Biomass Production for the Removal of Heavy Metals ... - Science Direct

InternationalB~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+.~ ELSEVIER

Biomass Production for the Removal of Heavy Metals From Aqueous Solutions at Low pH Using Growth-decoupled Cells of a Citrobacter 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

ABSTRACT A Citrobacter sp. accumulates heavy metals via the activity of an acid-type phosphatase that produces inorganic phosphate, HP04 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 overproduction 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 for a high biomass yield. The latter requirement wasfulfilled with enhanced enzyme production by the use of fed-batch culture, with substrate addition regulated via feedback analysis of the offgases. 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 biosorption); alternatively the use of metal-resistant living cells for metal bioaccumulation 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 recovery utilized biomass of Rhizopus arrhizus for the removal of uranium from acidic run-off waters at Denison Mines, USA (Tsezos et al., 1989); subsequent studies have investigated the engineering parameters of this immobilized 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 metabolicallygenerated 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 periplasmic space (Diels et al., 1991). Both processes are metabolically-mediated, 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 ~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 challenged with the metal-laden flow in the presence of an organic phosphate ‘donor’ molecule (phosphatase substrate). Enzymically-mediated liberation of inorganic phosphate (HP04 2-) allows the metal phosphate solubility 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 reproducible 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 carbonlimitation (Macaskie, 1990; Butler et al., 1991) or their maintenance in a chemostat under carbon limitation (Macaskie, 1990; Jeong, 1992; Macaskie 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 operation and the chemical constraints described above. In the present study it is shown that phosphatase overproduction can compensate for the pHinhibitory effect, and a method is presented for the production of high-

76


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 immobilized 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

Microorganisms and growth conditions The C~t~~~acterstrain N14 was used by kind permission of Isis Innovation, 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 online 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 bioreactor (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

TABLE 1 Media’ Used in this Study (Con~ntrations Medium

N source

(NH&SO4 (O-96) (NH&SO4 (0.96) (NH&HP04 (NH~)~HP~~ ~H4)2H~4 (NH4)2HP04 (NH4)2HP04 (NH4)&IP04

P source G2P (0.67)’ G2P (0.67) (0.96) (O-96) (l-92) (1.92) (1.92) (1.92)

sp.

in g/l)

C source

Glycerol Glycerol Glycerol Lactose Lactose Lactose Lactose Lactose

II

(3.00) (2.00) (2.00) (2-13) (4.26) (10.65 initially, 10.65 fed) (5.32 initially, 15.98 fed) (3.20 initially, 18.11 fed)

Fig.

1 2 NS 3a 3b, 4 5c 5b, 5c 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 to 7.0 (me~um A) or 7.2 (all other media) with 2M HCI. In fed batch cultures (F-H) the lactose provided initially (concentrations as shown) was supplemented by feeding when the concentration of CO, in the offgas started to fall. The rate of lactose addition was controlled by the CO* concentration in the off-gas. bG2P: 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 sixbladed Rushton turbines (diameter ‘of O-262 m) at 150 rpm, which correlated to a gassed power output of 0.3 kW/m3. The DO2 concentration, 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 600of the bacterial suspension via a conversion factor (Jeong, 1992).

78

L. E. ~ae~k~e ef al.

Preparation of immobilized ceil columns, and challenge with metal solution For a test system 5 g wet weight of cells was harvested and immobilized in a poIyacrylamide gel as described previously (Tolfey et al., 1995). The set and shredded gel was divided into five aliquots by weight (1 g wet weight of cells~aliquot). Each was washed in isotonic saline to remove unpolymerized gel components, packed into a column (25 ml) and ehalfenged with a flow that consisted of 5 mM citrate buffer (pH 7*0 or 5.0) supplemented with substrate (5 mM glycerol 2-phosphate) and lanthanum or uranyf nitrate (1 mM), at a flow rate controlled by an external peristaltic pump (Watson Marlow). Bioreactor activity is defined in terms of the FAri value, which is that flow rate giving 50% removal of metal from the flow, or that flow rate giving 50% cleavage of the substrate to product in metal-unsupplemented columns. Subsequent tests used cells immobilized within “Hypol’ foams and on various solid supports held within plug flow reactors and challenged with flows of waste water from the ENUSA mine (Roig et al,, 1995). Metal contacting tests at low pH using free cells Biomass harvested from the 5 1 cultures (see Fig. 5) was resuspended in 100 ml of isotonic saline and added to a contactor vessel containing 2 1 of challenge solution of composition 3.4 mM glycerol 2-phosphate and 0.2 mM many1 nitrate, with the pH adjusted to 4.5 with HCl. Agitation was maintained at 100 rpm., and aeration at 0.33 vol~vol/min. Samples (20 ml) were withdrawn at intervals and assayed for residual uranyl ion following removal of the cells by ~eut~fugation. When the uranyi ion was depleted, 3 1 of further challenge solution was added at a flow rate of 10 or 20 ml/h. Assay of metal ions L~nthan~ and uranyl ions were assayed using arsenazo III with appropriately diluted samples of challenge solutions, spent liquor and column outflows (Tolley et al., 19%). The arsenazo III solution was prepared by adding 0.038 g of Arsenazo III to 25 ml of distilled water and mixing for at least 1 h. This was filtered through a Whatman No. 1 filter paper to give a saturated solution that could be stored for up to 1 month before use. For assay the samples (final volume 2 ml) were acidified with 0.3 ml of 0.75 M HCl and the metal ion was visualized as the metal-Arsenazo III complex at 652 nm. Metal uptake by the cells was expressed as a percentage of the input metal removed (bow-through column experiments) or percentage of the initial metal remaining in solution (batch contactor studies).

Removal of heavy metals using

Citrobactersp.

79

RESULTS AND DISCUSSION The effect of pH on phosphatase activity It was shown previously that the optimum phosphatase activity was at pH values above 5.0; at pH 4.0 the retention of activity was only 55% of the value at neutral pH. This inhibition also occurred using polyacrylamidc gel-immobilized cells that were challenged with p-nitrophenyl phosphate in a metal-unsupplemented flow-through system (Tolley et al., 1995). Little uranyl ion was removed at pH 4.0 from test solutions (Tolley et al., 1995), and pH 4.5 was considered as a useful compromise between the requirement for optimal enzyme activity and the pH constraint imposed by acidic ‘target’ wastewaters. In the present study the latter was the acidic waste water from a Spanish uranium mine (see Roig et al., 1995). The retention of phosphatase activity at pH 4.5 was 70-80% of the activity at pH 7.0 (Tolley et al., 1995). Pre~minary studies on the effect of pH on metal removai from solution Although a pH value of 5.0 gave optimal phosphatase activity, and a rate of p-nitrophenol release identical to that at pH 7 using both purified enzyme (Jeong, 1992) and free and immobilized cells (Tolley et al., 1995) the removal of uranyl ion and lanthanum from test flows was pH-sensitive. Pre~~nary tests showed that the removal of uranyl ion was shown at a lower FA’,* value at a given pH. The respective FA ‘12values for uranyl ion and lanthanum were 1.2 and 5.2 ml/mm for a batch of cells of phosphatase specific activity of 206 units at harvest and challenged at pH 7.0 using polyacrylamide geli~obilized cells. This difference was attributed to the greater solubi~ty of HUOzP04 as compared to LaPO4 as shown in an ipl vitro test system (Macaskie et al., 1995), and in accordance with the solubility products of the metal phosphates (2.1 x 10-l’ and 3.7 x 1O-23for HU02P04 and LaP04, respectively: see Yong & Macaskie, 1995, for discussion). Identity of the pr~ipitated metal species, and imp~cations for bioprocess operation at low pH The identity of the metal phosphates (above) was confirmed by X-ray diffraction (XRD), solid-state nuclear magnetic resonance (NMR), energy dispersive X-ray analysis (EDAX) and proton induced X-ray emission (PIXE) with a Rutherford backscattering correction (Macaskie et d., 1992a, 6; Tolley et al., 1995; Yong & Macaskie, 1995; Lin & Macaskie, unpublished). It is not possible to differentiate using XRD between

80


HU02P04 and NaU02P04, since these have a very similar crystal structure. However EDAX and PIXE analyses have demonstrated significant amounts of Na associated with the crystal of HU02P04 (Yong & Macaskie, unpublished), and a mixed crystal of HUO2PO4 and NaU02P04 is very likely, given that the challenge solution contained 5 mM Na+ (provided as the sodium salt of glycerol 2-phosphate). The solubility product of alkali metal (potassium) uranyl phosphate (is 6.4 x 10-24: see Yong & Macaskie, 1995) and it would be anticipated that as the pH falls the proportion of protons within the crystal may rise, and with this the solubility product (c.f. above}, i.e. uranyl phosphate would be less soluble than lanthanum phosphate at low pH. The ease of metal phosphate precipitation would also be in~uenced by the pH-dependent solution chemistry of inorganic phosphate (at low pH HzPO; would tend to predominate over HP04 ‘-: see Yong & Macaskie, 1995 for discussion). In order to decouple the biochemical factors and phosphate solution chemistry effects from the additional crystallographic constraints associated with the deposition of uranyl phosphate (above), initial tests were done using lanthanum as the test metal. Flow rate-activity relationships of lanthanum removal by immobilize ceils The flow rate-dependent removal of lanthanum from a challenge flow is shown in Fig. 1. The phosphatase specific activity of Citrobacter sp. strain N14 is variable between preparations (Macaskie et al., 1988); this can be used as a tool in modelling studies. Data are shown for two preparations grown in medium A, together with a preparation of cells of the phosphatase-ove~rodu~ing mutant dc5c. The respective phosphatase activities and the FA,,, values for each strain at a challenge pH of 7-Oare shown in Table 2. The ratio of phosphatase activities to the ratio of FAi,,. values is highly conserved in each case, and the FA iI value for the removal of La is taken to be an accurate reflection of the phosphatase activity. The effect of pH was investigated using strain d&c. The FAi,, value for this strain at pH 5.0 was 312 ml/h (Fig. l), which is very similar to the FA,,, value for the low activity strain at pH 7-O (297 ml/h: Table 2) and it can be concluded that a 3.25-fold increase in phosphatase activity is necessary to compensate for a reduction in pH of 2 units (i.e. a lOO-fold increase in the concentration of H+ as the pH is reduced from 7-O to 5.0). Prediction of requirements for a bioprocess to treat uranyl wastes at pH 4.5, and specific objectives of this study The above data may have potentially serious implications for the use of this technique to treat waste solutions at pH 4-5; these can be expressed


Ln flow

sp.

81

rate (mliminf

Fig. 1. Flow rate-activity relationships for the removal of l~thanum from flows presented to polyacrylamide gel-immobilized cells at pH 7.0 (filled symbols) or 50 (open symbols). Two cultures of Citrobacter strain N14 of phosphatase specific activities of 206 (A) and 325 (v) units, gave corresponding FA 1,~values of 297 and 466 ml/h (arrowed at points A and C). The phosphatase-overproducing strain Citrobacter dc5c (phosphatase specific activity 670 units) was challenged at pH 7.0 (a) and 5.0 (0); the corresponding FA,,, values were 1090 and 312 ml/min (arrowed at points D and B). Biomass pre-growth was in medium A.

TABLE 2 Phosphatase Activities of Three Preparations of Citrobacter sp., and Corresponding Values for I~obili~d Cells A~mulating Lanthanum Preparation

I (Citrobacter N14)

II (Citrobacter N14) III (Citrobacter dc5c)

FA,,,

Phosphatase specific activity (units)

Relative value

FAljz value for La removal by bioreactor (mllmin) (Fig. I)

Relative value

206 325

1.oo I.58

297 466

670

3.25

1090

I .oo 1.57 3.67

terms of the phosphatase activity of the biomass, and the biomass yield required (Table 3). This calculation should be regarded as approximate, since the exact composition of the uranyl phosphate crystal at low pH, and the solubility product, is not known (see above), but the target is for a culture of phosphatase specific activity of approx. 3600 units. This may be difficult to achieve; furthermore, other studies have suggested that at very high phosphatase specific activities the rate of formation of precipitation foci, and the rate of metal phosphate crystal growth are rate limiting, i.e. a very high phosphatase activity is not expressed in terms of more efficient metal removal (Macaskie et al., 1994). An alterin

La, La, La,

relative vaZueis 1.0 (Table 2) relative value is 1.0 (above), x 3.25 (Table 2)b relative value is above, (1.0 x 3.25), x 5.0’

of a

1801 units; 3602 units.

14,405 f 25% = 18,006 units.

3350 x 4.3 = 14,405.

thus, by incorporation

206 206 x 3.25 = 670; 670 x 50 = 3350.

“This is a typical ODW value at harvest in media A and B (see Butler et al., 1991, and Fig. 3). bData from Table 2: this value is the increase in enzyme activity required to compensate for the effect of pH. “A change of pH from 5.0 to 4.5 represents a five-fold increase in the proton ~on~ntration; this extrapolation from the effect observed on shifting the pH from 7.0 to 5.0 (Fig. 2) is assumed to be valid since a shift of pH from 7-O to 6-O produced a small decrease in the FArf2 value (Strachan & Macaskie, unpublished). dCalculated from the ratio of FAr,, values of bioreactors removing uranyl ion and lanthanum (Tolley et al., 1995). This is 5.2/1.2 = 4.3 (see text). Note that the actual ratio has not been determined at acidic pH, and is difficult to predict, since the actual composition of the uranyl phosphate crystal, and hence its solubility product, is not known. This value is probably an underestimate (see text). “Tolley et al. (1995).

Assuming a concentration factor of cells of tenfold (i.e. OD err0z 14 units) this would be: and for an ODGee = 7 units the requirement would be The latter formed the target phosphatase activity and target final ODm of this investigation.

These values were calculated for lanthanum removal. However uranyl ion is the target metal, not lanthanum, correction factor: Discrepancy factor due to metal phosphate relative value is above (1.0 x 3.25 x 5.0), x 4.3 solubility factor = 4.3 (see texOd Assuming a loss of enzyme activity of 25% relative value is above (1.0 x 3.25 x 5-O x 4-3) + 25%: at sub-optimal pH (see text)e

At pH 7.0, accumulating At pH 5.0, accumulating At pH 4.5, accumulating

Specific activity (units)

TABLE 3 for Biomass and Enzyme Activity for the Removal of Uranyl Ion at pH 4.5, Calculated on the Basis of Values Obtained by Experiment

Assuming a ‘basal’ activity of 206 units in medium A at ODW = 1.4” units at harvest: Conditions (the actual phosphatase activities are as shown in Table 2, and see footnotes)

Requirement

!$ 8. rrt R

;

.P


Citrobactersp.

83

native approach could be to increase the final biomass content per unit of volume of the pre-culture (Table 3). Although the enzyme specific activity required could be only approx. 1800 units with a lo-fold increase in the culture biomass density, this may be difficult to achieve whilst maintaining the DO2 concentration suffjciently high to support the maximal growth rate (the Citrobacter sp. does not grow anaerobically on glycerol; Macaskie & Hewitt, unpublished). Additionally, the ‘sticky’ nature of the strain (which can be exploited for the formation of biofilms for metal accumulation: Macaskie, 1990; Roig et al., 1995) would be likely to impose viscosity and agitation constraints. A target OD6m of 7-O units and a phosphatase specific activity of 3600 units were set as a working compromise (Table 3). Since treatment of uranyl wastes at low pH is likely to be difficult for reasons outlined above (i.e. the calculation of Table 3 is likely to be an ~derestimate of the biomass and enzyme activity needed), it is foreseen that preparation of larger quantities will be required for incorporation into larger immobilized cell bioreactors to compensate. Although the unknown metal precipitation factors cannot yet be estimated (dete~ination of the relative amounts of protons and Na in the uranyl phosphate crystal is likely to be problematic and outside the immediate scope of this study), the opportunity was taken to check that the behaviour of the cultures was not adversely affected by scale-up of growth from a working volume of 550 ml to 600 1. Growth and phosphatase production in small and large scale hatch cultures in glycerol-glycerol 2- phosphate-based minimal medium Preliminary tests done in medium B con~~ed the low phosphatase activity in carbon-sufficient medium. Similar results were obtained irrespective of the culture volume (Fig. 2a, b). The phosphatase specific activities attained peak values of approx. 275 and 425 units, respectively, but these were transient values in the late-logarithmic phase of growth. The activity fell to a final value of approx. 100-200 units (Table 1). This fall, as noted previously (Butler et al., 1991) coincided with a decrease in the pH of the medium (Fig. 2), probably attributable to the production of acidic metabolites, and the removal of NH,f ions from the medium for amino acid biosynthesis. Tris buffer is a poor choice in this situation (plu, for Tris = 8.3-8.5). The use of phosphate buffer was precluded since phosphatase activity is co-regulated by both the carbon and the phosphate status of the medium (Butler et al., 199 1). Organic acid-based buffers would introduce additional utilizable carbon, while the widely-used nonmetabolizable buffers (Good et al., 1966) are uneconomic for large-scale use.

84


(4 300 ‘50

F

0.X 1l.h

01 Time

(h)

Fig. 2. Growth, and phosphatase activity of Citrobacter sp. N14 in medium B in culture volumes of 550 ml (A) and 600 1 (B). (a): Biomass growth. (0): Culture pH. (a): Dissolved oxygen concentration. (A): Phosphatase specific activity.

The mechanism of loss of activity of the phosphatase is unknown. A simple cessation of production (down-regulation) and dilution-out is discounted, since loss of enzyme activity was more rapid than biomass growth. It is possible that the enzyme was unstable at low pH (although substantial function at pH was retained: Tolley et al., 1995) or was post-translationally modified as a control mechanism. The existence of two distinct, but very similar, isoenzymes (the relative proportions of which varied according to the stage of growth) is documented (Jeong, 1992), but the degree of interconversion between them, if any, is not known. It was possible to chill and harvest small cultures rapidly, but an attempt to chill the large-scale culture rapidly, (before loss of phosphatase activity) by cold water circulation was unsuccessful. Rapid centrifugation using an on-line continuous separator (Sharples tubularbowl centrifugal separator) was not possible; this required several hours and gave a final biomass of low phosphatase specific activity (Fig. 2b).


Citrobactersp.

85

Development of an improved culture medium for biomass growth

A phosphatase constitutive variant of Citrobacter sp. strain N14, isolated by C.J. Hewitt (unpublished), was used in further studies. This strain produced phosphatase in the presence of inorganic phosphate and enabled substitution of ammonium phosphate as the nitrogen and phosphate source (medium C: Table 1). As well as providing a cheap and readily available nutrient source, this represented an improvement in the medium for investigation of the effect of the carbon source on phosphatase production. Cleavage of glycerol 2-phosphate as the phosphate source for growth would, in addition to providing phosphate, have co-liberated approx. 2 mM glycerol as a supplementary, readily-utilized carbon source: removal of this enabled meaningful evaluation of other carbon sources as growth substrates. Although the Citrobacter sp. showed growth on complex polysaccharides (starch and cellulose) with conservation of phosphatase activity (C.J. Hewitt, unpublished), the growth rate was too slow to be of realistic use for industrial biomass production. Maltodextrins, although a potentially useful alternative that supported good growth and a high level of phosphatase activity, would require debranching of starch for production, and attention was confined to carbon sources that are readily available without need for pre-treatment. Initial tests using lactose as a substitute (medium D: Table 1) showed that the growth and oxygen consumption profiles were similar to the results seen in medium B, except that growth on lactose followed a short lag; this extended the total culture time from 10 to 14 h. Lactose uptake, or its hydrolysis by the activity of P-galactosidase, was possibly rate-limiting (although this was not tested); the cells may have been physiologically under carbon ‘starvation’ by limitation of the intracellular monosaccharide supply. Growth was accompanied by phosphatase production to a level of 130~1400 units, which was maintained in the stationary phase (Fig. 3a). Notably, the pH did not fall below 6.5. Scale-up was done in medium E; here the increased concentration of lactose (Table 1) increased the final OD6e0 substantially, but at the expense of phosphatase activity, which peaked at approx. 1100 units and fell concomitantly with a fall in the medium pH to below 5.8 (Fig. 3b; cf. the glycerol-cultures, Fig. 2). A compromise between high biomass yield and retention of high phosphatase activity was sought. Use of a fed-batch system for continuous addition of lactose to growing cultures

The experiment of Fig. 3(b) was repeated in 5 1 batch cultures, which gave a similar conclusion (Fig. 4). Data from two batches are shown. The final


86

7.4 ---A

Phosphatase

~ I.4 - I.2

4

6

X

IO

4

6

8

I(1

I2

I4

I6

I?

I4

I6

().(I

7.0 6.X

6.2

5.X

Time (h)

activity of Citrobacter sp. N14 in medium D (A) and E (B) in culture volumes of 550 ml (A) and 600 1 (B). (0): B’lomass growth. (0): Culture pH. (A): Dissolved oxygen concentration. (A): Phosphatase specific activity.

Fig. 3. Growth and phosphatase

ODem was approx. 6 units, while the enzyme activity was only 700 units at the peak value, stabilizing at approx. 400 units in the stationary phase (Fig. 4). The use of fed-batch culture was evaluated. Here, the exit concentration of carbon dioxide was monitored, and as this fell (corresponding to a drop in the respiratory activity of the cells) the residual lactose was added gradually to the culture. A preliminary trial utilized 50% of the lactose initially, followed by addition of the residual 50% (medium F). The final OD6a0 was in excess of 5 units, with a final phosphatase activity of approx 1200 units (Fig. 5b). A second trial was done in medium G; here 25% of the lactose was added initially and the residual 75% was fed-in at the point arrowed in Fig. 5a (at 7-7.5 h approx.). The final biomass yield was similar to that of the previous experiment, but the final phosphatase specific activity was increased to approx. 1400 units (Fig. 5b). A third fed-batch culture in medium H (Table 1) gave similar results.


Time

sp.

87

(h)

Fig. 4. ILegend as Fig. 3. The medium was medium E, the culture volume was 5 1. Data are shown for two cultures, with (culture 1: tilled symbols, subscript 1) and without (culture 2: open symbols, subscript 2) pH control via an autotitrator. (e), (0): Biomass growth. (A), (a): Phosphatase specific activity. The data are plotted on separate scales to allow superimposition of the phosphatase specific activity data.

Use of the harvested biomass for uranyl removal at pH 4.5 in batch and flow-through contactors The preliminary studies (above) used 1 mM uranyl ion at pH 7-O in a carrier of citrate (ratio of uranyl to citrate was 1:2) in order to suppress hydroxylation and maintain the many1 ion in a constant speciation, as the uranyl citrate complex (Yong & Macaskie, 1994). Free uranyl ion, UO2 *+, is stable in acid solution, and for treatment of acidic solutions no citrate was required. For comparison with actual mine run-off waters (see Roig et ai., 1995), the cells were resuspended in a solution of citrate-free uranyl ion in a batch contactor, The UOz*+ was completely removed after 20 min, with activity sustained for a further 90 min before breakthrough under continuous-flow conditions (Fig. 6). In conlirmation of metal removal, lactose-grown cells were immobilized on suitable carrier matrices and challenged with simulated ENUSA wastewater in a flow-through column. Initially the uranyl ion was removed; the column activity fell, but subsequently recovered, to remove uranyl ion with more than 90% efficiency, concomitantly with the deposition of yellow precipitate within the columns (Hewitt & Paterson, unpublished). This is reported in detail in the companion paper (Roig et al., 1995). The reason for the apparent inhibition and recovery is not confirmed, but may be interpreted in terms of inhibition of the enzyme by free U02 *+ (Tolley, 1993; Yong & Macaskie, 1994). In the absence of rapid precipitation of uranyl phosphate at low pH (see earlier) substantial

88


!

Time (h)

activity of Citrobacter N14 in medium G (open symbols) and H (filled symbols). (a) Composition of the culture off-gases (culture in medium H). Addition of the lactose supplement was at the point (arrowed) where the concentration of CO2 in the exit gas started to fall. DO2 dissolved oxygen con~ntration (% of saturation); OU: oxygen uptake (% of off-gas depletion), (b) (e), (0): Biomass growth. (A), (a): Phosphatase specific activity.

Fig. 5. Growth, and phosphatase

enzyme activity is lost. Following nucleation, and consolidation of the uranyl phosphate crystals via the residual enzyme activity, it is proposed that the rate of uranyl phosphate deposition can accelerate as the crystalline area of deposit increases (Macaskie et al., 1995). Thus, although much of the enzyme activity may be lost, once precipitation has been initiated this can proceed maximally even with greatly reduced enzyme activity. This has been confirmed experimentally (Macaskie et al., 199%). Addi-


89

Time (min)

Fig. 6. Removal of many1 ion from solution in a batch contactor at pH 4.5. Biomass grown in fed-batch culture (Fig. 5) was resuspended in uranyl-suppl~ented (to 0.2 mM) solution at pH 4.5, with the residual uranyl ion in the supernatant monitored following removal of the cells by centrifugation. At the point arrowed the pump was switched on to give a flow rate of 20 (e) or 10 (0) ml/min.

tionally, recent studies (Bonthrone & Macaskie, unpublished) using 31P NMR have shown that the nucleation sites are localized on available phosphate groups within the exocellular polymeric material. The localized pH is likely to differ from that at the cell surface, while phosphate leaving the cells would tend to have a buffering effect. Thus continued function at a pH lower than that optimal for enzyme activity might be anticipated,

CONCLUSIONS This study has shown that it is possible to obtain high biomass yields of Citrobacter sp. by growth of the cells in a lactose-based minimal medium which requires only ammonium phosphate, iron and magnesium as additional supplements. This medium is likely to economic for the reproducible large-scale production of biomass for the treatment of metalcontaminated wastewaters. Large-scale growth of the organism has been demonstrated, with conservation of the characteristics seen in small-scale batch cultures. One identified problem of instability of phosphatase production in media of high carbon content was overcome by the use of fed-batch cultures, where the final phosphatase activity at harvest was high. The use of ammonium phosphate was possible using a phosphatase constitutive variant isolated in this investigation.

90

L.. E. Macaskie et al.

Theoretical predictions of the cellular phosphatase activity, and the biomass yield required for the treatment of uranyl-contaminated wastewaters at pH 4.5, were calculated from available data, obtained from this and previous studies. Use of fed-batch culture gave the required final biomass concentration, with a phosphatase activity of approx. 50% of that predicted necessary for wastewater decontamination under acidic conditions. A doubling of the pre-growth culture volume to increase the biomass yield would be sufficient to compensate. Other studies in test systems using polyacrylamide gel as the immobilizing matrix (Butler et al., 1991) or using biofilms immobilized on carrier supports (polyurethane reticulated foams and porous glass raschig rings - Macaskie et al., 1995) have shown that the increase in activity obtained by doubling the biomass content of the immobilized cell bioreactor obeys the relations~p predicted by the integrated form of the Mi~haelis-Menten equation, which defines bioreactor efficiency in terms of the biomass loading, input substrate concentration and flow rate through the column (Fulbrook, 1983): I!!&& = F[SoX+ Km. In (l/(1 -x))], where iu, and K, are kinetic constants of the enzyme as operating within the immobilized cells and are calculable by a substitution method (Macaskie et al., 1995), I& is the biomass loading of the bioreactor, So is the input substrate concentration and X is the efficiency of conversion of substrate to product (or of removal of the presented metal) within the fiow residence time (min) within the bioreactor at a given flow rate, F (ml/min) (Macaskie, 1990; Tolley et al., 1995; Macaskie et al., 1995). Using this relationship it is possible to predict the increase in biomass and/or decrease in the flow rate required to maintain bioreactor activity at a desired level. One particular challenge lies in the choice of carrier for the biomass such that all of the nominally available biomass is available for interaction with the perfusing flow. Evaluation of suitable carrier materials is in progress and is reported in a companion study which demonstrates the possible application of this system to bioremediation of an acidic mine wastewater (Roig et al., 1995). Crystallographic factors may play a role in decoupling the absolute phosphatase activity from the efficiency of uranyl phosphate deposition, once biomineralization processes have been initiated.

ACKNOWLEDGEMENTS The authors wish to acknowledge the financial support of the European Community (Third Framework Programme in the field of Environment:


91

grant no EVSV-CT93-0251) in support of this work, and the collaboration of the European partners (Drs J. F. Kennedy and M. Paterson: Birmingham, UK and Drs M. G. Roig, M. Diaz and T. Manzano, Salamanca, Spain). They are also indebted to Dr H. Eccles of British Nuclear Fuels plc (BNFL, UK) and Dr J. Josa of Empresa National de1 Uranio S.A. (ENUSA, Spain) for invaluable discussions and assistance, and the kind provision of wastewaters from ENUSA for analysis and testing in this bioremediation programme. The assistance of the technical staff of the pilot plant in the School of Chemical Enginee~ng, University of Birmingham, is acknowledged with thanks, and also the collaboration of Dr K. M. Bonthrone in the large-scale cultures. Dr Bonthrone was funded by the European Community (Brite-Euram Programme: grant no. BE5350).

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