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Analytica Chimica Acta 823 (2014) 25–31

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Analytica Chimica Acta
journal homepage: www.elsevier.com/locate/aca

Electrochemical monitoring of citric acid production by
Aspergillus niger
Anna Kutyła-Olesiuk, Urszula E. Wawrzyniak, Patrycja Ciosek, Wojciech Wróblewski *
Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

H I G H L I G H T S

G R A P H I C A L A B S T R A C T 

Citric acid fermentation process
(production) by Aspergillus niger. 
Qualitative/quantitative monitoring
of standard culture and culture
infected with yeast. 
Electronic tongue based on potentiometric and voltammetric sensors. 
Evaluation of the progress and the
correctness of the fermentation process. 
The highest classification abilities of
the hybrid electronic tongue.

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 20 January 2014
Received in revised form 24 March 2014
Accepted 25 March 2014
Available online 26 March 2014

Hybrid electronic tongue was developed for the monitoring of citric acid production by Aspergillus niger.
The system based on various potentiometric/voltammetric sensors and appropriate chemometric
techniques provided correct qualitative and quantitative classification of the samples collected during
standard Aspergillus niger culture and culture infected with yeast. The performance of the proposed
approach was compared with the monitoring of the fermentation process carried out using classical
methods. The results obtained proved, that the designed hybrid electronic tongue was able to evaluate
the progress and correctness of the fermentation process.
ã 2014 Elsevier B.V. All rights reserved.

Keywords:
Citric acid production
Fermentation process
Aspergillus niger
Process monitoring
Electrochemical sensors
Hybrid electronic tongue

1. Introduction
Citric acid (2-hydroxy-1,2,3-propanetricarboxylic acid) is involved in the metabolic process of energy conversion such as the
citric acid cycle (Krebs cycle, tricarboxylic acid cycle) [1,2]. Due to
its acidic and antioxidant properties, the citric acid is widely
applied as an acidity regulator, preservative and flavoring agent in
food industry [3] but also in chemical industry, pharmacy,

* Corresponding author. Tel.: +48 22 2345631; fax: +48 22 2345631.
E-mail address: wuwu@ch.pw.edu.pl (W. Wróblewski).
http://dx.doi.org/10.1016/j.aca.2014.03.033
0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

medicine [4–6]. Although lemon juice is a rich source of citric
acid [7], its industrial-scale production by fermentation has been
established for decades. Many microorganisms were exploited to
obtain citric acid, however, filamentous fungus Aspergillus
(especially Aspergillus niger) are the main producers [8]. The
biochemical mechanism by which A. niger accumulates citric acid
has attracted much scientific attention [9]; nevertheless, further
methods of biosynthesis of this acid by other fungi such as Candida
lipolytica, Yarrowia lipolytica were also developed [10,11].
The fermentation production can be carried out by the surface
method (on trays), submerged method, on solid media and in
dynamic conditions [12]. Fermentation process of a substrate to

After four days one of the cultures had been infected with baker’s yeast (Saccharomyces cerevisiae). it should be emphasized. appropriate analytical methods of culture monitoring are selected to assure the proper breeding of Aspergillus niger (pH. The initial pH of the medium was 3. and 31–33 wt% high-molecular-weight PVC (the membranes based on an ion-exchanger contained 3. It should be taken into account that the contamination by other microorganisms may limit the efficiency of citric acid production. this approach enabled the observation of spore germination. alcohol and polyphenol content. niger (before the inoculation. It was observed that the electronic tongue enabled to detect the evolution of taste and aroma profile [28]. whey [17] and glycerol [18] was also investigated. simple devices based on chemical sensor arrays coupled with chemometric analysis – electronic tongues (ET) – were also employed for the monitoring of biotechnological processes [26]. which contains sucrose [14]. the mycelium was proliferated on standard Petri dish on agar medium). dependent on culture method and on the selection of optimal parameters (optimum pH range. a novel hybrid electronic tongue system was developed for the monitoring of the citric acid fermentation process by A. oxygen content. Since the formation of small amounts of others carboxylic acids (especially oxalic and gluconic acid) is evidenced during the synthesis of citric acid. The quantification of citric acid produced during the fermentation process is commonly performed by HPLC [22]. optical). citrate and oxalate was achieved with good precision [35]. Potentiometric and voltammetric electronic tongue was demonstrated to be a promising tool for the qualitative and quantitative analysis of samples collected periodically during the beer fermentation process [32]. The samples were collected in 3 successive weeks. All studied samples were analyzed in five replicates. bis(2-ethylhexyl) sebacate (DOS). All experiments were performed in bulk solutions at room temperature.20-tetra-phenylporphyrinato) zirconium(IV) (Zr(IV)[TPP]Cl2) was synthesized via a metallation of the free porphyrin (Porphyrin Products) using ZrCl4 (Fluka) [36]. High-molecular-weight poly(vinyl chloride) (PVC). lipophilic salts: potassium tetrakis [3. that only a single attempt was made to control the fermentation process involving A. the samples were subsequently thawed and filtered through double filter paper and then analyzed using the hybrid electronic tongue. niger is strongly influenced by the composition of the fermentation medium. The medium was sterilized by triple sterilization at 100  C for 30 min. Other hybrid ET systems were introduced for the quantification of grape variety in red wines [33] as well as for the differentiation of fermented milks [34]. Therefore.15%). such methods require time-consuming steps of derivatization and sample pre-treatment. Kutyła-Olesiuk et al. On the other hand. As an example. Before vaccination. The membranes contained: 1–2 wt% appropriate ionophores. niger cultures were cultivated on synthetic medium. Flow-through sensor array consisted of 10 miniaturized ionselective electrodes (two electrode specimens were prepared for each membrane composition). the side-products are removed in a purification step. system based on an array of potentiometric sensors has been proposed for the monitoring of batch fermentation process of starting culture for light cheese production. niger using ET system. acidity.3%). tridodecylamine (hydrogen ionophore I). Probably. were purchased from Fluka (Selectophore). the increase in organic acids concentration. A potentiometric electronic tongue was also used as a screening tool for the analysis of different types of beer (prediction of real extract. plasticizers: o-nitrophenyl octyl ether (o-NPOE). The addition of inorganic salts is required for fungal growth and to reach high yield of citric acid [15]. the proposed potentiometric electronic tongue has been applied for the analysis of simulated fermentation complex media. Just before the measurement. samples contained only the medium were collected.1%) in deionised water (total volume 1000 mL).5 wt% TDMAC or 1 wt% KTFPB. 20– 50 mol% versus ionophore lipophilic salt. The control of alcoholic fermentation was conducted using near and mid infrared spectroscopies combined with electronic nose and electronic tongue. sulfuric acid and sodium hydroxide were of analytical grade and were purchased from Fluka. changes in media composition were monitored and correlated with biomass growth [31]. 64–66 wt% plasticizer. combining several types of (bio) sensors (including potentiometric.10. The multi-wavelength fluorescence spectroscopy was proposed for the on-line monitoring of the most relevant process variables in fungal cultures i. CO2 and O2 are the basic parameters controlled during the fermentation [21]). / Analytica Chimica Acta 823 (2014) 25–31 this carboxylic acid is related directly to the quality and quantity of the sugar source [13]. Another potentiometric sensor array was tested during the batch fermentations of Escherichia coli i. Medium consisted of sucrose (20%). The reliability of such device was compared with classic HPLC technique [27].26 A. and quantitative product formation [25]. one sample per day during 5 days a week.6. The production of citric acid by A. The possibility of cultivating the mycelium on ethanol [16]. 2. However.1. The stock solutions of salts (0. Hybrid electronic tongues. Nevertheless. metabolic activity. The ionophore dichloro(5. Finally. Chemicals. Flow-through array of miniaturized ion-selective electrodes was developed for the monitoring of periodic anaerobic digestion on the basis of chemical oxygen demand and volatile fatty acid analysis [30].5-bis(trifluoromethyl) phenyl] borate (KTFPB) and tridodecylmethyl-ammonium chloride (TDMAC). ionic additives: NH4NO3 (0. 2. this effect results from an inadequately sterilized medium. where a simultaneous determination of ammonium. GC–MS/29Si NMR [23] and in some cases by pyrolysis mass spectrometry [24]. ionophores: 4-tert-butylcalix [4] arene-tetraacetic acid tetraethyl ester (sodium ionophore X). MgSO4 (0. Preparation of samples Mycelium of A. niger. [30].e. The method of the membranes preparation and the electrodes conditioning were the same as for the standard ISEs. KH2PO4 (0. A detailed architecture of the miniaturized ion-selective electrodes compatible with a single flow-through module was presented in Ref. Moreover.1 M) were prepared in deionised water. changes of CO2 concentration. The constructed . Breeding was carried out for three weeks. The membrane components (200 mg in total) were dissolved in 2 mL of THF. Freshly distilled tetrahydrofuran (Fluka) was used as a solvent for the membrane components. Experimental 2. A system based on potentiometric and voltammetric sensors was applied to assess the progress and correctness of the fermentation process carried out in standard fungal culture and culture infected by yeast. cell dry mass and sugar concentration. were also designed to analyse biotechnological samples. eliminating the necessity of sample preparation. membrane materials and potentiometric sensor array preparation All inorganic salts used. which is contaminated by airspread microorganisms or contaminated equipment and water [20]. In this work. 1-morpholinoethanesulfonic acid (MES). The basic raw material for the production of citric acid is molasses. All samples were frozen and stored in 20  C. see Table 1). voltammetric. Then the medium was inoculated with mycelium A. conductivity. cultivation temperature and time) [19].15.e.2. and bitterness) [29].

niger (standard fungal culture and culture infected after four days by addition of yeast). A classical breeding monitoring. Satisfactory results.5.1 M solution of sodium hydroxide (in the presence of phenolphthalein) in order to quantify the acidity changes during the process.3 and 0. Eurosensor. Partial Least Squares – Discriminant Analysis (PLS-DA) and Partial Least Squares (PLS). Titrimetric analysis 1 mL sample solutions.1 M solutions of corresponding salts [37].. Natick.15 wt% KTFPB 1. 1) and. The working electrode was sequentially mechanically polished with 1. The introduction of the baker’s yeast into the culture (4 days after starting the culture). the standard and infected culture were studied.1 M NaCl/0. Voltammetric measurements Voltammetry measurements were carried out using a CHI 1040 potentiostat (CH Instrument.1 V s 1). To verify the performances of the electrodes. Conducting the fermentation process The fermentation process was carried out according to the scheme described in the Section 2. The steady-state responses of the sensors were recorded.5 V (scan rate 0. 0.  Phase III – termination of the citric acid production in the standard culture. Cole Parmer. Lawson Labs Inc.1 M phosphate buffer solution (pH 7. However. Northampton.05 mm alumina powder.00 wt% KTFPB 3. 2. Ag. USA) as the working electrode.7 wt% sodium ionophore X 1. Elmetron. whereas the pH was stabilized at a level of 2.025 M KH2PO4/Na2HPO4 0. involving the measurement of the pH changes and the determination of the total acidity of the sample solutions collected from both fermentation cultures. were obtained for all the electrodes. In order to remove remaining powder. The analyzed sample solutions (2. potentiometric selectivity coefficients were determined by the Separate Solution Method (SSM). the electrode has been sonicated for 5 min in methanol and water respectively.4). Successive production and accumulation of citric acid resulted in the gradual decrease of pH (Fig. Hydromet. collected from the fermentation culture according to the scheme presented above. The measurements of the electrical conductivity were performed using a portable conductivity meter (CPC-551. USA) and Origin (Microcal Software.. / Analytica Chimica Acta 823 (2014) 25–31 27 Table 1 Components used for preparation of potentiometric sensors. Metler Toledo. Two types of A.50 wt% TDMAC 65 wt% DOS 0. one of the culture was infected with yeast). Poland) with a conductivity cell (EPS-2.  Phase II – stabilization of citric acid production during the standard process or interruption of the fermentation process in the case of the infected culture.01 M KCl 0. 2. after the first phase. influencing the total acidity of the medium. USA) in the three electrode arrangement. a weaker acid than the citric acid was probably produced since the pH remained unchanged after the infection (pH was maintained by the citric acid formed during the I phase). almost linear growth of the total acidity of the samples (Fig. Austin. 3. and rinsed thoroughly with methanol and water. niger cultures i.01 M NaCl/0.0 wt% tridodecylamine Cationselective Anionselective – – Lipophilic salt Plasticizer Internal filling solution Conditioning solution 0. Poland) were applied for such purpose. 2. KCl 3 MCH3COOLi 1 Msample solutionmembrane internal filling solution. platinum rod as the counter electrode and the glassy carbon disk electrode (BASi. the measured values of pH and total acidity were similar for both cultures before the yeast infection.75 wt% KTFPB 1. 2.1.15 wt% KTFPB 0.01 M NaCl 0. with silver/silver chloride (Ag/AgCl) as the reference. After this procedure. Electrode type Ionophore + Na CH3COO 1. ORP and pH values of sample solutions (5 mL) were measured using laboratory pH-meter (Metler Delta 350. Kutyła-Olesiuk et al. Only the forward scans were considered for further data processing. were diluted to 5 mL and titrated with 0. Switzerland).5 M KH2P04/0.1 V to 1. Moreover. The results suggested that another organic acid was produced after the culture infection. pH electrode (Cole Parmer EW-5991-61. Sensors signals were recorded in two different fermentation processes by fungus A. Chemical images of the samples were processed using Principal Component Analysis (PCA).6. Malvern.0 wt% Zr(IV)[TPP]Cl2 H+ 2. USA) and two redox electrodes: Au and Pt (ERAu-13 and ERPt-13. AgCl. USA) was used for EMF measurements.0.01 M NaCl sensors were preconditioned for at least 24 h (the components of internal filling and conditioning solutions were presented in Table 1). Inc. AgCl. Potentiometric multiplexer (EMF 16 Interface. .3. USA) software. The fermentation process can be divided into three phases:  Phase I – proliferation of mycelium and start of citric acid production (after this phase. The reference electrode potential was calibrated by using a ferricyanide electrode process in 0.e. Data analysis Data analysis and calculations were carried out in MatLab (The MathWorks. 2) during the standard process. Potentiometric and conductometric measurements All measurements were carried out in flow-through mode with cells of the following type: Ag.A. Poland). the substrates were transferred to 1 mL sample solutions diluted to 5 mL with 0. consistent with previous studies [32].0001 M CH3COONa 66 wt% oNPOE 64 wt% DOS 0.4. according to our expectations.01 M NaCl 0.1 M phosphate buffer solution (pH 7.5 (higher than for standard culture). Results and Discussion 3. was carried out.5 mL) were diluted twice and pumped by a peristaltic pump to the flow-through sensor array.1.001 M CH3COONa 66 wt% DOS 64 wt % oNPOE 0.25 M Na2HPO4 0. using 0.. Argon was used to deaerate the solutions. Cyclic voltammograms were registered in the potential range from 0.4). caused an abrupt increase of acid concentration in the samples. Inc.

Determination of the total acidity of the samples collected during the standard and infected fermentation process.1 0. pH measurements of the samples collected during the standard and infected fermentation process.1 1. Especially.0 0.5 0.9 1. Kutyła-Olesiuk et al. the proper classification shown in Fig. rbio3._1)TD$IG] A.7 0. Nevertheless. Qualitative monitoring of the fermentation process Principal Component Analysis (PCA) technique enabled the preliminary evaluation of the recognition ability of the hybrid electronic tongue.1 to 1.0x10 phase III -5 4.1.3 0. Due to the large number of data points. Next.5 V comprised 1400 points (exemplary voltammograms recorded in the samples collected during the standard culture were plotted in Fig. Finally. 5).3 1. The extracted coefficients (used as inputs of the PLS model) allowed to form a data matrix of the voltammetric electronic tongue – Volt-ET (i. the qualitative monitoring of citric acid production was attempted using Partial Least Squares-Discriminant Analysis . db6. Exemplary cyclic voltammograms (forward scans) registered in the samples collected during the standard fermentation process. On the other hand.2. 2. a = 5) were selected for further calculations. The results presented in Figs. initial voltammetric signal compression involving discrete wavelet transform was applied to reduce the amount of data. the hybrid system was able to estimate the progress of the fermentation in the case of standard culture. the chemical images of the infected samples from the II and III phase were located close together. 3). the parameters of the final data compression method (rbio3.0x10 phase I 0. Such results were well correlated with those gained by the classical breeding control (see Figs. Moreover.5 -5 3. / Analytica Chimica Acta 823 (2014) 25–31 [(Fig.2 standard culture infection 0. based on the mentioned above potentiometric and voltammetric measurements as well as on pH and ORP values). The chemical images of the samples collected during standard fermentation process created separated clusters. the level of decomposition a = 5 led to a reduction of the data from 1400 points to 54 coefficients. as well as the loadings of the coefficients obtained after discrete wavelet transform of voltammetric data. Since the results of the preprocessing depend on the selected parameters of [(Fig. where in contrast to the standard culture the pH and total acidity changes were negligible during the II and III phase of the infected culture.5. while maintaining a waveform characteristics.2. 3. whereas 32 coefficients were obtained after decomposition at a = 6).0x10 3.5 Current [A] 6.1 1.4 0. 1 and 2). sym6) and different decomposition levels were employed for data compression (as an example. 4 and 5 indicated the potential usefulness of the developed hybrid electronic tongue for the differentiation between standard and infected fermentation process. five wavelets from different families (bior3. Time [h] 0._2)TD$IG] phase I phase II phase III infected culture cHA [mol/L] 0._3)TD$IG] phase I phase II phase III -5 8.0 infection pH infected culture 2. based only on potentiometric data).5.e. a clear linear separability between the clusters corresponding to different phases of the infected culture was not observed (Fig. the wavelet compression.5 0 100 200 300 400 Fig. based only on voltammetric data).e. since in the PCA model the loadings of the outputs of all potentiometric sensors were comparable.0 0 100 200 0.28 [(Fig. Additionally.0 standard culture 2. the data recorded by various electrochemical techniques were merged in a data matrix of the hybrid electronic tongue – Hybrid-ET (i.e.5 wavelet. The forward scans of cyclic voltammograms registered in the potential range from 0. Monitoring of the citric acid production by an electronic tongue An electronic tongue system based on miniaturized ionselective electrodes and classical 3-electrode electrochemical cell with glassy-carbon electrode as working electrode was proposed for the monitoring of the fermentation process. the clusters representing consecutive fermentation samples were arranged according to the phase order (Fig. On the basis of visual inspection of clusters’ separation on PLS plots.5 Potential [V] Fig. 4). 3.0x10 phase II -5 2. The steady-state signals recorded for the ion-selective electrode array were used to create a data matrix of the potentiometric electronic tongue – PotET (i. coif2. 1. 4 did not result solely from the pH measurement. 3.3 300 400 Time [h] Fig.

_7)TD$IG] 0. In accord with the results presented in Figs. R =0. The obtained measurement data were divided into train and test matrixes. R =0.1%) PC 2 (18.15 0. PCA plot of chemical images of the samples collected during infected fungal culture (data obtained for Hybrid-ET). the samples corresponding to the first phase formed one cluster for both fermentation processes. PLS-DA plot of chemical images of the samples collected during the standard and infected fermentation process (data obtained for Hybrid-ET). Moreover.04. comparable linear trends and correlation coefficients were attained for the train and the test set. 6.07. were presented in Fig. .phase I standard culture .25 0. the chemical images representing the phase II and III were plotted by the same marker).05 0. The model performance was characterized after building comparison graphs and performing the linear fitting of the expected data versus the data predicted by the PLS model. whereas the two subsequent columns informed about the occurrence of infection. 7.2._4)TD$IG] 29 [(Fig. 14 latent variables were chosen to create the PLS-DA model. it was necessary to build a proper target matrix embracing the duration of the process (counted from the inoculation moment of A. Quantitative monitoring of the fermentation process The developed electronic tongue combined with Partial Least Squares (PLS) technique was employed for the quantitative [(Fig.phase I infected culture . serving as inputs for PLS model (the data representing 4 replicates for each sample formed the train matrix.30 REAL CHA [mol/L] Fig. 6 (for the sake of clarity.10 -2 phase I phase II phase III -4 -6 -15 -10 -5 0 5 10 0. 1 and 2.2. It should be also stressed. On the basis of minimization of RMSE value.25 PRED CHA [mol/L] 6 PC 2 (9. 5.30 [(Fig. Fig.05 15 Fig. PLS-DA established the correlation of data matrix with target matrix. Therefore.phase II+III infected culture . The values of the parameters: slope (a). PCA plot of chemical images of the samples collected during standard fungal culture (data obtained for Hybrid-ET).15 train 2 y=0.7%) Fig. enabling to estimate the progress of the fermentation process) and the value of the total acidity (enabling to estimate the correctness of the process). comparing the determined total acidity and the values provided by the PLS model.3%) -10 -5 0 5 10 15 LV 1 (74.phase II+III -4 -6 -20 25 -15 PC 1 (67. / Analytica Chimica Acta 823 (2014) 25–31 [(Fig. 3.978 test 2 y=0. 0. 4.A. intercept (b) and determination coefficient (R2) indicated that the hybrid system permitted a correct quantitative modeling of the fermentation run. Exemplary results provided by the hybrid electronic tongue (Hybrid-ET) during the standard culture. The first two columns indicated the process duration. Model performances characterized after linear fitting of the real values of the total acidity for standard culture to the predicted data by the PLS model (data obtained for Hybrid-ET).980 0. whereas the fifth sample replicates were used to create the test matrix).98x+0._6)TD$IG] 8 10 6 8 6 LV 2 (9.20 0.3%) 4 2 0 -2 -6 -15 -10 -5 0 5 10 15 20 4 2 0 -2 phase I phase II phase III -4 standard culture ._5)TD$IG] 8 0.20 0.5%) 4 2 0 0. the chemical images of the samples from the II + III phase exhibited different changes of the position in the pattern space related to the occurrence of infection. (PLS-DA) technique. Kutyła-Olesiuk et al.94x+0.10 0. The obtained results confirmed the suitability of the hybrid ET to assess the correct course of fermentation. Moreover. In our case. monitoring of the studied fermentation process. that the larger scattering of the chemical images representing the II + III phase of the standard culture was in good agreement with the previous conclusions. 7. a data matrix containing chemical images of samples and its corresponding four-column target matrix (coded information on sample classification) were created. The results obtained by processing of the data by PLS-DA were presented in Fig. niger mycelium.

using high-performance liquid chromatography and/ or gas chromatography. D. D.993 0. in: A. Panda. London. Weinheim. Iwase. [16] G. [7] K. Verma. b and R2) for the standard and infected culture. The presented results led to the conclusion. determining the contribution of the individual electrochemical techniques to the final result.770 0.g. Advances in citric acid fermentation by Aspergillus niger: biochemical aspects. b. [8] D.98 4. An overview of citric acid production. Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose. Total acidity Process duration Pot-ET a b R2 Volt-ET Hybrid-ET Train Test Train Train Test 0. Journal of Endourology 22 (2008) 567–570. [35] reported the use of a potentiometric sensor array for the analysis of simulated Volt-ET Hybrid-ET Test Train Test Test Train Test 0. 2003. and commercially available fruit juice products.W.921 0.07 0. Citric acid production by yeasts: fermentation conditions.92 16.97 0. S. [15] A. Boston.996 fermentation samples. K. D. that to date. the fusion of various electrochemical techniques. vol. data matrixes composed of chemical images of samples created on the basis of separate potentiometric (Pot-ET) and voltammetric (Volt-ET) measurements as well as the data matrix corresponding to the hybrid system (Hybrid-ET) used above were correlated with the target matrix. [9] M. Ozbas. when the chemical images recorded applying various electrochemical techniques were processed. Kalra. Madrid.99 0.Y.A. and R2 were not acceptable for the test samples. Bende. Conclusions The qualitative and quantitative monitoring of the citric acid fermentation process by A. T.Y. Academic Press. Biotechnology Advances 25 (2007) 244–263.66 0. R.39 0.L. Recent advances in citric acid bioproduction and recovery.92 14.e. pp. [12] G. Ullmann’s Encyclopedia of Industrial Chemistry.8 0. Z. niger was reported in this work. D. in: R. Wiley-VCH.996 0. Lowenstein.20 0. the electronic tongue systems were not applied in the monitoring and especially in the detection of microbial infection during the citric acid production by A. niger culture.02 0. 130–135. Granner. Felse. improved the classification ability of the system for both standard and infected culture. Papagianni. Kutyła-Olesiuk et al. New perspectives for citric acid production and application.H.K. Yalcin. Bozdemir.2 0. J.94 20. 13. M.30 A.35 0. Citric Acid.61 0.99 0. niger.921 Table 3 Parameters of linear fitting of real and PLS-predicted total acidity and process duration for infected culture.970 0. [13] A. the use of the hybrid electronic tongue. C. A hybrid electronic tongue combining various electrochemical sensors was exploited for the recognition of the samples collected during standard and infected A. Rodwell (Eds.7 0. / Analytica Chimica Acta 823 (2014) 25–31 Table 2 Parameters of linear fitting of real and PLS-predicted total acidity and process duration for standard culture.P. In general. [14] M.875 0. Mendez-Vilas (Ed. Fungial production of citric acid.976 0.993 Pot-ET Train 1.P. 2009. For this purpose.91 0. lime juice.01 0. Production of critic acid: a review.98 0.967 0. 138–1374. [10] H. Current Separations 20 (2004) 127–131. LWT – Food Science and Technology 50 (2013) 367–370.92 16. Acknowledgement This work has been financially supported by the project LIDER/ 17/202/L-1/09/NCBiR/2010.99 12. The McGraw-Hill Companies.. Significantly better results were obtained for Pot-ET (see e.2 0. P. Therefore. Verhoff. Inc.D.880 0. Pazouki.996 0. The influence of type and concentration of the carbon source on production of citric acid by Aspergillus niger. respectively.04 0. membrane transport and modeling. Brar. Dhillon. Mayes. Angumeenal.M. Agricultural Wastes 9 (1984) 51–76. M.B. Venkappayya. Technology and Education Topics in Applied Microbiology and Microbial Biotechnology.02 0. Penniston. Biotechnology Advances 13 (1995) 209–234. Ashy. Xu. [6] H.83 0.718 1.99 1.P. Mayes.2 0. References [1] J.68 0. West. Food Technology and Biotechnology 44 (2006) 141–149. A. Biotechnology Annual Review 13 (2007) 303–343. [2] P. [3] F.99 1. It should be noticed. the classification properties of the developed electronic tongue were analyzed in details.K. 4. . Current Research. the total acidity determination for standard culture).05 0. 1969.975 0. process optimization and strain improvement. Xie. could be a simple and useful tool for the monitoring of the progress and the correctness (e.88 24. Methods in Enzymology: Citric Acid Cycle. Pandey.35 0. based on automated measurements with sensor array systems. Food and Bioprocess Technology 4 (2011) 505–529. i. Grewal. Abou-Zeid. Assimos. the proposed approach can replace the commonly performed off-line process monitoring.R.g.991 1. Sinha. M.A. Research Journal of Microbiology 2 (2007) 678–683.12 0. V.A.S. Use of citric acid as mobile phase and sample medium for the determination of ascorbic acid by liquid chromatography/electrochemistry.980 0. [5] C. Quantitative assessment of citric acid in lemon juice. Rohr. Vandenberghe.). Tyagi. detection of infection occurrence) of the fermentation process. T.99 12. Berovic.27 0. pp.993 0.). World Scientific. Citric acid production. Total acidity Process duration Pot-ET a b R2 Volt-ET Hybrid-ET Pot-ET Volt-ET Hybrid-ET Train Test Train Test Train Test Train Test Train Test Train Test 0. R. Holmes. L.02 0. Bioprocess Engineering 22 (2000) 353–361.90 0.61 0.978 0. Kubicek.886 0. 2005.89 0.G.A.e. Harper’s Illustrated Biochemistry.S.00 0. S. which could not reflect the processes occurring in the real conditions and therefore the complex composition of the medium. Nakada. The highest classification abilities were achieved in the case of the hybrid system (Hybrid-ET) i. Tables 2 and 3 summarize the obtained regression parameters (a.T.K. It should be also stated.02 0.992 Finally. that the developed device. Soccoll. Legisa.7 0. C. [4] M. similar dependences were notices in the case of the prediction of process duration and total acidity of the samples.L.S.A. [11] S. that the processing of the separate voltammetric data (Volt-ET) did not ensure a proper quantitative monitoring of the fermentation process – the values of a. Murray.P.00 0. M.P.R. M. Rodrigues.94 0. Applied Microbiology & Biotechnology 30 (1989) 553–558. The citric acid cycle: the catabolism of Acetyl-CoA. Citric acid production by Asperigillus niger on the ethanol dry milling coproduct thin stillage. P.02 2. The single approach described in Ref.765 0.7 0.87 0. C.K.997 1.A.993 0.

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