High rate treatment of terephthalic acid production wastewater in a two-stage anaerobic bioreactor

The feasibility was studied of anaerobic treatment of wastewater generated during purified terephthalic acid (PTA) production in two-stage upflow anaerobic sludge blanket (UASB) reactor system. The artificial influent of the system contained the main organic substrates of PTA-wastewater: acetate, benzoate, and terephthalate. Three parallel operated reactors were used for the second stage, and seeded with a suspended terephthalate degrading culture, with and without additional methanogenic granular sludge (two different types). The first stage UASB-reactor was seeded with methanogenic granular sludge. Reactors were operated at 37 degrees C and pH 7. During the first 300 days of operation a clear distinction between the biomass grown in both reactor stages was obtained. In the first stage, acetate and benzoate were degraded at a volumetric loading rate of 40 g-COD/L . day at a COD-removal efficiency of 95% within the first 25 days of operation. No degradation of terephthalate was obtained in the first stage during the first 300 days of operation despite operation of the reactor at a decreased volumetric loading rate with acetate and benzoate of 9 g-COD/L . day from day 150. Batch incubation of biomass from the reactor with terephthalate showed that the lag-phase prior to terephthalate degradation remained largely unchanged, indicating that no net growth of terephthalate degrading biomass occurred in the first stage reactor. From day 300, however, terephthalate degradation was observed in the first stage, and the biomass in this reactor could successfully be enriched with terephthalate degrading biomass, resulting in terephthalate removal capacities of 15 g-COD/L . day. Even though no single reason could be identified why (suddenly) terephthalate degradation was obtained after such a long period of operation, it is suggested that the solid retention time as well the prevailing reactor concentrations acetate and benzoate may have played an important role. From day 1 of operation, terephthalate was degraded in the second stage. In presence of methanogenic granular biomass, high terephthalate removal capacities were obtained in these reactors (15 g-COD/L . day) after approximately 125 days of operation. From the results obtained it is concluded that terephthalate degradation is the bottleneck during anaerobic treatment of PTA-wastewater. Pre-removal of acetate and benzoate in staged bioreactor reduces the lag-phase prior to terephthalate degradation in latter stages, and enables high rate treatment of PTA-wastewater.     

Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.     

Decreased Levels of Circulating IL17-Producing CD161+CCR6+ T Cells Are Associated with Graft-versus-Host Disease after Allogeneic Stem Cell Transplantation

The C-type lectin-like receptor CD161 is a well-established marker for human IL17-producing T cells, which have been implicated to contribute to the development of graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (allo-SCT). In this study, we analyzed CD161⁺ T cell recovery, their functional properties and association with GVHD occurrence in allo-SCT recipients. While CD161⁺CD4⁺ T cells steadily recovered, CD161^hiCD8⁺ T cell numbers declined during tapering of Cyclosporine A (CsA), which can be explained by their initial growth advantage over CD161^neg/lowCD8⁺ T cells due to ABCB1-mediated CsA efflux. Interestingly, occurrence of acute and chronic GVHD was significantly correlated with decreased levels of circulating CD161⁺CD4⁺ as well as CD161^hiCD8⁺ T cells. In addition, these subsets from transplanted patients secreted high levels of IFNγ and IL17. Moreover, we found that CCR6 co-expression by CD161⁺ T cells mediated specific migration towards CCL20, which was expressed in GVHD biopsies. Finally, we demonstrated that CCR6⁺ T cells indeed were present in these CCL20⁺ GVHD-affected tissues. In conclusion, we showed that functional CD161⁺CCR6⁺ co-expressing T cells disappear from the circulation and home to GVHD-affected tissue sites. These findings support the hypothesis that CCR6⁺CD161-expressing T cells may be involved in the immune pathology of GVHD following their CCL20-dependent recruitment into affected tissues.     

Reinerantha foliicola,a new genus and species of Lejeuneaceae subtribe Cololejeuneinae (Marchantiophyta) from Ecuador

Cololejeuneinae is the largest subtribe of the liverwort family Lejeuneaceae and the crown group of the family. The species often grow on the surfaces of living leaves and twigs or in swiftly running waters and many of them possess neotenic gametophytes, considered an adaptation to these extreme habitats. We describe a new, epiphyllous genus and species in Cololejeuneinae from the Western Cordillera of Ecuador, Reinerantha foliicola Gradst. & R.L.Zhu. Analysis of combined molecular datasets with parsimony, maximum likelihood and Bayesian methods revealed Reinerantha in a sister relationship to the large pantropical genus Diplasiolejeunea. Reinerantha differs from Diplasiolejeunea in leaf segmentation, epidermal wall thickening, stylus, male bracts and perianth keels, and is morphologically more similar to the genus Tuyamaella. In the molecular analysis, however, Reinerantha and Tuyamaella were resolved in different clades and not closely related. Tuyamaella is shown to be restricted to Asia; the report of Tuyamaella from South America (Peru) was erroneous and a case of mislabeling.     

O2 versus N2O respiration in a continuous microbial enrichment

Despite its ecological importance, essential aspects of microbial N₂O reduction—such as the effect of O₂ availability on the N₂O sink capacity of a community—remain unclear. We studied N₂O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N₂O and O₂ can occur, (ii) the mechanism governing the competition for N₂O and O₂, and (iii) how the N₂O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N₂O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O₂. When supplied simultaneously, N₂O reduction to N₂ was only detected when the O₂ concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N₂O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (K_S) for O₂ was found to be two orders of magnitude lower than the value for N₂O, explaining the preferential use of O₂ over N₂O under most environmentally relevant conditions.

     

Determination of CTP synthetase activity in crude cell homogenates by a fast and sensitive non-radiochemical assay using anion-exchange high-performance liquid chromatography

A non-radiochemical assay procedure for CTP synthetase was developed in which CTP is detected at 280 nm after separation with anion-exchange HPLC. A complete separation of all nucleoside triphosphates was achieved within 11 min and the minimum amount of CTP which could be accurately determined proved to be 5 pmol. Therefore, our assay procedure is ten-fold more sensitive compared to the frequently used radiochemical assays. The assay was linear with time and protein concentration, although at low protein concentration a lag phase was observed. An amount of 2×10⁶ cells was already sufficient to determine the specific activity of CTP synthetase in HL-60 cells, lymphocytes and in lymphoblasts obtained from pediatric patients suffering from acute lymphoblastic leukemia.     

Influence of silicate on enrichment of highly productive microalgae from a mixed culture

Microalgae have the potential to supply a biobased society with essential feedstocks like sugar and lipids. Besides being productive, strains used for this purpose should grow fast, be resistant to predators, and have good harvestability properties. Diatoms, a class of siliceous algae, have these and other preferred characteristics. In this paper, we describe the enrichment of microalgae in sequencing batch reactors with and without supply of silicate. Both reactors were operated with a light–dark cycle. To maximize storage compound production, carbon fixation and nitrogen uptake were uncoupled by limiting the availability of nitrate to the dark phase. After ten cycles, a stable culture was established in both reactors. The diatom Nitzschia sp. dominated the silicate-rich reactor, and the green algae Chlamydomonas sp. dominated the silicate-depleted reactor. Over the remaining 27 cycles of the experiment, the microalgal community structure did not change, indicating a highly stable system. Although the dominant microalga was highly dependent on the presence of silicate, the performance of both microalgal enrichments was similar. Polymers of glucose were stored during the nitrogen-limited light period. On organic matter dry weight basis, the sugar content of the biomass increased during the light period from 17?±?4 to 53?±?4 % for the silicate-limited culture, and from 14?±?4 to 43?±?4 % (w w ^?1) for the silicate excess culture. These results show that storage compound production can be achieved under various conditions, as long as a selective environment is maintained.     

Seabird metapopulations: searching for alternative breeding habitats

Today, many seabird species nest in port areas, which are also necessary for human economic activity. In this paper, we evaluate, using a metapopulation model, the possibilities for creating alternative breeding sites for the Common Tern (Sterna hirundo) in the Rhine–Meuse–Scheldt estuary. We explore 22 scenarios that differ with respect to (1) loss of breeding habitat in port areas, (2) location and size of newly created habitat, and (3) coexistence of old and new habitat. Results indicate that loss of port area habitats results in a serious 41% decline in the breeding population. When the loss in ports is compensated for within the ports, the decline was negligible. Fourteen scenarios result in an increase of the Common Tern metapopulation. In these, extra breeding habitat is created outside the ports in fish-rich waters, resulting in a potential metapopulation increase of 25%. However, the period of overlap between lost and newly created habitat strongly affects the results. A gap between the removal of old and the creation of new breeding areas might cause a drop in the metapopulation level of 30%. The population recovery from this drop might take more than 100 years due to slow recolonization. Our results suggest that conservation of seabird species should be evaluated on a metapopulation scale and that the creation of new habitat may help to compensate for habitat loss in other areas. Furthermore, the results indicate that overlap between the existence of old and newly created breeding habitats is crucial for the success of compensation efforts. However, new locations should be carefully selected, because not only is the suitability of the breeding grounds important, but ample fish availability nearby is also key. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ubiquitous expression of the rtTA2S-M2 inducible system in transgenic mice driven by the human hnRNPA2B1/CBX3 CpG island

Background  

A sensitive, ubiquitously expressed tetracycline inducible system would be a valuable tool in mouse transgenesis. However, this has been difficult to obtain due to position effects observed at different chromosomal sites of transgene integration, which negatively affect expression in many tissues. The aim of this study was to test the utility of a mammalian methylation-free CpG island to drive ubiquitous expression of the sensitive doxycycline (Dox) inducible rtTA2S-M2 Tet-transactivator in transgenic mice.