Scale-up of a pan-coating process

The purpose of this work was to develop a practical scale-up model for a solvent-based pan-coating process. Practical scale-up rules to determine the key parameters (pan load, pan speed, spray rate, air flow) required to control the process are proposed. The proposed scale-up rules are based on a macroscopic evaluation of the coating process. Implementation of these rules does not require complex experimentation or prediction of model parameters. The proposed scale-up rules were tested by conducting coating scale-up and scale-down experiments on 24-inch and 52-inch Vector Hi-coaters. The data demonstrate that using these rules led to similar cumulative drug release profiles (f2≫50; and P Analysis of Variance [P _ANOVA]≫0.05 for cumulative percentage of drug released after 12 hours [Cum 12] from tablets made at 24- and 52-inch scales. Membrane characteristics such as opacity and roughness were also similar across the 2 scales. The effects of the key process variables on coat weight uniformity and membrane characteristics were also studied. Pan speed was found to be the most significant factor related to coating uniformity. Spray droplet size was found to affect the membrane roughness significantly, whereas opacity was affected by the drying capacity.     

A process for extracellular thermostable lipase production by a novel Bacillus thermoamylovorans strain

A lipolytic enzyme-producing thermophilic microorganism, recently isolated from a hot spring in Galicia (North Western Spain), has been investigated. First, the strain was genetically identified and tentatively named Bacillus thermoamylovorans CH6B. It produced significant levels (around 450 U/L) of extracellular lipolytic activity in shake flask cultures, and the most suitable conditions for this biological process were found at temperatures between 50 and 55 °C, and an initial pH value around 7.0. Next, a preliminary scaling up of the process was carried out in a 5-L stirred tank bioreactor, and it was concluded that operation at agitation and aeration rates of 300 rpm and 0.33 vvm, respectively, were advisable. In both type of cultures, the results were successfully fitted to logistic equations, and the relationship between lipase production and cell growth was investigated. Furthermore, some relevant properties of the crude lipolytic enzyme extracts were assessed. The crude biocatalyst preferentially hydrolysed p-nitrophenyl esters of medium and long-chain fatty acids. Thermal stability in aqueous solution of the produced enzyme was also promising, and the deactivation profiles were fitted to a series-type deactivation model.     

Solid‐state fermentation for the production of debittering enzyme naringinase using Aspergillus niger MTCC 1344

Aspergillus niger produced high levels of naringinase using easily available, inexpensive industrial waste residues such as rice bran, wheat bran, sugar cane bagasse, citrus peel, and press mud in solid‐state fermentation (SSF). Among these, rice bran was found to be the best substrate. Naringinase production was highest after 96 h of incubation at 27°C and at a substrate‐to‐moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused maximum induction. An inoculum age of 72 h and an inoculum level of 15% resulted in maximum production of naringinase. Enzyme production was stimulated by the addition of nutrients such as naringin and peptone. Thus, A. niger produced a very high level of naringinase within a short time in solid‐state fermentation using inexpensive agro‐residues, a level that is much higher than reported for any other microbes.     

Shake-flask and bench-scale stirred tank bioreactor production optimization of a thermoalkaline protease from Bacillus cereus SIU1 using one-factor-at-a-time and response surface (statistical) methodologies

Abstract

We report the optimization of production of a halotolerant, thermoalkaline protease by Bacillus cereus SIU1, at shake-flask and bench-scale bioreactor level, using conventional and response surface methods. The basal medium supplemented with optimized (w/v) 0.8% glucose, 1.5% peptone, and 0.4% yeast extract produced 224 Uml^{? 1} alkaline protease after 20 h incubation. Enzyme yield was further increased to 491 Uml^{? 1} when the fermentation broth was supplemented with 0.02% (w/v) Ca²⁺. Optimization of physical factors resulted in still higher protease level of 651 Uml^{? 1} within 18 h fermentation at initial pH 9.0, 50°C, and 150 rpm agitation. Statistically designed experiments revealed significant effects of peptone and CaCl₂ on protease production. A maximum of 749 protease Uml^{? 1} was produced at optimum factor levels (w/v) of peptone 1.75%, yeast extract 0.4%, CaCl₂ 0.025%, and pH 9.0 after 18 h incubation. Optimization of agitation and aeration rates in bench-scale bioreactors further enhanced the enzyme yield to 941 protease Uml^{? 1} at 125 rpm and 2.0 vvm aeration. Optimization of protease production by conventional and statistical approaches resulted in a ～10.7-fold increase (941 Uml^{? 1}) compared to un-optimized conditions (88 Uml^{? 1}).     

Isolation and evaluation of a Bacillus methylotrophicus strain for control of corn stalk rot

Bacteria were isolated from corn plants and efficacy of a selected isolate for control of corn stalk rot caused by Fusarium graminearum was evaluated. The bacterial isolate provided the greatest suppression of F. graminearum in lab studies and reduced corn stalk rot significantly in 2013 and 2014 field experiments.     

Use of the glucose oxidase system for estimation of oxygen transfer rate in a solid-state bioreactor

The glucose oxidase system was adapted for estimation of the overall oxygen transfer rate in a periodic pressure oscillating, solid-state bioreactor. Enzyme concentration of 40 ml enzyme preparation L⁻¹ was found adequate to give linear gluconic acid production and attain maximal oxygen absorption rates. At 4 atm and 30°C, the oxygen transfer rate reached 892 mmol kg⁻¹ initial dry matter h⁻¹ in this system, while only 121 mmol kg⁻¹ initial dry matter h⁻¹ was obtained in a conventional static tray bioreactor.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Reactor comparison and scale-up for the microaerobic production of 2,3-butanediol by Enterobacter aerogenes at constant oxygen transfer rate

Stirred tank (STR), bubble column (BCR) and airlift (ALR) bioreactors of 0.05 and 1.5 m³ total volume were compared for the production of 2,3-butanediol using Enterobacter aerogenes under microaerobic conditions. Batch fermentations were carried out at constant oxygen transfer rate (OTR=35 mmol/lh). At 0.05 m³ scale, the STR reactor achieved much higher biomass and product concentrations than the BCR and ALR reactors. At 1.5 m³ scale, however, exactly the same biomass and product concentrations could be obtained in both STR and ALR reactors. The 1.5 m³ ALR reactor performed also much better than its counterpart at small scale, achieving a productivity 2.4-fold as high as that of the 0.05 m³ BCL and ALR reactors. No differences in performances were observed between BCR and ALR. As compared to STR the tower reactors have a 12 time higher energetic efficiency (referred to product formation) and thus should be the choice for large scale production of 2,3-butanediol.The criterion of constant OTR or constant k _L a is not applicable for the scale-up of this oxygen-sensitive culture due to strong influence of reactor hydrodynamics under microaerobic conditions. The effects of mixing and circulation time on growth and metabolism of E. aerogenes were quantitatively studied in scaled-down experiments with continuous culture. For a successful scale-up of this microaerobic culture it is necessary to have an homogeneous oxygen supply over the entire reactor volume. Under conditions of inhomogeneous oxygen supply an optimum liquid circulation time exists which gives a maximum production of 2,3-butanediol.List of Symbols BD 2,3-butanediol - [mmol/l] saturation value of dissolved oxygen - D [h^–1] dilution rate - D [mm] reactor diameter - D _K [mm] top section diameter - D _R [mm] stirrer diameter - D _S [mm] draft tube diameter - EtOH ethanol - E _P [kg/kWh] energy efficiency refered to product formation - H [mm] height of reactor - HAc acetate - H _L [mm] height of liquid - k _L a [h^–1] volumetric oxygen transfer coefficient - N [rpm=min^–1] stirrer speed - OTR [mmol/lh] oxygen transfer rate - OUR [mmol/lh] oxygen uptake rate - p [Pa] pressure - P [kW] power input - P/V _L [kW/m³] specific power input - [mmHg] oxygen partial pressure (mmHg) or - [mmol/l] dissolved oxygen (mmol/l) - [mmol/gh] specific oxygen uptake rate - q _P [mmol/gh] specific productivity - R [Nm/kgK] gas constant, R = 287.06 - RQ respiration quotient - t _c [s] liquid circulation time - T [°C or K] temperature - TCA tricarboxylic acid - u _G [cm/s] mean superficial gas velocity - v _G [m/s] gas velocity at nozzels of gas distributor - V_G [l/h] aeration rate at inlet - V [m³ or l] total volume - V _L [m³ or l] liquid volume - V _N [l/mol] gas mole volume under normal conditions, V _N = 24.4116 - X [g/l] biomass concentration - CO₂ mole fraction in the effluent gas - O₂ mole fraction in the effluent gas - inlet (above the gas distributor) - ratio of oxygen consumed through TCA cycle to the total oxygen uptake rate - [g/l or kg/m³] density - [%] degree homogeneity - outlet of fermenter or top of the dispersion phase Dedicated to the 65th birthday of Professor Fritz Wagner.We thank Dr. C. Posten and T. Gabel for support with the computer control system UBICON. T.-G. Byun gratefully acknowledges financial support by DAAD.     

A novel three-compartmented electrochemical bioreactor for enrichment of strict anaerobes based on metabolite production

In this study, a novel three-compartmented electrochemical bioreactor (3-CEB) was designed in an effort to overcome the disadvantages of the two-compartmented electrochemical bioreactor (2-CEB) separated with a cation-selective membrane for enrichment of strict anaerobes. The 3-CEB was comprised of an anode, outlet, and a cathode compartment. The outlet compartment was positioned between the anode and cathode compartment, and it was separated with the anode side by a rubber plate and with the cathode side by a porous glass membrane. A platinum wire bridging the anode and outlet compartment operated as a redox passage, however, through which no material could permeate. Butyrate fermentation bacteria were enriched on the basis of the metabolite production. Butyrate generated by strict anaerobes was significantly more abundant in the 3-CEB than in the 2-CEB. Acetic acid and lactic acid generated by facultative anaerobes was relatively higher in the 2-CEB than in the 3-CEB. Meanwhile, butyrate was not generated in the bioreactor utilized for the control test, to which the electrochemical potential was not charged. In a continuous culture using the 3-CEB, the majority of the glucose was fermented to butyrate, and the acetate additionally supplied to the bacterial culture was metabolically reduced to butyrate. More lactate than butyrate was generated from glucose in the 2-CEB.     

Scale-up performance of a partitioning bioreactor for the degradation of polyaromatic hydrocarbons by Sphingomonas aromaticivorans

A partitioning bioreactor, consisting of an aqueous phase containing Sphingomonas aromaticivorans and an immiscible organic phase (dodecane), loaded with naphthalene and phenanthrene, was operated at two scales, 5 l and 150 l. Complete degradation of 15 g and 300 g, respectively, of these polyaromatic hydrocarbon (PAH) mixtures was achieved in 21 h in both cases resulting in a volumetric PAH degradation rate of 238 mg l^–1 h^–1 based on reactor aqueous volumes.     

Scale-up studies on a defined medium process for pilot plant production of illicicolin by Gliocladium roseum

Illicicolin was cultivated at the 600-L pilot scale for purposes of material generation and process development. The initial medium containing oat flour was difficult operationally as a result of excessive foaming during sterilization, so a new defined medium process (with either glucose or sucrose as the carbon source), developed at the 23-L scale, was scaled up and improved for pilot scale needs. Pilot scale media development efforts focused on exploring the highest concentration of media (1.0 x to 3.0 x) that could be cultivated at the pilot scale and not be limited by mixing or oxygen mass transfer. The process was scaled up successfully and peak titers improved 7.5-fold, from about 200 mg/L in the initial complex medium to 1500 mg/L in the final defined medium.     

High density culture of mammalian cells with dynamic perfusion based on on-line oxygen uptake rate measurements

In a continuous culture with cell retention the perfusion rate must be adjusted dynamically to meet the cellular demand. An automated mechanism of adjusting the perfusion rate based on real-time measurement of the metabolic load of the bioreactor is important in achieving a high cell concentration and maintaining high viability. We employed oxygen uptake rate (OUR) measurement as an on-line metabolic indicator of the physiological state of the cells in the bioreactor and adjusted the perfusion rate accordingly. Using an internal hollow fiber microfiltration system for total cell retention, a cell concentration of almost 10⁸ cells/mL was achieved. Although some aggregates were formed during the cultivation, the viability remained high as examined with confocal microscopy after fluorescent vital staining. The results demonstrate that on-line OUR measurement facilitates automated dynamic perfusion and allows a high cell concentration to be achieved.     

Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32^T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, k_La, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at k_La condition of 0.006 s^?1.     

Two-compartment method for determination of the oxygen transfer rate with electrochemical sensors based on sulfite oxidation

The dissolved oxygen concentration is a crucial parameter in aerobic bioprocesses due to the low solubility of oxygen in water. The present study describes a new method for determining the oxygen transfer rate (OTR) in shaken-culture systems based on the sodium sulfite method in combination with an electrochemical oxygen sensor. The method replaces the laborious titration of the remaining sulfite by an on-line detection of the end point of the reaction. This method is a two-step procedure that can be applied in arbitrary flasks that do not allow the insertion of electrodes. The method does not therefore depend on the type of vessel in which the OTR is detected. The concept is demonstrated by determination of the OTR for standard baffled 1-L shake flasks and for opaque Ultra Yield™ flasks. Under typical shaking conditions, k_La values in the standard baffled flasks reached values up to 220 h^-1, whereas the k_La values of the Ultra Yield flasks were significantly higher (up to 422 h^-1).     

Characterization and improvement of oxygen transfer in pilot plant external air-lift bioreactor for mycelial biomass production

The oxygen transfer dynamics in a pilot plant external air-lift bioreactor (EALB) during the cultivation of mycelial biomass were characterized with respect to hydrodynamic parameters of gas holdup (), oxygen transfer coefficient (K_La) and superficial gas velocity (U _g), and dissolved oxygen (DO). An increased flow rate of air supply was required to meet the increased oxygen demand with mycelial biomass growth. Consequently, an increase in air flow rate led to an increase in , K_La and the DO level. The enhancement of oxygen transfer rate in the cultivated broth system, however, was limited with highly increased viscosity of the mycelial broth. An increase in air flow rate from 1.25 to 2.00 v/v/m resulted in a low increment of oxygen transfer. The newly designed pilot plant EALB with two air spargers significantly improved processing reliability, aeration rate and K_La. The pilot plant EALB process, operated under a top pressure from 0 to 1.0 bars, also demonstrated a significant improvement of oxygenation efficiency by more than 20% in DO and K_La. The performance of the two sparger EALB process under top pressure demonstrated an efficient and economical aerobic system with fast mycelial growth and high biomass productivity in mycelial biomass production and wastewater treatment.     

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

Mathematical model based estimation of volumetric oxygen transfer coefficient(s) in the production of proteolytic enzymes in Bacillus amyloliquefaciens

Alkaline protease is a class of important hydrolytic enzymes having wide applications in bioprocess industries. Their optimum pH in the alkaline range and stability at higher temperatures make them ideal in detergent and leather processing industries. These enzymes have excellent depilating capacity. The present study aims at process optimization for the production of alkaline protease from Bacillus amyloliquefaciens ATCC 23844. Information on the optimal operating temperature and pH were elicited from specific growth rates and alkaline protease yields. It was also observed that besides pH and temperature, the oxygen transfer rate is another important limiting variable for the production of protease. Volumetric oxygen transfer coefficient (k _L a) was estimated at various impeller speeds and aeration rates. The optimal impeller speed and aeration rates were determined from k _L a and the relative protease yield data. It was understood that the oxygen transfer rate is one of the crucial parameters for the production of proteolytic enzymes by B. amyloliquefaciens.     

Identification of optimal flow rate for culture media,cell density,and oxygen toward maximization of virus production in a fed-batch baculovirus-insect cell system

In recent times, it has been realized that novel vaccines are required to combat emerging disease outbreaks, and faster optimization is required to respond to global vaccine demands. Although, fed-batch operations offer better productivity, experiment-based optimization of a new fed-batch process remains expensive and time-consuming. In this context, we propose a novel computational framework that can be used for process optimization and control of a fed-batch baculovirus-insect cell system. Since the baculovirus expression vector system (BEVS) is known to be widely used platforms for recombinant protein/vaccine production, we chose this system to demonstrate the identification of optimal profile. Toward this, first, we constructed a mathematical model that captures the time course of cell and virus growth in a baculovirus-insect cell system. Second, the proposed model was used for numerical analysis to determine the optimal operating profiles of control variables such as culture media, cell density, and oxygen based on a multiobjective optimal control formulation. Third, a detailed comparison between batch and fed-batch culture was perfromed along with a comparison between various alternatives of fed-batch operation. Finally, we demonstrate that a model-based quantification of controlled feed addition in fed-batch culture is capable of providing better productivity as compared to a batch culture. The proposed framework can be utilized for the estimation of optimal operating regions of different control variables to achieve maximum infected cell density and virus yield while minimizing the substrate/media, uninfected cell, and oxygen consumption.     

一株替代粪肠球菌的生产用芽胞杆菌筛选及其性质

目的 筛选1株能够产业化、替代粪肠球菌的芽胞杆菌。方法 从健康鸡、鸭、仔猪的粪便与肠道内容物中筛选,采用选择性培养基和耐酸耐胆盐发酵,通过耐人工肠液和人工胃液试验与粪肠球菌比较得到1株产酸能力较好的替代粪肠球菌的芽胞杆菌,并对其性质进行研究。结果 所筛选的芽胞杆菌（GY0520）对人工胃液、人工肠液有很好耐受性,90 ℃水浴15 min存活率为97%,能够产生大量有机酸,有利于提高动物机体的抗病能力及改善其生理性能,但对抗生素有一定的敏感性,不能配伍使用。结论 所筛选的芽胞杆菌能够替代粪肠球菌用于生产,为养殖业的微生态产品提高稳定性提供参考。