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Enhancement of methane fermentation in the presence of Ni2+ chelators
Biochemical Engineering Journal 38 (2008) 98–104
Short communication
Enhancement of methane fermentation in the presence of Ni2+ chelators Hu Qing-Hao, Li Xiu-Fen ∗ , Liu He, Du Guo-Cheng, Chen Jian ∗ Laboratory of Environmental Biotechnology, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi City, Jiangsu 214122, China Received 9 March 2007; received in revised form 15 June 2007; accepted 3 July 2007
Abstract The enhancement of methane fermentation in the presence of Ni2+ chelators, citric acid (CA), nitrilotriacetic acid (NTA), and ethylene diamine tetraacetic acid (EDAT) respectively, was investigated in this study. The results showed that the addition of chelating agents promoted methane fermentation of acetate. At a sodium acetate concentration of 7 g/L, temperature of 35 ◦ C, and Ni2+ concentration of 20 M, methane production was enhanced 34.1%, 49.2%, and 38.6%, by the addition of 10 M CA, NTA and EDTA, respectively. The formation of soluble complexes between chelating agents and Ni2+ favored the dissolution of Ni2+ from their sulfides, and the bioavailability of Ni2+ was dramatically increased. By the addition of 10 M NTA, the concentration of dissolved Ni2+ was greatly increased from 58 to 1020 g/L. Accompanying with that, the concentrations of F430 and coenzyme M of the biomass increased 37% and by four-fold, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Anaerobic; Chelators; Bioavailability; Methane; Coenzyme; Nickel
1. Introduction The effect of the addition of trace metals on the performance of bioreactors has been an important study field in anaerobic processes, as metals are involved in the enzymatic activities of acidogenesis and methanogenesis [1]. Microbial regeneration time is a function of the concentrations of nutrients present. Although ideal nutrients concentrations are not essential, lack of even a single trace metal may severely limit anaerobic conversion processes [2,3]. Consequently, trace metal elements are generally supplied to the influent of full-scale anaerobic bioreactors to maintain a good reactor performance, or process compensation must be made in either lower loading rates or lower treatment efficiency as a trade-off for non-ideality of nutrients [4]. However, in the case of anaerobic reactors for the treatment of wastewaters, due to the ubiquitous presence of sulfide in most anaerobic bioreactors and the concomitant metal sulfide precipitation, the availability of the metal ions added may be limited [5,6]. In practice, trace metals are also added in excessive amounts to full-scale installations to ensure the functioning of the treatment reactors. Metal chelating agents can
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Corresponding authors. Tel.: +86 510 85918307/85913661. E-mail addresses: [email protected] (X.-F. Li), [email protected] (J. Chen). 1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.07.002
form soluble complexes with heavy metals and they have been used in phytoremediation of heavy-metal-contaminated soils and groundwater [7]. These researches demonstrated that the addition of chelator significantly promoted bioavailability of total metal for the plant and chelator enhancement was plantand metal-specific. However, it is still uncertain how chelator affects the bioavailability of metal elements for anaerobic microorganisms during wastewater treatment. Nickel is an important element for anaerobic microorganisms and plays a key role during methane formation. Methanogenic Archaea uses several pathways to utilize the various carbon substrates (e.g., methanol, acetate, and H2 /CO2 ), but all pathways converge on the common intermediate, methyl-S-CoM [8]. Methyl-S-CoM contains a nickel-harbouring tetrapyrolic structure, coenzyme F430 , which exists in all methanogens and exclusively found in methanogens. In addition, carbon monoxide dehydrogenase (CODH), which possesses two nickel-containing metallocenters, is present in both acetoclastic methanogens and acetogenic microorganisms [9]. Previous studies showed that methane fermentation could be enhanced by addition of Ni2+ [10,11]. Ni2+ added in concentrations as low as 10 M significantly increased biogas production in a laboratory poultry waste digester utilizing excreta from laying hens as the organic energy source. Canovas-Diaz also found that butyric acid conversion by a mixed methanogenic population in a pilot scale anaerobic downflow fixed-film reactor (DFFR), was increased by the addition of nickel (30 mg/L NiCl2 ·6H2 O) [11].
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In this study the enhancement of methane fermentation by the addition of metal chelating agents and Ni2+ is investigated according to the changes of methane production, the contents of F430 and CoM. The Ni2+ bioavailability for methanogenic microorganisms is also explored. It is expected that the results obtained from this study could provide valuable information for the application of chelators on the enhancement of methane fermentation. 2. Materials and methods 2.1. Biomass Biomass was collected from a full-scale anaerobic reactor treating beer wastewater (Wuxi, China). It had a pH value of 7.2 and VSS/TSS of 0.72. The synthetic wastewater was supplemented for acclimation of the biomass in a 2 L continuous stirred tank reactor (CSTR) digester, which had been operated at 35 ± 2 ◦ C. Components of the synthetic wastewater were described in 2.2 (except that Ni2+ concentration was 20 M). The hydraulic retention time was 5 days and the acclimation period was two months. Prior to an experimental run, several (3–4) successive subcultures in nickel deficient synthetic wastewater were generated for the acclimated biomass. 2.2. Synthetic wastewater Synthetic wastewater was used in the study. It contained the following components in ultrapure water: 85 mM sodium acetate, 0.50 mM KH2 PO4 , 0.50 mM Na2 HPO4 , 3.7 mM NH4 Cl, 0.50 mM MgCl2 , 0.89 mM CaCl2 , and 1.0 mM Na2 S. Acid and alkaline solutions of trace elements (1 mL of each per liter) were used. The acid trace element solution contained the following components: 7.5 mM FeCl2 , 1.0 mM H3 BO4 , 0.50 mM ZnCl2 , 0.10 mM CuCl2 , 0.50 mM MnCl2 , 8.5 mM CoCl2 ·6H2 O and 50 mM HCl. The alkaline trace element solution contained the following components: 0.10 mM Na2 SeO3 , 0.10 mM Na2 WO4 , 0.10 mM Na2 MoO4 ·2H2 O, and 10 mM NaOH. Nickel was added as required in the form of Ni2 Cl2 ·6H2 O. 2.3. Operating conditions All experiments were carried out in 150 mL glass serum bottles. The bottles were properly capped and connected to liquid displacement system (in which the liquid was 15% NaOH solutions to remove CO2 or other acidic gas from biogas) for recording methane production with time. Schematic view of experimental set-up was presented in Fig. 1. Required amount of biomass was added to serum bottles which contained 100 mL synthetic wastewater to get VSS concentration of 5 g/L. After seeding and adjusting pH to 7.0 ± 0.2, the bottles were flushed with N2 gas for 5 min to provide anaerobic conditions, then sealed with natural rubber stoppers and plastic screw-caps. They were incubated in a temperaturecontrolled room at 35 ± 2 ◦ C and gas production in each bottle was measured daily with the liquid displacement device. After
Fig. 1. Schematic of batch experiment set-up.
gas measurement the bottles were manually shaken. In serum bottles with biomass and 100 mL tap water (purged of oxygen with nitrogen gas), control experiments were conducted in parallel to determine the background gas production. Each experiment was performed in triplicate, and the averaged results were presented in this manuscript. 2.4. Analysis TSS and VSS were measured according to standard methods [12]. Before the total dissolved Ni2+ concentrations were determined by atomic absorption spectrometry (SpectraAA220, Varian, USA), the samples were acidified with 0.1 M HNO3 . These samples were taken from bottles without biomass, which were left to equilibrate for 7 days. Sampling was performed in an anaerobic chamber (Sheldon Manufacturing Inc. USA). Precipitates were separated from the dissolved phase by centrifugation at 10,000 × g for 10 min. F430 was extracted from the precipitate, which was obtained by centrifugation of a 50 mL aliquot of culture broth from the bottle as described by Kida et al. [13]. F430 concentrations in the samples were estimated based on nickel concentrations determined using atomic absorption spectrophotometry (SpectraAA220, Varian, USA). Coenzyme M (CoM) was extracted from 5 g wet sludge, which was obtained by centrifugation of a 20 mL aliquot of culture broth from the bottle, mixed with 2 mL 1% tri-(N)-butylphosphine in 2-propanol (1% TBP) and incubated for 1 h at room temperature. Then the concentration of CoM was analyzed by HPLC with an Econosphere C18 column as described by Elias [14]. The standard substance of CoM was purchased from Sigma. 3. Results and discussion 3.1. Effect of Ni2+ addition on methane fermentation Batch tests were performed with required amount of biomass, 100 mL synthetic wastewater, 0–200 M Ni2+ , and 1 mM sulfide. Methane production at various Ni2+ concentrations was shown in Fig. 2.
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Q.-H. Hu et al. / Biochemical Engineering Journal 38 (2008) 98–104 Table 1 Stable constants Kf of CA, NTA and EDTA with metal ions and solubility products Ksp of S2− with metal ions Chelators/S2−
CA NTA EDTA S2−
Fig. 2. Methane production due to the anaerobic conversion of acetate at various Ni2+ concentrations without NTA. Sodium sulfide was added at 1 mM. Ni was added at 0 (), 1 (×), 20 (), and 200 M ().
At the initial phase (0–48 h) at various Ni2+ concentrations, methane production was nearly 40 mL for all cases. After 48 h, methane production increased with increasing Ni2+ concentration (Fig. 2). At 168 h the methane production increased from 112 to 162 mL as Ni2+ concentration was elevated from nil to 200 M, indicating that a higher concentration of Ni2+ might promote the methane production, and nickel seemed to limit the rate of methanogenesis from acetate. When the Ni2+ concentration was 1, 20 and 200 M, methane production was 119, 132 and 162 mL at last, which increased 6.30%, 17.9% and 44.6%, respectively, compared with the control. Previous results [10,11,15] indicated that Ni2+ was required for the methane production. Nickel is structural constituents of the cofactor F430 , which are key components of the enzyme complexes that catalyze several reactions of the methanogenic pathway [8]. Kida et al. [13] investigated the requirement of Ni2+ and Co2+ for methanogenic activity in anaerobic CSTR reactor by the concentrations of coenzymes F430 and corrinoids. The methanogenic activity and concentrations of F430 and corrinoids in the biomass increased with increasing added amounts of Ni2+ and Co2+ . Our results described here also confirmed that the methanogenic activity was related to the concentration of Ni2+ . 3.2. Effect of chelating agent addition on methane fermentation Sulfide had dual effect on methane fermentation. On the one hand, the availability of sulfide was of great importance to the growth of methanogens. However, when sulfide was in excess, it precipitated with metal ions and prevented the direct uptake of trace metals by methanogens [5,6]. It was necessary to balance the addition of both trace metals and sulfide. The chemical form of the metals (speciation) will determine their availability for the microorganisms [16]. Metal sulfide precipitates were not directly available for methanogens and therefore in practice, in order to increase metal availability, a large amount of metal was usually needed in the reactors to meet the demand of methanogens.
Log stability constant/solubility products Ksp Ni
Co
Fe
6.9 11.5 18.6 2 × 10−21
5.0 10.6 16.2 4 × 10−21
4.4 8.8 14.3 1 × 10−18
Gonzalez-Gil et al. [17] found that the addition of yeast extract could improve the bioavailability of Co and Ni when using methanol as substrate. This suggested that the formation of strong metal-yeast extract complexes played an important role in methane fermentation. Callendar and Bradford [5] measured soluble metal concentrations in an anaerobic digester which treated the wastewater containing amino acids and polypeptides. The level of soluble metals in the presence of S2− was 104 -fold higher than the predicted concentrations using available solubility data. So, soluble metal complexes, which may be formed with amino acids and polypeptides, possibly increased the concentration of soluble metal. The mode of metal addition should be optimized such that the rate and extent of metal precipitation were minimized. In order to promote the dissolution of metal ions from precipitates, such as metal sulfide, which was unavailable for methanogens, CA, NTA and EDTA, which had different stable constant shown in Table 1 [18], were selected as chelating agents of metal ions. Under 20 M of Ni2+ , the time profiles of methane production with the addition of different chelating agents in the range of 1–1000 M were shown in Fig. 3. Two-hundred micromolar nickel may provide relatively sufficient bio-available nickel for anaerobic microorganism. As a result of that, the influence of chelating agents added on the bioavailability of nickel may be slight or little. Also, low dosage can save cost. Therefore, 20 M of Ni2+ (not 200 M) was selected here. The results in Fig. 3 revealed that the effect of metal ion chelators on methane production during the anaerobic digestion of acetate was significant. The methane production was 132 mL for the control, while it was 177 mL for the bottle with 10 M of CA. The corresponding value was 197 mL with 10 M of NTA and 183 mL with 10 M of EDTA, increasing 34.1%, 49.2% and 38.6%, respectively, compared with the control. EDTA was the strongest chelator with the highest stable constant, but its stimulating effect did not seem to be the highest. NTA with the middle stable constant was the best stimulant in our experiments. Methane production was enhanced by 49.2% in NTA amended bottles, 10.6% higher than that in EDTA amended ones (Fig. 3). Speece [19] also reported that, although chelators could greatly increase the availability of metal ions, excessively strong complexing ability may not enhance the bioavailability of metals. Maybe metal complexes formed with too strong chelator could not be combined with free surface sites on the cell membrane, therefore its uptake was influenced [20]. In addition, metal chelator concentration had obvious effect on methane fermentation. It can be seen from Fig. 3 that, for CA,
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NTA and EDTA, the methane production obtained the maximum under the added concentration of 10 M and the difference in the simulating effect for 100 M and 1000 M of chelating agent were not obvious, indicating that high chelator concentration may not stimulate methane production. The best dosage in our experiments was 10 M. It should be noted that the chelator was not simply serving as an additional carbon source of methane production according to the magnitude of increased methane production. Theoretically, 1 mol of NTA could produce about 2.3 mol of methane, and 1 mol of CA could do about 2 mol of methane. Accordingly, the complete conversion of 1 mol NTA and CA would produce 0.052 and 0.045 mL (at standard state condition) of methane. However, our experiments reveal the 10 M of CA and NAT in 100 mL synthetic wastewater present an increase in methane production of about 48 and 65 mL over the control. Therefore, the increase in methane production is not due to metabolism of chelators to methane, but possibly to the enhancement in the solubility of essential metal nutrients. With regard to the addition cost, it was firstly assumed that the wastewater contained 5000 mg COD/L. The methane production increased from 1.32 L/L wastewater to 1.77, 1.97, and 1.83 L/L wastewater by addition of 10 M CA, NTA and EDTA, respectively (according to the results in Fig. 3). Because the heat energy of 1 m3 methane was equal to 1 m3 natural gas, the methane price was equal to natural gas price. The cost and benefit analysis were conducted and listed in Table 2. As listed in Table 2, the net benefit for CA, NTA and EDTA were 0.98, 1.38 and 1.08 yuan/m3 , respectively. The net benefit of NTA was the highest. To sum up, NTA was the optimal chelating agent based on either the promoting efficiency to methane production or the addition cost. 3.3. Effect of chelating agent on total dissolved concentration of Ni2+
Fig. 3. Methane production due to the anaerobic conversion of acetate at various chelator concentrations. Sodium sulfide was added at 1 mM. Ni was added at 20 M. (a) CA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M (). (b) NTA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M (). (c) EDTA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M ().
The experiments were conducted in bottles only with synthetic wastewater for 7 days. The total dissolved Ni2+ concentration was determined and the results were listed in Table 3. The results clearly indicated that due to the chelating effect, the dissolution of metal sulfide precipitates in anaerobic digestion processes was promoted, nutrient limitations were overcomed and their availabilities could meet the requirements for biomass activity and growth. The total dissolved Ni2+ concentrations in the presence of 10 M NTA were indeed higher than that in control bottle. When the added Ni2+ concentration was 20 M, the total dissolved
Table 2 The cost and benefit analysis for the use of chelating agents Chelating agent
Price (yuan/t)
Dosage (g/m3 )
Cost (yuan/m3 )
methane price (yuan/m3 )
Methane production increase (m3 /m3 )
Benefit (yuan/m3 )
Net benefit (yuan/m3 )
CA NTA EDTA
6700 22000 15000
1.92 1.91 2.92
0.013 0.053 0.044
2.20 2.20 2.20
0.45 0.65 0.51
0.99 1.43 1.12
0.98 1.38 1.08
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Table 3 Effect of chelating agents on total dissolved concentrations of Ni2+ Medium
Dissolved concentrations of Ni2+ (g/L)
20 M Ni2+ + synthetic wastewater 20 M Ni2+ + synthetic wastewater + 10 M CA 20 M Ni2+ + synthetic wastewater + 10 M NTA 20 M Ni2+ + synthetic wastewater + 10 M EDTA
58 ± 0.32 338 ± 12 1020 ± 21 1026 ± 18
Fig. 4. Methane production due to the anaerobic conversion of acetate at various Ni2+ concentrations. Sodium sulfide was added at 1 mM. NTA was added at 10 M. Ni was added at 0 (), 1 (×), 20 (), and 200 M ().
Ni2+ concentration was 1020 g/L, which was only 58 g/L for bottles without NTA. CA has weaker chelating ability than NTA because its stable constant with Ni2+ is 6.9, which is lower than that of NTA (11.5). When 10 M CA was added to the bottle, the total dissolved Ni2+ concentration was only 338 g/L. It increased only by 4.8-fold, but 16.6-fold increase was obtained with addition of 10 M NTA. EDTA has the strongest complexing ability and the total dissolved Ni2+ concentration was 1026 g/L, appreciably higher than that in NTA amended bottle. 3.4. Effect of NTA on methane fermentation As the best chelating agent of metal ion, effect of NTA on methane fermentation under different Ni2+ concentration was investigated, shown in Fig. 4, in this study. When 10 M NTA was added in the batch tests with 1 mM sulfide, the methane production seemed to be independent of the added Ni2+ concentration. Although the results of Fig. 2 clearly showed that the metabolic activity of the anaerobic biomass was Ni-limited,
the effect of Ni2+ addition concentration on the methane production shown in Fig. 4 became slight. Gas production finally achieved 196, 195 and 185 mL, respectively, when Ni2+ addition concentration was 1, 20 and 200 M. One micromolar Ni2+ was sufficient for acetate digestion in the presence of sulfide. In contrast, the methane production always increased with the increase of Ni2+ dosage if NTA was absent. Gonzalez-Gil [15] found that methane production rate increased sharply in anaerobic conversion of methanol with increasing Ni2+ concentration from 0 to 400 M. He also calculated equilibrium dissolved metal concentrations with different initial dosage of Ni and Co. The calculated concentrations of free Ni and Co should increase sharply only when the initial dosage of Ni and Co were more than 100 M, corresponding to the moment that the amount of sulfide added became limiting for precipitation reactions, which prevented Ni and Co uptake by methanogens. The solubility products Ksp for Ni with S2− is 2 × 10−21 . NTA was a kind of metal ion chelating agent and it could form soluble complexes with Fe, Co, Ni, etc. Stability constant Kf of NTA for Ni2+ is 11.5 [18]. So the formation of dissolved complexes between metal and NTA could promote the dissolution of metals from their sulfides. It may provide an attractive approach to keep additions of essential metals at a minimum dosage while assisting in their bioavailability. Ni2+ was beneficial for the methane production. On the other hand, Ni2+ was also a potent inhibitor of macromolecular synthesis such as RNA and proteins in S. cerevisiae [21] as well as in Escherichia coli [22]. It was toxic to microorganism if its concentration was too high. As can be seen from Fig. 4, under 200 M of nickel, the methane production was slightly lower than those under 1 and 20 M when NTA was present. A modified Gompertz equation has been used to describe the product formation in the hydrogen production process by Mu et al. [23,24]. It was: Rmax × e P = Pmax exp − exp (λ − t) + 1 (1) Pmax where P is the product formed at fermentation time t; Pmax is the potential maximum product formed; Rmax is the maximum rate of product formed; λ is the lag time to exponential product formed. In order to describe the kinetics of methane production from acetate by anaerobic cultures with or without NTA addition, the equation above was also tried in this manuscript. The fitted equations and its parameters for the formation of methane were summarized in Table 4. Correlation coefficients of non-linear analysis by Eq. (1) was over 98.6%, suggesting that the modified Gompertz equation was able to describe the methane formation for anaerobic digestion of acetate. It also can be seen from Table 4 that the potential maximum methane
Table 4 Calculated results using the modified Gompertz equation for methane formation NTA
The modified Gompertz equation
Pmax (mL)
Rmax (mL/h)
λ (h)
R2
None 10 M NTA
P = 164.01 × exp{−exp[1.32 × e/164.01 × (17.52 − t) + 1]} P = 208.95 × exp{−exp[1.88 × e/164.01 × (27.29 − t) + 1]}
164.01 208.95
1.32 1.88
17.52 27.29
0.990 0.986
Ni2+ addition concentration was 20 M.
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Table 5 Measured cofactor contents in the presence and absence of 10 M NTA
Acknowledgement
Samples
F430 (mol Ni/gVSS)
CoM (mol/gVSS)
NTA presence NTA absent
0.30 ± 0.05 0.22 ± 0.03
1.05 ± 0.12 0.21 ± 0.04
The authors thank the Hi-tech research and development program of China (863 program, Grant No: 2006AA06Z315) for the partial financial support of this study.
production Pmax and the maximum methane production rate Rmax under 20 M of Ni2+ were indeed higher with 10 M NTA than without NTA. This also suggested that methane fermentation could be enhanced by addition of chelating agent NTA. 3.5. Cofactors in the biomass The coenzyme content in digested sludge has been suggested as a means for the assessment of potential methanogenic acitivity [25]. Several metal ion- containing enzymes involved in methanogenesis have been identified. The Nickel tetrapyrrole, coenzyme F430 , was known to bind to methyl-S-CoM reductase which catalyzed methane formation from methyl-S-CoM in all methanogens [8]. CoM, one of the few naturally occurring sulfonic acids, was discovered and identified in the mid-1970s. With the exception of an aerobic alkene-degrading bacterium, CoM was found only in methanogens, and the content of CoM varied only by a small factor in different species [26]. So F430 and CoM contents in the biomass were determined and shown in Table 5. The results in Table 5 revealed that the contents of two coenzymes in the 10 M NTA amended biomass were higher than those in control. The concentrations of F430 and coenzyme M of the biomass increased 37% and by 4 fold respectively. Combined with the results in Fig. 4, it suggested that the level of methanogenic activity be related to the contents of F430 and CoM. The bioavailability of Ni2+ was enhanced when NTA was in presence. Elias et al. [14] suggested CoM could be used as a biomarker to quantify methanogen biomass in a variety of environmental matrices. The increased CoM content confirmed that the addition of NTA could increase the solubility and availability of essential metal nutrients, and promote the growth of the methanogenic population. 4. Conclusions It showed that the methane fermentation was enhanced with the addition of CA, NTA and EDTA. Addition of chelators led to the formation of dissolved complexes and consequently facilitated the dissolution of metals from their sulfides. Ten micromolar NTA could increase the level of soluble Ni2+ in the presence of S2− by a factor of 16.6. The bioavailability of Ni2+ was dramatically increased and the coenzyme content also raised from 0.22 to 0.30 mol Ni/gVSS for F430 , from 0.21 to 1.05 mol/gVSS for CoM. Thus it may provide an attractive approach to keep the additions of essential metals to anaerobic reactors at a minimum.
References [1] P. Scherer, H. Lippert, G. Wolff, Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry, Biol. Trace Elem. Res. 5 (1983) 149–163. [2] M.H. Zandvoort, R. Geerts, G. Lettinga, P. Lens, Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge reactor, Biotechnol. Prog. 18 (2002) 1233–1239. [3] Z.B. Yue, H.Q. Yu, Z.L. Wang, Anaerobic digestion of cattail with rumen culture in the presence of heavy metals, Bioresour. Technol. 98 (2007) 781–786. [4] Y.S. Zhanga, Z.Y. Zhang, K. Suzukic, T. Maekawa, Uptake and mass balance of trace metals for methane producing bacteria, Biomass Bioenergy 25 (2003) 427–433. [5] I.J. Callander, J.P. Bradford, Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. I. Methodology, Biotechnol. Bioeng. 15 (1983) 1947–1957. [6] I.J. Callander, J.P. Bradford, Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. II. Applications, Biotechnol. Bioeng. 15 (1983) 1959–1972. [7] H. Chen, T. Cutright, EDTA and HEDTA effects on Cd, Cr and Ni uptake by Helianthus annuus, Chemosphere 45 (2001) 21–28. [8] R.K. Thauer, Biochemistry of methanogenesis: a tribute to Majory Stephenson, Microbiology 144 (1998) 2377–2406. [9] P. Hausinger, Nickel utilization by microorganisms, Microbiol. Rev. 51 (1987) 22–42. [10] C.M. Williams, J.C.H. Shih, J.W. Spears, Effect of nickel on biological methane generation from a laboratory poultry waste digester, Bioresour. Technol. 28 (1985) 1608–1610. [11] M. Canovas-Dlaz, J.A. Howell, Effect of nickel on methane production and butyric acid utilization in a downflow fixed-film reactor, Biotechnol. Lett. 8 (1986) 287–292. [12] APHA, AWWA, WEF, Standard Methods for the Examination of Water and Wastewater, nineteenth ed., American Public Health Association, Washington, DC, 1995. [13] K. Kida, T. Shigematsu, J. Kijima, Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis, J. Biosci. Bioeng. 91 (2001) 590–595. [14] D.A. Elias, L.R. Krumholz, R.S. Tanner, J.M. Suflita, Estimation of methanogen biomass by quantitation of coenzyme M, Appl. Environ. Microbiol. 65 (1999) 5541–5545. [15] G. Gonzalez-Gil, R. Kleerebezem, G. Lettinga, Effects of nickel and cobalt on kinetics of methanol conversion by methanogenic sludge as assessed by on-line CH4 monitoring, Appl. Environ. Microbiol. 65 (1999) 1789–1793. [16] M.H. Zandvoort, E.D. van Hullebusch, F.G. Fermoso, P.N.L. Lens, Trace metals in anaerobic granular sludge reactors: bioavailability and dosing Strategies, Eng. Life Sci. 3 (2006) 293–301. [17] G. Gonzalez-Gil, S. Jansen, M.H. Zandvoort, H.P. van Leeuwen, Effect of yeast extract on speciation and bioavailability of nickel and cobalt in anaerobic bioreactors, Biotechnol. Bioeng. 82 (2003) 134–142. [18] H. Yue, Advanced Inorganic Chemistry, second ed., Beijing, 2002. [19] R.E. Speece, Anaerobic Biotechnology for Industrial Wastewaters, Archac, USA, 1996. [20] P.L. Brown, S.J. Markich, Evaluation of the free ion activity model of metalorganism interaction: extension of the conceptual model, Aquat. Toxicol. 51 (2000) 177–194. [21] M. Joho, Y. Imada, T. Murayama, The isolation and characterization of Ni resistant mutants of Saccharomyces cerevisiae, Microbios 51 (1987) 183–190.
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[22] C. Guha, A. Mookerjee, Effect of nickel on macromolecular synthesis in Escherichia coli K-12, Nucleus 22 (1979) 45–47. [23] Y. Mu, G. Wang, H.Q. Yu, Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures, Bioresour. Technol. 97 (2006) 1302–1307. [24] Y. Mu, H.Q. Yu, G. Wang, A kinetic approach to anaerobic hydrogenproducing process, Water Res. 41 (2007) 1152–1160.
[25] M.J. Delafontaine, H.P. Naveau, E.J. Nyns, Fluorimetric monitoring of methanogensis in anaerobic digestors, Biotechnol. Lett. 1 (1979) 71–74. [26] W.E. Balch, R.S. Wolfe, Specificity and biological distribution of coenzyme M (2-mercaptoethanesulfonic acid), J. Bacteriol. 137 (1979) 256–263.
Short communication
Enhancement of methane fermentation in the presence of Ni2+ chelators Hu Qing-Hao, Li Xiu-Fen ∗ , Liu He, Du Guo-Cheng, Chen Jian ∗ Laboratory of Environmental Biotechnology, School of Biotechnology, Jiangnan University, Lihu Road 1800, Wuxi City, Jiangsu 214122, China Received 9 March 2007; received in revised form 15 June 2007; accepted 3 July 2007
Abstract The enhancement of methane fermentation in the presence of Ni2+ chelators, citric acid (CA), nitrilotriacetic acid (NTA), and ethylene diamine tetraacetic acid (EDAT) respectively, was investigated in this study. The results showed that the addition of chelating agents promoted methane fermentation of acetate. At a sodium acetate concentration of 7 g/L, temperature of 35 ◦ C, and Ni2+ concentration of 20 M, methane production was enhanced 34.1%, 49.2%, and 38.6%, by the addition of 10 M CA, NTA and EDTA, respectively. The formation of soluble complexes between chelating agents and Ni2+ favored the dissolution of Ni2+ from their sulfides, and the bioavailability of Ni2+ was dramatically increased. By the addition of 10 M NTA, the concentration of dissolved Ni2+ was greatly increased from 58 to 1020 g/L. Accompanying with that, the concentrations of F430 and coenzyme M of the biomass increased 37% and by four-fold, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Anaerobic; Chelators; Bioavailability; Methane; Coenzyme; Nickel
1. Introduction The effect of the addition of trace metals on the performance of bioreactors has been an important study field in anaerobic processes, as metals are involved in the enzymatic activities of acidogenesis and methanogenesis [1]. Microbial regeneration time is a function of the concentrations of nutrients present. Although ideal nutrients concentrations are not essential, lack of even a single trace metal may severely limit anaerobic conversion processes [2,3]. Consequently, trace metal elements are generally supplied to the influent of full-scale anaerobic bioreactors to maintain a good reactor performance, or process compensation must be made in either lower loading rates or lower treatment efficiency as a trade-off for non-ideality of nutrients [4]. However, in the case of anaerobic reactors for the treatment of wastewaters, due to the ubiquitous presence of sulfide in most anaerobic bioreactors and the concomitant metal sulfide precipitation, the availability of the metal ions added may be limited [5,6]. In practice, trace metals are also added in excessive amounts to full-scale installations to ensure the functioning of the treatment reactors. Metal chelating agents can
∗
Corresponding authors. Tel.: +86 510 85918307/85913661. E-mail addresses: [email protected] (X.-F. Li), [email protected] (J. Chen). 1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2007.07.002
form soluble complexes with heavy metals and they have been used in phytoremediation of heavy-metal-contaminated soils and groundwater [7]. These researches demonstrated that the addition of chelator significantly promoted bioavailability of total metal for the plant and chelator enhancement was plantand metal-specific. However, it is still uncertain how chelator affects the bioavailability of metal elements for anaerobic microorganisms during wastewater treatment. Nickel is an important element for anaerobic microorganisms and plays a key role during methane formation. Methanogenic Archaea uses several pathways to utilize the various carbon substrates (e.g., methanol, acetate, and H2 /CO2 ), but all pathways converge on the common intermediate, methyl-S-CoM [8]. Methyl-S-CoM contains a nickel-harbouring tetrapyrolic structure, coenzyme F430 , which exists in all methanogens and exclusively found in methanogens. In addition, carbon monoxide dehydrogenase (CODH), which possesses two nickel-containing metallocenters, is present in both acetoclastic methanogens and acetogenic microorganisms [9]. Previous studies showed that methane fermentation could be enhanced by addition of Ni2+ [10,11]. Ni2+ added in concentrations as low as 10 M significantly increased biogas production in a laboratory poultry waste digester utilizing excreta from laying hens as the organic energy source. Canovas-Diaz also found that butyric acid conversion by a mixed methanogenic population in a pilot scale anaerobic downflow fixed-film reactor (DFFR), was increased by the addition of nickel (30 mg/L NiCl2 ·6H2 O) [11].
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In this study the enhancement of methane fermentation by the addition of metal chelating agents and Ni2+ is investigated according to the changes of methane production, the contents of F430 and CoM. The Ni2+ bioavailability for methanogenic microorganisms is also explored. It is expected that the results obtained from this study could provide valuable information for the application of chelators on the enhancement of methane fermentation. 2. Materials and methods 2.1. Biomass Biomass was collected from a full-scale anaerobic reactor treating beer wastewater (Wuxi, China). It had a pH value of 7.2 and VSS/TSS of 0.72. The synthetic wastewater was supplemented for acclimation of the biomass in a 2 L continuous stirred tank reactor (CSTR) digester, which had been operated at 35 ± 2 ◦ C. Components of the synthetic wastewater were described in 2.2 (except that Ni2+ concentration was 20 M). The hydraulic retention time was 5 days and the acclimation period was two months. Prior to an experimental run, several (3–4) successive subcultures in nickel deficient synthetic wastewater were generated for the acclimated biomass. 2.2. Synthetic wastewater Synthetic wastewater was used in the study. It contained the following components in ultrapure water: 85 mM sodium acetate, 0.50 mM KH2 PO4 , 0.50 mM Na2 HPO4 , 3.7 mM NH4 Cl, 0.50 mM MgCl2 , 0.89 mM CaCl2 , and 1.0 mM Na2 S. Acid and alkaline solutions of trace elements (1 mL of each per liter) were used. The acid trace element solution contained the following components: 7.5 mM FeCl2 , 1.0 mM H3 BO4 , 0.50 mM ZnCl2 , 0.10 mM CuCl2 , 0.50 mM MnCl2 , 8.5 mM CoCl2 ·6H2 O and 50 mM HCl. The alkaline trace element solution contained the following components: 0.10 mM Na2 SeO3 , 0.10 mM Na2 WO4 , 0.10 mM Na2 MoO4 ·2H2 O, and 10 mM NaOH. Nickel was added as required in the form of Ni2 Cl2 ·6H2 O. 2.3. Operating conditions All experiments were carried out in 150 mL glass serum bottles. The bottles were properly capped and connected to liquid displacement system (in which the liquid was 15% NaOH solutions to remove CO2 or other acidic gas from biogas) for recording methane production with time. Schematic view of experimental set-up was presented in Fig. 1. Required amount of biomass was added to serum bottles which contained 100 mL synthetic wastewater to get VSS concentration of 5 g/L. After seeding and adjusting pH to 7.0 ± 0.2, the bottles were flushed with N2 gas for 5 min to provide anaerobic conditions, then sealed with natural rubber stoppers and plastic screw-caps. They were incubated in a temperaturecontrolled room at 35 ± 2 ◦ C and gas production in each bottle was measured daily with the liquid displacement device. After
Fig. 1. Schematic of batch experiment set-up.
gas measurement the bottles were manually shaken. In serum bottles with biomass and 100 mL tap water (purged of oxygen with nitrogen gas), control experiments were conducted in parallel to determine the background gas production. Each experiment was performed in triplicate, and the averaged results were presented in this manuscript. 2.4. Analysis TSS and VSS were measured according to standard methods [12]. Before the total dissolved Ni2+ concentrations were determined by atomic absorption spectrometry (SpectraAA220, Varian, USA), the samples were acidified with 0.1 M HNO3 . These samples were taken from bottles without biomass, which were left to equilibrate for 7 days. Sampling was performed in an anaerobic chamber (Sheldon Manufacturing Inc. USA). Precipitates were separated from the dissolved phase by centrifugation at 10,000 × g for 10 min. F430 was extracted from the precipitate, which was obtained by centrifugation of a 50 mL aliquot of culture broth from the bottle as described by Kida et al. [13]. F430 concentrations in the samples were estimated based on nickel concentrations determined using atomic absorption spectrophotometry (SpectraAA220, Varian, USA). Coenzyme M (CoM) was extracted from 5 g wet sludge, which was obtained by centrifugation of a 20 mL aliquot of culture broth from the bottle, mixed with 2 mL 1% tri-(N)-butylphosphine in 2-propanol (1% TBP) and incubated for 1 h at room temperature. Then the concentration of CoM was analyzed by HPLC with an Econosphere C18 column as described by Elias [14]. The standard substance of CoM was purchased from Sigma. 3. Results and discussion 3.1. Effect of Ni2+ addition on methane fermentation Batch tests were performed with required amount of biomass, 100 mL synthetic wastewater, 0–200 M Ni2+ , and 1 mM sulfide. Methane production at various Ni2+ concentrations was shown in Fig. 2.
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Q.-H. Hu et al. / Biochemical Engineering Journal 38 (2008) 98–104 Table 1 Stable constants Kf of CA, NTA and EDTA with metal ions and solubility products Ksp of S2− with metal ions Chelators/S2−
CA NTA EDTA S2−
Fig. 2. Methane production due to the anaerobic conversion of acetate at various Ni2+ concentrations without NTA. Sodium sulfide was added at 1 mM. Ni was added at 0 (), 1 (×), 20 (), and 200 M ().
At the initial phase (0–48 h) at various Ni2+ concentrations, methane production was nearly 40 mL for all cases. After 48 h, methane production increased with increasing Ni2+ concentration (Fig. 2). At 168 h the methane production increased from 112 to 162 mL as Ni2+ concentration was elevated from nil to 200 M, indicating that a higher concentration of Ni2+ might promote the methane production, and nickel seemed to limit the rate of methanogenesis from acetate. When the Ni2+ concentration was 1, 20 and 200 M, methane production was 119, 132 and 162 mL at last, which increased 6.30%, 17.9% and 44.6%, respectively, compared with the control. Previous results [10,11,15] indicated that Ni2+ was required for the methane production. Nickel is structural constituents of the cofactor F430 , which are key components of the enzyme complexes that catalyze several reactions of the methanogenic pathway [8]. Kida et al. [13] investigated the requirement of Ni2+ and Co2+ for methanogenic activity in anaerobic CSTR reactor by the concentrations of coenzymes F430 and corrinoids. The methanogenic activity and concentrations of F430 and corrinoids in the biomass increased with increasing added amounts of Ni2+ and Co2+ . Our results described here also confirmed that the methanogenic activity was related to the concentration of Ni2+ . 3.2. Effect of chelating agent addition on methane fermentation Sulfide had dual effect on methane fermentation. On the one hand, the availability of sulfide was of great importance to the growth of methanogens. However, when sulfide was in excess, it precipitated with metal ions and prevented the direct uptake of trace metals by methanogens [5,6]. It was necessary to balance the addition of both trace metals and sulfide. The chemical form of the metals (speciation) will determine their availability for the microorganisms [16]. Metal sulfide precipitates were not directly available for methanogens and therefore in practice, in order to increase metal availability, a large amount of metal was usually needed in the reactors to meet the demand of methanogens.
Log stability constant/solubility products Ksp Ni
Co
Fe
6.9 11.5 18.6 2 × 10−21
5.0 10.6 16.2 4 × 10−21
4.4 8.8 14.3 1 × 10−18
Gonzalez-Gil et al. [17] found that the addition of yeast extract could improve the bioavailability of Co and Ni when using methanol as substrate. This suggested that the formation of strong metal-yeast extract complexes played an important role in methane fermentation. Callendar and Bradford [5] measured soluble metal concentrations in an anaerobic digester which treated the wastewater containing amino acids and polypeptides. The level of soluble metals in the presence of S2− was 104 -fold higher than the predicted concentrations using available solubility data. So, soluble metal complexes, which may be formed with amino acids and polypeptides, possibly increased the concentration of soluble metal. The mode of metal addition should be optimized such that the rate and extent of metal precipitation were minimized. In order to promote the dissolution of metal ions from precipitates, such as metal sulfide, which was unavailable for methanogens, CA, NTA and EDTA, which had different stable constant shown in Table 1 [18], were selected as chelating agents of metal ions. Under 20 M of Ni2+ , the time profiles of methane production with the addition of different chelating agents in the range of 1–1000 M were shown in Fig. 3. Two-hundred micromolar nickel may provide relatively sufficient bio-available nickel for anaerobic microorganism. As a result of that, the influence of chelating agents added on the bioavailability of nickel may be slight or little. Also, low dosage can save cost. Therefore, 20 M of Ni2+ (not 200 M) was selected here. The results in Fig. 3 revealed that the effect of metal ion chelators on methane production during the anaerobic digestion of acetate was significant. The methane production was 132 mL for the control, while it was 177 mL for the bottle with 10 M of CA. The corresponding value was 197 mL with 10 M of NTA and 183 mL with 10 M of EDTA, increasing 34.1%, 49.2% and 38.6%, respectively, compared with the control. EDTA was the strongest chelator with the highest stable constant, but its stimulating effect did not seem to be the highest. NTA with the middle stable constant was the best stimulant in our experiments. Methane production was enhanced by 49.2% in NTA amended bottles, 10.6% higher than that in EDTA amended ones (Fig. 3). Speece [19] also reported that, although chelators could greatly increase the availability of metal ions, excessively strong complexing ability may not enhance the bioavailability of metals. Maybe metal complexes formed with too strong chelator could not be combined with free surface sites on the cell membrane, therefore its uptake was influenced [20]. In addition, metal chelator concentration had obvious effect on methane fermentation. It can be seen from Fig. 3 that, for CA,
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NTA and EDTA, the methane production obtained the maximum under the added concentration of 10 M and the difference in the simulating effect for 100 M and 1000 M of chelating agent were not obvious, indicating that high chelator concentration may not stimulate methane production. The best dosage in our experiments was 10 M. It should be noted that the chelator was not simply serving as an additional carbon source of methane production according to the magnitude of increased methane production. Theoretically, 1 mol of NTA could produce about 2.3 mol of methane, and 1 mol of CA could do about 2 mol of methane. Accordingly, the complete conversion of 1 mol NTA and CA would produce 0.052 and 0.045 mL (at standard state condition) of methane. However, our experiments reveal the 10 M of CA and NAT in 100 mL synthetic wastewater present an increase in methane production of about 48 and 65 mL over the control. Therefore, the increase in methane production is not due to metabolism of chelators to methane, but possibly to the enhancement in the solubility of essential metal nutrients. With regard to the addition cost, it was firstly assumed that the wastewater contained 5000 mg COD/L. The methane production increased from 1.32 L/L wastewater to 1.77, 1.97, and 1.83 L/L wastewater by addition of 10 M CA, NTA and EDTA, respectively (according to the results in Fig. 3). Because the heat energy of 1 m3 methane was equal to 1 m3 natural gas, the methane price was equal to natural gas price. The cost and benefit analysis were conducted and listed in Table 2. As listed in Table 2, the net benefit for CA, NTA and EDTA were 0.98, 1.38 and 1.08 yuan/m3 , respectively. The net benefit of NTA was the highest. To sum up, NTA was the optimal chelating agent based on either the promoting efficiency to methane production or the addition cost. 3.3. Effect of chelating agent on total dissolved concentration of Ni2+
Fig. 3. Methane production due to the anaerobic conversion of acetate at various chelator concentrations. Sodium sulfide was added at 1 mM. Ni was added at 20 M. (a) CA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M (). (b) NTA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M (). (c) EDTA was added at 0 (♦), 1 (), 10 (), 100 (×), and 1000 M ().
The experiments were conducted in bottles only with synthetic wastewater for 7 days. The total dissolved Ni2+ concentration was determined and the results were listed in Table 3. The results clearly indicated that due to the chelating effect, the dissolution of metal sulfide precipitates in anaerobic digestion processes was promoted, nutrient limitations were overcomed and their availabilities could meet the requirements for biomass activity and growth. The total dissolved Ni2+ concentrations in the presence of 10 M NTA were indeed higher than that in control bottle. When the added Ni2+ concentration was 20 M, the total dissolved
Table 2 The cost and benefit analysis for the use of chelating agents Chelating agent
Price (yuan/t)
Dosage (g/m3 )
Cost (yuan/m3 )
methane price (yuan/m3 )
Methane production increase (m3 /m3 )
Benefit (yuan/m3 )
Net benefit (yuan/m3 )
CA NTA EDTA
6700 22000 15000
1.92 1.91 2.92
0.013 0.053 0.044
2.20 2.20 2.20
0.45 0.65 0.51
0.99 1.43 1.12
0.98 1.38 1.08
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Table 3 Effect of chelating agents on total dissolved concentrations of Ni2+ Medium
Dissolved concentrations of Ni2+ (g/L)
20 M Ni2+ + synthetic wastewater 20 M Ni2+ + synthetic wastewater + 10 M CA 20 M Ni2+ + synthetic wastewater + 10 M NTA 20 M Ni2+ + synthetic wastewater + 10 M EDTA
58 ± 0.32 338 ± 12 1020 ± 21 1026 ± 18
Fig. 4. Methane production due to the anaerobic conversion of acetate at various Ni2+ concentrations. Sodium sulfide was added at 1 mM. NTA was added at 10 M. Ni was added at 0 (), 1 (×), 20 (), and 200 M ().
Ni2+ concentration was 1020 g/L, which was only 58 g/L for bottles without NTA. CA has weaker chelating ability than NTA because its stable constant with Ni2+ is 6.9, which is lower than that of NTA (11.5). When 10 M CA was added to the bottle, the total dissolved Ni2+ concentration was only 338 g/L. It increased only by 4.8-fold, but 16.6-fold increase was obtained with addition of 10 M NTA. EDTA has the strongest complexing ability and the total dissolved Ni2+ concentration was 1026 g/L, appreciably higher than that in NTA amended bottle. 3.4. Effect of NTA on methane fermentation As the best chelating agent of metal ion, effect of NTA on methane fermentation under different Ni2+ concentration was investigated, shown in Fig. 4, in this study. When 10 M NTA was added in the batch tests with 1 mM sulfide, the methane production seemed to be independent of the added Ni2+ concentration. Although the results of Fig. 2 clearly showed that the metabolic activity of the anaerobic biomass was Ni-limited,
the effect of Ni2+ addition concentration on the methane production shown in Fig. 4 became slight. Gas production finally achieved 196, 195 and 185 mL, respectively, when Ni2+ addition concentration was 1, 20 and 200 M. One micromolar Ni2+ was sufficient for acetate digestion in the presence of sulfide. In contrast, the methane production always increased with the increase of Ni2+ dosage if NTA was absent. Gonzalez-Gil [15] found that methane production rate increased sharply in anaerobic conversion of methanol with increasing Ni2+ concentration from 0 to 400 M. He also calculated equilibrium dissolved metal concentrations with different initial dosage of Ni and Co. The calculated concentrations of free Ni and Co should increase sharply only when the initial dosage of Ni and Co were more than 100 M, corresponding to the moment that the amount of sulfide added became limiting for precipitation reactions, which prevented Ni and Co uptake by methanogens. The solubility products Ksp for Ni with S2− is 2 × 10−21 . NTA was a kind of metal ion chelating agent and it could form soluble complexes with Fe, Co, Ni, etc. Stability constant Kf of NTA for Ni2+ is 11.5 [18]. So the formation of dissolved complexes between metal and NTA could promote the dissolution of metals from their sulfides. It may provide an attractive approach to keep additions of essential metals at a minimum dosage while assisting in their bioavailability. Ni2+ was beneficial for the methane production. On the other hand, Ni2+ was also a potent inhibitor of macromolecular synthesis such as RNA and proteins in S. cerevisiae [21] as well as in Escherichia coli [22]. It was toxic to microorganism if its concentration was too high. As can be seen from Fig. 4, under 200 M of nickel, the methane production was slightly lower than those under 1 and 20 M when NTA was present. A modified Gompertz equation has been used to describe the product formation in the hydrogen production process by Mu et al. [23,24]. It was: Rmax × e P = Pmax exp − exp (λ − t) + 1 (1) Pmax where P is the product formed at fermentation time t; Pmax is the potential maximum product formed; Rmax is the maximum rate of product formed; λ is the lag time to exponential product formed. In order to describe the kinetics of methane production from acetate by anaerobic cultures with or without NTA addition, the equation above was also tried in this manuscript. The fitted equations and its parameters for the formation of methane were summarized in Table 4. Correlation coefficients of non-linear analysis by Eq. (1) was over 98.6%, suggesting that the modified Gompertz equation was able to describe the methane formation for anaerobic digestion of acetate. It also can be seen from Table 4 that the potential maximum methane
Table 4 Calculated results using the modified Gompertz equation for methane formation NTA
The modified Gompertz equation
Pmax (mL)
Rmax (mL/h)
λ (h)
R2
None 10 M NTA
P = 164.01 × exp{−exp[1.32 × e/164.01 × (17.52 − t) + 1]} P = 208.95 × exp{−exp[1.88 × e/164.01 × (27.29 − t) + 1]}
164.01 208.95
1.32 1.88
17.52 27.29
0.990 0.986
Ni2+ addition concentration was 20 M.
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Table 5 Measured cofactor contents in the presence and absence of 10 M NTA
Acknowledgement
Samples
F430 (mol Ni/gVSS)
CoM (mol/gVSS)
NTA presence NTA absent
0.30 ± 0.05 0.22 ± 0.03
1.05 ± 0.12 0.21 ± 0.04
The authors thank the Hi-tech research and development program of China (863 program, Grant No: 2006AA06Z315) for the partial financial support of this study.
production Pmax and the maximum methane production rate Rmax under 20 M of Ni2+ were indeed higher with 10 M NTA than without NTA. This also suggested that methane fermentation could be enhanced by addition of chelating agent NTA. 3.5. Cofactors in the biomass The coenzyme content in digested sludge has been suggested as a means for the assessment of potential methanogenic acitivity [25]. Several metal ion- containing enzymes involved in methanogenesis have been identified. The Nickel tetrapyrrole, coenzyme F430 , was known to bind to methyl-S-CoM reductase which catalyzed methane formation from methyl-S-CoM in all methanogens [8]. CoM, one of the few naturally occurring sulfonic acids, was discovered and identified in the mid-1970s. With the exception of an aerobic alkene-degrading bacterium, CoM was found only in methanogens, and the content of CoM varied only by a small factor in different species [26]. So F430 and CoM contents in the biomass were determined and shown in Table 5. The results in Table 5 revealed that the contents of two coenzymes in the 10 M NTA amended biomass were higher than those in control. The concentrations of F430 and coenzyme M of the biomass increased 37% and by 4 fold respectively. Combined with the results in Fig. 4, it suggested that the level of methanogenic activity be related to the contents of F430 and CoM. The bioavailability of Ni2+ was enhanced when NTA was in presence. Elias et al. [14] suggested CoM could be used as a biomarker to quantify methanogen biomass in a variety of environmental matrices. The increased CoM content confirmed that the addition of NTA could increase the solubility and availability of essential metal nutrients, and promote the growth of the methanogenic population. 4. Conclusions It showed that the methane fermentation was enhanced with the addition of CA, NTA and EDTA. Addition of chelators led to the formation of dissolved complexes and consequently facilitated the dissolution of metals from their sulfides. Ten micromolar NTA could increase the level of soluble Ni2+ in the presence of S2− by a factor of 16.6. The bioavailability of Ni2+ was dramatically increased and the coenzyme content also raised from 0.22 to 0.30 mol Ni/gVSS for F430 , from 0.21 to 1.05 mol/gVSS for CoM. Thus it may provide an attractive approach to keep the additions of essential metals to anaerobic reactors at a minimum.
References [1] P. Scherer, H. Lippert, G. Wolff, Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry, Biol. Trace Elem. Res. 5 (1983) 149–163. [2] M.H. Zandvoort, R. Geerts, G. Lettinga, P. Lens, Effect of long-term cobalt deprivation on methanol degradation in a methanogenic granular sludge reactor, Biotechnol. Prog. 18 (2002) 1233–1239. [3] Z.B. Yue, H.Q. Yu, Z.L. Wang, Anaerobic digestion of cattail with rumen culture in the presence of heavy metals, Bioresour. Technol. 98 (2007) 781–786. [4] Y.S. Zhanga, Z.Y. Zhang, K. Suzukic, T. Maekawa, Uptake and mass balance of trace metals for methane producing bacteria, Biomass Bioenergy 25 (2003) 427–433. [5] I.J. Callander, J.P. Bradford, Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. I. Methodology, Biotechnol. Bioeng. 15 (1983) 1947–1957. [6] I.J. Callander, J.P. Bradford, Precipitation, chelation, and the availability of metals as nutrients in anaerobic digestion. II. Applications, Biotechnol. Bioeng. 15 (1983) 1959–1972. [7] H. Chen, T. Cutright, EDTA and HEDTA effects on Cd, Cr and Ni uptake by Helianthus annuus, Chemosphere 45 (2001) 21–28. [8] R.K. Thauer, Biochemistry of methanogenesis: a tribute to Majory Stephenson, Microbiology 144 (1998) 2377–2406. [9] P. Hausinger, Nickel utilization by microorganisms, Microbiol. Rev. 51 (1987) 22–42. [10] C.M. Williams, J.C.H. Shih, J.W. Spears, Effect of nickel on biological methane generation from a laboratory poultry waste digester, Bioresour. Technol. 28 (1985) 1608–1610. [11] M. Canovas-Dlaz, J.A. Howell, Effect of nickel on methane production and butyric acid utilization in a downflow fixed-film reactor, Biotechnol. Lett. 8 (1986) 287–292. [12] APHA, AWWA, WEF, Standard Methods for the Examination of Water and Wastewater, nineteenth ed., American Public Health Association, Washington, DC, 1995. [13] K. Kida, T. Shigematsu, J. Kijima, Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis, J. Biosci. Bioeng. 91 (2001) 590–595. [14] D.A. Elias, L.R. Krumholz, R.S. Tanner, J.M. Suflita, Estimation of methanogen biomass by quantitation of coenzyme M, Appl. Environ. Microbiol. 65 (1999) 5541–5545. [15] G. Gonzalez-Gil, R. Kleerebezem, G. Lettinga, Effects of nickel and cobalt on kinetics of methanol conversion by methanogenic sludge as assessed by on-line CH4 monitoring, Appl. Environ. Microbiol. 65 (1999) 1789–1793. [16] M.H. Zandvoort, E.D. van Hullebusch, F.G. Fermoso, P.N.L. Lens, Trace metals in anaerobic granular sludge reactors: bioavailability and dosing Strategies, Eng. Life Sci. 3 (2006) 293–301. [17] G. Gonzalez-Gil, S. Jansen, M.H. Zandvoort, H.P. van Leeuwen, Effect of yeast extract on speciation and bioavailability of nickel and cobalt in anaerobic bioreactors, Biotechnol. Bioeng. 82 (2003) 134–142. [18] H. Yue, Advanced Inorganic Chemistry, second ed., Beijing, 2002. [19] R.E. Speece, Anaerobic Biotechnology for Industrial Wastewaters, Archac, USA, 1996. [20] P.L. Brown, S.J. Markich, Evaluation of the free ion activity model of metalorganism interaction: extension of the conceptual model, Aquat. Toxicol. 51 (2000) 177–194. [21] M. Joho, Y. Imada, T. Murayama, The isolation and characterization of Ni resistant mutants of Saccharomyces cerevisiae, Microbios 51 (1987) 183–190.
104
Q.-H. Hu et al. / Biochemical Engineering Journal 38 (2008) 98–104
[22] C. Guha, A. Mookerjee, Effect of nickel on macromolecular synthesis in Escherichia coli K-12, Nucleus 22 (1979) 45–47. [23] Y. Mu, G. Wang, H.Q. Yu, Kinetic modeling of batch hydrogen production process by mixed anaerobic cultures, Bioresour. Technol. 97 (2006) 1302–1307. [24] Y. Mu, H.Q. Yu, G. Wang, A kinetic approach to anaerobic hydrogenproducing process, Water Res. 41 (2007) 1152–1160.
[25] M.J. Delafontaine, H.P. Naveau, E.J. Nyns, Fluorimetric monitoring of methanogensis in anaerobic digestors, Biotechnol. Lett. 1 (1979) 71–74. [26] W.E. Balch, R.S. Wolfe, Specificity and biological distribution of coenzyme M (2-mercaptoethanesulfonic acid), J. Bacteriol. 137 (1979) 256–263.