Electrochimica Acta 128 (2014) 41–47

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Electrochimica Acta
journal homepage: www.elsevier.com/locate/electacta

Voltammetric investigation of complex growth media at a bare glassy

carbon electrode: A case study of oxytetracycline
Jan Kruid 1 , Ronen Fogel 1 , Janice Limson ∗,1
Department of Biochemistry, Microbiology and Biotechnology, Rhodes University, South Africa

a r t i c l e i n f o a b s t r a c t

Article history: Reports regarding the voltammetric properties of microbiological growth media are scarce in the litera-
Received 10 June 2013 ture and limited focus has been placed towards the application of electroanalysis for analyte monitoring
Received in revised form 28 August 2013 in these complex media. This work aims to investigate the viability of voltammetry as a quantification
Accepted 31 August 2013
method for analytes in microbiological growth media, using oxytetracycline (OTC) as a model analyte.
Available online 16 September 2013
Analysis of both commercially available and laboratory prepared growth media indicated the presence
of interfering media components which produced anodic peaks at potentials ranging from ∼+0.85 V
Keywords:
to ∼+1.30 V (vs. Ag/AgCl) under acidic conditions and ∼+0.62 V to ∼+1.35 V (vs. Ag/AgCl) under neu-
Voltammetry
Electrochemical impedance spectroscopy
tral pH. These peaks were identified as originating from proteinaceous components of growth media
Microbiological growth media and correlated to the presence of peptone, malt extract and yeast extract. Electrochemical impedance
Protein spectroscopy demonstrated significant increases in the charge transfer resistance for Fe(CN)6 3−/4− redox
Oxytetracycline probes at glassy carbon electrodes in the presence of peptone-comprised media (130.3 ) compared to
media-free buffer (50.4 ). Adsorption of the aforementioned media components to the electrode surface
thus contributes to analytical interference through faradaic and non-faradaic processes. By adapting the
growth media for analyte detection purposes, this study proves the feasibility of detecting OTC, as well
as the use of dilution of the media to further decrease the interferent effects of growth media. A 50-fold
dilution of the media provided a 96.7% recovery of the OTC peak current at 20 ␮M concentration. The
empirical detection limit of OTC in 50-fold diluted media was determined to be 0.5 ␮M, which makes it
applicable to current industrial OTC fermentation processes.
© 2013 Elsevier Ltd. All rights reserved.

1. Introduction colourimetric methods are employed. One of the distinct advan-

The bioprocess industry directed at the production of high value
primary and secondary metabolites of microorganisms, such as
anticancer agents, amino acids and antibiotics [1–3] is ever more
reliant on sensor technology. These include sensors for monitor-
ing of factors vital to the regulation of optimal microorganism
growth such as pH, temperature, dissolved oxygen and nutrient lev-
els as well as monitoring of the production of the drug or product.
Whether through off-line sampling, or on-line analysis, the ability
of the diagnostic measures [4,5] to permit analysis with limited to
no pre-treatment is a key determinant for rapid decision making.
The turbidity of fermentation media [6] in which microor-
ganisms are grown necessitates separate pretreatment steps
prior to sample analysis where traditional chromatographic or
∗ Corresponding author at: Department of Biochemistry, Microbiology and
Biotechnology, Rhodes University, Grahamstown, Box 94, 6140, Eastern Cape, South
Africa. Tel.: +27 46 603 8441; fax: +27 466037576.
E-mail address: j.limson@ru.ac.za (J. Limson).
1
ISE member.
tages of voltammetric methods of analysis is that it is not limited
by solution turbidity [7,8]. Surprisingly few studies have explored
the application of voltammetric methods as a viable alternative
for bioprocess control measurement.
A wide range of different microbiological growth media is used
in the bioprocess industry; either purchased in a preformulated
mixture, adapted or custom made. The selection of the media is
often guided by the particular components (including salts, amino
acids, vitamins) required by the microorganism for optimal growth.
In particular, scant information exists in the literature examin-
ing the voltammetric profile of such different growth media used in
biological processes. A reference to an assessment of tissue culture
media (Dulbecco’s modified Eagle’s medium; DMEM) by Lawrence
et al. [9] indicated the potential for interference from electroactive
components in certain media, with anodic peaks at +0.8 V (vs. SCE),
attributed by the author to the oxidation of a tyrosine phenolic
group (a common component of growth media).
Through impedimetric and voltammetric methods, this study
aimed to probe the feasibility and limitations associated with uti-
lising electrochemical methods for analytical purposes in a range of
commonly used complex microbiological growth media. The study

0013-4686/$ – see front matter © 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.electacta.2013.08.188
42 J. Kruid et al. / Electrochimica Acta 128 (2014) 41–47
Fig. 1. Molecular structure of oxytetracycline [18].
also sought to identify particular media components which may
hinder voltammetric analysis.
The antibiotic oxytetracycline (OTC, Fig. 1) is widely used as
both a human and veterinary drug in the treatment of various
diseases caused by gram positive and gram negative bacteria and
pathogenic Rickettsia [3,10,11]. OTC has also shown promise as a
low-dose/long term growth promoter for livestock [11,12]. Pro-
duction of OTC by Streptomyces rimosus in fermentation vessels
in the bioprocess industry has been explored in a wide range of
different microbiological growth media for optimal generation of
OTC [13–17] which, coupled with its wide-spread use, provides a
relevant case study for this work.
Currently, quantification of OTC production during fermen-
tation occurs mainly through two off-line processes: high
performance liquid chromatography (HPLC) of pre-treated samples
[19,20], or variations of the disc-diffusion method which, based on
the required incubation period of 16–24 h, appears an impracti-
cal method for the OTC production process which itself spans only
5–10 days [13,17,21]. A rapid monitoring system for OTC during
production in bioprocesses would greatly improve on current pro-
cess monitoring for this industry [22].
2. Experimental
2.1. Instrumentation
Cyclic voltammetry (CV), square-wave voltammetry (SWV) and
electrochemical impedance spectroscopy (EIS) studies were per-
formed using a PGSTAT 302 potentiostat (Metrohm Autolab B.V.,
Netherlands) coupled to a Voltammetric Analytical stand (VA 663,
Metrohm Autolab B.V). A three electrode electrochemical cell was
used throughout the study, consisting of a glassy carbon working
electrode (GCE, 3 mm diameter) from BioAnalytical Systems, a plat-
inum wire and a Ag|AgCl electrode (BioAnalysital Systems, 3 M KCl,
NaCl saturated) serving as the counter- and reference electrode
respectively. Electrodes were equidistantly spaced for each analysis
at RT. All CV analyses were performed at 100 mV s−1 , and SWV was
performed with a step potential of 5 mV and a frequency of 20 Hz.
The GCE was cleaned between analyses, using a sequence of rinsing
with MilliQ water (18 M cm−1 ), absolute ethanol, MilliQ water,
polishing on a Buëhler pad (BAS) with aluminium oxide slurry
(<10 ␮m, Sigma–Aldrich) and finally, repeating the rinsing proce-
dure above with absolute ethanol and MilliQ water. The electrode
was scanned cyclically (10 times) in the absence of oxytetracycline
and growth media prior to each analysis in order to establish a
stable baseline.
2.2. Analysis of data and statistical treatment
Data analysis was performed using General Purpose Electro-
chemical Systems (GPES version 4.7.9, Eco Chemie B.V.), Frequency
Response analyser (FRA, version 4.9.007, Metrohm Autolab B.V.)
and Microsoft® Excel 2007 software packages. Data was ana-
lysed using Student’s t-test, with statistical significance assigned
at p < 0.05. CV and SWV data was background subtracted [23] prior
to analysis and is displayed as such in this work.
2.3. EIS analysis
EIS was performed in the presence of the selected growth media.
The electrode was scanned cyclically (10 scans) followed by a sin-
gle scan after the addition of 1 mM Fe(CN)6 3−/4− to confirm the
open-circuit potential (OCP). A frequency range of 100 mHz–15 kHz
was scanned logarithmically at an applied potential equal to the
observed OCP for the respective sample, with an alternating voltage
of 5 mV rms.
2.4. Buffer and analyte preparation
For analysis of oxytetracycline and growth media,
Britton–Robinson (BR) buffer (0.2 M) was prepared using equimo-
lar solutions of boric acid, phosphoric acid, and acetic acid, and
titrated to pH 2 using NaOH. Reference standards for Fe(CN)6 3−/4−
were analysed in 0.1 M KCl. Oxytetracycline (OTC) hydrochloride
(VETRANAL® grade, ≥98% pure) stocks were prepared in 0.2 M
BR buffer (pH 2). Fe(CN)6 3−/4− (1 mM) was prepared in MilliQ
prior to each experiment using equimolar solutions of K4 [Fe(CN)6 ]
and K3 [Fe(CN)6 ] sourced from Fluka and Merck, respectively. For
voltammetric studies in growth media, BR buffer was used in a
volumetric ratio of 49:1 (v/v) BR:growth media.
2.5. Preparation of growth media
For this work, three commercially available growth media i.e.
clostridium nutrient medium (CNM, Fluka), Luria Bertani broth (LB,
Biolab) and nutrient broth (NB, Biolab), and three laboratory made
media i.e. minimal salts media (MSM), a growth media optimised
for OTC production (referred to here as AZ media, modified from
Abou-Zeid et al. [24]) and finally laboratory derived media (LDM)
(formulated in this study), were analysed. All growth media were
prepared in MilliQ water. The composition of the growth media
tested can be found in Table 1. Growth media concentrations were
standardised for each experiment, and are provided within the rel-
evant sections.
3. Results and discussion
3.1. Anodic profile of growth media and selected media
components
Fig. 2(a) and (b) illustrates the potential for anodic interfer-
ence of commercially available (CNM, LB and NB) and laboratory
prepared growth media (MSM, LDM and AZ), respectively at low
pH.
The anodic profile of the growth media (at different pH values)
typically displayed the presence of two distinct groups of peaks,
denoted Group A which starts at ∼+0.85 V and Group B which starts
at ∼+1.15 V (vs. Ag|AgCl) as seen in Fig. 2(a) and (b) at pH 2. Anal-
ysis of MSM revealed no observable anodic peaks in the potential
window studied. The peak groups (A and B) shifted consistently
cathodically with increasing pH as observed in Fig. 2(c) and (d) with
a third group, Group C, becoming visible at potentials more positive
than Group B at pH 7. For the purposes of this study voltammet-
ric data was collected at pH 2 to permit comparison with studies
performed at this pH with OTC analysis (Section 3.3).
As observed the anodic wave for AZ media in Group A (Fig. 2(b))
is larger than that observed for LDM at the same potential. As the
 J. Kruid et al. / Electrochimica Acta 128 (2014) 41–47 43

Table 1
Growth media composition and components concentrations.

Components Component concentration in selected microbial growth media (g/l)

CNM LB NB MSM AZ media LDM

Glucose 5 10 10 10
Glycerol 2.5 2.5
Meat extract 10 1
Peptone 5 5 5
Yeast extract 3 6 2 0.002
Malt extract 5
Starch 1
NaCl 5 12 8 5 5
Sodium acetate 3
l-cysteine HCl 0.5
Agar 0.5
Tryptone 12
KNO3 5
NH4 Cl 0.1
Na2 HPO4 1
NaH2 PO4 0.6
MgSO4 ·7H2 O 0.2
KCl 0.2

Trace elements Concentration of trace elements(␮g/l)

H3 BO3 0.05
CaSO4 0.2
CoSO4 0.1
CuSO4 0.2
FeSO4 3
MnCl2 0.002
ZnSO4 ·7H2 O 0.03

CNM: Clostridium nutrient media (Fluka); LB: Luria Bertani broth (Biolab); NB: nutrient broth (Biolab); MSM: minimal salts media (lab prepared); AZ: Abou-Zeid media
(media proposed by Abou-Zeid et al. (1997) for OTC production by Streptomyces rimosus); LDM: lab derived media (derived from AZ media replacing peptone with malt
extract).

16 16
45 45
Current/μA

Group B
Current/μA

Group B
12 Group A 12 Group A
8 8
30 30
Current/μA

Current/μA

4 4
0.8 0.9 1 1.1 1.2 1.3 1.4 0.8 0.9 1 1.1 1.2 1.3 1.4
Potential vs. (Ag/AgCl)/V Group B Potential vs. (Ag/AgCl)/V
15 Group A 15 Group A Group B

0 0

-15 -15
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
(a) Potential vs. (Ag/AgCl)/V (b) Potential vs. (Ag/AgCl)/V
LB NB CNM Buffer AZ LDM MSM Buffer
140 20 Group B 140 20
Current/μA
Current/μA

16 16 Group B
120 12
Group A 120 12 Group A
Current/μA

Current/μA

100 8 100 8
4 4
80 0.6 0.7 0.8 0.9 1 1.1 80 0.6 0.7 0.8 0.9 1 1.1
Potential vs. (Ag/AgCl)/V Potential vs. (Ag/AgCl)/V
60 60
Group C Group C
40 40
Group B Group A Group B
20 Group A 20
0 0
-20 -20
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
(c) Potential vs. (Ag/AgCl)/V (d) Potential vs. (Ag/AgCl)/V
LB NB CNM Buffer AZ LDM MSM Buffer

Fig. 2. Anodic analysis of (a) commercially available growth media (LB, NB and CNM) and (b) laboratory prepared growth media (AZ, LDM, MSM) analysed in 0.2 M BR buffer
(pH 2), and at pH 7 (c) and (d) for commercially available and laboratory prepared media, respectively. Final growth media concentration was 0.1 g/l. Insets: magnification of
peak-rich areas, labelled Groups A and B, respectively, with Group C labelled in the main figures. Dotted line shows background scans performed in buffered solution only.
44 J. Kruid et al. / Electrochimica Acta 128 (2014) 41–47

(a) 20 AZ medium (0.55 g/L) (b) 20 Peptone (0.10 g/L)

Peptone free AZ medium (0.55 g/L) Yeast extract (0.10 g/L)
BR buffer blank Malt extract (0.10 g/L)
Current/μA 16 16

Current/μA
BR buffer blank

12 12

8 8

4 4
0.5 0.7 0.9 1.1 1.3 1.5 0.5 0.7 0.9 1.1 1.3 1.5
Potential vs. (Ag/AgCl)/V Potential vs. (Ag/AgCl)/V
Fig. 3. SWV overlay of (a) AZ media with and without peptone and (b) peptone, yeast extract and malt extract suspended in 0.2 M BR buffer (pH 2). Dotted line shows
background scans performed in buffered solution only.

only difference between these media is peptone (present only in
AZ media), malt extract (present only in LDM), and KNO3 (present
only in LDM and electrochemically inert [25]), proteinaceous com-
ponents appear to dictate the voltammetric profile of the growth
media in question. Examining the CV for both commercial media
(Fig. 2(a) and (c)) and laboratory derived (Fig. 2(b) and (d)), the
highest current responses for peaks at Group A are for LB, CNM, NB
and AZ. The close proximity of peaks and the formation of distinct
groupings observed for the range of growth media also suggests
that similar oxidisable components were present in each of the
tested media. Peptone is an ingredient common to CNM, NB and AZ
with LB containing yeast extract instead. Based on the composition
of the growth media tested, as reported in Table 1, several protein-
aceous components frequently used in growth media preparations
(malt extract, yeast extract and peptone) were selected for further
analysis and detailed in Fig. 3(a) and (b). Additionally, AZ media
was analysed with and without peptone to confirm the origin of
the anodic peaks.
It is shown by SWV in Fig. 3(a) that a complex media, such as AZ,
shows no appreciable anodic signal in the potential range tested
when peptone, the amino acid source for this media, is omitted
from the sample mixture. Yeast extract and malt extract are shown
to exhibit similar oxidation patterns to that of peptone, with anodic
peaks at ∼0.96 V and ∼1.21 V (Fig. 3(b)). These peaks correspond to
Group A and Group B, identified in Fig. 2. Similar to the voltam-
metric pattern observed for the different growth media (Fig. 2), the
anodic peaks of peptone, yeast extract and malt extract shift to a less
positive potential with increasing pH value. As shown in Fig. 3(b)
the lowest current response was observed with malt extract corre-
sponding to studies at Fig. 2(b) for LDM which utilises malt extract
as its proteinaceous source for microbial growth. These studies thus
(vs. SCE) under neutral conditions [32], as well as the irreversible
oxidation of primary amines in the range of 1.36–1.44 V (vs. SCE)
under neutral conditions [33] further supports the conclusion that
amino acids contribute to the anodic profile of the various growth
media. Therefore, based on the similar compositional breakdown
of peptone, yeast extract and malt extract, as well as their similar
oxidation profiles (Fig. 3(b)), growth media components which con-
tain small peptides, amino acids and vitamins are proposed as the
main oxidisable components and hence voltammetric interferents
of the tested growth media.
3.2. Interaction between growth media and electrode surface
For EIS analysis, three microbiological growth media were
selected: NB, as it is a commonly used growth media for non-
fastidious microorganisms, LDM for its proposed use as a media
for OTC fermentative production, and MSM as it contains a rela-
tively low concentration of yeast extract, shown to be an interferent
in Fig. 3(b) and was shown to produce voltammograms with mini-
mum anodic peaks in Fig. 2. As shown in an example of Nyquist plots
generated for this work, EIS data for BR buffer containing NB does
not correspond with that of media free BR buffer (Fig. 4). An under-
lying semi-circle is observed for BR containing NB, in the higher
frequency range, which is absent for the buffer control.
1000
800
600
- Z''/Ω

attribute the waveforms in Group A and B largely to the different

proteinaceous components present 400
In a compositional breakdown of malt extract, Fluckiger-Isler
et al. [26] reports at least 17 amino acids (including tyrosine and 200 0.2 M BR
cysteine) to be present in the formulation. In an earlier report, lab- NB in BR
oratory grade peptone, yeast extract and malt extract were shown
0
to contain 8 vitamin B-complex members, namely B1 , B2 , B3 , B5 ,
B7 , pyridoxine, folic acid and p-aminobenzoic acid at levels ran-
0 200 400 600 800 1000
ging from ng/l to mg/l [27]. At a neutral pH, amino acids such as Z'/Ω
tyrosine [28,29] and l-cysteine [30], as well as vitamin B6 [31]
have displayed oxidation peaks in the potential range tested in Fig. 4. Magnified Nyquist plot for a bare GCE in 0.2 M BR buffer in the absence and
Fig. 2, which shifted towards less positive potentials with increasing presence of 0.1 g/l NB, using the redox probes Fe(CN)6 3−/4− at 1 mM final concentra-
tion. Frequency range displayed from 900 mHz to 15 kHz. Inset: Randles equivalent
pH value, corresponding to the oxidation profile of peptone, yeast
circuit with CPE, adapted from Suni [34]. Rs is the solution resistance, Rct the
extract and malt extract under similar conditions in this study. The charge-transfer resistance, CPE the constant phase element, Q, and W the Warburg
irreversible oxidation of the phenolic group of tyrosine at +0.80 V impedance.
 J. Kruid et al. / Electrochimica Acta 128 (2014) 41–47 45

Table 2
EIS parameters for data modelled using Circuit A of Fe(CN)6 3−/4− in buffer and selected growth media.

Detection matrix Rs () Q (␮Mho) n CCPE (␮F) Rct () W (S.s1/2 × 10−4 ) 2 (×10−2 )

0.1 M KCl 187.8 (±3.3) 25.45 (±4.14) 0.764 (±0.008) 2.09 12.2 (±1.0) 3.303 (±0.027) 0.174 (±0.057)
Control in BR 254.6 (±13.6) 76.39 (±12.4) 0.697 (±0.024) 6.81 50.4 (±6.4) 2.172 (±0.129) 0.704 (±0.163)
NB in BR 221.7 (±9.0) 25.64 (±2.78) 0.726 (±0.018) 2.98 130.3 (±9.5) 3.177 (±0.041) 0.765 (±0.149)
LDM in BR 244.2 (±9.4) 38.66 (±1.45) 0.671 (±0.014) 1.37 28.5 (±1.2) 3.037 (±0.025) 0.706 (±0.120)
MSM in BR 246.5 (±9.5) 32.25 (±0.54) 0.731 (±0.026) 2.04 17.2 (±8.4) 3.118 (±0.030) 0.578 (±0.132)

Note: Analysis in 0.1 M KCl was performed in the absence of any growth media. Control in BR refers to EIS in 0.2 M BR buffer (pH 2) in the absence of growth media. Final
growth media concentration was 0.1 g/l.

EIS analysis of electrodes in the presence and absence of growth
media is summarised in Table 2. As experimental data could not
be effectively modelled using a conventional Randles circuit, data
was modelled using a modification of the classic Randles equiv-
alent circuit (inset to Fig. 4) where Cdl is replaced by a constant
phase element (CPE). CPE is representative of the electrical double
layer which exists between the interface of the electrode and the
electrolyte [35]. ‘True’ estimates of capacitance (CCPE ) for Q were
calculated to make the data comparable [36] using Eq. (1):
(Yo Rct )1/˛
CCPE =
Rct
where Yo is the value obtained for Q (Mho), ˛ the dimensionless
exponent of Q, and Rct the charge-transfer resistance ().
A decrease in CCPE was observed in all cases where growth media
was present in the solution, compared to the BR buffer control
(Table 2). Overall, CCPE of the GCE exposed to different growth
media decreased, with concomitant increases in modelled Rct val-
ues [34,35]. The dimensionless exponent of Q, n, was less than 1 in
all measured cases, indicating that the electrode does not behave as
an ideal capacitor, as is widely noted for carbonaceous electrodes
within literature [37,38].
Decreases in Cdl have been observed for different metal elec-
trodes onto which proteins were absorbed. Examples of this
phenomenon include the adsorption of bovine serum album (BSA)
to a platinum electrode [39] and foetal bovine serum onto titanium
and CoCrMo alloy electrodes [40,41]. Moulton et al. [41] demon-
strated that solution pH and adsorption time all play significant
roles in protein adsorption onto a gold electrode.
Subsequently, Moulton et al. [35] used EIS to show decreases in
capacitance for their gold electrode following adsorption of human
serum albumin and Immunoglobin G at pH 7, accompanied by an
increase in Rct . This is similar to results presented in Table 2. Moul-
ton et al. concluded that an inert layer of proteins had adsorbed onto
the electrode surface, hindering the passage of electrons between
the redox probe and gold electrode [35].
For more pronounced differences in CCPE and Rct between the
growth media, longer exposure time might be necessary [35,39].
It is important to note that the proteinaceous components studied
3.3. Voltammetry of OTC in complex microbiological growth
media.
OTC has been previously characterised by voltammetry
[11,42,43]. Based on published findings, an electrolyte pH of 2 was
selected for all analyses. Analysis of OTC using SWV (between +0.75
and +1.25 V) OTC produced two distinct oxidation peaks at ∼1.04 V
and ∼1.19 V for peaks denoted pa1(OTC) and pa2(OTC) , respectively,
in Fig. 5(a). Based on structural similarities of different tetracycline
antibiotics and their similar anodic profiles, oxidation of the pheno-
lic moiety at C10 (Fig. 1) was proposed as the source of pa1(OTC) [44].
The origin of pa2(OTC) is believed to be a consequence of products
(1)
formed during the primary oxidation process which yields pa1(OTC)
(data not shown). When analysed independently, LDM is seen to
produce three distinct oxidation peaks denoted paa1(LDM) , paa2(LDM)
and pab(LDM) , respectively (Fig. 5(c)).
Upon the addition of LDM to the working solution, the oxida-
tion peaks of OTC are shown to shift anodically (Fig. 5(b)). The peak
shifts ∼55 mV towards a more positive potential, while the current
response of pa1(OTC) , which is commonly used for OTC quantifica-
tion [11,42,43], decreases by ∼3.2%.
However, in AZ media which has been used for the production
of OTC by S. rimosus [17], the OTC current (Ipa1(OTC) ) response was
decreased by as much as 90% (data not shown). This is attributed to
the oxidation of peptone which, as shown in Section 3.1, oxidises in
the potential window used for OTC quantification. LDM, which was
formulated here as a possible alternative to AZ media, substitutes
peptone for malt extract, the latter previously having being used for
OTC production [20]. To further investigate the effect of LDM on the
primary anodic peak of OTC, analysis was performed in the pres-
ence of a range of LDM dilutions (Fig. 6), with the OTC concentration
remaining unchanged.
Fig. 6(a) demonstrates how increasing dilutions of LDM non-
linearly leads to a cathodic potential shift for pa1(OTC) explaining the
potential shift for pa1(OTC) observed in Fig. 5(b), while the current
6 pa2 (OTC)
pa1 (OTC)
5 a
b
Current/μA

4
here, i.e. peptone, yeast extract and malt extract, are products of
enzymatically or otherwise digested proteins. This would result in 3
smaller oligopeptide molecules with likely different electrode sur-
face coverage properties than those of larger proteins mentioned 2
c
in literature [35,39–41].
Decreases in Rct in the order NB > LDM > MSM correlated with 1
the voltammetric results in Section 3.1, where the size of anodic paa1 (LDM) paa2 (LDM) pab (LDM)
peaks in both groups decreased in the same order for the respective 0
media with no observable peaks for MSM, which had a low yeast 0.75 0.80 0.85 0.90 0.95 1.00 1.05 1.10 1.15 1.20 1.25
extract content. This finding suggests that proteinacious compo- Potential vs. (Ag/AgCl)/V
nents provide interference in the form of oxidation peaks in anodic
Fig. 5. SWV overlay of OTC stock solutions prepared in (a) 0.2 M BR buffer and (b)
voltammetry, as well as adsorbing to electrode surfaces leading to LDM in 0.2 M BR buffer, while (c) shows the analysis of LDM without OTC in 0.2 M
electrode passivation. BR buffer. OTC concentration was 20 ␮M, LDM was 0.55 g/l. pH 2 for all scans.
46
1.12
1.10
Epa1 vs. (Ag/AgCl)/V 1.08
1.06
1.04
1.02
1.00
0 10 20 30
(a) LDM (fold dilution)
Fig. 6. Effect of LDM dilution on (a) the position (V) and (b) signal recovery (%) of pa1(OTC) of 20 ␮M OTC in 0.2 M BR buffer (pH 2) by SWV. Dilutions 10, 25 and 50 fold equate
to 2.75, 1.10 and 0.55 g/l LDM, respectively. Dashed lines represent Epa1 and Ipa1 of OTC in the absence of LDM for (a) and (b), respectively.
response, Ipa1(OTC) , calculated as the percentage signal recovered
compared to analysis in pure BR buffer, is shown to increase in
Fig. 6(b) to 96.7 ± 3.1% at 50-fold dilution of media. This is sig-
nificant as OTC signal recovery in 50-fold diluted AZ media was
<10% due to peptone interference. Sample dilution is thus a viable
option to improve current signal recovery, having been previously
used to reduce/dilute interferents for biosensor applications [45]. In
the presence of 50-fold diluted LDM, an OTC detection limit (LOD)
was experimentally established to be 0.5 ␮M (data not shown). This
is suitably low for practical OTC determination, as OTC fermenta-
tion yields have been reported to vary between 0.24 and 3.71 mM
[14,15,17,21] which remains above the LOD even after 50-fold sam-
ple dilution.
4. Conclusions
The tested growth media NB, CNM and AZ generated anodic
responses at pH 2 and 7 in the potential window 0.0–1.6 V (Fig. 2)
which were attributed, in this study, to the oxidation of pro-
teinacious media components peptone and yeast extract. Under
identical conditions, MSM, used frequently for the cultivation of
bacteria, exhibited no faradaic current response, attributed to its
low yeast extract concentration. This makes MSM a good candidate
media for voltammetric analysis, subject to its use being warranted.
Through EIS analysis, adsorption of proteinacious components to
the electrode surface was observed, leading to increases in Rct of up
to 130.3  for analysis in the presence of NB, compared to 50.4 
in media free buffer (Table 2).
In the case study of OTC analysis in complex growth media, LDM
is offered as an alternative media to AZ, though the substitution of
peptone with malt extract, thereby decreasing anodic interference.
The detection of OTC was also improved though LDM sample dilu-
tion, allowing for nearly full recovery of the OTC peak at a 50-fold
dilution. Thus, the anodic detection current of OTC is reduced by
only 3.2% in the presence of diluted LDM, compared to >90% for
AZ media. The LOD of 0.5 ␮M was deemed suitable for real world
analysis of OTC in fermentation media. This study represents the
first focussed investigation to interrogate the feasibility of using
voltammetry for analysis in complex growth media of relevance to
the bioprocess industry, as part of a continuing study to address
this need in the literature.
Acknowledgement
The authors would like to acknowledge the DST/Mintek NIC for
funding Jan Kruid.
J. Kruid et al. / Electrochimica Acta 128 (2014) 41–47
120
100
Ipa1 signal recovery (%)
80
60
40
20
0
40 50 60 0 10 20 30 40 50 60
(b) LDM (fold dilution)
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