Contributions of the C-terminal domain to poly(A)-specific ribonuclease (PARN) stability and self-association

Poly(A)-specific ribonuclease (PARN) catalyzes the degradation of mRNA poly(A) tail to regulate translation efficiency and mRNA decay in higher eukaryotic cells. The full-length PARN is a multi-domain protein containing the catalytic nuclease domain, the R3H domain, the RRM domain and the C-terminal intrinsically unstructured domain (CTD). The roles of the three well-structured RNA-binding domains have been extensively studied, while little is known about CTD. In this research, the impact of CTD on PARN stability and aggregatory potency was studied by comparing the thermal inactivation and denaturation behaviors of full-length PARN with two N-terminal fragments lacking CTD. Our results showed that K⁺ induced additional regular secondary structures and enhanced PARN stability against heat-induced inactivation, unfolding and aggregation. CTD prevented PARN from thermal inactivation but promoted thermal aggregation to initiate at a temperature much lower than that required for inactivation and unfolding. Blue-shift of Trp fluorescence during thermal transitions suggested that heat treatment induced rearrangements of domain organizations. CTD amplified the stabilizing effect of K⁺, implying the roles of CTD was mainly achieved by electrostatic interactions. These results suggested that CTD might dynamically interact with the main body of the molecule and release of CTD promoted self-association via electrostatic interactions.     

Effective bioreactor pH control using only sparging gases

pH control is critical in bioreactor operations, typically realized through a two-sided control loop, where CO₂ sparging and base addition are used in bicarbonate-buffered media. Though a common approach, base addition could compromise culture performance due to the potential impact from pH excursions and osmolality increase in large-scale bioreactors. In this study, the feasibility of utilizing control of sparge gas composition as part of the pH control loop was assessed in Chinese hamster ovary (CHO) fed-batch cultures. Fine pH control was evaluated in multiple processes at different setpoints in small-scale ambr®250 bioreactors. Desired culture pH setpoints were successfully maintained via air sparge feedback control. As part of the pH control loop, air sparging was increased to improve CO₂ removal automatically, hence increase culture pH, and vice versa. The effectiveness of this pH control strategy was seamlessly transferred from ambr®250 to 200 L scale, demonstrating scalability of the proposed methodology. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2743, 2019     

Inorganic nanoparticles for transfection of mammalian cells and removal of viruses from aqueous solutions

Owing to their small size, synthetic nanoparticles show unprecedented biophysical and biochemical properties which may foster novel advances in life-science research. Using flame-spray synthesis technology we have produced non-coated aluminum-, calcium-, cerium-, and zirconium-derived inorganic metal oxide nanoparticles which not only exhibit high affinity for nucleic acids, but can sequester such compounds from aqueous solution. This non-covalent DNA-binding capacity was successfully used to transiently transfect a variety of mammalian cells including human, reaching transfection efficiencies which compared favorably with classic calcium phosphate precipitation (CaP) procedures and lipofection. In this straightforward protocol, transfection was enabled by simply mixing nanoparticles with DNA in solution prior to addition to the target cell population. Transiently transfected cells showed higher production levels of the human secreted glycoprotein SEAP compared to isogenic populations transfected with established technologies. Inorganic metal oxide nanoparticles also showed a high binding capacity to human-pathogenic viruses including adenovirus, adeno-associated virus and human immunodeficiency virus type 1 and were able to clear these pathogens from aqueous solutions. The DNA transfection and viral clearance capacities of inorganic metal oxide nanoparticles may provide cost-effective biopharmaceutical manufacturing and water treatment in developing countries.     

A general kinetic model for biological nutrient removal activated sludge systems: model development

In this article, a kinetic model is developed and presented for biological nutrient removal (BNR) activated sludge (BNRAS) systems in general, but for external nitrification (EN) BNRAS (ENBNRAS) systems in particular. The model is based on the UCTPHO model, but includes some significant modifications, such as anoxic P uptake and associated denitrification by phosphorus accumulating organisms (PAOs). Some key features of the model are described and discussed before the model is presented. Model evaluation will be addressed in another article (Hu et al., 2007).     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

Redox-stratification controlled biofilm (ReSCoBi) for completely autotrophic nitrogen removal: the effect of co- versus counter-diffusion on reactor performance

A multi-population biofilm model for completely autotrophic nitrogen removal was developed and implemented in the simulation program AQUASIM to corroborate the concept of a redox-stratification controlled biofilm (ReSCoBi). The model considers both counter- and co-diffusion biofilm geometries. In the counter-diffusion biofilm, oxygen is supplied through a gas-permeable membrane that supports the biofilm while ammonia (NH(4)(+)) is supplied from the bulk liquid. On the contrary, in the co-diffusion biofilm, both oxygen and NH(4)(+) are supplied from the bulk liquid. Results of the model revealed a clear stratification of microbial activities in both of the biofilms, the resulting chemical profiles, and the obvious effect of the relative surface loadings of oxygen and NH(4)(+) (J(O(2))/J(NH(4)(+))) on the reactor performances. Steady-state biofilm thickness had a significant but different effect on T-N removal for co- and counter-diffusion biofilms: the removal efficiency in the counter-diffusion biofilm geometry was superior to that in the co-diffusion counterpart, within the range of 450-1,400 microm; however, the efficiency deteriorated with a further increase in biofilm thickness, probably because of diffusion limitation of NH(4)(+). Under conditions of oxygen excess (J(O(2))/J(NH(4)(+)) > 3.98), almost all NH(4)(+) was consumed by aerobic ammonia oxidation in the co-diffusion biofilm, leading to poor performance, while in the counter-diffusion biofilm, T-N removal efficiency was maintained because of the physical location of anaerobic ammonium oxidizers near the bulk liquid. These results clearly reveal that counter-diffusion biofilms have a wider application range for autotrophic T-N removal than co-diffusion biofilms.     

Concurrent nitrite oxidation and aerobic denitrification in activated sludge exposed to volatile fatty acids

The goal of this research was to investigate the simultaneous occurrence of nitrification and denitrification by activated sludge exposed to volatile fatty acids (VFAs) during aerobic wastewater treatment using a single-stage reactor. A mixture of VFAs was spiked directly into a continuous-stirred tank reactor (CSTR) to assess subsequent impacts on nitrite removal, nitrate formation, CO(2) fixation, total bacterial density, and dominant nitrite oxidizing bacteria (NOB) concentration (i.e., Nitrospira). The activity of the periplasmic nitrate reductase (NAP) enzyme and the presence of nap gene were also measured. A rapid decrease in the nitrate formation rate (>70% reduction) was measured for activated sludge exposed to VFAs; however, the nitrite removal rate was not reduced. The total bacterial density and Nitrospira concentration remained essentially constant; therefore, the reduction in nitrate formation rate was likely not due to heterotrophic uptake of nitrogen or to a decrease in the dominant NOB population. Additionally, VFA exposure did not impact microbial CO(2) fixation efficiency. The activity of NAP enzyme increased in the presence of VFAs suggesting that nitrate produced as a consequence of nitrite oxidation was likely further reduced to gaseous denitrification products via catalysis by NAP. Little, if any, nitrogen was discharged in the aqueous effluent of the CSTR after exposure to VFAs demonstrating that activated sludge treatment yielded compounds other than those typically produced solely by nitrification.     

Wax removal for accelerated cotton scouring with alkaline pectinase

A rational approach has been applied to design a new environmentally acceptable and industrially viable enzymatic scouring process. Owing to the substrate specificity, the selection of enzymes depends on the structure and composition of the substrate, i.e. cotton fibre. The structure and composition of the outer layers of cotton fibre has been established on the basis of thorough literature study, which identifies wax and pectin removal to be the key steps for successful scouring process. Three main issues are discussed here, i.e. benchmarking of the existing alkaline scouring process, an evaluation of several selected acidic and alkaline pectinases for scouring, and the effect of wax removal treatment on pectinase performance. It has been found that the pectinolytic capability of alkaline pectinases on cotton pectin is nearly 75% higher than that of acidic pectinases. It is concluded that an efficient wax removal prior to pectinase treatment indeed results in improved performance in terms of hydrophilicity and pectin removal. To evaluate the hydrophilicity, the structural contact angle (theta) was measured using an auto-porosimeter.     

Comparison of quasisteady-state performance of the DEAMOX process under intermittent and continuous feeding and different nitrogen loading rates

The recently developed denitrifying ammonium oxidation (DEAMOX) process combines the anammox reaction with autotrophic denitrifying conditions using sulfide as an electron donor for the production of nitrite from nitrate within an anaerobic biofilm. This paper compares a quasisteady-state performance of this process for treatment of baker's yeast wastewater under intermittent and continuous feeding and increasing nitrogen loading rate (NLR) from 300 till 858 mg N/L/d. The average total nitrogen removal slightly decreased on increasing the NLR: from 86 to 79% (intermittent feeding) and from 87 to 84% (continuous feeding). The better performance under continuous feeding was due to a more complete nitrate removal in the former case whereas the ammonia removal was similar for both feeding regimes under the comparable NLR. A possible explanation can be that, during continuous feeding (simultaneous supply of nitrate and sulfide), there were less mass transfer limitations for sulfide oxidizing denitrifiers presumably located in the outer layer of sludge aggregates. On the contrary, the ammonia oxidisers presumably located inside the aggregates apparently suffered from nitrite mass transfer limitations under both the feedings. The paper further describes some characteristics of the DEAMOX sludge.