Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota.

AIMS: The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota. METHODS AND RESULTS: A 10-day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide-degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum-probiotic capsules was detected a significant increase in Lactobacillus-Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples. CONCLUSIONS: Exopolysaccharides constitute an interesting approach for colon-targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier. SIGNIFICANCE AND IMPACT OF STUDY: This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted-delivery coating material.

Bioremediation and biovalorisation of olive-mill wastes.

Olive-mill wastes are produced by the industry of olive oil production, which is a very important economic activity, particularly for Spain, Italy and Greece, leading to a large environmental problem of current concern in the Mediterranean basin. There is as yet no accepted treatment method for all the wastes generated during olive oil production, mainly due to technical and economical limitations but also the scattered nature of olive mills across the Mediterranean basin. The production of virgin olive oil is expanding worldwide, which will lead to even larger amounts of olive-mill waste, unless new treatment and valorisation technologies are devised. These are encouraged by the trend of current environmental policies, which favour protocols that include valorisation of the waste. This makes biological treatments of particular interest. Thus, research into different biodegradation options for olive-mill wastes and the development of new bioremediation technologies and/or strategies, as well as the valorisation of microbial biotechnology, are all currently needed. This review, whilst presenting a general overview, focus critically on the most significant recent advances in the various types of biological treatments, the bioremediation technology most commonly applied and the valorisation options, which together will form the pillar for future developments within this field.

Molecular microbial and chemical investigation of the bioremediation of two-phase olive mill waste using laboratory-scale bioreactors.

Two-phase olive mill waste (TPOMW) is a semisolid effluent that is rich in contaminating polyphenols and is produced in large amounts by the industry of olive oil production. Laboratory-scale bioreactors were used to investigate the biodegradation of TPOMW by its indigenous microbiota. The effect of nutrient addition (inorganic N and P) and aeration of the bioreactors was studied. Microbial changes were investigated by PCR-temperature time gradient electrophoresis (TTGE) and following the dynamics of polar lipid fatty acids (PLFA). The greatest decrease in the polyphenolic and organic matter contents of bioreactors was concomitant with an increase in the PLFA fungal/bacterial ratio. Amplicon sequences of nuclear ribosomal internal transcribed spacer region (ITS) and 16S rDNA allowed identification of fungal and bacterial types, respectively, by comparative DNA sequence analyses. Predominant fungi identified included members of the genera Penicillium, Candida, Geotrichum, Pichia, Cladosporium, and Aschochyta. A total of 14 bacterial genera were detected, with a dominance of organisms that have previously been associated with plant material. Overall, this work highlights that indigenous microbiota within the bioreactors through stimulation of the fungal fraction, is able to degrade the polyphenolic content without the inoculation of specific microorganisms.

Halomonas sabkhae sp. nov., a moderately halophilic bacterium isolated from an Algerian sabkha.

A novel moderately halophilic bacterium belonging to the genus Halomonas was isolated from brine samples collected from Ezzemoul sabkha in north-eastern Algeria. The cells of strain 5-3(T) were Gram-negative, rod-shaped and non-motile. The strain was catalase- and oxidase-positive and produced an exopolysaccharide. Growth occurred at NaCl concentrations of 5-25% (optimum at 7.5%), at 30-50 degrees C (optimum at 37-40 degrees C) and at pH 6.0-9.0 (optimum at pH 7.5). The major fatty acids were C(12:0) 3-OH, C(16:1)omega7c/iso-C(15:0) 2-OH, C(16:0), C(18:1)omega7c and C(19:0)omega8c cyclo. The G+C content of the genomic DNA was 57.0 mol% (T(m)). The affiliation of strain 5-3(T) with the genus Halomonas was confirmed by 16S rRNA gene sequence comparisons. The most closely related species was Halomonas halmophila, which showed a 16S rRNA gene sequence similarity of 99.7%. However, the level of DNA-DNA relatedness between the novel isolate and the related Halomonas species was less than 31.4%. On the basis of the data from this polyphasic study, strain 5-3(T) represents a novel species of the genus Halomonas, for which the name Halomonas sabkhae sp. nov. is proposed. The type strain is 5-3(T) (=CECT 7246(T)=DSM 19122(T)=LMG 24084(T)).

Estudio de aplicabilidad como aditivo alimentario de un nuevo exopolisacárido obtenido de Paenibacillus jamilae / Study of the use of a new exopolysaccharide obtained from Paenibacillus jamilae in food additives

Paenibacillus jamilae es una nueva especie bacteriana formadora de esporas, recientemente descrita por nuestro grupo de investigación y aislada a partir de una muestra de compost de maíz tratado con alpechín. P. jamilae es capaz de producir un nuevo exopolisacárido (EPS) en medio con sacarosa como única fuente de carbono. Este nuevo EPS ofrece algunas características reológicas de interés. La suspensión de este EPS en leche entera al 1 por ciento (p/V) presentó una viscosidad 5.7 veces mayor a la ofrecida en agua destilada a la misma concentración y mostró un claro carácter pseudoplástico. El gel formado con carragenanos, polímeros muy utilizados en la industria alimentaria, presentó buenas características en su aplicación como posible aditivo alimentario por una menor pérdida de agua (AU)

Paenibacillus jamilae sp. nov., an exopolysaccharide-producing bacterium able to grow in olive-mill wastewater.

Endospore-forming strains were isolated from corn-compost treated with olive-mill wastewater ('alpechin'). The strains were taxonomically studied and proposed as a novel Paenibacillus species. These organisms (strains B.3T, B.7 and B.9) were particularly distinguishable from other aerobic spore-forming species by their ability to grow optimally in 100% (v/v) olive-mill wastewater at 30 degrees C and pH 7.0 and concomitant production of an interesting exopolysaccharide. Chemotaxonomic analysis revealed that MK-7 was the predominant menaquinone, the major fatty acid was anteiso C15:0 and the cell wall contained meso-diaminopimelic acid. The DNA G+C content was 40.7 mol%. Comparative sequence analysis of 16S rDNA with different reference species from the genera Bacillus, Paenibacillus, Brevibacillus, Aneurinibacillus, Alicyclobacillus, Halobacillus, Virgibacillus, Amphibacillus, Coprobacillus and Gracilibacillus indicated that the isolated strains were highly related to the genus Paenibacillus. Strain B.3T formed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus. Strain B.3T was a close relative of Paenibacillus polymyxa, but DNA-DNA relatedness data with this species was very low (relative binding ratio < 16%). Based on the morphological and physiological characteristics, as well as on the phylogenetic position determined by 16S rDNA analysis and DNA-DNA relatedness data, it is concluded that these strains should be designated a novel species, for which the name Paenibacillus jamilae sp. nov. is proposed. The type strain is B.3T (= CECT 5266T = DSM 13815T).

Biological response modifier activity of an exopolysaccharide from Paenibacillus jamilae CP-7.

An extracellular polysaccharide was purified from culture supernatants of Paenibacillus jamilae CP-7, a gram-positive bacillus that was isolated from compost prepared with olive mill wastewaters. The extracellular polysaccharide was produced under aerobic conditions in a medium containing olive mill wastewaters (80% [vol/vol]). This exopolymer had a low level of acute toxicity when it is administered to BALB/c mice by the intraperitoneal route. Interesting immunomodulatory effects were detected when mice were given 10 mg of exopolysaccharide per kg of body weight; the proliferative responses of splenocytes to B-cell and T-cell mitogens were suppressed, the in vitro levels of production of gamma interferon and granulocyte-macrophage colony-stimulating factor by unstimulated and lipopolysaccharide-stimulated splenocytes were enhanced, and the levels of resistance to the intracellular pathogen Listeria monocytogenes was increased in mice. Also, the exopolysaccharide was able to induce lymphocyte proliferation in vitro. We conclude that P. jamilae produces an exopolysaccharide with interesting immunomodulatory properties.

The effect of olive mill wastewaters variability on xanthan production.

AIMS: Xanthan production by Xanthomonas campestris from several olive mill wastewaters (OMW) was investigated. METHODS AND RESULTS: Maximum xanthan production of 4 g l(-1) was obtained in media with 50% OMW as sole source of nutrients. OMW storage decreased effluent quality for xanthan production. The range of effluent concentration for X. campestris growth and xanthan production varied depending on OMW extraction METHOD: Wastewaters from press and two-phase extraction methods required higher dilution rates (< 10%) than those from the three-phase extraction method (50%). Nitrogen supplementation improved xanthan production in press and two-phase OMW. CONCLUSION: Factors affecting wastewaters composition, namely, waste storage, time of olive harvesting, and method for oil extraction, were found to influence xanthan production in shake-flask cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: Conditions for xanthan production from OMW should be optimized in accordance with the nature of the waste material.

Xanthomonas campestris strain selection for xanthan production from olive mill wastewaters.

Four Xanthomonas campestris strains were tested in olive mill wastewaters (OMW) for xanthan production. Differences among strains were found in the range of tolerance to OMW concentration and xanthan amount obtained. X. campestris NRRL B-1459 S4LII was chosen by its capability for xanthan production from 50-60% OMW as the sole nutrient source.

Taxonomic study of extreme halophilic archaea isolated from the "Salar de Atacama", Chile.

A large number of halophilic bacteria were isolated in 1984-1992 from the Atacama Saltern (North of Chile). For this study 82 strains of extreme halophilic archaea were selected. The characterization was performed by using the phenotypic characters including morphological, physiological, biochemical, nutritional and antimicrobial susceptibility test. The results, together with those from reference strains, were subjected to numerical analysis, using the Simple Matching (S(SM)) coefficient and clustered by the unweighted pair group method of association (UPGMA). Fifteen phena were obtained at an 70% similarity level. The results obtained reveal a high diversity among the halophilic archaea isolated. Representative strains from the phena were chosen to determine their DNA base composition and the percentage of DNA-DNA similarity compared to reference strains. The 16S rRNA studies showed that some of these strains constitutes a new taxa of extreme halophilic archaea.

Precipitation of carbonates by Nesterenkonia halobia in liquid media.

We investigated the precipitation of carbonates by Nesterenkonia halobia in a liquid medium at different concentrations of salts and incubation times. N. halobia only produced crystals at salt concentrations of 2.5%, 7.5% and 15%. At 20% salt concentration no crystal formation was observed. Calcite, aragonite and dolomite were precipitated in different quantities, depending on the salinity of the medium and incubation time. Scanning and transmission electron microscopy, microanalysis and electron diffraction were all used to study in detail the morphology, composition and internal structure of the bioliths. We propose a mechanism for biolith formation involving both biological and inorganic processes.

Microorganismos halófitos: potencial biotecnológico e interés farmacéutico / Biotechnological potential and pharmaceutical interest of Halophilic Microorganisms

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Effect of Halococcus morrhuae and Halobacterium saccharovorum on the activation of human peripheral blood lymphocytes.

The immunomodulator properties of two species of halophilic Archaebacteria, Halobacterium saccharovorum and Halococcus rnorrhuae, were analysed by the study of lymphocyte activation. Two methods were used to detect activation in lymphocytes, namely incorporation of the radioactive nucleotide [3H]-thymidine, and CD25 expression. H. morrhuae had a stimulatory effect on human lymphocytes, but this action was observed only with the [3H]-thymidine uptake method, whereas H. saccharovorum produced no immunomodulator effect.

Biomineralization of carbonates by Marinococcus albus and Marinococcus halophilus isolated from the Salar de Atacama (Chile).

We studied the precipitation of carbonates in 17 strains of moderately halophilic, Gram-positive cocci belonging to two species: Marinococcus halophilus and Marinococcus albus, isolated from the Salar de Atacama (Chile). They were cultivated in solid and liquid laboratory media for 42 days at salt concentrations (wt/vol) of 3%, 7.5%, 15%, and 20%. The bioliths precipitated were studied by X-ray diffraction and scanning electron microscopy. M. halophilus formed crystals at each of the salt concentrations, with a maximum number of strains capable of precipitating carbonates at 7.5% and 15% salt concentrations. M. albus did not precipitate at 20% and showed a maximum at 7.5%. This behavior is similar to that of other gram-positive bacteria and differs from that found in gram-negative bacteria. The bioliths precipitated were spherical, generally isolated, with a size of 10-100 microm, varying with salinity. They were of magnesium calcite (CO3 Ca1-x Mgx) with Mg content increasing with increasing salinity and Mg/Ca molar ratio of the culture medium. These results demonstrate the active role played by M. halophilus and M. albus in the precipitation of carbonates.

A study of struvite precipitation and urease activity in bacteria isolated from patients with urinary infections and their possible involvement in the formation of renal calculi.

Struvite precipitation and urease activity were studied in 72 bacterial strains isolated from patients with an urinary infection. The results revealed struvite precipitation by urease-positive, urease-negative and weakly urealitic bacteria. However, in some strains of each of these groups, no precipitation was observed. Variations in pH within the cultures were also investigated as were the mineralogy and morphology of the crystals precipitated using X-ray diffraction and scanning electron microscopy, respectively. The role of urease activity and alkalinization of the medium in struvite precipitation is discussed, as is the possible involvement of bacteria in nucleation processes. We concluded that urease-negative bacteria or those with weak urease activity may also be involved in the formation of struvite renal calculi although urease-positive bacteria seem to make a greater contribution.

Influence of salt concentration on the cellular fatty acid composition of the moderately halophilic bacterium Halomonas salina.

The cellular fatty acid composition of Halomonas salina, a moderately halophilic bacterium grown at different salt concentrations, is reported. Fatty acids C16:0 and C18:1 were major components and significant amounts of C16:1, C18:0 and cyc-C19:0 were also detected. The results showed clear chemotaxonomic relationships with recognized members of the genus Halomonas. The salt concentration greatly influenced the fatty acid composition, suggesting activation of cyclopropane synthetase at high levels of salt, since increases in cyclopropane fatty acids with decreases in monounsaturated fatty acids were observed as the salt concentration in the medium rose.

Biomineralization of carbonates by Halomonas eurihalina in solid and liquid media with different salinities: crystal formation sequence.

Carbonate precipitation by 20 strains of the moderately halophilic species Halomonas eurihalina in both solid and liquid media was studied. The influence of salinity and temperature on the quantity and type of crystals precipitated was also investigated. Some strains of H. eurihalina formed crystals in all conditions tested. The mineral phases precipitated were magnesium calcite, aragonite and monohydrocalcite in variable proportions depending on various factors such as the type of growth medium employed and its salinity. Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the crystal formation sequence. The process of biolith formation was sequential. It started with chains or filaments of bacteria, giving way to discs which finally produced spherical forms of approximately 50 microns in diameter. We suggest a mechanism of carbonate crystal formation by H. eurihalina.

Antimicrobial efficiency of hydrogel contact lens soaking solutions marketed in Spain.

The antimicrobial efficiency of 20 commercially available solutions for soaking and rinsing soft contact lenses was studied in relation to 5 bacteria (Escherichia coli, Staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus subtilis, and Serratia marcescens) and 1 fungus (Candida albicans). Each product was separately inoculated with each of six microorganisms, and samples of the inoculated contact lens solutions were taken at predetermined times, placed in a recovery medium, and incubated. Where there was growth, the colonies were counted. There were differences in performance even between solutions labeled as having the same antimicrobial content. One of the solutions marketed in Spain to soak hydrogel contact lenses failed to inactivate all six test strains.

Bacterial contamination of hydrophilic contact lens solutions marketed in spain.

Bacterial contamination of hydrophilic contact lens solutions may play an important role in contact lens-associated ocular infections. This study investigated bacterial contamination in 52 different hydrophilic contact lens solutions marketed in Spain by 12 different companies. We filtered the entire contents of 5 new, factory-sealed bottles from each of the 52 brands and cultured the fitter on a neutralizing broth plate. Bacteria were cultured, isolated, and identified from 29 of the 260 bottles tested (11.15%). Eight of the 52 brands had at least 1 of the 5 bottles contaminated (15.38%). Contaminated solutions originated from four different companies. One manufacturer contributed most of the positive cases due, presumably, to an industrial contamination by Pseudomonas fluorescens. The rest of the culture-positive bottles were contaminated by Bacillus spp. and Oerskovia spp.

Process of Carbonate Precipitation by Deleya halophila

Scanning electron microscopy and X-ray dispersive energy microanalysis were used to investigate the formation of carbonate crystals by Deleya halophila. The formation of calcium carbonate crystals (polymorphous aragonite) by D. halophila is a sequential process that commences with a nucleus formed by the aggregation of a few calcified bacterial cells and the subsequent accumulation of more calcified cells and carbonate, which acts to weld the bacteria together. The process leads to the formation of spherical bioliths measuring approximately 50 &mgr;m in diameter. The mechanism of carbonate precipitation by D. halophila under our working conditions represents a process of induced biomineralization.