Enrichment and characterization of bacteria capable of degrading diesel and HiQ diesel hydrocarbons from the microbial consortium “Enzyveba”

The biotreatability of soils contaminated by organic toxic compounds is often characterized by the lack of microorganisms able to mediate a rapid and extensive pollutants degradation. Thus, the addition of exogenous specialized biomass (bioaugmentation) can improve the bioremediation of such soils. However, several failures of the bioaugmentation approach have been reported in the literature, mostly due to the inability of inoculated microbes to compete with autochthonous microflora and to face the toxicity and the scarcity of nutrients occurring in the contaminated biotope. Complex microbial systems, such as compost or sludge, normally consisting of a large variety of robust microorganisms and essential nutrients, would have better chances to succeed in colonizing degraded contaminated soils. Enzyveba, a stable consortium of bacteria and fungi produced by Marcopolo Engineering S.p.A. (Italy), was recently found to significantly increase the biological remediation of diesel-, PCB- and PAH-contaminated soils. In this work, its bacterial biomass able to degrade Diesel and HiQ Diesel hydrocarbons was enriched and characterized. Enzyveba’s bacterial biomass able to degrade Diesel and HiQ Diesel hydrocarbons was enriched through a serial transfer approach (2% v/v inoculum) on a mineral medium containing 1000 mg/L of Diesel or HiQ Diesel as the only source of carbon and energy performed in aerobic bioreactors incubated at 30°C. After 5 transfers, the microbial populations enriched on the 2 diesel fuels were subjected to molecular characterization via PCR-DGGE analysis followed by band extraction and sequencing of 16S rRNA genes. In addition, colonies having different morphology on TSA agar plates were randomly picked and isolated from both mixed cultures on mineral medium containing 500 mg/L of Diesel or HiQ Diesel as the sole carbon and energy source. Isolates were characterized for their Diesel fuel hydrocarbons biodegradation capability and phylogenetically identified via 16S rDNA amplification and sequencing. The mixed cultures enriched on Diesel and HiQ Diesel displayed a remarkable and comparable hydrocarbon biodegradation activity. Initially occurring Diesel and HiQ Diesel hydrocarbons (1000 mg/l) were biodegraded by 63.3% and 70.2% after 3 days of incubation, respectively, and by up to 90% at the end of the experiment (10 days). A remarkable increase in biomass concentration, from 106 to 109 CFU/ml, was concomitantly detected in both enrichments during the first 3 days of incubation. According to DGGE analysis of 16S rDNA, both enriched cultures were composed by 6 dominant bacteria, namely Acinetobacter baumanii, Chryseobacterium taichungense, Achromobacter denitrificans, Pseudomonas stutzeri, Stenotrophomonas sp. and Paracoccus verutus. With the only exception of Paracoccus verutus, all major members of two microbial communities identified by DGGE were isolated. However, only Acinetobacter baumanii displayed a modest diesel hydrocarbons biodegradation capability, whereas the other isolates failed to grow on diesel fuel hydrocarbons as sole energy and carbon source after repeated transfers under selective conditions.