Isolation of Resistant Organisms. The study of complexation of trace metals in soils is extremely difficult due to the complexity of the soil system (Keeney and Wildung, 1976). In fact, although much information is available regarding organic ligands in soil (previous section), an organometal complex has never been isolated intact from soils. A logical approach to the study of microbial transformations of the transuranic elements is to isolate, from soil, resistant organisma most likely to alter transuranic form, study the transformation tn vitro and validate the results in the soil system using techniques specifically tailored to metabolites identified from the simpler in vitro systems. Application of enrichment techniques to the isolation of Pu resistant fungi, which have been demonstrated (previous section) to be the most resistant class of microorganisms, and actinomycetes from soil using Starch as a C-source (Schnefderman et al., 1975) resulted in the isolation of 14 fungal cultures and 13 cultures of actinomycetes distinct in colonial morphology. Of these, 7 of the actinomycetes and 5 of the fungal isolates were capable of growth at 100 ug/ml Pu as the soluble DTPA complex. There appeared to be a succession of actinomycetes types in the soil during incubation as indicated by the different colony morphologies obtained from enrichmenta after 4 and 25 days incubation. Although thia phenomenon may have resulted from changes in the eoil arising from the production of metabolites or chemical degradation products, it may alao have resulted from a responee to the presence of Pu. Only one actinomycete isolate waa found which was common to enrichments from both incubation periods and this organism was present at all Pu concentrations in the media. In contrast, the fungal isolatee exhibited 6 common morphological types regardiess of incubation period. Subsequent enrichment atudies (Pelroy, 1976; persona) communication) have resulted in the isolation of 30 distinct cultures of bacteria from soil. Of these 11 were resistant to Pu at concentrations as high as 100 jig/ml. These studies aleo indicated that C source as well as soil Pu concentration will play a role in determining the types and numbers of Pu resistant microorganisms present in soil, providing presumptive evidence that microbial metabolites, which will differ with C source, may play a role in Pu resistance. This subject will be discussed in the next section. The presence of Pu resistant organisms is apparently related to factors that tay be expected to vary with soil type and environmental conditions, Again, similar studies have not been conducted with other transuranic elements. Microbial Transformations As previously discussed, there are several means whereby microorganisms May transform trace metals in soil. These may be generalized to (1) direct mechanisms euch as alteration in valence state or alkylation (2) indirect mechanisms such as interactions with normal metabolites or microbial alterations of the physicochemical environment and (3) cycling mechanisms such as uptake during cell growth and release on ceil decomposition. In the latter case, any combination of indirect and direct methods of alteration may be operational. Although there have been no studies conducted to 152 date which would allow the unequivocal separation of these mechanisms, studies have been conducted which demonstrate the alteration of Pu form in vitro by soil microorganisms and provide evidence for both direct and indirect transformation of Pu. Direct Transformations. The potential for direct transformation of the transuranic elements through alteration of valence state or alkylation is difficult to assess. Although, as previously discussed, the transuranics have the potential for existing in aqueous solution in several valence states, information is not available to assess the role of soil microflora in direct alteration of valence. More information is available regarding the mechanism of metal alkylation. Alkylation of metals involving the alkyl donor methyl cobalamine and other alkyl cobalamines has been clearly demonstrated for Hg, As, and Pt (Wood et al., 1968; McBride and Wolfe, 1971; Taylor and Hanna, 1977). The methyl derivative of Hg may be present in significant quantities in soils (Beckert et al., 1974). Wood (1974) suggested that methylated derivates of Hg and As are important in governing their behavior in the environment. McBride (1977) also suggested that these reactions occur abiotically. The process of biochemical methylation of metals may be described as an overlap between the chemistry of methyl cobalamine (an intermediate in methane synthesis by anaerobic bacteria and methionine synthesis in aerobic bacteria) and the chemistry of the metals. In the case of the transuranics, particularly Pu, it is the complexity of the aqueous chemistry that has limited research into alkylation phenomena. It ta unknown whether an ionic species of Pu is capable of reacting in vitro with an alkyl cobalamine. Further, if a mechanism for biological alkylation of Pu, similar to the Hg, As, Pt alkylation reaction did exist, it would be of importance in influencing environmental behavior only if the alkylated molecule exhibited stability (Wood, 1974), i.e., a half-life in soils and sediments of hours rather than seconds. Considering the coordination chemistry of the actinides, Marks (1976) noted that U- and Th-C linkages are formed in organic solvents and the complexes are relatively stable thermally though highly sensitive to oxygen. Meaningful microbial studies await the development of an understanding of the chemical speciation of transuranics in aqueous solutions at environmental concentration levels. Indirect Transformations. The potential for indirect transformation of the transuranic elements may be greater than for direct transformation. The potential for Pu interaction with microbial celle and metabolites has been demonstrated and many of the other transuranics form stable complexes with known microbial metabolites. Beckert and Au (1976) demonstrated the uptake of 7°*Pu, applied initially to malt agar in nitrate, citrate and dioxide forms, by a common soil fungus, Aspergillus niger. Using a specialized spore collection method, the Pu was shown te be present in the fruiting bodies. Subsequent washing to remove external contamination indicated that the major portion of the 239py was incorporated into the spores. Similar results were obtained in preliminary studies with U, Th, and Po in mine tailings. 153 The order of