One pot facile green synthesis of crystalline bio-ZrO2 nanoparticles using Acinetobacter sp. KCSI1 under room temperature.

In this study, crystalline ZrO2 nanoparticles were synthesized in one pot at room temperature using an extremophilic Acinetobacter sp. KCSI1 and characterized using various techniques to study its structural, optical and crystalline properties. The average size of the ZrO2 nanoparticles was found to be 44 ±â€¯7 nm. The XRD and Raman spectra showed the crystalline structure of ZrO2. HRTEM and SAED images showed well aligned ordered crystal lattice nanoparticles. The zeta potential of Bio-NP of ZrO2 has been found to be 36.5 ±â€¯5.46 mV. The mechanical behaviour such as hardness and Young's modulus of Bio-ZrO2 NPs were determined using atomic force microscopy (AFM) and found to be 9.206 ±â€¯2.22 and 0.285 ±â€¯0.13 GPa, respectively. No significant cytotoxicity for ZrO2 nanoparticles was observed when tested with mouse fibroblast cells (L929), suggesting that the synthesized ZrO2 nanoparticle is biocompatible and safe for environmental applications.

High phenol degradation capacity of a newly characterized Acinetobacter sp. SA01: Bacterial cell viability and membrane impairment in respect to the phenol toxicity.

An efficient phenol-degrading bacterial strain, belonging to Acinetobacter genus, was isolated and selected to study the impact of different environmentally relevant phenol concentrations on the degradation process. The bacterial isolate, labeled as Acinetobacter sp. SA01 was able to degrade the maximum phenol concentration of 1 g/l during 60 h at optimum condition of pH 7, 30 °C and 180 rpm. Aeration and initial cell density, the two important factors, were carefully examined in the optimal growth conditions. The results showed that these two variables related proportionally with phenol degradation rate. Further investigations showed no effect of inoculum size on the enhancement of degradation of phenol at over 1 g/l. Flow cytometry (FCM) study was performed to find out the relationship between phenol-induced damages and phenol degradation process. Single staining using propidium iodide (PI) showed increased cell membrane permeability with an increase of phenol concentration, while single staining with carboxyfluorescein diacetate (cFDA) demonstrated a considerable reduction in esterase activity of the cells treated with phenol at more than 1 g/l. A detailed investigation of cellular viability using concurrent double staining of cFDA/PI revealed that the cell death increases in cells exposed to phenol at more than 1 g/l. The rate of cell death was low but noticeable in the presence of phenol concentration of 2 g/l, over time. Phenol at concentrations of 3 and 4 g/l caused strong toxicity in living cells of Acinetobacter sp. SA01. The plate count method and microscopy analysis of the cells treated with phenol at 1.5 and 2 g/l confirmed an apparent reduction in cell number over time. It was assumed that the phenol concentrations higher than 1 g/l have destructive effects on membrane integrity of Acinetobacter sp. SA01. Our results also revealed that the toxicity did not reduce by increasing initial cell density. Scanning electron microscopy (SEM) examination of bacterial cells revealed the surface morphological changes following exposure to phenol. The bacterial cells, with wizened appearance and wrinkled surface, were observed by exposing to phenol (1 g/l) at lag phase. A morphological change occurred in the mid-logarithmic phase as the bacterial cells demonstrated coccobacilli form as well as elongated filamentous shape. The wrinkled cell surface were totally disappeared in mid-stationary phase, suggesting that the complete degradation of phenol relieve the stress and direct bacterial cells toward possessing smoother cell membrane.

Human neutrophils phagocytose and kill Acinetobacter baumannii and A. pittii.

Acinetobacter baumannii is a common cause of health care associated infections worldwide. A. pittii is an opportunistic pathogen also frequently isolated from Acinetobacter infections other than those from A. baumannii. Knowledge of Acinetobacter virulence factors and their role in pathogenesis is scarce. Also, there are no detailed published reports on the interactions between A. pittii and human phagocytic cells. Using confocal laser and scanning electron microscopy, immunofluorescence, and live-cell imaging, our study shows that immediately after bacteria-cell contact, neutrophils rapidly and continuously engulf and kill bacteria during at least 4 hours of infection in vitro. After 3 h of infection, neutrophils start to release neutrophil extracellular traps (NETs) against Acinetobacter. DNA in NETs colocalizes well with human histone H3 and with the specific neutrophil elastase. We have observed that human neutrophils use large filopodia as cellular tentacles to sense local environment but also to detect and retain bacteria during phagocytosis. Furthermore, co-cultivation of neutrophils with human differentiated macrophages before infections shows that human neutrophils, but not macrophages, are key immune cells to control Acinetobacter. Although macrophages were largely activated by both bacterial species, they lack the phagocytic activity demonstrated by neutrophils.

An approach to study ultrastructural changes and adaptive strategies displayed by Acinetobacter guillouiae SFC 500-1A under simultaneous Cr(VI) and phenol treatment.

Acinetobacter guillouiae SFC 500-1A, a native bacterial strain isolated from tannery sediments, is able to simultaneously remove high concentrations of Cr(VI) and phenol. In this complementary study, high-resolution microscopy techniques, such as atomic force microscopy (AFM) and transmission electron microscopy (TEM), were used to improve our understanding of some bacterial adaptive mechanisms that enhance their ability to survive. AFM contributed in gaining insight into changes in bacterial size and morphology. It allowed the unambiguous identification of pollutant-induced cellular disturbances and the visualization of bacterial cells with depth sensitivity. TEM analysis revealed that Cr(VI) produced changes mainly at the intracellular level, whereas phenol produced alterations at the membrane level. This strain tended to form more extensive biofilms after phenol treatment, which was consistent with microscopy images and the production of exopolysaccharides (EPSs). In addition, other exopolymeric substances (DNA, proteins) significantly increased under Cr(VI) and phenol treatment. These exopolymers are important for biofilm formation playing a key role in bacterial aggregate stability, being especially useful for bioremediation of environmental pollutants. This study yields the first direct evidences of a range of different changes in A. guillouiae SFC 500-1A which seems to be adaptive strategies to survive in stressful conditions.

New insights on the palate, lung, and nasal epithelium clone (PLUNC) proteins: Based on molecular and functional analysis of its homolog of YH1/SPLUNC1.

The palate, lung, and nasal epithelium clone (PLUNC) proteins are intricate immune molecules and arisen questions from them are still unresolved. In order to identify the role of PLUNC family proteins, we had analyzed its homolog protein YH1/SPLUNC1, which highly expresses in nontumor nasopharyngeal epithelium while expresses weakly in nasopharyngeal carcinoma (NPC) tissues. It is found that YH1/SPLUNC1 protein expression level was higher in chronic normal nasopharynx inflammatory cells compared to NPC tissue cells. An approach to produce active YH1/SPLUNC1 protein had been established and recombinant YH1/SPLUNC1 protein could bind to all four Gram-positive and four Gram-negative bacteria we tested, and triggered the aggregation of those bacteria. Interestingly, YH1/SPLUNC1 protein has antimicrobial activity, and it can directly kill Escherichia coli and Acinetobacter haemolyticus. The microorganism cell showed morphological changes in cell wall such as cell damage and cytoplasmic leakage after exposure to YH1/SPLUNC1 protein, indicating that YH1/SPLUNC1 directly killed the microorganisms by cell wall permeabilization. All these results indicated that YH1/SPLUNC1 might be an important antimicrobial protein involved in innate immunity defense.

Biodegradation of Azo Dye Disperse Orange S-RL by a Newly Isolated Strain Acinetobacter sp. SRL8.

The strain SRL8, which could decolorize the azo dye disperse orange S-RL (S-RL), was first isolated from sludge and identified as Acinetobacter sp. through physiobiochemical identification and 16S rRNA gene sequences. The effects of temperature, pH, dye concentration, O2, and glucose concentration on S-RL decolorization by the strain SRL8 were studied. The optimal conditions were 30 °C, pH 7.0, 4g·L(-1) of inoculation (wet cells), and microaerophilic incubation. The decolorization percentage for S-RL by the strain SRL8 could reach 90.2% under optimal conditions. The strain SRL8 was highly tolerant to the azo dye SRL up to 300 mg·L(-1) and it had a broad decolorizing spectrum. According to the Monod equation, kinetic parameters of decolorization by SRL8 were calculated. The vmax and Km were 5.57×10(-3) h(-1) and 14.53 mg·L(-1), respectively.

The characteristics of a novel heterotrophic nitrifying and aerobic denitrifying bacterium, Acinetobacter junii YB.

A novel heterotrophic nitrifying bacterium was isolated from activated sludge and was identified as Acinetobacter junii YB. The strain exhibited efficient heterotrophic nitrification-aerobic denitrification ability at a broad range of ammonium loads and had the capability to utilize hydroxylamine, nitrite and nitrate as a sole nitrogen source. Based on the nitrogen removal and enzyme assay, the nitrogen removal pathway was speculated to be achieved through heterotrophic nitrification coupled with aerobic denitrification. In addition, single-factor experiments showed that efficient heterotrophic nitrification and growth of strain YB occurred with succinate as the carbon source, pH 7.5, 37 °C, and high C/N ratio and dissolved oxygen. Furthermore, the new isolate showed capacities for aggregation and hydrophobicity. Regular variations of the flocculating ability and relative hydrophobicity were observed during the whole cultivation. The ability to perform heterotrophic nitrification-aerobic denitrification and cell aggregation demonstrated the great potential of the strain YB for future applications.

Isolation and characterization of a newly isolated pyrene-degrading Acinetobacter strain USTB-X.

The pryene-degradation bacterium strain USTB-X was newly isolated from the polycyclic aromatic hydrocarbon (PAH)-contaminated soil in Beijing Coking Plant, China. The strain was identified as Acinetobacter with respect to its 16S rDNA and morphological and physiological characteristics. The strain was Gram-negative, non-mobile, non-acid-fast, and non-spore-forming, short rods in young culture and 0.8-1.6 µm in diameter and 1.2-2.5 µm long in the stationary phase of growth. Strain USTB-X could utilize pyrene, naphthalene, fluorene, phenanthrene, benzene, toluene, ethylbenzene, ethanol, methanol, and Tween 80 as sole source of carbon and energy. The strain could produce biosurfactants which enhanced the removal of pyrene and could remove 63 % of pyrene with an initial concentration of 100 mg·L-1 in 16 days without other substrates. Based on the intermediates analyzed by gas chromatography-mass spectrometry, we also deduced the possible metabolic pathway of strain USTBX for pyrene biodegradation. Results indicated that the strain USTB-X had high potential to enhance the removal of PAHs in contaminated sites.

Evaluation of Acinetobacter sp. B9 for Cr (VI) resistance and detoxification with potential application in bioremediation of heavy-metals-rich industrial wastewater.

Present work demonstrates Cr (VI) detoxification and resistance mechanism of a newly isolated strain (B9) of Acinetobacter sp. Bioremediation potential of the strain B9 is shown by simultaneous removal of major heavy metals including chromium from heavy-metals-rich metal finishing industrial wastewater. Strain B9 tolerate up to 350 mg L(-1) of Cr (VI) and also shows level of tolerance to Ni (II), Zn (II), Pb (II), and Cd (II). The strain was capable of reducing 67 % of initial 7.0 mg L(-1) of Cr (VI) within 24 h of incubation, while in presence of Cu ions 100 % removal of initial 7.0 and 10 mg L(-1) of Cr (VI) was observed with in 24 h. pH in the range of 6.0-8.0 and inoculum size of 2 % (v/v) were determined to be optimum for dichromate reduction. Fourier transform infrared spectroscopy and transmission electron microscopy studies suggested absorption or intracellular accumulation and that might be one of the major mechanisms behind the chromium resistance by strain B9. Scanning electron microscopy showed morphological changes in the strain due to chromium stress. Relevance of the strain for treatment of heavy-metals-rich industrial wastewater resulted in 93.7, 55.4, and 68.94 % removal of initial 30 mg L(-1) Cr (VI), 246 mg L(-1) total Cr, and 51 mg L(-1) Ni, respectively, after 144 h of treatment in a batch mode.

Cloning and characterization of an antibacterial L-amino acid oxidase from Crotalus durissus cumanensis venom.

An L-amino acid oxidase (LAAO) from Crotalus durissus cumanensis venom (CdcLAAO) was purified to homogeneity using a combination of size-exclusion and ion exchange chromatographies. CdcLAAO is a monomeric protein exhibiting an apparent molecular mass of 55 kDa and a calculated pI of 8. Its complete 498-amino-acid sequence was deduced through cDNA and protein sequencing. The enzyme oxidized L-Leu with K(m) and a V(Max) of 9.23 µM and 0.46 µM/min respectively, and exhibited Kcat and a Kcat/K(m) of 1.8 s(-1) and 195 mM(-1)s(-1). CdcLAAO inhibited in a dose-dependent manner the growth of Staphylococcus aureus and Acinetobacter baumannii. The inhibitory effect was more significant on S. aureus, with a Minimal Inhibitory Concentration (MIC) of 8 µg/mL and Minimal Bactericidal Concentration (MBC) of 16 µg/mL, than against A. baumannii, with a MIC of 16 µg/mL and MBC of 32 µg/mL. However, against Escherichia coli CdcLAAO did not show inhibitory capacity at the concentrations tested (2-128 µg/mL). CdcLAAO did not exhibit cytotoxic activity on the mouse myoblast cell line C(2)C(12) and on peripheral blood mononuclear cell (PBMC).

Molecular basis for different levels of tet(M) expression in Streptococcus pneumoniae clinical isolates.

Seventy-four unrelated clinical isolates of Streptococcus pneumoniae harboring the tet(M) gene were studied. Seven strains with low tetracycline (Tc) MICs (0.25 to 0.5 µg/ml) were found to harbor truncated tet(M) alleles that were inactivated by different frameshift mutations. In contrast, five strains bore deletions in the tet(M) promoter region, among which four displayed increased Tc MICs (16 to 64 µg/ml). The same promoter mutations were detected in Tc-resistant mutants selected in vitro from various susceptible strains. Sequence analysis revealed that these deletions might impede the formation of the transcriptional attenuator located immediately upstream of tet(M). Expression in Enterococcus faecalis of a tet(M) reporter gene transcribed from these promoter mutants conferred a level of Tc resistance similar to that observed in the parental S. pneumoniae strains. These results show that different levels of Tc susceptibility found in clinical isolates of S. pneumoniae can be explained by frameshift mutations within tet(M) and by alterations of the upstream transcriptional attenuator.

Isotherm kinetics of Cr(III) removal by non-viable cells of Acinetobacter haemolyticus.

The potential use of non-viable biomass of a Gram negative bacterium i.e. Acinetobacter haemolyticus to remove Cr(III) species from aqueous environment was investigated. Highest Cr(III) removal of 198.80 mg g(-1) was obtained at pH 5, biomass dosage of 15 mg cell dry weight, initial Cr(III) of 100 mg L(-1) and 30 min of contact time. The Langmuir and Freundlich models fit the experimental data (R(2)>0.95) while the kinetic data was best described using the pseudo second-order kinetic model (R(2)>0.99). Cr(III) was successfully recovered from the bacterial biomass using either 1M of CH(3)COOH, HNO(3) or H(2)SO(4) with 90% recovery. TEM and FTIR suggested the involvement of amine, carboxyl, hydroxyl and phosphate groups during the biosorption of Cr(III) onto the cell surface of A. haemolyticus. A. haemolyticus was also capable to remove 79.87 mg g(-1) Cr(III) (around 22.75%) from raw leather tanning wastewater. This study demonstrates the potential of using A. haemolyticus as biosorbent to remove Cr(III) from both synthetic and industrial wastewater.

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process.

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 µm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.

Performance of enhanced biological SBR process for aniline treatment by mycelial pellet as biomass carrier.

Mycelial pellet of Aspergillus niger Y3 was used as a biomass carrier to immobilize the aniline-degrading bacterium, Acinetobacter calcoaceticus JH-9 and the mix culture of the COD rapid degradation bacteria. In order to investigate its removal effect on aniline and COD, the combined mycelial pellets were applied in the SBR. Comparison of the performances was conducted between another SBR inoculated with sole strain JH-9 and the above SBR. The results showed that the stable degradations of aniline and COD were observed in both reactors. In the SBR with combined mycelial pellet, the biological removal efficiency was about 0.9 mg aniline/(L·d). It was much higher than that in the activated sludge reactor. Meanwhile, the performances of the sedimentation velocity, liquid-solid phase separation and the effluent quality were better in the SBR. According to SEM images and PCR-DGGE analysis, the species immobilized on the biomass carrier were more predominant in this system.

Protection against diesel oil toxicity by sodium chloride-induced exopolysaccharides in Acinetobacter sp. strain DR1.

Acinetobacter sp. strain DR1 is capable of growth on diesel oil. Interestingly, the degradation of diesel oil by the strain DR1 is enhanced in the presence of sodium chloride (NaCl). However, the growth rate of strain DR1 is not affected by the presence of NaCl. Northern blot analysis has also demonstrated that the effect of NaCl on the degradation of diesel oil is not attributable to increased levels of alkane hydroxylase (AlkM-type) gene expression. Rather, we have noted an increase in the exopolysaccharide (EPS) yields of strain DR1 under high NaCl conditions (9-fold). The lag-time of diesel oil biodegradation was significantly shorter in the strain DR1 with exogenous EPS than in the controls, although EPS alone does not support the growth of strain DR1. The recovery of strain DR1 when exposed to diesel oil was accelerated when exogenous EPS was added to the medium. The overproduction of EPS was also noted in the presence of diesel oil and n-hexadecane. The data indicated that EPS overproduction might play a protective role against diesel oil toxicity. Along with the results of the soil microcosm tests, the data presented herein demonstrated that NaCl-induced EPS is associated with a reduction in diesel oil toxicity, and thus increases diesel oil biodegradation in Acinetobacter sp. strain DR1.

Acinetobacter sp. Ud-4 efficiently degrades both edible and mineral oils: isolation and characterization.

A novel Acinetobacter strain, Ud-4, possessing a strong capacity to degrade edible, lubricating, and heavy oil was isolated from seawater in a fishing port located in Toyama, Japan. It was identified by morphological and physiological analyses and 16S rDNA sequencing. This strain could utilize five types of edible oils (canola oil, olive oil, sesame oil, soybean oil, and lard), lubricating oil, and C-heavy oil as the sole carbon source for growth in M9 medium. The strain grew well and heavily degraded edible oils in Luria-Bertani medium during a 7-day culture at 25 degrees C; it also degraded all kinds of oils in artificial seawater medium for marine bacteria. Furthermore, this strain was capable of degrading almost all C10-C25 n-alkanes in C-heavy oil during a 4-week culture. Oligonucleotide primers specific to two catabolic genes involved in the degradation of n-alkanes (Acinetobacter sp. alkM) and triglyceride (Acinetobacter sp. lipA) allowed amplification of these genes in strain Ud-4. To our knowledge, this is the first report on the isolation of a bacterium that can efficiently degrade both edible and mineral oils.

Analysis of force interactions between AFM tips and hydrophobic bacteria using DLVO theory.

Microbial adhesion to surfaces and interfaces is strongly influenced by their structure and physicochemical properties. We used atomic force microscopy (AFM) to measure the forces between chemically functionalized AFM tips and two bacterial species exhibiting different cell surface hydrophobicities, measured as the oil/water contact angle (theta): Acinetobacter venetianus RAG-1 (theta = 56.4 degrees ) and Rhodococcus erythropolis 20S-E1-c (theta = 152.9 degrees ). The forces were measured as the AFM tips, coated with either hydrophobic (octadecane) or hydrophilic (undecanol) groups, approached the bacterial cells in aqueous buffer. The experimental force curves between the two microbial cells and functionalized AFM probes were not successfully described by the classical Derjaguin-Landau-Verwey-Overbeek (DLVO) theory of colloid stability. To reconcile the discrepancy between theory and experiments, two types of extended DLVO models were proposed. The first modification considers an additional acid-base component that accounts for attractive hydrophobic interactions and repulsive hydration effects. The second model considers an additional exponentially decaying steric interaction between polymeric brushes in addition to the acid-base interactions. These extended DLVO predictions agreed well with AFM experimental data for both A. venetianus RAG-1, whose surface consists of an exopolymeric capsule and pili, and R. erythropolis 20S-E1-c, whose surface is covered by an exopolymeric capsule. The extended models for the bacteria-AFM tip force-distance curves were consistent with the effects of steric interactions.

Optimizing emulsan production of A. venetianus RAG-1 using response surface methodology.

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH(2)PO(4), 23.32 g/L K(2)HPO(4), 5.77 g/L (NH(4))(2)SO(4) and 0.354 g/L MgSO(4)*7H(2)O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.

Atomic force microscopy measurement of heterogeneity in bacterial surface hydrophobicity.

The structure and physicochemical properties of microbial surfaces at the molecular level determine their adhesion to surfaces and interfaces. Here, we report the use of atomic force microscopy (AFM) to explore the morphology of soft, living cells in aqueous buffer, to map bacterial surface heterogeneities, and to directly correlate the results in the AFM force-distance curves to the macroscopic properties of the microbial surfaces. The surfaces of two bacterial species, Acinetobacter venetianus RAG-1 and Rhodococcus erythropolis 20S-E1-c, showing different macroscopic surface hydrophobicity were probed with chemically functionalized AFM tips, terminating in hydrophobic and hydrophilic groups. All force measurements were obtained in contact mode and made on a location of the bacterium selected from the alternating current mode image. AFM imaging revealed morphological details of the microbial-surface ultrastructures with about 20 nm resolution. The heterogeneous surface morphology was directly correlated with differences in adhesion forces as revealed by retraction force curves and also with the presence of external structures, either pili or capsules, as confirmed by transmission electron microscopy. The AFM force curves for both bacterial species showed differences in the interactions of extracellular structures with hydrophilic and hydrophobic tips. A. venetianus RAG-1 showed an irregular pattern with multiple adhesion peaks suggesting the presence of biopolymers with different lengths on its surface. R. erythropolis 20S-E1-c exhibited long-range attraction forces and single rupture events suggesting a more hydrophobic and smoother surface. The adhesion force measurements indicated a patchy surface distribution of interaction forces for both bacterial species, with the highest forces grouped at one pole of the cell for R. erythropolis 20S-E1-c and a random distribution of adhesion forces in the case of A. venetianus RAG-1. The magnitude of the adhesion forces was proportional to the three-phase contact angle between hexadecane and water on the bacterial surfaces.