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The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to L. crispatus and L. gasseri species exert crucial protective functions against pathogens, although a total comprehension of factors that influence their dominance in healthy women is still lacking. Here we investigated the complete genome sequence and comprehensive phenotypic profile of L. crispatus strain BC5 and L. gasseri strain BC12, two vaginal strains featured by anti-bacterial and anti-viral activities. Phenotype microarray (PM) results revealed an improved capacity of BC5 to utilize different carbon sources as compared to BC12, although some specific carbon sources that can be associated to the human diet were only metabolized by BC12, i.e. uridine, amygdalin, tagatose. Additionally, the two strains were mostly distinct in the capacity to utilize the nitrogen sources under analysis. On the other hand, BC12 showed tolerance/resistance towards twice the number of stressors (i.e. antibiotics, toxic metals etc.) with respect to BC5. The divergent phenotypes observed in PM were supported by the identification in either BC5 or BC12 of specific genetic determinants that were found to be part of the core genome of each species. The PM results in combination with comparative genome data provide insights into the possible environmental factors and genetic traits supporting the predominance of either L. crispatus BC5 or L. gasseri BC12 in the vaginal niche, giving also indications for metabolic predictions at the species level.

Staphylococcus aureus is the major causative agent of skin and soft tissue infections, whose prevention and treatment have become more difficult due to the emergence of antibiotic-resistant strains. In this regard, the development of an effective treatment represents a challenge that can be overcome by delivering new antibiofilm agents with appropriate nanocarriers. In this study, a biosurfactant (BS) isolated from Lactobacillus gasseri BC9 and subsequently loaded in liposomes (LP), was evaluated for its ability to prevent the development and to eradicate the biofilm of different methicillin resistant S. aureus (MRSA) strains. BS from L. gasseri BC9 was not cytotoxic and was able to prevent formation and to eradicate the biofilm of different MRSA strains. BS loaded liposomes (BS-LP) presented a mean diameter (lower than 200 nm) suitable for topical administration and a low polydispersity index (lower than 0.2) that were maintained over time for up 28 days. Notably, BS-LP showed higher ability than free BS to inhibit S. aureus biofilm formation and eradication. BS-LP were loaded in lyophilized matrices able to quickly dissolve (dissolution time lower than 5 s) upon contact with exudate, thus allowing vesicle reconstitution. In conclusion, in this work, we demonstrated the antibiofilm activity of Lactobacillus-derived BS and BS-LP against clinically relevant MRSA strains. Furthermore, the affordable production of lyophilized matrices containing BS-LP for local prevention of cutaneous infections was established.

The vaginal microbiota, normally characterized by lactobacilli presence, is crucial for vaginal health. Members belonging to

The outbreak of SARS-CoV-2 pandemic highlighted the worldwide lack of surgical masks and personal protective equipment, which represent the main defense available against respiratory diseases as COVID-19. At the time, masks shortage was dramatic in Italy, the first European country seriously hit by the pandemic: aiming to address the emergency and to support the Italian industrial reconversion to the production of surgical masks, a multidisciplinary team of the University of Bologna organized a laboratory to test surgical masks according to European regulations. The group, driven by the expertise of chemical engineers, microbiologists, and occupational physicians, set-up the test lines to perform all the functional tests required. The laboratory started its activity on late March 2020, and as of the end of December of the same year 435 surgical mask prototypes were tested, with only 42 masks compliant to the European standard. From the analysis of the materials used, as well as of the production methods, it was found that a compliant surgical mask is most likely composed of three layers, a central meltblown filtration layer and two external spunbond comfort layers. An increase in the material thickness (grammage), or in the number of layers, does not improve the filtration efficiency, but leads to poor breathability, indicating that filtration depends not only on pure size exclusion, but other mechanisms are taking place (driven by electrostatic charge). The study critically reviewed the European standard procedures, identifying the weak aspects; among the others, the control of aerosol droplet size during the bacterial filtration test results to be crucial, since it can change the classification of a mask when its performance lies near to the limiting values of 95 or 98%.

In an aquifer-aquitard system in the subsoil of the city of Ferrara (Emilia-Romagna region, northern Italy) highly contaminated with chlorinated aliphatic toxic organics such as trichloroethylene (TCE) and tetrachloroethylene (PCE), a strong microbial-dependent dechlorination activity takes place during migration of contaminants through shallow organic-rich layers with peat intercalations. The in situ microbial degradation of chlorinated ethenes, formerly inferredby the utilization of contaminant concentration profiles and Compound-Specific Isotope Analysis (CSIA), was here assessed using Illumina sequencing of V4 hypervariable region of 16S rRNA gene and by clone library analysis of dehalogenase metabolic genes. Taxon-specific investigation of the microbial communities catalyzing the chlorination process revealed the presence of not only dehalogenating genera such as Dehalococcoides and Dehalobacter but also of numerous other groups of non-dehalogenating bacteria and archaea thriving on diverse metabolisms such as hydrolysis and fermentation of complex organic matter, acidogenesis, acetogenesis, and methanogenesis, which can indirectly support the reductive dechlorination process. Besides, the diversity of genes encoding some reductive dehalogenases was also analyzed. Geochemical and 16S rRNA and RDH gene analyses, as a whole, provided insights into the microbial community complexity and the distribution of potential dechlorinators. Based on the data obtained, a possible network of metabolic interactions has been hypothesized to obtain an effective reductive dechlorination process.

The purpose of this work was to prepare and characterize an innovative formulation for vaginal delivery of econazole nitrate, commonly used for the treatment of Candida infections. A novel biosurfactant isolated from a vaginal Lactobacillus strain was used to prepare phosphatidylcholine based mixed vesicles. Biosurfactant was produced by Lactobacillus gasseri BC9, isolated from the vagina of a healthy premenopausal woman, and was chemically characterized by FT-IR and ESI-MS. Mixed vesicles, obtained through film rehydration and extrusion method, were characterized in terms of size, zeta potential, encapsulation efficiency, mucoadhesion properties and econazole release. Moreover, the antimicrobial activity of the mixed vesicles was tested towards both planktonic cultures and biofilms of Candida albicans. Biosurfactant produced by L. gasseri BC9 was composed by peptide-like molecules containing hydrocarbon chains and possessed a high surface activity together with a low critical micelle concentration. All the mixed vesicles presented optimal diameter range (226-337nm) for topical vaginal administration. Econazole-loaded mixed vesicles containing biosurfactant showed higher encapsulation efficiency and mucoadhesion ability with respect to vesicles containing Tween 80. Further, they allowed a sustained release of econazole nitrate, maintaining the antifungal activity against C. albicans planktonic culture. Notably, biosurfactant-based vesicles were significantly more active than free econazole in the eradication of Candida biofilm. In conclusion, mixed vesicles are promising new vaginal delivery systems for the potential employment in the treatment of chronic infections.

BACKGROUND 1,1,2,2-Tetrachloroethane (TeCA) has been generally considered as non-biodegradable under aerobic conditions, while its complete biodegradation was reported with microbial consortia growing anaerobically. This study describes TeCA aerobic cometabolic degradation by the propanotroph Rhodococcus aetherivorans strain TPA isolated from a TeCA-degrading consortium. RESULTS R. aetherivorans TPA was able to grow on aliphatic hydrocarbons from propane to pentane and on gaseous n-alkanes metabolic intermediates. The Michaelis–Menten model allowed a satisfactory fit of the TPA propane utilization rates under resting cell conditions, while the TeCA degradation rates were successfully interpolated with Andrew's inhibition model. A significant propane - TeCA mutual inhibition was observed, although the results did not allow to distinguish between competitive and non-competitive inhibition. Among different bioreactor options for the on-site bioremediation of TeCA-contaminated groundwater, a single suspended-cell continuous stirred-tank reactor (CSTR) appeared to be the optimal one. CONCLUSIONS This study provides for the first time the kinetic and microbiological characterization of a bacterial strain capable to degrade TeCA under aerobic conditions.

Microbial degradation of 1,1,2,2-tetrachloroethane has been rarely analysed under aerobic conditions. In this work, the catabolic potential of a TeCA-degrading aerobic propanotroph consortium (C2) and the optimal bioreactor configuration for an on-site TeCA-bioremediation strategy with C2 were defined. More specifically, the diversity of alkane-oxidizing bacteria in C2 was assessed by means of clone libraries of genes coding for alkane monooxygenases (MOs) of different families (AlkB-like alkane hydroxylase, soluble di-iron MO and cytochromes P450). A large number of alkane MO sequences retrieved in this study showed the highest similarity with reference sequences belonging to Rhodococcus genus, suggesting a key role of this genus in TeCA/propane co-metabolism, while the remaining alkane MO sequences were mainly attributed to other Actinobacteria, to Bradhyrizobiaceae, and Cupriavidus genus. Further, the feasibility of an on-site TeCA bioremediation strategy with C2 was evaluated by simulating a continuous-flow aerobic co-metabolic process with different bioreactor configurations. Our results show that the configuration with a suspended-cell continuous stirred-tank reactor (CSTR) followed by a suspended-cell plug-flow reactor (PFR) was the one giving the best performance with consortium C2.

This is the final version of the following paper: CROGNALE S, STAZI SR, FIRRINCIELI A, PESCIAROLI L, FEDI S, PETRUCCIOLI M, D'ANNIBALE A 2019. Time-dependent changes in morpho-structural properties and relative abundances of contributors in Pleurotus ostreatus/Pseudomonas alcalliphila mixed biofilms. Frontiers in Microbiology, 10, p.1819 (ISSN: 1664-302X) DOI: https://doi.org/10.3389/fmicb.2019.01819 Scopus ID: 2-s2.0-85072733107 Web of Science ID: 000480249900001 PubMed ID: 31447819 URL of published article: https://www.frontiersin.org/articles/10.3389/fmicb.2019.01819/full, This study was supported by the Ministero dell'Istruzione dell'Università e della Ricerca (MIUR) within the project 2008P7K379. This work was also partially supported by the "Departments of Excellence-2018" Program (Dipartimenti di Eccellenza) of the Italian Ministry of Education, University and Research (MIUR), DIBAF-Department of University of Tuscia, Project "Landscape 4.0 – food, wellbeing, and environment".

Pleurotus ostreatusdual biofilms with bacteria are known to be involved in rock phosphate solubilization, endophytic colonization, and even in nitrogen fixation. Despite these relevant implications, no information is currently available on the architecture ofP. ostreatus-based dual biofilms. In addition to this, there is a limited amount of information regarding the estimation of the temporal changes in the relative abundances of the partners in such binary systems. To address these issues, a dual biofilm model system with this fungus was prepared by usingPseudomonas alcaliphila34 as the bacterial partner due to its very fast biofilm-forming ability. The application of the bacterial inoculum to already settled fungal biofilm on a polystyrene surface coated with hydroxyapatite was the most efficient approach to the production of the mixed system the ultrastructure of which was investigated by a multi-microscopy approach. Transmission electron microscopy analysis showed that the adhesion of bacterial cells onto the mycelial cell wall appeared to be mediated by the presence of an abundant layer of extracellular matrix (ECM). Scanning electron microscopy analysis showed that ECM filaments of bacterial origin formed initially a reticular structure that assumed a tabular semblance after 72 h, thus overshadowing the underlying mycelial network. Across the thickness of the mixed biofilms, the presence of an extensive network of channels with large aggregates of viable bacteria located on the edges of their lumina was found by confocal laser scanning microscopy; on the outermost biofilm layer, a significant fraction of dead bacterial cells was evident. Albeit with tangible differences, similar results regarding the estimation of the temporal shifts in the relative abundances of the two partners were obtained by two independent methods, the former relying on qPCR targeting of 16S and 18S rRNA genes and the latter on ester-linked fatty acid methyl esters analysis.

This paper presents a route for the treatment of MSWI fly (FA) and bottom ashes (BA) using microorganisms to critically assess whether bioleaching is within reach of effective industrial application. The leaching of metals from BA and FA was investigated in a controlled laboratory environment using a culture isolated from a natural system where the dominant strains are acidophilic bacteria, mainly Acidothiobacillus thiooxidans and Acidothiobacillus ferrooxidans. The community of microorganisms (mostly acidophilic, S- and Fe-oxidizing bacteria) was collected directly from overflows and ponds at the sediment–water interface of a natural system near a post-mining site. Pre-cultivation was done in 250 mL flasks followed by the adaptation to the different substrates (both FA and BA). The effect of different material pre-treatment and elemental sulphur concentrations were evaluated for both BA and FA, at a starting pH of 4. The bioleaching of BA and FA substrates experienced good yields of metal extraction with an optimum duration of two weeks. The results showed that more than 90% Zn, Cu, and 10% Pb are removed from FA; while 100% Cu, 80% Zn and 20% Pb are removed from BA samples. Batch experiments with regenerating ion-exchange resins did not perform well for metal recovery, but could serve as a valuable decontamination step. The techniques used here with FA and BA can be used for urban mining purposes (e.g. ashes and other meal-rich anthropogenic wastes), but also low-grade ores in the mining industry, contributing to resource recovery or decontamination agendas.

Alkanes are widely distributed in the environment as they not only constitute the large fraction of crude oil but are also produced by many living organisms. They are saturated hydrocarbons of different sizes and structures, which pose a variety of challenges to degradative microorganisms due to their physicochemical properties, i.e., the extremely limited solubility and the high energy required for activation. The hydrophobic cell surface of Rhodococcus spp., the ability to produce biosurfactants, and the possession of a wide range of oxygenases allow coping with such challenges. In particular, monooxygenase enzymes are involved in the activation of alkanes by converting them into alcohols, which undergo a series of oxidation steps before being converted to fatty acids. Rhodococcus alkane monooxygenases belong to different families (i.e., AlkB-like monooxygenase, soluble di-iron monooxygenase, cytochrome P450), have different genetic organization, and are subject to different regulatory mechanisms, which are poorly known. Because of their long-term survival capacity, broad catabolic abilities, and effective contact mechanisms with hydrocarbon molecules, alkanotrophic Rhodococcus strains have biotechnology applications and potential in bioremediation and biotransformation reactions.

"Terre Calde di Medolla" (TCM) (literally, "Hot Lands of Medolla") refers to a farming area in Italy with anomalously high temperatures and diffuse emissions of biogenic CO2, which has been linked to CH4 oxidation processes from a depth of 0.7 m to the surface. We herein assessed the composition of the total bacterial community and diversity of methane-oxidizing bacteria (MOB) in soil samples collected at a depth at which the peak temperature was detected (0.6 m). Cultivation-independent methods were used, such as: i) a clone library analysis of the 16S rRNA gene and pmoA (coding for the α-subunit of the particulate methane monooxygenase) gene, and ii) Terminal Restriction Fragment Length Polymorphism (T-RFLP) fingerprinting. The 16S rRNA gene analysis assessed the predominance of Actinobacteria, Acidobacteria, Proteobacteria, and Bacillus in TCM samples collected at a depth of 0.6 m along with the presence of methanotrophs (Methylocaldum and Methylobacter) and methylotrophs (Methylobacillus). The phylogenetic analysis of pmoA sequences showed the presence of MOB affiliated with Methylomonas, Methylocystis, Methylococcus, and Methylocaldum in addition to as yet uncultivated and uncharacterized methanotrophs. Jaccard's analysis of T-RFLP profiles at different ground depths revealed a similar MOB composition in soil samples at depths of 0.6 m and 0.7 m, while this similarity was weaker between these samples and those taken at a depth of 2.5 m, in which the genus Methylocaldum was absent. These results correlate the anomalously high temperatures of the farming area of "Terre Calde di Medolla" with the presence of microbial methane-oxidizing bacteria.

Kimura and co-workers (Kimura N et al. Int J Syst Evol Microbiol 2018;68:1429-1435) recently proposed renaming the obligate aerobe Pseudomonas pseudoalcaligenes KF707 as Pseudomonas furukawiisp. nov. type strain KF707. Since the first quasi-complete genome sequence of KF707 was reported in 2012 (accession number: PRJNA83639) numerous reports on chemotaxis and function/composition of the respiratory redox chain of KF707 have been published, demonstrating that KF707 contains three cheA genes for aerobic motility, four cytochrome oxidases of c(c)aa3- and cbb3-type and one bd-type quinol oxidase. With this background in mind, it has been quite a surprise to read within Table 1 of the paper by Kimura et al. that strain KF707 is phenotypically characterized as cytochrome oxidase-negative. Further, Table 1 also reports that KF707 is β-galactosidase-positive, an affirmation that is not consistent with results documented in the current literature. In this present 'Letter to the Editor' we show that Kimura et al. have contradicted themselves and provided inaccurate information in respect to the respiratory phenotypic features of P. furukawii. Based on this, an official corrigendum is requested since the publication, as it is, blurs the credibility of the International Journal of Systematic and Evolutionary Microbiology.

Naphthenic acids (NAs) are an important group of toxic organic compounds naturally occurring in hydrocarbon deposits. This work shows that Rhodococcus aetherivorans BCP1 cells not only utilize a mixture of eight different NAs (8XNAs) for growth but they are also capable of marked degradation of two model NAs, cyclohexanecarboxylic acid (CHCA) and cyclopentanecarboxylic acid (CPCA) when supplied at concentrations from 50 to 500 mgL-1. The growth curves of BCP1 on 8XNAs, CHCA, and CPCA showed an initial lag phase not present in growth on glucose, which presumably was related to the toxic effects of NAs on the cell membrane permeability. BCP1 cell adaptation responses that allowed survival on NAs included changes in cell morphology, production of intracellular bodies and changes in fatty acid composition. Transmission electron microscopy (TEM) analysis of BCP1 cells grown on CHCA or CPCA showed a slight reduction in the cell size, the production of EPS-like material and intracellular electron-transparent and electron-dense inclusion bodies. The electron-transparent inclusions increased in the amount and size in NA-grown BCP1 cells under nitrogen limiting conditions and contained storage lipids as suggested by cell staining with the lipophilic Nile Blue A dye. Lipidomic analyses revealed significant changes with increases of methyl-branched (MBFA) and polyunsaturated fatty acids (PUFA) examining the fatty acid composition of NAs-growing BCP1 cells. PUFA biosynthesis is not usual in bacteria and, together with MBFA, can influence structural and functional processes with resulting effects on cell vitality. Finally, through the use of RT (Reverse Transcription)-qPCR, a gene cluster (chcpca) was found to be transcriptionally induced during the growth on CHCA and CPCA. Based on the expression and bioinformatics results, the predicted products of the chcpca gene cluster are proposed to be involved in aerobic NA degradation in R. aetherivorans BCP1. This study provides first insights into the genetic and metabolic mechanisms allowing a Rhodococcus strain to aerobically degrade NAs.

none 7 si Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. Martina Cappelletti;Alessandro Presentato;Giorgio Milazzo;Raymond J. Turner;Stefano Fedi;Dario Frascari;Davide Zannoni Martina Cappelletti;Alessandro Presentato;Giorgio Milazzo;Raymond J. Turner;Stefano Fedi;Dario Frascari;Davide Zannoni

The role of anaerobic digestion (AD) on the decontamination and biomethanization of a PCB-spiked sludge obtained from a Membrane Biological Reactor (MBR) pilot plant was investigated throughout a 10-month batch experiment. The study was carried out under mesophilic (35 °C) and thermophilic (55 °C) conditions and was monitored by means of an integrated chemical, microbiological and molecular biology strategy. Remarkable PCB depletions (higher than 50% of the overall spiked PCBs) and dechlorinations were achieved under methanogenic conditions. The process was not affected by yeast extract addition. Both acetoclastic and hydrogenotrophic methanogens, together with some fermentative eubacteria, were found to persist in all PCB biodegrading microcosms. This finding, together with those obtained from parallel microcosms where specific populations were selectively inhibited, suggested that native methanogens played a key role in the biodegradation and dechlorination of the spiked PCBs. Taken together, the results of this study indicate that AD is a feasible option for the decontamination and the efficient disposal (with the production of a CH 4 -rich biogas) of contaminated MBR sludge, which can be then employed as a fertilizer for agricultural purposes.

In this work the aerobic cometabolic biodegradation of 1,1,2,2-tetrachloroethane (TeCA) by propane-utilizing bacteria was studied in slurry bioreactors containing soil and groundwater from 5 aquifers as well as in soil-free bioreactors. The main goals were: (a) to identify and calibrate a kinetic model of TeCA cometabolism; (b) to select and characterize a TeCA-degrading bacterial consortium; (c) to compare the results obtained in slurry and in soil-free bioreactors. The results showed that 4 of the 5 tested aquifers contain TeCA-degrading bacteria, indicating that aerobic cometabolism is a potentially effective approach for TeCA-contaminated aquifers. In bioaugmentation tests, a TeCA-cometabolizing consortium developed in the slurry bioreactors induced a strong reduction of the lag-time for the onset of TeCA cometabolism. The soil-free tests yielded a satisfactory TeCA degradation performance, indicating that on-site soil-free bioreactors represent an interesting technical solution for the aerobic cometabolic bioremediation of CAH-contaminated groundwaters. The mineralization of the organic Cl was equal to about 97%. The prolonged TeCA biodegradation determined a progressive selection of the bacterial strains more effective in TeCA degradation and less affected by degradation product toxicity. The tested Michaelis–Menten-based kinetic model proved an effective tool to interpret the experimental data of TeCA aerobic cometabolism.

Pseudomonas pseudoalcaligenes KF707 is a polychlorinated biphenyls (PCBs) degrader, also tolerant to several toxic metals and metalloids. The work presented here examines for the first time the chemotactic response of P. pseudoalcaligenes KF707 to biphenyl and intermediates of the PCB biodegradation pathway in the presence and absence of metals. Chemotaxis analyses showed that biphenyl, benzoic acid and chlorobenzoic acids acted as chemoattractants for KF707 cells and that metal cations such as Ni2+ and Cu2+ strongly affected the chemotactic response. Toxicity profiles of various metals on KF707 cells grown on succinate or biphenyl as planktonic and biofilm were determined both in the presence and in the absence of PCBs. Notably, KF707 cells from both biofilms and planktonic cultures were tolerant to high amounts (up to 0.5 g L−1) of Aroclor 1242, a commercial mixture of PCBs. Together, the data show that KF707 cells are chemotactic and can form a biofilm in the presence of Aroclor 1242 and specific metals. These findings provide new perspectives on the effectiveness of using PCB-degrading bacterial strains in bioremediation strategies of metal-co-contaminated sites.

Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that can be co-metabolically biotransformed by biphenyl-utilizing bacteria. In this study, terminal-restriction fragment length polymorphism (T-RFLP) was applied to the substrate specificity-determining region of the 2,3-biphenyl dioxygenase encoding genes of a microbial community found in a PCB-polluted soil. Notably, both the total biphenyl/PCB-utilizing community and its members actively expressing the 2,3-biphenyl dioxygenase gene were analyzed. T-RFLP fingerprinting along with gene library construction allowed us not only to detect biphenyl dioxygenases related to the well characterized catabolic patterns of Pseudomonas pseudoalcaligenes KF707 and Burkholderia xenovorans LB400, but also to reveal novel environmental enzyme classes displaying amino acid substitutions that may be related to broader specificity and improved catalytic properties. Furthermore, space and time of sampling along with bioavailability conditions of different PCBs were considered possible sources of profile variability.

Biofilms are communities of microorganisms adhering to a surface and embedded in an extracellular polymeric matrix, frequently associated with disease and contamination, and also used for engineering applications such as bioremediation. A mixed biofilm formed by bacteria and fungi may provide an optimal habitat for addressing contaminated areas. To exploit the potential of natural microbial communities consisting of bacteria and fungi, it is essential to understand and control their formation. In this work, a method to discriminate among bacteria of genera Bacillus, Pseudomonas, Rhodococcus with respect to the fungus Pleorotus in a biofilm by means of pyrolysis-gaschromatography-mass spectrometry and multivariate analysis is reported. Methylated fatty acids were chosen as biomarkers of microorganisms in the pyrolysates. In situ thermal hydrolysis and methylation was applied. Pyrograms were used as fingerprints, thus allowing for the characterization of whole cells analyzed without any sample pretreatment. Normalized pyrographic peak areas were chosen as variables for chemometric data processing. Principal components analysis was applied as a data exploration tool. Satisfactory results were obtained in analyzing a real biofilm. The influence of growth medium on whole bacteria fatty acid cell composition was also explored.

Tellurite (TeO3(2-)) is the most toxic and soluble oxyanion among tellurium (Te) compounds. The effects of the metalloid anion on the oxidative stress response of the obligate aerobe Pseudomonas pseudoalcaligenes KF707 were investigated. Cells treated with sub-lethal concentrations of TeO3(2-) showed neither adaptation to it nor cross-protection against oxidants such as 1,1'-4,4'-bipyridinium dichloride (paraquat, PQ2+), diazenedicarboxylic acid bis-N,N-dimethylamide (diamide), tert-butyl hydroperoxide (tBH) and hydrogen peroxide (H2O2). Notably, TeO3(2-) exerted a synergic effect on the toxicity of these latter oxidants. Tellurite was shown to decrease the cellular content of reduced thiols (RSH) with a consequent increase in the production of reactive oxygen species (ROS) and stimulation of the superoxide dismutase (SOD) activity. However, since the time course of ROS production by TeO3(2) (t1/2 > 30 min) was much slower than that with PQ2+ and/or diamide (t1/2

Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO(3)(2-); minimal inhibitory concentration, 250 microg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50 microg of K(2)TeO(3) per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several "needlelike" shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c(2)). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN(-)). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN(-) in the micromolar range) accounts for 35-40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system.

Bioremediation implies the use of living organisms, primarily microorganisms, to convert environmental contaminants into less toxic forms. The impact of the consequences of hydrocarbon release in the environment maintain a high research interest in the study of microbial metabolisms associated with the biodegradation of aromatic and aliphatic hydrocarbons but also in the analysis of microbial enzymes that can convert petroleum substrates to value-added products. The studies described in this Thesis fall within the research field that directs the efforts into identifying gene/proteins involved in the catabolism of n-alkanes and into studying the regulatory mechanisms leading to their oxidation. In particular the studies were aimed at investigating the molecular aspects of the ability of Rhodococcus sp. BCP1 to grow on aliphatic hydrocarbons as sole carbon and energy sources. We studied the ability of Rhodococcus sp. BCP1 to grow on gaseous (C2-C4), liquid (C5-C16) and solid (C17-C28) n-alkanes that resulted to be biochemically correlated with the activity of one or more monooxygenases. In order to identify the alkane monooxygenase that is involved in the n-alkanes degradation pathway in Rhodococcus sp. BCP1, PCR-based methodology was applied by using degenerate primers targeting AlkB monooxygenase family members. As result, a chromosomal region, including the alkB gene cluster, was cloned from Rhodococcus sp. BCP1 genome. We characterized the products of this alkB gene cluster and the products of the orfs included in the flanking regions by comparative analysis with the homologues in the database. alkB gene expression studies were carried out by RT-PCR and by the construction of a promoter probe vector containing the lacZ gene downstream of the alkB promoter. B-galactosidase assays revealed the alkB promoter activity induced by n-alkanes and by n-alkanes metabolic products. Furthermore, the transcriptional start of alkB gene was determined by primer extension procedure. A proteomic approach was subsequently applied to compare the protein patterns expressed by BCP1 growing on n-butane, n-hexane, n-hexadecane or n-eicosane with the protein pattern expressed by BCP1 growing on succinate. The accumulation of enzymes specifically induced on n-alkanes was determined. These enzymes were identified by tandem mass spectrometry (LC/MS/MS). Finally, a prm gene, homologue to the gene family coding for soluble di-iron monooxygenases (SDIMOs), has been isolated from Rhodococcus sp. BCP1 genome. This gene product could be involved in the degradation of gaseous n-alkanes in this Rhodococcus strain. The versatility in utilizing hydrocarbons and the discovery of new remarkable metabolic activities outline the potential applications of this microorganism in environmental and industrial biotechnologies.

The respiratory chain of Pseudomonas pseudoalcaligenes KF707 in membranes isolated from cells grown in the presence or absence of the toxic oxyanion tellurite ( \(TeO_{3}^{2{-}}\) ) was examined. Aerobic growth in the absence of tellurite shows an NADH-dependent respiration which is 80% catalysed by the cytochrome (cyt) bc 1-containing pathway leading to two terminal membrane-bound cyt c oxidases inhibited by different concentrations of KCN (IC50 0·2 and 1 μM). A third oxidase, catalysing the remaining 20% of the cyanide-resistant respiration and fully inhibited by 2–3 mM KCN, is also present; this latter pathway accounts for 60–70% of the total NADH-dependent respiration in membranes from cells grown in LB medium supplemented with potassium tellurite (35 μg ml−1). Two high-potential b-type haems (E m,7 +395 and 318 mV) are redox centres of a membrane-bound cyt c oxidase (possibly of the cbb 3 type) which shows a 50% decrease of its activity in parallel with a similar decrease of the c-type haem content (mostly soluble cyt c) in membranes from tellurite-grown cells; the latter type of cells specifically contain a cyt b type at +203 mV (pH 9·0) which is likely to be involved in cyanide-resistant respiration. Comparison of the growth curve of KF707 cells in parallel with tellurite uptake showed that intracellular accumulation of tellurium (Te0) crystallites starts from the mid-exponential growth phase, whereas tellurite-induced changes of the respiratory chain are already evident during the early stages of growth. These data were interpreted as showing that reduction of tellurite to tellurium and tellurite-dependent modifications of the respiratory chain are unrelated processes in P. pseudoalcaligenes KF707.

Fifteen bacterial strains using biphenyl as sole carbon and energy source, obtained from different positions and depths of a polychlorinated biphenyl (PCB)-contaminated area, were analyzed for their basic metabolic phenotypes and subjected to genomic DNA hybridization screening for the presence of well characterized bph operons such as those of Pseudomonas pseudoalcaligenes KF707 and Rhodococcus globerulus P6. Most of the isolates belonged to the gamma-subdivision (Pseudomonas stutzeri, P. plutida, P. fluorescens and Vibrio logei species) and to the beta-subdivision (genera Alcaligenes, Comamonas, Ralstonia) of the Proteobacteria. All the isolates were able to cometabolize different low chlorinated PCB congeners. Among the dichlorinated biphenyls tested, a lower degradation capacity was observed for the di-ortho substituted congeners, whereas high levels of degradation were observed for the di-meta and di-para isomers, whether they were chlorinated on one or on both rings. The PCB congeners nonsubstituted in the 2,3 or 2,3 and 3,4 positions were also degraded by most of the isolated strains, which were, however, unable to significantly metabolize PCBs with more than 3 chlorine atoms. Five of the isolated strains were also able to degrade some of the tri- and tetrachlorobiphenyls tested. Southern hybridization analysis showed a strong homology between four of the fifteen isolated strains and the bph operon obtained from P. pseudoalcaligenes strain KF707. Conversely, none of the isolates here examined showed homology with the bph operon of R. globerulus strain P6. In line with this, the KF707 bph probe strongly hybridized with DNA of a significant number of bacterial colonies obtained from selected locations in the contaminated area using biphenyl-supplemented minimal medium agar plates.

Rhodococcus sp. strain BCP1 (DSM 44980) co-metabolizes chlorinated compounds and mineralizes a broad range of alkanes being highly tolerant to these toxic chemicals. Here, we present the high-quality draft genome sequence of strain BCP1 consisting of 6,231,823 bp, with a G+C content of 70.4%, 5,902 protein-coding genes, and 58 RNAs genes. Rhodococcus genus comprises Gram-positive, non-sporulating, aerobic bacteria that are widely distributed in the environment (1). Rhodococcus sp. strain BCP1 (formerly: Rhodococcus aetherovorans strain BCP1, DSM 44980) was selected from an aerobic butane-utilizing consortium as the prevailing isolate able to co-metabolize chloroform, vinyl chloride and trichloroethylene (2, 3). As BCP1 also catabolizes a wide range of aliphatic, alicyclic and carboxylated alkanes, it represents a strain of considerable environmental and industrial interest (4). The genome sequencing of Rhodococcus sp. BCP1 was performed using 454 sequencing technology (Roche GS FLX Titanium). The total number of sequence reads were 668,686 from one shotgun library and 353,744 from one paired-end library (8-kb inserts). All the reads were assembled using Newbler 2.6 into 123 contigs with an N50 length of 237,787 bp and an average genome coverage of 65x. Based on paired-end directional information, the contigs were further ordered into 3 scaffolds, giving a total genome size of 6.2 Mb with a G+C content of 70.4%.

The adequacy of the Calgary biofilm device, often referred to as the MBEC system, as a high-throughput approach to the production and subsequent characterization of Pleurotus ostreatus biofilms was assessed. The hydroxyapatite-coating of pegs was necessary to enable biofilm attachment, and the standardization of vegetative inocula ensured a uniform distribution of P. ostreatus biofilms, which is necessary for high-throughput evaluations of several antimicrobials and exposure conditions. Scanning electron microscopy showed surface-associated growth, the occurrence of a complex aggregated growth organized in multilayers or hyphal bundles, and the encasement of hyphae within an extracellular matrix (ECM), the extent of which increased with time. Chemical analyses showed that biofilms differed from free-floating cultures for their higher contents of total sugars (TS) and ECM, with the latter being mainly composed of TS and, to a lesser extent, protein. Confocal laser scanning microscopy analysis of 4-day-old biofilms showed the presence of interspersed interstitial voids and water channels in the mycelial network, the density and compactness of which increased after a 7-day incubation, with the novel occurrence of ECM aggregates with an α-glucan moiety. In 4- and 7-day-old biofilms, tolerance to cadmium was increased by factors of 3.2 and 11.1, respectively, compared to coeval free-floating counterparts.

A process of chloroform (CF) aerobic cometabolic biodegradation by a butane-growing consortium was studied for 354 days in a 2-m continuous-flow sand-filled reactor. The study was aimed at (a) investigating the oxygen/substrate pulsed injection as a tool to control the clogging of the porous medium, to attain a wide bioreactive zone and to reduce substrate inhibition on CF cometabolism; (b) developing a reliable model of CF cometabolism in porous media. While the continuous supply of butane rapidly led to the clogging of the porous medium due to excessive biomass growth, the testing of six types of oxygen/substrate pulsed feeding led to the identification of a feeding schedule capable to prevent aquifer clogging and to lead to the development of a long bioreactive zone and to satisfactory CF degradation performances. The tested model of aerobic cometabolism allowed a suitable interpretation of the experimental data as long as the ratio of CF degraded to butane consumed was ≤0.27 mg CF mg butane −1 . A long-term 1-D simulation of the best-performing schedule of pulsed oxygen/substrate supply extended to a 30-m aquifer length resulted in a 20-m bioreactive zone and in a 96% CF removal.

Today there is growing a search for renewable, low-cost and low-emission energy sources. This work is aimed at assessing the technical and economic feasibility of a biological process of H2 production from food industry waste in thermophilic conditions. The production of H2 by 4 strains of the genus Thermotoga (T. neapolitana, T. petrophila, T. naphtophila, T. maritima) was studied at 77°C in batch bioreactors (120 mL) with glucose, molasses and whey as carbon sources. With all the substrates tested, T. neapolitana gave the best results, with an initial H2 production rate of 1.2–1.9 mmol Lmedium-1 h-1 and a selectivity of 1.6–2.6 mmolH2 mmolmonosaccharide consumed-1. Using T. neapolitana, further studies were conducted with cells immobilized on 4 types of porous supports (3 vitreous ones and a ceramic one). The best results were obtained with the ceramic support, characterized by the maximum interfacial area (2.1 m2 g−1). Further tests, aimed at simplifying the composition of the growth medium, gave encouraging results. For example, in the case of molasses a minimal medium composed only of NH4Cl, K2HPO4, NaCl, buffer and cysteine was identified. In comparison with the complete medium, the minimal medium was characterized by a 73% cost reduction and a 12% increase of the H2/substrate yield. Finally, the process scale-up to a 19-L reactor is in progress. Preliminary results indicate that, in the scale-up of the production of H2 from glucose with T. neapolitana, the values of the three monitored yields (H2/substrate, organic acids/substrate and biomass/substrate) showed no significant changes.

BACKGROUND: Bio-hydrogen production from organic residues is an attractive process that combines energy generation with waste treatment. This work describes hydrogen production from molasses and cheese whey by biofilm and suspended-cells of four hyperthermophilicThermotoga spp. For the best performing strain, a preliminary process optimization was performed. RESULTS: In experiments with glucose-fed T.neapolitana, HEPES was the best-performing buffer, whereas the highest H2 rate was obtained with the biofilm support exposing the highest specific surface. All the Thermotoga strains tested (T.neapolitana, T.maritima, T.naphtophila, T.petrophila) were able to produce H2 from glucose, molasses and cheese whey under both suspended- and attached-cell conditions. T.neapolitana was the best H2 producer with all three substrates. The best H2 production rate was higher on molasses (1.7 mmolH2 L −1 medium h −1 ) than on cheese whey (0.94 mmolH2 L −1 medium h −1 ), whereas the highestH2/substrateyieldsweresimilarforthetwosubstrates(2.95 molH2 mol −1 monosaccharide consumed onmolassesand2.50 molH2 mol −1 monosaccharide consumed on cheese whey). With both wastes, the progressive growth medium simplification allowed a drastic reduction of medium cost, without significant losses of process performance. CONCLUSION: Both molasses and cheese whey are suitable substrates for H2 production by suspended- and attached-cells of Thermotoga species. c � 2012 Society of Chemical Industry Supportinginformationmaybefoundintheonlineversionofthisarticle.

Chloroform (CF) is largely produced by both anthropogenic and natural sources. It is detected in ground- and surface-water sources and it represents the most abundant halocarbon in the atmosphere. Microbial CF degradation occurs under both aerobic and anaerobic conditions. Apart from a few reports describing the utilization of CF as a terminal electron acceptor during growth, CF degradation was mainly reported as a cometabolic process. CF aerobic cometabolism is supported by growth on short-chain alkanes (i.e. methane, propane, butane and hexane), aromatic hydrocarbons (i.e. toluene and phenol) and ammonia via the activity of monooxygenases operatively divided into different families. The main factors affecting CF cometabolism are i) the inhibition of CF degradation exerted by the growth substrate, ii) the need for reductant supply to maintain monooxygenase activity, and iii) the toxicity of CF degradation products. Under anaerobic conditions, CF degradation was mainly associated to the activity of methanogens, although some examples of CF-degrading sulfate-reducing, fermenting, and acetogenic bacteria are reported in the literature. Higher CF toxicity levels and lower degradation rates were shown by anaerobic systems in comparison to the aerobic ones. Applied, physiological and genetic aspects of microbial cometabolism of CF will be presented along with bioremediation perspectives.

Pseudomonas pseudoalcaligenes KF707 is a soil polychlorinated biphenyl (PCB) degrader, able to grow both planktonically and as a biofilm in the presence of various toxic metals and metalloids. Here we report the genome sequence (5,957,359 bp) of P. pseudoalcaligenes KF707, which provides insights into metabolic degradation pathways, flagellar motility, and chemotaxis.

This work focuses on chloroform (CF) cometabolism by a butane-grown aerobic pure culture (Rhodococcus aetherovorans BCP1) in continuous-flow biofilm reactors. The goals were to obtain preliminary information on the feasibility of CF biodegradation by BCP1 in biofilm reactors and to evaluate the applicability of the pulsed injection of growth substrate and oxygen to biofilm reactors. The attached-cell tests were initially conducted in a 0.165-L bioreactor and, then, scaled-up to a 1.772-L bioreactor. Glass cylinders were utilized as biofilm carriers. The continuous supply of growth substrate (butane), which led to the attainment of the highest CF degradation rate (8.4 mg(CF) day(-1) m (biofilm surface)(-2)), was compared with four schedules of butane and oxygen pulsed feeding. The pulsed injection technique allowed the attainment of a ratio of CF mass degraded per unit mass of butane supplied equal to 0.16 mg(CF) mg (butane)(-1), a value 4.4 times higher than that obtained with the continuous substrate supply. A procedure based on the utilization of integral mass balances and of average concentrations along the bioreactors resulted in a satisfactory match between the predicted and the experimental CF degradation performances, and can therefore be utilized to provide a guideline for optimizing the substrate pulsed injection schedule.

A histidine-kinase cheA gene in Pseudomonas pseudoalcaligenes KF707 plays a central role in the regulation of metabolic responses as well as in chemotaxis. Non-chemotactic mutants harboring insertions into the cheA gene were screened for their ability to form biofilms in the Calgary biofilm device. Notably, ≥95% decrease in the number of cells attached to the polystyrene surface was observed in cheA mutants compared to the KF707 wild-type biofilm phenotype. The ability to form mature biofilms was restored to wild-type levels, providing functional copies of the KF707 cheA gene to the mutants. In addition, phenotype micro-arrays and proteomic analyses revealed that several basic metabolic activities and a few periplasmic binding proteins of cheA mutant cells differed compared to those of wild-type cells. These results are interpreted as evidence of a strong integration between chemotactic and metabolic pathways in the process of biofilm development by P. pseudoalcaligenes KF707.

Rhodococcus sp. strain BCP1, known for its capacity to grow on short-chain n -alkanes (C 2 to C 7 ) and to cometabolize chlorinated solvents, was found to also utilize medium- and long-chain n -alkanes (C 12 to C 24 ) as energy and carbon sources. To examine this feature in detail, a chromosomal region which includes the alkB gene cluster encoding a non-heme di-iron monooxygenase ( alkB ), two rubredoxins, and one rubredoxin reductase was cloned from the BCP1 genome. Furthermore, the activity of the alkB gene promoter (P alkB ) was examined in the presence of gaseous, liquid, and solid n -alkanes along with intermediates of the putative n -alkane degradation pathway. A recombinant plasmid, pTP alkB LacZ, was constructed by inserting the lacZ gene downstream of P alkB , and it was used to transform Rhodococcus sp. strain BCP1. Measurements of β-galactosidase activity showed that P alkB is induced by C 6 to C 22 n -alkanes. Conversely, C 2 to C 5 and >C 22 n -alkanes and alkenes, such as hexene, were not inducers of alkB expression. The effects on P alkB expression induced by alternative carbon sources along with putative products of n -hexane metabolism were also evaluated. This report highlights the great versatility of Rhodococcus sp. strain BCP1 and defines for the first time the alkB gene transcriptional start site and the alkB promoter-inducing capacities for substrates different from n -alkanes in a Rhodococcus strain.

Pseudomonas pseudoalcaligenes KF707 is a polychlorinated biphenyls (PCBs) degrader, also tolerant to several toxic metals and metalloids. The work presented here examines for the first time the chemotactic response of P. pseudoalcaligenes KF707 to biphenyl and intermediates of the PCB biodegradation pathway in the presence and absence of metals. Chemotaxis analyses showed that biphenyl, benzoic acid and chlorobenzoic acids acted as chemoattractants for KF707 cells and that metal cations such as Ni(2+) and Cu(2+) strongly affected the chemotactic response. Toxicity profiles of various metals on KF707 cells grown on succinate or biphenyl as planktonic and biofilm were determined both in the presence and in the absence of PCBs. Notably, KF707 cells from both biofilms and planktonic cultures were tolerant to high amounts (up to 0.5 g L(-1)) of Aroclor 1242, a commercial mixture of PCBs. Together, the data show that KF707 cells are chemotactic and can form a biofilm in the presence of Aroclor 1242 and specific metals. These findings provide new perspectives on the effectiveness of using PCB-degrading bacterial strains in bioremediation strategies of metal-co-contaminated sites.

Summary Seventeen strains belonging to the genus Azospirillum were isolated from crop plant roots and characterized following routine morphological and physiological methods without obtaining a definite species assignment. DNA restriction pattern analysis was therefore performed on those strains and on 9 reference strains belonging to Azospirillum lipoferum, Azospirillum brasilense and Azospirillum amazonense. Fragments obtained with endonuclease BglII digestion were separated with PAGE and stained with silver nitrate. An internal standard was used to rescale the separation profiles, thereby permitting the comparison of samples from different gels. Each pattern was found to be unique, allowing the distinction of single strains. UPGMA analysis, yielding clusters of band patterns, was utilized to assign strains to a species. Restriction pattern analysis is therefore suggested for the routine identification of azospirilla.

An aerobic bacterial strain, named PB1, was microbiologically characterized and subjected to assays of cometabolic biodegradation of chlorinated aliphatic hydrocarbons (CAHs) aimed at (i) investigating its capacity to biodegrade vinyl chloride (VC), cis-1,2-dichloroethylene (cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), chloroform (CF) and 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA); and (ii) evaluating the suitability of a kinetic model with inhibition and transformation product toxicity to describe CAH biodegradation by PB1. The strain proved to be closely related to the genus Rhodococcus. It biodegraded VC, cis-DCE, 1,1,2-TCA and CF via aerobic cometabolism on propane, but not 1,1,2,2-TeCA. CAH biodegradation by PB1 was satisfactorily described by the tested kinetic model. The maximum specific biodegradation rates were in the 1-11 micromol/mg protein/day range. The transformation capacities varied from 0.6 to 3 micromol/mg protein. Propane exerted a significant inhibition on both CF and cis-DCE biodegradation. CF, at 52 microM, did not inhibit propane utilization. An analysis of different inhibition models showed that cis-DCE inhibition on propane utilization was satisfactorily simulated with the competitive model, with an inhibition constant equal to 70% of the cis-DCE half-saturation constant. A final assay specifically aimed at validating the kinetic model and the verifying the estimates of the kinetic parameters yielded a satisfactory result. The results provide positive indications on the feasibility of utilizing strain PB1 for CAH biodegradation in aquifers and wastewaters.

The susceptibility to various biocides was examined in planktonic cells and biofilms of the obligate aerobe, PCBs degrader, Pseudomonas pseudoalcaligenes KF707. The toxicity of two antibiotics, amikacin and rifampicin, three metalloid oxyanions (AsO2 −, SeO3 2−, TeO3 2−) and three metal cations (Cd2+, Ni2+, Al3+) was tested at two stages of the biofilm-development (4 and 24 h) and compared to planktonic cells susceptibility. Mature biofilms formed in rich (LB, Luria–Bertani) medium were thicker (23 μm) than biofilms grown in minimal (SA saccarose-arginine) medium (13 μm). Early grown (4 h) SA-biofilms, which consisted of a few sparse/attached cells, were 50–100 times more resistant to antibiotics than planktonic cells. Conversely, minor changes in tolerance to metal(loid)s were seen in both SA- and LB-grown biofilms. In contrast to planktonic cells, no reduction of TeO3 2− to elemental Te(0) or SeO3 2− to elemental Se(0) was seen in KF707 biofilms. The data indicate that: (a) metal tolerance in KF707 biofilms, under the growth and exposure conditions described here, is different than antibiotic tolerance; (b) KF707 planktonic cells and biofilms, are almost equally susceptible to killing by metal cations and oxyanions, and (c) biofilm-tolerance to TeO3 2− and SeO3 2− is not linked to metalloid reduction; this means that KF707 planktonic cells and biofilms differ in their physiology and strategy to counteract metalloid toxicity.

Surfactant-aided soil washing is often proposed for the restoration of aged organic pollutant-contaminated soils. As many of commercial surfactants have been found to be toxic and recalcitrant, the opportunity to use in this process cheap, non-toxic, and biodegradable pollutant-mobilizing agents, such as deoxycholic acid (DA), bovine bile (BB), and the residue resulting from DA extraction from BB (BBR), was studied in this work. A soil historically contaminated by chlorinated anilines and benzenes, thiophenes, and several polycyclic aromatic hydrocarbons was suspended at 15% w/v and washed in water or water amended at 1.0% (w/v) with DA, BB, BBR, or Triton X-100 (TX). The resulting effluents were supplemented with nutrients and subjected to aerobic bioremediation. The biogenic agents enhanced the water pollutant elution potential by 230/440%. TX enhanced the same parameter by about 540%; however, it mediated a lower depletion of the initial soil ecotoxicity and a more extensive mobilization of soil constituents with respect to the biogenic agents. Furthermore, TX adversely affected the biotreatability of resulting effluents, by adversely affecting the growth of cultivable bacterial biomass and the structure of eubacterial community of the effluent. On the contrary, the biogenic agents, and in particular DA and BB, enhanced the effluents bioremediation, by sustaining the growth and increasing the complexity of the effluent eubacterial communities. Thus, DA and BB are very promising additives for an effective and environmental friendly soil washing treatment of aged (chloro)organics contaminated soils. © 2005 Wiley Periodicals, Inc.

The aerobic cometabolic biodegradation of a mixture of chlorinated aliphatic hydrocarbons (CAHs) including vinyl chloride (VC), cis - and trans -1,2-dichloroethylene ( cis -DCE, trans -DCE), trichloroethylene (TCE), 1,1,2-trichloroethane (1,1,2-TCA) and 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA) was investigated at both 25 and 17 °C by means of bioaugmented and non-bioaugmented sediment-groundwater slurry microcosm tests. The goals of the study were (i) to study the long-term aerobic biodegradation of a CAH mixture including a high-chlorinated solvent (1,1,2,2-TeCA) generally considered non-biodegradable in aerobic conditions; (ii) to investigate the efficacy of bioaugmentation with two types of internal inocula obtained from the indigenous biomass of the studied site; (iii) to identify the CAH-degrading bacteria. VC, methane and propane were utilized as growth substrates. The non-bioaugmented microcosms were characterized, at 25 °C, by an average 18-day lag-time for the direct metabolism of VC (accompanied by the cometabolism of cis - and trans -DCE) and by long lag-times (36–264 days) for the onset of methane or propane utilization (associated with the cometabolism of the remaining CAHs). In the inoculated microcosms the lag-phases for the onset of growth substrate utilization and CAH cometabolism were significantly shorter (0–15 days at 25 °C). Biodegradation of the 6-CAH mixture was successfully continued for up to 410 days. The low-chlorinated solvents were characterized by higher depletion rates. The composition of the microbial consortium of a propane-utilizing microcosm was determined by 16s rDNA sequencing and phylotype analysis. To the best of our knowledge, this is the first study that documents the long-term aerobic biodegradation of 1,1,2,2-TeCA.

The ability of a Rhodococcus aetherovorans strain, BCP1, to grow on butane and to degrade chloroform in the 0-633 microM range (0-75.5 mg l(-1)) via aerobic cometabolism was investigated by means of resting-cell assays. BCP1 degraded chloroform with a complete mineralization of the organic Cl. The resulting butane and chloroform maximum specific degradation rates were equal to 118 and 22 micromol mg(protein)(-1)day(-1), respectively. Butane inhibition on chloroform degradation was satisfactorily interpreted by means of a model of competitive inhibition, with an inhibition constant equal to 38 % of the estimated butane half-saturation constant, whereas chloroform (at 11 microM) did not inhibit butane utilization. Acetylene (1,720 microM) induced an almost complete inactivation of the degradation of both butane and chloroform, indicating that the studied cometabolic process is mediated by a monooxygenase enzyme. BCP1 proved capable of degrading vinyl chloride and 1,1,2-trichloroethane, but not 1,2-trans-dichloroethylene. BCP1 could grow on the intermediates of the most common butane metabolic pathways and on the aliphatic hydrocarbons from ethane to n-heptane. After growth on n-hexane, it was able to deplete chloroform (13 microM) with a degradation rate higher than that obtained, at the same chloroform concentration, after growth on butane.

The focus of this microcosm study was to monitor the performances of 17 butane-utilizing microcosms during a long-term (100-250 days) aerobic cometabolic depletion of chloroform (CF). The depletion of the contaminant began after a lag-time variable between 0 and 23 days. All microcosms quickly reached a pseudo steady-state condition, in terms of biomass concentration (with an average of 9.3 x 106 CFU ml(-1)), chloroform depletion rate (5 micromol l(-1) d(-1)) and butane utilization rate (730 micromol l(-1) d(-1)). After about 100 days of CF depletion, a sudden 5- to 7-fold increase of the chloroform rate was observed in two microcosms, where the highest amount of contaminant had been depleted. In one of these high-performing microcosms, an experiment of chloroform depletion in the absence of butane resulted in the depletion of a surprisingly high amount of contaminant (765 micromolCF kg(-1) dry soil in 2 months) and in a marked selection of a single bacterial strain. Bioaugmentation assays conducted with the biomass selected in this microcosm and with a pure culture of the selected strain immediately resulted in very high chloroform depletion rates. Preliminary results of a study conducted with resting cells of the selected strain indicated that it can degrade chloroform concentrations up to 119 microM (14.2 mg l(-1)) without any sign of substrate toxicity, and that it is able to transform vinyl chloride and 1,1,2-trichloroethane.

In this study, T-RFLP analysis was used to determine the structure and spatial distribution of the indigenous bacterial community of an actual-site PCB-contaminated soil treated in aerobic packed-bed loop reactors (PBLRs) in the absence or in the presence of a mixture of randomly methylated beta-cyclodextrins (RAMEB) at 0.5 or 1% w/w. RAMEB was found to significantly enhance the aerobic bioremediation of soil with effects that increased proportionally with the concentration at which it was applied. At the end of treatment (180 days), T-RFLP analysis of the soil samples collected from the top and bottom regions of the PBLRs showed a series of 50 single T-RFs. Remarkably, the number of T-RFs was significantly lower (13-22) in samples collected from different sections of the RAMEB-amended bioreactors with respect to equivalent samples collected from the RAMEB-free reactor. Cluster analysis based on the presence or the absence of T-RFs peaks revealed high similarity, inside each reactor, between the top and bottom parts of its soil bed. Soil samples collected at the top and bottom regions of the two bioreactors amended with RAMEB, clustered together while the equivalent samples of the bioreactor without RAMEB formed a separate cluster which was distantly related to the soil samples obtained from the parallel amended bioreactor. Notably, T-RFLP analyses combined with extensive sequencing of 16S rDNA allowed us to tentatively allocate a series of bacterial species corresponding to specific peaks of the T-RFLP profiles and to determine their phylogenetic affiliation.