Note: Descriptions are shown in the official language in which they were submitted.
D19254 JPW
NOVEL STRAINS OF AZOSPIRILL~M, METHODS OF
GROWING THE STRAINS, COMPOSITIONS CONTAINING
THEM AND USE THERE
Background of the Invention
Nitrogen is an essential plant nutrient Unfortunately,
it is usually not present in soil at concentxations
sufficient for agricultural production of commercial
crops. It therefore must be provided to the crops in
the form o fertilizer. Of the commercially important
crops, cereals such as corn, wheat, rice and barley
require particularly large amounts of fertilizer.
Leguminous crops, for example, soybeans, peas, beans
and clover are able to obtain part o~ their nitrogen
requirements from atmospheric nitrogen. This is accom-
plished b~ virtue of a symbiotic relationship between
legumes and soil bacteria of the genus Rhizobium. Such
bacteria synthesize assimilable nitrogen compounds from
atmospheric nitrogen and make them available to legum-
inous plants.
In recent years, naturally-occurring bacteria of the
genus Azospirillum (formerly known as Spirillum) have
been found in association with cereals~ Azospirillum
bacteria are able to make atmospheric nitrogen avail-
able to cereals, thus reducing fertilizer requirements.
This association may also result in increased yields.
For example, Azospirillum has been shown to increase
total ear yield and average number of ears per plant in
sweet corn; to increase panicle weight per hectare,
percentage nitrogen present in seeds and panicle number
per plant in Sorghum bicolor; and to increase leaf
weight~ ear yield, seed dry weight and total nitrogen
~k ~
4~f~3
-- 2 --
yield in Zea ma~s. [Rapulnik, Y., et al., Experimental
Agric. 17: 179-187 (1981).]
Naturally occurring Azospirillum bacteria are free-
living, aerobic, gram-negative, motile and nitrogen
fixing. They generally pre~er organic acids as carbon
sources, e.g. malic or lactic acid, and fi~ nitrogen in
the absence of a combined nitrogen source, under micro-
aerophilic conditions (low oxyyen tension). They also
have relatively short shelf lives and generally are not
resistant to commonly used, commercial fungicides,
herbicides and other pesticides or to antibiotics re-
leased by different soil microorganisms. Finally, they
have relatively low pectinolytic activity, that is,
relatively little ability to break down pectin present
in the cell walls of cereal plants in order to render
their cell walls more permeable to minerals, hormones
and the like and possibly to the bacteria themselves.
In order to overcome certain disadvantages associated
with naturally-occurring Azospirillum strains, the
present invention proves novel strains having rela-
tively improved survivabilityl or resistance to common-
ly used pesticides or increased pectinolytic activity
or a combination of these traits. Such strains are
able to enhance crop yields or reduce nitrogen fer-
tilizer requirements, or both.
6B3
-- 3
Summar of the Invention
y
Bacteria of the genus Azos~irillum may be used as bio-
fertilizers for cereal crops to provide enhanced crop
yields or reduced fertilizer requirements or both.
Azospirillum bacteria which may be so used include
those having pectinolytic activity relative to the
pectinolytic activity of wild type strains in the range
from about 1.5:1 to 20:1, preferably in the range from
about 5:1 to 15:1. Presently available bacteria in
accordance with the invention are of the species
brasilense and include the preferred strain deposited
with the American Type Culture Collection, ~c;ckville,
Maryland 20852, United States of America, pursuant to
the provisions of the Budapest Treaty on the International
Recognition of the Deposit oE Microorganisms for the
purposes oE Patent Procedure under accession number
ATCC No. 39199. Additional bacteria within the scope
of the present invention are of the species lipoferum.
Such bacteria which additionally possess resistance to
amounts of herbicides or fungicides which would kill or
substantially retard the growth of wild type
Azospirillum bacteria are particularly useful as bio-
fertilæers~ One such strain which is presently preferred
has been deposited with the American Type CultureCollection pursuant to the provisions of the Budapest
Treaty under accession number ATTC No. 39200.
In addition, bacteria of the genus Azosperillum which
possess pectinolytic activity in the aforementioned
range and contain above about 1 percent dry weight
poly-beta-hydroxybutyrate, preferably from about 15
percent to about 35 percent dry weight based upon the
weight of the bacterium, are particularly useful.
33
-- 4 ~
Desirabl~, the bacteria possess all three characteristics,
i.e., pectinolytic activity in the stated range,
resistance to amounts of herbicides, funyicides, or
both, which would kill or substantially retard the
growth of wild type Azospirillum bacteria and poly-
beta-hydroxybutyrate content in the amounts set forth~
In addition to the individual bacteria of the preceding
types the invention also contemplates mixtures of the
bacteria. Such mixtures may include two or more types
of bacteria, e.g., bacteria having increased pectino-
lytic activity and bacteria having resistance
to herbicides or fungicides. ~lternatively, the mixtures
may be of bacteria of the same type but of different
specific characteristics, e.g., bacteria having pectino-
lytic activity in a given range admixed with bacteriahaving pectinolytic activity in a diEferent range or
bacteria having resistance to certaln ungicides and
herbicides admixed with bacteria having resistance to
other fungicides and herbicides.
Bacteria of the genus Azospirillum useful as biofertil-
izers for cereal crops may be prepared by treating a
naturally-occurring strain of an Azospirillum species
to induce mutation therein, growing the treated bacteria
under suitable conditions so as to select for bacteria
having pectinolytic activity relative to that of wild
type strains in the range rom about 1.5:1 to 20:1 and
recovering the resulting bacteria.
By growing the bacteria so recovered in the presence
of one or more herbicides, fungicides, or both, to
which resistance is desired, the herbicides or ungi-
cides being present in amounts sufficient to substan-
tially retard the growth of or kill bacteria lacking
resistance, recovering the bacteria which grow under
these conditions, growing them in the presence of in-
creased amounts of the same herbicides or fungicides,
and recovering the then-growing bacteria, one may prepare
bacteria which also possess resistance to various amounts
of herbicides, fungicides, or both.
Additional bacteria also having increased poly-beta-
hydroxybutyrate conten~ may be prepared by fermenting
bacteria of the genus Azosprillum which have both enhanced
pectinolytic activity and resistance to herbicides,
fungicides, or both, on a suitable carbon source in the
absence of nitrogen and at about 0 percent dissolved
nitrogen for a period of time sufficient to permit an
increase to a desired level in poly-beta-hydroxybutyrate
content~
Further bacteria in accordance with this invention have
resistance to amounts oE herbicides, fungicides, or
both, which substantially retard the growth of or kill
wild type Azospirillum strains. Presently available
bacteria are of the species brasilense and include the
preferred strain deposited with the American Type Culture
Collection pursuant to the provisions of the Budapest
Treaty under accession number ATCC No. 39201. Additional
strains within the scope o~ the invention are oE the
species lipoferum.
Such bacteria which additionally contain above about 1
percent dry weight poly-beta-hy~roxybutyrate, preferably
from about 15 percent to about 35 percent dry weight
poly-beta-hydroxybutyrate based upon the dry weight of
the bacterium, are particularly useful as bio~ertilizers.
Bacteria having resistance to amounts of a herbicide or
~2~4~33
fungicide which substantially retard the growth of or
kill wild type Azospirillum strains may be prepared by
growing bacteria of an Azosprillum species in the pres-
ence of amounts of the herbicide or fungicide sufficient
to substantially retard the growth of or kill the bac-
teria lacking the desired resistance, recovering the
bacteria which grow under these conditions, growing the
bacteria in the presence of increased amounts of the
same herbicide or fungicide and recovering the then-
growing bacteria.
Bacteria having resistance to amounts of herbicides orfungicides which substantially retard the growth of or
kill wild type Azospirillum strains and also having
poly-beta-hydroxybutyrate contents greater than about
1 percent, preferably in the range Erom about 15 percent
to about 35 percent, dry weight based upon the dry
weight of the bacteria may be prepared by irst preparing
bacteria having resistance to the herbicides or fungi-
cides using the method described hereinabove and fermenting the resultin~ bacteria on a suitable carbon source
in the absence of nitrogen and at about 0 percent dis-
solved oxygen for a period of time sufficient to permit
an increase in the amount of poly-beta-hydroxybutyrate
present in the bacteria.
The novel Azospirillum strains of this invention, singly
or in combinations, may be combined or admixed in effec-
tive biofertilizing amounts with a suitable agronomically
acceptable carrier to produce biofertilizer compositions.
They can also be used to coat cereal seeds prior to
planting. By using the bacteria, bio~ertilizer composi-
tions containing the bacteria or bacterial-coated cereal
seeds, one may reduce fertilizer requirements or obtain
increased yields, or both, in cereals such as maize,
corn, sorghum, wheat, setaria and panicum.
-- 7 --
The invention also provides a method for growing a
desired strain of Azospirillum comprising inoculating a
single colony of the desired bacterial strain onto a
suitable liquid culture medium, vigorously aerating the
culture medium for an appropriate period of time, trans-
ferring the culture medium containing the bacterial `
strain into a fermentor containing the liquid culture
medium while the bacterial strain is in its logarithmic
growth phase, growing the bacteria to a desired cell
density and recovering the resulting bacteria from the
fermentor.
~L2~ 33
- 8 -
Detailed Description of the Invention
Naturally-occurring strains of Azospirillum, particu-
larly strains of Azospirillum brasilense and Azospirillum
lipoferum, are useful as biofertilizers. When included
in or applied onto soil in which cereal crops are grown,
they can reduce the amount of externally provided nitro-
gen fertilizer necessary or provide enhanced crop yields,
or both. However, these naturally-occurring strains
are characterized by certain disadvantages. They are
generally not resistant to herbicides, fun~icides and
other pesticides. They are also generally not resis-
tant to antibiotics produced by different soil micro-
organisms and excreted into the soil. E'urthermore,
their relatively short shelf lives prevent them from
obtaining the maximum commercial advantages which might
otherwise be realized.
Pectinolytic activity, that is, the ability to break
down pectin present in the cell walls of cereal plants
so as to render the cell walls more permeable to minerals,
hormones and the lilce present in soil and possibly also
to nitrogen-fixing bacteria such as those of the genus
Azospirillum is relatively low in naturally-occurring
Azospirillum strains. Since pectinolytic activity can
be correlated with usefulness as biofertilizer,
novel strains of Azospirillum having pectinolytic
activity relative to the pectin~lytic activity of
naturally-occurring strains in the range from about
1.5:1 to 20:1 have been created. Presently, the pre-
ferred strains are of the species brasilense. Option-
ally, they are available as biologically pure, stable
cultures. Particularly preferred are strains having
pectinolytic activity relative to the pectinolytic
activity of the wild type strain in the range from
l33
g
about 5:1 to 15:1 of which the strain ATCC No. 39199 is
presently preferred.
Such strains may be produced by treating bacteria of a
naturally-occurring strain of an A spirillum species,
e.g.,brasilense, to induce mutation in the bacteria and
growing the treated bacteria under suitable conditions
such that those having pectinolytic activity in the de-
sired range can be identified or selected for. The
novel bacteria having the desired level of pectinolytic
activity are then recovered. Although the pectinolytic
activity relative to that present in wild type strains
may be in the range from about 1.5:1 to 20:1, it is
preferred that it be in the range from about 5:1 to
15:1. Various methods of inducing genetic mutation are
known and may be employed, including irradiation with
ultraviolet light or contact with mutagenic ~hemicals
such as nitrous acid.
Preferred strain ATCC No. 39199 was prepared as a mutant
of A. brasilense. It exhibits pectinolytic activity
about 10 times that found in wild type Azosperillum
strains. It also has 2 times the nitrogenase activity
(i.e., ability to fi~ nitrogen) of wild-type strains.
More particularlyr this strain was produced by muta-
genesis of colonies of naturally-occurring A bra-
silense strain cd [Nur, I. et al., J. Gen. Microbiol.
722:27-32 (1981)~ using nitrous acid t50mM sodium nitrite
in O.lM Na acetate buffer, pH 4.6, for 20 minutes at
35C), and screening resulting mutants for pectinolytic
activity. Mutants having pectinolytic activity within
the desired range were isolated. Pure cultures were
grown on solid media. This procedure is described in
greater detail in Example 1.
~2~ 3
-- 10 --
The nitrogenase activity of this and other strains was
measured by the acetylene reduction assay using conven-
tional methods. Both nitrogenase and the acetylene
reduction assay are described in detail in ~lardy, R.W.F.
and Holsten, R.D., (1977), "Methods fcr measurement of
dinitrogen fixation," pp. 451-486 ln: E~ardy, R.W.F.
and A.H. Gibson (eds.) A Treatise on Dinitro~en Fixation
IV: Agronomy and Ecolo~, J. Wiley and Sons, New York
and London.
Although bacteria having pectinolytic activities higher
than those o wild type Azospirillum strains would
render cell walls permeable, it is undesirable for the
pectinolytic activity to exceed about 20 times the
activity of wild type strains. Otherwise, the bacteria
may completely destroy the cell walls of the cereal
plants whose growth i5 desired. In general, pectinolytic
activity about 10 times that of wild type Azospirillum
strains is preferred.
Bacteria having pectinolytic activity relative to the
activity of wild type strains in the range from about
1.5:1 to about 20:1 which additionally possess resistance
to amounts of pesticides, e.g., herbicides or fungicides,
which substantially retard the growth of or kill wild
type Azospirillum bacteria are generally preferred over
those having only the desired levels of pectinolytic
activity. Presently preferred is A. brasilense ATCC
No. 39200, a strain which also has twice the nitrogenase
activity of wild type strains.
It is desirable that the strains of Azospirillum be
resistant to both fungicides and herbicides and desirably
to antibiotics as well. Such s~rains may be isolated
by genetic selection. A naturally-occurriny or mutant
-- 11 --
strain of Azospirillum is cultured in the presence of
an amount of a chemical antagonist such as a pesticide
to which resistance is desired for a period of time
sufficient to substantially retard the growth of or
kill Azospirillum bacteria lacking resistance to the
antagonist. Fast growing Azospirillum colonies emerging
on the medium are isolated and recultured in the presence
of an increased amount of the chemical antagonist. The
amount of the chemical antagonist and the period of
time for which the bacteria are cultured is again suf-
ficient to substantially retard the growth of or kill
the members of the bacterial population lacking resis-
tance to the increased amount of the antagonist. This
procedure may be repeated as needed, with increasing
amounts of chemical antagonist, to provide an Azospirillum
strain suEficiently resistant to the antagonist that it
is capable o~ cell multiplication and of entering into
a nitrogen-fixing symbiosis in the presence of agricul-
turally effective amounts of one or more such chemical
antagonists.
To produce strains resistant to a plurali-ty of such
chemical antagonistsj the same procedure is followed
except that at each step the Azospirillum strain is
cultured in the presence of differing amounts of each
of the various chemical antagonists.
rrhus, bacteria possessing both higher pectinolytic
activity and resistance to fungicides, herbicides, or
both, may be prepared by treating bacteria of a
naturally- occurring strain of an Azospirillum species
to induce mutation in the bacteria, growing the treated
bacteria under suitable conditions so as to select
bacteria having pectinolytic activity relative to that
`~ 35 of naturally-occurring strains in the range from about
~Z~4fi~3
1.5:1 to 20:1, preferably from about 5:1 to 15:1, re-
covering the resulting bacteria, growlng the resulting
bacteria in the presence of amounts of fungicides or
herbicides sufficient to substantially retard the growth
of or kill the bacteria lacking the desired resistance,
recovering the resulting growing bacteria, growing
these bacteria in the presence of increased amounts o
the same herbicides or fungicides, recovering the then-
growing bacteria and reculturing the bacteria so recoveredO
Additional bacterial strains useful in accordance with
the teachings of this invention include novel Azospirillum
strains which, in addition to pectinolytic activity
within the ~esired range and resistance to specific
pesticides, also contain more than about 1 percent dry
weight poly-beta-hydroxybutyrate based upon the dry
weight oE the bacterium, preferably from about 15 to
about 35 percent dry weight poly-beta-hydroxybutyrate.
Such bacteria can be prepared by first producing bac-
teria having the desired level of pectinolytic activityand optionally desired resistance to specific chemical
antagonists and then fermenting these bacteria so chara-
cterized on a suitable carbon source, e.g. malic acid,
lactic acid, succinic acid, fumaric acid, salts thereof,
or glycerol, in the absence of nitrogen and at about
0 percent dissolved oxygen for a period of time suffi-
cient to permit an increase in the amount of poly-beta-
hydroxybutyrate present in the bacteria.
Other Azospirillum strains useful as biofertilizer are
characterized by their resistance to amounts of herbi-
cides, fungicides, or both, which substantially retard
the growth of or kill wild type zospirillum bacteria.
Although resistance to fungicides or herbicides is
desirable, resistance to both herbicides and fungicides
3L2~ 3
- 13 -
and optionally to antibiotics as well i5 most desirable.
Presently, the preferred strains are of the species
brasilense. Desirably, they are in the form of bio-
logically pure, stable cultures. An example of such
a strain is A. brasilense ATCC No. 39201, which is a
nitrogen fixing strain.
Such bacteria may be prepared by growing naturally-
occurring bacteria o~ an Azospirillum strain in the
presence of amounts of herbicides, fungicides, or both,
sufficient to substantially retard the growth of or
kill bacteria lacking resistance thereto, growing the
bacteria in the presence of an increased amo~nt of the
same herbicides, fungicides, or both, recovering the
then-growing bacteria and reculturing the bacteria so
recovered.
Using this approach, strains o Azospirillum have been
isolated which are resistant to the following: (Details
20 of the procedure are presented in Example II.)
FUNGICIDES : Captan ~1,2,3,6-tetryhydro-N-
~trichloromethylthio)-Phthalirnide)
[Cp]; TMTD ~Tetramethyl thiuram
disulphide) [TMTD]; Caspan (ethyl-
mercuro-chloride) [Cs]; Benlate or
Benomyl (l-(butylamino) carbonyl-
l~l-benzimidozo (-2-yl) Carbamic
acid methyl ester) [B].
HERBICIDES : Atrazine (6-chloro-N-ethyl-N'-(l-
methylethy~)-1,3,5-triazine-2,4-
diamine) [At]; Trybonyl (1,3-
dimethyl-3-(2-benzo-thiazolyl)-
urea) [T]; Illoxan (2-(4-(2'4'-
dichlorophenozyl) Phenoxyl-methyl-
propionate) [I).
- 14 -
ANTIBIOTICS : Ampicillin [A]; Kanamycin [K];
Streptomycin [S].
BACTERICIDE/
BACTERIOSTAT : Sodium Azide [Na]
Illustrative strains including several having particu-
larly desirable resistance characteristics, and their
respective levels of resistance to specific chemicals
follow. The figures in parentheses are in parts per
million (ppm) of the compound, except for Na, which is
expressed in millimoles (mM).
Strain Resistant To
-
1 A(100); T(50); I(50); At(50)
2 S~250); T~50); I(50); At(50)
3 K~100); T~50); I~50); At(50)
4 A~100); S(250); K(100); T(50); I(50~;
At(50)
Na(0.2); T(50); I(50); At(50)
6 Na(0.2); S(200); B(20); T(50); I(50);
At(50)
7 Na(0.2); S(200); B( 2n); cp ( 50-100);
Cs(20); T(50); I(50); At(50)
~* A~100); S(250); K(50)
9* At~50); B(20); Cp(50)
10* Cs(20)
ATCC No. 39200* Ak(50); Cp(50); TMTD(50)
ATCC No. 39201 At(50); At(50); TMTD(50)
*Derivative of ATCC No. 39199 possessing enhanced pectino-
lytic activity as well as the chemical resistancesindicated.
~Z~4~33
It will be appreciated by those skilled in the art that
the foregoing resistance characteristics are exemplary
of resistance to other chemicals within the same or
similar chemical classes. Thus, resistance to Captan
is representative of resistance to chlorinated hydro-
carbons generally. Resistance to Caspan is repre-
sentative of resistance to organic mercury compounds;
to Benlate of carbamate compounds; to Atrazine of
triazine-~eterocyclic nitrogen derivatives; and to
Tribonyl of urea derivatives, etc. The invention is
therefore not limited to resistance to specific chemicals
but embraces the classes o commonly used, agricultural
chemicals including pesticides, e.q., herbicides and
fungicides.
In addition to resistance to herbicides, fungicides,
or both, strains are useful which additionally contain
more than about 1 percent dry weight poly-beta-
hydroxybutyrate based upon the dry weight of the bac-
terium, preerably from about 15 percent to about 35percent.
This latter type strain may be prepared from strains
having the desired levels of resistance to agricultural
chemicals prepared using methods already described.
The resistant strains are then fermented on a suitable
carbon source in the absence of nitrogen at about 0 per-
cent dissolved oxygen for a period of time suEficient
to permit an increase to the desired level in the amount
of poly-beta-hydroxybutyrate present in the bacteria.
Suitable carbon sources for use in this method include
malic acid, lactic acid, succinic acid, fumaric acid,
salts thereo, i.e., malate, lactate, succinate or
fumarate, or glycerol.
~g~
- 16 -
In addition to individual strains, mixtures of strains
haviny different desirable properties may be employed.
Alternatively, mixtures of strains having the same
property but in differing degree may be used. For
example, a mixture of two strains having different
levels of pectinolytic activity and two strains each
having resistance to different levels of different
chemical antagonists may be preferred for some applica-
tions.
Effective biofertilizing amounts of the various novel
Azospirillum strains of the present invention may be
incorporated into biofertilizer compositions together
with a suitable agronomically acceptable carrier. Gen-
lS erally, efective biofertilizing amounts of microorganismwill be in the range from about 106 to 101 bacteria
per gram oE the final composi.tion or formulation,
preEerably from about lx109 to 5x109 bacteria per
gram of composition or formulation.
Various conventional agronomically acceptable carriers
may be employed. One such carrier is peat, e.g. finely
ground peat. The carrier may contain additional compo-
nents, including carbonate, a wetting agent, a suspending
agent or an absorbing agent.
In addition to such biofertilizer compositions, cereal
seeds may be coated with an efective biofertilizing
amount of a bacterium in accordance with this invention.
Effective biofertilizing amounts of microorganism
typically range from about 106 to 101 bacteria per
gram of coating formulation, preferably from about
1 x 109 to ~ x 109 bacteria per gram~ Me~hods for seed
coating are well known to those skilled in the art
and are therefore not discussed in detail herein.
~Z~6~33
Fertilizer requirements may be reduced or increased
crop yields made possible, or both, by incorporating
into or applying onto soil in which cereals such as
maize, sorghum, corn, wheat, setaria or panicum are to
be grown effective amounts of one or more Azospirillum
bacterium according to the present invention or a
biofertilizer composition containing one or more such
bacterium or a cereal seed coated therewith~ Cereals
are then grown in a conventional manner.
A microbial culture of Azospirillum produced or grown
according to the above methods may be applied to crops
by any of a number of known methods, including seed
coating, wettable powder and granular formulations.
One particularly successful formulation suitable for
direct application to the soil as a spray is a wettable
powder of finely ground peat. ~his formulation comprises:
Finely ground or granular peat;
CaCO3 which alters the pH of the peat to about
pH 7.0;
A wetting agent: for example Nonyl phenol ethoxy-
late - 10 Ethylene oxide (commercially available
under the tradenames 9M10 [Dow Chemical Co.], NP-10
or Lanco). Since it is a liquid, it is generally
mixed with equal parts of milled silica before
being mixed with the other dry ingredients;
A suspending agent: e.g. Sodium ligno--sulfonate;
An absorbing agent: e.g. a milled silica such as
that marketed under the tradename Wessalon S;
[Degussa, W. Germany]; and
~L2~ 3
- 18 -
A microbial culture of the desired Azospirillum
strain or strains.
Alternatively, a microbial culture of an Azospirillum
strain may be applied to corn or other relatively large
seeds as a coating with an adhesive such as Pelgel (the
Nitragin Co., Clearwater, Florida~ U.S.A.). Pelgel
contains calcium carbonate, sugar and gum arabic.
Example III provides a further example of a biofertilizer
composition in accordance with the present invention.
Most strains of Azospirillum are nitrogen-fixing. Some
persons skilled in the art believe that the nitrogen-
fixing capability is responsible for the beneficial ef-
ects on cereal crop yield or fertilizer requirements.
Others skilled in the art maintain that there is no
causal relationship between nitrogen-fixing capability
and the ability to increase crop yield and reduce fertil-
izer requirements in cereal crops. The present inventioncontemplates both nitrogen-fixing and non-nitrogen-
fixing strains useful as biofertilizers and is not to
be construed as limited except in so far as the strains
are limited by the claims set forth hereinafter.
While Azospirillum strains have been previously grown
in the laboratory, there has existed no suitable manufac-
turing process for producing the bacteria in commercial-
ly useful quantities while retaining the desired bacteri-
al characteristics. Such a process has been developed.It is described in detail in Example IV.
Desired strains of Azospirillum may be grown as follows.
A single colony of the desired bacterial strain is
inoculated onto a suitable liquid culture medium which
- lg -
is then vigorously aerated for an appropriate period of
time. The culture medium containing the bacterial
strain is transferred into a fermentor containing the
liquid culture medium while the bacterial strain is in
its logarithmic growth phase. The bacteria are grown
to the desired cell density and the resulting bacteria
are recovered from the fermentor.
One suitable liquid culture medium includes appropriate
2 4I KH2PO4, MgSO4 7H2O, ferric ammonium
citrate, NH4Cl and a carbon source. Another suitable
medium contains appropriate amounts of K2HPO4, KH2PO4,
MgSO4 7H2O, FeC13, a nitrogen source and a carbon source.
Carbon sources useful in the invention include malate,
lactate, succinate, fumarate salts or the acids from
which they are derived, or glycerol. Nitrogen sources
include ammonia or an ammonium salt.
One method of growing desired strains of Azospirillum
comprises growing cells of a chosen or desired strain
on a solid medium. After dilution plating, a single
colony is inoculated onto a liquid culture medium in
baffled erlenmeyer flasks with glycerol as the carbon
source. A particularly successful li~uid culture medium
comprises: K2~IPO4, 6.Og/l; KH2PO4, 4.Og/l; MgSO4 7H2O,
0.2g/1; Ferric ammonium citrate, 0.01g/1; glycerol in a
concentration from 5.0 to 10.0g/1 with NH~Cl preferably
in the concentration 1.5 to 3.Og/l.
The inoculated flasks are vigorously aerated, as on a
rotary shaker, until a desired concentration of organisms
is reached. It is essential that the contents of these
flasks, which are to be used as inoculum for ~he follow-
ing fermentation stage be kept in the logarithmic growth
` 35 phase and not reach the stationary phase. The inoculum
~2~133
- 20 -
is inoculated into a fermentor having the same liquid
culture medium to which a carbon source and a nitrogen
source is continuously added. The carbon source
preferably comprises glycerol or may comprise malate,
lactate, succinate or fumarate. The nitrogen source
preferably comprises ammonia or an ammonium salt. The
pH and temperature are controlled during this growth
phase. The first stage of this growth takes about 20-
2~ hours, and terminates when the dissolved oxygen
~D.O.) begins to rise after all the glycerol has been
consumed. Additional carbon source is then added to
the culture in the fermentor. This carbon source may
be of any suitable organic acid, such as a concentrated
solution of malic acid, succinic acid or fumaric acid,
lS or additional glycerol. The addition of this carbon
source results in decreased D.O. content and increased
p~I. Growth is continued until the desired concentration
of cells is reached. This generally coincides with the
point at which the D.O. begins to increase again, indi-
cating that all the NH4+ in the fermentor has beenexhausted. The absence of N~4~ n~ay be confirmed by the
Nessler test. [Dawson, R.M.C., et al., Data for
Biochemical Research, Oxford Press, page 619.]
It is a particular feature of the present invention
that it yields extremely high cell densities of
Azospirillum, while utilizing an optimal carbon/nitrogen
ratio for organism growth.
About 0.3% of the dry weight of a naturally-occurring
Azospirillum strain comprises a preservative material,
poly-beta-hydroxybutyrate. It has been shown in labora-
tory tests that the presence of increased amounts of
poly-beta-hydroxybutyrate enhances long term bacterial
survival. [Daws, E.A. and Senior, P.J., Advances in
- 21 -
Microbial Physiology 10O 135-266 (1973)] In other
words, both shelf life of the stored product prior to
use and survival of the bacterium in the soil during
the initial period of plant growth are enhanced by the
presence of poly-beta-hydroxybutyrate in the organism.
It was therefore desirable to obtain a method for in-
creasing the amount of poly-beta-hydroxybutyrate within
a desired Azospirillum bacterium in order to enhance
its long term survival.
It has recently been found that poly-beta-hydroxybutyrate
is also useful as a biopolymer, and has piezoelectric
properties useful in various electrical apparatus. It
will be appreciated that the following method of enrich-
ing Azospirilla with poly-beta-hydroxybutyrate is also
useful for the production of such material itself, to
be followed by its removal from the bacterium.
A method of increasing the percentage of poly-beta-
hydroxybutyrate in an Azospirillum strain has been
developed. It is described in detail in Example V. It
involves continuing the growth of a desired culture of
an Azospirillum strain in the absence of nitrogen at
about 0 percent dissolved oxygen and with a continuously
fed carbon source, such as malic acid. Alternativelyl
a ripe culture of Azospirillum previously grown may be
returned to the fermentor and cultured under these
conditions. Suitable organic acids useful as carbon
sources include succinic acid, lactic acid, fumaric
acid and malic acid. Alternatively, the salts of these
acids may be used, i.e., succinate, lactate, fumarate
and malate. In the absence of nitro~en, the culture
does not continue to grow. Instead the carbon source
is converted into poly-beta-hydroxybutyrate within the
individual Azospirillum bacterium. At the end of this
~2~ 33
- 22 -
stage, the mature micro-organisms are ready for incor-
poration into a soil inoculant mixture~ Desirably,
more than 0.3% of ~he dry weight of the resulting
Azospirillum or bacterium will be poly-beta-hydroxy-
5 butyrate. Preferably, from about 15-35~ of the organisms'
dry weight will be poly-beta-hydroxybutyrate.
It is a particular feature of the present invention
that growth of a desired strain of Azospirillum and
enrichment thareof according to the above-described
methods not only yields extremely high cell densities
of Azospirillum, but also allows the harvesting of
cells containing a high poly-beta-hydroxybutyrate con-
centration which is important for survival of the bac-
teria and may be important to their ability to overcome
competing soil organisms.
E%AMPLE I
Isolation o~ Azos~irillum haviny higher pectinolytic
activit~.
Colonies of naturally-occurring A. brasilense strains
were treated with 50 mM sodium nitrite in 0.1 M Na
acetate buffer, pH 4.6, for 20 minutes at 35C to cause
mutagenesis. The resulting mutants were screened for
pectinolytic activity.
A culture of mutant A. brasilense strains was plated on
a solid medium containing 0.5~ pectin as the sole carbon
source. Of the few colonies that developed, a particu-
larly large;colony was selected and restreaked on fresh
solid medium. Of the colonies that then grew, tha
largest was picked. This is strain ATCC No. 39199. Its
is pectinolytic enzyme activity was confirmed to be higher
~Z~46~3
- 23 -
than the wild type's by a standard cup-plate assay.
[Dingle, J. et al., Sci. Food Agric. 3-4:149-155 (1953)].
This involved growing A. brasilense ATCC No. 39199 on
pectin (0.5~) in liquid culture, centrifuging the culture
broth, and applying an aliquot of supernatant to a well
in an agar plate containing pectin. The pectinases,
when present, attack the pectin and leave a clear zone
on the plate surrounding the well. Although this is a
semiquantitive assay, the results clearly indicated
that ATCC No.39199 had activity about 10-fold greater
than the activity associated with naturally-occurring
Azospirillum.
EXAMPLE II
Isolation of Azospirillum strains resistant to various
agricultural chemicals.
-
A naturally-occurring (wild type) strain oE A. brasilense
(cd) and A. brasilense ATCC No.39199 were each grown on
liquid minimal media. Single colonies of each strain
were then plated on solid minimal media of the following
composition (g/1): K2HPO4, 6.0; KH2PO4, 4.0; NH4Cl,
1.5; MgSO~ 7H2O, 0.2; ferric ammonium citrate, 0.01;
malic acid, 5.0; and Agar, 20.0, containing a low concen-
tration of one or more agricultural chemicals or anti-
biotics, on the order oE about 5 ppm. The chemicals
were each dissolved in :L ml 95% ethanol at a concentra-
tion sufEicient to produce the desired final concentra-
tion in the medium when the 1 ml was added to 1 liter
of medium. The ethanol solution was added to the medium
after the medium was autoclaved and allowed to cooI.
Fast growing colonies emerging on the plates were
transferred onto the solid minimal media containing an
6~3
- 24 -
increased amount of the agricultural chemical, on the
order of about 15 ppm, depending upon the amount of
resistance e'xhibited by the Azospirillum to the low
concentration of chemical. Again, fast emerging colonies
were isolated, purified and replated on media containing
an increased amount of the chemical, for example, 30
ppm.
These steps were repeated at increasing concentrations
of the chemical until the desired resistance was achieved.
EXAMPLE III
Formulation for application of Azospirillum to crops.
An inoculan~ formulation was prepared in the following
manner. The figures in parenthesis are in percent w/w
calculated on the final product.
The following ingredients were mixed:
Peat which, after drying at 105C, was milled so
that at least 80% passed a 325 mesh [particle size
below 44 microns~ (57);5
CaCO3 (3); [In the event this amount does not correct
the pH of the peat to 7.0, the percentages may be
altered to obtain a total of 60% for peat and CaCO3
combined~0
Wetting agent 9M10 (1);
Sodium ligno- sulfonate (3); and
~2~
- 25 -
Wessalon S (11), a portion of which was mixed with
the NP10 before addition to the other dry ingredients.
The mixture o the above ingredients ~as sterilized by
Gamma rays at a dose of 5 Megarad. To the sterile
mixture, the culture of ~zospirillum, as obtained at
the end of the growth process, was added without centri-
fugation or other manipulation at a concentration of
25%. As the culture had a cell concentration of about
1 to 3 x 101/ml, the final formulated inoculant had a
concentration of about 5 x 109 cells per gram.
The inoculant was stored in aseptic sealed packages and
maintained at room temperature until use. The sprayed
inoculant was obtained by adding lOOOg of inoculant to
100 1 of water. This quantity is suEficient to spray
one hectare of soil.
In applications of Azospirillum strains to crops in a
granular formulation, between 5 and 60 kilograms are
required per hectare depending upon the particular
crop and the plant stand.
Cereal crops on which formulations comprising a microbial
culture of Azospirillum according to the present inven-
tion are particularly useful include maize, sorghum
corn, setaria, panicum and wheat.
EXAMPLE IV
Growth of agriculturally useful quantities of
Azospirillum.
The cells of a chosen strain of A. brasilense were
grown in Petri dishes on a solid medium of the following
~20P4~
- 26 -
composition ~g/l): K2HPO4, 6.0; KH2PO~, 4.0; NH4Cl,
1.5; MgSO4 7H20, 0.2; Ferric ammonium citrate, 0.01;
Malic acid, 5.0; Agar, 20Ø
After dilution plating, a single colony was inoculated
on~o liquid culture medium in baffled ~rlenmeyer flasks
filled to 20% of nominal capacity. The medium had the
following composition (g/l): K2HPO4, 6.0; KH2PO4, 4.0;
MgSO4 7H2O, 0.2; Ferric ammonium citrate, 0.01. The
carbon source was glycerol rather than malic acid in a
concentration from 5.0 to 10.0, while the NH4Cl added
was in the range from 1.5 to 3~0.
The medium was adjusted to pH 7.2 and the flasks were
aerated Otl a rotary shaker at 200 rpm. The temperature
was maintained at 35C. After about 20-24 hours, the
Optical Density (O~D.) measured at 660 nm was 5.0 to
9.0, corresponding to an organism concentration of 1 to
5 x 109/ml.
A quantity of inoculum from the culture flasks was
inoculated into liquid culture medium in a fermentor,
nominal volume 16 liters. The inoculum was 5 to 10% of
the final volume of the fermentor, so that the initial
Optical Density was 0.1. The composition of the
culture medium was: K2HPO4, 2.0; KH2PO4, 1.3;
MgSO4 7H2O, 0.2; H2O, 0.2; Ferric ammonium citrate,
0.01; Glycerol, 10.0; NH~Cl, 3Ø
The pH of the medium was adjusted, ater sterilization,
to 7.0 by addition of 5N NaOE. A Silicone antifoam
agent, Sigma Antifoam C Emulsion, was added by means of
a peristaltic pump controlled by a foam sensing probe.
The growth temperature was maintained at 35C. Dissolved
oxygen (D.O.) was maintained at between 30 and ~0~
~L2~
- 27 -
saturation (although D.O. up to 100% saturation at this
stage will produce the desired results) by adjustment
of both agltation and sparging. This adjustment may be
made automatically by a D.Q. controller or manually
when only a D.O. analyzer is present. The pH is regulated
at 7.0 by a pH regulator which adds 5N NaOH when necessary.
The first stage of fermentor growth required about 20
to 24 hours and terminated when the D.O. began to rise,
because all the glycerol had been consumed. Malic acid
as a carbon source was added in a 500g/1 solution. The
initial addition was made equivalent to 7.5g/1 of malic
acid. This solution was adjusted to pH 5.5 with NaOH.
As a result of the malic acid addition, the D.O. de-
creased and the pH rose to 6.0-6.2. The concentrated
malic acid was continuatly fed into the fermentor through
the acid peristaltic pump oE the pH regulator. The
utilïzation of the malic acid by the bacteria raised
the pH, which the regulator maintained at 7.0 by adding
malic acid.
Growth continued until all the NH4~ in the fermentor
was exhausted, at which point the D.O. again began to
rise. The absence-of NH4~ was determined by the Nessler
2S test on a sample of the culture from which the cells
had been removed. At this stage the Optical Density
was between 22 and 28 and the organism concentration
about l to 3 x 101/ml.
EXAMPLE V
Enrichment for poly-beta-hydroxybutyrate.
Fermentation of the culture produced according to the
- 28 -
method of Example IV was continued and the dissolved
oxygen reduced to about 0 percent, bu~t avoiding anaerobic
conditions. The pH controller maintained the flow of
malic acid for 3 to 4 more hoursS during which there
was no further growth and the malic acid was converted
to poly-be-ta-hydroxybu~yrate in the absence of nitrogen,
all the nitrogen in the culture medium having been
previously consumed. The culture broth so produced was
utilized in formulations for application to plants.
The amount of poly-beta-hydroxybutyrate accumulated in
the bacteria was determined to be in the range 15-35~
using known methods [Williamson and Wilkinson, J. ~en.
Microbiol. 19-198-209 tl958); Law and Slepcky, J.
Bacteriol. 82:33-36 (1961)].
