Characterization of trehalolipid biosurfactant produced by the novel marine strain Rhodococcus sp. SP1d and its potential for environmental applications.

BACKGROUND: Biosurfactants are surface-active compounds with environmental and industrial applications. These molecules show higher biocompatibility, stability and efficiency compared to synthetic surfactants. On the other hand, biosurfactants are not cost-competitive to their chemical counterparts. Cost effective technology such as the use of low-cost substrates is a promising approach aimed at reducing the production cost. This study aimed to evaluate the biosurfactant production and activity by the novel strain Rhodococcus sp. SP1d by using different growth substrates. Therefore, to exploit the biosurfactant synthesized by SP1d for environmental applications, the effect of this compound on the bacteria biofilm formation was evaluated. Eventually, for a possible bioremediation application, the biosurfactant properties and its chemical characteristics were investigated using diesel as source of carbon. RESULTS: Rhodococcus sp. SP1d evidence the highest similarity to Rhodococcus globerulus DSM 43954T and the ability to biosynthesize surfactants using a wide range of substrates such as exhausted vegetable oil, mineral oil, butter, n-hexadecane, and diesel. The maximum production of crude biosurfactant after 10 days of incubation was reached on n-hexadecane and diesel with a final yield of 2.38 ± 0.51 and 1.86 ± 0.31 g L- 1 respectively. Biosurfactants produced by SP1d enhanced the biofilm production of P. protegens MP12. Moreover, the results showed the ability of SP1d to produce biosurfactants on diesel even when grown at 10 and 18 °C. The biosurfactant activity was maintained over a wide range of NaCl concentration, pH, and temperature. A concentration of 1000 mg L- 1 of the crude biosurfactant showed an emulsification activity of 55% towards both xylene and olive oil and a reduction of 25.0 mN m- 1 of surface tension of water. Eventually, nuclear magnetic resonance spectroscopy indicated that the biosurfactant is formed by trehalolipids. CONCLUSIONS: The use of low-cost substrates such as exhausted oils and waste butter reduce both the costs of biosurfactant synthesis and the environmental pollution due to the inappropriate disposal of these residues. High production yields, stability and emulsification properties using diesel and n-hexadecane as substrates, make the biosurfactant produced by SP1d a sustainable biocompound for bioremediation purpose. Eventually, the purified biosurfactant improved the biofilm formation of the fungal antagonistic strain P. protegens MP12, and thus seem to be exploitable to increase the adherence and colonization of plant surfaces by this antagonistic strain and possibly enhance antifungal activity.

Influence of Bacterial Physiology on Processing of Selenite, Biogenesis of Nanomaterials and Their Thermodynamic Stability.

We explored how Ochrobactrum sp. MPV1 can convert up to 2.5 mM selenite within 120 h, surviving the challenge posed by high oxyanion concentrations. The data show that thiol-based biotic chemical reaction(s) occur upon bacterial exposure to low selenite concentrations, whereas enzymatic systems account for oxyanion removal when 2 mM oxyanion is exceeded. The selenite bioprocessing produces selenium nanomaterials, whose size and morphology depend on the bacterial physiology. Selenium nanoparticles were always produced by MPV1 cells, featuring an average diameter ranging between 90 and 140 nm, which we conclude constitutes the thermodynamic stability range for these nanostructures. Alternatively, selenium nanorods were observed for bacterial cells exposed to high selenite concentration or under controlled metabolism. Biogenic nanomaterials were enclosed by an organic material in part composed of amphiphilic biomolecules, which could form nanosized structures independently. Bacterial physiology influences the surface charge characterizing the organic material, suggesting its diverse biomolecular composition and its involvement in the tuning of the nanomaterial morphology. Finally, the organic material is in thermodynamic equilibrium with nanomaterials and responsible for their electrosteric stabilization, as changes in the temperature slightly influence the stability of biogenic compared to chemogenic nanomaterials.

Ochrobactrum sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles.

BACKGROUND: Bacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles-both inside and outside the cells-characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences. RESULTS: In the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO32-) and tellurite (TeO32-) to their respective elemental forms (Se0 and Te0) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO32- and 0.5 mM TeO32- to the corresponding Se0 and Te0 in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO32- and TeO32- bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO32- bioreduction, while TeO32- bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs. CONCLUSIONS: In conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.

A comparison of the response of two Burkholderia fungorum strains grown as planktonic cells versus biofilm to dibenzothiophene and select polycyclic aromatic hydrocarbons.

In natural environments, bacteria often exist in close association with surfaces and interfaces by establishing biofilms. Here, we report on the ability of Burkholderia fungorum strains DBT1 and 95 to survive in high concentrations of hydrocarbons, and we compare their growth as a biofilm vs. planktonic cells. The 2 compounds tested were dibenzothiophene (DBT) and a mixture of naphthalene, phenanthrene, and pyrene (5:2:1) as representative compounds of thiophenes and polycyclic aromatic hydrocarbons (PAHs), respectively. The results showed that both strains were able to degrade DBT and to survive in the presence of up to a 2000 mg·L-1 concentration of this compound both as a biofilm and as free-living cells. Moreover, B. fungorum DBT1 showed reduced tolerance towards the mixed PAHs (2000 mg·L-1 naphthalene, 800 mg·L-1 phenanthrene, and 400 mg·L-1 pyrene) both as a biofilm and as free-living cells. Conversely, biofilms of B. fungorum 95 enhanced resistance against these toxic compounds compared with planktonic cells (P < 0.05). Visual observation through confocal laser scanning microscopy showed that exposure of biofilms to DBT and PAHs altered their structure: high concentrations of DBT triggered an aggregation of biofilm cells. These findings provide new perspectives on the effectiveness of using DBT-degrading bacterial strains in bioremediation of hydrocarbon-contaminated sites.

Bioremediation of diesel contamination at an underground storage tank site: a spatial analysis of the microbial community.

The present study reports on a real case of contamination due to the chronic leakage of diesel fuel from an underground tank at a dismissed service station. Speciation of the microbial community according to both lateral and vertical gradients from the origin of the contaminant release was analyzed by means of the PCR-DGGE technique. Moreover, the effects of a landfarming treatment on both the microbial community structure and the abatement of contamination were analyzed. The concentration of total petrol hydrocarbons (TPHs) decreased along the horizontal gradient (from 7042.2 ± 521.9 to 112.2 ± 24.3 mg kg(-1)), while increased downwards from the position of the tank (from 502.6 ± 43.7 to 4972.5 ± 275.3 mg kg(-1)). PCR-DGGE analyses and further statistical treatment of the data indicated a correlation between structure of the bacterial communities and amount of diesel fuel contamination. On the other hand, level of contamination, soil texture and depth were shown to affect the fungal community. Chloroflexi and Ascomycota were the most abundant microbes ascertained through culture-independent procedures. Landfarming promoted 91.6 % reduction of TPHs in 75 days. Furthermore, PCR-DGGE analyses evidenced that both bacterial and fungal communities of the treated soil were restored to the pristine conditions of uncontaminated topsoil. The present study demonstrated that bacterial and fungal communities were affected differently by soil factors such as level of hydrocarbon contamination as well as soil depth and texture. This report shows that a well-planned landfarming treatment can drive the restoration of the soil in terms of both abatement of the contaminants and resilience of the microbial community structure.

Bioaugmentation and biostimulation as strategies for the bioremediation of a burned woodland soil contaminated by toxic hydrocarbons: a comparative study.

In this work, the natural attenuation strategy (no soil amendments done) was compared with two different bioremediation approaches, namely bioaugmentation through soil inoculation with a suspension of Trichoderma sp. mycelium and biostimulation by soil addition with a microbial growth promoting formulation, in order to verify the effectiveness of these methods in terms of degradation efficiency towards toxic hydrocarbons, with particular attention to the high molecular weight (HMW) fraction, in a forest area impacted by recent wildfire in Northern Italy. The area under investigation, divided into three parcels, was monitored to figure out the dynamics of decay in soil concentration of C12₋40 hydrocarbons (including isoalkanes, cycloalkanes, alkyl-benzenes and alkyl-naphthalenes besides PAHs) and low molecular weight (LMW) PAHs, following the adoption of the foregoing different remediation strategies. Soil hydrocarbonoclastic potential was even checked by characterizing the autochthonous microbial cenoses. Field experiments proved that the best performance in the abatement of HMW hydrocarbons was reached 60 days after soil treatment through the biostimulation protocol, when about 70% of the initial concentration of HMW hydrocarbons was depleted. Within the same time, about 55% degradation was obtained with the bioaugmentation protocol, whilst natural attenuation allowed only a 45% removal of the starting C12-40 hydrocarbon fraction. Therefore, biostimulation seems to significantly reduce the time required for the remediation, most likely because of the enhancement of microbial degradation through the improvement of nutrient balance in the burned soil.

Delayed formation of zero-valent selenium nanoparticles by Bacillus mycoides SeITE01 as a consequence of selenite reduction under aerobic conditions.

BACKGROUND: Selenite (SeO32-) oxyanion shows severe toxicity to biota. Different bacterial strains exist that are capable of reducing SeO32- to non-toxic elemental selenium (Se0), with the formation of Se nanoparticles (SeNPs). These SeNPs might be exploited for technological applications due to their physico-chemical and biological characteristics. The present paper discusses the reduction of selenite to SeNPs by a strain of Bacillus sp., SeITE01, isolated from the rhizosphere of the Se-hyperaccumulator legume Astragalus bisulcatus. RESULTS: Use of 16S rRNA and GyrB gene sequence analysis positioned SeITE01 phylogenetically close to B. mycoides. On agarized medium, this strain showed rhizoid growth whilst, in liquid cultures, it was capable of reducing 0.5 and 2.0 mM SeO32- within 12 and 24 hours, respectively. The resultant Se0 aggregated to form nanoparticles and the amount of Se0 measured was equivalent to the amount of selenium originally added as selenite to the growth medium. A delay of more than 24 hours was observed between the depletion of SeO32 and the detection of SeNPs. Nearly spherical-shaped SeNPs were mostly found in the extracellular environment whilst rarely in the cytoplasmic compartment. Size of SeNPs ranged from 50 to 400 nm in diameter, with dimensions greatly influenced by the incubation times. Different SeITE01 protein fractions were assayed for SeO32- reductase capability, revealing that enzymatic activity was mainly associated with the membrane fraction. Reduction of SeO32- was also detected in the supernatant of bacterial cultures upon NADH addition. CONCLUSIONS: The selenite reducing bacterial strain SeITE01 was attributed to the species Bacillus mycoides on the basis of phenotypic and molecular traits. Under aerobic conditions, the formation of SeNPs were observed both extracellularly or intracellularly. Possible mechanisms of Se0 precipitation and SeNPs assembly are suggested. SeO32- is proposed to be enzymatically reduced to Se0 through redox reactions by proteins released from bacterial cells. Sulfhydryl groups on peptides excreted outside the cells may also react directly with selenite. Furthermore, membrane reductases and the intracellular synthesis of low molecular weight thiols such as bacillithiols may also play a role in SeO32- reduction. Formation of SeNPs seems to be the result of an Ostwald ripening mechanism.

Identification of aldolase and ferredoxin reductase within the dbt operon of Burkholderia fungorum DBT1.

Burkholderia fungorum DBT1, first isolated from settling particulate matter of an oil refinery wastewater, is a bacterial strain which has been shown capable of utilizing several polycyclic aromatic hydrocarbons (PAHs) including dibenzothiophene (DBT). In particular, this microbe is able to efficiently degrade DBT through the Kodama pathway. Previous investigations have lead to the identification of six genes, on a total of eight, required for DBT degradation. In the present study, a combined experimental/computational approach was adopted to identify and in silico characterize the two missing genes, namely a ferredoxin reductase and a hydratase-aldolase. Thus, the finding of all enzymatic components of the Kodama pathway in B. fungorum DBT1 makes this bacterial strain amenable for possible exploitation in soil bioremediation protocols.

Biomolecular composition of capping layer and stability of biogenic selenium nanoparticles synthesized by five bacterial species.

Biogenic metal/metalloid nanoparticles of microbial origin retain a functional biomolecular capping layer that confers structural stability. Little is known about the composition of such capping material. In this study, selenium nanoparticles (SeNPs) synthesized by five different bacterial strains underwent comparative analysis with newly proposed protocols for quantifying the concentration of carbohydrates, proteins and lipids present in capping layers. SeNPs were therefore treated with two different detergents to remove portions of the surrounding caps in order to assess the resulting effects. Capping material quantification was carried out along with the measure of parameters such as hydrodynamic diameter, polydispersity and surface charge. SeNPs from the five strains showed differences in their distinct biomolecule ratios. On the other hand, structural changes in the nanoparticles induced by detergents did not correlate with the amounts of capping matrix removed. Thus, the present investigation suggests a hypothesis to describe capping layer composition of the bacterial SeNPs: some biomolecules are bound more strongly than others to the core metalloid matrix, so that the diverse capping layer components differentially contribute to the overall structural characteristics of the nanoparticles. Furthermore, the application of the approach here in combining quantification of cap-associated biomolecules with the measurement of structural integrity-related parameters can give the biogenic nanomaterial field useful information to construct a data bank on biogenically synthesized nanostructures.

Untargeted Metabolomics Investigation on Selenite Reduction to Elemental Selenium by Bacillus mycoides SeITE01.

Bacillus mycoides SeITE01 is an environmental isolate that transforms the oxyanion selenite ( SeO 3 2 - ) into the less bioavailable elemental selenium (Se0) forming biogenic selenium nanoparticles (Bio-SeNPs). In the present study, the reduction of sodium selenite (Na2SeO3) by SeITE01 strain and the effect of SeO 3 2 - exposure on the bacterial cells was examined through untargeted metabolomics. A time-course approach was used to monitor both cell pellet and cell free spent medium (referred as intracellular and extracellular, respectively) metabolites in SeITE01 cells treated or not with SeO 3 2 - . The results show substantial biochemical changes in SeITE01 cells when exposed to SeO 3 2 - . The initial uptake of SeO 3 2 - by SeITE01 cells (3h after inoculation) shows both an increase in intracellular levels of 4-hydroxybenzoate and indole-3-acetic acid, and an extracellular accumulation of guanosine, which are metabolites involved in general stress response adapting strategies. Proactive and defensive mechanisms against SeO 3 2 - are observed between the end of lag (12h) and beginning of exponential (18h) phases. Glutathione and N-acetyl-L-cysteine are thiol compounds that would be mainly involved in Painter-type reaction for the reduction and detoxification of SeO 3 2 - to Se0. In these growth stages, thiol metabolites perform a dual role, both acting against the toxic and harmful presence of the oxyanion and as substrate or reducing sources to scavenge ROS production. Moreover, detection of the amino acids L-threonine and ornithine suggests changes in membrane lipids. Starting from stationary phase (24 and 48h), metabolites related to the formation and release of SeNPs in the extracellular environment begin to be observed. 5-hydroxyindole acetate, D-[+]-glucosamine, 4-methyl-2-oxo pentanoic acid, and ethanolamine phosphate may represent signaling strategies following SeNPs release from the cytoplasmic compartment, with consequent damage to SeITE01 cell membranes. This is also accompanied by intracellular accumulation of trans-4-hydroxyproline and L-proline, which likely represent osmoprotectant activity. The identification of these metabolites suggests the activation of signaling strategies that would protect the bacterial cells from SeO 3 2 - toxicity while it is converting into SeNPs.

On the Ability of Perfluorohexane Sulfonate (PFHxS) Bioaccumulation by Two Pseudomonas sp. Strains Isolated from PFAS-Contaminated Environmental Matrices.

PFASs (perfluoroalkyl and polyfluoroalkyl substances) are highly fluorinated, aliphatic, synthetic compounds with high thermal and chemical stability as well as unique amphiphilic properties which make them ingredients in a range of industrial processes. PFASs have attracted consideration due to their persistence, toxicity and bioaccumulation tendency in the environment. Recently, attention has begun to be addressed to shorter-chain PFASs, such as perfluorohexane sulfonate [PFHxS], apparently less toxic to and more easily eliminated from lab animals. However, short-chain PFASs represent end-products from the transformation of fluorotelomers whose biotic breakdown reactions have not been identified to date. This means that such emergent pollutants will tend to accumulate and persist in ecosystems. Since we are just learning about the interaction between short-chain PFASs and microorganisms, this study reports on the response to PFHxS of two Pseudomonas sp. strains isolated from environmental matrices contaminated by PFASs. The PFHxS bioaccumulation potential of these strains was unveiled by exploiting different physiological conditions as either axenic or mixed cultures under alkanothrofic settings. Moreover, electron microscopy revealed nonorthodox features of the bacterial cells, as a consequence of the stress caused by both organic solvents and PFHxS in the culturing substrate.

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms.

Arabidopsis halleri has the rare ability to colonize heavy metal-polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant-microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal-contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere-derived microorganisms. The results of this analysis emphasized the role of plant-microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2-DE and MS, indicated a general upregulation of photosynthesis-related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy-demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross-talk between heavy metal signaling and defense signaling.

Pseudomonas protegens MP12: A plant growth-promoting endophytic bacterium with broad-spectrum antifungal activity against grapevine phytopathogens.

Pseudomonas sp. MP12 was isolated from a soil sample collected in a typical warm-temperate deciduous forest near Brescia, Northern Italy. Phylogenetic analysis identified the species as Pseudomonas protegens. We evidenced in this strain the presence of the genes phlD, pltB and prnC responsible for the synthesis of the antifungal compounds 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin and pyrrolnitrin, respectively. P. protegens MP12 was also shown to produce siderophores and ammonia, yielded positive results with the indole-3-acetic acid test, and was capable of phosphate solubilization. Moreover, P. protegens MP12 exhibited inhibitory effects on in vitro mycelial growth of prominent grapevine (Vitis vinifera) phytopathogens such as Botrytis cinerea, Alternaria alternata, Aspergillus niger, Penicillium expansum and Neofusicoccum parvum. The strain showed activity even against Phaeomoniella chlamydospora and Phaeoacremonium aleophilum, which cause the devastating tracheomycosis/esca disease of grapevine trunks for which no efficacious control methods have been demonstrated so far. Furthermore, the MP12 strain manifested in vivo antifungal activity against B. cinerea on grapevine leaves. Culture-dependent and culture-independent analysis revealed the ability of P. protegens MP12 to efficiently and permanently colonize inner grapevine tissues. These results suggest that P. protegens MP12 could be worth of exploitation as an antifungal biocontrol agent for applications in viticulture.

Physical-Chemical Properties of Biogenic Selenium Nanostructures Produced by Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1.

Stenotrophomonas maltophilia SeITE02 and Ochrobactrum sp. MPV1 were isolated from the rhizosphere soil of the selenium-hyperaccumulator legume Astragalus bisulcatus and waste material from a dumping site for roasted pyrites, respectively. Here, these bacterial strains were studied as cell factories to generate selenium-nanostructures (SeNS) under metabolically controlled growth conditions. Thus, a defined medium (DM) containing either glucose or pyruvate as carbon and energy source along with selenite () was tested to evaluate bacterial growth, oxyanion bioconversion and changes occurring in SeNS features with respect to those generated by these strains grown on rich media. Transmission electron microscopy (TEM) images show extra- or intra-cellular emergence of SeNS in SeITE02 or MPV1 respectively, revealing the presence of two distinct biological routes of SeNS biogenesis. Indeed, the stress exerted by upon SeITE02 cells triggered the production of membrane vesicles (MVs), which surrounded Se-nanoparticles (SeNPsSeITE02-G_e and SeNPsSeITE02-P_e with average diameter of 179 ± 56 and 208 ± 60 nm, respectively), as highlighted by TEM and scanning electron microscopy (SEM), strongly suggesting that MVs might play a crucial role in the excreting mechanism of the SeNPs in the extracellular environment. On the other hand, MPV1 strain biosynthesized intracellular inclusions likely containing hydrophobic storage compounds and SeNPs (123 ± 32 nm) under pyruvate conditioning, while the growth on glucose as the only source of carbon and energy led to the production of a mixed population of intracellular SeNPs (118 ± 36 nm) and nanorods (SeNRs; average length of 324 ± 89). SEM, fluorescence spectroscopy, and confocal laser scanning microscopy (CLSM) revealed that the biogenic SeNS were enclosed in an organic material containing proteins and amphiphilic molecules, possibly responsible for the high thermodynamic stability of these nanomaterials. Finally, the biogenic SeNS extracts were photoluminescent upon excitation ranging from 380 to 530 nm, whose degree of fluorescence emission (λem = 416-640 nm) was comparable to that from chemically synthesized SeNPs with L-cysteine (L-cys SeNPs). This study offers novel insights into the formation, localization, and release of biogenic SeNS generated by two different Gram-negative bacterial strains under aerobic and metabolically controlled growth conditions. The work strengthens the possibility of using these bacterial isolates as eco-friendly biocatalysts to produce high quality SeNS targeted to possible biomedical applications and other biotechnological purposes.

Biogenic selenium nanoparticles synthesized by Stenotrophomonas maltophilia SeITE02 loose antibacterial and antibiofilm efficacy as a result of the progressive alteration of their organic coating layer.

Increasing emergence of drug-resistant microorganisms poses a great concern to clinicians; thus, new active products are urgently required to treat a number of infectious disease cases. Different metallic and metalloid nanoparticles have so far been reported as possessing antimicrobial properties and proposed as a possible alternative therapy against resistant pathogenic microorganisms. In this study, selenium nanoparticles (SeNPs) synthesized by the environmental bacterial isolate Stenotrophomonas maltophilia SeITE02 were shown to exert a clear antimicrobial and antibiofilm activity against different pathogenic bacteria, either reference strains or clinical isolates. Antimicrobial and antibiofilm capacity seems to be strictly linked to the organic cap surrounding biogenic nanoparticles, although the actual role played by this coating layer in the biocidal action remains still undefined. Nevertheless, evidence has been gained that the progressive loss in protein and carbohydrate content of the organic cap determines a decrease in nanoparticle stability. This leads to an alteration of size and electrical properties of SeNPs along with a gradual attenuation of their antibacterial efficacy. Denaturation of the coating layer was proved even to have a negative effect on the antibiofilm activity of these nanoparticles. The pronounced antimicrobial efficacy of biogenic SeNPs compared to the denatured ones can - in first instance - be associated with their smaller dimensions. This study showed that the native organic coating layer of biogenic SeNPs functions in avoiding aggregation and maintaining electrostatic stability of the nanoparticles, thus allowing them to maintain efficient antimicrobial and antibiofilm capabilities.

Effect of the length of the cycle on biodegradable polymer production and microbial community selection in a sequencing batch reactor.

The effect of the length of the cycle on the enrichment and selection of mixed cultures in sequencing batch reactors (SBRs) has been studied, with the aim of biodegradable polymers (namely, polyhydroxyalkanoates (PHAs)) production from organic wastes. At a fixed feed concentration (20 gCOD/L) and organic loading rate (20 gCOD/L/day), the SBR was operated at different lengths of the cycle, in the range 1-8 h. Process performance was measured by considering the rates and yields of polymer storage and of the competing phenomenon of growth. The selected biomass was enriched with microorganisms that were able to store PHAs at high rates and yields only when the length of the cycle was 2 or 4 h, even though in these conditions the process was unstable. On the other hand, when the length of the cycle was 1 or 8 h, the dynamic response of the selected microorganisms was dominated by growth. The best process performance was characterized by storage rates in the range 500-600 mgCOD/gCOD/h and storage yields of 0.45-0.55 COD/COD. The corresponding productivity of the process was in the range 0.25-0.30 gPHA/L/h, the highest values obtained until now for mixed cultures. The microbial composition of the selected biomasses was analyzed through denaturing gradient gel electrophoresis (DGGE) and reverse-transcriptase denaturing gradient gel electrophoresis (RT-DGGE). The instability of the runs characterized by high storage rate was associated with a higher microbial heterogeneity compared to the runs with a stable growth response.

Insights into selenite reduction and biogenesis of elemental selenium nanoparticles by two environmental isolates of Burkholderia fungorum.

Microorganisms capable of transforming toxic selenium oxyanions into non-toxic elemental selenium (Se°) may be considered as biocatalysts for the production of selenium nanoparticles (SeNPs), eventually exploitable in different biotechnological applications. Two Burkholderia fungorum strains (B. fungorum DBT1 and B. fungorum 95) were monitored during their growth for both capacity and efficiency of selenite (SeO32-) reduction and elemental selenium formation. Both strains are environmental isolates in origin: B. fungorum DBT1 was previously isolated from an oil refinery drainage, while B. fungorum 95 has been enriched from inner tissues of hybrid poplars grown in a soil contaminated by polycyclic aromatic hydrocarbons. Our results showed that B. fungorum DBT1 is able to reduce 0.5mM SeO32- to Se° when cultured aerobically in liquid medium at 27°C, while B. fungorum 95 can reduce more than 1mM SeO32- to Se° within 96h under the same growth conditions, with the appearance of SeNPs in cultures of both bacterial strains. Biogenic SeNPs were spherical, with pH-dependent charge and an average hydrodynamic diameter of 170nm and 200nm depending on whether they were produced by B. fungorum 95 or B. fungorum DBT1, respectively. Electron microscopy analyses evidenced that Se nanoparticles occurred intracellularly and extracellularly. The mechanism of SeNPs formation can be tentatively attributed to cytoplasmic enzymatic activation mediated by electron donors. Biogenic nanoparticles were then probably released outside the bacterial cells as a consequence of a secretory process or cell lysis. Nevertheless, formation of elemental selenium nanoparticles under aerobic conditions by B. fungorum DBT1 and B. fungorum 95 is likely due to intracellular reduction mechanisms. Biomedical and other high tech sectors might exploit these biogenic nanoparticles in the near future, once fully characterized and tested for their multiple properties.

Antimicrobial activity of biogenically produced spherical Se-nanomaterials embedded in organic material against Pseudomonas aeruginosa and Staphylococcus aureus strains on hydroxyapatite-coated surfaces.

In an effort to prevent the formation of pathogenic biofilms on hydroxyapatite (HA)-based clinical devices and surfaces, we present a study evaluating the antimicrobial efficacy of Spherical biogenic Se-Nanostructures Embedded in Organic material (Bio Se-NEMO-S) produced by Bacillus mycoides SelTE01 in comparison with two different chemical selenium nanoparticle (SeNP) classes. These nanomaterials have been studied as potential antimicrobials for eradication of established HA-grown biofilms, for preventing biofilm formation on HA-coated surfaces and for inhibition of planktonic cell growth of Pseudomonas aeruginosa NCTC 12934 and Staphylococcus aureus ATCC 25923. Bio Se-NEMO resulted more efficacious than those chemically produced in all tested scenarios. Bio Se-NEMO produced by B. mycoides SelTE01 after 6 or 24 h of Na2 SeO3 exposure show the same effective antibiofilm activity towards both P. aeruginosa and S. aureus strains at 0.078 mg ml-1 (Bio Se-NEMO6 ) and 0.3125 mg ml-1 (Bio Se-NEMO24 ). Meanwhile, chemically synthesized SeNPs at the highest tested concentration (2.5 mg ml-1 ) have moderate antimicrobial activity. The confocal laser scanning micrographs demonstrate that the majority of the P. aeruginosa and S. aureus cells exposed to biogenic SeNPs within the biofilm are killed or eradicated. Bio Se-NEMO therefore displayed good antimicrobial activity towards HA-grown biofilms and planktonic cells, becoming possible candidates as new antimicrobials.

Selenite biotransformation and detoxification by Stenotrophomonas maltophilia SeITE02: Novel clues on the route to bacterial biogenesis of selenium nanoparticles.

A putative biosynthetic mechanism for selenium nanoparticles (SeNPs) and efficient reduction of selenite (SeO32-) in the bacterial strain Stenotrophomonas maltophilia SeITE02 are addressed here on the basis of information gained by a combined approach relying on a set of physiological, chemical/biochemical, microscopy, and proteomic analyses. S. maltophilia SeITE02 is demonstrated to efficiently transform selenite into elemental selenium (Se°) by reducing 100% of 0.5mM of this toxic oxyanion to Se° nanoparticles within 48h growth, in liquid medium. Since the selenite reducing activity was detected in the cytoplasmic protein fraction, while biogenic SeNPs showed mainly extracellular localization, a releasing mechanism of SeNPs from the intracellular environment is hypothesized. SeNPs appeared spherical in shape and with size ranging from 160nm to 250nm, depending on the age of the cultures. Proteomic analysis carried out on the cytoplasmic fraction identified an alcohol dehydrogenase homolog, conceivably correlated with the biogenesis of SeNPs. Finally, by Fourier Transformed Infrared Spectrometry, protein and lipid residues were detected on the surface of biogenic SeNPs. Eventually, this strain might be efficaciously exploited for the remediation of selenite-contaminated environmental matrices due to its high SeO32- reducing efficiency. Biogenic SeNPs may also be considered for technological applications in different fields.

Combined application of Triton X-100 and Sinorhizobium sp. Pb002 inoculum for the improvement of lead phytoextraction by Brassica juncea in EDTA amended soil.

The process of EDTA-assisted lead phytoextraction from the Bovisa (Milan, Italy) brownfield soil was optimized in microcosms vegetated with Brassica juncea. An autochthonous plant growth-promoting rhizobacterium (PGPR), Sinorhizobium sp. Pb002, was isolated from the rhizosphere of B. juncea grown on the Pb-contaminated soil in presence of 2 mM EDTA. The strain was augmented (10(8) CFU g(-1) soil) in vegetated microcosms to stimulate B. juncea biomass production and, hence, its phytoextraction potential. Triton X-100 was also added to microcosms at 5 and 10 times the critical micelle concentration (cmc) to increase the permeability of root barriers to the EDTA-Pb complexes. Triton X-100 amendment determined an increase in Pb concentration within plant tissues. However it contextually exerted a phytotoxic effect. Sinorhizobium sp. Pb002 augmentation was crucial to plant survival in presence of both bioavailable lead and Triton X-100. The combination of the two treatments produced up to 56% increase in the efficiency of lead phytoextraction by B. juncea. The effects of these treatments on the structure of the soil bacterial community were evaluated by 16S rDNA denaturing gradient gel electrophoresis (DGGE).