Continuous cultivation of Dehalococcoides mccartyi with brominated tyrosine avoids toxic byproducts and gives tight reactor control.

Dehalococcoides mccartyi strain CBDB1 is a strictly anaerobic organohalide-respiring bacterium with strong application potential to remediate aquifers and soils contaminated with halogenated aromatics. To date, cultivation of strain CBDB1 has mostly been done in bottles or fed-batch reactors. Challenges with such systems include low biomass yield and difficulties in controlling the growth conditions. Here, we report the cultivation of planktonic D. mccartyi strain CBDB1 in a continuous stirring tank reactor (CSTR) that led to high cell densities (∼8 × 108 cells mL-1) and dominance of strain CBDB1. The reactor culture received acetate, hydrogen, and the brominated amino acid D- or L-3,5-dibromotyrosine as substrates. Both D- and L-3,5-dibromotyrosine were utilized as respiratory electron acceptors and are promising for biomass production due to their decent solubility in water and the formation of a non-toxic debromination product, tyrosine. By monitoring headspace pressure decrease which is indicative of hydrogen consumption, the organohalide respiration rate was followed in real time. Proteomics analyses revealed that the reductive dehalogenase CbdbA238 was highly expressed with both D- and L-3,5-dibromotyrosine, while other reductive dehalogenases including those that were previously suggested to be constitutively expressed, were repressed. Denaturing gradient gel electrophoresis (DGGE) of amplified 16S rRNA genes indicated that the majority of cells in the community belonged to the Dehalococcoides although the CSTR was operated under non-sterile conditions. Hence, tightly controlled CSTR cultivation of Dehalococcoides opens novel options to improve biomass production for bioaugmentation and for advanced biochemical studies.

Glycosylated amyloid-like proteins in the structural extracellular polymers of aerobic granular sludge enriched with ammonium-oxidizing bacteria.

A new type of structural extracellular polymers (EPS) was extracted from aerobic granular sludge dominated by ammonium-oxidizing bacteria. It was analyzed by Raman and FTIR spectroscopy to characterize specific amino acids and protein secondary structure, and by SDS-PAGE with different stains to identify different glycoconjugates. Its intrinsic fluorescence was captured to visualize the location of the extracted EPS in the nitrifying granules, and its hydrogel-forming property was studied by rheometry. The extracted EPS is abundant with cross ß-sheet secondary structure, contains glycosylated proteins/polypeptides, and rich in tryptophan. It forms hydrogel with high mechanical strength. The extraction and discovery of glycosylated proteins and/or amyloids further shows that conventionally used extraction and characterization techniques are not adequate for the study of structural extracellular polymers in biofilms and/or granular sludge. Confirming amyloids secondary structure in such a complex sample is challengeable due to the possibility of amyloids glycosylation and self-assembly. A new definition of extracellular polymers components which includes glycosylated proteins and a better approach to studying them is required to stimulate biofilm research.

Stable long-term operation of an upflow anammox sludge bed reactor at mainstream conditions.

The efforts for implementing the anammox process at mainstream conditions with high nitrogen removal rates have gained much attention in the race for achieving an energy-positive urban wastewater treatment plant. Here, the successful and stable long-term operation of an Upflow Anammox Sludge Bed (UAnSB) reactor treating a low-strength synthetic influent amended with ammonium and nitrite for 420 days, and a nitrite-amended pre-treated real urban wastewater for 110 days at temperatures as low as 11 °C is presented. The short and long-term effects of temperature on anammox activity were assessed when the synthetic influent was treated, and the UAnSB reactor was demonstrated to be a robust reactor to confront low temperatures, typically found at mainstream conditions. In fact, a nitrogen loading rate as high as 1.8 ± 0.1 g N L-1 d-1 with 82 ± 4% of nitrogen removal was achieved at 11 °C treating the low-strength synthetic influent. Furthermore, the effect of treating a nitrite-amended pre-treated real urban wastewater at 11 °C at long-term in the UAnSB reactor was evaluated, and a stable operation was achieved with a high average nitrogen removal rate (1.2 ± 0.5 g N L-1 d-1). The relative abundance of anammox bacteria was maintained higher than 70% according to fluorescence in situ hybridization during the whole operation, being Candidatus Brocadia anammoxidans the predominant microbial species. The presence of heterotrophs in the sludge bed was surmised through heterotrophic batch tests, but anammox activity was demonstrated to be higher than heterotrophic activity, even when the synthetic influent was replaced by the nitrite-amended pre-treated real wastewater. The feasibility of operating an enriched anammox reactor at high nitrogen removal rate at long-term at mainstream conditions was demonstrated in this study.

Effect of temperature on N2O emissions from a highly enriched nitrifying granular sludge performing partial nitritation of a low-strength wastewater.

In the race to achieve a sustainable urban wastewater treatment plant, not only the energy requirements have to be considered but also the environmental impact of the facility. Thus, nitrous oxide (N2O) emissions are a key-factor to pay attention to, since they can dominate the total greenhouse gases emissions from biological wastewater treatment. In this study, N2O production factors were calculated during the operation of a granular sludge airlift reactor performing partial nitritation treating a low-strength synthetic influent, and furthermore, the effect of temperature on N2O production was assessed. Average gas emission relative to conversion of ammonium was 1.5 ± 0.3% and 3.7 ± 0.5% while the effluent contained 0.5 ± 0.1% and 0.7 ± 0.1% (% N-oxidized) at 10 and 20 °C, respectively. Hence, temperature increase resulted in higher N2O production. The reasons why high temperature favoured N2O production remained unclear, but different theoretical hypotheses were suggested.

Kinetic and microbiological characterization of aerobic granules performing partial nitritation of a low-strength wastewater at 10 °C.

A granular airlift reactor enriched in ammonia oxidizing bacteria (AOB) was operated at 10 °C performing stable partial nitritation in the long-term. The reactor treated a synthetic low-strength influent during 250 days with an average nitrogen loading rate of 0.63 ± 0.06 g N L(-1) d(-1). Nitrate production was barely detected, being the average concentration in the effluent of 0.6 ± 0.3 mg N-NO3 L(-1). Furthermore, a suitable effluent for a subsequent reactor performing the anammox process was achieved. A maximum specific growth rate as high as 0.63 ± 0.05 d(-1) was determined by performing kinetic experiments with the granular sludge in a chemostat and fitting the results to the Monod model. Pyrosequencing analysis showed a high enrichment in AOB (41 and 65% of the population were identified as Nitrosomonas genus on day 98 and 233, respectively) and an effective repression of nitrite oxidizing bacteria in the long-term. Pyrosequencing analysis also identified the coexistence of nitrifying bacteria and heterotrophic psychrotolerant microorganisms in the granular sludge. Some psychrotolerant microorganisms are producers of cryoprotective extracellular polymeric substances that could explain the better survival of the whole consortia at cold temperatures.

Stable partial nitritation for low-strength wastewater at low temperature in an aerobic granular reactor.

Partial nitritation for a low-strength wastewater at low temperature was stably achieved in an aerobic granular reactor. A bench-scale granular sludge bioreactor was operated in continuous mode treating an influent of 70 mg N-NH4(+) L(-1) to mimic pretreated municipal nitrogenous wastewater and the temperature was progressively decreased from 30 to 12.5 °C. A suitable effluent nitrite to ammonium concentrations ratio to a subsequent anammox reactor was maintained stable during 300 days at 12.5 °C. The average applied nitrogen loading rate at 12.5 °C was 0.7 ± 0.3 g N L(-1) d(-1), with an effluent nitrate concentration of only 2.5 ± 0.7 mg N-NO3(-) L(-1). The biomass fraction of nitrite-oxidizing bacteria (NOB) in the granular sludge decreased from 19% to only 1% in 6 months of reactor operation at 12.5 °C. Nitrobacter spp. where found as the dominant NOB population, whereas Nitrospira spp. were not detected. Simulations indicated that: (i) NOB would only be effectively repressed when their oxygen half-saturation coefficient was higher than that of ammonia-oxidizing bacteria; and (ii) a lower specific growth rate of NOB was maintained at any point in the biofilm (even at 12.5 °C) due to the bulk ammonium concentration imposed through the control strategy.

Novel synthetic approach to 1alpha,25-dihydroxyvitamin D3 and analogues.

A mild and stereoconvergent synthesis of 1alpha,25-dihydroxyvitamin D(3) (calcitriol, 1a) is described. The key step is a cascade process consisting of two consecutive transformations: An initial palladium-catalyzed 6-exo-cyclocarbopalladation of vinyl triflate 4 followed by a Negishi cross-coupling reaction with alkenyl zinc 3. This approach is of interest for the rapid synthesis of a variety of new vitamin D(3) analogues of therapeutic potential, especially those modified at the triene and ring-A. The mildness of the method also allows the preparation of thermal sensitive vitamin D(3) analogues.

Pd-catalyzed carbocyclization-Negishi cross-coupling cascade: a novel approach to 1alpha,25-dihydroxyvitamin D3 and analogues.

[reaction: see text] A mild palladium-catalyzed cascade has been used for the synthesis of the hormone 1alpha,25-dihydroxyvitamin D3 (calcitriol, 1a) and its analogues 1b and 1c. This one-pot process involves two consecutive transformations at room temperature: An initial palladium-catalyzed 6-exo-cyclocarbopalladation of vinyl triflates followed by a Negishi cross-coupling reaction with an alkenyl zinc. This novel strategy opens new possibilities for the preparation of a variety of new vitamin D analogues of therapeutic potential, particularly with modifications at the triene and/or ring-A.