新型生物反应器结构研究进展

生物反应器是生物工程的核心设备,其结构的合理性直接决定反应器生物加工的效率。生物反应器的研究一直是生物工程的核心问题之一。随着青霉素的工业化生产,机械搅拌式生物反应器应运而生,此后,随着动植物细胞培养,高等真菌培养,藻类培养等生物过程的发展,人们相应开发了大量的生物反应器,其中以机械搅拌式生物反应器和气升式样生物反应器尤为突出,本文总结了近年来文献报道的新型生物反应器,主要阐述了机械搅拌式和气升式两类生物反应器结构的研究进展,对目前国内外报道的11种新型反应器典型结构进行了总结与分析。     

Use of Chicken Manure Extract for Biostimulation and Enhancement of Perchlorate Rhizodegradation in Soil and Water Media

The influence of biostimulation using dissolved organic carbon (DOC) on rhizodegradation of perchlorate and plant uptake was studied under greenhouse conditions using soil and hydroponic bioreactors. One set of bioreactors planted with willow (Salix babylonica) plants was spiked with 300 mg L^?1 DOC in the form of chicken manure extract, whereas a second set was not treated with DOC. A similar experiment without willow plants was run in parallel to the planted bioreactors. The planted soil bioreactors amended with DOC reduced perchlorate from 65.85 to 2.67 mg L^?1 in 21 days for humic soil (95.95% removal) and from 68.99 to 0.06 mg L^{? 1} for sandy loam (99.91% removal) in 11 days. Nonplanted DOC treated soil bioreactors achieved complete perchlorate removal in 6 and 8 days for humic and sandy loam, respectively. Both planted and nonplanted soil bioreactors without DOC removed > 95% perchlorate within 8 days. Planted soil bioreactors respiked with perchlorate reduced perchlorate to nondetectable levels in 6 days. Hydroponics experiment amended with DOC reduced perchlorate from approximately 100 mg L^{? 1} to nondetectable levels within 7 to 9 days. Hydroponic bioreactors without DOC had low perchlorate removal rates, achieving 30% removal in 42 days. Leaf samples from sandy loam soil bioreactors without DOC had four times perchlorate phytoaccumulation than the DOC-treated plants. Similar results were obtained with the nonplanted bioreactors. Persistence of perchlorate in solution of planted hydroponic bioreactors without DOC amendment suggested that natural DOC from the plant exudates was not enough to biostimulate perchlorate reducing microbes. The hydroponic bioreactor study provided evidence that DOC is a limiting factor in the rhizodegradation of perchlorate.     

Long-term performance of bioreactors cleaning mercury-contaminated wastewater and their response to temperature and mercury stress and mechanical perturbation

The long-term performance of bioreactors retaining mercury from contaminated industrial wastewater was analyzed at the laboratory scale, and its response to mechanical perturbations (gas bubbles and shaking) as well as to physical (increased temperature and hydraulic load) and chemical stresses (increased mercury concentration) likely to occur during on site operation was studied. Two packed-bed bioreactors with 80-cm(3) lava chips as biofilm carrier were inoculated with nine Hg(II)-resistant natural isolates of alpha- and gamma-proteobacteria. Chloralkali wastewater containing ionic mercury (3.0 to 9.7 mg/L Hg(2+)), amended with sucrose and yeast extract, flowed through the bioreactors at 160 mL/h. During the 16-month investigation the bioreactors showed no sign of depleted performance in terms of mercury-retaining capacity. After 16 months, both bioreactors still retained 96% of the mercury load. The performance of the bioreactors was sensitive to mechanical perturbations (e.g., sheer forces of gas bubbles). Shifts to higher Hg(2+) inflow concentrations initially decreased the mercury retention efficacy slightly. However, the bioreactors could adapt to Hg(2+) concentrations of up to 7.6 mg/L within several days. Old biofilms were less affected than the younger ones. The performance of the bioreactors was not affected by an increase in temperature up to 41 degrees C and an increased volumetric load (up to 240 mL/h). The bioreactors regained activity spontaneously after the stress had stopped. Recovery could be accelerated by increased nutrient concentration, although this may lead to blocking of the packed bed.     

Three-dimensional culture and bioreactors for cellular therapies

A bioreactor is defined as a specifically designed vessel to facilitate the growth of organisms and cells through application of physical and/or electrical stimulus. When cells with therapeutic potential were first discovered, they were initially cultured and expanded in two-dimensional (2-D) culture vessels such as plates or T-flasks. However, it was soon discovered that bioreactors could be used to expand and maintain cultures more easily and efficiently. Since then, bioreactors have come to be accepted as an indispensable tool to advance cell and tissue culture further. A wide array of bioreactors has been developed to date, and in recent years businesses have started supplying bioreactors commercially. Bioreactors in the research arena range from stirred tank bioreactors for suspension culture to those with various mechanical actuators that can apply different fluidic and mechanical stresses to tissues and three-dimensional (3-D) scaffolds. As regenerative medicine gains more traction in the clinic, bioreactors for use with cellular therapies are being developed and marketed. While many of the simpler bioreactors are fit for purpose, others fail to satisfy the complex requirements of tissues in culture. We have examined the use of different types of bioreactors in regenerative medicine and evaluated the application of bioreactors in the realization of emerging cellular therapies.     

Disposable bioreactors: the current state-of-the-art and recommended applications in biotechnology

Disposable bioreactors have increasingly been incorporated into preclinical, clinical, and production-scale biotechnological facilities over the last few years. Driven by market needs, and, in particular, by the developers and manufacturers of drugs, vaccines, and further biologicals, there has been a trend toward the use of disposable seed bioreactors as well as production bioreactors. Numerous studies documenting their advantages in use have contributed to further new developments and have resulted in the availability of a multitude of disposable bioreactor types which differ in power input, design, instrumentation, and scale of the cultivation container. In this review, the term “disposable bioreactor” is defined, the benefits and constraints of disposable bioreactors are discussed, and critical phases and milestones in the development of disposable bioreactors are summarized. An overview of the disposable bioreactors that are currently commercially available is provided, and the domination of wave-mixed, orbitally shaken, and, in particular, stirred disposable bioreactors in animal cell-derived productions at cubic meter scale is reported. The growth of this type of reactor system is attributed to the recent availability of stirred disposable benchtop systems such as the Mobius CellReady 3 L Bioreactor. Analysis of the data from computational fluid dynamic simulation studies and first cultivation runs confirms that this novel bioreactor system is a viable alternative to traditional cell culture bioreactors at benchtop scale.     

Development and efficiency of a new generation of bioreactors

The development and investigation of high-efficiency bioreactors is one of the primary problems bioprocess engineers have to solve. With increasing efficiency of the bioreactor the downstream processes will be much simplified, energy and costs will be substantially reduced. According to fundamental research on the properties of stirred tank bioreactors the following rules for the design of bioreactors should be observed:

1. Prevent rotational motion of the biosuspension.
2. Generate motion of the biosuspension in the radial and axial direction.
3. Prevent fluid flow in big spaces.

These design rules are in general observed in the development of high-efficiency bioreactors. A selected group of such bioreactors is described.     

Development and efficiency of a new generation of bioreactors

The development and investigation of high-efficiency bioreactors is one of the primary problems bioprocess engineers have to solve. With increasing efficiency of the bioreactor the downstream processes will be much simplified, energy and costs will be substantially reduced. According to fundamental research on the properties of stirred tank bioreactors the following rules for the design of bioreactors should be observed:

1. Prevent rotational motion of the biosuspension.
2. Generate motion of the biosuspension in the radial and axial direction.
3. Prevent fluid flow in big spaces.

These design rules are in general observed in the development of high-efficiency bioreactors. A selected group of such bioreactors is described.     

植物生物反应器优化策略与最新应用*

随着分子生物学和植物基因工程的迅猛发展以及分子医药和现代农业等学科的交叉融合,植物生物反应器已成为分子医药农业的核心内容。利用植物生物反应器生产抗体、疫苗和功能性食品,具有规模化、成本低、安全性高、周期短等优势。2022年2月,加拿大卫生部批准了新型冠状病毒疫苗Covifenz^®,这是世界首款植物源人体疫苗,标志着以植物生物反应器为代表的分子医药农业时代的来临。综述植物叶片和种子等代表性的植物生物反应器类型,分析瞬时表达系统和稳定表达系统的构建原理和应用,探讨通过启动子和密码子优化、糖基化过程“人源化”、基因沉默抑制和蛋白酶作用抑制等优化植物生物反应器的策略,总结国内外抗体、疫苗和功能性食品等植物源产品的开发进展,以期为我国植物生物反应器的研究及其在分子医药领域的应用提供参考。     

Sparged animal cell bioreactors: mechanism of cell damage and Pluronic F-68 protection.

Pluronic F-68 is a widely used protective agent in sparged animal cell bioreactors. In this study, the attachment-independent Spodoptera frugiperda Sf9 insect cell line was used to explore the mechanism of this protective effect and the nature of cell damage in sparged bioreactors. First, bubble incorporation via cavitation or vortexing was induced by increasing the agitation rate in a surface-aerated bioreactor; insect cells were rapidly killed under these conditions of the absence of polyols. Supplementing the medium with 0.2% (w/v) Pluronic F-68, however, fully protected the cells. Next, cell growth was compared in two airlift bioreactors with similar geometry but different sparger design; one of these bioreactors consisted of a thin membrane distributor, while the other consisted of a porous stainless steel distributor. The flow rates and bubble sizes were comparable in the two bioreactors. Supplementing the medium with 0.2% (w/v) Pluronic F-68 provided full protection to cells growing in the bioreactor with the membrane distributor but provided essentially no protection in the bioreactor with the stainless steel distributor. These results strongly suggest that cell damage can occur in the vicinity of the gas distributor. In addition, these results demonstrate that bubble size and gas flow rate are not the only important considerations of cell damage in sparged bioreactors. A model of cell death in sparged bioreactors is presented.     

Efficient degradation of rice straw in the reactors packed by carbon fiber textiles

We have reported for the first time that agricultural and cellulosic waste, i.e., rice straw was directly applied to methanogenic bioreactors containing carbon fiber textiles (CFT) as supporting material. Addition of CFT to the methanogenic bioreactors enhanced the conversion of dichromate chemical oxygen demand of the substrate to methane (41%) to a greater extent than bioreactors without CFT (9%). In addition, removal of rice straw as a suspended solid was increased from 31% (in bioreactors without CFT) to 57% (in those with CFT). Methanogenic 16S rRNA gene analysis showed that the abundance of acetoclastic methanogen, genus Methanosarcina, was about 11 times higher in bioreactors with CFT (suspended fraction plus retained fraction to CFT) than in bioreactors without CFT (suspended fraction), resulting in lower concentration of acetate in bioreactors with CFT (0.4 mM) than in those without CFT (29.7 mM). On the other hand, the abundance of hydrogenotrophic methanogen, genus Methanobacterium, in bioreactors with CFT was similar to those without CFT. Bacterial communities in bioreactors with CFT were different from those in bioreactors without CFT. Our results indicated that specific microbial community and cooperative relationships between microorganisms in reactors containing CFT facilitated efficient decomposition of rice straw and its conversion to methane.     

Membranes and bioreactors: a technical challenge in biotechnology

Integrating the properties of synthetic membranes with biological catalysts such as cells and enzymes forms the basis of an exciting new technology called membrane bioreactors. The impetus behind this marriage comes from the recent spectacular advances in recombinant DMA and cell fusion technologies and the need to develop competitive bioprocessing schemes to produce complex and active biological molecules. The advantages and limitations of using membrane bioreactors for entrapping whole cells and enzymes are reviewed. Various membrane configurations such as microcapsules, hollow fibers, and flat sheets are compared. Several different entrapped membrane bioreactors, including single, laminated and microporous, for the conversion of optically active enantiomers are described. As with new and exciting technologies, the future of membrane bioreactors in biotechnology will depend on their ability to produce desired molecules at competitive costs.     

Tubular bioreactors: case study of bioreactor performance for industrial production and scientific research

Application of bioreactors is dominated by industrial production with the consequence that bioreactors also for scientific purposes are mainly used following an empiric pragmatic approach. For the sake of a breakthrough in biotechnology in general, and especially for advanced process development, a more systematic approach is emphasized here. This methodology in bioreactor performance studies is explained and the meaning clarified in a case study of a new type of tubular bioreactor. The central role of so-called "model bioreactors" in bench-scale applications is illustrated as a powerful contribution to the optimal design of bioreactors in technical scale. Pilot plant data in case of a tubular reactor for the production of ethanol with Zymomonas and biopesticides with Bacillus thuringiensis are presented.     

动物细胞培养用生物反应器及相关技术

动物细胞大量培养是生产生物制品的重要途径,它用到的关键设备是生物反应器。根据培养细胞、培养载体、培养液混合方式的不同,生物反应器主要有搅拌式、气升式、中空纤维式、回转式等,其中搅拌式规模最大。回转式是NASA于20世纪90年代中期开发的一种新型生物反应器,被誉为空间生物反应器,可用于组织工程研究。与生物反应器配套的技术主要有灌注、微载体、多孔微球、转入抗凋亡基因等,可以有效地提高细胞密度,增加生物制品产量,提高质量。今后生物反应器研制主要朝两个方向发展:一是,以高密度培养动物细胞生产蛋白质药物为目的,二是以三维培养动物细胞(主要是人类细胞)再生组织或器官为目的。     

Methods and milliliter scale devices for high-throughput bioprocess design

Based on electromagnetic simulations as well as on computational fluid dynamics simulations gas-inducing impellers and their magnetic inductive drive were optimized for stirred-tank reactors on a 10 ml-scale arranged in a bioreaction block with 48 bioreactors. High impeller speeds of up to 4,000 rpm were achieved at very small electrical power inputs (63 W with 48 bioreactors). The maxima of local energy dissipation in the reaction medium were estimated to be up to 50 W L⁻¹ at 2,800 rpm. Total power input and local energy dissipation are thus well comparable to standard stirred-tank bioreactors. A prototype fluorescence reader for 8 bioreactors with immobilized fluorometric sensor spots was applied for online measurement of dissolved oxygen concentration making use of the phase detection method. A self-optimizing scheduling software was developed for parallel control of 48 bioreactors with a liquid-handling system for automation of titration and sampling. It was shown on the examples of simple parallel batch cultivations of Escherichia coli with different media compositions that high cell densities of up to 16.5 g L⁻¹ dry cell mass can be achieved without pH-control within 5 h with a high parallel reproducibility (standard deviation<3.5%, n=48) due to the high oxygen transfer capability of the gas-inducing stirred-tank bioreactors.     

Description and evaluation of reciprocating plate bioreactors

The environment in which live microorganisms has a major impact on their productivity. One important factor is the mechanical mixing that is used to promote good heat and mass transfer in bioreactors. In this paper, the performance of reciprocating plate bioreactors is first evaluated for their ability to produce high oxygen transfer coefficients. Pure water and a glycerol water (5050 wt%) solution are used for this evaluation. Then, the performance of reciprocating plate bioreactors for the production of an exocellular polysaccharide (pullulan) by yeast Aureobasidium pullulans is analyzed in terms of quantity and quality of the polysaccharide. Results clearly show that a more efficient substrate utilisation is achieved with reciprocating plate bioreactors.     

Activity of Nitrifying Biofilms Constructed on Low-Density Polyester Enhances Bioremediation of a Coastal Wastewater Effluent

Summary Nitrifying biofilms were constructed on low density polyester Dacron for the bioremediation of nitrogen from wastewater effluent of a municipal treatment plant. Dacron disks were inoculated with wastewater sludge enriched for 15 days for either ammonia- or nitrite-oxidizing bacteria (AOB or NOB, respectively) and packed into glass bioreactors. Wastewater effluent containing high levels of ammonia, nitrite, and phosphate was collected and fed to inoculated and uninoculated bioreactors. Both inoculated bioreactors showed stable nitrification efficiencies, removing 96 and 76% of the ammonia and 12 and 35% of the nitrite for AOB- and NOB-inoculated bioreactors, respectively. Efficiencies of phosphate removal were similar in both inoculated and uninoculated bioreactors, indicating that nitrifiers were not required for this process. AOB-inoculated bioreactors accumulated nitrite mid-way through the experiment and had low rates of conversion to nitrate, suggesting slow nitrite oxidizer growth. DGGE and sequence analysis of AOB 16S rRNA genes showed enrichment of Nitrosomonas spp. in both inoculated bioreactors, and a dominance of Nitrosospira spp. in non-inoculated bioreactors. This study describes an inexpensive and efficient technology for removing ammonia and nitrite from wastewater effluents of municipal treatment plants before its release to the environment.     

Computational fluid dynamics analysis of mixing and gas–liquid mass transfer in wave bag bioreactor

Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (k_La) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors.     

Industrial wastewater bioreactors: sources of novel microorganisms for biotechnology

Microorganisms exist in nature as members of complex, mixed communities. The microbial communities in industrial wastewater bioreactors can be used as model systems to study the evolution of new metabolic pathways in natural ecosystems. The evolution of microbial metabolic capability in these bioreactors is presumably analogous to phenomena that occur in natural ecosystems. The microorganisms in these bioreactors compete for different carbon sources and constantly have to evolve new metabolic capabilities for survival. Thus, industrial bioreactors should be a rich source of novel biocatalysts.     

Two new disposable bioreactors for plant cell culture: The wave and undertow bioreactor and the slug bubble bioreactor

The present article describes two novel flexible plastic-based disposable bioreactors. The first one, the WU bioreactor, is based on the principle of a wave and undertow mechanism that provides agitation while offering convenient mixing and aeration to the plant cell culture contained within the bioreactor. The second one is a high aspect ratio bubble column bioreactor, where agitation and aeration are achieved through the intermittent generation of large diameter bubbles, "Taylor-like" or "slug bubbles" (SB bioreactor). It allows an easy volume increase from a few liters to larger volumes up to several hundred liters with the use of multiple units. The cultivation of tobacco and soya cells producing isoflavones is described up to 70 and 100 L working volume for the SB bioreactor and WU bioreactor, respectively. The bioreactors being disposable and pre-sterilized before use, cleaning, sterilization, and maintenance operations are strongly reduced or eliminated. Both bioreactors represent efficient and low cost cell culture systems, applicable to various cell cultures at small and medium scale, complementary to traditional stainless-steel bioreactors.     

Introduction to advantages and problems of shaken cultures

Shaking bioreactors are the most frequently used reaction vessels in biotechnology and have been so for many decades. In spite of their large practical importance, very little is known about the characteristic properties of shaken cultures from an engineering point of view. The few publications available contain to some extent contradicting statements and conflicting advice concerning the correct operating conditions of shaking bioreactors. Depending on the investigated microbial system, the engineering parameters may more or less significantly influence the experimental results in a quantitative as well as in a qualitative manner. Unfortunately, these kind of interactions are often overlooked or ignored by scientists. Precise knowledge about the controlling hydrodynamic phenomena in shaking bioreactors and quantitative information about the physical parameters influencing the cultures are needed to assure reproducible and meaningful operating conditions. In this introduction, the state of the art of culturing microorganisms in shaking bioreactors is reviewed and some issues of their practical application in screening and process development projects are addressed.