Molecular characterization of bacterial communities mineralizing benzene under sulfate-reducing conditions

The microbial communities of in situ reactor columns degrading benzene with sulfate as an electron acceptor were analyzed based on clone libraries and terminal restriction fragment length polymorphism fingerprinting of PCR-amplified 16S rRNA genes. The columns were filled with either lava granules or sand particles and percolated with groundwater from a benzene-contaminated aquifer. The predominant organisms colonizing the lava granules were related to Magnetobacterium sp., followed by a phylotype affiliated to the genera Cryptanaerobacter/Pelotomaculum and several Deltaproteobacteria. From the sand-filled columns, a stable benzene-degrading consortium was established in sand-filled laboratory microcosms under sulfate-reducing conditions. It was composed of Delta- and Epsilonproteobacteria, Clostridia, Chloroflexi, Actinobacteria and Bacteroidetes. The most prominent phylotype of the consortium was related to the genus Sulfurovum, followed by Desulfovibrio sp. and the Cryptanaerobacter/Pelotomaculum phylotype. The proportion of the latter was similar in both communities and significantly increased after repeated benzene-spiking. During cultivation on aromatic substrates other than benzene, the Cryptanaerobacter/Pelotomaculum phylotype was outcompeted by other community members. Hence, this organism appears to be specific for benzene as a growth substrate and might play a key role in benzene degradation in both communities. Based on the possible functions of the community members and thermodynamic calculations, a functional model for syntrophic benzene degradation under sulfate-reducing conditions is proposed.     

Cloning, sequencing and mutational analysis of the cytochrome c552 gene (cycB) from Bradyrhizobium japonicum strain 110

We report the cloning and nucleotide sequence analysis of the cytochrome c552 gene (cycB) of Bradyrhizobium japonicum strain 110. The gene was identified with help of an oligonucleotide that was designed on the basis of the amino acid sequence determined for purified cytochrome c552 of B. japonicum strain CC705. The cycB gene product has an N-terminal 23-amino acid signal peptide that is missing in the mature cytochrome c552 protein. A B. japonicum cycB insertion mutant was constructed which had no observable phenotypic defects in denitrification and symbiotic nitrogen fixation. Thus, the function of c552 remains unknown.     

Truncation Error Estimates in Process Life Cycle Assessment Using Input‐Output Analysis

Process life cycle assessment (PLCA) is widely used to quantify environmental flows associated with the manufacturing of products and other processes. As PLCA always depends on defining a system boundary, its application involves truncation errors. Different methods of estimating truncation errors are proposed in the literature; most of these are based on artificially constructed system complete counterfactuals. In this article, we review the literature on truncation errors and their estimates and systematically explore factors that influence truncation error estimates. We classify estimation approaches, together with underlying factors influencing estimation results according to where in the estimation procedure they occur. By contrasting different PLCA truncation/error modeling frameworks using the same underlying input‐output (I‐O) data set and varying cut‐off criteria, we show that modeling choices can significantly influence estimates for PLCA truncation errors. In addition, we find that differences in I‐O and process inventory databases, such as missing service sector activities, can significantly affect estimates of PLCA truncation errors. Our results expose the challenges related to explicit statements on the magnitude of PLCA truncation errors. They also indicate that increasing the strictness of cut‐off criteria in PLCA has only limited influence on the resulting truncation errors. We conclude that applying an additional I‐O life cycle assessment or a path exchange hybrid life cycle assessment to identify where significant contributions are located in upstream layers could significantly reduce PLCA truncation errors.     

Development and Application of a Selective PCR-Denaturing Gradient Gel Electrophoresis Approach To Detect a Recently Cultivated Bacillus Group Predominant in Soil

The worldwide presence of a hitherto-nondescribed group of predominant soil microorganisms related to Bacillus benzoevorans was analyzed after development of two sets of selective primers targeting 16S rRNA genes in combination with denaturing gradient gel electrophoresis (DGGE). The high abundance and cultivability of at least some of these microorganisms makes them an appropriate subject for studies on their biogeographical dissemination and diversity. Since cultivability can vary significantly with the physiological state and even between closely related strains, we developed a culture-independent 16S rRNA gene-targeted DGGE fingerprinting protocol for the detection of these bacteria from soil samples. The composition of the B. benzoevorans relatives in the soil samples from The Netherlands, Bulgaria, Russia, Pakistan, and Portugal showed remarkable differences between the different countries. Differences in the DGGE profiles of these communities in archived soil samples from the Dutch Wieringermeer polder were observed over time during which a shift from anaerobic to aerobic and from saline to freshwater conditions occurred. To complement the molecular methods, we additionally cultivated B. benzoevorans-related strains from all of the soil samples. The highest number of B. benzoevorans relatives was found in the soils from the northern part of The Netherlands. The present study contributes to our knowledge of the diversity and abundance of this interesting group of microbes in soils throughout the world.     

Cilia‐localized LKB1 regulates chemokine signaling,macrophage recruitment,and tissue homeostasis in the kidney

Polycystic kidney disease (PKD) and other renal ciliopathies are characterized by cysts, inflammation, and fibrosis. Cilia function as signaling centers, but a molecular link to inflammation in the kidney has not been established. Here, we show that cilia in renal epithelia activate chemokine signaling to recruit inflammatory cells. We identify a complex of the ciliary kinase LKB1 and several ciliopathy‐related proteins including NPHP1 and PKD1. At homeostasis, this ciliary module suppresses expression of the chemokine CCL2 in tubular epithelial cells. Deletion of LKB1 or PKD1 in mouse renal tubules elevates CCL2 expression in a cell‐autonomous manner and results in peritubular accumulation of CCR2⁺ mononuclear phagocytes, promoting a ciliopathy phenotype. Our findings establish an epithelial organelle, the cilium, as a gatekeeper of tissue immune cell numbers. This represents an unexpected disease mechanism for renal ciliopathies and establishes a new model for how epithelial cells regulate immune cells to affect tissue homeostasis.     

How Honeybees Defy Gravity with Royal Jelly to Raise Queens

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Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Phylogenetic and Functional Diversity Within Toluene-Degrading,Sulphate-Reducing Consortia Enriched from a Contaminated Aquifer

Three toluene-degrading microbial consortia were enriched under sulphate-reducing conditions from different zones of a benzene, toluene, ethylbenzene and xylenes (BTEX) plume of two connected contaminated aquifers. Two cultures were obtained from a weakly contaminated zone of the lower aquifer, while one culture originated from the highly contaminated upper aquifer. We hypothesised that the different habitat characteristics are reflected by distinct degrader populations. Degradation of toluene with concomitant production of sulphide was demonstrated in laboratory microcosms and the enrichment cultures were phylogenetically characterised. The benzylsuccinate synthase alpha-subunit (bssA) marker gene, encoding the enzyme initiating anaerobic toluene degradation, was targeted to characterise the catabolic diversity within the enrichment cultures. It was shown that the hydrogeochemical parameters in the different zones of the plume determined the microbial composition of the enrichment cultures. Both enrichment cultures from the weakly contaminated zone were of a very similar composition, dominated by Deltaproteobacteria with the Desulfobulbaceae (a Desulfopila-related phylotype) as key players. Two different bssA sequence types were found, which were both affiliated to genes from sulphate-reducing Deltaproteobacteria. In contrast, the enrichment culture from the highly contaminated zone was dominated by Clostridia with a Desulfosporosinus-related phylotype as presumed key player. A distinct bssA sequence type with high similarity to other recently detected sequences from clostridial toluene degraders was dominant in this culture. This work contributes to our understanding of the niche partitioning between degrader populations in distinct compartments of BTEX-contaminated aquifers.     

Carbon Conversion Efficiency and Limits of Productive Bacterial Degradation of Methyl tert-Butyl Ether and Related Compounds

The utilization of the fuel oxygenate methyl tert-butyl ether (MTBE) and related compounds by microorganisms was investigated in a mainly theoretical study based on the Y_ATP concept. Experiments were conducted to derive realistic maintenance coefficients and K_s values needed to calculate substrate fluxes available for biomass production. Aerobic substrate conversion and biomass synthesis were calculated for different putative pathways. The results suggest that MTBE is an effective heterotrophic substrate that can sustain growth yields of up to 0.87 g g⁻¹, which contradicts previous calculation results (N. Fortin et al., Environ. Microbiol. 3:407-416, 2001). Sufficient energy equivalents were generated in several of the potential assimilatory routes to incorporate carbon into biomass without the necessity to dissimilate additional substrate, efficient energy transduction provided. However, when a growth-related kinetic model was included, the limits of productive degradation became obvious. Depending on the maintenance coefficient m_s and its associated biomass decay term b, growth-associated carbon conversion became strongly dependent on substrate fluxes. Due to slow degradation kinetics, the calculations predicted relatively high threshold concentrations, S_min, below which growth would not further be supported. S_min strongly depended on the maximum growth rate μ_max, and b and was directly correlated with the half maximum rate-associated substrate concentration K_s, meaning that any effect impacting this parameter would also change S_min. The primary metabolic step, catalyzing the cleavage of the ether bond in MTBE, is likely to control the substrate flux in various strains. In addition, deficits in oxygen as an external factor and in reduction equivalents as a cellular variable in this reaction should further increase K_s and S_min for MTBE.