Biotechnology Letters 26: 331–336, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands.
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Lead biosorption by waste biomass of Corynebacterium glutamicum generated from lysine fermentation process S.B. Choi & Y.-S. Yun∗ Division of Environmental and Chemical Engineering and Research Institute of Industrial Technology, Chonbuk National University, Chonju, Chonbuk 561-756, Korea ∗ Author for correspondence (Fax: +82-63-270-2306; E-mail:
[email protected]) Received 20 November 2003; Revisions requested 27 November 2003; Revisions received 17 December 2003; Accepted 18 December 2003
Key words: biosorption, Corynebacterium glutamicum, fermentation wastes, lead, lysine
Abstract Biomass waste, mainly Corynebacterium glutamicum, is generated from large-scale lysine fermentation process. In this study, protonated C. glutamicum biomass was evaluated as a biosorbent for the removal of lead from synthetic wastewater. As Pb2+ were bound to the biomass, the solution pH deceased, indicating that protons in the biomass were exchanged with lead ions. The Corynebacterium biomass bound Pb2+ at up to 2.74 mmol g−1 at pH 5, where lead does not precipitate. Compared with other biosorbents and conventional sorbents, such as natural zeolite, activated carbon and synthetic ion exchange resin, the protonated C. glutamicum biomass was considered to be a useful biomaterial for lead biosorption.
Introduction Biosorption is an economically feasible means for the removal and/or recovery of heavy metals from industrial wastewaters (Volesky 1990). The low cost of biosorbents is a tangible advantage over other technologies, such as ion exchange and reverse osmosis (Bailey et al. 1999). Furthermore, biosorption processes do not generate the chemical sludge that is a major problem in chemical precipitation methods for the removal of heavy metals. Therefore, extensive efforts have been made to explore new types of biosorbent materials capable of effectively sequestering heavy metals (Bailey et al. 1999, Volesky & Holan 1995). Among various types of biosorbents, fermentation byproducts along with biological sludge could be one of the cheapest and most abundant (Kuyucak 1990). Lysine fermentation industries generate a huge amount of biological solid waste, which is mainly the biomass of Corynebacterium glutamicum. Although this fermentation byproduct is potentially recyclable,
until now most has been dumped at sea. Therefore, the feasibility for reusing it as a value-added biosorbent deserves to be assessed. Because of its severe toxicity, lead is a major environmental concern (Volesky 1990) and has been classified in the USEPA’s Group B2 (probable human carcinogen) (Evangelou 1998). Therefore, effective (bio)sorbents for the removal of lead have been extensively evaluated. In this study, C. glutamicum biomass from an industrial lysine fermentation process was evaluated as a biosorbent material for the treatment of leadbearing wastewater. To our knowledge, this biomass has not been evaluated as a biosorbent of heavy metals. The dynamics of lead biosorption were studied under pH-varying and pH-static conditions. Isotherm experiments were carried out at various pHs and initial lead concentrations. The capacity of lead uptake by this biomass was compared with that by other sorbents previously reported.
332 Materials and methods Materials and experimental system The fermentation wastes (Corynebacterium glutamicum biomass) were obtained in dried powder form from a lysine fermentation industry (BASF Korea, Kunsan, Korea). Two types of biomass (acid-washed and water-washed) were used for sorption experiments. The protonated biomass was prepared by treating the raw biomass with a 1 M HNO3 solution for 24 h, thereby replacing the natural mix of ionic species with protons. The acid-treated biomass, designated as protonated biomass, was washed with deionized distilled water several times and thereafter dried at 60 ◦ C in an oven for 24 h. The water-washed biomass was not treated with acid but only washed with deionized distilled water under the same conditions. The resulting dried C. glutamicum biomass was stored in a desiccator and used as a biosorbent in the sorption experiments. All chemicals used in this study were of analytical grade. The lead stock solution was prepared using Pb(NO3 )2 . In order to adjust the pH, 1 M NaOH and HNO3 solutions were used. A multi-purpose auto-titration system (PC-Titration Plus, Man-Tech Associates Inc., Guelph, Canada) was used for lead sorption dynamics and equilibrium experiments. This system was able to control simultaneously the solution pH of a maximum of 28 reaction vessels. In all experiments the vessels were rigorously mixed using a stirrer equipped in the system. Sorption dynamics experiments
19 mM. Suspensions were agitated at room temperature (20 ± 2 ◦ C) and the pH was controlled using 1 M NaOH. Samples were taken periodically to analyse the lead concentration. Detailed standard procedure for determination of the sorption isotherm has been reported elsewhere (Volesky 1990). Measurements of lead uptake Before analysis of lead concentration, all samples were filtered through 0.45 µm pore size hydrophilic membranes (HVLP01300, Millipore, USA). The dissolved lead concentration of samples was analyzed using an atomic absorption spectrometer. In order for the change of working volume (up to 5%) by added Pb2+ stock and NaOH solutions, the metal uptake (q) was calculated from the mass balance as follows: q=
Vo Co − Vf Cf , M
(1)
where Vo and Vf are the initial and final (initial plus added base solution) volumes, respectively. Co and Cf are the initial and final lead concentrations, respectively. M stands for the weight of biomass used. Desorption experiments To evaluate the desorption efficiency the Pb2+ -loaded biomass was dried at 60 ◦ C for 24 h after equilibrium sorption experiments at pH 5. The dried biomass was contacted with 1 M HNO3 for 24 h to allow lead to be released from the biomass. Thereafter, the desorbed lead was analyzed and the desorption efficiency was calculated as follows: Desorption efficiency (%) =
To determine the contact time required for the sorption equilibrium experiments, biomass (5 g l−1 ) was mixed with synthetic wastewater (80 ml containing 10 or 20 mM Pb2+ ). Samples were intermittently removed from the vessels to analyze the lead concentration. The total volume of withdrawn samples did not exceed 2% of the working volume.
Results and discussion
Equilibrium sorption experiments
Biosorption dynamics
To evaluate the sorption capacity of the biomass, lead biosorption isotherms were obtained at different pHs and at different concentrations of background ions such as Ca2+ , Na+ , and Mg2+ . The isotherm experiments were carried out with 0.25 g biomass in 50 ml with the initial lead concentration ranging from 0 to
During the contact between C. glutamicum biomass and the Pb2+ -containing aqueous solution, the concentrations of lead ions and protons were measured without pH control. As shown in Figure 1, the solution pH decreased as the Pb2+ ions were bound to the biomass. This indicates that the protons attached to the protonated biomass were being exchanged for
released lead (mg) × 100. initially sorbed lead (mg)
(2)
333 Table 1. Effects of background ions (Ca2+ , Na+ , and Mg2+ ) on the lead uptake. Ions
Concentration levels (m M)
Lead uptake (mmol g−1 )
Ca2+
0.33 5 10.2
118 115 101
Na+
0.23 230 468
121 119 118
0.15 25 53.2
120 114 123
Mg2+
Fig. 2. Dynamics of lead biosorption in pH-static experiment. Error bars indicate standard deviations of replicate experiments. Conditions used: biomass concentration = 5 g l−1 , initial concentration of lead = 10 m M (open symbols) and 20 m M (closed symbols), pH = 4. Lead concentration (, ), lead uptake (, ). Fig. 1. Lead-proton exchange in pH-varying experiment. Conditions used: biomass concentration = 5 g l−1 , initial concentration of lead = 10 m M, initial pH = 4. Lead concentration (), proton concentration ().
Pb2+ . However, since a constant ratio of lead-proton exchange was not found, other mechanisms must have been coupled with ion exchange, e.g. complexation (Yun et al. 2001). To examine the contact time required to reach the equilibrium state under pH-static condition, the dynamics of lead biosorption were studied at constant pH 4 (Figure 2). Just after the biomass was contacted with the lead-bearing solution, the lead concentration sharply decreased over the initial 10 min. Thereafter, the biosorption decreased to a very slow rate until approximately 150 min. This tendency typically occurs in biosorption with various types of biomass (Volesky 1990, Yun et al. 2001). As shown in Figure 2, a contact
time of 2 h was sufficient with both 10 and 20 mM Pb2+ to achieve equilibrium. Effect of pH on lead uptake An isotherm is an equilibrium relationship of sorbate distribution between aqueous solution and sorbent phase at the pH-static condition. It is useful for evaluating the capacity of (bio)sorbents. As can be seen in Figure 3, the uptake of Pb2+ by the C. glutamicum biomass was obtained by varying the initial concentration of lead. As the solution pH was increased up to pH 5, uptake was enhanced, probably because of proton competition to Pb2+ binding (Yun & Volesky 2003). However, at pH 6 uptake was lower than that at pH 5. To explain this, the aquatic chemistry of Pb2+ was studied using the computer software MINEQL plus (Schecher 1991). Lead can exist in various forms depending on pH. At pH < 5, approx. 79% of Pb2+ and 21% of PbNO+ 3 are at
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Fig. 3. Isotherms of lead biosorption by protonated and water-washed biomass at various solution pHs. At pH 4, the sorption experiments were carried out in triplicate and the standard deviations of final concentrations and equilibrium uptakes are presented with 2-dimentional error bars. Conditions used: biomass concentration = 5 g l−1 . Protonated biomass: pH 3 (), pH 4 (), pH 5 (), pH 6 (); water-washed biomass: pH 5 ().
equilibrium. However, as the solution pH increases above 5, insoluble lead [Pb(OH)2] begins to precipitate, and at pH > 6, Pb2+ is essentially insoluble. When solid Pb(OH)2 exists in the solution, the lead that is able to be effectively bound to the negatively charged groups of the biomass decreases and thus the uptake should be reduced. This was considered to be why the lead uptake in the isotherms at pH 6 was lower than that at pH 5 (Figure 3). Therefore, to evaluate (bio)sorbents for lead removal, the solution pH needs to be maintained at pH 5 or lower, to avoid lead precipitation. If the biosorption takes place under conditions that lead may precipitate, a chemical sludge is generated which should be treated via appropriate solid waste management methods. From a practical point of view, therefore, lead biosorption processes are better operated at pH under 5. Effect of protonation From a practical point of view, protonation of biomass may increase the cost of biosorbent. However, the protonated biomass showed a higher uptake than the raw biomass (Figure 3), probably because protonation plays a role not only in cleaning-up ionic impurities in the raw biomass but also in activating binding sites (Kapoor & Viraraghavan 1998).
Fig. 4. Desorption efficiency of lead from the biomass when different concentrations of lead are present in synthetic wastewater to be treated by biosorption.
Effect of background ions Some cations, such as Ca2+ , Na+ , Mg2+ , and K+ , occur in wastewaters contaminated with heavy metals. These ions may affect the uptake of lead by electrostatic interaction and/or direct competition for negatively charged binding sites (Yun & Volesky 2003). Therefore, the effects of the three cations (Ca2+ , Na+ , Mg2+ ) on lead biosorption by C. glutamicum biomass were investigated (Table 1). Concentration levels of these ions ranged from those found in river water ([Ca2+ ] = 0.33, [Na+ ] = 0.23, and [Mg2+ ] = 0.15 mM) to those found in seawater ([Ca2+ ] = 10.2, [Na+ ] = 468, and [Mg2+ ] = 53.2 mM) (Berner & Berner 1987). As can be seen in Table 1, concentrations of Na+ and Mg2+ up to average concentrations found in seawater did not diminish Pb2+ uptake. Pb2+ uptake with 10.2 mM Ca2+ was 83% of that in Ca-free water. However, considering that Ca2+ has a strong affinity to the binding sites of other biosorbents (Volesky 1990), C. glutamicum biomass was less affected by Ca2+ than other systems. Desorption efficiency To recover heavy metals or reuse the biosorbents, the desorption efficiency should be considered. In this study, the lead-bearing biomass was contacted with 1 M nitric acid (Figure 4). When a low initial concentration of Pb2+ (2 mM) was used in the sorption step, the desorption efficiency was 71%. However, as the initial concentration of lead in the sorption experiment was increased, the desorption efficiency decreased.
335 Table 2. Comparison of lead uptake capability of various types of sorbents. Type of sorbent
Uptake capacity (mmol g−1 )
Remark
Reference
Natural zeolite Powdered activated carbon Granular activated carbon Pseudomonas sp. Sphaerotilus natans Penicillum oligosporus Arthobacter sp. Sphaerotilus natans Streptomyces noursei Padium pavonia Sargassum natans Ecklonia rachata Ion exchange resin (Dulite GT-73) Sargassum hystrix Durvillaea potatorum C. glutamicum Alginic acid C. glutamicum Ion exchange resin (IR-120 plus) Murcor rouxii
0.08 0.1 0.15 0.33 0.39 0.59 0.63 0.65 0.18 1.05 1.15 1.26 1.37 1.38 1.55 1.89 2.32 2.74 3.32 3.71
Zeolite, pH 5
Matheickal & Yu (1996a) Matheickal & Yu (1996b) Muraleedharan et al. (1995) Chang et al. (1997) Pagnanelli et al. (2003) Ariff et al. (1999) Veglio et al. (1997) Pagnanelli et al. (2003) Mattuschka & Straube (1993) Jalali et al. (2002) Jalali et al. (2002) Matheickal & Yu (1999) Volesky (1994) Jalali et al. (2002) Matheickal & Yu (1999) This study Jeon (2002) This study Jeon (2002) Lo et al. (1999)
Bacterium, pH 5 Bacterium, pH 4 Fungus, pH 5 Bacterium, pH 5–5.5a Bacterium, pH 5 Bacterium, pH 6.1a Seaweed, pH 4.5 Seaweed, pH 4.5 Chemically modified seaweed, pH 4.5 Synthetic resin Seaweed, pH 4.5 Chemically modified seaweed, pH 4.5 Bacterium, pH 4 Extracted from seaweed, pH 4 Bacterium, pH 5 Synthetic resin, pH 4 Fungus, pH 6a
a Sorption experiments were carried out at pH > 5 where lead might precipitate. Therefore, the reported values are not appropriate for
evaluating sorption capability in comparison with that of this study.
Therefore, further studies to choose the proper eluents, other than nitric acid, and to optimize the desorption conditions are needed for the repeated reuse of the biomass.
ent of Pb2+ and it deserves further investigations into the details of practical application, for example on the development of desorption methods and on sorption process optimization.
Discussion on sorption capability The protonated biomass of C. glutamicum bound up up to 1.9 mmol Pb2+ g−1 at pH 4 and at 2.7 mmol g−1 at pH 5. Since the isotherms did not reach saturation levels, the values of Pb2+ uptake were not maximum, however. The sorption capability of the C. glutamicum biomass was compared with that of other sorbents previously reported. As summarized in Table 2, the C. glutamicum biomass was superior to other biosorbents, except Murcor rouxii. Although the M. rouxii biomass had a larger uptake than that of C. glutamicum, its uptake was evaluated at pH 6, where lead precipitates, and a direct comparison was therefore not possible. Interestingly C. glutamicum biomass bound more Pb2+ than commercial synthetic ion exchange resin, Dulite GT-73 which is much more expensive. Consequently, this biomass may have a significant potential for use as a high-value biosorb-
Acknowledgements This work was financially supported by the Korea Science and Engineering Foundation through the Advanced Environmental Biotechnology Research Center at Pohang University of Science and Technology.
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