Bioremediation of 60Co from simulated spent decontamination solutions

Science of the Total Environment 328 (2004) 1–14

Bioremediation of

60

Co from simulated spent decontamination solutions

K. Rashmia, T. Naga Sowjanyaa, P. Maruthi Mohana, V. Balajib, G. Venkateswaranb,* a Department of Biochemistry, Osmania University, Hyderabad 500007, India Applied Chemistry Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

b

Received 30 June 2003; received in revised form 10 November 2003; accepted 23 February 2004

Abstract Bioremediation of 60Co from simulated spent decontamination solutions by utilizing different biomass of (Neurospora crassa, Trichoderma viridae, Mucor recemosus, Rhizopus chinensis, Penicillium citrinum, Aspergillus niger and, Aspergillus flavus) fungi is reported. Various fungal species were screened to evaluate their potential for removing cobalt from very low concentrations (0.03–0.16 mM) in presence of a high background of iron (9.33 mM) and nickel (0.93 mM) complexed with EDTA (10.3 mM). The different fungal isolates employed in this study showed a pickup of cobalt in the range 8–500 ngyg of dry biomass. The wFex ywCox and wNix ywCox ratios in the solutions before and after exposure to the fungi were also determined. At micromolar level the cobalt pickup by many fungi especially the mutants of N. crassa is seen to be proportional to the initial cobalt concentration taken in the solution. However, R. chinensis exhibits a low but iron concentration dependent cobalt pickup. Prior saturating the fungi with excess of iron during their growth showed the presence of selective cobalt pickup sites. The existence of cobalt specific sorption sites is shown by a model experiment with R. chinensis wherein at a constant cobalt concentration (0.034 mM) and varying iron concentrations so as to yield wFeyCoxinitial ratios in solution of 10, 100, 1000 and 287 000 have all yielded a definite Co pickup capacity in the range 8–47 ngyg. The presence of Cr(III)EDTA (3 mM) in solution along with complexed Fe and Ni has not influenced the cobalt removal. The significant feature of this study is that even when cobalt is present in trace level (sub-micromolar) in a matrix of high concentration (millimolar levels) of iron, nickel and chromium, a situation typically encountered in spent decontamination solutions arising from stainless steel based primary systems of nuclear reactors, a number of fungi studied in this work showed a good sensitivity for cobalt pickup. 䊚 2004 Elsevier B.V. All rights reserved. Keywords: Bioremediation; Fungi; Nuclear reactor; Chemical decontamination

1. Introduction

*Corresponding author. Tel.: q91-22-2559-3866; fax: q9122-2550-5151yq91-22-2551-9613. E-mail address: [email protected] (G. Venkateswaran).

The spent decontamination solution from a nuclear power reactor contains dissolved corrosion deposits from the system surfaces along with significant amounts of radioactivity (typically of

0048-9697/04/$ - see front matter 䊚 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2004.02.009

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K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

Co). This spent solution has to be processed before disposal. The liquid after processing is practically free from the radionuclides and the decontamination chemicals added to the system. Hence it is recycled or otherwise diluted and dispersed into large water bodies according to the regulatory requirements. The radioactive solid mass (typically ion-exchange resin and insolubles) are sent for storage in trenchesytileholes (these are typcially 700 mm internal diameter CS pipe lined inside and outside with concrete). Among the various radionuclides, the major concern is of 60Co because of its long half life (5.26 years) and high gamma energies (1.17 and 1.33 MeV). Treatment of contaminated low oxidation state metal ion solution by using the ion exchange process (Bradbury et al., 1986) and by a flocculation process using Al (OH)3 has been reported earlier (Letschert et al., 1987; Segal et al., 1986; Wood, 1986). Conventional polystyrene-based strong acid cation and strong base-anion exchangers and co-precipitation methods are employed for retention of 60Co from the decontamination formulation. Cation exchange treatment results in decontamination factors (DFs) of 8–40 with respect to 60Co if the spent V(II)yV(III) picolinate solution is treated immediately after decontamination but yields lower DFs of 3–4 if it is stored for )24 h under ambient conditions (Gokhale et al., 1994). However, due to the limited ion-exchange capacity of ion-exchange resin, a large volume of the resin (solid waste) is generated for disposal after the treatment of spent decontamination formulation. As described earlier, such a disposal requires a number of ion-exchange columns and subsequently tile-holes for the safe burial of the resin bearing columns. This requirement increases the cost of such waste treatment. Bacterial remediation of solid resin is also reported (Tusa, 1992). The bacteria feed on the synthetic organic ion-exchanger. However, typical feed ratios in such cases are ;1–2 kgyday in model plants and this requires long remediation periods. Microbial biomass has a high affinity for the actinide elements, heavy metals and also radionuclides, as observed both in laboratory studies and in natural environments (Gadd and White, 1989). Microorganisms generally take up the metal ions

by two different mechanisms (a) biosorption; and (b) bioaccumulation. While the former is a metabolically independent physical adsorption process at cell surface, the latter includes an energy dependent process involving intracellular accumulation. The wild type Neurospora crassa was found to remove cobalt from solutions having a cobalt content of 10 mgyl. The cobalt resistant mutant of N. crassa was shown to remove more than 90% of cobalt even from solution having Co concentrations as high as 500 mgyl. In this case, the contribution of biosorption was shown to be approximately 40% while the remaining 60% was by bioaccumulation (Karna et al., 1996). Nonliving biomass of the common seaweed Ascophyllum nodosum capable of accumulating cobalt from aqueous solutions to the extent of 160 mgyg was reported earlier (Kuyucak and Volesky, 1989). Accumulation of Uranium by live cultures of Sacharomyces cerevesiae and Pseudomonas aeroginosa was also demonstrated. Cell bound uranium concentration reached up to 10–15% dry cell weight in these cases (Strandberg et al., 1981). Microbial bioremediation of radioactive waste is increasingly considered as a potential alternative to the conventional organic ion-exchanger based treatment. A radiation resistant bacterium Deinococcus radiodurans was engineered for mercury remediation from mixed radioactive waste environments (Hassan and Daly, 2000). Micrococcus luteus and Pseudomonas putidae were used for mobilization 137Cs and 85Sr in contaminated soils (Navaric et al., 1997). Our interest in bio-remediation of metals from spent decontamination solution stems from a desire to minimize the waste volume generated during decontamination. The conventional synthetic organic ion-exchange process based radioactive waste treatment is only an interim measure. Though radioactive liquid volume reductions are achieved by trapping the metals in the ion-exchanger, still the minimization of waste volume could not be realized. Minimization will result if we can recover the sorbed corrosion product metals (a few tens of kilogram) from the ion-exchanger and free the resin. Then treatment of such a small amount of waste becomes advantageous in many respects. Alternatively, solution bio-remediation can be tried to achieve the above

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

objective of waste minimization. If suitable fungal biomass capable of hyper-accumulating Co in presence of excess Fe, Ni and Cr is employed, then one recovers the metals sorbed by these microbes by the process of incineration and minimize the decontamination waste volume. The present study is directed towards evaluating a number of fungal biomass for their cobalt uptake under a variety of conditions and zero-in on an hyperaccumulator under the simulated conditions of solution metal loading encountered during chemical decontamination. Hence bio-remediation of 60Co from spent chemical decontamination solution using fungal biomass appears to be worth investigating as little information exists in this area. 2. Materials and methods 2.1. Fungal strains The fungal cultures (Trichoderma viridae, Mucor recemosus, Aspergillus niger, Aspergillus flavus, Rhizopus chinensis and Penicillium citrinum) were obtained from the mycology and plant pathology section, Department of Botany, Osmania University, Hyd. N. crassa wild type (噛4200 a) was obtained from Fungal genetics stock center, Kansas city, USA. Cobalt-sensitive mutants (CSMI and CSM-II) were isolated in the lab (to be published elsewhere) and cobalt-resistant mutant was earlier characterized strain (Sajani and Maruthi Mohan, 1997). 2.2. Media and growth conditions The fungal isolates were cultured in 20 ml basal medium with Ammonium-N source in 100 ml conical flasks for 72 h at 27 8C. Both stationary and shaking conditions (150 rev.ymin) were employed separately in a shaker-incubator (Sajani and Maruthi Mohan, 1997). 2.3. Growth in the presence of excess Fe(III)EDTA The fungi were cultured in 20 ml basal medium with Ammonium-N source containing 0, 1, 3, and 6 mM Fe(III)EDTA to saturate the fungi with iron during growth.

3

2.4. Removal of Cobalt The mycelial biomass obtained was washed extensively with tap water followed by distilled demineralized water and pressed free of excessive moisture under the folds of the filter paper, weighed and suspended in 19.2 ml aqueous solution containing 0.03–0.16 mM cobalt traced with 60 Co, iron f9.33 and 0.9 mM of nickel complexed with EDTA (pH 5.1) and incubated for 44 h. The dry fungal biomass weights employed in the studies were in the range 0.1–0.4 g. This variation in the weights of the fungi employed was because in the 72 h culture period employed in the present studies the yields of the mutant varieties such as CSM-I, CSM-II and cor were less than that of the normal fungi. The incubation period of 44 h was based on the fact that in trial experiments with inactive cobalt in solution at ppm levels, the metal uptake by the fungal microorganism reached a saturation in 24 h equilibration time under rotary shaking as well as stationary conditions. With radioactive cobalt wherein total cobalt is present in a few parts per billion chemical concentration, a period of 44 h was uniformly followed in all the experiments with different biomass to ensure that metal pickup reached a saturation condition. After equilibration, the suspension was filtered through a 0.45 mm membrane filter and the filtrate was counted for radioactivity. The cobalt remaining after the exposure with fungi was determined by an integral g-counter coupled to a 30=30 NaI Tl detector after filtering the solutions through a 0.45 m membrane filter. Thus, in this work the initial radioactivity of solution before introducing the fungal microorganism and the solution radioactivity after exposure to the fungi and filtration were used to determine the Cobalt uptake by the fungi. A radioactivity balance was done in a few cases between the initial amount of 60Co activity before the introduction of fungi, the activity in solution after treatment with fungi and the activity in the residue left in the filter paper. A recovery of radioactivity )95% could be ascertained. 2.5. Effect of weight of biomass employed In order to know the effect of employing different weights of biomass (in the same volume of

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K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

solution) on the pickup capacity (expressed as nanograms of Coygram of biomass) and arrive at an optimum solution volumeymass of biomass, a case evaluation was done with R. chinensis. Different weights namely 0.3, 0.5, 0.8 and 1.0 g of this fungi were suspended in a constant volume of simulated solution (19.2 ml) containing 0.034 mM of Cobalt traced with 60Co and the pickup capacities in nanogram per gram were determined as described earlier. 2.6. Effect of varying iron concentrations on Co pickup The Co pickup capacity was additionally determined when the initial concentration of Fe(III)EDTA in solution was varied from 0.34 mM to 9.6 mM. This was done to know the variation of Co pickup capacity over a wide initial wFeyCox ratios ranging from 10 to 2.87=105. R. chinensis was employed as a model fungi for this evaluation. 2.7. Effect of the presence of Cr(III)EDTA along with Fe(III)EDTA and Ni(II)EDTA In order to evaluate the influence of the presence of Cr in solution as Cr(III)EDTA along with Fe(III)EDTA and Ni(II)EDTA, an experiment was carried out under stationary conditions with R. chinensis exposed to a medium containing 9.46 mM Fe(III)EDTA, 0.86 mM NI(II)EDTA, 3 mM Cr(III)EDTA along with 0.03 mM Co(II)EDTA. 2.8. Estimation of iron after exposure to fungi: This was carried out by the visible spectrophotometric method employing o-phenanthroline as complexing agent. A volume of 0.5 ml of the sample was treated with 0.5 ml of conc. HCl, followed by the addition of 5 ml of 10% Hydroxylamine Hydrochloric acid, 1.5 ml of 1:1 Ammonia solution, 2.5 ml of sodium acetate, pH 4 buffer, 0.25% of 1,10-phenanthroline. The solutions were made up to 50 ml with distilled deionized water. The optical density at 510 nm was measured against a reagent blank using a spectrophotometer.

2.9. Estimation of nickel after exposure to fungi This was carried out by the visible spectrophotometric method employing DMG (dimethylgloxime) as complexing agent in the presence of iron (1:10 wNix:wFex). Before complexation with DMG, the samples were subjected to acid digestion with conc HNO3 and evaporating to dryness and repeating this cycle twice so as to completely oxidize all the EDTA. Iron interference by way of increased absorbance was observed. This absorbance was corrected for actual concentration of nickel. Some of the samples were crosschecked with standard method of nickel estimation in Niq Fe mixture by using the extraction technique (Fe– Ni separation method). Note: All experiments were done in duplicate and average values of metal pickup are shown. This is essentially because in the present study the interest is to test a number of fungi both naturaly mutant types for their sensitivity to cobalt pickup in presence of a large background concentration of iron and identify the ones with a high capacity. It is to be noted that the two replicates showed within "13% deviation from the mean. 3. Results and discussion Tables 1 and 2 show the cobalt and iron pick up capacities by the different fungi as well as wFey Cox, wNiyCox ratios in solution before and after exposure under stationary and shaking conditions (150 rev.ymin). Pickup capacity is defined as micrograms of cobalt taken up per gram of dry biomass. The pickup as described earlier is evaluated from the initial radioactivity of 60Co in solution and residual radioactivity of 60Co solution after exposure to the fungal microorganism followed by filtration through a 0.45 mm membrane filter. The iron and nickel capacities were evaluated by estimating these metals in solution by Visibile spectrophotometry before and after exposure. At an initial FeyCo solution concentration ratio of f6=104 (Table 1) T. viridae, M. recemosus, A. flavus, mutants of N. crassa (CSM-I, CSM-II and cor) and Penicillium showed cobalt pick up capacity between 51 and 331 ngyg under stationary condition and between 62 and 246 ngyg under

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

5

Table 1 Cobalt, iron and nickel pickup capacities of certain fungi in solution with an initial wFeyCox ratio of f6=104 Type of fungi

Fe (mM)

Co (mM)

wFeyCoxini y wNiyCoxini*

wFeyCoxRem y wNiyCoxRema

Fe pick up capacity (mgyg)

Co pick up capacity (ngyg)

Ni pick up capacity (mgyg)

Stationary condition CSM-I

9.45

0.160

295.3

0.24

9.45

0.160

13.6

331

0.02

cor

9.45

0.160

P. citrinum

9.45

0.160

T. viridae

9.25

0.157

M. recemosus

9.25

0.157

A. flavus

9.25

0.157

58781y 5273 62746y 5411 58434y 4667 54299y 4762 65287y 5280 57849y 5417 56900y 4173

17.3

CSM-II

59 312y 4375 59 312y 4375 59 312y 4375 59 312y 4375 58 981y 4140 58 981y 4140 58 981y 4140

9.45

0.160

CSM-II

9.45

0.160

cor

9.45

0.160

P. citrinum

9.45

0.160

T. viridae

9.25

0.157

M. recemosus

9.25

0.157

A. flavus

9.25

0.157

59 312y 4375 59 312y 4375 59 312y 4375 59 312y 4375 58 981y 4140 58 981y 4140 58 981y 4140

58199y 4745 59460y 5173 57563y 5091 64676y 5877 56419y – 54818y – 56859y –

Shaking condition CSM-I

* a

6.5

95.4

nil

13.6

126.4

nil

3.6

83.2

0.14

3.7

61.6

0.02

3.2

50.8

0.2

4.6

66.2

0.05

9.1

167.6

0.19

8

122.2

0.03

246

0.16

10.8 4.6

70.5

–

4.6

64

–

4.1

62.4

–

Initial value. Ratio remaining at the end of run.

shaking condition. Under these conditions (stationary and shaking) iron removal capacity was observed to be between 3 and 17 mgyg. The cobalt pickup capacities of CSM-I and CSM-II have shown a 75% and 50% decrease respectively, under shaking condition as compared to the capacities observed under stationary condition. This indicates a possible less binding of cobalt to the cell walls of these fungi. The cobalt and iron pickup capacities of T. viridae, M. recemosus, A. flavus and cor have shown only marginal change (with respect to "13% deviation from the mean obtained from

duplicate determinations) between stationary and shaking conditions. Comparing the stationary and shaking conditions with respect to iron pickup capacity of CSMI and CSM-II, it is seen that CSM-I showed a decrease similar in magnitude to the decrease of its cobalt pickup capacity while CSM-II showed a much less decrease. As compared to its cobalt pickup capacity under stationary condition, Penicillium has shown a two-fold increase in its cobalt pickup under shaking condition, which indicates the influence of increased area of contact in bind-

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

6

Table 2 Cobalt, iron and nickel pickup capacities of certain fungi in solution with an initial wFeyCox ratio of f2.87=105 Type of fungi

Fe (mM)

Stationary condition CSM-I 9.46

Co (mM)

wFeyCoxini y wNiyCoxini*

wFeyCoxRem y wNiyCoxRema

Fe pick up capacity (mgyg)

Co pick up capacity (ngyg)

Ni pick up capacity (mgyg)

0.033

287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140

281 544y 26 174 212 621y 24 272 231 169y 27 273 243 849y 27 129 240 729y 26 140 248 629y 26 791 282 675y 26 140

14.3

44

1.13

38.7

28.6

1.55

31.4

29

0.28

23.2

16.9

nil

20.5

nil

nil

19.8

11.5

nil

2.1

nil

nil

287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140 287 538y 26 140

255 973y 29 351 235 709y 26 014 251 222y 24 437 225 251y 33 205 255 756y 27 652 228 939y 27 652 251 033y 26 140

9.9

19.4

nil

12.7

17.9

0.48

8.4

9.8

0.54

18.5

37.4

nil

7.7

9.6

nil

9.6

nil

CSM-II

9.46

0.033

cor

9.46

0.033

P. citrinum

9.46

0.033

T. viridae

9.46

0.033

R. chinensis

9.46

0.033

N. crassa (wild) Shaking condition CSM-I

9.46

0.033

9.46

0.033

CSM-II

9.46

0.033

cor

9.46

0.033

P. citrinum

9.46

0.033

T. viridae

9.46

0.033

R. chinensis

9.46

0.033

N. crassa (wild)

9.46

0.033

* a

12 6.1

nil

nil

Initial value. Ratio remaining at the end of run.

ing the cobalt to more available sites. The iron pickup capacity of Penicillium, however, has not shown any significant change under these two conditions. When the wFex y wCox ratio in the solution was increased to 2.85=105 (by a factor of 4.8) (Table 2), there was a drastic reduction, i.e. by a factor of 3–8 in the cobalt pick up capacities (f20–40 ngyg) of the mutant strains of N. crassa (CSM-I, CSM-II and cor) and penicillium both under stationary and shaking conditions. Thus, the Co pickup of these fungi appears to be proportional to the initial concentration of Cobalt. It is to be noted that when the wFeyCox initial concentration

ratio was increased from 6=104 to 2.87=105, the Fe concentration was kept at about the same level and only the cobalt concentration was decreased by a factor of 4.8. Thus with a decrease in initial wCox there was a concomitant decrease in cobalt pick up capacity. It is significant that even at a low cobalt concentration (0.033 mM), the cobalt uptake is observed. The cobalt pick up of CSM-I, CSM-II and cor showed a decrease by a factor in the range 1.5–3 under shaking condition as compared to the capacity observed under stationary condition. As mentioned earlier this may be due to the loose binding of the cobalt to the cell wall (bio-sorption). T. viridae at high wFeyCoxini ratio

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

7

Table 3 Effect of growth of fungi in Fe(III)EDTA bearing basal medium on the cobalt and iron pickup capacities under stationary conditions in solutions with initial FeyCo ratio of f6.5=104 Conc. of Fe in growth medium (mM) CSM-I 0 3 6 CSM-II 0 3 6 R. chinensis 0 3 6 * a

Fe (mM)

Co (mM)

wFeyCoxini*

wFeyCoxRema

Fe pick up capacity (mgyg)

Co pick up capacity (ngyg)

10.28 10.28 10.28

0.158 0.158 0.158

65 063 65 063 65 063

67 686 73 809 75 846

22.3 10.5 4.5

414.5 359 315.3

10.28 10.28 10.28

0.158 0.158 0.158

65 063 65 063 65 063

73 171 68 811 68 966

22.9 7.9 4

490.6 279.4 240.2

10.28 10.28 10.28

0.158 0.158 0.158

65 063 65 063 65 063

38 904 21 986 66 511

9.9 6.9 0.8

28 23.2 20.9

Initial value. Ratio remaining at the end of run.

(low cobalt concentration) does not exhibit any capacity for cobalt under stationary conditions while under shaking condition it exhibits a low capacity of 10 ngyg. In P. citrinum the cobalt pick up was increased by a factor of two under shaking conditions. CSM-I, CSM-II and P. citrinum were relatively exhibiting better cobalt pick up capacity than the other fungi. CSM-II, cor and P. citrinum have higher iron capacity under higher wFeyCoxini level in solution both under stationary and shaking conditions as compared to their capacities observed under lower wFeyCoxini in solution. In the case of CSM-I the iron capacity has not changed much under stationary conditions at low and high initial wFeyCox concentration ratios (the reproducibility level in duplicate runs observed in these experiments is "13% from mean. Hence if one puts an error (deviation) bar around 17.3 it will read as 17.3"2.2 (i.e. covering the range 15.1–19.5). Similarly the value 14.3 with the error bar will become 14.3"1.9 and can cover the range 12.4– 16.2. The upper error band of the lower value of 14.3 and the lower error band of the upper value of 17.3 are seen to merge. This is why we state that the iron pickup capacity has not changed much) while under shaking conditions it has

shown a higher Fe pickup (9.9 mgyg) at higher wFeyCoxini than that (4.6 mgyg) at lower ratio. Generally, shaking conditions have yielded lower iron pickup at both the wFeyCox concentration ratios for CSM-I, CSM-II mutants (9.9, 12.7 under shaking condition Vs 14.3 and 38.7 mgyg under stationary condition at high ratio; 4.6, 9.1 under shaking condition Vs 17.3 and 13.6 mgyg under stationary condition at low ratio) and in P. citrinum. (13.6–10.8 mgyg and 23.2–18.5 mgygchange in Fe pickup observed on changing stationary to shaking condition (Tables 1 and 2)). Table 3 shows the effect of pre-saturating the iron pickup capacity of the fungi by growing them in the basal medium containing Fe(III)EDTA. This was done to see whether their cobalt pickup capacity would improve and whether the specificity of cobalt pickup sites would be preserved. The results in Table 3 indicate that when the w{Fe} y {Co}xini ratio in solution was f6.5=104, there was a decrease of approximately 24–25% in the cobalt pickup capacity of CSM-I and R. chinensis when they were saturated with 6 mM Fe(III)EDTA during their growth. In the case of CSM-II, however, there was f50% decrease. However, when the wFeyCoxini ratio was f2.5=105 (Table 4), presaturation with 6 mM Fe bearing solution had

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

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Table 4 Effect of growth of fungi in Fe(III)EDTA bearing basal medium on the cobalt and iron pickup capacities under stationary conditions in solutions with initial FeyCo ratio of f2.5=105 Conc. of Fe in growth medium (mM) CSM-I 0 3 6 CSM-II 0 3 6 cor 0 3 6 R. chinensis 0 3 6 P. citrinum 0 3 6 * a

Fe (mM)

Co (mM)

wFeyCoxini*

wFeyCoxRema

Fe pick up capacity (mgyg)

Co pick up capacity (ngyg)

8.3 8.3 8.3

0.033 0.033 0.033

251 515 251 515 251 515

278 333 281 200 292 000

19.5 3.6 9.6

82.7 81.8 149.6

8.3 8.3 8.3

0.033 0.033 0.033

251 515 251 515 251 515

266 071 273 462 275 600

Nil 11.2 18.1

50.1 51.6 94.1

8.3 8.3 8.3

0.033 0.033 0.033

251 515 251 515 251 515

278 846 273 846 261 538

8.3 1.8 7.5

47 45.1 43

8.3 8.3 8.3

0.033 0.033 0.033

246 970 246 970 246 970

176 800 221 154 254 074

20 12.9 4.6

47 37.4 33.6

8.3 8.3 8.3

0.033 0.033 0.033

246 970 246 970 246 970

236 296 252 000 262 759

4.7 1.6 1.4

18.2 9.6 12.1

Initial value. Ratio remaining at the end of run.

increased the cobalt pickup capacity (by 81% and 88%, respectively) of CSM-I and CSM-II while cor showed approximately 9% decrease. In the case of R. chinensis and P. citrinum approximately 30% decrease in cobalt pickup capacity was noticed. While the decrease in cobalt pickup capacities could be explained by some competition effect from Fe for the Co sites, these results nevertheless indicate that even when the fungal microorganisms are grown in presence of millimolar concentration of iron, the nanomolar level of binding of cobalt is still exhibited by the fungi. Such a behavior also implies that there are specific cobalt pickup sites which are active even under an onerous competition from the relatively high concentration of iron in the solution In fact at high wFeyCox ratios, growth in 6 mM Fe(III)EDTA helped in increasing the Co pickup capacities of CSM-I and CSM-II which is advantageous.

With or without iron in growth medium, the cobalt removal capacity of the mutant varieties of N. crassa was found to be proportional to the Co concentration used (0.03 or 0.16 mM). Without iron in growth medium, for example, under stationary condition the mutant varieties of N. crassa namely CSM-I, CSM-II and cor have shown Co pickup capacities of 295.3 ngyg, 331 ngyg and 95 ngyg, respectively, when the wFeyCox initial concentration ratio was 9.45 mMy0.160 mM, i.e. a ratio of 59 312 (Table 1). However, when the initial Co concentration in solution was reduced to 0.033 mM and initial Fe concentration in solution was same, i.e. the wFeyCox ratio was high, 287 538, the Co pickup capacities of these species got lowered to 44 ngyg, 28.6 ngyg and 29 ngyg, respectively (Table 2). Similarly under shaking conditions also, a Co concentration dependent cobalt pickup capacity of these species was

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

observed: CSM-I, CSM-II and cor under shaking condition have shown 66 ngyg, 167.6 ngyg, 122.2 ngyg Co pickup, respectively, when initial Co concentration in solution was 0.160 mM (Table 1) but the capacities fell to 19.4 ngyg, 17.9 ngyg, 9.8 ngyg, respectively, at an initial Co concentration of 0.033 mM (Table 2). With iron in growth medium, at 6mM Fe(III), under stationary condition, CSM-I and II and R. chinensis have shown 315, 240 and 20.9 ngyg Co pickup when the initial cobalt concentration was 0.158 mM and Fe was 10.28 mM with wFeyCox initial concentration ratio being 65 063 (Table 3). Under the same initial Fe and Co concentrations, when CSM-I, CSM-II and R. chinensis were not grown in iron bearing medium, they, respectively, exhibited Co pickup capacities of 414.5 ngyg, 490.6 ngyg and 28 ngyg. Thus, growth in iron medium had resulted in Co pickup capacity reduction by 50% in the case of CSM-II and 25% in cases of CSM-I and R. chinensis. When the initial Co and Fe concentrations were 0.033 mM and 8.3 mM, respectively, and wFeyCox ratio was 251515 (Table 4), CSM-I and CSM-II and R. chinensis grown in 6 mM Fe(III)EDTA growth medium had shown 149.6 ngyg and 94.1 ngyg and 33.6 ngyg Co pickup, respectively, against corresponding capacities of 82.7, 50.1 and 47 ngyg shown by them when they were not grown in Fe medium. Thus, when the initial wFeyCox ratio was 2.51=105 with wCoxs0.033 mM, growth in 6 mM iron containing medium increased the Co pickup (with respect to growth in normal medium) in the case of CSM-I and II by 85% whereas in the case of cor, R. chinensis and P. citrinum (Table 4) the Co pickup capacities got reduced by 9%, 30% and 33%, respectively. These observations have shown that these microorganisms have cobalt specific sites available and are able to sorb cobalt at trace levels in the presence of excess iron concentration. Only in the case of R. chinensis such a concentration dependent pickup of cobalt was not observed. The Co pickup by R. chinensis seems to be somewhat critically dependent on the initial iron concentration. For, e.g. when the initial Fe concentration was 9.46 mM and Co was at 0.033 mM, R. chinensis grown in the absence of Fe in growth medium exhibited a Co pickup of 11.5 ngy

9

g under stationary condition (Table 2). However, at the same Co concentration, when the wFex was 8.3 mM and was not grown in Fe containing medium, it showed a Co pickup of 47 ngyg (Table 4). When the initial Co concentration was 0.158 mM but initial wFex was 10.28 mM and fungi was not grown in iron medium, its Cobalt pickup was only 28 ngyg. Nevertheless it can be stated R. chinensis yields a Co pickup capacity, which is independent of initial Co concentration and yields a higher Co pickup at lower initial iron concentrations. It should be noted here that the cobalt used in the experiments was complexed to EDTA. Since fungi taking up cobalt can recognize only the free ionic form of cobalt, the effective cobalt ion concentration is four orders of magnitude lower (1.84=10y6 mM). Fungi are known to take up metal ions like Zn, Cu and Mg from low concentrations with the help of high affinity transporter (Dances et al., 1994; Komeda et al., 1997; Eitinger and Freidrich, 1991; Mobley et al., 1995; Galagan, 2003; Gadd, 1993). These high affinity systems only perform at low levels of metal ions; however, they do not function in micromolar to millimolar concentrations. Recent studies suggested the presence of two separate transporter systems for zinc in Saccharomyces cerevisiae, one system has high affinity for zinc with an apparent Km of 500 nM (functioning at low concentrations). The high affinity system has induced-in activity )100 in response to zinc limited conditions. The second system for zinc uptake in yeast has a lower affinity for substrate (Kms10 mM) and is active at high concentrations (Zhao and Eide, 1996). Table 5 shows the cobalt and iron pick up capacity when the different amounts of R. chinensis biomass were suspended in the same volume of simulated solution. When 0.3 and 0.5 g of the fungal biomass was taken, the cobalt pick up capacity was in the range 17–19 ngyg. However, with 0.8 and 1 g biomass the cobalt pick up capacity decreased to 13 ngyg. While cobalt removal decreased from 19 to 13 ngyg, iron removal increased 1.1 to 7 mgyg (fseven-fold). This experiment suggests that using more amount of biomass to remediate certain volume of the solution is not advisable. As shown in Table 5 maximum capacities were obtained when the vol-

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

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Table 5 Cobalt and Iron pickup specific capacities with different initial amounts of fungal biomass in the constant volume of solution (study with R. chinensis under stationary conditions) Fresh weight (g)

Fe (mM)

Co (mM)

wFeyCoxIni*

wFeyCoxRema

Fe pick up capacity (mgyg)

Co pick up capacity (ngyg)

0.3 0.5 0.8 1.0

10.28 10.28 10.28 10.28

0.034 0.034 0.034 0.034

302 353 302 353 302 353 302 353

498 000 298 333 200 000 187 000

1.14 2.85 5.73 7.01

19.2 17.04 12.96 13.05

* a

Initial value. Ratio remaining at the end of run.

ume of the simulated solution to mass of bio-mass ratio G30 cm3 yg is advantageous and lower volume to mass ratios are not beneficial. Table 6 shows the results of cobalt pickup by R. chinensis by keeping a constant initial Co concentration and varying the iron concentration so as to get initial wFeyCox concentration ratios of 10, 100, 1000, 246 970 and 287 538. The Co pickup of R. chinensis shows a dependence on initial concentration of iron taken in a rather complex manner. The fact that in presence of high concentrations of iron (orders of magnitude higher as compared to the Co concentration considered) the fungal microorganism shows capacity to Co pickup points to the presence of specific Co sorption sites in the fungi. However, this Co pickup capacity seems to be influenced by the presence of iron. At a constant initial Co concentration of 0.034 mM and when the initial iron concentration was increased from 3.4 mm to 8.3 mM, the Co pick up increased from 8.3 to 47 ngyg. This observation shows in the medium concentration range (micromolar to a few millimolar) solution

iron instead of competing for the sites of Co, seems to aid the sorption of Co with the result that increasing concentrations of iron within this range leads to higher Co sorption. However, at 9.5 mM Fe concentration, the Co pickup drastically decreased to 11.5 ngyg. Hence there appears to be a critical iron concentration below which increasing iron concentration increases the Co pickup. However, at very low iron concentration (0.34 mM), a higher Co pickup (21.3 ngyg) is observed which is nearly the Co sorption value observed in presence of 34 mM iron. These observations from the data in Table 6 point out clearly that there are highly specific sites for Co sorption and in the case of R. chinensis the solution iron also influences the Co uptake, i.e. decreases the cobalt uptake in presence of increasing iron in the very low concentration range (fraction of a micromolar), increases the Co uptake with increasing iron in the medium concentration range (a few micromolar to a few millimolar) and again decreases the Co uptake at very high solution concentrations of iron (say 9.5 mM).

Table 6 Cobalt pickup by R. chinensis under stationary conditions from solutions with different initial wFeyCox ratios but with constant cobalt concentration Initial concentration of Fe

Initial concentration of Co (mM)

wFeyCoxini*

Co pickup capacity (ngyg)

0.34 mM 3.4 mM 34 mM 8.3 mM 9.46 mM

0.034 0.034 0.034 0.033 0.033

10 100 1000 246 970 287 538

21.3 8.3 20.6 47 11.5

*

Initial value.

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14 Table 7 Influence of the presence of Cr(III)EDTA along with Fe(III)EDTA and Ni(II)EDTA on cobalt pickup by R. chinensis under stationary conditions Conc. of. Cr (mM)

Concentration of Co (mM)

Co pick up capacity (ngyg)

0 3

0.03 0.03

17.04 14.59

The fungi studied in this work has shown a poor Ni uptake as compared to their iron uptake even after allowing for the fact initial Ni concentration in solutions employed was an order of magnitude lower to the Fe solution concentration. At an initial concentration of 0.9 mM which is uniformly employed in these studies, the maximum Ni pickup observed was 1.13 mgyg and 1.55 mgyg for CSMI and CSM-II, respectively; all other fungi have shown nil or extremely low Ni pickup (Tables 1 and 2). This shows the fungi prefer iron over nickel. Table 7 shows the influence of chromium on the cobalt pick up by Rhizopus. The results indicate chromium does not influence the cobalt pick up capacity of Rhizopus. 3.1. Relative performance of different biomass types Metal transport and homeostasis in microorganisms is beginning to be understood with the recent advances in complete genome sequencing of a large number of microorganisms (Radisky and Kaplan, 1999). Metal specific transporters have been identified for Zn and Cu in S. cerevisiae (Zhao and Eide, 1996; Dances et al., 1994), Co in Rhodococcus rhodochrous (Komeda et al., 1997), for Ni in Alcaligenes eutrophous (Eitinger and Freidrich, 1991) and Helicobacter pylori (Mobley et al., 1995). Neurospora genome sequence has been reported recently (Galagan, 2003) and we have identified a number of putative metal transporter genes based on comparison with established metal transporter genes from bacteria and yeast. The cobalt-sensitive mutants of N. crassa (CSM-I and CSM-II) used in the present

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study were shown to be hyperaccumulators. In these mutants the cell wall binding capacity for cobalt was decreased when compared to wild type N. crassa (unpublished data). Cell wall is known to filter toxic metal ions from entering the cell and hence is a protective barrier (Gadd, 1993) and when this function is decreased due to mutation, could lead to accumulation of cobalt ions. Thus, apart from metal transporters located on plasma membrane, cell wall also plays an important role in metal transport. Most of the cobalt removed in the present study is by transport into cells rather than passive binding to cell wall fraction and hence longer incubation times are required. Based on the above knowledge it is reasonable to interpret the good performance of cobalt removal by some fungi to be due to the presence of high affinity transporters, while their absence in others results in lower removal capacity. The physiology of the fungus in general is known to be different under stationary and shaker culture conditions. It is a well-known fact that fungi in general grow under stationary conditions and hence a better performance was observed in the present study. 3.2. A comparison of conventional techniques applied in the treatment of spent decontamination solutions Spent chemical solutions from decontamination are radioactive and typically contain metal complexes with or without some buffering chemicals. In a typical stainless steel based primary system decontamination of a boiling water reactor, the dissolved corrosion products can be of 5–6 mM concentration. The main radioactivity is typically 60 Co and the chemical concentration of Co (Co59qCo-60) can be in the range 1–2 ppb (0.017– 0.034 mM). The liquid waste is normally treated using synthetic organic ion-exchange resins for reduction in waste volume and its solidification. Both radioactive and non-radioactive metals as well as the added decontamination chemicals are removed and the system water chemistry is returned to its pre-decontamination status. A liter of resin typically can treat approximately 10–30 l of spent decontamination solution when a multi-

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cycle mode of decontamination involving a oxidative pre-treatment stage followed by a dissolution stage is employed (Venkateswaran et al., 2003). Since the concentrations of chemicals employed can vary from very dilute to less dilute so that uncertainties in the estimation of the amount of oxide are taken care of, a range of solution volume between 10 and 30 l for a liter of the resin is involved. Letschert et al. (Letschert et al., 1987) have carried out an experimental flocculation process for treatment of vanadous based low-oxidation state metal ion (LOMI) waste wherein the pH of the solution was adjusted to 9–10 and an aluminium hydroxide wAl(OH)3) floc was produced with alum KAl(SO4)2.12H2O. One kilogram of alum was used for treating 1000 l of LOMI waste solution yielding 25 l sludge. In the precipitation method investigated for treating the LOMI waste on a laboratory scale, Gokhale et al. (Gokhale et al., 1994) have estimated that for treating 1000 l of 4 mM spent vanadous LOMI, approximately 0.6 kg of KMnO4 is required producing approximately 0.7 kg of solid comprising of MnO2 and V2O5. Such a co-precipitation and subsequent filtration through a sub-micron (0.5 mm) filter paper yielded 96% removal of cobalt from solution. Recently, resin decomposition using specially developed strains of microbes is reported (Tusa, 1992). The resins used for the treatment of spent decontamination solution are subsequently decomposed using resin-eating microbes. A typical resin decomposition rate achieved is 3 kgyd on a pilot plant scale in a 1.5 m3 sized container. The solution left after the resin decomposition will be radioactive containing the corrosion product metals and the decontamination chemicals. A few hundred kilograms of the solid radioactive waste is expected upon concentration of such a solution. In the present study the mutated varieties of N. crassa designated as CSM-I and CSM-II had yielded a cobalt pickup capacity of 44 ngyg and 29 ngyg of dry biomass, respectively. CSM-I and CSM-II had shown iron pickup capacities of 14.3 mgyg and 38.7 mgyg, respectively. These capacities were obtained when wFex y wCox initial concentration ratio in solution was kept at f2.8=105, a

ratio which closely simulates that in the spent decontamination solution from a BWR system. Using these capacity values, in order to bioremediate a liter of spent decontamination solution containing 0.017 mM Coq6 mM Fe, it is estimated that approximately 35 g dry biomass will be required. Hence 1 kg of biomass will bio-remediate f29 l of decontamination solution when employed in proper volume of solution treatedymass of biomass as found in this work. This figure is comparable to that obtained in the case of treatment with synthetic organic ion-exchangers. However, it has to be noted that in bio-remediation we only bio-remediate the metals. The solution after filtration to remove the biomass will be practically free of metals (both radioactive and non-radioactive) but will contain the decontamination chemicals as dissolved solids. One can recover these chemicals by different methods like evaporation in solar ponds or in an evaporator. The waste volume of bio-mass can be incinerated and all the metals can be recovered thus reaching the minimum waste volume. This method thus has an advantage over resin-decomposition methods discussed earlier in that chemicals and metals and the water are separated. The chemicals and water will be nonradioactive while the metals will be radioactive thus achieving an ideal minimum radioactive waste volume. The solution bio-remediation method is also kinetically more attractive than solid resin decomposition by microbes. 4. Conclusions The bio-remediation of 60Co simulated spent decontamination solutions by using different fungi reported in this work revealed that 1. The mutant varieties of N. crassa have exhibited the highest Co and Fe pickup capacities among the different fungii employed. 2. The Co pickup capacities exhibited by these mutant fungi depends on the metal ion concentration of cobalt in solution. The organism adjusts itself to the concentration of metal ion in solution. 3. Cobalt specific sites are available. Growth of these mutant fungi in iron bearing medium

K. Rashmi et al. / Science of the Total Environment 328 (2004) 1–14

before exposure to the simulated decontamination solution containing 0.033 mM Co(II)EDTA has increased or only marginally reduced (10%) the cobalt pick up in spite of the presence of high concentration of iron in the growth medium at initial wFeyCox of 2.5=105. 4. Suspending more fungal biomass for a certain volume of solution in order to bioremediate the solution has limitations. This is because extrapolated capacity from measurements with less biomass of fungi is not realized when actually the corresponding mass of fungi is taken. There appears to be an optimum solution volume (milliliter)yfungal biomass (gram) to realize the maximum capacity. Typically a ratio G30 is preferable. 5. Presence of Cr along with Fe and Ni in simulated spent decontamination solution has not affected Co pick up capacity. Acknowledgments The authors gratefully acknowledge Dr N.M. Gupta, Head, Applied Chemistry Division, BARC for the keen interest shown and helpful discussions during the course of this work. The work was supported by Grant from the Board of Research in Nuclear sciences, Department of Atomic Energy (No.2000y37y20yBRNS) and University Grants Commission (UGC-SAP(DRS-I)), Government of India. References Bradbury D, Smee TL, Williams, MR. 1986. Recent reactor decontamination experience with LOMIyCANDECON and related processes. In: Proceedings of International Conference on water chemistry of Nuclear reactor systems (4), British Nuclear Energy Society (BNES) London, 257. Dances A, Haile D, Yuan DS, Klausner RD. The Saccharomyces cerevisiae copper transport protein (ctrlp). Biochemical characterization, regulation by copper and physilogic role in copper uptake. J. Biol. Chem. 1994;269:25 660 – 25 667. Eitinger T, Freidrich B. Cloning, nucleotide sequence and heterologous expression of a high-affinity nickel transport gene from Alcaligenes eutrophus. J. Biol. Chem. 1991;266:3222 –3227. Gadd GM. Interactions of fungi with toxic metals. New Phytol. 1993;124(25):25 –60.

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