Biodegradation of novel amino acid derivatives suitable for complexing agents in pulp bleaching applications

Science of the Total Environment 377 (2007) 45 – 51 www.elsevier.com/locate/scitotenv

Biodegradation of novel amino acid derivatives suitable for complexing agents in pulp bleaching applications Sirpa Metsärinne a,⁎, Erja Ronkainen a , Tuula Tuhkanen b , Reijo Aksela c , Mika Sillanpää a a

b

University of Kuopio, Department of Environmental Sciences, P.O. Box 1627, FI-70211 Kuopio, Finland Tampere University of Technology, Environmental Engineering and Biotechnology, P.O. Box 541, FI-33101 Tampere, Finland c Kemira Oyj, Espoo Research Centre, P.O. Box 44, FI-02271 Espoo, Finland Received 1 March 2006; received in revised form 15 January 2007; accepted 28 January 2007 Available online 7 March 2007

Abstract The biodegradability of four novel diethanolamine derivative complexing agents was examined by using two biodegradation tests standardised by OECD (301B and 301F). Ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA) were employed as reference substances. Biodegradation of the new complexing agents was studied both with unacclimated and acclimated inocula as well as by simulating wastewater treatment in sequencing batch reactors (SBRs). These new complexing agents were of technical grade, and therefore, the results are only indicative but these new compounds hold promise for use as complexing agents in the pulp and paper industry. The novel complexing agents were not readily biodegradable but they showed slight biodegradation. Around 10–30% degradation was found in the SBR where degradation was followed by measurement of concentration. Moreover the novel complexing agents did not have any negative impact on reactor performance as measured by chemical oxygen demand reduction. In the standardised biodegradation tests at best around 50% degradation was observed with the acclimated inoculum and in the prolonged test whereas EDTA and DTPA exhibited no biodegradation. The elevated degradation in acclimated sludge indicates that the water treatment plant microbes are capable of decomposing these molecules under favourable conditions. The total concentration of novel complexing agents decreased slightly during biodegradation tests, while the EDTA and DTPA concentrations remained stable. © 2007 Published by Elsevier B.V. Keywords: Biodegradation test; Complexing agents; Diethanolamine derivatives; DTPA; EDTA; Sequencing batch reactor

1. Introduction During the past decade, bleaching methods based on oxygen chemicals, e.g., hydrogen peroxide and peroxy ⁎ Corresponding author. Present address: Orion Corporation, P.O. Box 1780, FI-70701 Kuopio, Finland. E-mail address: [email protected] (S. Metsärinne). 0048-9697/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.scitotenv.2007.01.097

acids became popular for environmental reasons. Since transition metal ions, especially iron (Fe 3+ ) and manganese (Mn2+) ions, can catalyse the decomposition of hydrogen peroxide and peroxy acids, the removal of transition metal ions from the bleaching liquors by complexation is an essential step in the elemental chlorine free (ECF) or total chlorine free (TCF) bleaching processes. Ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are two

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commonly used complexing agents in these kinds of chelation processes. Neither EDTA nor DTPA have shown any signs of breaking down in biodegradability tests (Gerike and Fischer, 1979; Means et al., 1980; Boatman et al., 1986; Allard et al., 1996; Kari and Giger, 1996) though under specific environmental conditions degradation can occur (van Ginkel et al., 1997). Therefore, it is evident that significant quantities of EDTA and DTPA are being released into natural water sources. Several authors have investigated the occurrence of EDTA in the European aquatic environment, whereas DTPA has received much less attention. EDTA is commonly found in a concentration range about 10–60 μg/l in surface waters. DTPA has been found at concentrations of 2–15 μg/l in the River Rhine, Germany (Wanke and Eberle, 1992) and at 9–18 μg/l in Lake Saimaa, Finland, a water course polluted by the effluents of three pulp and paper mills (Sillanpää and Oikari, 1996). EDTA and DTPA are not expected to be acutely toxic to aquatic organisms, but it is thought that they evoke long-term adverse effects (Sillanpää, 1997). They can certainly desorb heavy metals bound to sediments and also prevent heavy metal sedimentation, thus increasing the cycling of these compounds in water. EDTA and DTPA are a major source of nitrogen in the effluents emerging from pulp mills and after their slow biochemical degradation in the surface waters these complexing agents constitute a source of nutrient nitrogen for aquatic algae (Belly et al., 1975).

A series of new diethanolamine derivatives as complexing agents capable of stabilising peroxy compounds in bleaching conditions have been designed (Aksela et al., 1997): N-bis[2-(carboxymethoxy)ethyl] glycine (BCA3), N-bis[2-(methyl-carboxymethoxy) ethyl]glycine (MBCA3), N-bis[2-(1,2-dicarboxyethoxy)ethyl]glycine (BCA5) and N-bis[2-(1,2-dicarboxyethoxy)ethyl] aspartic acid (BCA6). The molecular structures of these complexing agents are presented in Fig. 1. The performance of BCA5 and BCA6 was shown to be comparable to that of EDTA and DTPA in bleaching sequences of TCF pulp (Aksela et al., 2001; Hyvönen et al., 2006). BCA5 and BCA6 have proven to be the only known phosphorus-free complexing agents capable of deactivating iron and manganese ions in the presence of strong oxidants like chlorine dioxide or peroxy acids (Jäkärä et al., 1999; Parén et al., 1997). They can efficiently deactivate Fe and Mn ions by forming inactive metal complexes, whereas Mn–EDTA and Mn–DTPA complexes decompose chlorine dioxide or peracetic acid. In addition, they have a substantially lower nitrogen content compared to EDTA or DTPA. The aim of this study was to determine the biodegradability of these diethanolamine derivatives. Two biodegradation tests standardised by OECD (301B and 301F) were applied. EDTA and DTPA were used as reference substances. The biodegradation of the new complexing agents was studied both with unacclimated

Fig. 1. The molecular structures of the four new alternative complexing agents investigated in the present study.

S. Metsärinne et al. / Science of the Total Environment 377 (2007) 45–51 Table 1 Contents of tested new complexing agents

BCA3 Glycolic acid Oxalic acid N-bis(2-hydroxyethyl)glycine Diethanolamine MBCA3 “Monoalkylated product”: N-[2(1-carboxyethoxy)]-[N-(2-hydroxyethyl)] glycine Lactic acid BCA5 Oxybis(butanedioic acid) Maleic acid Malic acid BCA6 “Monoalkylated product”: N-[2(1,2-dicarboxy-ethoxy)ethyl-N(2-hydroxyethyl)-aspartic acid N-bis[2-(1,2-dicarboxy-ethoxy)ethyl]amine Sodium fumarate Diethanolamine Maleic acid Malic acid Oxalic acid

Batch A (%)

Batch B (%)

62.9 9.9 8.5 18.7 0 55.0 36.7

52.8 6.1 14.0 25.2 1.9 63.0 31.4

8.3 82.1 5.6 11.5 0.8 89.5 2.9

5.6

65.4 0

5.1 1.0 1.5 0 0 0

11.5 4.3 10.1 2.9 3.9 1.9

and acclimated inoculum. Biodegradation was also studied by simulating wastewater treatment in sequencing batch reactors. 2. Materials and methods 2.1. Chemicals The new complexing agents were prepared by at Kemira Oyj Espoo research centre. They were of technical grade, pure substances were not available. The purity of the complexing agents was determined by 13C NMR, and the values are presented in Table 1. There were two different batches of BCA3, MBCA3 and BCA6: Batch A of BCA3 and MBCA3 was used in the unacclimated tests and Batch B in acclimated tests. Batch B of BCA6 was used in the CO2 evolution test where the inoculum was taken from municipal wastewater treatment plant; all other tests were carried out with Batch A. Due to the molecular asymmetry and due to the non-selective preparation method, BCA5 contains three conformational isomers, SS-, SR- and RR-BCA5. Similarly, the samples of BCA6 consist of a mixture of six conformational isomers, SSS-, SSR-, SRR-, RRR-, SRS- and RSR-BCA6.

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Reference substances, EDTA disodium salt dihydrate (Titriplex® III, pro analyse) were purchased from Merck KgaA, Germany, and DTPA was from Sigma. Mineral medium for biodegradation tests was prepared in distilled water in accordance with the OECD Guideline for Ready Biodegradability Tests (OECD, 1992) and all other chemicals were of analytical grade. 2.2. Sequencing batch reactors The biodegradation of all new complexing agents was studied by simulating wastewater treatment in a laboratory-scale SBR (Fig. 2). SBR was run by using municipal wastewater and activated sludge. SBR is a fill-and-draw activated sludge treatment system. The unit processes involved in the SBR and conventional activated sludge systems are similar with aeration and sedimentation being carried out in both systems. The only important difference is that in conventional plants, the processes are carried out simultaneously in separate tanks, whereas in the SBR operation, the processes are carried out sequentially in the same tank. The laboratory-scale SBR operated in four sequences: (1) fill, (2) aeration, (3) settle and (4) draw (decant). The total volume of the reactor was 2.0 l, the liquid volume was 1.5 l and the volume of fill and draw was 1.0 l. The hydraulic retention times used were 12 h or 24 h, mixed-liquor suspended solids (MLSS) was from 1.5 to 2.5 g/l, sludge loading of 0.4–0.5 g chemical oxygen demand (COD)/g MLSS-day, and sludge age of 19 days. The system included four reactors: one was control and three for biodegradation testing. Complexing agents were added one at a time to the influent. The concentration of complexing agent was gradually increased to 30 mg/l before degradation studies were started. The degradation of complexing agent was studied by analyzing complexing agent concentration in the influent and in the corresponding effluent. Performance of SBR was also followed by measuring COD reduction. The complexing agent concentrations were analysed by high performance liquid chromatography; the analytical procedure will be described elsewhere (Metsärinne et al., 2005). The sludge volume index (SVI) was used as an indication of the settling characteristics of the sludge. 2.3. Biodegradation tests The biodegradation tests, CO2 evolution test (OECD 301B) and Manometric respirometry test (OECD 301F) were carried out adhering to the respective OECD guidelines. Test duration was 28 days as defined in

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Fig. 2. A schematic diagram of the SBR including three reactors for test chemical and one as control.

OECD guidelines, but also prolonged CO2 evolution tests (around 50 days) were carried out. The Manometric respirometry test was performed by using an automatic BOD analyzer, OxiTop® Control AN12 (WTW, Weilheim, Germany). The inoculum for the biodegradation tests was taken from the SBR. The acclimated inoculum for biodegradation tests was taken at least 2 weeks after adding the complexing agent to the influent. The sludge for tests without acclimation was taken before addition of the complexing agent. Biodegradation of EDTA, BCA5 and BCA6 was also studied with inoculum taken from a municipal wastewater treatment plant. The test performance was assured with readily biodegradable S, S-ethylenediaminedisuccinic acid (S,S-EDDS) both in CO2 evolution and in the Manometric respirometry test. The CO2 evolution test with unacclimated inoculum was carried out with the novel complexing agents and with the reference substances, EDTA and DTPA. The new complexing agents were also tested with the acclimated inoculum. The Manometric respirometry test was carried out with BCA3 and MBCA3 with acclimated inoculum. BCA3 contained N,N-bis(2-hydroxyethyl) glycine in significant quantities, and its biodegradability was tested in the CO2 evolution test. It was found to be readily biodegradable. Since all of the novel complexing agents were only of technical quality, degradation was also followed by measuring the concentration of the

complexing agents in both biodegradation tests. The samples for HPLC analysis were taken at the beginning and at the end of the tests. 3. Results and discussion 3.1. Sequencing batch reactors The biodegradation of all novel complexing agents was studied by simulating wastewater treatment in SBR. As shown in Fig. 3, no significant reduction of these complexing agents was observed in SBR; only about 10–30% became degraded. On the other hand, these complexing agents caused no negative effect on SBR performance since the COD reduction was rather similar in both control and test units. During conventional biological wastewater treatment, EDTA is neither biodegraded nor absorbed onto activated sludge (Alder et al., 1990; Saunamäki, 1995; Kari and Giger, 1996). Previous studies have shown that DTPA has a negative impact on the microbial community in an activated sludge reactor as well as on reactor performance treating totally chlorine-free (TCF) and elemental chlorine-free (ECF) bleached pulp mill effluent (Larisch and Duff, 1997, 2000). DTPA can solubilise cell capsular material at a concentration 50–100 mg/l which is only slightly higher than the concentration used in this study, 30 mg/l.

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The solubilisation of capsular material may render the sludge more susceptible to other toxic effects. Higher concentrations of DTPA have decreased substrate uptake kinetics, disrupted flock structure and decreased BOD removal efficiency.

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Table 2 Results of the biodegradation tests Compound

BCA3 MBCA3 BCA5 BCA6 EDTA

DTPA

CO2 evolution test

Manometric respirometry test

Unacclimated inoculum, (%) degraded

Acclimated inoculum, (%) degraded

Acclimated inoculum, (%) degraded

4 42 53 (47 days) 22 20 a 7 29 a 4 3a 7 (57 days) 0

19 47

37 45

27

n.a.

8 15 (57 days) n.a.

n.a. n.a.

n.a.

n.a.

n.a.: not analysed. CO2 evolution test was carried out both with unacclimated and acclimated inoculum; the Manometric respirometry test, only with acclimated inoculum. Test duration was 28 days, prolonged durations are declared in parenthesis. a Inoculum taken from municipal wastewater treatment plant.

3.2. Biodegradation tests

Fig. 3. Reduction of tested complexing agents and COD in sequencing batch reactors.

It is important to bear in mind that the present results are only indicative since these tested new complexing agents were of technical grade. The biodegradation of these new alternative complexing agents was compared to that of EDTA and DTPA by using the CO2 evolution test. The results are presented in Table 2. The most biodegradable compounds with unacclimated inoculum taken from SBRs were MBCA3 and BCA5, while degradation of BCA3 and BCA6 was similar to that seen with EDTA and DTPA i.e. less than 10% was degraded after incubation for 28 days. However, BCA6 was much better degraded by the inoculum taken from a municipal wastewater treatment plant though it must be noted that the Batch B of BCA6 used in this test contained more biodegradable organic impurities than the Batch A used in other tests. The inoculum, the source of microorganisms for the test, is one important factor which cannot be standardised (Pagga, 1997). The composition and the quantity of inoculum are the major factors which affect the reproducibility of results in ready biodegradability tests (Vazguez-Rodriguez et al., 2000). Acclimated inoculum was used in the Manometric respirometry test and in the CO2 evolution test for all compounds except EDTA and DTPA. The biodegradation of BCA3 improved markedly after inoculum was acclimated to the compound before the test (Table 2). However, acclimation had no effect on the biodegradation of BCA6, and only a slight impact on the

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degradation of MBCA3 and BCA5. According to the ready biodegradation tests standardised by OECD, the acclimation of inoculum prior to biodegradation tests is not allowed. This might lead to underestimation of biodegradability of many chemicals which are sufficiently broken down, especially in adapted wastewater treatment plants, but these compounds do not meet the criteria for ready biodegradability. The Manometric respirometry test gave higher biodegradabilities than the CO2 evolution test in the case of BCA3. The acclimation time of the inoculum was longer in the Manometric respirometry test (31 days) than in CO2 evolution test (16 days), which might explain the difference in the results between these two tests. In prolonged tests, better biodegradabilities were obtained with MBCA3 and BCA6 which was probably due to acclimation to the test chemical. MBCA3 was degraded even better in the prolonged test with unacclimated inoculum than with inoculum acclimated to it before the test in SBRs. It seems that acclimation was more efficient in test conditions than in SBRs. Other studies have shown over 35% degradation of BCA6 in a prolonged (75 days) incubation under similar test conditions (Itävaara and Vikman, 1997). Complexing agent concentrations were studied before and after every biodegradation test. Only BCA5 with unacclimated inoculum was degraded by 10% as assayed via its concentration. The difference in BCA5 degradation results is due to degradation of organic impurities. Concentration of the other novel complexing agents did not decrease significantly. EDTA and DTPA concentrations remained stable during the tests. Even though these new alternative complexing agents were not readily biodegradable, our future aim is to study the biodegradability of different stereoisomers of the molecules containing asymmetric centres. It has been shown that the isomers exhibit different biodegradabilities. For example, ethylenediaminedisuccinic acid (EDDS) possesses three stereoisomers (S,S-, R,R-and R,S-EDDS) but only S,S-EDDS is readily biodegradable, whereas R,R-EDDS is very stable (Schowanek et al., 1997). On the other hand, even partial degradation of complexing agents in the natural environment is desirable so that they lose their ability to mobilise heavy metals from sediments. 4. Conclusions Based on these results, it seems that none of these new alternative complexing agents is readily biodegradable. Around 10–30% degradation of these new complexing agents was observed in the wastewater

treatment simulation in SBR where degradation was followed by measurement of concentration. In the standardised biodegradation tests, MBCA3 appeared to be the most biodegradable compound but also BCA5 was more biodegradable than EDTA and DTPA. However, it must be remembered that these new complexing agents were of technical quality and included other organic substances which may complicate the interpretation of our results. The preliminary results obtained in this study will need to be confirmed by using purified molecules. Moreover, we need to define the biodegradability of the separate optical isomers of the molecules containing asymmetric centres. There is a clear longterm need to replace the conventional complexing agents EDTA and DTPA, with better compounds, which not only contain less nitrogen and are more biodegradable but which also provide an improved technical performance in the actual bleaching process. Acknowledgements This study was financially supported by The National Technology Agency of Finland (Tekes), Maa-ja vesitekniikan tuki ry, Maj and Tor Nessling Foundation and Kuopio University Foundation. The technical assistance of Mr Matti Pessi with the sequencing batch reactors and of Mr Arto Moilanen with the COD analysis is gratefully acknowledged. References Aksela R, Renvall I, Parén A. N-Bis- or N-tris-[(1,2-dicarboxyethoxy)-ethyl]-amine derivatives and preparation and use of the same. Pat Appl 1997 [WO9745396]. Aksela R, Parén A, Jäkärä J, Renvall I. The overall performance of new diethanolamine derivatives as complexing agents in peroxide and peracetic acid bleaching of TCF pulp. 11th International symposium on wood and pulping chemistry. Nice, France. Conference proceeding; 2001. p. 127. Alder AC, Siegrist H, Gujer W, Giger W. Behaviour of NTA and EDTA in biological wastewater treatment. Water Res 1990;24:733–42. Allard A-C, Renberg L, Neilson AH. Absence of 14CO2 evolution from 14C-labelled EDTA and DTPA and the sediment/water partition ratio. Chemosphere 1996;33:577–83. Belly RT, Lauff JJ, Goodhue CT. Degradation of ethylenetrieminetetraacetic acid by microbial populations from aerated lagoon. Appl Microbiol 1975;29:787–94. Boatman RJ, Cunningham SL, Ziegler DA. A method for measuring biodegradation of organic chemicals. Environ Tox Chem 1986;5: 233–43. Gerike P, Fischer WK. A correlation study of biodegradability determinations with various chemicals in various tests. Ecotoxicol Environ Saf 1979;3:159–73. Hyvönen H, Orama M, Arvela R, Henriksson K, Saarinen H, Aksela R, Parén A, Jäkärä I, Renvall I. Studies on three new environmentally friendly chelating ligands. Appita J 2006;59:142–9.

S. Metsärinne et al. / Science of the Total Environment 377 (2007) 45–51 Itävaara M, Vikman M. Test report BEL 235/97, VTT, Bio and Food Technology, Finland; 1997. Jäkärä J, Aksela R, Parén A, Renvall I. Bleaching of chemical pulp and treatment with chelating agents. PCT Int Appl 1999 [WO 9925919]. Kari FG, Giger W. Speciation and fate of ethylenediaminetetraacetate (EDTA) in municipal wastewater treatment. Water Res 1996;30: 122–34. Larisch BC, Duff SJB. Effect of H2O2 and DTPA on the characteristics and treatment of TCF (totally chlorine-free) and ECF (elementally chlorine-free) kraft pulping effluents. Water Sci Technol 1997;35:163–71. Larisch BC, Duff SJB. Effect of DTPA and EDTA on activated sludge reactors treating bleached kraft mill effluent. Tappi J 2000;83:54. Means JL, Kucak T, Crerar DA. Relative degradation rates of NTA, EDTA and DTPA and environmental implications. Environ Pollut Ser B Chem Phys 1980;1:45–60. Metsärinne S, Ronkainen E, Aksela R, Tuhkanen T, Sillanää M. Determination of novel complexing agents in pulp and paper mill effluents and in humic lake water by liquid chromatography. J Chromatogr A 2005;1094:56–9. OECD. Guidelines for testing of chemicals; 1992. Pagga U. Testing biodegradability with standardized methods. Chemosphere 1997;35:2953–72.

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Parén A, Renvall I, Aksela R. N-Bis- or N-tris-[(1,2-dicarboxyethoxy)-ethyl]-amine derivatives and preparation and use of the same. Pat Appl 1997 [WO9745396]. Saunamäki R. Treatability of wastewaters from totally chlorine free bleaching. Tappi 1995;78:185–92. Schowanek D, Feijtel TCJ, Perkins CM, Hartman FA, Federle TW, Larson RJ. Biodegradation of [S,S], [R,R] and mixed stereoisomers of ethylene diamine disuccinic acid (EDDS), a transition metal chelator. Chemosphere 1997;34:2375–91. Sillanpää, M., 1997. Analysis and environmental fate of EDTA and DTPA. Dissertation, Helsinki University of Technology, Finland. Sillanpää M, Oikari A. Transportation of complexing agents released by pulp and paper industry: a Finnish lake case. Toxicol Environ Chem 1996;57:79–91. van Ginkel CG, Vandenbroucke KL, Stroo CA. Biological removal of EDTA in conventional activated sludge plants operated under alkaline conditions. Bioresour Technol 1997;59:151–5. Vazguez-Rodriguez G, Goma G, Rols JL. Toward a standardization of the microbial inoculum for ready biodegradability testing of chemicals. Water Sci Technol 2000;42:43–6. Wanke VT, Eberle SH. Die gaschromatographische Bestimmung von diethylentriaminpentaessigsäure in oberflächenwasser. Acta Hydrochim Hydrobiol 1992;20:192–6.