Accepted Manuscript Title: Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers Author: Bruno Rodr´ıguez-Morgado Isidoro G´omez Juan Parrado Carlos Garc´ıa Teresa Hern´andez Manuel Tejada PII: DOI: Reference:
S0304-3894(15)00441-0 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.05.045 HAZMAT 16847
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
13-2-2015 29-4-2015 26-5-2015
Please cite this article as: Bruno Rodr´iguez-Morgado, Isidoro G´omez, Juan Parrado, Carlos Garc´ia, Teresa Hern´andez, Manuel Tejada, Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.05.045 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.
1
Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers
Bruno Rodríguez-Morgadoa, Isidoro Gómezb, Juan Parradoa, Carlos Garcíac, Teresa Hernándezc, Manuel Tejadab*[email protected]
a
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de
Sevilla, C/ Prof. García González 2, 41012 Sevilla, Spain b
Grupo de Investigación “Edafología Ambiental”, Departamento de Cristalografía,
Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain c
Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Centro
de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain
*
Corresponding author. Tel.: +34954486468, fax.: +34954486436
2
Highlight The number of aromatic rings in the PAHs is closely related to the soil toxicity The application of organic wastes decreased the toxic action of PAHs The low molecular weight protein of wastes increased the degradation of PAHs
Abstract We studied in the laboratory the bioremediation effects over a 100-day period of three edaphic biostimulants (BS) obtained from sewage sludge (SS) and from two different types of chicken feathers (CF1 and CF2), in a soil polluted with three polycyclic aromatic hydrocarbons (PAH) (phenanthrene, Phe; pyrene, Py; and benzo(a)pyrene, BaP), at a concentration of 100 mg kg-1 soil. We determined their effects on enzymatic activities and on soil microbial community. Those BS with larger amounts of proteins and a higher proportion of peptides (< 300 daltons), exerted a greater stimulation on the soil biochemical properties and microbial community, possibly because low molecular weight proteins can be easily assimilated by soil microorganisms. The soil dehydrogenase, urease, β-glucosidase and phosphatase activities and microbial community decreased in PAH-polluted soil. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe. The application of the BS to PAH-polluted soils decreased the inhibition of the soil biological properties, principally at 7 days into the experiment. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe, and was higher in polluted soils amended with CF2, followed by SS and CF1 respectively.
Keywords Phenanthrene; pyrene; benzo(a)pyrene; edaphic biostimulants; soil biochemical properties; soil microbial community
3
1. Introduction The technique of applying organic matter to soils contaminated with polycyclic aromatic hydrocarbons (PAHs) is widespread among scientists and engineers. It is a good environmental strategy which aims to eliminate or reduce the negative effects that these chemicals cause on soil microorganisms [1, 2, 3, 4, 5]. Because the microbial degradation is the most important process for these pollutants, for some authors this second role of organic matter has a greater importance than the first in the bioremediation of PAHs-polluted soils [6, 7, 8]. Organic matter mineralization releases nutrients continuously or intermittently over a period of time and therefore has been applied to PAH-contaminated soils to stimulate and maintain indigenous biodegradation rates. It is, however, a slow process that depends on several factors such as the PAH type, soil microorganisms tolerant to this PAH, and the quantity and chemical composition of the M.O. added to soil [4, 5, 9]. In recent years there has been increasing use of organic fertilizers or edaphic biostimulants (BS), obtained by hydrolysis from various organic materials, in the bioremediation of soils contaminated with organic xenobiotics such as PAHs and plaguicides. Unlike other sources of organic matter, these organic compounds are usually constituted by low-molecular-weight proteins and amino acids. This aspect is very important, since these compounds can be used directly by the soil microorganisms and therefore accelerates degradation of xenobiotic in soil. These products are also characterized by high polysaccharides content, and humic-like molecules that stimulate soil microorganisms, and thus, promote the degradation of the xenobiotic in soil [5, 10, 11, 12, 13]. Recently, the use of BS obtained from sewage sludge and chicken feathers has become very common in the degradation of plaguicides, mainly oxyfluorfen and chlorpyrifos [11, 13],
4
with a notable increase in the degradation of the above xenobiotics after the applying both BS to the polluted soils.Since nostudies have been reported using these BS on PAH-contaminated soil, we hypothesize that both protein hydrolysates can be very useful in remedying different PAH-contaminated soils. The objective of this study, therefore, was to investigate, under laboratory conditions, the influence of BS obtained from sewage sludge and chicken feathers in soils polluted with different PAH andits effect on soil biochemical properties and microbial community.
2. Materials and methods 2.1. Soil, biostimulants and PAH characteristics The soil used in this experiment is a Calcaric Regosol [14]. The main soil characteristics are reported elsewhere [11, 13], and summarized in Table 1. The methodology used to the determine the physical and chemical parameters in soil and organic wastes is also described in Tejada et al. [11] and Rodriguez-Morgado et al. [13]. Three edaphic BS were used: (1) SS, derived from sewage sludge; (2) CF1, derived from chicken feathers provided by a poultry industry located in Murcia (Spain); and (3) CF2, derived from chicken feathers provided by a local poultry slaughterhouse (TG-SL) located in Seville (Spain). All BS were obtained by enzymatic hydrolysis. Sewage sludge and both feathers were hydrolysed according to the pH-stat method [15], using an endoprotease obtained by liquid fermentation of Bacillus licheniformis ATCC 21415 as the hydrolytic agent in a bioreactor operating under controlled temperature and pH, agitation and NaOH consumption [16]. The enzymatic hydrolysis process is detailed in Rodriguez-Morgado et al. [13]. Three hydrocarbons were utilized to artificially contaminate the soil: (1) phenanthrene (Phe), a PAH consisting of three fused benzene rings; (2) pyrene (Py), a PAH consisting of
5
three fused benzene rings; and (3) benzo(a)pyrene (BaP), a PAH consisting of five fused benzene rings.
2.2. Incubation procedure Prior to being treated, seven hundred grams of soil were pre-incubated at 25 °C for 7 days at 30-40% of their water-holding capacity, according to Tejada [17]. After this preincubation period, soil samples were mixed with Phe, Py or BaP respectively. According to Mueller and Shann [18] and Smith et al. [19], each PAH was dissolved in acetone and added to soil at 100 mg kg-1soil. Acetone was also added to the non-polluted soils (control treatment). Each treatment was mixed thoroughly and allowed to evaporate two days in a fume hood. During this period, the soil was mixed at intervals to ensure homogeneous distribution of the PAHS in each treatment. Subsequently, the three experimental BS was added to the soil. Soil samples were mixed with CF1 and CF2 to a concentration of 1%, 1.3% and 1.6%, respectively, in order to apply the same amount of organic matter with each BS to the soil. According to Rodriguez-Morgado et al. [13], all biostimulants were liquid and were solubilized in distilled water (500 l ha-1) before applying. An unamended and non-polluted soil was used as control. The incubation treatments are described in Table 3. Triplicate treatments were kept in semi-closed microcosms at 20 ± 2 °C for 100 days, respectively.
2.3. Soil PAH determinations The Phe and Py were extracted from 5 g samples of soil. These samples were introduced into a 50 ml centrifuge tube with 4 ml of deionized water and shaken for one minute, after which, 10 ml of acetonitrile were added to the tube and the mix was shaken for a further 30 seconds. Finally, a QuEChERS dSPE Phenomenex ref. KSO-8912 extraction kit was added to the centrifuge tube. It was vigorously shaken for one minute and then
6
centrifuged for 5 minutes at 4000 rpm [20]. The extracts were passed through a 0.45-µm syringe filter and the filtered extracts were concentrated to 1 ml. They were then analyzed by gas chromatograph equipped with autosampler, programmable splitless inlet temperature of deactivated fused silica pre-column of intermediate polarity (1-3m x 0.25 to 0.32 mm internal diameter), connected by connector to a fused silica column with 5% phenylmethylsilicone (5MS type) and dimensions 30m x 0.25 mm x 0.1 µm, for mass spectrometry detector. Analysis conditions were as follows: Autosampler: Wash the syringe with hexane; Volume injected: 2 µl; Injection speed: 1.0 µl s-1; Washing time of the syringe: 20 seconds; Pause: 2 seconds; Shooting speed: µl s-1 Injector program: Initial temperature: 70 ° C; Ramp: hold for 0.1 minutes, up to 300 ºC to 200 ºC min-1, keep up to 40 minutes Gas chromatograph (separation): Temperature of the transfer line 280 ºC; Initial temperature: 70 °C; Ramp: hold for 1.5 minutes, up to 300 ° C at 5 °C min-1, hold 4 minutes. Flow of carrier gas (He): 1.5 ml min-1 measured at baseline. Mass
Detector
(purchase):
Acquisition
time:
45
minutes;
Delay in
the
filament/multiplier: 2.50 minutes. The extraction of BaP from soil was performed using the Song et al. [21] method. A sample of 1.5 g of soil was put in a 15-ml Pyrex tube and10 ml acetone was added, shaken on a vortex and sonicated for20 min. Subsequently, the soil sample was centrifuged at 13700×g for 15 min, the supernatant was added to 20 ml glass flasks and the acetone used to extract BaP was left to evaporate. The same procedure was repeated again twice and the extracts were added to a 20-ml flask. The extracts were passed through a 0.45-µm syringe filter. The methodology used to measure the BaP is described in Tejada et al. [5].
7
2.4. Soil biological determinations The activity levels of four soil enzymes for each treatment were measured at days 1, 5, 7, 12, 20, 40, 70 and 100 during the incubation period. Dehydrogenase activity was measured as the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl) 5-phenyl tetrazoliumchloride to iodonitrophenylformazan [22]. Urease activity was determined by the buffered method of Kandeler and Gerber [23], using urea as the substrate. β-glucosidase activity was determined using p-nitrophenyl-β-D-glucopyranoside as the substrate [24]. Phosphatase activity was measured using p-nitrophenyl phosphate as the substrate [25]. For each treatment, phospholipids were extracted at days 5, 12, 40 and 100 during the incubation period, (three replicates per treatment) using a chloroform–methanol extraction method based on Bligh and Dyer [26]. Phospholipids were transformed by alkaline methanolysis into fatty acid methyl esters (FAMEs), which were quantified by a gas chromatograph (TRACE GC Ultra, Thermo Scientific) fitted with a 60-m capillary column (BPX70 60 m X 0.25 mm ID X 0.25 µm film), using helium as carrier gas. The initial temperature was 150 °C for 0.5 min and it was increased to 180 °C at 2 °C min-1 and then to 240 °C at 4 °C min-1. The Gram-positive representative fatty acids were: i15:0, a15:0, i16:0, and i17:0 and the Gram-negative specific fatty acids were: 18:1u9c, 18:1u9t, cy17:0, and cy19:0. The fatty acids i15:0, a15:0, 15:0, i16:0, i17:0, 18:1u9c, 18:1u9t, cy17:0, and cy19:0 were chosen to represent the biomass of bacterial community [27, 28, 29]. The fatty acid 18:2u6 was taken to indicate the fungal biomass [30, 31, 32]. All results are given in nmol g-1. For each treatment and each experimental day, 20 g of soil was taken. Soil samples were stored in sealed polyethylene bags at 4 °C for 15 days, prior to analysis of the enzymatic activities and at -20 °C prior to phospholipid analysis [11, 13].
8
2.5. Statistical analysis Data were submitted to two-way ANOVA with treatment and sampling time as factors followed by Tukey’s significant difference as a post hoc test, considering a significance level of p0.05). Incubation days 5 C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
2.8b ± 0.9 2.1b ± 0.7 19.7c ± 1.4 21.9c ± 1.8 16.6c ± 1.4 12.0c ± 1.5 14.9c ± 1.1 11.1c ± 1.3
12 40 Bacterial Gram+ PLFA 2.6b ± 0.7 2.8b ± 0.7 1.4a ± 0.4 0.96a ± 0.18 38.0d ± 2.7 3.9b ± 0.9 39.6d ± 2.5 4.4b ± 1.1 35.8d ± 3.2 3.1b ± 0.6 31.4d ± 3.3 1.9b ± 0.6 33.9d ± 2.4 2.1b ± 0.6 28.6d ± 3.0 1.7ab ± 0.4
C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
1.2b ± 0.3 0.92b ± 0.15 3.9d ± 1.0 4.3d ± 1.2 3.1c ± 0.9 2.5c ± 0.8 2.9c ± 0.9 2.2c ± 0.6
Bacterial Gram- PLFA 1.1b ± 0.2 1.2b ± 0.2 0.62a ± 0.17 0.43a ± 0.11 7.3d ± 2.2 1.8c ± 0.5 7.9e ± 2.5 2.1c ± 0.3 6.3d ± 2.0 1.5b ± 0.4 6.4d ± 1.6 0.83b ± 0.15 6.9d ± 2.1 0.91b ± 0.13 5.2d ± 1.5 0.74b ± 0.16
1.0b ± 0.2 0.30a ± 0.10 1.2b ± 0.2 1.1b ± 0.2 1.1b ± 0.3 0.43a ± 0.11 0.47a ± 0.16 0.34a ± 0.09
4.0b ± 1.1 3.0b ± 0.9 23.6c ± 2.5 26.2c ± 3.1 19.7c ± 2.4 14.5c ± 2.4 17.8c ± 2.0 13.3c ± 2.0
Total bacterial PLFA 3.7b ± 1.2 4.0b ± 1.1 2.0a ± 0.6 1.4a ± 0.4 45.1d ± 4.6 5.7b ± 1.2 47.5d ± 4.8 6.5b ± 1.5 42.1d ± 5.1 4.6b ± 1.1 37.8d ± 4.0 2.7b ± 0.9 40.8d ± 3.3 3.0b ± 0.8 33.8d ± 4.4 2.4ab ± 0.6
3.7b ± 1.1 1.0a ± 0.2 3.8b ± 0.5 3.7b ± 0.9 3.8b ± 1.0 1.5a ± 0.3 1.8a ± 0.4 1.2a ± 0.2
0.53b ± 0.12 0.43b ± 0.15 0.92c ± 0.19 0.96c ± 0.15 0.77c ± 0.12 0.57bc ± 0.09 0.65c ± 0.10 0.50b ± 0.09
Fungal PLFA 0.48b ± 0.10 0.49b ± 0.11 0.29b ± 0.11 0.18a ± 0.07 1.9c ± 0.4 1.1c ± 0.1 2.1d ± 0.2 1.2c ± 0.1 1.6c ± 0.3 0.95c ± 0.13 1.6c ± 0.2 0.32b ± 0.08 1.9c ± 0.3 0.35b ± 0.07 1.3c ± 0.2 0.28b ± 0.07
0.51b ± 0.08 0.12a ± 0.08 0.54b ± 0.09 0.55b ± 0.08 0.48b ± 0.07 0.22a ± 0.07 0.26b ± 0.07 0.18a ± 0.04
C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
100 2.7b ± 0.8 0.74a ± 0.11 2.6b ± 0.4 2.6b ± 0.6 2.7b ± 0.5 1.1a ± 0.2 1.3a ± 0.2 0.89a ± 0.15
30
Table 10 Evolution of bacterial Gram+, bacterial Gram-, total bacterial and fungal PLFAs (nmol g-1) in soils amended with the experimental edaphic biostimulants and polluted with benzo(a)pyrene (BaP) during the experimental period. Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p>0.05). Incubation days 5 C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
2.8c ± 0.9 1.9c ± 0.7 19.7e ± 1.4 21.9f ± 1.8 16.6e ± 1.4 11.4e ± 1.1 13.1e ± 1.5 9.0d ± 1.3
12 40 Bacterial Gram+ PLFA 2.6c ± 0.7 2.8c ± 0.7 1.1b ± 0.4 0.75ab ± 0.16 38.0f ± 2.7 3.9c ± 0.9 39.6f ± 2.5 4.4d ± 1.1 35.8f ± 3.2 3.1c ± 0.6 13.9e ± 1.4 1.6b ± 0.3 14.6e ± 1-3 1.9b ± 0.2 10.8e ± 1.1 1.0b ± 0.2
C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
1.2c ± 0.3 0.80c ± 0.15 3.9d ± 1.0 4.3e ± 1.2 3.1d ± 0.9 2.3d ± 0.3 2.7d ± 0.4 1.7c ± 0.3
Bacterial Gram- PLFA 1.1c ± 0.2 1.2c ± 0.2 0.43b ± 0.11 0.32b ± 0.07 7.3e ± 2.2 1.8d ± 0.5 7.9e ± 2.5 2.1d ± 0.3 6.3e ± 2.0 1.5c ± 0.4 5.5e ± 1.5 0.63b ± 0.12 6.0e ± 1.3 0.69b ± 0.16 4.3e ± 1.3 0.45b ± 0.11
1.0c ± 0.2 0.12a ± 0.04 1.2c ± 0.2 1.1c ± 0.2 1.1c ± 0.3 0.37b ± 0.09 0.48b ± 0.10 0.26a ± 0.08
4.0c ± 1.1 2.7b ± 0.8 23.6e ± 2.5 26.2ef ± 3.1 19.7e ± 2.4 13.7e ± 1.5 15.8e ± 2.0 10.7e ± 1.5
Total bacterial PLFA 3.7c ± 1.2 4.0c ± 1.1 1.5b ± 0.4 1.1b ± 0.2 45.1f ± 4.6 5.7d ± 1.2 47.5f ± 4.8 6.5d ± 1.5 42.1f ± 5.1 4.6d ± 1.1 19.4e ± 3.0 2.2c ± 0.6 20.6e ± 2.7 2.6c ± 0.4 15.1e ± 2.3 1.5b ± 0.4
3.7c ± 1.1 0.45a ± 0.11 3.8c ± 0.5 3.7c ± 0.9 3.8c ± 1.0 1.2b ± 0.3 1.4b ± 0.3 0.9a ± 0.2
0.53c ± 0.12 0.37b ± 0.06 0.92d ± 0.19 0.96d ± 0.15 0.77c ± 0.12 0.53c ± 0.11 0.60c ± 0.14 0.44c ± 0.12
Fungal PLFA 0.48c ± 0.10 0.49c ± 0.11 0.20b ± 0.06 0.12a ± 0.09 1.9de ± 0.4 1.1d ± 0.1 2.1e ± 0.2 1.2d ± 0.1 1.6d ± 0.3 0.95d ± 0.13 0.88c ± 0.20 0.37b ± 0.15 0.94d ± 0.21 0.43c ± 0.14 0.61c ± 0.13 0.25b ± 0.09
0.51c ± 0.08 0.07a ± 0.02 0.54c ± 0.09 0.55c ± 0.08 0.48c ± 0.07 0.18b ± 0.05 0.25b ± 0.06 0.13a ± 0.03
C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
100 2.7c ± 0.8 0.33a ± 0.09 2.6c ± 0.4 2.6c ± 0.6 2.7c ± 0.5 0.86b ± 0.15 0.93b ± 0.13 0.64a ± 0.13
31
Figures 100
a a a a
Soil Phe (mg kg-1)
a 80
b b ab
a b b
b ab
b
b
60
b b
b
c
b c
c
40
bc
b c
c
c
b c
c
c d
20 0 1
5
7
S+Phe
Soil Py (mg kg-1)
100
a a a a
a b b a
80
12
S+Phe+SS a
20
S+Phe+CF1
a b
b
b
40
b
b b
c
60
100
S+Phe+CF2
a b
70
c
b
b c
c
b c
c
c
c
c
c
d
40 20 0 1
5
7 S+Py
Soil BaP (mg kg-1 )
100
a a a a
a
a a a
12
S+Py+SS a ab
80
a
20
S+Py+CF1
a
b
40
b
a
b
b
100
S+Py+CF2
a b
70
b
b
a b b b
a b
b
60
b bc
b c
40 20 0 1
5 S+BaP
7 S+BaP+SS
12
20
S+BaP+CF1
40 S+BaP+CF2
70
100
32
Figure 1. Evolution of phenanthrene (Phe), pyrene (Py) and benzo(a)pyrene (BaP) (mean ± standard error) in soils during the experimental period. Data are the means of three samples. Columns followed by the same letter(s) are not significantly different (p> 0.05).
S0304-3894(15)00441-0 http://dx.doi.org/doi:10.1016/j.jhazmat.2015.05.045 HAZMAT 16847
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
13-2-2015 29-4-2015 26-5-2015
Please cite this article as: Bruno Rodr´iguez-Morgado, Isidoro G´omez, Juan Parrado, Carlos Garc´ia, Teresa Hern´andez, Manuel Tejada, Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2015.05.045 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.
1
Accelerated degradation of PAHs using edaphic biostimulants obtained from sewage sludge and chicken feathers
Bruno Rodríguez-Morgadoa, Isidoro Gómezb, Juan Parradoa, Carlos Garcíac, Teresa Hernándezc, Manuel Tejadab*[email protected]
a
Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de
Sevilla, C/ Prof. García González 2, 41012 Sevilla, Spain b
Grupo de Investigación “Edafología Ambiental”, Departamento de Cristalografía,
Mineralogía y Química Agrícola, E.T.S.I.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spain c
Departamento de Conservación de Suelos y Agua y Manejo de Residuos Orgánicos, Centro
de Edafología y Biología Aplicada del Segura, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain
*
Corresponding author. Tel.: +34954486468, fax.: +34954486436
2
Highlight The number of aromatic rings in the PAHs is closely related to the soil toxicity The application of organic wastes decreased the toxic action of PAHs The low molecular weight protein of wastes increased the degradation of PAHs
Abstract We studied in the laboratory the bioremediation effects over a 100-day period of three edaphic biostimulants (BS) obtained from sewage sludge (SS) and from two different types of chicken feathers (CF1 and CF2), in a soil polluted with three polycyclic aromatic hydrocarbons (PAH) (phenanthrene, Phe; pyrene, Py; and benzo(a)pyrene, BaP), at a concentration of 100 mg kg-1 soil. We determined their effects on enzymatic activities and on soil microbial community. Those BS with larger amounts of proteins and a higher proportion of peptides (< 300 daltons), exerted a greater stimulation on the soil biochemical properties and microbial community, possibly because low molecular weight proteins can be easily assimilated by soil microorganisms. The soil dehydrogenase, urease, β-glucosidase and phosphatase activities and microbial community decreased in PAH-polluted soil. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe. The application of the BS to PAH-polluted soils decreased the inhibition of the soil biological properties, principally at 7 days into the experiment. This decrease was more pronounced in soils contaminated with BaP than with Py and Phe, and was higher in polluted soils amended with CF2, followed by SS and CF1 respectively.
Keywords Phenanthrene; pyrene; benzo(a)pyrene; edaphic biostimulants; soil biochemical properties; soil microbial community
3
1. Introduction The technique of applying organic matter to soils contaminated with polycyclic aromatic hydrocarbons (PAHs) is widespread among scientists and engineers. It is a good environmental strategy which aims to eliminate or reduce the negative effects that these chemicals cause on soil microorganisms [1, 2, 3, 4, 5]. Because the microbial degradation is the most important process for these pollutants, for some authors this second role of organic matter has a greater importance than the first in the bioremediation of PAHs-polluted soils [6, 7, 8]. Organic matter mineralization releases nutrients continuously or intermittently over a period of time and therefore has been applied to PAH-contaminated soils to stimulate and maintain indigenous biodegradation rates. It is, however, a slow process that depends on several factors such as the PAH type, soil microorganisms tolerant to this PAH, and the quantity and chemical composition of the M.O. added to soil [4, 5, 9]. In recent years there has been increasing use of organic fertilizers or edaphic biostimulants (BS), obtained by hydrolysis from various organic materials, in the bioremediation of soils contaminated with organic xenobiotics such as PAHs and plaguicides. Unlike other sources of organic matter, these organic compounds are usually constituted by low-molecular-weight proteins and amino acids. This aspect is very important, since these compounds can be used directly by the soil microorganisms and therefore accelerates degradation of xenobiotic in soil. These products are also characterized by high polysaccharides content, and humic-like molecules that stimulate soil microorganisms, and thus, promote the degradation of the xenobiotic in soil [5, 10, 11, 12, 13]. Recently, the use of BS obtained from sewage sludge and chicken feathers has become very common in the degradation of plaguicides, mainly oxyfluorfen and chlorpyrifos [11, 13],
4
with a notable increase in the degradation of the above xenobiotics after the applying both BS to the polluted soils.Since nostudies have been reported using these BS on PAH-contaminated soil, we hypothesize that both protein hydrolysates can be very useful in remedying different PAH-contaminated soils. The objective of this study, therefore, was to investigate, under laboratory conditions, the influence of BS obtained from sewage sludge and chicken feathers in soils polluted with different PAH andits effect on soil biochemical properties and microbial community.
2. Materials and methods 2.1. Soil, biostimulants and PAH characteristics The soil used in this experiment is a Calcaric Regosol [14]. The main soil characteristics are reported elsewhere [11, 13], and summarized in Table 1. The methodology used to the determine the physical and chemical parameters in soil and organic wastes is also described in Tejada et al. [11] and Rodriguez-Morgado et al. [13]. Three edaphic BS were used: (1) SS, derived from sewage sludge; (2) CF1, derived from chicken feathers provided by a poultry industry located in Murcia (Spain); and (3) CF2, derived from chicken feathers provided by a local poultry slaughterhouse (TG-SL) located in Seville (Spain). All BS were obtained by enzymatic hydrolysis. Sewage sludge and both feathers were hydrolysed according to the pH-stat method [15], using an endoprotease obtained by liquid fermentation of Bacillus licheniformis ATCC 21415 as the hydrolytic agent in a bioreactor operating under controlled temperature and pH, agitation and NaOH consumption [16]. The enzymatic hydrolysis process is detailed in Rodriguez-Morgado et al. [13]. Three hydrocarbons were utilized to artificially contaminate the soil: (1) phenanthrene (Phe), a PAH consisting of three fused benzene rings; (2) pyrene (Py), a PAH consisting of
5
three fused benzene rings; and (3) benzo(a)pyrene (BaP), a PAH consisting of five fused benzene rings.
2.2. Incubation procedure Prior to being treated, seven hundred grams of soil were pre-incubated at 25 °C for 7 days at 30-40% of their water-holding capacity, according to Tejada [17]. After this preincubation period, soil samples were mixed with Phe, Py or BaP respectively. According to Mueller and Shann [18] and Smith et al. [19], each PAH was dissolved in acetone and added to soil at 100 mg kg-1soil. Acetone was also added to the non-polluted soils (control treatment). Each treatment was mixed thoroughly and allowed to evaporate two days in a fume hood. During this period, the soil was mixed at intervals to ensure homogeneous distribution of the PAHS in each treatment. Subsequently, the three experimental BS was added to the soil. Soil samples were mixed with CF1 and CF2 to a concentration of 1%, 1.3% and 1.6%, respectively, in order to apply the same amount of organic matter with each BS to the soil. According to Rodriguez-Morgado et al. [13], all biostimulants were liquid and were solubilized in distilled water (500 l ha-1) before applying. An unamended and non-polluted soil was used as control. The incubation treatments are described in Table 3. Triplicate treatments were kept in semi-closed microcosms at 20 ± 2 °C for 100 days, respectively.
2.3. Soil PAH determinations The Phe and Py were extracted from 5 g samples of soil. These samples were introduced into a 50 ml centrifuge tube with 4 ml of deionized water and shaken for one minute, after which, 10 ml of acetonitrile were added to the tube and the mix was shaken for a further 30 seconds. Finally, a QuEChERS dSPE Phenomenex ref. KSO-8912 extraction kit was added to the centrifuge tube. It was vigorously shaken for one minute and then
6
centrifuged for 5 minutes at 4000 rpm [20]. The extracts were passed through a 0.45-µm syringe filter and the filtered extracts were concentrated to 1 ml. They were then analyzed by gas chromatograph equipped with autosampler, programmable splitless inlet temperature of deactivated fused silica pre-column of intermediate polarity (1-3m x 0.25 to 0.32 mm internal diameter), connected by connector to a fused silica column with 5% phenylmethylsilicone (5MS type) and dimensions 30m x 0.25 mm x 0.1 µm, for mass spectrometry detector. Analysis conditions were as follows: Autosampler: Wash the syringe with hexane; Volume injected: 2 µl; Injection speed: 1.0 µl s-1; Washing time of the syringe: 20 seconds; Pause: 2 seconds; Shooting speed: µl s-1 Injector program: Initial temperature: 70 ° C; Ramp: hold for 0.1 minutes, up to 300 ºC to 200 ºC min-1, keep up to 40 minutes Gas chromatograph (separation): Temperature of the transfer line 280 ºC; Initial temperature: 70 °C; Ramp: hold for 1.5 minutes, up to 300 ° C at 5 °C min-1, hold 4 minutes. Flow of carrier gas (He): 1.5 ml min-1 measured at baseline. Mass
Detector
(purchase):
Acquisition
time:
45
minutes;
Delay in
the
filament/multiplier: 2.50 minutes. The extraction of BaP from soil was performed using the Song et al. [21] method. A sample of 1.5 g of soil was put in a 15-ml Pyrex tube and10 ml acetone was added, shaken on a vortex and sonicated for20 min. Subsequently, the soil sample was centrifuged at 13700×g for 15 min, the supernatant was added to 20 ml glass flasks and the acetone used to extract BaP was left to evaporate. The same procedure was repeated again twice and the extracts were added to a 20-ml flask. The extracts were passed through a 0.45-µm syringe filter. The methodology used to measure the BaP is described in Tejada et al. [5].
7
2.4. Soil biological determinations The activity levels of four soil enzymes for each treatment were measured at days 1, 5, 7, 12, 20, 40, 70 and 100 during the incubation period. Dehydrogenase activity was measured as the reduction of 2-(p-iodophenyl)-3-(p-nitrophenyl) 5-phenyl tetrazoliumchloride to iodonitrophenylformazan [22]. Urease activity was determined by the buffered method of Kandeler and Gerber [23], using urea as the substrate. β-glucosidase activity was determined using p-nitrophenyl-β-D-glucopyranoside as the substrate [24]. Phosphatase activity was measured using p-nitrophenyl phosphate as the substrate [25]. For each treatment, phospholipids were extracted at days 5, 12, 40 and 100 during the incubation period, (three replicates per treatment) using a chloroform–methanol extraction method based on Bligh and Dyer [26]. Phospholipids were transformed by alkaline methanolysis into fatty acid methyl esters (FAMEs), which were quantified by a gas chromatograph (TRACE GC Ultra, Thermo Scientific) fitted with a 60-m capillary column (BPX70 60 m X 0.25 mm ID X 0.25 µm film), using helium as carrier gas. The initial temperature was 150 °C for 0.5 min and it was increased to 180 °C at 2 °C min-1 and then to 240 °C at 4 °C min-1. The Gram-positive representative fatty acids were: i15:0, a15:0, i16:0, and i17:0 and the Gram-negative specific fatty acids were: 18:1u9c, 18:1u9t, cy17:0, and cy19:0. The fatty acids i15:0, a15:0, 15:0, i16:0, i17:0, 18:1u9c, 18:1u9t, cy17:0, and cy19:0 were chosen to represent the biomass of bacterial community [27, 28, 29]. The fatty acid 18:2u6 was taken to indicate the fungal biomass [30, 31, 32]. All results are given in nmol g-1. For each treatment and each experimental day, 20 g of soil was taken. Soil samples were stored in sealed polyethylene bags at 4 °C for 15 days, prior to analysis of the enzymatic activities and at -20 °C prior to phospholipid analysis [11, 13].
8
2.5. Statistical analysis Data were submitted to two-way ANOVA with treatment and sampling time as factors followed by Tukey’s significant difference as a post hoc test, considering a significance level of p0.05). Incubation days 5 C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
2.8b ± 0.9 2.1b ± 0.7 19.7c ± 1.4 21.9c ± 1.8 16.6c ± 1.4 12.0c ± 1.5 14.9c ± 1.1 11.1c ± 1.3
12 40 Bacterial Gram+ PLFA 2.6b ± 0.7 2.8b ± 0.7 1.4a ± 0.4 0.96a ± 0.18 38.0d ± 2.7 3.9b ± 0.9 39.6d ± 2.5 4.4b ± 1.1 35.8d ± 3.2 3.1b ± 0.6 31.4d ± 3.3 1.9b ± 0.6 33.9d ± 2.4 2.1b ± 0.6 28.6d ± 3.0 1.7ab ± 0.4
C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
1.2b ± 0.3 0.92b ± 0.15 3.9d ± 1.0 4.3d ± 1.2 3.1c ± 0.9 2.5c ± 0.8 2.9c ± 0.9 2.2c ± 0.6
Bacterial Gram- PLFA 1.1b ± 0.2 1.2b ± 0.2 0.62a ± 0.17 0.43a ± 0.11 7.3d ± 2.2 1.8c ± 0.5 7.9e ± 2.5 2.1c ± 0.3 6.3d ± 2.0 1.5b ± 0.4 6.4d ± 1.6 0.83b ± 0.15 6.9d ± 2.1 0.91b ± 0.13 5.2d ± 1.5 0.74b ± 0.16
1.0b ± 0.2 0.30a ± 0.10 1.2b ± 0.2 1.1b ± 0.2 1.1b ± 0.3 0.43a ± 0.11 0.47a ± 0.16 0.34a ± 0.09
4.0b ± 1.1 3.0b ± 0.9 23.6c ± 2.5 26.2c ± 3.1 19.7c ± 2.4 14.5c ± 2.4 17.8c ± 2.0 13.3c ± 2.0
Total bacterial PLFA 3.7b ± 1.2 4.0b ± 1.1 2.0a ± 0.6 1.4a ± 0.4 45.1d ± 4.6 5.7b ± 1.2 47.5d ± 4.8 6.5b ± 1.5 42.1d ± 5.1 4.6b ± 1.1 37.8d ± 4.0 2.7b ± 0.9 40.8d ± 3.3 3.0b ± 0.8 33.8d ± 4.4 2.4ab ± 0.6
3.7b ± 1.1 1.0a ± 0.2 3.8b ± 0.5 3.7b ± 0.9 3.8b ± 1.0 1.5a ± 0.3 1.8a ± 0.4 1.2a ± 0.2
0.53b ± 0.12 0.43b ± 0.15 0.92c ± 0.19 0.96c ± 0.15 0.77c ± 0.12 0.57bc ± 0.09 0.65c ± 0.10 0.50b ± 0.09
Fungal PLFA 0.48b ± 0.10 0.49b ± 0.11 0.29b ± 0.11 0.18a ± 0.07 1.9c ± 0.4 1.1c ± 0.1 2.1d ± 0.2 1.2c ± 0.1 1.6c ± 0.3 0.95c ± 0.13 1.6c ± 0.2 0.32b ± 0.08 1.9c ± 0.3 0.35b ± 0.07 1.3c ± 0.2 0.28b ± 0.07
0.51b ± 0.08 0.12a ± 0.08 0.54b ± 0.09 0.55b ± 0.08 0.48b ± 0.07 0.22a ± 0.07 0.26b ± 0.07 0.18a ± 0.04
C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py C Py SS CF1 CF2 SS+Py CF1+Py CF2+Py
100 2.7b ± 0.8 0.74a ± 0.11 2.6b ± 0.4 2.6b ± 0.6 2.7b ± 0.5 1.1a ± 0.2 1.3a ± 0.2 0.89a ± 0.15
30
Table 10 Evolution of bacterial Gram+, bacterial Gram-, total bacterial and fungal PLFAs (nmol g-1) in soils amended with the experimental edaphic biostimulants and polluted with benzo(a)pyrene (BaP) during the experimental period. Data are expressed as mean values ± standard error. Columns followed by the same letter(s) are not significantly different (p>0.05). Incubation days 5 C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
2.8c ± 0.9 1.9c ± 0.7 19.7e ± 1.4 21.9f ± 1.8 16.6e ± 1.4 11.4e ± 1.1 13.1e ± 1.5 9.0d ± 1.3
12 40 Bacterial Gram+ PLFA 2.6c ± 0.7 2.8c ± 0.7 1.1b ± 0.4 0.75ab ± 0.16 38.0f ± 2.7 3.9c ± 0.9 39.6f ± 2.5 4.4d ± 1.1 35.8f ± 3.2 3.1c ± 0.6 13.9e ± 1.4 1.6b ± 0.3 14.6e ± 1-3 1.9b ± 0.2 10.8e ± 1.1 1.0b ± 0.2
C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
1.2c ± 0.3 0.80c ± 0.15 3.9d ± 1.0 4.3e ± 1.2 3.1d ± 0.9 2.3d ± 0.3 2.7d ± 0.4 1.7c ± 0.3
Bacterial Gram- PLFA 1.1c ± 0.2 1.2c ± 0.2 0.43b ± 0.11 0.32b ± 0.07 7.3e ± 2.2 1.8d ± 0.5 7.9e ± 2.5 2.1d ± 0.3 6.3e ± 2.0 1.5c ± 0.4 5.5e ± 1.5 0.63b ± 0.12 6.0e ± 1.3 0.69b ± 0.16 4.3e ± 1.3 0.45b ± 0.11
1.0c ± 0.2 0.12a ± 0.04 1.2c ± 0.2 1.1c ± 0.2 1.1c ± 0.3 0.37b ± 0.09 0.48b ± 0.10 0.26a ± 0.08
4.0c ± 1.1 2.7b ± 0.8 23.6e ± 2.5 26.2ef ± 3.1 19.7e ± 2.4 13.7e ± 1.5 15.8e ± 2.0 10.7e ± 1.5
Total bacterial PLFA 3.7c ± 1.2 4.0c ± 1.1 1.5b ± 0.4 1.1b ± 0.2 45.1f ± 4.6 5.7d ± 1.2 47.5f ± 4.8 6.5d ± 1.5 42.1f ± 5.1 4.6d ± 1.1 19.4e ± 3.0 2.2c ± 0.6 20.6e ± 2.7 2.6c ± 0.4 15.1e ± 2.3 1.5b ± 0.4
3.7c ± 1.1 0.45a ± 0.11 3.8c ± 0.5 3.7c ± 0.9 3.8c ± 1.0 1.2b ± 0.3 1.4b ± 0.3 0.9a ± 0.2
0.53c ± 0.12 0.37b ± 0.06 0.92d ± 0.19 0.96d ± 0.15 0.77c ± 0.12 0.53c ± 0.11 0.60c ± 0.14 0.44c ± 0.12
Fungal PLFA 0.48c ± 0.10 0.49c ± 0.11 0.20b ± 0.06 0.12a ± 0.09 1.9de ± 0.4 1.1d ± 0.1 2.1e ± 0.2 1.2d ± 0.1 1.6d ± 0.3 0.95d ± 0.13 0.88c ± 0.20 0.37b ± 0.15 0.94d ± 0.21 0.43c ± 0.14 0.61c ± 0.13 0.25b ± 0.09
0.51c ± 0.08 0.07a ± 0.02 0.54c ± 0.09 0.55c ± 0.08 0.48c ± 0.07 0.18b ± 0.05 0.25b ± 0.06 0.13a ± 0.03
C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP C BaP SS CF1 CF2 SS+BaP CF1+BaP CF2+BaP
100 2.7c ± 0.8 0.33a ± 0.09 2.6c ± 0.4 2.6c ± 0.6 2.7c ± 0.5 0.86b ± 0.15 0.93b ± 0.13 0.64a ± 0.13
31
Figures 100
a a a a
Soil Phe (mg kg-1)
a 80
b b ab
a b b
b ab
b
b
60
b b
b
c
b c
c
40
bc
b c
c
c
b c
c
c d
20 0 1
5
7
S+Phe
Soil Py (mg kg-1)
100
a a a a
a b b a
80
12
S+Phe+SS a
20
S+Phe+CF1
a b
b
b
40
b
b b
c
60
100
S+Phe+CF2
a b
70
c
b
b c
c
b c
c
c
c
c
c
d
40 20 0 1
5
7 S+Py
Soil BaP (mg kg-1 )
100
a a a a
a
a a a
12
S+Py+SS a ab
80
a
20
S+Py+CF1
a
b
40
b
a
b
b
100
S+Py+CF2
a b
70
b
b
a b b b
a b
b
60
b bc
b c
40 20 0 1
5 S+BaP
7 S+BaP+SS
12
20
S+BaP+CF1
40 S+BaP+CF2
70
100
32
Figure 1. Evolution of phenanthrene (Phe), pyrene (Py) and benzo(a)pyrene (BaP) (mean ± standard error) in soils during the experimental period. Data are the means of three samples. Columns followed by the same letter(s) are not significantly different (p> 0.05).
