RAMOS® for the Bioprocess Optimisation
RAMOS® (Respiration Activity MOnitoring System) has established itself worldwide as the standard measuring system for the online determination of the breathing activity of prokaryotic and eukaryotic cultures in shake flasks
The respiration rate is directly proportional to the aerobic metabolic activity of living cells and thus allows a continuous monitoring of the cell growth and, indirectly, of the product formation rate.
Through the use of RAMOS® in screening for efficient production strains and optimum process control, amortisation may be achieved in a short time through savings in labour-hours and the shortening of development times, leading to a sustainable and decisive reduction in production costs.
The Advantages
- More information on your microbiological processes
- Differentiation of process-based and biological effects
- Replaces expensive tests in a fermenter
- Culture conditions identical to standard shake flask
- Virtual non-stop operation thanks to short set-up times and simple handling
- Ensures optimal screening conditions
- Fast and specific media optimisation
- Reduction of the effective test duration
- Shortened "time to market"
RAMOS® supplies the required information for
- Bioprocess development
- Production strain screening
- Process optimisation
- Media optimisation
- Fermentation validation
- Scale-up
Potential
By default, investigations to determine the breathing activity in stirred tank reactors are carried out by separate exhaust gas analysis. Statistically reliable results, however, demand many parallel tests which considerably increase the amount of experimental effort required. By using RAMOS® eight tests may be carried out during the same period in parallel and recorded online, thus making the amount of time and costs only a fraction of that of conventional methods.
Media optimisation is a typical screening application with a correspondingly high number of experiments. Due to the considerable experimental effort in general complete or complex media (e.g. LB medium for E. coli) are used for the cultivation. The heterogeneous composition of complex media often leads to limitations in growth of product formation, as one or more substances are stoichiometrically or simply insufficiently available. Using the combination of RAMOS® with statistical experimental technique (DOE - Design Of Experiments), the influence of each substance on growth and product formation can be determined in a very short time.
Basic biological phenomena can be easily detected with the aid of the measurement curves, examples being:
- Substrate/oxygen limitation
- Product inhibition
- Diauxia
- Fermentation
- Overflow metabolism
Key insights were obtained in this way during a number of tested processes, thus giving decisive impetus for many years of development work.
Areas of Application
- Continuous monitoring of the growth of prokaryotic and eukaryotic
- cultures including that of mammalian cells
- Eightfold parallel online measurement of breathing activity under sterile conditions
- Easy determination of typical characteristics (OTR, CTR, RQ, μ max )
- Bioprocess optimisation through the early recognition of oxygen and substrate limitation or product inhibition
- Bioassays (toxicity test, proliferation assay)
- Determination of suitable operating parameters for primary and secondary screening (media, test duration, operating conditions)
- Reduction of the development times for media and process optimisation
- Process balancing and quantification
- Quality control
Measurement of Oxygen Transfer Rate
The oxygen transfer rate (OTR) is the most suitable and most universally measured variable for the characterization and quantification of the physiological condition and the activity of a culture of aerobic micro-organisms, because almost every metabolic activity is coupled to oxygen consumption.
The online-measurement of the OTR in stirred bioreactors is nowadays state-of-the-art technology but is technically rather complex.
The determination of the OTR has, on the other hand, not been possible until now under sterile conditions in shaken bioreactors (shake flasks). RAMOS® fills this gap and combines the advantages of a shake flask (low space requirement, inexpensive, can be parallelized etc.) with those of the stirred bioreactor (online-measurement technology).
Measurement of Carbon Dioxide Transfer Rate
In addition to the OTR, the carbon dioxide transfer rate (CTR) and respiration quotient (RQ) are important sources of information for micro-biologists and cell-biologists.
The CTR (in the case of RAMOS® the "gas development rate") enables the additional use in many cases of RAMOS® for anaerobic and micro aerophilic organisms.
Using the RQ permits us to detect, among other things, which type of nutrient source of the microorganisms should be assimilated, for example
- RQ(Glucose) = 1
- RQ(triglycerides/lipids) = 0,65
The RAMOS® shake flask system was developed in cooperation with the Faculty for Bioprocess Engineering at the RWTH Aachen. The demand for equivalent culture conditions for microorganisms in regular shake flasks and in RAMOS® flasks was the top priority in the development of the system. For this reason, the volumetric flask was designed in such a way that in the lower liquid-filled section it matches a standard stirred flask in terms of gas and hydrodynamics.
Furthermore, a method was developed which makes it possible to determine the oxygen transfer rate solely from the change in the oxygen partial pressure in the gas section of the volumetric flask (no invasive electrodes). The system is protected by patent.
The gas composition above the culture liquid in the measuring system flasks is set identically to that of a normal flask in order to ensure equivalent culture conditions.
The handling of the system is simple and can be quickly learnt. After only a short time, like numerous users before you, you will not be able to imagine working without RAMOS®.
The RAMOS® system fits on a standard benchtop. It is supplied fully assembled with a shaker, incubator, control PC, USV and software.
Successful examinations carried out using RAMOS® include those with the following organisms
- Bacillus sp.
- Botrytis cinerea
- Catharanthus roseus
- CHO Zellen
- Corynebacterium glutamicum
- Escherichia coli
- Nicotiana tabacum
- Pichia pastoris
- Pichia stipitis
- Pseudoalteromonas haloplanctis
- Pseudomonas fluorescens
- Pseudomonas putida
- Saccharomyces cerevisiae
- Hybridoma Zellen
- Streptomyces lividans
- Vibrio natregiens
- Xanthomonas campestris
- Arthrospira platensis
- Wolffia australiana
- Gluconobacter oxydans
- Hansenula polymorpha
| Incubator dimensions (D x W x H) | 750 x 690 x 490 mm |
|---|---|
| Weight | approx. 66 kg (device without cooling system) |
| Size of the tray (W x D) | 420 x 420 mm |
| Standard sized volumetric flasks | 8 volumetric flasks (250 ml), bracket for 6 additional standard shake flasks (250 ml) for sample taking (reference flasks) or 8 shake flasks (500 ml). |
| Electrical connections | 230 V AC, 3 A, other voltages on request |
| Gas connections | None, air intake from ambient air, optional upstream gas mixer |
| Resolution | Oxygen transfer rate ≤ 0.001 mol/l/h (20 ml culture volume, 10 min measurement time), better resolution at greater volumes and longer measurement time |
| Error OTR | < 5 % |
| Temperature control | A water cooling system is necessary for temperatures under +30 °C (optional) and a cooling unit for under +20 °C |
| Temperature range | Without cooling RT +30 °C to +50 °C, with a water cooling system RT -5 °C to +50 °C on request +4 to +50 °C |
| Uncertainty T-control | ± 0.2 °C |
| Product code | Description |
|---|---|
| VA-RAMOSOTR | RAMOS®-system for OTR measurement with 8 volumetric flasks, without incubator shaker and control PC |
| VA-RAMOSINKSHA | RAMOS® incubator shaker, up to 300 rpm |
| CG-RAMOSABK | OS for the controlling of a RAMOS® system (for equipment cf. CG-ABK2) |
| VA-RAMOSRPM | Increase in the shaker rotation speed up to a max. of 400 rpm including balancing |
| VA-RAMOSCTR | RAMOS® system extension CTR/RQ measurement for 8 volumetric flasks |
| VA-RAMOSTKSE | RAMOS® cooler, P max = 1 bar, T min cooling medium = 2 °C (1, 2) |
| VA-RAMOSTKSDM | Pressure regulator for RAMOS® cooler down to 5 °C below the ambient temperature (with cooling water at 1...5 bar) |
| VA-RAMOSTKSKA | Cooling unit for RAMOS® cooler down to 10 °C below ambient temperature (2) |
| VA-RAMOST500 | Modified tray for 8 x 500 ml volumetric flasks |
| VA-RAMOSMK250 | RAMOS® replacement volumetric flasks 250 ml (without fittings and filters) |
| VA-RAMOSMKX | RAMOS® volumetric flasks, customized production |
| VA-RAMOSFI1 | Replacement sterile diaphragm filters for the O 2 sensors, 8 pcs |
| VA-RAMOSMTP4 | Extension for microtiter plates, special tray with 4 measuring chambers |
Please note:
The cooler VA-RAMOSTKSE can be used as follows:
1. For the compensation of an elevated laboratory temperature which is less than 5 degrees below the required incubator temperature. Here it is sufficient to use tap or cooling water as a cooling medium in conjunction with the pressure regulator VA-RAMOSTKSDM. A VA-RAMOSTKSKA cooling thermostat is required if the system is intended to work independently from a water supply system.
2. A cooling thermostat is required for the achievement of incubator temperatures which are up to 10 degrees under the laboratory temperature.
RAMOS®-fb Fed-Batch Extension
The RAMOS® system Fed-Batch Extension enables fully automated fed-batch fermentation in shake flasks or stirred vessels.
RAMOS®-cl Light Exposure Expansion
Light energy is a necessary condition for the cultivation of plant cells or general phototrophic organisms (reactions to light and darkness). For this purpose a light module was developed for the RAMOS® which enables an individual light exposure for each flask.