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Lab-scale biopile assessment for the evaluation of diesel fuel soil remediation
No full textA soil of north-eastern Italy was interested by a strong diesel fuel spill. A wetting system was used to periodically release a microbial inoculant, containing a dried consortium of bacterial strains proven effective by previous microcosm evaluation. Investigations indicated that oil degrading bacterial strains were shown not to enhance significantly the remediation when the polluting agent was present in the soil for many years (Basaglia et al., 2003).
To make a distinction between the role of the remediation activity of oil degrading bacterial inoculants and that of the aeration and nutrients application, four lab-scale biopiles were set up and monitored. This assessment was also necessary to dispose of suitable controls and make the right comparison between inoculated and not inoculated soil (Aichberger et al., 2005).
Two different soil types were used in two different sets of experiments:
set 1 - two lab-scale biopiles loaded with an aged polluted soil coming from a 30 years contaminated site: this soil was supposed to contain a well established oil tolerant and oil degrading microflora.
set 2 - two lab-scale biopiles loaded with an unpolluted soil, artificially contaminated with diesel fuel: this soil was supposed not to contain a well established oil tolerant and oil degrading microflora.
In both the above experiments the control was represented by a non inoculated column.
Soil microbial community composition, as well as that of the commercial inoculant used, was mainly determined by denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1998). A number of microorganisms capable of living in the presence of diesel as the only carbon source were also isolated and identified by 16s rDNA analysis. Among the bacterial species isolated and identified in the active biopiles Sphingomonas sp. was almost constantly detected.
DGGE analysis performed on set 1 showed that the profile related to the inoculant did not cluster with any of the profiles obtained from the two biopiles, at any time. Since gas-chromatographic analysis indicated that both the biopiles were fully active in the remediation process, the biodegradation occurred was to be ascribed to the autochthonous microflora that experienced a strong and continuous selective pressure during the long period (more than 30 years) of continuous pollution. After so long time the microbial community evolved in a hydrocarbon resistent and actively degrading population.
The results obtained from the experiment set 2, instead, indicated that the community profile of the inoculated biopile clustered with that obtained from the inoculant. This indicates that the inoculated bacteria did colonize the soil. While GC analysis demonstrated that they effectively metabolized the hydrocarbons, very reduced degradation activity was detected in the not inoculated control.
The above results clearly suggest that while bioventing technology always stimulates the natural in situ biodegradation, bioaugmentation could significantly contribute to the remediation process only if rapidly applied after the contamination occurs
A possibile role of nirK in Rhizobium sullae HCNT1.
No full textIn response to a number of environmental stresses many bacterial species, including Vibrio
vulnificus, Sinorhizobium meliloti, Micrococcus luteus, Escherichia coli and Helicobacter pylori,
enter the viable-but-not-culturable (VBNC) status (McDougald et al., 1998). In this metabolic state
they lose their ability to grow on media that usually sustain them and undergo such physiological
and morphological changes as increased resistance to several physic and chemical factors, and
changes in protein and lipid content. The recent increasing use of specific fluorescent dyes such as
Syto 9, CTC (5-cyano-2,3-di-4-tolyl-tetrazolium chloride), AO (Acridine Orange), propidium
iodide, made possible a proper identification of viability and the metabolic state of microbes
(Basaglia et al. 1997).
Sinorhizobium meliloti 41, a rhizobium nodulating Medicago sativa, enters VBNC status in
liquid microcosms when O2 is depleted from the atmosphere of the incubation mixture (Toffanin et
al., 2000; Casella et al. 2001). Plasmid-borne, firefly-derived, luciferase gene (luc) was inserted and
stably inherited in Sinorhizobium meliloti 41 (pRP4-luc) as a reporter gene. The strain obtained, S.
meliloti 41 pRP4-luc and its parental strain, served as a model system for VBNC experiments both
in vitro and in soil samples.
Rhizobium sullae, formerly Rhizobium ’hedysari’, is a nitrogen fixing bacterium that induces
symbiotic nodule formation on the legume Hedysarum coronarium (Squartini et al., 2002). Strain
HCNT1, expressing a copper-containing nitrite reductase encoded by nirK, which is closely related
to nitrite reductases in true denitrifiers, enters the same VBNC status when oxygen is limiting, but
only when nitrite is present and converted to NO. Since HCNT1 cannot grow as a denitrifier and
inactivation of nirK only resulted in the loss of NO production (Toffanin et al, 1996), the hypothesis
that expression of nirK may induce the VBNC status has been investigated.
Therefore, a comparison between the two systems, S. meliloti and R. sullae, is presented in order
to verify the possible connection of nirK with the VBNC status.
McDougald D. et al. 1998. FEMS Microbiology Ecology 25: 1-9
Basaglia M. et al. 1997. In: The biotechnology and ecological interactions of microbial inoculants".
Granada, Spain. pp. 19-20
Toffanin A. et al. 2000. Biol. Fertil. Soils. 31 (6): 484-488
Casella S. et al. 2001. ISME-9, Amsterdam (The Netherlands), p. 191
Squartini A. et al. 2002. Int. J. Syst. Evol. Microbiol. 52: 1267-1276
Toffanin A. et al. 1996. Appl. Environ. Microbiol. 62 (11): 4019-402
Insights into the grapevine inner tissue life: characterization of the bacterial endophyte community
No full textValutazione di proprietà biochimiche di endofiti batterici e del loro ruolo nella promozione della crescita in Arabidopsis thaliana
No full textMultiple oxyanion reducing capacity of the copper-containing nitrite reductase of R. sullae
No full textRhizobium sullae is an unusual and unique bacterium due to the radical truncation of its denitrification electron transport chain. This bacterium has only one of the four terminal reductases required for complete denitrification. The sole reductase present, nitrite reductase, produces a toxic end product, nitric oxide. This enzyme cannot be used to support growth under anoxic conditions. Expression of the gene encoding nitrite reductase, nirK, is also unusual in that it does not require the presence of a nitrogen oxide. Expression only requires a decrease in oxygen concentration. The role of nitrite reductase in R. sullae has yet to be elucidated. The presence and expression of nitrite reductase has been investigated as a detoxification strategy or as a means to reduce the energy content in the bacterial cell in order to induce dormancy.
Since nitrite reductase expression only occurs under microoxic conditions it is possible the enzyme plays some role within the root nodule, which is a low oxygen environment. However, comparisons of nodulation efficiency, plant growth and nitrogen fixation have not revealed any significant differences between wild type and nitrite reductase-deficient strains, so far. Some evidence has been provided suggesting a link between nitrite reductase activity and the viability and culturability of the cells. Nitrite reductase activity may reduce energy content but it remains uncertain whether this is an indirect consequence of generation of nitric oxide or a desired result intended to prolong cell viability under certain conditions.
Recently, several bacterial species have been found to contain reductases with the capacity to reduce multiple oxyanions. The oxyanion reducing capacity activity of the copper-containing nitrite reductase of R. sullae was tested to determine its substrate range. In addition, the oxyanion-reducing capacity of several nirK-plus and nirK-minus strains of R. sullae was compared with that of other denitrifying and non-denitrifying bacterial strains belonging to the genera Stenotrophomonas, Bacillus, Agrobacterium and Rhodobacter. The results suggest that the nitrite reductase of R. sullae can reduce other physiologically important oxyanions, which may provide new insight into the role of this enzyme
Analisi della comunità di endofiti batterici isolata da Vitis vinifera per la selezione di potenziali agenti di biocontrollo.
No full textSimulating a biopile for understanding the bioremediation activity in a polluted soil
No full textTo evaluate the remediation activity of oil degrading bacterial strains, contained in a dried microbial consortium inoculant, it is required to distinguish between degradation activities operated by native microbiota and that due to the newly introduced strains. The effect due to chemical compounds contained into the inoculant as a support should also be considered. Previous studies showed that in an aged polluted soil the autochthonous microbial population seems to be highly specialized for diesel fuel degradation, probably due to the long period of time under such a selective pressure. Therefore, the use of microbial inoculants might fail to improve the rate of soil remediation. Previous experiments indicated that using formulation specifically designed for diesel degradation, the oil content in the soil was strongly and rapidly reduced under lab-scale if promptly applied after the contamination occurrence. The aim of this study was to investigate on the effect of both the microbial inoculant, and the chemicals compounds , on the rate of oil degradation in a newly contaminated soil. This was attained by the construction of lab-scale biopiles artificially contaminated with diesel fuel and equipped with recycling systems to simulate a field scale structure. The results obtained showed that in the inoculated biopile the diesel oil content decreased approximately to 20% of the initial value, while it remained constant in the non inoculated one. No effects were due to the chemicals contained in the formulation. DNA based techniques confirmed an effective colonization of soil by the bacterial consortium present in the inoculan
Degradation of Diesel fuel in an Aged and in a Newly Polluted Soil using a Bacterial Consortium.
No full textIn order to evaluate the remediation activity of oil degrading bacterial strains it is required to clearly distinguish between the intrinsic microbial activity of the soil under investigation and the activity of newly introduced inoculant strains. Previous investigations indicated that oil degrading bacterial strains, proven highly effective in lab-scale experiments, were shown not to enhance significantly the remediation of soil when the polluting agent was present in the soil for many years. The aim of this study was to evaluate, in areated soil laboratory columns, the biodegradation rate of diesel fuel in both a newly (system I) and a 30 years aged (system II) polluted soil inoculated and non inoculated with a dried consortium of bacterial strains.
Total Recoverable Petroleum Hydrocarbons (TRPH) and Diesel Recoverable Oil (DRO) were constantly analysed by GC-FID and a study on the bacterial communities was performed by DNA soil extraction and ARDRA profiles. Total heterothrophs were also determined at different times by plating on Total count Agar (TCA) and TCA supplemented with 1% diesel fuel. Moreover, after characterization of the main bacterial strains released with the inoculant, their soil colonization was evaluated using specific antisera and immunological techniques coupled to 16S rDNA sequence analysis.
A clear difference between the two systems was found in terms of rapidity and effectiveness of degradation, and dynamics of indigenous and newly introduced bacterial populations are presented and discussed.
Acknowledgements
This work was supported by Provincia di Belluno. The authors wish to thank Serenambiente Sr
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