In the recent decades, some American, European and Asian experts, such as Hammer (1980), Wildeman (1998), Sucha and Sottnik (1999), Zaluski (2000), Yang (2002), Fujita and Kashiwa (2003) applied successfully microorganisms to remove heavy metal from wastewater.

The basic activity of microbial treatment is returning mobile and toxic contaminants to their stable immobile mineral forms (oxides, hydroxides, carbonates, sulfides…). In anaerobic condition the sulfate-reducing bacteria (SRB) reduce SO₄^2- to H₂S and form sulfide precipitation. Thiobacillus and others in aerobic condition are relatively effective in forming hydroxides, oxides…

Zaluski and Canty [13] demonstrated the ability of SRB to reduce Cr VI to Cr III, a much less toxic form of chromium by a series of experiments. Hydrogen sulfide produced by the SRB reacts with Cr VI according to the following chemical reaction:

3 HS^- + 6FeSO₄ + 4CrO₄^2- + 13H₂O + OH^- đ 3So + 6Fe(OH)₃ + 4Cr(OH)₃ + 6SO₄^2-

In Việt Nam, there are many manufacting-craft-settlements, which produce metal equipment, especially concentrated in the Nam Định province up to 71 settlements. Because of using old technology, lack of treatment technical planning knowledge of environment protection, almost wastewater is discharged directly into the environment without treatment. The polluted environment influences seriously to human health. The portion of people getting the cancer disease, nervous disease and borning abnormal baby in these settlements is very high. Average longevity of people here is only 55-57 years old, 10-12 years lower than the state average.

In order to solve the heavy metal wastewater pollution of craft-settlements, we carried out studying on microorganisms in heavy metal contaminated wastewater in the Vân Chàng settlement.

Metal contaminated water and mud samples such as Cr, Ni, Al and Fe were collected from different places in the Vân Chàng village, Nam Định province.

Desulfobulbus: Na₂SO₄ 3, KH₂PO₄ 0.2, NH₄Cl 0.3, NaCl 1, KCl 0.5, MgCl₂ O.4, CaCl₂.2H₂O 0.15, FeSO₄.7H₂O 0.5, yeast extract 1, vitamins 1, trace element 0.1, water up to 1 liter, pH 7.2-7.4.

Desulfovibrio: KH₂PO₄ 0.5, NH₄Cl 1, NaCl 1, CaSO₄ 1, MgSO₄.7H₂O 2, sodium lactate 3.5, FeSO₄.7H₂O 0.5, yeast extract 1, vitamins 0.1, trace elements 0.1, water up to 1 liter, pH 7.2-7.4.

Thiobacillus ferooxidans: KH₂PO₄ 0.2, (NH₄)₂SO₄ 0.3, KCl 1, Ca(NO₃)₂ 0.02, MgSO₄ 1, FeSO₄ 1, water up to 1 liter, pH 3.5-4.0 .

Thiobacillus thiooxidans: Na₂S₂O₃.5H₂O 5, KH₂PO₄ 3, NH₄Cl 0.1, CaCl₂ 0.25, MgCl₂ 0.1, water up to 1 liter, pH 3.

Thiobacillus thioparus: Na₂S₂O₃. 5H₂O 5, NaHCO₃ 1, Na₂HPO₄ 0.2, MgCl₂ 0.1, NH₄Cl 0.1, FeSO₄ trace, water up to 1 liter, pH 7.4-8.5.

Morphology of bacteria and the heavy metal inside the cells are observed by optical microscope Olympus and electronic microscope JEM 1010

Electronic microscope procedure: the culture incubated for 7 days is harvested by centrifugation at 5000 rpm for 15 min., washed with PBS buffer (0.1 M, pH 7.4). Washed cells are fixed in glutaraldehyde (2.5%) for 20 min. Washed twice by centrifugation. The specimens are mounted on collodion coated 200 mesh copper grids and steam covered with Cr and then observed under JEM 1010 instrument. The instrument is operated at 100 kV. Thin sections are cut with LKB 8800 Ultramicrotome and mounted on carbon-coated 200-mesh copper grids. Then, thin sections are stained according to Renold. The specimens are examined by JEM 1010 instrument. The instrument is operated at 100 KV.

From mud and water samples taken from different places in Vân Chàng village, we isolated and collected some strains, which were capable of removing heavy metal (Table 1). Metal-oxidizing bacteria in aerobic condition are in highest number up to 10³ CFU/ml (gram of mud). SRB in all samples was estimated from 10² to 10⁷ CFU/ml;

Table 1. Number of bacteria in mud and water in Vân Chàng village

We carried out the study on cell morphology and their physiological, biochemical properties and analysis of the 16s rDNA. Results are presented in Table 2.

Table 2. Morphological properties of some strains isolated from Vân Chàng settlement.

Among the identified species, Comamonas testosteroni, Rhodococcus equi and Desulfovibrio desulfuricans dominate in Vân Chàng wastewater. Their ability of heavy metal removal is shown in Table 3, 4.

Table 3. Effect of heavy metal to aerobic bacteria growing

The data in table 3 show that all of 5 aerobic strains have not played directly the role of heavy metal removing, they might use metabolite of heavy metal removal bacteria.

We selected two species of SRB isolated from deposit of trial model for assessment of heavy metal removal (Table 4). Activity of these strains is determined by number of bacteria, Cr and Ni concentration before and after treatment. In anaerobic condition two studied strains grew well. Number of bacteria increased from 10⁴ to 10⁸ CFU/ml. And concentration of Cr decreased to 99%, concentration of Ni reduced 60% in comparison with the control.

Table 4. Heavy metal removal ability of Desulfovibrio desulfuricans

Above results show that SRB could minimize heavy metal concentration of wastewater in Vân Chàng village. So, these native bacteria could be applied to heavy metal treatment model in craft-settlements.

From mud and wastewater of the Vân Chàng craft-settlement, Nam Định province, we isolated different species of bacteria (Alcaligenes defragans, Pseudomonas chlororaphis, Sphingomonas spec, Comamonas testosteroni, Rhodococcus equi, Rhodococcus facians, Bacillus pumilus, Bacillus cereus, Desulfovibrio desulfuricans (1), Desulfovibrio desulfuricans (2), among them Desulfovibrio, Desulfobulbus, Thiobacillus are capable of removing heavy metal.

Experiment of single strain at laboratory scale showed that Desulfovibrio desulfuricans grows well in Cr, Ni, Fe wastewater and it could reduce Cr from 30 to 0.32 mg/l (90%); Ni from 97 to 37mg/l (60%). SRB have been used successfully in lab scale and pilot scale for removing heavy metal from Vân Chàng wastewater.

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