Function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress

Journal Pre-proof Function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress

Huiqing Sun, Meng Meng, Lingran Wu, Xiaomin Zheng, Zhenyuan Zhu, Shuhan Dai PII:

S0141-8130(19)40490-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.02.207

Reference:

BIOMAC 14824

To appear in:

International Journal of Biological Macromolecules

Received date:

19 December 2019

Revised date:

14 February 2020

Accepted date:

19 February 2020

Please cite this article as: H. Sun, M. Meng, L. Wu, et al., Function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress, International Journal of Biological Macromolecules(2020), https://doi.org/10.1016/ j.ijbiomac.2020.02.207

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© 2020 Published by Elsevier.

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Function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress Huiqing Suna,b,c, Meng Menga,b,c, Lingran Wua,b,c, Xiaomin Zhenga,b,c, Zhenyuan Zhua,b,c,∗, Shuhan Daia,b,c a

State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University

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b

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Technology, Tianjin 300457, PR China

College of Food Science and Biotechnology, Tianjin University of Science and

Technology, Tianjin 300457, PR China

Corresponding author： Tel.: +86 2260912390, Fax: +86 2260912390.

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*

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c

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of Science and Technology, Tianjin 300457, PR China

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E-mail address: [email protected]. (Z.Y. Zhu).

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Abstract Trichoderma asperellum ZZY had good tolerance to Pb2+. The polysaccharide contains a functional group which can be effectively combined with metal ions. So in this manuscript, the function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum were further explored. The results indicated that

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the polysaccharide plays vital role in Pb2+ tolerance of Trichoderma asperellum. Most

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lead ions adsorbed on and transferred into mycelia were accumulated in the pure polysaccharide. The proportion of uronic acid and the ratio of main chain in pure

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polysaccharide were increased when the strain under Pb2+ stress. These changes

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increase the contact area of polysaccharides with Pb2+ and the ratio of carboxyl groups

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to provide more binding sites for Pb2+, which is beneficial to reduce the amount of

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free Pb2+ and slow down the toxicity. The response changes in surface morphology and advanced structure of polysaccharide also support the conclusion. The manuscript

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provided theoretical basis for the application on the remediation of lead pollution. It also had contributions to the remediation of heavy metal pollution in the environment and the environmental safety.

Keywords: Trichoderma asperellum; Polysaccharide; Pb2+ tolerance; Mechanism

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1. Introduction The heavy metal, with a density exceeding 5 g/cm3, can inhibit the growth and metabolism of microorganisms and even cause death when the concentration increases to a certain extent [1]. Some microorganisms have tolerance to heavy metal ions and also can absorb and transform heavy metals and their compounds [2, 3].

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Microorganisms can survive in high concentrations of heavy metals, suggesting, that

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they can form an effective defense system to reduce the toxicity of heavy metals. These

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defense systems are based on the metabolites synthesized by the cells in the

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extracellular and intracellular and can be chelated with heavy metal ions [4, 5]. The

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microorganism contains various biological active substances, such as polysaccharides, proteins and so on. These biological active substances contain functional groups such as

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a carboxyl, phosphate, hydroxyl, thiol and amino which can combine with heavy metal

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ions around through electrostatic adsorption, complexation, chelation, ion exchange and covalent adsorption [6, 7], or form extracellular precipitates with heavy metal ions to prevent them from entering the cell [8]. The polysaccharides and proteins play vital roles in the resistance the toxicity [5, 9, 10]. In previous work, it found that the strain of Trichoderma asperellum ZZY, isolated from heavy metal contaminated soil has good tolerance and adsorption to Pb2+ [11]. Trichoderma asperellum ZZY was applied in the National Spark Key Program of China (2015GA610001). It proved that the Trichoderma asperellum ZZY can effectively reduce the contents of heavy metals in grains, fruits and vegetables through competitive

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adsorption on heavy metals with plants. The Tolerance mechanism of Trichoderma asperellum to Pb2+ from response changes of related active ingredients under Pb2+ stress. It revealed that the polysaccharides have significance response changes under Pb2+ stress [12]. But the specific function and mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum on Pb2+ are not clearly clarified.

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In this manuscript, the purpose was to research the function and mechanism of

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polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress.

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The functions of polysaccharide in enhancing the tolerance of Trichoderma asperellum

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on Pb2+ were explored firstly. Then the researches of mechanism were performed with

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researching the response changes of polysaccharide on chemical composition, primary structure, and advanced structure to enhancing the tolerance of Trichoderma asperellum.

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All the experiments are devoted to clarify the mechanism of tolerance of Trichoderma

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asperellum on heavy metals. And it aimed to provide scientific explanations of reducing the contents of heavy metals in grains, fruits and vegetables. 2. Materials and methods

2.1 Materials and chemicals Trichoderma asperellum ZZY, isolated from heavy metal contaminated soil, is a strain with resistant to heavy metals. The deposit number of ZZY is CGMCC No.12071. The monosaccharides standards (D-glucose, D-xylose, D-galactose, L-rhamnose, D-mannose, D-arabinose, D-galacturonic acid and D-glucuronic acid) was bought from Sigma Chemical Co. (St. Louis, MO, USA). The chemical reagents utilized in

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instrumental analysis were chromate-graphically grade. Other chemical reagents in other experiments were analytically grade. 2.2 Preparation of polysaccharide from Trichoderma asperellum mycelia under Pb2+ stress The strain, stored in 4 ◦C, needs to be activated for three generations. Then the

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spore suspension, concentration of 4×108 cells/mL, was prepared with activated strain

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and sterile water and applied to the liquid fermentation as seed liquid. The fermentation

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medium were composed of Potato Dextrose Medium with and Pb(NO3)2. The

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fermentation was performed with seed liquid of 2% at 26◦C and 160 rpm for 72 h. After

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the liquid fermentation, the mycelia of Trichoderma asperellum under stress with Pb2+ of 0 mg/L, 60 mg/L and 200 mg/L were obtained, dried and grinded. The extraction of

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polysaccharides was referred to the followed steps in Fig. S1. The dried powder of

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mycelia were mixed with distilled water (1:20 ratio of raw material to water, w/v) in a round-bottom flask at 80 ◦C for 2 h [13]. The suspension was centrifuged with a centrifugal machine (Sorvall ST16R, Thermo, USA) and the insoluble residue was handled twice as mentioned above. The above supernatant was combined and concentrated with a rotary evaporator (Sy-2000, ShanghaiYa Rong, China) at 60◦C. The extract was disposed by precipitation with 4 times volume of 99% (v/v) ethanol at 4 ◦C for 24 h [14] . The precipitation was collected and dissolved in distilled water. The polysaccharide was deproteinizated with the Sevag composed of trichloromethane and butyl alcohol (v:v=4:1). The solutions of polysaccharide and Sevag with ratio of 5:1

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(v:v) were mixed and shaken for 20 min to remove the proteins [13]. After 12 h, the organic phase contained protein was abandoned. The process of deproteinization was repeated and terminated when there is no protein in organic phase. The solution after deproteinization was lyophilizated to obtain the crude polysaccharides. The crude polysaccharides from mycelia of Trichoderma asperellum under stress with Pb2+ of 0

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mg/L, 60 mg/L and 200 mg/L were named as TAPS-I-0, TAPS-I-60 and TAPS-I-200,

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respectively. The molecular weight (MW) distributions of three crude polysaccharides

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were measured with HPGPC (Agilent-1200) accordance with literature [13]. According

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to the MW distributions, the three crude polysaccharides were further purified by

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ultrafiltration tube with cut-off molecular weight of 100 KDa at 4000 rpm for 15 min [15-17]. The process was repeated to obtain pure polysaccharides. The purity of pure

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polysaccharides was detected with HPGPC. After the peak in HPGPC spectrum was

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single and symmetry, the solution in upper layer was collected to obtain pure polysaccharide. The pure polysaccharides were lyophilized and named as TAPS-II-0, TAPS-II-60 and TAPS-II-200, respectively. The molecular weight of three polysaccharides were calculated with standard curve established with T-series Dextrans [13].

The

three

polysaccharides

were

also

detected

by using

UV-visible

spectral-scanning method [13]. 2.3 Determination of Pb content in mycelia, crude polysaccharide and pure polysaccharide of Trichoderma asperellum without and under Pb2+ stress

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The Pb contents in mycelia without and under Pb2+ stress, crude polysaccharides and pure polysaccharide were measured by atomic absorption spectrophotometry (Shimadzu, Japan). The Pb content in sample was calculated with the equation as followed. 

C  A  50 m  1000

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Where α, C, A, m represented the Pb content in sample (mg/g), the concentration of

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Pb2+ in measured solution (mg/L), the diluted times, the quality of sample (g),

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respectively.

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2.4 Response changes of chemical composition of pure polysaccharide under Pb2+

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stress

The total sugar, reducing sugar, protein and uronic acid in TAPS-II-0, TAPS-II-60

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and TAPS-II-200 were measured with phenol-sulfuric acid method, 3, 5-dinitrosalicylic

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acid (DNS) assay, Bradford (G-250) method and carbazole-sulfuric acid method, respectively [14].

2.5 Response changes of structure of pure polysaccharide under Pb2+ stress 2.5.1 FT-IR analysis The sample of 1.00 mg and dried KBr of 150.00 mg were accurately weighed and mixed. The FT-IR spectrophotometer (Perkin Elmer Corp., USA) was applied to conduct FT-IR analysis according to Sun [13]. 2.5.2 NMR analysis

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The freeze-dried sample (15 mg) was dissolved with D2O of 500 μL. Put the sample in nuclear magnetic tube. The 1H NMR and 13C NMR analysis of sample were performed by using Bruker spectrometer (400 MHz) (Bruker, Germany) [18]. 2.5.3 Monosaccharide composition analysis The monosaccharide analysis of sample was referred to the literature of Tang [14].

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The sample was hydrolyzed with TFA. The products were reduced with NaBH4. Then

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the solution was desalted and the pyridine-Ac2O was used to acetylate the sample. The

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acetylated sample was further analyzed with GC-MS (VARIAN, USA). The standards

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were derivative prepared with above method of D-glucose, D-xylose, D-galactose,

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L-rhamnose, D-mannose, D-arabinose, D-galacturonic acid and D-glucuronic acid. 2.5.4 Periodate oxidation and Smith degradation analysis

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The experiment was referred to the literature [13]. NaIO4 of 0.03 M was used to

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oxidize the sample (25 mg). The experiment was performed in the dark at 4 ◦C. Every 8 h, 0.1 mL of solution was diluted to 25 mL with distilled water and detected by UV spectrophotometer at 223 nm. When the absorbance stabled, the reaction was quenched with ethylene glycol (0.2 mL). The consumption of NaIO4 was calculated with the absorbance. And the production of formic acid was determined by titration with 0.01 M NaOH. The reaction mixture was dialyzed for 36 h and reduced with NaBH4 (30 mg) for 12 h in dark. Adjust the pH of reaction mixture to 5.0-6.0. The reaction mixture was dialyzed and lyophilized. The sample was hydrolyzed and acetylated according literatures [13, 14] The product was measured by GC-MS (VARIAN, USA) according

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to method of Ge [19]. The six monosaccharide standards (D-glucose, D-xylose, D-galactose, L-rhamnose, D-mannose, D-arabinose), erythritol and glycerin were used as standards [20]. 2.5.5 Methylation analysis Before

methylated,

the

sample

was

reacted

with

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1-(3-Dimethyaminopropyl)-3-ethylcarbodiimide Hydrochloride to reduce the carboxyl

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accordance with the literature [14]. Then the products were performed with methylation

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analysis by referring Needs and Selvendran [21]. The degree of methylation was

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detected by FT-IR [22]. After the methylation, the sample was used to hydrolyzation and

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acetylation accordance with Sun [23]. Then the product was dissolved in chloroform and analyzed with GC-MS (VARIAN, USA). The search library was NIST05.

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2.5.6 Congo-red test analysis

The experiment was referenced to Liu [24]. 0.5 mg/mL of the polysaccharide

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samples were mixed with the same volume of 50 mmol/L Congo red solution, and then 1 mol/L NaOH was added to the mixed solutions until the final concentration of NaOH solutions were 0, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 and 0.40 mol/L, respectively. After standing for 10 minutes, the maximum absorption wavelength was recorded by the UV-visible spectrometer in wavelength range of 400-600 nm. 2.5.7 SEM analysis The copper stub was used to fix the sample with conductive plastic. Then the sample was sputtered with gold with vacuum coating instrument and examined with scanning electron microscope (SU1510, Hitachi) according to the literature [25]. 9

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2.5.8 AFM analysis The sample was dissolved and diluted with distilled water to 0.01 μg/mL. The solution of 1 μL was placed on the mica plates and dried in a dry, clean environment. The mica plate was placed on an atomic force microscope (JSPM-5200) for observation. The probe length, elastic constant and scanning head are 225 μm, 20-70 N/m, AC type,

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respectively. The contact force is controlled within the order of 3-4 nN [26].

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2.6 Statistical analysis

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The SPSS 19.0 software was used to the statistical analysis. The data from three

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independent experiments are presented as means ± SD.

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3. Results and Discussion

under Pb2+ stress

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3.1 Extraction and purification of polysaccharide in Trichoderma asperellum mycelia

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To study the mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum under Pb2+ stress, the polysaccharide was extracted and purified from Trichoderma asperellum mycelia. The crude polysaccharides (TAPS-I-0, TAPS-I-60 and TAPS-I-200) were derived from Trichoderma asperellum mycelia under Pb2+ stress of 0 mg/L, 60 mg/L and 200 mg/L. The three crude polysaccharides were all powdery solid with brown. The yields of TAPS-I-0, TAPS-I-60 and TAPS-I-200 from mycelia were 7.31%, 7.54% and 7.83%, respectively. The three crude polysaccharides were further purified to obtain pure polysaccharides (TAPS-II-0, TAPS-II-60 and TAPS-II-200). The HPGPC spectra of pure polysaccharides were presented in Fig. 1(a-c). The peaks were

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both single and symmetrical, which indicate that the three pure polysaccharides were all pure polysaccharide. After lyophilization, the TAPS-II-0, TAPS-II-60 and TAPS-II-200 were both white powdery solid with yields of 68.67%, 67.43% and 70.32% (from crude polysaccharides), respectively, from crude polysaccharides. The final yields of TAPS-II-0, TAPS-II-60 and TAPS-II-200 from mycelia were 5.02%, 5.08 % and 5.51%,

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respectively. The UV-visible scanning spectra of three pure polysaccharides were

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demonstrated in Fig. S2(a-c). The three pure polysaccharides all had absorption peak in

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260-280 nm which can prove that there are carbonyl groups, peptides or protein in

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them.

under Pb2+ stress

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3.2 Function of polysaccharides on enhancing tolerance of Trichoderma asperellum

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The Pb content in mycelia, crude polysaccharides and pure polysaccharide of

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Trichoderma asperellum without and under Pb2+ stress were measured to study the function of polysaccharides on enhancing tolerance of Trichoderma asperellum under Pb2+ stress. The results were illustrated in Table 1. From the results of detection, the Pb had not been detected in the mycelia, crude polysaccharides and pure polysaccharide without stress of Pb2+ which is the opposite of that under Pb2+ stress. Under Pb2+ stress, the adsorption capacity of Pb in mycelia increased gradually with the increase of Pb2+ concentration. Moreover, Pb was founded in both crude polysaccharides and pure polysaccharide under Pb2+ stress. From the previously results, the yields of TAPS-I-60 and TAPS-II-60, TAPS-I-200 and TAPS-II-200 were 7.54% and 67.43%, 7.83% and

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70.32%, respectively. After calculation, when Pb2+ concentration was 60 mg/L, 55.22% of Pb in mycelia was accumulated to TAPS-I-60 and 86.49% of Pb in TAPS-I-60 was accumulated to TAPS-II-60. When Pb2+ concentration was 200 mg/L, 31.29% of Pb in mycelia was accumulated to TAPS-I-200 and 89.70% of Pb in TAPS-I-200 was accumulated to TAPS-II-200. All the results indicated that the crude polysaccharides

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can interact with Pb2+ to adsorb or transform it when Pb2+ transferred into mycelia.

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Most of the Pb contained in TAPS-I-60 and TAPS-I-200 was accumulated to

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TAPS-II-60 and TAPS-II-200. It indicated that the pure polysaccharide was the main

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ingredient which can interact with Pb2+ in crude polysaccharide under Pb2+ stress. The

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polysaccharide contains functional groups such as hydroxyl, carboxyl and amino group which can be effectively combined with metal ions [27]. Li et al. [9] analyzed the

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biochemical changes of polysaccharides and proteins in extracellular polysaccharide

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under Pb (II) stress in Rhodotorula mucilaginosa. It concluded that the survived cells displayed a stress response and secreted more polysaccharides to resist Pb 2+ toxicity at high Pb level. Kopycinska et al. [28] explored the role of extracellular polysaccharide in the response of Rhizobium leguminosarum, free-living and during symbiosis to zinc stress. The zinc stress can stimulate extracellular polysaccharide synthesis and it effectively protected the cells to against the zinc stress. The polysaccharides can provide more adsorption sites for Pb2+ and reduce the toxicity on cells. All results indicated that the polysaccharide plays an important role in Pb2+ tolerance of Trichoderma asperellum. 3.3 Mechanism of polysaccharide on enhancing tolerance of Trichoderma asperellum

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under Pb2+ stress The polysaccharides can be effectively combined with metal ion through electrostatic adsorption, complexation, chelation, ion exchange and chemical adsorption due to the functional-groups [29]. And the advanced structure also has influence on the combination of polysaccharide and metal ions [30]. To further clarify the mechanism of

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polysaccharide enhancing the tolerance of Trichoderma asperellum to Pb2+, the

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responses changes of polysaccharide in chemical components and structure were

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studied.

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3.3.1 Response changes in molecular weight of pure polysaccharide under Pb2+ stress

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The HPGPC spectra of three pure polysaccharides were presented in Fig. 1(a-c). The retention times of TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 8.902 min, 8.928

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min and 9.186 min, respectively. The equation of standard curve was: y = -0.334x +

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9.4008，R² = 0.993 (y = lg MW，x = Retention time). The MW of TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 2.673×103 kDa, 2.618×103 kDa and 2.147×103 kDa, respectively which indicated that the MW of polysaccharide was decreased when the Trichoderma asperellum under stress of Pb2+.

3.3.2 Response changes of chemical composition of pure polysaccharide under Pb2+ stress The contents of total sugar, reducing sugar, protein and uronic acid in three pure polysaccharides were measured. The Table 1 indicated that the contents of polysaccharides, uronic acid and protein were in TAPS-II-0, TAPS-II-60 and

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TAPS-II-200 were as follows: 62.81%±0.45%, 20.65%±0.50% and 13.78%±0.66%; 68.87%±0.37%, 21.56%±0.62% and 16.89%±0.57%; 70.84%±0.44%, 28.78%±0.44% and 14.34%±0.56%. All the results demonstrated that the three polysaccharides were glycopeptide which also contain uronic acid. The results were consistent with the results of purity examination. Comparing the chemical compositions, when the strain under

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stress of Pb2+, the contents of polysaccharides and uronic acid of polysaccharides were

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increased, and protein content decreased. The increase of uronic acid content and ratio

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can increase the proportion of carboxyl groups in the polysaccharides. The carboxyl

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groups can provide more binding sites for Pb2+.

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3.3.3 Response changes in functional group and glycosidic bond configuration of pure polysaccharide under Pb2+ stress

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The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were analyzed with FT-IR analysis,

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NMR analysis. The FT-IR spectra of TAPS-II-0, TAPS-II-60 and TAPS-II-200 were demonstrated in Fig. 2. The signals of -OH stretching vibration, C-H stretching vibration, C-H bending vibration and C-H bending vibration, characteristic bands of polysaccharides, can be obviously observed at 3405 cm-1 (3416 cm-1, 3416 cm-1), 2974 cm-1 ( 2923 cm-1 , 2924 cm-1), 2925 cm-1 (2853 cm-1, 2853 cm-1) and 1383 cm-1 (1383 cm-1, 1383 cm-1), respectively [31, 32]. The bands at 1647 cm-1 (1644 cm-1 , 1651 cm-1) were attributed to the stretching vibration of C=O from -COOH and CO-NH [33, 34]. The band of C-N stretching vibration (1414 cm-1/1415 cm-1 /1416 cm-1) indicates the presence of CO-NH. The bands at 1160-950 cm-1 demonstrated that the TAPS-II-0,

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TAPS-II-60 and TAPS-II-200 all contained pyran configuration [35]. The peak at 879 cm-1 (880 cm-1) revealed the presence of β-anomeric configuration [36]. All the results indicated that the TAPS-II-0, TAPS-II-60 and TAPS-II-200 were glycopeptide with pyranose and β-anomeric configuration. The spectra of 1H NMR and 13C NMR of TAPS-II-0, TAPS-II-60 and TAPS-II-200

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were demonstrated in Fig. S3 (a-f). In the spectrum of 1H NMR, the chemical shift of

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the proton at the C-1 in α-glycosidic bond is greater than 5.0 ppm, while in β-glycosidic

-p

bond is less than 5.0 ppm [24, 37]. The spectrum of 1H NMR of TAPS-II-0 was

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demonstrated in Fig. S3a. The signal of deuterium oxide (solvent) was at 4.787 ppm.

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The chemical shifts of the H-1 proton in TAPS-II-0 were distributed at 4.353-4.899 ppm, 5.064-5.491 ppm, which indicates that the TAPS-II-0 contains both α-glycosidic bond

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configuration and β-glycosidic bond configuration. The peaks at 0.500-1.500 ppm and

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2.000 ppm were attributed to protons on alkanes, alkenes, ester bonds, and amide bonds. The spectrum of 13C NMR of TAPS-II-0 was shown in Fig. S3b. The peaks at low field from 160-180 ppm are attributed to carboxyl which indicates that the TAPS-II-0 is an acidic polysaccharide [14]. The signals at 129.58 ppm and 127.84 ppm indicated the presence of aromatic ring in TAPS-II-0 [38]. There are multiple signals at not only at 90-103 ppm (98.13-102.88 ppm), but also at 103-110 ppm (103.72-107.88 ppm), which proves that TAPS-II-0 contains glycosidic bonds of α-type and β-type. In the spectrum of 13C NMR, the signals of the anomeric carbon in α-type glycoside are at 90 ppm-103 ppm in the polysaccharide, while in β-type glycoside at 103 ppm-110 ppm [24]. The

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peaks at 60 ppm to 90 ppm were characteristic signals of carbons except for anomeric carbon. The multiple signals at 20-40 ppm indicated that the existence of methyl, methylene, etc. in TAPS-II-0 [38]. In conclusion, the TAPS-II-0 is a glycopeptide containing aromatic hydrocarbon and acidic sugars, and both with α-type glycosidic bonds and β-type glycosidic bonds. The signals in 1H NMR and

13

C NMR of

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TAPS-II-60 and TAPS-II-200 (Fig. S3(b-c)) were also analyzed and the results indicated

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that TAPS-II-60 and TAPS-II-200 are glycopeptides containing aromatic hydrocarbon

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and acidic sugars, and both with α-type glycosidic bonds and β-type glycosidic bonds.

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From above, it revealed that the functional group and glycosidic bond configuration of

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pure polysaccharide had no significant changes under Pb2+ stress. 3.3.4 Response changes in monosaccharide of pure polysaccharide under Pb2+ stress

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The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were degraded, acetylated and

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analyzed with GC-MS. The GC-MS spectra of standards and samples were illustrated in Fig. 3a, Fig. 3b, Fig. 3c and Fig. 3d, respectively. Compared with Fig. 3a, the Fig. 3b, Fig. 3c and Fig. 3d indicated that the TAPS-II-0, TAPS-II-60 and TAPS-II-200 were both composed of D-mannose, D-glucose, D-galactose, D-galacturonic acid and D-glucuronic acid with ratios of 26.14 : 14.50 : 36.25 : 12.15 : 10.95, 39.52 : 14.60 : 16.96 : 16.33 : 12.59 and 47.95 : 4.28 : 9.59 : 24.42 : 13.77, respectively. The contents of uronic acid in TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 23.10%, 28.92% and 38.19%, respectively. It revealed that, under Pb2+ stress the kinds of monosaccharide have no response changes but the ratio was changed. The content of uronic acid was

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increased in the high concentration of Pb2+. 3.3.5 Response changes in glycosidic bonds of pure polysaccharide under Pb2+ stress The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were analyzed with periodate oxidation and Smith degradation analysis and methylation analysis. The regression equation of standard curve of periodate oxidation was as followes: y = 0.0091x - 0.0111,

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R2 = 0.9991, (x = the concentration of sodium periodate, y = absorbance in 223 nm).

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The absorbance of reaction system reaches equilibrium after 15 days which indicated

-p

the termination of reaction. After calculating, the sodium periodate remaining amounts

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in the reaction of TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 0.7327 mmoL, 0.7169

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mmoL and 0.7190 mmoL, respectively. The consumptions of sodium periodates were 0.0173 mmoL, 0.0331 mmoL and 0.0310 mmoL with the saccharides residues of 0.0377

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mmoL (TAPS-II-0), 0.0494 mmoL（TAPS-II-60）and 0.0407 mmoL (TAPS-II-200),

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respectively. It means that the consumptions of sodium periodate with per mole of sugar residue were both less than 1 mol in three polysaccharides. The results indicated that the presence of glycosidic bonds of (1→3), (1→3, 6), (1→2, 3), (1→2, 4), (1→3, 4) or (1→2, 3, 4) in three polysaccharides [39]. The yields of formic acid were 0.0065 mmoL, 0.0063 mmoL and 0.0046 mmoL, respectively, which indicated that the existence of glycosidic bonds of (1→6) or (1→) in TAPS-II-0, TAPS-II-60 and TAPS-II-200 [13]. The sodium periodate consumption and formic acid was over 2 : 1. It indicated there may exist glycosidic bonds of (1→2), (1→4) and (1→4, 6) in three polysaccharides [13]. The GC-MS spectra of derivatives of glycerol, erythritol, monosaccharides,

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TAPS-II-0, TAPS-II-60 and TAPS-II-200 were illustrated in Fig. S4a, Fig. S4b, Fig. S4c, Fig. 4a, Fig. 4b and Fig. 4c, respectively. Compared with the standards, the products of TAPS-II-0, TAPS-II-60 and TAPS-II-200 both contained glycerin, erythritol, mannose, glucose,

galactose

with

3.18:55.92:5.74:12.62:22.54

and

ratio

of

6.97:16.82:7.73:37.42:31.06,

3.43:60.81:3.85:12.69:19.21,

respectively.

The

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appearance of erythritol and glycerol demonstrated the existence of (1→2) or (1→2, 6),

ro

(1→4), (1→4, 6), (1→6) or (1→) glycosidic bonds in three polysaccharides [24]. The

-p

appearance of mannose, glucose and galactose indicated that the existence of (1→3),

re

(1→2, 3), (1→3, 4), (1→3, 6) or (1→ 2, 3, 4) glycosidic bonds in three polysaccharides

lP

[40]. The results were consistent with periodate oxidation. The methylations of TAPS-II-0, TAPS-II-60 and TAPS-II-200 repeated 10, 10 and

na

12 times, respectively. The FT-IR spectra were illustrated in Fig. S5(a-c). The shape of

Jo ur

the peak of -OH at 3000 cm-1-3600 cm-1 changed broad-flat to narrow and which indicated that the characteristic absorption peak caused by C-H stretching vibration in -CH3 at 2900 cm-1 is obviously enhanced. All the results indicated that the TAPS-II-0, TAPS-II-60 and TAPS-II-200 were completely methylated. The fully methylated sample was degraded, reduced, derivatizated and detected with GC-MS. Five main products were both matched from the GC-MS spectra of three polysaccharides and the results were analyzed and summarized in Table 2. It indicated the main products of TAPS-II-0, TAPS-II-60 and TAPS-II-200 all contained 2, 4, 6-tri-O-Me-D-galactitol, 2, 3, 4, 6-tetra-O-Me-D-glucitol, 2, 4, 6-tri-O-Me-D-glucitol, 2, 3, 6-tri-O-Me-D-mannitol, 2,

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6-di-O-Me-D-mannitol

with

molar

ratios

of

2.87:0.71:2.21:2.33:0.79,

2.27:0.41:2.21:3.33:0.59 and 2.47:0.23:1.31:3.53:0.33, respectively. It indicated that the three polysaccharides were composed of (1→3)-galactose, (1→3)-galacturonic acid, (1→)-glucose, (1→3)-glucose, (1→3)-glucuronic acid, (1→4)-mannose, (1→3, 4)-mannose. The results were unanimous with periodate oxidation and Smith

of

degradation. The main chain was (1→4)-mannose and the branches were composed of

ro

glucuronic acid, mannose, glucose, galactose, and galacturonic acid linked with (1→3)

-p

in three polysaccharides. The ratio of main chain was proportional to the concentration

re

of Pb2+. The ratio of main chains in TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 2.33,

lP

3.33 and 3.53, respectively. The results indicated that the length of the main chain

under Pb2+ stress.

na

increased, but the amounts of branches decreased when the Trichoderma asperellum

Jo ur

3.3.6 Response changes in surface morphology and advanced structure of pure polysaccharide under Pb2+ stress

The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were analyzed with Congo-red test, SEM and AFM. The results of Congo-red test analysis were demonstrated in Fig. 5. When the NaOH is 0.10 M, the maximum wavelengths of Congo red + sample were all higher than Congo red. It indicates that the combination of sample and Congo red has caused a red shift in the absorbance of Congo red. However, with the increase of NaOH concentration, the maximum wavelengths of the Congo red + TAPS-II-0 complex, Congo red + TAPS-II-60 complex and Congo red + TAPS-II-200 complex gradually

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decreased. When the NaOH is 0.15 M, the maximum wavelengths of Congo red + sample complex were gradually approached to Congo red. It revealed that the TAPS-II-0, TAPS-II-60 and TAPS-II-200 have a triple helix structure in a weakly alkaline solution [40]. The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were observed by using SEM with magnification of ×500 and ×5000. The images were illustrated in Fig.

of

S6. The TAPS-II-0 was loose lump structure with numerous voids when the

ro

magnification is ×500. It can be more clearly observed that spherical particles adhere to

-p

the surface in TAPS-II-0 when the magnification is ×5000. Different with the block with

re

rough and honeycombed surface of TAPS-II-0, the surface of TAPS-II-60 and

lP

TAPS-II-200 were both relatively smooth sheet attached with spherical particles. The results of AFM were demonstrated in Fig. 6. The Fig. 6(a, c, e), Fig. 6(b, d, f) and Fig.

na

S7(a, b, c) were the planar image, three-dimensional image and linear image of

Jo ur

TAPS-II-60 (TAPS-II-200), respectively. The Fig. 6(a, c, e) indicated that the morphology of three polysaccharides in mica flakes were both irregular linear forms. The Fig. 6(b, d, f) indicated that the surface of three polysaccharides were rugged and connected by island-like protrusions which tightness degree increased with the concentration of Pb2+. From the above results, it can be inferred that the polysaccharide chains of three polysaccharides were all curled and rotated and the trend is very similar to that of the spiral. In conclusion, the TAPS-II-0, TAPS-II-60 and TAPS-II-200 were both glycopeptide and also contain aromatic hydrocarbon and acidic sugars. The

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monosaccharides in three polysaccharides were pyranose linked with α-type and β-type glycosidic bonds. The TAPS-II-0, TAPS-II-60 and TAPS-II-200 were composed of D-mannose, D-glucose, D-galactose, D-galacturonic acid and D-glucuronic acid with ratio of 26.14 : 14.50 : 36.25 : 12.15 : 10.95, 39.52 : 14.60 : 16.96 : 16.33 : 12.59 and 47.95 : 4.28 : 9.59 : 24.42 : 13.77, respectively. The contents of uronic acid in

of

TAPS-II-0, TAPS-II-60 and TAPS-II-200 were 23.10%, 28.92% and 38.19%,

ro

respectively. In three polysaccharides, the main chains were both (1→4)-mannose and

-p

the branches were composed of mannose, glucose, glucuronic acid, galactose,

re

galacturonic acid linked with (1→3). The ratio of main chains in TAPS-II-0,

lP

TAPS-II-60 and TAPS-II-200 were 2.33, 3.33 and 3.53, respectively. After the comparison of related dates of TAPS-II-0, TAPS-II-60 and TAPS-II-200,

na

the proportion of uronic acid was increased with the increase of Pb2+ concentration

Jo ur

which was consistent with chemical components determination of polysaccharide. With the increase of Pb2+, the proportion of (1→4)-mannose in polysaccharides increased but proportions of (1→3, 4)-mannose and (1→)-glucose decreased. It indicated that the length of the main chain increased, but the amount of branches decreased when the Trichoderma asperellum under Pb2+ stress. The increase of uronic acid content and ratio can increase the proportion of carboxyl groups in the polysaccharides. The carboxyl groups can provide more binding sites for Pb2+ [41]. Under Pb2+ stress, the increase of main chain and uronic acid content of polysaccharide can increase the contact area of polysaccharides with Pb2+ and the ratio of carboxyl groups to provide more binding

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sites for Pb2+, which is beneficial to reduce the amount of free Pb2+ and slow down the toxicity of Pb2+ to the Trichoderma asperellum. In conclusion, under Pb2+ stress, the response changes in primary structure of polysaccharide can enhance the tolerance of Trichoderma asperellum on Pb2+. The response changes in surface morphology and advanced structure of polysaccharide under Pb2+ stress also support the conclusion. The

of

results of AFM revealed that the surface of TAPS-II-0, TAPS-II-60 and TAPS-II-200

ro

were rugged and connected by island-like protrusions which tightness degree increased

-p

with the concentration of Pb2+. This response changes may be caused by the interaction

re

of polysaccharide and Pb2+. The interaction of polysaccharide and Pb2+ can neutralize

lP

the negative charge on the surface of polysaccharide, reduce the mutual repulsion between different groups and sugar chains and cause “concentration” phenomenon in

na

advanced structure. All the results revealed that the polysaccharide plays an important

Jo ur

role and has significant importance on enhancing the tolerance of Trichoderma asperellum on Pb2+. The results were consistent with the literatures. Cho et al. [42] reported that the -COOH in the polysaccharides was the primary components/sites of divalent metal. The carboxyl groups can provide more binding sites for Pb2+. And Gutsch et al. [43] studied the influence of long-term cadmium exposure on the composition of pectic polysaccharides in the cell wall of Medicago sativa stems. It revealed that the long-term cadmium exposure increased presence of xylogalacturonan in pectic polysaccharides. And the changes in the composition and properties of cell wall polysaccharides which can alter the function of the cell wall to protect the plant

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from Cd-induced damages. Enhancing the tolerance of microorganisms can increase the survival rates under higher concentrations of heavy metal ions and expand the application range in remediating heavy metal pollution. It has contributions to the remediation of heavy metal pollution in the environment and the environmental safety. 4. Conclusion

of

In conclusion, the polysaccharide played a vital role on enhancing tolerance of

ro

Trichoderma asperellum under Pb2+ stress. The high concentration of Pb2+ can promote

-p

the synthesis of polysaccharide in Trichoderma asperellum mycelia. And the pure

re

polysaccharide can interacte with Pb2+ to adsorb or transform it. Under Pb2+ stress, the

lP

polysaccharide had response changes in chemical composition, primary structure and advance structure to reduce the amount of free Pb2+ and enhance the tolerance of

na

Trichoderma asperellum. The manuscript clarified the mechanism of polysaccharide on

Jo ur

enhancing tolerance of Trichoderma asperellum on Pb2+ and provided scientific explanations of reducing the contents of heavy metals in grains, fruits and vegetables. It has theoretical significance to the remediation and prevention of heavy metal pollution in water, soil and food. It also provided theoretical basis for the application on the repair of lead pollution. With the study of response changes of polysaccharide, it will be significant to further research the regulation of polysaccharide synthesis from perspective of gene expression and enzymology. Declarations of Interest None.

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Acknowledgments This work was financially supported by the National Natural Science Foundation of China (31871791), Technology Program of Tianjin, China, (18ZYPTJC00020), the key program of the Foundation of Tianjin Educational Committee (2018ZD06) and the National Spark Key Program of China (2015GA610001).

of

Appendix A. Supplementary data

ro

Reference

environment:

A

review

of

co-toxic

mechanisms

leading

to

re

aquatic

-p

[1] P.T. Gauthier, W.P. Norwood, E.E. Prepas, G.G. Pyle, Metal-PAH mixtures in the

lP

more-than-additive outcomes, Aquat. Toxicol. 154(5) (2014) 253-269. [2] S. Choudhary, P. Sar, Characterization of a metal resistant Pseudomonas sp. isolated

na

from uranium mine for its potential in heavy metal (Ni2+, Co2+, Cu2+, and Cd2+)

Jo ur

sequestration, Bioresource Technology 100(9) (2009) 2482-2492. [3] M.H. Abd-Alla, F.M. Morsy, A.W.E. El-Enany, T. Ohyama, Isolation and characterization of a heavy-metal-resistant isolate of Rhizobium leguminosarum bv. viciae potentially applicable for biosorption of Cd

2+

and Co

2+

, International

Biodeterioration & Biodegradation 67(2) (2012) 48-55. [4] G.M. Zeng, D.L. Huang, G.H. Huang, T.J. Hu, X.Y. Jiang, C.L. Feng, H.L. Liu, Composting of lead-contaminated solid waste with inocula of white-rot fungus, Bioresource Technology 98(2) (2007) 320-326. [5] Z. Teng, W. Shao, K. Zhang, Y. Huo, J. Zhu, M. Li, Pb biosorption by Leclercia

24

Journal Pre-proof

adecarboxylata: Protective and immobilized mechanisms of extracellular polymeric substances, Chemical Engineering Journal 375 (2019) 122113. [6] M. Rajkumar, N. Ae, M.N.V. Prasad, H. Freitas, Potential of siderophore-producing bacteria for improving heavy metal phytoextraction, Trends in Biotechnology 28(3) (2010) 142-149.

of

[7] J.J. Harrison, H. Ceri, R.J. Turner, Multimetal resistance and tolerance in microbial

ro

biofilms, Nat. Rev. Microbiol. 5(12) (2007) 928-938.

-p

[8] M.R. Bruins, S. Kapil, F.W. Oehme, Microbial resistance to metals in the

re

environment, Ecotoxicology & Environmental Safety 45(3) (2000) 198-207.

lP

[9] Li, Z.Q. Jiang, S.S. Chen, T. Wang, L. Jiang, M.X. Wang, Z. Li, Biochemical changes of polysaccharides and proteins within EPS under Pb(II) stress in

na

Rhodotorula mucilaginosa, Ecotoxicology and environmental safety 174 (2019)

Jo ur

484-490.

[10] S. Kumari, S. Mahapatra, S. Das, Ca-alginate as a support matrix for Pb(II) biosorption with immobilized biofilm associated extracellular polymeric substances of Pseudomonas aeruginosa N6P6, Chemical Engineering Journal 328 (2017) 556-566. [11] Z.Y. Zhu, Q.Y. Song, F.Y. Dong, Taxonomy characterization and plumbum bioremediation of novel fungi, Journal of Basic Microbiology 58(4) (2018) 368-376. [12] H. Sun, L. Wu, Y. Hao, C. Liu, L. Pan, Z. Zhu, Tolerance mechanism of

25

Journal Pre-proof

Trichoderma asperellum to Pb2+: response changes of related active ingredients under Pb2+ stress, RSC Advances 10(9) (2020) 5202-5211. [13] H.Q. Sun, Z.Y. Zhu, Y.L. Tang, Y.Y. Ren, Q.Y. Song, Y. Tang, Y.M. Zhang, Structural characterization and antitumor activity of a novel Se-polysaccharide from selenium-enriched Cordyceps gunnii, Food & function 9(5) (2018)

of

2744-2754.

ro

[14] Y. Tang, Y. Zhu, Y. Liu, H.Q. Sun, Y.M. Zhang, Chemical structure and anti-aging

-p

bioactivity of an acid polysaccharide from Rose buds, Food & function 9(2) (2018)

re

2300-2312.

lP

[15] L. Zhang, M. Wang, Polyethylene glycol-based ultrasound-assisted extraction and ultrafiltration separation of polysaccharides from Tremella fuciformis (snow

na

fungus), Food and Bioproducts Processing 100 (2016) 464-468.

Jo ur

[16] G. Ma, W. Yang, Y. Fang, N. Ma, F. Pei, L. Zhao, Q. Hu, Antioxidant and cytotoxicites

of

Pleurotus

eryngii

residue

polysaccharides

obtained

by

ultrafiltration, Lwt 73 (2016) 108-116. [17] J.H. Xie, M.Y. Shen, S.P. Nie, Q. Zhao, C. Li, M.Y. Xie, Separation of water-soluble polysaccharides from Cyclocarya paliurus by ultrafiltration process, Carbohydr Polym 101 (2014) 479-83. [18] S.S. Van Leeuwen, B.R. Leeflang, G.J. Gerwig, J.P. Kamerling, Development of a 1 H

NMR

structural-reporter-group

concept

for

the

primary

structural

characterisation of α-d-glucans, Carbohydrate Research 343(6) (2012) 1114-1119.

26

Journal Pre-proof

[19] Y. Ge, Y. Duan, G. Fang, Y. Zhang, S. Wang, Polysaccharides from fruit calyx of Physalis alkekengi var. francheti: Isolation, purification, structural features and antioxidant activities, Carbohyd. Polym. 77(2) (2009) 188-193. [20] J.S. Dixon, D. Lipkin, Spectrophotometric determination of vicinal glycols: Application to the determination of ribofuranosides, Anal. Chem. 26(6) (1954)

of

1092-1093.

ro

[21] P.W. Needs, R.R. Selvendran, Avoiding oxidative degradation during sodium

-p

hydroxide-methyl iodide-mediated carbohydrate methylation in dimethyl sulfoxide,

re

Carbohydrate Research 245(1) (1993) 1-10.

lP

[22] J. Gooneratne, P.W. Needs, P. Ryden, R.R. Selvendran, Structural features of cell wall polysaccharides from the cotyledons of mung bean Vigna radiata, Carbohyd.

na

Res. 265(1) (1994) 61-77.

Jo ur

[23] H.Q. Sun, Z.Y. Zhu, X.Y. Yang, M. Meng, L.C. Dai, Y.M. Zhang, Preliminary characterization

and

immunostimulatory activity of

a

novel

functional

polysaccharide from Astragalus residue fermented by Paecilomyces sinensis, RSC Advances 7(38) (2017) 23875-23881. [24] X.C. Liu, Z.Y. Zhu, Y.L. Tang, M.F. Wang, Z. Wang, A.J. Liu, Y.M. Zhang, Structural properties of polysaccharides from cultivated fruit bodies and mycelium of Cordyceps militaris, Carbohydrate Polymers 142 (2016) 63-72. [25] L.S. Lai, D.H. Yang, Rheological properties of the hot-water extracted polysaccharides in Ling-Zhi (Ganoderma lucidum), Food Hydrocolloids 21(5-6)

27

Journal Pre-proof

(2007) 739-746. [26] Z.Y. Zhu, W. Pang, Y.Y. Li, X.R. Ge, L.J. Chen, X.C. Liu, Y.M. Zhang, Effect of ultrasonic

treatment

on

structure

and

antitumor

activity

of

mycelial

polysaccharides from Cordyceps gunnii, Carbohydrate Polymers 114 (2014) 12-20. [27] D.R. Crist, R.H. Grist, J.R. Martin, J.R. Watson, Ion exchange systems in

of

proton-metal reactions with algal cell walls, Fems Microbiol. Rev. 14(4) (1994)

ro

309-313.

-p

[28] M. Kopycinska, P. Lipa, J. Ciesla, M. Koziel, M. Janczarek, Extracellular

re

polysaccharide protects Rhizobium leguminosarum cells against zinc stress in vitro

lP

and during symbiosis with clover, Env. Microbiol. Rep. 10(3) (2018) 355-368. [29] R. De Philippis, G. Colica, E. Micheletti, Exopolysaccharide-producing

na

cyanobacteria in heavy metal removal from water: molecular basis and practical

697-708.

Jo ur

applicability of the biosorption process, Appl. Microbiol. Biotechnol. 92(4) (2011)

[30] K. Nowak, A. Wiater, A. Choma, D. Wiacek, A. Bieganowski, M. Siwulski, A. Wasko, Fungal (1-->3)-alpha-d-glucans as a new kind of biosorbent for heavy metals, International journal of biological macromolecules 137 (2019) 960-965. [31] M.B. Romdhane, A. Haddar, I. Ghazala, K.B. Jeddou, C.B. Helbert, S. Ellouz-Chaabouni, Optimization of polysaccharides extraction from watermelon rinds: Structure, functional and biological activities, Food Chemistry 216 (2017) 355-364.

28

Journal Pre-proof

[32] M. Mujtaba, A.M. Salaberria, M.A. Andres, M. Kaya, A. Gunyakti, J. Labidi, Utilization of flax (Linum usitatissimum) cellulose nanocrystals as reinforcing material for chitosan films, International journal of biological macromolecules 104(Pt A) (2017) 944-952. [33] F. Nejatzadeh-Barandozi, S. Enferadi, FT-IR study of the polysaccharides isolated

of

from the skin juice, gel juice, and flower of Aloe vera tissues affected by fertilizer

ro

treatment, Organic and Medicinal Chemistry Letters 2(1) (2012) 33-41.

-p

[34] T. Wang, Q. Hu, M. Zhou, Y. Xia, M.P. Nieh, Y. Luo, Development of "all natural"

re

layer-by-layer redispersible solid lipid nanoparticles by nano spray drying

lP

technology, European journal of pharmaceutics and biopharmaceutics : official

(2016) 273-285.

na

journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V 107

Jo ur

[35] J. Liu, Y.P. Zhao, Q.X. Wu, A. John, Y.M. Jiang, J.L. Yang, B. Yang, Structure characterisation of polysaccharides in vegetable "okra" and evaluation of hypoglycemic activity, Food Chemistry 242 (2018) 211-216. [36] W.F. Yang, Y. Wang, X.P. Li, P. Yu, Purification and structural characterization of Chinese yam polysaccharide and its activities, Carbohydrate Polymers 117(5) (2015) 1021-1027. [37] H.K. Jeong, D. Lee, H.P. Kim, S.H. Baek, Structure analysis and antioxidant activities of an amylopectin-type polysaccharide isolated from dried fruits of Terminalia chebula, Carbohyd. Polym. 211 (2019) 100-108.

29

Journal Pre-proof

[38] N. YC, Structural Identification of Organic Compounds with Spectroscopic Techniques, Science Press, Beijing, China, 2010. [39] H.Q. Sun, W.W. Song, L.J. Zhang, X.Y. Yang, Z.Y. Zhu, R.C. Ma, D.Y. Wang, Structural characterization and inhibition on α-glucosidase of a novel oligosaccharide from barley malt, Journal of Cereal Science 82 (2018) 82-93.

of

[40] Y. Zhang, M. Gu, K.P. Wang, Z.X. Chen, L.Q. Dai, J.Y. Liu, F. Zeng, Structure,

-p

edodes, Fitoterapia 81(8) (2010) 1163-1170.

ro

chain conformation and antitumor activity of a novel polysaccharide from Lentinus

re

[41] H. Li, M. Wei, W. Min, Y. Gao, X. Liu, J. Liu, Removal of heavy metal Ions in

lP

aqueous solution by Exopolysaccharides from Athelia rolfsii, Biocatalysis and Agricultural Biotechnology 6 (2016) 28-32.

na

[42] D.H. Cho, K.H. Chu, E.Y. Kim, Lead uptake and potentiometric titration studies

Jo ur

with live and dried cells of Rhodotorula glutinis, World Journal of Microbiology & Biotechnology 27(8) (2011) 1911-1917. [43] A. Gutsch, K. Sergeant, E. Keunen, E. Prinsen, G. Guerriero, J. Renaut, A. Cuypers, Does long-term cadmium exposure influence the composition of pectic polysaccharides in the cell wall of Medicago sativa stems?, BMC Plant Biol. 19(1) (2019) 10.1186/s12870-019-1859-y.

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Table 1 Pb contents in samples without and under Pb2+ stress and contents of polysaccharides, uronic acids and proteins in pure polysaccharide Polysaccharide

Uronic acid content

Protein content in

in pure

pure polysaccharide

polysaccharide (%)

(%)

--

--

--

--

--

--

62.81±0.45

20.65±0.50

13.78%±0.66

6.70±0.23

--

--

--

49.03±0.53

--

--

--

Pure polysaccharide (TAPS-II-60)

62.04±0.32

68.87±0.37

21.56%±0.62

16.89%±0.57

Mycelia

21.30±0.96

--

--

--

Cure polysaccharides (TAPS-I-200)

87.08±0.65

--

--

--

Pure polysaccharide (TAPS-II-200)

110.10±0.90

70.84±0.44

28.78±0.44

14.34±0.56

Concentration of Pb2+ (mg/L)

f o

Pb content in Sample

content in pure sample (mg/g)

o r p

polysaccharide (%)

0

Mycelia

Not detected

Crude polysaccharides (TAPS-I-0)

Not detected

Pure polysaccharide (TAPS-II-0)

Not detected

l a

Mycelia 60

200

n r u

Crude polysaccharides (TAPS-I-60)

o J

r P

e

--: The date of the sample was not detected.

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Table 2 Methylation analysis of TAPS-II-0, TAPS-II-60 and TAPS-II-200 Retention time

Methylated sugars

Linkage types

(min)

Molar ratios

Mass fragments (m/z)

TAPS-II-0 →3)Gal(1→

2,4,6-tri-O-Me-D-Gal

7.678

2,3,4,6-tetra-O-Me-D-Glc

8.076

2,4,6-tri-O-Me-D-Glc

0.71

→3)Glc(1→

→4)Man (1→

-p

2,3,6-tri-O-Me-D-Man

→3, 4)Man(1→

2,6-di-O-Me-D-Man

TAPS-II-60

43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205 43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205

43, 45, 59, 71, 75, 87, 101, 113,

2.21

ro

→3)GlcA(1→

re

8.789

T-

2.33

117, 129, 145, 161, 205

43, 45, 59, 71, 85, 87, 103, 113, 117, 129, 131, 142, 159, 173, 187, 233

0.79

43, 45, 59, 87, 99, 117, 129, 143, 171, 185, 203, 261, 305

lP

8.543

→3)GalA(1→

2.87

of

6.346

2,4,6-tri-O-Me-D-Gal

→3)Gal(1→ →3)GalA(1→

2.27

43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205

7.784

2,3,4,6-tetra-O-Me-D-Glc

T-

0.41

43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205

8.098

2,4,6-tri-O-Me-D-Glc

→3)Glc(1→ →3)GlcA(1→

2.21

43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205

3.33

43, 45, 59, 71, 85, 87, 103, 113, 117, 129, 131, 142, 159, 173,

8.803

Jo ur

8.589

na

6.464

2,3,6-tri-O-Me-D-Man

2,6-di-O-Me-D-Man

→4)Man (1→

→3, 4)Man(1→

0.59

187, 233 43, 45, 59, 87, 99, 117, 129, 143, 171, 185, 203, 261, 305

TAPS-II-200 6.423

2,4,6-tri-O-Me-D-Gal

→3)Gal(1→ →3)GalA(1→

2.47

7.695

2,3,4,6-tetra-O-Me-D-Glc

T-

0.23

8.091

2,4,6-tri-O-Me-D-Glc

→3)Glc(1→ →3)GlcA(1→

1.31

32

43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205 43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205 43, 45, 59, 71, 75, 87, 101, 113, 117, 129, 145, 161, 205

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43, 45, 59, 71, 85, 87, 103, 113, 2,3,6-tri-O-Me-D-Gal

→4)Man (1→

3.53

8.789

2,6-di-O-Me-D-Man

→3，4)Man(1→

0.33

117, 129, 131, 142, 159, 173, 187, 233 43, 45, 59, 87, 99, 117, 129, 143, 171, 185, 203, 261, 305

Jo ur

na

lP

re

-p

ro

of

8.553

33

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Figure captions: Fig. 1. HPGPC spectra of TAPS-II-0 (a), TAPS-II-60 (b) and TAPS-II-200 (c). Fig. 2. FT-IR spectra of TAPS-II-0, TAPS-II-60 and TAPS-II-200. Fig. 3. GC-MS chromatogram of standard monosaccharides (a), TAPS-II-0 (b), TAPS-II-60 (c), TAPS-II-200 (d) (1: D-rhamnose, 2: D-arabinose, 3: D-xylose, 4:

of

D-mannose, 5: D-glucose, 6: D-galactose, 7: D-galacturonic acid, 8: D-glucuronic

ro

acid.).

-p

Fig. 4. GC-MS spectrum of TAPS-II-0 derivatives (0), TAPS-II-60 derivatives (b)

re

and TAPS-II-200 derivatives (c) (1: glycerol, 2: erythritol, 3: D-mannose, 4: D-glucose,

lP

5: D-galactose, 6: inositol).

Fig. 5. Maximum wavelength of Congo red, Congo red +TAPS-II-0, Congo red

Jo ur

solution.

na

+TAPS-II-60, Congo red +TAPS-II-200 at various concentrations of sodium hydroxide

Fig. 6 AFM images of TAPS-II-0 (a: Planar image, b: Three-dimensional image), TAPS-II-60 (c: Planar image, d: Three-dimensional image) and TAPS-II-200 (e: Planar image, f: Three-dimensional image)

34

Jo ur

na

lP

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of

Journal Pre-proof

35

Journal Pre-proof

Highlights: The polysaccharide plays a vital role in Pb2+ tolerance of Trichoderma asperellum. The polysaccharide was the main ingredient interacted with Pb2+. The ratio of uronic acid in polysaccharide was increased to enhance Pb2+ tolerance.

Jo ur

na

lP

re

-p

ro

of

The ratio of main chain in polysaccharide was increased to enhance Pb2+ tolerance.

36

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6