A filtration probe-free on-line monitoring of glycerol during fermentation by a biosensor device

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Enzyme and Microbial Technology 42 (2008) 434–439

A filtration probe-free on-line monitoring of glycerol during fermentation by a biosensor device c , Vladim´ır Mastihuba d , ˇ coviˇcov´a a , Jaroslav Katrl´ık b,∗ , Vladim´ır Stefuca ˇ Jana Sefˇ Igor Voˇstiar e , Gabriel Greif e , Marek Buˇcko a , Jan Tkac a , Peter Gemeiner a a

Department of Glycobiotechnology, Institute of Chemistry, Slovak Academy of Sciences, D´ubravsk´a cesta 9, SK-845 38 Bratislava, Slovakia b Department of Pharmaceutical Analysis and Nuclear Pharmacy, Faculty of Pharmacy, Comenius University, Odboj´arov 10, SK-832 32 Bratislava, Slovakia c Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinsk´eho 9, SK-812 37 Bratislava, Slovakia d Department of Glycochemistry, Institute of Chemistry, Slovak Academy of Sciences, D´ubravsk´a cesta 9, SK-845 38 Bratislava, Slovakia e Institute of Biotechnology and Food Science, Faculty of Chemical and Food Technology, Slovak University of Technology, Bratislava, Radlinsk´eho 9, SK-812 37 Bratislava, Slovakia Received 22 October 2007; received in revised form 30 December 2007; accepted 2 January 2008

Abstract A novel on-line monitoring approach implementing an improved sampling methodology is presented here. A sample is directly withdrawn from the bioreactor without a need for a filtration or a dialysis probe, extensively diluted in a flow-through fashion and mixed with a mobile phase consisting of a buffer and a soluble mediator. The developed sampling system efficiently controls dilution of the sample prior to biosensor assay covering full dynamic range of glycerol concentrations during its fermentation to 1,3-propanediol. The new sampling protocol minimizes problems associated with the use of traditional on-line sampling methods implementing membrane separation or filtration techniques. Concentration of glycerol in the sample is determined by a flow injection analysis (FIA) with an amperometric biosensor using glycerol dehydrogenase and diaphorase with a throughput of nine samples per hour. The biosensor performance was validated by a reference analytical method (HPLC) with a very good agreement. © 2008 Elsevier Inc. All rights reserved. Keywords: Biosensor; On-line bioprocess monitoring; Probe-free sampling; Glycerol; 1,3-Propanediol; Biodiesel

1. Introduction A sustainable development is a necessary task for mankind, what is more obvious than ever due to change of a global climate. Worldwide demand for environment-friendly chemical processes and products requires the development of novel and costeffective approaches to pollution prevention. The fundamental challenge is the shift of the production of energy and carbonbased products from fossil fuels to renewable resources [1,2]. One of such examples might be a green and sustainable production of polyester based plastics (>24,000 t daily worldwide). More of the effort in making polyesters has been

∗

Corresponding author. E-mail address: [email protected] (J. Katrl´ık).

0141-0229/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2008.01.006

focused on 1,4-butandiol than on 1,3-propanediol, mainly due to the non-availability of sufficient quantity and quality of 1,3propanediol (1,3-PD). 1,3-PD-based polyesters are expected to be more biodegradable compared to 1,4-butandiol based ones [3]. Polyester made of 1,3-PD can be prepared with an environmentally-benign manufacturing process with a current considerable industrial interest in microbial 1,3-PD production [4–6]. The process of microbial production of 1,3-PD is more efficient, when glycerol is used instead of traditional fermentation substrate glucose. Green production of biodiesel by transesterification of seed oil forms a large volume of a byproduct – glycerol. There are currently many ways how this “waste” glycerol is transformed into value added fine products [7]. One of such ways is microbial transformation of glycerol into 1,3-PD, what makes production of 1,3-PD economically and environmentally even more attractive [8].

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Efficient real-time bioprocess monitoring is needed to get a direct insight to the bioprocess state. Moreover, it allows immediate action to be taken in order to resolve the situation while the process is being carried out [9]. FIA systems are used for on-line process monitoring, because of their short response time, high flexibility and inexpensive components [10]. On-line monitoring is usually done by withdrawing a sample from a bioreactor by a sampling device with subsequent analyte analysis by a selective and sensitive analytical device. On-line aseptic sampling is not a trivial task, because of real risk of contamination. Moreover, reliability of analysis can be negatively influenced by changes in transmembrane permeation rate as a result of membrane biofouling during cultivation [11–13]. Biosensors are highly powerful analytical devices fusing the exquisite sensitivity and specificity of living systems with the processing power of microelectronics [14]. Electrochemical biosensors have been proved to deliver targeted information in a fast, simple, and low-cost fashion by intimately coupling of a biological recognition element to an electrode transducer [15]. This is the reason why biosensors are so often used for on-line bioprocess monitoring [16,17]. At least four different enzymatic systems are applicable for construction of a glycerol biosensor [18], but only two of them, based on oxidase or dehydrogenase system, are of common use for specific glycerol detection [19]. Although glycerol biosensors have been successfully used for off-line bioprocess monitoring [19–22], only two examples describing on-line monitoring of glycerol have been published [12,23]. The main goal of the paper is to present a simple on-line biosensor system for continuous monitoring of glycerol concentration in the course of the fermentation process. The presented approach illustrates a simple way to avoid problems associated with the commonly used filtration sampling devices (membrane clogging, formation of air bubbles, etc.) [13]. 2. Experimental 2.1. Enzymes, chemicals and materials Enzymes glycerol dehydrogenase (GDH, E.C. 1.1.1.6, 90 U/mg) from Cellulomonas sp., diaphorase (DP, E.C. 1.8.1.4, 59 U/mg) from Clostridium sp. and oxidised ␤-nicotinamide adenine dinucleotide (NAD+ ) were supplied from Sorachim (Paris, France). The enzymes were used without further purification. Polycarbamoyl sulfonate (PCS) prepolymer was acquired from SensLab (Leipzig, Germany). Polyethyleneimine and glycerol were from Merck (Darmstadt, Germany). Potassium ferricyanide was obtained from Centralchem (Bratislava, Slovakia) and components used for preparation of electrolytes and buffers were provided from Mikrochem (Pezinok, Slovakia). Dialysis membrane Servapor® 44144 (cut-off 6–8 kDa) was supplied from Serva (Heidelberg, Germany).

2.2. Fermentation conditions and sampling Bacterial strain Clostridium sp. IK 124 screened and provided by Prof. K.-D. Vorlop (Institute of Technology and Biosystems Engineering and Federal Agricultural Institute, Braunschweig, Germany) metabolizes glycerol preferentially to 1,3-PD. The cells were grown in a 2-L bioreactor (B. Braun Biostat, Germany) with pH 7.0, temperature 32 ◦ C and stirring rate of 400 rpm in the fermentation media as published previously [8]. The strain was maintained by reinoculation once a week and stored in sealed vials at 30 ◦ C. Bacterial suspension at expo-

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nential phase was used for inoculation of the bioreactor. Samples for glycerol analysis were withdrawn directly through a plastic tube (ID = 0.5 mm) inserted into the bioreactor and were analyzed on-line by the FIA system described below.

2.3. Electrodes and biosensor preparation The electrochemical experiments were performed with a tailor-made cylindrical combined electrode (Elektrochemick´e detektory, Turnov, Czech Republic) containing both glassy carbon electrode (d = 3 mm) and Ag/AgCl reference electrode (d = 2 mm) embedded in a Teflon casing (OD = 9 mm). The electrode was fitted into the flow cell of the FIA system (dead volume ∼150 ␮L). The electrode was polished with a fine alumina suspension (particle size of 0.05 ␮m, Buehler, Lake Bluff, IL, USA) and thoroughly rinsed with distilled water before modification. The electrode surface was covered by the enzyme membrane containing both GDH and DP giving the following reaction cascade: glycerol is dehydrogenated by NAD+ in the presence of GDH to 1,3-dihydroxyacetone and re-oxidation of formed NADH in the presence of ferricyanide is catalysed by DP. Ferricyanide is reduced by DP to ferrocyanide, which is oxidised by the voltage applied to the working electrode and the current is proportional to the concentration of the glycerol in the sample/standard [19]. The enzymes were immobilised using PCS prepolymer and polyethyleneimine directly on the dialysis membrane with details published recently [19].

2.4. Instrumentation and on-line measurement procedures The FIA system (FIAlab Instruments, Bellevue, WA, USA) used for glycerol online monitoring is depicted in Fig. 1. The electrodes of the biosensor were biased to +350 mV vs. Ag/AgCl by a potentiostat PST-3 (FEI STU Bratislava, Slovakia) and data were acquired and evaluated by custom made application developed in LabVIEW environment (LabVIEW, National Instruments, Austin, TX, USA). For the measurements, carrier buffer was a mix of two streams, 0.1 M PBS pH 7.8 containing 4 mM NAD+ and 0.1 M PBS pH 7.8 containing 4 mM ferricyanide, mixed in 1:1 ratio just before reaching the flow cell. The overall set up of the FIA system is shown in Fig. 1. A sample or standard was pumped by a peristaltic pump 7-1 (0.6 mL/min) depending on the position of the switching valve 6-1, and injected into the water stream by the injection valve 6-2 (20 ␮l loop). The peristaltic pump 7-2 was used for pumping of distilled water through the injection valve 6-2 and for both streams of carrier buffers at flow rate of 0.54 mL/min. The water and buffer solutions were continuously degassed with a degasser unit 9 (L-7614, Merck, Darmstadt, Germany). After the sample/standard passed the first injection valve 6-2, the sample/standard was washed out by the distilled water flowing into the mixing chamber 8 (magnetically stirred plexiglas chamber with a height of 0.3 mm and ID = 1 cm) from which it was passed into the injection loop of the injection valve 6-3 (20 ␮L loop) and injected into the biosensor flow cell 10. As the actual concentration of standard/sample in the loop of valve 6-3 varied dynamically during its washing from the mixing chamber, the glycerol concentration injected into the system depended on the time delay between switching of valves 6-2 and 6-3. In the flow cell with amperometric biosensor, the diluted sample reacted in the enzyme membrane on the electrode surface. The current of electrochemical oxidation of ferrocyanide formed during the oxidation of glycerol by both enzymes in the presence of NAD+ and ferricyanide was measured and used for the determination of glycerol concentration.

2.5. HPLC assays Reference HPLC assays of glycerol and 1,3-PD were run on a DeltaChromTM liquid chromatograph (Watrex, Bratislava, Slovakia) equipped with WellChrom K-2301 refractive index detector (Knauer, Berlin, Germany). The analytical conditions were as follows: column, Polymer IEX in H+ form, 250 mm × 8 mm, 5 ␮m (Watrex, Bratislava, Slovakia); guard column, Polymer IEX in H+ form, 10 mm × 4 mm, 8 ␮m (Watrex, Bratislava, Slovakia); mobile phase, H2 SO4 , 9 mM in demineralized water; flow rate, 1.0 mL/min; data were collected and processed by Clarity chromatography station (DataApex, Prague, Czech Republic). Samples were diluted in mobile phase and filtered through 0.45 ␮m Chromafil AO filters (Macherey-Nagel, D¨uren, Germany) prior to analysis.

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Fig. 1. Experimental set-up of the on-line FIA system: (1) bioreactor, (2) glycerol standard solution, (3) water for dilution, (4) buffer solution containing NAD+ , (5) buffer solution containing ferricyanide, (6-1) switch valve, (6-2 and 6-3) injection valves, (7-1 and 7-2) peristaltic pumps, (8) mixing chamber, (9) degasser, (10) flow cell containing biosensor and (11) waste.

3. Results and discussion We have previously described two different approaches for preparation of glycerol biosensors. The first one was based on a tri-enzymatic system containing glycerol kinase, glycerol-3phosphate oxidase and peroxidase, while the second, a dehydrogenase based one, used only two enzymes – GDH and DP [19]. Dehydrogenase-based glycerol biosensor proved to be more robust compared to its oxidase-based opponent. The main advantages are higher selectivity in the presence of electrochemically active compounds and a better precision for measurement of real samples [19]. This is why in this study a GDH-DP based glycerol biosensor system was tested for its ability to continuously monitor changes of glycerol concentration during fermentation. The response of previously described GDH-DP biosensor in the FIA system [19] was linear up to 0.1 g/L of glycerol with limit of detection of 1.0 mg/L. However, the maximum glycerol concentration occurring in the initial phase of fermentation can be thousand times higher. To address this problem, a dilution component was implemented (Fig. 1) into the FIA setup as described in our previous work [23]. Dilution of sample was based on a sequential injection of the sample through two injection valves (6-2, 6-3 in Fig. 1) separated by a mixing chamber (8 in Fig. 1). Change of the time delay between injections through individual valves (6-2 and 6-3) in combination with an adjustment of the flow rate, volume of sample loops of injection valves and dead-volume of a mixing chamber allows efficient control of the extent of sample dilution. The effect of the change in time delay between injections through individual valves (6-2 and 6-3) was characterised in order to cover a full dynamic range of glycerol during fermentation with a reasonable throughput of analysis (Fig. 2). The injection time delay of 120 s was selected for further experiments with approximate dilution factor of 500. Such a dilution set-up allows sample throughput of 9 samples/standards per hour and the dynamic range of the biosensor device was between 0.2 and 50 g/L of glycerol (Fig. 3) with limit of detection of 0.1 g/L. The sampling procedure is a very important step for on-line monitoring of a biotechnological process. To obtain cor-

rect analytical results in time, the sampling should be rapid, reproducible with the smallest possible dead volume of a sampling device. The most common sampling methodology is the use of various filtration or dialysis probes integrated directly into the bioreactor. The presented approach shows possibility of direct sampling from the bioreactor without a pre-separation of a fermentation broth through filtration or a dialysis. Continuous filtration and dialysis can be accompanied with significant problems associated with membrane clogging or fouling due to the presence of a pressure gradient or adsorption effects. Two different filtration probes with a ceramic or a polypropylene membrane (Flownamics Analytical Instruments Inc. and Eppendorf) have been unsuccessfully tested by the group. The main reasons for failed use of filtration probes are membrane clogging and formation of bubbles in flow stream negatively influencing performance of on-line analysis [23]. Our system utilises an advantage of using the sensitive biosensor detection, which allows extensive dilution of analysed samples. A high lateral flow of extensively diluted sample

Fig. 2. Effects of injection delay (valves 6-2 and 6-3) on the response of glycerol biosensor. Measurement is performed at a flow rate of 0.54 mL/min with a glycerol standard (20 g/L).

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Fig. 3. Calibration curve of the amperometric GDH-DP biosensor mounted into FIA system.

over the biosensor surface further reduces fouling of the dialysis membrane used to mechanically stabilise and protect the enzyme layer of the biosensor. Incorporation of the dilution components prevents satisfactorily the problems with fouling of the dialysis membrane. The decrease in sensitivity of glycerol detection (40 g/L) during on-line fermentation monitoring was faster (I = 375 nA − 14.5 × t (h), R2 = 0.973 with average decrease of 3.9%/h, Fig. 4) than previously observed for a glycerol standard solutions (0.4%/h) [19]. Possible reasons for faster decrease in sensitivity of detection can be either inactivation/inhibition of enzymes by a fermentation broth or fouling of the dialysis membrane used to cover the enzyme layer of the biosensor. Decrease in the biosensor sensitivity during on-line monitoring cannot be easily prevented, but it can be efficiently compensated by a current recalibration promoting precision of the on-line monitoring. Storage stability of the biosensor was studied previously at 4 ◦ C and was excellent, when 90% or 66% of the initial sensitivity, after 2 or 11 months of storage, respectively, was reported [19].

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Fig. 5. Time course of the glycerol fermentation monitored on-line with the FIA biosensor system (filled circles) compared with the results obtained from off-line HPLC assay (open circles). Arrow indicates the exchange of the recalibration standard used during the on-line monitoring.

Reproducibility of the sample detection by the biosensor was 2.3%. The on-line monitoring was performed with alternate injections of samples and standards. The concentration of glycerol in each sample was calculated by linear interpolation of responses to glycerol standards before and after sample injection. To further enhance accuracy of detection of glycerol in the sample, the concentration of glycerol standard was changed two times during fermentation (Fig. 5). When the glycerol concentration in the fermentation broth reached 30 g/L, the glycerol standard (40 g/L) was replaced by a glycerol standard with lower glycerol concentration (20 g/L) and when glycerol in the sample reached 10 g/L the standard with 5 g/L of glycerol was introduced. Fermentation product and by-products like 1,3-propanediol, acetic acid, butyric acid, glucose, and ethanol present in fermentation broth do not interfere with glycerol detection as shown in our recent study [19].

Fig. 4. Section of the real-time output from the on-line FIA assay of glycerol. Unmarked peaks–40 g/L glycerol standards (A). Real-time dynamics of the biosensor response (B).

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The on-line monitoring system worked efficiently during the whole fermentation (13 h). The molar yield of glycerol conversion to 1,3-PD was 64%, which is in a good agreement with a pilot-scale microbial production of 1,3-PD from glycerol [24]. The results obtained on-line by the biosensor were validated by off-line HPLC measurements. The reproducibility of off-line sample detection by the same biosensor set-up expressed as an average RSD was 1.8% [19] similar to average RSD of off-line HPLC of 2.1%. The reproducibility of glycerol detection in a sample by an on-line FIA detection was 2.3%. The time of analysis by the biosensor is less than 2 min (Fig. 4B), while in case of HPLC it is 20 min. The limit of detection is 0.11 mM for glycerol and 0.22 mM for 1,3-PD by HPLC and the biosensor detection of glycerol without a dilution step is more sensitive (11 ␮M). Besides lower sensitivity and longer analysis time HPLC has another drawback compared to an amperometric biosensor – rather costly equipment is needed. These are all reasons why HPLC is not routinely used for on-line bioprocess monitoring [25]. The main outcome of the study is the conclusion that results obtained by the on-line monitoring of glycerol by the biosensor without using any sampling probe were in a good agreement with results obtained by reference off-line HPLC assays (Fig. 5). Higher sensitivity of the biosensor system described here compared to HPLC allows extensive on-line dilution of the sample prior analysis and the sample can be analysed without any sampling probe. The current biosensor set-up can be further upgraded by a modification of a dialysis membrane via grafting of polyelectrolytes or oligo(ethylene glycol) to form a hydrophilic protein/cell resistant thin film coating [26–28]. Furthermore a flexibility and robustness of fermentation monitoring can be enhanced by implementing a second biosensor system (e.g., recently developed 1,3-PD microbial biosensor [29]) for measurement the product of the fermentation, if such a biosensor system is known. Acknowledgements This work was supported by the European Commission, under the scope of the BIODIOL project. The BIODIOL project (QLK5-CT-2002-01343) is funded by the European Commission within the 5th Framework Program. The integrated bioprocess development and economical evaluation of BIODIOL also includes a partner’s work, which is not presented here. The EU is not responsible for the content of this publication. This work was also supported by a grant from the Slovak Grant Agency for Science VEGA under the Project Nos. 1/4299/07 and 1/4452/07. References [1] Jenck JF, Agterberg F, Droescher MJ. Products and processes for a sustainable chemical industry: a review of achievements and prospects. Green Chem 2004;6:544–56. [2] Ranke J, Stolte S, Stormann R, Arning J, Jastorff B. Design of sustainable chemical products – the example of ionic liquids. Chem Rev 2007;107:2183–206.

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