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Electrical Impedance for Bacterial Metabolic Activity Screening-Evaluation of Single and Mixed Bacterial Consortia for Wastewater Biodegradation

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Electrical Impedance for Bacterial Metabolic Activity Screening-Evaluation of Single and Mixed Bacterial Consortia for Wastewater Biodegradation International Food Research Journal 17: 591-599 (2010) Electrical impedance for bacterial metabolic activity screening-evaluation of single and mixed bacterial consortia for wastewater biodegradation *Lasik, M. and Nowak, J. Poznań University of Life Sciences, Faculty of Food Science and Nutrition ul. Wojska Polskiego 31, 60-624 Poznan, Poland Abstract: The use of an electrical impedance measurement method for selection of bacteria with high metabolic activity, specific for treatment of high loaded potato industry wastewaters was investigated. The dynamic and effectiveness of wastewater biodegradation were evaluated in two ways: by the analysis of electrical impedance changes caused by bacterial metabolism during bioremediation process as well as by the reduction of chemical oxygen demand (COD). The course of impedance signals were described with Gompertz mathematical model and a parameter of maximal impedance changes rate (Imax), expressed as % of impedance changes h-1, was proposed for bacterial metabolic activity screening tests. A significantly higher (p<0.05) metabolic activity of bacteria occurring in the mixed cultures in comparison with single isolates coming from them was determined. A linear correlation (r=0.89), found between the wastewater COD reduction and the values of the maximal impedance changes rate (Imax) allowed the use of electrical impedance method and the proposed parameter for easier and quicker, in comparison with classic COD measurement, evaluation of the microbial wastewater treatment capability. Keywords: Electrical impedance, single and mixed bacteria populations, potato processing wastewater, biodegradation Introduction (Surucu, 1999). The microorganisms occurring in mixed cultures are characterized by higher biological Food industry produces one of the largest streams activity and smaller nutritive requirements in of industry wastewater. A high organic load, quite comparison with single isolates (Sakazawa et al., frequently campaign character (occurring temporarily 1981; Surucu, 1999; Lim et al., 2001). Except that, in large quantities) of the wastewater, cause that in the case of processes carried out by use of mixed despite many solutions there is still an urgent cultures there were observed higher product yields, need to look for other new methods of rapid and higher growth rates and lower contamination dangers effective ways of utilization. Most study is focused in confrontation with processes of single cultures on the control of the process, modification of the (Yang et al., 2003; He et al., 2004). technologies etc. On the other hand, little is known A successful biodegradation process may require about the microflora operating in the processes. the use of multiple microorganism consortium with Microorganisms employed in the wastewater complementary metabolic capabilities. It can bring utilization include bacteria mainly from the genus numerous potential benefits, including the ability Bacillus. They are used most commonly in systems of to degradation of specific, toxic compounds and mixed populations (Sonnleitner and Fiechter, 1983; establishing catabolic pathways that have not been Surucu, 1999; Lapara et al., 2001; Lim et al., 2001; occurred by single cultures (Sakazawa, et al., 1981; Cibis et al., 2002; Krzywonos et al., 2008). Mixed Shimao et al., 1984; Ramos et al., 1996; Oh and cultures taking part in wastewater biodegradation are Bartha, 1997; Park et al., 1999; Gilbert et al., 2003). formed generally in a natural way, in the result of The objective of the presented study was to select their adaptation to environmental conditions as well bacteria isolates characterized by high metabolic as microbial synergistic and antagonistic interactions activity and capability for biodegradation and Corresponding author. Email: [email protected] © All Rights Reserved Tel: +48 61 848 72 88; Fax: +48 61 848 73 14 592 Lasik, M. and Nowak, J. afterwards to develop from them a mixed culture wastes derived from the fruit and vegetable industry specific for the type of the treated wastewater. The (Lammas Resources Ltd., Corby, Northamptonshire, selection of the isolates, characterized by high the United Kingdom) and Mixed Culture C2 metabolic activity, was performed by employing (Bacillus laterosporus, Bacillus circulans, Bacillus the electrical impedance measurement method. fillocolonicus, Bacillus acidocaldarius, Bacillus The principle of the impedimetric technique is the stearothermophilus, Bacillus licheniformis) utilized measure of the electrical impedance changes caused for the process of hot potato slops biodegradation by the metabolic processes of microorganisms in the (Department of Bioprocess Engineering, Wroclaw culture medium. Molecules of high molecular weight University of Economics, Poland). The individual present in the medium (e.g. proteins, carbohydrates isolates obtained from the two mixed cultures were and fats) are degraded during biochemical processes identified by the Bergey key, API tests and the PCR of microorganisms to ionized metabolic products. method (unpublished data). Smaller, charged molecules result in the increase of All the microorganisms, mixed cultures and electric current conductivity and, consequently lead to single isolates coming from them were previously the decrease in the electrical impedance of the culture adapted to both types of wastewater, separately. environment (Noble, 1999; Gomez et al., 2002; The acclimatization was carried out by regularly Yunus et al., 2002). Primarily, this method is used passaging (every 3 days) on wastewater media by for rapid detection or quality and quantity analyses of shake culturing (130 rpm) at 55ºC (thermophilic microorganisms, especially in the quality control of conditions). food, pharmaceutical and cosmetic articles (Wawerla The inoculum was prepared using a nutritional et al., 1999). However, attempts were made to employ broth (BTL, Poland) as a growth medium. The impedance measurements for characterization of culture was carried out for 24 h in shaking conditions metabolic activities of microorganisms. Curda and (130 rpm) at the temperature of 55 °C. The inoculum Plockova (1995) investigated inhibiting properties used for the inoculation constituted 10% (v/v) of the of honey and its influence on the metabolic activities treated medium. of lactic acid bacteria, while Paquet et al. (2000) applied the measurement of electrical impedance to Evaluation of the metabolic activity of bacteria assess the activity of starter cultures in the process of The metabolic activity was performed by the cheese production. method of electrical impedance changes measurement In this study, the electrical impedance method is by using the Automatic Analyzer of Microorganisms proposed for selection of microorganisms consortia Growth BacTrac 4100 (Sy-Lab, Austria). Special with high metabolic activity for biological wastewater 10 ml measuring test tubes (Sy-Lab), equipped in utilization. The specific mixed bacteria cultures were 4 electrodes were used in the experiment. The test developed for the biodegradation of high loaded tubes were filled with 9 ml of wastewater, sterilized wastewater from a potato processing plant. at 121°C for 15 min and then inoculated with 1 ml inoculum of the tested bacteria population. As the Materials and Methods impedance changes strictly depend on the number of microorganisms in the sample, the inoculum was Wastewater diluted to the range from 3⋅103 to 4⋅103 cfu ml-1 and Two types of wastewater obtained from potato the number of viable bacteria cells in the inoculum processing plant (Wielkopolskie Przedsiebiorstwo was determined employing the classical plate method Przemysłu Ziemniaczanego, Lubon, Poland) were by using Plate Count Agar (Merck). The measuring used in the performed investigations: first, wastewater test tubes were incubated in the thermostat of the manufactured in the process of protein recovery from Automatic Analyzer of Microorganisms Growth potato juices by means of acid-thermal coagulation BacTrac 4100 for 24 h at 55°C. Changes in the – named hot wastewater, and second, wastewater electrical impedance of the treated wastewater, created during the process of starch production – caused by metabolic processes of bacteria, were named starch wastewater (Table 1). calculated in relation to the initial value according to the following formula: Microorganisms Two thermophilic Bacillus mixed cultures were used in the performed investigations: Mixed Culture C1 (Bacillus coagulans, Bacillus licheniformis, Bacillus laterosporus, Bacillus cereus) isolated from International Food Research Journal 17: 591-599 Electrical impedance for bacterial metabolic activity screening - evaluation of single and mixed bacterial consortia for wastewater593 Table 1. Chemical composition of potato starch wastewaters Concentration Parameters Unit Hot wastewater Starch wastewater Temperature ºC 80-85 10-20 pH - 5.3 ± 0.2 5.6 ± 0.3 COD [gO2 l-1] 31 ± 5 21 ± 5 BOD [gO2 l-1] 18 ± 3 10 ± 3 Total organic carbon (TOC) [g l-1] 9.4 ± 2.8 5.4 ± 1.2 Total nitrogen [g l-1] 2.1 ± 0.3 1.2 ± 0.1 Total phosphate [g l-1] 0.4 ± 0.1 0.2 ± 0.1 (y − y ) curves (Figure 1). The proposed parameters constitute y = 0 i ⋅100% authors own modification of those elaborated by y0 Zwietering (1993) and describe the dynamic of y – changes in the electrical impedance of the wastewater microbial growth (Lasik, 2004; Nowak et al., 2005). y0 – value of the electrical impedance at the beginning of culturing. The process of wastewater biodegradation y – value
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