Mycelium-Enhanced Bacterial Degradation of Organic Pollutants under Bioavailability Restrictions.

This work examines the role of mycelia in enhancing the degradation by attached bacteria of organic pollutants that have poor bioavailability. Two oomycetes, Pythium oligandrum and Pythium aphanidermatum, were selected as producers of mycelial networks, while Mycobacterium gilvum VM552 served as a model polycyclic aromatic hydrocarbon (PAH) degrading bacterium. The experiments consisted of bacterial cultures exposed to a nondisturbed nonaqueous phase liquid (NAPL) layer containing a heavy fuel spiked with 14C-labeled phenanthrene that were incubated in the presence or absence of the mycelia of the oomycetes in both shaking and static conditions. At the end of the incubation, the changes in the total alkane and PAH contents in the NAPL residue were quantified. The results revealed that with shaking and the absence of mycelia, the strain VM552 grew by utilizing the bulk of alkanes and PAHs in the fuel; however, biofilm formation was incipient and phenanthrene was mineralized following zero-order kinetics, due to bioavailability limitations. The addition of mycelia favored biofilm formation and dramatically enhanced the mineralization of phenanthrene, up to 30 times greater than the rate without mycelia, possibly by providing a physical support to bacterial colonization and by supplying nutrients at the NAPL/water interface. The results in the static condition were very different because the bacterial strain alone degraded phenanthrene with sigmoidal kinetics but could not degrade alkanes or the bulk of PAHs. We suggest that bacteria/oomycete interactions should be considered not only in the design of new inoculants in bioremediation but also in biodegradation assessments of chemicals present in natural environments.

Key high molecular weight PAH-degrading bacteria in a soil consortium enriched using a sand-in-liquid microcosm system.

A novel biphasic system containing mineral medium and sand coated with a biologically weathered creosote-PAH mixture was developed to specifically enrich the high molecular weight polycyclic aromatic hydrocarbon (HMW PAH)-degrading community from a creosote-polluted soil. This consortium (UBHP) removed 70% of the total HMW PAHs and their alkyl-derivatives in 12 weeks. Based on a combined culture-dependent/independent approach, including clone library analysis, detection of catabolic genes, metabolomic profiles, and characterization of bacterial isolates, 10 phylotypes corresponding to five major genera (Sphingobium, Sphingomonas, Achromobacter, Pseudomonas, and Mycobacterium) were pointed out as key players within the community. In response to exposure to different single PAHs, members of sphingomonads were associated to the utilization of phenanthrene, fluoranthene, benzo[a]anthracene, and chrysene, while the degradation of pyrene was mainly associated to low-abundance mycobacteria. In addition to them, a number of uncultured phylotypes were detected, being of special relevance a group of Gammaproteobacteria closely related to a group previously associated with pyrene degradation that were here related to benzo(a)anthracene degradation. The overall environmental relevance of these phylotypes was confirmed by pyrosequencing analysis of the microbial community shift in the creosote-polluted soil during a lab-scale biostimulation.

Bacterial PAH degradation in marine and terrestrial habitats.

Cycling of pollutants is essential to preserve functional marine and terrestrial ecosystems. Progress in optimizing these natural biological processes relies on the identification of the underlying microbial actors and deciphering their interactions at molecular, cellular, community, and ecosystem level. Novel advances on PAH biodegradation are built on a progressive approach that span from pure cultures to environmental communities, illustrating the complex metabolic networks within a single cell, and their further implications in higher complexity systems. Recent analytical chemistry and molecular tools allow a deeper insight into the active microbial processes actually occurring in situ, identifying active functions, metabolic pathways and key players. Understanding these processes will provide new tools to assess biodegradation occurrence and, as a final outcome, predict the success of bioremediation thus reducing its uncertainties, the main drawback of this environmental biotechnology.

Community structure and PAH ring-hydroxylating dioxygenase genes of a marine pyrene-degrading microbial consortium.

Marine microbial consortium UBF, enriched from a beach polluted by the Prestige oil spill and highly efficient in degrading this heavy fuel, was subcultured in pyrene minimal medium. The pyrene-degrading subpopulation (UBF-Py) mineralized 31 % of pyrene without accumulation of partially oxidized intermediates indicating the cooperation of different microbial components in substrate mineralization. The microbial community composition was characterized by culture dependent and PCR based methods (PCR-DGGE and clone libraries). Molecular analyses showed a highly stable community composed by Alphaproteobacteria (84 %, Breoghania, Thalassospira, Paracoccus, and Martelella) and Actinobacteria (16 %, Gordonia). The members of Thalasosspira and Gordonia were not recovered as pure cultures, but five additional strains, not detected in the molecular analysis, that classified within the genera Novosphingobium, Sphingopyxis, Aurantimonas (Alphaproteobacteria), Alcanivorax (Gammaproteobacteria) and Micrococcus (Actinobacteria), were isolated. None of the isolates degraded pyrene or other PAHs in pure culture. PCR amplification of Gram-positive and Gram-negative dioxygenase genes did not produce results with any of the cultured strains. However, sequences related to the NidA3 pyrene dioxygenase present in mycobacterial strains were detected in UBF-Py consortium, suggesting the representative of Gordonia as the key pyrene degrader, which is consistent with a preeminent role of actinobacteria in pyrene removal in coastal environments affected by marine oil spills.