GB1563392A - Production of single cell protein by continuous fermentation - Google Patents

Description

(54) IMPROVEMENTS IN OR RELATING TO THE PRODUCTION OF SINGLE CELL PROTEIN BY CONTINUOUS FERMENTATION (71) We, IMPERIAL CHEMICAL INDUSTRIES LIMITED, Imperial Chemical House, Millbank, London, SWIP 3JF, a British Company, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to improvements in or relating to the production of single cell protein by continuous fermentation.

In a continuous fermentation process for the production of single cell protein it is important that the rate of production of dried cells after harvesting and drying should be maintained at a high level in order to avoid excessive liquid handling and recycling costs. However the total productivity of a process for the producing single cell protein is limited by the oxygen transfer capacity of the system in which the process is operated. Thus in operating such a process upon a commercial scale the system must have an oxygen transfer capacity sufficiently great to avoid excessive liquid handling and recycling costs.

According to the present invention we provide a fermentation process for continuously culturing micro-organisms upon a medium comprising a source of assimilable carbon and inorganic nutrients which comprises the steps of continuously withdrawing a proportion of the culture from the main body thereof, treating the withdrawn culture to kill micro-organisms present therein and returning the treated culture to the main body of the culture, the proportion of the micro-organisms killed being such that, when the fermentation is proceeding in its steady state, 20 ', to 700,',, of the micro-organisms present in the main body of the culture are dead.

Preferably, the fermentation process is a process for the production of single cell protein, particularly by culturing yeasts or bacteria upon media containing hydrocarbons or alcohols such as methanol as sources of assimilable carbon. The present invention is particularly suitable for carrying out the process of our UK Specification No. 1,370,892 in which bacteria belonging to the species Pseudomonas methylotropha, Hvphomicrobium variabile, Microcyclus polvmorphum or Pseudomonas rosea are cultured. The characteristics of these species are described in our UK Specification No. 1,370,892. Preferred micro-organisms for use in the process of this specification are Pseudomonas methylotropha strains, particularly those strains deposited at the following culture collections: a. National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, UK (NCIB) b. US Department of Agriculture, Peoria, Illinois, USA (NRRL) c. Fermentation Research Institute, Japan (FERM) and given the following culture collection numbers: a. NCIB 10508-15 and 10592-6 b. NRRL B 5352-64 c. FERM 1215-27.

The proportion of micro-organisms killed is such that, when the fermentation is proceeding in its steady state, 200/, to 70?t, particularly about 50 X", of the microorganisms present in the culture are dead.

Any known means may be used for killing the micro-organisms. Suitable means include a high temperature shock at for example 5O650C, subjection to yradiation, a pH shock at for example pH < 4.5 or > 8.0 or treatment with formaldehyde. Treatment with formaldehyde is preferred since this reagent is an efficient sterilant at low temperatures of both vegetative cells and spores and can be metabolised by many micro-organisms and can thus be kept at a limiting concentration. e.g. 1 to 2 ppm, in a carbonlimited system. For example formaldehyde can be co-metabolised. i.e. it can be metabolised in the presence of another carbon source, by strains of Pseudomonas methllotropha which convert methanol via formaldehyde to formic acid and then to carbon dioxide. Preferably cells which are treated are killed absolutely and 'mained' cells are either not produced or are produced in minimal proportions. When formaldehyde is used it is preferably added to the medium entering the fermentation process in proportions between 0.1 ,', and 5.0", by volume (levels of 0.5 /" to 1.0% will usually be sufficient to kill a satisfactory proportion of the micro-organisms).

The invention is illustrated by the Example which is described with the aid of the accompanying drawing.

EXAMPLE The drawing shows in diagrammatic form a process operated in accordance with the present invention. The fermentation is performed in a fermenter 1 of our UK Specification Nos. 1,353,008; 1,417,486 or 1,417,487 and has a riser 2 and downcomer 3. In the fermenter which has a volume of 1000 litres the circulation rate of the culture is about 30 M3/hr, the dilution rate D is 0.1 hrs~l and the culture contains 30 gms/l of live cells plus 30 gms/l of dead cells. The micro-organisms belong to a strain of Pseudomonas methylotropha and the carbon source is methanol.

From the fermenter culture containing 6?,, solids is removed along line 4 to be harvested at 6. Harvesting may be performed by the method of our UK Specification No. 1,381,306. From harvesting stage 6 30 litres/hr of liquid containing 200 litre of cells passes along line 7 to a drier (not shown in the drawing) whilst 70 litres/hr of recycle medium containing no cells passes along line 8. At point 9 on line 8 there is added to the recycle medium fresh medium containing methanol and inorganic nutrients together with formaldehyde. The fresh medium is added in proportions such that the flow of recycle and fresh medium along line 5 is at the rate of 100 litres/hr and the combined media contain 1 " formaldehyde and 1 1% methanol by volume.

From the fermenter there is a culture recycle along line 11 whereby culture at the rate of 100 litres/hr is fed into recycle line 5.

Thus the concentration of formaldehyde in part 10 of the recycle line is half its concentration in line 5, namely 0.50,:,.

In this system equal volumes of medium and fermenter culture are mixed at a point where the medium is already sterile. During passage along line 10 the formaldehyde kills the micro-organisms. When the fermenter is being operated at a steady state this leads to 50"" of the micro-organisms present in the culture in the fermenter being dead. So long as the formaldehyde is fully metabolised by the micro-organisms none will remain in the culture medium and the product will be the same as that produced in the absence of the formaldehyde.

A 60 zmsil feed of cells into line 4 gives a maximum recycle of 70 n (cf 85"" on a 30 gm/l feed). This is half the recycle achieved in a conventional system and leads to a much reduced chance of a build up of inorganic nutrients in the culture medium.

We believe that such a system will produce the following advantages:- 1. No necessity for heat sterilization once culturing is continuous.

2. Reduced heat exchanger fouling problems.

3. Liquid volume flows halved.

4. Reduced /n recycle.

5. The same dissolved oxygen tension (DOT) profile as in a conventional system.

6. Marginally reduced ash content of final product.

WHAT WE CLAIM IS:- 1. A fermentation process for continuously culturing micro-organisms upon a medium comprising a source of assimilable carbon and inorganic nutrients which comprises the steps of continuously withdrawing a proportion of the culture from the main body thereof, treating the withdrawn culture to kill micro-organisms present therein and returning the treated culture to the main body of the culture, the proportion of the micro-organisms killed being such that, when the fermentation is proceeding in its steady state, 20% to 70% of the micro-organisms present in the main body of the culture are dead.

2. A process according to Claim 1, wherein the carbon source is a hydrocarbon or an alcohol.

3. A process according to Claim 2, wherein the carbon source is methanol.

4. A process according to Claim 3, wherein the micro-organisms are bacteria belonging to the species Pseudomonas methvlotuopha.

5. A process according to Claim 4, wherein the bacteria belong to one of the strains NClB Nos. 10508-15 and 10592-6.

6. A process according to any of the preceding claims, wherein the microorganisms which are killed are killed by treatment with formaldehyde.

7. A process according to Claim 6, wherein formaldehyde is added to the medium entering the fermentation process in proportions between 0.1?,,,, and 5.0 , by volume.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (8)

**WARNING** start of CLMS field may overlap end of DESC **. metabolised in the presence of another carbon source, by strains of Pseudomonas methllotropha which convert methanol via formaldehyde to formic acid and then to carbon dioxide. Preferably cells which are treated are killed absolutely and 'mained' cells are either not produced or are produced in minimal proportions. When formaldehyde is used it is preferably added to the medium entering the fermentation process in proportions between 0.1 ,', and 5.0", by volume (levels of 0.5 /" to 1.0% will usually be sufficient to kill a satisfactory proportion of the micro-organisms). The invention is illustrated by the Example which is described with the aid of the accompanying drawing. EXAMPLE The drawing shows in diagrammatic form a process operated in accordance with the present invention. The fermentation is performed in a fermenter 1 of our UK Specification Nos. 1,353,008; 1,417,486 or 1,417,487 and has a riser 2 and downcomer 3. In the fermenter which has a volume of 1000 litres the circulation rate of the culture is about 30 M3/hr, the dilution rate D is 0.1 hrs~l and the culture contains 30 gms/l of live cells plus 30 gms/l of dead cells. The micro-organisms belong to a strain of Pseudomonas methylotropha and the carbon source is methanol. From the fermenter culture containing 6?,, solids is removed along line 4 to be harvested at 6. Harvesting may be performed by the method of our UK Specification No. 1,381,306. From harvesting stage 6 30 litres/hr of liquid containing 200 litre of cells passes along line 7 to a drier (not shown in the drawing) whilst 70 litres/hr of recycle medium containing no cells passes along line 8. At point 9 on line 8 there is added to the recycle medium fresh medium containing methanol and inorganic nutrients together with formaldehyde. The fresh medium is added in proportions such that the flow of recycle and fresh medium along line 5 is at the rate of 100 litres/hr and the combined media contain 1 " formaldehyde and 1 1% methanol by volume. From the fermenter there is a culture recycle along line 11 whereby culture at the rate of 100 litres/hr is fed into recycle line 5. Thus the concentration of formaldehyde in part 10 of the recycle line is half its concentration in line 5, namely 0.50,:,. In this system equal volumes of medium and fermenter culture are mixed at a point where the medium is already sterile. During passage along line 10 the formaldehyde kills the micro-organisms. When the fermenter is being operated at a steady state this leads to 50"" of the micro-organisms present in the culture in the fermenter being dead. So long as the formaldehyde is fully metabolised by the micro-organisms none will remain in the culture medium and the product will be the same as that produced in the absence of the formaldehyde. A 60 zmsil feed of cells into line 4 gives a maximum recycle of 70 n (cf 85"" on a 30 gm/l feed). This is half the recycle achieved in a conventional system and leads to a much reduced chance of a build up of inorganic nutrients in the culture medium. We believe that such a system will produce the following advantages:- 1. No necessity for heat sterilization once culturing is continuous. 2. Reduced heat exchanger fouling problems. 3. Liquid volume flows halved. 4. Reduced /n recycle. 5. The same dissolved oxygen tension (DOT) profile as in a conventional system. 6. Marginally reduced ash content of final product. WHAT WE CLAIM IS:-

1. A fermentation process for continuously culturing micro-organisms upon a medium comprising a source of assimilable carbon and inorganic nutrients which comprises the steps of continuously withdrawing a proportion of the culture from the main body thereof, treating the withdrawn culture to kill micro-organisms present therein and returning the treated culture to the main body of the culture, the proportion of the micro-organisms killed being such that, when the fermentation is proceeding in its steady state, 20% to 70% of the micro-organisms present in the main body of the culture are dead.

2. A process according to Claim 1, wherein the carbon source is a hydrocarbon or an alcohol.

3. A process according to Claim 2, wherein the carbon source is methanol.

4. A process according to Claim 3, wherein the micro-organisms are bacteria belonging to the species Pseudomonas methvlotuopha.

5. A process according to Claim 4, wherein the bacteria belong to one of the strains NClB Nos. 10508-15 and 10592-6.

6. A process according to any of the preceding claims, wherein the microorganisms which are killed are killed by treatment with formaldehyde.

7. A process according to Claim 6, wherein formaldehyde is added to the medium entering the fermentation process in proportions between 0.1?,,,, and 5.0 , by volume.

8. A process for continuously culturing

micro-organisms substantially as described and as shown in the Example.