Irma Susana Morelli

Archaeosomes made of Halorubrum tebenquichense total polar lipids: a new source of adjuvancy

BackgroundArchaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from selected genera and species from the Archaea domain, elicit both antibody and cell-mediated immunity to the entrapped antigen, as well as efficient cross priming of exogenous antigens, evoking a profound memory response. Screening for unexplored Archaea genus as new sources of adjuvancy, here we report the presence of two new Halorubrum tebenquichense strains isolated from grey crystals (GC) and black mood (BM) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular transit and immune response induced by two subcutaneous (sc) administrations (days 0 and 21) with BSA entrapped in ARC made of TPL either form BM (ARC-BM) and from GC (ARC-GC) at 2% w/w (BSA/lipids), to C3H/HeN mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with 25 μg of bare BSA, were determined.ResultsDNA G+C content (59.5 and 61.7% mol BM and GC, respectively), 16S rDNA sequentiation, DNA-DNA hybridization, arbitrarily primed fingerprint assay and biochemical data confirmed that BM and GC isolates were two non-previously described strains of H. tebenquichense. Both multilamellar ARC mean size were 564 ± 22 nm, with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). ARC inner aqueous content remained inside the phago-lysosomal system of J-774 cells beyond the first incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon subcutaneous immunization of C3H/HeN mice, BSA entrapped in ARC-BM or ARC-GC elicited a strong and sustained primary antibody response, as well as improved specific humoral immunity after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers could be demonstrated in long-term (200 days) recall suggesting induction of a mixed Th1/Th2 response.ConclusionWe herein report the finding of new H. tebenquichense non alkaliphilic strains in Argentinean Patagonia together with the adjuvant properties of ARC after sc administration in mice. Our results indicate that archaeosomes prepared with TPL from these two strains could be successfully used as vaccine delivery vehicles.

Assessment of the toxic potential of hydrocarbon containing sludges

A short-time period microbial toxicity test-battery was used for the investigation of acute toxicity and genotoxicity of five hydrocarbon containing sludges. Four sludges were obtained from a petrochemical industry and the fifth from a petroleum refinery. Some of the sludges had been stored for long periods. Bioremediation potential assays for soils polluted with each of the sludges were also considered. The sludges did not show acute toxicity in any of the microbial tests performed. However, when the diethylether soluble fractions of these sludges were analyzed some of them showed acute toxicity, for which the clearest results were obtained with the resazurin reduction method. The greatest toxicity detected with the Resazurin based method was found in the diethylether extracts of the freshly collected (not stored) sludges. On the other hand, the diethylether soluble fraction of those sludges that had been stored showed genotoxicity when analyzed with the Salmonella/microsome assay. After the incorporation of the sludges into the soil, increased bacterial counts were noted and substantial hydrocarbon elimination was achieved in 30 days, showing that bioremediation may be a possible technology for cleaning soils polluted with these sludges.

Bioremediation of PAH-Contaminated Soil by Fungi

Polycyclic aromatic hydrocarbons (PAHs) are by-products of the incomplete combustion of organic materials. They are considered to be priority pollutants in the environment due to their recalcitrance and mutagenic properties. The principal PAH loss process from soil is through microbial degradation; therefore, the bioremediation is considered as an efficient, financially affordable, and adaptable alternative for the recuperation of PAH-contaminated soil. Several microorganisms, such as bacteria, yeasts, and filamentous fungi, are capable of degrading different types of PAHs. The ability of the fungi to degrade the high-molecular-weight PAHs, together with their physiological versatility, converts the fungal remediation in a promising technique for the cleanup of PAH-contaminated soil. This chapter summarizes the recent information on the metabolic pathway of the fungal transformation of PAHs and provides a critical review of previous work about fungal bioremediation of PAH-contaminated soil. Also, some of the most recently used fungal technology to enhance PAHs bioremediation processes is discussed.

Assigning ecological roles to the populations belonging to a phenanthrene-degrading bacterial consortium using omic approaches

The present study describes the behavior of a natural phenanthrene-degrading consortium (CON), a synthetic consortium (constructed with isolated strains from CON) and an isolated strain form CON (Sphingobium sp. AM) in phenanthrene cultures to understand the interactions among the microorganisms present in the natural consortium during phenanthrene degradation as a sole carbon and energy source in liquid cultures. In the contaminant degradation assay, the defined consortium not only achieved a major phenanthrene degradation percentage (> 95%) but also showed a more efficient elimination of the intermediate metabolite. The opposite behavior occurred in the CON culture where the lowest phenanthrene degradation and the highest HNA accumulation were observed, which suggests the presence of positive and also negative interaction in CON. To consider the uncultured bacteria present in CON, a metagenomic library was constructed with total CON DNA. One of the resulting scaffolds (S1P3) was affiliated with the Betaproteobacteria class and resulted in a significant similarity with a genome fragment from Burkholderia sp. HB1 chromosome 1. A complete gene cluster, which is related to one of the lower pathways (meta-cleavage of catechol) involved in PAH degradation (ORF 31–43), mobile genetic elements and associated proteins, was found. These results suggest the presence of at least one other microorganism in CON besides Sphingobium sp. AM, which is capable of degrading PAH through the meta-cleavage pathway. Burkholderiales order was further found, along with Sphingomonadales order, by a metaproteomic approach, which indicated that both orders were metabolically active in CON. Our results show the presence of negative interactions between bacterial populations found in a natural consortium selected by enrichment techniques; moreover, the synthetic syntrophic processing chain with only one microorganism with the capability of degrading phenanthrene was more efficient in contaminant and intermediate metabolite degradation than a generalist strain (Sphingobium sp. AM).

Effect of storage on the toxic potential of hydrocarbon containing sludges

In many cases, petroleum‐derived sludges are stored in open air ponds. In this condition, weathering may produce changes that can be important when choosing a treatment and/or disposal method. The effect of weathering on chemical and toxicological characteristics of sludges were studied in two different sludges, one from a petrochemical API‐separator (API2N) and the other from a refinery biological treatment unit. Starting determinations were performed with end‐of‐the‐pipe composite samples. Determinations were repeated after a 6‐month weathering period. Physico‐chemical characterization included pH, water content, extractable organics, and class fractionation into aliphatics, aromatics, and asphaltics. Microbiological concentration was done by enumeration of viable bacteria in plate count agar and solid mineral medium with the sludge as the only source of carbon and energy, and fungi in Cooke rose bengal agar. For toxicological assessment a battery of bioassays was performed that included: Bacillus cereus spot test, Resazurine reduction test, Bioluminiscence test with Photobacterium, effects on viable soil bacteria counts, Latuca sativa seed germination and root elongation test. The Ames test (TA‐98 with S9 microsomal activation) was used to assess genotoxicity changes. Comparison of starting results of both sludges with those obtained after 6 months showed a minimal change in the chemical characteristics, a significant increase in the starting low level of bacteria in API2N, reduction in acute toxicity and increase in the mutagenic effect in the dimethyl sulfoxide (DMSO)‐sludges fraction, but not in the DMSO‐ethylether organic fraction in both sludges. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 227–233, 1999