Class of Kinetic Hydrate Inhibitors with Good Biodegradability - Energy

Jul 29, 2008 - Telephone: +47-51831823. ... Current commercial kinetic hydrate inhibitors are used for field applications ... Kinetic Hydrate Inhibito...
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Energy & Fuels 2008, 22, 3143–3149

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Class of Kinetic Hydrate Inhibitors with Good Biodegradability Luca Del Villano, Roald Kommedal, and Malcolm A. Kelland* Department of Mathematics and Natural Sciences, Faculty of Science and Technology, UniVersity of StaVanger, 4036 StaVanger, Norway ReceiVed March 3, 2008. ReVised Manuscript ReceiVed May 14, 2008

Kinetic hydrate inhibitors have been used successfully in the field for about the last 13 years to prevent gas hydrate formation mostly in gas- and oilfield production lines. They work by delaying the nucleation and often also the growth of gas hydrate crystals for periods of time dependent upon the subcooling in the system. Current commercial kinetic hydrate inhibitors are used for field applications where the subcooling is as high as about 10 °C. In the Norwegian sector of the North Sea, very few of the commercial kinetic hydrate inhibitors are available for use offshore because of poor environmental properties usually related to biodegradability. We have designed and synthesized a class of kinetic hydrate inhibitor, which appears to show good biodegradability (OECD306, >20% in 28 days). Inhibitor performance tests have been carried out in stirred autoclaves (titanium and sapphire) using a natural gas blend and saline water giving structure II hydrates. In the presence of solvents, we have obtained a fairly good performance of the new inhibitors but a little lower than that of a current commercial inhibitor Luvicap 55W.

Introduction Kinetic hydrate inhibitors (KHIs) are a class of low-dosage hydrate inhibitor (LDHI) that have been in commercial use in the oil and gas industry for about 14 years.1 KHIs are watersoluble polymers, often with added synergists that improve their performance. KHIs delay the nucleation and usually also the crystal growth of gas hydrates. The nucleation delay time (induction time), which is the most critical factor for field operations, is dependent upon the subcooling (∆T) in the system: the higher the subcooling, the lower the induction time. The absolute pressure is also a factor.2–4 There are currently only two main classes of polymers, which are used in KHI formulations in oil and gas field operations. They are (1) homopolymers and copolymers of vinyl caprolactam and (2) hyperbranched poly(ester amide)s. These polymers are being successfully used to prevent gas hydrate formation in pipelines in locations, such as the U.K. sector of the North Sea, the Gulf of Mexico, South America, and the Middle East.5–11 At the time of writing, only one * To whom correspondence should be addressed. Telephone: +4751831823. Fax: +47-51831750. E-mail: [email protected]. (1) Kelland, M. A. Energy Fuels 2006, 20, 825. (2) Arjmandi, M.; Tohidi, B.; Danesh, A.; Todd, A. C. Chem. Eng. Sci. 2005, 60, 1313–1321. (3) Peytavy, J.-L.; Gle´nat, P.; Bourg, P. In Proceedings of the International Petroleum Technology Conference, Dubai, United Arab Emirates, Dec 4-6, 2007; IPTC 11233. (4) Kelland, M. A.; Mønig, K.; Iversen, J. E.; Lekvam, K. A feasibility study for the use of kinetic hydrate inhibitors in deep water drilling fluids. In Proceedings of the 6th International Conference on Gas Hydrates, Vancouver, British Columbia, Canada, July 6-10, 2008. (5) Argo, C. B.; Blaine, R. A.; Osborne, C. G.; Priestly, I. C. In Proceedings of the SPE International Symposium on Oilfield Chemistry, Houston, TX, Feb 1997; SPE 37255. (6) Talley, L. D.; Mitchell, G. F. In Proceedings of the 30th Annual Offshore Technology Conference, Houston TX, May 3-6, 1998; OTC 11036. (7) Fu, S. B.; Cenegy, L. M.; Neff, C. In Proceedings of the SPE International Symposium on Oilfield Chemistry, Houston, TX, Feb 1316, 2001; SPE 65022.

polymer from these classes is available for use offshore Norway because the environmental authorities demand at least 20% biodegradability for new oilfield chemicals and that the products of degradation are also environmentally friendly. We have endeavored to find a new polymer class that is both high performing and has at least 20% biodegradation in the Organization for Economic Cooperation and Development (OECD) 306 test. Polyaspartates are already used as scale and corrosion inhibitors in the oil and gas industry because of their high biodegradability.12–15 A related class of polypeptide polymer that we have investigated as potentially biodegradable KHIs is polyaspartamides.16 Instead of pendant carboxylate groups that are found in polyaspartates, polyaspartamides have pendant N-alkylamide or N,N-dialkylamide groups. Thus, both the peptide backbone and side groups have hydrolyzable linkages that could lead to good biodegradation. The use of polyaspartate (8) Phillips, N. J.; Grainger, M. In Proceedings of the Annual Gas Technology Symposium, Calgary, Alberta, Canada, March 15-18, 1998; SPE 40030. (9) Leporcher, E. M.; Fourest, J. P.; Labes-Carrier, C.; Lompre, M. In Proceedings of the SPE European Petroleum Conference, The Hague, The Netherlands, Oct 20-22, 1998; SPE 50683. (10) MacDonald, A. W. R.; Petrie, M.; Wylde, J. J.; Chalmers, A. J.; Arjmandi M. In Proceedings of the SPE Gas Technology Symposium, Calgary, Alberta, Canada, May 15-17, 2006; SPE 99388. (11) Gle´nat, P.; Peytavy, J. L.; Holland-Jones, N., Grainger, M. In Proceedings of the 11th Abu Dhabi International Petroleum Exhibition and Conference, Abu Dhabi, United Arab Emirates, Oct 10-13, 2004; SPE 88751. (12) Little, B. J.; Sikes, C. S. Corrosion inhibition by thermal polyaspartate. In Surface ReactiVe Peptides and Polymers: DiscoVery and Commercialization; Sikes, C. S., Wheeler, A. P., Eds.; American Chemical Society: Washington, D.C., 1990; ACS Symposium Series 444. (13) Fan, J. C.; Fan, L.-D. G.; Mazo, J. Composition for inhibition of metal corrosion. U.S. Patent 6,620,338, 2003. (14) Kohler, N.; Courbinm, G.; Estievenart, C.; Ropital, F. Polyaspartates: Biodegradable alternatives to polyacrylates or noteworthy multifunctional inhibitors? NACE CORROSION Conference, 2002; paper 02411. (15) Ross, R. J.; Low, K. C.; Shannon, J. E. Mater. Perform. 1997, p53. (16) Kelland, M. A. Additives for inhibiting gas hydrate formation. International Patent Application WO/2008/023989.

10.1021/ef800161z CCC: $40.75  2008 American Chemical Society Published on Web 07/29/2008

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Figure 2. Sapphire cell high-pressure test equipment. Figure 1. Synthesis of polyaspartamides from polysuccinimide.

and related polymers with recurring succinyl units has been reported.17 A small amount of work on polyaspartamides with pendant pyrrolidinyl groups has been published, but N-alkylamide derivatives were not investigated as KHIs nor their biodegradability.18,19 Experimental Section Design and Synthesis of Polyaspartamides. Polyaspartamides can be made most conveniently by ring-opening of polysuccinimide (PSI) with alkylamines (Figure 1).20,21 The ring can open in two ways to produce both R and β aspartamide units. If PSI is hydrolyzed in aqueous base (e.g., NaOH) the ratio R/β is approximately 7:3.22 We obtained two different samples of polysuccinimide from Nanochem Solutions: (1) GF70-48A, fairly branched, molecular weight (Mw) ) 5000; and (2) GF70-58A, less branched, molecular weight (Mw) ) 3000. Both samples were made from aspartic acid. We made polyaspartamides from both these PSI samples, first by dissolving the PSI in a polar organic solvent and then adding the required amount of amines to open all of the succinimide rings. The polyaspartamide could then be isolated in quantitative yield by precipitation by adding excess diethyl ether. Alternatively, the polyaspartamide can be left in the polar solvent, and the whole solution can be tested as a KHI. This latter method is the one that we primarily used in our studies. Suitable solvents for PSI include dimethylformamide (DMF), N-methyl pyrrolidone (NMP), and N-ethyl pyrrolidone (NEP). PSI is not soluble in N-butyl pyrrolidone. We concentrated on using NEP because this is categorized as nontoxic and allowed for offshore use in the North Sea. Later, we found that we could dissolve PSI in a blend of NEP and 2-butoxyethanol (BGE, a cheaper solvent than NEP) and used this to make many of our polyaspartamides. (17) Lehmann, B.; Moritz, R. International Patent Application WO/98/ 16719. (18) Kelland, M. A.; Svartaas, T. M.; Øvsthus, J.; Namba, T. A new class of kinetic hydrate inhibitor. In 3rd International Conference on Gas Hydrates, Annals of the New York Academy of Science, 2000; Vol. 912, p 281. (19) Namba, T. F.; Saeki, Y.; Kobayashi, T. International Patent Application WO/1996/038492, 1996. (20) Tachibana, Y.; Kurisawa, M.; Uyama, H.; Kakuchi, T.; Kobayashi, S. Chem. Commun. 2003, 106. (21) Xu, W.; Li, L.; Yang, W.; Hu, J.; Wang, C.; Fu, S. J. Macromol. Sci., Part A 2003, 40, 511. (22) Roweton, S.; Huang, S. J.; Swift, G. J. EnViron. Polym. Degrad. 1997, 5, 175.

It is known from previous work on N-alkylacrylamide polymers18,23 and amide derivatives of maleic copolymers24 that the most likely pendant N-alkylamides that would give high-performance KHI polymers were N-isopropylamide and N-isobutylamide groups. These alkyl groups are of optimum size to interact with structure II 51264 cages. We first synthesized a polyaspartamide with 100% pendant N-isopropylamide groups. We also prepared polymers with varying amounts of isobutylamide groups. We found that polymers with 100% N-isobutylamide groups were not fully water-soluble at room temperature. However, a polyaspartamide made from PSI using 75 mol % isobutylamine and 25 mol % methylamine (using 40 wt % methylamine in water) was fully water-soluble. Another polyaspartamide that we synthesized contained N-isopentyl groups. We found that 60% N-isopentyl and 40% N-methylamide gave a watersoluble polyaspartamide, but higher percentages of the larger alkylamide gave water-insoluble polymers. We also prepared polymers with pendant, carbonylpyrrolidine, and N,N-diethylamide groups for comparison. Finally, we made a polyaspartamide using PSI and 3-dimethylamino-1-propylamine. The pendant groups in this polymer are similar to those found in polymers containing dimethylaminoethylmethacrylate (DMAEMA), such as Gaffix VC713, a N-vinyl caprolactam/N-vinyl pyrrolidone/DMAEMA terpolymer from International Specialty Products (ISP), a known KHI.25 DMAEMA-based copolymers without N-vinyl lactams have also been reported as KHIs.26 KHI Test Procedure. KHI performance tests were carried out in high-pressure autoclave equipment shown in Figure 2. Initially, the autoclave was a single titanium cell, but later, we switched to a single sapphire cell because this latter cell appeared to give more reproducible results, which was especially useful to determine the ranking of inhibitors with fairly similar performance. In all of the experiments, we used the same standard natural gas (SNG) mixture that gives structure II hydrates (Table 1). The aqueous phase was a 3.6 wt % NaCl solution. In the titanium cell, we also added decane as a liquid hydrocarbon phase at 60% water cut, but this was omitted in the sapphire cell experiments. At the onset of each experiment, the start of the induction time before gas hydrate began to form, the pressure was 90 bar. The equilibrium temperature at this pressure was calculated using Calsep’s PVTSim software. For the titanium cell system with added decane, the equilibrium temperature was calculated to be 17.8 °C, while for the sapphire cell system without decane, the equilibrium temperature was 19.6 °C. (23) Colle, K. S.; Costello, C. A.; Oelfke, R. H.; Talley, L. D.; Longo, J. M.; Berluche, E. U.S. Patent 5,600,044, 1997. (24) Klug, P.; Kelland, M. A. International Patent Application WO/1998/ 023843, 1998. (25) Sloan, E. D., Jr. U.S. Patent 5,880,319, 1999. (26) Freeman, D. J.; Irvine, D. J.; Kitching, J.; Rogers, C. S. International Patent Application WO/2006/051265.

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Table 1. Composition of SNG component

mole %

methane ethane propane iso-butane n-butane N2 CO2

80.67 10.20 4.90 1.53 0.76 0.10 1.84

The same procedure for preparation of the KHI experiment and filling of the cell was followed in all experiments. (1) The additive to be tested was dissolved in the brine to the desired concentration. (2) The magnet housing of the cell was filled with the aqueous solution containing the additive to be tested. The magnet housing was then mounted in the bottom end piece of the cell, which thereafter was attached to the titanium or sapphire tube and the cell holder. (3) The desired amount of the aqueous solution was filled in the cell (above the cell bottom) using a pipet; the top end piece was mounted; and the cell was placed into the cooling bath (plastic cylinder). For experiments in the titanium cell, a fixed volume of decane was added at this stage. (4) The temperature of the cooling bath was adjusted to 2-3 °C outside the hydrate region at the pressure conditions to be used in the experiment. (5) The cell was purged twice with the SNG used, and then the cell was loaded with SNG to the desired pressure stirring at 600 rpm. (6) The stirring was stopped, and the cell was cooled to the experimental temperature (2-10 °C) When the temperature and pressure (90 bar) in the cell had stabilized, the stirring was started. The induction time, ti, for hydrate formation was measured from the time of start of stirring at the experimental temperature. The time from start of hydrate formation to the time when rapid growth of hydrate ensues, usually with the formation of a hydrate plug in the cell, is called the crystal growth delay time, st-1. The results of all experiments were recorded by plotting the gas consumption (bar) as a function of time (min). An example of a typical plot is given in Figure 3 for a commercial KHI, Luvicap 55W (a 1:1 vinyl caprolactam/vinyl pyrrolidone copolymer) from BASF (Figure 4). In this work, we only interested in measuring induction times. In general, for a set of identical experiments, we have found that induction times vary by (30-40% of the average induction time in our equipment. KHI Performance Test Results. Results in the Titanium Cell. The first polyaspartamide KHIs that we tested were made using PSI GF70-48A. The results are given in Table 2. We found that a polymer with only isopropyl groups gave a poor performance, giving induction times (17 and 54 min) that were not much better than no additive. We then tested a polyaspartamide with theoretically 75% isobutyl groups and 25% methyl groups (iBu/Me 3:1 polyaspartamide). This gave much better performance as judged by an average induction time of 313 min. In fact, several polymers with varying percentages of isobutyl groups clearly performed better than the isopropyl polymer, although it was statistically impossible to tell which isobutyl polymer was best. In addition, iBu/Me 3:1 polyaspartamide did not appear to perform significantly better in NEP (10 000 ppm), indicating that this solvent is not a good synergist for this polymer. For comparison, we also tested a polymer with pyrrolidine groups. This gave a performance within the same statistical window as the isobutyl polymers. A polymer with diethyl groups performed considerably worse, only a little better than the isopropyl polyaspartamide. We obtained the same trends with polymers made with PSI GF70-48B, which is a less branched and a higher molecular-weight (Table 3) polymer. Thus, a polyaspartamide with isopropyl groups was clearly worse than polymers with a high percentage of isobutyl groups. The results suggested that the 3:1 iBu/Me polymer

Figure 3. Example of KHI test result: Luvicap 55W at 90 bar, 7 °C, 12.6 °C subcooling, and SII hydrate. Induction time (ti) and slow growth phase (st-1) are shown. The axis “time zero” is the time since the start of stirring at time 0 min.

Figure 4. Structure of 1:1 vinyl caprolactam/vinyl pyrrolidone copolymer in Luvicap 55W. Table 2. Selected KHI Results in the Titanium Cell with 5000 ppm Polyaspartamides Made from GF70-48A at 90 bar and 10.9 °C Subcooling polymer R groups

number of experiments

average induction time ti (min)

no additive ipropyl 100% ppta iBu 100% ppt iBu/Me 3:1 ppt iBu/Me 3:1 in NEP iBu/Me 1:1 ppt pyrrolidine 100% ppt diethyl 100% ppt

5 2 2 2 5 2 2 2

18 35 419 313 386 476 340 73

a

ppt ) precipitated in Et2O.

Table 3. Selected KHI Results in the Titanium Cell with 5000 ppm Polyaspartamides Made from GF70-58A at 90 bar and 10.9 °C Subcooling polymer R groups

number of experiments

average induction time ti (min)

ipropyl 100% ppta iBu/Me 3:1 ppt iBu/Me 1:1 ppt

2 2 2

54 237 170

a

ppt ) precipitated in Et2O.

performed a little better than the 1:1 iBu/Me polymer. In addition, polyaspartamides made from PSI GF70-48A appeared to perform marginally better than polyaspartamides made from GF70-58A. Thus, we concentrated on making KHIs from the former PSI, particularly the 1:1 and 3:1 iBu/Me polyaspartamides, which had

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Figure 5. Induction times for Luvicap 55W tested at 5000 ppm (active polymer) with SNG and 3.6% NaCl in the sapphire cell.

Del Villano et al.

Figure 7. Comparison of Luvicap 55W with 75:25 iBu/Me polyaspartamide (KHI055). Table 4. KHI Results in the Sapphire Cell with 5000 ppm Polyaspartamides Made from GF70-48A at 90 bar and 10.6 °C Subcooling polyaspartamide R groups iBu/Me iBu/Me iBu/Me iBu/Me

3:1 3:1 1:1 1:1

in in in in

induction time ti (min)

BGE/NEP BGE/NEP BGE/NEP BGE/NEP

1334 1346 596 641

Table 5. KHI Results in the Sapphire Cell with 5000 ppm Polyaspartamides (28 wt % in BGE/NEP) Made from GF70-48A at 90 bar polyaspartamide R groups ∆T (°C) number of tests average ti (min)

Figure 6. Luvicap 55W at 90 bar at 5000 ppm (active polymer) on a logarithmic scale.

100% water solubility and we assumed had optimum performance with a high number of large hydrophobic side groups. Results in the Sapphire Cell. We switched to carrying out tests in a sapphire cell without the use of decane because tests on other polymer KHIs appeared to indicate somewhat more reliable results with this test system. By changing two parameters, the type of cell and no decane, this did not allow us to compare results but this was unnecessary because we only used the titanium cell equipment to determine that polyaspartamides with isobutyl groups performed better as KHIs than those with isopropyl groups and that GF7048A was the PSI that gave the best KHIs. We first carried out sapphire cell tests on Luvicap 55W, a 1:1 vinyl caprolactam/vinyl pyrrolidone copolymer in water, a sample of which was kindly given to us by BASF (Figure 4). This is a commercial low-molecularweight polymer used in KHI formulations by service companies. The induction time results of KHI tests on this polymer at various subcoolings are plotted in Figure 5. Figure 6 shows the same data plotted on a logarithmic scale. We found that there was some scattering in the results but that by running experiments at a range of subcoolings we could obtain a reasonable calculated line of best fit. Using this technique, we could compare Luvicap 55W with the performance of what we assumed was our best polyaspartamide, the 3:1 iBu/Me derivative made from PSI GF70-48A. This and all polyaspartamide samples tested in the sapphire cell were made as a 28 wt % polymer solution in NEP (44 wt %) and BGE (28 wt %). Figure 7 shows the results plotted on a logarithmic scale for tests at 5000 ppm (active polymer). The calculated lines of best fit indicate that Luvicap 55W gives the same induction times as the best polyaspartamide at approximately 1.0-1.5 °C higher subcooling dependent upon the absolute subcooling. The results that we have obtained with the 3:1 iBu/Me polyaspartamide indicate that the induction times for KHI tests in the titanium cell are shorter than tests in the sapphire cell at the same subcooling conditions. This may be due to greater heteronucleation by the titanium surface than sapphire surfaces and a steel stirrer

iBu/Me 3:1 iPe/Me 6:4 iBu/Me 3:1 iPe/Me 6:4 iBu/Me 3:1 iPe/Me 6:4 iBu/Me 6:4

14.6 14.6 12.6 12.6 10.6 10.6 12.6

3 2 3 3 2 1 2

103 44 324 219 1340 596 200

blade present in both cells, or it may be related to using a decane/ brine mixture in the titanium cell and only brine in the sapphire cell. The tests in the titanium cell were inconclusive as to whether a 1:1 iBu/Me polyaspartamide was better than the 3:1 iBu/Me polyaspartamide; therefore, we conducted tests on these polymers in the sapphire cell. The results in Table 4 at 10.6 °C subcooling confirm that the 3:1 iBu/Me polyaspartamide is the better KHI of the two polymers. Having shown that polyaspartamides with isopropyl groups performed worse than KHIs than polyaspartamides with a high proportion of isobutyl groups, we were interested to compare the performance of a polymer with isopentyl (iPe) groups. A polymer with up to 60% isopentyl groups, the rest being methyl groups, was found to be water-soluble. A 6:4 iPe/Me polyaspartamide was tested at three subcoolings. The results are given in Tables 5. The general trend was that the 6:4 iPe/Me polyaspartamide performed worse than the 3:1 iBu/Me polyaspartamide but about the same as a iBu/Me 6:4 polyaspartamide. We also carried out tests on a 28 wt % solution of 3:1 iBu/Me polyaspartamide made in NEP only (i.e., without BGE). This polymer gave results in the same statistical range as the same 28 wt % solution of polymer made in a mixture of NEP and BGE. Because we knew that NEP was not a synergist from studies in the titanium cell, this confirmed that BGE is also not a KHI synergist for this polymer. Thus, neither solvent, NEP or BGE, is necessary to improve the KHI performance of the polyaspartamides but they are useful high boiling point, high flash point solvents. We also wondered if we could use water as a solvent to make polyaspartamides from PSI and alkylamines. Although PSI is insoluble in water, we found that a slurry of PSI in water would react easily with alkylamines to give a soluble product. However, all of these products made using a variety of alkylamines had almost no effect as KHIs in sapphire cell tests. The reason is probably that

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Table 6. KHI Test Results with 3:1 iBu/Me Polyaspartamide (28 wt % in BGE/NEP) at Varying Concentration in the Sapphire Cell at 90 bara concentration (ppm) 2500 5000 10 000 2500 5000* 10 000*

∆T (°C)

average ti (mins)

12.6 12.6 12.6 10.6 10.6 10.6

336 324 380 712 1340 2046

a The average induction time (ti) is based on three experiments, except those indicated with an asterisk (/), which are based on two experiments.

alkylamines in water are basic, and the preferred reaction is attack of hydroxide ion on PSI to give an anionic polyaspartate with alkylammonium counterions, neither of which are expected to be good KHIs. The best polyaspartamide KHI, 3:1 iBu/Me polyaspartamide (28 wt % in BGE/NEP) was tested at three different concentrations, 2500, 5000, and 10 000 ppm (active polymer). The results are summarized in Table 6. At 10.6 °C subcooling, the performance did increase with KHI concentration. However, at 12.6 °C subcooling, induction times were very similar at the two lowest concentrations and only slightly higher at 10 000 ppm. The last polyaspartamide that we tested, made from PSI and 100% 3-dimethylamino-1-propylamine, gave very poor results at both 10.6 and 12.6 °C subcooling, almost no better than no additive at all. Biodegradation Tests. Test Procedure. Marine biodegradation was experimentally measured according to the OECD 306 closed bottle method.27 Briefly, fresh seawater samples were taken from 70 m depth at Byfjord, Stavanger, Norway, transported to the laboratory within the hour and kept at room temperature in the dark. Aliquots (350 or 250 mL, depending upon the initial concentration) were transferred to prewashed 510 mL dark bottles, spiked with 2 mL non-ammonium (ammonium replaced by nitrate) Bushnell-Haas nutrient solution (contained per liter: 0.2 g of MgSO4, 0.02 g of CaCl2, 2.0 g of K2HPO4, 2.0 g of NaNO3, and 0.05 g of FeCl3, adjusted to pH 8.0), and volumes of sample bringing the initial concentration of test compounds to 100 mg/L. Sample bottles were closed by OxiTop-C screw cap measuring heads (WTW, Wissenschaftlich-Technische, Germany), equipped with saturated NaOH headspace solution for CO2 trapping, and incubated at 20 °C under continuous mixing. BOD bottles were prepared according to the vendors guidelines for a BOD 28 day test. The sample heads correlate the oxygen consumption with the partial pressure reduction in the closed headspace, providing continuous oxygen readings without opening the system. All samples received nutrient solution, and glucose (initial concentration of 100 mg/L) was used for a positive control, while acidified (addition of 4 M H2SO4 to pH < 2) and autoclaved sample bottles (containing test sample) served as negative controls. Positive controls and KHIs were tested in duplicates. Theoretical oxygen demand (ThOD) was calculated according to the literature method based on the chemical composition of the tested chemicals.28

Results Figure 8 shows the dissolved oxygen consumption during degradation of the positive control (glucose) in the seawater samples used to study the KHIs. The two parallels are shown together with the sample blank. Similarily, dissolved oxygen (DO) consumption during the two 3:1 iBu/Me polyaspartamide (27) Organization for Economic Cooperation and Development (OECD). Guideline for testing of chemicals, biodegradability in seawater. Adopted by the council on July 17, 1992; p 27. (28) Rittmann, B. E.; McCarty, P. L. EnVironmental Biotechnology: Principles and Applications, International ed.; McGraw-Hill, Inc.: Singapore, 2001; p 128.

Figure 8. Oxygen demand during glucose (positive control) degradation.

Figure 9. Oxygen demand during 3:1 iBu/Me polyaspartamide (KHI055) degradation by the seawater sample, including the uptake of the blank.

(KHI055) parallels are shown in Figure 9. This polymer is pure and does not contain organic solvents. On the basis of the measured oxygen consumed and the calculated ThOD, the percent biodegradation was calculated according to the OECD 306 method. Table 7 shows the results of the ThOD and the measure BOD values for the tested chemicals, standards and controls, and the degradation degree. Because this method is based on a degradation model not including growth,29 Table 7 also shows that the theoretical biological oxygen demand (ThBOD) estimated heterotrophic growth yields (YX/S) of 0.67 g of COD/g of COD for glucose30 and 0.5 g of COD/g of COD for tested chemicals (estimated on the basis of the degree of reduction of carbon in substrates according to the literature31,32). On the basis of the degradation model including growth, Table 7 shows the ThBOD and the percent degradation based on the BOD/ThBOD ratio. According to the OECD 306 method, only the DMF is biodegradable (i.e., 0.6 < BOD/ThOD) when leaving the assimilative uptake of substrate out of the stoichiometric calculation. By including growth (using the above given biomass yield coefficients), ready degradation is found also for the control (glucose) and the KHI055 inhibitor. The biodegradation (BOD) time lag observed is 2-3 days for KHI055, while no time lag is involved in the glucose batch (29) Painter, H. A. Detailed review paper on biodegradability testing. Environment monograph 98, OCDE/GD(95)43, Paris, France, 1995. (30) Dircks, K.; Beun, J. J.; van Loosdrecht, M. C. M.; Heijnen, J. J.; Henze, M. Biotechnol. Bioeng. 2001, 73, 85. (31) Heijnen, J. J. A thermodynamically based description of chemotrophic microbial growth stoichiometry and kinetics. In Encyclopedia of Bioprocess Technology: Fermentation Biocatalysis and Biodeparation; Flickinger, M. C., Drew, S. W., Eds.; John Wiley and Sons: New York, 1999. (32) Ro¨els, J. A. Energetics and Kinetics in Biotechnology, Elsevier: Amsterdam, The Netherlands, 1983.

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Table 7. Measured BOD and Estimated ThOD and ThBOD Values for Tested Chemicals in This Studya compound

ThOd28 no growth (mg/L)

ThBOD28 including growth (mg/L)

BOD28 (mg/L)

% 28 day biodegradation according to OECD306

% 28 day biodegradation including growth

seawater plus nutrients glucose plus nutrients glucose plus nutrients KHI055 plus nutrients KHI055 plus nutrients BGE plus nutrients BGE plus nutrients NEP plus nutrients NEP plus nutrients DMF plus nutrients DMF plus nutrients DMF plus H2SO4 Luvicap 55W plus nutrients Luvicap 55W plus nutrients S1200 plus nutrients S1200 plus nutrients PEA 02 plus nutrients PEA 02 plus nutrients

0 107 107 155 155 126 126 59 59 153 153 153 217 217 169 169 220 220

0 35 35 77 77 60 60 30 30 74 74 77 104 104 83 83 106 106

1.9 33.3 31.0 47.5 45.0 33.9 28.2 14.1 14.1 117.4 112.6 3.8 8.6 8.6 0.6 6.2 4.3 4.4

0 29 27 29 28 21 17 12 12 75 72 1 3 3 -10 -7 1 1

0 89 83 60 57 45 35 24 24 157 151 0 6 6 -21 -14 2 2

a KHI055, 3:1 iBu/Me polyaspartamide without solvents; BGE, 2-butoxyethanol; NEP, N-ethyl pyrrolidone; DMF, dimethylformamide; 55W, Luvicap 55W from BASF; S1200, a pure hyperbranched poly(ester amide) made from succinic anhydride and diisopropanolamine with Mw ) 1200 from DSM; PEA02, a poly(ester amide) made from cis-1,2-cyclohexanedicarboxylic anhydride, diisopropanolamine, and 3,3′-iminobis(N,N-dimethylpropylamine) in a 7:3:5 ratio.

test. Following onset of biodegradation at day 3, the oxygen plateau level is reached on day 17. Discussion OECD 306 closed bottle method provides an efficient and cost-effective experimental approach to biodegradation testing with equivalent sensitivity and accuracy as the closed bottle method. In comparison to the closed bottle method that relies on measurements of primary biodegradation (i.e., removal of the test chemical), it provides quantitative information on mineralization.29 However, inferring the degree of biodegradation only involves the ratio to the ThOD (or the measured COD), without any growth stoichiometric evaluation. This has been discussed as a limitation of the method by some authors.29 Assuming biomass growth on the test chemical, the measured oxygen consumption accounts for the dissimilated substrate (test chemical) only, represented by the 1 - YX/S fraction of the COD of the tested chemical. The effect of growth yield on the measured oxygen consumption is partly compensated by the 60% BOD/ThOD ratio; however, for many easily biodegradable compounds the aerobic heterotrophic growth yield is as high as 0.5-0.8 g of COD/g of COD.33 In these cases, the measured BOD will only reach 30-50% of ThOD (or COD), resulting in a nonreadily biodegradable classification. This is obviously errorous for a known easily biodegradable compound such as glucose, whose ThBOD is 30-40% of the ThOD using published growth yields of 0.6-0.7 g of COD/g of COD.30 Because of the observations by several authors that know easily biodegradable compounds do not satisfy the 60% requirement, it has been suggested to reduce this to 50%.29,34 Also, the approach applied in the OECD methods (all respirometric methods) does not take endogenous respiration by the newly formed biomass into account. Endogenous respiration by the blank is subtracted from the test BOD curves, but the additional oxygen demand caused by growth on the test chemical is not included. This is especially problematic because the test period is as long as 28 days and test compounds may easily be (33) Strotmann, U. J.; Geldern, A.; Kuhn, A.; Gendig, C.; Klein, S. Chemosphere 1999, 38, 3555. (34) Boethling, R. S.; Lynch, D. G.; Thom, G. C. EnViron. Toxicol. Chem. 2003, 22, 837.

biodegraded (and mineralized) in substantially shorter time. A long endogenous period following the growth phase of the batch test may compensate for the difference between the ThBOD and ThOD. A structured model including growth and endogenous respiration should therefore be applied when analyzing biodegradation based on respirometric data, similar to the methods used to respirometrically characterize wastewater BOD35 or the modeling approach described elsewhere.36 In this work, we used published stoichiometric yields to calculate the oxygen consumption during growth on the test chemicals. As seen in Figure 1, the measured BOD of glucose is 30-35 mg/L after 28 days, accounting for less than 30% of the ThOD. When growth is included, this accounts for close to 90% of the ThBOD. The kinetic behavior of the batch, with no clear exponential phase, indicates uptake and storage, a process often seen during growth of a mixed culture on glucose.37 As seen from Table 1, using the ThOD instead of the ThBOD as the reference oxygen consumption leads to very different conclusions. While only DMF may be classified as readily biodegradable using the ThOD as the reference, both glucose and KHI055 are degradable after 28 days of exposure. The high degradation of the DMF test indicates prolonged endogenous respiration. Negative BOD as found in the S1200 test is most likely due to high initial background respiration found for the inoculum used during that particular test (data not shown). The good results with KHI055 are possibly due to the polymer containing some carboxylate groups. The reaction of PSI in organic solvents to make this polyaspartamide is carried out with isobutylamine and 40 wt % methylamine in water, the latter of which will contain hydroxide ions. Attack of hydroxide ions on PSI will lead to polymers with carboxylate groups. In comparison to KHI055, Luvicap 55W gave very low biodegradation. The two water-soluble hyperbranched poly(ester

(35) Ekama, G. A.; Dold, P. L.; Marais, G. v. R. Water Sci. Technol. 1986, 18, 91. (36) Smets, B. F.; Jobba´gy, A.; Cowan, R. M.; Leslie Grady, C. P., Jr Ecotoxicol. EnViron. Saf. 1996, 33, 88. (37) Krishna, C.; van Loosdrecht, M. C. M. Water Res. 1999, 33, 3149.

Class of KHIs with Good Biodegradability

amide)s, S1200 from DSM and PEA02, the latter made in house, also gave low biodegradation in 28 days.38,39 Conclusions We have synthesized and tested a range of polyaspartamides as KHIs. A polymer with a 3:1 ratio of isobutyl/methyl pendant (38) Klomp, U. C. U.S. Patent 6,905,605, 2005. (39) Froehling, P. E. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 3110.

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groups appeared to perform best but somewhat worse than a commercial KHI polymer, Luvicap 55W. The 3:1 iBu/Me polyaspartamide did however show greater than 20% 28 day biodegradability by the closed bottle method according to the OECD306 test procedure. This value becomes 57-60% if growth assimilation is taken into account. Acknowledgment. We thank StatoilHydro, Total, and NanoChem Solutions, Inc. for financial support of this work. EF800161Z