# US 8,785,180 B2 · Biochemical reactor

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- Number: US 8,785,180 B2
- Document ID: US8785180B2
- Status: Granted
- Kind: B2
- Application number: 12/871,539
- Filed: 2010-08-30
- Published: 2012-01-19
- Granted: 2014-07-22
- Earliest claimed: 2007-06-18
- Assignees: Shanghai Guoqiang Bioengineering Equipment Co., Ltd.; Shanghai Cohere Electronics Technology Co., Ltd.; East China University of Science and Technology
- Applicants: Siliang Zhang; Yuefang Zhang; Jian Liu; Xujun Yuan; Meijin Guo; Liqing He; Gongjian Chen; Ming Zhang; Guoqiang Sun; Wenfeng Ma; Ju Chu; Yingping Zhuang; Yonghong Wang; Mingzhi Huang; Haifeng Hang; Jianye Xia
- Inventors: Siliang Zhang; Yuefang Zhang; Jian Liu; Xujun Yuan; Meijin Guo; Liqing He; Gongjian Chen; Ming Zhang; Guoqiang Sun; Wenfeng Ma; Ju Chu; Yingping Zhuang; Yonghong Wang; Mingzhi Huang; Haifeng Hang; Jianye Xia
- Web page: https://patentgrep.com/patent/US8785180B2

## CPC

- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- C12M 27/02: {Stirrer or mobile mixing elements}
- C12M 41/06: {of illumination}
- C12M 41/36: {of biomass, e.g. colony counters or by turbidity measurements (electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404)}
- C12M 41/46: {of cellular or enzymatic activity or functionality, e.g. cell viability}
- G01N 21/253: {for batch operation, i.e. multisample apparatus (analytical automats G01N35/00)}
- G02B 21/0016: {Technical microscopes, e.g. for inspection or measuring in industrial production processes}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS}
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS

## Abstract

A biochemical reactor involves an online cell examination microscope thereon. The online cell examination microscope comprises a main body, an objective lens, an observation entrance window, a sampling device, an external light source system. Said observation entrance window lies in front of said main body and the sampling device lies in front of said observation entrance window. An objective lens and said external light source system lie behind the observation entrance window in the main body. A reflector prism is installed between the external light source system and the objective lens. A reflector lies behind the objective lens and in front of the observation entrance window. An annular diaphragm is placed in front of the reflecting prism. A CCD or an area image sensor is arranged on the upper side of the reflectance prism.

## Claims

### Claim 1

A biochemical reactor, wherein the biochemical reactor is equipped with an in-situ cell examination microscope,
wherein the in-situ cell examination microscope comprises a main body of the microscope, an object lens, an observation entrance window, a sampling device and an exterior light source system,
wherein the observation entrance window is arranged in front of the main body of the microscope, the sampling device is arranged in front of the observation entrance window, and an object lens and an exterior light source system are both arranged behind the observation entrance window in the main body of the microscope,
wherein a reflecting prism is arranged between the exterior light source system and the object lens, a reflector is arranged behind the object lens while in front of the observation entrance window, an annular diaphragm plate is arranged in front of the reflecting prism, and a CCD or an area array image sensor is arranged above one side of the reflecting prism; and
wherein the sampling device consists of a sampling piece, a flexible element and a movable device,
wherein the sampling piece and a driving axle of the movable device are connected by the flexible element, and the space between the front end face of the sampling piece and the observation entrance window forms a sampling pool;
wherein a living cell sensor is further arranged on a body of the biochemical reactor, and
wherein the biochemical reactor is capable of measuring physiological metabolic parameters to optimize and scale up a fermentation process, and
wherein the flexible element is compressible or stretchable to adjust a distance between the front end face of the sampling piece and the observation entrance window.

### Claim 2 (depends on 1)

A biochemical reactor of claim 1, wherein the exterior light source system of the in-situ cell examination microscope consists of a light source, a condenser and a replaceable color filter, wherein the light source is a halogen lamp or a LED lamp, and light beams emitted by the lamp are focused by the condenser to give parallel beams from which beams in a desired band are obtained after they pass through the replaceable color filter.

### Claim 3 (depends on 1)

A biochemical reactor of claim 1, wherein the movable device of the in-situ cell examination microscope consists of the driving axle, a connecting rod, a pulling rod for sampling and a flexible element, wherein the connecting rod equipped with the driving axle is connected to the pulling rod for sampling through a screw, the pulling rod for sampling and the flexible element are welded together at a position to give a sealed joint, and the main body of the microscope is connected to the other end face of the flexible element at a position by welding; or
the movable device of the in-situ cell examination microscope consists of the driving axle, an electric motor and a sampling tube, wherein the sampling tube is connected to the front end of the microscope body, the electric motor is arranged at the back of the sampling tube, and the output axle of the electric motor is connected to the driving axle.

### Claim 4 (depends on 1)

A biochemical reactor of claim 1, wherein the following components for process optimization and data scale-up are arranged on the body of the biochemical reactor:
a sensing system with apparatuses, instruments and sensors for detecting and controlling multiple parameters, a process pipeline system with installing supports, and an electric control cabinet with industrial personal computers and executing components.

### Claim 5 (depends on 4)

A biochemical reactor of claim 4, wherein:
the sensing system comprises a temperature sensor, a pH sensor, a dissolved oxygen sensor, a whole-tank weight sensor, an exhaust gas CO2 interface, an exhaust gas O2 interface, a speed sensor, a pressure sensor and a defoaming sensor, wherein the multiple parameters include temperature, agitation speed, gas flow rate, tank pressure, defoaming, pH, dissolved oxygen concentration, real volume and weight of fermentation broth, amount of the feed including substrate, precursor, oil, and acidic/basic substance, exhaust gas CO2 and exhaust gas O2;
the process pipeline system with installing supports comprises a feed flask, a preheater, a stand for whole-tank weighing, a special support for sampling, a tank body assembly, a quick detachable bedplate, an electric motor, a pipe rack, an oil-water separator, a pressure reducing valve, a filter, a flowmeter, an air filter, a pressure gauge, a cooler, a pipe sight glass, a water heater, a sampling valve with no dead volume, a defoaming sensor interface, an exhaust gas CO2 interface, an exhaust gas O2 interface, a temperature sensor, a pH sensor interface and a DO sensor interface; and
the electric control cabinet with industrial personal computers and executing components comprises a thermal mass flowmeter, a high-precision peristaltic pump, an electronic balance for supplementing substrate, a circulating pump, an electromagnetic valve, a digital-to-analog converter, an analog-to-digital converter, a local computer, an upper computer, a modem and a rare-earth motor.

### Claim 6

An in-situ cell examination microscope, comprising a main body of the microscope, an object lens, an observation entrance window, a sampling device and an exterior light source system,
wherein the observation entrance window is arranged in front of the main body of the microscope, the sampling device is arranged in front of the observation entrance window, and an object lens and an exterior light source system are both arranged behind the observation entrance window in the main body of the microscope,
wherein a reflecting prism is arranged between the exterior light source system and the object lens, a reflector is arranged behind the object lens while in front of the observation entrance window, an annular diaphragm plate is arranged in front of the reflecting prism, and a CCD or an area array image sensor is arranged above one side of the reflecting prism;
wherein the sampling device consists of a sampling piece, a flexible element and a movable device,
wherein the sampling piece and a driving axle of the movable device are connected by the flexible element, and the space between the front end face of the sampling piece and the observation entrance window forms a sampling pool,
wherein a living cell sensor is further arranged on a body of the biochemical reactor, and
wherein the flexible element is compressible or stretchable to adjust a distance between the front end face of the sampling piece and the observation entrance window.

### Claim 7 (depends on 4)

A fermentation process using the biochemical reactor of claim 4, comprising the following steps:
(a) analysis of physiological metabolic parameters, and characteristics associated with the physiological metabolic parameters or combinations thereof,
wherein the physiological metabolic parameters are selected from dissolved oxygen, oxygen uptake rate, pH, CO2 evolution rate, respiratory quotient, the amount of living cells or cell morphology, metabolic product(s) to be measured, substrate consumption, or combinations thereof;
(b) comparing the physiological metabolic parameters, the characteristics associated with the physiological metabolic parameters or combinations thereof as measured in step (a) with those having predetermined values, choosing a biochemical reactor bearing measurements closest to the predetermined values, and thus determining a reactor that is optimized and scaled-up accordingly;
wherein the physiological metabolic parameters are defined in step (a).

### Claim 8 (depends on 7)

A process of claim 7, wherein in step (a), the physiological metabolic parameters, the characteristics associated with the physiological metabolic parameters or combinations thereof are obtained by detecting several process control parameters of the biochemical reactor,
wherein the several process control parameters are temperature, agitation speed, gas flow rate, tank pressure, defoaming, pH, dissolved oxygen concentration, real volume and weight of fermentation broth, amount of the feed including substrate, precursor, oil, and acidic/basic substance, exhaust gas CO2 and exhaust gas O2.

### Claim 9 (depends on 6)

The in-situ cell examination microscope of claim 6, wherein the flexible element extends between a first end and a second end opposite the first end, the flexible element connects the sampling piece at the first end and connects the driving axle at the second end.

### Claim 10 (depends on 1)

The biochemical reactor of claim 1, wherein the flexible element extends in a direction generally perpendicular to the observation entrance window.

### Claim 11 (depends on 6)

The in-situ cell examination microscope of claim 6, wherein the flexible element extends in a direction generally perpendicular to the observation entrance window.

### Claim 12

A biochemical reactor, wherein the biochemical reactor is equipped with an in-situ cell examination microscope,
wherein the in-situ cell examination microscope comprises a main body of the microscope, an object lens, an observation entrance window, a sampling device and an exterior light source system,
wherein the observation entrance window is arranged in front of the main body of the microscope, the sampling device is arranged in front of the observation entrance window, and an object lens and an exterior light source system are both arranged behind the observation entrance window in the main body of the microscope,
wherein a reflecting prism is arranged between the exterior light source system and the object lens, a reflector is arranged behind the object lens while in front of the observation entrance window, an annular diaphragm plate is arranged in front of the reflecting prism, and a CCD or an area array image sensor is arranged above one side of the reflecting prism,
wherein the sampling device consists of a sampling piece, a flexible element and a movable device, wherein the sampling piece and a driving axle of the movable device are connected by the flexible element, the space between the front end face of the sampling piece and the observation entrance window forms a sampling pool, the flexible element extends between a first end and a second end opposite the first end, and the flexible element connects the sampling piece at the first end and connects the driving axle at the second end,
wherein a living cell sensor is further arranged on a body of the biochemical reactor, and
wherein the flexible element is compressible or stretchable to adjust a distance between the front end face of the sampling piece and the observation entrance window.