Fixed-bed Bioreactor Research Articles

Conversion Mechanisms of Carbon, Nitrogen, and Phosphorus in Ozone-Fixed-Bed and Membrane Bioreactors for Deep Treatment of Municipal Tail Water

An ozone-oyster shell fixed-bed bioreactor (OFBR) and a membrane bioreactor (MBR) were constructed and operated for about half a year for the deep treatment of municipal tail water. Pilot-scale test results showed that the combined OFBR-MBR had high removal efficiencies for carbon, ammonium, and phosphorus, and mean removal efficiencies for chemical oxygen demand, ammonium, total nitrogen, and total phosphorus (TP) were 75%, 99%, 20%, and 40%, respectively. Refractory organics in the municipal tail water were transformed to biodegradable organics in the OFBR, and the MBR effectively intercepted the surplus micro-molecular organics and microbes. Ammonium was mostly converted to nitrate, ∼10% of which was released as nitrogen gas through nitrification and denitrification by commonly known aerobic ammonia-oxidizing bacteria, nitrite-oxidizing bacteria, and denitrifying bacteria in the OFBR-MBR. The sludge was enriched in TP, which could be removed via surplus sludge discharge when sludge loading increased to overload amounts. Total removal depended on the uptake of phosphorus-accumulating organisms in the aerobic phase. Conversion mechanisms of carbon, nitrogen, and phosphorus in the OFBR-MBR system might be further adjusted to optimize process operation parameters, which might result in greater application of this system.

Hexavalent chromium bioreduction and chemical precipitation of sulphate as a treatment of site-specific fly ash leachates

Most of the power generation globally is by coal-fired power plants resulting in large stockpiles of fly ash. The trace elements associated with the ash particles are subjected to the leaching effects of precipitation which may lead to the subsequent contamination of surface and groundwater systems. In this study, we successfully demonstrate an efficient and sustainable dual treatment remediation strategy for the removal of high levels of Cr6+ and SO42- introduced by fly ash leachate generated by a power station situation in Mpumalanga, South Africa. The treatment consisted of a primary fixed-bed bioreactor kept at a reduction potential for Cr6+ reduction. Metagenome sequencing clearly indicated a diverse bacterial community containing various bacteria, predominantly of the phylum Proteobacteria which includes numerous species known for their ability to detoxify metals such as Cr6+. This was followed by a secondary BaCO3/dispersed alkaline substrate column for SO42- removal. The combination of these two systems resulted in the removal of 99% Cr6+ and 90% SO42-. This is the first effective demonstration of an integrated system combining a biological and chemical strategy for the remediation of multi-contaminants present in fly ash leachate in South Africa.

Removal of ammonium and organic carbon from leachate by the anammox process in a fixed bed bioreactor

One of the inevitable drawbacks of sanitary landfilling of municipal solid waste (MSW) is the production of leachate. This study aimed to assess the treatment of leachate for the purpose of removing ammonium and organic carbon using the anammox process. To grow autotrophic bacteria in the reactor, the carbon source was gradually decreased from 500 mg L–1 to less than 10 mg L–1. NH4 and nitrite concentrations from 5 and 6.6 to 300 and 396 mg L–1, respectively, were injected into the reactor and the removal was investigated. Finally, in order to assess carbon and nitrogen removal simultaneously, the concentrations of chemical oxygen demand, ammonia, and nitrite were increased to 250, 500, and 660 mg L–1, respectively. The highest efficiencies for ammonium and nitrite removal that were obtained were 76.69 and 91.12, respectively, at the volumetric loading rate of 0.15 and 0.132 kg m–3 d–1 in the hydraulic retention time (HRT) of 24 h. As HRT decreased from 24 to 6 h and the loading rate increased, the highest efficiencies achieved for the removal of ammonia and NO2 were 95.19 and 80.56, respectively. Eventually, ammonium removal efficiency did not exceed 22.28 while the removal efficiency for organic matters was 72.63. Contrary to nitrite, ammonium removal is dependent on the appropriate performance of the anammox process. © 2017 Desalination Publications. All rights reserved.

Strategy of mitigating ammonium-rich waste inhibition on anaerobic digestion by using illuminated bio-zeolite fixed-bed process

Intermittent illumination combined with bio-zeolite fixed-bed process was utilized to improve the efficiency of anaerobic digestion with ammonium-rich substrate. The batch experiments were carried out at NH4+-N concentration of 2211mg/L under intermittent illumination and dark (as control) conditions, respectively. The illuminated bioreactor achieved higher methane production (287mL/g-DOC) and ATP value (0.38μmol/L) than that under dark condition. Then the bio-zeolite fixed-bed bioreactor (NH4+-N concentration: 3000mg/L) was used to study the additional efficiency on the illuminated ammonium-rich anaerobic digestion process. The result showed that the illuminated fixed-bed bioreactor presented the greatest methane concentration (70%), methane yield (283mL/g-DOC) and quantity of methanogens comparing with no-bed bioreactor. Furthermore, the illuminated fixed-bed bioreactor achieved better performance during 118-day semi-continuous fermentation. The combination of the intermittent illumination and bio-zeolite fixed-bed strategy contributed to the higher efficiency and stability of the ammonium-rich anaerobic digestion process.

BIODEGRADACIÓN DE VINAZAS DE CAÑA DE AZÚCAR MEDIANTE EL HONGO DE PUDRICIÓN BLANCA Pleurotus ostreatus EN UN REACTOR DE LECHO EMPACADO

<p>Sugarcane vinasses are considered a complex effluent because of its organic load, low pH, high temperature, and by the presence of recalcitrant substances such as melanoidins and phenolic compounds. The aim of this work was to evaluate the potential of the fungus <em>Pleurotus ostreatus</em> to carry out the biodegradation of sugarcane vinasses in a fixed-bed bioreactor. The experiments evidence the potential of the fungus <em>Pleurotus ostreatus</em> to carry out the decolorization (83%), the removal of the Chemical Oxygen Demand (COD=87%) and the Biochemical Oxygen Demand (BOD<sub>5</sub>=92%), the reduction of total suspended solids (83%) and volatile suspended solids (72%) of vinasses. The technical simplicity of the proposed alternative as well as process performance reveals the potential of the fungus <em>Pleurotus ostreatus</em> for the treatment of sugarcane mill effluents.</p>

Intrinsic kinetics for fixed bed bioreactor in hospital wastewater treatment.

Variation in hospital wastewater (HWW) pollutants and differences with municipal wastewater (MWW), make the use of biokinetic coefficients obtained from activated sludge in the MWW treatment unprofitable for designing, modeling and evaluation of biological processes for HWW treatment. Since this study was conducted to evaluate the performance and biokinetic coefficients of a fixed bed bioreactor (FBBR) using rice husks as fixed media in HWW treatment, a new modified method was also proposed for biokinetic estimation in FBBR processes. For these purposes, five hydraulic retention times along with five sludge retention times were introduced to a pilot setup and the required data were attained. The performance process for chemical oxygen demand (COD) removal was significant (87.8-97.5%) in different conditions. The values of biokinetic coefficients k, Ks, Y and Kd were obtained as 2.42 (day-1), 55.5 (mgCOD/L), 0.2929 (mgBiomass/mgCOD) and 0.0164 (day-1), respectively. The rice husks with high surface area and high affinity for biomass accumulation on its surface are promising media for a green and environmentally friendly FBBR process. The kinetics parameters values are utilizable for modeling of FBBR using rice husks as fixed media in HWW treatment.

High Laccase Expression by Trametes versicolor in a Simulated Textile Effluent with Different Carbon Sources and PHs.

Textile effluents are highly polluting and have variable and complex compositions. They can be extremely complex, with high salt concentrations and alkaline pHs. A fixed-bed bioreactor was used in the present study to simulate a textile effluent treatment, where the white-rot fungus, Trametes versicolor, efficiently decolourised the azo dye Reactive Black 5 over 28 days. This occurred under high alkaline conditions, which is unusual, but advantageous, for successful decolourisation processes. Active dye decolourisation was maintained by operation in continuous culture. Colour was eliminated during the course of operation and maximum laccase (Lcc) activity (80.2 U∙L−1) was detected after glycerol addition to the bioreactor. Lcc2 gene expression was evaluated with different carbon sources and pH values based on reverse transcriptase-PCR (polymerase chain reaction). Glycerol was shown to promote the highest lcc2 expression at pH 5.5, followed by sucrose and then glucose. The highest levels of expression occurred between three and four days, which corroborate the maximum Lcc activity observed for sucrose and glycerol on the bioreactor. These results give new insights into the use of T. versicolor in textile dye wastewater treatment with high pHs.

Development and Optimization of AAV hFIX Particles by Transient Transfection in an iCELLis(®) Fixed-Bed Bioreactor.

Adeno-associated virus (AAV) vectors are increasingly popular in gene therapy because they are unassociated with human disease, replication dependent, and less immunogenic than other viral vectors and can infect a variety of cell types. These vectors have been used in over 130 clinical trials, and one AAV product has been approved for treatment of lipoprotein lipase deficiency in Europe. To meet the demand for the increasing quantities of AAV required for clinical trials and treatment, a scalable high-capacity technology is required. Bioreactors meet these requirements but limited options are available for adherent HEK 293T/17 cells. Here we optimize the transient transfection of HEK293T/17 cells for the production of AAV human factor IX in a disposable fixed-bed bioreactor, the iCELLis(®) Nano (PALL Corporation). A fixed bed in the center of the iCELLis bioreactor is surrounded by culture medium that is pumped through the bed from the bottom of the bioreactor so that a thin film of the medium overflows the bed and is replenished with oxygen and depleted of CO2 as it returns to the surrounding medium reservoir. We show that this fixed-bed bioreactor can support as many as 2.5 × 10(8) cells/ml of fixed bed (1.9 × 10(6) cells/cm(2)). By optimizing culture and transfection parameters such as the concentration of DNA for transfection, day of harvest, size of PEI/DNA particles, and transfection medium, and adding an additional medium change to the process, we increased our yield to as high as 9.0 × 10(14) viral particles per square meter of fixed bed. We also show an average GFP transfection of 97% of cells throughout the fixed bed. These yields make the iCELLis a promising scalable technology for the clinical production of AAV gene therapy products.

706. Consistent Viral Vector Manufacturing for Phase III Using iCELLis 500 Fixed-Bed Technology

Single use disposable technology faces further challenges in producing viral vectors in sufficient large quantities especially where adherent cells are needed. Scaling up the adherent system has proven to be troublesome. The PALL iCELLis® disposable fixed-bed bioreactors offer a novel option for viral vector manufacturing in large quantities in an adherent environment. We have made process development in iCELLis Nano, where the cultivation area varies between 0.53 - 4 m2, after which we went forward to iCELLis 500, where the cultivation volume can be upgraded to 500 m2 (66 - 500 m2). iCELLis 500 has proven to be ideal to satisfy upstream demand and large-scale downstream purification process was developed to supply high quality recombinant adenovirus based gene products in our fully-licensed GMP manufacturing facility for pre-clinical and clinical trials The process is initiated by HEK293 cell mass expansion in suspension mode using Biostat® CultiBag RM (Sartorius Stedim Biotech S.A.) bioreactor. The expanded suspension cell mass is inoculated into iCELLis 500 for further expansion in adherent mode. This is infected by Working Viral Seed Stock and subsequent virus is released from the infected cells by chemical lysis. Downstream process contains Benzonase digestion, clarification, concentration and conditioning by crossflow ultrafiltration, capture and polishing by anion exchange chromatography, and final concentration and formulation is achieved using crossflow filtration. All product contact parts are fully disposable. Several batches have been produced with consistent results. Further validation of the process for commercial manufacturing is currently ongoing. We established a scalable, large-scale manufacturing process to supply high quality recombinant adenovirus based gene products in our fully-licensed GMP manufacturing facility for clinical trials. Alongside this we have validated assays which are providing a relevant quantitative measure of biologic function of the vector and demonstrating the quality and comparability of drug product batches in Phase 3 and commercial use.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

Influence of salinity on fungal communities in a submerged fixed bed bioreactor for wastewater treatment

Salinity is known to influence the performance of biological wastewater treatment plants. While its impact on bacterial communities has been thoroughly studied, its influence on fungal communities has been largely overlooked. To address this knowledge gap, we assessed the effect of saline influents (0, 3.7, 24.1 and 44.1gNaCl/L) on the community structure and diversity of fungi in a submerged fixed bed bioreactor (SFBBR). For this purpose, denaturing gradient gel electrophoresis (DGGE) and 454-pyrosequencing of PCR-amplified fungal 18S rRNA gene fragments and ITS regions, respectively, were used. Significant differences in the fungal community structure were found in relation to the NaCl concentration. Fungal diversity increased as salinity increased to a concentration up to 24.1gNaCl/L, but was significantly reduced at 44.1gNaCl/L. Basidiomycota dominated the fungal community in the absence of NaCl but decreased in relative abundance with increasing salinity, being replaced progressively by Ascomycota.

Insect cell entrapment, growth and recovering using a single-use fixed-bed bioreactor. Scaling up and recombinant protein production

Insect cells are largely used for industrial production of vaccines, viral vectors and recombinant proteins as well as in research and development as an important tool for biology and bioprocess studies. They grow in suspension and are semi-adherent cells. Among the cell culture systems enabling scalable bioprocess the single-use fixed-bed iCELLis® bioreactors offer great advantages. We have established the conditions for Drosophila melanogaster Schneider 2 (S2) and Spodoptera frugiperda (Sf9) cells entrapment into the fixed-bed, cell growth and recover from the fixed-bed once high cell densities were attained. Our established protocol allowed these cells, at a cell seeding of 2×1E5 cells/microfiber carriers (MC) (3.5×1E6cells/mL; 1.7×1E4cells/cm2), to grow inside a 4m2/200mL fixed-bed attaining a concentration of 5.3×1E6 cells/MC (9.5×1E7cells/mL; 4.7×1E5 cells/cm2) for S2 cells or 4.6×1E6 cells/MC (8×1E7cells/mL; 4.1×1E5cells/cm2) for Sf9 cells. By washing the fixed-bed, entrapped cells could then be recovered from the fixed-bed at a high rate (>85%) with high viability (>95%) by increasing the agitation to 1200/1500rpm. Although the cell yields in the fixed-bed bioreactor were comparable to those obtained in a stirred tank (respectively, 1.3×1E10 and 2.5×1E10 total cells), S2 cells stably transfected with a cDNA coding for the rabies virus glycoprotein (RVGP) showed a 30% higher preserved rRVGP production (2.5±0.1 and 1.9±0.1μg/1E7 cells), as evidenced by a conformational ELISA evaluation. These findings demonstrate not only the possibility to entrap, cultivate to high densities and recover insect cells using a single-use fixed-bed bioreactor, but also that this system provides suitable physiological conditions for the entrapped cells to produce a cell membrane associated recombinant protein with higher specific biological activity as compared to classical suspension cell cultures.

Performance Characteristics of Anaerobic Biodegradation of Synthetic Phenolic Industrial Wastewater

The treatment of toxic and inhibitory phenolic compounds using biological techniques have been pursued vas a promising and widely accepted treatment process due to its easiness of handling, a greater level of stabilization of waste and properly operated to prevent the production of secondary pollutants. Up-flow Anaerobic Bio-Reactors (UAFB) has been widely applied for the handling of high organic load industrial wastewater. The treatment of synthetic phenolic wastewater by a single stage anaerobic fixed bed bioreactor with granite stones packing at four different temperatures was studied. The effect of hydraulic retention time on COD reduction and other steady state characteristics and kinetic parameters, which form these characteristics, was also studied. A recirculated single stage up-flow anaerobic bioreactor operated at all the above-given temperatures with initial BOD 1462 mg/l and initial COD 5720 mg/l for a digestion period of 25 days with a working volume of 1000 ml. The performance of the reactor was monitored after every five days and analyzed in terms of percentage (COD, BOD, TS, TDS, VS removal and biogas production). The removal efficiency of BOD, COD, TS, TDS and VS reached a maximum value of 63.20%, 61.24%, 44.88%, 47.67% and 53.12% respectively. With the change in HRT, the maximum COD reduction was 66.04% at 24 hrs HRT at 400C with initial COD of 5000 mg/l. Specific biogas yield increased up to 0.0162 ml/mg CODr.

Biocatalytic methanation of hydrogen and carbon dioxide in a fixed bed bioreactor

Biocatalytic methanation of H2 and CO2 was studied in a fixed bed reactor system consisting of two solid state bioreactors in series connected to a recirculation system. Bioreactors were packed with a mixture of vermiculite shales and granular perlite material as a support material. A maximal methane productivity of 6.35l/lreactord was achieved at a hydrogen feed rate of 25.2l/lreactord, while hydrogen conversion rate was 100%. However, stable operation of the reactor at this efficiency remains to be achieved. Very simple reactor design, constructed from low cost materials, and the idea of exploiting waste material as a robust source of nutrients for methanogens makes this study very interesting regarding the overall usability and suitability of the system as part of a decentralized energy system.

Fixed-bed degradation of histamine in fish sauce by immobilized whole cells of Natrinema gari BCC 24369

Fish sauce often contains histamine at high levels, which may cause its rejection for export and is also associated with the food poisoning. Therefore, this study aimed to apply the ability of immobilized whole cells of Natrinema gari BCC 24369 to degrade histamine in fish sauce. Under static condition, the optimum conditions for histamine degradation in fish sauce were using 10 % immobilized cells of Nnm. gari BCC 24369 for fish sauce (pH 6.5) in the presence of 10 mg/100 ml of riboflavin for 2 h, in which the immobilized cells exhibited a maximum histamine removal efficiency of 24 ± 1 %. The operation of immobilized cells in a fixed-bed bioreactor was more efficient than static batch operation for histamine removal. At the optimal feeding flow rate of 0.5 ml/min, a significant amount of histamine in fish sauce (51 ± 3 %) was removed as the result of histamine-degrading activity of the cells. After treatment with immobilized cells, not only was histamine effectively removed, but color darkening was also reduced. Additionally, no detrimental effects on the unique quality of fish sauce were observed during 6 months of storage.

Process Development of Adenoviral Vector Production in Fixed Bed Bioreactor: From Bench to Commercial Scale.

Large-scale vector manufacturing for phase III and beyond has proven to be challenging. Upscaling the process with suspension cells is increasingly feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome. The iCELLis(®) disposable fixed-bed bioreactors offer a possible option for viral vector manufacturing in large quantities in an adherent environment. In this study, we have optimized adenovirus serotype 5 manufacturing using iCELLis Nano with a cultivation area up to 4 m(2). HEK293 cell cultivation, infection, and harvest of the virus (by lysing the cells inside the bioreactor) proved possible, reaching total yield of up to 1.6×10(14) viral particles (vp)/batch. The iCELLis 500 is designed to satisfy demand for large-scale requirements. Inoculating a large quantity of cell mass into the iCELLis 500 was achieved by first expanding the cell mass in suspension. Upscaling the process into an iCELLis 500/100 m(2) cultivation area cassette was practical and produced up to 6.1×10(15) vp. Flask productivity per cm(2) in iCELLis Nano and iCELLis 500 was in the same range. As a conclusion, we showed for the first time that iCELLis 500 equipment has provided an effective way to manufacture large batches of adenoviral vectors.

Development of a bio-zeolite fixed-bed bioreactor for mitigating ammonia inhibition of anaerobic digestion with extremely high ammonium concentration livestock waste

A novel bio-zeolite fixed-bed bioreactor combining with physicochemical and biological methods for mitigating ammonia inhibition was developed to improve the anaerobic digestion of ammonium-rich livestock wastes. The fixed-bed bioreactor was constructed by zeolite that was fixed using three different kinds of polymer materials including chlorinated polyethylene (CPE), polymer foaming sponge (PFS) and porous nylon (PN). Using ammonium-rich swine wastes (NH4+-N: 7511mg/l) as substrate, a series of batch anaerobic fermentation experiments were carried out under 35°C. The bioreactor suspending with CPE fixed zeolite showed the shortest start-up period (2days) and the highest methane production (about 96 times higher than that of the Control) after 30-days anaerobic digestion. Furthermore, the higher and stable methane concentration and yield during 100-day of semi-continuous anaerobic digestion clearly indicated the long-term practical effectiveness of CPE-fixed zeolite bioreactor. Synergy of ammonia adsorption and microorganism immobilization by CPE fixed zeolite contributed to the enhanced anaerobic digestion efficiency. The developed novel CPE fixed zeolite bioreactor was suggested to be a favorable system for improve the anaerobic digestion of ammonium-rich agricultural wastes.

Enzymatic hydrolysis of non-treated sugarcane bagasse using pressurized liquefied petroleum gas with and without ultrasound assistance

In this work the enzymatic hydrolysis of sugarcane bagasse using pressurized liquefied petroleum gas (LPG) and ultrasound assisted pressurized LPG (US assisted-LPG) to obtain fermentable sugar in a unique step was investigated. For this purpose, non-treated sugarcane bagasse was used for enzymatic hydrolysis in a fixed-bed bioreactor. Several experiments were carried out to investigate the influence of moisture content, enzyme load and pressure on the yield of fermentable sugar after the hydrolysis. Maximum yield of fermentable sugar for hydrolysis carried out using pressurized LPG and US assisted-LPG was 245 and 217 g kg−1, respectively. The main contribution of this work was to demonstrate that the use of alternative technologies (US assisted-LPG) has the potential to increase the efficiency of enzymatic hydrolysis of lignocellulosic materials without the needs of hard pre-treatment of the material and high enzyme loading.

672. iCELLisTM Fixed-Bed Technology for Adherent Cells Is an Efficient Scalable System for Viral Vector Production Applications

To date many early phase gene therapy trials have been successful. However, phase III and commercial phase have brought further challenges in producing viral vectors in sufficient large quantities. Upscaling the process on suspension cells is feasible, but many viral production applications are still applicable only in adherent settings. Scaling up the adherent system has proven to be troublesome and costly. The PALL iCELLisTM disposable fixed-bed bioreactors offer an efficient option for viral vector manufacturing in large quantities in an adherent environment. In iCELLisTM Nano, the cultivation area 0.53-4 m2 for smaller batches, is ideal for process development purposes. In iCELLIs500 the cultivation area varies between 66 and 500 m2 and is ideal to satisfy demand for phase3/commercial requirements.We have optimized adenovirus type 5 manufacturing using iCELLisTM Nano. HEK293 cell cultivation, infection and harvest of the virus by lysing the cells inside the bioreactor were efficient, reaching total yield of 3.4 × 10^14 vp/batch. When upscaling the process into 100 m2 cultivation area with iCELLis500, 1.2 × 10^16 vp/batch were produced.iCELLisTM technology is applicable also to other vector types which require for example plasmid transfection. Virus can also be harvested by perfusion from the medium. Lentiviral vector production in 293T cells was tested in iCELLisTM Nano and we achieved high plasmid transfection efficiency, leading to the comparable titers and productivity as in flasks. To conclude, iCELLisTM equipment has provided us an efficient way to manufacture large batches of different kinds of gene therapy products suitable for large preclinical animal models and up to phase III trial and beyond.

Pulp and paper mill effluent treatment by hybrid anaerobic upflow fixed-bed bioreactor combined with slow sand filter

In the present work, pollutant removal study of Century Pulp and Paper Mill effluent was done. A hybrid unit of upflow fixed-bed anaerobic bioreactor (UFBAB) along with slow sand filter (SSF) was compared with single unit UFBAB for pollutant removal at different hydraulic retention time (HRT). The hybrid system showed better removal efficiency which can be attributed to SSF. It was observed that SSF provides a polishing effect to the effluent treated by UFBAB. The biodegradation rate in both filters was observed to be increased in first six weeks of experiment, prior to the acclimatization of microbial communities. The relative removal efficiency of biological oxygen demand and chemical oxygen demand was found to between 0.90 and 0.99, respectively, at 24 h HRT, demonstrating very high removal efficiency of the hybrid system. The relative removal efficiency of total dissolved solids and total suspended solids was around 1 at 16 h HRT indicating 100% efficient and within the limits as prescribed by Central Pollution Control Board India (CPCB) for treated effluent discharge in surface water bodies. Best removal efficiency was observed in case of sulfate at low HRT of 8 h. Relative removal efficiency for phenols was found to be greater than 1 at 12 h HRT. SSF was found to be a suitable substrate for polishing of treated effluent of pulp and paper mill wastewater, demonstrating promising relative efficiency.