Isolation and characterization of a novel nicotinophilic bacterium, Arthrobacter sp. aRF-1 and its metabolic pathway.

Nicotine is a potent parasympathomimetic stimulant and an important natural alkaloid mainly found in the Nicotiana genus of plants. It can directly threaten ecological security and human health in tobacco waste, wastewater, and other forms of tobacco production. Therefore, it is the basis of nicotine pollution prevention and of great application value to explore efficient and a wide range of nicotinophilic bacteria for tobacco industry and environmental protection. In this study, one nicotinophilic bacterium was isolated from the soil, which accumulated tobacco waste over 50 years at a Hefei cigarette factory. The strain was named aRF-1, which was identified as Arthrobacter sp. by analysis. The nicotine degradation tests showed that the optimum temperature for cell growth and metabolism of nicotine of Arthrobacter sp. aRF-1 was 30 °C, and the optimum initial pH value was about 7.0. Under the optimum experimented conditions, it can tolerance nicotine concentration as high as 8 g·L-1 . The highest removal rate of nicotine was 93.8% in 72 H in nonsterilization contaminated soil by Arthrobacter sp. aRF-1. LC-MS/MS was used to analyze the nicotine metabolic intermediates of strain Arthrobacter sp. aRF-1. A total of nine major metabolites that were detected were able to metabolize nicotine along a variant pathway of pyridine and pyrrolidine, and there may be more than two nicotine metabolic pathways for Arthrobacter sp. aRF-1 through the analysis of the main intermediate products.

Screening micro-organisms for cadmium absorption from aqueous solution and cadmium absorption properties of Arthrobacter nicotianae.

To obtain basic information on how microbial cells absorb cadmium from aqueous solution, we examined cadmium absorption in various micro-organisms. Of 51 micro-organism strains tested, we found that some Gram-positive bacteria, such as, Arthrobacter nicotianae and Bacillus subtilis, and some actinomycetes, such as, Streptomyces flavoviridis and S. levoris were highly capable of absorbing cadmium from an aqueous solution. A. nicotianae absorbed the largest amount of cadmium, over 800 µmol cadmium per gram of dry wt. cells. However, cadmium absorption by A. nicotianae was affected by the solution pH, cadmium concentration, and cell density. The absorption of cadmium was very rapid. Some factors that affected cadmium absorption by A. nicotianae cells were also discussed.

Formulations for freeze-drying of bacteria and their influence on cell survival.

Cellular water can be removed to reversibly inactivate microorganisms to facilitate storage. One such method of removal is freeze-drying, which is considered a gentle dehydration method. To facilitate cell survival during drying, the cells are often formulated beforehand. The formulation forms a matrix that embeds the cells and protects them from various harmful stresses imposed on the cells during freezing and drying. We present here a general method to evaluate the survival rate of cells after freeze-drying and we illustrate it by comparing the results obtained with four different formulations: the disaccharide sucrose, the sucrose derived polymer Ficoll PM400, and the respective polysaccharides hydroxyethyl cellulose (HEC) and hydroxypropyl methyl cellulose (HPMC), on two strains of bacteria, P. putida KT2440 and A. chlorophenolicus A6. In this work we illustrate how to prepare formulations for freeze-drying and how to investigate the mechanisms of cell survival after rehydration by characterizing the formulation using of differential scanning calorimetry (DSC), surface tension measurements, X-ray analysis, and electron microscopy and relating those data to survival rates. The polymers were chosen to get a monomeric structure of the respective polysaccharide resembling sucrose to a varying degrees. Using this method setup we showed that polymers can support cell survival as effectively as disaccharides if certain physical properties of the formulation are controlled.

Biosorption of arsenite (As(+3)) and arsenate (As(+5)) from aqueous solution by Arthrobacter sp. biomass.

In this study we investigated the role of arsenic-resistant bacteria Arthrobacter sp. biomass for removal of arsenite as well as arsenate from aqueous solution. The biomass sorption characteristics were studied as a function of biomass dose, contact time and pH. Langmuir, Freundlich and Dubinin-Radushkevich (D-R) models were applied to describe the biosorption isotherm. The Langmuir model fitted the equilibrium data better than the Freundlich isotherm. The biosorption capacity of the biomass for As(+3) and As(+5) was found to be 74.91 mg/g (pH 7.0) and 81.63 mg/g (pH 3.0), respectively using 1 g/L biomass with a contact time of 30 min at 28 degrees C. The mean sorption energy values calculated from the D-R model indicated that the biosorption of As(+3) and As(+5) onto Arthrobacter sp. biomass took place by chemical ion-exchange. The thermodynamic parameters showed that the biosorption of As(+3) and As(+5) ions onto Arthrobacter sp. biomass was feasible, spontaneous and exothermic in nature. Kinetic evaluation of experimental data showed that biosorption of As(+3) and As(+5) followed pseudo-second-order kinetics. Fourier transform infrared spectroscopy (FT-IR) analysis indicated the involvement of possible functional groups (-OH, -C=O and -NH) in the As(+3) and As(+5) biosorption process. Bacterial cell biomass can be used as a biosorbent for removal of arsenic from arsenic-contaminated water.

A novel galactofuranan from the cell wall of Arthrobacter sp. VKM Ас-2576.

The investigated cell wall polysaccharide of Arthrobacter sp. VKM Ac-2576 (phylum Actinobacteria) is a (1→6)-linked ß-D-galactofuranan with the α-D-GlcpNAc3NAc residues that glycosylate virtually all (~90%) hydroxyl groups at C-2 of galactose. Its repeating unit has the following structure: →6)D-ß-Galf-(1→2) ↑ α-D-GlcpNAc3NAc-(1 (90%). The structure of the polysaccharide was established by chemical and NMR spectroscopic methods; it is identified for the first time.

Effects of hydroxypropyl-ß-cyclodextrin on cell growth, activity, and integrity of steroid-transforming Arthrobacter simplex and Mycobacterium sp.

A comparative investigation was performed on the effects of hydroxypropyl-ß-cyclodextrin (HP-ß-CD) on the growth, biocatalytic activity, and cell integrity of Arthrobacter simplex TCCC 11037 (ASP) and Mycobacterium sp. NRRL B-3683 (MSP). The addition of HP-ß-CD to ASP medium improved its cell wall permeability for lipophilic compounds but significantly inhibited its growth and biocatalytic activity. On the other hand, the addition of HP-ß-CD to MSP broth had lesser effects. Atomic force microscopy scanning analysis revealed that HP-ß-CD damaged the cell integrity in ASP, especially the outermost cell surface structure, but not in MSP, which remained intact, owing to the differences in their cell wall and cell membrane composition. Protein leaking and lipid content in ASP increased with increased HP-ß-CD concentration, indicating possible alterations in ASP cell membrane features caused by HP-ß-CD. These alterations may also explain the slow cell growth and decreased cell ΔΨm in ASP upon the addition of HP-ß-CD.

Impact of matrix properties on the survival of freeze-dried bacteria.

BACKGROUND: Disaccharides are, in general, the first choice as formulation compounds when freeze-drying microorganisms. Although polysaccharides and other biopolymers are considered too large to stabilise and interact with cell components in the same beneficial way as disaccharides, polymers have been reported to support cell survival. In the present study we compare the efficiency of sucrose and the polymers Ficoll, hydroxyethylcellulose, hydroxypropylmethylcellulose and polyvinylalcohol to support the survival of three bacterial strains during freeze drying. The initial osmotic conditions were adjusted to be similar for all formulations. Formulation characterisation was used to interpret the impact that different compound properties had on cell survival. RESULTS: Despite differences in molecular size, both sucrose and the sucrose-based polymer Ficoll supported cell survival after freeze drying equally well. All formulations became amorphous upon dehydration. Scanning electron microscopy and X-ray diffraction data showed that the discerned differences in structure of the dry formulations had little impact on the survival rates. The capability of the polymers to support cell survival correlated with the surface activity of the polymers in a similar way for all investigated bacterial strains. CONCLUSION: Polymer-based formulations can support cell survival as effectively as disaccharides if formulation properties of importance for maintaining cell viability are identified and controlled.

Complete degradation of di-n-octyl phthalate by biochemical cooperation between Gordonia sp. strain JDC-2 and Arthrobacter sp. strain JDC-32 isolated from activated sludge.

Two bacterial strains were isolated from activated sludge using mixtures of phthalic acid esters (PAEs) as the sole source of carbon and energy. One of the isolates was identified as Gordonia sp. strain JDC-2 and the other as Arthrobacter sp. strain JDC-32, mainly through 16S rRNA gene sequence analysis. Gordonia sp. strain JDC-2 rapidly degraded di-n-octyl phthalate (DOP) into phthalic acid (PA), which accumulated in the culture medium. Arthrobacter sp. strain JDC-32 degraded PA but not DOP. The co-culture of Gordonia sp. strain JDC-2 and Arthrobacter sp. strain JDC-32 degraded DOP completely by overcoming the degradative limitations of each species alone. The biochemical pathway of DOP degradation by Gordonia sp. strain JDC-2 was proposed based on the identified degradation intermediates. The results suggest that DOP is completely degraded by the biochemical cooperation of different microorganisms isolated from activated sludge.

Hexavalent chromium reduction by free and immobilized cell-free extract of Arthrobacter rhombi-RE.

In the present study, hexavalent chromium (Cr(VI)) reduction potential of chromium reductase associated with the cell-free extracts (CFE) of Arthrobacter rhombi-RE species was evaluated. Arthrobacter rhombi-RE, an efficient Cr(VI) reducing bacterium, was enriched and isolated from a chromium-contaminated site. Chromium reductase activity of Arthrobacter rhombi-RE strain was associated with the cell-free extract and the contribution of extracellular enzymes to Cr(VI) reduction was negligible. NADH enhanced the chromium reductase activity. The enzyme activity was optimal at a pH of 5.5 and a temperature of 30 degrees C. Among the ten electron donors screened, sodium pyruvate was the most effective one followed by NADH and propionic acid. Michaelis-Menten constant, K(m), and maximum reaction rate, V(max), obtained from the Lineweaver-Burk plot were 48 microM and 4.09 nM/mg protein/min, respectively, in presence of NADH as electron donor and 170.5 microM and 4.29 nM/mg protein/min, respectively, in presence of sodium pyruvate as electron donor. Ca(2+) enhanced the enzyme activity while Hg(2+), Cd(2+), Ba(2+), and Zn(2+) inhibited the enzyme activity. Among the various immobilization matrices screened, calcium alginate beads seemed to be the most effective one. Though immobilized enzyme system was able to reduce Cr(VI), the performance was not very encouraging in continuous mode of operation.

Optimization of physical parameters for lipase production from Arthrobacter sp. BGCC#490.

The physical parameters for the production of thermostable, alkaline lipase from Arthrobacter sp. BGCC# 490 were optimized using response surface methodology (RSM), employing face centered central composite design (FCCCD). The design was employed by selecting pH, temperature and incubation period as the model factors and to achieve maximum yield, interaction of these factors was studied by RSM. A second-order quadratic model and response surface method showed that the optimum conditions for lipase production (pH 10.0, temperature 40 degrees C and incubation period 48 h) resulted in 1.6-fold increase in lipase production (13.75 EUml(-1)), as compared to the initial level (8.6 EUml(-1)) after 48 h of incubation, whereas its value predicted by the quadratic model was 12.8 EUml(-1). Lipase showed stability in the pH range 8-10 and temperature range 40-60 degrees C, with maximum activity at pH 9.0 and temperature 50 degrees C. Lipase activity was enhanced in the presence of K+, Ca2+ and Mg2+ ions, but inhibited by Hg2+ ions. The enzyme exhibited high activity in the presence of acetone, isopropanol and ethanol, but was unaffected by methanol. These properties suggest that the lipase may find potential applications in the detergent industry. The present work also demonstrated the feasibility of using experimental design tools to optimize physical parameters for lipase production by an indigenous Arthrobacter sp.

Plasmon microscopy and imaging ellipsometry of Artrobacter oxydans attached on polymer films.

The attachment of Artrobacter oxydans 1,388 on a newly synthesized biodegradable copolymer of poly-(hexanlactam)-co-block-poly-(delta-valerolactone) is investigated by optical, microscopic and biochemical methods. The potentials of surface plasmon microscopy and imaging ellipsometry for detecting microorganisms when Al films are used to excite plasmons is assessed by comparing images obtained by these methods with dark field microscopy pictures. The experimental results demonstrate that in this case imaging ellipsometry and plasmon microscopy in transmission are promising methods and can be used in optical sensors for monitoring cell adhesion.

Complete degradation of butyl benzyl phthalate by a defined bacterial consortium: role of individual isolates in the assimilation pathway.

Two bacterial strains, in consortium, were isolated by enrichment techniques from municipal waste-contaminated soil, which utilized butyl benzyl phthalate (BBP) as the sole carbon source. One of the isolates was identified as Arthrobacter sp. strain WY and the other one as Acinetobacter sp. strain FW based on the morphological, nutritional and biochemical characteristics and 16S rRNA sequence analysis. Various metabolites of BBP engendered by Arthrobacter sp. strain WY were isolated and identified by a combination of chromatographic and spectrophotometric analyses, which revealed a pathway involving monobutylphthalate (MBuP), monobenzyl phthalate (MBzP), phthalic acid and protocatechuic acid. The protocatechuic acid in turn was processed by ortho-cleavage dioxygenase to form beta-carboxy-cis,cis-muconate, ultimately leading to the TCA cycle. The Arthrobacter sp. strain WY could not utilize the hydrolyzed alcohols of BBP. On the other hand, the Acinetobacter sp. strain FW, which by itself could not utilize BBP as the sole carbon source, is capable of utilizing the hydrolyzed alcohols of BBP. Benzyl alcohol was found to be metabolized by the Acinetobacter sp. strain FW via benzaldehyde, benzoic acid and catechol. Catechol was further degraded by ortho-cleavage dioxygenase to cis,cis-muconic acid and subsequently to muconolactone leading to beta-ketoadipate pathway. Moreover, the Acinetobacter sp. strain FW metabolized 1-butanol through butyraldehyde and butyric acid leading to the tricarboxylic acid cycle via beta-oxidation pathway. This is the first report on the complete degradation of BBP by a defined consortium describing the role of its individual constituents in the BBP assimilation pathway.

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized Arthrobacter sp. and Delftia acidovorans.

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l(-1)) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l(-1)) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l(-1 )h(-1) vs. 0.06 mg l(-1 )h(-1) with free cells in co-culture).

Growth and physiological response of Arthrobacter protophormiae RKJ100 toward higher concentrations of o-nitrobenzoate and p-hydroxybenzoate.

Bioremediation of sites that are heavily contaminated with pollutant chemicals is a challenge as most of the microorganisms cannot tolerate higher concentrations of toxic compounds. Only a few strains of the genus Pseudomonas have been studied for their tolerance toward the higher concentrations of aromatic pollutant compounds, a phenomenon that is accompanied by various physiological changes. In the present study we have characterized the growth response and physiological changes (adaptations) of a Gram-positive bacterium, Arthrobacter protophormiae RKJ100, toward the higher concentrations of two aromatic compounds, viz. o-nitrobenzoate (ONB) and p-hydroxybenzoate (PHB). Arthrobacter protophormiae RKJ100 could utilize 30 mM ONB and 50 mM PHB as sole sources of carbon and energy. It was capable of growth on higher concentrations of ONB (up to 200 mM) and PHB (up to 150 mM) when the cells were pre-exposed to lower concentrations of these compounds. The adaptive responses shown by the organism during growth on higher concentrations of these compounds were evident from significant changes in cellular fatty acid profiles. In addition, Bacterial Adhesion To Hydrocarbon (BATH) assay and scanning electron microscopy showed substantial increase in cell surface hydrophobicity and decrease in cell size of A. protophormiae RKJ100 when grown on ONB and PHB as compared to succinate-grown cells.

Microbiological degradation of pentane by immobilized cells of Arthrobacter sp.

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42 days of incubation at 20 degrees C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36 h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85-95% pentane degradation during a 2 month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.

Arthrobacter psychrophenolicus sp. nov., isolated from an alpine ice cave.

On the basis of phenotypic, genotypic and chemotaxonomic characteristics, a novel species belonging to the genus Arthrobacter is described. A facultatively psychrophilic bacterium, strain AG31(T), was isolated from an alpine ice cave. The aerobic, Gram-positive, non-spore-forming, non-motile strain exhibited a rod-coccus growth cycle and produced a yellow pigment. Good growth and phenol biodegradation occurred at a temperature range of 1-25 degrees C. Up to 10 mM phenol was utilized as a sole carbon source. Glucose was not assimilated. Analysis of the 16S rRNA gene revealed that strain AG31(T) represents a distinct lineage within the genus Arthrobacter, being most closely related to Arthrobacter sulfureus. The level of DNA-DNA relatedness to the type strain of A. sulfureus was 29.9 %. Anteiso-C(15 : 0) was the predominant fatty acid (72 %). Strain AG31(T) exhibited A4alpha l-lys-l-Glu-type peptidoglycan and contained glucose as the only cell-wall sugar. MK-10 was the predominant menaquinone, and the polar lipid pattern consisted of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol and an unidentified glycolipid. Strain AG31(T) (=DSM 15454(T)=LMG 21914(T)) is assigned as the type strain of a novel Arthrobacter species, Arthrobacter psychrophenolicus sp. nov.

Influence of Al on growth, cell size and content of intracellular water of Arthrobacter sp. PI/1-95.

A distinct inhibition of the growth of a typical soil-born bacterium, Arthrobacter sp. PI/1-95, by aluminium was established. The content of intracellular water and (consequently) the cell size of the bacterium significantly increased when cells came into contact with aluminium. Both parameters were also increased when applying HCl, but Al caused the greater change, so that a distinct discrimination between the effects of aluminium on the one hand and a sole pH-effect on the other hand was possible. The presented results point to an osmo-regulative disorder connected with a malfunction of cell membrane and cell wall, which seems to be a probable mechanism for an Al-toxicity in connection with Arthrobacter sp. PI/1-95.

[Studies on hydantoinase producing conditions of Arthrobacter K1108].

The hydantoinase-producing conditions from strain Arthrobacter K1108 were investigated. It is shown that the hydantoinase in the strain is intracellular and inducible. Its optimal inducer is 5-benzylhydantoin, while 5-indolylmethylhydantoin and 5-phenylhydantoin cannot induce the production of hydantoinase in K1108. A gratuitous inducer was designed, with which the hydantoinase production is 343% as much as that with 5-benzylhydantoin. The media for culturing the bacteria were screened and optimized. Under optimal conditions, the specific activity of K1108 cells reached 10.8 U/mL.