WO2008040365A1

WO2008040365A1 - Method and system for fed-batch cultivation of hydrogen-oxidizing bacteria

Info

Publication number: WO2008040365A1
Application number: PCT/EE2007/000022
Authority: WO
Inventors: Kalju Vanatalu; Kalle Tiismaa
Original assignee: Tallinn University Of Technology
Priority date: 2006-10-06
Filing date: 2007-10-08
Publication date: 2008-04-10
Also published as: EE200900029A

Abstract

The method describes the fed-batch cultivation of hydrogen-oxidizing bacteria in an open system where the effluent gas is discharged at atmospheric pressure. The economical use of CO2 is achieved by keeping the carbon dioxide concentration in the effluent gas at very low values. This is achieved by the following means: 1) decreasing the CO2 concentration in the feeding gas lower than evaluated by the stoichiometry of its consumption with respect to H2 and O2, 2) circulating the gas back to the cultivation media and 3) feedback regulation of the feeding rate of the fresh gas mixture to keep the CO2 concentration in the effluent at low level. The method is particularly useful in growing hydrogen oxidizing bacteria on isotopically 13C labeled carbon dioxide.

Description

METHOD AND SYSTEM FOR FED-BATCH CULTIVATION OF HYDROGEN- OXIDIZING BACTERIA

Background of the invention

Technical field

The invention describes a method and a system of fed-batch cultivation of hydrogen-oxidizing bacteria with economical use of carbon dioxide.

Background art

Hydrogen-oxidizing bacteria can grow chemolithoautotrophically on the mixture of gaseous hydrogen, oxygen and carbon dioxide in simple inorganic salt media. The bacteria get energy by oxidation H₂ gas with O₂ and carbon is fixed from CO₂. The best characterized species among the hydrogen-oxidizing bacteria is Ralstonia eutropha. R. eutropha and other hydrogen-oxidizing bacteria are of commercial interest because of several reasons. They can serve as organisms for fixing carbon dioxide from industrial wastes; the biomass can be used as animal food. It has been suggested that hydrogen oxidizing bacteria can be used in space- ships in closed environment to reutilize CO₂ and to use the H₂ which is a by-product of electrolysis when oxygen is produced from water. One of the major interests, however, is the ability of R. eutropha to accumulate poly-D-hydroxybutyric acid (P(3HB)) which is a raw material for biodegradable plastics.

One of the fields is to use hydrogen-oxidizing bacteria as an economical means of producing isotopically labeled biomass (Heumann, 2003). Isotopically labeled (for example, with ¹³C) carbon dioxide enables to produce labeled R. eutropha biomass much more cheaply during autotrophic growth than when growing the cells heterotrophically on isotopically labeled other carbon sources like ¹³C-glucose or ¹³C-acetate. The reason for this is that gaseous ¹³CO₂ is much cheaper than other ¹³C-labeled carbon sources. Also, carbon from CO₂ is exclusively switched into biomass and the P(3HB) while only 30-50% of carbon of a carbon source is switched into biomass during heterotrophic growth.

The cultivation of hydrogen-oxidizing bacteria differs from conventional fermentation systems mainly because of the need to apply gaseous compounds (Ishizaki et al, 2001). The peculiarities are connected with the low solubility of H₂ in water and the danger of explosion of the "Knallgas" (a mixture of H₂ and O₂). The chemolithoautotrophic growth can be carried out in batch, fed-batch or continuous cultivation. The gas supply can be carried out either in closed or open systems. In the closed (or dead-end) system the cultivation media and the gas- space are hermetically isolated from the atmosphere; usually, the system is under overpressure. In a simple closed system the growth very soon slows down because the deviation of gases' concentrations from the initially set values. Besides, if there emerges even a minor leakage in the system the whole gas mixture is lost into the surrounding. Nevertheless, good results with respect to growth rates and thorough utilization of the gases have been achieved in closed systems in conjunction with gas-recycling inside the system (Ishizaki and Tanaka, 1990; Ishizaki et al, 1975). The drawback is the need to use a gas mixing chamber about 300 times the volume of the liquid in the fermentor. In the open cultivation system the exhausted gas is discharged from the fermentation system. As the gases can be applied at high flow rate this supports good growth rate, however, the drawback is that the utilization of substrate gases is not economical as they are discharged at high concentration (Repaske, 1976).

For isotopic labeling of hydrogen-oxidizing bacterial biomass with isotopes of carbon like ¹³C it is necessary to apply a cultivation system where virtually all the CO₂ is used up by the cells. Another pre-requisite is to afford reasonable growth rate and presumably also high cell densities. Up to now only closed systems have been developed where attention has been drawn to economical use of substrate gases (Ishizaki and Tanaka, 1990). Such closed systems work under overpressure with respect to atmosphere and therefore need very careful specific measures to avoid leakage. Even minor leakage can lead to rapid discharge of the gas mixture and thus to loss of the very expensive isotopically labeled ¹³CO₂. Closed systems are therefore complex and expensive due to the need to make it resistant to leakage under constant overpressure.

Thus, there is a need for a new open system with controlled discharge of gases, where virtually all CO₂ is used up by the cells. Also, there is a need for a new method of fed-batch cultivation of hydrogen-oxidizing bacteria in an open system. Disclosure of the invention

One aspect of the invention is an open cultivation system where the gaseous space is connected with the surrounding atmosphere and at the same time avoid the discharge of CO₂ by regulating it's concentration to nearly zero.

Another aspect of the invention is a a method for fed-batch cultivation of hydrogen-oxidizing bacteria in an open system.

Yet another aspect of the invention is a software product comprising an algorithm for controlling the system and performing the method.

The idea of the cultivation method is as following. It is known that during autotrophic growth R. eutropha uses CO₂, O₂ and H₂ in ratio of about 1 :2:7. Thus in our system the percentage of CO₂ in the gas mixture fed to the fermentor is slightly reduced (from 10% to 5-7%) with the aim to achieve CO₂ limiting conditions. The relative loss of carbon dioxide to the CO₂ input equals the ratio of effluent to inflow of gases multiplied by the ratio of CO₂ concentrations in effluent to the inflow gas mixture. As a result, cultivation conditions can be achieved such that the CO₂ concentration in the gaseous space above the fermentation liquid is close to zero and the rest of the remaining gases contain only O₂ and H₂ and can be continuously discharged from the system. The amount and content ratio of the H₂ and O₂ remaining depends then on the ratio of the three different gases in the feeding mixture. For better dissolution of the substrate gases the gas mixture above the fermentation liquid is directed back to the fermentation medium by vigorous circulation using a diaphragm pump. The CO₂ concentration in the gas mixture above the fermentation medium is evaluated by a simple infrared (IR) analyzer and the flow rates of the effluent as well as of the inflow gas estimated by flowmeters. The growth algorithm regulates the inflow of the gas mixture in such a way that the CO₂ concentration measured by the IR analyzer is kept at very low values (preferably below 0.1%). When the CO₂ concentration falls below the setpoint value (usually about 0.05%), the inflow rate is increased. Thus the culture is automatically fed by the fresh gas mixture in a manner where the gas feeding rate is balanced to the CO₂ use by the cells. The method enables fed-batch growth of hydrogen-oxidizing bacteria to high cell densities with economic use of CO₂. The features and advantages described herein are not all-inclusive and, in particular, many additional features and advantages will be apparent to one of ordinary skill in the art in view of the drawings, specification, and claims. Moreover, it should be noted that the language used in the specification has been principally selected for readability and instructional purposed, and not to limit the scope of the inventive subject matter.

Brief description of the drawings

The technical essence of the invention is described in details by following figure Fig. 1 that depicts a scheme of the cultivation set-up according to one embodiment of the invention.

Modes for carrying out the invention

Fig.l depicts a scheme of the cultivation set-up according to one embodiment of the invention, where arrows with dotted line show flow of control and measurement signals and arrows with continuous line show gas flow. 1, 2, 3 are, respectively, gas bottles for CO₂, O₂, H₂, 4 - feeding gas mixing reservoir; 5, 6, 7,8 -electromagnetic valves; 9- electronic manometer; 10 - controller of electromagnetic valves, 11- gas pressure reducer; 12- flowmeter of gas feed; 13 -flow control valve; 14- fermentor vessel; 15- perforated gas inlet tube; 16- impeller; 17- cooled moisture condensor; 18- infrared (IR) gas analyzer; 19- low speed diaphragm pump; 20- diaphragm pump for gas circulation; 21- flowmeter of effluent gas; 22- personal computer; 23- open atmosphere.

For the cultivation of R. eutropha in fermentor first the inoculum culture is prepared in a shaker flask. The inoculum can be grown up in heterotrophic conditions using fructose as the carbon source. Cell suspension from the stock culture in glycerol kept at -2O⁰C is streaked on a Petri dish with Luria-Bertani (LB) medium to obtain single colonies. The single colony is transferred to a shaker flask containing minimal medium M9 supplemented with 4g/l of fructose and cultivated at 3O⁰C overnight until the optical density at 600nm (OD) rises above 1-2.

The fermentation system for autotrophic fed-batch cultivation of Fig. 1 is operated as follows. The feeding gases CO₂, O₂ and H₂ are kept under high pressure (at least above 8-10 bar) in separate gas bottles (respectively 1, 2 and 3 in fig.l). The gas bottles have electromagnetic valves 5, 6 and 7 that can be closed and opened independently from each other by the controller 10. When the pressure in the mixing reservoir 4 falls below threshold value the controller sequentially opens and closes the valves of CO₂, O₂ and H₂ bottles and refills the mixing reservoir. The partial pressures of each component in the mixture can be regulated by pregiving the setpoint pressure values where the controller opens and closes the addition of each gas component. For example, CO₂ is added at 0.72 to 1.03 bar, O₂ is added at 1.03 to 2.2 bar and H₂ is added at 2.3 to 6.02 bar. The volume of the mixing reservoir may be around 2 liters. The gas mixture is passed through a reducer 11 (reduces the pressure to about 0.5 bar) to a gas-flow meter 12 and flow-controller 13, controlled according to an algorithm, run on a computer 22. Thus the flow rate of gas mixture into the fermentor could be measured and controlled.

The fermentor vessel 14 can be made of stainless steal. The gas mixture can be introduced through stainless steel perforated rounded tube 15 under the impeller 16 and the effluent gas is passed out through a cooled condensor 17 on the top of the vessel. After passing through the condensor the gas flow is divided into three paths: 1) circulation path, driven by a diaphragm pump 19 (e.g., with output between 10 and 50ml/min) back to the fermentor through the IR gas analyzer 18, 2) vigorous circulation path, driven by a high-speed diaphragm pump 20 (e.g., with output between 10 to 30 1/min) back to the fermentor with the aim to totally redissolve the remaining CO₂ in the growth medium and 3) free flow path (discharge) of the gas into the open atmosphere 23 outside the building.

Another embodiment of the invention is a method for controlling a concentration of CO₂ in a system as described in Fig. 1. The method comprises the following stages:

introducing a mixture of gases into said growth medium in said fermentor vessel, said mixture comprising CO2, 02 and H2 in a predetermined ratio, wherein the concentration of CO2 in said mixture is lower that its consumption rate relative to H2 and 02;

monitoring the CO2 concentration in said gas space and keeping said concentration below setpoint value by changing an inflow rate of said mixture into said growth medium;

recirculating at least a first portion of effluent gas from said gas space back into said growth medium; and

discharging at least a second portion of effluent gas from said gas space into the atmosphere. The concentration of CO₂ in the mixture is less than 10 per cent, preferably between 5 to 7 per cent. Isotopically labeled ¹³CO₂ and ¹³C-depleted ¹²CO₂ can be used.

Another embodiment of the invention is a computer program comprising an algorithm for controlling the system as shown in Fig. 1. The CO₂ concentration in the gas mixture above the fermentation medium is evaluated by a simple infrared (IR) analyzer and the flow rates of the effluent as well as of the inflow gas estimated by flowmeters. The growth algorithm regulates the inflow of the gas mixture in such a way that the CO₂ concentration measured by the IR analyzer is kept at very low values (preferably below 0.1%). When the CO₂ concentration falls below the setpoint value (usually about 0.05%), the inflow rate is increased. Thus the culture is automatically fed by the fresh gas mixture in a manner where the gas feeding rate is balanced to the CO₂ use by the cells. The method enables fed-batch growth of hydrogen- oxidizing bacteria to high cell densities with economic use of CO₂.

EXAMPLE

A system as described above was set up. The total volume of the fermentor vessel was 7 liters with working volume 2 to 4 liters. It was equipped with temperature and agitation control and with pH and pθ₂ electrodes. The system was put under a hood to ensure that the gases could not leak into the ambient atmosphere.

According to the example, the fermentor was autoclaved at 121 ⁰C for 20 min. The fermentor was filled with 4 liters of autoclaved media containing KH₂PO₄ 3 g/1, Na₂HPO₄* 12H₂O 15 g/1 and NH₄Cl 4 g/1. To this solution were added 1) 2 ml of 1 M MgSO₄*7H₂O, 2) 2 ml of 0.1 M CaCl₂*2H₂O, 3) 2 ml of 0.05 % (w/v) Fe(III)NH₄ citrate and 4) 2 ml of trace element solution of the following composition (g/1): H₃BO₃ 0.3, CoCl₂*6H₂O 0.2, CuCl₂*2H₂O 0.01, MnCl₂*4H₂O 0.03, Na₂MoO₄*2H₂O 0.03, NiCl₂*6H₂O 0.02, ZnSO₄*7H₂O 0.148.

The pH was automatically kept above 6.8 by titration with 2N NaOH. Fermentation temperature was 32⁰C.

The fermentation in 4 liter of media was started by inoculation 20 ml of heterotrophically grown R. eutropha (a mutant incapable- of accumulating P(3HB)). After the inoculation the fermentation was started by adding the gas mixture of composition 5.7% (v/v) CO₂, 22.1% (v/v) of O₂ and 72.3% (v/v) of H₂ and used throughout the cultivation. In the initiation of the growth a computer controlled peristaltic pump was used instead of the flow controller to achieve more precise addition of small amounts of the feeding gas. The gas was added automatically by a peristaltic pump at speed 20 ml/min. until the CO₂ concentration rose to the setpoint value (usually 0.05-0.1%). Then the algorithm added by the pump a certain amount of gas mixture (usually about 50 ml) every time the CO₂ concentration measured by the IR gas analyzer fell below the setpoint value. After 4Oh from the start when OD had reached 1.0 the gas mixture was started to add by the control valve instead of the peristaltic pump. The algorithm controlled the inflow rate of the feeding gas. The gas inflow was changed proportionally to the deviation of the measured CO₂ concentration from the setpoint value: if the CO₂ concentration exceeded the setpoint value then the feeding was decreased and vice versa. Every time the feeding was regulated to a new value there was a lag period (usually 3 minutes) before a new regulation was executed. Such algorithm is applied to the end of fed-batch cultivation. Along with the increase of cell density other nutrients are added batchwise to the fermentation medium as following. Per increase of OD of 10 units should be added per liter of fermentation medium: 3 g ammonium chloride, 3 ml of 1 M magnesium sulfate, 3 ml of 0.1 M calcium chloride, 3 ml of 0.05% (w/v) solution of Fe(III)NH₄-citrate and 3ml of the trace element solution described above.

The OD reached 6 by the 70th h and 53 by the 115th h of cultivation. By measuring the inflow and effluent flow rates of the gases and the CO₂ content in the effluent it was evaluated that 99-99.5% of the carbon dioxide was used by the cells. On the 115th h the most of the cultivaton medium was harvested and fermentor was filled with a fresh medium and the fed- batch cultivation was repeated. Then the third harvest and repetition of fed-batch was started on the 160th h.

Although this invention is described with respect to a set of aspects and embodiments, modifications thereto will be apparent to those skilled in the art. The foregoing description of the embodiments of the invention has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Many modifications and variations are possible in light of this disclosure. It is intended that the scope of the invention be limited not by this detailed description, but rather by the claims appended hereto.

Claims

1. A method for controlling a concentration of CO₂ for cultivation of hydrogen oxidizing bacteria in an open system, comprising a fermentor vessel, comprising a growth medium and a gas space above said growth medium, said gas space connected with the atmosphere, said method comprising the stages of:

introducing a mixture of gases into said growth medium in said fermentor vessel, said mixture comprising CO₂, O₂ and H₂ in a predetermined ratio, wherein the concentration of CO₂ in said mixture is lower that its consumption rate relative to H₂ and O₂;

monitoring the CO₂ concentration in said gas space and keeping said concentration below setpoint value by changing an inflow rate of said mixture into said growth medium;

discharging at least a second portion of effluent gas from said gas space into the atmosphere.

2. A method as in claim 1, wherein the concentration of CO₂ in said mixture is less than 10 per cent.

3. A method as in claim 1, wherein the concentration of CO₂ in said mixture is between 5 to 7 per cent.

4. A method as in claims 1 to 3, wherein said method is used for cultivating hydrogen- oxidizing bacteria on isotopically labeled ¹³CO₂.

5. A method as in claim 1 to 3, wherein said method is used for cultivating hydrogen oxidizing bacteria on ¹³C-depleted ¹²CO₂.

6. A method as in claims 1 to 4, using IR analyzer for monitoring the CO₂ concentration.

7. An open system for fed-batch cultivation of hydrogen oxidizing bacteria, the system comprising: a fermentor vessel, comprising a growth medium and a gas space above said growth medium, said gas space connected with the atmosphere; a source of gas mixture, said gas mixture comprising CO₂, O₂ and H₂ in a predetermined ratio, wherein the concentration of CO₂ in said mixture is lower than its consumption rate relative to H₂ and O₂;

a gas inflow tube for supplying said gas mixture from said source of gas mixture into said growth medium;

a gas flow control device on said gas inflow tube;

a subsystem for removing the gas from said gas space, said subsystem comprising a slow circulation path for gas mixture back to the fermentor vessel, said slow circulation path comprising a gas analyzer for determining the concentration of CO₂ in said gas mixture, a vigorous circulation path for pumping said gas mixture back into said growth medium in said fermentor vessel, said vigorous circulation path comprising a high speed pump, and a free flow path for discharging effluent gas into the atmosphere, said free flow path comprising an effluent gas flowmeter; and a processor device for controlling said gas flow control device in response to data from said gas analyzer and said effluent gas flowmeter.

8. A system as in claim 7, wherein said gas flow control device comprises a flow control valve and a flowmeter of gas feed.

9. A system as in claims 7 to 8, wherein said high speed pump is a diaphragm pump.

10. A system as in claim 9, wherein an output of said high speed pump is from about 1 to about 8 liters per minute per liter of liquid in the fermentor.

11. A system as in claims 7 to 10, wherein said slow circulating path comprises a low speed diaphragm pump.

12. A system as in claims 11, wherein an output of said low speed diaphragm pump is from about 1 milliliter per minute to about 15 milliliter per minute per liter of liquid in the fermentor.

13. A system as in claims 7 to 12, wherein said processor is a standard PC.

14. A system as in claims 7 to 13, wherein said gas inflow is changed proportionally in response to readings of said gas analyzer, and where the concentration of CO₂ is kept between from about 0.05 to 0.5 percent.

15. A system as in claims 7 to 14, wherein said gas analyzer is an IR gaz analyzer.

16. A system as in claim 7 to 15, said source of gas mixture comprising a first gas bottle for a first gas, a second gas bottle for a second gas, and a third gas bottle for a third gas, each said bottle comprising a valve, said first, said second and said third bottle connected with a mixing reservoir, said mixing reservoir comprising a manometer, and a controller for controlling said valves in response to a reading from said manometer, the composition of the gas mixture in said mixing reservoir is controlled by supplying gas from said first, said second and said third gas bottles sequentially so that the gas from said first gas bottle is supplied until the pressure in said mixing reservoir reaches a first predetermined value, then the second gas from said second gas bottle is supplied until the pressure in said mixing reservoir reaches a second predetermined value and then the third gas from said third gas bottle is supplied until the pressure in said mixing reservoir reaches a third predetermined value.

17. A system as in claim 16, wherein the first predetermined value is from about 0.72 to about 1.03 bar, the second predetermined value is from about 1.03 to 2.2 bar and the third predetermined value is from about 2.3 to 6.02 bar.

18. A software product to be downloaded into and executed in a computer for controlling a system of claims 7 to 17.