Indian Journal of Chemical Technology Vol. 10, November 2003, pp. 607-6 1 0

Articles

Microorganism based biosensor for monitoring of microbiologically influenced

corrosion caused by fungal species

R S Dubey* & S N Upadhyay

Centre of Advanced Study, Department of Chemical Engineering and Technology, Banaras Hindu University, Varanasi 22 1 005, India

Received 11 January 2002; revised received 19 August 2003; accepted 30 August 2003

>" ' <The microfu.!Jgi are a group�micrOorgaIiisrris, which obtain their. ene�gy, either' by �etobiC Qxtdajl§ilor by:Ji'rieio,hi�'t'er': • ""'. _ • . I - ( . 1 • • • • • _ _ . . . ... .- . • - • " .. . . .. \ __ • • • .1 - � - • . , • • '-:' . ' . '.s - � . :� ' J >.

inentatiori of orgariic m;lterial!r arid release 'organic aci3 metabolite as;�{end�prOduc:k·w.hich:is corrosive' W·1ial\i.ie� Xlieime:: ?b9lites .c�uld be detected , 9nli�e ;by mi�robial' �iosensor: An �.Rer�.�eiric.;.�9"o�!al:biosens�;. 'foi:ti;:���f;�#:��ti� of metabohtes .w.as developed'on a modlfied ,oxygen electrode WIth lffiffioblhze4 AcetQb(lCter.:sp>. o� l!,�tylct: o��J;Jlem.: br�e. The serisor 'signai a'nd fesponse time'wer� Qptimi:ied with:respect to pH, �mpefa�, Q(l.nce-l)tnW9.ij: .:�" ·j>qfitii� !�pb�lized �crobial cells. The< �st. �nSiti�it� :,�� re�pon� tiine : by� }��q�i�( bl�s�����{f�.�: '

" , " j';����4 :ffilc�oblal cells a�. 4.3 pH of the. cgrro&lve «n,vrronment, at.room tem�rature.X�9.!1C), ·�t: �������t>;� B",,-, ,;re:; 'sponSe time difference were exanuitect; using; fh'e,t$ain'tn:orrosive e.nviionmerit ... .. 'y _ �F - .- . < ��:;i:';;-l,·;�'::{'Si:';>�1ti • ..-

The metallic corrosion by organic acids, produced by metabolic activities of microfungi, have been reported by many investigators 1 -3 . Microbial corrosion can be influenced either by aerobic or anaerobic activity which is dependent upon the presence or absence of oxygen in the environment4-7. It has been well­recognized that microfungi produce organic acids such as, acetic acid, citric acid, glutamic acid, carbox­ylic acid, etc. which create problems in many indus­tries8•9•

Corrosion is influenced by the presence of micro­organisms on the metallic surface, through the elec­trochemically active species secreted by microorgan­isms in the corrosion process. It is well-known that corrosion reactions are subject to electrochemi.cal po­larization phenomenon, which be monitored system­atically by electrochemical techniques9, 1 O. However, in the case of microbiologically influenced corrosion (MIC), electrochemical and surface analytical tech­niques are not adequate in explaining the extent and type of microorganisms involved in the MIC prob­lems. Due to lack of knowledge about exact microbial species involved in microbial corrosion process, the monitoring of MIC is very difficult The products se­creted during MIC are not thoroughly understood. Therefore, the mechanisms involved are still contro-

*For correspondence (E-mail : dubey_rsagar@rediffmail.com; snu@banaras.ernet.in)

versial and conflicting about microbes and their meta­bolic activities.

In view of the above constraints, the online meas­urement of corrosive species secreted by the fungal species during microbe-metal interaction is rather dif­ficult. In this regard, microbial biosensors are very useful for monitoring different microbial metabolites, which directly participate in MIC. Over the last few years, several biosensors have been developed for the detection of organic substrates I 1 , 1 2, Many techniques have been reported13- 1 5 for the immobilization of mi­croorganisms'. Enzyme based biosensors are generally specific and more expensive. Many microbial biosen­sors consist of immobilized microorganisms placed under suitable environment, with electrochemical de­vice and thus, are suitable for online monitoring of biochemical processes.

In the present investigation, a microorganism-based biosensor consisting of immobilized Acetobacter sp, on porous cellulose acetate membrane attached with oxygen electrode has been developed and its applica­tion in monitoring of microbiologically influenced corrosion caused by the group of microfungi has been investigated.

Experimental Procedure

Culture of bacterin The Acetobacter sp. was isolated from the corroded

metal surface. Details of isolation and identification

Articles

are given elsewhere '8 . These bacteria were used for the construction of microbial biosensor. The bacteria were cultured in growth medium [dextrose, 2; casein, 0.2; K2HP04, 0.05 ; FeCh, 0.06 and MgS04.7H20, 0.20 g per l itre in triple distilled water] under aerobic conditions at pH 6.5-6.8.

Immobilization of the microorganism Fifteen mL of the culture broth of Acetobacter sp.

grown for 48 h was centrifuged at 1 0,000 rpm for 1 0 min. The supernatant was discarded. The pellet -was washed three times with sterile triple distilled water, and finally suspended in 5 mL distilled water, and 4 mL of suspension was dripped onto a porous acetyl­celullose membrane (Genei Co., 0.45 /lm pore size, 4.7 mm diameter, 1 50 /lm, thickness). The microbes were allowed to be retained over the membrane.

Assembly of the microbial electrode The scheme of the microbial biosensor is shown in

Fig. 1 . The oxygen electrode (Elico Instrumentation Ltd., Hyderabad, India) consisted of gold cathode and a silver anode. The porous acetylcelullose membrane retaining Acetobacter sp. was cut into circular disc and soaked in the alkaline buffer (0. 1 M KH2P04-NaOH and pH), adjusted to pH 8.2 for better perform­ance. The circular membrane was attached to the ca­thodic surface of the oxygen probe. The microbe layer was then covered with gas permeable synthetic net and held in place by the help of an O-ring.

Experimental set�up and method The experimental set-up is depicted in Fig. 2. All

measurements were performed in 250 mL Borosil glass-cell containing microbial electrode and mag­netic stirrer ( 1 000 rpm). The cell was directly attached to interface (Wenking POS-73) and recorder (Servo­gor XY 733).

The working volume of the solution containing acetic acid was 1 00 mL and temperature of the cell was maintained by the electronically controlled ther­mostat at 25±0.5°C. The pH of the solution was ad­justed with 0.05 M H2S04 and saturated with air. Then, it was added periodically in the cell for meas­uring the current value.

Results and Discussion

Response of microbial biosensor The experiments were performed to investigate the

effect of various parameters on the response time. Fig. 3 shows typical response curves of microbial bio-

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Indian J. Chern. Techno!., November 2003

Anode (silver)

Separator

Acetyl celullose ....-:1MW.MlJlt'-' membrane

Synthetic net

Fig. l -Scheme of the microorganism-based biosensor

Microbial

biosensor

Buffer

solution

Interface Recorder

Fig. 2-Schematic diagram of the amperometric microbial bio­sensor

sensor based on the immobilized Acetobacter sp. for acetic acid. At beginning period (zero stage) the cur­rent was obtained with oxygen saturated water and showed endogeneous respiration in immobilized Acetobacter sp. on porous acetylcelullose membrane. During endogeneous respiration the inherent oxygen present in situ caused oxidation of substrate present in cytoplasm of bacterial cell, and there was equilibrium between the oxygen dissolved in the medium and the current level. When the sample solution was added into the cell it permeated through the acetylcelullose membrane. Immobilized microbe present on mem­brane assimilated it. Microorganisms then consumed oxygen for respiration, so that the concentration of dissolved oxygen around the immobilized microbial membrane decreased.

Dubey & Upadhyay: Microorganism based biosensor for monitoring of microbiologically influenced corrosion Articles

The steady state of the current revealed, that, the consumption of oxygen by the microorganism and diffusion of the oxygen to the immobilized microbe from the cell containing test solution were in equilib­rium state, which depends on the concentration of acetic acid, injected into the cell and the amount of the microorganism immobilized on the membrane surface. The pH of the sample solution containing acetic acid had to be kept below pH value for acetic acid (4.74 at 28°C), because acetic ions cannot pass through the membrane. When microbial biosensor was inserted in distilled water, the sensor returned to its normal state. These results are in good agreement with the observations reported by other workers 19•2 1 •

When sample solution was added to test solution during the experiment, a calibration curve was ob­tained as shown in Fig. 4. A linear relationship was obtained between the concentration of acetic acid and the decrease in current. The total decrease in oxygen current is 0.4 /lA and response time is 1 0 min. The minimum concentration of acetic acid for measure­ment was 8 mg/L. The sensitivity of microbial biosen­sor for acetic acid is 0.04 /lA/mM. The reproducibility of the current difference was determined under influ­enced response of the microbial biosensor.

Monitoring of MIC by microbial biosensor

The microbiologically influenced corrosion (MIC) is a common problem in many industries. Electro­chemical process of corrosion is accelerated by meta­bolic activities of microorganisms. If biosensors and electrochemical techniques are combined together it can play a significant role for monitoring of MIC. In the case of Acetobacter sp. and other microfungi in­volved in the MIC, microorganisms produce organic acids, which influence corrosion process. According to Coponhegen2, most of the organic acids produced by microfungi are more detrimental to metals.

It is well-known that organic acids are more corro­sive for all types of metallic structures. When micro­bial biosensor was inserted into the culture medium contaminated by fungal strains for the determination of organic acids, the response was found to be very good in the presence of fungal species. In other envi­ronments (culture medium) affected by bacteria Pseu­domonas sp., Thiobacillus sp., Desulphovibrio sp., and nitrifying bacteria, etc. , however, the response of biosensor was very poor. The response of microbial biosensor depends on the assimilation of organic acid by immobilized microbes. The results obtained by

0.8 0 ·4

·3 1 . 3 x 1 0 M. CH3COOH

·3 2. 6 x 10 M. CH3COOH ·3

3. '9 x 1 0 M. CH3COOH

0.0 L....._--...JL...-._---L __ � __ _' o 1 0 2 0

Time (min) 3 0

Fig. 3-Response curves of microbial biosensor

4 0

0.6 r----------------,

� 0 .5 III � 0.4 CIl 'C 1 °·3 � 0 .2 '" 0 0 . 1

O . O ���-L-��-L-��-�� o 2 3 4 5 6 7 8 9 1 0 Concentration x 163(M)

Fig. 4-Calibration curves of microbial electrode

microbial biosensor and those obtained by conven­tional methods were found to be in good agreement and were not affected by other metabolic products and organic materials. The selectivity of microbial biosen­sor was determined. The sensor did not respond to other compounds produced by microorganisms or nu­trients present in the environment. Since, the work still is under progress, the detailed data has not been given here. For investigations of actual cause (type of microorganisms), which influences MIC, microbial biosensor may be very helpful. The microbial elec­trodes are stored below 4°C in order to prolong the life of the electrode for more than two weeks.

Conclusions The amperometric microbial biosensor was devel­

oped using immobilized Acetobacter sp. The micro­bial biosensor has good stability, response time, re­producibility and is cheaper than enzyme based bio-

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sensors. This may prove to be an ideal analytical tool for online monitoring of MIC. It may be applied for the identification of microorganisms and microbial problems caused by fungal species in many industries.

Acknowledgement One of the authors (RSD) thanks Dr P C Pandey,

Department of Chemistry, B.H.U. for experimental support. The financial support provided by the De­partment of Science and Technology, New Delhi un­der Fast Track Scheme is gratefully acknowledged.

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