Wild soil fungi able to degrade the herbicide isoproturon
Hongos de suelo nativos con potencial degradador del herbicida isoproturon
María Cristina Romero1*, María Inés Urrutia2, Enso Hugo Reinoso1 and Alejandro Moreno Kiernan2
1 Micología Médica e Industrial, Dep. Microbiología, Facultad Ciencias Veterinarias.
2 Facultad Cinecias Agrarias y Forestales – Universidad Nacional de La Plata, Argentina.
]]>* Autor para correspondencia:
M. Cristina Romero cmriar@yahoo.com.ars.
Recibido 30 de abril 2008
Aceptado 11 de octubre 2008.
Resumen
El herbicida isoproturon (IPU) es un contaminante fuerte debido a su elevada solubilidad acuosa y bajo potencial para la degradación microbiana. El problema ambiental se debe a su amplio empleo en la agricultura convencional, áreas urbanas, algicida y aditivo de pinturas. Especies fúngicas degradaron el herbicida en cultivos in–vitro, produciendo derivados hidroxilados frecuentes en areas agrícolas. Los objetivos planteados fueron aislar las especies fúngicas representativas de suelos tratados con IPU, evaluar su potencial transformador, e identificar los derivados. La mayoría de los aislamientos produjeron 1 –OH–IPU y 2–OH–IPU, indicando que los hongos pueden ser la causa de estos compuestos en las muestras ambientales. De los 35 aislamientos, 10 de ellos fueron especies dominantes y activas degradadoras. Aspergillus ochraceus, Fusarium flocciferum, Talaromyces helicus, Acremonium strictum, Mucor hiemalis, Paecilomyces lilacinus y Penicillium frequentans transformaron el herbicida produciendo cantidades significativas de diversos derivados. Esta es la primera mención de dicha actividad para estas especies.
Palabras clave: bioremediación, hongos filamentosos de suelo, potencial degradador, metabolitos hidroxilados.
]]>Abstract
The herbicide isoproturon (IPU) is a strong contaminant due to it water solubility and low microbial degradation. Environmental concerns arised from the worldwide use of this herbicide in conventional agriculture, urban areas and algicide in antifouling paints. Several fungi have shown the ability to degrade IPU and its derivatives; however, hydroxylated metabolites were frequently detected in agricultural areas. The aims of this study were to isolate representatives fungi from polluted soils, to study their IPU–degradation potential and to identify the transformation products. Aspergillus ochraceus, Fusarium flocciferum, Talaromyces helicus, Acremonium strictum, Mucor hiemalis, Paecilomyces lilacinus and Penicillium frequentans uptook the herbicide and produced significant amounts of diverse derivatives. The fungi produced 1–OH–IPU and 2–OH–IPU, being the source of the hydroxylated IPU–derivatives detected in environment. Therefore, this study demonstrated that selected fungi could be used for the polluted soils bioremediation.
Key words: bioremediation, filamentous species, biodegradation potential, hydroxylated metabolites.
Introduction
The herbicide isoproturon, 3–(4–isopropylphenyl)–1,1 ,dimethylurea (IPU) and other phenylurea compounds had been detected as priority pollutants (Environment Agency, 2000; Environment Agency, 2001) due to their high water solubility and low microbial degradation potential that determined the levels exceeding the limit concentrations (Stig et al., 2005). Environmental concerns arised from the widespread IPU used being the most extensively pesticide in conventional agriculture (Suhadolc et al., 2004). IPU and its derivatives had shown detrimental effects to algae (Pérés et al., 1996; Mostafa and Helling, 2001) invertebrates, ciliated protozoan (Perrin–Ganier et al., 2001) and microbial activity as 50% methanogenesis reduction (Attaway et al., 1982; Remde and Traunspurger, 1994) also genotoxic effects had also been revealed (Behera and Bhunya, 1990; Hoshiya et al., 1993). Degradation can involve abiotic and biotic processes, where microbial transformation is especially interesting, since it is the only known pathways to complete mineralisation of aromatic compounds (Alexander, 1981; Mansour et al., 1999). Slow natural biodegradation rate, 5–25% within 2–3 months was more frequent than quick and extensive IPU–mineralisation only observed in previously field–treated soils (Sørensen et al., 2002).
Most of the studies used enrichment–culture techniques to isolate phenylurea–degrading microorganisms (Stangroom et al., 1998), but some isolates from agricultural soils failed to extensively degrade the herbicide (Bending et al., 2001). The addition of IPU–metabolite, 3–(4–isopropylphenyl)–1–methylurea (MDIPU) as carbon and energy source yielded a mixed culture able to mineralized MDIPU and 4–isopropyl–aniline (4IA), with enhanced herbicide degradation (Sørensen and Aamand, 2001), evenmore, others studies indicated that co–operative metabolic activities may be involved in the IPU–transformation (Sebai et al., 2004). In soils, however, hydroxylated metabolites have frequently been observed; therefore, the aims of this study were: 1) to identify wild filamentous soil fungi as organisms that potentially play a role in the formation of these hydroxylated intermediates in soils treated with isoproturon, 2) to confirm their IPU–potential uptake and 3) to identify the transformation products.
Materials and methods
]]> Soil samples. Samples were taken from the upper 10 cm of an agricultural area near La Plata, Argentina. Soil borne fungi were obtained from organic soil particles by sieving through a 2 mm sieve, and 5 g soil was added to a L–liter sterile screw–cap bottle with 500 mL of 0.1% (wt/vol) Na4O7P2·10H2O to disperse clumps and colloids, and shaken horizontally for 1 h at 4°C (Thorn et al., 1996).The suspension was poured through a stack of two sieves with grids of 0.5 mm and 63 µm. After a brief rinse with cold tap water, the 0.5 mm sieve was removed, and the contents of the 63 µm sieve were washed for 2 min under cold tap water. When the mineral fraction settled and most of the water run out, 1 mL was collected from the dense suspension of organic particles and used for isolation of fungi.
Fungal isolation. Filamentous fungi were isolated by plating 100 µL of a 1x 102 dilution of the soil organic particles on mineral medium (MM) (Romero et al., 2005) with 5 g IPU /L and 2% agar. The isoproturon was allowed to dissolve overnight before use. pH was adjusted to 6.5 with 1 M HCl, after autoclaving and cooling to 50°C, the IPU–MM was supplemented with 40 mg/L tetracycline, 20 mg penicillin/L and 20 mg streptomycin/L. The dishes were incubated in the dark at 27 °C and screened for 3 weeks. Hyphae growing from particles were transferred to other IPU–MM plates to confirm the biodegradation potential.
Fungal isolates were characterized on the basis of colony morphology, spore structures microscopy and nucleotide sequences of the internal transcribed spacers (ITS) of rRNA genes. Mycelium from cultures was extracted in 40 µLTris–EDTAbuffer (pH 8.0) with 10 µL20% Chelex. A2 µL aliquot of the extract was used as the template for a PCR using primers ITS1F and ITS4 (White et al., 1990). Amplification was performed with a thermocycler (PCT–200; MJ Research Inc., Massachusetts) using the PCR conditions described by Gardes and Bruns (1993). PCR products were purified (StrataPrep PCR purification kit; catalog no. 211189–1; Stratagene) and sequenced by MWG–Biotech AG.
Degradation analysis. Three filamentous fungi were examined for IPU–degradation. The isolates were inoculated as 1 mL portions of spore suspensions of each culture, prepared by vortexing sporulating mycelium in 3 mL distilled water containing 1 g/L Tween 80 and 8.5 g/L NaCl. Each fungus was inoculated into 100 mL bottles with IPU–MM, and incubated in the dark, at 27°C, 150 rpm for 30 days, by triplicate. Three different control flasks were incubated in the same conditions, one without IPU, another non–inoculated bottle and a third one inoculated and sterilized flask, in order to allow differentiation between fungal exudates, to assess IPU–uptake and its metabolites production in the chromatographic analysis. One mL sample was collected every fifth day with a syringe and passed through a polytetraflouroethylene filter (diam 17 mm; Titan 2 HPLC filter; 0.20 µm membrane; no. 42213–PC) into glass vials for the chromatographic analysis. An HPLC system (1050 HP; Hewlett–Packard) with a UV/VIS detector was used with a Hypersil 5 µm C18 column (250 by 2 mm; Phenomenex).
The biomass of the fungi was determined at the end of the experiment, 30th day, by filtering the mycelia onto filter paper disks; then samples were air dried to a constant weight in a fume hood. The assays were done by triplicate, and the results are reported as arithmetic means with 5 % standard deviation.
Routine HPLC analysis of IPU, MDIPU (3–(4–isopropylphenyl)–1–methylurea), DDIPU (3–(4–isopropylphenyl)–urea) and 4–isopropyl–aniline (4IA) was performed using the isocratic method (Juhler et al., 2001) and identified by comparison with authenthic standards and literature data. Hydroxylated intermediates, 1–OH–IPU, 2–OH–IPU and 1–OH–MDIPU, were detected with a gradient method using acetonitrile and water as eluents at 0.3 mL/min flow rate. For the first 8 min 15% (by volume) acetonitrile was used, then it was raised linearly to 45% between 8–12 min., and between 23–25 min the acetonitrile level decreased to 15%. Equilibration time before the next injection was 7 min, with 4 µL injection volume and the 45°C column temperature. Phenylureas were detected and quantified at 245 nm, and 4IA was detected and quantified at 200 nm. The retention times were: 7.0 min for 1–OH–MDIPU; 9.8 min for 2–OH–IPU; 11.1 min for 1–OH–IPU; 17.8 min for DDIPU; 18.7 min for MDIPU; 20.0 min for IPU; and 22.0 min for 4IA (Del Pilar Castillo et al., 2001; Gerecke et al., 2001). The reported IPU–derivatives are the arithmetic means of three separated experiments with replicated batch cultures; standard deviation was no more than 5 %. The analytical standards were purchased from Ehrenstorfer GmbH, Augsberg, Germany: isoproturon (CAS no. 34123–59–6), MDIPU (CAS no. 34123–57–4), DDIPU (CAS no. 56046–17–4) and 4IA (CAS no. 99–88–7).
Results and discussion
About 35 isolates were obtained from the IPU–polluted soils using the isolation strategies, then, the 10 dominants fungi, that represented 70 % of the isolates, were selected and identified to the genus level when possible. Acremonium strictum, Alternaria alternata, Aspergillus ochraceus, Fusarium flocciferum, Mortierella spp., Mucor hiemalis, Phoma spp., Paecilomyces lilacinus, Penicillium frequentans, Talaromyces wortmannii and Trametes versicolor were identified as IPU–degrading isolates. A. ochraceus, A. strictum, F. flocciferum, M. hiemalis, P. lilacinus, P. frequentans and T. helicus were used in further IPU–bioassays to isolate and identify the derivatives as they showed the higher metabolic activity againt the herbicide.
]]> A significant uptake was observed in T. helicus cultures, approximatly 80% IPU disappeared within 15 days, considering as 100% the initial IPU–level (Figure 1). Demethylation to MDIPU and DDIPU, and two hydroxylation position, at the first and second points on the isopropyl side chain, yielding 1–OH–UPI, 2–OH–IPU and 1–OH–MDIPU metabolites with this fungi (Figure 2). The integrated area of the detected compounds mirrored the area of the residual IPU–peak, and the total integrated area was almost constant in the cultures.A significant IPU–uptake was also detected in the other fungi cultures, as 63 and 54% of the herbicide had disappeared at the end of the 30th incubation day in A. ochraceus and F. flocciferum assays, respectively. A. ochraceus produced the same derivatives as T. helicus but in minor amounts and within 25th days; while in the F. flocciferum cultures brief 1–OH–MDIPU and non DDIPU levels were observed. A. strictum, M. hiemalis, P. lilacinus and P. frequentans cultures exhibited a transient IPU–transformation, as a significant residual IPU–levels were detected at 30th day. Moreover, demethylation was dominant for A. strictum, and M. hiemalis produced more hydroxylated intermediates at the first position; while P. lilacinus and P. frequentans produced similar amounts of 1–OH–IPU, 2–OH–IPU and low MDIPU concentrations.
In the remaining fungal cultures, the herbicide initial levels were quite similar to the controls, suggesting that these species are not active degradors of the studied pesticide.
IPU was not degrade in the three abiotic controls, a decreae of only 0.65% was observed in the aninoculated bottles, 0.48% in the control without IPU as substrate, but a slight higher value (0.95%) was observed in the inoculated and sterilized flasks. The first values could be to IPU–absortion to the soil particles, the last one could be explained by the present of diverse substances produced during the sterilization processes.
Other researches had pointed out that MDIPU was the main metabolite produced by pure cultures of soil fungi (Sandermann et al., 1998; Sørensen and Aamand., 2001) and 1–OH–IPU had only been measured in cultures derived from soils (Elkhattabi et al, 2004; Spliid and Køppen, 1998). In the present study, demethylation and two hydroxylation position of IPU were detected in the fungal assays, with a highly significant IPU transformation in the T. helicus and A ochraceus experiments, as only 20 and 37% residual IPU–levels were detected at the final incubation time.
The fungal hydroxylated metabolites may be important from an environmental point of view as field data suggest that 2–OH–IPU had a greater tendency than MDIPU to leach from the soil (Glässgen et al., 1999). The higher mobility of the hydroxylated intermediates underlined the need to evaluate the production of these compounds, their ecotoxicity and their fate in soils (Walker et al., 2002; Bolte, 2004).
Growth was observed in A. ochraceus, F. flocciferum, T. helicus, as these fungal species were able to uptake the herbicide and produced significant and diverse intermediates; also in the A. strictum, M. hiemalis, P. lilacinus and P. frequentans experiments, a slight growth were obtained. The biomasses ranged from 2.0 0.1 to 0.14 0.04 mg/mL for the first and second fungi group, respectively. Therefore, the fungal species were able to grow in this medium using IPU as carbon and energy sources, and without a second substrate.
The fungi able to partialy degrade phenylurea herbicides cover a broad range of different species including Cunninghamella elegans, Mortierella isabellina (Tixier et al., 2000), Talaromyces wortmanii (Vroumsia et al., 1996), Rhizopus japonicus, Rhizoctonia solani and Aspergillus niger (Vroumsia et al., 1996). Among other authors results, Mortierella spp., Phoma spp. and Alternaria spp. hydroxylated isoproturon at the first position of the isopropyl side chain 3 (Sandermann et al., 1998). This study is the first mention as IPU–degraders for the following filamentous soil fungi A. ochraceus, F. flocciferum, T. helicus, A. strictum, M. hiemalis, P. lilacinus and P. frequentans, they uptook the herbicide and produced significant amounts and diverse derivatives. Therefore, enhancing the possibility to detoxify polluted areas by natural bioremediation strategies, as the mentionated micoflora are frequent and active species in polluted sediments. These fungi could be used to accelerate the detoxification of polluted areas (Schuelein et al., 1996; Bending et al., 2003).
In conclusion, thirty–five isolates were obtained from IPU–polluted soils and 10 dominat of them were active to degradate the herbicide. We suggested that soil filamentous fungi were responsible for the hydroxylated metabolites detected in the environmental polluted samples. The advantage of the fungal activity could be explained by easy metabolized intermediates produced by fungi, and the subsequent degradation by other microorganisms (Sørensen et al., 2003). This could determine the hydroxylated derivatives persistence and may provide new insight into interactions between fungi and bacteria in the IPU–degradation (Lehr et al., 1996; Sørensen et al., 2001).
]]> Acknowledgements
This work was supported by grants from the National Council of Scientific and Technological Research – CONICET and from the National University of La Plata (UNLP), Fac. Ciencias Veterinarias, Departamento de Microbiología, Cát. Micología Médica e Industrial, Argentina.
References
Alexander, M., 1981. Biodegradation of chemicals of environmental concern. Science 211:132–138. [ Links ]
Attaway, H.H., M.J.B. Paynter, N.D. Camper, 1982. Degradation of selected phenylurea herbicides by anaerobic pond sediment. Journal of Environmental Sciences and Health B17: 683–699. [ Links ]
Bending, G.D., E. Shaw, A. Walker, 2001. Spatial heterogeneity in the metabolism and dynamics of isoproturon degrading microbial communities in soil. Biology and Fertility of Soils 33:484–489. [ Links ]
Bending, G.D., S.D. Lincoln, S.R. Sørensen, J.A.W. Morgan, J. Aamand, A. Walker, 2003. In–field spatial variability in the degradation of the phenyl–urea herbicide isoproturon is the result of interactions between degradative Sphingomonas spp. and soil pH. Applied and Environmental Microbiology 69: 827–834. [ Links ]
Behera, B.C., S.P. Bhunya, 1990. Genotoxic effect of isoproturon (herbicide) as revealed by three mammalian in–vivo mutagenic bioassays. Industrial Journal of Experimental Biology 28:862– 867. [ Links ]
Bolte, M., 2004. Environmental fate of pollutants: example of phenylurea pesticides. Actual Chemestry 278:33–39. [ Links ]
Del Pilar Castillo, M., S. von Wire'n–Lehr, I. Scheunert, L. Torstensson, 2001. Degradation of isoproturon by the white rot fungus Phanerochaete chrysosporium. Biology and Fertility of Soils 33: 521–528. [ Links ]
Elkhattabi, K., A. Bouhaouss, C. Perrin–Ganier, M. Sciavon, 2004. Fate of isoproturon in two Moroccan soils. Agronomie 24: 177–183. [ Links ]
Environment Agency, 2000. Monitoring of pesticides in the environment. Environmental Agency. www.environment–agency.gov.uk. [ Links ]
Environment Agency, 2001. Pesticides 2000: a summary of monitoring of the aquatic environment in England and Wales. Environmental Agency. www.environment–agency.gov.uk. [ Links ]
Gardes, M., T.D. Bruns, 1993. ITS primers with enhanced specificity for basidiomycetes—application for the identification of mycorrhizae and rusts. Molecular Ecolology 2: 113–118. [ Links ]
Gerecke, A.C., C. Tixier, T. Bartels, R.P. Schwarzenbach, S.R. Muller, 2001. Determination of phenylurea herbicides in natural waters at concentrations below 1 ng l31 using solid–phase extraction, derivatization, and solid–phase microextraction gas chromatographymass spectrometry. Journal of Chromatography 930: 9–19. [ Links ]
Glässgen, W. E., D. Komossa, O. Bohnenkamper, M. Haas, N. Hertkorn, R. G. May, W. Szymczak, H. Sandermann, 1999. Metabolism of the herbicide isoproturon in wheat and soybean cell suspension cultures. Pesticide Biochemestry and Physiology 63: 97–113. [ Links ]
Hoshiya, T., R. Hasegawa, K. Hakoi, L. Cui, T.Ogiso, R. Cabral, N. Ito, 1993. Enhancement by nonmutagenic pesticides of GST–Ppositive hepatic foci development initiated with diethyl– nitrosamine in the rat. Cancer Letters 72: 59–64. [ Links ]
Juhler, R. K., S.R. Sørensen, L. Larsen, 2001. Analysing transformation products of herbicide residues in environmental samples. Water Research 35: 1371–1378. [ Links ]
Lehr, S., Glässgen, W.E., Sandermann, H., Beese, Jr.F. and Scheunert, I. 1996. Metabolism of isoproturon in soils originating from diferent agricultural management systems and in cultures of isolated soil bacteria. International Journal of Environmental and Analitycal Chemestry 65: 231–243. [ Links ]
Mansour, M., E.A. Feicht, A. Behechti, K.W. Schramm, A. Kettrup, 1999. Determination photostability of selected agrochemicals in water and soil. Chemosphere 39: 575–585. [ Links ]
Mostafa, F.I.Y., C.S. Helling, 2001. Isoproturon degradation as affected by the growth of two algal species at different concentrations and pH values. Journal of Environmental Sciences and Health (Part B) 36:709–727. [ Links ]
Pérés, F., D. Florin, T. Grollier, A. Feurtet–Mazef, M. Coste, F. Ribeyre, M. Ricard, A. Boudou, 1996. Effect of the phenyl–urea herbicide isoproturon on periphytic diatom communities in fresh water indoor microcosms. Environmental Pollution 94: 141–152. [ Links ]
Perrin–Ganier, C., F. Schiavon, J.–L. Morel, M. Schiavon, 2001. Effect of sludge–amendment or nutrient addition on the biodegradation of the herbicide isoproturon in soil. Chemosphere 44: 887–892. [ Links ]
Remde, A., W. Traunspurger, 1994. A method to assess the toxicity of pollutants on anaerobic microbial degradation activity in sediments. Environmental and Toxicology Water Quality 9: 293–298. [ Links ]
Romero, M.C., E. Hammer, R. Hanschke, A.M. Arambarri, F. Schauer, 2005. Biotransformation of biphenyl by filamentous fungus Talaromyces helicus. World Journal of Microbiology and Biotechnology 21: 101–106. [ Links ]
Sandermann, H., W. Heller, N. Hertkorn, E. Hoque, D. Pieper, R. Winkler, 1998. A new intermediate in the mineralization of 3,4–dichloroaniline by the white rot fungus Phanerochaete chrysosporium. Applied and Environmental Microbiology 64: 3305–3312. [ Links ]
Schuelein, J., W.E. Glaessgen, N. Hertkorn, P. Schroeder, H. Sandermann, A. Kettrup, 1996. Detection and identification of the herbicide isoproturon and its metabolites in field samples after a heavy rainfall event. International Journal of Environmental and Analitycal Chemestry 65:193–202. [ Links ]
Sebai, T.E., B. Lagacherie, G. Soulas, F. Martin–Laurent, 2004. Isolation and characterisation of an isoproturon–mineralising Methylopila spp. TES from French agricultural soil. FEMS Microbiological Letters 239:103–110. [ Links ]
Sørensen, S.R., J. Aamand., 2001. Biodegradation of the phenylurea herbicide isoproturon and its metabolites in agricultural soils. Biodegradation 12: 69–77. [ Links ]
Sørensen, S.R., Z. Ronen, J. Aamand, 2001. Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon. Applied and Environmental Microbiology 67:5403–5409. [ Links ]
Sørensen, S.R., Z. Ronen, J. Aamand, 2002. Growth in coculture stimulates metabolism of phenylurea herbicides isoproturon by Sphingomonas sp. SRS2. Applied and Environmental Microbiology 68: 3478–3485. [ Links ]
Sørensen, S.R., G.D. Bending, C.S. Jacobsen, A. Walker, J. Aamand, 2003. Microbial degradation of isoproturon and related phenylurea herbicides in and below agricultural fields. FEMS Microbial Ecology 45: 1–11. [ Links ]
Spliid, N.H., B. Køppen, 1998. Occurrence of pesticides in Danish shallow ground water. Chemosphere 37: 1307–1316. [ Links ]
Stangroom, S.J., C.D. Collins, J.N. Lester, 1998. Sources of organic micropollutants to lowland rivers. Environmental Technology 19:643–666. [ Links ]
Stig R., B. Jensen, S. Rosendahl, B.B. Kragelund, R.K. Juhler, J. Aamand, 2005. Hydroxylation of the herbicide isoproturon by fungi isolated from agricultural soil. Applied and Environmental Microbiology 71:7927–7932. [ Links ]
Suhadolc, M., R. Schroll, A. Gattinger, M. Schloter, J.C. Munch, D. Lestan, 2004. Effects of modified Pb, Zn and Cd–availability on the microbial communities and on the degradation of isoproturon in a heavy metal contaminated soil. Soil Biology and Biochemestry 36:1943–1954. [ Links ]
Thorn, R.G., C.A. Reddy, D. Harris, E.A. Paul, 1996. Isolation of saprophytic basidiomycetes from soil. Applied and Environmental Microbiology 62: 4288– 4292. [ Links ]
Tixier, C., P. Bogaerts, M. Sancelme, F. Bonnemoy, T. Twagilimana, A. Cuer, J. Bohatier, H. Veschambre, 2000. Fungal biodegradation of a phenylurea herbicide, diuron: structure and toxicity of metabolites. Pesticide Managing Science 56:455–462. [ Links ]
Vroumsia, T., R. Steiman, F. Seigle–Murandi, J.–L. Benoit–Guyod, A. Khadrani, 1996. Biodegradation of three substituted phenylurea herbicides (chlortoluron, diuron, and isoproturon) by soil fungi. A comparative study. Chemosphere 33:2045–2056. [ Links ]
Walker, A., R.H. Bromilow, P.H. Nicholls, A.A. Evans, V.J.R. Smith, 2002. Spatial variability in the degradation rates of isoproturon and chlorotoluron in a clay soil. Weed Research 42:39–44. [ Links ]
White, T.J., T. Bruns, P. Leeflang, J. Taylor, 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis, M.A., D.H. Gelfand, J.J. Sninsky, T.J. White (eds.), PCR protocols: a guide to methods and applications. Academic Press, New York, N.Y. pp. 315–322. [ Links ] ]]>