Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online.

Biotechnology Journal

Biotechnol. J. 2015, 10, 1259–1268

DOI 10.1002/biot.201400534

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Research Article

Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online Bettina Luchterhand1,*, Jannis Nolten1,*, Sadik Hafizovic2, Tino Schlepütz1, Sandra Janine Wewetzer1, Elke Pach1, Kristina Meier1, Georg Wandrey1 and Jochen Büchs1 1 AVT

– Biochemical Engineering, RWTH Aachen University, Aachen, Germany Instruments AG, Zurich, Switzerland

2 Zurich

In microtiter plates, conventional online monitoring of biomass concentration based on optical measurements is limited to transparent media: It also cannot differentiate between dead or viable biomass or suspended particles. To address this limitation, this study introduces and validates a new online monitoring setup based on impedance spectroscopy for detecting only viable biomass in 48- and 96-well microtiter plates. The setup was first validated electronically and characterized by determining the cell constants of the measuring geometry. Defined cell suspensions of Ustilago maydis, Hansenula polymorpha, Escherichia coli and Bacillus licheniformis were characterized to find, among other parameters, the most suitable frequency range and the characteristic frequency of β-dispersion for each organism. Finally, the setup was exemplarily applied to monitor the growth of Hansenula polymorpha online. As reference, three different parallel cultures were performed in established cultivation systems. This new online monitoring setup based on impedance spectroscopy is robust and enables precise measurements of microbial biomass concentration. It is promising for future high-throughput applications.

Received Revised Accepted Accepted article online

15 DEC 2014 13 MAR 2015 19 MAY 2015 26 MAY 2015

Supporting information available online

Keywords: Biomass determination · High-throughput system · Impedance spectroscopy · Microtiter plate · Online-monitoring

1 Introduction Bioprocess development and biotechnological screenings are typically performed in small scale bioreactors, such as shake flasks and microtiter plates (MTPs) [1–3]. From standard shake flasks and MTPs usually samples are taken and important fermentation parameters, such as pH, metabolites or biomass, are analyzed offline. However, devices to measure these parameters online are highly

Correspondence: Prof. Jochen Büchs, Department of Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany E-mail: [email protected] Abbreviations: CTR, carbon dioxide transfer rate; DCW, dry cell weight; DOT, dissolved oxygen tension; GPIO, general purpose input/output; IC, integrated circuit; MTP, microtiter plate; NADH, nicotinamide adenine dinucleotide; OD600, optical density at 600 nm wavelength; OTR, oxygen transfer rate; RAMOS, respiration activity monitoring system; RQ, respiration quotient; TB, terrific broth; YPG, yeast-pepton-glycerol

© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

demanded for high-throughput applications [1]. A variety of online monitoring devices for shaken culture systems have been developed [4–6] and are commercially available, e.g. the Respiration Activity MOnitoring System (RAMOS) (HitecZang, Herzogenrath, Germany), the gas analyzing system BCpreFerm for shake flasks (BlueSens gas sensor GmbH, Herten, Germany), and the BioLector technology for MTPs (m2p-labs, Baesweiler, Germany). Biomass is frequently measured by turbidity measurements either in transmitted or scattered light configuration. However, the determination of biomass by optical means is limited to nonturbid transparent media, in particular, because suspended particles inherently interfere with the measurement. Furthermore, it is not possible to distinguish between viable and dead biomass with optical measuring techniques [7]. Impedance spectroscopy can be used to address these limitations [8, 9]. This technique is based on the polarization of intact cell mem-

* These authors contributed equally to this work.

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branes when an alternating electrical field is applied to the cell suspension within a frequency range of 0.1 to maximal 50 MHz [9–13]. Within this frequency range, the capacitance decreases from a high capacitance plateau at low frequencies to a low capacitance plateau at high frequencies. This phenomenon is called β-dispersion and can be fitted with the Cole–Cole equation [7, 14, 15]. The frequency, where half of the β-dispersion is reached, is called characteristic frequency fc. Since the cell size significantly contributes to the sensitivity of the capacitance signal, this technique has been mainly used for large entities, such as cell cultures [9, 12, 16] and fungi [10, 12, 17, 18, 19]. Today, impedance-measuring probes for application in stirred bioreactors are commercially available. Nevertheless, for cultivations in high-throughput systems, i.e. microtiter plates (MTPs), there are no commercial options available. Hofmann et al. [10] proposed a system for impedance measurements of cell suspensions within a frequency range of 0.1–10 MHz in 96-well MTPs. In their work, rotation-symmetric four-electrode configurations on polyimide foils were used to measure cultivations of Hansenula polymorpha and Escherichia coli in complex media. For the small E. coli cells, the applied high frequency limit of 10 MHz was insufficient, due to E. coli´s higher characteristic frequency (fc) of around 14 MHz [12]. Furthermore, the detection of bacteria in comparison to larger microorganisms is challenging, since the cell radius influences the measurement signal with 4th power [20]. Thus, impedance measuring devices with maximum frequencies exceeding 10 MHz are required to detect cell growth of small bacterial cells in high-throughput applications. The aim of this work is to develop and validate an impedance spectroscopy measuring setup for onlinemonitoring of diverse microbial cultures in 48- and 96-well MTPs. This new system ought to be cost-effective, mechanically robust and yield precise measurements at a maximum frequency above 10 MHz. The new system was first validated and characterized by measuring circuit boards with defined electronic components (capacitors and resistances) and KCl solutions. Afterwards, defined cell suspensions of four different fungal and bacterial strains were investigated. Finally, the measuring system was used to monitor the growth of H. polymorpha in a 48-well MTP. At the same time, three parallel cultivations were conducted as references in established bioreactor systems, i.e. in a RAMOS device, BioLector device and a 3 L-bioreactor equipped with an impedance probe.

2 Materials and methods 2.1 Chemicals Chemicals were obtained from Merck (Darmstadt, Germany), Sigma-Aldrich (Hamburg, Germany), Carl Roth

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(Karlsruhe, Germany) and Boehringer Mannheim (Mannheim, Germany). All chemicals were of analytical grade.

2.2 Microorganisms and media Cultivation experiments were conducted with recombinant Hansenula polymorpha RB11 harboring the plasmid pC10-FMD (PFMD-GFP) encoding for GFP under the control of the FMD promoter [21], wildtype Ustilago maydis MB215, Escherichia coli K12 and Bacillus licheniformis ATCC 9945. All strains were maintained in 15% w/v glycerol cryo stocks at -80°C. H. polymorpha RB11 pC10-FMD (PFMD-GFP) was cultivated in complex YPG medium containing 10 g L–1 glycerol, 20 g L–1 tryptone (6681.3, Carl Roth GmbH, Karlsruhe, Germany) and 10 g L–1 yeast extract (2363.2 Carl Roth GmbH, Karlsruhe, Germany). U. maydis MB215 was cultivated in modified Tabuchi medium [20] containing 60 g L–1 glucose, 1 g L–1 yeast extract (2363.2, Carl Roth GmbH, Karlsruhe, Germany), 4 g L–1 (NH4)2SO4, 1 g L–1 KH2PO4, 0.2 g L–1 MgSO4 · 7 H2O, 0.01 g L–1 FeSO3· 7 H2O and 33 g L–1 CaCO3. E. coli K12 was cultivated in Terrific Broth (TB) medium containing 5 g L–1 glycerol, 12 g L–1 tryptone (6681.3, Carl Roth GmbH, Karlsruhe, Germany), 24 g L–1 yeast extract (2363.2, Carl Roth GmbH, Karlsruhe, Germany), 12.5 g L–1 K2HPO4, and 2.3 g L–1 KH2PO4. B. licheniformis ATCC 9945 was cultivated in IFO 702 medium containing of 10 g L–1 peptone (6681.3, Carl Roth GmbH, Karlsruhe, Germany), 2 g L–1 yeast extract (2363.2, Carl Roth GmbH, Karlsruhe, Germany), 1 g L–1 MgSO4 · 7 H2O. The pH-value of 7 for the IFO 702 medium was adjusted with 1 M NaOH and 1 M HCl. The pH-values of the other media were not adjusted.

2.3 Impedance spectroscopy setup 2.3.1 Measuring setup The microtiter plate impedance measuring device basically consists of a HF2IS impedance analyzer, a HF2TA current-to-voltage (I/U) converter (both Zurich Instruments, Zurich, Switzerland), and a self-constructed multiplexer with an integrated preamplifier (AD8130ARZ, Analog Devices, Munich, Germany) (Fig. 1A). The impedance analyzer generates an alternating voltage (Uin) of 100 mV within a frequency range from 0.1–30 MHz. The voltage is applied on the center electrode of the rotationsymmetric four-electrode configuration at the bottom of each well (Fig. 1A and 1B). The resulting current Iout is discharged via the outer electrode and turned into the corresponding voltage by the I/U converter. This signal is fed back to the impedance analyzer. The voltage difference ΔU between the two inner annular electrodes is amplified and led to the impedance analyzer as well. With the voltage difference ΔU, the resulting current Iout (Fig. 1A and 1C), and the phase shift between these signals, the impedance Z of the sample can be calculated as



Biotechnol. J. 2015, 10, 1259–1268


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Figure 1. Overview of the measurement setup for impedance measurement in microtiterplates and the different devices used for validation of the system and online monitoring of fermentations. The general measurement setup (A) consists of an impedance analyzer (HF2IS, Zurich instruments), an I/U converter (HF2TA, Zurich Instruments), a selfmade multiplexer with a preamplifier and a computer. To measure impedance of cell suspensions, in eight wells of a 48-well or a 96-well MTP, four circular electrodes are rotation-symmetrically aligned on a circuit board which makes up the bottom of the respective MTP (B). The modified microtiter plate is adjusted on the multiplexer, which is fixed on a shaker device and covered by a gas hood for aeration with humidified air (C). For the validation of the measurement setup, circuit plates with defined electronic elements (capacitors and resistors) were fabricated (D) and measured instead of a MTP. One circuit board only with resistances (zero capacity) (D, right) was used for calibration, after which another circuit board with additional capacitors of 4.7, 10, 15 and 22 pF (D, left) was measured.

described in the Supporting information. Since the multiplexer consecutively switches between the different wells, the impedance of eight wells of a 48-well or 96-well MTP can be measured one after the other with the fabricated setup. The measured capacitances [pF] and conductances [mS] depend on the electrode geometry. But these capacitances and conductances can be converted into the universally valid capacity [pF cm–1] and conductivity [mS cm–1] by multiplication with the respective cell constant K [cm–1] of the applied electrode configuration. Hence, the cell constant is defined as the relation between conductivity [mS cm–1] and conductance [mS] and is determined by evaluating KCl solutions with defined conductivities. Monitoring and control of the impedance measurements was realized in a self-developed LabVIEW (National Instruments, Munich, Germany) program, containing sub-programs provided by Zurich Instruments (Zurich, Switzerland). Figure 1B illustrates circuit boards manufactured by multi-cb (Brunnthal-Hofolding, Germany) with circular


electrode configurations for eight wells of a 48- and 96-well MTP. The circuit boards were manufactured from a copper-laminated (35 μm) fiber-glass reinforced epoxy resin base (thickness of 1.55 mm). The copper layer was chemically etched to build the electrode design which is based on the layout of Hofmann et al. [10] described as “Design 3”. The four electrodes were covered with gold (thickness of 25–100 nm) and have a width of 0.4 mm. The outer diameters of the four electrodes of the 96-well MTP were 6 mm, 4.8 mm, 2.2 mm, and 1 mm. For the 48-well MTP these outer diameters were 10 mm, 8.8 mm, 2.2 mm, and 1 mm, respectively. The electrodes were contacted from the backside of the circuit boards via IC-sockets. Thus, the circuit boards (Fig. 1B) were manufactured via the so-called “via-in-pad” technology. The circuit boards were fixed to MTP bodies with double-sided adhesive tape (Duplobond 3605.2 Plus, Lohmann, Neuwied, Germany) in which the eight measuring positions were punched out before. Therefore, the circuit boards serve as liquid-tight bottoms of the MTPs. These MTPs were

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connected to the multiplexer and mounted onto a shaker platform using two adapter plates (Fig. 1C). The respective MTP was covered by a hood for aeration with humidified air, to prevent excessive evaporation during cultivation. To validate the measuring device, two test circuit boards were constructed that could be connected to the multiplexer via IC-sockets instead of the microtiter plate. One of these test circuit boards was equipped only with resistors (120 Ω and 0 pF) and the other one additionally with parallel-wired capacitors of 4.7, 10, 15 and 22 pF (Fig. 1D).

To perform impedance measurements of H. polymorpha, U. maydis, E. coli, and B. licheniformis with cell suspensions of defined optical densities, the respective cells were harvested from precultures by centrifugation at 4000 rpm in a Rotina 35 R centrifuge (Hettich Lab Technology, Mulheim a. d. Ruhr, Germany) for 15 min and were resuspended in smaller amounts of supernatant. The pure supernatant was used to calibrate the impedance measuring device prior to the measurements with the concentrated cell suspensions of defined optical density and dilutions thereof. Three measuring cycles were conducted for each cell suspension.

2.3.2 Validation and characterization of the measuring setup

2.4 Fermentations

The impedances of defined electronic resistors or capacitors (Fig. 1D) and KCl solutions were measured to validate and characterize the developed measuring device. All impedance measurements of liquids were conducted at a shaking frequency of 900 rpm and a shaking diameter of 3 mm. The two circuit boards with defined electronic resistors and capacitors were connected to the multiplexer and the background compensation procedure was performed to remove any parasitic capacities and set the initial measurement values to zero. Prior to the measurement, both circuit boards were exchanged with each other, so that the defined electronic capacities on the one circuit board were removed on four measurement positions but added on the opposite four measurement positions. Thus, the four measurement positions where the capacitors were removed ought to show negative capacities, whereas the other four measurement positions where the capacitors were added ought to show positive values equal of value. To determine the cell constants K, five differently concentrated KCl solutions with conductivities of 1.7, 4.55, 9, 12.9, and 18.75 mS cm–1 were filled one after the other into all eight measuring wells of a 48- and 96-well MTP. Filling volumes were 0.2 mL and 1 mL for the 96-well MTP and the 48-well MTP, respectively. Before each measurement of conductance [mS], a system compensation with the respective KCl solution was conducted.

Six parallel fermentations of H. polymorpha were conducted in a 48-deep well MTP (Greiner Bio-One, Frickenhausen, Germany) in the experimental impedance spectroscopy setup. As references, additional parallel fermentations were conducted in a 48-deep well MTP in a selfmade BioLector device, in 250 mL shake flasks in a self-made RAMOS device and in a 3 L-bioreactor (Applikon Biotechnology, Schiedam, Netherlands). Experimental conditions for the MTP devices, impedance spectroscopy and BioLector, were 30°C, 1 mL filling volume, 3 mm shaking diameter and 900 rpm shaking frequency. In the impedance spectroscopy system, capacitance and conductance were determined online. The biomass (as scattered light) and NADH fluorescence in six parallel cultivations were monitored online in a BioLector device [22] in a 48-deep well MTP with transparent bottom and optodes for pH-value and dissolved oxygen (m2p-labs, Baesweiler, Germany), whereas the optodes were not used. For biomass detection, the scattered light intensity at a wavelength of 650 nm was measured. NADH fluorescence was measured at an excitation and emission wavelength of 360 nm and 440 nm, respectively. To enable sterile cultivation conditions, the MTPs were covered with a gas-permeable sealing film (m2p-labs, Baesweiler, Germany) with a silicone layer perforated for 48-well MTPs. Respiration activities, i.e. oxygen transfer rate (OTR), carbon dioxide transfer rate (CTR), and respiratory quotient (RQ), were online-monitored with a RAMOS device [5] in eight parallel shake flasks. The culture conditions were 30°C, 10 mL filling volume, 50 mm shaking diameter and 300 rpm shaking frequency. A batch cultivation of H. polymorpha was conducted in a 3 L stirred bioreactor filled with 1 L YPG medium at 30°C without pH control. The aeration rate was set to 1.5 vvm. The dissolved oxygen tension (DOT) was maintained above 30% air saturation by automatic control of the stirrer speed in a range of 200–800 rpm. Foaming was prevented by adding 1 mL L–1 Plurofac LF1200 (BASF, Ludwigshafen, Germany) prior to the start of cultivation. The bioreactor was equipped with a Standard Remote

2.3.3 Preparation and impedance measurement of defined cell suspensions To prepare cell suspensions with defined OD600-values (Supporting information, Fig. S1), the four studied microbial strains were cultivated in the aforementioned media (see section 2.2). Ten milliliter of the medium were inoculated with at least 200 μL of a cryo stock culture in a 250 mL shake flask and incubated for 15–17 h overnight at 30°C and 300 rpm (H. polymorpha and U. maydis) or 37°C and 350 rpm (E. coli and B. licheniformis) on a LS-Kühner shaker (Birsfelden, Switzerland) with 50 mm shaking diameter.

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Futura probe (Aber Instruments, Aberystwyth, UK) to measure capacity and conductivity within a frequency range of 0.45 to 20 MHz. After inoculation of 1.1 L medium in the bioreactor, 100 mL of the cell broth was removed under sterile conditions and aliquoted to the two MTPs for the impedance measurements and the BioLector, and the eight RAMOS shake flasks. A filling volume of 1 L remained in the bioreactor for cultivation. During the cultivation, samples were taken out of the stirred bioreactor at t = 0 h, 5.2 h, 9 h and 22.7 h for offline analysis.

2.5 Offline analysis Cultivation samples were analyzed with respect to optical density (OD600), pH, dry cell weight (DCW), cell size and cell density. The OD600 of cell broths was determined in semi-micro cuvettes (PS, Carl Roth, Karlsruhe, Germany) with 1 mL filling volume in a Genesys 20 photometer (Thermo Scientific, Dreieich, Germany) at a wavelength of 600 nm. For pH-analyses, a pH 510 probe (Eutech Instruments, Nijkerk, Netherlands) was used. Dry cell weight was determined twice according to Meier et al. (2013) [23]. Cell size and density were determined using a Multisizer 4 Coulter Counter (Beckman Coulter, Krefeld, Germany) equipped with a 100 μm capillary. Furthermore, conductivities of cell-free fresh media or of KCl solutions were measured with a WTW LF 340 probe (Wissenschaftlich-Technische Werkstätten, Weilheim, Germany).

3 Results 3.1 Validation of the measuring setup and system compensation procedure with defined electronic components To test the functionality and accuracy of the new MTP impedance spectroscopy system, two circuit boards with defined electronic elements (Fig. 1D) were measured. As illustrated in Supporting information, Fig. S2, the capacitances of 4.7, 10, 15, and 22 pF were correctly measured up to a maximal frequency of 10 MHz. The mean deviations from the known capacitances were 3.8% at 10 MHz, 16.9% at 20 MHz, and 19% at 30 MHz. This increasing deviation towards higher frequencies is presumably caused by reaching the critical frequency of the measurement setup with its electronic elements.

3.2 Determination of the cell constants with KCl solutions of defined conductivity In Fig. 2, the measured conductances [mS] of five different KCl solutions in 48- and 96-well MTPs with electrodes are plotted against the corresponding conductivities [mS cm–1]. The cell constants of 1.35 cm–1 (48-well MTP)


Figure 2. Conductance and mean capacitance between 0.4 and 30 MHz of KCl solutions with different concentrations. Calibration was performed with the lowest concentration. Measurements of conductance and capacitance were conducted in eight wells in parallel. Each measurement was repeated three times. The mean values of the eight wells with respective standard deviations were plotted against the specific conductivity of the KCl solution. Experimental conditions: T = 30°C, d0 = 3 mm, n = 900 rpm, VL = 0.2 mL (96-well MTP), VL = 1 mL (48-well MTP)

and 1.1 cm–1 (96-well MTP) were obtained from the inverse slopes of the linear fits. The capacitance during the system compensation procedure of each KCl solution plateaued in the frequency range of 0.4 MHz to 30 MHz (data not shown). Thus, Fig. 2 illustrates the mean capacitance [pF] in this range as a function of the conductivity [mS cm–1]. The higher the conductivities of the KCl solutions were, the lower were the measured mean capacitances. The mean deviations of conductance and capacitance of eight independent measurements were maximal ±0.25 mS (1.33%) and ±11.1 pF (10.88%) for the 48-well MTP, and ±0.37 mS (1.97%) and ±23.16 pF (22.91%) for the 96-well MTP, respectively. The smaller mean deviations for the 48-well MTP might be attributed to the larger distance between the measurement electrodes of the 48-well MTP, whereby the electrical field lines penetrate through larger amounts of liquid during the measurements.

3.3 Measurement of defined cell suspension and correlation of capacity and optical density After the validation and characterization of the MTP impedance spectroscopy setup, defined cell suspensions of Hansenula polymorpha RB11 pC10 FMD (PFMD-GFP) in the corresponding medium were measured up to a frequency of 30 MHz. As illustrated in Fig. 3A, the capacitance of a cell suspension of H. polymorpha with an OD600-value of 29, measured in a 48-well MTP, decreased from 4.99 pF at 0.5 MHz to -0.5 pF at 30 MHz. Thus, the corresponding

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Figure 3. Offline measurements of defined cell suspensions of H. polymorpha RB11 pC10-FMD (PFMD-GFP) in a 48-well MTP (A, C, E, G) and a 96-well MTP (B, D, F, H). Defined OD600-values were adjusted with a concentrated cell suspension and the supernatant of the same cell broth. Conductances and capacitances were measured in eight wells in parallel. Mean capacitance/conductance values of the eight wells are depicted with their standard deviations. Each measurement was repeated three times. The crosses in (E) symbolize the online measured capacity differences at the end of the fermentation. Experimental conditions: T = 30°C, d0 = 3 mm, n = 900 rpm, VL = 0.2 mL (96-well MTP), VL = 1 mL (48-well MTP).

capacitance difference ΔC was 5.49 pF. With decreasing OD600-values, ΔC decreased linearly (Fig. 3E). The slope of the linear fit was 0.179 pF/OD600 for H. polymorpha in the 48-well MTP. For the 96-well MTP (Fig. 3B and 3F), the same behavior was obtained as for the 48-well MTP, but the slope of ΔC as a function of OD600 was steeper with a value of 0.222 pF/OD600. Considering the cell constants, capacity values of 2.42 pF cm–1 (48-well MTP) and 2.44 pF cm–1 (96-well MTP) were obtained. Comparing Fig. 3A with 3B and Fig. 3E with 3F, the standard deviations were smaller for the 48-well MTP (max. ±0.79 pF) than those for the 96-well MTP (max. ±1.24 pF). The crosses in Fig. 3E symbolize the online measurement results for the subsequent cultivation experiment, as described in section “Fermentation of H. polymorpha”.

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The measured conductances of the cell suspensions in the frequency range of 0.5–30 MHz in the 48- and 96-well MTP are shown in Fig. 3C and 3D, respectively. In the frequency range from 0.5 MHz up to approximately 10 MHz the measured conductances remain almost constant (48-well MTP: around 2.05 mS; 96-well MTP: around 2.5 mS). By multiplying the respective cell constants, conductivities of 2.77 mS cm–1 (48-well MTP) and 2.75 mS cm–1 (96-well MTP) were obtained. At frequencies higher than 10 MHz, the conductances increased considerably to values of 3.4 mS and 4.13 mS for the 48- (Fig. 3C) and 96-well MTP (Fig. 3D), respectively. The conductance of the medium was independent of the cell density, indicated by the conductance remaining nearly constant for different OD600-values of the cell suspensions (Fig. 3G and 3H).





Table 1. Overview of characteristic impedance spectroscopy parameters for four investigated microbial strains

ΔC/OD600 [pF] 48-well MTP 96-well MTP

fc [MHz] a)

Cole–Cole α [–]a)

0.79 ± 0.08 (0.87 pF cm–1)

0.1 0.4

1–2

0.18 ± 0.01 (0.24 pF cm–1)

0.22 ± 0.02 (0.24 pF cm–1)

1–5

1–3

30

0.05 ± 0.01 (0.07 pF cm–1)

0.06 ± 0.01 (0.07 pF cm–1)

6–10

-0.5–0.1

30

0.10 ± 0.00 (0.13 pF cm–1)

0.11 ± 0.01 (0.12 pF cm–1)

15–20

-2.5–0

Strain

Medium (conductivity)

flow [MHz]

fhigh [MHz]

U. maydis MB215

Tabuchi (2.7 mS cm–1)

0.1

30

0.6 ± 0.03 (0.81 pF cm–1)

H. polymorpha RB11 pC10FMD (PFMD-GFP)

YPG (2.8 mS cm–1)

0.5

30

E. coli K12

TB (18 mS cm–1)

1

B. licheniformis ATCC 9945

IFO-702 (5.8 mS cm–1)

2

a) These parameters were calculated by approximizing the dispersions with the Cole–Cole equation (10) in Supporting information.

Identical experiments as for H. polymorpha were performed for Ustilago maydis MB215, Escherichia coli K12 and Bacillus licheniformis ATCC 9945. In Supporting information, Fig. S3–S5 the corresponding results are shown. In Table 1, the conductivities of the fermentation media, measured in the new MTP impedance spectroscopy setup (48- and 96-well MTP), and the correlations between capacitances and OD600 (ΔC/OD600) are summarized for the four investigated strains. The conductivities of the fermentation media for E. coli and B. licheniformis with respective values of 18 mS cm–1 and 5.8 mS cm–1 were much higher than those obtained for both fungi. While the capacitance measurements for U. maydis and H. polymorpha were evaluated in a frequency range of 0.1–30 MHz and 0.5–30 MHz, those for E. coli and B. licheniformis were evaluated from 1–30 MHz and 2–30 MHz, respectively. For the two fungal strains clearly higher ΔC/OD600-values were obtained than for the two bacterial strains. Additionally, approximations for all obtained β-dispersions were conducted according to the Cole–Cole equation (Supporting information) [15]. The characteristic frequencies fc of the β-dispersions of E. coli and B. licheniformis (6–10 MHz and 15–20 MHz), according to approximations with the Cole–Cole equation, were considerably higher than those of U. maydis and H. polymorpha (0.1–0.4 MHz and 1–5 MHz) (Table 1). Moreover, the Cole–Cole a-values for the bacterial strains are around zero, while the values for the fungal strains are between one and three (Table 1).

3.4 Fermentation of H. polymorpha to prove the online monitoring capability To finally prove the applicability of the new impedance spectroscopy system for online-monitoring of microbial cultures in MTPs, cultivations of H. polymorpha were performed. Besides the cultivation with impedance spectroscopy in MTP (Fig. 4A), three parallel cultivations were


performed in established cultivation systems as a reference: (i) a MTP-based BioLector cultivation (Fig. 4B); (ii) a 250 mL shake flask-based RAMOS cultivation (Fig. 4C); and (iii) a stirred 3 L bioreactor cultivation with integrated biomass probe (Fig. 4D). Figure 4A illustrates, the courses of the curves for the capacity differences and conductivities during the cultivation of H. polymorpha measured in a 48-well MTP with the new MTP impedance spectroscopy setup. Cultivations were performed in six wells of the 48-well MTP in parallel. The measured signals were averaged and plotted together with their corresponding standard deviations. As a control, pure medium was measured in two wells in parallel (Fig. 4A, dotted curves). Within the first hour of cultivation, the capacity difference value of between 0.5 and 30 MHz and the conductivity increased slightly to values of 0.4 pF cm–1 and 5.1 mS cm–1, respectively. From 1 h to 8 h, the capacity difference increased exponentially from 0.4 pF cm–1 to 4.1 pF cm–1, indicating an exponential growth of H. polymorpha. Subsequently, the capacity difference slowly increased to 4.9 pF cm–1 at 23.5 h. The conductivity decreased from 5.1 mS cm–1 to 4.8 mS cm–1 between 1 h and 6 h. Then, between 6 h and 8 h, the conductivity increased up to a value of 5.3 mS cm–1, followed by a slight increase up to a value of 6 mS cm–1 at the end of cultivation. The capacity difference values and conductivities of the sterile medium remained constant during the entire fermentation, obtaining values of 0.1 pF cm–1 and 5.1 mS cm–1, respectively. Growth of H. polymorpha was monitored via scattered light and NADH fluorescence measurements in the BioLector device (Fig. 4B). From the beginning of cultivation until 9 h, the scattered light as well as NADH fluorescence intensity increased exponentially up to a value of 5.24 × 106 and 1 × 106 arbitrary units, respectively. Afterwards the scattered light signal remained almost constant until the end of cultivation. By contrast, after 9 hours, the NADH fluorescence intensity decreased

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Figure 4. Online measurements of a cultivation of H. polymorpha RB11 with vector pC10-FMD (PFMD-GFP). The cultivation was conducted in four different systems running in parallel: new impedance spectroscopy setup for MTPs (A), BioLector microtiter plate culture device (B), RAMOS shake flask culture device (C), Bioreactor with impedance probe (D). Experimental conditions: Impedance spectroscopy device: d0 = 3 mm, n = 900 rpm, VL = 1 mL (48 well MTP). Cultivation was performed in six wells in parallel and the mean values and standard deviations are shown. Two wells were filled with medium only for reference purpose and shown as dotted lines; Biolector: d0 = 3 mm, n = 900 rpm, VL = 1 mL (48 well MTP). Cultivation was performed in six wells in parallel for each scattered light and NADH. Two wells were filled with medium only for reference purpose;RAMOS: 250 mL shake flasks, d0 = 50 mm, n = 300 rpm, VL = 10 mL. Cultivation was performed in eight flasks in parallel. The mean values with their respective standard deviations are plotted; Bioreactor: DOTmin = 30%, VL = 1 L, without pH control, T = 30°C.

steadily to a minimal value of around 0.4 × 106 at the end of cultivation. In the shake flask RAMOS device (Fig. 4C), the OTR and CTR were measured during the cultivation of H. polymorpha. The OTR and CTR values increased exponentially until 9 h of cultivation and reached maximal values of 44 mmol L–1 h–1 and 34 mmol L–1 h–1, respectively. From that maximal value, both transfer rates dropped to 5 mmol L–1 h–1 between 9 and 11 h. Then, the OTR and CTR curves decreased slowly to zero until the end of cultivation. During the cultivation in the stirred bioreactor (Fig. 4D), the OTR, CTR, capacity and conductivity values were measured online. Samples were analyzed offline for OD600 (Fig. 4D), cell density, and cell dry weight. The OTR and CTR curves increased exponentially to values of 36 mmol L–1 h–1 and 29 mmol L–1 h–1 within the first 9 h of cultivation. Then, after a sharp drop, these transfer rates slightly decreased to zero until the end of cultivation. The capacity difference signal reached a maximal value of

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3.8 pF cm–1 at 9 h. Finally, the value slightly decreased to 2.2 pF cm–1 at the end of fermentation. The conductivity decreased from a value of 5.3 mS cm–1 to 4.5 mS cm–1 within the first 9 h. After 9 h, the conductivity increased to a final value of 5.4 mS cm–1. Offline samples showed that the cultivation started with an OD600-value of 3 which increased to a final value of 19.75. The final cell density was 2.92 108 cells mL–1, which corresponds to a cell dry weight of 9.6 g L–1 (data not shown).

4 Discussion 4.1 Validation of the measurement setup with defined electronic components As shown in Supporting information, Fig. S2, the system compensation procedure was successful. The measured capacitances are precise up to 10 MHz, when defined electronic elements (capacitors and resistors) were meas-





ured (Supporting information, Fig. S2B). At frequencies of around 20 MHz, the measured capacitances increasingly differ from the known values. The negative capacitances for four measuring positions are caused by the previous system compensation with the circuit board containing capacitors. During the subsequent measurement, the circuit board without capacitors was measured, thereby resulting in negative capacitance signals.

4.2 Determining the cell constants To determine the cell constants for the 48-well and 96-well MTP that consider both different electrode geometries, various concentrated KCl solutions were measured (Fig. 2). This procedure is well established in literature to characterize measurement devices for impedance spectroscopy [24, 25]. It was found that the capacitance level between 0.4 and 30 MHz decreased with increasing conductivities. This phenomenon will be investigated in further studies. The standard deviations of capacitances and conductances calculated for the 48-well MTP were smaller than those for the 96-well MTP. Consequently, the measurement error decreases in relation to the height of the measurement signal.

4.3 Correlation of measured capacitance and optical density and determination of the characteristic frequencies To prove the functionality of the new impedance spectroscopy setup for online monitoring of biomass in MTPs, defined cell suspensions of U. maydis, H. polymorpha, B. licheniformis, and E. coli were investigated (Table 1, Fig. 3; Supporting information, Fig. S3–S5). In comparison to the MTP impedance spectroscopy device reported by Hofmann et al. (max. 10 MHz), the extension of the upper frequency is a clear improvement. Within a frequency range of 0.1 to 30 MHz, β-dispersions were obtained for all four investigated strains. The differences in the height of the β-dispersion (ΔC) in relation to the OD600 (ΔC/OD600) between the fungi and bacterial strains are caused by the different cell sizes. The mean cell diameter influences the capacitance signal by the 4th power [20]. Since fungal cells are bigger than bacterial cells, their ΔC/OD600-values are also higher. This result agrees with the observations made by Markx and Davey [26]. In this current study, H. polymorpha shows a similar characteristic frequency as S. cerevisiae with values of 1–5 MHz (Table 1) and 1.4–3.4 MHz [27], respectively. Likewise, Hofmann et al. [10] determined a characteristic frequency of 5.4 MHz for H. polymorpha. The characteristic frequency of around 10 MHz for E. coli is in good agreement with the observation made by Hauttmann and Müller [12]. Furthermore, for the two investigated bacterial strains, the entire β-dispersion could not be evaluated, since the low capacitance plateau was not yet reached at 30 MHz (Supporting infor-


mation, Fig. S4A, S4B, S5A and S5B). Consequently, the values for ΔC/OD600 would presumably have been higher, if the entire β-dispersion could have been evaluated. Thus, for measurements of bacteria, the frequency range should ideally be extended to higher frequencies, e.g. by using a more appropriate high-end I/U converter. Additionally, in the range of low frequencies, standard deviations were higher. Presumably, this is caused by the electrode configuration that was used [24]. This effect is even more pronounced in the case of low biomass and high conductivities [28]. One solution to reduce electrode polarization could be to cover the electrodes with platinum [25]. If the capacitances and conductances of the fungal strains are compared to those of the bacterial strains (Table 1, Fig. 3; Supporting information, Fig. S3–S5), it becomes clear that, in accordance to Hofmann [29], the accuracy decreased for higher conductivities.

4.4 Online monitoring of cell growth by cultivation of H. polymorpha For the cultivation experiments with online monitoring of cell growth, the yeast H. polymorpha was chosen. Prior to these cultivations, biocompatibility tests in the RAMOS device had proven that the plate material had no inhibiting effect on the growth of H. polymorpha (data not shown). An increase in capacity difference and conductivity is observable in Fig. 4A during the first hour of cultivation, which cannot be explained by microbial growth. Presumably, this increase is caused by the activation of the gold electrodes [30]. Furthermore, measured capacity difference values among the six wells containing cell suspensions show standard deviations of around 0.5 pF cm–1 (Fig. 4A). This may be caused by unequal or insufficient thickness of the gold layer on the electrodes. Thus, the electrodes will be optimized in a further study by electroplating of a thicker gold layer on the electrodes. After 1 h of cultivation the capacity difference values increased exponentially (Fig. 4A), demonstrating the exponential growth of H. polymorpha. The curves for scattered light intensity (Fig. 4B), OTR, CTR (Fig. 4C), capacity difference measured with the impedance probe, and OD600 (Fig. 4D) likewise change exponentially until around 9 h of cultivation, thereby proving the applicability of the new impedance spectroscopy device. Comparing the capacity difference measured with the new impedance spectroscopy setup for MTPs with the impedance probe in the stirred bioreactor, indicates excellent agreement between both methods (Fig. 4A and 4D) in terms of time, value and course of the signals. The slightly lower capacity difference value obtained with the impedance probe in the stirred bioreactor might be caused by the limited frequency range of up to 20 MHz of that probe. Additionally, the capacity differences measured in the MTP (Fig. 4A) showed less scattering com-

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pared to the values of the impedance probe in the stirred bioreactor (Fig. 4D). Since gas bubbles influence the measurement of capacity in the bioreactor, the absence of bubbles in the MTP resulted in the desirable smooth capacity signals. Furthermore, the conductivities measured in the MTP and in the bioreactor were comparable.

4.5 Summary and outlook In summary, the impedance spectroscopy measurement technique was successfully implemented on MTP scale. Its applicability for monitoring microbial cultures could be proven in particular for fungi, because the entire β-dispersion could be detected up to a frequency of 30 MHz. The new setup for MTPs is an appropriate system for monitoring viable cells. The order of magnitude was similar to that of a wellestablished impedance probe for stirred bioreactors. This proposed setup can be improved by optimizing the electrodes, and increasing the number of measured wells to 48 or 96, respectively. By implementing these improvements, this technique will be well suited for high-throughput applications. In future studies, it might for example be interesting to investigate the growth of filamentous organisms to detect morphology changes by the change of the shape of the β-dispersion curve.

This work was performed within the research network “Genomik Transfer”, funded by the Federal Ministry of Research and Education, Germany (FKZ 0315632B). The authors declare no financial or commercial conflict of interest.

5 References [1] Büchs, J., Introduction to advantages and problems of shaken cultures. Biochem. Eng. J. 2001, 7, 91–98. [2] Rao, G., Moreira, A., Brorson, K., Disposable bioprocessing: The future has arrived. Biotechnol. Bioeng. 2009, 102, 348–356. [3] Duetz, W. A., Microtiter plates as mini-bioreactors: Miniaturization of fermentation methods. Trends Microbiol. 2007, 15, 469–475. [4] Klöckner, W., Büchs, J., Advances in shaking technologies. Trends Biotechnol. 2012, 30, 307–314. [5] Anderlei, T., Büchs, J., Device for sterile online measurement of the oxygen transfer rate in shaking flasks. Biochem. Eng. J. 2001, 7, 157–162. [6] Anderlei, T., Zang, W., Papaspyrou, M., Büchs, J., Online respiration activity measurement (OTR, CTR, RQ) in shake flasks. Biochem. Eng. J. 2004, 17, 187–194. [7] Maskow, T., Röllich, A., Fetzer, I., Yao, J., Harms, H., Observation of non-linear biomass–capacitance correlations: Reasons and implications for bioprocess control. Biosens. Bioelectron. 2008, 24, 123–128. [8] Asami, K., Gheorghiu, E., Yonezawa, T., Real-time monitoring of yeast cell division by dielectric spectroscopy. Biophys. J. 1999, 76, 3345–3348. [9] Justice, C., Brix, A., Freimark, D., Kraume, M. et. al., Process control in cell culture technology using dielectric spectroscopy. Biotechnol. Advances. 2011, 4, 391–401.

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ISSN 1860-6768 · BJIOAM 10 (8) 1091–1326 (2015) · Vol. 10 · August 2015

Systems & Synthetic Biology · Nanobiotech · Medicine

8/2015 Bioengineering Industrial biotechnology Bioprocess development


Cover illustration Special Issue: ESBES. This issue of BTJ highlights a selection of articles presented at the 10th European Symposium on Biochemical Engineering Sciences (ESBES), which was held in Lille, France, September 7–10, 2014. The issue is edited by Guilherme Ferreira and Philippe Jacques and includes articles on metabolic engineering, protein expression and bioprocess development. The cover shows the Grande Place in Lille. © bbsferrari – Fotolia.com

Biotechnology Journal – list of articles published in the August 2015 issue. Editorial: Bioengineering for a better quality of life Guilherme Ferreira and Philippe Jacques

http://dx.doi.org/10.1002/biot.201500419 Forum Process analytical technologies in food industry – challenges and benefits: A status report and recommendations Bernd Hitzmann, Ralph Hauselmann, Andreas Niemoeller, Daryoush Sangi, Jens Traenkle and Jarka Glassey

http://dx.doi.org/10.1002/biot.201400773 Commentary A new use for existing technology – continuous precipitation for purification of recombination proteins Veena Warikoo and Rahul Godawat

http://dx.doi.org/10.1002/biot.201400840 Review Clinical-scale purification of pluripotent stem cell derivatives for cell-based therapies Gonçalo M. C. Rodrigues, Carlos A. V. Rodrigues, Tiago G. Fernandes, Maria Margarida Diogo and Joaquim M. S. Cabral

http://dx.doi.org/10.1002/biot.201400535

Review Towards plant protein refinery: Review on protein extraction using alkali and potential enzymatic assistance Yessie W. Sari, Wilhelmus J. Mulder, Johan P. M. Sanders and Marieke E. Bruins

http://dx.doi.org/10.1002/biot.201400569 Review Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats Ruben R. G. Soares, Ana M. Azevedo, James M. Van Alstine and M. Raquel Aires-Barros

http://dx.doi.org/10.1002/biot.201400532 Review Engineering microbial cell factories: Metabolic engineering of Corynebacterium glutamicum with a focus on non-natural products Sabine A. E. Heider and Volker F. Wendisch

http://dx.doi.org/10.1002/biot.201400590 Review Current progress in high cell density yeast bioprocesses for bioethanol production Johan O. Westman and Carl Johan Franzén

Review Bioreactor control improves bioprocess performance


Rimvydas Simutis and Andreas Lübbert

Research article Continuous precipitation of IgG from CHO cell culture supernatant in a tubular reactor

http://dx.doi.org/10.1002/biot.201500016 Mini-Review Photobioreactors with internal illumination – A survey and comparison

Nikolaus Hammerschmidt, Beate Hintersteiner, Nico Lingg and Alois Jungbauer


Martin Heining and Rainer Buchholz




Research Article Fermentation broth components influence droplet coalescence and hinder advanced biofuel recovery during fermentation Arjan S. Heeres, Karin Schroën, Joseph J. Heijnen, Luuk A. M. van der Wielen and Maria C. Cuellar

Research Article Maximizing the utilization of Laminaria japonica as biomass via improvement of alginate lyase activity in a two-phase fermentation system Yuri Oh, Xu Xu, Ji Young Kim and Jong Moon Park



Research Article Modeling leucine’s metabolic pathway and knockout prediction improving the production of surfactin, a biosurfactant from Bacillus subtilis

Research Article Improving recombinant protein production in the Chlamydomonas reinhardtii chloroplast using vivid Verde Fluorescent Protein as a reporter

François Coutte, Joachim Niehren, Debarun Dhali, Mathias John, Cristian Versari and Philippe Jacques

Stephanie Braun-Galleani, Frank Baganz and Saul Purton

http://dx.doi.org/10.1002/biot.201400541 Research Article A xeno-free microcarrier-based stirred culture system for the scalable expansion of human mesenchymal stem/stromal cells isolated from bone marrow and adipose tissue Joana G. Carmelo, Ana Fernandes-Platzgummer, Maria Margarida Diogo, Cláudia Lobato da Silva and Joaquim M. S. Cabral

http://dx.doi.org/10.1002/biot.201400586 Research Article Dynamic flux balancing elucidates NAD(P)H production as limiting response to furfural inhibition in Saccharomyces cerevisiae

http://dx.doi.org/10.1002/biot.201400566 Research Article Regulation of the NADH pool and NADH/NADPH ratio redistributes acetoin and 2,3-butanediol proportion in Bacillus subtilis Teng Bao, Xian Zhang, Xiaojing Zhao, Zhiming Rao, Taowei Yang and Shangtian Yang

http://dx.doi.org/10.1002/biot.201400577 Research Article Engineering a branch of the UDP-precursor biosynthesis pathway enhances the production of capsular polysaccharide in Escherichia coli O5:K4:H4

Pornkamol Unrean and Carl J. Franzen

Donatella Cimini, Elisabetta Carlino, Alfonso Giovane, Ottavia Argenzio, Ileana Dello Iacono, Mario De Rosa and Chiara Schiraldi



Research Article Newly designed and validated impedance spectroscopy setup in microtiter plates successfully monitors viable biomass online

Research Article Phenotypic variability in bioprocessing conditions can be tracked on the basis of on-line flow cytometry and fits to a scaling law

Bettina Luchterhand, Jannis Nolten, Sadik Hafizovic, Tino Schlepütz, Sandra Janine Wewetzer, Elke Pach, Kristina Meier, Georg Wandrey and Jochen Büchs

http://dx.doi.org/10.1002/biot.201400534 Research Article A versatile, non genetically modified organism (GMO)-based strategy for controlling low-producer mutants in Bordetella pertussis cultures using antigenic modulation

Jonathan Baert, Romain Kinet, Alison Brognaux, Anissa Delepierre, Samuel Telek, Søren J. Sørensen, Leise Riber, Patrick Fickers and Frank Delvigne


Philippe Goffin, Thomas Slock, Vincent Smessaert, Philippe De Rop and Philippe Dehottay




A temperature regulator for microtiter plates.

Rapid Determination of Ionization Constants (pK a) by UV Spectroscopy Using 96-Well Microtiter Plates.

Mini-transfections on 96-well microtiter plates.

96-well microtiter plates for biofouling simulation in biomedical settings.

Revisiting the electrochemical impedance spectroscopy of magnesium with online inductively coupled plasma atomic emission spectroscopy.

Impedance spectroscopy.

Development of stable and reproducible biosensors based on electrochemical impedance spectroscopy: three-electrode versus two-electrode setup.

Newly designed anterolateral and posterolateral locking anatomic plates for lateral tibial plateau fractures: a finite element study.

Evaporation as a cause of positional differences in cell plating and growth in microtiter plates.

Antibody Microarray for E. coli O157:H7 and Shiga Toxin in Microtiter Plates.

Growth, morphology and chemosensitivity studies on postconfluent cells cultured in 'V'-bottomed microtiter plates.

Copper iodide staining and determination of proteins adsorbed to microtiter plates.

Measuring oxidative stress resistance of Caenorhabditis elegans in 96-well microtiter plates.

Fed-batch operation in special microtiter plates: a new method for screening under production conditions.

Filamentous fungi in microtiter plates-an easy way to optimize itaconic acid production with Aspergillus terreus.

Newly designed occluder pin for presacral hemorrhage.

A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates.

A sensitive sandwich enzyme immunoassay for measurement of insulin on microtiter plates.

Impedance spectroscopy to monitor fracture healing.

Sensing Estrogen with Electrochemical Impedance Spectroscopy.

High-throughput impedance spectroscopy biosensor array chip.

Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein A and neomycin phosphotransferase II in two-chamber-well microtiter plates.

"Just Another Tool for Online Studies" (JATOS): An Easy Solution for Setup and Management of Web Servers Supporting Online Studies.

Mandibular distraction osteogenesis with newly designed electromechanical distractor.