Changes in the Potential Activity of Nitrite Reducers and the Microbial Community Structure After Sediment Dredging and Plant Removal in the Empuriabrava FWS-CW.

In constructed wetlands (CW), denitrification usually accounts for > 60% of nitrogen removal and is supposedly affected by wetland management practices, such as dredging (and plant removal). These practices cause an impact in sediment properties and microbial communities living therein. We have quantified the effects of a sediment dredging event on dissimilatory nitrite reduction by analysing the structure and activities of the microbial community before and after the event. Potential rates for nitrate reduction to ammonia and denitrification were in accordance with changes in the physicochemical conditions. Denitrification was the predominant pathway for nitrite removal (> 60%) and eventually led to the complete removal of nitrate. On the contrary, dissimilatory nitrite reduction to ammonia (DNRA) increased from 5 to 18% after the dredging event. Both actual activities and abundances of 16S rRNA, nirK and nirS significantly decreased after sediment dredging. However, genetic potential for denitrification (qnirS + qnirK/q16S rRNA) remained unchanged. Analyses of the 16S rRNA gene sequences revealed the importance of vegetation in shaping microbial community structures, selecting specific phylotypes potentially contributing to the nitrogen cycle. Overall, we confirmed that sediment dredging and vegetation removal exerted a measurable effect on the microbial community, but not on potential nitrite + nitrate removal rates. According to redundancy analysis, nitrate concentration and pH were the main variables affecting sediment microbial communities in the Empuriabrava CWs. Our results highlight a high recovery of the functionality of an ecosystem service after a severe intervention and point to metabolic redundancy of denitrifiers. We are confident these results will be taken into account in future management strategies in CWs.

High reactivity of deep biota under anthropogenic CO2 injection into basalt.

Basalts are recognized as one of the major habitats on Earth, harboring diverse and active microbial populations. Inconsistently, this living component is rarely considered in engineering operations carried out in these environments. This includes carbon capture and storage (CCS) technologies that seek to offset anthropogenic CO2 emissions into the atmosphere by burying this greenhouse gas in the subsurface. Here, we show that deep ecosystems respond quickly to field operations associated with CO2 injections based on a microbiological survey of a basaltic CCS site. Acidic CO2-charged groundwater results in a marked decrease (by ~ 2.5-4) in microbial richness despite observable blooms of lithoautotrophic iron-oxidizing Betaproteobacteria and degraders of aromatic compounds, which hence impact the aquifer redox state and the carbon fate. Host-basalt dissolution releases nutrients and energy sources, which sustain the growth of autotrophic and heterotrophic species whose activities may have consequences on mineral storage.

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.

Mass spectrometry identification of alkyl-substituted pyrazines produced by Pseudomonas spp. isolates obtained from wine corks.

We investigated the pyrazine production of 23 Pseudomonas isolates obtained from cork in order to assess their implications in off-flavour development. Off-flavour development in cork stoppers is a crucial process in maintaining the high quality of some wines. Pyrazine production was analyzed by headspace solid-phase-microextraction (HS-SPME) and gas chromatography coupled with mass spectrometry (GC-MS). Five out of the 23 isolates, i.e. Pseudomonas koreensis TCA20, Pseudomonas palleroniana TCA16, Pseudomonas putida TCA23 and N7, and Pseudomonas stutzeri TRA27a were able to produce branched alkyl-substituted pyrazines. For isolates N7 and TCA16, 14 compounds could be identified as pyrazines. The use of mineral media supplemented with different carbon and nitrogen sources resulted in changes in the pyrazine production capacity. In the two strains the amount of pyrazines produced was higher with glucose and decreased significantly with lactate. In all cases, 2,5-di(1-methylethyl)pyrazine was found to be dominant and independent of amino acid addition, suggesting a completely de novo synthesis. Aroma descriptions of most alkyl substituted pyrazines include mild vegetal aromas, not necessarily undesirable for the cork manufacturing industry. Methoxypyrazines, exhibiting earthy and musty aromas, could not be detected in any of the strains analysed.

Emergent macrophytes act selectively on ammonia-oxidizing bacteria and archaea.

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were quantified in the sediments and roots of dominant macrophytes in eight neutral to alkaline coastal wetlands. The AOA dominated in most samples, but the bacterial-to-archaeal amoA gene ratios increased with increasing ammonium levels and pH in the sediments. For all plant species, the ratios increased on the root surface relative to the adjacent bulk sediment. This suggests that root surfaces in these environments provide conditions favoring enrichment of AOB.

Abundance and composition of epiphytic bacterial and archaeal ammonia oxidizers of marine red and brown macroalgae.

Ammonia-oxidizing bacteria (AOB) and archaea (AOA) are important for nitrogen cycling in marine ecosystems. Little is known about the diversity and abundance of these organisms on the surface of marine macroalgae, despite the algae's potential importance to create surfaces and local oxygen-rich environments supporting ammonia oxidation at depths with low dissolved oxygen levels. We determined the abundance and composition of the epiphytic bacterial and archaeal ammonia-oxidizing communities on three species of macroalgae, Osmundaria volubilis, Phyllophora crispa, and Laminaria rodriguezii, from the Balearic Islands (western Mediterranean Sea). Quantitative PCR of bacterial and archaeal 16S rRNA and amoA genes was performed. In contrast to what has been shown for most other marine environments, the macroalgae's surfaces were dominated by bacterial amoA genes rather than those from the archaeal counterpart. On the basis of the sequences retrieved from AOB and AOA amoA gene clone libraries from each algal species, the bacterial ammonia-oxidizing communities were related to Nitrosospira spp. and to Nitrosomonas europaea and only 6 out of 15 operational taxonomic units (OTUs) were specific for the host species. Conversely, the AOA diversity was higher (43 OTUs) and algal species specific, with 17 OTUs specific for L. rodriguezii, 3 for O. volubilis, and 9 for P. crispa. Altogether, the results suggest that marine macroalgae may exert an ecological niche for AOB in marine environments, potentially through specific microbe-host interactions.

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.

Off-odor compounds produced in cork by isolated bacteria and fungi: a gas chromatography-mass spectrometry and gas chromatography-olfactometry study.

The risk of development of specific olfactory profiles in cork was evaluated after inoculation of cork granules and agglomerated and natural cork stoppers with isolated bacteria and fungi. The highest incidence of off-odor development was found in assays when fungi were inoculated. Cork granules with musty-earthy, musty-earthy-TCA, and vegetative deviations were inspected by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). Sixteen odor zones were clearly recognized in the GC-O analyses. Among these, octanal, 2-methoxy-3,5-dimethylpyrazine (MDMP), Z-2-nonenal, 2-methylisoborneol, 2,4,6-trichloroanisole (TCA), geosmin, and guaiacol were the most significant odorants and helped in the discrimination of sensory deviations. Only TCA and guaiacol were detected above their respective detection limits by HS-SPME-GC-MS. The fungi Cryptococcus sp. isolate F020, Rhodotorula sp. isolate F025, Penicillium glabrum isolate F001, and Pennicillium variabile F003A and the bacterium Pseudomonas jessenii isolate A1 were found to produce TCA to a greater extent. Additionally, 13 of 38 isolated microorganisms (2 bacteria and 11 fungi) proved able to produce unpleasant musty-earthy or vegetative odors that were not related to a significant TCA accumulation.

Molecular fingerprinting by PCR-denaturing gradient gel electrophoresis reveals differences in the levels of microbial diversity for musty-earthy tainted corks.

The microbial community structure of cork with marked musty-earthy aromas was analyzed using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Cork stoppers and discs were used for DNA extraction and were analyzed by using selective primers for bacteria and fungi. Stoppers clearly differed from discs harboring a different fungal community. Moreover, musty-earthy samples of both types were shown to have a specific microbiota. The fungi Penicillium glabrum and Neurospora spp. were present in all samples and were assumed to make only a small contribution to off-odor development. In contrast, Penicillium islandicum and Penicillium variabile were found almost exclusively in 2,4,6-trichloroanisole (TCA) tainted discs. Conversely, Rhodotorula minuta and Rhodotorula sloofiae were most common in cork stoppers, where only small amounts of TCA were detected. Alpha- and gammaproteobacteria were the most commonly found bacteria in either control or tainted cork stoppers. Specific Pseudomonas and Actinobacteria were detected in stoppers with low amounts of TCA and 2-methoxy-3,5-dimethylpyrazine. These results are discussed in terms of biological degradation of taint compounds by specific microorganisms. Reliable and straightforward microbial identification methods based on a molecular approach provided useful data to determine and evaluate the risk of taint formation in cork.

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.

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.

Diversity of the nitrite reductase gene nirS in the sediment of a free-water surface constructed wetland.

The diversity of the nitrite reductase gene nirS was studied in the bulk sediment of a free-water surface constructed wetland (FWS-CW) located next to the Empuriabrava wastewater treatment plant (WWTP), in Castelló d'Empúries (Girona, NE Spain). The study period extended from the inception of the treatment wetland, in June 1998, until March 1999 and comprised periods of relatively high nitrate and ammonium concentrations at the influent and low nitrate-removal efficiencies. To evaluate nirS diversity, partial gene sequences were obtained by cloning of the respective PCR products. Rarefaction curves based on DOTUR analyses of the deduced amino-acid sequences predicted a greater diversity of nirS genes in samples containing higher ammonium concentrations. Estimated Shannon-Weaver indices of the four cloned samples showed a positive relationship with the N-NH4 +/N-NO3 - ratios measured at the FWS-CW inlet. Identities between the deduced amino-acid sequences and those previously deposited in public databases ranged from 72 to 97%. Phylogenetic analysis based on these deduced sequences grouped 165 nirS clones in seven main clusters according to high similarity indices. Up to 60% of the clones clustered together in a highly homogeneous group with little homologies to any sequence retrieved from cultured representatives. Moreover, prevailing environmental conditions appeared to select for particular denitrifying populations, e.g., with respect to ammonium load and nitrogen removal efficiencies. This observation is of particular interest for the management of treatment wetlands, in which only slight variations in the theoretical denitrification potential of the system can occur.