Jourrial of General Microbiology (1980), 117, 89-96.
Printed in Greut Britain
89
Utilization of Methyl Amines as Nitrogen Sources by
Non-methylotrophs
By B. BICKNELLT A N D J. D. OWENSS*
Department of Microbiology, University of Sydney, N.S. W. 2006, Australia
(Received I3 August 1979)
Sixteen non-methylotrophic bacteria able to grow with methylamine as sole nitrogen source
in the presence of a mixture of organic compounds but unable to grow with methylamine as
sole carbon source were isolated. They included representatives of Arthrobacter, Bacillus,
Pseudomonas and Enterobacteriaceae. Other compounds used as sole nitrogen sources but
not as sole carbon sources were ammonium salts (16 strains), dimethylamine (I), trimethylamine (I), trimethylamine N-oxide ( I), ethylamine (2), p-alanine (2), L-serine (6) and
betaine (6). The ecological and evolutionary significance of the results is discussed.
INTRODUCTION
Mineralization of organic nitrogen is a process accorded great importance since production in natural ecosystems is often limited by availability of inorganic nitrogen (Strickland,
I97 1 ; Alexander, 1977). Generally, it is assumed that organic nitrogenous compounds serve
as sources of carbon, energy and nitrogen for heterotrophic microbes with the concomitant
release, as ammonium, of the nitrogen surplus to the requirements of the growing microbes
(Alexander, 1977; Fenchel & Jarrgensen, 1977). Little consideration has been given to the
possibility that some microbes might use nitrogenous organic compounds (other than urea)
as sources of nitrogen without being able to use them as principal sources of carbon. However, there are reports of bacteria and/or fungi able to use methyl amines (Budd & Spencer,
1968; van Dijken et al., 1979), ethyl amines (Budd & Spencer, 1968), pyrimidines (Vogels &
Drift, 1976), purines (Rouf & Lomprey, 1968; Vogels & Drift, 1976; Aretz et al., 1978),
amides (Ennis et al., 1978), imides (Ennis et al., 1978) or amitrole (3-amino-1,2,3-triazole)
(Campacci et al., 1977) as sole sources of nitrogen but unable to use the same compounds
as sole carbon sources.
Under conditions of a high ratio of organic carbon to nitrogen, heterotrophic microorganisms compete with primary producers for the available nitrogen (Alexander, 1977). If
much of the nitrogen is present in organic form, it seems likely that the ability to use organic
nitrogenous compounds as nitrogen sources would confer some advantage to organisms
with this ability over those lacking it. Since the direct utilization of organic nitrogen constitutes a by-passing of mineralization it may render an organic nitrogen source totally
unavailable to the primary producers. Hence, it is desirable to know how widespread is the
ability to use nitrogenous organic compounds as sole nitrogen sources among microorganisms and the significance of such organisms in the nitrogen cycle in natural habitats.
-f Present address : Department of Chemical Engineering, University of Sydney, N.S.W. 2006, Australia,
j: Present address: National College of Food Technology, University of Reading, St George’s Avenue,
Weybridge, Surrey KT13 ODE.
0022-1287/80/0000-8911 $02.00 GI 1980 SGM
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90
B. B I C K N E L L A N D J. D. OW E N S
This report describes the isolation and characterization of bacteria able to use methylamine, dimethylamine, trimethylamine or trimethylamine N-oxide as sole source of nitrogen,
but unable to use these compounds as sole carbon sources.
METHODS
Culture media. Mineral salts were provided as base E or base E( - N) (Owens & Keddie, 1969) at pH 6-8,
made up at single or double strength and sterilized by autoclaving at 121 "C for 20 min. Agar (Oxoid L28)
was washed as described by Meynell & Meynell(l970) but without the final washing with ethanol. The agar
was added to distilled water to give 24 g 1-I and sterilized by autoclaving at 121 "C for 20 min. Carbon source
mixture contained (gl-l in distilledwater) : ethanol, 20; sodium acetate, 20; sodium succinate, 20; D-ribose,20;
D-glucose, 20. It was sterilized by membrane filtration and added to media at 1 % (v/v). Methyl amines mixture contained (mM in distilled water) : methylamine. HCI, 60; dimethylamine. HCI, 30; trimethylamine .HCI,
20; trimethylamine N-oxide, 20; tetramethylammonium chloride, 15. It was sterilized by membrane
filtration and added to media at 1 or 10 % (v/v) as required. Stock solutions of growth factors were prepared
at 100 times the concentration required in the culture medium and sterilized by membrane filtration. Vitamins
solution contained (mg 1-1 in distilled water) : nicotinic acid, 10; calcium D-pantothenate, 10; thiamin. HCI,
10; riboflavin, 10; folic acid, 1 ; p-aminobenzoic acid, 1 ; pyridoxal.HC1, 1; biotin, 0.1 ; vitamin B12, 0.1.
L-Methionine solution was 10 mg 1-1 in distilled water.
Culture media were prepared aseptically by mixing the appropriate sterile ingredients and dispensing into
sterile containers. To make solidified media, sterile double-strength liquid medium was mixed with an equal
volume of sterile molten agar. The media used included: medium EM [base E (includes ammonium) + growth
factorst methyl amines mixture (10 %, v/v)]; medium EMA [as medium EMfagar]; medium EC [base
E+ carbon source mixture]; medium ECA [as medium EC agar] ;medium EGA [as medium ECA +growth
factors]; medium E-NX [base E(- N) (contains no nitrogen) growth factors + carbon source mixture +
methyl amines mixture (1 % (v/v)]; medium E-NXA [as medium E-NX+agar].
Source of water sample. Samples of water were collected from 10 sites around the circumference of an
artificial pond in Victoria Park, Chippendale, Sydney, Australia. In the laboratory, 50 ml of each sample
were combined to provide a composite sample which was used in subsequent work.
Isolation and enumeration ojnon-methylotrophic methyl amine-utilizing bacteria. To remove large particulate
matter the sample was membrane filtered (pore size 5 pm). Suitable volumes of the filtrate were then added to
50 ml sterile base E( - N) and passed through sterile membrane filters (47 mm diam., 0-2pm pore size). The
retained material was washed with 10 ml sterile base E( - N). The filter was then placed on a sterile absorbent
paper pad (Gelman Instrument Co., Ann Arbor, Mich., U.S.A.)soaked in medium E-NX in a Petri dish.
Duplicate cultures were prepared for each dilution of the water sample. Cultures were incubated at 25 "C
for 5 d and then all the colonies from measured segments of filters with well-separated colonies were picked
offto medium E-NXA. The isolates were purified by streaking growth from isolated colonies on plates of
the same medium, and checked for purity by streaking on plates of medium EGA and of nutrient agar
(Oxoid CM3). The isolates were tested, using the methods described below, for their ability to use the methyl
amines mixture as sole carbon and/or sole nitrogen source; those using the methyl amines mixture as sole
nitrogen source but unable to use it as sole carbon source were retained for further study. The number of
such non-methylotrophic methyl amine-utilizing bacteria in the original water sample was estimated by
relating the number of isolates obtained to the area of the filter picked and the dilution of the sample filtered.
Enumeration of methylotrophic bacteria. The method used was similar to that for non-methylotrophic
bacteria except that absorbent pads were soaked in medium EM and isolates were picked to and purified on
plates of medium EMA.
Characterization of isolates. Cell morphology, Gram-stain reaction, oxidase reaction and production of
catalase were examined on growth from 24 h cultures on medium ECA. Cell morphology and motility were
examined using phase contrast microscopy.
Mode of attack on glucose was tested by the method of Hugh & Leifson (1953). Anaerobic growth was
examined by incubating cultures on medium ECA in an atmosphere of H2 and COB(BBL GasPak) in the
dark. Growth in the presence of NaCl was tested on medium ECA+NaCl(20 g 1-I) and on medium ECA+
NaCl (50 g 1-l). Starch hydrolysis was examined on medium ECA+soluble starch (10 g 1-l).
All the above cultures were incubated at 25 "C and examined after 2 to 6 d incubation.
To determine the range of temperature supporting growth, cultures on medium ECA were incubated at 4,
30, 37 and 44 "C.
Determination of compounds used as sole nitrogen sources. Growth from a 24 or 48 h culture on medium
ECA was suspended in base E(- N) to form a just visibly turbid suspension. One drop of this suspension
+
+
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Non-rnethylotrophic use of methyl arnines
91
was added to each of three test-tubes (13 x 125 mni) containing 5 ml of one of the following culture media:
base E( - N) carbon source mixture; base E( - N) carbon source mixture+ 0.75 mM-(NH,),SO,; base
E( - N) + carbon source mixture+ test compound at a concentration providing 1.5 mM-N. To reduce
contamination by extraneous nitrogen, freshly collected distilled deionized water was used in the preparation
of these media. The compounds tested as nitrogen sources are shown in Table 2. They were prepared as
concentrated stock solutions and sterilized by filtration.
Cultures were incubated in air at 25 "C in a vertical position on an orbital shaker (2.4 cm diam. orbit;
175 rev. min-l). Growth was assessed visually and any tube showing turbidity after incubation was subcultured with a wire loop into a sterile tube of the same medium to confirm that growth was due to utilization of the added nitrogen source rather than to carry-over of nitrogen with the original inoculum. Cultures
showing good growth with the test compound but little or none in the absence of added nitrogen were deemed
to use the compound as sole nitrogen source.
Determination of a requirement for growth factors. Growth in the nitrogen test media with (NH&S04 or
with a test compound, as described above, also indicated that an isolate did not have a requirement for
vitamins or L-methionine. However, the technique used was not capable of detecting requirements for
vitamins, such as biotin, that are required in very small amounts.
Determination of compounds used as sole carbon sources. Each organism was inoculated on to a plate of
basal medium containing base ESgrowth factors+agar and on to plates of the same medium+test compound (1 gl-l). Concentrated stock solutions of the test compounds were either sterilized by membrane
filtration or used without treatment except for storage at -20 "C. Plates were inoculated, using a multipoint
inoculator, with drops of a just visibly turbid suspension in base E( - N) of bacteria from a 24 or 48 h culture
on medium EGA. Enhanced growth, after incubation at 25 "C for 3 or 6 d, on the medium with the test
compound over that on the basal medium was taken as evidence for utilization of the compound as a carbon
and energy source.
Determination of methanol, formaldehyde and forniic acid in culture filtrates. Cultures in base E( -N) +
17 mwglucose+3 m-methylamine were grown with shaking at 30 "C for about 18 h and then filtered
through a membrane filter (Millipore HA). The culture filtrate was tested for the presence of methanol with
the permanganate oxidation method, for formic acid with the magnesium powder reduction method and for
formaldehyde using the chromotropic acid reagent (Feigl, 1966). Culture filtrates of strain NB4 in base
E(- N) I7 nm-glucose+ 3 m-dimethylamine and in base E( - N)+ 17 mM-glucose+ 3 m-trimethylamine
were similarly tested. In control tests using solutions of pure chemicals the minimum concentrations giving
positive colour reactions were about 2 m-methanol, I mwfornialdehyde and 1 m-formic acid.
+
+
+
RESULTS
Bacteria isolated
A total of 44 cultures able to use the methyl anlines mixture as sole nitrogen source but
unable to use methyl amines as sole carbon source were isolated. This corresponded to
2.8 x lo3 colony-forming units (c.f.u.) ml-l in the original water sample. The number of
methylotrophs able to use the same mixture of methyl amines as sole carbon source in the
same water sample was 3-5x lo4 c.f.u. inlkl.
Sixteen representative cultures of the non-methylotrophs were selected for further study.
All grew in air, possessed catalase and grew at 25 and 30 "C. None grew at 44 "C or in the
presence of 5 "/o NaC1 or required vitamins or L-methionine. Other morphological and
physiological properties of the organisms are shown in Table 1. Fifteen of the cultures can,
on the basis of these properties, be assigned to Arthrobacter, Bacillus, Pseudomonas or to
Enterobucteriaceae. Culture N C 1 3 is at present unassigned.
Nitrogen nutrition
Various compounds were utilized as sole nitrogen sources by the cultures and some of
these could also be used as sole carbon sources (Table 2). All the cultures grew with ammonium or methylamine as sole nitrogen source but only one culture utilized dimethylamine,
trimethylamine or trimethylamine N-oxide as nitrogen sources and none utilized tetramethylammonium salts. No methyl amine compound was used as sole carbon source by
any of the 16 cultures.
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NR
+
+
+
-
NR
+
+
+-
w
I
-
-
+
-
C/R
N3
C/R
+
DT35
+
+
-
SR
NR
-
pink
+
+
pale
SR
0
-
+
+
-
SR
NA41 NA44 NA50
F
-
--
R
~
NB2
-
+
+
F
-
-
+
-
R
NCl
Y
+
+
-+
+
F
-
+
-
R
NB13
-2
+
+
+
F
pale
yellow
-
+
-
R
CE8
Bacterial strain
0
+
++
+
-
-
0
Y
CA4
+
0
NA19 NA21
~~
0
+
+
+
-
-
+
NB4
0
+
-
-
+
CB15
~~
0
+
-
+
d
-
+
-
+
-
-
NR
-
-
pale
yellow
+
-
R
NC13
R
NA8
Enterobacteriaceae
Probable genus/family
A rthrobacter
Pseudomonas
?
* Details are given in Methods: + , positive result; - , negative result.
t C/R, Cocci or coccal rods in simple media with methyl amines and in 4 d cultures on nutrient agar, rods in 24 h cultures on nutrient agar; SR, spore-forming rods;
R, rod-shaped.
$ NR, No reaction; F, fermentative; 0,oxidative.
Oxidase
O / F test on glucose$
Anaerobic growth
Starch hydrolysis
Growth in 2 % (w/v) NaCl
Growth at 4 "C
Growth at 37 "C
Cell morphology?
Gram reaction
Motility
Colony pigmentation
Property*
~~
Table 1. Some morphological and physiological properties of I6 bacterial cultures able to use methylamine as sole nitrogen source but not as sole
carbon source
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+
+
+
-
Methylamine
+-- I
Dimethylamine
Tr imethylamine
*
Tr imet hy 1amine
N-oxide
A
Ethylamine
L-Serine
--
P-Alanine
Utilization? of compound as sole carbon (C) or nitrogen (N) source
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
I
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Bacterial
rJ--strain
NH4+ NOz- NO,N
C
N
C
N
C
N
C
N
C
N
C
N
C
+
DT35
+
+
+
N3
+
+
f
+
NA41
ND
ND
+
+
+
NA44
+
+
+
NA50
ND
ND
+
+
NB2
+
NC 1
+
+
+
+
NB13
ND
ND
CE8
ND
NA19
ND
NA2 1
+
- 3 .
ND
ND
CA4
NB4
CB15
ND
NA8
ND
+
+
+
NCI 3
ND
ND
f
No culture grew with tetramethylammonium salts, diethylamine, triethylamine or isopropylamine as carbon or nitrogen source.
* A mixture of ethanol, acetate, succinate, D-ribose and D-glucose was provided as carbon source.
? +, Compound utilized; -, compound not utilized; ND, not determined.
f
Table 2. lnorgmic and organic nitrogenous compounds used as sole nitrogen sources" and organic nitrogenous compounds used
sources by bacteria able to use methylamine as sole nitrogen source but not as sole carbon source
Betaine
>
sole carbon
-
+
+
ND
ND
ND
-
-
+
-
-
-
*
N
C
US
3
4,
%*
a
3'
h
2
2
3
0
B. B I C K N E L L A N D J. D. O W E N S
94
Several of the cultures also utilized other nitrogenous organic compounds as sole nitrogen
sources but not as sole carbon sources. Such compounds includd L-serine (utilized by 6
strains), betaine (6 strains), /7-alanine (2 strains) and ethylamine (2 strains). L-Serine, betaine.
/?-alanine and ethylamine were also used as sole carbon sources by one or more isolates. In
every case where a culture grew with a nitrogenous organic compound as sole carbon and
energy source, the same compound could also serve as sole nitrogen source.
Carbon nutrition
Table 3 lists some compounds that were utilized as sole carbon sources by one or more
cultures and some nitrogenous organic compounds used as carbon sources are shown in
Table 2. It is evident that these bacteria, with the possible exception of the Bacillus strains
NA41, NA44 and NA50, are versatile organisms able to utilize a wide range of different
compounds singly as sole carbon sources. Compounds also tested but not used as carbon
source by any of the 16 cultures included methylamine, dimethylamine, trimethylamine,
trimethylamine N-oxide, tetramethylammonium salts, methanol, formate, 1,l-dimethylurea,
dimethylolurea, dimethylformamide, dimethylsulphoxide, dimethylsulphone, trimethylphosphate, oxalate, diethylamine, triethylamine, isopropylamine and L-methionine.
Tests f o r metabolites in culture Jiltrates
Culture filtrates of strains DT35, NB13 and NA8 grown on glucose with methylamine as
sole nitrogen source gave positive reactions for formic acid but not for methanol or formaldehyde. Culture filtrates of strain NB4 grown on dimethylamine and glucose or trimethylamine and glucose gave similar results, but when strain NB4 was grown on methylamine and
glucose, formaldehyde was detected in the culture filtrate. Due to the nature of the tests used,
it was not possible to determine whether formate and/or methanol were also present.
Uninoculated control media gave negative reactions for the presence of all three compounds.
DISCUSSION
It is evident that the ability to use methyl amines as nitrogen sources but not as carbon
sources occurs in representatives of diverse bacterial genera. Budd & Spencer (1968)
assigned their marine isolates to Micrococcus, Achromobacter and Pseudomonas, while the
freshwater isolates obtained in the present work include representatives of Arthrobacter,
Bacillus, Pseudomonas and Enterobacteriaceae. The widespread occurrence of this ability
suggests that methyl amines might commonly be utilized in this way in natural habitats.
Some of the isolates also utilized ethylamine, P-alanine, L-serine and/or betaine as
nitrogen but not as sole carbon sources. Their inability to use these compounds as sole
carbon sources is surprising since the same cultures could grow with other C, and C, compounds such as ethanol, acetate, glycine or glycerol. The reasons for this are unknown but
its occurrence suggests that the mechanisms used by bacteria to obtain nitrogen from these
compounds are specialized and perhaps distinct from the pathways of carbon metabolism.
Methylotrophic bacteria able to use methyl amines as sole carbon and energy sources
first convert the methyl groups to formaldehyde which is then either assimilated or oxidized
to CO, (Anthony, 1975; Lin & Wagner, 1975). Since our isolates cannot use methyl amines
as sole carbon sources, they presumably lack the specialized pathways for the assimilation
of C1 compounds. Hence, the formaldehyde (or other product of the deamination reaction)
has three possible fates. Firstly, it might be partially or totally assimilated as a minor carbon
source; secondly, it might be discarded and excreted as formaldehyde; or thirdly, it might
be further oxidized to formic acid or CO,.
Budd (1969) obtained evidence for the formation of formaldehyde during growth with
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+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
N3
NA41
+
-
+
+-
+-
NA50
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
-I-
+
-
+
+
+
+
+
-
f
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
ND
+
++
+
+
+
k
+
+
+
+
-
k
ND
+
+
+-
-
NA19
+
+
+
+
+
+
+
NA2 1
+
+
+
++
+
+
+
+
+
+
+
ND
+
+
-
-
+
-
CA4
ND
+
+
+
-I+
+
+
+
+
+
+
+
ND
+
+
+
+
+-
NB4
k , enhanced growth visible only after 6 d incubation; - , no enhancement of growth;
NA44
* +, Enhanced growth visible after 3 d incubation;
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
D-Ribose
D-Xylose
D-Glucose
D-Fructose
Cellobiose
Ethanol
Glycerol
Mannitol
Acetate
Malate
Succinate
Fumarate
Benzoate
G 1yci ne
L-Alanine
L- Aspartate
L- GIutama te
L-Arginine
L-Proline
L-Phenylalanine
+
DT35
Compound
Utilization" by bacterial strain
+
+
+
ND,
+
+
+
+
+
+
+
+
-I+
+
+
+
+
ND
k
+
+
+
+
+
+
+
+
+
+
+
+
+
-
ND
+
+
-
NA8
+
+
+
NC13
not determined.
CB15
Table 3. Some compounds utilized as carbon and energy sources by bacteria able to use methylamine as sole nitrogen source but not as sole
carbon source
E
a
96
B. B I C K N E L L A N D J. D. O W E N S
trimethylamine as a nitrogen source and suggested that this was further oxidized to C 0 2
which was assimilated by the cell. Van Dijken et al. (1979) reported evidence that suggested
that methylamine was oxidized 10 CO, in yeasts able to use it as a nitrogen source but not as
a carbon source. In the present work, cultures of three strains accumulated formic acid and
one strain accumulated formaldehyde. This suggests that in these organisms at least some of
the methyl carbon is excreted in a partially oxidized form rather than assimilated or oxidized
t o CO,. If such compounds were excreted by organisms in nature they would constitute
potential carbon sources for methylotrophs and represent part of the microbial food chain.
The existence of micro-organisms able to use a nitrogenous organic compound as nitrogen
source only while using a different organic compound as principal carbon and energy source
raises some interesting questions regarding their evolutionary ecology. If, as seems possible,
the carbon moiety of the nitrogenous compound is discarded to the environment one might
ask why should an organism carry out the apparently wasteful process of taking in a compound and then discarding a major part of it. A possible interpretation is that this represents
a n adaptation that supports faster growth than either using the nitrogenous organic compound as carbon and nitrogen source or using the nitrogenous and non-nitrogenous organic
compounds simultaneously as carbon sources. Such an interpretation requires that the nonnitrogenous organic compound be a more favourable energy and carbon source than the
carbon moiety of the nitrogenous compound.
We have some evidence to support this view. Enrichment cultures provided with glucose
and with methylamine as the only nitrogen source are dominated by bacteria able to use
glucose as sole carbon source and methylamine as sole nitrogen source but unable to use
methylamine as sole carbon source (unpublished observations).
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H. & KLEMME,
J. H. (1978).
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