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

none8siA 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 polyhydrox- yalkanoates (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.mixedF. Abbondanzi; G. Biscaro; G. Carvalho; L. Favaro; P. Lemos; M. Paglione; C. Samorì; C. TorriF. Abbondanzi; G. Biscaro; G. Carvalho; L. Favaro; P. Lemos; M. Paglione; C. Samorì; C. Torr

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

none7noThis 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).mixedMoretto, L.M.; Silvestri, S.; Ugo, P; Zorzi, G.; Abbondanzi, F.; Baiocchi, C.; Iacondini, A.Moretto, L.M.; Silvestri, S.; Ugo, P; Zorzi, G.; Abbondanzi, F.; Baiocchi, C.; Iacondini, A

Chemical, microbiological and ecotoxicological monitoring of biodegradation in PAH-contaminated sediments

This study was performed in order to evaluate factors that influence the magnitude and rates of PAH biodegradation processes in contaminated sediments at a laboratory scale. Several factors influence the feasibility and effectiveness of sediment biodegradation: (1) the existence of an indigenous sediment microbial population capable of degrading PAH contaminants, (2) the availability of the contaminants to sediment microbial population (bioavailability), (3) the optimization of environmental factors such as temperature and oxygen, and nutrients availability, (4) the influence of a natural surfactant on PAH degradation velocity. The rate and extent of PAH biodegradation were evaluated by sediment-slurry experiments by monitoring chemical, microbiological and ecotoxicological parameters. The last focus of our work was to investigate the source and the extent of toxicity in slurry experiments using the Microtox\uae test both on solid and liquid phases, in order to determine any enhancement or shift in toxicity over the time and/or between the solid and liquid phases due to release or increased bioavailability of the target contaminants

Sediment Toxicity Identification Evaluation (TIE) of a coastal lagoon (Pialassa Baiona, Italy) using Microtox\uae system.

The Pialassa Baiona is a shallow coastal lagoon covering a surface of about 1100 ha, which lies parallel to the Northern Adriatic Sea shoreline, approximately 10 km north of the town of Ravenna. Natural and man-made changes over the time lead to existing physical features resulting in a number of different basins of less than 1 m in depth, broad and irregular in shape, separated by levees and crossed by a network of artificial channels. From the fifties, the delicate balance of life in Pialassa Baiona lagoon has been threatened by the presence of a petrochemical industrial complex, fuel oil power generation, inland intensive agriculture, as well as to the close contact with the densely populated town and coast, and human negligence. As industrial and urban development increased, so did the input of chemical contamination in the lagoon. During the last decades, various chemicals from municipal and industrial wastewaters, and agriculture runoff have been discharged into the lagoon, depending upon treatment levels, chemicals common to the time period (i.e. DDT, mercury). These sediment-borne chemicals, may, in turn, cause toxicity to aquatic organisms. As a part of an on-going study, pore water extracts were collected and evaluated with the Microtox\uae system using a procedure similar to the Toxicity Identification Evaluation (TIE) protocol for acute and chronic marine TIEs according to the United States EPA (US EPA/600/R-96/054). The TIE approach Phase I, namely Toxicity Characterisation, is based on the principle of sequential removal of various chemical fractions coupled with toxicity testing of the obtained fractions. When removal of a class of chemicals also removes toxicity, it is assumed that those compounds were responsible for some of the toxicity observed in the original sample. In the present study the USEPA Phase I approach was followed, and the manipulation chosen were filtration/centrifugation, metal chelation with EDTA, aeration, and passage through a C18 cartridge column. These treatments were thought to yield information on compounds responsible for toxicity. In this TIE protocol, the toxicity of the initial extract and of the various treated pore water extracts was assessed using Vibrio fischeri in the Microtox\uae test system, which is a rapid, low-cost toxicity test widely used in screening tests for assessing sediment toxicity. Pore water, rather than whole sediment, was chosen for evaluation because the interstitial phase has been found to be representative of the bioavailable fraction of the contaminants of concern (i.e., metals and ammonia). To this end, in tandem analysis of nitrogen ions and metals, and Microtox\uae test were performed on pore water samples. The addition of EDTA and the aeration step removed toxicity only in some samples. Passage through the C18 column removed toxicity in all samples, but no toxicity was found in most of the organic eluates. This may indicate either that the organic compounds responsible for toxic effects were irreversibly bound to the C18 resin, or that the organic solvent, commonly used for eluting C18 columns, was not efficient in removing all bound organic compounds. In summary, the sediment pore water toxicity is likely to be due to joint effects of confounding factors such as ammonia and sulphide, as well as metals and organic compounds

Evaluación de la calidad del sedimento en puertos españoles mediante un bioensayo con algas verdes

9 páginas, 4 figuras, 2 tablas.[EN] Coastal zones are under environmental pressure due to human activities, and ports are the areas that probably present the highest levels of contaminants in their sediments. In the present study we have combined chemical data and biological effects to determine the sediment quality of six Spanish ports. Trace metals and organic contaminants have been analyzed, and an acute bioassay was carried out in sediment elutriate using the unicellular green alga Dunaliella tertiolecta. The toxicological parameter employed was EC50, which was estimated after exposure to different concentrations of elutriates during 72 h. The development of this alga may be affected when there is a xenobiotic in the environment, and growth inhibition can be quantified and linked to the levels of contaminants in the sediment in order to determine its quality. The results showed that the metals (Cd, Cr, Hg, and Zn) and organic contaminants (PAHs and PCBs) bound to the sediments reduced the normal growth of the alga, but no alterations were detected due to the presence of Cu. The main disadvantage observed was the hormesis produced by the presence of high levels of organic material in the sediment that might hide the toxicity of xenobiotics.[ES] En la actualidad las áreas costeras están sometidas a una presión ambiental por parte de las actividades humanas y los puertos son las zonas que probablemente presentan los mayores niveles de contaminantes en sus sedimentos. En el presente estudio se han combinado datos químicos y efectos biológicos para determinar la calidad ambiental de los sedimentos de seis puertos españoles. Se llevó a cabo el análisis de metales traza y contaminantes orgánicos además de un ensayo de toxicidad con la microalga marina Dunaliella tertiolecta. El parámetro toxicológico utilizado fue el EC50, calculado a partir de una exposición a distintas concentraciones de lixiviado de sedimento durante 72 h. El desarrollo de este alga verde puede verse afectado con la presencia ambiental de xenobióticos de forma que la inhibición del crecimiento de la población puede ser cuantificada y relacionada con los niveles de contaminantes en el sedimento con el fin de determinar su calidad. Los resultados demostraron que tanto la presencia de metales (Cd, Cr, Hg y Zn) como de contaminantes orgánicos (PAHs y PCBs) en el sedimento afectaron al crecimiento normal del alga mientras que no se detectaron alteraciones por la presencia de Cu. La principal desventaja observada fue el fenómeno de hormesis que ocurre con concentraciones elevadas de materia orgánica en el sedimento enmascarando la toxicidad de los xenobióticos.Este trabajo fue financiado parcialmente por los proyectos PET2006_0685_00, PET2006_0685_01 y PHB2005-0100-PC del Ministerio de Innovación y Ciencia de España. C Morales-Caselles y Á Rico fueron financiadas por el Ministerio Español de Educación y Ciencia en el programa ARGO.Peer reviewe

A new microbial assay for the toxicity detection of contaminated soils

A comparative study to detect toxicity prior to bioremediation treatment was set in order to investigate dehydrogenase activity inhibition of a common soil bacterium caused by soil contaminated with Cu, Pb, and As. A spectrophotometric test with Pseudomonas fluorescens strain ATCC 13525 utilising the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction by microbial dehydrogenase has been adapted for this purpose. Soil samples are incubated for 48 hours at 30±1°C in 18-ml tubes in the presence of TTC as an artificial electron-acceptor. The reduced TTC forms a reddish colour substance named triphenyl formazan (TPF), which can be extracted from the microbial cells and measured colorimetrically. The rapid response of biological activity in microorganisms and the reported sensitivity to the toxicants in the contaminated samples are reflected by the TTC reduction method, which is a sensitive tool for toxicity screening of contaminated sites, routine monitoring of bioremediation processes, as well as for feasibility studies of bioremediation treatments, in order to assess whether a specific pollutant or any other substance at a site location could inhibit the microbiological processes

Sediment intrinsic turbidity influence on Microtox® basic solid-phase test

In this work sediment samples collected from a coastal lagoon (Italy), were tested with a new Microtox® assay designed for solid samples, the Basic Solid Phase Test (Basic SPT). According to this method, bacteria are exposed to sediment suspension dilutions and light production is directly measured on suspended sediments without any further manipulation. The purpose of the experiments here described was to evaluate the performance of the Microtox® Basic SPT taking into account the colour and turbidity of the sample during the whole measurement. Firstly, bacteria are dispensed into the sediment sample dilutions, and the maximum signal received immediately after dispensing (I 0) is compared to the signal after 5, 15 and 30 minute contact times (I5, I15, and I30 respectively). Secondly, absorbance of sediment samples was spectrophotometrically measured in triplicates at 490 nm, and EC50 values were corrected according to the manufacturer. EC50 data obtained with the two correction procedures compared to those of the standard Microtox® Basic SPT test suggest that sample colour affects the results at high EC50 values; however this effect is generally negligible at low EC50 values

Sediment intrisic turbidity influence on Microtox Basic Solid-Phase test.

In this work sediment samples collected from a coastal lagoon (Italy), were tested with a new Microtox\uae assay designed for solid samples, the Basic Solid Phase Test (Basic SPT). According to this method, bacteria are exposed to sediment suspension dilutions and light production is directly measured on suspended sediments without any further manipulation. The purpose of the experiments here described was to evaluate the performance of the Microtox\uae Basic SPT taking into account the colour and turbidity of the sample during the whole measurement. Firstly, bacteria are dispensed into the sediment sample dilutions, and the maximum signal received immediately after dispensing (I0) is compared to the signal after 5, 15 and 30 minute contact times (I5, I15, and I30 respectively). Secondly, absorbance of sediment samples was spectrophotometrically measured in triplicates at 490 nm, and EC50 values were corrected according to the manufacturer. EC50 data obtained with the two correction procedures compared to those of the standard Microtox 2 Basic SPT test suggest that sample colour affects the results at high EC50 values; however this effect is generally negligible at low EC50 values

Test con Microtox: determinazione della tossicit\ue0 dei sedimenti in un'area costiera.

La Pialassa della Baiona costituisce un complesso lagunare costiero di circa 1100 ha di superficie che si \ue8 formato naturalmente ed \ue8 stato modificato nel tempo da una serie di interventi effettuati sul territorio circostante [1]. Il sistema ha subito un profondo deterioramento a partire dagli anni \u201950, sostanzialmente per tre ragioni: (1) la diminuzione dell\u2019apporto di acqua dolce conseguente all\u2019inalveamento del fiume Lamone, che ha inciso in modo significativo sulla salinit\ue0 di alcune aree; (2) lo sviluppo industriale dell\u2019area portuale e l\u2019aumento della popolazione della citt\ue0, che hanno provocato un aumento del carico inquinante nella zona; e (3) l\u2019evolversi dell\u2019agricoltura locale. L\u2019obiettivo di questo lavoro di ricerca \ue8 stato quello di determinare le concentrazioni di idrocarburi policiclici aromatici (IPA) e bifenili clorurati (PCB) in campioni di sedimento superficiale raccolti nell\u2019area di studio, e di verificarne contemporaneamente la potenziale tossicit\ue0 tramite test su un batterio naturalmente luminescente, Vibrio fisheri. A tale scopo i test sono stati condotti sia sulla fase solida, alla quale si trovano legati gli inquinanti di interesse, sia sull\u2019estratto organico del sedimento, in modo da avere una risposta pi\uf9 chiara circa l\u2019influenza della componente organica sugli effetti tossici rilevati nel batterio. I risultati ottenuti hanno mostrato che le aree di campionamento localizzate in prossimit\ue0 degli insediamenti industriali sono quelle che presentano le concentrazioni pi\uf9 elevate di IPA e PCB, come precedentemente ritrovato da altri autori [2, 3], mentre i risultati ottenuti con il test Microtox\uae effettuato sul sedimento tal quale, hanno mostrato che anche campioni prelevati in aree dove le concentrazioni degli inquinanti oggetto di studio erano risultate molto pi\uf9 basse, hanno comunque riportato effetti tossici elevati. Una possibile spiegazione del fenomeno pu\uf2 essere ricercata nei cos\uec detti confounding factors (granulometria, solfuri, ammonio) ma anche nella presenza di altri inquinanti quali i metalli, che possono provocare effetti tossici rilevanti sul batterio [4]. Riferimenti [1] La Pialassa della Baiona. Qualit\ue0 dell\u2019ambiente e attivit\ue0 di ricerca. A cura di Centro Interdipartimentale di Ricerca per le Scienze Ambientali, Universit\ue0 di Bologna in Ravenna; Comune di Ravenna, Assessorato Ambiente. Editrice La Madrangola (Imola), 2003. [2] G. Matteucci, P. Rossini, A. Arcangeli, G. De Falco, P. Fonti e S. Guerzoni. \u201cOrganic pollution in a Ramsar site (Pialassa Baiona, Northern Adriatic Sea)\u201d. Annali di Chimica, 91, 445-, 2001. [3] D. Fabbri, I. Vassura, C.G. Sunb, C.E. Snape, C. McRaec, A.E. Fallick, \u201cSource apportionment of polycyclic aromatic hydrocarbons in a coastal lagoon by molecular and isotopic characterisation\u201d. Marine Chemistry, 84, 123-,2003. [4] C.Y. Hsieh, M.H. Tsai, D.K. Ryan e O.C. Pancorbo. \u201cToxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox chronic toxicity test\u201d. Science of the Total Environment, 320, 37-, 2004

Assessing degradation capability of aerobic indigenous microflora in PAH-contaminated brackish sediments

A study was conducted to determine polycyclic aromatic hydrocarbons (PAHs) distribution and microbial population changes in brackish sediments from an Italian lagoon included in the Ramsar List of Wetlands of International Importance. The presence and level of PAH-degrading bacteria were estimated by the most probable number (MPN) enumeration technique, whereas degradation capability towards target compounds was checked against loss of spiked PAHs (Phenanthrene, Anthracene and Fluoranthene) in MPN tubes after incubation in optimal conditions. Chemical analyses and microbiological counts suggested a potential for PAHs biodegradation by natural occurring populations of sediment microorganisms, thus indicating an \u2018\u2018optimal range\u2019\u2019 in sediment PAHs concentrations, outside of which the natural selection of the indigenous microflora did not occur. The MPN procedure here described, provided an effective and reliable way to simultaneously determine microbial population densities and subsequent confirmation of the biodegradation capability of sediment indigenous microflora when exposed to laboratory and environmental concentrations of PAHs