Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System.

The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1-2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.

Ex Vivo Modeling of Human Neuroendocrine Tumors in Tissue Surrogates.

Few models exist for studying neuroendocrine tumors (NETs), and there are mounting concerns that the currently available array of cell lines is not representative of NET biology. The lack of stable patient-derived NET xenograft models further limits the scientific community's ability to make conclusions about NETs and their response to therapy in patients. To address these limitations, we propose the use of an ex vivo 3D flow-perfusion bioreactor system for culturing and studying patient-derived NET surrogates. Herein, we demonstrate the utility of the bioreactor system for culturing NET surrogates and provide methods for evaluating the efficacy of therapeutic agents on human NET cell line xenograft constructs and patient-derived NET surrogates. We also demonstrate that patient-derived NET tissues can be propagated using the bioreactor system and investigate the near-infrared (NIR) dye IR-783 for its use in monitoring their status within the bioreactor. The results indicate that the bioreactor system and similar 3D culture models may be valuable tools for culturing patient-derived NETs and monitoring their response to therapy ex vivo.

Enhanced anaerobic degradation of nitrogen heterocyclic compounds with methanol, sodium citrate, chlorella, spirulina, and carboxymethylcellulose as co-metabolic substances.

The aim of the study was to explore the feasibility of methanol, sodium citrate, chlorella, spirulina, and carboxymethylcellulose (CMC) as co-metabolic substances in strengthening the anaerobic degradation of selected nitrogen heterocyclic compounds (NHCs). Chlorella, spirulina, and CMC as co-metabolic substances were first introduced into the enhanced anaerobic treatment of refractory compounds. With the addition of 300 µg/L sodium citrate, chlorella, spirulina, and CMC, reactor 3, reactor 4, reactor 5, and reactor 6 had higher degradation ratios than reactor 2 with methanol as co-metabolic substance. The addition of sodium citrate, chlorella, spirulina, and CMC increased the number of bacterial sequences, promoted the richness and diversity of the bacterial community structure, and enriched the functional genera (Levilinea and Longilinea) responsible for the degradation of quinoline and indole.

Operational parameters in H2S biofiltration under extreme acid conditions: performance, biomass control, and CO2 consumption.

This paper reports the treatment of gaseous hydrogen sulfide, H2S, in a biotrickling filter (BTF) under extreme acidic pH conditions (≈ 1.2). The effect of adding thiosulfate (Na2S2O3.5H2O) to promote biomass growth, feeding low concentrations of ozone to control excess biomass, and the carbon dioxide, CO2, consumption by the chemolithoautotrophic consortium were evaluated. The results showed a global removal efficiency over 98.0% with loads of H2S > 50 g m-3 h-1 (at 639 ppmv) and a linear relation between H2S elimination capacity with the CO2 consumption rate of around 0.1 gCO2/gH2S. Supplementing sulfur in the medium with 2 g L-1 thiosulfate resulted in negative effect performance. Respirometry tests proved that the consortium could not utilize this sulfur form at this pH. Additionally, continuous and intermittent O3 feeding to the BTF in gaseous concentrations of 98 ± 5.4 mg m-3 caused a slight decreased in the performance but the biomass activity in the BTF was only slightly affected allowing a quick performance recovery once O3 addition was suspended.

Effect of waste compaction density on stabilization of aerobic bioreactor landfills.

Landfill stabilization contributes to the safe operation and maintenance of landfills. This study used a simulated aerobic bioreactor landfill to investigate the impact of different compaction densities on its stabilization to provide a basis for optimal parameter selection during landfill design. Samples of municipal solid waste were tested with compaction densities of 450, 500, 550, 600, and 650 kg/m3 during the experiment. The optimum compaction density was obtained by periodically monitoring the temperature of the waste pile, the water quality of leachate, and the composition of the waste. The impacts of waste compaction density on waste pile temperature and leachate were investigated and coupled with the analysis of waste composition to discuss the possible reaction mechanism. Results showed that the most complete waste degradation occurred at 550 kg/m3 compaction density, which was effective at accelerating stabilization of the simulated aerobic bioreactor landfill. Limitations of the experiment are given to lay foundations for further study.

Modelling biofilm anaerobic reactor with effluent from hydrolytic/acidogenic reactor as substrate.

This work presents modelling of an anaerobic biofilm reactor using ceramic bricks as support. The results were compared with the experimental data. It was observed that the substrate concentration curves showed the same tendency. The methane formation curves showed significant differences. The substrate removal efficiency was 83%. In the steady state, the experimental data were higher than the model, from the result the substrate degrading bacteria grew enough to reach biofilm and that the effect of the shear stress was more significant as the biofilm increased in thickness. To the methane production, the model in steady state reached a maximum value of 0.56 m3 CH4/m3 *d and the experimental data reached 0.42 (m3 CH4/m3 * d). The biofilm thickness calculated by the model was 14 µm.

Simultaneous p-nitrophenol and nitrogen removal in PNP wastewater treatment: Comparison of two integrated membrane-aerated bioreactor systems.

The chemical p-nitrophenol (PNP) is a priority pollutant, and PNP wastewater is highly toxic and resistant to biodegradation. The traditional physical and chemical methods (adsorption, extraction, and oxidation) for treating PNP wastewater have the disadvantages of complicated processes, high costs and secondary pollution generation. In this study, two integrated membrane-aerated bioreactor systems (RA and RB) with anoxic and aerated zones were constructed to enhance PNP biodegradation. The results showed that a helical silicone rubber membrane module displayed a high oxygen supply rate under a low membrane aeration pressure, and the hydraulic flow state of the reactor approached ideal mixing. At an influent PNP concentration of 500 mg/L, the average removal rates of PNP, chemical oxygen demand (COD) and total nitrogen (TN) reached 95.86%, 89.77%, and 94.81%, respectively, for RA and 89.48%, 74.26% and 64.78%, respectively, for RB, indicating efficient simultaneous PNP and nitrogen removal. Compared with that of RB, the pre-anoxic zone in RA not only performed detoxification pretreatment but also enhanced PNP degradation and denitrification effects, which relieved the biological treatment burden of the subsequent aerated zone. Based on these comprehensive analyses of reactor performance, the hydroquinone pathway might be the main route in the aerobic degradation of PNP.

Dynamic optimization of fed-batch bioprocesses using flower pollination algorithm.

There exist several optimization strategies such as sequential quadratic programming (SQP), iterative dynamic programing (IDP), stochastic-based methods such as differential evolution (DE), genetic algorithm (GA), particle swarm optimization (PSA), and ant colony optimization (ACO) for finding optimal feeding profile(s) during fed-batch fermentations. Here in the present study, flower pollination algorithm (FPA) which is inspired by the pollination process in terrestrial flowering plants has been used for the first time to find the optimal feeding profile(s) during fed-batch fermentations. Single control variable, two control variables and state variable bounded problems were chosen to test the robustness of the FPA for optimal control problems. It was observed that FPA is computationally less intensive in comparison with other stochastic strategies. Thus, obtained results were compared to other studies and it has been found that the FPA converged either to newer optima or closer to the established global optimum for the cases studied.

A stoichiometric organic matter decomposition model in a chemostat culture.

Biodegradation, the disintegration of organic matter by microorganism, is essential for the cycling of environmental organic matter. Understanding and predicting the dynamics of this biodegradation have increasingly gained attention from the industries and government regulators. Since changes in environmental organic matter are strenuous to measure, mathematical models are essential in understanding and predicting the dynamics of organic matters. Empirical evidence suggests that grazers' preying activity on microorganism helps to facilitate biodegradation. In this paper, we formulate and investigate a stoichiometry-based organic matter decomposition model in a chemostat culture that incorporates the dynamics of grazers. We determine the criteria for the uniform persistence and extinction of the species and chemicals. Our results show that (1) if at the unique internal steady state, the per capita growth rate of bacteria is greater than the sum of the bacteria's death and dilution rates, then the bacteria will persist uniformly; (2) if in addition to this, (a) the grazers' per capita growth rate is greater than the sum of the dilution rate and grazers' death rate, and (b) the death rate of bacteria is less than some threshold, then the grazers will persist uniformly. These conditions can be achieved simultaneously if there are sufficient resources in the feed bottle. As opposed to the microcosm decomposition models' results, in a chemostat culture, chemicals always persist. Besides the transcritical bifurcation observed in microcosm models, our chemostat model exhibits Hopf bifurcation and Rosenzweig's paradox of enrichment phenomenon. Our sensitivity analysis suggests that the most effective way to facilitate degradation is to decrease the dilution rate.

The environmental footprint of a membrane bioreactor treatment process through Life Cycle Analysis.

This study includes an environmental analysis of a membrane bioreactor (MBR), the objective being to quantitatively define the inventory of the resources consumed and estimate the emissions produced during its construction, operation and end-of-life deconstruction. The environmental analysis was done by the life cycle assessment (LCA) methodology, in order to establish with a broad perspective and in a rigorous and objective way the environmental footprint and the main environmental hotspots of the examined technology. Raw materials, equipment, transportation, energy use, as well as air- and waterborne emissions were quantified using as a functional unit, 1m(3) of urban wastewater. SimaPro 8.0.3.14 was used as the LCA analysis tool, and two impact assessment methods, i.e. IPCC 2013 version 1.00 and ReCiPe version 1.10, were employed. The main environmental hotspots of the MBR pilot unit were identified to be the following: (i) the energy demand, which is by far the most crucial parameter that affects the sustainability of the whole process, and (ii) the material of the membrane units. Overall, the MBR technology was found to be a sustainable solution for urban wastewater treatment, with the construction phase having a minimal environmental impact, compared to the operational phase. Moreover, several alternative scenarios and areas of potential improvement, such as the diversification of the electricity mix and the material of the membrane units, were examined, in order to minimize as much as possible the overall environmental footprint of this MBR system. It was shown that the energy mix can significantly affect the overall sustainability of the MBR pilot unit (i.e. up to 95% reduction of the total greenhouse gas emissions was achieved with the use of an environmentally friendly energy mix), and the contribution of the construction and operational phase to the overall environmental footprint of the system.

Load limit of a UASB fed septic tank-treated domestic wastewater.

Performance of a 250 L pilot-scale up-flow anaerobic sludge blanket (UASB) reactor, operated at ambient temperatures, fed septic tank effluents intermittently, was monitored for hydraulic retention time (HRT) from 18 h to 4 h. The total suspended solids (TSS), total chemical oxygen demand (CODT), dissolved chemical oxygen demand (CODdis) and suspended chemical oxygen demand (CODss) removal efficiencies ranged from 20 to 63%, 15 to 56%, 8 to 35% and 22 to 72%, respectively, for the HRT range tested. Above 60% TSS and 47% CODT removal were obtained in the combined septic tank and UASB process. The process established stable UASB treatment at HRT≥6 h, indicating a hydraulic load design limit. The tested septic tank-UASB combined system can be a low-cost and effective on-site sanitation solution.

Regression based state space adaptive model of two-phase anaerobic reactor.

In this paper, a linear state space model for the two-phase anaerobic reactor system was developed based on historical data. Subsequently, the model was used to predict its future behavior. The state space model developed involved correlation analysis and model development. The model would be updated at every time point when a new data set became available, giving it an "adaptive" feature. The model was then applied to monitor two-phase anaerobic co-digestion of a feed comprising 2 industrial secondary sludges and 2 industrial wastewaters. The case study showed the proposed model was able to provide good predictions of various process parameters. In addition, it also predicted impending process failure and this would have allowed the operator to take necessary measures to prevent or reduce impact of such failure during plant operation.

Nitritating-anammox biomass tolerant to high dissolved oxygen concentration and C/N ratio in treatment of yeast factory wastewater.

Maintaining stability of low concentration (< 1 g L(-1)) floccular biomass in the nitritation-anaerobic ammonium oxidation (anammox) process in the sequencing batch reactor (SBR) system for the treatment of high COD (> 15,000 mg O2 L(-1)) to N (1680 mg N L(-1)) ratio real wastewater streams coming from the food industry is challenging. The anammox process was suitable for the treatment of yeast factory wastewater containing relatively high and abruptly increased organic C/N ratio and dissolved oxygen (DO) concentrations. Maximum specific total inorganic nitrogen (TIN) loading and removal rates applied were 600 and 280 mg N g(-1) VSS d(-1), respectively. Average TIN removal efficiency over the operation period of 270 days was 70%. Prior to simultaneous reduction of high organics (total organic carbon > 600mg L(-1)) and N concentrations > 400 mg L(-1), hydraulic retention time of 15 h and DO concentrations of 3.18 (+/- 1.73) mg O2 L(-1) were applied. Surprisingly, higher DO concentrations did not inhibit the anammox process efficiency demonstrating a wider application of cultivated anammox biomass. The SBR was fed rapidly over 5% of the cycle time at 50% volumetric exchange ratio. It maintained high free ammonia concentration, suppressing growth of nitrite-oxidizing bacteria. Partial least squares and response surface modelling revealed two periods of SBR operation and the SBR performances change at different periods with different total nitrogen (TN) loadings. Anammox activity tests showed yeast factory-specific organic N compound-betaine and inorganic N simultaneous biodegradation. Among other microorganisms determined by pyrosequencing, anammox microorganism (uncultured Planctomycetales bacterium clone P4) was determined by polymerase chain reaction also after applying high TN loading rates.

Design and operating experiences of full-scale municipal membrane bioreactors in Japan.

In Japan, membrane bioreactor (MBRs) have been installed in 17 small-scale municipal wastewater treatment plants (WWTPs) in the past 8 years, together with two recently installed MBRs for larger-scale WWTPs. In this study, design and operating data were collected from 17 of them as part of a follow-up survey, and aspects including system design, biological treatment, membrane operation, problems and costs were overviewed. Because most of the MBRs were designed according to standardized guidance, system configuration of the plants was similar; pre-denitrification using the Modified Ludzack-Ettinger (MLE) process with membrane units submerged in aerobic tanks, following a fine screen and flow equalization tank. This led to effluent quality with biochemical oxygen demand and T-N of less than 3.5 and 7.4 mg/L, respectively, for nine plants on an annual average basis. It was a common practice in extremely under-loaded plants to operate the membrane systems intermittently. Frequency of recovery cleaning events was plant-specific, mostly ranging from 1 to 5 times/year. Cost evaluation revealed that specific construction costs for the small-scale MBRs were no more than for oxidation ditch plants. Although specific energy consumption values tended to be high in the under-loaded plants, the demonstration MBR, where several energy reducing measures had been incorporated, attained specific energy consumption of 0.39 kWh/m(3) under full-capacity operation.

On-line prediction of the feeding phase in high-cell density cultivation of rE. coli using constructive neural networks.

Streptococcus pneumoniae (pneumococcus) is a bacterium responsible for a wide spectrum of illnesses. The surface of the bacterium consists of three distinctive membranes: plasmatic, cellular and the polysaccharide (PS) capsule. PS capsules may mediate several biological processes, particularly invasive infections of human beings. Prevention against pneumococcal related illnesses can be provided by vaccines. There is a sound investment worldwide in the investigation of a proteic antigen as a possible alternative to pneumococcal vaccines based exclusively on PS. A few proteins which are part of the membrane of the pneumococcus seem to have antigen potential to be part of a vaccine, particularly the PspA. A vital aspect in the production of the intended conjugate pneumococcal vaccine is the efficient production (in industrial scale) of both, the chosen PS serotypes as well as the PspA protein. Growing recombinant Escherichia coli (rE. coli) in high-cell density cultures (HCDC) under a fed-batch regime requires a refined continuous control over various process variables where the on-line prediction of the feeding phase is of particular relevance and one of the focuses of this paper. The viability of an on-line monitoring software system, based on constructive neural networks (CoNN), for automatically detecting the time to start the fed-phase of a HCDC of rE. coli that contains a plasmid used for PspA expression is investigated. The paper describes the data and methodology used for training five different types of CoNNs, four of them suitable for classification tasks and one suitable for regression tasks, aiming at comparatively investigate both approaches. Results of software simulations implementing five CoNN algorithms as well as conventional neural networks (FFNN), decision trees (DT) and support vector machines (SVM) are also presented and discussed. A modified CasCor algorithm, implementing a data softening process, has shown to be an efficient candidate to be part of an on-line HCDC monitoring system for detecting the feeding phase of the HCDC process.

Application of dynamic models to estimate greenhouse gas emission by wastewater treatment plants of the pulp and paper industry.

Greenhouse gas (GHG) emission in wastewater treatment plants of the pulp-and-paper industry was estimated by using a dynamic mathematical model. Significant variations were shown in the magnitude of GHG generation in response to variations in operating parameters, demonstrating the limited capacity of steady-state models in predicting the time-dependent emissions of these harmful gases. The examined treatment systems used aerobic, anaerobic, and hybrid-anaerobic/aerobic-biological processes along with chemical coagulation/flocculation, anaerobic digester, nitrification and denitrification processes, and biogas recovery. The pertinent operating parameters included the influent substrate concentration, influent flow rate, and temperature. Although the average predictions by the dynamic model were only 10 % different from those of steady-state model during 140 days of operation of the examined systems, the daily variations of GHG emissions were different up to ± 30, ± 19, and ± 17 % in the aerobic, anaerobic, and hybrid systems, respectively. The variations of process variables caused fluctuations in energy generation from biogas recovery by ± 6, ± 7, and ± 4 % in the three examined systems, respectively. The lowest variations were observed in the hybrid system, showing the stability of this particular process design.

Operational experience with a seasonally operated full-scale membrane bioreactor plant.

A seasonally operated full-scale membrane bioreactor plant (flat sheet, 0.03 µm) treating municipal wastewater from a recreation facility was monitored for 2 years. In particular, membrane bioreactor operation characteristics and development and changes in extracellular polymeric substances and soluble microbial product concentrations were observed, which were both dependent on volume and quality of incoming wastewater. Microbiological effluent quality, nutrient removal efficiency and activated sludge characteristics were analysed on a regular basis. Correlations between activated sludge quality, extracellular polymeric substance and soluble microbial product concentrations were identified. Pathogen related changes in effluent quality during plant operation were also observed. Nutrient removal efficiency was very good, despite fluctuations in influent flow.

Fluid and mass transport modelling to drive the design of cell-packed hollow fibre bioreactors for tissue engineering applications.

A model for fluid and mass transport in a single module of a tissue engineering hollow fibre bioreactor (HFB) is developed. Cells are seeded in alginate throughout the extra-capillary space (ECS), and fluid is pumped through a central lumen to feed the cells and remove waste products. Fluid transport is described using Navier-Stokes or Darcy equations as appropriate; this is overlaid with models of mass transport in the form of advection-diffusion-reaction equations that describe the distribution and uptake/production of nutrients/waste products. The small aspect ratio of a module is exploited and the option of opening an ECS port is explored. By proceeding analytically, operating equations are determined that enable a tissue engineer to prescribe the geometry and operation of the HFB by ensuring the nutrient and waste product concentrations are consistent with a functional cell population. Finally, results for chondrocyte and cardiomyocyte cell populations are presented, typifying two extremes of oxygen uptake rates.

Mass transport in a microchannel bioreactor with a porous wall.

A two-dimensional flow model has been developed to simulate mass transport in a microchannel bioreactor with a porous wall. A two-domain approach, based on the finite volume method, was implemented. For the fluid part, the governing equation used was the Navier-Stokes equation; for the porous medium region, the generalized Darcy-Brinkman-Forchheimer extended model was used. For the porous-fluid interface, a stress jump condition was enforced with a continuity of normal stress, and the mass interfacial conditions were continuities of mass and mass flux. Two parameters were defined to characterize the mass transports in the fluid and porous regions. The porous Damkohler number is the ratio of consumption to diffusion of the substrates in the porous medium. The fluid Damkohler number is the ratio of the substrate consumption in the porous medium to the substrate convection in the fluid region. The concentration results were found to be well correlated by the use of a reaction-convection distance parameter, which incorporated the effects of axial distance, substrate consumption, and convection. The reactor efficiency reduced with reaction-convection distance parameter because of reduced reaction (or flux), and smaller local effectiveness factor due to the lower concentration in Michaelis-Menten type reactions. The reactor was more effective, and hence, more efficient with the smaller porous Damkohler number. The generalized results could find applications for the design of bioreactors with a porous wall.

The Plackett-Burman design in optimization of media components for biomass production of Lactobacillus rhamnosus OXY.

The central composite design was developed to search for an optimal medium for the growth of Lactobacillus rhamnosus OXY. The effect of various media components, such as carbon sources, simple and complex nitrogen sources, mineral agents, and growth factors (vitamins B, amino acids) was examined. The first-order model based on Plackett-Burman design showed that glucose, sodium pyruvate, meat extract and mineral salts significantly influenced the growth of the examined bacteria. The second-order polynomial regression confirmed that maximum biomass production could be achieved by the combination of glucose (12.38 g/l), sodium pyruvate (3.15 g/l), meat extract (4.08 g/l), potassium phosphate (1.46 g/l), sodium acetate (3.65 g/l) and ammonium citrate (1.46 g/l). The validation of the predicted model carried out in bioreactor conditions confirmed the usefulness of the new medium for the culture of L. rhamnosus OXY in large scale. The optimal medium makes the culture of the probiotic bacterium L. rhamnosus OXY more cost effective.