IE42634B1 - Improvements in and relating to the continuous culture of micro-organisms - Google Patents

Abstract

1528011 Continuous cultivation of bacteria &c on methanol &c SHELL INTERNATIONALE RESEARCH MAATSCHAPPIJ BV 9 Feb 1976 [11 Feb 1975] 5747/75 Heading C6F In a process for the production of microorganisms involving continuously cultivating one or more hydrocarbon or oxygenated hydrocarbon utilizing micro-organisms under aerobic conditions in a liquid growth medium comprising assimilable sources of nitrogen and essential mineral salts in the presence of a hydrocarbon or oxygenated hydrocarbon, withdrawing from the growing culture, liquid growth medium containing micron organisms, separating micro-organisms from the withdrawn liquid growth medium and recycling at least part of the withdrawn liquid growth medium to the growing culture, the withdrawn liquid growth medium is chilled to a temperature in the range of -5‹ to +20‹C prior to, or during, the separation of the micro-organisms. It is stated that any buildup of growth-inhibiting material during the separation stage is avoided and contamination of the separation and recycle lines by undesirable anaerobic micro-organisms is discouraged as a result of the chilling. The hydrocarbon or oxygenated hydrocarbon may for example be natural gas, methane or methanol. The Examples relate to the continuous cultivation of a mixed culture of microorganisms comprising a methanol-utilizing bacterium and four non-methanol utilizing bacteria in methanolic culture medium, with recycle of the culture medium and recovery of the micro-organisms produced. The cultivation temperature was 40‹ or 42‹C and the chilling temperature 0‹C. Growth inhibition was encountered when recycling took place without the chilling according to the invention.

Description

This invention relates to a process for the production of micro-organisms. Many micro-organisms are known which can utilise hydrocarbons or certain derivatives thereof as their carbon and/or energy source. The dried biomass obtainable by the cultivation of such micro-organisms, often referred to as single cell protein, is rich in protein and can be used as a possible food.-Btuff or food supplement for man and animals.

A process for the cultivation of hydrocarbon - utilising 10 micro-organisms usually comprises the growth of such organisms under aerobic conditions in ah aqueous growth medium comprising, in addition to the carbon Bource, various nutrients such as assimilable sources of nitrogen and essential mineral salts. Such a process may be carried out batch-wise, semi-continuously or preferably, in continuous flow culture.

For. continuous flow culture the micro-organiBms may be grown in any suitable adapted fermentation vessel (fermenter). The micro-organism cells may be harvested from the growth medium by any of the standard techniques commonly used. In a continuous flow culture liquid growth medium ie regularly withdrawn from the fermenter, the cells are separated from fche liquid medium, and the liquid medium may subsequently br. wholly or partially recycLed to the fontHjnter. The nutrients which after separation of the cell mass are still present iri the liquid growth medium are thus recycled and, together with the liquid, re-employed in the cultivation stage.

Fresh nutrients may, of course, be added to the recycled - 2 42634 medium or, alternatively, may be added separately to the fermenter.

The invention provides a process for the production of micro-organisms which comprises continuously cultivating one or more hydrocarbon - utilising micro-organisms (as hereinafter defined), optionally together with one or more non-hydrocarbon utilising micro-organisms, which is/are capable of metabolising organic substances produced by the hydrocarbon - utilising microorganism^), under aerobic conditions in a liquid growth medium JO comprising assimilable sources of nitrogen and essential mineral salts in the presence of a hydrocarbon (as oxygenated derivative thereof), withdrawing from the growing culture liquid growth medium containing micro-organisms, separating micro-organisms from the withdrawn liquid growth medium and recycling at least part of the withdrawn liquid growth medium to the growing culture, in which process the withdrawn liquid growth medium is chilled to a temperature in the range of -5 to 20°C prior to, or during, the separation of the micro-organisms.

The term hydrocarbon - utilising micro-organism as used herein is meant to include not only micro-organisms capable of using hydrocarbons, but also micro-organisms capable of using oxygenated derivatives of hydrocarbons, and non-hydrocarbon utilising micro-organisms shall be construed accordingly.

The term micro-organism is used herein in a broa1 sense to include not only bacteria, but also yeasts, fi Lamentous fungi, actinomycetes and protozoa.

The main advantage of the process of the invention is that, generally, build-up of growth-inhibiting material during the separation stage is avoided: without chilling the withdrawn liquid growth medium, materials might he formed in this medium which, when returned to the growing culture, will inhibit the growth of the micro organisms in the culture and eventually might wash-out the entire culture. An additional advantage of the process of the invention is that chilling of the withdrawn liquid growth medium discourages the contamination of the separation and recycle lines hy undesired anaerobic organism.

The process of the invention is particularly useful in processes including the cultivation of hydrocarbon - utilising bacteria, optionally in the presence of non-hydrocarbon utilising bacteria, for example processes producing microbial protein from methanol, methane or natural gas.

Suoh processes are described in, for example. Patent Specification No, 390Ϊ5 · and British Patent Specification No.1,467,022. In these processes the nutrients can be supplied in concentrated aqueous streams which enable the recycle of up to $0% of the water. The oarbon source may be supplied as a pure liquid, a concentrated solution in water or as a gas.

The withdrawn liquid growth medium is preferably chilled to a temperature in tlie range of -5 to 15°0, and more preferably in the range of -5 to 10°C.

The liquid growth medium comprises a nitrogen-containing & 48634 compound which may for example be ammonia, urea, an ammonium salt, for example ammonium sulphate or ammonium chloride, or a nitrate, for example an alkali metal nitrate. Nitrogen ia suitably present in a concentration from 5 to g/l.

Other elements which may be present in the medium include phosphorus, sulphur, magnesium and iron. Phosphorus may be present in the form of one or more phosphates, for example K^HPO^, KH^PO^, NagHPO^, or (ΝΗ^)^ΗΡΟ^, or phosphoric acid, and preferably is present in a concentration from to 20 g/l. The sulphur source may be sulphuric acid or a sulphate, for example S0^, and suitably is present in a concentration from 0.5 to 10.0 g sulphur/l. The two metals are suitably provided as one or other of their salts, for example MgSO^.THgO in a concentration from 0.2 to 20 g/l and FeCly6HgO in a concentration from 0.01 to 5 g/l.

The medium may also contain trace amounts of other elements In the form of suitable salts, for example calcium, manganese, zinc, cobalt, molybdenum and boron.

The process of the invention is carried out in continuous flow culture, for which the micro-organisms may be grown in any suitably adapted fermentation vessel, for example a stirred baffled fermenter or sparged tower fermenter, which is provided either with internal cooling or an external recycle cooling loop and with adequate recycle lines. If methane or natural gas is used as the carbon source, a gas mixture containing methane and oxygen and possibly carbon dioxide or other gases is contacted with the medium preferably by - 5 4 bubbling continuously through a sparger at the base of the vessel. The source of oxygen for the culture may be air, oxygen or oxygen enriched air. Spent gas is removed from the head of the vessel. Spent gas may be recycled either through an external loop or internally by means of a gas inducer impeller. The gas flows and recycle should be arranged to give maximum growth of organism and maximum utilisation of methane.

The temperature of the culture is generally maintained 10 between 30 to 60°C and preferably between 40 and 50°C. The pH of the culture is controlled at a pH between 6.0 and 8.0 and preferably between 6.4 and 7.4 by the appropriate addition of an alkali, for example NaOH, KOH, NHjOH, and/or an acid, for example H^SO^ or H^PO^.

The micro-organism cells may be harvested from the liquid growth medium by any of the standard techniques commonly used, for example flocculation, sedimentation, and/or precipitation, followed by centrifugation and/or filtration. The biomass is then dried e.g. by freeze, flaeh and/or spray drying and may be used in this form as a protein food stuff or food supplement for man or animals. The invention ie illustrated further in the following examples.

Example X A. (comparison). A mixed culture of micro-organisms (comprising a methanol -utilising bacteria and four non-methanolutilising bacteria) designated JMS 72 and fully described in Patent Specification No. 39015 was grown in a - 6 42634 continuous fermenter (volumes 2.6 litres) under sterile conditions using the following defined medium:Concentrated nutrient stream:- CH^OH 192.0 g/l (lffl4)2S04 56.0 „ H5P04(90?0 7.4 „ MgSO4.7H2O 2.4 „ PeS04.7H20 0.3. „ + trace elements The pH was controlled hy addition of an equimolar mixture of NaOH and KOH. Prior to the initiation of recycle this concentrated stream was fed to the fermenter mixed with water. The steady state conditions were as follows:- Temperature 42°C PH 6.9 flow of water 480 ml/h Plow of nutrients 96 ml/h Dilution rate 0.22 h-1 Cell density 10.3 e/i Gas supply air/oxygen The effluent from the fermenter (i.e. liquid growth medium plus oells) was fed to a sterile centrifuge (Sharpies: (Trade Mark) Type 1AP) operating at a speed of 30,000 r.p.m. from which it was discharged as a clear aqueous phase. When steady state conditions had teen achieved in the fermenter the water stream was substituted hy the aqueous phase from the centrifuge and fed at the same rate. The recycle ratio H (i.e. flow of recycle water: flow of nutrients) was - 7 14 400 : 96 = 5·0· After 90 minutes inhibition was apparent by build up of methanol in the fermenter and a decrease in the respiration rate. After 3 hours respiration had stopped and the culture began to washout.

B. The experiment of Example IA was repeated With the difference that the effluent and the centrifuge were chilled to 0°C. Recycle was maintained at R = 5 for 12 hours when the bowl of the centrifuge had reached its capacity and the effluent leaving the centrifuge was no longer clear. During this time there was no discernible build up of methanol or change in the respiration rate. Example II The mixed culture JMS 72 was grown in a continuous fermenter (volume = 2.1 l) under the following steady state conditions:- Temperature 40.0 c° pH 6.0 Cell density 10.6 g/l Gas supply air/oxygen rates :- Methanol (625 g/l) 20.34 ml/h Ammonia solution (6.95% w/v) 13.00 ml/h Concentrated salts solution 15.14 ml/h Total nutrient flow rates 48.56 ml/h Water flow rate 450.no ml/h Total liquid flow rate 498-56 ml/h Dilution rate 0.237 h1 Concentrated salts composition (in mM):HjPO4 - 400 ; MgSO4 - 60 ; - 80 5 Na2S04 - 80 j FeSO. - 8 1 CaCl_ - 6 ; ZnSO. - 2 ; CuSO. - 0.5 1 4 d 4 4 κι - 0.5 ; coci„ - 0.5 ; h,bo, - 0.6 ; Na.MoO, - 0.5 ; 3 5 2 4 The effluent from the fermenter was chilled to 0°C and supplied to a sterile centrifuge chilled to 8°C. The clear aqueous phase from the centrifuge was reoycled to the fermenter at 45° ffll/h in place of the water flow giving a recycle ratio of 450/48.56 = 9·27. These conditions were maintained for hours with no Aiscernable change in the respiration rate or cell density.

Claims (6)

1. A process for the production of micro-organisms which comprises continuously cultivating one or more hydrocarbon utilising micro-organisms (as hereinbefore defined) under aerobic conditions in a liquid growth medium comprising assimilable 5 sources of nitrogen and essential mineral salts in the presence of a hydrocarbon, (or oxygenated derivative thereof), withdrawing from the growing culture liquid growth medium containing microorganisms, separating micro-organisms from the withdrawn liquid growth medium and recycling at least part of the withdrawn liquid 10 growth medium to the growing culture in which process the withdrawn liquid growth medium is chilled to a temperature in the range of -5 to 20 e C prior to, or during, the separation of the micro-organisms.

2. A process as claimed in claim 1, wherein the hydrocarbon 15 utilising micro-organism(s) is(are) cultivated in the presence of one or more non-hydrocarbon - utilising micro-organisms which is/are capable of metabolising organic substances produced by the hydrocarbon -utilising micro-organism(s). 5. A process as claimed in claim 1 or 2, wherein 20 cultivation of the micro-organisms is carried out at a temperature in the range of 30 to 60°C.

3. 4. A process as claimed in any one of claims 1-3, wherein the withdrawn liquid growth medium is chilled to a temperature in the range of -

4.

5 to 10°C. >5 5· A process as olaimed in claim 1, substantially as described in Examples IB and II herein. 10 42634

6. Micro-organisms when produced by a process as claimed in any one of claims 1-5·