Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.     

Anaerobic digestion and wastewater treatment systems

Upflow Anaerobic Sludge Bed (UASB) wastewater (pre-)treatment systems represent a proven sustainable technology for a wide range of very different industrial effluents, including those containing toxic/inhibitory compounds. The process is also feasible for treatment of domestic wastewater with temperatures as low as 14–16° C and likely even lower. Compared to conventional aerobic treatment systems the anaerobic treatment process merely offers advantages. This especially is true for the rate of start-up. The available insight in anaerobic sludge immobilization (i.e. granulation) and growth of granular anaerobic sludge in many respects suffices for practice. In anaerobic treatment the immobilization of balanced microbial communities is essential, because the concentration of intermediates then can be kept sufficiently low.So far ignored factors like the death and decay rate of organisms are of eminent importance for the quality of immobilized anaerobic sludge. Taking these factors into account, it can be shown that there does not exist any need for phase separation when treating non- or slightly acidified wastewaters. Phase separation even is detrimental in case the acidogenic organisms are not removed from the effluent of the acidogenic reactor, because they deteriorate the settleability of granular sludge and also negatively affect the formation and growth of granular sludge. The growing insight in the role of factors like nutrients and trace elements, the effect of metabolic intermediates and end products opens excellent prospects for process control, e.g. for the anaerobic treatment of wastewaters containing mainly methanol.Anaerobic wastewater treatment can also profitably be applied in the thermophilic and psychrophilic temperature range. Moreover, thermophilic anaerobic sludge can be used under mesophilic conditions.The Expanded Granular Sludge Bed (EGSB) system particularly offers big practical potentials, e.g. for very low strength wastewaters (COD 1 g/l) and at temperatures as low as 10° C. In EGSB-systems virtually all the retained sludge is employed, while compared to UASB-systems also a substantially bigger fraction of the immobilized organisms (inside the granules) participates in the process, because an extraordinary high substrate affinity prevails in these systems. It looks necessary to reconsider theories for mass transfer in immobilized anaerobic biomass.Instead of phasing the digestion process, staging of the anaerobic reactors should be applied. In this way mixing up of the sludge can be significantly reduced and a plug flow is promoted. A staged process will provide a higher treatment efficiency and a higher process stability. This especially applies for thermophilic systems.     

Use of the upflow sludge blanket (USB) reactor concept for biological wastewater treatment,especially for anaerobic treatment

In recent years considerable effort has been made in the Netherlands toward the development of a more sophisticated anaerobic treatment process, suitable for treating low a strength wastes and for applications at liquid detention times of 3–4 hr. The efforts have resulted in new type of upflow anaerobic sludge blanket (UASB) process, which in recent 6 m³ pilot-plant experiments has shown to be capable of handling organic space loads of 15–40 kg chemical oxygen demand (COD)·m^?3/day at 3–8 hr liquid detention times. In the first 200 m³ full-scale plant of the UASB concept, organic space loadings of up to 16 kg COD·m^?3/day could be treated satisfactorily at a detention times of 4 hr, using sugar beet waste as feed. The main results obtained with the process in the laboratory as well as in 6 m³ pilot plant and 200 m³ full-scale experiments are presented and evaluated in this paper. Special attention is given to the main operating characteristics of the UASB reactor concept. Moreover, some preliminary results are presented of laboratory experiments concerning the use of the USB reactor concept for denitrification as well as for the acid formation step in anaerobic treatment. For both purposes the process looks feasible because very satisfactory results with respect to denitrification and acid formation can be achieved at very high hydraulic loads (12 day^?1) and high organic loading rates, i.e., 20 kg COD·m^?3/day in the denitrification and 60–80 kg COD·m^?3/day in the acid formation experiments.     

Anaerobic bioprocessing of organic wastes

Anaerobic digestion of dissolved, suspended and solid organics has rapidly evolved in the last decades but nevertheless still faces several scientific unknowns. In this review, some fundamentals of bacterial conversions and adhesion are addressed initially. It is argued in the light of G-values of reactions, and in view of the minimum energy quantum per mol, that anaerobic syntrophs must have special survival strategies in order to support their existence: redistributing the available energy between the partners, reduced end-product fermentation reactions and special cell-to-cell physiological interactions. In terms of kinetics, it appears that both reaction rates and residual substrate thresholds are strongly related to minimum G-values. These new fundamental insights open perspectives for efficient design and operation of anaerobic bioprocesses. Subsequently, an overview is given of the current anaerobic biotechnology. For treating wastewaters, a novel and high performance new system has been introduced during the last decade; the upflow anaerobic sludge blanket system (UASB). This reactor concept requires anaerobic consortia to grow in a dense and eco-physiologically well-organized way. The microbial principles of such granular sludge growth are presented. Using a thermodynamic approach, the formation of different types of aggregates is explained. The application of this bioprocess in worldwide wastewater treatment is indicated. Due to the long retention times of the active biomass, the UASB is also suitable for the development of bacterial consortia capable of degrading xenobiotics. Operating granular sludge reactors at high upflow velocities (5–6 m/h) in expanded granular sludge bed (EGSB) systems enlarges the application field to very low strength wastewaters (chemical oxygen demand < 1 g/l) and psychrophilic temperatures (10°C). For the treatment of organic suspensions, there is currently a tendency to evolve from the conventional mesophilic continuously stirred tank system to the thermophilic configuration, as the latter permits higher conversion rates and easier sanitation. Integration of ultrafiltration in anaerobic slurry digestion facilitates operation at higher volumetric loading rates and at shorter residence times. With respect to organic solids, the recent trend in society towards source separated collection of biowaste has opened a broad range of new application areas for solid state anaerobic fermentation.W. Verstraete and D. de Beer are with the Center for Environmental Sanitation, University of Gent, Coupure L 653, B-9000 Gent, Belgium; D. de Beer is also with the Max Plank Institut für Marine Mikrobiologie-Microzensor Group, Fahrenstrasse 1, 28359 Bremen, Germany. M. Pena is with the Groupo de Biotechnologia Ambiental, Departamento de Ingenieria Quimica, Universidad de Valladolid, Prado de la Magdalena, 47005 Valladolid, Spain. G. Lettinga is with the Department of Environmental Technology, Wageningen Agricultural University, Bomenweg 2, 6703 HD Wageningen, The Netherlands. P. Lens is with the Environmental Research Unit. Department of Microbiology, University College Galway, Galway, Ireland.     

Review of the in vitro activity and potential clinical efficacy of levofloxacin in the treatment of anaerobic infections

The activity of levofloxacin against aerobic bacteria has been well documented both in vitro and clinically, but its anaerobic activity has been infrequently studied. This new fluoroquinolone exhibits good in vitro activity (MIC(S) < or =2.0 microg/mL) against many anaerobic pathogens associated with acute sinusitis, bite wounds, and other soft-tissue infections. It is less active against Bacteroides fragilis (MIC (90)=2-4 microg/mL ) and has poor inhibitory activity against non-fragilis B. fragilis group species that are associated with gastrointestinal and genitourinary tract infections. Levofloxacin does not antagonize the in vitro activity of clindamycin and metronidazole and often provides additive or synergistic activity against anaerobic bacteria with these agents. In pharmacodynamic models, levofloxacin exhibits rapid bactericidal activity at 2-4 times the MIC of anaerobic bacteria. Prolonged killing is observed when the area-under-the concentration-time-curve to MIC ratio is greater than 40. In clinical efficacy trials, levofloxacin has been effective in the treatment of patients with gynecologic, skin and skin-structure, and bone infections involving anaerobic pathogens. Both micro-biologic and pharmacodynamic studies support further evaluations of levofloxacin in the treatment of selective mixed aerobic/anaerobic infections.     

Treatment Strategies and Survival of Older Breast Cancer Patients – An International Comparison between the Netherlands and Ireland

ObjectivesForty percent of breast cancers occur among older patients. Unfortunately, there is a lack of evidence for treatment guidelines for older breast cancer patients. The aim of this study is to compare treatment strategy and relative survival for operable breast cancer in the elderly between The Netherlands and Ireland.ResultsOverall, 41,055 patients from The Netherlands and 5,826 patients from Ireland were included. Overall, more patients received guideline-adherent locoregional treatment in The Netherlands, overall (80% vs. 68%, adjusted p<0.001), stage I (83% vs. 65%, p<0.001), stage II (80% vs. 74%, p<0.001) and stage III (74% vs. 57%, P<0.001) disease. On the other hand, more systemic treatment was provided in Ireland, where endocrine therapy was prescribed to 92% of hormone receptor-positive patients, compared to 59% in The Netherlands. In The Netherlands, only 6% received chemotherapy, as compared 24% in Ireland. But relative survival was poorer in Ireland (5 years relative survival 89% vs. 83%), especially in stage II (87% vs. 85%) and stage III (61% vs. 58%) patients.ConclusionTreatment for older breast cancer patients differed significantly on all treatment modalities between The Netherlands and Ireland. More locoregional treatment was provided in The Netherlands, and more systemic therapy was provided in Ireland. Relative survival for Irish patients was worse than for their Dutch counterparts. This finding should be a strong recommendation to study breast cancer treatment and survival internationally, with the ultimate goal to equalize the survival rates for breast cancer patients across Europe.     

Evaluation for anaerobic culture system: Anoxomat Mart II]

Anoxomat Mart II (Mart Microbiology BV, Lichtenvooorde, Netherlands, Central Scientific Commerce Inc., Tokyo, Japan) is an anaerobic jar apparatus which uses a vacuum pump in combination with catalyst as gas replacement procedure to remove all traces of oxygen. As we had a chance to use Anoxomat Mart II, we compared it with other two anaerobic culture methods; namely AnaeroPack anaero (Mitsubishi Gas Chemical Co., Tokyo, Japan) which employs anaerobic jar method, and Concept400 (RUSKINN TECHNOLOGY LTD, England; Central Scientific Commerce INc., Tokyo, Japan) which uses anaerobic chamber method. We used 10 different species of anaerobic bacteria obtained from ATCC. One strain each of 10 species was cultured and examined for measurement of the sensitibity of an anaerobic indicator, th number of bacteria after 48 hour culture, the diameter of colonies, and MIC value. As a result, the time to reach the anaerobic condition was around 30 minutes by the Mart II against around 60 minutes by the AnaeroPack anaero. There was no difference concerning the number of bacteria after 48 hour culture among three methods. But anaerobic bacteria cultured by Mart II tended to make bigger colonies compared to other two methods in the 5 strains out of 9, except for one strain in which the diameter of colonies could not be measured. On the other hand, the comparison of MIC value showed good correlation in 11 antibiotics out of 12 among three methods. The MIC value of 11 antibiotics fitted within 1-fold difference, and 2-fold difference was observed in only one antibiotic. Mart II is so small that it does cheep consumables. From these reasons, we concluded that Mart II can be one of the useful anerobic culture methods.     

Limitations of thermophilic anaerobic wastewater treatment and the consequences for process design

Thermophilic anaerobic digestion offers an attractive alternative for the treatment of medium- and high-strength wastewaters. However, literature reports reveal that thermophilic wastewater treatment systems are often more sensitive to environmental changes than the well-defined high-rate reactors at the mesophilic temperature range. Also, in many cases a poorer effluent quality is experienced while the carry over of suspended solids in the effluent is relatively high. In this paper recent achievements are discussed regarding the process stability of thermophilic anaerobic wastewater treatment systems. Laboratory experiments reveal a relatively low sensitivity to temperature changes if high-rate reactors with immobilized biomass are used. Other results show that if a staged process is applied, thermophilic reactors can be operated for prolonged periods of time under extreme loading conditions (80–100 kg chemical oxygen demand.m^-3.day^-1), while the concentrations of volatile fatty acids in the effluent remain at a low level.     

The anammoxosome: an intracytoplasmic compartment in anammox bacteria

Anammox bacteria belong to the phylum Planctomycetes and perform anaerobic ammonium oxidation (anammox); they oxidize ammonium with nitrite as the electron acceptor to yield dinitrogen gas. The anammox reaction takes place inside the anammoxosome: an intracytoplasmic compartment bounded by a single ladderane lipid-containing membrane. The anammox bacteria, first found in a wastewater treatment plant in The Netherlands, have the potential to remove ammonium from wastewater without the addition of organic carbon. Very recently anammox bacteria were also discovered in the Black Sea where they are responsible for 30-50% of the nitrogen consumption. This review will introduce different forms of intracytoplasmic membrane systems found in prokaryotes and discuss the compartmentalization in anammox bacteria and its possible functional relation to catabolism and energy transduction.     

Anaerobic methanethiol degradation in upflow anaerobic sludge bed reactors at high salinity (> or =0.5 M Na(+))

The feasibility of anaerobic methanethiol (MT) degradation at elevated sodium concentrations was investigated in a mesophilic (30 degrees C) lab-scale upflow anaerobic sludge bed (UASB) reactor, inoculated with estuarine sediment originating from the Wadden Sea (The Netherlands). MT was almost completely degraded (>95%) to sulfide, methane and carbon dioxide at volumetric loading rates up to 37 mmol MT x L(-1) x day(-1), 0.5 M sodium (NaCl or NaHCO(3)) and between pH 7.3 and 8.4. Batch experiments revealed that inhibition of MT degradation started at sodium (both NaCl and NaHCO(3)) concentrations exceeding 0.8 M. Sulfide inhibited MT degradation already around 3 mM (pH 8.3).     

Relationship between intestinal microecology and the translocation of intestinal bacteria

It is now well known that endogenous bacteria can translocate from the intestinal tract and cause many of the complicating infections seen in severely ill, hospitalized patients. Of the hundreds of bacterial species in the intestinal tract, relatively few aerobic/facultative species appear to translocate with any frequency. Van der Waaij and colleagues (1971, 1972a, 1972b) originally proposed that, by a process termed colonization resistance, strictly anaerobic bacteria prevented the intestinal overgrowth and subsequent translocation of these potentially pathogenic aerobic/facultative bacteria. Selective antimicrobial decontamination, designed to maintain colonization resistance, has been effective in reducing the incidence of infectious morbidity in high risk patients. However, the mechanisms controlling bacterial translocation remain unclear, but appear to depend on host factors, as well as on factors inherent in the microbe itself. There is both clinical and experimental evidence supporting the concept that strictly anaerobic bacteria do not readily translocate. Bacteria that are able to survive within macrophages (e.g., Salmonella species and Listeria monocytogenes) translocate easier than others, and there is recent experimental evidence that normal intestinal bacteria may translocate to the draining mesenteric lymph node within host phagocytes. There is also evidence that anaerobic bacteria translocate along with facultative species in situations associated with intestinal epithelial damage, i.e., burn trauma, oral ricinoleic acid, and acute mesenteric ischemia. In contrast, recent experimental evidence demonstrates that facultative bacteria can translocate across a histologically intact intestinal epithelium, and that the ileal absorptive cell may be at least one portal of entry prior to transport into deeper tissues. It is anticipated that further clarification of the routes and mechanisms involved in bacterial translocation will provide new insights into the treatment and prevention of a significant proportion of the infectious morbidity seen in severely ill, hospitalized patients. Antoni van Leeuwenhoek Lecture presented at the Annual Meeting of the Netherlands Society of Microbiology, Utrecht, 23 November, 1989.     

"Candidatus Cloacamonas acidaminovorans": genome sequence reconstruction provides a first glimpse of a new bacterial division

Many microorganisms live in anaerobic environments. Most of these microorganisms have not yet been cultivated. Here, we present, from a metagenomic analysis of an anaerobic digester of a municipal wastewater treatment plant, a reconstruction of the complete genome of a bacterium belonging to the WWE1 candidate division. In silico proteome analysis indicated that this bacterium might derive most of its carbon and energy from the fermentation of amino acids, and hence, it was provisionally classified as "Candidatus Cloacamonas acidaminovorans." "Candidatus Cloacamonas acidaminovorans" is probably a syntrophic bacterium that is present in many anaerobic digesters. This report highlights how environmental sequence data might provide genomic and functional information about a new bacterial clade whose members are involved in anaerobic digestion.     

Benzene Degradation at a Site Amended with Nitrate or Chlorate

Aromatic hydrocarbons are widespread in nature and often contribute to the pollution of soils, sediments, and groundwater. The contamination of soil with mobile aromatic compounds, generally termed BTEX (benzene, toluene, ethylbenzene, xylene) is observed at many industrial sites, especially those associated with the petrochemical industry. In situ bioremediation of sites that are contaminated with BTEX can be applied both aerobically and anaerobically. The use of anaerobic in situ bioremediation is advantageous because supply of oxygen is not needed. Nevertheless, anaerobic in situ bioremediation is less commonly used for BTEX contaminated sites. This paper describes push-pull experiments in order to stimulate the degradation of benzene by the addition of nitrate or chlorate. Deuterated benzene was subjected with nitrate-amended groundwater to the aquifer, and the mineralization was traced by the enrichment of deuterium in the groundwater. Nitrate can be used as electron acceptor, and the addition of nitrate at a site in The Netherlands resulted in partial degradation of benzene. This was demonstrated by comparing various push-pull experiments, benzene concentration measurements, stable isotope analyses of benzene and water, and modeling. Chlorate can be used for the in situ production of oxygen, followed by degradation of benzene with oxygen as electron acceptor. The addition of chlorate at the site resulted in the complete removal of benzene demonstrating a complete degradation within 4 weeks. A pull phase was not needed during this run.     

A dynamic phosphate budget model for a eutrophic lake

The relations between the external nutrient loading of lakes, recycling through sediments and the resulting productivity are complicated by feed-back mechanisms, seasonal variations and trends. Simulation is a useful tool for the identification of controlling factors and the assessment of the effects of management measures, supplementary to experimental research. The model variables in our dynamic phosphate budget model include inorganic and organic particulate phosphate and dissolved o-phosphate, in both sediments and overlying water. Sediments may be aerobic or anaerobic, depending on topography, temperature and composition. The major processes described are primary production, mineralisation, sedimentation, adsorption and diffusion. Several model parameters have been estimated directly for Lake Brielle (Netherlands). The sediment dilution rate, the extent of anaerobic conditions and the number and character of adsorption sites are important controlling factors.     

Detoxification and partial mineralization of the azo dye mordant orange 1 in a continuous upflow anaerobic sludge-blanket reactor

In batch toxicity assays, azo dye compounds were found to be many times more toxic than their cleavage products (aromatic amines) towards methanogenic activity in anaerobic granular sludge. Considering the ability of anaerobic microorganisms to reduce azo groups, detoxification of azo compounds towards methanogens can be expected to occur during anaerobic wastewater treatment. In order to test this hypothesis, the anaerobic degradation of one azo dye compound, Mordant orange 1 (MO1), by granular sludge was investigated in three separate continuous upflow anaerobic sludge-blanket reactors. One reactor, receiving no cosubstrate, failed after 50 days presumably because of a lack of reducing equivalents. However, the two reactors receiving either glucose or a volatile fatty acids (acetate, propionate, butyrate) mixture, could eliminate the dye during operation for 217 days. The azo dye was reductively cleaved to less toxic aromatic amines (1,4-phenylenediamine and 5-aminosalicylic acid) making the treatment of MO1 feasible at influent concentrations that were over 25 times higher than their 50% inhibitory concentrations. In the reactor receiving glucose as cosubstrate, 5-aminosalicylic acid could only be detected at trace levels in the effluent after day 189 of operation. Batch biodegradability assays with the sludge sampled from this reactor confirmed the mineralization of 5-aminosalicylic acid to methane. Received: 11 July 1996 / Received revision: 18 September 1996 / Accepted: 18 September 1996     

Aerobic and Anaerobic Biodegradation of Aged Pentachlorophenol by Indigenous Microorganisms

Pentachlorophenol (PCP) use as a general biocide, particularly for treating wood, has led to widespread environmental contamination. Biodegradation has emerged as the main mechanism for PCP degradation in soil and groundwater and a key strategy for remediation. Examining the microbial biodegrading potential for PCP at a contaminated site is crucial in determining its fate. Hundreds of studies have been published on PCP microbial degradation, but few have described the biodegradation of PCP that has been in contact with soils for many years. The bioavailability of “aged” hydrophobic organics is a significant concern. PCP- and 2,3,4,6-tetrachlorophenol (2,3,4,6-TeCP)-contaminated soil samples from several depths at a former wood treatment site were placed under varying conditions in the laboratory to determine the anaerobic and aerobic potential for biodegradation of chlorophenols at the site. PCP biodegradation occurred in both anaerobic and aerobic soil samples. Rapid aerobic degradation occurred in samples spiked with 2- and 4-chlorophenol, but not with 3-chlorophenol. Reductive dechlorination of PCP in anaerobic samples resulted in the accumulation of 3-chlorophenol. In most anaerobic replicates, 3-chlorophenol was degraded with the appearance of detectable, but not quantifiable amounts of phenol. These results indicate excellent potential for remediation at the site using the indigenous microorganisms under both aerobic and anaerobic conditions. However, a fraction of the PCP was unavailable for degradation.     

Advanced directives. Legal and practical aspects of negative instructions concerning medical treatment

The right of a competent patient to stipulate in advance of possible non-competence, medical treatment he does not want, is based on the fundamental legal principle that a person cannot be medically treated without his informed consent. It is in Dutch law a strong and almost unqualified right. There are indications in the international literature, however, that advance directives in many cases may not have much influence on medical treatment at the end of life. Very little is known about the social practice of advance directives in the Netherlands.     

Use of the Static Granular Bed Reactor (SGBR) with anaerobic sludge to treat poultry slaughterhouse wastewater and kinetic modeling

Poultry slaughterhouses discharge very high amount of wastewaters and these wastewaters can be treated successfully at a very low cost using anaerobic treatment. In this study, the Static Granular Bed Reactor (SGBR), a newly developed anaerobic process which is fully anaerobic granule, and another Static Granular Bed Reactor containing both anaerobic granular biomass and non-granular biomass were employed for the treatment of poultry slaughterhouse wastewater. The objective of the use of two reactors having different types of anaerobic biomass is to evaluate whether anaerobic sludge could be used effectively instead of anaerobic granule, which is much more difficult to obtain than the other during the start up period. Average COD removal efficiencies were greater than 95% for both of the reactors. Furthermore, Grau second-order and modified Stover–Kincannon models were successfully used to develop a kinetic model of the experimental data with a high correlation coefficient (R² > 0.95).     

Analysis of Microbial Communities in a Landfill Leachate Polluted Aquifer using a New Method for Anaerobic Physiological Profiling and 16S rDNA Based Fingerprinting

Abstract Databases containing information regarding presence and activity of microbial communities will be very useful for determination of the potential for intrinsic bioremediation in landfill leachate polluted aquifers. Simple analyses such as community-level physiological profiling (CLPP) and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments yield large sets of data for inclusion into such databases. In this study we describe the development of a method for anaerobic CLPP, using commercially available Biolog plates. Incubation at the in situ temperature of the aquifer (10°C) for 28 days was optimal for obtaining a specific, reproducible physiological profile. Anaerobic incubation was essential for profiling anaerobic communities. The anaerobic cultivation-dependent CLPP method and cultivation-independent DGGE were applied to groundwater and sediment samples from the aquifer near the Coupépolder landfill in The Netherlands. A combination of computer-assisted CLPP and DGGE analysis of both groundwater and sediment samples yielded the best separating power for characterizing microbial communities in the aquifer. Communities in groundwater were significantly different from those in the corresponding sediment. Microbial communities present in subsamples from sediment cores usually were similar for the various sampling locations. Variation was observed for the heterogeneous sediment beneath the landfill. Both anaerobic CLPP and DGGE analysis clearly separated microbial communities from the polluted aquifer underneath the landfill from those in the less or not polluted aquifer downstream and upstream of the landfill. Received: 3 January 2000; Accepted: 21 March 2000; Online Publication: 28 August 2000