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Leaching Of Harbour Sediments By Estuarine Iron-Oxidising Bacteria

A.G. Crane and P.J. Holden

Australian Nuclear Science and Technology Organisation, Menai, Australia, 2234.

As part of a program to develop remedial technologies for harbour sediments, salt-tolerant autotrophic and mixotrophic Fe and S oxidising bacteria were isolated from Sydney Harbour sediment using artificial seawater medium. Leaching of metals from 4 sediments was investigated in shake flasks and stirred reactors (2-10 L)in media containing 0-5% NaCI. The sediments contained Fe (21.3-42.5 g/kg), Cu (320- 840 mg/kg), Zn (380-4200 mg/kg), Pb (190-1370 mg/kg) and Cd (3-15.4 mg/kg), predominantly as sulfides, as well as 30-60 mg/kg PAHs and 200-1400 mg/kg total petroleum hydrocarbons. Two of the sediments were particularly acid consuming and leaching could only be established after addition of acid or sulfur. Good extraction of metals was obtained with sediment microflora but inoculation with the enrichment cultures enhanced extraction rates. Leaching using iron-oxidising bacteria was comparable or superior to that obtained with addition of elemental S (5 g/L) and inoculation with S-oxidisers. Autotrophic and mixotrophic bacteria gave comparable extraction rates and proved superior to Thiobaci/ lus ferrooxidans which would not grow at 2% NaCI. Comparison with Thiobaci l lus prosperus - hitherto the only described salt tolerant iron oxidising Thiobacillus, showed that the autotrophic isolates gave superior Cu and Cr extraction and similar Zn and Cd extraction (acid mediated). At 25% pulp density 72.5% Cu, 93.4% Zn, 95.8% Cd and 0.1% Pb was leached after 14 days. Semi-continuous reactor tests at 15% w/v produced Cu, Zn, and Cd removal after 3 days which was sufficient to approximate regulatory requirements for disposal as clean fill. Lead removal required an additional step to separate lead sulfate, which is largely insoluble.

1. INTRODUCTION

The generic problem of how to effectively detoxify sediments and soils co- contaminated with heavy metals and toxic organic chemicals is one that is currently receiving much attention world-wide. The disposal of contaminated harbour sediments after dredging constitutes a difficult environmental problem, particularly in Australia where landfill sites able to receive large volumes of contaminated material are non-existent. A program of research aimed at developing remedial technologies, utilising a combination of biological, chemical and physical remediation techniques to

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treat co-contaminated harbour and estuarine sediments from Sydney Harbour, Australia, was undertaken. It involved a multidisciplinary integrated approach that started at the undisturbed sediment bed and finished with the disposal of waste streams. It incorporated chemical and ecotoxicological characterisation of sediments before and after treatment as well as treatment technology development.

A major component of that program was the evaluation of bioleaching of metals from sediments in saline media as a remedial option. Prior to our work, the only report of iron-oxidising marine, estuarine, or salt tolerant, bacteria known to these authors was that of Thiobacillus prosperus isolated and described by Huber and Stetter (1). Their work included leaching of conventional/synthetic mineral ores but did not examine leaching of metals in polluted sediments. This paper describes the enrichment of autotrophic and mixotrophic iron-oxidising marine bacteria that proved to be effective in mobilising metals from sediments under aerobic conditions.

2. MATERIALS & METHODS

2.1 Sediments Preliminary sampling and analysis of cores from 21 sites in Sydney Harbour

and Cooks River, Sydney, Australia, facilitated selection of sites for extensive sampling and analysis. The sediments used in these investigations were obtained from Rozelle Bay and Canada Bay, in Sydney Harbour, and from Alexandra Canal and Cooks River. Approximately 140-800 L of sediment was removed from each site to a depth of roughly 1 m and immediately transferred into 200 L drums and stored at 2~ They were analysed for total petroleum hydrocarbons and polynuclear aromatic hydrocarbon (PAH) by Gas Chromatography-Mass Spectrometry and metal content by Microwave Assisted Acid Extraction/Flame Atomic Absorption Spectroscopy.

2.2 Enrichment of Iron and Sulfur Oxidising Bacteria Samples (1-5 g) of sediment from the top 20 cm of cores obtained from five

sites at Rozelle Bay, Canada Bay and Duck River within Sydney Harbour and the vicinity of Princes Highway Bridge and Alexandra Canal in Cooks River were enriched using thiosulfate, elemental sulfur, and ferrous sulfate as substrates. These sites were known by prior analyses to be contaminated with heavy metals. Sediment was inoculated into the artificial seawater medium, Nine Salts Solution (NSS) (2), modified by the addition of 0.2 g/L NH4CI, 0.2 g/L Na2HPO 4 and 0.01 g/L FeSO4.7H20, and containing either Na2S203.5H20 (5 g/L) or gamma sterilised elemental sulfur (5 g/L). The pH was adjusted to pH 7.0 or 5.5 for thiosulfate media, and pH 4.0 in the case of sulfur, using HCI. Inoculated shake flasks were incubated at 21~ and 26~ respectively. Ferrous sulfate enrichment was conducted in ISP medium (3) modified by the addition of 10 or 20 g/L NaCI and adjusted to pH 2.1 with H2SO 4. Inoculated shake flasks were incubated at 21~ and transferred to fresh media after 7 days.

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Iron-oxidising cultures were routinely cultured at 28~ and were maintained in modified ISP with or without the addition of yeast extract (0.1 g/L). Mixotrophic cultures were purified on modified ISP media containing 13.9 g/L FeSO4.7H20 and yeast extract, solidified with agarose (7 g/L). Autotrophs were purified by serial dilution.

2.3 Shake Flask Leach Tests The ability of cultures to leach metals from the sediments was determined in

250 or 500 mL shake flasks containing wet sediment and either ISP (FeSO4.7H20 deleted) or NSS supplemented with 0.2 g/L NH4CI. Some flasks were inoculated (1 x 106 cell/mL) with the various isolates or with Thiobaci//us ferrooxidans TFI-35 (4), at various salt concentrations. For each inoculated flask a corresponding uninoculated treatment was established to compare the metal leaching capabilities of the sediment microflora plus an inoculant, with the sediment microflora alone. For sterile controls the sediment in the flask was autoclaved prior to the addition of medium. Metal leaching was quantified by ICPAES of leachates and residual sediments.

2.4 Batch Reactor Tests Comparison was also made between leaching by the marine autotroph A19-

16 and Thiobaci//us prosperus DSM 5130 in batch reactor tests. Quickfit vessels containing 10% w/v Rozelle Bay sediment and NSS supplemented with 0.2 g/L NH4CI in a total volume of 2L were stirred at 400 rprn by Janke & Kunkel RW20 overhead stirrers and sparged with air (1 IEmin.). The vessels were inoculated with 2 xl07 cell/mL of culture raised on sediment in shake flasks. The pH of the slurry was maintained at pH 3 for the first 48 hours following inoculation by addition of H2SO 4 controlled by a Leeds & Northrup 7260 pH controller and a Masterflex pump drive.

To examine the effect of scale and pulp density on metal dissolution rates, leaching of 10% w/v sediment in 2L total volume was compared with leaching of 10 and 25% w/v sediment in 10 L volumes. These tests were conducted in 15 L stirred vessels which were sparged with air at 4 L/min. and pH controlled at pH 4 for the first 48 h. Inoculation (6 x 107 cells/mL) was made with A19-16 raised on autoclaved sediment (5% w/v).

2.5 Semi-Continuous Reactors Due to the difficulty of slowly pumping small volumes of slurry containing very

fine solids at 15% w/v, the effect of residence time on leaching rates by A19-16 was determined in semi-continuous flow reactors (2 L) which periodically had a volume of slurry removed and replaced with fresh sediment and media. Reactors were initially established as in batch mode and after a flourishing culture had developed and the pH had decreased to pH 1.8-1.9, hourly slurry replacement was initiated. Residence times of 20, 5 and 3 days were simulated with rates being quantified from the average of 7 samples over a 6 hour period. An accommodation period of 2- 4 days was allowed between each shift in residence time.

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3. RESULTS

3.1 Sediments The sediments contained quantities of most trace metals well in excess of

background values. The metals that consistently exceeded regulatory limits were copper, lead, zinc and cadmium, and their concentrations are given in Table 1. Chromium and nickel occasionally also exceeded the limit in horizons of limited depth (results not shown). The sediments also contained 30-60 mg/kg PAHs and 200 -1400 mg/kg total petroleum hydrocarbons, but little chlorinated hydrocarbons.

Table 1 Metal content of Australian harbour sediments

Cu Zn Pb Fe Cd SITE (mg/kg) (mg/kg) (mg/kg) (mg/kg) (mg/kg)

CANADA BAY 358 380 189 26,400 2.9 ROZELLE BAY 471 3440 1370 42,500 6.6

COOKS RIVER 319 2050 705 21,300 9.0

ALEXANDRA CANAL 841 3770 1200 32,700 15.4

3.2 Bacteria 28 cultures were isolated and purified from enrichment of sediments from the

five sites with thiosulfate at pH 5.5 and 7.0. All but four of these cultures could grow in the presence of glucose but none in the absence of NaCI. However, only four cultures were able to utilise elemental sulfur as a substrate. Seven consortia of bacteria were isolated from elemental sulfur enrichments. Transfer of these cultures into media containing ferrous sulfate and yeast extract lead to the isolation and purification of five mixotrophic iron-oxidising bacteria, designated A2-8, A8-4, A l l - 13, A19-22 and A20-24 representing cultures from each of the five sites. All of these cultures consisted of Gram positive rods, some of which were observed to form spores. Autotrophic iron-oxidising bacteria were only isolated from sediments from the Cooks River site. Strain A19-16 was enriched on ferrous sulfate at 1% NaCI and A19-17 at 2% NaCI. Both cultures were subsequently maintained at 2% NaCI and were capable of growth between 0.5 and 4% NaCI. They were purified by endpoint dilution. The original enrichment culture of A19-17 also contained Penic i l l ium minioluteum.

3.3 Leaching Efficacy Batch shake flask tests of sterile and non-sterile sediment (2.5% w/v) from

Rozelle Bay showed that the sediment microflora was capable of leaching zinc, copper, lead and chromium. No copper or chromium was leached in sterile controls and the zinc removal was markedly less. Sterile controls were conducted in all

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subsequent experiments with leaching of sediments but the results are not shown as the conclusions were as above. Increasing the salt concentration from 1 to 2 % NaCI in the medium did not improve leaching. Leaching at 3% NaCI improved copper leaching by 16% and zinc by 7%. In the case of lead, far higher values were obtained due to the higher chlorine concentration improving solubility (Table 2).

Tests of leaching of all four sediments (non-sterile) using sediment microflora with and without the addition of elemental sulfur (5 g/I) in media containing 2% NaCI demonstrated that some sediments were more acid consuming than others. The addition of sulfur, or sulfuric acid was necessary to initiate leaching of metals in the case of Canada Bay and Cooks River - presumably because of their high shell grit (carbonate) content (Table 3). The addition of sulfur led to acid production and leaching in all cases. However, in the case of Alexandra Canal and Rozelle Bay sediment, sulfur addition was only of benefit in regard to slightly improving copper extraction with zinc extraction be essentially complete in both treatments. These trends were also observed when leaching Rozelle Bay sediment with the sulfur- oxidising consortia isolated above.

The mixotrophic iron oxidiser A20-24 was tested for leaching capability on all four sediments (non-sterile). Leaching could not be established on Canada Bay sediments without pH control due to high acid consumption. A20-24 greatly improved leaching of Cooks River sediment over that obtained with microflora. In the case of Rozelle Bay and Alexandra Canal sediments, similar results were obtained with both microflora and A20-24 (Table 4) with the exception that inoculation improved chromium extraction from Rozelle Bay sediment. Comparison with four other mixotrophic cultures indicated that they all mobilised metals from Rozelle Bay sediment to a similar extent (results not shown).

Table 2 Leaching of Rozelle Bay sediment after 21 days by microflora at 1%, 2%, And 3% NaCI

NaCI Sediment Cu Zn Pb Cr (~g/mL) (#g/mL) (~g/mL) (~g/mL)

1% Sterile 0 36.9 0.95 0

2% Sterile 0 6.10 1.25 0

3% Sterile 0.05 14.1 1.10 0

1% Non-sterile 7.20 82.2 1.10 0.50

2% Non-sterile 7.60 83.4 1.10 0.60

3% Non-sterile 8.85 89.2 6.65 0.50

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Table 3 Bioleaching by microflora in NSS medium (2% NaCI) with and without S addition

Sediment Solids 5 g/I S Day 0 Day 31 % Cu % Zn (%) pH pH

CANADA BAY 2.5 - 5.21 7.05 0 0

CANADA BAY 2.5 + 2.69 2.27 98.0 100

ROZELLE BAY 2.5 - 6.04 3.39 62.3 89.7

ROZELLE BAY 2.5 + 6.05 2.46 94.0 93.3

COOKS RIVER 2 - 4.59 3.77 0 3.4

COOKS RIVER 2 + 4.65 2.35 75.3 88.0 ALEXANDRA

CANAL 2 - 4.90 2.89 68.8 91.6 ALEXANDRA

CANAL 2 + 5.03 2.33 79.4 92.1

Table 4 Leaching by mixotrophic iron oxidising bacterium A20-24 after 31 days

SEDIMENT INOCULUM pH % Cu % Zn % Cd % Cr

CANADA BAY - 7.05 0 0 N/A 0.7

CANADA BAY A20-24 6.63 0 0.3 N/A 0

ROZELLE BAY - 3.39 62.3 89.7 61.4 1.9

ROZELLE BAY A20-24 3.19 63.2 87.0 52.1 7.9

COOKS RIVER - 3.77 0 3.4 5.8 0.4

COOKS RIVER A20-24 2.58 75.4 89.8 83.0 46.9

ALEXANDRA C. - 2.89 68.8 91.6 76.7 44.2

ALEXANDRA C. A20-24 2.75 66.8 87.2 83.3 36.8

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Table 5 Metal extraction from Rozelle Bay sediments after 31 Days leaching by iron oxidising bacteria

Inoculum pH % Cu % Zn % Cd % Cr

None 3.39 62.3 89.7 61.4 1.9

A20-24 3.19 63.2 87.0 52.1 7.9

A19-16 2.51 68.3 99.7 61.2 7.2

A19-17 2.77 59.9 90.2 62.9 5.2

T. ferrooxidans TFI-35

A comparison of leaching of sterile Rozelle Bay sediment by the microflora, mixotrophic iron oxidisers (A20-24), and autotrophic iron oxidisers (A19-16, A19-17 and Thiobacillus ferrooxidans TFI-35) after 20 days confirmed that inoculation improved total chromium extraction. In the case of copper, zinc and cadmium, significant differences were not observed in final extraction between the three cultures (Table 5). However, a comparison of rates indicated that inoculation vastly decreased the lag period and leaching rates were more rapid as exemplified with the results for copper (Figure 1). T. ferrooxidans TFI 35 could not be established at 2% NaCI content and hence no biological leaching was observed in that treatment.

3.4 Comparison With Thiobacillus Prosperus: Leaching of Rozelle Bay sediment observed in batch reactor (2 I) tests at

10% w/v sediment using the marine autotroph A19-16 was compared with that obtained with Thiobaci//us prosperus. Zinc and cadmium leaching rates, largely mediated by acid dissolution, were similar. However, copper and chromium leaching by A19-16 was distinctly superior (Figure 2).

3.5 Scale And Pulp Density: The effect of increasing the solids loading in reactor tests to 25% w/v and the

scale to 10 L was investigated using Rozelle Bay sediment and culture A19-16. At 10% w/v sediment, increasing the reaction volume from 2 to 10 L did not adversely affect leaching and final extraction values (Table 6) were comparable or exceeded regulatory targets (Cu 82.3%; Zn 84.2%; Cd 15.3%; Ni 23.9%; Pb 84.1%) with the exception of lead which has very limited solubility in sulfated systems. Subsequent tests demonstrated that lead sulfate could be removed by washing the leached sediment with acetate/NaCI solutions. Increasing pulp density reduced copper extraction from 80.3 to 72.5%. Similar results were obtained with Alexandra Canal sediments (results not shown).

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80

60 z O m I- o < rr l-- 40 x iii IT" W n 13. O 20 O

h---

0 10 20 30 40

TIME (DAYS)

MICROFLORA A19-16 A19-17 A20-24

Figure 1. Batch shake flask leaching of Rozelle Bay sediment (2.5% w/v)

100

80

g ,

5 ~ ,

w 40" l ~ ._1 , . , , , . . - - I

p - ,

W

20"

0 10 20 30

TIME (DAYS)

A19-16 Cr T. PROSPERUS Cr A19-16 Cu T.PROSPERUS Cu

Figure 2. Leaching of Rozelle Bay sediment (10% w/v) in a 2 L batch reactor

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Table 6 Metal extraction from Rozelle Bay sediment by A19-16 after 15 Days

BATCH WN % Cu % Zn % Cd % Cr % Pb % Ni REACTOR SOLIDS

2L 10% 81.2 97.4 100 35.4 2.3 80.5

10 L 10% 80.3 100 100 38.2 0.2 100

10L 25% 72.5 93.4 95.8 36.3 0.1 81.1

REGULATORY TARGET 82.3 88.2 15.3 0 84.1 23.9

3.6 Residence Time The effect of residence (treatment) time of sediment in the leach system on

final extraction by culture A19-16 in a semi-continuous feed reactor at 15% w/v sediment was investigated. The residence time was sequentially reduced from 20 days, to 5, and finally, 3 days. Figure 3 shows the mean total metal extraction values (n=7: Coefficient of Variance <1.2 %) for each residence time.

o4 z O u

o <

x w

<

w

100

80

60

40

20

0 10 20 30

RESIDENCE (DAYS)

Figure 3. Metal extraction by A19-16 at different residence times

= Cd m--. Zn : Ni

Cu l, Cr

m, Fe

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As the residence time decreased, the extraction values for copper, zinc and chromium remained relatively stable and iron increased. Cadmium and nickel declined slightly but extraction was far greater than required by regulatory limits. With a residence time of 3 days, the reactor leachate metal concentrations indicated leaching of 59.1% Cu, 92.1% Zn, 91.4% Cd, 31.7% Cr and 75.5% Ni. The copper extraction values obtained in these tests were lower than previously observed under similar conditions. However, the conclusions regarding effect of residence time are valid as they were all obtained in same reactor run. Acid consumed maintaining pH of the reactors at pH 1.9 was 4.4 mL/day for the three day residence time and zero for the longer periods.

4. CONCLUSIONS

1. Salt tolerant iron and sulfur oxidising bacteria are readily enriched from Sydney Harbour sediments 2. Good extraction of metals from sediments was obtained with sediment microflora but inoculation with enrichment cultures enhanced rates 3. Autotrophic and Mixotrophic bacteria gave comparable rates of extraction 4. The autotrophic isolates obtained produced superior rates of sediment leaching compared with Thiobacillus prosperus. 5. The mixotrophic iron oxidising isolates were clearly different from Thiobacillus prosperus as they are Gram Positive bacteria capable of spore formation. 6. Final extraction values approximating regulatory targets were obtained in reactor tests (25%w/v sediment) with the exception of lead which required a further separation process. 7. Reducing sediment treatment time to 3 days did not compromise achievement of extraction targets.

ACKNOWLEDGMENTS The work reported here was partially funded by a GIRD grant from the Dept.

Industry, Science & Technology, Australian Federal Government, which was held collaboratively with Rio Tinto Research & Technology Development, Patterson Britton and Partners Ltd, and Sydney Water.

REFERENCES

1. H. Huber and K.O. Stetter, Arch. Microbiol., 151 (1989) 479. 2. A.E. Goodman, E. Hild, K.C. Marshall and M. Hermansson, Appl. Environ. Microbiol., 59 (1993) 1035. 3. H.L. Manning, Appl Microbiol., 30 (1975) 1010. 4. P.A.W. Martin, P.R. Dugan, and O.H. Tuovinen, Can. J. Microbiol., 27 (1981) 850.