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Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency., (© 2023. The Author(s).)

Microbial electrosynthesis (MES) is a promising carbon utilization technology, but the low-value products (i.e., acetate or methane) and the high electric power demand hinder its industrial adoption. In this study, electrically efficient MES cells with a low ohmic resistance of 15.7 mΩ m 2 were operated galvanostatically in fed-batch mode, alternating periods of high CO 2 and H 2 availability. This promoted acetic acid and ethanol production, ultimately triggering selective (78% on a carbon basis) butyric acid production via chain elongation. An average production rate of 14.5 g m -2  d -1 was obtained at an applied current of 1.0 or 1.5 mA cm -2 , being Megasphaera sp. the key chain elongating player. Inoculating a second cell with the catholyte containing the enriched community resulted in butyric acid production at the same rate as the previous cell, but the lag phase was reduced by 82%. Furthermore, interrupting the CO 2 feeding and setting a constant pH 2 of 1.7-1.8 atm in the cathode compartment triggered solventogenic butanol production at a pH below 4.8. The efficient cell design resulted in average cell voltages of 2.6-2.8 V and a remarkably low electric energy requirement of 34.6 kWh el kg -1 of butyric acid produced, despite coulombic efficiencies being restricted to 45% due to the cross-over of O 2 and H 2 through the membrane. In conclusion, this study revealed the optimal operating conditions to achieve energy-efficient butyric acid production from CO 2 and suggested a strategy to further upgrade it to valuable butanol., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

It has been recently suggested that Alcaligenes use a previously unknown pathway to convert ammonium into dinitrogen gas (Dirammox) via hydroxylamine (NH 2 OH). This fact alone already implies a significant decrease in the aeration requirements for the process, but the process would still be dependent on external aeration. This work studied the potential use of a polarised electrode as an electron acceptor for ammonium oxidation using the recently described Alcaligenes strain HO-1 as a model heterotrophic nitrifier. Results indicated that Alcaligenes strain HO-1 requires aeration for metabolism, a requirement that cannot be replaced for a polarised electrode alone. However, concomitant elimination of succinate and ammonium was observed when operating a previously grown Alcaligenes strain HO-1 culture in the presence of a polarised electrode and without aeration. The usage of a polarised electrode together with aeration did not increase the succinate nor the nitrogen removal rates observed with aeration alone. However, current density generation was observed along a feeding batch test representing an electron share of 3% of the ammonium removed in the presence of aeration and 16% without aeration. Additional tests suggested that hydroxylamine oxidation to dinitrogen gas could have a relevant role in the electron discharge onto the anode. Therefore, the presence of a polarised electrode supported the metabolic functions of Alcaligenes strain HO-1 on the simultaneous oxidation of succinate and ammonium., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)

Hydrogen is a key intermediate element in microbial electrosynthesis as a mediator of the reduction of carbon dioxide (CO 2 ) into added value compounds. In the present work we aimed at studying the biological production of hydrogen in biocathodes operated at - 1.0 V vs. Ag/AgCl, using a highly comparable technology and CO 2 as carbon feedstock. Ten bacterial strains were chosen from genera Rhodobacter, Rhodopseudomonas, Rhodocyclus, Desulfovibrio and Sporomusa, all described as hydrogen producing candidates. Monospecific biofilms were formed on carbon cloth cathodes and hydrogen evolution was constantly monitored using a microsensor. Eight over ten bacteria strains showed electroactivity and H 2 production rates increased significantly (two to eightfold) compared to abiotic conditions for two of them (Desulfovibrio paquesii and Desulfovibrio desulfuricans). D. paquesii DSM 16681 exhibited the highest production rate (45.6 ± 18.8 µM min -1 ) compared to abiotic conditions (5.5 ± 0.6 µM min -1 ), although specific production rates (per 16S rRNA copy) were similar to those obtained for other strains. This study demonstrated that many microorganisms are suspected to participate in net hydrogen production but inherent differences among strains do occur, which are relevant for future developments of resilient biofilm coated cathodes as a stable hydrogen production platform in microbial electrosynthesis.

The benefits of plant-microbe interactions have been exploited extensively for nutrient removal. Radial oxygen loss in aquatic macrophytes potentially promotes nitrification and accelerates nitrogen removal through coupled nitrification-denitrification process. Nitrification is likely the limiting activity for an effective nitrogen removal in wetlands. In this work, we have quantified the effect of radial oxygen losses in Typha angustifolia plants in environments of contrasting salinities, including a temporary lagoon, a constructed wetland, and a river estuary. In all sites, radial oxygen diffusion occurred mainly at a narrow band, from 1 to 5 cm from the root tip, and were almost absent at the tip and basal sections of the root (> 5 cm). Root sections with active oxygen diffusion tended to show higher bacterial and archaeal densities in the rhizoplane according to 16S rRNA gene abundance data, except at higher salinities. Archaeal amoA /bacterial amoA gene ratios were highly variable among sites. Archaeal nitrifiers were only favoured over bacteria on the root surface of Typha collected from the constructed wetland. Collectively, radial oxygen loss had little effect on the nitrifying microbial community at the smaller scale (differences according to root-section), and observed differences were more likely related to prevailing physicochemical conditions of the studied environments or to long-term effects of the root microenvironment (root vs sediment comparisons).

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.

Organosilicon compounds are the most undesirable compounds for the energy recovery of biogas. These compounds are still resistant to biodegradation when biotechnologies are considered for biogas purification. Herein we isolated 52 bacterial species from anaerobic batch enrichment cultures (BEC) saturated with D4 and from an anaerobic lab-scale biotrickling filter (BTF) fed with a gas flow containing D4 as unique carbon source. Among those Methylibium sp. and Pseudomonas aeruginosa showed the highest capacity to remove D4 (53.04% ± 0.03 and 24.42% ± 0.02, respectively). Contrarily, co-culture evaluation treatment for the biodegradation of siloxanes together with volatile organic compounds removed a lower concentration of D4 compared to toluene and limonene, which were completely removed. Remarkably, the siloxane D5 proved to be more biodegradable than D4. Substrates removal values achieved by Methylibium sp. suggested that this bacterial isolate could be used in biological removal technologies of siloxanes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Electromethanogenesis is the bioreduction of carbon dioxide (CO2) to methane (CH4) utilizing an electrode as electron donor. Some studies have reported the active participation of Methanobacterium sp. in electron capturing, although no conclusive results are available. In this study, we aimed at determining short-time changes in the expression levels of [NiFe]-hydrogenases (Eha, Ehb and Mvh), heterodisulfide reductase (Hdr), coenzyme F420-reducing [NiFe]-hydrogenase (Frh), and hydrogenase maturation protein (HypD), according to the electron flow in independently connected carbon cloth cathodes poised at- 800 mV vs. standard hydrogen electrode (SHE). Amplicon massive sequencing of cathode biofilm confirmed the presence of an enriched Methanobacterium sp. population (>70% of sequence reads), which remained in an active state (78% of cDNA reads), tagging this archaeon as the main methane producer in the system. Quantitative RT-PCR determinations of ehaB, ehbL, mvhA, hdrA, frhA, and hypD genes resulted in only slight (up to 1.5 fold) changes for four out of six genes analyzed when cells were exposed to open (disconnected) or closed (connected) electric circuit events. The presented results suggested that suspected mechanisms for electron capturing were not regulated at the transcriptional level in Methanobacterium sp. for short time exposures of the cells to connected-disconnected circuits. Additional tests are needed in order to confirm proteins that participate in electron capturing in Methanobacterium sp., Competing Interests: The authors have declared that no competing interests exist.

Microbial fuel cells (MFCs) can be designed to combine water treatment with concomitant electricity production. Animal manure treatment has been poorly explored using MFCs, and its implementation at full-scale primarily relies on the bacterial distribution and activity within the treatment cell. This study reports the bacterial community changes at four positions within the anode of two almost identically operated MFCs fed swine manure. Changes in the microbiome structure are described according to the MFC fluid dynamics and the application of a maximum power point tracking system (MPPT) compared to a fixed resistance system (Ref-MFC). Both external resistance and cell hydrodynamics are thought to heavily influence MFC performance. The microbiome was characterised both quantitatively (qPCR) and qualitatively (454-pyrosequencing) by targeting bacterial 16S rRNA genes. The diversity of the microbial community in the MFC biofilm was reduced and differed from the influent swine manure. The adopted electric condition (MPPT vs fixed resistance) was more relevant than the fluid dynamics in shaping the MFC microbiome. MPPT control positively affected bacterial abundance and promoted the selection of putatively exoelectrogenic bacteria in the MFC core microbiome (Sedimentibacter sp. and gammaproteobacteria). These differences in the microbiome may be responsible for the two-fold increase in power production achieved by the MPPT-MFC compared to the Ref-MFC., Competing Interests: The authors have declared that no competing interests exist.

Gasification of organic wastes coupled to syngas fermentation allows the recovery of carbon in the form of commodity chemicals, such as carboxylates and biofuels. Acetogenic bacteria ferment syngas to mainly two-carbon compounds, although a few strains can also synthesize four-, and six-carbon molecules. In general, longer carbon chain products have a higher biotechnological (and commercial) value due to their higher energy content and their lower water solubility. However, de-novo synthesis of medium-chain products from syngas is quite uncommon in acetogenic bacteria. An alternative to de-novo synthesis is bioproduction of short-chain products (C2 and C4), and their subsequent elongation to C4, C6, or C8 through reversed β-oxidation metabolism. This two-step synergistic approach has been successfully applied for the production of up to C8 compounds, although the accumulation of alcohols in these mixed cultures remained below detection limits. The present work investigates the production of higher alcohols from syngas by open mixed cultures (OMC). A syngas-fermenting community was enriched from sludge of an anaerobic digester for a period of 109 days in a lab-scale reactor. At the end of this period, stable production of ethanol and butanol was obtained. C6 compounds were only transiently produced at the beginning of the enrichment phase, during which Clostridium kluyveri, a bacterium able to carry out carbon chain elongation, was detected in the community. Further experiments showed pH as a critical parameter to maintain chain elongation activity in the co-culture. Production of C6 compounds was recovered by preventing fermentation pH to decrease below pH 4.5-5. Finally, experiments showed maximal production of C6 compounds (0.8 g/L) and alcohols (1.7 g/L of ethanol, 1.1 g/L of butanol, and 0.6 g/L of hexanol) at pH 4.8. In conclusion, low fermentation pH is critical for the production of alcohols, although detrimental to C. kluyveri. Fine control of fermentation pH to final values around 4.8 could allow sustained production of higher alcohols.

Microorganisms are vital in mediating the earth's biogeochemical cycles; yet, despite our rapidly increasing ability to explore complex environmental microbial communities, the relationship between microbial community structure and ecosystem processes remains poorly understood. Here, we address a fundamental and unanswered question in microbial ecology: 'When do we need to understand microbial community structure to accurately predict function?' We present a statistical analysis investigating the value of environmental data and microbial community structure independently and in combination for explaining rates of carbon and nitrogen cycling processes within 82 global datasets. Environmental variables were the strongest predictors of process rates but left 44% of variation unexplained on average, suggesting the potential for microbial data to increase model accuracy. Although only 29% of our datasets were significantly improved by adding information on microbial community structure, we observed improvement in models of processes mediated by narrow phylogenetic guilds via functional gene data, and conversely, improvement in models of facultative microbial processes via community diversity metrics. Our results also suggest that microbial diversity can strengthen predictions of respiration rates beyond microbial biomass parameters, as 53% of models were improved by incorporating both sets of predictors compared to 35% by microbial biomass alone. Our analysis represents the first comprehensive analysis of research examining links between microbial community structure and ecosystem function. Taken together, our results indicate that a greater understanding of microbial communities informed by ecological principles may enhance our ability to predict ecosystem process rates relative to assessments based on environmental variables and microbial physiology.

Up to date a few electroactive bacteria embedded in biofilms are described to catalyze both anodic and cathodic reactions in bioelectrochemical systems (i.e. bidirectional electron transfer). How these bacteria transfer electrons to or from the electrode is still uncertain. In this study the extracellular electron transfer mechanism of bacteria within an electroactive biofilm was investigated by using cyclic voltammetry (CV) and differential pulse voltammetry (DPV). First, a mature anodic electroactive biofilm was developed from an activated sludge sample (inoculum), acetate as electron donor and a poised electrode (+397mV vs. SHE). Later, this biofilm was "switched" to biocathodic conditions by feeding it with a medium containing nitrates and poising the electrode at -303mV vs. SHE. The electrochemical characterization indicated that both, acetate oxidation and nitrate reduction took place at a similar formal potential of -175±05 and -175±34mV vs. SHE, respectively. The biofilm was predominantly composed by Geobacter sp. at both experimental conditions. Taken together, the results indicated that both processes could be catalyzed by using the same electron conduit, and most likely by the same bacterial consortium. Hence, this study suggests that electroactive bacteria within biofilms could use the same electron transfer conduit for catalyzing anodic and cathodic reactions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity., (Copyright© by the Spanish Society for Microbiology and Institute for Catalan Studies.)

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.

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.

Coastal wetlands, as transient links from terrestrial to marine environments, are important for nitrogen removal by denitrification. Denitrification strongly depends on both the presence of emergent plants and the denitrifier communities selected by different plant species. In this study, the effects of vegetation and habitat heterogeneity on the community of denitrifying bacteria were investigated in nine coastal wetlands in two preserved areas of Spain. Sampling locations were selected to cover a range of salinity (0.81 to 31.3 mS/cm) and nitrate concentrations (0.1 to 303 μM NO3-), allowing the evaluation of environmental variables that select for denitrifier communities in the rhizosphere of Phragmites sp., Ruppia sp., and Paspalum sp. Potential nitrate reduction rates were found to be dependent on the sampling time and plant species and related to the denitrifier community structure, which was assessed by terminal restriction fragment length polymorphism analysis of the functional genes nirS, nirK and nosZ. The results showed that denitrifier community structure was also governed by plant species and salinity, with significant influences of other variables, such as sampling time and location. Ruppia sp. and Phragmites sp. selected for certain communities, whereas this was not the case for Paspalum sp. The plant species effect was strongest on nirK-type denitrifiers, whereas water carbon content was a significant factor defining the structure of the nosZ-harboring community. The differences recognized using the three functional gene markers indicated that different drivers act on denitrifying populations capable of complete denitrification, compared to the overall denitrifier community. This finding may have implications for emissions of the greenhouse gas nitrous oxide.

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.

To highlight the effects of a variety of chlorophenols (CP) in relation to the response of an indigenous bacterial community, an agricultural Mediterranean calcareous soil has been studied in microcosms incubated under controlled laboratory conditions. Soil samples were artificially polluted with 2-monochlorophenol (MCP), 2,4,6-trichlorophenol (TCP) and pentachlorophenol (PCP), at concentrations ranging from 0.1 up to 5000 mg kg(-1). Both activity and composition of the microbial community were assessed during several weeks, respectively, by respirometric methods and PCR-DGGE analysis of extracted DNA and RNA. Significant decreases in soil respirometric values and changes in the bacterial community composition were observed at concentrations above 1000 mg kg(-1) MCP and TCP, and above 100 mg kg(-1) PCP. However, the persistence of several active bacterial populations in soil microcosms contaminated with high concentration of CP, as indicated by DGGE fingerprints, suggested the capacity of these native bacteria to survive in the presence of the pollutants, even without a previous adaptation or contact with them. The isolation of potential CP degraders was attempted by culture plating from microcosms incubated with high CP concentrations. Twenty-three different isolates were screened for their resistance to TCP and PCP. The most resistant isolates were identified as Kocuria palustris, Lysobacter gummosus, Bacillus sp. and Pseudomonas putida, according to 16S rRNA gene homology. In addition, these four isolates also showed the capacity to reduce the concentration of TCP and PCP from 15% to 30% after 5d of incubation in laboratory assays (initial pollutant concentration of 50 mg L(-1)). Isolate ITP29, which could be a novel species of Bacillus, has been revealed as the first known member in this bacterial group with potential for CP bioremediation applications, usually wide-spread in the soil natural communities, which has not been reported to date as a CP degrader., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Phosphorus deficiency was analysed in the oxic-anoxic gradient of the karstic sulphurous lakes Vilar and Sisó during the stratification period. The distribution of planktonic photosynthetic populations along a vertical gradient coincided with an increase in alkaline phosphatase activity (APA). A multiple stepwise correlation analysis of data yielded a positive correlation of APA with planktonic phototrophic populations. MUF-P hydrolysis saturation curves were used to estimate the enzyme kinetics. High-affinity phosphatases (i.e. low K(M) saturation constant) coincided with the oxic-anoxic gradient and progressively declined through both the epi- and the hypolimnion. Changes in the K(M) values are likely due to phosphate inhibition and the contribution of different planktonic populations in the induction of alkaline phosphatases. Extremely low organic phosphorus turnover times (as short as 0.37 h) were also estimated in the gradient zone, indicating a high dependence of the bacterial populations on organic phosphate esters. Phosphatase saturation kinetics revealed K(M) values from 0.53 to 8.45 microM MUF-P, perfectly matching those found in the isolates Thiocapsa sp. UdG3513, Chlorobium limicola UdG6050 and UdG6055 and Chlorobium phaeobacteroides CL1401. The results obtained indicate that a relevant adaptation of sulphur phototrophic bacteria may occasionally face periods of phosphate limitation despite thriving in nutrient-rich anoxic waters.

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.

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.

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.

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.

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 division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-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.