Nitrogen fixing rhizobia associated with the Medicago L. genus belong to two closely related species Sinorhizobium medicae and S. meliloti. To investigate the symbiotic requirements of different Medicago species for the two microsymbionts, 39 bacterial isolates from nodules of eleven Medicago species growing in their natural habitats in the Mediterranean basin plus six historical Australian commercial inocula were symbiotically characterized with Medicago hosts. The bacterial species allocation was first assigned on the basis of symbiotic proficiency with M. polymorpha. PCR primers specific for 16S rDNA were then designed to distinguish S. medicae and S. meliloti. PCR amplification results confirmed the species allocation acquired in the glasshouse. PCR fingerprints generated from ERIC, BOXA1R and nif-directed RPO1 primers revealed that the Mediterranean strains were genetically heterogenous. Moreover PCR fingerprints with ERIC and BOX primers showed that these repetitive DNA elements were specifically distributed and conserved in S. meliloti and S. medicae, clustering the strains into two divergent groups according to their species. Linking the Sinorhizobium species with the plant species of origin we have found that S. medicae was mostly associated with medics well adapted to moderately acid soils such as M. polymorpha, M. arabica and M. murex whereas S. meliloti was predominantly isolated from plants naturally growing on alkaline or neutral pH soils such as M. littoralis and M. tornata. Moreover in glasshouse experiments the S. medicae strains were able to induce well-developed nodules on M. murex whilst S. meliloti was not infective on this species. This feature provides a very distinguishing characteristic for S. medicae. Results from the symbiotic, genotypic and cultural characterization suggest that S. meliloti and S. medicae have adapted to different Medicago species according to the niches these medics usually occupy in their natural habitats.

The ability of activated sludge obtained from a local wastewater treatment plant to dechlorinate hexachloro-1,3-butadiene (HCBD) in the presence of either acetate or lactate and cyanocobalamin was investigated. Results from headspace analysis indicated complete dechlorination of HCBD by the accumulation of fully dechlorinated C4 gases (1-buten-3-yne, 1,3-butadiene, and 1,3-butadiyne). Dechlorination products were not observed in the control cultures without cyanocobalamin. Examination of control cultures revealed that the disappearance of HCBD from the headspace was partly due to adsorption into the biomass. However, the key for dechlorination was the shuttle (cyanocobalamin) rather than specific microbial enzymatic activity. The hypothesis that the bacteria reduced cyanocobalamin, which in turn reductively dechlorinated HCBD, was supported by the finding that cyanocobalamin reduced by zero-valent zinc resulted in complete dechlorination. The significance of the findings is that, in contrast to prior work where specific anaerobic bacteria (enrichments or pure cultures) were believed to be necessary for dechlorination resulting in only partly dechlorinated products, the currect data show that nonspecific aerobic activated sludge bacteria can be employed for complete HCBD dechlorination at rates sufficiently high to be considered for bioremediation projects.

The microbial reductive dechlorination of chlorinated solvents is a redox reaction in which the chlorinated carbon receives electrons from a suitable electron donor. Hexachloro-1,3-butadiene (HCBD) is a particularly recalcitrant chlorinated solvent. Its slow, cyanocobalamin-dependent biochemical dechlorination by mixed microbial consortia had been previously demonstrated. This study shows that the reductive dechlorination reaction of HCBD can be monitored in situ by recording the redox potential (E Ag/AgCl ). The addition of HCBD to mixed anaerobic consortia triggered a rise of E Ag/AgCl and the formation of dechlorinated endproducts. This indicated that the change in E Ag/AgCl was linked to the dechlorination of HCBD. Total concentration of dechlorinated endproducts (C4 gases) equated to approximately 50% of HCBD added. The rise and subsequent fall in E Ag/AgCl after the addition of HCBD caused a peak. Peak areas obtained, from the change in E Ag/AgCl , provided an indication of the amount of HCBD dechlorinated. Moreover, the saturation concentration of HCBD can be estimated from peak heights. This online monitoring of HCBD reductive dechlorination could potentially be used for improved process control of bioremediation reactors and on-site as online biosensors. Online monitoring of HCBD dechlorination via redox potential offers both reliability and portability at a low cost. In addition, this novel and innovative technique could potentially be used to monitor the dechlorination of contaminants other than HCBD. AbbreviationsC4 gases, dechlorinated endproducts (1,3-butadiene,1-buten-3-yne and 1,3-butadiyne); E Ag/AgCl , redox potential (referenced to Ag/AgCl electrolyte); EBCRC, environmental biotechnology co-operative research