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The effect of photochemical treatment of water on algal growth
Water Research Vol. 14, pp. 539 to 543 O Pergamon Press Ltd 1980. Printed in Great Britain
0043-1354/80]0501-0539102.00/0
THE EFFECT O F P H O T O C H E M I C A L TREATMENT OF WATER ON ALGAL GROWTH* A. J. ACHER~" Institute of Soils and Water, The Volcani Center, Bet Dagan, Israel and ADA ELGAVISH Department of Isotopes, The Weizmann Institute of Science, Rehovot, Israel (Received September 1978) Abstraet--A new method for the destruction of algae in surface waters by dye-sensitized photooxidation is described. The algae (Peridiniura, Pediastrura and Cosmarium) used as test organisms were inoculated in an artificial culture medium, containing a dye-sensitizer, with subsequent incubation in a controlled environment (20 + 2°C, 14 h light, 10 h darkness~ Water samples from Peridinium bloom in Lake Kinneret underwent similar treatment. The algicidal effect of various sensitizer concentrations and of different sunlight exposure times was investigated. Complete destruction of algae was obtained in about 2 weeks of incubation after.exposure to solar radiation for 30-60 min in the presence of 0.25, 0.15 and 0.75 nag 1-1 methylene blue or 0.5, 0.8 and 0.8 mg 1-1 rose bengal in Peridinium, Pediastrum and Cosmarium cultures, respectively.
INTRODUCTION A massive occurrence of algae, called water bloom, has been reported to appear in many eutrophicated lakes (Huber-Pestalozzi, 1950; Jurgens, 1953). In Lake Kinneret (Israel) there is a bloom of the dinoflagellate algae Peridinium each year from about January to June (Berman& Rodbe, 1971). During the other seasons of the year, the phytoplankton population is dominated by Chlorophyta algae (Pollingher & Kimor, 1969). The Peridinium bloom is especially harmful to the quality of the lake water, which is the main reservoir for Israel's National Water Carder. Several methods have been considered for the removal of algae at the intake of the National Water Carrier (Eren, 1972), but with only limited satisfactory results: microstraining was found to be too expensive, CuSO4 treatment resulted in the release of odorous metabolites, while effective chlorination raised the amount of toxic chlorine derivatives in the treated water to an unacceptable level (Brungs, 1973). Other methods used for removal or destruction of algae, particularly from oxidation ponds or stabilization lagoons, are based on destabilization of algae suspension by chemical coagulation and flocculation followed by sedimentation or flotation (Friedman et al., 19771 Recently a new method for the treatment of organic matter in sewage effluent has been proposed (Acher & Rosenthal, 1977). The treatment consists of the combined action of visible light and atmospheric oxygen * Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 222-E, 1978 series. t" To whom correspondence should be addressed. 539
upon organic matter via an exogeneous photosensitizer (Kautsty, 19391 The high efficiency of this photochemical method in water disinfection (Acher & Juven, 1977) led us to investigate the influence of dyesensitizer on algal growth. The goal of this research was to determine the effects of various concentrations of methylene blue and rose bengal at different sunlight exposure times on the reduction of the algae population in batch cultures of three algae from Lake Kinneret; the dinoflagellate Peridinium cincture fa westii arid two Chloro. phyta algae: Pediastrum duplex, a colonial green algae, and Cosmarium sp., a desmid. The aim of this study was to develop a new approach to algal destruction in surface water. MATERIALS AND METHODS
Test organism The experiments were performed in batch cultures of. Peridinium cinctum fa westii, Pediastrum duplex and Cosmarium sp., three algae which have been isolated from Lake Kinneret at the Kinneret Limnological Laboratory, Tabgha, Israel. The three algae were grown in a minimal artificial medium (Table 1), at 20 :t: 2°C and an alternating light cycle of 14h light (white cool light of about 900/rE m -2 seC -1) and 10 h darkness. The effect of the treatment on Peridinium was also studied on a lake water sample from the May 1977 Peridiniura bloom (more than 90% of the phytoplankton consisted of Peridinium). A summary of the lake water characteristics has been published (german, 1973). Chemicals The sensitizers tested were methylene blue (British Drug House Ltd., No. 26132Q) and rose bengal (British Drug House Ltd., No. 26172E). They were added to the culture samples as 0.5% solution in distilled water.
A J. A(HFR and ADA ELGAVISH
540
Table 1, Chemical composition of the culture medium* Component
mg I ~
NaHCO3 Ca(NO3) 2 CaCI 2 MgSOa 7H20 NH4CI NaCI ZnCI: FeCI 3 -6H20 (NH4)6MoTO24'4H20
Component
20.0 40.0 27.7 25.0 20.0 228.0 0.10 0.20 1.00
mg 1-
MnCI 2 "4H20 COC12"6H2 H2SeO 3 H3BO 3 KI KBr Sr(NO3)2 Li2SO 4 EDTA
0.80 0.02 0.0l 4.00 0.002 20.0 0.044 0.1 20.0
* The pH of the culture medium = 7.40.
Solar radiation In the experiments in which samples were exposed to sunlight, the average light intensities were 1850 + 100/iE m -2 sec -t, as measured by a quantum sensor (Acher & Rosenthal, 1977). Biomass raeasurement The fluorescence emitted by the chlorophyll in intact algae cells 0-cmlut** = 660 mr0 upon excitation by a light beam (/ltx¢luttioa -~" 405--436 m#), was used as a method for algal biomass determination (Udenfriend, 19621 The fluorescence emitted in a 2 rnl culture sample was measured relative to the fluorescence of a standard filter (using water as a blank). A linear standard curve of dry algal weight vs. relative fluorescence in Peridinium, Pediastrum and Cosmarium cultures enabled us to translate the relative fluorescence measured in the experiments to dry biomass in mg (El$avish, 1978). The populatio n density of Peridinium was also determined by filtering 1 ml of culture or water-diluted culture through a millipore filter and counting the cells under a binocular microscope,
The desired amount of dye solution was added to the culture medium prior to the addition of the inoculum (2 ml). Control experimeaat~ without dye, were run simultaneously. Irnnu~ately after the inoculation, some of the samples were sunlight-irradiated for various intervals of time ranging from I0 to 120rain. During the solar radiation the flask-samples were kept immexsed in a water bath at 20 4-2°C. Both sunlight-exposed and non-exposed samples were then incubated in a controlled environment for about 35 days, The size of the algal population was measured immediately after the inoculation and then at intervals of about 7 days. All the experiments were performed in duplicate. Samples of lake water (10 ml) taken from the 1977 Peridinium bloom areas were transferred into sterile Erlenmeyer flasks and were treated like the Peridinium batch cultures.
RESULTS AND DISCUSSION
Aloal destruction treatment The experiments were performed in 25 mi Erlenmeyer flasks containing 8 ml of culture medium. The flasks were sterilized in an autoclave at 121°C for 20rain, All subsequent handling demanding sterile conditions was performed in a laminar air flow unit (class 100 gelaire, Gelman Instrument).
The effect of dye type Since algae can be described as hydrophilic biocolloids with a p p a r e n t negative charges (Ires, 1956}, different molecular charges of ionic-type dyes (cationic or anionic) may influence the intimate contact of the
Table 2. The minimum conditions for total algal destruction Algae
Type
Pediastrum
MB MB RB RB MB MB RB RB MB MB RB RB MB MB RB RB
Cosmarium
Peridinium
Peridinium (in lake water)
Dye mg 1- ~ 0.40 0.15 t.50 0.80 0.75 0.75 1.20 0.80 0.50 0.25 2.00 0.60 0.50 0.30 1.60 0.80
Solar exposure (min)
Incubation time (days)*
0 40 0 40 0 30 0 60 0 30 0 30 0 60 0 60
7 10 7 I0 20 10 10 10 25 14 35 35 14 14 14 14
* Minimum incubation time when algal population count was zero, without subsequent recovery.
Photochemical t r e a ~ t
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The effect of dye concentrations
; o..~......_ o.~7
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0
-
..-':
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-
.
,-
7 14 Incubation time, days
21
Fig. 1. The effect of dye concentration on Pediastrum growth. The figures on the curves represent dye concentration, in nag dye l-m. - - experiments with MB; - - experiments with RB; . . . . . control experiments. dye with the algae surface and contribute to the algicidal effectiveness of dyes. Consequently, two types of dye were used in the study: methylene blue (MB), a phenothyazine derivative of cationic type; and rose bengal (RB), a fluoresceine derivative of anionic type. The algicidal efficiency was checked by measuring their effect on algal biomass production in cultures. Results given in Table 2 and Figs. 1-3 show that both MB and RB affect algal growth. However, in all the experiments performed, MB showed a greater algicidai effect than RB. Therefore the complete destruction of algae (no algal recovery observed afterwards) was achieved at lower concentrations of MB, which represented 26, 62, 25 and 32% of the RB concentrations which exhibited the same algicidal effects on Pediastrum, Cosmarium, Peridinium in culture medium and
All the algae investigated were affected by the presence of dye in culture medium but their sensitivities are different and increase in the following order: Cosmarium < Peridinium < Pediastrum. The presence of a low concentration of MB (0.04 mg 1-1) in Pediastrum culture medium inhibited its growth (Fig. 1); the population size determined after incubation periods of 7, 14 and 21 days represented about 60, 63 and 70% of the population size in control samples, respectively. In the presence of 0.3 rag RB l- 1 the above figures were 35, 45 and 60~/~ respectively. The inhibition effect of 0.25 mg MB 1-1 on Peridinium growth was demonstrated by a reduction of the initial population size from 40 cells ml- 1 to 25 and 10 cells ml- ~ after 15 and 25 incubation days, while the algal population in the control samples had increased to 160 and 230 cells ml-~ in the same incubation periods, respectively (Fig. 2). In the case of Cosmarium (Fig. 3), addition of 0.30 rag RB l- 1 or 0.40 mg MB l- ~ stimulated the algal growth, and the biomass determination after 20 incubation days showed a relative increase of about 180 and 130~/~ respectively, as compared with control cultures. At higher dye concentrations the algicidal effect of MB and RB caused the complete destruction of the
240~
I
200 F
o=
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F,o
541
Peridinium in lake water, respectively (Table 2, samples not exposed to sunlight)~ Approximately the same figures were obtained by comparison of MB and RB concentrations used in samples submitted to solar radiation (Table 2l This greater effectiveness of MB may be attributed to factors like: chemical and photochemical stability, penetration power into algae cell, etc. It has already been reported that MB is readily taken up by algae and that it is non-toxic, e.g. the oxygen consumption of Chlorella pyrenoidosa increased upon addition to its culture medium of as much as 320 mg MB 1-1 (James, 1953).
m
,,AI,p"
30.5-
= o.o I-
I
of water on algal growth
i -/"'I=
t
//
8°1- ,-" .- . . . . . . . . . IZ.:--"'-
/-I
4 o ~ _ _ ~ o . ~ o z
0
0
15 Incubation time, days
25
Fig. 2. The effect of dye concentration on Peridinium growth. The figures on the curves represent dye concentration, in mg dye l-1. - - , experiments with MB; - - - experiments with RB; . . . . . control
experiments.
542
A.J. ACHERand ADA ELGAVISH I
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.~_---,', 0.30
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/
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,
/
=
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/ /
-
.¢/ -
Eo
I,'/I
186 /i/~ ~ - a - -
0
0.80
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:>0
Incubofion time, duys Fig. 3. The effect of dye concentration on Cosmarium growth. The figures on the curves represent dye concentration, in mg dye 1-1. - experiments with MB; - - experiments with RB; . . . . . control experiments.
algal cultures. In Pediastrum cultures, addition of 0.40 mg MB 1-1 or 1.50 nag RB 1-1 resulted in the complete destruction of the algae and no algal biomass could be detected after 7 incubation days. The Peridinium cultures were destroyed with 0,50 mg MB i - 1 or 2.00 nag RB 1-1 during an incubation period of 15 to 25 days. In Cosmarium cultures 0.75 mg MB 1-1 or 1.20 mg RB l- t was already effective for algal destruction after an incubation period of 20 days. In experiments carried out with Peridinium bloom in lake water, the addition of 0.50 mg MB l - t or 1.60 mg RB 1- ~ led to complete destruction of algae. The control samples of Peridinium bloom in lake water showed in the same time (I0 days) an increase in algal population from 120 to 150 cells m1-1. The effect of sunlight exposure
The exposure of the algae in the dye containing cultures to sunlight radiation affected significantly the biomass production in the following incubation days. Table 2 shows that for Pediastrum and Peridinium the dye concentrations necessary to achieve a complete algicidal effect in the cultures exposed to sunlight were about half those required in the cultures not submitted to solar radiation, In the case of Cosmarium cultures, the MB concentrations were the same with or without sunlight exposure, but the incubation time until the algal population was completely destroyed was reduced to half. Peridinium bloom cultures in lake water containing 0.30 mg MB 1-1 were less sensitive to solar radiation than Peridinium in artificial culture medium (0.25 mg MB 1- t and required a longer solar radiation time than the latter for ~ compl0te destruction (60 min as compared to 30rain). Control samples of Pediastrura, Cosmarium, Peridinium and
Peridinium in lake water submitted to solar radiation for 120 min at 20 + 2°C showed, after incubation of 10, 20, 25 and 14 days, relative algal growth of 730, 790, 350 and 250%, respectively, as compared with the initial populations. (Control samples not exposed to solar radiation showed about the same relative algal growth.) The synergistic effect of solar exposure and MB or RB on algal growth indicates the dye-sensitized photooxidation reaction are probably involved in the algicidal mechanism. In the case of algae living in surface waters in the presence of singlet oxygen (Zepp et al., 1977), the algae might have developed a biological defence system against the damaging effect of photodynamic action (Krinsky, 1968). However, the results presented in this paper suggested that the algae investigated are sensitive to such reactions and may undergo lethal damage. In the case of coliforms, it has been shown that the bactericidal effect was obtained by a destructive photooxidation and was not a mere dye inhibition of coliform growth (Acher & Juven, 1977). The working conditions (2 mg MB 1-~ and 30 rain exposure to sunlight) which were described above as having a bactericidal effect are well above those needed for complete destruction of algae. Thus, the present study demonstrates that the dye-sensitized photooxidation which had previously been proposed as an efficient method for the destruction of coliforms in water and sewage effluents, is adequate also for algal destruction, without suffering from the drawbacks of the conventional methods previously mentioned. Furthermore, as the dye-sensitizers are eventually photooxidized to uncolored compounds, no detrimental environmental impact is to be expected. REFERENCES
Acher A. J. & Rosenthal I. (1977) Dye-sensitized-photooxidation--a new approach to the treatment of organic matter in sewage effluents. Water Res. 11,557-562. Acher A. J. & Juven B. J. (1977) Destruction of coliforms in water and sewage water by dye-sensitized photooxidation. Appl. envir. Microbiol. 33, 1019-1022. Berman T. (ed.) (1973) Interaction between Hydrographic Physical and Biological Factors in Warm Lakes. Lake Kinneret Data Record, Kinneret Linmological Laboratory, Tabgha, Israel. Berman T. & Rodhe W. (1971) Distribution and migration of Peridinium in Lake Kinneret. Mitt. int. Verein. LimnoL 19, 266-276. Brungs W. A. (1973) Effects of residual chlorine on aquatic life. J. War. Pollut. Control Fed. 45, 2180-2193. Elgavish A. (1978) A comparative study of phosphorus utilization and storage in throe algae from Lake Kinncret: Peridinium cinctumfawestii, Pediastrum duplex and Cosmarium sp. Ph.D. Thesis, The Weizmann Institute of Science, Rehovot, Israel. Eren J. (1972) Effects of algal blooms on water quality in Israel National Water System. Prec. Adv. Water Pollut. Res., 6th Int. Conf, Jerusalem, June 18-23, A/5/10/1A/5/10/6. Friedman A. A., Peaks D. A. & Nichols R. L. (1977) Algae separation from oxidation pond effluents, J. Wat. Pollut. Control Fed. 49, 111-119.
Photochemical treatment of water on algal growth Huber-Pestalozzi G. (1950) Das phytoplankton des Siisswiissers: Systematik und Biologie Cryptophycea, Chloromonadien, Peridineen. Binnenoewa'sser 16, Teil 3. Ires K. J. (1956) Eiectrokinetic phenomena of planktonic algae. Proc. Soc. Wat. Treat. Examiners 5, 473-479. James W. O. (1953) Plant Respiration, p. 188. Oxford University Press, London. lurgens K. C. (1953) The red tide of Lake Austin Texas. Game and Fish 11, 8-13. Kautsky H. (1939) Quenching of luminiscenc¢ by oxygen. Trans. Faraday Soc. 35, 216-219. Krinsky M. I. (1968) Photophysiology, Vol. III, pp. 123-196. Academic Press, New York.
543
Pollingher U. & Kimor B. (1969) Seasonal and bathymetrical changes in the composition of the phytoplankton populations of Lake Tiberias based on biomass estimation during the years 1964-1967. Sea Fish Res. Bull. 55, 3-20. Udenfriend S. (1962) Fluorescence assay in biology and medicine, in Molecular Biology, Vol. 3, edited by Kaplan N. O. & Sehcraga H. A. Academic Press, New York. Zepp R. G., Wolfe N. L., Baughman G. L. & Hollis R. C. (1977) Singlet oxygen in natural waters. Nature, Lond. 267, 421-423.
0043-1354/80]0501-0539102.00/0
THE EFFECT O F P H O T O C H E M I C A L TREATMENT OF WATER ON ALGAL GROWTH* A. J. ACHER~" Institute of Soils and Water, The Volcani Center, Bet Dagan, Israel and ADA ELGAVISH Department of Isotopes, The Weizmann Institute of Science, Rehovot, Israel (Received September 1978) Abstraet--A new method for the destruction of algae in surface waters by dye-sensitized photooxidation is described. The algae (Peridiniura, Pediastrura and Cosmarium) used as test organisms were inoculated in an artificial culture medium, containing a dye-sensitizer, with subsequent incubation in a controlled environment (20 + 2°C, 14 h light, 10 h darkness~ Water samples from Peridinium bloom in Lake Kinneret underwent similar treatment. The algicidal effect of various sensitizer concentrations and of different sunlight exposure times was investigated. Complete destruction of algae was obtained in about 2 weeks of incubation after.exposure to solar radiation for 30-60 min in the presence of 0.25, 0.15 and 0.75 nag 1-1 methylene blue or 0.5, 0.8 and 0.8 mg 1-1 rose bengal in Peridinium, Pediastrum and Cosmarium cultures, respectively.
INTRODUCTION A massive occurrence of algae, called water bloom, has been reported to appear in many eutrophicated lakes (Huber-Pestalozzi, 1950; Jurgens, 1953). In Lake Kinneret (Israel) there is a bloom of the dinoflagellate algae Peridinium each year from about January to June (Berman& Rodbe, 1971). During the other seasons of the year, the phytoplankton population is dominated by Chlorophyta algae (Pollingher & Kimor, 1969). The Peridinium bloom is especially harmful to the quality of the lake water, which is the main reservoir for Israel's National Water Carder. Several methods have been considered for the removal of algae at the intake of the National Water Carrier (Eren, 1972), but with only limited satisfactory results: microstraining was found to be too expensive, CuSO4 treatment resulted in the release of odorous metabolites, while effective chlorination raised the amount of toxic chlorine derivatives in the treated water to an unacceptable level (Brungs, 1973). Other methods used for removal or destruction of algae, particularly from oxidation ponds or stabilization lagoons, are based on destabilization of algae suspension by chemical coagulation and flocculation followed by sedimentation or flotation (Friedman et al., 19771 Recently a new method for the treatment of organic matter in sewage effluent has been proposed (Acher & Rosenthal, 1977). The treatment consists of the combined action of visible light and atmospheric oxygen * Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 222-E, 1978 series. t" To whom correspondence should be addressed. 539
upon organic matter via an exogeneous photosensitizer (Kautsty, 19391 The high efficiency of this photochemical method in water disinfection (Acher & Juven, 1977) led us to investigate the influence of dyesensitizer on algal growth. The goal of this research was to determine the effects of various concentrations of methylene blue and rose bengal at different sunlight exposure times on the reduction of the algae population in batch cultures of three algae from Lake Kinneret; the dinoflagellate Peridinium cincture fa westii arid two Chloro. phyta algae: Pediastrum duplex, a colonial green algae, and Cosmarium sp., a desmid. The aim of this study was to develop a new approach to algal destruction in surface water. MATERIALS AND METHODS
Test organism The experiments were performed in batch cultures of. Peridinium cinctum fa westii, Pediastrum duplex and Cosmarium sp., three algae which have been isolated from Lake Kinneret at the Kinneret Limnological Laboratory, Tabgha, Israel. The three algae were grown in a minimal artificial medium (Table 1), at 20 :t: 2°C and an alternating light cycle of 14h light (white cool light of about 900/rE m -2 seC -1) and 10 h darkness. The effect of the treatment on Peridinium was also studied on a lake water sample from the May 1977 Peridiniura bloom (more than 90% of the phytoplankton consisted of Peridinium). A summary of the lake water characteristics has been published (german, 1973). Chemicals The sensitizers tested were methylene blue (British Drug House Ltd., No. 26132Q) and rose bengal (British Drug House Ltd., No. 26172E). They were added to the culture samples as 0.5% solution in distilled water.
A J. A(HFR and ADA ELGAVISH
540
Table 1, Chemical composition of the culture medium* Component
mg I ~
NaHCO3 Ca(NO3) 2 CaCI 2 MgSOa 7H20 NH4CI NaCI ZnCI: FeCI 3 -6H20 (NH4)6MoTO24'4H20
Component
20.0 40.0 27.7 25.0 20.0 228.0 0.10 0.20 1.00
mg 1-
MnCI 2 "4H20 COC12"6H2 H2SeO 3 H3BO 3 KI KBr Sr(NO3)2 Li2SO 4 EDTA
0.80 0.02 0.0l 4.00 0.002 20.0 0.044 0.1 20.0
* The pH of the culture medium = 7.40.
Solar radiation In the experiments in which samples were exposed to sunlight, the average light intensities were 1850 + 100/iE m -2 sec -t, as measured by a quantum sensor (Acher & Rosenthal, 1977). Biomass raeasurement The fluorescence emitted by the chlorophyll in intact algae cells 0-cmlut** = 660 mr0 upon excitation by a light beam (/ltx¢luttioa -~" 405--436 m#), was used as a method for algal biomass determination (Udenfriend, 19621 The fluorescence emitted in a 2 rnl culture sample was measured relative to the fluorescence of a standard filter (using water as a blank). A linear standard curve of dry algal weight vs. relative fluorescence in Peridinium, Pediastrum and Cosmarium cultures enabled us to translate the relative fluorescence measured in the experiments to dry biomass in mg (El$avish, 1978). The populatio n density of Peridinium was also determined by filtering 1 ml of culture or water-diluted culture through a millipore filter and counting the cells under a binocular microscope,
The desired amount of dye solution was added to the culture medium prior to the addition of the inoculum (2 ml). Control experimeaat~ without dye, were run simultaneously. Irnnu~ately after the inoculation, some of the samples were sunlight-irradiated for various intervals of time ranging from I0 to 120rain. During the solar radiation the flask-samples were kept immexsed in a water bath at 20 4-2°C. Both sunlight-exposed and non-exposed samples were then incubated in a controlled environment for about 35 days, The size of the algal population was measured immediately after the inoculation and then at intervals of about 7 days. All the experiments were performed in duplicate. Samples of lake water (10 ml) taken from the 1977 Peridinium bloom areas were transferred into sterile Erlenmeyer flasks and were treated like the Peridinium batch cultures.
RESULTS AND DISCUSSION
Aloal destruction treatment The experiments were performed in 25 mi Erlenmeyer flasks containing 8 ml of culture medium. The flasks were sterilized in an autoclave at 121°C for 20rain, All subsequent handling demanding sterile conditions was performed in a laminar air flow unit (class 100 gelaire, Gelman Instrument).
The effect of dye type Since algae can be described as hydrophilic biocolloids with a p p a r e n t negative charges (Ires, 1956}, different molecular charges of ionic-type dyes (cationic or anionic) may influence the intimate contact of the
Table 2. The minimum conditions for total algal destruction Algae
Type
Pediastrum
MB MB RB RB MB MB RB RB MB MB RB RB MB MB RB RB
Cosmarium
Peridinium
Peridinium (in lake water)
Dye mg 1- ~ 0.40 0.15 t.50 0.80 0.75 0.75 1.20 0.80 0.50 0.25 2.00 0.60 0.50 0.30 1.60 0.80
Solar exposure (min)
Incubation time (days)*
0 40 0 40 0 30 0 60 0 30 0 30 0 60 0 60
7 10 7 I0 20 10 10 10 25 14 35 35 14 14 14 14
* Minimum incubation time when algal population count was zero, without subsequent recovery.
Photochemical t r e a ~ t
I
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et/'
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i. j
.,is,"/
0.0o,,.,..le
~
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.,.~r /
,-I.20
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_.--zx
The effect of dye concentrations
; o..~......_ o.~7
0
0
-
..-':
T
-
.
,-
7 14 Incubation time, days
21
Fig. 1. The effect of dye concentration on Pediastrum growth. The figures on the curves represent dye concentration, in nag dye l-m. - - experiments with MB; - - experiments with RB; . . . . . control experiments. dye with the algae surface and contribute to the algicidal effectiveness of dyes. Consequently, two types of dye were used in the study: methylene blue (MB), a phenothyazine derivative of cationic type; and rose bengal (RB), a fluoresceine derivative of anionic type. The algicidal efficiency was checked by measuring their effect on algal biomass production in cultures. Results given in Table 2 and Figs. 1-3 show that both MB and RB affect algal growth. However, in all the experiments performed, MB showed a greater algicidai effect than RB. Therefore the complete destruction of algae (no algal recovery observed afterwards) was achieved at lower concentrations of MB, which represented 26, 62, 25 and 32% of the RB concentrations which exhibited the same algicidal effects on Pediastrum, Cosmarium, Peridinium in culture medium and
All the algae investigated were affected by the presence of dye in culture medium but their sensitivities are different and increase in the following order: Cosmarium < Peridinium < Pediastrum. The presence of a low concentration of MB (0.04 mg 1-1) in Pediastrum culture medium inhibited its growth (Fig. 1); the population size determined after incubation periods of 7, 14 and 21 days represented about 60, 63 and 70% of the population size in control samples, respectively. In the presence of 0.3 rag RB l- 1 the above figures were 35, 45 and 60~/~ respectively. The inhibition effect of 0.25 mg MB 1-1 on Peridinium growth was demonstrated by a reduction of the initial population size from 40 cells ml- 1 to 25 and 10 cells ml- ~ after 15 and 25 incubation days, while the algal population in the control samples had increased to 160 and 230 cells ml-~ in the same incubation periods, respectively (Fig. 2). In the case of Cosmarium (Fig. 3), addition of 0.30 rag RB l- 1 or 0.40 mg MB l- ~ stimulated the algal growth, and the biomass determination after 20 incubation days showed a relative increase of about 180 and 130~/~ respectively, as compared with control cultures. At higher dye concentrations the algicidal effect of MB and RB caused the complete destruction of the
240~
I
200 F
o=
It .tl/"
F,o
541
Peridinium in lake water, respectively (Table 2, samples not exposed to sunlight)~ Approximately the same figures were obtained by comparison of MB and RB concentrations used in samples submitted to solar radiation (Table 2l This greater effectiveness of MB may be attributed to factors like: chemical and photochemical stability, penetration power into algae cell, etc. It has already been reported that MB is readily taken up by algae and that it is non-toxic, e.g. the oxygen consumption of Chlorella pyrenoidosa increased upon addition to its culture medium of as much as 320 mg MB 1-1 (James, 1953).
m
,,AI,p"
30.5-
= o.o I-
I
of water on algal growth
i -/"'I=
t
//
8°1- ,-" .- . . . . . . . . . IZ.:--"'-
/-I
4 o ~ _ _ ~ o . ~ o z
0
0
15 Incubation time, days
25
Fig. 2. The effect of dye concentration on Peridinium growth. The figures on the curves represent dye concentration, in mg dye l-1. - - , experiments with MB; - - - experiments with RB; . . . . . control
experiments.
542
A.J. ACHERand ADA ELGAVISH I
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.~_---,', 0.30
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/
-
,
/
=
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/ /
-
.¢/ -
Eo
I,'/I
186 /i/~ ~ - a - -
0
0.80
I0
:>0
Incubofion time, duys Fig. 3. The effect of dye concentration on Cosmarium growth. The figures on the curves represent dye concentration, in mg dye 1-1. - experiments with MB; - - experiments with RB; . . . . . control experiments.
algal cultures. In Pediastrum cultures, addition of 0.40 mg MB 1-1 or 1.50 nag RB 1-1 resulted in the complete destruction of the algae and no algal biomass could be detected after 7 incubation days. The Peridinium cultures were destroyed with 0,50 mg MB i - 1 or 2.00 nag RB 1-1 during an incubation period of 15 to 25 days. In Cosmarium cultures 0.75 mg MB 1-1 or 1.20 mg RB l- t was already effective for algal destruction after an incubation period of 20 days. In experiments carried out with Peridinium bloom in lake water, the addition of 0.50 mg MB l - t or 1.60 mg RB 1- ~ led to complete destruction of algae. The control samples of Peridinium bloom in lake water showed in the same time (I0 days) an increase in algal population from 120 to 150 cells m1-1. The effect of sunlight exposure
The exposure of the algae in the dye containing cultures to sunlight radiation affected significantly the biomass production in the following incubation days. Table 2 shows that for Pediastrum and Peridinium the dye concentrations necessary to achieve a complete algicidal effect in the cultures exposed to sunlight were about half those required in the cultures not submitted to solar radiation, In the case of Cosmarium cultures, the MB concentrations were the same with or without sunlight exposure, but the incubation time until the algal population was completely destroyed was reduced to half. Peridinium bloom cultures in lake water containing 0.30 mg MB 1-1 were less sensitive to solar radiation than Peridinium in artificial culture medium (0.25 mg MB 1- t and required a longer solar radiation time than the latter for ~ compl0te destruction (60 min as compared to 30rain). Control samples of Pediastrura, Cosmarium, Peridinium and
Peridinium in lake water submitted to solar radiation for 120 min at 20 + 2°C showed, after incubation of 10, 20, 25 and 14 days, relative algal growth of 730, 790, 350 and 250%, respectively, as compared with the initial populations. (Control samples not exposed to solar radiation showed about the same relative algal growth.) The synergistic effect of solar exposure and MB or RB on algal growth indicates the dye-sensitized photooxidation reaction are probably involved in the algicidal mechanism. In the case of algae living in surface waters in the presence of singlet oxygen (Zepp et al., 1977), the algae might have developed a biological defence system against the damaging effect of photodynamic action (Krinsky, 1968). However, the results presented in this paper suggested that the algae investigated are sensitive to such reactions and may undergo lethal damage. In the case of coliforms, it has been shown that the bactericidal effect was obtained by a destructive photooxidation and was not a mere dye inhibition of coliform growth (Acher & Juven, 1977). The working conditions (2 mg MB 1-~ and 30 rain exposure to sunlight) which were described above as having a bactericidal effect are well above those needed for complete destruction of algae. Thus, the present study demonstrates that the dye-sensitized photooxidation which had previously been proposed as an efficient method for the destruction of coliforms in water and sewage effluents, is adequate also for algal destruction, without suffering from the drawbacks of the conventional methods previously mentioned. Furthermore, as the dye-sensitizers are eventually photooxidized to uncolored compounds, no detrimental environmental impact is to be expected. REFERENCES
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