Microbial community and extracellular polymeric substances analysis of anaerobic granular sludge exposed to selenate,cadmium and zinc

The microbial community and extracellular polymeric substances composition of anaerobic granular sludge exposed to selenate (~10 mg/L), cadmium (Cd) and zinc (Zn) (~2 and 5 mg/L) were investigated by high-throughput sequencing and fluorescence excitation emission matrix (FEEM) spectra, respectively. As a response to selenate, Cd and/or Zn exposure, significant fluorescence quenching of fulvic-like acids and humic-like substances was observed. With selenate, Cd and/or Zn in the influent with respective concentrations of 10, 5 and 5 mg/L, the abundance of the phyla Proteobacteria, Firmicutes, Spirochaetae, Cloacimonetes and Synergistetes increased significantly, and the dominant taxa in the anaerobic granular sludge exposed to Se, Cd and/or Zn were Halothiobacillaceae (10.2%), Pseudomonas (8.8%), Synergistaceae (7.7%), Spirochaetaceae (7.2%), Blvii28 wastewater sludge group (6.7%), Telmatospirillum (4.6%), Veillonellaceae (4.3%), Geobacter (4.0%) and Enterobacteriaceae (3.0%). Compared with the inoculum, the abundance of the archaea Methanobacterium and Methanosaeta decreased to below detection limit in the UASB reactor after 116 days exposure to Se, Cd and Zn.     

Cwl0971, a novel peptidoglycan hydrolase,plays pleiotropic roles in Clostridioides difficile R20291

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from cell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary/late phase of cell growth. Moreover, sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the defect of Cwl0971 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which affects toxin release, sporulation, germination, and pathogenicity of R20291, indicating that Cwl0971 could be an attractive target for C. difficile infection therapeutics and prophylactics.     

Biogeography of Australasian flightless weevils (Curculionidae,Celeuthetini) suggests permeability of Lydekker's and Wallace's Lines

The Indo‐Australian region was formed by the collision of the Australian and Asian plates, and its fauna largely reflects this dual origin. Lydekker's and Wallace's Lines represent biogeographic transition boundaries between biotas although their permeability through geological times was rarely assessed. Here, we explore the evolutionary history of flightless weevils of the tribe Celeuthetini in this geologically highly complex region. We generated a DNA sequence data set of 2236 bp comprising two nuclear and two mitochondrial markers for 62 species of the Indo‐Australian tribe Celeuthetini. We used Bayesian Inference and Maximum Likelihood to reconstruct the first molecular phylogeny of the group. Based on this phylogenetic tree, we employed the program BioGeoBEARS to infer the biogeographical history of Celeuthetini in the region. The group's radiation begun east of Wallace's Line, probably during the mid‐Eocene. We unveil multiple transgressions of Lydekker's and Wallace's Lines mostly during the Miocene with a significant role of founder‐event speciation. The phylogeny of Celeuthetini is geographically highly structured with the first lineages occurring in New Guinea and the Moluccas, and a deep divergence between two clades largely confined to Sulawesi and their respective sister clades of the Lesser Sunda Islands. Wallace's Line was crossed once from Sulawesi and three times from the Lesser Sunda Islands to Java whilst Lydekker's Line was crossed once from New Guinea to the Moluccas. Although this beetle group shows extensive local diversification with little dispersal, the biogeographical demarcations of the Australasian region appear to have been rather porous barriers to dispersal.     

ULTRASTRUCTURAL CHANGES IN HAPLOID EMBRYOIDS OF LARIX DECIDUA DURING EARLY EMBRYOGENESIS

Cultures of haploid tissue derived from megagametophyte tissue were investigated at various stages of embryoid development. At the earliest stage of development the cells were vacuolate and plastids and mitochondria were simple in structure. Eventually, embryonal domes were formed which in turn, produced suspensor cells. As the embryoid developed, its apical cells showed increased complexity of plastids, eventually developing chloroplasts and amyloplasts. In addition, the vacuolar volume in the cells was reduced, while vesicle production increased. Cristae in mitochondria became more apparent. In the suspensor cells, plastids remained as leucoplasts and amyloplasts without any internal membrane structures. Mitochondria became more developed and vesicle number increased in the long cells. Smooth endoplasmic reticulum, which was the most common form of this class of membrane, was reduced by the cotyledonary stage and replaced by rough endoplasmic reticulum. Phenolic deposits appeared with maturation of the suspensor, and an extracellular matrix of PATAg (Thiery) positive polysaccharide was detected between the cells.     

Implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) for advanced bioprocess monitoring

We report on the implementation of proton transfer reaction‐mass spectrometry (PTR‐MS) technology for on‐line monitoring of volatile organic compounds (VOCs) in the off‐gas of bioreactors. The main part of the work was focused on the development of an interface between the bioreactor and an analyzer suitable for continuous sampling of VOCs emanating from the bioprocess. The permanently heated sampling line with an inert surface avoids condensation and interaction of volatiles during transfer to the PTR‐MS. The interface is equipped with a sterile sinter filter unit directly connected to the bioreactor headspace, a condensate trap, and a series of valves allowing for dilution of the headspace gas, in‐process calibration, and multiport operation. To assess the aptitude of the entire system, a case study was conducted comprising three identical cultivations with a recombinant E. coli strain, and the volatiles produced in the course of the experiments were monitored with the PTR‐MS. The high reproducibility of the measurements proved that the established sampling interface allows for reproducible transfer of volatiles from the headspace to the PTR‐MS analyzer. The set of volatile compounds monitored comprises metabolites of different pathways with diverse functions in cell physiology but also volatiles from the process matrix. The trends of individual compounds showed diverse patterns. The recorded signal levels covered a dynamic range of more than five orders of magnitude. It was possible to assign specific volatile compounds to distinctive events in the bioprocess. The presented results clearly show that PTR‐MS was successfully implemented as a powerful bioprocess‐monitoring tool and that access to volatiles emitted by the cells opens promising perspectives in terms of advanced process control. Biotechnol. Bioeng. 2012; 109: 3059–3069. © 2012 Wiley Periodicals, Inc.     

Attrition-free pyrolysis to produce bio-oil and char

Experiments are performed on a laboratory scale setup where beech wood chips are heated by gas convection and walls radiation. This study shows that it is possible to obtain high bio-oil and char yields with relatively low external heat transfer coefficients. The main advantage of this convection/radiation heat transfer mode compared to solid–solid collisions, applied in fluidized bed or twin screw reactors, is the reduction of solid attrition (char and sand). Thus tricky gas–solid separation through hot cyclones and/or hot filters could be avoided or reduced. It should be possible to recover directly bio-oil with less char particles and char free of sand dust. These qualities would allow easier use of these bio-products in different applications.     

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells: Relationship to cellular quiescence and senescence in culture

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin. Cells treated with hematoporphyrin and a lysosomal umbelliferone probe show a fast-rising umbelliferone emission, plus a fluorescent photoproduct. Studies in rapidly growing versus quiescent L, early passage/late passage WI-38 cells, suggest accumulation of fluorescence Schiff bases (i.e., their association with granular regions of cells in stationary phase, spectral properties, fast increase in photosensitized cells) and a possible lysosomal membrane permeabilization in quiescent or senescent cells.