Chlorinated biphenyl degradation by wild yeasts pre-cultured in biphasic systems
M. Cristina Romero*
Micología Médica e Industrial, Microbiología
Facultad de Ciencias Veterinarias
Universidad Nacional de
Av. 60 e/ 119 y 120 s/nro., 1900
La Plata, Argentina
Tel: 0054 221 4247642
Facultad de Ciencias Agrarias y Forestales
Universidad Nacional de
Av. 60 e/ 119 y 120 s/nro., 1900
Tel: 0054 221 4835934
Financial support: 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- Facultad de Ciencias Veterinarias), Argentina.
Keywords: biphasic system, fungal adaptation, increased solubilities, transformation rates, yeasts communities.
Environmental biotechnology has developed as an offshoot from sanitary engineering, and only recently the biological component of the ecosystems had been recognized as relevant when bioremediation strategies must be chosen to solve environmental problems. Yeasts were isolated on 2,4-dichlorobiphenyl, 2,3',4- and 2,4',5-trichlorobiphenyl, poorly soluble compounds in water, as carbon sources. Debaryomyces castelli, Debaryomyces maramus and Dipodascus aggregatus composed the mixed culture and represented 72% of the isolates; their degradation potential were studied in biphasic and monophasic systems. The biphasic cultures were obtained with phenol as the organic phase and MSM as the aqueous ones, the monophasic medium only with MSM. Both cultures were supplied with 50, 100, 150 and 200 ppm DCB, TCB-
Successful detoxification of polluted areas is the result of interrelated factors, such as the contaminant toxicity, organism's degradation potential and ecological conditions. The first and major problem in environmental biotechnology is the microbial selection, as the organisms must transform significant amount of toxicants in a short conversion times and produce no-toxic metabolites. Up to date, the research on fungal degradation of pollutants has focused mostly on basidio- and zygomycetes species, but in heavy polluted sites, increasing yeast standing stock had been observed (Oudin et al. 1999; Sietmann et al. 2001; Romero et al. 2004).
This situation leads to consider wild yeasts as a promising organism to clean-up chemicals, and to assess them to develop environmental biotechnologies. Few researches had pointed out these advantages (Sietmann et al. 2002; Romero et al. 2002; Romero et al. 2005).
Polychlorinated biphenyls (PCBs) had been widely used as plasticizers, flame retardants, heat transfer, hydraulic and dielectric fluids, due to their physical-chemical inertness, but they are mutagenic and strongly toxic, so listed as priority pollutants by the US Environmental Protection Agency (USEPA, 2003). Bioremediation is an effective strategy to detoxify many contaminants, and efforts to engineer degradation-systems for PCBs polluted areas had been studied (Abramowicz, 1990; Swannell et al. 1996; Stringer and Johnston, 2001).
When a low-soluble toxicant, like PCBs congeners, is mixed with an aqueous medium given a byphasic system, the cells in the water-phase are in contact with less xenobiotic concentration than the yeasts adhered to the organic drops, therefore with high pollutant bioavailability (Hammer et al. 1998). Moreover, fungal adaptation is preceded by an acclimation time, requiring the low-soluble substrates longer periods, in these cases the organic presence improve the transformation rates (Havel and Reineke, 1995; Löser et al. 1999). So, the aims of this study were to evaluate the biodegradation of DCB, TCB-
Surface sediments were taken from natural and artificial channels that drain to Rio de
The yeast community was selected by enriched cultures performed in biphasic, water-phenol, and aqueous monophasic systems, amended with 50 ppm DCB, TCB-
The biphasic system was obtained with phenol as the organic phase and MSM as the aqueous ones, the monophasic medium consisted only of MSM. Both systems were supplied with 50, 100, 150 and 200 ppm 2,4-dichlorobiphenyl; or 2,3',4- or 2,4',5-trichlorobiphenyl as substrates and 50 ml phenol as co-substrate, in the biphasic cultures. The flasks were inoculated with 5 x 105 cells of each species, incubated at
Growth occurred in both phases, so, the kinetics in the multiphase liquor were described by the Monod-Haldane equation, where: µmax was the maximum specific growth rate; S was the substrate level; Ks was the saturation constant and Ki was the inhibition constant. The specific growth rates were obtained by using the OD600-data and the kinetic parameters were estimated by linear and nonlinear regression analyses:
The ration of adhered cells to phenol to cells in the aqueous phase was obtained by centrifuged cultures at
Ten-ml cultures were homogenized with Na2SO4 and extracted with 400 ml CH2Cl2 in a glass Soxhlet apparatus, to analyze the chlorinated compounds. The CH2Cl2 extract was reduced and exchanged to hexane using a concentrator, and treated with acid-activated copper powder to remove elemental sulfur. PCBs were eluted with 100 ml hexane, then these extracts were reduced to 1 ml in a TurboVap (Zymark Corp.) (Kannan et al. 1997).
Extracts were analyzed on Varian 3400CX -GC with electron capture detector (GC/ECD) for primary quantitation, and ion trap mass spectrometric (GC/ITMS) detector to congeners confirmations. The columns (
Growth in chlorinated substrates presence was measured by chloride release with an specific and reference electrode (Microprossesin pMX 2000/ION; Wissenchaftlich-Technische Werkstatten GmbH,
Means, standard deviations, regressions and variance analysis (ANOVA) were performed using SPSS/PC + Version 5.0 (
Debaryomyces castelli Capriotti (1958), Debaryomyces maramus di Menna (1954) and Dipodascus aggregatus Francke-Grosmann (1952) represented 72% of the isolated yeasts. The mixed culture was composed by equal numbers of these dominant and more efficient species. Indeed, their abilities to grow on each PCBs as carbon sources were evaluated by the µ-values (Table 1).
The DCB, TCB-
The µ-data, specific growth rates, increased from 0.11 to 0.65 h-1 in the biphasic medium and from 0.07 to 0.28 h-
The µ-values were twice as high in the water-organic assays, with µ = 0.65 h-1 in this culture and 0.28 h-1
During the incubation period, the yeasts distribution in the aqueous and phenolic phases were significantly different (P < 0.05), the cells partitioning showed that cells adhered to the organic drops when the shaker stopped. These data and the microscopic observations revealed that yeasts grew in both phases just like in the liquid-liquid interface (Figure 4).
Significantly better adaptation was observed in the consortium in relation to each species alone in the biphasic assays; moreover, phenol presence incremented the interfacial associated cells and the biodegradation of hydrophobic organic pollutants as was shown by other authors (Köhler et al. 1994; Owen et al. 1997).
In two-phase systems, the compounds diffuse from the organic to the aqueous compartment, yeasts bioconverted the pollutants in the interfacial and water-phase and the metabolites accumulated in the organic ones. So, the relevance of this culture type was based on the avoiding of the substrate inhibition and increasing tolerance to great xenobiotic concentrations in contact with the fungi, finally, the high bioavailability improved the detoxification rates.
Biphasic system with water-heptamethylnonane to evaluate naphthalene degradation by Artrobacter spp. (Focht, 1997), and water-silicone oil for di- and trichlorobenzene by bacteria and yeasts spp. (Ascon-Cabrera and Lebeault, 1993; Kim and Picardal, 2001) had been performed. Wild yeasts with n-hexadecane and dichloro- and trichlorobenzene as pollutants were assessed (Romero et al. 2002), but in these reports with biphasic assays, other pollutants and species were tested.
Scarce studies were realized on yeast biodegradation potential of biarilic compounds, only Trichosporon spp., Debaryomyces vanrijiae, Candida spp. and Rhodotorula spp. had been referred as efficient species (Hammer et al. 1998; Lange et al. 1998; Romero et al. 2001). Indeed, total yeast number and the ratio between red to other yeasts were suggested as pollution indexes (Spencer and Spencer, 1997). In this study, two different Debaryomyces species and Dipodascus aggregatus were confirmed as effective PCBs-degraders, being the first reference on their conspicuous degradation potential.
The feasibility of bioremediation is based on the presence of efficient microbial populations and pollutant bioavailability (Madsen, 1997; Head, 1998). The tested yeasts and the organic-water system assayed in this research put forward the possibility that hydrophobic substrates could be mineralized in natural habitats by wild yeast consortium (Dighton et al. 2005). This event aimed towards the applications of the results by environmental biotechnologies to detoxify polluted areas (Dighton, 2003; Arora, 2004).
In conclusion, we confirmed that the yeast consortium bio-transformed the substrates and revealed that a significant cell percentage fixed to the organic drops, pointing out the relevance of the interfacial area in the biodegradation processes. The presence of a water-organic system increased the adaptation of degrading yeasts and pollutants biotransformation; so, this system could improve microbial selection and the bioremediation of poorly water-soluble pollutants.
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