An experiment was performed in a carbon tetrachloride (CT)- and nitrate-contaminated aquifer at Schoolcraft, MI, to evaluate bioaugmentation with Pseudomonas stutzeri KC, a denitrifying bacterium that degrades CT without producing chloroform (CF). A test section of the aquifer was treated to create pH conditions favorable for KC and then inoculated with culture grown aerobically on site. Activity was sustained with pulses of acetate-amended groundwater, followed by “chase” pulses of acetate-free water. In regions with effective substrate delivery, KC was detected, nitrate levels fell by 85%, pH levels increased, and CT levels decreased by ∼65%, with no significant increase in CF. After 3 weeks, denitrification and CT transformation activity decreased, and KC was no longer detected in groundwater from four wells. Loss of denitrification was attributed to the acetate-free chase. Upon eliminating the chase, CT transformation resumed, and KC was detected, but CF production was also observed, implicating indigenous organisms as agents of transformation. Final sediment analyses indicated 60−88% CT removal, little CF, and persistent KC. This work demonstrates subsurface pH adjustment, subsurface transport of KC, assimilation of KC into the aquifer community, CT removal without CF production after inoculation, and CF formation when KC activity declined.

A copper-containing nitrite reductase gene (nirU) from Pseudomonas sp. strain G-179 was found in a 1.9-kb EcoRI-BamHI DNA fragment. The coding region contained information for a polypeptide of 379 amino acids. The encoded protein had 78% identity in amino acid sequence to the nitrite reductase purified from Achromobacter cycloclastes. The ligands for type 1 copper-and type 2 copper-binding sites found in A. cycloclastes were also found in Pseudomonas sp. strain G-179, suggesting that these binding sites are conserved. Upstream from the promoter, two putativeJfr boxes were found, suggesting that an FNR-like protein may be involved in regulation of the nitrite reductase gene under anaerobic conditions. When the 1.9-kb clone was used to probe Southern blots for similar sequences in DNAs from different denitrifiers, hybridization bands were seen for 15 of 16 denitrifiers known to have nitrite reductases containing copper. Except for Pseudomonas stutzeri JM300, all denitrifiers tested that have nitrite reductases containing heme c,d, showed no or weak hybridization to this probe. Thus, this structural gene may be useful as a probe to detect denitrifiers with copper-containing nitrite reductases.

Approximately equal numbers of bacteria were isolated from primarily tropical soils by growth on biphenyl and naphthalene to compare their competence in polychlorinated biphenyl (PCB) degradation. The strains isolated by growth on biphenyl catalyzed more extensive PCB degradation than the strains isolated by growth on naphthalene, suggesting that naphthalene cocontamination may be only partially effective in stimulating the cometabolism of lower chlorinated PCBs. Probes were made from the bph, nah, and tod genes encoding the large iron sulfur protein of the dioxygenase complex and hybridized to 19 different strains. The hybridization patterns did not correlate well with the substrates of isolation, suggesting that there is considerable diversity in these genes in nature and that probe hybridization is not a reliable indication of catabolic capacity. The strains with the most extensive PCB degradation capacity did strongly hybridize to the bph probe, but a few strains that exhibited strong hybridization had poor PCB-degrading ability. Of the 19 strains studied, 5 hybridized to more than one probe and 2, including one strong PCB degrader, hybridized to all three probes. Southern blots showed that the bph and nah probes hybridized to separate bands, suggesting that multiple dioxygenases were present. Multiple dioxygenases may be an important feature of competitive decomposers in nature and hence may not be rare. Most of the isolates identified were members of the beta subgroup of the Proteobacteria, a few were gram positive, and none were true Pseudomonas species.