Investigation of lead bioimmobilization and transformation by Penicillium oxalicum SL2

Accepted Manuscript Investigation of lead bioimmobilization and transformation by Penicillium oxalicum SL2 Binhui Ye, Yating Luo, Junyu He, Lijuan Sun, Bibo Long, Qinglin Liu, Xiaofeng Yuan, Peibin Dai, Jiyan Shi PII: DOI: Reference:

S0960-8524(18)30726-0 https://doi.org/10.1016/j.biortech.2018.05.066 BITE 19972

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

20 March 2018 15 May 2018 17 May 2018

Please cite this article as: Ye, B., Luo, Y., He, J., Sun, L., Long, B., Liu, Q., Yuan, X., Dai, P., Shi, J., Investigation of lead bioimmobilization and transformation by Penicillium oxalicum SL2, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.05.066

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Investigation of lead bioimmobilization and transformation by Penicillium oxalicum SL2 Binhui Yea, b, Yating Luoa, Junyu Hed, Lijuan Sune, Bibo Longa, Qinglin Liua, Xiaofeng Yuanc, Peibin Daif, Jiyan Shia, b, *

a

Department of Environmental Engineering, College of Environmental and Resource Science,

Zhejiang University, Hangzhou, 310058, China. b

MOE Key laboratory of Environmental Remediation and Ecological Health, College of

Environmental and Resource Science, Zhejiang University, Hangzhou, 310058, China. c

College of Life Science, Zhejiang Chinese Medical University, Hangzhou 310053, China

d

Institute of Islands and Coastal Ecosystems, Ocean College, Zhejiang University, Zhoushan,

China e

Institute of ECO-Environment and Plant Protection, Shanghai Academy of Agricultural

Sciences, Shanghai, 2014303, China f

Department of Applied Engineering, Zhejiang Economic and Trade Polytechnic, Hangzhou

310018, China *

Corresponding author: Jiyan Shi

Tel.: +86-0571-88982019 Fax: +86-0571-88982019 E-mail: [email protected] Postal address: Department of Environmental Engineering, Zhejiang University, Hangzhou, Zhejiang Province, 310058, PR China 1

Abstract: Fungi Penicillium oxalicum SL2 was applied for Pb2+ bioremediation in aqueous solution in this study. After 7 days of incubation at different initial concentrations of Pb2+ (0, 100, 500 and 2500 mg L-1), most of Pb2+ were removed (90, 98.3, and 86.2 %), the maximum Pb content in mycelium reached about 155.6 mg g-1 dw. Meanwhile, the formation of extracellular secondary minerals and intracellular Pb-complex were observed and identified, the speciation of Pb in mycelium was also detected by X-ray absorption near-edge structure (XANES) spectroscopy, i.e., Pb-oxalate, Pb-citrate, Pb-hydrogen phosphate and Pb-glutathione analogues. In addition, content of glutathione and oxidized glutathione was increased under the exposure of Pb2+, which implied that glutathione might play a key role in Pb immobilization and detoxification in P. oxalicum SL2. This study elucidated partial mechanisms of Pb immobilization and speciation transformation of this strain, providing an alternative biomaterial in the bioremediation of Pb-contaminated wastewater.

Keywords: Penicillium oxalicum SL2; Pb; Bioimmobilization; Transformation; Glutathione 1. Introduction Since the unique properties of lead (Pb), such as low melting point, good ductility and corrosion resistance, it has been widely used in various fields, like leaded petrol, automobiles, lead-acid batteries, lead-based paint, recreational shooting, etc (Flora et al., 2012; Sullivan et al., 2012). It’s inevitable that massive lead ions and lead compounds will enter the environment, threatening ecological environment and human health. As is reported, irreversible and insidious damages of nervous system, hematopoietic system, renal 2

dysfunction, cardiovascular, reproductive health and bone system in human race would be induced due to the chronic or acute toxicity of Pb (Flora et al., 2012). The non-biodegradable characteristics of Pb determined its long-term persistence in environment, thus immobilization of metal ions via physicochemical precipitation can be an effective approach to reduce the mobility and bioavailability of Pb in environment, which is one of the most popular methods of the emerging microbial remediation technology (Liu et al., 2017). Compared to other microbe groups, fungi frequently have higher affinity and tolerance to metal ions, and considered as a bioremediation materials with great potential (Ma et al., 2016). First of all, low molecular organic acids secreted by fungi are essential to the stress resistance against metals, and impact the metal transformation and precipitation, for examples, oxilic, citric acid, etc (Li et al., 2011; Liang et al., 2016). Secondly, increasingly secreting organic acids is an important extracellular defense mechanisms against the biotoxicity induced by heavy metals; in the meantime, it is helpful for the formation of metal-chelation and the detoxification in the extracellular microenvironment (Jarosz-Wilkolazka and Gadd, 2003; Gadd et al., 2014). Moreover, during the metabolic process, the immobilization of metals by fungi often involved biomineralization, like the formation of metal carbonates, pyromorphite, hydrocerussite, etc, (Li et al., 2014; Li and Gadd, 2017; Liang et al., 2016), and these secondary minerals are always identified by energy dispersive spectrometer (EDS), X-ray diffraction (XRD), which is meaningful for better understanding of the metal transformation process (Li et al., 2016). On the other side, it is known that the intracellular detoxification capability of fungi against the biotoxic metals is the foundation of bioremediation. So far, some functional ligands have 3

been found in fungi, like metallothioneins, glutathione, γ-glutamylcysteine, phytochelatins, etc (Bellion et al., 2006). Furthermore, excess heavy metals will stimulate the activity of intracellular antioxidant system (including GSH system). In this system, positive sulfydryl is considered as one of the most important free-radical scavengers and plays an important role in detoxification mechanism against toxic metals (Gharieb and Gadd, 2004; Chen et al., 2014). Thus, more attention needs to be paid on the effects of intracellular active ligands on metal ions, and its role in metal bioimmobilization and detoxification. A new type of Pb-resistance filamentous fungi named Penicillium oxalicum SL2 was obtained, which may has potential to apply in the field of Pb contamination bioremediation. The main objectives of this work were, therefore, to assess the Pb2+ removal ability of living fungi P. oxalicum SL2 and demonstrate the possible mechanisms of lead bioimmobilization and transformation in liquid medium. After 7 days incubation of P. oxalicum SL2 with or without Pb2+ in liquid media, the removal efficiency and immobilization effect of Pb2+ was first tested. Then, scanning electron microscopy equipped with energy-dispersive spectroscopy (SEM-EDS) and transmission electron microscopy equipped with energy-dispersive spectroscopy (TEM-EDS) were both adopted to investigate the morphological of mycelia structure and the elemental composition changes of intracellular and extracellular products. Besides, X-ray absorption near-edge structure (XANES) spectroscopy was employed to further determine the Pb speciation transformation in mycelium. At last, GSH-GSSG analysis was adopted as a proving test to detect the changes of GSH and GSSG levels in mycelium treated with different initial concentrations of Pb2+.

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2. Materials and methods 2.1 Organisms and culture media Filamentous fungus Penicillium oxalicum SL2 was isolated and used for bioremediation of wastewater and soils contaminated by heavy metals, which was preserved in China Center for Type Culture Collection (CCTCC). Potato dextrose agar (PDA) solid medium comprised 20 g dextrose, 20 g agar and 1000 mL filtered soup of 200 g potatoes boiled for about 30 min previously, medium was autoclaved at 121 ℃ for 30 min. The formula of potato dextrose liquid (PDL) medium was similar to PDA but without agar, and autoclave sterilization was taken before the experiments.

2.2 Cultivation of P. oxalicum SL2 in liquid medium amended with Pb2+ After 5 days period of slant cultivation on PDA medium, fresh conidia in tests tubes were rinsed with 20 mL sterile water contained tween 20 (0.05 %) and harvested as spores suspension (107 spores ml-1). Conidia suspension (1 % v/v) were added into sterilized PDL medium and incubated in 250 mL conical flasks at 30 ℃ in a rotary speed of 180 rpm. After 48 h preculture, groups were supplemented with 0, 100, 500 or 2500 mg L-1 Pb2+ (Pb(NO3)2), (Sinopharm chmical reagent Co., Ltd), respectively. Each group carried out at least triplicate, and continuously incubated for 7 days. Supernatant fluid of different groups was filtered by filter membrane (0.45 μm) after 7 days cultivation, and the content of Pb was determined by atomic absorption spectroscopy (AAS, MKII M6, Thermo Electron, USA). All the fungi mycelium in different groups were taken out from the flasks and washed by distilled water for three times, then dried to constant weight in an oven at 80 ℃ and weighed. 5

After that, 0.140-0.150 g dry weight mycelium was accurately weighed and placed in a teflon tube, acid digestion was conducted as follows: 4.5 mL of nitric acid was added to each tube, after 30 min at room temperature, tubes were placed at a digest instrument (DigiBlock EHD36, LabTech, USA) and heated to 60 ℃ for 30 min, then 120 ℃ for 30 min. Once cooled down to indoor temperature, 0.6 mL of hydrogen peroxide (H2O2) was added to each tube and stayed at 120 ℃ for 15 min (Muñoz et al., 2005). At last, the cooled mixture was transferred to a volumetric flask (100 mL) and the quantification of Pb2+ was carried out by AAS.

2.3 Scanning electron microscopy and energy-dispersive spectroscopy (SEM-EDS) analysis After 7 days incubation of P. oxacilium SL2 and Pb2+ in PDL, some mycelium pellets from different groups were removed out from the flasks using sterilized forceps and fixed with 2.5 % glutaraldehyde in phosphate buffer (0.1 M, pH 7) at 4 ℃ over night. Then stationary liquid was discarded, and samples were rinsed for three times by phosphate buffer (0.1 M, pH 7) for 15 min per rinse. Then postfixed with 1 % OsO4 in phosphate buffer for 1 h and washed three times in the phosphate buffer for 15 min at each step. The fixated samples were dehydrated by a graded series of ehanol (30, 50, 70, 80, 90, 95 and 100 %) for 15 min at each step. Prior to the examination, dehydrated samples were coated with gold-palladium using a Hitachi Model E-1010 ion sputter for 4-5 min and observed in an environmental scanning electron microscope (ESEM, SU-8010, Hitachi, Japan) under an accelerating voltage of 15 kV. To explore the elemental composition of visible and regular crystals formed by P. oxacilium SL2 during the period of incubated with Pb2+, energy dispersive spectrometer (EDS, X-MaxN 80, 6

Oxford instrument, UK) was also used.

2.4 Transmission electron microscopy and energy-dispersive spectroscopy (TEM-EDS) analysis Transmission electron microscope (TEM, H-7650, Hitachi, Japan) observation was also conducted. Samples after double fixation and dehydration were transferred to absolute acetone for another 20 min. Then the specimen was placed in 1: 1 mixture of absolute acetone and the final Spurr resin mixture for 1 h at room temperature, then transferred to the 1: 3 mixture of acetone and the final spurr resin for 3 h, after that, specimen was placed in absolute Spurr resin at room temperature overnight. Infiltrated specimen was imbedded and heated about 70 ℃ overnight, then sliced by an ultramicrotome (EM UC7, LEICA, Germany) with the thickness of 70-90 nm. Ultrathin sections were put on the nickel grids and elemental composition analysis was also conducted by energy dispersive spectrometer (EDS, PV77-47510 ME, AMETEK, USA).

2.5 X-ray absorption near-edge structure (XANES) analysis Synchrotron radiation based X-ray absorption near edge structure (XANES) analysis was conducted due to its accuracy and sensitivity in heavy metal speciation determination in situ. Mycelia of P. oxalicum SL2 treated with different initial concentrations of Pb2+ were collected and rinsed in phosphate buffer saline for three times, filtered and pre-frozen at -80 ℃ overnight, and lyophilized for 3 days (Alpha1-4LSC, Marin Christ Ltd., Germany). The dried mycelia were grounded by agate mortar supplemented with liquid nitrogen, the powdered 7

samples were pressed into slices (diameter 10 mm), adhered to sample holders by 3M tape (Scotch 810, 3M, USA). The Pb reference samples including PbNO3, PbCl3, PbSO4, PbCO3, PbS, Pb(OH)2, PbHPO4, PbO, Pb-acetate, Pb-citrate, Pb-oxalate (Sinopharm chemical reagent Co., Ltd; Sigma-aldrich Co., Ltd; Alfa Aesar Co., Ltd), Pb3(PO4)2 and Pb5(PO4)3Cl were synthesized according to the method of previous study (Kopittke et al., 2008), Pb-glutathione was synthesized as described in previous study (Duan et al., 2014). The Pb L3-edge XANES spectra were collected on the beamline 14W1 at the Shanghai Synchrotron Radiation Facility (SSRF, Shanghai, China) with a storage-ring energy of 3.5 GeV and a beam current of 130-210 mA. A Si (311) double-crystal monochromator was adopted in the station, and the the photon energy was calibrated with the first inflection point of the Pb L3-edge Pb (13,035 eV) XANES spectra of Pb foil. Pb L3-edge spectra of Pb-glutathione was collected in the fluorescence mode using a Lytle detector, while the spectra of other reference samples were recorded in the transmission mode according to the purity of the samples. And the mycelia samples treated with different initial concentrations of Pb2+ were detected in a transmission mode. All the XANES data were processed using the Athena software, the data processing method was described in prior study (Peng et al., 2015).

2.6 Glutathione (GSH) and oxidized glutathione (GSSG) assay The GSH-GSSG kit was purchased from Nanjing Jiancheng Bioengineering Institute. Samples preparations were as follows: fungi mycelium was filtrated and blotted up by filter +

paper after the experiment of Pb2 immobilization immediately. Then, the mycelium samples were weighed accurately (0.1-0.2 g) and transferred to glass homogenizers for completely 8

homogenate in ice bath, the mass liquid ratio was controlled at 1: 9. The mycelium homogenate was centrifuged with the rotate speed of 3500 rpm at 4 ℃. And the supernatant fluid was extracted carefully and processed according to manufacturer's specification for the next determination of GSH and GSSG by a microplate reader (SpectraMax i3x, Molecular Devices, USA).

2.7 Data analysis The Pb2+ removal and immobilized data in Table 1 and the GSH and GSSG content data for the mycelium were reported as the mean value ± standard deviation (SD) of at least three replicates for each treatment, and one way ANOVA was followed by the Tukey-HSD test (SPSS Version 20.0, SPSS Inc., USA), differences of p < 0.05 were considered to be statistically significant.

3. Results and discussion 3.1 Pb2+ removal efficiency and immobilization ability by P. oxalicum SL2 Nowadays, many kinds of microbes, especially fungi, have been reported to possess the potential capacity to handle the metal contaminated wastewater or soils due to the fact that these kinds of microbial materials are cost-effective (Wang and Chen, 2009; Xu et al., 2015). In this study, the effect of different Pb2+ levels on the removal efficiency, percentage of bioimmobilized Pb in mycelium and Pb content in mycelium after 7 days of bioimmobilization by P. oxalicum SL2 was shown in Table 1. The removal efficiency at different initial concentrations (100, 500 and 2500 mg L-1) of Pb2+ were 90.0, 98.3 and 86.2 %, 9

respectively. And about 20.3 ± 3.2 % Pb2+ was removed in the control group (without fungi) when the initial concentration of Pb2+ was 500 mg L-1. More than 50 % of Pb2+ in liquid media was bioimmobilized in mycelium even at the concentration of 2500 mg L-1. In addition, the Pb content in mycelium was increased with the concentration of the Pb2+. In the groups that contained 100, 500 mg L-1 of Pb2+, 9.5, 49.9 mg g-1 biomass were seen; The maximal value of Pb accumulation and adsorption was seen as 155.6 mg g-1 dw at the extremely high initial concentration of 2500 mg L-1. Based on these findings, this strain has outstanding ability in Pb2+ removal and bioimmobilization.

3.2 Observation and identification of Pb secondary mineral intracellular and extracellular A layer of legible particles were found on the mycelium surface of P. oxalicum SL2 with or without Pb2+ treatments by both TEM and SEM observation, which were probably extracellular polymeric substances (EPS) (Gazzè et al., 2013), and playing an irreplaceable role in the metal immobilization and detoxification due to the effective biosorption, chelation and complexation to the toxic metals (Nouha et al., 2016; Sheng et al., 2010). Besides, under Pb2+ treatments, amount of regular crystals at the diameter of 10-20 μm and obviously nanocrystals were found extracellular of mycelium by SEM and TEM observations, which were confirmed to be Pb compounds (main elements were Pb, O, C, P) according to the EDS results. It was reported that heavy metals could be immobilized outside the hyphae or precipitated on the cell wall, and in some cases, biomineralization were also observed (Li and Gadd, 2017; Qian et al., 2017). It is speculated that biomineralization may be the main reason for the remarkable Pb2+ removal efficiency and immobilization ability of this strain in liquid 10

medium (Table 1). On the other hand, intracellular Pb compounds were also determined, although the Pb counts was low, indicating that Pb could permeate the fungi cell wall and cell membrane, thus inducing the biotoxicity effect.

3.3 Pb transformation in the mycelium Recently, metal transformation during the process of bioremediation mediated by fungi metabolism has attracted more and more attentions, which is significant for understanding the removal mechanism of metals and promoting the development of bioremediation technology (Sheng et al., 2013; Wu et al., 2015). In order to further understand Pb speciation transformation in P. oxalicum SL2, Pb L3-edge XANES analysis was conducted. Principal component analysis (PCA) and linear combination fitting (LCF) were taken, and a series of internal references and mycelium samples of P. oxalicum SL2 treated with different concentrations of Pb2+ for 7 days were determined. The results showed that the Pb-complex in fungi mycelium treated with Pb2+ (100, 500 and 2500 mg L-1) were consisted of Pb-oxalate, Pb-citrate, Pb-hydrogen phosphate and Pb-glutathione analogues. The Pb-oxalate and Pb-citrate were the main Pb speciation (Pb-oxalate: 52.8 %, 49.6 %; Pb-citrate: 28.2 %, 28.9 %) in the samples treated with Pb2+ (500, 2500 mg L-1), and the main Pb species in the samples treated with Pb2+ at 100 mg L-1 were Pb-hydorogen phosphate (40.5 %) and Pb-citrate (40.2 %). At last, the proportion of Pb-glutathione in fungi samples of all different treatments was 10 %, approximately. This finding implies that the complex metabolism and detoxification system against the biotoxicity induced by Pb in fungi P. oxalicum SL2. Oxalic and citric acids are commonly 11

secreted by fungi in environment, including P. oxalicum SL2 in this study, which have profound implications for metal speciation, physiology and biogeochemical cycles (Gadd, 1999). Besides, citrate has been reported as the main metal-complex former in soil solution from forest soils (van Hees et al., 2001). In particular, many researchers have demonstrated the speciation of metal-oxalates by various fungi, for instance, Trametes versicolor (Adeyemi and Gadd, 2005), Aspergillus niger (Wei et al., 2013), Serpula himantioides (Gadd et al., 2014), Penicillium corylophilum (Fomina et al., 2010), etc. The insoluble Pb-oxalates analogue formed with different shapes or dimensions, therefore, providing an effective mechanism to metal immobilization and detoxification (Wu et al., 2010), although a few fungi and bacteria are capable to degrade metal-oxalates (Morris and Allen, 1994; Sayer and Gadd, 1997). In addition, not only organic acids, but also some anionic groups (e.g. phosphate, carbonate) are related to the metal immobilization and insolubilization properties of some fungi (Li et al., 2014; Liang et al., 2016). Phosphorus is one of the essential nutrient elements during the fungi growth and metabolism. In some studies, the formation of stable mineral pyromorphite was detected, and this process of biomineralization was supposed to depend on the hydrolysis of organic phosphate (Rhee et al., 2014). However, in this study, the secondary minerals formed by P. oxalicum SL2 were certified to possess a certain proportion of phosphorus elements by EDS determination, then one of the principal Pb speciations was further confirmed to be Pb hydrogen phosphate, rather than pyromorphite, it is speculated to be caused by the different fungi stains and culture conditions. Moreover, some fungi were reported to be able to degrade pyromorphite to Pb-oxalate dehydrate first, and then to be 12

anhydrous Pb-oxalate (Adeyemi and Gadd, 2005). It is noteworthy that, during the cultivation of P. oxalicum SL2 and Pb2+, the concentration of HPO42- in liquid media was dynamic changed, which may be related to the hydrolysis of organic phosphate and the formation of Pb hygrogen phosphate (Rhee et al., 2014).

3.4 Glutathione and oxidized glutathione in mycelium Glutathione (GSH), a kind of cysteine-rich peptides with a low redox potential, is regarded as a reductant, and intracellular dissolved metals can induce the formation of oxidized glutathione (GSSG) during the detoxification processes (Brunet et al., 2009), in some cases, form metal-GSH complex, like the Cu-glutathione complex in Saccharomyces. cerevisiae (Lin et al., 1993). In this work, the intracellular accumulation of Pb-complex was detected, and Pb-glutathione analogue was determined as one of the principal components according to XANES result, indicating that GSH system may play an important role in the immobilization and transformation of Pb2+ by P. oxalicum SL2. After 7 days of incubation, contents of GSH and GSSG in mycelium were both at low level of about 12.7, 3.4 μmol L-1 in control group (without Pb2+), and a series of initial concentrations of Pb2+ exposure resulted in the gradually increasing of GSH and GSSG contents (p < 0.01, Fig. 1). The contents of GSH and GSSG in the samples treated with 2500 mg L-1 Pb2+ were 49.3 and 40.9 folds that of the control treatment. In addition, the ratio of GSH/GSSG in different treatments were also shown in Fig. 1, it was decreased in the groups that treated with Pb at 100 and 500 mg L-1, but a remarkably elevation was discovered at the Pb concentration of 2500 mg L-1 compared to control group. Acceleration of GSH synthesis were also observed under the induction of metal 13

ions in other fungi, such as Phanerochaete chrysosporium (Chen et al., 2014), Pleurotus ostreatus HAU-2 (Zhang et al., 2016), etc. These results indicated the sensitive effect of GSH-system in P. oxalicum SL2 against the biotoxicity induced by Pb2+. Therefore, the increased biosynthesis of GSH facilitated the speciation transformation of Pb, which is probably one of the intracellular Pb detoxification mechanisms of P. oxalicum SL2.

5. Conclusions Pb2+ could be removed effectively by P. oxalicum SL2 via bioimmobilization, and the extracellular Pb secondary minerals and intracellular Pb-complex were observed and identified. Furthermore, Pb-oxalate, Pb-citrate, Pb-hydrogen phosphate and Pb-glutathione analogues were determined as the principal components of Pb compounds in mycelium, indicating a complicated Pb speciation transformation during the cultivation. At last, GSH content was increased with Pb2+ treatment, suggesting a key role of intracellular GSH system in alleviating Pb-initiated cell injuries. This study indicated that P. oxalicum SL2 could be an alternative biomaterial in dealing with Pb2+ contaminated wastewater.

Acknowledgements This work was supported by National Natural Science Foundation of China (U1532103, 41721001), National Key Research and Development Program of China (2016YFD0800401). Thanks to Jingyuan Ma and Lijuan Zhang at Shanghai Synchrotron Radiation Facility (SSRF) for their generous help.

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Appendix A. Supplementary data Supplementary data include SEM-EDS, TEM-EDS and XANES analysis associated with this article can be found in the online version.

References [1] Adeyemi, A.O., Gadd, G.M., 2005. Fungal degradation of calcium-, lead- and silicon-bearing minerals. Biometals 18, 269-281. [2] Bellion, M., Courbot, M., Jacob, C., Blaudez, D., Chalot, M., 2006. Extracellular and cellular mechanisms sustaining metal tolerance in ectomycorrhizal fungi. FEMS Microbiol. Lett. 254, 173-181. [3] Brunet, J., Varrault, G., Zuily-Fodil, Y., Repellin, A., 2009. Accumulation of lead in the roots of grass pea (Lathyrus sativus L.) plants triggers systemic variation in gene expression in the shoots. Chemosphere 77, 1113-1120. [4] Chakraborty, S., Mukherjee, A., Khuda-Bukhsh, A.R., Das, T.K., 2014. Cadmium-induced oxidative stress tolerance in cadmium resistant Aspergillus foetidus: its possible role in cadmium bioremediation. Ecotox. Environ. Safe. 106, 46-53. [5] Chen, A.W., Zeng, G.M., Chen, G.Q., Liu, L., Shang, C., Hu, X.J., Lu, L.H., Chen, M., Zhou, Y., Zhang, Q.H., 2014. Plasma membrane behavior, oxidative damage, and defense mechanism in Phanerochaete chrysosporium under cadmium stress. Process. Biochem. 49, 589-598. [6] Duan, D.C., Peng, C., Xu, C., Yu, M.G., Sun, L.J., Worden, N., Shi, J.Y., Hu, T.D., 2014. Lead phytoavailability change driven by its speciation transformation after the addition of tea polyphenols (TPs): Combined selective sequential extraction (SSE) and XANES analysis. Plant.Soil. 382, 103-115. [7] Flora, G., Gupta, D., Tiwari, A., 2012. Toxicity of lead: A review with recent updates. Interdiscipl. Toxicol. 5, 47-58. [8] Fomina, M., Burford, E.P., Hillier, S., Kierans, M., Gadd, G.M., 2010. Rock-building fungi. Geomicrobiol. J. 27, 624-629. [9] Gadd, G.M., 1999. Fungal production of citric and oxalic acid: importance in metal speciation, physiology and biogeochemical processes. Adv. Microb. Physiol. 41, 47-92. [10] Michael,G., Bahri-Esfahani, J., Li, Q.W., Rhee, Y.J., Wei, Z., Fomina, M., Liang, X.J., 2014. Oxalate production by fungi: significance in geomycology, biodeterioration and bioremediation. Fungal. Biol. Rev. 28, 36-55. [11] Gazzè, S.A., Saccone, L., Smits, M.M., Duran, A.L., Leake, J.R., Banwart, S.A., Ragnarsdottir, K.V., McMaster, T.J., 2013. Nanoscale observations of extracellular polymeric substances deposition on phyllosilicates by an ectomycorrhizal fungus. Geomicrobiol. J. 30, 721-730. [12] Gharieb, M.M., Gadd, G.M., 2004. Role of glutathione in detoxification of metal (loid)s by Saccharomyces cerevisiae. Biometals 17, 183-188. [13] Jarosz-Wilkolazka, A., Gadd, G.M., 2003. Oxalate production by wood-rotting fungi growing in toxic metal-amended medium. Chemosphere 52, 541-547. 15

[14] Kopittke, P.M., Asher, C.J., Blamey, F.P.C., Auchterlonie, G.J., Guo, Y.N., Menzies, N.W., 2008. Localization and chemical speciation of Pb in roots of signal grass (Brachiaria decumbens) and rhodes grass (Chloris gayana). Environ. Sci. Technol. 42, 4595-4599. [15] Li, N.J., Zeng, G.M., Huang, D.L., Hu, S., Feng, C.L., Zhao, M.H., Lai, C., Huang, C., Wei, Z., Xie, G.X., 2011. Oxalate production at different initial Pb 2+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium. Bioresour. Technol. 102, 8137-8142. [16] Li, Q.W., Csetenyi, L., Gadd, G.M., 2014. Biomineralization of metal carbonates by Neurospora crassa. Environ. Sci. Technol. 48, 14409-14416. [17] Li, Q.W., Gadd, G.M., 2017. Fungal nanoscale metal carbonates and production of electrochemical materials. Microb. Biotechnol. 10, 1131-1136. [18] Li, Z., Wang, F.W., Bai, T.S., Tao, J.J., Guo, J.Y., Yang, M.Y., Wang, S.M., Hu, S.J., 2016. Lead immobilization by geological fluorapatite and fungus Aspergillus niger. J. Hazard. Mater. 320, 386-392. [19] Liang, X.J., Kierans, M., Ceci, A., Hillier, S., Gadd, G.M., 2016. Phosphatase-mediated bioprecipitation of lead by soil fungi. Environ. Microbiol. 18, 219-231. [20] Lin, C.M., Crawford, B.F., Kosman, D.J., 1993. Distribution of cerevisiae: cellular locale and metabolism.

64

Cu in Saccharomyces

J. Gen. Microbiol. 139, 1605-1615.

[21] Liu, S.H., Zeng, G.M., Niu, Q.Y., Liu, Y., Zhou, L., Jiang, L.H., Tan, X.f., Xu, P., Zhang, C., Cheng, M., 2017. Bioremediation mechanisms of combined pollution of PAHs and heavy metals by bacteria and fungi: A mini review. Bioresour. Technol. 224, 25-33. [22] Ma, Y., Rajkumar, M., Zhang, C., Freitas, H., 2016. Beneficial role of bacterial endophytes in heavy metal phytoremediation. J. Environ. Manage. 174, 14-25. [23] Morris, S.J., Allen, M.F., 1994. Oxalate-metabolizing microorganisms in sagebrush steppe soil. Biol. Fert. Soils. 18, 255-259. [24] Muñoz, A.H.S., Corona, F.G., Wrobel, K., Soto, G.M., Wrobel, K., 2005. Subcellular distribution of aluminum, bismuth, cadmium, chromium, copper, iron, manganese, nickel, and lead in cultivated mushrooms (Agaricus bisporus and Pleurotus ostreatus). Biol. Trace. Elem. Res. 106, 265-277. [25] Nouha, K., Kumar, R.S., Tyagi, R.D., 2016. Heavy metals removal from wastewater using extracellular polymeric substances produced by Cloacibacterium normanense in wastewater sludge supplemented with crude glycerol and study of extracellular polymeric substances extraction by different methods. Bioresour. Technol. 212, 120-129. [26] Peng, C., Duan, D.C., Xu, C., Chen, Y.S, Sun, L.J., Zhang, H., Yuan, X.F., Zheng, L.R., Yang, Y.Q., Yang, J.J., Zhen, X.J., Chen, Y.X., Shi, J.Y., 2015. Translocation and biotransformation of CuO nanoparticles in rice (Oryza sativa L.) plants. Environ. Pollut. 197, 99-107. [27] Qian, X.Y., Fang, C.L., Huang, M.S., Achal, V., 2017. Characterization of fungal-mediated carbonate precipitation in the biomineralization of chromate and lead from an aqueous solution and soil. J. Clean. Prod. 164, 198-208. [28] Rhee, Y.J., Hillier, S., Pendlowski, H., Gadd, G.M., 2014. Fungal transformation of metallic lead to pyromorphite in liquid medium. Chemosphere 113, 17-21. [29] Sayer, J.A., Gadd, G.M., 1997. Solubilization and transformation of insoluble inorganic metal compounds to insoluble metal oxalates by Aspergillus niger. Mycol. Res. 101, 653-661. [30] Sheng, G.P., Yu, H.Q., Li, X.Y., 2010. Extracellular polymeric substances (EPS) of microbial 16

aggregates in biological wastewater treatment systems: A review. Biotechnol. Adv. 28, 882-894. [31] Sheng, G.P., Xu, J., Luo, H.W., Li, W.W., Li, W.H., Yu, H.Q., Xie, Z., Wei, S.Q., Hu, F.C., 2013. Thermodynamic analysis on the binding of heavy metals onto extracellular polymeric substances (EPS) of activated sludge. Water Res. 47, 607-614. [32] Sullivan, T.S., Gottel, N.R., Basta, N., Jardine, P.M., Schadt, C.W., 2012. Firing range soils yield a diverse array of fungal isolates capable of organic acid production and Pb mineral solubilization. Appl. Environ. Microb. 78, 6078-6086. [33] Tsekova, K., Christova, D., Ianis, M., 2006. Heavy metal biosorption sites in Penicillium cyclopium. J. Appl. Sci. Environ. Manage. 10, 117-121. [34] van Hees, P.A.W., Tipping, E., Lundström, U.S., 2001. Aluminium speciation in forest soil solution-

modelling the contribution of low molecular weight organic acids. Sci. Total Environ.

278, 215-229. [35] Wang, J.L., Chen, C., 2009. Biosorbents for heavy metals removal and their future. Biotechnol. Adv. 27, 195-226. [36] Wei, Z., Liang, X.J., Pendlowski, H., Hillier, S., Suntornvongsagul, K., Sihanonth, P., Gadd, G.M., 2013. Fungal biotransformation of zinc silicate and sulfide mineral ores. Environ. Microbiol. 15, 2173-2186. [37] Wu, G., Kang, H.B., Zhang, X.Y., Shao, H.B., Chu, L.Y., Ruan, C.J., 2010. A critical review on the bio-removal of hazardous heavy metals from contaminated soils: Issues, progress, eco-environmental concerns and opportunities. J. Hazard. Mater. 174, 1-8. [38] Wu, S.L., Zhang, X., Sun, Y.Q., Wu, Z.X., Li, T., Hu, Y.J., Su, D., Lv, J.T., Li, G., Zhang, Z.S., Zheng, L.L., Zhang, J., Chen, B.D., 2015. Transformation and immobilization of chromium by arbuscular mycorrhizal fungi as revealed by SEM-EDS, TEM-EDS, and XAFS. Environ. Sci. Technol. 49, 14036-14047. [39] Xu, P., Leng, Y., Zeng, G.M., Huang, D.L., Lai, C., Zhao, M.H., Wei, Z., Li, N.J., Huang, C., Zhang, C., Li, F.L., Cheng, M., 2015. Cadmium induced oxalic acid secretion and its role in metal uptake and detoxification mechanisms in Phanerochaete chrysosporium. Appl. Microbiol. Biot. 99, 435-443. [40] Zhang, S.M., Zhang, X.L., Chang, C., Yuan, Z.Y., Wang, T., Zhao, Y., Yang, X.T., Zhang, Y.T., La, G.X., Wu, K., Zhang, Z.M., Li, X.Z., 2016. Improvement of tolerance to lead by filamentous fungus Pleurotus ostreatus HAU-2 and its oxidative responses. Chemosphere 150, 33-39.

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Tables Table. 1. Total removal efficiency of Pb2+ (%), immobilized Pb in mycelium (%) and the Pb content in mycelium (mg g-1 dw) after 7 days of immobilization by fungi P. oxalicum SL2. Values are means ± SD, n = 3. dw, dry weight of biomass. Different letters indicate significant differences among the treatment means (p < 0.05). Pb level

Pb removal efficiency

Pb in mycelium

Pb content in mycelium

(mg L-1)

(%)

(%)

(mg g-1 dw)

100

90.0 ± 2.0 b

72.4% ± 7.7 a

9.5 ± 1.3 c

500

98.3 ± 0.2 a

63.7% ± 3.1 b

49.9 ± 1.0 b

2500

86.2 ± 1.0 c

50.9% ± 4.0 c

155.6 ± 8.0 a

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Figure captions

Fig. 1. The contents of GSH and GSSG in fungi mycelium treated with different levels of Pb2+ (0, 100, 500 and 2500 mg L-1). All experiments were conducted at least in triplicate, the error bars represent the standard deviation, and the different letters represent the significance of different treatments (p < 0.05).

19

20

HIGHLIGHTS



P. oxalicum SL2 has high ability of Pb bioimmobilization.



Secondary minerals were formed during the process of bioimmobilization.



Intracellular Pb-complex were detected by TEM-EDS.



Pb-oxalate, Pb-citrate, PbHPO4 and Pb-GSH analogues was detected by XANES.



Glutathione might play a key role in elimination of the Pb-initialed toxicity.

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