Abstract
BackgroundThe bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity. In such cases, the soil inoculation with cultures with the desired catabolic capabilities (bioaugmentation) is an essential option. The use of consortia of microorganisms obtained from rich sources of microbes (e.g., sludges, composts, manure) via enrichment (i.e., serial growth transfers) on the polluting hydrocarbons would provide bioremediation enhancements more robust and reproducible than those achieved with specialized pure cultures or tailored combinations (co-cultures) of them, together with none or minor risks of soil loading with unrelated or pathogenic allocthonous microorganisms.ResultsIn this work, two microbial consortia, i.e., ENZ-G1 and ENZ-G2, were enriched from ENZYVEBA (a complex commercial source of microorganisms) on Diesel (G1) and HiQ Diesel (G2), respectively, and characterized in terms of microbial composition and hydrocarbon biodegradation capability and specificity.ENZ-G1 and ENZ-G2 exhibited a comparable and remarkable biodegradation capability and specificity towards n-C10 to n-C24 linear paraffins by removing about 90% of 1 g l-1 of diesel fuel applied after 10 days of aerobic shaken flask batch culture incubation at 30°C. Cultivation dependent and independent approaches evidenced that both consortia consist of bacteria belonging to the genera Chryseobacterium, Acinetobacter, Psudomonas, Stenotrophomonas, Alcaligenes and Gordonia along with the fungus Trametes gibbosa. However, only the fungus was found to grow and remarkably biodegrade G1 and G2 hydrocarbons under the same conditions. The biodegradation activity and specificity and the microbial composition of ENZ-G1 and ENZ-G2 did not significantly change after cryopreservation and storage at -20°C for several months.ConclusionsENZ-G1 and ENZ-G2 are very similar highly enriched consortia of bacteria and a fungus capable of extensively degrading a broad range of the hydrocarbons mainly composing diesel fuels. Given their remarkable biodegradation potential, stability and resistance to cryopreservation, both consortia appear very interesting candidates for bioaugmentation operations on Diesel fuel impacted soils and sites.
Highlights
The bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity
Diesel fuel-degrading consortia obtained from ENZYVEBA The two microbial consortia ENZ-G1 and ENZ-G2 were obtained from ENZYVEBA through 5 consecutive culture transfers (2% v/v) on minimum mineral medium (MMM) for bacteria amended with Diesel (G1) and HiQ Diesel (G2) respectively applied at 1 g l-1 as the main carbon and energy source
The bacterial biomass able to grow on Tryptic Soy Agar (TSA) medium grew rapidly during the first 3 days of incubation by increasing from 5.4 × 106 ± 7.5 × 104 and 6.9 × 106 ± 6.0 × 105 CFU ml-1 observed at the beginning of incubation in ENZ-G1 and ENZ-G2 cultures, respectively, to values of 1.0 × 109 ± 1.5 × 107 and 1.3 × 109 ± 1.8 × 108 CFU ml-1, respectively (Figure 1B)
Summary
The bioremediation of soils impacted by diesel fuels is very often limited by the lack of indigenous microflora with the required broad substrate specificity. Diesel fuels are complex mixtures of saturated hydrocarbons (primarily paraffins including n, iso, and cycloparaffins), and aromatic hydrocarbons (including naphthalenes and alkylbenzenes) obtained from the middle-distillate, gas-oil fraction during petroleum separation Due to their massive production and use as fuels for transportation, they are among the most common sources of organic pollutants for the surface soil. The mineralization of complex hydrocarbon mixtures such as those composing diesel fuels, usually requires the co-existence and effective cooperation of several specialized microorganisms with complementary substrate specificity [5,6,7] Microbial consortia with such physiological and metabolic features might not exist in a soil, in particular if only recently impacted by a diesel fuel release, and this is often the main cause of the poor bio-treatability of diesel fuel contaminated soils and sites. The inoculation of the impacted soil with high concentrations of characterized cultures of highly specialized microbes (bioaugmentation) is one of the most promising options for getting its sustainable remediation
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