Lactobacilli extracellular vesicles: potential postbiotics to support the vaginal microbiota homeostasis.

BACKGROUND: Lactobacillus species dominate the vaginal microflora performing a first-line defense against vaginal infections. Extracellular vesicles (EVs) released by lactobacilli are considered mediators of their beneficial effects affecting cellular communication, homeostasis, microbial balance, and host immune system pathways. Up to now, very little is known about the role played by Lactobacillus EVs in the vaginal microenvironment, and mechanisms of action remain poorly understood. RESULTS: Here, we hypothesized that EVs can mediate lactobacilli beneficial effects to the host by modulating the vaginal microbiota colonization. We recovered and characterized EVs produced by two vaginal strains, namely Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12. EVs were isolated by ultracentrifugation and physically characterized by Nanoparticle Tracking Analysis (NTA) and Dynamic Light Scattering (DLS). EVs protein and nucleic acids (DNA and RNA) content was also evaluated. We explored the role of EVs on bacterial adhesion and colonization, using a cervical cell line (HeLa) as an in vitro model. Specifically, we evaluated the effect of EVs on the adhesion of both vaginal beneficial lactobacilli and opportunistic pathogens (i.e., Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, and Enterococcus faecalis). We demonstrated that EVs from L. crispatus BC5 and L. gasseri BC12 significantly enhanced the cellular adhesion of all tested lactobacilli, reaching the maximum stimulation effect on strains belonging to L. crispatus species (335% and 269% of average adhesion, respectively). At the same time, EVs reduced the adhesion of all tested pathogens, being EVs from L. gasseri BC12 the most efficient. CONCLUSIONS: Our observations suggest for the first time that EVs released by symbiotic Lactobacillus strains favor healthy vaginal homeostasis by supporting the colonization of beneficial species and preventing pathogens attachment. This study reinforces the concept of EVs as valid postbiotics and opens the perspective of developing postbiotics from vaginal strains to maintain microbiota homeostasis and promote women's health.

Some facts about the respiratory enzymes of Pseudomonas pseudoalcaligenes KF707 recently renamed as Pseudomonas furukawaii sp. nov., type strain KF707.

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 ß-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.

Characterization of Pleurotus ostreatus biofilms by using the calgary biofilm device.

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.

Genome sequence of the polychlorinated-biphenyl degrader Pseudomonas pseudoalcaligenes KF707.

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.

Molecular characterization of microbial communities in a peat-rich aquifer system contaminated with chlorinated aliphatic compounds.

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 inferred by 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.

Insight into phenotypic and genotypic differences between vaginal Lactobacillus crispatus BC5 and Lactobacillus gasseri BC12 to unravel nutritional and stress factors influencing their metabolic activity.

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.

Time-Dependent Changes in Morphostructural Properties and Relative Abundances of Contributors in Pleurotus ostreatus/Pseudomonas alcaliphila Mixed Biofilms.

Pleurotus ostreatus dual 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 of P. 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 using Pseudomonas alcaliphila 34 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.

Liposomes containing biosurfactants isolated from Lactobacillus gasseri exert antibiofilm activity against methicillin resistant Staphylococcus aureus strains.

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.

Novel mixed vesicles containing lactobacilli biosurfactant for vaginal delivery of an anti-Candida agent.

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.

Aerobic Growth of Rhodococcus aetherivorans BCP1 Using Selected Naphthenic Acids as the Sole Carbon and Energy Sources.

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.

Biphenyl Modulates the Expression and Function of Respiratory Oxidases in the Polychlorinated-Biphenyls Degrader Pseudomonas pseudoalcaligenes KF707.

Pseudomonas pseudoalcaligenes KF707 is a soil bacterium which is known for its capacity to aerobically degrade harmful organic compounds such as polychlorinated biphenyls (PCBs) using biphenyl as co-metabolite. Here we provide the first genetic and functional analysis of the KF707 respiratory terminal oxidases in cells grown with two different carbon sources: glucose and biphenyl. We identified five terminal oxidases in KF707: two c(c)aa3 type oxidases (Caa3 and Ccaa3), two cbb3 type oxidases (Cbb31 and Cbb32), and one bd type cyanide-insensitive quinol oxidase (CIO). While the activity and expression of both Cbb31 and Cbb32 oxidases was prevalent in glucose grown cells as compared to the other oxidases, the activity and expression of the Caa3 oxidase increased considerably only when biphenyl was used as carbon source in contrast to the Cbb32 oxidase which was repressed. Further, the respiratory activity and expression of CIO was up-regulated in a Cbb31 deletion strain as compared to W.T. whereas the CIO up-regulation was not present in Cbb32 and C(c)aa3 deletion mutants. These results, together, reveal that both function and expression of cbb3 and caa3 type oxidases in KF707 are modulated by biphenyl which is the co-metabolite needed for the activation of the PCBs-degradation pathway.

Long-term aerobic cometabolism of a chlorinated solvent mixture by vinyl chloride-, methane- and propane-utilizing biomasses.

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 degrees 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 degrees 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 degrees 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.

Diversity of Methane-Oxidizing Bacteria in Soils from "Hot Lands of Medolla" (Italy) Featured by Anomalous High-Temperatures and Biogenic CO2 Emission.

"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.

The Role of cheA Genes in Swarming and Swimming Motility of Pseudomonas pseudoalcaligenes KF707.

A genome analysis of Pseudomonas pseudoalcaligenes KF707, a PCBs degrader and metal-resistant soil microorganism, revealed the presence of two novel gene clusters named che2 and che3, which were predicted to be involved in chemotaxis-like pathways, in addition to a che1 gene cluster. We herein report that the histidine kinase coding genes, cheA2 and cheA3, have no role in swimming or chemotaxis in P. pseudoalcaligenes KF707, in contrast to cheA1. However, the cheA1 and cheA2 genes were both necessary for cell swarming, whereas the cheA3 gene product had a negative effect on the optimal swarming phenotype of KF707 cells.

T-RFLP analysis of bacterial communities in cyclodextrin-amended bioreactors developed for biodegradation of polychlorinated biphenyls.

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.

Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes.

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.

Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance.

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

Use of potassium tellurite for testing the survival and viability of Pseudomonas pseudoalcaligenes KF707 in soil microcosms contaminated with polychlorinated biphenyls.

This study shows that the oxyanion tellurite TeO3(2-) can be used as a tool to detect and quantify the release in soil microcosms of Pseudomonas pseudoalcaligenes KF707, a strain spontaneously resistant to tellurite with a minimal inhibitory concentration (MIC) of 150 microg ml(-1). KF707 cells which carry the genes for degradation of a wide range of polychlorinated biphenyl congeners (PCBs) were used for inoculation of laboratory microcosms prepared with two different PCB-contaminated soils (Ci/s and Di/s) in the presence or absence of biphenyl as carbon source. In all microcosms supplemented with biphenyl, significant survival of strain KF707 was noted over a time period of 35 days; conversely, in microcosms containing Ci/s soil without biphenyl addition a rapid decrease in KF707 inoculated cells was observed. By comparing the number of inoculated KF707 cells with the number of indigenous bacteria growing on biphenyl (IBGB) of both Ci/s and Di/s microcosms, it could be concluded that the KF707/IBGB ratio is a relevant parameter in determining the fate of the added strain. The efficacy of potassium tellurite as a selective marker to monitor strain KF707 in laboratory microcosms was confirmed by ARDRA analyses of the 16S rDNA, while the isolated indigenous bacteria growing on biphenyl were identified as members of three different species of the genus Pseudomonas. We also report that in microcosms inoculated with KF707 cells in the absence of biphenyl, only low chlorinated biphenyls were degraded.

Application of pyrolysis-gas chromatography-mass spectrometry and multivariate analysis to study bacteria and fungi in biofilms used for bioremediation.

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.

A kinetic study of biohydrogen production from glucose, molasses and cheese whey by suspended and attached cells of Thermotoga neapolitana.

Batch tests of H2 production from glucose, molasses and cheese whey by suspended and immobilized cells of Thermotoga neapolitana were conducted to develop a kinetic model of the process. H2 production was inhibited by neither H2 (up to 0.7 mg L(-1)) nor O2 (up to 0.2 mg L(-1)). The H2 specific rates obtained at different substrate concentrations were successfully interpolated with Andrew's inhibition model. With glucose and molasses, biofilms performed better than suspended cells. The suspended-cell process was successfully scaled-up to a 19-L bioreactor. Assays co-fed with molasses and cheese whey led to higher H2 productivities and H2/substrate yields than the single-substrate tests. The simulation of the suspended-cell continuous-flow process indicated the potential attainment of H2 productivities higher than those of the batch tests (up to 3.6 mmol H2 h(-1) L(-1) for molasses and 0.67 mmol H2 h(-1) L(-1) for cheese whey) and allowed the identification of the optimal dilution rate.