CA2069712A1 - Pleomorphism - Google Patents
PleomorphismInfo
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- CA2069712A1 CA2069712A1 CA 2069712 CA2069712A CA2069712A1 CA 2069712 A1 CA2069712 A1 CA 2069712A1 CA 2069712 CA2069712 CA 2069712 CA 2069712 A CA2069712 A CA 2069712A CA 2069712 A1 CA2069712 A1 CA 2069712A1
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Abstract
ABSTRACT OF THE DISCLOSURE
Pleomorphic agents may be introduced into a culture medium for a microorganism which produces granules of product in side the cell. The pleomorphic agent alters the nature of the cell wall permitting one or more of the following benefits: increase in cell size; increase in the number of granules per cell; and the excretion of the product into the medium.
Pleomorphic agents may be introduced into a culture medium for a microorganism which produces granules of product in side the cell. The pleomorphic agent alters the nature of the cell wall permitting one or more of the following benefits: increase in cell size; increase in the number of granules per cell; and the excretion of the product into the medium.
Description
æo~97~2 Pleomorphi~m T~CHNICA~ FI~L~
This invention relates to the industrial application of pleomorphism induced by the presence of certain peptones, and particularly fish peptone in the production of polymers by culturing microorganisms. In particular the present invention relates to the application of pleomorphism in the hyperproduction of certain poly-hydroxyalkanoates during growth of mutant strains of Azotobacter vinelandii.
The production of polymers by culturing microorganisms is becoming more important. There are a number of reasons for this trend including a concern 1~5 about producing polymers from renewable resources, the production of "bio-polymers" which are perceived as more environmentally friendly than "synthetic polymers" and the use of industrial processes which are less energy demanding and have a lower potential for pollution.
Currently one class of polymer produced by such processes is the polyhydroxy alkanoates.
Poly-hydroxyalkanoates may be characterized as polymers of one or more monomers residues o~ the ~ormula -0CRIR2(cR3~4)n-cO
wherein n is an integer from 1 to 5; and R~, ~2, R3 and ~
are independently selected from the group consisting of a hydrogen atom and C~s alkyl radicals. The polymers include poly-B-hydroxybutyrate (PHB) and copolymers of 3-hydroxybutyrate and 3- or ~-hydroxyvalerate (the PHB~'s).
Poly-~-hydroxybutyrate (PHB) is a biodegradable, biocompatible, thermoplastic made by microorganisms [Baptist, ~.N., 1962, U.S. Patent 3,036,959]. In the cell, PHB is an intracellular storage material synthesized and accumulated during unbalanced growth. It accumulates as distinct white granules that are clearly visible in the cytoplasm of the cell. Under conditions of nutrient starvation, PHB is used by the cell as an . . ~ .,; . ,:
. . . :, ` ~
internal reserve of carbon and energy. Many bacteria including those in the soil, are capable of PHB
production and breakdown. Animal cells do not form PHB
but are able to break down the polymer.
PHB is a homopolymer of repeating 3-hydroxybutyric acid units. Copolymers with hydroxyvaleric acid (PHBV) can be made by "precursoring" (e.g.) adding propionic acid to the culture during growth [Holmes et al, 1981, European Patent 0,052,459 and 1984 U.S0 Patent 4,477,654]. This modification to the PHB homopolvmer reduces the crystallinity and melting point of the plastic, allowing film formation and melt-extrusion applications. PHB plastics also are used in microelectronics applications exploiting the piezoelectric properties of PHB. An immediate market for PHB plastics will be in high value added products (e.g.) biodegradable surgical pins, plates, pegs and sutures, implants for drug delivery, and possibly meshes can be used as artificial skin materials. PHB/PHBV derived plastics also have considerable potential application as biodegradable bulk plastics, replacing non-biodegradable products formed from polypropylene or polyethylene.
Development of many of these produ~ts is ongoing in view of their potential uses.
There are a number of approaches to the production of poly-hydroxy alkanoates by culturing microorganisms.
Commercially Imperial Chemical Industries PLC. ("ICI") produces PHBV by culturing Alcaliaenes eutrophus. There have been a number of publications and patents, including 30 WP ~9/01323, based on work by Dr. W. Page and O. Knosp relating to the use of a mutant species of Azotobacter vinelandii for the production of PHB/PHBV.
At present, PHB/PHBV is produced commercially by ICI
in tha U.K., using a strain of Alcaliaenes eutrophus growing in a glucose salts medium. Their fermentation involves a rapid growth phase (60h), followed by phosphate limitation and glucose feeding (an additional ; ~
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This invention relates to the industrial application of pleomorphism induced by the presence of certain peptones, and particularly fish peptone in the production of polymers by culturing microorganisms. In particular the present invention relates to the application of pleomorphism in the hyperproduction of certain poly-hydroxyalkanoates during growth of mutant strains of Azotobacter vinelandii.
The production of polymers by culturing microorganisms is becoming more important. There are a number of reasons for this trend including a concern 1~5 about producing polymers from renewable resources, the production of "bio-polymers" which are perceived as more environmentally friendly than "synthetic polymers" and the use of industrial processes which are less energy demanding and have a lower potential for pollution.
Currently one class of polymer produced by such processes is the polyhydroxy alkanoates.
Poly-hydroxyalkanoates may be characterized as polymers of one or more monomers residues o~ the ~ormula -0CRIR2(cR3~4)n-cO
wherein n is an integer from 1 to 5; and R~, ~2, R3 and ~
are independently selected from the group consisting of a hydrogen atom and C~s alkyl radicals. The polymers include poly-B-hydroxybutyrate (PHB) and copolymers of 3-hydroxybutyrate and 3- or ~-hydroxyvalerate (the PHB~'s).
Poly-~-hydroxybutyrate (PHB) is a biodegradable, biocompatible, thermoplastic made by microorganisms [Baptist, ~.N., 1962, U.S. Patent 3,036,959]. In the cell, PHB is an intracellular storage material synthesized and accumulated during unbalanced growth. It accumulates as distinct white granules that are clearly visible in the cytoplasm of the cell. Under conditions of nutrient starvation, PHB is used by the cell as an . . ~ .,; . ,:
. . . :, ` ~
internal reserve of carbon and energy. Many bacteria including those in the soil, are capable of PHB
production and breakdown. Animal cells do not form PHB
but are able to break down the polymer.
PHB is a homopolymer of repeating 3-hydroxybutyric acid units. Copolymers with hydroxyvaleric acid (PHBV) can be made by "precursoring" (e.g.) adding propionic acid to the culture during growth [Holmes et al, 1981, European Patent 0,052,459 and 1984 U.S0 Patent 4,477,654]. This modification to the PHB homopolvmer reduces the crystallinity and melting point of the plastic, allowing film formation and melt-extrusion applications. PHB plastics also are used in microelectronics applications exploiting the piezoelectric properties of PHB. An immediate market for PHB plastics will be in high value added products (e.g.) biodegradable surgical pins, plates, pegs and sutures, implants for drug delivery, and possibly meshes can be used as artificial skin materials. PHB/PHBV derived plastics also have considerable potential application as biodegradable bulk plastics, replacing non-biodegradable products formed from polypropylene or polyethylene.
Development of many of these produ~ts is ongoing in view of their potential uses.
There are a number of approaches to the production of poly-hydroxy alkanoates by culturing microorganisms.
Commercially Imperial Chemical Industries PLC. ("ICI") produces PHBV by culturing Alcaliaenes eutrophus. There have been a number of publications and patents, including 30 WP ~9/01323, based on work by Dr. W. Page and O. Knosp relating to the use of a mutant species of Azotobacter vinelandii for the production of PHB/PHBV.
At present, PHB/PHBV is produced commercially by ICI
in tha U.K., using a strain of Alcaliaenes eutrophus growing in a glucose salts medium. Their fermentation involves a rapid growth phase (60h), followed by phosphate limitation and glucose feeding (an additional ; ~
..
. : i : . i ;
:~ : . ~' .. :
2~69712 48-60h). During phosphate-limited growth, PHB is formed and may account for 75% of the total cell weight. The yield per litre is dependent on the initial cell mass and theoretical yields of 0.33 t PHB t-~ glucose have been calculated [Byrom, D., Trends in Biotechnology 5, 246-250, 1987~.
Generally in the ICI production of PHB/PHBV in the cell occurs during imbalanced growth. Usually this is the stationary phase of bacterial growth, but this can be induced in an actively growing culture by imposing a nutrient (2~ nitrogen, phosphate, or sulfate) limitation in the presence of excess carbon source. During this imbalance, NADH accumulates and exerts a feedback repression on various enzymes whose activities are essential for the continued growth of the cell. NADH can be oxidized to NAD~, eliminating this growth inhibition, by the action of acetoacetyl CoA reductase and the polymerization of acetoacetyl CoA into PHB.
NAD+ is nicotinamide adenine dinualeotide and NADH
is its reduced form. NAD~ is a major electron acceptor in the oxidation of fuel molecules in the cell. NAD~
fulfills this function by accepting two electrons and two hydrogen ions from substances its oxidizes. Thus, NAD+
becomes NADH, Stryer, Biochemistry (2d ed.), W.H. Freeman and Company, San Francisco, pp 244-246.
Azotobacter vinelandii is a harmless soil micro~e that has an obligate 2 requirement for growth and can use N2 as a nitrogen source via nitrogen fixation. A.
yinelandii normally produces PHB by the methods noted above and much of the early work concerning PH~ synthesis was conducted in Azotobacter species. To the best of applicants knowledge the production of PHBV from A.
vinelandii has naver been reported. Azotobacter species that produce large amounts of PHB have been reported, but these cells have been unstable and also produce large amounts of capsule and slime, which interfere with PHB
extraction and decrease the efficiency of conversion of ::
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carbon substrate to PHB. Page and Knosp have overcome this drawback by using a mutant strain o~ A. vinelandii which may be gro~n on readily commercially available substrates and which does not produce significant amounts slime.
United States patent 4,138,291 issued February 6, 1979 in the name of Lafferty, assigned to Agroferm AG of Switzerland also discloses a process for selecting microorganisms which may be used to produce poly-hydroxy alkanoates.
One of the difficulties in these processes for producing polymers by culturing strains of microorganisms is the problem of separating the polymer from the microorganism. The cell wall must be ruptured, destroying the microorganism, and the polymer granules must be extracted. Accordingly one of the limitations in the current processes is how much polymer may be incorporated into a cell because the cell cytoplasm, where the granules form, is contained within a rigid cell wall that will grow to dimensions characteristic of that organism (and no larger). The excretion of solid, cell byproducts such as polymers for example, those of PHB/PHBV through the living cell wall and into the culture medium has never been reported in the literature.
This concept has very significant implications for the entire field of biofermentation and production of products such as polymers, inclusions or crystals and their recovery.
Pleomorphic agents are agents which weaken the cell wall of a microorganism. Generally the agents cause the cell wall to rupture, usually due to an osmotic imbalance between the ambient environment and the contents of the cell. Some bacteria are more sensitive to the induction of pleomorphism than others. An agent that will induce pleomorphism in many cultures is complex mixtures of amino acids commercially available as peptones prepared by the enzymatic or acidic digestion of animal, plant, ~ .
20~9~12 milk or fish proteins. The susceptibility of an organism to pleomorphic agents can be determined ~y non-inventive experimentation and is frequently documented in the literature relating to the cultivation and identification of microorganisms (e.g. "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J. G. Holt (ed) Williams and Williams, Baltimore, 1984). In these cases, the addition of pleomorghic agents to the growth medium is avoided, because the cells will lose osmotic stability and rupture (lyse) in normal growth media. However, in other cases, peptones will not induce pleomorphism (or will ~o so only at high concentrations, 1 - 5%) and the peptone may serve as an excellent source of organic nitrogen and carbon for cell growth. However, even in these cases addition of higher concentrations of certain amino acids, notably 0.5 M glycine to the growth medium will induce pleomorphism tN. Welsh ~ P. Osterrieth, Antonie van Leeuwenhoek, Journal of Microbiology and Serology, 1958, 24, 257-273; J.L. Strominger & C.H.
Birge, ~ournal of Bacteriology 1965, 89, 1124-1127; P.H.
Fung ~ A.J. Winter, Journal of Bacteriology, 1968, 1~89 1894).
The paper "Effect of Peptone on Azotobacter Morphology" by G.R. Vela and R.S. Rosenthal, Journal of Bacteriology, July 197~, 260-266, describes the effect of Difco peptone on the morphology of Azotobacter. The paper teaches that the cells become osmotically fragile and pleomorphic or fungoid in appearance. It is noted that one strain of A. vinelandii will yield at least several types of morphology and cells are much larger than normal. The paper does not mention the production of PHB under these conditions and granules of said polymer are not demonstrated in the micrographs presented.
Furthermore, the paper "Polymer Production by a Mucoid Strain of Azotobacter vinelandii in Batch Culture"
by A.C. Brivonese and I.W. Sutherland, Applied .
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Microbiology and Biotechnology, 1989, 30, 97-102, shows that the production of PHB by A. vinelandii strain AX (a mucoid strain) in medium containing 0.05 - 0.2% peptone and 4% glucose reached 1 mg/ml after 48 hours incubation with a low rate of aeration (120 rpm shaking~. If the shaking rate was increased to 280 rpm, the yield of PHB
was not significantly increased (about 1.5 mg/ml) after 48 hours incubation. The authors did note a positive effect on alginate formation by these A. vinelandil strains, but the biosynthesis of PHB and alginate are unrelated.
Similarly, the paper "Correlation of Ultrastructure in ~zotobacter vinelandil with Nitrogen Source for Growth" by J. Oppenheim and L. Marcus, Journal of l'; Bacteriology, 1970, 101, 286-291, instructs that when 0.5% casamino acids (an acid digest of casein) was present as a source of amino acids for the growth of strain OP (ATCC 13705) "PHB does not seem to be synthesized when nitrate or amino acids are used as the nitrogen source". Thus, the prior art teaches away from the incorporation of pleomorphic agents (peptones or glycine) into the culture medium and teaches that peptones or casamino acids do not promote the formation of PHB.
A variety of microbes are susceptible to pleomorphic agents resulting in the formation of enlarged and osmotically fragile cells. The paper "Effect of Yeast Extract Concentration on Viability and Cell Distortion in Rhizobium spp." by F.A. Skinner, R.J. Roughley, and M~riel R. Chandler, Journal of Applied Bacteriology, 1977, ~3, 287-297 demonstrates that yeast extract acts as a pleomorphic agent in the culture of Rhizobium spp. The thrust of the paper is on the morphology of the cells and not on the production of polymers using such agents in the culture medium. The paper "A Comparative Study of the Transformation of Gram-Negative Rods into "Protoplasts" Under the Influence of Penicillin and Glycine" by M. Welsch & P. Osterrieth, Antoine van Leeuwenhoek, Journal of Microbiology and Serology, 1958, 2~, 257-273, demonstrates that a number of Gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa, are susceptible to the pleomorphic agent glycine. The pleomorphic cells formed appeared to have remnants of cell wall still attached to the cell and have varied osmotic fragility, in some cases even 20~ sucrose in the growth medium would not stabilize the cells. The paper "Nucleotide Accumulation Induced in Staphyloccus aureus by Glycine" by J.L. Strominger & C.H. Birge, Journal of Bacteriology, 1965, 89, 1124-1127, and the references show that the pleomorphic agent glycine interfer~s with cell wall synthesis in Gram-positive organisms as well. Thus, the prior art teaches that representatives of both Gram staining groups (Gram-positive and Gram-negative) are susceptible to pleomorphic agents.
The paper "Microbial Growth on Peptones from Fish Industrial Wastes" by Susan E. Vecht-Lifshitz, ~.A.
Almast and E. Aomer, Letters in Applied Microbiolo~y, 1990, 10, 183-186, discloses growing Streptomyces tendae ATTC 31160, Gibberella fulikuroi, ~3acillus thurinqiensis var. israliensis 1884, Escherichia coli B, Salmonella typhi, Staphylococcus aureus, Serratia marcescens and Proteus vulgaris in the presence of fish peptones to produce toxins. The paper does not suggest the use of such a procedure to produce polymers.
Accordingly, the prior art teaches away from incorporating pleomorphic agents into a culture medium.
In the production of polymers by culturing microorganisms, especially in the case of PHB and PHBV
which is an intracellular accumulation, there is a need for a method for either increasing the size of cell which may be grown thus providing for an increase cell volume and hence polymer volume, or better still to provide a :
206~71~
mechanism to permit the cell to excrete or extrude polymer into the culture medium.
The present invention seeks to provide such a mechanism.
DT8C~OBURE OF THE I~VENTION
Accordingly, the present invention provides in a process of producing a polymer by culturing a strain of microorganism having a hyperproduction of polymer during exponential growth in a medium comprising at least a source of assimilable carbon, the improvement comprising subjecting the bacteria to a controlled pleomorphism by introducing into the culture from 0.05 to 0.20 weight ~
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture medium.
A further embodiment of the present invention provides a process for increasing the cell size during the culturing of a microorganism in a medium comprising at least one source of assimilable carbon which comprises introducing i~to the culture medium from 0.05 to 0.20 weight % of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
The present invention further provides a process for increasing the number of granules per cell in the culturing of a microorganism which produces granules of polymer, when cultured in a medium comprising at least one source of assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight %
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance -between the microorganism and the culture.
The present invention further provides a process whiah permits the excretion of granules through the cell wall of a microorganism cultured in a medium comprising at least one source o~ assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight ~
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" i, 'i 2~69712 of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
BRIEF DE8C~IPTION OF DRAWIN~S
Figures 1 A, B and C are electron micrograph~ of cultures of UWD cells of Azotobacter vinelandii which have been cultured in the absence of pleomorphic agents (Fig. lA), in the presence of proteose peptone #3 (Fig.
lB) and in the presence of fish peptone (Fig. lC). The electron micrographs show that fish peptone stabiilizes the cells, prevents cell lysis, and promotes the extrusion of the polymer ~rom the cells without a loss of cell integrity.
BBS~ MODE FOR CARRYING OUT THE INVENTION
A number of polymers may be produced by the culturing of microorganisms. However, particularly useful polymers include the poly-hydroxyalkanoates. The poly-hydroxyalkanoates which may be produced in accordance with the present invention may be co or homopolymers of one or more monomers residues of the formula -OCRIR2(cR3~)n-cO
wherein n is 1 or 2; and Rl, R2, R3 and ~ are independently selected from the group consisting of a hydrogen atom and Cl2 alkyl radicals. Preferably in formula 1, n is 1 or 2 and Rl, R2, R3 and ~ are independently selected from the group consisting of a hydrogen atom, a methyl radical and an ethyl radical. If the polymer is a homopolymer and n is 1 and one of Rl and R2 is a methyl radical and the other is a hydrogen atom and both R3 and ~ are hydrogen atoms, the polymer is poly-B-hydroxybutyrate (PHB). If the polymer is a copolymer and n, R~, R2, R3 and ~ are as described immediately above in one monomer residue and in the other monomer residue, n is 2, one or R~ and R2 is a methyl radical and the other is hydrogen and R3 and ~ (both occurrences) are hydro~en the polymer is PHBV.
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Preferably in the above copolymer the mole ratio of the butyrate residues to the valerate residues is from about 70:30 to about 9~:1, most preferably above 95:5.
If it is desired to produce a copolymer in accordance with the present invention it is necessary to add a precursor. The precursor may be a Csg odd numbered alkanoic acid or a salt thereof such an alkali metal salt such as sodium or potassium salts. Preferably the precursor is sodium valerate or valeric acid. The precursor may be present in the growth medium in a concentration from 5 to 45, preferably 10 millimolar ("mM"). The precursor should be added during the time when the cells are actively forming polymer. The percent comonomer (e.g. hydroxyvalerate; HV) in the polymer may be increased by adding higher concentrations of precursor, or by repeated addition of precursor, or by addition of the precursor shortly (between 4 to 8 hours) before harvesting.
The present invention may be practised ~ith microorganisms which under go exponential growth in a culture medium. The application of the principles of this disclosure to other microbial systems is expected to involve some period of microbial growth, because the pleomorphic agents exert their effect through interference ~ith cell wall growth. The number of cell divisions and extent of growth would be determined by non-inventive experimenttation in each case.
A bacterial transformant, use~ul in accordance with the present invention, which for the purpose of reference in the detailed description is identified as UWD, was derived from the species Azotobacter vinelandii. It is understood that the invention encompasses not only the particular UWD, but all derivatives thereof and other related microorganisms having similar taxonomic 3~ descriptions. The Azotobacter genus is described in "Bergey's Manual of Determinative Bacteriology" b~v N.R.
Krieg and J.G. Holt (ed.), Williams and Wilkins, . .
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ll Baltimore, 1984, 1, 219-229; and "Azotobacteraceae: The taxonomy and ecology of the aerobic nitrogen-fixing bacteria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp. 168-69, pp. 178-79.
Azotobacter vinelandii wild-type, which for the purpose of reference in the detailed description is identified as OP, is readily available and has been investigated by various groups. Azotobacter vinelandii OP is deposited at American Type Culture Collection under accession number ATCC 13705. It is understood that throughout the specification, the generally accepted nomenclature "ATCC" for the American Type Culture Collection will be used. ATCC is located at 12301 Parklawn Drive, Rockville, Maryland, U.S.A. All deposits as ATCC are given accession numbers which are referred to throughout the specification. The University of Wisconsin subculture of Azotobacter vinelandii OP is identified as UW for the purpose of reference in the detailed description. The UWD transformant, according to this invention, was developed and :isolated by transforming UW cells with DNA preparPd from the mutant Azotobacter vinelandil ATCC 63800 Itwhich for the purpose of reference in the detailed descrlption is identified as strain 113) such as defined in the following Example. It is appreciated that there are many techniques available for inducing such mutation and th~t: there are many techniques available for transforming bacterial cells or otherwise changing their genetic composition and such other techniques are contemplated herein although not specifically exemplified.
The isolated genetic transformant has been characterized in the following Examples and has been deposited at th~ American Type Culture Collection. The deposit was made on August 10, 1988 under accession number ATCC 53799~ The taxonomical characteristics of the UWD strain are as follows.
.
.
.... .
~06g~ 2 The UWD strain shares characteristics with strain UW
(OP, ATCC 13705) except that it forms excess PHB during exponential growth, it is resistant to rifampicin (20~g/ml), it has a white colony colour, and it forms the fluorescent-green pigment under iron-sufficient conditions.
Basic characteristics: Large ovoid cells, Gram-negative, pleomorphic (in this sense meaning polymorphic eg. multiple forms and not necessarily swollen or osmotically fragile) ranging from rods to cocci. The cells are motile by peritrichous flagella, substantial capsules are not formed and the cells form fragile cysts only after very long incubation (several months to a year). Growth is strictly aerobic and nitrogen is fixed aerobically. Substrate utilization characteristics have been well defined and agree with "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J.G. Holt (ed.), Williams and Wilkins, Baltimore, 1984, 1, pp.
219-229; and "Azobacteraceae: The taxonomy and ecology of the aerobic nitrogen-fixing bact:eria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp.
168-69, pp. 178-179.
The taxomonic description of s~train 113 is as follows.
This strain is derived from A. vinelandii ATCC 12837 by NTG mutaqenesis. It shares characteristics with ATCC
12837 except it is resistant to rifampicin (20~g/ml) and it forms poly-B-hydroxybutyrate (PHB) during exponential growth.
Basic characteristics: Large ovoid cells, Gram-negative, pleomorphic (in this sense meaning polymorphic e.g. multiple forms and not necessarily swollen or osmotically fragile) ranging from rods to cocci. The cells are motile by peritrichous flagella, form capsules and form cysts in older cultures. Iron-limited cultures produce a fluorescent yellow-green pigment. Growth is strictly aerobic and nitrogen is fixed aerobically.
: . -"~ '' . -, 2~69712 Substrate utilization characteristics have been welldefined and agree with "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J.G. Holt (ed.), Williams and Wilkins, Baltimore, 1984, 1, pp. 219-229; and I'Azotobacteraceae: The taxonomy and ecoL~ o~ the aerobic nitrogen~fixing bacteria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp. 168-~9, pp. 178-79.
The UWD transformant was developed and isolated in accordance with the following preferred method. Cells of strain UW were obtained from the Department of Bacteriology at University of Wisconsin. This strain of cells is a capsule-negati~e wild-type, which were genetically transformed with DNA prepared from cells of stain 113 Azotobacter vinelandii 53800, which is a rifampin-resistant strain derived by NTG metagenesis of the capsule-positive strain ATCC 12837.
Strain 113 ~ATCC 53~00) was produced by exposing Azotobacter vinelandii strain ATCC 12837 to 100 ~g/ml N-methyl-N'-nitro-N-nitrosoguanidine (NTG) in Burk buffer, pH 7.2, for 30 minutes. Survivors were plated on Burk medium containing 1% glucose, 1.8% agar and 20 ~g rifampin/ml. Strain 113 was, therefore, selected as a nitrogen-fixing, rifampin-resistant strain of ATCC 12837.
The hyperproduction of PHB by strain 113 was an unselected mutation that also has occurred during the NTG - -mutagenesis procedure DNA for transformation was prepared as a crude lysate material. A thick suspension of strain 113 was prepared in 15 mM saline-15 mM sodium citrate buffer, pH
7.0, containing 0.05% sodium dodecyl sulfate. This suspension was heated at 60C for 60 minutes in a water bath. When cool, this lysate containing crude (unpurified) DNA was used directly in transformation assays. Optimal conditions for generation of competent strain UW (or Azotobacter vinelandii in general) which can take up this crude DNA, are documented in Page and . .
' ~ ' 20697~2 von Tigerstrom, 1978, Canadian Journal of Microbiology, 24, 1590-1594. Optimal conditions for the transformation of these competent cells by the crude DNA are documented in Page and von Tigerstrom, 1979, Journal of Bacteriology, 139, 1058-1061.
The preferred procedure used to transform UW cells with strain 113 DNA is described in Page, W.J., 1985, Canadian Journal of Microbiology, 31, 659-662. While almost any strain of Azotobacter vinelandii can be transformed by strain 113 DNA, the most successful transformations for the purpose of producing maximal amounts of readily extractable PHB are the transformations of capsule-negative strains. Of these, Azotobacter vinelandii strain OP (ATCC 13705) is a readily available strain held in culture collections.
The strain that was transformed to create the UWD
transformant was strain UW, the University of Wisconsin copy of strain OP.
Transformation of strain UW w;ith strain 113 DNA
results in a rifampin-resistance transformation frequency of about 1.0 x 104 to 8.7 x 10-3 per viable cell plated (i.e. at best 8.7 transformants per. 1000 cells plated).
This is a readily reproducible fre~uency of transformation. Of these rifampin~-resistant colonies, about 13% appeared to have streaks of white or white sectors within the normal pinkish-tan cell mass. When these sec~oring colonies were restreaked, they gave rise to sectoring colonies and dense white colonies at a ratio of about 1:1. The dense white colonies were then selected and designated UWD. This procedure and these rasults are reproducible and will readily ~enerate UWD
cells of this invention.
UWD is readily separated from the UW stock because UWD is resistant to rifampin and UW is not resistant.
Therefore, on plates containing solid medium and 20 ~g rifampin/ml, only colonies of UWD ~or other rifampin resistant cells) will grow.
'~
0697~2 The separation of PHB hyperproducing cells from the general population of transformed cells is readily reproducible, because the sectoring colony phenotype is quite distinctive and upon subculture it readily generates solid white colonies. Non-hyperproducing rells result in pinkish-tan colonies under the same conditions.
The cells from these dense white colonies (the UWD
cells) were packed with PHB granules, while the wild-type (strain UW) contained only a few small granules of PHB.
It is believed that in theory the production of PHB/PHBV in the mutant is due to a genetic defect in strain 113 and the UWD cells concerning the NADH oxidase that normally recycles NAD~ via respiratory oxidation of NADH. As a result of this defect, the cell accumulates NADH and must turn-on PHB/PHBV synthesis in order to grow. Therefore, PHB/PHBV are formed during active (balanced; exponential) growth, the exact opposite of the normal conditions promoting PHB/PHBV formation in the wild-type. Although this theory appears to be borne out by the examples, it is understood that the principles of the invention should not be limited to this theory.
Because PHB/PHBV type polymers are formed during exponential growth, conditions which enhance growth also increase PHB/PHBV type polymer formation. For example, 2S vigorous aeration (rather than 02-limitation) promotes faster use of glucose and faster production of PHB/PHBV
type polymers. Nitrogen-fixing UWD cells also produce PHB.
Strain 113 also produces large amounts of PHB, but also produces large amounts of capsule and slime. The UWD cells of this invention do not form substantial capsule and slime and therefore only convert the bulk of the sugar into cell mass (like strain UW) and PHB.
In summary, the genetically transformed bacterium of the species Azotobacter vinelandil has the identifying characteristics of:
(i) extremely abundant PHB granules in cells;
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- 2~69712 (ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of PHB during the exponential growth of the mutant microorganism.
Production of PHB was demonstrated in batch cultures. The volumes of culture were in the range 20-50% culture volume: flask or vessel volume with rotary shaking (to increase aeration and mixing) at 175 to 300 rpm (normally 250 rpm was used). Incubation temperatures were found to be 28-30~C for optimal yields. -The nitrogen source used in the cell culture is preferably N2 (from air) or ammonium acetate at 1.1 to 2.2 g/L-Various carbon sources were added to the culture medium at 1 to 5% (w/v) concentration. PHB production was best with reduced hexose, mono and disaccharide carbon sources (glucose, sucrose, maltose) or with sodium gluconate or glycerol, and was much lower with more oxid:lzed or short-chain carbon sources (acetate, ethanol). Glucose and sucrose are relatively expensive refined substrates for PHB production. Sucrose does not have to be "invertedl' before use by the UWD cells but fructose is poorly used for PHB production. Because PHB
formation is not dependent ~n nutrient limitation, cheaper unrefined carbon sources can be used by the UWD
cells. Good production of PHB has been obtained using 2%
(wtv) corn syrupt sugar-beet molasses, blackstrap and cooking molasses, and more refined grades of refiner's molasses. The UWD cells grow well in media containing at least 5% (w/v) molasses or corn syrup, however, the yield of PHB ml~~ is usually not greater than that obtained with 2 or 3% molasses or corn syrup except for a further preferred embodiment of this invention as noted below.
.
~-On these impure carbon sources the wild-type strains did not produce detectable or significant amounts (<0.1 mg/ml) of PHB.
Growth of UWD cells and yield of PHB under intense aeration conditions (culture volume 10% of vessel volume) were unexpectedly increased when sugar beet molasses was used as the carbon source. Under intense aeration conditions (as established by the culture volume being 10% of the vessel volume during shaker flask culture), a yield of S.42 PHB/ml was obtained with 5% sugar beet molasses, a 42-fold increase over the yield obtained with 2% sucrose as carbon source under the same conditions.
Sugar beet molasses concentrations greater than 5%, or increases in sucrose only, had no further beneficial effect. This growth promotional effect of sugar beet molasses under intense aeration conditions was observed with all ten different samples of sugar beet molasses examined.
Increasing PHB yields by use of an unrefined carbon source such as sugar beet molasses offers a significant reduction in production costs.
This increase in PHBV production at high aeration was only observed in beet molases. This improved method (i.e. increased a~ration of a culture containing 5% w/v sugar substrate) did not improve PHBV yields in cultures containing other impure and inexpensive sugar sources or other pure sugar sources. A typical approximate analysis of beet molasses is 81-84~ w/v) solids comprised of 50-52% sucrose, 12-20~ nitrogenous compounds (mostly amino acids and betaine), 12-13% non-nitrogenous compounds (mostly organic acids) and 11-12% ash (salts) (R. A.
McGinnis "Sugar beet Technology", 3rd Edition, 1982.
Beet Sugar Development Foundation, Fort Collins, Colorado~. Of the 100's of compounds present, it seemed most likely that the organic nitrogenous compounds may have been responsible for the observed PHBV yield promoting effect. This was suggest~d by the fact that , :
~;
.~
,,: ' .:, ' 206971~
sugar was not limiting in these other cultures and organic acids are known not to promote PHB formation in this organism (W. J. Page, 198g. Applied Microbiology and Bio-technology 31, 329-333). Extensive tests, demonstraed that the addition of a variety of complex nitrogen sources to well aerated cultures containing pure or unrefined sugars increased the yield of PHBV. This is demonstrated in the examples presented. Furthermore, these complex nitrogen sources, when added into the culture medium in an amount of about 0.05 to 0.20~
(weight %) acted as pleomorphic agents, causing the cells to swell, allowing them to fill with many polymer granules and extrude polymer through the weakened cell wall.
After reading the present disclosure, suitable pleomorphic agents may be identified using non-inventive experimentation. What is required is an agent which will at a concentration of from 0.05 to 0.20 weight % in the culture medium weaken the cell wall permitting it to distend but not result in cell wall rupture under the conditions of use.
A skilled biologist or biochemist would realize that the critical balance is between th~l osmotic pressure within the cell and the osmotic pressure of the culture medium. Accordingly one may have to incorporate one or more agents which balance the osmotic pressure in the cell and the culture medium. Generally such agents would comprise salts. Additionally, such agents could include agents which are capable of acting as surface active agents such as complex polysaccharides including sugars, and lipo`ropic substances such a betaine, choline, taurine and other members of the choline/methionine cycle.
Thus beet molasses per se may be an effective pleomorphic agent because it contains a variety of nitrogen compounds, some of which appear to promote pleomorphism in A. vinelandii, plus a mixture of salts, sucrose and betaine to stabilize the cells. Similarly fish peptone contains a ~ariety of amino acids which appear very effective in promoting pleomorphism, plus a mixture of salts, taurine and choline for stabilizing the cells.
Some useful pleomorphic agents include enzymatic or acid digests of one or more proteins or protein extracts.
For example, suitable pleomorphic agents in this application includa proteose peptone #3 ~Difco), Bacto-casitone, tryptone, casamino acids, proteose peptone andthe fish peptones commercially available from Marine Biochemicals a.s. in Tromso, Norway. Fish peptones have the advantage of being a relatively inexpensive peptone that is readily a~ailable for large scale fermentations, they ha~e a beneficial effect on promoting good cell growth (Vecht-Lifshitx, Almas & Zomer. Letters in Applied Microbiology, 1990, 10, 183-186), and are effective at low concentrations (0~1-0.2%) in promoting pleomorphism and enhancing PHB or PHBV yield.
Other useful pleomorphic agent:s include fish fertilizer, yeast extract, casein and lactalbumin solubles, and mixtures thereof. As noted above, there are many types of pleomorphic agent:s available which may be identified in the conte~t of the~ present disclosure using routine non-inventive experimentation. However, Applicant has found these agents particularly useful in the above noted ranges.
Typically the fish peptone is an enzymatic or autolytic digest of Atlantic cod. Howaver, other fish species can be used. The digests may be further standardized by ultrafiltration and may be supplied as powders, syrups or sterile solutions. Usually they are 100% soluble. Fish peptone comprises from about 65 to 80, preferably from 70 to 80 weight ~ of protein and has a total nitrogen content from about 10 to 15 weight %, and an amino nitrogen content from about 3 to ~ weight %.
The amino acid content is comprised of common amino - : :,, ,; . :
:;:.
. : :
2~697~2 acids, taurine, and choline. Other ingredients may include salts of phosphate, magnesium, calcium, sulfate, sodium, and potassium. A description of the production of fish peptones is found for example in the trade literature of Protan Biopolymers of Norway whose post office address is P.O. Box 3271 Gronnasen, 9001 Tromso, Norway.
In addition to the above elements, it is beneficial if the medium contains a non-ammonium ion nitrogsn source. In the paper "Cyst Formation and Poly-~-hydroxybutyric Acid Accumulation in Azotobacter" by L.H.
Stevenson and M.D. Socolofsky, Journal of Bacteriology, 1966, 91, 304-310, it is clearly demonstrated that ammonium ions decrease the amount of PHB formed by three strains of A. vinelandii. The reason for this was speculated to relate to the cell's ability to oxidize tha carbon and energy source faster than it could synthesize protein in the absence of ammonium ions. In this situation the cells will reduce the nitrogen in the air by the action of nitrogenase enzyme, to form amino acids.
The result is a highly reducing en~rironment inside the cell (from the oxidation of the carbon source), an environment that is well suited to the synthesis of PHB.
In the paper "Influence of Nitrogen Source on Growth and Nitrogenase Activity in Azotobacter vinelandii" by D.
Gadkari and H~ Stolp, Archives of Microbiology, 1974, 96, 135-144, it is shown that yeast extract increased the growth of Azotobacter by 25% and the cells retained 64%
of the nitrogenase activity of the cells incubated without additions. This paper did not examine the effects of yeast extract on PHB formation. Similarly, the paper "Amino Acid Transport in Azotobacter vinelandii:
Implications of Nonavailability of Amino Acid Auxotrophs"
by P. Mishra, B. Roy, R. Prasad; and H.K. Das, FEMS, Microbiology Letters, 1991, 79, 41-44, demonstrates that this organism has active transport systems for the accumulation of a variety of amino acids. Thus, the . . . ~
prior art teaches that optimal PHB production should occur in the absence of ammonium ions, a well known repressor of nitrogenase activity. Complex nitrogen sources like yeast extract may promote cell growth, since A. vinelandii has transport activities for the accumulation of a variety of amino acids, but nitrogenase activity need not be repressed. Thus, the selection of a suitable pleomorphic agent is likely to be influenced by the nitrogen compounds therein and their ability to promote cell growth without adversely affecting the reducing environment within the cell.
Accordingly, it may be helpful if the medium contains a nitrogen source which is protein based.
Suitable nitrogen sources may be selected from the group consisting of C2 6 amino acids, proteose peptone #3, yeast extract, bacto-casitone, trytone, casamino acids, proteose peptone, phytone, cacto-peptone, gelatone, beef extract, and neopeptone. The nitrogen source may be used in amounts up to about 0.5 weight ~. Various aspects of the invention are demonstrated in the following examples. In the examples unless otherwise indicated parts are parts by weight and % is weight %. The examples are not intended, nor do t:hey, limit the present invention.
25 CULTURE: MEDIUM `:
In all the experiments the base culture medium consisted of a glucose culture which consisted of Burk's minimal salts medium (e.g. g/l KH2P04, 0.2; K2HP04, 0.8;
MgSO4.7H20, 0.2; CaSO4.2H20, 0.1; FeSO4.7H2O, 0.005; and 30 Na2MoO4.2H2O, 0.0025 at pH 7.2) supplemented with ferric citrate to bring the total iron content to 50-60 ~M, 3 w/v glucose, and 15 mM ammonium acetate.
Ammonium acetate is added to the medium to promote initial cell growth, but at the time of PHB or PHBV
formation the ammonium and the acetate in the medium have been depleted. Thus, the A. vinelandii cells are growing on the carbon sources noted in the Examples and are , , ............ :.~ :
:
2~697.~2 fixing nitrogen (using nitrogen from the air, by the action of nitrogenase).
~xample 1 A standard culture medium (3% glucose) was prepared as above. To the culture medium was added 0.1% w/v of a fish based peptone or 0.4% v/v fish fertilizer. The fish peptone comprised 100% fish peptone ("FP"), fish peptone plus 60% yeast extract ~+60% YE), fish peptone plus 30%
yeast extract (+30% YE), fish peptone plus 30% casein extract (+30% CAS), fish peptone plus 30% lactalbumin solubles (+30% LACT) or fish fertilizer ("FF").
The above glucose medium was inoculated with 4%
(v/v) of UWD strain of Azotobacter vinelandii. Fifty t50) ml of culture were placed in a 500 ml Erlenmyer flask and the flasks were incubated at 28-30C with shaking in a rotary shaker at 250 rpm to provide high aeration. After 24 hours the cultures were analyzed for PHB content using the method described by J.H. Law and R.A. Slepecky, 1961, Journal of Bacteriology, 82, 33-66.
The results of the analysis for PHB in the different cultures is set forth in Table 1.
:', ;
-20~97~2 _~ . _ _ _ _ PHB mg PHB PHB
Peptone~ mg/ml mg protein % dry weight Efficiency _ _ ___ ____ .___ ¦ 1. FP 9.31 8.15 76 0.33 ¦ 2. +60% YE 7.82 4.34 75 0.33 ¦
_ 3 FP 7.94 S.S1 74 0.32 4. +30% YE 7.49 3.90 75 0.32 10S. +30% Cas9.10 7.78 80 0.35 _ . . 11 6. +30% Lact~.69 4.80 74 0.31 ¦
7. FP 8.22 6.63 77 0.23 . . . _ 11 8. FP 8.42 8.67 79 0.28 ¦
_ _ _ 11 9. FF 8.77 6.09 76 0.30 ¦
~ . . _ 11 ' .
1510. Control (No Peptone) 1.79 1.44 53 _ .
A Each line presents representative data obtained with a diferent fish peptone product (from Protan Bioppolyers A/S, Drammen, Norway) 1. - HOlOOBT; 2. -H0260BT; 3. - HO100; 4. - H0230; 5. - H0330; 6. - H0430;
7. - POlOOLS; 8. - L-130; 9. - Fish Fertilizer (from Alaska Fish Fertilizer Co., Reuton, Washington); 10. - 3%
glucose culture only.
b mg PHB produced per mg glucose consumed.
This experiment shows a significant increase in the production of PHB from UWD strain of Azotobactar vinelandii in the presence of fish peptones and peptone mixtures.
;
, 2~9712 Exam~le 2 Same growth conditions as in Example 1, except that the media contained pure sugars at 3% plus or minus the addition of 0.1% fish peptone:
~ABLE 2 PHB mg PHB PHB
Carbon Sollrce FP mg/ml mg protein % dry weight _________ ___ ~ `
Glucose 2.59 2.75 64 + 9.89 11.1 87 ~ ... I.. _._._ .... _ _ _ . . __ Sucrose 1.30 1.86 48 ___ + 7.70 10.4 . 78 Maltose 3.50 3.37 70 _ + 6.80 9.07 77 The, experiment as set out in Table 2 shows that significant improvements in PHB yield can be obtained from pure sugars other than glucose in the presence of 0.1% fish peptone.
Bxample 3 Same growth conditions, as in Example 1, except that the media contained impure, unrefined sugar.
TAB~E 3 _ PHB mg PHB PHB ¦
Car~on Source FP mglrnl mg protein % dry weight l ___ _____ __I
2 5Cane Molasses 0.90 0.58 8 ¦
+ 5.40 3.07 61 l _ .. . __ I
Malt Extract 6.00 3.95 71 :
+ 8.s0 5.4? 72 .
Corn Syrup 5.48 4.22 73 ~`
+ 9.06 7.74 83 . . . . ~
The experiment shows that significant improvements in PHB yield can be obtained from impure, unrefined sugar sources in the presence of 0.1% fish peptone.
, Example 4 Same growth conditions as in Example 1, except that sodium valerate is added to a final concentration of 10 mM to media containing 3% glucose and 0.1% proteose peptone ~3 as a pleomorphic agent.
_ . ~
PHA lEfficiency % HV ~t harvest tim~ of Time of Yalerate m~ PHA _ Addition (h) mg/ml % mg/mgmg glucose 16 h 20 h 24 h _ ~_ _~ protein _ __ _ _ No addn. 6.73 70 5.43 0.35 ND ND O
Generally in the ICI production of PHB/PHBV in the cell occurs during imbalanced growth. Usually this is the stationary phase of bacterial growth, but this can be induced in an actively growing culture by imposing a nutrient (2~ nitrogen, phosphate, or sulfate) limitation in the presence of excess carbon source. During this imbalance, NADH accumulates and exerts a feedback repression on various enzymes whose activities are essential for the continued growth of the cell. NADH can be oxidized to NAD~, eliminating this growth inhibition, by the action of acetoacetyl CoA reductase and the polymerization of acetoacetyl CoA into PHB.
NAD+ is nicotinamide adenine dinualeotide and NADH
is its reduced form. NAD~ is a major electron acceptor in the oxidation of fuel molecules in the cell. NAD~
fulfills this function by accepting two electrons and two hydrogen ions from substances its oxidizes. Thus, NAD+
becomes NADH, Stryer, Biochemistry (2d ed.), W.H. Freeman and Company, San Francisco, pp 244-246.
Azotobacter vinelandii is a harmless soil micro~e that has an obligate 2 requirement for growth and can use N2 as a nitrogen source via nitrogen fixation. A.
yinelandii normally produces PHB by the methods noted above and much of the early work concerning PH~ synthesis was conducted in Azotobacter species. To the best of applicants knowledge the production of PHBV from A.
vinelandii has naver been reported. Azotobacter species that produce large amounts of PHB have been reported, but these cells have been unstable and also produce large amounts of capsule and slime, which interfere with PHB
extraction and decrease the efficiency of conversion of ::
:
.
. . ~ .
.. ..
- . . : : : ::: .
'~..., ... ';
20~97t2 . .
carbon substrate to PHB. Page and Knosp have overcome this drawback by using a mutant strain o~ A. vinelandii which may be gro~n on readily commercially available substrates and which does not produce significant amounts slime.
United States patent 4,138,291 issued February 6, 1979 in the name of Lafferty, assigned to Agroferm AG of Switzerland also discloses a process for selecting microorganisms which may be used to produce poly-hydroxy alkanoates.
One of the difficulties in these processes for producing polymers by culturing strains of microorganisms is the problem of separating the polymer from the microorganism. The cell wall must be ruptured, destroying the microorganism, and the polymer granules must be extracted. Accordingly one of the limitations in the current processes is how much polymer may be incorporated into a cell because the cell cytoplasm, where the granules form, is contained within a rigid cell wall that will grow to dimensions characteristic of that organism (and no larger). The excretion of solid, cell byproducts such as polymers for example, those of PHB/PHBV through the living cell wall and into the culture medium has never been reported in the literature.
This concept has very significant implications for the entire field of biofermentation and production of products such as polymers, inclusions or crystals and their recovery.
Pleomorphic agents are agents which weaken the cell wall of a microorganism. Generally the agents cause the cell wall to rupture, usually due to an osmotic imbalance between the ambient environment and the contents of the cell. Some bacteria are more sensitive to the induction of pleomorphism than others. An agent that will induce pleomorphism in many cultures is complex mixtures of amino acids commercially available as peptones prepared by the enzymatic or acidic digestion of animal, plant, ~ .
20~9~12 milk or fish proteins. The susceptibility of an organism to pleomorphic agents can be determined ~y non-inventive experimentation and is frequently documented in the literature relating to the cultivation and identification of microorganisms (e.g. "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J. G. Holt (ed) Williams and Williams, Baltimore, 1984). In these cases, the addition of pleomorghic agents to the growth medium is avoided, because the cells will lose osmotic stability and rupture (lyse) in normal growth media. However, in other cases, peptones will not induce pleomorphism (or will ~o so only at high concentrations, 1 - 5%) and the peptone may serve as an excellent source of organic nitrogen and carbon for cell growth. However, even in these cases addition of higher concentrations of certain amino acids, notably 0.5 M glycine to the growth medium will induce pleomorphism tN. Welsh ~ P. Osterrieth, Antonie van Leeuwenhoek, Journal of Microbiology and Serology, 1958, 24, 257-273; J.L. Strominger & C.H.
Birge, ~ournal of Bacteriology 1965, 89, 1124-1127; P.H.
Fung ~ A.J. Winter, Journal of Bacteriology, 1968, 1~89 1894).
The paper "Effect of Peptone on Azotobacter Morphology" by G.R. Vela and R.S. Rosenthal, Journal of Bacteriology, July 197~, 260-266, describes the effect of Difco peptone on the morphology of Azotobacter. The paper teaches that the cells become osmotically fragile and pleomorphic or fungoid in appearance. It is noted that one strain of A. vinelandii will yield at least several types of morphology and cells are much larger than normal. The paper does not mention the production of PHB under these conditions and granules of said polymer are not demonstrated in the micrographs presented.
Furthermore, the paper "Polymer Production by a Mucoid Strain of Azotobacter vinelandii in Batch Culture"
by A.C. Brivonese and I.W. Sutherland, Applied .
- ::
Microbiology and Biotechnology, 1989, 30, 97-102, shows that the production of PHB by A. vinelandii strain AX (a mucoid strain) in medium containing 0.05 - 0.2% peptone and 4% glucose reached 1 mg/ml after 48 hours incubation with a low rate of aeration (120 rpm shaking~. If the shaking rate was increased to 280 rpm, the yield of PHB
was not significantly increased (about 1.5 mg/ml) after 48 hours incubation. The authors did note a positive effect on alginate formation by these A. vinelandil strains, but the biosynthesis of PHB and alginate are unrelated.
Similarly, the paper "Correlation of Ultrastructure in ~zotobacter vinelandil with Nitrogen Source for Growth" by J. Oppenheim and L. Marcus, Journal of l'; Bacteriology, 1970, 101, 286-291, instructs that when 0.5% casamino acids (an acid digest of casein) was present as a source of amino acids for the growth of strain OP (ATCC 13705) "PHB does not seem to be synthesized when nitrate or amino acids are used as the nitrogen source". Thus, the prior art teaches away from the incorporation of pleomorphic agents (peptones or glycine) into the culture medium and teaches that peptones or casamino acids do not promote the formation of PHB.
A variety of microbes are susceptible to pleomorphic agents resulting in the formation of enlarged and osmotically fragile cells. The paper "Effect of Yeast Extract Concentration on Viability and Cell Distortion in Rhizobium spp." by F.A. Skinner, R.J. Roughley, and M~riel R. Chandler, Journal of Applied Bacteriology, 1977, ~3, 287-297 demonstrates that yeast extract acts as a pleomorphic agent in the culture of Rhizobium spp. The thrust of the paper is on the morphology of the cells and not on the production of polymers using such agents in the culture medium. The paper "A Comparative Study of the Transformation of Gram-Negative Rods into "Protoplasts" Under the Influence of Penicillin and Glycine" by M. Welsch & P. Osterrieth, Antoine van Leeuwenhoek, Journal of Microbiology and Serology, 1958, 2~, 257-273, demonstrates that a number of Gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa, are susceptible to the pleomorphic agent glycine. The pleomorphic cells formed appeared to have remnants of cell wall still attached to the cell and have varied osmotic fragility, in some cases even 20~ sucrose in the growth medium would not stabilize the cells. The paper "Nucleotide Accumulation Induced in Staphyloccus aureus by Glycine" by J.L. Strominger & C.H. Birge, Journal of Bacteriology, 1965, 89, 1124-1127, and the references show that the pleomorphic agent glycine interfer~s with cell wall synthesis in Gram-positive organisms as well. Thus, the prior art teaches that representatives of both Gram staining groups (Gram-positive and Gram-negative) are susceptible to pleomorphic agents.
The paper "Microbial Growth on Peptones from Fish Industrial Wastes" by Susan E. Vecht-Lifshitz, ~.A.
Almast and E. Aomer, Letters in Applied Microbiolo~y, 1990, 10, 183-186, discloses growing Streptomyces tendae ATTC 31160, Gibberella fulikuroi, ~3acillus thurinqiensis var. israliensis 1884, Escherichia coli B, Salmonella typhi, Staphylococcus aureus, Serratia marcescens and Proteus vulgaris in the presence of fish peptones to produce toxins. The paper does not suggest the use of such a procedure to produce polymers.
Accordingly, the prior art teaches away from incorporating pleomorphic agents into a culture medium.
In the production of polymers by culturing microorganisms, especially in the case of PHB and PHBV
which is an intracellular accumulation, there is a need for a method for either increasing the size of cell which may be grown thus providing for an increase cell volume and hence polymer volume, or better still to provide a :
206~71~
mechanism to permit the cell to excrete or extrude polymer into the culture medium.
The present invention seeks to provide such a mechanism.
DT8C~OBURE OF THE I~VENTION
Accordingly, the present invention provides in a process of producing a polymer by culturing a strain of microorganism having a hyperproduction of polymer during exponential growth in a medium comprising at least a source of assimilable carbon, the improvement comprising subjecting the bacteria to a controlled pleomorphism by introducing into the culture from 0.05 to 0.20 weight ~
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture medium.
A further embodiment of the present invention provides a process for increasing the cell size during the culturing of a microorganism in a medium comprising at least one source of assimilable carbon which comprises introducing i~to the culture medium from 0.05 to 0.20 weight % of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
The present invention further provides a process for increasing the number of granules per cell in the culturing of a microorganism which produces granules of polymer, when cultured in a medium comprising at least one source of assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight %
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance -between the microorganism and the culture.
The present invention further provides a process whiah permits the excretion of granules through the cell wall of a microorganism cultured in a medium comprising at least one source o~ assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight ~
" ;"` ~ "
" i, 'i 2~69712 of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
BRIEF DE8C~IPTION OF DRAWIN~S
Figures 1 A, B and C are electron micrograph~ of cultures of UWD cells of Azotobacter vinelandii which have been cultured in the absence of pleomorphic agents (Fig. lA), in the presence of proteose peptone #3 (Fig.
lB) and in the presence of fish peptone (Fig. lC). The electron micrographs show that fish peptone stabiilizes the cells, prevents cell lysis, and promotes the extrusion of the polymer ~rom the cells without a loss of cell integrity.
BBS~ MODE FOR CARRYING OUT THE INVENTION
A number of polymers may be produced by the culturing of microorganisms. However, particularly useful polymers include the poly-hydroxyalkanoates. The poly-hydroxyalkanoates which may be produced in accordance with the present invention may be co or homopolymers of one or more monomers residues of the formula -OCRIR2(cR3~)n-cO
wherein n is 1 or 2; and Rl, R2, R3 and ~ are independently selected from the group consisting of a hydrogen atom and Cl2 alkyl radicals. Preferably in formula 1, n is 1 or 2 and Rl, R2, R3 and ~ are independently selected from the group consisting of a hydrogen atom, a methyl radical and an ethyl radical. If the polymer is a homopolymer and n is 1 and one of Rl and R2 is a methyl radical and the other is a hydrogen atom and both R3 and ~ are hydrogen atoms, the polymer is poly-B-hydroxybutyrate (PHB). If the polymer is a copolymer and n, R~, R2, R3 and ~ are as described immediately above in one monomer residue and in the other monomer residue, n is 2, one or R~ and R2 is a methyl radical and the other is hydrogen and R3 and ~ (both occurrences) are hydro~en the polymer is PHBV.
.
.,:
' - . ~
Preferably in the above copolymer the mole ratio of the butyrate residues to the valerate residues is from about 70:30 to about 9~:1, most preferably above 95:5.
If it is desired to produce a copolymer in accordance with the present invention it is necessary to add a precursor. The precursor may be a Csg odd numbered alkanoic acid or a salt thereof such an alkali metal salt such as sodium or potassium salts. Preferably the precursor is sodium valerate or valeric acid. The precursor may be present in the growth medium in a concentration from 5 to 45, preferably 10 millimolar ("mM"). The precursor should be added during the time when the cells are actively forming polymer. The percent comonomer (e.g. hydroxyvalerate; HV) in the polymer may be increased by adding higher concentrations of precursor, or by repeated addition of precursor, or by addition of the precursor shortly (between 4 to 8 hours) before harvesting.
The present invention may be practised ~ith microorganisms which under go exponential growth in a culture medium. The application of the principles of this disclosure to other microbial systems is expected to involve some period of microbial growth, because the pleomorphic agents exert their effect through interference ~ith cell wall growth. The number of cell divisions and extent of growth would be determined by non-inventive experimenttation in each case.
A bacterial transformant, use~ul in accordance with the present invention, which for the purpose of reference in the detailed description is identified as UWD, was derived from the species Azotobacter vinelandii. It is understood that the invention encompasses not only the particular UWD, but all derivatives thereof and other related microorganisms having similar taxonomic 3~ descriptions. The Azotobacter genus is described in "Bergey's Manual of Determinative Bacteriology" b~v N.R.
Krieg and J.G. Holt (ed.), Williams and Wilkins, . .
.. . .
~ ,:
-, . ~ :
2~6g71~
ll Baltimore, 1984, 1, 219-229; and "Azotobacteraceae: The taxonomy and ecology of the aerobic nitrogen-fixing bacteria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp. 168-69, pp. 178-79.
Azotobacter vinelandii wild-type, which for the purpose of reference in the detailed description is identified as OP, is readily available and has been investigated by various groups. Azotobacter vinelandii OP is deposited at American Type Culture Collection under accession number ATCC 13705. It is understood that throughout the specification, the generally accepted nomenclature "ATCC" for the American Type Culture Collection will be used. ATCC is located at 12301 Parklawn Drive, Rockville, Maryland, U.S.A. All deposits as ATCC are given accession numbers which are referred to throughout the specification. The University of Wisconsin subculture of Azotobacter vinelandii OP is identified as UW for the purpose of reference in the detailed description. The UWD transformant, according to this invention, was developed and :isolated by transforming UW cells with DNA preparPd from the mutant Azotobacter vinelandil ATCC 63800 Itwhich for the purpose of reference in the detailed descrlption is identified as strain 113) such as defined in the following Example. It is appreciated that there are many techniques available for inducing such mutation and th~t: there are many techniques available for transforming bacterial cells or otherwise changing their genetic composition and such other techniques are contemplated herein although not specifically exemplified.
The isolated genetic transformant has been characterized in the following Examples and has been deposited at th~ American Type Culture Collection. The deposit was made on August 10, 1988 under accession number ATCC 53799~ The taxonomical characteristics of the UWD strain are as follows.
.
.
.... .
~06g~ 2 The UWD strain shares characteristics with strain UW
(OP, ATCC 13705) except that it forms excess PHB during exponential growth, it is resistant to rifampicin (20~g/ml), it has a white colony colour, and it forms the fluorescent-green pigment under iron-sufficient conditions.
Basic characteristics: Large ovoid cells, Gram-negative, pleomorphic (in this sense meaning polymorphic eg. multiple forms and not necessarily swollen or osmotically fragile) ranging from rods to cocci. The cells are motile by peritrichous flagella, substantial capsules are not formed and the cells form fragile cysts only after very long incubation (several months to a year). Growth is strictly aerobic and nitrogen is fixed aerobically. Substrate utilization characteristics have been well defined and agree with "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J.G. Holt (ed.), Williams and Wilkins, Baltimore, 1984, 1, pp.
219-229; and "Azobacteraceae: The taxonomy and ecology of the aerobic nitrogen-fixing bact:eria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp.
168-69, pp. 178-179.
The taxomonic description of s~train 113 is as follows.
This strain is derived from A. vinelandii ATCC 12837 by NTG mutaqenesis. It shares characteristics with ATCC
12837 except it is resistant to rifampicin (20~g/ml) and it forms poly-B-hydroxybutyrate (PHB) during exponential growth.
Basic characteristics: Large ovoid cells, Gram-negative, pleomorphic (in this sense meaning polymorphic e.g. multiple forms and not necessarily swollen or osmotically fragile) ranging from rods to cocci. The cells are motile by peritrichous flagella, form capsules and form cysts in older cultures. Iron-limited cultures produce a fluorescent yellow-green pigment. Growth is strictly aerobic and nitrogen is fixed aerobically.
: . -"~ '' . -, 2~69712 Substrate utilization characteristics have been welldefined and agree with "Bergey's Manual of Determinative Bacteriology" by N.R. Krieg and J.G. Holt (ed.), Williams and Wilkins, Baltimore, 1984, 1, pp. 219-229; and I'Azotobacteraceae: The taxonomy and ecoL~ o~ the aerobic nitrogen~fixing bacteria" by J.P. Thompson and V.B.D. Skerman, 1979, Academic Press, New York, pp. 168-~9, pp. 178-79.
The UWD transformant was developed and isolated in accordance with the following preferred method. Cells of strain UW were obtained from the Department of Bacteriology at University of Wisconsin. This strain of cells is a capsule-negati~e wild-type, which were genetically transformed with DNA prepared from cells of stain 113 Azotobacter vinelandii 53800, which is a rifampin-resistant strain derived by NTG metagenesis of the capsule-positive strain ATCC 12837.
Strain 113 ~ATCC 53~00) was produced by exposing Azotobacter vinelandii strain ATCC 12837 to 100 ~g/ml N-methyl-N'-nitro-N-nitrosoguanidine (NTG) in Burk buffer, pH 7.2, for 30 minutes. Survivors were plated on Burk medium containing 1% glucose, 1.8% agar and 20 ~g rifampin/ml. Strain 113 was, therefore, selected as a nitrogen-fixing, rifampin-resistant strain of ATCC 12837.
The hyperproduction of PHB by strain 113 was an unselected mutation that also has occurred during the NTG - -mutagenesis procedure DNA for transformation was prepared as a crude lysate material. A thick suspension of strain 113 was prepared in 15 mM saline-15 mM sodium citrate buffer, pH
7.0, containing 0.05% sodium dodecyl sulfate. This suspension was heated at 60C for 60 minutes in a water bath. When cool, this lysate containing crude (unpurified) DNA was used directly in transformation assays. Optimal conditions for generation of competent strain UW (or Azotobacter vinelandii in general) which can take up this crude DNA, are documented in Page and . .
' ~ ' 20697~2 von Tigerstrom, 1978, Canadian Journal of Microbiology, 24, 1590-1594. Optimal conditions for the transformation of these competent cells by the crude DNA are documented in Page and von Tigerstrom, 1979, Journal of Bacteriology, 139, 1058-1061.
The preferred procedure used to transform UW cells with strain 113 DNA is described in Page, W.J., 1985, Canadian Journal of Microbiology, 31, 659-662. While almost any strain of Azotobacter vinelandii can be transformed by strain 113 DNA, the most successful transformations for the purpose of producing maximal amounts of readily extractable PHB are the transformations of capsule-negative strains. Of these, Azotobacter vinelandii strain OP (ATCC 13705) is a readily available strain held in culture collections.
The strain that was transformed to create the UWD
transformant was strain UW, the University of Wisconsin copy of strain OP.
Transformation of strain UW w;ith strain 113 DNA
results in a rifampin-resistance transformation frequency of about 1.0 x 104 to 8.7 x 10-3 per viable cell plated (i.e. at best 8.7 transformants per. 1000 cells plated).
This is a readily reproducible fre~uency of transformation. Of these rifampin~-resistant colonies, about 13% appeared to have streaks of white or white sectors within the normal pinkish-tan cell mass. When these sec~oring colonies were restreaked, they gave rise to sectoring colonies and dense white colonies at a ratio of about 1:1. The dense white colonies were then selected and designated UWD. This procedure and these rasults are reproducible and will readily ~enerate UWD
cells of this invention.
UWD is readily separated from the UW stock because UWD is resistant to rifampin and UW is not resistant.
Therefore, on plates containing solid medium and 20 ~g rifampin/ml, only colonies of UWD ~or other rifampin resistant cells) will grow.
'~
0697~2 The separation of PHB hyperproducing cells from the general population of transformed cells is readily reproducible, because the sectoring colony phenotype is quite distinctive and upon subculture it readily generates solid white colonies. Non-hyperproducing rells result in pinkish-tan colonies under the same conditions.
The cells from these dense white colonies (the UWD
cells) were packed with PHB granules, while the wild-type (strain UW) contained only a few small granules of PHB.
It is believed that in theory the production of PHB/PHBV in the mutant is due to a genetic defect in strain 113 and the UWD cells concerning the NADH oxidase that normally recycles NAD~ via respiratory oxidation of NADH. As a result of this defect, the cell accumulates NADH and must turn-on PHB/PHBV synthesis in order to grow. Therefore, PHB/PHBV are formed during active (balanced; exponential) growth, the exact opposite of the normal conditions promoting PHB/PHBV formation in the wild-type. Although this theory appears to be borne out by the examples, it is understood that the principles of the invention should not be limited to this theory.
Because PHB/PHBV type polymers are formed during exponential growth, conditions which enhance growth also increase PHB/PHBV type polymer formation. For example, 2S vigorous aeration (rather than 02-limitation) promotes faster use of glucose and faster production of PHB/PHBV
type polymers. Nitrogen-fixing UWD cells also produce PHB.
Strain 113 also produces large amounts of PHB, but also produces large amounts of capsule and slime. The UWD cells of this invention do not form substantial capsule and slime and therefore only convert the bulk of the sugar into cell mass (like strain UW) and PHB.
In summary, the genetically transformed bacterium of the species Azotobacter vinelandil has the identifying characteristics of:
(i) extremely abundant PHB granules in cells;
. , :, -, ~ :
! . , ~
~,~
' , . - ' ' . " ""
- 2~69712 (ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of PHB during the exponential growth of the mutant microorganism.
Production of PHB was demonstrated in batch cultures. The volumes of culture were in the range 20-50% culture volume: flask or vessel volume with rotary shaking (to increase aeration and mixing) at 175 to 300 rpm (normally 250 rpm was used). Incubation temperatures were found to be 28-30~C for optimal yields. -The nitrogen source used in the cell culture is preferably N2 (from air) or ammonium acetate at 1.1 to 2.2 g/L-Various carbon sources were added to the culture medium at 1 to 5% (w/v) concentration. PHB production was best with reduced hexose, mono and disaccharide carbon sources (glucose, sucrose, maltose) or with sodium gluconate or glycerol, and was much lower with more oxid:lzed or short-chain carbon sources (acetate, ethanol). Glucose and sucrose are relatively expensive refined substrates for PHB production. Sucrose does not have to be "invertedl' before use by the UWD cells but fructose is poorly used for PHB production. Because PHB
formation is not dependent ~n nutrient limitation, cheaper unrefined carbon sources can be used by the UWD
cells. Good production of PHB has been obtained using 2%
(wtv) corn syrupt sugar-beet molasses, blackstrap and cooking molasses, and more refined grades of refiner's molasses. The UWD cells grow well in media containing at least 5% (w/v) molasses or corn syrup, however, the yield of PHB ml~~ is usually not greater than that obtained with 2 or 3% molasses or corn syrup except for a further preferred embodiment of this invention as noted below.
.
~-On these impure carbon sources the wild-type strains did not produce detectable or significant amounts (<0.1 mg/ml) of PHB.
Growth of UWD cells and yield of PHB under intense aeration conditions (culture volume 10% of vessel volume) were unexpectedly increased when sugar beet molasses was used as the carbon source. Under intense aeration conditions (as established by the culture volume being 10% of the vessel volume during shaker flask culture), a yield of S.42 PHB/ml was obtained with 5% sugar beet molasses, a 42-fold increase over the yield obtained with 2% sucrose as carbon source under the same conditions.
Sugar beet molasses concentrations greater than 5%, or increases in sucrose only, had no further beneficial effect. This growth promotional effect of sugar beet molasses under intense aeration conditions was observed with all ten different samples of sugar beet molasses examined.
Increasing PHB yields by use of an unrefined carbon source such as sugar beet molasses offers a significant reduction in production costs.
This increase in PHBV production at high aeration was only observed in beet molases. This improved method (i.e. increased a~ration of a culture containing 5% w/v sugar substrate) did not improve PHBV yields in cultures containing other impure and inexpensive sugar sources or other pure sugar sources. A typical approximate analysis of beet molasses is 81-84~ w/v) solids comprised of 50-52% sucrose, 12-20~ nitrogenous compounds (mostly amino acids and betaine), 12-13% non-nitrogenous compounds (mostly organic acids) and 11-12% ash (salts) (R. A.
McGinnis "Sugar beet Technology", 3rd Edition, 1982.
Beet Sugar Development Foundation, Fort Collins, Colorado~. Of the 100's of compounds present, it seemed most likely that the organic nitrogenous compounds may have been responsible for the observed PHBV yield promoting effect. This was suggest~d by the fact that , :
~;
.~
,,: ' .:, ' 206971~
sugar was not limiting in these other cultures and organic acids are known not to promote PHB formation in this organism (W. J. Page, 198g. Applied Microbiology and Bio-technology 31, 329-333). Extensive tests, demonstraed that the addition of a variety of complex nitrogen sources to well aerated cultures containing pure or unrefined sugars increased the yield of PHBV. This is demonstrated in the examples presented. Furthermore, these complex nitrogen sources, when added into the culture medium in an amount of about 0.05 to 0.20~
(weight %) acted as pleomorphic agents, causing the cells to swell, allowing them to fill with many polymer granules and extrude polymer through the weakened cell wall.
After reading the present disclosure, suitable pleomorphic agents may be identified using non-inventive experimentation. What is required is an agent which will at a concentration of from 0.05 to 0.20 weight % in the culture medium weaken the cell wall permitting it to distend but not result in cell wall rupture under the conditions of use.
A skilled biologist or biochemist would realize that the critical balance is between th~l osmotic pressure within the cell and the osmotic pressure of the culture medium. Accordingly one may have to incorporate one or more agents which balance the osmotic pressure in the cell and the culture medium. Generally such agents would comprise salts. Additionally, such agents could include agents which are capable of acting as surface active agents such as complex polysaccharides including sugars, and lipo`ropic substances such a betaine, choline, taurine and other members of the choline/methionine cycle.
Thus beet molasses per se may be an effective pleomorphic agent because it contains a variety of nitrogen compounds, some of which appear to promote pleomorphism in A. vinelandii, plus a mixture of salts, sucrose and betaine to stabilize the cells. Similarly fish peptone contains a ~ariety of amino acids which appear very effective in promoting pleomorphism, plus a mixture of salts, taurine and choline for stabilizing the cells.
Some useful pleomorphic agents include enzymatic or acid digests of one or more proteins or protein extracts.
For example, suitable pleomorphic agents in this application includa proteose peptone #3 ~Difco), Bacto-casitone, tryptone, casamino acids, proteose peptone andthe fish peptones commercially available from Marine Biochemicals a.s. in Tromso, Norway. Fish peptones have the advantage of being a relatively inexpensive peptone that is readily a~ailable for large scale fermentations, they ha~e a beneficial effect on promoting good cell growth (Vecht-Lifshitx, Almas & Zomer. Letters in Applied Microbiology, 1990, 10, 183-186), and are effective at low concentrations (0~1-0.2%) in promoting pleomorphism and enhancing PHB or PHBV yield.
Other useful pleomorphic agent:s include fish fertilizer, yeast extract, casein and lactalbumin solubles, and mixtures thereof. As noted above, there are many types of pleomorphic agent:s available which may be identified in the conte~t of the~ present disclosure using routine non-inventive experimentation. However, Applicant has found these agents particularly useful in the above noted ranges.
Typically the fish peptone is an enzymatic or autolytic digest of Atlantic cod. Howaver, other fish species can be used. The digests may be further standardized by ultrafiltration and may be supplied as powders, syrups or sterile solutions. Usually they are 100% soluble. Fish peptone comprises from about 65 to 80, preferably from 70 to 80 weight ~ of protein and has a total nitrogen content from about 10 to 15 weight %, and an amino nitrogen content from about 3 to ~ weight %.
The amino acid content is comprised of common amino - : :,, ,; . :
:;:.
. : :
2~697~2 acids, taurine, and choline. Other ingredients may include salts of phosphate, magnesium, calcium, sulfate, sodium, and potassium. A description of the production of fish peptones is found for example in the trade literature of Protan Biopolymers of Norway whose post office address is P.O. Box 3271 Gronnasen, 9001 Tromso, Norway.
In addition to the above elements, it is beneficial if the medium contains a non-ammonium ion nitrogsn source. In the paper "Cyst Formation and Poly-~-hydroxybutyric Acid Accumulation in Azotobacter" by L.H.
Stevenson and M.D. Socolofsky, Journal of Bacteriology, 1966, 91, 304-310, it is clearly demonstrated that ammonium ions decrease the amount of PHB formed by three strains of A. vinelandii. The reason for this was speculated to relate to the cell's ability to oxidize tha carbon and energy source faster than it could synthesize protein in the absence of ammonium ions. In this situation the cells will reduce the nitrogen in the air by the action of nitrogenase enzyme, to form amino acids.
The result is a highly reducing en~rironment inside the cell (from the oxidation of the carbon source), an environment that is well suited to the synthesis of PHB.
In the paper "Influence of Nitrogen Source on Growth and Nitrogenase Activity in Azotobacter vinelandii" by D.
Gadkari and H~ Stolp, Archives of Microbiology, 1974, 96, 135-144, it is shown that yeast extract increased the growth of Azotobacter by 25% and the cells retained 64%
of the nitrogenase activity of the cells incubated without additions. This paper did not examine the effects of yeast extract on PHB formation. Similarly, the paper "Amino Acid Transport in Azotobacter vinelandii:
Implications of Nonavailability of Amino Acid Auxotrophs"
by P. Mishra, B. Roy, R. Prasad; and H.K. Das, FEMS, Microbiology Letters, 1991, 79, 41-44, demonstrates that this organism has active transport systems for the accumulation of a variety of amino acids. Thus, the . . . ~
prior art teaches that optimal PHB production should occur in the absence of ammonium ions, a well known repressor of nitrogenase activity. Complex nitrogen sources like yeast extract may promote cell growth, since A. vinelandii has transport activities for the accumulation of a variety of amino acids, but nitrogenase activity need not be repressed. Thus, the selection of a suitable pleomorphic agent is likely to be influenced by the nitrogen compounds therein and their ability to promote cell growth without adversely affecting the reducing environment within the cell.
Accordingly, it may be helpful if the medium contains a nitrogen source which is protein based.
Suitable nitrogen sources may be selected from the group consisting of C2 6 amino acids, proteose peptone #3, yeast extract, bacto-casitone, trytone, casamino acids, proteose peptone, phytone, cacto-peptone, gelatone, beef extract, and neopeptone. The nitrogen source may be used in amounts up to about 0.5 weight ~. Various aspects of the invention are demonstrated in the following examples. In the examples unless otherwise indicated parts are parts by weight and % is weight %. The examples are not intended, nor do t:hey, limit the present invention.
25 CULTURE: MEDIUM `:
In all the experiments the base culture medium consisted of a glucose culture which consisted of Burk's minimal salts medium (e.g. g/l KH2P04, 0.2; K2HP04, 0.8;
MgSO4.7H20, 0.2; CaSO4.2H20, 0.1; FeSO4.7H2O, 0.005; and 30 Na2MoO4.2H2O, 0.0025 at pH 7.2) supplemented with ferric citrate to bring the total iron content to 50-60 ~M, 3 w/v glucose, and 15 mM ammonium acetate.
Ammonium acetate is added to the medium to promote initial cell growth, but at the time of PHB or PHBV
formation the ammonium and the acetate in the medium have been depleted. Thus, the A. vinelandii cells are growing on the carbon sources noted in the Examples and are , , ............ :.~ :
:
2~697.~2 fixing nitrogen (using nitrogen from the air, by the action of nitrogenase).
~xample 1 A standard culture medium (3% glucose) was prepared as above. To the culture medium was added 0.1% w/v of a fish based peptone or 0.4% v/v fish fertilizer. The fish peptone comprised 100% fish peptone ("FP"), fish peptone plus 60% yeast extract ~+60% YE), fish peptone plus 30%
yeast extract (+30% YE), fish peptone plus 30% casein extract (+30% CAS), fish peptone plus 30% lactalbumin solubles (+30% LACT) or fish fertilizer ("FF").
The above glucose medium was inoculated with 4%
(v/v) of UWD strain of Azotobacter vinelandii. Fifty t50) ml of culture were placed in a 500 ml Erlenmyer flask and the flasks were incubated at 28-30C with shaking in a rotary shaker at 250 rpm to provide high aeration. After 24 hours the cultures were analyzed for PHB content using the method described by J.H. Law and R.A. Slepecky, 1961, Journal of Bacteriology, 82, 33-66.
The results of the analysis for PHB in the different cultures is set forth in Table 1.
:', ;
-20~97~2 _~ . _ _ _ _ PHB mg PHB PHB
Peptone~ mg/ml mg protein % dry weight Efficiency _ _ ___ ____ .___ ¦ 1. FP 9.31 8.15 76 0.33 ¦ 2. +60% YE 7.82 4.34 75 0.33 ¦
_ 3 FP 7.94 S.S1 74 0.32 4. +30% YE 7.49 3.90 75 0.32 10S. +30% Cas9.10 7.78 80 0.35 _ . . 11 6. +30% Lact~.69 4.80 74 0.31 ¦
7. FP 8.22 6.63 77 0.23 . . . _ 11 8. FP 8.42 8.67 79 0.28 ¦
_ _ _ 11 9. FF 8.77 6.09 76 0.30 ¦
~ . . _ 11 ' .
1510. Control (No Peptone) 1.79 1.44 53 _ .
A Each line presents representative data obtained with a diferent fish peptone product (from Protan Bioppolyers A/S, Drammen, Norway) 1. - HOlOOBT; 2. -H0260BT; 3. - HO100; 4. - H0230; 5. - H0330; 6. - H0430;
7. - POlOOLS; 8. - L-130; 9. - Fish Fertilizer (from Alaska Fish Fertilizer Co., Reuton, Washington); 10. - 3%
glucose culture only.
b mg PHB produced per mg glucose consumed.
This experiment shows a significant increase in the production of PHB from UWD strain of Azotobactar vinelandii in the presence of fish peptones and peptone mixtures.
;
, 2~9712 Exam~le 2 Same growth conditions as in Example 1, except that the media contained pure sugars at 3% plus or minus the addition of 0.1% fish peptone:
~ABLE 2 PHB mg PHB PHB
Carbon Sollrce FP mg/ml mg protein % dry weight _________ ___ ~ `
Glucose 2.59 2.75 64 + 9.89 11.1 87 ~ ... I.. _._._ .... _ _ _ . . __ Sucrose 1.30 1.86 48 ___ + 7.70 10.4 . 78 Maltose 3.50 3.37 70 _ + 6.80 9.07 77 The, experiment as set out in Table 2 shows that significant improvements in PHB yield can be obtained from pure sugars other than glucose in the presence of 0.1% fish peptone.
Bxample 3 Same growth conditions, as in Example 1, except that the media contained impure, unrefined sugar.
TAB~E 3 _ PHB mg PHB PHB ¦
Car~on Source FP mglrnl mg protein % dry weight l ___ _____ __I
2 5Cane Molasses 0.90 0.58 8 ¦
+ 5.40 3.07 61 l _ .. . __ I
Malt Extract 6.00 3.95 71 :
+ 8.s0 5.4? 72 .
Corn Syrup 5.48 4.22 73 ~`
+ 9.06 7.74 83 . . . . ~
The experiment shows that significant improvements in PHB yield can be obtained from impure, unrefined sugar sources in the presence of 0.1% fish peptone.
, Example 4 Same growth conditions as in Example 1, except that sodium valerate is added to a final concentration of 10 mM to media containing 3% glucose and 0.1% proteose peptone ~3 as a pleomorphic agent.
_ . ~
PHA lEfficiency % HV ~t harvest tim~ of Time of Yalerate m~ PHA _ Addition (h) mg/ml % mg/mgmg glucose 16 h 20 h 24 h _ ~_ _~ protein _ __ _ _ No addn. 6.73 70 5.43 0.35 ND ND O
5.57 63 4.22 0.33 ND ND 3.6 4 5.23 64 3.74 0.33 ND ND 4.5 : ' 8 4.94 52 3.53 0.53 20.1 ND 11.2 .. _ _ . .. _ 12 6.61 72 5.46 0.48 18.3 20.616.6 16 7.07 73 6.09 0.53 ND 14.5t5.3 These results, as set out in Table 4, show that HV
contents between 5 - 21% can be readily generated by the addition of sodium valerate to cells growing on glucose in the presen~e of a pleomorphic agent. The timing of addition is important to coincide with active PHB
formation (8 - 16 h period, under these conditions).
There appears to be a slight lag between valerate uptake into the cell and valerate appearance in the polymer (=
4 h~. ;
Example S
Same growth conditions,as Example 1, except that sodium valerate is added to a final concentration of 10 mM after 14 h incubation of a medium containing 3~
glucose and 0.1% fish peptone, or the medium consists of .
.
- 2~9712 30-45 mM sodium valerate and 0.1% fish peptone, without glucose addition~
TAB~E 5 _ l Valerate PHA % HV at 5 I Carbon SourceConsists (mM) I mg/ml % mg/mg protein 22 h I
~~~ __ _~ __ __ 3% Glucose 0 7.71 79 ND 0 S 749 76 S.24 8.9 8.02 79 7.7013.0 7.22 68 7.5215.8 _ . _ 20 6.04 70 6.~8 164 30 mM Valerate 069 33 1.38 285 l 45 mM Valerate 1.13 42 1.79 45.4 . _ The experiment shows, as set out in Table 5, that copolymer is readily formad in pure sugar culture by the addition of sodium valerate. In addition, the combination of fish peptone and sodium valerate as a carbon source promotes some growth and a high % copolymer formation. The production of commercially useful copolymers (butyrate: valerate ratios between 70:30) can be formed in this growth medium.
ExamplQ _6 According to a recent review "Occurrence, Metabolism, Metabolic Role, and Inclustrial Uses of Bacterial Polyhydroxyalkanoates" by A.J. Anderson and E.A. Dawes, Microbiology Reviews, 1990, 54, 450-472, the p~B producing cell normally contains 6-8 granules of pHB
per cell, with the granules being initiated when the synthesis of polymer is initiated. However, examination of A. vinelandii strain UWD growing in 3% glucose medium containing 0.1% fish peptone by light microscopy reveals large swollen cells, filled with many PHB granules.
These cells may be up to 7 ~m in diameter (that is at least 3 times the normal diameter of 2 ~m). When the UWD
.~
' :
20~712 cells being incubated in this medium are examined in the electron microscope, the following observations are set out in Table 6:
TAsLE 6 ;
5Incub$ion CellCellVolume % # ~r~uleSi~e Time Type ~3) Coverageb Granul~/Cell ~) ____ ___ _ ___ ____ 18h Div 7.3 77 14 041 24h Div 6~ 61 41 0.10 A~D 18.0 62 115 0.10 _ 48h Div 68 73 14 0.36 _ ATD 17.8 99 45 0.39 ~ Cell types are Div = cell has just divid~d; ATD =
cell about to divide.
b % cell area covered by granules.
All values are means based on actual measurements of 2 50 cell sections examined in the electron microscope.
The results show that the cells contain 14 to >lO0 granules per cell, depending on the growth phase. It appears that the largest number of granules are in the biggest cells (ATD cells), which upon division pass about hal~ of the granules to each daughter cell. As the cell grows again, the granules increase in number because more granules are initiated enzymatically by the PHB
biosynthetic enzymes. As a result, the A. vinelandii UWD
cells growing in the fish peptone medium contain many more granules per cell than is considered normal.
Example 7 The production of PHB in other organisms may benefit from the addition of fish peptone. In this case Bacillus megaterium NCIB 9521 (NCIB - National Collection of Industrial Bacteria, UK) was cul~ured in a medium containing (g/L) Na2HP04, 0.6; KH2P04, 0.3; NaCl, 0.3; ;
NH4Cl, O.l; Na2S04, O.Ol; MgCl2.6H20, O.Ol; MnCl2.4H20, O.Ol;
Casamino acids, O.Ol with 3% glucose and O.l M sodium :
:
.
2~697~2 acetate. The medium was inoculated and incubated for 24 h with vigorous aeration (50 ml medium per 500 ml flask, 225-250 rpm, at 28-30C). PHB was extracted as described by Law & Slepecky (1961).
S
~ABLE 7 . .. _._ ¦ % FPTotal CellPHB PHB Cell Protein mg PHB
Dry Weight mg/ml% Dry Weight mg/ml mg Protein I 0 3 8S 1.55 40 0.47 3 32 I _ _ 0.1 5 19 3.67 71 0.49 7.52 _ .. _ ~ I
I 0.2 __ 7 28 3.98 5~ 0.43 9 22 0 35.76 _ 2.67 46 0.45 5.97 0.44.32 1 21 28 0 41 2 92 0 52 70 0.99 _ 37 0.49 2.04 15 Table 7 clearly shows an increased PHB yield in the Gram positive organism, Bacillus megaterium, in the presence of 0.1-0.2% fish peptone. This yield increase is not merely the result of increased growth (i.e. the cell protein/ml does not increase at these levels of fish peptone) but is a enhancement of PHB biosynthesis, such that the PHB per ml, P~B per total dry weight and PHB per cell protein are increased. The results also show that higher concentrations of fish peptone are inhibitory to PHB production. Thus, with any given organism an amount of experimentation may be necessary to determine optimal concentrations of peptone to be used.
Examination of the A. vinelandii UWD cells producing PHB in medium containing 3% glucose and 0.1%
fish peptone (from Example 5) reveals that the polymer is being extruded into the extracellular medium. This is an active cell wall extrusion process, not merely the result of cell lysis. Such an active cell extrusion process has never been reported in the literature. The hypothesis for this extrusion process, although not proven, suggests that the cell drives the accumulation of polymer to the ~97~ 2 extent that it exerts considerable pressure in the cell.
The polymer inside the granules inside the cell is present as a fluid lipid (G.N. Barnard & J.K.M. Sanders.
The Poly-~-hydroxybutyrate Granule in vivo. A New Insight Based on NMR Spectroscopy of Whole Cells. Journal of Biological Chemistry, 1989, 264, 3286-3291). The PHB
granule is able to fuse with the cytoplasmic membrane, as demonstrated in the paper " Cellular Incorporation of Poly-~-hydroxybutyrate into Plasma Membranes of -Escherichia coli and Azotobacter vinelandii Alters NativeMembrane Structure" by R. Reusch, T. Hiske, H. Sadoff, ~.
Harris and T. Beveridge, Canadian Journal of Micro~iology, 1~87, 33, 435-444. However, in this present application, the internal pressure of the cell, or the pressure created by polymer syn~h sis, allows the polymer also to be extruded through the weakened cell -wall into the aqueous medium. When the PHB gets out of the cell it is no longer a granule, but an irregular blob of polymer, usually still attached to the cell. This blob of polymer starts to solidify (solidification is known to occur, see Barnard & Sanders, 1989) and effectively plugs the "lipid neck" through which the polymer has been forced. An analoS~y would be the solidification of tar being pushed through a crack in an underwater pipe, which congeals an~l plugs the exit.
However, in the case of the bacterial cell, there is no need for a rupture in the cell membrane or cell wall, only a ~usion of similar materials in the membranes and a diffusion of the polymer through the weakened cell wall (peptidoglycan) structure.
The results show the cultivation in medium containing fish peptone promotes the formation of large numbers of PHB granules and the extrusion of polymer into the medium. Also, when UWD cells that have been grown in the absence of fish peptone are examined, it appears that the cells are fragile and subject to lysis. The presence ~697~2 of fish peptone appears to stabilize the cells, permitting more extensive growth and polymer formation.
Figure 1 shows the stabilization and extrusion of polymsr from strain UWD cells grown in fish peptone. In Figr lA, strain UWD was grown overnight in medium without peptone. Although large cells are seen, these cells are unstable and there is evidence of a loss of cell integrity (the cells loose electron density and membranes curl up, arrows) and lysis (the debris and PHB granules in the background of the photograph). When cells were grown with 0.5% proteose peptone #3 (Fig. !B), there also is loss of cell integrity (arrows) and lysis (debris in background). When grown in 0.2% fish peptone, the cells are s~able (uniformly electron dense) (Fig. !C), and the cells are able to extrude the polym~r into the surrounding medium without cell wall rupture and ysis.
(The size market in all cases is 1 ~m.) INDUS~RIAL APPLICABILITY
The present invention is usefu:L to increase the production of polymers by culturing microorganisms in a variety of growth media containing pure or unrefined sugars. More particularly, the pr~sent invention is applicable to the production of polymers of hydroxybutyrate, (e.g. PHB) and copolymers of hydroxybutyrate and hydroxyvalerate (e.g. PHBV).
Although prPferred embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
contents between 5 - 21% can be readily generated by the addition of sodium valerate to cells growing on glucose in the presen~e of a pleomorphic agent. The timing of addition is important to coincide with active PHB
formation (8 - 16 h period, under these conditions).
There appears to be a slight lag between valerate uptake into the cell and valerate appearance in the polymer (=
4 h~. ;
Example S
Same growth conditions,as Example 1, except that sodium valerate is added to a final concentration of 10 mM after 14 h incubation of a medium containing 3~
glucose and 0.1% fish peptone, or the medium consists of .
.
- 2~9712 30-45 mM sodium valerate and 0.1% fish peptone, without glucose addition~
TAB~E 5 _ l Valerate PHA % HV at 5 I Carbon SourceConsists (mM) I mg/ml % mg/mg protein 22 h I
~~~ __ _~ __ __ 3% Glucose 0 7.71 79 ND 0 S 749 76 S.24 8.9 8.02 79 7.7013.0 7.22 68 7.5215.8 _ . _ 20 6.04 70 6.~8 164 30 mM Valerate 069 33 1.38 285 l 45 mM Valerate 1.13 42 1.79 45.4 . _ The experiment shows, as set out in Table 5, that copolymer is readily formad in pure sugar culture by the addition of sodium valerate. In addition, the combination of fish peptone and sodium valerate as a carbon source promotes some growth and a high % copolymer formation. The production of commercially useful copolymers (butyrate: valerate ratios between 70:30) can be formed in this growth medium.
ExamplQ _6 According to a recent review "Occurrence, Metabolism, Metabolic Role, and Inclustrial Uses of Bacterial Polyhydroxyalkanoates" by A.J. Anderson and E.A. Dawes, Microbiology Reviews, 1990, 54, 450-472, the p~B producing cell normally contains 6-8 granules of pHB
per cell, with the granules being initiated when the synthesis of polymer is initiated. However, examination of A. vinelandii strain UWD growing in 3% glucose medium containing 0.1% fish peptone by light microscopy reveals large swollen cells, filled with many PHB granules.
These cells may be up to 7 ~m in diameter (that is at least 3 times the normal diameter of 2 ~m). When the UWD
.~
' :
20~712 cells being incubated in this medium are examined in the electron microscope, the following observations are set out in Table 6:
TAsLE 6 ;
5Incub$ion CellCellVolume % # ~r~uleSi~e Time Type ~3) Coverageb Granul~/Cell ~) ____ ___ _ ___ ____ 18h Div 7.3 77 14 041 24h Div 6~ 61 41 0.10 A~D 18.0 62 115 0.10 _ 48h Div 68 73 14 0.36 _ ATD 17.8 99 45 0.39 ~ Cell types are Div = cell has just divid~d; ATD =
cell about to divide.
b % cell area covered by granules.
All values are means based on actual measurements of 2 50 cell sections examined in the electron microscope.
The results show that the cells contain 14 to >lO0 granules per cell, depending on the growth phase. It appears that the largest number of granules are in the biggest cells (ATD cells), which upon division pass about hal~ of the granules to each daughter cell. As the cell grows again, the granules increase in number because more granules are initiated enzymatically by the PHB
biosynthetic enzymes. As a result, the A. vinelandii UWD
cells growing in the fish peptone medium contain many more granules per cell than is considered normal.
Example 7 The production of PHB in other organisms may benefit from the addition of fish peptone. In this case Bacillus megaterium NCIB 9521 (NCIB - National Collection of Industrial Bacteria, UK) was cul~ured in a medium containing (g/L) Na2HP04, 0.6; KH2P04, 0.3; NaCl, 0.3; ;
NH4Cl, O.l; Na2S04, O.Ol; MgCl2.6H20, O.Ol; MnCl2.4H20, O.Ol;
Casamino acids, O.Ol with 3% glucose and O.l M sodium :
:
.
2~697~2 acetate. The medium was inoculated and incubated for 24 h with vigorous aeration (50 ml medium per 500 ml flask, 225-250 rpm, at 28-30C). PHB was extracted as described by Law & Slepecky (1961).
S
~ABLE 7 . .. _._ ¦ % FPTotal CellPHB PHB Cell Protein mg PHB
Dry Weight mg/ml% Dry Weight mg/ml mg Protein I 0 3 8S 1.55 40 0.47 3 32 I _ _ 0.1 5 19 3.67 71 0.49 7.52 _ .. _ ~ I
I 0.2 __ 7 28 3.98 5~ 0.43 9 22 0 35.76 _ 2.67 46 0.45 5.97 0.44.32 1 21 28 0 41 2 92 0 52 70 0.99 _ 37 0.49 2.04 15 Table 7 clearly shows an increased PHB yield in the Gram positive organism, Bacillus megaterium, in the presence of 0.1-0.2% fish peptone. This yield increase is not merely the result of increased growth (i.e. the cell protein/ml does not increase at these levels of fish peptone) but is a enhancement of PHB biosynthesis, such that the PHB per ml, P~B per total dry weight and PHB per cell protein are increased. The results also show that higher concentrations of fish peptone are inhibitory to PHB production. Thus, with any given organism an amount of experimentation may be necessary to determine optimal concentrations of peptone to be used.
Examination of the A. vinelandii UWD cells producing PHB in medium containing 3% glucose and 0.1%
fish peptone (from Example 5) reveals that the polymer is being extruded into the extracellular medium. This is an active cell wall extrusion process, not merely the result of cell lysis. Such an active cell extrusion process has never been reported in the literature. The hypothesis for this extrusion process, although not proven, suggests that the cell drives the accumulation of polymer to the ~97~ 2 extent that it exerts considerable pressure in the cell.
The polymer inside the granules inside the cell is present as a fluid lipid (G.N. Barnard & J.K.M. Sanders.
The Poly-~-hydroxybutyrate Granule in vivo. A New Insight Based on NMR Spectroscopy of Whole Cells. Journal of Biological Chemistry, 1989, 264, 3286-3291). The PHB
granule is able to fuse with the cytoplasmic membrane, as demonstrated in the paper " Cellular Incorporation of Poly-~-hydroxybutyrate into Plasma Membranes of -Escherichia coli and Azotobacter vinelandii Alters NativeMembrane Structure" by R. Reusch, T. Hiske, H. Sadoff, ~.
Harris and T. Beveridge, Canadian Journal of Micro~iology, 1~87, 33, 435-444. However, in this present application, the internal pressure of the cell, or the pressure created by polymer syn~h sis, allows the polymer also to be extruded through the weakened cell -wall into the aqueous medium. When the PHB gets out of the cell it is no longer a granule, but an irregular blob of polymer, usually still attached to the cell. This blob of polymer starts to solidify (solidification is known to occur, see Barnard & Sanders, 1989) and effectively plugs the "lipid neck" through which the polymer has been forced. An analoS~y would be the solidification of tar being pushed through a crack in an underwater pipe, which congeals an~l plugs the exit.
However, in the case of the bacterial cell, there is no need for a rupture in the cell membrane or cell wall, only a ~usion of similar materials in the membranes and a diffusion of the polymer through the weakened cell wall (peptidoglycan) structure.
The results show the cultivation in medium containing fish peptone promotes the formation of large numbers of PHB granules and the extrusion of polymer into the medium. Also, when UWD cells that have been grown in the absence of fish peptone are examined, it appears that the cells are fragile and subject to lysis. The presence ~697~2 of fish peptone appears to stabilize the cells, permitting more extensive growth and polymer formation.
Figure 1 shows the stabilization and extrusion of polymsr from strain UWD cells grown in fish peptone. In Figr lA, strain UWD was grown overnight in medium without peptone. Although large cells are seen, these cells are unstable and there is evidence of a loss of cell integrity (the cells loose electron density and membranes curl up, arrows) and lysis (the debris and PHB granules in the background of the photograph). When cells were grown with 0.5% proteose peptone #3 (Fig. !B), there also is loss of cell integrity (arrows) and lysis (debris in background). When grown in 0.2% fish peptone, the cells are s~able (uniformly electron dense) (Fig. !C), and the cells are able to extrude the polym~r into the surrounding medium without cell wall rupture and ysis.
(The size market in all cases is 1 ~m.) INDUS~RIAL APPLICABILITY
The present invention is usefu:L to increase the production of polymers by culturing microorganisms in a variety of growth media containing pure or unrefined sugars. More particularly, the pr~sent invention is applicable to the production of polymers of hydroxybutyrate, (e.g. PHB) and copolymers of hydroxybutyrate and hydroxyvalerate (e.g. PHBV).
Although prPferred embodiments of the invention are described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
Claims (43)
1. In a process of producing a polymer by culturing a strain of microorganism having a hyperproduction of polymer during exponential growth in a medium comprising at least a source of assimilable carbon, the improvement comprising subjecting the microorganism to a controlled pleomorphism by introducing into the culture from 0.05 to 0.20 weight % of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture medium.
2. The process according to claim 1, wherein said medium further comprises an assimilable source of nitrogen.
3. The process according to claim 2, wherein said medium comprises up to 5% (w/v) of one or more members selected from the group consisting of cane molasses, sugar-beet molasses, malt extract, maltose, corn syrup, glucose, and sucrose.
4. The process according to claim 3, wherein said culture optionally further comprises one or more substances to maintain the osmotic balance between the microorganism and the culture medium selected from the group consisting of salts, sugars, betaines, taurine, and choline.
5. The process according to claim 4, wherein said pleomorphic agent is selected from the group consisting of fish peptone, fish fertilizer, yeast extract, protease peptone #3, bactocasitone, typtone, casamino acids, or proteose peptone.
6. The process according to claim 5, wherein said culture is subjected to intense aeration.
7. The process according to claim 6, wherein said microorganism is a genetically transformed bacteria of the species Azotobacter vinelandii having the characteristics:
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
8. The process according to claim 7, wherein said microorganism is a microorganism of the species Azotobacter vinelandii having all the identifying characteristics of ATCC 53799.
9. The process according to claim 8, wherein said polymer comprises one or more monomer residues of the formula:
-OCR1R2(CR3R4)n-C I
wherein n is 0 or an integer from 1 to 5 and R1, R2, R3, and R4 are independently selected from the group consisting of a hydrogen atom and C1-4 alkyl radicals.
-OCR1R2(CR3R4)n-C I
wherein n is 0 or an integer from 1 to 5 and R1, R2, R3, and R4 are independently selected from the group consisting of a hydrogen atom and C1-4 alkyl radicals.
10. The process according to claim 9, wherein said polymer is a homopolymer.
11. The process according to claim 10, wherein in formula I n is 1, one of R1, and R2, is a hydrogen atom and the other is a methyl radical and R3, and R4 are hydrogen atoms.
12. The process according to claim 11, wherein said pleomorphic agent is fish peptone.
13. The process according to claim 9, wherein there is added to said medium from 8 to 16 hours following inoculation of said medium with said microorganism one or more odd numbered C5-9 aliphatic carboxylic acids and alkali or alkaline earth metal or ammonium salts thereof or C1-4 alkyl amine derivatives thereof.
14. The process according to claim 13, wherein said medium contains up to 45 mM of one or more odd numbered C5-9 carboxylic acids or alkali or alkaline earth metal or ammonium salts thereof.
15. The process according to claim 14, wherein said odd numbered C5-9 carboxylic acid is valeric acid.
16. The process according to claim 15, wherein said polymer is a copolymer and in the first monomer residue of formula I n is 1, one of R1, and R2, is a hydrogen atom and the other is a methyl radical and R3, and R4 are hydrogen atoms and in the second monomer residue of formula I n is 2, and one or R1, and R2, is a hydrogen atom and the other is a ethyl radical and R3, and R4 are hydrogen atoms.
17. The process according to claim 16, wherein said pleomorphic agent is fish peptone.
18. A process for increasing the cell size during the culturing of a microorganism in a medium comprising at least one source of assimilable carbon which comprises introducing into the culture medium from 0.05 to 0.20 weight % of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
19. The process according to claim 18, wherein said medium further comprises an assimilable source of nitrogen.
20. The process according to claim 19, wherein said medium comprises up to 5% (w/v) of one or more members selected from the group consisting of cane molasses, sugar-beet molasses, malt extract, maltose, corn syrup, glucose, and sucrose.
21. The process according to claim 20, wherein said culture optionally further comprises one or more substances to maintain the osmotic balance between the microorganism and the culture medium selected from the group consisting of salts, sugars, betaines, taurine, and choline.
22. The process according to claim 21, wherein said pleomorphic agent is selected from the group consisting of fish peptone, fish fertilizer, yeast extract, protease peptone #3, bactocasitone, typtone, casamino acids, or proteose peptone.
23. The process according to claim 22, wherein said culture is subjected to intense aeration.
24. The process according to claim 23, wherein said microorganism is a genetically transformed bacteria of the species Azotobacter vinelandii having the characteristics:
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
25. The process according to claim 24, wherein said microorganism is a microorganism of the species Azotobacter vinelandii having all the identifying characteristics of ATCC 53799.
26. A process for increasing the number of granules per cell in the culturing of a microorganism which produces granules, when cultured in a medium comprising at least one source of assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight %
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
27. The process according to claim 26, wherein said medium further comprises an assimilable source of nitrogen.
28. The process according to claim 27, wherein said medium comprises up to 5% (w/v) of one or more members selected from the group consisting of cane molasses, sugar-beet molasses, malt extract, maltose, corn syrup, glucose, and sucrose.
29. The process according to claim 28, wherein said culture optionally further comprises one or more substances to maintain the osmotic balance between the microorganism and the culture medium selected from the group consisting of salts, sugars, betaines, taurine, and choline.
30. The process according to claim 29, wherein said pleomorphic agent is selected from the group consisting of fish peptone, fish fertilizer, yeast extract, protease peptone #3, bactocasitone, typtone, casamino acids, or proteose peptone.
31. The process according to claim 30, wherein said culture is subjected to intense aeration.
32. The process according to claim 31, wherein said microorganism is a genetically transformed bacteria of the species Azotobacter vinelandii having the characteristics:
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
33. The process according to claim 32, wherein said microorganism is a microorganism of the species Azotobacter vinelandii having all the identifying characteristics of ATCC 53799.
34. A process for to permit the excretion of granules through the cell wall of a microorganism cultured in a medium comprising at least one source of assimilable carbon which comprises introducing into said medium from 0.05 to 0.20 weight % of one or more pleomorphic agents and where required one or more substances to maintain the osmotic balance between the microorganism and the culture.
35. The process according to claim 34, wherein said medium further comprises an assimilable source of nitrogen.
36. The process according to claim 35, wherein said medium comprises up to 5% (w/v) of one or more members selected from the group consisting of cane molasses, sugar-beet molasses, malt extract, maltose, corn syrup, glucose, and sucrose.
37. The process according to claim 36, wherein said culture optionally further comprises one or more substances to maintain the osmotic balance between the microorganism and the culture medium selected from the group consisting of salts, sugars, betaines, taurine, and choline.
38. The process according to claim 37, wherein said pleomorphic agent is selected from the group consisting of fish peptone, fish fertilizer, yeast extract, protease peptone #3, bactocasitone, typtone, casamino acids, or proteose peptone.
39. The process according to claim 38, wherein said culture is subjected to intense aeration.
40. The process according to claim 39, wherein said microorganism is a genetically transformed bacteria of the species Azotobacter vinelandii having the characteristics:
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
(i) extremely abundant polymer granules in cells;
(ii) dense white colonies of cells;
(iii) very turbid culture which reaches an O.D.620 of 10 and looks like cow's milk after 24 hours of growth;
(iv) no formation of substantial capsule or slime;
and (v) hyperproduction of polymer during the exponential growth of the mutant microorganism.
41. The process according to claim 40, wherein said microorganism is a microorganism of the species Azotobacter vinelandii having all the identifying characteristics of ATCC 53799.
42. The process according to claim 41, wherein said granule comprises a polymer.
43. The process according to claim 42, wherein said polymer comprises one or more monomer residues of the formula:
-OCR1R2(CR3R4)n-CO I
wherein n is 0 or an integer from 1 to 5 and R1, R2, R3, and R4 are independently selected from the group consisting of a hydrogen atom and C1-4 alkyl radicals.
-OCR1R2(CR3R4)n-CO I
wherein n is 0 or an integer from 1 to 5 and R1, R2, R3, and R4 are independently selected from the group consisting of a hydrogen atom and C1-4 alkyl radicals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2069712 CA2069712A1 (en) | 1992-05-27 | 1992-05-27 | Pleomorphism |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2069712 CA2069712A1 (en) | 1992-05-27 | 1992-05-27 | Pleomorphism |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2069712A1 true CA2069712A1 (en) | 1993-11-28 |
Family
ID=4149919
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2069712 Abandoned CA2069712A1 (en) | 1992-05-27 | 1992-05-27 | Pleomorphism |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2069712A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513548A (en) * | 2017-10-31 | 2017-12-26 | 荣成市日鑫水产有限公司 | A kind of method that peptone is prepared using fish meal pressed liquor |
-
1992
- 1992-05-27 CA CA 2069712 patent/CA2069712A1/en not_active Abandoned
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107513548A (en) * | 2017-10-31 | 2017-12-26 | 荣成市日鑫水产有限公司 | A kind of method that peptone is prepared using fish meal pressed liquor |
| CN107513548B (en) * | 2017-10-31 | 2021-01-22 | 荣成市日鑫水产有限公司 | Method for preparing peptone by using fish meal squeezed liquid |
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