Environ Sci Pollut Res (2015) 22:14932–14939 DOI 10.1007/s11356-015-4708-z
RESEARCH ARTICLE
Effects of electrolysis by low-amperage electric current on the chlorophyll fluorescence characteristics of Microcystis aeruginosa Li Lin 1,2 & Cong Feng 1,2 & Qingyun Li 1,2 & Min Wu 1,2 & Liangyuan Zhao 1,2
Received: 11 September 2014 / Accepted: 13 May 2015 / Published online: 22 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Effects of electrolysis by low-amperage electric current on the chlorophyll fluorescence characteristics of Microcystis aeruginosa were investigated in order to reveal the mechanisms of electrolytic inhibition of algae. Threshold of current density was found under a certain initial no. of algae cell. When current density was equal to or higher than the threshold (fixed electrolysis time), growth of algae was inhibited completely and the algae lost the ability to survive. Effect of algal solution volume on algal inhibition was insignificant. Thresholds of current density were 8, 10, 14, 20, and 22 mA cm−2 at 2.5×107, 5×107, 1×108, 2.5×108, and 5×108 cells mL−1 initial no. of algae cell, respectively. Correlativity between threshold of current and initial no. of algae cells was established for scale-up and determining operating conditions. Changes of chlorophyll fluorescence parameters demonstrated that photosystem (PS) II of algae was damaged by electrolysis but still maintained relatively high activity when algal solution was treated by current densities lower than the threshold. The activity of algae recovered completely after 6 days of cultivation. On the contrary, when current density was higher than the threshold, connection of phycobilisome (PBS) and PS II core complexes was destroyed, PS II system of algae was damaged irreversibly, and algae could not survive thoroughly. The inactivation of M. aeruginosa by electrolysis can be Responsible editor: Philippe Garrigues * Li Lin [email protected] 1
Department of Basin Water Environmental Research, Changjiang River Scientific Research Institute, Wuhan 430010, China
2
Key Laboratory of Water Resources in River Basins and Eco-Environmental Science of Hubei Province, Wuhan 430010, China
attributed to irreversible separation of PBS from PS II core complexes and the damage of PS II of M. aeruginosa. Keywords Algae . Electrolysis . Chlorophyll fluorescence . Mechanism . Thresholds of current density . Eutrophication
Introduction Cyanobacteria bloom, one of the most serious environmental problems around the world, seriously threatens the drinking water safety worldwide (Paerl and Otten. 2013). Electrochemical method is an environmental-friendly technology for water treatment (Feng et al. 2004; Martinez-Huitle and Brillas 2008; Yuan et al. 2010). In recent years, there has been an increasing interest in the use of electrochemical method to inhibit and inactivate algae (Shi et al. 2005; Xu et al. 2007; Nanayakkara et al. 2011; Lin et al. 2012). Electrochemical method has the advantage that there is no need to add any harmful chemicals to the water (Nanayakkara et al. 2011). The ability of electrolysis to inhibit and inactivate algae has been reported to be effective (Liang et al. 2005; Xu et al. 2007; Feng et al. 2003). Our previous study (Lin et al. 2012) demonstrated that electrolytic inhibition of Microcystis aeruginosa was very effective by low-amperage electric current. However, the thresholds of current density for scale-up and operation of electrolytic inhibition of M. aeruginosa were still not clear. This blocked the improvement of efficiency of energy utilization of electrochemical method badly. The electrolytic inhibition mechanisms were considered as (i) the generated active substances (e.g., active oxygen and free chlorine) can inhibit algae (Xu et al. 2007) and (ii) changes of algal surface morphological structure caused by electrochemical treatment (Liang et al. 2005). Besides, the active oxygen species electrochemically generated at the anode were
Environ Sci Pollut Res (2015) 22:14932–14939
proved playing the most important role in producing the significant residual effect for controlling the growth of M. aeruginosa (Xu et al. 2007). Parts of oxidants produced from the destruction of radicals had quite a long life time, and they could diffuse into the area away from the electrodes continuing the oxidation process for continuous algal inhibition (Xu et al. 2007) However, the damage mechanism of algal cells by electrolysis was not fully understood. Chlorophyll fluorescence is a good measure of the photosynthetic activities of algae and higher plants (Macinnis-Ng and Ralphsd 2003; Muller et al. 2008). It is well known that when photosynthetic events related to biochemical or physiological processes are inhibited, the yield and kinetics of dissipated fluorescence are significantly changed. Therefore, chlorophyll fluorescence can serve as a reliable, noninvasive indicator of photosynthetic processes in algae. The pulseamplitude-modulation (PAM) fluorometric approach can provide useful information concerning photosynthetic electron transport and energy dissipation processes associated with photosystem (PS) II and PS I activity. Parameters linked to maximum quantum yield of PS II (Fv/Fm), effective quantum yield of PS II (Y(II)), rate of photosynthetic electron transport through PS II (ETR), and non-regulated energy dissipation of PS II (Y(NO)) are sensitive bioindicators of damage in algae and plants (Li et al. 2011; Maxwell and Johnson 2000). It has been used to detect the changes of photosynthetic activities of algae and higher plants in adversities including high temperature (Yamane et al. 1997), low temperature (Rizza et al. 2001), drought (Massacci et al. 2008), UV light (Heraud and Beardall 2000), toxic pollutants (Schreiber et al. 2007), etc. Nevertheless, according to our knowledge, there are no reports on the effect of electrolysis by low-amperage electric current on the chlorophyll fluorescence parameters of M. aeruginosa. In this study, electrolysis by low-amperage electric current was used to inhibit the growth of M. aeruginosa in water. The objectives are (1) to investigate the thresholds of current density for scale-up and operation of electrolytic inhibition of M. aeruginosa and (2) to explore the changes of chlorophyll fluorescence of M. aeruginosa for the mechanisms of electrolytic inhibition of M. aeruginosa.
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growth phase to a freshly sterilized flask with BG-11 media. The algal seeds were cultured in an illumination incubator with illumination (Model SPX-250B-G, China). The continuous light was provided by incandescent lamp with an automated 14-h/10-h light/dark cycle. The light intensity during the light phase was 30 μmol photons m−2 s−1. The temperature was controlled at 25±1 °C. Electrolysis cell The schematic diagram of the experimental apparatus was shown in Fig. 1. The experiment was carried out in beakers with different volumes (100 and 500 mL), with a vertical arrangement of electrodes. According to our previous study (Lin et al. 2012), Ti-RuO2 and stainless steel were chosen as anode and cathode, respectively. The dimensions of electrodes were about 2.5 cm× 7.5 cm. The geometrical area immersed in the algal solution was 13.75 cm2. The electrodes’ distance was 4 cm. Agitation during electrolysis was provided by a magnetic stirrer (Model 85-2, China), and agitation rate was 200 rpm. A direct current power source (Model WYK-305B2, China, 30 V/5 A) was employed to provide the electric power during electrochemical experiments. The room temperature was controlled at 20 °C approximately by an air conditioner. Experimental procedure Beakers with 100- and 500-mL volumes were filled with 100and 500-mL algal solutions, respectively, at different initial algae cell densities. The electrolytic time was set as 15 min.
Materials and methods Algal culture In this study, M. aeruginosa FACHB-905 was obtained from the Wuhan Institute of Hydrology Chinese Academy of Sciences. The composition of the BG-11 (Olvera-Ramírez et al. 2000) was added to each Erlenmeyer flask equipped with a gauze stopper and autoclaved (Mode BXM-30R, China) (121 °C, 30 min). Stock cultures were prepared daily by transfer of a known number of algal cells from a culture in the log
Fig. 1 Schematic diagram of the experimental apparatus (1 anode, 2 cathode, 3 direct current power, 4 thermometer, 5 sampling valve, 6 thermostat, 7 magnetic stirring bar)
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Environ Sci Pollut Res (2015) 22:14932–14939
The current densities and algal solution volumes used were shown in Table 1. In order to know whether the remaining algae cells had the potential to survive and grow, the algal solution after electrolysis was poured to a 100-mL conical flask with a gauze stopper and put into an illumination incubator to culture. Twelvemilliliter samples were taken from the conical flask at 0, 4, 8, 15, and 20 days or 0, 2, 4, 6, 8, and 15 days (depended on the initial cell densities of algal solutions). Control samples with no electrolysis were also exposed to the same conditions as the test samples. Optical density (OD)680 of algal solution samples which was used as the indirect index of cell viability of algal solution was measured. Chlorophyll fluorescence parameters were measured to determine the damage of algal cells.
Optical density measurement The optical densities at 680 nm of algal cell solutions (OD680) were measured by Lambda 25 spectrophotometer (PE, America). Chlorophyll fluorescence measurement Chlorophyll fluorescence parameters were recorded by the method described by Perreault et al. (2010) using a prototype of a Multi-ColorPAM (Heinz Walz GmbH, Effeltrich, Germany) at 20 °C. Initially, algal solution samples were dark-adapted for 5 min. The maximal PS II quantum yield (Fv/Fm), effective quantum yield of PS II (Y(II)), electron transport rate through PS II (ETR), and non-regulated energy dissipation of PS II (Y(NO)) were recorded automatically. Data analysis and statistics
Analysis methods Cell density measurement M. aeruginosa cells were counted on a compound microscope (Mode 44X3A, Shanghai, China) in the counting chamber. Chlorophyll-a measurement The samples were filtered through GF/C filter papers under low vacuum, and the chlorophylls were extracted using 5 mL of 90 % acetone. The OD of the extracts at 663, 645, 630, and 750 nm were determined using a Lambda 25 ultraviolet spectrophotometer (PE, America). The chlorophyll-a concentration was calculated using the equations given by the Chinese National Standard methods (China Environmental Science Press 2002):
Results and discussion
Chlorophyll‐aðmg=LÞ ¼ ½11:64ðA1 −A4 Þ−2:16ðA2 −A4 Þ þ 0:10ðA3 −A4 ÞV =V g where A1, A2, A3, and A4 are the absorbance at 663, 645, 630, and 750 nm, respectively; V is the volume of the extract (5 mL); and Vg is the volume of filtered water (10 mL). Table 1
Experiment conditions for thresholds of current density
No. Initial no. of Initial cell Algal solution Current densities/ algae cells/ density/cells volume/mL mA cm−2 cells mL−1 1
2.5×107
4
2 3 4 5 6 7 8 9 10
2.5×105
5×107 1×108 2.5×108 5×108
5×10 5×105 1×105 1×106 2×105 2.5×106 5×105 5×106 1×106
All the experiments were performed in triplicate, and means and standard deviations were calculated for each treatment. For the chlorophyll fluorescence measure, five experiments were done. Significant differences between control and treated samples were determined by analysis of variance (ANOVA) using the software SPSS. Multiple comparisons were undertaken using the least significant difference (LSD) test, homogeneity of variance test, and Duncan’s multiple range tests for differences between means. The selected level of significant was p
RESEARCH ARTICLE
Effects of electrolysis by low-amperage electric current on the chlorophyll fluorescence characteristics of Microcystis aeruginosa Li Lin 1,2 & Cong Feng 1,2 & Qingyun Li 1,2 & Min Wu 1,2 & Liangyuan Zhao 1,2
Received: 11 September 2014 / Accepted: 13 May 2015 / Published online: 22 May 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract Effects of electrolysis by low-amperage electric current on the chlorophyll fluorescence characteristics of Microcystis aeruginosa were investigated in order to reveal the mechanisms of electrolytic inhibition of algae. Threshold of current density was found under a certain initial no. of algae cell. When current density was equal to or higher than the threshold (fixed electrolysis time), growth of algae was inhibited completely and the algae lost the ability to survive. Effect of algal solution volume on algal inhibition was insignificant. Thresholds of current density were 8, 10, 14, 20, and 22 mA cm−2 at 2.5×107, 5×107, 1×108, 2.5×108, and 5×108 cells mL−1 initial no. of algae cell, respectively. Correlativity between threshold of current and initial no. of algae cells was established for scale-up and determining operating conditions. Changes of chlorophyll fluorescence parameters demonstrated that photosystem (PS) II of algae was damaged by electrolysis but still maintained relatively high activity when algal solution was treated by current densities lower than the threshold. The activity of algae recovered completely after 6 days of cultivation. On the contrary, when current density was higher than the threshold, connection of phycobilisome (PBS) and PS II core complexes was destroyed, PS II system of algae was damaged irreversibly, and algae could not survive thoroughly. The inactivation of M. aeruginosa by electrolysis can be Responsible editor: Philippe Garrigues * Li Lin [email protected] 1
Department of Basin Water Environmental Research, Changjiang River Scientific Research Institute, Wuhan 430010, China
2
Key Laboratory of Water Resources in River Basins and Eco-Environmental Science of Hubei Province, Wuhan 430010, China
attributed to irreversible separation of PBS from PS II core complexes and the damage of PS II of M. aeruginosa. Keywords Algae . Electrolysis . Chlorophyll fluorescence . Mechanism . Thresholds of current density . Eutrophication
Introduction Cyanobacteria bloom, one of the most serious environmental problems around the world, seriously threatens the drinking water safety worldwide (Paerl and Otten. 2013). Electrochemical method is an environmental-friendly technology for water treatment (Feng et al. 2004; Martinez-Huitle and Brillas 2008; Yuan et al. 2010). In recent years, there has been an increasing interest in the use of electrochemical method to inhibit and inactivate algae (Shi et al. 2005; Xu et al. 2007; Nanayakkara et al. 2011; Lin et al. 2012). Electrochemical method has the advantage that there is no need to add any harmful chemicals to the water (Nanayakkara et al. 2011). The ability of electrolysis to inhibit and inactivate algae has been reported to be effective (Liang et al. 2005; Xu et al. 2007; Feng et al. 2003). Our previous study (Lin et al. 2012) demonstrated that electrolytic inhibition of Microcystis aeruginosa was very effective by low-amperage electric current. However, the thresholds of current density for scale-up and operation of electrolytic inhibition of M. aeruginosa were still not clear. This blocked the improvement of efficiency of energy utilization of electrochemical method badly. The electrolytic inhibition mechanisms were considered as (i) the generated active substances (e.g., active oxygen and free chlorine) can inhibit algae (Xu et al. 2007) and (ii) changes of algal surface morphological structure caused by electrochemical treatment (Liang et al. 2005). Besides, the active oxygen species electrochemically generated at the anode were
Environ Sci Pollut Res (2015) 22:14932–14939
proved playing the most important role in producing the significant residual effect for controlling the growth of M. aeruginosa (Xu et al. 2007). Parts of oxidants produced from the destruction of radicals had quite a long life time, and they could diffuse into the area away from the electrodes continuing the oxidation process for continuous algal inhibition (Xu et al. 2007) However, the damage mechanism of algal cells by electrolysis was not fully understood. Chlorophyll fluorescence is a good measure of the photosynthetic activities of algae and higher plants (Macinnis-Ng and Ralphsd 2003; Muller et al. 2008). It is well known that when photosynthetic events related to biochemical or physiological processes are inhibited, the yield and kinetics of dissipated fluorescence are significantly changed. Therefore, chlorophyll fluorescence can serve as a reliable, noninvasive indicator of photosynthetic processes in algae. The pulseamplitude-modulation (PAM) fluorometric approach can provide useful information concerning photosynthetic electron transport and energy dissipation processes associated with photosystem (PS) II and PS I activity. Parameters linked to maximum quantum yield of PS II (Fv/Fm), effective quantum yield of PS II (Y(II)), rate of photosynthetic electron transport through PS II (ETR), and non-regulated energy dissipation of PS II (Y(NO)) are sensitive bioindicators of damage in algae and plants (Li et al. 2011; Maxwell and Johnson 2000). It has been used to detect the changes of photosynthetic activities of algae and higher plants in adversities including high temperature (Yamane et al. 1997), low temperature (Rizza et al. 2001), drought (Massacci et al. 2008), UV light (Heraud and Beardall 2000), toxic pollutants (Schreiber et al. 2007), etc. Nevertheless, according to our knowledge, there are no reports on the effect of electrolysis by low-amperage electric current on the chlorophyll fluorescence parameters of M. aeruginosa. In this study, electrolysis by low-amperage electric current was used to inhibit the growth of M. aeruginosa in water. The objectives are (1) to investigate the thresholds of current density for scale-up and operation of electrolytic inhibition of M. aeruginosa and (2) to explore the changes of chlorophyll fluorescence of M. aeruginosa for the mechanisms of electrolytic inhibition of M. aeruginosa.
14933
growth phase to a freshly sterilized flask with BG-11 media. The algal seeds were cultured in an illumination incubator with illumination (Model SPX-250B-G, China). The continuous light was provided by incandescent lamp with an automated 14-h/10-h light/dark cycle. The light intensity during the light phase was 30 μmol photons m−2 s−1. The temperature was controlled at 25±1 °C. Electrolysis cell The schematic diagram of the experimental apparatus was shown in Fig. 1. The experiment was carried out in beakers with different volumes (100 and 500 mL), with a vertical arrangement of electrodes. According to our previous study (Lin et al. 2012), Ti-RuO2 and stainless steel were chosen as anode and cathode, respectively. The dimensions of electrodes were about 2.5 cm× 7.5 cm. The geometrical area immersed in the algal solution was 13.75 cm2. The electrodes’ distance was 4 cm. Agitation during electrolysis was provided by a magnetic stirrer (Model 85-2, China), and agitation rate was 200 rpm. A direct current power source (Model WYK-305B2, China, 30 V/5 A) was employed to provide the electric power during electrochemical experiments. The room temperature was controlled at 20 °C approximately by an air conditioner. Experimental procedure Beakers with 100- and 500-mL volumes were filled with 100and 500-mL algal solutions, respectively, at different initial algae cell densities. The electrolytic time was set as 15 min.
Materials and methods Algal culture In this study, M. aeruginosa FACHB-905 was obtained from the Wuhan Institute of Hydrology Chinese Academy of Sciences. The composition of the BG-11 (Olvera-Ramírez et al. 2000) was added to each Erlenmeyer flask equipped with a gauze stopper and autoclaved (Mode BXM-30R, China) (121 °C, 30 min). Stock cultures were prepared daily by transfer of a known number of algal cells from a culture in the log
Fig. 1 Schematic diagram of the experimental apparatus (1 anode, 2 cathode, 3 direct current power, 4 thermometer, 5 sampling valve, 6 thermostat, 7 magnetic stirring bar)
14934
Environ Sci Pollut Res (2015) 22:14932–14939
The current densities and algal solution volumes used were shown in Table 1. In order to know whether the remaining algae cells had the potential to survive and grow, the algal solution after electrolysis was poured to a 100-mL conical flask with a gauze stopper and put into an illumination incubator to culture. Twelvemilliliter samples were taken from the conical flask at 0, 4, 8, 15, and 20 days or 0, 2, 4, 6, 8, and 15 days (depended on the initial cell densities of algal solutions). Control samples with no electrolysis were also exposed to the same conditions as the test samples. Optical density (OD)680 of algal solution samples which was used as the indirect index of cell viability of algal solution was measured. Chlorophyll fluorescence parameters were measured to determine the damage of algal cells.
Optical density measurement The optical densities at 680 nm of algal cell solutions (OD680) were measured by Lambda 25 spectrophotometer (PE, America). Chlorophyll fluorescence measurement Chlorophyll fluorescence parameters were recorded by the method described by Perreault et al. (2010) using a prototype of a Multi-ColorPAM (Heinz Walz GmbH, Effeltrich, Germany) at 20 °C. Initially, algal solution samples were dark-adapted for 5 min. The maximal PS II quantum yield (Fv/Fm), effective quantum yield of PS II (Y(II)), electron transport rate through PS II (ETR), and non-regulated energy dissipation of PS II (Y(NO)) were recorded automatically. Data analysis and statistics
Analysis methods Cell density measurement M. aeruginosa cells were counted on a compound microscope (Mode 44X3A, Shanghai, China) in the counting chamber. Chlorophyll-a measurement The samples were filtered through GF/C filter papers under low vacuum, and the chlorophylls were extracted using 5 mL of 90 % acetone. The OD of the extracts at 663, 645, 630, and 750 nm were determined using a Lambda 25 ultraviolet spectrophotometer (PE, America). The chlorophyll-a concentration was calculated using the equations given by the Chinese National Standard methods (China Environmental Science Press 2002):
Results and discussion
Chlorophyll‐aðmg=LÞ ¼ ½11:64ðA1 −A4 Þ−2:16ðA2 −A4 Þ þ 0:10ðA3 −A4 ÞV =V g where A1, A2, A3, and A4 are the absorbance at 663, 645, 630, and 750 nm, respectively; V is the volume of the extract (5 mL); and Vg is the volume of filtered water (10 mL). Table 1
Experiment conditions for thresholds of current density
No. Initial no. of Initial cell Algal solution Current densities/ algae cells/ density/cells volume/mL mA cm−2 cells mL−1 1
2.5×107
4
2 3 4 5 6 7 8 9 10
2.5×105
5×107 1×108 2.5×108 5×108
5×10 5×105 1×105 1×106 2×105 2.5×106 5×105 5×106 1×106
All the experiments were performed in triplicate, and means and standard deviations were calculated for each treatment. For the chlorophyll fluorescence measure, five experiments were done. Significant differences between control and treated samples were determined by analysis of variance (ANOVA) using the software SPSS. Multiple comparisons were undertaken using the least significant difference (LSD) test, homogeneity of variance test, and Duncan’s multiple range tests for differences between means. The selected level of significant was p
