Hydrocyclones as cell retention device for CHO perfusion processes in single-use bioreactors.

In this study, a hydrocyclone (HC) especially designed for mammalian cell separation was applied for the separation of Chinese hamster ovary cells. The effect of key features on the separation efficiency, such as type of pumphead in the peristaltic feed pump, use of an auxiliary pump to control the perfusate flow rate, and tubing size in the recirculation loop were evaluated in batch separation tests. Based on these preliminary batch tests, the HC was then integrated to 50-L disposable bioreactor bags. Three perfusion runs were performed, including one where perfusion was started from a low-viability late fed-batch culture, and viability was restored. The successive runs allowed optimization of the HC-bag configuration, and cultivations with 20-25 days duration at cell concentrations up to 50 × 106 cells/ml were performed. Separation efficiencies up to 96% were achieved at pressure drops up to 2.5 bar, with no issues of product retention. To our knowledge, this is the first report in literature of high cell densities obtained with a HC integrated to a disposable perfusion bioreactor.

Process intensification for production of Streptococcus pneumoniae whole-cell vaccine.

The available pneumococcal conjugate vaccines provide protection against only those serotypes that are included in the vaccine, which leads to a selective pressure and serotype replacement in the population. An alternative low-cost, safe and serotype-independent vaccine was developed based on a nonencapsulated pneumococcus strain. This study evaluates process intensification to improve biomass production and shows for the first time the use of perfusion-batch with cell recycling for bacterial vaccine production. Batch, fed-batch, and perfusion-batch were performed at 10 L scale using a complex animal component-free culture medium. Cells were harvested at the highest optical density, concentrated and washed using microfiltration or centrifugation to compare cell separation methods. Higher biomass was achieved using perfusion-batch, which removes lactate while retaining cells. The biomass produced in perfusion-batch would represent at least a fourfold greater number of doses per cultivation than in the previously described batch process. Each strategy yielded similar vaccines in terms of quality as evaluated by western blot and animal immunization assays, indicating that so far, perfusion-batch is the best strategy for the intensification of pneumococcal whole-cell vaccine production, as it can be integrated to the cell separation process keeping the same vaccine quality.

Environmental evaluation of flocculation efficiency in the separation of the microalgal biomass of Scenedesmus sp. cultivated in full-scale photobioreactors.

In this paper the environmental evaluation of the separation process of the microalgal biomass Scenedesmus sp. from full-scale photobioreactors was carried out at the Research and Development Nucleus for Sustainable Energy (NPDEAS), with different flocculants (iron sulfate - FeCl3, sodium hydroxide - NaOH, calcium hydroxide - Ca(OH)2 and aluminum sulphate Al2(SO4)3, by means of the life cycle assessment (LCA) methodology, using the SimaPro 7.3 software. Furthermore, the flocculation efficiency by means of optical density (OD) was also evaluated. The results indicated that FeCl3 and Al2(SO4)3 were highly effective for the recovery of microalgal biomass, greater than 95%. Though, when FeCl3 was used, there was an immediate change in color to the biomass after the orange colored salt was added, typical with the presence of iron, which may compromise the biomass use according to its purpose and Al2(SO4)3 is associated with the occurrence of Alzheimer's disease, restricting the application of biomass recovered through this process for nutritional purposes, for example. Therefore, it was observed that sodium hydroxide is an efficient flocculant, promoting recovery around 93.5% for the ideal concentration of 144 mg per liter. It had the best environmental profile among the compared flocculant agents, since it did not cause visible changes in the biomass or compromise its use and had less impact in relation to acidification, eutrophication, global warming and human toxicity, among others. Thus, the results indicate that it is important to consider both flocculation efficiency aspects and environmental impacts to identify the best flocculants on an industrial scale, to optimize the process, with lower amount of flocculant and obtain the maximum biomass recovery and decrease the impact on the extraction, production, treatment and reuse of these chemical compounds to the environment. However, more studies are needed in order to evaluate energy efficiency of the process coupled with other microalgal biomass recovery technologies. In addition, studies with natural flocculants, other polymers and changes in pH are also needed, as these are produced in a more sustainable way than synthetic organic polymers and have the potential to generate a biomass free of undesirable contaminants.

Production of Monoclonal Antibody That Recognizes Zika Virus and Other Flaviviruses in Serum-Free Conditions.

With the recent outbreaks of Zika and Dengue virus infections in various countries worldwide, production of vaccines or diagnostic kits is an urgent public health demand. Production of a monoclonal antibody (mAb) that specifically binds to a common antigen shared by the Flavivirus genus will be necessary for new diagnostic kits or characterization and viral identity tests during vaccine development. This study aimed to cultivate, in serum-free conditions, the 4G2 hybridoma that produces an mAb, which recognizes a shared epitope from the Flavivirus genus. We compared 4G2 hybridoma growth and biochemical profiles between cells cultivated in batch mode over 10 days in roller bottles containing Dulbecco's modified Eagle's medium high glucose containing 10% fetal bovine serum medium or hybridomas directly adapted to Ex-Cell serum-free medium. Cellular parameters such as specific growth rate (µ), maximum cell concentration, specific l-lactate, and glucose and IgG rates were evaluated. Thereafter, we also compared total mAb volumetric productivity, purification yield, and mAb staining of Vero cells infected with Zika and Dengue-2 virus. Direct adaptation to serum-free conditions did not change hybridoma growth rate and mAb production under the conditions tested. Instead, serum-free mAb purification showed a higher yield with no alterations on mAb structure or mAb staining of Zika and Dengue Vero-infected cells.

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells.

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017.

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L-1 · h-1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L-1 · h-1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc.

Simultaneous Production of Amyloglucosidase and Exo-Polygalacturonase by Aspergillus niger in a Rotating Drum Reactor.

Simultaneous production of amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) was carried out by Aspergillus niger in substrate of defatted rice bran in a rotating drum bioreactor (RDB) and studied by a 31 × 22 factorial experimental design. Variables under study were A. niger strains (A. niger NRRL 3122 and A. niger t0005/007-2), types of inoculum (spore suspension and fermented bran), and types of inducer (starch, pectin, and a mix of both). Solid-state fermentation process (SSF) was conducted at 30 °C under 60-vvm aeration for 96 h in a pilot scale. Production of AMG and exo-PG was significantly affected by the fungal strain and the type of inoculum, but inducers did not trigger any significant effect, an evidence of the fact that these enzymes are constitutive. The maximum activity of exo-PG was 84 U gdm-1 whereas the maximum yield of AMG was 886.25 U gdm-1.

Startup and oxygen concentration effects in a continuous granular mixed flow autotrophic nitrogen removal reactor.

The startup and performance of the completely autotrophic nitrogen removal over nitrite (CANON) process was tested in a continuously fed granular bubble column reactor (BCR) with two different aeration strategies: controlling the oxygen volumetric flow and oxygen concentration. During the startup with the control of oxygen volumetric flow, the air volume was adjusted to 60mL/h and the CANON reactor had volumetric N loadings ranging from 7.35 to 100.90mgN/Ld with 36-71% total nitrogen removal and high instability. In the second stage, the reactor was operated at oxygen concentrations of 0.6, 0.4 and 0.2mg/L. The best condition was 0.2 mgO2/L with a total nitrogen removal of 75.36% with a CANON reactor activity of 0.1149gN/gVVSd and high stability. The feasibility and effectiveness of CANON processes with oxygen control was demonstrated, showing an alternative design tool for efficiently removing nitrogen species.

Nitrogen removal in a SBR operated with and without pre-denitrification: effect of the carbon:nitrogen ratio and the cycle time.

The effects of cycle time (CT) (8, 12 and 16h) and C/N ratio (3, 6 and 9) on nitrogen removal efficiencies in a bench top sequencing batch reactor treating slaughterhouse wastewater were investigated under different operating conditions: in condition 1, the reaction comprises an aerobic/anoxic phase and in condition II, the reaction comprises anoxic I/aerobic/anoxic II phases (with pre-denitrification). The greatest percentages of nitrogen removal were obtained in the CT range from 12 to 16 h and C/N ratios from 3 to 6, with mean efficiency values of 80.76% and 85.57% in condition I and 90.99% and 91.09% in condition II. Although condition II gave a higher removal of total inorganic nitrogen (NH4+ - N + NO2- - N + NO3- - N) than condition I, only condition I showed statistically significant and predictive regression for all the steps of nitrogen removal.

Treatment of wastewater using a sequencing batch reactor.

The aim of this study was to evaluate the efficiency of two sequencing batch reactors (R1 and R2) at removing nutrient (N and P) and chemical oxygen demand (COD). The two reactors (R1 and R2) were of the same design, operating under identical cycles and had a sludge retention time of 5 d. In R1, the substrate was sewage enriched with cooked and triturated cereals. In R2, the substrate was raw sewage mixed with triturated discarded excess sludge. Respirometry tests were performed to compare the biodegradability of the substrates used during the experimental period. The efficiency of R1 in removing soluble P and N-ammonia was considerably higher (90.4 and 97.2%, respectively) than reactor R2 (60 and 39.2%, respectively). While the effluent generated by R1 contained only minor amounts of N-nitrite and N-nitrate (0.5 +/- 0.4 and 1.7 +/- 0.8 mg L(-1), respectively). The concentrations of nitrite and nitrate in the effluent from R2 were 2 and 7 times higher. The lack of biodegradable COD available for denitrification was responsible for the high concentrations of nitrite and nitrate in the effluent of R2.

HPLC analysis of midodrine and desglymidodrine in culture medium: evaluation of static and shaken conditions on the biotransformation by fungi.

A high-performance liquid chromatography (HPLC) method is presented for the simultaneous determination of midodrine and desglymidodrine (DMAE) in Czapek-Dox culture medium, to be used in biotransformation studies by fungi. The HPLC analysis was conducted using a Lichrospher 100 RP18 column, acetonitrile-40 mmol/L formic acid solution (60:40, v/v) as mobile phase, and ultraviolet detection at 290 nm. The sample preparation was conducted by liquid-liquid extraction using ethyl acetate as extractor solvent. The method was linear over the concentration range of 0.4-40.0 µg/mL for midodrine (r ≥ 0.9997) and DMAE (r ≥ 0.9998). Within-day and between-day precision and accuracy were evaluated by relative standard deviations (≤ 8.2%) and relative errors (-7.3 to 7.4%), respectively. The validated method was used to assess midodrine biotransformation by the fungi Papulaspora immersa Hotson SS13, Botrytis cinerea UCA 992 and Botrytis cinerea 2100 under static and shaken conditions. Under shaken conditions, the biotransformation of midodrine to DMAE was more efficient for all studied fungi, especially for the fungus Botrytis cinerea 2100, which converted 42.2% of midodrine to DMAE.

Catalytic and thermodynamic properties of a tannase produced by Aspergillus niger GH1 grown on polyurethane foam.

Tannase is an inducible enzyme with important applications in the food and pharmaceutical industries. This enzyme was produced by the fungus Aspergillus niger GH1 under solid-state fermentation using polyurethane foam as solid support and tannic acid as sole carbon source and tannase inducer. Physicochemical properties of A. niger tannase were characterized, and the kinetic and thermodynamics parameters on methyl gallate hydrolysis were evaluated. The enzyme was stable in a pH range of 2-8 and a functional temperature range of 25-65 °C. The highest k(cat) value was 2,611.10 s(-1) at 65 °C. Tannase had more affinity for methyl gallate at 45 °C with a K(M) value of 1.82 mM and an efficiency of hydrolysis (k(cat)/K(M)) of 330.01 s(-1) mM(-1). The lowest E(a) value was found to be 21.38 kJ/mol at 4.4 mM of methyl gallate. The lowest free energy of Gibbs (ΔG) and enthalpy (ΔH) were found to be 64.86 and 18.56 kJ/mol, respectively. Entropy (ΔS) was -0.22 kJ/mol K. Results suggest that the A. niger GH1 tannase is an attractive enzyme for industrial applications due its catalytic and thermodynamical properties.