Degradation of Lead-Contaminated Lignocellulosic Waste by

May 21, 2008 - Danlian HuangXuelei YanMing YanGuangming ZengChengyun ZhouJia WanMin ChengWenjing Xue. ACS Applied Materials & Interfaces ...
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Environ. Sci. Technol. 2008, 42, 4946–4951

Degradation of Lead-Contaminated Lignocellulosic Waste by Phanerochaete chrysosporium and the Reduction of Lead Toxicity DAN-LIAN HUANG,† G U A N G - M I N G Z E N G , * ,† CHONG-LING FENG,† SHUANG HU,† XIAO-YUN JIANG,† LIN TANG,† FENG-FENG SU,† YU ZHANG,† W E I Z E N G , † A N D H O N G - L I A N G L I U †,‡ College of Environmental Science and Engineering, Hunan University, Changsha 410082, China, and Chinese Research Academy of Environmental Science, Beijing 100084, China

Received January 8, 2008. Revised manuscript received April 7, 2008. Accepted April 9, 2008.

Lead, as one of the most hazardous heavy metals to the environment, interferes with lignocellulosic biomass bioconversion and carbon cycles in nature. The degradation of leadpolluted lignocellulosic waste and the restrain of lead hazards by solid-state fermentation with Phanerochaete chrysosporium were studied. Phanerochaete chrysosporium effectively degraded lignocellulose, formed humus and reduced active lead ions, even at the concentration of 400 mg/kg dry mass of lead. The highest lignocellulose degradation (56.8%) and organic matter loss (64.0%) were found at the concentration of 30 mg/kg of lead, and at low concentration of lead the capability of selective lignin biodegradation was enhanced. Microbial growth was delayed in polluted substrate at the initial stage of fermentation, and organic matter loss is correlated positively with microbial biomass after 12 day fermentation. It might be because Phanerochaete chrysosporium developed active defense mechanism to alleviate the lead toxicity. Scanning electron micrographs with energy spectra showed that lead was immobilized via two possible routes: adsorption and cation exchange on hypha, and the chelation by fungal metabolite. The present findings will improve the understandings about the degradation process and the lead immobilization pathway, which could be used as references for developing a fungibasedtreatmenttechnologyformetal-contaminatedlignocellulosic waste.

as logging residues, agricultural waste, and plant residues are difficult to degrade, and the degradation process is often slow in nature (2). Therefore, much attention has currently been drawn to the development of solid-state fermentation (SSF) with fungi for the efficient treatment of lignocellulosic waste (3, 4). In many countries and districts there are lead (Pb) mine areas and Pb-contaminated soils, accordingly the plants and crops nearby are polluted to some extent. High concentrations of Pb are found in the leaves, stems, and other parts of the plants grown in the Pb-polluted sites (5, 6). Therefore the wastes from these plants, such as fallen leaves and agricultural residues also contain high concentration of Pb, which is the main source of Pb-contaminated lignocellulosic waste. Meanwhile lignocellulosic waste is used as biosorbent to remove Pb from wastewater, and therefore needs proper disposal (7, 8). Pb in substrate is a challenge in the degradation of lignocellulosic waste, because Pb affects fungal colonization and biodegradation ability. So the understanding on the SSF process of lignocellulosic waste in the presence of Pb is necessary for the research. White-rot fungi are characterized by their unique ability to degrade lignin (9, 10). The finding that white-rot fungus Phanerochaete chrysosporium (Pc) could efficiently bridge the lignin barrier has attracted particular attention, and Pc is studied as a characteristic species (9, 11, 12). Previous studies confirmed that Pc could tolerate the low and middle concentration of Pb and remove Pb from wastewater with its mycelium (13, 14). So Pc could be adapted to the complex polluted environment, and the application of Pc in the treatment of Pb-polluted lignocellulosic waste was supposed to be a promising strategy. Although experiments with the organic matter degradation and the Pb accumulation from liquid media by Pc can provide useful information about the relationship between fungal activities and metal (13, 15), they could not exactly reflect the actual situation in SSF. Until now, there is limited and scattered information about the SSF process with Pc in the presence of Pb. We found that Pc maintained active and degraded organic matter significantly at the total Pb concentration of 105 mg/kg and 400 mg/kg (dry weight) (16, 17). But no details on lignocellulose degradation and Pb immobilization pathway by Pc under different initial Pb concentrations have been reported in literature so far. In this study, we investigated the fungal degradation of lignocellulosic waste at different concentrations of Pb and the effect of Pb on biotreatment with chemical and microbiological analyses. The transformation of Pb fractions and the immobilization pathway of Pb in biotreatment process were also studied.

Experimental Section Introduction Lignocellulose is a macromolecular complex consisting of lignin, cellulose, and hemicellulose. Lignin is a highly irregular and insoluble polymer and chemically bonded by covalent linkages with hemicellulose and the lignin-carbohydrate complexes enwrap cellulose in plant cell wall (1). This intricate association constitutes accessibility barriers to the lignocellulose transformation, so the lignocellulosic wastes, such * Corresponding author phone: (86)-731-8822754; fax: (86)-7318823701; e-mail: [email protected]. † College of Environmental Science and Engineering, Hunan University, China. ‡ Chinese Research Academy of Environmental Science. 4946

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Strain and Chemicals. The fungus Pc strain BKMF-1767 was obtained from China Center for type Culture Collection (Wuhan, China). Fungal cultures were maintained on potato dextrose agar (PDA) slants at 4 °C, and then transferred to PDA plates at 37 °C for several days. The spores on the agar surface were gently scraped and blended in the sterile distilled water as spore suspension. The spore concentration was measured and adjusted to 2.0 × 106 spores per mL. All the chemicals used in this work were of analytical reagent grade. Solid-State Fermentation Conditions. The straw as lignocellulosic waste from uncontaminated soil was air-dried and ground to pass through a 2 mm nylon screen. The concentration of total Pb in the straw was 2.1 mg/kg. A 33 g amount of straw powder, contained in each 500 mL fer10.1021/es800072c CCC: $40.75

 2008 American Chemical Society

Published on Web 05/21/2008

mentation flask labeled as A(control), B(30), C(200) and D(400), respectively, was supplemented and mixed thoroughly with Pb(NO3)2 solutions by adding total Pb(II) 0, 30, 200, and 400 mg/kg straw (dry weight). They were used to simulate different degrees of Pb pollution. Each flask was stoppered and autoclaved for 60 min at 121 °C. Then 2 mL spore suspension was inoculated. The fermentation experiments were performed at 37 °C for 42 days. The humidity was maintained at the initial level (75%) in the entire fermentation period. The additional batch of four flasks were prepared in the same way, which were sampled only on day 0 and 42 for better indication of the loss of lignocellulose and straw dry mass. To make a better comparison, the noninoculated control flasks were used. All experiments were performed in three replicates. Sampling and Analytical Methods. The fungus Pc was tested for its ability to degrade straw in the presence of Pb in terms of the loss of straw dry mass and lignocellulose component (lignin, cellulose and hemicellulose) content. The change in fungal biomass, total organic matter (TOM), and humus carbon (CHE) during SSF process were also considered. Samples taken from the additional batch of flasks on day 0 and 42 were dried, homogenized, and analyzed. The loss of straw dry mass was calculated as the difference against the mass on day 0. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were determined according to a rather rough method (18). Hemicellulose was estimated as the difference between NDF and ADF. Cellulose was estimated as the difference between ADF and ADL. Lignin was estimated as the difference between ADL and ash content. The degradation rate of lignin, cellulose, and hemicellulose was calculated by the difference between the control and the inoculated flasks. Capability of selective lignin degradation estimated by selective index is ascertained by comparing the degradation rate of lignocelluolse with that of lignin. The fungal biomass carbon was measured by fumigation of the fresh sample with ethanol-free chloroform and extraction with 0.5 M K2SO4, according to Wu et al. (19). TOM in samples could be estimated in the same way as described by Huang et al. (20). CHE was determined by Kononova method (21). The humification of straw was evaluated by the percentage of CHE to total organic carbon in the sample (CHE/Corg). For a detailed description of the analytical procedures, see the Supporting Information. Analysis of Pb fractions was performed at every 6 days in SSF. Pb fractions were analyzed as introduced by Huang et al. (16). Five fractions of Pb were extracted in turn as follows: soluble-exchangeable, carbonate-bound, Fe-Mn oxidesbound, organic-bound, and residual Pb (see the Supporting Information). To confirm the adsorption of Pb by fungal mycelia, microscopic surface and composition analysis of mycelia were performed. On day 42, the fungal mycelia selected from the colonized straw were washed with deionized water and freeze-dried. Then they were observed by using a JEOL-5600LV (Japan) scanning electron microscope (SEM) equipped with an EDS-Microanalysis System (Vantage DI 4105, American). The back scattering electron (BSE) image was used. Statistical Analysis. The results to be presented were the mean value of the three replicates, and the standard deviations were used to analyze experimental data. Statistical analyses were performed to obtain more comprehensive and useful information, using the software package SPSS 13.0 for Windows (SPSS, Germany). These tests included (i) a oneway analysis of variance (ANOVA) for straw dry mass, lignin, cellulose, and hemicellulose degradation, respectively and (ii) correlation analysis used to determine relationships between organic carbon loss and microbial biomass.

FIGURE 1. Degradation of lignocellulose components in A, B, C, and D straws after fermentation. (A) Control without Pb addition. (B) Treatment with Pb 30 mg/kg dry straw. (C) Treatment with Pb 200 mg/kg dry straw. (D) Treatment with Pb 400 mg/kg dry straw.

Results and Discussion Biodegradation of Lignocelluloses. In A(control), most cellulose was transformed, with the degrading rate of 55.90%, whereas the most recalcitrant polymer (lignin) was decomposed by 42.14% (Figure 1). Cellulose degradation was restrained by Pb(II), and the higher initial Pb(II) concentration resulted in the greater inhibition. Except for Pb(II) addition of 400 mg/kg, other additional amounts were not found to inhibit hemicellulose and lignin degradation by Pc. The lignin degradation rate (48.44%), hemicellulose degradation rate (67.53%), and the selectivity index as 0.85 in C(200) showed the highest value in all these trials. The higher lignocellulose loss rates were obtained in B(30) and C(200) than that in control, especially the former reached 56.8%. Lignocellulose was still degraded effectively in D(400) under inhibition. Straw dry mass in each flask decreased obviously after 42 day SSF (see Figure S1, Supporting Information). The highest dry mass loss reaching 49.03% was found in B(30), whereas 47.42% of total straw dry mass in C(200) was degraded. In contrast, the loss of straw dry mass in A(control) and D(400) were 40.12 and 38.02%, respectively. There was insignificant difference (P ) 0.156 and 0.105, respectively) between the dry mass loss and the hemicellulose degradation rate in D(400) and those in the control. But the degradation rate of lignin and cellulose, and the selectivity index in D(400) were significantly (P ) 0.001, 0.002, and 0.001, respectively) lower than those in control. It is found that lignocellulose was degraded effectively at high concentration of Pb by Pc during SSF, which indicated Pc is resistant to the toxicity of Pb. Low or no inhibition of Pb to lignocellulose degradation by Pc in this study might be because that the availability of Pb was limited during SSF due to nonspecific binding, which has also been found for other white-rot fungi by Baldrian et al. (22) and Tuomela et al. (23). Promotion of lignocellulose decay by Pc at low concentration of Pb might be because Pb affected the carbon and energy supplying system of ligninase, cellulases, and hemicellulases (14), but the mechanism needed further study. Several researchers found that fungi VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Changes of microbial biomass carbon in the control without Pb addition and the treatments with Pb in concentrations of 30, 200, and 400 mg/kg dry straw, respectively. The bars represent the standard deviations of the means (n ) 3). mainly utilized organic carbon to synthesize the fungal cell, but tended to transform it into CO2 to maintain energy in substrate containing heavy metal at moderate concentration levels (24). This might be another reason for the increase of lignocellulose loss at low concentration of Pb. Since lignin is chemically bonded by covalent linkages with hemicellulose (see Figure S2, Supporting Information), we also found in this work that the hemicellulose degrading rate was high when lignin was degraded effectively. Dynamic Changes in Microbial Biomass. The fluctuating curves of microbial biomass in all treatments were similar (Figure 2). Microbial biomass increased rapidly during the initial stage followed by the fast decline along with the time shift, and return to rise obviously after 24 day SSF. After 36 day SSF, microbial biomass declined again. The main degraded materials in substrate at the different stages of SSF by fungi were in turn hemicellulose, cellulose, and lignin according to their dissimilar structure and availability (see Figure S3 for the classification of different stages of SSF), whereas the growth of fungi may be affected by the degradation of hemicellulose, cellulose, and lignin. This could be a reason for the fluctuation of biomass. Microbial biomass carbon showed the highest peak value (13.8 mg/g) on day 3 in A(control), while in B(30) and C(200) it reached a peak (13.5 mg/g and 12.8 mg/g, respectively) on day 6. The microbial biomass in D(400) did not reach a peak until day 9, and the maximum was 12.0 mg/g. The results indicated that the growth rate of Pc was slowed in the presence of Pb ion in the initial stage of SSF, and the lag phase of growth became longer due to the inhibition of Pb. Lengthening of the lag phase was also recorded on media containing metal in the case of Pleurotus ostreatus, Pycnoporus cinnabarinus, and Serpula lacrymans in the research of Mandal et al. (25). No inhibition of fungal growth by Pb was observed in Pb-supplemented substrate after 15-day SSF in this study, and Pc could colonize successfully even in the presence of Pb. This might be because Pc is resistant to Pb, and the toxicity of Pb is weakened due to the transformation of toxic Pb ions into inactive forms during incubation by fungal activity (26, 27). Relationship between Organic Matter Loss and Microbial Biomass. TOM decreased obviously during SSF (Table S1). The changing trend of TOM loss corresponded to the variation in microbial biomass. TOM decreased rapidly from day 0 to day 6, and then decreased slowly from day 6 to day 24, and afterward the fast decline appeared after day 24. After 36 days TOM was only decreasing slowly. Although the microbial 4948

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biomass in Pb-supplemented straw was not higher than those in the control, slightly higher degradation of TOM reaching 64.0% was observed in B(30) after 42 day SSF. The loss of TOM was primarily due to the fungal consumption of carbohydrates mostly associated with cell wall (28). Fewer TOM losses were found in treatments than in A(control) in the initial stage, which was probably due to the weakening fungal activity and modest fungal growth in the presence of active Pb ions until day 3. But the total loss of TOM was not reduced even at high concentration of Pb during 42 day SSF in this study. Baldrian et al. (29) also found that high concentrations of heavy metals did not result in the decrease of organic matter degradation although they caused the reduction of fungal colonization rate or incomplete colonization. The correlation between TOM loss and microbial biomass carbon content was analyzed (Figure 3). During the whole SSF process (from 0 to 42 days), the remarkable linear correlation between TOM loss and the content of microbial biomass carbon was found in A(control) as shown in Figure 3(a). But no obvious relationship was established between the TOM loss and the content of microbial biomass carbon in B(30), C(200), and D(400), presented in Figure 3(b-d). The results indicated that the Pb ions disturbed the degradation of TOM by Pc. During the period from day 12 to 42, the TOM loss significantly correlated to the content of microbial biomass in B(30), C(200), and D(400). The reason might be that the effect of Pb on fungi turned weak due to the immobilization of Pb ions after 12 days of SSF. Formation of humus. CHE and CHE/Corg were much higher in three treatments compared to those in A(control) before 18-day SSF, and then the difference among all trials decreased greatly (see Table S1, Supporting Information). The humus degradation always exists concurrently with its formation, so the CHE measured in samples is in fact the carbon content of the remaining humus subtracting the degraded humus from the formed humus. It was confirmed that humus could chelate Pb ions, and these chelated compounds hampered the humus degradation by fungi (30). Therefore, the higher CHE and CHE/Corg observed in Pb-supplemented treatments might be due to the great reduction of the degraded humus. Transformation of Pb Fractions. Pb concentrations in five fractions varied after SSF by Pc (Table 1). After 42 day SSF, in all flasks, the content of the soluble-exchangeable Pb and the carbonate-bound Pb decreased, whereas that of the other three forms increased. The amount of the immobilized Pb during SSF by Pc decreased with the initial metal concentrations increasing. The bioavailability and transfer ability of five Pb-fractions decreases in the order of extraction. Free forms of metals in solution are generally supposed to be more toxic to microorganisms than complexed or sorbed forms. Therefore the results in this study revealed that most of the active Pb ions had been transformed into inactive Pb forms, which indicated the reduction of toxicity and bioavailability of Pb after 42 day SSF by Pc. Reasons for these results could be as follows: (i) white-rot fungi could chelate Pb with the carboxyl, hydroxyl, or other active functional groups on cell wall surface to reduce Pb activity (14, 31) and (ii) white-rot fungi could promote the formation of humus by accelerating the organic matter decomposition, and the chelation of Pb by humus is the mechanism responsible for Pb immobilization, as we reported previously (16, 17). Immobilization Pathway of Pb by Fungal Mycelia. The BSE image of fungal mycelia shows the formation of granular particle along with the precipitation of strip-shaped crystals (Figure 4a). The granular particle was presented on the cell wall surface of fungal hyphae, while the strip-shaped crystal precipitates were wrapped in the mycelia. The EDS microanalyses indicated that the granular particle on surface

FIGURE 3. Relationship between total organic matter loss and microbial biomass carbon in different treatments. (a) Control without Pb addition. (b) Treatment with Pb 30 mg/kg dry straw. (c) Treatment with Pb 200 mg/kg dry straw. (d) Treatment with Pb 400 mg/kg dry straw. The three small figures in Figures (b), (c), and (d) are presented results from day 12 to 42.

TABLE 1. Pb Content of Five Fractions in the Control without Pb Addition and the Treatments with Pb in Concentration of 30, 200, and 400 mg/kg dry Straw, Respectively, Before and after Solid-State Fermentation Pb-fractions

control

treatment (30 mg/kg)

treatment (200 mg/kg)

treatment (400 mg/kg)

a

a

day 0 day 42

0.16 (0.02) 0.01 (0.01)

day 0 day 42

0.21 (0.02) 0.13 (0.01)

day 0 day 42

0.56 (0.03) 0.62 (0.03)

day 0 day 42

0.88 (0.05) 1.01 (0.06)

day 0 day 42

0.29 (0.02) 0.43 (0.02)

soluble-exchangeable Pb (mg/kg) 23.6 (0.21) 155.6 (1.09) 0.02 (0.01) 0.09 (0.01) carbonate-bound Pb (mg/kg)a 4.64 (0.09) 37.51 (0.54) 4.01 (0.10) 34.68 (0.43) Fe-Mn oxides-bound Pb (mg/kg)a 1.25 (0.05) 3.47 (0.08) 6.03 (0.11) 30.15 (0.27) organic-bound Pb (mg/kg)a 1.77 (0.06) 3.59 (0.07) 15.52 (0.13) 99.34 (0.88) residual Pb (mg/kg)a 0.94 (0.03) 2.13 (0.06) 6.82 (0.12) 38.24 (0.31)

307.35 (1.27) 0.11 (0.02) 76.53 (0.61) 75.39 (0.52) 6.24 (0.10) 58.5 (0.56) 7.72 (0.12) 204.02 (1.14) 4.06 (0.07) 64.08 (0.59)

Values are means (n ) 3) with standard deviations in parentheses.

of fungal hyphae was strongly enriched in lead, potassium, phosphorus, and carbon (Figure 4b). The EDS microanalyses also showed that the strip-shaped crystals observed around hyphae of Pc from Pb-polluted substrate had a Pb-rich composition (Figure 4c). So the fungus Pc was proved to be good at immobilizing metal ions with its mycelium, as shown in Figure 4 and Table 1. White-rot fungi can accumulate metals from substrate. It has been reported that the intracellular absorption of metal is limited for white-rot fungi, and the sorption of metal to polysaccharides, proteins, or other molecules on the cell wall surface probably plays the most important role (26, 32).

Therefore we applied SEM-EDS to reveal the Pb immobilization by the extracellular and cell wall-associated binding according to refs 33 and 34. From our findings by SEM-EDS analysis and some known interaction mechanism (14), two hypotheses could be made: (i) Pb ions were adsorbed on cell walls, because cell walls of fungus Pc consist mostly of polysaccharides, peptides, and pigments with good capacity to bind heavy metals, or (ii) Pb ions were chelated by the carboxyl, hydroxyl, or other active functional groups on cell wall surface accompanied with ion exchange. They coincided with the conclusions reported by Say et al. (26) and Saglam et al. (35). Falih (27) confirmed that Pc was able to survive VOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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on the immobilization of heavy metals using extracellular chelating compounds (14). One of the typical metal chelators produced by white-rot fungi is oxalic acid, and higher amounts of oxalic acid produced by Pc than by other whiterot fungi could facilitate the immobilization of soluble metal ions by chelating metal ions as insoluble oxalates (14, 36). This might explain the precipitation of strip-shaped crystals containing both lead and carbon around mycelia. Results from this study are expected to offer useful references on alleviating the environmental impact of metal-contaminated lignocellulosic waste by fungi. Further studies are needed to understand the related mechanism more deeply and improve the biotreatment technology for contaminated lignocellulosic waste.

Acknowledgments The study was financially supported by the National 863 High Technology Research Program of China (no. 2004AA649370), the Chinese National Basic Research Program (973 Program) (no. 2005CB724203), the Program for Changjiang Scholars and Innovative Research Team in University (IRT0719), the Environmental Protection Technology Research Program of Hunan (no. 2007185) and the National Natural Science Foundation of China (no. 50608029).

Supporting Information Available More details of the analytic procedures, the loss of straw dry mass, the structure of lignocellulose, the classification of the SSF stage by cluster analysis and the detailed data of TOM, CHE and CHE/Corg. This material is available free of charge via the Internet at http://pubs.acs.org.

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FIGURE 4. SEM image (a) of fungal mycelium from Pb-polluted straw after 42 day solid-state fermentation. Energy spectra of the granular particle (b) and the strip-shaped crystals (c) observed in SEM image. and grow in the liquid medium containing up to 400 mg/kg of Pb ions and accumulate considerable amounts of Pb from the growth medium. The interaction of fungi with heavy metals causes severe changes in the physiological processes and under certain circumstances it can even kill the mycelium. Therefore, fungi evolved active defense mechanism to alleviate the toxicity of metals. The defense is usually based 4950

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