Micro bioreactor and high-throughput strains screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Authoritative report

1, there is a gap between domestic and foreign bacteria production level smaller than the world's microbial fermentation product market size of about 20 billion U.S. dollars, of which antibiotics accounted for 46%, from the world trend, the antibiotic market is growing at an average annual growth rate of 8%, and China's market annual growth is more than 20%. the production of raw materials for several major antibiotic varieties
is mainly concentrated in China.
, such as China's annual production of penicillin more than 60,000 tons, accounting for about 90% of the world's share, apinew drug sales of more than 60% of the export.
, such as vitamin C, the global production capacity of more than 130,000 tons, the demand is generally maintained at about 90,000 tons, mainly controlled by six major enterprises: Shijiazhuang Pharmaceutical Group, North China Pharmaceutical Group, Northeast Pharmaceutical Group, Jiangshan Pharmaceutical Co., Ltd. and The Netherlands DSM and Germany BASF.
and the domestic four major enterprises have more than 90,000 tons of production capacity, China has become the world's major base of vitamin C suppliers, China's vitamin C exports to more than 150 countries and regions.
China has become a veritable fermentation country, but due to various reasons, China's fermentation technology level is still in a backward position, in which the fermentation level of bacteria compared with foreign advanced technology has a considerable gap.
such as penicillin foreign bacteria fermentation efficiency price per milliliter of fermentation liquid nearly 200,000 units, and China is currently only about 100,000 units;
the production capacity of other fermented products in China is basically the same situation that lags behind the international level.
therefore, improving the fermentation capacity of industrial production strains is an important basis for improving the overall level of China's fermentation industry.
2, the development of the current situation of the development of my country's strain selection technology (strain improve) according to its principle can be divided into random selection (random screening) and rational selection (rationalized selection) two major methods.
Although the research of genetic engineering, metabolic engineering and systems biology consisting of various histologys based on molecular biology provides a broad prospect for the directed construction of functional cell lines, it is extremely difficult to achieve the goal of transforming with these functional cell cultures.
due to the complexity and multi-junctionity of the metabolic network in the microorganism, the metabolic flow of the strain after gene manipulation often does not shift in the direction of the expected design.
especially in the complex system of producing secondary metabolites such as antibiotics, in order to obtain excellent industrial production strains, the main use of random breeding techniques, including natural breeding, mutagenic breeding and hybrid breeding techniques represented by protoplasm fusion, but the screening workload is heavy and time-consuming.
this is because the yield is quantitative, genetics is determined by multiple genes, a mutagenic and screening is difficult to have a substantial increase, the need for multiple rounds of mutagenic screening to gradually accumulate to a certain high yield characteristics, which is different from the discovery of compounds as the target of the systle screening.
therefore, from the practice of strains selection, in addition to some primary metabolites modified by metabolic engineering methods to produce strains, regardless of the use of random breeding or rational selection, can not bypass a large number of cumbersome strain screening work.
on the other hand, whether it is random or rational selection, the classic sieve and resision of the classic strains are in the shaker or test tube without any test parameters, the screening process of the external culture environment of microorganisms and industrial fermentation production there is a huge contrast, many really in line with the actual production environment, excellent characteristics of bacteria tend to be in At the beginning of the screening was missing from the screen, the design and optimization of the fermentation process after the serological screening and screening were carried out separately and sequentially, and the lack of technical parameters between the two was not related to the excellent strains screened by the shaker bottle and the actual process conditions were not corresponding, so the high yield performance of many good strains was difficult to reflect in industrial production.
this screening model has been used in our country for more than half a century without significant improvement.
it can be seen that in the field of germ selection, it is urgent to carry out multi-parameter, combined with the fermentation process of high-throughput bacteria screening technology and methods.
3. International research progress in the screening of high-throughput strains Has been developed and matured in recent years for the device and related technologies for high-throughput screening of microbial strains.
invented a variety of automatic high-throughput screening system, can be carried out in the medium sterilization, reverse plate, pick single colonies, sub-assembled fermentation media, inoculation, extraction, HPLC (high-efficiency liquid chromatography) analysis and data automatic collection and processing processing process, that is, combined into a continuous set of automated system, to achieve efficient automatic screening, thereby greatly improving screening efficiency.
international serological screening technology is developing towards high-throughput, miniaturization, automation and instrumentation, of which bioreactor miniaturization is an important trend of current development.
, such as the University of Maryland, the Massachusetts Institute of Technology, the University of Regensburg, Germany, and the University of London, are stepping up the development of microbioreactors with high throughput characteristics.
some products have been commercialized by a number of large pharmaceutical companies, such as Bioscreen C MBR High-throughput fully automatic growth curve analysis system (Figure 4).
3.1, micro bioreactors currently appear in the international microbioreactor in the structure of the following categories: plate plate (microtiter plate, MTP, see Figure 1, see Figure 1), based on existing shaker or fermentation tank prototype miniature split microreactor (biominiature reactor, MBR, see Figure 2), Microfluidcontrol Chip Lab (microfluid-basedlab-on-a-chip, see Figure 3), analytical system for dynamic measurement of sample turbidity (optical density, light density) based on existing shaker and plate microreactor prototypes.
(Biolien C MBR) Micro bioreactors, although in different forms, have the following functions and features: (1) multi-parameter on-line detection function, can simultaneously measure pH, DO (dissvloed oxygen, dissolved oxygen), Pco2, OD (optical density, light density) and other important parameters; High-throughput analysis function, the number of micro-fermentation tanks integrated on a microreactor can range from 6, 12, 24, 48, 96;
in addition, there are low cost, reduce expensive raw material consumption, reduce labor intensity and other advantages. the miniaturization of
bioreactors is mainly based on the successful application of photochemical sensing technology in fermentation.
photochemical sensor is measured by: a specific wavelength of excitation light irradiated to a fluorescent dye indicator previously added to the medium or fixed to the inner wall of the bioreactor containing fluorescent dye indicator (patch, i.e. chemical sensor), the indicator produces emission light or light absorption, and is detected by the detector, according to the different oxygen or carbon dioxide content of the medium (or pH changes), detected in the different fluorescent intensity.
unlike sensors designed in the past based on electrochemical principles, photochemical sensors are non-non-non-persensors, thus minimizing on-line detection interference with fermentation states, also solving the problem of contamination, and making it easy to reduce the reactor.
and photochemical sensors are inexpensive, costing 1/20 to 1/10 times the cost of conventional electrochemical electrodes.
3.2, perforated plate microreactor (MTP) microplate is the standard way of automatic and efficient screening: screening automation, quantitative results, can be matched with automated machinery, liquid processing system, optical board reader, etc.
MTP is available in a variety of formats (6 to 1536 holes), but the 24-well, 48-well and 96-well formats are the most commonly used.
MTP was first used in the field of analysis and is now widely used in combination chemistry, biotransformation, microbial fermentation and cell culture screening research.
Bioscreen-C of Finland has developed a high-throughput growth curve analysis and test system based on microplate microreactors (MTP) (Bioscreen C MBR Figure 4), which is a screening system that integrates microbial fermentation and cell culture processes.
the system uses microorganisms (bacteria, fungi, yeast and algae) to reproduce and grow in the culture liquid to produce turbidity, through dynamic measurement of sample turbidity, the test process is fully automated, the system simultaneously provides up to 200 sample tests simultaneously, the sample in the temperature range of 1.0 to 60.0 degrees C can be up to 1600 hours long.
the culture system can quickly complete the optimization of culture conditions in a short period of time, can detect 3 different wavelengthS of OD value changes at the same time, to achieve the target strain or microalgae sample high-throughput screening.
Bioscreen C MBR has been used in the field of microbial fermentation and cell metabolism, and in the field of microbial fermentation and cell metabolism, and in the field of microbiome measurement in food.
turbidity due to the reproduction of all microorganisms (bacteria, fungi, yeast and algae) in the culture.
test tube test, all turbidity and color changes, can be done with the honeycomb plate "micro-test tube", Bioscreen C MBR to achieve the continuous growth and fermentation of microorganisms to achieve a rapid analysis, improve the efficiency of microbial culture process, save a lot of human, material, financial resources, and save a lot of media and consumables.
research shows that this growth curve analysis system is very promising in improving analytical performance, reducing laboratory safety requirements, reducing costs, reducing analysis time and reducing reagent usage.
the U.S. company Biolog has developed a phenotythal analytical microarray (PM) system.
the system can simultaneously provide more than 2000 different culture conditions to detect different phenotypes such as microbial growth, breathing, metabolism, etc.
the high-throughput culture system contains 200 carbon sources, 400 nitrogen sources, more than 100 phosphorus and sulfur sources, 100 nutritional supplements, and different ranges of pH, osmosis pressure, ion concentrations and other growth environments.
these preset growth conditions enable rapid and high-throughput detection of physiological or genetic characteristics of different siline cells or mutants.
, the system has been successfully applied in the rapid screening of thousands of different bacteria, fungi and mammalian cells.
this has had a huge impact on drug development, microbiological testing, food safety and other related fields.
M24 is a micro reactor system in a deep plate format (see Figure 1) with 24 cylindrical plate-free deep holes (microreactors) with a working volume of 5mL.
a non-contact pH, DO and temperature sensor, thermal conductor, 0.2mm jet film (can be used through mixed air, oxygen, nitrogen, carbon dioxide and ammonia, see Figure 5) in each hole.
the upper end of each hole with a breathable cap sealed, play the role of septic.
M24 deep-hole plate can be cultured in a temperature tank.
Chen and others have reported for the first time the development of animal cell suspension culture processes using the M24 as a reduced model of conventional laboratory-scale fermentation tanks.
examined the accuracy of pH, DO measurements, and temperature control (see Figures 6 and 7).
compared with the conventional benchtop reactor of 2L working volume in terms of pH, DO, temperature, cell growth, product formation and protein volume parameters, the results show edicomplace, indicating that the system is suitable for the application of the scaled-down model as cell culture process research.
3.3, Split Micro Bioreactor (MBR) Harms, etc., reported a 24-bit split MBR system (see Figure 2), each reactor with a working volume of 1mL. In addition to a separate non-contact in-line DO and pH sensor for each micro reactor, the
is equipped with a stirring control driven by a stepper motor, which can provide a maximum speed of 1000 r/min.
the advantages of this system is that each microreactor can be sterilized, allowed to operate on a purification table, and each reactor can control different speeds.
the system exhibits good DO, pH, OD reproducibility and the possibility of controlling DO in E.coli fermentation.
Ge et al. report an MBR system consisting of 12 micro-fermentation tanks with a diameter of 30mm, a height of 78mm and a working volume of approximately 30mL (see Figure 8).
the bottom of each reactor is also equipped with a disposable optical sensor, with SP20/0 myeloma/ rat hybrid tumor cell line as the test object, through transcription analysis shows that the optical sensing system does not affect the physiological characteristics of the cell.
3.4, micro-flow control chip system in recent years, the development of microflow control technology for microbial strains (cell strains) screening and fermentation process (cell culture) conditions of high throughput, rapid optimization of screening provides a good platform.
microfluidic control system can use trace amounts of medium and strains of several hundred fermentation tests in parallel at the same time, the results are highly comparable, so that with less time and cost for more refined, broader fermentation optimization (cell culture) condition screening.
micro-flow-controlled fermentation reactor can also simulate the actual production of replenishment strategy research, such as the method, timing, quantity and rate of replenishment and the composition of the material, concentration, etc. , micro-flow control fermentation reactor throughout the control process can maintain the added nutrients and fermentation process in the closed micro-flow control pipe and cavity, reduce the possibility of dyeing bacteria, and micro-flow-controlled fermentation reactor easy to automate, reduce the tedious labor, save a lot of tedious labor.