Rapid determination of microorganisms using a flow-injection system

Analytrca Chrmrca Acta, 234 (1990) 247-251 Elsevler Science Pubhshers B.V., Amsterdam

247 - Pnnted

m The Netherlands

Rapid determination of microorganisms using a flow-injection system THOMAS GBF - Gesellschaft fur Blotechnologwhe

DING

* and ROLF

D SCHMID

Forschung, Department of Enzyme D-3300 Braunschwerg (F R G) (Received

7th November

Technology, Mascheroder

Weg I,

1989)

ABSTRACT An automated method for the rapid determmatlon of microorgamsms using a flow-mJectlon system IS presented Electrochenncal measurement of a mediator reduced by nucroblal metabohsm allowed the determmatlon of fungi and bactena m a few mmutes The lowest detection hnut was 5 X lo6 colony-forming umts (cfu) ml-’ for Escherrchra cob Correlation between the flow-lqectlon method and standard nucroblologJca1 methods was excellent (r = 0 997, n = 4 for Beauoena bassrana, r = 0 997, n = 7 for E cob) The flowqectlon system was applied to the on-hne control of an E co11 cultlvatlon

The deterrmnation of nncroorganisms is very important in medicine, water analyis, biotechnology and food and pharmaceutical processing. Conventional rmcrobiological methods are labonous and time consuming; a standard plate count takes 3 days and the identification of selected microorganisms requires even more time. Faster mtcrobiologtcal analysis would permit a rapid response to mtcrobtal contamination in process lines, which in turn would prevent high costs caused by spoilage of food and by storage of the products until delivery. Faster diagnosis of human diseases caused by microbial infections would permit more effective therapy. Therefore, modern industrial processes and further improvements in medicine require rapid and automated systems for microbiological control. For this purpose, several mstruments have been developed during the last decade using various prmciples of determinatton, e.g., bio luminescence [l], epifluorescence [2], flow cytometry [3], tmpedimetry [4,5], microcalortmetry [6], radiometry [7], turbtdtmetry [B] and many others [9-111. None of them completely covers the demand for short response times (m the range of 0003-2670/90/$03

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mmutes), high sampling frequency and cheap equipment. Bioelectrochemtcal methods based on electron transfer from living microorganisms or free enzymes to artificial electron acceptors (mediators) have been described for different applicattons [12-211. Recently, bioelectrochemical instrumentation has been developed [22] which allows the rapid determination of mrcroorgarnsms in a stmple way; a mediator IS reduced by the mtcroorganisms and the amount of reduced mediator is detected amperometrically (see Fig. 1). The current obtained is correlated with the number of

mediator reduced

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mediator oxidized

Fig 1 Prmclple nucroorganisms

of the bloelectrochemlcal

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Flow-inJectIon system The flow-injection manifold for bioprocess control is shown in Fig. 2. The sample and carrier stream were propelled by penstaltic pumps (Jungkeit, N&ten Hardenberg, F.R.G.). The flow-injection tubing (Omnifit, Cambridge, Great Britain) was made of Teflon and had an i.d. of 0.5 or 0.8 mm. The sampling frequency was 1 h-‘. Sterile potassium hexacyanoferrate(II1) (25 mM) and glucose (5 mM) solution in potassium phosphate buffer (0.1 M, pH 7.0) were mixed with the sample m a mixing chamber. The flow was stopped for 4 min to allow the reaction between mediator and microorganisms to proceed to a sufficient extent, then 30 ~1 of this mixture were injected into the carrier stream (potassium phosphate buffer, 0.1 M, pH 7.0) by a V 200 injection valve (Tecator, Hiiganas, Sweden). The pumps and the valve were switched automatically by a PT 810 S process timer (Alphotromc, Stutensee, F.R.G.). The electrochenncal detector consisted of a small-wall jet system constructed at the GBF and a potentiostat (Bank Elektronik, G8ttmgen, F.R.G.). Detection

Mlcrobtologlcal methods E cob K 12 wild type (DSM - Deutsche Sammlung fiir Mikroorganismen, Braunschweig, F.R.G.) were grown in liquid culture, harvested by centrifugation (10 min at 4°C at 4800 min-‘), washed and resuspended in potassium phosphate buffer (0.1 M, pH 7.0). Standard colony plate count was performed by incubating dilutions of the microorganisms on nutrient agar containing 5 g 1-l yeast extract, 10 g 1-l peptone, 10 g 1-l NaCl and 15 g 1-l agar. The agar plates were incubated at 37 o C for 24 h and the colonies were counted visually. Beauvena basszana IMI 13929 and Pseudomonas aerugmosa were collected from fermentations at the Cranfield Institute of Technology (Cranfield, Great Britain) and diluted with potassium phosphate buffer (0.1 M, pH 7.0). On-line bioprocess control was investigated dunng a 5-l cultivation of E. cob K12 DH5 in a Bioflo III fermenter (New Brunswick, Heusenstamm, F.R.G.). The liquid medium contained 50 miring

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R D SCHMID

g 1-l glucose, 2 g 1-l yeast extract, 1 g 1-l KH,PO,, 2 g l-’ (NH,),HPO,, 0.8 g 1-l MgSO,, 5 mgl-’ EDTA, 1 mgl -I thiamine, trace element solution and 0.2 ml 1-l Ucolub anti-foam reagent. The absorbance was monitored by taking samples hourly, diluting them properly and measunng the absorbance at 546 nm by a Nova Spec II spectrophotometer (Pharmacia LKB, Freiburg, F.R.G.) at 546 nm. The biomass dry weight was controlled hourly by drying the samples for 12 h at 40 “C under vacuum (0.005 bar).

microorganisms [lo]. This system can be further optirmzed by combination with flow-injection analysis (FIA), which provides the possibility of full automation and precise measurement of small sample volumes [23].

Chemicals All chemicals were of analytical-reagent from Merck (Darmstadt, F.R.G.) and (Munich, F.R.G.).

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detector

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was effected with a three-electrode system with a platinum working electrode at a potential of + 400 mV vs. Ag/AgCl. The buffer, reagent solution and reaction loop were maintained at 30°C. The whole flow system was sterilized by overmght incubation with 5% sodium azide solution followed by rinsing with the same solution and sterile water. Calibration graphs were plotted by comparison of the flow-inJection signal with the results of standard methods using a slightly modified system. Instead of mixing the samples with the reagents m a mixing chamber, suspensions of microorganisms were mixed with potassium hexacyanoferrate(II1) and glucose in a beaker to fmal concentrations of 25 and 5 mM, respectively. Samples of this mixture were mjected mto the carrier stream every 30 s and measured amperometrically as described above.

RESULTS

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DISCUSSION

Determrnatlon of fungi For the deterrmnation of fungi using the flowmJection system, several dilutions of a Beauuena basslana suspension were mixed with mediator and glucose solution in a beaker and stirred. Samples were drawn from the beaker every 30 s and measured electrochenncally in a flow-through detector. As reference the biomass dry weight was determined. The increase m current due to the reoxidation of the reduced mediator at the workmg electrode is shown m Fig. 3. The relationship between current and time was linear. The rate of current increase was proportional to biomass dry weight and followed the equation y = 1.617x + 1.912 (r = 0.997; n = 4), where y is the dry weight m g l-‘, x is the slope of the current increase in nA min-’ and r 1s the correlation coefficient. The detection hrmt was 1.9 g 1-i; lower concentrations could not be detected owing to the background noise of the electrochemical detection system. These results demonstrated the basic suitability of the flow-injection method for the rapid deterrnmation of fungi. According to Jarvis et al. [24], no rapid and automated methods are available for the assay of moulds in food and feedmg

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time (mln) Fig. 3 Determmatlon of Beauuerrn bass~ana at different concentratlons usmg the flow-mqectlon system. Current: peak heights of flow-mJectlon signals. Time: reactlon time between mediator and sample. Correspondmg dry we&s v, 15 4, v, 7 7, n, 3.9; 0, 1.9 g-1.

stuffs. Therefore, the use of the flow-inJection method is an interestmg approach to the detection of fungal contammation in a few minutes. The application of the method is still limited to fungi that are small enough to be propelled through the tubes and valves of the flow-inJection system. For example, experiments with spores of Aspergihs niger were unsuccessful because they blocked the inJection valve. Determrnatron of bactena E. coli could be determined using the flow-inJection system in the range 4.7 X 106-2.4 x lo9 colony-forming units (cfu) ml-’ when the microorganisms were not separated from the cultivation medium (see Fig. 4). The corresponding calibration graph followed the equation y = 1.137x +

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Fig 4. Determmatlon of E co11 at different concentrations using the flow-uqectton system Current and time as m Fig. 3 Correspondmg cell counts’ v, 2 4X 109; v, 9 4X lo*, n, 4 7 x 10s. 0,94X10’; 0,47X10’; 0, 47X106cfu ml-’

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Fig 5 Determmatlon of Pseudomonas aerugrnosa at two dlfferent concentrattons usmg the flow-mqectlon system. Current and time as m Fig 3 Correspondmg absorbances T, 0 05; v, 0 11

1.406 (r = 0.997; n = 7) where y is the standard colony count in lo6 cfu ml-‘, x is the slope of the current increase in nA mm’ and r is the correlation coefficient. The results correlate with recently published work [20] in which a linear relationship between cell concentration and current rate was reported for a bioelectrochemical method using tlnonme as mediator. When the bacterial cells were harvested by centrifugation and then washed and resuspended in phosphate buffer, a detection limit of 7 x 10’ cfu ml-’ was obtained. This decrease in sensitivity was probably due to damage of the bacterial cells during isolation. Further, the metabolic activity of the bacteria decreased on storage m phosphate buffer at 4°C. A maximum reaction time of 20 mm was chosen in both mstances because the aim of this work was to develop a rapid method. A better sensitivity of the system should be obtained by a longer reaction time between the microorganisms and the mediator. Figure 5 shows the results of measurements with Pseudomonas aerugznosa for two different concentrations. No linear relationship between current and absorbance was found for the higher concentration and the signals were similar for the two samples. Therefore, the bioelectrochemical flow-injection method with potassium hexacyanoferrate(II1) as mediator was not applicable to this rmcroorganism. The mediator was obviously not suitable as an electron acceptor in the metabolic

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pathways of Pseudomonas aeruginosa. This demonstrated a fundamental problem with the bioelectrochermcal method, i.e., there are differences in the acceptance of a certain mediator by different microorganisms. To overcome this problem, other workers used a mediator mixture of potassium hexacyanoferrate(II1) and benzoquinone [25]. More microorganisms were detectable and a more even signal was achieved with different microorganisms by use of this mixture. The use of benzoquinone m the flow-injection system was impossible because of its instability in aqueous solution. Further applications of the flow-injection method for the detection of other microorganisms will probably include the use of other mediators. However, at present there are no details available about the reaction mechanism between the mediator and the living cells. Hence the sunability of a mediator for the detection of a microorganism can only be demonstrated experimentally. Bzoprocess control

The suitability of the automated flow-injection system for on-line bioprocess control was investigated during an E colz batch fermentation. The results are shown in Fig. 6 in comparison with the reference methods. In the first 8 h the standard methods gave an exponential curve whereas the flow-injection signal showed a logarithmic response. A very good correlation was demonstrated

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Fig 6 Control of an E co11 K12 DH5 cultlvatlon usmg the flow-mjectton system and standard methods. Ttme culttvatton ttme V, flow-mJectton signal, v, dry we&t, n, absorbance at 546 nm.

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between the electrochemical signals, biomass dry we&t and absorbance after 8 h of cultivation. The standard methods for determining biomass in fermenters are the photometnc measurement of the absorbance and the determination of the biomass dry weight. Neither method is suitable for automation and on-line control of bioprocesses owmg to the need for dilution steps or prolonged heat treatment, respectively. Further, the measurement of physical parameters, such as absorbance and dry mass, does not take mto consideratton the metabolic state of the microorganisms. These disadvantages are overcome m the flow-mjection system. It provtdes full automation and free variation of the sampling frequency up to 10 h-‘. Reduction of the mediator by the sample is dependent on the metabolism of the microorganisms. Therefore, the flow-mjectton signals indicate the metabolic state of the microbial cells. In btoprocesses used for the production of substances, the metabolic actwrty is a more important parameter than the increase in biomass. The flow-mjectton system is a cheap and time-saving alternative for the control of bioprocesses. Applications to microorganisms other than E. colr should be possible by using other mediators. The authors thank I.J. Higgins, A.P.F. Turner, A. Swam, J. Dicks and all co-workers at the Cranfield Institute of Technology for their helpful support. Special thanks are due to D. Korz for domg the fermentation experiments.

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