Environ. Sci. Technol. 2010, 44, 6202–6208
Cr(VI) Removal on Fungal Biomass of Neurospora crassa: the Importance of Dissolved Organic Carbons Derived from the Biomass to Cr(VI) Reduction L.C. HSU,† S.L. WANG,† Y.C. LIN,† M.K. WANG,‡ P.N. CHIANG,§ J.C. LIU,| W.H. KUAN,⊥ C.C. CHEN,# AND Y . M . T Z O U * ,† Department of Soil & Environmental Sciences, National Chung Hsing University, Taichung, TW, 40227, Department of Agricultural Chemistry, National Taiwan University, The Experimental Forest, National Taiwan University, Nantou, TW, 55743, Agricultural Research Institute No.189, Jhongjheng Rd., Wufong, Taichung County, TW 41301, Department of Safety, Health, and Environmental Engineering, Ming Chi University, Taishan, Taipei 24301, and Department of Life Science, National Taiwan Normal University, Taipei, TW 116
Received May 19, 2010. Revised manuscript received July 8, 2010. Accepted July 8, 2010.
Interactions of toxic Cr(VI) with renewable biomaterials are considered an important pathway for Cr(VI) removal in ecosystems. Biomaterials are susceptible to dissolution, and their dissolved derivatives may provide an alternative to surface-involved pathway for scavenging of Cr(VI). In this study, dissolved organic carbon (DOC) derived from Neurospora crassa biomass was investigated. The proportion of Cr(VI) reduction by DOC to that on biomass was determined to evaluate the importance of DOC to Cr(VI) reduction. A rapid increase in DOC concentration from 145.6 to 193.7 mg L-1 was observed when N. crassabiomass was immersed in 0.01 M KCl solution at pH of 1-5, and polysaccharides, peptides, and glycoproteins with carboxyl, amide, and -NH functional groups, are the major compositions of DOC. On reaction of 96.2 µM Cr(VI) with N. crassabiomass or DOC, it was estimated that DOC contributed ∼53.8-59.5% of the total Cr(VI) reduction on biomass in the dark. Illumination enhanced Cr(VI) reduction via photo-oxidation of biomass/DOC under aeration conditions, which formed superoxide for Cr(VI) reduction. At pH 1, photoinduced Cr(VI) reduction by DOC proceeded more rapidly than reduction on the biomass surface. However, at pH >3, with a decrease in Cr(VI) reduction by DOC, photon-excited biomass may become an important electron source for Cr(VI) photoreduction.
Introduction Intentional or unintentional discharges of heavy-metalcontaining wastewaters from various industries such as * Corresponding author phone: 886-4-22840373(x4206); fax: 8864-22855167; e-mail:
[email protected]. † National Chung Hsing University. ‡ Department of Agricultural Chemistry, National Taiwan University. § The Experimental Forest, National Taiwan University. | Agricultural Research Institute No.189. ⊥ Ming Chi University. # National Taiwan Normal University. 6202
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
electroplating, tanning, and dyeing have resulted in serious impacts on the environment worldwide (1, 2). Chromium is one of the most commonly found metals in a waste-stream because of its extensive use (3). Chromium, particularly in hexavalent form [Cr(VI)], is of great scientific concerns owing to its high toxicity to living organisms (4–6) and its potential carcinogenicity to humans (7, 8). Cr(VI) can be reduced to less toxic Cr(III) by various inorganic materials, such as Fe(0), Fe(II), and S(II) (9–11), and organic matters, including humic substances, black carbon, and artificial organic compounds (12–16). After Cr(VI) is reduced to Cr(III), the mobility and toxicity of Cr(VI) decrease greatly because the resultant Cr(III), can be readily adsorbed on negatively charged soil minerals or precipitated in solutions of pH > 5.5 (17). It has recently reported that nonhumified biomaterials (NHBs) such as bacteria, yeast, seaweed, algae, and rotten wood are capable of removing Cr(VI) from aqueous media (18–23). Cr(VI) removal was mainly attributed to reduction by specific functional groups, such as carboxyl and amino groups on NHBs over a wide pH range (3, 23). Park et al. (3) proposed that Cr(VI) reduction on fungal biomass included direct and indirect pathways, emphasizing the important role of fungal surfaces involving Cr(VI) reduction, as observed by other scientists using various NHBs as reductants (19–21). However, unlike most rigid inorganic solids, biomaterials are susceptible to a certain degree of dissolution, forming dissolved organic carbon (DOCNHB) in soil solutions or natural water bodies. Considering its mobility, DOCNHB is more likely to interact with Cr(VI) and control its fate in an environment containing both NHB and Cr(VI). There has been less of a focus in the literature on the contribution of DOCNHB to Cr(VI) reduction than that of the corresponding biomass surfaces. The role and importance of DOCNHB in Cr(VI) reduction need to be clarified for two reasons. First, NHB materials such as fungi are widely distributed in soils and natural water bodies, and thus may be a dominant source of DOC within soil pores and localized aquatic environments containing Cr(VI). Second, previous studies on DOC derived from humic substances suggested that the aromatic moieties in DOC structure is responsible for reducing Cr(VI) in water and soil solution (14, 24). DOCNHB derived from fungi is rich in aliphatic carbons such as polysaccharides, peptides, chitin, glucan and glycoporteins and the kinetics and mechanisms for DOCNHB reaction with Cr(VI) remain unknown. This study first addresses the important role of DOCNHB enriched in aliphatic carbons for Cr(VI) reduction, and thus, it provides complementary results to the known reactions of Cr(VI) with dissolved humic acids containing both aromatic and aliphatic moieties (24) The primary objective of this study is to investigate the Cr(VI) reduction by DOCNHB derived from easily cultured Neurospora crassa fungus, which is commonly found in the nature environment, to addresses the important role of DOCNHB derived from biomaterials for Cr(VI) reduction. In addition, upon its dissolution from biomaterials, DOCNHB transported to the surface environment can ultimately be exposed to sunlight. Thus, light-induced Cr(VI) reduction by DOCNHB was also investigated in this work to clarify its contribution to the Cr(VI) removal in the environment.
Materials and Methods Culture and Preparation of N. crassa Biomass. The 74A strain of N. crassa was provided by the Fungal Physiology and Molecular Biology Laboratory, National Taiwan University. The culture and preparation of the N. crassa-biomass were as previously described (25). 10.1021/es1017015
2010 American Chemical Society
Published on Web 07/29/2010
Preparation of DOC from N. crassa Biomass. A sample of 1 g of freeze-dried N. crassa-biomass was suspended in a reaction vessel with 1 L of deionized water containing 0.01 M KCl as an electrolyte, preadjusted to pH 1, 3, or 5 at 25 ( 1 °C. Aliquots of 10 mL of the suspension were periodically extracted and passed through a 0.2-µm pore-size membrane filter. Approximately 500 µL of the filtrate was transferred to a TOC analyzer (Multi N/C 2100S, Analytik Jena) to determine DOC derived from N. crassa-biomass. The dissolution of N. crassa-biomass as a function of suspension time was recorded. The dissolution reached an apparent equilibrium at ca. 6 h for each tested pH, so DOC was collected after 6 h by passing suspended N. crassa-biomass through a 0.2-µm pore-size membrane filter for subsequent experiments. The final pH of each collected DOC was slightly increased, particularly at pH 5, by about 0.2-0.3 pH unit. Reduction of Cr(VI) by N. crassa-Biomass and DOC. Changes in DOC and Cr concentrations on the reaction of 96.2 µM Cr(VI) with 1 g L-1 N. crassa-biomass were measured at pH 1, 3, or 5 in the dark and under illumination. During the experiments, the pH was maintained constantly using 0.01 M HCl or NaOH. Experiments were conducted in a waterjacketed reaction vessel containing a 100 W medium-pressure mercury UV lamp in the center. The lamp was inserted in a borosilicate well to filter out λ e 290 nm, the wavelength maximum for UVC radiation. Average light intensity of 2.35 × 10-4 ( 5.61 × 10-5 einstein s-1 L-1 (n ) 16) was measured in the vessel using an actinometric technique (24). The vessel and the borosilicate well were both connected to a circulating water bath to maintain a temperature of 25 ( 1 °C. At specific reaction times, the suspension was extracted and passed through a 0.2-µm pore-size membrane filter and filtrate was collected. The Cr(VI) concentration in the filtrate was determined using a UV/vis spectrophotometer (Variance Cary 50) at 540 nm after reaction with 1,5-diphenylcarbazide indicator (DPC) (26). Total chromium was determined by atomic absorption spectroscopy (AAS, HITACHI Z-2000) at λ ) 359.3 nm. The difference between total Cr and Cr(VI) concentrations obtained by AAS and DPC methods, respectively, was attributed to Cr(III). Changes in DOC concentrations during the reaction time were analyzed on a TOC analyzer. Dark experiments were conducted using the same system without light application. Each experiment was performed in triplicate. In another set of experiments, 240 mL DOC derived from N. crassa-biomass at pH 1 and 3 was transferred into a reaction vessel containing 10 mL of 125 mg L-1 Cr(VI). Thus, the final solution contained 96.2 µM Cr(VI) and 139.8 and 185.9 mg L-1 DOC at pH 1 and 3, respectively. Aliquots of 10 mL was periodically extracted and the concentrations of Cr(VI), Cr(III), and DOC were analyzed as described above. Dark control experiments were also conducted under these experimental conditions to evaluate the influence of light energy on Cr(VI) reduction by DOC. Spectroscopic Analyses of Cr-Loaded Biomass and DOC. The oxidation state and speciation of Cr on Cr-loaded N. crassa-biomass were determined using K-edge X-ray absorption near-edge structure (XANES) at the X17C beamline of the National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan. Details of the procedure are provided in Supporting Information (SI). The morphology of biomass treated with or without Cr and the Cr distribution on the biomass were observed using a field-emission scanning electron microscope (FE-SEM, JEOL JSM-6700F), coupled to an X-ray energy dispersive spectrometer (EDS). To determine the chemical structures, DOC suspensions were condensed to an appropriate volume using a rotary evaporator coupled to a VR600 vacuum controller (Yamato Science, RE 600). DOC sample was then freeze-dried and analyzed using 13C CP-MAS NMR (Bruker DSX400WB NMR)
FIGURE 1. Reduction of 96.2 µM Cr(VI) on 1 g L-1 N. crassa-biomass and changes in DOC concentration at (a) pH 1, (b) pH 3, and (c) pH 5 in the dark and under illumination. and FTIR (Thermo Nicolet Nexus) spectrometers. Details for the procedures of spectroscopic analysis are provided in the SI. 13C CP-MAS NMR and infrared spectra were also measured for Cr(VI)-treated DOC to determine the functional groups involved in Cr(VI) reduction.
Results and Discussions Cr(VI) removal on N. crassa-Biomass. The interactions of Cr(VI) with N. crassa-biomass were investigated at pH 1, 3, and 5. At pH e 3, a rapid decrease in Cr(VI) concentration was observed in the first 10 min, followed by a slower decline in Cr(VI) concentration (Figure 1). Cr(VI) removal on N. crassa-biomass is a first-order reaction with respect to Cr(VI) concentrations; the rate constants varied from 0.0005 to 0.0176 min-1, depending on solution pH and the presence of light (Table 1). The initially rapid decrease in Cr(VI) concentration was not observed at pH 5 (Figure 1c) or in the absence of N. crassa-biomass, so Cr(VI) adsorption may dominate the initial reaction of Cr(VI) on positively charged surfaces of N. crassa-biomass, which has a ZPC of 6.55 (25). VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6203
TABLE 1. First-Order Rate Constant (k) for the Reduction of 96.2 µM Cr(VI) by DOC or N. crassa-Biomass at pH 1-5 in the Dark or under Illumination DOCa
N. crassa-Biomass -1
-1
2b
k (min )
r2
pH
k (min )
r
dark 1 3 5
2.37 × 10-3 ( 1.95 × 10-4 7.74 × 10-4 ( 6.75 × 10-5 5.05 × 10-4 ( 2.13 × 10-5
0.84 0.86 0.99
1.23 × 10-3 ( 1.19 × 10-4 2.49 × 10-4 ( 2.90 × 10-5 -c
0.93 0.96 -
light 1 3 5
1.76 × 10-2 ( 8.44 × 10-4 1.28 × 10-2 ( 1.30 × 10-4 1.48 × 10-3 ( 1.20 × 10-4
0.99 0.99 0.98
8.95 × 10-3 ( 5.98 × 10-4 2.78 × 10-3 ( 2.87 × 10-4 -
0.99 0.98 -
a The initial DOC concentration is 139.8 and 185.9 mg L-1 at pH 1 and 3, respectively. measured indicated by -.
The slow decrease in Cr(VI) concentration was attributed to Cr(VI) reduction on biomass, as evidenced by the disappearance of a strong pre-edged feature at ∆E ) 3.0 eV in XANES spectrum, indicating Cr(VI) characteristic absorption (SI Figure S1). After Cr(VI) reduction by N. crassabiomass, the resultant Cr(III) was either adsorbed on the biomass (SI Figure S2) or released into solution. It was estimated from the results of Figure 3 that after 360 min reaction in the dark, Cr(VI) reduction contributed to about 91.3 and 70.3% of total Cr(VI) removal at pH 1 and 3, respectively. At pH e 3, less than 29.7% of Cr(VI) removal on biomass was attributed to Cr(VI) adsorption, which became indiscernible at pH 5 and under illumination with an increase in reaction time (details please see below). Liberation of DOC on N. crassa-Biomass Oxidation by Cr(VI). As Cr(VI) was reduced on N. crassa-biomass, the DOC concentration gradually increased, particularly at higher pH under illumination (Figure 1). The increase in DOC concentration can be attributed to a combination of two processes: oxidation/dissolution of biomass by Cr(VI) and simple dissolution of N. crassa-biomass in an acidic solution. At this stage, it was not clear which functional groups on biomass were liberated upon its oxidation by Cr(VI). Structural domains associated with amide, -NH, or carboxyl groups, which are susceptible to oxidation by Cr(VI) (25), may become part of the DOC. However, the increase in DOC concentration upon the oxidation of biomass by Cr(VI) was small compared with that in the absence of Cr(VI). Thus, we believe that DOC derived from simple dissolution of N. crassabiomass should dominate the DOC concentration in our current system. Simple Dissolution of N. crassa-Biomass. On adding N. crassa-biomass to a solution, a proportion of it readily dissolved. At pH 1, the DOC content remained constant at 145.6 ( 1.2 mg L-1 for over 6 h. When the solution pH was increased to 3 or 5, the DOC concentration gradually increased to 193.7 ( 1.45 mg L-1 (SI Figure S3). The observations indicated that N. crassa-biomass dissolved when immersed in an acidic solution regardless of any oxidationdissolution mechanisms in the presence of Cr(VI). N. crassa-biomass is a filamentous fungus with cell walls mainly composed of aminopolysaccharides and glycorpoteins with minor amounts of proteins, lipids, carbohydrates, and inorganic salts (27). These components establish a two-layer cell wall system. The outer layer consists of glucanpeptide-galactosamine and the inner layer of β-1,3-glucan and fibrils of chitin (28). On suspension of N. crassa-biomass in an acidic solution, water-soluble compounds such as proteins and polysaccharides and peptide on the outer layer materials were dissolved. Thus, FTIR spectra of DOC and N. crassa-biomass exhibited very similar peaks at 2926, 1737, 1655, and 1040-1152 cm-1, assigned to -CH2/-CH3, car6204
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
b
The correlation coefficients.
c
Not
FIGURE 2. (a) FTIR and (b) NMR spectra of N. crassa-biomass, DOC, and Cr-loaded DOC. boxyl, amide I band, and -C-O bands of polysaccharides, respectively (Figure 2a) (15, 29–31). Plisko et al (32) suggested that chitin, a major skeletal polysaccharide, could be hydrolyzed in a concentrated hydrochloric acid solution. The hydrolytic products of chitin, glucosamine and acetic acid, may also contribute to DOC. However, no integral chitin structures were detected in DOC as evidenced by the absence of 13C CP-MAS NMR signals at 50-120 ppm corresponding to 6C in the cycloaliphatic structure of chitin (Figure 2b). The small peak at 72 ppm was attributed O-alkyl C, resulting from dissolved polysaccharides. Even though chitin can be partially hydrolyzed in an acidic solution, SEM analysis of acid-treated N. crassa-biomass revealed that the framework
FIGURE 3. Removal of 96.2 µM Cr(VI) on biomass (curves A) and by DOC (curves B) in the dark at pH 1 (a) and pH 3 (b), and under illumination at pH 1 (c) and pH 3 (d). Curves C represent Cr(VI) adsorbed on biomass and long dash lines denote the sum of initially adsorbed Cr(VI) on biomass and curve B. of filamentous N. crassa-biomass had no discernible change (SI Figure S2). Comparisons of Cr(VI) Reduction by DOC and N. crassaBiomass. As mentioned previously, Cr(VI) removal on biomass was attributed to two combined processes: (1) adsorption and reduction of Cr(VI) on the biomass surfaces and (2) reduction of Cr(VI) by DOC. Differentiation between these processes was difficult in a system containing biomass, so another method was used to evaluate the contribution of each process to Cr(VI) removal. Adsorbed Cr(VI) was determined using 0.1 M K2HPO4/KH2PO4 buffer solution to extract Cr(VI) from Cr-loaded biomass (33, 34). Cr(VI) reduction by DOC in the presence of biomass (denoted as DOCwb) was indirectly evaluated by reacting Cr(VI) with DOC (denoted as DOCnb) obtained according to the following reaction: 0.01MKCl
biomass 98 solid phase + DOC 98 DOCnb dissolution
filtration
(1) This evaluation is reasonable because of the similarity of initial DOCwb and DOCnb concentrations, and the DOC resulting from the oxidation/dissolution of biomass by Cr(VI) is small comparable to DOCnb/DOCwb. The results for Cr(VI) reduction by DOCnb at pH 5 are not presented because the redox reaction proceeded extremely slowly.
In the Dark. Changes in Cr(VI) concentration over the reaction time in the presence of biomass and DOCnb were shown in Figure 3. At pH 1, approximately 51.9 and 28.2 µM Cr(VI) was removed by biomass (Figure 3a, curve A) and DOCnb (Figure 3a, curve B), respectively, after a reaction time of 360 min. Only 24.6 and 9.3 µM Cr(VI) was removed by biomass and DOCnb, respectively, over the same reaction time at pH 3 (Figure 3b). It was estimated that Cr(VI) reduction by DOCnb was approximately 37.8 and 54.3% of the overall Cr(VI) removal on biomass at pH 3 and 1, respectively, at the end of the reaction. Accordingly, the Cr(VI) reduction rate of DOC decreased as pH was increased. With increasing reaction time to 360 min, adsorbed Cr(VI) on biomass gradually decreased from 26.7 to 4.5 µM and from 13.8 to 7.3 µM at pH 1 and 3, respectively (curves C in Figure 3a, b). When adsorbed Cr(VI) was deducted from the overall Cr(VI) removal on biomass, Cr(VI) reduction by DOCnb reached 53.8 and 59.5% of the total Cr(VI) reduction on biomass at pHs 3 and 1, respectively, after 360 min. The results demonstrate the importance of DOC in the overall Cr(VI) removal in a system containing a filamentous fungus. The decrease in adsorbed Cr(VI) was time-dependent and it has been confirmed that this is a result of Cr(VI) reduction on the biomass surface (25). Summation of the curves B and C in Figure 3a and b, representing Cr(VI) reduction by DOCnb and residual adsorbed Cr(VI) on biomass, respectively, yielded VOL. 44, NO. 16, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
6205
SCHEME 1. Cr(VI) Reduction by DOC or N. crassa-Biomass over Timea
a Pathways 1 and 2 indicate Cr(VI) reduction by DOC wb with biomass and Cr(VI) adsorption/reduction on biomass, respectively; pathway 3 indicates Cr(VI) reduction by DOCnb without biomass. Light can enhance each pathway, including pathway 2a, DOC release from biomass due to Cr(VI) oxidation. Pathway 4 indicates photo-induced Cr(VI) reduction by excited biomass.
a constant value over the reaction time. This result suggested that the rate of Cr(VI) reduction by DOCnb was apparently equal to the reduction of adsorbed Cr(VI) on biomass. The Cr(VI) reduction by both DOCnb and biomass obeyed a firstorder reaction, although the latter showed a slight deviation (i.e., lower r2 in Table 1) from the first-order reaction. This deviation was due to adsorption of Cr(VI) on biomass, which was also responsible for the higher reaction rates in a system with biomass (Table 1). Interestingly, when we added up the initial values for adsorbed Cr(VI) (∼26.7 and 13.8 µM Cr(VI) at pH 1 and 3, respectively) to curves B in Figure 3a, b, a curve close to curve A was obtained (Figure 3a, b, long-dash lines). The results suggested that for a fixed amount of adsorbed Cr(VI) (Scheme 1, pathway 2), Cr(VI) reduction by DOCnb (Scheme 1, pathway 3) could represent Cr(VI) reduction by DOCwb (Scheme 1, pathway 1). That is, the kinetics of Cr(VI) reduction by DOCwb could be approximately estimated by a system with DOCnb. Accordingly, the DOC, resulting from oxidation/ dissolution of biomass by Cr(VI) (pathway 2a in scheme 1) seemed not to contribute greatly to Cr(VI) reduction. This is probably because of its relatively lower concentration compared with DOCnb or DOCwb. Under Illumination. Upon exposure these systems to light, a significant enhancement of Cr(VI) reduction by DOCnb was found at both pH values (Figure 3c, d, curves B) and the first-order rate constants were approximately 7-fold larger than the values obtained in the dark (Table 1). The interactions of excited DOC/DOC•+ with dissolved O2 in an aerated environment, forming O2•- radicals via reactions 2 and 3, may be responsible for the acceleration of Cr(VI) reduction (reaction 4). 6206
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 16, 2010
dissolved biomass(DOC) + hv + O2DOC·+ + O·2
(2)
DOC·++O2 f decomposed DOC/CO2+O·-2
(3)
Cr(VI) + 3O·2 f Cr(III) + 3O2
(4)
Although the production of superoxide was not measured during our experiments, an enhanced Cr(VI) reduction on biomass was indeed observed when O2 was present. Therefore, Cr(VI) reduction by superoxide, which was photoinduced in the presence of excited DOC and O2 as proposed by Gaberell et al. (24), might also occur in our systems, representing an alternative pathway for Cr(VI) removal under illumination. Accompanied with DOC oxidation by Cr(VI), peaks in the FTIR spectrum disappeared (Figure 2a), indicating that each of the major groups on DOC was susceptible to Cr(VI) attack. The strong peak at 1640 cm-1 can be attributed to a shift of sCdO band upon Cr(III) adsorption on residual carboxyl groups (22, 25). Even though Cr(VI) was continuously reduced by DOC up to 360 min (Figure 3), the DOC concentrations rapidly decreased within the first 10 min, followed by a slow decrease up to 60 min, and then maintained constant thereafter (Figure 4). The results demonstrated that DOC was not completely converted to CO2 on oxidation by Cr(VI). That is, only a small proportion of DOC (
