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ABSTRACT

NITROSOMONAS EUROPAEA AND NITROBACTER ASILISx
PURE CULTURE STUDIES, GEL: ENUMERATION
AND THE EFFECT OF PESTICIDES ON NITRIFICATION

 

 

by Aline L. Garretson

Readily reproducible growth conditions in 250 ml sta-
tionary flask cultures were obtained for Nitrobacter agilis
and Nitrosomonas europaea. For both organisms a minimal
inoculum of 3.13% (by volume) was required for the initiation
of growth in Medium A with a lag period of less than 48 hrs.
3. agilis completely oxidized the energy substrate during
the exponential growth phase. However, 3. euroEaea cultures
terminated the eXponential growth phase leaving 12 to zug of

the NHu-N unoxidized to NO -N, depending on the growth condi-

2
tions. 3. agilis grew equally well in Medium AA, containing
no insoluble components, at a pH of 10.0 after autoclaving.
g. euroEaea did not survive in this medium when Ca012 was
added and NOZ-N was replaced by NHu-N. The removal of
amines from distilled water used in the preparation of media
had no gross effect on the rate of nitrification by
Nitrobacter.

Three bacterial and 2 streptomyces cultures were isola-
ted from a contaminated g. agilis culture. These isolates
were heterotrOphic and incapable of utilizing Nan-N, NOZ-N or

NO -N. When grown along with pure cultures of either

3

Aline L. Garretson
fl. agilis or 3. euro aea, they exerted no adverse effect
on either growth or nitrification of the nitrifiers.

A membrane filter technique was attempted for the
quantitation of cell pepulations by a modified colonial
plate count method. However, colonial develOpment was
inadequate on the filter to permit easy enumeration and
further work will be necessary to enhance colonial size
or to facilitate visualization.

The nitrification process of g. agilis was sensitive
to aldrin, lindane, and rhothane at final concentrations
of 1 ug/ml. Complete inhibition for 1a days was obtained
with rhothane, aldrin, and parathion at 10 ug/ml. Delayed
nitrification occurred with baygon at 10 ug/ml and with
lindane at l ug/ml. Malathion caused delayed nitrification
at 1000 ug/ml. Lindane, malathion, and baygon were at least
100 times more toxic for Nitrosomonas cultures than for

Nitrobacter.

NITHOSOMONAS EUROPAEA AND fllTROBACTER AGILIS:
PURE CULTURE STUDIES, CELL ENUMERATION, AND THE
EFFECT OF PESTICIDES ON NITHIFICATION

by

Aline L. Garretson

A THESIS

Submitted to -
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Microbiology and Public Health

1967

407

{3/92

To members of my family, for their constant encourage-
ment, assistance, and understanding during the course of

my master's program.

11

ACKNOWLEDGMENTS

I wish to thank Dr.'Charles L. San Clemente under
whose guidance this thesis was prepared, for his kind atten-
tion, interest, and valuable advice in all matters concerned
with this study.

A portion of this research was supported under a sub-
project of the Public Health Pesticide Research program under
the guidance of A. R. Holcott of the Department of Soil
Science. Sincere thanks are extended to Dr. Wolcott for the
many stimulating and enlightening discussions and helpful
advice.

Sincere appreciation is also extended to Dr. Hans A.
Lillevik of the Department of Biochemistry who served on my
guidance committee.

I also wish to acknowledge my gratitude to Mr. J. L.
Davenport and Chas. Pfizer & Co., Inc. for granting me a

leave of absence during the preparation of this thesis.

iii

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . iii
LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . viii

INTRODUCTION. 0 e e e e e e e e e e e e e e e e e e e 1
HISTORICAL e e e e e e e e e e e e e e e e e e e e e h
3101081031 Nitrification Concept 0 e e e e e e e #
Metab011° ACthithS e e e e e e e e e e e e e e 5
Nutrition 0 e e e e e e e e e e e e e e e e e e 7
Pure Culture Methods 0 e e e e e e e e e e e e e 10
Biochemistry of Nitrification. . . . . . . . . . 12
P'Bticides e e e e e e e e e e e e e e e e e e e 17

EXPERIMENTAL. O O O O O O O O O O O O O O O O O O O O 19

PART I. MAINTENANCE AND PROPAGATION IN PURE
CULTURE O O O O O O O O O O O O O O O O 19

Introduction. 0 e . e e e e e e e e e e e e 19
Materials and MCthOds e e e e e e e e e e e 19

 

ROSU1t8 e e e e e e e e e e e e e e e e e 23
Rates or Nitrification e e e e e e e e 23
Incomplete oxidation by Nitrosomonas . 26
MGdIaSDUdIQSOeeeeeeeeeeee 29
Influence or pH. 0 e e e e e e e e e e 32
Removal of amines from distilled water 34
HeterotrOphic contamination. . . . . . 35

Preliminary characterization. . . 37

Non-utilization of NHu-N and
NOz'N e e e e e e e e e e e e e 44

DISCUSSIOneeeeeeeeeeeeeeeee ‘50
PART II. ATTEMPT TO DEVELOP AN IMPROVED CELL

ENUMERATION METHOD USING MEMBRANE
FILTENS O O O O O O O O O O O O O O O O 55

Introduction. 0 e e e e e e e e e e e e e e 55
Materials and MGthO’j S e e e e e e e e e e e 56
ReSUIDS e e e e e e e e e e e e e e e e e e 58

iv

TABLE OF CONTENTS (continued)

Deve10pment of technique . . . . . . .
Quantitation procedure . . . . . . . .

Discu5810neeeeeeeeeeeeeeeeee

PART III. EFFECT OF PESTICIDES ON NITHIFICA-

TION. O O O O O O O O O O O O O O O O 0

Introduction. e e e e e e e e e e e e e e e 0
Materials and Methods . . . . . . . . . . . .
338111138 0 e e e e e e e e e e e e e e e e e e

The effect of pesticides on Nitrobacter

a 11130 e e e e e e e e e e e e e e

The ef%ect of pesticides on Nitrosomonas
eurOEEeae e e e e e e e e e e e e e
DLSCUSSIOne e e e e e e e e e e e e e e e e 0

SUMMARY 0 O O O O O O O O O O O O O O O O O O O O O O 0

LITERATURE CITED 0 O O O O O O O O O O O O O O O O O O O

Page

53
59

62

65
67
67
72
75

LIST OF TABLES

Table Page

1. The incomplete utilization of NH -N in Medium
A by Nitrobacter eurOpaea (Schmigt) after 10
days incubation e e e e e e e e e e e e e e e e 27
2. The availability of NH -N for conversion to
NOZ-N in Medium A during the late exponential
growth phase of Nitrosomonas eurOpaea (Schmidt) 28

3. Similar rates of nitrification by two
Nitrobacter agilis cultures in Medium A and
MedIum AA 0 e e e e e e e e e e e e e e e e e e 30

u. Dissimilar rates of nitrification by
Nitrosomonas eurOpaea (Schmidt) in Medium A
and Medium AA 0 e e e e e e e e e e e e e e e e 31

5. Influence of pH on nitrification by Nitrobacter
881113 (Fischer) in Medium AA 0 e e e e e e e e 33

6. The influence of amine-free water on
nitrification by NItTObfiCtere e e e e e e e e e 36

7. Cultural characteristics of heterotrophs
isolated from Nitrobacter a 1113 (Fischer)
and grown in Nutrient Broth at 0 C for
48 ane e e e e e e e e e e e e e e e e e e e e 38

8. Characterization of heterotrOphs isolated from
Nitrobacter agilis (Fischer) in various media . 39

9. Growth at 1h days of heterotrophic bacteria
isolated from Nitrobacter agilis (Fischer)
without carbohydrate fermentation at 37 C . . . #0

10. Colonial characteristics on Nutrient Agar of
heterotrOphic bacteria isolated from

Nitrobacter agilis (Fischer). . . . . . . . . . 43

11. Failure of heterotrOphic contaminants to oxidize
NO%-N in Medium AA containing 106 ug Noz-N/ml
(& ter 13 dayS) e e e e e e e e e e e e e e e e “j

12. Failure of heterotrophic contaminants to effect
NOz-N oxidation by Nitrobacter a ilis (Fischer) ‘
in Medium AA containing 106 ug NOZ-N7m1 . . . . A6

LIST OF TABLES (continued)

Table Page

13. Failure of heterotrOphic contaminants to
oxidize NH -N in Medium A containing 66 ug
NHu-N/ml (after 111' d8YS). e e e e e e e e e e e ’4'?

lu. Influence of heterotrOphic contaminants on
NBA-N oxidation by Nitrosomonas euro aea
(Schmidt) in Medium A oonEaInIng 66 ug NHu-N/ml QB

15. Quantitation of various types of micro—
organisms in a liquid culture of Nitrobacter
agilis (Fischer) by membrane filter
technique 0 e e e e e e e e e e e e e e e e e e 60

16. Inhibition of nitrification by Nitrobacter
a 1118 (Fischer) in Medium AA containing
chIorinated hydrocarbons. . . . . . . . . . . . 66

17. Inhibition of nitrification by Nitrobacter
agilis (Fischer) in Medium AA containing
organic phosphates and a-methylcarbamate. . . . 68

18. Inhibition of nitrification by Nitrosomonas

euroEaea (Schmidt) in Medium A containing
various peSDICIdeSe e e e e e e e e e e e e e e 69

vii

Figure Page

1. Logarithmic plot of % nitrite-nitrOgen
oxidized by various % inocula of Nitrobacter
£51118(Pramer)................. 214'

2. Logarithmic plot of % nitrite-nitrogen

produced by 3.13 and 12.5 % inocula of
Nitrosomonas europaea (Schmidt). . . . . . . . . 25

viii

INTRODUCTION

Soil nitrification has long been of interest to both the
agronomist and theoretical microbiologist. This process is
important in agriculture since nitrate or ammonium is the
major nitrogen form assimilated by higher plants. Nitrogen
assimilated in the inorganic state is then incorporated in the
proteinaceous matter of all living tissue. The nitrogenous
materials available in the soil exist largely in the organic
form. The release of the organically bound element is accom-
plished by two separate and distinct microbiological processes,
ammonification and nitrification. The latter process is limited
to a relatively small number of nitrifying organisms. Their
importance in the nitrogen cycle lies in their capacity to
produce nitrate. The dominant microbial Species concerned in
the oxidation of ammonium to nitrite, and nitrite to nitrate
are members of two genera of the family Nitrobacteriaceag,
Nitrosomonas and Nitrobacter. Only two species of each genera
are recognized, Nitrosomonas eurOpaea, Nitrosomonas monocella,
Nitrobacter winogradskyi, and Nitrobacter agilis.

Up to the present time few reports have appeared concern-
ing metabolic studies on the chemolithotrOphic nitrifiers.

The carbon metabolism of these organisms still remains to
be elucidated. The study of these bacteria not only has
practical application to agriculture but also offers a stim-

ulating challenge to biochemical investigations.

2

My initial objective was to study the effect of various
chemical factors, including pesticides, on the nutrition and
physiology of these organisms. Because of their unique prop-
erties ordinary microbiological methods were not readily
utilizable and the methods applicable were cumbersome and
time consuming.

The size of colonies formed on a solid medium is so
small that visualization without optical magnification is not
possible. Turbidimetric measurement of cell numbers is also
impossible unless repeated supplementation of both nutrients
and buffer are made to prolong the exponential growth phase.
Therefore, these methods are not applicable for the prepara-
tion of large numbers of replicate cultures. With the excep-
tion of an analytical chemical analysis for the three states
of nitrogen (NHJ', NOZ', or N037, no other assay method is
available for the accurate quantitation of small numbers of
microorganisms. Consequently, it_was deemed advisable not
only to develop an accurate method of cell quantitation but
also to improve on c611 culture conditions for faster rates
of reproduction and more efficient utilization of metabolites.

During the course of this study, difficulties were
encountered in the maintenance of pure cultures. Hetero-
trophic contamination therefore became an additional area for
attention.

There subsequently evolved three major areas for

research: (a) pure culture methods, (b) cell enumeration,

and (3) the effect of pesticides on nitrification. Although

3
closely interrelated, each area represents a distinct phase
of activity and the judgment was made to treat each as a
separate unit in this dissertation.
The results described in this dissertation have estab-
lished a necessary foundation for future study of the biochem-
istry and physiology of these microorganisms. The need for

an effective cell enumeration method also warrants further‘

study.

HI STORICAL

Biological Nitrification Concept

Despite its great economic importance in agriculture,
the process of nitrification has received relatively little
attention and then only spasmodically during the past eighty-
five years. Although Pasteur in 1862 was the first to
suggest that the formation of nitrate in soil was micro-
biological and perhaps similar to the conversion of alcohol
to acetic acid it was not until 1877 that Schloessing and
Mfihtz demonstrated that the oxidation of ammonia was indeed
microbiological. The experimental evidence was obtained by
passing sewage daily through a column of sterile sandy soil
and chalk. After twenty days, the ammonia input appeared in
the effluent almost completely as nitrate-nitrOgen. This
process after inhibition by the addition of chloroform or
heating the column could be restored to its former activity
by the addition of fresh garden soil washings.

Between 1890-1891 the oxidizing organisms were isolated
apart from the soil by three independent workers (Frankland
and Frankland, 1890; Harrington, 1891; Winogradsky, 1890a,b,c;
l891a,b). It followed from the work of Harrington that two
kinds of special, aerobic organisms must be responsible,'
respectively, for the two stage oxidation, ammonia to nitrite
and nitrite to nitrate. His attempts to subculture these
organisms from enrichment cultures failed. Winogradsky,

however, was able to isolate colonies on an inorganic medium

a

5
solidified with silica gel. Two types of nitrifiers were
isolated, Nitrosomonas, oxidizing ammonia to nitrite, and

Nitrobacter, oxidizing nitrite to nitrate.
Metabolic Activities

The next advance came in 1916 when Meyerhof extensively
investigated the respiratory activity of these organisms
and the effect of various inhibitors. By measuring oxidation
rates the Optimal pH of Nitrobacter was found to be about the
same as that of Nitrosomonas, 8.5 to 8.8 (Meyerhof, 19l6a,b).
Meyerhof's early studies (1916a, 1917) also threw light on
many other factors influencing the metabolism of these
organisms. The extreme sensitivity of these organisms to
hydrogen ion concentration requires a bicarbonate buffer.
Growth inhibition of Nitrobacter occurs as nitrate accumulates
or in the presence of free ammonia, while on the other hand
Nitrosomonas is inhibited by both ammonia and nitrite. Both
forms are inhibited by divalent metal ions and lowered oxygen
pressure (Meyerhof, 1916; Amer and Bartholomew, 1951). This
fact suggests that at least one stage of the oxidation involves
an oxidase with much less affinity for 02 than cytochrome
oxidase. Meyerhof's investigations indicate that ions must
freely enter the Nitrobacter cell, since at the Optimal pH
both nitrite and bicarbonate are very largely dissociated,
and non-ionized-NOZ and -NO groups in the form of metallic
complexes or organic nitroso-compounds (Meyerhof, 1917)

could not be used.

6

Glaring gaps in the literature on the metabolic activ-
ities of the nitrifiers continued until the advent of the
first soil perfusion apparatus described by Lees and Quastel
(1944) and subsequently modified by Audus (1946) and Lees
(1947). In this apparatus, a metabolite is continuously
percolated through a soil column, the repeated perfusion
permitting direct study of the kinetics of soil nitrifica-
tion under controlled environmental conditions. The sub-
sequent use of this technique for the study of organic and
inorganic conversions was published intermittingly in several
reports from 1944 to 1954. It is to be expected that in soil,
with its complex microflora and its special physicochemical
conditions, the kinetics, possibly even the mechanism of
nitrification, could differ materially from what takes place
in pure cultures of the nitrifiers. A significant difference
between growth in soil and in pure culture is the response
to the effect of organic matter. Peptone is far less inhib-
itory in sand than in solution cultures (Winner, 1904), and
cottonseed meal and ammonium sulfate both are more rapidly
nitrified in soil than in solution. The presence of certain
heterotrophs develOping symbiotically in soil greatly influences
the nitrification process (Desai and Fazal-un-Din, 1937;
Pandalai, 1946). The stimulation by colloids in culture
media on bacterial behavior (Albrecht and McCalla, 1937,
1938; Conn and Conn, 1940; Nommik, 1957; Zobel, 1943) suggests
the possibility of similar effects in soils.

7
Nutrition

Although considerable attention has been given to the
nutrition of the nitrifying chemolithotrOphs these studies
often have been incomplete and ambiguous. Many of the
earlier investigations performed in impure media and in
mixed culture should be verified under improved cultural
conditions now available. Most studies were concerned with
the influence of organic compounds on both growth and nutri-
tion of the nitrifiers as a group, and the effect upon the
overall oxidation of ammonium ion to nitrate (Bomeke, 1950;
Lees and Quastel, 1946a,b,c,; Meiklejohn, 1951, 1952a;
Quastel and Scholefield, 1949). Using pure culture methods
in a defined medium recent studies have been sharply focused
on the possible effects of organic supplements. In the light
of these recent experiments no sugar, organic acid or other
organic molecule will serve as sources of either carbon or
energy for the growth of N. agilis (Delwiche and Finstein,
1965; Ida and Alexander, 1965). A stimulatory effect on
nitrification and growth of this strain is obtained with
supplements of yeast extract, Vitamionree Casamino Acids,
and certain amino acids, while a strong stimulation limited
to growth is obtained with acetate (Delwiche and Finstein,
1965). Both acetate and glycine contribute to cell carbon.
These same workers demonstrated that the organism is permeable
to certain organic acids. At least some of the enzymes for
organic metabolism must also be present. No stimulation of

nitrite production by N. eurogaea by either B vitamins or

8
amino acids (Gundersen, 19553) has been found and similar .
negative responses have been obtained with N. agilis (Aleem
and Alexander, 1960). Krulwich and Funk (1965), however,
have more recently reported enhancement of both nitrite
utilization and growth with biotin using four strains of
N. agilis.

It is quite certain that Nitrosomonas and Nitrobacter
do develOp in inorganic solutions from which all traces of
organic matter is removed. The sole energy sources support-
ing growth are nitrogenous, typically ammonium or nitrite
salts. The nitrogen of amino acids, amides, proteins or urea
is not oxidized by N. eurOpaea although some of the nitrogen
in certain purines may be converted to nitrite by some strains;
the purines are likely deaminated prior to nitrification
(Ruban, 1958). Engle and Alexander (1960) and Silver (1960)
have demonstrated hydroxylamine and formic acid oxidation by
this bacterium but there is no evidence that these compounds
are energy sources for proliferation.

The mineral requirements, except for the carbon and
energy sources, resemble those known for heterotrOphs. The
nutrient demand in laboratory studies is low since the num-
ber of cells is relatively small. Specific requirements for
magnesium, phOSphate, and nitrite have been reported (Bomeke,
1950; Meiklejohn, 1952b; Meyerhof, 1916a) but under question-
able cultural conditions. The optimal iron concentration for
growth of N. winograd sky}. is reported to be about 6 ug/ml for
the oxidation of 200 ug/ml nitrite-nitrogen (Meiklejohn,

1953) but this iron level exceeds the amount of cell carbon

9
formed (Meyerhof, 1916a). The use of well-defined culture
methods has been recently initiated for nutritional studies
of N. agilis (Aleem and Alexander, 1960). Using an alumina
purified inorganic medium, the requirements for each nutrient
is studied by omitting the nutrient under study and adding
graded amounts to flasks in a standard series. Using these
methods the Optimal nutrient levels now appear to be approx-
imately 5 ug/ml for both phOSphorus and magnesium and at
least 0.005 ug/ml for iron with N. agilis. These data con-
flict with the findings of Meiklejohn who reported the iron
requirement as high as the P and K levels. It is suggested
that the data of Meiklejohn may result from reactions with
CaCOB rendering most of the iron unavailable to the
microorganisms. The investigations of Aleem and Alexander
(1960) also reveal that the effect of nitrate on rate of
nitrite oxidation is one of retarding the initiation of
growth, results which are similar to those obtained in soil
(Stojanonic and Alexander, 1958). The toxicity of ammonium
salts in alkaline environments seems to be a generic property
since N. gg;lis is markedly inhibited as are other Nitrobacter
isolates (Bomeke, 1950; Boullanger and Massol, 1903; Meyerhof,
1916b). Cell-mass development and concomitant nitrite oxida-
tion is enhanced in freshly inoculated cultures if the medium
contains small amounts of molybdenum (Finstein and Delwiche,
1965; Zavarzin, 1958). Zavarzin postulated that a molybdo-

flavOprotein is concerned in the energy yielding reaction of

these autotrOphs. According to investigations of Finstein

10
and Delwiche the enhancement may simply be a response to the
development of greater cell mass and not a direct molybdenum
function in enzymatic nitrite oxidations. A function of
molybdenum in nitrate reductase (Nicholas, Nason and McElroy,
1953) does suggest, but does not establish, a function in the
‘reverse direction. Winogradsky (1890a, 1891) used MgCOB,
instead of CaCOB, in his original medium. When made up with
purified reagents this medium gave poor growth and nitrifica-
tion. Kingma Boltjes (1935) established Ca ion as the missing
factor which was essential for NNtrosomonas but not necessary
for Nitrobacter (Meyerhof, 1916a,b). However, Alexander (1965)
believes there is no valid evidence for a requirement for
significant quantities Of calcium and cited several reports
in support of his argument (Aleem and Alexander, 1960; Bomeke,
1950; Lees and Meiklejohn, 1948). Potassium.(Welch and Scott,
1959) and sulfur are also required elements, and cOpper is
reported to be stimulatory to Nitrobacter (Kiesow, 1962;
Zavarzin, 1958).

Pure Culture Methods

After gelatin proved to be useless for the colonial
develOpment of the nitrifiers from enrichment cultures,
Winogradsky (1891a,b), using an inorganic medium solidified
by silicic acid, finally isolated Nitrosomonas and Nitgobacter
in monoculture. Since Winogradsky's time the isolation and
pure culture of nitrifying bacteria were repeatedly reported

(Bisset and Grace, 1954; Bomeke, 1939; Gibbs, 1919; Gould and

11
Lees, 1960; Kingma Boltjes, 1935; Lees, 1952; Lewis and
Pramer, 1958; Meiklejohn, 1954; Nelson, 1931). Considerable
difficulty still exists, however, in obtaining monocultures
free of contaminating microorganisms. These contaminants
often remain undetected because of the morphological similar-
ity to the predominant organism and some may be incapable of
developing on conventional laboratory media. Common contam-
inants in the final enrichments include species of Pseudomonas,
Nyphomicrobium, Mycobacterium, Flavobacterium and Serratia as
well as an occasional myxobacterium (Gundersen, 1955b; Stapp,
1940; Ulyanova, 1960). A symbiotic relationship may exist
which might explain the growth of the autotrOph in an unfa-
vorable soil environment.

Many methods have been described to separate the auto-
trOphs from their contaminants. In some of these procedures,
the autotrOphic pOpulation is increased by addition of succes-
sive increments of nitrogen and thereby increasing the ratio
of autotrOphs to heterotrOphs. The enrichment culture is
then diluted, and pure cultures made by single-cell technique,
either by plating or by tube dilution to a heterotroph free
endpoint. Several modifications have been devised, one
modification entails bubbling carbon dioxide through the
cultures to remove the cells from the carbonate particles
(Meiklejohn, 1950). Inhibitory compounds such as antibiotics
and dyes are also employed (Gould and Lees, 1958, 1960;

Prouty, 1929) for suppression of contaminating Species.

12
Lewis and Pramer (1958) eliminated the problem of adsorption
of contaminant and nitrifier<m1 the carbonate particles by
using soluble carbonates in their medium.

The problem of contaminants is not ended once a nitri-
fier is obtained in pure culture. The corollary problem of
maintaining a culture contaminant free is perhaps more impor-
tant when studying the biochemistry of these organisms. The
recent study of Clark and Schmidt (1966) on the effect of
mixed culture on N. eurogaea serves to emphasize this point,
and indicates the ease in which stock laboratory cultures

become contaminated with chemcorganotrOphs.

Biochemistry of Nitrification

Until recently, effective investigations on the mech-
ansim of nitrification were hindered because of the lack of
cell culture methods for the production of large quantities
of active cells which could be easily freed from insoluble
medium ingredients. Improved culture methods are now avail-
able (Engle and Alexander, 1958a,b) for N. eurogaea in which
this chemolithotrOph grows in a medium free of insoluble
carbonates with a relatively short generation time. The
yield of metabolically active cells is 72.4 mg (dry weight)
per liter when grown in a 25 L pyrex fermentor fitted with a
stainless steel heating and sparging assembly. Similar
apparatus for large scale aerated batch cultures are described
for Nitrobacter (Aleem and Alexander, 1958; Butt and Lees,
1958; Gould and Lees, 1960; Lees and Simpson, 1957; Skinner
and Walker, 1961).

13

Since the develOpment of these large batch cultures, a
number of investigations have been reported on the biochemical
reactions of washed cell suSpensions and cell-free prepara-
tions. These studies have resulted in numerous reports on
the intermediary metabolism of nitrification.

All considerations of the nitrogenous intermediates in
the mechanisms of nitrification originates with Kluyver and
Donker (1926). The conversion of ammonia to nitrate involves
a sequential conversion of nitrOgen from the -3 to the +5
oxidation state. The compounds of nitrogen known for all the
possible oxidation states in this sequence are: hydrazine
(-2), hydroxylamine (-l), molecular nitrOgen (0), hyponitrous
acid or nitrous oxide (#1), nitric oxide (#2), nitrous acid
(#3), and nitrogen dioxide (#4). Subsequent investigators
have attempted to provide either confirmatory evidence in
support of the prOposed pathway by Kluyver and Donker, or
evidence for a satisfactory alternative. Kluyver and Donker
proposed a sequence consisting of a series of oxidations

each representing a loss of two electrons:

H H 1/2 c F 1/2 02 on '93
v’-os _ ’-OH H-N-O a-n 1/2 c, hC-I=0
“1365.) “T" E \ "

H H OH “:KE5S>
Ammonium Hydroxyl- Nitroxyl Dihydroxy- Nitrite
hydroxide amine ammonia

Hydroxylamine (NH2OH) is commonly considered to be the initial
product of ammonia oxidation. Some weak evidence in support

of hydroxylamine as an intermediate is provided by several

1c
investigators. Added hydroxylamine is oxidized by N.
europaea cell suspensions to nitrite rapidly and stoichiomet-
rically without any time lag (Engle and Alexander, 1958a).
Hydroxylamine is identified as the accumulated nitrOgenous
compound when Nitrosomonas is incubated with ammonium sulfate
in the presence of hydrazine (NZHQ), which inhibits the oxida-
tion of hydroxylamine (Yoshida and Alexander, 1964). N.
europaea cells contain an enzyme catalyzing the oxidations
of hydroxylamine (Burge, Malavolta, and Delwiche, 1963; Engle
and Alexander, 1959; Nicholas and Jones, 1960). These enzyme
preparations show no detectable oxidation of ammonium but in
the presence of some suitable electron acceptor produced
about three-fourths of the eXpected quantity of nitrite. In
this reaction, neither nicotinamide adenine dinucleotide
(NAP) nor nicotinamide adenine dinucleotide phosphate (NADP)
is reduced.

The intermediate at the next higher oxidation state
remains completely obscure. Although hyponitrous acid
(HO-NZN-OH) is a possible intermediate, no microbial conver-
sion of hyponitrite to nitrite is noted with either cell
suspension or enzyme extracts prepared from Nitrosomonas
(Lees, 1954; Nicholas and Jones, 1960).

Reduction of cytochrome c, in the absence of oxygen,
takes place during hydroxylamine oxidations with cell-free
extracts (Nicholas and Jones, 1960). In this process nitrous
oxide is evolved which is probably a degradation product of

the unknown intermediate formed between hydroxylamine and

15

nitrite (Falcone, Shug, and Nicholas, 1962). Aleem 23 ai.
(1962) propose that this intermediate is nitrohydroxylamine
(NOZ'NHOH). These investigators observed that one mole of
nitrite is formed for each mole of nitrohydroxylamine metab-
olized. It is suggested that nitrohydroxylamine is generated
in a reaction between hydroxylamine and endogenous nitrite,
and the resulting product is then oxidized by the bacterium

to 2 moles of nitrite.

1/2 0 1/2 0
NH 01—1 ,1 HNO —_—$ NO 'NHOH __..._._...2..;., ZHNO
2 2 .H20 2 2

Anderson (1964) reports negligible amounts of nitrite
formed from hydroxylamine anaerobically in the presence of
Nitrosomonas extract and methylene blue. Nitric oxide and
nitrous oxide are produced in amounts equivalent to the
hydroxylamine added.

Nitrite is rapidly metabolized by extracts of N. agilis
cells with complete conversion to nitrate and the consumption
of oxygen is essentially equal to the theoretical amount

expected according to the following equation:

N02- g 1/2 02 .___.._; ”03‘

The enzyme isassociated with the particulate constit-
uents of the cell (Aleem and Alexander, 1953). Laudelot and
Van Tiechelen (1960) report the Michaelis constant, Km, to
be 6.7 x 10‘“ M at 320C. Aleem and Nason (1959) report the
enzymic oxidation of nitrite is catalyzed by a cytochrome-

containing electron transport particle via cytOchrome c and

16
cytochrome oxidase-like components. This oxidation is
coupled with the generation of high-energy phOSphate bonds
identified as adenosine triphosphate, ATP (Aleem and Nason,
1960). The overall reaction is :

cytochrome electron

n62 / 1/2 02 / nADP # nPi transport chain 7— N03 {.nATP

 

The energy liberated by the above equation is available
for the reduction and assimilation of carbon dioxide (Aleem,
1965) and for the reduction of pyridine nucleotides essential
for the Operation of the carbon reduction cycle (Aleem, 1965;
Aleem, Lees and Nicholas, 1963). This process of chemosyn-
thesis appears to be analogous to photosynthesis.

As in photosynthesis, water must be the hydrogen donor
for the pyridine nucleotide reduction. Aleem, Hoch, and
Varner (1965) report that water, and not molecular oxygen
participates in the nitrite oxidation and that the hydrOgen
donor for the concomitant reduction of pyridine nucleotides

is also water:

- . 18 -;s 18
N02 / HZO / A <2- No3 / AH2

From their results it appears that a hydrated nitrite molecule,
or some activated form of it, may be the substrate for a

dehydrogenation:

( OH)-2H

H o
HO-N=O l.§.__s> EHO'MQ~§""""*> HO-‘
:1

<f‘ho

17
Iron, but not zinc, molybdenum, magnesium or cOpper,
is stimulatory to nitrite oxidation by enzyme preparations
(Aleem and Alexander, 1958). This observation suggests a

role for iron in this reaction.

Pesticides

Since World War II the use of synthetic organic biocides
has resulted in a marked increase in crOp production and
decrease in incidence of insect-borne diseases. More
recently, however, there has been a growing concern with the
effects of their distribution in the environment. This
interest has been mainly centered on the presence of pesticides,
in or on foods for human consumption, and the contamination
of air and surface waters. The pesticide residues in soil
have been well studied but relatively few investigations have
been directed toward their effect on soil microbial inter-
actions.

Among the possible toxic effects of pesticides on the
normal microbial flora in the soil, in addition to the inhibi-
tion of specific members of the population, there are sec-
ondary effects changing microbial dominance and concommitant
'alteration of the nitrogen cycle. Since nitrate is the major
source of nitrogen assimilated by higher plants one would
expect that an imbalance in the microbial pOpulation could
ultimately affect the rates of nitrification and influence
plant nutrition.

Approximately 60,000 pesticidal products have been

registered in the United States. A minimum of #90 basic

13
chemicals are on the market, 200 of which are of first-rate
importance. These economic poisons include the insecticides,
herbicides, fungicides, rodenticides, and nematocides. These
substances fall into one of several groups of chemical com-
pounds: chlorinated hydrocarbons, organic phosphates, carbam-
ates, dinitrOphenols, organic mercurials, organic sulfur
compounds, natural products, and inorganics.

A beneficial use of inhibitors is the prevention of
nitrogen losses following fertilization. Surveys have been
aimed at finding non-phytotoxic chemicals which would selec-
tively inhibit the nitrifying bacteria and thereby maintain
the fertilizers in a reduced form for longer periods. One
such compound, 2-chloro-6-(trichloromethyl)-pyridine, causes
a marked deleterious effect on nitrifying organisms (Goring,
1962a,b) and this action was not noted with other groups of

organisms (Shattuck and Alexander, 1963).

EXPERIMENTAL

PART I. MAINTENANCE AND PROPAGATION IN PURE CULTURE

Introduction

Methods for the continuous propagation of pure cultures
of g. eurogaea and g. agilis in stationary culture have
received little attention in the past and the more recent
publications on the growth of these nitrifiers have been
limited to large scale aerated batch cultures, shake flask
cultures or soil perfusions.

Since none of these culture systems was applicable to
our intended investigations requiring large numbers of repet-
itive cultures, it was first necessary to establish readily
reproducible growth conditions. Small volume, stationary
flask cultures were utilized in which pH, aeration, and sub-
strate nitrOgen closely approximated the optimal conditions

for nitrification in the soil.

Materials and Methods

Culture strains. Cultures of g. eurogaea were received

on two different occasions, 7/25/65 and 4/15/66, from Dr.

E. L. Schmidt. This strain was identified as ATCC 122h8.

g. agilis was obtained as follows: from Dr. I. Fischer a
garden soil isolate of Dr. C. C. Delwiche, and an unidentified
culture from Dr. David Pramer. All of these cultures were
maintained at 25-30 C by continuous subculture in fluid media.

19

20

Growth media. For cell culture maintenance and
eXperimentation the organisms were cultivated in 250 ml
flasks containing 50-75 ml of the apprOpriate growth
medium. This resulted in an air space to culture fluid
volume ratio ranging from 2 to h.

u. europaea was grown in a basal medium, referred
to as Medium A in the text, supplemented with 0.3 or 0.5 g
(NH“)ZSOu/liter. The concentration of NHu-N in the
complete medium was 63.6 and 106 ug/ml, respectively.
Medium A is described in detail in the appendix (page 83)
and is a modification of the medium of Pramer and Schmidt
(1965).

n. agilis was propagated in one of two media:
either Medium A, Just described, but containing 102 ug
N02-N/ml in the form of NaNO2 instead of (NHu)280h, or a
modification of the medium described by Aleem and
Alexander (1960). The latter basal medium, designated
as Medium AA in the text, is also described in the appen-
dix (page 83). The complete medium contained 0.6 KNOZ/
liter (98.8 ug NOZ-N/ml).

All of the components of the media were reagent grade.
Distilled, deionized water was routinely used throughout
the course of this study. A further reduction of water impur-
ities was effected in most of the experiments by the passage

of distilled water through a mixed cation and anion resin

21
(Barnstead Still and Sterilizer Co., Boston, Mass.) and then
a cation removal resin (Barnstead Still and Sterilizer Co.,
Boston, Mass.). This second resin was for the removal of
amines known to be commonly present in the condensed "boiler"
water.

The pH of all fluid media prior to sterilization ranged
from 8.0 to 9.0 depending upon the specific reagents in the
medium. The pH was independent of the quality of the water
used to dissolve the reagents.

Sterilization of the complete medium was accomplished
by autoclaving unless otherwise indicated. In certain eXper-
iments sterilization was accomplished by passage through
sterile membrane filters (Millipore Filter Corp., Bedford,
Mass.) with a pore size of 0.45 u. Stock cultures were incuba-
ted at 25-30 C, and experimental cultures were incubated at
28-30 C.

Nitrification analysis. Analysis of culture fluids was
routinely made on the basis of the appearance of nitrite in
the case of Nitgosomonas, or the disappearance of nitrite
in the case of Nitrobacter, by the alpha-naphthylamine-
sulfanilic acid procedure of Rider and Mellon (19b6) which
was performed Spectrophotometrically at a wave length of 520

1111.10

22
Culture purity. The presence of heterotrOphic contam-
inants in autotrOphic cultures was routinely evaluated by
the addition of a 5 to 10% inoculum to each of the following
organic fluid media: Nutrient Broth, Lactose Broth, Czapek
Dox Broth, and ThiOglycollate Broth. Incubations were
carried out at 28 to 30 C for 1 month. The absence of

turbidity was considered an indication of culture purity.

RESULTS

 

Rates of Nitrification
I It was noted in initial experimentation that the rates
of nitrification differed greatly with the two types of
chemolithotrOphs. In order to evaluate these differences
accurately, duplicate experiments for Nitrobacter agilis
(Pramer) and Nitrosomonas eurqpaea (Schmidt) were conducted
in Medium A. A standard series of flasks for each strain
was inoculated at twofold dilutions ranging from 25 to 1.56%
(by volume). Samples of 1 ml were taken daily over a 7 day
incubation period from the Nitrobacter cultures and over an
8 day incubation period from the Nitrosomonas cultures. The
rates of nitrification were determined by chemical analysis.
The % N02-N oxidized by various levels of inoculum was plotted
against time for Nitrobacter (Fig. l). The % NOZ-N produced
by the 3.13 and 12.5% inocula is similarly represented (Fig.
2) for the Nitrosomonas series. The 25 and 6.25% inocula
were omitted from this graph as they approximated the 12.5
and 3.13% inocula, respectively.

The results of both experiments demonstrated a short lag
period of oxidation, or growth, followed by a rapid period of
exponential growth with the 3.13% inoculum. In the case of
the Nitrobacter cultures, this was the minimum level of inoc-
ulum initiating exponential growth within #8 hours.

The Nitrobacter culture series effected the complete

A conversion of NOZ-N to NOB-N during the exponential growth
23

% NITRITE-NITROGEN OXIDIZED

21+

9—125 ‘7. INOCULUM
o—o. 25% -

13—1137. /@// O/ D A‘

4-1. 56% ;A/

F'
100

50-

30-

10-

 

 

 

A L .' . .

2 3 4
DAYS

Fig. l. Logarithmic plot of S nitrite-nitrogen

oxidized by various % inocula of Nitrobacter agilis
(m0r)e '

25

I1'
I

100

50-

 

% NITRITE-NITROGEN PRODUCED
5
I
4——()=====u.

 

 

 

Fig. 2. Logarithmic plot of % nitrite-nitrogen
produced by 3.13 and 12.5% inocula of Nitrosomonas

europaea (Schmidt).

26
phase. A similar response was also obtained with the
Fischer strain of N. agilis. For Nitrosomonas, however,
the exponential growth phase was terminated when 70 to 80%
of the theoretical amount of NHu-N appeared in the form of
N0 -N. These patterns of nitrogen oxidation have been

2
repeatedly encountered in this laboratory.

Igggmplete Oxidationgby Nitrosomonas

A more detailed analysis of the data obtained in the
previous growth experiment showed that approximately 14.8%
(average value) of the NHu-N was not converted to NOZ-N after
10 days incubation (Table 1). Although slightly higher %
values were obtained with the 25 and 12.5% inocula, this was

not considered significant. The amounts of N0 -N initially

2
present in the media were considerably lower than concentra-
tions reported to be inhibitory to Nitrosomonas (Engle and
Alexander, 1958).

In order to determine if NHb-N became inaccessible during
the incubation period another series of duplicate cultures
was seeded in Medium A containing 66.3 ug NHu-N/ml. After

a 7 day incubation period assays for both N02-N and NH -N

n
were conducted on the culture fluids (Table 2). The combined
values for NHu-N and NOZ-N after 7 days incubation were sum-
ilar to the initial concentration of NHu-N for the two inoc-
ula of 6.25 and 3.13%. These results demonstrate that most
of the NHu-N not converted to Noz-N was still available in

the medium for the ultimate oxidation to Noz-N.

27

Table l. The incomplete utilization of NH -N in Medium A
by Nitrosomonas eurOpaea (Schmid?) after 10
days incubation

 

 

 

Inoculum Oxidative state of nitrOgen
(fl)
Initial After 10 days incubation
NOZ-N NHu-Nb NO -N NHu-N not
from prcdvced converted to
inoculuma (ug/ml) NO -N
(us/m1) (us/m1) (i)
25 50.0 82.5 70.0 15.2
12.5 25.8 93-3 77-2 17.3
6.25 11.3 97.8 84.7 13.3
3.13 5.90 103.0 90.1 12.6

 

a Values obtained by assay.

b Calculations based on the dilution of the medium which
contained 106 ug NHg-N/ml.

Table 2.

28

The availability of NHu-N for conversion to N02-N
in Medium A during the late exponential growth
phase of Nitrosomonas europaea (Schmidt)

 

Culture description

Oxidative state of nitrogen

 

 

 

Inoculum Growth phase Initial After 7 days
(f) incubation
I: NHg-N NHi-N NHu-N NEH-N
+
(us/m1)
6.25 Late 62.3 66.6 19.8 63.3
exponential
3.13 Early 64.3 66.9 53.8 62.5

exponential

 

29
Media Studies

Medium A was routinely used for the maintenance of stock
cultures of both nitrifiers, the only composition difference
being in the nitrogen source. This medium contained a large
amount of a fine precipitate which settled out rapidly and
under certain conditions aggregated into larger particles.

For pure culture isolation and plate counting methods
the even dispersal of microorganisms is an essential require-
ment. Since the nitrifiers as well as possible heterotrOphic
contaminants tend to adhere to the insoluble particles,uniform
dispersion and separation of the microorganisms are not pos-
sible in this type of medium. A completely soluble medium
is also desirable for the preparation of cell suspensions
free of media constituents.

Growth of these microorganisms was therefore attempted
in Medium AA, which contained no insoluble components and
which was originally described by Aleem and Alexander (1960)
for the growth of N. agilis in flasks incubated on a 285-rpm
rotary shaker. The results obtained in this laboratory with
N. agilis are given in Table 3. Equal, if not slightly
better rates of oxidation were obtained in Medium AA with
two isolates. In spite of the fact that the inoculum
consisted of microorganisms grown in Medium A, no period of
adjustment to the new Medium AA was observed.

Attempts to culture N. euroEaea in Medium AA (Table 4)

containing 63.4 ug NHu-N/ml in place of NOZ-N and with

30

Table 3. Similar rates of nitrification by two
Nitrobacter agilis cultures in Medium A
and Medium AA

 

 

 

Medium Culture NOZ-N remaining (ug/ml)a
Days
2 3 4
A Pramer 97.0 68.0 0
Fischer 96.5 66.0 0
AAb Pramer 79.5 51-5 0
Fischer 75.5 33.0 0

 

a 105.0 ug NOZ-N/ml present initially in both media
as determined by chemical analysis.

1b Medium AA sterilized by filtration through a membrane
fi ter.

31

 

 

 

Table 4. Dissimilar rates of nitrification by Nitrosomonas
euroggea (Schmidt) in Medium A and Med um AA
Medium NOZ-N produceda
(us/m1)
Days
3 4 5 11 14 17 21
A 0 8e8 lOeS 18.0 28e5 34.5 ‘
0 10.2 16.5 33.0 41.5 - -
AAb 0 6.4 7.0 12.5 13.0 13.5 13.0
0 7.6 10.8 12.5 15.0 15.0 16.0

 

a NH -N contained in medium was approximately equal to
63.5 ug/mI.

b See text (page 29) for modification of Medium AA.

32
calcium chloride added (0.136 g/L), were unsuccessful. In
Medium A, the end of the exponential growth phase was reached
at about 14 days after the conversion of most of the NHu-N to
NOZ-N. In Medium AA, however, only 15 ug/ml NOZ-N were detected
after a similar incubation period, and no further oxidation

was noted after a subsequent period of 7'days.

W

Meyerhof (1916a,b) clearly defined the effect of pH by
the bicarbonate ion upon the respiration of Nitrosomonas and
Nitrobacter. The rate of respiration reached a maximum at pH
8.5 to 8.7 for both microorganisms and a steep decrease in
rate was initiated on the alkaline side at a pH value slightly
above these values. Complete inhibition of oxygen uptake
occurred between pH 9.5 and 10.5 for Nggrgggxgnag and
NNErobacter. Less dramatic drOps in rate of respiration were
observed on the acid side.

Since the effect of pH would thus appear to be very
critical, studies were made on the influence of pH on
Nitrobacter in Medium AA. The pH of this medium was found
to vary considerably depending on various physical factors.
The initial pH at time of media preparation was 9.0 but upon
refrigeration at 10°C for 18 hours the pH drOpped to 8.53.
Attempts to lower the initial pH by the addition of KHZP°4
were made and replicate cultures prepared (Table 5). The

observed rates of nitrification over a 3 day growth period

were equal for the three levels of KHZII’OI+ added to the medium.

Table 5. Influence of pH on nitrification by Nitrobacter
agilis (Fischer) in Medium AA

 

 

 

Initial pH Amount NO -N remaining
pH Post KH P0; ing/m1)a
autoclave added
(S/L) Days
2 3
8.53 10.0 - 59.0 0
8.05 10.0 0.08 62.0 0
7.63 10.0 0.20 59.0 0

 

a 105.0 ug NOZ-N/ml in medium initially.

34

The added amounts of KHZPO had no measurable effect on the

a
final pH since after autoclaving the pH rose to 10.0 in each
case. It appears that the buffering capacity of this medium
was too great to be altered by the relatively small amounts
of added KHZPO4' It is interesting to note that in spite

of the high pH at the time of inoculation no inhibition of
Nitrobacter occurred.

Further attempts to regulate the pH nearer to 8.0 by
the use of increased amounts of NaHZPOu were also unsuccess-
ful. When the pH was adjusted to 7.6, 8.0 and 8.50 by varia-
tions in the amount of NaHZPOu, the resulting pH after over-
night exposure to air, increased to 8.9--9.0.

It is apparent that the pH rises as the bicarbonate

dissociates with the concommitant loss of CO and the

. 2’
phosphate buffer system is ineffective in its presence. A
more 93811! controlable suffer system would be required for

the preper evaluation of pH effects.

13....9.__.em valflmaof e Wists:

Because of the method of cleaning commercial steam
distillation apparatus contaminating amines are frequently
present in the distillate. The high pH of the distilled
water encountered in this laboratory indicated that such
contamination was probably taking place. Values of pH as
high as 8.5 were not unusual. Because of the known sensitiv-
ities of the nitrifiers to amines, measures were undertaken

either to remove the amines from the distilled water or

35
eliminate their presence entirely by the use of glass distilled
tap water. A growth experiment was then performed to deter-
mine if the absence of amines grossly affected the rate of
nitrification (Table 6). From the results obtained no such

unfavorable effect was evident.

geterotrophic Contaminatign

Periodic checks for microbial contaminants were routinely
made since it had been our experience that both Nitrosomonas
and Nitrobacter fluid cultures readily became contaminated
with heterotrOphic bacteria, as well as with Species of molds
commonly encountered in microbiological laboratories.
Frequently, contaminants went unnoticed in the stock cultures
of the nitrifiers because of insufficient growth in the
inorganic medium. In most cases of contamination, however,
growth was initiated within 1 to 2 days in all subcultures
made in test broths except Czapek Box.

A particular stock culture of N. agilis was fc: i
to contain a variety of bacterial contaminants when plated
on membrane filters (refer to Part II of this thesis). Since
these contaminants had no apparent toxic effect on the
Nitrobacter, studies were undertaken to determine the role
of these organisms in the growth and nitrification of the
chemolithotrOph. Preliminary isolation of the contaminants
was accomplished by transfer of a portion of the membrane
filter containing representative types of the various colonies
to fresh Medium AA containing 106 ug-N/ml. After 14 days,

2 successive subcultures were :ade in Nutrient Broth followed

'Table 6. The influence of amine-free water on
nitrification by Nitrobacter

 

 

 

Media Water Culture NOZ-N remaining (ug/ml)
source
Days
0 3 4 5
a b
A Reg. Pramer 99e0 97e0 38.0 0.0

FISCher 103e0 74.4 26e6 OeO

Aa Glassc Pramer 99.0 90.0 43.2 0.1
Fischer 102.0 73.0 30.0 0.0
AA Reg Pramer 104.0 78.4 12.0 0.0
Fischer 105.0 74.4 4.9 0.0
AA Glass Pramer 100.0 74.4 22.6 0.0
Fischer 104.0 56.4 3.4 0.0

 

a Calcium chloride omitted.

b Regular distilled water passed over standard deionizer.

0 Glass distilled tap water passed over standard
deionizer.

37
by cross streaking on Nutrient Agar. Eight individual
colonies were then isolated. Upon later examination only
4, or perhaps 5, different morphological types were recognized,
including 2 different streptomyces.

Nutrient Agar plates were then prepared from broth
suspensions of the bacterial isolates and individual colonies
were again cultivated. When cross streaking of these isolates
an agar gave colonies of a single type, subcultures were
made on Nutrient Agar slants. These cultures were designated
isolates 34-1 to 34-7. At this time all of the isolates
differed morphologically. Three bacterial and 2 streptomyces
species were readily distinguiShable while the remaining 2
isolates were only slightly different from the bacterial
isolates. The bacteria were gram negative rods of varying
size. With the exception of isolate 34-3, all remained viable
after repeated subculture in Nutrient Broth.

Preliminary Characterization. In an attempt to distin-
guish among the various isolates, their oxygen requirements
and growth characteristics in Nutrient Broth were investigated
(Table 7). Further characterization of the isolates was
attempted by growth in various media.(Table 8). Additional
tests on the bacterial isolates were performed using
Phenol Red Broth Base Hedi-Disc (Pennsylvania Biological
Laboratories, Inc., Philadelphia, Penn.) with 23 different
carbohydrates (Table 9). In all media excluding Nutrient

Broth growth was slow and Sparse

Table 7 e

K.)
CI)

Cultural characteristics of heterotrOphs

isolated from Nitrobacter agilis (Fischer)

and grown in nutrient

rot. at 0

C for 48 hrs

 

Isolate Surface Sediment Turbidity Media Relative

No. growth appear- growth
ance

34-1 None Pellets None Brown Sparse
34-2 + + + Cloudy Abundant
34-4 + + + Cloudy Abundant
34-5 None Pellets None Unchanged Sparse
34-6 None + + Cloudy Abundant
33-7 None + + Cloudy Abundant

 

39

Table 8. Characterization of heterotrophs isolated from
Nitrobacter agilis (Fischer) in various mediaa

 

Media Growth and reaction isolate

34-1 34-2 34-4 34-5 34-6 34-7

 

Nutrient Broth (24 hr)

30 C Wk + + - + +
37 C Wk + + - + +

0 e 5% 111111111
(Phenol Bed Broth) - - - - - -

0.5% Sorbitol
(Phenol fled Broth) - - - - - -

Litmus Milk

2 day -
10 day - + + - + +

Simmons Citrate Agar

Growth Wk - + wk + +
“it, Gele 1n NeBe

Growth Wk + + — + +

Liquifaction - + - - - -
(4% Gel. 1n BHI

Growth + + + Wk + +

Liquifaotion - + - - - -

 

a All results based on a 48 hour test period at 30 C
unless otherwise indicated.

40

Table 9. Growth at 14 days of heterotrOphic bacteria
isolated from Nitrobacter agilis (Fischer)
without carbohydrate fermentation at 37 C

 

 

Carbohydrate Isolate
34-2 34-6 34-7
Dextrose + - +
Sucrose + - -
Sorbitol + - -
Lactose + - -
Salicin + - -
Mannitol + - -
Xylose + + Slight
Maltose + Slight "
Rhamnose + - "
Dulcitol + Slight "
Arabinose + u "
Galactose + " "
Baffinose + " Slight
Levulose + + +
Inositol + Slight Slight
Trehalose + " "
Adonitol + " "
Mannose + " "
So rbo se + - "
Starch + Slight "
Inulin + " ”
Melibiose + " "

Melizitose

+
+

 

41
for the bacterial isolates compared to the abundant growth
obtained in Nutrient Broth using an identical inoculum. The
Streptomyces isolates 34-1 and 34-5 even grew poorly in
Nutrient Broth. For this reason the carbohydrate fermenta-
tion study (Table 9) was not utilized for the streptomyces
isolates.

From the foregoing tests certain basic similarities
among the heterotrOphs were rec0gnized. Maximum growth was
obtained in Nutrient Broth at 30 or 37 C within 48 hrs and
growth in Phenol Red Broth containing either 0.5% inulin or
0.5% sorbitol was Sparse even after 10 days. As one would
suspect the streptomyces isolates grew poorly in all of the
media tested, and the mycelia formed pellets in the liquid
media. A positive reSponse in Litmus Milk was obtained with
the bacteria and a negative response was obtained with the
streptomyces at 10 days.

Distinction between 2 of the bacterial isolates was
made on the basis of the following: A

Isolate 34-2 Gelatin liquifaction at 48 hours,
negative test on Simmons Citrate
Agar, surface growth on Nutrient
Broth, and growth in 23 carbohydrate
media;

Isolate 34-6 No gelatin liquifaction, positive
test on Simmons Citrate Agar, no
surface growth in Nutrient Broth,
and varied growth reaponse in

carbohydrate media.

42
Isolates 34-4 and 34-7 were similar to 34-2 and 34-6, reSpec-
tively. Isolates 34-1 and 34-5 were readily distinguishable
on the basis of morphology.

It was clearly evident that a minimum of 4 different
heterotrOphs had been isolated from the contaminated
Nitrobacter stock culture. Isolate 34-7 was suSpected of
containing a mixture of bacteria.

These cultures were subsequently rechecked microscOp-
ically for purity using the Gram stain. Isolates 34-4 and
34-7 were indeed found to be mixed with the 34-2 and 34-6
types. Pour plate colony isolations were again made in an
attempt to confirm the purity of the 34-2 and 34-6 isolates
and to separate the bacteria in the mixed cultures. Isolates
34-2 and 34-6 had remained pure while 34-4 was found to be a
mixture of these two. Isolate 34-7 contained the 34-6 type
in small amounts as well as a type differing from the pure
cultures. The 3 different bacterial isolates are described
in Table 10. Gram Stains of these colonies confirmed the
purity of the 34-2 and 34—6 cultures and the impurity of the
34-7 isolate. The 34-2 culture consisted of very short gram
negative rods, less than 0.? u in length. The 34-6 gram
negative rods were considerably larger. Isolate 34-7
contained a mixture of gram negative rods and the predominant
type was larger than those of the 34-6 isolate. It was
impossible to determine if this new colony type still contained

a mixture of bacteria or if the rods were pleomorphic.

Table 10.

Colonial characteristics on nutrient agar of

heterotrOphic bacteria isolated from Nitrobacter
agilis (Fischer)

 

 

Colonial Isolate
characteristic
34-2 34-6 34-7
Surface
colonies
Size 10-16 mm 2-4 mm 5-7 mm
Form Circular Circular Circular
Surface Smooth Smooth Bough
(Radiately
ridged)
Edge Entire Entire Undulate
Elevation Flat Convex Flat
Optical
characters Opaque Transparent Transparent
Lustre Dull Glistening Glistening
Consistency Membranous Membranous Butyrous
Color Milky white Tan Tan
(reverse-
large yell.
center)
Deep colonies + + -
Size Extend to 1 mm
surface
Form Ellipsoidal
Pigmentation - Pink Golden brown

(21 days)

 

44

Non-utilization of NHNTN and_N02-N. Since the
heterotrOphic contaminants had no apparent toxic effect on
the cultivation of N. aging various studies were undertaken
to determine if they played a role in the nitrification
process.

The heterotrOphic isolates were seeded singly or combined
in the Nitrobacter medium (Medium AA containing 106 ug NOZ-N/ml)
in the absence of Nitrobagggg. After 13 days, no loss in
NOZ-N content was detected (Table 11). This fact clearly
indicated that the inorganic form of nitrOgen remained
unchanged.

No effect on the rate of nitrification by a pure culture
of N. agilis was demonstrated when the contaminants were
added individually or all together (Table 12).

The continuous presence of one or more of these contam-

inants in the stock culture of Nitrobacter was followed over

 

a 7 month period. No change in the culture behavior or rate
of nitrification was observed during this entire period.

The effect on the oxidation of NH4+ by N. euroEaga was
also investigated under similar experimental conditions.
The heterotrOphic isolates were not only unable to convert
the NH“+ to N02” during a 14 day incubation period (Table 13)
but their presence had no overall effect on the oxidation by
Nitrosomonas (Table 14). The presence of two of the isolates,
34-1 and 34-2, may have slightly delayed the oxidation process.
However, other factors could have been reaponsible for the.

slight difference in the results.

 

 

Table 11. Failure of heterotrophic contaminants to
oxidize N0 -N in Medium AA containing 106 ug
NOZ-N/ml (after 13 days)
Heterotrophic Glucose N0 -N
isolate added rema ning
(fl) (us/m1)
34-1 None 107
1.0 102
34-2 None 106
1.0 105
34-5 None 109
1.0 100
34-6 None 104
1.0 100
39-7 None 114
1.0 104
All None 110
1.0 106

 

 

 

 

Table 12. Failure of heterotrophic contaminants

to effect NOZ-N oxidation by N. agilis

(Fischer) in Medium AA containing ug

NOZ-N/ml
Heterotrophic NOZ-N (ug/ml)

isolate
Days
4 5
None 34.8 6.1
34-1 3.5 0.0
34-2 13.5 0.0
314—5 Bites 107
34-6 1‘00 0.0
Alla 27.0 0.7
b

Nutrient broth 31.5 2.8

 

a

All above heterotrophic isolates added.

b Equivalent amount of Nutrient Broth added
as in heterotrOph inoculations.

47

Table 13. Failure of heterotronhic contaminants
to oxidize NE "E in Medium A containing
I J - v
66 ug NquN/mi (after 14 days}.

 

 

1.0

Heterotroph (%) N0 -N
isolate Glucose produced
(us/m1)
34-1 None 0.25
1.0 0.25
34-2 None 0.0
1.0 1.0
34-5 None 0.0
1.0 0.75
39-6 None 0.0
1.0 0.0
39-7 None 0.0
1.0 0.0
All None 0.0

0.0

 

48

Table 14. Influence of heterotrOphic contaminants on
NHu-N oxidation by Nitrosomonas eurOpaea
(Schmidt) in Medium A containing 66 ug NHu-N/ml

4 —

 

 

Hetfggizgghic N02-N produced (ug/ml)
Days
6 7 9 14
None 34.5 39.4 41.0 42.0
34—1 9.6 9.3 11.5 41.0
34-2 6.1 5-5 7.0 37-3
34-5 38.0 45.8 46.0 46.5
34-6 32.5 37.5 44.0 45.5

49
Test tube experiments in Nutrient Broth containing
nitrate were negative in all cases for nitrate reductase
activity. Therefore, none of the isolates were capable of
reducing nitrate to nitrite, when tested in a medium giving

Optimal growth.

DISCUSSION

For lack of a better method of estimation of cell
growth, the rate of oxidation of the energy substrate was
regularly determined colorimetrically using the a-naphthyl-
amine and sulfanilic acid method. Engle and Alexander (1958a)
presented evidence that the rates of nitrification by
N. egggpgea paralleled cell synthesis as estimated by viable
cell counts by the dilution tube method. It is assumed that
a similar relationship between viable cell count and rate of

oxidation also exists for Nitrobacter. Consequently, the

 

rate of oxidation of the energy substrate was considered
synonymous to cell growth, and all growth studies were made
using the criterion of oxidation rate.

Short term stationary cultures prepared in media contain-
ing limiting amounts of energy substrate were used throughout
the course of this study. This type of culture system permit-
ted the handling of large numbers of replicate cultures with
a minimum amount of manipulation. This cell culture method,
however, did impose serious restrictions in the use of cell
enumeration methods which could have been employed had larger
cell pOpulations been produced. These methods include
turbidimetric and direct microscopic cell counting techniques.
Since no such method of cell enumeration was applicable,
inoculum size had to be quantitated on a % basis. This was

done for both nitrifiers.

50

51

The results indicated that a 3.13% inoculum taken from
a culture which had recently completed the oxidation of 100
ug (NHu-N or NOZ-N)/ml was the minimal inoculum for both
nitrifiers for the initiation of growth within a reasonably
short lag period.

Recent grOWth Studies cited in the literature have all
been for large scale continuous culture systems in Which
amounts far greater than 100 ug N/ml were oxidized. The
growth curves contained in Fig. l and 2, however, are typ-
ical of the growth reSponses obtained by other investigators,
except for the incomplete utilization of NHu-N by Nitrosomonas.

The % utilization of energy substrate by N. eurogaea

differed markedly from N. agilis. Unlike Nitrobacter,

 

Nitrosomonas cultures regularly terminated the eXponential
growth phase prior to 100% conversion of NHQ-N to Noz-N. In
fact, cessation of the eXponential phase ocCurred when 12.6
to 31.7% of the NHu-N still remained in the unoxidized form.
Because of this wide range among various eXperiments it would
appear that multiple factors were involved.

The experimental data in Table 2 precluded the inacces-
sibility of NH),+ for oxidation to nitrite. Only 3.3 and 3.4
ug N, for the 2 inocula levels respectively, could not be
accounted for as NHu-N and NOZ-N. These amounts of nitrogen
may have been present as an intermediate oxidative state and

not detected by NHu-N and N0 -N assay. In the case of the

2
cultures in the late exponential phase of growth (6.25% inoc-

ulum), 24.7% of the initial NHu-N added still remained

52
unoxidized.

Engle and Alexander (1958a) reported a slight diminution
in the rate of nitrite formation at the end of the exponential
growth phase in a 25 L pyrex fermentor culture with a final
nitrite concentration of 1162 ug NOZ-N/ml. A perfunctory
re-analysis of their data showed that the lagarithmic phase
for nitrite production was maintained for approximately 70
hours during which time only 700 ug NOZ-N/ml were detected.
In view of the results obtained in this laboratory concerning
the incomplete oxidation of NHn-N, it is possible that their
results were, at least partly, a reflection of a similar
phenomenon.

Comparisons were made on the growth of 2 cultures of
N. agilis in Medium A containing a large amount of a fine
precipitate, and Medium AA, containing no insoluble compo-
nents. Rates of nitrification were identical for both cul-
tures in the 2 media. A similar comparison was made for
N. eggogaea (Schmidt) in the same basal media containing
NHu-N in place of NOZ-N. Medium AA did not support the
growth of this nitrifier even with CaCl2 added.

The growth of N. agilis was not affected by initial
pH as high as 10.2. This contrasts markedly with the
findings of Meyerhof (l916a,b) who demonstrated a steep
decrease in rate of reSpiration for Nitrobacter on the
alkaline side of pH 8.7. It is apparent that the effect
described by Meyerhof is not obtained in fluid cultures of

N. agilis during active growth.

53

Deionized distilled water used in the preparation of
media was routinely passed over a cation removal resin for
the removal of amines. This was done as a precautionary
measure since continuous monitoring of the distilled water
supply was not feasible, and it was decided to eliminate
the possibility of the presence of inhibitory levels of
amines.

The use of impure strains of the chemolithotrOphs,
although widely practiced in the past, is extremely unwise,
particularly for biochemical investigations. Nevertheless,

‘ r ‘v- e V n. -,A ‘ + ‘ .I n a ‘ n 3 ~ ‘. e A 9-
only a 12w -e-sn- studies have SpGCi.AZH‘ : ,ited he use

-— d.

s V
v ' . ..-..,,__, .w. +-
t. :11“ cal.

Jre techniques.
The use of an unsterilized 48 L carboy containing uneter-
ilized deionized tap water also was reported (Anderson, 1964).

Another investigator reported the use of a Nitrobacter cul-

 

ture containing known quantities of heterotrOphic contaminants
(Fischer, 1965).

Since methods for obtaining pure cultures of the
nitrifiers have been available for some time, there is no
good reason for the use Of impure strains. An attempt was
made in this laboratory to define the role of heterotrOphic

contaminants which had no apparent toxic effect on Nitrobacter.

 

The heterotrOphs found as contaminants in our stock cul-
ture of N. agilis and previously described in detail, were
unique in several respects. They produced no toxic effect
on either the Nitrobacter or on one another. They were

maintained at a low Optical density in Nitrobacter cultures

 

sh

J

for a period of 7 months, and they were incapable of utiliz-

Z-N’ 0r N03-NO

Their nitrogen requirements must have been satisfied indirectly.

ing inorganic nitrogen in the form of NHu-N, NO

The presence of these organisms also had no effect on the
growth or nitrification of the Nitrobacter.
One can readily appreciate from these interactions with
the nitrifier, that any biochemical investigations, aside
from the nitrification process itself, performed on this
mixed culture could lead to possible erroneous interpretation.
So far as the identity of the bacterial heterotrOphs is
concerned, insufficient differential tests were performed for
accurate classification, and further testing is beyond the
ScOpe of this thesis. It is most probably that the bacterial

contaminants represent different species of Pseudomonas.

PART II. ATTEMPT TO DEVELOP AN IMPROVED CELL ENUMERATION
METHOD USING MEMBRANE FILTERS.

Introduction

It can not be assumed that cultures oxidizing an equal
amount of an inorganic nitrogen substrate have the same
activity since it has been shown by several investigators
that growth and oxidative capacity are not simultaneously
inhibited. Meyerhof (1916a) and Gould and Lees (1958)
demonstrated nitrate inhibition of growth first and then
oxidation of Nitrobacter. Lees (1952) found that streptomy-
cin at concentrations inhibitory to growth had no direct
effect on ammonia oxidation.

Such dual type of inhibition could be extremely complica-
ted. A direct method of growth evaluation in addition to
the quantitative estimation of products of oxidation would
be desirable in the study of the effects of various inhibitory
or stimulatory substances.

The methods available for the quantitative detection of
cellular growth of nitrifiers in liquid media are seriously
restricted due to the nature of these microorganisms. Colony
growth on solid media is limited to silica gels (Allen, 1957;
Funk and Krulwich, 1964; Pramer, 1958; Temple, 1949;
WinOgradSKy, 1891a), or an inorganic medium supplemented with.
washed agar such as Noble's Agar (Difco) (Gould and Lees,
1960; Kingma-Boltjes, 1935; Meiklejohn, 1950). The silica

gels are unsuitable as a rapid means of obtaining plate

55

56

counts. The use of Noble Agar is at present the only reli-
able means for colony growth but does not allow macrosccpic
identification of these organisms. The colonies formed are
so minute, less than 0.1 mm, that they must be viewed under
low power magnification of 20 to 30 X. Having to employ a
magnification device seriously hampers the use of the plate
dilution technique, by requiring standardization of the
microscOpic field and the plate area observed.

The use of a membrane filter was therefore investigated.
Membrane filters offered the advantage over agar plates in
that they could be obtained with grid markings facilitating
the counting of colonies with the aid of an ordinary dissect-
ing microscOpe. The ease with which the membrane filters
could be stained, and indicators applied also offered addi-
tional advantages over colony identification on an agar sur-
face. Furthermore, the filtration of low bacterial pOpula-
tions permits the concentration of organisms on the membrane.
And lastly, the stained membranes could be easily preserved
and stored for later evaluation. The methods develOped for
the use of membrane filters are described and evaluated in

this section.

Materials and Methods
Membrane filters. MF-Millipore filters of HA type
(0.45 u pore size) and 47 mm in diameter were obtained
presterilized and grid-marked. Each grid-square (3.1 mmz)

equals l/lOOth of the 9.6 cm2 effective filtering area of a

57
47 mm filter used in the Millipore filter holder.

Base media. Fluid media identical to that used in the
maintenance of stock laboratory cultures (Part I) served as
the source of nutrients. The filters were supported by
either an absorbent pad saturated with 1.8 to 2.0 ml of fluid
media or Noble Agar medium consisting of 1.5 per cent agar
in the culture media Just described.

Plating procedure. Serial dilutions were prepared in
growth media from fluid cultures of the nitrifiers in the
eXponential phase of growth. Colony growth directly on the
agar surface was obtained by spreading 0.1 to 0.2 m1 of the
culture dilution over the agar surface. In the membrane
filter technique 5 to 10 m1 of the diluted cell suspension
was filtered over the membrane filter previously washed by
the addition of 10 m1 of fresh culture medium. The sides of
the filtration funnel were then washed with 10 ml of fresh
medium and the membrane filter transferred to either an agar
plate or a petri dish containing an absorbent pad saturated
with fluid medium. After incubation at 28 to 30 C for 8--12
days the membrane filter was then observed microscopically
for colony formation, stained and colony counts made.

Preparation of methylene blue stain and_indicators.
Methylene blue chloride (National aniline Co.) solution was
prepared by dissolving 1.5 gm of dye in 150 m1 of ethyl
alcohol and mixed with 500 ml KOH solution (0.01% Wt/Vol).

The membrane was stained for 3 minutes by placing the filter

58
on an absorbent pad saturated with 1.8 m1 of the staining
solution.

TTC (2, 3, 5-triphenyl-ZH-tetrazolium chloride, Eastman
Organic Chemical) was prepared as a 2% solution in culture
medium.

Resazurin solutions were prepared by dissolving 58 mg

in 100 m1 of M/15 phosphate buffer solution, pH 7.0 at 100°C.

Results

Deve10pment of Technique

A series of plating experiments was devised to deter-
mine the efficacy of using grid-marked membrane filters for
the enumeration of cells in suspension culture. In the
first experiment, a nine day old suspension culture of
g. agilis (Fischer) was used. Suitable portions 0f dilutions
ranging from 1/10 to 1/1280 were applied directly to the
surface of agar plates and to the filters which were incuba-
ted over an agar or absorbent pad. After 12 days the plates
contained several types of colonies and attempts were made to
identify those formed by the Nitrobacter. Plates seeded with
a 1/1280 dilution were too heavily pOpulated on the agar
surface for the estimation of colony number. The colony
types present were morphologically of the same type obtained
on the filter. Examination revealed at least 4 colony types,
two of submacroscOpic size and two readily visible without

the aid of a microscope.

59

Staining of membrane filters with methylene blue greatly
enhanced visualization of the colonies both macroscOpically
and microscopically. All 4 colony types clearly stood out
as darkly.stained areas on a lightly stained background.

Within minutes after the application of the TTC solution
to membrane filters only a few colonies appeared bright red
while most appeared unchanged. Further observations over a
24 hour period showed no additional changes except for a
decrease in color intensity in the colonies originally reduc-
ing the tetrazolium salt.

The bacteria did no: p:o;uce a iatsctable change in the

*9

A1

dazurin.

Quantitation Procedure

After staining with methylene b1ue,colony counts were
obtained by counting the number of colonies within one or
more 3.1 mm squares. Approximatedly 60 colonies were counted
per filter. The number of cells in the undiluted sample was
calculated from the average number of cells observed at each

dilution by applying the formula:

(100) (organisms counted)¥(reciproca1 of dilution)

Cells/m1 a (grid-squares exam.) (m1 of sample)

 

Using this method of calculation the data from experiment I
was evaluated on the basis of the numbers of macroscOpic and
microscOpic colonies present in the original culture. The

results are included in Table 15.

60

Table 15. Quantitation of various types of micro-organisms
in a fluid culture of Nitrobacter agilis (Fischer)
by membrane filter technique

!‘

 

Type Nutrient Dilutions Averagea
colony base evaluated no. of
cells/m1
microsc0pic Agar l/320,1/640 5.8 x 105
1/1280
Absorbent 6.0 x 105
pad
MacroscOpic Agar 1/20, 1/40, 1.35 x 103
1/80
Absorbent 3
pad 1.04 x 10

 

A The average of cell counts on 3 dilutions run in
triplicate.

 

5111!. ‘III- I [I'll I l . |

61

A maximum of 60 colonies/filter grid—square (3.1 mm)
could be accuratedly counted when the colony size was that
of the nitrifiers, around 0.1 mm. As many as 6000 such
colonies could be effectively evaluated on a single membrane
filter using this technique.

The same plating procedure was repeated using a second
subculture of the same 3. agilis stock culture. Identical
results were obtained as in the first experiment, that is the
presence of four colony types was again noted. It appeared
that 3 or even 4 different contaminants were present. The
possibility that both of the microscOpic type colonies were
other than 3. agilis was considered.

To determine the presence of viable g. agilis cells on
the filter membrane, filter strips were taken after colony
development, by aseptically cutting the filter, and were
transferred to fresh nutrient media in test tubes. This
type of subculture was prepared from areas of the membrane
filter in which the macroscOpic colonies were absent. Within
a 14 day period nitrification occurred in 21 out of 21 such
subcultures. The ability of g. agilis to grow on membrane
filters was thus established.

The use of the membrane filter technique was then applied
to contaminant-free fl. agilis and g. euroRaea fluid cultures.
When these cultures were plated on a suitable medium contain-
ing Noble Agar colonies develOped within 8 days. No further
increase in colony size was obtained after 8 days. Parallel

studies were performed on the membrane filters but

02
the colony size was much smaller than those obtained growing
directly on the agar surface. In fact, the size of the
colonies was so close to that of media debris that distinc-

tion was not possible.

Discussion

A reliable membrane filter technique would greatly facil-
itate the enumeration of small numbers of viable cells in
fluid cultures of gigggbggtgg and gitrosomonas. Evidence
was obtained for the growth of g. agilis either on the filter
surface directly or in the thin film of medium on the filter
surface.

The reason for not obtaining colonies like those which
appear on an inorganic medium solidified with Nonie Agar
is not understood. The possibility exists that Noble Agar
contains certain substances conducive to growth enhancement.
Several factors which increase colony size on washed agar are
known. Trace amounts of peptone (Fred and Davenport, 1921)
caused enlarged colonies of both nitrifiers, while biotin
was found to accelerate colony formation by Nitrosomonas
(Gundersen, 1955a). These as well as other compounds reported
to be stimulatory to growth or nitrification should be inves-
tigated as potential stimulants of colony growth.

The inhibitory effect of membrane filters on colony
formation should not be overlooked. Membrane filters produced
by several manufactures are reported to contain 2 to 3% of
their dry weight as detergent (Cahn, 1967). The same inves-

tigator found that the use of tissue culture medium filtered

63

through unwashed Millipore filters, containing Triton X-100
or a similar detergent, considerably lowered the plating
efficiency of clonal cultures of cartilage cells‘ig 2&322'

The use of membrane filters in many areas of sterility
testing is widely accepted, but the more specific application
for fastidious microorganisms has been reported infrequently.
The continuation of efforts leading to the application of

this technique to the nitrifiers is warranted.

cu

PART III. EFFECT OF PESTICIDES ON NITRIFICATION.

Introduction

The persistence of pesticides in soils for many months
or even years may affect the role of the nitrifying bacteria
in the maintenance of soil fertility.

The results of many investigators have indicated that
many herbicides and insecticides when applied at the
recommended field rates generally have no harmful effects
upon the microscOpic pOpulation or upon its biochemical activ-
ities (Alexander, 1964).

At the present time, however, little information is
available on the effect of various pesticides on nitrifica-
tion in pure cultures of flitrosomonas and gitrobacteg.

Caseley and Luckwell (1965) have shown that monuron, a
substituted-urea herbicide, inhibits Nitrobacter at concentra-
tions similar to those applied to soils. Campbell and Aleem
(1965a,b) have shown that another substance, N-Serve, inhib-
its Nitrosomonas at extremely low concentrations.

Studies on the effects of other pesticides on nitrification

by both of these chemolithotrOphs were undertaken in this
laboratory.

Materials and Methods

Replicate cultures for pesticide assays were prepared.
as previously described under Part I or this thesis. Each
culture flask contained a final volume of 50 ml after the

apprOpriate additions of both test material and inoculum.

65
Medium A containing 64 ug NHu-N/ml was used for the
N. europaea (Schmidt) experiments while Medium AA contain-
ing 100 ug NOZ—N/ml was used for the N. agilis (Fischer)
experiments.

Solutions of the pesticides were prepared in the appro-
priate solvent, either acetone or ethyl alcohol, and ster-
ilized by passage through a sterile UF sintered glass filter.
These preparations were then added aseptically in the apgrc~
priate volume to their respective assay flasks at the time of
culture inoculation. Cultures in all espsriments were incuba»

ted at 29-33 a for a period up to 14 days.

Results
Thaw“. 9.9.12.0: Pest i0 id 8 s omdigsasclsaasilis

The effect of pesticides at final medium concentra-
tions ranging from 10"1 to 103 ug/ml on the growth of this
organism was followed by determining the decrease in nitrite
in the culture medium. Nitrite determinations were made as
described previously.

N. agilis was sensitive to all 3 chlorinated hydrocarbons
at concentrations as low as l ug/ml (Table 16). Lindane
appears to be the least toxic and only produced a delay in
nitrate production even at the highest level tested, 1000 ug/ml.
Both rhothane, the most toxic, and aldrin caused complete
inhibition over a 14 day period at concentrations of 10 ug/ml.

Delayed nitrate production over the same period resulted from

exposure to 0.1 and 1.0 ug/ml.

66

 

 

 

 

 

Table 16. Inhibition of nitrification by Nitrobacter 25ll£§
(Fischer) in Medium AA containing chlorinated
hydrocarbons

Pesticide NOZ-N remaining (ug/ml)a
Name Final Days

(3:?51) 3 4 6 14

Aldrin 103 88.8 90.0 83.0 98.5
102 86.8 85.5 83.3 100.0
101 83.8 79.0 87.5 80.0
100 73.5 64.3 34.5 0.0
10‘1 1.5 0.0 - -

Lindane 103 71.3 62.0 69.0 44.5
102 69.3 2.0 56.8 0.0
101 69.9 69.0 65.5 0.0
100 44.2 19.9 0.3 -
10'1 18.8 0.4 - -

fihothane 103 87.5 90.0 88.8 100.0
102 75.0 81.3 90.0 103.0
101 85.0 83.0 87.5 96.5
100 80.3 85.0 81.3 88.0
10"1 42.3 31.5 0.0 -

Control - 18.0 0.0 - -

a ._

Medium contained 100 ug NOZ-N/ml.

67
0f the two organic phosphate_compounds (Table 17)
malathion was the less toxic, in fact the least toxic of all
the compounds tested, wuile parathion was as toxic as the
chlorinated hydrocarbon, aldrin. Baygon, a methyl carbamate,
had lower toxicity (Table 17), and was similar to lindane as
it only caused delayed nitrification.

... -m.---.--. I'm” 9.-

 

Three of the same pesticides tested against N. agilis
were also tested against N. europaea using the identical
pesticide stock solutions. Each substance was representative
of one of the three types of chemical compounds included in
the previous study. With these compounds, lindane, malathion,
and.baygon, the sensitivity of this bacterium (Table 18) was
greater than that of N. agilis. Based on complete inhibition
dosage levels for a 14 day period, N. eurogaea was at least
100 times more sensitive to each pesticide than N. agilis.

At all the concentrations tested, this bacterium gave an all
or none response, that is either no inhibition, or complete
inhibition was obtained. Delayed nitrification was not

evident under these test conditions.

Discussion

It is evident from these studies and those reported by
others that certain pesticides have a selective action on the
nitrifiers (Campbell and Aleem, 1965a,b; Caseley and Luckvmll,
1965; Goring, l962a,b). In the case of lindane, malathion,
and baygon they are more toxic to Nitrosomonas then to

Nitrobacter.

Table 1?. Inhibition of nitrification by Nitrobacter
agilis (Fischer) in Medium AA containing
organic phosphates and a-methylcarbamate.

 

 

 

 

Pesticide Roz-N remaining (ug/ml)a
Kane Final Days

cone. 3 4 6 9 l4
(us/m1)

Malathion 103 54.5 39.5 13.5 9.3 0.0
102 17.7 0.0 - - -
101 37.3 0.0 - - -
10° 18.0 0.0 - - -
10"1 23.9 0.0 - - -

Parathion 103 81.3 82.0 80.3 89.5 92.0
10‘ 79.0 83.3 85.3 82.5 95.0
101 76.8 75.5 74.5 79.0 75.0
10° 0.0 - - - -
10"1 1.0 - - - - .

Baygon 103 82.5 82.0 83.3 55.3 44.5
102 74.5 70.5 55.8 28.5 0.0
101 52.8 31.2 0.0 - -
100 28.5 2.0 - _ -
10’1 14.3 0.0 - - -

Control - 14.7 0.0 ’ - - -

 

aMedium contained 100 ug NOZ-N/ml.

69

Table 18. Inhibition of nitrification by Nitrosomonas

 

eurogaea (Schmidt) in Medium A contaIning
various pesticides.

 

 

Pesticide NOZ-N produced (ug/ml)a

Name Final Days
COHCe
(“S/m1) 7 8 11 14

Lindane 103 1.8 1.5 4.0 1.8
101 2.5 1.8 3.3 1.0
100 38.3 40.8 43.0 -
10‘1 41.3 43.8 49.3 -

Malathion 102 4.0 3.8 5.8 4.0
101 2.5 3.0 3.8 1.0
10° 28.8 31.3 34.0 30.3
10"1 40.3 44.3 48.3 -

Baygon 103 2.0 2.5 4.0 2.3
102 4.3 6.8 5.8 7.0
101 1.8 2.8 4.0 2.5
100 35.0 36.5 40.8 40.5
10‘1 38.5 42.5 41.0 40.0

Control - 41.0 45.5 43.5 46.5

 

a Medium contained 66 ug NHu-N/ml.

70
The level of toxicity causing complete inhibition of
Nitrgsomonas was 10 ug/ml for lindane, malathion and baygon.
Nitrgbacter was similarly inhibited with equal amounts of
parathion, aldrin, and rhothane. The application of these
pesticides at rates of 1-10 pounds/acre would result in
similar concentrations in the soil which could cause inhibi-

tion or even death to the nitrifiers.

Casely and Luokwill (1965) reported that monuron
applied at rates of l and 3 lbs/acre delayed the start of
nitrification by 6 and 14 days respectively, while applica-
tion at 10 lbs/acre completely inhibited nitrification.
Their pure culture studies showed monouron to be an inhib-
itor of N. eurogaea at 100 ug/ml and of N. agilis at 25 ug/ml.
It is conceivable therefore that those compounds showing
inhibitory prOperties at concentrations of 25 ug/ml or less
could have a detrimental effect on the rates of nitrifica-
tion in the soil. All six of the compounds included in this
study were within this concentration range for the inhibition
of one or the other nitrifier. On the basis of results
obtained from pure culture studies, one could predict those
compounds likely to cause inhibition of nitrification under
field conditions in the absence of such factors as the absorp-
tion on soil colloids and degradation of the inhibitor. It
is also conceivable that degradation products might be even
more toxic than the original substance, as in the case of
aldrin which degrades to dieldrin. It has also been shown

(Caseley and Luckwill, 1965) that the formulated products in

71
pure culture eXperiments are far more toxic to Nitrosomonas
and Nitrobacter than the pure unformulated herbicides.

These observations were attributed to the wetting agents

ability to also inhibit nitrification.

SUMMARY

This dissertation has been primarily concerned with the
develOpment of methods for the study of the effects of various
chemical factors, including pesticides, on the nutrition and

physiology or the nitrifiers: Nitrobacter agilis, and

 

Nitrosomonas eurogaea.

Readily reproducible growth conditions using 250 ml
stationary flask cultures were obtained in an inorganic medium
(Medium A) by inoculation with a 3.13% (by volume) inoculum.
Inoculations were made from liquid cultures which had
completed the exponential phase or growth in a medium contain-
ing 100 ug of energy substrate per ml.

-

During the eXponential growth phase, L. ar“'; g ,f3g3;

_ . _.
a L'L—A. I—o-o-tJ-Q

effected 100% oxidation of NOZ-N. For N. egregaea, however,
the exponential growth phase was terminated when 70 to 80%
of the theoretical amount of NHu-N appeared in the form of

NOZ-N. The incomplete oxidation of NHQ-N by Nitrosomonas

 

cultures could not be attributed to either the Spontaneous

loss of N88, or a binding of N84 ions. Nor was it attrib-
./

J)

utable to intermediate oxidation state” as all of the NH -N
supplied to the medium initially could be accounted for as

either NEH-N or NCP-N.

It)

80th the Pramer and ischer cultures of Nitrobacter

arilis grew equally well in Medium AA, a modification of the

 

B

edium described by Aleem and Alexander (1960), which
contained no insoluble components. This same basal mediL

72

73

but with NH -N replacing NO -N, did not support the growth of

4 2
N. eurogaea. Quantitative, but not qualitative, differences
in the substances present in the medium probably were the
cause of the differences in growth reaponse of the two
nitrifiers. N. euroEaea may have been less tolerant to a
high hydrogen ion concentration (pH 10).

The removal of amines from distilled water through the
use of a cation exchange resin had no gross effect on the
rate of nitrification by Nggrobacter.

All stock cultures of the nitrifiers were repeatedly
checked for the presence of contaminants. From one of the
stock culturesof N. agilis (Fischer), 2 streptomyces and 3
bacterial strains were obtained. All except 1 bacterial
strain was isolated in pure culture. The bacterial isolates
were tentatively identified as various species of ggggdomonag.

An attempt was made to quantitate fluid culture pOpula-
tions using a Millipore membrane filter in a modified colo-
nial plate count method. Growth of N. Efiillé."33 observed
in or on the filter membrane but there was no easily recogniz-
able colonial develOpment.

An evaluation was made on the effect of various pes—

ticides on nitrification by both N. agilis (Fischer)
and N. egrepaea (Schmidt). N. agilig was sensitive to all 3

 

chlorinated hydrocarbons, aldrin, lindane, and rhothane at
final medium concentrations of 1 ug/ml. Complete inhibition
after 14 days was obtained with both rhothane and aldrin at

concentrations of 10 ug/ml. Lindane was considerably less

74

toxic, causing only a delay in nitrate production over a
concentration range of 1 to 1000 ug/ml. Malathion and
parathion, two organic phOSphate compounds, differed widely
in their toxicity for Nitrobacter. Malathion was the least
toxic of all the compounds tested causing only delayed
nitrification at 1000 ug/ml. Parathion, as toxic as aldrin,
gave complete inhibition at 10 ug/ml. Baygon, a methyl
carbamate, delayed nitrification by Nitrobacter at concentra-
tions of 10 ug/ml or greater.

Lindane, malathion, and baygon, were also tested against
N. europaea. All three of these compounds were at least 100
times more toxic for the Nitrosomonas cultures than for
Nitrobacter. Delayed nitrification was not evident in
Nitrosomonas cultures as either no inhibition, or complete

inhibition was obtained.

LITERATURE CITED

Albrecht, W. A. and McCalla, T. M. 1937. Nitrification of
ammonia adsorbed on colloidal clay. Proc. Soil Sci. Soc.
Ame £8263-267e '

Albrecht, W. A., and T. M. McCalla. 1938. The colloidal
claz fraction of soil as a cultural medium. Am. J. Bot.
gi' 03-407.

Aleem, M. I. H. 1965. Path of carbon and assimilatory power
ifi.chemosynthetic bacteria. Biochim. BiOphys. Acta. 102:
1 28.

Aleem, M. I. H., and M. Alexander. 1958. Cell-free nitrifica-
tion by Nitrobacter. J. Bacteriol. 168510-514.

Aleem, M. I. H., and M. Alexander. 1960. Nutrition and
ghysiology of Nitrobacter agilis. Appl. Microbiol. 8:

Aleem, Me 10 He, Ge E0 HOOh, and Jo Be varner. 1965. Water
as the source of oxidant and reductant in bacterial
chemosynthesis. Proc. Nat. Acad. Sci. 538869-873.

Aleem, M. I. H., H. Lees, B. Lyric, and D. Weiss. 1962.
Nitrohydroxylamine: The unknown intermediate in
nitrification? Biochem. BiOphys. Research Comm. 1!
126-127.

Aleem, Me I. Be, Le L688, and De<Je De NIOhOIBSe 19630 .
Adenosine triphosphate-dependent reduction of nicotinamide
adenine dinucleotide by ferro-cytochrome c in chemo-
autotrOphic bacteria. Nature £9Q8759-761.

Aleem, M. I. H., and A. Nason. 1959. Nitrite oxidase, a
particulate cytochrome electron transport system from
Nitrobacter. Biochem. BiOphys. Research Comm. lfl323'327-

Aleem, M. I. H., and Mason, A. 1960. Phosphorylation coupled
to nitrite oxidation by particles from the chemoautotroph,
Nitrobacter 851118e PTOOe Nflte Acad. 3010 fléfi763-7680

Alexander, M. 1964. Introduction to soil microbiology.
John Wiley 8 Sons, Inc., New York.

Alexander, M. 1965. Nitrification, p. 307-343. In W. V.
Bartholomew and F. E. Clark (ed.), Soil NitrOgen,
Agronomy Monograph 10. Amer. Soc. Agronomy, Inc.,
Madison.

75

r‘
. - \
f \J

Allen, O. N. 1957. EXperiments in soil bacteriology. 3rd
Ed., Burgess Publishing Co., Minneapolis.

Amer, F. M., and W. V. Bartholomew. 1951. Influence of oxygen
concentration in soil air on nitrification. Soil. Sci.,
1;:215-219.

American Public Health Association. 1955. Standard methods
for the examination of water, sewage, and industrial
wastes. 10th ed., New York, New York.

Anderson, J. H. 1964. The metabolism of hydroxylamine to
nitrite by Nitrosomonas. Biochem. J. 2;:8-17.

Audus, L. J. 1946. A new soil perfusion apparatus. Nature
l§88419.

Bisset, K. A., and J. B. Grace. 1954. The nature and
relationships of autotrOphic bacteria. £3 E. A. Fry
and J. L. Peel (ed.), p. 28-53. AutotrOphic Microorganisms
(IVth Symp. Soc. Gen. Microbiol.) Cambridge Univ. Press,
Cambridge.

Bbmeke, H. 1939. Zeitraqe zur Physiologic Nitrifizierender
Bakterien. Arch. Mikrobiol. $93385-445.

ngeke, H. 1950. Uber die Erhnahrungs und Wachstumfaktoren
der Nitrifikationsbakterien. Arch. Mikrobiol. $3363-98.

Boullanger, E., and L. Massol. 1903. Etudes sur les microbes
nitrificateurs. Ann. Inst. Pasteur $13492-515.

Burge, W. D., E. Malavolta, and C. C. Delwiche. 1963.
Phosphorylation by extracts of Nitrosomonas eurOpaea.
Jo Bacteriol. §j3106-110e

BUtt, W. D., and H. Lees. 1958. Cytochromes of Nitrobacter.
Nature 182:732—733.

Cahn, Robert D. 1967. Detergents in membrane filters.
Science 155:195-196.

Campbell, N. E. R., and M. I. H. Aleem. 1965a. The effect
of 2-ch1oro, 6-(trichloromethy1) pyridine on the chemo-
autotrOphic metabolism of nitrifying bacteria. I. Ammonia
and Hydroxylamine Oxidation by.Ning§gmgna§, Antonie
van Leeuwenhoek, J. Microbiol. and Serol. 1;:124-136.

, and . 1965b. The effect

of Z-chloro, 6-(trichloromethyl) pyridine on the chemo-
autotrophic metabolism of nitrifying bacteria. II. Nitrite
oxidation by_NLnghagtez, Antonie van Leewenhoek,

J. Microbiol. and Serol. 2;:137-144.

 

 

Caseley, J. C., and L. C. Luckwill. 1965. The effect of some
residual herbicides on soil—nitrifying bacteria. Ann.
hep. Long. Ashton Res. Sta. for 1964:78-88.

Clark, C., and E. L. Schmidt. 1966. Effect of mixed culture
on Nitrosomonas eurOpaea simulated by uptake and utiliza-
tion of pyruvate. J. of Bacteriol. 2;:367-373.

Conn, H. J., and J. E. Conn. 1940. The stimulating effect
of colloids upon the growth of certain bacteria.
Jo BaCtCIIOle 22399-100.

Delwiche, C. C., and M. S. Finstein. 1965. Carbon and energy
sources for the nitrifying autotrOph Nitrobacter.
Jo Bacteriol. 293102-1070

Desal, S. V., and Fazal-Ud-Din. 1937. Influence of non-

nitrifying organisms on nitrification. Indian J. Agric.
SCI. 23895-905e

Engle, M. S., and M. Alexander. 1958a. Growth and autotrOphic
metabolism of Nitrosomonas eurOpaea. J. of Bacteriol.
Zé3217-2220

, and . 1958b. Culture of Nitrosomonas

euro sea in mediawfree of insoluble constituents. Nature
IBIIE§60

 

 

 

 

 

, and . 1959. Enzymatic activity of
Nitrosomonas extracts. J. Bacteriol. 18:796-799.

, and . 1960. AutotrOphic oxidation
of ammonium and hydroxylamine. Soil Sci. Soc. Am. Proc.
£3348-50.

Falcone, A. Be, A. Le Shug, and De Jo De NIChOIBSe 19620
Oxidation of hydroxylamine by particles from Nitrosomonas.
Biochem. BiOphys. Research Comm. 21126-131.

Finstein, M. S., and C. C. Delwiche. 1965. Molybdenum as a
microngtrient for Nitrobacter. J. of Bacteriol. 823
123-12 . '

Fischer, Imre. 1965. Phosphorus metabolism in Nitrobacter

WinOgradskyi. Ph. D. Thesis, Universite Catholique De
Louvain, Louvain.

Frankland, P. F., and G. Frankland. 1890. The nitrifying
process and its Specific ferment. Trans. Roy. Soc.
(London), 21813107.

Fred, E. B., and A. Davenport. 1921. The effect of organic

nitrOgenous compounds on the nitrate-forming organism.
Soil Sci. ;;:389-404.

78

Funk, Helen B., and Terry A. Krulwich. 1964. Preparation of
clear silica gels that can be streaked. J. of Bacteriol.
@31200-12010

Gibbs, W. M. 1919. The isolation and study of nitrifying
bacteria. 3011 3010 Q8427-471.

Goring, C. A. I. 1962a. Control of nitrification by 2-chloro-6
(trichloromethyl) pyridine. Soil Sci. 218211-218.

. 1962b. Control of nitrification of ammonium
fertIIIzers and urea by 2-chloro-6 (trichloromethyl)
pyridine. Soil Sci. 22!431'“39-

Gould, G. W., and Lees, H. 1958. The intensive culture of
NitrObaCtBre BiochCme J. @338 p.

Gould, G. W., and H. Lees. 1960. The isolation and culture

of the nitrifying organisms. I. Nitrobacter. Can. J.
Microbiol. 68299-307.

Gunderson, K. 1955a. Effects of B-vitamins and amino-acids
on nitrification. Physiol. Plantarum 83136-141.

Gunderson, K. 1955b. Observations on mixed cultures of
Nitrosomonas and heterotrOphic soil bacteria. Plant and

Ida, 3., and M. Alexander. 1965. Permeability of Nitrobacter
agilis to organic compounds. J. Bacteriol. 2§;IBI-I56.

Kiesow, L. 1962. ‘Uber den mechanismus der chemosynthese.
Zeitschr. Naturforschung lzbs455-465.

Kingma BoltJes, T. I. 1935. Untersuchungenflflber die nitrifi-
zierenden bakterien. Arch. Mikrobiol. éfi79'138-

Kluyver, A. J., and H. J. L. Donker. 1926. Die Elnheit in
der Biochemie. Chem. Zelle u. Gewebe N28134-l90

Krulwich, T. A., and R. B. Funk. 1965. Stimulation of
Nitrobacter agilis by biotin. J. of Bacterial. 291729-733.

Laudelout, H., and L. van Tichelen. 1960. Kinetics of the
nitrite oxidation by Nitrobacter winogradskyl. J. of
Bacteriol. 12:39-42.

Lees, H. 1947. A simple automatic percolator. J. Agric.
3610 32-3270

Lees, H. 1952. The biochemistry of the nitrifying organisms.

I. The ammonia-oxidizing systems of Nitrosomonas.
Biochem. J. 528134-139.

7

Lees, R. 1954. The biochemistry of the nitrifying bacteria.
13 B. A. Fry and J. L. Peel, eds. Autotrophic Micro-
organisms. Cambridge Univ. Press, Cambridge. pp. 84-98.

Lees, H., and Jane Meiklejohn. 1948. Trace elements and
nitrification. Nature 1613398-399.

Lees, H., and J. H. Quastel. 1944. A new technique for the
study of soil sterilization. Chem. and Ind., No. 26,
238-239.

Lees, H. and J. H. Quastel. 1946a. Biochemistry of nitrifica-
tion in soil. I. Kinetics of, and the effects of
poisons on, soil nitrification, as studied by a soil
perfusion technique. Biochem. J. 3&3803-814.

. 1946b. Biochemistry of nitrifica-
tionan in soiI. II. The site of soil nitrification.
BIOCheTHe Jo 2:98 815-823e

and _ . 1946:. Biochemistry of nitrifica-

tfgn in soif.‘"III. Nitrification of various organic
nitrogen compounds. Biochem. J. 423824-828.

Lees, H. and Simpson, J. R. 1957. The biochemistry of the
nitrifying organisms. 5. Nitrite oxidation by
Nitrobacter. Biochem. J. 65:297-305.

Lewis, B. F., and D. Pramer. 1958. Isolation of Nitrosomonas
in pure culture. J. of Bacteriol. 263524-528.

Meiklejohn, J. 1950. The isolation of Nitrosomonas europaea
in pure culture. J. Gen Microbiol. 33185-191.

MeikleJohn, J. 1951. The effects of glucose on impure
cultures of nitrifying bacteria. Plant Soil 2388-93.

Meiklejohn, J. 1952a. Some organic substances and the
nitrifying bacteria. Proc. Soc. Appl. Bacteriol. £53
77-81.

. 1952b. Minimum phOSphate and magnesium
requirements of nitrifying bacteria. Nature 12031131.

 

Meiklejohn, J. 1953. Iron and the nitrifying bacteria.
J. Gen. Microbiol. 8358-65.

MeikleJohn, J. 1954. Some aSpects of the physiology of the
ggtgifying bacteria. Symp. Soc. Gen. Microbiol. 33
- 3,

80

Meyerhof, O. 1916a. Untersuchungen fiber den Atmungsvorgang
Nitrifizierenden Bakterien. I. Die Atmung des
Nitratbildners. Pfluegers Arch. Ges. Physiol. 1643353-
427.

. 1916b. Untersuchungen.fiber den Atmungsvorgang
Nitrifizierenden Bakterien. II. Beeinflussung der
Atmungdes Nitratbildners dorch Chemische Substanzen.
Idluegers Arch. Ges. Physiol. 8883229-284.

 

Meyerhof, 0. 1917. Untersuchunger uber den Atmungsvorgang
Nitrifizierenden Bakterien. IV. Die Atmung des
Nitritbildners und ihve Beeinfltssung durch Chemische
Substanzen. Pfluegers Arch. Ges. Physiol. $883240-280.

Nelson, D. H. 1931. Isolation and characterization of
Nitrosomonas and Nitrobacter. Zentr. Bakt. II 823280-

Nicholas, D. J. D., and O. T. G. Jones. 1960. Oxidation of
hydroxylamine in cell-free extracts of Nitrosomonas

eurogaea. Nature N853512-514.

Nicholas,D.J’. D., A. Nason, and W. D. McElroy. 1953. The
essentiality of molybdenum for the enzymatic reduction
of nitrate. Amer. Inst. Biol. Sci. Bull. 8364.

Nommik, H. 1957. Fixation and defixation of ammonium in
soils. Acta Agr. Scand. 13395-436.

Pandalai, K. M. 1946. Symbiotic aspects of nitrification.
Nature 1583484-485.

Pramer, D. 1957. The influence of physical and chemical
factors on the preparation of silica gel media. Appl.
Microbiol.Ԥ3392-395.

Pramer, De and Be Le Schmidt. 1965e EIperimental 8011
microbiology. Burgess Publishing Co., Minneapolis.

Prouty, C. C. 1929. The use of dyes in the isolation of
a nitrite oxidizing organism. Soil Sci. 883125-136.

Quastel, J. H., and P. G. Scholefield. 1949. Influence of
organic nitrogen compounds on nitrification in soil.
Nature 16431068-1072.

Rider, B. F., and M. G. Mellon. 1946. Colorimetric determina-
tion of nitrite. Ind. Eng. Chem. Anal. Ed. 88396-99.

Ruban, Eugeniya L. 1958. Nitrogen metabolism of Nitrosomonas
euroEgea. (In Russian). Mikrobiologia 81353 -5 1.

81

Shattuck, G. E., Jr., and M. Alexander. 1963. A differential
inhibitor of nitrifying microorganisms. Soil Sci. Soc.
Am. Proc. 813600-601.

Silver, W. S. 1960. Exogenous respiration in Nitrobacter.
Nature 1853555-556.

Skinner, F. A., and N. Walker. 1961. Growth of Nitrosomonas
euro aea in batch and continuous culture. Arcfi. MIEroBIol.
25 3339-349.

Stapp, C. 1940. Uber Begleitorganismen der Nitrifikations-
bakterien. Zent. BakterIOIe, II Abte 1023193-214.

Stqlanovic, B. J., and M. Alexander. 1958. Effect of
inorganic nitrogen on nitrification. Soil Sci. 883
208-215.

Temple, K. L. 1949. A new method for the preparation of
silica gel plates. J. Bacteriol. 813383.

Ulyanova, O. M. 1960. Isolation and characteristics of
pure cultures of Nitrosomonas from various natural
substrates. (In Russian). Mikrobiologia 823813-819.

Warrington, R. 1891. On nitrification. Part 4. J. Chem.
SOC e 23484-529 e

Welch, L. F., and A. D. Scott. 1959. Nitrification in
nutrient solutions with low levels of potassium.
Can. J. Microbiol. 53425-430.

Wimmer, G. 1904. Beitrag zur Kenrtnis der Nitrifications-
bakterien. z. Hyg. Infektionekrankh, 883135-174.

Winogradsky, S. 1890a. Recherches sur les organismes de
la nitrification. Ann. Inst. Pasteur 83213-231.

. 1890b. Recherches sur les organismes de
la nitrification. Ann. Inst. Pasteur 83257-275.

 

. 18900. Recherches sur les organismes de,
la nitrification. Ann. Inst. Pasteur 83760-771.

 

Winogradsky, S. l891a. Recherches sur les organismes de la
nitrification. Ann. Inst. Pasteur 5392-100

. 1891b. Recherches sur les or anismes de la
nitrIfIcation. Ann. Inst. Pasteur 83577- 16.

 

Yoshida, T., and M. Alexander. 1964. Hydroxylamine forma-
tion by Nitrosomonas europaea. Can. J. Microbiol. 183
923-926-

Zavarzin, G. A. 1958. The-incitant 7f ins :s;:g* j ass of
nitrification. II. The effect of heavy metals on
nitrification. (In Russian). Mikrobiologia 813542-546.

Zobell, C. E. 1943. The effect of solid surfaces upon
bacterial activity. J. 333t9T194- 1&239-56-

83

APPENDIX

Formulas for basal mediaa

 

 

 

Components Medium Ab Medium AAc
g/liter
CaCl2 0.136
NaCl 0.1
MgSOu‘7H20 0.20 0.20
FeSOu’7H20 3.5 x 10"5 3.5 x 10"5
KHZPOu 0.7
NaZHPO4 13.5
KZHP04 0.175
NaHCO3 0.5
KHCO3 1.5
NaMoOu'ZHZO 2.5 x 10'5
Deionized distilled q.s. liter q.s. liter
water

a Does not contain nitrogen. For the addition of
nitrogen to the basal medium consult the text (page 20).

b Modification of the medium described by Pramer
and Schmidt (1965) which contained 0.1 g MgSOn'HZO, 0.014 g
FeCL3'6H20 (no FeSOu), and distilled H20.

° Modification of the medium described by Aleem and
Alexander (1960) which contained 0.175 g MgSOu‘7H20, and

7 iii ‘l‘l1l’.5 II". 31.1... I! u.' VI .‘1 dbl.- .‘ 11... ii I ‘ t '3 l t f I

"71111111"11711111174