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Applied and Environmental Microbiology, February 2001, p. 617-622, Vol. 67, No. 2
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.617-622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Cultivation of Bacteria Producing Polyamino Acids
with Liquid Manure as Carbon and Nitrogen Source
Markus
Pötter,
Fred
Bernd
Oppermann-Sanio, and
Alexander
Steinbüchel*
Institut für Mikrobiologie der
Westfälischen Wilhelms-Universität Münster, 48149 Münster, Germany
Received 18 July 2000/Accepted 2 November 2000
 |
ABSTRACT |
Poly(
-D-glutamic acid) (PGA)-producing strains of
Bacillus species were investigated to determine their
ability to contribute to reducing the amount of ammonium nitrogen in
liquid manures and their ability to convert some of the ammonium into
this polyamino acid as a transient depot for nitrogen. Organisms that
do these things should help solve the serious environmental
problems which are caused by the use of large amounts of liquid
manure resulting from intensified agriculture; these problems are
mainly due to the high content of ammonium nitrogen. Bacillus
licheniformis ATCC 9945 and Bacillus subtilis were
able to grow in liquid manure and to produce PGA in the presence of
sodium gluconate. On artificial liquid manure these two strains were
able to produce 0.85 and 0.79 g of PGA per liter, respectively.
Under conditions that are found in intensified farming situations the
ammonia content was reduced within 48 h from 1.3 to 0.75 g/liter.
One mutant of B. subtilis 1551 impaired in the catabolism
of PGA was obtained after nitrosoguanidine mutagenesis. This mutant
produced PGA at a final concentration of 4.8 g/liter, whereas the wild
type produced only 3.7 g/liter.
| |
INTRODUCTION |
As a result of intensified
agriculture, huge amounts of liquid manure are produced. On a typical
pig-fattening farm (700 animals) in northwest Germany 2.8 m3 is produced daily (European Communities website,
http://europa.eu.int). Liquid manure is a highly heterogeneous liquid
mixture of fodder, litter, and animal cells, and the composition is
extremely dependent on the age of the pigs, the type of fodder, the age
of the liquid manure, and the storage conditions (8).
Average values for the major parameters of this manure have been shown
to be follows (20): dry weight, 60 to 80 g/liter; chemical
oxygen demand, 50 to 80, g/liter; biological oxygen demand, 15 to 32 g/liter; nitrogen concentration, 5.0 to 6.0 g/liter; ammonium nitrogen concentration, 2.5 to 4.5 g/liter; potassium concentration, 2.1 to 2.5 g/liter; calcium concentration, 1.8 to 2.1 g/liter; phosphorus concentration, 0.7 to 0.9 g/liter; and magnesium concentration, 0.5 to
0.6 g/liter.
The traditional and most widely used way to dispose of liquid manure is
by spreading it onto agricultural fields. Up to 80% of the ammonia is
released into the atmosphere, and up to 56% (wt/vol) of the remaining
ammonia can be utilized by plants (12, 19). In addition,
depending on the type of soil and the microflora, the ammonium ions are
partially oxidized to nitrate. Up to 40% (wt/wt) of the nitrate is
released into the groundwater or washed into the surface water, which
leads to unacceptably high concentrations of nitrate and eutrophication
(1, 6). This causes serious health and environmental
problems and has forced legislators to restrict the amount of nitrogen
in liquid manure that is spread onto fields; in Germany the amount is
restricted to 210 kg per ha per year (3). Therefore, new
methods for disposal of liquid manure from intensified animal-fattening
farms are needed.
Several gram-positive bacteria, such as Bacillus
licheniformis, Bacillus subtilis, Bacillus
megaterium, Bacillus anthracis, Sporosarcina
halophila, and Planococcus halophila, are able to synthesize the polyamino acid (PAA) poly(-D-glutamic
acid) (PGA) as a capsular substance or as a water-soluble slime
(4, 22). In the presence of excess amounts of a suitable
carbon source and ammonia, B. licheniformis excretes up to
20 g of PGA per liter into the medium (2). As PGA is
remarkably resistant to proteolytic attack, degradation of this polymer
by a nonadapted microflora proceeds very slowly (13).
In this study, we investigated conversion of the ammonia in liquid
manure into PAAs by known PAA-producing bacteria. The PAAs should
function as a transient depot for ammonia. Slow microbial degradation
of the PAAs should then result in only low concentrations of free
ammonia as a nutrient for plants. In addition, PAAs have a fertilizing
function. Due to their polyanionic character, divalent cationic ions
(i.e., Ca2+ and Mg2+) bind to the polymer, and
by this process they are obviously concentrated and more efficiently
transferred to the rhizosphere of plants (7).
| |
MATERIALS AND METHODS |
Bacterial strains.
All bacteria investigated in this study
are listed in Table 1.
Growth of bacteria.
Bacillus strains were cultivated
at different temperatures in 0.8% (wt/vol) nutrient broth, in mineral
salts medium (15), in liquid manure, in artificial liquid
manure, or in particle-free liquid manure supplemented with carbon
sources by using different rates of aeration as indicated below.
Artificial liquid manure contained (per liter) 16.52 g of
(NH4)2SO4, 0.23 g of
Na2HPO4 · 2H2O, 0.71 g
of MgSO4 · 7H2O, and 1.34 g of KCl
(19). The pH was adjusted to 7.5 with 1.0 N
H2SO4. Liquid manure was obtained from the
Landwirtschafliche Untersuchungs- und Forschungsanstalt (Oldenburg,
Germany) and was stored at 4°C. Prior to use the liquid manure was
filtered, centrifuged for 10 min at 3,000 × g to
remove solid particles, and autoclaved. The chemical composition of the liquid manure was as follows: 0.46% (wt/wt) nitrogen, 0.32% (wt/wt) ammonium N, 0.27% (wt/wt) P2O5, 0.29% (wt/wt)
K2O, and 3.9% (wt/wt) dry matter. For PGA production
medium E was also used (11). To obtain solid media, 1.5%
(wt/vol) agar agar was added. Growth was monitored photometrically at
600 nm or with a Klett-Summerson photometer by using a green no. 54 filter (520 to 580 nm).
Nitrosoguanidine mutagenesis.
N-Methyl-N'-nitro-N-nitrosoguanidine
mutagenesis of B. subtilis 1551 was performed as described
previously by Reh and Schlegel (14).
Electrophoretic methods.
Proteins were separated under
denaturing conditions in 11.5% (wt/vol) polyacrylamide gels by the
method of Laemmli (10) and were stained with Serva Blue R.
Quantitative determination of ammonia.
The ammonia content
was determined with a type 15 230 3000 gas-sensitive electrode (Mettler
Toledo GmbH, Steinbach/Ts, Germany) according to the instructions
provided by the manufacturer.
Qualitative and quantitative determination of PAAs.
The
amount of isolated PAAs was analyzed after precolumn derivatization of
PAA hydrolysates with ortho-phthaldialdehyde reagent on a
reversed-phase high-performance liquid chromatography column as
recommended by the manufacturer (Merck, Darmstadt, Germany) (21). Hydrolysis was carried out by adding 1 ml of 6 N HCl
to 1 mg of lyophilized PAAs and incubating the mixture for 6 h at 105°C.
| |
RESULTS |
Search for suitable strains able to produce PAAs in liquid
manure.
In order to identify organisms suitable for conversion of
the ammonia that is present in liquid manure into PAAs, strains of
bacterial species which had been found previously to excrete PGA,
including B. licheniformis ATCC 9945 and B. subtilis natto IFO3335, as well as polylysine-producing
Streptomyces albulus strain 346, were tested for PAA
production under the appropriate optimal conditions (Table
2). Whereas B. licheniformis ATCC 9945 and B. subtilis
(natto) IFO3335 showed the expected PGA production, S. albulus 346 did not excrete any detectable polylysine. In
addition, six other Bacillus strains from the culture
collection of our laboratory were analyzed for PAA production. These
studies identified two of these six strains as PGA producers.
The PGA-producing strains
B. licheniformis ATCC 9945,
B. subtilis (
natto) IFO3335, and
B. subtilis 1551 and b'5 were chosen
to investigate the
influence of liquid manure on polymer production.
To do this, the
production medium was replaced stepwise by increasing
the fraction (33, 50, 66, or 100%, [vol/vol]) of liquid manure.
Although all strains
produced reasonable amounts of PGA in the
absence of liquid manure, the
amounts of PGA were significantly
reduced in the presence of increasing
concentrations of manure,
as shown in Fig.
1.
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FIG. 1.
Cultivation of several Bacillus strains with
different concentrations of liquid manure. The strains were cultivated
at 35°C for 96 h in 500-ml conical flasks equipped with three
baffles containing a total volume of 100 ml of medium. Medium E was
replaced stepwise by increasing the fraction of liquid manure, as
follows: 0% (vol/vol) ( ), 33% (vol/vol) ( ), 50% (vol/vol)
( ), 66% (vol/vol) ( ), and 100% (vol/vol)
( ).
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Effects of different carbon sources on PGA production.
In
liquid manure, the nitrogen-to-carbon ratio is approximately 11:1
(16). In medium E, which provides the optimal amounts of
carbon and nitrogen for PGA production by B. licheniformis ATCC 9945 (22), the nitrogen-to-carbon
ratio is 1:13.8. Therefore, liquid manure was supplemented with
different relatively inexpensive carbon sources to improve the
conditions for PGA production.
As Table
3 shows, good growth and PGA
production were obtained with both
B. licheniformis
ATCC 9945 and
B. subtilis 1551
on liquid manure in the
presence of sodium gluconate. Glucose
and sucrose also had positive
effects on growth and PGA production.
Beet molasses stimulated
growth only slightly and did not or almost
did not result in
production of PGA. Addition of whey also had
a negligible effect. Since
these carbon sources had no effect
or only a negligible effect on
growth of and PGA production by
the other two strains of
B. subtilis and also had no effect on
growth of and polylysine
production by
S. albulus,
B. licheniformis ATCC 9945 and
B. subtilis 1551 were chosen for further
detailed
studies of the growth kinetics.
Determination of growth kinetics by using artificial and
particle-free liquid manure.
Differences in the composition of
liquid manure made it impossible to obtain reproducible quantitative
data for growth and conversion of ammonia into PGA. Furthermore, liquid
manure contains up to 4.7% (wt/vol) solid material (e.g., hairs,
straw, etc.), which prevented continuous measurement of growth and PGA
content. Therefore, at the beginning of the experiments, artificial
liquid manure was used as the medium for comparative studies of the
growth kinetics of B. licheniformis ATCC 9945 and
B. subtilis 1551. The composition of artificial liquid
manure corresponded to the average values for the main soluble mineral
constituents of liquid manure (19).
On artificial liquid manure containing 0.5% (wt/vol) sodium gluconate,
both
B. licheniformis ATCC 9945 (Fig.
2A) and
B. subtilis 1551 (Fig.
2B) exhibited relatively fast growth, with doubling
times of 3.2 and 2.4 h, respectively. PGA production started after
the cells
entered the stationary growth phase, and the concentrations
reached
maximum values of 0.85 and 0.79 g/liter, respectively,
after 96 h
of cultivation. Slower growth (doubling times, 13.0
and 10.8 h,
respectively) and less PGA (0.58 and 0.39 g/liter,
respectively) were
obtained with these strains in the presence
of 0.5% (wt/vol) glucose.
No PGA production was detected during
cultivation of either strain on
sucrose, beet molasses, or whey.
Whereas
B. licheniformis ATCC 9945 exhibited weak growth on sucrose
(doubling
time, 6.6 h) and even less growth on molasses,
B. subtilis 1551 was not able to use either substrate for growth.
With both
species, no growth occurred in the presence of whey.
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FIG. 2.
PGA production and cultivation of B. licheniformis ATCC 9945 (A) and B. subtilis 1551 (B) in artificial liquid manure. The strains were cultivated at 35°C
for 96 h in 500-ml conical flasks equipped with three baffles
containing 100 ml of artificial liquid manure. The concentration of
each carbon source was 0.5% (wt/vol). Symbols:  , sodium gluconate;
 , glucose;  , sucrose;  , beet molasses;  , whey; , no
carbon source.
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|
In order to verify these results under conditions more likely to occur
in the field, natural liquid manure was used, but it
was pretreated by
filtration and centrifugation to obtain particle-free
liquid manure.
During cultivation of
B. licheniformis ATCC 9945
and
B. subtilis 1551 in particle-free liquid manure, which
was
supplemented with 0.5% (wt/vol) sodium gluconate, PGA production
and reduction of ammonium occurred at rates which were 50% of
those
obtained with artificial liquid manure (Fig.
3). As revealed
by comparison with a
sterile control that was incubated under
identical conditions,
approximately 40% of the reduction was due
to evaporation of
NH
3. Therefore, based on a PGA yield of approximately
0.4 g/liter, only 5% of the ammonia in each case was converted
into PGA.
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FIG. 3.
Cultivation of, ammonium reduction by, and PGA
production by B. licheniformis ATCC 9945 (A) and
B. subtilis 1551 (B) in particle-free liquid manure.
The strains were cultivated at 35°C for 96 h in 500-ml conical
flasks equipped with three baffles containing 100 ml of particle-free
liquid manure. The concentration of sodium gluconate was 0.5%
(wt/vol). Symbols: , optical density at 600 nm;  , ammonium
concentration; , PGA concentration.
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Conversion of ammonia into PGA under conditions that occur in
intensified farming situations.
In order to test the ability of
the PGA-producing bacteria to convert ammonia into the PGA polymer
under conditions similar to those that occur in intensified agriculture
situations, growth experiments were performed in a medium composed of
54% (vol/vol) tap water, 33% (vol/vol) medium E, and 13% (vol/vol)
particle-free liquid manure. This composition resembled those that were
used in a pilot plant at the Landwirtschaftliche Untersuchungs- und Forschungsanstalt. In this pilot plant, experiments to reduce the
ammonia content of liquid manure by nitrification were done, and to
0.13 volume of liquid manure 0.33 volume of activated sludge and 0.54 volume of water were added. Once a week 0.13 volume of the culture
fluid was replaced by fresh liquid manure.
As shown in Fig.
4,
B. licheniformis ATCC 9945 and
B. subtilis 1551 exhibited similar growth under the conditions mentioned
above. After
inoculation with
B. licheniformis ATCC 9945, the
ammonia content was reduced from 1.3 to 0.75 g/liter within 48
h.
In two steps after 48 and 96 h of cultivation, 0.13 volume
of the
culture volume was replaced by fresh liquid manure. During
the
following 2 days of incubation, the ammonia concentration
was reduced
to 0.74 g/liter. Net production of PGA occurred only
during the first
96 h, and the maximum value was 2.2 g/liter.
During the following
2 days, the concentration of PGA decreased
to 1.6 g/liter.
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FIG. 4.
Cultivation of B. licheniformis ATCC
9945 (A) and B. subtilis 1551 (B) under fed batch
conditions. The strains were cultivated at 35°C for 144 h in
500-ml conical flasks equipped with three baffles containing 100 ml of
medium composed of 54% (vol/vol) tap water, 33% (vol/vol) medium E,
and 13% (vol/vol) particle-free liquid manure. At the times indicated
by arrows, 0.13 volume of particle-free liquid manure was added.
Symbols: , optical density at 600 nm; , ammonium concentration;
, PGA concentration.
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The results obtained with
B. subtilis 1551 differed
from those obtained with
B. licheniformis; both
reduction of ammonia and
production of PGA were lower (Fig.
4).
Isolation and characterization of a B. subtilis
mutant affected in degradation of PGA.
PGA-producing bacteria are
generally also able to utilize the excreted PGA polymer as a nutrient
(13). Therefore, mutants of PGA-producing strains affected
in degradation of PGA should give higher yields of the polymer during
batch cultivation in liquid manure. After mutagenization of
B. licheniformis ATCC 9945 and B. subtilis 1551 with
N-methyl-N'-nitro-N-nitrosoguanidine, the cells were incubated in 50 ml of nutrient broth in the presence of
penicillin (75 mg/ml) to enrich those mutants which were defective in
degrading PGA. To identify and isolate the desired mutants, the cells
were plated on medium E, on which slimy colonies indicated the wild
type by the ability to excrete PGA. In parallel, the cells were also
plated onto mineral medium with 0.5% (wt/vol) PGA as the sole carbon
source, on which no or reduced growth indicated defective PGA degradation.
Although no mutant of
B. licheniformis ATCC 9945 with
the desired phenotype was obtained, one stable mutant of
B. subtilis 1551 out of 1,500 candidates investigated showed reduced
growth
on PGA. Cultivation in liquid medium containing PGA as the sole
carbon source revealed that this mutant, which was referred to
as MP5,
was able to grow on the polymer, but the rate of degradation
was
significantly lower than that of the wild type (Fig.
5). Thus,
mutant MP5 exhibited the
phenotype PGA leaky. This reduced capacity
resulted in faster polymer
production and a higher yield during
cultivation in medium E, in which
the mutant produced PGA at final
concentrations up to 4.8 g/liter,
whereas the final concentration
of PGA obtained with the wild type was
only 3.7 g/liter (Fig.
6).
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FIG. 5.
Consumption of PGA by B. subtilis 1551 (A) and the PGA-degrading mutant B. subtilis MP5 (B) in
mineral salts medium. The strains were cultivated at 35°C for 11 days
in 500-ml conical flasks equipped with three baffles containing 100 ml
of mineral salts medium and 0.5% (wt/vol) PGA as the sole carbon
source. Symbols: , optical density as measured with a
Klett-Summerson colorimeter; , PGA concentration.
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FIG. 6.
Production of PGA by B. subtilis 1551 (A) and the PGA-degrading mutant B. subtilis MP5 (B) in
medium E. The strains were cultivated done at 35°C for 11 days in
500-ml conical flasks equipped with three baffles containing 100 ml of
medium E. Symbols: , optical density as measured with a
Klett-Summerson colorimeter; , PGA concentration.
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Polyacrylamide gel electrophoresis of denatured crude extracts of
mutant MP5 and the wild type revealed a clear difference.
Whereas the
protein pattern for cells of the wild type grown on
PGA had a weak but
distinct band representing a protein with an
apparent
Mr of 35,000 ± 1,000, which was absent in
wild-type cells
after growth in PGA production medium, this protein was
absent
in the electropherograms of mutant MP5 cells in the early growth
phase and in the late stationary growth
phase.
| |
DISCUSSION |
The purpose of this study was to describe a process for conversion
of the ammonia of liquid manure into PAAs. In this study, it was shown
that PGA-producing species of the genus Bacillus are able to
convert a significant fraction of the ammonium nitrogen present in
liquid manure transiently into this PAA. By this process, the
concentration of free ammonium in the manure could be reduced, and the
PGA produced acted as a transient depot for ammonium. However, the
conversion occurred only to a low extent. In the case of B. licheniformis ATCC 9945 and B. subtilis 1551 this
was mainly due to the carbon-to-nitrogen ratio in liquid manure, which is the inverse of that in medium E, which is known to favor PGA production. Consequently, an increase in PGA production with a corresponding reduction in the amount of ammonia was achieved by adding
a suitable carbon source or medium E to liquid manure. Another strategy
involving a better nitrogen sink is application of organisms producing
the proteinlike polymer cyanophycin, which consists of equimolar
amounts of arginine and aspartic acid (nitrogen to carbon ratio, 1:2)
(18). Cyanophycin is unique to cyanobacteria and has been
reported to occur in many species of cyanobacteria. At present, the use
of genetically engineered microorganisms would be necessary to produce
such a nitrogen storage compound in manure. Further systematic
optimization of the culture conditions would surely lead to improvement
of the conversion rate. Whether such a strategy can be
biotechnologically applied will depend on further optimization of the
process and also on isolation of PGA-producing bacteria that are better
adapted to the conditions prevailing at agricultural sites, including
high concentrations of manure and relatively low temperatures.
However, any technical process for refinement of liquid manure must
comply with the requirements of agriculture and consequently should be
as simple and cheap as possible. Both treatment of liquid manure to
improve the culture conditions and control of the process may be too
costly and too sophisticated to be accepted by farmers.
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ACKNOWLEDGMENTS |
We are indebted to Landwirtschaftliche Untersuchungs- und
Forschungsanstalt for providing the liquid manure and analytic data on the composition of the manure.
This study was supported by a grant from Bayer AG (Leverkusen, Germany).
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FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Mikrobiologie, Westfälische
Wilhelms-Universität Münster, Corrensstraße 3, 48149 Münster, Germany. Phone: 49-251-8339821. Fax:
49-251-8338388. E-mail: steinbu{at}uni-muenster.de.
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REFERENCES |
| 1.
|
Archer, J. R., and R. J. Nicholson.
1992.
Liquid wastes from farm animal enterprises, p. 325-343.
In
C. Phillips, and D. Piggins (ed.), Farm animals and the environment. C.A.B. International, Wallingford, United Kingdom.
|
| 2.
|
Birrer, G. A.,
A. M. Cromwick, and R. A. Gross.
1994.
-Poly(glutamic acid) formation by Bacillus licheniformis ATCC9945a: physiological and biochemical studies.
Int. J. Biol. Macromol.
16:265-275[CrossRef][Medline].
|
| 3.
| Bundesgesetzblatt. 1996. Verordnung über die
Grundsätze der guten fachlichen Praxis beim Düngen
(Düngerverordnung). 1(6):118-121.
|
| 4.
|
Hara, T., and S. Ueda.
1982.
Regulation of polyglutamate production in Bacillus subtilis (natto): transformation of high -D-PGA productivity.
Agric. Biol. Chem.
46:2275-2281.
|
| 5.
|
Heyndrickx, M.,
K. Vandermeulebroecke,
K. Kersters,
P. De Vos,
N. A. Logan,
M. A. Aziz,
N. Ali, and R. C. W. Berkeley.
1996.
A polyphasic reassessment of the genus Paenibacillus, reclassification of Bacillus lautus as Paenibacillus lautus comb. nov. and of Bacillus peoriae as Paenibacillus peoriae comb. nov., and emended description of P. lautus and of P. peoriae.
Int. J. Syst. Bacteriol.
46:988-1003[Abstract/Free Full Text].
|
| 6.
|
Isermann, K.
1990.
Ammoniakemission der Landwirtschaft als Bestandteil ihrer Stickstoffbilanz und Lösungsansätze zur hinreichenden Minderung, p. 1.1-1.76.
In
Kuratorium für Technik und Bauwesen in der Landwirtschaft e.V. (KTBL), Verein Deutscher Ingenieure (VDI): Ammoniak in der Umwelt. KTBL-Schriften-Vertrieb im Landwirtschaftsverlag GmbH, Münster-Hiltrup, Germany.
|
| 7.
| Kinnersley, A., D. Strom, A. R. Y. Meah, and
C. P. Koskan. May 1994. Composition and method for enhanced
fertilizer uptake by plants. Patent WO94/09628
|
| 8.
|
Kirchgessner, M.,
M. Kreuzer, and F. X. Roth.
1991.
Bestimmungsfaktoren der Güllecharakteristik beim Schwein. 1. Einfluß von Leistungsrichtung, -stadium und - intensität sowie Futteraufnahme und Geschlecht unter üblichen Fütterungsbedingungen.
Agribiol. Res.
44:299-324.
|
| 9.
|
Kunioka, M., and A. Goto.
1994.
Biosynthesis of poly(-glutamic acid) from L-glutamic acid, citric acid, and ammonium sulfate in Bacillus subtilis IFO3335.
Appl. Microbiol. Biotechnol.
40:867-872[CrossRef].
|
| 10.
|
Laemmli, U. K.
1970.
Cleavage of structural protein during the assembly of the head of bacteriophage T4.
Nature (London)
227:680-685[CrossRef][Medline].
|
| 11.
|
Leonard, C. G.,
R. D. Housewright, and C. B. Thorne.
1958.
Effects of some metallic ions on glutamyl polypeptide synthesis by Bacillus subtilis.
J. Bacteriol.
76:499-503[Free Full Text].
|
| 12.
|
Miflin, B. J., and P. J. Lea.
1982.
Ammonia assimilation and amino acid metabolism, p. 5-64.
In
D. Boulter, and B. Parthier (ed.), Nucleic acids and proteins in plants, vol. 1. Springer-Verlag, Berlin, Germany.
|
| 13.
|
Opperman, F. B.,
S. Pickartz, and A. Steinbüchel.
1998.
Biodegradation of polyamides.
Polym. Degrad. Stabil.
59:337-344[CrossRef].
|
| 14.
|
Reh, M., and H. G. Schlegel.
1969.
Anreicherung und Isolierung auxotropher Mutanten von Hydrogenomas H16.
Arch. Mikrobiol.
67:99-109[CrossRef][Medline].
|
| 15.
|
Schlegel, H. G.,
H. Kaltwasser, and G. Gottschalk.
1961.
Ein Submersverfahren zur Kultur wasserstoffoxidierender Bakterien: Wachstumsphysiologische Untersuchungen.
Arch. Mikrobiol.
38:209-222[CrossRef][Medline].
|
| 16.
|
Schulze-Rettmer, R.
1980.
Nitrogen in liquid manure.
Wiss. Umwelt.
4:171-174.
|
| 17.
|
Shima, S., and H. Sakai.
1977.
Polylysine produced by Streptomyces.
Agric. Biol. Chem.
41:1807-1809.
|
| 18.
|
Simon, R. D., and P. Weathers.
1976.
Determination of the structure of the novel polypeptide containing aspartic acid and arginine which is found in cyanobacteria.
Biochim. Biophys. Acta
420:165-176[Medline].
|
| 19.
|
Syrett, P. J.
1988.
Uptake and utilization of nitrogen compounds, p. 23-39.
In
L. J. Rogers, and J. R. Gallon (ed.), Biochemistry of the algae and cyanobacteria. Oxford Science Publications Clarendon Press, Oxford, United Kingdom.
|
| 20.
|
Thomas, P.
1984.
Aerob mikrobielle Behandlung von Schweinegülle mit anschließender Hefebiomassebildung. Ph.D. thesis.
Westfälische Wilhelms-Universität, Münster, Münster, Germany.
|
| 21.
|
Tran, H.,
F. B. Oppermann-Sanio, and A. Steinbüchel.
1999.
Purification and characterization of cyanophycin synthetase from the thermophilic Synechococcus sp. MA19.
FEMS Microbiol. Lett.
181:229-236[CrossRef][Medline].
|
| 22.
|
Troy, F. A.
1973.
Chemistry and biosynthesis of the poly(-D-glutamyl) capsule in Bacillus licheniformis. I. Properties of the membrane-mediated biosynthetic reaction.
J. Biol. Chem.
248:305-315[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, February 2001, p. 617-622, Vol. 67, No. 2
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.2.617-622.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
