Reactivation of aerobic and anaerobic ammonium oxidizers in OLAND biomass after long-term storage

The biomass of an oxygen-limited autotrophic nitrification/denitrification (OLAND) biofilm reactor was preserved in various ways to find a storage method for both aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). Storage occurred at −20°C with and without glycerol as cryoprotectant and at 4 and 20°C with and without nitrate as redox buffer. After 2 and 5 months, reactivation of AerAOB and AnAOB was achieved with the biomass stored at 4°C with and without nitrate and at 20°C with nitrate. Moreover, the presence of the AerAOB and AnAOB was confirmed with fluorescent in situ hybridization (FISH). Preservation in a nitrate environment resulted in a lag phase for the AnAOB reactivation. The supplied nitrate was denitrified during storage, and a real-time polymerase chain reaction with nitrifying and denitrifying genes allowed to estimate that at least 1.0 to 6.0% of the OLAND biofilm consisted of denitrifiers. It was concluded that reactivation after long-term storage is possible and that preservation at 4°C without nitrate addition is the recommended storage technique. The possibility to store OLAND biomass will facilitate research on AnAOB and can overcome larger-scale start-up and inhibition problems of novel nitrogen processes involving AnAOB.     

Presence of Acyl-Homoserine Lactone in Subtidal Biofilm and the Implication in Larval Behavioral Response in the Polychaete <Emphasis Type="Italic">Hydroides elegans</Emphasis>

Quorum sensing (QS) signals have been considered to play important roles in biofilm development and in the attractiveness of biofilms to higher organisms in marine ecosystem. In this study, bacterial QS signalsacylated homoserine lactone derivatives (AHLs) were detected in 2-, 4-, and 6-day-old subtidal biofilms by using AHLs reporter strains. N-dodecanoyl-homoserine lactone (C12-HSL) was identified in 6-day-old biofilm at a concentration of 9.04 μg cm^−minus;2 (3.36 mmol l^−minus;1). To investigate the possible role of AHLs in the consequent eventlarval settlement of the polychaete Hydroides elegans onto subtidal biofilmsseven biofilm-derived bacteria that effectively induced larval settlement of H. elegans, were screened for AHL production. One of them, the Vibrio sp. UST950701-007, produced N-hexanoyl-homoserine lactone (C6-HSL). Larval settlement bioassay showed that C6-HSL, C12-HSL, and 3-oxo-octanoyl-homoserine lactone (3-oxo-C8-HLS) at certain concentrations induced some initial larval settlement behaviors such as reducing swimming speed, crawling on the bottom. However, these AHLs did not effectively induce larval settlement in comparison to the effective settlement inducer 3-isobutyl-1-methylxanthine. The possible chemokinetic mechanism and indirect effects of AHLs on larval settlement are suggested.     

Vertical migration of aggregated aerobic and anaerobic ammonium oxidizers enhances oxygen uptake in a stagnant water layer

Ammonium can be removed as dinitrogen gas by cooperating aerobic and anaerobic ammonium-oxidizing bacteria (AerAOB and AnAOB). The goal of this study was to verify putative mutual benefits for aggregated AerAOB and AnAOB in a stagnant freshwater environment. In an ammonium fed water column, the biological oxygen consumption rate was, on average, 76 kg O₂ ha⁻¹ day⁻¹. As the oxygen transfer rate of an abiotic control column was only 17 kg O₂ ha⁻¹ day⁻¹, biomass activity enhanced the oxygen transfer. Increasing the AnAOB gas production increased the oxygen consumption rate with more than 50% as a result of enhanced vertical movement of the biomass. The coupled decrease in dissolved oxygen concentration increased the diffusional oxygen transfer from the atmosphere in the water. Physically preventing the biomass from rising to the upper water layer instantaneously decreased oxygen and ammonium consumption and even led to the occurrence of some sulfate reduction. Floating of the biomass was further confirmed to be beneficial, as this allowed for the development of a higher AerAOB and AnAOB activity, compared to settled biomass. Overall, the results support mutual benefits for aggregated AerAOB and AnAOB, derived from the biomass uplifting effect of AnAOB gas production.     

A low volumetric exchange ratio allows high autotrophic nitrogen removal in a sequencing batch reactor

Sequencing batch reactors (SBRs) have several advantages, such as a lower footprint and a higher flexibility, compared to biofilm based reactors, such as rotating biological contactors. However, the critical parameters for a fast start-up of the nitrogen removal by oxygen-limited autotrophic nitrification/denitrification (OLAND) in a SBR are not available. In this study, a low critical minimum settling velocity (0.7 m h⁻¹) and a low volumetric exchange ratio (25%) were found to be essential to ensure a fast start-up, in contrast to a high critical minimum settling velocity (2 m h⁻¹) and a high volumetric exchange ratio (40%) which yielded no successful start-up. To prevent nitrite accumulation, two effective actions were found to restore the microbial activity balance between aerobic and anoxic ammonium-oxidizing bacteria (AerAOB and AnAOB). A daily biomass washout at a critical minimum settling velocity of 5 m h⁻¹ removed small aggregates rich in AerAOB activity, and the inclusion of an anoxic phase enhanced the AnAOB to convert the excess nitrite. This study showed that stable physicochemical conditions were needed to obtain a competitive nitrogen removal rate of 1.1 g N L⁻¹ d⁻¹.     

Characterization of an Autotrophic Nitrogen-Removing Biofilm from a Highly Loaded Lab-Scale Rotating Biological Contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH₄⁺ wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% ± 5% of the influent N at the highest surface load of approximately 8.3 g of N m⁻² day⁻¹, with N₂ as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 ± 7.6 and 76.5 ± 6.4 mg of NH₄⁺-N g of volatile suspended solids [VSS]⁻¹ day⁻¹, respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS⁻¹ day⁻¹). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH₄⁺ wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.     

Acyl-homoserine lactones suppresses IEC-6 cell proliferation and increase permeability of isolated rat colon

We investigated to determine whether a variety of acyl-homoserine lactones (AHLs) influences epithelial cell proliferation and mucosal permeability. 3-Oxo-C₁₂-homoserine lactone (HSL) and 3-oxo-C₁₄-HSL significantly suppressed IEC-6 cell proliferation. A significant increase in mucosal permeability was observed in isolated rat colon tissue exposed to C₁₂-HSL, 3-oxo-C₁₂-HSL, and 3-oxo-C₁₄-HSL. These data indicate that AHLs suppress epithelial proliferation and disrupt barrier function in intestinal mucosa.     

Phaeobacter sp. Strain Y4I Utilizes Two Separate Cell-to-Cell Communication Systems To Regulate Production of the Antimicrobial Indigoidine

The marine roseobacter Phaeobacter sp. strain Y4I synthesizes the blue antimicrobial secondary metabolite indigoidine when grown in a biofilm or on agar plates. Prior studies suggested that indigoidine production may be, in part, regulated by cell-to-cell communication systems. Phaeobacter sp. strain Y4I possesses two luxR and luxI homologous N-acyl-l-homoserine lactone (AHL)-mediated cell-to-cell communication systems, designated pgaRI and phaRI. We show here that Y4I produces two dominant AHLs, the novel monounsaturated N-(3-hydroxydodecenoyl)-l-homoserine lactone (3OHC_12:1-HSL) and the relatively common N-octanoyl-l-homoserine lactone (C₈-HSL), and provide evidence that they are synthesized by PhaI and PgaI, respectively. A Tn5 insertional mutation in either genetic locus results in the abolishment (pgaR::Tn5) or reduction (phaR::Tn5) of pigment production. Motility defects and denser biofilms were also observed in these mutant backgrounds, suggesting an overlap in the functional roles of these systems. Production of the AHLs occurs at distinct points during growth on an agar surface and was determined by isotope dilution high-performance liquid chromatography–tandem mass spectrometry (ID-HPLC-MS/MS) analysis. Within 2 h of surface inoculation, only 3OHC_12:1-HSL was detected in agar extracts. As surface-attached cells became established (at ∼10 h), the concentration of 3OHC_12:1-HSL decreased, and the concentration of C₈-HSL increased rapidly over 14 h. After longer (>24-h) establishment periods, the concentrations of the two AHLs increased to and stabilized at ∼15 nM and ∼600 nM for 3OHC_12:1-HSL and C₈-HSL, respectively. In contrast, the total amount of indigoidine increased steadily from undetectable to 642 μM by 48 h. Gene expression profiles of the AHL and indigoidine synthases (pgaI, phaI, and igiD) were consistent with their metabolite profiles. These data provide evidence that pgaRI and phaRI play overlapping roles in the regulation of indigoidine biosynthesis, and it is postulated that this allows Phaeobacter sp. strain Y4I to coordinate production of indigoidine with different growth-phase-dependent physiologies.     

Biofilm Formation and Quorum-Sensing-Molecule Production by Clinical Isolates of Serratia liquefaciens

Serratia spp. are opportunistic human pathogens responsible for an increasing number of nosocomial infections. However, little is known about the virulence factors and regulatory circuits that may enhance the establishment and long-term survival of Serratia liquefaciens in the hospital environment. In this study, two reporter strains, Chromobacterium violaceum CV026 and VIR24, and high-resolution triple-quadrupole liquid chromatography–mass spectrometry (LC-MS) were used to detect and to quantify N-acyl-homoserine lactone (AHL) quorum-sensing signals in 20 S. liquefaciens strains isolated from clinical samples. Only four of the strains produced sufficient amounts of AHLs to activate the sensors. Investigation of two of the positive strains by high-performance liquid chromatography (HPLC)-MS confirmed the presence of significant amounts of short-acyl-chain AHLs (N-butyryl-l-homoserine lactone [C₄-HSL] and N-hexanoyl-l-homoserine lactone [C₆-HSL]) in both strains, which exhibited a complex and strain-specific signal profile that included minor amounts of other short-acyl-chain AHLs (N-octanoyl-l-homoserine lactone [C₈-HSL] and N-3-oxohexanoyl-l-homoserine lactone [OC₆-HSL]) and long-acyl-chain (C₁₀, C₁₂, and C₁₄) AHLs. No correlation between biofilm formation and the production of large amounts of AHLs could be established. Fimbria-like structures were observed by transmission electron microscopy, and the presence of the type 1 fimbrial adhesin gene fimH in all strains was confirmed by PCR. The ability of S. liquefaciens to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment, increasing the probability of causing nosocomial infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.     

OLAND is feasible to treat sewage-like nitrogen concentrations at low hydraulic residence times

Energy-positive sewage treatment can, in principle, be obtained by maximizing energy recovery from concentrated organics and by minimizing energy consumption for concentration and residual nitrogen removal in the main stream. To test the feasibility of the latter, sewage-like nitrogen influent concentrations were treated with oxygen-limited autotrophic nitrification/denitrification (OLAND) in a lab-scale rotating biological contactor at 25°C. At influent ammonium concentrations of 66 and 29 mg N L⁻¹ and a volumetric loading rate of 840 mg N L⁻¹ day⁻¹ yielding hydraulic residence times (HRT) of 2.0 and 1.0 h, respectively, relatively high nitrogen removal rates of 444 and 383 mg N L⁻¹ day⁻¹ were obtained, respectively. At low nitrogen levels, adapted nitritation and anammox communities were established. The decrease in nitrogen removal was due to decreased anammox and increased nitratation, with Nitrospira representing 6% of the biofilm. The latter likely occurred given the absence of dissolved oxygen (DO) control, since decreasing the DO concentration from 1.4 to 1.2 mg O₂ L⁻¹ decreased nitratation by 35% and increased anammox by 32%. Provided a sufficient suppression of nitratation, this study showed the feasibility of OLAND to treat low nitrogen levels at low HRT, a prerequisite to energy-positive sewage treatment.     

Quorum Sensing in <Emphasis Type="Italic">Aeromonas</Emphasis> Species Isolated from Patients in Malaysia

Bacterial quorum sensing signal molecules called N-acylhomoserine lactone (AHL) controls the expression of virulence determinants in many Gram-negative bacteria. We determined AHL production in 22 Aeromonas strains isolated from various infected sites from patients (bile, blood, peritoneal fluid, pus, stool and urine). All isolates produced the two principal AHLs, N-butanoylhomoserine lactone (C4-HSL) and N-hexanoyl homoserine lactone (C6-HSL). Ten isolates also produced additional AHLs. This report is the first documentation of Aeromonas sobria producing C6-HSL and two additional AHLs with N-acyl side chain longer than C₆. Our data provides a better understanding of the mechanism(s) of this environmental bacterium emerging as a human pathogen.     

An extractive membrane biofilm reactor for degradation of 1,3-dichloropropene in industrial waste water

A bacterial biofilm, capable of mineralising a technical mixture of cis- and trans-1,3-dichloropropene (DCPE), was enriched on the biomedium side of an extractive membrane biofilm reactor (EMBR). The membrane separates the biomedium from the industrial waste water, in terms of pH, ionic strength and the concentration of toxic chemicals. The biofilm, attached to a silicone membrane, is able to mineralise DCPE after its diffusion through the membrane. Five bacterial strains with degradation capabilities were isolated from the metabolically active biofilm and further investigated in batch experiments. Two of them, Rhodococcus erythropolis strains EK2 and EK5, can grow with DCPE as the sole carbon source. Pseudomonas sp. EK1 utilises cis-3-chloroallylalcohol and cis-3-chloroacrylic acid, whereas the metabolite trans-3-chloroacrylic acid represents a dead-end product of the pathway of this strain. The other two strains, Delftia sp. EK3 and EK4, although unable to grow with DCPE as the carbon source, can transform DCPE and its upper-pathway intermediates at reasonable conversion rates. They may represent helper functions of the biofilm consortium, which mineralised up to 12.5 mmol DCPE per hour per gram of biomass protein. Higher feed rates in the EMBR (up to 15 mmol per hour per 100-l bioreactor volume) and shock loads corresponding to concentrations up to 1.8 mmol l⁻¹ led to a significant increase in the freely floating bacterial biomass in the reactor medium (OD₅₄₆= 0.2). At the standard operating feed rate of 1.8 mmol h⁻¹, the free biomass concentration was very low (OD₅₄₆= 0.04). Received: 23 April 1999 / Received revision: 1 July 1999 / Accepted: 5 July 1999     

Complete degradation of tetrachloroethene in coupled anoxic and oxic chemostats

Anaerobic tetrachloroethene(C₂Cl₄)-dechlorinating bacteria were enriched in slurries from chloroethene-contaminated soil. With methanol as electron donor, C₂Cl₄ and trichloroethene (C₂HCl₃) were reductively dechlorinated to cis-1,2-dichloroethene (cis-C₂H₂Cl₂), whereas, with l-lactate or formate, complete dechlorination of C₂Cl₄ via C₂HCl₃, cis-C₂H₂Cl₂ and chloroethene (C₂H₃Cl) to ethene was obtained. In oxic soil slurries with methane as a substrate, complete co-metabolic degradation of cis-C₂H₂Cl₂ was obtained, whereas C₂HCl₃ was partially degraded. With toluene or phenol both of the above were readily co-metabolized. Complete degradation of C₂Cl₄ was obtained in sequentially coupled anoxic and oxic chemostats, which were inoculated with the slurry enrichments. Apparent steady states were obtained at various dilution rates (0.02–0.4 h⁻¹) and influent C₂Cl₄-concentrations (100–1000 μM). In anoxic chemostats with a mixture␣of␣formate and glucose as the carbon and electron source, C₂Cl₄ was transformed at high rates (above␣140 μmol l⁻¹ h⁻¹, corresponding to 145 nmol Cl⁻ min⁻¹ mg protein⁻¹) into cis-C₂H₂Cl₂ and C₂H₃Cl. Reductive dechlorination was not affected by addition of 5 mM sulphate, but strongly inhibited after addition of 5 mM nitrate. Our results (high specific dechlorination rates and loss of dechlorination capacity in the absence of C₂Cl₄) suggest that C₂Cl₄-dechlorination in the anoxic chemostat was catalysed by specialized dechlorinating bacteria. The partially dechlorinated intermediates, cis-C₂H₂Cl₂ and C₂H₃Cl, were further degraded by aerobic phenol-metabolizing bacteria. The maximum capacity for chloroethene (the sum of tri-, di- and monochloro derivatives removed) degradation in the oxic chemostat was 95 μmol l⁻¹ h⁻¹ (20 nmol min⁻¹ mg protein⁻¹), and that of the combined anoxic → oxic reactor system was 43.4 μmol l⁻¹ h⁻¹. This is significantly higher than reported thus far. Received: 17 April 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Characterization of an autotrophic nitrogen-removing biofilm from a highly loaded lab-scale rotating biological contactor

In this study, a lab-scale rotating biological contactor (RBC) treating a synthetic NH(4)(+) wastewater devoid of organic carbon and showing high N losses was examined for several important physiological and microbial characteristics. The RBC biofilm removed 89% +/- 5% of the influent N at the highest surface load of approximately 8.3 g of N m(-2) day(-1), with N(2) as the main end product. In batch tests, the RBC biomass showed good aerobic and anoxic ammonium oxidation (147.8 +/- 7.6 and 76.5 +/- 6.4 mg of NH(4)(+)-N g of volatile suspended solids [VSS](-1) day(-1), respectively) and almost no nitrite oxidation (< 1 mg of N g of VSS(-1) day(-1)). The diversity of aerobic ammonia-oxidizing bacteria (AAOB) and planctomycetes in the biofilm was characterized by cloning and sequencing of PCR-amplified partial 16S rRNA genes. Phylogenetic analysis of the clones revealed that the AAOB community was fairly homogeneous and was dominated by Nitrosomonas-like species. Close relatives of the known anaerobic ammonia-oxidizing bacterium (AnAOB) Kuenenia stuttgartiensis dominated the planctomycete community and were most probably responsible for anoxic ammonium oxidation in the RBC. Use of a less specific planctomycete primer set, not amplifying the AnAOB, showed a high diversity among other planctomycetes, with representatives of all known groups present in the biofilm. The spatial organization of the biofilm was characterized using fluorescence in situ hybridization (FISH) with confocal scanning laser microscopy (CSLM). The latter showed that AAOB occurred side by side with putative AnAOB (cells hybridizing with probe PLA46 and AMX820/KST1275) throughout the biofilm, while other planctomycetes hybridizing with probe PLA886 (not detecting the known AnAOB) were present as very conspicuous spherical structures. This study reveals that long-term operation of a lab-scale RBC on a synthetic NH(4)(+) wastewater devoid of organic carbon yields a stable biofilm in which two bacterial groups, thought to be jointly responsible for the high autotrophic N removal, occur side by side throughout the biofilm.     

N-acylhomoserine lactone-degrading bacteria isolated from hatchery bivalve larval cultures

Quorum sensing (QS) systems, which depend on N-acylhomoserine lactone (AHL) signal molecules, mediate the production of virulence factors in many pathogenic microorganisms. One hundred and forty-six bacterial strains, isolated from a bivalve hatchery, were screened for their capacity to degrade five synthetic AHLs [N-butyryl-dl-homoserine lactone (C₄-HSL), N-hexanoyl-dl-homoserine lactone (C₆-HSL), N-octanoyl-dl-homoserine lactone (C₈-HSL), N-decanoyl-dl-homoserine lactone (C₁₀-HSL) and N-dodecanoyl-dl-homoserine lactone (C₁₂-HSL)] using well diffusion agar-plate assays with three biosensors, Chromobacterium violaceum CV026, C. violaceum VIR07 and Agrobacterium tumefaciens NTL4 (pZLR4). The results of these assays led to our choosing four strains (PP2-67, PP2-459, PP2-644 and PP2-663) that were able to degrade all five synthetic AHLs, thus showing a wide spectrum of quorum quenching (QQ) activity. We subsequently confirmed and measured the QQ activity of the four strains by high-performance liquid chromatography plus mass-spectrometry analysis (HPLC–MS). One of the strains which showed the highest AHL-degrading activity, PP2-459, identified as being a member of the genus Thalassomonas was chosen for further study. Finally, using thin-layer chromatography (TLC), we went on to confirm this strain's capacity to degrade the AHLs produced by other non-pathogenic and pathogenic bacteria not taxonomically related.     

Effects of nitrogen limitation on biofilm formation in a hydrocarbon-degrading trickle-bed filter

The effect of nitrogen limitation on young and mature steady-state biofilm in a trickle-bed filter was studied. Toluene and n-heptane were the sole carbon source. Biomass concentration, respiration, substrate-induced respiration, metabolic quotient, and total hydrocarbon degradation efficiency were measured. The aim of the experiment was to control excess biomass production in the trickle-bed filter by limiting the mineral nutrients and to achieve increased mineralization of the carbon source. Biofilm growth responded strongly to the amount of available nitrogen, whereas hydrocarbon degradation efficiency reached a maximum of 60% and could not be increased even by further addition of nitrogen. The experiments showed that 95% of the adsorbed carbon was mineralized completely and only 5% was used for biofilm formation. This complete mineralization can also be concluded from the metabolic quotient. The value of the latter was about 6–10 mg CO₂-C g⁻¹C_mic h⁻¹, indicating an expanded energy demand due to stress effects in the presence of nutrient deficiency. It was postulated that determination of the metabolic quotient could be an simple instrument to measure the rate of mineralization of carbon sources and also the rate of biomass formation in trickle-bed filters or biofilters. Received: 11 November 1998 / Accepted: 5 December 1998     

Browsing and grazing in elephants: the isotope record of modern and fossil proboscideans

The diet of extant elephants (Loxodonta in Africa, Elephas in Asia) is dominated by C₃ browse although some elephants have a significant C₄ grass component in their diet. This is particularly noteworthy because high-crowned elephantid cheek teeth represent adaptation to an abrasive grazing diet and because isotopic analysis demonstrates that C₄ vegetation was the dominant diet for Elephas in Asia from 5 to 1 Ma and for both Loxodonta and Elephas in Africa between 5–1 Ma. Other proboscideans in Africa and southern Asia, except deinotheres, also had a C₄-dominated diet from about 7 Ma (when the C₄ biomass radiated in tropical and subtropical regions) until their subsequent extinction. Received: 1 July 1998 / Accepted: 16 February 1999     

Comparison of Nitrogen Fixation for North- and South-facing <Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> Stands in Central Korea

The nitrogenase activity, root nodule biomass, and rates of nitrogen (N) fixation were measured in 25-year-old pure north- and south-facing Robinia pseudoacacia stands in an urban forest of Seoul (Kkachisan Mountain) in central Korea. The nitrogenase activity was estimated using an acetylene reduction (AR) assay, which showed an increasing trend during the early growing season, with sustained high rates from June through to September with a decrease thereafter. July had the highest nitrogenase activity rate (micromoles C₂H₄ per gram dry nodule per hour), averaging 95.8 and 115.1 for the north- and south-facing stands, respectively. The maximum root nodule biomass (kilograms per hectare) was 45.7 and 9.1 for the north- and south-facing stands in July, respectively. The AR rate appeared to be strongly correlated to the soil temperature (r ² = 0.68, P < 0.001) and soil pH (r ² = 0.59, P < 0.001) while root nodule biomass was correlated to the soil temperature (r ² = 0.36, P < 0.01) and water content (r ² = 0.35, P < 0.05). The soil temperature showed clear differences between seasons, while there was a significant difference in soil pH, organic matter, total N concentrations, and available phosphorus between the north- and south-facing stands. The N₂ fixation rates during the growing season varied from 0.1 to 37.5 kg N ha⁻¹ month⁻¹ depending on the sampling location and time. The annual N₂ fixation rate (kg N per hectare per year) was 112.3 and 23.2 for the north- and south-facing stands, respectively. The differences in N₂ fixation rate between the two stands were due mainly to the differences in total nodule biomass.     

A field study of the effects of elevated CO2 on plant biomass and community structure in a calcareous grassland

The effects of elevated CO₂ on plant biomass and community structure have been studied for four seasons in a calcareous grassland in northwest Switzerland. This highly diverse, semi-natural plant community is dominated by the perennial grass Bromus erectus and is mown twice a year to maintain species composition. Plots of 1.3 m² were exposed to ambient or elevated CO₂ concentrations (n = 8) using a novel CO₂ exposure technique, screen-aided CO₂ control (SACC) starting in March 1994. In the 1st year of treatment, the annual harvested biomass (sum of aboveground biomass from mowings in June and October) was not significantly affected by elevated CO₂. However, biomass increased significantly at elevated CO₂ in the 2nd (+20%, P = 0.05), 3rd (+21%, P = 0.02) and 4th years (+29%, P = 0.02). There were no detectable differences in root biomass in the top 8 cm of soil between CO₂ treatments on eight out of nine sampling dates. There were significant differences in CO₂ responsiveness between functional groups (legumes, non-leguminous forbs, graminoids) in the 2nd (P = 0.07) and 3rd (P < 0.001) years of the study. The order of CO₂ responsiveness among functional groups changed substantially from the 2nd to the 3rd year; for example, non-leguminous forbs had the smallest relative response in the 2nd year and the largest in the 3rd year. By the 3rd year of CO₂ exposure, large species-specific differences in CO₂ response had developed. For five important species or genera the order of responsiveness was Lotus corniculatus (+271%), Carex flacca (+249%), Bromus erectus (+33%), Sanguisorba minor (no significant CO₂ effect), and six Trifolium species (a negative response that was not significant). The positive CO₂ responses in Bromus and Carex were most closely related to increases in tiller number. Species richness was not affected by CO₂ treatment, but species evenness increased under elevated CO₂ (modified Hill ratio; P = 0.03) in June of the 3rd year, resulting in a marginally significant increase in species diversity (Simpson's index; P = 0.09). This and other experiments with calcareous grassland plants show that elevated atmospheric CO₂ concentrations can substantially alter the structure of calcareous grassland communities and may increase plant community biomass. Received: 12 July 1997 / Accepted: 14 September 1998     

Autoinducer production and quorum-sensing dependent phenotypes of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> vary according to isolation site during colonization of intubated patients

Background  

Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C₁₂-HSL and C₄-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear.     

Simultaneous nutrients and carbon removal during pretreated swine slurry degradation in a tubular biofilm photobioreactor

The biodegradation potential of an innovative enclosed tubular biofilm photobioreactor inoculated with a Chlorella sorokiniana strain and an acclimated activated sludge consortium was evaluated under continuous illumination and increasing pretreated (centrifuged) swine slurry loading rates. This photobioreactor configuration provided simultaneous and efficient carbon, nitrogen, and phosphorous treatment in a single-stage process at sustained nitrogen and phosphorous removals efficiencies ranging from 94% to 100% and 70–90%, respectively. Maximum total organic carbon (TOC), NH₄ ⁺, and PO₄ ³⁻ removal rates of 80 ± 5 g C m_r ⁻³ day⁻¹, 89 ± 5 g N m_r ⁻³ day⁻¹, and 13 ± 3 g P m_r ⁻³ day⁻¹, respectively, were recorded at the highest swine slurry loadings (TOC of 1,247 ± 62 mg L⁻¹, N–NH₄ ⁺ of 656 ± 37 mg L⁻¹, P–PO₄ ³⁺ of 117 ± 19 mg L⁻¹, and 7 days of hydraulic retention time). The unusual substrates diffusional pathways established within the phototrophic biofilm (photosynthetic O₂ and TOC/NH₄ ⁺ diffusing from opposite sides of the biofilm) allowed both the occurrence of a simultaneous denitrification/nitrification process at the highest swine slurry loading rate and the protection of microalgae from any potential inhibitory effect mediated by the combination of high pH and high NH₃ concentrations. In addition, this biofilm-based photobioreactor supported efficient biomass retention (>92% of the biomass generated during the pretreated swine slurry biodegradation).