Lluís Bañeras

Identification of and Spatio-Temporal Differences between Microbial Assemblages from Two Neighboring Sulfurous Lakes: Comparison by Microscopy and Denaturing Gradient Gel Electrophoresis

ABSTRACT The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the divisionProteobacteria. Sequences obtained from Lake Vilar samples were related to members of theCytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.

Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria.

Lactic acid bacteria were isolated from fresh vegetables and fruit and its ability to inhibit the growth of foodborne human pathogens (Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus) was tested using the agar spot assay. Eighteen isolates showed a strong antagonistic capacity and were further characterised and identified using 16S rDNA sequencing and API 50CH. Most of them pertained to Leuconostoc spp. and Lactobacillus plantarum, and a few corresponded to Weissella spp. and Lactococcus lactis. Growth and efficacy of control of foodborne pathogen test bacteria by selected strains were tested in wounded Golden Delicious apples and Iceberg lettuce leaf cuts. The strains grew on the substrates and did not cause negative effects on the general aspect of tissues of apple or lettuce. Treatment of apple wounds and lettuce cuts with the antagonistic strains reduced the cell count of S. typhimurium and E .coli by 1 to 2 log cfu/wound or g, whereas the growth of L. monocytogenes was completely inhibited. Results support the potential use of lactic acid bacteria as bioprotective agents against foodborne human pathogens in ready-to-eat fresh fruit and vegetable products.

Lactic acid bacteria from fresh fruit and vegetables as biocontrol agents of phytopathogenic bacteria and fungi.

This study evaluated the efficacy of lactic acid bacteria (LAB) isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 LAB strains was tested in vitro and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also analyzed for the inhibition of P. expansum infection in wounds of Golden Delicious apples. Four strains (TC97, AC318, TM319, and FF441) reduced the fungal rot diameter of the apples by 20%; only Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred mediators of inhibition but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against E. carotovora. While previous reports of antifungal activity by LAB are scarce, our results support the potential of LAB as biocontrol agents against postharvest rot.

Structure and function of denitrifying and nitrifying bacterial communities in relation to the plant species in a constructed wetland.

The community structure and potential activities of nitrifying and denitrifying bacteria were studied in the rhizosphere of Typha latifolia and Phragmites australis present in a free water system constructed wetland (CW). Potential nitrate reduction and nitrification activities were shown to be significantly higher in the rhizosphere when compared with the nonvegetated sediment. Higher rates were generally obtained for P. australis. The community structure of denitrifying bacteria in the rhizosphere differed from that found at the bulk sediment, as revealed by PCR-denaturing gradient gel electrophoresis (DGGE) of the nitrous oxide reductase encoding gene nosZ. Results also show a greater nosZ genotype diversification and suggest a plant species effect in rhizosphere samples obtained during events of low hydraulic retention times. Ammonia-oxidizing communities were less complex on the basis of PCR-DGGE analysis of the 16S rRNA gene. Retrieved sequences were all related to Nitrosomonas marina and Nitrosomonas ureae, being both present in rhizosphere and bulk sediment regardless of environmental changes. The results demonstrate the effect of vegetation on the functioning and structure of bacterial communities involved in the removal of nitrogen in the treatment cells of a CW and point to the use of vegetation coverage to promote nitrification or denitrification in particular areas.

Autotrophic nitrite removal in the cathode of microbial fuel cells.

Nitrification to nitrite (nitritation process) followed by reduction to dinitrogen gas decreases the energy demand and the carbon requirements of the overall process of nitrogen removal. This work studies autotrophic nitrite removal in the cathode of microbial fuel cells (MFCs). Special attention was paid to determining whether nitrite is used as the electron acceptor by exoelectrogenic bacteria (biologic reaction) or by graphite electrodes (abiotic reaction). The results demonstrated that, after a nitrate pulse at the cathode, nitrite was initially accumulated; subsequently, nitrite was removed. Nitrite and nitrate can be used interchangeably as an electron acceptor by exoelectrogenic bacteria for nitrogen reduction from wastewater while producing bioelectricity. However, if oxygen is present in the cathode chamber, nitrite is oxidised via biological or electrochemical processes. The identification of a dominant bacterial member similar to Oligotropha carboxidovorans confirms that autotrophic denitrification is the main metabolism mechanism in the cathode of an MFC.

Genetic potential for N2O emissions from the sediment of a free water surface constructed wetland.

Removal of nitrogen is a key aspect in the functioning of constructed wetlands. However, incomplete denitrification may result in the net emission of the greenhouse gas nitrous oxide (N(2)O) resulting in an undesired effect of a system supposed to provide an ecosystem service. In this work we evaluated the genetic potential for N(2)O emissions in relation to the presence or absence of Phragmites and Typha in a free water surface constructed wetland (FWS-CW), since vegetation, through the increase in organic matter due to litter degradation, may significantly affect the denitrification capacity in planted areas. Quantitative real-time PCR analyses of genes in the denitrification pathway indicating capacity to produce or reduce N(2)O were conducted at periods of different water discharge. Genetic potential for N(2)O emissions was estimated from the relative abundances of all denitrification genes and nitrous oxide reductase encoding genes (nosZ). nosZ abundance was invariably lower than the other denitrifying genes (down to 100 fold), and differences increased significantly during periods of high nitrate loads in the CW suggesting a higher genetic potential for N(2)O emissions. This situation coincided with lower nitrogen removal efficiencies in the treatment cell. The presence and the type of vegetation, mainly due to changes in the sediment carbon and nitrogen content, correlated negatively to the ratio between nitrate and nitrite reducers and positively to the ratio between nitrite and nitrous oxide reducers. These results suggest that the potential for nitrous oxide emissions is higher in vegetated sediments.

Bioprotective Leuconostoc strains against Listeria monocytogenes in fresh fruits and vegetables

Ten Leuconostoc mesenteroides and one Ln. citreum strains isolated from fresh fruit and vegetables were tested for their antagonistic capacity against Listeria monocytogenes. Genetic differences among strains were analyzed by Random Amplified Polymorphic DNA (RAPD). All the isolates clustered together and differed from the type strain Ln. mesenteroides ATCC 8293 as well as from Ln. fallax and Ln. citreum. Organic acids, hydrogen peroxide and bacteriocins were detected as main inhibition mechanisms. Characterization of culture supernatants from the bacteriocinogenic strains, CM135 and CM160 revealed a high resistance of antibacterial activity to temperature and pH, and a bactericidal mode of action against L. monocytogenes. Produced bacteriocins belonged to the Class IIa and sequencing of genes showed complete homology with mesentericin Y105. A study of the effect of the relative dose of pathogen and LAB on control of L. monocytogenes in wounds of Golden Delicious apples and Iceberg lettuce leaf cuts was performed. A comparison of the dose of bioprotective strain needed for a ten fold reduction of the viable pathogen concentration (ED90) revealed that strain CM160 was the most effective against L. monocytogenes. ED90 values varied from 1.3.10(4) to 5.0.10(5) cfu.g(-1) or wound, at ranges of pathogen levels from 1.0.10(3) to 5.0.10(4) cfu.g(-1) of lettuce or wound of apple. The efficiency of the strains was also calculated as the ratio of the ED90 value to the pathogen dose inoculated. The lowest ratio was found for strain CM160 at 5 to 50 cells of LAB per cell of pathogen. The strain offers potential application for prevention of the presence of L. monocytogenes in fresh fruit and vegetables.

Denitrifying bacterial communities affect current production and nitrous oxide accumulation in a microbial fuel cell.

The biocathodic reduction of nitrate in Microbial Fuel Cells (MFCs) is an alternative to remove nitrogen in low carbon to nitrogen wastewater and relies entirely on microbial activity. In this paper the community composition of denitrifiers in the cathode of a MFC is analysed in relation to added electron acceptors (nitrate and nitrite) and organic matter in the cathode. Nitrate reducers and nitrite reducers were highly affected by the operational conditions and displayed high diversity. The number of retrieved species-level Operational Taxonomic Units (OTUs) for narG, napA, nirS and nirK genes was 11, 10, 31 and 22, respectively. In contrast, nitrous oxide reducers remained virtually unchanged at all conditions. About 90% of the retrieved nosZ sequences grouped in a single OTU with a high similarity with Oligotropha carboxidovorans nosZ gene. nirS-containing denitrifiers were dominant at all conditions and accounted for a significant amount of the total bacterial density. Current production decreased from 15.0 A·m−3 NCC (Net Cathodic Compartment), when nitrate was used as an electron acceptor, to 14.1 A·m−3 NCC in the case of nitrite. Contrarily, nitrous oxide (N2O) accumulation in the MFC was higher when nitrite was used as the main electron acceptor and accounted for 70% of gaseous nitrogen. Relative abundance of nitrite to nitrous oxide reducers, calculated as (qnirS+qnirK)/qnosZ, correlated positively with N2O emissions. Collectively, data indicate that bacteria catalysing the initial denitrification steps in a MFC are highly influenced by main electron acceptors and have a major influence on current production and N2O accumulation.

Novel bacteriochlorophyll e structures and species-specific variability of pigment composition in green sulfur bacteria

Abstract. The relative composition of bacteriochlorophyll (BChl) homologs in five different strains of brown-colored green sulfur bacteria was investigated by HPLC-MS/MS and NMR analyses. In addition, the effect of incubation light intensities on homolog distribution was studied in one of the strains (strain Dagow III). A total of 23 different BChl e structures were detected and comprise four homologous porphyrin ring systems and eight different esterifying alcohols. Several BChl e structures are novel. These include a C-8 ethyl, C-12 methyl [E, M] BChl eF homolog which was identified by 1H-NMR analyses of the isolated, main farnesyl homologs (BChl eF). In addition, five previously unknown homolog series with dodecanol, pentadecenol, tetradecanol, hexadecenol and phytol as the esterifying alcohols were detected. The composition of BChl e homologs from the five strains of green sulfur bacteria differed with respect to the relative abundance of the homologs (BChl eF : 25.6–67.0% of total BChl e content in stationary cultures). In strain Dagow III, the abundance of BChl eF homologs decreased upon entry into the stationary phase. In all free-living strains, the abundance of BChl eF was increased when the relative carotenoid content was low. The present results provide a detailed picture of pigment composition in chlorosomes and thus will help to elucidate their structure and function. Furthermore, the newly discovered BChl e molecules are valuable biomarkers for the study of the occurrence and metabolism of green sulfur bacteria in past and present ecosystems.

Fingerprinting the genetic diversity of the biotin carboxylase gene (accC) in aquatic ecosystems as a potential marker for studies of carbon dioxide assimilation in the dark

We designed and tested a set of specific primers for specific PCR amplification of the biotin carboxylase subunit gene (accC) of the Acetyl CoA carboxylase (ACCase) enzyme. The primer set yielded a PCR product of c. 460 bp that was suitable for denaturing gradient gel electrophoresis (DGGE) fingerprinting followed by direct sequencing of excised DGGE bands and sequence analysis. Optimization of PCR conditions for selective amplification was carried out with pure cultures of different bacteria and archaea, and laboratory enrichments. Next, fingerprinting comparisons were done in several aerobic and anaerobic freshwater planktonic samples. The DGGE fingerprints showed between 2 and 19 bands in the different samples, and the primer set provided specific amplification in both pure cultures and natural samples. Most of the samples had sequences grouped with bacterial accC, hypothetically related to the anaplerotic fixation of inorganic carbon. Some other samples, however, yielded accC gene sequences that clustered with Crenarchaeota and were related to the 3-hydroxypropionate/4-hydroxybutyrate cycle of autotrophic crenarchaeota. Such samples came from oligotrophic high mountain lakes and the hypolimnia of a sulfide-rich lake, where crenarchaeotal populations had been previously reported by 16S rRNA surveys. This study provided a fast tool to look for presence of accC genes in natural environments as potential marker for studies of carbon dioxide assimilation in the dark. After further refinement for better specificity against archaea, the new and novel primers could be very helpful to establish a target for crenarchaeota with implications for our understanding of archaeal carbon biogeochemistry.