Registration Dossier

Environmental fate & pathways

Additional information on environmental fate and behaviour

Administrative data

Endpoint:
additional information on environmental fate and behaviour
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase
Author:
Cokesa, Z. et al.
Year:
2004
Bibliographic source:
Applied and Environmental microbiology 70 (7), July 2004, pp. 3941–3947

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 301F
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Test material form:
solid: granular

Results and discussion

Any other information on results incl. tables

Biodegradation tests. A standardized OECD test (301F) was carried out to analyze the biodegradability of the three IDS isomers, technical-grade IDS, and the metal-IDS complexes. As shown in Fig. 1, biodegradation of all IDS isomers and technical IDS attained approximately the level of the reference compound sodium benzoate. Interestingly, for the R,R-IDS isomer, a significant degradation rate was not observed before day 3.

The IDSdegrading microorganism in this study was isolated from activated sludge. It was enriched in batch culture with buffered mineral salts medium supplemented with 5 mM R,R-IDS as the sole source of carbon, nitrogen, and energy.

Isolation and identification of microorganisms. The IDSdegrading microorganism in this study was isolated from activated sludge. It was enriched in batch culture with buffered mineral salts medium supplemented with 5 mM R,R-IDS as the sole source of carbon, nitrogen, and energy. The enrichment cultures were incubated in a baffled Erlenmeyer flask on a rotary shaker at 125 rpm and room temperature. The cultures were subcultivated daily for 5 days. Subsequently, the cultures were repeatedly transferred on agar plates also containing 5 mMR,R-IDS until a pure culture (BY6) was isolated. Strain BY6 grew with R,R-IDS as the sole source of carbon, nitrogen, and energy. It was gram negative and oxidase positive and formed motile cocci of 1 to 1.5 m. Furthermore, strain BY6 was identified by 16S ribosomal DNA sequencing. Sequence

alignment with BLAST (1) achieved highest homologies (99%) with A. tumefaciens (accession number D14500) and A. tumefaciens strain CIP111-78 (AJ389897). Thus, the organism was named A. tumefaciens BY6. Growth of A. tumefaciens BY6 on IDS. A. tumefaciens BY6 grew in mineral salts medium with each of the three IDS isomers as the sole source of carbon, nitrogen, and energy. The doubling times on R,S-IDS, S,S-IDS, and R,R-IDS at 23°C were 3, 3.3, and 3.8 h, respectively. During growth on R,S-IDS, R,R-IDS, and S,S-IDS, the pH increased as a result of ammonia excretion by the cells. The amount of ammonia released into the medium after 5 days corresponded to approximately 30% 5% of the nitrogen contained in IDS (data shown for R,R-IDS in Fig. 3).

In addition, growth of A. tumefaciens BY6 was also possible on different metal-IDS chelates, namely, Mg2-, Ca2-, Fe2-, Cu2-, and Mn2-IDS. The complexes with the metal ions were generated by adding their sulfate salts in equimolar amounts to technical IDS. The degradation of metal-IDS complexes was monitored by measuring dissolved organic carbon.

Mg2- and Ca2-IDS complexes were degraded completely within 3 days, while only 75% of Fe2-IDS was mineralized in

this time span. Complete mineralization of Fe2-IDS and about 75% mineralization of Mn2-IDS were achieved within 10 days. In contrast, only about 25% of Cu2-IDS was mineralized within 10 days (data not shown). Metal hydroxide precipitation was noted during growth on Fe2-, Cu2-, and Mn2-IDS. These results correspond with the observations made during biodegradation tests with metal-IDS complexes.

Applicant's summary and conclusion

Conclusions:
The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Mg-IDS complexes as the sole carbon, nitrogen, and energy source.
Executive summary:

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F

(manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all

stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated

sludge. The strain was able to grow on each IDS isomer as well as on Fe2-, Mg2-, and Ca2-IDS complexes

as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn2-IDS were

rather scant and very slow on Cu2-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated

that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this

organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of

R,S-IDS, generating D-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation

is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any

cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified

lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy

metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete

mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all

stereoisomers into a catabolic pathway initiated by a stereoselective lyase.