Fast method for the determination of short-chain-length polyhydroxyalkanoates (scl-PHAs) in bacterial samples by In Vial-Thermolysis (IVT).

A new method based on the GC-MS analysis of thermolysis products obtained by treating bacterial samples at a high temperature (above 270°C) has been developed. This method, here named "In-Vial-Thermolysis" (IVT), allowed for the simultaneous determination of short-chain-length polyhydroxyalkanoates (scl-PHA) content and composition. The method was applied to both single strains and microbial mixed cultures (MMC) fed with different carbon sources. The IVT procedure provided similar analytical performances compared to previous Py-GC-MS and Py-GC-FID methods, suggesting a similar application for PHA quantitation in bacterial cells. Results from the IVT procedure and the traditional methanolysis method were compared; the correlation between the two datasets was fit for the purpose, giving a R2 of 0.975. In search of further simplification, the rationale of IVT was exploited for the development of a "field method" based on the titration of thermolyzed samples with sodium hydrogen carbonate to quantify PHA inside bacterial cells. The accuracy of the IVT method was fit for the purpose. These results lead to the possibility for the on-line measurement of PHA productivity. Moreover, they allow for the fast and inexpensive quantification/characterization of PHA for biotechnological process control, as well as investigation over various bacterial communities and/or feeding strategies.

Polycyclic aromatic hydrocarbons degradation by composting in a soot-contaminated alkaline soil.

This study deals with the biodegradation of the polycyclic aromatic hydrocarbons (PAH)s present in a soil contaminated by soot waste, characterised by a total PAHs content in the 200 mg kg(-1) range. A challenging characteristic of the waste soil treated was its high alkalinity, with a pH of about 12.8. The waste came from a soot-contaminated area located in the industrial zone of Porto Marghera, Venice (Italy). The biodegradation process employed was the composting of the waste with sewage sludge and yard waste. The process was carried out on a pilot scale using a closed tank with forced aeration for a period of 60 days, followed by 70 days with natural aeration. The time evolution of the process was monitored by following the time change in the concentration of the 16 US-EPA PAHs, as well as temperature, pH, electrical conductivity, C and N contents. Also phytotoxicity parameters, such as the growth and respiration indexes, were monitored. An induction time of about 30 days was observed, which corresponded to the time required before observing a significant self-drop in the waste pH and an increase in mass temperature. Afterward, a progressive drop in the PAHs concentration was observed, up to reaching after 130 days an overall degradation percentage in the order of 68%. The degradation was more effective on rather low molecular weight PAHs (2-4 rings).

Effect of sediment turbidity and color on light output measurement for Microtox Basic Solid-Phase Test.

In this work, sediment samples collected from several Spanish harbours were tested with two toxicity procedures, designed for solid samples: the Microtox Basic Solid-Phase Test (BSPT) and a modified procedure of the previous test protocol (mBSPT). According to the BSPT procedure, after initial light readings, pure bacteria were exposed to sediment suspension dilutions and light production was directly measured on suspended sediments without any further manipulation. As measurements are likely to be affected by sediment turbidity and color, a variation in initial light measurement has been here suggested, in order to consider the sample effect at all time readings during the test. Firstly, when sediment suspensions at different concentrations were added to bacteria suspension, immediately the initial light output drastically decayed by more than 50% in signal difference, resulting in a false inhibition, as effect of sample turbidity/color. This effect was more evident at high EC50 values, when slightly or not toxic samples were assessed. Secondly, the comparison of the EC50 obtained with both procedures, demonstrated that the mBSPT produced higher EC50 values (less toxic) than those obtained with the standard procedure. The mBSPT procedure resulted rapid and effective and it could be applied simultaneously with BSPT, in order to better evaluate the toxicity.

Pilot plant study of the microbial flora in a diesel fuel contaminated soil.

This study focused on the impact of a recent soil pollution of diesel fuel on a site and its indigenous microbial flora. A pilot plant (0.5 m3) was set up and filled with a soil (about 700 kg), artificially and uniformly polluted with diesel fuel (7 g/kg). This plant was then chemically and biologically monitored during the whole experiment (about two years). During the monitoring, a morphological change of the microbial colonies was observed. This was probably due to the acclimation phenomena to the pollution. With batch kinetic studies (10 ml) and increasing the selective pressure of the pollutant, it was possible to select and isolate a microbial consortium and a single strain that developed the ability to use different diesel fuel fractions as carbon sources. GC-MS analytical techniques were used. Results showed that different fractions were degraded at different times. In the batch system, in 7 days, the microbial consortium degraded some aromatic hydrocarbons. The isolate strain, in 20 days, degraded linear hydrocarbons. After a two years acclimation, it was possible to obtain, from a pilot plant, a microbial consortium and a strain able to degrade diesel fuel, for a future bioremediation in situ process.