The effects of calcium and magnesium carbonates on some biological transformations of nitrogen in soils

UNIVERSITY OF CALIFORNIA PUBLICATIONS

IN

AGRICULTURAL SCIENCES

Vol. 1, No. 3, pp. 39-49 December 14, 1912

THE EFFECTS OF CALCIUM AND
MAGNESIUM CARBONATES ON SOME BIO-
LOGICAL TRANSFORMATIONS OF
NITROGEN IN SOILS

BY
W. P. KELLEY

HISTORICAL INTRODUCTION

Loew1 and his co-workers found, some years ago, that the
growth of a number of plants may be markedly influenced by
variations in the ratio of calcium to magnesium, both in solution
and soil cultures. Osterhout2 also showed that a more or less
definite relation between other elements in culture solutions is
necessary for maximum growth. These and other researches have
drawn attention to certain long neglected phases of plant physi-
ology and strengthen the view that in addition to the mere
presence of the necessary elements, plants also demand a physio-
logically balanced relation between the elements in solution if
maximum growth is to be produced. By means of artificial cul-
ture solutions principles of great importance are being worked
out, but in generalizing from culture solutions to natural soils,
many difficulties arise. The great complexity of the factors in-
volved and the difficulties inherent in the question necessitate
the greatest care in making broad generalizations regarding soils.

iLoew and May, Bur. Plant Tnd. U. S. D. A., Bui. No. 1; Aso, Bui.
Col. Agr. Tokyo, vol. 4, pp. 361-370; vol. 5, p. 495; vol. 6, p. 97; Loew and
Aso, vol. 7, pp. 395-407.

2Bot. Gaz. 42, 127-134; 44, 259-272; 48, 98-104.

40 University of California Publications in Agricultural Sciences [Vol. 1

In this connection the "lime-magnesia ratio" has become a
matter of general interest and is being extensively investigated
at the present time. Some recent experiments by Lemmermann3
and others seem to indicate that a wide variation in this ratio is
of no consequence to plants. It is well known, however, that the
effects produced by natural limes and limestones are not alwaj^s
equal. In certain instances dolomitic limes are known to produce
less favorable results than non-magnesian limes. During recent
years additional light on the action of lime in soils has been
found in the fact that calcium carbonate enhances certain bio-
logical activities through supplying an active base by means of
which the essential neutral condition is maintained. In this con-
nection the question of the effects on bacterial activity brought
about by different sources of lime and limestone naturally sug-
gests itself.

In regard to physiologically balanced solutions for bacteria,
Dr. C. B. Lipman4 has shown that the ammonification of peptone
by pure cultures of B. sitbtilis is favored on the one hand by a
certain ratio of calcium to potassium, magnesium to sodium and
potassium to sodium ; while on the other hand, he failed to observe
any antagonism between calcium and magnesium or calcium and
sodium. In his investigations Lipman found that a certain con-
centration of magnesium chloride proved toxic to the development
and activity of B. subtilis and at the same time the addition of
certain amounts of calcium chloride failed to overcome this tox-
icity. Likewise, magnesium or sodium was ineffective in over-
coming the toxicity of calcium. While it is probably true that
calcium is not necessary for the normal development of bacteria,
the importance of these observations, if found to apply in soils, is
at once obvious.

From a study of the effects of various carbonates on the
nitrification of ammonium sulphate in solutions, Owen5 in 1908
concluded that magnesium carbonate is better suited to the stimu-
lation and growth of nitrifying organisms than calcium, potas-

3 Landw. Jahrb., 40 (1911), pp. 173-254; Also see Gile, Porto Rico Sta.
Ann. Rept., 1911.

4Bot. Gaz., 48 (1908), pp. 105-125; 49 (1909), pp. 41-50.
■• Ota,. Sta. Bui. 81 (Technical Series No. 1), 1908.

1912] Kelley : Biological Transformations of Nitrogen in Soils 41

sium or ammonium carbonates. It is but fair to mention in this
connection, however, that great dilution of these carbonates was
employed.

In 1910 Dr. J. G. Lipman6 observed that the addition of one
gram of calcium carbonate per one hundred grams of a New Jer-
sey soil stimulated the ammonification of dried blood but de-
pressed the formation of ammonia from cotton seed meal. In
parallel experiments he observed that an equal amount of mag-
nesium carbonate caused a depression in the ammonification of
dried blood but stimulated the ammonification of cotton seed
meal. In other words, the ammonification of dried blood and cot-
ton seed meal in one and the same soil were affected by calcium
and magnesium carbonates in opposite ways, both as regards the
carbonates and the nitrogenous substances employed. These re-
sults are interesting and suggestive and point to the complexity
of this single step in the preparation of available nitrogen from
the organic substances occurring in soils.

In the same year Kellerman and Robinson7 pointed out that
the addition of magnesium carbonate to a highly magnesian soil
in quantities above 0.25 per cent greatly depressed the formation
of nitrates while the application of calcium carbonate in quanti-
ties up to 2 per cent markedly stimulated nitrification. The
growth of crops on this soil had been found to be much more
favorably influenced by the application of ground oyster shells
than by magnesium limestone. The authors inferred from their
experiments that the inferior effects on crops following the appli-
cation of dolomitic limestone may be due, in part, to retarded
nitrification.

AMMONIFICATION

In the course of some studies on soil bacteriology at the Uni-
versity of California, the writer undertook a study of certain
biological transformations, as affected in two different sandy soils
from California by varying amounts and combinations of calcium
and magnesium carbonates. On account of the striking nature
of the results obtained in the preliminary ammonification ex-

e N. J. Sta. Kept., 1910.
7 Science, 32, p. 159.

42 University of California Publications in Agricultural Sciences [Vol. 1

periments a systematic study of this question was undertaken.
The soil employed in the ammonification experiments presently
to be described was of a light sandy character having been taken
from near Oakley in the upper part of San Joaquin Valley and
represents a large area now devoted to the growth of peaches
and other fruits. With suitable moisture conditions this land
produces excellent growth of the crops suited to it. The followT-
ing analysis furnished by the courtesy of Dr. Lipman sets forth
the composition of this soil.

Table I. Composition of Soil Used in Ammonification Experiments

Per cent
Insoluble matter 80.45

Soluble silica 6.15

Potash (K20) 0.35

Soda (Na20) 0.15

Lime (Ca O) 1.41

Magnesia (Mg O) 0.33

Br. Ox. Manganese (Mn:.04) 0.09

Ferric Oxide (Fe2Oy) 3.96

Alumina (AL03) 4.45

Phosphoric Acid (P20.) 0.10

Sulphuric Acid (S 0:;) 0.06

H20 at 110° C 0.80

Volatile matter 2.02

Total 100.32

In the ammonification experiments dried blood was used as a
source of nitrogen. Five grams of this material and varying
amounts of calcium and magnesium carbonates were thoroughly
mixed with 100 gram portions of sifted soil, placed in tumblers
and then optimum moisture conditions provided by the addition
of sterile water. The tumblers were covered with Petri dishes
and after an incubation period of seven days the ammonia was
distilled into standard acid by the use of magnesium oxide and
measured in the usual way. The results are recorded in the
following table.

1912] Kelley: Biological Transformations of Nitrogen in Soils 43

Table II. Effects of Calcium and Magnesium Carbonates on the
Ammonification of Dried Blood

Ammonia nitrogen
Treatment mgs.

None 81.4

1 Gram Calcium Carbonate 84.3

2 Gram Calcium Carbonate 85.0

4 Gram Calcium Carbonate 91.0

6 Gram Calcium Carbonate 91.0

8 Gram Calcium Carbonate 87.8

12 Gram Calcium Carbonate 87.8

1 Gram Magnesium Carbonate 53.2

2 Gram Magnesium Carbonate 53.9

4 Gram Magnesium Carbonate 50.0

These data, as all others submitted in this paper, represent
averages of closely agreeing duplicates. In examining the above
data we note a slight stimulation in ammonia formation from the
use of the several amounts of calcium carbonate employed, the
maximum stimulation being reached with from 4 to 6 grams per
100 grams of soil. With the use of magnesium carbonate a marked
depression in ammonia accumulation occurred, there having been
found to be a falling off of approximately one-third as compared
with the amounts found without the use of carbonate. It is also
noteworthy that one gram of magnesium carbonate proved to be
about as toxic to ammonification as larger amounts.

A second series was prepared with the use of still smaller
amounts of magnesium carbonate for the purpose of determining
the concentration at which toxic effects begin and also to deter-
mine the minimum amount of this carbonate necessary to produce
maximum toxicity. The results follow.

Table III. Ammonification of Dried Blood as Affected by Small
Amounts of Magnesium Carbonate

Ammonia nitrogen
Treatment mgs.

None 93.1

0.1 Gram Magnesium Carbonate 77.4

0.2 Gram Magnesium Carbonate 70.6

0.4 Gram Magnesium Carbonate 65.6

0.6 Gram Magnesium Carbonate 65.2

0.8 Gram Magnesium Carbonate 64.6

1.0 Gram Magnesium Carbonate 62.0

44 University of California Publications in Agricultural Sciences [Vol. 1

These data are instructive as showing the marked depression
of ammonification in the soil employed, even with the small
amount of .1 per cent of magnesium carbonate. The toxicity in-
creased with greater amounts of the magnesium carbonate added
reaching a practical maximum with from 0.8 to 1 gram per 100
grams of soil.

According to Loew the toxic effects of an excess of magnesia
in soils can be overcome or antagonized by the application of
lime. While this theory was proposed and held for the higher
plants, it was thought to be of some interest to study the question
with reference to the ammonification process. Accordingly the
following series of experiments was arranged. In these trials one
gram of magnesium carbonate per 100 grams of soil was used
throughout, this quantity having been found to be the lowest
that produced maximum toxicity.

Table IV. Ammonification of Dried Blood in the Presence of Both

CaCo and MgCo

3 3

Ammonia nitrogen
Treatment mgs.

1 Gram Calcium Carbonate 84.3

1 Gram Magnesium Carbonate 53.9

1 Gram Magnesium Carbonate 4- 0.5 Grams Calcium Carbonate 51.1
1 Gram Magnesium Carbonate 4- 1. Grams Calcium Carbonate 53.9
1 Gram Magnesium Carbonate 4- 2. Grams Calcium Carbonate 53.2
1 Gram Magnesium Carbonate 4- 3. Grams Calcium Carbonate 50.6
1 Gram Magnesium Carbonate 4- 4. Grams Calcium Carbonate 51.1
1 Gram Magnesium Carbonate 4- 5. Grams Calcium Carbonate 50.7
1 Gram Magnesium Carbonate 4- 6. Grams Calcium Carbonate 50.3
1 Gram Magnesium Carbonate 4- 8. Grams Calcium Carbonate 50.7
1 Gram Magnesium Carbonate 4-12. Grams Calcium Carbonate 50.4

From these data it is at once seen that no antagonism was
produced. Even the very large amount of 12 grams of calcium
carbonate in no way reduced the toxic effects produced by one
gram of magnesium carbonate. The results, therefore, are in
harmony with the observations made by Dr. C. B. Lipman8 in his
studies on the physiology of B. subtilis.

* Loc. cit.

1912] Kelley : Biological Transformations of Nitrogen in Soils 45

NITRIFICATION

Having failed to observe any antagonism between calcium and
magnesium in the complex process of ammonification in the soil
under investigation, attention was directed to a study of nitrifica-
tion under similar conditions. A sandy soil from Anaheim, Cali-
fornia, that contained a vigorous nitrifying flora, was employed
in these studies. The following table of analyses furnished by
the kindness of Dr. Lipman shows the chemical composition of
this soil.

Table V. Composition of Soil Used in Nitrification Experiments

Per cent

Insoluble matter 73.59

Soluble Silica 11.17

Potash (K,0) 64

Soda (Na20) 15

Lime (Ca O) 1.39

Magnesia (Mg O) 93

Br. Ox. Manganese (Mn304) 04

Ferric Oxide (Fe203) 5.10

Alumina (Al263) 3.92

Phosphoric Acid (PA,) 12

Sulphuric Acid (SO,) 02

Volatile matter

I

H20 at 110° C

Total 99.95

The nitrification experiments were carried out in tumblers,
two grams of dried blood being mixed with each 100 gram portion
of soil. The amounts of calcium and magnesium carbonates added
are shown in the table. Optimum moisture conditions were main-
tained throughout the 21 day incubation period during which
time a temperature of 27 to 28 degrees was maintained. The
results are shown in the following table.

46 University of California Publications in Agricultural Sciences [Vol. 1

Table VI. Effects of Calcium and Magnesium Carbonates on the
Nitrification of Dried Blood

Nitrate nitrogen
Treatment found mgs.

None 14.5

1.0 Gram Calcium Carbonate 23.5

2.0 Gram Calcium Carbonate 19.2

4.0 Gram Calcium Carbonate 21.2

8.0 Gram Calcium Carbonate 20.2

0.1 Gram Magnesium Carbonate 3.6

0.2 Gram Magnesium Carbonate 2.9

0.4 Gram Magnesium Carbonate 2.8

0.8 Gram Magnesium Carbonate 5.1

1.0 Gram Magnesium Carbonate 1.0

2.0 Gram Magnesium Carbonate 2.0

4.0 Gram Magnesium Carbonate 2.9

8.0 Gram Magnesium Carbonate 3.3

Original soil 5.0

It will be observed that while approximately a 50 per cent
stimulation in nitrate formation was effected by the addition of
calcium carbonate, nitrification was totally inhibited by the addi-
tion of one-tenth of one gram of magnesium carbonate. Before
further discussing these results the data obtained from the
effects of calcium and magnesium carbonates acting synchron-
ously will be presented.

Table VII. The Lack of Antagonism Between Calcium and Magnesium
Carbonates as Shown in the Nitrification of Dried Blood

Nitrate nitrogen
Treatment found mgs.

None 14.5

1.0 gram Calcium Carbonate 23.5

0.1 gram Magnesium Carbonate 3.6

0.1 gram Magnesium Carbonate and 1. gram Calcium Carbonate 4.1

0.1 gram Magnesium Carbonate and 2. gram Calcium Carbonate 3.4

0.1 gram Magnesium Carbonate and 3. gram Calcium Carbonate 2.6

0.2 gram Magnesium Carbonate and 1. gram Calcium Carbonate 1.9

0.2 gram Magnesium Carbonate and 2. gram Calcium Carbonate 1.4

0.2 gram Magnesium Carbonate and 3. gram Calcium Carbonate 2.0

0.4 gram Magnesium Carbonate and 1. gram Calcium Carbonate 2.2

0.4 gram Magnesium Carbonate and 2. gram Calcium Carbonate 1.8

0.4 gram Magnesium Carbonate and 3. gram Calcium Carbonate 3.1

0.8 gram Magnesium Carbonate and 1. gram Calcium Carbonate 2.9

0.8 gram Magnesium Carbonate and 2. gram Calcium Carbonate 3.5

0.8 gram Magnesium Carbonate and 3. gram Calcium Carbonate 4.1

1912] Kelley : Biological Transformations of Nitrogen in Soils 47

Here again it is shown that .1 gram of magnesium carbonate
per 100 grams of soil entirely prevented nitrification. Neither
do we observe any effective antagonism through the use of cal-
cium carbonate.

On the one hand it was found that ammonification of dried
blood was seriously interfered with by the presence of small
amounts of magnesium carbonate, and on the other, nitrification
was completely prevented by its presence. In neither case was
there any evidence of an antagonism between magnesium and
calcium carbonates. In the above nitrification experiments, mag-
nesium carbonate not only prevented the formation of nitrates
but at the same time induced a reduction in the amounts of
nitrates originally present in the soil. It was observed that with
the addition of magnesium carbonate a much more abundant
growth of moulds took place than in the tumblers receiving cal-
cium carbonate.

With a view of throwing further light on this question, total
nitrogen was determined, both before and after the incubation
period of 21 days, in a similar set of experiments to which one
gram of magnesium carbonate had been added. The result showed
that during the period of bacterial action, similar to that in the
preceeding nitrification experiments, the soil sustained a loss
equal to about 20 per cent of the combined nitrogen originally
present.

Two factors suggest themselves as bearing on this question.
The first and probably most important is that of volatilization
and, therefore, loss of ammonia. J. G. Lipman9 in his numerous
researches found that the dilution of a heavy silt loam with
silica sand caused a loss of ammonia in ammonification experi-
ments. The loss began to manifest itself with the use of 30 per
cent of sand but greatly increased with larger amounts. This
loss was attributed to the volatilization of ammonia and was
sufficiently great to give an appreciable odor of ammonia above
the tumblers. The soils employed in the experiments herein
described were largely composed of sand and contained very
small amounts of silt and clay. The substances capable of fixing
large amounts of ammonia are, therefore, largely absent from

N. J. Sta. Kept., 1909.

48 University of California Publications in Agricultural Sciences [Vol. 1

these soils and consequently considerable loss may have been
sustained through the volatilization of ammonia. The data on
ammonification, therefore, should be considered as representing
the ammonia accumulated rather than the absolute amounts
formed. The relative effects of calcium and magnesium carbon-
ates on the loss of ammonia were not investigated.

A second factor in the loss of nitrogen is that of denitrifica-
tion. It was recently shown by Vogel10 that calcium carbonate
under certain conditions can bring about a considerable loss of
nitrogen as nitrates in soils through denitrification. In the pre-
vious experiments it was observed that with the use of magnesium
carbonate a decided reaction for nitrites could be obtained. Deni-
trification, therefore, took place and an actual loss of nitrogen
is probably traceable to this cause. From the preceding data
(Table VI) it is seen that the use of small amounts of magnesium
carbonate not only inhibited nitrification but, as previously men-
tioned, also caused a considerable loss of the nitrates already in
the soil. We have here, therefore, still further evidence of deni-
trification having taken place. "With the use of larger amounts
of magnesium carbonate, nitrification and denitrification were
both inhibited but no considerable loss of the nitrates originally
present in the soil took place.

It seems probable, therefore, that the smaller amounts of mag-
nesium carbonate were toxic to the nitrifying bacteria while still
allowing the denitrifiers to act, but under the influence of larger
amounts of magnesium carbonate both the nitrifying and deni-
trifying groups were rendered inactive.

The striking nature of the results obtained in the previous
ammonification and nitrification experiments suggested a study
of nitrogen fixation under similar conditions. For this purpose
the Anaheim soil was employed since it contains a vigorous nitro-
gen fixing flora. Mannite was used in these experiments and
the usual method followed. The results obtained proved to be so
irregular and discordant that their publication is withheld at this
time. In one series a slight decrease in the amount of nitrogen
fixed followed the use of magnesium carbonate, while in still
another series no effects were observed.

"><VhlU. lisikt. ,'J4, pp. 540-561.

1912] Kelley: Biological Transformations of Nitrogen in Soils 40

DISCUSSION

The experimental data presented above show that under the
conditions employed and in the soils studied, calcium carbonate
stimulated the ammonification of dried blood to a limited extent
but exercised a more noteworthy stimulating effect on nitrifica-
tion. With magnesium carbonate a pronounced toxic effect was
produced. In the ammonification of dried blood there was sus-
tained a loss of about one-third as compared with the experiments
without the use of carbonates, while in the nitrification experi-
ments magnesium carbonate completely inhibited nitrate forma-
tion. It is also noteworthy that no evidence of antagonism be-
tween calcium and magnesium carbonates was observed. It is
not intended, however, to generalize from these results. It does
not follow that similar results would be obtained from any soil.
In fact, data already obtained from other soils show that the
phenomena observed in the two soils above discussed are not of
universal occurrence under similar conditions.

A further study of the lime-magnesia ratio in reference to
nitrogen transformations in soils is now under way with the
use of several types of Hawaiian soils and interesting results have
already been obtained. A more complete interpretation of the
results obtained is reserved for a subsequent publication after a
wider range of observations have been made. Before a satis-
factory understanding of the lime-magnesia question in regard
to field crops is presented it is imperative that we have more
specific knowledge concerning the effects produced on the various
organisms of soils, now generally admitted to be of fundamental
importance in plant growth, and it is especially important that
the effects produced on the organisms affecting nitrogen trans-
formations be more fully understood. It is hoped that this work
may stimulate other investigations along this line.

The author wishes to extend his thanks to Dr. C. B. Lipman,
in whose laboratory this work was carried out, for many valuable
suggestions offered from time to time and the great interest
shown.