Abstract
White rot fungi have been extensively reported to have strong adsorption capacity to heavy metal ions, whereas the knowledge of extracellular polymeric substances (EPS) from the fungus has been rarely involved. In this study, the contribution characteristics of ‘the in situ EPS in Phanerochaete chrysosporium to Pb immobilization were investigated. First of all, the component and amount of EPS were investigated. It was found that the main component of EPS was carbohydrates, and highest EPS amount was produced at 5 days. In the Pb2+ immobilization experiments, EPS was demonstrated to play a more important role in immobilizing Pb2+ at lower initial Pb concentration. pH increase was beneficial for EPS to immobilize Pb. Higher EPS amount increased the Pb removal efficiency at a certain extent, while the specific uptake decreased. The Pb2+ immobilization by EPS produced at 7 days was most successful.
Similar content being viewed by others
Explore related subjects
Discover the latest articles, news and stories from top researchers in related subjects.Avoid common mistakes on your manuscript.
Introduction
In recent years, many studies have demonstrated that the white rot fungi have strong adsorption capacity to heavy metals [1, 2]. Along with the research, more scholars began to study the interaction mechanism of white rot fungi with heavy metals [3,4,5,6]. White rot fungi can form effective defense systems against the stress of toxic heavy metals. These defense systems are based on the intracellular and extracellular material which has the ability to complex heavy metals [7]. As the first interaction environment, the extracellular defense system is the most important for the passivation of heavy metal ions [8, 9].
In the biotreatment, extracellular polymeric substances (EPS) could be excreted by microorganisms produced from lysis and hydrolysis and adsorbed organic matters from wastewater [10,11,12]. Generally, the main components of EPS were comprised of polysaccharides, proteins, nucleic acids and other cellular components [13]. Part of EPS is dispersed in the solution (known as soluble polymer), the other part is located around the surface of microorganisms (known as the adsorbed polymers) [13]. The adsorbed polymers have multiple physiological functions for the living cells, including enhancing the cell’s adhesion, protecting cells from the harsh external environment, as well as adsorbing and storing the nutrient [14,15,16]. EPS is rich in a variety of functional groups, such as hydroxyl, amide, amino, carboxyl, thiol, etc., providing important basis for the removal of heavy metal ions in the environment [17, 18]. A number of studies have demonstrated that directly or indirectly proved the contribution of EPS to heavy metal immobilization [19, 20]. However, those studies were mainly focused on EPS produced by bacteria, biofilm and the activated sludge, knowledge of EPS from fungi, including white rot fungi, has been rarely involved.
Until now, the researches on the mechanisms of white rot fungi to remove heavy metal displayed some findings related with EPS. For example, heavy metal has always been explained to be ion exchanged by the fungal cell wall where EPS probably also participated in metal ion exchange [21]. Previous reports revealed that heavy metal oxalate crystals were found outside the mycelium of white rot fungi indicating that EPS probably plays a role in immobilizing metal chelate [4, 22]. However, the effect of the fungal EPS has not been directly studied. Those ambiguous descriptions would lead to inaccurate assessment of EPS contribution to heavy metal sorption or immobilization, and would hinder the further use of white rot fungi, in particular, some direct application of the fungal EPS in heavy metal treatment. Therefore, it is necessary to study the contribution characteristics of EPS when white rot fungi coping with toxic heavy metal.
In this study, the basic property of EPS produced by the model white rot fungi Phanerochaete chrysosporium (P. chrysosporium) was researched, including the component and the amount changes of EPS. Investigations on the contribution characteristic of the in situ EPS to Pb immobilization was carried out in batches of Pb contact experiment, including the effect of Pb concentration, pH, EPS amount and EPS age.
Materials and methods
Culture experiment
Phanerochaete chrysosporium (BKMF-1767) (CCTCC AF96007) was grown on potato dextrose agar plates at 37 °C for several days. Spores on the agar surface were diluted in sterile distilled water and controlled at 2.5 × 106 spores per mL [8]. 1.5 mL of spore suspension was inoculated into 200 mL of sterile potato dextrose broth in 500 mL flasks at room temperature. The culture process was undertaken in a constant temperature incubator shaker at 150 rpm, 30 °C.
EPS extraction
Mycelia in one flask were separated from the culture medium with filter paper and washed three times with ultrapure water. The obtained mycelia were re-suspended in 50 mL of ultrapure water and then centrifuged at 10,000 rpm for 15 min. The centrifuged suspension was separated from the fungal mycelia and used as EPS solution. The mycelia after centrifugation were seen as mycelia without EPS. There were additional compounds to EPS in the solution even if EPS might be the major component, such as oxalate as reported in the previous study [8]. However, that kind of oxalate was most likely to be oxalate metal which nearly has no ability to immobilize Pb2+. From this point, the additional compounds to EPS in the solution had little influence on the calculation of all the results in the manuscript.
Characterization of EPS
The carbohydrate content in EPS solution was determined with the phenol–sulfuric acid method, using glucose as standard. The protein content was determined by coomassie brilliant blue G-250 method, using bovine serum albumin as standard.
Pb2+ immobilization by mature mycelia with EPS and mature mycelia without EPS
The white rot fungal mycelia were cultured for some days and then separated from the medium for Pb2+ immobilization experiment. Mycelia with EPS or mycelia without EPS were exposed to Pb2+. Pb(NO3)2 was used as the source of Pb2+.
Choose of exposure time between mycelia and Pb2+ in the preliminary experiment
In the preliminary experiment with mycelia exposed to 50 mg L−1 of Pb2+, Pb concentration in the solution was sampled at different exposure time. It was found out the saturation point existed at 4 h which was used as the exposure period for the following experiments (Online Resource 1).
Effect of Pb2+ concentration
Mycelia with EPS or mycelia without EPS were exposed to different concentrations of Pb2+, i.e. 10, 50 and 100 mg L−1.
Effect of pH
pH may influence the protonated state of the functional groups in EPS, which further affect the contribution of EPS to Pb2+ immobilization [23]. Therefore, the effect of pH was considered in this study. Mycelia with EPS or mycelia without EPS were exposed to 50 mg L−1 of Pb2+ for 4 h. The pH condition was adjusted to a range from 3.0 to 7.0 with 0.1 mol L−1 of NaOH or 0.1 mol L−1 of HCl.
Effect of EPS amount
Different amount of 5-day-old fungal mycelia with EPS were exposed to 50 mg L−1 of Pb2+ for 4 h. With the same original amount of mycelia with EPS, EPS extraction was carried out and obtained the corresponding portion of mycelia without EPS, which were also exposed to 50 mg L−1 of Pb2+ for 4 h. EPS amount was calculated with the content summation of carbohydrates and protein and expressed with the unit of mg L−1.
Effect of EPS age
Based on the knowledge of EPS composition during the fungal growth, a certain amount of 3-, 5- and 7-day-old fungal mycelia was used for Pb2+ immobilization experiment, ensuring the same EPS amount in each treatment.
Analysis of Pb concentration
The sample was acidified with 3% (v:v) HON for the estimation of Pb concentration, which was determined with atomic absorption spectrophotometer. The instrument was calibrated with Pb2+ standard solutions. The final results were expressed in relation to the dry weight (dw) of the harvested mycelia, which was measured after the harvested mycelia were freeze dried.
Statistical analysis
All experiments in this study were performed in triplicates and mean values were used in the analysis.
Results and discussion
Characteristic of EPS
The compositions of EPS extracted from the mycelia are shown in Fig. 1. The results revealed that carbohydrates were always the main component of EPS during the whole fungal culture period. By comparison, there was much less protein which was out of our initial expectation. This may be due to the physical property of carbohydrates made them more easily attached to the cell wall, while most protein secreted by the fungus probably was soluble in solution. We further detected the soluble protein content at 5 days and found it was 40.6 mg L−1, much more than the biosorbed protein when both the two parts of protein were converted into mg as a unit. As displayed in Fig. 1, most of time both of the two compositions of EPS continuously decreased, which was due to the increase of EPS amount was much slower than the increase of fungal mycelia weight. Mycelia used for the batches of Pb2+ immobilization experiment was 5-day-old, adsorbed 92.02 mg g−1 of carbohydrates and 5.26 mg g−1 of protein. The fungal EPS production at different growth stage was different. EPS amount produced by fungus increased during the previous 2-day culture and then kept at a constant level from 3 to 4 days or from 5 to 6 days. At 7 days, the EPS amount decreased. As the two main compositions in EPS, carbohydrates and protein contain functional groups, like carboxyl (–COOH), hydroxyl (–OH) or amino (–NH2) groups, which provide negative charges for the molecules [20] and is important for EPS to bind cations, such as heavy metal ions.
Role of EPS in the immobilization of Pb
As shown in Fig. 2, with initial Pb2+ content of 10 and 50 mg L−1, the immobilization rate of Pb2+ by mycelia with EPS was close (57.4 and 57.7%, respectively), so was that by mycelia without EPS (41.7 and 41.6%, respectively). However, with 100 mg L−1 of Pb2+, the immobilization rate of Pb2+ by the two kinds of mycelia decreased obviously (39.7 and 27.6%, respectively). It is clear that the Pb2+ immobilization rates by mycelia with EPS were always higher at the three different initial Pb2+ concentrations. It was proved that the absence of EPS affected the Pb2+ immobilization absolutely, while the mycelia without EPS still displayed efficient removal ability of Pb2+.
Those findings demonstrate that EPS is important for P. chrysosporium to immobilize Pb2+. The most significant difference in the immobilization rate of Pb2+ between the two kinds of mycelia was 15.7% at 10 mg L−1 of Pb2+, with 16.1 and 12.1% at 50 and 100 mg L−1 of Pb2+, respectively. It seems that EPS was proportionally more important for Pb2+ immobilization at lower initial Pb2+ concentration. When initial Pb2+ concentration increased, the probability of binding sites in EPS to contact with Pb2+ in solution was greater. If there were enough binding sites in EPS, the immobilization rate of Pb2+ would be kept at a constant level around 16%. Considering the above result, we speculate that the binding ability of EPS probably reached saturation when mycelia faced with more than 50 mg L−1 of Pb2+ and in that situation the fungal intracellular immobilization of Pb2+ became more vital.
Effect of pH on the EPS contribution to Pb immobilization
Figure 3 presents the immobilization of Pb2+ by mature mycelia with EPS and mature mycelia without EPS as a function of pH. For the mycelia with EPS, the removal of Pb2+ was more efficient at higher pH. However, the situation for the mycelia without EPS was apparently different, with highest removal rate of Pb2+ at pH 5.0. Comparison between the two kinds of mycelia found out that Pb2+ was more efficiently removed by mycelia with EPS, and the difference as presented in Fig. 3 became more obvious as the increase of pH.
In aquatic system, pH is considered to be the main parameter affecting the affinity of metal ions to biological surfaces due to the competition between the proton and metal ions for available binding sites [24]. The results obtained above proved that the binding sites in EPS contributed to the immobilization of Pb in the whole pH range of 3.0–7.0 and the contribution became greater when pH increased. Functional groups, such as carboxylic (pK 4.0–6.0), phosphoric (pK 7.0–7.4), thiol and amino (pK 7.0–9.0), hydroxyl groups (pK 11.0) [25], display different deprotonated or protonated states with pH changes. From the pH range investigated in the study, we can suppose carboxylic group in EPS is probably the main functional group involved in Pb immobilization. Within the pH range from 4 to 6.0, most carboxylic units are deprotonated and present high metal retention ability. When pH in the solution was higher than 6.0, the functional groups seemed not to be so effective from the aspect of pK values. Even though, the mass removal of Pb2+ by EPS was also obtained. At pH 7.0, Pb2+ was prone to react with hydroxide ions in the solution and form lead hydroxide particles, which cannot be ignored. Because of the mucilaginous characteristic of EPS, the original thought Pb2+ immobilization probably was accomplished by the adhesion of lead hydroxide particles to EPS.
Effect of EPS amount on Pb immobilization
The results of Pb2+ immobilization on 5-day-old EPS when the in situ EPS amounts ranging from 146 to 1171 mg L−1 are presented in Fig. 4. The increase of the in situ EPS amount led to a higher Pb immobilization efficiency by EPS, increased from 10.8 to 26.7%, which probably is mainly due to the increase of immobilization sites and the surface area. Nevertheless, the Pb2+ specific immobilization decreased from 37 to 11.4 mg g−1 EPS. That is because that with constant initial Pb2+ amount and the increase of EPS amount, the proportion of immobilization sites to contact with Pb2+ became lower. Similar trend in EPS sorption characteristics was also reported by Dogru et al. [26].
Effect of EPS age
In this study, we chosen same amount of EPS at 3, 5 and 7 days and analyzed the Pb2+ immobilization characteristic by in situ EPS produced at different period. The result is presented in Fig. 5. It can been seen that the immobilization rate of Pb2+ was least for EPS at 3 days (13.9%), that by EPS at 5 days was 16.1% and at 7 days was 17.3%. As the growing curve of P. chrysosporium reported before [8], the 3-day-old fungus was at its accelerate phrase, the 5-day-old fungus was at its stationary phrase. At 7 days the fungal biomass began to decrease, which was at the decline phrase and the sign of mycelia hydrolysis due to the lack of nutrient in the environment. The result implied that EPS resourced from mycelia hydrolysis was most beneficial to the immobilization of Pb2+, EPS from resting mycelia the second and EPS from accelerate phrase the last. The main reason for the effect of EPS age on Pb immobilization probably lies in the composition change of EPS. On the one hand, the proportion of carbohydrates and protein in EPS changed during fungal growth as proved in Fig. 1. On the other hand, the specific material species of EPS probably changed and affected its contribution to Pb removal. The secondary metabolite was secreted by wood rooting fungi at decline phrase, such as polyketides, nonribosomal peptides, and terpenoids, which play a critical role for fungal survival and success, and the array of secondary metabolism genes has also been reported [27].
Conclusion
This study investigated the contribution characteristics of the in situ EPS in P. chrysosporium to Pb immobilization. Highest EPS amount was produced after 5-day culture. The main component of EPS was carbohydrates, with only a small part of protein and the ratio of carbohydrates to protein changed during the fungal growth. EPS was demonstrated to play a more important role in immobilizing Pb2+ at lower initial Pb concentration. The increase of pH in the solution was beneficial to the Pb immobilization in EPS. Higher EPS amount increased the Pb removal efficiency at a certain extent, while the specific uptake decreased. EPS produced at 7 days was most beneficial to the Pb2+ immobilization, which probably lies in the composition change of EPS, including not only the proportion of carbohydrates and protein, but also the existence of the secondary metabolite. This investigation could provide detailed information for the biology of white rot fungi.
References
Vigneshwaran N, Kathe AA, Varadarajan PV, Nachane RP, Balasubramanya RH (2006) Biomimetics of silver nanoparticles by white rot fungus, Phanerochaete chrysosporium. Colloids Surf B 53(1):55–59
Gabriel J, Vosahlo J, Baldrian P (1996) Biosorption of cadmium to mycelial pellets of wood-rotting fungi. Biotechnol Tech 10(5):345–348
Xu P, Zeng GM, Huang DL, Lai C, Zhao MH, Wei Z, Li NJ, Huang C, Xie GX (2012) Adsorption of Pb(II) by iron oxide nanoparticles immobilized Phanerochaete chrysosporium: equilibrium, kinetic, thermodynamic and mechanisms analysis. Chem Eng J 203:423–431
Zeng G, Li N, Huang D, Lai C, Zhao M, Huang C, Wei Z, Xu P, Zhang C, Cheng M (2015) The stability of Pb species during the Pb removal process by growing cells of Phanerochaete chrysosporium. Appl Microbiol Biotechnol 99(8):3685–3693
Li NJ, Zeng GM, Huang DL, Hu S, Feng CL, Zhao MH, Lai C, Huang C, Wei Z, Xie GX (2011) Oxalate production at different initial Pb2+ concentrations and the influence of oxalate during solid-state fermentation of straw with Phanerochaete chrysosporium. Biores Technol 102(17):8137–8142
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
Huang DL, Zeng GM, Feng CL, Hu S, Jiang XY, Tang L, Su FF, Zhang Y, Zeng W, Liu HL (2008) Degradation of lead-contaminated lignocellulosic waste by Phanerochaete chrysosporium and the reduction of lead toxicity. Environ Sci Technol 42(13):4946–4951
Li N, Zeng G, Huang D, Huang C, Lai C, Wei Z, Xu P, Zhang C, Cheng M, Yan M (2015) Response of extracellular carboxylic and thiol ligands (oxalate, thiol compounds) to Pb2+ stress in Phanerochaete chrysosporium. Environ Sci Pollut Res 22:12655–12663
Pointing S (2001) Feasibility of bioremediation by white-rot fungi. Appl Microbiol Biotechnol 57(1–2):20–33
Pal A, Paul A (2008) Microbial extracellular polymeric substances: central elements in heavy metal bioremediation. Indian J Microbiol 48(1):49–64
Tang L, Zeng G-M, Shen G-L, Li Y-P, Zhang Y, Huang D-L (2008) Rapid detection of picloram in agricultural field samples using a disposable immunomembrane-based electrochemical sensor. Environ Sci Technol 42(4):1207–1212
Gong J-L, Wang B, Zeng G-M, Yang C-P, Niu C-G, Niu Q-Y, Zhou W-J, Liang Y (2009) Removal of cationic dyes from aqueous solution using magnetic multi-wall carbon nanotube nanocomposite as adsorbent. J Hazard Mater 164(2–3):1517–1522
Meng L, Xi J, Yeung M (2016) Degradation of extracellular polymeric substances (EPS) extracted from activated sludge by low-concentration ozonation. Chemosphere 147:248–255
Long G, Zhu P, Shen Y, Tong M (2009) Influence of extracellular polymeric substances (EPS) on deposition kinetics of bacteria. Environ Sci Technol 43(7):2308–2314
Bitton G, Freihofer V (1977) Influence of extracellular polysaccharides on the toxicity of copper and cadmium toward Klebsiella aerogenes. Microb Ecol 4(2):119–125
Zhang W, Cao B, Wang D, Ma T, Xia H, Yu D (2016) Influence of wastewater sludge treatment using combined peroxyacetic acid oxidation and inorganic coagulants re-flocculation on characteristics of extracellular polymeric substances (EPS). Water Res 88:728–739
Yin Y, Hu Y, Xiong F (2011) Sorption of Cu(II) and Cd(II) by extracellular polymeric substances (EPS) from Aspergillus fumigatus. Int Biodeterior Biodegrad 65(7):1012–1018
Chen Y-P, Zhang P, Guo J-S, Fang F, Gao X, Li C (2013) Functional groups characteristics of EPS in biofilm growing on different carriers. Chemosphere 92(6):633–638
Sheng G-P, Xu J, Li W-H, Yu H-Q (2013) Quantification of the interactions between Ca2+, Hg2+ and extracellular polymeric substances (EPS) of sludge. Chemosphere 93(7):1436–1441
Joshi PM, Juwarkar AA (2009) In vivo studies to elucidate the role of extracellular polymeric substances from azotobacter in immobilization of heavy metals. Environ Sci Technol 43(15):5884–5889
Yetis U, Dolek A, Dilek FB, Ozcengiz G (2000) The removal of Pb(II) by Phanerochaete chrysosporium. Water Res 34(16):4090–4100
Jarosz-Wilkolazka A, Gadd GM (2003) Oxalate production by wood-rotting fungi growing in toxic metal-amended medium. Chemosphere 52(3):541–547
Zhang W, Cao B, Wang D, Ma T, Yu D (2016) Variations in distribution and composition of extracellular polymeric substances (EPS) of biological sludge under potassium ferrate conditioning: effects of pH and ferrate dosage. Biochem Eng J 106:37–47
Horsfall MJ, Spiff AI (2004) Studies on the effect of pH on the sorption of Pb2+ and Cd2+ ions from aqueous solutions by Caladium bicolor (Wild Cocoyam) biomass. Electron J Biotechnol 7:313–323
Wang L-L, Wang L-F, Ren X-M, Ye X-D, Li W-W, Yuan S-J, Sun M, Sheng G-P, Yu H-Q, Wang X-K (2011) pH dependence of structure and surface properties of microbial EPS. Environ Sci Technol 46(2):737–744
Dogru M, Gul-Guven R, Erdogan S (2007) The use of Bacillus subtilis immobilized on amberlite XAD-4 as a new biosorbent in trace metal determination. J Hazard Mater 149(1):166–173
Fox EM, Howlett BJ (2008) Secondary metabolism: regulation and role in fungal biology. Curr Opin Microbiol 11(6):481–487
Acknowledgements
The study was jointly supported by the National Natural Science Foundation of China (51608142 and 51638006), the Natural Science Foundation of Guangxi Province (2016GXNSFBA380076), and the Foundation of Guilin University of Technology. We are also grateful for the help from Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, and Guangxi Scientific Experiment Center of Mining, Metallurgy and Environment.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Li, N., Zhang, X., Wang, D. et al. Contribution characteristics of the in situ extracellular polymeric substances (EPS) in Phanerochaete chrysosporium to Pb immobilization. Bioprocess Biosyst Eng 40, 1447–1452 (2017). https://doi.org/10.1007/s00449-017-1802-2
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00449-017-1802-2