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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

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0 w

ere

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0 t

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cell

conce

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atio

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we

re o

bta

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n the b

act

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com

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qual v

olu

me o

f liq

uid

culti

vatio

n (

volu

me

equal t

o c

om

bin

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agar

and b

roth

volu

mes

in a

bip

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stem

).

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me

tho

d.

Applic

able

for

various

mic

roorg

anis

ms

and

nu

trie

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bro

ths.

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ontr

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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

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d r

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of

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m.

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the g

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th m

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as

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y ch

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the e

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me a

mo

unt. M

ediu

m w

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als

o “

boost

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with

com

ple

x

additi

ves

for

impro

ved r

eco

mbin

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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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