CA1050457A - Microbiological process - Google Patents

Microbiological process

Info

Publication number
CA1050457A
CA1050457A CA228,704A CA228704A CA1050457A CA 1050457 A CA1050457 A CA 1050457A CA 228704 A CA228704 A CA 228704A CA 1050457 A CA1050457 A CA 1050457A
Authority
CA
Canada
Prior art keywords
hydrocarbon
utilising
bacterium
bacteria
process according
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA228,704A
Other languages
French (fr)
Inventor
John H. Harwood
Lionel J. Barnes
Harmannus J. Doddema
David E. F. Harrison
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shell Canada Ltd
Original Assignee
Shell Canada Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shell Canada Ltd filed Critical Shell Canada Ltd
Application granted granted Critical
Publication of CA1050457A publication Critical patent/CA1050457A/en
Expired legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P39/00Processes involving microorganisms of different genera in the same process, simultaneously
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/26Processes using, or culture media containing, hydrocarbons
    • C12N1/28Processes using, or culture media containing, hydrocarbons aliphatic
    • C12N1/30Processes using, or culture media containing, hydrocarbons aliphatic having five or less carbon atoms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/32Processes using, or culture media containing, lower alkanols, i.e. C1 to C6

Abstract

ABSTRACT OF THE DISCLOSURE
A process for the production of bacteria in which a hydrocarbon-utilising bacterium is grown under aerobic conditions in a liquid growth medium comprising urea and essential mineral in the presence of a hydro-carbon and in the presence of a non-hydrocarbon-utilising urease positive bacterium which is capable of metabolising organic substances produced by the growing hydrocarbon-utilising bacterium.

Description

~(~S0457 ~ lis invention relates to a process for the production of bac-teria. Many bacteria are known which can utilise hydrocarbons as their carbon and/or energy source. The dried biomass obtainable by the cultivation of such bacteria, often referred to as single cell protein, is rich in pro-tein and can be used as a possible food-stuff or food supplement for man and animals.
A process for the cultivation of hydrocarbon-utilising bacteria usually comprises the growth of such bacteria under aerobic conditions in a liquid growth medium comprising, in addition to the carbon source, assimilable sources of nitrogen, such as nitrogen (N2) gas, nitrate-ions, amn~onium-ions or urea. Although nitrogen gas is cheaply available, its use as source of nitrogen is disadvantageous in that much energy is required to metabolise it which makes it rather expensive on the carbon-source utilised by the micro-organisms. Nitrate-ions are also expensive on the carbon source as they have to be reduced to ammonium-ions in order to become metabolisable by the bac-teria. Ammonium-ions are both cheaply available and not expensive on the carbon-source but have the draw-back of inhibiting the growth of many bacteria, particularly certain methanol- and methane-utilising bacteria, when present in the liquid growth medium in too high a concentration. Finally urea, which is also cheaply available and is not expensive on the carbon-source, can only be utilized by a restricted number of bacterial species.
It is an object of the present invention to provide an improved process for the cultivation of hydrocarbon-utilizing bacteria whereby the dis-advantages inherent in the use of certain sources of nitrogen are overcome.
Accordingly the invention provides a process for the production of bacteria in which one or more bacteria which utilises hydrocarbons or oxygenated derivatives of hydrocarbons is or are grown under aerobic condi-tions in a liquid growth medium comprising urea and essential mineral salts in the presence of a hydrocarbon or an oxygenated derivative of a hydrocarbon and in the presence of one or more non-hydrocarbon-utilizing urease positive - 1 - ~ : .

, .. . . - . . .
~ . . , .- , . - ~

:1050457 bacteria which is or are capable of me~abolising organic substances produced by the growing hydrocarbon-utilising bac~eria.
The term "hydrocarbon" as used herein is meant to include not only hydrocarbons, but also oxygenated derivatives thereof.
According to ~he process of the invention the urease positive bacteria gradually break down urea thereby forming ammonium-ions in the liquid growth medium which ions become then available to the hydrocarbon-utilising bacteria as an easily assimilable source of nitrogen. One of the advantages of the p~ocess of the invention is the fact that the ammonium ions become available to the hydrocarbon-utilising bacteria whilst the con-centration thereof is checked by the non-hydrocarbon-utilising urease posi-tive bacteria thus preventing the formation of inhibitory concentrations of ammonium ions in the liquid growth medium.
When the hydrocarbon-utilising bacteria grow quickly the rate of produc~ion of extracellular products is high and consequently the rate of growth of the non-hydrocarbon-utilising urease positive bacteria which meta-bolize these extracellular products, is high. Therefore, the latter bacteria are able to break down the supplied urea into ammonium ions at a fast rate and thus ammonium ions are made available to the hydrocarbon-utilising bac teria at a rapid rate to suit their rapid growth.
When the hydrocarbon-utilising bacteria grow slowly, however, the rate of production of extracellular products is low and consequently the rate of growth of the non-hydrocarbon utilising urease positive bacteria is low. Therefore, the latter bacteria are unable to break down the supplied urea into ammonium ions at a fast rate. This low rate of production of am-monium ions matches the slow growth rate of the hydrocarbon-utilising bacteria.
Thus ammonium ions are available to the hydrocarbon-utilising bacteria at a rate proportional to the requirement9 i.e. proportional to the -;
growth rate of the hydrocarbon-utilising bacteria. Consequently the concen-tration of the ammonium ions in the liquid growth medium remains relatively A~
.. . . - -- .. . ~ . . . .
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. . ~ . . ~ . .

105(~57 constant and in practice will virtually never exceed the level at which inhibitory effects on growth occur.
In its simplest form the culture of bacteria used in the proccss of the invention comprises a hydrocarbon-utilising bacterium which cannot normally utilise urea but can utilise ammonium ions as nitrogen source, to-gether with one or more urease positive non-hydrocarbon-utilising species which can break down urea and release ammonium ions into the medium.
The hydrocarbon-utilising bacterium preferably is a methane-utilising bacterium which grows in the presence of methane or a methanol-utilising bacterium which grows in the presence of methanol. Good results are obtained if the culture contains both a methane-utilising bacterium and a methanol-utilising bacterium and is grown in the presence of methane.
However, other bacteria may also be present in the culture, for example one or more non-hydrocarbon-utilising urease negative bacteria which is/are capable of metabolising organic substances produced by the growing hydrocarbon-utilising bacteria, and also further hydrocarbon-utilising bac-teria may be present.
Methane-utilising bacteria which can suitably be used in the process of the invention are for example Methylococcus capsulatus, Methyl-ococcus minimus, Methylobacter vinelandii, Methylosinus trichosporium, Methylocystis parnis and the organism SM3 (NCIB 11084) which has been des-cribed in detail in U. K. Patent Specification 1,467,022 (Canadian Patent Applica~ion Serial No. 214,308). Suitable methanol-utilising bacteria are for example Hyphomicrobium sp. (NCIB No. 11040), Pseudomonas extorquens, Pseudomonas methylotropha (NCIB Nos. 10,508 - 10,515 and 10,592 - 10,596) and the bacterium OML (NCIB 11112) which has been fully described in the above men-tioned U. K. Patent. Any non-hydrocarbon-utilising bacterium capable of utilising urea and capable of growing in mixed culture with hydrocarbon-utilising bacteria may be used, for example the bacteria ~1 (NCIB 11062), M2 (NCIB 110613 and M4 ~NCIB 11065) which have all been fully described in the .' ~05~)4S~
above-mentioned U. K. patent. A preferred mlxcd culture for use in the process of the invention in the presence of methane is the culture designated T3 (NCIs 11085) which comprises the methane-utilising bacterium SM3 (NCIB
11084), the methanol-utilising bacterium OML (NCIB 11112) and four non-methylotrophic bacteria Ml, M2, M4 and M3 (NCIB 11063). M3 is a urease negative bacterium. The culture T3 ~nd its components have been fully des-cribed in U. K. Patent Specification No. 1,467,022.
Urea is suitably present in the liquid growth medium in a con-centration from 3-50 g/l. It will be obvious that no substantial amounts of ammonium ions should be supplied to the liquid medium other than through the break down of urea.
Other elements which are usually present in the medium are phos-phorus, sulphur, magnesium and iron. The phosphorus source is preferably one or more phosphates, for example K2HP04, KH2P04 or Na2HP04 or phosphoric acid, preferably present in a concentration from 3-20 g/l. The sulphur source may be sulphuric acid or a sulphate suitably in a concentration from 0.5-5.0 g/l. The two metals are provided as one or other of their salts, for example MgS04.7H20 in a concentration from 0.2-2.0 g/l and FeCl3.6H20 in a concentration from 0.01-0.1 g/l.
The medium may also contain trace amounts of other elements in the form of suitable salts, for example, calcium, mangan~se, zinc, cobalt, molybdenum and boron. An example of a suitable medium is given in the Example.
The process of the invention may be carried out batch-wise, semi-continuously but preferably in continuous flow culture. To obtain growth the bacteria are inoculated into the medium which is contacted with oxygen. For continuous flow culture the bacteria 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 re-cycle cooling loop. Fresh medium is pumped continuously into the culture at rates equivalent to 0.02 to 1.00 culture volumes per hour and culture is re-L~ ..,~

~050457 moved at a rate such that the volume of culture remains constant. Oxygen and possibly carbon dioxide is contacted with the medium preferably by 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 o~ the vessel. Spent gas may be recycled either through an external loop or internally by means of a gas inducer impeller.
The temperature of the culture is generally maintained between 30 to 50C and preferably from 38 to 45C. The pH of the culture is usually 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, and/or an acid, for example H2S4 or H3PO4.
The bacterium cells may be harvested from the growth medium by any of the standard techniques commonly used, for example flocculation, sedi-mentation, and/or precipitation, followed by centrifugation and/or filtration.
The biomass is then dried e.g. by freeze or spray drying and may be used in this form as a protein food stuff or food supplement for man or animals. The invention is illustrated further in the following Example.
Example A Biotec ~trade mark) fermenter with a working volume of 2.5 litre was charged with medium and inoculated with 100 ml of culture T3 (com-prising the bacteria SM3, OML, Ml, M2, M3 and M4). This culture was prepared as described in our U. K. Patent No. 1,467,022. The medium contained the following ingredients:
KH2PO4 1.6 grams/litre Na2HP4 1.16 "
Urea 1.122 "
MgS4 7H2 0.08 "
FeSO4.7H2O 0.014 "

Ca~NO3)2.4H2O 0.025 "
S 4.5H2 8x10 6 "

. ~ , . .
~ ~ ' . ' `' ~ :

~1~S0~57 ZnSO4.7~120 6.8x10 7 grams/litre MnSO4.4H2O 6.0x10
2 4.2H2O 4.8x10-7 ~, The fermenter temperature was controlled at 42C and the pH at 7Ø A methane (25%)/air ~75%~ mixture was bubbled into the base of the fermenter at a rate of 600 ml/min. The fermenter was stirred by means of a single 6 bladed im-peller at 1200 rpm.
Growth occurred within 24 hours and when the dissolved oxygen tension reached ~ero, continuous culture was carried out, using medium of the above composition. The dilution rate was increased at intervals of about 2 hours by steps of about 0.02h 1 until a dilution rate of 0.175 h 1 was reached.
The fermenter was then operated under the following conditions:
Absolute inlet gas pressure 770mmHg Inlet gas temperature 24~C
Measured inlet gas flow rate 116.8 sec 1 Inlet nitrogen ~percentage) 67.65 Inlet methane (percentage) 11.79 Inlet carbon dioxide ~percentage) 0.50 Inlet oxygen (percentage) 20.06 Outlet nitrogen (percentage) 80.13 Outlet methane ~percentage) 4.20 Outlet carbon dioxide ~percentage) 6.08 Outlet oxygen dioxide ~percentage) 9.59 Liquid medium flsw rate 0.356 lh 1 Fermenter volume ~litre) 1.9 pH 7.1 Biomass concentration 3.22 gl 1 Agitation speed 1380 rpm Fermenter temperature 41.8C

Under these csnditions a methane-limited steady state was ob-105~1457 tained.

Analysis for urea and ammonium ions (expressed in NH3) revealed the follow-ing informatlon.

Concentration in the medium Concentration in the c~lture .
Urea1.122 gl~l 0.250 gl 1 NH3 0.051 gl Thus it can be seen that urea is utilised. Ammonium ions are released into the medium and are available to the methane-utilising bacteria as the nitro-gen source. The level of ammonium ions in solution in the culture broth is low (51 mg 1 1) allowing good growth of the methane-utiliser with no inhibi-tion. The total amount of residual nitrogen source in the culture, if it were in the form of ammonium ions, would be sufficient to inhibit growth and cause washout of the culture.

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Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the production of bacteria in which one or more bacteria which utilises hydrocarbons or oxygenated derivatives of hydrocarbons is or are grown under aerobic conditions in a liquid growth medium comprising urea and essential mineral salts in the presence of a hydrocarbon or an oxy-genated derivative of a hydrocarbon and in the presence of one or more non-hydrocarbon-utilizing urease positive bacteria which is or are capable of metabolising organic substances produced by the growing hydrocarbon-utilising bacteria.
2. A process according to claim 1, wherein the hydrocarbon-utilis-ing bacterium is a methane-utilising bacterium and the hydrocarbon is methane.
3. A process according to claim 1, wherein the hydrocarbon-utilis-ing bacterium is a methanol-utilising bacterium and the oxygenated derivative of a hydrocarbon is methanol.
4. A process according to claim 2, wherein in addition to the methane-utilising bacterium a methanol-utilising bacterium is employed.
5. A process according to claim 1, wherein in addition to the hydrocarbon-utilising bacterium and the non-hydrocarbon-utilising urease positive bacterium a non-hydrocarbon utilising urease negative bacterium is employed which is capable of metabolising organic substances produced by the growing hydrocarbon-utilising bacterium.
6. A process according to claim 1, wherein the mixed culture desig-nated T3 and having the NCIB Accession No. 11085 is grown in the presence of methane.
7. A process according to claim 1, 2 or 3, wherein the temperature is maintained in the range 30 - 50°C.
8. A process according to claim 4, 5 or 6, wherein the temperature is maintained in the range 30 - 50°C.
9. A process according to claim 1, 2 or 3, wherein the pH is con-trolled in the range 6.0 - 8.0
10. A process according to claim 4, 5 or 6, wherein the pH is con-trolled in the range 6.0 - 8.0
CA228,704A 1974-08-12 1975-06-06 Microbiological process Expired CA1050457A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
GB35435/74A GB1514485A (en) 1974-08-12 1974-08-12 Regulation of assimilable nitrogen in microbiological processes

Publications (1)

Publication Number Publication Date
CA1050457A true CA1050457A (en) 1979-03-13

Family

ID=10377691

Family Applications (1)

Application Number Title Priority Date Filing Date
CA228,704A Expired CA1050457A (en) 1974-08-12 1975-06-06 Microbiological process

Country Status (10)

Country Link
US (1) US4003790A (en)
JP (1) JPS5141488A (en)
BE (1) BE832105A (en)
CA (1) CA1050457A (en)
DE (1) DE2535782A1 (en)
FR (1) FR2281981A1 (en)
GB (1) GB1514485A (en)
NL (1) NL7509531A (en)
NO (1) NO752792L (en)
SU (1) SU603348A3 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5395514A (en) * 1977-02-01 1978-08-21 Nippon Telegr & Teleph Corp <Ntt> Test system for subscriber line
US4288554A (en) * 1979-12-20 1981-09-08 Euteco Impianti S.P.A. Process for the cultivation of yeast cells
JP2603182B2 (en) * 1993-02-25 1997-04-23 環境庁国立環境研究所長 Culture method for organochlorine compound degrading bacteria
US6835560B2 (en) * 2001-10-18 2004-12-28 Clemson University Process for ozonating and converting organic materials into useful products
US7651615B2 (en) * 2005-12-23 2010-01-26 Clemson University Research Foundation Process for reducing waste volume

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS4937273B1 (en) * 1970-03-03 1974-10-07
BE787963A (en) * 1971-08-24 1973-02-26 British Petroleum Co PROCESS FOR THE PRODUCTION OF MICRO-ORGANISMS
GB1467022A (en) * 1974-01-07 1977-03-16 Shell Int Research Cultivating of methane-utilising micro-organisms

Also Published As

Publication number Publication date
FR2281981B1 (en) 1978-04-28
US4003790A (en) 1977-01-18
JPS5141488A (en) 1976-04-07
DE2535782A1 (en) 1976-02-26
GB1514485A (en) 1978-06-14
FR2281981A1 (en) 1976-03-12
SU603348A3 (en) 1978-04-15
NL7509531A (en) 1976-02-16
BE832105A (en) 1976-02-04
NO752792L (en) 1976-02-13

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