Environ Sci Pollut Res DOI 10.1007/s11356-015-4429-3
RESEARCH ARTICLE
Response of extracellular carboxylic and thiol ligands (oxalate, thiol compounds) to Pb2+ stress in Phanerochaete chrysosporium Ningjie Li 1,2 & Guangming Zeng 1,2 & Danlian Huang 1,2 & Chao Huang 1,2 & Cui Lai 1,2 & Zhen Wei 1,2 & Piao Xu 1,2 & Chen Zhang 1,2 & Min Cheng 1,2 & Ming Yan 1,2
Received: 16 January 2015 / Accepted: 20 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract When exposed to higher Pb2+ concentration, Phanerochaete chrysosporium secreted higher content of oxalate and thiol compounds. An earlier and faster increase in oxalate was observed after short-term exposure, comparing with a gentle increase in the thiol compounds. In the extracellular polymeric substances (EPS) extract, more oxalate and T-SH were detected when the initial Pb2+ was higher, and the variations were different from the situation in the culture medium. In EPS solution, the oxalate amount was more closely related with Pb than that of thiol compounds. pH condition in the whole Pb removal process by P. chrysosporium ranged from 4 to 6.5 and was more beneficial for the binding of Pb2+ to carboxylic groups in the oxalic acid. More Pb2+ induced more EPS amount, and the increase of EPS amount influenced the immobilized oxalate more seriously. The present study can supply more comprehensive information about the metal passivation mechanism in white-rot fungi and provide meaningful references for an enhanced removal of heavy metals.
Responsible editor: Philippe Garrigues * Guangming Zeng [email protected] * Danlian Huang [email protected] 1
College of Environmental Science and Engineering, Hunan University, Changsha, Hunan 410082, China
2
Ministry of Education, Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Changsha, Hunan 410082, China
Keywords Phanerochaete chrysosporium . Heavy metal . Oxalate . Thiol compounds . Stress response . Extracellular ligands
Introduction Heavy metals have been increasingly concerned for their toxicity. To find effective and environmentally friendly technologies for heavy metal removal was always a hot topic, because of the drawbacks of traditional techniques (Fu and Wang 2011; Gong et al. 2009; Hu et al. 2013; Tang et al. 2014; Xu et al. 2012). In the new technologies, white-rot fungi have been reported to play a very important role as a biologic metal remover in environmental bioremediation (Huang et al. 2010; Zeng et al. 2013a, b; Yetis et al. 2000) or as a metal transformer in geochemical process or bionics (Rhee Young et al. 2012; Vigneshwaran et al. 2006). An understanding of the essence of those roles in the biotechnologies is critical to predicting and controlling the environmental fate of heavy metals. It has been demonstrated that white-rot fungi could survive in the surroundings with high metal contamination (Huang et al. 2008; Zeng et al. 2007), indicating the help of effective defence systems to alleviate metal toxicity. The defence systems are usually based on the immobilization of heavy metals using extracellular and intracellular chelating compounds, which is defined as functional substances here. With the limited intracellular uptake of metal for white-rot fungi (Baldrian 2003), the extracellular and cell wall-associated binding of metal onto the cell wall surface probably plays the most important role. Therefore, the extracellular functional substances of white-rot fungi are vital and necessary to obtain more specific knowledge. The two functional groups (i.e., –SH and –COOH) have been found to be responsible for the distribution of heavy
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metals between particles and solutions (Schuster 1991). The research into the extracellular carboxylic and thiol ligands would be meaningful for the enhanced removal of heavy metal by white-rot fungi. Organic acids produced by fungi are important sources of carboxylic group and have profound implications for metal speciation, physiology, and biogeochemical cycles. It has been reported that fungi could produce oxalic acid, malic acid, fumaric acid, formic acid, glyoxylic acid, and so on (Galkin et al. 1998; Hofrichter et al. 1999; Mäkelä et al. 2002). Among various organic acids, oxalate is the primary metal chelator produced by white-rot fungi (Baldrian 2003; Li et al. 2011). Thiol compounds are one kind of compounds containing sulfydryl group. The binding of metals to –SH in thiol compounds or –COOH in organic acids could decrease the toxic effects of free metal ions. So the main objective of this study is to investigate the effect of the two functional ligands (oxalate and thiol compounds) on the bioremoval of Pb by white-rot fungi. Previous studies proved the oxalate secretion and the oxalate crystals formation by white-rot fungi (JaroszWilkolazka and Gadd 2003). Moreover, the biogenic production of lead oxalate dihydrate was observed during the fungal transformation of pyromorphite (Gadd 2000; Rhee Young et al. 2012). Therefore, the extracellular response mechanism of lead oxalate has significant consequences for metal mobility and transfer between environmental compartments and organisms. Until now, the relationship between the oxalate production and the metal bio-removal has never been proved. Sulfydryl-containing compounds has also aroused interests of many researchers because of its high affinity to metals (Guimarães-Soares et al. 2007; Jarosz-Wilkołazka et al. 2006; Vacchina et al. 2002). It has been elucidated that thiols production in the cells was enhanced when white-rot fungi resisted against metal toxicity (Jarosz-Wilkołazka et al. 2006), while the metal binding by intracellular thiol compounds was insignificant (Vacchina et al. 2002). It seems that the extracellular metal binding by thiols is more important. So far as we know, studies on metal-induced thiol compounds were focused on its intracellular production (Guimarães-Soares et al. 2007; Jarosz-Wilkołazka et al. 2006). The secretion of thiol compounds in white-rot fungi is still uncertain and even less is known about the secretion of extracellular thiols under heavy metal stress. Thus, the present study can supply more detailed and comprehensive information about the role of oxalate and thiol compounds in the biotechnological decontamination processes. The study was accomplished by tracing the responses of oxalic acid and thiol compounds in the solution against different initial Pb 2 + concentrations in Phanerochaete chrysosporium. The extracellular compounds formed by interactions of functional substances with heavy metals are immobilized around the fungal cell wall or entrapped in the
mycelium matrix, with the help of extracellular polymeric substances (EPS). In this study, the relationship between oxalate and Pb or that between thiol compounds and Pb in the EPS extract was also studied to facilitate the study of the contribution of oxalate or thiol compounds to Pb2+ bioremoval by P. chrysosporium.
Material and methods Inoculum preparation P. chrysosporium (BKMF-1767) (CCTCC AF96007) was obtained from China Center for type Culture Collection (Wuhan, China) and maintained on potato dextrose agar (PDA) slants at 4 °C. The fungus was grown on PDA plates at 37 °C for several days and spores on the agar surface were diluting in the sterile distilled water. The spore solution was controlled at 2.5×106 spores/ml (Pellinen et al. 1989) and used as inocula. Culture condition Batch culture experiments of 200 ml sterile potato dextrose broth (PDB) were conducted in 500 ml flasks, and the flasks were labeled as A(0), B(25), C(50), D(100), E(200), and F(400), respectively. With inoculation of 1.5 ml spore suspensions at room temperature, the culture process was undertaken in a constant temperature incubator shaker at 150 rpm, 30 °C. Pb2+ was added in the form of Pb(NO3)2 to the 41-h-old cultures at concentrations of 0, 25, 50, 100, 200, and 400 mg l−1, respectively. For a better comparison, control flasks containing 200 mg l−1 Pb2+ without inoculation of fungus were also prepared under the same conditions. The Pb2+ exposure period was terminated at 205 h. All experiments were performed in triplicates and mean values were used in the analysis. Sampling and analytical methods After Pb2+ addition, all the fungal mycelia in each treatment were separated from the supernatant with filter paper at the given time. The filtrate was used for the determination of pH, Pb concentration, oxalate, and thiol compounds in the culture medium. The fungal mycelia were blotted dry between layers of filter paper to decrease the influence of adsorbed culture medium, before they were resuspended in 200 ml of ultrapure water. After 15 min of centrifugation at 10,000 rpm, the suspension was separated from the fungal mycelia and used as EPS solution. There were additional compounds to EPS in the solution even if EPS might be the major component. The EPS solution was used for the estimation of Pb concentration, oxalate, thiol compounds, and EPS amount. The final results were expressed in relation to the dry weight (dw) of the
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harvested mycelia, which was measured after the harvested mycelia were dried at 80 °C for 4 h to a constant weight. pH determination pH in the environment would affect the operation of Pb2+ immobilization by functional groups (Horsfall and Spiff 2004). Therefore, pH of the culture medium was measured with a Mettler Toledo FE 20 pH meter. Estimation of Pb in the culture medium and in the EPS solution The culture medium filtrate or the EPS solution was acidified with 3 % (v/v) HNO3 and stored at 4 °C for Pb2+ analysis. Pb concentrations in the solutions were determined with an atomic absorption spectroscopy (AAS). The instrument was calibrated with Pb2+ standard solutions. High-performance liquid chromatography (HPLC) analysis of oxalate Oxalate in the culture medium filtrate or in the EPS solution was analyzed using an Agilent 1100 HPLC apparatus (Dutton et al. 1993) equipped with UV–visible variable wavelength detector (VWD). Phosphoric acid (0.15 % v/v) was used as the solvent at a flow rate of 0.5 ml min−1, and the detection wavelength was 210 nm. The column was maintained at 30 °C. Oxalic acid was used as the external standard for quantization. Before used for HPLC analysis of oxalate, the culture medium filtrate or the EPS solution was filtered through 0.45 μm filter paper. Assays of thiol-containing compounds (T-SH) The concentration of T-SH was measured with 5,5′dithio-bis(2-nitrobenzoic acid) (DTNB) as described by Sedlak and Lindsay (1968). To determine T-SH, 150 μl aliquot of the culture medium filtrate or the EPS solution was mixed with 450 μl of 0.2 M Tris (pH 8.2) and 30 μl of 0.01 M DTNB, and the reaction mixture was brought to 3.0 ml with absolute methanol. After 15 min, the mixture was centrifuged (1,833×g, 10 min), and then its absorbance was examined at 412 nm (Shimadzu 2550 UV–visible spectrophotometer) against a reagent blank where the culture medium filtrate or the EPS solution was replaced accordingly with the solvent of the culture medium filtrate or the EPS solution. The reduced glutathione was used as the standard.
et al. 2009), i.e., the phenol-sulfuric acid assay. 0.2 ml of 80 % phenol was added into 1.2 ml of the obtained EPS solution. Then 8 ml of concentrated sulfuric acid was added into the mixture. The samples were allowed to cool to room temperature, and the absorbance of the solution was measured at 490 nm.
Results and discussion Changes of biomass weight As shown in Fig. 1, the biomass weights in all groups with Pb2+ addition display similar variations. The biomass weight increased rapidly from 42 to 61 h and the increase became slower till 157 h, followed with a tiny decline at 205 h. The biomass in E(200) and F(400) at 157 h were 0.306 and 0.242 g, respectively. The biomass weights in other groups at 157 h were around 0.33 g. It is obvious that less biomass was detected in the group with more Pb2+ addition. It is suggested that the growth of P. chrysosporium was partially affected in the presence of less than 400 mg l−1 of Pb ions and was inhibited in the medium containing 400 mg l−1 of Pb2+. Changes of Pb content and removal rate in the culture medium Changes of Pb content in the culture medium during the 164-h assay are displayed in Fig. 2. After 1-h Pb2+ exposure, Pb amount in the culture medium of F(400) decreased the most, with a Pb removal value of 72 mg l−1. In the treatments with initial Pb2+ content lower than 100 mg l−1, the removal rate of Pb was about 40 %, while it was 23.5 % in E(200). In the following 43-h assay, Pb concentrations were diminished constantly and quickly. 40 % more of Pb in B(25) and C(50) was
Estimation of EPS amount in the EPS solution The quantity of EPS was preliminarily estimated according to the Dubois method by using glucose as standard (Braissant
Fig. 1 Variations of the dry weight of the biomass obtained from 42 to 205 h
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Fig. 2 Changes of Pb content and removal rate in the culture medium during the 164-h assay
removed, and about 30, 60, and 67 % more of Pb in D(100), E(200), F(400), respectively. Later on, the decrease of Pb concentrations slowed down. At 121 h, a faint decrease of Pb removal rate was detected in E(200) and F(400). At the end of this study, removal rates of Pb in the medium were about 95 % in treatments except that no Pb was detected in B(25). The limited intracellular absorption of metal in white-rot fungi has been reported (Baldrian 2003). To protect the cells from poisonous metals, the fungi replied with a variety of
extracellular defence mechanisms (Connolly and Jellison 1995). The steep descendant of Pb concentrations within the 1-h Pb2+ exposure implies prompt and effective extracellular responses in P. chrysosporium to Pb2+ pollution (Fig. 2), especially when the initial Pb2+ concentration was up to 400 mg l−1. The extracellular responses were indicated by the production of potential Pb removers. Similar removal rate of Pb in the treatments with less than 100 mg l−1 of Pb2+ is probably due to the tolerance of the existing Pb removers
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produced in the former 41-h culture. However, when initial Pb2+ content was higher, the previously produced Pb removers probably were not enough to tolerate so much Pb2+ so that more Pb removers were needed to ensure the efficient Pb removal. Most of the time in the experiment, Pb levels in the culture medium were lowering except at 121 h in E(200) and F(400) (Fig. 2). That might be due to the more decomposition or desorption of adsorbed Pb complexes than newly adsorbed ones. To learn the detailed performance of extracellular responses in Pb2+ biosorption, a further study on the production of oxalic acid and thiol-containing compounds (Fig. 3) under different Pb2+ stresses was carried out, both of which were important materials in the metal detoxification (Guimarães-Soares et al. 2007; Jarosz-Wilkolazka and Gadd 2003). Oxalate changes in the culture medium Oxalate contents in the culture medium are displayed in Fig. 3a. At 42 h, oxalate contents in B(25) and C(50) were 0.43 and 0.21 mM, respectively. They were both lower than
0.56 mM of oxalate in A(0), in contrast with that 0.72 mM in D(100), 3.03 mM in E(200), and 2.91 mM in F(400), which was much higher than that in A(0) (Fig. 3a). Oxalate content in A(0) and B(25) decreased during the whole determination period. Oxalate content in C(50), D(100), and E(200) displayed the highest level at 49 h (0.21, 0.72, and 3.03 mM, respectively), while in F(400), it did not reach a peak value until 85 h, i.e., 3.56 mM. Nearly no oxalate was detected in A(0) at 49 h, in B(25) and C(50) at 85 h, while oxalate in D(100), E(200), and F(400) decreased rapidly till 121 h. After 1-h exposure to higher initial levels of Pb 2+ , P. chrysosporium secreted more oxalate in all the treatments except B(25) and C(50) (Fig. 3a), probably because the Pb content under 50 mg l−1 did not form acute threat to the mycelia cells on the basis of the existed oxalate produced in the 41 h. The secretion of oxalic acid in the culture medium could tolerate a certain amount of Pb2+ by the formation and biosorption of Pb-oxalate complexes (Jarosz-Wilkolazka and Gadd 2003). At 42 h, the rapid rise of oxalate in E(200) and F(400) illustrated the quick initiation of the extracellular resistance mechanisms when P. chrysosporium contacted with high level of Pb2+. Oxalate concentrations in E(200) and F(400) during the former 8-h Pb2+ exposure were approximate. The reason probably is that excess oxalate was regulated by oxalate decarboxylase, which can directly decompose oxalate into formic acid and carbon dioxide (Mäkelä et al. 2002). And that also could explain the decrease of oxalate contents at 41 h in A(0) and B(25), at 49 h in C(50), D(100), and E(200), and at 85 h in F(400). It can be seen that the existence of more Pb2+ led to a delayed decrease of oxalate (Fig. 3a). T-SH content in the culture medium
Fig. 3 Variations of oxalate (a) and T-SH content (b) in the culture medium during the 164-h assay
T-SH content in the culture medium of all treatments showed similar variations (Fig. 3b). In A(0), T-SH content at 42 h was apparently different from that at 41 h. In the treatments with Pb2+ addition, T-SH content was kept at a relatively stable level during the first-hour exposure period. During the following 7-h assay, T-SH content decreased slowly, followed with a faint increase from 49 to 61 h in all the Pb2+ added treatments except B(25). In B(25), T-SH content decreased until 85 h. In A(0), T-SH content was kept at a stable level from 49 to 85 h. A second increase of T-SH content was detected from 85 to 157 h in Pb2+ treatments and from 85 to 121 h in A(0). T-SH content fluctuated around 0.3-0.5 mM in the previous 116-h Pb2+ exposure and dropped quickly to about 0.1 mM at 205 h. Data displayed in Fig. 3b suggests that extracellular thiol compounds was detectable (Fig. 3b), implying the secretion of free thiol compounds outside the cell. Similar phenomenon was obtained in other fungi, for instance, Thermomyces lanuginosus (Hasnain et al. 1992). The T-SH concentrations in Pb2+-added treatments were all higher than that in A(0) (Fig. 3b), similar with the enhanced intracellular synthesis of
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thiols by fungi under Pb stress reported by Guimarães-Soares et al. (2007). The reason for the higher T-SH concentration in Pb2+-added treatments may lie in the interference of Pb2+ in the metabolism system of P. chrysosporium (Baldrian 2003). It is interesting that the fluctuation trend of T-SH is opposite to the variation of oxalate. Thiols was proved to mediate the oxidation of nonphenolic lignin model compounds by manganese peroxidase of P. chrysosporium and that also may be involved in the variations of T-SH (Wariish et al. 1989). In the quantity perspective, metal immobilization requires compounds containing metal-binding functional groups, thus higher production of functional substances implies effective response to metal exposure. The obtained results indicate that P. chrysosporium utilized oxalic acid as a sensitive functional substance against Pb2+ toxicity and thiol compounds as a gentle response to Pb2+ in extracellular defence systems (Fig. 3). Changes of oxalate content and T-SH content in EPS solution As shown in Fig. 4a, the oxalate content in EPS solution of A(0) decreased continuously from 0.83 mmol g−1 at 41 h to 0 mmol g−1 at 121 h. The variations of oxalate in B(25) and C(50) were similar with that of A(0), except that the oxalate at 61 h increased a little. Oxalate content in EPS solution of D(100) and E(200) increased to a highest level at 42 h (0.93 and 1.03 mmol g−1, respectively), while in F(400), the peak displayed at 49 h (2.04 mmol g−1). From 121 to 157 h, the decrease of oxalate was relieved. The result implied that more oxalate was immobilized in the EPS layer when more Pb2+ was initially added. In Fig. 4b, the changes of T-SH content were different from that of the oxalate content. The T-SH contents in all treatments at 41 h and that at 42 h were close. After 42 h, more T-SH content was determined in treatment with higher initial Pb2+ concentration. The T-SH content showed similar variations, increasing at the beginning and following with a continued decrease until 205 h. The highest T-SH content in A(0) and B(25) appeared at 49 h (0.155 and 0.161 mmol g−1). The peak of T-SH in C(50), D(100), E(200), and F(400) was displayed at 61 h (0.172, 0.186, 0.231, and 0.282 mmol g−1, respectively). There are mucilaginous polymer substances distributed around the white-rot fungal cells, which are important materials for the removal of metal from aqueous solutions. Centrifugation is one useful method to extract the EPS from the cell wall. In Fig. 4, it is shown that oxalate and thiols were both detectable in EPS extract during the entire estimation period. Variations of oxalate in the EPS extract were different from the situation in the medium where no oxalate was estimated after 85 h in C(50) or 121 h in F(400) (Figs. 3a and 4a), and it implies that not all the adsorbed oxalate was degraded. The finding consolidated the decreasing Pb removal rate at 121 h in E(200) and F(400), implying that the degradation
Fig. 4 Variations of oxalate (a) and T-SH content (b) in the EPS solution during the 164-h assay
of oxalate affected the removal of Pb more seriously in the treatments with more than 200 mg l−1 of Pb2+. It also illustrates that when P. chrysosporium was exposed to high stress of Pb2+, oxalate played a very important role in Pb removal. Even the T-SH content in the culture medium fluctuated, T-SH in EPS solution increased continuously from 42 to 121 h. More T-SH in EPS solution of treatment with higher initial Pb2+ concentration probably implies that more Pb2+ would be removed and immobilized in the EPS of P. chrysosporium. The decrease of T-SH in EPS after 121 h maybe lies in the fast decrease of T-SH in the culture medium, maintaining the balance between the amount of T-SH in the solution and that on the surface of fungal mycelium. The decrease of the two kinds of functional substances did not restrict the Pb removal by P. chrysosporium, which is probably due to the participation of other Pb-binding substances. White-rot fungi can produce other organic acids except oxalic acid which can precipitate Pb ions and immobilize Pb in the mucilaginous polymer layer, such as malic acid, even the oxalic acid accumulation by P. chrysosporium F1767 was reported to be maximal during the fungal growth (Galkin et al. 1998, Hofrichter et al. 1999).
Environ Sci Pollut Res Table 1 Correlation between Pb and oxalate and that between Pb and T-SH in the EPS solution of C(50) and F(400)
R2 (oxalate/Pb) R2 (T-SH/Pb)
C(50)
F(400)
0.6853 0.2439
0.7685 0.266
Correlation between Pb and oxalic acid and that between Pb and T-SH The correlations between Pb and oxalate and that between Pb and T-SH in the EPS extract of the treatments were analyzed. Considering the first decreased and then increased variation of oxalate in C(50) and the similar variations of oxalate when initial Pb2+ content was over 50 mg l−1, the analysis results of the treatments C(50) and F(400) are presented in Table 1. During the whole Pb2+ exposure period, the linear correlation coefficient square between Pb and oxalate in the EPS extract of C(50) or F(400) was 0.6853, 0.7685, respectively, much higher than that between Pb and T-SH (0.2639, 0.266). The two kinds of coefficient in the F(400) were respectively slightly higher than those in C(50). The analysis of correlation between Pb and functional substances would provide some information on the contribution of the functional substance to the Pb removal. The results of correlation analysis suggest that oxalate was more significantly correlated to the Pb removal than thiol compounds. Nevertheless, no obvious relationship was found between adsorbed Pb and oxalate.
pH variations during the operation of functional substances As shown in Fig. 5, pH decreased in the previous 20-h exposure period, and then increased in the following detecting period from 61 to 205 h. pH values in the culture medium of all the treatments ranged from 4 to 6 before 205 h (Fig. 5). The
Fig. 6 Changes of EPS amount during the 164-h assay
pH in the culture medium in A(0), B(25), C(50), and D(100) at 205 h was in the range of 6.0–6.5. In the treatments with higher initial Pb2+ content, pH value is lower. The functional groups used for metal binding in oxalate and thiol compounds are carboxylic groups and thiol groups, respectively. The deprotonated or protonated states of the two functional groups, which may vary considerably with pH changes (Wang et al. 2011), can significantly influence metal binding and organic adsorption (Say et al. 2001). In the environment with a pH range from 4 to 6.5, most carboxylic units are deprotonated and present high metal retention ability (Rivas et al. 2003), while the thiol groups with pK values of 7.0–9.0 are prone to change in protonation state (Braissant et al. 2007). These may result in the more significant correlation between adsorbed Pb and oxalate than that between adsorbed Pb and T-SH (Fig. 5). Changes of EPS amount during the operation of functional substances As shown in Fig. 6, the EPS amount in the groups with Pb2+ addition was similar, increasing from 41 to 61 h and decreasing continually till 205 h. In A(0), the amount of EPS decreased continually. After 8-h exposure, higher and faster production of EPS was detected when more Pb2+ threatened the fungal cells. Even the fungal growth was inhibited in F(400), the EPS amount was not affected (Figs. 1 and 6). In general, EPS are produced from lysis and hydrolysis of adsorbed organic matters from the medium. More EPS in F(400) maybe Table 2 Correlation between oxalate and EPS and that between T-SH and EPS in the EPS solution
Fig. 5 pH variations in the culture medium of all the treatments during the 164-h detecting period
R2 (oxalate/EPS) R2 (T-SH/EPS)
B(25)
C(50)
E(200)
F(400)
0.522 0.918
0.58 0.861
0.666 0.831
0.738 0.637
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originated from the cell’s lysis, which was caused by the reactive oxygen species induced by the stress of Pb ions (Kim et al. 2008). Roles of EPS include enhancing cellular adhesion, providing a protective barrier, as well as adsorbing and storing nutrients for microbial growth (Long et al. 2009). Besides, EPS plays an important role in immobilizing the metal complex which is vital for the removal of heavy metal from the fluid environment. Therefore, the amount of EPS may be an important factor affecting the proceedings of metal removal by functional substances. The correlation between oxalate and EPS and that between T-SH and EPS in the EPS solution was analyzed, and the result is presented in Table 2. It is interesting to find that the R2 (oxalate/EPS) became higher in the group of more Pb2+, while the R2 (T-SH/EPS) displayed an opposite trend. The result probably indicates that the increase of EPS amount influenced the immobilized oxalate more seriously.
Conclusion In conclusion, P. chrysosporium was found to utilize oxalic acid as a sensitive functional substance against Pb2+ toxicity, while thiol compounds as a gentle response to Pb2+ in extracellular defence systems. In the treatment with higher initial Pb2+ concentration, higher content of oxalate and thiol compounds were both detected in the culture medium and in the EPS solution, but their variations in medium and that in EPS were different. Nevertheless, oxalate was found to contribute more to the Pb removal by P. chrysosporium than thiol compounds, with a more significant correlation with the removed Pb. The difference probably resulted from the pH variation and the EPS amount in the experiment. A pH range from 4 to 6.5 was beneficial for the operation of Pb2+ binding by carboxylic groups in the oxalic acid. More Pb2+ induced more EPS amount, and the increase of EPS amount influenced the immobilized oxalate more seriously. The present findings would provide useful references for the metal passivation mechanism in white-rot fungi and the transfer between environmental compartments and organisms.
Acknowledgments The study was financially supported by the National Natural Science Foundation of China (51278176, 51378190, and 51408206), the Environmental Protection Technology Research Program of Hunan (2007185), the New Century Excellent Talents in University (NECT-13-0186), the Young Teacher Growth Program of Hunan University, the Scientific Research Fund of Hunan Provincial Education Department (521293050), the Fundamental Research Funds for the Central Universities, the Hunan University Fund for Multidisciplinary Developing (531107040762), the Hunan Provincial Innovation Foundation for Postgraduate (CX2014B141), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17).
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Tang WW, Zeng GM, Gong JL, Liang J, Xu P, Zhang C, Huang BB (2014) Removal of heavy metals from aqueous solutions using nanomaterials affected by humic/fulvic acid: a review. Sci Total Environ 468–469:1014–1027 Vacchina V, Baldrian P, Gabriel J, Szpunar J (2002) Investigation of the response of wood-rotting fungi to copper stress by size-exclusion chromatography and capillary zone electrophoresis with ICP MS detection. Anal Bioanal Chem 372:453–456 Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH (2006) Biomimetics of silver nanoparticles by white rot fungus, Phaenerochaete chrysosporium. Colloids Surf B Biointerfaces 53:55–59 Wang LL, Wang LF, Ren XM, Ye XD, Li WW, Yuan SJ, Sun M, Sheng GP, Yu HQ, Wang XK (2011) pH dependence of structure and surface properties of microbial EPS. Environ Sci Technol 46:737–744 Wariish H, Valli K, Renganathan V, Gold MH (1989) Thiol-mediated oxidation of nonphenolic lignin model compounds by manganese peroxidase of Phanerochaete chrysosporium. J Biol Chem 264: 14185–14191 Xu P, Zeng GM, Huang DL, Feng CL, Hu S, Zhao MH, Lai C, Wei Z, Huang C, Xie GX, Liu ZF (2012) Use of iron oxide nanomaterials in wastewater treatment: a review. Sci Total Environ 424:1–10 Yetis U, Dolek A, Dilek FB, Ozcengiz G (2000) The removal of Pb(II) by Phanerochaete chrysosporium. Water Res 34:4090–4100 Zeng GM, Huang DL, Huang GH, Hu TJ, Jiang XY, Feng CL, Chen YN, Tang L, Liu HL (2007) Composting of lead-contaminated solid waste with inocula of white-rot fungus. Bioresour Technol 98: 320–326 Zeng GM, Chen M, Zeng ZT (2013a) Risks of neonicotinoid pesticides. Science 340:1403 Zeng GM, Chen M, Zeng ZT (2013b) Shale gas: surface water also at risk. Nature 499:154
RESEARCH ARTICLE
Response of extracellular carboxylic and thiol ligands (oxalate, thiol compounds) to Pb2+ stress in Phanerochaete chrysosporium Ningjie Li 1,2 & Guangming Zeng 1,2 & Danlian Huang 1,2 & Chao Huang 1,2 & Cui Lai 1,2 & Zhen Wei 1,2 & Piao Xu 1,2 & Chen Zhang 1,2 & Min Cheng 1,2 & Ming Yan 1,2
Received: 16 January 2015 / Accepted: 20 March 2015 # Springer-Verlag Berlin Heidelberg 2015
Abstract When exposed to higher Pb2+ concentration, Phanerochaete chrysosporium secreted higher content of oxalate and thiol compounds. An earlier and faster increase in oxalate was observed after short-term exposure, comparing with a gentle increase in the thiol compounds. In the extracellular polymeric substances (EPS) extract, more oxalate and T-SH were detected when the initial Pb2+ was higher, and the variations were different from the situation in the culture medium. In EPS solution, the oxalate amount was more closely related with Pb than that of thiol compounds. pH condition in the whole Pb removal process by P. chrysosporium ranged from 4 to 6.5 and was more beneficial for the binding of Pb2+ to carboxylic groups in the oxalic acid. More Pb2+ induced more EPS amount, and the increase of EPS amount influenced the immobilized oxalate more seriously. The present study can supply more comprehensive information about the metal passivation mechanism in white-rot fungi and provide meaningful references for an enhanced removal of heavy metals.
Responsible editor: Philippe Garrigues * Guangming Zeng [email protected] * Danlian Huang [email protected] 1
College of Environmental Science and Engineering, Hunan University, Changsha, Hunan 410082, China
2
Ministry of Education, Key Laboratory of Environmental Biology and Pollution Control, Hunan University, Changsha, Hunan 410082, China
Keywords Phanerochaete chrysosporium . Heavy metal . Oxalate . Thiol compounds . Stress response . Extracellular ligands
Introduction Heavy metals have been increasingly concerned for their toxicity. To find effective and environmentally friendly technologies for heavy metal removal was always a hot topic, because of the drawbacks of traditional techniques (Fu and Wang 2011; Gong et al. 2009; Hu et al. 2013; Tang et al. 2014; Xu et al. 2012). In the new technologies, white-rot fungi have been reported to play a very important role as a biologic metal remover in environmental bioremediation (Huang et al. 2010; Zeng et al. 2013a, b; Yetis et al. 2000) or as a metal transformer in geochemical process or bionics (Rhee Young et al. 2012; Vigneshwaran et al. 2006). An understanding of the essence of those roles in the biotechnologies is critical to predicting and controlling the environmental fate of heavy metals. It has been demonstrated that white-rot fungi could survive in the surroundings with high metal contamination (Huang et al. 2008; Zeng et al. 2007), indicating the help of effective defence systems to alleviate metal toxicity. The defence systems are usually based on the immobilization of heavy metals using extracellular and intracellular chelating compounds, which is defined as functional substances here. With the limited intracellular uptake of metal for white-rot fungi (Baldrian 2003), the extracellular and cell wall-associated binding of metal onto the cell wall surface probably plays the most important role. Therefore, the extracellular functional substances of white-rot fungi are vital and necessary to obtain more specific knowledge. The two functional groups (i.e., –SH and –COOH) have been found to be responsible for the distribution of heavy
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metals between particles and solutions (Schuster 1991). The research into the extracellular carboxylic and thiol ligands would be meaningful for the enhanced removal of heavy metal by white-rot fungi. Organic acids produced by fungi are important sources of carboxylic group and have profound implications for metal speciation, physiology, and biogeochemical cycles. It has been reported that fungi could produce oxalic acid, malic acid, fumaric acid, formic acid, glyoxylic acid, and so on (Galkin et al. 1998; Hofrichter et al. 1999; Mäkelä et al. 2002). Among various organic acids, oxalate is the primary metal chelator produced by white-rot fungi (Baldrian 2003; Li et al. 2011). Thiol compounds are one kind of compounds containing sulfydryl group. The binding of metals to –SH in thiol compounds or –COOH in organic acids could decrease the toxic effects of free metal ions. So the main objective of this study is to investigate the effect of the two functional ligands (oxalate and thiol compounds) on the bioremoval of Pb by white-rot fungi. Previous studies proved the oxalate secretion and the oxalate crystals formation by white-rot fungi (JaroszWilkolazka and Gadd 2003). Moreover, the biogenic production of lead oxalate dihydrate was observed during the fungal transformation of pyromorphite (Gadd 2000; Rhee Young et al. 2012). Therefore, the extracellular response mechanism of lead oxalate has significant consequences for metal mobility and transfer between environmental compartments and organisms. Until now, the relationship between the oxalate production and the metal bio-removal has never been proved. Sulfydryl-containing compounds has also aroused interests of many researchers because of its high affinity to metals (Guimarães-Soares et al. 2007; Jarosz-Wilkołazka et al. 2006; Vacchina et al. 2002). It has been elucidated that thiols production in the cells was enhanced when white-rot fungi resisted against metal toxicity (Jarosz-Wilkołazka et al. 2006), while the metal binding by intracellular thiol compounds was insignificant (Vacchina et al. 2002). It seems that the extracellular metal binding by thiols is more important. So far as we know, studies on metal-induced thiol compounds were focused on its intracellular production (Guimarães-Soares et al. 2007; Jarosz-Wilkołazka et al. 2006). The secretion of thiol compounds in white-rot fungi is still uncertain and even less is known about the secretion of extracellular thiols under heavy metal stress. Thus, the present study can supply more detailed and comprehensive information about the role of oxalate and thiol compounds in the biotechnological decontamination processes. The study was accomplished by tracing the responses of oxalic acid and thiol compounds in the solution against different initial Pb 2 + concentrations in Phanerochaete chrysosporium. The extracellular compounds formed by interactions of functional substances with heavy metals are immobilized around the fungal cell wall or entrapped in the
mycelium matrix, with the help of extracellular polymeric substances (EPS). In this study, the relationship between oxalate and Pb or that between thiol compounds and Pb in the EPS extract was also studied to facilitate the study of the contribution of oxalate or thiol compounds to Pb2+ bioremoval by P. chrysosporium.
Material and methods Inoculum preparation P. chrysosporium (BKMF-1767) (CCTCC AF96007) was obtained from China Center for type Culture Collection (Wuhan, China) and maintained on potato dextrose agar (PDA) slants at 4 °C. The fungus was grown on PDA plates at 37 °C for several days and spores on the agar surface were diluting in the sterile distilled water. The spore solution was controlled at 2.5×106 spores/ml (Pellinen et al. 1989) and used as inocula. Culture condition Batch culture experiments of 200 ml sterile potato dextrose broth (PDB) were conducted in 500 ml flasks, and the flasks were labeled as A(0), B(25), C(50), D(100), E(200), and F(400), respectively. With inoculation of 1.5 ml spore suspensions at room temperature, the culture process was undertaken in a constant temperature incubator shaker at 150 rpm, 30 °C. Pb2+ was added in the form of Pb(NO3)2 to the 41-h-old cultures at concentrations of 0, 25, 50, 100, 200, and 400 mg l−1, respectively. For a better comparison, control flasks containing 200 mg l−1 Pb2+ without inoculation of fungus were also prepared under the same conditions. The Pb2+ exposure period was terminated at 205 h. All experiments were performed in triplicates and mean values were used in the analysis. Sampling and analytical methods After Pb2+ addition, all the fungal mycelia in each treatment were separated from the supernatant with filter paper at the given time. The filtrate was used for the determination of pH, Pb concentration, oxalate, and thiol compounds in the culture medium. The fungal mycelia were blotted dry between layers of filter paper to decrease the influence of adsorbed culture medium, before they were resuspended in 200 ml of ultrapure water. After 15 min of centrifugation at 10,000 rpm, the suspension was separated from the fungal mycelia and used as EPS solution. There were additional compounds to EPS in the solution even if EPS might be the major component. The EPS solution was used for the estimation of Pb concentration, oxalate, thiol compounds, and EPS amount. The final results were expressed in relation to the dry weight (dw) of the
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harvested mycelia, which was measured after the harvested mycelia were dried at 80 °C for 4 h to a constant weight. pH determination pH in the environment would affect the operation of Pb2+ immobilization by functional groups (Horsfall and Spiff 2004). Therefore, pH of the culture medium was measured with a Mettler Toledo FE 20 pH meter. Estimation of Pb in the culture medium and in the EPS solution The culture medium filtrate or the EPS solution was acidified with 3 % (v/v) HNO3 and stored at 4 °C for Pb2+ analysis. Pb concentrations in the solutions were determined with an atomic absorption spectroscopy (AAS). The instrument was calibrated with Pb2+ standard solutions. High-performance liquid chromatography (HPLC) analysis of oxalate Oxalate in the culture medium filtrate or in the EPS solution was analyzed using an Agilent 1100 HPLC apparatus (Dutton et al. 1993) equipped with UV–visible variable wavelength detector (VWD). Phosphoric acid (0.15 % v/v) was used as the solvent at a flow rate of 0.5 ml min−1, and the detection wavelength was 210 nm. The column was maintained at 30 °C. Oxalic acid was used as the external standard for quantization. Before used for HPLC analysis of oxalate, the culture medium filtrate or the EPS solution was filtered through 0.45 μm filter paper. Assays of thiol-containing compounds (T-SH) The concentration of T-SH was measured with 5,5′dithio-bis(2-nitrobenzoic acid) (DTNB) as described by Sedlak and Lindsay (1968). To determine T-SH, 150 μl aliquot of the culture medium filtrate or the EPS solution was mixed with 450 μl of 0.2 M Tris (pH 8.2) and 30 μl of 0.01 M DTNB, and the reaction mixture was brought to 3.0 ml with absolute methanol. After 15 min, the mixture was centrifuged (1,833×g, 10 min), and then its absorbance was examined at 412 nm (Shimadzu 2550 UV–visible spectrophotometer) against a reagent blank where the culture medium filtrate or the EPS solution was replaced accordingly with the solvent of the culture medium filtrate or the EPS solution. The reduced glutathione was used as the standard.
et al. 2009), i.e., the phenol-sulfuric acid assay. 0.2 ml of 80 % phenol was added into 1.2 ml of the obtained EPS solution. Then 8 ml of concentrated sulfuric acid was added into the mixture. The samples were allowed to cool to room temperature, and the absorbance of the solution was measured at 490 nm.
Results and discussion Changes of biomass weight As shown in Fig. 1, the biomass weights in all groups with Pb2+ addition display similar variations. The biomass weight increased rapidly from 42 to 61 h and the increase became slower till 157 h, followed with a tiny decline at 205 h. The biomass in E(200) and F(400) at 157 h were 0.306 and 0.242 g, respectively. The biomass weights in other groups at 157 h were around 0.33 g. It is obvious that less biomass was detected in the group with more Pb2+ addition. It is suggested that the growth of P. chrysosporium was partially affected in the presence of less than 400 mg l−1 of Pb ions and was inhibited in the medium containing 400 mg l−1 of Pb2+. Changes of Pb content and removal rate in the culture medium Changes of Pb content in the culture medium during the 164-h assay are displayed in Fig. 2. After 1-h Pb2+ exposure, Pb amount in the culture medium of F(400) decreased the most, with a Pb removal value of 72 mg l−1. In the treatments with initial Pb2+ content lower than 100 mg l−1, the removal rate of Pb was about 40 %, while it was 23.5 % in E(200). In the following 43-h assay, Pb concentrations were diminished constantly and quickly. 40 % more of Pb in B(25) and C(50) was
Estimation of EPS amount in the EPS solution The quantity of EPS was preliminarily estimated according to the Dubois method by using glucose as standard (Braissant
Fig. 1 Variations of the dry weight of the biomass obtained from 42 to 205 h
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Fig. 2 Changes of Pb content and removal rate in the culture medium during the 164-h assay
removed, and about 30, 60, and 67 % more of Pb in D(100), E(200), F(400), respectively. Later on, the decrease of Pb concentrations slowed down. At 121 h, a faint decrease of Pb removal rate was detected in E(200) and F(400). At the end of this study, removal rates of Pb in the medium were about 95 % in treatments except that no Pb was detected in B(25). The limited intracellular absorption of metal in white-rot fungi has been reported (Baldrian 2003). To protect the cells from poisonous metals, the fungi replied with a variety of
extracellular defence mechanisms (Connolly and Jellison 1995). The steep descendant of Pb concentrations within the 1-h Pb2+ exposure implies prompt and effective extracellular responses in P. chrysosporium to Pb2+ pollution (Fig. 2), especially when the initial Pb2+ concentration was up to 400 mg l−1. The extracellular responses were indicated by the production of potential Pb removers. Similar removal rate of Pb in the treatments with less than 100 mg l−1 of Pb2+ is probably due to the tolerance of the existing Pb removers
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produced in the former 41-h culture. However, when initial Pb2+ content was higher, the previously produced Pb removers probably were not enough to tolerate so much Pb2+ so that more Pb removers were needed to ensure the efficient Pb removal. Most of the time in the experiment, Pb levels in the culture medium were lowering except at 121 h in E(200) and F(400) (Fig. 2). That might be due to the more decomposition or desorption of adsorbed Pb complexes than newly adsorbed ones. To learn the detailed performance of extracellular responses in Pb2+ biosorption, a further study on the production of oxalic acid and thiol-containing compounds (Fig. 3) under different Pb2+ stresses was carried out, both of which were important materials in the metal detoxification (Guimarães-Soares et al. 2007; Jarosz-Wilkolazka and Gadd 2003). Oxalate changes in the culture medium Oxalate contents in the culture medium are displayed in Fig. 3a. At 42 h, oxalate contents in B(25) and C(50) were 0.43 and 0.21 mM, respectively. They were both lower than
0.56 mM of oxalate in A(0), in contrast with that 0.72 mM in D(100), 3.03 mM in E(200), and 2.91 mM in F(400), which was much higher than that in A(0) (Fig. 3a). Oxalate content in A(0) and B(25) decreased during the whole determination period. Oxalate content in C(50), D(100), and E(200) displayed the highest level at 49 h (0.21, 0.72, and 3.03 mM, respectively), while in F(400), it did not reach a peak value until 85 h, i.e., 3.56 mM. Nearly no oxalate was detected in A(0) at 49 h, in B(25) and C(50) at 85 h, while oxalate in D(100), E(200), and F(400) decreased rapidly till 121 h. After 1-h exposure to higher initial levels of Pb 2+ , P. chrysosporium secreted more oxalate in all the treatments except B(25) and C(50) (Fig. 3a), probably because the Pb content under 50 mg l−1 did not form acute threat to the mycelia cells on the basis of the existed oxalate produced in the 41 h. The secretion of oxalic acid in the culture medium could tolerate a certain amount of Pb2+ by the formation and biosorption of Pb-oxalate complexes (Jarosz-Wilkolazka and Gadd 2003). At 42 h, the rapid rise of oxalate in E(200) and F(400) illustrated the quick initiation of the extracellular resistance mechanisms when P. chrysosporium contacted with high level of Pb2+. Oxalate concentrations in E(200) and F(400) during the former 8-h Pb2+ exposure were approximate. The reason probably is that excess oxalate was regulated by oxalate decarboxylase, which can directly decompose oxalate into formic acid and carbon dioxide (Mäkelä et al. 2002). And that also could explain the decrease of oxalate contents at 41 h in A(0) and B(25), at 49 h in C(50), D(100), and E(200), and at 85 h in F(400). It can be seen that the existence of more Pb2+ led to a delayed decrease of oxalate (Fig. 3a). T-SH content in the culture medium
Fig. 3 Variations of oxalate (a) and T-SH content (b) in the culture medium during the 164-h assay
T-SH content in the culture medium of all treatments showed similar variations (Fig. 3b). In A(0), T-SH content at 42 h was apparently different from that at 41 h. In the treatments with Pb2+ addition, T-SH content was kept at a relatively stable level during the first-hour exposure period. During the following 7-h assay, T-SH content decreased slowly, followed with a faint increase from 49 to 61 h in all the Pb2+ added treatments except B(25). In B(25), T-SH content decreased until 85 h. In A(0), T-SH content was kept at a stable level from 49 to 85 h. A second increase of T-SH content was detected from 85 to 157 h in Pb2+ treatments and from 85 to 121 h in A(0). T-SH content fluctuated around 0.3-0.5 mM in the previous 116-h Pb2+ exposure and dropped quickly to about 0.1 mM at 205 h. Data displayed in Fig. 3b suggests that extracellular thiol compounds was detectable (Fig. 3b), implying the secretion of free thiol compounds outside the cell. Similar phenomenon was obtained in other fungi, for instance, Thermomyces lanuginosus (Hasnain et al. 1992). The T-SH concentrations in Pb2+-added treatments were all higher than that in A(0) (Fig. 3b), similar with the enhanced intracellular synthesis of
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thiols by fungi under Pb stress reported by Guimarães-Soares et al. (2007). The reason for the higher T-SH concentration in Pb2+-added treatments may lie in the interference of Pb2+ in the metabolism system of P. chrysosporium (Baldrian 2003). It is interesting that the fluctuation trend of T-SH is opposite to the variation of oxalate. Thiols was proved to mediate the oxidation of nonphenolic lignin model compounds by manganese peroxidase of P. chrysosporium and that also may be involved in the variations of T-SH (Wariish et al. 1989). In the quantity perspective, metal immobilization requires compounds containing metal-binding functional groups, thus higher production of functional substances implies effective response to metal exposure. The obtained results indicate that P. chrysosporium utilized oxalic acid as a sensitive functional substance against Pb2+ toxicity and thiol compounds as a gentle response to Pb2+ in extracellular defence systems (Fig. 3). Changes of oxalate content and T-SH content in EPS solution As shown in Fig. 4a, the oxalate content in EPS solution of A(0) decreased continuously from 0.83 mmol g−1 at 41 h to 0 mmol g−1 at 121 h. The variations of oxalate in B(25) and C(50) were similar with that of A(0), except that the oxalate at 61 h increased a little. Oxalate content in EPS solution of D(100) and E(200) increased to a highest level at 42 h (0.93 and 1.03 mmol g−1, respectively), while in F(400), the peak displayed at 49 h (2.04 mmol g−1). From 121 to 157 h, the decrease of oxalate was relieved. The result implied that more oxalate was immobilized in the EPS layer when more Pb2+ was initially added. In Fig. 4b, the changes of T-SH content were different from that of the oxalate content. The T-SH contents in all treatments at 41 h and that at 42 h were close. After 42 h, more T-SH content was determined in treatment with higher initial Pb2+ concentration. The T-SH content showed similar variations, increasing at the beginning and following with a continued decrease until 205 h. The highest T-SH content in A(0) and B(25) appeared at 49 h (0.155 and 0.161 mmol g−1). The peak of T-SH in C(50), D(100), E(200), and F(400) was displayed at 61 h (0.172, 0.186, 0.231, and 0.282 mmol g−1, respectively). There are mucilaginous polymer substances distributed around the white-rot fungal cells, which are important materials for the removal of metal from aqueous solutions. Centrifugation is one useful method to extract the EPS from the cell wall. In Fig. 4, it is shown that oxalate and thiols were both detectable in EPS extract during the entire estimation period. Variations of oxalate in the EPS extract were different from the situation in the medium where no oxalate was estimated after 85 h in C(50) or 121 h in F(400) (Figs. 3a and 4a), and it implies that not all the adsorbed oxalate was degraded. The finding consolidated the decreasing Pb removal rate at 121 h in E(200) and F(400), implying that the degradation
Fig. 4 Variations of oxalate (a) and T-SH content (b) in the EPS solution during the 164-h assay
of oxalate affected the removal of Pb more seriously in the treatments with more than 200 mg l−1 of Pb2+. It also illustrates that when P. chrysosporium was exposed to high stress of Pb2+, oxalate played a very important role in Pb removal. Even the T-SH content in the culture medium fluctuated, T-SH in EPS solution increased continuously from 42 to 121 h. More T-SH in EPS solution of treatment with higher initial Pb2+ concentration probably implies that more Pb2+ would be removed and immobilized in the EPS of P. chrysosporium. The decrease of T-SH in EPS after 121 h maybe lies in the fast decrease of T-SH in the culture medium, maintaining the balance between the amount of T-SH in the solution and that on the surface of fungal mycelium. The decrease of the two kinds of functional substances did not restrict the Pb removal by P. chrysosporium, which is probably due to the participation of other Pb-binding substances. White-rot fungi can produce other organic acids except oxalic acid which can precipitate Pb ions and immobilize Pb in the mucilaginous polymer layer, such as malic acid, even the oxalic acid accumulation by P. chrysosporium F1767 was reported to be maximal during the fungal growth (Galkin et al. 1998, Hofrichter et al. 1999).
Environ Sci Pollut Res Table 1 Correlation between Pb and oxalate and that between Pb and T-SH in the EPS solution of C(50) and F(400)
R2 (oxalate/Pb) R2 (T-SH/Pb)
C(50)
F(400)
0.6853 0.2439
0.7685 0.266
Correlation between Pb and oxalic acid and that between Pb and T-SH The correlations between Pb and oxalate and that between Pb and T-SH in the EPS extract of the treatments were analyzed. Considering the first decreased and then increased variation of oxalate in C(50) and the similar variations of oxalate when initial Pb2+ content was over 50 mg l−1, the analysis results of the treatments C(50) and F(400) are presented in Table 1. During the whole Pb2+ exposure period, the linear correlation coefficient square between Pb and oxalate in the EPS extract of C(50) or F(400) was 0.6853, 0.7685, respectively, much higher than that between Pb and T-SH (0.2639, 0.266). The two kinds of coefficient in the F(400) were respectively slightly higher than those in C(50). The analysis of correlation between Pb and functional substances would provide some information on the contribution of the functional substance to the Pb removal. The results of correlation analysis suggest that oxalate was more significantly correlated to the Pb removal than thiol compounds. Nevertheless, no obvious relationship was found between adsorbed Pb and oxalate.
pH variations during the operation of functional substances As shown in Fig. 5, pH decreased in the previous 20-h exposure period, and then increased in the following detecting period from 61 to 205 h. pH values in the culture medium of all the treatments ranged from 4 to 6 before 205 h (Fig. 5). The
Fig. 6 Changes of EPS amount during the 164-h assay
pH in the culture medium in A(0), B(25), C(50), and D(100) at 205 h was in the range of 6.0–6.5. In the treatments with higher initial Pb2+ content, pH value is lower. The functional groups used for metal binding in oxalate and thiol compounds are carboxylic groups and thiol groups, respectively. The deprotonated or protonated states of the two functional groups, which may vary considerably with pH changes (Wang et al. 2011), can significantly influence metal binding and organic adsorption (Say et al. 2001). In the environment with a pH range from 4 to 6.5, most carboxylic units are deprotonated and present high metal retention ability (Rivas et al. 2003), while the thiol groups with pK values of 7.0–9.0 are prone to change in protonation state (Braissant et al. 2007). These may result in the more significant correlation between adsorbed Pb and oxalate than that between adsorbed Pb and T-SH (Fig. 5). Changes of EPS amount during the operation of functional substances As shown in Fig. 6, the EPS amount in the groups with Pb2+ addition was similar, increasing from 41 to 61 h and decreasing continually till 205 h. In A(0), the amount of EPS decreased continually. After 8-h exposure, higher and faster production of EPS was detected when more Pb2+ threatened the fungal cells. Even the fungal growth was inhibited in F(400), the EPS amount was not affected (Figs. 1 and 6). In general, EPS are produced from lysis and hydrolysis of adsorbed organic matters from the medium. More EPS in F(400) maybe Table 2 Correlation between oxalate and EPS and that between T-SH and EPS in the EPS solution
Fig. 5 pH variations in the culture medium of all the treatments during the 164-h detecting period
R2 (oxalate/EPS) R2 (T-SH/EPS)
B(25)
C(50)
E(200)
F(400)
0.522 0.918
0.58 0.861
0.666 0.831
0.738 0.637
Environ Sci Pollut Res
originated from the cell’s lysis, which was caused by the reactive oxygen species induced by the stress of Pb ions (Kim et al. 2008). Roles of EPS include enhancing cellular adhesion, providing a protective barrier, as well as adsorbing and storing nutrients for microbial growth (Long et al. 2009). Besides, EPS plays an important role in immobilizing the metal complex which is vital for the removal of heavy metal from the fluid environment. Therefore, the amount of EPS may be an important factor affecting the proceedings of metal removal by functional substances. The correlation between oxalate and EPS and that between T-SH and EPS in the EPS solution was analyzed, and the result is presented in Table 2. It is interesting to find that the R2 (oxalate/EPS) became higher in the group of more Pb2+, while the R2 (T-SH/EPS) displayed an opposite trend. The result probably indicates that the increase of EPS amount influenced the immobilized oxalate more seriously.
Conclusion In conclusion, P. chrysosporium was found to utilize oxalic acid as a sensitive functional substance against Pb2+ toxicity, while thiol compounds as a gentle response to Pb2+ in extracellular defence systems. In the treatment with higher initial Pb2+ concentration, higher content of oxalate and thiol compounds were both detected in the culture medium and in the EPS solution, but their variations in medium and that in EPS were different. Nevertheless, oxalate was found to contribute more to the Pb removal by P. chrysosporium than thiol compounds, with a more significant correlation with the removed Pb. The difference probably resulted from the pH variation and the EPS amount in the experiment. A pH range from 4 to 6.5 was beneficial for the operation of Pb2+ binding by carboxylic groups in the oxalic acid. More Pb2+ induced more EPS amount, and the increase of EPS amount influenced the immobilized oxalate more seriously. The present findings would provide useful references for the metal passivation mechanism in white-rot fungi and the transfer between environmental compartments and organisms.
Acknowledgments The study was financially supported by the National Natural Science Foundation of China (51278176, 51378190, and 51408206), the Environmental Protection Technology Research Program of Hunan (2007185), the New Century Excellent Talents in University (NECT-13-0186), the Young Teacher Growth Program of Hunan University, the Scientific Research Fund of Hunan Provincial Education Department (521293050), the Fundamental Research Funds for the Central Universities, the Hunan University Fund for Multidisciplinary Developing (531107040762), the Hunan Provincial Innovation Foundation for Postgraduate (CX2014B141), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17).
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