Traditional Bioreactor Research Articles

Research Progress on the Configurations and Performance of Reducing Pollution and Carbon Emissions by Bacterial–Algal Reactor

Currently, the water ecological environment is severely polluted and traditional bioreactors have issues with high energy consumption and greenhouse gas emissions. However, a promising solution is the bacterial–algal reactor, which is a green bioreactor that can simultaneously treat sewage and fix CO2. The main configurations of bacterial–algal reactors, including several types, activated sludge, biofilm, batch biofilm–sludge reactor coupled with activated sludge method, and bacterial–algal open reactor, have been reviewed. The performance of these reactors in reducing pollutants and carbon emissions during wastewater treatment has been investigated. Additionally, the technical advantages of coupling a bacterial–algal symbiosis system with a conventional bioreactor have been analyzed. The interaction mechanism of the bacterial–algal system in various reactors has also been elaborated. The bacterial–algal reactor improves pollutant removal efficiency through assimilation and absorption of pollutants by microalgae, and reduces aeration by releasing oxygen through photosynthesis of microalgae. Finally, the existing problems in the practical application of bacterial–algal reactors have been summarized, and future research directions have been suggested, providing theoretical support for the future application of bacterial–algal reactors and directions for optimal design and development of bacterial–algal symbiotic reactors.

Fluid dynamics and power consumptions in a single use stirred tank adopted in the pharmaceutical industry

Single use bioreactors (SUB) are recurrently adopted within the pharmaceutical industry since they offer several advantages with respect to traditional bioreactors. Generally, they consist of a stainless-steel container with a bag and an integrated impeller for handling (mixing stage), storage (post mixing stage I) and transfer (post mixing stage II) of formulated products. In this work, one of the single use bioreactor types, the Flexel-Levmixer by Sartorius, is investigated. It is a vessel with a square section stirred with a bottom impeller, for which little information is available in the literature, thus making troubleshooting and optimization difficult. To address this issue, a combination of experimental techniques, namely Particle Image Velocimetry, and torque sensors based on strain gauges, are selected for the investigation aimed at the understanding of the fluid dynamics and power consumption with different filling levels and impeller speeds.

Designing of an Advanced Compression Bioreactor with an Implementation of a Low-Cost Controlling System Connected to a Mobile Application

(1) Background: Bioreactors mimic the natural environment of cells and tissues by providing a controlled micro-environment. However, their design is often expensive and complex. Herein, we have introduced the development of a low-cost compression bioreactor which enables the application of different mechanical stimulation regimes to in vitro tissue models and provides the information of applied stress and strain in real-time. (2) Methods: The compression bioreactor is designed using a mini-computer called Raspberry Pi, which is programmed to apply compressive deformation at various strains and frequencies, as well as to measure the force applied to the tissue constructs. Besides this, we have developed a mobile application connected to the bioreactor software to monitor, command, and control experiments via mobile devices. (3) Results: Cell viability results indicate that the newly designed compression bioreactor supports cell cultivation in a sterile environment without any contamination. The developed bioreactor software plots the experimental data of dynamic mechanical loading in a long-term manner, as well as stores them for further data processing. Following in vitro uniaxial compression conditioning of 3D in vitro cartilage models, chondrocyte cell migration was altered positively compared to static cultures. (4) Conclusion: The developed compression bioreactor can support the in vitro tissue model cultivation and monitor the experimental information with a low-cost controlling system and via mobile application. The highly customizable mold inside the cultivation chamber is a significant approach to solve the limited customization capability of the traditional bioreactors. Most importantly, the compression bioreactor prevents operator- and system-dependent variability between experiments by enabling a dynamic culture in a large volume for multiple numbers of in vitro tissue constructs.

Time scale analysis & characteristic times in microscale-based bio-chemical processes: Part II – Bioreactors with immobilized cells, and process flowsheet analysis

Time-Scale Analysis and Characteristics Times are suggested as a novel and useful tool for analyzing the performance of microscale-based bioreactors with immobilized bioactive material (e.g. enzymes or microbial species), and plant flow-sheet diagrams of chemical processes. Transport rates, reaction kinetics, and phase contacting can be easily represented by unique time constants, which facilitate understanding and representation of these processes via the ‘heat-map tableau’ of Characteristic Times. Details related to the development and the origin of Characteristic Times are presented in Part I of this paper; Jovanovic et al. (2020). Time Scale Analysis & Characteristic Times is a technical approach germane to process improvement, where it facilitates the discovery of areas in need of process intensification. The feasibility and usefulness of this novel tool is demonstrated by considering a microbial biochemical reaction process performed in a traditional bioreactor vs. performance in a microscale-based bioreactor design and evaluating the balance of characteristic times associated with each technology. We believe that this technical approach will confidently find its place in the toolbox of practicing chemical reaction engineers.

Efficient purification of toluene gas by anoxic denitrification.

As a part of volatile organic compounds (VOCs), toluene does harm to human health. This paper reported the purification of toluene waste gas by liquid-phase absorption and anoxic denitrification. In this work, two same biofilm reactors were set to treat toluene waste gas, one of which was added into nitrate as an electron acceptor. Then, the purification properties of toluene waste gas treated by anoxic denitrification and biological methods were compared. The results indicate that with the increase of toluene mass concentration, the toluene removal rate of the denitrifying reactor decreased slightly. When the inlet concentration increased from 50mgm-3 to 3440mgm-3, the removal efficiency of the denitrifying reactor remained over 94.1% while the removal efficiency of the traditional bioreactor decreased to 82.9%. The highest removal capacity of the denitrifying reactor was 127.2gm-3h-1, which was 11.8% higher than that of the traditional bioreactor. Toluene was degraded by denitrification inside the biofilm microenvironment hypoxia; the denitrification rate increased along with the increase of inlet concentration within certain limits and reached a maximum of 2.4mg NO3--NL-1h-1. Nitrate can act as an electron acceptor, and denitrification can promote aerobic degradation and intensify the concentration gradient inside the biofilm to strengthen the toluene transfer process.

C-Stem, a new technology fro scale up of cell therapy production

Background & Aim Most stem cells, including Human pluripotent stem cells (hPSCs) are adherent cells. The historical standard of culture is in 2D at the bottom of Petri dishes. This technology allowed for the first trials in cell therapy but is limited by: i) reproducibility issues, ii) scalability, iii) high cost due to labor intensive manipulation of the cells, and iv) genomic instability. For cell therapy applications, cell quality reflects the culture process and large quantities of cells are required for allogenic strategies. 2D automation enables reproducible clinical batch production but commercial batch sizes will require further scale up. Suspension culture in bioreactors is a straightforward choice for scale up with two main techniques: i) microcarriers, where the cells grow attached to their surface or ii) spheroids, where cells grow in 3D aggregates. In both culture conditions the cells’ exposure to shear stress in the bioreactors limits further scale up. Methods, Results & Conclusion The C-Stem™ technology addresses this challenge by encapsulating cells within hollow alginate hydrogel capsules, 250 µm in diameter with 30 µm thick walls. Capsules both protect cells form shear stress and let small molecules (smaller than 150 kDa), gas, nutrients and waste in and out. Inside the capsule, hPSCs are seeded in extracellular matrix to mimic in vivo conditions. The capsules are produced at high throughput (5000 per second) with proprietary microfluidics. The C-Stem™ technology allows for 100x fold expansion per week of hPSCs and their differentiation in traditional bioreactors. At the PSC stage, C-Stem™ significantly improves cell viability as compared to standard 2D PSC culture. C-Stem™ promotes pluripotency, yields biomimetic hPSC “epiblastoids” and maintains state of the art genomic integrity. Addressing current manufacturing issues, the process is fully integrated from seeding to harvest, scalable, single use, closed and aligned with cGMP requirements. Most 2D and 3D protocols can be transposed in C-Stem™. Preclinical results will be presented for Parkinson's disease cell therapy. Fully mature dopaminergic neuronal microtissues are differentiated from hPSCs within capsules. They are then engrafted in Parkinson's disease rat model striata. This results in behavioral correction of rats within 6 weeks of surgery, thereby reducing by a factor 2-3 the recovery time as compared to standard progenitor engraftment.

3D MSC Culture in Saint-Gobain VueLife® FEP Bags Using Microcarriers

Background & Aim MSCs are anchorage-dependent cells that require interaction with a substrate for cell survival and active proliferation. Achieving clinically relevant autologous cell doses in vitro requires significant surface area. Microcarriers are a commonly used, high surface area 3D culture solution combined with traditional bioreactors for allogeneic therapies; however, bioreactors are operated at a much larger scale than that required for personalized medicine. Therefore, a solution combining mid-size, fully closed, single use bags from Saint-Gobain's VueLife® product line with commercially available microcarriers is promising for autologous scale expansion of MSCs. As demonstrated here, VueLife® FEP bags are particularly attractive for this application due to the inherent non-stick properties of the FEP, preventing cell and microcarrier adhesion to the bag surface. Methods, Results & Conclusion For this study, human bone marrow-derived MSCs were purchased from RoosterBio Inc. and cultured in RoosterNourish™ MSC-XF medium. To assess donor-dependent variability in the cell culture performance, three individual donors were compared. MSC expansion was performed in Saint-Gobain's VueLife® 32-C bags with Corning® CellBIND® polystyrene microcarriers and Corning® Synthemax™ II-coated dissolvable microcarriers. Protocols for MSC attachment, expansion and harvest were developed for both microcarrier systems. Cell expansion results after 6 days of culture using the microcarriers are summarized in Tables 1+2. Strong donor-dependent variability was observed in the outcome, with expansion results ranging from 4- to 21-fold increase. However, because the CellBIND® beads are not transparent, cell attachment and harvest efficiency cannot be easily tracked by microscopy and may have also impacted the outcome. Cell attachment and expansion was easier to observe on the dissolvable microcarriers, with clear attachment after four hours and full confluence after four days of culture (Figure 1). MSCs showed 24-26-fold expansion for the three donors (Table 2) using the Corning® dissolvable microcarriers. MSC surface marker expression levels were not affected by culture on either microcarrier system in VueLife® bags and comparable to expression in T-175 flasks, as shown by flow cytometry. To support further expansion towards clinically-relevant doses, additional beads and media may be added to the bags. In summary, MSC expansion can be facilitated using microcarriers in VueLife® FEP bags for patient specific therapies. Results of MSC expansion on Corning's CellBIND® and Synthemax™ II-coated microcarriers after 6 days in culture in Saint-Gobain VueLife® 32-C bags. Results are shown for three tested MSC donors (#164, #172 and #227).

Design and Application of an Electrochemical Sensor for Ammonium Monitoring in Bioprocesses

IntroductionMonitoring and control of manufacturing processes is of great importance in all industries. Bioprocesses use living microorganisms to produce a broad range of valuable goods such as proteins, hormones, enzymes or fermented food products. In a bioprocess, metabolite concentrations vary with the different fermentation phases. Non-ideal mixing conditions further contribute to the formation of local concentration gradients. These conditions render process monitoring a challenging task, specifically in large bioreactors. To ensure high product yields, product quality and process consistency, it is essential to monitor and control the bioreactor, which is tightly connected with the metabolism of the microorganisms. Therefore, being able to measure physico-chemical processes from a quantitative and qualitative perspective is key to achieving successful process control [1]. At many production sites nowadays, simple real-time monitoring systems are already implemented. These systems rely on measurements providing a limited number of variables such as pH, temperature, dissolved oxygen concentration, and carbon dioxide evolution rate (CER). Contrary to that, information concerning biologically important variables such as the concentration of different nutrients, metabolites, and biomass is mainly available by laborious off-line analyses [2]. Over the past decades, there has been an increasing focus on process analytical techniques (PAT) that allow rapid and non-destructive measurements in the frame of fermentation based manufacturing. Therefore, the sensing techniques employed need to be rapid to facilitate this deeper understanding of the process. Rapid quantification is also required for improved process control. On-line monitoring of key primary and secondary metabolites enables fast decision-making through applying dynamic feeding strategies that are tailored to the process conditions. Electrochemical (bio)sensors offer the possibility of on-line monitoring in combination with the advantages of low cost, high sensitivity and selectivity [3].This work focuses on the development and automation of electrochemical sensors for on-line monitoring of ammonium ions in the fermentation broth. Ammonium is one of the central nutrients in fermentation media and needs to be present at relevant levels to promote growth and enzyme production. Besides, there are also major challenges associated with ammonium. It imposes additional costs on downstream wastewater treatment if more ammonium is added to the medium than is actually consumed by the microorganisms during the fermentation. Usually, the quantification of ammonium in a fermentation broth is performed by spectroscopic methods which are rather time-consuming, considering that a sample preparation is needed in most cases [1]. Microfabricated electrochemical sensors allow for an improved spatial surveillance of production units with sensors placed at several critical locations, and even with the option to incorporate the electrochemical sensors into free-floating sensor particles for data collection in large bioreactors.MethodAn amperometric ammonium sensor is fabricated by immobilization of three different layers on top of a graphene working electrode in a screen-printed electrode (SPE), including electrodeposited copper oxide (Cu2O), a Nafion membrane and electropolymerized polyaniline (PANi). Prior to use, the electrodes were soaked in phosphate buffer for 30 min at room temperature to equilibrate the sensing composite.The addition of ammonium ions results in the formation of a complex between Cu(I) and neutral ammonia, which is easily oxidized by dissolved oxygen to Cu(II) with subsequent electrochemical reduction back to Cu(I), observed as a cathodic current increase [4]. Four electron reduction of oxygen down to water illustrates the high reactivity of the Cu(I)-based catalyst. In other words, ammonium complex formation triggers an oxygen reduction reaction (ORR) at the Cu(I)-based species, which in turn, acts to enhance the binding. The whole process is explained in terms of ammonia-associated ORR electrocatalysis, as illustrated in Fig. 1.Results and ConclusionsAmperometry at −0.45 V was utilized for the measurement of the response to an increase of the ammonium concentration at the PANi-Nafion-Cu-modified electrode in the range of 0.05 to 1.0 mM (Fig. 2). A consistent increase in the cathodic current was observed as a result of the increased ammonium ion concentration. The presence of ammonium causes complex formation with copper, and this causes electroreduction of oxygen to result in an increased current.The results set the frame for an innovative measuring concept which will be presented. In a traditional bioreactor set-up, data about each measured variable is collected by a single sensor that is mounted in a fixed position close to the reactor wall. In the novel concept, miniaturized biosensors will be mounted on free-floating sensor particles. Data are transmitted by wireless technology as the sensor particle surfaces the fermentation broth. This emerging technology offers great potential for accurate on-line monitoring of fermentation processes. An outlook is given on how novel electrochemical sensors will contribute to monitoring nutrients and metabolites in large scale bioreactors.

The Influence of Submerged Fermentation of Inonotus obliquus with Control Atmosphere Treatment on Enhancing Bioactive Ingredient Contents.

This study led in the pioneering technique incubated in a bioreactor with the forced air injection system. The purpose of this study was to establish the optimal incubation conditions for this technique. The results showed that the speed at which Inonotus obliquus was incubated with the forced air injection system was superior to that with a normal bioreactor. A nitrogen to oxygen ratio of 50:50 provided the best results with the forced air injection system, including in terms of the achievement of biomass, total triterpenes, betulinic acid content, and the scavenging activities of DPPH radicals, which reached up to 21.3g/1000mL, 2.1g/1000mL, 1.9g/1000mL, and 87.3%, respectively. The results showed that the bioreactor with the forced air injection system could more effectively incubate I. obliquus by using less vapor while still utilizing a model close to that of a traditional bioreactor. The innovative bioreactor fermentation model was thus more economical than the traditional bioreactor model.

Dynamic Bioreactors with Integrated Microfabricated Devices for Mechanobiological Screening.

Biomechanical stimulation is a common strategy to improve the growth, maturation, and function of a variety of engineered tissues. However, identifying optimized biomechanical conditioning protocols is challenging, as cell responses to mechanical stimuli are modulated by other multifactorial microenvironmental cues, including soluble factors and biomaterial properties. Traditional bioreactors lack the throughput necessary for combinatorial testing of cell activity in mechanically stimulated engineered tissues. Microfabricated systems can improve experimental throughput, but often do not provide uniform mechanical loading, are challenging to use, lack robustness, and offer limited amounts of cells and tissue for analysis. To address the need for higher-throughput, combinatorial testing of cell activity in a tissue engineering context, we developed a hybrid approach, in which flexible polydimethylsiloxane microfabricated inserts were designed to simultaneously generate multiple tensile strains when stretched cyclically in a standard dynamic bioreactor. In the embodiment presented in this study, each insert contained an array of 35 dog bone-shaped wells in which cell-seeded microscale hydrogels can be polymerized, with up to eight inserts stretched simultaneously in the bioreactor. Uniformity of the applied strains, both along the length of a microtissue and across multiple microtissues at the same strain level, was confirmed experimentally. In proof-of-principle experiments, the combinatorial effects of dynamic strain, biomaterial stiffness, and transforming growth factor (TGF)-β1 stimulation on myofibroblast differentiation were tested, revealing both known and novel interaction effects and suggesting tissue engineering strategies to regulate myofibroblast activation. This platform is expected to have wide applicability in systematically probing combinations of mechanobiological tissue engineering parameters for desired effects on cell fate and tissue function. Impact Statement In this study, we introduce a dynamic bioreactor system incorporating microfabricated inserts to enable systematic probing of the effects of combinations of mechanobiological parameters on engineered tissues. This novel platform offers the ease of use, robustness, and well-defined mechanical strain stimuli inherent in traditional dynamic bioreactors, but significantly improves throughput (up to 280 microtissues can be tested simultaneously in the embodiment presented in this study). This platform has wide applicability to systematically probe combinations of dynamic mechanical strain, biomaterial properties, biochemical stimulation, and other parameters for desired effects on cell fate and engineered tissue development.

Improved energy utilization efficiency via adding solar radiant heating mode for traditional bioreactor to dispose straw: Experimental and numerical evaluation

Energy utilization efficiency of heating for the operation process of biogas reactor is an important factor limiting its development and popularization. A novel mode of solar radiant heating combined with the conventional heating mode was proposed to reduce the power loss and improve the utilization cycle of heat exchanger. In present work, experimental and numerical researches about the anaerobic fermentation process under two heating modes were made to investigate the effect of temperature fluctuation on non-isothermal fermentation process under solar radiant heating. The results show that the methane production capacity of non-isothermal process under solar radiant heating reduces by up to 14% compared with the constant temperature condition in three seasons; increasing the total solid concentration of bioreactor is helpful for improving the effect of solar radiant heating; the effects of temperature fluctuation coefficient on acid and methane productions are bigger than the one on pH of slurry.

Enhancement of the denitrification activity by exoelectrogens in single-chamber air cathode microbial fuel cells

Single-chamber microbial fuel cells (MFCs) can efficiently treat wastewater containing nitrate, probably because the interaction between exoelectrogens and denitrifying bacteria may enhance the denitrification activity of MFCs. In this study, the denitrification of nitrate with a wide range of concentrations was investigated by using single-chamber air cathode MFCs. The maximum average denitrification rate of the MFCs inoculated and operated under closed-circuit conditions (Group N-CC) was up to 12.2 ± 0.6 kg NO3−-N m−3 d−1 at a high nitrate concentration of 2000 mg NO3-N L−1, which was 74.3% higher than that of the MFCs inoculated and operated under open-circuit conditions and which was significantly higher than those of other MFC systems and many traditional bioreactors. The high denitrification activity of the MFCs of Group N-CC was attributed to the significant reduction of nitrite accumulation through the possible bioelectrochemical nitrite reduction by exoelectrogens that were only enriched at the anodes of the MFCs of Group N-CC. In addition, the MFCs of Group N-CC showed good stability (over 3.5 years) and low apparent activation energy (34.0 kJ mol−1) of the denitrification, indicating the good coexistence of exoelectrogens (Geobacter) and denitrifying bacteria (Thauera) with high performance on denitrification during the long-term operation.

Analysis of process intensification and performance assessment for fermentative continuous production of bioethanol in a multi-staged membrane-integrated bioreactor system

The work analyses process intensification in production of fermentative biofuel (bioethanol) in a multi-staged membrane integrated bioreactor system from sugarcane juice (SCJ) using Saccharomyces cerevisiae (NCIM 3205). Membrane based clarification, sterilization and concentration of sugar cane juice could replace several energy-intensive and polluting steps of conventional production schemes. Integration of traditional bioreactor with membrane-based devices for downstream purification of bioethanol enabled the system for recycling of cells and residual sugars to the fermentation unit through largely fouling free cross flow microfiltration and nanofiltration membrane modules. The new process design is characterized by high yield (0.48 g/g), productivity (14.6 g/L/h) and concentration capability (97.6 g/L) using concentrated SCJ of 22 wt% under high cell density in a flexible, compact, eco-friendly and modular plant. The final membrane distillation stage concentrated ∼98% pure ethanol in a solar-driven direct contact membrane distillation configuration using PTFE/PET hydrophobic membrane with enhanced flux with vis-à-vis the existing processes. A rectangular flat sheet cross flow membrane module in counter-current flow mode of hot and cold streams was employed. The membrane-based system achieves quite high levels of process intensification essential to sustainable operation at industrial scale. The achieved process intensification in the new system has been analysed vis-a-vis the existing conventional systems in terms of eco-friendliness, flexibility, cost of equipment and production, consumption of energy and material, profitability, E-factor, atom efficiency and business sustainability.

Biogas production from a native beverage vinasse using a modified UASB bioreactor

This work evaluated the redesign of an Upflow Anaerobic Sludge Blanket (UASB) bioreactor to increase biogas production from anaerobic digestion of vinasse and compared the redesign UASB bioreactor with a conventional UASB design. Vinasse contains a high level of organic matter, 120.2±18.4gO2/L of chemical organic demand (COD), and a 0.8 biodegradability index (BI). Thus, vinasse is a potential nutrient source for biogas production by anaerobic digestion. The modified UASB bioreactor has an innovation that consisted in a second leach bed sludge, which improved the methane proportion in biogas producing 2140mLCH4/day as compared to the traditional bioreactor (1840mLCH4/day).

Integration of cell line and process development to overcome the challenge of a difficult to express protein.

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi-prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench-top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10-fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening.

A novel flat plate algal bioreactor with horizontal baffles: structural optimization and cultivation performance.

A novel flat plate photobioreactor with horizontal baffles was developed. The effects of aeration intensity and aeration site were investigated using computational fluid dynamics. The effects of baffle structural parameters, including the ratio of clearance between the baffle and the bioreactor wall (d) to bioreactor width (D) and the ratio of distance between two adjacent baffles (h) to D on the flow and mixing performance were assessed. A good light/dark cycle and strong, uniform mixing performance were created at d/D=0.5 and h/D=1 when the aeration site was located at the bottom center. The cultivation performance was assessed by culturing Chlorella vulgaris 31. The maximum biomass productivity in the optimized bioreactor was 1.88 times than that of a traditional bioreactor without baffles. For a light path length of 80 mm, the optimized baffles offer a large economical advantage in improving algal productivity and reduce growth condition difference.

Increasing Ethanol Titer and Reducing Enzyme Dosage via Fed-Batch, Simultaneous Saccharification and Fermentation in a High Solids Bioreactor

Abstract- Fed-batch, solid state, simultaneous saccharification and fermentation (SSF) was evaluated as an approach to reduce enzyme use in converting cellulose to ethanol, and to maximize ethanol titer. Kraft pulp, an intermediate in paper production, was used to represent a fractionated cellulose feedstock. Following a literature survey, average dosages were determined as 34 FPU of cellulase (Celluclast 1.5L) and 135 CBU o f β -glucosidase (Novozyme 188) per gram glucan, and were set as 100% dosages. Initially, submerged fed-batch SSF trials were conducted in a traditional bioreactor using enzyme dosages of 17, 33, 67, and 133%, with a final solids loading rate (SLR) of 14%. Ethanol production was similar (77.3-83.4% of theoretical yield) for trials with 33-133% enzyme dosages, but fell to 36% of theoretical at 17% enzyme dosage. Fed-batch saccharification and fed-batch SSF were then performed in a solid state bioreactor, achieving a 34.8% SLR. This reduced the initial 133% enzyme dosage to 19%. In saccharification trials the glucose yield was only 35% of theoretical (103.6 g/L), due to feedback inhibition of enzymes. Companion SSF trials achieved an ethanol yield of only 20% of theoretical (30.1 g/L ethanol). While some ethanol was lost due to evaporation, yeast inhibition by low water activity was presumed to be the primary limitation. Performance at lower solid loading rates were evaluated, and even diluted the fermented slurry and conducted a secondary fermentation. These trials suggest that enzyme inactivation via irreversible binding may be the primary limitation, instead of low water activity or nutrient limitation.

Disposable bioreactors: from process development to production

Single-use bioreactors are commonly used for seeding stainless steel bioreactors or for producing material. The profitability of these equipments has been well demonstrated on more that decade. But few data on its scalability have been published. In 2010-2011, Merck Serono Biodevelopment performed a study in order to evaluate the performance of several disposable bioreactors. As different technologies and scales were available, this study compared the performance of several types of mixing in single-use bioreactors for process development and pilot scale production. In addition, the evaluation was performed for both seeding applications and for clinical material production. Thus the feature of this study is the comparison of 3 to 50L disposable bioreactors with internal mixing system or external agitation (Table ​(Table1).1). In order to compare their performance with traditional bioreactors, gas flow rate applied in disposable bioreactors was scaled-down from seeding and production stainless steel bioreactors. Furthermore the ratio working volume on total volume and the power input applied to the 3L disposable bioreactor were similar to those in glass bioreactors. Table 1 Description of the different disposable bioreactors assessed. In order to compare the 5 different types of disposable bioreactors, a fed-batch process producing a highly glycosylated molecule was performed. The cell growth during the amplification phase was similar reflected by the doubling time of the cells. It was similar between traditional bioreactors (21h in 250L stainless steel seeding bioreactor) and disposable bioreactors (from 17h in the Nucleo and the Orbital to 19h in the STR) The quality of the molecule together with the molecule titer and the cell growth were compared in the 5 single use technologies during a production process. The process performance was also compared in 250L and 1.25kL bioreactors and to a 3.6L glass development bioreactor. The overall production process performance was similar in traditional and disposable bioreactors (Figure ​(Figure11). Figure 1 Quantity of highly glycosylated molecule produced in different bioreactors This study was completed by a characterization of liquid/liquid and gas/liquid transfers inside each disposable bioreactor in order to estimate their potential in terms of cell culture. These cells did not require a lot of oxygen. A low kLa (0.44h–1 in the Nucleo) was sufficient to run this process. The air-flow rates applied in the orbital shaking bioreactor during the process were defined according to Merck Serono Biodevelopment and supplier experience. These flow rates should have been decreased as kLa was higher (3.6h-1) than in traditional bioreactors (2.6 h-1). Mixing time was measured in phosphate saline buffer at the maximum working volume and the agitation speed applied during production process. Mixing time was measured by injecting concentrated sodium hydroxide from the top of the bag and from the bottom of the bag. The Nucleo and the CellReady 3L system have classical probes, while the Orbital, the RM and the STR bioreactors have optical probes. Optical probes were more stable because they took a measure every five seconds. Classical probes were less stable and showed variations of around 0.03 pH unit even a few minutes after injection in the Nucleo. Nevertheless, mixing times on all disposable bioreactors (from 15s in CellReady 3L to 100s in Orbital) were higher than in traditional bioreactors (12s in 3.6L glass bioreactor to 28s in 1250L stainless steel bioreactor). For the STR, the mixing time was close to one minute, which is the recommended maximum for mammalian cell culture. The Orbital and the Nucleo had a mixing time below two minutes. Howerver, after sodium hydroxide injection, pH measured below or close to the final pH value. The disposable bioreactor which has a mixing time closest to traditional bioreactors is the CellReady 3L. Finally, an evaluation grid was applied to choose the best disposable bioreactor. All these comparisons allowed Merck Serono Biodevelopment to come to a conclusion about disposable bioreactor use and on the scalability (up and down) of these disposable systems. It also enabled us to highlight critical points for disposable bioreactor implementation such as tubing size and length, use of disposable or reusable probes or factory.

Study on Temperature Field of L-Phenylalanine Fermentation in Heat Pipe Bioreactor

L-phenylalanine fermentation (L-Phe) is produced by the preparation method of PPA-transamination using the ferment liquor, which is the production of the E.coli No.1 fermented in heat pipe bioreactor (HPBR), the technical indexes are superior. The temperature profile of ferment liquor in the period of microbe growth is well distributed, the mean difference in temperature is 0.08°C, while the maximum value is 0.4°C.Base on operational principle of heat pipe, HPBR solves many problems on heat transfer in traditional bioreactor: nonuniformity of temperature profile, inefficiency of heat transfer etc. The study in-depth on transfer process in HPBR will effectively accelerate exploitation for new-style bioreactor and advancement of biotechnology.

Experiment of L-Phenylalanine Fermentation in New Bioreactor

L-phenylalanine fermentation (L-Phe) is produced by the preparation method of PPA-transamination using the ferment liquor, which is the production of the E.coli No.1 fermented in heat pipe bioreactor (HPBR), the technical indexes are superior. Compared with in the traditional agitated bioreactor, the biomass(taken its measure by OD value) of microbe fermented in new bioreactor is enhanced from 0.40 to 0.54, moreover, the average rate of L-Phe production by the preparation method of PPA-transamination is raised from 70% to 75%.