cadmium bioremediation by recombinant bacteria

8/11/2019 cadmium bioremediation by recombinant bacteria

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Electroporation of expressionvectors into rhizobium strains

Expression vectors (pMPnolBMTL4nifHPCS andpMPnifHPCS) were transformed into strain b3

and mutant strain B3::nifHMTL4



Bacteria Selection

Selection on TY agar medium supplementedwith tetracycline (20μg/mL).



Bacteria Selection

Several clones were isolated and the plasmidDNA in the clones was confirmed by restrictionenzyme digestion.



Microaerobic culture of free-livingrhizobia and quantitation of Cd

Cd accumulation in free-living rhizobia was

analyzed under microaerobic conditions. A 10 ml cell culture was transferred to a

sealed 100 ml bottle.

The sample was flushed with a mixture of 1% O2 and 99% N2 at 0.551/ min for 5 minat room temperature.

CdCl2 was added to the bottle to obtain afinal concentration of 30 μM and cells weregrown at 28°C with gentle shaking (100 rpm) for 40 h.

For Cd extraction from bacterial cells, thecells were pelleted, washed twice in 5 mM

HEPES(pH 7.1)containing 0.85% NaCl, driedat 65 °C for 24 h, and suspended overnightin 70% nitric acid.

The concentration of Cd was measureddirectly in the soluble fraction using anatomic absorption spectrophotometer (AAS)



Nodule formation and Cd accumulationtest in hydroponic culture

A. sinicus seeds were surface-sterilized, sown on0.7% agar plate and incubated for three d in thedark at 25 °C.

Seedlings were transferred to vermiculate soil inscrewcapped glass bottles containing a nitrogen-free (NFR) medium, and then inoculated with approximately 106 cells of wild-type B3 or recom-binant B3.

After two weeks of cultivation under light (16hphotoperiod per a day) at 25 –28 °C, nodule formation observed.

Plants with nodules were transferred to the NFRmedium supplemented with 50 μM CdCl2 for hydroponic culture.

After two weeks of hydroponic culture, plantswere harvested, washed in 10 mM EDTA andthen in 0.1 N HCl, dried at 105 °C for 24 h, andsuspended in 70% nitric acid.

Cd concentration was measured directly from

the soluble fraction using AAS.



Phytoremediation of Cd polluted soil

The pots (7.5 cm depth, 6 cm bottom di-ameter) contained a mixture of each 200 g

of (wet weight) rice paddy soil (without Cdcontamination which was supplied withCdCl2 solution to obtain a Cd concentrationof 1 mg/kg dry weight) and approximately106 cells of wild-type B3 or recombinantB3.

Six seedlings of A. sinicus, which were 3 dold after germination, were transferred toone pot and grown under light (16 h photoperiod/day) at 25 –28°C for two months.

Shoots, roots and nodules were harvestedfrom two month old plants, washed in 10mM EDTA (and further washed in 0.1 NHCl for roots and nodules), dried at 105 Cfor 24 h and suspended overnight in 70%nitric acid.

Cd concentration was measured directlyfrom the soluble fraction using AAS.

l i f d i



Accumulation of Cd infree-living cells

l i f Cd i d l



Accumulation of Cd in nodulesin hydroponic culture

Ph di i f Cd



Phytoremediation of Cdcontaminated soil

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cadmium bioremediation by recombinant bacteria