3 Aims and Progress of the Work
3.3 AUTOTROPHIC LEACHING
Application of Microbial Products
commonly obtained by bacterial growth in iron-deficient substrates (Schwyn and Neilands, 1987; Alexander and Zuberer, 1991). Combined with the fact that the Pb-concentrations applied in the studies documenting siderophore-enhanced dissolution of Pb from soil-minerals was several size-orders below that in contaminated soils (Neubauer et al., 2000; Kraemer et al., 2002; Neubauer et al., 2002), this fact made a doubt rise on the applicability of siderophores for promotion of EDR of Pb-contaminated soil.
In order to exclude any unaccounted positive effects, the track was, however, followed a little further by making a laboratory-scale EDR experiment, where the ability of P. fluorescens DS178 to produce siderophores and enhance Pb-remediation was tested. Glucose consumption was documented by the negative detection after less than 24 hours after addition. Siderophore-production could not be detected at any point of time. After termination of the experiment < 1% of the Pb had been remediated. Consequently no further research in this topic was completed, and the reasons behind the glucose depletion and lacking siderophore production were therefore not elucidated. Possible reasons could be: inherent soil-microorganisms oust P. fluorescens DS178; sufficient iron is released form the soil to make siderophore production unfeasible; growth is impeded in the electric field and glucose is oxidized at the anode.
Application of Microbial Products Complete acidification of neutral material may therefore be obtained by successive growth of less acidophilic and extremely acidophilic sulfur-oxidizing bacteria.
Because it was shown early that A. ferrooxidans and A. thiooxidans are sensitive to even low concentrations of a wide variety of organic substances often present in soils (Tuttle and Dugan, 1976), the efforts on application of this technique to decontamination of soil was low until it was later shown that some strains are tolerant to organic substances (Blais et al., 1993; Zagury et al., 1994). Since, a number of studies on bioleaching of heavy metals from contaminated soils and sediments were made, however being ambiguous in their results on the leachability of Pb: Leaching of Pb by incubation with elemental sulfur and in some cases specific species was successfully obtained from storm water detention pond sediments (Anderson et al., 1997; Anderson et al., 1998); wastewater sludge (Du et al., 1995; Shanableh and Omar, 2003); sewage sludge (Ravishankar et al., 1994) and anaerobically digested sludge (Xiang et al., 2000; Wong et al., 2004). In many works, however, dissolution of Pb by sulfur-oxidizing bacteria was excluded due to precipitation of leadsulfate, which has a very low solubility compared to other metal-sulfates (Ksp = 1.6 x 10-8).
Bioleaching experiments with various industrially contaminated soils successfully dissolved most of the heavy metals present, but not Pb (Gourdon and Funtowicz, 1995; White et al., 1998; Gomez and Bosecker, 1999), and in a highly contaminated river sediment Zn, Cd, Mn, Co, Cu and Ni were leachable, while Pb and Cr were nearly immobile (Seidel et al., 2004).
The referred results suggest that bioleaching of Pb from sediments in general is more feasible than from soils. This hypothesis was supported by a study, where 96%
of the Pb was leached from contaminated wastewater sludge while from a mixture of contaminated soil and wastewater sludge only 10-33% of the Pb was leached (Shanableh and Omar, 2003). In other words: the low solubility of lead-sulfate may not be the only limiting factor. The different compositions of soil and sediments also seem to play an important role.
Indigenous presence of sulfur-oxidizing species was documented in several soil and sediment samples: In sewage sludge it was shown that T. thioparus and A.
thiooxidans dominated the acidification amendment with sulfur (Blais et al., 1993). A.
ferrooxidans was found indigenous in stormwater detention pond sediment, while A.
thiooxidans and the less acidophilic species were not (Anderson et al., 1997). In two contaminated soils and a river sediment both A. thiooxidans and A. ferrooxidans were found to be indigenous, while in a rainwater-collection basin sludge only A.
thiooxidans was identified (Gomez and Bosecker, 1999).
Direct current was found to be detrimental to low cell densities of the bacteria A.
ferrooxidans and an Acidiphilium sp. in liquid culture. In contrast, bacterial metabolism was stimulated by the current in soil slurries (Jackman et al., 1999), which support the feasibility of enhanced remediation by simultaneous bioleaching and EDR of contaminated soil. One work showed how energy consumption by EKR of copper from soil was reduced by an integrated method incorporating sulfur-oxidizing bacteria (Maini et al., 2000).
3.3.2 Materials and Methods
An industrially contaminated Danish soil of unknown origin (referred to as soil 10 in chapters 4 and 5), obtained from a pile after excavation, was used as experimental soil. The soil fines were obtained by simple wet-sieving of the original soil with distilled water through a 0.063 mm sieve. Concentrated slurry of fines was obtained by centrifugation at 3000 rpm for 10 min. and decantation of the supernatant. The soil
Application of Microbial Products
fines were kept in slurry and stored at 5ºC in access of oxygen. The Pb-content of the soil-fines was 1300 mg/kg. Acid-enhanced extraction of Pb from the soil was investigated by extraction of 5.00g dry, crushed soil with 25.00ml reagent at 200rpm for 7 days. The reagents were as follows: 1.0M NaOH, 0.5M NaOH, 0.1M NaOH, 0.05M NaOH, 0.01M NaOH, distilled water, 0.01M HNO3, 0.05M HNO3, 0.1M HNO3, 0.5M HNO3, 1.0M HNO3, 0.01M H2SO4, 0.05M H2SO4, 0.1M H2SO4, 0.5M H2SO4, 1.0M H2SO4. pH was measured after 10min settling, after which the liquid was filtered through a 0.45 m filter for subsequent Pb-analysis AAS. Non acidic samples were preserved with one part of conc. HNO3 to four parts of liquid in autoclave at 200 kPa and 120ºC for 30 minutes prior to AAS measurement
3.3.3 Results and discussion
In figure 3.10 and 3.11 the acid-enhanced desorption of Pb from soil-fines is illustrated. Extraction with nitric acid was efficient below pH 2, and some extraction was seen by sodium-hydroxide above pH 13. Extraction with sulfuric acid was very limited. Only 3% of the Pb was extracted even at pH below 1 (figure 3.11). In figure 3.12 the speciation of Pb in the presence of sulfate is illustrated. According to these equilibrium calculations, the majority of the Pb will precipitated as crystalline lead-sulfate. The limited extraction of Pb with sulfuric acid is therefore likely to be due to precipitation of lead-sulfate. Under influence of a DC current field, the equilibrium would however be shifted by constantly removing soluble species. Therefore enhancement of EDR by sulfur-induced heterotrophic leaching can not be excluded on basis of these batch-extraction results.
0 20 40 60 80 100 120
0 2 4 6 8 10 12 14
pH
Pb extracted [%]
NaOH/HNO3 H2SO4
Figure 3.10: Acid-enhanced extraction of Pb from contaminated soil-fines.
0.0 1.0 2.0 3.0 4.0
0 2 4 6 8
pH
Pb extracted [%]
Application of Microbial Products Figure 3.11: Extraction of Pb from contaminated soil-fines with sulfuric acid.
Gry Pedersen investigated the possibility of enhancing EDR of Pb-contaminated soil-fines by sulfur-amendment (Pedersen, 2005). She found that indigenous bacteria were able to acidify the soil-fines slightly upon sulfur-amendment, however no acidification below pH 4 was obtained, indicating that only the least acidophilic sulfur oxidizers like Paracoccus versutus were indigenous. In experiments where pH was adjusted manually to pH 4, the lack of further acidification upon sulfur-amendment indicated that extremely acidophilic sulfur-oxidizing species were not present in the soil. Identical EDR-experiments with contaminated soil-fines left 32% of the Pb in the soil subjected to acidification by sulfur-amendment and only 6% after remediation without sulfur-amendment. Direct addition of sulfuric acid gave even worse results with 98% of the Pb left in the soil-fines after experimental remediation. Apart from Pb, the soil was contaminated with Zn, and in order to evaluate the feasibility of autotrophic leaching for toxic metals, which do not precipitate easily as a sulphate, the removal of Zn was monitored in the same experiments. In that case addition of sulfuric acid and sulfur-amendment gave significantly better results: 18 and 23% of the Zn was left in the soil after remediation with sulfuric acid and sulfur-amendment respectively. In comparison, an average of 37% of the Zn was left in the soil after remediation in the reference experiments. The results suggest that precipitation of crystalline lead-sulfate impedes EDR of Pb-contaminated soil-fines significantly, and that heterotrophic leaching in combination with EDR of Pb-contaminated soil-fines in suspension is not a viable technology, while it may be for toxic metals which do not precipitate as sulphates.