Biodegradation of L-Glutamatediacetate by Mixed Cultures

Chapter 10

Biodegradation of L-Glutamatediacetate by Mixed Cultures and an Isolate Cornelis G. van Ginkel, Roy Geerts, and Phuong D. Nguyen

Downloaded by EMORY UNIV on January 27, 2016 | http://pubs.acs.org Publication Date: July 21, 2005 | doi: 10.1021/bk-2005-0910.ch010

Akzo Nobel Chemicals Research, Velperweg 76, 6824 B M Arnhem, The Netherlands ([email protected])

The aerobic biodegradation of both enantiomers of glutamate-N,N-diacetate (GLDA) was studied according to official O E C D test guidelines. L - G L D A was found to be readily biodegradable. Degradation of D - G L D A was demonstrated in an inherent biodegradability test. Analysis of L - G L D A as well as monitoring the change of dissolved organic carbon demonstrate that L - G L D A degrades extensively following a short acclimatization period in activated sludge treatment plants. A bacterium strain BG-1 was isolated from activated sludge on the basis of its capacity to use L - G L D A as sole nitrogen, carbon and energy source. The isolated strain was identified as a Rhizobium radiobacter. Strain BG-1 also utilized glyoxylate, L-glutamate, and NTA. D - G L D A , S,S-EDDS, and E D T A did not support growth of the strain. Significant oxygen uptake rates by L-GLDA-grown washed cell suspensions were observed with glyoxylate, L-glutamate, and oxoglutarate. Iminodiacetate did not stimulate oxygen consumption by L - G L D A -grown cells. This substrate utilization pattern suggests that L - G L D A is degraded by the successive removal of the two carboxymethyl groups, resulting in the formation of L-glutamate. The results of this study strongly indicate that L - G L D A readily undergoes complete degradation by microbial cultures.

© 2005 American Chemical Society In Biogeochemistry of Chelating Agents; Nowack, Bernd, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Introduction Anthropogenic chelating agents are of great importance for different purposes. They have a wide variety of uses, from household cleaners, pulp and paper bleaching, water treatment, industrial and institutional cleaning to photography. L-Glutamate-N,N-diacetate (L-GLDA), a new chelating agent, is composed of two carboxymethyl groups linked to the nitrogen atom of L-glutamate. L - G L D A is primarily used in detergents. After use this chelating agent will end up in wastewater. Therefore, it is important to understand whether or not L - G L D A is biodegradable in the environment and technosphere. When the issue of biodégradation became a matter of public interest, tests to assess the biodegradability were developed. These biodegradability tests were internationally harmonized by the Organization of Economic Cooperation and Development (OECD). Not only the biodegradability of the parent compound is of importance but also the possible formation of recalcitrant metabolites. Biodégradation without the formation of recalcitrant metabolites can be demonstrated by determining the biodégradation pathway. The use of pure cultures of microorganisms is a valuable tool in elucidating the biodégradation pathway of chelating agents. Many bacteria that degrade chelating agents, i.e. nitrilotriacetate (NTA), ethylenediaminetetraacetate (EDTA), and 5,5-ethylenediaminedisuccinate (5,5-EDDS), have been isolated ( i , 2). Two metabolic pathways have been described by which microorganisms degrade aminocarboxylates. Evidence on microbial degradation of 5,5-EDDS demonstrates that microorganisms catalyze the cleavage of fumarate from ethylenediamine (5, 4, 5). Biodégradation of E D T A and N T A is initiated by a cleavage of the C - N bond, releasing glyoxylate as product (7, 2). We report here the biodégradation of L - G L D A and D - G L D A in wastewater treatment plants, using O E C D biodegradability tests. In addition, L - G L D A degradation by an isolate is described to provide evidence of complete mineralization.

Materials and methods Chemicals: L-Glutamate-N,N-diacetate ( L - G L D A ) (Dissolvine®) and D - G L D A were obtained from Akzo Nobel Chemicals, B U Functional Chemicals, Amersfoort, the Netherlands. A l l other chemicals used were purchased. The metal-chelate solutions were prepared by adding equimolar amounts of the respective metals as salts to L - G L D A or N T A solutions with a concentration of 2 g/L. Subsequently, the metal-chelate solutions were diluted to a concentration of 1 g/L of L - G L D A .

In Biogeochemistry of Chelating Agents; Nowack, Bernd, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Inocula and bacteria: The activated sludge for the biodégradation experiments was from the aeration tank of the municipal wastewater treatment plant Nieuwgraaf, Duiven, the Netherlands. The plant consists of mechanical and biological stages for the treatment of mainly domestic wastewater. Rhizobium radiobacter D S M Z 30147, Aminobacter aminovorans D S M Z 6449, and Chelatococcus asaccharovorans D S M Z 6461 were purchased from D S M Z , Braunschweig, Germany. Biodegradability tests: The capability of microorganisms to degrade L - G L D A and D - G L D A was determined, using O E C D biodegradability tests. The closed-bottle test was performed according to O E C D Test Guideline 301 D with some minor modifications (6, 7). Activated sludge from a municipal treatment plant and an S C A S unit used as inocula were diluted to a concentration of 2 mg/L dry weight in the bottles. The SCAS tests were performed in accordance with the O E C D Test Guideline 302 A (8). The daily fill and draw cycle involved the addition of domestic wastewater spiked with 110 mg/L of G L D A . The concentration of the activated sludge was initially 2 g/L dry weight. The removal of G L D A in the SCAS units was followed by nonpurgeable organic carbon (NPOC) and G L D A analyses. A standardized method was used for evaluating the behavior of L - G L D A in continuous activated sludge (CAS) systems (9). The reactor consisted of an aeration vessel capable of holding 0.35-Liter from which the liquor was passed continuously to a settler of 0.15-Liter capacity. Aeration was achieved through a capillary on the bottom of the aeration vessel at a rate of approximately 10 L/hour. Domestic wastewater spiked with 50 mg/L of L - G L D A was supplied with a pump. A n SRT of 10 days and an H R T of 10 hours were maintained in the C A S reactor. The liquor passed through the aeration vessel and settler was analyzed for N P O C , and L - G L D A . Removal of L - G L D A was assessed at 10 and 20°C. Isolation and characterization: The bacterium used in this study was isolated by selective enrichment on L - G L D A as the sole source of nitrogen, carbon, and energy. Activated sludge from a plant treating primarily domestic wastewater was used as inoculum. After growth was obtained, dilutions of the cell suspensions were streaked on agar plates containing L - G L D A in a nitrogenfree mineral salts medium (10). Colonies began to appear on these plates upon incubation at 30°C. One colony was picked and streaked to purity on L - G L D A agar plates. Analyses to identify the isolate were carried out by D S M Z (Braunschweig, Germany), using fatty acid methyl ester analysis and 16S r D N A sequencing. Growth, and washed cell suspensions: The isolate, Rhizobium radiobacter D S M Z 30147, Aminobacter aminovorans D S M Z 6449, and Chelatococcus asaccharovorans D S M Z 6461, were grown in 5-L Erelnmeyer flasks with


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500 mL of nitrogen-free mineral salts medium with 1 g/L L - G L D A , or N T A . The isolate was also grown on various other organic compounds (1 g/L) in a mineral salts medium containing ammonium as nitrogen source (10). The cultures were shaken at 200 rpm in an orbital incubator at 30°C. Growth on L - G L D A was followed by removing samples at various time intervals and analyzed for L - G L D A and ammonium. Cell suspensions of the batch cultures were harvested by centrifiigation at 10,000 χ g for 5 min at 4°C, washed three times with 100 m M phosphate buffer, p H 7.0 or with 50 m M HEPES buffer, p H 7.0. The washed cell suspensions were stored at 4°C. Respiration experiments: Oxygen uptake was measured with a Biological Oxygen Monitor (Yellow Springs Instruments, Yellow Springs, Ohio), which consisted of an electrode and a water-jacketed vessel (5 mL). Washed cell suspensions were incubated in the vessel at 30°C for at least five minutes to allow determination of the endogenous respiration rate. Subsequently, 0.1 m L of a substrate solution (1 g/L) was injected, and the increase in the respiration rate was determined. Influence of the counter ions on the oxidation of L - G L D A and N T A was assessed, using a H E P E S buffer ( / i ) . Analysis: L - G L D A was determined by high performance liquid chromatography (HPLC). The H P L C system consisted of a high precision pump model 300 (Separations, H.I. Ambacht, the Netherlands) a Spark autosampler model basic marathon (Separations, H.I. Ambacht, the Netherlands), an Ionpac AS7 column with an AG7 guard-column (Dionex, Bavel, the Netherlands), and a U V / V I S detector model A B I 759A (Separations, H.I. Ambacht, the Netherlands). The concentration of L - G L D A was measured at a wavelength of 330 nm. The mobile phase was 50 m M nitrate in demineralized water containing 50 m M sodium acetate, p H = 2.75 ± 0.2. This solution was filtered over a 0.45 μιη cellulose nitrate filter prior to use. The flow rate was 0.4 mL/min. Samples were prepared by mixing 4 mL of the sample with 1 m L of an iron nitrate solution (12.4 m M F e ( N 0 ) solution containing 151.2 m M nitric acid). Samples were shaken and allowed to stand for 15 minutes. Samples (50 μΐ) were injected after particles were removed by filtration through a 0.45-μπι filter. The accumulation of ammonium in the medium during the transformation of L - G L D A was determined colorimetrically at 690 nm by the formation of indophenol blue with hypochlorite and salicylate in the presence of sodium nitroferricyanide as catalyst (72). Samples were passed through a membrane filter (0.45-μιη) prior to analysis. 3

3

Dissolved organic carbon was determined using a T O C analyzer (Shimadzu Corporation, Kyoto, Japan). Samples were passed through a membrane filter


187 (0.45 μπι pore diameter) prior to analysis. Samples were acidified prior to injection in the T O C apparatus.


Results and discussion Assessment of the biodegradability: The most important aspect with regard to the environmental fate of G L D A is its biodegradability. G L D A contains one asymmetric carbon atom. Although only L - G L D A is marketed, the biodegradability of both enantiomers, i.e., L - G L D A and D - G L D A , was studied because of known enantio-selective degradation of other chelates (13,14). The closed-bottle test (OECD 301 D) has been established for the investigation of the ready biodegradability by mixed microbial cultures. For degradation to occur in the closed-bottle test, microorganisms must be present that are capable of utilizing the compound as an energy and carbon source. In the closed-bottle test, growth of microorganisms on L - G L D A is accompanied by oxygen consumption, exhibiting lag, exponential, and stationary phases (Figure 1). A high number of L-GLDA-degrading microorganisms present in the activated sludge probably account for a lag period of approximately 10 days. L G L D A exhibited a high degradation percentage exceeding the pass level within 22 days (Figure 1). L - G L D A does therefore meet the O E C D test criteria for ready biodegradability. D - G L D A did not degrade when incubated with activated sludge in the closed-bottle test. Failure to achieve biodégradation in the closedbottle test may either be due to recalcitrance of the test substance or to the severe conditions imposed during the ready biodegradability testing. Inherent biodegradability tests, therefore, are to satisfy conditions required to enable biodégradation, thus permitting the assumption that any failure to observe biodégradation is due to recalcitrance. The S C A S test (OECD 302 A ) was employed to demonstrate inherent biodegradability of D - G L D A . In the S C A S test, D - G L D A was degraded after an incubation period of approximately 8 weeks (Figure 2). For comparison an SCAS test was also carried out with L G L D A . L - G L D A and D - G L D A were shown to be almost completely removed within 3 and 10 weeks, respectively (Figure 2). After acclimatization in the S C A S units more than 95% of both L - and D-GLDA-carbon was removed from the influent. High removal of the organic carbon demonstrates that no recalcitrant water-soluble intermediates are formed during the degradation of both L - G L D A and D - G L D A . Inoculation of the closed-bottle test with acclimated sludge, which was derived from the SCAS unit fed with D - G L D A , resulted in 80% biodégradation of D - G L D A within 4 weeks. Acclimatization of sludge to D - G L D A also affected the biodégradation of L - G L D A . No lag phase was detected and biodégradation of L - G L D A up to 80% took place within 2 weeks (data not shown).



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Figure 1. Biodégradation of L-GLDA (Π) and D-GLDA (M) in Closed Bottle tests inoculated with activated sludge



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Ο

15

30

45

60

75

90

Time (days) Figure 2. Biodégradation of L-GLDA (Π ) and D-GLDA (U) in SCAS tests

The findings demonstrate that the biodegradability of L - and D - G L D A differs. In the literature many examples of the preferred usage of one of the enantiomers are found. Examples of enantio-selective biodégradation of chelates are E D D S and iminodisuccinate (IDS). The three enantiomers of IDS are biodegradable; R,R-1DS being the least biodegradable enantiomer (14). One enantiomer of E D D S , i.e., R R-EDOS, is not biodegradable, whereas &S-EDDS is readily biodegradable (13). Finally, a test simulating conventional activated sludge treatment was performed. In continuously-fed activated sludge (CAS) test, activated sludge was fed domestic wastewater spiked with L - G L D A . Biodégradation was followed by specific analysis of L - G L D A and by monitoring the change of dissolved organic carbon present in the effluent. At a temperature of 20°C, during an initial 10-day period little or no degradation of L - G L D A was observed due to the need for acclimatization of microorganisms. Lowering the temperature in the C A S test from 20° to 10°C, increased the lag period and the time required to obtain almost complete removal significantly (Figure 3). t

The temperature did not influence the extent of biodégradation (Figure 3). Additional analysis of the dissolved organic carbon demonstrated that L - G L D A was not converted into a recalcitrant organic substance. Consequently, L - G L D A will be removed almost completely under conditions prevailing in conventional activated sludge plants.



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Figure 3. Removal of L-GLDA in continuously-fed activated sludge units operated at an ERT and SRToflO hours and 20 days, respectively. The tests were carried out according to OECD guideline 303 A at incubation temperatures of 10 °C (Ώ)) and 20 °C (U)

Pure culture studies: Of general importance is the determination of the complete (ultimate) biodegradability of a substance as already indicated by the O E C D test results. To prove complete degradation a bacterium designated as strain BG-1 was isolated from activated sludge. The isolation of strain BG-1 capable of degrading L - G L D A from activated sludge without a history of L - G L D A discharge suggests that L - G L D A degrading microorganisms are widespread in the environment. Strain BG-1 is an aerobic, Gram-negative, rodshaped bacterium. The isolated strain was identified as a Rhizobium radiobacter. The type strain was unable to utilize L - G L D A . L - G L D A served as sole nitrogen, carbon and energy source for Rhizobium radiobacter B G - 1 . L - G L D A biodégradation was observed by virtue of its total disappearance from the growth medium as detected by H P L C . The consumption of L - G L D A by Rhizobium radiobacter strain BG-1 led to the release of ammonium. A t an initial substrate concentration of 4.0 m M the molar ratio of L - G L D A degraded to ammonia produced was found to be 1:0.5. The observed maximum doubling time of Rhizobium radiobacter growing on L - G L D A was 8 h. Rhizobium radiobacter



191 strain BG-1 was also able to grow on the aminoearboxylate N T A . Aminobacter aminovorans D S M Z 6449 and Chelatococcus asaccharovorans D S M Z 6461 (15) capable of utilizing N T A as sole carbon and energy source were also capable of growing on L - G L D A . Other aminocarboxylates did not support growth of Rhizobium radiobacter strain B G - 1 . These included D - G L D A , E D T A , 5,5-EDDS and diethylenetriaminepentaacetate. Oxidation of L - G L D A complexes was studied with washed cell suspensions of L-GLDA-grown Rhizobium radiobacter B G - 1 . Only limited oxidation of L - G L D A complexed with N i Co, Cu, Fe, and Z n with washed cell suspensions of L-GLDA-grown cells was detected. Washed cell suspensions of L - G L D A grown Rhizobium radiobacter BG-1 were capable of oxidizing L - G L D A in the form of Ca, M g , and M n complexes as well as uncomplexed L - G L D A . M g - N T A , Ca-NTA and M n - N T A were oxidized by washed cell suspensions of NTA-grown Rhizobium radiobacter BG-1 at high rates. The presence of these counter ions increased the oxygen uptake rate. Metal-NTA complexes with N i , Co, C u , Fe and Z n were not or slowly oxidized (Table I). Firestone and Tiedje (16) found comparable results with a bacterium capable of

Table I. Increase of oxygen uptake rates in percentages by washed cells suspensions grown on L-GLDA, and NTA at 30°C in the presence of the respective growth substrate and various counter ions (1:1 molar ratio). Rates of oxygen uptake are expressed as percentages increase compared to the endogenous respiration Substrate Counter ion none Ca Mg Mn Zn Fe Cu Co Ni 2 +

2 +

2 +

2 +

3+

2 +

2 +

2 +

L·GLDA 570 660 520 460 0 50 20 30 20

NTA 220 860 530 660 90 50 0 20 0

degrading N T A . For example, they found no oxygen uptake with C u - N T A and with N i - N T A and an intermediate rate with Zn-NTA. High rates were found for complexes with Ca and M n . VanBriessen et al (17) found the concentration of C a - N T A was rate limiting for biodégradation of N T A by C. heintzii and removal



192 of other species was dependent on the concentration of C a - N T A in equilibrium with other metal-chelate forms. Rhizobium radiobacter BG-1 was grown on a number of different substrates as sole source of carbon, after which respiration rates of washed cell suspensions were determined with a variety of substrates. Cells grown on L - G L D A respired D - G L D A and N T A . Washed cell suspensions of NTA-grown Rhizobium radiobacter strain BG-1 were able to oxidize L - G L D A but not D - G L D A . Further substrates oxidized by strain BG-1 grown on N T A were intermediates of N T A catabolism, i.e. iminodiacetate (IDA), glycine, and glyoxylate. Washed cell suspensions of L-GLDA-grown cells oxidized L - G L D A , glutamate, oxoglutarate, and glyoxylate. Trans-ketoglutaconate and I D A did not increase the rate of oxygen uptake by the washed cells above the endogenous rate. Glycine enhanced the respiration of L-GLDA-grown cells slightly (Table II). This substrate utilization pattern indicates strongly that Rhizobium Table II. Increase of oxygen uptake rates of washed cells suspensions grown on L-GLDA, NTA, and acetate at 30°C. Rates of oxygen uptake are expressed as percentages increase compared to the endogenous respiration

Substrate L·GLDA D-GLDA L-Glutamate D-Glutamate trans Ketoglutaconate IDA Glyoxylate Oxoglutarate Glycine Acetate NTA

L-GLDA 540 100 420 0 0 0 30 80 50 80 50

Growth substrate NTA Acetate 70 40 10 0 800 600 0 0 0 0 0 1500 20 30 120 120 80 160 470 150 0 1400

radiobacter BG-1 has evolved enzymes that convert L - G L D A into glyoxylate and L-glutamate. B y analogy with N T A metabolism, a pathway for L - G L D A degradation involving the successive removal of the carboxymethyl groups is proposed (Figure 4). Acetate-grown cells were not able to oxidize N T A and L - G L D A at high rates demonstrating that the enzymes catalyzing the degradation of the aminocarboxylates are not constitutively expressed.


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Figure 4. Proposed biodégradation route of L-GLDA

In conclusion, biodegradability test results and the isolation of strain BG-1 from activated sludge without a history of L - G L D A discharge demonstrate that L G L D A degrading microorganisms are widespread in the environment and biological treatment systems. These microorganisms degrade L - G L D A completely as strongly indicated by the Rhizobium radiobacter strain B G - 1 .

References 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11.

Nortemann, Β. Appl. Microbiol. Biotechnol. 1999, 51, 751-759. Bucheli-Witschel,M.;Egli, T. FEMS Microbiol. Rev. 2001, 25, 69-106. Takahashi, R.; Fujimoto, N . ; Suzuki, M.; Endo, T. Biosci. Biotechnol. Biochem. 1997, 61, 1957-1959. Takahashi, R.; Yamayoshi, K . N . ; Fujimoto, N . ; Suzuki, M . Biosci. Biotechnol. Biochem. 1999, 63, 1269-1273. Bucheli-Witschel,M.;Egli, T. Biodegradation. 1998, 8, 419-428. O E C D . Guidelines for testing of chemicals; Degradation and accumulation, No. 301: Ready biodegradability, Paris, France. 1992. Ginkel, C . G . van; Stroo, C . A . Ecotoxicol. Environ. Saf., 1992, 24, 319-327. O E C D Guidelines for testing chemicals; Degradation and accumulation No 302 A , Inherent biodegradability; modified SCAS test, Paris, France. 1981. O E C D Guidelines for testing chemicals; Simulation test - Aerobic sewage treatment. No 303 A , Paris, France. 1981. Kroon, A . G . M . ; Ginkel, C.G. van. Environ. Microbiol. 2001, 3, 131-136. Kluner, T.; Hempel, D . C . ; Nortemann, Β. Appl. Microbiol. Biotechnol. 1998, 49, 194-201.


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12. Verdouw, H.; Echteld van, C.J.A; Dekkers E.M.J. Water Res. 1978, 12, 399-402. 13. Schowanek, D . ; Feijtel, T.C.J.; Perkins, C.M.; Harman, F . A . ; Ferderle, T.W.; Larson, R.J. Chemosphere. 1997, 34, 2375-2391. 14. Reinecke, F.; Groth, T.; Heise, K.P.; Joentgen, W.; Muller, N.; Steinbuchel, A . FEMS Microbiol. Lett. 2000, 18, 41-46. 15. Auling, G . ; Busse, H.J.; Egli, T.; El-Banna, T.; Stackebrandt, E . System Appl. Microbiol. 1993, 16, 104-116. 16. Firestone, M . K . ; Tiedje, J.M. Appl Microbiol. 1975, 29, 758-764. 17. vanBriessen, J . M . ; Rittmann, B . E . ; Girvin, D.; Bolton, H . Environ. Sci. Technol. 2000, 3346-3353.


Biodegradation of L-Glutamatediacetate by Mixed Cultures

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