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Full text of "Ammonification and nitrification in a strip mine spoil"

Digitized by the Internet Archive 

in 2010 with funding from 

Lyrasis IVIembers and Sloan Foundation 



http://www.archive.org/details/ammonificationni379wils 






BULLETIN 379T 

June 1955 



Ammonification and Nitrification 
In a Strip Mine Spoil 



WEST VIRGINIA UNIVERSITY AGRICULTURAL EXPERIMENT STATION 



THE AUTHORS 

Harold A. Wilson is Associate Bacteriol- 
ogist at the West Virginia University Agri- 
cultural Experiment Station and Associate 
Professor of Bacteriology in the College of 
Agriculture, Forestry, and Home Economics. 
Gwendolyn Stewart was Assistant in Bacteriol- 
ogy at the West Virginia University Agricul- 
tural Experiment Station. 



West Virginia University' 

Agricultural Experiment Station 

College of Agriculture, Forestry, and Home Economics 

H. R. Varney, Director 

Morgantown 



Ammonification and Nitrification 
In a Strip Mine Spoil 

H. A. WILSON and GWENDOLYN STEWART 

THE revegetation of spoils which result from strip or open pit mining 
of coal is a relatively new endeavor. Most of our knowledge dealing 
with revegetation is based upon work done on recognized soil types, 
and not upon a spoil, which is a mixture of soil and parent material 
such as limestone, sandstone, shale, and glacial till. 

When vegetation is once established its continued presence will 
depend upon numerous physical and chemical factors. One of the 
important chemical factors is a continuous supply of nitrogen. Since 
only a few species are capable of utilizing nitrogen in the ammonium 
form satisfactorily, the nitrate form is the most important. 

Ammonification and nitrification in the soil are biological processes. 
Both are necessary in transforming the nitrogen of organic materials 
first to ammonium and then to the nitrite and finally to the nitrate 
form. Knowledge concerning these processes in spoil is limited. 

Laboratory studies of the ammonification of various organic nitro- 
genous compounds and the nitrification of ammonium sulphate in 
spoil samples from a single area are reported in this bulletin. 

The ammonification process is brought about by several groups of 
microorganisms. Many fungi and actinomycetes, as well as numerous 
aerobic and anaerobic bacteria, according to Waksman and Starkey (14), 
are capable of liberating ammonium nitrogen from various organic 
nitrogenous compounds. 

Since many fungi, actinomycetes, and bacteria are capable of carry- 
ing out the ammonification process, the soil reaction, within limits, will 
have little effect upon this action. Ammonification in an acid environ- 
ment would be by microorganisms tolerant of acid conditions. Muntz and 
Coudon, and Marchal, as reported by Russell (9), showed that some 
species of Mucor, Fusarium, Aspergillus and Cephalothecium, and 
other soil fungi were active ammonifiers in acid soil. Conversely, 
bacteria and actinomycetes play the major role in ammonium nitrogen 
fonnation in the alkaline soils of the arid region. 

Cornfield (2) reported that the accumulation of ammonium nitrogen 
was generally high in acid and low in neutral and alkaline soils. From 



a study of some Connecticut soils having pH values of 5.30 and 5.50, 
Dorsey (5) found that when CaCOg was added the ammonifying power 
of the soil organisms increased. 

According to Waksman and Starkey (14), some organic nitrogenous 
compounds are more readily decomposed than others; also, micro- 
organisms vary in their ability to transform nitrogen compounds. Pulley 
(8) also showed that microorganisms differ in their ammonifying power. 

Nitrification, the oxidation of ammonium nitrogen to nitrite and 
nitrate, is a function of a certain few autotrophic bacteria. Consequently, 
this process is limited by some conditions which would have little or 
no effect upon the process of ammonification. Five genera of bacteria, 
Nitrosomonas, Nitrosocystis, Nitrospira, Nitrosococcus, and Nitrosogloea, 
possess the ability of oxidizing ammonium to nitrite (13), while only the 
bacterial genus, Nitrohacter, seems capable of oxidizing the nitrite to 
nitrate. However, not all of the nitrite-forming bacteria are found in all 
soils. 

Although different workers have reported slightly different degrees 
of acidity below which the nitrification process fails to function, it 
seems that a pH value of 3.7-4.0 (13) is the minimum. The process has 
an optimum pH of 6.5 to 7.5 

Cornfield (2), and others (6, 15), have noted that the addition of a 
neutralizing material, as CaCOg, to acid soils favors the nitrification 
process. Coville (3) concluded that nitrification is active in acid soils 
around lime concentrations but that the production of nitrate in acid 
soil is low. Stevenson and Chase (10) also found that the addition of 
CaCOg to an acid soil increased the nitrification rate, but they did not 
believe that the increase was due entirely to the decrease in the H-ion 
concentration. They concluded that it could be due to a stimulating 
effect of the CaCOg upon the nitrifiers or to an increase in numbers of 
the nitrifying bacteria. This last view agrees with the findings of 
Allison and Sterling (1), and of Walker, Thorne and Brown (16). 

Materials and Methods 

Spoil from the Canyon area, near Morgantown, West Virginia, was 
used in this work. Previously, a part of this area had been limed, 
fertilized, and seeded to grasses and legumes by Tyner and associates 
(11, 12). An adjacent area of the spoil was untreated and remained 
devoid of plant growth. Near-by soil which appeared not to have been 
modified by the stripping operations was available as a control. 

Composite bulk samples of the nonvegetated spoil, the vegetated 
spoil, and the undisturbed soil were collected. These samples were 
immediately passed through a quarter-inch wire mesh to remove bits of 



rock, coal and shale and then brought to the laboratory and spread 
out until air dry. 

One-hundred-gram portions (oven-dry basis) of the samples were 
weighed into 250-ml. beakers. The samples were properly divided and 
the following investigations made: (a) ammonification of different or- 
ganic nitrogenous compounds, with and without added Ca (OH)2; (b) 
the ammonification rate of the samples when supplied with urea, 
peptone, and egg albumin; and (c) the nitrification of (NH4)2SO^ when 
added to the samples with and without Ca (OH),. 

After the various materials had been thoroughly mixed into the 
samples, distilled water was added to bring the moisture content of each 
sample to 45 per cent of its water-holding capacity. The beakers were 
then placed in an incubator room maintained at 25° ± 1°C. Moisture 
lost during the incubation period was restored at semi-weekly intervals 
by bringing the samples back to their original weight by the addition of 
distilled water. The samples were thoroughly stirred each week. 

At the end of an incubation period the entire sample was removed 
from its beaker and thoroughly mixed. An amount of the sample 
equivalent to 10 gm. (oven-dry basis) was removed and placed in a small 
beaker for pH determination. Sufficient water was added to make a 
soil: water ratio of 1:1.5 and after standing, with frequent shakings for 
one hour, the pH was determined, using a glass electrode. The remainder 
of the sample was placed in a quart milk bottle and 2N KCl (acidified 
with HCl to pH 1.5) was added as an extracting fluid. The bottle and 
its contents were shaken on a Precision equipoise shaker for 30 minutes 
and the contents filtered through two E & D No. 613 filter papers in a 
Biichner funnel. 

Ammonium and nitrate nitrogen were determined upon an aliquot 
of the filtrate. The ammonium nitrogen was determined by the MgO 
method and the nitrate nitrogen by the DeVarda's alloy method. All 
data are reported as ammonium or nitrate nitrogen in terms of milli- 
grams of nitrogen per 100 gm. of spoil on an oven-dry basis. Each 
result is the average of duplicate samples. Each sample was an inde- 
pendent sample, therefore the results are more accurate than an average 
obtained from two aliquots from the same sample. 

The theoretical amount of Ca (OH)2 required to neutralize each 
sample was determined from the bufiEer curves developed according to 
the method of Dunn (4) . 

To eliminate needless repetition, the term spoil often will include 
the soil sample. The samples from the nonvegetated part of the spoil 
area will be designated as "non-vegetated"; from the vegetated part as 
"vegetated" and the soil as "undisturbed." Individually or collectively 
these terms will be referred to as treatment. 

5 



Results 



AMMONIFICATION OF VARIOUS NITROGENOUS 
ORGANIC SUBSTANCES 

The organic nitrogen sources used in studying ammonification by 
the nonvegetated, vegetated, and undisturbed spoils were urea, asparagine, 
peptone, casein hydrolysate, tryptone, and egg albumin. Each nitrogen 
source was added to the spoil samples to supply 100 mg. o£ nitrogen 
per 100 gm. of spoil. Two series of samples were run. In one series 
the pH of the spoil was unaltered. In the other the theoretical amount 
of Ca(OH)2 required to neutralize (pH 7.0) the spoil was added to 
the samples. This quantity was determined from titration values 
shown in Figure 1. 

The results obtained after seven days incubation at 25° C are tabu- 
lated in Table 1 and shown graphically in Figure 2. 

In the nonvegetated spoil asparagine was ammonified more readily 
than any other nitrogenous source used, and egg albumin the least 

/ 
/ 
/ 
/ . 



9.0 
8.0 



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I 6.0 
a. 



5.0 



4-0 



3.0 



NONVEGETATED 
VEGETATED 
UNDISTURBED y^ 








10 15 20 25 30 35 
ML. OF 0.04 N Ca(0H)2 



FIGURE 1. Buffer Curves of the Canyon Area Nonvegetated and Vegetated 
Spoils and Undisturbed Soil. 

6 



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readily. This was true with or without the added Ca (OH)2, even though 
at the end of seven days the pH of the spoil was 4.25 without Ca (OH)2 
and 6.73 with Ca (OH)^. 

In most instances the addition of Ca (OH), resulted in less am- 
monium nitrogen being recovered. The greatest reduction occurred 
in the nonvegetated samples with casein hydrolysate and tryptone. 



Rate of Ammonification 

The preceding data indicated differences in the amount of am- 
monium as nitrogen obtained from these spoil samples after a given 
period of incubation but no indication as to the rate of the ammonifica- 
tion process. Determinations of rate of ammonium nitrogen production 
were made on another series of samples, using three nitrogen sources, 
egg albumin, peptone and urea. Since it was evident from the data in 
Table 1 that the addition of Ca (OH), depressed the amount of am- 
monium nitrogen recovered, none was added to this series. All other 
conditions were the same. Ammonium as nitrogen was determined on 
samples every two days. These results are given in Table 2 and shown 
graphically in Figure 3. 

The data show that the rate of ammonification of egg albumin, 
peptone and urea by the nonvegetated spoil is slower than the cor- 
responding rates for the vegetated and the undisturbed spoils. The 
curves representing the rate of ammonification of egg albumin and 
peptone by the vegetated and the nonvegetated spoils are roughly 
parallel, and the amount of nitrogen represented is not greatly different. 
However, the curve representing the rate of ammonification of these 



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FIGURE 2. Ammonification of Various Organic Nitrogenous iVIaterials Added 
to Spoil. (Incubated at 25 ± 1° C.) 



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two substances by the undisturbed soil is difiEerent in shape and represents 
a considerably greater amount of nitrogen. 

The rate of ammonification of urea by the three samples varies 
greatly. Ammonification of urea by the nonvegetated sample is slow 
and the amount of ammonium nitrogen eventually liberated is small. 
The rate of ammonification by the vegetated sample is much more rapid, 
and the amount of ammonium nitrogen eventually liberated is nearly 
as great as that of the undisturbed sample. Ammonification of urea by 



100 
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a. 



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a- 100 

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NONVEGETATED 



60 



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VEGETATED 

UNDISTURBED 



CONTROL 



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^^ ALBUMIN 




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X 



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INCUBATION TIME 
DAYS 
FIGURE 3. The Ammonification Rate of Three Organic Nitrogen Sources 
Added to Spoil. (Incubated at 25 ± 1° C.) 

10 



-J 



the undisturbed sample reached its maximum in less than 10 days. After 
16 days the amount of ammonium nitrogen began to decrease, so deter- 
minations were discontinued. 

NITRIFICATION 

Nitrification by the three samples was determined by adding 
(NH4)2S04, equivalent to 50 mg. of nitrogen, to 100 gram samples of 
spoil and incubating for varying periods of time. Because of the low 
pH, particularly of the nonvegetated spoil, one series received the 
theoretical amount of Ca (OH), to neutralize the spoil, and the other 
received no Ca (OH), Conditions of incubation were as previously 
described. The results are shown in Table 3. 

It was soon evident that determinations at 5-day intervals would 
quickly exhaust the number of incubating samples and little information 
would be obtained since nitrification occurred so slowly. Because of this 
no fixed time pattern was followed. 

After 159 days of incubation, 45.24 mg. of ammonium nitrogen were 
obtained from the nonvegetated spoil without Ca (OH),. Only traces 
of nitrate nitrogen were found during the first 35 days of incubation 
and after that time none. The pH during the entire incubation 
period varied; increasing from 3.48 at days to a maximum of 3.73 on 
the 47th day and down to 3.55 after 159 days of incubation. 

Only traces of nitrate nitrogen were found in the nonvegetated spoil 
with added Ca (OH), until the 97th day of incubation, but by the 
159th day nitrification of the (NHJ^SO^ was definitely taking place. 
The pH values of these samples which received Ca (OH), ranged from 
3.70 at days to 4.58 after 159 days of incubation with two maxima of 
pH 6.20 after 15 and 35 days of incubation. 

Although only 2.21 mg. of nitrogen as nitrate were found in the 
vegetated sample without added Ca (OH), after 159 days of incubation, it 
is probable that nitrate nitrogen was beginning to accumulate. Nitrifi- 
cation of the (NH4)2SO^ in the vegetated spoil with added Ca (OH), 
began between the 15th and the 25th days of incubation as only 0.37 
mg. of nitrate nitrogen were found after 15 days of incubation but 4.05 
mg. after 25 days. A gradual increase in the amount of nitrate nitrogen 
was apparent from 25 through 77 days of incubation; reaching a high 
of 26.85 mg. at that time. This increase in nitrate nitrogen was ac- 
companied by a decrease of ammonium nitrogen, decreasing from 47.26 
mg. at days to 12.32 mg. after 77 days incubation. No samples were 
available to continue the determinations for a longer incubation period. 

In the undisturbed samples, without added Ca(OH),, only traces 
of nitrate nitrogen were found in samples incubated for any period 

11 



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through 62 days. After 97 days of incubation, 2.21 mg. of nitrate 
nitrogen were found, indicating that nitrates were beginning to accumu- 
late. After 159 days of incubation, 46.42 mg. of nitrogen as nitrates 
were found; accompanied by a decrease of ammonium nitrogen from 
49.95 mg. at days to 14.70 mg. after 159 days of incubation. 

Nitrification in the undisturbed sample, with added Ca (OH)^, 
became evident after 35 days incubation and seemed nearly complete 
after 77 days, as judged from the increase of nitrate and the decrease 
of ammonium nitrogen. 

Discussion 

The transformation of organic nitrogen to ammonium nitrogen 
from different materials incorporated into an acid spoil occur at varying 
rates. In the nonvegetated spoil, with a pH of 3.29, the availability of 
the compounds in terms of ammonium nitrogen accumulated in seven 
days was in the following decreasing order: asparagine, casein hydrolysate, 
tryptone, peptone, urea, and egg albumin. The addition of the theoretical 
amount of Ca (OH), to neutralize this spoil did not change the order of 
accumulation, although less ammonium nitrogen accumulated from two 
of the six sources with added Ca (OH)2. 

Under vegetated and undisturbed conditions more ammonium nitro- 
gen was released from urea after seven days incubation than from any 
other material; asparagine was second in yield and egg albumin last. 

In all cases the H-ion concentration decreased as ammonium nitro- 
gen was released from the organic material, and in most cases the 
sample which contained the largest amount of ammonium nitrogen 
had the highest pH value. 

The addition of Ca (OH)^ to the spoil probably made conditions 
favorable for the increase in microorganisms, and this may account for 
the smaller amount of ammonium nitrogen in samples with added 
Ca (OH),; apparently some of the ammonium nitrogen had been utilized 
by the microorganisms. 

The nonvegetated spoil appears to be somewhat deficient in those 
microorganisms capable of transforming the nitrogen of urea to am- 
monium nitrogen. As shown in Table 2, even after 30 days incubation, 
only 27 mg. of ammonium nitrogen were available. This amount is 
practically the same as that found after only seven days incubation. 
On the other hand, urea is readily ammonified in the vegetated and 
undisturbed samples. It seems apparent that a nonvegetated spoil, as 
acid as 3.29, is not a favorable environment for these microorganisms. 

The nitrogen of peptone is readily transformed into ammonium 
nitrogen in the nonvegetated, vegetated, and undisturbed samples; the 

13 



transformation was slowest with the nonvegetated sample. Egg albumin, 
the most difficult of the organic materials to decompose, became rather 
quickly ammonified after a short lag period. This lag period was 
approximately six days in the nonvegetated and vegetated samples but 
only two days in the undisturbed sample. 

These data indicate that the availability of ammonium nitrogen 
from organic nitrogenous materials added to spoil will be satisfactory 
even at the relatively high H-ion concentrations which exist. If such 
spoil areas are planted to pine seedlings, the ammonium form of nitro- 
gen will be used (7). 

The nitrification data indicate that without a reduction of the 
H-ion concentration no oxidation of the ammonium to nitrate nitrogen 
will take place, particularly in the nonvegetated spoil. Even in the 
vegetated spoil with a somewhat higher pH, 4.13 as compared to 3.48 
in the nonvegetated spoil, only 2.21 mg. of nitrogen as nitrate was 
found after 159 days of incubation. This was the same as in the 
undisturbed sample after 97 days. In the undisturbed soil however, 
considerable nitrification had taken place within five months. In all 
three samples, with added Ca (OH),, nitrification does take place, but 
at different rates. It has been shown that the number of nitrifiers are 
low as determined by MPN method (17), even in the undisturbed 
samples. It is apparent from these data that strongly acid spoils must 
be limed before the nitrification process will take place. Although 
such spoils may be supporting vegetation the amount of nitrate forma- 
tion may be small. 

Summary 

A laboratory study of the ammonifying and nitrifying powers of 
nonvegetated and vegetated spoil samples from one coal strip-mined 
area were compared with those of samples taken of a near-by soil. 

The ammonifying power of the samples was measured after adding 
urea, asparagine, tryptone, casein hydrolysate, peptone, and egg albumin, 
equivalent to 100 mg. nitrogen per 100 gm. of sample, by determining 
the amount of ammonium nitrogen produced after seven days incuba- 
tion at 25° C. The moisture content was maintained at 45 per cent of 
the samples' water-holding capacity during incubation. Two series 
of samples were run. One received the theoretical amount of Ca (OH)., 
required to neutralize the acidity, and the other received no Ca (OH),. 

All of the nitrogen sources were ammonified in all samples but at 
varying rates. In the vegetated spoil and soil samples, urea and 
asparagine were ammonified most rapidly and egg albumin most slowly. 
In the nonvegetated spoil samples, asparagine was most rapidly am- 

14 



monified but urea was ammonified only slightly more rapidly than 
egg albumin. In general, the additional Ca (OH), resulted in a smaller 
residue of ammonium nitrogen after incubation. 

The ammonification of the urea, peptone, and egg albumin nitrogen 
was determined at two-day intervals during a 30-day incubation period, 
when added to the samples of spoil and soil without added Ca (OH),. 
The release of ammonium nitrogen from all three sources was slower 
in the nonvegetated spoil than in the vegetated spoil and undisturbed 
soil. However, the total amount of ammonium nitrogen released from 
peptone or egg albumin after 30 days incubation was not greatly differ- 
ent in the nonvegetated and vegetated samples. On the other hand, 
urea, when added to the nonvegetated spoil, was ammonified not only at 
a much slower rate than in the vegetated spoil and undisturbed soil 
samples, but the total amount of ammonium nitrogen found after 30 days 
incubation was much less. 

The nitrifying power of the samples was measured, after adding 
ammonium sulfate equivalent to 50 mg. of nitrogen per 100 gm. of 
sample, by determining the amount of nitrate nitrogen produced after 
incubating the samples at 25° C. for varying lengths of time. The samples 
were maintained at 45 per cent of their water-holding capacity. Because 
of the low pH values, particularly of the nonvegetated spoil sample, 
one series received the theoretical amount of Ca (OH), required to 
neutralize the pH and the other series received no Ca (OH),. 

In the absence of Ca (OH),, nitrification failed to take place in the 
nonvegetated spoil samples, even after 159 days of incubation, but was 
beginning in the vegetated spoil and undisturbed soil samples after 97 
days incubation. In the presence of Ca (OH),, however, all samples 
were capable of nitrifying the ammonium of added ammonium sulfate. 
The lag period preceding the appearance of nitrates in the series with 
added Ca (OH), ranged from 25 days incubation with the vegetated 
spoil samples to 97 days with the nonvegetated spoil samples. 



15 



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16