich cultivation medium than in the minimal EnBase
medium (Fig. 11c). However, the relative amount of soluble TIM protein from total
TIM was higher in EnBase, resulting in higher volumetric productivity than in the
conventional culture (Fig. 11b). Protein expression is slower in a minimal medium
due to different metabolic requirements as discussed above, and the product has
enough time to fold properly. If the product is prone to proteolysis, slowing down
the growth rate may increase the product yield.
Interestingly, recombinant protein expression was not very sensitive in relation
to the time of induction in the fed-batch method (Fig. 11). The highest soluble
protein amount was obtained with induction at an OD600 of 5. However, the amount
of protein harvested deviated less than 20% from average when inductions between
OD600 of 5 to 12.1 (induction at 16.4 h to 32.8 h after inoculation, respectively)
were compared, showing the robustness of the system in relation to the time of
induction. This indicates the possibility to select the induction time more freely
compared to standard methods, relieving the planning of the cultivation. In addition,
it also indicates that the method could be used to obtain the higher induction cell
densities necessary for expression of toxic proteins.
70
Fig. 11. Expression of recombinant TbTIM in 96-well plates with EnBase at 25 °C and
induction at different cell densities. Expression levels of TbTIM are shown as a ratio of
insoluble protein (white) and the target soluble protein (black). a) Growth curves after
induction, b) Total concentrations of TbTIM, and c) product amounts expressed per cell
unit. The protein concentrations after induction are shown for four different OD600
levels in the novel cultivation system and for the reference culture (M9ZB, induction at
OD600 = 0.3). Cultures were performed with 3 AGU l-1 of glucoamylase. Product synthesis
was induced by addition of 0.5 mM IPTG at the indicated times and cell densities, and
the cultivation was continued for up to 18 hours after induction. Additional ammonia (at
26.2 h) and magnesium (at 36.85 h) were added during the cultivations. (Research article
III, reprinted with permission of BioMed Central)
5.0
0 1 4 18
TIM
[µg
ml-1
, OD
600
=1]
0
20
40
60
80 Soluble
7.5
0 1 4 18
Insoluble
9.8
0 1 4 18
12.1
0 1 4 18
OD600 at induction
Time after induction [h]
TIM
[µg
ml-1
]
0
200
400
600
800SolubleInsoluble
Control (0.3)
0 4 18
OD
600
0
10
20a
b
c
71
4.4.2 Use of enzymatic glucose feeding in the cultivation of methylotrophic Pichia pastoris producing heterologous lipase
Lipases (EC 3.1.1.3) hydrolyze triacylglycerols at the interface between water and
the insoluble substrate (see e.g. a review by Minning et al. (1998)). Lipases also
catalyze the hydrolysis or synthesis of a wide range of natural and unnatural esters.
Takahashi et al. (1998) concluded that Rhizopus lipases have high 1,3-
regiospecificity towards triacylglycerols, and are therefore useful in production of
structured lipids. As versatile enzymes, they are of great interest for industrial
applications. They have been used for example in the enzymatic production of
biodiesel from renewable biological material like tung oil and soy-bean oil (Chen
et al. 2006, Li et al. 2011, Yu et al. 2013). However, the high production costs of
the native enzymes limit industrial use (Houde et al. 2004), and therefore
heterologous production of the enzyme has been intensively studied (Valero 2012).
The small-scale fed-batch method was used for production of heterologous
Rhizopus oryzae lipase in the methylotrophic Pichia pastoris X33 pPICZA-ROL.
The company BioSilta Oy had further developed the small-scale fed-batch system
into a totally soluble form where glucose is fed to the cultivation from a soluble
polymer, later referred to as a soluble substrate (Krause et al. 2010). However, the
principle of the system remained the same as in the gel based-method; the growth
of the cells was controlled by adjusting the amount of polymer-degrading enzyme.
The soluble substrate, kindly provided by BioSilta Oy, was added in a buffered
minimal medium (Invitrogen) to act as a carbon source. Small-scale fed-batch
cultivations in this medium were compared to cultivations in the same minimal
medium where the carbon source was glycerol prior to induction and methanol after
induction.
Even though the expression system based on the AOX1 promoter is generally
thought to be repressed by glucose, successful heterologous protein production in
deepwell plate cultivations was obtained with the slow glucose release method.
Product activities as much as three times higher were observed compared with the
conventional method (Fig. 12). The glucose concentrations in the cultivation
medium were low enough not to repress the AOX1 promoter.
Mixed feeding fed-batch strategies have been used in bioreactors to improve
the final cell densities and/or product yields with P. pastoris (Abad et al. 2010,
Katakura et al. 1998, Mcgrew et al. 1997). In this study, the continuous enzymatic
glucose feed clearly increased the cell densities obtained compared to pure
methanol feed, especially when 2 U l-1 of the enzyme was used (Fig. 12 A1). An
72
increase in the final product concentration of up to three times was obtained. The
methanol addition was found to increase the cell mass when compared to the
cultivation having the enzymatic glucose feed alone. The final cell densities with
1 U l-1 and 2 U l-1 were about 5 and 9 OD600 units higher in methanol-induced slow
glucose release cultivations compared to enzymatic glucose feed alone (Fig. 12 A1
and B1, respectively). The pH change remained at relatively low levels. A pH
decrease of ~ 0.7 units was observed with 2 U l-1 while in the conventional
cultivation no pH decrease was detected (Fig. 12 A2).
Fig. 12. Utilization of enzymatic glucose release in production of fungal lipase with P.
pastoris X33 pPICZαA-ROL in deepwell plates. a) Comparison of conventional
(BMG/BMM) and fed-batch media (BMEB). Conventional cultivation with buffered
minimal methanol medium (BMG/BMM) without glucose polymer or glucose-releasing
enzyme. Fed-batch cultivation with buffered minimal medium including the glucose
polymer (BMEB) and different glucose-releasing enzyme concentrations. Methanol was
added to a final concentration of 0.5% for induction at the time points shown by arrows.
b) Non-induced P. pastoris X33 pPICZαA-ROL cultivations with buffered minimal
medium including the glucose polymer (BMEB) and different glucose-releasing enzyme
concentrations. The error bars were calculated from three parallel cultivations for a),
and from two parallel cultivations for b). (Different representation of data presented in
research article IV)
The highest product activities per cell were observed with the enzymatic
glucose production combined with methanol addition, up to 67 h of cultivation (Fig.
Methanol induction
OD
600
0
10
20
30
Time [h]0 20 40 60 80 100
pH
4
5
6
7 B2
Enzymatic glucose releaseNo induction
Time [h]0 20 40 60 80 100
Act
ivity
[U
ml-1
]
0
2
4
6
Time [h]0 20 40 60 80 100
Glu
cose
[g
l-1]
0.0
0.2
0.4
0.6
0.8
A1
A2
B1
B2
3 AGU l-1
OD600
pH Glucose0.5 AGU l-1
1 AGU l-1
2 AGU l-1
BMG/BMM
Lipase activity
A2, B2A1, B1
73
13). The activity per ml measured in the conventional cultivation reached the
activities measured in the mixed feeding cultivations after 88 h, except in the
cultivation with the smallest enzyme amount. Interestingly, the optical cell densities
in the BMG cultivation were at the same level as the cultivation in BMEB using
1 U l-1 of glucose-releasing enzyme, except at the 88 h time point (Fig. 12 A1). The
higher measured ROL activities in BMEB indicate that the higher carbon amount
available in BMEB is directed to recombinant protein production rather than to
growth, up to 67 h of cultivation.
Fig. 13. ROL activities in relation to biomass. The activities of expressed ROL were
compared to biomass (OD600) in induced BMM cultivations, and to cultivations in BMEB
with different glucose-releasing enzyme concentrations (0.5 U l-1, 1 U l-1 or 2 U l-1). The
error bars were calculated from three parallel cultivations. (Different representation of
data presented in research article IV)
No ROL expression was observed when over-optimal amount of the glucose-
releasing enzyme (5 U l-1) was used with 0.5% methanol addition every 24 h (data
Time [h]
0 40 60 80 100
Activ
ity [U
ml-1
] /O
D60
0
0.00
0.05
0.10
0.15
0.20
0.25
BMMBMEB 0.5U BMEB 1U BMEB 2U
74
not shown). The glucose concentration after 24 h was 3.8 g l-1, and 1.4 g l-1 after 48
h. These results together with the data from non-repressed cultivations indicate that
the repressing glucose concentration is somewhere between 0.2 g l-1 (Fig. 12A2)
and 1.4 g l-1. The non-repressing concentration of glucose seems to be in the range
of the KS value (~ 0.1 g l-1 for glycerol, Jahic et al. (2002), when the same range is
assumed for glucose). As the KS value is the glucose concentration that supports a
growth rate of half the maximum, the non-repressing concentration is clearly in the
growth-limiting range.
Zhang et al. (2010) suggest that the glucose transporter Pphxt1 is directly
involved in the catabolite repression of the AOX1 promoter by taking part in the
signal transduction system of the repression/derepression mechanism. In the low
glucose concentrations (< 1 g l-1), the Pphxt1 transporter mRNA levels were one
third from the levels expressed at the glucose concentration of 5 g l-1, while the
glucose transporter Pphxt2 was fully expressed in the low glucose concentrations.
They also observed that the deletion of Pphxt1 relieved AOX1 repression.
Consequently, if the Pphxt1 is not fully expressed at a low glucose concentration,
one factor in the signal transduction system of total AOX1 repression is missing,
relieving the repression. This could be one explanation for the measured ROL
activities detected in non-induced cultivations (Fig. 12B): Pphxt1 may have not
been expressed due to low glucose levels and therefore AOX1 was not fully
repressed. However, this should be studied further.
The conventional cultivation protocol for methylotrophic P. pastoris includes
the pulse feeding of methanol usually once or twice a day to keep protein
expression ongoing. Since methanol is the only carbon source in the cultivation,
carbon starvation occurs between the pulses when all of the added methanol has
been consumed, as observed in Research article II and later by Ruottinen et al.
(2008). With the small-scale fed-batch method, these starvation phases do not occur
since glucose is continuously produced for the growth medium. The added
methanol is consumed between the pulses, but complete carbon starvation is
avoided due to the constant glucose feeding. Although not proven, the small amount
of glucose may down-regulate the methanol metabolism relieving some of the
oxidative stress in cells, as was suggested for sorbitol by Zhu et al. (2013). However,
this should be studied further.
There are several unanswered questions in the regulation of simultaneous
methanol and glucose utilization. However, the derepression of the AOX1 promoter
at low glucose concentrations is clear and opens the possibility to utilize enzyme-
based glucose delivery in methylotrophic P. pastoris cultivations. When the method
75
is utilized in well plate formats it simplifies the process by avoiding the medium
change prior to induction. The carbon starvation that occurs between the methanol
pulses in the conventional method is also avoided. However, the optimal amount
of the glucose-releasing enzyme should be screened in order to obtain the highest
productivity.
4.4.3 Applications benefitting from enzymatically controlled glucose feeding
The developed enzymatic glucose feeding has been implemented for several other
research purposes (Siurkus et al. 2010, Taskila et al. 2009, Taskila et al. 2010).
Taskila et al. (2009) utilized the method for improving the enrichment cultivation
of beer-spoiling lactic acid bacteria. The growth of the contaminants was
accelerated by applying enzymatic glucose feed, allowing faster detection. Later,
Taskila et al. (2010) significantly decreased the analysis time for beer-spoiling
lactic acid bacteria by utilizing enzyme-controlled glucose feed and a beer-MRS
medium. Siurkus et al. (2010) utilized the small-scale fed-batch method in the high-
throughput multifactorial screening of a clone library of a ribonuclease inhibitor
with 45 vectors in E. coli in microwell plates. The small-scale fed-batch process
was finally scaled up to shake flasks and to a 10 liter fermentor operated in fed-
batch mode. They found that the medium composition and the specific growth rate
before induction had a higher effect on active product formation than the common
factors: temperature or the inducer concentration. They also noticed more variation
in the amount of active protein obtained in scaling up from batch cultures than from
the enzymatic fed-batch process.
The fully soluble cultivation system developed by Krause et al. (2010) has
provided improved product yields as well as cell densities with E. coli (Ehrmann et
al. 2010, Imaizumi et al. 2013, Krause et al. 2010, Li et al. 2014, Mahboudi et al.
2013, Nguyen et al. 2011, Peck et al. 2014, Ukkonen et al. 2011), improved growth
for different yeast strains (Grimm et al. 2012), and improved recovery of heat-
injured Salmonella typhimurium for faster detection (Taskila et al. 2011). This
method improves pH control since its complex additives enable intrinsic pH control
via the deamination principle observed in research article I. The method has also
been successfully utilized in a lactose autoinduction system in E. coli instead of the
conventional glycerol usage, resulting in a more robust small-scale process (Mayer
et al. 2014, Ukkonen et al. 2013a). In a biosorbent preparation study, the Cd2+ and
76
Pb2+ uptake capacity of Bacillus sp. grown with enzymatic glucose release was
enhanced (Palela et al. 2013).
Enzyme-based glucose feeding has resulted in improved cell and/or product
concentrations or yields compared to conventional methods. However, the success
depends on the application used. For example, Hortsch & Weuster-Botz (2011)
compared a completely soluble enzymatic glucose feeding system, complex
medium, Luria-Bertani broth, and terrific broth in the expression of recombinant
alcohol dehydrogenase and formate dehydrogenase in E. coli using milliliter-scale
stirred tank reactors with dissolved oxygen control. They obtained the best growth
in terrific broth, the maximum specific activity of alcohol dehydrogenase in the
complex medium after 6 h of induction, and the highest specific activity of formate
dehydrogenase with enzyme-based glucose delivery after 24 h induction. Clearly,
the results are protein-specific, and some proteins are not as sensitive for the fast
production typical for a complex medium as others. It has been suggested that the
reduced product synthesis rate reduces aggregate formation by reducing the amount
of aggregation-prone folding intermediates inside the cell (Georgiou & Valax 1996).
However, the cultivation conditions in the milliliter-scale stirred reactors used by
Hortsch & Weuster-Botz (2011) are much better in relation to aeration and thus the
results could be completely different in shake flasks or well plates with poor oxygen
transfer rates. The unlimited growth in a complex medium can easily lead to oxygen
depletion, which may have a negative effect on product yield. The variation of the
responses to cultivation conditions indicates the importance of screening for the
optimal conditions for recombinant protein production.
The use of fed-batch-like cultivation conditions on small scale and thus
utilizing controlled growth has clear benefits for cell and product yields, as
discussed above. When the amount of parallel cultivations is high, well plate
formats ease the handling. However, one premise for enzyme-based glucose
delivery was to develop a method applicable for high-throughput systems. Siurkus
et al. (2010) utilized the method for 45 different vectors in E. coli as discussed
above. Tegel et al. (2011) demonstrated the utilization of the soluble version of the
method in the screening of 96 different recombinant human proteins in E. coli and
concluded that the method (150 µl cultures in microwell plates) can replace the
standard shake flask protocol (100 ml cultures in 1 l shake flasks). They obtained
improved volumetric product yields, and applied a high-throughput small-scale
purification system for efficient product analysis.
The developed in situ glucose feeding method has shown high potential for
improving cultivation conditions and production rate in small-scale cultivations.
77
By selecting a suitable amount of the glucose-releasing enzyme, it was possible to
avoid oxygen depletion and acetate accumulation in E. coli cultivations in shake
flasks. An increased amount of soluble protein was obtained with recombinant TIM
compared to the conventional method. Successful deepwell plate cultivations of the
yeast P. pastoris with improved measured product activities showed that the
method is suitable even for the methylotrophic strain. The need for medium
exchange prior to induction, required in conventional cultures, was eliminated,
enabling high-throughput cultivations. The limitations of the developed method
were the gel poured onto the bottom of the cultivation vessel, and the fact that the
pH control still relied mostly on the buffering capacity of the medium. However,
the observed pH drop was not as severe as that observed in conventional shake
flasks. These issues have been solved in other research by utilizing soluble glucose
polymers and pH control via the deamination of amino acids. However, the addition
of amino acids or complex medium component reduces the controllability of the
system.
The possibility to control growth rate and increased cell densities gives new
screening possibilities and flexibility for small-scale cultivations. Higher cell
densities at the time of induction will be useful in production of proteins that are
toxic to the cells. The possibility to select the growth rate by the amount of glucose-
releasing enzyme enables screening of the optimal growth rate for product
formation in a high-throughput manner. Especially when successful protein folding
requires a lower growth rate, the method will surpass the conventional uncontrolled
batch cultivation. Also, as the induction cell density is not as important as in the
conventional method, laborious monitoring of cell density can be avoided, and the
researcher is released for a more flexible timetable. However, based on the
literature survey, it is obvious that the best cultivation protocol has to be screened
for each protein separately. High-throughput process development is becoming
more and more important in the development of new biopharmaceuticals and
industrial enzymes. The cultivation protocol should provide sufficient cell mass and
product for further analysis. For that purpose, the enzymatic glucose release system
may serve as the enabling technology.
78
79
5 Conclusions and future perspectives
When the SenBit system was developed, no other wireless pH and oxygen
measurement system existed for shake flasks. This system is still valid today, since
it can be utilized for wireless on-line measurements in normal non-modified
incubators. The alternative systems are based on measurement of fluorescence or
exhaust gas analysis, and require specialized systems for the cultivation vials or
shaker. Even though the invasive sensors utilized may influence the oxygen transfer
in the flask, the system gives information on how the pH and pO2 responses
correlate to growth, shaking stops, glucose concentrations, optical densities,
organic acid concentrations, and even to induction. The next step could be the
development of a flexible non-invasive wireless monitoring system for accurate
measurements of the parameters. This would require input from sensor and
monitoring system developers to solve the problems of transferring the measured
signal from the shake flask to the transmitter without the large measuring devices
needed, for example, in fluorescence-based devices.
The small-scale glucose feeding method developed in this work fulfilled most
of the expectations set for a fed-batch mimicking system. The cell growth was
controlled without external feeding devices and high cell densities and increased
product amounts compared to conventional methods were obtained. Acetate
accumulation as well as oxygen depletion was avoided in E. coli cultivations. The
method was suitable also for organisms other than E. coli. By utilizing the method,
the starvation phases between methanol pulses in methylotrophic P. pastoris
cultivations were avoided without repressing the AOX1 promoter and losing
productivity.
The use of enzymatic substrate release is not limited only to starch processing.
As was seen in the case with L. salivarius ssp. salicinius, hydrolytic degradation of
proteins for improved utilization was also successful. The limitation of amino acids
was relieved and increase in the product (lactic acid) amount was observed. There
are probably several other applications where a similar in situ hydrolysis principle
could be utilized. As the pH control in mineral medium cultivation has still not been
solved, one interesting application would be the enzymatic feeding of a pH-
controlling agent. However, a pH-responsive pH control should be developed to
exclude pH changes, especially in mineral medium cultivations.
Nowadays, more and more data per experiment can be handled due to the
development of computer data processing capabilities, robotics and bioinformatics.
In addition, more and more of the development of new biological products is being
80
done by utilizing high-throughput methods. Consequently, development of these
methods will continue. The fed-batch-mimicking small-scale system based on
enzymatic glucose release is suitable for high-throughput cultivations, as has been
demonstrated in several studies in the literature. The enzymatic glucose feeding
method is highly suited for protein overexpression in well plate formats. However,
the same methods and conditions are not optimal for all cases, even when the same
strain is used. For the best results, the optimal growth rate, and thus the appropriate
enzyme concentration, should be determined for each protein separately.
81
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Original publications
I Vasala A, Panula J, Neubauer P (2005) Efficient lactic acid production from high salt containing dairy by-products by Lactobacillus salivarius ssp. salicinius with pre-treatment by proteolytic microorganisms. J Biotechnol 117: 421–431.
II Vasala A, Panula J, Bollók M, Illmann L, Hälsig C, Neubauer P (2006) A new wireless system for decentralised measurement of physiological parameters from shake flasks. Microb Cell Fact 5: 8.
III Panula-Perälä J, Šiurkus J, Vasala A, Wilmanowski R, Casteleijn M, Neubauer P (2008) Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks. Microb Cell Fact 7: 31.
IV Panula-Perälä J, Vasala A, Karhunen J, Ojamo H, Neubauer P, Mursula A (2014) Small-scale slow glucose feed cultivation of Pichia pastoris without repression of AOX1 promoter: towards high throughput cultivations. Bioproc Biosyst Eng 37: 1261–1269.
Reprinted with kind permission from Elsevier (I), BioMed Central (II, III), and
Springer Science and Business Media (IV).
Original publications are not included in the electronic version of the dissertation.
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DEVELOPMENT AND APPLICATION OF ENZYMATIC SUBSTRATE FEEDING STRATEGIES FOR SMALL-SCALE MICROBIAL CULTIVATIONSAPPLIED FOR ESCHERICHIA COLI, PICHIA PASTORIS, AND LACTOBACILLUS SALIVARIUS CULTIVATIONS
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