Appl Microbiol Biotechnol DOI 10.1007/s00253-015-6690-2

ENVIRONMENTAL BIOTECHNOLOGY

Characterization of a novel β-cypermethrin-degrading Aspergillus niger YAT strain and the biochemical degradation pathway of β-cypermethrin Weiqin Deng 1 & Derong Lin 1 & Kai Yao 2 & Huaiyu Yuan 1 & Zhilong Wang 1 & Jianlong Li 1 & Likou Zou 3 & Xinfeng Han 1 & Kang Zhou 1 & Li He 1 & Xinjie Hu 1 & Shuliang Liu 1

Received: 18 March 2015 / Revised: 8 May 2015 / Accepted: 12 May 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Aspergillus niger YAT strain was obtained from Chinese brick tea (Collection number: CGMCC 10,568) and identified on the basis of morphological characteristics and internal transcribed spacer (ITS) sequence. The strain could degrade 54.83 % of β-cypermethrin (β-CY; 50 mg L−1) in 7 days and 100 % of 3-phenoxybenzoic acid (3-PBA; 100 mg L−1) in 22 h. The half-lives of β-CY and 3-PBA range from 3.573 to 11.748 days and from 5.635 to 12.160 h, respectively. The degradation of β-CY and 3-PBA was further described using first-order kinetic models. The pathway and mechanism of β-CY degraded by YAT were investigated by analyzing the degraded metabolites through high-performance liquid chromatography (HPLC) and liquid chromatographymass spectrometry (LC-MS). Relevant enzymatic activities and substrate utilization were also investigated. β-CY degradation products were analyzed. Results indicated that YAT

Weiqin Deng and Derong Lin contributed equally to this article. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6690-2) contains supplementary material, which is available to authorized users. * Shuliang Liu [email protected] 1

College of Food Science, Sichuan Agricultural University, Ya’an, Sichuan 625014, People’s Republic of China

2

College of Light Industry and Food, Sichuan University, Chengdu, Sichuan 610065, People’s Republic of China

3

The Laboratory of Microbiology, Dujiangyan Campus, Sichuan Agricultural University, Dujiangyan, Sichuan 611830, People’s Republic of China

strain transformed β-CY into 3-PBA. 3-PBA was then gradually transformed into permethric acid, protocatechuic acid, 3hydroxy-5-phenoxy benzoic acid, gallic acid, and phenol gradually. The YAT strain can also effectively degrade these metabolites. The results indicated that YAT strain has potential applications in bioremediation of pyrethroid insecticide (PI)contaminated environments and fermented food. Keywords β-cypermethrin . 3-phenoxybenzoic acid . Biodegradation . Degradation pathway . Aspergillus niger YAT strain

Introduction β-cypermethrin (β-CY) is one of the important pyrethroid insecticides (PIs) used to control insects. β-CY is widely used worldwide to control pests infesting cotton, fruits, and vegetables as well as domestic control of cockroaches, fleas, and termites (Tallur et al. 2008). It has become a major class of insecticides used against pests as a replacement of more toxic organophosphorus pesticides (Katsuda 1999). PIs are generally considered safe for humans; however, continuous and excessive use of PIs may cause environmental and health problems (Cuthbertson and Murchie 2010; Cuthbertson et al. 2010). PIs may elicit cumulative toxic (Wang et al. 2009), neurotoxic (Shafer et al. 2005), reproductive (Lavado et al. 2014), and genotoxic effects (Ansari et al. 2011) on nontarget organisms. Furthermore, β-CY is a potential carcinogen in humans (Shukla et al. 2002). The reported half-life of β-CY under different conditions ranges from 4 to more than 100 days (Gu et al. 2008), yielding

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3-phenoxybenzoic acid (3-PBA) as the major degradation product (Hirosawa et al. 2011; McCoy et al. 2012). 3-PBA exhibits a smaller molecular weight, stronger polarity, and faster migration rate in the environment than the parent compound; the half-life of 3-PBA can reach up to 180 days (Halden et al. 1999). 3-PBA was classified as an endocrine disruptor owing to its estrogenic properties and could be detected in human urine causing different kinds of toxicity to human (Fortes et al. 2013; Dewailly et al. 2014; Sun et al. 2014). With the massive use of β-CY, safety and health risks have been severe. Thus, effective remediation methods should be developed to eliminate β-CY and 3-PBA in environments and agricultural products. Biodegradation is generally considered to be an effective and safe way to detoxify and degrade contaminants found in the environment (Lovley 2011) and widely used in degrading environmental pollutants such as pesticides, petroleum, plastics, dyes, pigments, and surface active agents, respectively. Until now, most studies focused on the isolation of PIdegrading strains such as Cladosporiumg (Chen et al. 2011a), Streptomyces aureus (Chen et al. 2012a), Penicillium pinophilum (Peng et al. 2012), Sphingomonas sp. (Tang et al. 2013), and Bacillus sp. (Liu et al. 2014). However, many strains have been isolated from pesticide plant sewage or PI-contaminated soils. Besides, the degrading strains tended to transform PIs by hydrolysis to 3-PBA, which may accumulate in the culture media or soils and could not be further degraded. To overcome these problems, in this study, we investigated Aspergillus niger, a β-CY and 3-PBA-degrading fungus isolated from Chinese brick tea. In this study, optimized biodegradation conditions of β-CY and 3-PBA were identified, and the degradation pathways of β-CY and 3-PBA by A. niger YAT strain were analyzed.

Mineral salt medium (MSM) containing 1.5 g of (NH4)2SO4, 0.5 g of K2HPO4, 1.5 g of KH2PO4, 0.2 g of MgSO4, and 0.5 g of NaCl per 1000 mL of distilled water was used. The solution was adjusted to pH 7.5 and sterilized at 121 °C for 15 min. Enrichment, isolation, and screening of the β-CY-degrading strain The vinegar-fermented grains, liquor starter, fermented bean curd, and brick tea from factories located in Sichuan Province of China were used for the isolation of β-CY-degrading strain. The sample (5 g) was transferred into a 250-mL Erlenmeyer flask containing 30 mL of PD and 25 mg L−1 of β-CY and then incubated at 30 °C in a rotary shaker at 180 rpm for 7 days. The strain was successively transferred to potato dextrose medium (PD) containing 50, 100, 200, and 300 mg L−1 of βCY. Final cultures were transferred (5 %, v/v) to 30 mL of fresh PD containing 100 mg L−1 β-CY; the control was prepared with the same amount of sterile saline solution and incubated for 5 days. The concentrations of β-CY residues in each culture were detected through (HPLC, LC-10A2010C HT, ShimadzuJapan) according to the methods of Liu et al. (2012). Final cultures of strains that could effectively degrade βCY were serially diluted, spread on potato dextrose agar (PDA) containing 100 mg L−1 of β-CY, and incubated for 3 days. Strains that grew on the plates were selected and further purified. These strains were inoculated on PDA slants and washed with sterile saline solution to obtain inocula. The inocula were transferred (5 %, v/v) to 30 mL of fresh PD containing 100 mg L−1 β-CY; the control was prepared with the same amount of sterile saline solution. The concentrations of β-CY residues in various cultures were determined through HPLC, and the strain that showed the highest degradation ability was selected for the succeeding experiments. Identification and characterization of the YAT strain

Material and methods Chemicals and media β-CY (99.7 % purity) and 3-PBA standard (98 % purity) were purchased from Sigma-Aldrich, USA. Phenol (99.9 % purity), protocatechuic acid (98 % purity), 3-phenoxybenzaldehyde (97 % Acros Organics), catechol (99.9 % Accus standard), deltamethrin (98.5 % purity), fenvalerate (96.0 % purity), bifenthrin (97.0 % purity), cyfluthrin (95.2 % purity), permethric acid (95 % purity), permethrin (98 % purity), and gallic acid (99 % purity) standards were obtained from Nanjing Rongcheng Chemical Co., Ltd., Nanjing, China. High-performance liquid chromatography (HPLC)-grade acetonitrile was purchased from CNW Technologies GmbHDusseldorf, Germany.

The colony morphological characteristics of the YAT strain were documented after the strains were inoculated on a PDA plate and incubated at 30 °C. The size, color, texture, surface ornamentation, edge, and exudates were observed for 5 days. The strains were dyed and examined under a light microscope (Olympus-BH-2, Tokyo, Japan). Total genomic DNA was extracted using DNA extraction kits (Tianze Genetic Engineering Co., Ltd., Dalian, China). The internal transcribed spacer (ITS) sequence was amplified through polymerase chain reaction (PCR; C1000 Thermal Cycler, Bio-RAD, USA) by using the universal primers ITS1 (5′-TCCGTAGGT GAACCTGCGG-3′) and ITS4 (5′TCCTCCGCTTA TTGATATGC-3′) produced by Biological Engineering Co., Ltd., Dalian, China. T carrier cloning and sequencing of the cloned insert were performed by Biological

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Engineering Co., Ltd., Dalian, China. The ITS sequence (GenBank Accession No. JQ366070) was compared with genes available in the Genbank Nucleotide Library through a BLAST search in the National Center for Biotechnology Information (NCBI) website. The phylogenetic tree was analyzed and constructed with the neighbor-joining method. Biodegradation of β-CY Growth characteristics of the YAT strain and degradation characteristics of β-CY The inocula were carefully transferred (5 %, v/v) to 30 mL of fresh PD and PD-β-CY (the concentration of β-CY was 50 mg L−1) and incubated at 30 °C in a rotary shaker at 180 rpm. The culture was extracted daily for 7 days, and the samples were filtered to obtain mycelia. The obtained mycelia were dried at 80 °C to analyze YAT strain growth and to detect any residual β-CY to analyze β-CY degradation. Dynamic characteristics of β-CY The significant factors and their optimized ranges that were selected as independent variables in this experiment were substrate concentration (25, 50, and 100 mg L−1), temperature (25, 30, and 35 °C), and medium pH (6.0, 7.0, and 8.0). The entire cultures were taken at 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, and 5 days, respectively. The residual β-CY concentrations were determined, and the data were fitted to a first-order dynamics equation.

PI degradation Sterilized PD medium was supplemented with 50 mg L−1 of deltamethrin, fenvalerate, bifenthrin, cyfluthrin, and permethrin at 50 mg L−1 with 5 % (v/v) inocula. An equal amount of sterile saline solution was prepared as a control and incubated for 5 days. Growth and degradation were measured daily as described above.

Identification of β-CY and 3-PBA metabolites A small amount of 3-PBA, which was quickly degraded, was produced through β-CY decomposition. Therefore, the detection or analysis of 3-PBA and its metabolites remain a challenge. We investigated the degradation of β-CY into 3-PBA and the subsequent degradation of 3-PBA to elucidate the pathway of β-CY degradation by the YAT strain. The inocula were carefully transferred (5 %, v/v) to 30 mL of fresh PD-β-CY (50 mg L−1) and incubated at 30 °C in a rotary shaker at 30 °C set at 180 rpm. The culture was extracted daily for 7 days. The samples were reprocessed as follows: (1) β-CY and its metabolites were subjected to HPLC as previously described except the gradient elution program (acetonitrile percentage was linearly increased from 45 to 85 % in 10 min and retained at 85 % for 8 min). (2) The acetonitrile extraction liquids obtained at different times were combined, dried in a pressure-blowing concentrator, and dissolved in a moderate amount of acetonitrile. The solution was subsequently centrifuged at 12, 000 r/min for 10 min, and the liquid supernatant was filtered using a 0.45-μm organic phase membrane filter. The filtrate was detected by HPLC and liquid chromatography-mass spectrometry (LC-MS). (3) The feasible products were added to the mixed samples and tested by HPLC as the proportion of mobile phase was changed continually. The metabolites during 3-PBA degradation were identified in the same manner. LC-MS investigations were carried out under the conditions listed in the following section by using Agilent Technologies 1200 Series-HPLC/Agilent 6210 Time-ofFlight MS, USA, and the chromatographic column was an Agilent ZORBAX XDB-C18 (1.8 μm, 2.1 × 100 mm, i.d.). The mobile phases were acetonitrile and 0.5 % formic acid aqueous solution (9:11, v/v). Column temperature was maintained at 25 °C, and injection volume was 5.0 μL with a flow rate of 0.3 mL min−1. A DAD detector with an ion source temperature of 325 °C and a voltage of 3500 V was used. Dry gas flow rate was 9.0 L min−1 with an atomizer pressure of 45 psi and cataclastic voltage of 175 V.

Degradation of 3-PBA β-CY and its metabolite 3-PBA are simultaneously degraded by the YAT strain. The experiments to elucidate the characteristics of 3-PBA degradation were analogous to those in β-CY, although significant differences are notable. These differences included detection method, mobile phase [buffer solution (pH 2.5)/acetonitrile = 9:11 (v/v)], flow rate (0.7 mL min−1), and injection volume (10 μL); the samples were detected at 210 nm and 25 °C (Ding et al. 2004).

Degradation rates of the metabolites Sterilized PD and MSM were supplemented with 50 mg L−1 of 3-phenoxybenzaldehyde, 3-PBA, permethric acid, gallic acid, protocatechuic acid, phenol, and catechol. The samples were prepared with the YAT strain inocula and the growth of the YAT strain, and the residual substrate concentrations were measured after this strain was incubated 72 h.

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Enzyme activities of the YAT strain Phosphate buffer solution (50 mmol L−1, pH 7.0) was added to 10 mg L−1 of each of β-CY, 3-phenoxybenzaldehyde, 3-PBA, permethric acid, gallic acid, protocatechuic acid, phenol, and catechol followed by addition of 2.0 g of YAT mycelial pellets. The reaction occurred at 30 °C for 12 h in a rotary shaker at 180 rpm, and 10 mL of acetonitrile was added to terminate the reaction. The residues of the substrates were detected. The amount of mycelial pellets required to degrade 1.0 nmol of substrates per hour under this experimental condition was defined as one unit of enzyme activity (U).

PBA. Thus, YAT was chosen for further experiments in this study. Identification and characterization of the YAT strain

where Ct is the residual concentration of β-CY or 3-PBA in the sample solution (mg L−1) and C0 is the initial concentration of β-CY or 3-PBA (mg L−1) at time zero. Degradation rate constant (Kd) was determined by the firstorder kinetic model expressed as Eq. (2).

The colony and morphology characteristics of the strain YAT were shown in Figure S1 and Figure S2 in supplementary materials. The colonies of YAT strain were white with radial mycelia, and dark brown spores were produced after 2 days. The central region of the colonies gradually turned black with a thick and velvet-like texture; by contrast, the irregular edges remained white. The diameters of the 5-day-old colonies were 39.2 mm, and the colonies were covered with dark brown spores. Pigment was not produced during growth. In addition, the upper layer of the spore sac was spherical or almost spherical with diameters ranging from 40 to 50 μm. The sequenced fragment length of YAT was 598 bp (GenBank Accession No. JQ366070). BLAST indicated that the YAT sequence is highly similar (99 %) to the ITS sequence of GU082483 and JF436883 strains of the A. niger group. Thus, a phylogenetic tree based on the ITS sequences of YAT and related strains was constructed (Fig. 1). Based on morphological characteristics and on the ITS sequence alignments, the strain was eventually identified as A. niger. This strain was deposited in China General Microbiological Culture Collection Center (Collection Number: CGMCC 10,568).

C t ¼ C 0 e−K d t ;

Biodegradation of β-CY

Data analysis Degradation rate was calculated according to the following equation: Degradation rate ð%Þ ¼

ðC 0 −C t Þ  100%; C0

ð1Þ

ð2Þ

where Kd is the degradation rate constant [mg (L day/h)−1], t is the degradation time (day/h), Ct is the residual concentration of the substrate (mg L−1) for some time t, and C0 is the initial concentration of β-CY or 3-PBA (mg L−1) at time zero. The half-life (T1/2) of β-CY was calculated by using Eq. (3). T 1=2 ¼ ln2=k;

ð3Þ

where k is the rate constant (day−1/h−1).

Degradation results of β-CY in different media by the A. niger YAT strain Growth and degradation of β-CY of A. niger YAT strain in PD, 1/5 PD, 1/10 PD, and MSM indicated that the YAT strain could degrade β-CY more rapidly when more nutrients were available. However, it did not grow in MSM and showed no degradation of β-CY. Growth characteristics of the YAT strain and degradation of β-CY

Results Isolation and screening of β-CY-degrading fungi CP2, a fungus isolated from vinegar-fermented grains, degraded 20.05 % of β-CY, whereas YAT, a fungus isolated from Chinese brick tea, displayed a 26.31 % β-CY degradation rate. No significant difference between the amount of 3PBA and its predicted concentration was observed in CP2 fermentation, whereas 3-PBA was not detected in the YAT fermentation broth. The results showed that YAT can degrade not only β-CY but also its intermediate product, namely, 3-

The growth of YAT in PD and PD-β-CY medium showed some differences (Fig. 2). The basic biomass did not increase after 5 days; ultimately, the cell dry weight was determined to be 6.92 g L−1. The growth pattern of the YAT strain in PD-βCY was essentially similar to that in PD, with a final dry cell weight at 7.20 g L−1. The results showed that β-CY did not significantly affect the growth of YAT in PD. Although β-CY prolonged the lag phase, it did not affect the final biomass. βCY was degraded rapidly on the first day where the degradation rate reached 41.32 % after 5 days of cultivation. Subsequently, the strain did not show growth and the degradation rate reached 54.83 % after 7 days.

Appl Microbiol Biotechnol YAT(JQ366070)

Fig. 1 Phylogenetic tree of the YAT strain constructed by the neighbor-joining method based on ITS sequences of YAT and related strains. Bootstrap values are given at branching points

Aspergillus niger (GU082483) Aspergillus niger (HQ014690.1)

99

Aspergillus niger (JF040211.1) 61

Aspergillus niger (JF436883) Aspergillus sp. LZ1(GU258410)

100

Aspergillus sp. HZ-35(EU301661) Aspergillus auricomus (AY373839) Aspergillus sclerotiorum (EF634389) 95 98

Aspergillus sulphureus (EF661409) Aspergillus roseoglobulosus (EF661406) Cochliobolus sativus (AY372677)

73

Pleospora tarda (AF071345) Nimbya alternanthera (AY372674)

100

Macrospora scirpicola (AY359887)

83

Ulocladium atrum (AY372683)

38

Alternaria alternata (FJ455502)

50 99

Alternaria tenuissima (AB369436)

Trichoderma longibrachiatum (SWFC8690) 100

Hypocrea lixii (SWFC8726)

49

Trichoderma atroviride (SWFC10168)

0.1

Degradation kinetics of β-CY by A. niger YAT strain

55

9

50

8 7

45

6

40

5 35 4 30

3

25

2

20 15

Dry cell weight (g/L)

β-CY concentration (mg/L)

In the tested substrate concentrations, reaction temperatures, and initial medium pH in this study, the reactions followed

first-order kinetics and T1/2 of β-CY varied from 3.573 to 11.748 days (Table 1). At substrate concentrations of 25 to 100 mg L−1, T1/2 of β-CY increased as substrate concentration increased. However, T1/2 slightly changed as temperature increased from 25 to 35 °C or as medium pH varied from 6.0 to 8.0. Temperature at 35 °C was more conducive to β-CY degradation. The results showed that the YAT strain could efficiently degrade βCY at 25 to 35 °C and at medium pH of 6.0 to 8.0; thus, PI-degrading microorganisms, such as the YAT strain, possess survival advantage in a highly variable environment because these microorganisms can utilize xenobiotics even when exposed to adverse conditions (Chen et al. 2012b). Degradation of different PIs

1 0

1

2

3

4

5

6

7

8

0

Incubation time (d) gowth curve: PD PD- β -CY blank contral degradation curve:

YAT

Fig. 2 Growth characteristics and β-CY degradation curves of the A. niger YAT strain

The degradation spectrum of the YAT strain showed no evidence of bifenthrin degradation, although degradation of deltamethrin, fenvalerate, cyfluthrin, and permethrin acid was evident at degradation rates of 27.53, 58.00, 53.23, and 25.34 %, respectively. These results indicated wide degradation spectrum of YAT strain for PIs.

Appl Microbiol Biotechnol Table 1 Kinetic parameters of βCY degradation by YAT under the effects of different variables

Factors

Kinetic equation

C0 (mg L−1)

Temperature (°C)

pH

T1/2 (day)

Kd (mg (L day)−1)

R2

25 50 100 25 30

Ct Ct Ct Ct Ct

= = = = =

25.78e−0.194t 50.14e−0.110t 100.81−0.059t 50.25e−0.103t 50.14e−0.110t

3.573 6.301 11.748 6.730 6.301

0.194 0.110 0.059 0.103 0.110

0.924 0.921 0.950 0.945 0.921

35 6.0 7.0 8.0

Ct Ct Ct Ct

= = = =

50.21e−0.118t 50.37e−0.094t 50.14e−0.110t 50.09e−0.120t

5.874 7.374 6.301 5.776

0.118 0.094 0.110 0.120

0.990 0.910 0.921 0.915

Biodegradation of 3-PBA

Analysis of β-CY biodegradation metabolites by HPLC

Growth characteristics of the YAT strain and degradation of 3-PBA

Metabolites produced from β-CY degradation into 3-PBA

The growth results of the YAT strain and degradation of 3PBA (Fig. 3) showed that 3-PBA exhibited some influences on the growth of YAT. 3-PBA not only prolonged the lag phase of YAT strain but also affected the final biomass of the strain. The YAT strain could totally degrade 100 mg L−1 3PBA in 22 h, indicating that the YAT strain could effectively degrade 3-PBA.

Degradation kinetics of 3-PBA by A. niger YAT strain T1/2 of 3-PBA varied from 5.635 to 12.160 h (Table 2) within a range of substrate concentrations, reaction temperatures, and medium pH. The obtained T1/2 was shorter than normal T1/2 (180 days). This observation further confirmed that A. niger YAT strain could effectively degrade 3-PBA and β-CY. 110

9

100

8

The results showed that no new chromatographic peak appeared in the PD culture systems, but two distinct peaks (B, C) appeared in the PD-β-CY culture systems (Fig. 4a). The chromatograms of the mixed samples obtained from PD-βCY culture systems at different times (Fig. 4b) showed that substance B accumulated with β-CY-degradation and substance C could be assayed on the 3rd but not on the 5th day. Substance C was possibly produced through β-CY degradation but was quickly degraded. Thus, substances B and C were hypothesized as β-CY metabolites. After comparing the chromatograms with that of the mixed sample of β-CY, permethric acid, and 3-PBA, we hypothesized that substance B and C were permethric acid and 3-PBA, respectively. Permethric acid and 3-PBA references were added to the PD-β-CY hybrid samples and run through HPLC using different proportions of mobile phase. The peak areas of B and C increased, and the chromatographic peaks were still unimodal under these conditions. These results further supported our hypothesis that substances B and C were permethric acid and 3-PBA, respectively.

7

80 70

6

60

5

50 4

40 30

3

20

2

Dry cell weight (g/L)

3-PBA concentration (mg/L)

90

10 1

0 -10

0

0

24

48

72 96 120 Incubation time (h)

gowth curve: PD PD- 3-PBA degradation curve: blank contral

144

168

192

YAT

Fig. 3 Growth characteristics and 3-PBA degradation curves of the A. niger YAT strain

Metabolites produced from 3-PBA degradation by the YAT strain Samples obtained at different time points from PD and PD-3PBA cultivation systems were analyzed through HPLC. No new chromatographic peaks were observed in the PD cultivation system, whereas two new evident peaks appeared in PD3-PBA systems (Fig. 5a). Samples obtained at different time points from the PD-3-PBA cultivation systems were evaluated through HPLC (Fig. 5b). The peak areas of substance D gradually increased from 0 to 20 h and then diminished throughout 3-PBA degradation; by contrast, the peak area of substance E remained constant from 0 to 22 h. Thus, substance D was assumed as 3-PBA metabolite that can be degraded by the

Appl Microbiol Biotechnol Table 2 Kinetic parameters of 3PBA degradation by YAT under the effects of different variables

Factors

Kinetic equation

C0 (mg L−1)

Temperature (°C)

pH

Kd (mg (L h)−1)

R2

50 100 150 25 30

Ct Ct Ct Ct Ct

= = = = =

50.22e−0.123t 101.24e−0.101t 150.51e−0.057t 100.87e−0.062t 101.24e−0.101t

5.635 6.863 12.160 11.180 6.863

0.123 0.101 0.057 0.062 0.101

0.935 0.924 0.957 0.943 0.924

35 6.0 7.0 8.0

Ct Ct Ct Ct

= = = =

100.41e−0.107t 100.87e−0.093t 101.24e−0.101t 100.97e−0.106t

6.478 7.453 6.863 6.539

0.107 0.093 0.101 0.106

0.963 0.902 0.924 0.970

permethric acid, dihydroxy benzoic acid, hydroxy-3phenoxy benzoic acid, trihydroxybenzoic acid, and phenol. Dihydroxy benzoic acid, hydroxy-3-phenoxy benzoic acid, and trihydroxybenzoic acid may have multiple isomers. On the basis of the results of retention time analysis and ChemBio3D Ultra 12.0., we assumed that these compounds are protocatechuic acid, 3-hydroxy-5-phenoxy benzoic acid, and gallic acid.

YAT strain. Compound D was identified as phenol by comparing its retention time with that of the standard. However, we cannot easily determine whether the other peaks were products of 3-PBA degradation; further analysis through LC-MS should be performed. LC/MS analysis of β-CY biodegradation metabolites The samples obtained at different time points from the PD-βCY cultivation systems were evaluated through LC-MS to further identify the unknown products of β-CY degradation. Seven different substances were detected (Table 3). Aside from 3-PBA and β-CY, the five other substances were

Fig. 4 a HPLC chromatogram of the mixed samples taken from PD and PD-β-CY medium inoculated with A. niger YAT culture systems. b HPLC chromatograms of samples obtained at different incubation times from the PD-βCY medium inoculated with A. niger YAT culture systems

T1/2 (h)

Degradation of metabolites Table 4 shows that the YAT strain could effectively degrade βCY metabolites except permethric acid. The YAT strain could

uV 60000

a

PD β-CY

50000

A

40000 30000

C

20000

B

10000 0 0.0

2.5

5.0

7.5

10.0

12.5

15.0

min

15.0

min

uV 30000

0d

b

A

3d

25000

5d 20000

7d

15000

B 10000

C

5000 0 0.0

2.5

5.0

7.5

10.0

12.5

Appl Microbiol Biotechnol Fig. 5 a HPLC chromatograms of mixed samples obtained from PD and PD-3-PBA media inoculated with A. niger YAT culture systems. b HPLC chromatograms of samples obtained at different incubation times from the PD-3-PBA medium inoculated with A. niger YAT culture systems

uV

125000

PD

a

PD-3-PBA

100000

E 75000

D

C

50000 25000 0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

min

uV

125000

0h 8h 10 h 12 h 16 h 20 h 22 h

100000

75000

50000

b D E C

25000

0 0.0

1.0

2.0

degrade gallic acid, protocatechuic acid, and benzoic acid in MSM media; the YAT strain could also degrade 3phenoxybenzaldehyde, 3-PBA, and phenol only in PD media. These results indicated that gallic acid, protocatechuic acid, and benzoic acid were degraded through mineralization and 3phenoxybenzaldehyde, 3-PBA, and phenol were degraded through co-metabolism. Besides, the degradation rates of phenol, 3-phenoxybenzaldehyde, and 3-PBA were higher than those of other metabolites, and the substrates (50 mg L−1) could be degraded completely within 72 h. Enzymes activities of A. niger YAT strain on β-CY and its metabolites The enzymatic activities of A. niger YAT strain on β-CY and its metabolites were determined; the results showed that YAT

3.0

4.0

5.0

6.0

7.0

8.0

min

cells could not degrade permethric acid, and this result is consistent with that of other study (Saikia et al. 2005). These enzymes showed high degradation activities of 3phenoxybenzaldehyde, 3-PBA, gallic acid, protocatechuic acid, phenol, and catechol. Degradation pathway of β-CY by A. niger YAT strain The pathway of β-CY degradation by A. niger YAT strain (Fig. 6) can be inferred from the results obtained in this study. Esterase degraded β-CY into permethric acid and α-cyano-3phenoxy benzyl alcohol. α-cyano-3-phenoxy benzyl alcohol is very unstable and can spontaneously rearrange to form 3phenoxybenzaldehyde. In addition, 3-phenoxybenzaldehyde may undergo further modification with dehydrogenase to form 3-PBA. This result is similar to that in Micrococcus sp.

Table 3 Substances detected by LC-MS from the samples obtained at different incubation times from PD-β-CY medium inoculated with A. niger YAT culture systems Number

Retention time (min)

m/z

Molecular weight

Chemical formula

Compounds

Cpd1 Cpd2 Cpd3 Cpd4 Cpd5 Cpd6 Cpd7

1.666 2.323 10.834 16.986 19.194 19.514 24.722

171.029 155.034 95.049 231.069 215.069 209.013 416.080

170.026 154.026 94.042 230.058 214.062 208.006 415.073

C7H6O5 C7H6O4 C6H6O C13H10O4 C13H10O3 C8H10Cl2O2 C22H19Cl2NO3

Trihydroxy benzoic acid Dihydroxy benzoic acid Phenol Hydroxy-3-phenoxy benzoic acid 3-PBA Permethric acid β-CY

Appl Microbiol Biotechnol Table 4

Degradation rates of metabolites by the A. niger YAT strain

Substrates

3-phenoxybenzaldehyde 3-PBA permethric acid gallic acid protocatechuic acid phenol catechol a

MSM

PD

Biomas (dry cell weight g L−1)

Degradation rate (%)

Biomas (dry cell weight g L−1)

Degradation rate (%)

0.03 0.02 0.03 1.69 1.33 0.05 1.93

– – – 90.65 48.79 – >99.99

5.13 5.65 6.27 / / 6.61 /

>99.99 >99.99 – / / 43.12 /

Note: ‘–’means no degradation, ‘/’means not detected

strain CPN 1 (Tallur et al. 2008) but differs from that described in a previous report on β-CY (Chen et al. 2012a). 3-PBA may be degraded in two ways: (1) 3-PBA can be directly degraded into protocatechuic acid and phenol and (2) 3-PBA is modified into 3-hydroxy-5-phenoxy benzoic acid through the action of hydroxylase and then ether bond is cleaved, producing the metabolites phenol and gallic acid. The phenol produced in both pathways can then be converted into catechol under hydroxylase; catechol, protocatechuic acid, and gallic acid are cleaved to form a straight-chain olefin acid, which is oxidized to carbon dioxide.

Discussion Fungal oxidases can degrade various compounds, including xenobiotic compounds, pesticides, and dyes (Diez 2010; Pinto et al. 2012). The most commonly reported β-CY-degrading strains were isolated from soil and from pesticide-treated plant sludge; most of these strains are bacteria. The application of β-CY-degrading strains in the food is limited because of safety considerations associated with bacterial source, among other factors. To overcome these difficulties, we isolated A. niger, a β-CY and 3-PBA-degrading fungus from traditional fermented foods as sources; A. niger was included in the list of 42 types of Bmicroorganisms which are allowed to be fed directly and generally recognized as safe^ by the US Food and Drug Administration and the Association of American Feed Control Officials in 1989. For the first time, a β-CY-degrading strain was isolated from Chinese brick tea, which is a good source of the strain that could degrade β-CY residues in environments and food. A. niger YAT strain could rapidly degrade a wide range of PIs, such as β-CY, deltamethrin, fenvalerate, cyfluthrin, and permethrin acid. PI-degrading strains show a certain degradation spectrum (Ruan et al. 2013; Yu et al. 2013). Differences in the degradation rates of various PIs by the YAT strain may be attributed to the effects of substituents (i.e., –CN, −Cl, −F,

−Br) on the breakage of the ester bonds by strains (Maloney et al. 1993). The particular strain was also found to be highly effective in degrading 3-PBA that was a major metabolite of PIs. It is a very important characteristic because 3-PBA is persistent towards degradation by microorganisms and may limit the biodegradation of PIs because 3-PBA exhibits antimicrobial activities (Topp and Akhtar 1991; Xie et al. 2008). Some fungal species can degrade contaminants with a diphenyl ether bond; however, these species are rarely reported. The YAT strain could degrade 54.83 % of β-CY (50 mg L −1 ) in 7 days and could completely degrade 100 mg L−1 of 3-PBA within 22 h, which is considerably higher than the reported degradation rates of some bacteria, such as Ochrobactrum lupini DG-S-01 (Chen et al. 2011b) and Stenotrophomonas sp. ZS-S-01 (Chen et al. 2011c). The degradation rates of β-CY and 3-PBA could be described using a first-order kinetic model. The degradation of β-CY into 3-PBA and the metabolites produced from the process were detected through HPLC and LC-MS. HPLC results revealed that permethric acid and 3PBA were metabolites of β-CY. The changes in 3-PBA chromatographic peak area (Fig. 4b) indicated that 3-PBA was formed during β-CY degradation and could be degraded further by the YAT strain. The peak area of phenol gradually increased from 0 to 20 h of the 3-PBA degradation and then decreased thereafter (Fig. 5b)]. In addition, HPLC reflected the dynamic process of these intermediate products. LC/MS analysis further validated and supplemented the HPLC results on β-CY and 3-PBA metabolites. LC/MS results showed that 3-PBA, permethric acid, protocatechuic acid, 3-hydroxy-5phenoxy benzoic acid, gallic acid, and phenol were metabolites of β-CY. Some of these metabolites were not detected in previous studies (Chen et al. 2012 c; Tang et al. 2013). Table 4 shows that the YAT strain could utilize gallic acid, protocatechuic acid, and catechol as sole carbon and energy sources for growth and could completely degrade these substances. Moreover, the YAT strain could degrade 3-

Appl Microbiol Biotechnol Fig. 6 Proposed pathway of βCY degradation by A. niger YAT

CN

O

Cl CH

C

C O

CH

CH

Cl

O

CH

C H3C

β -CY

CH3

esterase

CN O

Cl C

CH

OH

CH

CH

Cl

α -cyano-3-phenoxybenzyl alcohol

C H3C

O

CH

HO

C

CH3

spontaneous reaction

permethric acid OHC

O

3-phenoxybenzaldehyde dehydrogenase HOOC

O

OH

HOOC

hydroxylase

3-hydroxy-5-phenoxy benzoic acid

O

3-phenoxybenzoic acid dioxygenase

dioxygenase

OH HO

OH

HO OH

HO

phenol gallic acid

hydroxylase COOH

COOH protocatechuic acid

HO HO

catechol

straight chain olefin acid

phenoxybenzaldehyde, 3-PBA, and phenol through co-metabolism. This result indicated that mineralization and cometabolism co-existed throughout the degradation of β-CY and its intermediate products by the YAT strain. The ability of the YAT strain to effectively degrade these metabolites indicated that YAT has potential applications in the bioremediation of PI-contaminated environments and fermented food. Enzymatic activities of A. niger YAT strain on β-CY and βCY metabolites were measured. The results showed that YAT cells effectively degraded β-CY and its metabolites except permethric acid. Trihydroxybenzoic acid and dihydroxy benzoic acid were actually gallic acid and protocatechuic acid,

respectively. We hypothesized that the β-CY-degrading enzymes were esterases and 3-phenoxybenzaldehydedegrading enzymes were dehydrogenases. Moreover, 3PBA, protocatechuic acid, and catechol degradation may be attributed to the effect of dioxygenases, and phenol degradation enzymes may be hydroxylases. These assumptions were deduced from the chemical structures of these compounds and from reports of previous studies (Guo et al. 2009; Tallur et al. 2008). Reports on β-CY and 3-PBA biodegradation pathways and mechanisms are relatively rare. We speculated that 3-PBA was initially degraded to phenol and protocatechuic acid; this

Appl Microbiol Biotechnol

speculation is based on the analysis of the degradation products of PIs or 3-PBA in a research on cypermethrin degradation by Micrococcus sp. CPN1 (Tallur et al. 2008), fenpropathrin degradation by Sphingobium sp. JZ-2 (Guo et al. 2009), and 3PBA degradation by Pseudomonas ET1(Gaskin et al. 2013). Furthermore, Chen et al. (2012c) discovered that 3-PBA forms 3-(2-hydroxy phenoxy) benzoic acid through the action of oxygenase and then subsequently broke into protocatechuic acid, phenol, and 3,4-dimethoxy-phenol. Based on the analysis of the products of 3-PBA degradation by Sphingomonas sp. SC1, Tang et al. (2013) speculated that 3-PBA is degraded into hydroxy diphenyl ether and then its ether bond is cleaved to produce phenol and catechol. Thus far, degradation of β-CY and 3-PBA by fungi were rarely reported. This research verified that 3-PBA was converted into 3-hydroxy-5-phenoxy benzoic acid via oxidation and then subsequently oxidized into gallic acid, a result that is quite different from that of previous studies (Liu et al. 2014; Chen et al. 2012a). The extent of our analysis on the degradation of β-CY by the YAT strain and on the metabolite pathway determination is vast and completely relative to that of other studies on PI-degrading strains. In conclusion, A. niger YAT strain obtained from Chinese brick tea showed high degradation efficiency of β-CY and 3PBA. The YAT strain was capable of degrading four types of PIs, which suggested that this strain might be useful in bioremediation of PI-contaminated environments and fermented food. Moreover, we revealed a novel microbial metabolic pathway for β-CY and 3-PBA by oxidization and cleavage of the diaryl ether bond by the YAT strain, which plays an important role in β-CY and 3-PBA biodegradation. Acknowledgments The authors extend their gratitude to the National Natural Science Foundation of China (31371775) for the financial support to this research. Conflict of interest We declare that no conflict of interest exists in the submission of this manuscript. Ethical statement All authors agreed to publish this manuscript. I would like to declare on behalf of my co-authors that this original work has not been previously published and is not under consideration for publication elsewhere, in whole or in part.

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