Bioresource Technology 369 (2023) 128508 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech Biotransformation of chloramphenicol by white-rot-fungi Trametes versicolor under cadmium stress Zewen Tan a, b, Diana Losantos a, Yongtao Li b, Montserrat Sarrà a, * a Departament d′ Enginyeria Química, Biologica i Ambiental, Escola d′ Enginyeria, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain b College of Natural Resources and Environment, Joint Institute for Environment & Education, South China Agricultural University, Guangzhou 510642, PR China H I G H L I G H T S G R A P H I C A L A B S T R A C T • Trametes versicolor is capable of simul­ taneous biodegrading CAP and removing Cd. • Nine transformation products (TPs) were identified, including six novel TPs. • Two novel CAP biodegradation path­ ways were proposed through the TPs. • Biodegradation and adsorption were the main mechanism of CAP removal by T. versicolor. • Cellular accumulation and surface adsorption were the main mechanism of Cd removal. A R T I C L E I N F O A B S T R A C T Keywords: The recalcitrant chloramphenicol (CAP) combined with heavy metals cadmium (Cd) commonly co-existed in the Trametes versicolor environment, posing threat to environment health. The capacity of Trametes versicolor to remove/biodegrade CAP Chloramphenicol biotransformation in air-pulse fluidized-bed reactor was evaluated, even under Cd stress. T. versicolor could remove 44 % CAP of 5 Cadmium stress mg/L in 15 days, even 51 % CAP under 1 mg/L Cd stress. Sustained Cd stress inhibited CAP biodegradation and Biotransformation pathway Fluidized-bed reactor Cd removal in a 5-batches of a 5-days cycle sequential batch reactor. Nine transformation products and two novel pathways were proposed, with initial multi-step transformation reaction into CP2 and allylic alcohol, respec­ tively. Furthermore, the main mechanism of Cd removal by T. versicolor was extracellular surface bioadsorption and intracellular accumulation. This study filled the gap of the mechanism of simultaneous CAP removal/ biodegradation and Cd removal by white-rot fungi T. versicolor, which offer a theoretical basis for future application of biological removal of CAP containing wastewater. 1. Introduction growth and human health (Carvalho and Santos, 2016; Wang et al., 2020a,b). However, its over-application might generate environmental Antibiotics has been widely applied in human medical and livestock risks and human health threats (Kumar et al., 2019; Wang et al., 2016). and aquaculture breeding industry to meet the demand of population Chloramphenicol (CAP) is a broad-spectrum antibiotic, which has been * Corresponding author. E-mail address: montserrat.sarra@uab.cat (M. Sarrà). https://doi.org/10.1016/j.biortech.2022.128508 Received 23 November 2022; Received in revised form 14 December 2022; Accepted 15 December 2022 Available online 19 December 2022 0960-8524/© 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Z. Tan et al. Bioresource Technology 369 (2023) 128508 extensively administrated as a veterinary antibiotic (Hanekamp and treatment process of real wastewater containing CAP with T. versicolor. Bast, 2015). Due to the incompletely metabolism characteristics by human beings or animals, most un-metabolized CAP (30–90 %) were 2. Materials and methods excreted out into the environment through urine and excrement (Van Boeckel et al., 2015). So, those environmental CAP residues threatened 2.1. Strain and media the environment health, for example, decline of soil fertility and mi­ crobial diversity (Machowska and Stålsby Lundborg, 2019; Zhang et al., Trametes versicolor ATCC 42,530 was obtained from American Type 2020a,b), promotion of antibiotics resistance genes spreading (Lu et al., Culture Collection. The subcultures, blended mycelial suspension and 2020), even threaten human health through food chain (Pan and Chu, pellets were maintained following Hu et al. (2020a,b). 2017). Due to the stable structure, recalcitrant and anti-bacteria prop­ Defined medium (glucose 8 g/L, NH4Cl 1.42 g/L, micronutrients 10 erties, CAP can exist in a long time and accumulated in the environment mL/L, macronutrients 100 mL/L, pH 4.5) was prepared as described by (Hanekamp and Bast, 2015). So, CAP residues have been detected in Hu et al. (2020). many environmental media, such as livestock manure, municipal sludge, aquaculture wastewater, river sediment, farm soil, and even in 2.2. Chemicals and reagents some food resources, such as radish, rape, coriander, eggs, rabbit, and etc (Chung et al., 2017; Song et al., 2021; Wang et al., 2020a,b). Thus, it The standard of chloramphenicol (CAP, purity ≥ 98 %), commercial is urgent to seek a highly-efficient strategy to reduce or eliminate CAP laccase (20 AU/mg, purified from T. versicolor), the laccase mediator residues in the environment. 2,2′ -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium In addition, cadmium (Cd), a common heavy metal and toxic salt (ABTS, purity ≥ 98 %) were purchased from Sigma-Aldrich (Bar­ metallic element for all organism, was commonly detected in animal celona, Spain). HPLC (high performance liquid chromatography) feeds and manure in low concentration (Kirkham, 2006; Shahid et al., gradient grade acetonitrile was purchased from Carlo Erba Reagents S.A. 2017). So, Cd commonly entered into the environment with antibiotics S (Barcelona, Spain). Methanol was obtained from Merck (Darmstadt, residue through livestock manure and wastewater (Adamse et al., 2017; Germany). Stock solution of CAP (1 g/L) was prepared by appropriate Liang and Hu, 2021; Shao et al., 2019), which means that Cd and CAP dilution of the substances in ethanol and sterilized through 0.22 μm often co-exist in livestock waste. Traditional method to remove organic/ sterilized nylon filter. inorganic pollutants include physical, chemical and biological methods, such as chemical precipitation, ion exchange, adsorption, advanced 2.3. Fungal degradation of CAP under Cd stress oxidation processes, electrochemical oxidation, photocatalytic oxida­ tion, microbial biodegradation/immobilization, plant/microbe accu­ T. versicolor pellets were transferred into air-pulsed fluidized-bed mulation, and etc (Teng et al., 2019; Uddin, 2017; Wang et al., 2018; Xu bioreactor, with the initial biomass amount of 2.5 g dry weight. The et al., 2020; Li et al., 2022). Compared with chemical and physical bioreactor has at the bottom, a ceramic plate as air diffuser, and a cy­ methods, microbial biodegradation is a promising, low cost, and envi­ lindrical vertical center body which is connected diametrically wider ronmentally friendly methods. But most microbial biodegradation head. The valid volume of the bioreactor was 1.5 L, and 1 L defined methods focused on bacterial CAP biodegradation and microbial Cd medium was added into the bioreactor. As described in Hu et al. (2021), accumulation (Ma et al., 2020; Ma et al., 2019; Tan et al., 2022). There is the medium pH was controlled at a constant value of 4.5 ± 0.1, aeration scarce information about CAP biodegradation by fungi even under Cd rate was maintained at 0.8 L/min (1 s air pulse every 3 s). stress, especially by white rot fungi (WRF). Two kinds of experiment were operated using air-pulse fluidized-bed WRF are a group of basidiomycetous fungus, including many species, bioreactor: (a) four different experiments were carried out where the such as Trametes versicolor, Ganoderma lucidum, Pleurotus ostreatus, defined medium was spiked with 0 mg/L, 5 mg/L, 10 mg/L CAP, and Stropharia rugosoannulata, Irpex lacteus etc. WRF, as is known to all, can both 5 mg/L CAP and 1 mg/L Cd. At the beginning and end of each aerobically decompose lignin and other organic pollutants using its non- batch, samples were collected for laccase activity, glucose concentra­ specific intracellular and extracellular enzymes, such as ligninolytic tion, CAP concentration, Cd concentration analyses, and all the pellets enzymes, cytochrome P450 enzyme system, laccases (Mir-Tutusaus were harvested to quantify the biomass at the end of batch; (b) according et al., 2018). For example, white rot fungus P. ostreatus could biodegrade to the previous result of CAP and Cd removal by T. versicolor, 5 batches di(2-ethyl hexyl) phthalate (endocrine-disruptor) through de- of a 5-days cycle sequential batch reactor were operated at the same esterification, oxidation and oxidation-hydrolysis pathway (Ahuactzin- conditions of initial biomass, pH value, aeration rate. The defined me­ Pérez et al., 2018). Phanerochaete chrysosporium could biodegrade 49 % dium was spiked with both 5 mg/L CAP and 1 mg/L Cd at the beginning. of insecticide thiamethoxam in 15 days at the initial concentration of 10 At the end of each batch, 200 mL supernatant were withdrawn, and mg/L (Chen et al., 2021). And P. chrysosporium (MTCC-787) could same volume of new concentrated defined medium was added into the biodegrade 71.5 % of cyclophosphamide and 98.4 % of etoposide in 6 bioreactor to keep each medium component with the same concentra­ days at the initial concentration of 0.3 mg/L, respectively (Yadav et al., tion at the beginning. And at the beginning of each batch, CAP and Cd 2022). T. versicolor is a common species of WRF, and it has been verified were added. Samples were collected at the beginning and end of each to biodegrade various of organic pollutants, such as cyclophosphamide, batch, and laccase activity, glucose concentration, CAP concentration, bentazone, etoposide, diuron, chlorpyrifos, dicofol, cypermethrin and Cd concentration were tested. At the end of the experiment, all the fluoroquinolone, etc (García-Vara et al., 2021; Hu et al., 2020a,b; pellets were harvested to quantify the biomass. Manasfi et al., 2020; Yadav et al., 2022). The aim of this study was to evaluate the ability of biodegrading CAP 2.4. In vitro CAP biodegradation by laccase by T. versicolor as WRF in an air-pulse fluidized bioreactor, even under Cd stress, and was to reveal the biodegradation pathway of CAP by According to García-Vara et al. (2021), commercial laccase was identifying the transformation products (TPs) formed during the added into the laccase reaction solution (250 mM sodium malonate biodegradation process. Furthermore, the enzymatic system related to dibasic monohydrate, pH 4.5) with the final enzyme activity at 500 AU/ the biodegradation process was explored. The biodegradation pattern of L. Laccase Treatment: CAP was added into the reaction solution with a CAP by T. versicolor even under Cd stress provides a new insight of the final concentration at 5 mg/L. Laccase + ABTS Treatment: laccase CAP transformation fate in heavy metal contaminated environments, mediator ABTS was added into Laccase Treatment with a final concen­ and also provide a new candidate for both CAP and Cd bioremediation in tration at 1 mM. All experiment was incubated on dark at 135 rpm the future. So, this study is essential before developing a wastewater orbital shaker and 25 ◦ C. 1 mL sample was collected at 0, 3, 6, 12, 24 h, 2 Z. Tan et al. Bioresource Technology 369 (2023) 128508 2.5.3. Cd concentration The collected liquid samples were filtered through 0.22 μm filter before Cd concentration testing. To quantify the Cd concentration of cellular surface adsorption, the fungi cell pellets were collected and washed with 10 mM EDTA in three times, and the washing solution was combined together. Subsequently the washed pellets were dried at 105 ◦ C to constant weight for cellular accumulated Cd concentration. 1 mL liquid sample and 0.25 g solid sample were digested with HNO3, and then the Cd concentration was measured by ICP-MS (7900, Agilent, USA). The instrument conditions were set as follows: S/C temperature, 2℃; RF matching, 1.10 w; RF power, 1600 w; 77 carrier gas, 0.82 L/min; Smpl Depth, 10.00 mm; Nebulizer pump, 0.1 rps; Pump rate, 40 r/min. 2.5.4. Identification of transformation products The transformation products of CAP were identified by UPLC (Agi­ lent, USA)-Q-TOF-MS spectrometer (Acquity, Bruker, Germany). The UPLC was equipped with a DAD detector (1200RR, Agilent) and with a C18 reversed-phase column (Phenomenex®, Luna Omega C18 100 Å, 4.6 mm × 250 mm, 5 μm). And the UPLC separation conditions were set as follows: mobile phase at 0.1 % formic acid in H2O (solvent A) and 0.1 Fig. 1. Time-course of CAP remaining and laccase activity of T. versicolor under % formic acid in methanol (solvent B), flow rate at 0.8 mL/min, column no CAP and 5/10 mg/L CAP conditions. temperature at 40 ◦C, injection volume at 2 μL, detection wavelength at 278 nm. The separation gradient properties followed at: maintained 10 % B in 1 min initially, increased to 95 % over 22 min and maintained at 95 % B for 3 min, decreased to 10 % B over 1 min, and then maintained at 10 % B for 13 min, for a total runtime of 40 min. The tandem HRMS (microTOFQ, Bruker) analysis was performed using an electrospray ionization source in positive and negative polarity with a spray voltage at 4.0 KV (in ESI + ) or 3.5KV (in ESI-), dry heater at 210 ◦ C, a dry gas at 8.0 L/min, nebulizer at 4.0 bar. The MS acquisition was performed in full scan mode 50–1000 Da. The molecular structure for CAP and their degradation products were tentatively proposed by the detection of predicted mass and isotope pattern. 3. Results and discussion 3.1. CAP biodegradation at different initial concentration The chloramphenicol (CAP) removal and biodegradation ability by Trametes versicolor in different concentration was evaluated in an air- pulse fluidized-bed bioreactor. As it is shown in Fig. 1, the removal ef­ ficiency in 5 mg/L CAP was slightly higher than that in 10 mg/L in 11 days incubation, and the CAP removal efficiency were 42 % and 33 %, Fig. 2. Time-course of CAP remaining, Cd remaining and laccase activity of respectively. But the CAP removal ability was nearly the same at day 13, T. versicolor under both 5 mg/L CAP and 1 mg/L Cd condition. and the removal efficiency of CAP were 42 % and 42 % at 5 and 10 mg/L CAP, respectively. At the end of the batch treatment, the removal effi­ and then mixed with 0.1 mL of 1 M HCl to stop the reaction. The con­ ciency of CAP in 10 mg/L CAP was higher than that in 5 mg/L CAP, the centration of CAP was then tested by HPLC. efficiency was 53 % and 44 %. Furthermore, the removal kinetics of CAP by T. versicolor fitted well with the first-order kinetic model under 5 and 2.5. Analytical methods 10 mg/L CAP conditions (see supplementary material), with a correla­ tion coefficient R2 of 0.938 and 0.967 (see supplementary material), 2.5.1. Laccase activity and glucose concentration testing respectively. The kinetic of removal constant under 5 mg/L CAP con­ According to the conventional methods our lab, laccase activity was dition was higher than that under 10 mg/L CAP condition, the constant k measured through the oxidation of 2,6-dymetoxyphenol (DMP) by lac­ was 0.039 d− 1 and 0.037 d− 1 (see supplementary material), respec­ case without the presence of a cofactor (Hu et al., 2021). The glucose tively, which mean that high antibiotics concentration inhibit the anti­ concentration was tested by biochemistry analyzer (2700 select, Yellow biotic biodegradation by fungi. The inhibition effect of high antibiotic Springs Instrument, USA). concentration on antibiotic biodegrading strain has been commonly observed. Holanda et al. (2019) found that strains Aspergillus sp. BIORG 2.5.2. CAP concentration 9 and Trichoderma sp. BIORG 7 could biodegrade 29.3 % and 25.2 % of CAP concentration was detected by HPLC (Ultimate 3000, Dionex, 100 mg/L CAP in 9 days, respectively, and CAP concentration was the USA), which equipped with a UV detector and with a C18 reversed- main factor with the highest influence over biodegradation. Further­ phase column (Phenomenex®, Kinetex® EVO C18 100 Å, 4.6 mm × more, the adsorption of organic pollutants was inevitable, so the 150 mm, 5 μm). The running conditions were set as follows: injection adsorption of CAP by T. versicolor were determined using killed volume at 2 μL, mobile phase at 60 % methanol and 40 % water, flow T. versicolor. The adsorption of CAP stabilized in 1 day, and then pla­ rate at 0.8 mL/min, column temperature at 30 ◦C, detection wavelength teaued between day, so the adsorption efficiency of CAP was nearly 14 at 287 nm. % (0.25 mg/g dry weight) (see supplementary material). 3 Z. Tan et al. Bioresource Technology 369 (2023) 128508 Fig. 3. Remaining of CAP and Cd, and laccase activity, and glucose concentration by T. versicolor in sequencing batch bioreactor (SBR) under initial 5 mg/L CAP and 1 mg/L Cd. increased from 0 to 10 AU/L in 3 days incubation, and decreased slightly to 5 AU/L at day 7, then increased to 23 AU/L at day 11 and plateaued between at day 11 to day 15. It was reported that if fungal laccase participated in pollutants biodegradation, pollutants concentration corresponded to the time-course laccase activity (Hu et al., 2020a,b; Wang et al., 2022). But, the laccase activity of T. versicolor didn’t fit with CAP concentration changing, which implied that the laccase of T. versicolor may not participate in CAP biodegradation. Furthermore, the biomass of T. versicolor with no CAP and with 5/10 mg/L CAP conditions were 7.8, 7.2 and 6.2 g (see supplementary material), respectively. All those results indicated that high CAP concentration (10 mg/L) was more toxic to T. versicolor, which represented in inhibiting fungal growth, extracellular enzyme production and CAP biodegrada­ tion ability. Same toxic situation was observed in thiamethoxam biodegradation by white-rot fungus Phanerochaete chrysosporium, and high concentration of thiamethoxam inhibit its biodegradation rate and catalase and peroxidase activity (Chen et al., 2021). 3.2. CAP biodegradation under Cd stress As shown in Fig. 2, the effluence of Cd stress on CAP removal and Cd Fig. 4. Cd distribution in T. versicolor in bioreactor batch and sequencing batch removal of T. versicolor was assessed. At the initial concentration of both under initial 5 mg/L CAP and 1 mg/L Cd. 5 mg/L CAP and 1 mg/L Cd condition, the CAP removal pattern of T. versicolor was nearly same with that under only 5 mg/L CAP condition The laccase activity and biomass of T. versicolor were also determined at day 0 to day 5. Then the CAP removal efficiency increased and was under no CAP, and 5/10 mg/L CAP conditions (Fig. 1). With the absence higher than other conditions at day 7 to day 13, and the highest removal of CAP, the extracellular laccase activity maintained low (24 ± 2 AU/L) efficiency was 51 % at day 15, which indicated that 1 mg/L Cd promoted until day 7 when it increased sharply to 119 AU/L at day 11 and pla­ CAP removal of T. versicolor. As described in previous researches that teaued between at day 11 to day 13. For the low laccase activity at the low concentration of metal had different impact on organic biodegra­ beginning, it is may due to the metabolism strategies of many fungi that dation by WRF. For example, Liu et al. (2020) reported that low con­ high concentration of carbon source stimulated the biomass accumula­ centration of Cr (VI) (1 mM) greatly promoted reactive black 5 tion, but inhibit the laccase production. It is reported that the synthesis decolorization, from 57 % to 84 %, by white-rot-fungus T. hirsute. of laccase was activated by carbon or nitrogen depletion, and the laccase Furthermore, the removal kinetics of CAP by T. versicolor fitted well with production was also influenced by some metal ions and xenobiotics the first-order kinetic model under both 5 mg/L CAP and 1 mg/L Cd (Yang et al., 2017). Interestingly, the trends of laccase activity changes conditions (see supplementary material), with a correlation coefficient under 5 mg/L CAP condition were the same with that under no CAP R2 of 0.957 and the removal constant k of 0.047 d− 1 (see supplementary condition. But under 10 mg/L CAP condition, the laccase activity material). The removal constant k under 10 mg/L CAP condition was increased to 21 AU/L at day 3, and then decreased to 6 AU/L from day 3 lower than that under 5 mg/L CAP condition and both 5 mg/L CAP and to day 13. And with the presence of 5 mg/L CAP, the laccase activity 1 mg/L Cd condition, which indicated that higher CAP concentration 4 Z. Tan et al. Bioresource Technology 369 (2023) 128508 Table 1 The identified CAP biodegradation transformation products by T. versicolor using UPLC-QTOF-MS. Peak number Elemental composition Measured mass (m/z) Possible structural formula CAP C11H12O5N2Cl2 323.02 OH Cl H N Cl O O2N OH CP2 C10H9O3N2Cl2 228.1468 OH H N OH H2 N OH TP182 C9H12O3N 182.0801 OH COOH H2N TP136 C8H10ON 136.0744 O H2N TP165 C9H9O3 165.0536 O COOH TP121 C7H5O2 121.0291 O OH CHA C11H14O3N2Cl2 221.0292 COOH OH COOH O COOH Allylic alcohol C11H12O2N2Cl2 275.0349 Cl H N Cl O H2N OH TP205 C11H13O2N2 205.0956 H N O H2N O TP188 C11H10O2N 188.0701 H N O O was more toxic than low CAP concentration and even under both Cd The laccase activity of T. versicolor was also tested, and the changing condition to T. versicolor. trend was similar with other conditions at day 0 to day 3, but lower than Different WRF species has different strategies in heavy metals, such under other conditions. Then, the laccase activity deceased at day 5 to as adsorption by mycelium, extracellular EPS and other extracellular day 15 (Fig. 2). Furthermore, the biomass of T. versicolor was 6.8 g (see substrates; immobilization by extracellular/intracellular chelating supplementary material), which was lower than that under 5 mg/LCAP compounds (such as metallothioneins, oxalate, etc); cell wall adsorption and higher than that under 10 mg/LCAP. These results indicated that (melanins and phenolic molecules); intracellular accumulation (Bal­ high concentration CAP was more toxic than both low concentration drian, 2003). For example, Noormohamadi et al. (2019) found that CAP and Cd stress. white-rot fungi Phanerochaete chrysoporium could remove Cd and Ni through surface biosorption on fungi mycelia in high removal efficiency (96.23 % and 89.48 % respectively). Schlunk et al. (2015) verified that 3.3. CAP biodegradation assay by laccase Tricholoma vaccinum could encode Mte1 to transport heavy metal into the vacuole, which mediated detoxification. In this research, the Cd Fungal laccase, a kind of extracellular enzyme, has been reported to concentration decreased sharply, and the removal efficiency were 85 % have the ability to degrade recalcitrant pollutants, such as olsalazine and 100 % at day 5 and day 11, respectively. But the Cd concentration (Bankole et al., 2021), melanin (Park et al., 2021), bentazone (García- then increased at day 13, which may due to incident with pH controller Vara et al., 2021), and etc. So, whether the laccase of T. versicolor and leading Cd de-adsorption from fungi cellular surface at the end of participated in CAP biodegradation was investigated. The CAP concen­ the treatment. The concentration of cellular accumulation and cellular tration didn’t change with or without the presence of laccase mediator surface adsorption were 89 μg/g and 13.8 μg/g biomass, respectively ABTS, which indicated that the laccase of T. versicolor was not involved (Fig. 4), which means Cd removal of T. versicolor was mainly attributed in CAP biodegradation (see supplementary material). But, Navada and to cellular accumulation (61 %). Kulal (2019) reported that laccase from T. hirsute could efficiently degrade 10 mg/L CAP in 48 h with the presence of laccase mediator 5 Z. Tan et al. Bioresource Technology 369 (2023) 128508 Fig. 5. The proposed CAP biodegradation pathways by T. versicolor. system, such as vanillin and ABTS. The reason for different activity of efficiency in each batch were fluctuated between batches, but the Cd laccase from different fungi on CAP biodegradation is that fungal laccase removal efficiency was lower than that in batch 1, and the Cd removal has several gene families (5–17 family members) and the function be­ efficiency in each batch were 94, 24, 67, 15 and 40 %, respectively. It tween them are vary, for example, Lcc3/5, Lac4, LacA, LccA and etc was reported that heavy metal stress could induce reactive oxygen (Iimura et al., 2018; Yang et al., 2017). So, in conclusion, there was no species (ROS), low concentration of ROS could promote heavy metal relationship between CAP biodegradation and laccase activity among removal, but high concentration of ROS could result in membrane T. versicolor. damage and enzyme inactivation (Sharma et al., 2020; Zhang et al., 2015). The irregular removal rate of Cd in each batch may due to the lagging production or different production amount of ROS. 3.4. CAP biodegradation and Cd removal in sequencing batch bioreactor The laccase activity increased in batch 1 and 2, and reached a maximum value of 58 AU/L during the second batch of day 3, but it To investigate the sustained Cd stress on CAP removal/biodegrada­ decreased to nearly 0 at batch 3 to 5. The reason for the laccase activity tion by T. versicolor, 5 batches of 5-days cycle were operated (Fig. 3), and trends during the experiment maybe that Cd and CAP supply promoted CAP and Cd in fresh medium were supplemented at the end of each T. versicolor laccase secretion at the beginning of batch 2, but the batch according with the previous results (Fig. 2). In the first batch, the accumulated Cd and CAP in the medium and T. versicolor cell poisons CAP removal efficiency and Cd removal efficiency were nearly the same cell growth and metabolism in bath 2 to bath 5. As Liu et al. (2020) with previous results in Fig. 2, 31 % and 94 %, respectively (Fig. 3). And reported that the decolorization ratio of azo dye (Reactive Black 5) by with the following batches (batches 2 to 5), the inhibition of CAP Trametes hirsuta promoted by the increased hexavalent chromium (Cr removal efficiency increased with the increasing batches by the sus­ (VI)) concentration (0–1 mM), then inhibited with the increased Cr(VI) tained Cd stress. It was reported that low heavy metal concentration concentration (0–5 mM); and the laccase activity also promoted with the could promote the growth of fungi, but high concentration of heavy presence of Cr(VI), which was responsible for azo dye decolorization. metal (especially in sustained Cd stress) could inhibit their growth, such The glucose concentration was nearly 0 at day3 of each batch (Fig. 3), as Sharma et al. (2020) reported that low Cd and Pb concentration (1–2 and the fungal biomass after 5 batches was 5 g (see supplementary μM) promoted the growth and its removal efficiency of Phlebia brevis­ material), which was lower than all other treatments. It means that pora, but high concentration of Cd (100 μM) was toxic to its growth and T. versicolor could metabolize glucose to grow and synthesize other inhibited their removal efficiency. Interestingly, the final Cd removal 6 Z. Tan et al. Bioresource Technology 369 (2023) 128508 substances, but the growth rate was lower than other treatments and the Declaration of Competing Interest cell was still active. Furthermore, the cellular surface adsorbed and cellular accumulated Cd was 45.6 and 13.4 μg/g (Fig. 4), respectively, The authors declare that they have no known competing financial which were totally different with that in Section 3.2. So, sustained Cd interests or personal relationships that could have appeared to influence and CAP addition may change the strategies of T. versicolor to remove the work reported in this paper. Cd, which mean T. versicolor may pump out more Cd from cell and synthesis more extracellular polymeric substances and cellular surface Data availability substances to adsorb Cd. These results indicated that sustained Cd stress in 5 batches made T. versicolor adsorbing more Cd on its surface, those Data will be made available on request. adsorbed and accumulated Cd by T. versicolor are more toxic and inhibited CAP removal/biodegradation and Cd removal. Acknowledgements This work was supported by MCIN/AEI/10.13039/501100011033 3.5. CAP transformation products and pathway (Project PID2019-103989RB-100). Zewen Tan acknowledges support from Chinese Scholarship Council (CSC No. 202108440365). The CAP transformation products by T. versicolor were identified through HPLC-QTOF-MS, and nine transformation products were Appendix A. Supplementary data determined, including 6 novel products (Table 1 and see supplementary material). And two novel CAP bio transformation pathways by Supplementary data to this article can be found online at https://doi. T. versicolor were proposed through the transformation products in org/10.1016/j.biortech.2022.128508. Fig. 5, which haven’t been reported before. To our acknowledge, four initial CAP biodegradation steps were reported, including amide bond References hydrolysis, acetylation, reduction of nitro group, and oxidation of hy­ droxyl group; and all of them were focused on bacterial biodegradation Adamse, P., Van der Fels-Klerx, H.J.I., de Jong, J., 2017. Cadmium, lead, mercury and (Ma et al., 2020; Tan et al., 2022; Zhang et al., 2020a,b). Navada and arsenic in animal feed and feed materials – trend analysis of monitoring results. Food Addit. Contam. A 34 (8), 1298–1311. https://doi.org/10.1080/ Kulal (2019) reported that fungi could biodegrade CAP, and only one 19440049.2017.1300686. biodegradation product was identified, chloramphenicol aldehyde, Ahuactzin-Pérez, M., Tlecuitl-Beristain, S., García-Dávila, J., Santacruz-Juárez, E., which formed after dehalogenation. In pathway 1, CAP was transformed González-Pérez, M., Gutiérrez-Ruíz, M.C., Sánchez, C., 2018. A novel biodegradation pathway of the endocrine-disruptor di(2-ethyl hexyl) phthalate by Pleurotus ostreatus into CP2 through multi-step reaction, including reduction of nitro group based on quantum chemical investigation. Ecotox. Environ. Safe. 147, 494–499. at C4 and dechlorination reaction of chloride at C2′ ’. Same multiple Baldrian, P., 2003. Interactions of heavy metals with white-rot fungi. Enzyme Microb. reaction step of nitro group reduction was observed in bacterial CAP Technol. 32 (1), 78–91. https://doi.org/10.1016/S0141-0229(02)00245-4. 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