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Influence of microwave radiation on bacterial community structure in biofilm
Process Biochemistry 42 (2007) 1250–1253 www.elsevier.com/locate/procbio
Short communication
Influence of microwave radiation on bacterial community structure in biofilm Marcin Zielin´ski *, Sławomir Ciesielski, Agnieszka Cydzik-Kwiatkowska, Jarosław Turek, Marcin De˛bowski University of Warmia and Mazury in Olsztyn, Faculty of Environmental Sciences and Fisheries, Department of Environmental Protection Engineering, Prawochen´skiego St. 1, 10-957 Olsztyn, Poland Received 9 June 2006; received in revised form 9 May 2007; accepted 10 May 2007
Abstract Organic matter exposed to microwave radiation triggers standard thermal effects as well as a range of so called non-thermal effects. The present work examined the non-thermal effects of microwave radiation on the biofilm in bioreactors with immobilised biomass. Microwave-exposed and conventionally heated reactors were used and both groups of reactors operated on analogous technological parameters. The analysis of the treated sewage demonstrated a significant increase in nitrification and denitrification efficiency in the bioreactors treated with microwave radiation. An analysis of bacteria diversity based on DGGE method showed significantly different bacterial communities developed in the reactors exposed to the microwave radiation in comparison to the control reactors. Moreover, bacterial richness measured by Shannon index was significantly higher in the microwave treated samples (Mann–Whitney’s test, p < 0.05). These findings indicate that microwave radiation can affect the structure and function of bacterial communities independent of thermal effects. # 2007 Elsevier Ltd. All rights reserved. Keywords: PCR-DGGE; Richness index; Bacterial communities; Nitrogen compounds removal; Organic compounds removal
1. Introduction Microwaves are part of the electromagnetic spectrum with a wavelength ranging from 1 mm to 1 m and a corresponding frequency from 300 MHz to 300 GHz. Two types of microwaves effects have been recognized, thermal and non-thermal [1]. Thermal effects relate to processes which generate heat as a result of the absorption of the microwave energy by water, or organic complexes marked by either constant or induced polarization. The microwave energy is transformed into heat derived from the internal resistance of rotation. Non-thermal effects (also known as ‘athermal effects’ or ‘specific effects of electromagnetic irradiation’) relate to several microwaveinduced phenomena unrelated to temperature rise [2]. The kinetic reaction rate of wastewater treatment in a biofilm reactor depends on several phenomena and parameters such as biofilm biomass density and activity, wastewater–biofilm contact area and diffusion rate between the biofilm and
* Corresponding author. Tel.: +48 89 523 41 24; fax: +48 89 523 41 24. E-mail address: [email protected] (M. Zielin´ski). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.05.008
wastewaters. Wastewater treatment end effects depend largely on the efficiency of external and internal diffusion rates and the rate of biochemical conversion of organic compounds by cellular metabolism. While these processes are significantly affected by temperature, microwave heating may provide not only focused heating of the biofilm, but also generate nonthermal effects that may affect biofilm structure and function. The objectives of this research were to isolate non-thermal effects in biofilms exposed to microwave electromagnetic radiation and to determine microwave radiation effects on bioreactor efficiency as a result changes in the structure of bacterial community in biofilm. 2. Materials and methods 2.1. Reactors design and chemical analysis Eight trickling bioreactors were used in the experiment, four of which were located inside a microwave-exposed chamber whereas the other four were placed in a chamber heated with warm air as a control group. Identical temperature of the bioreactor media (21 1 8C) was maintained in each case. The four research columns were irradiated with microwave radiation at 2.45 GHz at 18 W or 0.01 W cm3 of the reactor’s media. A control system
M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253 Table 1 Composition of synthetic wastewaters (PN-87/C–04616/10) Component
Unit
Dry broth concentrate stock Meat concentrate Peptone NaCl Pepton K Yeast concentrate NH2CONH2 CH3COONa C6H10O5 CaCl22H2O MgSO47H2O NaCl KCl
150 mg l1 0.40 g l1 4.00 g l1 3.50 g l1 5.40 g l1 1.70 g l1 30 mg l1 10 mg l1 50 mg l1 7 mg l1 50 mg l1 30 mg l1 7 mg l1
automatically synchronized the temperature of all eight trickling bioreactors. The temperature was measured with HI 98804 Thermometer with K-type thermocouple probe (Hanna Instruments). Both the reactor’s cover and the media were made of material permeable to microwaves. The trickling towers bioreactor’s active volume (V) was 565 cm3 with a media specific surface area(s) of 202 m2/m3 giving a theoretical active surface (F) of 0.114 m2. A magnetron was used as the source of microwave radiation, which was transmitted through a waveguide to four of the bioreactor’s column. Table 1 presents the composition of synthetic wastewaters used in the experiment. The ratio between the COD (chemical oxygen demand) and the BOD (biochemical oxygen demand) was calculated as 0.71. The biofilm depth ranged from 0.05 mm to 1.5 mm. A mean dry weight of the biofilm was 0.8433 g and 0.8426 g for the microwave and the conventionally heated bioreactors, respectively. The biofilm increment was 0.3403 g/g COD in the microwave heated bioreactors and 0.3454 g/g COD in the control bioreactors. The bioreactors operated on a time cycle, where once every 24 h the wastewaters (V = 0.5 l) in a sump tank was exchanged. The artificial wastewater was pumped from the retention tank to biological reactor, and then returned to the retention tank (Fig. 1). The hydraulic loading rate used in the experiment (q) was 0.20 m3 m2 h1, thus the wastewaters flowed through the bioreactors 6.8 times every 24 h. The organic pollutant load (COD) was 1.29 g m2 d1.
Fig. 1. Schematic of the research system.
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The bioreactors were inoculated by pumping activated sludge through them for 24 h. This was followed by a 60-day period of bioreactor operation with artificial wastewater, followed by a 13-day test period at the end of which samples for genetic analysis were collected. During the test period, samples were collected daily to determine the concentration of both organic compounds total organic carbon (TOC) and total nitrogen (TN) with a TOC THERMO 1200 analyzer. In addition, the concentration of ammonia-nitrogen and the oxidized nitrogen forms (nitrate-nitrogen and nitrite-nitrogen) were analyzed in the treated sewage, using a HACH DR 2010 spectrophotometer and HACH analysis procedures based on standard methods [3]. Data were tested for significance by one-way ANOVA, at the significance level of p < 0.05 followed by Tukey’s test to ascribe differences. The normality of the distribution was confirmed by Szapiro–Wilk’s test, and homogeneity of variances verified by Levene’s test.
3. PCR-DGGE The biofilm samples were obtained from the polyethylene media by scraping. The samples (approximately 0.5 g) were taken from eight reactors (four treated and four control) from layers situated 5 cm below the top of the column and stored at 20 8C prior to molecular analysis. Genomic DNA was extracted using the method of Tsai and Olson [4]. For DGGE analysis extracted DNA was amplified by PCR using 16S rRNA primer pair specific for eubacteria (341F: 50 -cgc ccg ccg cgc gcg gcg ggc ggg gcg ggg gca cgg ggg g cct acg gga ggc agc ag-30 , 515R: 50 -att acc gcg gct gct gg-30 ), spanning V region of 16S rDNA [5]. A touchdown thermocycling program was used for PCR as described by Murray et al. [6]. DGGE was performed with a D-CODE Universal Mutation System (Bio-Rad, Hercules, USA). The PCR products (20 ml) were applied directly to 8% polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) in a 0.5 TAE buffer (40 mM Tris, 40 mM acetic acid, 1 mM EDTA; pH 7.5) with a denaturing gradient ranging from 30% to 70%. Electrophoresis was run at a constant voltage of 60 V at 60 8C. After 16 h of electrophoresis, the gel was stained with SYBR gold (Molecular Probes, USA). Bands were detected automatically from digital images of the gel using KODAK 1D 3.6 Image Analysis Software (Eastman Kodak Company, USA). The structural diversity of the microbial community was examined by the Shannon index of general diversity H [7]: H = S(ni/N)log(ni/N), where ni is the height of density of gel bonds peaks and N the sum of all peaks’ heights of the densitometric curve. Mann–Whitney’s U-test was applied at the probability level of 95% to test for significant differences. The DGGE patterns were converted to a binary matrix, using presence–absence data. A pairwise similarity of the banding patterns of the different samples was calculated applying NeiLi distance [8] and an UPGMA cluster analysis was conducted by utilizing DGGEStat software [Erik van Hannen; the Netherlands Institute of Ecology]. Bootstrap values were calculated for each dichotomy. The statistical significance of the differences in community structure due to microwave irradiation was determined by using the Nei-Li distances. Two groups (microwave irradiated and control) were considered to be different if their Nei-Li distance was significantly greater than the Nei-Li distances between replicates in this same group [9]. Statistical significance between inter- and intra-group
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M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253
distances were investigated using Student’s t-test at the probability level of 95%. 4. Results Organic compounds concentration in the treated sewage did not exceed 6.3 mg TOC l1 and 9.13 mg TOC l1 in microwave- and conventionally heated reactors, respectively (Fig. 2). Such a low concentration of organic compounds in the treated sewage suggested that the organic feed had been greatly reduced. Nitrification of the available ammonia-nitrogen was facilitated in the microwave treatment. In the case of conventionally heated reactors, the amount of ammonianitrogen in the treated effluent was significantly higher (Fig. 3) and nitrification efficiency was lower. The amount of nitrates and nitrites in the treated effluent reached on average 34 mg N-NOx l1 whereas ammonia nitrogen concentration reached 15 mg N-NH4 l1, which comprised nearly 30% of total nitrogen in the treated effluent (Fig. 3). The reduction of nitrogen oxidized forms (N-NOx) was calculated as the difference between the amount of total nitrogen in the nontreated and treated sewage. In case of microwave heating the amount of total nitrogen was reduced on average by 35.9%, whereas in case of conventional heating by 19.2% (Fig. 4). Analysis of DGGE profiles revealed differences in the banding patterns and bands’ intensities between groups and samples. The number of bands in the DGGE profiles varied from 15 to 18 in microwave treated group and from 9 to 11 in control group. The H index values calculated for each sample ranged from 1.0631 to 1.1720 in the group treated with microwaves (mean 1.0955 0.0516), and from 0.7203 to 0.982 in the control group (mean 0.8424 0.130). The observed H index values were significantly higher in the treated group than in the control (Mann–Whitney’s test, p < 0.05). Cluster analysis indicated that the DGGE patterns for microwaves were distinct from conventional heated reactor
Fig. 3. Participation of different form of nitrogen compounds in sewage.
Fig. 4. Differences between total nitrogen NTOT concentration in irradiate and control reactors.
(bootstrap values of 100 and 99.3%). The genetic distance was significantly higher between the microwaves treated and the conventional heated reactors than between replicates (Student’s t-test, p < 0.05). 5. Discussion
Fig. 2. Differences between TOC concentration in irradiate and control reactors.
Although numerous studies have been published on the possible non-thermal effects of microwave exposure on all kinds of living systems, at present little is known about the modes of action of microwaves at sublethal doses [10]. The destruction of microorganisms by microwaves at temperatures lower than the thermal killing point has been observed [11] indicating significant non-thermal effects. Electromagnetic fields have been shown to affect biofilms on the basis of either direct or indirect influence on the properties of receptors, which bind ligands such as Ca2+, neurotransmitters or hormones [12]. However, numerous researchers have questioned the existence of microwave-induced non-thermal effects [13,14]. Nonthermal effects on catalytic activity have been observed in isolated enzymes exposed to in vitro irradiation. For instance, Parker et al. [15] claimed that microwave-treated enzymatic
M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253
reaction rates increased two to three times in comparison to those reaction rates for enzymes exposed to conventional heating. In contrast, other enzymatic systems such as protein thermophilic enzymes respond to various intensities of microwave field with inhibition of activity [1]. Dreyfuss and Chipley [11] characterized effects of microwave irradiation on cells of Staphylococcus aureus. Upon exposure to microwave irradiation, the activity of some metabolic enzymes (e.g. glucose-6-phosphate dehydrogenase, alkaline phosphatase, lactate dehydrogenase) were altered in a way different from conventionally heated cells. Banik et al. [16] found microwave irradiation increased the growth and biomethanation of Methanosarcina barkeri. Rebrova [17] reported frequency-dependent exposure suppressed the colhicin synthesis in Escherichia coli and stimulated the synthesis of fibrinolytic enzymes in Bacillus firmus. In the gram-positive bacterium Bacillus mucilaginous increase in DNA, RNA and peptides synthesis was noticed in response to microwave radiation. Woo et al. [18] found that surface morphology of E. coli cells was more sensitive than Bacillus subtilis to microwaves. Furthermore, microwave-injured E. coli cells were more easily lysed by sodium dodecyl sulfate then to B. subtilis cells. Bacterial community structure and function was significantly affected by non-thermal effects of microwave. The differences in microwaves treated and hot air treated systems could be due to differential response of bacteria to microwaves. In our experiments these difference were manifest in the community activity. Microwaves noticeably improved nitrification efficiency. 6. Conclusion The results obtained in this experiment show that microwave radiation can have non-thermal effects on microbial community structure and the function in bioreactors. Microwave heating triggered alterations within the biofilm, which increased the efficiency of both nitrification and denitrification. Thus using microwaves as a means to increase bioreactor temperatures may have benefits exceeding intended thermal effects on metabolic activity. Acknowledgements The study was financed under the projects nos. 805.0810 and 809.0801 of the University of Warmia and Mazury in Olsztyn, Poland. This publication was supported by the Foundation for the Polish Science.
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References [1] Porcelli M, Cacciapuoti G, Fusco S, Massa R, d’Ambrosio G, Bertoldo C, et al. Non-thermal effects of microwaves on proteins: thermophilic enzymes as model system. FEBS Lett 1997;402:102–6. [2] Hong SM, Park JK, Lee YO. Mechanisms of microwave irradiation involved in the destruction of fecal coliforms from biosolids. Water Res 2004;38:1615–25. [3] Hach Company, 1997. Procedures manual for DR/2010 instruments. (Nitrogen, Ammonia, saliculate method, Test ’N Tube method 10031, Nitrite, Diazotizationing method, Test ‘N Tube method 8507, Nitrate, Chromatotropic Acid Method, Test ‘N Tube, method 10020), Colorado, USA. [4] Tsai GL, Olson BH. Rapid method for direct extraction of DNA from soil and sediments. Appl Environ Microbiol 1991;57:1070–4. [5] Muyzer G, de Waal EC, Uitterlinden AG. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes encoding for 16S rRNA. Appl Environ Microbiol 1993;59:695–700. [6] Murray AE, Hollibaugh JT, Orrego G. Phylogenetic composition of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments. Appl Environ Microbiol 1996;62:2676–80. [7] Shannon CE, Weaver W. The mathematical theory of communication. Urbana: University of Illinois Press; 1963. [8] Nei M, Li WH. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 1979;76: 5269–73. [9] van Hannen EJ, Mooij W, van Agterveld MP, Gons HJ, Laanbroek HJ. Detritus-dependent development of the microbial community in an experimental system: qualitative analysis by denaturing gradient gel electrophoresis. Appl Environ Microbiol 1999;65:2478–84. [10] Mileva K, Georgieva B, Radicheva N. About the biological effects of high and extremely high frequency electromagnetic fields. Acta Physiol Pharmacol Bulg 2003;27:89–100. [11] Dreyfuss MS, Chipley JR. Comparison of effects of sublethal microwave radiation and conventional heating on the metabolic activity of Staphylococcus aureus. Appl Environ Microbiol 1980;39:13–6. [12] Geletyuk VI, Kazachenko VN, Chemeris NK, Fasenko EE. Dual efects of microwaves on single Ca2+-activated K+ in cultured kidney cells Vero. FEBS Lett 1995;359:85–8. [13] Jeon JY, Kim HY. Microwave irradiation effect on diffusion of organic molecules in polymer. Eur Polym J 2000;36:895–9. [14] Ponne CT, Bartels P. Interaction of electromagnetic energy with biological material—relation to food processing. Radiat Phys Chem 1995;45:591–607. [15] Parker MC, Besson T, Lamare S, Legoy MD. Microwave radiation can increase the rate of enzyme catalysed reaction in organic media. Tetrahedron Lett 1996;37:8383–6. [16] Banik S, Bandyopadhyaya S, Ganguly S, Dan D. Effect of microwave irradiated Methanosarcina barkeri DSM-804 on biomethanation. Bioresour Technol 2006;97:819–23. [17] Rebrova TB. Effect of millimeter-range electromagnetic radiation on the vital activity of microorganisms. Milimetrovie Volni v Biologii i Med 1992;1:37–47. [18] Woo IS, Rhee IK, Park HD. Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure. Appl Environ Microbiol 2000;66:2243–7.
Short communication
Influence of microwave radiation on bacterial community structure in biofilm Marcin Zielin´ski *, Sławomir Ciesielski, Agnieszka Cydzik-Kwiatkowska, Jarosław Turek, Marcin De˛bowski University of Warmia and Mazury in Olsztyn, Faculty of Environmental Sciences and Fisheries, Department of Environmental Protection Engineering, Prawochen´skiego St. 1, 10-957 Olsztyn, Poland Received 9 June 2006; received in revised form 9 May 2007; accepted 10 May 2007
Abstract Organic matter exposed to microwave radiation triggers standard thermal effects as well as a range of so called non-thermal effects. The present work examined the non-thermal effects of microwave radiation on the biofilm in bioreactors with immobilised biomass. Microwave-exposed and conventionally heated reactors were used and both groups of reactors operated on analogous technological parameters. The analysis of the treated sewage demonstrated a significant increase in nitrification and denitrification efficiency in the bioreactors treated with microwave radiation. An analysis of bacteria diversity based on DGGE method showed significantly different bacterial communities developed in the reactors exposed to the microwave radiation in comparison to the control reactors. Moreover, bacterial richness measured by Shannon index was significantly higher in the microwave treated samples (Mann–Whitney’s test, p < 0.05). These findings indicate that microwave radiation can affect the structure and function of bacterial communities independent of thermal effects. # 2007 Elsevier Ltd. All rights reserved. Keywords: PCR-DGGE; Richness index; Bacterial communities; Nitrogen compounds removal; Organic compounds removal
1. Introduction Microwaves are part of the electromagnetic spectrum with a wavelength ranging from 1 mm to 1 m and a corresponding frequency from 300 MHz to 300 GHz. Two types of microwaves effects have been recognized, thermal and non-thermal [1]. Thermal effects relate to processes which generate heat as a result of the absorption of the microwave energy by water, or organic complexes marked by either constant or induced polarization. The microwave energy is transformed into heat derived from the internal resistance of rotation. Non-thermal effects (also known as ‘athermal effects’ or ‘specific effects of electromagnetic irradiation’) relate to several microwaveinduced phenomena unrelated to temperature rise [2]. The kinetic reaction rate of wastewater treatment in a biofilm reactor depends on several phenomena and parameters such as biofilm biomass density and activity, wastewater–biofilm contact area and diffusion rate between the biofilm and
* Corresponding author. Tel.: +48 89 523 41 24; fax: +48 89 523 41 24. E-mail address: [email protected] (M. Zielin´ski). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.05.008
wastewaters. Wastewater treatment end effects depend largely on the efficiency of external and internal diffusion rates and the rate of biochemical conversion of organic compounds by cellular metabolism. While these processes are significantly affected by temperature, microwave heating may provide not only focused heating of the biofilm, but also generate nonthermal effects that may affect biofilm structure and function. The objectives of this research were to isolate non-thermal effects in biofilms exposed to microwave electromagnetic radiation and to determine microwave radiation effects on bioreactor efficiency as a result changes in the structure of bacterial community in biofilm. 2. Materials and methods 2.1. Reactors design and chemical analysis Eight trickling bioreactors were used in the experiment, four of which were located inside a microwave-exposed chamber whereas the other four were placed in a chamber heated with warm air as a control group. Identical temperature of the bioreactor media (21 1 8C) was maintained in each case. The four research columns were irradiated with microwave radiation at 2.45 GHz at 18 W or 0.01 W cm3 of the reactor’s media. A control system
M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253 Table 1 Composition of synthetic wastewaters (PN-87/C–04616/10) Component
Unit
Dry broth concentrate stock Meat concentrate Peptone NaCl Pepton K Yeast concentrate NH2CONH2 CH3COONa C6H10O5 CaCl22H2O MgSO47H2O NaCl KCl
150 mg l1 0.40 g l1 4.00 g l1 3.50 g l1 5.40 g l1 1.70 g l1 30 mg l1 10 mg l1 50 mg l1 7 mg l1 50 mg l1 30 mg l1 7 mg l1
automatically synchronized the temperature of all eight trickling bioreactors. The temperature was measured with HI 98804 Thermometer with K-type thermocouple probe (Hanna Instruments). Both the reactor’s cover and the media were made of material permeable to microwaves. The trickling towers bioreactor’s active volume (V) was 565 cm3 with a media specific surface area(s) of 202 m2/m3 giving a theoretical active surface (F) of 0.114 m2. A magnetron was used as the source of microwave radiation, which was transmitted through a waveguide to four of the bioreactor’s column. Table 1 presents the composition of synthetic wastewaters used in the experiment. The ratio between the COD (chemical oxygen demand) and the BOD (biochemical oxygen demand) was calculated as 0.71. The biofilm depth ranged from 0.05 mm to 1.5 mm. A mean dry weight of the biofilm was 0.8433 g and 0.8426 g for the microwave and the conventionally heated bioreactors, respectively. The biofilm increment was 0.3403 g/g COD in the microwave heated bioreactors and 0.3454 g/g COD in the control bioreactors. The bioreactors operated on a time cycle, where once every 24 h the wastewaters (V = 0.5 l) in a sump tank was exchanged. The artificial wastewater was pumped from the retention tank to biological reactor, and then returned to the retention tank (Fig. 1). The hydraulic loading rate used in the experiment (q) was 0.20 m3 m2 h1, thus the wastewaters flowed through the bioreactors 6.8 times every 24 h. The organic pollutant load (COD) was 1.29 g m2 d1.
Fig. 1. Schematic of the research system.
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The bioreactors were inoculated by pumping activated sludge through them for 24 h. This was followed by a 60-day period of bioreactor operation with artificial wastewater, followed by a 13-day test period at the end of which samples for genetic analysis were collected. During the test period, samples were collected daily to determine the concentration of both organic compounds total organic carbon (TOC) and total nitrogen (TN) with a TOC THERMO 1200 analyzer. In addition, the concentration of ammonia-nitrogen and the oxidized nitrogen forms (nitrate-nitrogen and nitrite-nitrogen) were analyzed in the treated sewage, using a HACH DR 2010 spectrophotometer and HACH analysis procedures based on standard methods [3]. Data were tested for significance by one-way ANOVA, at the significance level of p < 0.05 followed by Tukey’s test to ascribe differences. The normality of the distribution was confirmed by Szapiro–Wilk’s test, and homogeneity of variances verified by Levene’s test.
3. PCR-DGGE The biofilm samples were obtained from the polyethylene media by scraping. The samples (approximately 0.5 g) were taken from eight reactors (four treated and four control) from layers situated 5 cm below the top of the column and stored at 20 8C prior to molecular analysis. Genomic DNA was extracted using the method of Tsai and Olson [4]. For DGGE analysis extracted DNA was amplified by PCR using 16S rRNA primer pair specific for eubacteria (341F: 50 -cgc ccg ccg cgc gcg gcg ggc ggg gcg ggg gca cgg ggg g cct acg gga ggc agc ag-30 , 515R: 50 -att acc gcg gct gct gg-30 ), spanning V region of 16S rDNA [5]. A touchdown thermocycling program was used for PCR as described by Murray et al. [6]. DGGE was performed with a D-CODE Universal Mutation System (Bio-Rad, Hercules, USA). The PCR products (20 ml) were applied directly to 8% polyacrylamide gels (37.5:1 acrylamide:bisacrylamide) in a 0.5 TAE buffer (40 mM Tris, 40 mM acetic acid, 1 mM EDTA; pH 7.5) with a denaturing gradient ranging from 30% to 70%. Electrophoresis was run at a constant voltage of 60 V at 60 8C. After 16 h of electrophoresis, the gel was stained with SYBR gold (Molecular Probes, USA). Bands were detected automatically from digital images of the gel using KODAK 1D 3.6 Image Analysis Software (Eastman Kodak Company, USA). The structural diversity of the microbial community was examined by the Shannon index of general diversity H [7]: H = S(ni/N)log(ni/N), where ni is the height of density of gel bonds peaks and N the sum of all peaks’ heights of the densitometric curve. Mann–Whitney’s U-test was applied at the probability level of 95% to test for significant differences. The DGGE patterns were converted to a binary matrix, using presence–absence data. A pairwise similarity of the banding patterns of the different samples was calculated applying NeiLi distance [8] and an UPGMA cluster analysis was conducted by utilizing DGGEStat software [Erik van Hannen; the Netherlands Institute of Ecology]. Bootstrap values were calculated for each dichotomy. The statistical significance of the differences in community structure due to microwave irradiation was determined by using the Nei-Li distances. Two groups (microwave irradiated and control) were considered to be different if their Nei-Li distance was significantly greater than the Nei-Li distances between replicates in this same group [9]. Statistical significance between inter- and intra-group
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M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253
distances were investigated using Student’s t-test at the probability level of 95%. 4. Results Organic compounds concentration in the treated sewage did not exceed 6.3 mg TOC l1 and 9.13 mg TOC l1 in microwave- and conventionally heated reactors, respectively (Fig. 2). Such a low concentration of organic compounds in the treated sewage suggested that the organic feed had been greatly reduced. Nitrification of the available ammonia-nitrogen was facilitated in the microwave treatment. In the case of conventionally heated reactors, the amount of ammonianitrogen in the treated effluent was significantly higher (Fig. 3) and nitrification efficiency was lower. The amount of nitrates and nitrites in the treated effluent reached on average 34 mg N-NOx l1 whereas ammonia nitrogen concentration reached 15 mg N-NH4 l1, which comprised nearly 30% of total nitrogen in the treated effluent (Fig. 3). The reduction of nitrogen oxidized forms (N-NOx) was calculated as the difference between the amount of total nitrogen in the nontreated and treated sewage. In case of microwave heating the amount of total nitrogen was reduced on average by 35.9%, whereas in case of conventional heating by 19.2% (Fig. 4). Analysis of DGGE profiles revealed differences in the banding patterns and bands’ intensities between groups and samples. The number of bands in the DGGE profiles varied from 15 to 18 in microwave treated group and from 9 to 11 in control group. The H index values calculated for each sample ranged from 1.0631 to 1.1720 in the group treated with microwaves (mean 1.0955 0.0516), and from 0.7203 to 0.982 in the control group (mean 0.8424 0.130). The observed H index values were significantly higher in the treated group than in the control (Mann–Whitney’s test, p < 0.05). Cluster analysis indicated that the DGGE patterns for microwaves were distinct from conventional heated reactor
Fig. 3. Participation of different form of nitrogen compounds in sewage.
Fig. 4. Differences between total nitrogen NTOT concentration in irradiate and control reactors.
(bootstrap values of 100 and 99.3%). The genetic distance was significantly higher between the microwaves treated and the conventional heated reactors than between replicates (Student’s t-test, p < 0.05). 5. Discussion
Fig. 2. Differences between TOC concentration in irradiate and control reactors.
Although numerous studies have been published on the possible non-thermal effects of microwave exposure on all kinds of living systems, at present little is known about the modes of action of microwaves at sublethal doses [10]. The destruction of microorganisms by microwaves at temperatures lower than the thermal killing point has been observed [11] indicating significant non-thermal effects. Electromagnetic fields have been shown to affect biofilms on the basis of either direct or indirect influence on the properties of receptors, which bind ligands such as Ca2+, neurotransmitters or hormones [12]. However, numerous researchers have questioned the existence of microwave-induced non-thermal effects [13,14]. Nonthermal effects on catalytic activity have been observed in isolated enzymes exposed to in vitro irradiation. For instance, Parker et al. [15] claimed that microwave-treated enzymatic
M. Zielin´ski et al. / Process Biochemistry 42 (2007) 1250–1253
reaction rates increased two to three times in comparison to those reaction rates for enzymes exposed to conventional heating. In contrast, other enzymatic systems such as protein thermophilic enzymes respond to various intensities of microwave field with inhibition of activity [1]. Dreyfuss and Chipley [11] characterized effects of microwave irradiation on cells of Staphylococcus aureus. Upon exposure to microwave irradiation, the activity of some metabolic enzymes (e.g. glucose-6-phosphate dehydrogenase, alkaline phosphatase, lactate dehydrogenase) were altered in a way different from conventionally heated cells. Banik et al. [16] found microwave irradiation increased the growth and biomethanation of Methanosarcina barkeri. Rebrova [17] reported frequency-dependent exposure suppressed the colhicin synthesis in Escherichia coli and stimulated the synthesis of fibrinolytic enzymes in Bacillus firmus. In the gram-positive bacterium Bacillus mucilaginous increase in DNA, RNA and peptides synthesis was noticed in response to microwave radiation. Woo et al. [18] found that surface morphology of E. coli cells was more sensitive than Bacillus subtilis to microwaves. Furthermore, microwave-injured E. coli cells were more easily lysed by sodium dodecyl sulfate then to B. subtilis cells. Bacterial community structure and function was significantly affected by non-thermal effects of microwave. The differences in microwaves treated and hot air treated systems could be due to differential response of bacteria to microwaves. In our experiments these difference were manifest in the community activity. Microwaves noticeably improved nitrification efficiency. 6. Conclusion The results obtained in this experiment show that microwave radiation can have non-thermal effects on microbial community structure and the function in bioreactors. Microwave heating triggered alterations within the biofilm, which increased the efficiency of both nitrification and denitrification. Thus using microwaves as a means to increase bioreactor temperatures may have benefits exceeding intended thermal effects on metabolic activity. Acknowledgements The study was financed under the projects nos. 805.0810 and 809.0801 of the University of Warmia and Mazury in Olsztyn, Poland. This publication was supported by the Foundation for the Polish Science.
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