The ability of Escherichia coli MV1184 to accumulate inorganic phosphate (Pi) was enhanced by manipulating the genes involved in the transport and metabolism of Pi. The high-level Pi accumulation was achieved by modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate-inducible transport system (pstS, pstC, pstA, and pstB). Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. Recombinant strains, which contained either pBC29 (carrying ppk) or pEP02.2 (pst operon), removed approximately twoand threefold, respectively, more P; from minimal medium than did the control strain. The highest rates of Pi removal were obtained by strain MV1184 containing pEP03 (ppk and ackA). However, unlike the control strain, MV1184(pEP03) released Pi to the medium after growth had stopped. Drastic changes in growth and Pi uptake were observed when pBC29 (ppk) and pEP02.2 (pst operon) were introduced simultaneously into MV1184. Even though growth of this recombinant was severely limited in minimal medium, the recombinant could remove approximately threefold more Pi than the control strain. Consequently, the phosphorus content of this recombinant reached a maximum of approximately 16% on a dry weight basis (49%o as phosphate). * Corresponding author. also has been found downstream of the ppk gene, constituting a polyP operon (2a). Under ordinary operating conditions, activated sludges are capable of removing an average of only 20 to 40% of the

Wastewaters containing hexavalent chromium (chromate: Cr042 ) are generated in many industrial processes, including chromium plating, metal cleaning and processing, wood preservation, and alloy preparation (6). Since Cr042 is harzardous to fauna and flora in natural ecosystems, these wastewaters must be treated before being discharged into the environment (7,8,10,11).In our previous studies (2, 4, 5, 9), we demonstrated the potential of a chromate-reducing bacterium, Enterobacter cloacae (strain HO 1), to remove Cr042 from wastewaters. To develop the biological treatment process, however, it must be considered that chromate-bearing wastewaters from industrial processes generally contain heavy metal cations, such as Cu2', Cd2~, Zn2, and Ni2+ (6). Since the heavy metal cations are known to be toxic for most organisms, they may greatly influence the performance of the biological process for treating Cr042 . However, there are presently no data published as to the effects of heavy metal cations on the bacterial reduction of Cr042 . In this communication, we demonstrate how heavy metal cations affect Cr042 reduction by E, cloacae strain HOI.Growth and reduction experiments were conducted anaerobically in KSC medium, as previously described (6,13). The pH of the KSC medium was 7.2. The heavy metal cations tested were Ag`' , Cd2 , Cot +, Cu`D , Hg2 +, Mn2D , Nit +, and Zn2' . The metal salts used were of the chloride type, except for ZnSO4, and the metal solution was added to the culture at various final concentrations just before the start of incubation. The concentration of viable cells was determined by plating 100,u1 of appropriately diluted culture onto nutrient agar and incubating the plates at 37°C for 16--20 h.

Hexavalent chromium (Cr6+) is toxic for most organisms. We isolated a bacterial strain that is capable of reducing toxic Cr6+ to trivalent chromium ( Cr3+ ). The reduced chromium formed insoluble hydroxides at a neutral pH and precipitated, thus making it less available to biological systems. The bacterial ability to reduce Cr6+ was proved to be useful for treating a Cr6+-contaminated industrial effluent.

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of P1 to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4°C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.

Genetic improvement of bacterial ability to accumulate phosphate (Pi) was investigated using Escherichia coli as a test organism. High levels of Pi accumulation were achieved by (i) modifying the genetic regulation and increasing the dosage of the E. coli genes encoding polyphosphate kinase (ppk), acetate kinase (ackA), and the phosphate inducible transport system (pstS, pstC, pstA, and pstB) and (ii) genetically inactivating ppx encoding exopolyphosphatase. Acetate kinase was employed as an ATP regeneration system for polyphosphate synthesis. The best recombinant strain, which contained both pBC29 (ppk) and pEP02.2 (pst genes) accumulated approximately 10-fold more Pi than did the control strain. The phosphorus content of this recombinant reached a maximum of 16 % on the dry weight basis (49 % as phosphate). About 65 % of the cellular phosphorus was stored as polyphosphate.