Biodegradation of p-chloroaniline by fungus Isaria fumosorosea SP535

Biodegradation of p-chloroaniline by fungus Isaria fumosorosea SP535 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Biodegradation of p-chloroaniline by fungus Isaria fumosorosea SP535 Shicong Huang, Jiahui Gao, Lin Zhou, Liujian Gao, Mengke Song, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4840476/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Efficient methods to remediate PCA (p-chloroaniline) polluted environment are urgent due to the widespread, persistence and toxic of PCA in the environment. Biodegradation facilitated by microbes presents a promising approach for remediating PCA pollution. However, the PCA-degrading fungi still yet to be explored. This study confirmed the highly PCA degrading efficiency of an isolated fungus, Isaria fumosorosea SP535. This fungus can achieve a PCA degradation efficiency of 100% under optimal conditions characterized by initial PCA concentration of 1.0 mM, pH of 7.0, and temperature of 25 ℃. SEM and TEM analyses revealed that the toxicity of PCA resulted in roughened surfaces of SP535 hyphae, voids in the cytoplasm, and thickened cell walls. PCA addition significantly elevated the activities of cytochrome P450 monooxygenase in both cell-free extracts and microsomal fractions in the media, suggesting the important role of P450 system in PCA metabolization by SP535. The results provide microbial resource and fundamental knowledge for addressing PCA pollution. p-chloroaniline fungus Biodegradation Toxicity Cytochrome P450 monooxygenase Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction PCA (p-chloroaniline), widely used as a chemical raw material in the production of dyes, cosmetics, pesticides, and herbicides, can enter the environment through wastewater and waste generated during chemical production processes (Khusnun et al. 2016 ; Silambarasan et al. 2016; Dong et al. 2017 ). Reports indicated that PCA has been detected in various environments, including water bodies, sediments, agricultural soils, and living organisms (Tongarun et al. 2008 ; Zhu et al. 2012 ; Dantzger et al. 2018 ). Due to its toxicity and adverse effects on human health, PCA was classified as a persistent organic pollutant and has been designated as a priority pollutant by the US Environmental Protection Agency (EPA) and European Union legislation (Nitisakulkan et al. 2014 ; Kumar al. 2020). Consequently, the development of effective methods for removing PCA residues from the environment has drawn great attention. Bioremediation relying on microorganisms is an efficient method to remove organic pollutants in environment (Li et al. 2020 ; Wang et al. 2019 ). Therefore, it is crucial to identify microorganisms with degradation capabilities from the environment. Numerous bacteria have been isolated from various environmental media for the remediation of PCA contamination. Brevibacillus S-618, isolated from sludge, was able to completely degrade PCA at a concentration of 180 mg·L –1 within 72 h (Li et al. 2020 ). Thauera sp. M9, isolated from soil, completely degraded PCA in culture medium within 30 h (Kumar et al. 2020 ). Bacillus sp., isolated from textile wastewater, demonstrated a 76% degradation of PCA at a concentration of 100 mg·L –1 , attaining complete degradation in the presence of lipopeptide surfactant (Carolin et al. 2021 ). However, current studies have predominantly focused on bacteria, and PCA-degrading fungi have yet to be reported. Fungi have been demonstrated with the remarkable capacity of adapting to harsh environments and exhibiting a strong ability to degrade organic pollutants (Coleine et al. 2022; Sen et al. 2022). The degradation efficiency of Aspergillus LS-1, isolated from the sludge of a pharmaceutical plant, was 95.41% for 80.14 mg L –1 CTC (chlortetracycline) within 3 d (He et al. 2023). A yeast strain, Cutaneotrichosporon dermatis M503, isolated from the sediment tank of a sewage treatment system at a tetracycline manufacturing facility, achieved a degradation efficiency of 86.62% for TC (tetracycline) within 7 d under optimal conditions (Tan et al. 2022 ). Aspergillus sydowii W1, isolated from contaminated soil, demonstrated an 84.05% degradation efficiency for 100 mg·L –1 erythromycin at a concentration within 7 d (Ren et al. 2023 ). Eight lignin-degrading fungi being capable of degrading polychlorinated biphenyls (PCBs) have been reported, with Pleurotus ostreatus removing 98.4% and 99.6% of a PCB mixture in complex and mineral media, respectively, after 6 weeks (Čvančarová et al. 2012 ). Furthermore, some filamentous fungi have been shown to degrade environmental pollutants via cytochrome P450 monooxygenase (Črešnar et al. 2011). With the involvement of cytochrome P450, Marasmiellus sp. CBMAI 1062 could nearly completely degrade pyrene (0.08 mg·L –1 ) after 48 h under saline condition (Vieira et al. 2018 ). The degradation efficiency of the herbicide diuron by Phanerochaete chrysosporium reached 94% after 10 d cultivation, while the degradation of diuron was inhibited by the addition of 1 mmol/L cytochrome P450 inhibitor ABT (1-aminobenzotriazole) (Coelho-Moreira et al. 2013 ). Sun et al. (2022) confirmed that cytochrome P450 is involved in the biodegradation of dichlorvos by Trichoderma atroviride T23, and identified five relevant genes including TaCyp548 , TaCyp620 , TaCyp52 , TaCyp528 , and TaCyp504 . Therefore, the isolation of fungi with high degradation efficiency for PCA is urgent and necessary for the remediation of PCA pollution. In this study, the filamentous fungus Isaria fumosorosea strain SP535, isolated from soil (Xu 2018 ), was selected for experiments on PCA degradation. The objectives of this research were (1) to investigate the toxic effects PCA on SP535 by assessing biomass, cell morphology, and cellular ultras; (2) to evaluate the degradation efficiency of SP535 on PCA and elucidate the degradation mechanism by analyzing cytochrome P450 monooxygenase activity in free extracts and microsomal fractions; and (3) to examine the influence of cultural factors on PCA degradation by SP535. 2.Materials and methods 2.1 Chemicals and growth medium p-chloroaniline (PCA) (CAS# 106-47-8, purity > 99.5%), purchased from Sigma-Aldrich USA, was dissolved in sterilized deionized water to obtain a stock solution of 10 mM. The other chemicals and reagents, including K 2 HPO 4 , KH 2 PO 4 , NH 4 Cl, MgSO 4 .7H 2 O, MnSO 4 , and glucose, were supplied by Tianjin Kemiou Chemical Reagent Co., Ltd., China. The mineral growth medium was prepared with some modifications according to the methods reported by Różalska et al. ( 2010 ). Specifically, its composition was as follows (g/L): K 2 HPO 4 (4.36), KH 2 PO 4 (1.7), NH 4 Cl (2.1), MgSO 4 ·7H 2 O (0.2), MnSO 4 (0.05), FeSO 4 ·7H 2 O (0.01), CaCl 2 ·2H 2 O (0.03), glucose (20) and dH 2 O (up to 1000 mL). Integer 25.0 mL was added to each 100 mL Erlenmeyer flask followed by sterilization at 121 ℃ for 20 min. 2.2 Preparation of Fungal conidia inoculum Fungus, Isaria fumosorosea strain SP535, originally isolated from soil and deposited to the collection at Key Laboratory of Biopesticides Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou, P. R. China, was used during these studies. SP535 was incubated on PDA slants for 14 d at 25 ℃ to allow sufficient spore production. Then the fungal conidia cultured on PDA slants were harvested with sterilized deionized water containing 0.01% Tween 80 and sieved using filter paper (Whatman No. 2; Science Kit & Boreal Laboratories, New York, NY, USA) into sterile vials. Conidia were counted using a compound microscope and a hemocytometer (0.0625 m 2 ; Fuchs-Rosenthal Merck Euro Lab, Darmstadt, Germany) to calibrate a suspension of 1×10 8 conidia mL –1 . 2.3 Determination of the degradation capacity of SP535 on PCA To determine the degradation capacity of SP535 to PCA, the growth medium prepared as per section 2.1 was added into 100 mL conical bottle, and the PCA stock solution was added to make the final PCA concentration reach 1.0 mM, the pH was adjusted to 7.0, 2 mL fungal conidia inoculum prepared as per section 2.2 was added. Then the conical bottle was cultured in the constant temperature shaker at 180 rpm and 25 ℃ for 120 h. PCA concentration was measured with 2 mL samples taken every 24 h. The culture samples (2.0 mL) were taken from each treatment at 24 h interval for PCA quantification. 2.4 Optimization studies of isolate SP535 Three factors affecting PCA degradation were studied, including initial PCA concentration (0.5, 1.0, 2.0 mM), initial pH (3, 5, 7, 9, 11) and culture temperature (15, 25, 35, 45, 55 ℃). Unless otherwise stated, the above experiments were performed at an initial pH of 7.0, an initial PCA concentration of 1.0 mM, and a culture temperature of 25 ℃. All cultures were incubated in a thermostatic oscillator for 120 h. PCA concentrations were measured by sampling 2.0 mL solution every 24 h to investigate the influence of culture conditions on the degradation of PCA by SP535. 2.5 Estimation of biomass To investigate the effects of PCA on SP535 growth, fungal conidia inoculum was inoculated in the medium without PCA and the medium with PCA of 1.0 mM, and cultured at 180 rpm and 25 ℃ in a constant temperature shaker for 120 h. Culture samples (2.0 mL) were taken from each treatment every 24 h to measure the biomass of strain SP535. Total biomass produced by strain SP535 was quantified by following Ali et al. ( 2009 ) with some modifications. The whole cultures were filtered through Whatman filter papers (No.1), which were dried at 80 ℃ until constant weight and values were expressed as g/L. 2.6 Determination of cytochrome P450 monooxygenase Mycelia obtained after 10 d of culture were converted to spheroplasts by following the method used by Ali et al. ( 2014 ). Briefly, 50 mg of mycelia were washed and concentrated 10-fold in 1.0 M sorbitol. The cells were briefly suspended in a mixture solution, including 1.4 M sorbitol, 40 mM HEPES (pH 7.5), 0.5 mM MgCl 2 and a trace of β-mercaptoetahnol. The suspension was shaken for 15 min at 20 ℃ and then 5 mg/mL lyticase was added to lyse the cells. This suspension was shaken for 45 min at 20 ℃ and then the samples were checked microscopically for the presence of spheroplasts. The spheroplasts were separated from the suspension by centrifugation at 10,000 rpm for 15 min at 4 ℃. The spheroplasts from different treatments were suspended in 200 mL fractionation medium with pH at 7.4 (20 mM Tris, 20 mM KH 2 PO 4 , 0.33 M sucrose,1 mM EDTA and 0.2% bouvine serum albumin). The suspension was homogenized to prevent the aggregation of sub-cellular particles (Kovác et al. 1968; Mauersberger et al. 1980 ). The spheroplast lysate was diluted up to 350 mL with fractionation medium and pH was adjusted to 7.4. This homogenate was then fractionated by differential centrifugation. Intact spheroplast, nuclei and large debris were removed by centrifugation at 10,000 rpm for 10 min. The pellet was homogenized for 1 min, diluted and centrifuged as above. The supernatant obtained after centrifugation will be referred as cell free extract. The remaining pellet was centrifuged at 20,000 rpm for 30 min; the supernatant was carefully discarded leaving the mitochondrial peroxisomal fraction. The pellets were re-suspended in fractionation medium and centrifuged at 20,000 rpm for 20 min. The supernatant was then decanted leaving the post-mitochondrial pellet and centrifuged at 20,000 rpm for 60 min after re-suspension in fractionation medium. This centrifugation step resulted in microsomal pellet which was re-suspended in fractionation buffer. The concentration of functional cytochrome P450 monooxygenase was determined by CO difference spectra (Estabrook et al. 1978). Samples of microsomes containing 1.5 to 2 mg of protein in 1.0 mL of 50 mM Tris-HCl, pH 8.0, in a stopper cuvette were gently sparged with CO for 1 to 2 min, at which time several fine grains of solid sodium dithionite were added. Sparging was continued for 1 to 2 min more, and the cuvette was stoppered. Spectra (400 to 500 nm) were recorded at 20 ℃ using an extinction coefficient of 91 mm –1 ·cm –1 . The sample was scanned repeatedly; the maximum development of the difference spectrum occurred 5 to 10 min after addition of the sodium dithionite and was recorded. 2.7 Sample preparation for scanning electron microscopy and transmission electron microscopy To investigate the effects of PCA on the morphology and cell structure of SP535, SEM and TEM were performed on SP535 cultured for 120 h according to section 2.3 . The culture solution was centrifuged at 5,000 rpm for 20 min and supernatant was removed and the fungal mycelia were left. The fungal mycelia were washed thrice with 0.1 M PBS (pH 7.2). The material was then fixed with 2.5% glutaraldehyde in PBS buffer for 3 h at 4 ℃ then rinsed twice with PBS for 10 min each time followed by rinsing with ddH2O. The samples were then placed on glass cover slip (5*5 mm) and freeze dried in refrigerator at − 80 ℃ for 3 h followed by overnight drying at 4 ℃. The samples were then gold sprayed. The surface morphology of mycelia grown in the presence or absence of PCA was observed with SU8010 (Hitachi) scanning electron microscope (SEM) being operated at accelerated voltage of 5.0 kV. For transmission electron microscope (TEM), the fungal material, fixed with 2.5% glutaraldehyde in PBS buffer for 3 h at 4 ℃ as above, was rinsed with same buffer and post-fixed in 1% osmium tetraoxide for 2 h. After dehydration in graded ethanol series, then propylene oxide, the samples were gradually infiltrate and finally embedded in an Epon-spurr’s resin mixture. Ultrathin section, prepared using Leica CM1950 microtome, were stained with uranyl acetate and lead citrate (Reynolds 1963 ; Różalska et al. 2014 ) and examined in a JEM 1400 TEM (Hitachi, Japan) at 80 kV. 2.8 Chemical analysis Samples obtained in section 2.4 were centrifuged at 10,000 rpm for 10 min and supernatant was used for PCA quantification. PCA concentration in supernatant was measured by HPLC following Hussain et al. ( 2012 ) with some modifications. HPLC (Shimazdu LC-20) equipped with a UV detector at 254 nm and a reverse phase C18 column (250 mm × 4.6 mm) was used. The temperature of column was maintained at 40 ℃, the mobile phase was methanol-water (60:40, v/v) with a flow efficiency of 0.5 mL/min. A range of different concentrations of PCA standard were run along with the samples and PCA concentrations were quantified by PCA standard curve. 3.Results and discussion 3.1 Effect of PCA on the growth and cellular structure of SP535 Figure 1 showed the influence of PCA on the growth of strain SP535. During the first 72 h, the biomass of SP535 in the media containing PCA was lower than that in the media without PCA, with values of 6.8 g/L and 6.5 g/L respectively. However, at 96 h, the biomass of SP535 in the media containing PCA surpassed that in the media without PCA, with values of 7.6 g/L and 7.1 g/L, respectively. After 120 h, the biomass of SP535 in the two media reached 7.4 g/L (without PCA) and 8.9 g/L (with PCA), respectively. This observation suggested that SP535 may utilize PCA as a carbon source to enhance its growth in the later stages of culture. Figure 2 and Fig. 3 showed the effect of PCA on hyphal morphology and cell ultrastructure of SP535 during biodegradation. In batch culture without PCA, the outer surface morphology of SP535 was smooth and clean (Fig. 2 a ~ d), while the surface of fungal mycelia growing in the presence of PCA was rough and the deposition of PCA on the outer surface of mycelia was clear (Fig. 2 e ~ h). At the same time, the fungal mycelia in PCA treated samples had obvious space in the cytoplasm and the cell wall was thickened (Fig. 3 ). The toxicity of environmental pollutants usually leads to changes in the cellular structure of microorganisms. The toxicity of alkyl phenols caused similar morphological changes in the cells of Espergillus tubingensis , with tight mycelium arrangement resulting in rough surface and some oval-shaped vacuoles in the cytoplasm (Kuzikova et al. 2020 ). Similarly, when exposed to tributyltin, the protoplasts of fungal cells are destroyed and some gaps appear between the cell membrane and the cell wall (Soboń et al. 2018 ). Phanerochaete chrysosporium exposed to 100 mg·L –1 PFOS (perfluorooctane sulfonate) resulted in the formation of a large number of intracellular cavities, which was attributed to the fact that PFOS can bind to membrane phospholipids, thus preventing the process of membrane synthesis and leading to the formation of cavities (Qiao et al. 2019 ). The increase in fungal cell wall thickness may be a response to adverse environmental conditions. Wallemia can adapt to high salinity environments by significantly increasing cell wall thickness (Kralj et al. 2010). Cell wall thickness increased significantly in Metarhizium robertsii cultured in the presence of nonylphenol (Różalska et al. 2014 ). In this study, the toxicity of PCA caused the mycelium surface of SP535 to become rough, the cytoplasm appeared obvious gaps, and the cell wall thickened. 3.2 The biodegradation of PCA by SP353 Control treatment without Fungal conidia inoculum was performed in all experiments, and the loss rate of PCA was lower than 2%. Figure 4 showed the biodegradation of PCA by SP535. Following the addition of fungal conidia inoculum, concentration of PCA gradually diminished from the initial 1.0 mM to 0 over 120 h, indicating that strain SP535 possesses a robust degradation capacity for PCA. Over the past few decades, several PCA-degrading bacteria have been isolated and utilized in studies focused on PCA contamination remediation. For instance, a novel strain, Delftia tsuruhatensis H1, isolated by Zhang et al. ( 2010 ), demonstrated the ability to completely degrade PCA at a concentration of 400 mg·L –1 within 25 h. Acinetobacter baylyi strain GFJ2 has been employed for the degradation of aniline and halogenated aniline, achieving a 97% reduction of PCA at a concentration of 0.2 mM within 72 h (Hongsawat et al. 2011). Thauera sp. M9, isolated from contaminated soil by Kumar et al. ( 2020 ), exhibited a degradation efficiency of 100% for PCA at a concentration of 300 mg·L –1 in 30 h, alongside significant degradation capacity for 2-CA (2-Chloroaniline), 3-CA (3-Chloroaniline), and 3,4-DCA (3, 4-Dichloroaniline). Brevibacillus S-618, isolated from effluent by Li et al. ( 2020 ), accomplished complete degradation of PCA at a concentration of 180 mg·L –1 at a temperature of 30 ℃, pH 7, and an air-water ratio of 0.3 m 3 /m 3 ·min within 72 h. Bacillus sp., isolated by Carolin et al. ( 2021 ), was able to completely degrade PCA at an initial concentration of 100 mg·L –1 within 72 h in the presence of a lipopeptide surfactant. However, fungi capable of degrading PCA have not been reported previously. In this study, Isaria fumosorosea strain SP535, a filamentous fungus isolated from soil, successfully degraded PCA at a concentration of 1.0 mM under pH 7.0 and a temperature of 25 ℃ within 120 h. To the best of our knowledge, this study represents the first instance of isolated PCA-degrading fungi, thereby enriching the biological resources available for PCA biodegradation. Table 1 Activities of cytochrome P450 monooxygenase in cell free extract and microsomal fraction of SP535 incubated for 120 h in the medium without PCA and containing PCA. Treatment Cell free extract Microsomal fraction Control 0.02 ± 0.00 b 0.03 ± 0.01 b PCA 0.31 ± 0.02 a 0.84 ± 0.04 a Data represents the mean (± S.E) of three independent replicates. The values are expressed as mean ± standard deviation. Cytochrome P450 enzymes were investigated in this study to elucidate the potential degradation mechanism of PCA by the isolated fungus. As heme monooxygenases that are widely present in filamentous fungi, cytochrome P450 enzymes were believed to be involved in the transformation processes of organic matter by these organisms (van Gorcom et al. 1998; Lah et al. 2008 ; Behrendorff et al. 2021). The role of the cytochrome P450 system in PCA biodegradation was assessed by measuring the cytochrome P450 monooxygenase activity in the free cell extracts and microsomal components derived from fungal mycelia cultivated in media with and without PCA. The activities of cytochrome P450 monooxygenase in the cell-free extract and microsomal fraction were enhanced by the addition of PCA, exhibiting concentrations of 0.02 and 0.03 nmol/mg protein in the control group, and 0.31 and 0.84 nmol/mg protein in the PCA treatment group, respectively (Table 1 ). This finding suggested that the cytochrome P450 system may contribute to PCA degradation by SP353. Similarly, Al-Hawash et al. ( 2018 ) reported that the expression of oxidation-related cytochrome P450 genes increased from 0.94-fold to 5.45-fold under n-hexadecane (HXD) conditions, indicating that HXD stimulates cytochrome P450 production. Furthermore, the expression of P450 in the HBCD (hexabromocyclododecane) degrading bacterium Rhodopseudomonas palustris increased fivefold after 12 h of treatment with HBCD at 35 ℃ (Li et al. 2021 ). Wu et al. ( 2018 ) documented that cytochrome P450 monooxygenase activity rose from 0 to nearly 10 U·mg –1 following the inoculation of Rhodopseudomonas marshes in soil contaminated with the herbicide butachlor, and the timing of EthB regulatory gene expression correlated with butachlor degradation, suggesting that butachlor induces EthB gene expression to synthesize cytochrome P450 monooxygenase, thereby facilitating the degradation of butachlor by Rhodopseudomonas marshes . 3.3 Factors affecting the degradation of PCA by SP353 3.3.1 Effects of initial PCA concentrations on biodegradation The effects of different initial concentrations of PCA on biodegradation were shown in Fig. 5 . The biodegradation of PCA by SP535 decreased with the increase of PCA concentration within the experimental concentration range. When the initial concentration of PCA was 0.5 mM (6.38 mg·L –1 ) and 1.0 mM (12.76 mg·L –1 ), the degradation efficiency of PCA by SP535 reached 100% within 120 h. When the initial concentration of PCA was further increased to 2.0 mM (25.51 mg·L –1 ), the degradation efficiency of PCA by SP535 decreased to 79%. This indicated that SP535 can effectively degrade PCA at concentration below 1.0 mM. Since the toxicity of PCA may inhibit the growth of degrading bacteria and even lead to the death of degrading bacteria, the concentration of PCA will affect the degradation effect of degrading bacteria on PCA. The PCA degrading bacteria Bacillus sp had a degradation efficiency of 100% for PCA with a concentration lower than 100 mg·L –1 , but the degradation efficiency of PCA dropped to less than 85% when the concentration of PCA increased to 150 mg·L –1 Carolin et al. ( 2021 ). After 30 h culture, when the concentration of PCA in the medium was 300 mg/L, the OD 600 of Thauera sp.M9 increased from 0.1 to 0.372 and the degradation efficiency was 100%, while when the concentration of PCA in the medium was increased to 400 mg·L –1 and 500 mg·L –1 , the OD 600 increased from 0.1 to 0.321 and 0.265, and the degradation efficiencies were 28% and 15%, respectively (Kumar et al. 2020 ). Similarly, Vangnai and Petchkroh ( 2007 ) reported that the growth of Acinetobacter baumannii CA2, Pseudomonas putida CA16 and Klebsiella sp. CA17 was almost completely inhibited when the concentration of PCA in the medium was increased from 0.2 mM to 1.6 mM. When PCA concentration increased from 180 mg·L –1 to 270 mg·L –1 , the growth of Brevibacillus S-618 was inhibited, the cell dry weight decreased from 1.6 g/L to 0.25 g/L, and the degradation efficiency of PCA decreased from 86.7–9% (Li et al. 2020 ). 3.3.2 Effects of pH on biodegradation The effects of different pH values on biodegradation were shown in Fig. 6 . As shown in Fig. 6 , the degradation efficiency of PCA by SP535 increased with the increase of pH in acidic environment, while the degradation efficiency of PCA by SP535 decreased with the increase of pH in alkaline environment. The optimal pH for PCA degradation by SP535 was 7, and the degradation efficiencies at 96 h and 120 h PCA were 73% and 100%, respectively. At initial pH values of 3 and 11, PCA degradation efficiencies were 32% and 42%, indicating that SP535 was more adapted to alkaline environment. pH affected microbial growth and the enzymatic activity of the catabolic system (Wu et al. 2019 ; He et al. 2020 ), thereby impacting the biodegradation of PCA. Brevibacillus S-618, isolated by Li et al. ( 2020 ), exhibited the highest PCA degradation efficiency of 86.3% at pH 7; however, its degradation efficiency decreased when the pH was either increased or decreased. Similarly, Carolin et al. ( 2021 ) found that Bacillus sp. demonstrated optimal PCA degradation at pH 7. Bacillus licheniformis strain ycsd02, isolated by Ding et al. ( 2011 ), could degrade 94% of 20 mg·L –1 4-CA after 100 h of culture at pH 7.0, with degradation efficiency dropping to 25% at pH 9.0. Acinetobacter baylyi strain GFJ2 can completely degrade 25 mg·L –1 PCA at pH 7 (Hongsawat and Vangnai 2011 ). Additionally, the optimal pH for the cytochrome P450 enzyme produced by Isaria fumosorosea ranges from 5.7 to 7.0, facilitating PCA biodegradation by SP535 (Ali et al. 2014 ). Furthermore, pH affects the bioavailability of certain pesticides, influencing their absorption and degradation by microorganisms. For example, the bioavailability of the piscicide TFM (3-trifluoromethyl-4-nitrophenol) increases at lower pH levels, making it more easily absorbed by organisms (Wilkie et al. 2021 ). The bioavailability of four neonicotinoids including imidacloprid, acetamiprid, clothianidin, and thiamethoxam, was positively correlated with soil pH, with higher bioavailability noted at elevated pH levels (Hua et al. 2023 ). 3.3.3 Effects of temperature on biodegradation The effects of SP535 on the biodegradation of PCA at different temperatures were shown in Fig. 7 . When the temperature increased from 15 ℃ to 25 ℃, the degradation efficiency of PCA by SP535 increased, while the degradation efficiency of PCA by SP535 decreased when the temperature increased from 25 ℃ to 55 ℃. When the temperature is 15 ℃, the degradation efficiency of PCA by SP535 is 4%, and SP535 hardly degrade PCA at low temperature. At the temperature of 25 ℃, SP535 achieved complete degradation of PCA within 120 h. With the further increase of temperature 35, 45 and 55 ℃, the degradation efficiencies of PCA by SP535 were 86%, 47% and 32%, respectively. The effects of temperature on PCA biodegradation in previous studies indicated an initial increase followed by a decrease, likely due to the inhibitory effects of both high and low temperatures on strain growth and enzyme activity. Brevibacillus S-618 showed the highest degradation efficiency of PCA (85.9%) at an incubation temperature of 30 ℃, and the dry cell weight of the strain was more than 1.5 g/L, whereas the growth of Brevibacillus S-618 was inhibited at incubation temperatures of 22 ℃ and 38 ℃, and the growth efficiency of Brevibacillus S 618 were both significantly lower than 0.75 g/L, resulting in PCA degradation efficiencies of less than 40% in both cases (Li et al. 2020 ). Ding et al. ( 2011 ) reported similar findings, indicating that the optimal degradation temperature for the Bacillus licheniformis strain ycsd02 was between 30–32 ℃, with rapid declines in degradation efficiency at temperatures above or below this range. Additionally, Deltia tsuruhatensis H1 was capable of completely degrading PCA at a concentration of 300 mg·L –1 at 30 ℃ (Zhang et al. 2010 ). The degradation efficiency of Thauera sp. M9 for 300 mg·L –1 PCA reached 99.29% at 30 ℃, while at 25 ℃, 35 ℃, 40 ℃, and 45 ℃, the efficiencies were only 30%, 22.58%, 2.92%, and 2.39%, respectively (Kumar et al. 2020 ). Furthermore, Wang et al. ( 2023 ) demonstrated that high temperatures (39 ℃) inhibited the expression of cytochrome P450 family 11 subfamilies A member 1, revealing the possibility that high temperature might inhibit the expression of cytochrome P450 and thus inhibit the biodegradation of PCA by SP535. 4. Conclusion This study reported an isolate of filamentous fungus Isaria fumosorosea SP535 with highly degrading ability of PCA for the first time. The optimal degradation parameters were: initial PCA concentration of 1.0 mM; initial pH of 7.0; and growth temperature of 25 ℃. The ability of SP535 to degrade PCA displays its potential use in the remediation of PCA contaminated sites. Fungal cells grown on PCA showed high cytochrome P450 enzymes activities suggesting that SP535 may metabolize PCA through the P450 system. Declarations Author Contributions Shicong Huang: Experimentation, Data analysis, Manuscript writing; Jiahui Gao, Lin Zhou, Liujian Gao: Experimental assistance; Mengke Song: Review and edit of manuscript, Corresponding author; Qiaoyun Zeng: Review and edit of manuscript, Corresponding author, funding acquisition. Funding This work was funded by the Science and Technology Planning Project of Guangzhou (202206010162), the Guangdong Basic and Applied Basic Research Foundation (2022A1515010703), National Natural Science Foundation of China (No. 42377209), Natural Science Foundation of Guangdong Province (No. 2022A1515010890). Data availability The data are available on request. 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Int Biodeterior Biodegrad 178: 105545. https://doi.org/10.1016/j.ibiod.2022.105545 Reynolds E S (1963) The use of lead citrate at high pH as an electron-opaque stain in electron microscopy . J Cell Biol 17(1): 208. https://doi.org/10.1083/jcb.17.1.208 Różalska S, Glińska S, Długoński J (2014) Metarhizium robertsii morphological flexibility during nonylphenol removal . Int Biodeterior Biodegrad 95: 285-293. https://doi.org/10.1016/j.ibiod.2014.08.002 Różalska S, Szewczyk R, Długoński J (2010) Biodegradation of 4-n-nonylphenol by the non-ligninolytic filamentous fungus Gliocephalotrichum simplex: a proposal of a metabolic pathway . J Hazard Mater 180: 323-331. https://doi.org/10.1016/j.jhazmat.2010.04.034 Sen SK, Raut S, Bandyopadhyay P et al (2016) Fungal decolouration and degradation of azo dyes: a review . Fungal Biol Rev 30: 112-133. https://doi.org/10.1016/j.fbr.2016.06.003 Silambarasan S, Vangnai AS (2016) Biodegradation of 4-nitroaniline by plant-growth promoting Acinetobacter sp. AVLB2 and toxicological analysis of its biodegradation metabolites . J Hazard Mater 302: 426-436. https://doi.org/10.1016/j.jhazmat.2015.10.010 Soboń A, Szewczyk R, Różalska S et al (2018) Metabolomics of the recovery of the filamentous fungus Cunninghamella echinulata exposed to tributyltin . Int Biodeterior Biodegrad 127: 130-138. https://doi.org/10.1016/j.ibiod.2017.11.008 Tan H, Kong D, Ma Q, et al (2022) Biodegradation of tetracycline antibiotics by the yeast strain Cutaneotrichosporon dermatis M503 . Microorganisms 10: 565. https://doi.org/10.3390/microorganisms10030565 Tongarun R, Luepromchai E, Vangnai A S (2008) Natural attenuation, biostimulation, and bioaugmentation in 4-chloroaniline-contaminated soil . Curr Microbiol 56: 182-188. https://doi.org/10.1007/s00284-007-9055-y van Gorcom RFM, van den Hondel C A, Punt P J (1988) Cytochrome P450 enzyme systems in fungi . Fungal Genet Biol 23: 1-17. https://doi.org/10.1006/fgbi.1997.1021 Vangnai A S, Petchkroh W (2007) Biodegradation of 4-chloroaniline by bacteria enriched from soil . FEMS Microbiol Lett 268: 209-216. https://doi.org/10.1111/j.1574-6968.2006.00579.x Vieira GAL, Magrini MJ, Bonugli-Santos RC, et al (2018) Polycyclic aromatic hydrocarbons degradation by marine-derived basidiomycetes: optimization of the degradation process . Braz J Microbiol 49: 749-756. https://doi.org/10.1016/j.bjm.2018.04.007 Wang K, Li Z, Li Y et al (2023) Impacts of elevated temperature on morphology, oxidative stress levels, and testosterone synthesis in ex vivo cultured porcine testicular tissue . Theriogenology 212: 181-188. https://doi.org/10.1016/j.theriogenology.2023.09.015 Wang X, Miao J, Pan L et al (2019) Toxicity effects of p-choroaniline on the growth, photosynthesis, respiration capacity and antioxidant enzyme activities of a diatom, Phaeodactylum tricornutu. Ecotox Environ Safe 169: 654-661. https://doi.org/10.1016/j.ecoenv.2018.11.015 Wilkie MP, Tessier LR, Boogaard M et al (2021) Lampricide bioavailability and toxicity to invasive sea lamprey and non-target fishes: the importance of alkalinity, pH, and the gill microenvironment . J Gt Lakes Res 47: S407-S420. https://doi.org/10.1016/j.jglr.2021.09.005 Wu P, Xie L, Li J et al (2018) The removal of butachlor from soil by wastewater‐derived Rhodopseudomonas marshes. Soil Use Manage 36: 153-156. https://doi.org/10.1111/sum.12538 Wu X, Wu X, Shen L et al (2019) Whole genome sequencing and comparative genomics analyses of Pandoraea sp. XY-2, a new species capable of biodegrade tetracycline . Front Microbiol 10: 33. https://doi.org/10.3389/fmicb.2019.00033 Xu J (2018) Studies on the Extraction, Characterization and toxicity of toxins produced by different isolates of entomopathogenic fungus Isaria fumosorosea . Dissertation, South China Agriculture University Zhang L, He D, Chen J et al (2010) Biodegradation of 2-chloroaniline, 3-chloroaniline, and 4-chloroaniline by a novel strain Delftia tsuruhatensis H1 . J Hazard Mater 179: 875-882. https://doi.org/10.1016/j.jhazmat.2010.03.086 Zhu L, Lv M, Dai X et al (2012) Reaction kinetics of the degradation of chloroanilines and aniline by aerobic granule . Biochem Eng J 68: 215-220. https://doi.org/10.1016/j.bej.2012.07.015 Additional Declarations No competing interests reported. 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(a) ~ (d) SEM images of SP535 incubated for 120 h in medium without PCA; (e) ~ (h) SEM images of SP535 incubated for 120 h in medium containing PCA.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/ce12d71404cae8dc0cea54a0.png"},{"id":63602894,"identity":"a0f902ee-6cc9-4f1c-9726-c367bc606c99","added_by":"auto","created_at":"2024-08-30 05:48:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1197525,"visible":true,"origin":"","legend":"\u003cp\u003eTEM images of SP535 after 120 h of incubation. (a) ~ (d) TEM images of SP535 incubated for 120 h in medium without PCA; (e) ~ (h) TEM images of SP535 incubated for 120 h in medium containing PCA.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/a8c7e4f6ff0cbe157bc97d81.png"},{"id":63602887,"identity":"55804a4d-40a1-4fc1-9598-b7bc54e4dec9","added_by":"auto","created_at":"2024-08-30 05:48:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94033,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of PCA in the medium after 120 h incubation with SP535 at 25 ℃, pH 7.0.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/9fbf211b241e10e70ca36ec2.png"},{"id":63602890,"identity":"2c02bd17-6d78-4278-b89d-5e5202811377","added_by":"auto","created_at":"2024-08-30 05:48:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":176556,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation efficiencies of PCA in the medium after incubating SP535 for 120 h incubation at 25 ℃ and pH 7.0 at different initial PCA concentrations\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/f3e25b51f3e1a70e0d11a885.png"},{"id":63602891,"identity":"e4caa751-37fd-4f83-9711-65b6958375a3","added_by":"auto","created_at":"2024-08-30 05:48:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":207112,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation efficiencies of PCA in the medium after incubating SP535 for 120h at PCA concentration of 1.0 mM and 25 ℃ at different pH.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/cd848f1ce84c7ab76fc0db40.png"},{"id":63603621,"identity":"6fc3230b-a13d-49a5-ab47-d6e427637bdc","added_by":"auto","created_at":"2024-08-30 05:56:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":198450,"visible":true,"origin":"","legend":"\u003cp\u003eDegradation efficiencies of PCA in the medium after incubating SP535 for 120 h at PCA concentration of 1.0 mM and pH 7.0 at different temperature.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/5273c7832f4f8bd3be2ab6d4.png"},{"id":67890090,"identity":"808308e7-6471-4e3b-8f30-59945305c2c3","added_by":"auto","created_at":"2024-10-30 19:46:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3819843,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4840476/v1/58ff9725-92a2-47c5-bf2d-80b98b9aef3d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Biodegradation of p-chloroaniline by fungus Isaria fumosorosea SP535","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePCA (p-chloroaniline), widely used as a chemical raw material in the production of dyes, cosmetics, pesticides, and herbicides, can enter the environment through wastewater and waste generated during chemical production processes (Khusnun et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Silambarasan et al. 2016; Dong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Reports indicated that PCA has been detected in various environments, including water bodies, sediments, agricultural soils, and living organisms (Tongarun et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Zhu et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Dantzger et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Due to its toxicity and adverse effects on human health, PCA was classified as a persistent organic pollutant and has been designated as a priority pollutant by the US Environmental Protection Agency (EPA) and European Union legislation (Nitisakulkan et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kumar al. 2020). Consequently, the development of effective methods for removing PCA residues from the environment has drawn great attention.\u003c/p\u003e \u003cp\u003eBioremediation relying on microorganisms is an efficient method to remove organic pollutants in environment (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Therefore, it is crucial to identify microorganisms with degradation capabilities from the environment. Numerous bacteria have been isolated from various environmental media for the remediation of PCA contamination. \u003cem\u003eBrevibacillus\u003c/em\u003e S-618, isolated from sludge, was able to completely degrade PCA at a concentration of 180 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e within 72 h (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eThauera\u003c/em\u003e sp. M9, isolated from soil, completely degraded PCA in culture medium within 30 h (Kumar et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e sp., isolated from textile wastewater, demonstrated a 76% degradation of PCA at a concentration of 100 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, attaining complete degradation in the presence of lipopeptide surfactant (Carolin et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). However, current studies have predominantly focused on bacteria, and PCA-degrading fungi have yet to be reported.\u003c/p\u003e \u003cp\u003eFungi have been demonstrated with the remarkable capacity of adapting to harsh environments and exhibiting a strong ability to degrade organic pollutants (Coleine et al. 2022; Sen et al. 2022). The degradation efficiency of \u003cem\u003eAspergillus\u003c/em\u003e LS-1, isolated from the sludge of a pharmaceutical plant, was 95.41% for 80.14 mg L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e CTC (chlortetracycline) within 3 d (He et al. 2023). A yeast strain, \u003cem\u003eCutaneotrichosporon dermatis\u003c/em\u003e M503, isolated from the sediment tank of a sewage treatment system at a tetracycline manufacturing facility, achieved a degradation efficiency of 86.62% for TC (tetracycline) within 7 d under optimal conditions (Tan et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eAspergillus sydowii\u003c/em\u003e W1, isolated from contaminated soil, demonstrated an 84.05% degradation efficiency for 100 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e erythromycin at a concentration within 7 d (Ren et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Eight lignin-degrading fungi being capable of degrading polychlorinated biphenyls (PCBs) have been reported, with \u003cem\u003ePleurotus ostreatus\u003c/em\u003e removing 98.4% and 99.6% of a PCB mixture in complex and mineral media, respectively, after 6 weeks (Čvančarov\u0026aacute; et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Furthermore, some filamentous fungi have been shown to degrade environmental pollutants via cytochrome P450 monooxygenase (Črešnar et al. 2011). With the involvement of cytochrome P450, \u003cem\u003eMarasmiellus\u003c/em\u003e sp. CBMAI 1062 could nearly completely degrade pyrene (0.08 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) after 48 h under saline condition (Vieira et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The degradation efficiency of the herbicide diuron by \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e reached 94% after 10 d cultivation, while the degradation of diuron was inhibited by the addition of 1 mmol/L cytochrome P450 inhibitor ABT (1-aminobenzotriazole) (Coelho-Moreira et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Sun et al. (2022) confirmed that cytochrome P450 is involved in the biodegradation of dichlorvos by \u003cem\u003eTrichoderma atroviride\u003c/em\u003e T23, and identified five relevant genes including \u003cem\u003eTaCyp548\u003c/em\u003e, \u003cem\u003eTaCyp620\u003c/em\u003e, \u003cem\u003eTaCyp52\u003c/em\u003e, \u003cem\u003eTaCyp528\u003c/em\u003e, and \u003cem\u003eTaCyp504\u003c/em\u003e. Therefore, the isolation of fungi with high degradation efficiency for PCA is urgent and necessary for the remediation of PCA pollution.\u003c/p\u003e \u003cp\u003eIn this study, the filamentous fungus \u003cem\u003eIsaria fumosorosea\u003c/em\u003e strain SP535, isolated from soil (Xu \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), was selected for experiments on PCA degradation. The objectives of this research were (1) to investigate the toxic effects PCA on SP535 by assessing biomass, cell morphology, and cellular ultras; (2) to evaluate the degradation efficiency of SP535 on PCA and elucidate the degradation mechanism by analyzing cytochrome P450 monooxygenase activity in free extracts and microsomal fractions; and (3) to examine the influence of cultural factors on PCA degradation by SP535.\u003c/p\u003e"},{"header":"2.Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and growth medium\u003c/h2\u003e \u003cp\u003ep-chloroaniline (PCA) (CAS# 106-47-8, purity\u0026thinsp;\u0026gt;\u0026thinsp;99.5%), purchased from Sigma-Aldrich USA, was dissolved in sterilized deionized water to obtain a stock solution of 10 mM. The other chemicals and reagents, including K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, NH\u003csub\u003e4\u003c/sub\u003eCl, MgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, MnSO\u003csub\u003e4\u003c/sub\u003e, and glucose, were supplied by Tianjin Kemiou Chemical Reagent Co., Ltd., China.\u003c/p\u003e \u003cp\u003eThe mineral growth medium was prepared with some modifications according to the methods reported by R\u0026oacute;żalska et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Specifically, its composition was as follows (g/L): K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e (4.36), KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e (1.7), NH\u003csub\u003e4\u003c/sub\u003eCl (2.1), MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (0.2), MnSO\u003csub\u003e4\u003c/sub\u003e (0.05), FeSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO (0.01), CaCl\u003csub\u003e2\u003c/sub\u003e\u0026middot;2H\u003csub\u003e2\u003c/sub\u003eO (0.03), glucose (20) and dH\u003csub\u003e2\u003c/sub\u003eO (up to 1000 mL). Integer 25.0 mL was added to each 100 mL Erlenmeyer flask followed by sterilization at 121 ℃ for 20 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation of Fungal conidia inoculum\u003c/h2\u003e \u003cp\u003eFungus, \u003cem\u003eIsaria fumosorosea\u003c/em\u003e strain SP535, originally isolated from soil and deposited to the collection at Key Laboratory of Biopesticides Innovation and Application of Guangdong Province, South China Agricultural University, Guangzhou, P. R. China, was used during these studies. SP535 was incubated on PDA slants for 14 d at 25 ℃ to allow sufficient spore production. Then the fungal conidia cultured on PDA slants were harvested with sterilized deionized water containing 0.01% Tween 80 and sieved using filter paper (Whatman No. 2; Science Kit \u0026amp; Boreal Laboratories, New York, NY, USA) into sterile vials. Conidia were counted using a compound microscope and a hemocytometer (0.0625 m\u003csup\u003e2\u003c/sup\u003e; Fuchs-Rosenthal Merck Euro Lab, Darmstadt, Germany) to calibrate a suspension of 1\u0026times;10\u003csup\u003e8\u003c/sup\u003e conidia mL\u003csup\u003e\u0026ndash;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Determination of the degradation capacity of SP535 on PCA\u003c/h2\u003e \u003cp\u003eTo determine the degradation capacity of SP535 to PCA, the growth medium prepared as per section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e2.1\u003c/span\u003e was added into 100 mL conical bottle, and the PCA stock solution was added to make the final PCA concentration reach 1.0 mM, the pH was adjusted to 7.0, 2 mL fungal conidia inoculum prepared as per section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e was added. Then the conical bottle was cultured in the constant temperature shaker at 180 rpm and 25 ℃ for 120 h. PCA concentration was measured with 2 mL samples taken every 24 h. The culture samples (2.0 mL) were taken from each treatment at 24 h interval for PCA quantification.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Optimization studies of isolate SP535\u003c/h2\u003e \u003cp\u003eThree factors affecting PCA degradation were studied, including initial PCA concentration (0.5, 1.0, 2.0 mM), initial pH (3, 5, 7, 9, 11) and culture temperature (15, 25, 35, 45, 55 ℃). Unless otherwise stated, the above experiments were performed at an initial pH of 7.0, an initial PCA concentration of 1.0 mM, and a culture temperature of 25 ℃. All cultures were incubated in a thermostatic oscillator for 120 h. PCA concentrations were measured by sampling 2.0 mL solution every 24 h to investigate the influence of culture conditions on the degradation of PCA by SP535.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Estimation of biomass\u003c/h2\u003e \u003cp\u003eTo investigate the effects of PCA on SP535 growth, fungal conidia inoculum was inoculated in the medium without PCA and the medium with PCA of 1.0 mM, and cultured at 180 rpm and 25 ℃ in a constant temperature shaker for 120 h. Culture samples (2.0 mL) were taken from each treatment every 24 h to measure the biomass of strain SP535. Total biomass produced by strain SP535 was quantified by following Ali et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) with some modifications. The whole cultures were filtered through Whatman filter papers (No.1), which were dried at 80 ℃ until constant weight and values were expressed as g/L.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Determination of cytochrome P450 monooxygenase\u003c/h2\u003e \u003cp\u003eMycelia obtained after 10 d of culture were converted to spheroplasts by following the method used by Ali et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Briefly, 50 mg of mycelia were washed and concentrated 10-fold in 1.0 M sorbitol. The cells were briefly suspended in a mixture solution, including 1.4 M sorbitol, 40 mM HEPES (pH 7.5), 0.5 mM MgCl\u003csub\u003e2\u003c/sub\u003e and a trace of β-mercaptoetahnol. The suspension was shaken for 15 min at 20 ℃ and then 5 mg/mL lyticase was added to lyse the cells. This suspension was shaken for 45 min at 20 ℃ and then the samples were checked microscopically for the presence of spheroplasts. The spheroplasts were separated from the suspension by centrifugation at 10,000 rpm for 15 min at 4 ℃. The spheroplasts from different treatments were suspended in 200 mL fractionation medium with pH at 7.4 (20 mM Tris, 20 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 0.33 M sucrose,1 mM EDTA and 0.2% bouvine serum albumin). The suspension was homogenized to prevent the aggregation of sub-cellular particles (Kov\u0026aacute;c et al. 1968; Mauersberger et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1980\u003c/span\u003e). The spheroplast lysate was diluted up to 350 mL with fractionation medium and pH was adjusted to 7.4. This homogenate was then fractionated by differential centrifugation. Intact spheroplast, nuclei and large debris were removed by centrifugation at 10,000 rpm for 10 min. The pellet was homogenized for 1 min, diluted and centrifuged as above. The supernatant obtained after centrifugation will be referred as cell free extract.\u003c/p\u003e \u003cp\u003eThe remaining pellet was centrifuged at 20,000 rpm for 30 min; the supernatant was carefully discarded leaving the mitochondrial peroxisomal fraction. The pellets were re-suspended in fractionation medium and centrifuged at 20,000 rpm for 20 min. The supernatant was then decanted leaving the post-mitochondrial pellet and centrifuged at 20,000 rpm for 60 min after re-suspension in fractionation medium. This centrifugation step resulted in microsomal pellet which was re-suspended in fractionation buffer.\u003c/p\u003e \u003cp\u003eThe concentration of functional cytochrome P450 monooxygenase was determined by CO difference spectra (Estabrook et al. 1978). Samples of microsomes containing 1.5 to 2 mg of protein in 1.0 mL of 50 mM Tris-HCl, pH 8.0, in a stopper cuvette were gently sparged with CO for 1 to 2 min, at which time several fine grains of solid sodium dithionite were added. Sparging was continued for 1 to 2 min more, and the cuvette was stoppered. Spectra (400 to 500 nm) were recorded at 20 ℃ using an extinction coefficient of 91 mm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e\u0026middot;cm\u003csup\u003e\u0026ndash;1\u003c/sup\u003e. The sample was scanned repeatedly; the maximum development of the difference spectrum occurred 5 to 10 min after addition of the sodium dithionite and was recorded.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Sample preparation for scanning electron microscopy and transmission electron microscopy\u003c/h2\u003e \u003cp\u003eTo investigate the effects of PCA on the morphology and cell structure of SP535, SEM and TEM were performed on SP535 cultured for 120 h according to section \u003cspan refid=\"Sec5\" class=\"InternalRef\"\u003e2.3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe culture solution was centrifuged at 5,000 rpm for 20 min and supernatant was removed and the fungal mycelia were left. The fungal mycelia were washed thrice with 0.1 M PBS (pH 7.2). The material was then fixed with 2.5% glutaraldehyde in PBS buffer for 3 h at 4 ℃ then rinsed twice with PBS for 10 min each time followed by rinsing with ddH2O. The samples were then placed on glass cover slip (5*5 mm) and freeze dried in refrigerator at \u0026minus;\u0026thinsp;80 ℃ for 3 h followed by overnight drying at 4 ℃. The samples were then gold sprayed. The surface morphology of mycelia grown in the presence or absence of PCA was observed with SU8010 (Hitachi) scanning electron microscope (SEM) being operated at accelerated voltage of 5.0 kV.\u003c/p\u003e \u003cp\u003eFor transmission electron microscope (TEM), the fungal material, fixed with 2.5% glutaraldehyde in PBS buffer for 3 h at 4 ℃ as above, was rinsed with same buffer and post-fixed in 1% osmium tetraoxide for 2 h. After dehydration in graded ethanol series, then propylene oxide, the samples were gradually infiltrate and finally embedded in an Epon-spurr\u0026rsquo;s resin mixture. Ultrathin section, prepared using Leica CM1950 microtome, were stained with uranyl acetate and lead citrate (Reynolds \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1963\u003c/span\u003e; R\u0026oacute;żalska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and examined in a JEM 1400 TEM (Hitachi, Japan) at 80 kV.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Chemical analysis\u003c/h2\u003e \u003cp\u003eSamples obtained in section \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e were centrifuged at 10,000 rpm for 10 min and supernatant was used for PCA quantification. PCA concentration in supernatant was measured by HPLC following Hussain et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) with some modifications. HPLC (Shimazdu LC-20) equipped with a UV detector at 254 nm and a reverse phase C18 column (250 mm \u0026times; 4.6 mm) was used. The temperature of column was maintained at 40 ℃, the mobile phase was methanol-water (60:40, v/v) with a flow efficiency of 0.5 mL/min. A range of different concentrations of PCA standard were run along with the samples and PCA concentrations were quantified by PCA standard curve.\u003c/p\u003e \u003c/div\u003e"},{"header":"3.Results and discussion","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of PCA on the growth and cellular structure of SP535\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e showed the influence of PCA on the growth of strain SP535. During the first 72 h, the biomass of SP535 in the media containing PCA was lower than that in the media without PCA, with values of 6.8 g/L and 6.5 g/L respectively. However, at 96 h, the biomass of SP535 in the media containing PCA surpassed that in the media without PCA, with values of 7.6 g/L and 7.1 g/L, respectively. After 120 h, the biomass of SP535 in the two media reached 7.4 g/L (without PCA) and 8.9 g/L (with PCA), respectively. This observation suggested that SP535 may utilize PCA as a carbon source to enhance its growth in the later stages of culture.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e showed the effect of PCA on hyphal morphology and cell ultrastructure of SP535 during biodegradation. In batch culture without PCA, the outer surface morphology of SP535 was smooth and clean (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea\u0026thinsp;~\u0026thinsp;d), while the surface of fungal mycelia growing in the presence of PCA was rough and the deposition of PCA on the outer surface of mycelia was clear (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee\u0026thinsp;~\u0026thinsp;h). At the same time, the fungal mycelia in PCA treated samples had obvious space in the cytoplasm and the cell wall was thickened (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe toxicity of environmental pollutants usually leads to changes in the cellular structure of microorganisms. The toxicity of alkyl phenols caused similar morphological changes in the cells of \u003cem\u003eEspergillus tubingensis\u003c/em\u003e, with tight mycelium arrangement resulting in rough surface and some oval-shaped vacuoles in the cytoplasm (Kuzikova et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, when exposed to tributyltin, the protoplasts of fungal cells are destroyed and some gaps appear between the cell membrane and the cell wall (Soboń et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). \u003cem\u003ePhanerochaete chrysosporium\u003c/em\u003e exposed to 100 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e PFOS (perfluorooctane sulfonate) resulted in the formation of a large number of intracellular cavities, which was attributed to the fact that PFOS can bind to membrane phospholipids, thus preventing the process of membrane synthesis and leading to the formation of cavities (Qiao et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The increase in fungal cell wall thickness may be a response to adverse environmental conditions. \u003cem\u003eWallemia\u003c/em\u003e can adapt to high salinity environments by significantly increasing cell wall thickness (Kralj et al. 2010). Cell wall thickness increased significantly in \u003cem\u003eMetarhizium robertsii\u003c/em\u003e cultured in the presence of nonylphenol (R\u0026oacute;żalska et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In this study, the toxicity of PCA caused the mycelium surface of SP535 to become rough, the cytoplasm appeared obvious gaps, and the cell wall thickened.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 The biodegradation of PCA by SP353\u003c/h2\u003e \u003cp\u003eControl treatment without Fungal conidia inoculum was performed in all experiments, and the loss rate of PCA was lower than 2%. Figure\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e showed the biodegradation of PCA by SP535. Following the addition of fungal conidia inoculum, concentration of PCA gradually diminished from the initial 1.0 mM to 0 over 120 h, indicating that strain SP535 possesses a robust degradation capacity for PCA.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOver the past few decades, several PCA-degrading bacteria have been isolated and utilized in studies focused on PCA contamination remediation. For instance, a novel strain, \u003cem\u003eDelftia tsuruhatensis\u003c/em\u003e H1, isolated by Zhang et al. (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), demonstrated the ability to completely degrade PCA at a concentration of 400 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e within 25 h. \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e strain GFJ2 has been employed for the degradation of aniline and halogenated aniline, achieving a 97% reduction of PCA at a concentration of 0.2 mM within 72 h (Hongsawat et al. 2011). \u003cem\u003eThauera\u003c/em\u003e sp. M9, isolated from contaminated soil by Kumar et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), exhibited a degradation efficiency of 100% for PCA at a concentration of 300 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in 30 h, alongside significant degradation capacity for 2-CA (2-Chloroaniline), 3-CA (3-Chloroaniline), and 3,4-DCA (3, 4-Dichloroaniline). \u003cem\u003eBrevibacillus\u003c/em\u003e S-618, isolated from effluent by Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), accomplished complete degradation of PCA at a concentration of 180 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e at a temperature of 30 ℃, pH 7, and an air-water ratio of 0.3 m\u003csup\u003e3\u003c/sup\u003e/m\u003csup\u003e3\u003c/sup\u003e\u0026middot;min within 72 h. \u003cem\u003eBacillus\u003c/em\u003e sp., isolated by Carolin et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), was able to completely degrade PCA at an initial concentration of 100 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e within 72 h in the presence of a lipopeptide surfactant. However, fungi capable of degrading PCA have not been reported previously. In this study, \u003cem\u003eIsaria fumosorosea\u003c/em\u003e strain SP535, a filamentous fungus isolated from soil, successfully degraded PCA at a concentration of 1.0 mM under pH 7.0 and a temperature of 25 ℃ within 120 h. To the best of our knowledge, this study represents the first instance of isolated PCA-degrading fungi, thereby enriching the biological resources available for PCA biodegradation.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eActivities of cytochrome P450 monooxygenase in cell free extract and microsomal fraction of SP535 incubated for 120 h in the medium without PCA and containing PCA.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCell free extract\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMicrosomal fraction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00 b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePCA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eData represents the mean (\u0026plusmn;\u0026thinsp;S.E) of three independent replicates. The values are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003cp\u003eCytochrome P450 enzymes were investigated in this study to elucidate the potential degradation mechanism of PCA by the isolated fungus. As heme monooxygenases that are widely present in filamentous fungi, cytochrome P450 enzymes were believed to be involved in the transformation processes of organic matter by these organisms (van Gorcom et al. 1998; Lah et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Behrendorff et al. 2021). The role of the cytochrome P450 system in PCA biodegradation was assessed by measuring the cytochrome P450 monooxygenase activity in the free cell extracts and microsomal components derived from fungal mycelia cultivated in media with and without PCA. The activities of cytochrome P450 monooxygenase in the cell-free extract and microsomal fraction were enhanced by the addition of PCA, exhibiting concentrations of 0.02 and 0.03 nmol/mg protein in the control group, and 0.31 and 0.84 nmol/mg protein in the PCA treatment group, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This finding suggested that the cytochrome P450 system may contribute to PCA degradation by SP353. Similarly, Al-Hawash et al. (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported that the expression of oxidation-related cytochrome P450 genes increased from 0.94-fold to 5.45-fold under n-hexadecane (HXD) conditions, indicating that HXD stimulates cytochrome P450 production. Furthermore, the expression of P450 in the HBCD (hexabromocyclododecane) degrading bacterium \u003cem\u003eRhodopseudomonas palustris\u003c/em\u003e increased fivefold after 12 h of treatment with HBCD at 35 ℃ (Li et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Wu et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) documented that cytochrome P450 monooxygenase activity rose from 0 to nearly 10 U\u0026middot;mg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e following the inoculation of \u003cem\u003eRhodopseudomonas marshes\u003c/em\u003e in soil contaminated with the herbicide butachlor, and the timing of \u003cem\u003eEthB\u003c/em\u003e regulatory gene expression correlated with butachlor degradation, suggesting that butachlor induces \u003cem\u003eEthB\u003c/em\u003e gene expression to synthesize cytochrome P450 monooxygenase, thereby facilitating the degradation of butachlor by \u003cem\u003eRhodopseudomonas marshes\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Factors affecting the degradation of PCA by SP353\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Effects of initial PCA concentrations on biodegradation\u003c/h2\u003e \u003cp\u003eThe effects of different initial concentrations of PCA on biodegradation were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. The biodegradation of PCA by SP535 decreased with the increase of PCA concentration within the experimental concentration range. When the initial concentration of PCA was 0.5 mM (6.38 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) and 1.0 mM (12.76 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), the degradation efficiency of PCA by SP535 reached 100% within 120 h. When the initial concentration of PCA was further increased to 2.0 mM (25.51 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), the degradation efficiency of PCA by SP535 decreased to 79%. This indicated that SP535 can effectively degrade PCA at concentration below 1.0 mM.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince the toxicity of PCA may inhibit the growth of degrading bacteria and even lead to the death of degrading bacteria, the concentration of PCA will affect the degradation effect of degrading bacteria on PCA. The PCA degrading bacteria \u003cem\u003eBacillus\u003c/em\u003e sp had a degradation efficiency of 100% for PCA with a concentration lower than 100 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, but the degradation efficiency of PCA dropped to less than 85% when the concentration of PCA increased to 150 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e Carolin et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). After 30 h culture, when the concentration of PCA in the medium was 300 mg/L, the OD\u003csub\u003e600\u003c/sub\u003e of \u003cem\u003eThauera\u003c/em\u003e sp.M9 increased from 0.1 to 0.372 and the degradation efficiency was 100%, while when the concentration of PCA in the medium was increased to 400 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 500 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, the OD\u003csub\u003e600\u003c/sub\u003e increased from 0.1 to 0.321 and 0.265, and the degradation efficiencies were 28% and 15%, respectively (Kumar et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Similarly, Vangnai and Petchkroh (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) reported that the growth of \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e CA2, \u003cem\u003ePseudomonas putida\u003c/em\u003e CA16 and \u003cem\u003eKlebsiella\u003c/em\u003e sp. CA17 was almost completely inhibited when the concentration of PCA in the medium was increased from 0.2 mM to 1.6 mM. When PCA concentration increased from 180 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e to 270 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, the growth of \u003cem\u003eBrevibacillus\u003c/em\u003e S-618 was inhibited, the cell dry weight decreased from 1.6 g/L to 0.25 g/L, and the degradation efficiency of PCA decreased from 86.7\u0026ndash;9% (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Effects of pH on biodegradation\u003c/h2\u003e \u003cp\u003eThe effects of different pH values on biodegradation were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, the degradation efficiency of PCA by SP535 increased with the increase of pH in acidic environment, while the degradation efficiency of PCA by SP535 decreased with the increase of pH in alkaline environment. The optimal pH for PCA degradation by SP535 was 7, and the degradation efficiencies at 96 h and 120 h PCA were 73% and 100%, respectively. At initial pH values of 3 and 11, PCA degradation efficiencies were 32% and 42%, indicating that SP535 was more adapted to alkaline environment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003epH affected microbial growth and the enzymatic activity of the catabolic system (Wu et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; He et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), thereby impacting the biodegradation of PCA. \u003cem\u003eBrevibacillus\u003c/em\u003e S-618, isolated by Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), exhibited the highest PCA degradation efficiency of 86.3% at pH 7; however, its degradation efficiency decreased when the pH was either increased or decreased. Similarly, Carolin et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that \u003cem\u003eBacillus\u003c/em\u003e sp. demonstrated optimal PCA degradation at pH 7. \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain ycsd02, isolated by Ding et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), could degrade 94% of 20 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e 4-CA after 100 h of culture at pH 7.0, with degradation efficiency dropping to 25% at pH 9.0. \u003cem\u003eAcinetobacter baylyi\u003c/em\u003e strain GFJ2 can completely degrade 25 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e PCA at pH 7 (Hongsawat and Vangnai \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Additionally, the optimal pH for the cytochrome P450 enzyme produced by \u003cem\u003eIsaria fumosorosea\u003c/em\u003e ranges from 5.7 to 7.0, facilitating PCA biodegradation by SP535 (Ali et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, pH affects the bioavailability of certain pesticides, influencing their absorption and degradation by microorganisms. For example, the bioavailability of the piscicide TFM (3-trifluoromethyl-4-nitrophenol) increases at lower pH levels, making it more easily absorbed by organisms (Wilkie et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The bioavailability of four neonicotinoids including imidacloprid, acetamiprid, clothianidin, and thiamethoxam, was positively correlated with soil pH, with higher bioavailability noted at elevated pH levels (Hua et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Effects of temperature on biodegradation\u003c/h2\u003e \u003cp\u003eThe effects of SP535 on the biodegradation of PCA at different temperatures were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e. When the temperature increased from 15 ℃ to 25 ℃, the degradation efficiency of PCA by SP535 increased, while the degradation efficiency of PCA by SP535 decreased when the temperature increased from 25 ℃ to 55 ℃. When the temperature is 15 ℃, the degradation efficiency of PCA by SP535 is 4%, and SP535 hardly degrade PCA at low temperature. At the temperature of 25 ℃, SP535 achieved complete degradation of PCA within 120 h. With the further increase of temperature 35, 45 and 55 ℃, the degradation efficiencies of PCA by SP535 were 86%, 47% and 32%, respectively.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe effects of temperature on PCA biodegradation in previous studies indicated an initial increase followed by a decrease, likely due to the inhibitory effects of both high and low temperatures on strain growth and enzyme activity. \u003cem\u003eBrevibacillus\u003c/em\u003e S-618 showed the highest degradation efficiency of PCA (85.9%) at an incubation temperature of 30 ℃, and the dry cell weight of the strain was more than 1.5 g/L, whereas the growth of \u003cem\u003eBrevibacillus\u003c/em\u003e S-618 was inhibited at incubation temperatures of 22 ℃ and 38 ℃, and the growth efficiency of \u003cem\u003eBrevibacillus\u003c/em\u003e S 618 were both significantly lower than 0.75 g/L, resulting in PCA degradation efficiencies of less than 40% in both cases (Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ding et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) reported similar findings, indicating that the optimal degradation temperature for the \u003cem\u003eBacillus licheniformis\u003c/em\u003e strain ycsd02 was between 30\u0026ndash;32 ℃, with rapid declines in degradation efficiency at temperatures above or below this range. Additionally, \u003cem\u003eDeltia tsuruhatensis\u003c/em\u003e H1 was capable of completely degrading PCA at a concentration of 300 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e at 30 ℃ (Zhang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The degradation efficiency of \u003cem\u003eThauera\u003c/em\u003e sp. M9 for 300 mg\u0026middot;L\u003csup\u003e\u0026ndash;1\u003c/sup\u003e PCA reached 99.29% at 30 ℃, while at 25 ℃, 35 ℃, 40 ℃, and 45 ℃, the efficiencies were only 30%, 22.58%, 2.92%, and 2.39%, respectively (Kumar et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Furthermore, Wang et al. (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) demonstrated that high temperatures (39 ℃) inhibited the expression of cytochrome P450 family 11 subfamilies A member 1, revealing the possibility that high temperature might inhibit the expression of cytochrome P450 and thus inhibit the biodegradation of PCA by SP535.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThis study reported an isolate of filamentous fungus \u003cem\u003eIsaria fumosorosea\u003c/em\u003e SP535 with highly degrading ability of PCA for the first time. The optimal degradation parameters were: initial PCA concentration of 1.0 mM; initial pH of 7.0; and growth temperature of 25 ℃. The ability of SP535 to degrade PCA displays its potential use in the remediation of PCA contaminated sites. Fungal cells grown on PCA showed high cytochrome P450 enzymes activities suggesting that SP535 may metabolize PCA through the P450 system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e Shicong Huang: Experimentation, Data analysis, Manuscript writing; Jiahui Gao, Lin Zhou, Liujian Gao: Experimental assistance; Mengke Song: Review and edit of manuscript, Corresponding author; Qiaoyun Zeng: Review and edit of manuscript, Corresponding author, funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was funded by the Science and Technology Planning Project of Guangzhou (202206010162), the Guangdong Basic and Applied Basic Research Foundation (2022A1515010703), National Natural Science Foundation of China (No. 42377209), Natural Science Foundation of Guangdong Province (No. 2022A1515010890).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data are available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe article does not involve human participants and/or animal studies.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAl-Hawash AB, Zhang J, Li S et al (2018) Biodegradation of n-hexadecane by \u003cem\u003eAspergillus\u003c/em\u003e sp. 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Dissertation, South China Agriculture University\u003c/li\u003e\n\u003cli\u003eZhang L, He D, Chen J et al (2010) Biodegradation of 2-chloroaniline, 3-chloroaniline, and 4-chloroaniline by a novel strain \u003cem\u003eDelftia tsuruhatensis\u003c/em\u003e H1\u003cem\u003e.\u003c/em\u003e J Hazard Mater 179: 875-882. https://doi.org/10.1016/j.jhazmat.2010.03.086\u003c/li\u003e\n\u003cli\u003eZhu L, Lv M, Dai X et al (2012) Reaction kinetics of the degradation of chloroanilines and aniline by aerobic granule\u003cem\u003e.\u003c/em\u003e Biochem Eng J 68: 215-220. https://doi.org/10.1016/j.bej.2012.07.015\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"p-chloroaniline, fungus, Biodegradation, Toxicity, Cytochrome P450 monooxygenase","lastPublishedDoi":"10.21203/rs.3.rs-4840476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4840476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEfficient methods to remediate PCA (p-chloroaniline) polluted environment are urgent due to the widespread, persistence and toxic of PCA in the environment. Biodegradation facilitated by microbes presents a promising approach for remediating PCA pollution. However, the PCA-degrading fungi still yet to be explored. This study confirmed the highly PCA degrading efficiency of an isolated fungus, \u003cem\u003eIsaria fumosorosea\u003c/em\u003e SP535. This fungus can achieve a PCA degradation efficiency of 100% under optimal conditions characterized by initial PCA concentration of 1.0 mM, pH of 7.0, and temperature of 25 ℃. SEM and TEM analyses revealed that the toxicity of PCA resulted in roughened surfaces of SP535 hyphae, voids in the cytoplasm, and thickened cell walls. PCA addition significantly elevated the activities of cytochrome P450 monooxygenase in both cell-free extracts and microsomal fractions in the media, suggesting the important role of P450 system in PCA metabolization by SP535. The results provide microbial resource and fundamental knowledge for addressing PCA pollution.\u003c/p\u003e","manuscriptTitle":"Biodegradation of p-chloroaniline by fungus Isaria fumosorosea SP535","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-30 05:48:33","doi":"10.21203/rs.3.rs-4840476/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cb80da4a-26be-4347-a2fe-75b321c846ab","owner":[],"postedDate":"August 30th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-30T19:38:35+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-30 05:48:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4840476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4840476","identity":"rs-4840476","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}