RTS-1C, Personal bioreactor

You are correct, in reality, there are many types of reaction vessels and bioreactors that the market can offer for the cultivation of microorganisms, yet we think that RTS technology has its unique niche.

The most important issue when choosing a bioprocess technology is whether it is scalable. The impossibility of direct transfer of optimal conditions from thermoshakers to bioreactors became a common issue. Many engineers are in search for the middle ground, which is so that the cultivation in the bioreactor would be as simple and accessible as in shaker flasks and so that the process could be carried out in parallel from one hand, and on the other hand so that the bioprocess would be scalable. Lower aeration values using shaker flasks in comparison to bioreactors or, in other words, low oxygen mass transfer into the culture medium does not allow getting as high cell densities as quickly as possible like in bioreactors.

Yes, and from the point of view of its place in the market of disposable bioreactors this smart mini-bioreactor RTS-1 is unique. It occupies an intermediate link between shake flask reactors and typical bioreactors and laboratory bioreactors, while being similar to shake flasks because of the possibility of interdependent matrix cultivation, and high kLa values makes RTS-1 friendlier for further scalability.

The contradiction between the shaker and bioreactor way of realization of bioprocess implementation is not only in the impossibility of matching the data obtained but also that the principle of agitation (mixing to maximize the interaction of the reactants and microorganisms in the bioreactor) is quite different. Mass transfer, including oxygen saturation of the medium, the speed of incubation in a shaker incubator are incomparably lower than that in the bioreactor. Bioengineers of world's leading companies are in search of an intermediary, and there are hybrid solutions like installing bioreactor on a shaker or non-invasive monitoring of parameters of cultivation, but they are destined for failure because the most important parameter of bioprocess is mass transfer and shakers maximum k_La is 100 h^-1 while average bioreactor k_La is 1000 h^-1.

RTS-1 oxygen mass transfer into the broth medium is up to 200 h^-1 and RTS mixing technology can occupy an intermediate stage of macroevolution of bioprocess and can be between shaker-incubators and bioreactors because of high k_La values and scalability. Moreover, when optimization bioprocess is required by matrix type cultivation where study of several interdependent physicochemical factors is required in configurations like, 2x2 = 4 RTS, 3x3 = 9 RTS, 4x4 = 16RTS, 5x5 = 25RTS, and even 10x10 = 100 concurrent bioreactors, the RTS can be perfectly applied. This kind of RTS technology bioreactor amount is acceptably imaginable on a laboratory bench or in case of the need of higher biological safety in Laminar Cabinets class II and III and this kind of solution can be cost efficient and consequently, promising.

As can be observed from image. Biosan produces most of devices for noninvasive mixing which in turn can be used for bioprocesses.  Moreover, we would like to introduce a new mixing principle into this table called Reverse Spinning. In the future, we will develop multi-channel RTS devices with higher and smaller volumes. In our estimates, the mixing technology can be scaled up to 20 litres and scaled down to 5ml. With a possibility to add vital bioprocess parameters noninvasive measurement such as pH and pO₂

Initiation of the Vortex Type Mixing (VTM) and depth of the Vortex cave depend on 1) angular speed of RS–Reactor 2) time from initiating rotation of RS–Reactor 3) growth media viscosity 4) temperature. These parameters, also, determine the angular speed of rotating Vortex Layer (VL) and transition state from the Irrotational Vortex (IRV), when angular speed of the VL is proportional to the radius, to the Rotational Vortex, when the angular speed of the VL is the same and VL looks like a monolithic Vortex cavity, as shown below. Common rules regulating VTM processes may be stated as follows: the more time has passed since Vortex formation, the more obvious is a transition from IRV to the RV. The concept of the Reverse–Spin (RS) mixing is based on these assumptions.

The essential elements of RTS-1 measurement system consist, as shown below, of a light source, a detector (photodiode), a falcon tube compartment in a rotating state. The falcon tube accelerates to 2000 min^-1 creating a monolayer of cell suspension shortening the optical path of the sample. By shortening the optical path, it is possible to measure biomass in the range of 0-19 OD_600nm at 10-20ml and 0-15.2 OD_600nm at 10-30ml.

Thin layer increases OD measurement by mechanical non-invasive dilution.

O₂ k_La into the medium is extremely important indicator to assess the possible RTS-1 prospects to obtain high output growth rates of biomass.

Stirring of the liquid medium is happening due to Vortex Type Mixing (VTM) and Reverse-Spin (RS). Due to multiple increase of the contact surface liquid and gaseous medium during the rotation and the emergence of waves (see fig. 1) at the time of movement direction change, the mixing efficiency is substantially increased both in liquid and liquid-gas phase.  As a result, the saturation of the liquid phase by gas and its further dissolution in it occurs with greater efficiency than in most standard mixing devices.

As shown on Fig. 2, spread of broth media inside of the tube depends on rotation intensity, which in turn affects the efficiency of VTM and rate of O₂ supply.

Figure 1

Figure 2

Since we are dealing with a non-invasive method of measuring light scattering in cell suspension of growing cells, then the standardization of the vessel in which the fermentation takes place is very difficult. Instead of using a spectrophotometric cuvette and a spectrophotometer, we offer to use a standard falcon tube that is popular in growing microorganisms in the early stages of scale-up processes – a disposable falcon tube with respiratory membrane, which is slowly becoming a gold standard. Yet, falcon tubes are not 100% transparent and the degree of its turbidity depends on the temperature and the wavelength at which you are making the measurement. With this in mind, we have transitioned to NIR spectra wavelength to avoid the optical characteristics of the plastic material that is affected by temperature.

Factory calibration of the instrument is designed for specific microorganism size of 0.4-0.8 x 1-3 μm and a cell volume of approximately 0.6-0.7 μm3. In case of exceeding the allowable size, the measurement system using factory calibration will not work correctly.

Optical density OD (λ = 850 nm) to OD (λ = 600 nm) conversion rate coefficient is equal to 1.9 (cells taken for measurement from stationary phase using a spectrophotometer and 1mm optical path cuvette).

Example of calculation: to convert 3.5 OD (λ = 850 nm) to OD (λ = 600 nm), multiply the result by 1.9, resulting in 6.65 OD (λ = 600 nm).

The microorganism that is used for factory calibration is E.coli BL21. The cells are taken from shake flask night culture at stationary phase of growth.

During the growth transition of Escherichia coli culture from the exponential growth to the stationary phase, a number of morphological and physiological changes take place, including cell volume decrease and cell shape change. Therefore, if cells taken for referent measurement using spectrophotometer at different stages from stationary phase then the correctness of measurement will be worse than specified.

Firstly, Rotation intensity influences air transfer introduced to the tube from the outside environment. Secondly, the ambient temperature influences the temperature set in the bioreactor, and therefore can cool or heat the sample. Thirdly, contact between liquid and falcon tube compartment is less at higher rotation intensities. See image below, for breakdown of temperature differences (Δ t°C) between tube sample and set thermoblock temperature, depending on aerobe and microaerophilic rotation intensities at ambient temperature 23 °C ± 2 °C and 45% ± 10% RH.

OD measurement accuracy is affected by moisture that can appear on the outside wall of the tube because of dew point temperature. Relative humidity and temperature affect the dew point, therefore the temperature of the tube must be higher than the dew point temperature for the measurement system to work correctly.

Finding dew point temperature for the user.

Find the corresponding curve on image that matches the humidity of the room in which the device is located.

The horizontal axis indicates the temperature of the room in which device is located.

Using that information, make a projection on the vertical axis. The found point on the graph is the temperature of the dew point.

If the dew point temperature reached, moisture will disrupt correct measurement of the system. To avoid dew point temperature, decrease the temperature difference between the ambient temperature and bioreactor and sample temperature. By placing the bioreactor to an environmental chamber, the temperature difference can be significantly lower.

Example of calculation of environmental chamber temperature selection:

If the range of temperature profiling is from +10°C to +40°C, calculate the environmental chamber temperature:

(40-10) / 2 = 15°C

It is possible to place the device in a cold room or environmental chamber, but the temperature of thermoblock and temperature of the sample will not be accurate at specific temperature ranges. Temperature measurements of the sample and corrections at maximum and minimum temperature ranges or 4°C-10°C and 60°C-70°C must be performed by the user. If any additional questions appear, please contact Biosan RTS support team directly for assistance.

When temperature of the plastic material is changing, i.e. during temperature cycling from +37°C to +10°C and back every hour, the plastic material of the tube changes optical characteristics in a range of ±0.1 OD. This influence can be observed on all the ranges of temperature profiling that are exceeding 15°C or more.

Get cell suspension samples in falcon tubes with typical optical densities of your experiments. If the maximal OD of your experiment (stationary phase) is 5 OD_600nm then the recommended samples are 0 (ddH₂O water or broth media) 1, 2, 3, 4, 5, 6 OD_600nm. Volume accuracy of the samples must be ±0.05.

Measure OD at desired wavelength of each cell suspension using a spectrophotometer with proper prior dilutions. The proportionality between OD and cell density exists only for OD ≤ 0.4 (approximately), we recommend diluting samples to the range of 0.1-0.2 OD.

Multiply the dilution factor values to get the OD of the samples.

Proceed to the software calibration module described in the software manual.