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Biomass production for the removal of heavy metals from aqueous solutions at low pH using growth-decoupled cells of a Citrobacter sp

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Biomass production for the removal of heavy metals from aqueous solutions at low pH using growth-decoupled cells of a Citrobacter sp
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  ELSEVIER zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA International ~o~teriar~tion & 3io~r~tio~ (1995) 73-92 Copyright 0 1995 Elsetier Science Limited Printed in Great Britain. All rights reserved ~~g3~5~95/$9.50+.~ zyxwvutsrqponm Biomass Production for the Removal of Heavy Metals From Aqueous Solutions at Low pH Using Growth-decoupled Cells of a zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQP itrobacter sp. L. E. Macaskie,** C. J. Hewitt,a J. A. Shearerb & C. A. Kentb “School of Biological Sciences and ‘BBSRC Centre for Biochemical Engineering, School of Chemical Engineering, The University of Bi~ingham, Edg~aston, Bi~ingham B15 2TT, UK zyxwvutsrqponmlkjihgfedcbaZYXWVUT ABSTRACT A Citrobacter sp. accumulates heavy metals via the activity of an acid-type phosphatase that produces inorganic phosphate, zyxwvutsrqponmlkjihgfedcbaZYXW P04 2-. This ligand precipitates with heavy metals (A4) as MHPOd, which is retained at the cell surface. Continuous metal deposition has been coupted to the removal of heavy metals from metal-laden solution. The pH optimum of the mediating phos~hatase is 5G8.0, with 55 and W80 retention of activity at pH 4-O and 4.5, respectively. Metal ac~mu~ation was reduced at pH 5.0, ~trib~tab~e to increased metal phosphate sol~i~ity and reduced metal phosphate precipitation, but this was overcome using ceBs of higher phosphatase activity. A 3.25-fold overproduction of enzyme compensated for a l&?-fold increase in the concentration of H . Preliminary tests enabled prediction of the increased phosphatase activity required to treat a target waste stream containing uranyl ion at pH 4.5. Enzyme over- production was achieved by growth of a phosphatase constitutive variant in a lactose-based medium, but enzyme activity was reduced at the high carbon concentration required zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONML or a high biomass yield. The latter require- ment was fulfilled with enhanced enzyme production by the use of fed-batch culture, with substrate addition regulated via feedback analysis of the off- gases. The biomass removed uranyl ion efficientiy from a challenge solution at pH 4.5 in a batch contactor. Lactose-grown immobilized cells also removed uranyl ion from an acidic simulated industrial wastewater. *To whom co~spondence should be addressed. 73  74 L. E. Macaskie et al. The use of microbial cells and biomass for the removal of heavy metals from contaminated solutions and industrial wastes is receiving increased attention in the light of increasingly stringent legislative constraints on permissible discharge levels of toxic metals worldwide. Bioremediative technologies often utilize inert biomasses to sorb metals (metal biosorp- tion); alternatively the use of metal-resistant living cells for metal bioac- cumulation has been described (Macaskie & Dean, 1989; Volesky, 1990; Macaskie, 1991; Gadd, 1992). The choice of technology may be imposed by the constraints of the ‘target’ waste. In particular, many industrial wastes and mine run-offs may be chemically aggressive, precluding the use of live biomass. Although biosorptive techniques are well-established, bioso~tion at low pH is often reduced due to the speciation of the metals in solution and to competitive protonation of the sorbing sites; indeed, a reduction in pH has been used to elute and recover metals from loaded biomass (see Volesky, 1990). The efficiency of metal removal is governed by the final equilibrium between metal-loaded biomass and residual free metal in solution, while the longevity of a system in continuous use is ultimately governed by saturation constraints. Continuous processes based on sorption-desorption cycling have been described by several authors (see Eccles & Hunt, 1986; Volesky, 1990 for reviews). One notable example of the use of biosorption for effluent treatment and metal recov- ery utilized biomass of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH hizopus arrhizus for the removal of uranium from acidic run-off waters at Denison Mines, USA (Tsezos et al., 1989); subse- quent studies have investigated the engineering parameters of this immo- bilized biomass system (Tsezos, 1990; Tsezos & Deutschmann, 1990). An alternative approach to the continuous treatment of wastes relies upon the continuous deposition of heavy metals with metabolically- generated ligands, a process that has been termed biomineralization. Examples include the deposition of metal sulphides by sulphate reducing bacteria (Barnes et al., 1991), and the crystallization of metal hydroxides and carbonates by Alcaligenes eutrophus. In the latter case this is an indirect result of the metal resistance mechanism of the organism, whereby metal efflux from the cells is concomitant with uptake of protons to maintain electroneutrality across the cell membrane. Metal precipitation occurs as a result of the localized shift to an alkaline pH within the peri- plasmic space (Diels et al., 1991). Both processes are metabolically-medi- ated, and would find only limited application at growth-inhibitory, low pH values; application to aggressive wastes and acidic mine run-off waters is problematic. A technique using growth-decoupled biomineralization has been  Removal of heavy metals using Citrobacter sp. 7.5 described (Macaskie & Dean, 1989; Macaskie, 1990; Ma~s~e et al., 1992~~). This utilizes pre-grown cells of a zyxwvutsrqponmlkjihgfedcbaZYXWVUTS itro~acfer sp., which produce a heavy metal-resistant periplasmic phosphatase that retains activity upon subsequent immobilization of the cells within a ‘cartridge’ which is chal- lenged with the metal-laden flow in the presence of an organic phosphate ‘donor’ molecule (phosphatase substrate). Enzymically-mediated libera- tion of inorganic phosphate (HP04 2-) allows the metal phosphate solu- bility product to be exceeded locally, at low bulk solution concentration of metal, with stoichiometric precipitation of insoluble metal phosphate at the cell surface via nucleation sites in juxtaposition to areas of enzymic activity (Tolley, 1993; Macaskie et al., 1994). The advantage of this approach is that very high metal loads can be achieved (e.g. 9 g metal/g of dry biomass), the final solid waste is of low biomass content and the metals can be recovered if necessary as a concentrated slurry with the potential for biomass recycle (Macaskie, 1990). Although the enzyme is an acid-type phosphatase with a broad pH optimum of 5N.O (Jeong, 1992), in practice the working pH may be limited to a minimum of 4.0, at which pII 45% of the enzyme activity was lost (Tolley et al., 1995). Adaptation of the cells to a low pH environment by continuous culture resulted in a loss of phosphatase production (low specific activity). In addition, increased metal phosphate solubility at low pH (Macaskie, 1990) and retardation of the onset of metal precipitation (Yong & Macaskie, 1995) imposed further constraints in application of this approach to the treatment of acidic wastewaters on a high throughput basis. For a working process it could be envisaged that cartridges of immobilized biomass would be prepared off-site and transported to the site of use. The cartridges should be compact, and of known and repro- ducible activity. Previous studies have shown that production of the phosphatase is regulated by the carbon status of the pre-growth medium. Maximal enzyme production occurred upon entry of batch cultures into carbon- limitation (Macaskie, 1990; Butler et al., 1991) or their maintenance in a chemostat under carbon limitation (Macaskie, 1990; Jeong, 1992; Macas- kie et al., 1995). Since the yield of biomass is related to the concentration of utilizable carbon provided, this may impose a paradox whereby a high yield under carbon-sufficiency will result in low phosphatase specific activity. High yields are necessary for economic biomass production at the industrial scale, but for treatment of wastes at low pH a high phosphatase titre (specific activity) is necessary to compensate for sub-optimal opera- tion and the chemical constraints described above. In the present study it is shown that phosphatase overproduction can compensate for the pH- inhibitory effect, and a method is presented for the production of high-  76 L. E. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPON acaskie et al. activity cells with high yield, using a fed-batch system. The effectiveness of batch-grown cells in the removal of uranyl ion from solution in a batch contactor at pH 4.5 was investigated in preliminary tests, which were confirmed and extended in parallel studies using flow-through immobi- lized cell reactors. Initial tests utilized synthetic solutions prepared in the laboratory, while later studies investigated the activity of immobilized biomass cartridges in the removal of uranyl ions from a synthetic waste water based on the composition of acidic mine wastewaters produced by the Empresa National de1 Uranio (ENUSA) mine in the municipality of Saelices el Chico, Spain. This application is described briefly in the present communication, and is detailed fully in the companion paper (Roig et al., 1995). MATERIALS AND METHODS zyxwvutsrqponmlkjihgfedcbaZYXW Microorganisms and growth conditions The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA ~t~~~acter strain N14 was used by kind permission of Isis Innova- tion, Oxford. This, and the phosphatase ove~rodu~ing strain dc5c, were as described previously (Macaskie et aE., 1988). The organisms were maintained on solid nutrient agar media, and grown in tris-based minimal medium of composition as appropriate to individual experiments (Table 1). All cultures were grown at 30°C. Batches were routinely grown in shake-flask cultures with a starter culture in the appropriate minimal medium to adapt the cells to the medium prior to experiment. Small-scale batch cultures were in a New Brunswick ‘Bioflo’ apparatus with the outflow clipped-off to give a culture volume of 550 ml, with on- line monitoring of pH (Ingold pH electrode) and dissolved oxygen content, which was expressed as DO2 (Oh of saturation). The input compressed air was constant at 0.33 vol/vol/min; the turbine speed was 400 rpm. Cultures for immobilized cell preparations (3 1) were as described previously (Tolley et al., 1995 . For larger scale cultures an LSL bior- eactor (working volume of 5 1) was used. The DO2 concentration was measured with an Ingold polarographic oxygen electrode and the pH was monitored with an Ingold combination pH electrode. Off-gas was analysed by mass spectrometry and the data produced by the experiment was logged and stored using the SETCON industrial process management system (SETPOINT corporation). The agitation speed was maintained at 500 rpm (gassed power input of O-4 kW/m3, using two Rushton turbines (tube diameter/3)]. The aeration rate was constant at 0.33 vol~vol~min. Visible foaming was controlled manually by the dropwise addition of 2%  Removal of heavy metals using Citrobacter sp. II TABLE 1 Media’ Used in this Study (Con~ntrations in g/l) Medium N source P source C source Fig. (NH&SO4 (O-96) G2P (0.67)’ Glycerol (3.00) 1 (NH&SO4 (0.96) G2P (0.67) Glycerol (2.00) 2 (NH&HP04 (0.96) Glycerol (2.00) NS (NH~)~HP~~ (O-96) Lactose (2-13) 3a ~H4)2H~4 (l-92) Lactose (4.26) 3b, 4 (NH4)2HP04 (1.92) Lactose (10.65 initially, 10.65 fed) 5c (NH4)2HP04 (1.92) Lactose (5.32 initially, 15.98 fed) 5b, 5c (NH4)&IP04 (1.92) Lactose (3.20 initially, 18.11 fed) NS “All media contained tris buffer (12.0 g/l), MgS04.7Hz0 (O-63 g/i), FeS04.7H20 (0.~032 g/l) and KC1 (O-62 g/l), with the pH adjusted zyxwvutsrqponmlkjihgfedcbaZYXWVUTS o 7.0 (me~um A) or 7.2 (all other media) with 2M HCI. In fed batch cultures (F-H) the lactose provided initially (concen- trations as shown) was supplemented by feeding when the concentration of CO, in the off- gas started to fall. The rate of lactose addition was controlled by the CO* concentration in the off-gas. b G2P: glyceroI2-phosphate. NS: not shown. polypropylene glycol antifoam. For the fed-batch mode of operation a sterile lactose solution was fed-in at a known flow rate via a peristaltic pump. Lactose was added at the point where the percentage of CO2 in the off-gas began to decline. Pump speeds were modified throughout the fermentation to add lactose continuously via feedback analysis of the CO2 concentration in the off-gas. Large-scale batch cultures were grown in a purpose-built pilot plant based on an LSL Biolafitte bioreactor with a total volume of 800 1. Cultures were grown in 600 1 with an air input of 0.33 vol~vol~min, with antifoam (as above) added as necessary. Agitation was using two six- bladed Rushton turbines (diameter ‘of O-262 m) at 150 rpm, which correlated to a gassed power output of 0.3 kW/m3. The DO2 concen- tration, and the culture pH were monitored as described for the 5 1 cultures. Samples were withdrawn from the cultures as appropriate, harvested by centrifugation, washed in isotonic saline (8.5 g/l of NaCI) and assayed for phosphatase by the release of p-nitrophenol from the chromogenic substrate p-nitrophenyl phosphate in MOPS or MES buffer (Good et al., 1966) at appropriate pH values as described previously (Tolley et al., 1995). Phosphatase specific activity (unit) is expressed as nmol product released/min/mg bacterial protein, with protein assayed by the method of Lowry, and calculated from the OD 600 of the bacterial suspension via a conversion factor (Jeong, 1992).
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