Symbiotic relationship between <Emphasis Type="Italic">Microbacterium</Emphasis> sp. SK0812 and <Emphasis Type="Italic">Candida tropicalis</Emphasis> SK090404

A bacterium growing inside yeast cytoplasm was observed by light microscope without staining. The bacterium was separately stained from yeast cell by a fluorescent dye, 4′,6-diamidino-2-phenylindole (DAPI). The bacterium actively moved inside yeast cytoplasm and propagated in company with the yeast growth. The bacterium was separated from the yeast cytoplasm by selective disruption of yeast cells and the yeast without the intracellular bacterium (YWOB) was obtained by selective inactivation of bacterial cells. The yeast and the intracellular bacterium were identified as Candida tropicalis and Microbacterium sp., respectively. The length of Microbacterium sp. and C. tropicalis measured with SEM image was smaller than 0.5 μm and was larger than 5 μm, respectively. The yeast with the intracellular bacterium (YWIB) grew in a starch-based medium but the YWOB was not C. tropicalis has neither extracellular nor intracellular saccharification enzyme. Glucose was produced from starch by the extracellular crude enzyme (culture fluid) of Microbacterium sp. YWIB produced significantly more ethanol from glucose than YWOB but did not from starch. Conclusively, C. tropicalis is thought to catabolize starch dependent upon Microbacterium sp. growing in its cytoplasm and furnish stable habitat for the Microbacterium sp.     

Metabolites produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. enable a Gram-positive bacterium to achieve extracellular electron transfer

Previous studies revealed the abundance of Pseudomonas sp. in the microbial community of a microbial fuel cell (MFC). These bacteria can transfer electrons to the electrode via self-produced phenazine-based mediators. A MFC fed with acetate where several Pseudomonas sp. were present was found to be rich in a Gram-positive bacterium, identified as Brevibacillus sp. PTH1. Remarkably, MFCs operated with only the Brevibacillus strain in their anodes had poor electricity generation. Upon replacement of the anodic aqueous part of Brevibacillus containing MFCs with the cell-free anodic supernatants of MFCs operated with Pseudomonas sp. CMR12a, a strain producing considerable amounts of phenazine-1-carboxamide (PCN) and biosurfactants, the electricity generation was improved significantly. Supernatants of Pseudomonas sp. CMR12a_Reg, a regulatory mutant lacking the ability to produce PCN, had no similar improvement effect. Purified PCN, together with rhamnolipids as biosurfactants (1 mg L⁻¹), could clearly improve electricity generation by Brevibacillus sp. PTH1, as well as enable this bacterium to oxidize acetate with concomitant reduction of ferric iron, supplied as goethite (FeOOH). When added alone, PCN had no observable effects on Brevibacillus’ electron transfer. This work demonstrates that metabolites produced by Pseudomonas sp. enable Gram-positive bacteria to achieve extracellular electron transfer. Possibly, this bacterial interaction is a key process in the anodic electron transfer of a MFC, enabling Brevibacillus sp. PTH1 to achieve its dominance. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Statistic evaluation of the influence of species variation,culture conditions,surface wettability and fluid shear on attachment and biofilm development of marine bacteria

A budding coccoid bacterium, (CH1), a Vibrio sp. and a Pseudomonas sp. were investigated for factors governing their attachment to glass surfaces in static batch culture and laminar flow continuous culture systems. An analysis of variance showed that the three species exhibited very different responses. For CH1 attachment was dependent on cell density, incubation time and nutrient concentration. The Vibrio sp. was affected by nutrient concentration while the attachment of the Pseudomonas sp. was independent of cell density, incubation time and nutrient concentration. A comparison of attachment to hydrophilic and hydrophobic surfaces showed that attachment of the Vibrio sp. and CH1 to hydrophilic surfaces was 3 and 10 times greater respectively than to hydrophobic surfaces while Pseudomonas attached in equal numbers to both surfaces. The continuous culture system with defined flow hydrodynamics and growth conditions at steady state revealed a random sampling effect 3 times smaller than the batch culture system did. When the biofilm development of Pseudomonas sp. was followed during 46 h at different fluid shear under laminar and turbulent flow conditions, the former biofilm reached 3.3·10⁸ cells·cm^-2 and the latter 8.2·10⁷ cells·cm^-2.Non-common abbreviation NSS Nine salt solution     

Utilization of dibenzothiophene as sulfur source by <Emphasis Type="Italic">Microbacterium</Emphasis> sp. <Emphasis Type="Italic">NISOC-06</Emphasis>

Oil-polluted soils were sampled from National Iranian South Oil Company (NISOC) for isolation and screening of C–S and not C–C targeted Dibenzothiophene (DBT) degrading microorganisms. Microbacterium sp. NISOC-06, a C–S targeted DBT degrading bacterium, was selected and its desulfurization ability was studied in aqueous phase and water-gasoline biphasic systems. The 16srRNA gene was amplified using universal eubacteria-specific primers, PCR product was sequenced and the sequence of nearly 1,500 bp 16srDNA was studied. Based on Gas Chromatography results Microbacterium sp. NISOC-06 utilized 94.8% of 1 mM DBT during the 2 weeks of incubation. UV Spectrophotometry and biomass production measurements showed that the Microbacterium sp. NISOC-06 was not able to utilize DBT as a carbon source. There was no accumulation of phenolic compounds as Gibb’s assay showed. Biomass production in a biphasic system for which DBT-enriched gasoline was used as the sulfur source indicated the capability of Microbacterium sp. NISOC-06 to desulfurize gasoline.     

Detoxification process of tolaasins,lipodepsipeptides, by Microbacterium sp. K3-5

Tolaasins are antimicrobial lipodepsipeptides. Here, we report the tolaasins-detoxifying properties of Microbacterium sp. K3-5 (K3-5). The detoxification of tolaasins by K3-5 was performed by hydrolyzation of cyclic structure of tolaasins depending on the tolaasin-K3-5 cell interaction. Our data suggest that the cyclic structure of tolaasins is critical for its interaction to target cells.     

Molecular cloning, expression and characterization of a chitosanase from Microbacterium sp.

A gene encoding a chitosanase (mschito) was cloned from Microbacterium sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.     

Characterization of bacterial communities associated with Brassica napus L. growing on a Zn-contaminated soil and their effects on root growth

The interaction between plant growth-promoting bacteria (PGPB) and plants can enhance biomass production and metal tolerance of the host plants. This work aimed at isolating and characterizing the cultivable bacterial community associated with Brassica napus growing on a Zn-contaminated site, for selecting cultivable PGPB that might enhance biomass production and metal tolerance of energy crops. The effects of some of these bacterial strains on root growth of B. napus exposed to increasing Zn and Cd concentrations were assessed. A total of 426 morphologically different bacterial strains were isolated from the soil, the rhizosphere, and the roots and stems of B. napus. The diversity of the isolated bacterial populations was similar in rhizosphere and roots, but lower in soil and stem compartments. Burkoholderia, Alcaligenes, Agrococcus, Polaromonas, Stenotrophomonas, Serratia, Microbacterium, and Caulobacter were found as root endophytes exclusively. The inoculation of seeds with Pseudomonas sp. strains 228 and 256, and Serratia sp. strain 246 facilitated the root development of B. napus at 1,000 µM Zn. Arthrobacter sp. strain 222, Serratia sp. strain 246, and Pseudomonas sp. 228 and 262 increased the root length at 300 µM Cd.     

Removal of malachite green from aqueous solution by immobilized Pseudomonas sp. DY1 with Aspergillus oryzae

Triphenylmethane dyes are considered to be one of the most recalcitrant pollutants in the environment. Malachite Green (MG) was successfully removed from aqueous solution by Pseudomonas sp. DY1 immobilization with Aspergillus oryzae. Inhibition test in the presence of sodium azide and nystatin indicated that A. oryzae was a natural immobilization reagent, and removal of MG by the immobilized cell pellets was attributed to the biodegradation by Pseudomonas sp. DY1. Optimum conditions of immobilization for maximum biodegradation were obtained using Taguchi design at 37 °C, inoculation size of Pseudomonas sp. DY1 (dry cell mass) 0.01 g, of A. oryzae (spore number) 1.0 × 10⁹, initial pH 6.5. Decolorization and biodegradation of MG by immobilized pellets under optimum conditions were 99.5% and 93.3%, respectively. Immobilized pellets exhibited more than 96% decolorization after 16 days in batch condition, indicating it had stable and high biodegradation capabilities when immobilized for long-term operation.     

Use of a microbial fuel cell for the rapid enumeration of bacteria

Summary The detection of bacteria using a thionine mediated microbial fuel cell was examined. On addition of bacteria to the anode compartment of a fuel cell, a rapid increase in the current output was observed. Both the total change in the steady state current (mA) and the initial rate of change of current were proportional to the numbers of bacteria added. Regression analysis of plots of log₁₀ mA against log₁₀ bacteria ml^-1 (final concentration) upon the addition of E. coli K12, Lactococcus lactis, coliform sp. A1, Micrococcus sp. M3 but not Pseudomonas sp. P5 gave reasonable correlation coefficients. Determination of the rates of respiration and thionine reduction by E. coli indicated that the transfer of metabolic electrons from the bacteria to the mediator was reasonably efficient (approx. 50%). These results are discussed with respect to the potential application of this technique for the rapid estimation of the bacterial contamination of foods.     

Elicitation of alkaloids in in vitro PLB (protocorm-like body) cultures of Pinellia ternata

The objective of this study was to determine the effect of two endophytic bacterial elicitors (Pseudomonas sp. and Enterobacter sp.) on the production of alkaloids in protocorm-like bodies (PLBs) of Pinellia ternata Breit. Both bacterial strains increased the growth rate of P. ternata PLBs. Pseudomonas sp. promoted the differentiation of the PLBs, whereas Enterobacter sp. inhibited PLB differentiation. The bacterial strains increased guanosine production in PLBs by 9–166%, inosine production by 2–33%, and trigonelline production by 114–1140% compared to the control. For Pseudomonas sp., guanosine and trigonelline production was greater when bacterial extracts were added to the PLB suspension cultures rather than living cells (co-culture treatment). Inosine production was similar in both the bacterial extract and co-culture treatments. For the Enterobacter sp., guanosine, inosine, and trigonelline production tended to be greatest when living cells were added to the PLB suspension cultures rather than bacterial extracts. These results suggest that Pseudomonas sp. and Enterobacter sp. could increase alkaloid yield from P. ternata under field or tissue culture conditions. We also observed that Pseudomonas sp. and Enterobacter sp. produced some of the same alkaloids as their host plants. Additional study needs to be done to determine if these endophytic bacteria could be used to produce alkaloids in the fermentation industry.     

Synergistic interaction between two bacterial isolates in the degradation of carbofuran

The dominant bacteriaPseudomonas sp. andArthrobacter sp. were isolated from the standing water of carbofuran-retreatedAzolla plot.Arthrobacter sp. hydrolysed carbofuran added to the mineral salts medium as a sole source of carbon and nitrogen while no degradation occurred withPseudomonas sp. Interestingly, when the medium containing carbofuran was inoculated with bothArthrobacter sp. andPseudomonas sp., a synergistic increase in its hydrolysis and subsequent release of CO₂ from the side chain was noticed. This synergistic interaction was better expressed at 25° C than at 35° C. Likewise, related carbamates, carbaryl, bendiocarb and carbosulfan were more rapidly degraded in the combined presence of both bacterial isolates.     

Adhesive properties of predominant bacteria in raw cow’s milk to bovine mammary gland epithelial cells

Various bacteria have been found in raw cow’s milk, and identifying milk microflora and its functions is critical for maintaining cow health and farm hygiene. Although studies on pathogens and spoilage bacteria in milk have been widely reported, the relationship between milk bacteria, including nonpathogenic bacteria, and the bovine udder is poorly understood. We investigated milk microflora over 1 year using a culture-dependent method and culture-independent analysis by denaturing gradient gel electrophoresis. Among 240 isolates, Lactococcus lactis (81/240) was predominant. The predominant genera were Lactococcus, Stenotrophomonas, Microbacterium, Chryseobacterium, Serratia and Pseudomonas. Among seven strains belonging to these predominant genera, two strains of L. lactis (ssp. lactis and ssp. cremoris) exhibited the highest adherence to bovine mammary gland epithelial cells (BMECs) derived from the bovine udder; 3.4 % of the inoculated bacteria adhered to BMECs. This was followed by Serratia sp. (1.6 %), Microbacterium sp. (0.8 %), Stenotrophomonas maltophilia (0.5 %), Pseudomonas sp. (0.3 %) and Chryseobacterium sp. (0.1 %). The two L. lactis isolates exhibited higher adherence to BMECs than type strains and isolates of various origins.     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Reduction of Cr(VI) by Desulfovibrio vulgaris and Microbacterium sp.

Many industrial wastes contain Cr(VI), a carcinogen and mutagen, the toxicity of which can be ameliorated by reduction to Cr(III). Microbacterium sp. NCIMB 13776 andDesulfovibrio vulgaris NCIMB 8303 reduced Cr(VI) to Cr(III) anoxically using 25 mM sodium citrate buffer (pH 7), with 25 mM sodium acetate and 25 mM sodium formate as electron donors at 30 °C, under which conditions the rates of reduction of 500 M sodium chromate were 77 and 6 nmol h^–1 mg dry cell wt for D. vulgaris and Microbacterium sp., respectively, these being increased to 127 and 17 nmol h^–1 mg dry cell wt in the presence of 20 mM MOPS/NaOH buffer.     

Microbial biodiversity and in situ bioremediation of endosulfan contaminated soil

Molecular characterization based on 16s rDNA gene sequence analysis of bacterial colonies isolated from endosulfan contaminated soil showed the presence of Ochrobacterum sp, Burkholderia sp, Pseudomonas alcaligenes, Pseudomonas sp and Arthrobacter sp which degraded 57–90% of α-endosulfan and 74–94% of β-endosulfan after 7days. Whole cells of Pseudomonas sp and Pseudomonas alcaligenes showed 94 and 89% uptake of α-isomer and 86 and 89% of β-endosulfan respectively in 120 min. In Pseudomonas sp, endosulfan sulfate was the major metabolite detected during the degradation of α-isomer, with minor amount of endosulfan diol while in Pseudomonas alcaligenes endosulfan diol was the only product during α-endosulfan degradation. Whole cells of Pseudomonas sp also utilized 83% of endosulfan sulfate in 120 min. In situ applications of the defined consortium consisting of Pseudomonas alcaligenes and Pseudomonas sp (1:1) in plots contaminated with endosulfan showed that 80% of α-endosulfan and 65% of β-endosulfan was degraded after 12 weeks of incubation. Endosulfan sulfate formed during endosulfan degradation was subsequently degraded to unknown metabolites. ERIC-PCR analysis indicated 80% survival of introduced population of Pseudomonas alcaligenes and Pseudomonas sp in treated plots.     

Biodegradation kinetics of the nitramine explosive CL-20 in soil and microbial cultures

The cyclic nitramine explosive CL-20 (C₆H₆N₁₂O₁₂, 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12 -hexaazaisowurtzitane) is a relatively new energetic compound which could be a persistent organic pollutant. To follow its biodegradation dynamics, CL-20 was added to soil alone or together with organic co-substrates and N-source and incubated under oxic and anoxic conditions. Without co-substrates, the CL-20 degradation was detectable only under anoxic conditions. The highest degradation rate was found under aerobic conditions and with the addition of co-substrates, succinate and pyruvate being more efficient than acetate, glucose, starch or yeast extract. When added to intact soil, CL-20 degradation was not affected by the N content, but in soil serially diluted with N-free succinate-mineral medium, the process became N-limited. About 40% of randomly selected bacterial colonies grown on succinate agar medium were able to decompose CL-20. Based on 16S rDNA gene sequence and cell morphology, they were affiliated to Pseudomonas, Rhodococcus, Ochrobactrum, Mycobacterium and Ralstonia. In the pure culture of Pseudomonas sp. MS-P grown on the succinate-mineral N(+) medium, the degradation kinetics were first order with the same apparent kinetic constant throughout growth and decline phases of the batch culture. The observed kinetics agreed with the model that supposes co-metabolic transformation of CL-20 uncoupled from cell growth, which can be carried out by several constitutive cellular enzymes with wide substrate specificity. The GenBank accession numbers for the 16S rRNA gene sequences obtained on this study are AY773005–AY773010. Pseudomonas sp. MS-P (=B-41417) was deposited with Agriculture Research Service Culture Collection, USA.     

Genotypic Characterization and Phylogenetic Relations of Pseudomonas sp. (Formerly P. stutzeri) OX1

Pseudomonas sp. OX1, an aromatic compound-degrading bacterium that was tentatively identified by conventional biochemical methods as P. stutzeri, has now been investigated at the molecular level to clarify its taxonomic position. Amplified ribosomal DNA restriction analysis and multiple enzyme restriction fragment length polymorphism (MERFLP) analysis suggested that Pseudomonas sp. OX1 could not be classified as P. stutzeri. Phylogenetic analyses based on 16S rRNA and gyrB genes further confirmed that this strain belongs to the Pseudomonas (sensu stricto) genus, but not to the stutzeri species. The data obtained demonstrated that Pseudomonas sp. OX1 belongs to intrageneric cluster II and is related to the P. fluorescens–P. syringae complex.     

The structure of a pyoverdine from Pseudomonas sp. CFML 96.188 and its relation to other pyoverdines with a cyclic C-terminus

From Pseudomonas sp. CFML 96.188 a pyoverdine was isolated and its primary structure was elucidated by spectroscopic methods and degradation reactions. This strain is of interest as it accepts the structurally different pyoverdines from several other Pseudomonas strains. They all have in common as a specific structural feature a C-terminal cyclic substructure, the importance of which for the recognition of a pyoverdine at the cell surface of a given strain will be discussed.     

Biological Control of Chickpea Collar Rot by Co-inoculation of Antagonistic Bacteria and Compatible Rhizobia

Two hundred and seven bacteria were isolated from composts and macrofauna and screened for plant growth promoting and antagonistic traits. Seven of the 207 isolates showed antagonistic activity against Sclerotium rolfsii in plate culture. Inhibition of S. rolfsii by the bacterial isolates ranged between 61 and 84%. Two of the seven isolates were Bacillus sp. and rest belonged to Pseudomonas sp. Two isolates, Pseudomonas sp. CDB 35 and Pseudomonas sp. BWB 21 was compatible with chickpea Rhizobium sp. IC 59 and IC 76 in plate culture conditions. Increase in plant biomass (dry weight) ranged between 18 and 30% on application of these bacteria by seed coating and seed priming methods. However, by seed-priming there was an increase in plant biomass by 5–7% compared to seed coating. Number of nodules and the nodule weight was similar by both seed coating and seed priming methods. Disease incidence was reduced up to 47% in treatments where captan (fungicide) or antagonistic Pseudomonas sp. CDB 35 was applied. Increase in shoot weight was 36% by seed coating with Rhizobium sp. IC 59 and Pseudomonas sp. CDB 35 when compared to captan application. Whereas by seed priming with IC 59 and CDB 35 increased shoot weight by 3 and 39% increase in nodulation was observed.     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.