Aerobic Biodegradation of Hydrocarbons in High Temperature and Saline Groundwater

A series of laboratory microcosm experiments and a field pilot test were performed to evaluate the potential for aerobic biodegradation of aromatic hydrocarbons and methyl tert‐butyl ether (MtBE; a common oxygenate additive in gasoline) in saline, high temperature (>30° C) groundwater. Aquifer, sediment, and groundwater samples from two sites, one in Canada and another in Saudi Arabia, were incubated for 106 days to evaluate the changes in select hydrocarbon and MtBE concentrations and microbial community structure. Almost complete biodegradation of the aromatic hydrocarbons was found in the Saudi Arabian microcosm samples whereas the Canadian microcosm samples showed no significant biodegradation during the laboratory testing. MtBE degradation was not observed in either set of microcosms. Denaturing gradient gel electrophoresis analyses showed that, while the Canadian microorganisms were the most diverse, they showed little response during incubation. The microbial communities for the Saudi Arabian sample contained significant numbers of microorganisms capable of hydrocarbon degradation which increased during incubation. Based on the laboratory results, pilot‐scale testing at the Saudi Arabian field site was carried out to evaluate the effectiveness of enhanced aerobic biodegradation on a high temperature, saline petroleum hydrocarbon plume. Dissolved oxygen was delivered to the subsurface using a series of oxygen diffusion emitters installed perpendicular to groundwater flow, which created a reactive zone. Results obtained from the seven‐month field trial indicated that all the target compounds decreased with removal percentages varying between 33 percent for the trimethylbenzenes to greater than 80 percent for the BTEX compounds. MtBE decreased 40 percent on average whereas naphthalene was reduced 85 percent on average. Examination of the microbial population upgradient and downgradient of the emitter reactive zone suggested that the bacteria population went from an anaerobic, sulfate‐reducing dominated population to one dominated by a heterotrophic aerobic bacteria dominant population. These studies illustrate that field aerobic biodegradation may exceed expectations derived from simple laboratory microcosm experiments. Also, high salinity and elevated groundwater temperature do not appear to inhibit in situ aerobic biorestoration. © 2014 Wiley Periodicals, Inc.     

Identification of pyrene‐degradation pathways: Bench‐scale studies using Pseudomonas fluorescens 29L

This article summarizes the bench‐scale studies to identify pyrene‐degradation pathways using an environmental microbial isolate, Pseudomonas fluorescens 29L. Strain 29L was grown on 50 mg of pyrene per liter of mineral medium. At a pyrene biodegradation rate of 14.7 mg/L of medium/day, 82.38 percent of pyrene was degraded in six days. Naphthalene and phenanthrene accounted for 1.09 percent and 3.69 percent, respectively, of the carbon mass from pyrene in the late log phase. Substituted benzene compounds accounted for 26.10 percent of the carbon mass from pyrene in the late log phase. In the stationary phase, carboxylic acids accounted for 10.44 percent of the carbon mass from pyrene. Strain 29L mutants were used for enzyme assays. Pyrene is oxidized by monoxygenases and dioxygenases, and the oxidized ring is cleaved. These enzymes were induced in the presence of pyrene and their activities peaked in the late log phase. No gentisate 1,2‐dioxygenase activity was detected in Strain 29L wild type (WT), whereas mutant M15 did not show any catechol 2,3‐dioxygenase activity. This indicates the possibility of multiple branchings in the pyrene‐biodegradation pathways. In conclusion, multiple degradative pathways are operating concurrently in this strain. The study shows the versatility of Pseudomonas fluorescens Strain 29L for pyrene degradation. It also emphasizes the need to study pyrene‐degradation pathways in other microorganisms so as to enhance the bioremediation potential for the in situ treatment of pyrene‐contaminated sites. © 2008 Wiley Periodicals, Inc.     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in compost

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or copolymers with 10% [P(3HB-co-10%3HV)] and 20% [P(3HB-co-20%3HV)] 3-hydroxyvaleric acid was studied in small household compost heaps. Degradation was measured through loss of weight (surface erosion) and changes in molecular weight and mechanical strength. It was concluded, on the basis of weight loss and loss of mechanical properties, that P(3HB) and P(3HB-co-3HV) plastics were degraded in compost by the action of microorganisms. No decrease inM _w could be detected during the degradation process. The P(3HB-co-20%3HV) copolymer was degraded much faster than the homopolymer and P(3HB-co-10%3HV). One hundred nine microbial strains capable of degrading the polymersin vitro were isolated from the samples used in the biodegradation studies, as well as from two other composts, and identified. They consisted of 61 Gram-negative bacteria (e.g.,Acidovorax facilis), 10 Gram-positive bacteria (mainlyBacillus megaterium), 35Streptomyces strains, and 3 molds.     

Biodegradation of Epoxy and MF Modified Polyurethane Films Derived from a Sustainable Resource

Mesua ferrea L. seed oil (MFLSO) modified polyurethanes blends with epoxy and melamine formaldehyde (MF) resins have been studied for biodegradation with two techniques, namely microbial degradation (broth culture technique) and natural soil burial degradation. In the former technique, rate of increase in bacterial growth in polymer matrix was monitored for 12 days via a visible spectrophotometer at the wavelength of 600 nm using McFarland turbidity as the standard. The soil burial method was performed using three different soils under ambient conditions over a period of 6 months to correlate with natural degradation. Microorganism attack after the soil burial biodegradation of 180 days was realized by the measurement of loss of weight and mechanical properties. Biodegradation of the films was also evidenced by SEM, TGA and FTIR spectroscopic studies. The loss in intensity of the bands at ca. 1735 cm⁻¹ and ca. 1050 cm⁻¹ for ester linkages indicates biodegradation of the blends through degradation of ester group. Both microbial and soil burial studies showed polyurethane/epoxy blends to be more biodegradable than polyurethane/MF blends. Further almost one step degradation in TG analysis suggests degradation for both the blends to occur by breakage of ester links. The biodegradation of the blends were further confirmed by SEM analyses. The study reveals that the modified MFLSO based polyurethane blends deserve the potential to be applicable as “green binders” for polymer composite and surface coating applications.     

Assessment of Bacterial Degradation of Aromatic Hydrocarbons in the Environment by Analysis of Stable Carbon Isotope Fractionation

¹³C/¹²C stable carbon isotope fractionation was used to assess biodegradation in contaminated aquifers with toluene as a model compound. Different strains of anaerobic bacteria (Thauera aromatica, Geobacter metallireducens, and the sulfate-reducing strain TRM1) showed consistent ¹³C/¹²C carbon isotope fractionation with fractionation factors between C = 1.0017 and 1.0018. In contrast, three cultures of aerobic organisms, using different mono- and dioxygenase enzyme systems to initiate toluene degradation, showed variable isotope fractionation factors of C = 1.0027 (Pseudomonasputida strain mt-2), C = 1.0011 (Ralstonia picketii), andC = 1.0004 (Pseudomonas putida strain F1). The great variability of isotope fractionation between different aerobic bacterial strains suggests that interpretation of isotope data in oxic habitats can only be qualitative. A soil column was run as a model system for contaminated aquifers with toluene as the carbon source and sulfate as the electron acceptor and samples were taken at different ports along the column. Microbial toluene degradation was calculated based on the ¹³C/¹²C isotope fractionation factors of the batch culture experiments together with the observed ¹³C/¹²C isotope shifts of the residual toluene fractions. The calculated percentage of biodegradation, B, correlated well with the decreasing toluene concentrations at the sampling ports and indicated the increasing extent of biodegradation along the column. The theoretical toluene concentrations as calculated based on the isotope values matched the measured concentrations at the different sampling ports indicating that the Rayleigh equation can be used to calculate biodegradation in quasi closed systems based on measured isotope shifts. A similar attempt was performed to assess toluene degradation in a contaminated, anoxic aquifer. A transect of groundwater wells was monitored along the main direction of the groundwater flow and revealed decreasing concentrations accompanied with an increase in the ¹³C/¹²C stable carbon isotope ratio of the residual toluene. Calculation of the extent of biodegradation based on the isotope values and laboratory derived isotope fractionation factors showed that the residual toluene was degraded to more than 99% by microbial activity. Calculation of the theoretical residual toluene concentrations based on the measured isotope values described the strongly decreasing concentrations along the plume. Other aromatic hydrocarbons like benzene and naphthalene which were analysed in the same course also showed decreasing concentrations along the groundwater flow path accompanied by increasing ¹³C values indicating biodegradation.     

Fungal Degradation of Poly(l-lactide) in Soil and in Compost

The biodegradability of polymers by microorganisms is generally studied in a real environment that contains a natural mixture of fungi and bacteria. The present research mainly focused on the purely fungal degradation of poly(l-lactide), PLLA, to enclose the part of fungi in a real process of biodegradation and to understand the kinetics of biodegradation. Respirometric tests were realized in soil at 30?°C, and in compost at 30?and 58?°C. Results indicated that temperature is the predominant parameter governing the fungal degradation of PLLA. Moreover, in real compost, the biodegradation kinetics of the PLLA revealed a synergy between bacteria and fungi. The curves of PLLA and cellulose biodegradation were modeled by Hill sigmo?d. Fungal degradation was completed by investigating the physical and the chemical properties of the polymer during the process of degradation using several analytical methods such as matrix assisted laser desorption ionization-time of fly spectroscopy, infrared spectroscopy, size exclusion chromatography, and differential scanning calorimetry. These experiments led to a better understanding of the various stages of fungal degradation of PLLA: hydrolysis as well as mineralization. Furthermore, metabolizing products (by-products) of PLLA was investigated also.     

Biodegradability and biodegradation rate of poly(caprolactone)-starch blend and poly(butylene succinate) biodegradable polymer under aerobic and anaerobic environment

The biodegradability and the biodegradation rate of two kinds biodegradable polymers; poly(caprolactone) (PCL)-starch blend and poly(butylene succinate) (PBS), were investigated under both aerobic and anaerobic conditions. PCL-starch blend was easily degraded, with 88% biodegradability in 44 days under aerobic conditions, and showed a biodegradation rate of 0.07 day⁻¹, whereas the biodegradability of PBS was only 31% in 80 days under the same conditions, with a biodegradation rate of 0.01 day⁻¹. Anaerobic bacteria degraded well PCL-starch blend (i.e., 83% biodegradability for 139 days); however, its biodegradation rate was relatively slow (6.1 mL CH₄/g-VS day) compared to that of cellulose (13.5 mL CH₄/g-VS day), which was used as a reference material. The PBS was barely degraded under anaerobic conditions, with only 2% biodegradability in 100 days. These results were consistent with the visual changes and FE-SEM images of the two biodegradable polymers after the landfill burial test, showing that only PCL-starch blend had various sized pinholes on the surface due to attack by microorganisms. This result may be use in deciding suitable final disposal approaches of different types of biodegradable polymers in the future.     

Biodegradation of Fuel Oil Under Laboratory and Arctic Marine Conditions

The aerobic biodegradation of the components of a fuel oil under Arctic summer conditions follows a pattern that is indistinguishable from that exhibited under temperate conditions. Straight chain alkanes and small aromatics are degraded first, followed by branched alkanes and larger and alkylated aromatics. We present data on the biodegradation of heptadecane as a representative n-alkane, pristane as a representative iso-alkane, and naphthalene, phenanthrene, and chrysene and their alkylated forms as representative two-, three- and four-ring aromatic hydrocarbons. In particular, the pattern of degradation of the alkylated aromatics allows the identification of biodegradation in samples collected from the field and the estimation of the extent of biodegradation that occurred in the In-Situ Treatment of Oiled Sediment Shorelines Field Trials.     

Biodegradation of Polycaprolactone Powders Proposed as Reference Test Materials for International Standard of Biodegradation Evaluation Method

Polycaprolactone (PCL) powders were prepared from PCL pellets using a rotation mechanical mixer. PCL powders were separated by sieves with 60 and 120 meshes into four classes; 0–125 μm, 125–250 μm, 0–250 μm and 250–500 μm. Biodegradation tests of PCL powders and cellulose powders in an aqueous solution at 25°C were performed using the coulometer according to ISO 14851. Biodegradation tests of PCL powders and cellulose powders in controlled compost at 58°C were performed by the Mitsui Chemical Analysis and Consulting Service, Inc. according to ISO 14855-1 and by using the Microbial Oxidative Degradation Analyzer (MODA) instrument according to ISO/DIS 14855-2. PCL powders were faster biodegraded than cellulose powders. The reproducibility of biodegradation of PCL powders is excellent. Differences in the biodegradation of PCL powders with different class were not observed by the ISO 14851 and ISO/DIS 14855-2. An enzymatic degradation test of PCL powders with different class was studied using an enzyme of Amano Lipase PS. PCL with smaller particle size was faster degraded by the enzyme. PCL powders with regulated sizes from 125 μm to 250 μm are proposed as a reference material for the biodegradation test.     

Kinetic Study of Naphthalene Biodegradation in Aerobic Slurry Phase Microcosms for the Optimisation of the Process

The research was focused on the slurry-phase biodegradation of naphthalene. The biodegradation process was optimised with preliminary experiments in slurry aerobic microcosms. From soil samples collected on a contaminated site, a Pseudomonas putida strain, called M8, capable to degrade naphthalene was selected. Microcosms were prepared with M8 strain by mixing non-contaminated soil and mineral M9 medium. Different experimental conditions were tested varying naphthalene concentration, soil:water ratio and inoculum density. The disappearance of hydrocarbon, the production of carbon dioxide, and the ratio of total heterotrophic and naphthalene-degrading bacteria were monitored at different incubation times. The kinetic equation that best fitted the disappearance of contaminant with time was determined. The results showed that the isolated strain enhanced the biodegradation rate with respect to the natural biodegradation.     

Biodegradable Polyester-Based Blend Reinforced with Curauá Fiber: Thermal,Mechanical and Biodegradation Behaviour

Biodegradable composites can be produced by the combination of biodegradable polymers (BP) as matrix and vegetal fibers as reinforcement. Composites of a commercial biodegradable polymer blend and curauá fibers (loaded at 5, 15 and 20 wt%) were prepared by melt mixing in a twin-screw extruder. Chemical treatments such as alkali treatment of the fiber and addition of maleic anhydride grafted polypropylene (MA-g-PP) as coupling agent were performed to promote polymer/fiber interfacial adhesion so that mechanical performance can be improved. The resulting composites were evaluated through hardness, melt flow index and tensile, flexural and impact strengths as well as water absorption. Thermal analysis and Fourier transform infrared spectroscopy were also employed to characterize the composites. The polymer/fiber interface was investigated through scanning electron microscopy analysis. The biodegradability of composites was evaluated by compost-soil burial test. The addition of curauá fiber promoted an increase in the mechanical strengths and composites treated with 2 wt% MA-g-PP with 20 wt% curauá fiber showed an increase of nearly 75% in tensile and 56% in flexural strengths besides an improvement in impact strength with respect to neat polymer blend. Nevertheless, treated composites showed an increase in water absorption and biodegradation tests showed that the addition of fiber retards degradation time. The retained mass of BP/20 wt% fiber composite with MA-g-PP and neat BP was 68 and 26%, respectively, after 210 days of degradation test.     

Biodegradation of Poly(ε-caprolactone) (PCL) Film by <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

The biodegradation behavior of PCL film with high molecular weight (80,000 Da) in presence of bacterium Alcaligenes faecalis and the analysis of degraded polymer film have been carried out. Thin Films of PCL were prepared by means of solution casting method and the bacterial degradation behavior was carried in basal medium, in presence of bacteria with time variation after UV treatment. It was observed that after UV treatment the degradation of polymer film was increased and the degradation rate followed a three steps degradation mechanism. The degraded polymer film was analyzed by means of Differential Scanning Calorimeter (DSC), Thermo Gravimetric Analyzer (TGA) and Fourier Transform Infrared Spectroscope (FTIR). DSC results revealed that at the initial stages of the degradation up to 15–20 days, the bacterium preferentially degrades the amorphous parts of the polymer film over the crystalline zone. Thermo gravimetric analysis highlighted the low temperature stability of degraded films with extent of degradation. FTIR results showed the chain scission mechanism of the polymer chains and also supported the preferential degradation of amorphous phase over crystalline phase in the initial stages of the degradation.     

Comparison of the food waste degradation rate and microbial community in the matrix between two biodegradation agents in a food waste disposer

To reduce the proportion of food waste in municipal solid waste, a food waste biodegradation experiment with two biodegradation agents was conducted for seven weeks with 500 g of food waste added every day into each disposer. The agent containing four biodegradation bacterial strains showed higher degradation rates and matrix temperatures than that containing two. Furthermore, significant differences in the microbiological community structures of the matrixes were found not only between the two biodegradation systems but also among different stages in the same degradation system based on DGGE profiles. The F2 strain exhibited the highest DGGE optical density (OD) value among biodegradation systems and at all experimental stages, suggesting it was a dominant strain during food waste degradation.     

Fungal Degradation of Poly(Butylene Adipate-Co-Terephthalate) in Soil and in Compost

The present work mainly dedicated to fungal degradation of poly(butylene adipate-co-terephthalate) [PBAT], to enclose the role of fungi in a real process of biodegradation, the degree of degradation, and to understand the kinetics of PBAT biodegradation. Respirometer tests were realized in soil at 30 °C, and in compost at 30 and 58 °C. Results have shown that temperature is one of the essential parameters governing the fungal degradation of PBAT. Moreover, the final rates of PBAT biodegradation in an inoculated compost with fungi and in a real compost were found comparable, which means that the selected fungi were efficient as much as a mixture of bacteria and fungi. The curves of PBAT biodegradation were modeled by Hill sigmoid. Fungal degradation was completed by investigating the physical and the chemical properties of the polymer during the process of degradation using several analytical methods such as matrix assisted laser desorption ionization-time of fly spectroscopy, size exclusion chromatography, and differential scanning calorimetry. These experiments led to a better understanding of the various stages of fungal degradation of PBAT: hydrolysis as well as mineralization. Furthermore, the analysis of metabolizing products was investigated also.     

Novel synthesis blends between bacterial polyesters and acrylic rubber: A study on enzymatic biodegradation

The biodegradability of a multicomponent system based on biotechnological occurring polyester (poly(-hydroxybutyrate-co--hydroxyvalerate) (PHBV)) with inclusion of acrylate elastomer (polybutylacrylate) (PBA) was investigated. A bacterium which produced extracellular enzymes that degrades PHBV even when blended with PBA was isolated and tentatively designated asAureobacterium saperdae. It was observed, by morphological investigation, that, while the bacterial degradation was permitted for PBA content of 20% by weight, it was inhibited for PBA content of 30%, owing to the occurrence of a rubbery layer that prevents to the bacteria an easy accessibility in the PHBV-rich regions. In fact, owing the bacterial growth, only PHBV was metabolized, whereas no degradation of PBA was detected for blend samples. It was confirmed that the degradation proceeded via surface erosion of PHBV also in the blends. Finally, mechanical tests on PHBV/PBA specimens as a function of degradation extent have shown different behavior of the blends at different the PBA content. Thermal analysis of blends and PHBV has been reported, too     

Biodegradability of Poly (lactic acid), Preparation and Characterization of PLA/Gum Arabic Blends

In this study, the biodegradation of PLA films using microorganisms from Lake Bogoria (Kenya) were investigated. The biodegradation tests done using certain strains of thermophilic bacteria showed faster biodegradation rates and demonstrated temperature dependency. The biodegradation of the PLA films was studied using Gel Permeation Chromatography (GPC) and light microscopy. The biodegradation of PLA was demonstrated by decrease in molecular weight. The preparation and characterization of PLA/Gum Arabic blends were also investigated using DSC, TGA, TMA and NMR. In summary, the results obtained in this research show that PLA films undergo fast biodegradation using thermophiles isolated from Lake Bogoria. The PLA/GA blends studies show it is possible to prepare films of varying hydrophobic–hydrophilic properties for various applications.     

Biochemical Cooperation Between Klebsiella oxytoca Sc and Methylobacterium mesophilium Sr for Complete Degradation of Dimethyl Isophthalate

Klebsiella oxytoca Sc and Methylobacterium mesophilicum Sr were isolated from enrichment cultures using dimethyl isophthalate (DMI) as the sole carbon and energy source and mangrove sediment as an inoculum. Complete degradation of DMI required both species of bacteria at different biochemical transformation steps. The biochemical degradation pathway was DMI to monomethyl isophthalate (MMI) by K. oxytoca Sc, MMI to isophthalate (IPA) by M. mesophilium Sr, and IPA by both K. oxytoca Sc and M. mesophilium Sr sequentially. The consortium comprising of K. oxytoca Sc and M. mesophilicum Sr was effective in complete degradation of DMI in eight days. Our results suggest that a consortium of microorganisms indigenous to the mangrove environment is responsible for mineralization of environmental pollutant DMI through biochemical cooperation.     

Intrinsic and Stimulated In Situ Biodegradation of Hexachlorocyclohexane (HCH)

The feasibility of the biodegradation of HCH and its intermediates has been investigated. A recent characterisation of two sites in The Netherlands has shown intrinsic biodegradation of HCH. At one site, breakdown products (monochlorobenzene, benzene and chlorophenol) were found in the core of the HCH-plume, whereas the HCH-concentration decreased over time and space. Characterisation of a second, industrial site indicated less intrinsic biodegradation and the need to stimulate biodegradation. In the laboratory, enhanced HCH degradation was tested with soil and groundwater material from both sites, and the required conversion to the intermediates benzene and monochlorobenzene was demonstrated. Furthermore, the biodegradation of these intermediates could be initiated by adding low amounts of oxygen (<5%). Adding nitrate enhanced this degradation. We hypothesise that this occurs through anaerobic nitrate reducing conversion of oxidised intermediates.At the non-industrial other site, intrinsic degradation took place, as shown in the laboratory experiments. Interpretation of the field data with computer codes Modflow and RT3D was performed. As a result of the modelling study, it has been proposed to monitor natural attenuation for several years before designing the final approach.At the industrial site, the results of the batch experiments are applied. Anaerobic HCH degradation to monochlorobenzene and benzene is stimulated via the addition of an electron donor.Infiltration facilities have been installed at the site to create an anaerobic infiltration zone in which HCH will be degraded, and these facilities are combined with the redevelopment of the site.     

Effects of physicochemical factors on PAH degradation by Planomicrobium alkanoclasticum

Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that are mutagenic, carcinogenic, and toxic to living organisms. Here, the ability and effectiveness of selected bacteria isolated from an oil‐contaminated area in biodegrading PAHs were evaluated, and the optimal conditions conducive to bacterial PAH biodegradation were determined. Of six bacterial isolates identified based on their 16S rRNA sequences, Planomicrobium alkanoclasticum could subsist on and consume nearly all hydrocarbons according to the 2,6‐dichlorophenolindophenol assay. The efficacy of this isolate at PAH biodegradation was then empirically confirmed. After 30 days of incubation, P. alkanoclasticum degraded 90.8% of the 16 PAH compounds analyzed and fully degraded eight of them. The optimum P. alkanoclasticum growth conditions were 35°C, pH 7.5, and NaNO₃ as the nitrogen source. Under these biostimulant conditions, P. alkanoclasticum degraded 91.4% of the total PAH concentration and completely decomposed seven PAHs after 15 days incubation. Hence, P. alkanoclasticum is an apt candidate for the biodegradation of PAHs and the bioremediation of sites contaminated by them.     

Fungal Degradation of the Bioplastic PHB (Poly-3-hydroxy- butyric acid)

PHB (poly-3-hydroxybutyric acid) is a thermoplastic polyester synthesized by Ralstonia eutropha and other bacteria as a form of intracellular carbon and energy storage and accumulated as inclusions in the cytoplasm of these bacteria. The degradation of PHB by fungi from samples collected from various environments was studied. PHB depolymerization was tested in vials containing a PHB-containing medium which were inoculated with isolates from the samples. The degradation activity was detected by the formation of a clear zone below and around the fungal colony. In total, 105 fungi were isolated from 15 natural habitats and 8 lichens, among which 41 strains showed PHB degradation. Most of these were deuteromycetes (fungi imperfecti) resembling species of Penicillium and Aspergillus and were isolated mostly from soils, compost, hay, and lichens. Soil-containing environments were the habitats from which the largest number of fungal PHB degraders were found. Other organisms involved in PHB degradation were observed. A total number of 31 bacterial strains out of 67 isolates showed clear zones on assay medium. Protozoa, possible PHB degraders, were also found in several samples such as pond, soil, hay, horse dung, and lichen. Lichen, a fungi and algae symbiosis, was an unexpected sample from which fungal and bacterial PHB degraders were isolated.