Endophytic colonization of olive roots by the biocontrol strain Pseudomonas fluorescens PICF7

Confocal microscopy combined with three-dimensional olive root tissue sectioning was used to provide evidence of the endophytic behaviour of Pseudomonas fluorescens PICF7, an effective biocontrol strain against Verticillium wilt of olive. Two derivatives of the green fluorescent protein (GFP), the enhanced green and the red fluorescent proteins, have been used to visualize simultaneously two differently fluorescently tagged populations of P. fluorescens PICF7 within olive root tissues at the single cell level. The time-course of colonization events of olive roots cv. Arbequina by strain PICF7 and the localization of tagged bacteria within olive root tissues are described. First, bacteria rapidly colonized root surfaces and were predominantly found in the differentiation zone. Thereafter, microscopy observations showed that PICF7-tagged populations eventually disappeared from the root surface, and increasingly colonized inner root tissues. Localized and limited endophytic colonization by the introduced bacteria was observed over time. Fluorescent-tagged bacteria were always visualized in the intercellular spaces of the cortex region, and no colonization of the root xylem vessels was detected at any time. To the best of our knowledge, this is the first time this approach has been used to demonstrate endophytism of a biocontrol Pseudomonas spp. strain in a woody host such as olive using a nongnotobiotic system.     

Influence of environmental conditions, bacterial activity and viability on the viral component in 10 Antarctic lakes

The influence of biotic and environmental variables on the abundance of virus-like particles (VLP) and lysogeny was investigated by examining 10 Antarctic lakes in the Vestfold Hills, Antarctica, in the Austral Spring. Abundances of viruses and bacteria and bacterial metabolic activity were estimated using SYBR Gold (Molecular Probes), Baclight (Molecular Probes) and 6-carboxy fluorescein diacetate (6CFDA). Total bacterial abundances among the lakes ranged between 0.12 and 0.47 x 10(9) cells L(-1). The proportion of intact bacteria (SYTO 9-stained cells) ranged from 13.5% to 83.5% of the total while active (6CFDA-stained) bacteria ranged from 33% to 116%. Lysogeny, as determined with Mitomycin C, was only detected in one of the lakes surveyed, indicating that viral replication was occurring predominantly via the lytic cycle. Principal component analysis and confirmatory correlation analysis of individual variables showed that high abundances of VLP occurred in lakes of high conductivity with high concentrations of soluble reactive phosphorus and dissolved organic carbon. These lakes supported high concentrations of chlorophyll a, intact bacteria, rates of bacterial production and virus to bacteria ratios. Thus, it was suggested that viral abundance in the Antarctic lakes was determined by the trophic status of the lake and the resultant abundance of intact bacterial hosts.     

First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA

Nitrite oxidoreductase (NXR) is the key enzyme responsible for the oxidation of NO(2)(-) to NO(3)(-) in nitrite-oxidizing bacteria. For the first time a molecular approach for targeting the nxrA gene was developed, encoding the catalytic subunit of the NXR, to study diversity of Nitrobacter-like organisms based on the phylogeny of nxrA gene sequences in soils. NxrA sequences of the Nitrobacter strains analysed (Nitrobacter hamburgensis, Nitrobacter vulgaris, Nitrobacter winogradskyi, Nitrobacter alkalicus) by PCR, cloning and sequencing revealed the occurrence of multiple copies of nxrA genes in these strains. The copy number and similarity varied among strains. The diversity of Nitrobacter-like nxrA sequences was explored in three soils (a French permanent pasture soil, a French fallow soil, and an African savannah soil) using a cloning and sequencing approach. Most nxrA sequences found in these soils (84%) differed from nxrA sequences obtained from Nitrobacter strains. Moreover, the phylogenetic distribution and richness of nxrA-like sequences was extremely variable depending on soil type. This nxrA tool extends the panel of functional genes available for studying bacteria involved in the nitrogen cycle.     

Sulphur-oxidizing extracellular bacteria in the gills of Mytilidae associated with wood falls

Six morphotypes of small mussels (Bivalvia: Mytilidae) were found attached to naturally sunken wood collected in the Bohol Sea (Philippines). These specimens are related to the large Bathymodiolus mussels that are found worldwide at cold seeps and hydrothermal vents. In these habitats, the mytilids harbour sulphur- and methane-oxidizing endosymbionts in their gills and depend on the energy and carbon provided by the symbionts. In this study, bacteria associated with the gills of wood-associated mussels are characterized using molecular and microscopic techniques. The existence of bacteria in the lateral zone of gill filaments in all specimens is demonstrated. Comparative analyses of 16S rRNA gene and adenosine 5'-phosphosulphate (APS) reductase gene sequences indicate that the bacteria are closely related to sulphur-oxidizing endosymbionts of Bathymodiolus. FISHs using specific probes confirm that sulphur oxidizers are by far the most abundant, if not the only bacteria present. Electron micrographs displayed mostly extracellular bacteria located between microvilli at the apical surface of host gill epithelial cells all along the lateral zone of each gill filament. In some specimens, occasional occurrence of intracellular bacteria with similar morphology was noted. This study provides the first molecular evidence for the presence of possible thiotrophic symbiosis in sunken wood ecosystems. With their epibiotic bacteria, wood-associated mussels display a less integrated type of interaction than described in their seep, vent and whale fall relatives.     

Phylogenetic analysis of epiphytic marine bacteria on Hole-Rotten diseased sporophytes of <Emphasis Type="Italic">Laminaria japonica</Emphasis>

During an occurrence of Hole-Rotten Disease of Laminaria japonica in a cultivating farm in Ma Shan Shandong province, China, 42 Gram-negative epiphytic marine bacteria were isolated and purified on Zobell 2216E marine agar medium. Morphological and biochemical characteristics of each isolated bacterium were studied, and molecular identification of bacterial strains was conducted with polymerase chain reaction amplification to 16S rRNA gene sequence analysis. Based on nearly full length of 16S rRNA gene sequence analysis, the isolated strains were bacteria that belong to genus Pseudoalteromonas, Vibrio, Halomonas and Bacillus. The percentage of each group was 61.9%, 28.6%, 7.1% and 2.4% respectively. The results of pathogenicity assay showed that 12 strains could cause the disease symptoms in sporophytes of L. japonica. They belonged to the genera Pseudoalteromonas, Vibrio and Halomonas with 58.3%, 33.3%, 8.3% respectively. The results suggest that these bacteria are the dominant marine bacteria on diseased sporophytes of L. japonica and may be the potential pathogenic bacteria associated with Hole-Rotten Disease of L. japonica.     

A novel moderately halophilic bacterium for decolorizing azo dye under high salt condition

Halomonas sp strain GTW was newly isolated from coastal sediments contaminated by chemical wastewater and was identified to be a member of the genus Halomonas by 16S rDNA sequence analysis and physical and biochemical tests. The optimal decolorization conditions were as follows: temperature 30°C, pH 6.5.0–8.5, NaCl 10–20% (w/v) and the optimal carbon source was yeast exact. The results of experiments demonstrated that the bacteria could decolorize different azo dyes under high salt concentration conditions, and the decolorization rate of five tested azo dyes could be above 90% in 24 h. The exploitation of the salt-tolerant bacteria in the bio-treatment system would be a great improvement of conventional biological treatment systems and the bio-treatment concept.     

The new incorporation bio-treatment technology of bromoamine acid and azo dyes wastewaters under high-salt conditions

The accelerating effect of quinones has been studied in the bio-decolorization processes, but there are no literatures about the incorporation bio-treatment technology of the bromoamine acid (BA) wastewater and azo dyes wastewaters under high-salt conditions (NaCl, 15%, w/w). Here we described the BA wastewater as a redox mediator in the bio-decolorization of azo dye wastewaters. Decolorization of azo dyes was carried out experimentally using the salt-tolerant bacteria under the BA wastewater and high-salt conditions. The BA wastewater used as a redox mediator was able to increase the decolorization rate of wastewater containing azo dyes. The effects of various operating conditions such as dissolved oxygen, temperature, and pH on microbial decolorization were investigated experimentally. At the same time, BA was tested to assess the effects on the change of the Oxidation–Reduction Potential (ORP) values during the decolorization processes. The experiments explored a great improvement of the redox mediator application and the new bio-treatment concept.     

TNT biodegradation and production of dihydroxylamino-nitrotoluene by aerobic TNT degrader <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain TM15 in an anoxic environment

Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10⁹ cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10¹⁰ cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of lactic acid bacteria isolated from sourdoughs for Cornetto, a traditional bread produced in Basilicata (Southern Italy)

A total of 41 strains of lactic acid bacteria (LAB) isolated from durum wheat sourdoughs used to produce Cornetto di Matera bread, were identified by SDS-PAGE of whole cell proteins (WCP) and screened for acid production ability, antimicrobial activity and exopolysaccharide (EPS) production. The isolates were identified as Lactobacillus plantarum (49%), Leuconostoc mesenteroides (17%), Lactobacillus curvatus (15%), Lactobacillus paraplantarum (12%), Weissella cibaria (5%) and Lactobacillus pentosus (2%). Several strains of Lb. plantarum and Leuc. mesenteroides showed a high acid production ability. The antagonistic activity was tested using an agar-spot deferred antagonism assay against a set of five indicators. The species had different profiles of inhibition. Lb. plantarum had the largest spectrum of inhibition, while no isolates of W. cibaria and Leuc. mesenteroides showed antimicrobial activity. No strains had antimicrobial activity against Bacillus cereus. The inhibitory activity of five strains was confirmed to be sensitive to proteolytic enzymes and thus potentially due to bacteriocin production. All Leuc. mesenteroides and W. cibaria strains produced EPS from sucrose. Some Lb. plantarum and Lb. paraplantarum strains produced EPS from different sugars in solid media. EPS production in liquid media was different within the species, with the highest production in liquid media containing glucose and maltose. A defined strain starter culture (W. cibaria DBPZ1006, Lb. plantarum DBPZ1015 and S. cerevisiae MTG10) was selected on the basis of technological properties and tested in model sourdough fermentations.     

Enhancement of biogenic sulfide production in a packed-bed bioreactor via critical inoculum design and carrier material selection

Sulfate reducing bacteria (SRB) are commonly used in environmental bioprocesses for the treatment of acid mine drainage and sulfate wastewaters. Biogenic H(2)S is also a potential source of H(2) fuel with the recent development of H(2)S splitting technologies. In this study, a sulfate reducing packed bed bioreactor (PBR) capable of rapidly achieving high volumetric productivities was developed using a novel method of rational inoculum design and the selection of improved biomass carrier materials. An inoculum with initial composition of approximately 95% Desulfovibrio desulfuricans (ATCC 7757) and 5% SRB consortium was designed based on the pure strain's superior immobilization potential and the SRB consortium's superior kinetics. Diatomaceous earth (DE) pellets, porous glass beads, polyurethane foam and bone char were evaluated as potential biomass carrier materials. The DE pellets immobilized the most biomass and were employed in two packed bed bioreactor fermentations. Using the designed inoculum and DE pellets, a packed bed bioreactor achieved a volumetric productivity of 493 mol H(2)S m(-3) day(-1) (based on a 308 mL working volume) with a dissolved sulfide concentration of 9.9 mM. This occurred after 8.3 days of operation and represents a tenfold reduction in the start-up period compared to other sulfate reducing PBRs described in the literature.