Nitrification in hybrid bioreactors treating simulated domestic wastewater

Aim

To provide deeper insights into nitrification process within aerobic bioreactors containing supplemental physical support media (hybrid bioreactors).

Methods and Results

Three bench‐scale hybrid bioreactors with different media size and one control bioreactor were operated to assess how biofilm integrity influences microbial community conditions and bioreactor performance. The systems were operated initially at a 5‐day hydraulic retention time (HRT), and all reactors displayed efficient nitrification and chemical oxygen demand (COD) removal (>95%). However, when HRT was reduced to 2·5 days, COD removal rates remained high, but nitrification efficiencies declined in all reactors after 19 days. To explain reduced performance, nitrifying bacterial communities (ammonia‐oxidizing bacteria, AOB; nitrite‐oxidizing bacteria, NOB) were examined in the liquid phase and also on the beads using qPCR, FISH and DGGE. Overall, the presence of the beads in a reactor promoted bacterial abundances and diversity, but as bead size was increased, biofilms with active coupled AOB–NOB activity were less apparent, resulting in incomplete nitrification.

Conclusions

Hybrid bioreactors have potential to sustain effective nitrification at low HRTs, but support media size and configuration type must be optimized to ensure coupled AOB and NOB activity in nitrification.

Significance and Impact of the Study

This study shows that AOB and NOB coupling must be accomplished to minimize nitrification failure.     

High cell density media for <Emphasis Type="Italic">Escherichia coli</Emphasis> are generally designed for aerobic cultivations – consequences for large-scale bioprocesses and shake flask cultures

Background  

For the cultivation of Escherichia coli in bioreactors trace element solutions are generally designed for optimal growth under aerobic conditions. They do normally not contain selenium and nickel. Molybdenum is only contained in few of them. These elements are part of the formate hydrogen lyase (FHL) complex which is induced under anaerobic conditions. As it is generally known that oxygen limitation appears in shake flask cultures and locally in large-scale bioreactors, function of the FHL complex may influence the process behaviour. Formate has been described to accumulate in large-scale cultures and may have toxic effects on E. coli.     

Bioreactor mixing efficiency modulates the activity of a prpoS::GFP reporter gene in E. coli

Background  

Extensive studies have shown that up-scaling of bioprocesses has a significant impact on the physiology of the microorganisms. Among the factors associated with the fluid dynamics of the bioreactor, concentration gradients induced by loss of the global mixing efficiency associated with the increasing scale is the main phenomena leading to strong physiological modifications at the level of the microbial population. These changes are not fully understood since they involve complex physiological mechanisms. In this work, we intend to investigate, at the single cell level, the expression of the rpoS gene associated with the stress response of E. coli. The cultures of the reporter strain have been performed in a small scale reactor as well as in a series of scaled-down bioreactors able to induce extracellular perturbations with increasing level of magnitude.     

An ant colony optimisation algorithm for the 2D and 3D hydrophobic polar protein folding problem

Background  

The protein folding problem is a fundamental problems in computational molecular biology and biochemical physics. Various optimisation methods have been applied to formulations of the ab-initio folding problem that are based on reduced models of protein structure, including Monte Carlo methods, Evolutionary Algorithms, Tabu Search and hybrid approaches. In our work, we have introduced an ant colony optimisation (ACO) algorithm to address the non-deterministic polynomial-time hard (NP-hard) combinatorial problem of predicting a protein's conformation from its amino acid sequence under a widely studied, conceptually simple model – the 2-dimensional (2D) and 3-dimensional (3D) hydrophobic-polar (HP) model.     

Correlation between mass transfer coefficient k<Subscript>L</Subscript>a and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale

Background

Among disposable bioreactor systems, cylindrical orbitally shaken bioreactors show important advantages. They provide a well-defined hydrodynamic flow combined with excellent mixing and oxygen transfer for mammalian and plant cell cultivations. Since there is no known universal correlation between the volumetric mass transfer coefficient for oxygen k_La and relevant operating parameters in such bioreactor systems, the aim of this current study is to experimentally determine a universal k_La correlation.

Results

A Respiration Activity Monitoring System (RAMOS) was used to measure k_La values in cylindrical disposable shaken bioreactors and Buckingham’s π-Theorem was applied to define a dimensionless equation for k_La. In this way, a scale- and volume-independent k_La correlation was developed and validated in bioreactors with volumes from 2 L to 200 L. The final correlation was used to calculate cultivation parameters at different scales to allow a sufficient oxygen supply of tobacco BY-2 cell suspension cultures.

Conclusion

The resulting equation can be universally applied to calculate the mass transfer coefficient for any of seven relevant cultivation parameters such as the reactor diameter, the shaking frequency, the filling volume, the viscosity, the oxygen diffusion coefficient, the gravitational acceleration or the shaking diameter within an accuracy range of +/? 30%. To our knowledge, this is the first k_La correlation that has been defined and validated for the cited bioreactor system on a bench-to-pilot scale.

     

Optimization of codon composition and regulatory elements for expression of human insulin like growth factor-1 in transgenic chloroplasts and evaluation of structural identity and function

Background  

Transgenic chloroplasts are potential bioreactors for recombinant protein production, especially for achievement of high levels of protein expression and proper folding. Production of therapeutic proteins in leaves provides transgene containment by elimination of reproductive structures. Therefore, in this study, human Insulin like Growth Factor-1 is expressed in transgenic chloroplasts for evaluation of structural identity and function.     

Use of historic metabolic biotransformation data as a means of anticipating metabolic sites using MetaPrint2D and Bioclipse

Background  

Predicting metabolic sites is important in the drug discovery process to aid in rapid compound optimisation. No interactive tool exists and most of the useful tools are quite expensive.     

Development of patatin knockdown potato tubers using RNA interference (RNAi) technology,for the production of human-therapeutic glycoproteins

Background  

Patatins encoded by a multi-gene family are one of the major storage glycoproteins in potato tubers. Potato tubers have recently emerged as bioreactors for the production of human therapeutic glycoproteins (vaccines). Increasing the yield of recombinant proteins, targeting the produced proteins to specific cellular compartments, and diminishing expensive protein purification steps are important research goals in plant biotechnology. In the present study, potato patatins were eliminated almost completely via RNA interference (RNAi) technology to develop potato tubers as a more efficient protein expression system. The gene silencing effect of patatins in the transgenic potato plants was examined at individual isoform levels.     

Engineering characterisation of a single well from 24-well and 96-well microtitre plates

The detailed engineering characterisation of shaken microtitre-plate bioreactors will enhance our understanding of microbial and mammalian cell culture in these geometries and will provide guidance on the scale-up of microwell results to laboratory and pilot scale stirred bioreactors. In this work computational fluid dynamics (CFD) is employed to provide a detailed characterisation of fluid mixing, energy dissipation rate and mass transfer in single well bioreactors from deep square 24-well and 96-well microtitre plates. The numerical predictions are generally found to be in good agreement with experimental observation of the fluid motion and measured values of the key engineering parameters. The CFD simulations have shown that liquid mixing is more intensive in 96-well than in 24-well bioreactors due to a significant axial component to the fluid velocity. Liquid motion is strongly dependent on the orbital shaking amplitude which generally has a greater impact than the shaking frequency. Average power consumptions of 70–100 W m⁻³ and 500–1000 W m⁻³, and overall mass transfer coefficient, k_La, values of 0.005–0.028 s⁻¹ and 0.056–0.10 s⁻¹ were obtained for 24-well and 96-well bioreactors respectively at an orbital shaking amplitude of 3 mm and shaking frequencies ranging from 500 rpm to 1500 rpm. The distribution of energy dissipation rates within each bioreactor showed these to be greatest at the walls of the well for both geometries. Batch culture kinetics of E. coli DH5 showed similar maximum specific growth rates and final biomass yields in shaken 24-well and shake flask bioreactors and in stirred miniature and 20 L bioreactors at matched k_La values. The CFD simulations thus give new insights into the local and overall engineering properties of microwell bioreactor geometries and further support their use as high throughput tools for the study and optimisation of microbial and mammalian cell culture kinetics at this scale.     

Denitrifying bioreactors—An approach for reducing nitrate loads to receiving waters

Low-cost and simple technologies are needed to reduce watershed export of excess nitrogen to sensitive aquatic ecosystems. Denitrifying bioreactors are an approach where solid carbon substrates are added into the flow path of contaminated water. These carbon (C) substrates (often fragmented wood-products) act as a C and energy source to support denitrification; the conversion of nitrate (NO₃^?) to nitrogen gases. Here, we summarize the different designs of denitrifying bioreactors that use a solid C substrate, their hydrological connections, effectiveness, and factors that limit their performance. The main denitrifying bioreactors are: denitrification walls (intercepting shallow groundwater), denitrifying beds (intercepting concentrated discharges) and denitrifying layers (intercepting soil leachate). Both denitrifcation walls and beds have proven successful in appropriate field settings with NO₃^? removal rates generally ranging from 0.01 to 3.6 g N m^?3 day^?1 for walls and 2–22 g N m^?3 day^?1 for beds, with the lower rates often associated with nitrate-limitations. Nitrate removal is also limited by the rate of C supply from degrading substrate and removal is operationally zero-order with respect to NO₃^? concentration primarily because the inputs of NO₃^? into studied bioreactors have been generally high. In bioreactors where NO₃^? is not fully depleted, removal rates generally increase with increasing temperature. Nitrate removal has been supported for up to 15 years without further maintenance or C supplementation because wood chips degrade sufficiently slowly under anoxic conditions. There have been few field-based comparisons of alternative C substrates to increase NO₃^? removal rates but laboratory trials suggest that some alternatives could support greater rates of NO₃^? removal (e.g., corn cobs and wheat straw). Denitrifying bioreactors may have a number of adverse effects, such as production of nitrous oxide and leaching of dissolved organic matter (usually only for the first few months after construction and start-up). The relatively small amount of field data suggests that these problems can be adequately managed or minimized. An initial cost/benefit analysis demonstrates that denitrifying bioreactors are cost effective and complementary to other agricultural management practices aimed at decreasing nitrogen loads to surface waters. We conclude with recommendations for further research to enhance performance of denitrifying bioreactors.     

Module detection in complex networks using integer optimisation

Background  

The detection of modules or community structure is widely used to reveal the underlying properties of complex networks in biology, as well as physical and social sciences. Since the adoption of modularity as a measure of network topological properties, several methodologies for the discovery of community structure based on modularity maximisation have been developed. However, satisfactory partitions of large graphs with modest computational resources are particularly challenging due to the NP-hard nature of the related optimisation problem. Furthermore, it has been suggested that optimising the modularity metric can reach a resolution limit whereby the algorithm fails to detect smaller communities than a specific size in large networks.     

Antifoam addition to shake flask cultures of recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> increases yield

Background  

Pichia pastoris is a widely-used host for recombinant protein production. Initial screening for both suitable clones and optimum culture conditions is typically carried out in multi-well plates. This is followed by up-scaling either to shake-flasks or continuously stirred tank bioreactors. A particular problem in these formats is foaming, which is commonly prevented by the addition of chemical antifoaming agents. Intriguingly, antifoams are often added without prior consideration of their effect on the yeast cells, the protein product or the influence on downstream processes such as protein purification. In this study we characterised, for the first time, the effects of five commonly-used antifoaming agents on the total amount of recombinant green fluorescent protein (GFP) secreted from shake-flask cultures of this industrially-relevant yeast.     

Parameters for the Mathematical Modelling of Clostridium difficile Acquisition and Transmission: A Systematic Review

Introduction

Mathematical modelling of Clostridium difficile infection dynamics could contribute to the optimisation of strategies for its prevention and control. The objective of this systematic review was to summarise the available literature specifically identifying the quantitative parameters required for a compartmental mathematical model of Clostridium difficile transmission.

Methods

Six electronic healthcare databases were searched and all screening, data extraction and study quality assessments were undertaken in duplicate. Results were synthesised using a narrative approach.

Results

Fifty-four studies met the inclusion criteria. Reproduction numbers for hospital based epidemics were described in two studies with a range from 0.55 to 7. Two studies provided consistent data on incubation periods. For 62% of cases, symptoms occurred in less than 4 weeks (3-28 days) after infection. Evidence on contact patterns was identified in four studies but with limited data reported for populating a mathematical model. Two studies, including one without clinically apparent donor-recipient pairs, provided information on serial intervals for household or ward contacts, showing transmission intervals of <1 week in ward based contacts compared to up to 2 months for household contacts. Eight studies reported recovery rates of between 75% - 100% for patients who had been treated with either metronidazole or vancomycin. Forty-nine studies gave recurrence rates of between 3% and 49% but were limited by varying definitions of recurrence. No study was found which specifically reported force of infection or net reproduction numbers.

Conclusions

There is currently scant literature overtly citing estimates of the parameters required to inform the quantitative modelling of Clostridium difficile transmission. Further high quality studies to investigate transmission parameters are required, including through review of published epidemiological studies where these quantitative estimates may not have been explicitly estimated, but that nonetheless contain the relevant data to allow their calculation. [Systematic review reference: CRD42012003081]     

Response coefficient analysis of a fed-batch bioreactor to dissolved oxygen perturbation in complementary cultures during PHB production

Background  

Although the production of poly-β-hydroxybutyrate (PHB) has many biological, energetic and environmental advantages over chemically synthesized polymers, synthetic polymers continue to be produced industrially since the productivities of fermentation processes fr PHB are not yet economically competitive. Improvement of a PHB fermentation requires good understanding and optimization under the realistic conditions of large bioreactors.     

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Background

The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A_2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.

Results

Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A_2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A_2AR, and therefore more suitable for further functional and structural studies.

Conclusion

Large-scale expression of the A_2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures.     

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

Background  

In cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.     

Rate variation in parasitic plants: correlated and uncorrelated patterns among plastid genes of different function

Background  

The analysis of synonymous and nonsynonymous rates of DNA change can help in the choice among competing explanations for rate variation, such as differences in constraint, mutation rate, or the strength of genetic drift. Nonphotosynthetic plants of the Orobanchaceae have increased rates of DNA change. In this study 38 taxa of Orobanchaceae and relatives were used and 3 plastid genes were sequenced for each taxon.     

Glycerol utilisation for the production of chemicals: Conversion to succinic acid,a combined experimental and computational study

An effective method for the valorisation of the main by-product of biodiesel production, i.e. glycerol is investigated in this work. It involves the biological conversion of glycerol to succinic acid, a top added-value material, which can be used as a building block for the production of various commodity and specialty chemicals. Our aim is to give new insights into this bioprocess, which has so far received little attention and is open for further investigation, through a combination of experimental and computational studies. The microorganism employed here was Actinobacillus succinogenes in batch bioreactors where glycerol was used as the sole carbon source.The highest obtained product yield, final succinate concentration and productivity were found to be equal to 1.23 g-succinate/g-glycerol, 29.3 g-succinate/L and 0.27 g-succinate/L/h, respectively. Furthermore, an unstructured model of the batch experiments was developed by considering both substrate and product inhibition. Kinetic parameters of the model were estimated by minimising the difference between experimental and predicted values. The corresponding optimisation problem was solved by using a combination of stochastic and deterministic methodologies, with the goal to probabilistically compute global minima and the resulting parameter values. The model developed can be utilised to successfully predict the concentration profiles of the five most important state variables (biomass, glycerol, succinic acid, formic acid, and acetic acid) with different initial glycerol concentrations. Scaled-up experiments in larger-scale bioreactors were used for further validation purposes. Our model can be further used to compute optimal operating/parametric conditions, which maximise yield, productivity and/or the final succinic acid concentration.     

Evidence of accelerated ageing in clinical drug addiction from immune, hepatic and metabolic biomarkers

Background  

Drug addiction is associated with significant disease and death, but its impact on the ageing process has not been considered. The recent demonstration that many of the items available in routine clinical pathology have applicability as biomarkers of the ageing process implies that routine clinical laboratory parameters would be useful as an initial investigation of this possibility.