- Email: [email protected]
Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by micronucleus test and comet assay
Accepted Manuscript Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by micronucleus test and comet assay
Guoming Zeng, Maolan Zhang, Pu Wang, Xiang Li, Pei Wu, Da Sun PII:
S0045-6535(18)32256-2
DOI:
10.1016/j.chemosphere.2018.11.148
Reference:
CHEM 22632
To appear in:
Chemosphere
Received Date:
02 October 2018
Accepted Date:
23 November 2018
Please cite this article as: Guoming Zeng, Maolan Zhang, Pu Wang, Xiang Li, Pei Wu, Da Sun, Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by micronucleus test and comet assay, Chemosphere (2018), doi: 10.1016/j.chemosphere. 2018.11.148
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by
2
micronucleus test and comet assay
3
Guoming Zeng* 1, Maolan Zhang*2,Pu Wang3,Xiang Li 1,Pei Wu1 , Da Sun4,
4
1Guoming
5
2Maolan
6
1Xiang
7
1Pei
8
3Chongqing
9
4Institute
Zeng ,Chongqing University of Science and Technology, Chong Qing, P. R. China,401331
Zhang *,Sichuan University of Science and Engineering,Si Chuan, P. R. China, 643000
Li ,Chongqing University of Science and Technology, Chong Qing, P. R. China, 401331
Wu,Chongqing University of Science and Technology, Chong Qing, P. R. China, 401331 University, Chong Qing, P. R. China, 400044;
of Life Sciences & Biomedicine Collaborative Innovation Center, Wenzhou University,
10
Wenzhou, P. R. China,325000
11
These two authors contributed equally to this work.
12
*Corresponding
13
R. China, 643000, [email protected],+86 18708392487;
14
Guoming Zeng ,Chongqing University of Science and Technology, Chong Qing, P. R.
15
China,401331,[email protected],+86 13996471404;
16
Abstract: Algal blooms and toxins have become serious ecological problems. White-rot fungi have
17
been demonstrated to be a feasible means of control, but the genotoxicity mechanisms involved have
18
not been reported. In this study, Cryptomonas obovata FACHB-1301, Oscillatoria sp. FACHB-1083,
19
and Scenedesmus quadricauda FACHB-507 were co-cultured with Phanerochaete chrysosporium
20
under optimal conditions of 250 mg-l at 25 °C with DO 7.0 mg-l for 1, 3, 5 and 7 d.Compared to the
21
control groups, the values for tadpoles exposed to algae treated with Phanerochaete chrysosporium
22
were only increased from 1.95±0.09, 2.78±0.08 and 2.37±0.13 to 2.45±0.07, 3.56±0.08 and 2.54±0.10,
author:Maolan Zhang *,Sichuan University of Science and Engineering,Si Chuan, P.
1
ACCEPTED MANUSCRIPT 23
and the frequency of nuclear anomalies reached 6.45±0.06, 11.14±0.05 and 7.85±0.10 to 7.68±0.08,
24
13.12±0.06 and 8.57±0.12 in the experimental groups after 7 d. What’s more, the tail lengths were only
25
increased to 36.77±0.54, 41.58±0.78 and 35.38 ± 0.66, and the comet length reached 55.67±0.68,
26
68.56±0.85 and 51.43±0.82 . The results demonstrated that Phanerochaete chrysosporium effectively
27
decreased genotoxicity effects in Fejervarya multistriat tadpoles. These results could provide new ideas
28
for inhibiting water blooms, and lay a theoretical foundation for promoting the deepening of water
29
eutrophication.
30
Keywords:Algal bloom;Phanerochaete chrysosporium;Fejervarya multistriat tadpoles; Genotoxicity;
31
1.Introduction
32
Algal blooms, characterized by abnormal reproduction of algae and other aquatic organisms in
33
aquatic ecosystems, are one of the most serious environmental problems in lakes, reservoirs and other
34
natural bodies of water(Campbell et al.,2011;Su et al., 2007). These blooms not only deteriorate water
35
quality, block aquatic organism physiological development and damage the structure of aquatic
36
ecosystems, they also destroy the function of bodies of water and the ecological environment (Acero et
37
al.,2005;Zamyadi et al.,2012;Zong et al.,2013). In addition, blooms seriously affect the utilization of
38
water resources for industry, agriculture, aquaculture and water transportation, and a variety of toxins
39
are released during algae growth that can be detrimental to animals, plants and humans.
40
Micronucleus(MCN) (Ai et al.,1995;Çavas et al.,2011;Ma et al.,2011) is a form of chromosomal
41
aberration that can be used to monitor pollution through the observation of the rate of occurrence of
42
cell micronuclei in plant and animal tissues. This technique has been widely used in environmental
43
monitoring, tumor formation and chemical evaluation. The comet assay (Crasta et al.,2012;Jackson et
44
al.,2009;Lavelle et al.,2002) can be used for the quantitative determination of DNA damage at the
2
ACCEPTED MANUSCRIPT 45
single-cell level, and it is suitable for monitoring genetic damage. Moreover, the amphibian larval
46
tadpoles are susceptible to the influence of their surroundings for their own physiological and structural
47
characteristics, such as amphibians and bare skin. When the concentration of poisons in water reaches a
48
certain level or the pollution lasts to a certain period, amphibians will show a series of poisoning
49
symptoms, including abnormal behavior, physiological disorders, histopathological changes and even
50
death.Therefore, it is very sensitive to environmental pollution and as model animal to study the
51
toxicity effects(Smith, 2001).
52
White-rot fungi (Han et al.,2011;Jia et al.,2010;Zeng et al.,2015) can degrade or decrease various
53
environmental pollutants, including dyes, lignin, and toxic wastes, and previous studies have indicated
54
that a suppressive effect on algal species can be caused by white-rot fungi secretions during co-
55
incubation.However,Some algae-lysing microorganisms can produce endotoxins or exotoxins,and the
56
biological toxins would be released in the process of algae cell death (Hashimoto et al.,2009), which
57
posed a serious threat to the safety of water. Therefore, the toxicity of the algae-control system of white
58
rot fungi determined whether the algae-control system of white rot fungi can be successfully and safely
59
applied to the treatment of eutrophic water .Although the antioxidant response in white-rot fungi
60
(Xie et al., 2016;Huang et al., 2017) have reported, the genotoxicity effects of algal species co-cultured
61
with white-rot fungi have not been investigated.
62
The aims of the present study were to evaluate the genotoxicity effects of Phanerochaete
63
chrysosporium on three algal species in Fejervarya multistriat tadpoles using the MCN test and comet
64
assay and provide a reference for controlling algal blooms.
65
2. Materials and methods
66
2.1 Algal strains and cultivation
3
ACCEPTED MANUSCRIPT 67
Cryptomonas obovata FACHB-1301, Oscillatoria sp. FACHB-1083, and Scenedesmus
68
quadricauda FACHB-507 were provided by the Freshwater Algae Culture Collection of the Chinese
69
Academy of Sciences. All strains were maintained in an illuminated incubator for 15 d at 25 °C on a 12
70
h/12 h light/dark cycle with approximately 90 µmol photons m−2s−1 provided by cool-white fluorescent
71
lamps to achieve exponential growth. The growth media used for three algal were described in Tables
72
1,2, and 3, respectively.
73
74
75
76
2.2 Fungal strains
77
Phanerochaete chrysosporium was provided by the Center of Industrial Culture Collection, China.
78
The strain was maintained on potato dextrose agar (PDA) plates for 5 d, stored at 4 °C, and then
79
subcultured every month. Batch liquid tests were conducted in 5000-ml beakers containing 250 mg dry
80
weight of Phanerochaete chrysosporium. Controls throughout the experiments were the same cultures
81
as the test groups but without Phanerochaete chrysosporium.
82
2.3 Animal experimental design
83
Fejervarya multistriat tadpoles were collected from the Chongqing farmland suburbs and
84
placed in a no-pollution eco-pond (27-28 °C). Healthy tadpoles with uniform size were selected at stage
85
32-36, and their body length and weight were 26.28 ± 0.34 mm and 0.19 ± 0.019 g, respectively.
86
Fejervarya multistriat tadpoles were fed the three algae and the algae treated with Phanerochaete
87
chrysosporium under optimal conditions for Phanerochaete chrysosporium 250 mg-l at DO 7.0 mg-l
88
with 25 °C and 12:12 h (light:dark) cycle for 1, 3, 5 and 7 d, respectively. The concentration of
4
ACCEPTED MANUSCRIPT 89
chlorophyll-a were 185 mgL-1,192 mgL-1and 187 mgL-1,respectively.Controls throughout the
90
experiments were the same cultures as the test groups but without Phanerochaete chrysosporium. All
91
experiments were conducted in triplicate.
92
2.4. Analytical methods
93
2.4.1. MCN test
94
The surface of the tadpoles was dried with filter paper, cut the tail by sterilized scalpel and the
95
tail blood used to make blood smears. The blood was allowed to dry naturally and fixed with methanol
96
for 15 min. Wright stain was applied for 15-20 min and washed with PBS (pH 6.8). Finally, the smears
97
were dyed for 25 min with Gimsa solution, washed with deionized water three times, and allowed to
98
dry naturally for observation by microscope. A total of 1000 cells were observed in each blood smear,
99
and the number of micronuclei and abnormal nuclei were recorded.All experiments were conducted in
100
triplicate.
101
2.4.2. Comet assay
102
The alkaline version of the comet assay was performed according to guidelines proposed by
103
Singh(Singh et al.,1988). The heads of Fejervarya multistriat tadpoles were removed, and blood cells
104
were collected and diluted in PBS (pH 6.8) to a cell density of 105-106. Blood cells were used directly
105
for the comet assay. The buffer containing blood cells was transferred into a 2-ml centrifuge tube.
106
Completely frosted microscopic slides were covered with 90 µl 0.7% normal melting agarose (NMA)
107
to form a thin layer and allowed to solidify at 4 °C for 30 min. Low Melting Point Agarose (LMA, 75
108
µl at 1%) containing 25 µl blood cells was placed on the NMA and allowed to rest at 4 °C for 30 min.
109
Afterwards, the slides were placed in lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-
110
HCl, 1% Triton X-100 and 10% DMSO, pH 10) at 4 °C for 1.5 h in the dark. The slides were then
5
ACCEPTED MANUSCRIPT 111
washed with chilled distilled water and placed in a horizontal gel electrophoresis chamber in an
112
alkaline buffer (300 mM NaOH, 1 mM Na2EDTA, pH>13) at 20 °C using 20 V and 200 mA for 30
113
min in the dark to avoid DNA damage. After electrophoresis, each slide was neutralized with
114
neutralization buffer (0.4 M Tris-HCI, pH 7.3) three times, washed with chilled distilled water and
115
placed in ethanol for 5 min at 4 °C. Finally, the samples were stained with Godview for 20 min in the
116
dark and observed with a fluorescence microscope (BX51, Olympus, Japan) at 490 nm excitation. Each
117
group yielded three parallel samples, and 100 cells were observed on each slide. The images of comets
118
were analyzed using Comet Assay Software Project, CASP software.
119
2.4.3. Statistical analysis
120 121
The data from the control and treatment groups were analyzed by Student's t-test, and analysis of variance (ANOVA) was performed using the commercial software SPSS 12.0.
122 123
3. Results and Discussion
124
3.1. MCN
125
126
Fig. 1.Micronuclei and nuclear abnormalities from erythrocytes of Fejervarya multistriat tadpoles(10 *
127
100) (a)normal nuclear (b) micronucleus; (c) nuclear concavity; (d) nuclear protrusion;(e)nuclear and
128
micronucleus linked by silk; (f)three micronucleus (g) nuclear vacuole;(h) nuclear pycnosis.
129
130
Fig. 2. Micronuclei analysis of the three algal cultures: Cryptomonas obovata FACHB-1301 (a),
131
Oscillatoria sp. FACHB-1083 (c) and Scenedsmus quadricauda FACHB-507 (e) and corresponding
132
algal cultures treated with Phanerochaete chrysosporium ((b), (d), and (f), respectively).
6
ACCEPTED MANUSCRIPT 133
Guoming Zeng, Maolan Zhang, Pu Wang, Xiang Li, Pei Wu, Da Sun PII:
S0045-6535(18)32256-2
DOI:
10.1016/j.chemosphere.2018.11.148
Reference:
CHEM 22632
To appear in:
Chemosphere
Received Date:
02 October 2018
Accepted Date:
23 November 2018
Please cite this article as: Guoming Zeng, Maolan Zhang, Pu Wang, Xiang Li, Pei Wu, Da Sun, Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by micronucleus test and comet assay, Chemosphere (2018), doi: 10.1016/j.chemosphere. 2018.11.148
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Genotoxicity effects of Phanerochaete chrysosporium against harmful algal bloom species by
2
micronucleus test and comet assay
3
Guoming Zeng* 1, Maolan Zhang*2,Pu Wang3,Xiang Li 1,Pei Wu1 , Da Sun4,
4
1Guoming
5
2Maolan
6
1Xiang
7
1Pei
8
3Chongqing
9
4Institute
Zeng ,Chongqing University of Science and Technology, Chong Qing, P. R. China,401331
Zhang *,Sichuan University of Science and Engineering,Si Chuan, P. R. China, 643000
Li ,Chongqing University of Science and Technology, Chong Qing, P. R. China, 401331
Wu,Chongqing University of Science and Technology, Chong Qing, P. R. China, 401331 University, Chong Qing, P. R. China, 400044;
of Life Sciences & Biomedicine Collaborative Innovation Center, Wenzhou University,
10
Wenzhou, P. R. China,325000
11
These two authors contributed equally to this work.
12
*Corresponding
13
R. China, 643000, [email protected],+86 18708392487;
14
Guoming Zeng ,Chongqing University of Science and Technology, Chong Qing, P. R.
15
China,401331,[email protected],+86 13996471404;
16
Abstract: Algal blooms and toxins have become serious ecological problems. White-rot fungi have
17
been demonstrated to be a feasible means of control, but the genotoxicity mechanisms involved have
18
not been reported. In this study, Cryptomonas obovata FACHB-1301, Oscillatoria sp. FACHB-1083,
19
and Scenedesmus quadricauda FACHB-507 were co-cultured with Phanerochaete chrysosporium
20
under optimal conditions of 250 mg-l at 25 °C with DO 7.0 mg-l for 1, 3, 5 and 7 d.Compared to the
21
control groups, the values for tadpoles exposed to algae treated with Phanerochaete chrysosporium
22
were only increased from 1.95±0.09, 2.78±0.08 and 2.37±0.13 to 2.45±0.07, 3.56±0.08 and 2.54±0.10,
author:Maolan Zhang *,Sichuan University of Science and Engineering,Si Chuan, P.
1
ACCEPTED MANUSCRIPT 23
and the frequency of nuclear anomalies reached 6.45±0.06, 11.14±0.05 and 7.85±0.10 to 7.68±0.08,
24
13.12±0.06 and 8.57±0.12 in the experimental groups after 7 d. What’s more, the tail lengths were only
25
increased to 36.77±0.54, 41.58±0.78 and 35.38 ± 0.66, and the comet length reached 55.67±0.68,
26
68.56±0.85 and 51.43±0.82 . The results demonstrated that Phanerochaete chrysosporium effectively
27
decreased genotoxicity effects in Fejervarya multistriat tadpoles. These results could provide new ideas
28
for inhibiting water blooms, and lay a theoretical foundation for promoting the deepening of water
29
eutrophication.
30
Keywords:Algal bloom;Phanerochaete chrysosporium;Fejervarya multistriat tadpoles; Genotoxicity;
31
1.Introduction
32
Algal blooms, characterized by abnormal reproduction of algae and other aquatic organisms in
33
aquatic ecosystems, are one of the most serious environmental problems in lakes, reservoirs and other
34
natural bodies of water(Campbell et al.,2011;Su et al., 2007). These blooms not only deteriorate water
35
quality, block aquatic organism physiological development and damage the structure of aquatic
36
ecosystems, they also destroy the function of bodies of water and the ecological environment (Acero et
37
al.,2005;Zamyadi et al.,2012;Zong et al.,2013). In addition, blooms seriously affect the utilization of
38
water resources for industry, agriculture, aquaculture and water transportation, and a variety of toxins
39
are released during algae growth that can be detrimental to animals, plants and humans.
40
Micronucleus(MCN) (Ai et al.,1995;Çavas et al.,2011;Ma et al.,2011) is a form of chromosomal
41
aberration that can be used to monitor pollution through the observation of the rate of occurrence of
42
cell micronuclei in plant and animal tissues. This technique has been widely used in environmental
43
monitoring, tumor formation and chemical evaluation. The comet assay (Crasta et al.,2012;Jackson et
44
al.,2009;Lavelle et al.,2002) can be used for the quantitative determination of DNA damage at the
2
ACCEPTED MANUSCRIPT 45
single-cell level, and it is suitable for monitoring genetic damage. Moreover, the amphibian larval
46
tadpoles are susceptible to the influence of their surroundings for their own physiological and structural
47
characteristics, such as amphibians and bare skin. When the concentration of poisons in water reaches a
48
certain level or the pollution lasts to a certain period, amphibians will show a series of poisoning
49
symptoms, including abnormal behavior, physiological disorders, histopathological changes and even
50
death.Therefore, it is very sensitive to environmental pollution and as model animal to study the
51
toxicity effects(Smith, 2001).
52
White-rot fungi (Han et al.,2011;Jia et al.,2010;Zeng et al.,2015) can degrade or decrease various
53
environmental pollutants, including dyes, lignin, and toxic wastes, and previous studies have indicated
54
that a suppressive effect on algal species can be caused by white-rot fungi secretions during co-
55
incubation.However,Some algae-lysing microorganisms can produce endotoxins or exotoxins,and the
56
biological toxins would be released in the process of algae cell death (Hashimoto et al.,2009), which
57
posed a serious threat to the safety of water. Therefore, the toxicity of the algae-control system of white
58
rot fungi determined whether the algae-control system of white rot fungi can be successfully and safely
59
applied to the treatment of eutrophic water .Although the antioxidant response in white-rot fungi
60
(Xie et al., 2016;Huang et al., 2017) have reported, the genotoxicity effects of algal species co-cultured
61
with white-rot fungi have not been investigated.
62
The aims of the present study were to evaluate the genotoxicity effects of Phanerochaete
63
chrysosporium on three algal species in Fejervarya multistriat tadpoles using the MCN test and comet
64
assay and provide a reference for controlling algal blooms.
65
2. Materials and methods
66
2.1 Algal strains and cultivation
3
ACCEPTED MANUSCRIPT 67
Cryptomonas obovata FACHB-1301, Oscillatoria sp. FACHB-1083, and Scenedesmus
68
quadricauda FACHB-507 were provided by the Freshwater Algae Culture Collection of the Chinese
69
Academy of Sciences. All strains were maintained in an illuminated incubator for 15 d at 25 °C on a 12
70
h/12 h light/dark cycle with approximately 90 µmol photons m−2s−1 provided by cool-white fluorescent
71
lamps to achieve exponential growth. The growth media used for three algal were described in Tables
72
1,2, and 3, respectively.
73
74
75
76
2.2 Fungal strains
77
Phanerochaete chrysosporium was provided by the Center of Industrial Culture Collection, China.
78
The strain was maintained on potato dextrose agar (PDA) plates for 5 d, stored at 4 °C, and then
79
subcultured every month. Batch liquid tests were conducted in 5000-ml beakers containing 250 mg dry
80
weight of Phanerochaete chrysosporium. Controls throughout the experiments were the same cultures
81
as the test groups but without Phanerochaete chrysosporium.
82
2.3 Animal experimental design
83
Fejervarya multistriat tadpoles were collected from the Chongqing farmland suburbs and
84
placed in a no-pollution eco-pond (27-28 °C). Healthy tadpoles with uniform size were selected at stage
85
32-36, and their body length and weight were 26.28 ± 0.34 mm and 0.19 ± 0.019 g, respectively.
86
Fejervarya multistriat tadpoles were fed the three algae and the algae treated with Phanerochaete
87
chrysosporium under optimal conditions for Phanerochaete chrysosporium 250 mg-l at DO 7.0 mg-l
88
with 25 °C and 12:12 h (light:dark) cycle for 1, 3, 5 and 7 d, respectively. The concentration of
4
ACCEPTED MANUSCRIPT 89
chlorophyll-a were 185 mgL-1,192 mgL-1and 187 mgL-1,respectively.Controls throughout the
90
experiments were the same cultures as the test groups but without Phanerochaete chrysosporium. All
91
experiments were conducted in triplicate.
92
2.4. Analytical methods
93
2.4.1. MCN test
94
The surface of the tadpoles was dried with filter paper, cut the tail by sterilized scalpel and the
95
tail blood used to make blood smears. The blood was allowed to dry naturally and fixed with methanol
96
for 15 min. Wright stain was applied for 15-20 min and washed with PBS (pH 6.8). Finally, the smears
97
were dyed for 25 min with Gimsa solution, washed with deionized water three times, and allowed to
98
dry naturally for observation by microscope. A total of 1000 cells were observed in each blood smear,
99
and the number of micronuclei and abnormal nuclei were recorded.All experiments were conducted in
100
triplicate.
101
2.4.2. Comet assay
102
The alkaline version of the comet assay was performed according to guidelines proposed by
103
Singh(Singh et al.,1988). The heads of Fejervarya multistriat tadpoles were removed, and blood cells
104
were collected and diluted in PBS (pH 6.8) to a cell density of 105-106. Blood cells were used directly
105
for the comet assay. The buffer containing blood cells was transferred into a 2-ml centrifuge tube.
106
Completely frosted microscopic slides were covered with 90 µl 0.7% normal melting agarose (NMA)
107
to form a thin layer and allowed to solidify at 4 °C for 30 min. Low Melting Point Agarose (LMA, 75
108
µl at 1%) containing 25 µl blood cells was placed on the NMA and allowed to rest at 4 °C for 30 min.
109
Afterwards, the slides were placed in lysis solution (2.5 M NaCl, 100 mM Na2 EDTA, 10 mM Tris-
110
HCl, 1% Triton X-100 and 10% DMSO, pH 10) at 4 °C for 1.5 h in the dark. The slides were then
5
ACCEPTED MANUSCRIPT 111
washed with chilled distilled water and placed in a horizontal gel electrophoresis chamber in an
112
alkaline buffer (300 mM NaOH, 1 mM Na2EDTA, pH>13) at 20 °C using 20 V and 200 mA for 30
113
min in the dark to avoid DNA damage. After electrophoresis, each slide was neutralized with
114
neutralization buffer (0.4 M Tris-HCI, pH 7.3) three times, washed with chilled distilled water and
115
placed in ethanol for 5 min at 4 °C. Finally, the samples were stained with Godview for 20 min in the
116
dark and observed with a fluorescence microscope (BX51, Olympus, Japan) at 490 nm excitation. Each
117
group yielded three parallel samples, and 100 cells were observed on each slide. The images of comets
118
were analyzed using Comet Assay Software Project, CASP software.
119
2.4.3. Statistical analysis
120 121
The data from the control and treatment groups were analyzed by Student's t-test, and analysis of variance (ANOVA) was performed using the commercial software SPSS 12.0.
122 123
3. Results and Discussion
124
3.1. MCN
125
126
Fig. 1.Micronuclei and nuclear abnormalities from erythrocytes of Fejervarya multistriat tadpoles(10 *
127
100) (a)normal nuclear (b) micronucleus; (c) nuclear concavity; (d) nuclear protrusion;(e)nuclear and
128
micronucleus linked by silk; (f)three micronucleus (g) nuclear vacuole;(h) nuclear pycnosis.
129
130
Fig. 2. Micronuclei analysis of the three algal cultures: Cryptomonas obovata FACHB-1301 (a),
131
Oscillatoria sp. FACHB-1083 (c) and Scenedsmus quadricauda FACHB-507 (e) and corresponding
132
algal cultures treated with Phanerochaete chrysosporium ((b), (d), and (f), respectively).
6
ACCEPTED MANUSCRIPT 133